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

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STUDIES RELATED TO THE. BIOSYNTHESIS OF INDOLE ALKALOIDS  BY  RATTAN SAGAR SOOD B.Sc. Honours, Panjab University, India, 1964 M.Sc, The University of B r i t i s h Columbia, 1968  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Chemistry  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1970  In presenting  this thesis in partial fulfilment of the requirements for  an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may by his representatives.  be granted by the Head of my Department or It is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver 8, Canada  Date  o  ABSTRACT  The Strychnos skeleton postulated  (e.g. preakuammicine, 56) has been  i n the l i t e r a t u r e to rearrange to Aspidosperma (e.g.  vindoline, 5) and Iboga (e.g. catharanthine,  6) bases v i a the i n t e r -  4 21 vention of A '  -dehydrosecodine (76).  Part A of the thesis describes  the syntheses and biosynthetic evaluation of two close r e l a t i v e s , 16,17dihydrosecodin-17-ol (90) and secodine (107), of the f u g i t i v e a c r y l i c ester  (76). In the synthetic sequence, condensation of 3-ethylpyridine with  2-carboethoxy-3-(3-chloroethyl)-indole (80) followed by the of the r e s u l t i n g pyridinium  chloride (82) gave N[g-{3-(2-hydroxymethylene)-  indolyl}-ethyl]-3'-ethyl-3'^piperideine of alcohol (84) was  reduction  (84).  The benzoate ester  (85)  treated with potassium cyanide to afford N-[g-{3-(2-  cyanomethylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine  (86).  This  l a t t e r compound upon treatment with methanol and hydrogen chloride gas gave N-J 0-{3- (2-carbomethoxymethylene)-indolyl}-ethyl]-3 -ethyl-3'1  piperideine C88).  Formylation of the ester (88) with methyl formate  followed by reduction of the r e s u l t i n g enol (89) gave 16,17-dihydrosecodin -17-61 (90). C90)  Feeding of [ C00CH ]-16,17-dihydrosecodin-17-ol 14  3  into Vinca minor L. revealed no_ s i g n i f i c a n t a c t i v i t y into the  isolated a l k a l o i d s .  The substance i n fact appeared to be a toxic  component with marked deterioration of the plant occurring within  24  hours. In another investigation,, synthetic 16,17-dihydrosecodin-17-ol 3 was  dehydrated to secodine (107).  Feeding of [ar- Hj-secondine  (90)  (107)  into Virica minor L. showed low but p o s i t i v e incorporation into vincamine  (72) and minovine (73).  "Blank" experiments revealed that after the  maximum period required for the plant to absorb a solution of the l a b e l l e d compound, 61% remained as monomer (107) while 32% had been converted to the dimers (presecamine and secamine). In conclusion this study while providing some preliminary informat i o n on the l a t e r stages of indole alkaloid biosynthesis has also created an entry into more sophisticated biosynthetic experiments.  This  s i t u a t i o n w i l l hopefully lead to a better understanding of the manner i n which this large family of natural products i s synthesized  in'Lthe  l i v i n g plant. In Part B of the thesis some preliminary studies leading to the biosynthesis of vincamine (ebumamine family) are described. intermediacy  The  of a t e t r a c y c l i c pyruvic ester (12) was invoked by  Wenkert several years ago to r a t i o n a l i s e the rearrangement of Aspidosperma skeleton to vincamine (2) .  To confirm this ^speculationa short  synthesis of a close r e l a t i v e ( i . e . , 24) of the postulated  precursor  was contemplated. In the synthetic sequence reaction of tryptophyl bromide (18), produced by the action of phosphorus tribromide on tryptophol  (17), with  3-acetylpyridine ethylene ketal (16) gave N-{g-(3-indolyl)-ethyl]-3'acetylpyridinium ethylene k e t a l bromide (19).  The pyridinium bromide  (19) on c a t a l y t i c reduction and acid hydrolysis furnished N-[g-(3indolyl)-ethyl]-3'-acetylpiperidine (21).  A l k y l a t i o n of the ketone (21)  using t r i t y l sodium and a l l y l bromide gave N-[g-(3-indolyl)-ethyl]-3'a l l y l - 3 ' - a c e t y l p i p e r i d i n e (22).  Osmylation of the a l l y l i c double bond  i n C22) d i d not give the desired d i o l  (23).  Tentative assignment i s  given i n structure (34) to the polar compound obtained  i n this manner.  TABLE OF CONTENTS Page TITLE PAGE  ........  ABSTRACT  •  '  TABLE OF CONTENTS  ........  •  LIST OF TABLES  PART A  •. .  iv  .......  .. .. •  i i  .  '  ..  ACKNOWLEDGEMENTS  i  . . . •  . ...  LIST OF FIGURES  •.  v  '  . • • .  v  '  •  •. .  •  EXPERIMENTAL  . .. . .. ..  34 ..................  ............  II ........ ..\  REFERENCES • •••••••• •.•**•  •  .  ......  91 93  ....  109  •' •' • • .•*•-•*"•*•'••  • • •  INTRODUCTION  EXPERIMENTAL  73  •  PART B ........ .....  DISCUSSION  • . . .•  118 123 124 129  .  REFERENCES ..............  x  31  .'  PART I ............................... PART  l  2  •..  .... .  PART I I  i  1  INTRODUCTION  PART I .  i  • •  ....  DISCUSSION  i  •  '  '  ... . '  •  142 150  LIST OE FIGURES Figure  Page  PART A 1  Scheme portraying! the; proposed relationship between three main classes of indole alkaloids  •  6  2  Barger-Hahn-Robinson-Woodward hypothesis ..........  8  3  Wenkert's prephenic acid hypothesis  9  4  The acetate hypothesis  10  5  The Thomas-Wenkert hypothesis  13  6  A summary of geraniol  16  7  A summary of the secologanin ->- geissoschlzine pathway .................. The rearrangement of Corynanthe -> Strychnos skeleton  19 21  Scott's scheme for the rearrangement of Corynanthe -»Strychnos skeleton  23  8 9 10  secologanin pathway .......  Wenkert's proposal for the biosynthesis of Aspidosperma and Iboga alkaloids  25  11  Some l a t e r stages of indole a l k a l o i d biosynthesis .  28  12  Synthesis of 16,17-dihydrosecodi.n-17-ol C90)  35  13  Synthesis of 2^carboethoxy-3"C3'"chloroethyl)^ indole C80)  ........  •  36  14  Synthesis of 6,7-diazasteroid (95)  37  15  Nmr spectrum of alcohol 84  41  16  Mass spectrum of alcohol 84  43  17  Nmr  spectrum of n i t r i l e 86  18  Nmr  spectrum ?of).*carbomethoxy ester 88  51  19  Mass spectrum of carbomethoxy ester 88 ...  52  20  Nmr  spectrum of 16,17-dihydrosecodin-17-ol (90) ...  56  21  Mass spectrum of 16,17-dihydrosecodin-17-ol (90) ..  57  ....  48  Figure 22  Page Postulated fragmentation of 16,17-dihydrosecodin17-ol (90) i n the mass spectrometer  58  23  Synthesis of [ar- H]-16,17-dihydrosecodin-17-ol (90)  61  24  Synthesis of [ C00CH ]-16,17-dihydrosecodin-17-ol (90) .  64  3  14  ,- ^ '  .  25 26  Battersby's'.'synthesis.-of 16,17-dihydrosec6din-17-ol (90) Smith's synthesis of tetrahydrosecodine (110)  69 70  27  Some of the compounds derivable i n vivo from 16,17dihydrosecodinsl7-ol (90)  71  Rearrangement (119)  of presecamine (type a) to secamine ...  77  Rearrangement (120)  of presecamine (type b) to secamine  28 29 30  78  A summary of data supporting the structure of secodine Q-07) ....  . . . .  80  31  Nmr spectrum of secodine (107)  83  32 33  Mass spectrum of secodine O-07) Proposed elaboration of secodine into vincamine and minovine • ............................  84 88  PART B 1  Various eburnamine-vincamine type alkaloids  124  2  Scheme showing similar rearrangement of nontryptophan portion i n vincamine and vincadifformine  125  Fenkert's proposal f o r the rearrangement of Aspidosperma skeleton to vincamine ......................  126  4  Synthesis of vincamine  127  5  Synthesis of eburnamine  6  Proposed synthesis of intermediate 24  131  7  Synthesis of some model piperidine systems ........  132  3  . . . .•  128  Figure  Page  >  8  Nmr  spectrum of 28  133  9  Nmr  spectrum of 29  135  10  Nmr  spectrum of o l e f i n 22  11  Mass spectrum of o l e f i n 22  , 12 "  .. •  Postulated fragmentation of 22 i n the mass spectrometer ....... . . . .  137 138  139  LIST OF TABLES Table  Page  1  Isolation of alkaloids from Vinca rosea seedlings..  2  Results of the "blank" experiment  18 117  ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e thanks and a p p r e c i a t i o n t o P r o f e s s o r James P. Kutney.  H i s encouragement and e x p e r t g u i d a n c e ,  b o t h as a t e a c h e r and a s c i e n t i s t throughout t h e c o u r s e o f t h i s r e s e a r c h have made t h i s t h e s i s p o s s i b l e . I am a l s o g r a t e f u l t o Mr. John Beck f o r h i s c o l l a b o r a t i o n w i t h me i n t h i s r e s e a r c h and f o r many h e l p f u l d i s c u s s i o n s . The f o r m i d a b l e t a s k o f l e t t e r i n g i n t h e formulae and t y p i n g o f the m a n u s c r i p t was s k i l l f u l l y executed by M i s s Diane Johnson. would l i k e t o e x p r e s s my deep sense o f g r a t i t u d e t o h e r .  I  PART A STUDIES RELATED TO THE BIOSYNTHESIS OF INDOLE ALKALOIDS  INTRODUCTION During the past f i f t y or sixty years, tremendous advances have been made i n the laboratory preparation of complex naturally occurring organic substances.  As early as the f i r s t part of this  present  century pioneering work was being carried out i n various laboratories. Among the early successes i n this vastly challenging f i e l d may be cited the syntheses of numerous terpenes, alkaloids including nicotine, dihydroquinine  and the'simple opium'bases; porphyrins  including the  blood pigments, the common hexoses, as well as many amino acids and peptides. Yet, while the dramatic announcements of multistage  syntheses  appeared successively during the l a s t several decades, a f a i n t e r appeal could be heard emanating from a smaller group of individuals interested i n knowing how these complex natural products are formed i n nature.  I t seemed that with the structure of so many natural  products having been established, i t should be possible to perceive some relations between them.  Such considerations brought numerous  suggestions about the possible biosynthetic pathways which may be involved. The f i r s t forays into the biogenetic speculations began with the recognition of common structure features among the compounds produced by closely related natural organisms.  This recognition led to i n f e r -  ences of a r e l a t i v e l y simple common o r i g i n for these compounds. Fortunately, i t appears that this fantastic array of naturally.occurring compounds are indeed b u i l t up from a r e l a t i v e l y small number of fundamental  templates.  For example, i n the f i e l d of alkaloids the  common building blocks are acetic acid, ornithine and lysine for the reduced systems and tyrosine, phenyl alanine, 3,4-dihydroxyphenylpyruvic acid, and tryptophan for the many bases containing aromatic n u c l e i . Once the fundamental building block for a certain group of compounds i s established this allows one to speculate on the sequence of transformations regarded as feasible i n the l i v i n g c e l l .  Most  fruitful  among the e f f o r t s to v e r i f y these speculations have been the feeding experiments with radioactive precursors.  This i s followed by i s o l a t i o n  of-radioactive natural compound and chemical degradation to i s o l a t e p a r t i c u l a r atoms and examine their r a d i o a c t i v i t y .  I t i s a tribute  to the authors of these biogenetic schemes that they have been so often proved correct. When we examine how> the study of biogenesis of natural products has helped i n our understanding, the following two g r a t i f y i n g features always come to one's mind:  a) f i r s t of a l l , with the emergence of  pathways of biogenesis, i t became possible to organise and divide the natural products into families according to their biogenetic groups. For example, steroids and terpenes, which at one time looked a bewildering array of compounds, now allow convenient c o r r e l a t i o n through their isoprenoid derivation.  Although the present d i v i s i o n  of natural products into biogenetic groups i s sometimes rough and arbitrary and often speculative, i t provides a convenient organization  f o r l e a r n i n g and o r i e n t a t i o n w h i c h i s b e t t e r than has been p o s s i b l e b e f o r e ; b) the b i o g e n e t i c s p e c u l a t i o n s l e d t o the s u c c e s s f u l  construc-  t i o n o f some remarkably s i m p l e l a b o r a t o r y s y n t h e s e s of complex n a t u r a l compounds.  These s y n t h e s e s were modeled on b i o g e n e t i c  lines  and the s y n t h e t i c schemes are t o many c h e m i s t s , more e s t h e t i c a l l y p l e a s i n g and s a t i s f y i n g .  More i m p o r t a n t  however, the  biogenetic  s y n t h e s e s are o f t e n n e a t e r , s h o r t e r and more e f f i c i e n t than normal r o u t e s i n w h i c h no a t t e n t i o n i s p a i d to the n a t u r a l  processess.  Indeed i t i s sometimes found t h a t the most s a t i s f a c t o r y r o u t e t o a p a r t i c u l a r n a t u r a l p r o d u c t i s the b i o g e n e t i c t y p e .  The  Robinson  tropinone  s y n t h e s i s i s an e a r l y but s t i l l an e x c e l l e n t example of  approach.  A d i r e c t method of c o n s t r u c t i n g the complex s t r y c h n i n e  this  2  skeleton,  summarized i n the f o l l o w i n g scheme, i s a n o t h e r i l l u s t r a t i o n . 0  OHC  I t must be emphasized here t h a t our r e a l b i o s y n t h e t i c e v i d e n c e from t r a c e r and enzyme study i s y e t i n i t s i n f a n c y and some of  the  b i o g e n e t i c schemes c o u l d be d i s c r e d i t e d or s e r i o u s l y a l t e r e d .  It is  u n d e n i a b l e t h a t w i t h the study of b i o g e n e s i s , the s c i e n c e of n a t u r a l p r o d u c t s has  r a i s e d to a new  level.  In the annals of biogenetic theory perhaps no single class of natural products has enjoyed more ingenious speculations from the organic chemists than the family of indole a l k a l o i d s , which are formally derived from tryptamine  and a "C_-C y  " unit (for reviews see 1U  references 3-5). Not only the biochemical o r i g i n of the l a t t e r species but i t s appearance i n the well known Corynanthe-Strychnos pattern (10, Figure 1) has provoked stimulating comments ever since Barger^ drew attention to a possible biogenesis of yohimbine i n 1934. Recent s t r u c t u r a l studies have increased the number of these alkaloids to more than 800.^  A tryptamine  residue (2) appears almost 8 9  invariably and i n the few cases examined by the tracer method, ' this residue has been found to be derived i n the expected way from tryptophan  (1).  Tryptamine^ ^  (2) has recently been shown to be  s p e c i f i c a l l y incorporated into several alkaloids of Vinca rosea with considerable v a r i a t i o n i n e f f i c i e n c y , suggesting that decarboxylation may be delayed i n some cases.  Figure 1.  Scheme portraying the proposed relationship between three main classes of indole a l k a l o i d s .  The r e m a i n i n g n i n e o r t e n carbon atoms ( C - C 0  y  xu  "r u n i t ) appear i n  what a t f i r s t s i g h t seems a b e w i l d e r i n g v a r i e t y of d i f f e r e n t arrangements but c l o s e r i n s p e c t i o n a l l o w s t h r e e main groups t o be 13 discerned.  Together t h e s e t h r e e main groups account f o r the v a s t  m a j o r i t y of i n d o l e a l k a l o i d s .  We can c o n v e n i e n t l y r e f e r t o them as  (a) C o r y n a n t h e - S t r y c h n o s t y p e w h i c h p o s s e s s the C -C • .  e.g.  u n i t as  n  c o r y n a n t h e i n e ( 7 ) , a j m a l i c i n e ( 3 ) ; (b) the Aspidosperma  having the  C^-C^Q  (10)  1U  y  type  u n i t as (11) e.g. v i n d o l i n e (5) and (c) the Iboga  s e r i e s where t h e C -C y  u n i t appears as (12) e.g. lu  catharanthine (6). >_ .  I n t h o s e a l k a l o i d s where o n l y n i n e carbon atoms.are p r e s e n t i n a d d i t i o n t o the t r y p t a m i n e r e s i d u e , i t i s i n v a r i a b l y t h e carbon atom i n d i c a t e d by the d o t t e d l i n e t h a t has been l o s t O r i g i n of Cg-C Unit  (Figure 1).  Q  I n c o n t r a s t t o the g e n e r a l agreement by d i f f e r e n t workers w i t h r e g a r d s t o the " t r y p t o p h a n " p o r t i o n of the i n d o l e a l k a l o i d s , the b i o g e n e t i c o r i g i n o f t h e " n o n - t r y p t o p h a n " or C_-C y  - u n i t , has been the xu  s u b j e c t o f much c o n t r o v e r s y . A number of t h e o r i e s d e a l i n g w i t h  this  a s p e c t have been proposed o v e r the y e a r s . 6 I t was suggested many y e a r s ago by B a r g e r i n d o l e a l k a l o i d s such as^ yohimbine  14 and Hahn  t h a t the  (13) a r e formed by a Mannich  between t r y p t a m i n e and 3 , 4 - d i h y d r o x y p h e n y l a c e t y l a l d e h y d e . p r o d u c t o f t h i s r e a c t i o n (14) then undergoes a second  The  reaction initial  Mannich  r e a c t i o n w i t h formaldehyde y i e l d i n g the p e n t a c y c l i c system ( 1 5 ) .  It  was t h e n suggested"*" t h a t the e x t r a carbon atom, w h i c h i s o f t e n a t i n r i n g E ( F i g u r e 2 ) , i s a l s o d e r i v e d from formaldehyde.  A contribution  Corynantheine (7)  Figure 2.  Barger-Hahh-Robinson-Woodward hypothesis.  of Woodward''""'  was t h e s u g g e s t i o n t h a t the c a t e c h o l type r i n g E c o u l d  undergo f i s s i o n t o y i e l d an i n t e r m e d i a t e such as (16). c h a i n s a t t a c h e d t o r i n g D can then undergo p l a u s i b l e w i t h each o t h e r  (e.g. a j ^ m l i c i n e , 3; c o r y n a n t h e i n e ,  The two s i d e condensations  7) o r w i t h o t h e r p a r t s  of t h e m o l e c u l e (e.g. a j ^ m l i n e 7) t o g i v e r i s e t o v a r i o u s types.  structural  W i t h t h i s scheme Woodward was a l s o a b l e t o r a t i o n a l i s e t h e  biogenesis of strychnine (8, Figure 2 ) . However, a number of d e f i c i e n c i e s a r o s e w i t h t h e above t h e o r y 17-19 and i n 1959 Wenkert that prephenic The  acid  l a t t e r rearranges  proposed an e l e g a n t a l t e r n a t i v e .  He suggested  (16) a c t s as a p r o g e n i t o r o f the i n d o l e a l k a l o i d s . a c c o r d i n g t o t h e scheme shown i n F i g u r e 3 t o  a f f o r d a c r u c i a l i n t e r m e d i a t e , t h e seco-prephenate-formaldehyde (SPF) unit  ( 2 0 ) , w h i c h can be i n c o r p o r a t e d i n t o yohimbine (13) and  F i g u r e 3. ' Wenkert's p r e p h e n i c  acid  hypothesis.  corynantheine  (7).  The most a t t r a c t i v e features of this hypothesis are that i t  accounted for the presence of carboxyl group at C^g and also r a t i o n a l i s e s the fact that the hydrogen attached at  i n yohimbine  (13) and related alkaloids almost always has an a-configuration. Wenkert suggested that the a-configuration of the hydrogen atom at C._  i n the intermediate  (17) i s the result of s t e r e o s p e c i f i c migration  of the pyruvate side chain i n compound (16). l i t t l e chance for randomization  at  Furthermore, there i s  i n the subsequent modifications  of (17) to y i e l d the various indole alkaloids. S c h l i t t e r and Taylor  13  i n 1960  and Leete  20-22  i n 1961  postulated  that the "non-tryptophan" portion of the indole alkaloids was v i a the acetate pathway.  The suggestion was  derived  that a s i x carbon chain  derived from three acetate u n i t s , condenses with malonic acid and a one carbon unit ( b i o l o g i c a l l y equivalent to formaldehyde) y i e l d i n g the desired  _ unit (Figure 4). 0 II H-C-H  3CH-C00H  Figure 4.  The acetate hypothesis.  Another hypothesis based on s t r u c t u r a l relationships was suggested independently by Wenkert  17—19  23 and Thomas.  The s t r i k i n g s i m i l a r i t i e s  between the s k e l e t a l features of various monoterpenes, verbenalin (21), g e n t i o p i c r i n (22), bakankasin (23), swertiamarin (24), genipin (25), aucubin (26) andthe seco-prephenate-formaldehyde unit (20, the "C C^Q" -  y  unit) led these authors to suggest that the non-tryptophan portion of the indole alkaloids i s "monoterpenoid" i n o r i g i n  OGlu  OGlu OGlu  CH 00C 3  21  22  23 CH 0H  CH 0H  2  2  OGlu  OGlu  CH 00  CH 00C  3  24  3  25  26  The i n i t i a l biosynthetic experiments using radioactive disproved  precursors-  a l l the hypotheses concerning the genesis of "Cy-C^Q  M  portion  24 of the indole a l k a l o i d s .  It was not u n t i l 1965 that Scott  and  coworkers were the f i r s t to report a successful incorporation of mevalonate (27) into vindoline (5). 25—28 several groups of workers mevalonic acid was incorporated  Subsequent publications by  established that s p e c i f i c a l l y l a b e l l e d into the indole alkaloids i n a manner  consistent with the monoterpene hypothesis.  The next l o g i c a l  precursor  29-32 g e r a n i o l (28) was  found t o be i n c o r p o r a t e d  as an I n t a c t u n i t  into  v i n d o l i n e ( 5 ) , c a t h a r a n t h i n e (6) and a j m a l i c i n e (3) i n V i n c a r o s e a L. shoots.  Each o f these a l k a l o i d s i s r e p r e s e n t a t i v e of one of the t h r e e  types of a l k a l o i d f a m i l i e s found i n t h i s and o t h e r p l a n t s . 4 These f i n d i n g s a l l o w e d B a t t e r s b y  to r a t i o n a l i s e the t h r e e  c a t e g o r i e s of i n d o l e a l k a l o i d s as shown i n F i g u r e 5.  The Corynanthe -  s k e l e t o n (10) i s f i r s t d e r i v e d f o r m a l l y by c l e a v a g e of the g e n e r a l i z e d i r i d o i d p a t t e r n (29).  Loss of one carbon atom ( i n d i c a t e d by b r o k e n  l i n e i n 10) r a t i o n a l i s e s the S-trychnos akuammicine ( 4 ) .  The  "C " u n i t (30) , as found i n  Corynanthe s k e l e t o n i s s c h e m a t i c a l l y r e l a t e d to  the Aspidosperma (11) and the Iboga s e r i e s (12) by c l e a v a g e of the C. _-C^g  bond i n (10) and f o r m a t i o n of the ^2_7~^20 ^ P ' at  1  ^  o r  ^17 ^14 -(  (path B) bonds. The f o r m a t i o n of the yohimbine c l a s s (31) i s a l s o reached from 10, t h i s time by r i n g c l o s u r e v i a C - C bond f o r m a t i o n . 1 7  1 0  1 /  The  f i r s t e v i d e n c e f o r the c y c l o p e n t a n e  lo  i n t e r m e d i a t e i n the 33  pathway was  o b t a i n e d by B a t t e r s b y and coworkers  l o g a n i n (32) was  who  showed t h a t  i n c o r p o r a t e d i n t o a j m a l i c i n e ( 3 ) , v i n d o l i n e (5) and  c a t h a r a n t h i n e (6) i n V i n c a r o s e a L. p l a n t s . c o n f i r m e d by o t h e r workers i n V i n c a r o s e a H0„,  CH 00C 3  These f i n d i n g s were l a t e r and R a u w o l f i a s e r p e n t i n a  F i g u r e 5.  The Thomas-Wenkert  hypothesis.  plants.  The next step forward  i n the continuing search to unfold  the b i o s y n t h e t i c pathway o f i n d o l e a l k a l o i d s , had to w a i t u n t i l t h e 37 s t r u c t u r e of another a l k a l o i d s , i p e c o s i d e  (33) was u n r a v e l e d .  The-  n o n - n i t r o g e n o u s p o r t i o n o f i p e c o s i d e was a l s o found t o be d e r i v e d from  OGlu  CH OOC 3  33 loganin (32).  This l e d Battersby t o suspect that l o g a n i n i s cleaved  to y i e l d secologanin  (36) and the i l l u s t r a t e d p r o c e s s by way o f hydroxy  l o g a n i n ( 3 4 ) , perhaps as i t s phosphate e s t e r ( 3 5 ) , i s a p l a u s i b l e ' one  4,38  OGlu CH 00C 3  CH-OOC  34, X = H 35, X = phosphate  36  Confirmatory evidence f o r the existence o f the h y p o t h e t i c a l  intermediate  39 40 (36) came w i t h t h e i s o l a t i o n (37) , d i h y d r o f o l i a m e n t h i n  '  o f t h r e e new g l u c o s i d e s ,  foliamenthin  ( 3 8 ) , and m e n t h i a f o l i n ( 3 9 ) . Not o n l y a r e  these g l u c o s i d e s o f g r e a t b i o s y n t h e t i c i n t e r e s t i n t h e i r own r i g h t , but they a l s o c o n t a i n s e c o l o g a n i n  (36) i n a masked l a c t o l form.  i n t e r e s t i n g to point out that secologanin  i s a biointermediate  ponding t o (10) i n t h e Thomas-Wenkert h y p o t h e s i s  It is corres-  (Figure 5 ) .  The s y n t h e s i s o f r a d i o a c t i v e ( d o u b l y - l a b e l l e d ) s e c o l o g a n i n and i t s  feeding to Vinca rosea resulted i n the positive incorporation without scrambling ^' "'" of l a b e l . 3  4  A most g r a t i f y i n g outcome of a l l these  elegant l a b e l l i n g experiments was  the finding that the stereochemical  OGlu  37 38, 7' ,8'-reduced  39  i n t e g r i t i e s of -C^ i n the loganin (32) and i t s secoderivative (36) aresmaintained  at C^,. i n the Corynanthe alkaloids.  Furthermore C^ of  the loganin (32) i s carried through to the alkaloids at the aldehyde l e v e l , i . e . , the proton marked with an a s t e r i s k i n geraniol (28) (see Figure 6) survives a l l subsequent rearrangements. requirement  The  latter  assumes prime importance i n formulating and testing the  mechanisms developed  below.  The summary of the geraniol  seco-  38 loganin pathway  i s shown i n Figure 6 and i t includes the recently A- 2 ^\ 3 described i r i d o i d (40) as well as the "secolactone" sweroside (41) which has been b i o l o g i c a l l y converted into the three main classes of indole alkaloids. 4 It has been further argued some years ago  that i f secologanin  (36) condenses with tryptamine, a 0-carboline (e.g. 42) would be formed  OHC  OGlu  OGlu CH.OOC  Figure 6.  36  A summary of geraniol •+ secologanin pathway.  