UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies related to the synthesis and biosynthesis of indole alkaloids Hanssen, Harald Wilhelm 1978

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STUDIES RELATED TO THE  SYNTHESIS  AND B I O S Y N T H E S I S OF INDOLE ALKALOIDS  by  HARALD WILHELM M.Sc,  University  HANSSEN  of B r i t i s h  A T H E S I S SUBMITTED  Columbia,  1972  I N P A R T I A L F U L F I L L M E N T OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF  PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Dept. We  accept this to  of  Chemistry)  t h e s i s as  the required  conforming  standard  THE U N I V E R S I T Y OF B R I T I S H  COLUMBIA  M a r c h , 1978  H a r a l d W i l h e l m H a n s s e n , 1978  In  presenting  an  advanced  the  Library  I  this  degree shall  f u r t h e r agree  for  scholarly  by h i s of  this  written  at make  that  thesis  it  freely  permission  purposes  for  in p a r t i a l  the U n i v e r s i t y  may  representatives.  financial  University  of  British  MTQV  ¥ 7 2  British  by  for  gain  shall  Columbia  the  that  not  requirements  Columbia,  I  agree  r e f e r e n c e and copying  t h e Head o f  understood  CZL-/^2^?^c.,r^J  of  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  is  of  for extensive  be g r a n t e d  It  fulfilment of  available  permission.  Department The  thesis  of  this  or  that  study. thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  - i i -  ABSTRACT In Part and  I , a modified  i t s incorporation  synthesis  into vindoline  In a model s t u d y , f o r the 3-ethylpyridine  (74),  the  correspondingly  the  intermediate  e t h a n e was  of  (7)  r a d i o - l a b e l l e d secodine is  synthesis  described. of s i d e - c h a i n l a b e l l e d  2 [2- H ] - ( 3 ' - p y r i d y l ) - e t h a n e  labelled 3-acetylpyridine  t h i o k e t a l (93).  synthesized  by  (68)  was  by  achieved  from  desulphurization  of  3  In a second s t u d y ,  [1- H ] - ( 3 ' - p y r i d y l ) -  treating 3-acetylpyridine with  sodium borohydride-  3 H.  The  resulting alcohol  the d e s i r e d  (95)  was  a c e t y l a t e d , and  hydrogenolysis  achieved  product.  3  The e s t e r a l c o h o l (74) was c o u p l e d t o [ 1 H]-(3'-pyridyl)-ethane t h e r e s u l t i n g p y r i d i n u m s a l t (90) was r e d u c e d t o t h e c o r r e s p o n d i n g  and  piperdeine  ester  (80)  i n a "one-pot" s y n t h e s i s .  The  conversion  of  (80)  3 to  [ 1 9 - H ] - s e c o d i n e was I n two  3.00  and  experiments,  1.54)  vindoline from the  was  3 [ 1 9 - H,  administered  ( 7 ) w h i c h was  the N-methyl group of u l e i n e  results  f r o m an  olivacine  14  C0 CH ]-secodine 2  i s o l a t e d was  I I , a degradation  a known p r o c e d u r e .  and  3 14 ( 6 8 ) ( H/ C ratios =  shown t o h a v e b e e n  3 14 ( H/ C = 3.31  and  1.35  The  biosynthesized respectively).  scheme d e s i g n e d t o a c h i e v e t h e (1)  i s described  as w e l l as  i n v e s t i g a t i o n i n t o the b i o s y n t h e s i s  isolation  preliminary  of u l e i n e  (1)  and  (4).  V a r i o u s l y r a d i o - l a b e l l e d forms of acid  3  to Catharanthus roseus p l a n t s .  e n t i r e secodine molecule  In Part of  a c h i e v e d by  secodine  s e g m e n t s and  (18)  were a d m i n i s t e r e d  whole p l a n t s .  The  uleine  tryptophan  (15), a n t h r a n i l i c  to Aspidosperma p y r i c o l l u m ( 1 ) w h i c h was  i s o l a t e d was  root  found  to  iii  be  i n a c t i v e i n a l l experiments. Variously  r a d i o - l a b e l l e d forms of  tryptophan  (15), a n t h r a n i l i c  14 acid  and  secodine  to Aspidosperma isolated.  The  (18)  as w e l l  australe only  as  CH^-methionine  (30)  Uleine  olivacine  plants.  incorporation  that  (1)  could  and  be  was  administered  demonstrated  (4)  was  was  that  14 of  CK^-methionine  0.147%.  The  contained In  isolation  97%  and  Part  A new  the of  and  of  for  reduction  aldehyde  the  (37)  the  p o s i t i o n C-16  of  synthesis  was  converted  indolenine  curenol  corresponding  indole  product  aldehyde  could  be  (75)  showed  and  that i t  (60),  (7), was (5)  or  (76).  (39)  (50)  was into  improved  the  C-16  of  yields  a base  position and  i n t r o d u c t i o n of  or  indole  acetate  cyanide (57)  via  unsuccessful. i s believed  achieved.  This  stemmadenine  (73)  derivatives  developed.  using  the  (52)  which  The  the  a modification  to achieve  Also,  was  curan  Also,  group  and  alcohol  obtained.  and  (1)  0.168%  described.  (36)  (44)  thioacetal derivative  thioacetals  is  a carbomethoxy  i n t o akuammicine  (72)  of  compounds o f  achieved.  unsuccessful.  of  from  of  nor-fluorocurarine  the  extent  deoxygenation of  chloroindolenines  preakuammicine  indole  synthesis  curan aldehyde d e r i v a t i v e s  The  be  C-18  the  i n t r o d u c t i o n of  corresponding  with  of  the  activity.  attempted  and  m e t h y l c h l o r o f o r m a t e was into  the  the  to  N-methyl group  conditions  Oppenauer o x i d a t i o n the  the  (1)  stemmadenine-series  method  Birch  The of  98%  into uleine  of  I I I , the  preakuammicine-  using  (30)  yielded  material  (4).  ring-opening the  t o be  Only  identical could the  reaction of  not  dehydrated the  decarboxylated  iv  TABLE OF CONTENTS Page T i t l e Page Abstract  i i i  Table of Contents  iv  L i s t of Figures  v  L i s t of Tables  viil  Acknowledgements  x  Part I Introduction  2  Discussion  31  Experimental  44  Bibliography  59  Part I I Introduction  67  Discussion  75  Experimental  93  Bibliography  ^03  Part I I I Introduction  106  Discussion  112  Experimental  168  Bibliography  202  V  LIST OF FIGURES PART I  Figure 1.  Page The Barger-Hahn Robinson-Woodward  hypothesis  for indole alkaloid biosynthesis  7  2.  The Wenkert p r e p h e r i c  9  3.  The Thomas-Wenkert monoterpene h y p o t h e s i s  4.  Leete's modified  5.  The monoterpene o r i g i n o f the Cg-C^^ u n i t  12  6.  Summary scheme o f t h e e a r l y s t a g e s o f a l k a l o i d b i o s y n t h e s i s as proven by experiment  17  The p o s s i b l e b i o g e n i s i s o f the Corynanthe a l k a l o i d s as s u p p o r t e d by experiment  20  Wenkert's h y p o t h e s i s f o r the o r i g i n o f the Strychnos f a m i l y o f a l k a l o i d s  21  The S c o t t h y p o t h e s i s f o r the b i o s y n t h e s i s o f the S t r y c h n o s a l k a l o i d s  23  The Wenkert h y p o t h e s i s f o r the b i o s y n t h e s i s o f the Aspidosperma and Iboga a l k a l o i d s  24  H y p o t h e t i c a l b i o s y n t h e s i s o f the Aspidosperma and Iboga a l k a l o i d s from the S t r y c h n o s f a m i l y v i a d e h y d r o s e c o d i n e (64)  29  12.  The s y n t h e s i s o f the c h l o r o - i n d o l e . (73)  32  13.  The Kutney s y n t h e s i s o f s e c o d i n e  33  14.  The s y n t h e s i s o f e s t e r a l c o h o l (84)  15.  An a l t e r n a t e s y n t h e s i s o f p i p e r d e i n e  7.  8.  9.  10.  11.  a c i d hypothesis .'  acetate hypothesis  10 11  (68)  36 ester  (80) ....  37  vi  PART I I Figure  1.  2.  3.  4.  5.  Page The Wenkert h y p o t h e s i s f o r t h e b i o s y n t h e s i s o f e l l i p t i c i n e ( 6 ) , u l e i n e (1) and o l i v a c i n e (4)  69  The D j e r a s s i - G i l b e r t r a t i o n a l f o r t h e b i o s y n t h e s i s of a p p a r i c i n e (2)  70  The P o t i e r - J a n o t p o s t u l a t e f o r t h e b i o s y n t h e s i s o f u l e i n e ( 1 ) , a p p a r i c i n e (2) and the pyridocarbazole a l k a l o i d s  73  E x p e c t e d p o s i t i o n s o f r a d i o - l a b e l i n u l e i n e (1) and o l i v a c i n e (4) from a v a i l a b l e p r e c u r s o r s as s u g g e s t e d by the P o t i e r - J a n o t h y p o t h e s i s ( F i g u r e 3) -.  77  D e g r a d a t i o n o f u l e i n e (1) by means o f Hofmann elimination reactions  83  PART I I I 1.  The p r o p o s e d b i o s y n t h e s i s o f t h e S t r y c h n o s a l k a l o i d s from t h e Corynanthe f a m i l y  108  The i m p l i c a t i o n o f t h e d e h y d r o s e c o d i n e J stemmadenine b i o - e q u i l i b r i u m i n i n d o l e a l k a l o i d b i o s y n t h e s i s as s u p p o r t e d by e x p e r i m e n t  109  3.  Some b i o m i m e t i c c o n v e r s i o n s o f stemmadenine (4)  110  4.  Synthesis  116  5.  The d e g r a d a t i o n o f s t r y c h n i n e G u m l i c h a l d e h y d e (22)  2.  6.  7.  o f 16-epi-stemmadenine (28) (29) t o W i e l a n d -  117  Two methods f o r d e o x y g e n a t i o n o f C-18 o f W i e l a n d - G u m l i c h a l d e h y d e (22)  119  A p o s s i b l e mechanism f o r t h e two-step r e d u c t i o n o f W i e l a n d - G u m l i c h a l d e h y d e (22) t o 23,16a-cur-19-ene1 7 - o l (36)  120  vii Page 8.  9.  10.  The o x i d a t i o n o f a l c o h o l f l u o r o c u r a r i n e (39)  (36) t o n o r 122  The s y n t h e s i s o f N - f o r m y l - 1 8 - d e o x y - W i e l a n d G u m l i c h aldehyde (44) A r a t i o n a l e f o r the fragmentation of (44) i n t h e mass s p e c t r o m e t e r  . 127  aldehyde 129  11.  A t t e m p t e d f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) and the proposed p l a n f o r i t s c o n v e r s i o n to preakuammicine and stemmadenine d e r i v a t i v e s 131  12.  The s y n t h e s i s o f N - c a r b o m e t h o x y - 1 8 - d e o x y W i e l a n d G u m l i c h a l d e h y d e (50)  137  The p l a n t o s y n t h e s i z e s t e m m a d e n i n e ( 4 ) v i a t h e c h l o r o i n d o l e n i n e o f des-carbomethoxy stemmadenine (52)  143  The p l a n t o s y n t h e s i z e s t e m m a d e n i n e the c h l o r o i n d o l e n i n e a c e t a t e (58)  146  13.  14.  15.  16.  The n u c l e o p h i l i c systems  reactions of  (4) v i a  enaminocarbonyl 149  The p o s s i b l e r e a c t i o n s o f n o r - f l u o r o c u r a r i n e (38) w i t h m e t h y l c h l o r o f o r m a t e  150  17.  The u v s p e c t r u m o f t h e p r o d u c t f r o m t h e r e a c t i o n o f n o r - f l u o r o c u r a r i n e ( 3 9 ) w i t h m e t h y l c h l o r o f o r m a t e . . . 152  18.  The c a r b o n y l r e g i o n o f t h e i n f r a r e d  spectrum of  product  (60)  19.  The pmr  s p e c t r u m o f compound  20.  Some e x p e c t e d r e a c t i o n s o f p r e a k u a m m i c i n e derivatives  157  21.  A p o s s i b l e mechanism compound ( 7 2 )  159  22.  153 (60)  155  f o r the formation of  Reactions of thioacetal  (73)  165  viii  L I S T OF TABLES PART I  Table  Page  1.  3 14 The i n c o r p o r a t i o n o f [ 1 9 - H, C0 CH.lsecodine i n t o v i n d o l i n e (7)  43  2.  A d d i t i o n a l data  58  2  associated with  Table 1  PART I I 1  Incorporation  studies  into  apparicine  (2) i n  A. p y r i c o l l u m p l a n t s  78  2.  Incorporation  studies  i n A. p y r i c o l l u m p l a n t s  86  3.  Incorporation  s t u d i e s i n A. a u s t r a l e p l a n t s  4.  A d d i t i o n a l data  associated with  Table 2  100  5.  A d d i t i o n a l data  associated with  Table 3  102  90  PART I I I 1.  Attempted  2.  F u r t h e r s t u d i e s on t h e f u n c t i o n a l i z a t i o n o f aldehyde (44)  135  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f N - c a r b o m e t h o x y aldehyde (50)  139  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a the c h l o r o i n d o l e n i n e o f i n d o l e (52)  144  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a t h e c h l o r o i n d o l e n i n e o f i n d o l e a c e t a t e (57)  147  6.  Reactions  161  7.  F u r t h e r s t u d i e s on t h e m e t a l h y d r i d e o f compound ( 6 0 )  3.  4.  5.  8.  C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e  (44)....  o f compound ( 6 0 ) w i t h NaBH^  134  reduction  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l data associated with Table 1  161  186  ix  Table 9.  10.  11.  12.  Page A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e a d d i t i o n a l data a s s o c i a t e d w i t h Table 2  (44):  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e a d d i t i o n a l data a s s o c i a t e d w i t h Table 3  (50):  187  190  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l data a s s o c i a t e d w i t h Table 4  194  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l data a s s o c i a t e d w i t h Table 5  195  X  ACKNOWLEDGMENTS  The him of  author i s g r a t e f u l to Prof.  the opportunity natural  t o do r e s e a r c h  J . P. K u t n e y f o r  i n several  p r o d u c t s c h e m i s t r y and a l s o  giving  i n t e r e s t i n g areas  f o r h i s encouragement and  supervision. Many h e l p f u l d i s c u s s i o n s gratefully and  acknowledged.  Special  J . Beck f o r i n t r o d u c i n g  I also  with  many h e l p f u l c o l l e a g u e s  thanks a r e due t o D r s .  are  R. S o o d  the author to techniques i n biosynthesis.  t h a n k A n n a Wong f o r t y p i n g  the manuscript.  xi  To Brenda, who  endured.  -1-  PART  I  S t u d i e s R e l a t e d to the B i o s y n t h e s i s of V i n d o l i n e  -2-  INTRODUCTION  Man's w o n d e r a n d c u r i o s i t y illustrated  i n his earliest  necessity, h i s early  about h i s n a t u r a l world i s  records.  attempts  Out o f c u r i o s i t y ,  at understanding  m a t e r i a l s p r o v i d e d by n a t u r e was, i n f a c t , compounds. chemistry  From these b e g i n n i n g s  and u s i n g t h e  the study  t h e modern f i e l d  of organic  of organic  evolved.  Up u n t i l was e x c l u s i v e l y  the 19th century, the study  the study  materials  t h a t were o b t a i n e d  classical  systematic study  identification  chemistry  I n most c a s e s ,  of purified  organic  perfumes o r c o l o u r i n g  from p l a n t s or micro-organisms.  The  n a t u r a l compounds b e g a n w i t h  of f u n c t i o n a l groups,  s k e l e t o n and t h e n a t t e m p t e d proposed molecular  of organic  of n a t u r a l products.  e x t r a c t s had been used as f o l k m e d i c i n e s ,  the  and o f t e n  d e l i n e a t i o n of the  s y n t h e s i s o f t h e compound  carbon  to c o n f i r m the  structure.  A bewildering array of molecular  s t r u c t u r e s were d i s c o v e r e d .  T e r p e n e s , s t e r o i d s , amino a c i d s , p e p t i d e s , n u c l e o t i d e s , s u g a r s a l k a l o i d s w e r e some c l a s s e s o f c o m p o u n d s w h i c h e m e r g e d a n d s o o n their  own s p e c i a l t i e s .  unsatisfying.  struct  demanded  JThese c l a s s d i v i s i o n s were o f t e n a r b i t r a r y  and  C h e m i s t s and b o t a n i s t s began t o wonder about t h e i n t e r -  r e l a t i o n s h i p s between these molecules.  and  c l a s s e s and t h e o r i g i n s  o f t h e move c o m p l e x  Were t h e r e a n y " b u i l d i n g b l o c k s " w h i c h n a t u r e  the molecules  of each c l a s s ,  common t o s e v e r a l c l a s s e s o f n a t u r a l  used  o r were t h e r e " b u i l d i n g products?  to  con-  blocks"  -3-  P e r h a p s some o f t h e m o s t e l e g a n t e a r l y s p e c u l a t i o n s biogenesis  o f n a t u r a l p r o d u c t s were those of S i r Robert  who  r e g a r d e d as t h e f a t h e r  i s now  study i n organic  chemistry.  of structures  e x a m p l e , we now  a re-classification  according  know t h a t  monoterpene through t o the t h i r t y the  isoprene  often  unit.  shown t h e way  (C^Q)  One  into  family  as  carbon  pathways have synthesis  than approaches t h a t have n o t  taken  consideration.  o f n a t u r a l p r o d u c t s w h i c h h a v e p r e s e n t e d many o f  t h e more c h a l l e n g i n g  and s u b t l e  e l u c i d a t i o n , and i n s y n t h e s i s ,  chemical problems i n s t r u c t u r a l has been the i n d o l e a l k a l o i d s .  compounds w h o s e m a i n s t r u c t u r a l f e a t u r e s  indoline  For  up f r o m t h e f i v e  and more d i r e c t l a b o r a t o r y  w h i c h have p r o v e n more e f f i c i e n t natural routes  origin.  