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Studies on the biosynthesis of indole alkaloids Beck, John Frank 1971

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STUDIES ON THE BIOSYNTHESIS OF INDOLE ALKALOIDS BY JOHN F. BECK B.Sc. Honors, Loyola of Montreal, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY ' i We accept t h i s t h e s i s as conforming to the •j required standard THE UNIVERSITY OF BRITISH COLUMBIA Jul y , 1971 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ivers i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying o f th i s thes is fo r s cho la r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or pub l i ca t i on o f th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Department of STUDIES ON THc BIOSYNTHESIS OF INDOLE ALKALOIDS ABSTRACT In Section A of t h i s t hesis an i n v e s t i g a t i o n of the intermediacy of g l y c i n e i n the biosynthesis of the monoterpenoid C^^ unit of the i n d o l e a l k a l o i d s i s described. U t i l i z i n g the carbon-14 l a b e l l e d forms of g l y c i n e and plants of Vinca rosea Linn., experiments were c a r r i e d out on the Corynanthe a l k a l o i d ajmalicine [56]. Degradation of t h i s compound revealed the r e l a t i v e non-importance of g l y c i n e i n the b i o s y n t h e s i s of the u n i t . The incorporation of t h i s amino acid i n t o tryptophan [11] and hence i n t o the i n d o l e a l k a l o i d s was postulated. This p o s t u l a t e has since been confirmed by another laboratory. Sec t i o n B, provides i n i t i a l l y a resume of the synthesis of 16,17-dihydrosecodin-I7-ol [114] and of secodine [115]. The b i o l o g i c a l evaluation of these compounds as p o t e n t i a l intermediates in. the l a t e r stages of i n d o l e a l k a l o i d b i osynthesis i s described i n Sections B, C, and D o Experiments were c a r r i e d out with the various l a b e l l e d forms of these two compounds and the plant systems V. rosea, V_. minor Linn, and Aspidosperma p y r i c o l l u m Muell. Arg. Degradation of the a l k a l o i d s v i n d o l i n e [3] and apparicine [144] e s t a b l i s h e d the s p e c i f i c incorpora-t i o n of the i n d o l e p o r t i o n of secodine[115] i n t o these substances. A d d i t i o n a l information provided by double isotope studies with a i l three plant systems i n d i c a t e d support f o r the s p e c i f i c i n c o r p o r a t i o n of the e n t i r e secodine molecule [115] i n t o s e v e r a l indole a l k a l o i d f a m i l i e s . In a d d i t i o n , the evidence obtained strongly suggests the existence of a c e n t r a l common intermediate, which by appropriate enzymatic c o n t r o l at the l a t e r stages of the pathway, allows elaboration to the various f a m i l i e s of i n d o l e a l k a l o i d s . - i i i -TABLE OF CONTENTS Page TITLE PAGE 1 ABSTRACT ± i TABLE OF CONTENTS 1 1 1 LIST OF FIGURES i v LIST OF TABLES v i ACKNOWLEDGEMENT v l i i INTRODUCTION 1 DISCUSSION 30 Section A 30 Section B 50 Section C 94 Section D 103 EXPERIMENTAL 119 Section A .. 121 Section B • 129 Section C 140 Section D 144 BIBLIOGRAPHY 149 - i v -LIST OF FIGURES Figure Page 1 Examples of Indole A l k a l o i d s 2 2 The Barger-Hahn-Robinson-Woodward Hypothesis f o r Indole A l k a l o i d Biosynthesis 6 3 The Wenkert Prephenate Postulate of A l k a l o i d Biosynthesis 8 4 The Thomas^Wenkert Monoterpene Postulate of A l k a l o i d Biosynthesis ^ 5 The Acetate Postulates of A l k a l o i d Biosynthesis I 3 6 The Biosynthesis of Tryptophan v i a the Shikimate-Chorismate Pathway . 16 7 The Biosynthesis of the Cg_^Q Unit of the Indole A l k a l o i d s 2 0 8 The Postulated Origins of the Strychnos Family of A l k a l o i d s 2 3 9 The Wenkert Postulate f o r the Biogenesis of the 25 Aspidosperma and Iboga Systems 10a The Biosynthetic Pathway to the Major Indole A l k a l o i d o o Families as Confirmed by Experimentation 10b The Postulated Pathway of the Latter Stages of the 29 Indole A l k a l o i d Biosynthesis 31 11 The Acetate Biogenesis of Mevalonate J X 12a The Photosynthetic F i x a t i o n of C0 2 3 2 12b The G l y c o l y t i c Pathway to Pyruvate 3 3 13 The Shah and Rogers C h l o r o p l a s t i d i c Acetyl-CoA 35 Biogenesis . - v -Figure Page 14 The Degradation of Ajmalicine 41 15 The Postulated Incorporation of Glycine i n t o Tryptophan 47 16 Grbger, Maier, Simchen Degradation of Strychnine .... 48 17 The Synthesis of 16,17-Dihydrosecodin-17-ol 61 18 The Synthesis of 2-Carboethoxy-3-(g-chloroethyl)-i n d o l e 6 2 19 The Battersby Synthesis of 16,17-Dihydrosecodin-17-ol ^8 20 The Smith Synthesis of 16,17,15,20-Tetrahydrosecodine 7 0 21 The Proposed Synthetic Conversion of the Corynanthe Skeleton to the Strychnos Skeleton 73 22a The Acid Catalyzed Rearrangement of the Presecamine Skeleton to the Secamine Skeleton (Type A) 7 ^ 22b The Acid Catalyzed Rearrangement of the Presecamine Skeleton to the Secamine Skeleton (Type B) 7 7 23 The Degradation of Vindoline from the Incorporation of [3,14,15,21- 3H, 1 4COOCH 3] Secodine 9 0 24 The Wenkert Postulate f o r the Biogenesis of the 96 Hunteria A l k a l o i d s 25 The Wenkert Postulate f o r the Biogenesis of Uleine .. 26 The D j e r a s s i - G i l b e r t Postulate for the Biogenesis of Apparicine 106 27 The Postulated Conversion of Stemmadenine to Apparicine 108 28 The Degradation of Apparicine 114 - v i -LIST OF TABLES Preliminary Results of the Incorporation of Glycine i n t o V_. rosea and V. minor  The Results of the Incorporation of Glycine i n t o rosea  The D i s t r i b u t i o n of Label i n 1 4 C - A j m a l i c i n e from 14 Glycine-2- C Incorporation The Results of the Incorporation of DL-Tryptophan-3-14 C i n t o _V. minor at Various Time Int e r v a l s The Ratio of Monomer to Dimer Produced i n a T y p i c a l Feeding Experiment The Results of the Incorporation of 16,17-Dihydro-secodin-17-ol and Secodine i n t o V. rosea  The S p e c i f i c A c t i v i t i e s Associated with the Experiments i n Table 6 The Results of Incorporation of Various Intermediates i n t o V. minor-1 The Results of Incorporation of Various Intermediates i n t o V. minor-II The S p e c i f i c A c t i v i t i e s Associated with the E x p e r i -ments i n Table 9 3 14 The Results of Incorporating f a r - H, COOCH^]Secodine into V. minor  The Results of the Incorporation of 16,17-Dihydro-secodin-17-ol and Secodine i n t o A. pyricollum  - v i i -Table Page 13 The S p e c i f i c A c t i v i t i e s Associated with the Experiments i n Table 12 H I 14 The Incorporation of Labelled Glycine i n t o V. rosea . 124 15a,b The Feeding of Labelled Secodine to V. rosea 135 16a,b, Feeding Experiments 4, 5, 6, 7, 8, 9, 10 to V. minor 143 16c 17a,b The Feeding of Labelled Compounds to A. pyricollum .. 147 - v l i i -ACKNOWLEDGEMENT I wish to express my thanks to Professor J.P. Kutney f o r h i s e x c e l l e n t guidance throughout the course of t h i s research. The support provided by h i s unbounded optimism and u n f a i l i n g encouragement gr e a t l y f a c i l i t a t e d the succe s s f u l completion of t h i s work. I must also acknowledge the gratitude owing to my wife for her support and understanding throughout t h i s study, and for her help i n the preparation of t h i s manuscript. I am g r a t e f u l to the National Research Council of Canada f o r the f i n a n c i a l support provided. INTRODUCTION Man's search f o r knowledge has taken him to a l l corners of the world. I t has taken him out of i t , and wit h i n the next two decades i t w i l l take him to the corners of other worlds to personally supervise the continuing quest. The true measure of t h i s i n s a t i a b l e t h i r s t can be gauged more completely, however, by studying man's preoccupation with d e t a i l s , with the minute bases of h i s world, and h i s stubborn endeavours to understand and c o n t r o l these e n t i t i e s . This t h e s i s i s , i n r e a l i t y , a study of t h i s preoccupation and stubborness with, i n t h i s case, the i n t r i c a c i e s of synthesis of indole a l k a l o i d s w i t h i n the c e l l s of plant t i s s u e . I t i s estimated that 1 0 - 2 0 % of a l l plants produce alkaloids,''' and that at l e a s t one quarter of these a l k a l o i d s contain the indole 2 or reduced dihydroindole nucleus. Although the d i s t r i b u t i o n of these compounds i s f a i r l y general i n flowering plants, the family, Apocynacae, i s p a r t i c u l a r l y r i c h i n them, e s p e c i a l l y the genera Rauwolfia, Vinca (catharanthus), and Aspidosperma, which have as a 3 4 consequence, been extensively i n v e s t i g a t e d . ' Interest i n the indole a l k a l o i d s has been stimulated by the fac t that many of them are b i o l o g i c a l l y a c t i v e compounds of use i n medicine. Their number include the hypotensive and c e n t r a l nervous system - 2 -[ 7 ] Figure 1 : Examples of Indole A l k a l o i d s . - 3 -depressant reserpine [1], the cardiac p r i n c i p l e and stimulant strychnine [2], the veterinary aphrodisiac yohimbine [7] and the more recently discovered anti-cancer agents v i n b l a s t i n e - VLB [9] and v i n c r i s t i n e - VCR [10],whose complex structures can be seen to be the dimeric form of an indole and a dihydroindole a l k a l o i d . R-N As the examples i n Figure 1 i n d i c a t e , the indole compounds would seem to have l i t t l e i n common. However t h i s confusion of structure w i l l soon be c l a r i f i e d and the fac t established that most phyto-indole a l k a l o i d s have a great deal more i n common than j u s t the l-benzo(b) py r r o l e r i n g system. In addi t i o n to t h i s common denominator, the great majority of these a l k a l o i d s contain a 0-aminoethyl group. This, and the fac t that tryptophan [11] i s recognized as a main constituent of plant proteins, prompted P i c t e t i n 1906 to postulate that t h i s amino ac i d i s the progenitor of indole a l k a l o i d s . I t was not u n t i l the l a s t decade, however, that t h i s hypothesis received experimental backing. Labelled tryptophan, when administered to plants or plant [10], R = CHO - 4 -cuttings by a v a r i e t y of means, re s u l t e d i n p o s i t i v e i n c orporation. Ajmaline [4],^ v i n d o l i n e [ 3 ] , ^ ' ^ ' ^ reserpine [ 1 ] , " ^ and catharanthine [8 ] " ^ are only a few of the a l k a l o i d s which were i s o l a t e d and shown to contain r a d i o a c t i v e tracer a f t e r such experiments. To date, t h i s postulate has received only support. In f a c t , i t has never r e a l l y been 00H , _ NH H 2 [11] [12] 2 questioned; however, nothing else i s known concerning the b i o -transformation of the amino acid u n t i l i t appears i n the a l k a l o i d . Tryptamine [12], the decarboxylation product of tryptophan [11] and the exact r e p l i c a of the moiety contained i n most indole a l k a l o i d s , i s incorporated with only mixed success. 1^,12 At the present time i t i s d i f f i c u l t to make a d e f i n i t i v e i n t e r p r e t a t i o n of t h i s r e s u l t , but i t may suggest that decarboxylation of tryptophan i s delayed i n some cases u n t i l a f t e r the amino acid i s united with the remainder of the a l k a l o i d . Whatever the exact nature of the ultimate precursor, tryptophan [11] i s generally considered as a precursor of the indole portion of the a l k a l o i d s . In contrast to the agreement on the b i o - o r i g i n s of the (3-aminoethyl indole moiety, the source(s) of the remaining nine or ten carbon atoms present i n most of these a l k a l o i d s was, for a long time, a bone of - 5 -contention among nat u r a l products chemists. In a 1933 p u b l i c a t i o n on the s t r u c t u r e of yohimbine [7], Barger and Scholz i n d i c a t e very b r i e f l y that the molecule i s very probably the product of tryptophan [11] and a tyrosine [13] derived aldehyde [14], condensing i n much the 13 same manner as i n the postulated biosynthesis of berberine [15]. The following year, Hahn and coworkers used t h i s postulate f o r the f i n a l determination of the hydroxyl p o s i t i o n i n the E-ring of yohimbine 14 [7] and then expanded on the postulate by s t i p u l a t i n g that the missing unit necessary to complete the D-ring came from glycine v i a form-aldehyde."^ Robinson postulated, some time l a t e r , that the carboxyl carbon at p o s i t i o n 16 of yohimbine [7] came from an expansion of r i n g E to a tropolone system followed by extrusion of the carboxyl on 16 co l l a p s e of the r i n g . Woodward r e f i n e d the theory, making i t f l e x i b l e enough to accommodate the biosynthesis of other f a m i l i e s of a l k a l o i d s . He postulated the f i s s i o n of the aromatic E r i n g between the hydroxyl functions, to y i e l d [16] which could act i n a v a r i e t y of ways, e i t h e r to include an a d d i t i o n a l u n i t or not, to lead to such a l k a l o i d s as yohimbine [7] or cinchonamine [17]. In a d d i t i o n , he demonstrated that, with an i n i t i a l condensation at the g-position of the indole system, 17 18 19 the Barger-Hahn-Robinson theory could account f o r strychnine [2]. ' ' This combined theory i s out l i n e d i n Figure 2. In 1959, Wenkert and B r i n g i published a paper c r i t i c i s i n g t h i s 20 longstanding hypothesis. They pointed out that, i n the f i r s t place, only one yohimbine a l k a l o i d , a l s t o n i n e , was known to contain an aromatic E - r i n g . Secondly, they themselves, had shown the strong p o s s i b i l i t y that a l l indole and phytochemically r e l a t e d a l k a l o i d s - 6 -Figure 2: The Barger-Hahn-Robinson-Woodward Hypothesis f or Indole A l k a l o i d Biosynthesis. - 7 -possessed the same absolute stereochemistry at the carbon corresponding to C ^ , - i n yohimbine [ 7 ] ( i . e . C^^-H, a-configuration). The Barger-Hahn-Robinson-Woodward theory could not account f o r t h i s s i t u a t i o n . F i n a l l y , the o r i g i n of the carbomethoxy group at of yohimbine [ 7 ] was f e l t to be mechanistically u n l i k e l y and i n o r d i n a t e l y s p e c i f i c according to the older theory. Instead, Wenkert advanced the opinion 2 0 2 1 2 2 that hydrated prephenic a c i d , ' l a t e r ammended to prephenic acid [ 2 0 ] , was the progenitor of the or C ^ Q u n i t . This compound a r i s e s from the carbohydrate metabolism v i a shikimic acid [ 1 8 ] and pyruvic acid . . [ 1 9 ] . The s t e r e o s p e c i f i c rearrangement, oxidation, hydration and r e t r o a l d o l i z a t i o n of t h i s molecule lead to intermediates [ 2 3 ] and [ 2 4 ] which are r e a d i l y d i s c e r n i b l e s t r u c t u r a l units i n indole a l k a l o i d s . Mannich type condensation, with the tryptamine unit plus an a d d i t i o n a l formyl methylene to close the D-ring, completes the b i o s y n t h e t i c pathway (Figure 3 ) . This hypothesis, therefore, presents a p l a u s i b l e o r i g i n of the predominantly a l i p h a t i c u n i t . The s t e r e o s p e c i f i c rearrangement of the prephenate molecule f i x e s the absolute configuration at the carbon atom destined to become C ^ < . i n the yohimbine-type [ 7 ] skeleton, and, the carbomethoxy group, instead of being attached whenever necessary, i s an i n t e g r a l part of the hydroaromatic progenitor even i n the e a r l y stages of the pathway. I f necessary, i t can be removed by a process which i s more e a s i l y r a t i o n a l i z e d than tropolone expansion and contraction. The a d d i t i o n of the formyl residue to the u n i t [ 2 4 ] y i e l d s [ 2 6 ] , which Wenkert named the seco-prephenate-formaldehyde (SPF) u n i t . - 8 -coo" 3 - 9 -The repeated appearance of t h i s unit i n non-nitrogeneous plant glycosides such as g e n t i o p i c r i n [27] and oleuropeine [28] led him to conclude "that the SPF moiety i s an important metabolic c e l l constituent of higher plants whose i n t e r a c t i o n with a v a r i e t y of simple organic substances, e.g. amino acids and carbohydrates, i s responsible f o r the 2 formation of a wide v a r i e t y of s u p e r f i c i a l l y unrelated plant products". 22 In the same p u b l i c a t i o n , Wenkert commented on the e l u c i d a t i o n among the plant glucosides of cyclopentane-monterpenic structures. Compounds such as verbenalin [29], genipin [30], aucubin [31] and asperuloside [32] can a l l be seen to contain the carbon skeleton of the SPF unit [26]. In a d d i t i o n the absolute stereochemistry of these monoterpenes, at the s t a r r e d carbon atom i n genipin [30], i s the same as that of the C ^ p o s i t i o n of most indole a l k a l o i d s . In view of these obvious s i m i l a r i t i e s , Wenkert adopted the p o s i t i o n that e i t h e r these monoterpenes were prephenic acid derived, or the indole a l k a l o i d units were obtained from monoterpenic b u i l d i n g blocks. [31] [32] - 10 -23 Thomas, i n an independent postulate, maintained that the l a t t e r of these p o s s i b i l i t i e s was indeed the case. He suggested that the cyclopentano-monoterpenes and the non-tryptamine u n i t of the indole a l k a l o i d s shared a common synthesis which began with two units of mevalonic acid [33]. The synthesis proceeded v i a a cyclopentanoid monoterpene [34] to the a c y c l i c compound [35] and f i n a l l y the c y c l i c unit [36] (Figure 4). In contrast to the prephenic acid theory, the a c y c l i c unit [35] i s the precursor of the c y c l i c system [36], but as before the moiety can be obtained from the C^^unit v i a decarboxylation. In t h i s case, a l l the carbon atoms f o r the unit of the indole a l k a l o i d s are included and the need f o r an a d d i t i o n a l from form-a l d e h y d e ^ or glycine'*" 4 i s absent. In a d d i t i o n , the monoterpenes and the a l k a l o i d s were known to occur together i n some n a t u r a l sources thus strengthening the supposition that they were of common o r i g i n . OOC [34] OOC [35] OOC [36] Figure 4: The Thomas-Wenkert Monoterpene Postulate of A l k a l o i d Biosynthesis. - 11 -To t h i s point, the hypotheses concerning the biosynthesis of the indole a l k a l o i d s were purely speculative and unsupported by any sort of experimentation. The ready a v a i l a b i l i t y of r a d i o a c t i v e isotopes, which came about i n the 1950's, provided the r e a l opportunities f o r t h e i r evaluation. Postulated b i o l o g i c a l intermediates could now be synthesized containing a r a d i o a c t i v e isotope as a l a b e l or t r a c e r . Upon administering these l a b e l l e d intermediates to the b i o l o g i c a l system under consideration, allowing f o r a growth period, and then i s o l a t i n g the appropriately l a b e l l e d or non-labelled n a t u r a l product, experimenters could obtain evidence to confirm or r e j e c t the various postulates. During the l a s t decade, these r a d i o a c t i v e l a b e l l i n g techniques were applied to the problem of the C y _ i o u n i t of the indole a l k a l o i d s . 24 Leete, i n an attempt to evaluate the previous theories concerning the o r i g i n of t h i s C_ i n u n i t , fed a s e r i e s of l a b e l l e d compounds to y—lu Rauwolfia serpentina, a plant species of the Apocynaceae family, known 14 to contain the appropriate a l k a l o i d s . When tyrosine-2- C [13] was administered, the ajmaline [4] and reserpine [1] i s o l a t e d were completely i n a c t i v e . This cast doubt on the Earger-Hahn-Robinson-Woodward postulate (Figure 2). In order to examine the Wenkert prephenic acid 14 proposal (Figure 3), alanine-2- C was fed to the R. serpentina plants. 25 This amino a c i d had been shown to be a precursor of pyruvic a c i d [19]. The ajmaline [4J i s o l a t e d t h i s time was of very low a c t i v i t y with only 2% of i t s a c t i v i t y at the postulated p o s i t i o n . Leete f e l t that t h i s f i n d i n g was incompatible with the proposal. In order to evaluate the Thomas-Wenkert monoterpene postulate (Figure 4), mevalonic acid-2-"*"4C [33] - 12 -14 and sodium acetate-1- C were fed to the plant system. The mevalonic-acid-fed plants y i e l d e d i n a c t i v e ajmaline [4]. The acetate-fed plants y i e l d e d a c t i v e ajmaline [4], i n which one quarter of the a c t i v i t y was s i t u a t e d at and of the a l k a l o i d . (See Figure 5 f o r numbering sequence). The Thomas-Wenkert postulate required a c t i v i t y at C 1 / , C 1 Q , C 0 1 , and C , of the a l k a l o i d (starred p o s i t i o n s 14 i y / l l b i n Figure 4). These r e s u l t s were again incompatible with the experi-mental f i n d i n g s . Leete, however, f e l t that an acetate derived ^ moiety could be supported by these f i n d i n g s . He, therefore, adopted and expanded 26 the acetate theory o r i g i n a l l y put f o r t h by Schlittler and Taylor. This theory (Figure 5a) considered the biosynthesis of the u n i t i n terms of three acetyl-coenzyme A u n i t s , a malonyl-coenzyme A unit and 27 a u n i t derived from formate. Once more the skeleton of the C ^ Q unit [37] derived i n t h i s way i s nearly i d e n t i c a l to Wenkert's SPF u n i t [26] of shikimate-prephenate o r i g i n . In t h i s case, however, 14 acetate-1- C would be expected to l a b e l carbons 3, 15, 17, and 19 of ajmaline [4J. In other words, and should each contain one quarter of the t o t a l a c t i v i t y i n the a l k a l o i d . In a f u r t h e r communi-28 que, Leete reported the i s o l a t i o n of a c t i v e ajmaline [4] and reserpine [1] upon feeding l a b e l l e d malonic acid to R. serpentina plants. In a d d i t i o n , these a l k a l o i d s were i n a c t i v e at the C ^ g and C ^ g but 74% of the t o t a l a c t i v i t y resided at C ^ . 