and this l a t t e r substance was suggested as the f i r s t  nitrogenous  intermediate i n the biosynthetic sequence leading to indole alkaloids. Evidence f o r this kind of intermediate was obtained with the discovery of s t r i c t o s i d i n e  (42) (stereochemistry not established) i n  44 Rhazya species  and i t s presence was subsequently demonstrated i n 45  Vinca rosea by d i l u t i o n with radioactive tryptophan and loganin. A mixture of the radioactive isomers, vincoside (43) and i s o vincoside (44) (enantiomeric at C-) prepared from secologanin of known stereochemistry and tryptamine, when fed to Vinca rosea resulted i n the i s o l a t i o n of radioactive ajmalicine (3), vindoline (5), cathar46 47 anthine (6) and perivine (45). ' D i l u t i o n analysis i n Vinca rosea  COOCH  CH^OOC 42  45  4 3 , a«C H44, B-C.H 3 3 plants which had previously taken up [5- H]-loganin, confirmed  that  secologanin (36), vincoside (43) and isovincoside (44) are natural products of the plant.  Subsequently N-acetylvincoside was isolated  from the glycoside f r a c t i o n of Vinca r o s e a ^ i n good y i e l d 1.5 kg) and vincoside (43) was shown to be converted types of indole alkaloids.  (19 mg/  to the three  Isovincoside (44) was not an e f f e c t i v e  47 precursor. With this knowledge i n hand that indole alkaloids are i n fact elaborated monoterpenoids, the next problem was to find out how 3 carboline (42) i s converted into various indole alkaloids.  A major  problem inherent i n these unknown steps concerns the timing and mechanisms of transformations whereby the g-carboline system i s sequent i a l l y transformed  not only to form Corynanthe and Strychnos alkaloids  but how i t rearranges  to the Aspidosperma and Iboga bases.  In this  regard i t i s relevant to mention that Scott's study of the sequential appearance of various alkaloids i n short term (1-300 hours) germinating Vinca rosea has helped considerably i n suggesting the dynamics of the biosynthetic mechanisms.  An interesting account of this work has been  r e v i e w e d v e r y r e c e n t l y by S c o t t  and a summary of t h e s e r e s u l t s i s  shown i n T a b l e I .  Table I .  I s o l a t i o n of A l k a l o i d s from V i n c a r o s e a S e e d l i n g s  Germination time, hr 0 26  28-40  40-50  72 100-160 200  Alkaloids  isolated  Type  None V i n c o s i d e (43) A j m a l i c i n e (3) C o r y n a n t h e i n e (7)  "Corynanthe"  C o r y n a n t h e i n e aldehyde (50) G e i s s o s c h i z i n e (51) 3 - H y d r o x y i n d o l e n i n e (57) " D i o l " (58) G e i s s o s c h i z i n e O x i n d o l e (59)  Corynanthe  Preakuammicine (56)" Akuammicine (4) Stemmadenine (55) T a b e r s o n i n e (71) 11-Methoxytabersonine  Corynanthe "Corynanthe-Strychnos" "Strychnos "Corynanthe-Strychnos"  (77)  Aspidosperma  C a t h a r a n t h i n e (6) C o r o n a r i d i n e (78)  Iboga  V i n d o l i n e (5)  Aspidosperma  The b i o l o g i c a l c o n v e r s i o n o f v i n c o s i d e (43) i n t o v a r i o u s i n d o l e ' a l k a l o i d s c o u l d r e a s o n a b l y i n v o l v e c l e a v a g e o f t h e g l u c o s e u n i t t o form v i n c o s i d e aglucone  (46) which would be i n e q u i l i b r i u m w i t h , o r con-  v e r t i b l e i n t o aldehyde  (47,48).  R i n g c l o s u r e t o N(b) (see 49, F i g u r e  7) and r e d u c t i o n c o u l d then l e a d t o c o r y n a n t h e i n e aldehyde  (50) and/or  g e i s s o s c h i z i n e ( 5 1 ) . A j m a l i c i n e ( 3 ) , an abundant a l k a l o i d o f V i n c a r o s e a  could be reached by c y c l i z a t i o n of the species 51 or 49 since the C 3 proton of the ajmalicine (3) i s not l a b e l l e d by loganin-2E a r l i e r feeding experiments i n Vinca r o s e a ^  20  49 H.  established the intact  incorporation of geissoschizine (51) into ajmalicine (3), akuammicine (4), vindoline (5), and catharanthine  (6).  The incorporation of  corynantheine aldehyde (50) into vindoline (5) and catharanthine  (6)  has been reported i n Vinca rosea s e e d s . T h i s finding i s i n sharp contrast to the i n s i g n i f i c a n t incorporation of this substance i n mature plants.50>51  fj  observations,  has suggested that i t could be possible that seedings  o w e v e r  are able to convert  Battersby,"^  (50) to (51).  i n trying to reconcile these  Recently  geissoschizine (51)  has  been shown to be a component present both i n Vinca rosea plants"*^ and 52 Vinca rosea seeds (28-40 hours f r a c t i o n ) .  This study therefore  suggests that geissoschizine (51) stands as a key Corynanthe a l k a l o i d beyond vincoside (43) on the biosynthetic pathway.' A most g r a t i f y i n g outcome of these elegant l a b e l l i n g studies  was  the rearrangement (a ->- 3) of geissoschizine (51) to form the 'Strychnos skeleton of akuammicine (4)."*^' This observation was  particularly  important because this rearrangement generates the bond between C^g  (see 51 and 56 i n Figure 8).  Since i t i s necessary to oxidize the  Corynanthe series i n order to reach the'Strychnos l e v e l , - i t 19 suggested  and  was  53 '  that this process when applied to geissoschizine  (51)  involves one electron oxidative coupling to give strictamine (52^ R = CHO).  '••  Precedent for the rearrangement of compounds such as (53) 54  to the Strychnos representative, akuammicine (4) i s available, that the indolenine (54) or i t s reduced form (56)  so  (Figure 8) could be  COOCH 4  Figure 8.  The rearrangement of Corynanthe -> Strychnos skeleton.  reached by such a mechanism.  An alternative to this mechanism, also  an i n v i t r o a n a l o g y , i s a-protonation of the indole nucleus followed by the a •* g rearrangement summarized i n Figure 8. this study was  Consistent with  the i s o l a t i o n of " C ^ Q " and "C^" Strychnos alkaloids  preakuammicine (56) and akuammicine (4) i n the 45-50 hours f r a c t i o n of Vinca rosea seeds. More recently Scott"^ has suggested  a t h i r d mechanism (Figure 9)  which imputes an intermediary role.to an unknown a l k a l o i d geissoschizine oxindole (59).  The formation of such an oxihdole from geissoschizine  (51) has ample i n v i t r o precedence and might take place i n vivo by the steps indicated i n Figure 9, where the g-hydroxy indolenine (57) i s rearranged d i r e c t l y or v i a the dihydroxyindoline ("diol" 58) to (59). Conversion of 59 to the imino ether (60, R = a l k y l or enzyme bound functionality) would endow 60 with r e a c t i v i t y required"^ to form preakuammicine (56), as shown. Im.support of this mechanism Scott"^ was very g r a t i f i e d to find geissoschizine oxindole (59) ( i d e n t i c a l with the synthetic material) i n the 45-hour f r a c t i o n of Vinca rosea seeds. To summarise a l l the work up to here, i t i s safe to say that a l l the steps involved i n the biochemical conversion of secologanin to Corynanthe alkaloids have become clear.  With the study of sequential  i s o l a t i o n of various biointermediates, the steps involved i n the rearrangement of Corynanthe skeleton to Strychnos  skeleton have just  begun to unfold themselves.  The Biogenesis of Aspidosperma and Iboga Alkaloids A most ingenious idea was  adduced by Wenkert"*"^ '"^ to r a t i o n a l i s e  the transformation of the Corynanthe skeleton to the Aspidosperma and  Figure 9.  Scott's scheme for the rearrangement of Corynanthe •> Strychnos skeleton.  Iboga type  ( F i g u r e 10)  l e v e l i n Figure 5).  (type A and B t r a n s f o r m a t i o n s a t the a l k a l o i d  The  rearrangements suggested  r e q u i r e d the presence of the 1 , 5 - d i c a r b o n y l  f o r p a t h s A and  f u n c t i o n i n order to  B operate  the r e v e r s e M i c h a e l r e a c t i o n i m p l i c i t i n the c l e a v a g e o f C-^-C^g. the c l e a v a g e p r o d u c t  Thus  (63) undergoes o r d i n a r y o x i d a t i o n - r e d u c t i o n  changes and i n t h i s manner p i p e r i d i n e s o f v a r i o u s o x i d a t i o n s t a t e s are formed.  I n t r a m o l e c u l a r M i c h a e l and Mannich r e a c t i o n s of the  l e a d to Aspidosperma (65) and Iboga l i k e  (68) s k e l e t o n s .  latter  However,  f e e d i n g experiments w i t h r a d i o a c t i v e p r e c u r s o r s r a i s e d some s e r i o u s doubts r e g a r d i n g some of the s t e p s d e p i c t e d i n F i g u r e 10.  For example  t r a n s a n n u l a r c y c l i z a t i o n u t i l i z e d by Wenkert to p r o v i d e an e n t r y i n t o Aspidosperma (64 ->- 65) and Iboga (67 -»• 68) s k e l e t o n s , i n s p i t e of having e x c e l l e n t analogies i n v i t r o " ^ ^ an i n s i g n i f i c a n t b i o c h e m i c a l r e a c t i o n .  was  shown by K u t n e y ^ ' ^ 3  4  to be  T h i s l a t t e r study suggested  that  the g e n e s i s o f p e n t a c y c l i c a l k a l o i d s (Aspidosperma type) e\g. v i n c a d i f f o r m i n e (70) i s c o m p l e t e l y independent of the nine-membered a l k a l o i d s e.g. v i n c a d i n e ( 6 9 ) .  C00CH„ 69  COOCH. 70  Aspidosperma-type alkaloids Figure 10.  68  0  Wenkert's proposal for the biosynthesis of Aspidosperma and Iboga alkaloids.  Returning to the chronological i s o l a t i o n of various alkaloids . from Vinca rosea seeds (Table I) Scott"^ found another stemmadenine (55) i n the 50-hour experiment.  alkaloid  It i s very interesting to  point out here that the possible intermediacy of units similar i n structure to stemmadenine e.g. 62 was  invoked i n the sequence between  the Corynantheinoid and Aspidosperma bases by Wenkert"^ '"^ (Figure 10) many years ago.  When the germination was allowed to proceed further  j (72 hours), i t led to the i s o l a t i o n of an Aspidosperma a l k a l o i d , tabersonine (71).  Catharanthine  (6), the p r i n c i p a l Iboga a l k a l o i d of  Vinca, although isomeric with tabersonine, does not appear to be formed u n t i l the germination has proceeded for 100 hours.  This was  a  v i t a l piece of evidence i n suggesting a rough sequence of alkaloid formation i n nature i . e. I stemmadenine (55) -> tabersonine catharanthine (6).  (71)  The biochemical conversion of tabersonine  (71)  to vindoline (5) and most i n t e r e s t i n g l y to the Iboga a l k a l o i d 64 catharanthine (6) has been demonstrated i n our laboratories independently by Scott"'"'" i n Vinca rosea seeds.  and  These l a t t e r results  suggest a possible relationship between the Aspidosperma and Iboga alkaloids.  Similarly b e l i e f i n stemmadenine (55) as a true b i o i n t e r -  mediate was  further strengthened by i t s incorporation into tabersonine  (71) and catharanthine (6) i n Vinca rosea seeds"'"'" and into vincamine (72) and minovlne (73) i n Vinca minor plants i n our l a b o r a t o r i e s . ^ 66 Scott and Qureshi  reported the rearrangement of tabersonine (71) to  (J)-rcatharanthine (6) and (±)-pseudocatharanthine (74) i n refluxing acetic acid.  Stemmadenine (55) under similar conditions (refluxing  acetic acid)rearranged to (_)-tabersonine  (71), (i)-catharanthine (6),  and Gt)-pseudocatharanthine  (74).  These results were portrayed as  a laboratory simulation of the biochemical results described above. 67 However, l a t e l y Smith  i n s p i t e of many repeated attempts f a i l e d to  duplicate Scott's i n v i t r o results. A most a t t r a c t i v e mechanism l i n k i n g stemmadenine (55), tabersonine 64 (71), and catharanthine (6) was  advanced by.Kutney.  This involves  the a c h i r a l intermediate 76a=76b, which can, i n p r i n c i p l e be generated by migration of the double bond i n stemmadenine (55) to (75), followed by the i l l u s t r a t e d fragmentation  (Figure 11).  A similar  postulate for the formation of the a c r y l i c ester (76) has been independently advanced by Scott*^.  i n order to explain the observed  71, T a b e r s o n i n e  .I  (R=H)  77, 11-Methoxytabersonine  •1  5, V i n d o l i n e  F i g u r e 11.  (R=0CH~)  6, C a t h a r a n t h i n e ^ 15 2 0 ^ 78, C o r o h a r i d i n e ; A '. - — r e d u c e d  Some l a t e r s t a g e s of i n d o l e a l k a l o i d  biosynthesis.  sequence, i t i s suggested that the enzymatic folding of 76 i n mode A would give tabersonine (71), and l a t e r , at an other enzymatic  site,  c y c l i z a t i o n iniwmode B forms catharanthine (6) . Yet a t h i r d p o s s i b i l i t y C explains the genesis of the vincadine (69) series.  The r e l a t i v e  63 64 i n s i g n i f i c a n c e of the transannular c y c l i z a t i o n  '  now  suggest that  the process 7 ->• 21 occurs prior to or simulataneously with 17  20  i n the elaboration of the putative intermediate (76) to vincadifformine (70).  In a similar fashion the conversion of the u n i t (76) to the  a l k a l o i d catharanthine (6) i s unlikely to proceed i n i t i a l l y v i a the process 17 -> 14, system (79).  since this would lead to a carbomethoxycleavamine 63 Previous results i n our laboratory have suggested that  carbomethoxycleavamine (79) i s not a progenitor of this Iboga alkaloid (6).  This theory therefore places stemmadenine (55) i n a key position  between the Strychnos and other families not only i n yinca rosea but predictably i n a l l species, and furthermore r a t i o n a l i z e s the formation of racemic Aspidosperma alkaloids such as (+)-vincadifformine (70) mediated by the a c h i r a l ester (76).  The absolute minimum of function-  a l i t y has been used for a l l of these postulated interconversions, and  i t i s g e n e r a l l y b e l i e v e d t h a t t h e proposed b i o g e n e s i s species.  Thus i t so t u r n e d o u t t h a t t h e g a l a x y  i s common t o a l l  o f complex, oxygenated,  f r a g m e n t a t e d , and r e a r r a n g e d s t r u c t u r e s w h i c h c o n s t i t u t e s t h e complex series of indole a l k a l o i d s i n fact.stem alkaloids.  from these few fundamental  I t must be emphasized h e r e t h a t a l t h o u g h a l l t h e e v i d e n c e  i n d i c a t e d t o d e v e l o p * t h e above t h e o r y r e p r e s e n t  an i m p o r t a n t m o d i f i c a t i o n  of Wenkert's o r i g i n a l t h e o r y p a r t i c u l a r l y w i t h r e g a r d s t o sequence, o x i d a t i o n l e v e l and mechanisms, t h e s e r e s u l t s do n o t d e t r a c t from t h e e s s e n t i a l correctness  o f h i s v i e w s on t h e i n t e r r e l a t i o n s h i p o f t h e  main c l a s s e s o f i n d o l e a l k a l o i d s . I n summary, a l l t h e a v a i l a b l e r e s u l t s suggest v e r y  emphatically  t h a t t h e a c y l i c e s t e r (76) would f u l f i l a p i v o t a l r o l e i n t h e g e n e s i s of v a r i o u s f a m i l i e s o f i n d o l e a l k a l o i d s .  Laboratory  analogies f o r  almost a l l o f t h e suggested p r o c e s s e s a r e now a v a i l a b l e . establishment ship  With the  o f t h e Corynanthe-Strychnos-Aspidosperma-Iboga r e l a t i o n -  Chased on s e q u e n t i a l i s o l a t i o n and f e e d i n g e x p e r i m e n t s ) t h e 4  various f u r t h e r subclasses  should  f a l l into place.  I n 1967 B a t t e r s b y  made t h e statement "The problem i s a t a most f a s c i n a t i n g s t a g e where the r e s e a r c h e r  can see t h a t t h e p r e c i s e d e t a i l o f t h e pathways t o t h e  i n d o l e a l k a l o i d s cannot now escape him". -  a s  summarized above p r o v i d e s  The r a p i d l y e v o l v i n g scene  ample p r o o f i n s u p p o r t o f t h i s v i e w .  DISCUSSION With the knowledge that indole alkaloids are i n fact elaborated monoterpenoids, we were intrigued by the second major problem posed by the structures before us; how are the rearrangement of Corynanthe to Aspidosperma and Iboga skeletons carried out i n nature  (summarized i n Figure 11), and  where would we begin to test the v i r t u a l myraid of possible substrates designed  to undergo the A and B transformations?  I t was f u l l y revealed i n  53 the introduction that Wenkert's  speculations on the mechanisms of  rearrangements involve the 'acrylic acid (66) and i t s dihydro-derivative (63) as intermediates, whilst Kutney's^  4  and S c o t t ' s ^ results led them to  propose a d i f f e r e n t mechanism making use of acrylic.- ester .(76) and the corresponding  enamine.  The intermediacy of .acrylic'j ester (76) was again  66 invoked by Scott  to explain the i n v i t r o transformation of tabersonine  (71) to (+)-catharanthine  (6) and (+)-pseudocatharanthine (74); and of  stemmadenine (55) to (i)-tabersonine (71), (+)-catharanthine  (6) and (+)-  pseudocatharanthine (74) (as already mentioned e a r l i e r , these l a t e r trans67 formations have now been questioned  ). In summation, a l l these results  provided a strong suggestion that the acrylic"ester (76) may be a true biointermediate.  We decided that i n spite of many problems associated with  the synthesis and feeding of this putative intermediate  (76) , the knowledge  gained by i t s evaluation as a biogenetic intermediate would be of greatest value i n suggesting the dynamics of the biosynthesis of Aspidosperma and  Iboga a l k a l o i d s . To examine the b i o g e n e t i c r o l e of any i n t e r m e d i a t e , the s t e p i n v o l v e s the s y n t h e s i s o f the p o s t u l a t e d p r e c u r s o r .  first  T h i s i s then  f o l l o w e d by f e e d i n g the a c t i v e p r e c u r s o r i n t o the a p p r o p r i a t e p l a n t system and i s o l a t i o n o f the a l k a l o i d s a f t e r a c e r t a i n p e r i o d t o examine the amount o f r a d i o a c t i v i t y i n them.  I t was  o f time  c l e a r to us at  the o u t s e t o f our s y n t h e t i c work t h a t (76) because o f the presence of a d i h y d r o p y r i d i n e segment i n i t would be a v e r y u n s t a b l e compound. There was  c o n s i d e r a b l e p r e c e d e n t a v a i l a b l e i n the l i t e r a t u r e w h i c h  could lend support  to our i n i t i a l doubts.  For example, i t i s w e l l  known t h a t d i h y d r o p y r i d i n e s r e a d i l y o x i d i z e to the pyridines.  This process  corresponding  o c c u r s so r a p i d l y t h a t even c o n t a c t w i t h  a t m o s p h e r i c oxygen i s s u f f i c i e n t to b r i n g about the  transformation.  T h i s p r o p e r t y t h e r e f o r e makes the c h a r a c t e r i z a t i o n and study of  the  68 p r o p e r t i e s of the d i h y d r o p y r i d i n e s r a t h e r d i f f i c u l t .  Another r e a c t i o n  c h a r a c t e r i s t i c of d i h y d r o p y r i d i n e s i s t h e i r r a p i d i s o m e r i z a t i o n . Although  complete e x p e r i m e n t a l  d e t a i l s a r e hot known,, . any  reagent  t h a t can a s s i s t i n the removal of a p r o t o n , hydrogen atom, or 68 i o n may  cause i s o m e r i z a t i o n .  i s o m e r i z a t i o n of 1,2-  The  f o l l o w i n g scheme i l l u s t r a t e s  and 1 , 4 - d i h y d r o p y r i d i n e s  +  1  R  the  through a p y r i d i n i u m i o n . + R  R  hydride  R'  I n v e s t i g a t i o n s d i r e c t e d towards e l u c i d a t i n g t h e s t r u c t u r e s and mode o f b i o l o g i c a l a c t i o n o f coenzymes NAD and NADP have f r e q u e n t l y engendered s t u d i e s on t h e c h e m i s t r y  of dihydropyridines.  On s e v e r a l  69 occasions,  t h e l a t t e r have been found t o d i m e r i z e .  W i t h a l l t h i s knowledge in.'hand, i t became o b l i g a t o r y t o make some model compound as our s y n t h e t i c t a r g e t .  We u n d e r s t o o d t h a t  this  model compound w h i l e s u f f i c i e n t l y s t a b l e i n i t s own r i g h t ( t o a l l o w c h a r a c t e r i z a t i o n ) s h o u l d be c a p a b l e o f t r a n s f o r m a t i o n putative intermediate  i n vivo to the  (76) v i a b i o l o g i c a l l y f e a s i b l e r e a c t i o n s .  One  such compound w h i c h m e t ^ a l l t h e s e p r e r e q u i s i t e s was 1 6 , 1 7 - d i h y d r o s e c o d i n 17-ol  (90, see F i g u r e 12) ( t h e name f o r t h i s compound was suggested by  Battersby,^  numbered a c c o r d i n g  compound upon d e h y d r a t i o n  to b i o g e n e t i c p r i n c i p l e s ^ ) .  (COOCH^-C H  y-^ OH  o x i d a t i o n i n t h e p i p e r i d i n e r i n g (H-C-Nthe d e s i r e d i n t e r m e d i a t e .  This  •-—• C00CH.-C=CH-) and +  —>- -C=N-) c o u l d  generate  Since both these r e a c t i o n s are b i o g e n e t i c a l l y .  f e a s i b l e , we made t h e a l c o h o l (90) as o u r i n i t i a l s y n t h e t i c t a r g e t . For t h e sake o f convenience and ease of p r e s e n t a t i o n , t h e d i s c u s s i o n has been d i v i d e d i n t o two p a r t s .  The f i r s t p a r t d e s c r i b e s  the synthesis 3  of 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l (90) as w e l l as s y n t h e s e s o f [ a r - H]-16,1714 dihydrosecodin-17-ol (90) and [ COOCH ] - 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l ( 9 0 ) . 14 F i n a l l y feeding of the C - p r e c u r s o r i n t o V i n c a minor L. i s p r e s e n t e d . The second p a r t d e s c r i b e s t h e s y n t h e s e s o f s e c o d i n e ( 1 0 7 , t h e name f o r ..96.i - 3 t h i s compound was suggested by Smith- ') , [ a r - H]-secodine (107) and feeding of the a c t i v e precursor  i n t o V i n c a m i n o r L.  PART I The  d e s i r e d a l c o h o l (90) was amenable t o s y n t h e s i s by t h e r o u t e  shown i n F i g u r e 12.  The c h o i c e o f t h i s r o u t e was d i c t a t e d by t h e f a c t  t h a t i t was p o s s i b l e t o s y n t h e s i z e  2-carboethoxy-3-(g-chloroethyl)-  i n d o l e (80) i n t h e l a b o r a t o r y i n r e a s o n a b l e  quantities.  This m a t e r i a l  i n t r i n s i c a l l y i n c o r p o r a t e d a l l the s t r u c t u r a l requirements s t a r t o u r sequence.  needed t o  F o r example t h e p r e s e n c e o f the a-carboethoxy  group a c t e d as a h a n d l e i n a l l o w i n g us t o expand the s i d e c h a i n a t t h e a - p o s i t i o n o f t h e i n d o l e i n (80) t o t h e d e s i r e d f u n c t i o n a l i t y .  On t h e  o t h e r hand presence o f c h l o r i n e i n t h e 3 - ( g - c h l o r o e t h y l ) s i d e c h a i n a l l o w e d us t o a t t a c h t h e a p p r o p r i a t e d s u b s t i t u t e d p y r i d i n e t o t h e indole nucleus. The  s y n t h e s i s o f t h e d e s i r e d c h l o r o i n d o l e (80) was d e v i s e d a few  y e a r s ago i n our l a b o r a t o r i e s i n c o n n e c t i o n w i t h some o t h e r work on the t o t a l s y n t h e s i s o f i n d o l e a l k a l o i d s ^ and t h e sequence i s f u l l y 72 r e v e a l e d i n F i g u r e 13. prepared  Diethyl-y-chloropropylmalonate  (91) was  i n 70% y i e l d by t r e a t i n g t h e monosodium s a l t o f t h e d i e t h y l -  malonate w i t h 1,3-bromochloropropane. (91) was c o n v e r t e d  The c h l o r o m a l o n a t e d e r i v a t i v e  i n t o the corresponding  arylhydrazone 73 74  the agency o f a Jap'p-Klingemann r e a c t i o n .  '  (93) through  This procedure  i n v o l v e d t h e slow a d d i t i o n o f anhydrous benzenediazonium c h l o r i d e ^ to t h e a n i o n o f (91) i n e t h a n o l a t -5°. The r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d o v e r n i g h t i n t h e r e f r i g e r a t o r and t h e crude r e a c t i o n p r o d u c t was s u b j e c t e d t o a F i s c h e r i n d o l e s y n t h e s i s ^ u s i n g  sulfuric  a c i d as c a t a l y s t . We would l i k e h e r e t o make c e r t a i n remarks r e g a r d i n g t h e p r e p a r a t i o n  OH <j)COCl  Figure 12.  Synthesis of 16,17-dihydrosecodin-17-ol (90).  COOEt I  Cl-(CH_)_-Br z 3  +  CI-(CH ) -CH(COOEt)  CH_ j z COOEt  3  91 COOEt EtOOC i ) NaOEt  -  +  i i ) C , H j L c i or C,H N BF. 6 5 2 6 5 2 4 C  >  0  92  COOEt  EtOH  COOEt 80  93 F i g u r e 13.  Synthesis of 2 - c a r b o e t h o x y - 3 - ( 3 - c h l o r o e t h y l ) - i n d o l e  o f anhydrous benzenediazonium c h l o r i d e .  (80)  The u s u a l method f o r making  t h i s d i a z o s a l t i s g i v e n by Smith and W a r i n g . ^  These a u t h o r s  t h a t t h e i r method g i v e s the s a l t i n c r y s t a l l i n e form.  report  However our  e x p e r i e n c e w i t h t h i s procedure i s i n sharp c o n t r a s t to t h i s c l a i m . I n s p i t e of h a v i n g f o l l o w e d the r e p o r t e d p r o c e d u r e v e r y we  carefully,  c o n t i n u a l l y ended up w i t h lumps of the s o l i d d i a z o n i u m c h l o r i d e .  S i n c e the r e a c t i o n c o n d i t i o n s demanded t h a t we add the d i a z o n i u m i n s m a l l p o r t i o n s , t h e lumps had nitrogen).  to be b r o k e n i n t o s m a l l p i e c e s  salt (under  T h i s d i d not pose much o f a problem i n s m a l l s c a l e  r e a c t i o n s , but i t s t a r t e d r a i s i n g  i t s u g l y head when the r e a c t i o n was  scaled up to obtain larger quantities of the chloroindole (80).  In  one of the instances the crushing of the lumps into small pieces resulted i n a very (Violent explosion.  A l l of these unfortunate incidents  kept on thwarting our progress for a long time as the supply of chloroindole (80) remained very limited. 6,7-diazasteroid  In the meantime a new  synthesis of  (95, see Figure 14) appeared i n the l i t e r a t u r e ^  the use of m-methoxybenzenediazonium fluoroborate was  reported.  and This  78 led us to wonder i f we could also use the more stable fluoborate i n our sequence.  Indeed coupling of the anion of (91) with  benzenediazonium f l u o b o r a t e ^ ' ^ gave a deep red o i l .  Figure 14.  