into  t r i t e r p e n e s as w e l l  The d e l i n e a t i o n o f b i o s y n t h e t i c to simpler  of natural  f r o m t h e t e n c a r b o n (C-^Q)  s t e r o i d s , have a l l been u n i v e r s a l l y b u i l t  (C,.)  are  carbon  1  o f some c l a s s e s  to t h e i r biogenetic  terpenes ranging  Robinson ,  large area of  The s t u d y o f t h e b i o g e n e s i s  compounds h a s made p o s s i b l e families  of this r e l a t i v e l y  on t h e  (2) n u c l e u s p l u s  one b a s i c n i t r o g e n  are the i n d o l e  (1) o r  atom e l s e w h e r e i n t h e  molecule.  (1)  These  (2)  -4-  Indole and  a l k a l o i d s have been found  sub-tropical plant  Rubiaceae. productive  The  f a m i l i e s s u c h as Apocynaceae,  Apocynaceae p l a n t  i n providing  used  been  particularily  i s now  Some  i s a cardiac  and  (b) s t r y c h n i n e  c e n t r a l nervous  (c) v i n b l a s t i n e (5), a b i s - i n d o l e a l k a l o i d  Catharanthus which  and  (3) f r o m t h e g e n u s R a u w o l f i a w h i c h i s  strychnos species  and  tropical  Loganaceae  importance.  i n the t r e a t m e n t of m e n t a l d i s o r d e r s ;  from v a r i o u s stimulant;  f a m i l y has  a l k a l o i d s of m e d i c i n a l  examples a r e : (a) r e s e r p i n e widely  i n a l a r g e number o f  extremely important i n cancer  (4) system  from the  genus  chemotherapy.  (7)  -5-  These a l k a l o i d s are c l a s s i f i c a t i o n of  representative  of  i n d o l e a l k a l o i d s i n t o the  the  four major  w h i c h have been most a r d e n t l y  studied.  illustrated  i s r e f e r r e d t o as  by  resperpine  Corynanthe f a m i l y . alkaloids. skeleton  The  (3)  Strychnine  (4)  b i s - i n d o l e (5) has  a d e r i v a t i v e of  The  as  The  first  some o r d e r  of  biogenetic  i n the  great  groups  skeleton  being  of  of  the  the  Strychnos  i t s " u p p e r " h a l f an  catharanthine  i s representative  carbon  is illustrative  (6) w h i l e  a n a t u r a l l y o c c u r r i n g monomeric a l k a l o i d latter  historical  the  Iboga  "lower" part  called vindoline  (7).  is The  the Aspidosperma f a m i l y of a l k a l o i d s . speculations  v a r i e t y of  which attempted  structures  to e s t a b l i s h  found i n the  indole  2 alkaloid  a r e a w e r e o f f e r e d by  structures to the that  established at  3-position  the  of  amino a c i d  become t r y p t a m i n e C^g  and  C^Q  tryptaphan,  c a r b o n 14  that  t i m e had  tryptophan ( 9 ) , and  i n the  given  the  h a l f century support.  ajmaline,  reserpine  and  serpentine  i n t o three 4 ' .  specific  i n c o r p o r a t i o n of  initiated A  a d e l u g e of work i n the  area of  l a r g e number o f r e v i e w s o f  These v e r y  into  the  this  showed radioactive  Rauwolfia  doubt  about  important r e s u l t s  indole alkaloid  indole alkaloid  to  That l a b e l l e d carbon  i s o l a t e d f r o m t h e s e a l k a l o i d s , t h u s l e a v i n g no precursor.  speculate  Leete  a t o m was  the  attached  passed before  labelled with  incorporated  indolic  decarboxylated  I n 1960,  3 alkaloids:  to  then elaborated  been s p e c i f i c a l l y  s i d e c h a i n , was  logical  1) was  l a t t e r was One  a l l the  a 3-aminoethyl group  (8, Figure  experimental  w h i c h had  Since  i n d o l e p o r t i o n , i t was  indole alkaloids.  s p e c u l a t i o n was that  the  P i c t e t , i n 1906.  biosynthesis.  biosynthesis  have  -6-  appeared  over  the  informative^ standing in  this  job  of  area.  the  t h e more r e c e n t  Two  these  of  instructive  h i g h l i g h t s of  work to be  described  With  to  the  Another review at be  the  this  later  this i n the  present  exciting  story  establishment  1)  the  of  tryptophan  complex a l k a l o i d s remained  Figure  origin  Scholz''""'" t h o u g h t condensed w i t h  elaborated  that  a  of  justified.  work to i f only  (13)  the a  (8)  as  a  summarize to put  the  (9)  fundamental  other  nine  great  puzzle.  tyrosine derived  tryptamine  i n t o yohimbine  out-  i n t o some p e r s p e c t i v e .  atoms o f  and  have done an  time c a n n o t be  precursor,  Barger  particularly  t o t a l h i s t o r y o f work done  biosynthetic the  being  r e v i e w s ^ '"^  putting order  However, i t w i l l briefly  years,  and  the  a representative  or  ten In  1933,  aldehyde  product of  carbon  the  was  (10, then  then w e l l 12  known C o r y n a n t h e - t y p e . and  later  biogenesis dihydroxy indolenine of  the  by  Woodward"^'and  of  the  Strychnos  derivative (15).  (16)  to  Robinson"'  family of  (14)  Inherent  o x i d a t i o n and  intermediate  M o d i f i c a t i o n s of  plus  a  in this  subsequent achieve  allowed  -  In  1959,  deficiencies alkaloids,  f o r some  W e n k e r t and  scheme was  daring  time. 15  i n this hypothesis.  characterized at  that  Hahn  hypothetical  unit  of  Strychnos  Bringi  the  single acetate  "Barger-Hahn-Robinson-Woodward" h y p o t h e s i s , remained unchallenged  p o s t u l a t e by  i n d o l e a l k a l o i d s from  cleavage  the  this  the  to give  an  speculation  the p h e n o l i c  system  carbon skeleton illustrated  3,4-  via  (4).  The  i n Figure  16 '  pointed  Firstly,  out  a l l the  time, possessed  the  three  major  yohimbine-like same  absolute  1,  (13)  F i g u r e 1.  The B a r g e r - H a h n - R o b i n s o n - W o o d w a r d indole alkaloid biosynthesis.  hypothesis f  -8-  stereo-chemistry was i n d e e d  a t C - 1 5 , a f a c t w h i c h seemed u n l i k e l y  aromatic  at that l e v e l  Secondly,  the  i n t r o d u c t i o n o f a c a r b o m e t h o x y g r o u p a t C-16 was m e c h a n i s t i c a l l y  c o u l d be p r e p h e n i c  carbohydrate  acid  metabolism.  prephenate moiety  thirdly,  (17, Figure  2 ) , a known p r o d u c t  of  The s t e r e o s p e c i f i c r e a r r a n g e m e n t o f t h e  to the seco-prephenate-formaldehyde u n i t  the stereochemistry  (SPF,20)  a t C-15 i n t h e C o r y n a n t h e a l k a l o i d s a n d  t h e C-16 c a r b o m e t h o x y g r o u p t o b e a n i n a t e p a r t o f t h e p r o -  genitor.  This  Not  scheme i s s u m m a r i z e d i n F i g u r e  2.  o n l y d i d t h e SPF u n i t a c c o u n t w e l l f o r t h e n o n - t r y p t a m i n e  part of the yohimbine-like of  And  They p r o p o s e d i n s t e a d t h a t t h e p r o g e n i t o r o f t h e C^-C^Q  unlikely.  allows  r i n g E.  only  compound o f t h i s  fixes  p o s s e s s e d an a r o m a t i c  E  one  unit  type  of biosynthesis.  i fring  the Strychnos  a l k a l o i d s , but could  alkaloids,  a l s o be s e e n as p a r t  and by a more complex mechanism i t c o u l d  a l s o a c c o u n t f o r t h e C^-C^Q p a r t o f t h e I b o g a and A s p i d o s p e r m a Moreover, t h i s  s t r u c t u r e was s e e n i n a n u m b e r o f m o n o t e r p e n e s , some  of w h i c h had been i s o l a t e d led  from Strychnos  Thomas'*"^ t o p r o p o s e t h a t  could  into  The l a t t e r  i n the condensation  (21) t o g i v e a c y c l i c  then be e l a b o r a t e d  species.  t h e C ^ - C ^ Q u n i t was i n f a c t d e r i v e d  monoterpenes and had i t s o r i g i n acid moieties  alkaloids.  from  o f two m e v a l o n i c  s t r u c t u r e (22).  the cyclopentanoid  feature  This  structure  monoterpenes and t h e  Cg-C^ progenitor. About t h i s  same t i m e two l a b o r a t o r i e s b e g a n t o c o n s i d e r 18  acetate  d e r i v e d C -C, u n i t .  three acetate  Q  n  Schlittler  an  19-21 and L e e t e  u n i t s condensed w i t h mavalonic a c i d  suggested  that  (18) p l u s a one-carbon  -9-  Figure  2.  The  Wenkert prephenic  acid  hypothesis.^'  -10-  unit the  t o g i v e a C^Q s t r u c t u r e w i t h SPF u n i t  (20).  This  proposal  t h e same c a r b o n s k e l e t o n a s t h a t o f s o o n l e d t o some i m p o r t a n t  investi-  gations . I n the e a r l y 1960's,  the r e s u l t s  f r o m many r a d i o a c t i v e t r a c e r  e x p e r i m e n t s began t o c a s t d o u b t s on a l l t h e h y p o t h e s i s o r i g i n o f t h e u b i q u i t o u s C^-C^Q u n i t .  Leete  20  concerning  21 ' , working  the  with  14 R a u w o l f i a S e r p e n t i n a from the Apocynaceae f a m i l y , f e d [ 2 - C ] - t y r o s i n e , 14 14 14 [2C ] - a l a n i n e , [ 2 - C ] - m e v a l o n i c a c i d and sodium [ 1 - C ] - a c e t a t e , and isolated  ajmaline  (26) and r e s e r p i n e  family.  A l l o f these  feedings  except f o r t h e sodium a c e t a t e  (3), both representing  resulted i n a negative experiment.  One q u a r t e r  the Corynanthe  incorporation of the a c t i v i t y  (22) Figure  3.  The T h o m a s - W e n k e r t m o n o t e r p e n e  hypothesis.  -11-  i s o l a t e d was  f o u n d a t p o s i t i o n s C-3  Thomas-Wenkert h y p o t h e s i s C-16.  required  Leete then modified  s a t i o n of  three  unit  and  (24)  W e n k e r t ' s SPF (Figure  the  and  C-19  activity  acetate  C^-unit  unit  (20)  to give  but  a t C-14,  hypothesis  acetyl-coenzyme-A u n i t s  one  i n ajmaline  4.  conden-  (25), which i s s i m i l a r to  better explains  the  experimental  results  (25)  Leete's modified  Then i n 1965, successful  Scott  and  acetate  hypothesis.  his collaborators  i n c o r p o r a t i o n of mevalonate  i n terpene b i o s y n t h e s i s ,  vindoline  (7) b i o s y n t h e s i z e d  (21),  i n t o the  i n C.  a true progenitor quick  '  reported  of  roseus. 24—28  geraniol  a precursor  the  the w e l l - k n o w n " b u i l d i n g  Soon the  alkaloid, r e s u l t s of  e s t a b l i s h e d mevalonate  i n indole alkaloid biosynthesis.  establishment  t h e m o n o t e r p e n e s , as  23  Aspidosperma  experiments from s e v e r a l l a b o r a t o r i e s  the  the  malonyl-conenzyme-A  22  was  and  4).  Figure  as  C-21  C-19,  (26)  block"  The  to i n c l u d e  ( 2 3 ) , one  structure  (26).  (27),  i n several  the  Equally  tracer  (2l)  exciting  immediate precursor  f a m i l i e s of  to  these a l k a l o i d s .  26  '  29  '  F i g u r e 5 s u m m a r i z e s t h i s new h y p o t h e s i s a s s u p p o r t e d evidence.  The m o n o t e r p e n e  F i g u r e 5.  h y p o t h e s i s was  now  by  on f i r m  The m o n o t e r p e n e o r i g i n o f t h e C - C Q  _  experimental ground.  unit.  -13-  Th e n e x t s t e p  i n the b i o s y n t h e s i s of  .the i d e n t i f i c a t i o n o f (28).  As  the h y p o t h e t i c a l c y c l o p e n t a n o i d  mentioned e a r l i e r ,  t h e r e w e r e a number o f  ( o r i r i d o i d ) mono t e r p e n e s known w h i c h had and,  i n some c a s e s ,  the C^-C^Q u n i t had  co-existed with  the  to  be  intermediate cyclopentanoid  correct  stereochemistry  indole alkaloids i n several 32-34  plant species. venbanalin  Battersby  (31), genipin  and  h i s co-workers  ( 3 2 ) and  loganin  fed r a d i o - l a b e l l e d  ( 3 3 ) t o C.  r o s e u s and  found  14  that only  [CG*2  CH^]-loganin  f a m i l i e s of a l k a l o i d s . group  35  who  alkaloids achieved  were a b l e i n the  loganin  3  to i n c o r p o r a t e  [C H ^ ] - l o g a n i n  same p l a n t s p e c i e s . no  The  doubt as  (33) i n several a l k a l o i d from mevalonate  i n t o three  q u i c k l y confirmed  Subsequent work i n t h i s area  biosynthesis The  incorporated  f i n d i n g was  i n these s t u d i e s l e f t  progenitor. of  This  ( 3 3 ) was  high  i n t o the  l e v e l s of  to the  by  demonstrated the  ipecoside  Swiss  same  importance of  g e r a n i o l ( 2 7 ) was  b i o s y n t h e t i c i n v e s t i g a t i o n of  a  three  incorporation this  occurrence  containing plant species  ( 2 1 ) and  major  and  its  confirmed.  ( 3 4 ) , the  first  3 6  "  3 8  tetra-  -14-  h y d r o i s o q u i n o l i n e m o n o t e r p e n e t o be the next  step  i n indole alkaloid  39  discovered  biosynthesis.  , provided Battersby  a clue  to  speculated  (34)  (35)  that i f hydroxyloganin  e l i m i n a t e d t h e e l e m e n t s o f H^O  the c y c l o p e n t a n e  then  ring,  one  with cleavage  would have a m o i e t y which  of  could 40  condense d i r e c t l y w i t h a structure, crucial The  called  intermediate  isolation  and  tryptamine  secologanin (29)  (9)  to give  ipecoside  (35), would c l o s e l y  (34) .  correspond  i n the Thomas-Wenkert h y p o t h e s i s  structural  e l u c i d a t i o n of  t h r e e new  Such to  the  (Figure 5).  glucosides  lent  ^ 1 ^-2 great c r e d i b i l i t y  to t h i s  idea.  '  Menthiafolin(36a) , foliamenthin  (36a)  (36b)  0  -15-  (36b)and i t s d i h y d r o - d e r i v a t i v e contained-the secologanin  (35) i n a masked l a c t o l  Battersby loganin labelled into  skeleton of  form.  and h i s c o - w o r k e r s w e r e s u c c e s s f u l i n c l e a v i n g  (33) t o s e c o l o g a n i n precursor  (35) and f e e d i n g  t o C. r o s e u s  plants.  the l a t t e r  A high  serpentine, ajmalicine (30), catharanthine  found.  level  the presence of secologanin  Thus a l l t h e r e q u i r e m e n t s  for this  doubly-  of i n c o r p o r a t i o n  ( 6 ) a n d p e r i v i n e was  In addition, a large scale extract of this  revealed  as a  plant  (35) as a n a t u r a l  species  product.  compound t o b e a t r u e  precursor  43 44 to  the three  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 were met.  The n e x t  step  look f o r the product (35)  by  i n d e l i n e a t i n g t h i s b i o s y n t h e t i c p a t h w a y was resulting  and the n i t r o g e n o u s m o i e t y 45  the Battersby  with secologanin  group  from the condensation tryptamine  of  ( 9 ) . An i n v i t r o  (35) to g i v e as a major product  (38) as the m i n o r p r o d u c t .  experiments,  r e p o r t e d by these w o r k e r s , a n d f e d them t o C. r o s e u s  vincoside  I n an e l e g a n t both  to  secologanin  s h o w e d t h a t , a t pH 4.5, t r y p t a m i n e  isovincoside  radio-labelled  '  experiment  (9) condensed (37) and  s e r i e s of  o f t h e s e compounds w e r e 4 6 47 plants. ' They f o u n d t h a t  -16-  only  the  36(R)  isomer,  three indole a l k a l o i d that both  isomers  vincoside families.  (37),  c o u l d be  They were a l s o a b l e  e x i s t e d as n a t u r a l p r o d u c t s  p r o b l e m , h o w e v e r , was  that vincoside  r a t i o n was  of opposite  alkaloids,  as  (37)  stereochemistry  represented  by  incorporated into to  demonstrate  i n C. r o s e u s .  having  the  possessed  a j m a l i c i n e (30).  by  3B(R) the  I t was  the  The configu-  Corynanthe  suggested  that  48 C-3  underwent i n v e r s i o n w i t h r e t e n t i o n of  the hydrogen.  This  point  4 9-51 has  b e e n t h e s u b j e c t o f much c o n t r o v e r s y .  g a t i o n showed, i n f a c t ,  t h a t C-3  A laboratory  investi-  o f v i n c o s i d e d e r i v a t i v e s was  easily  52-54 epimerized  by  an  o x i d a t i v e - r e d u c t i v e mechanism.  great credence to B a t t e r s b y ' s carbon-13 m a g n e t i c r e s o n a n c e d e r i v a t i v e s and the e a r l i e r An  assigned  3a(S)  in  the  to t h i s  on v i n c o s i d e  c o u l d be  formation  of  1977  by  cell  a West German  vincoside  Rhazia  stricta,  convincing evidence  f o r i s o v i n c o s i d e (38)  T h i s compound i s now S m i t h who  first  V i n c a m i n o r and  m o r e commonly c a l l e d  isolated  i t f r o m R.  C.  being  the  three  of only (37)  the  was  roseus.  the  indole that  their  3a(S)  never  observed  Rhazia  This  i s very  the o b l i g a t e p r e c u r s o r .  strictosidine,  stricta  that  cultures, only  They a l s o r e p o r t e d  formation  t h e 33(R)  confirmed  They r e p o r t e d  incorporated into  c a t a l y z e d the  (37)  compounds.  controversy. roseus  lent  secologanin  f i v e Apocynaceae s p e c i e s Amsonia tabernaemontianum,  orientalis,  by  6  these  found i n that s p e c i e s .  cell-free preparations the  of  enzyme e x t r a c t f r o m C.  families  i s o m e r and  study"'  finding  More r e c e n t l y , a  of v a r i o u s  r e s u l t made p u b l i c i n l a t e  i s o v i n c o s i d e (38)  alkaloid  proposal.  