29 In 1964 Hendrickson suggested a m o d i f i c a t i o n of t h i s theory (Figure 5b) to bring i t more i n l i n e with the then popular acetogenin (polyketide) postulate. In t h i s l a t t e r sequence a ten carbon chain - 13 -Figure 5 : The Acetate Postulates of A l k a l o i d Biosynthesis. - 14 -undergoes loss of the terminal methyl group, condensation with a one carbon moiety, an a l d o l c y c l i z a t i o n and a r e t r o a l d o l i z a t i o n to y i e l d [38] which i s e s s e n t i a l l y the same as Leete's intermediate [37]. The acetate theory was not to l a s t however. Researchers, under the d i r e c t i o n of A.R. Battersby, repeated the work of Leete and h i s 30 31 co-workers and disagreed with the o r i g i n a l f i n d i n g s . ' Working not only with R. serpentina plants, but also with those of Cephaelis ipecacuanha, Battersby was able to show that the incorporation of l a b e l l e d sodium acetate l e d to s c a t t e r i n g of the l a b e l throughout the Cg u n i t of the a l k a l o i d s i n v e s t i g a t e d . On v e r i f y i n g these d i s c o v e r i e s , 32 Leete withdrew h i s precursor. In a d d i t i o n , the administration of sodium 14 formate- C l ed to i s o l a t i o n of l a b e l only at the indolic-N-methyl of ajmaline [4] and the aromatic 0-methyl of cephaeline [39]. Thus, the formate had merely l a b e l l e d the pool of the p l a n t s , rather than taking part i n the s k e l e t a l buildup of the a l k a l o i d s . This l a t t e r 33 f i n d i n g was also subsequently confirmed. The a l k a l o i d s of C_. ipecacuanha, cephaeline [39] and emetine [40], also afforded the opportunity to confirm Leete's work regarding the e a r l i e r postulates on the o r i g i n of the C^ ^ u n i t . Although these compounds belong to the i s o q u i n o l i n e family of a l k a l o i d s , they contain OCH J [39], R = H - 15 -the Cg_^Q u n i t (shown i n dark outline) i n the form characterized by 22 14 Wenkert as the SPF u n i t . Thus, when tyrosine-2- C [13] was fed to (3. ipecacuanha, the a l k a l o i d s i s o l a t e d , exhibited r a d i o a c t i v i t y i n the i s o q u i n o l i n e moieties, s p e c i f i c a l l y at p o s i t i o n C^, and no a c t i v i t y at the p o s i t i o n , the p o s i t i o n t h e o r e t i c a l l y derived from of tyrosine (see Figure 2). This experiment showed that the plant system could convert tyrosine [13] to i t s 3,4-dihydroxy d e r i v a t i v e , as t h i s i s required f o r the i s o q u i n o l i n e u n i t s , but that i t d i d not u t i l i z e t h i s m a terial i n the buildup of the Cg_^Q group. These f i n d i n g s l a i d to r e s t the t h i r t y - y e a r - o l d postulate of Barger and Hahn. Although the only evidence obtained by Battersby against the Wenkert prephenic a c i d proposal was the non-incorporation of a unit as required (see Figure 3), t h i s postulate was f i n a l l y discounted when i t was found that l a b e l l e d shikimic a c i d [18] r e s u l t e d i n the tagging of only the indole p o r t i o n and not the ubiquitous a l i p h a t i c unit of 34 the a l k a l o i d s i n v e s t i g a t e d . That the indole moiety should become l a b e l l e d was expected, as the progenitor of t h i s group, the aromatic amino acid tryptophan [11], i s i t s e l f a product of the shikimate [18]-chorismate [41] pathway. Although the complex biosynthesis of t h i s compound, as o u t l i n e d i n Figure 6, was painstakingly elaborated using 35 36 b a c t e r i a l mutants, ' work i n the l a t e I960's showed that t h i s route 37 was also a p p l i c a b l e to the higher plant systems. A l l of the above r e s u l t s now l e f t only the monoterpene postulate f o r serious consideration. I n i t i a l t r i a l s by Battersby u t i l i z i n g 14 mevalonolactone-2- C on R. serpentina and C^. ipecacuanha led r e s p e c t i v e l y to r a d i o - i n a c t i v e ajmaline [4] and cephaeline [39]. However, furth e r - 16 -COO COO Carbohydrate metabolism COO PO' OH -> > HO' COO OH [18] COO O H P O " O H O H coo" COO" COO OH [41] „ C H 9 0 P 0. / 2 > [42] OH OH C 0 ° " J H - C H - C H 2 O P OH OH C O O " O H fl ?H ^ I ^ C H - C H - C H 2 O P COOH = - t o 0. [11] Figure 6: The Biosynthesis of Tryptophan v i a the Shikimate-Chorismate Pathway. - 17 -14 experimentation y i e l d e d low i n c o r p o r a t i o n of sodium mevalonate-2- C [33] i n t o these a l k a l o i d s . These r e s u l t s were q u i c k l y confirmed by two other research groups working w i t h the p l a n t species V i n c a rosea 38 39 39 L i n n . ' and V i n c a major L i n n . W i t h i n a year the Thomas-Wenkert monterpene p o s t u l a t e was f u l l y s u b s t a n t i a t e d . From separate experiments u t i l i z i n g sodium mevalonate [33] l a b e l l e d w i t h carbon-14 at d i f f e r e n t s i t e s and s e v e r a l p l a n t systems, i t was demonstrated by means of a r i g o r o u s degradation of the a l k a l o i d s i s o l a t e d , that t h i s p r e c u r s o r was s p e c i f i c a l l y i n c o r p o r a t e d i n t o these a l k a l o i d s , w i t h no p r i o r c a t a b o l i s i s . ^ In a d d i t i o n , deuterium l a b e l l e d mevalonolactone fed to V. rosea y i e l d e d a l k a l o i d s whose mass s p e c t r a l fragmentation p a t t e r n s s u b s t a n t i a t e d the r a d i o a c t i v e t r a c e r f i n d i n g s . With the search f o r the p r o g e n i t o r s of the c y _^o n o w headed i n the c o r r e c t d i r e c t i o n , e f f o r t s to uncover f u r t h e r p r e c u r s o r s provided f r u i t f u l r e s u l t s . The intermediacy of the monoterpene, 41 g e r a n i o l , as i t s pyrophosphate d e r i v a t i v e [45a] was demonstrated and 42-46 s u b s t a n t i a t e d . L a t e r , B a t t e r s b y showed that n e r o l [45b], the c i s 43 44 isomer of g e r a n i o l , could act as an i n t e r m e d i a t e w i t h equal e f f i c i e n c y . ' Subsequent experiments revealed the p a r t played by the monoterpene, l o g a n i n [48]. This compound, a p p r o p r i a t e l y l a b e l l e d , was s p e c i f i c a l l y 47-49 i n c o r p o r a t e d as an i n t a c t u n i t i n t o the a l k a l o i d s of y_. rosea, 49 50 R. s e r p e n t i n a , and C. epicauanha. In a d d i t i o n , the b i o s y n t h e s i s of t h i s compound was demonstrated to be v i a mevalonate [33] and g e r a n i o l and/or n e r o l , again presumably as t h e i r pyrophosphates [45a], [45b]^7,49,51 58 that i t co-occurred w i t h the i n d o l e a l k a l o i d s i n V_. r o s e a 4 ^ ' " ^ and Strychnos nux v o m i c a , a n o t h e r p l a n t species r i c h - 18 -i n indole a l k a l o i d s . The stereochemistry of the molecule (as shown i n [48]) and i t s mode of formation i n d i c a t e that: (a) i t i s i d e n t i c a l i n stereochemistry to the Corynanthe and Strychnos a l k a l o i d s (e.g. yohimbine [7] and strychnine [2]) at the center corresponding to C^ ,. of yohimbine, and (b) that i t s biosynthesis i s highly s t e r e o s p e c i f i c , reminiscent P ^ - K- u • , * +u + 51-53,55-57 of the terpene bxochemistry of other systems. Thus, the p r e r e q u i s i t e of stereochemistry f o r the progenitor of the 20 Cg u n i t , as f i r s t pointed out by Wenkert, i s completely f u l f i l l e d by t h i s monoterpene. Deoxyloganin [47] has also been established to be s p e c i f i c a l l y incorporated i n t o loganin [48] and the a l k a l o i d s of V_. rosea, as w e l l as being a constituent of V_. rosea and s . nux vomica. ' ^ The cleaved monoterpene d e r i v a t i v e of loganin [48], secologanin [49], has also been i s o l a t e d from V . rosea p l a n t s . ^ I t s intermediacy i n the sequence from loganin [48] to the f u n c t i o n a l i z e d form [50] of the Thomas-Wenkert c e n t r a l unit [35] has been con c l u s i v e l y p r o v e n . ^ The most recent a d d i t i o n to the b i o s y n t h e t i c sequence has been 62 63 the u t i l i z a t i o n of the hydroxy g e r a n i o l d e r i v a t i v e [46]. ' This compound i s incorporated i n t o the a l k a l o i d s of V . rosea with randomization of the l a b e l from the p o s i t i o n s i n d i c a t e d as 2,6 i n [46]. This would i n d i c a t e that oxidation at both of these carbon atoms i s a necessary part of the sequence to deoxyloganin [47], as w e l l as oxidative removal of the pyrophosphate group. A resume of a l l t h i s work i s o u t l i n e d i n Figure 7. The numbering system maintained throughout i s that of the o r i g i n a l mevalonate groups - 19 -i n order to i l l u s t r a t e the exact l o c a t i o n of the mevalonic carbon atoms i n the Corynanthe or unrearranged a l k a l o i d system, t y p i f i e d by corynantheine [25], and i n the rearranged units of the Aspidosperma and Iboga systems, exemplified by vi n d o l i n e [3] and catharanthine [8] r e s p e c t i v e l y . The arrows, designated as A and B schematically demonstrate the routes of these rearrangements as determined by degradative studies of the representative r a d i o a c t i v e a l k a l o i d s , a f t e r 44 the incorporation of ra d i o a c t i v e mevalonate and g e r a n i o l . I t i s of i n t e r e s t to note that although Wenkert's prephenic acid 22 2A 31 33 3^ postulate (Figure 3) was unsupported by experimental evidence, ' ' ' the nature of h i s c e n t r a l group, the SPF unit [26] was, i n f a c t , almost i d e n t i c a l i n structure and oxidation l e v e l to that of what has been shown to be the actu a l c r i t i c a l intermediate [50]. There i s no attempt made i n Figure 7, to i n d i c a t e the point at which the un i t i s joined to the aminoethyl indole moiety. In f a c t , the i s o l a t i o n of s t r i c t o s i d i n e 153] i n 1968 from Rhazya s t r i c t a 65 and vincoside £54] from V_. rosea i n d i c a t e s that t h i s union takes place at the secologanin [49] stage. No stereochemistry has been put 64 OGluc. CH300C CH 300C OGluc. [53] [54] - 2 0 -Figure 7 : The Biosynthesis of the C Q i n Unit of the Indole A l k a l o i d s . - 21 -66 f o r t h f o r the R. s t r i c t a metabolite*, however, f u r t h e r work showed that i t occurred i n V_. rosea and was tryptophan [11] and l o g a n i n [48] d e r i v e d . B a t t e r s b y , i n h i s examination of v i n c o s i d e [54], determined i t s s tereochemistry as shown, by s y n t h e s i s and by comparison of the molecular r o t a t i o n d i f f e r e n c e s r e s u l t i n g from N - a c e t y l a t i o n of v i n c o s i d e [54] and i s o v i n c o s i d e [54] ( i s o m e r i c at the s t a r r e d p o s i t i o n ) against standards of known stereochemistry. In a d d i t i o n , v i n c o s i d e [54] was shown to be s p e c i f i c a l l y i n c o r p o r a t e d i n t o the three main f a m i l i e s of a l k a l o i d s i n V.rosea and that i t i s , i t s e l f , tryptophan {11] and l o g a n i n 65 148] d e r i v e d . The a c t u a l comparison of the v i n c o s i d e s and s t r i c t o -s i d i n e [53] has not been reported i n the l i t e r a t u r e and so i t i s d i f f i c u l t to determine which p a r t i c u l a r stereochemistry i s to be a p p l i e d to the l a t t e r . I t appears l i k e l y t hat v i n c o s i d e occupies a c e n t r a l r o l e i n the b i o s y n t h e t i c pathway of the v a r i o u s a l k a l o i d f a m i l i e s . For example, enzymatic cleavage of the glucose r e s i d u e of v i n c o s i d e 154], r o t a t i o n about the a p p r o p r i a t e C-C bond, S c h i f f base formation and r e d u c t i o n l e a d to the Corynanthe f a m i l y of a l k a l o i d s which i n c l u d e s corynantheine [25], g e i s s o s c h i z i n e [55], a j m a l i c i n e [56] and s e r p e n t i n e [57]. [55] OH [56] [57] r i n g C aromatized - 22 -That the more f u n c t i o n a l i z e d Corynanthe a l k a l o i d s can be derived from the l e s s f u n c t i o n a l i z e d members ( i . e . geissoschizine [55]) was shown by the incorporation of the l a t t e r i n t o ajmalicine [56] and serpentine [ 5 7 ] . ^ In a d d i t i o n , g e i s s o s c h i z i n e [55] was shown to be s p e c i f i c a l l y incorporated i n t o members of the Strychnos, Aspidosperma, and Iboga f a m i l i e s . Subsequent large scale e x t r a c t i o n of V_. rosea plants revealed i t s occurrence along with the members of the above f a m i l i e s . ^ ' ^ Thus, the genesis of these higher order a l k a l o i d s would seem to be v i a the s t r u c t u r a l l y simpler types, rather than through any d i r e c t transformation of the tryptamine-[50] e n t i t y . That t h i s might be the case was recognized by Wenkert at an e a r l y stage. In a 1965 p u b l i c a t i o n . the p o s s i b l e route from the Corynanthe to the Strychnos family was put 69 f o r t h . (Figure 8 - Pathway A). The transformation involved a one e l e c t r o n oxidation of the aldehyde form of geissoschizine [55a], attack by the r a d i c a l [58] at the g-position of the indole nucleus, and subsequent rearrangement u t i l i z i n g the free p a i r of electrons associated with the second nitrogen atom, to y i e l d the Strychnos skeleton [61] as t y p i f i e d by the a l k a l o i d , akuammicine 165]. A second pathway has since been p r o p o s e d ^ i n v o l v i n g oxidation of the aldehyde [55a] and n u c l e o p h i l i c attack at the a - p o s i t i o n of the i n d o l i n e 162] to form the spiro-imino compound J63]. The l a t t e r , upon subsequent i n t r a -molecular n u c l e o p h i l i c displacement by the r e a d i l y formed anion at the oxygenated side chain generates the Strychnos skeleton [61], and the a l k a l o i d , preakuammicine [64], which has been reported to form akuammicine [65] spontaneously i n v i t r o ^ (Figure 8 - Pathway B) . To a r r i v e at the more complex a l k a l o i d systems, rearrangements of - 23 -Figure 8: The Postulated Origins of the Strychnos Family of A l k a l o i d s . - 24 -the C n i n unit [50] are required. The f i r s t proposal to account f o r y—IU 22 these transformations was put f o r t h by Wenkert i n 1962. As o u t l i n e d i n Figure 9, the proposal begins with a charged precursor [66], which i s e s s e n t i a l l y equivalent to imine [60], and of analogous o r i g i n . A retro-Michael r e a c t i o n , reduction, and dehydration of t h i s intermediate lead to the intermediate piperidienes [68] and [69]. These intermediates v i a intramolecular Michael and Mannich condensations lead to the nine-membered r i n g systems [70] and [71] which on transannular c y c l i z a t i o n lead to Aspidosperma [72] and Iboga [73]-type skeletons. P e r i p h e r a l oxidations, reductions, and a l k y l a t i o n s could then account fo r a l l the a l k a l o i d s of these various f a m i l i e s . Since i t s p u b l i c a t i o n , t h i s proposal has received consideration i n s everal l a b o r a t o r i e s . The f i r s t reported work along these l i n e s was c a r r i e d out by Scott and co-workers and involved the i n c o r p o r a t i o n of stemmadenine 174] and tabersonine 175] into the a l k a l o i d s of the CH300C CH20H [74] [75] germinating seeds of r o s e a . ^ Concurrent work by the Kutney research group involved examination of the transannular c y c l i z a t i o n required by the Wenkert postulate both i n v i t r o ^ ^ and i n v i v o . " ^ ' ^ The i j i vivo experiments included the feeding of l a b e l l e d t e t r a c y c l i c indole compounds such as the 6,7-dehydrovincadine d e r i v a t i v e [76], - 25 -[ 7 2 ] Figure 9 : The Wenkert Postulate f o r the Biogenesis of the Aspidosperma and Iboga Systems. - 2 6 -carbomethoxycleavamine [ 7 7 ] , quebrachamine [ 7 8 ] and vincaminoreine [ 7 9 ] , as w e l l as the p e n t a c y c l i c tabersonine [ 7 5 ] to two plant systems, V. rosea and V. minor Linn. [ 7 8 ] [ 7 9 ] Since these r e s u l t s w i l l be dealt with more f u l l y i n Section B of the Discussion, i t i s s u f f i c i e n t to state here that the work of Scott and Kutney substantiated some of Wenkert's hypotheses but also suggested that some mo d i f i c a t i o n was required to accommodate t h e i r experimental f i n d i n g s . In conclusion, the biosynthesis of the major classes of indole a l k a l o i d s may be summed up as i n Figures 1 0 a and 1 0 b . Figure 1 0 a represents the pathway based on experimental f i n d i n g s . With the exception of the stemmadenine 1 7 5 ] preakuammicine 1 6 4 ] -> akuammicine [ 6 5 ] sequence, the pathway has been v e r i f i e d through the s p e c i f i c - 27 -incorporation of a l k a l o i d s i n t o succeeding classes, i . e . Corynanthe Strychnos Aspidosperma Iboga. The stemmadenine [75] <t pre-akuanmiicine [64] ->• akuamraicine [65] sequence has been demonstrated to 12 be a f a c i l e i n v i t r o process. Figure 10b r e l a t e s to the l a t t e r stages of the biosynthesis and i s postulated i n order to accomodate the experimental findings of Scott and Kutney. The intermediacy of the a c y l i c ester [80] i s as yet s t r i c t l y t h e o r e t i c a l . I t s relevance remains to be proven. Pertinent experiments concerning i t s r o l e are described i n Sections B, C and D of the Discussion. - 28 -COOH CH3OOC Catharanthine [8] (Iboga) Figure 10a: The Biosynthetic Pathway to the Major Indole A l k a l o i d Families, as Confirmed by Experimentation. - 29 -Vindoline [3] (Aspidosperma) Figure 1 0 b : The Postulated Pathway of the L a t t e r Stages of the Indole A l k a l o i d Biosynthesis. - 3 0 -DISCUSSION The biosynthesis of indole and dihydroindole a l k a l o i d s , as portrayed i n the Introduction, has received considerable c l a r i f i c a t i o n i n recent years. To a large extent t h i s meant the determination of the biogenesis of the u n i t . The e l u c i d a t i o n of the pathway a f t e r the union of a tryptophan residue and th i s C ^ Q unit has also received considerable a t t e n t i o n , but i s , as yet, no more than a broad o u t l i n e . Our work, as presented below, deals with various aspects of the biosynthesis of these a l k a l o i d s . For purposes of c l a r i t y and ease of presentation, the work discussed here w i l l be divided i n t o four sections. The f i r s t of these w i l l deal with the p o s s i b l e intermediacy of g l y c i n e as a precursor of the C ^ Q u n i t . The remaining sections w i l l deal b r i e f l y with the synthesis of compounds resembling the a c r y l i c ester [ 8 0 ] , and the subsequent b i o s y n t h e t i c studies i n v o l v i n g these compounds, i n various plant systems. Section A As documented i n the Introduction,there are numerous elegant experiments i n hand to prove that the o r i g i n of the C ^ Q unit i s terpenoid i n nature. These terpenoids are, i n turn, derived from - 31 -mevalonate [33], as ou t l i n e d i n Figure 7. Mevalonic acid i s i t s e l f the product r e s u l t i n g from the combination of three units of a c e t y l 78 CoA, and as shown i n Figure 11, i t s biosynthesis i s accomplished v i a two C l a i s e n - l i k e condensations and reduction of the resultant g-hydroxy, 3-methyl g l u t a r y l CoA [82]. 0 II 3CH3C0S-CoA • CH3-C-CH2-COS-CoA CH3-COS-CoA OH OH I CH -C-CH -COS-CoA \ c COO CH2COO ^ OH [82] [33] Figure 11. The Acetate Biogenesis of Mevalonate. The p r i n c i p a l c e l l u l a r source of acetyl-CoA i s the intramito-79 chondrial oxidation of pyruvate [83], which i s i t s e l f the major 79 product of glucose c a t a b o l i s i s i n most c e l l s (Figure 12b). Thus the o r i g i n of the C ^ Q unit can be d i r e c t l y retraced to the carbohydrate products of photosynthesis. This process of carbon dioxide f i x a t i o n i s o u t l i n e d i n Figure 12a. The starred atom i l l u s t r a t e s the path of the carbon dioxide carbon atom from i t s i n i t i a l f i x a t i o n by 80 ribulose-l,5-diphosphate [84] through to glucose-6-phosphate [93]. In Figure 12b, the g l y c o l y t i c pathway from glucose-6-phosphate [93] to pyruvate [83] i s presented. I f the phospho-3-glycerate [87] i s d i r e c t l y derived from the carbon dioxide f i x a t i o n process, then the - 32 -CH.O-P I 2 c=o I H-C-OH I H-C-OH I CH2-0-P [84] CH2-0-P C-OH II C-OH I HC-OH CH20-P [85] CO, CH.-O-P * I c-c-c OOC-C-OH I C=0 HC-OH CH2-0-I [86] H 20 CHn-0-P *CO-OP *CHO CH -OH COO I HC-OH [87] ' 2 1 J. HO-C-H H-C-OH H-C-OH C=0 *C00~ ' CH -O-P m„„„ CH^O-P CH„-0-I ATP ~* 2 TPNH 2 2 ->- >• ADP [88] T p N + [89] [90] CH20-P \ / It ? P=-P=0 I 0 CHO I H-C-OH * l HO-C-H * l H-C-OH H-C-OH CH2-0-I [93] CHo0H I 2 C=0 *l HO-C-H *! H-C-OH I H-C-OH I CH20-P [92] CH„-0-P I 2 C=0 * l HO-C-H * l H-C-OH I H-C-OH CH20-P [91] Figure 12a: The Photosynthetic F i x a t i o n of CO - 33 -CHO H-j>OH HO-C-H I H-C-OH I H-C-OH t C [93] CH2-0-P CH2OH [92] CH.-O-P I 2 r HO-C-H H-C-H H-C-I OH H-C-OH I CH20-P [91] CO-OP H-C-C -OH CH20-P [88] ADP ATP TPN TPNH r CHO I H-C-OH I CH20-P [89] \ CH.OH I 2 C=0 I CH20-P [90] COO I H-C-OH I CH2OP [87] C0 2 + CH3COS-CoA COO I H-C-O-P I CH2OH [99] CoA-SH COO I C-O-P II CH 2 [100] ADP COO~ I c=o I CH 3 [83] Figure 12b: The G l y c o l y t i c Pathway to Pyruvate - 34 -shortest route to pyruvate [83], and thereby to a c e t y l CoA, i s : carbon dioxide ->- 2-carboxy-3-ketopentitol [86] -»• phospho-3-glycerate [87] phospho-2-glycerate [99] phosphoenolpyruvate [100] pyruvate [83] ->• a c e t y l CoA. The purpose of t h i s precursor d e l i n e a t i o n i s to show that a u n i t becomes involved with the biosynthesis of the C ^ Q unit only at the acetate stage of the mevalonate synthesis. I t was, therefore, of some i n t e r e s t when Shah and Rogers postulated the intermediacy of g l y c o l l a t e [94] v i a g l y c i n e [96] and serine [44a] i n the biosynthesis of a c e t y l -81 CoA. This proposal was based on work with greening e t i o l a t e d maize seedlings, and involved i n t r a - and e x t r a c h l o r o p l a s t i d i c terpenoid synthesis. In the maize seedlings, the tetraterpenoid, 3-carotene,is produced w i t h i n the c h l o r o p l a s t , while several s t e r o l s are e x t r a c h l o r o p l a s t i d i c i n o r i g i n . By means of a wide range of r a d i o i s o t o p i c - i n c o r p o r a t i o n studies, r a d i o i s o t o p i c - d i l u t i o n studies, and experiments with i n h i b i t o r s of the proposed pathway, Shah and Rogers were able to demonstrate the existence of the pathway, carbon dioxide -»- g l y c o l l a t e [94] -*• glyoxylate [95] -*• g l y c i n e [96] -»- serine [44a] -*• pyruvate [83] -*• acetyl-CoA, w i t h i n the c h l o r o p l a s t s (Figure 13). E x t r a c h l o r o p l a s t i d i c s t e r o l s displayed i n c o r p o r a t i o n patterns consistent with the established sequence carbon dioxide ->• glucose [93] -> pyruvate [83] ->- acetate -* mevalonate [33]. The authors, however, terminated t h e i r work with a note of caution: "The g l y c o l l a t e pathway for acetyl-CoA formation may well be only s i g n i f i c a n t i n young r a p i d l y developing seedlings. As the c h l o r o p l a s t matures to a chromoplast i t s metabolism may be correspondingly a l t e r e d . This i s i n d i c a t e d by an increased - 35 -CO, - 2CH 2OH °coo [ 9 4 ] CHO 'coo [ 9 5 ] - ° C O , * + CH 2NH 3 °COO _ [ 9 6 ] Figure 13: The Shah and Rogers C h l o r o p l a s t i d i c Acetyl-CoA Biogenesis. incorporation of mevalonic acid i n t o 0-carotene i n tomato chromoplasts; i n contrast the incorporation of intermediates of the g l y c o l l a t e pathway i n t o c h l o r o p l a s t terpenoids becomes prog r e s s i v e l y l e s s e f f i c i e n t as the c h l o r o p l a s t ..81 senesces. Nevertheless, the authors f e l t that t h e i r work offe r e d strong evidence f o r a number of proposals, s p e c i f i c a l l y the r e l a t i v e l y d i r e c t synthesis of amino acids from carbon dioxide by-passing the carbohydrate intermediates, and the involvement of these amino acids i n terpenoid, i . e . acetate, biosynthesis. Both of these proposals are supoorted i n the i .. „ 82,83 l i t e r a t u r e . In an attempt to determine a s p e c i f i c a l l y incorporated ( i . e . non-randomized) precursor of mevalonate and the terpenoid C ^ Q u n i t , Gear 84 and Garg administered l a b e l l e d g l y c o l l i c acid and glycine to three-14 year-old C^. ipecacuanha plants. The 1- C - l a b e l l e d forms of both these compounds gave e i t h e r i n a c t i v e or only s l i g h t l y a c t i v e cephaeline 81 [39j. This was not unexpected as Shah and Rogers had likewise 14 reported negative incorporation f o r g l y o x y l i c acid-1- C [95] i n t o 3-carotene. The r a t i o n a l e f o r t h i s f i n d i n g can be seen i n the biogenesis of acetyl-CoA as postulated by these authors (Figure 13). 14 14 When, however, glycine-2- C [96] and g l y c o l l i c acid-2- C [94] were fed to the aforementioned plant system, a c t i v e cephaeline [39] was i s o l a t e d . In the case of g l y c o l l i c a c i d , the degradation of the a l k a l o i d molecule by the Kuhn-Roth oxidation revealed only 4.8% of the t o t a l a c t i v i t y d i v i d e d between C ^ and C^ ,.. This was i n t e r p r e t e d by - 37 -Gear and Garg to represent randomization of the l a b e l l e d compound. The 14 Kuhn-Roth oxidation of the glycine-2- C derived a l k a l o i d revealed 15.1% of the t o t a l a c t i v i t y of the compound s p e c i f i c a l l y at C^ ,.. T h i s , the authors f e l t , was i n d i c a t i v e of "glycine acting as a s p e c i f i c two 84 carbon unit precursor of mevalonic a c i d and the ^^_^Q u n i t . " 14 14 In subsequent p u b l i c a t i o n s , glycine-2- C and acetate-2- C were 85 86 fed to JC. acumentata and R. serpentina plants. In the former, cephaeline and the p h y t o s t e r o l , g - s i t o s t e r o l [101], were i s o l a t e d and examined. I t was found that, whereas acetate was s p e c i f i c a l l y incorporated i n t o the s t e r o l , i t s a c t i v i t y was randomized and the unit was incorporated to a l e s s e r extent i n t o the C y u n i t of the a l k a l o i d . This l a t t e r f i n d i n g i n i t s e l f was not new. Three other research groups 9 31 87 had already reported s i m i l a r paradoxical r e s u l t s . ' ' However, 14 glycine-2- C was again reported to be s p e c i f i c a l l y incorporated i n t o the C y u n i t of cephaeline [39] but not at a l l into the phytosterol. This r e s u l t was taken to i n d i c a t e that g l y c i n e and acetate "act as s p e c i f i c and exclusive precursors of d i f f e r e n t monoterpene moieties, i n OCH, [39] [101] - 38 -85 d i f f e r e n t compounds, i n the same plant". In t h e i r l a s t p u b l i c a t i o n , Gear and Garg turned to the un i t i n indole a l k a l o i d s . Ajmaline [4] and reserpine [1] were i s o l a t e d from 14 14 glyci n e - 2 - C and acetate-2- C fed R.. serpentina plants. The acetate r e s u l t s were i n t e r p r e t e d as i n d i c a t i n g randomization of the l a b e l , 14 as had been previously reported. On the other hand, the glycine-2- C was found to be s p e c i f i c a l l y incorporated into ajmaline [4]. In the case of reserpine [1], 15% of the a c t i v i t y of the molecule was reported to reside i n the 3,4,5-trimethoxybenzoate portion while 83% was found i n the r e s e r p i c a c i d moiety. This was taken to mean that some of the 14 glycine-2- C l a b e l was entering the pool of the plant and hence the O-methyl groups of the benzoate moiety. This process had been previously reported i n plant systems, and found to proceed with great 88 f a c i l i t y . To account f o r the remaining a c t i v i t y , Gear and Garg concluded, "Since the indole p o r t i o n of reserpine (less the O-methyl group) a r i s e s from tryptophan, most of the a c t i v i t y found i n the 86 r e s e r p i c a c i d derived from g l y c i n e must reside i n the Cg_^Q u n i t . " In order to evaluate these f i n d i n g s , a study of the question of glycine involvement i n the biosynthesis of indole a l k a l o i d s was under-taken. For the study, y_. rosea and V. minor were chosen as s u i t a b l e plant substrates. Not only do these plants biosynthesize an abundant v a r i e t y of indole a l k a l o i d s , but both species are also hardy and su s t a i n the various feeding methods w e l l . In ad d i t i o n , a large body of knowledge based on previous experiences had accummulated within our research group concerning the administration of precursor to, and the e x t r a c t i o n of - 39 -a l k a l o i d s from these plants. With V. rosea plants, the most convenient procedure of incorporating a compound has been found to be by the cotton wick method u t i l i z i n g i n t a c t potted plants; with V_. minor, a 89 hydroponic feeding to cut stems has proven e f f i c i e n t . 