benzenediazonium  Synthesis of 6,7-diazasteroid  subjected to a Fischer indole synthesis.  Thisvproduct  was  (95).  P u r i f i c a t i o n of the crude  product by chromatography on alumina gave a c r y s t a l l i n e compound which showed the same R  f  value on thin layer chromatography ( t . l . c . ) and  spectral properties as the chloroindole (80) obtained when benzenediazonium chloride was used. undertaking were that:  The most gratifyingAoutcomesof  this  (a) benzenediazonium fluoborate, when dry was  always a very f i n e powder (like talcum face powder), a s i t u a t i o n which remarkably s i m p l i f i e d our technical problem i n the Japp-Klingemann reaction namely the slow addition of diazonium s a l t to the anion of ( 9 1 ) ; (b) the fact that the fluoborate s a l t was f a i r l y stable and easy to handle, allowed us to scale up the preparation of this s a l t to 120 gm/batch, thereby allowing large scale preparation of the chloroindole ( 8 0 ) . W i t h a l l these problems unraveled, we were able to s t a r t our synthetic sequence with  confidence.  For various reasons we thought the best reaction to s t a r t ( i n i t i a l l y was the coupling of 3-ethylpyridine to the chloroindole ( 8 0 ) . F o r this purpose 3-ethylpyridine (81, bp 162-163°) was r e a d i l y obtained from commercially available 3-acetylpyridine by means of W o l f f - K i s h n e r 80 reduction.  C o n d e n s a t i o n of chloroindole (80) with 3-ethylpyridine  gave a white amorphous s o l i d (82) i n 91% y i e l d . was used d i r e c t l y for the succeeding step.  I n general this s a l t  However a small amount of  material was c r y s t a l l i z e d for a n a l y t i c a l purposes, mp 87-89°. The spectral data compared favourably with the assigned  structure ( 8 2 ) .  The infrared spectrum indicated a strong ester absorption at 1701 cm I n the nmr spectrum the resonances of the two ethyl groups (-CH^CH^, -C00C H,_) were c l e a r l y separated; two t r i p l e t s at x 8.98 (3H, -CH^S'CH^) 2  and 8.63 (3H, -COOCH^CH^) ; two quartets at T 7.40 (2H, -CH_ CH ) and 2  5.74  (2H, -COOCH^CH^).  3  I n the spectrum the resonances corresponding to  the aromatic protons of the pyridinium nucleus (4H, x 1.4-2.34) and of  the i n d o l e n u c l e u s  (4H,:;T 2.56-3.18) were a l s o c l e a r l y  discerned.  Because o f the o v e r l a p p i n g a b s o r p t i o n s of the p y r i d i n i u m and n u c l e i , the u l t r a v i o l e t spectrum was the m o l e c u l a r metry (M  +  not too i n f o r m a t i v e .  f o r m u l a , '-'20^23^2^2^''"'  W a S  s u  PP  o r t e <  * by  m  a  s  indole  Finally spectro-  s  358).  W i t h the p y r i d i n i u m s a l t (82) i n hand, r e d u c t i o n t o the t e t r a h y d r o p y r i d i n e (83) was i f we  then c o n s i d e r e d .  At' t h i s stage i t was  c o u l d use l i t h i u m aluminum h y d r i d e  (LAH)  r e d u c t i o n o f the p y r i d i n i u m segment i n s a l t  thought t h a t  f o r t h i s purpose,  (82) to the  tetrahydro-  p y r i d i n e s t a g e would be accompanied by c o n v e r s i o n o f the e s t e r f u n c t i o n to the d e s i r e d p r i m a r y a l c o h o l (84).  However a survey of the  literature  r e v e a l e d t h a t w h i l e t h e r e i s a g e n e r a l agreement between v a r i o u s w o r k e r s t h a t sodium b o r o h y d r i d e  always reduces N - a l k y l p y r i d i u m  salts  81—83 to t h e i r c o r r e s p o n d i n g the LAH  tetrahydropyridines,  r e d u c t i o n s are a t v a r i a n c e .  the r e s u l t s  For example, i t was  from  found by  81 Panouse  t h a t N - a l k y l p y r i d i n i u m s a l t s a r e reduced by LAH  1,2-dihydropyridines.  Reduction  to  1-alkyl-  of N - [ 6 - ( 3 - i n d o l y l ) - e t h y l ] - p y r i d i n i u m 82  bromide (96) by means of sodium b o r o h y d r i d e - o r  LAH  to l e a d e x c l u s i v e l y t o . t h e t e t r a h y d r o p y r i d i n e ( 9 7 ) .  has been r e p o r t e d 83 L a t e r Wenkert  r e p o r t e d the f o r m a t i o n of t h r e e compounds (97-99) upon r e d u c t i o n of  3' 98, A  -reduced  (96) w i t h LAH.  A l l these c o n t r a d i c t o r y f i n d i n g s diminished  e n t h u s i a s m f o r LAH c h l o r i d e (82) was  reduction.  Indeed when a s m a l l amount of  exposed to LAH,  the crude p r o d u c t was  pyridinium  we were not s u r p r i s e d to f i n d  a complicated mixture  more s u c c e s s f u l a l t e r n a t i v e i n v o l v i n g two desirable results.  our  that  of s e v e r a l components.  r e d u c t i v e steps provided  Thus sodium b o r o h y d r i d e  the  r e d u c t i o n of the s a l t  t o the t e t r a h y d r o p y r i d i n e (83) f o l l o w e d by LAH  r e d u c t i o n of the  A  (82)  carbo-  ethoxy group i n the l a t t e r y i e l d e d a l c o h o l ( 8 4 ) . The  nmr  informative.  spectrum o f the crude sodium b o r o h y d r i d e The  resonance c o r r e s p o n d i n g  p r o d u c t was  t o the f o u r p r o t o n s of  p y r i d i n i u m n u c l e u s i n (82)  (x 1.2-2.34) had  completely  i n s t e a d a b r o a d one p r o t o n  s i n g l e t a t x 4.5  was  p r e s e n c e of an o l e f i n i c p r o t o n a t C ^ v - -  very  In t h i s  the  disappeared  reconcilable with  and the  tetrahydropyridine  ( 8 3 ) , the i s o l a t e d double bond has been t e n t a t i v e l y p l a c e d i n the - 3' A  4' '  . The  I t s p o s i t i o n would be made unambiguous i n the next s t e p . crude t e t r a h y d r o p y r i d i n e e s t e r (83) o b t a i n e d above  was  s u b j e c t e d d i r e c t l y to the l i t h i u m aluminum h y d r i d e r e d u c t i o n . r e s u l t a n t l i g h t y e l l o w gum alumina provided  w h i c h was  p u r i f i e d by chromatography on  a c r y s t a l l i n e compound (84) , mp  o v e r a l l y i e l d of 70% from the c h l o r o i n d o l e (80). was  no-.absorption  The  structure  (84)  I n the i n f r a n  i n ; t h e - e s t e r r e g i o n , and i n s t e a d a broad  band a t 3340 cm ^ (-CH^OII) was 15)  108^-110°, i n an  a s s i g n e d on the b a s i s of the f o l l o w i n g s p e c t r a d a t a .  t h e r e was  The  now  evident.  The nmr  spectrum  ^  (Figure  showed the newly formed hydroxymethylene .'.group as a sharp s i n g l e t  a t T.5.17.  The  o l e f i n i c proton  ( C ^ i - H ) was  l o c a t e d a t x 4.45 i n good 83 agreement w i t h the assignment r e p o r t e d by Wenkert. The mass spectrum  Figure 15.  Nmr spectrum of alcohol 84.  ( F i g u r e 16) showed a m o l e c u l a r i o n peak at m/e by two s i g n i f i c a n t peaks a t m/e  160 and m/e  284 and was  124.  dominated  These peaks were  a t t r i b u t e d t o the s i m p l e f r a g m e n t a t i o n of the p a r e n t m o l e c u l e to the i o n s (100) and  (101) r e s p e c t i v e l y .  C. H .ON , was l o _4 _  c o n f i r m e d by h i g h r e s o l u t i o n mass s p e c t r o m e t r y  0  284.184; C a l c . : Now  F i n a l l y the m o l e c u l a r f o r m u l a ,  (Found:  284.188) and e l e m e n t a l a n a l y s i s .  to e l a b o r a t e the s i d e c h a i n a t the a - p o s i t i o n of the i n d o l e i n  a l c o h o l (84) to the d e s i r e d f u n c t i o n a l i t y , i t was n e c e s s a r y a t t h i s stage to i n c o r p o r a t e an e x t r a carbon atom. proved most d e s i r a b l e f o r t h i s purpose. (84) to benzoate  (85) was  The sequence, 84 -> 86,  The c o n v e r s i o n of the a l c o h o l  a c c o m p l i s h e d by d i s s o l v i n g the a l c o h o l i n  d r y p y r i d i n e and t r e a t i n g i t w i t h b e n z o y l c h l o r i d e . was  The whole r e a c t i o n  over w i t h i n t h r e e hours and the crude p r o d u c t o b t a i n e d was homo-  geneous on t i c .  T h i s a l l o w e d i t s u t i l i z a t i o n i n the s u c c e e d i n g s t e p  w i t h o u t any p u r i f i c a t i o n . of m a t e r i a l was  However f o r a n a l y t i c a l purposes  a s m a l l amount  f u r t h e r p u r i f i e d by chromatography on alumina  and  r e c r y s t a l l i z a t i o n from methylene c h l o r i d e and p e t r o l e u m e t h e r , mp 110.5-112.5°.  The  s p e c t r a l d a t a of the benzoate  a c c o r d w i t h the f o r m u l a t i o n . benzoate  e s t e r a t 1715  The  (85) was  in  complete  i n f r a r e d showed the c a r b o n y l of the  cm ^ w h i l e i n the nmr  spectrum,  the a r o m a t i c  CN  ALCOHOL (84)  in. >-  f—  CO  s !  ' /.I  UJ  I—in. Ijj  ct:  £75  50  i ""i  100  " i—r  150  200  M/E Figure 16.  Mass spectrum of alcohol 84.  i—i  250  r^-r  "i—i—i  300  r  i  350  i  i  r  400  protons of the benzoyl group although overlapping with the protons of the indole nucleus, appeared between x 2-3 indicated a molecular ion peak at m/e  388;  (9H).  The mass spectrum  F i n a l l y the molecular  formula, C „ H 0 „ N , was confirmed by high resolution mass spectrometry zo  (Found:  o  oc  ZJ  z  o  z  388.215; Calc.:  388.216) and elemental analysis.  Although the above procedure for making the benzoate derivative (85) was quite s a t i s f a c t o r y , i t was observed that i f a l l the; pyridine r  (used as solvent) was not c a r e f u l l y removed from the crude product, the y i e l d i n the succeeding reaction was the known s u s c e p t i b i l i t y of benzoates removed at room temperature  i n vacuo.  somewhat lower.  In«view of  to heat, the pyridine had to be This process was  tedious and  generally required leaving the compound under vacuo for considerable period//,' of time.  In order to obviate this d i f f i c u l t y several alternative  procedures were studied.  The optimum conditions involved dissolving  the alcohol (84) i n tetrahydrofuran and treating the mixture with benzoyl chloride i n the presence of s o l i d potassium carbonate. crude product thus obtained was chromatographed on alumina.  The  Elution  with chloroform gave a c r y s t a l l i n e compound which had the same spectral properties as the benzoate obtained above.  and  The o v e r a l l  conversion of alcohol (84) to benzoate (85) by this second  procedure  was e s s e n t i a l l y quantitative and the quality of the product obtained was much superior. With benzoate (85) i n hand, the next step forward demanded the nucleophilic displacement of the benzoate group with cyanide anion. This reaction contrary to our expectation, turned out to be a very temperamental process.  This conversion posed many problems and some  of them are delineated below: sensitive to temperature.  (a) the reaction proved extremely  After running a few s m a l l - s c a l e • t r i a l  reactions i t was clear that the temperature had to be raised very slowly from room temperature to 105° otherwise considerable tarring of  the reaction mixture occurred; (b) we found that the desired compound  (86) was also very sensitive to temperature. the  This implied that when  temperature of the reaction mixture reached 105°, i t had to be  maintained at this elevated temperature f o r a minimum amount of time. If the reaction mixture was l e f t longer than required at high temperature obtained.  (105°), an o v e r a l l y i e l d of 20% as compared to 65% was Furthermore the cyano compound (86) obtained was also  contaminated with many spurious side products.  At this stage i t i s not  possible to define the various side reactions that occurred at this high temperature.  For the purpose of control i n this reaction thin  layer chromatography  played an important r o l e .  Fortunately i t so  happened that the benzoate (85) and the cyano compound (86) showed very d i s t i n c t colors when the t i c plate was sprayed with antimony pentachloride.  Benzoate  (85) appeared as a dark blue spot while the  cyano compound (86) appeared as l i g h t green.  Upon disappearance of the  benzoate i n the mixture (as monitored by t i c ) the reaction was immediately terminated; (c) f i n a l l y we experienced some trouble i n the p u r i f i c a t i o n of the cyano compound (86) by chromatography.  I t was  found that when a large amount of alumina was used (ratio of compound to alumina 1:100) i n a n t i c i p a t i o n of achieving better separation, i t led to considerable polymerization of the desired compound.  This  fact was even more pronounced when the columns employed were slow running or i n other words when the cyano compound (86) was l e f t on the  column f o r a l o n g e r p e r i o d of t i m e .  I n a l l these cases o n l y v e r y dark  p o l a r gums were o b t a i n e d from the columns.  However when the columns  employed were v e r y s h o r t and f a s t r u n n i n g , the cyano compound was  (86)  e l u t e d i n c r y s t a l l i n e form and i n a h i g h l y s a t i s f a c t o r y .yield.'.  summation, i t c o u l d be s a i d t h a t t h i s d i s p l a c e m e n t r e a c t i o n critical  was.very  ( i n terms of t e m p e r a t u r e , t i m e , and p u r i f i c a t i o n by  graphy) but the whole p r o c e s s t u r n e d out to be f a i r l y  In  chromato-  e f f i c i e n t when  p r o p e r l y executed. The optimum c o n d i t i o n s f o r the above r e a c t i o n r e q u i r e d the benzoate cyanide (  (85) i n dimethylformamide  10 f o l d e x c e s s ) to i t .  and adding s o l i d  T h i s heterogeneous  s t i r r e d a t room temperature f o r one hour.and the o i l b a t h was g r a d u a l l y r a i s e d to 105°  dissolving  potassium  mixture  was  then the temperature of  over a p e r i o d o f 45 m i n u t e s .  The r e a c t i o n m i x t u r e was m a i n t a i n e d a t t h i s e l e v a t e d temperature f o r about one hour.  At t h i s time t i c i n d i c a t e d no more s t a r t i n g m a t e r i a l .  The r e a c t i o n was  i m m e d i a t e l y stopped and the pure cyano compound  was  i s o l a t e d by chromatography  benzene-petroleum  on a l u m i n a .  The i n i t i a l e l u t i o n s w i t h  e t h e r (1:1) p r o v i d e d a c r y s t a l l i n e compound  (55%)  w h i l e the l a t t e r f r a c t i o n s (benzene e l u t i o n ) were gummy ( 1 0 % ) . e x a m i n a t i o n of t h i s gum  (86)  Tic  i n d i c a t e d t h a t t h i s p o r t i o n of the cyano^compound  c o n t a i n e d a v e r y minute i m p u r i t y but the compound was q u a l i t y t o be u t i l i z e d i n the next r e a c t i o n . a s m a l l amount o f t h e m a t e r i a l was  of r e a s o n a b l e  For a n a l y t i c a l  purposes  r e c r y s t a l l i z e d from d i c h l o r o m e t h a n e -  p e t r o l e u m ether,.mp 135-137°, and l a t e r s u b l i m e d a t 100°/0.01 mm.  The  s p e c t r a l d a t a compared f a v o u r a b l y w i t h the a s s i g n e d s t r u c t u r e ( 8 6 ) . The i n f r a r e d was  d i a g n o s t i c f o r the p r e s e n c e of a n i t r i l e group  (2256 cm  w h i l e t h e s t r o n g e s t e r peak p r e s e n t i n t h e benzoate (85) a t 1715 cm ^ had c o m p l e t e l y d i s a p p e a r e d .  Ins t h e nmr spectrum ( F i g u r e 17) t h e  methylene group a d j a c e n t t o t h e n i t r i l e s i n g l e t a t x 6.2.  (-CH^-CN) appeared as a sharp  The mass spectrum i n d i c a t e d a m o l e c u l a r i o n peak a t  m/e 293 and was dominated by two peaks a t m/e 124 and m/e 169. F i n a l l y t h e m o l e c u l a r f o r m u l a , C H . N , was c o n f i r m e d by h i g h 1Q  iy  0  ZJ J  r e s o l u t i o n t m a s s s p e c t r o m e t r y (Found:  293.186; C a l c . :  293.189) and  elemental a n a l y s i s . Now t h a t a l l t h e problems a s s o c i a t e d i n o b t a i n i n g t h e cyano compound (86) had been u n r a v e l e d , we c o n s i d e r e d i t s c o n v e r s i o n t o t h e carbomethoxy e s t e r ( 8 8 ) . purpose a r e :  Two r e a c t i o n s u t i l i s e d most w i d e l y f o r t h i s  (a) h y d r o l y s i s o f n i t r i l e s t o c o r r e s p o n d i n g c a r b o x y l i c  a c i d s and subsequent e s t e r i f i c a t i o n o f t h e l a t t e r t o e s t e r s ; (b) t r e a t i n g t h e n i t r i l e s w i t h methanol and h y d r o c h l o r i c a c i d .  Both o f t h e s e  p r o c e d u r e s were t r i e d f o r our own purpose. Treatment o f t h e n i t r i l e  (86) w i t h a l c o h o l i c p o t a s s i u m h y d r o x i d e  gave t h e c o r r e s p o n d i n g c a r b o x y l i c a c i d ( 8 7 ) . We i n i t i a l l y had some "fears  t h a t t h e c a r b o x y l i c a c i d ( 8 7 ) , because o f i t s a m p h o t e r i c  c h a r a c t e r might pose some problem d u r i n g i t s workup.  But c o n t r a r y t o  our e x p e c t a t i o n t h e r e a c t i o n p r o d u c t s c o u l d be e x t r a c t e d  quantitatively  from t h e aqueous l a y e r once t h e pH o f t h e r e a c t i o n medium was c a r e f u l l y brought t o 7.  The impure a c i d (87) o b t a i n e d i n t h i s way was e s t e r i f i e d  w i t h diazomethane.  U n f o r t u n a t e l y t i c o f t h e crude p r o d u c t showed i t  to be a m i x t u r e o f many components.  However, t h e d e s i r e d compound was  o b t a i n e d pure by v e r y c a r e f u l chromatography on a l u m i n a ( y i e l d 25-30%). When t h e c a r b o x y l i c a c i d (87) was e s t e r i f i e d w i t h methanol and s u l f u r i c a c i d , a g a i n a y i e l d o f 25% was o b t a i n e d .  The poor,yield obtained above necessitated an i n v e s t i g a t i o n of the a l t e r n a t i v e procedure mentioned e a r l i e r , namely the methanolysis of n i t r i l e s .  For this purpose the cyano compound (86) was dissolved  i n a mixture of methanol and 12 N HC1 (1:1) and the contents were s t i r r e d at room temperature for three days. the crude product  We were very happy when  indicated e s s e n t i a l l y one spot on t i c .  But when  this product was flushed through a small alumina column to get r i d of what was apparently a minor polar baseline contaminant, there was a considerable loss of material.  The pure compound obtained  only a 30% y i e l d i n the conversion, 86  88.  represented  This implied that the  baseline material was either the bulk i n the crude product or else decomposition of ester was occurring on the column. A l l our i n i t i a l attempts to improve the y i e l d of the conversion (86  88) f a i l e d completely.  A considerable amount of time was spent 84  with no apparent success.  Fortunately during this time Wenkert  published a synthesis of dl-dihydrogambirtannine  (102).  The most  interesting part of the synthesis, which was pertinent to our own work, was the conversion of n i t r i l e  OH  (103) to chloroester (104) by treatment  'CI OCH  103  CN  C00CH 104  3  w i t h methanolic h y d r o c h l o r i c a c i d .  The h i g h y i e l d of c h l o r o e s t e r (104)  o b t a i n e d i n t r i g u e d us t o u t i l i z e t h e same r e a c t i o n c o n d i t i o n as i n d i c a t e d by Wenkert.  So the cyano compound (86) was  methanol c o n t a i n i n g 1% water.  T h i s s o l u t i o n was  gas and the r e s u l t i n g m i x t u r e was hours.  dissolved i n  saturated with  s t i r r e d a t room temperature  HC1  f o r 60  The crude p r o d u c t i n d i c a t e d e s s e n t i a l l y one spot on t i c ( a l u m i n a ,  c h l o r o f o r m / e t h y l a c e t a t e , 1:1).  When t h i s p r o d u c t was  f l u s h e d through  an a l u m i n a column, we were v e r y s u r p r i s e d as w e l l as g r a t i f i e d t o f i n d the carbomethoxy e s t e r (88) o b t a i n e d as w h i t e c r y s t a l l i n e n e e d l e s i n s t e a d of the dark brown gum  obtained e a r l i e r .  I t i s important to  emphasize the f a c t t h a t i n the above r e a c t i o n , the amount of water p r e s e n t i n the methanol was v e r y c r i t i c a l i n terms of y i e l d . happened t h a t 1% water gave the optimum y i e l d . i n the r e a c t i o n m i x t u r e the y i e l d was the b e s t way  methanol w i t h 1% w a t e r . m a t e r i a l was mp  I f more water was  c o n s i d e r a b l y lowered.  t o o b t a i n r e p r o d u c i b l e r e s u l t s was  I t just  We  present found  to d i l u t e a b s o l u t e  F o r ' a n a l y t i c a l purposes a s m a l l amount o f  r e c r y s t a l l i z e d from d i c h l o r o m e t h a n e and p e t r o l e u m e t h e r ,  85-87.5°.  The s p e c t r a l d a t a compared f a v o u r a b l y w i t h the a s s i g n e d  s t r u c t u r e (88).  The presence of an e s t e r group was  i n d i c a t e d by the  infrared  (1728 cm ^) and a sharp s i n g l e t i n the nmr  ( F i g u r e 18) a t  T 6.34.  The methylene group a d j a c e n t t o the e s t e r c a r b o n y l (-CH^-COOCH^)  appeared  as a sharp s i n g l e t a t x 6.27.  The mass spectrum  i n d i c a t e d the m o l e c u l a r i o n a t m/e.326 and was dominated s i g n i f i c a n t peaks a t m/e formula, 20 26 2°2' C  (Found:  H  N  W a S  124 and m/e c o n  326.202; C a l c . :  ^^  v m e  202.  ( F i g u r e 19) by  two  F i n a l l y the m o l e c u l a r  ' ^ by h i g h r e s o l u t i o n mass s p e c t r o m e t r y  326.199) and e l e m e n t a l a n a l y s i s .  o-  ESTER •a-  CD  (88) 8  CM  CO  UJ |—LI.  LU  1  ct:  JlLjl 50  100  "IT  JUi 150  i  i  1  i  I  200  i  i  J I i — i — r — i — i  250  M/E Figure 19. Mass spectrum of carbomethexy ester 88.  1—i—I  300  r—r-"i  1—i—i  350  1—i—i—i  400  With the synthesis of the basic ring skeleton accomplished, i t remained to complete the synthesis of the desired  16,17-dihydrosecodin-  17-ol (90) by incorporating the hydroxymethylene (-CH^OH) side chain at C^Q.in the ester (88).  It was envisaged to put the said reaction  into practice by formylating the carbomethoxy ester (88) to enol (89) and then reducing the l a t t e r with sodium borohydride.  Formylation of  the ester (88) was done using sodium hydride and methyl formate.  Tic  examination of the crude product indicated extremely l i t t l e starting material •(< 5%) and one huge/streak r i s i n g from just above the baseline. This heavy spot we believed was the desired enol (89).  This crude  product could be separated i n e f f i c i e n t l y (poor separation and.considerable loss of material on column) into i t s components (88 and 89) by chromatography on s i l i c a gel.  The p u r i f i e d enol (89) indicated a  parent molecular ion peak at m/e  354 which was i n agreement with the  molecular formula, C„.. H.,.N„0,.. 21 26 2 3 The d i f f i c u l t i e s mentioned above prompted us to u t i l i z e the crude enol i n the next reaction, consequently the product obtained above was dissolved i n methanol and the solution was exposed to sodium borohydride at 0°.  We were surprised when the t i c examination of the crude  product indicated the presence of one very polar compound along with the major component.  It was found that the amount of this polar material  eventually decreased to a minimum as the temperature of the reaction mixture was lowered to -30°.  In view of the peripheral nature of this  "polar component", we made no attempt to characterize i t .  However,  85 Battersby  while working independently on the synthesis of 16,17-  dihydrosecodin-17-ol (90) observed the same result (a complete discussion of  B a t t e r s b y ' s work i s ' d e f e r r e d u n t i l a l a t e r p o r t i o n o f t h i s According  thesis).  t o him t h i s p o l a r compound i s t h e " d i o l " . ( 1 0 5 ) . R e c e n t l y  105  a  CH 0H 2  s i m i l a r o b s e r v a t i o n has been made d u r i n g t h e r e d u c t i o n o f e n o l  (106).  Thus t h e p o s s i b l e r e d u c t i o n of b o t h t h e e s t e r and e n o l f u n c t i o n s i n (89) c a u t i o n e d us t o e x e r c i s e some c a r e . C00CH  M o n i t o r i n g o f t h e r e a c t i o n by  COOCH  3  CH 0H 2  CH 0H  HOH  2  106  t i c was c a r e f u l l y conducted and t h e f i r s t appearance o f " d i o l " a s i g n a l to immediately  t e r m i n a t e t h e r e d u c t i o n by quenching t h e excess  of b o r o h y d r i d e w i t h a few drops o f 2 N HC1. be observed  (105) was  The o t h e r p r e c a u t i o n t o  i n t h i s r e a c t i o n was t o always keep t h e temperature l o w  (around -30°).  Once t h e p r e c a u t i o n s i n d i c a t e d were o b s e r v e d ,  p r o c e s s c o u l d be performed v e r y e f f i c i e n t l y .  The crude a l c o h o l  o b t a i n e d was p u r i f i e d by chromatography on alumina t i o n p r o v i d e d an a n a l y t i c a l sample, mp 131.5-132°. of t h e r e a c t i o n i . e . 88 -> 90 was 40%.  t h e whole  and a f t e r c r y s t a l l i z a The o v e r a l l y i e l d  87—89 There a r e  ^number of i n s t a n c e s i n the l i t e r a t u r e  where the  f o r m y l a t i o n o f a c t i v a t e d methylene groups a d j a c e n t t o e s t e r c a r b o n y l f u n c t i o n s have been done u s i n g t r i p h e n y l m e t h y l sodium as a base.  (trityl  sodium)  T h i s tempted us t o s u b s t i t u t e t r i t y l sodium f o r sodium  h y d r i d e i n our sequence.  I t must be emphasized h e r e t h a t t h i s  g a t i o n was u n d e r t a k e n t o improve the y i e l d of a l c o h o l ( 9 0 ) .  investi-  However,  i t was found t h a t f o r m y l a t i o n of e s t e r (88) u s i n g t r i p h e n y l m e t h y l and m e t h y l formate f o l l o w e d by r e d u c t i o n o f the r e s u l t i n g e n o l gave the same y i e l d of a l c o h o l (90) as o b t a i n e d e a r l i e r .  sodium  (89)  The c o m p l i c a -  t i o n of s e p a r a t i n g t r i p h e n y l m e t h a n e from the r e a c t i o n p r o d u c t e t c . f o r c e d us t o d i s c o n t i n u e the use of t h i s base. The p u r i f i e d a l c o h o l i n d i c a t e d s p e c t r a l d a t a w h i c h was i n complete a c c o r d w i t h the a s s i g n e d s t r u c t u r e ( 9 0 ) .  