study^  stereochemistry  added more f u e l  in a cell-free  (CMR)  an X - r a y d i f f r a c t i o n  intriguing  g r o u p " ^ has  original  This  a name  p l a n t s i n 1968.  suggested "^'"^  -17-  The divided  story  between the e a r l y  strictosidine acid  of indole  Figure  stages which  form  can a t t h i s p o i n t  the condensation  ( 3 8 ) , a r i s i n g from monoterpene b i o g e n i s i s  biogenisis,  functionalized  alkaloid biosynthesis  and  and  the l a t e r stages  rearranged  6 summarizes the e a r l y  into  i n which  product, amino  t h i s compound i s  the v a r i o u s a l k a l o i d  s t a g e s as p r o v e n  and  be  by  families.  experiment.  (38) F i g u r e 6.  Summary scheme o f t h e e a r l y a s p r o v e n by e x p e r i m e n t .  stages of a l k a l o i d  biosynthesis  -18-  A number o f i n v e s t i g a t o r s s o o n b e g a n u n r a v e l l i n g t h e •by w h i c h s t r i c t o s i d i n e families. achieved  ( 3 8 ) was  I t s conversion by  into  the v a r i o u s  to the Corynanthe s k e l e t o n c o u l d  a l o s s o f a one  Subsequent e l a b o r a t i o n of members o f  converted  c a r b o n u n i t and  this  Figure  alkaloid simply  be  r i n g c l o s u r e to form r i n g  structure could  the Corynanthe f a m i l y .  pathways  r e s u l t i n the  D.  other  7 s u m m a r i z e s some o f  these  proposals.. The  n a t u r a l product  c a t h e n a m i n e ( 3 9 ) ^ a p p e a r e d t o be  immediate rearrangement product to accumulate i n c e l l - f r e e deprived  of s t r i c t o s i d i n e  enzyme p r e p a r a t i o n s  cleavage  of  g r o u p and  the b a s i c n i t r o g e n .  roseus  found  t h a t were  ajmalicine  (30)  and  a trivial  (40)  corynantheine s t r u c t u r e (38)  formaldehyde  between the n o w - r e l e a s e d  Subsequent d e h y d r a t i o n  carbonolamine would give cathenamine  and  i t was  the e t h y l i d e n e group w i t h l o s s o f  from the l a t t e r would a l l o w condensation  natively  o f C.  as  most  o f NADPH ( t h e w i d e s p r e a d n a t u r a l r e d u c t i o . n - o x i d a t i o n s y s t e m ) ."^  H y d r o l y s i s and  or  (38)  the  (39).  Reduction  of  the  aldehyde  intermediate  of r i n g C  gives  e l a b o r a t i o n of r i n g E would g i v e g e i s s o c h i z i n e  aldehyde c o u l d be  (42a)  and  elaborated  corynantheine to the  immonium  r e d u c t i o n o f t h e l a t t e r w o u l d g i v e g e i s s o c h i z i n e (40)  of r i n g E would r e s u l t i n a j m a l i c i n e  (42b).  and  Alter-  species cyclization  (30).  62 I n 1969, present  i n C.  the B a t t e r s b y  r o s e u s and  was  group  showed t h a t g e i s s o c h i z i n e ( 4 0 )  a good p r e c u r s o r  not  only of  the  was  Corynanthe,  -19-  I b o g a and  Aspidosperma a l k a l o i d s but  represented indicated  i n t h a t p l a n t by  akuamicine  that corynantheine  ajmalicine  (30)  (45).  Strychnos  Earlier  a l d e h y d e ( 4 2 a ) was  or catharanthine  However, s i g n i f i c a n t  a l s o of the  not  reported  work^'^  a precursor  (6) i n m a t u r e C.  i n c o r p o r a t i o n was  family  of  roseus plants.  in  seedlings  6 3 of  this plant.  that C.  In fact,  t h e s e compounds a r e  roseus s e e d l i n g s . ' ^  s u b s e q u e n t work by  a l l actively  S c o t t ' s group  involved  i n biosynthesis  on  of b i o s y n t h e s i s  i n t h i s a r e a m u s t r e m a i n an o p e n  place  i n the  and  the  One  of  evidence from t h i n - l a y e r chromatography the  e l u c i d a t i o n of  other  o f g e i s s o c h i z i n e (40)  Strychnos a l k a l o i d s provided  the o r i g i n a l p r o p o s a l s  attack at  (Figure 8).  the  3 - p o s i t i o n of  aldehydo-ester  ( p o s s i b l y by  indolenine  (41).  concerning  the  The  an  details  take  to akuammicine  intriguing  Strychnos biosynthesis  crucial  i n d o l e by  step  the  epimer  was  i n t h i s scheme i s  a c t i v a t e d carbon of  the the  a r a d i c a l mechanism) to g i v e a p e n t a c y c l i c  stemmadenine ( 4 3 ) .  of the i n d o l e n i n e aldehyde g i v e akuammicine (45). The 68  (45)  challenge.  Subsequent rearrangement c o u l d r e s u l t i n the  known S t r y c h n o s a l k a l o i d  con-  question.  the b i o s y n t h e t i c e v e n t s w h i c h must  transformation  that of W e n k e r t . ^  in  However, s i n c e t h i s group based t h e i r  clusions mainly  The  suggested  A transannular  little-  cyclization  and l o s s o f t h e a l d e h y d e f u n c t i o n a l i t y , w o u l d natural products formylstryctamine (41)^  and  i t s C-16  w h i c h were i s o l a t e d r e c e n t l y from Rhazya  add  i n t e r e s t i n g support to the Wenkert  hypothesis.  stricta  -20-  0H (30)  Figure  7.  (40)  The p o s s i b l e b i o g e n i s i s o f t h e C o r y n a n t h e a s s u p p o r t e d by e x p e r i m e n t .  alkaloids  -21-  -22-  6y A second hypothesis was  offered by Scott and Quereshi i n  They proposed that the hydroxyindolenine  (46) could rearrange  1969. to an  oxindole (47) which i n turn could c y c l i z e to preakuamicine aldehyde (44), a structure which possess the Strychnos i s interesting  skeleton.  However, i t  to note that this type of c y c l i z a t i o n i s a very d i f f i c u l t  one to perforin i n the laboratory.  Compound (44) could then be elaborated  to stemmadenine (43) v i a a ring-opening reaction of a type already known i n the laboratory (this reaction w i l l be discussed i n d e t a i l i n Part III of this thesis). was  A great deal of evidence  provided by Scott's laboratory.^  to support  this hypothesis  He reported the i s o l a t i o n of the  oxindole (47) and i t s incorporation into akuammicine (45) i n C. seedlings.  Also reported was 69  and preakuammicine (48)  .  roseus  the i s o l a t i o n of the hydroxyindolenine  The co-occurrence  great credence to the Scott hypothesis.  (46)  of these structures lends  More recently, Heimberger and  Scott were able to incorporate geissoschizine (40) into Wieland-Gumlich aldehyde (49) and strychnine ( 4 ) . ^  (40)  (4)  (49)  -23-  OH (45)  Figure  9.  The the  S c o t t h y p o t h e s i s f o r the b i o s y n t h e s i s Strychos a l k a l o i d s .  (48)  of  -24Wenkert's  hypothesis  Iboga a l k a l o i d s  is illustrated  Strychnos skeleton the  crucial pivotal  (53)  Figure  f o r the b i o s y n t h e s i s i n Figure  (50), similar precursor  10.  to structure  He  proposed  (42)(Figure  t o t h e A s p i d o s p e r m a and  (52)  10.  of the Aspidosperma that  and  the  8) c o u l d  Iboga  be  families.  (56)  The W e n k e r t h y p o t h e s i s f o r t h e b i o s y n t h e s i s A s p i d o s p e r m a and Iboga a l k a l o i d s .  of the  -25-  The  important  feature  in this  scheme i s t h e  z a t i o n used to convert  s t r u c t u r e (52)  A great  i n our  d e a l of e f f o r t  hypothesis  i n C.  to  (53)  and  l a b o r a t o r i e s was  roseus plants.  Despite  the  transannular  cycli-  s t r u c t u r e (55)  made t o t e s t  success  of  to  (56).  this  the  corres-  72-74 ponding laboratory transformation that  t h i s was  (57)  and  failed  not  a viable route.  cleavamine  (58)  to incorporate  , the ? 7  '  I  7 8  n  i n v i v o study the  both l a b e l l e d with  into  catharanthine  (57)  latter  tritium  ( 6 ) and  of  t h i s w o r k came t h e  Aspidosperma a l k a l o i d  catharanthine  (6).  catharanthine  (6)  Strychnos the  tabersonine  into  reverse  (59)  i n the aromatic  6 3  At  '^  incorporated  this  i s incorrect.  the  that  into  the Iboga  transformation  c o u l d not  be  C.  Sequential  roseus seedlings  p o i n t , one  feeding  the  the  more  alkaloid of  demonstrated.  t h e b i o s y n t h e t i c s e q u e n c e t o be Iboga.  using  (59)  ring,  vindoline (7).  surprising result  was  process,  tabarsonine  Aspidosperma  Scott g r o u p  hypothesis  The  s t r o n g l y suggests  this proposal.  quebrachamine  (59)  e l a b o r a t e A s p i d o s p e r m a a l k a l o i d v i n d o l i n e ( 7 ) and  fact  study,  (58)  However, out  This  suggested  C o r y n a n t h e ->•  experiments  provided  evidence  must c o n c l u d e t h a t the  Wenkert  by for  -26-  Another the  important  above d e s c r i b e d  Vinca  minor  ratios  of  plants  (62)  and  minovine  R = H  (61)  R = CH  annular  of  14  t i m e and  C]-tryptophan  observing  alkaloids vincadine  during to  the  (60)  and  "ring-closed" alkaloids vincadifformine that  the  r a t i o s were r e l a t i v e l y  to  i n d i c a t e the  convertable  to  the  (62)  R  (63)  R =  existence  a l k a l o i d s without  going  constant.  =H  of  "ring-opened"  of  earlier  results  from  biosynthetic intermediate.  experiments using  [3-  laboratory  CH  a  3  pivotal  and  through  the a  trans-  reaction.  Rationalization a new  lengths  revealed  Aspidosperma  cyclization  demanded  two  b e e n made i n o u r  A d m i n i s t r a t i o n of  "ring-open"  appeared  w h i c h was  "ring-closed"  had  3  observation  intermediate  and  (63)  7 7  varying  i n two  (61),  (60)  This  study. over  activity  vincaminoreine  observation  the  Strychnos  alkaloid  Scott's  In v i v o  laboratory also  and  stemmadenine  in  vitro  (43)indicated  the  80—82 latter The  was  converted  into  s t r u c t u r e favoured  w h i c h was  closely  necessarily  of  both  for this  related  a different  to  Aspidosperma interemdiate  the Wenkert  oxidation  and was  Iboga the  alkaloids. acrylic  intermediates  level.  (51)  ester and  (64),  (54)  but  -27-  (64)  The  isolation  alkaloids,  called 83  acrylic (66) -v, the  ester.  and  from Rhazya  the secamines,  8 >4 '  Later,  (67), which s p e c i• e s .  same  species  8  5  >  three  a new  family  related directly  of dimeric  to the  r e l a t e d monomeric  8  hypothetical  alkaloids  were o f p a r t i c u l a r i m p o r t a n c e , were  indole  (65),  isolated  from  6  (65)  (66)  (67)  87  The called the  Manchester  presecamines, which  previously  secamines acrylic  group  underwent  characterized  were D i e l s - A l d e r  ester  discovered  function.  acid  secamines.  type dimers  The  possible  another  group  catalysed They  also  of a l k a l o i d s ,  rearrangement found  that  the  of secodines possessing presence of secodine  (68)  an  to prea-  and  -28-  C0 CH 2  C0 CH 2  3  (68)  its  that  from which  the  This the with  current  Strychnos  alkaloid  to The  the  the  synthesis  imperative  synthesis that  give  of  for  carbon and  structure  dihydro-pyridine pyridinium  via  could  the  system  (64)  may  the  (43)  skeletons  of  has  (78)  or  privotal  i n Figure  of  not (71)  of  this  precursor  11,  suggests  (70).  between atoms,  Aspidosperma  that  equilibrium  iso-stemmadenine  formation  the  biosynthesized.  is in biological  the  radiolabelling evaluation  strongly supports  the  intermediate bond  moieties  be  illustrated  undergo  (64)  plant  indole alkaloids i s  stemmadenine  (64)  (64)  i n the  f a m i l i e s of  hypothesis,  dehydrosecodine  shown,  (67)  dehydrosecondine various  Dehydrosecodine  was  (69)  15,20-dihydroderivative  hypothesis  3  and  as  Iboga  types.  dehydrosecodine d e r i v a t i v e s new  hypothesis.  b e e n p o s s i b l e as undergo very  date  i t is well  facile  r a p i d rearrangements  To  and  the  known  oxidation  to  polymerization:  (71)  R  *  This  biogenetic  numbering  (72)  s y s t e m was  suggested  by  L e M e n and  Taylor.  -29-  (60)  Figure  11.  (58)  H y p o t h e t i c a l b i o s y n t h e s i s o f t h e A s p i d o s p e r m a and Iboga a l k a l o i d s from the Strychnos f a m i l y v i a dehydrosecodine.  -30-  Therefore,  a more p r a c t i c a l synthetic target was  ester, secodine (68) and 17-ol  (69).  the tetrahydro-acrylic  i t s hydrated d e r i v a t i v e 16,17-dihydrosecodine-  These two compounds soon became a v a i l a b l e for biosynthetic  investigation. 89 Battersby's group  synthesized  the hydroxy ester (69)  determined by i s o t o p i c d i l u t i o n that i t was  present i n Rhazya b r i e n t a l i s  plants and possibly present i n (3. roseus seedlings. and  (69) were synthesized  and  Both structures 90  i n our laboratories by Drs. R. Sood  (68)  and  91 J. Beck.  In the next section of this t h e s i s , this work and i t s  biosynthetic implications w i l l be  described.  Part II of this thesis w i l l discuss investigations into two  other  families of indole a l k a l o i d s for which no biosynthetic evidence has been obtained.  These two groups, the ulein-type and  of indole a l k a l o i d s are considered  the  pyridocarbazole-type  somewhat anomalous i n that they do  not possess the normal two-carbon bridge between the indole portion the basic nitrogen atom.  In the uleine-type  absent, while i n the pyridocarbazole found.  and  structures this bridge i s  a l k a l o i d s a three-carbon bridge i s  C l e a r l y , the evaluation of the biosynthesis of these compounds  provides an i n t e r e s t i n g challenge. Part I I I of this thesis describes work directed towards the synthesis of the r a d i o - l a b e l l e d precursors  preakuammicine aldehyde  (44),  preakuammicine (48), stemmadenine (43) and  i t s i s o - d e r i v a t i v e (70).  As  previously described  these structures are deeply implicated i n the l a t e r  stages of biosynthesis of a l l indole a l k a l o i d s and  the possession  i n suitable r a d i o - l a b e l l e d form would be extremely desirable.  of them  -31-  DISCUSSION  A s was r e v e a l e d the  Cg-C^  i n the I n t r o d u c t i o n , the b i o s y n t h e s i s  u n i t f r o m t h e monoterpene pathway and t h e  n  p o r t i o n of the s t r u c t u r e s of the four has been f i r m l y the  established.  by S c o t t  dihydrosecodin-17-ol present of  The i m m e d i a t e c h a l l e n g e ,  and K u t n e y .  families  h o w e v e r , was  ( F i g u r e 11) p u t  forward  As mentioned e a r l i e r ,  ( 6 9 ) was s y n t h e s i z e d  16,17-  a n d shown t o p o s s i b l y b e  i n (3. r o s e u s p l a n t s b y t h e B a t t e r s b y  (69) and i t s d e h y d r a t i o n  tryptamine  large indole alkaloid  t e s t i n g of the dehydrosecodine hypothesis  independently  of both  product secodine  group.  89  The  synthesis  ( 6 8 ) was a l s o a c c o m p l i s h e d i n  90 9 1 our  laboratories.  synthesis  '  I n t h i s s e c t i o n there w i l l be d e s c r i b e d  of r a d i o - l a b e l l e d secodine  (68) and i t s s u c c e s s f u l  i n t o A s p i d o s p e r m a a l k a l o i d s i n _C. r o s e u s  Already  Figure (69)  8.  Using  and s e c o d i n e  to o b t a i n  this  prepared according  sufficient  was  incorporation  the 2-carboethoxy  t o t h e s e q u e n c e shown i n  compound a s s t a r t i n g m a t e r i a l  (68) were s y n t h e s i z e d ,  modified  plants. 90  a v a i l a b l e i n our l a b o r a t o r i e s  i n d o l e d e r i v a t i v e (73)  a  16,17-dihydrosecodin-17-ol  b y t h e s e q u e n c e shown i n F i g u r e  quantities for radio-labelling  and p l a n t  13,  feeding  experiments. The c h l o r o e t h y l i n d o l e ( 7 3 ) was c o u p l e d a sealed  t u b e a t 120° t o g i v e  Sodium b o r o h y d r i d e  reduction  the pyridinium  alcohol  3-ethylpyridine  salt  (75) i n h i g h  to the t e t r a h y d r o p y r i d i n e  f o l l o w e d by l i t h i u m aluminum h y d r i d e the  with  ( 7 7 ) , a l s o i n good  yield.  (74) i n  yield.  d e r i v a t i v e (76)  r e d u c t i o n o f t h e e s t e r group gave  -32-  (73)  90 Figure  The chain  12. T h e S y n t h e s i s o f t h e c h l o r o - i n d o l e ( 7 3 )  next  i n this  by one c a r b o n  carbon  s e q u e n c e was  i n a manner w h i c h would  by d i s p l a c i n g  t o g i v e compound  hydrochloric  acid  (79) .  gave  introduced  at this  tritiated  benzoate  Subsequent h y d r o l y s i s  secodine  i n the s y n t h e s i s .  trifluoroacetic  acid  of  This  was  (78) w i t h (79) w i t h  cyanide methanolic  ester (80).  ( 6 8 ) was  required, tritium  The e s t e r  (prepared  the s i d e -  the i n t r o d u c t i o n o f  reagent.  