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Studies related to the biosynthesis of indole alkaloids Sood, Rattan Sagar 1970

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STUDIES RELATED TO THE. BIOSYNTHESIS OF INDOLE ALKALOIDS BY RATTAN SAGAR SOOD B.Sc. Honours, Panjab University, India, 1964 M.Sc, The University of British Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8 , Canada Date o ABSTRACT The Strychnos skeleton (e.g. preakuammicine, 56) has been postulated i n the literature to rearrange to Aspidosperma (e.g. vindoline, 5) and Iboga (e.g. catharanthine, 6) bases via the inter-4 21 vention of A ' -dehydrosecodine (76). Part A of the thesis describes the syntheses and biosynthetic evaluation of two close relatives, 16,17-dihydrosecodin-17-ol (90) and secodine (107), of the fugitive acrylic ester (76). In the synthetic sequence, condensation of 3-ethylpyridine with 2-carboethoxy-3-(3-chloroethyl)-indole (80) followed by the reduction of the resulting pyridinium chloride (82) gave N[g-{3-(2-hydroxymethylene)-indolyl}-ethyl]-3'-ethyl-3'^piperideine (84). The benzoate ester (85) of alcohol (84) was treated with potassium cyanide to afford N-[g-{3-(2-cyanomethylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine (86). This latter compound upon treatment with methanol and hydrogen chloride gas gave N-J 0-{3- (2-carbomethoxymethylene)-indolyl}-ethyl]-3 1-ethyl-3'-piperideine C88). Formylation of the ester (88) with methyl formate followed by reduction of the resulting enol (89) gave 16,17-dihydro-secodin -17-61 (90). Feeding of [14C00CH3]-16,17-dihydrosecodin-17-ol C90) into Vinca minor L. revealed no_ significant activity into the isolated alkaloids. The substance in fact appeared to be a toxic component with marked deterioration of the plant occurring within 24 hours. In another investigation,, synthetic 16,17-dihydrosecodin-17-ol (90) 3 was dehydrated to secodine (107). Feeding of [ar- Hj-secondine (107) into Virica minor L. showed low but positive incorporation into vincamine (72) and minovine (73). "Blank" experiments revealed that after the maximum period required for the plant to absorb a solution of the labelled compound, 61% remained as monomer (107) while 32% had been converted to the dimers (presecamine and secamine). In conclusion this study while providing some preliminary informa-tion on the later stages of indole alkaloid biosynthesis has also created an entry into more sophisticated biosynthetic experiments. This situation w i l l hopefully lead to a better understanding of the manner in which this large family of natural products is synthesized in'Lthe livi n g plant. In Part B of the thesis some preliminary studies leading to the biosynthesis of vincamine (ebumamine family) are described. The intermediacy of a tetracyclic pyruvic ester (12) was invoked by Wenkert several years ago to rationalise the rearrangement of Aspido-sperma skeleton to vincamine (2) . To confirm this ^speculationa short synthesis of a close relative (i.e., 24) of the postulated precursor was contemplated. In the synthetic sequence reaction of tryptophyl bromide (18), produced by the action of phosphorus tribromide on tryptophol (17), with 3-acetylpyridine ethylene ketal (16) gave N-{g-(3-indolyl)-ethyl]-3'-acetylpyridinium ethylene ketal bromide (19). The pyridinium bromide (19) on catalytic reduction and acid hydrolysis furnished N-[g-(3-indolyl)-ethyl]-3'-acetylpiperidine (21). Alkylation of the ketone (21) using t r i t y l sodium and a l l y l bromide gave N-[g-(3-indolyl)-ethyl]-3'-allyl-3'-acetylpiperidine (22). Osmylation of the a l l y l i c double bond in C22) did not give the desired diol (23). Tentative assignment is given in structure (34) to the polar compound obtained in this manner. TABLE OF CONTENTS Page TITLE PAGE ........ • •. i ABSTRACT ' . . . • i i TABLE OF CONTENTS . ... ........ . iv LIST OF FIGURES • ' ....... v LIST OF TABLES .. .. .. ' v i i i ACKNOWLEDGEMENTS • •. . . • • . ' • • l x PART A .... 1 INTRODUCTION • •. . 2 DISCUSSION • •.. 31 PART I . .... . .' 34 PART II . .. . .................. 73 EXPERIMENTAL .. .. ............ • ...... 91 PART I ............................... • 93 PART II ........ ..\ . .... 109 REFERENCES • •••••••• •.•**• •' •' • • .•*•-•*"•*•'•• • • 118 PART B ........ ..... • 123 INTRODUCTION • 124 DISCUSSION . . .• 129 EXPERIMENTAL . • ' ' ... . 142 REFERENCES .............. ' • 150 LIST OE FIGURES Figure Page PART A 1 Scheme portraying! the; proposed relationship between three main classes of indole alkaloids • 6 2 Barger-Hahn-Robinson-Woodward hypothesis .......... 8 3 Wenkert's prephenic acid hypothesis 9 4 The acetate hypothesis 10 5 The Thomas-Wenkert hypothesis 13 6 A summary of geraniol secologanin pathway ....... 16 7 A summary of the secologanin ->- geissoschlzine pathway .................. 19 8 The rearrangement of Corynanthe -> Strychnos skeleton 21 9 Scott's scheme for the rearrangement of Corynanthe -»-Strychnos skeleton 23 10 Wenkert's proposal for the biosynthesis of Aspido-sperma and Iboga alkaloids 25 11 Some later stages of indole alkaloid biosynthesis . 28 12 Synthesis of 16,17-dihydrosecodi.n-17-ol C90) 35 13 Synthesis of 2^carboethoxy-3"C3'"chloroethyl)^ indole C80) ........ • 36 14 Synthesis of 6,7-diazasteroid (95) 37 15 Nmr spectrum of alcohol 84 41 16 Mass spectrum of alcohol 84 43 17 Nmr spectrum of n i t r i l e 86 .... 48 18 Nmr spectrum ?of).*carbomethoxy ester 88 51 19 Mass spectrum of carbomethoxy ester 88 ... 52 20 Nmr spectrum of 16,17-dihydrosecodin-17-ol (90) ... 56 21 Mass spectrum of 16,17-dihydrosecodin-17-ol (90) .. 57 Figure Page 22 Postulated fragmentation of 16,17-dihydrosecodin-17-ol (90) in the mass spectrometer 58 23 Synthesis of [ar-3H]-16,17-dihydrosecodin-17-ol (90) 61 24 Synthesis of [14C00CH ]-16,17-dihydrosecodin-17-ol (90) . 64 ,- ^ ' . 25 Battersby's'.'synthesis.-of 16,17-dihydrosec6din-17-ol (90) 69 26 Smith's synthesis of tetrahydrosecodine (110) 70 27 Some of the compounds derivable in vivo from 16,17-dihydrosecodinsl7-ol (90) 71 28 Rearrangement of presecamine (type a) to secamine (119) ... 77 29 Rearrangement of presecamine (type b) to secamine (120) 78 30 A summary of data supporting the structure of secodine Q-07) .... . . . . 80 31 Nmr spectrum of secodine (107) 83 32 Mass spectrum of secodine O-07) 84 33 Proposed elaboration of secodine into vincamine and minovine • ............................ 88 PART B 1 Various eburnamine-vincamine type alkaloids 124 2 Scheme showing similar rearrangement of non-tryptophan portion in vincamine and vincadifformine 125 3 Fenkert's proposal for the rearrangement of Aspido-sperma skeleton to vincamine ...................... 126 4 Synthesis of vincamine 127 5 Synthesis of eburnamine . . . .• 128 6 Proposed synthesis of intermediate 24 131 7 Synthesis of some model piperidine systems ........ 132 Figure > Page 8 Nmr spectrum of 28 133 9 Nmr spectrum of 29 135 10 Nmr spectrum of olefin 22 .. • 137 11 Mass spectrum of olefin 22 138 , 12 Postulated fragmentation of 22 in the mass spectro-" meter ....... . . . . 139 LIST OF TABLES Table Page 1 Isolation of alkaloids from Vinca rosea seedlings.. 18 2 Results of the "blank" experiment 117 ACKNOWLEDGEMENTS I wish to express my si n c e r e thanks and a p p r e c i a t i o n to Prof e s s o r James P. Kutney. His encouragement and expert guidance, both as a teacher and a s c i e n t i s t throughout the course of t h i s research have made t h i s t h e s i s p o s s i b l e . I am a l s o g r a t e f u l to Mr. John Beck f o r h i s c o l l a b o r a t i o n w i t h me i n t h i s research and f o r many h e l p f u l d i s c u s s i o n s . The formidable task of l e t t e r i n g i n the formulae and typing of the manuscript was s k i l l f u l l y executed by Miss Diane Johnson. I would l i k e to express my deep sense of g r a t i t u d e to her. PART A STUDIES RELATED TO THE BIOSYNTHESIS OF INDOLE ALKALOIDS INTRODUCTION During the past f i f t y or sixty years, tremendous advances have been made in the laboratory preparation of complex naturally occurring organic substances. As early as the f i r s t part of this present century pioneering work was being carried out in various laboratories. Among the early successes in this vastly challenging f i e l d may be cited the syntheses of numerous terpenes, alkaloids including nicotine, dihydroquinine and the'simple opium'bases; porphyrins including the blood pigments, the common hexoses, as well as many amino acids and peptides. Yet, while the dramatic announcements of multistage syntheses appeared successively during the last several decades, a fainter appeal could be heard emanating from a smaller group of individuals interested in knowing how these complex natural products are formed in nature. It seemed that with the structure of so many natural products having been established, i t should be possible to perceive some relations between them. Such considerations brought numerous suggestions about the possible biosynthetic pathways which may be involved. The f i r s t forays into the biogenetic speculations began with the recognition of common structure features among the compounds produced by closely related natural organisms. This recognition led to infer-ences of a relatively simple common origin for these compounds. Fortunately, i t appears that this fantastic array of naturally.occurring compounds are indeed built up from a relatively small number of fundamental templates. For example, in the f i e l d of alkaloids the common building blocks are acetic acid, ornithine and lysine for the reduced systems and tyrosine, phenyl alanine, 3,4-dihydroxyphenylpyruvic acid, and tryptophan for the many bases containing aromatic nuclei. Once the fundamental building block for a certain group of compounds is established this allows one to speculate on the sequence of transformations regarded as feasible in the living c e l l . Most f r u i t f u l among the efforts to verify these speculations have been the feeding experiments with radioactive precursors. This is followed by isolation of-radioactive natural compound and chemical degradation to isolate particular atoms and examine their radioactivity. It is a tribute to the authors of these biogenetic schemes that they have been so often proved correct. When we examine how> the study of biogenesis of natural products has helped in our understanding, the following two gratifying features always come to one's mind: a) f i r s t of a l l , with the emergence of pathways of biogenesis, i t became possible to organise and divide the natural products into families according to their biogenetic groups. For example, steroids and terpenes, which at one time looked a bewildering array of compounds, now allow convenient correlation through their isoprenoid derivation. Although the present division of natural products into biogenetic groups is sometimes rough and arbitrary and often speculative, i t provides a convenient organization f o r l e a r n i n g and o r i e n t a t i o n which i s b e t t e r than has been p o s s i b l e before; b) the b i o g e n e t i c s p e c u l a t i o n s l e d to the s u c c e s s f u l construc-t i o n of some remarkably simple l a b o r a t o r y syntheses of complex n a t u r a l compounds. These syntheses were modeled on b i o g e n e t i c l i n e s and the s y n t h e t i c schemes are to many chemists, more e s t h e t i c a l l y p l e a s i n g and s a t i s f y i n g . More important however, the b i o g e n e t i c syntheses are o f t e n neater, shorter and more e f f i c i e n t than normal routes i n which no a t t e n t i o n i s paid to the n a t u r a l processess. Indeed i t i s sometimes found that the most s a t i s f a c t o r y route to a p a r t i c u l a r n a t u r a l product i s the b i o g e n e t i c type. The Robinson tropinone s y n t h e s i s i s an e a r l y but s t i l l an e x c e l l e n t example of t h i s approach. A d i r e c t method of c o n s t r u c t i n g the complex str y c h n i n e 2 s k e l e t o n , summarized i n the f o l l o w i n g scheme, i s another i l l u s t r a t i o n . 0 OHC I t must be emphasized here that our r e a l b i o s y n t h e t i c evidence from t r a c e r and enzyme study i s yet i n i t s i n f a n c y and some of the b i o g e n e t i c schemes could be d i s c r e d i t e d or s e r i o u s l y a l t e r e d . I t i s undeniable that w i t h the study of b i o g e n e s i s , the science of n a t u r a l products has r a i s e d to a new l e v e l . In the annals of biogenetic theory perhaps no single class of natural products has enjoyed more ingenious speculations from the organic chemists than the family of indole alkaloids, which are formally derived from tryptamine and a "C_-C " unit (for reviews see y 1 U references 3-5). Not only the biochemical origin of the latter species but i t s appearance in the well known Corynanthe-Strychnos pattern (10, Figure 1) has provoked stimulating comments ever since Barger^ drew attention to a possible biogenesis of yohimbine in 1934. Recent structural studies have increased the number of these alkaloids to more than 800.^ A tryptamine residue (2) appears almost 8 9 invariably and in the few cases examined by the tracer method, ' this residue has been found to be derived in the expected way from tryptophan (1). Tryptamine^ ^ (2) has recently been shown to be specifically incorporated into several alkaloids of Vinca rosea with considerable variation in efficiency, suggesting that decarboxylation may be delayed in some cases. Figure 1. Scheme portraying the proposed relationship between three main classes of indole alkaloids. The remaining nine or ten carbon atoms ( C 0 - C "r u n i t ) appear i n y x u what at f i r s t s i g h t seems a b e w i l d e r i n g v a r i e t y of d i f f e r e n t arrangements but c l o s e r i n s p e c t i o n allows three main groups to be 13 discerned. Together these three main groups account f o r the vast m a j o r i t y of i n d o l e a l k a l o i d s . We can conveniently r e f e r to them as (a) Corynanthe-Strychnos type which possess the C -C n u n i t as (10) • . y 1 U e.g. corynantheine ( 7 ) , a j m a l i c i n e (3); (b) the Aspidosperma type having the C ^ - C ^ Q u n i t as (11) e.g. v i n d o l i n e (5) and (c) the Iboga s e r i e s where the C -C u n i t appears as (12) e.g. catharanthine (6). y l u >_ . In those a l k a l o i d s where only nine carbon atoms.are present i n a d d i t i o n to the tryptamine r e s i d u e , i t i s i n v a r i a b l y the carbon atom i n d i c a t e d by the dotted l i n e that has been l o s t (Figure 1). O r i g i n of Cg-C Q U n i t In c o n t r a s t to the general agreement by d i f f e r e n t workers w i t h regards to the "tryptophan" p o r t i o n of the i n d o l e a l k a l o i d s , the b i o g e n e t i c o r i g i n of the "non-tryptophan" or C_-C - u n i t , has been the y x u subject of much controversy. A number of t h e o r i e s d e a l i n g w i t h t h i s aspect have been proposed over the years. 6 14 I t was suggested many years ago by Barger and Hahn that the i n d o l e a l k a l o i d s such as^ yohimbine (13) are formed by a Mannich r e a c t i o n between tryptamine and 3,4-dihydroxyphenylacetylaldehyde. The i n i t i a l product of t h i s r e a c t i o n (14) then undergoes a second Mannich r e a c t i o n w i t h formaldehyde y i e l d i n g the p e n t a c y c l i c system (15). I t was then suggested"*" that the e x t r a carbon atom, which i s o f t e n at i n r i n g E (Figure 2 ) , i s a l s o derived from formaldehyde. A c o n t r i b u t i o n Corynantheine (7) Figure 2. Barger-Hahh-Robinson-Woodward hypothesis. of Woodward''""' was the suggestion that the c a t e c h o l type r i n g E could undergo f i s s i o n to y i e l d an intermediate such as (16). The two si d e chains attached to r i n g D can then undergo p l a u s i b l e condensations w i t h each other (e.g. a j ^ m l i c i n e , 3; corynantheine, 7) or w i t h other p a r t s of the molecule (e.g. aj^mline 7) to give r i s e to various s t r u c t u r a l types. With t h i s scheme Woodward was al s o able to r a t i o n a l i s e the biogenesis of st r y c h n i n e (8, Figure 2). However, a number of d e f i c i e n c i e s arose w i t h the above theory 17-19 and i n 1959 Wenkert proposed an elegant a l t e r n a t i v e . He suggested that prephenic a c i d (16) acts as a progenitor of the i n d o l e a l k a l o i d s . The l a t t e r rearranges according to the scheme shown i n Figure 3 to a f f o r d a c r u c i a l i n t e r m e d i a t e , the seco-prephenate-formaldehyde (SPF) u n i t (20), which can be incorporated i n t o yohimbine (13) and corynantheine Figure 3. ' Wenkert's prephenic a c i d hypothesis. (7). The most attractive features of this hypothesis are that i t accounted for the presence of carboxyl group at C^g and also rationalises the fact that the hydrogen attached at in yohimbine (13) and related alkaloids almost always has an a-configuration. Wenkert suggested that the a-configuration of the hydrogen atom at C._ in the intermediate (17) is the result of stereospecific migration of the pyruvate side chain in compound (16). Furthermore, there is l i t t l e chance for randomization at in the subsequent modifications of (17) to yield the various indole alkaloids. 13 20-22 Schlitter and Taylor in 1960 and Leete in 1961 postulated that the "non-tryptophan" portion of the indole alkaloids was derived via the acetate pathway. The suggestion was that a six carbon chain derived from three acetate units, condenses with malonic acid and a one carbon unit (biologically equivalent to formaldehyde) yielding the desired _ unit (Figure 4). 0 II H-C-H 3CH-C00H Figure 4. The acetate hypothesis. Another hypothesis based on structural relationships was suggested 17—19 23 independently by Wenkert and Thomas. The striking similarities between the skeletal features of various monoterpenes, verbenalin (21), gentiopicrin (22), bakankasin (23), swertiamarin (24), genipin (25), aucubin (26) andthe seco-prephenate-formaldehyde unit (20, the "Cy-C^Q" unit) led these authors to suggest that the non-tryptophan portion of the indole alkaloids is "monoterpenoid" in origin CH300C OGlu OGlu OGlu 21 22 23 CH20H OGlu CH300 CH300C CH20H OGlu 24 25 26 The i n i t i a l biosynthetic experiments using radioactive precursors-disproved a l l the hypotheses concerning the genesis of "Cy-C^QM portion 24 of the indole alkaloids. It was not until 1965 that Scott and coworkers were the f i r s t to report a successful incorporation of mevalonate (27) into vindoline (5). Subsequent publications by 25—28 several groups of workers established that specifically labelled mevalonic acid was incorporated into the indole alkaloids in a manner consistent with the monoterpene hypothesis. The next logical precursor 29-32 g e r a n i o l (28) was found to be incorporated as an I n t a c t u n i t i n t o v i n d o l i n e ( 5 ) , catharanthine (6) and a j m a l i c i n e (3) i n Vinca rosea L. shoots. Each of these a l k a l o i d s i s r e p r e s e n t a t i v e of one of the three types of a l k a l o i d f a m i l i e s found i n t h i s and other p l a n t s . 4 These f i n d i n g s allowed Battersby to r a t i o n a l i s e the three cat e g o r i e s of i n d o l e a l k a l o i d s as shown i n Figure 5. The Corynanthe -s k e l e t o n (10) i s f i r s t derived f o r m a l l y by cleavage of the g e n e r a l i z e d i r i d o i d p a t t e r n (29). Loss of one carbon atom ( i n d i c a t e d by broken l i n e i n 10) r a t i o n a l i s e s the S-trychnos "C " u n i t (30) , as found i n akuammicine (4). The Corynanthe s k e l e t o n i s s c h e m a t i c a l l y r e l a t e d to the Aspidosperma (11) and the Iboga s e r i e s (12) by cleavage of the C. _-C^g bond i n (10) and formation of the ^2_7~^20 ^P a t' 1 ^ o r ^17-(^14 (path B) bonds. The formation of the yohimbine c l a s s (31) i s a l s o reached from 10, t h i s time by r i n g c l o s u r e v i a C 1 7 - C 1 0 bond formation. 1 / l o The f i r s t evidence f o r the cyclopentane intermediate i n the 33 pathway was obtained by Battersby and coworkers who showed that l o g a n i n (32) was incorporated i n t o a j m a l i c i n e ( 3 ) , v i n d o l i n e (5) and catharanthine (6) i n Vinca rosea L. p l a n t s . These f i n d i n g s were l a t e r confirmed by other workers i n Vinca rosea and Rauwolfia serpentina H0„, CH300C Figure 5. The Thomas-Wenkert hypothesis. p l a n t s . The next step forward i n the contin u i n g search to u n f o l d the b i o s y n t h e t i c pathway of i n d o l e a l k a l o i d s , had to wait u n t i l the 37 s t r u c t u r e of another a l k a l o i d s , i p e c o s i d e (33) was unraveled. The-non-nitrogenous p o r t i o n of i p e c o s i d e was al s o found to be derived from OGlu CH3OOC 33 l o g a n i n (32). This l e d Battersby to suspect that l o g a n i n i s cleaved to y i e l d secologanin (36) and the i l l u s t r a t e d process by way of hydroxy l o g a n i n (34), perhaps as i t s phosphate e s t e r (35), i s a p l a u s i b l e ' one 4,38 OGlu CH300C CH-OOC 34, X = H 35, X = phosphate 36 Confirmatory evidence f o r the existence of the h y p o t h e t i c a l intermediate 39 40 (36) came w i t h the i s o l a t i o n ' of three new g l u c o s i d e s , f o l i a m e n t h i n (37) , di h y d r o f o l i a m e n t h i n (38), and m e n t h i a f o l i n (39). Not only are these glucosides of great b i o s y n t h e t i c i n t e r e s t i n t h e i r own r i g h t , but they a l s o c o n t a i n secologanin (36) i n a masked l a c t o l form. I t i s i n t e r e s t i n g to po i n t out that secologanin i s a bi o i n t e r m e d i a t e c o r r e s -ponding to (10) i n the Thomas-Wenkert hypothesis (Figure 5 ). The synthesis of r a d i o a c t i v e ( d o u b l y - l a b e l l e d ) secologanin and i t s feeding to Vinca rosea resulted in the positive incorporation without scrambling3^'4"'" of label. A most gratifying outcome of a l l these elegant labelling experiments was the finding that the stereochemical OGlu 37 38, 7' ,8'-reduced 39 integrities of -C^  in the loganin (32) and i t s secoderivative (36) aresmaintained at C^ ,. in the Corynanthe alkaloids. Furthermore C^ of the loganin (32) is carried through to the alkaloids at the aldehyde level, i.e., the proton marked with an asterisk in geraniol (28) (see Figure 6) survives a l l subsequent rearrangements. The latter requirement assumes prime importance in formulating and testing the mechanisms developed below. The summary of the geraniol seco-38 loganin pathway is shown in Figure 6 and i t includes the recently A- 2 \^ 3 described ir i d o i d (40) as well as the "secolactone" sweroside (41) which has been biologically converted into the three main classes of indole alkaloids. 