I n the i n f r a r e d ; a b r o a d peak  at  3050 cm ^ i m p l i e d the presence of a h y d r o x y l group.  A sharp peak  at  3400 cm ^ was a t t r i b u t e d t o i n d o l i c - N H w h i l e the e s t e r group  appeared at.1718 cm \  The nmr spectrum ( F i g u r e 20) e x i b i t e d t h e  f o l l o w i n g resonances.  The m e t h y l group of the e s t e r appeared as a  sharp s i n g l e t a t x 6.37. comparison t o t h e nmr  Prominant f e a t u r e s of t h i s spectrum i n  spectrum of the e s t e r (88) '(Figure 18) was  the  appearance of a braod m u l t i p l e t c e n t e r e d a t x 6.0.  This multiplet  i n t e g r a t e d f o r f o u r p r o t o n s (-CH^-OH + -C-CH^.OH) . H  I n the mass spectrum  ( F i g u r e 21) the a l c o h o l (90) i n d i c a t e d a m o l e c u l a r i o n a t m/e r e a d i l y l o s t a m o l e c u l e o f water t o g i v e t h e r a d i c a l i o n m/e  356.  It  338, w h i c h  corresponded t o t h e m o l e c u l a r i o n of secodine.(107). As t o be expected ion  (107) fragmented t o the i o n s (108, m/e  Furthermore an i o n m/e  326  214) and t o (101, m/e  124).  (109) c o r r e s p o n d i n g t o l o s s of CH 0 s t r o n g l y  16.17-DIHYDRGSECGDIN-17 CM  to u  CO  8 CM  i  50  100  150  1  Tj  i  200  M/E  250  ~i  1  1  Figure 21. Mass spectrum of 16,17-dihyirosecodin-17-ol  1  1  I  300  (90).  1—T  T  350  1  1  1  1  400  COOCH m/e Figure 22.  202  J  m/e  124  Postulated fragmentation of 16,17-dihydrosecodin-17-ol (90) i n the mass spectrometer.  suggested p o s i t i o n 17 for the hydroxyl group. mass spectrometric  A scheme portraying the  fragmentations has been summarized i n Figure  F i n a l l y the molecular formula, C H 01  Zl  o  N 0 , was  Zo  resolution mass spectrometry (Found:  o  Z  22.  confirmed by high  j  356.207; C a l c :  356.209) and  elemental analysis. This marked the end of our i n i t i a l synthetic endeavor.  We now .  were i n a p o s i t i o n to investigate whether (90) played a r o l e i n the biosynthesis of the various families of indole alkaloids mentioned previously.  Evaluation of 16,17-Dihydrosecodinr-17-ol (90) as Two  Bio-intermediate  radioisotopes most widely u t i l i s e d i n biosynthetic studies  for making radioactive "precursors" from inactive alkaloids are 3 tritium ( H) and  14 C.  It i s well known that tritium l a b e l l i n g  although r e l a t i v e l y l e s s expensive can sometimes give  erroieous  results due  to exchange between the protons and the t r i t i u m atoms 14 i n vivo. On the other hand C l a b e l l i n g i s very r e l i a b l e i n the sense that i n general no exchange of the l a b e l can occur.. However, 14 the much.ijhigher costs often associated with the synthesis of  C-  l a b e l l e d materials sometimes require at least i n i t i a l reliance on t r i t i u m as the tracer.  In our instance i t was  considered  advantageous  f i r s t to make the tritium l a b e l l e d synthetic alcohol (90) since we believed that this could f u l f i l our immediate needs.,-. If we were ' 3 fortunate i n obtaining p o s i t i v e incorporation with ',[ar-^Hj-alcohol (90), 14 i t was envisaged to check the extent of incorporation with [ COOCH^]3 alcohol (90). For this purpose preparation of [ar- H]-16,17-dihydro-  secodin-17-ol (90) and '[  COOCH ]-16,17-dihydrosecodin-17-ol (90)  were considered and the syntheses of both of these active compounds are .^described below. The method u t i l i s e d f o r making t r i t i u m labelled indole precursors 90 was developed i n our laboratories  a few years ago.  I t involves acid  catalysed exchange of aromatic protons of the indole nucleus with t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c acid. by reacting  equimolar quantities  t r i t i u m l a b e l l e d water.  The l a t t e r reagent i s prepared  of t r i f l u o r o a c e t i c anhydride and  A simple vacuum transfer  system i s used to  bring the tritium l a b e l l e d t r i f l u o r o a c e t i c acid into contact with the alkaloid.  The acid i s subsequently removed after the reaction i s  complete.  I t was soon realised that this method f o r the formation of  radioactive  alkaloids possessed some s i g n i f i c a n t features:  (a) the  alkaloids were recovered v i r t u a l l y unchanged.from the a c i d i c medium, (b) the method appeared general to e s s e n t i a l l y a l l indole  alkaloids,  (c) since a large excess of acid was-used, the d i l u t i o n of r a d i o a c t i v i t y i n the reaction was very small and the recovered t r i f l u o r o a c e t i c acid was suitable f o r reuse, and (d) the experimental procedure was very simple i n i t s operation. With a l l this knowledge i n hand, we exposed the synthetic 16,17dihydrosecodin-17-ol (90) to t r i t i u m labelled t r i f l u o r o a c e t i c acid. Unfortunately when the reaction mixture was worked up, we were very surprised  to f i n d the crude product as a complicated mixture of  several components.  I t must be emphasised  here that even before the  above reaction was performed, we were a l i t t l e iskeptical that some of the alcohol C90) might dehydrate to the corresponding a c r y l i c ester (107). But this l a t t e r reaction  and then subsequent  transformation of the  resulting a c r y l i c ester (107) considered  to other spurious products were not  to be predominating under the mild conditions employed.  In  view of the small amount of alcohol (90) at hand during the course of the active synthesis, i t was  not possible to define the various  products formed i n the above reaction. quickly unraveled.  However this problem  It was mentioned on page 50  solution of n i t r i l e (86) i s saturated with HC1 transformed into the carbomethoxy ester (88) suggested to us that the ester (88) was i n a c i d i c medium.  that when a methanol gas, the former i s  (Figure 12).  This result  a r e l a t i v e l y stable compound  We planned to c a p i t a l i s e on this observation  exchanging the aromatic protons of ester (88) with t r i t i u m . to secure the desired radioactive alcohol (90), i t was to formylate  was  by  In order  then necessary  the "hot" ester (88) and reduce the r e s u l t i n g enol.  postulated scheme (Figure 23) when put into practice proved highly satisfactory.  The  B e f o r e d e s c r i b i n g the a c t i v e s y n t h e s e s i t s h o u l d be mentioned here t h a t w h i l e w o r k i n g w i t h the r a d i o a c t i v e compounds as i n Figures 23 and 24, t h i n l a y e r chromatography proved helpful.  presented  extremely  F o r t u n a t e l y a l l the compounds s t a r t i n g from benzoate  (85)  to a l c o h o l (90) showed v e r y c h a r a c t e r i s t i c c o l o r s when t i c p l a t e s were sprayed w i t h antimony p e n t a c h l o r i d e .  Therefore w h i l e  the a c t i v e s y n t h e s e s ( F i g u r e s 23 and 24) i t was  not  pursuing;  considered  i m p e r a t i v e to o b t a i n any f o r m a l s p e c t r a l d a t a s i n c e i t was t o compare the  v a l u e s and  c o l o r s of the r a d i o a c t i v e compounds w i t h  t h e i r c o l d c o u n t e r p a r t s a l r e a d y a v a i l a b l e and c o m p l e t e l y ized  sufficient  character-  (Figure 12). To s t a r t the sequence o u t l i n e d i n F i g u r e 23, the carbomethoxy 3  e s t e r (88) was product  treated with  H-trifluoroacetic acid.  The  crude  a l t h o u g h homogeneous on t i c , showed some b a s e l i n e m a t e r i a l .  The m i x t u r e was  f l u s h e d t h r o u g h a s m a l l a l u m i n a column t o a f f o r d the  pure r a d i o a c t i v e e s t e r ( 8 8 ) .  This a c t i v e ester was.formylated using  sodium h y d r i d e and m e t h y l f o r m a t e and the r e s u l t i n g e n o l was w i t h sodium b o r o h y d r i d e .  reduced  Chromatography of the crude product 3  on  a l u m i n a a f f o r d e d the d e s i r e d [ a r - H ] - 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l The most g r a t i f y i n g outcome of t h i s v e n t u r e was  the f a c t t h a t  (90). the  o b t a i n e d a c t i v e a l c o h o l (90) had a v e r y h i g h s p e c i f i c a c t i v i t y  (dpm/mg).  T h i s r e s u l t a l l o w e d us to conduct,numerous e x p e r i m e n t s b o t h i n V i n c a r o s e a and V i n c a minor p l a n t s . 14 While contemplating secodin-17-ol displacement  the s y n t h e s i s of [  (90) our a t t e n t i o n was  C00CR,j]-16,17-dihydro-  o b v i o u s l y drawn to the n u c l e o p h i l i c  r e a c t i o n where the benzoate group was  d i s p l a c e d by  cyanide  anion (85 -> 86, Figure 12).  For a while our preconceived goal looked  very easy with the thought that by substituting radioactive potassium 14 cyanide (K  CN) for potassium cyanide i n the above reaction, we could  obtain the a c t i v e - n i t r i l e  (86).  In order to obtain the desired  14 [  COOCH^]-alcohol (90), i t would be merely necessary to carry the  active n i t r i l e  (86) through a similar sequence of reactions as done  previously on i t s cold counterpart i n Figure 12.  However, i t was soon  r e a l i s e d that in the conversion, 85 -* 86, we were using approximately a ten-fold excess of potassium cyanide.  In view of the high cost of  radioactive potassium cyanide, i t became imperative to reinvestigate this reaction.  This investigation was directed at finding out the minimum  amount of potassium cyanide required i n the displacement of the benzoate while s t i l l maintaining a reasonable conversion to the n i t r i l e (86).  For this purpose a series of reactions were run with decreasing  amounts of potassium cyanide.  It immediately became apparent that the  displacement reaction required a minimum of 5 fold excess of potassium cyanide.  Under these conditions a 40% y i e l d of n i t r i l e  (86) was  obtained as compared to 65% when a 10 fold excess was employed.  I f the  amount of potassium cyanide was reduced any further the y i e l d of n i t r i l e was cut down very d r a s t i c a l l y .  For example a two f o l d excess  of potassium cyanide gave less than 20% of n i t r i l e . In the displacement reaction (85 -»- 86, Figure 24) the benzoate (85) was dissolved i n dimethylformamide and the solution was exposed 14 to radioactive potassium cyanide (K CN). The pure active n i t r i l e 6 9 (86, s p e c i f i c a c t i v i t y 4.32 x 10 dpm/mg or 1.27 x 10 dpm/mmole) obtained by chromatography on alumina was dissolved i n methanol  containing 1% water.  The solution was then saturated with hydrogen  chloride gas and the resultant crude product upon chromatography furnished the desired active carbomethoxy ester (88).  The l a t t e r  substance was formylated as before to y i e l d the crude enol (89) which without further p u r i f i c a t i o n was reduced with sodium borohydride at ^30°. Chromatography of the crude product on alumina furnished the desired I C00CH ]-16,17-dihydrosecodin-17-ol (90). 14  With the completion of the synthesis of both t r i t i u m and  C-  alcohol (90$, i t became necessary to investigate the incorporation i f any, of this substance into the appropriate  plant systems.  • > Interest i n Vinca rosea has been considerable i n i t of antileukemic extensive  alkaloids.  since the discovery  As a result of this finding an  investigation of i t s a l k a l o i d a l constituents has been 91-94  conducted i n various laboratories.  The structures ..of more than  s i x t y alkaloids are known and these represent many s t r u c t u r a l types. Vindoline  (5), catharanthine (6) and ajmalicine  (3) are three of the  major alkaloids present and possess the Aspidosperma (11), Iboga (12) and Corynanthe (10) systems respectively. 95 On the other hand the tiny green plant Vinca minor a wonderful array of Aspidosperma a l k a l o i d s .  possesses  Of the more than twenty  alkaloids i s o l a t e d , structures of about twenty are known.  Minovine  (73, Aspidosperma type) and vincamine (72, eburnamine family) are two major alkaloids which could be i s o l a t e d , p u r i f i e d and r e c r y s t a l l i s e d with great ease. campus.  In addition Vinca minor grows i n abundance around our  A l l these factors made this plant an excellent choice for. our  biosynthetic studies. Before describing the feeding r e s u l t s i t would be relevant to mention that at t h i s stage our whole research project became very diversified..  We r e a l i s e d • that now: the-problem would involve  feeding  14 the t r i t i u m and  C-labelled, alcohols (90) to both Vinca rosea and  Vinca minor f o r various i n t e r v a l s of time.  Regardless whether the active  substances showed p o s i t i v e or negative incorporation, these results would require r e p e t i t i o n to confirm the i n i t i a l findings.  In order to  carry out these requirements with optimum accuracy and e f f i c i e n c y , the various incorporation:? experiments were performed simultaneously by three of us, John Beck, N e i l Westcott and myself.^-  In this thesis the  result of only those experiments which were performed by me are described.  Whenever relevant or necessary  i n the l a t e r discussion,  the results of the other workers w i l l be mentioned. 14 For the purpose of the biosynthetic study, dihydrosecodin-17-ol  [  COOCH ]-16,173  (90, t o t a l a c t i v i t y 9.89 x 10  dpm) made soluble  with 0.1 N acetic acid and a few drops of ethanol was incorporated v i a the hydroponic technique, to Vinca minor shoots.  After four days,  the plants were k i l l e d and the isolated a l k a l o i d a l material was shown to contain 31% of the t o t a l a c t i v i t y fed.  Vincamine (72) and minovine  (73) were isolated by a chromatographic separation developed e a r l i e r 90 i n these laboratories.  Vincamine showed one spot on t i c and i n most  of the various experiments conducted the isolated amount was s u f f i c i e n t to allow several c r y s t a l l i z a t i o n s without  the addition of cold material.  Minovine (73) however; required further p u r i f i c a t i o n by preparative layer chromatography and then further d i l u t i o n with the cold a l k a l o i d to allow 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 . revealed that vincamine (72) possessed corresponding  Several c r y s t a l l i z a t i o n s  an a c t i v i t y of 102 dpm  to a s p e c i f i c incorporation of < 0.001%.  (total)  Unfortunately  this amount of r a d i o a c t i v i t y was so small that i t was d i f f i c u l t to ascertain the s i g n i f i c a n c e i f any of this r e s u l t .  A minute trace of  a radioactive impurity present i n the a l k a l o i d could be responsible. On the other hand v i r t u a l l y no a c t i v i t y could be; detected - i n the p u r i f i e d minovine (73).  In a p a r a l l e l series of experiments, [ar- Hj-16, 17-dihydrosecodin17-ol  (90) was  fed to Vinca minor by my  colleague, John Beck.  s u f f i c i e n t here to state that he also could not detect any  It i s  significant  a c t i v i t y i n the two a l k a l o i d s , vincamine (72) and minovine (73). <-. In another concurrent din-17-ol  (90) was  3  investigation, [ar- H]-16,17-dihydroseco-  fed to Vinca rosea by another colleague N e i l  Westcott, no s i g n i f i c a n t a c t i v i t y could be detected i n vindoline(5) and catharanthine  (6).  The most f r u s t r a t i n g aspect of a l l these results was to delineate what might be construed  the i n a b i l i t y  as a p o s i t i v e incorporation of  alcohol (90) into any of those alkaloids i s o l a t e d by us.  We,  of  course, were f u l l y aware of the fact that negative r e s u l t s i n biosynthetic studies have to be interpreted with great care.  It i s  well..known that success i n a biosynthetic experiment depends upon such factors as absorption and permeability i n the plant as well as the a b i l i t y of the plant to carry out the desired biosynthesis.  Thus the  age of the plant, length of incorporation, the method of feeding etc. become very c r i t i c a l factors.  In this regard i t i s pertinent to  mention that the e a r l i e r workers i n our laboratories had established. conditions during which large molecular weight substances were 63 64 incorporated into the plant systems. reinforced our p r e v a i l i n g impression  '  This s i t u a t i o n therefore  that the apparently  negative  incorporations of alcohol (90) were not due to technical d i f f i c u l t i e s with the experimental method. It was  indicated i n the early part of this discussion (page 33)  that we prepared 16,17-dihydrosecodin-17-ol (90) as our synthetic target only with the hope that i t would be transformed i n vivo to the  p u t a t i v e i n t e r m e d i a t e (76) by a p p r o p r i a t e d e h y d r a t i o n and o x i d a t i o n ( i n the p i p e r i d i n e r i n g ) . (90) suggest  However n e g a t i v e i n c o r p o r a t i o n of a l c o h o l  t h a t the p l a n t systems u t i l i s e d may be i n c a p a b l e of  c a r r y i n g out e i t h e r one o r b o t h o f these r e a c t i o n s . 85 A f t e r we had completed t h e above r e s u l t s , B a t t e r s b y the presence  of 16,17-dihydrosecodin-17-ol  Rhazya o r i e n t a l i s .  I n these experiments  reported  (90) i n t h e p l a n t / / he f e d to the shoots  [0-methyl-  3 H ] - l o g a n i n and from t h e i s o l a t e d " a c t i v e a l c o h o l , 90, was a b l e t o show an i n c o r p o r a t i o n o f 0;013%. Jn-. t h e same manner, when the  experiment  was r e p e a t e d w i t h shoots of V i n c a r o s e a , r a d i o a c t i v e (90) was i s o l a t e d but o f v e r y low s p e c i f i c a c t i v i t y .  again  To use i t as a c a r r i e r  85 Battersby  s y n t h e s i s e d t h e a l c o h o l (90) by t h e g e n e r a l r o u t e o u t l i n e d  i n F i g u r e 25.  Although Battersby's route i s q u i t e d i f f e r e n t  i n the e a r l i e r stages  from ours  (see F i g u r e 1 2 ) , t h e l a t e r s t e p s a r e e s s e n t i a l l y  identical. I n summary of h i s independent s t u d y B a t t e r s b y s t a t e d t h a t "16,17d i h y d r o s e c o d i n - 1 7 - o l (90) i s . a n a t u r a l product p r e s e n t i n Rhazya o r i e n t a l i s p r o b a b l y a r i s i n g from a b i o s y n t h e t i c i n t e r m e d i a t e b l o c k e d by r e d u c t i o n (e.g. 113) o r by h y d r a t i o n and r e d u c t i o n (e.g. 7 6 ) " . 96 R e c e n t l y Smith  r e p o r t e d t h e i s o l a t i o n of  and 16,17-dihydrosecodine(111)  tetrahydrosecodine(110)  from Rhazya s t r i c t a and t e t r a h y d r o s e c o d i n -  1 7 - o l (112) from Rhazya o r i e n t a l i s .  These a l k a l o i d s were i s o l a t e d i n  v e r y s m a l l amounts and t h e i r s t r u c t u r e s were d e r i v e d m a i n l y from mass s p e c t r o m e t r i c measurements. The presence of t e t r a h y d r o s e c o d i n e ( 1 1 0 ) was a g a i n demonstrated i n Rhazya o r i e n t a l i s by d i l u t i o n s t u d i e s when 14 97 [2- C ] - t r y p t o p h a n was a d m i n i s t e r e d t o Rhazya o r i e n t a l i s . The  Figure 25.  Battershy's synthesis of 16,17-dihydrosecodin-17^ol.  plants were worked up for the alkaloids with the addition of synthetic (110) as c a r r i e r (Figure 26).  The constant  110 Figure 26.  Smith's synthesis of  a c t i v i t y found for the  COOCH tetrahydrosecodine(110).  rigorously p u r i f i e d tetrahydrosecodine(110) corresponded to incorporation.  This high incorporation was  0.5%  quite remarkable and  this  led Smith to suggest that tetrahydrosecodine (110) i s on a metabolic side track very close to the main a l k a l o i d biosynthetic route.  This  fact f i t s well with the notion that simple reduction of the putative a c r y l i c ester (76) takes tetrahydrosecodine(110) out of c i r c u l a t i o n . It i s interesting to note that a similar type of explanation ( i . e .  Figure 27.  Some of the compounds derivable i n vivo from 16,17-dihydrosecodin-17-ol  (90).  hydration and reduction of a c r y l i c ester, 76) was used by Battersby when 16,17-dihydrosecodin-17-ol (90) was i s o l a t e d from the plants. The question now i s how the above r e s u l t s f i t into the biosynthetic story which i s rapidly evolving from the combined data of.the laboratories:  various  F i r s t of a l l , these r e s u l t s give further support to the  suggested cleavage process f o r the biosynthesis of indole alkaloids i n the Aspidosperma and Iboga families as mentioned previously.  It  i s further evident that compounds 107, 76, 89, 113, 114 (not yet isolated) 110, 111, 112 arid the dimeric secamine (119) and presecamine 98 (116),  a l l are derivable i n p r i n c i p l e from the alcohol (90) i n vivo  whether any of these compounds (Figure 27) w i l l turn out to be the correct biointermediate  remains an open question.  Some of our own  experiments which could be r e a d i l y extended into t h i s area are. discussed i n the next section of this thesis.  PART II  The negative  incorporation obtained by feeding the synthetic  16,17-dihydrosecodin-17-ol (90) into Vinca minor and Vinca rosea plants suggested to us that the former may not be capable of acting as a progenitor of the a c r y l i c ester (76) i n the plant.  I t therefore  became evident that to persue our preconceived goal the alcohol (90) required synthetic modification to some other model compound.  At this  stage our whole research project entered a very perplexing phase. Among the many avenues which were a v a i l a b l e , i t was very d i f f i c u l t to decide unequivocally which route to explore f i r s t .  In this part of the  discussion we w i l l portray some of our attempts to obtain some.of the other close r e l a t i v e s of the f u g i t i v e a c r y l i c ester (76). The two compounds bearing structures 107 and 115 appeared to us to represent convert  templates which could under reasonable biochemical  modification  to the a c r y l i c ester (76) and thereby i n turn to the Aspido-  sperma and Iboga bases.  Our choice was obviously dictated by the fact  that these compounds (107 and 115) were amenable to syntheses from + X  the available alcohol (90).  I t shouldi/be noted that i n comparison to  the alcohol (90), the pyridinium alcohol (115) which would be s u f f i c i e n t l y stable for i s o l a t i o n represented oxidation i n the piperidine ring.  a much higher l e v e l of  I t seemed reasonable that some  reducing  system i n the plant l i k e NADPH would convert +  ring to the desired dihydropyridine system.  the pyridinium  On the other hand the 96  tetrahydropyridine derivative, 107, named secodine by Smith  who  has recently i s o l a t e d the close r e l a t i v e s of this compound i n h i s work on Rhazya species, was i n a lower l e v e l of oxidation than the a c r y l i c ester (76).  Perhaps an oxidative process i n the plant  would lead i n vivo to 76.  The discussion which follows describes our  attempts i n the laboratory syntheses of these substances. In connection with transannular tories,"^ ^  c y c l i z a t i o n work i n our labora-  mercuric acetate was found to be an excellent reagent f o r  oxidising the piperidine ring of several alkaloids to the corresponding  + tetrahydropyridines  Ci-e. C-N- ->- C=N-).  We also envisaged to  u t i l i s e the same•reaction f o r oxidising 16,17-dihydrosecodin-17-ol (90) to the pyridinium alcohol (115).  This aspect of the problem was  undertaken by two of'my colleagues, N e i l Westcott and John Beck. Although the d e t a i l s of a l l t h i s work cannot be properly discussed • here, s u f f i c e i t to say that a l l our attempts with mercuric acetate oxidation reaction were very disappointing.  In a l l instances poor  y i e l d s and products of l i t t l e u t i l i t y were obtained.  These results  therefore l e f t us l i t t l e a l t e r n a t i v e except to concentrate  our e f f o r t s  i n obtaining secodine (107) for biosynthetic evaluation. To secure secodine (107) from alcohol (90) i t was obligatory to. dehydrate the l a t t e r substance.  For several reasons to be presented  l a t e r we preferred the base catalysed dehydration alternative of acid c a t a l y s i s .  rather than the  For this purpose a small amount of  alcohol C90) dissolved i n benzene was exposed to sodium hydride as the base.  After the reaction was over, the excess of the hydride was  d e s t r o y e d by t h e a d d i t i o n o f a few drops o f 2 N h y d r o c h l o r i c a c i d . T i c e x a m i n a t i o n o f t h e crude p r o d u c t i n d i c a t e d two s p o t s w i t h v e r y similar  values.  The whole crude p r o d u c t was r a p i d l y f l u s h e d  a s m a l l column o f alumina and t h e m i x t u r e exposed examination.  through  to a spectroscopic  The nmr spectrum i n d i c a t e d two s i g n a l s f o r NH (x  0.63  and 1.23) and two e s t e r (COOCH^) peaks a t x 6.29 and 6.49 r e s p e c t i v e l y . S i m i l a r l y i n t h e i n f r a r e d two e s t e r peaks were e v i d e n t and these c o u l d be c o n v e n i e n t l y a s s i g n e d t o s a t u r a t e d (1730 cm "*") and u n s a t u r a t e d (1680 cm "*") e s t e r groups.  T h i s r e s u l t i m m e d i a t e l y suggested t h a t we were  d e a l i n g w i t h a m i x t u r e which c o n t a i n e d a t l e a s t one d i m e r i c compound^.  