the corresponding methyl  stage  to l e n g t h e n  allow  the corresponding  When a r o m a t i c a l l y l a b e l l e d  with  designed  14 u s i n g a n i n e x p e n s i v e a n d c o n v e n i e n t  accomplished ion  step  from  ( 8 0 ) was  was  stirred  trifluoroacetic  3 anhydride  and  H^O) f o r 48 h o u r s .  After  c h r o m a t o g r a p h y and  recrystalli-  -33-  'N  n (73)  (74)  NaBH,  (75)  -N LiAlH, N H  OH (77)  X0 Et 2 L  (76)  0CCC1  -N KCN  OCOC H 6  CN  5  (78)  (79) HC1/CK 0H 3  HC0 Et 2  II ll N' H  CO2CH3 (81)  CO2CH3  (80) NaBH,  KaH  CO2CH3  CO2CH3  (69)  Figure  1 3 . The K u t n e y S y n t h e s i s  (68)  of secodine  (68)  9 0  '  9 1  -34-  zation  to c o n s t a n t  o b t a i n a b l e i n 80% To  radioactivity,  aromatically labelled  secodine  s k e l e t o n , i n t r o d u c t i o n of  m e t h y l e n e g r o u p a t o t h e e s t e r g r o u p was (80) w i t h m e t h y l f o r m a t e  and  (81) w i t h s o d i u m b o r o h y d r i d e .  a t -30°  and  monitored  by  thin-layer  e s t e r a l c o h o l (69) were o b t a i n e d r e d u c t i o n to the c o r r e s p o n d i n g of  for  the a l c o h o l (69)  feeding experiments  store  precursor  reducing  hydroxy-  the  the  resulting  t o be  performed  Good y i e l d s  o n l y when p r e c a u t i o n s  against  of  the  over-  d i o l were e x e r c i s e d .  (68).  u s i n g sodium h y d r i d e  I t was  found  t h a t i t was  as a c r y s t a l l i n e m a t e r i a l a n d immediately  for conversion  the  treating  chromatography.  before  [ar- H]-16,17-dihydrosecodin-17-ol  secodin-17-ol  by  T h i s r e a c t i o n had  the a l c o h o l e s t e r (69)  t h e somewhat u n s t a b l e s e c o d i n e store  achieved  carefully  product  Dehydration  was  yield.  complete the  anion of  (80)  use.  and  [  prepare  T h u s , we  gave  best  the  to  secodine  were a b l e  to  C0 CH ]-16,17-dihydro2  3  to the c o r r e s p o n d i n g l y l a b e l l e d  secodine  (68). 90  The  initial  were done by  Dr.  feeding experiments Sood i n o u r  laboratories,  t h i s p l a n t system could not u t i l i z e however, converted definite  into vincamine  incorporation.  C0 CH^\ 2  (82)  with  these  new  i n Vinca minor revealed  the a l c o h o l (69).  (82)  I n C_. r o s e u s  and,  precursors, which  minovine  p l a n t s a low  Secodine (83)  was,  i n a low  i n c o r p o r a t i o n of  CH  3  CO2CH3  (83)  that  but  1  -35-  only  secodine  (68) i n t o v i n d o l i n e  (7) and c a t h a r a n t h i n e  (6) c o u l d  be  obtained. Studies  with  radioactivity  doubly-labelled  from t r i t i u m  precursors  with  a known r a t i o o f  i n t h e a r o m a t i c r i n g and carbon-14 i n t h e  carbomethoxy group o f secodine  (68), provided  the important  information  t h a t a t l e a s t t h e i n d o l i c - e s t e r p a r t o f t h e m o l e c u l e was b e i n g  utilized  90 91 by  the p l a n t .  '  Therefore,  i t w o u l d be h i g h l y d e s i r a b l e  to also  have t h e s e c o d i n e m o l e c u l e r a d i o - l a b e l l e d i n the p i p e r i d e i n e p o r t i o n , as well,  to confirm  secodine  utilization  The s y n t h e s i s  of  ( 6 8 ) l a b e l l e d i n t h i s way b e c a m e t h e n e x t s t a g e i n t h e e v a l u a t i o n  of  s e c o d i n e as a p r e c u r s o r  of  indole alkaloids. Clearly,  the  of the whole s t r u c t u r e .  common t o t h e b i o s y n t h e s i s  i t i s of great  completion of a lengthy  advantage to introduce  synthesis  of a desired  of the major f a m i l i e s  r a d i o - l a b e l s near  precursor  i n con-  s i d e r a t i o n o f t h e expense o f t h e e x p e r i m e n t and o f the s a f e t y o f t h e workers involved. a higher  t o t a l a c t i v i t y must be h a n d l e d d u r i n g  manipulations. the  piperdeine  (Figure of  The  to The  Thus, i f one d e s i r e d portion of secodine  ( 6 8 ) was  basic  introduction of a radio-label into  favorable.  synthetic  i f the piperdeine  i n Figure  route  An a l t e r n a t e  requirement i n a second s y n t h e t i c route  alcohol ester  number o f  synthesis  required. to s e c o d i n e (68)  u n i t c a r r i e d a r a d i o - l a b e l i t should  the t r y p t a m i n e p o r t i o n by a s i m p l e  outlined  e a r l y i n the sequence,  a greater  (68) the f i r s t  13) w o u l d n o t be p a r t i c u l a r l y  secodine  was t h a t  I f a r a d i o - l a b e l i s introduced  procedure near  be c o u p l e d  t h e end o f t h e sequence.  ( 8 4 ) was a v a i l a b l e i n o u r l a b o r a t o r i e s b y t h e s e q u e n c e 14.  Due t o t h e i n i t i a l  development of t h i s  sequence by  -36-  92  Westcott and  , bulk  i t was  q u a n t i t i e s of  c h o s e n t o be  the  the  alcohol ester  starting  commercially  available indole ester  alcohol  and  (86)  i n t r o d u c t i o n of  converted c y a n i d e and  homologated e s t e r the as  presence of a  to  (89) .  stannic  stable material  the  material (85)  was  methanolysis of the  new  reduced  to  be  the  0  LiAlH,  H  obtained  synthesis.  The  corresponding  Subsequent  cyano group y i e l d e d  alcohol  conveniently  be  the  (87).  latter with  c h l o r i d e gave the could  could  i n the  benzoate ester  Treatment of  that  (84)  ethylene  (84)  the  oxide  i n good y i e l d  and  stored.  C0C1  0C0C H  OH  (85)  in  5  (86)  5  (87) KCN  HCl CH 0H 3  O2CH3  (84)  (89)  Figure  With for  the  14.  this  coupling  a t i o n was  CN  The  Synthesis  a l c o h o l i n hand  of  (88) ester  alcohol  (84)  s t u d i e s were i n i t i a t e d  with  the  3-ethylpyridine  to d i s p l a c e  the  hydroxyl  with  unit  (74).  92  to develop Our  chloride ion using  first  methodology consider-  methanolic  hydrogen  93 chloride  according  to a p r o c e d u r e p u b l i s h e d  by  Wenkert.  T h i s method  met  -37-  with  only  other  p a r t i a l s u c c e s s a s t h e p r o d u c t was c o n t a m i n a t e d w i t h  components. Our  a t t e n t i o n turned  tosylate.  to consideration  I t i s w e l l known t h a t p r i m a r y  derivative could  be formed u s i n g  of the corresponding  pyridinium  s a l t d e r i v a t i v e w o u l d be t h e e x p e c t e d  was i n d e e d  successful.  c h l o r i d e and c o o l e d added.  After  first  ( 8 4 ) was d i s s o l v e d  (tic) indicated  with  i n methylene  to r e f l u x  base-line  until  material  was d i s s o l v e d i n  the  tetrahydropyridine from the  13.  (90) 15.  This  An a l t e r n a t e s y n t h e s i s  sequence allowed  3-ethylpyridine operation,  approach  the corresponding m a t e r i a l  o f s e c o d i n e shown i n F i g u r e  (84)  to give  This  t o s y l c h l o r i d e was  the residue  sodium borohydride  ( 8 0 ) , w h i c h was i d e n t i c a l  synthesis  Figure  that only  A f t e r removal of the solvent  m e t h a n o l and t r e a t e d w i t h structure  product.  t h i s m i x t u r e was s l o w l y warmed  l a y e r chromatography  was p r e s e n t .  as the base, the d e s i r e d  t o 0° a n d f r e s h l y r e c r y s t a l l i z e d  two h o u r s  groups  Therefore,, i f t h e t o s y l a t e  3-ethylpyridine  The a l c o h o l  primary  t o s y l a t e s a r e good l e a v i n g  i n n u c l e o p h i l i c displacement reactions.  thin  several  and o b t a i n  i n 21% y i e l d .  of piperdeine  us, then,  to couple  the reduced product Subsequently,  this  ester (80).  the i n d o l i c  (80) i n a  p o r t i o n to  "one-pot"  s e q u e n c e was s t u d i e d  by  others  -38-  in  our group The  94  a n d t h e y i e l d was r a i s e d  next step  introducing  t o 35%.  i n our i n v e s t i g a t i o n s required  a method f o r  a r a d i o - l a b e l i n t o the p i p e r i d e i n e moiety of secodine (68).  T r i t i u m was c h o s e n o v e r c a r b o n 14 f o r t h i s p u r p o s e s i n c e the  l a t t e r would r e q u i r e  skeleton.  I f t r i t i u m were t o be u s e d  instance, into  to introduce  this  isotope  synthesis  the biosynthesis  a significant Our  i t was d e s i r a b l e , i n t h e f i r s t  i n t o the e t h y l side chain  labile  of the various  alkaloid  rather  skeletons  than  elaboration  and, therefore,  l o s s o f t h e l a b e l would be e x p e c t e d .  first  consideration  protons  fortritium  i n t r o d u c t i o n was t o e x c h a n g e  i n the methyl group o f 3 - a c e t y l p y r i d i n e  remove t h e o x y g e n f u n c t i o n under c o n d i t i o n s back  of the 3-ethylpyridine  the p y r i d i n e r i n g because the l a t t e r undergoes extensive  during  the  a more e l a b o r a t e  introduction of  to the o r i g i n a l  i n a c t i v e system.  (.91) a n d t h e n  which would not a l l o w  I n a model study u s i n g  exchange  deuterium i t 2  was f o u n d  that,  i n a two-phase system o f t e t r a h y d r o f u r a n  and  s o d i u m c a r b o n a t e , more t h a n 9 5 % o f t h e m e t h y l p r o t o n s c o u l d measured by p r o t o n n u c l e a r  magnetic resonance  was made b y i n t e g r a t i n g t h e s i g n a l a t T 7.4. material  H^O  be exchanged as  (p m r ) .  This  With  deuterated  this  containing  determination  i n h a n d we b e g a n a s t u d y d i r e c t e d a t t h e r e m o v a l o f t h e o x y g e n  function.  Or*** (91)  (92)  -39-  The  conventional  procedure f o r deoxygenation of aldehydes and  ketones which would probably enolization thioketal of  concerned  followed  the deuterated  etherate  the formation  ketone  (92) w i t h  gave t h e t h i o k e t a l  ring.  the deuterated  deuterated  tothe  o f deuterium from the  (93) u s i n g  i n s u c h a way a s t o r e t a r d was w o r k e d up w i t h  thioketal  3-ethylpyridine  Raney n i c k e l (94).  i t s liberation.  a 5% s o l u t i o n  ( 9 3 ) was a c h i e v e d ,  gave  I t was r e a s o n e d  o f t h e p y r i d i n e r i n g was c o o r d i n a t i n g  o f aqueous  h y d r o x i d e a g o o d y i e l d o f t h e p r o d u c t was o b t a i n e d . of  trifluoride  Integration of the  of deutero-3-ethylpyridine  perhaps the b a s i c n i t r o g e n  reaction mixture  and b o r o n  at T6.6 corresponding  1.4% l o s s  of the thioketal  a poor recovery  Raney n i c k e l  signals  that only  Treatment  occurred.  Desulphurization  the  ethylene  E x a m i n a t i o n o f t h e pmr s p e c t r u m o f  i n the t h i o k e t a l  a t T7.9revealed  initially  ethane d i t h i o l  (93).  multiplet  four methylene protons  methyl group had  of the corresponding  by d e s u l p h u r i z a t i o n w i t h Raney n i c k e l .  t h i s product revealed  signal  n o t i n v o l v e a s i g n i f i c a n t degree o f  with  When t h e sodium  When d e s u l p h u r i z a t i o n  t h e pmr s p e c t r u m o f t h e  ( 9 4 ) showed t h a t a p p r o x i m a t e l y  2.1%  loss  of lable  that  -40-  had  occurred  be e a s i l y  on t h e s u r f a c e  o f t h e Raney n i c k e l .  t o l e r a t e d i n a subsequent t r i t i a t i o n  Our s e c o n d c o n s i d e r a t i o n chain  d 3-ethylpyridine  studies using  for introducing  involved  labelling  s o d i u m b o r o h y d r i d e showed  acetylpyridine could  This  with  proton  Aprotic  the methylene group.  that  product obtained  first  was  t h e k e t o n e f u n c t i o n o f 3manner,  through  exchange  of the a l c o h o l group i n the  from sodium borohydride r e d u c t i o n  converting  R =  In this  t r i t i u m i n t o the  minimized loss of l a b e l  the hydrogenolysis  the hydroxyl  t o make t h e c o r r e s p o n d i n g  (99)  Initial  source.  The n e x t s t e p  involved  could  t r i t i u m i n t o the s i d e -  be r e d u c e d u n d e r a p r o t i c c o n d i t i o n s .  conditions  loss  procedure.  s o d i u m b o r o t r i t i i d e was e f f i c i e n t l y u s e d t o i n t r o d u c e side-chain.  small  - C —  CH  into a better tosylate  o f (.91).  l e a v i n g group.  (96),mesylate  Our We  plan attempted  (97) and b e n z o a t e  3  d e r i v a t i v e s , b u t i n each case o n l y  the s t a r t i n g  t h i n l a y e r chromatography or recovered  alcohol could  be s e e n b y  from the r e a c t i o n mixture.  The  (98)  -41-  corresponding The  acetate  identity  of  (99)  could,  t h i s m a t e r i a l was  which exhibited a molecular w h i c h showed  the  presence of  more, the  pmr  signal  T 7 . 9 3 w h i c h was  at The  achieved gas  spectrum of  by  i n the  t r e a t i n g the presence of  65%  obtained  e s t a b l i s h e d by  i o n p e a k a t m/e a carbonyl  165  expected  f o r the  acetate  of  (99)  In  and  the  with  t h i s way,  an at  yield.  spectrum  i n f r a r e d spectrum 1725  cm  three-proton  p r e s e n c e o f an  ^.  Further-  singlet  acetate  group.  o x y g e n f u n c t i o n a l i t y was an  palladium-on-charcoal  acid solution.  i n high  i t s mass  absorption  t h i s p r o d u c t showed a  subsequent hydrogenolysis  hydrochloric in  h o w e v e r , be  atmosphere of  hydrogen  catalyst in dilute  3-ethylpyridine  (74)  aqueous was  obtained  overall yield. When t h i s  s e q u e n c e was  used  to produce r a d i o - l a b e l l e d m a t e r i a l ,  3 sodium borohydride-  H was  an  extended p e r i o d  of  to  the  The  substrate.  allowed  of  radio-label in this 3-ethylpyridine Of  the  two  the  carbonyl  time to ensure a complete t r a n s f e r of r e a c t i o n was  i n a c t i v e sodium borohydride. the  to r e a c t w i t h  (74)  The  t h e n c o m p l e t e d by  tritium  the  the  87%. l a b e l l e d forms of  3-ethylpyridine  (74)  which  a v a i l a b l e , t h e m e t h y l e n e l a b e l l e d d e r i v a t i v e was  chosen  coupling  to  reaction  elaborated, (69) dpm  with per  as  indole alcohol  (84).  previously described,  a specific  millimole.  of  radioactivity  w e r e now  the  of  retention  specific  for  tritium  addition  o v e r a l l i n t r o d u c t i o n and  s e q u e n c e a s m e a s u r e d by was  the  compound  radioactivity  The  product of  to g i v e  i n the  this  for was  16,17-dihydrosecodin-17-ol  piperdeine  side-chain  of  2.17x10"^  -42-  With side-chain l a b e l l e d the  entire  precursor  biosynthesize  utilized  the b i o s y n t h e s i s of  (68)  by  i n hand, proof  the p l a n t system  c o u l d be  to  obtained.  this precursor,  that  To  doubly-labelled  14  [ 1 9 - H, bon  being  the Aspidosperma a l k a l o i d s  further evaluate 3  was  secodine  14,  feeding  CC^CK^]-secodine having equal  t o 3.00  technique.  i n a Waring blender  was  fed  a radioactivity  r a t i o , of  t o a C_. r o s e u s p l a n t u s i n g  A f t e r nine  days,  t h e p l a n t was  t r i t i u m over  the  cotton  mascerated  car-  wick  i n methanol  v i n d o l i n e (7) was i s o l a t e d i n a m a n n e r s i m i l a r 90 91 t o t h a t d e s c r i b e d by S o o d and B e c k . T h i s m a t e r i a l was recrystalized 91 and r e d u c e d w i t h l i t h i u m a l u m i n u m h y d r i d e t o v i n d o l i n o l (100) . The l a t t e r was 3  and  then r e c r y s t a l i z e d  to a c o n s t a n t  radioactivity with a  ratio  14 H/  in  C e q u a l t o 3.31. I n v i e w o f this area,  this  However, to v e r i f y  ratio  was  the r e s u l t s considered  this result,  o f many f e e d i n g  experiments  t o be w i t h i n e x p e r i m e n t a l  a s e c o n d e x p e r i m e n t was  carried  error.  out.  In a second f e e d i n g experiment u s i n g d o u b l y - l a b e l l e d secodine a radioactivity a manner s i m i l a r  ratio to  o f 1.54  was  that described  (68)  administered  t o a C.  roseus plant.  In  above, the  isolated  v i n d o l i n e (7)  was  -43-  converted ratio  to v i n d o l i n o l  of radioactivity  ( 1 0 0 ) a n d t h e l a t t e r was f o u n d equal  t o 1.35.  t o have a  constant  These r e s u l t s a r e summarized i n  T a b l e 1. The  important  t h e s e two f e e d i n g is  indeed  conclusion  t h a t c a n be drawn f r o m t h e r e s u l t s o f  experiments i s that  utilized  by the p l a n t system f o r the b i o s y n t h e s i s  Aspidosperma a l k a l o i d v i n d o l i n e ( 7 ) .  T a b l e 1.  