4 It has been further argued some years ago that i f secologanin (36) condenses with tryptamine, a 0-carboline (e.g. 42) would be formed OHC CH.OOC OGlu 36 OGlu Figure 6. A summary of geraniol •+ secologanin pathway. and this latter substance was suggested as the f i r s t nitrogenous intermediate in the biosynthetic sequence leading to indole alkaloids. Evidence for this kind of intermediate was obtained with the discovery of strictosidine (42) (stereochemistry not established) i n 44 Rhazya species and i t s presence was subsequently demonstrated in 45 Vinca rosea by dilution with radioactive tryptophan and loganin. A mixture of the radioactive isomers, vincoside (43) and iso-vincoside (44) (enantiomeric at C-) prepared from secologanin of known stereochemistry and tryptamine, when fed to Vinca rosea resulted in the isolation of radioactive ajmalicine (3), vindoline (5), cathar-46 47 anthine (6) and perivine (45). ' Dilution analysis in Vinca rosea COOCH CH^ OOC 42 45 43, a«C H-44, B-C.H 3 3 plants which had previously taken up [5- H]-loganin, confirmed that secologanin (36), vincoside (43) and isovincoside (44) are natural products of the plant. Subsequently N-acetylvincoside was isolated from the glycoside fraction of Vinca rosea^ in good yield (19 mg/ 1.5 kg) and vincoside (43) was shown to be converted to the three types of indole alkaloids. Isovincoside (44) was not an effective 47 precursor. With this knowledge in hand that indole alkaloids are in fact elaborated monoterpenoids, the next problem was to find out how 3 -carboline (42) is converted into various indole alkaloids. A major problem inherent in these unknown steps concerns the timing and mechanisms of transformations whereby the g-carboline system is sequen-t i a l l y transformed not only to form Corynanthe and Strychnos alkaloids but how i t rearranges to the Aspidosperma and Iboga bases. In this regard i t is relevant to mention that Scott's study of the sequential appearance of various alkaloids in short term (1-300 hours) germinating Vinca rosea has helped considerably in suggesting the dynamics of the biosynthetic mechanisms. An interesting account of this work has been reviewed very r e c e n t l y by Scott and a summary of these r e s u l t s i s shown i n Table I . Table I . I s o l a t i o n of A l k a l o i d s from Vinca rosea Seedlings Germination time, hr A l k a l o i d s i s o l a t e d Type 0 None 26 Vincoside (43) A j m a l i c i n e (3) Corynantheine (7) "Corynanthe" 28-40 Corynantheine aldehyde (50) G e i s s o s c h i z i n e (51) 3-Hydroxyindolenine (57) " D i o l " (58) G e i s s o s c h i z i n e Oxindole (59) Corynanthe Corynanthe 40-50 Preakuammicine (56)" Akuammicine (4) Stemmadenine (55) Tabersonine (71) "Corynanthe-Strychnos" "Strychnos "Corynanthe-Strychnos" 72 11-Methoxytabersonine (77) Aspidosperma 100-160 Catharanthine (6) Coronaridine (78) Iboga 200 V i n d o l i n e (5) Aspidosperma The b i o l o g i c a l conversion of v i n c o s i d e (43) i n t o v a r i o u s i n d o l e ' a l k a l o i d s could reasonably i n v o l v e cleavage of the glucose u n i t to form v i n c o s i d e aglucone (46) which would be i n e q u i l i b r i u m w i t h , or con-v e r t i b l e i n t o aldehyde (47,48). Ring c l o s u r e to N(b) (see 49, Figure 7) and r e d u c t i o n could then lead to corynantheine aldehyde (50) and/or g e i s s o s c h i z i n e (51). A j m a l i c i n e ( 3 ) , an abundant a l k a l o i d of Vinca rosea could be reached by cyclization of the species 51 or 49 since the C 3 49 proton of the ajmalicine (3) is not labelled by loganin-2- H. 20 Earlier feeding experiments in Vinca rosea^ established the intact incorporation of geissoschizine (51) into ajmalicine (3), akuammicine (4), vindoline (5), and catharanthine (6). The incorporation of corynantheine aldehyde (50) into vindoline (5) and catharanthine (6) has been reported in Vinca rosea s e e d s . T h i s finding i s in sharp contrast to the insignificant incorporation of this substance in mature plants.50>51 f j o w e v e r Battersby,"^ in trying to reconcile these observations, has suggested that i t could be possible that seedings are able to convert (50) to (51). Recently geissoschizine (51) has been shown to be a component present both in Vinca rosea plants"*^ and 52 Vinca rosea seeds (28-40 hours fraction). This study therefore suggests that geissoschizine (51) stands as a key Corynanthe alkaloid beyond vincoside (43) on the biosynthetic pathway.' A most gratifying outcome of these elegant labelling studies was the rearrangement (a ->- 3) of geissoschizine (51) to form the 'Strychnos skeleton of akuammicine (4)."*^' This observation was particularly important because this rearrangement generates the bond between and C^g (see 51 and 56 in Figure 8). Since i t is necessary to oxidize the Corynanthe series in order to reach the'Strychnos l e v e l , - i t was 19 53 suggested ' that this process when applied to geissoschizine (51) involves one electron oxidative coupling to give strictamine (52^ '•• R = CHO). Precedent for the rearrangement of compounds such as (53) 54 to the Strychnos representative, akuammicine (4) is available, so that the indolenine (54) or i t s reduced form (56) (Figure 8) could be COOCH 4 Figure 8. The rearrangement of Corynanthe -> Strychnos skeleton. reached by such a mechanism. An alternative to this mechanism, also an in vitro a n a l o g y , i s a-protonation of the indole nucleus followed by the a •* g rearrangement summarized in Figure 8. Consistent with this study was the isolation of "C ^ Q" and "C^" Strychnos alkaloids preakuammicine (56) and akuammicine (4) in the 45-50 hours fraction of Vinca rosea seeds. More recently Scott"^ has suggested a third mechanism (Figure 9) which imputes an intermediary role.to an unknown alkaloid geissoschizine oxindole (59). The formation of such an oxihdole from geissoschizine (51) has ample in vitro precedence and might take place in vivo by the steps indicated in Figure 9, where the g-hydroxy indolenine (57) is rearranged directly or via the dihydroxyindoline ("diol" 58) to (59). Conversion of 59 to the imino ether (60, R = alkyl or enzyme bound functionality) would endow 60 with reactivity required"^ to form preakuammicine (56), as shown. Im.support of this mechanism Scott"^ was very gratified to find geissoschizine oxindole (59) (identical with the synthetic material) in the 45-hour fraction of Vinca rosea seeds. To summarise a l l the work up to here, i t is safe to say that a l l the steps involved in the biochemical conversion of secologanin to Corynanthe alkaloids have become clear. With the study of sequential isolation of various biointermediates, the steps involved in the rearrangement of Corynanthe skeleton to Strychnos skeleton have just begun to unfold themselves. The Biogenesis of Aspidosperma and Iboga Alkaloids A most ingenious idea was adduced by Wenkert"*"^  '"^ to rationalise the transformation of the Corynanthe skeleton to the Aspidosperma and Figure 9. Scott's scheme for the rearrangement of Corynanthe •> Strychnos skeleton. Iboga type (Figure 10) (type A and B transformations at the a l k a l o i d l e v e l i n Figure 5). The rearrangements suggested f o r paths A and B r e q u i r e d the presence of the 1,5-dicarbonyl f u n c t i o n i n order to operate the reverse Michael r e a c t i o n i m p l i c i t i n the cleavage of C-^-C^g. Thus the cleavage product (63) undergoes or d i n a r y o x i d a t i o n - r e d u c t i o n changes and i n t h i s manner p i p e r i d i n e s of various o x i d a t i o n s t a t e s are formed. Intramolecular Michael and Mannich r e a c t i o n s of the l a t t e r l e a d to Aspidosperma (65) and Iboga l i k e (68) s k e l e t o n s . However, feeding experiments w i t h r a d i o a c t i v e precursors r a i s e d some ser i o u s doubts regarding some of the steps depicted i n Figure 10. For example transannular c y c l i z a t i o n u t i l i z e d by Wenkert to provide an entry i n t o Aspidosperma (64 ->- 65) and Iboga (67 -»• 68) s k e l e t o n s , i n s p i t e of having e x c e l l e n t analogies i n v i t r o " ^ ^ was shown by Kutney^ 3'^ 4 to be an i n s i g n i f i c a n t biochemical r e a c t i o n . This l a t t e r study suggested that the genesis of p e n t a c y c l i c a l k a l o i d s (Aspidosperma type) e\g. v i n c a -d i f f o r m i n e (70) i s completely independent of the nine-membered a l k a l o i d s e.g. vincadine (69). C00CH„ COOCH. 69 70 Aspidosperma-type alkaloids 68 0 Figure 10. Wenkert's proposal for the biosynthesis of Aspidosperma and Iboga alkaloids. Returning to the chronological isolation of various alkaloids . from Vinca rosea seeds (Table I) Scott"^ found another alkaloid stemmadenine (55) in the 50-hour experiment. It is very interesting to point out here that the possible intermediacy of units similar in structure to stemmadenine e.g. 62 was invoked in the sequence between the Corynantheinoid and Aspidosperma bases by Wenkert"^ '"^ (Figure 10) many years ago. When the germination was allowed to proceed further j (72 hours), i t led to the isolation of an Aspidosperma alkaloid, tabersonine (71). Catharanthine (6), the principal Iboga alkaloid of Vinca, although isomeric with tabersonine, does not appear to be formed until the germination has proceeded for 100 hours. This was a v i t a l piece of evidence in suggesting a rough sequence of alkaloid formation in nature i . e. I stemmadenine (55) -> tabersonine (71) catharanthine (6). The biochemical conversion of tabersonine (71) to vindoline (5) and most interestingly to the Iboga alkaloid 64 catharanthine (6) has been demonstrated in our laboratories and independently by Scott"'"'" in Vinca rosea seeds. These latter results suggest a possible relationship between the Aspidosperma and Iboga alkaloids. Similarly belief in stemmadenine (55) as a true biointer-mediate was further strengthened by i t s incorporation into tabersonine (71) and catharanthine (6) in Vinca rosea seeds"'"'" and into vincamine (72) and minovlne (73) in Vinca minor plants in our laboratories.^ 66 Scott and Qureshi reported the rearrangement of tabersonine (71) to (J)-rcatharanthine (6) and (±)-pseudocatharanthine (74) in refluxing acetic acid. Stemmadenine (55) under similar conditions (refluxing acetic acid)rearranged to (_)-tabersonine (71), (i)-catharanthine (6), and Gt)-pseudocatharanthine (74). These results were portrayed as a laboratory simulation of the biochemical results described above. 6 7 However, lately Smith inspite of many repeated attempts failed to duplicate Scott's in vitro results. A most attractive mechanism linking stemmadenine (55), tabersonine 64 (71), and catharanthine (6) was advanced by.Kutney. This involves the achiral intermediate 76a=76b, which can, in principle be generated by migration of the double bond in stemmadenine (55) to (75), followed by the illustrated fragmentation (Figure 11). A similar postulate for the formation of the acrylic ester (76) has been independently advanced by Scott*^. in order to explain the observed 71, Tabersonine (R=H) . I 77, 11-Methoxytabersonine (R=0CH~) 6, Catharanthine •1 ^ 15 2 0 ^ -5, V i n d o l i n e 78, Coroharidine; A '. -—reduced Figure 11. Some l a t e r stages of i n d o l e a l k a l o i d b i o s y n t h e s i s . sequence, i t is suggested that the enzymatic folding of 76 in mode A would give tabersonine (71), and later, at an other enzymatic site, cyclization iniwmode B forms catharanthine (6) . Yet a third possibility C explains the genesis of the vincadine (69) series. The relative 63 64 insignificance of the transannular cyclization ' now suggest that the process 7 ->• 21 occurs prior to or simulataneously with 17 20 in the elaboration of the putative intermediate (76) to vincadifformine (70). In a similar fashion the conversion of the unit (76) to the alkaloid catharanthine (6) is unlikely to proceed i n i t i a l l y via the process 17 -> 14, since this would lead to a carbomethoxycleavamine 63 system (79). Previous results in our laboratory have suggested that carbomethoxycleavamine (79) is not a progenitor of this Iboga alkaloid (6). This theory therefore places stemmadenine (55) i n a key position between the Strychnos and other families not only in yinca rosea but predictably in a l l species, and furthermore rationalizes the formation of racemic Aspidosperma alkaloids such as (+)-vincadifformine (70) mediated by the achiral ester (76). The absolute minimum of function-a l i t y has been used for a l l of these postulated interconversions, and i t i s g e n e r a l l y b e l i e v e d that the proposed biogenesis i s common to a l l species. Thus i t so turned out that the galaxy of complex, oxygenated, fragmentated, and rearranged s t r u c t u r e s which c o n s t i t u t e s the complex s e r i e s of i n d o l e a l k a l o i d s i n fact.stem from these few fundamental a l k a l o i d s . I t must be emphasized here that although a l l the evidence i n d i c a t e d to develop*the above theory represent an important m o d i f i c a t i o n of Wenkert's o r i g i n a l theory p a r t i c u l a r l y w i t h regards to sequence, o x i d a t i o n l e v e l and mechanisms, these r e s u l t s do not de t r a c t 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 of the main c l a s s e s of i n d o l e a l k a l o i d s . In summary, a l l the a v a i l a b l e r e s u l t s suggest very emphatically that the a c y l i c e s t e r (76) would f u l f i l a p i v o t a l r o l e i n the genesis of v a r i o u s f a m i l i e s of i n d o l e a l k a l o i d s . Laboratory analogies f o r almost a l l of the suggested processes are now a v a i l a b l e . With the establishment of the Corynanthe-Strychnos-Aspidosperma-Iboga r e l a t i o n -ship Chased on s e q u e n t i a l i s o l a t i o n and feeding experiments) the 4 v a r i o u s f u r t h e r subclasses should f a l l i n t o p l a c e . In 1967 Battersby made the statement "The problem i s at a most f a s c i n a t i n g stage where the researcher can see that the p r e c i s e d e t a i l of the pathways to the in d o l e a l k a l o i d s cannot now escape him". The r a p i d l y e v o l v i n g scene - a s summarized above provides ample proof i n support of t h i s view. DISCUSSION With the knowledge that indole alkaloids are in fact elaborated mono-terpenoids, we were intrigued by the second major problem posed by the structures before us; how are the rearrangement of Corynanthe to Aspidosperma and Iboga skeletons carried out in nature (summarized in Figure 11), and where would we begin to test the virtual myraid of possible substrates designed to undergo the A and B transformations? It was fu l l y revealed in 53 the introduction that Wenkert's speculations on the mechanisms of rearrangements involve the 'acrylic acid (66) and i t s dihydro-derivative (63) as intermediates, whilst Kutney's^4 and Scott's^ results led them to propose a different mechanism making use of acrylic.- ester .(76) and the corresponding enamine. The intermediacy of .acrylic'j ester (76) was again 66 invoked by Scott to explain the in vitro transformation of tabersonine (71) to (+)-catharanthine (6) and (+)-pseudocatharanthine (74); and of stemmadenine (55) to (i)-tabersonine (71), (+)-catharanthine (6) and (+)-pseudocatharanthine (74) (as already mentioned earlier, these later trans-6 7 formations have now been questioned ). In summation, a l l these results provided a strong suggestion that the acrylic"ester (76) may be a true biointermediate. We decided that in spite of many problems associated with the synthesis and feeding of this putative intermediate (76) , the knowledge gained by i t s evaluation as a biogenetic intermediate would be of greatest value in suggesting the dynamics of the biosynthesis of Aspidosperma and Iboga a l k a l o i d s . To examine the b i o g e n e t i c r o l e of any i n t e r m e d i a t e , the f i r s t step i n v o l v e s the s y n t h e s i s of the p o s t u l a t e d precursor. This i s then followed by feeding the a c t i v e precursor i n t o the appropriate p l a n t system and i s o l a t i o n of the a l k a l o i d s a f t e r a c e r t a i n p e r i o d of time to examine the amount of r a d i o a c t i v i t y i n them. I t was c l e a r to us at the outset of our s y n t h e t i c work that (76) because of the presence of a d i h y d r o p y r i d i n e segment i n i t would be a very unstable compound. There was considerable precedent a v a i l a b l e i n the l i t e r a t u r e which could lend support to our i n i t i a l doubts. For example, i t i s w e l l known that d i h y d r o p y r i d i n e s r e a d i l y o x i d i z e to the corresponding p y r i d i n e s . This process occurs so r a p i d l y that even contact w i t h atmospheric oxygen i s s u f f i c i e n t to b r i n g about the t r a n s f o r m a t i o n . This property t h e r e f o r e makes the c h a r a c t e r i z a t i o n and study of the 68 p r o p e r t i e s of the d i h y d r o p y r i d i n e s r a t h e r d i f f i c u l t . Another r e a c t i o n c h a r a c t e r i s t i c of d i h y d r o p y r i d i n e s i s t h e i r r a p i d i s o m e r i z a t i o n . Although complete experimental d e t a i l s are hot known,, . any reagent that can a s s i s t i n the removal of a proton, hydrogen atom, or hydride 68 i o n may cause i s o m e r i z a t i o n . The f o l l o w i n g scheme i l l u s t r a t e s the i s o m e r i z a t i o n of 1,2- and 1,4-dihydropyridines through a p y r i d i n i u m i o n . + R + R1 R R' I n v e s t i g a t i o n s d i r e c t e d towards e l u c i d a t i n g the s t r u c t u r e s and mode of b i o l o g i c a l a c t i o n of coenzymes NAD and NADP have f r e q u e n t l y engendered st u d i e s on the chemistry of d i h y d r o p y r i d i n e s . On s e v e r a l 69 occasions, the l a t t e r have been found to dimerize. With a l l t h i s knowledge in.'hand, i t became o b l i g a t o r y to make some model compound as our s y n t h e t i c t a r g e t . We understood that t h i s model compound w h i l e s u f f i c i e n t l y s t a b l e i n i t s own r i g h t (to allow c h a r a c t e r i z a t i o n ) should be capable of transformation i n v i v o to the p u t a t i v e intermediate (76) v i a b i o l o g i c a l l y f e a s i b l e r e a c t i o n s . One such compound which m e t ^ a l l these p r e r e q u i s i t e s was 16,17-dihydrosecodin-17-ol (90, see Figure 12) (the name f o r t h i s compound was suggested by B a t t e r s b y , ^ numbered according to b i o g e n e t i c p r i n c i p l e s ^ ) . This compound upon dehydration (COOCH^-C y-^ •-—• C00CH.-C=CH-) and H OH + o x i d a t i o n i n the p i p e r i d i n e r i n g (H-C-N- —>- -C=N-) could generate the d e s i r e d intermediate. Since both these r e a c t i o n s are b i o g e n e t i c a l l y . f e a s i b l e , we made the a l c o h o l (90) as our i n i t i a l s y n t h e t i c t a r g e t . For the sake of convenience and ease of p r e s e n t a t i o n , the d i s c u s s i o n has been d i v i d e d i n t o two p a r t s . The f i r s t p art describes the synth e s i s 3 of 16,17-dihydrosecodin-17-ol (90) as w e l l as syntheses of [ar- H]-16,17-14 dihydrosecodin-17-ol (90) and [ COOCH ]-16,17-dihydrosecodin-17-ol (90). 