r  96-98 At a time when we were s t i l l e n t a n g l e d i n t h i s problem,  Smith  p u b l i s h e d a s e r i e s o f papers w h i c h q u i c k l y u n r a v e l l e d our problems. r e p o r t e d t h e i s o l a t i o n o f t h r e e new d i m e r i c a l k a l o i d s , (116a o r 116b), d i h y d r o p r e s e c a m i n e  He  presecamine  (117a o r 117b) , t e t r a h y d r o p r e s e c a m i n e  (118a o r 118b) from Rhazya s t r i c t a and t e t r a h y d r o p r e s e c a m i n e from 98 Rhazya o r i e n t a l i s . 116b)  Smith  f u r t h e r observed t h a t presecamine  (116a o r  r e a r r a n g e s q u a n t i t a t i v e l y a t room temperature i n 2 N h y d r o c h l o r i c  a c i d t o one o f t h e secamines  (119, F i g u r e 28; 120 F i g u r e 29).  This  i m p o r t a n t o b s e r v a t i o n r e p r e s e n t e d one o f t h e main arguments f o r t h e s t r u c t u r e s suggested and would  tend t o f a v o u r s t r u c t u r e type ( a ) .  The type (b) dimers was however n o t e x c l u d e d on m e c h a n i s t i c grounds ( F i g u r e 29) s i n c e i t c o u l d ( a l t h o u g h l e s s p l a u s i b l y ) l e a d t o t h e o t h e r p o s s i b l e secamine  s t r u c t u r e (120).  convenience i n t h e subsequent  I t s h o u l d be noted t h a t f o r  d i s c u s s i o n presecamine and secamine  be c o n s i d e r e d i n terms o f s t r u c t u r e s 116a and 119 r e s p e c t i v e l y .  will  Figure 28.  Rearrangement of presecamine (type a) to secamine  (119).  Figure 29.  Rearrangement of presecamine (type b) to secamine  (120).  It was further observed that on attempted sublimation presecamine (116) undergoes a f a c i l e retro-Diels-Alder reaction to y i e l d secodine 98 (107).  A b r i e f resume of the data obtained by Smith  i n support of  structure, 107, f o r secodine i s f u l l y revealed i n Figure 30.  Not only  was this data of great importance i n i t s own right but i t was  directly  pertinent to our work as well. We s h a l l now t r y to portray how our work on dehydration of 16,17dihydrosecodin-17-ol (90) converges with Smith's experiments i n a most g r a t i f y i n g way.  Our crude mixture from the sodium hydride reaction;  indicated u l t r a v i o l e t absorption (A 224, 285 ( i n f ) , 292, 326 max  my)  and nmr signals (two ester singlets at x 6.29 and 6.49) which was reminiscent of the spectroscopic properties (^  max  227, 228 ( i n f ) , 295,  329 my and singlet at x 6.23 and 6.42) reported for the dimeric presecamine (116).  With this knowledge i n hand i t became possible to  speculate on the nature of at least three compounds present i n the crude reaction mixture.  The presence of the desired secodine (107)  was indicated by a very pronounced unsaturated methoxycarbonyl singlet at T 6.29 i n the nmr spectrum.  The r a t i o between the integrations of  the unsaturated and saturated methoxycarbonyl singlets was 3:1 ( i n pureipresecamine this r a t i o should be 1:1).  The other two compounds  present i n the crude mixture were obviously presecamine (116) and secamine (119).  The l a t t e r substance would arise from the rearrangement  of presecamine CL1"6) when 2 N hydrochloric acid was used i n the work up of the reaction. Returning to Smith's study i t was clear that the reaction mixture should not be exposed to hydrochloric acid to avoid the rearrangement of  Figure 30.  A summary of data supporting the structure of secodine (107).  presecamine (116) to secamine (119).  Furthermore Smith  had found  that secodine (107) reacted very slowly (over a period of 10 days) with methanol to give 17-methoxy-16,17-dihydrosecodine (121).  Finally  i n the dimerization of secodine (107) to presecamine (116, Figure 30) i t was s p e c i f i c a l l y indicated that this reaction occurs at 0° i n the absence of solvent. that secodine  These observations c l e a r l y pointed to the fact  (107) could perhaps be isolated free from dimer i f the  reaction mixture was kept cold and i n solution.  I t was indeed found  i n our work that secodine (107) could be stored f o r several hours i n dry benzene at 0° without any appreciable dimerization. The optimum conditions f o r the dehydration of the alcohol (90) involved dissolving this substance i n dry benzene and exposing i t to sodium hydride.  The- reaction -mixture was s t i r r e d under nitrogen at  40° f o r 15 minutes.  At this time t i c examination of the mixture  indicated three spots.  The front running spot which seemed to represent  the bulk of -material, was due to secodine (107).  The other two minor  spots were due to the dimer presecamine (116) and the starting compound (90).  The crude mixture was flushed through a small alumina column  using benzene f o r the e l u t i o n . immediately  under vacuum.  The fraction collected was freezer-dried  We were very surprised as well as g r a t i f i e d  to find that the gummy product obtained i n this manner was homogeneous on t i c .  Further elution of the column with chloroform afforded  presecamine (116) and unreacted alcohol (90).  In small scale reactions  the y i e l d i n this dehydration procedure varied but the most favourable reaction provided 61% of secodine.  I t i s now appropriate to discuss  some of the evidence i n support of the s t r u c t u r a l assignment.  In the  nmr spectrum (Figure 31) the o l e f i n i c protons of the a c r y l i c ester were represented as a pair of doublets at x 3.55 (J = 1 Hz), respectively. singlet at x 6.20.  (J = 1 Hz) and  The methoxycarbonyl was indicated by a sharp  These signals compared favourably with those  reported f o r 15,20-dihydrosecodine (107, A ^ ' ^ - r e d u c e d ) . the  3.91  o l e f i n i c protons were indicated at x 3.54 and 4.01;  In the l a t t e r  methoxycarbonyl  at x 6.18.  The mass spectrum (Figure\32) indicated a molecular ion  peak at m/e  338 i n agreement with the molecular formula, -'21^26^2^2" (  The spectrum was dominated by\ ^ s i g n i f i c a n t peak at m/e  124(101). while a  weak peak-at-m/e 214 showing-ra metastable peak at 135 ,.5 was_ consistent with'-thec-fragment 108,, both formed i n the manner i l l u s t r a t e d .  107  108  101  It i s pertinent to mention here that when sodium hydride i n the dehydration reaction was substituted by t r i t y l sodium  (triphenylmethyl  sodium)!, i t led to products of l i t t l e u t i l i t y . TLC>indicated thatthe  crude product was a mixture of ..three components. Out of these, two  compounds separated i n pure form by chromatography very strong end absorption i n the uv spectrum. a second aromatic system.  on alumina indicated  This was i n d i c a t i v e of  I t was f e l t that these two compounds arise  RELATIVE INTENSITY 25  50  _L_  75  _J_  C  n ID  o P3 cn cn  124 (4x)  cn  135  x)  ID n rt  e on •  n  c  3 o Hi cn . CD n o  m  ro• a CD s—214  ro  or  o  CO  m CI Q  a o-  a  m •338 CO  o  a  from the condensation of the t r i t y l anion with the a c r y l i c ester f u n c t i o n a l i t y of secodine (107).  Due to the very small amount of  material at hand, no attempt was made to rigorously prove the structures of  these compounds but tentative assignments  are given i n structures  (122) and (123), respectively. \Although "this i s riot usual for triphenyl99 methyl sodium to react i n this manner with an ester,  an example of  0=C  COOCH,  C(<{,).  X  123  122  the formation of a . t r i t y l ketone v i a a similar process has been observed In our case however, the molecule  (107) because of the presence of  the a c r y l i c ester group was endowed with much greater r e a c t i v i t y to allow Micheal addition of the t r i t y l anion. With the completion of the long sought secodine (107) we now were i n a position to investigate i t s possible role i n the biosynthesis of the Aspidosperma and Iboga alkaloids.  Evaluation of secodine (107) as bio-intermediate It shouldi-be mentioned here that some e a r l i e r workers i n our laboratories have tentatively found that the best way to incorporate any given precursor into the plant system i s to convert i t into the acetate 90 . salt.  Normally the acetate s a l t i s made by dissolving the compound  1D0,  i n 0.1 N acetic acid and a few drops of ethanol.' So i t was perfectly clear to us i n the beginning that no matter how much care was taken i n i s o l a t i n g pure secodine (107), the necessary conversion to the s a l t and eventual incorporation into the plant would allow some dimerization to presecamine of the presecamine (119).  (116).  Under the influence of acid some  (116) would then obviously rearrange to secamine  In summary we understood that we would be required under  normal circumstances to incorporate a mixture of these three compounds. However i t was envisaged that the presence of the dimeric compounds presumably would not jeopardise the feeding experiment provided the composition of the mixture could be determined at the time of feeding. For this purpose a "blank" experiment was.conducted  i n such a manner  that a clear d i s t i n c t i o n between the amount of secodine (107) and the dimeric compounds could be made.  The d e t a i l s of this experiment are  deferred, u n t i l a l a t e r portion of the discussion.  It i s s u f f i c i e n t to  emphasize presently that the procedure proved highly s a t i s f a c t o r y . 3 For the biosynthetic investigation Jar- R]-16,17-dihydrosecodin17-ol (90, s p e c i f i c a c t i v i t y 7.94 x .10^ dpm/mg) was dehydrated with sodium hydride i n exactly the same manner as indicated previously. 3 8 The pure l a r - H]-secodine (107, t o t a l a c t i v i t y 2.65 x 10 dpm) was made soluble with 0.1 N acetic acid and a few drops of ethanol and the solution was administered to the shoots of Vinca minor L.  The plants  were allowed to grow f o r four days and then the alkaloids were i s o l a t e d . The crude extract contained 22% of the t o t a l a c t i v i t y fed.  Vincamine  (72) and minovine (73) were isolated i n i t i a l l y by chromatography and then further p u r i f i e d by preparative thin layer chromatography followed  by several c r y s t a l l i z a t i o n s .  Liquid s c i n t i l l a t i o n counting  that the i s o l a t e d alkaloids contained  revealed  a very low l e v e l of r a d i o a c t i v i t y .  Vincamine (72) showed an a c t i v i t y of 261 dpm/mg corresponding  to a  s p e c i f i c incorporation of 0.0013% while minovine (73) indicated an a c i t i v i t y of c|5625 dpm/mg corresponding to a s p e c i f i c incorporation of < 0.001%.  I t i s pertinent to c a l l attention to the fact that i n spite  of the very low incorporation observed, the alkaloids were showing appreciable counts (cpm) above the normal background.  In order to  confirm the r e l i a b i l i t y of the above r e s u l t s another colleague of mine, John Beck, repeated this.biosynthetic experiment employing a new series of plants.  Fortunately his r e s u l t s for vincamine (72) and  minovine (73) turned out to be i n good agreement with the figures quoted above for these two a l k a l o i d s .  In spite of the fact that the  l e v e l of incorporation was extremely low,,the most important to emerge from these experiments were that:  observations  (a) i n comparison to our  l a s t biosynthetic experiment when 16,17-dihydrosecodin-17-ol (90) was fed, .the Vinca mirtor shoots remained very healthy for the duration of the experiment (four days).  In the former case the plants had  started collapsing just a f t e r one day of feeding;  (b) we were rather  surprised when minovine (73) indicated almost twice as much radioa c t i v i t y (dpm/mg) i n comparison to vincamine (72);  This factor was not  s e l f evident i n considering the r e s u l t s of s p e c i f i c incorporation because minovine (73) i s usually i s o l a t e d i n much smaller than vincamine.  quantity  This r e s u l t could be due to the fact that for the  secodine skeleton to incorporate into minovine (73), a few r e l a t i v e l y . straightforward r i n g closures are required  (Figure 33) while for  +  Figure 33.  Proposed elaboration of secodine into vincamine and minovine.  vincamine (72L the secodine 0-07). molecule must undergo numerous 'rearrangements (Figure 33). A l a t e r discussion concerning studies on vincamine w i l l present this aspect i n more d e t a i l .  We have no firm  basis f o r this explanation and obviously additional experiments w i l l be necessary before any more d e f i n i t e statement can be made.  3 In a complimentary series of experiments [ar- H]-secodine (107) was fed to Vinca rosea L. by John Beck i n our laboratory.  The radioactive  vindoline (5) isolated (0.02% incorporation) was.shown to be radiochemically pure by further conversion of this a l k a l o i d into v i n d o l i n e t r i o l having the same constant molar a c t i v i t y .  Somewhat surprisingly Iboga  a l k a l o i d catharanthine (6) which co-occurs with vindoline (5) i n  Vinca rosea indicated no a c t i v i t y . was  S i m i l a r l y [ar- H]-secodine  fed to Aspidosperma pyrricollum plants by Dr. Ken  laboratory.  I s o l a t i o n of radioactive apparicine  (107)  Stuart i n our  (124)  indicated  0.01%  incorporation.  124 A l l these incorporation results indicated above leave  little  doubt i n our minds that secodine (107) or some.closely related derivative which may 107 may  be obtained by reaction of the enzyme systems on  turn out to be a c r u c i a l bio-intermediate  biosynthesis.  It was  now  i n indole a l k a l o i d  necessary to determine what percentage of the  compound fed got into the plant i n i t s monomeric state and what percentage of i t was  converted to the dimeric systems during the period 14  of incorporation.  A blank experiment was  conducted i n which [  secodine (107) was  converted into the acetate s a l t by dissolving i t .  i n 0.1 N acetic acid and a few drops of ethanol. was  COOCH^]-  The cloudy solution  l e f t at room.temperature for 2 hours (this i s the maximum time  the- plants require to absorb the above s o l u t i o n ) .  The contents were  freeze-^dried and a portion of the resulting, gum was  run on a  Eastman  Kodak neutral alumina s t r i p plate employing a system which had been previously established for t h i s purpose by means of the "cold" materials  (for complete d e t a i l s see page  l i 7 ).  The a c t i v i t i e s i n  the  two  spots  c o r r e s p o n d i n g to secondine  compounds (presecamine and  (107)  and  the  dimeric  secamine) were>counted w i t h a s t r i p  counter.  I t immediately became obvious t h a t i n the m i x t u r e the r a t i o between the s e c o d i n e and corrected (72)  the d i m e r i c  be  0.002% and  assumes t h a t the d i m e r i c  f o r minovine  (73)  m o l e c u l e s do not  first  The  incorporated  This c a l c u l a t i o n  back to the monomers  g r a t i f y i n g s i n c e they  next important q u e s t i o n as a i n t a c t u n i t w i l l 3  doubly l a b e l l e d p r e c u r s o r  provided  tetrahydropyridine  as to s e c o d i n e  (107)  is  require the preparation  of  C00CH./J-secodine (107)  l a b e l i n the i n d o l e u n i t and  portion.  various  14  i . e . [ a r - H;  even b e t t e r , s e c o d i n e w i t h one i n the  f o r vincamine  < 0.0015%.  convert  the  p o s i t i v e i n c o r p o r a t i o n of a s y n t h e t i c substance i n t o the  plant species. being  Therefore  plant.  A l l of the above r e s u l t s were v e r y our  61:32.  s p e c i f i c i n c o r p o r a t i o n i n t o V i n c a minor L.  should  i n the  compounds was  Such i n v e s t i g a t i o n s are  the  or other  currently  underway i n our l a b o r a t o r i e s . In c o n c l u s i o n  i t i s c l e a r t h a t the above work has  preliminary  information  on the l a t e r  synthesis.  M o s t , i m p o r t a n t l y i t has  provided  some  stages of i n d o l e a l k a l o i d s b i o created  an e n t r y  i n t o the more  s o p h i s t i c a t e d experiments which w i l l h o p e f u l l y l e a d to a b e t t e r u n d e r s t a n d i n g of the b i o s y n t h e s e s of t h i s l a r g e f a m i l y of products.  natural  EXPERIMENTAL M e l t i n g p o i n t s were determined on a K o f l e r b l o c k and a r e u n c o r r e c t e d . The u l t r a v i o l e t  (uv) s p e c t r a were r e c o r d e d i n methanol on a Cary 11  r e c o r d i n g s p e c t r o m e t e r , and the i n f r a r e d  ( i r ) s p e c t r a were taken on  a P e r k i n Elmer Model 21, Model 137 and Model 457 s p e c t r o m e t e r s as KBr d i s c s ( u n l e s s o t h e r w i s e s t a t e d ) .  N u c l e a r magnetic  resonance  (nmr)  s p e c t r a were r e c o r d e d i n d e u t e r i o c h l o r o f o r m ( u n l e s s o t h e r w i s e s t a t e d ) at  100 megacycles per second  ( u n l e s s o t h e r w i s e s t a t e d ) on a V a r i a n  HA-100 i n s t r u m e n t and t h e l i n e p o s i t i o n s o r c e n t r e o f m u l t i p l e t s a r e g i v e n i n T i e r s T s c a l e w i t h r e f e r e n c e to t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d ; m u l t i p l i c i t y , i n t e g r a t e d a r e a and the type ofprotons are i n d i c a t e d i n parentheses.  Mass s p e c t r a were r e c o r d e d on  an A t l a s CH.-4 mass s p e c t r o m e t e r and h i g h r e s o l u t i o n m o l e c u l a r weight d e t e r m i n a t i o n s were c a r r i e d , out on an AE--MS'-9 mass s p e c t r o m e t e r . v  A n a l y s e s 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 L a b o r a t o r y , The U n i v e r s i t y o f B r i t i s h Columbia.  Woelm n e u t r a l a l u m i n a  and s i l i c a g e l c o n t a i n i n g 2% by weight o f G e n e r a l E l e c t r i c Retma p-1, {'"--Type 188-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and p r e p a r a t i v e t h i n l a y e r chromatography ( t i c ) .  Chromatoplates were developed u s i n g  the s p r a y reagent carbon t e t r a c h l o r i d e - a n t i m o n y p e n t a c h l o r i d e ( 2 : 1 ) .  Woelm neutral alumina ( a c t i v i t y  III) was used for column  chromatography  (unless otherwise indicated). Radioactivity was measured with a Nuclear Chicago Mark I Model 6860 Liquid S c i n t i l l a t i o n  Counter i n counts per minute  r a d i o a c t i v i t y of a sample i n disintegrations per minute  (cpm).  The  (dpm) was  calculated using the counting e f f i c i e n c y which was determined for each sample by the external standard technique u t i l i s i n g the b u i l t i n barium133 gamma source.  The r a d i o a c t i v i t y of the sample was determined  using a s c i n t i l l a t i o n solution made up of the following composition: toluene (1 l i t r e ) , 2,5-diphenyloxazole (4 gm) and 1,4-bis[2-(5phenyloxazolyl)]benzene (0.05 gm).  In practice, a sample of an alkaloid  as a free base was dissolved i n benzene (1 ml) i n a counting v i a l . In the case of the s a l t of an a l k a l o i d , the sample was dissolved i n methanol. the  Then i n both cases, the volume was made up to 15 ml with  a b o v e ' s c i n t i l l a t o r solution.  background  For each sample counted, the  (cpm) 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 s c i n t i l l a t o r solution and counting (3 x 40 min).  The counting-vial was emptied, r e f i l l e d with the sample to be  counted and the s c i n t i l l a t o r solution,, and counted again (3 x 40 min). The difference i n cpm between the background count and the sample count was used for the subsequent For  calculations.  the sake of convenience and ease of presentation, the  experimental has been divided i n t o two portions.  The f i r s t part  describes the syntheses of 16,17-dihydrosecodin-17-ol (90) and secodine ; (107).  The second portion describes the syntheses of radioactive  precursors and their subsequent feeding into Vinca minor Linn.  ^  PART I 72 D i e t h y l - y ^ c h l o r b p r o p y l m a l o n a t e (91) To a s o l u t i o n o f sodium e t h o x i d e p r e p a r e d by d i s s o l v i n g sodium (23 gm, 1 mole). i n e t h a n o l (350 ml) was added i n one p o r t i o n a s o l u t i o n o f d i e t h y l malonate (160 gm, 1 mole) and 1,3-bromochloropropane (160 gm, 1 mole) i n d r y e t h e r (200 m l ) . was m a i n t a i n e d temperature  The r e a c t i o n m i x t u r e  a t 35° f o r 4 hours and then a l l o w e d t o s t a n d a t room  f o r 24 h o u r s .  Then t h e m i x t u r e was poured i n t o w a t e r  (700 ml) and e x t r a c t e d w i t h e t h e r .  The e x t r a c t was washed w i t h w a t e r ,  s a t u r a t e d sodium c h l o r i d e s o l u t i o n , d r i e d over anhydrous sodium s u l f a t e and c o n c e n t r a t e d under reduced p r e s s u r e . was d i s t i l l e d a t reduced 4 8 % ) ; bp 115°/0.5 mm, cm ; nmr  The r e s u l t i n g o i l  p r e s s u r e t o g i v e t h e d e s i r e d m a t e r i a l (112 gm,  ( l i t . bp 142°/10mm);  v  (film):  1730 (-C00C„H )  (60 mc/s) : x 5.75 ( q u a r t e t , 4H, 2 x C00CH_ CH ), 6.56  1  2  2H, -CR -CH -C1), 2  2  6.75  ( t r i p l e t , 1H, -CH -CH-(COOEt) ), 8.10 2  ( m u l t i p l e t , 4H, -CH -CH -CH -C1), 8.80 ( t r i p l e t , 6H, 2  Benzenediazonium  (triplet,  3  2  2  2  2x-C00CH CH ). 2  3  chloride^  A n i l i n e h y d r o c h l o r i d e (50 gm, 0.350 mole) was suspended i n a mixture of g l a c i a l a c e t i c acid (300 m l ) . nitrile  (300 ml) and d r y p e r o x i d e f r e e d i o x a n  The m i x t u r e was c o o l e d i n a i c e - s a l t b a t h and i s o a m y l  (50 gm, 0.420 mole) was added s l o w l y , the temperature  h e l d below 0°.  A f t e r t h e a d d i t i o n was complete the m i x t u r e was  f o r 30 m i n u t e s d u r i n g w h i c h t h e s o l i d s u s p e n s i o n d i s s o l v e d . dioxan  being stirred  Dry  (1500 ml) o r d r y e t h e r (1500 ml) was added i n t o n e p o r t i o n and t h e  w h i t e p r e c i p i t a t e o f benzenediazonium c h l o r i d e was c o l l e c t e d , washed s e v e r a l times w i t h f r e s h s o l v e n t and d r i e d i n a vacuum d e s s i c a t o r  (Yield 52 gm).  Synthesis of 2-carboethoxy-3-(g-chloroethyl)-indole (80) using benzenediazonium  chloride  To a solution of sodium ethoxide, prepared by dissolving sodium (8.25 gm, 0.360 mole) i n dry ethanol (1000 ml),'" was added diethyl-ychloropropymaionate ... (91, 85.0 gm, 0.360 mole) and the mixture was s t i r r e d under nitrogen f o r 30 minutes at room temperature.  After  cooling the reaction mixture i n a i c e - s a l t bath, the benzenediazonium chloride (52 gm, 0.370 mole) was added i n small portions.  During the  addition the temperature of the reaction mixture was held below -2°. After the addition of the diazo s a l t was complete, the mixture was s t i r r e d f o r 30 minutes and then l e f t i n the r e f r i g e r a t o r f o r 12 hours. The contents were poured into water (1000 ml) and the dark red o i l thus separated was extracted into ether.  The extract was washed with  water, saturated brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure.  The crude reaction product  (93, 106 gm) was immediately subjected to a Fisher indole synthesis described below. The material obtained above was dissolved i n dry ethanol (750 ml). To this concentrated s u l f u r i c acid (100 ml) was added slowly and the mixture was refluxed f o r 12 hours.  After cooling to room temperature,  the volume of the reaction mixture was reduced to half under reduced pressure.  The contents were poured onto i c e and the resulting  mixture was extracted with chloroform.  The extract was washed several  times with water, then with sodium carbonate•solution, and again with  water, dried over anhydrous sodium sulfate and evaporated under reduced^ pressure.  The crude dark semicrystalline material was p u r i f i e d by  chromatography  on alumina (Shawinigan, a c t i v i t y I I I , 3 kg).  ,.,  The  desired material was eluted, f i r s t with benzene and l a t e r with benzene-chloroform (1:1), as a c r y s t a l l i n e s o l i d .  Recrystallization  from chloroform-petroleum ether gave a white c r y s t a l l i n e s o l i d (11.2 19%), (log  mp 130-132°; v^ "*": max 0  e):  3250 (-NH) , 1670  229 (4.40), 296 (4.27) my; nmr  s i n g l e t , 1H, -NH), 2.60 -C00CH -CH ), 6.50 2  3  (-C00C H ) cm" ; - 2 5 1  o  c  A max  (60 mc/s): T 0.75 (broad  (multiplet, 4H, aromatic), 5.54  (multiplet, 4H, -CH_ -CH_ -C1), 8.60 2  gm,  2  (quartet, 2H,  (triplet,  3H,  -C00CH -CH ). Anal. Calc. for C CI.  14.12.  Found:  Benzenediazonium  H^O^Cl:  C, 62.03; H, 5.57; N, 5.57; 0. 12.75;  C, 62.07; H, 5.53; N, 5.60; 0. 12.64; CI, 14.08.  fluoroborate^'^  A n i l i n e hydrochloride (108 gm, 0.83 mole) was dissolved i n water (275 ml) and cone. HC1  (140 ml) i n a three l i t r e beaker.  was cooled to 0° and sodium n i t r i t e  The solution  (69 gm, 1 mole) i n water (150 ml)  was added dropwise, maintaining the temperature a l l the while below 5°.  The addition of the sodium n i t r i t e solution was stopped when a  drop from the reaction mixture gave a blue coloration with starch iodide paper.  A solution of 48% HBF^  (183 ml) was cooled to 0° and  added slowly to the diazonium s a l t solution.  P r e c i p i t a t i o n was  immediate but the s t i r r i n g was continued for an additional 10 minutes. About half of this suspension was transferred to a sintered glass funnel and washed with i c e cold water (50 ml), cold methanol  (25 ml) and ether  (50 ml). The  The  s a l t was  overnight.  s o l i d was  sucked as dry as possible after each washing.  transferred to a beaker and dried i n a vaccum dessicator The other half was  of s o l i d material was  (108  treated s i m i l a r l y .  