14 14 Commercially a v a i l a b l e glycine-2- C and glycine-1- C were administered to the plants and these preliminary feedings y i e l d e d the 90 r e s u l t s l i s t e d i n Table 1. Experiments 2 and 3 c l e a r l y i n d i c a t e the TABLE 1. Preliminary Results of the Incorporation of Glycine i n t o V. rosea and V. minor. Expt. No. Compound fed Feeding Method Feeding Period % Incorporation Ajmalicine Catharanthine Vindoline [56] [8] [3] V. rosea 1 14 glycine-2- C wick 9 days 0.26% 0.011% 0.0032% V. minor Vincamine [102] 2 14 glycine-2- C hydroponic to stems 24 hrs. 0.02% 3 14 glycine-2- C hydroponic to stems 3 days 0.06% 4 14 g l y c i n e - 1 - C hydroponic to stems 3 days 0.0004% incor p o r a t i o n of the a-carbon of g l y c i n e i n t o the Hunterea a l k a l o i d , vincamine [102], whereas the carboxyl carbon of g l y c i n e , as demonstrated by experiment 4, was not incorporated. Experiment 1 shows p o s i t i v e - 4 0 -[ 1 0 2 ] incorporation of the a-carbon of g l y c i n e i n t o the three main indole a l k a l o i d f a m i l i e s , with a p a r t i c u l a r l y e f f i c i e n t i n c o r p o r a t i o n i n t o the Corynanthe group as evidenced by ajmalicine [ 5 6 ] . In view of t h i s 1 4 rather favorable incorporation of g l y c i n e - 2 - C, ajmalicine [ 5 6 ] was chosen for further study. Further feedings of l a b e l l e d glycine to V_. rosea were c a r r i e d out, not only to v e r i f y the i n i t i a l r e s u l t s , but also to accummulate ajmalicine for degradation purposes. These a d d i t i o n a l precursor administrations are l i s t e d i n Table 2 . TABLE 2 . The Results of the Incorporation of Glycine i n t o V. rosea. Expt. No. Compound Fed A c t i v i t y Fed Feeding Period % Incorporation ajmalicine [ 5 6 ] 5 9 1 14 g l y c i n e - 2 - C 5 . 5 5 x 1 0 7 dpm 9 days 0 . 1 7 % 6 1 4 g l y c i n e - 2 - C 4 . 9 9 x 1 0 8 dpm 9 days 0 . 4 8 % 7 glycine -2-"'' 4C 5 . 5 4 x 1 0 8 dpm 9 days 0 . 3 1 % 8 g l y c i n e - 1 - C Q 5 . 0 0 x 1 0 dpm 9 days 0 . 0 0 0 8 % 8 1 8 4 — 8 6 As i n the work of Shah and Rogers, and Gear and Garg, we found that i n both plant substrates, the carboxylic carbon of glycine i s - 41 -not s i g n i f i c a n t l y incorporated into any part of the compounds i s o l a t e d , whereas, the a-carbon i s incorporated r e l a t i v e l y w e l l (compare experiments 4 and 8 vs. experiments 1-3 and 5-7. However, i n order to 14 determine whether glycine-2- C had been u t i l i z e d as a s p e c i f i c precursor f o r the terpenoid u n i t of these a l k a l o i d s , the exact l o c a t i o n of the l a b e l was required. To t h i s end, a s e r i e s of reactions was c a r r i e d out on the various samples of ajmalicine [56] obtained as a r e s u l t of experiments 1, 5, 6 and 7. This degradation scheme i s i l l u s t r a t e d i n Figure 14. The d i s t r i b u t i o n of l a b e l determined as a [104] Figure 14: The Degradation of Ajmalicine. - 42 -r e s u l t of t h i s scheme i s l i s t e d i n Table 3. TABLE 14 3. The D i s t r i b u t i o n of Label i n C-Ajmalicine from Glycine-2-14 C Incorporation. Expt. No. A j m a l i c i n e 3 [56] CH 3C00Na b Harman [104] CH 3C00Na C A j m a l i c i n o l [103] I 9 0 100% 0.41% - - -5 9 1 100% 0.86% 69% - -6 100% 0.58% 59% - 63% 7 100% - 78% 0.78% 79% The t o t a l a c t i v i t y i n the a l k a l o i d i s a r b i t r a r i l y set at 100%. These values r e f e r to the sodium acetate derived from the Kuhn-Roth oxidation of ajmalicine [56]. This value r e f e r s to the sodium acetate derived from the Kuhn-Roth oxidation of harman [104]. The experiment numbers therein correspond to those found above for the 14 glycine-2- C feedings. The r a d i o a c t i v e ajmalicine [56] was i n i t i a l l y subjected to a Kuhn-Roth oxidation by r e f l u x i n g the sample i n an aqueous chromic a c i d -32 s u l f u r i c a c i d s o l u t i o n . The a c e t i c a c i d thus formed and representing the C 1 0 - C i n atoms of the a l k a l o i d was then i s o l a t e d as the sodium s a l t i o i y and c r y s t a l l i z e d to constant a c t i v i t y . I f g l y c i n e functioned as a s p e c i f i c two carbon precursor of the C ^ Q u n i t , the l a b e l l i n g pattern i n d i c a t e d by the marked carbons of the heavy l i n e d terpenoid unit of ajmalicine [56] i n Figure 14 would be expected. This i s i d e n t i c a l to the l a b e l d i s t r i b u t i o n speculated by Gear and Garg for cephaeline [39] - 43 -(p. 37 ) i n t h e i r studies with g l y c i n e ^ - ^ C . It i s to be noted that the C ^ Q unit i n both of these a l k a l o i d s i s e s s e n t i a l l y i d e n t i c a l . On the basis of t h e i r r e s u l t s the acetate derived from C, 0-C,„ of ajmalicine 18 19 J [56] should have accounted for one-sixth the a c t i v i t y of the a l k a l o i d . Instead, l e s s than 1% of t h i s a c t i v i t y was found to be present i n t h i s two carbon fragment as shown i n each of the three oxidations c a r r i e d out on samples of the a l k a l o i d (Table 3). During the search for precursors of the C ^ Q u n i t , Battersby had 44 employed a base cleavage of serpentine [57] to a f f o r d harman [104]. The s i m i l a r i t y of serpentine [57] and ajamlicine [56] prompted us to attempt t h i s cleavage on the l a t t e r compound and our e f f o r t s were rewarded with a small but adequate y i e l d of t h i s g-carboline. In order to v e r i f y t h i s synthesis of harman [104], a supply of authentic m a t e r i a l was produced according to the procedure of Hernack, Perkin and 91 92 Robinson. ' Both samples proved to be i d e n t i c a l with respect to mixed melting point, u l t r a v i o l e t , and i n f r a r e d spectra. When the ajmalicine £56] derived sample was p u r i f i e d and c r y s t a l l i z e d to constant a c t i v i t y , i t was found to contain from 59% to 78% of the o r i g i n a l a l k a l o i d r a d i o a c t i v i t y . In order to d i f f e r e n t i a t e the carbon atoms of the C ^ Q unit from that of the methyl ester, the l a t t e r was removed u t i l i z i n g a l i t h i u m aluminum hydride reduction of the carbomethoxy 93 ester [56] to y i e l d the hydroxymethylene, a j m a l i c i n o l [103]. This primary a l c o h o l when p u r i f i e d accounted f o r 63% and 79% of the a c t i v i t y of the parent, ajmalicine [56]. When viewed i n conjunction with the corresponding a c t i v i t y figures f o r the harman [104] derived from the same samples of ajmalicine [56] , the above percentages reveal that the - 44 -a c t i v i t i e s l o s t i n degrading the parent a l k a l o i d to harman [104] were located completely (i.e., w i t h i n experimental error) i n the methyl group of the carbomethoxy ester (Table 3). These r e s u l t s completely eliminated the p o s s i b i l i t y that g l y c i n e was entering, to any s i g n i f i c a n t degree, into the biosynthesis of mevalonate [33], and hence the terpenoid u n i t . For the sake of completeness, the accumulated samples of a c t i v e ajmalicine [56] were combined and the l e v e l of r a d i o a c t i v i t y determined. The a l k a l o i d was then converted to harman [104] as previously described. The p u r i f i e d harman was sub-sequently subjected to a Kuhn-Roth oxidation i n order to examine the remaining two carbon atoms of the u n i t , and C ^ . As expected, the acetate thus derived accounted f o r l e s s than 1% the r a d i o a c t i v i t y of the parent a l k a l o i d . The degradation of ajmalicine [56], therefore, revealed that high l e v e l s of r a d i o a c t i v i t y r e s i d e i n two portions of the molecule: the tryptophan residue containing 59% to 78%, and the methyl group of the ester, containing 37% to 21% of the a c t i v i t y . The unit contained very l i t t l e , i f any, a c t i v i t y , a maximum value of only 4% being allowed from our various experiments. This r e s u l t may i n d i c a t e an aberrant b i o s y n t h e t i c route f o r the i n c o r p o r a t i o n of g l y c i n e i n t o the terpenoid skeleton. However, t h i s pathway must be considered i n s i g n i f i c a n t as compared to the incorporation of g l y c i n e i n t o the other parts of the molecule. The claim, therefore, that g l y c i n e i s a s p e c i f i c two carbon precursor of the u n i t of indole 86 a l k a l o i d s cannot be supported by t h i s work. Furthermore, the concept that g l y c i n e may be a n o n - s p e c i f i c precursor, ( i . e . precursor - 45 -units derived from catabolized glycine) must also be r e j e c t e d , i n 14 l i g h t of the extremely low incorporation of both glycine-1- C and 14 glycine-2- C i n t o the a l k a l o i d a l u n i t . Whether the postulated involvement of g l y c i n e i n the cephaeline [39] b i o s y n t h e s i s 8 4 ' 8 " ' reveals a d i f f e r e n t b i o s y n t h e t i c pathway i n that plant system r e l a t i v e to V. rosea remains an open question. 81 Regarding the studies of Shah and Rogers on terpenoid biosynthesis, i t i s evident that our findings can neither support nor r e f u t e t h i s work. These authors stated s p e c i f i c a l l y that the pathway, carbon dioxide -y g l y c o l l a t e [94] ->- glyoxylate [95] -> g l y c i n e [96] serine [44a] ->• pyruvate [83] ->• a c e t y l CoA (Figure 13) may only be s i g n i f i c a n t i n very young plants. Our studies were a l l c a r r i e d out with two year o l d V. rosea plants. Therefore, our r e s u l t s can only be i n t e r p r e t e d as i n d i c a t i v e of the i n s i g n i f i c a n c e of t h i s g l y c o l l a t e -»• a c e t y l CoA pathway i n mature plants. The question remained, however, as to the exact l o c a t i o n of the 14 l a b e l and the manner i n which glycine-2- C was being incorporated i n t o the 3-ethyl indole moiety of the a l k a l o i d with such r e l a t i v e e f f i c i e n c y . As mentioned i n the Introduction, the biosynthesis of tryptophan [11] i n micro-organisms has been shown to proceed v i a the shikimate-35 36 chorismate pathway (Figure 6). ' The f i n a l operation of t h i s sequence i s the replacement of the g l y c e r o l phosphate moiety of indolyl-3-glycerol,3-phosphate [43] by serine [44a] i n a two step procedure to form the side chain of the amino acid. The e n t i r e 37 sequence has been shown to be operative i n higher plant systems and, i n p a r t i c u l a r , the a c t i v i t y of tryptophan synthetase, the enzyme - 46 -c a t a l y z i n g the f i n a l operation, has been shown to be present i n 94 p l a n t s . Furthermore, the a c t i v i t y of the enzyme, serine aldolase, responsible i n mammalian systems f o r the interconversion of g l y c i n e 94 [96] and serine [44a] has also been shown i n plant systems. Since 95 both glycine and serine are known to be present i n Vinca species, i t i s a t t r a c t i v e to postulate that g l y c i n e can be u t i l i z e d i n the biosynthesis of the tryptophan unit i n V. rosea. This postulate, o u t l i n e d i n Figure 15, accounts f o r the high l e v e l of r a d i o a c t i v i t y found i n the degradation product, harman [104]. I t also accounts f o r 14 the non-incorporation of g l y c i n e - 1 - C, as the carboxyl group of glycine becomes the carboxyl group of tryptophan [11], and t h i s moiety i s l o s t i n the formation of ajamlicine [56]. The a c t i v i t y encountered i n the methyl carbon of the ester of ajmalicine [56] i s e x p l i c a b l e i n terms of the degradation of g l y c i n e 88 to a u n i t , a process observed previously. Byerrum and coworkers showed that i n plant systems the of glycine could function as a unit as e f f i c i e n t l y as the methyl groups of methionine and choline, and ten times as e f f i c i e n t l y as formate, whereas the carboxyl carbon of g l y c i n e showed no such a c t i v i t y . Our studies are consistent with both these f i n d i n g s . Some time a f t e r the p u b l i c a t i o n of our f i n d i n g s , a report by a 96 German research group appeared. GrOger, Maier, and Simchen had 14 also administered glycine-2- C to V_. rosea as w e l l as to S^. nux vomica. U t i l i z i n g a hydroponic feeding method to cut stems and a shorter feeding period, they were able to s u b s t a n t i a l l y increase the incorporation 14 of glycine-2- C i n t o v i n d o l i n e [3] r e l a t i v e to our own feeding method. - 47 -Shikimate-Chorismate Pathway tryptophan [44] synthetase [56] Figure 15: The Postulated Incorporation of Glycine i n t o Tryptophan. - 48 -On degrading the molecule, they found that 30% of the a c t i v i t y resided i n the N- and O-methyl groups of the molecule, that 4% was located i n the ester methyl group, and that 61% was located i n the tryptophan derived portion of v i n d o l i n e [3]. From S_. nux vomica, r a d i o a c t i v e strychnine [2] was i s o l a t e d . This molecule was degraded, as o u t l i n e d i n Figure 16, to the i n d o l i n e base [105], which was subjected to a Kuhn-Roth oxidation. The acetate and propionate i s o l a t e d from t h i s r e a c t i o n Figure 16: Grb'ger, Maier, Simchen Degradation of Strychnine. were devoid of r a d i o a c t i v i t y . When the N-methyl-tetrahydro-4,5-chanostrychnine {106] obtained from the a c t i v e strychnine [2] was s i m i l a r l y oxidized, the acetate i s o l a t e d accounted f o r 77.4% of the - 49 -a c t i v i t y of the molecule. The authors conclude that i n V. rosea and i n S^ . nux vomica, 14 glycine-2- C does not function as a precursor of the C y _ i o u n l t > D u t e i t h e r contributes to the plant pool, or i s transformed i n t o serine and i s incorporated v i a tryptophan i n t o the indole a l k a l o i d s . These findings are completely consistent with our own r e s u l t s , and bear out our postulated incorporation of g l y c i n e i n t o tryptophan i n plant systems. - 50 -Section B As o u t l i n e d i n Figure 9, Wenkert postulated that the Aspidosperma-type [72] and Iboga-type [73] skeletons arose v i a rearrangements of the 22 Strychnos skeleton [66]. In order to evaluate t h i s proposal, Scott and coworkers fed l a b e l l e d stemmadenine [74] and tabersonine [75] to the germinating seeds of _V. r o s e a . B o t h compounds were s p e c i f i c a l l y incorporated i n t o the Aspidosperma and Iboga a l k a l o i d s of t h i s plant system. The s i m i l a r i t y of stemmadenirie [74] and Wenkert's precursor [66] [ 74 ] [75 ] i s obvious, and the r e l a t i v e l y high, s p e c i f i c incorporation of t h i s compound i n t o v i n d o l i n e [3] and catharathine [8] v e r i f i e d the primary aspect of Wenkert's proposal concerning the d e r i v a t i o n of the Aspido-sperma and Iboga f a m i l i e s from the Strychnos s e r i e s . The i n c o r p o r a t i o n of tabersonine [75] i n t o v i n d o l i n e [3], although expected, confirmed the premise that minor f u n c t i o n a l i t i e s were introduced i n t o the molecule a f t e r the major s k e l e t a l modifications were complete. However, the in c o r p o r a t i o n of t h i s Aspidosperma a l k a l o i d into the Iboga compound, catharanthine [8], was e n t i r e l y unexpected, and necessitates the proposal of a f a c i l e r e v e r s i b l e r e a c t i o n at the branching point of Wenkert's postulate. Labelled catharanthine [8] when administered to V_. rosea seeds, f a i l e d to be incorporated i n t o any other a l k a l o i d , - 51 -i n d i c a t i n g a p o s s i b l e lack of r e v e r s i b i l i t y on the Iboga side of the branching point, or simply that catharanthine [8] i s too f a r from the main pathway to c o n s t i t u t e an e f f e c t i v e precursor. In a d d i t i o n to the incorporation of p o s s i b l e intermediates, a sequential a l k a l o i d formation study was c a r r i e d out u t i l i z i n g germinating 71 12 V_. rosea seeds. ' The seeds themselves contain no a l k a l o i d ; however, wi t h i n twenty-eight hours a f t e r germination, Cornanthe-type compounds (e.g. vincoside [54] and cornantheine [ 2 5 ] ) f i r s t appear. These are followed by the Strychnos s e r i e s (e.g. preakuammicine [64], stemmadenine [74], and akuammicine [65]) which i n turn i s followed by the Aspidosperma (e.g. tabersonine [75]) and f i n a l l y by the Iboga (e.g. catharanthine [8]) a l k a l o i d s . This sequence, therefore, i n a d d i t i o n to the evidence supplied by the incorporation of stemmadenine [74] and tabersonine [75], and supported by the B a t t e r s b y ^ incorporation of g e i s s o s c h i z i n e [55] i n t o Aspidosperma and Iboga compounds, suggests, but does not prove that the order: Cornanthe -> Strychnos -*• Aspidosperma -> Iboga as o r i g i n a l l y postulated by Wenkert i s correct. In our own laboratory, evaluation of the 1962 Wenkert proposal (Figure 9) was approached from a d i f f e r e n t stand-point. The proposed transannular c y c l i z a t i o n required to form the p e n t a c y c l i c systems of the Aspidosperma £72] and Iboga [73] f a m i l i e s was examined and found to be 72—76 a v i a b l e process s y n t h e t i c a l l y . Aspidospermidine [107] was formed from quebrachamine [ 7 8 ] ^ v i a mercuric acetate oxidation of the t e r t i a r y nitrogen of quebrachamine [78] to the iminium s a l t [108] followed by n u c l e o p h i l i c attack by the ir-electron system of the indole chromophore to y i e l d the i n d o l i n e [109], which on reduction affords aspidospermidine [107]. - 52 -[109] [107] In an analogous manner, carbomethoxycleavamine [77] y i e l d s 74 catharanthine [8]. In t h i s case, the C, _ p o s i t i o n of carbomethoxy-cleavamine [77] i s s u f f i c i e n t l y a c t i v a t e d to serve as the nucleophile for the transannular c y c l i z a t i o n . However, when fed to mature V. rosea p l a n t s , neither carbomethoxycleavamine [77] nor 6,7-dehydrovincadine [76] were converted to Iboga or Aspidosperma compounds. In s i m i l a r fashion, neither did quebrachamine [78] nor vincaminoreine [79] r e s u l t i n any incorporation into the Aspidosperma a l k a l o i d s of V_. minor when l a b e l l e d forms of these were fed to the plant system."^ - 53 -[79] To lessen the hazard involved i n i n t e r p r e t i n g negative incorpora-t i o n s , s e v e r a l a d d i t i o n a l feedings were c a r r i e d out. A v a r i e t y of methods were employed to administer r a d i o a c t i v e carbomethoxycleavamine [77J to _V. rosea p l a n t s . In no experiment, however, was the a c t i v i t y of catharanthine [8] derived from the plant equal to or higher than that found i n a blank experiment. Tabersonine [75] was administered to mature y_;_ rosea plants and was found to incorporate i n t o both catharanthine [8] and v i n d o l i n e [3] with equal e f f i c i e n c y . This 97 f i n d i n g , which has r e c e n t l y been confirmed, establishes two points: f i r s t , that the high molecular weight compounds being fed were reaching the s i t e s of biosynthesis and, thereforej that the feeding technique was e f f e c t i v e ; and secondly, that there e x i s t s a f a c i l e r e v e r s i b i l i t y between the Aspidosperma ( i . e . tabersonine [75]) and the Iboga ( i . e . catharanthine [8]) f a m i l i e s . In the l i g h t of the former, i t was f e l t that the negative incorporations of the nine membered r i n g intermediates were more l i k e l y due to the lack of any r e a l importance of the trans-annular c y c l i z a t i o n process i n biosynthesis, rather than an experimental d i f f i c u l t y . To add strength to t h i s argument, a time sequence study 14 77 was c a r r i e d out u t i l i z i n g V_. minor plants and tryptophan-3- C. - 54 -The i n c o r p o r a t i o n s of t h i s known p r e c u r s o r of i n d o l e a l k a l o i d s was determined f o r two groups of these compounds, the t e t r a c y c l i c f a m i l y represented by v i n c a d i n e [110] and vincaminoreine [79], and the p e n t a c y c l i c f a m i l y represented by v i n c a d i f f o r m i n e [111] and minovine [112], over a p e r i o d of time. The r e s u l t s of these f i n d i n g s are summarized i n Table 4. As i s e v i d e n t , the r a t i o of a c t i v i t y (B/A) of Table 4. The R e s u l t s of the I n c o r p o r a t i o n of DL-Tryptophan-3-C i n t o V. minor at Various Time I n t e r v a l s 7 7 [111] [110] [112] 14 Time T o t a l % I n c o r p o r a t i o n R a t i o of A c t i v i t y B/A Vin c a d i n e [110] + vinc a m i n o r e i n e [79] (A)  Vi n c a d i n e [110] + minovine [112] : m 4 hrs 1 day 2 days 4 days 7 days 14 days 0.003 0.015 0.010 0.010 0.009 0.003 0.057 0.24 0.21 0.22 0.13 0.06 19 16 21 22 14 20 - 55 -the p e n t a c y c l i c versus the t e t r a c y c l i c a l k a l o i d s remained f a i r l y stable over the period, four hours to fourteen days. This i s i n d i c a t i v e of the lack of conversion of the l a t t e r to the former and v i c e versa. To eliminate the p o s s i b i l i t y of there being an e q u i l i b r i u m between the two a l k a l o i d forms, thereby maintaining the r a t i o at a constant f i g u r e , r a d i o a c t i v e minovine [112] was administered to the plant system, and the same groups of a l k a l o i d s i s o l a t e d a f t e r a seven day feeding period. The t e t r a c y c l i c compounds showed no incorporation of l a b e l , while the p e n t a c y c l i c demonstrated high l e v e l s of r a d i o a c t i v i t y ( i . e . B/A = 2500). There was, therefore, no longer any .doubt that the 22 transannular c y c l i z a t i o n as postulated by Wenkert had l i t t l e b i o -synthetic relevance. Moreover, these r e s u l t s when considered i n conjunction with the previous f i n d i n g s " ^ s u g g e s t e d the existence of a common intermediate capable of generating Iboga and Aspidosperma t e t r a c y c l i c and p e n t a c y c l i c compounds without invoking the transannular c y c l i z a t i o n process. A general o u t l i n e summarizing these r e s u l t s and u t i l i z i n g the a c r y l i c ester structure [80], as an a t t r a c t i v e postulate, i s presented below. Thus, bond formation between a -* b leads to Aspidosperma t e t r a c y c l i c compounds; between a b and c -*• d, to the Aspidosperma p e n t a c y c l i c compounds. Analogously, bond formation between a -> e leads to t e t r a c y c l i c members of the Iboga family, while a -»• e and f •+ d lead to the Iboga p e n t a c y c l i c a l k a l o i d s . The enamine form [80a] of t h i s intermediate was invoked by 98 Qureshi and Scott to explain i n v i t r o transformation of tabersonine [75] to catharanthine [8] and of stemmadenine [74] to tabersonine [75] 99 and catharanthine [8]. Although t h i s work has been questioned, the - 56 -COOCH [8] evidence f o r the existence of the intermediate [80] or i t s enamine [80a] had been a r r i v e d at independently by two research groups u t i l i z i n g e n t i r e l y d i f f e r e n t approaches to the problem of indole a l k a l o i d biogenesis. I t i s also pertinent to note the [80a] resemblance of the a c r y l i c ester [80] to Wenkert's a c r y l i c 22 ester [69] contained i n h i s o r i g i n a l proposal. F i n a l l y the - 57 -i s o l a t i o n , from Tabernamontaria cummjnsij, of the compound, 2-ethyl-3-[2-(3-ethyl p i p e r i d i n e ) - e t h y l ] i n d o l e [ 1 1 3 ] * ^ which can be regarded as the reduced no r - d e r i v a t i v e of the a c r y l i c ester [80], provided a [113] strong i n vivo suggestion of the existence of t h i s c r i t i c a l intermediate. Confronted with t h i s evidence f o r the intermediacy of the a c r y l i c ester [80], we decided that the synthesis of t h i s compound f o r purposes of b i o l o g i c a l evaluation was of prime importance to the unraveling of the pathway of biosynthesis of the Aspidosperma and Iboga bases. Before embarking on t h i s synthesis, however, i t was r e a l i z e d that dihydropyridines tend to be unstable e n t i t i e s . In many cases, the presence of molecular oxygen i s s u f f i c i e n t to e f f e c t t h e i r conversion to the p y r i d i n e system. This tendency to form aromatic systems has been used i n the synthesis of pyridines i n which a dihydropyridine i s the d i r e c t r e s u l t s of the p a r t i c u l a r r e a c t i o n , but only the desired aromatized compound i s a c t u a l l y i s o l a t e d t h a t i s : - 58 -R' H R' H R' Dihydropyridines are also known to isomerize r a p i d l y , i . e . , 1,2-dihydro-102 p y r i d i n e isomerizes to the 1,4-compound, or to the 1,6-derivative. Furthermore, dihydropyridines are known to disproportionate such that three molecules of the dihydropyridine lead to two of the py r i d i n e plus one of the saturated piperidine.'