14 F i n a l l y feeding of the C-precursor i n t o Vinca minor L. i s presented. The second p a r t describes the syntheses of secodine(107, the name f o r ..96.i - 3 t h i s compound was suggested by Smith- ') , [ar- H]-secodine (107) and feeding of the a c t i v e precursor i n t o Vinca minor L. PART I The d e s i r e d a l c o h o l (90) was amenable to synth e s i s by the route shown i n Figure 12. The choice of t h i s route was d i c t a t e d by the f a c t that i t was p o s s i b l e to synthesize 2-carboethoxy-3-(g-chloroethyl)-i n d o l e (80) i n the l a b o r a t o r y i n reasonable q u a n t i t i e s . This m a t e r i a l i n t r i n s i c a l l y incorporated a l l the s t r u c t u r a l requirements needed to s t a r t our sequence. For example the presence of the a-carboethoxy group acted as a handle i n a l l o w i n g us to expand the si d e chain at the a - p o s i t i o n of the i n d o l e i n (80) to the de s i r e d f u n c t i o n a l i t y . On the other hand presence of c h l o r i n e i n the 3-(g-chloroethyl) s i d e chain allowed us to a t t a c h the appropriated s u b s t i t u t e d p y r i d i n e to the i n d o l e nucleus. The s y n t h e s i s of the d e s i r e d c h l o r o i n d o l e (80) was devised a few years ago i n our l a b o r a t o r i e s i n connection w i t h some other work on the t o t a l s y n t h e s i s of i n d o l e a l k a l o i d s ^ and the sequence i s f u l l y 72 revealed i n Figure 13. Diethyl-y-chloropropylmalonate (91) was prepared i n 70% y i e l d by t r e a t i n g the monosodium s a l t of the d i e t h y l -malonate w i t h 1,3-bromochloropropane. The chloromalonate d e r i v a t i v e (91) was converted i n t o the corresponding arylhydrazone (93) through 73 74 the agency of a Jap'p-Klingemann r e a c t i o n . ' This procedure i n v o l v e d the slow a d d i t i o n of anhydrous benzenediazonium c h l o r i d e ^ to the anion of (91) i n ethanol at -5°. The r e a c t i o n mixture was allowed to stand overnight i n t h e r e f r i g e r a t o r and the crude r e a c t i o n product was subjected to a F i s c h e r i n d o l e s y n t h e s i s ^ using s u l f u r i c a c i d as c a t a l y s t . We would l i k e here to make c e r t a i n remarks regarding the p r e p a r a t i o n OH <j)COCl Figure 12. Synthesis of 16,17-dihydrosecodin-17-ol (90). COOEt I Cl-(CH_)_-Br + CH_ z 3 j z COOEt i ) NaOEt + - > i i ) C , H j L c i or C,HCN0BF. 6 5 2 6 5 2 4 CI-(CH ) 3-CH(COOEt) 91 EtOOC COOEt 92 COOEt 93 EtOH COOEt 80 Figure 13. Synthesis of 2-carboethoxy-3-(3-chloroethyl)-indole (80) of anhydrous benzenediazonium c h l o r i d e . The usual method f o r making t h i s diazo s a l t i s given by Smith and W a r i n g . ^ These authors report that t h e i r method gives the s a l t i n c r y s t a l l i n e form. However our experience w i t h t h i s procedure i s i n sharp c o n t r a s t to t h i s c l a i m . In s p i t e of having followed the reported procedure very c a r e f u l l y , we c o n t i n u a l l y ended up w i t h lumps of the s o l i d diazonium c h l o r i d e . Since the r e a c t i o n c o n d i t i o n s demanded that we add the diazonium s a l t i n s m a ll p o r t i o n s , t h e lumps had to be broken i n t o s m all pieces (under n i t r o g e n ) . This d i d not pose much of a problem i n small s c a l e r e a c t i o n s , but i t s t a r t e d r a i s i n g i t s ugly head when the r e a c t i o n was scaled up to obtain larger quantities of the chloroindole (80). In one of the instances the crushing of the lumps into small pieces resulted in a very (Violent explosion. A l l of these unfortunate incidents kept on thwarting our progress for a long time as the supply of chloro-indole (80) remained very limited. In the meantime a new synthesis of 6,7-diazasteroid (95, see Figure 14) appeared in the l i t e r a t u r e ^ and the use of m-methoxybenzenediazonium fluoroborate was reported. This 78 led us to wonder i f we could also use the more stable benzenediazonium fluoborate in our sequence. Indeed coupling of the anion of (91) with benzenediazonium f l u o b o r a t e ^ ' ^ gave a deep red o i l . Thisvproduct was Figure 14. Synthesis of 6,7-diazasteroid (95). subjected to a Fischer indole synthesis. Purification of the crude product by chromatography on alumina gave a crystalline compound which showed the same Rf value on thin layer chromatography (t.l.c.) and spectral properties as the chloroindole (80) obtained when benzene-diazonium chloride was used. The most gratifyingAoutcomesof this undertaking were that: (a) benzenediazonium fluoborate, when dry was always a very fine powder (like talcum face powder), a situation which remarkably simplified our technical problem in the Japp-Klingemann reaction namely the slow addition of diazonium salt to the anion of (91); (b) the fact that the fluoborate salt was f a i r l y stable and easy to handle, allowed us to scale up the preparation of this salt to 120 gm/batch, thereby allowing large scale preparation of the chloro-indole (80). With a l l these problems unraveled, we were able to start our synthetic sequence with confidence. For various reasons we thought the best reaction to s t a r t ( i n i t i a l l y was the coupling of 3-ethylpyridine to the chloroindole (80). For this purpose 3-ethylpyridine (81, bp 162-163°) was readily obtained from commercially available 3-acetylpyridine by means of Wolff-Kishner 80 reduction. Condensation of chloroindole (80) with 3-ethylpyridine gave a white amorphous solid (82) in 91% yield. In general this salt was used directly for the succeeding step. However a small amount of material was crystallized for analytical purposes, mp 87-89°. The spectral data compared favourably with the assigned structure (82). The infrared spectrum indicated a strong ester absorption at 1701 cm In the nmr spectrum the resonances of the two ethyl groups (-CH^CH^, -C00C2H,_) were clearly separated; two triplets at x 8.98 (3H, -CH^S'CH^) and 8.63 (3H, -COOCH^CH^) ; two quartets at T 7.40 (2H, -CH_2CH3) and 5.74 (2H, -COOCH^CH^). In the spectrum the resonances corresponding to the aromatic protons of the pyridinium nucleus (4H, x 1.4-2.34) and of the i n d o l e nucleus (4H,:;T 2.56-3.18) were a l s o c l e a r l y discerned. Because of the overlapping absorptions of the p y r i d i n i u m and i n d o l e n u c l e i , the u l t r a v i o l e t spectrum was not too i n f o r m a t i v e . F i n a l l y the molecular formula, '-'20^23^2^2^''"' W a S s u P P o r t e < * by m a s s s p e c t r o -metry (M + 358). With the p y r i d i n i u m s a l t (82) i n hand, r e d u c t i o n to the t e t r a h y d r o -p y r i d i n e (83) was then considered. At' t h i s stage i t was thought that i f we could use l i t h i u m aluminum hydride (LAH) f o r t h i s purpose, r e d u c t i o n of the p y r i d i n i u m segment i n s a l t (82) to the t e t r a h y d r o -p y r i d i n e stage would be accompanied by conversion of the e s t e r f u n c t i o n to the d e s i r e d primary a l c o h o l (84). However a survey of the l i t e r a t u r e revealed that w h i l e there i s a general agreement between v a r i o u s workers that sodium borohydride always reduces N - a l k y l p y r i d i u m s a l t s 81—83 to t h e i r corresponding t e t r a h y d r o p y r i d i n e s , the r e s u l t s from the LAH reductions are at v a r i a n c e . For example, i t was found by 81 Panouse that N - a l k y l p y r i d i n i u m s a l t s are reduced by LAH to 1 - a l k y l -1,2-dihydropyridines. Reduction of N - [ 6 - ( 3 - i n d o l y l ) - e t h y l ] - p y r i d i n i u m 82 bromide (96) by means of sodium borohydride-or LAH has been reported 83 to l e a d e x c l u s i v e l y to.the t e t r a h y d r o p y r i d i n e (97). L a t e r Wenkert reported the formation of three compounds (97-99) upon r e d u c t i o n of 3' 98, A -reduced (96) w i t h LAH. A l l these c o n t r a d i c t o r y f i n d i n g s diminished our enthusiasm f o r LAH r e d u c t i o n . Indeed when a small amount of p y r i d i n i u m c h l o r i d e (82) was exposed to LAH, we were not s u r p r i s e d to f i n d that the crude product was a complicated mixture of s e v e r a l components. A more s u c c e s s f u l a l t e r n a t i v e i n v o l v i n g two r e d u c t i v e steps provided the d e s i r a b l e r e s u l t s . Thus sodium borohydride r e d u c t i o n of the s a l t (82) to the t e t r a h y d r o p y r i d i n e (83) followed by LAH r e d u c t i o n of the carbo-ethoxy group i n the l a t t e r y i e l d e d a l c o h o l (84). The nmr spectrum of the crude sodium borohydride product was very i n f o r m a t i v e . The resonance corresponding to the four protons of the p y r i d i n i u m nucleus i n (82) (x 1.2-2.34) had completely disappeared and i n s t e a d a broad one proton s i n g l e t at x 4.5 was r e c o n c i l a b l e w i t h the presence of an o l e f i n i c proton at C ^ v - - In t h i s t e t r a h y d r o p y r i d i n e (83), the i s o l a t e d double bond has been t e n t a t i v e l y placed i n the - 3' 4' A ' . I t s p o s i t i o n would be made unambiguous i n the next step. The crude t e t r a h y d r o p y r i d i n e e s t e r (83) obtained above was subjected d i r e c t l y to the l i t h i u m aluminum hydride r e d u c t i o n . The r e s u l t a n t l i g h t y e l l o w gum which was p u r i f i e d by chromatography on alumina provided a c r y s t a l l i n e compound (84) , mp 108^-110°, i n an o v e r a l l y i e l d of 70% from the c h l o r o i n d o l e (80). The s t r u c t u r e (84) was assigned on the b a s i s of the f o l l o w i n g spectra data. In the i n f r a n there was no-.absorption i n ; the-ester region, and i n s t e a d a broad ^ band at 3340 cm ^ (-CH^OII) was now evident. The nmr spectrum (Figure 15) showed the newly formed hydroxymethylene .'.group as a sharp s i n g l e t at T.5.17. The o l e f i n i c proton (C^ i-H) was l o c a t e d at x 4.45 i n good 83 agreement w i t h the assignment reported by Wenkert. The mass spectrum Figure 15. Nmr spectrum of alcohol 84. (Figure 16) showed a molecular i o n peak at m/e 284 and was dominated by two s i g n i f i c a n t peaks at m/e 160 and m/e 124. These peaks were a t t r i b u t e d to the simple fragmentation of the parent molecule to the ions (100) and (101) r e s p e c t i v e l y . F i n a l l y the molecular formula, C.0H .ON , was confirmed by high r e s o l u t i o n mass spectrometry (Found: l o _4 _ 284.184; Calc.: 284.188) and elemental a n a l y s i s . Now to elaborate the s i d e chain at the a - p o s i t i o n of the i n d o l e i n a l c o h o l (84) to the d e s i r e d f u n c t i o n a l i t y , i t was necessary at t h i s stage to i n c o r p o r a t e an e x t r a carbon atom. The sequence, 84 -> 86, proved most d e s i r a b l e f o r t h i s purpose. The conversion of the a l c o h o l (84) to benzoate (85) was accomplished by d i s s o l v i n g the a l c o h o l i n dry p y r i d i n e and t r e a t i n g i t w i t h benzoyl c h l o r i d e . The whole r e a c t i o n was over w i t h i n three hours and the crude product obtained was homo-geneous on t i c . This allowed i t s u t i l i z a t i o n i n the succeeding step without any p u r i f i c a t i o n . However f o r a n a l y t i c a l purposes a s m a l l amount of m a t e r i a l was f u r t h e r p u r i f i e d by chromatography on alumina and r e c r y s t a l l i z a t i o n from methylene c h l o r i d e and petroleum ether, mp 110.5-112.5°. The s p e c t r a l data of the benzoate (85) was i n complete accord w i t h the f o r m u l a t i o n . The i n f r a r e d showed the carbonyl of the benzoate e s t e r at 1715 cm ^ while i n the nmr spectrum, the aromatic CN in. >-f — CO U J I—in. Ijj ct: £75 50 i " " i " i — r 100 150 200 M/E Figure 16. Mass spectrum of alcohol 84. ALCOHOL (84) s ! ' /.I i — i r^-r " i — i — i r i i i r 250 300 350 400 protons of the benzoyl group although overlapping with the protons of the indole nucleus, appeared between x 2-3 (9H). The mass spectrum indicated a molecular ion peak at m/e 388; Finally the molecular formula, C„ oH o c0„N o, was confirmed by high resolution mass spectrometry z o Z J z z (Found: 388.215; Calc.: 388.216) and elemental analysis. Although the above procedure for making the benzoate derivative (85) was quite satisfactory, i t was observed that i f a l l the;rpyridine (used as solvent) was not carefully removed from the crude product, the yield in the succeeding reaction was somewhat lower. In«view of the known susceptibility of benzoates to heat, the pyridine had to be removed at room temperature in vacuo. This process was tedious and generally required leaving the compound under vacuo for considerable period//,' of time. In order to obviate this d i f f i c u l t y several alternative procedures were studied. The optimum conditions involved dissolving the alcohol (84) in tetrahydrofuran and treating the mixture with benzoyl chloride in the presence of solid potassium carbonate. The crude product thus obtained was chromatographed on alumina. Elution with chloroform gave a crystalline compound which had the same and spectral properties as the benzoate obtained above. The overall conversion of alcohol (84) to benzoate (85) by this second procedure was essentially quantitative and the quality of the product obtained was much superior. With benzoate (85) in hand, the next step forward demanded the nucleophilic displacement of the benzoate group with cyanide anion. This reaction contrary to our expectation, turned out to be a very temperamental process. This conversion posed many problems and some of them are delineated below: (a) the reaction proved extremely sensitive to temperature. After running a few small-scale•trial reactions i t was clear that the temperature had to be raised very  slowly from room temperature to 105° otherwise considerable tarring of the reaction mixture occurred; (b) we found that the desired compound (86) was also very sensitive to temperature. This implied that when the temperature of the reaction mixture reached 105°, i t had to be maintained at this elevated temperature for a minimum amount of time. If the reaction mixture was l e f t longer than required at high temperature (105°), an overall yield of 20% as compared to 65% was obtained. Furthermore the cyano compound (86) obtained was also contaminated with many spurious side products. At this stage i t is not possible to define the various side reactions that occurred at this high temperature. For the purpose of control in this reaction thin layer chromatography played an important role. Fortunately i t so happened that the benzoate (85) and the cyano compound (86) showed very distinct colors when the t i c plate was sprayed with antimony pentachloride. Benzoate (85) appeared as a dark blue spot while the cyano compound (86) appeared as light green. Upon disappearance of the benzoate in the mixture (as monitored by tic) the reaction was immediately terminated; (c) f i n a l l y we experienced some trouble in the purification of the cyano compound (86) by chromatography. It was found that when a large amount of alumina was used (ratio of compound to alumina 1:100) in anticipation of achieving better separation, i t led to considerable polymerization of the desired compound. This fact was even more pronounced when the columns employed were slow running or in other words when the cyano compound (86) was l e f t on the column f o r a longer p e r i o d of time. In a l l these cases only very dark p o l a r gums were obtained from the columns. However when the columns employed were very short and f a s t running, the cyano compound (86) was e l u t e d i n c r y s t a l l i n e form and i n a h i g h l y s a t i s f a c t o r y .yield.'. In summation, i t could be s a i d that t h i s displacement r e a c t i o n was.very c r i t i c a l ( i n terms of temperature, time, and p u r i f i c a t i o n by chromato-graphy) but the whole process turned out to be f a i r l y e f f i c i e n t when pr o p e r l y executed. The optimum c o n d i t i o n s f o r the above r e a c t i o n r e q u i r e d d i s s o l v i n g the benzoate (85) i n dimethylformamide and adding s o l i d potassium cyanide ( 10 f o l d excess) to i t . This heterogeneous mixture was s t i r r e d at room temperature f o r one hour.and then the temperature of the o i l bath was g r a d u a l l y r a i s e d to 105° over a p e r i o d of 45 minutes. The r e a c t i o n mixture was maintained at t h i s elevated temperature f o r about one hour. At t h i s time t i c i n d i c a t e d no more s t a r t i n g m a t e r i a l . The r e a c t i o n was immediately stopped and the pure cyano compound (86) was i s o l a t e d by chromatography on alumina. The i n i t i a l e l u t i o n s w i t h benzene-petroleum ether (1:1) provided a c r y s t a l l i n e compound (55%) w h i l e the l a t t e r f r a c t i o n s (benzene e l u t i o n ) were gummy (10%). T i c examination of t h i s gum i n d i c a t e d that t h i s p o r t i o n of the cyano^compound contained a very minute impurity but the compound was of reasonable q u a l i t y to be u t i l i z e d i n the next r e a c t i o n . For a n a l y t i c a l purposes a small amount of the m a t e r i a l was r e c r y s t a l l i z e d from dichloromethane-petroleum ether,.mp 135-137°, and l a t e r sublimed at 100°/0.01 mm. The s p e c t r a l data compared favourably w i t h the assigned s t r u c t u r e (86). The i n f r a r e d was d i a g n o s t i c f o r the presence of a n i t r i l e group (2256 cm w h i l e the strong e s t e r peak present i n the benzoate (85) at 1715 cm ^  had completely disappeared. Ins the nmr spectrum (Figure 17) the methylene group adjacent to the n i t r i l e (-CH^-CN) appeared as a sharp s i n g l e t at x 6.2. The mass spectrum i n d i c a t e d a molecular i o n peak at m/e 293 and was dominated by two peaks at m/e 124 and m/e 169. F i n a l l y the molecular formula, C 1 QH 0.N , was confirmed by high i y Z J J resolutiontmass spectrometry (Found: 293.186; C a l c . : 293.189) and elemental a n a l y s i s . Now that a l l the problems associated i n o b t a i n i n g the cyano compound (86) had been unraveled, we considered i t s conversion to the carbomethoxy e s t e r (88). Two r e a c t i o n s u t i l i s e d most widely f o r t h i s purpose are: (a) h y d r o l y s i s of n i t r i l e s to corresponding c a r b o x y l i c acids and subsequent e s t e r i f i c a t i o n of the l a t t e r to e s t e r s ; (b) t r e a t -ing the n i t r i l e s w i t h methanol and h y d r o c h l o r i c a c i d . Both of these procedures were t r i e d f o r our own purpose. Treatment of the n i t r i l e (86) w i t h a l c o h o l i c potassium hydroxide gave the corresponding c a r b o x y l i c a c i d (87). We i n i t i a l l y had some "fears that the c a r b o x y l i c a c i d (87), because of i t s amphoteric character might pose some problem during i t s workup. But contrary to our e x p e c t a t i o n the r e a c t i o n products could be ex t r a c t e d q u a n t i t a t i v e l y from the aqueous l a y e r once the pH of the r e a c t i o n medium was c a r e f u l l y brought to 7. The impure a c i d (87) obtained i n t h i s way was e s t e r i f i e d w i t h diazomethane. Unfortunately t i c of the crude product showed i t to be a mixture of many components. However, the d e s i r e d compound was obtained pure by very c a r e f u l chromatography on alumina ( y i e l d 25-30%). When the c a r b o x y l i c a c i d (87) was e s t e r i f i e d w i t h methanol and s u l f u r i c a c i d , again a y i e l d of 25% was obtained. The poor,yield obtained above necessitated an investigation of the alternative procedure mentioned earlier, namely the methanolysis of n i t r i l e s . For this purpose the cyano compound (86) was dissolved in a mixture of methanol and 12 N HC1 (1:1) and the contents were stirred at room temperature for three days. We were very happy when the crude product indicated essentially one spot on t i c . But when this product was flushed through a small alumina column to get r i d of what was apparently a minor polar baseline contaminant, there was a considerable loss of material. The pure compound obtained represented only a 30% yield in the conversion, 86 88. This implied that the baseline material was either the bulk in the crude product or else decomposition of ester was occurring on the column. A l l our i n i t i a l attempts to improve the yield of the conversion (86 88) failed completely. A considerable amount of time was spent 84 with no apparent success. Fortunately during this time Wenkert published a synthesis of dl-dihydrogambirtannine (102). The most interesting part of the synthesis, which was pertinent to our own work, was the conversion of n i t r i l e (103) to chloroester (104) by treatment OH 'CI C00CH OCH 103 CN 104 3 w i t h methanolic h y d r o c h l o r i c a c i d . The high y i e l d of c h l o r o e s t e r (104) obtained i n t r i g u e d us to u t i l i z e the same r e a c t i o n c o n d i t i o n as i n d i c a t e d by Wenkert. So the cyano compound (86) was d i s s o l v e d i n methanol c o n t a i n i n g 1% water. This s o l u t i o n was saturated w i t h HC1 gas and the r e s u l t i n g mixture was s t i r r e d at room temperature f o r 60 hours. The crude product i n d i c a t e d e s s e n t i a l l y one spot on t i c (alumina, chlor o f o r m / e t h y l a c e t a t e , 1:1). When t h i s product was f l u s h e d through an alumina column, we were very s u r p r i s e d as w e l l as g r a t i f i e d to f i n d the carbomethoxy e s t e r (88) obtained as white c r y s t a l l i n e needles i n s t e a d of the dark brown gum obtained e a r l i e r . I t i s important to emphasize the f a c t that i n the above r e a c t i o n , the amount of water present i n the methanol was very c r i t i c a l i n terms of y i e l d . I t j u s t happened that 1% water gave the optimum y i e l d . I f more water was present i n the r e a c t i o n mixture the y i e l d was considerably lowered. We found the best way to o b t a i n r e p r o d u c i b l e r e s u l t s was to d i l u t e absolute methanol w i t h 1% water. F o r ' a n a l y t i c a l purposes a small amount of m a t e r i a l was r e c r y s t a l l i z e d from dichloromethane and petroleum ether, mp 85-87.5°. The s p e c t r a l data compared favourably w i t h the assigned s t r u c t u r e (88). The presence of an e s t e r group was i n d i c a t e d by the i n f r a r e d (1728 cm ^) and a sharp s i n g l e t i n the nmr (Figure 18) at T 6.34. The methylene group adjacent to the e s t e r carbonyl (-CH^-COOCH^) appeared as a sharp s i n g l e t at x 6.27. The mass spectrum (Figure 19) i n d i c a t e d the molecular i o n at m/e.326 and was dominated by two s i g n i f i c a n t peaks at m/e 124 and m/e 202. F i n a l l y the molecular formula, C20 H26 N2°2' W a S c o n^^ v m e'^ by high r e s o l u t i o n mass spectrometry (Found: 326.202; C a l c . : 326.199) and elemental a n a l y s i s . o-CD •a-CM ESTER (88) 8 CO UJ |—LI . 1 L U ct: JlLjl " I T J U i J I i i 1 i I i i i — i — r — i — i 1 — i — I r — r - " i 1 — i — i 1 — i — i — i 50 100 150 200 M/E 250 300 350 400 Figure 19. Mass spectrum of carbomethexy ester 88. With the synthesis of the basic ring skeleton accomplished, i t remained to complete the synthesis of the desired 16,17-dihydrosecodin-17-ol (90) by incorporating the hydroxymethylene (-CH^ OH) side chain at C^Q.in the ester (88). It was envisaged to put the said reaction into practice by formylating the carbomethoxy ester (88) to enol (89) and then reducing the latter with sodium borohydride. Formylation of the ester (88) was done using sodium hydride and methyl formate. Tic examination of the crude product indicated extremely l i t t l e starting material •(< 5%) and one huge/streak rising from just above the baseline. This heavy spot we believed was the desired enol (89). This crude product could be separated in e f f i c i e n t l y (poor separation and.considera-ble loss of material on column) into i t s components (88 and 89) by chromatography on s i l i c a gel. The purified enol (89) indicated a parent molecular ion peak at m/e 354 which was in agreement with the molecular formula, C„.. H.,.N„0,.. 