The t o t a l weight  gm).  Synthesis of 2-carboethoxy-3-(g-chloroethyl)-indole (80) benzenediazonium  using  fluoroborate  To a solution of sodium ethoxide prepared by dissolving sodium (12 gm,  0.51  mole) i n dry ethanol (1000 ml) was  chloropropylmalonate (91, 120 gm,  0.50  added diethyl-y-  mole) and the mixture  s t i r r e d under nitrogen for 30 minutes at room temperature. cooling the mixture i n a i c e - s a l t bath, the fluoroborate 0.55  mole) was  always below -2°.  complete, the mixture was  poured into water (1000 ml) and extracted  salt  (105  gm,  After the addition of the s t i r r e d at 0° for 2 hours  and then l e f t i n the cold room (-10°) for 12 hours.  was  After  added i n small portions so that the temperature of the  reaction mixture was diazo s a l t was  was  into ether.  The contents were  the dark red o i l which separated out  The extract was  washed with water and with  saturated brine solution, dried over anhydrous sodium sulfate and then concentrated under vacuum. was  immediately subjected  The  crude reaction product (93, 190  to a Fisher indole synthesis  as  gm)  described  below. The thick red o i l was  dissolved i n dry ethanol (1000 ml).  this cone, s u l f u r i c acid (200 ml) was refluxed for 12 hours. mixture was 94 .  The  added and the mixture  To  was  After cooling to room temperature the reaction  worked up i n exactly the same manner as indicated on page  crude semicrystalline compound (140 gm)  was  p u r i f i e d by  chromatography  on alumina (Shawinigan, a c t i v i t y I I I , 3 kg).  Elution  with benzene-chloroform (1:1) afforded the desired compound.  This  was r e c r y s t a l l i s e d from chloroform-petroleum ether as white c r y s t a l l i n e plates (14 gm, y i e l d 13%), mp 131-132°. same  This material had the  value on t i c and spectral properties as the chloroindole (80)  obtained e a r l i e r when diazonium chloride was used.  3-Ethylpyridine (81)  on  A mixture of 3-acetylpyridine (60 gm), potassium hydroxide (50 gm), triethylene g l y c o l (400 ml) and 85% hydrazine (90 ml) was heated for 1 hour at 110-125°.  The reaction mixture was cooled and gradually  reheated with a take-off condenser to a bath temperature of 180-190°. When the evolution of the nitrogen i n the reaction mixture had ceased, the  volume collected from the take-off condensor  extracted with ether.  (about 200 ml) was  The extract was dried over anhydrous  sulfate and concentrated under reduced pressure.  sodium  The resulting o i l  was d i s t i l l e d using an e f f i c i e n t fractionating column and 3-ethylpyridine was collected at 162-163° ( l i t . value 162-165°) (36 gm, y i e l d 68%). v ^ ^ : m  no absorption i n the carbonyl region.  N-[g-{3-(2-carboethoxy)-indolyl}-ethyl]-3'-ethyl-pyridinium chloride (82) Chloroindole (80, 6.431  gm) was dissolved i n 3-ethylpyridine (22 ml)  and the mixture was heated i n a sealed tube at 120°- for 24 hours.  After  cooling to room temperature the sealed tube was opened and•the contents were poured into anhydrous ether with s t i r r i n g . l e f t at room temperature for 3 hours.  The mixture was then  During this time a l l the occluded  3-ethylpyridine was extracted from the s a l t into the ether.  The  white amorphous s o l i d was f i l t e r e d under suction, , washed several times with dry ether and f i n a l l y dried i n a vacuum dessicator (8.343 gm, y i e l d 91%); mp 87-89° ( r e c r y s t a l l i s e d from methanol-ether);  v  m a x  :  3120-2880 (several bands, ,vC-H, aromatic), 1701 (vC=0) and 1250 (vC-O-C) cm" ; X 1  220 (4.3) my;  (log e): 296 (4.2), 276 (inf)(3.9), 226 (4.35) and  nmr (CD 0D): x 1.4-2.34 (multiplet, 4H, pyridinium 3  protons), 2.56-3.18 (multiplet, 4H, indole protons), 5.74 (quartet, 2H, -C00CH -CH ), 7.40 (quartet, 2H, -CH_ -CH ), 8.63 ( t r i p l e t , 3H, 2  3  2  3  -C00CH -CH_ ), 8.98 ( t r i p l e t , 3H, -CH -CH ); mass spectrum: 2  3  main peaks:  2  3  M  +  358;  m/e 215, 187, 169, 129.  N-[g-{3-(2-Carboethoxy)-indolyl}-ethyl]-3'-ethyl-3'-piperideine To a solution of the pyridinium s a l t  (83)  (82, 7.67 gm) i n methanol  (275 ml) and triethylamine (8 ml) was added slowly a solution of sodium borohydride  (25 gm) i n methanol (400 ml).  The yellow color of the  s a l t solution was discharged when the addition of the borohydride was complete.  After s t i r r i n g at room temperature for 2.5 hours, the  reaction mixture was diluted with water (100 ml) and methanol was evaporated under reduced pressure.  The remaining aqueous solution was  a c i d i f i e d with 6 N HC1 to pH 2, s t i r r e d at room temperature f o r 20 minutes and then made basic with 10% sodium carbonate basic solution was extracted with methylene chloride.  solution.  The  The extract  was washed with water, dried over anhydrous sodium sulfate and evaporated to give a thick gum (7.003 gm).  This material was homogeneous on t i c  and was used i n the next reaction as such.  Nmr (60 mc/s) of the crude  p r o d u c t : T 4.5  (broad s i n g l e t , 1H,  C y - H ) , 5.60  ( q u a r t e t , - 2H,-C00CH -CH ) 2  N-[g-{3-(2-Hydroxymethylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine A s o l u t i o n of t e t r a h y d r o p y r i d i n e was  (83, 7.003 gm)  i n dry THF  3  (84) (70  ml)  dropped s l o w l y over a p e r i o d of 40 minutes i n t o a s u s p e n s i o n of  l i t h i u m aluminum h y d r i d e (9 gm) the a d d i t i o n was  i n THF  (350 ml)  under n i t r o g e n .  c o m p l e t e , the r e a c t i o n m i x t u r e was  temperature f o r 20 minutes and  After  s t i r r e d a t room  then r e f l u x e d f o r 2 h o u r s .  After  the m i x t u r e to i c e t e m p e r a t u r e , the excess of the h y d r i d e was  cooling  destroyed  by c a r e f u l a d d i t i o n of w a t e r (9 m l ) , 15% sodium h y d r o x i d e s o l u t i o n (9 ml)  and w a t e r a g a i n (27 m l ) .  under s u c t i o n and  The  organic  s u l f a t e and m a t e r i a l was  The  the r e s u l t i n g gum l a y e r was  f i l t r a t e was was  r e d i s s o l v e d i n methylene c h l o r i d e .  (5.390 gm).  chromatographed on a l u m i n a (150 gm).  :  3340 (vOH), 3180  (vNH)  (80) was  70%.  The  gm).  alcohol  l a t e r s u b l i m e d at 98°/0.01 mm, cm" ; 1  X  ITlcLX  (log e):  This  Elution with  gave the d e s i r e d a l c o h o l (84, 4.40  r e c r y s t a l l i s e d from . Me0H and  292  (3.73),  Yield  was mp  108-110°;  284  ITlclX  (3.81),  274  (shoulder)(3.76),  T 2.40-3.09 ( m u l t i p l e t , 4H, 5.17  e v a p o r a t e d under reduced  e v a p o r a t e d to a f f o r d a l i g h t y e l l o w gum  of the r e a c t i o n from c h l o r o i n d o l e  :  filtered  washed w i t h w a t e r , d r i e d over anhydrous sodium  b e n z e n e - c h l o r o f o r m (1:1)  v  r e a c t i o n m i x t u r e was  the p r e c i p i t a t e d h y d r o x i d e s were washed s e v e r a l times  w i t h methylene c h l o r i d e . p r e s s u r e and  The  Csinglet, 2H,  and  +  (4.45) mp;  indole protons),  -CH^-OH), 9.00  spectrum ( F i g u r e 1 6 ) : M  223  284;  4.45  ( t r i p l e t , 3H,  main peaks:  m/e  nmr  (CT^OD) ( F i g u r e  ( m u l t i p l e t , 1H,  174,  3  160,  h i g h r e s o l u t i o n mass s p e c t r o m e t r y : C a l c . f o r C^gH^N^O: Found:  284.184.  C ,-H),  -CH -CH_ ) ; mass 2  142,  124;  284.188.  15):  3  Anal. Calc. for (C,H N 0)CH.0H: o  o/  lo  Found:  C, 72.09; H, 8.93;  o  Z  ZH  C, 72.05; H, 9.13;  N,  8.85.  J  N,  8.22.  Benzoate ester of alcohol (84) The alcohol (84, 1.50 pyridine (15 ml).  gm,  5.4 mmole) was  The reaction mixture was  chloride (5.5 ml, 47 mmole) was minutes.  The mixture was  dissolved i n dry  cooled to 0° and  benzoyl  added dropwise over a period of 10  s t i r r e d at 0° for three hours, d i l u t e d with  water (15 ml), made basic with 10% aqueous sodium carbonate and extracted with methylene chloride.  solution  The extract was washed several  times with water, dried over anhydrous sodium sulfate and  then  evaporated very c a r e f u l l y (bath temperature not exceeding  40°) under  reduced pressure to afford a thick gum pyridine).  (2.370 gm,  contained traces of  This material was homogeneous on t i c and was used as such  for the succeeding reaction.  For a n a l y t i c a l purposes, a small amount  of benzoate (1 gm) was p u r i f i e d by chromatography on alumina (50 Elution with benzene gave the desired material.  This was  gm).  recrystallised  from methylene chloride-petroleum ether, mp 110.5-112.5°; v : • max  3000  J  (several bands, vC-H, (phenyl r i n g ) , 1260  aromatic), 1715 (vC-O-C) cm" ; 1  (vC=0 of benzoyl group),  X  (log e):  293  1455  (3.8), 284  (3.96),  nicLx  274  (3.94), 2.24  (4.61) mp;  nmr:  T 1.36  (singlet, 1H, indole NH),  (multiplet, 9H, 0^-0=0 + 4 indole protons), 4.58 -CH_-0-C-cj)) , 9.0 0 main peaks: metry:  m/e  (singlet, 3H, Cy-H  ( t r i p l e t , 3H, -CH -CH_ ) ; mass spectrum:  2  2  2-3  3  M  +  388;  266, 170, 143, 124, 122; high resolution mass spectro-  Calc. for C.X.N.O.: ZD  Zo  Z  388.216.  Anal. Calcd. for C „ H „ N 0 „ : c  ZJ  C, 77.05; H, 7.29;  N,  Found:  388.215.  Z o  ZO  7.05.  o  Z  Z  C, 77.27; H, 7.28;  N, 7.21.  Found:  +  A l t e r n a t i v e s y n t h e s i s o f benzoate (85) The a l c o h o l (84, 2.82 gm, 0.01 mole) was d i s s o l v e d i n d r y THF (50 ml) and t h e r e a c t i o n m i x t u r e was c o o l e d w i t h i c e . To t h i s anhydrous p o t a s s i u m c a r b o n a t e (5 gm) was added and t h e heterogeneous m i x t u r e was t r e a t e d , d r o p w i s e , w i t h b e n z o y l c h l o r i d e (5 m l , 0.042 mole) under n i t r o g e n .  T h e u n i x t u r e was s t i r r e d a t 0° f o r 1 hour and then a t  room temperature f o r 3 h o u r s .  The r e a c t i o n was worked up by a d d i n g  w a t e r (50 ml) f o l l o w e d by m i l d warming o f the m i x t u r e i n warm water bath.  A few minutes l a t e r s a t u r a t e d - sodium c a r b o n a t e s o l u t i o n (50 ml)  was added and t h e m i x t u r e was e x t r a c t e d u s i n g benzene and methylene chloride.  The o r g a n i c phase was washed w i t h w a t e r , d r i e d over  anhydrous  sodium s u l f a t e and evaporated.. The r e s u l t i n g m a t e r i a l was put on a column o f a l u m i n a (100 gm). benzoate  E l u t i o n w i t h chloroform afforded the  (85) as w h i t e c r y s t a l l i n e s o l i d  foam (1.5 gm, one s p o t on t i c ) . benzoate showed t h e same  (2.3 gm) as w e l l as a y e l l o w i s h  The o v e r a l l y i e l d was 99%.  This  v a l u e and s p e c t r a l p r o p e r t i e s as t h e  benzoate o b t a i n e d e a r l i e r .  N - [ 3 - { 3 - ( 2 - C y a n o m e t h y l e n e ) - i n d o l y l } - e t h y l ] - 3 ' - e t h y l - 3 ' - p i p e r i d e n i n e (86) The benzoate formamide (60 m l ) .  ( 8 5 , 2 gm, .005 mole) was d i s s o l v e d i n d i m e t h y l To t h i s s o l i d p o t a s s i u m c y a n i d e (3.3 gm, 0.050 mole)  was added and t h e heterogeneous m i x t u r e was s t i r r e d under n i t r o g e n a t room temperature f o r 1 hour.  The temperature o f t h e r e a c t i o n m i x t u r e  was now g r a d u a l l y r a i s e d t o 105-110° o v e r a p e r i o d o f 45 m i n u t e s . r e a c t i o n was m o n i t o r e d by t i c and a f t e r 1 hour a t t h e e l e v a t e d t u r e , t i c i n d i c a t e d the completion of the r e a c t i o n . c o o l e d down t o room t e m p e r a t u r e , d i l u t e d w i t h water  The  tempera-  The m i x t u r e was (100 ml) and  e x t r a c t e d w i t h methylene c h l o r i d e .  The  e x t r a c t was  washed several*".  times w i t h w a t e r , d r i e d over anhydrous sodium s u l f a t e and to a f f o r d a dark t h i c k o i l .  T h i s o i l y m a t e r i a l was  l e f t under vaccum  u n t i l a l l the dimethylformamide was  removed.  c r y s t a l l i n e compound (1.450 gm)  chromatographed on a l u m i n a .  w i t h benzene-petroleum e t h e r the pure n i t r i l e  was  The  evaporated  r e s u l t a n t dark Elution  (1:1) and l a t e r w i t h benzene f u r n i s h e d  (86) as a c r y s t a l l i n e compound (0.825 gm,  55%).  L a t e r f r a c t i o n s of e l u t i o n w i t h benzene and b e n z e n e - c h l o r o f o r m a f f o r d e d a s m a l l amount of a d d i t i o n a l n i t r i l e as gum T h i s l a t t e r m a t e r i a l was but the compound was reaction.  The  (0.125 gm,  of r e a s o n a b l e  q u a l i t y to be u t i l i s e d i n the next  o v e r a l l y i e l d of the r e a c t i o n was  -65%.  (86) o b t a i n e d was  For  135-137°; v  :  3160  analytical  recrystallised  from methylene c h l o r i d e - p e t r o l e u m e t h e r and l a t e r sublimed mp  10%).  c o n t a m i n a t e d w i t h a v e r y minute r e d i m p u r i t y  p u r p o s e s , a s m a l l amount of n i t r i l e  .01 mm,  (9:1)  at  100°/  (vN-H), = 2900 ( s e v e r a l bands,  vC-H,  max a r o m a t i c ) , 2256 (VCEN) cm" ; 1  (3.84)i  221  (4.69)my; nmr  2.46-3.00 ( m u l t i p l e t , 4H, 6.2 M  +  A  (log e):  ( F i g u r e 1 7 ) : x 1.63  spectrometry:  m/e  267,  in  C, 77.65; H,  7.86;  N, 14.16.  (3.85),  274  indole-NH),  ( m u l t i p l e t , 1H,  C ,-H), 3  ( t r i p l e t , 3H, -CH -CH ); mass spectrum: 2  169,  C a l c . f o r C^H^N.^:  A n a l . C a l c . f o r C H„„N„:  ( 3 . 7 7 ) , 281 ( s i n g l e t , 1H,  i n d o l e p r o t o n s ) , 4.58  ( s i n g l e t , 2H, -CH^-CN), 8.99 293; main peaks:  291  156,  3  124; h i g h r e s o l u t i o n mass  293.189.  C, 77.75; H,  Found: 7.92;  293.186.  N, 14.32.  Found:  N-[3-{3-(3-Carbomethoxymethylene)-indolyl}-ethyl]-3'-ethyl-3'piperideine  (88)  Crystalline n i t r i l e  (86, 0.746 gm,  2.5 mmole) was  dissolved i n  dry  methanol (20 ml) and to t h i s a s m a l l amount of water  was  added.  The m i x t u r e was  After stirring  c o o l e d i n i c e and s a t u r a t e d w i t h HC1  at room temperature  f o r 60 hours, the s o l u t i o n  taken to dryness under vacuum and the r e s i d u e was bicarbonate s o l u t i o n . chloride.  The b a s i c s o l u t i o n was  The e x t r a c t was  (0.2 ml o r  gas.  was  t r e a t e d w i t h sodium  e x t r a c t e d w i t h methylene  washed w i t h water,  sodium s u l f a t e and evaporated.  1%)  d r i e d over anhydrous  The crude p r o d u c t so o b t a i n e d  was  d i s s o l v e d i n a s m a l l amount of benzene and put on a column of alumina (40 gm).  E l u t i o n w i t h petroleum ether-benzene  benzene f u r n i s h e d the pure carbomethoxy e s t e r compound.  The compound was  petroleum e t h e r (0.574 gm,  recrystallised 70%), mp  (2:8) and l a t e r w i t h (88) as white  crystalline  from methylene c h l o r i d e -  85-87.5°;  v  :  3000 ( s e v e r a l bands,  max vC-H;., a r o m a t i c ) , 1728 (vC-O-C) cm" ;  X  1  (vC=0 of e s t e r ) , 1460  (log e):  292  (6C-H  (3.83), 283  , CH^-CO-),  (3.92), 274  1245  (3.87),  ITlctX  223  (4.43) mu;  nmr  ( F i g u r e 18): x 1.46  2.42-3.00 ( m u l t i p l e t , 4H, 6.27  i n d o l e p r o t o n s ) , 4.58  ( s i n g l e t , 2H, -CH -C00CH ), 6.34 2  (triplet,  (broad s i n g l e t , 1H,  3  3H, -CR^-CH.^); mass spectrum  indole-NH),  ( m u l t i p l e t , 1H,  C ,-H), 3  ( s i n g l e t , 3H, -CH^-COOCH^), ( F i g u r e 19):  M  +  9.00  326; main  peaks: m/e 267, 202, 156, 144, 124; h i g h r e s o l u t i o n mass s p e c t r o m e t r y : Calc. f o r C H N 0 : 326.199. Found: 326.202. 20 26 2 2 o o  o r  o  o  Anal. Calc. f o r C H „ , N 0 • 20 26 2 2 o n  C, 73.47; H,  8.05;  N,  8.71.  o  C, 73.50; H,  8.04;  N,  8.58.  Found:  Formylation of ester (88) using sodium hydride as base A 25-ml three necked flask was equipped with a magnetic a reflux condenser, a dropping funnel and a nitrogen i n l e t .  stirrer, A l l the  glassware was flame dried then thoroughly flushed with dry nitrogen. To the reaction flask a 65% suspension of sodium hydride i n p a r a f f i n o i l (0.050 gm, 1.3 mmole) was added.  This suspension was. washed three times  with 1-ml portions of dry benzene under nitrogen.  The o i l free sodium  hydride was suspended i n a fresh portion of dry benzene'(2 ml) and to this freshly d i s t i l l e d methyl formate (dried f i r s t over calcium chloride and then over ^2^5^ ^  m  ^  w  a  s  a  ^ded.  The carbomethoxy  ester (88, 0.050 gm, 0.15 mmole) was dissolved i n dry benzene (3 ml) and added dropwise to the above suspension.  The reaction mixture was  s t i r r e d at room temperature for.15 minutes and at 35° for 2 hours. t h i s time t i c indicated the completion of the reaction.  At  The excess of  hydride i n the reaction mixture was destroyed by cooling the mixture to 0°,  adding a few drops of methanol, followed by the addition of some  crushed i c e . The mixture was made a c i d i c with 2 N HC1. the  The excess of  acid was neutralised with aqueous sodium bicarbonate solution and  the heterogeneous mixture was extracted with methylene chloride.  The  extract was washed with water, dried over anhydrous sodium sulfate and evaporated to afford the crude enol. (89) as white foam (0.070 gm, contained some mineral o i l ) .  This material was used as such for the  next reaction.  16,17-Dihydrosecodin-17-ol (90) The crude enol (89) obtained above was dissolved i n methanol  (3 ml).  The solution was cooled to -30° i n a dry ice-acetone bath and sodium  borohydride  (0.050 gm) was added t o t h i s i n s m a l l p o r t i o n s .  After  s t i r r i n g f o r 40 minutes a t -30°, an a d d i t i o n a l amount o f sodium b o r o h y d r i d e (0.040 gm) was a g a i n added i n s m a l l p o r t i o n s t o t h e r e a c t i o n m i x t u r e . Ten/,minutes l a t e r t h e m i x t u r e i n d i c a t e d no more u n r e a c t e d on t i c and i n s t e a d t h e p o l a r " d i o l "  (105) had j u s t s t a r t e d  enol  appearing.  The excess o f b o r o h y d r i d e i n the c o l d r e a c t i o n m i x t u r e was t h e r e f o r e immediately  quenched by c a r e f u l a d d i t i o n o f 2-3 drops of 2 N HC1.  The m i x t u r e was d i l u t e d w i t h water (5 ml) and t h e methanol was evaporated  under reduced p r e s s u r e .  The r e m a i n i n g m i x t u r e was a c i d i f i e d  w i t h 2 N HC1, made b a s i c w i t h sodium b i c a r b o n a t e s o l u t i o n and e x t r a c t e d with chloroform.  The o r g a n i c phase a f t e r d r y i n g and e v a p o r a t i o n  a w h i t e foam (68 mg).  T h i s m a t e r i a l was d i s s o l v e d i n a s m a l l amount  o f benzene and p u t on a column of alumina benzene-chloroform  (2.5 gm).  r e a c t i o n from t h e e s t e r (88) was 40%.  vC-H, A  :  3  4  0  0  (  s h a r  P>  v N _ H  )»  3  0  5  0  The y i e l d of t h e  F o r a n a l y t i c a l purposes t h e  a l c o h o l (90) was c r y s t a l l i s e d from d i c h l o r o m e t h a n e , max  Elution with  i n t h e o r d e r ( 9 : 1 ) , ( 7 : 3 ) , (1:1) and f i n a l l y w i t h  c h l o r o f o r m a f f o r d e d t h e pure a l c o h o l (90, 22 mg).  V  left  mp 131.5-132°;  (broad, vO-H), ^ 2900 ( s e v e r a l bands,  a r o m a t i c ) , 1718 ( vC=0), 1465 (6C-H, CH^CO-), 1235 (vC-O-C) cm" ; 1  ( l o g e ) : 292 (3.86),'284 ( 3 . 9 3 ) , 274 ( s h o u l d e r ) (3.87) , 222 (4.49) my;  nmr ( F i g u r e 2 0 ) :  1.16 ( s i n g l e t , 1H, i n d o l e N-H), 2.48-3.00 ( m u l t i p l e t ,  4H, i n d o l e p r o t o n s ) , 4.61 (broad s i n g l e t , 1H, C^,_-H) , 6.00  (multiplet,  4H, -CH_ -0H + -C-H), 6.37 ( s i n g l e t , 3H, -C00CH_ ) , 9.04 ( t r i p l e t , 3H, 2  3  - C ^ - C H ^ ) ; mass spectrum ( F i g u r e 2 1 ) : M  +  356; main peaks:  214, 202, 124; h i g h r e s o l u t i o n mass s p e c t r o m e t r y :  m/e 338, 326,  Calc. f o r C H 0 1  z l  356.209.  Found:  356.207.  N 0 : zo z 3 C  o  Anal. Calc. f o r i 2 8 2 ° 3 c  a  N  2  :  C  ' • "' » ' - '> > 70  24  E  7  93  N  7  8 6  «  Found:  C, 70.20; H, 7.83; N, 7.35. .  Formylation of carbomethoxy ester C88) using t r i t y l sodium as base A 25-ml three necked flask was equipped with a magnetic a r e f l u x condenser, a dropping funnel and a nitrogen i n l e t .  stirrer, A l l the  glassware was flame dried and then thoroughly flushed with dry nitrogen. To a solution of the ester (88, 0.050 gm, 0.155 mmole) i n dry t e t r a hydrofuran (3 ml) was added dropwise a solution of t r i t y l (2.2 ml, 0.18 N, 0.387 mmole) under nitrogen.  sodium  The f i r s t half of the  t r i t y l sodium solution decolorized very rapidly as the proton from the  indole nitrogen reacted. . The other half of the solution decolorized  very slowly u n t i l f i n a l l y the last 1-2 drops were added, the red color of the base stayed i n the reaction mixture.  The solution was s t i r r e d  at room temperature f o r about 2 minutes and' then methyl formate (2 ml, dried f i r s t over calcium chloride and then freshly d i s t i l l e d over V^O^), was added dropwise.  The red color of the base disappeared immediately  and the resulting yellow solution was s t i r r e d at room temperature f o r 1.5 hours.  The solution was evaporated to dryness under vacuum and the  residue was a c i d i f i e d with 2 N HC1.  The excess acid was neutralised  with sodium bicarbonate solution and the mixture was extracted with chloroform.  The extract was washed with water, dried over anhydrous  sodium sulfate and evaporated under reduced pressure. (89) was u t i l i s e d as such for the next reaction.  The crude enol  R e d u c t i o n of the crude e n o l (89) o b t a i n e d by u s i n g t r i t y l sodium as base The crude p r o d u c t o b t a i n e d above ( c o n t a i n i n g crude e n o l and t r i phenylmethane) was did  d i s s o l v e d i n methanol.  not d i s s o l v e i n methanol and was  medium.  l e f t f l o a t i n g i n the r e a c t i o n  A f t e r c o o l i n g the heterogeneous  b o r o h y d r i d e . (50 mg) minutes.  was  Some of the t r i p h e n y l m e t h a n e  m i x t u r e down to -30°,  added i n s m a l l p o r t i o n s over a p e r i o d o f 10  The m i x t u r e was  s t i r r e d a t -30°  f o r 40 m i n u t e s .  time an a d d i t i o n a l amount of sodium b o r o h y d r i d e (40 mg) was added to the r e a c t i o n m i x t u r e . c o m p l e t i o n of the r e a c t i o n .  At t h i s again  10 minutes l a t e r t i c i n d i c a t e d the  The excess of b o r o h y d r i d e was  quenched by c a r e f u l a d d i t i o n o f 2-3 drops o f 2 N HC1 reaction mixture.  sodium  immediately  to the c o l d (-30°)  The m i x t u r e was worked up i n e x a c t l y the same  manner as i n d i c a t e d p r e v i o u s l y (page 105)• chromatographed on a l u m i n a  (2; 5 gm).  Elution with  e t h e r (1:1) gave the t r i p h e n y l m e t h a n e . e l u t e d w i t h benzene-chloroform f i n a l l y with chloroform.  The crude p r o d u c t  was.  benzene-petroleum  The d e s i r e d compound (90)  i n the o r d e r ( 9 : 1 ) , ( 7 : 3 ) , (1:1)  The pure a l c o h o l (90, 20 mg)  was  and  obtained  r e p r e s e n t e d a y i e l d of 37% from the e s t e r ( 8 8 ) .  Secodihe  (107)  A 10-ml  f l a s k was  equipped w i t h a magnetic  condenser, and a n i t r o g e n i n l e t .  stirrer, a reflux  A l l the g l a s s w a r e was  d r i e d and then t h o r o u g h l y f l u s h e d w i t h d r y n i t r o g e n .  first  To the r e a c t i o n  f l a s k a 65% s u s p e n s i o n of sodium h y d r i d e i n m i n e r a l o i l (25 mg, mmole) was  added.  flame  0.65  T h i s s u s p e n s i o n was washed t h r e e times w i t h 0.5-ml  p o r t i o n s of d r y benzene and f i n a l l y  the o i l f r e e sodium h y d r i d e was  suspended i n a fresh portion of dry benzene (0.5 ml). 16,17-dihydrosecodin-17-ol (90, 20 mg, (2.5 ml) was nitrogen.  0.06  mmole) i n dry benzene  dropped very-rapidly into the above suspension under  The reaction mixture was  s t i r r e d at 40° for 15 minutes.  the meantime a column of alumina (2 gm, benzene.  A solution of  In  a c t i v i t y IV) was made i n dry  The crude reaction mixture was-flushed through t h i s column  using benzene as eluent. desired material, was  This f r a c t i o n (40 ml) which contained  collected i n a cold receiver.  It was  the  frozen  with l i q u i d nitrogen and freeze-dried under vacuum to afford secodine (107)  as a l i g h t yellow gum  (broad s i n g l e t , 1H,  (9.1: mg, ;  50%).  Nmr  (Figure 31): T  indole-NH), 2.40-3.00 (multiplet 4H, indole  3.55  (doublet, J  3.91  (doublet, J  4.58  (multiplet, "1H,  m/e  307,  protons),  1 Hz, 1H, o l e f i n i c proton of the a c r y l i c e s t e r ) , 1 Hz, 1H, o l e f i n i c proton of the a c r y l i c ester), C^-H), 6.20  ( s i n g l e t , 3H, CH =C-C00CH_ ), 2  ( t r i p l e t , 3H, -CTL^-GH^); mass spectrum (Figure 32 )'• peaks:  0.89  251,  214,  154,  124.  3  M  +  9.00  338; main  PART I I 3 T r i f l u o r o a c e t i c acid-[.H] T r i f l u o r o a c e t i c anhydride (1.17 gm, 5.55 mmoles) was added to 3 water- H (0.10 gm, 5.50 mmoles, 100 mcurie/gm) using a vacuum 3 transfer system.  The r e s u l t i n g t r i f l u o r o a c e t i c acid- H (1.27 gm, 0.9  mcurie/mmole) was stored under an atmosphere of nitrogen at -10° u n t i l required. 3 [ar- H]-Carbomethoxy ester (88) 3 T r i f l u o r o a c e t i c acid- H (1.27 gm, 0.9 mcurie/mmole) was added to the c r y s t a l l i n e ester (88, 0.1887 gm) using a vac'uum transfer system.  The solution was allowed to stand at room temperature for  48 hours under nitrogen atmosphere.  After this time the t r i f u l o r o a c e t i c  3 acid- R was removed with a vacuum transfer system and concentrated ammonium hydroxide solution (10 ml) was added c a r e f u l l y to the above gummy residue.  