^''"'''"^ We f e l t , therefore, that the a c r y l i c ester [80], or i t s enamine isomer [80a], could provide experimental d i f f i c u l t i e s i n the i s o l a t i o n , l a b e l l i n g and/or incorpora-t i o n studies i n the various plant systems. The obvious a l t e r n a t i v e which would overcome these problems involved a r e l a t i v e l y s t a b l e analogue which v i a reasonable i n vivo conversions would provide the desired intermediate [80]. The compound which seemed most l i k e l y to possess these q u a l i t i e s was the g-hydroxyester [114], f o r which the 103 name 16,17-dihydrosecodin-17-ol could be assigned, since the a c r y l i c ester [115] was given the name secodine by S m i t h . T h u s , appropriate dehydration of the hydroxyester moiety and oxidation of the pi p e r i d e i n e r i n g , i n the plants, would y i e l d the desired a c r y l i c ester [80]. Since both of these reactions are b i o l o g i c a l l y f e a s i b l e , we focused our a t t e n t i o n on the preparation of the intermediate [114]. - 59 -[115] It i s necessary to mention, at t h i s point, that t h i s s y n t h e t i c project was c a r r i e d out by several members of our research group, of which I was a member. A f u l l y d e t a i l e d account of the synthesis of 16,17-dihydrosecodin-17-ol [114] can be found i n the doctoral thesis 104 of R.S. Sood. Consequently, a b r i e f d e s c r i p t i o n of t h i s synthesis w i l l be presented here together with f u l l d e t a i l s on any de v i a t i o n from t h i s sequence as described i n the aforementioned t h e s i s . It i s also pertinent, at t h i s point, to present the numbering scheme which w i l l be used i n dealing with these multi-atom molecules throughout the d e s c r i p t i o n of the synthesis and subsequent b i o l o g i c a l evaluation of 16,17-dihydrosecodin-17-ol [114] and i t s d e r i v a t i v e s . The scheme i s that adopted by Smith"'"^"' f o r secodine [115]. I t i s based on the biogenetic assumption that the secodine-type molecule [115] i s derived from the Cornanthe family of alkaloids, i n which the C ^ Q u n i t i s unrearranged, and f o r which i d e n t i c a l numbering schemes have been - 60 -proposed by Le Men and Taylor 106 and by Trojanek and Blaha. 107 The d e r i v a t i o n of the scheme and i t s present a p p l i c a t i o n can best be demonstrated by u t i l i z i n g the Corynanthe a l k a l o i d , cornantheine [25], that i s : The synthesis of the intermediate [114] was accomplished by the pathway presented i n Figure 17. This route was chosen f o r a number of reasons. F i r s t l y , the c h l o r o e t h y l indole [116] was a v a i l a b l e i n our laboratory i n m u l t i p l e gram q u a n t i t i e s . This compound incorporated the f u n c t i o n a l i t i e s required to' elaborate the structure to the desired product. Secondly, the steps involved i n the synthesis were simple chemical reactions, c a r r i e d out under r e l a t i v e l y mild conditions. The presence of the l a b i l e indole system necessitated t h i s precaution, as experience wi t h i n our research group had amply demonstrated the high s u s c e p t i b i l i t y of t h i s moiety to oxidation, reduction and various s u b s t i t u t i o n reactions. T h i r d l y , the sequence as presented provides the opportunity of introducing a r a d i o a c t i v e t r a c e r , e i t h e r t r i t i u m or carbon-14, at a number of s i t e s i n the molecule. The synthesis of the c h l o r o e t h y l indole [116] had been achieved i n our laboratory i n connection with work toward the syntheses of 108 indole a l k a l o i d s . The synthetic route as o u t l i n e d i n Figure 18 - 61 -CI +C0C1 [119] 0°C [114] Figure 17: The Synthesis of 16,17-Dihydrosecodin-17-ol. - 62 -^COOCH 2CH 3 C l - ( C H 2 ) 3 - B r + CH 2 > Cl-(CH 2> 3-CH(COOCH 2CH 3) 2 ^COOCl^CH.j [116] 108 F i g u r e 18: The Synthe s i s of 2 - C a r b o e t h o x y - 3 - ( B - c h l o r o e t h y l ) - i n d o l e . p r o v i d e d f o r the p r o d u c t i o n of t h i s compound i n batches of approximately 108 ten grams. The only change from the o r i g i n a l scheme i n v o l v e d the s u b s t i t u t i o n of benzenediazonium c h l o r i d e by benzenediazonium f l u o -b o r a t e . This was done because of the n e c e s s i t y of o b t a i n i n g the diazonium s a l t i n a dry form p r i o r to formation of the arylhydrazone [125]. The e x p l o s i v e nature of benzenediazonium c h l o r i d e i n t h i s c o n d i t i o n i s w e l l known, and a f t e r a p a r t i c u l a r l y violent e x p l o s i o n at t h i s p o i n t , the s u b s t i t u t i o n of the f l u o b o r a t e was i n i t i a t e d . 3 - E t h y l p y r i d i n e [117] was obtained by means of a W o l f f - K i s h n e r r e d u c t i o n of the commercially - 63 -a v a i l a b l e 3-acetylpyridine. The condensation of the s t a r t i n g materials was e f f e c t e d i n near q u a n t i t a t i v e y i e l d s by u t i l i z i n g the 3-ethyl-pyr i d i n e as solvent as well as the reagent i n the r e a c t i o n . The pyridinium c h l o r i d e [118] was obtained as a white c r y s t a l l i n e s o l i d (m.p. 87-89°) a f t e r a twenty-four hour r e a c t i o n period, during which time the c h l o r o e t h y l indole [116] and e t h y l p y r i d i n e [117] were sealed i n a Carius tube and kept at a temperature of 118-120°C. The pyr.idium c h l o r i d e [118] was subsequently converted to the p i p e r i d e i n e [119] by sodium borohydride reduction i n methanol at 0°C, and the product, without p u r i f i c a t i o n , was then reduced i n r e f l u x i n g tetrahydrofuran s o l u t i o n with l i t h i u m aluminum hydride to N-[g-(3-[2-hydroxymethylene]-indolyl)-ethyl]-3'-ethyl-3'-piperideine [120]. Once again, the product was used, without further p u r i f i c a t i o n , to form the benzoate d e r i v a t i v e [121]. This conversion was accomplished i n a heterogeneous mixture, of anhydrous tetrahydrofuran, anhydrous potassium carbonate and the a l c o h o l [120] , to which benzoyl c h l o r i d e was added. A f t e r one hour at 0°C, under a nitrogen atmosphere, the r e a c t i o n mixture provided a yellow o i l which c r y s t a l l i z e d on standing. Further p u r i f i c a t i o n of the benzoate [121] by column chromatography on alumina r e s u l t e d i n y i e l d s as high as 70% f o r the o v e r a l l sequence from the c h l o r o e t h y l i n d o l e [116] to the benzoate [121]. The p u r i f i e d benzoate [121] (mp 110.5-112.5°C) was then converted to the n i t r i l e [122], i n 69% y i e l d by performing the displacement i n N,N-dimethylformamide and a t e n - f o l d molar excess of potassium cyanide. Several methods of hydrolyzing t h i s compound to the e s t e r , N-[g-(3-[2-carbomethoxymethylene]-indolyl)-ethyl]-3'-ethyl-3'-piperideine [123] - 64 -were attempted. The one which proved most favourable i n terms of y i e l d and p u r i t y of product was the procedure used by Wenkert and associates f o r the h y d r o l y s i s of a compound anlogous to the one under consideration 109 here. This method f i r s t involved the d i s s o l u t i o n of the n i t r i l e [122] i n anhydrous methanol followed by a d d i t i o n of 1% by volume of water. The s o l u t i o n was then cooled i n i c e and saturated with anhydrous hydrogen c h l o r i d e gas. On sa t u r a t i o n , the r e a c t i o n f l a s k was stoppered and the mixture maintained at room temperature f o r approximately s i x t y hours. The r e s u l t a n t crude methyl ester [123], a f t e r column chromato-graphy on alumina, c r y s t a l l i z e d from methylene c h l o r i d e (mp 131.5-132°C). The optimum y i e l d of t h i s r e a c t i o n was found to be 69%. Formylation at of the ester [123] was c a r r i e d out i n anhydrous benzene employing methyl formate and sodium hydride as the base. The expected enol 1124] was c a r e f u l l y reduced u t i l i z i n g sodium borohydride i n methanol s o l u t i o n at -30°C to a f f o r d the secodinol d e r i v a t i v e [114]. At higher temperatures or on using a large excess of sodium borohydride the y i e l d of 16,17-dihydrosecodin-17-ol [114] was s i g n i f i c a n t l y decreased and concomitant formation of a compound whose i n f r a r e d spectra showed no carbonyl absorption was noted. The nuclear magnetic resonance spectrum of t h i s compound was s i m i l a r to that of the desired 3-hydroxyester 1114] except that no -OCH^ s i g n a l was apparent at x 6.37. Instead, a broad two proton s i n g l e t appeared at x 5.16 which completely vanished on the a d d i t i o n of deuterium oxide to the sample. The by-product was, therefore, t e n t a t i v e l y assigned the d i o l s tructure [126] although fur t h e r work for complete c h a r a c t e r i z a t i o n was not c a r r i e d out. - 65 -Oil [126] Characterization of a l l the intermediates i n the above sequence, i n c l u d i n g melting points, u l t r a v i o l e t , i n f r a r e d , nuclear magnetic resonance spectra as w e l l as mass spectra, high r e s o l u t i o n mass spectra and elemental a n a l y s i s , confirmed the structures as shown above. reported here. In order to evaluate the b i o l o g i c a l p o t e n t i a l of the compound now i n hand, r a d i o i s o t o p i c incorporation studies were comtemplated. This required the intr o d u c t i o n of e i t h e r t r i t i u m or carbon-14 i n t o 16,17-dihydrosecodin-17-ol [114J. The t r i t i a t i o n of indole compounds had been developed wi t h i n our 89 research group and consisted of e q u i l i b r a t i n g the compound i n t r i t i a t e d t r i f l u o r o a c e t i c a c i d . E s s e n t i a l l y , the a c i d i c proton of the acid acts as an e l e c t r o p h i l i c agent, attacking the aromatic r i n g of the indole. When t h i s proton i s a t r i t i u m cation incorporation of the l a b e l i n t o the unsubstituted aromatic p o s i t i o n s of the molecule ensues. The s i m p l i c i t y of the method, i t s high incorporation of the ra d i o a c t i v e isotope, and i t s high chemical y i e l d made t h i s technique the preferred choice with our p a r t i c u l a r system. However, 16,17-dihydrosecodin-17-ol These data are reported elsewhere 104 and w i l l consequently not be - 66 -[114] was found to be unstable to acid conditions and, therefore N-[3-(3-[2-carbomethoxymethylene]-indolyl)-ethyl]-3'-ethyl-3'-piperideine [123], the intermediate i n the synthetic sequence formed i n i t i a l l y i n an a c i d i c medium, was chosen as the point at which l a b e l was to be introduced. When subjected to the t r i t i a t i o n conditions, 80% of the methyl acetate d e r i v a t i v e [123] was recovered a f t e r p u r i f i c a t i o n , and was found to be of high s p e c i f i c r a d i o a c t i v i t y . This l a b e l l e d compound 3 was then converted to [ar- H]-16,17-dihydrosecodin-17-ol [114] as previously described. B i o l o g i c a l evaluation of the l a b e l l e d compound was c a r r i e d out using specimens of V_. rosea, V_. minor, and Aspidosperma pyricollum Muell.-Arg. as plant substrates. The compound [114] was fed, i n a l l cases, as i t s acetate s a l t , and the feeding periods and administration techniques v a r i e d with each plant species. The r e s u l t s with the two l a t t e r plant species w i l l be presented i n Sections C and D. I t i s s u f f i c i e n t to state here that they supported the r e s u l t s obtained with V_. rosea. As described i n Section A, the feeding technique u t i l i z e d f o r V. rosea plants involved the threading of a cotton wick through the 3 stems of whole potted plants. When the acetate s a l t of [ar- H]-16,17-dihydrosecodin-17-ol [114] was thus administered, the a l k a l o i d s , subsequently i s o l a t e d p u r i f i e d and examined f or r a d i o a c t i v i t y , showed no s i g n i f i c a n t i n corporation of l a b e l (Experiment 1, Tables 6 and 7). In dealing with the incorporation of compounds in t o plant systems, several f a c t o r s must be considered. These include the s o l u b i l i t y of - 67 -the compound with i n the plant, the transport of the compound with i n the plant, the permeability of c e l l walls with respect to the compound, and the rate of biosynthesis of the p a r t i c u l a r a l k a l o i d under i n v e s t i -gation. The feeding techniques developed i n our research group had been shown to deal e f f e c t i v e l y with these f a c t o r s i n the case of large molecular weight precursors and i t was known that the rates of biosynthesis of the a l k a l o i d s i s o l a t e d were s u f f i c i e n t l y rapid to have incorporated 89 an authentic precursor i n the feeding times employed. We therefore f e l t that the negative r e s u l t s encountered with 16,17-dihydrosecodin-17-ol [114] were not, i n a l l p r o b a b i l i t y , due to t e c h n i c a l d i f f i c u l t i e s associated with our experimental procedures. Instead, we f e l t that the a n t i c i p a t e d bio-transformation of the a l c o h o l [114] to the acrylic ester [80] was, i n f a c t , not taking place i n the plant systems studied. Whether t h i s implied an i n c a p a b i l i t y on the part of the plants to perform both the dehydration and the oxidation r e a c t i o n s , or merely one of these remained an open question, but one which we intended to answer. A f t e r obtaining the above negative r e s u l t s , a communication 103 appeared by Battersby and Bhatnagar. They described the synthesis of 16,17-dihydrosecodin-17-ol [114] as outlined i n Figure 19, as w e l l as b i o l o g i c a l experimentation i n v o l v i n g the a l c o h o l [114]. Their synthesis, i n i t s l a t e r stages, i . e . [123] -> [124] -> [114] i s p r a c t i c a l l y i d e n t i c a l to our sequence. They also found, for example, that the borohydride reduction of the enol [124], unless r i g i d l y c o n t r o l l e d , y i e l d e d large amounts of the d i o l [126], The f i r s t part of t h e i r synthesis i s markedly d i f f e r e n t from our own, and according to t h e i r p u b l i c a t i o n was s u i t a b l e only f o r the production of small q u a n t i t i e s of - 69 -the c h l o r o e t h y l i n d o l e acetate [129]. In t h i s respect, we f e e l that the synthesis of the a l c o h o l [114] as developed i n our laboratory i s superior to that of Battersby. Our sequence can produce r e l a t i v e l y large amounts of the product, e.g. two grams of the benzoate [121] y i e l d , on the average, one hundred milligrams of the a l c o h o l [114]. It was, however, the r e s u l t s of the b i o l o g i c a l experimentation of Battersby and Bhatnagar which i n t e r e s t e d us to a greater extent. 3 By feeding [O-methyl- H] loganin [48] to the shoots of plants of both Rhazya o r i e n t a l i s and V_. rosea, working these up f o r a l k a l o i d s , and adding synthetic [114] as c a r r i e r , the authors were able to demonstrate r e t e n t i o n of r a d i o a c t i v i t y i n the r i g o r o u s l y p u r i f i e d 16,17-dihydro-secodin-17-ol £114]. They conclude, therefore, that t h i s tetrahydro-p y r i d i n e £114] i s a n a t u r a l a l k a l o i d constituent of these plant systems, 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 reduction (e.g. £131]) or by hydration and reduction (e.g. [80]) ,H»103 6 5 C H 3 0 0 C C H 3 O O C [131] [80] 103 Further to t h i s p u b l i c a t i o n , several other papers appeared at t h i s time, i n which the authors report the i s o l a t i o n of 16,17,15,20-tetrahydrosecodine [132], 16,17-dihydrosecodine [133], and 16,17,15,20-tetrahydrosecodin-17-ol [134] from plant s o u r c e s . 1 0 5 ' 1 1 1 - 70 -[133] t 1 3 * ! - 71 -The f i r s t two bases were i s o l a t e d from R. s t r i c t a and i d e n t i f i e d spectro-s c o p i c a l l y . The t h i r d was i s o l a t e d from R_. o r i e n t a l i s and s i m i l a r l y i d e n t i f i e d . In a d d i t i o n , tetrahydrosecodine [132] was synthesized as o u t l i n e d i n Figure 20, and used as a c a r r i e r i n the a l k a l o i d a l extract 14 of tryptophan-2- C-fed R . o r i e n t a l i s shoots. The r i g o r o u s l y p u r i f i e d base [132] retained r a d i o a c t i v i t y corresponding to 0.5% i n c o r p o r a t i o n of the tryptophan. This led the authors to conclude "that 16,17,15,20-tetrahydrosecodine ([132])is on a metabolic side-track close to the main a l k a l o i d b i o s y n t h e t i c route". In the l i g h t of these r e s u l t s , and from the conclusions of two independent research groups, we f e l t that our i n i t i a l d e c i s i o n to synthesize and evaluate a compound resembling, the a c r y l i c ester [80] was, indeed, c o r r e c t , and would most c e r t a i n l y bear f r u i t , i f only a compound close enough to the l a b i l e intermediate [80] could be synthesized. To t h i s end, we considered two d e r i v a t i v e s of 16,17-dihydro-secodinol [114], the pyridinium system [314a] and the dehydrated analogue, secodine [115]. The f i r s t of these would require a dehydration, as before, and a reduction, e.g. by NADH to the dihydropyridine to y i e l d the desired intermediate [80] i n the plant. The second compound would require only the oxidation of the p i p e r i d e i n e r i n g to produce the same intermediate. [114a] [115] - 72 -Since the pyridinium system [U4a] w a s f e l t to be a st a b l e compound, i t s synthesis from the tetrahydropyridine alcohol [114] was inve s t i g a t e d f i r s t . In the work i n v o l v i n g the transannular c y c l i z a t i o n s of t e t r a -72—76 c y c l i c a l k a l o i d s , mercuric acetate had been found to be a valuable reagent i n forming the imine system. Such a r e a c t i o n i n t h i s case should lead to the i s o l a t i o n of the pyridinium s a l t [114a] i n view of the tendency of tetrahydropyridines to a r o m a t i z e . 1 0 1 , 1 0 ^ This, conversion proved unsuccessful. Extensive experimentation employing model compounds and the alcohol [114], i t s e l f , f a i l e d to y i e l d any s i g n i f i c a n t amounts of the pyridinium system. A d e t a i l e d account of t h i s p a r t i c u l a r study i s contained i n the doctoral thesis of N. Westcott. The d i f f i c u l t y associated with the preparation of the alcohol [114a] d i r e c t e d us to f i n d a route to the a c r y l i c e ster, secodine [115]. This synthetic study was, however, considerably shortened by two experimental fin d i n g s which came to l i g h t at the time when the dehydration of 16,17-dihydrosecodin-17-ol [114] was being considered as the simplest route to secodine [115]. The f i r s t involved a synthetic p r o j e c t i n our own laboratory. The rea c t i o n scheme, out l i n e d i n Figure 21, was being i n v e s t i g a t e d as a p o s s i b l e i n v i t r o transformation of the Corynanthe skeleton to that of the Strychnos family. D i h y d r o s i t s i r i k i n e [135] on treatment with t - b u t y l hypochlorite formed the chloroindolenine [136]. The l a t t e r substance undergoes rearrangement to the isomeric s p i r o i n d o l -enines [137] on rea c t i o n with sodium methoxide i n methanol. The f i n a l step to y i e l d dihydropreakuammicine [138] was envisioned as occurring v i a anion formation at C,, of the spiroindolenine [137] and attack of l o t h i s anion at the a-carbon of the indolenine system thereby allowing - 73 -CI CH3OOC CH2OH [138] Figure 21: The Proposed Synthetic Conversion of the Corynanthe Skeleton to the Strychnos Skeleton. - 74 -r i n g closure with concomitant extrusion of methoxide. The only product of t h i s r e a c t i o n , when sodium hydride was employed as base, was found 113 to be the a c r y l i c ester [137a]. In view of the s i m i l a r f u n c t i o n a l i t y possessed by 16,17-dihydrosecodin-17-ol [114], the base-catalyzed dehydration was attempted. The r e s u l t i n g r e a c t i o n mixture consisted of two compounds, the spectroscopic data of which revealed that at l e a s t one was dimeric i n nature. At t h i s time, the second experimental r e s u l t came to l i g h t i n the i n v e s t i g a t i o n s they describe the i s o l a t i o n and properties of the dimeric a l k a l o i d s , presecamine [139A or B], tetrahydropresecamine [140A or B] and dihydropresecamine [141A or B]. These compounds, on treatment with d i l u t e a c i d at room temperature, rearrange q u a n t i t a t i v e l y to t h e i r secamine d e r i v a t i v e s [142A or B] as portrayed i n Figures 22a and 22b. In add i t i o n , presecamine [139A or B] and tetrahydropresecamine [140A or B] when exposed to sublimation conditions y i e l d e d secodine [115] and 15,20-dihydrosecodine [143] r e s p e c t i v e l y as t h e i r sole r e a c t i o n products. These products on standing i n the absence of solvent [137a] form of a p u b l i c a t i o n by C o r d e l l , Smith, and Smith. 114 In t h e i r - 75 -[143] - 76 -[139A] [142A] Figure 22a: The Acid Catalyzed Rearrangement of the Presecamine Skeleton to the Secamine Skeleton (Type A). - 77 -[ 1 3 9 B ] Figure 2 2 b : The Acid Catalyzed Rearrangement of the Presecamine Skeleton to the Secamine Skeleton (Type B). - 78 -reconverted to the dimeric compounds presumably v i a a D i e l s - A l d e r r e a c t i o n to form the appropriate presecamine d e r i v a t i v e and i t s corresponding diastereoisomer. These r e s u l t s were invaluable i n allowing us to proceed with the necessary precautions f o r the preparation and incorporation of the l a b i l e secodine d e r i v a t i v e s . The r e a c t i o n of 16,17-dihydrosecodin-17-ol [114] with sodium hydride i n benzene followed by a rapid chromato-graphy of the r e a c t i o n mixture on a column of n e u t r a l alumina allowed f o r the separation of the monomer from the r a p i d l y forming dimeric compounds. The benzene s o l u t i o n of the secodine [115] thus obtained was then freeze-dried to a f f o r d the pure a c r y l i c ester as an amorphous s o l i d . The e n t i r e sequence avoids the use of a c i d and hydroxylic solvents, which, as reported by Smith, attack the monomer. Rapid manipu-l a t i o n of the appropriate experimental steps when coupled with the freeze drying technique allow for the i s o l a t i o n of the secodine [115] free of any dimeric materials. The s p e c t r a l c h a r a c t e r i s t i c s of the i s o l a t e d s o l i d agreed with those reported by Smith and coworkers. Complete c h a r a c t e r i z a t i o n of t h i s compound, as w e l l as a more d e t a i l e d d i s c u s s i o n of i t s synthesis can, as previously mentioned, be found i n the doctoral 104 thesis of R.S. Sood. The plant feeding method found to be most s u c c e s s f u l , i n our laboratory, when dealing with large molecular weight precursors, involves the formation of the acetate s a l t of these compounds. This water soluble s a l t i s then administered to the plants v i a the wick method described f o r V_. rosea, or hydroponically to the cut stems or to the roots of the other plant systems used. The maximum time required for the c a p i l l a r y a c t i o n of the plants to draw up the acetate - 79 -s o l u t i o n has been e m p i r i c a l l y determined as being two hours. The question arose, therefore, as to whether the secodine [115] could survive t h i s maximum feeding time i n aqueous, s l i g h t l y a c i d i c s o l u t i o n . In order to answer t h i s question and, thus, a c e r t a i n the u t i l i t y of t h i s 14 feeding technique i n the present circumstances, [ COOCH^] secodine [115] was synthesized. This synthesis was accomplished according to the scheme presented 1 4 i n Figure 17, with the s u b s t i t u t i o n of potassium cyanide- C i n the re a c t i o n to form the l a b e l l e d n i t r i l e [122]. This p a r t i c u l a r r e a c t i o n was s l i g h t l y modified to achieve the maximum incorporation of the ra d i o a c t i v e cyanide into the product. S p e c i f i c a l l y , t h i s involved 14 adding the potassium cyanide- C, made up to one molar equivalent with unlabelled cyanide, to the N,N-dimethyl formamide s o l u t i o n of the benzoate [121]. This r e a c t i o n mixture was then s t i r r e d at room tempera-ture under nitrogen f o r one hour. The temperature was then r a i s e d slowly to 90°C. When the potassium benzoate p r e c i p i t a t e became d i s t i n c t l y detectable, a d d i t i o n a l unlabelled potassium cyanide was added, to a t o t a l amount of f i v e molar equivalents. The re a c t i o n was maintained at 90°C f o r one hour and the subsequent work-up was then c a r r i e d out as previously reported. 