21 26 2 3 The d i f f i c u l t i e s mentioned above prompted us to u t i l i z e the crude enol in the next reaction, consequently the product obtained above was dissolved in methanol and the solution was exposed to sodium borohydride at 0°. We were surprised when the t i c examination of the crude product indicated the presence of one very polar compound along with the major component. It was found that the amount of this polar material eventually decreased to a minimum as the temperature of the reaction mixture was lowered to -30°. In view of the peripheral nature of this "polar component", we made no attempt to characterize i t . However, 85 Battersby while working independently on the synthesis of 16,17-dihydrosecodin-17-ol (90) observed the same result (a complete discussion of Battersby's work i s ' d e f e r r e d u n t i l a l a t e r p o r t i o n of t h i s t h e s i s ) . According to him t h i s p o l a r compound i s the " d i o l " . ( 1 0 5 ) . Recently a 105 CH20H s i m i l a r observation has been made during the re d u c t i o n of enol (106). Thus the p o s s i b l e r e d u c t i o n of both the e s t e r and enol f u n c t i o n s i n (89) cautioned us to e x e r c i s e some care. Monitoring of the r e a c t i o n by C00CH 3 HOH COOCH CH20H CH20H 106 t i c was c a r e f u l l y conducted and the f i r s t appearance of " d i o l " (105) was a s i g n a l to immediately terminate the re d u c t i o n by quenching the excess of borohydride w i t h a few drops of 2 N HC1. The other p r e c a u t i o n to be observed i n t h i s r e a c t i o n was to always keep the temperature low (around -30°). Once the precautions i n d i c a t e d were observed, the whole process could be performed very e f f i c i e n t l y . The crude a l c o h o l obtained was p u r i f i e d by chromatography on alumina and a f t e r c r y s t a l l i z a -t i o n provided an a n a l y t i c a l sample, mp 131.5-132°. The o v e r a l l y i e l d of the r e a c t i o n i . e . 88 -> 90 was 40%. 87—89 There are ^number of instances i n the l i t e r a t u r e where the f o r m y l a t i o n of a c t i v a t e d methylene groups adjacent to e s t e r carbonyl f u n c t i o n s have been done using t r i p h e n y l m e t h y l sodium ( t r i t y l sodium) as a base. This tempted us to s u b s t i t u t e t r i t y l sodium f o r sodium hydride i n our sequence. I t must be emphasized here that t h i s i n v e s t i -g a t i o n was undertaken to improve the y i e l d of a l c o h o l (90). However, i t was found that f o r m y l a t i o n of e s t e r (88) using t r i p h e n y l m e t h y l sodium and methyl formate followed by r e d u c t i o n of the r e s u l t i n g enol (89) gave the same y i e l d of a l c o h o l (90) as obtained e a r l i e r . The complica-t i o n of separating triphenylmethane from the r e a c t i o n product e t c . forced us to d i s c o n t i n u e the use of t h i s base. The p u r i f i e d a l c o h o l i n d i c a t e d s p e c t r a l data which was i n complete accord w i t h the assigned s t r u c t u r e (90). In the i n f r a r e d ; a broad peak at 3050 cm ^ i m p l i e d the presence of a hydroxyl group. A sharp peak at 3400 cm ^ was a t t r i b u t e d to i n d o l i c - N H w h i l e the e s t e r group appeared at.1718 cm \ The nmr spectrum (Figure 20) e x i b i t e d the f o l l o w i n g resonances. The methyl group of the e s t e r appeared as a sharp s i n g l e t at x 6.37. Prominant features of t h i s spectrum i n comparison to the nmr spectrum of the e s t e r (88) '(Figure 18) was the appearance of a braod m u l t i p l e t centered at x 6.0. This m u l t i p l e t i n t e g r a t e d f o r four protons (-CH^-OH + -C-CH^ .OH) . In the mass spectrum H (Figure 21) the a l c o h o l (90) i n d i c a t e d a molecular i o n at m/e 356. I t r e a d i l y l o s t a molecule of water to give the r a d i c a l i o n m/e 338, which corresponded to the molecular i o n of secodine.(107). As to be expected io n (107) fragmented to the ions (108, m/e 214) and to (101, m/e 124). Furthermore an i o n m/e 326 (109) corresponding to l o s s of CH 0 s t r o n g l y CM CM Tj 16.17-DIHYDRGSECGDIN-17 to u CO 8 50 100 150 i 1 i ~i 1 1 1 1 I 1—T 200 M/E 250 300 T 1 1 1 1 350 400 Figure 21. Mass spectrum of 16,17-dihyirosecodin-17-ol (90). COOCH m/e 202 J m/e 124 Figure 22. Postulated fragmentation of 16,17-dihydrosecodin-17-ol (90) in the mass spectrometer. suggested position 17 for the hydroxyl group. A scheme portraying the mass spectrometric fragmentations has been summarized in Figure 22. Finally the molecular formula, C 0 1H oN o0 , was confirmed by high Z l Z o Z j resolution mass spectrometry (Found: 356.207; C a l c : 356.209) and elemental analysis. This marked the end of our i n i t i a l synthetic endeavor. We now . were in a position to investigate whether (90) played a role in the biosynthesis of the various families of indole alkaloids mentioned previously. Evaluation of 16,17-Dihydrosecodinr-17-ol (90) as Bio-intermediate Two radioisotopes most widely util i s e d in biosynthetic studies for making radioactive "precursors" from inactive alkaloids are 3 14 tritium ( H) and C. It is well known that tritium labelling although relatively less expensive can sometimes give erroieous results due to exchange between the protons and the tritium atoms 14 in vivo. On the other hand C labelling is very reliable in the sense that in general no exchange of the label can occur.. However, 14 the much.ijhigher costs often associated with the synthesis of C-labelled materials sometimes require at least i n i t i a l reliance on tritium as the tracer. In our instance i t was considered advantageous f i r s t to make the tritium labelled synthetic alcohol (90) since we believed that this could f u l f i l our immediate needs.,-. If we were ' 3 fortunate in obtaining positive incorporation with ',[ar-^Hj-alcohol (90), 14 i t was envisaged to check the extent of incorporation with [ COOCH^]-3 alcohol (90). For this purpose preparation of [ar- H]-16,17-dihydro-secodin-17-ol (90) and '[ COOCH ]-16,17-dihydrosecodin-17-ol (90) were considered and the syntheses of both of these active compounds are .^described below. The method utili s e d for making tritium labelled indole precursors 90 was developed in our laboratories a few years ago. It involves acid catalysed exchange of aromatic protons of the indole nucleus with tritium labelled trifluoroacetic acid. The latter reagent is prepared by reacting equimolar quantities of trifluoroacetic anhydride and tritium labelled water. A simple vacuum transfer system is used to bring the tritium labelled trifluoroacetic acid into contact with the alkaloid. The acid is subsequently removed after the reaction is complete. It was soon realised that this method for the formation of radioactive alkaloids possessed some significant features: (a) the alkaloids were recovered virtually unchanged.from the acidic medium, (b) the method appeared general to essentially a l l indole alkaloids, (c) since a large excess of acid was-used, the dilution of radioactivity in the reaction was very small and the recovered trifluoroacetic acid was suitable for reuse, and (d) the experimental procedure was very simple in i t s operation. With a l l this knowledge i n hand, we exposed the synthetic 16,17-dihydrosecodin-17-ol (90) to tritium labelled trifluoroacetic acid. Unfortunately when the reaction mixture was worked up, we were very surprised to find the crude product as a complicated mixture of several components. It must be emphasised here that even before the above reaction was performed, we were a l i t t l e iskeptical that some of the alcohol C90) might dehydrate to the corresponding acrylic ester (107). But this latter reaction and then subsequent transformation of the resulting acrylic ester (107) to other spurious products were not considered to be predominating under the mild conditions employed. In view of the small amount of alcohol (90) at hand during the course of the active synthesis, i t was not possible to define the various products formed in the above reaction. However this problem was quickly unraveled. It was mentioned on page 50 that when a methanol solution of n i t r i l e (86) is saturated with HC1 gas, the former is transformed into the carbomethoxy ester (88) (Figure 12). This result suggested to us that the ester (88) was a relatively stable compound in acidic medium. We planned to capitalise on this observation by exchanging the aromatic protons of ester (88) with tritium. In order to secure the desired radioactive alcohol (90), i t was then necessary to formylate the "hot" ester (88) and reduce the resulting enol. The postulated scheme (Figure 23) when put into practice proved highly satisfactory. Before d e s c r i b i n g the a c t i v e syntheses i t should be mentioned here that w h i l e working w i t h the r a d i o a c t i v e compounds as presented i n Figures 23 and 24, t h i n l a y e r chromatography proved extremely h e l p f u l . F o r t u n a t e l y a l l the compounds s t a r t i n g from benzoate (85) to a l c o h o l (90) showed very c h a r a c t e r i s t i c c o l o r s when t i c p l a t e s were sprayed w i t h antimony p e n t a c h l o r i d e . Therefore w h i l e pursuing; the a c t i v e syntheses (Figures 23 and 24) i t was not considered imperative to o b t a i n any formal s p e c t r a l data s i n c e i t was s u f f i c i e n t to compare the values and c o l o r s of the r a d i o a c t i v e compounds w i t h t h e i r c o l d counterparts already a v a i l a b l e and completely c h a r a c t e r -i z e d (Figure 12). To s t a r t the sequence o u t l i n e d i n Figure 23, the carbomethoxy 3 e s t e r (88) was t r e a t e d w i t h H - t r i f l u o r o a c e t i c a c i d . The crude product although homogeneous on t i c , showed some b a s e l i n e m a t e r i a l . The mixture was f l u s h e d through a small alumina column to a f f o r d the pure r a d i o a c t i v e e s t e r (88). This a c t i v e e s t e r was.formylated using sodium hydride and methylformate and the r e s u l t i n g enol was reduced w i t h sodium borohydride. Chromatography of the crude product on 3 alumina af f o r d e d the d e s i r e d [ar- H]-16,17-dihydrosecodin-17-ol (90). The most g r a t i f y i n g outcome of t h i s venture was the f a c t that the obtained a c t i v e a l c o h o l (90) had a very high s p e c i f i c a c t i v i t y (dpm/mg). This r e s u l t allowed us to conduct,numerous experiments both i n Vinca  rosea and Vinca minor p l a n t s . 14 While contemplating the s y n t h e s i s of [ C00CR,j]-16,17-dihydro-secodin-17-ol (90) our a t t e n t i o n was obviously drawn to the n u c l e o p h i l i c displacement r e a c t i o n where the benzoate group was d i s p l a c e d by cyanide anion (85 -> 86, Figure 12). For a while our preconceived goal looked very easy with the thought that by substituting radioactive potassium 14 cyanide (K CN) for potassium cyanide in the above reaction, we could obtain the active-nitrile (86). In order to obtain the desired 14 [ COOCH^]-alcohol (90), i t would be merely necessary to carry the active n i t r i l e (86) through a similar sequence of reactions as done previously on i t s cold counterpart in Figure 12. However, i t was soon realised that in the conversion, 85 -* 86, we were using approximately a ten-fold excess of potassium cyanide. In view of the high cost of radioactive potassium cyanide, i t became imperative to reinvestigate this reaction. This investigation was directed at finding out the minimum amount of potassium cyanide required in the displacement of the benzoate while s t i l l maintaining a reasonable conversion to the n i t r i l e (86). For this purpose a series of reactions were run with decreasing amounts of potassium cyanide. It immediately became apparent that the displacement reaction required a minimum of 5 fold excess of potassium cyanide. Under these conditions a 40% yield of n i t r i l e (86) was obtained as compared to 65% when a 10 fold excess was employed. If the amount of potassium cyanide was reduced any further the yield of n i t r i l e was cut down very drastically. For example a two fold excess of potassium cyanide gave less than 20% of n i t r i l e . In the displacement reaction (85 -»- 86, Figure 24) the benzoate (85) was dissolved in dimethylformamide and the solution was exposed 14 to radioactive potassium cyanide (K CN). The pure active n i t r i l e 6 9 (86, specific activity 4.32 x 10 dpm/mg or 1.27 x 10 dpm/mmole) obtained by chromatography on alumina was dissolved in methanol containing 1% water. The solution was then saturated with hydrogen chloride gas and the resultant crude product upon chromatography furnished the desired active carbomethoxy ester (88). The latter substance was formylated as before to yield the crude enol (89) which without further purification was reduced with sodium borohydride at ^30°. Chromatography of the crude product on alumina furnished the desired I14C00CH ]-16,17-dihydrosecodin-17-ol (90). With the completion of the synthesis of both tritium and C-alcohol (90$, i t became necessary to investigate the incorporation i f any, of this substance into the appropriate plant systems. •> Interest in Vinca rosea has been considerable since the discovery in i t of antileukemic alkaloids. As a result of this finding an extensive investigation of i t s alkaloidal constituents has been 91-94 conducted in various laboratories. The structures ..of more than sixty alkaloids are known and these represent many structural types. Vindoline (5), catharanthine (6) and ajmalicine (3) are three of the major alkaloids present and possess the Aspidosperma (11), Iboga (12) and Corynanthe (10) systems respectively. 95 On the other hand the tiny green plant Vinca minor possesses a wonderful array of Aspidosperma alkaloids. Of the more than twenty alkaloids isolated, structures of about twenty are known. Minovine (73, Aspidosperma type) and vincamine (72, eburnamine family) are two major alkaloids which could be isolated, purified and recrystallised with great ease. In addition Vinca minor grows in abundance around our campus. A l l these factors made this plant an excellent choice for. our biosynthetic studies. Before describing the feeding results i t would be relevant to mention that at this stage our whole research project became very diversified.. We realised • that now: the-problem would involve feeding 14 the tritium and C-labelled, alcohols (90) to both Vinca rosea and Vinca minor for various intervals of time. Regardless whether the active substances showed positive or negative incorporation, these results would require repetition to confirm the i n i t i a l findings. In order to carry out these requirements with optimum accuracy and efficiency, the various incorporation:? experiments were performed simultaneously by three of us, John Beck, Neil Westcott and myself.^- In this thesis the result of only those experiments which were performed by me are described. Whenever relevant or necessary in the later discussion, the results of the other workers w i l l be mentioned. 14 For the purpose of the biosynthetic study, [ COOCH3]-16,17-dihydrosecodin-17-ol (90, total activity 9.89 x 10 dpm) made soluble with 0.1 N acetic acid and a few drops of ethanol was incorporated via the hydroponic technique, to Vinca minor shoots. After four days, the plants were k i l l e d and the isolated alkaloidal material was shown to contain 31% of the total activity fed. Vincamine (72) and minovine (73) were isolated by a chromatographic separation developed earlier 90 in these laboratories. Vincamine showed one spot on t i c and in most of the various experiments conducted the isolated amount was sufficient to allow several crystallizations without the addition of cold material. Minovine (73) however; required further purification by preparative layer chromatography and then further dilution with the cold alkaloid to allow crystallization to constant activity. Several crystallizations revealed that vincamine (72) possessed an activity of 102 dpm (total) corresponding to a specific incorporation of < 0.001%. Unfortunately this amount of radioactivity was so small that i t was d i f f i c u l t to ascertain the significance i f any of this result. A minute trace of a radioactive impurity present in the alkaloid could be responsible. On the other hand virtually no activity could be; detected - in the purified minovine (73). In a parallel series of experiments, [ar- Hj-16, 17-dihydrosecodin-17-ol (90) was fed to Vinca minor by my colleague, John Beck. It is sufficient here to state that he also could not detect any significant activity in the two alkaloids, vincamine (72) and minovine (73). <-. 3 In another concurrent investigation, [ar- H]-16,17-dihydroseco-din-17-ol (90) was fed to Vinca rosea by another colleague Neil Westcott, no significant activity could be detected in vindoline(5) and catharanthine (6). The most frustrating aspect of a l l these results was the ina b i l i t y to delineate what might be construed as a positive incorporation of alcohol (90) into any of those alkaloids isolated by us. We, of course, were ful l y aware of the fact that negative results in biosynthetic studies have to be interpreted with great care. It is well..known that success in a biosynthetic experiment depends upon such factors as absorption and permeability in the plant as well as the ab i l i t y of the plant to carry out the desired biosynthesis. Thus the age of the plant, length of incorporation, the method of feeding etc. become very c r i t i c a l factors. In this regard i t is pertinent to mention that the earlier workers in our laboratories had established. conditions during which large molecular weight substances were 63 64 incorporated into the plant systems. ' This situation therefore reinforced our prevailing impression that the apparently negative incorporations of alcohol (90) were not due to technical d i f f i c u l t i e s with the experimental method. It was indicated in the early part of this discussion (page 33) that we prepared 16,17-dihydrosecodin-17-ol (90) as our synthetic target only with the hope that i t would be transformed in vivo to the p u t a t i v e intermediate (76) by appropriate dehydration and o x i d a t i o n ( i n the p i p e r i d i n e r i n g ) . However negative i n c o r p o r a t i o n of a l c o h o l (90) suggest that the pl a n t systems u t i l i s e d may be incapable of c a r r y i n g out e i t h e r one or both of these r e a c t i o n s . 85 A f t e r we had completed the above r e s u l t s , Battersby reported the presence of 16,17-dihydrosecodin-17-ol (90) i n the plant// Rhazya o r i e n t a l i s . In these experiments he fed to the shoots [0-methyl-3 H]-loganin and from the i s o l a t e d "active a l c o h o l , 90, was able to show an i n c o r p o r a t i o n of 0;013%. Jn-. the same manner, when the experiment was repeated w i t h shoots of Vinca rosea, r a d i o a c t i v e (90) was again i s o l a t e d but of very low s p e c i f i c a c t i v i t y . To use i t as a c a r r i e r 85 Battersby synthesised the a l c o h o l (90) by the general route o u t l i n e d i n Figure 25. Although Battersby's route i s q u i t e d i f f e r e n t from ours i n the e a r l i e r stages (see Figure 12), the l a t e r steps are e s s e n t i a l l y i d e n t i c a l . In summary of h i s independent study Battersby s t a t e d that "16,17-dihydrosecodin-17-ol (90) i s . a n a t u r a l product present i n Rhazya o r i e n t a l i s probably a r i s i n g from a b i o s y n t h e t i c intermediate blocked by r e d u c t i o n (e.g. 113) or by h y d r a t i o n and r e d u c t i o n (e.g. 76)". 