C0„CH]-secodine  R a t i o o f A c t i v i t y Fed ( H/ C) 3  Ratio of A c t i v i t y  3.00  3.31  2  1.54  1.35  work d e s c r i b e d  i n this  dehydrosecodine hypothesis  to evaluate  the i m p l i c a t i o n s of  i n the biosynthesis  o f many f a m i l i e s o f  C u r r e n t l y work i s p r o g r e s s i n g  s t a b l e chromium complex o f d e h y d r o s e c o d i n e . these precursors  will  from s e v e r a l p l a n t The  be e v a l u a t e d  on t h e s y n t h e s i s  Also  i n cell-free  i n the near  enzyme  of a  future  preparations  species.  next part of this  thesis describes  from an i n v e s t i g a t i o n i n t o t h e b i o s y n t h e s i s and  Isolated  s e c t i o n forms p a r t o f an ongoing  program, i n our l a b o r a t o r i e s , designed  indole alkaloids.  i n t o v i n d o l i n e (7)  1 4  1  The  of the  95  The i n c o r p o r a t i o n o f [ 1 9 - H,  Exp.  the  the e n t i r e secodine molecule (68)  olivacine-types.  some p r e l i m i n a r y r e s u l t s  of a l k a l o i d s of the u l e i n e -  -44-  EXP ERIMENTAL  Thin  l a y e r c h r o m a t o g r a p h i c s t u d i e s were c a r r i e d  Merck s i l i c a  g e l o r Woelm n e u t r a l a l u m i n a a s  chromatoplates, an  0.3  mm  e l e c t r o n i c phosphor as  or a mixture  u n d e r a s h o r t and  The  C o l u m n c h r o m a t o g r a p h y was Woelm n e u t r a l a l u m i n a . at  The  the accepted  Throughout t h i s work, the Gas  liquid  plus  was  scanning  optimum r a t i o  the  w o r k was  of diameter  s o l v e n t s were d i s t i l l e d ( g l c ) was  Ultraviolet C a r y 15 recorded  recording  vapors.  as  a carrier  a column temperature of  gel  to h e i g h t  before  p e r f o r m e d on gas  6 0 / 8 0 mesh C h r o m s o r b W s u p p o r t  done u s i n g  and  as  a Varian  model  at a flow rate  of  i n c h x 10  consisted  packing  feet was  used.  A l l  175-205°.  i n m e t h a n o l on  a C a r y 11  s p e c t r o p h o t o m e t e r and  the a b s o r p t i o n  bands  (nm).  1:10.  use.  s p e c t r a were recorded  i n nanometers  or  columns were g e n e r a l l y  r o u t i n e a n a l y s i s a c o l u m n 1/4  c a r b o w a x on  examined  p e r f o r m e d u s i n g Woelm s i l i c a  80-85 m l / m i n . 20%  absorbent  lamp  or i o d i n e  using helium  of  In a l l cases,  2% m e t h a n o l , a n d  A-90-P i n s t r u m e n t , For  used.  thin  were developed i n e i t h e r  spray  dimensions of  chromatography  The  activated i n  added to the  long wavelength u l t r a v i o l e t  v i s u a l i z e d w i t h antimony p e n t a c h l o r i d e  maintained  mm  chromatoplates  of chloroform  either  In preparative s c a l e  ( a b o u t 2% b y w e i g h t ) was  fluorescent indicator.  chloroform,  hours.  t h i c k e r l a y e r o f 0.5  using  the absorbents.  i n t h i c k n e s s , w e r e a i r d r i e d and  o v e n a t 120°C f o r m o r e t h a n t h r e e  l a y e r chromatography a  out  I n f r a r e d s p e c t r a were o b t a i n e d  or  a  (A. ) are max on a P e r k i n -  -45-  E l m e r m o d e l 21 d o u b l e - b e a m s p e c t r o p h o t o m e t e r . i n KBr p e l l e t s o r i n c h l o r o f o r m s o l u t i o n . maxima  (v ) a r e quoted max Proton magnetic  i n wave numbers  resonance  resonance  spectrometer.  (cm  .  T h e s e w e r e m e a s u r e d a t 60  Where a d d i t i o n a l r e s o l u t i o n o r  MHz  double  s t u d i e s w e r e r e q u i r e d , t h e s p e c t r a w e r e m e a s u r e d a t 100  u s i n g a V a r i a n HA-100 i n s t r u m e n t . absorption signals as  The p o s i t i o n s o f a b s o r p t i o n  (p m r ) s p e c t r a w e r e m e a s u r e d i n  d e u t e r o c h l o r o f o r m a t room t e m p e r a t u r e . o n a V a r i a n T-60  Samples were measured  the i n t e r n a l  MHz  The p o s i t i o n s o f a l l p.m.r.  are g i v e n i n the T i e r s T s c a l e w i t h t e t r a m e t h y l s i l a n e  s t a n d a r d a t T 10.00.  For m u l t i p l e t  signals  the x-values  g i v e n represent the center of the s i g n a l . Mass s p e c t r a w e r e measured on an A s s o c i a t e d E l e c t r i c a l MS-9  d o u b l e - f o c u s i n g mass s p e c t r o m e t e r  meter.  Fragmentation  d a t a i s g i v e n i n mass t o c h a r g e  f o l l o w e d by p e r c e n t r e l a t i v e were a l s o determined of  known m o l e c u l a r  o r a n A t l a s CH-4  abundance.  o n t h e MS-9  High  minute  resolution  applying,  converted  f o r each  (m/e)  measurements  instrument using suitable  standards  weight.  Scintillation  (cpm) was  mass s p e c t r o -  ratios  R a d i o a c t i v i t y was m e a s u r e d u s i n g a N u c l e a r - C h i c a g o 6860 L i q u i d  Industries  Counter.  The m e a s u r e m e n t  Mark I Model  i n counts  t o d i s i n t e g r a t i o n s p e r m i n u t e (dpm)  sample, the counting e f f i c i e n c y  determined  per by by t h e 97  external standard The l i q u i d  technique which  uses  a barium  s c i n t i l l a t i o n s o l u t i o n consisted of  133 gamma s o u r c e . 2,5-diphenyloxazole  (4 g r a m s ) a n d l , 4 - b i s [ 2 - ( 5 - p h e n y l o x a z o l y ) ] b e n z e n e ( 0 . 5 g r a m s ) in  toluene  (1 l i t r e ) .  The b a c k g r o u n d  r a d i a t i o n o f each  dissolved  counting  vial  -46-  was  determined  or methanol then was  before  (1 ml)  added.  i t was  i n the v i a l  When d o u b l y  recalibrated  and  A s a m p l e was  and  scintillator  labelled  f o r each  Catheranthus roseus  d i s s o l v e d i n benzene solution  samples were counted  the o v e r l a p between the  measurements were o b t a i n e d The  used.  (14 ml)  the  t r i t i u m and  instrument  carbon  the  s u p e r v i s i o n of Dr.  p l a n t s used i n t h i s  P.  Salsbury of  M e l t i n g p o i n t s were determined Elemental  analyses  Laboratory,  Synthesis  of  The  Secodine  (68)  s y n t h e s i s of 90  laboratories  study  were grown i n  the Department of  a K o f l e r b l o c k and P.  Columbia,  Columbia,  Borda of  Chemistry.  are  uncorrected.  the M i c r o a n a l y t i c a l  Vancouver.  from 2 - C a r b o e t h o x y ) - 3 - ( B - c h l o r o e t h y l ) - i n d o l e  secodine  ( 6 8 ) was  p r e v i o u s l y worked out  in  (73)  our  91 '  and  q u a n t i t i e s u s e d and procedure w i l l  on  w e r e p e r f o r m e d b y Mr.  U n i v e r s i t y of B r i t i s h  14  sample.  a Department of H o r t i c u l t u r e greenhouse, U n i v e r s i t y of B r i t i s h under  was  be  has  yields  given  been d e s c r i b e d obtained,  for this  in detail.  as w e l l as  particular  Therefore,  only  some m i n o r c h a n g e s  in  sequence. 90  N-[B{3-(2-Carboethoxy)-indolyl}-ethyl]-3"-ethyl-pyridinium chloride The  c a r b o e t h o x y c h l o r o i n d o l e ( 7 3 ) ( 5 g ) was  (74,16 ml)  i n t h i c k - w a l l e d tube.  oil  bath  and  the contents were s t i r r e d  yield  kept  a white  a t 120°  f o r 24  s o l i d w h i c h was  dissolved i n 3-ethylpyridine  After sealing,  hours.  The  (75)  the  t u b e was  t u b e was  then  (6.3  in  cooled, broken  i n d i e t h y l e t h e r f o r 2 h o u r s and d r i e d i n h i g h vacuum  placed  g).  filtered  an  open to  -47-  N-[B-{3-(2-Carboethoxy)-indolyl}-ethyl]-3'-ethyl-3"-piperideine The  indole pyridinium  methanol (20  g) was  After (100  (400  2.5 ml)  ml)  slowly  containing  added.  hydrochloric  acid.  basified with  ( 7 5 ) ( 6 . 3 g) was  triethylamine  (6 m l ) .  the  The  s o l v e n t was  s a t . sodium bicarbonate The  s o l u t i o n and  combined o r g a n i c  amorphous m a t e r i a l  borohydride stirring. water  acidified with  f o r 30 m i n u t e s  the  2N  solution  extracted  was  with  e x t r a c t s were washed  water, d r i e d over anhydrous sodium sulphate an  Sodium  in  removed i n vacuo b e f o r e  r e s u l t i n g s o l u t i o n was  After stirring  methylene c h l o r i d e .  to y i e l d  dissolved  added a t room t e m p e r a t u r e w i t h v i g o r o u s  h o u r s most of was  chloride  (76)  and  with  evaporated i n vacuo  (76)(6g).  90 N-[g-{3-(2-hydroxymethylene)-indolyl}-ethyl]-3^-ethyl-3^-piperideine The freshly  p i p e r i d e i n e (76)(6g) from the  distilled  to a v i g o r o u s l y THF  (300  ml)  stirring  filtered,  c h r o m a t o g r a p h y on the  cooled  water again  s u s p e n s i o n was  The  of  and  was  atmosphere.  alumina  and  destroyed  A f t e r the  additions the  e v a p o r a t e d and (150  g,  act  the  material  a d d e d o v e r 40  At  the  end  sodium  excess reagent.  The  dried i n high  vacuum. the  in  minutes  (8g)  a d d i t i o n was  o f w a t e r , 15%  I I I ) permitted  piperideine alcohol from the  dissolved  l i t h i u m aluminum h y d r i d e  p i p e r i d e i n e a l c o h o l (77)(3.6g) using  Benzoylation  r e a c t i o n was  s l o w l y warmed t o r e f l u x f o r 2 h o u r s .  r e a c t i o n was  s o l u t i o n and  (THF,70 ml)  suspension of  under a n i t r o g e n  t h e m i x t u r e was time the  tetrahydrofuran  previous  (77)  in  complete, of  this  hydroxide  resulting Column  s o l u t i o n of  a benzene/chloroform  gradient.  (77)  above r e a c t i o n  (3.6  g) was  dissolved  in freshly  -48-  dried was  and d i s t i l l e d  added over  another  15 m i n u t e s a n d r e s u l t i n g  3 hours a t 0°.  sodium b i c a r b o n a t e mixture was in  p y r i d i n e (30 m l ) a t 0°.  Water  solution.  Benzoyl  s o l u t i o n was s t i r r e d  A f t e r t h e e v o l u t i o n o f gas ceased t h e The o r g a n i c  washed r e p e a t e d l y w i t h w a t e r , d r i e d , e v a p o r a t e d  (78)(5.2g).  for  ( 3 0 m l ) was added f o l l o w e d b y s a t u r a t e d  was e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e .  h i g h vacuum o v e r n i g h t  c h l o r i d e (11 ml)  to y i e l d  extract  i n vacuo and d r i e d  a gummy, d a r k c o l o u r e d  material  An a l t e r n a t e b e n z o y l a t i o n p r o c e d u r e w h i c h a v o i d s  p y r i d i n e was l a t e r w o r k e d o u t .  using  89  90 N - [ g - { 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 i r i e (79) The  benzoate p i p e r i d e i n e (78)(5.2g)  formamide stirred After  (135 ml) w i t h potassium  cyanide  was d i s s o l v e d i n d i m e t h y l (.9.0 g) .  a t room t e m p e r a t u r e f o r 1 h o u r b e f o r e  about 1 hour the mixture  w i t h water  (300  was c o o l e d  slowly heating  ml) and e x t r a c t e d w i t h methylene c h l o r i d e .  dried  and evaporated  chromatography on alumina  solid  (3.7 g ) .  diluted  The o r g a n i c  anhydrous  i n vacuo t o g i v e a t h i c k o i l .  i n h i g h vacuum t o g i v e a c o l o u r e d  t o 110°.  i n an ice-water bath,  e x t r a c t was washed r e p e a t e d l y w i t h w a t e r , d r i e d o v e r sulphate  T h i s m i x t u r e was  sodium  T h i s o i l was Subsequent  (150 g, a c t I I I ) y i e l d e d t h e n i t r i l e  (79)(2.1g).  N-[B-{3-(3-Carbomethoxy-methylene)-indolyl}-ethyl]-3'-ethy1-3"-piperideine  wo)  90  The (about  indole n i t r i l e  50 m l ,  distilled  (79)(2.1g)  was d i s s o l v e d i n f r e s h l y  from magnesium).  Water  d r i e d methanol  (.0.5 m l ) was a d d e d t o  -49-  c o n s t i t u t e 1% o f t h e s o l u t i o n a n d t h e m i x t u r e was c o o l e d water bath. mixture  Hydrogen  c h l o r i d e g a s was b u b b l e d i n t o  to achieve a saturated  solution.  i n an i c e -  the r e a c t i o n  T h i s m i x t u r e was  stirred  a t r o o m t e m p e r a t u r e f o r 2.5 d a y s a n d t h e n e v a p o r a t e d i n v a c u o . r e s i d u e was p a r t i t i o n e d b e t w e e n methylene c h l o r i d e .  s a t . sodium b i c a r b o n a t e s o l u t i o n and  The o r g a n i c e x t r a c t was w a s h e d w i t h w a t e r ,  over anhydrous sodium s u l p h a t e and e v a p o r a t e d jin vacuo amorphous m a t e r i a l alumina  (1.8 g ) .  w h i c h c o u l d be r e c r y s t a l l i z e d  ester  (80)  The r e c r y s t a l l i z e d  dried  to give an  T h i s p r o d u c t was c h r o m a t o g r a p h e d o n  (100 g, a c t I I I ) t o y i e l d  3 [Ar-H]-indole  The  the carbomethoxy  indole  ( 8 0 ) ( 1 . 4 5 g)  f r o m m e t h y l e n e c h l o r i d e , m.p.  85-88°.  90  and d r i e d  indole ester  ( 8 0 ) ( 2 1 0 mg) w a s  dissolved  3 i n previously prepared t r i f l u o r a c e t i c mmole) u s i n g a v a c u u m t r a n s f e r  a n h y d r i d e - [ H] ( 1 . 2 g , 0.9  technique.  T h i s s o l u t i o n was a l l o w e d  a t r o o m t e m p e r a t u r e f o r 2.5 d a y s u n d e r a n i t r o g e n a t m o s p h e r e . of  the solvent,  t h e r e s i d u e was p a r t i t i o n e d  h y d r o t i d e s o l u t i o n and methylene c h l o r i d e . several rated  times w i t h water, d r i e d  in.vacuo.  millicuries/  between  to s t i r  After  removal  a c o n e , ammonium  The o r g a n i c e x t r a c t was w a s h e d  o v e r anhydrous sodium s u l p h a t e and evapo-  S u b s e q u e n t c h r o m a t o g r a p h y on a l u m i n a y i e l d e d  (157 mg) w h i c h c o u l d b e r e c r y s t a l l i z e d  to a constant s p e c i f i c  the ester activity  o f 4.71 x 1 0 ^ dpm/mg.  [Ar- H j - S e c o d i n e a l d e h y d e (81) The a r o m a t i c a l l y  labelled  indole ester  ( 1 5 7 mg) was d i s s o l v e d  i n dry  -50-  b e n z e n e and ( 1 6 0 mg  add  dropwise  to a suspension of o i l - f r e e  sodium  o f 55% d i s p e r s i o n i n p a r a f f i n ) i n benzene ( 5 ml)  distilled  formate  (4 m l )  under  r e a c t i o n t e m p e r a t u r e was  raised  t o 35°  2 hours.  methyl  The  r e a c t i o n m i x t u r e was  treated with cold water. d r y i n g over anhydrous  a l i g h t y e l l o w f o a m ( 1 8 0 mg)  and  freshly  a n i t r o g e n atmosphere. and  s t i r r i n g was  The  continued f o r  c o o l e d i n an i c e - w a t e r b a t h  Subsequent  sodium  hydride  e x t r a c t w i t h methylene  s u l p h a t e and w h i c h was  and  chloride,  e v a p o r a t i o n i n vacuo  used  directly  i n the  yielded  next  reaction.  3 [Ar- H]-16-17-Dlhydrosecodln-17-ol The  amorphous m a t e r i a l  (69)  ( 1 5 0 mg)  90  from  the above r e a c t i o n  was t  dissolved  i n methanol  borohydride until  ( 2 5 0 mg)  thin-layer  T h i s m i x t u r e was  (15 m l )  over 1 hour  chromatography  between s a t . sodium  and  reduced  coloured yielded  foam  stirred  indicated  treated with  the absence  i n volume i n vacuo.  washed w i t h w a t e r  and  Column c h r o m a t o g r a p h y  30  of s t a r t i n g  yielded  on a l u m i n a  a  material.  partitioning  chloride over an  minutes  acid,  Subsequent  dried  Removal o f the s o l v e n t _in vacuo  ( 1 8 0 mg).  sodium  f o r an a d d i t i o n a l  b i c a r b o n a t e s o l u t i o n and m e t h y l e n e  o r g a n i c e x t r a c t w h i c h was sulfate.  and  and  t h e n t r e a t e d w i t h 3 d r o p s o f 2N h y d r o c h l o r i c  d i l u t e d with water  sodium  c o o l e d t o -30°  gave  an  hydrous  slightly ( 1 0 g, a c t I I I )  t h e s e c o d i n o l ( 6 9 ) ( 4 1 mg) f r o m a b e n z e n e / c h l o r o f o r m g r a d i e n t a s  w h i t e foam.  R e c r y s t a l l i z a t i o n of this material  afforded a specific  activity  o f 4.54  from  x io'' dpm/mg.  dichloromethane  a  -51-  Alternative  Synthesis  thyl}-3'-piperdeine  (15 m l )  of (80)  The  ester alcohol  and  3-ethylpyridine  atmosphere.  To  (600  a d d e d and  mg)  mixture off.  N-{g-[3-(2-Carbomethbxymethyl)-iriddlyl]-  was was  the  ( 8 4 ) ( 1 9 0 mg)  was  s t i r r i n g was  continued  e x c e s s 3 - e t h y l p y r i d i n e was  stirring  while  was  organic  e x t r a c t was  continued  extracted repeatedly  m a t e r i a l on  solvent had  gradient  p h y s i c a l and  after  with  yield  (15,  the  act.Ill)  ( l g ) was  desired  spectroscopic  previously prepared m a t e r i a l  slowly  the  was added  f o r a further 2 hours.  stirring  (340  using  distilled  the r e s i d u e  f o r 10  mg). a  Two  minutes  (125  d r i e d over anhydrous sodium sulphate  alumina  reaction  After cooling,  dichloromethane  residue  chloride  This  d i c h l o r o m e t h a n e was  Sodium b o r o h y d r i d e  a t 0° was  inhert  overnight.  