14 A 10 mg por t i o n of the [ C00CH 3]-16,17-dihydrosecodin-17-ol [114] subsequently produced was dehydrated by the established procedure. The secodine [115] a f t e r freeze-drying was immediately taken up i n f i v e drops of ethanol to which f i v e drops of 0.1 N a c e t i c a c i d were added, followed by a h a l f m i l l i l i t e r of water. This s l i g h t l y cloudy s o l u t i o n i s the normal medium used to administer precursors to pla n t s . This - 80 -s o l u t i o n was maintained at room temperature for two hours and then freeze-dried. The residue was taken up i n methanol and spotted on Eastman-Kodak prepared alumina p l a t e s . When the chromatography pla t e s were eluted, u t i l i z i n g benzene:chloroform - 1:1, the secodine [115] displayed an value of 0.6; the presecamine and secamine together displayed an R^ value of 0.45. In a d d i t i o n , a small amount of baseline was noted. To determine the r a t i o of the compounds thus separated, the Kodak t h i n layer s t r i p s were scanned for r a d i o a c t i v i t y on a Nuclear-Chicago Actigraph Scanner connected to a pen recorder and a d i g i t a l p r i n t out system. The r a t i o of the compounds present, as recorded on several runs for two separate t h i n layer s t r i p s , are l i s t e d i n Table 5. Table 5: The Ratio of Monomer Experiment to Dimer Produced i n a T y p i c a l Feeding S t r i p No. Secodine [115] R f 0.60 Presecamine [139] and Secamine [142] R f 0.45 Baseline 1 62.4 31.8 5.8 2 61.5 32.0 6.5 I t was evident from t h i s blank experiment that the major p o r t i o n of our precursor [115] would, indeed, reach the plant system as the monomeric u n i t . Whether a mechanism e x i s t s w i t h i n the plant to cleave the dimeric compound, reforming secodine [115], i s unknown, but i n view 114 of the i s o l a t i o n of the dimers from plant systems, i t may be i n f e r r e d that such i s not the case; at l e a s t , not f o r a f a c i l e cleavage. - 81 -We, therefore, proceeded on the assumption that i f secodine [115] can be u t i l i z e d by the plant system, i t , rather than i t s D i e l s - A l d e r type dimers, would be p r e f e r e n t i a l l y incorporated. In order to evaluate the b i o l o g i c a l p o t e n t i a l of secodine [115] , several r a d i o a c t i v e l a b e l l e d forms of t h i s a c r y l i c ester were synthesized. The f i r s t two, the aromatically t r i t i a t e d compound and the a c r y l i c ester bearing carbon-14 i n the carbomethoxy group have already been mentioned. Further q u a n t i t i e s of both these were prepared as w e l l as a t h i r d 3 l a b e l l e d form, [3,14,15,21- H]-secodine [115]. This l a t t e r r a d i o a c t i v e COOCH3 [115] d e r i v a t i v e was prepared by reducing the pyridinium c h l o r i d e [118] with sodium b o r o t r i t i d e , followed by sodium borohydride to complete the p a r t i c u l a r r e a c t i o n . The reduced compound [119] was then elaborated 3 to [3,14,15,21- H]-16,17-dihydrosecodin-17-ol [114] i n the usual manner, and dehydrated to the a c r y l i c ester [115] j u s t p r i o r to plant feedings. As with the al c o h o l [114], the inc o r p o r a t i o n studies were c a r r i e d out with three plant species, V_. rosea, V. minor and A. pyricollum. Each of these i n v e s t i g a t i o n s had somewhat complimentary, but yet d i f f e r e n t goals. The experiments with V_. rosea would provide information on Aspidosperma (vindoline [3]) and Iboga (catharanthine [8]) a l k a l o i d b i o s y n t h e s i s . Those on _V. minor would allow evaluation of the - 82 -eburnamine-vincamine (vincamine [102]) group, for which no experimental data was a v a i l a b l e , and f i n a l l y the a l k a l o i d s of A. pyricollum (apparicine [144] and uleine [145]) are d i s t i n c t l y i n t e r e s t i n g since they la c k the normal tryptophan side chain. Various postulates f o r the o r i g i n of these compounds have been put f o r t h , but again no e x p e r i -mental data was a v a i l a b l e . The r e s u l t s of the incorporations of the various r a d i o a c t i v e secodine [115] precursors f o r the l a t t e r two plant systems w i l l be discussed i n Section C and Section D r e s p e c t i v e l y . The present s e c t i o n w i l l concentrate on the r e s u l t s obtained with y_. rosea. The discussion dealing with the incorporation of g l y c i n e into the indole a l k a l o i d s (Section A) revealed our experience with V. rosea i n b i o s y n t h e t i c studies. Consequently t h i s species was once more chosen as the primary t e s t i n g medium f o r the synthetic a c r y l i c ester [115]. The feeding technique employed was the wick method already discussed i n Section A except that secodine [115] was fed as i t s acetate s a l t , as o u t l i n e d i n the d i s c u s s i o n of the blank feeding experiments. The r e s u l t s obtained by experimentation with _V. rosea plants are summarized i n Tables 6 and 7. The incorporation f i g u r e s for the a l c o h o l [114] (Experiment 1) are included as a comparison of the r e l a t i v e e f f i c a c y of the two indole compounds. [144] [145] Table 6: The Results of the Incorporation of 16,17-Dihydrosecodin-17-ol and Secodine i n t o V. rosea. Experiment Compound A c t i v i t y Fed Feeding % Incorporation Number Fed (dpm) Time Ajmalicine [56] Catharanthine [8] Vindoline [3] 3H 1 4 c (hrs) 3H 1 4 c 3H 1 4 c 3H 1 4 C j l l O [ar- 3H]-16,17-H2~secodin-17-o l [114] 1.89 x 10 7 - 216 < 0.001 — < 0.001 — < 0.001 2 3 [ar- H]-seco-dine [115] 3.31 x 10 8 - 216 < 0.001 — < 0.001 — 0.01 3 3 [ar- H]-seco-dine 1.23 x i o 8 - 216 < 0.001 — < 0.001 — 0.02 3 — 4 [ar- 3H, 1 4COOCH 3]-secodine 8.47 x 10 7 9.58 x i o 6 216 < 0.001 <o .001 < 0.001 < 0.001 0.042 0.043 5 [3,14,15,21-3H, 1 4COOCH 3]-seco-dine 3.50 x 10 7 1.01 x i o 7 216 0.01 0.03 a Base on the r a d i o a c t i v i t y of v i n d o l i n o l [147] derived from the i s o l a t e d a l k a l o i d . Table 7: The S p e c i f i c A c t i v i t i e s Associated with the Experiments i n Table 6. Experiment S p e c i f i c A c t i v i t y Fed Ratio S p e c i f i c A c t i v i t y Isolated Ratio Number (dpm/mM) 3 ^ (dpm/mM) 3 H / 1 4 C Ajmalicine [56] Catharanthine [8] Vindoline [3] Vindoline 3H U C 3H 1 4 C 3H 1 4 C 3H 1 4 C ™ j l l O 8.46 X i o 8 — — 627 — 913 000 — 2 2.83 X i o 1 0 — 4.25 x i o 4 - 1.28 x i o 5 1.10 x i o 6 — 3 2.83 X i o 1 0 — — 9.61 x 10 3 - 2.55 x i o 4 7.54 x i o 5 * 4 1.10 X i o 1 0 1.28 x i o 9 8.8 4.23 x i o 3 000 1.53 x i o 4 000 1.77 x i o 6 2.13 x i o 5 8.3 5 9.82 X i o 9 1.27 x i o 9 3.5 — — — 5.96 x i o 4 4.40 x i o 4 1.4 a oo 2^  Based on the r a d i o a c t i v i t y of the v i n d o l i n o l [147] derived from the i s o l a t e d a l k a l o i d . - 85 -The i n i t i a l r e s u l t s (experiments 2 and 3) obtained with the aromatically l a b e l l e d secodine [115] indic a t e d that the plant system seemed to be able to u t i l i z e the precursor i n order to synthesize indole a l k a l o i d s . As a check on the l a b e l found i n v i n d o l i n e [3], the a l k a l o i d was subjected to r e f l u x i n g concentrated hydrochloric a c i d , f o r a short period. This treatment achieves h y d r o l y s i s of the a c e t y l group, and also serves to exchange the protons of the aromatic rings i n a manner analogous to the t r i t i a t i o n of t h i s moiety with t r i t i a t e d t r i f l u o r o a c e t i c a c i d . The desacetylvindoline [146] thus obtained, when examined f o r r a d i o a c t i v i t y , contained only 10% of the l a b e l found i n v i n d o l i n e [3]. This i n d i c a t e s that the t r i t i u m contained by the a l k a l o i d was located i n the aromatic r i n g , as i t was i n the precursor, secodine [115]. To furth e r v e r i f y the a u t h e n t i c i t y of the incorporation i n t o v i n d o l i n e [3], the a l k a l o i d i s o l a t e d i n experiment 3 was reduced with l i t h i u m aluminum hydride. The product was p u r i f i e d by preparative t h i n l a y e r chromato-graphy and c r y s t a l l i z e d to constant a c t i v i t y from a d i e t h y l ether-petroleum ether s o l u t i o n . I t s p h y s i c a l and s p e c t r a l c h a r a c t e r i z a t i o n showed i t to be the t r i o l , v i n d o l i n o l [147] according to previously published d a t a . 4 ^ a ' 1 1 5 I t s r a d i o a c t i v i t y content corresponded to a [146] [147] - 86 -0.02% incorporation of secodine [115] i n t o v i n d o l i n e [3]. Encouraged by these r e s u l t s , experiment 4, employing the doubly l a b e l l e d secodine [115], was c a r r i e d out. The tritium/carbon-14 r a t i o of the secodine [115] was 8.8. The v i n d o l i n e [3] obtained from the experiment ind i c a t e d an 0.04% in c o r p o r a t i o n , but more importantly i t s tritium/carbon-14 r a t i o was found to be 8.3. This i n d i c a t e d , f i r s t l y , that s i g n i f i c a n t exchange or l o s s of t r i t i u m i n the indole r i n g does not occur during biosynthesis, confirming a previous r e s u l t observed with doubly l a b e l l e d tryptophan*""^ and e s t a b l i s h i n g the v a l i d i t y of experiments 1, 2 and 3. I t should be noted here that although v i n d o l i n e [3] possesses a methoxyl substituent at the p o s i t i o n (numbering according to secodine [115]) and the secodine [115] does not, s i g n i f i c a n t l o s s of t r i t i u m due to the i n t r o d u c t i o n of t h i s methoxyl moiety i s not expected. E l e c t r o p h i l i c s u b s t i t u t i o n of indole systems su b s t i t u t e d at the 2 and 3 p o s i t i o n s leads predominantly to 5 and/or 6 f u n c t i o n a l i z e d indoles'!""'"7 Therefore, t r i t i a t i o n of N-[3 -(3-[2-carbomethoxymethylene]-indolyl)-ethy l ] - 3 ' - e t h y l - 3 ' - p i p e r i d e i n e [123] by the t r i f l u o r o a c e t i c a c i d method discussed above, would be expected to a f f o r d t h i s indole acetate d e r i v a t i v e t r i t i a t e d p r i m a r i l y at the corresponding C i n and C... p o s i t i o n s . 4 H - 87 -During enzymatic hydroxylation of aromatic compounds, i t has been found that the in t r o d u c t i o n of the hydroxyl i s accompanied by a s h i f t of the 118 displaced proton to an adjacent p o s i t i o n . Thus Witkop and co-workers have found that the a c t i o n of tryptophan-5-hydroxylase on 5 - t r i t i o t r y p t o -phan r e s u l t s i n the retention of over 90% of the l a b e l to produce the product 5-hydroxy-4-tritiotryptophan, i . e . They have also shown that t h i s "NIH. s h i f t " i s a general phenomenon of enzymatically c o n t r o l l e d hydroxylations of aromatic systems. Since the methoxylation of the v i n d o l i n e progenitor presumably involves hydroxy-l a t i o n followed by methylation, l e s s than 10% of the t r i t i u m l a b e l i s expected to be l o s t . This i s i n f a c t the case, with a t o t a l d i f f e r e n c e of 5.7% i n the t r i t i u m l e v e l , based on the tritium/carbon-14 r a t i o found i n experiment 4. The second f a c t established by the re t e n t i o n of the tritium/carbon-14 r a t i o i n experiment 4 i s that the indole p o r t i o n of the secodine u n i t [115] i s not a l t e r e d to a s i g n i f i c a n t extent during i t s i n c o r p o r a t i o n i n t o the plant a l k a l o i d . A d d i t i o n a l information which establishes the correctness of t h i s statement, at l e a s t f o r v i n d o l i n e [3], comes from the degradation of t h i s a l k a l o i d as w i l l be discussed s h o r t l y . Although experiment 4 established the rather s p e c i f i c i n c o r p o r a t i o n of the indole p o r t i o n of secodine [115], thus suggesting that the e n t i r e - 88 -molecule was being u t i l i z e d by the plant system, unequivocal proof that the e t h y l p i p e r i d e i n e r i n g was also incorporated remained to be obtained. Although we f e l t i t to be extremely u n l i k e l y that the doubly l a b e l l e d secodine [115] molecule could be catabolized and b i o l o g i c a l l y recycled and yet r e t a i n the double isotope r a t i o i n the indole residue, we nonetheless wished to e s t a b l i s h beyond question the s p e c i f i c i n c o r p o r a t i o n of the e n t i r e secodine [115] molecule. To t h i s end, a secodine [115] molecule with r a d i o a c t i v e l a b e l i n the p i p e r i d e i n e moiety was required. The most r e a d i l y a v a i l a b l e i s o t o p i c d e r i v a t i v e 3 meeting t h i s requirement was [3,14,15,21- H.]-secodine [115], the synthesis of which was discussed p r e v i o u s l y . As evidenced by experiment 5 i n 14 Tables 6 and 7, when t h i s t r i t i a t e d compound was mixed with [ COOCH^]-secodine [115] and administered to V_. rosea p l a n t s , the tritium/carbon-14 r a t i o dropped from 3.5 to 1.4. This represents a 60% l o s s of the t r i t i u m l a b e l . A l o s s of such magnitude i s not unexpected, and y i e l d s several pieces of information. F i r s t l y , the incorporation of t r i t i u m l a b e l i n d i c a t e s that secodine [115] i s most probably being u t i l i z e d i n t a c t by the plant systems. Secondly, the los s of l a b e l must be r e l a t e d to the o v e r a l l a l t e r a t i o n s which must p r e v a i l i n the l a t e r stages of the b i o s y n t h e t i c pathway leading to the a l k a l o i d s . Obviously, more d e f i n i t e comments concerning these aspects must await fur t h e r experiments i n which, f i r s t of a l l , the l a b e l remains unaltered i n the l a t t e r stages of the b i o s y n t h e s i s , and then with secodine [115] d e r i v a t i v e s l a b e l l e d at s p e c i f i c carbon atoms of the p i p e r i d e i n e moiety. The former experiment would provide unequivocal proof of the intermediacy of secodine [115], should the double isotope r a t i o remain unaltered, as w e l l as f u r n i s h a basis f o r a mechanistic r a t i o n a l e , derivable from the r e s u l t s - 89 -of the l a t t e r experiments, f o r the bio-transformation of secodine [115] to the indo l e a l k a l o i d s . In order to e s t a b l i s h p o s i t i v e l y that a l a b e l l e d precursor i s being s p e c i f i c a l l y ( i . e . non-randomly) incorporated into a n a t u r a l product, a degradation of that natural product, designed to remove the l a b e l i s required. In our work, using l a b e l l e d secodine [115] with V_. rosea p l a n t s , t h i s was done i n two ways. The f i r s t method was the ac i d cata-lyzed removal of t r i t i u m from the v i n d o l i n e [3] i s o l a t e d i n experiment 2-This r e s u l t proved that the r a d i o a c t i v i t y of the a l k a l o i d was s p e c i f i c a l l y located i n the aromatic nucleus of the compound, as had the l a b e l i n the secodine [115] precursor. The second method involved the degradation 14 of v i n d o l i n e [3] and the i s o l a t i o n of the l a b e l . When [ COOC^]-secodine [115] i s incorporated i n t o v i n d o l i n e [3], the carbon-14 appears i n the carboxyl carbon of the a l k a l o i d . In order to i s o l a t e t h i s p a r t i c u l a r carbon atom, the vi n d o l i n e [3] from experiment 5, which had already been reduced to v i n d o l i n o l [147], was oxidized with p e r i o d i c a c i d . The oxidative cleavage of the v i c i n a l t r i o l released the primary a l c o h o l moiety as formaldehyde which was trapped as i t s b i s -dimedone d e r i v a t i v e [149]. This degradation sequence i s presented i n Figure 23, along with the s p e c i f i c a c t i v i t i e s f o r the various compounds. The expected and obtained r e t e n t i o n of a c t i v i t y i n the i s o l a t e d formaldehyde d e r i v a t i v e [149] proves that the ester function of secodine [115] becomes t h i s f u n c t i o n a l i t y i n vi n d o l i n e [3]. Thus, the statement made e a r l i e r , concerning the non-catabolysis of the indole p o r t i o n of secodine [115] i n going to v i n d o l i n e [3], i s borne out and the tritium/carbon-14 r a t i o obtained i n experiment 4 i s also - 90 -[149] 4.60 x 10 4 dpm/mM Figure 23: The Degradation of Vindoline from the Incorporation of [3,14,15,21- 3H, 1 4COOCH 3]-Secodine. - 91 -shown to be a true i n d i c a t i o n of s p e c i f i c i n c o r p o r a t i o n . As w e l l , the r a t i o obtained i n experiment 5 i s a true r e f l e c t i o n of the b i o - t r a n s -formations involved with the l a t t e r stages of indole a l k a l o i d b i o s y n t h e s i s . The above experiments have demonstrated that the p l a n t , V_. rosea, can u t i l i z e the a c r y l i c ester, secodine [115], i n i t s biosynthesis of the Aspidosperma a l k a l o i d s . The a c t u a l b i o l o g i c a l intermediate i s probably very s i m i l a r i n s t r u c t u r e to secodine [115] and the p o s t u l a t e ^ that the c r i t i c a l intermediate i n the biosynthesis of indole a l k a l o i d s i s the dihydropyridinium [80] i s i n accord with our f i n d i n g s . To t h i s point, the i n c o r p o r a t i o n of secodine [115] i n t o the other two prominent a l k a l o i d s of V. rosea has not been mentioned. Ajmalicine [56] and catharanthine [8] are Corynanthe and Iboga a l k a l o i d s r e s p e c t i v e l y . As can be seen from Figure 10a and 10b, the c r i t i c a l intermediate [80] [ 5 6 ] - 92 -i s postulated to give r i s e to the Aspidosperma and Iboga bases. Thus, ajmalicine [56] would not be expected to reveal s i g n i f i c a n t i n corporation i n the above experiments. As Tables 6 and 7 i n d i c a t e , t h i s i s indeed the case. The f i g u r e s i n Table 7 i n d i c a t i n g the s p e c i f i c a c t i v i t y of t h i s a l k a l o i d represent r a d i o a c t i v i t y l e v e l s which are only s l i g h t l y above background. However, the Iboga a l k a l o i d catharanthine [8] would be expected to incorporate the secodine molecule [115] i n view of the proposed biosyn-t h e t i c pathway (Figure 10b). As Tables 6 and 7 show, t h i s i s not the case. Although the s p e c i f i c r a d i o a c t i v i t y of the catharanthine [8] i s o l a t e d i n the various experiments i s c o n s i s t e n t l y higher than that of ajmalicine [56], i t i s also c o n s i s t e n t l y lower than that of v i n d o l i n e [3]. The reason f o r t h i s i s unknown, but postulates can be advanced to explain t h i s anomaly. For instance, since the feeding technique employed undoubtedly achieves i t s purpose of ge t t i n g the precursor to the s i t e of a l k a l o i d synthesis, perhaps secodine [115] i s incorporated i n t o the a l k a l o i d b i o s y n t h e t i c pathway at a very r a p i d r a t e . Then a f t e r the usual nine day feeding period, the normal turnover of a l k a l o i d s i n the plant could cause the only a l k a l o i d s r e t a i n i n g appreciable l a b e l to be those which take longer to be biosynthesized, e.g. the h i g h l y f u n c t i o n a l i z e d v i n d o l i n e [3]. Scott showed that catharanthine [8] i s produced one hundred hours a f t e r the germination of V. rosea seeds. Vindoline [ 3 ] , on the other hand, appears only two hundred hours a f t e r 12 germination. Thus, the turnover of a l k a l o i d s i n the p l a n t , combined with a rapid i ncorporation of secodine [115] into the pathway could then 87 a f f o r d the r e s u l t s obtained. The r e s u l t s of Goeggel i l l u s t r a t e t h i s point. In Table 1, Section A, our wick feeding technique i n v o l v i n g a - 93 -14 nine day period r e s u l t e d i n a 0.0032% incorporation of glycine-2- C int o v i n d o l i n e [3]. Goeggel and co-workers, using a hydroponic method with cut stems of V_. rosea and a two day feeding period achieved a 3.4% i n c o r p o r a t i o n of g l y c i n e - 2 - 1 4 C i n t o v i n d o l i n e [ 3 ] . * ^ A second explanation of the lower incorporation of secodine [115] in t o catharanthine [8] may l i e at the enzymatic l e v e l . Because of the diff e r e n c e s i n structure of the Aspidosperma and Iboga bases, two enzyme s i t e s probably e x i s t , which catalyze the conversion of the common intermediate to each of these systems. I f then secodine [115] c l o s e l y resembles t h i s common intermediate and can i n turn be adsorbed on the enzyme surface i n i t s place, the lower incorporation of t h i s synthetic precursor i n t o catharanthine [8] can be r a t i o n a l i z e d . For i f the Iboga producing s i t e cannot complex as w e l l i n i t i a l l y with secodine [115] as can the Aspidosperma s i t e , then there would be a lower conversion of secodine [115] to Iboga compounds. The evaluation of both of the above explanations, must await the i s o l a t i o n and examination of the enzyme systems responsible f o r t h i s b i o s y n t h e t i c pathway. Several other r a t i o n a l i z a t i o n s could perhaps also be advanced to explain t h i s low incorporation i n t o catharanthine [8]. However the explanation that the incorporation of secodine [115] i n t o v i n d o l i n e [3] i s only a f o r t u i t o u s and not a general occurrence must be discounted, i n view of the s p e c i f i c nature of the incorporation i n t o that a l k a l o i d and i n view of the r e s u l t s obtained with the a l k a l o i d s of V_. minor and A. pyricollum as discussed i n the following sections. - 94 -Section C The evergreen ground creeper V_. minor contains an i n t e r e s t i n g array of a l k a l o i d s . The s t r u c t u r a l e l u c i d a t i o n of these compounds, 119 120 c a r r i e d out p r i n c i p a l l y i n the laboratory of I. Kompis, ' has revealed that an overwhelming majority of them can be c l a s s i f i e d as ei t h e r Aspidosperma or Hunteria a l k a l o i d s . The former i s one of the 119 major indole a l k a l o i d f a m i l i e s , and about h a l f of the V. minor bases belong to t h i s s t r u c t u r a l grouping. The most abundant of these i s minovine [112]. The Hunteria family i s characterized by a pe n t a c y c l i c skeleton, i n which both nitrogen atoms are common to d i f f e r e n t p a i r s of r i n g s . The major Hunteria constituent of V. minor i s vincamine [102], but eburnamonine [6], eburnamine [150], and eburnamenine [151] as w e l l as oxygenated d e r i v a t i v e s of these (e.g. v i n c i n i n e [152]) are also indigenous to y_. minor and i n t o t a l account f o r almost as many 119 a l k a l o i d s i n t h i s p l a n t , as does the Aspidosperma family. Concerning the biogenesis of these Hunteria bases, no information i s presently a v a i l a b l e , and only one postulate has been put f o r t h which attempts to explain t h e i r o r i g i n . This hypothesis put f o r t h by Wenkert 69 and Wickberg i n a 1965 p u b l i c a t i o n c a l l e d f o r the d e r i v a t i o n of the [112] 3 C 0 0 C H 3 - 95 -[152] vincamine [102] structure from an Aspidosperma progenitor, resembling hydrated vincadifformine, the a l c o h o l [153]. The mechanism, presented i n Figure 24, would then lead to vincamine [102] which through a decarboxy-l a t i o n , a reduction and a dehydration would y i e l d eburnamonine [6], eburnamine [150], and eburnamenine [151] r e s p e c t i v e l y . The r e a l i t y of such a sequence would, as presented i n the Introduction, l i n k these a l k a l o i d s with the Corynanthe and Strychnos groups and would implicate the a c r y l i c ester intermediate [80] i n the biosynthesis of these p e n t a c y c l i c compounds. In order to examine t h i s p ostulate, Dr. V.R. Nelson, then of our research group, administered l a b e l l e d a l k a l o i d s of the Corynanthe ( i . e . 3 3 [ar- H]-geissoschizine [55]), Strychnos ( i . e . [ar- H]-stemmadenine [74])., 3 and Aspidosperma ( i . e . [ar- H]-tabersonine [75]) f a m i l i e s to _V. minor Figure 24 : The Wenkert Postulate for the Biogenesis of the Hunteria A l k a l o i d s . c u t t i n g s . The r e s u l t s of these feedings are summed-up i n Table 8. Table 8: The Results of Incorporation of Various Intermediates i n t o V. minor - I Exp. No. Compound Fed Feeding A c t i v i t y Time Fed (hr) (dpm) % Incorporation Vincamine Minovine [102] [112] 1 2 3 3 7 [ar- H] geissoschizine[55] 24 3.9 x 10 [ar- 3H] stemmadenine[74] 24 4.2 x 10 7 [ar- H] tabersonine[75] 24 1.6 x 10 0.005 <0.001 0.076 0.001 0.070 0.002 These r e s u l t s suggested that the eburnamine-vincamine a l k a l o i d s may be derived from the Corynanthe family v i a the Strychnos and Aspidosperma bases, as o u t l i n e d i n Figure 24-To f u r t h e r explore the proposed biosynthesis, feedings i n v o l v i n g the s y n t h e t i c a l l y obtained precursors, 16,17-dihydrosecodin-17-ol [114] and secodine [115] were i n i t i a t e d . The r e s u l t s of these experiments are tabulated i n Tables 9 and 10. A hydroponic feeding technique was u t i l i z e d , and i n most of the feedings, the plant cuttings absorbed an aqueous s o l u t i o n of the 3 acetate s a l t of the precursor. In experiment 11, [ar- H] secodine [115] was emu l s i f i e d i n an aqueous medium containing Tween 20, and thus administered to the p l a n t s , i n order to determine i f such a change of procedure could increase the incorporation of the precursor. As i s evident from Table 10, the reverse occurred and the incorporation of - 98 -Table 9: The Results of Incorporation of Various Intermediates i n t o V. minor - II Exp. No. Compound Fed Feeding A c t i v i t y Time Fed (hr) (dpm) % Incorporation Vincamine [102] Minovine [112] 4 3 [ar- H]-tryptophan[11] 24 7.41 X i o 7 0.089 0.080 5 [ar- 3H]-16,17-dihydro-secodin-17-ol [114] 24 1.86 X IO 7 £0.001 0.000 6 [ar- 3H]-16,17-dihydro-secodin-17-ol 96 2.40 X i o 7 £0.002 £0.001 7 [ar- 3H]-16,17-dihydro-secodin-17-ol 96 3.45 X i o 7 . <0.001 <0.001 8 [ar- 3H]-secodine[115] 24 3.40 X i o 8 <0.001 <0.001 9 3 [ar- H]-secodine 96 2.65 X i o 8 0.001 0.001 10 3 [ar- H]-secodine 96 2.10 X i o 8 •<0.001 £0.001 11 3 [ a r - H]-secodine 96 2.52 X i o 8 <0.001 <0.001 Table 10: The S p e c i f i c A c t i v i t i e s Associated with the Experiments i n Table 9. Exp. No. Feeding Method S p e c i f i c A c t i v i t y Fed (dpm/mM) S p e c i f i c A c t i v i t y Isolated (dpm/mM) Vincamine Minovine [102] [112] 4 Hydroponic i n a c e t i c 1.55 x 10' aci d 5 Hydroponic as acetate 7.33 x 10 6 Hydroponic as acetate 8.90 x 10 7 Hydroponic as acetate 8.90 x 10 8 Hydroponic as acetate 2.83 x 10 9 Hydroponic as acetate 2.83 x 10 10 Hydroponic as acetate 2.83 x 10 11 Hydroponic-Tween 20 2.83 x 10 8 8 8 10 10 10 10 2.33 x 10 2.12 x 10 4.34 x 10" 4.69 x 10" 4.67 x 10* 9.49 x 10 1 9.98 x 10* 5.31 x 10* 4.97 x 10 0.00 4.18 x 10" 3.55 x 10 J 2.76 x 10" 2.03 x 10 f 1.84 x 10 ! 