96 Recently Smith reported the i s o l a t i o n of tetrahydrosecodine(110) and 16,17-dihydrosecodine(111) from Rhazya s t r i c t a and tetrahydrosecodin-17-ol (112) from Rhazya o r i e n t a l i s . These a l k a l o i d s were i s o l a t e d i n very small amounts and t h e i r s t r u c t u r e s were derived mainly from mass spectrometric measurements. The presence of tetrahydrosecodine(110) was again demonstrated i n Rhazya o r i e n t a l i s by d i l u t i o n s t u d i e s when 14 97 [2- C]-tryptophan was administered to Rhazya o r i e n t a l i s . The Figure 25. Battershy's synthesis of 16,17-dihydrosecodin-17^ol. plants were worked up for the alkaloids with the addition of synthetic (110) as carrier (Figure 26). The constant activity found for the 110 COOCH Figure 26. Smith's synthesis of tetrahydrosecodine(110). rigorously purified tetrahydrosecodine(110) corresponded to 0.5% incorporation. This high incorporation was quite remarkable and this led Smith to suggest that tetrahydrosecodine (110) is on a metabolic side track very close to the main alkaloid biosynthetic route. This fact f i t s well with the notion that simple reduction of the putative acrylic ester (76) takes tetrahydrosecodine(110) out of circulation. It i s interesting to note that a similar type of explanation (i.e. Figure 27. Some of the compounds derivable in vivo from 16,17-dihydro-secodin-17-ol (90). hydration and reduction of acrylic ester, 76) was used by Battersby when 16,17-dihydrosecodin-17-ol (90) was isolated from the plants. The question now is how the above results f i t into the biosynthetic story which is rapidly evolving from the combined data of.the various laboratories: First of a l l , these results give further support to the suggested cleavage process for the biosynthesis of indole alkaloids in the Aspidosperma and Iboga families as mentioned previously. It is further evident that compounds 107, 76, 89, 113, 114 (not yet isolated) 110, 111, 112 arid the dimeric secamine (119) and presecamine 98 (116), a l l are derivable in principle from the alcohol (90) in vivo whether any of these compounds (Figure 27) w i l l turn out to be the correct biointermediate remains an open question. Some of our own experiments which could be readily extended into this area are. discussed in the next section of this thesis. PART II The negative incorporation obtained by feeding the synthetic 16,17-dihydrosecodin-17-ol (90) into Vinca minor and Vinca rosea plants suggested to us that the former may not be capable of acting as a progenitor of the acrylic ester (76) in the plant. It therefore became evident that to persue our preconceived goal the alcohol (90) required synthetic modification to some other model compound. At this stage our whole research project entered a very perplexing phase. Among the many avenues which were available, i t was very d i f f i c u l t to decide unequivocally which route to explore f i r s t . In this part of the discussion we w i l l portray some of our attempts to obtain some.of the other close relatives of the fugitive acrylic ester (76). The two compounds bearing structures 107 and 115 appeared to us to represent templates which could under reasonable biochemical modification convert to the acrylic ester (76) and thereby in turn to the Aspido-sperma and Iboga bases. Our choice was obviously dictated by the fact that these compounds (107 and 115) were amenable to syntheses from the available alcohol (90). It shouldi/be noted that in comparison to the alcohol (90), the pyridinium alcohol (115) which would be sufficiently stable for isolation represented a much higher level of oxidation in the piperidine ring. It seemed reasonable that some + X reducing system in the plant like NADPH+ would convert the pyridinium ring to the desired dihydropyridine system. On the other hand the 96 tetrahydropyridine derivative, 107, named secodine by Smith who has recently isolated the close relatives of this compound in his work on Rhazya species, was in a lower level of oxidation than the acrylic ester (76). Perhaps an oxidative process in the plant would lead in vivo to 76. The discussion which follows describes our attempts in the laboratory syntheses of these substances. In connection with transannular cyclization work in our labora-tories,"^ ^ mercuric acetate was found to be an excellent reagent for oxidising the piperidine ring of several alkaloids to the corresponding + tetrahydropyridines Ci-e. C-N- ->- C=N-). We also envisaged to u t i l i s e the same•reaction for oxidising 16,17-dihydrosecodin-17-ol (90) to the pyridinium alcohol (115). This aspect of the problem was undertaken by two of'my colleagues, Neil Westcott and John Beck. Although the details of a l l this work cannot be properly discussed • here, suffice i t to say that a l l our attempts with mercuric acetate oxidation reaction were very disappointing. In a l l instances poor yields and products of l i t t l e u t i l i t y were obtained. These results therefore l e f t us l i t t l e alternative except to concentrate our efforts in obtaining secodine (107) for biosynthetic evaluation. To secure secodine (107) from alcohol (90) i t was obligatory to. dehydrate the latter substance. For several reasons to be presented later we preferred the base catalysed dehydration rather than the alternative of acid catalysis. For this purpose a small amount of alcohol C90) dissolved in benzene was exposed to sodium hydride as the base. After the reaction was over, the excess of the hydride was destroyed by the a d d i t i o n of a few drops of 2 N h y d r o c h l o r i c a c i d . T i c examination of the crude product i n d i c a t e d two spots w i t h very s i m i l a r v alues. The whole crude product was r a p i d l y f l u s h e d through a small column of alumina and the mixture exposed to a spectroscopic examination. The nmr spectrum i n d i c a t e d two s i g n a l s f o r NH (x 0.63 and 1.23) and two e s t e r (COOCH^) peaks at x 6.29 and 6.49 r e s p e c t i v e l y . S i m i l a r l y i n the i n f r a r e d two es t e r peaks were evident and these could be conveniently assigned to saturated (1730 cm "*") and unsaturated (1680 cm "*") e s t e r groups. This r e s u l t immediately suggested that we were d e a l i n g w i t h a mixture which contained at l e a s t one dimeric compound^. r96-98 At a time when we were s t i l l entangled i n t h i s problem, Smith published a s e r i e s of papers which q u i c k l y u n r a v e l l e d our problems. He reported the i s o l a t i o n of three new dimeric a l k a l o i d s , presecamine (116a or 116b), dihydropresecamine (117a or 117b) , tetrahydropresecamine (118a or 118b) from Rhazya s t r i c t a and tetrahydropresecamine from 98 Rhazya o r i e n t a l i s . Smith f u r t h e r observed that presecamine (116a or 116b) rearranges q u a n t i t a t i v e l y at room temperature i n 2 N h y d r o c h l o r i c a c i d to one of the secamines (119, Figure 28; 120 Figure 29). This important observation represented one of the main arguments f o r the s t r u c t u r e s suggested and would tend to favour s t r u c t u r e type ( a ) . The type (b) dimers was however not excluded on mechanistic grounds (Figure 29) sin c e i t could (although l e s s p l a u s i b l y ) l e a d to the other p o s s i b l e secamine s t r u c t u r e (120). I t should be noted that f o r convenience i n the subsequent d i s c u s s i o n presecamine and secamine w i l l be considered i n terms of s t r u c t u r e s 116a and 119 r e s p e c t i v e l y . Figure 28. Rearrangement of presecamine (type a) to secamine (119). Figure 29. Rearrangement of presecamine (type b) to secamine (120). It was further observed that on attempted sublimation presecamine (116) undergoes a facile retro-Diels-Alder reaction to yield secodine 98 (107). A brief resume of the data obtained by Smith in support of structure, 107, for secodine is fu l l y revealed in Figure 30. Not only was this data of great importance in i t s own right but i t was directly pertinent to our work as well. We shall now try to portray how our work on dehydration of 16,17-dihydrosecodin-17-ol (90) converges with Smith's experiments in a most gratifying way. Our crude mixture from the sodium hydride reaction; indicated ultraviolet absorption (A 224, 285 (inf), 292, 326 my) max and nmr signals (two ester singlets at x 6.29 and 6.49) which was reminiscent of the spectroscopic properties (^ m a x 227, 228 (inf), 295, 329 my and singlet at x 6.23 and 6.42) reported for the dimeric presecamine (116). With this knowledge in hand i t became possible to speculate on the nature of at least three compounds present in the crude reaction mixture. The presence of the desired secodine (107) was indicated by a very pronounced unsaturated methoxycarbonyl singlet at T 6.29 in the nmr spectrum. The ratio between the integrations of the unsaturated and saturated methoxycarbonyl singlets was 3:1 (in pureipresecamine this ratio should be 1:1). The other two compounds present in the crude mixture were obviously presecamine (116) and secamine (119). The latter substance would arise from the rearrangement of presecamine CL1"6) when 2 N hydrochloric acid was used in the work up of the reaction. Returning to Smith's study i t was clear that the reaction mixture should not be exposed to hydrochloric acid to avoid the rearrangement of Figure 30. A summary of data supporting the structure of secodine (107). presecamine (116) to secamine (119). Furthermore Smith had found that secodine (107) reacted very slowly (over a period of 10 days) with methanol to give 17-methoxy-16,17-dihydrosecodine (121). Finally in the dimerization of secodine (107) to presecamine (116, Figure 30) i t was specifically indicated that this reaction occurs at 0° in the absence of solvent. These observations clearly pointed to the fact that secodine (107) could perhaps be isolated free from dimer i f the reaction mixture was kept cold and in solution. It was indeed found in our work that secodine (107) could be stored for several hours in dry benzene at 0° without any appreciable dimerization. The optimum conditions for the dehydration of the alcohol (90) involved dissolving this substance in dry benzene and exposing i t to sodium hydride. The- reaction -mixture was stirred under nitrogen at 40° for 15 minutes. At this time t i c examination of the mixture indicated three spots. The front running spot which seemed to represent the bulk of -material, was due to secodine (107). The other two minor spots were due to the dimer presecamine (116) and the starting compound (90). The crude mixture was flushed through a small alumina column using benzene for the elution. The fraction collected was freezer-dried immediately under vacuum. We were very surprised as well as gratified to find that the gummy product obtained in this manner was homogeneous on t i c . Further elution of the column with chloroform afforded presecamine (116) and unreacted alcohol (90). In small scale reactions the yield i n this dehydration procedure varied but the most favourable reaction provided 61% of secodine. It is now appropriate to discuss some of the evidence in support of the structural assignment. In the nmr spectrum (Figure 31) the olefinic protons of the acrylic ester were represented as a pair of doublets at x 3.55 (J = 1 Hz) and 3.91 (J = 1 Hz), respectively. The methoxycarbonyl was indicated by a sharp singlet at x 6.20. These signals compared favourably with those reported for 15,20-dihydrosecodine (107, A^'^-reduced). In the latter the olefinic protons were indicated at x 3.54 and 4.01; methoxycarbonyl at x 6.18. The mass spectrum (Figure\32) indicated a molecular ion peak at m/e 338 in agreement with the molecular formula, (-'21^ 26^ 2^ 2" The spectrum was dominated by\ ^ s i g n i f i c a n t peak at m/e 124(101). while a weak peak-at-m/e 214 showing-ra metastable peak at 135 ,.5 was_ consistent with'-thec-fragment 108,, both formed in the manner illustrated. 107 108 101 It is pertinent to mention here that when sodium hydride in the dehydration reaction was substituted by t r i t y l sodium (triphenylmethyl sodium)!, i t led to products of l i t t l e u t i l i t y . TLC>indicated that-the crude product was a mixture of ..three components. Out of these, two compounds separated in pure form by chromatography on alumina indicated very strong end absorption in the uv spectrum. This was indicative of a second aromatic system. It was f e l t that these two compounds arise RELATIVE INTENSITY 25 _L_ 50 75 _ J _ C n ID P3 cn cn cn x) ID n rt n c 3 o Hi cn . CD n o o 124 (4x) 135 en • o m ro a • CD s—214 ro or o a -o CO m C I Q a m • 3 3 8 CO o a from the condensation of the t r i t y l anion with the acrylic ester functionality of secodine (107). Due to the very small amount of material at hand, no attempt was made to rigorously prove the structures of these compounds but tentative assignments are given in structures (122) and (123), respectively. \Although "this is riot usual for triphenyl-99 methyl sodium to react in this manner with an ester, an example of COOCH, 0=C XC(<{,). 122 123 the formation of a . t r i t y l ketone via a similar process has been observed In our case however, the molecule (107) because of the presence of the acrylic ester group was endowed with much greater reactivity to allow Micheal addition of the t r i t y l anion. With the completion of the long sought secodine (107) we now were in a position to investigate i t s possible role in the biosynthesis of the Aspidosperma and Iboga alkaloids. 1D0, Evaluation of secodine (107) as bio-intermediate It shouldi-be mentioned here that some earlier workers in our laboratories have tentatively found that the best way to incorporate any given precursor into the plant system is to convert i t into the acetate 90 . salt. Normally the acetate salt i s made by dissolving the compound in 0.1 N acetic acid and a few drops of ethanol.' So i t was perfectly clear to us in the beginning that no matter how much care was taken in isolating pure secodine (107), the necessary conversion to the salt and eventual incorporation into the plant would allow some dimerization to presecamine (116). Under the influence of acid some of the presecamine (116) would then obviously rearrange to secamine (119). In summary we understood that we would be required under normal circumstances to incorporate a mixture of these three compounds. However i t was envisaged that the presence of the dimeric compounds presumably would not jeopardise the feeding experiment provided the composition of the mixture could be determined at the time of feeding. For this purpose a "blank" experiment was.conducted in such a manner that a clear distinction between the amount of secodine (107) and the dimeric compounds could be made. The details of this experiment are deferred, un t i l a later portion of the discussion. It is sufficient to emphasize presently that the procedure proved highly satisfactory. 3 For the biosynthetic investigation Jar- R]-16,17-dihydrosecodin-17-ol (90, specific activity 7.94 x .10^  dpm/mg) was dehydrated with sodium hydride in exactly the same manner as indicated previously. 3 8 The pure l a r - H]-secodine (107, total activity 2.65 x 10 dpm) was made soluble with 0.1 N acetic acid and a few drops of ethanol and the solution was administered to the shoots of Vinca minor L. The plants were allowed to grow for four days and then the alkaloids were isolated. The crude extract contained 22% of the total activity fed. Vincamine (72) and minovine (73) were isolated i n i t i a l l y by chromatography and then further purified by preparative thin layer chromatography followed by several crystallizations. Liquid s c i n t i l l a t i o n counting revealed that the isolated alkaloids contained a very low level of radioactivity. Vincamine (72) showed an activity of 261 dpm/mg corresponding to a specific incorporation of 0.0013% while minovine (73) indicated an ac i t i v i t y of c|5625 dpm/mg corresponding to a specific incorporation of < 0.001%. It is pertinent to c a l l attention to the fact that in spite of the very low incorporation observed, the alkaloids were showing appreciable counts (cpm) above the normal background. In order to confirm the r e l i a b i l i t y of the above results another colleague of mine, John Beck, repeated this.biosynthetic experiment employing a new series of plants. Fortunately his results for vincamine (72) and minovine (73) turned out to be in good agreement with the figures quoted above for these two alkaloids. In spite of the fact that the level of incorporation was extremely low,,the most important observations to emerge from these experiments were that: (a) in comparison to our last biosynthetic experiment when 16,17-dihydrosecodin-17-ol (90) was fed, .the Vinca mirtor shoots remained very healthy for the duration of the experiment (four days). In the former case the plants had started collapsing just after one day of feeding; (b) we were rather surprised when minovine (73) indicated almost twice as much radio-activity (dpm/mg) in comparison to vincamine (72); This factor was not self evident in considering the results of specific incorporation because minovine (73) is usually isolated in much smaller quantity than vincamine. This result could be due to the fact that for the secodine skeleton to incorporate into minovine (73), a few relatively . straightforward ring closures are required (Figure 33) while for + Figure 33. Proposed elaboration of secodine into vincamine and minovine. vincamine (72L the secodine 0-07). molecule must undergo numerous 'rearrangements (Figure 33). A later discussion concerning studies on vincamine w i l l present this aspect in more detail. We have no firm basis for this explanation and obviously additional experiments w i l l be necessary before any more definite statement can be made. 3 In a complimentary series of experiments [ar- H]-secodine (107) was fed to Vinca rosea L. by John Beck i n our laboratory. The radioactive vindoline (5) isolated (0.02% incorporation) was.shown to be radio-chemically pure by further conversion of this alkaloid into vindolinetriol having the same constant molar activity. Somewhat surprisingly Iboga alkaloid catharanthine (6) which co-occurs with vindoline (5) in Vinca rosea indicated no activity. Similarly [ar- H]-secodine (107) was fed to Aspidosperma pyrricollum plants by Dr. Ken Stuart i n our laboratory. Isolation of radioactive apparicine (124) indicated 0.01% incorporation. 124 A l l these incorporation results indicated above leave l i t t l e doubt in our minds that secodine (107) or some.closely related derivative which may be obtained by reaction of the enzyme systems on 107 may turn out to be a crucial bio-intermediate in indole alkaloid biosynthesis. It was now necessary to determine what percentage of the compound fed got into the plant in i t s monomeric state and what percentage of i t was converted to the dimeric systems during the period 14 of incorporation. A blank experiment was conducted in which [ COOCH^]-secodine (107) was converted into the acetate salt by dissolving i t . in 0.1 N acetic acid and a few drops of ethanol. The cloudy solution was l e f t at room.temperature for 2 hours (this i s the maximum time the- plants require to absorb the above solution). The contents were freeze-^dried and a portion of the resulting, gum was run on a Eastman Kodak neutral alumina strip plate employing a system which had been previously established for this purpose by means of the "cold" materials (for complete details see page l i 7 ). The activities in the two spots corresponding to secondine (107) and the dimeric compounds (presecamine and secamine) were>counted with a s t r i p counter. It immediately became obvious that i n the mixture the r a t i o between the secodine and the dimeric compounds was 61:32. Therefore the corrected s p e c i f i c incorporation into Vinca minor L. for vincamine (72) should be 0.002% and for minovine (73) < 0.0015%. This c a l c u l a t i o n assumes that the dimeric molecules do not convert back to the monomers i n the plant. A l l of the above r e s u l t s were very g r a t i f y i n g since they provided our f i r s t p o s i t i v e incorporation of a synthetic substance into the various plant species. The next important question as to secodine (107) i s being incorporated as a i n t a c t unit w i l l require the preparation of 3 14 doubly l a b e l l e d precursor i . e . [ar- H; C00CH./J-secodine (107) or even better, secodine with one l a b e l i n the indole unit and the other i n the tetrahydropyridine portion. Such i n v e s t i g a t i o n s are currently underway i n our l a b o r a t o r i e s . In conclusion i t i s clear that the above work has provided some preliminary information on the l a t e r stages of indole a l k a l o i d s bio-synthesis. Most,importantly i t has created an entry into the more sophisticated experiments which w i l l hopefully lead to a better understanding of the biosyntheses of t h i s large family of natural products. EXPERIMENTAL M e l t i n g p o i n t s were determined on a K o f l e r block and are uncorrected. The u l t r a v i o l e t (uv) s p e c t r a were recorded i n methanol on a Cary 11 recording spectrometer, and the i n f r a r e d ( i r ) s p e c t r a were taken on a P e r k i n Elmer Model 21, Model 137 and Model 457 spectrometers as KBr d i s c s (unless otherwise s t a t e d ) . Nuclear magnetic resonance (nmr) spec t r a were recorded i n deuteriochloroform (unless otherwise stated) at 100 megacycles per second (unless otherwise stated) on a Va r i a n HA-100 instrument and the l i n e p o s i t i o n s or centre of m u l t i p l e t s are given i n T i e r s T s c a l e w i t h reference to t e t r a m e t h y l s i l a n e as the i n t e r n a l standard; m u l t i p l i c i t y , i n t e g r a t e d area and the type of-protons are i n d i c a t e d i n parentheses. Mass spe c t r a were recorded on an A t l a s CH.-4 mass spectrometer and high r e s o l u t i o n molecular weight determinations were c a r r i e d , out on an AE--MS'-9 mass spectrometer. v Analyses were c a r r i e d out by Mr. P. Borda of the M i c r o a n a l y t i c a l Laboratory, The U n i v e r s i t y of B r i t i s h Columbia. Woelm n e u t r a l alumina and s i l i c a g e l c o n t a i n i n g 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 p r e p a r a t i v e t h i n l a y e r chromatography ( t i c ) . Chromatoplates were developed using the spray reagent carbon tetrachloride-antimony p e n t a c h l o r i d e (2:1). Woelm neutral alumina (activity III) was used for column chromatography (unless otherwise indicated). Radioactivity was measured with a Nuclear Chicago Mark I Model 6860 Liquid S c i n t i l l a t i o n Counter in counts per minute (cpm). The radioactivity of a sample in disintegrations per minute (dpm) was calculated using the counting efficiency which was determined for each sample by the external standard technique u t i l i s i n g the built in barium-133 gamma source. The radioactivity of the sample was determined using a s c i n t i l l a t i o n solution made up of the following composition: toluene (1 l i t r e ) , 2,5-diphenyloxazole (4 gm) and 1,4-bis[2-(5-phenyloxazolyl)]benzene (0.05 gm). In practice, a sample of an alkaloid as a free base was dissolved in benzene (1 ml) in a counting v i a l . In the case of the salt of an alkaloid, the sample was dissolved in methanol. Then in both cases, the volume was made up to 15 ml with the above'scintillator solution. For each sample counted, the background (cpm) was determined for the counting v i a l to be used by, f i l l i n g the v i a l with the s c i n t i l l a t o r solution and counting (3 x 40 min). The counting-vial was emptied, r e f i l l e d with the sample to be counted and the sc i n t i l l a t o r solution,, and counted again (3 x 40 min). The difference in cpm between the background count and the sample count was used for the subsequent calculations. For the sake of convenience and ease of presentation, the experimental has been divided into two portions. The f i r s t part describes the syntheses of 16,17-dihydrosecodin-17-ol (90) and secodine ; (107). The second portion describes the syntheses of radioactive ^ precursors and their subsequent feeding into Vinca minor Linn. PART I 72 Diethyl-y^chlorbpropylmalonate (91) To a s o l u t i o n of sodium ethoxide prepared by d i s s o l v i n g sodium (23 gm, 1 mole). i n ethanol (350 ml) was added i n one p o r t i o n a s o l u t i o n of d i e t h y l malonate (160 gm, 1 mole) and 1,3-bromochloro-propane (160 gm, 1 mole) i n dry ether (200 ml). The r e a c t i o n mixture was maintained at 35° f o r 4 hours and then allowed to stand at room temperature f o r 24 hours. Then the mixture was poured i n t o water (700 ml) and ex t r a c t e d w i t h ether. The e x t r a c t was washed w i t h water, saturated sodium c h l o r i d e s o l u t i o n , d r i e d over anhydrous sodium s u l f a t e and concentrated under reduced pressure. The r e s u l t i n g o i l was d i s t i l l e d at reduced pressure to give the d e s i r e d m a t e r i a l (112 gm, 48%); bp 115°/0.5 mm, ( l i t . bp 142°/10mm); v ( f i l m ) : 1730 (-C00C„H ) cm 1 ; nmr (60 mc/s) : x 5.75 (quartet, 4H, 2 x C00CH_2CH3), 6.56 ( t r i p l e t , 2H, -CR 2-CH 2-C1), 6.75 ( t r i p l e t , 1H, -CH 2-CH-(COOEt) 2), 8.10 ( m u l t i p l e t , 4H, -CH 2-CH 2-CH 2-C1), 8.80 ( t r i p l e t , 6H, 2x-C00CH 2CH 3). Benzenediazonium c h l o r i d e ^ A n i l i n e h y d r o c h l o r i d e (50 gm, 0.350 mole) was suspended i n a mixture of g l a c i a l a c e t i c a c i d (300 ml) and dry peroxide f r e e dioxan (300 ml). The mixture was cooled i n a i c e - s a l t bath and isoamyl n i t r i l e (50 gm, 0.420 mole) was added s l o w l y , the temperature being held below 0°. A f t e r the a d d i t i o n was complete the mixture was s t i r r e d f o r 30 minutes during which the s o l i d suspension d i s s o l v e d . Dry dioxan (1500 ml) or dry ether (1500 ml) was added intone p o r t i o n and the white p r e c i p i t a t e of benzenediazonium c h l o r i d e was c o l l e c t e d , washed s e v e r a l times w i t h f r e s h solvent and d r i e d i n a vacuum d e s s i c a t o r (Yield 52 gm). Synthesis of 2-carboethoxy-3-(g-chloroethyl)-indole (80) using  benzenediazonium chloride To a solution of sodium ethoxide, prepared by dissolving sodium (8.25 gm, 0.360 mole) in dry ethanol (1000 ml),'" was added diethyl-y-chloropropymaionate ... (91, 85.0 gm, 0.360 mole) and the mixture was stirred under nitrogen for 30 minutes at room temperature. After cooling the reaction mixture in a ice-salt bath, the benzenediazonium chloride (52 gm, 0.370 mole) was added in small portions. During the addition the temperature of the reaction mixture was held below -2°. After the addition of the diazo salt was complete, the mixture was stirred for 30 minutes and then l e f t in the refrigerator for 12 hours. The contents were poured into water (1000 ml) and the dark red o i l thus separated was extracted into ether. The extract was washed with water, saturated brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude reaction product (93, 106 gm) was immediately subjected to a Fisher indole synthesis described below. The material obtained above was dissolved in dry ethanol (750 ml). To this concentrated sulfuric acid (100 ml) was added slowly and the mixture was refluxed for 12 hours. After cooling to room temperature, the volume of the reaction mixture was reduced to half under reduced pressure. The contents were poured onto ice and the resulting mixture was extracted with chloroform. The extract was washed several times with water, then with sodium carbonate•solution, and again with water, dried over anhydrous sodium sulfate and evaporated under reduced^ pressure. The crude dark semicrystalline material was purified by ,., chromatography on alumina (Shawinigan, activity III, 3 kg). The desired material was eluted, f i r s t with benzene and later with benzene-chloroform (1:1), as a crystalline solid. Recrystallization from chloroform-petroleum ether gave a white crystalline solid (11.2 gm, 19%), mp 130-132°; v^ 0"*": 3250 (-NH) , 1670 (-C00CoHc) cm"1; A max - 2 5 max (log e): 229 (4.40), 296 (4.27) my; nmr (60 mc/s): T 0.75 (broad singlet, 1H, -NH), 2.60 (multiplet, 4H, aromatic), 5.54 (quartet, 2H, -C00CH2-CH3), 6.50 (multiplet, 4H, -CH_2-CH_2-C1), 8.60 (triplet, 3H, -C00CH -CH ). Anal. Calc. for C H^O^Cl: C, 62.03; H, 5.57; N, 5.57; 0. 