The mixture was extracted with dichloromethane.  The  extract was washed with water, dried over anhydrous sodium sulfate and evaporated.  The resulting gum was dissolved i n methanol (10 ml) and  then evaporated. l a b i l e tritium.  This process was repeated four times to remove any The crude product was put on a column of alumina (15  gm) and the desired compound was eluted with benzene (0.152 gm, 80% 8 y i e l d , s p e c i f i c a c t i v i t y 1.10 x 10 dpm/mg). 3 Formylation of [ar- H]-carbomethoxy ester (88) The t r i t i a t e d ester obtained above (0.152 gm) was formylated using sodium hydride (0.150 gm) and freshly d i s t i l l e d methyl formate (4 ml) (for complete d e t a i l s of the procedure see page 104).  Temperature of the  reaction mixture was maintained at 35° and the reaction took 2 hours for completion.  Evaporation of the solvent after work up gave the  crude radioactive enol (89, 180.5 mg). reaction as such.  This was used for the next.  '  [ar- H]-16,17-Dihydrosecodin-17-ol (90) 3  The crude active enol (89, 180.5 mg) was dissolved i n methanol (15 ml)i  After cooling the reaction mixture down to -30°, sodium  borohydride (180 mg) was added i n small portions over a period of 15 minutes.  The mixture was s t i r r e d at -30° for 40 minutes.  At this  time an additional amount of sodium borohydride (100 mg) was again added i n small portions.  15 Minutes l a t e r when the mixture indicated no  more enol on t i c , the excess of borohydride was quenched with a few drops of 2 N HC1 and the mixture was worked up i n exactly the same manner as indicated on page 105. The crude product (152 mg) was chromatographed  on alumina (8 gm).  Elution with benzene-chloroform  i n the order (9:1), (7:3), (1:1) and f i n a l l y with chloroform afforded the pure alcohol (90) which was c r y s t a l l i s e d from methylene  chloride-  petroleum ether (72 mg, 43% y i e l d , s p e c i f i c a c t i v i t y 2.83 x 10"^ dpm/ mmole or 7.94 x 10^ dpm/mg).  14 N-[g-{3-(2-Cyano( CN)methylene)-indolyl}-ethyl]-3'-ethyl-3'-piperideine (86) The benzoate (85, 1.09 gm, 0.25 mmole) was dissolved i n dimethylformamide (25 ml).  This was treated with a mixture of radioactive  14 C-potassium cyanide (0.072 gm, t o t a l a c t i v i t y 8 mcurie) and potassium cyanide (0.753 gm, t o t a l potassium cyanide (0.825 gm) used i n the reaction was 1.25 mmole or a f i v e f o l d molar excess).  The reaction  was s t i r r e d at room•• temperature for 1 hour under nitrogen.  The  temperature of the reaction mixture was now gradually raised to 105° over a period of 45 minutes.  After 1 hour at this elevated temperature,  t i c indicated the completion of the reaction.  The mixture was  to room temperature and the crude readioactive n i t r i l e  cooled  (86) was  isolated as a dark c r y s t a l l i n e compound (for d e t a i l s of the work up see page 102) . (30 gm).  This crude material was chromatographed  Elution with benzene and l a t e r with benzene-chloroform (9:1)  afforded the pure active n i t r i l e 4.32 x 10 was  on alumina  6  dpm/mg or 1.27 x 10  (86, 0.483 gm, s p e c i f i c a c t i v i t y  9* ^ dpm/mmole).  The y i e l d of the reaction  63%.  [ C00CR ]-Carbomethoxy ester (88) 14  3  C r y s t a l l i n e radioactive n i t r i l e dissolved i n dry methanol  (10 ml).  (86, 0.480 gm, 1.6 mmoles) was To this a small amount of water  (0.1 ml or 1%) was added and the solution was saturated with HC1 gas. After s t i r r i n g at room temperature for 60 hours, the crude ester was isolated £(for complete d e t a i l s of the work up see page 103).  It was  dissolved i n a small amount of benzene and put on a column of alumina (25 gm).  Elution with benzene-petroleum ether (2:8) and l a t e r with  benzene furnished the pure radioactive ester (86, 0.302 gm). y i e l d of the reaction was  The  65%.  14 Formylation of [ COOCH^]-carbomethoxy ester (88) Radioactive ester (88, 0.100 gm, 0.30 mmole) was formylated using sodium hydride (0.100 gm, 2.6 mmoles) and freshly d i s t i l l e d methyl formate (4 ml) (for complete>.details of the procedure see page  104).  The crude enol (89, 0.126  gm) isolated was used d i r e c t l y for the  next reaction.  [ C00CH ]-16,17-Dihydrosecodin-17-ol 14  3  The crude active enol (89, 0.126 i n methanol.  (90) gm) obtained above was dissolved  After cooling the solution downNto -30°, sodium  borohydride (100 mg) was added i n small portions over a period of 15 minutes.  The mixture was s t i r r e d at -30° for 40 minutes.  At this  time a small amount of sodium borohydride (0.040 gm) was again added slowly to the reaction mixture. Teh/minutes l a t e r when t i c indicated the completion of the reaction, the crude alcohol was isolated (for d e t a i l s of the work up see page 105) and chromatographed (5 gm).  on alumina  Elution with benzene-chloroform i n the order (9:1), (7:3),  (1:1) and f i n a l l y with chloroform afforded the pure alcohol (90, 0.044 6 mg,  s p e c i f i c activity" ,3. 56 x 10  from the ester (88) was  dpm/mg).  The y i e l d of the reaction  40%.  Extraction of alkaloids from Vinca minor Linn The following procedure was developed i n order to extract and purify the alkaloids of Vinca minor Linn plants.  This procedure was  used for a l l extractions of Vinca minor L. plants and was  scaled  according to the wet weight of the plants used. Vinca minor Linn plants (9 kg', wet weight), obtained from the gardens of the University of B r i t i s h Columbia, were •macerated  with  methanol i n a Waring blender, f i l t e r e d and .-re-macerated " u n t i l the  f i l t r a t e was c o l o r l e s s .  This green f i l t r a t e (8000 ml) was concentrated  to dryness under reduced pressure and the residue was dissolved i n 2 N HC1 (4500 ml).  The acid layer was extracted with benzene (2 x 2000  ml) and the benzene extracts were back extracted with 2 N HC1 (2 x 500 ml).  The combined aqueous phases were made basic with 15 N ammonium  hydroxide, taking care that the temperature of the solution did not r i s e above 25°, and extracted with chloroform (3 x 2400 ml). combined chloroform extracts  The  were washed with water, dried over sodium  sulfate, and concentrated under reduced pressure.  The resulting  alkaloids residue (13.6 gm) was dissolved i n benzene-methylene chloride (1:1) (100 ml) and chromatographed on alumina (700 gm).  The column  was eluted successively with petroleum ether, benzene, chloroform, and methanol; fractions of 700 ml were taken.  The fractions eluted  with petroleum ether and benzene were combined and subjected to an additional column chromatography on alumina (300 gm).  Elution with  petroleum ether-benzene (6:4) afforded minovine (73, 0.41  gm).  Successive f r a c t i o n s , eluted with petroleum ether-benzene (2:8) and f i n a l l y with benzene, were combined and c r y s t a l l i s e d from methanol affording vincamine (72, 0.65 gm).  Both these alkaloids were compared  on t i c with authentic samples.  14 Feeding of [  COOCH :]-16,17-dihydrosecodin-17-ol (90) to Vinca minor 3  Linn, (feeding experiment no. I) [ C00CH ]-16,17-bihydrosecodin-17-ol 14  3  6 10  (90 , 0.0065 gm, 9.89 x •*:  dpm) was made soluble i n 0.1 N acetic acid (1 ml) and ethanol (0.5 :  The cloudy.solution was diluted with' water (5 ml).  The.resulting clear  solution was distributed equally among ten test tubes and  three Vinca minor cuttings were placed into each of these test tubes (total weight of plants u t i l i s e d was 43 gm).  The plants were placed  under fluorescent lamp, illumination and the aqueous levels i n the test tubes were maintained with d i s t i l l e d water.  After four days the  cuttings were extracted to afford the crude alkaloids as dark foam, ft (0.092 gm, 3.06 x 10  dpm.or 31% of the total- a c t i v i t y fed).  The  crude extract was dissolved i n a small amount of benzene-methylene chloride (~ 2ml) and put on a column of alumina (15 gm).  Elution  with petrole um ether—benzene (1:1) and then further p u r i f i c a t i o n of the resulting gum by preparative thin layer chromatography  ( s i l i c a gel,  ethyl acetate-methanol, 2:1) afforded pure minovine (73, 0.00545 gm). It was diluted further with an authentic sample of minovine (15.70 mg). Several c r y s t a l l i s a t i o n s revealed that the a l k a l o i d contained v i r t u a l l y no a c t i v i t y . Further e l u t i o n of the column with petroleum ether-benzene (4:6) afforded pure vincamine (72) as a c r y s t a l l i n e compound (7.7 mg). After several c r y s t a l l i s a t i o n s from methanol, vincamine indicated an a c t i v i t y of 102 dpm  (total).  This represented a maximum incorpora-  t i o n of < 0.001%. 3 Feeding of [ar- H]-secodine(107) to Vinca minor Linn.  (feeding  experiment no. 2) In view of the rapid dimerization of secodine the following l  procedure was developed and i s t y p i c a l of a l l the feeding experiments done whenever secodinewas fed to the various plant species i n our laboratories.  A 10-ml  f l a s k was  equipped w i t h a magnetic s t i r r e r , a r e f l u x  condenser and a dry n i t r o g e n i n l e t . and  then t h o r o u g h l y  glassware  flushed with nitrogen.  h y d r i d e i n m i n e r a l o i l (10 mg, flask.  The  T h i s s u s p e n s i o n was  0.26  was  A 65%  mmole) was  flame d r i e d  suspension  of sodium  added to the r e a c t i o n  washed t h r e e times w i t h 0.5  ml-portions  of d r y benzene under n i t r o g e n and the o i l f r e e sodium h y d r i d e suspended i n a f r e s h p o r t i o n of d r y benzene (0.5  ml).  was  In a small dry  3 t e s t tube [ a r - H]-16,17-dihydrosecodin-17-ol (90,  10 mg,  0.03  79.4  ml) by  slightly  x 10^  dpm)  was  d i s s o l v e d i n dry benzene (1.2  warming the t e s t tube i n a hot water b a t h .  T h i s s o l u t i o n was  v e r y r a p i d l y ( i n about 35 seconds) i n t o the s u s p e n s i o n hydride.  The m i x t u r e was  s t i r r e d a t 40°  f r a c t i o n was  The  column was  d i l u t e d to 100 -3  l a t t e r s o l u t i o n (or 2.5  x 10  c o u n t i n g purposes.  remaining  The  f r a c t i o n was.transferred f r a c t i o n was vacuum. 2.65  x 10 The  o f sodium  gm,  a c t i v i t y IV) made  e l u t e d w i t h benzene and  c o l l e c t e d i n a c o l d 25r-ml v o l u m e t r i c f l a s k .  t h i s s o l u t i o n was  dropped  f o r 15 m i n u t e s and then q u i c k l y  f l u s h e d t h r o u g h a s m a l l column o f a l u m i n a (1.5 up w i t h benzene.  mmole,  the e l u t e d One  ml w i t h benzene and 1 ml of  o f the compound to be fed) was  ml  of  this used f o r  p o r t i o n (24 ml) o f the benzene  to a s p e c i a l l y designed  evaporator.  The  f r o z e n w i t h l i q u i d n i t r o g e n and f r e e z e - d r i e d under  T h i s f u r n i s h e d secodine(107) as a l i g h t y e l l o w gum  (3.2  mg,  dpm).  8  gum  and e t h a n o l  o b t a i n e d above was made s o l u b l e i n 0.1 (0.5  m l ) . T h e . c l o u d y / s o l u t i o n was  N a c e t i c a c i d (1  ml)  d i l u t e d w i t h water (5 ml)  and  the r e s u l t i n g c l e a r s o l u t i o n w a s ' d i s t r i b u t e d "equally, among t e n t e s t - t u b e s . Three V i n c a minor c u t t i n g s were i n s e r t e d i n t o each"""of. these t e s t tubes ( t o t a l w e i g h t of p l a n t s u t i l i s e d was  40 gm)  and  the  plants were placed under fluorescent lamp i l l u m i n a t i o n and the aqueous l e v e l s i n the test tube were maintained with d i s t i l l e d water.  After  four days, the cuttings were extracted to afford the crude alkaloids as a dark foam (90.2 mg, 5.8 x 10^ dpm representing a recovery of 22% of the t o t a l a c t i v i t y fed).  The crude product was dissolved i n a  small amount of benzene-methylene chloride and chromatographed on alumina (20 gm).  E l u t i o n with petroleum ether-benzene (7:3) and then  subsequent p u r i f i c a t i o n of the r e s u l t i n g gum by preparative t i c ( s i l i c a gel,  ethyl acetate-methanol, 2:1) afforded pure minovine (73, 4.8 mg).  This was d i l u t e d with cold minovine (10.4 mg) andthe t o t a l compound (15.2 mg) was c r y s t a l l i s e d to constant represented  a c t i v i t y (562 dpm/mg).  a maximum incorporation of 0.001%.  This  This incorporation was  corrected f o r the amount of secodine(107) that dimerized  at the time  of feeding by running a "blank" experiment (for d e t a i l s see pageill7) . The corrected incorporation should be < 0.0015%. Further e l u t i o n of the column with petroleum ether-benzene (1:1) afforded c r y s t a l l i n e vincamine (72, 12.7 mg).  This was further  p u r i f i e d by preparative t i c (alumina, ethylacetate-chloroform, 1:1). Pure vincamine (3.8 mg) obtained  i n this manner was.diluted  with cold  vincamine (4.65 mg) andthe mixture was c r y s t a l l i s e d to constant a c t i v i t y (261 dpm/mg). of 0.0013%.  This represented  a maximum incorporation  This extent of incorporation was again corrected f o r the  amount of secodine(107) that dimerized  at the time of feeding by c o r r e l a -  tion with the "blank" experiment (for d e t a i l s see page 117). The corrected incorporation should be 0.002%.  " B l a n k " f o r the f e e d i n g experiment no. 2 [ C O O C H ] - 1 6 , 1 7 - D i h y d r o s e c o d i n - 1 7 - o l (90, 10 mg, 14  3  20.7 x 1 0  dpm)  6  14 6 was dehydrated to [ COOCH^-secodine(107, 3.4 mg,7.03 x 10 dpm)  in  e x a c t l y the same manner as i n d i c a t e d i n the f e e d i n g experiment no.  2.  The r e s u l t i n g gum was made s o l u b l e i n 0.1 N a c e t i c a c i d (3 ml) e t h a n o l (0.5 m l ) . hours  The s o l u t i o n was  and  l e f t a t room temperature f o r 2  ( t h i s i s the maximum time the V i n c a minor c u t t i n g s t a k e to  absorb t h e above volume o f s o l u t i o n ) , f r o z e n w i t h l i q u i d n i t r o g e n , and f i n a l l y f r e e z e - d r i e d under vacuum. i n methanol  The r e s u l t i n g s o l i d was  and a s m a l l p o r t i o n of t h i s s o l u t i o n was  on two Kodak n e u t r a l alumina chromatogram s h e e t s . developed i n a m i x t u r e of b e n z e n e - c h l o r o f o r m  redissolved  spotted h o r i z o n t a l l y  The s h e e t s were  (1:1) and passed  through  a c a l i b r a t e d N u c l e a r - C h i c a g o A c t i g r a p h 11 Model 1039  t i c counter  connected to a r e c o r d e r ( N u c l e a r - C h i c a g o Model 8416)  and  ( N u c l e a r - C h i c a g o Model 8704).  integrator  The a c t i v i t i e s found i n t h r e e s p o t s  c o r r e s p o n d i n g t o the b a s e l i n e , d i m e r i c compounds (presecamine secamine) and s e c o d i n e and t h e i r r e l a t i v e p e r c e n t a g e s i n the 1  and initial  m i x t u r e are summarized i n T a b l e 2.  T a b l e 2.  R e s u l t s of the " b l a n k " experiment .  Sheets  Secodine activity  %  cpm  D i m e r i c Compounds  Baseline Material  activity  activity  %  cpm  %  cpm  Sheet no. 1  21,693.94  61.22  11.313  32  2,394.46  6.78  Sheet no. 2  32,650  61.52  16,871  32.03  3,422  6.45  REFERENCES  1.  R. Robinson, "The Structural Relations of Natural Products", Claredon Press, Oxford, (1955).  2.  E.E. van Tamelen, L.J. Dolby, and R.G. Lawton, Tetrahedron Letters, 30 (1960).  3.  E. Leete, i n "Biogenesis of Natural Products", p. 953, ed.- P. Bernfeld, Pergamon Press, (1967).  4.  A.R. Battersby, Pure Appl. Chem., 14_, 117 (1967).  5.  A.I. Scott, Acc. Chem. Res., 3_, 151 (1970).  6.  G. Barger and C. Sicholz, Helv. Chim. Acta, 16, 1343 (1933).  7.  M. Hesse, "Indolalkaloide i n Tabellen", Sprinzer, B e r l i n , Vol. 1, 1964; Vol. I I , 1968; Alkaloids, 11, 1 (1968).  8.  D. Groger, K. S t o l l e , and K. Mothes, Tetrahedron Letters, 2579 (1964)  9.  E. Leete, A. Ahmad, and I. Kompis, J . Amer. Chem. Soc., 87, 4168 (1965).  N  10.  E. Lette, Acc. Chem. Res., _2, 59 (1969).  11.  I.D. Spenser, Compr. Biochem., 20, 231 (1968).  12.  A.R. Battersby, A.R. Burnett, and P.G. Parsons, Chem. Comm., 137 (1968) and references therein.  13.  E. S c h l i t t e r and W.I. Taylor, Experientia, 16, 244 (1960).  14.  G. Hahn and H. Ludewig, Ber. , 67_, 203 (1934).  15.  R.B. Woodward, Nature, 162, 155 (1948).  16.  R.B. Woodward, Angew. Chem.,68, 13 (1956).  17. - E. Wenkert and N.V. B r i n g i , J . Am. Chem. Soc., 81_, 1474, .1535 (1959). 18.  E. Wenkert, Experientia, 1_5, 165 (1959).  19.  E. Wenkert, J . Am. Chem. S o c , 84, 98 (1962).  20.  P.N. Edwards and E. Leete, Chem. and Ind., 1666 (1960).  21.  E. Leete, S. Ghosal, and P.N. Edwards, J . Am. Chem. Soc.,84, 1068 (1962).  22.  E. Leete and S. Ghosal, Tetrahedron Letters, 1179 (1962).  23.  R. Thomas, Tetrahedron Letters, 544 (1961).  24.  T. Money, I.G. Wright, F. McCapra, and A.I. Scott, Proc. Nat. Acad.Sci., U.S.A., 53, 901 (1965).  25.  F. McCapra, T. Money, A.I. Scott, and I.G. Wright, Chem. Comm., 537 (1965).  26.  H. Goeggel and D. Arigoni, Chem. Comm., 538 (1965).  27.  A.R. Battersby, R.T. Brown, R.S. K a p i l , A.O. Plunkett, and J.B. Taylor, Chem. Comm., 46 (1966).  28.  A.R. Battersby, R.T. Brown, R.S. K a p i l , J.A. Knight, J.A. Martin, and A.O. Plunkett, Chem. Comm., 801 (1966).  29.  A.R. Battersby, R.T. Brown, J.A. Knight, J.A. Martin and A. O. Plunkett, Chem. Comm., 346 (1966).  30.  P. Leow, H. Goeggel, and D. Arigoni, Chem. Comm., 347 (1966).  31.  E.S. H a l l , F. McCapra, T. Money, K. Fukumato, J.R. Hanson, B. S. Moots; G.T. P h i l i p s ,i and A:;i. Scott, Chem. Comm. , 348 (1966) .  32.  E. Leete and S. Ueda, Tetrahedron Letters, 4915 (1966).  33.  A.R. Battersby, R.T. Brown, R.S. K a p i l , J.A. Martin, and A. O. Plunkett, Chem. Comm., 812 (1966). A.R. Battersby, R.S. K a p i l , J.A. Martin, and L. Mo, Chem. Comm.,  34.  133 0-968).  35.  A.R. Battersby, R.S. K a p i l , and R. Southgate, Chem. Comm., 131 (1968).  36.  S. Brechbuhler-Bader, C.J. Coscia, P. Leow, Ch. von Szczepanski and D. Arigoni, Chem. Comm., 136 (1968).  37.  M.M.^JanotP. Potier, P. Francois, J . L e v i s a l l e s , A.R. Battersby, B. Gregory^ H. Spenser and J.C. Turner, Chem. Comm., 219 (1967).  38.  A.R. Battersby, A.R. Burnett, and P.G. Parsons, J . Chem. S o c , 1187 (1969).  39.  P. Leow, Ch. von Szczepanski, C.J. Cosica, and D. Arigoni. Chem. Comm., 1276 (1968).  40.  A.R. Battersby, A.R. Burnett, G.D. Knowles, and P.G. Parsons, Chem. "Comm., 1277 (1968).  41.  A.R. Battersby, A.R. Burnett, and P.G. Parsons, Chem. Comm., 1280 (1968).  42.  H. Inouye, S. Ueda, Y. Aoki, and Y; Takeda, Tetrahedron Letters, 2351 (1969).  43.  H. Inouye, S. Ueda and Y..Takeda, Tetrahedron Letters, 3453 (1968).  44.  G.N. Smith, Chem. Comm., 912 (1968).  45.  R.T. Brown, G.N. Smith, K.S.J. Stapleford, Tetrahedron Letters, 4349 (1968).  46.  A.R. Battersby, A.R. Burnett, and P.G. Parsons, Chem. Comm., 1282 (1968).  47.  A.R. Battersby, A.R. Burnett, and P.G. Parsons, J . Chem. Soc.j 1193 (1969).  48.  A.R. Battersby, A.R. Burnett, E.S. H a l l , and P.G. Parsons, Comm., 1582 (1968) .  49.  A.R. Battersby, J.C. Byrne, R.S. K a p i l , J.A. Martin, T. Payne, D. Arigoni, and P. Leow,.Chem. Comm., 951 (1968).  50.  A.R. Battersby and E.S. H a l l , Chem. Comm., 793 (1969).  51.  A.A. Qureshi and A.I. Scott, Chem. Comm., 948 (1968).  52.  A.I. Scott, P.C. Cherry and A.A. Qureshi, J . Am. Chem. S o c , 91, 4932 (1969) .  53.  E. Wenkert, and B. Wickberg, J . Am. Chem. S o c , 87_, 1580 (1965).  54.  J.E. Saxton, A l k a l o i d s 1 0 , 501 (1968).  55.  J . Harley-Mason and W. Waterfield, Tetrahedron 19, 65 (1963); A.J. Gaskelle and J.A. Joule, i b i d ; 23, 4053 (1967).  56.  A.I. Scott, and A.A. Qureshi, J . Am. Chem. S o c , 91, 5874 (1969).  57.  T. Oishi, M. Nagai and Y. Ban, Tetrahedron Letters, 491 (1968).  58.  E. Wenkert and B. Wickberg, J . Am. Chem. S o c , 87_, 1580 (1965).  59.  J.P. Kutney and E. P i e r s , J . Am. Chem. S o c , 8j6, 953 (1964); J.P. Kutney, E. Piers and R.T. Brown, i b i d , 92, 1700 (1970).  60.  J.P. Kutney, R.T. Brown, and E. P i e r s , J . Am. Chem. S o c , J36, 2286, 2287 0-964); J.P. Kutney, R.T. Brown, E. Piers and J.R. Hadfield, i b i d , 92, 1708 (1970).  Chem.  61. .62.  J.P. Kutney, N. Abdhurahman, P. Le Quesne, E. Piers and I. Vlattas, J . Am. Chem. S o c , 88, 3656 (1966). J.P. Kutney, W.J. Cretney, P. Le Quesne, B. Makague and E. P i e r s , J. Am. Chem. S o c , 88, 4756 (1966); 92, 1712 (1970).  63.  J.P. Kutney, W.J. Cretney, J.R. Hadfield, E.S. H a l l , V.R. Nelson, and D.C. Wigfield, J . Am. Chem. S o c , 90, 3566 (1968).  64.  J.P. Kutney, C. Ehret, V.R. Nelson, and D.C. Wigfield, J . Am. Chem. S o c , 90, 5929 (1968).  65.  J.P. Kutney, J.F. Beck, V.R. Nelson, and R.S. Sood, submitted for publication.  66.  A.A. Qureshi and A.I. Scott, Chem. Comm., 945, 947 (1968).  67.  R.T. Brown, J.S. H i l l , G.F. Smith, K.S. Stapleford, J . Poisson, M. Muquet, and N. Kunesch, Chem. Comm., 1475 (1969).  68.  R.A. Barnes, Properties and Reactions ofvPyridines and I t s Hydrogenated Derv., i n "The Chemistry of Heterocyclic Comps.", Vol. 14 (Part 1), p. 1, ed. E. Klingberg, Interscience Pub. Inc., N.Y. (1960).  69.  D.L. C o f f i n , J . Org. Chem., J33_, 137 (1967) and references therein.  70.  J . Le Men and W.I. Taylor, Experientia, 21, 508 (1965).  71.  N. Abdurahman, Ph.D. Thesis, U .B .C .,• p. 35, (1967).  72.  T.A. Favorskaya and I.P. Yakoulev, Zhur. Obshchei Khim, _22, 113 0-952); C.A., 46, 1119 (1952).  73.  R. P h i l l i p s , Organic Reactions, V o l . X, p. 143, (1959).  74.  R.L. Frank and R.R. P h i l l i p s , J . Am. Chem. Soc. , 7_1, 2804 0-949).  75.  W. Smith and C.E. Waring, J . Am. Chem. Soc. , 64_, 469 (1942).  76.  R. Robinson, Chem. Rev., 63, 373 (1963).  77.  U.K. Pandit, M.J.M. Pollmann, and H.0. Huisman, Chem. Comm., 10, 527 (1969).  78.  D.T. Flood, Organic Syntheses, C o l l . Vol. I I , p. 295, ed. A.H. B l a t t , John Wiley and Sons, Inc., London, (1947).  79.  A.I. Vogel, P r a c t i c a l Organic Chemistry, p. 609, Longman, Green and Co. Inc., London (1961).  80.  R.L. Frank and C. Weatherbee, J . Am. Chem. S o c , 7_0, 3482 (1948).  81.  J . J . Panouse, B u l l . Soc. Chim. France, D60 (1953).  82.  R.C. E i n g e r f i e l d , B.C. Fischer, and J.M. Lagowski, J . Org. Chem., 22, 1376 (1957).  83.  E. Wenkert, R.A. Massy-Westropp, and R.G. Lewis, J . Am. Chem. S o c , 84, 3732 (1963).  84.  E. Wenkert, K.G. Dave, C.T. Bnewuch and P.W. Sprague, J . Am. Chem. S o c , 90, 5251 (1968). .  85.  A.R. Battersby and A.K. Bhatnagar,Chem. Comm., 193 (1970).  86.  T.R. Govindachari and N. Viswanathan, Tetrahedron, 26, 715 (1970).  87.  E.E. van Tamelen and C. Placeway, J . Am. Chem. S o c , j$3_, 2594 (1961); E.E. van Tamelen, C. Placeway, I.G. Wright, and G.P. Schiemenz, i b i d , 91, 7357 (1969).  88.  E.E. van Tamelen and J.B. Hester, J . Am. Chem. S o c , 81_, 3805 (1961).  89.  R.L. Autrey and P.W. Scullard, J . Am. Chem. S o c , 90, 4917 (1968).  90.  V.R. Nelson, Ph.D. Thesis, U.B.C., p. 92 (1969).  91.  R.L. Noble, C.T. Beer, and C.T. Cutts, Ann. N.Y. Acad. Science, 76, 882 (1958).  92.  B.K. Moza and J . Trojanek, C o l l . Czech. Chem. Comm.,28, 1419 0-963).  93.  N. Neuss, B u l l . Soc. Chim. France, 1509 (1963).  94.  N.J. Cone, R. M i l l e r , and N. Neuss, J . Pharm. S c i . , 52, 688 (1963).  95.  J . Mokry and I. Kompis, Lloydia, 27, 428 (1964).  96.  G.A. Cordell, G.F. Smith, and-G.N. Smith, Chem. Comm., 189 (1970).  97.  R.T. Brown, G.F. Smith, K.S.J. Stapleford, and D.A. Taylor, Chem. Comm., 190 (1970).  98.  G.A. Cordell, G.F. Smith, and G.N.Smith, Chem. Comm., 191 (1970).  99.  B.E. Hudson, J r . and C.R. Hauser, J . Am. Chem. S o c , 63, 3156 (1941).  100.  E.G. Lindstorm and W.D. McPhee, J . Am. Chem. S o c , 65, 2387 (1943).  101.  E.E. van Tamelen and J.B. Hester, J r . , J . Am. Chem. S o c , 91^, 7342 0-969). .  PART B STUDIES RELATED TO THE BIOSYNTHESIS OF VINCAMINE  INTRODUCTION Some y e a r s ago d u r i n g t h e c o u r s e o f an e x a m i n a t i o n o f t h e A f r i c a n apocynaceous p l a n t H u n t e r i a eburnea p i c h o n  1  and t h e t i n y  evergreen-  p l a n t Vinca minor L . , f o r substances'of p o s s i b l e / t h e r a p e u t i c v a l u e d 2  s e v e r a l new a l k a l o i d s were i s o l a t e d .  3 R  3  = H, R  4 R  3  = H, R  5 R  3  = OCH , R  'Figure 1.  3  ±  1  = OH, R  2  = C00CH  = COOCH , R 3  1  = OH, R  2  2  As t h e s t r u c t u r e o f t h e s e new  Vincamine  3  = OH  Epivincamine  = COOCH V i n c i n e 3  Various eburnamineT-vincamine type  alkaloids.  alkaloids were' unraveled, i t soon became evident that the unifying feature of a l l these compounds was the inheritance of a general pentacyclic structure (e.g.,'1-7-, see Figure 1). and eburnamine  Since vincamine (3)  (1) were the major alkaloids of the above mentioned  plants, these pentacyclic alkaloids are now referred to as "eburnaminevincamine" type alkaloids.  A recent review of the chemistry of this  3 family i s now  available.  The s t r i k i n g s i m i l a r i t y between the arrangement of the "nontryptophan or C ^ Q " portion of vincamine (3) and the Aspidosperma 4 a l k a l o i d ( l i k e vincadifformine, 8) (see Figure 2) led Wenkert to 3  21  Figure 2.  Scheme showing similar arrangement of non-tryptophan portion i n vincamine and vincadifformine.  suggest that vincamine (3) i s a rearranged Aspidosperma a l k a l o i d . attempting to bring this family into the main stream of the other groups (Corynanthe, Aspidosperma and Iboga) Wenkert advanced an  In  a t t r a c t i v e mechanism which i s shown i n Figure 3. ' This r a t i o n a l would explain the biosynthesis of the entire family i f i t was assumed that  9  . . .  