2.00 x 10 ! - 99 -[ar- H]-secodine [115] dropped, r e l a t i v e to that revealed i n experiments 9 and 10. A further attempt to increase the incorporation was the exten-s i o n of the feeding period from twenty-four to n i n e t y - s i x hours. This r e s u l t e d i n doubling the incorporation i n t o vincamine [102], but decreased s l i g h t l y the incorporation of precursor i n t o minovine [112] ( i . e . compare experiments 8 vs. 9 and 10). As to the r e s u l t s of the experiments themselves, tryptophan [11] was r e a d i l y incorporated i n t o both a l k a l o i d s as a n t i c i p a t e d . Experiments 5, 6 and 7 revealed that 16,17-dihydrosecodin-17-ol [114] was incorporated to a very minor extent, i f at a l l . The a c t u a l r a d i o a c t i v i t y of the samples i n these experiments r e g i s t e r e d only a few counts above background and i n the case of minovine [112] i n experiment 5, i t a c t u a l l y r e g i s t e r e d only background. This lack of s i g n i f i c a n t r a d i o a c t i v i t y combined with the abnormal d e t e r i o r a t i o n of the plants during the feeding periods, and the r e s u l t s obtained i n our feedings of V. rosea (see Section B) i n d i c a t e d to us that 16,17-dihydrosecodin-17-ol [114] was most probably not being incorporated by the plant, and, i n f a c t , could not be a s s i m i l a t e d by the plant system. This l a t t e r point was also borne out by the large recovery of r a d i o a c t i v i t y i n the a l k a l o i d a l entract of V_. minor. In the three experiments, 56%, 86% and 73% of the a c t i v i t y fed was recovered i n t h i s e x t r a c t . Experiments 8, 9, 10, and 11 i n d i c a t e a low but d e f i n i t e incorpora-t i o n of secodine [115] i n t o the a l k a l o i d s of V_. minor. In a d d i t i o n , very l i t t l e plant d e t e r i o r a t i o n was noted and although t h i s may be due to the smaller amounts of secodine [115] u t i l i z e d per feedings, r e l a t i v e to 16,17-dihydrosecodin-17-ol [114], there was also a marked reduction of r a d i o a c t i v i t y recovered i n the a l k a l o i d a l extract; that i s 39%, 22%, - 100 -30%, and 32% for experiments 8, 9, 10, and 11 r e s p e c t i v e l y . This i n d i c a t e s that the plant system was able to metabolize or use t h i s precursor. In ad d i t i o n , the two 96 hour feedings, experiments 9 and 10, afforded a l k a l o i d s of very s i m i l a r a c t i v i t y , i n d i c a t i n g that the secodine [115] in c o r p o r a t i o n was reproducible. I t i s also i n t e r e s t i n g to note that a pattern consistent with the Wenkert p r o p o s a l ^ 9 might be v i s u a l i z e d from the r e s u l t s of the l a t t e r four feedings. That i s , i n the twenty-four hour feeding, experiment 8, the Aspidosperma a l k a l o i d e x h i b i t s almost s i x times the a c t i v i t y of the Hunteria a l k a l o i d . In the feeding employing Tween 20, experiment 11, i n which the secodine [115] appeared not to be as r e a d i l y a v a i l a b l e to the plant system as i n i t s acetate form, t h i s r a t i o was reduced to about four; while i n the n i n e t y - s i x hour feedings, experiments 9 and 10, the Aspidosperma base i s only about twice as ac t i v e as the Hunteria base. This r e s u l t would suggest that the Aspidosperma compounds are biosynthesized i n i t i a l l y and act as precursors f o r the Hunteria a l k a l o i d s thereby 69 supporting the proposal of Wenkert. C l e a r l y a more d e f i n i t i v e conclusion must await further experiments. With the above information on hand we sought a d d i t i o n a l evidence f o r the incorporation of secodine [115] i n t o the a l k a l o i d s of V. minor. As i n the case of V_. rosea, t h i s evidence was obtained by employing the 3 14 doubly l a b e l l e d [ar- H, COOCH^-secodine [115]. Table 11 demonstrates that i n the case of vincamine [102] and probably also i n minovine [112], the doubly l a b e l l e d secodine [115] i s incorporated without a l t e r a t i o n of the major p o r t i o n of the compound. With respect to the minovine [112] sample, i t should be noted that e f f i c i e n t r a d i o a c t i v i t y a nalysis - 101 3 14 Table 11: The Results of Incorporating [ar- H, C00CH 3]-secodine i n t o V. minor. Exp. A c t i v i t y Fed (dpm) Ratio A l k a l o i d % Incorporation Ratio No. 3 U 14 n 3 U /14_ Isolated 3„ 14„ Isolated Vincamine 0.001 0.001 8.6 o 7 [102] 12 1.51 x 10 1.79 x 10 8.4 Minovine 0.001 0.0008 10.1 [112] of the a l k a l o i d was hampered by a.small recovery of the a l k a l o i d from the plant coupled with the already low incorporation of the precursor. Obviously, t h i s double l a b e l experiment must be repeated to insure the accuracy of the r e s u l t s . However, i n view of the r e s u l t s obtained here as w e l l as those obtained with _V. rosea, there appears l i t t l e doubt that secodine [115] can be u t i l i z e d by the plant system, and i s incorporated s p e c i f i c a l l y i n t o the a l k a l o i d s of V_. minor. A d d i t i o n a l experiments which w i l l hopefully shed more l i g h t i n t h i s d i r e c t i o n are now underway. The reasons f o r the low incorporation of secodine [115] by t h i s plant are more d i f f i c u l t to explain. Perhaps the c e l l walls are impermeable to i t s passage, or, there may be d i f f i c u l t y w i t h i n the plant system i n transporting the compound to the s i t e of synthesis. A d d i t i o n a l experimentation, varying the feeding techniques, may a f f o r d higher incorporations; however, for the present, there i s s u f f i c i e n t evidence to support the p o s i t i v e i ncorporation of secodine [115] in t o V_. minor. In summary, these r e s u l t s provide preliminary support f o r the 69 proposal of Wenkert (Figure 24) as to the o r i g i n of the Hunteria a l k a l o i d s , and they also supply a d d i t i o n a l support f o r the correctness - 102 -of the postulate that the a c r y l i c ester [80] or some close r e l a t i v e plays a c e n t r a l r o l e i n the l a t e r stages of the biosynthesis of these indole a l k a l o i d s . [80] - 103 -Section D The a l k a l o i d s of A. pyricollum represent bases whose s t r u c t u r a l type i s unique among the family of indole a l k a l o i d s . Compounds such as u l e i n e [145] and apparicine [144], lack the normal g-aminoethyl side chain, j o i n i n g the g-position of the indole nucleus to the t e r t i a r y nitrogen atom. I t can be seen, however, that they do possess the ubiquitous C y ^ u n i t , rearranged as i n the manner of the Strychnos family, with the a d d i t i o n a l l o s s of a C^ u n i t (e.g. compare stemmadenine [74]). The question of the biosynthesis of these [74] bases r e l a t i v e to the a l k a l o i d s containing the complete tryptamine moiety has been dealt with by several authors, but only two communications (from our laboratory) are present i n the l i t e r a t u r e which re v e a l any relevant experimental b i o s y n t h e t i c data. The a l k a l o i d , uleine 1145'],. was i s o l a t e d from A. u l e i by - 104 -121 Schmutz, Hunziker and H i r t i n 1957, and i t s structure was e l u c i -122 dated by Buchi and Warnhoff i n 1959. I t s presence i n pyricollum 123 was demonstrated i n 1960. Apparicine [144] was i s o l a t e d from 12 A 125 A. p y r i c o l l u m i n 1965 and i t s structure elucidated i n the same year. The f i r s t postulate put f o r t h to account f o r the o r i g i n of these 22 compounds was that of Wenkert (Figure 25). Uleine [145] was envisioned as a r i s i n g not from tryptophan [11] but from a precursor of the amino ac i d , g l y c o s y l i d e n e a n t h r a n i l i c acid [154]. This intermediate a f t e r a-oxidation and condensation with the SPF unit [26] could give r i s e to the complex 1155] which through the conventional manner would be meta-b o l i z e d to the a,g-disubstituted indole [156]. I f t h i s compound then undergoes c y c l i z a t i o n with an appropriate nitrogenous source the r e s u l t a n t h e t e r o c y c l i c product [157] could react i n the manner shown to extrude the g-glycosyl group, forming the uleine [145] skeleton. 125 D j e r a s s i , G i l b e r t and coworkers adopted t h i s postulate and adapted i t to explain the biosynthesis of apparicine [144]. As presented i n Figure 26, the d i s u b s t i t u t e d indole [157] a f t e r a p r o t o t r o p i c s h i f t , followed by the appropriate c y c l i z a t i o n could also give r i s e to the apparicine [144] skeleton. This l a t t e r postulate, however, was shown to be i n c o r r e c t by means 126 127 of the b i o s y n t h e t i c experiments of Kutney, Nelson, and W i g f i e l d . ' Tryptophan [11] and stemmadenine [74] were shown to be incorporated i n t o apparaicine [144] r e l a t i v e l y e f f i c i e n t l y while vallesamine [158] and 3-aminomethylindole [159] were shown to incorporate poorly or not 3 at a l l . In a d d i t i o n , by feeding [ar- H] tryptophan [11] along with the - 105 -[145] Figure 25: The Wenkert Postulate for the Biogenesis of Ul - 106 -[144] Figure 26: The D j e r a s s i - G i l b e r t Postulate f o r the Biogenesis of Apparaicine. [158] [159] and carbon-14 l a b e l l e d forms of the amino a c i d , i t was demonstrated that only the of tryptophan [11] i s retained i n apparicine [144], presumably as the methylene bridge between the indole nucleus and the t e r t i a r y nitrogen atom. The r e l a t i v e l y e f f i c i e n t i n corporation of - 107 -steramadenine [74] (0.55%) coupled with the lower i n c o r p o r a t i o n of vallesamine [158] (0.01%) and 3-aminomethylindole [159] (<0.001%) prompted these authors to postulate that: 1) stemmadenine [74] i s of c r u c i a l importance i n the biosynthesis of apparaicine [144]; 2) fragmentation of the aminoethyl side chain and loss of therefore, occurs as one of the f i n a l stages i n the b i o s y n t h e s i s ; and 3) e i t h e r l o s s of and decarboxylation of the ester function occur simultaneously or decarboxylation precedes l o s s of C^. 12< A communication pertinent to these observations appeared i n 1970. A French research group, i n v e s t i g a t i n g the fragmentation patterns of N-oxides i n t r i f l u o r o a c e t i c a n h y d r i d e / t r i f l u o r o a c e t i c a c i d , demonstrated that NjN-dimethyltryptamine oxide [160] y i e l d s [161] and [162] as the i n i t i a l fragmentation products. They extended t h i s observation to the [160] H CH 3 [161] [162] - 108 -indole a l k a l o i d f i e l d and postulated that a simultaneous los s of and decarboxylation of stemmadenine [74] could give r i s e to the apparicine [144] structure (Figure 27), i f a leaving group equivalent to the trifluoroacetoammonium ion i n t h e i r tryptamine oxide sequence could be invoked (represented by -X i n compound [163] of Figure 27). 00C CH20H [163] H [144] Figure 27: The Postulated Conversion of Stemmadenine to Apparicine. - 109 -In view of the r e s u l t s obtained with other plant substrates (Sections B and C), i t was of i n t e r e s t to us to extend the b i o l o g i c a l evaluation of our synthetic compounds [ I l k ] and [115] to A. pyricollum and the unique a l k a l o i d s contained therein. A hydroponic feeding technique to root cuttings was used to administer the l a b e l l e d compounds. This method was adopted i n view of the presence of apparicine [144] and u l e i n e 1145] i n the roots, and because of the slow growth rate of t h i s t r o p i c a l plant species making the feeding of an e n t i r e plant or the a e r i a l portions thereof c o s t l y i n terms of plants as w e l l as the time required to grow s u i t a b l e specimens. The l a b e l l e d compounds were fed as t h e i r acetate s a l t s , and l a b e l l e d secodine [115] was prepared j u s t p r i o r to feeding as i n the manner described i n Section B. The r e s u l t s of these feedings are presented i n Tables 12 and 13. They reve a l that the incorporation studies with apparicine [144] followed the same pattern set with the a l k a l o i d s of V. rosea and V_. minor. 16,17-Dihydrosecodin-17-ol [114] showed a very low incorporation while secodine [115] was incorporated i n t o apparicine [144] i n each of the feedings to the extent of about 0.01%. Uleine [145] demonstrated a much 3 lower in c o r p o r a t i o n (0.003%) of [ar- H] secodine [115] i n experiment 2, but t h i s i s probably i n d i c a t i v e of a much lower concentration of t h i s a l k a l o i d i n the plant roots rather than low r a d i o a c t i v i t y i n the compound. This i s borne out by the s p e c i f i c a c t i v i t y of uleine [145] which, as i s evident i n Table 12, experiment 2, i s a c t u a l l y higher than that of apparicine [144]. The explanation f o r t h i s may l i e i n the r e l a t i v e rates of biosynthesis of apparicine [144] and uleine [145]. If the l a t t e r i s biosynthesized at a slower rate than the former and i s retained i n the plant at a lower concentration, then such r e s u l t s could Table 12: The Results of the Incorporation of 16,17-Dihydrosecodin-17-ol and Secodine i n t o A. pyricollum Experiment Number Compound Fed A c t i v i t y Fed (dpm) H 14, Feeding Time (days) % Incorporation Apparicine[144] Uleine[145] H 14, H 14. 2 3 4 5 [ar- H]-16,17-dihydro-secodin-17-ol [114] 3 [ar- H] secodine [115] [ 1 4COOCH 3] secodine [ar- 3H, 1 4COOCH 3] secodine [3,14,15,21- 3H, 1 4COOCH 3J-secodine 9.20x10 2.57x10 8 1.11x10 4.42x10 8 7.06x10 1.28x10 1.07x10 5 5 5 5 <0.001 0.01 0.014 0.005 0.01 0.015 0.009 <0.001 0.003 o i Table 13: The S p e c i f i c A c t i v i t i e s Associated with the Experiments i n Table 12 Experiment S p e c i f i c A c t i v i t y Fed Ratio S p e c i f i c A c t i v i t y I s o l a t e d Ratio Number (dpm/mM) 3T1 ,140 . . . n / / i ni • n / n ^ . . l ^ ^ H/ C Apparicine [144] . Uleine [145] H/ C 3H 1 4 C 3H 1 4 C 3H 1 4 C A p p a r i c i [144] 1 1.82xl0 1 0 - - 1.43xl0 4 - 4.98xl0 4 2 1.82xl0 1 0 - - 7.68xl0 5 - l . l O x l O 6 3 - 1.19xl0 9 - - 1.06xl0 4 - -4 l . l O x l O 1 0 1.28xl0 9 8.7 1.62xl0 5 1.94xl0 4 - - 8.4 5 9.82xl0 9 1.27xl0 9 4.2 3.03xl0 4 1.35xl0 4 - - 2.2 - 112 -be expected. Regardless of the reason, our furth e r i n v e s t i g a t i o n s centered about apparicine [144]. Experiments 3, 4, and 5 a l l i n d i c a t e d a s i m i l a r incorporation (~0.01%) of carbon-14 i n t o apparicine [144] from the appropriate carbomethoxy l a b e l l e d form of secodine [115]. In ad d i t i o n , i t was shown, by means of experiment 4, i n which a doubly l a b e l l e d pre-cursor was employed, that the tritium/carbon-14 r a t i o of the precursor i s retained i n the a l k a l o i d ( i . e . 8.7 vs 8.4). This implies that the carbomethoxy ester group of secodine [115] becomes the e x o c y c l i c methylene moiety of apparicine [144]. The pi p e r i d e i n e l a b e l l e d form of the 3 precursor, [3,14,15,21- H]-secodine [115], when fed i n conjunction with r e l a t i v e to the carbon-14 l e v e l . As with the 60% loss of t r i t i u m encountered on incorporating t h i s double l a b e l l e d form i n t o v i n d o l i n e 13] (Section B, experiment 5), t h i s r e s u l t would i n d i c a t e that the precursor i s being u t i l i z e d i n t a c t by the plant system and that the lo s s of l a b e l i s re l a t e d to the o v e r a l l a l t e r a t i o n s which p r e v a i l i n the [115] (experiment 5) exhibited a 48% loss of t r i t i u m OOCCH 3 [115] [3] [144] - 113 -l a t t e r stages of the b i o s y n t h e t i c pathway leading to apparicine [144]. The d i f f e r e n c e of l a b e l r e t e n t i o n between apparicine [144] and v i n d o l i n e [3] i s not unexpected when the d i f f e r e n c e i n f u n c t i o n a l i t y of t h i s h e t e r o c y c l i c r i n g i n the two a l k a l o i d s i s noted. As before, more d e f i n i t e comments concerning the l a t t e r transformations of the precursor -must await fur t h e r experimentation. In order to demonstrate that secodine [115] was incorporated s p e c i f i c a l l y i n t o apparicine 1144], the a l k a l o i d sample obtained from experiment 3 was degraded i n such a manner as to i s o l a t e only the 129 e x o c y c l i c methylene group. This involved c a r e f u l ozonolysis of the a l k a l o i d , a reductive cleavage of the ozonide, and the trapping of the r e s u l t a n t formaldehyde and acetaldehyde as t h e i r dimedone d e r i v a t i v e s J149] and 1164], The acetaldehyde-dimedone d e r i v a t i v e [164] was then c y c l i z e d to i t s b i s - e n o l ether [165] by r e f l u x i n g the mixture i n an a c e t i c a c i d s o l u t i o n . Formaldehyde bis-dimedone 1149] i s s t a b l e under these conditions, and the above c y c l i z a t i o n thus enables the separation o£ these two d e r i v a t i v e s to be made by s i l i c a g e l preparative t h i n layer 130 131 chromatography and/or by chemical means. ' Figure 28 o u t l i n e s t h i s degradation scheme and presents as w e l l the r a d i o a c t i v i t y of the pertinent compounds. As can be seen the v i n y l methylene carbon atom contains a l l of the r a d i o a c t i v i t y present i n the a l k a l o i d . Thus, i t s d e r i v a t i o n must be from the carbomethoxy carbon atom of secodine [115] providing reasonable support for the s p e c i f i c incorporation of t h i s synthetic precursor i n t o the a l k a l o i d . Furthermore, by e s t a b l i s h i n g the non-random incorporation of the carbon-14 l a b e l , the r e s u l t of the degradation also confirms the v a l i d i t y of experiment 4 i n which - 114 -[165] Figure 28: The Degradation of Apparicine. - 115 -0 1/ *V T / [ar- H, COOCH^] secodine [115] was u t i l i z e d . The R/ C r a t i o therein obtained f o r apparicine [144] can thus be seen to be a true i n d i c a t i o n of the s p e c i f i c i ncorporation of the indole moiety of the secodine [115] in t o the a l k a l o i d , and an i n d i c a t i o n , as w e l l , that s i g n i f i c a n t exchange or l o s s of t r i t i u m i n the indole r i n g does not occur during biosynthesis. 3 14 Analogously, the r e s u l t s of experiment 5, i n which 13,14,15,21- H, COOCH^] secodine 1115] was administered, can be seen to be a probable i n d i c a t i o n that the e n t i r e secodine [115] molecule i s incorporated i n t o the 3 1A a l k a l o i d b i o s y n t h e t i c pathway and that the H/ C r a t i o obtained f o r apparicine I144J i s a r e f l e c t i o n of the a l t e r a t i o n s inherent i n the l a t e r stages of that pathway leading to that a l k a l o i d . As i n the case of v i n d o l i n e [3] from s i m i l a r feedings to _V. rosea (Section B, experi-ments 4 and 5) unequivocal confirmation of the s p e c i f i c i n c o r p o r a t i o n of the e n t i r e secodine [115] molecule into apparicine [144] must await experimentation i n v o l v i n g a doubly l a b e l l e d secodine d e r i v a t i v e [115] i n which the r a d i o a c t i v e isotopes are i n separate portions of the molecule 3 14 and f o r which the H/ C r a t i o of the precursor i s retained i n t a c t i n the a l k a l o i d . The data obtained with A. pyricdllum a l k a l o i d s thus i n d i c a t e s that secodine [115] can be u t i l i z e d by that plant i n a s p e c i f i c manner to produce apparicine [144] and po s s i b l y uleine 1145] (experiment 4). This r e s u l t again supports the postulate 7 7 that a compound such as the dihydropyridinium-acrylic ester system [80] or some c l o s e l y r e l a t e d analogue plays a c e n t r a l r o l e i n the biosynthesis of indole a l k a l o i d s . 125 The data also provides evidence against the D j e r a s s i - G i l b e r t postulate (Figure 26) for the biogenesis of apparicine [144]. In t h i s respect - 1 1 6 -C H 3 O O C [ 8 0 ] i t i s i n agreement w i t h the f i n d i n g s of Kutney, Nelson, and W i g f i e l d . ' I t can, t h e r e f o r e , be s t a t e d that a p p a r i c i n e [144] a r i s e s from a tryptophan r e s i d u e and a terpenoid C ^ Q u n i t , as do the m a j o r i t y of i n d o l e a l k a l o i d s , and that the b i o - t r a n s f o r m a t i o n s which impart the unique s t r u c t u r a l f e a t u r e s to t h i s a l k a l o i d take p l a c e i n the l a t e r stages of i t s b i o s y n t h e s i s . However, the f i n d i n g s presented above modify 127 the Kutney, Nelson, and W i g f i e l d proposal i n t h a t i f stemmadenine [74] i s regarded as a precursor of a p p a r i c i n e [144] , the e x t r u s i o n of a methylene group from the tryptophan r e s i d u e i s accompanied by l o s s of the hydroxymethylene moiety; not by d e c a r b o x y l a t i o n of the e s t e r f u n c t i o n . Whether these processes occur simultaneously or independently of each other remains to be determined. S i m i l a r l y the conversion of stemmadenine 1 2 8 174] to a p p a r i c i n e 1144] as proposed by P o t i e r and coworkers CFigure 27) must be m o d i f i e d to account f o r the conversion of a carbomethoxy [144] [74] - 117 -ester moiety to a v i n y l group. The r e s u l t s of experiment 2 i n which uleine [145] exhibits a higher s p e c i f i c a c t i v i t y than does apparicine [144] cast doubt on the 22 o r i g i n a l Wenkert postulate (Figure 25) f o r the o r i g i n of t h i s compound. This r e s u l t would i n d i c a t e that uleine [145] a r i s e s from the indole a l k a l o i d pathway i n a s i m i l a r manner to that of apparicine [144]. However, more d e f i n i t e comments i n t h i s d i r e c t i o n must await fur t h e r exp erimentation. In conclusion, i t has been found that secodine [115] i s incorporated i n t o the A. p y r i c o l l u m a l k a l o i d , apparicine [144], i n s i m i l a r fashion to i t s i n c o r p o r a t i o n into the V_. rosea a l k a l o i d , v i n d o l i n e [3], and i n agreement with the r e s u l t s of feedings to y_. minor. The s i n g l e most important f a c t a r i s i n g from t h i s r e s u l t i s that apparicine [144] i s derived not from a unique non-tryptophan route, but from tryptophan and a C ^ Q terpene unit as are the majority of indole a l k a l o i d s , and that the bio-transformations which impart to t h i s a l k a l o i d i t s unique s t r u c t u r a l features occur during the l a t e r stages of i t s biosynthesis. A l l of the r e s u l t s presented i n Sections B , C and D of t h i s t h e s i s strongly support the s u g g e s t i o n 7 7 that there e x i s t s an intermediate resembling secodine [115], be i t the compound [80] or some c l o s e l y r e l a t e d e n t i t y which plays a c e n t r a l r o l e i n the l a t e r stages of indole a l k a l o i d biosyntheses s i m i l a r to that played by squalene i n isoprenoid biosyntheses. Whether t h i s " a l k a l o i d a l squalene" i s as ubiquitous as i t s isoprene analogy awaits further experimentation. Such a l k a l o i d s , f or example, as o l i v a c i n e [166] and e l l i p t i c i n e [167] o f f e r the means to show such ub i q u i t y , thus demonstrating that the complex - 118 -[166] [167] S t r u c t u r a l features of indole a l k a l o i d s are i n r e a l i t y the products of two simple units and the rearrangements of those u n i t s . - 119 -EXPERIMENTAL Melting points 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) spectra were recorded i n methanol s o l u t i o n on a Cary 11 or Cary 15 recording spectrometer. The i n f r a r e d ( i r ) spectra were taken on a Perkin-Elmer Model 21 spectrometer u t i l i z i n g a potassium bromide d i s c . Nuclear magnetic resonance (nmr) spectra were recorded i n deuteriochloroform s o l u t i o n (unless otherwise indicated) at 100 megacycles per second on a Varian HA-100 or a Varian XL-100 instrument and at 60 megacycles per second on a Varian T-60 spectrometer. Chemical s h i f t s are given i n T i e r s T scale with reference to tetramethylsilane as the i n t e r n a l standard. The chemical s h i f t s f o r m u l t i p l e t s are reported with respect to the apparent center of these s i g n a l s . Mass spectra were recorded on an Atl a s CR-4 mass spectrometer and high r e s o l u t i o n mass spectra, to determine molecular formulae,were c a r r i e d out on an AEI-MS-9 instrument. 