12.75; CI. 14.12. Found: C, 62.07; H, 5.53; N, 5.60; 0. 12.64; CI, 14.08. Benzenediazonium fluoroborate^'^ Aniline hydrochloride (108 gm, 0.83 mole) was dissolved in water (275 ml) and cone. HC1 (140 ml) in a three l i t r e beaker. The solution was cooled to 0° and sodium n i t r i t e (69 gm, 1 mole) in water (150 ml) was added dropwise, maintaining the temperature a l l the while below 5°. The addition of the sodium n i t r i t e solution was stopped when a drop from the reaction mixture gave a blue coloration with starch iodide paper. A solution of 48% HBF^ (183 ml) was cooled to 0° and added slowly to the diazonium salt solution. Precipitation was immediate but the sti r r i n g was continued for an additional 10 minutes. About half of this suspension was transferred to a sintered glass funnel and washed with ice cold water (50 ml), cold methanol (25 ml) and ether (50 ml). The solid was sucked as dry as possible after each washing. The salt was transferred to a beaker and dried in a vaccum dessicator overnight. The other half was treated similarly. The total weight of solid material was (108 gm). Synthesis of 2-carboethoxy-3-(g-chloroethyl)-indole (80) using  benzenediazonium fluoroborate To a solution of sodium ethoxide prepared by dissolving sodium (12 gm, 0.51 mole) in dry ethanol (1000 ml) was added diethyl-y-chloropropylmalonate (91, 120 gm, 0.50 mole) and the mixture was stirred under nitrogen for 30 minutes at room temperature. After cooling the mixture in a ice-salt bath, the fluoroborate salt (105 gm, 0.55 mole) was added in small portions so that the temperature of the reaction mixture was always below -2°. After the addition of the diazo salt was complete, the mixture was stirred at 0° for 2 hours and then l e f t in the cold room (-10°) for 12 hours. The contents were poured into water (1000 ml) and the dark red o i l which separated out was extracted into ether. The extract was washed with water and with saturated brine solution, dried over anhydrous sodium sulfate and then concentrated under vacuum. The crude reaction product (93, 190 gm) was immediately subjected to a Fisher indole synthesis as described below. The thick red o i l was dissolved in dry ethanol (1000 ml). To this cone, sulfuric acid (200 ml) was added and the mixture was refluxed for 12 hours. After cooling to room temperature the reaction mixture was worked up in exactly the same manner as indicated on page 94 . The crude semicrystalline compound (140 gm) was purified by chromatography on alumina (Shawinigan, activity III, 3 kg). Elution with benzene-chloroform (1:1) afforded the desired compound. This was recrystallised from chloroform-petroleum ether as white crystalline plates (14 gm, yield 13%), mp 131-132°. This material had the same value on t i c and spectral properties as the chloroindole (80) obtained earlier when diazonium chloride was used. o n 3-Ethylpyridine (81) A mixture of 3-acetylpyridine (60 gm), potassium hydroxide (50 gm), triethylene glycol (400 ml) and 85% hydrazine (90 ml) was heated for 1 hour at 110-125°. The reaction mixture was cooled and gradually reheated with a take-off condenser to a bath temperature of 180-190°. When the evolution of the nitrogen in the reaction mixture had ceased, the volume collected from the take-off condensor (about 200 ml) was extracted with ether. The extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting o i l was d i s t i l l e d using an efficient fractionating column and 3-ethyl-pyridine was collected at 162-163° ( l i t . value 162-165°) (36 gm, yield 68%). v ^ ^ m : no absorption i n the carbonyl region. N-[g-{3-(2-carboethoxy)-indolyl}-ethyl]-3'-ethyl-pyridinium chloride (82) Chloroindole (80, 6.431 gm) was dissolved in 3-ethylpyridine (22 ml) and the mixture was heated in a sealed tube at 120°- for 24 hours. After cooling to room temperature the sealed tube was opened and•the contents were poured into anhydrous ether with st i r r i n g . The mixture was then l e f t at room temperature for 3 hours. During this time a l l the occluded 3-ethylpyridine was extracted from the salt into the ether. The white amorphous solid was fil t e r e d under suction, , washed several times with dry ether and f i n a l l y dried i n a vacuum dessicator (8.343 gm, yield 91%); mp 87-89° (recrystallised from methanol-ether); v m a x : 3120-2880 (several bands, ,vC-H, aromatic), 1701 (vC=0) and 1250 (vC-O-C) cm"1; X (log e): 296 (4.2), 276 (inf)(3.9), 226 (4.35) and 220 (4.3) my; nmr (CD30D): x 1.4-2.34 (multiplet, 4H, pyridinium protons), 2.56-3.18 (multiplet, 4H, indole protons), 5.74 (quartet, 2H, -C00CH2-CH3), 7.40 (quartet, 2H, -CH_2-CH3), 8.63 (triplet, 3H, -C00CH2-CH_3), 8.98 (triplet, 3H, -CH2-CH3); mass spectrum: M+ 358; main peaks: m/e 215, 187, 169, 129. N-[g-{3-(2-Carboethoxy)-indolyl}-ethyl]-3'-ethyl-3'-piperideine (83) To a solution of the pyridinium salt (82, 7.67 gm) in methanol (275 ml) and triethylamine (8 ml) was added slowly a solution of sodium borohydride (25 gm) in methanol (400 ml). The yellow color of the salt solution was discharged when the addition of the borohydride was complete. After stirring at room temperature for 2.5 hours, the reaction mixture was diluted with water (100 ml) and methanol was evaporated under reduced pressure. The remaining aqueous solution was acidified with 6 N HC1 to pH 2, stirred at room temperature for 20 minutes and then made basic with 10% sodium carbonate solution. The basic solution was extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium sulfate and evaporated to give a thick gum (7.003 gm). This material was homogeneous on t i c and was used in the next reaction as such. Nmr (60 mc/s) of the crude product: T 4.5 (broad s i n g l e t , 1H, C y - H ) , 5.60 (quartet, - 2H,-C00CH2-CH3) N-[g-{3-(2-Hydroxymethylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine (84) A s o l u t i o n of t e t r a h y d r o p y r i d i n e (83, 7.003 gm) i n dry THF (70 ml) was dropped sl o w l y over a p e r i o d of 40 minutes i n t o a suspension of l i t h i u m aluminum hydride (9 gm) i n THF (350 ml) under n i t r o g e n . A f t e r the a d d i t i o n was complete, the r e a c t i o n mixture was s t i r r e d at room temperature f o r 20 minutes and then r e f l u x e d f o r 2 hours. A f t e r c o o l i n g the mixture to i c e temperature, the excess of the hydride was destroyed by c a r e f u l a d d i t i o n of water (9 m l ) , 15% sodium hydroxide s o l u t i o n (9 ml) and water again (27 ml). The r e a c t i o n mixture was f i l t e r e d under s u c t i o n and the p r e c i p i t a t e d hydroxides were washed s e v e r a l times w i t h methylene c h l o r i d e . The f i l t r a t e was evaporated under reduced pressure and the r e s u l t i n g gum was r e d i s s o l v e d i n methylene c h l o r i d e . The organic l a y e r was washed w i t h water, d r i e d over anhydrous sodium s u l f a t e and evaporated to a f f o r d a l i g h t y e l l o w gum (5.390 gm). This m a t e r i a l was chromatographed on alumina (150 gm). E l u t i o n w i t h benzene-chloroform (1:1) gave the d e s i r e d a l c o h o l (84, 4.40 gm). Y i e l d of the r e a c t i o n from c h l o r o i n d o l e (80) was 70%. The a l c o h o l was r e c r y s t a l l i s e d from . Me0H: and l a t e r sublimed at 98°/0.01 mm, mp 108-110°; v : 3340 (vOH), 3180 (vNH) cm"1; X (log e ) : 292 (3.73), 284 ITlcLX ITlclX (3.81), 274 (shoulder)(3.76), and 223 (4.45) mp; nmr (CT^OD) (Figure 15): T 2.40-3.09 ( m u l t i p l e t , 4H, i n d o l e p r o t o n s ) , 4.45 ( m u l t i p l e t , 1H, C 3,-H), 5.17 Csinglet, 2H, -CH^-OH), 9.00 ( t r i p l e t , 3H, -CH2-CH_3) ; mass spectrum (Figure 16): M + 284; main peaks: m/e 174, 160, 142, 124; high r e s o l u t i o n mass spectrometry: Calc. f o r C^gH^N^O: 284.188. Found: 284.184. Anal. Calc. for (C, oHo/No0)CH.0H: C, 72.09; H, 8.93; N, 8.85. l o ZH Z J Found: C, 72.05; H, 9.13; N, 8.22. Benzoate ester of alcohol (84) The alcohol (84, 1.50 gm, 5.4 mmole) was dissolved in dry pyridine (15 ml). The reaction mixture was cooled to 0° and benzoyl chloride (5.5 ml, 47 mmole) was added dropwise over a period of 10 minutes. The mixture was stirred at 0° for three hours, diluted with water (15 ml), made basic with 10% aqueous sodium carbonate solution and extracted with methylene chloride. The extract was washed several times with water, dried over anhydrous sodium sulfate and then evaporated very carefully (bath temperature not exceeding 40°) under reduced pressure to afford a thick gum (2.370 gm, contained traces of pyridine). This material was homogeneous on t i c and was used as such for the succeeding reaction. For analytical purposes, a small amount of benzoate (1 gm) was purified by chromatography on alumina (50 gm). Elution with benzene gave the desired material. This was recrystallised from methylene chloride-petroleum ether, mp 110.5-112.5°; v : 3000 J • max (several bands, vC-H, aromatic), 1715 (vC=0 of benzoyl group), 1455 (phenyl ring), 1260 (vC-O-C) cm"1; X (log e): 293 (3.8), 284 (3.96), nicLx 274 (3.94), 2.24 (4.61) mp; nmr: T 1.36 (singlet, 1H, indole NH), 2-3 (multiplet, 9H, 0^-0=0 + 4 indole protons), 4.58 (singlet, 3H, Cy-H + -CH_2-0-C-cj)) , 9.0 (triplet, 3H, -CH2-CH_3) ; mass spectrum: M+ 388; 0 main peaks: m/e 266, 170, 143, 124, 122; high resolution mass spectro-metry: Calc. for C.X.N.O.: 388.216. Found: 388.215. ZD Zo Z Z Anal. Calcd. for C„ cH„ oN o0„: C, 77.27; H, 7.28; N, 7.21. Found: ZJ ZO Z Z C, 77.05; H, 7.29; N, 7.05. A l t e r n a t i v e s y n t h e s i s of benzoate (85) The a l c o h o l (84, 2.82 gm, 0.01 mole) was d i s s o l v e d i n dry THF (50 ml) and the r e a c t i o n mixture was cooled w i t h i c e . To t h i s anhydrous potassium carbonate (5 gm) was added and the heterogeneous mixture was t r e a t e d , dropwise, w i t h benzoyl c h l o r i d e (5 ml, 0.042 mole) under n i t r o g e n . Theunixture was s t i r r e d at 0° f o r 1 hour and then at room temperature f o r 3 hours. The r e a c t i o n was worked up by adding water (50 ml) followed by m i l d warming of the mixture i n warm water bath. A few minutes l a t e r saturated- sodium carbonate s o l u t i o n (50 ml) was added and the mixture was ex t r a c t e d using benzene and methylene c h l o r i d e . The organic phase was washed w i t h water, d r i e d over anhydrous sodium s u l f a t e and evaporated.. The r e s u l t i n g m a t e r i a l was put on a column of alumina (100 gm). E l u t i o n w i t h chloroform afforded the benzoate (85) as white c r y s t a l l i n e s o l i d (2.3 gm) as w e l l as a y e l l o w i s h foam (1.5 gm, one spot on t i c ) . The o v e r a l l y i e l d was 99%. This benzoate showed the same value and s p e c t r a l p r o p e r t i e s as the benzoate obtained e a r l i e r . N-[3-{3-(2-Cyanomethylene)-indolyl}-ethyl]-3'-ethyl-3'-piperidenine (86) The benzoate (85, 2 gm, .005 mole) was d i s s o l v e d i n dimethyl-formamide (60 ml). To t h i s s o l i d potassium cyanide (3.3 gm, 0.050 mole) was added and the heterogeneous mixture was s t i r r e d under n i t r o g e n at room temperature f o r 1 hour. The temperature of the r e a c t i o n mixture was now g r a d u a l l y r a i s e d to 105-110° over a pe r i o d of 45 minutes. The r e a c t i o n was monitored by t i c and a f t e r 1 hour at the elevated tempera-t u r e , t i c i n d i c a t e d the completion of the r e a c t i o n . The mixture was cooled down to room temperature, d i l u t e d w i t h water (100 ml) and e x t r a c t e d w i t h methylene c h l o r i d e . The e x t r a c t was washed several*". times w i t h water, d r i e d over anhydrous sodium s u l f a t e and evaporated to a f f o r d a dark t h i c k o i l . This o i l y m a t e r i a l was l e f t under vaccum u n t i l a l l the dimethylformamide was removed. The r e s u l t a n t dark c r y s t a l l i n e compound (1.450 gm) was chromatographed on alumina. E l u t i o n w i t h benzene-petroleum ether (1:1) and l a t e r w i t h benzene f u r n i s h e d the pure n i t r i l e (86) as a c r y s t a l l i n e compound (0.825 gm, 55%). L a t e r f r a c t i o n s of e l u t i o n w i t h benzene and benzene-chloroform (9:1) afforded a small amount of a d d i t i o n a l n i t r i l e as gum (0.125 gm, 10%). This l a t t e r m a t e r i a l was contaminated w i t h a very minute red i m p u r i t y but the compound was of reasonable q u a l i t y to be u t i l i s e d i n the next r e a c t i o n . The o v e r a l l y i e l d of the r e a c t i o n was -65%. For a n a l y t i c a l purposes, a small amount of n i t r i l e (86) obtained was r e c r y s t a l l i s e d from methylene chloride-petroleum ether and l a t e r sublimed at 100°/ .01 mm, mp 135-137°; v : 3160 (vN-H), = 2900 ( s e v e r a l bands, vC-H, max aromatic), 2256 (VCEN) cm"1; A (log e ) : 291 (3.77), 281 (3.85), 274 (3.84)i 221 (4.69)my; nmr (Figure 17): x 1.63 ( s i n g l e t , 1H, indole-NH), 2.46-3.00 ( m u l t i p l e t , 4H, i n d o l e p r o t o n s ) , 4.58 ( m u l t i p l e t , 1H, C 3,-H), 6.2 ( s i n g l e t , 2H, -CH^-CN), 8.99 ( t r i p l e t , 3H, -CH 2-CH 3); mass spectrum: M + 293; main peaks: m/e 267, 169, 156, 124; high r e s o l u t i o n mass spectrometry: Calc. f o r C^H^N.^: 293.189. Found: 293.186. Anal. Calc. f o r C i nH„„N„: C, 77.75; H, 7.92; N, 14.32. Found: C, 77.65; H, 7.86; N, 14.16. N - [3-{3-(3-Carbomethoxymethylene)-indolyl}-ethyl]-3'-ethyl-3'- piperideine (88) C r y s t a l l i n e n i t r i l e (86, 0.746 gm, 2.5 mmole) was dissolved i n dry methanol (20 ml) and to t h i s a small amount of water (0.2 ml or 1%) was added. The mixture was cooled i n i c e and saturated with HC1 gas. Aft e r s t i r r i n g at room temperature for 60 hours, the so l u t i o n was taken to dryness under vacuum and the residue was treated with sodium bicarbonate s o l u t i o n . The basic s o l u t i o n was extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium s u l f a t e and evaporated. The crude product so obtained was dissolved i n a small amount of benzene and put on a column of alumina (40 gm). E l u t i o n with petroleum ether-benzene (2:8) and l a t e r with benzene furnished the pure carbomethoxy ester (88) as white c r y s t a l l i n e compound. The compound was r e c r y s t a l l i s e d from methylene c h l o r i d e -petroleum ether (0.574 gm, 70%), mp 85-87.5°; v : 3000 (several bands, max vC-H;., aromatic), 1728 (vC=0 of e s t e r ) , 1460 (6C-H , CH^-CO-), 1245 (vC-O-C) cm"1; X (log e): 292 (3.83), 283 (3.92), 274 (3.87), ITlctX 223 (4.43) mu; nmr (Figure 18): x 1.46 (broad s i n g l e t , 1H, indole-NH), 2.42-3.00 (multiplet, 4H, indole protons), 4.58 (multiplet, 1H, C 3,-H), 6.27 ( s i n g l e t , 2H, -CH 2-C00CH 3), 6.34 ( s i n g l e t , 3H, -CH^-COOCH^), 9.00 ( t r i p l e t , 3H, -CR^-CH.^); mass spectrum (Figure 19): M + 326; main peaks: m/e 267, 202, 156, 144, 124; high r e s o l u t i o n mass spectrometry: Calc. f o r C o oH o rN o0 o: 326.199. Found: 326.202. 20 26 2 2 Anal. Calc. for C o n H „ , N o 0 • C, 73.50; H, 8.04; N, 8.58. Found: 20 26 2 2 C, 73.47; H, 8.05; N, 8.71. Formylation of ester (88) using sodium hydride as base A 25-ml three necked flask was equipped with a magnetic s t i r r e r , a reflux condenser, a dropping funnel and a nitrogen inlet. A l l the glassware was flame dried then thoroughly flushed with dry nitrogen. To the reaction flask a 65% suspension of sodium hydride in paraffin o i l (0.050 gm, 1.3 mmole) was added. This suspension was. washed three times with 1-ml portions of dry benzene under nitrogen. The o i l free sodium hydride was suspended in a fresh portion of dry benzene'(2 ml) and to this freshly d i s t i l l e d methyl formate (dried f i r s t over calcium chloride and then over ^2^5^ ^ m ^ w a s a^ded. The carbomethoxy ester (88, 0.050 gm, 0.15 mmole) was dissolved in dry benzene (3 ml) and added dropwise to the above suspension. The reaction mixture was stirred at room temperature for.15 minutes and at 35° for 2 hours. At this time t i c indicated the completion of the reaction. The excess of hydride in the reaction mixture was destroyed by cooling the mixture to 0°, adding a few drops of methanol, followed by the addition of some crushed ice. The mixture was made acidic with 2 N HC1. The excess of the acid was neutralised with aqueous sodium bicarbonate solution and the heterogeneous mixture was extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium sulfate and evaporated to afford the crude enol. (89) as white foam (0.070 gm, contained some mineral o i l ) . This material was used as such for the next reaction. 16,17-Dihydrosecodin-17-ol (90) The crude enol (89) obtained above was dissolved in methanol (3 ml). The solution was cooled to -30° in a dry ice-acetone bath and sodium borohydride (0.050 gm) was added to t h i s i n small p o r t i o n s . A f t e r s t i r r i n g f o r 40 minutes at -30°, an a d d i t i o n a l amount of sodium boro-hydride (0.040 gm) was again added i n s m a l l p o r t i o n s to the r e a c t i o n mixture. Ten/,minutes l a t e r the mixture i n d i c a t e d no more unreacted enol on t i c and i n s t e a d the polar " d i o l " (105) had j u s t s t a r t e d appearing. The excess of borohydride i n the c o l d r e a c t i o n mixture was th e r e f o r e immediately quenched by c a r e f u l a d d i t i o n of 2-3 drops of 2 N HC1. The mixture was d i l u t e d w i t h water (5 ml) and the methanol was evaporated under reduced pressure. The remaining mixture was a c i d i f i e d w i t h 2 N HC1, made b a s i c w i t h sodium bicarbonate s o l u t i o n and e x t r a c t e d w i t h chloroform. The organic phase a f t e r drying and evaporation l e f t a white foam (68 mg). This m a t e r i a l was d i s s o l v e d i n a small amount of benzene and put on a column of alumina (2.5 gm). E l u t i o n w i t h benzene-chloroform i n the order (9:1), (7:3), (1:1) and f i n a l l y w i t h chloroform a f f o r d e d the pure a l c o h o l (90, 22 mg). The y i e l d of the r e a c t i o n from the e s t e r (88) was 40%. For a n a l y t i c a l purposes the a l c o h o l (90) was c r y s t a l l i s e d from dichloromethane, mp 131.5-132°; Vmax : 3 4 0 0 ( s h a rP> v N _ H ) » 3 0 5 0 (broad, vO-H), ^ 2900 ( s e v e r a l bands, vC-H, aromatic), 1718 ( vC=0), 1465 (6C-H, CH^CO-), 1235 (vC-O-C) cm"1; A (log e): 292 (3.86),'284 (3.93), 274 (shoulder) (3.87) , 222 (4.49) my; nmr (Figure 20): 1.16 ( s i n g l e t , 1H, i n d o l e N-H), 2.48-3.00 ( m u l t i p l e t , 4H, i n d o l e p r o t o n s ) , 4.61 (broad s i n g l e t , 1H, C^ ,_-H) , 6.00 ( m u l t i p l e t , 4H, -CH_2-0H + -C-H), 6.37 ( s i n g l e t , 3H, -C00CH_3) , 9.04 ( t r i p l e t , 3H, -C^-CH^); mass spectrum (Figure 21): M + 356; main peaks: m/e 338, 326, 214, 202, 124; high r e s o l u t i o n mass spectrometry: Calc. f o r C 0 1H CN 0 o: z l z o z 3 356.209. Found: 356.207. Anal. Calc. for c 2i a28 N2°3 : C ' •70"'24» E ' 7-93'> N> 7 - 8 6 « Found: C, 70.20; H, 7.83; N, 7.35. . Formylation of carbomethoxy ester C88) using t r i t y l sodium as base A 25-ml three necked flask was equipped with a magnetic s t i r r e r , a reflux condenser, a dropping funnel and a nitrogen inlet. A l l the glassware was flame dried and then thoroughly flushed with dry nitrogen. To a solution of the ester (88, 0.050 gm, 0.155 mmole) in dry tetra-hydrofuran (3 ml) was added dropwise a solution of t r i t y l sodium (2.2 ml, 0.18 N, 0.387 mmole) under nitrogen. The f i r s t half of the t r i t y l sodium solution decolorized very rapidly as the proton from the indole nitrogen reacted. . The other half of the solution decolorized very slowly u n t i l f i n a l l y the last 1-2 drops were added, the red color of the base stayed in the reaction mixture. The solution was stirred at room temperature for about 2 minutes and' then methyl formate (2 ml, dried f i r s t over calcium chloride and then freshly d i s t i l l e d over V^O^), was added dropwise. The red color of the base disappeared immediately and the resulting yellow solution was stirred at room temperature for 1.5 hours. The solution was evaporated to dryness under vacuum and the residue was acidified with 2 N HC1. The excess acid was neutralised with sodium bicarbonate solution and the mixture was extracted with chloroform. The extract was washed with water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude enol (89) was utilised as such for the next reaction. Reduction of the crude enol (89) obtained by using t r i t y l sodium as base The crude product obtained above (c o n t a i n i n g crude enol and t r i -phenylmethane) was d i s s o l v e d i n methanol. Some of the triphenylmethane d i d not d i s s o l v e i n methanol and was l e f t f l o a t i n g i n the r e a c t i o n medium. A f t e r c o o l i n g the heterogeneous mixture down to -30°, sodium borohydride. (50 mg) was added i n small p o r t i o n s over a p e r i o d of 10 minutes. The mixture was s t i r r e d at -30° f o r 40 minutes. At t h i s time an a d d i t i o n a l amount of sodium borohydride (40 mg) was again added to the r e a c t i o n mixture. 