removed i n h i g h vacuum and  i n vacuo to g i v e a d a r k c o l o u r e d this  the  u n d e r an  p-toluene sulphonyl  continued  v o l u m e s o f w a t e r w e r e t h e n a d d e d and mixture  a t 0°  a t 95-98° f o r 8 h o u r s .  i n m e t h a n o l a t 0°.  o v e r 1 h o u r and  ml)  dissolved i n dichloromethane  cool reaction mixture  t h e n warmed t o 95°  Stirring  dissolved  (74)(0.5  was  ml).  and  this The  evaporated  Chromatography  of  benzene-dichloromethane  indole ester  (60 mg).  properties identical  with  This those  material of  (80).  1- (3 **-Pyr i d y l ) - 1 - t h i o - e t h y l e h e k e t a l - e t h a n e 3-Acetylpyridine dithiol  (5 m l )  and  boron t r i f l u o r i d e  a t room t e m p e r a t u r e . r e s i d u e was  ( 9 1 ) ( l g ) was  dissolved i n a mixture etherate  A f t e r removal of  the  p a r t i t i o n e d between s a t u r a t e d  (1 m l )  solvent  and  stirred  i n high  aqueous sodium  of  ethane overnight  vacuum,  the  bicarbonate  -52-  s o l u t i o n and with  dichloromethane.  sat. bicarbonate  s o l u t i o n and  anhydrous sodium s u l p h a t e . i n v a c u o and to y i e l d  the product  (singlet,  4H,  3H,  was  ( l g ) was  g ) , pmr:  the  washed t w i c e w i t h  t h e p r e s e n c e o f one  product  was  as  1.45, 4H,  over  evaporated (15  1.90,  g,  act.Ill)  2.78  -S-CT^CH^-S-),  7.85  M 197. +  was  Raney n i c k e l  stirred with  ethanol  and  distilled  to y i e l d  deuterium oxide  (3 m l )  closed vessel.  The  f o r 16  filtered  off.  sample of  the  ml)  hours. hydroxide The  A glc examination  residue  extracted sodium showed  same r e t e n t i o n t i m e  (6  3-ethyl p y r i d i n e (74).  The  a c o l o u r l e s s l i q u i d whose s p e c t r a l sample of  (74).  (92)  2  3-Acetylpyridine  (50  aqueous sodium  p r o p e r t i e s were a l s o i d e n t i c a l w i t h an a u t h e n t i c  [2"- H]-3-Acetylpyridine  g)  A f t e r d r y i n g over anhydrous  removed i n v a c u o .  authentic  (20  ethanol  the combined f i l t e r a t e s were  component w h i c h had  an  5%  i n o r g a n i c m a t e r i a l was  s o l v e n t was  m i n u t e s , 175°)  1.0,  alumina  twice  drying  then  dissolved i n absolute  repeatedly w i t h dichloromethane. the  s o l v e n t was  (multiplet,  f r e s h l y prepared  cooling t h i s mixture  sulphate  the  c h r o m a t o g r a p h e d on  6.6  washed  l-(3"-pyridyl)-l-thioethylerieketal-ethane  thioketal  f o r 15 m i n u t e s a n d  e x t r a c t was  once w i t h water b e f o r e  - C H ^ ) ; mass s p e c t r u m :  refluxed with  After  was  aromatic-H),  D e s u l p h u r i z a t i o n of The  organic  Most of  a c o l o u r l e s s o i l (1.55  (multiplets,  and  The  (2 g) was  and  s t i r r e d with dry  d r i e d sodium carbonate  reaction mixture  was  tetrahydrofuran  CO.5  g)  overnight  t h e n p a r t i t i o n e d and  the  (5  ml),  in a organic  -53-  l a y e r was solvent -CHIJ^)  d r i e d over anhydrous sodium s u l p h a t e . i n h i g h vacuum, the  integrated  p r o d u c t was  t o show 9 5 %  A f t e r removal of  examined by  exchange of  to  deuterated  i t s corresponding  examined by  pmr:  material  (1.5  g)  t h i o k e t a l (93)(2.1  7.9  7.40  (multiplet,  isotopes.  2 [2'- H ] - l - ( 3 " - P y r i d y l ) - l - t h i o e t h y l e n e k e t a l - e t h a n e The  pmr:  the  from the g)  as  (93)  a b o v e r e a c t i o n was  previously  ( m u l t i p l e t , -CHE^) i n t e g r a t e d  converted  described  and  t o show l o s s o f  1.4%  from s t a r t i n g m a t e r i a l .  [2- H]-(3"-Pyridyl)-ethane  (94)  2  The  above d e u t e r a t e d  3-ethylpyridine  t h i o k e t a l ( 9 3 ) ( 2 g ) was  ( 9 4 ) ( 0 . 8 6 g)  by  cedure p r e v i o u s l y described. (multiplet, the  -CHTJ^) r e v e a l e d  1-(3"-Pyridyl)-ethanol  hours.  (25 m l )  ( 9 1 ) (.3.5  g) was  The  of  8.8  label  i n anhydrous  sodium b o r o h y d r i d e  ( 2 6 5 m g ) a t 80°  then cooled  sodium bicarbonate  dichloromethane.  3.4%  pmr:  dissolved  a c i d f o r 5 m i n u t e s and  saturated  p r o d u c t by  pro-  from  (92).  treated with  T h i s m i x t u r e was  hydrochloric with  and  deuterated  (.95)  3-Acetylpyridine ethane  the  i n t e g r a t i o n a l o s s of  starting 3-acetylpyridine  to  the Raney n i c k e l d e s u l p h u r i z a t i o n  Examination of by  converted  organic  and the  stirred with  0.1  N  r e s u l t i n g m i x t u r e was  s o l u t i o n and  extracted  dimethoxyfor  aqueous made  basic  repeatedly  with  e x t r a c t s w e r e w a s h e d w i t h w a t e r and  over anhydrous sodium s u l p h a t e .  Removal of  the  solvent  24  i n vacuo  dried  yielded  -54-  a c o l o u r l e s s o i l w h i c h was This product d a t a ; uv: 2.22, 5.10  ( 9 5 ) ( 3 . 4 5 g, b.p.  similar  2.78  chromatographed  ( q u a r t e t , J=7,  mass s p e c t r u m :  M  +  4H,  heated  w i t h water  ( 2 . 8 g) was  ( q u a r t e t , J=7, ( d o u b l e t , 3H,  and  1.5,  2.30,  2.51  The  sodium  sulphate.  rapidly  -CH(0H)CH ); 3  then  - C H ( O A c ) C H ) ; mass s p e c t r u m : 3  sodium  then washed  After  removal on 1725  max  aromatic-H), 3H,  4.05  -0 CCH ), 2  8.44  3  M 165. +  (99)  p y r i d a l a c e t a t e ( 9 9 ) ( 2 . 0 g) was  t h e r e s i d u e was  4H,  and  removed  chromatographed  (singlet,  3  stirred  -OH),  anhydride  o r g a n i c e x t r a c t was  I H , - C H ( O A c ) C H ) , 7.93  dissolved  c h l o r i d e gas.  dissolved  palladium-on-charcoal catalyst m i x t u r e was  i n acetic  s o l v e n t was  (multiplets,  t r e a t e d b r i e f l y w i t h hydrogen  i n v a c u o and  IH,  3H,  t o g i v e a c o l o r l e s s o i l (3.0 g ) : v  C a t a l y t i c Hydrogenolysis of Acetate The  (broad s i n g l e t ,  dissolved  The  d r i e d over anhydrous  pmr:  1.56,  p a r t i t i o n e d b e t w e e n 5% a q u e o u s  dichloromethane.  (20 g, a c t . I I )  (acetate);  following  (99)  t h e s o l v e n t t h e r e s i d u e ( 3 . 2 g) was  alumina  the  act.III).  123.  t h e r e s i d u e was  and  had  ( d o u b l e t , J=7,  3  on a s t e a m b a t h f o r 45 m i n u t e s .  b i c a r b o n a t e and  mm)  (25 g,  3 2 5 0 (-0H); pmr:  I H , - C H ( 0 H ) C H ) , 8.55  above a l c o h o l  i n v a c u o and  of  v  a r o m a t i c - H ) , 4.0  1-Ethyl-(3'-pyridyl)-acetate The  105-120° a t 1.2  to 3 - e t h y l p y r i d i n e :  (multiplets,  on a l u m i n a  i n water  ( 1 0 % , 400  a t room t e m p e r a t u r e  mg)  was  The  in  dimethoxyethane  s o l v e n t was  (30 m l ) . a d d e d and  To  removed  this  the  solution,  resulting  i n an atmosphere o f hydrogen  gas.  -55-  After  one  mixture  e q u i v a l e n t o f h y d r o g e n had  been consumed,  was  filtered  and  made b a s i c w i t h  bicarbonate  solution  and  extracted with  organic the  e x t r a c t s were d r i e d  s o l v e n t was  then  distilled  with  an  carefully to y i e l d  authentic  over  s a t . aqueous  reaction  sodium  dichloromethane.  anhydrous  removed by  the  sodium  sulphate  distillation.  3 - e t h y l - p y r i d i n e (0.9  The  The  before  residue  g) w h i c h was  was  compared  sample.  [l- H]-l-(3"-Pyridyl)-ethanol 3  3-Acetyl ethane  (30 m l )  p y r i d i n e (4.5 and  g) was  treated with  dry  dissolved  i n anhydrous  sodium b o r o h y d r i d e  dimethoxy-  (10 mg)  at  85°  and  the  3 for  2 hours.  r e a c t i o n was then  added  silica was  gel  Sodium b o r o h y d r i d e continued  and  overnight.  stirring  (chloroform)  then worked  up  as  K  was  (40 mg, Dry  continued  indicated  200  mc)  was  added  sodium b o r o h y d r i d e until  examination  a complete  reaction.  (450  by  mg)  was  t i c on  This  mixture  previously described.  3 [1-  H]-l-Ethyl-(3'-pyridyl)-acetate The  acetic and  tritiated  anhydride  worked  up  as  a l c o h o l from  (25 m l )  at  the  100°.  above  reaction  This reaction  was  was  dissolved  monitored  by t i c  previously described.  [1- H]-1-(3'-Pyridyl)-ethane 3  The converted with  tritiated to  pyridyl  acetate  i t s hydrochloride salt,  palladium-on-charcoal  (10%,  80  from  the  above r e a c t i o n  dissolved mg).  After  i n water  and  in  was treated  hydrogenalysis  in  -56-  an atmosphere o f hydrogen.  The r e a c t i o n m i x t u r e was w o r k e d u p a s  3 previously described to y i e l d 2.18xl0  [1- H]-l-(3'-pyridyl)-ethane  (2.6 g,  dpm/mg).  8  [19- H]-N[6{3-(2-Carbomethoxymethyl)-indolyl}-ethyl]-3'-ethyl-3'3  piperdeine The  ester alcohol  ( 8 4 ) ( 2 0 0 mg) w a s d i s s o l v e d  methane (15 m l ) , t r i t i a t e d sulphonyl chloride  ethylpyridine  ( 6 5 0 mg) a t 0 ° .  out as p r e v i o u s l y d e s c r i b e d t o y i e l d  i n dry dichloro-  (0.5 ml) w i t h p - t o l u e n e  The r e a c t i o n was t h e n the t r i t i a t e d  carried  p i p e r d e i n e (80)  (65 mg).  3 [19-  H]-Secodine-17-al The  tritiated  alcohol  (10 m l , f r e s h l y d i s t i l l e d formate oil  ( 8 0 ) ( 6 5 g) was d i s s o l v e d  from l i t h i u m aluminium  (4 m l ) a n d t r e a t e d w i t h s o d i u m  suspension).  After  2.5 h o u r s  i n benzene  h y d r i d e ) and m e t h y l  h y d r i d e ( 6 5 mg o f a 5 5 %  t h e r e a c t i o n was w o r k e d up a s  previously described.  [19- H]-16,17-Dihydrosecodin-17-ol 3  The and to  a b o v e p r o d u c t was d i s s o l v e d  t r e a t e d w i t h sodium the procedure  recrystallized (6.56xlQ  7  barohydride  dpm/mg).  ( 1 0 m l ) a t -35°  ( 2 0 mg) o v e r 2 h o u r s  previously described.  from dichloromethane  i n methanol  The p r o d u c t  according  ( 3 0 mg) was  to give t r i t i a t e d secodinol  -57-  The I s o l a t i o n of Vindoline (7) from Feeding Experiments i n C. roseus Plants ' 9 0  9 1  Vindoline (7) was  i s o l a t e d from whole plants of C^. roseus by a  procedure s i m i l a r to that used by others i n our laboratories and w i l l only be b r i e f l y summarized.  The plant was up-rooted and cleaned of  s o i l before i t was mascerated  i n methanol i n a Waring blender.  The  r e s u l t i n g pulp was re-mascerated twice more with a d d i t i o n a l quantities of methanol.  The combined methanolic extracts were then evaporated  and the residue was p a r t i t i o n e d between 10% aqueous a c e t i c acid and benzene.  This aqueous extract  was made basic with saturated sodium  bicarbonate s o l u t i o n and extracted with chloroform. was  The organic extract  then washed with water and dried over anhydrous sodium sulphate  before the solvent was removed.  Chromatography of the residue on alumina  ( a c t . I l l ) yielded vindoline (7), using a benzene-chloroform  solvent  gradient.  3 14 90 91 The Administration of [19- H, CO^H^]-Secodine (68) to C. roseus ' In both of the following feeding experiments, the radioactive precursor was dissolved i n d i s t i l l e d water (0.5 ml) with 2 drops of IN acetic acid.  This s o l u t i o n was administered to the plant v i a cotton  wick (~5 cm) which had been passed through the stem of the potted plant. After the s o l u t i o n had been taken up by the plant d i s t i l l e d water (0.5 ml) with 1 drop of IN acetic acid was added to the container and allowed to be taken up by the plant.  After that, the plant was allowed to grow  under a r t i f i c i a l l i g h t s at room temperature for a t o t a l of 9 days.  -58-  The d a t a r e l e v a n t t o t h e f e e d i n g e x p e r i m e n t s u s i n g C. r o s e u s p l a n t s i s summarized i n T a b l e 2. was  I n b o t h cases the v i n d o l i n e  isolated  converted to v i n d o l i n o l before r e c r y s t a l l i z a t i o n to constant  radioactivity.  T a b l e 2.  3  A d d i t i o n a l data a s s o c i a t e d w i t h Table 1  S p e c i f i c A c t i v i t y Fed H,dpm/mmole C,dpm/mmole 14  3.48  x 10'  1.16  7.61 x 10'  a.  a  3  Specific Activity Isolated H,dpm/mmole ^ c ^ p m / m m o l e  x 10'  2.15  x 10"  6.50 x 10  4.94 x 10'  3.67  x 10"  2.72  ["''^C^CH.j]-Secodinol was a v a i l a b l e from the o r i g i n a l by Sood.  Vindolinol ( 1 0 0 )  x 10"  synthesis  9 1 , 9 8  R a d i o a c t i v e v i n d o l i n e (7) was c o n v e r t e d t o v i n d o l i n o l by the 91 p r o c e d u r e r e p o r t e d by Beck.  V i n d o l i n e (7) was d i s s o l v e d i n d r y  t e t r a h y d r o f u r a n and r e f l u x e d w i t h e x c e s s l i t h i u m aluminum h y d r i d e f o r 2 hours.  The excess r e d u c i n g r e a g e n t was d e s t r o y e d w i t h s a t . sodium  sulphate s o l u t i o n .  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I a general  i n t r o d u c t i o n to i n d o l e a l k a l o i d  U n t i l very  been d i r e c t e d toward  r e c e n t l y , a l l t h e work i n t h i s a r e a had  the four major 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 ,  namely t h e Corynanthe, t h e Strychnos, skeletons. being has  found i n nature  been a l t e r e d .  n i t r o g e n atom  i n which the tryptamine  between t h e i n d o l e n u c l e u s and the b a s i c  A l k a l o i d s of the uleine-type while  ( 1 ) , h o w e v e r , h a v e no  members o f t h e a p p a r i c i n e  f a m i l y have a one-carbon b r i d g e .  On t h e o t h e r  i s apparent i n the pyridocarbazole  (5)  (2) and hand, a  a l k a l o i d s of which  known e x a m p l e s a r e o l i v a c i n e ( 4 ) , g u a t a m b u i n e  (4)  p o r t i o n of the skeleton  Members o f t h e f o u r m a j o r f a m i l i e s j u s t m e n t i o n e d  (N, ) .  such carbon b r i d g e ,  bridge  the Aspidosperma and t h e Iboga  T h e r e i s , h o w e v e r , a n i n c r e a s i n g number o f i n d o l e a l k a l o i d s  possess a two-carbon bridge  (3)  biosynthesis  vallesamine three-carbon the best  (5) and e l l i p t i c i n e ( 6 ) .  (6)  -68-  The f i r s t of  tryptophan,  was t h a t  give an i n t e r m e d i a t e unit,  illustrated unit  to give  the  equivalent,  ellipticine Since  (8) has been s e t a s i d e moiety  the l a t t e r  (15)?  He s u g g e s t e d  Wenkert's that a  (7) condensed w i t h  (8, see a l s o Figure  (10) w h i c h w i t h  i n F i g u r e J..  seco-loganin Since  t h e y w e r e not."'"  (SPF) u n i t  or i t s b i o l o g i c a l  cyclizations  from tryptophan  glycosyleneanthranilic acid  prephenate-formaldehyde  SPF  t h a t had t o be a s k e d was: a r e t h e s e f a m i l i e s  indole alkaloids also derived  early hypothesis of  question  precursor the seco-  2 i n Part  the a d d i t i o n of a methylamine could  undergo  (6), uleine  the appropriate  (1) and o l i v a c i n e (4) as  the p u b l i c a t i o n of this hypothesis i n favour  of the s t r u c t u r a l l y  behave i n a s i m i l a r  SPF u n i t , t h e W e n k e r t h y p o t h e s i s  remains i n t a c t  fashion  the  similar  ( 9 ) , t h e d e v e l o p m e n t o f w h i c h was d e s c r i b e d  compound c o u l d  I) to  i n Part I .  to that of  and w o r t h y o f f u r t h e r  study. I n 1965, w i t h  the s t r u c t u r e of apparicine  (2) i n hand D j e r a s s i  2 and  Gilbert  cyclize units  to provide  to give  apparicine  two  suggested  that Wenkert's i n t e r m e d i a t e  the one-carbon bridge  the apparicine  skeleton.  (13) c o u l d  between t h e i n d o l e and p i p e r i d i n e The f a c t t h a t u l e i n e  ( 2 ) c o - o c c u r i n t h e same p l a n t s p e c i e s may  suggest 3  s t r u c t u r e s h a v e a common b i o s y n t h e t i c i n t e r m e d i a t e .  known i n b e r b e r i n e precursors  alkaloid  to methylene  biosynthesis  bridges.  also  (1) and that  Also,  these  i t is  t h a t N - m e t h y l groups c a n be  -69-  Figure  1.  The W e n k e r t h y p o t h e s i s f o r the b i o s y n t h e s i s of e l l i p t i c i n e ( 6 ) , u l e i n e ( 1 ) and o l i v a c i n e ( 4 ) .  -70-  OHC  COf  OHC  (13)  C0 " 2  (14)  Figure  2.  The f i r s t  (2)  The D j e r a s s i - G i l b e r t r a t i o n a l f o r t h e biosynthesis of apparicine (2).  