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 Gel G (acc. to Stahl) containing 1% by weight General E l e c t r i c Retma P - l , Type 188-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and preparative t h i n - l a y e r chromatography ( t i c ) , unless otherwise noted. Chromoplates were developed using the spray reagent carbon tetrachloride-antimony pentachloride (2:1), or iodine vapor. Woelm n e u t r a l alumina ( a c t i v i t y 111 - unless otherwise - 120 -indicated) was used f o r column chromatography. R a d i o a c t i v i t y was measured with a Nuclear-Chicago Mark 1 Model 6860 L i q u i d S c i n t i l l a t i o n counter i n counts per minute (cpm). The radioactivity of a sample i n d i s i n t e g r a t i o n s per minute (dpm) was subsequently c a l c u l a t e d using the counting e f f i c i e n c y which was 132 determined f o r each sample by the external standard technique u t i l i z i n g the b u i l t - i n barium-133 gamma source. The l i q u i d s c i n t i l l a t i o n s o l u t i o n used with the counter was made up of the following components: toluene (1 l i t r e ) , 2,5-diphenyloxazole (4 grams), and 1,4-bis[2-(5-phenyloxazoly)]benzene (0.05 gram). In p r a c t i c e , a sample was dissolved i n benzene (1 ml) or i n methanol i n the case of a s a l t , i n a counting v i a l . The volume was then made up to 15 ml with the above s c i n t i l l a t o r s o l u t i o n . For each sample counted, the background was determined f o r the counting v i a l to be used, by f i l l i n g the v i a l with the appropriate s c i n t i l l a t o r s o l u t i o n and counting (3 x 40 min. or 3 x 100 min.) to determine the background cpm. The v i a l was then emptied, r e f i l l e d with the sample to be counted and the s c i n t i l l a t o r s o l u t i o n and counted (3 x 40 min. or 3 x 100 min.). The di f f e r e n c e i n cpm between the background count and the sample count was used f o r subsequent c a l c u l a -t i o n s . In the case of doubly l a b e l l e d samples, the L i q u i d S c i n t i l l a t i o n counter was r e c a l i b r a t e d and set-up to operate following a procedure 133 adapted from Hendler. The r a d i o a c t i v i t y of l a b e l l e d compounds adsorbed on t i c pla t e s was measured using a Nuclear-Chicago Actigraph Scanner, Model 1036, connected to a Nuclear-Chicago Model 8416 pen recorder and a Nuclear-Chicago Model 8437 d i g i t a l p r i n t - o u t recorder. - 121 -The V_. rosea and A. pyricollum plants used i n t h i s study were obtained from the H o r t i c u l t u r a l Department greenhouse, The U n i v e r s i t y of B r i t i s h Columbia. V_. minor plants were obtained from the gardens 134 on the campus of The U n i v e r s i t y of B r i t i s h Columbia. Section A 89 The Administration of Labelled Compounds to V_. rosea The following procedure was u t i l i z e d to administer r a d i o a c t i v e compounds to V_. rosea plants. The procedure was used f or a l l V. rosea feedings discussed i n t h i s t h e s i s . The compound to be fed was dissol v e d i n ethanol (5-10 drops). To t h i s was added 0.1 N a c e t i c a c i d (5 drops) and d i s t i l l e d water (0.5 ml). (In the case of l a b e l l e d g l y c i n e , the commercial m a t e r i a l was obtained diss o l v e d i n 0.1 N hydrochloric a c i d (2.25 or 2.5 ml) and was fed as such.) This s o l u t i o n was administered to mature, 1-2 year o ld V_. rosea plants by the cotton wick method. This method required the threading of a cotton s t r i n g through the stem of the growing p l a n t s , at a point above the ground, but below the branching point. The intertwined ends of the wick were placed i n a small v i a l (capacity, 1 ml) located at the base of the plan t . This v i a l was f i l l e d with the precursor s o l u t i o n . When t h i s s o l u t i o n was absorbed i n t o the plant, the o r i g i n a l container of the l a b e l l e d compound was washed with 0.1 N a c e t i c a c i d (0.5 ml) and d i s t i l l e d water (2 ml), and t h i s l i q u i d was used to incorporate any remaining compound in t o the pla n t . Subsequent washings over the duration of the feeding u t i l i z e d d i s t i l l e d water. The plants were placed under fluorescent lamp i l l u m i n a t i o n and watered every second day. The feeding period f o r V_. rosea was nine days. - 122 -E x t r a c t i o n of A l k a l o i d s from \7. rosea The following procedure was employed to extract and p u r i f y the a l k a l o i d s of V_. rosea. This procedure was followed f o r a l l extractions of V. rosea discussed i n t h i s study. The V_. rosea plants (3 or 4 p l a n t s , approximately 50-200 gm f r e s h plant) were mascerated with methanol i n a Waring blender, f i l t e r e d and re-mascerated u n t i l the f i l t r a t e was c o l o r l e s s . The solvent was removed under reduced pressure, and the aqueous residue taken up i n 2 N hydrochloric a c i d (150 ml). This mixture was extracted with benzene (3 x 75 ml). The combined benzene extracts were washed with 2 N hydro-c h l o r i c acid (50 ml) and the combined aqueous extracts were cooled i n i c e , b a s i f i e d with 15 N ammonium hydroxide and extracted with chloroform (3 x 100 ml). The combined chloroform extracts were washed with water (100 ml), dri e d over sodium s u l f a t e and evaporated under reduced pressure to give a brown foam. The foam was d i s s o l v e d i n a small volume of methylene ch l o r i d e and chromatographed on alumina (25 gm). The column was eluted successively with petroleum ether 30/60°, benzene, chloroform, and methanol. Fractions of 25 ml i n volume were taken. The l a t t e r petroleum ether-benzene (1:1) f r a c t i o n s were combined and evaporated to dryness. The residue was then taken up i n methanol, over which anhydrous hydrogen c h l o r i d e was passed. Catharanthine [8], hydrochloride c r y s t a l l i z e d from t h i s s o l u t i o n on reducing the i n i t i a l volumn of solvent. The a l k a l o i d s a l t was r e c r y s t a l l i z e d from methanol-ether. The l a t t e r benzene f r a c t i o n s afforded ajmalicine [56]. If required, t h i s a l k a l o i d was fur t h e r p u r i f i e d by preparative t i c employing s i l i c a g e l as the - 123 -adsorbant and e t h y l acetate as the e l u t i n g solvent. The free base was then converted to i t s hydrochloride s a l t by adding one drop of concentrated hydrochloric acid to a methanol s o l u t i o n of the a l k a l o i d . The s a l t c r y s t a l l i z e d out on standing, and was r e c r y s t a l l i z e d from methanol. The a l k a l o i d , v i n d o l i n e [3], was eluted from the column with benzene-chloroform (1:1). This a l k a l o i d was handled as a free base. I t s furt h e r p u r i f i c a t i o n included a preparative t i c on s i l i c a g e l employing e t h y l acetate-methanol (99:1) as solvent, and subsequent c r y s t a l l i z a t i o n from ether. Feeding; Experiments 5, 6, 7j and 8. These experiments were c a r r i e d out as described above. In each case only ajmalicine [56] was i s o l a t e d , p u r i f i e d , and examined f o r r a d i o a c t i v i t y . The d e t a i l s of these feedings are l i s t e d i n Table 14. 32 Kuhn-Roth Oxidation of Ajmalicine [56] Radioactive ajmalicine hydrochloride (3.12 mg, 2.62 x 10 7 dpm/mM), from feeding experiment 6, was mixed with i n a c t i v e ajmalicine (52.01 mg). The s p e c i f i c a c t i v i t y of t h i s mixture was c a l c u l a t e d at 1.35 x 10^ dpm/mM. The mixture was oxidized using a s o l u t i o n (20 ml) of the follow-ing composition:chromium t r i o x i d e (20 gm), 98% s u l f u r i c a c i d (25 ml), and water (120 ml). A f t e r r e f l u x i n g t h i s s o l u t i o n f o r 2 hours, the v o l a t i l e r e a c t i o n products were steam d i s t i l l e d i n a micro-apparatus constructed f o r t h i s purpose. A 0.01 N sodium hydroxide s o l u t i o n was used to n e u t r a l i z e and s l i g h t l y b a s i f y (pH 7.9) the d i s t i l l a t e (150 ml) which was then evaporated under reduced pressure and dried under vacuum. Table 14: The Incorporation of Labelled Glycine i n t o V. rosea Experiment Compound Weight A c t i v i t y Wet . Ajmalicine T o t a l S p e c i f i c % Number Fed Fed Fed Plant [56] A c t i v i t y A c t i v i t y Incorporation Weight Isolated (mg) (dpm) (gm) (mg) (dpm) (dpm/mM) 5 9 1 G l y c i n e - 2 - 1 4 C 0.1 5.55 x 10 7 52.8 3.0 9.32 x 10 4 1.09 x 10 7 0.17% 6 Gl y c i n e - 2 - 1 4 C 0.9 4.99 x 10 8 103.7 16.6 2.41 x 10 6 2.62 x 10 7 0.48% i 7 Glycine-2- 1 4C 1.0 5.54 x 10 8 118.4 10.4 1.73 x 10 6 2.38 x 10 7 0.31% G 8 G l y c i n e - l - 1 4 C 0.7 5.00 x 10 8 147.4 4.4 4.06 x 10 3 9.47 x 10 4 0.0008% 1 - 125 -Sodium acetate (3.53 mg) was thus obtained. Radioinactive sodium acetate (4.67 mg). was added to the sodium acetate obtained from a c t i v e ajmalicine and t h i s was c r y s t a l l i z e d to constant a c t i v i t y from ethanol 3 (3.37 x 10 dpm/mM). This value a f t e r compensating for the unlabelled acetate added represented 0.58% of the a c t i v i t y of the ajmalicine. Ajmalicine obtained from feeding experiment 5 was s i m i l a r l y 91 oxidized. The mixture of l a b e l l e d and unlabelled a l k a l o i d had a s p e c i f i c a c t i v i t y of 7.84 x 10 dpm/mM. The acetate i s o l a t e d had a s p e c i f i c a c t i v i t y of 677 dpm/mM or 0.86% of the r a d i o a c t i v i t y of the ajmalicine oxidized. Harman 1 1 0 4 ] 9 2 * 1 3 5 Tryptophan [11] (0.6 gm) was dissolv e d i n water (150 ml), to which 98% s u l f u r i c a c i d (0.6 ml) and acetaldehyde (10 ml of a 20% aqueous solution) were added. A f t e r r e f l u x i n g f o r 10 minutes, the source of heat was removed and a 10% aqueous s o l u t i o n of potassium dichromate (30 ml) was slowly added while the r e a c t i o n mixture was s t i l l hot. A f t e r a 5 minute period, the s o l u t i o n was heated to b o i l i n g and then allowed to cool to room temperature. A f t e r being made basic with a concentrated sodium hydroxide s o l u t i o n , the mixture was extracted with e t h y l acetate (4 x 50 ml). The organic extract was dried over sodium s u l f a t e and evaporated to dryness, under vacuum, to y i e l d a brown c r y s t a l l i n e m a t e r i a l (0.20 gm). A f t e r c r y s t a l l i z i n g from benzene, the 136 harman was obtained as yellow prisms, mp 234-237°C. ( L i t . mp 238°C). The following s p e c t r a l evidence v e r i f i e d the a u t h e n t i c i t y of the mat e r i a l obtained by Dr. V.R. Nelson: X (log e ) : 214(4.28), 234(4.59), - 126 -239(4.57), 250(4.86), 281(4.00), 288(4.24), 335(3.67), 348(3.67) nm; JCRr _ 1 v : 3130(N-H), 1622 (C=N), 1565 (C=C) cm ; nmr: x 1.26 (broad max s i n g l e t , IH, N-H), 1.63 (doublet, J = 5 cps, IH, -CH=CH-N), 1.89 (doublet, J = 8 cps, 1H,|| I [j ), 2.19 (doublet, J = 5 cps, IH, H H -CH=CH-N) , 2.61 (mul t i p l e t , 3H, aromatic), 7.20 ( s i n g l e t , 3H, -N=C-CH_3). 44 135 Potassium Hydroxide Fusion of Ajmalicine [56] ' A mixture of ajmalicine (0.21 gm) and potassium hydroxide (2.0 gm) was fused at 300-350°C f o r one hour, under a slow stream of nitrogen, with occasional s t i r r i n g . A f t e r cooling, the melt was dis s o l v e d i n water (25 ml) and extracted with ether (5 x 10 ml). The ether layer was d r i e d over sodium s u l f a t e and evaporated under vacuum to a f f o r d a brown gum. This m a t e r i a l was chromatographed on alumina (9.5 gm). The harman [104] was eluted with chloroform. Further p u r i f i c a t i o n using preparative t i c on s i l i c a g e l with ethanol-ethyl acetate (1:2) afforded a pure sample of harman (13 mg, 12% y i e l d ) . The harman c r y s t a l l i z e d from benzene as yellow prisms (mp 234-237°C). The mixed melting point with authentic harman was undepressed at 234-237°C. As w e l l , the uv and i r spectra of both samples were i d e n t i c a l . Potassium Hydroxide Fusion of Radioactive Ajmalicine [56] Ajmalicine hydrochloride (5.5 9 mg, 2.62 x 10^ dpm/mM) from feeding experiment 6 was mixed with i n a c t i v e ajmalicine (199.12 mg). The cal c u l a t e d s p e c i f i c a c t i v i t y of t h i s mixture was 6.5 x 10~* dpm/mM. This mixture was fused with potassium hydroxide, and the harman 1104] was i s o l a t e d by chromatography following the procedure described - 127 -above f o r the i n a c t i v e sample. On c r y s t a l l i z a t i o n from benzene, harman (1.4 mg, 1.3%) was obtained as yellow prisms. This m a t e r i a l was d i l u t e d with r a d i o i n a c t i v e harman (5.62 mg) and the sample was c r y s t a l l i z e d to constant a c t i v i t y (3.87 x 10 4 dpm/mM or 59.35% of the r a d i o a c t i v i t y of the parent a j m a l i c i n e ) . Samples of the a l k a l o i d from experiments 5 and 7 were s i m i l a r l y 91 degraded. From experiment 5 , the ajmalicine a f t e r d i l u t i o n with 4 u n l a b e l l e d a l k a l o i d , had a s p e c i f i c a c t i v i t y of 3.58 x 10 dpm/mM. The harman obtained had a s p e c i f i c a c t i v i t y ( a f t e r a 31.1 f o l d d i l u t i o n with u n l a b e l l e d material) of 795 dpm/mM, representing a 69% ret e n t i o n of l a b e l . From experiment 7 the a l k a l o i d , a f t e r d i l u t i o n with unlabelled a j m a l i c i n e , had a s p e c i f i c a c t i v i t y of 5.77 x 10^ dpm/mM. The res u l t a n t harman, a f t e r a 4.2 f o l d d i l u t i o n with cold m a t e r i a l , had a s p e c i f i c a c t i v i t y of 1.07 x 10"* dpm/mM, representing a 78% re t e n t i o n of l a b e l . A j m a l i c i n o l [103] Ajmalicine [56] hydrochloride (100 mg) was suspended i n a s o l u t i o n (1:1) of d i e t h y l ether and tetrahydrofuran (10 ml). This mixture was added to a r a p i d l y s t i r r i n g suspension of l i t h i u m aluminum hydride (40 mg) i n d i e t h y l ether (10 ml). The reac t i o n mixture was refluxed under nitrogen atmosphere f o r one hour. I t was, then, cooled i n i c e , and the excess l i t h i u m aluminum hydride was destroyed by adding, dropwise, a 10% sodium hydroxide s o l u t i o n . The organic solvents were removed and the r e s u l t a n t aqueous mixture was extracted with methylene chl o r i d e (4 x 25 ml). The organic phase was subsequently dried over anhydrous sodium s u l f a t e and evaporated under vacuum to a white powder (90 mg, 98% y i e l d ) . This powder c r y s t a l l i z e d r e a d i l y from ethanol and was - 128 93 I d e n t i f i e d as tetrahydroserpentinol or a j m a l i c i n o l [103], mp 237°C 93 (with extensive decomposition) ( L i t . mp 244°C, with extensive decomposi-t i o n ) . X (log e): 225(4.56), 274 sh(3.89), 279 (3.90), 283 (3.91), m 3.x 291 (3.8 2) nm; v : 3460 (OH), 3290 (NH), 1654 (enol ether) cm ; max ' ' ' nmr: (deuteromethanol) x 2.87 (m u l t i p l e t , 4H, aromatic), 3.71 ( s i n g l e t , IH, enol e t h e r ) , 8.86 (doublet, J = 6 cps, 3H, C-CH 3); high r e s o l u t i o n mass spectrum: Calculated f o r c 2 o H 2 4 N 2 ° 2 : m 324.184. Found: 324.183. Lithium Aluminum Hydride Reduction of Radioactive Ajmalicine [56] Ajmalicine hydrochloride (0.5 mg, 2.62 x 10 7 dpm/mM) from feeding experiment 6 was mixed with i n a c t i v e ajmalicine (19.4 mg). The ca l c u l a t e d s p e c i f i c a c t i v i t y of t h i s mixture was 5.99 x 10^ dpm/mM. The. mixture was reduced as described above to y i e l d r a d i o a c t i v e a j m a l i c i n o l [103] (18.2 mg) of s p e c i f i c a c t i v i t y 3.79 x 10^ dpm/mM or 63.29% of the ra d i o -a c t i v i t y of the parent a l k a l o i d . A second sample of ajmalicine hydrochloride (1.23 mg, 2.38 x 10^ dpm/mM), from feeding experiment 7, was s i m i l a r l y mixed with i n a c t i v e ajmalicine (18.09 mg), to y i e l d a mixture of s p e c i f i c a c t i v i t y , 1.38 x 10^ dpm/mM. When reduced, the a j m a l i c i n o l retained 78.97% of t h i s a c t i v i t y (1.09 x 10 6 dpm/mM). Kuhn-Roth Oxidation of Labelled Harman [104] Ajmalicine [56] hydrochloride samples from feeding experiments 6 and 7 were combined and c r y s t a l l i z e d from methanol to a constant a c t i v i t y of 2.32 x 10 7 dpm/mM. This a l k a l o i d s a l t (22.5 mg) was mixed with r a d i o i n a c t i v e ajmalicine - 129 -(183.6 mg) to y i e l d a mixture of s p e c i f i c a c t i v i t y , 2.31 x 10 dpm/mM. This mixture was converted, i n the manner described above, to harman (1.58 mg). The a c t i v e (3-carboline (1.58 mg) was d i l u t e d with r a d i o i n a c t i v e harman (18.48 mg) and subjected to a Kuhn-Roth oxidation by r e f l u x i n g with a s o l u t i o n (20 ml) of the following composition: chromium t r i o x i d e (20 g), 98% s u l f u r i c acid (25 ml), and water (120 ml). A f t e r r e f l u x i n g t h i s s o l u t i o n f o r 2 3/4 hours, the v o l a t i l e r e a c t i o n products were steam d i s t i l l e d i n a micro-apparatus constructed f o r t h i s purpose. The d i s t i l l a t e (200 ml) was ne u t r a l i z e d (pH 7.2) with a 0.01 N sodium hydroxide s o l u t i o n . A f t e r evaporating to dryness under vacuum, a white powder (7.1 mg) remained. The sodium acetate was c r y s t a l l i z e d to constant a c t i v i t y from ethanol to y i e l d acetate of s p e c i f i c a c t i v i t y 1.41 x 3 10 dpm/mM. This value represented 0.78% of the r a d i o a c t i v i t y of the ajmalicine used. Section B 89 T r i t i a t e d T r i f l u o r o a c e t i c Acid T r i t i a t e d water (0.125 gm, 6.94 mM, lC/gm) was combined with t r i f l u o r o a c e t i c anhydride (1.45 gm, 6.94 mM) u t i l i z i n g a vacuum t r a n s f e r system. The re s u l t a n t t r i t i a t e d a c i d (1.57 gm, 36 mC/mM) was kept i n a sealed v i a l under dry nitrogen at -10°C u n t i l required. [ar- 3H]-N-[p-(3-[2-Carbomethoxymethylene]-indolyl)-ethyl]-3'-ethyl-3'- p i p e r i d e i n e [123] The c r y s t a l l i n e indole acetate d e r i v a t i v e [123] (188.9 mg) was combined with t r i t i a t e d t r i f l u o r o a c e t i c a c i d (1.57 gm, 36 mC/mM) by means of a - 130 -vacuum t r a n s f e r system. The acid s o l u t i o n was maintained under a dry nitrogen atmosphere at room temperature f o r 48 hours. The t r i t i a t e d t r i f l u o r o a c e t i c a c i d was then removed u t i l i z i n g the vacuum tr a n s f e r method. The res u l t a n t red gum was taken up i n concentrated ammonium hydroxide (1 ml) and the mixture was extracted with methylene c h l o r i d e ( 3 x 1 ml). The organic phase was evaporated and the residue taken up i n methanol (2 ml). This solvent was removed under a stream of nitrogen and the process was repeated four times. This procedure was employed to remove any r e a d i l y exchangeable t r i t i u m atoms i n the molecule. The residue was chromatographed on alumina. E l u t i o n with benzene provided the l a b e l l e d indole acetate d e r i v a t i v e (152 mg, 80% recovery). This 8 10 compound displayed an a c t i v i t y of 1.10 x 10 dpm/mg or 3.59 x 10 dpm/mM. 3 The synthesis of [ar- H]-16,17-dihydrosecodin-17-ol [114] was then completed i n the normal way. 1 0 4 I 1 4CNj-N-[g-(3-[2-Cyanomethyl]-indolyl)-ethyl]-3'-ethyl-3'-piperideine [122] The benzoate ester [121] of N-[g-(3-[2-hydroxymethylene]-indolyl)-et h y l ] - 3 ' - e t h y l - 3 ' - p i p e r i d e i n e [120] (1.25 gm, 3.2 mM) was dissolv e d i n N,N-dimethylformamide (20 ml). This was added at room temperature 14 to a mixture of potassium cyanide- C (131 mg, 10 mC) and potassium cyanide (65.9 mg, 3.2 mM t o t a l cyanide). The heterogeneous r e a c t i o n was maintained under a stream of nitrogen at room temperature with s t i r r i n g f o r one hour. A f t e r t h i s period, the re a c t i o n temperature was r a i s e d , over a 40 minute period, to 90°C. At approximately 65°C a heavy p r e c i p i t a t e of potassium benzoate was noted, and at t h i s point, potassium cyanide (736 mg, 11.3 mM) was added i n order to e f f e c t - 131 -completion of the reacti o n . The r e a c t i o n mixture was maintained at 90°C fo r one hour before cooling i n i c e . Water (10 ml) and a saturated sodium carbonate s o l u t i o n (10 ml) were then added and the mixture was extracted with methylene c h l o r i d e (3 x 20 ml). The organic phase was washed with water (1 x 30 ml), dri e d over sodium s u l f a t e and the solvent removed under vacuum. This procedure afforded a red gum. E l u t i o n of the l a t t e r by means of benzene-ether (9:1) from an alumina column yi e l d e d the c r y s t a l l i n e r a d i o a c t i v e n i t r i l e (301.3 mg) i n 32% y i e l d . The a c t i v i t y of t h i s compound was found to be 2.27 x 10 7 dpm/mg or 9 6.64 x 10 dpm/mM, and i t was i d e n t i c a l to the previously obtained n i t r i l e i n a l l other respects. [ 1 4C00CH 3]-16,17-Dihydrosecodin-17-ol [114] and [ 1 4C00CH 3J secodine 104 I115J were subsequently obtained following the normal procedure. J3,14,15,21- 3Rj-N-[g-(3-[2-Carboethoxy]-indolyl)-ethyl]-3'-ethy1-3'- p i p e r i d e i n e [119] The pyridinium c h l o r i d e [118] (1.5 gm) was diss o l v e d i n methanol (30 ml). This s o l u t i o n was cooled, under a nitrogen atmosphere, i n an i c e - s a l t bath. Sodium borohydride (100 mg) was added to t h i s r a p i d l y s t i r r e d s o l u t i o n , followed by sodium b o r o t r i t i d e (50 mg, 100 mC). This procedure was followed, one h a l f hour l a t e r , with a d d i t i o n a l sodium borohydride (890 mg). The r e a c t i o n was s t i r r e d at 0°C f o r three hours under nitrogen. The work-up involved a slow a c i d i f i c a t i o n with 2 N hydrochloric acid and d i l u t i o n with water (50 ml). The methanol was then removed under vacuum, and the resultant aqueous s o l u t i o n was made ba s i c with a - 132 -saturated sodium carbonate s o l u t i o n . The aqueous phase was then extracted with methylene ch l o r i d e (3 x 50 ml). The combined organic extracts were dried over sodium s u l f a t e and concentrated under vacuum to a golden-orange gum which c r y s t a l l i z e d on standing (1.37 gm). This product was used without further p u r i f i c a t i o n and elaborated i n the usual w a y 1 0 4 to [3,14,15,21- 3H]-16,17-dihydrosecodin-17-ol [114] and 3 9 [3,14,15,21- H]-secodine [115] of s p e c i f i c a c t i v i t y 9.82 x 10 dpm/mM. Blank Feeding Experiment [ 1 4COOCH 3]-16,17-Dihydrosecodin-17-ol [114] (10 mg, 7.37 x 10 7 dpm/mM) 14 was dehydrated to [ COOCH^-secodine [115] i n the manner described below i n preparation f o r a plant feeding. The resultant gum was taken up i n ethanol (5 drops) followed by 0.1 N a c e t i c ac i d (5 drops) and water (0.50 ml). This s o l u t i o n was maintained at room temperature f o r two hours a f t e r which time i t was frozen i n l i q u i d nitrogen and the solvents removed by vacuum d e s i c c a t i o n . The residue was taken up i n methanol (1 ml) and spotted on Eastman-Kodak prepared alumina plates (20 cm x 4 cm) con-t a i n i n g phosphor. These plates were developed i n a benzene-chloroform (1:1) s o l u t i o n . The a c r y l i c ester [115] displayed an value of 0.6 while the mixture of dimers ( i . e . presecamine [139] and secamine [142]) together displayed an R^ value of 0.45. The alumina plates were then scanned f o r r a d i o a c t i v i t y using a t i c p l a t e scanner connected to a pen recorder and a d i g i t a l p r i n t - o u t recorder. The r a t i o of secodine to the dimeric materials to the polar "baseline" substances was then obtained by f i r s t r e l a t i n g the peaks representing r a d i o a c t i v i t y , as traced out by the pen recorder, to the relevant counts per minute as obtained from the - 133 -d i g i t a l p r i n t e r . The sums of the counts per minute f o r each peak were then compared to a f f o r d the desired r a t i o . The r a t i o s ( i n percent) for the two alumina pla t e s are given i n Table 5. Table 5: The Ratio of Monomer to Dimer Produced i n a T y p i c a l Feeding Experiment S t r i p No. Secodine [115] Presecamine [139] and Baseline R f 0.60 Secamine [142] R f 0.45 1 62.4 31.8 5.8 2 61.5 32.0 6.5 The Feeding of Labelled Secodine [115] to V. rosea The l a b i l i t y of secodine [115] necessitated a sequence which would bring about the formation of t h i s compound and introduce i t i n t o the 104 plant system as r a p i d l y as p o s s i b l e . The following i s the procedure employed to form and administer the secodine i n a l l of i t s l a b e l l e d forms, to V_. rosea plants. The l a b e l l e d compound used for feeding experiment 2 w i l l be used to i l l u s t r a t e the procedure. A H glassware was dried overnight at 110°C. A 10 ml f l a s k equipped with a magnetic s t i r r e r , a r e f l u x condenser, and a dry nitrogen i n l e t was flame d r i e d and then thoroughly flushed with nitrogen. A 65% suspension of sodium hydride i n mineral o i l (10 mg) was added to the •reaction v e s s e l . The suspension was washed with dry benzene (3 x 0.5 ml) and the o i l free compound was then suspended i n a f r e s h p o r t i o n of dry 3 benzene (0.5 ml). In a small dry test tube [ar- H]-16,17-dihydrosecodin-17-ol [114] (10 mg, 2.83 x 10"*"^  dpm/mM) was disso l v e d i n dry benzene - 134 -(0.6 ml) by bringing the solvent to a b o i l i n a hot water bath. This s o l u t i o n was dropped r a p i d l y i n t o the sodium hydride-benzene suspension. An a d d i t i o n a l p o r t i o n of dry benzene (0.6 ml) was used to r i n s e the test tube and was added to the re a c t i o n . The reaction mixture was s t i r r e d at 40°C f o r 15 minutes and then r a p i d l y chromatographed on alumina Cl.5 gm, a c t i v i t y IV). The column was eluted with benzene which was c o l l e c t e d i n a c h i l l e d volumetric f l a s k (25 ml). Of t h i s measured volume, 1 ml was d i l u t e d to 100 ml with benzene and t h i s d i l u t e d s o l u t i o n was used f or r a d i o a c t i v e counting purposes. The remaining benzene f r a c t i o n (24 ml) was tr a n s f e r r e d to a s p e c i a l l y designed evaporator. 