10 minutes l a t e r t i c i n d i c a t e d the completion of the r e a c t i o n . The excess of borohydride was immediately quenched by c a r e f u l a d d i t i o n of 2-3 drops of 2 N HC1 to the c o l d (-30°) r e a c t i o n mixture. The mixture was worked up i n e x a c t l y the same manner as i n d i c a t e d p r e v i o u s l y (page 105)• The crude product was. chromatographed on alumina (2; 5 gm). E l u t i o n w i t h benzene-petroleum ether (1:1) gave the triphenylmethane. The d e s i r e d compound (90) was e l u t e d w i t h benzene-chloroform i n the order (9:1), (7:3), (1:1) and f i n a l l y w i t h chloroform. The pure a l c o h o l (90, 20 mg) obtained represented a y i e l d of 37% from the e s t e r (88). Secodihe (107) A 10-ml f l a s k was equipped w i t h a magnetic s t i r r e r , a r e f l u x condenser, and a n i t r o g e n i n l e t . A l l the glassware was f i r s t flame d r i e d and then thoroughly f l u s h e d w i t h dry n i t r o g e n . To the r e a c t i o n f l a s k a 65% suspension of sodium hydride i n m i n e r a l o i l (25 mg, 0.65 mmole) was added. This suspension was washed three times w i t h 0.5-ml po r t i o n s of dry benzene and f i n a l l y the o i l f r e e sodium hydride was suspended in a fresh portion of dry benzene (0.5 ml). A solution of 16,17-dihydrosecodin-17-ol (90, 20 mg, 0.06 mmole) in dry benzene (2.5 ml) was dropped very-rapidly into the above suspension under nitrogen. The reaction mixture was stirred at 40° for 15 minutes. In the meantime a column of alumina (2 gm, activity IV) was made in dry benzene. The crude reaction mixture was-flushed through this column using benzene as eluent. This fraction (40 ml) which contained the desired material, was collected in a cold receiver. It was frozen with liquid nitrogen and freeze-dried under vacuum to afford secodine (107) as a light yellow gum (9.1:; mg, 50%). Nmr (Figure 31): T 0.89 (broad singlet, 1H, indole-NH), 2.40-3.00 (multiplet 4H, indole protons), 3.55 (doublet, J 1 Hz, 1H, olefinic proton of the acrylic ester), 3.91 (doublet, J 1 Hz, 1H, olefinic proton of the acrylic ester), 4.58 (multiplet, "1H, C^-H), 6.20 (singlet, 3H, CH2=C-C00CH_3), 9.00 (tr i p l e t , 3H, -CTL^-GH^); mass spectrum (Figure 32 )'• M+ 338; main peaks: m/e 307, 251, 214, 154, 124. PART II 3 Trifluoroacetic acid-[.H] Trifluoroacetic anhydride (1.17 gm, 5.55 mmoles) was added to 3 water- H (0.10 gm, 5.50 mmoles, 100 mcurie/gm) using a vacuum 3 transfer system. The resulting trifluoroacetic acid- H (1.27 gm, 0.9 mcurie/mmole) was stored under an atmosphere of nitrogen at -10° until required. 3 [ar- H]-Carbomethoxy ester (88) 3 Trifluoroacetic acid- H (1.27 gm, 0.9 mcurie/mmole) was added to the crystalline ester (88, 0.1887 gm) using a vac'uum transfer system. The solution was allowed to stand at room temperature for 48 hours under nitrogen atmosphere. After this time the trifuloroacetic 3 acid- R was removed with a vacuum transfer system and concentrated ammonium hydroxide solution (10 ml) was added carefully to the above gummy residue. The mixture was extracted with dichloromethane. The extract was washed with water, dried over anhydrous sodium sulfate and evaporated. The resulting gum was dissolved in methanol (10 ml) and then evaporated. This process was repeated four times to remove any labile tritium. The crude product was put on a column of alumina (15 gm) and the desired compound was eluted with benzene (0.152 gm, 80% 8 yield, specific activity 1.10 x 10 dpm/mg). 3 Formylation of [ar- H]-carbomethoxy ester (88) The triti a t e d ester obtained above (0.152 gm) was formylated using sodium hydride (0.150 gm) and freshly d i s t i l l e d methyl formate (4 ml) (for complete details of the procedure see page 104). Temperature of the reaction mixture was maintained at 35° and the reaction took 2 hours for completion. Evaporation of the solvent after work up gave the crude radioactive enol (89, 180.5 mg). This was used for the next. reaction as such. ' [ar-3H]-16,17-Dihydrosecodin-17-ol (90) The crude active enol (89, 180.5 mg) was dissolved in methanol (15 ml)i After cooling the reaction mixture down to -30°, sodium borohydride (180 mg) was added i n small portions over a period of 15 minutes. The mixture was stirred at -30° for 40 minutes. At this time an additional amount of sodium borohydride (100 mg) was again added in small portions. 15 Minutes later when the mixture indicated no more enol on t i c , the excess of borohydride was quenched with a few drops of 2 N HC1 and the mixture was worked up in exactly the same manner as indicated on page 105. The crude product (152 mg) was chromatographed on alumina (8 gm). Elution with benzene-chloroform in the order (9:1), (7:3), (1:1) and fi n a l l y with chloroform afforded the pure alcohol (90) which was crystallised from methylene chloride-petroleum ether (72 mg, 43% yield, specific activity 2.83 x 10"^ dpm/ mmole or 7.94 x 10^ dpm/mg). 14 N-[g-{3-(2-Cyano( CN)methylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine (86) The benzoate (85, 1.09 gm, 0.25 mmole) was dissolved in dimethyl-formamide (25 ml). This was treated with a mixture of radioactive 14 C-potassium cyanide (0.072 gm, total activity 8 mcurie) and potassium cyanide (0.753 gm, total potassium cyanide (0.825 gm) used in the reaction was 1.25 mmole or a five fold molar excess). The reaction was stirred at room•• temperature for 1 hour under nitrogen. The temperature of the reaction mixture was now gradually raised to 105° over a period of 45 minutes. After 1 hour at this elevated temperature, t i c indicated the completion of the reaction. The mixture was cooled to room temperature and the crude readioactive n i t r i l e (86) was isolated as a dark crystalline compound (for details of the work up see page 102) . This crude material was chromatographed on alumina (30 gm). Elution with benzene and later with benzene-chloroform (9:1) afforded the pure active n i t r i l e (86, 0.483 gm, specific activity 6 9* ^  4.32 x 10 dpm/mg or 1.27 x 10 dpm/mmole). The yield of the reaction was 63%. [14C00CR3]-Carbomethoxy ester (88) Crystalline radioactive n i t r i l e (86, 0.480 gm, 1.6 mmoles) was dissolved in dry methanol (10 ml). To this a small amount of water (0.1 ml or 1%) was added and the solution was saturated with HC1 gas. After st i r r i n g at room temperature for 60 hours, the crude ester was isolated £(for complete details of the work up see page 103). It was dissolved in a small amount of benzene and put on a column of alumina (25 gm). Elution with benzene-petroleum ether (2:8) and later with benzene furnished the pure radioactive ester (86, 0.302 gm). The yield of the reaction was 65%. 14 Formylation of [ COOCH^]-carbomethoxy ester (88) Radioactive ester (88, 0.100 gm, 0.30 mmole) was formylated using sodium hydride (0.100 gm, 2.6 mmoles) and freshly d i s t i l l e d methyl formate (4 ml) (for complete>.details of the procedure see page 104). The crude enol (89, 0.126 gm) isolated was used directly for the next reaction. [14C00CH3]-16,17-Dihydrosecodin-17-ol (90) The crude active enol (89, 0.126 gm) obtained above was dissolved in methanol. After cooling the solution downNto -30°, sodium borohydride (100 mg) was added in small portions over a period of 15 minutes. The mixture was stirred at -30° for 40 minutes. At this time a small amount of sodium borohydride (0.040 gm) was again added slowly to the reaction mixture. Teh/minutes later when t i c indicated the completion of the reaction, the crude alcohol was isolated (for details of the work up see page 105) and chromatographed on alumina (5 gm). Elution with benzene-chloroform in the order (9:1), (7:3), (1:1) and f i n a l l y with chloroform afforded the pure alcohol (90, 0.044 6 mg, specific activity" ,3. 56 x 10 dpm/mg). The yield of the reaction from the ester (88) was 40%. Extraction of alkaloids from Vinca minor Linn The following procedure was developed in order to extract and purify the alkaloids of Vinca minor Linn plants. This procedure was used for a l l extractions of Vinca minor L. plants and was scaled according to the wet weight of the plants used. Vinca minor Linn plants (9 kg', wet weight), obtained from the gardens of the University of British Columbia, were •macerated with methanol in a Waring blender, fi l t e r e d and .-re-macerated " until the f i l t r a t e was colorless. This green f i l t r a t e (8000 ml) was concentrated to dryness under reduced pressure and the residue was dissolved in 2 N HC1 (4500 ml). The acid layer was extracted with benzene (2 x 2000 ml) and the benzene extracts were back extracted with 2 N HC1 (2 x 500 ml). The combined aqueous phases were made basic with 15 N ammonium hydroxide, taking care that the temperature of the solution did not rise above 25°, and extracted with chloroform (3 x 2400 ml). The combined chloroform extracts were washed with water, dried over sodium sulfate, and concentrated under reduced pressure. The resulting alkaloids residue (13.6 gm) was dissolved in benzene-methylene chloride (1:1) (100 ml) and chromatographed on alumina (700 gm). The column was eluted successively with petroleum ether, benzene, chloroform, and methanol; fractions of 700 ml were taken. The fractions eluted with petroleum ether and benzene were combined and subjected to an additional column chromatography on alumina (300 gm). Elution with petroleum ether-benzene (6:4) afforded minovine (73, 0.41 gm). Successive fractions, eluted with petroleum ether-benzene (2:8) and fi n a l l y with benzene, were combined and crystallised from methanol affording vincamine (72, 0.65 gm). Both these alkaloids were compared on t i c with authentic samples. 14 Feeding of [ COOCH3:]-16,17-dihydrosecodin-17-ol (90) to Vinca minor Linn, (feeding experiment no. I) [14C00CH3]-16,17-bihydrosecodin-17-ol (90 , 0.0065 gm, 9.89 x 6 •*: 10 dpm) was made soluble in 0.1 N acetic acid (1 ml) and ethanol (0.5 : The cloudy.solution was diluted with' water (5 ml). The.resulting clear solution was distributed equally among ten test tubes and three Vinca minor cuttings were placed into each of these test tubes (total weight of plants uti l i s e d was 43 gm). The plants were placed under fluorescent lamp, illumination and the aqueous levels in the test tubes were maintained with d i s t i l l e d water. After four days the cuttings were extracted to afford the crude alkaloids as dark foam, ft (0.092 gm, 3.06 x 10 dpm.or 31% of the total- activity fed). The crude extract was dissolved in a small amount of benzene-methylene chloride (~ 2ml) and put on a column of alumina (15 gm). Elution with petrole um ether—benzene (1:1) and then further purification of the resulting gum by preparative thin layer chromatography ( s i l i c a gel, ethyl acetate-methanol, 2:1) afforded pure minovine (73, 0.00545 gm). It was diluted further with an authentic sample of minovine (15.70 mg). Several crystallisations revealed that the alkaloid contained virtually no activity. Further elution of the column with petroleum ether-benzene (4:6) afforded pure vincamine (72) as a crystalline compound (7.7 mg). After several crystallisations from methanol, vincamine indicated an activity of 102 dpm (total). This represented a maximum incorpora-tion of < 0.001%. 3 Feeding of [ar- H]-secodine(107) to Vinca minor Linn. (feeding experiment no. 2) In view of the rapid dimerization of secodinel the following procedure was developed and is typical of a l l the feeding experiments done whenever secodinewas fed to the various plant species in our laboratories. A 10-ml f l a s k was equipped w i t h a magnetic s t i r r e r , a r e f l u x condenser and a dry n i t r o g e n i n l e t . The glassware was flame d r i e d and then thoroughly f l u s h e d w i t h n i t r o g e n . A 65% suspension of sodium hydride i n mineral o i l (10 mg, 0.26 mmole) was added to the r e a c t i o n f l a s k . This suspension was washed three times w i t h 0.5 ml-portions of dry benzene under n i t r o g e n and the o i l f r e e sodium hydride was suspended i n a f r e s h p o r t i o n of dry benzene (0.5 ml). In a small dry 3 t e s t tube [ar- H]-16,17-dihydrosecodin-17-ol (90, 10 mg, 0.03 mmole, 79.4 x 10^ dpm) was d i s s o l v e d i n dry benzene (1.2 ml) by s l i g h t l y warming the t e s t tube i n a hot water bath. This s o l u t i o n was dropped very r a p i d l y ( i n about 35 seconds) i n t o the suspension of sodium hydride. The mixture was s t i r r e d at 40° f o r 15 minutes and then q u i c k l y f l u s h e d through a s m a l l column of alumina (1.5 gm, a c t i v i t y IV) made up w i t h benzene. The column was e l u t e d w i t h benzene and the e l u t e d f r a c t i o n was c o l l e c t e d i n a c o l d 25r-ml volu m e t r i c f l a s k . One ml of t h i s s o l u t i o n was d i l u t e d to 100 ml w i t h benzene and 1 ml of t h i s -3 l a t t e r s o l u t i o n (or 2.5 x 10 of the compound to be fed) was used f o r counting purposes. The remaining p o r t i o n (24 ml) of the benzene f r a c t i o n was.transferred to a s p e c i a l l y designed evaporator. The f r a c t i o n was frozen w i t h l i q u i d n i t r o g e n and f r e e z e - d r i e d under vacuum. This f u r n i s h e d secodine(107) as a l i g h t y ellow gum (3.2 mg, 2.65 x 10 8 dpm). The gum obtained above was made s o l u b l e i n 0.1 N a c e t i c a c i d (1 ml) and ethanol (0.5 m l ) . The.cloudy/solution was d i l u t e d w i t h water (5 ml) and the r e s u l t i n g c l e a r s o l u t i o n w a s ' d i s t r i b u t e d "equally, among ten t e s t - t u b e s . Three Vinca minor c u t t i n g s were i n s e r t e d i n t o each"""of. these t e s t tubes ( t o t a l weight of p l a n t s u t i l i s e d was 40 gm) and the plants were placed under fluorescent lamp illumination and the aqueous levels in the test tube were maintained with d i s t i l l e d water. After four days, the cuttings were extracted to afford the crude alkaloids as a dark foam (90.2 mg, 5.8 x 10^ dpm representing a recovery of 22% of the total activity fed). The crude product was dissolved in a small amount of benzene-methylene chloride and chromatographed on alumina (20 gm). Elution with petroleum ether-benzene (7:3) and then subsequent purification of the resulting gum by preparative t i c ( s i l i c a gel, ethyl acetate-methanol, 2:1) afforded pure minovine (73, 4.8 mg). This was diluted with cold minovine (10.4 mg) andthe total compound (15.2 mg) was crystallised to constant activity (562 dpm/mg). This represented a maximum incorporation of 0.001%. This incorporation was corrected for the amount of secodine(107) that dimerized at the time of feeding by running a "blank" experiment (for details see pageill7) . The corrected incorporation should be < 0.0015%. Further elution of the column with petroleum ether-benzene (1:1) afforded crystalline vincamine (72, 12.7 mg). This was further purified by preparative t i c (alumina, ethylacetate-chloroform, 1:1). Pure vincamine (3.8 mg) obtained in this manner was.diluted with cold vincamine (4.65 mg) andthe mixture was crystallised to constant activity (261 dpm/mg). This represented a maximum incorporation of 0.0013%. This extent of incorporation was again corrected for the amount of secodine(107) that dimerized at the time of feeding by correla-tion with the "blank" experiment (for details see page 117). The corrected incorporation should be 0.002%. "Blank" f o r the feeding experiment no. 2 [ 1 4COOCH 3]-16,17-Dihydrosecodin-17-ol (90, 10 mg, 20.7 x 10 6 dpm) 14 6 was dehydrated to [ COOCH^-secodine(107, 3.4 mg,7.03 x 10 dpm) i n e x a c t l y the same manner as i n d i c a t e d i n the feeding experiment no. 2. The r e s u l t i n g gum was made s o l u b l e i n 0.1 N a c e t i c a c i d (3 ml) and ethanol (0.5 ml). The s o l u t i o n was l e f t at room temperature f o r 2 hours ( t h i s i s the maximum time the Vinca minor c u t t i n g s take to absorb the above volume of s o l u t i o n ) , f r o z en w i t h l i q u i d n i t r o g e n , and f i n a l l y f r e e z e - d r i e d under vacuum. The r e s u l t i n g s o l i d was r e d i s s o l v e d i n methanol and a small p o r t i o n of t h i s s o l u t i o n was spotted h o r i z o n t a l l y on two Kodak n e u t r a l alumina chromatogram sheets. The sheets were developed i n a mixture of benzene-chloroform (1:1) and passed through a c a l i b r a t e d Nuclear-Chicago A c t i g r a p h 11 Model 1039 t i c counter connected to a recorder (Nuclear-Chicago Model 8416) and i n t e g r a t o r (Nuclear-Chicago Model 8704). The a c t i v i t i e s found i n three spots corresponding to the b a s e l i n e , dimeric compounds (presecamine and secamine) and secodine 1 and t h e i r r e l a t i v e percentages i n the i n i t i a l mixture are summarized i n Table 2. Table 2. Results of the "blank" experiment . Sheets Secodine Dimeric Compounds Base l i n e M a t e r i a l a c t i v i t y % a c t i v i t y % a c t i v i t y % cpm cpm cpm Sheet no. 1 21,693.94 61.22 11.313 32 2,394.46 6.78 Sheet no. 2 32,650 61.52 16,871 32.03 3,422 6.45 REFERENCES 1. R. Robinson, "The Structural Relations of Natural Products", Claredon Press, Oxford, (1955). 2. E.E. van Tamelen, L.J. Dolby, andNR.G. Lawton, Tetrahedron Letters, 30 (1960). 3. E. 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McPhee, J. Am. Chem. Soc, 65, 2387 (1943). 101. E.E. van Tamelen and J.B. Hester, Jr., J. Am. Chem. Soc, 91^, 7342 0-969). . PART B STUDIES RELATED TO THE BIOSYNTHESIS OF VINCAMINE INTRODUCTION Some years ago during the course of an examination of the A f r i c a n apocynaceous pl a n t Hunteria eburnea p i c h o n 1 and the t i n y evergreen-p l a n t Vinca minor L. 2, f o r substances'of p o s s i b l e / t h e r a p e u t i c valued s e v e r a l new a l k a l o i d s were i s o l a t e d . As the s t r u c t u r e of these new 3 R 3 = H, R± = OH, R 2 = C00CH 3 Vincamine 4 R 3 = H, R 1 = COOCH3, R 2 = OH Epivincamine 5 R 3 = OCH3, R 1 = OH, R 2 = COOCH3 V i n c i n e 'Figure 1. Various eburnamineT-vincamine type alkaloids. alkaloids were' unraveled, i t soon became evident that the unifying feature of a l l these compounds was the inheritance of a general pentacyclic structure (e.g.,'1-7-, see Figure 1). Since vincamine (3) and eburnamine (1) were the major alkaloids of the above mentioned plants, these pentacyclic alkaloids are now referred to as "eburnamine-vincamine" type alkaloids. A recent review of the chemistry of this 3 family is now available. The striking similarity between the arrangement of the "non-tryptophan or C ^ Q" portion of vincamine (3) and the Aspidosperma 4 alkaloid (like vincadifformine, 8) (see Figure 2) led Wenkert to 3 21 Figure 2. Scheme showing similar arrangement of non-tryptophan portion in vincamine and vincadifformine. suggest that vincamine (3) is a rearranged Aspidosperma alkaloid. In attempting to bring this family into the main stream of the other groups (Corynanthe, Aspidosperma and Iboga) Wenkert advanced an attractive mechanism which is shown in Figure 3. ' This rational would explain the biosynthesis of the entire family i f i t was assumed that 9 . . . 10 V CH300C \ 3 Figure 3. Wenkert's proposal for the rearrangement of Aspidosperma skeleton to vincamine. vincamine (3) was the precursor of the alkaloids eburnamine (!), eburnamonine (2) etc. At a time when Wenkert put forward this proposal, the intermediate.