b i o s y n t h e t i c i n v e s t i g a t i o n s ^ of these a l k a l o i d s  by W i g f i e l d and N e l s o n i n o u r l a b o r a t o r i e s r e v e a l e d Aspidosperma p y r i c o l l u m p l a n t s , tryptophan aromatic  ring with  incorporated  t r i t i u m a n d i n t h e C-3  into apparicine  (2)  (15) l a b e l l e d position with  w h i c h was a l s o i s o l a t e d  (2)  vallesamine  could ever  i n these  o f i m p o r t a n c e was ( 1 6 ) was v e r y  to the extent  C a r b o n 1 4 , was  (2)  H o w e v e r , no a c t i v i t y  stemmadenine  i n the  (See T a b l e 1 ) .  (15)  Also  that, i n  i n u l e i n e (1)  experiments.  t h e d i s c o v e r y by o u r group  efficiently  o f 0.55%, w h i l e  ( 1 7 ) was  be d e t e c t e d  incorporated  incorporated  t h e more s i m i l a r  into  that apparicine  structure  into apparicine i n only  0.01%.  These r e s u l t s is  involved  atom o f of  the  the  s t r o n g l y suggest  i n the  later  original  pathway.  stages of b i o s y n t h e s i s  tryptamine bridge  A g a i n , no  "OH  that a stemmadenine-like  "  "  d e m o n s t r a t e d f r o m any  of  being  with  lost  the  i n the  incorporation into uleine  (1)  precursor C-2  carbon  later  stages  could  be  (17)  W i t h t h i s new  insight  p o r t i o n of a p p a r i c i n e , mechanistic with Their also  the  proposal^  extrusion  in vitro suffered  the  of  studies  Kim  precursors into  and  two  failed  origin  Erickson  which allowed the  the  tried.  the  modified  the an  nitrogenous earlier  involvement of  carbon bridge to s u p p o r t  of  to form the  this proposal.  f r o m a m a j o r d e f i c i e n c y i n t h a t i t was  tryptophan uleine The  specific  (15)  skeletons. proposal for  uleine  -72-  and  could not Our  and  their  take  early own  i n t o account  results  the  concerning  experience with  formation of a p p a r i c i n e (2). the i n c o r p o r a t i o n of  the P o l o n o v s k i - t y p e  stemmadenine  fragmentation  of  3 N-oxides s t i m u l a t e d a French apparicine  and  to propose a b i o s y n t h e s i s of  (2) w h i c h i n c l u d e d e x t r u s i o n o f  two-carbon b r i d g e . tryptamine  group  T h i s i d e a was  derivatives.  Fuller,  supported  by  F u r t h e r work i n our  using d o u b l y - l a b e l l e d secodine  m a t e r i a l was  t h e C-2  incorporated intact  into  carbon  in vitro  from  studies  l a b o r a t o r i e s , by  (18)  the  revealed that  a p p a r i c i n e ( 2 ) i n A.  on  Beck this  pyricollum  CH 0 2  C0 CH 2  3  (18)  (2)  9 plants with r e t e n t i o n of  the carbomethoxy group.  hypothesis  a concerted  had  fragmentation  in  1973  T h i s new  suggested reaction.  l o s s of  Subsequently,  this  10 '  The  group d u r i n g  experimental  evidence  a satisfying  to date  and  had  the "anomolous" a l k a l o i d s along v e r y i n accord with  their  French  the  a m o d i f i e d h y p o t h e s i s was  i n w h i c h l o s s o f t h e h y d r o x y m e t h y l e n e g r o u p was h y p o t h e s i s was  earlier  one  i n t h a t i t had  allowed similar  published 11 12  suggested. utilized  '  a l l the  f o r the b i o s y n t h e s i s of a l l pathways.  This suggestion i s  co-existence i n several plant species.  Figure  3.  The P o t i e r - J a n o t p o s t u l a t e f o r t h e b i o s y n t h e s i s o f a p p a r i c i n e (2)> u l e i n e (1) and t h e p y r i d o c a r b a z o l e alkaloids.  -74-  An i m p o r t a n t  p o i n t to recognize  i n v o l v e stemmadenine  i s t h a t these b i o s y n t h e t i c pathways  (16), the c r u c i a l precursor  Aspidosperma a l k a l o i d s  discussed  t o t h e I b o g a and  i n Part I . 13  A recent provides  synthesis of e l l i p t i c i n e  an i n t e r e s t i n g  support  (6) by t h e P o t i e r group  to t h e i r hypothesis.  the c y c l i z a t i o n o f s t r u c t u r e (21) v i a a P o l o n o v s k i give  (22) i n h i g h y i e l d .  This reaction i s p a r a l l e l  (19)->(20)->-(6) i n F i g u r e 3. S t r u c t u r e  ( 2 2 ) was  achieved  fragmentation to the  to  conversion  dehydrop.enated  (21)  to  They  (22)  ellipticine (6). To d a t e  have f a i l e d .  a l l attempts I t was  felt,  t o i n c o r p o r a t e any p r e c u r s o r  into  u l e i n e (1)  due t o t h e i m p o r t a n c e o f t h i s p r o b l e m ,  some r e - i n v e s t i g a t i o n s a n d e x t e n s i o n o f t h e e a r l i e r  s t u d i e s was  The f o l l o w i n g s e c t i o n d e s c r i b e s f u r t h e r w o r k d i r e c t e d t o w a r d s y n t h e s i s o f u l e i n e (1) and o l i v a c i n e ( 4 ) .  that  necessary.  the b i o -  -75-  DISCUSSION  The f a c t t h a t (18)  could  a l l be i n c o r p o r a t e d  that uleine since  tryptophan  (1) c o u l d  ( 1 5 ) , stemmadenine into apparicine  (16) and s e c o d i n e  (2) s t r o n g l y  suggests  a l s o a r i s e from a s i m i l a r b i o s y n t h e t i c pathway  they co-occur i n s e v e r a l Aspidosperma p l a n t s p e c i e s .  As p r e -  4 v i o u s l y mentioned, work i n our l a b o r a t o r i e s c a r b o n atom o f t r y p t o p h a n  was r e t a i n e d d u r i n g  the u n u s u a l s i n g l e - c a r b o n  bridge  showed  t h a t t h e C-3  biosynthesis  to form  b e t w e e n t h e i n d o l e n u c l e u s a n d N, i n b  apparicine  ( 2 ) . The f i r s t  to the b i o s y n t h e s i s  of uleine  possess such a bridge derived  the  t h a t h a d t o be a n s w e r e d w i t h  (1) was: i s t h i s m o l e c u l e w h i c h  also derived  from a p r o g e n i t o r  hypothesis^  question  from tryptophan  of tryptophan  (Figure 1)?  t h e C-2 a t o m i s r e t a i n e d  pyridocarbozole  (Figure 3 )  both uleine  while  ?  And  as revealed  (1) as  thirdly, i n Part I ,  o r i g i n of the  d e r i v a t i v e o l i v a c i n e ( 4 ) , a member o f t h e " a n o m o l o u s " a three-carbon  bridge  The P o t i e r - J a n o t h y p o t h e s i s  (1) and o l i v a c i n e (4) a r e d e r i v e d  f r o m w h i c h t h e C-3 c a r b o n a t o m o r i g i n a l l y The f o r m a t i o n  1 1  may b e a s k e d o f t h e b i o g e n e t i c  family of a l k a l o i d s having  n u c l e u s a n d N^.  i s i n v o l v e d , what i s  t o become t h e N - m e t h y l g r o u p o f u l e i n e  what r o l e does the d e h y d r o s e c o d i n e h y p o t h e s i s , play i n the b i o s y n t h e s i s of u l e i n e ( 1 ) . questions  (15) o r i s i t  I s t h e C-3 c a r b o n a t o m e x t r u d e d  suggested by the P o t i e r - J a n o t h y p o t h e s i s  Similar  doesn't  as suggested by the Wenkert  Secondly, i f tryptophan  f a t e o f c a r b o n s - 2 a n d 3?  regard  of the f u l l y  aromatic  between t h e i n d o l e 11  ( F i g u r e 3)  suggests  f r o m t h e same  i n tryptophan  that  intermediate,  has been  extruded.  s t r u c t u r e o f o l i v a c i n e (4) a l s o  -76-  requires  l o s s of  group.  Figure  uleine  ( 1 ) and  labelled  species, and  had A.  C-2  earlier  tryptophan  p y r i c o l l u m and  in  A.  in  Table  study of  A.  (16)  and  This  provided  ( 2 ) was  secodine  Aspidosperma  us w i t h  begun f i r s t ,  (1),  As  (18).  plant apparicine  a unique  these unusual a l k a l o i d s .  The  N-methyl  appropriately  a u s t r a l e , i n which uleine  apparicine  pyricollum plants.  stemmadenine  l a b o r a t o r i e s two  co-occur. of  the  expected p o s i t i o n s of r a d i o - l a b e l i n  (15),  a v a i l a b l e i n our  the b i o s y n t h e s i s the  b e e n i m p l i c a t e d as  o l i v a c i n e (4) as w o u l d a r i s e f r o m t h e  o l i v a c i n e (4)  to study  atom w h i c h had  4 summarizes the  precursors  We  (2)  the  opportunity  mentioned  i n our  laboratories,  b i o s y n t h e t i c evidence obtained  i s summarized  1. 4  E x p e r i m e n t s 1 and  2 i n T a b l e 1,  showed f o r t h e  first  apparicine  (2)  and  some p o i n t  i n the b i o s y n t h e s i s .  was  found  while  t o be  time that  that  the more s t r u c t u r a l l y  only  0.01%.  (18)  precursors  doubt that operative  Although  the  was  C-2 The  i n the  Strychnos a l k a l o i d s u r p r i s i n g l y high  s i m i l a r vallesamine  of  ( 3 ) was  at  stemmadenine  level  of  ,  (16)  0.55%,  incorporated  in  i n c o r p o r a t i o n of v a r i o u s l y l a b e l l e d secodine  generally  low  (experiments•5-8) there  biosynthesis  of  this molecule.  i t w o u l d be  of  great  can  understand  r e s u l t s obtained  possible biosynthesis  of  I n none of  be  little  these  experi-  demonstrated.  i n t e r e s t to expand the  (1)  negative  i n A.  (1) be  i n v e s t i g a t i o n of u l e i n e the  Wigfield  c a r b o n atom w h i c h i s e x t r u d e d  incorporation into uleine  Clearly,  N e l s o n and  proposed stemmadenine J s e c o d i n e b i o - e q u i l i b r i u m i s  i n the  ments c o u l d  the  by  tryptophan i s indeed a precursor  i t i s the  incorporated  reported  pyricollum plants  i n an  t o d a t e , and  then  biosynthetic  attempt  to  evaluate  t h i s molecule i n another Aspidosperma plant  species.  -77-  Figure  4.  Expected olivacine by  the  p o s i t i o n s of (4)  from  Potier-Janot  radio-label in uleine  available precursors hypothesis  (Figure  as 3).  (1)  and  suggested  -78-  Table  1  Incorporation studies  Entry(ref)  into apparicine  Precursor  ( 2 ) i n A. p y r i c o l l u m  Percent incorporation  plants  Ratio Activity Fed( H/^C)  Ratio Activity , , Isolated( H/C) 3  1(4)  [Ar- H, 3- C]tryptophan(15)  1.1  1.5  2(4)  [Ar- H, 2- C]tryptophan(15)  1.0  <0.03  3(5)  [Ar- H]-stemmadenine(16)  3  1 4  3  1 4  3  0.55  3  4(5)  [Ar- H]-vallesamine(3)  0.01  5(9)  [ C0 CH ]-secodine(18)  6(9)  [Ar- H, C0 CH ]secodine(is)  7(9)  [3,14,15,21- H, C0 CH ]secodine(18)  8(10)  [19- H, C0 CH ]secodine(is)  1 4  2  3  3  2  3  3  1 4  2  3  1 4  2  3  .01 0.014, 0.015  1 4  3  0 . 0 0 5 , 0.009  .024  8.7  8.4  4.2  2.2  3.98  2.05  -79-  I n A.  australe plants, s i m i l a r precusors could  .investigate t h e i r possible (1) of  and  the  the  The of  the  of  biosynthesis The  not  this discussion  of u l e i n e  (1)  and  The  t h a t an  deals  with  olivacine  the  current  bear a radio  label.  the  (1)  investigation  i n Figure  of  this  i n mind, the  4  molecule  adapted  c a r b o n atoms w h i c h a r e  With t h i s o b j e c t i v e  uleine  (4).  i n v e s t i g a t i o n i n t o the b i o s y n t h e s i s  i s o l a t i o n o f some o f  of  before.  g r e a t l y e n h a n c e d i f a d e g r a d a t i o n scheme c o u l d be  p e r m i t the  to  biosynthesis  been i n v e s t i g a t e d  h y p o t h e t i c a l l y l a b e l l e d s t r u c t u r e of u l e i n e  illustrates w o u l d be  a l k a l o i d o l i v a c i n e (4).  c l a s s o f a l k a l o i d s has  remaining part  administered  p a r t i c i p a t i o n i n the b i o s y n t h e s i s  pyridocarbazole  latter  be  to  expected  to  following plan  was  implemented. Our (1).  Uleine  The by  first  o b j e c t i v e was  was  first  i s o l a t e d by  s t r u c t u r e proposed at the  latter The  now  to i s o l a t e the  that  indole portion.  to p e r m i t the be  This  i s o l a t i o n of  useful in a biosynthetic To  obtain bulk  subsequent p l a n t o f A.  treated with  a 15%  total  synthesis  the  the  b a s e d on  removal of  scheme, t h e n , c o u l d  ulei  i n 1957 . was  years l a t e r . ^  The  X  of u l e i n e  in  a classical the  basic  replaced  1971.  1 6  Hofmann  nitrogen  p r o b a b l y be  adapted  N - m e t h y l g r o u p i n a manner w h i c h w o u l d  investigation.  /  q u a n t i t i e s of u l e i n e f o r our  feeding  australe plants  B u c h i two  t h i s m o l e c u l e was  d e g r a d a t i o n scheme w h i c h p e r m i t t e d  uleine  h o w e v e r , i n c o r r e c t and  p r o v e n s t r u c t u r e p r o p o s e d by  s t r u c t u r a l proof of  from the  S c h m u t z e t . a l . f r o m A.  t i m e was,  group a l s o succeeded i n the  N-methyl group of  e x p e r i m e n t s we  had  collected in Brazil.  d e g r a d a t i o n work  available ethanolic The  extracts  e x t r a c t s were d r i e d  aqueous a c e t i c a c i d s o l u t i o n , f i l t e r e d  to  and  remove  and  -80-  i n s o l u b l e m a t e r i a l , and t h e r e s u l t i n g petroleum ether This and  acidic  t o remove any r e m a i n i n g  s o l u t i o n was made b a s i c w i t h  extracted with  fraction. of u l e i n e  s o l u t i o n was washed  diethyl  ether  non-alkaloidal material. aqueous sodium  to yield  the a l k a l o i d  Subsequent chromatography on s i l i c a (1), apparicine  with  hydroxide containing  gelyielded quantities  (2) and o l i v a c i n e (4) and s e v e r a l  other  _ 17,18 components. A similar of u l e i n e  isolation  s c h e m e was u s e d  (1) and o l i v a c i n e (4) from A s p i d o s p e r m i a O l i v a c e u m  extracts.  Chromatagraphy o f the a l k a l o i d a l  alumina as adsorbent p r o v i d e d as  pure c r y s t a l l i n e Uleine  ultraviolet spectrum.  and  fraction using  plant  deactivated  an e a s i e r method f o r o b t a i n i n g u l e i n (1)  material.  ( 1 , m.p.75-80°) w a s i d e n t i f i e d  spectrum The l a t t e r  (^  by i t s c h a r a c t e r i s t i c  2 1 0 , 3 0 7 , 3 1 6 ) , m a s s s p e c t r u m ; a n d pmr  m a x  e x h i b i t e d two s i n g l e t  which are c h a r a c t e r i s t i c  absorptions  o f T 4 . 6 4 a n d 4.72  of e x o c y c l i c methylene protons.  a t T 7 , ^ 0 w h i c h was a s s i g n e d  singlet  to obtain further quantities  Also  to the N-methyl group.  p h y s i c a l data were found t o be i d e n t i c a l w i t h p u b l i s h e d  present  The s p e c t r a l data ^ 1  as  18 well  as w i t h  an a u t h e n t i c  Olivacine ultraviolet  the  sample o f u l e i n e ( 1 ) .  ( 4 , m.p.312-315°) was s i m i l a r i l y  spectrum  aromatic  spectrum e x h i b i t e d besides a t x 6 . 8 6 w h i c h was a s s i g n e d at T7.21  identified.  Its  (X 2 2 4 , 3 3 8 , 2 7 7 , 2 8 8 , 3 1 5 , 3 2 9 , 375) showed max  presence o f the f u l l y  pyridocarbazole  the seven aromatic  ring  system.  The pmr  protons,  a  singlet  t o t h e C - l m e t h y l group and a n o t h e r  was a t t r i b u t e d t o t h e C-5 m e t h y l g r o u p .  was a  singlet  The s p e c t r a l a n d  -81-  p h y s i c a l data"'"'' o n  this  compound w e r e i d e n t i c a l w i t h p u b l i s h e d  data  24 and  w i t h an a u t h e n t i c sample o f o l i v a c i n e The  involving  first two  degradation  o f u l e i n e (1)  iodide The as  Uleine  ( 1 ) was  converted  to g i v e a very h i g h y i e l d  mass s p e c t r u m o f  this  of  group to the u l e i n e m o l e c u l e . by  c o n s i d e r e d was  elemental  group from  the r e s t  to i t s quaternary  one  the methiodide  The  the methyl  d e r i v a t i v e m.p. peak  t o t h e a d d i t i o n o f one identity  of  salt with  compound e x h i b i t e d t h e p a r e n t  the base peak which corresponded  confirmed  t h a t we  c l a s s i c a l Hofmann e l i m i n a t i o n r e a c t i o n s w h i c h w o u l d  remove the b a s i c n i t r o g e n w i t h i t s m e t h y l molecule.  (4).  205-206°.  (m/e  281)  methyl  o f s t r u c t u r e (23)  was  analysis.  Uleine methiodide  ( 2 3 ) was  r e f l u x e d with potassium  hydroxide  aqueous e t h a n o l t o g i v e a s i n g l e p r o d u c t w h i c h had an u l t r a v i o l e t s p e c t r u m (A 2 3 5 ( s h ) , 2 4 8 , 2 6 2 , 2 8 7 , 2 9 3 , 307) c h a r a c t e r i s t i c o f max carbazole derivative. molecular  parent  triplet  in uleine (1). w h i c h was a  mass s p e c t r u m o f  p e a k a t m/e  groups of aromatic methyl  The  proton  a t T8.75 Also  assigned  280.  signals,  indicating  The  pmr  this  to a methyl  an  ethyl  group a t t a c h e d w h i c h was  t o an  assigned  system  Hofmann f r a g m e n t a t i o n  (24), w h i c h underwent f a c i l e (25)  i n 82%  yield.  