10  V  CH 00C 3  Figure 3.  \  3  Wenkert's proposal for the rearrangement of Aspidosperma skeleton to vincamine.  vincamine (3) was the precursor of the alkaloids eburnamine (!), eburnamonine (2) etc.  At a time when Wenkert put forward  proposal, the intermediate.(12)  this  had already been converted i n v i t r o  into vincamine (12-3, Figure 4)"\  This l a t t e r conversion had another  3 Figure 4.  Synthesis of vincamine.  very close p a r a l l e l i n the synthesis of eburnamine (13-14, Figure 5) . The laboratory syntheses however do,s. not provide any r e a l evidence for the biosyntheses of these alkaloids.  I t i s the purpose of this portion  of-" the thesis to present some preliminary studies which we hope w i l l allow us to obtain some information i n this d i r e c t i o n .  dl-Eburnamine •Figure 5.  Synthesis of eburnamine.  DISCUSSION  In spite of the fact that the genesis of these pentacyclic alkaloids (Figure i )  w a  s Proposed ^ s e v e r a l years ago, i t i s somewhat  surprising that u n t i l now the biogenetic proposal has received no support from feeding experiments with radioactive precursors. previous  Our  experience with Vinca minor L. as already noted i n the f i r s t  section of this thesis stimulated us to initiate some biosynthetic studies i n this d i r e c t i o n . _Before describing the r e s u l t s of our experiments i t i s pertinent to c a l l attention to the fact that i t had alreadysheen established 7 8 In our laboratory '  that ring opening of the pentacyclic Aspidosperma  type alkaloids to nine-membered intermediates the transannular  (e.g. 9-10, Figure 3) and  c y c l i z a t i o n reaction (e.g. 10-11, Figure 3) are not  s i g n i f i c a n t biochemical  reactions i n Vinca rosea and Vinca minor plants.  This study casts some doubt about some of the steps depicted i n Wenkert's proposal.  On the other hand there was no reason to doubt  the.possible v a l i d i t y of the other" steps proposed.  -  While i n i t i a t i n g the project we were faced with the problem as to which substrate we should select (Figure 3) for biosynthetic evaluation.'"  I t was indicated i n the Introduction that (12), a  bio-intermediate  i n Wenkert's proposal, has been converted into vincamine  (3) in-rvitro (Figure 4).  We decided  that the knowledge gained by  evaluation of this type of transformation  (12 •> 3, Figure 4) i n vivo  while simulating the i n , v i t r o r e s u l t s would also be great value i n suggesting  the dynamics of the biosynthesis of vincamine (3).  For  this purpose a synthesis of a close r e l a t i v e ( i . e . 24) of the proposed intermediate  (12) was  revealed i n Figure 6.  contemplated and the synthetic sequence i s f u l l y Our choice for making the compound (24) as  our i n i t i a l synthetic target was  dictated by the fact that the  proposed synthetic sequence was very short and i t was  easy to pursue  the sequence from commercially available s t a r t i n g materials acetylpyridine and tryptophol). C24) was  I t was  capable of transformation  (3-  assumed that the model compound  i n vivo to the putative  intermediate  (12) v i a b i o l o g i c a l l y feasible reactions. Before s t a r t i n g the synthetic sequence outlined i n Figure 6, following p i l o t route This work was  (Figure 7) on model piperidines was  the  investigated.  undertaken to obtain optimum conditions for most of the  reactions to be u t i l i s e d l a t e r i n the sequence i n Figure 6. 3-acetylpyridine (15) was  Accordingly  converted into the known k e t a l (16)  and  this compounds was.treated with methyl iodide i n ether to afford the c r y s t a l l i n e s a l t (25) i n 95% y i e l d . quaternary s a l t  C a t a l y t i c hydrogenation of the  (25) yielded the piperidine ketal (26) which on acid  hydrolysis furnished N-methyl-3-acetylpiperidine assigned was  derived from the following spectral data.  i n the infrared (1710 (x 7.85)  was  (27).  The  A sharp peak  cm ) and a three proton s i n g l e t i n the 1  structure  nmr  reconcilable with the presence of a methyl ketone where  as a three proton s i n g l e t for the N-methyl occurred i n the expected  Figure J .  Synthesis of some -model piperidine systems.  region (x 7.72).  F i n a l l y the molecular formula, CgH^NO, was  confirmed by elemental analysis. Attempts were now made to alkylate the ketone (27) using equivalent amounts of t r i t y l sodium and a l l y l bromide.  The chromato-  graphy of the crude product on alumina allowed the separation of two major components.  The desired compound (28), eluted f i r s t from the  column, indicated the following spectral data. In ;the nmr  spectrum  (Figure 8) the newly incorporated o l e f i n i c protons (-CH^-CH^CH^) were represented by a multiplet i n the region x 4.20-5.18 where as the three proton singlets for the methylketone  and the N-methyl groups  were s t i l l located i n the expected region (x 7.88  and 7.81).  The  molecular formula, C. H _N0, was confirmed by elemental analysis.  \  PPM(T)  Figure 8.  Nmr spectrum of 28.  The nmr spectrum (Figure 9) of the other compound isolated above showed the disappearance of the singlet corresponding ketone group.  to the methyl  With this result i t immediately became obvious that i n  this compound the a l k y l a t i o n had occurred at the methyl group of the methyl ketone.  Since the multiplet i n the o l e f i n i c region integrated  for three protons  (-C^-CH^CH^) , the p o s s i b i l i t y that this was a  dialkylated material was dismissed.  Onthe basis of this limited  information the compound has been tentatively assigned structure (29). Further work i s necessary before a more d e f i n i t e structure could be put forth.  0  Encouraged by the success on the inodel compounds we started the sequence i n Figure 6.  The synthesis of the carbon skeleton present 9  i n C241 was f a c i l i t a t e d by the reported synthesis of the ketone (21). Although this synthesis was reported, the experimental procedure of some of the steps were not completely  clear and the f i r s t attempts to  repeat the synthesis met with c e r t a i n minor d i f f i c u l t i e s .  However  these problems were quickly eliminated and the experimental  procedures  followed i n the present work are i n accord with the sequence indicated i n Figure 6.  The k e t a l i z a t i o n of 3-acetylpyridine (15) proceeded to  give a good y i e l d (78%) of the k e t a l ( 1 6 ) . T r y p t o p h y l  bromide (18)  was obtained from tryptophol (17) i n 80% y i e l d and was used  immediately  (owing to the i n s t a b i l i t y of 18) for reaction with ketal (16) to give the s a l t (19) i n quantitative y i e l d . over platinum oxide.  The s a l t was reduced  catalytically  The crude reduced product (20) on acid hydrolysis  followed by chromatography on alumina and c r y s t a l l i z a t i o n afforded the pure ketone  (21), mp 132.5-134°.  The spectral data of the ketone  compared favourably with the assigned structure. carbonyl region of the infrared spectrum  A sharp peak i n the  (1704 cm "*") and a three proton  singlet i n the nmr (x 7.91) were diagnostic f o r the presence of the methyl ketone i n (21).  The molecular formula, C ^ R ^ ^ O , was confirmed  by mass spectrometry (M 270). +  In an attempt to alkylate the ketone  (21), the anion of the ketone  was made by means of t r i t y l sodium and the anion so formed was immediately quenched with a l l y l bromide.  The crude product on chromato-  graphy on alumina afforded the desired compound (22) i n 30% y i e l d . For a n a l y t i c a l purposes a small amount of material was further p u r i f i e d by sublimation at 150°/0.05 mm.  Supporting evidence f o r the assigned  structure was derived from the following spectral data.. The .prominant feature of the nmr spectrum  (Figure 10) i n comparison  to the nmr of the  ketone (21) was the appearance of a multiplet i n the o l e f i n i c region Cx 4.40^-5.20, 3R,< -CH -CH=CH_ ) while the signals f o r the methyl 2  2  ketone  Cx 7.93) and the indolic-NH (x 1.93) were s t i l l located at the same regions as they were i n the ketone  (21).  This l a t t e r observation was  a v i t a l piece of evidence i n indicating that the reaction has indeed taken the desired course.  However f i n a l confirmation for the structure  (22) came from mass spectrometry.  In the mass.spectrum (Figure 11) the  '.. •  .W"*- - ft* Jv-»K  RELATIVE INTENSITY 25 •  c i-i ft!  cn cn cn  .  50  V. '•  75  •96  1 f: .'  (2.5x)  13?  rc o  ;•  •Si  rti-i g  180  O — I 1  ro 3  s—310  O Q  •  o l e f i n (22) indicated a molecular ion peak at m/e 310. agreement with the molecular formula, ^20^26^2^'  A  s  t  o  This was in. ^  e  expected  the parent ion of (22) fragmented to the ions 30 (m/e 130) and 31 (m/e 180).  Furthermore  ion (31) readily l o s t the acetyl side chain to  33, m/e 96  'F&pUra 12.  32, m/e 137  Postulated fragmentation of 22 i n the mass spectrometer.  give the r a d i c a l ion 32 (m/e 137).  This l a t t e r ion further l o s t the  a l l y l side chain to give the ion 33 (m/e 96, metastable peak at m/e 67). A scheme portraying the mass spectrometric fragmentations has been summarized i n Figure 12.  In spite of repeated attempts, the o l e f i n (22)  did not give the correct elemental analysis. With the completion of the basic carbon skeleton, i t was now considered appropriate to hydroxylate the a l l y l i c double bond i n (22). Of the many reactions a v a i l a b l y for this purpose, osmylation enjoys a reputation f o r s e l e c t i v i t y and was favoured a p r i o r i i n the present  connection.  Therefore the o l e f i n (22) was dissolved i n dry tetrahydro-  furan and treated with osmium t e t r o x i d e . the dark at room temperature for 2 days.  1 1  The mixture was l e f t i n  When the reaction mixture was  worked up, unfortunately we observed a considerable loss of material. The crude product isolated represented a recovery of only 50% of the starting o l e f i n (22). on alumina.  However the crude product was chromatographed  The small quantities of pure material so obtained i n this  p a r t i c u l a r investigation allowed only infrared and nmr spectral determinations.  The spectral data immediately  compound was not the desired d i o l (23).  revealed that the above  For example there was no  evidence f o r the presence of a methyl ketone.  The most interesting  feature of the nmr spectrum was the finding there was no absorption i n the o l e f i n i c region and instead a two proton multiplet and a three proton singlet were located at x 6.5 and T 8.64 respectively.  These  two signals being reconcilable with the presence of a hydroxymethylene group and a O-C-CH^ system suggested a tentative assignment of structure (34.1 to the above' compound. 5  However further work i s  necessary- before a d e f i n i t e structure can be established.  34 This unexpected c y c l i z a t i o n of the d i o l (.23) to the hemiacetal ;; (341 made i t undesirable to continue the sequence as outlined i n  Figure 6.  I t therefore became apparent  that to obviate the above  d i f f i c u l t y i t was necessary to reduce the acetyl group i n the o l e f i n (22) to the corresponding ethyl group ( i . e . -22-35) p r i o r to osmylation of the o l e f i n i c ^ l i n k a g e .  In this manner the eventual completion of the.  0  22  .35  syntheses of intermediates bearing the desired skeleton as portrayed i n 24 could Be envisaged.  Unfortunately time did not permit me to  carry t h i s work any further at this time but i t w i l l be continued by other workers i n our laboratory.  EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected.  The u l t r a v i o l e t (uv) spectra were recorded i n methanol on /''\  a Cary-11 recording spectrometer, andvthe, infrared ( i r ) spectra were taken on a Perkin Elmer Model 21 and Model 137 spectrometers. magnetic resonance  Nuclear  (nmr) spectra were recorded i n deuteriochloroform  at 100 megacycles per second (unless otherwise stated) on a Varian HA'rlOO instrument and the chemical s h i f t s are given i n Tiers T scale with reference to tetramethylsilane as the i n t e r n a l standard; m u l t i p l i c i t y , integrated area and type of protons are indicated i n parentheses.  Mass spectra were recorded on an Atlas CH-4 mass spectro-  meter and high resolution molecular weight determinations were carried out on an AE-MS-9 mass, spectrometer.  Analyses were carried out by  Mr. P. Borda of the Microanalytical Laboratory, The University of B r i t i s h Columbia.  Wbelm neutral alumina and s i l i c a g e l G (acc. to  Stahl) containing 2% by weight of General E l e c t r i c Retma p-1, Type 188-2-7 electronic phosphor were used for a n a l y t i c a l and preparative thin layer chromatography ( t i c ) .  Chromatoplates were developed using  the spray reagent carbon tetrachloride-antimony pentachloride (2:1) or iodine vapors.  Woelm neutral alumina  ( a c t i v i t y III) was used for  column chromatography (unless otherwise stated).  3-Acetylpyridine ethylene ketal  (16)  A solution of 3-acetylpyridine (15, 60 gm), (40 gm)  ethylene glycol  and p-toluenesulfonic acid hydrate (105 gm)  i n benzene (250 ml)  was heated under r e f l u x for 17 hr with a Dean-Stark apparatus to remove water.  The mixture was poured into excess aqueous sodium  bicarbonate solution, the layers separated and the aqueous phase was extracted with benzene.  The combined ^extracts were washed with  sodium bicarbonate solution water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. (63.3 gm);  bp 165/88 mm;  nmr:  (multiplet, 4H, k e t a l ) , 8.35  x 2.5  D i s t i l l a t i o n gave the product  (multiplet, 4H, aromatic),  6.1  (singlet, .3H, C-CH )_. 3  N-Methyl-3-acetylpyridinium iodide ethylene ketal A solution of methyl iodide (20 gm)  (25)  i n dry ether (50 ml)  was  added to a s t i r r e d ice-cold solution of 3-acetylpyridine ethylene k e t a l (16, 20 gm)  i n ether (50 ml).  The reaction mixture was  at room temperature overnight and the precipitated s a l t , 25, filtered  (34.5 gm,  methanol, mp  stirred was  95% yield) and p u r i f i e d by r e c r y s t a l l i z a t i o n from  190°.  Anal. 'Calc. for C^H^NO^: C, 39.12; R, 4.86;  N,  C, 39.11; H, 4.60;  N, 4.60.  Found:  4.55.  N-Methyl-3-acetylpiperidine ethylene k e t a l (26). The s a l t (25, 10 gm,  32.6 mmoles) was dissolved i n a mixture of  water and ethanol (100 ml, 1:1).  This pale yellow solution was  added dropwise to a suspension of hydrogen-activated platinum oxide  (800 mg) i n ethanol (200 ml) and the mixture was hydrogenated at atmospheric pressure.  The absorption of hydrogen was complete  2400 ml, 66 mmole) after 8 hours.  (about  The catalyst was f i l t e r e d off and  the solvent removed i n vacuo to afford a l i g h t yellow s o l i d .  This  product was dissolved i n 10% sodium carbonate solution and was extracted with chloroform.  The organic layer was washed with water,  dried over anhydrous sodium sulfate and evaporated to afford to pale f ilm yellow o i l . ( 5 . 8 gm)• - v nmr 8.75  m a x  : no absorption i n the aromatic region;  (60 mc/s): x 6.1 (singlet, 4H, k e t a l ) , 7.74 (singlet, 4H, N-CH + H(?)), 3  (singlet, 311, C-CH ). 3  NT-Methyl-3-racetylpiperidine (27) The crude k e t a l (26, 5.8 gm) was dissolved i n 2 N hydrochloric acid (50 ml) and the mixture was s t i r r e d at room temperature overnight. The solution was made basic with 10% sodium carbonate solution and extracted with chloroform,  The extract.was washed with water, dried  over anhydrous sodium sulfate and evaporated.  The resulting o i l was  d i s t i l l e d under reduced pressure, bp 116-117°/14 mm, to give a clear oil  (4.48 gm).  The y i e l d of the reaction f o r reduction and removal  of the k e t a l was 90%; v : 1710 (vC=0); nmr (60 mc/s): x 7.72 max Csinglet, 3E,. N-CH ), 7.85 (singlet, 3H, -C0CH_ ). 3  3  Anal. Calc. for C H N0: o  o  Found:  1c  C, 68.01; H, 10.63; N, 9.93; 0. 11.34.  15  C, 67.80; H, 10.61; N, 10.08; 0  ;  11.50-  N-Methyl-3-acetyl-3-allylpiperidine (28) A 5 0 - m l t h r e e necked f l a s k was equipped w i t h a magnetic s t i r r e r , a r e f l u x condensor, a d r o p p i n g  f u n n e l and a n i t r o g e n i n l e t .  ware was f l a m e d r i e d and then t h o r o u g h l y To a s o l u t i o n o f t r i p h e n y l m e t h y l sodium was  flushed with dry nitrogen. (23  added d r o p w i s e a s o l u t i o n o f t h e k e t o n e  i n anhydrous e t h e r  (2 ml).  by t h e i n s t a n t d i s a p p e a r a n c e  ml,  0.17  (27,  ml,  0.0039  N,  mole)  0.0038  gm,  0.540  mole)  0.0038  The f o r m a t i o n o f t h e c a r b a n i o n was i n d i c a t e d o f t h e r e d c o l o r o f t h e base.  m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 1 hour. (0.34  The g l a s s -  The  A l l y l bromide  mole) was t a k e n up i n anhydrous e t h e r and added  d r o p w i s e t o t h e above p a l e y e l l o w s o l u t i o n over a p e r i o d o f 5 m i n u t e s . D u r i n g t h e a d d i t i o n o f a l l y l bromide, sodium bromide s t a r t e d s e p a r a t i n g o u t a n d t h e m i x t u r e became c l o u d y . 1  a d d i t i o n a l 20 minutes. separated;  The m i x t u r e was s t i r r e d f o r an  Water ( 1 0 m l ) was added and t h e l a y e r s were  The aqueous l a y e r was e x t r a c t e d w i t h e t h e r .  l a y e r s were combined and e v a p o r a t e d  i n Vacuo.  The l i g h t  The two e t h e r yellow  semi s o l i d was t a k e n up i n benzene and t r e a t e d w i t h 10% aqueous a c e t i c (20 ml).  acid was  The two l a y e r s were s e p a r a t e d , and t h e benzene l a y e r  washed t w i c e w i t h w a t e r .  The combined aqueous l a y e r s were combined,  m a d e . b a s i c . w i t h 10% aqueous sodium c a r b o n a t e s o l u t i o n and e x t r a c t e d with, chloroform.  The e x t r a c t was washed w i t h w a t e r , d r i e d over  anhydrous sodium s u l f a t e and e v a p o r a t e d . oil  The r e s u l t a n t l i g h t  CO.450 gm) was chromatographed on a l u m i n a ( 4 0 gm).  benzene-petroleum e t h e r 0.105  gm),bp  T-CH=CI1 ), 2  7.81  125°/6  Elution with  CI:9) a f f o r d e d t h e d e s i r e d compound ( 2 8 ,  mm; nmr ( F i g u r e 8 ) : x  (singlet,  yellow  3H,  N-CH^),  7.88  4.20-5.18  (singlet,  (multiplet, 3H,  -C0CH_ ). 3  3H,  Anal. Calc. for C.-H-.-NO:  C, 72.85; H, 10.48; N, 7.72. Found:  ix i y  C, 72.83; H, 10.72; N, 7.55. Further elution of the column with petroleum provided another compound, (29, 60 mg). 7.78  ether-benzene (1:1)  3-(5'-keto-l'-pentenyl)-N-methylpiperidine  Nmr (Figure 9): T 4.10-5.2 (multiplet, 3H, ~CH=CH_) and 2  (singlet, 3H, N-CH^).  The loss of singlet corresponding  to the  methyl ketone was suggestive of a l k y l a t i o n at the methyl group, but the evidence at the time was.insufficient to assign a d e f i n i t e structure to this compound.  Tryptophyl bromide ( 1 8 )  9  A solution of phosphorus tribromide (1 ml) i n ether (20 ml) was added to an ice-cold solution of tryptophol (17, 4.8 gm) i n ether (250 ml).  After 15 hours, the supernatant was decanted, washed with sodium  bicarbonate solution, water and dried with sodium s u l f a t e .  Removal  of the solvent yielded the product as white crystals (4.5 gm, 80%), mp 95-100° ( l i t . mp 90-95°).  N^J g^ (3^Indolyl)_-ethylT, -3 -acetylpyridinium ethylene ketal bromide (19) . 1  Tryptophyl bromide (18, 4.5 gm) and 3-acetylpyridine ethylene ketal (16, 10 ml) were heated at 80° under nitrogen for 8 hours. Addition of ether (40 ml) to the cooled reaction mixture yielded a p r e c i p i t a t e whose c r y s t a l l i z a t i o n from methanol afforded pure s a l t (19, 7.2 gm), mp 208-211° ( L i t . mp 209-210°).  N- [ g - ( 3 - I n d o l y l ) - e t h y l ] - 3 ' - a c e t y l p i p e r i d i n e e t h y l e n e . k e t a l (20) The p y r i d i n i u m s a l t  (19, 7.0  gm)  was  T h i s y e l l o w s o l u t i o n was  added dropwise  a c t i v a t e d platinum oxide  (1 gm)  was. hydrogenated  at atmospheric  complete a f t e r 10 hours.  The  d i s s o l v e d i n e t h a n o l (250 m l ) .  to a suspension of hydrogen-  i n e t h a n o l (100 ml) pressure.  The  c a t a l y s t was  removed i n vacuo to a f f o r d a y e l l o w gum.  and  the  mixture  uptake of hydrogen  f i l t e r e d o f f and T h i s was  was  the s o l v e n t  d i s s o l v e d i n 10%  aqueous sodium c a r b o n a t e ' s o l u t i o n and e x t r a c t e d w i t h c h l o r o f o r m .  The  e x t r a c t was  and  evaporated  washed w i t h water, d r i e d over anhydrous sodium s u l f a t e to a f f o r d the p i p e r i d i n e k e t a l  (20) as a l i g h t y e l l o w  N-Ig-C3-Indolyl)-ethyl]-3'-acetylpiperidine The  crude p i p e r i d i n e e t h y l e n e k e t a l  (21)  (20, 7.5  gm)  was  dissolved i n  methanol (150 m l ) .  The s o l u t i o n s w a s a c i d i f i e d w i t h 4 N HC1  and the m i x t u r e was  heated  temperature,  methanol was  The r e s u l t a n t gum  was  at 85°  f o r 5 hours.  evaporated  The  e x t r a c t was  chromatographed on alumina  petroleum T h i s was 134°;  v  ether  A f t e r c o o l i n g to room  (400 gm).  and  washed w i t h water, d r i e d The r e s u l t i n g gum  (6.1  E l u t i o n w i t h benzene-  (1:1) and benzene a f f o r d e d the d e s i r e d ketone ( 2 1 ) .  crystallised (CEC1„):  C s i n g l e t , 1H, N-H),  from benzene-petroleum e t h e r 3400 (vN-H), 1704  (vC=0) cm" ; 1  (4.33 gm), nmr:  x  mp  132.5-  1.68  2.40-3.12 ( m u l t i p l e t , 5H, i n d o l e p r o t o n s ) , 7.91  ( s i n g l e t , 3H, -COCH^); mass spectrum: 130, 103.  ml)  from the r e a c t i o n m i x t u r e .  over anhydrous sodium s u l f a t e and evaporated. was  (100  made b a s i c w i t h sodium b i c a r b o n a t e s o l u t i o n  extracted with chloroform.  gm)  gum.  M  +  270; main peaks: •  m/e  140,  1  i N-[g-(3-Indolyl)-ethyl)-3'-acetyl-3'-allylpiperidine (22) A 500-ml three necked f l a s k was equipped with a magnetic s t i r r e r , a reflux condensor, a dropping funnel and an nitrogen i n l e t .  A l l the  glassware was flame dried and then thoroughly flushed with dry nitrogen. To a solution of the piperidine ketone (21, 4.00 gm, 0.014 mole) i n dry tetrahydrofuran (150 ml) was added dropwise a solution of triphenylmethyl sodium u n t i l the red color of the base just stayed i n the reaction mixture (150 ml, 0.2 N, 0.030 mole).  The solution was  s t i r r e d at room temperature for about 5 minutes and then a l l y l bromide CI.2 ml, 0.014 mole) i n dry tetrahydrofuran (15 ml) was added to the above solution.  The reaction mixture was s t i r r e d at room temperature  for 2 hours and then evaporated to dryness.  The residue was extracted  with chloroform. The organic layer was washed with water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was dissolved i n a small amount of benzene and put on a column of alumina (250 gm).  Elution with benzene-petroleum ether (1:1)  furnished the desired compound (22,'1.4 gm,.yield 30%).  For a n a l y t i c a l  purposes a small amount of a l l y ! compoxmd was sublimed at 140-150°/0.05 v TJlcLX  (CEC1 1:  3367 (yN-R), 1701  (vC=0) cm" ; 1  nmr  mm;  (Figure 10): x 1.93  j  (broad s i n g l e t , 1R,> indole-NH), 2.40-3.20 Cmultiplet, 5H, indole protons), 4.40-5.20 Cmultiplet, 3R, -CR=CH_) , 7.93 2  mass spectrum (Figure 11):  M  +  310; main peaks:  m/e  (singlet, 3H, -C0CH ); 3  180, 137, 130, 96,  67.  O.smylation of the o l e f i n  C22)  Osmium tetroxide (48 mg, 0.25 mmole) i n dry p u r i f i e d dioxane (3 ml) was added to the o l e f i n (22, 50 mg, 0.16 mmole) i n the same solvent  (2 ml).  The solution was l e f t at room temperature f o r 48 hours and  then saturated with hydrogen s u l f i d e gas. The black p r e c i p i t a t e was f i l t e r e d o f f and the dioxan solution was evaporated reduced pressure.  to dryness under  The residue (13 mg) was dissolved i n a small amount  of benzene and put on a column of alumina E l u t i o n with ether-methanol  (1 gm, a c t i v i t y IV).  (95:5) afforded a very polar compound.  Spectral data indicated that this was not the desired d i o l (23). v •- ' max r  (CKClg).:  no absorption i n the carbonyl region; nmr: x 1.8 (broad  s i n g l e t , .IE,, indole N-E) , 2.40-3.20 (multiplet, 5E, indole protons);, 6.50 (multiplet, ,2H, -CH oQ) and 8.64 (singlet, 3H -0-C-CH_ ). 3  On.the.basis  of this spectral data, the polar compound isolated  above has'been tentatively assigned structure 34.  Further work i s  necessary before a d e f i n i t e structure can be established.  REFERENCES 1.  M.F. B a r t l e t t and W.I. Taylor, J . Am. Chem. S o c , 82_, 5941 (1960).  2.  J . Mokry and I. Kompis, Lloydia, 2_7, 428 (1964).  3.  W.I. Taylor i n "Alkaloids" Vol. XI, p. 125, ed. R.H.F. Manske, Academic Press, New York (1969).  4.  E. Wenkert, and B. Wickberg, J . Am. Chem. S o c , 87_, 1580 (1965).  5.  M.E. Kuehne, J . Am. Chem. S o c , 86_, 2946 (1964); Lloydia, _2_7, 435 (1964).  6.  J.E.D. Barton and J. Harley-Mason, and K.C. Yates, Tetrahedron Letters, 3669 (1965).  7.  J.P. Kutney, W.J. Cretney, J.R. Hadfield, E.S. H a l l , V.R. Nelson, and D.C. Wigfield, J . Am. Chem. S o c , £0, 3566 (1968).  8. \ 9.  J.P. Kutney, C. Ehret, V.R. Nelson and D.C. Wigfield, J . Am. Chem. S o c , 90, 5929 (1968). E. Wenkert, R.A. Massy-Westropp and R.G. Lewis, J . Am. Chem. S o c , 84, 3732 (1962).  10.  S. Sugasawa and M. Kirisawa, Pharm. B u l l Japan, _3, 190 (1955).  11.  D.H.R. Barton and J. Flad, J . Chem. S o c , 2085 (1956).  

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