(12) had already been converted in vitro into vincamine (12-3, Figure 4)"\ This latter conversion had another 3 Figure 4. Synthesis of vincamine. very close parallel in the synthesis of eburnamine (13-14, Figure 5) . The laboratory syntheses however do,s. not provide any real evidence for the biosyntheses of these alkaloids. It i s the purpose of this portion of-" the thesis to present some preliminary studies which we hope w i l l allow us to obtain some information in this direction. dl-Eburnamine •Figure 5. Synthesis of eburnamine. DISCUSSION In spite of the fact that the genesis of these pentacyclic alkaloids (Figure i) w a s Proposed ^several years ago, i t is somewhat surprising that u n t i l now the biogenetic proposal has received no support from feeding experiments with radioactive precursors. Our previous experience with Vinca minor L. as already noted in the f i r s t section of this thesis stimulated us to initiate some biosynthetic studies in this direction. _Before describing the results of our experiments i t is pertinent to c a l l attention to the fact that i t had alreadysheen established 7 8 In our laboratory ' that ring opening of the pentacyclic Aspidosperma type alkaloids to nine-membered intermediates (e.g. 9-10, Figure 3) and the transannular cyclization reaction (e.g. 10-11, Figure 3) are not significant biochemical reactions in Vinca rosea and Vinca minor plants. This study casts some doubt about some of the steps depicted in Wenkert's proposal. On the other hand there was no reason to doubt the.possible validity of the other" steps proposed. -While i n i t i a t i n g the project we were faced with the problem as to which substrate we should select (Figure 3) for biosynthetic evaluation.'" It was indicated in the Introduction that (12), a bio-intermediate in Wenkert's proposal, has been converted into vincamine (3) in-rvitro (Figure 4). We decided that the knowledge gained by evaluation of this type of transformation (12 •> 3, Figure 4) in vivo while simulating the in,vitro results would also be great value in suggesting the dynamics of the biosynthesis of vincamine (3). For this purpose a synthesis of a close relative (i.e. 24) of the proposed intermediate (12) was contemplated and the synthetic sequence is f u l l y revealed in Figure 6. Our choice for making the compound (24) as our i n i t i a l synthetic target was dictated by the fact that the proposed synthetic sequence was very short and i t was easy to pursue the sequence from commercially available starting materials (3-acetylpyridine and tryptophol). It was assumed that the model compound C24) was capable of transformation in vivo to the putative intermediate (12) via biologically feasible reactions. Before starting the synthetic sequence outlined in Figure 6, the following pilot route (Figure 7) on model piperidines was investigated. This work was undertaken to obtain optimum conditions for most of the reactions to be u t i l i s e d later in the sequence in Figure 6. Accordingly 3-acetylpyridine (15) was converted into the known ketal (16) and this compounds was.treated with methyl iodide in ether to afford the crystalline salt (25) in 95% yield. Catalytic hydrogenation of the quaternary salt (25) yielded the piperidine ketal (26) which on acid hydrolysis furnished N-methyl-3-acetylpiperidine (27). The structure assigned was derived from the following spectral data. A sharp peak in the infrared (1710 cm 1) and a three proton singlet in the nmr (x 7.85) was reconcilable with the presence of a methyl ketone where as a three proton singlet for the N-methyl occurred in the expected Figure J . Synthesis of some -model piperidine systems. region (x 7.72). Finally the molecular formula, CgH^NO, was confirmed by elemental analysis. Attempts were now made to alkylate the ketone (27) using equivalent amounts of t r i t y l sodium and a l l y l bromide. The chromato-graphy of the crude product on alumina allowed the separation of two major components. The desired compound (28), eluted f i r s t from the column, indicated the following spectral data. In ;the nmr spectrum (Figure 8) the newly incorporated olefinic protons (-CH^ -CH^ CH^ ) were represented by a multiplet in the region x 4.20-5.18 where as the three proton singlets for the methylketone and the N-methyl groups were s t i l l located in the expected region (x 7.88 and 7.81). The molecular formula, C. H _N0, was confirmed by elemental analysis. \ PPM(T) Figure 8. Nmr spectrum of 28. The nmr spectrum (Figure 9) of the other compound isolated above showed the disappearance of the singlet corresponding to the methyl ketone group. With this result i t immediately became obvious that in this compound the alkylation had occurred at the methyl group of the methyl ketone. Since the multiplet in the olefinic region integrated for three protons (-C^-CH^CH^) , the possibility that this was a dialkylated material was dismissed. Onthe basis of this limited information the compound has been tentatively assigned structure (29). Further work is necessary before a more definite structure could be put forth. 0 Encouraged by the success on the inodel compounds we started the sequence in Figure 6. The synthesis of the carbon skeleton present 9 in C241 was facilitated by the reported synthesis of the ketone (21). Although this synthesis was reported, the experimental procedure of some of the steps were not completely clear and the 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 quickly eliminated and the experimental procedures followed in the present work are in accord with the sequence indicated in Figure 6. The ketalization of 3-acetylpyridine (15) proceeded to give a good yield (78%) of the ketal ( 1 6 ) . T r y p t o p h y l bromide (18) was obtained from tryptophol (17) in 80% yield and was used immediately (owing to the instability of 18) for reaction with ketal (16) to give the salt (19) in quantitative yield. The salt was reduced catalytically over platinum oxide. The crude reduced product (20) on acid hydrolysis followed by chromatography on alumina and crystallization afforded the pure ketone (21), mp 132.5-134°. The spectral data of the ketone compared favourably with the assigned structure. A sharp peak in the carbonyl region of the infrared spectrum (1704 cm "*") and a three proton singlet in the nmr (x 7.91) were diagnostic for the presence of the methyl ketone in (21). The molecular formula, C^R^^O, was confirmed by mass spectrometry (M+ 270). In an attempt to alkylate the ketone (21), the anion of the ketone was made by means of t r i t y l sodium and the anion so formed was immediately quenched with a l l y l bromide. The crude product on chromato-graphy on alumina afforded the desired compound (22) i n 30% yield. For analytical purposes a small amount of material was further purified by sublimation at 150°/0.05 mm. Supporting evidence for the assigned structure was derived from the following spectral data.. The .prominant feature of the nmr spectrum (Figure 10) in comparison to the nmr of the ketone (21) was the appearance of a multiplet in the olefinic region Cx 4.40^ -5.20, 3R,< -CH2-CH=CH_2) while the signals for the methyl ketone Cx 7.93) and the indolic-NH (x 1.93) were s t i l l located at the same regions as they were i n the ketone (21). This latter observation was a v i t a l piece of evidence in indicating that the reaction has indeed taken the desired course. However fi n a l confirmation for the structure (22) came from mass spectrometry. In the mass.spectrum (Figure 11) the '.. • .W"*- - ft* Jv-»K V. '• c i-i ft! cn cn cn rc o rt-i-i g O I—1 ro 3 RELATIVE INTENSITY 25 • . 50 1 f: .' s — 3 1 0 75 •96 (2.5x) 13? ;• • S i 180 O • Q olefin (22) indicated a molecular ion peak at m/e 310. This was in. agreement with the molecular formula, ^20^26^2^' A s t o ^ e expected the parent ion of (22) fragmented to the ions 30 (m/e 130) and 31 (m/e 180). Furthermore ion (31) readily lost the acetyl side chain to 33, m/e 96 32, m/e 137 'F&pUra 12. Postulated fragmentation of 22 in the mass spectrometer. give the radical ion 32 (m/e 137). This latter ion further lost the a l l y l side chain to give the ion 33 (m/e 96, metastable peak at m/e 67). A scheme portraying the mass spectrometric fragmentations has been summarized in Figure 12. In spite of repeated attempts, the olefin (22) did not give the correct elemental analysis. With the completion of the basic carbon skeleton, i t was now considered appropriate to hydroxylate the a l l y l i c double bond in (22). Of the many reactions availably for this purpose, osmylation enjoys a reputation for selectivity and was favoured a p r i o r i in the present connection. Therefore the olefin (22) was dissolved in dry tetrahydro-furan and treated with osmium tetroxide. 1 1 The mixture was l e f t in the dark at room temperature for 2 days. When the reaction mixture was worked up, unfortunately we observed a considerable loss of material. The crude product isolated represented a recovery of only 50% of the starting olefin (22). However the crude product was chromatographed on alumina. The small quantities of pure material so obtained in this particular investigation allowed only infrared and nmr spectral determinations. The spectral data immediately revealed that the above compound was not the desired diol (23). For example there was no evidence for the presence of a methyl ketone. The most interesting feature of the nmr spectrum was the finding there was no absorption in the olefinic region and instead a two proton multiplet and a three proton singlet were located at x 6.5 and T 8.64 respectively. These two signals being reconcilable with the presence of a hydroxymethylene group and a O-C-CH^ system suggested a tentative assignment of structure (34.15 to the above' compound. However further work is necessary- before a definite structure can be established. 34 This unexpected cyclization of the diol (.23) to the hemiacetal ;; (341 made i t undesirable to continue the sequence as outlined in Figure 6. It therefore became apparent that to obviate the above di f f i c u l t y i t was necessary to reduce the acetyl group in the olefin (22) to the corresponding ethyl group (i.e. -22-35) prior to osmylation of the olefinic^linkage. In this manner the eventual completion of the. 0 22 .35 syntheses of intermediates bearing the desired skeleton as portrayed in 24 could Be envisaged. Unfortunately time did not permit me to carry this work any further at this time but i t w i l l be continued by other workers in our laboratory. EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected. The ultraviolet (uv) spectra were recorded in methanol on / ' ' \ a Cary-11 recording spectrometer, andvthe, infrared (ir) spectra were taken on a Perkin Elmer Model 21 and Model 137 spectrometers. Nuclear magnetic resonance (nmr) spectra were recorded in deuteriochloroform at 100 megacycles per second (unless otherwise stated) on a Varian HA'rlOO instrument and the chemical shifts are given in Tiers T scale with reference to tetramethylsilane as the internal standard; multiplicity, integrated area and type of protons are indicated in parentheses. Mass spectra were recorded on an Atlas CH-4 mass spectro-meter and high resolution molecular weight determinations were carried out on an AE-MS-9 mass, spectrometer. Analyses were carried out by Mr. P. Borda of the Microanalytical Laboratory, The University of Br i t i s h Columbia. Wbelm neutral alumina and s i l i c a gel G (acc. to Stahl) containing 2% by weight of General Electric Retma p-1, Type 188-2-7 electronic phosphor were used for analytical and preparative thin layer chromatography ( t i c ) . Chromatoplates were developed using the spray reagent carbon tetrachloride-antimony pentachloride (2:1) or iodine vapors. Woelm neutral alumina (activity III) was used for column chromatography (unless otherwise stated). 3-Acetylpyridine ethylene ketal (16) A solution of 3-acetylpyridine (15, 60 gm), ethylene glycol (40 gm) and p-toluenesulfonic acid hydrate (105 gm) in benzene (250 ml) was heated under reflux for 17 hr with a Dean-Stark apparatus to remove water. The mixture was poured into excess aqueous sodium bicarbonate solution, the layers separated and the aqueous phase was extracted with benzene. The combined ^extracts were washed with sodium bicarbonate solution water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. D i s t i l l a t i o n gave the product (63.3 gm); bp 165/88 mm; nmr: x 2.5 (multiplet, 4H, aromatic), 6.1 (multiplet, 4H, ketal), 8.35 (singlet, .3H, C-CH3)_. N-Methyl-3-acetylpyridinium iodide ethylene ketal (25) A solution of methyl iodide (20 gm) in dry ether (50 ml) was added to a stirred ice-cold solution of 3-acetylpyridine ethylene ketal (16, 20 gm) in ether (50 ml). The reaction mixture was stirred at room temperature overnight and the precipitated salt, 25, was fi l t e r e d (34.5 gm, 95% yield) and purified by recrystallization from methanol, mp 190°. Anal. 'Calc. for C^H^NO^: C, 39.11; H, 4.60; N, 4.60. Found: C, 39.12; R, 4.86; N, 4.55. N-Methyl-3-acetylpiperidine ethylene ketal (26). The salt (25, 10 gm, 32.6 mmoles) was dissolved in a mixture of water and ethanol (100 ml, 1:1). This pale yellow solution was added dropwise to a suspension of hydrogen-activated platinum oxide (800 mg) in ethanol (200 ml) and the mixture was hydrogenated at atmospheric pressure. The absorption of hydrogen was complete (about 2400 ml, 66 mmole) after 8 hours. The catalyst was fi l t e r e d off and the solvent removed in vacuo to afford a light yellow solid. This product was dissolved in 10% sodium carbonate solution and was extracted with chloroform. The organic layer was washed with water, dried over anhydrous sodium sulfate and evaporated to afford to pale f ilm yellow oil.(5.8 gm)• - v m a x : no absorption in the aromatic region; nmr (60 mc/s): x 6.1 (singlet, 4H, ketal), 7.74 (singlet, 4H, N-CH3 + H(?)), 8.75 (singlet, 311, C-CH3). NT-Methyl-3-racetylpiperidine (27) The crude ketal (26, 5.8 gm) was dissolved in 2 N hydrochloric acid (50 ml) and the mixture was stirred at room temperature overnight. The solution was made basic with 10% sodium carbonate solution and extracted with chloroform, The extract.was washed with water, dried over anhydrous sodium sulfate and evaporated. The resulting o i l was d i s t i l l e d under reduced pressure, bp 116-117°/14 mm, to give a clear o i l (4.48 gm). The yield of the reaction for reduction and removal of the ketal was 90%; v : 1710 (vC=0); nmr (60 mc/s): x 7.72 max Csinglet, 3E,. N-CH3), 7.85 (singlet, 3H, -C0CH_3). Anal. Calc. for CoH1cN0: C, 68.01; H, 10.63; N, 9.93; 0. 11.34. o 1 5 Found: C, 67.80; H, 10.61; N, 10.08; 0 ; 11.50-N - M e t h y l - 3 - a c e t y l - 3 - a l l y l p i p e r i d i n e ( 2 8 ) A 5 0 - m l three necked f l a s k was equipped w i t h a magnetic s t i r r e r , a r e f l u x condensor, a dropping funnel and a n i t r o g e n i n l e t . The g l a s s -ware was flame d r i e d and then thoroughly f l u s h e d w i t h dry n i t r o g e n . To a s o l u t i o n of tr i p h e n y l m e t h y l sodium ( 2 3 ml, 0 . 1 7 N, 0 . 0 0 3 8 mole) was added dropwise a s o l u t i o n of the ketone ( 2 7 , 0 . 5 4 0 gm, 0 . 0 0 3 8 mole) i n anhydrous ether (2 ml). The formation of the carbanion was i n d i c a t e d by the i n s t a n t disappearance of the red c o l o r of the base. The mixture was s t i r r e d at room temperature f o r 1 hour. A l l y l bromide ( 0 . 3 4 ml, 0 . 0 0 3 9 mole) was taken up i n anhydrous ether and added dropwise to the above pale yellow s o l u t i o n over a p e r i o d of 5 minutes. During the a d d i t i o n of a l l y l bromide, sodium bromide s t a r t e d separating out 1and the mixture became cloudy. The mixture was s t i r r e d f o r an a d d i t i o n a l 2 0 minutes. Water ( 1 0 ml) was added and the l a y e r s were separated; The aqueous l a y e r was ex t r a c t e d w i t h ether. The two ether l a y e r s were combined and evaporated i n Vacuo. The l i g h t y ellow semi s o l i d was taken up i n benzene and t r e a t e d w i t h 10% aqueous a c e t i c a c i d ( 2 0 ml). The two l a y e r s were separated, and the benzene l a y e r was washed twice w i t h water. The combined aqueous l a y e r s were combined, made.basic.with 10% aqueous sodium carbonate s o l u t i o n and ex t r a c t e d with, chloroform. The e x t r a c t was washed w i t h water, d r i e d over anhydrous sodium s u l f a t e and evaporated. The r e s u l t a n t l i g h t y e l l o w o i l CO.450 gm) was chromatographed on alumina ( 4 0 gm). E l u t i o n w i t h benzene-petroleum ether CI:9) a f f o r d e d the des i r e d compound ( 2 8 , 0 . 1 0 5 gm),bp 1 2 5 ° / 6 mm; nmr (Figure 8 ) : x 4 . 2 0 - 5 . 1 8 ( m u l t i p l e t , 3 H , T -CH=CI1 2 ) , 7 . 8 1 ( s i n g l e t , 3 H , N - C H ^ ) , 7 . 8 8 ( s i n g l e t , 3 H , - C 0 C H _ 3 ) . Anal. Calc. for C.-H-.-NO: C, 72.85; H, 10.48; N, 7.72. Found: i x i y C, 72.83; H, 10.72; N, 7.55. Further elution of the column with petroleum ether-benzene (1:1) provided another compound, 3-(5'-keto-l'-pentenyl)-N-methylpiperidine (29, 60 mg). Nmr (Figure 9): T 4.10-5.2 (multiplet, 3H, ~CH=CH_2) and 7.78 (singlet, 3H, N-CH^). The loss of singlet corresponding to the methyl ketone was suggestive of alkylation at the methyl group, but the evidence at the time was.insufficient to assign a definite structure to this compound. Tryptophyl bromide (18) 9 A solution of phosphorus tribromide (1 ml) in ether (20 ml) was added to an ice-cold solution of tryptophol (17, 4.8 gm) in ether (250 ml). After 15 hours, the supernatant was decanted, washed with sodium bicarbonate solution, water and dried with sodium sulfate. Removal of the solvent yielded the product as white crystals (4.5 gm, 80%), mp 95-100° ( l i t . mp 90-95°). N^J g^ (3^Indolyl)_-ethylT, -31 -acetylpyridinium ethylene ketal bromide (19) . Tryptophyl bromide (18, 4.5 gm) and 3-acetylpyridine ethylene ketal (16, 10 ml) were heated at 80° under nitrogen for 8 hours. Addition of ether (40 ml) to the cooled reaction mixture yielded a precipitate whose crystallization from methanol afforded pure salt (19, 7.2 gm), mp 208-211° (Lit. mp 209-210°). N- [ g - ( 3 - I n d o l y l ) - e t h y l ] - 3 ' - a c e t y l p i p e r i d i n e ethylene.ketal (20) The pyridinium s a l t (19, 7.0 gm) was dissolved i n ethanol (250 ml). This yellow s o l u t i o n was added dropwise to a suspension of hydrogen-activated platinum oxide (1 gm) i n ethanol (100 ml) and the mixture was. hydrogenated at atmospheric pressure. The uptake of hydrogen was complete a f t e r 10 hours. The c a t a l y s t was f i l t e r e d o ff and the solvent removed i n vacuo to a f f o r d a yellow gum. This was dissolved i n 10% aqueous sodium carbonate'solution and extracted with chloroform. The extract was washed with water, drie d over anhydrous sodium s u l f a t e and evaporated to a f f o r d the p i p e r i d i n e k e t a l (20) as a l i g h t yellow gum. N-Ig- C 3-Indolyl)-ethyl]-3'-acetylpiperidine (21) The crude pip e r i d i n e ethylene k e t a l (20, 7.5 gm) was dissolved i n methanol (150 ml). The solutionswas a c i d i f i e d with 4 N HC1 (100 ml) and the mixture was heated at 85° for 5 hours. A f t e r cooling to room temperature, methanol was evaporated from the r e a c t i o n mixture. The resultant gum was made basic with sodium bicarbonate s o l u t i o n and extracted with chloroform. The extract was washed with water, dried over anhydrous sodium s u l f a t e and evaporated. The r e s u l t i n g gum (6.1 gm) was chromatographed on alumina (400 gm). E l u t i o n with benzene-petroleum ether (1:1) and benzene afforded the desired ketone (21). This was c r y s t a l l i s e d from benzene-petroleum ether (4.33 gm), mp 132.5-134°; v (CEC1„): 3400 (vN-H), 1704 (vC=0) cm"1; nmr: x 1.68 Csinglet, 1H, N-H), 2.40-3.12 (multiplet, 5H, indole protons), 7.91 1 ( s i n g l e t , 3H, -COCH^); mass spectrum: M + 270; main peaks: m/e 140, 130, 103. • i N-[g-(3-Indolyl)-ethyl)-3'-acetyl-3'-allylpiperidine (22) A 500-ml three necked flask was equipped with a magnetic s t i r r e r , a reflux condensor, a dropping funnel and an nitrogen i n l e t . A l l the glassware was flame dried and then thoroughly flushed with dry nitrogen. To a solution of the piperidine ketone (21, 4.00 gm, 0.014 mole) in dry tetrahydrofuran (150 ml) was added dropwise a solution of triphenyl-methyl sodium unt i l the red color of the base just stayed in the reaction mixture (150 ml, 0.2 N, 0.030 mole). The solution was stirred at room temperature for about 5 minutes and then a l l y l bromide CI.2 ml, 0.014 mole) in dry tetrahydrofuran (15 ml) was added to the above solution. The reaction mixture was stirred at room temperature for 2 hours and then evaporated to dryness. The residue was extracted with chloroform. The organic layer was washed with water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was dissolved i n a small amount of benzene and put on a column of alumina (250 gm). Elution with benzene-petroleum ether (1:1) furnished the desired compound (22,'1.4 gm,.yield 30%). For analytical purposes a small amount of a l l y ! compoxmd was sublimed at 140-150°/0.05 mm; v (CEC1 1: 3367 (yN-R), 1701 (vC=0) cm"1; nmr (Figure 10): x 1.93 TJlcLX j (broad singlet, 1R,> indole-NH), 2.40-3.20 Cmultiplet, 5H, indole protons), 4.40-5.20 Cmultiplet, 3R, -CR=CH_2) , 7.93 (singlet, 3H, -C0CH3); mass spectrum (Figure 11): M+ 310; main peaks: m/e 180, 137, 130, 96, 67. O.smylation of the olefin C22) Osmium tetroxide (48 mg, 0.25 mmole) in dry purified dioxane (3 ml) was added to the olefin (22, 50 mg, 0.16 mmole) in the same solvent (2 ml). The solution was l e f t at room temperature for 48 hours and then saturated with hydrogen sulfide gas. The black precipitate was fil t e r e d off and the dioxan solution was evaporated to dryness under reduced pressure. The residue (13 mg) was dissolved in a small amount of benzene and put on a column of alumina (1 gm, activity IV). Elution with ether-methanol (95:5) afforded a very polar compound. Spectral data indicated that this was not the desired diol (23). v r •- ' max (CKClg).: no absorption in the carbonyl region; nmr: x 1.8 (broad singlet, .IE,, indole N-E) , 2.40-3.20 (multiplet, 5E, indole protons);, 6.50 (multiplet, ,2H, -CH oQ) and 8.64 (singlet, 3H -0-C-CH_3). On.the.basis of this spectral data, the polar compound isolated above has'been tentatively assigned structure 34. Further work is necessary before a definite structure can be established. REFERENCES 1. M.F. Bartlett and W.I. Taylor, J. Am. Chem. Soc, 82_, 5941 (1960). 2. J. Mokry and I. Kompis, Lloydia, 2_7, 428 (1964). 3. W.I. Taylor in "Alkaloids" Vol. XI, p. 125, ed. R.H.F. Manske, Academic Press, New York (1969). 4. E. Wenkert, and B. Wickberg, J. Am. Chem. Soc, 87_, 1580 (1965). 5. M.E. Kuehne, J. Am. Chem. Soc, 86_, 2946 (1964); Lloydia, _2_7, 435 (1964). 6. J.E.D. Barton and J. Harley-Mason, and K.C. Yates, Tetrahedron Letters, 3669 (1965). 7. J.P. Kutney, W.J. Cretney, J.R. Hadfield, E.S. Hall, V.R. Nelson, and D.C. Wigfield, J. Am. Chem. Soc, £0, 3566 (1968). 8. J.P. Kutney, C. Ehret, V.R. Nelson and D.C. Wigfield, J. Am. \ Chem. Soc, 90, 5929 (1968). 9. E. Wenkert, R.A. Massy-Westropp and R.G. Lewis, J. Am. Chem. Soc, 84, 3732 (1962). 10. S. Sugasawa and M. Kirisawa, Pharm. Bull Japan, _3, 190 (1955). 11. D.H.R. Barton and J. Flad, J. Chem. Soc, 2085 (1956). 

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