3 The f r a c t i o n was frozen at -10°C and drie d under vacuum to y i e l d [ar- H.J g secodine (4.3 mg, 3.58 x 10 dpm) as a pale yellow gum. The gum was dissol v e d i n ethanol (5 drops) followed by a 0.1 N a c e t i c acid s o l u t i o n (5 drops) and water (0.5 ml). This s o l u t i o n was fed to V_. rosea plants (81.9 gm) i n the manner described i n Section A. The work-up of the plants afforded ajmalicine [56] (4.7 mg, 0.0002% incorp-o r a t i o n ) , catharanthine [8] (3.7 mg, 0.0004% in c o r p o r a t i o n ) , and v i n d o l i n e [3] (4.8 mg, 0.01% in c o r p o r a t i o n ) . Feeding Experiments 3, 4, and 5 Feeding experiments 3, 4, and 5 were c a r r i e d out as described above f o r experiment 2. The s p e c i f i c d e t a i l s of these experiments are l i s t e d i n Tables 15a and 15b. Table 15a: The Feeding of Labelled Secodine to V. rosea Experi-ment No. Compound Fed Weight Fed (mg) A c t i v i t y Fed (dpm) S p e c i f i c A c t i v i t y Ratio Fed (dpm/mM) 3 H / 1 4 C 14, H 14, H Wet Plant Weight (gm) Weight of A l k a l o i d Isolated (mg) Ajmali-cine [56] Catharan-thine [8] Vindol-ine[3] 2 3 [ar- H]secodine 4.3 - 3.31xl0 7 - 2.83xl0 1 0 - 81.9 4.7 3.7 4.8 3 3 [ar- H]secodine 1.6 - 1.23xl0 8 - 2.83xl0 1 0 - 98.2 6.9 17.1 13.6 4 [ar- 3H, 1 4COOCH 3] secodine 3.2 9.58xl0 6 8.47xl0 7 1.28xl0 9 l . l O x l O 1 0 8.8 124.7 27.7 15.9 9.2 5 [3,14,15,21- 3H, 14 COOH 3]secodine 4.2 l . O l x l O 7 3.50xl0 7 1.27xl0 9 9.82xl0 9 3.5 194.3 - - 17.7 Table 15b: Experi-ment No. S p e c i f i c A c t i v i t y Isolated (dpm/mM) % Incorporation Ratio H/ C Ajmalicine[56] Catharanthine[8] Vindoline[3] Ajmalicine[56] Catharanthine[8] Vindoline[3] Vindoline[3] 14, 14, 14, H 14, H 14, 14, H 2 3 4 5 4.25x10 9.61x10" 1.28x10" 2.55x10 1.10x10 7.54x10" <0.001 <0.001 <0.001 <0.001 0.00 4.23xl0 3 0.00 1.53xl0 4 2.13xl0 5 1.77xl0 6 0.00 <0.001 <0.001 <0.001 - - - 4.40xl0 4 5.96xl0 4 - - - -0.01 0.02 0.043 0.042 0.03 0.01 8.3 1.4 - 136 -Desacetylvlndoline [146] 1 1 5 Vindoline [3] (114 mg) was dissolv e d i n concentrated hydrochloric acid (7.5 ml, 12 N) and the s o l u t i o n refluxed for 10 minutes. The re a c t i o n was cooled i n i c e and b a s i f i e d with a saturated s o l u t i o n of sodium carbonate. Brine (10 ml) was added to t h i s and the aqueous phase was extracted with methylene c h l o r i d e (3 x 20 ml). The organic phase was dried over sodium s u l f a t e and the solvent removed under reduced pressure. The vacuum dried residue was taken up i n d i e t h y l ether, f i l t e r e d to remove i n s o l u b l e m a t e r i a l and concentrated. The desired compound c r y s t a l l i z e d from t h i s s o l u t i o n (61 mg, 59% y i e l d ) , mp 156-157°C ( L i t . 1 1 5 mp 156-157°C). v C H C 1 3 : 3553 (OH), 1725 (COOCH ) nicix o cm nmr: T 3.15 (doublet, J = 8 cps, IH,aromatic) , 3.73 (doublet of doublets, J - 2 cps, J = 8 cps, IH, aromatic), 3.95 (doublet, J = 2 cps, IH, aromatic), 6.16 ( s i n g l e t , 3H, -C00CH 3), 6.23 ( s i n g l e t , 3H, 0CH 3), 7.28 ( s i n g l e t , 3H, N-CH3>, 9.38 (poorly resolved t r i p l e t , 3H, -CH2-CH_3) . The above spectra were superimposable with those of authentic d e s a c e t y l v i n d o l i n e . Acid Treatment of Radioactive Vindoline [3] The v i n d o l i n e obtained from feeding experiment 2 was p u r i f i e d by column chromatography and by s i l i c a g e l preparative t i c . I t was then c r y s t a l l i z e d to a constant a c t i v i t y of 1.10 x 10 dpm/mM, representing 3 a 0.01% incorporation of [ar- H]-secodine [115]. The p u r i f i e d c r y s t a l s of a c t i v e v i n d o l i n e plus the various mother l i q u o r s were then recombined (8.4 mg) and converted to desacetylvindoline [146] as described above, by r e f l u x i n g the mat e r i a l i n concentrated - 137 -hydrochloric a c i d (1.5 ml, 12 N). The c r y s t a l l i n e compound thus obtained (4.6 mg) was d i l u t e d with r a d i o i n a c t i v e desacetylvindoline (4.6 mg). This mixture was taken up i n hydrochloric acid (5 ml, 2N) and extracted with benzene ( 3 x 5 ml). The aqueous phase was b a s i f i e d with a saturated sodium carbonate s o l u t i o n and extracted with methylene c h l o r i d e (3 x 10 ml). The organic phase was dried over sodium s u l f a t e , and the solvent removed i n vacuo. The residue was c r y s t a l l i z e d from d i e t h y l ether to a f f o r d a c t i v e desacetylvindoline (7.2 mg). This m a t e r i a l was f u r t h e r d i l u t e d with i n a c t i v e compound (3.6 mg) and then p u r i f i e d , once more, by s i l i c a g e l preparative t i c , by e l u t i n g with e t h y l acetate-methanol (95:5). The desacetylvindoline thus obtained (7.5 mg) was c r y s t a l l i z e d from d i e t h y l ether to a constant a c t i v i t y of 79.99 dpm/mg. When the d i l u t i o n f a c t o r s had been considered t h i s Value corresponded to a s p e c i f i c a c t i v i t y of 1.0 x 10^ dpm/mM or 9.6% of the a c t i v i t y of the o r i g i n a l a l k a l o i d . V l n d o l i n o l 11471 Vindoline [3] (97.6 mg) was d i s s o l v e d i n dry tetrahydrofuran (8 ml). To t h i s s o l u t i o n was added l i t h i u m aluminum hydride (61.9 mg). The suspension was gently refluxed under a nitrogen atmosphere f o r 2 hours. The r e a c t i o n was then cooled i n i c e and the excess l i t h i u m aluminum hydride was destroyed by slowly adding a saturated sodium s u l f a t e s o l u t i o n (15 ml). The r e a c t i o n mixture was then f i l t e r e d and the aqueous phase extracted with methylene c h l o r i d e (3 x 20 ml). The organic phase was d r i e d over sodium s u l f a t e and the solvent removed i n vacuo. The r e s u l t a n t white foam c r y s t a l l i z e d on adding d i e t h y l ether to a f f o r d the pure - 138 -v i n d o l i n o l (78 mg, 95% y i e l d ) , mp 171-174°C; ( L i t . 1 1 5 mp 172-176°C);\ in 3.x (log e ) : 214 (4.47), 254 (3.83), 306 (3.69) nm; v K B r : 3480, 1044 (OH) IH3X cm ^; nmr: T 3.19 (doublet, J = 8 cps, IH, aromatic); 3.78 (doublet of doublets, J = 8 cps, J = 2 cps, IH, aromatic), 3.95 (doublet, J = 2 cps, IH, aromatic), 6.28 ( s i n g l e t , 3H, -0CH 3), 6.52 ( s i n g l e t , 2H, hydroxymethylene), 7.06 ( s i n g l e t , 3H, N-CH 3), 9.40 (poor t r i p l e t , 3H, -C^-CH^); high r e s o l u t i o n mass spectrum: Calculated f o r (-'22^30^2^4: M.W. 386.221; Found: 386.220. Reduction of Radioactive Vindoline [3] The v i n d o l i n e (12.1 mg) i s o l a t e d i n experiment 3 was converted to v i n d o l i n o l [147] (9.5 mg) by the procedure o u t l i n e d above. C r y s t a l l i z a -t i o n from d i e t h y l ether afforded 6.4 mg of white c r y s t a l s which were d i l u t e d with r a d i o i n a c t i v e v i n d o l i n o l (6.42 mg). This substance was then p u r i f i e d by preparative t i c on s i l i c a g e l employing chloroform: methanol (1:3) as the e l u t i n g solvent. The r e s u l t a n t compound (9.7 mg) was c r y s t a l l i z e d to a constant a c t i v i t y (7.54 x 10 5 dpm/mM) thereby 3 representing a 0.02% incorporation of [ar- H] secodine [115] i n t o v i n d o l i n e . Formaldehyde-bisdimedone [149] A 36% formalin s o l u t i o n (1 ml) was added dropwise to an ethanol s o l u t i o n (1 ml) of dimedone [148] (100 mg). The r e a c t i o n v e s s e l was sealed and maintained at room temperature for 18 hours. The white p r e c i p i t a t e was then f i l t e r e d o f f , washed with water and c r y s t a l l i z e d from ethanol to a f f o r d formaldehyde-bisdimedone (110 mg) as white needles, mp 192-193°C ( L i t . mp 192°C). v : 2965 (C-H), 1610, - 139 -1593 (g-diketone) cm nmr: x 6.85 ( s i n g l e t , 2H, ex o c y c l i c methylene), 7.71 ( s i n g l e t , 8H, c y c l i c methylenes), 8.93 ( s i n g l e t , 12H, gem dimethyls). 42a Degradation of V i n d o l i n o l [147] V i n d o l i n o l (9.5 mg) was dissolv e d i n ethanol (3 drops). To t h i s s o l u t i o n was added an aqueous s o l u t i o n of 0.3 M p e r i o d i c acid (0.2 ml). The re a c t i o n v i a l was sealed and maintained at room temperature f o r 4 hours. The re a c t i o n was then cooled i n i c e and a saturated aqueous s o l u t i o n of dimedone [148] (2 ml, 0.0297 M) was added. The re a c t i o n v e s s e l was again sealed and maintained at room temperature f o r 18 hours. The r e s u l t a n t p r e c i p i t a t e was f i l t e r e d o f f , washed with water and a i r d r i e d . The l i g h t yellow s o l i d was then dissolved i n methanol (1 ml), f i l t e r e d to remove i n s o l u b l e m a t e r i a l and the methanol removed i n vacuo. The residue was c r y s t a l l i z e d from ethanol to a f f o r d a f t e r three such c r y s t a l l i z a t i o n s , formaldehyde-bisdimedone [149] (3.2 mg, mp 191°C). A t h i n l a y e r chromatogram, on alumina, eluted with e t h y l acetate-methanol (99:1), of the above product and authentic formaldehyde-bisdimedone reyealed the i d e n t i t y of the two compounds. Further confirmation was obtained by a mixed melting point determination. Degradation of Radioactive V i n d o l i n o l [147] V i n d o l i n o l (9.5 mg, 5.96 x 10 4 dpm/mM-3H, 4.40 x 10 4 dpm/mM-14C), derived, as pre v i o u s l y described, from the vi n d o l i n e [3] obtained from experiment 5, was degraded as described above to a f f o r d a f t e r three c r y s t a l l i z a t i o n s from ethanol, formaldehyde-bisdimedone [149] (1.6 mg). - 140 -On analyzing f o r r a d i o a c t i v i t y , the compound was found to have a s p e c i f i c a c t i v i t y of 4.99 x 10^ dpm/mM representing 113% of the a c t i v i t y of the v i n d o l i n o l . The remaining material (1.2 mg) was dissolv e d i n methylene c h l o r i d e (0.25 ml) and extracted with 2% sodium hydroxide (4 x 0.13 ml). The aqueous s o l u t i o n was a c i d i f i e d with concentrated hydrochloric acid (2 drops, 12 N) and extracted with chloroform (3 x 0.5 On evaporating the solvent and c r y s t a l l i z i n g the residue, formaldehyde-bisdimedone (1.06 mg) was again obtained. This sample on c r y s t a l l i z i n g to constant a c t i v i t y , displayed a s p e c i f i c a c t i v i t y of 4.6 x 10 4 dpm/mM or 105% of the a c t i v i t y of the v i n d o l i n o l . Section C 89 The Administration of Labelled Compounds to V. minor The procedure employed to administer r a d i o a c t i v e compounds to _V. minor consisted of a hydroponic feeding to cut stems of the plant. Unless otherwise i n d i c a t e d , the following procedure describes t h i s feeding technique. The compound to be administered was dissolved i n ethanol (5 drops). To t h i s s o l u t i o n was added a 0.1 N a c e t i c ac i d s o l u t i o n (5 drops) and water (5 ml). This s o l u t i o n was equally d i s t r i b u t e d among ten t e s t tubes (10 x 70 mm). Into each of these t e s t tubes were placed three f r e s h l y cut shoots (10-20 i n . i n length) of \/. minor. The tes t tubes were placed i n a rack and the plant stems were exposed to fluorescent i l l u m i n a t i o n . The o r i g i n a l container of the l a b e l l e d compound was rinsed with ethanol (0.25 ml), the 0.1 N a c e t i c a c i d s o l u t i o n (0.5 ml), and water (2 ml). This r i n s e was used to r e f i l l the feeder tubes a f t e r the i n i t i a l precursor s o l u t i o n had been absorbed by the shoots. - 141 -Following these washings, d i s t i l l e d water was used and the ends of the shoots were kept below the surface of the l i q u i d f o r the duration of the experiment. V_. minor feedings were c a r r i e d out for e i t h e r 24 or 96 hours. 89 E x t r a c t i o n of A l k a l o i d s from V. minor The following procedure was used i n a l l cases to extract and p u r i f y the a l k a l o i d s of V_. minor. The stems and leaves of V. minor (approximately 25-65 gm, wet weight) were mascerated with methanol i n a Waring blender, f i l t e r e d , and remacerated u n t i l the f i l t r a t e was c o l o r l e s s . The solvent was removed i n vacuo and the aqueous residue taken up i n 2 N hydrochloric acid (150 ml). This s o l u t i o n was extracted with benzene (3 x 75 ml). The combined benzene extracts were washed with 2 N hydroc h l o r i c acid (50 ml). The combined aqueous phases were cooled i n i c e , b a s i f i e d with 15 N ammonium hydroxide, and extracted with chloroform (3 x 100 ml). The combined chloroform extracts were washed with water (100 ml), dried over sodium s u l f a t e and evaporated under reduced pressure to give an orange foam. The foam was d i s s o l v e d i n a small volume of methylene c h l o r i d e and chromatographed on alumina (10 gm). The column was eluted successively with petroleum ether 30/60°, benzene, and methanol. Fractions of 35 ml i n volume were taken. Petroleum etherrbenzene (2:1) eluted minovine [112] while petroleum ether:benzene (1:1) eluted vincamine [102] from the column. The l a t t e r a l k a l o i d c r y s t a l l i z e d r e a d i l y from methanol and no further p u r i f i c a t i o n was required f o r t h i s compound. The former - 142 -compound was fur t h e r p u r i f i e d by preparative s i l i c a g e l t i c , employing e t h y l acetate:ethanol (4:1) as the e l u t i n g solvents. The re s u l t a n t m a terial was then c r y s t a l l i z e d from methanol. Feeding Experiments 4 to 10 These experiments were c a r r i e d out as described above. In the case of the secodine [115] feedings (experiments 8, 9, 10), the compound was synthesized j u s t p r i o r to feeding i n the manner described i n Section B. The experimental d e t a i l s f o r these feedings are presented i n Tables 16a, 16b, and 16c. Feeding Experiment 11 Q 1 0 [ar- H]-16,17-Dihydrosecodin-17-ol [114] (10 mg, 2.83 x 10 dpm/mM) was dehydrated as described above (Section B). The l a b e l l e d secodine [115] (3.1 mg) was di s s o l v e d i n ethanol (0.25 ml). To t h i s s o l u t i o n was added an aqueous s o l u t i o n (1 drop to 250 ml water) of Tween 20 (1 ml). The re s u l t a n t mixture was swirled and then vigorously shaken to form a uniform suspension. This suspension was fed to y_. minor cuttings (37.4 gm wet weight) following the procedure described above. A f t e r 96 hours, the plants were worked up f o r a l k a l o i d s . On p u r i f i c a t i o n and c r y s t a l l i z a t i o n to constant a c t i v i t y , minovine [112] (2.2 mg) was found to e x h i b i t 0.0005% incorporation of l a b e l with a s p e c i f i c a c t i v i t y of 2.0 x 10 5 dpm/mM, while vincamine [102] (6.9 mg) displayed a 0.0004% incorporation of l a b e l at 5.3 x 10 dpm/mM. - 143 -Table 16a: Feeding Experiments 4, 5, 6, 7, 8, 9, 10 to V. minor Exp. Compound Fed Feeding A c t i v i t y % Incorporation No. Time Fed 4 [ar - 3 H ] - t r y p t o p h a n [ l l ] 24 7.41 x 10 7 0.089 0.080 5 [ar- 3H]-16,17-dihydro- 24 1.86 x 10 7 $0,001 0.000 secodin-17-ol [114] 6 [ar- 3H]-16,17-dihydro- 96 2.40 x 10 7 $0,002 $0,001 secodin-17-ol 7 [ar- 3H]-16,17-dihydro- 96 3.45 x 10 7 <0.001 <0.001 secodin-17-ol 8 [ar- 3H]-secodine[115] 24 3.40 x 10 8 <0.001 <0.001 9 [ar- 3H]-secodine 96 2.65 x 10 8 0.001 0.001 10 [ar- 3H]-secodine 96 2.10 x 10 8 $0,001 $0,001 Table 16b: Exp. Feeding Method S p e c i f i c A c t i v i t y S p e c i f i c A c t i v i t y Isolated No. Fed (dpm/mM) (dpm/mM) Vincamine Minovine [102] [112] . - ^ 4 Hydroponic i n a c e t i c 1.55 x 10 2.33 x 10 4.97 x 10 acid 8 4 5 Hydroponic as acetate 7.33 x 10 2.12 x 10 0.00 6 Hydroponic as acetate 8.90 x 10 8 4.34 x 10 3 4.18 x 10 3 8 3 3 7 Hydroponic as acetate 8.90 x 10 4.69 x 10 3.55 x 10 8 Hydroponic as acetate 2.83 x 1 0 1 0 4.67 x 10 2.76 x IO 5 9 Hydroponic as acetate 2.83 x 1 0 1 0 9.49 x 10 4 2.03 x 10 5 10 Hydroponic as acetate 2.83 x 1 0 1 0 9.98 x 10 4 1.84 x 10 5 Table 16c: Exp. Weight Wet Plant Weight of Al k a l o i d s Isolated No. Compound Fed Weight (mg) (mg) (gm) Vincamine [102] Minovine [112] 4 9.8 64.0 10.0 4.0 5 9.1 52.9 11.6 5.6 6 9.7 25.3 10.0 5.1 7 14.0 37.6 10.0 3.4 8 4.1 31.3 4.3 1.7 9 2.4 40.0 12.7 4.8 10 2.5 56.5 5.7 3.4 - 144 -Feeding Experiment 12 [ar- 3H, 1 4COOCH 3]-16,17-Dihydrosecodin-17-ol [114] (12.1 mg, 1.1 x • 7 3 9 14 10 dpm/mM- H, 1.28 x 10 dpm/mM- C) was dehydrated as described above (Section B). The doubly l a b e l l e d secodine [115] (5 mg) displayed a 3H/ 1 4C r a t i o of 8.4 and the t o t a l a c t i v i t y fed was 1.79 x 10 7 dpm- 1 4C 8 3 and 1.51 x 10 dpm- H. This substance was fed to _V. minor cuttings (43.4 gm) for a 5 day period. The a l k a l o i d s were i s o l a t e d and p u r i f i e d to constant a c t i v i t y by the usual procedure. These exhibited the 3 following a c t i v i t i e s : minovine [112] (3.8 mg), H-0.001% inc o r p o r a t i o n at 1.36 x 10 5 dpm/mM, 1 4C-0.0008%. at 1.35 x 10 4 dpm/mM, 3H/ 1 4C r a t i o , 10.1; vincamine [102] (5.3 mg), 3H-0.001% at 5.74 x 10 5 dpm/mM, 1 4c-0.001% at 6.65 x 10 4 dpm/mM, 3H/ 1 4C r a t i o 8.6. Section D The Administration of Labelled Compounds to A. pyricollum The following procedure was u t i l i z e d to administer l a b e l l e d compounds to A. pyricollum. The procedure was used f o r a l l A. pyricollum feedings discussed i n t h i s t h e s i s . The compound to be fed was dissolv e d i n ethanol (5-10 drops). To t h i s s o l u t i o n was added 0.1 N a c e t i c a c i d (5 drops) and d i s t i l l e d water (0.5 ml). This s o l u t i o n was administered to root sections, cut from two-year-old plants, contained i n a tes t tube (25 x 110 mm). The precursor s o l u t i o n was absorbed i n t o the roots i n approximately 20 minutes. The o r i g i n a l container of the l a b e l l e d compound was washed with 0.1 N a c e t i c a c i d (0.5 ml) and d i s t i l l e d water (1 ml). These washings, plus a d d i t i o n a l d i s t i l l e d water as required, were used over - 145 -the feeding period to keep the roots moist. The feeding period for A. pyricollum was 5 days. E x t r a c t i o n of A l k a l o i d s from A. pyricollum The following procedure was u t i l i z e d to extract and p u r i f y the a l k a l o i d s of A. pyricollum. This procedure was followed f o r a l l extrac-tions of t h i s plant system discussed i n t h i s t h e s i s . The root segments of A. pyricollum (13-20 gm, wet weight) were mascerated with methanol i n a Waring blender, f i l t e r e d , and remascerated u n t i l the f i l t r a t e was c o l o r l e s s . The solvent was removed under reduced pressure and the aqueous residue taken up i n 2 N hydrochloric acid (150 ml). This s o l u t i o n was extracted with benzene (3 x 75 ml). The combined benzene extracts were washed with 2 N hydrochloric a c i d (50 ml). The combined aqueous extracts were cooled i n i c e , b a s i f i e d with 15 N ammonium hydroxide and extracted with chloroform (3 x 100 ml). The combined chloroform extracts were washed with water (100 ml), d r i e d oyer sodium s u l f a t e , and the solvent evaporated under reduced pressure to a f f o r d a pale yellow foam. The foam was d i s s o l v e d i n a small volume of methylene c h l o r i d e and chromatographed on alumina (30 gm). The column was eluted successively with petroleum ether 30/60°, benzene, chloroform, and methanol, f r a c t i o n s of 50 ml i n volume were taken. The l a t t e r benzene-chloroform, (1:11 f r a c t i o n as w e l l as the f i r s t chloroform f r a c t i o n eluted apparicine [144]. The chloroform f r a c t i o n eluted uleine [145] as w e l l . These a l k a l o i d s were f u r t h e r p u r i f i e d by preparative t i c using s i l i c a gel as adsorbant and e t h y l acetate-ethanol (2:1) as the e l u t i n g solvents. - 146 -Apparicine was c r y s t a l l i z e d from acetone s o l u t i o n , while the uleine c r y s t a l l i z e d from methanol. Feeding Experiments 1 to 5 These experiments were c a r r i e d out as described above. As described e a r l i e r , (Section B), secodine [115] was produced j u s t p r i o r to the a c t u a l feeding experiment by the base catalyzed dehydration of 16,17-dihydrosecodin-17-ol [114]. The experimental d e t a i l s f o r experiments 1-5 are presented i n Tables 17a and 17b. 129-131 Degradation of Apparicine [144]  Apparicine (20 mg) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (10 ml). Ozone was bubbled through t h i s s o l u t i o n at room temperature u n t i l the f a i n t yellow of the s o l u t i o n became a bluish-green i n d i c a t i n g an excess of the gas. Water (10 ml) was then added, the s o l u t i o n swirled, and then maintained at room temperature for 30 minutes. This s o l u t i o n was then steam d i s t i l l e d using an external steam source. The d i s t i l l a t e (65 ml) was deposited d i r e c t l y i n t o a saturated s o l u t i o n of dimedone [148] (15 ml, 0.0297M) which was then stored overnight at 10°C. To the r e s u l t a n t mixture, i n which some c r y s t a l l i n e m a t e r i a l was evident, was then added g l a c i a l a c e t i c a c i d (2 ml) and t h i s was refluxed f o r 8 hours. The solvents were removed under reduced pressure and the r e s u l t a n t white s o l i d was chromatographed on s i l i c a gel preparative p l a t e s . The formaldehyde bisdimedone [149] (R^ 0.60) was separated from the acetaldehyde bis-dimedone [164] (R^ 0.65) by e l u t i n g with chloroform-e t h y l acetate, (5:1). The formaldehyde bis-dimedone thus obtained was Table 17a: The Feeding of Labelled Compounds to A. pyricollum Experiment Number Compound Fed Weight A c t i v i t y Fed S p e c i f i c A c t i v i t y Fed Fed (dpm) (dpm/mM) (mg) 14 c 3 R 14 c 3 R Ratio 3H/ 1 4C Weight of A l k a l o i d Isolated (mg) Apparicine[144] Uleine[145] 1 [ar - 3 H ] -secodin-16,17-dihydro-17-ol[114] 1.8 9.20xl0 7 - 1.82xl0 1 0 - 7.0 1.4 2 [ar - 3 H ] - secodine[115] 4.7 - . 2.57xl0 8 - 1.82xl0 1 0 - 7.8 1.7 3 [14COOCH -]-secodine 2.0 7.06xl0 6 l . l l x l O 8 1.19xl0 9 l . l O x l O 1 0 - 25.4 4 [ar- H, secodine C00CH 3]- 3.7 1.28xl0 7 1.28xl0 9 8.7 24.9 i 5 [3,14,15 secodine 3 14 ,21- H, C00CH 3]4.7 1.07xl0 7 4.42xl0 7 1.27xl0 9 9.82xl0 9 4.2 17.5 Table 17b: Experiment Wet S p e c i f i c A c t i v i t y Isolated (dpm/mM) < Z Incorporation Ratio 3H/ 1 4C Number Plant Weight (gm) Apparicine[144] Uleine[145] 1 4 C 3H 1 4 C 3H Apparicine[144] 1 4 C 3H Uleine[145] Apparicine[144] 1 4 C 3H 1 19.5 - 1.43xl0 4 4.98xl0 4 - <0.001 - <0.001 2 13.5 - 7.68xl0 5 l . l O x l O 6 - 0.01 - 0.003 3 18.8 1.06xl0 4 ' - - 0.01 - - -4 20.0 1.94xl0 4 1.62xl0 5 - 0.015 0.014 - 8.4 5 13.2 1.35xl0 4 3.03xl0 4 — 0.009 0.005 — 2.2 - 1A8 -c r y s t a l l i z e d from ethanol to provide c o l o r l e s s needles (7 mg, mp 191-192°C) ( L i t . mp 192°C). Degradation of Radioactive Apparicine [144] Apparicine (23.3 mg, 1.06 x 10 dpm/mM), from experiment 3, was degraded as above. The m a t e r i a l obtained from the p u r i f i c a t i o n v i a preparative t i c was d i s s o l v e d i n methylene c h l o r i d e (2 ml) and extracted with 2% sodium hydroxide s o l u t i o n ( 4 x 1 ml). The aqueous extract was a c i d i f i e d with concentrated hydrochloric a c i d (20 drops) and extracted with chloroform ( 4 x 1 ml). This extract on drying over sodium s u l f a t e , and concentrating under reduced pressure produced a white s o l i d (6.7 mg) which was c r y s t a l l i z e d to constant a c t i v i t y from ethanol (mp 192-192.5°C). This sample exhibited an a c t i v i t y of 1.05 x 10 4 dpm/mM or 99% of the a c t i v i t y of the o r i g i n a l a l k a l o i d . - 1 4 9 -BIBLIOGRAPHY 1 . K. Mothes, The A l k a l o i d s , Vol. VI, R.F. Manske, Ed., Academic Press, New York ( 1 9 6 0 ) . 2 . T. 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This feeding, the subsequent plant work-up, and the examination of the a l k a l o i d s i s o l a t e d were c a r r i e d out by Dr. N.D. Westcott. 111. R.T. Brown, G.F. Smith, K.S. Stapleford, D.A. Taylor, Chem. Commun. 190 (1970). 112. N.D. Westcott, Studies Related to: Bark Extracts of Some F i r and  Spruce Species; and Synthesis and Biosynthesis of Indole A l k a l o i d s , Doctoral t h e s i s , The U n i v e r s i t y of B r i t i s h Columbia (1970). 113. G.A. Poulton, Research Report The Un i v e r s i t y of B r i t i s h Columbia (1970). 114. G.A. C o r d e l l , G.F. Smith, G.N. Smith, Chem. Commun. 191 (1970). 115. M. Gorman, N. Neuss, G.H. Svoboda, A.J. Barnes, N.J. Cone, J. Am. Pharm. Assoc., Scie. Ed. 4j8, 256 (1959). 116. J.P. Kutney, V.R. Nelson, D.C. W i g f i e l d , J . Am. Chem. Soc. 91, 4278 (1969). 117. R.J. Sundberg, The Chemistry of Indoles, Academic Press, New York (1970). 118. G. Guroff, J.W. Daly, D.M. J e r i n a , J . Renson, B. Witkop, S. Udenfriend, Science 157, 1524 (1967). - 155 -119. J . Mokry, I. Kompis, L l o y d i a 2j5, 428 (1964) and references contained therein. 120. J . Mokry, I. Kompis, G. S p i t e l l e r , C o l l e c t i o n Czech. Chem. Commun. 32, 2523 (1967). 121. J . Schmutz, F. Hunziker, R. H i r t , Hel. Chem. Acta. 4-0, 1189 (1957). 122. G. Buchi, E.W. Warnhoff, J . Am. Chem. Soc. 81, 4433 (1959). 123. B. G i l b e r t , L.D. Antonaccio, A.A.P.G. Archer, C. D j e r a s s i , E x p e r i e n t i a JL6, 61 (1960) . 124. R.R. Arndt, C. D j e r a s s i , E x p e r i e n t i a 23_, 566 (1965). 125. J.A. Joule, H. Monteiro, L.J; Durham, B. G i l b e r t , C. D j e r a s s i , J . Chem. Soc. 4773 (1965). 126. J.P. Kutney, V.R. Nelson, D.C. W i g f i e l d , J . Am, Chem. Soc. 91 4278 (1969). 127. J.P. Kutney, V.R. Nelson, D.C. W i g f i e l d , I b i d , 4279 (1969). 128. A. Ahond, A. Cave, C. Kan-Fan, Y. Langlois, P. P o t i e r , Chem. Commun. 517 (1970). 129. G. Kunesch, J . Polonsky, Phytochemistry 8^, 1221 (1969). 130. D. Vorlander, Z. Anal. Chem. 77_, 321 (1929). 131. Appreciation i s g r a t e f u l l y acknowledged to Mr. G.B. F u l l e r for determining the exact conditions f o r t h i s degradation. 132. Mark I L i q u i d S c i n t i l l a t i o n Systems I n s t r u c t i o n Manual, Section 1, Nuclear-Chicago Corporation, Des Pl a i n e s , I l l i n o i s (1966). 133. R.W. Hendler, Ana. Biochem. 7, 110 (1964). 134. Appreciation i s g r a t e f u l l y acknowledged to Dr. P. Salisbury f o r advice on and c u l t i v a t i o n of the plants used i n t h i s study. 135. Appreciation i s g r a t e f u l l y acknowledged to Dr. V.R. Nelson f o r carrying out t h i s r e a c t i o n . 136. P h y s i c a l Data of Indole and Dihydroindole A l k a l o i d s , V o l. 1, L i l l y Research Laboratories, E l i L i l l y and Company, Indianapolis 6, Indiana (1960). 

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