two and  side-chain, similar singlet  at  aromatic  t o two  a  to  that  x7.50 ring  N-methyl  Uleine methiodide  r e a c t i o n t o g i v e an  a r o m a t i z a t i o n to g i v e the  a  a  from  r e v e a l e d a q u a r t e t a t x7.10  These d a t a a r e c o n s i s t e n t w i t h s t r u c t u r e ( 2 4 ) . underwent the expected  showed  spectrum, apart  there appeared a three proton  s i x - p r o t o n s i n g l e t a t T7.61  mediate  product  in  and groups. (23) inter-  carbazole  -82-  The  carbazole derivative  ( 2 5 ) was  to g i v e i t s c o r r e s p o n d i n g methiodide formed  i n quantitative The  salt  ( 2 2 , m.p.  n e x t s t e p i n t h e d e g r a d a t i o n was  n i t r o g e n from  300-302°) w h i c h  the methiodide  (26)  t o p e r f o r m a Hofmann t o remove the  the c a r b a z o l e p a r t of the m o l e c u l e .  to a l i t e r a t u r e r e p o r t ,  failed  s o l u t i o n a t 200°C, was  employed.  basic  Initial  to a c h i e v e the  f r a g m e n t a t i o n when p o t a s s i u m h y d r o x i d e d i s s o l v e d water  iodide  yield.  e l i m i n a t i o n r e a c t i o n on  contrary  reacted with methyl  attempts,  desired  i n ethylene  glycol-  However, p o t a s s i u m _ t - b u t o x i d e  i n f r e s h l y d r i e d ^ - b u t a n o l a c h i e v e d t h e e l i m i n a t i o n a t 80°. (27)  from  292,  306  t h i s r e a c t i o n had  nm)  expected  a m o l e c u l a r i o n as two m e t h y l  groups  spectrum  f o r a c a r b a z o l e system.  t h e p a r e n t p e a k a t m/e to g i v e fragments  revealed a multiplet group  an u l t r a v i o l e t  a t m/e  235 w i t h a f a c i l e 220  and  205.  15-20  i t was  m i l l i g r a m s (mg)  obtainable  from p l a n t  of u l e i n e  in  the l a s t  collector  ( 1 ) , t h i s b e i n g the expected  sequence.  o f d r y n i t r o g e n gas  also  showed of spectrum vinyl  biosynthetic  through  ( 2 9 ) was  with  quantity  essential  T h i s r e q u i r e m e n t was  containing a methanolic solution  tetramethylammonium i o d i d e  I t was  t h e t r i m e t h y l a m i n e ( 2 8 ) w h i c h was  r e a c t i o n of t h i s  a slow passage  in a  to e v a l u a t e the sequence s t a r t i n g  feeding experiments.  a b l e to trap e f f i c i e n t l y  pmr  265,  ring.  necessary  be  loss  The  product  248,  assigned to a  I n o r d e r f o r t h i s d e g r a d a t i o n s c h e m e t o be u s e f u l investigation  228,  I t s mass s p e c t r u m  c e n t e r e d a t x 3 . 5 4 w h i c h was  bonded t o an a r o m a t i c  (A. max  The  to  liberated achieved  the r e a c t i o n m i x t u r e i n t o of methyl  iodide.  by a  Crystalline  o b t a i n e d i n an o v e r a l l y i e l d  of  70-75%.  -83-  (29)  F i g u r e 5.  D e g r a d a t i o n o f u l e i n e b y means o f H o f m a n n elimination reactions.  -84-  I t was a l s o d e s i r a b l e  to evaluate  t h i s d e g r a d a t i o n scheme  radioactive material  before proceeding with  the plant  feeding  i n order to discover  any f u r t h e r d i f f i c u l t i e s which might a r i s e .  using  experiments Uleine  14 ( l ) ( 2 0 m g ) was r e a c t e d ammonium s a l t activity  with  C - m e t h y l i o d i d e and t h e r e s u l t i n g q u a t e r n a r y  ( 1 9 ) was r e c r y s t a l l i z e d  to a constant s p e c i f i c radio-  o f 7.17x10"' d i s i n t e g r a t i o n s p e r m i n u t e  Degradation of this material tetramethyl  ammonium i o d i d e  (dpm) p e r m i l l i m o l e .  by the procedure d e s c r i b e d  above  ( 2 5 ) ( 7 2 % y i e l d ) , w h i c h was  recrystallized  t o a c o n s t a n t r a d i o a c t i v i t y o f 6 . 9 5 x 1 0 ^ dpm p e r m i l l i m o l e . of  9 7 % o f t h e s p e c i f i c a c t i v i t y was c o n s i d e r e d  experimental  plant  plant  i s a slow growing shrub, indigenous  be  species  maintained  We f o u n d  f o r subsequent  experiments using  A t t h e end o f t h e f e e d i n g  time, the roots  a crude e x t r a c t .  (l)(20mg) was a d d e d t o t h i s  extract  of  or tap  topologically to were masurated  i n methanol and the normal a l k a l o i d e x t r a c t i o n procedure, as e a r l i e r , was u s e d t o o b t a i n  described  I n each experiment  This  uleine  to a c t as a c a r r i e r f o r s m a l l  r a d i o a c t i v e u l e i n e w h i c h may h a v e b e e n p r e s e n t i n q u a n t i t i e s  to study d i r e c t l y .  could  experiments.  either fibrous  was a d m i n i s t e r e d  stores  roots or  experiments the remaining plant  the water s o l u b l i z e d precursor  system.  to South America, which  i n a s a t i s f a c t o r y growing s t a t e feeding  The  This  t h a t b y r e m o v i n g some f i b r o u s  f o ruse i n feeding  In our i n i t i a l  the  the l i m i t s of  c h o s e n f o r t h i s s t u d y was A. p y r i c o l l u m .  i n the roots.  tap root  roots  recovery  i n v e s t i g a t i o n o f u l e i n e was now i n i t i a t e d .  first  the  This  error.  A biosynthetic  uleine  within  yielded  technique of r a d i o - d i l u t i o n allows  amounts  insufficient  one t o s t u d y  -85-  the  total  i n c o r p o r a t i o n i n t o a compound  (defined  as r a d i o a c t i v i t y  obtained  from the p l a n t  but not s p e c i f i c  per^ m i l l i m o l e )  i s never  since  incorporation  t h e amount o f  compound  known.  Table 2 summarizes the f e e d i n g  experiments using  A.  pyricollum.  I n e x p e r i m e n t s 1 a n d 2, t r y p t o p h a n l a b e l l e d i n t h e a r o m a t i c r i n g t r i t i u m and w i t h hydroponically time period an  to tap root  the  salt  of secodine  into uleine  this precursor  (1) c o u l d  i s described  that  precursor  incorporation into uleine.  c a r b o n 14 i n  The  of uleine  The r e s o u r c e s  required  term f e e d i n g  using  (15) o r  t o do a c o m p l e t e time.  the wick  was  r e s u l t i n some time  In experiments  4 a n d 5, t r y p t o p h a n a n d a n t h r a n i l i c a c i d w e r e a d m i n i s t e r e d a c e t i c a c i d s o l u t i o n s to growing p l a n t s  to  (1) i n t h e p l a n t  t o a g r o w i n g p l a n t may  s t u d y o f i n c o r p o r a t i o n were n o t a v a i l a b l e a t t h a t  The r e s u l t s o f t h e s e l o n g  Again,  preparation  t o A. p y r i c o l l u m w e r e i n i t i a t e d  the a d m i n i s t r a t i o n of tryptophan  anthranilic acid  plant  to a tap r o o t .  n o t be d e m o n s t r a t e d .  the production  a s l o w p r o c e s s and t h e r e f o r e its  (18) l a b e l l e d w i t h  should  i n Part I.  A second s e r i e s o f feedings the p o s s i b i l i t y  until  incorporation of  experiment i n t h i s  administered  a  I t i s generally  that i f a t o t a l  I n the t h i r d  c a r b o m e t h o x y g r o u p was s i m i l a r i l y  test  i s o l a t e d and r e c r y s t a l l i z e d  of biosynthesis  negative.  the acetate  incorporation of  ( 1 ) was  After  i n t o a t a r g e t m o l e c u l e i s b e l o w 0.001% t h e r e s u l t s  be viewed as b e i n g species,  administered  amount o f r a d i o a c t i v i t y was d e t e c t e d .  i n the f i e l d  a precursor  ( C - 2 ) was  and f i b r o u s r o o t s , r e s p e c t i v e l y .  of 6 days, u l e i n e  insignificant  accepted  carbon-14 i n the s i d e c h a i n  with  i n very  feeding  experiments i n whole  dilute  technique.  plants  -86-  T a b l e 2.  Incorporation  E x p . No.  studies  i n A. p y r i c o l l u m  Precursor  Activity  [Ar- H, 2- C] tryptophan ( 1 5 ) 3  1 4  [Ar- H, 2- C]tryptophan ( 1 5 ) 3  [  1 4  2  3  1/f  3  a  1 4  a  A v a i l a b l e f r o m New  b  A v a i l a b l e from  7  H - 2.34xl0 C - 7.62xl0  8  8  3  9  3.70x10'  1 4  [Ar- H]anthranilic  H - 1.13xl0 C - 1.14xl0  b  [Ar- H, 2- C]tryptophan ( 1 5 )  Fed(dpm)  3  a  1 4  C0 CH ]secodine(18)  plants  3  3  1 4  H C  -  3.89xl0 4.92xl0  England  the o r i g i n a l  <  0.001  <  0.001  <  0.001  <  0.001  <  0.001  8  7  2.22x10' acid'  Percent incorporation i n t o U l e i n e (1)  Nuclear synthesis  b y Sood i n o u r l a b o r a t o r i e s .  -87-  appeared a l s o  t o be  e x p e r i m e n t s i n A.  negative.  p y r i c o l l u m appear i n Table  Another plant l a b o r a t o r i e s was biosynthesize  Additional data concerning  species  w h i c h had  (1) b u t  also  of  plants  used i n t h i s work were e i g h t e e n  twenty  to  the  twenty-five  precursor  p l a n t and  the  tube d u r i n g uleine  was  (1)  centimeters  administered  p l a n t was  a feeding and  extracts with  1 and  i n a c t i v e a l k a l o i d s and  experimental  conditions.  closer  to  the  end  of  t h e s e a l k a l o i d s two  to  As  this the  feeding The  before by  could  No  be  (1)  or  i n c o r p o r a t i o n of  p l a n t was  secodine  (18)  and  whole  in a  target  d i l u t i o n of  test  compounds  the  alkaloidal  isolation. Experiments  tryptophan could  be  o l i v a c i n e (4) u n d e r  hope t h a t a l a t e r  the  the  these experiments.  experiments using  to  The  experiments,  root of  the  the  these  stage precursor,  secodine same a s  into uleine  being  elaborated  doubly-labelled  procedure f o r preparing  to i t s a d m i n i s t r a t i o n I.  s e r i e s of  tap  subsequent  r e s u l t s of  the  plant  pyridocarbazole  to grow h y d r o p o n i c a l l y  into uleine In  this  t h e b i o s y n t h e t i c p a t h w a y , m i g h t be  were performed.  prior Part  In  2 show t h a t n e i t h e r a n t h r a n i l i c a c i d n o r significantly  did  t w e n t y - f o u r months o l d  o l i v a c i n e (4) w e r e o b t a i n e d  incorporated  (18)  to  directly  6 days.  Table 3 summarizes the  only  our  particular interest.  tall.  then allowed time of  Not  i t produced s e v e r a l  a l k a l o i d s o f w h i c h o l i v a c i n e (4) was  feeding  4.  become a v a i l a b l e i n  Aspidosperma a u s t r a l e .  uleine  the  into  secodine  (18)  immediately  that described  (1)  or o l i v a c i n e  in (4)  demonstrated.  Another b i o g e n i s i s of It  i s w e l l known t h a t  is  the  the  N-methyl group of  uleine  ( 1 ) was  considered.  the main s o u r c e of m e t h y l groups i n b i o l o g i c a l  amino a c i d m e t h i o n i n e .  Methionine  (30)  i n i t s a c t i v e f o r m as  systems S-  -88-  adenosylmethionine biological  (31)  equivalent  could  transfer  i t s S-methyl group to the  of des-N-methyluleine  (32).  The l a t t e r  (32)  COf H-C-NH  2  CH -S-CH CH CH-CO-,H 3  2  2  CH  3  CH  2  (30;  H  OH  (31)  could be  possibly  be  i n biological  on  the b i o s y n t h e t i c  equilibrium  (32)  .  with  route  to u l e i n e  the methylated  (1) o r i t c o u l d  derivative.  (1)  -89-  T h e r e f o r e i t was with methionine As  shown by  (30)  e x p e r i m e n t s 5 and  l a b e l i n the  the  a c t i v i t y obtained The s e c t i o n do  as  the  provide  Wenkert h y p o t h e s i s biosynthesis  (1) was  found.  the  or  1  the  To  " C ^ - p o o l " i n the i n the  a high  and  98%  incorporation  the  specific  radio-  (29).  investigations described  any  direct information  the  Potier-Janot  u l e i n e and  relating  hypothesis  of  The  l a t t e r would a l s o r e q u i r e has an way  already  the  A.  on  of  been demonstrated  (as d e s c r i b e d  i n Part  to  the  t h e A s p i d o s p e r m a and  described  australe  pathway.  to the The  to  the  administration  first  I) that  secodine i s  be  the  to  to  4).  by  the  necessarily  three  in interpreting problems  plants.  o v e r c o m e i s one plant.  by  (Figure  elaborated  exercised  i n v e s t i g a t i o n s due  within  The  since i t  equilibrium  could  C a u t i o n m u s t be  p r o b l e m t h a t m u s t be  s i t e of b i o s y n t h e s i s  (18)  t h e s e compounds a r e  of m a t e r i a l  the  Iboga type of a l k a l o i d s  these precursors  negative results i n biosynthetic inherent  is  hypothesis.  secodine  s t e m m a d e n i n e (16)  mean t h a t  the  incorporation  i n t o more c o m p l e x a l k a l o i d s u n d e r  does not  true biosynthetic  Z  (18)  f a c t t h a t none of  p y r i c o l l u m o r A.  conditions  Potier-Janot  incorporation  dehydrosecodine  However, the  to the  the  active precursor of  (15)  this  the  concerning  1 1  o l i v a c i n e type of a l k a l o i d s .  tryptophan  in  to e i t h e r  o f a n t h r a n i l i c a c i d i s e s s e n t i a l t o the Wenkert h y p o t h e s i s as incorporation  position  degradation  tetramethylammonium i o d i d e s a l t  the b i o s y n t h e t i c  of  precise  described of  plant  S-methyl group.  a s c e r t a i n the  previously  i s o l a t i o n o f 97%  r e s u l t s of not  c a r b o n 14  6 i n T a b l e 3,  isolated uleine  scheme p e r m i t t e d  to l a b e l the  labelled with  methionine into uleine of  decided  We  of  know t h a t  transportation the p l a n t  has  -90-  Table  3.  Incorporation  studies  Activity  Precursor  Exp.No.  [Ar- H]anthranilic  i n A.  australe  Fed(dpm)  2.22x10'  , acid'  [Ar- H, 2 - C ] tryptophan(15)' 3  H 14 C -  2.48xl0 7.44xlO  [19- H, C0 CH ]secodine(18)  14  1.19x10' 7.50xl0  3  3  1 4  2  3  D  3  1 4  2  3  c  [  a. b.  c.  CH ]methionine (30)  c  <  0.001  <  0.001  -<  0.001  <  0.001  <  0.001  <  0.001  <  0.001  <  0.001  3  <  0.001  0.168  CH ]methionine(30 )  <  0.001  0.147  6  8  a  2.22x10  1 4  3  a  England  [ " ^ C ^ C H ^ ] - s e c o d i n e was  7  c  2.22x10  1 4  A v a i l a b l e f r o m New  our  Percent Incorporation olivacine(4) uleine(1)  1 4  [Ar- H, C0 CH ]secodine(18)  [  plants  8  Nuclear  a v a i l a b l e from  the o r i g i n a l  synthesis  b y Sood i n  laboratories  Synthesis  o of [Ar-H]-secodine  and  3  [19- H]-secodine  i s described  i n Part  I  -91-  absorbed a l a r g e percent activity  the  the b i o s y n t h e s i s  precursor The  of  the  i s transported  more s e r i o u s w i t h  attached  takes  question  third  the  time of  again  only during  simply  slowly  stored  i t is likely  biodegradation  before  d o n ' t know w h e r e  This  nor  know i f  required. of  the c e l l  walls  problem i s b e l i e v e d  secodine  (18).  the  plant  to  I t may  be  be  are  unattached precursors  may  be  systems.  in a biosynthetic investigation i s  the  the  experiment.  very  s l o w l y and  I t i s possible that stored  c e r t a i n growth periods during that  other  times.  the p r e c u r s o r  a significant  or  i n the  t h e y may life  of  If biosynthesis t h a t was  amount o f  be  bioplant  i s occurring  fed would  i t could  these  be the  target  undergo  used to  synthesize  a l k a l o i d s under i n v e s t i g a t i o n . In order  t o o v e r c o m e some o f  these problems inherent  a d m i n i s t r a t i o n of precursors  to p l a n t systems a great  and  One  w o r k w o u l d be  with in  do we  in  the p l a n t i s a c t i v e l y b i o s y n t h e s i z i n g the  compounds a r e b i o s y n t h e s i z e d synthesized  be  residual radio-  the b i o s y n t h e t i c b u i l d i n g b l o c k s  problem a r i s i n g  of whether or not  compounds d u r i n g  the  place.  l a r g e r m o l e c u l e s s u c h as  f o r e i g n m a t e r i a l by  The  we  of p e r m e a b i l i t y  t o c a r r i e r p r o t e i n m o l e c u l e s and  t r e a t e d as  But  t o w h e r e i t may  that under normal c o n d i t i o n s  very  because the  a l k a l o i d s occurs  s e c o n d p r o b l e m i s one  behind which biosynthesis  and  precursor  i n the v e s s e l i s always measured.  the p l a n t our  of  cell-free  e x t r a c t s of  t h i s area w i l l The  required.  fact  be  the  initiated  that uleine  l a b e l l e d methionine  (30)  way  i n our  d e a l of  the  material  these problems i s to work  enzyme s y s t e m s o f  ( 1 ) was  casts  around  in  the  plant species.  l a b o r a t o r i e s i n the near  future.  e f f i c i e n t l y methylated with  g r a v e d o u b t on  the P o t i e r - J a n o t  Work  carbon  14  hypothesis  -92-  which required (15).  t h e N - m e t h y l g r o u p t o come f r o m C-2 c a r b o n o f t r y p t o p h a n  I t i s therefore  logical  i s an immediate precursor nitrogen bility  to speculate  to uleine  atom o c c u r s as t h e  last  (1) and t h a t m e t h y l a t i o n  step  i s supported by the d i s c o v e r y  m e t h y l d e r i v a t i v e (32) w i t h  uleine  t h a t d e s - N - m e t h y l u l e i n e /32)  i n the biosynthesis.  o f the co-e::istence  of the basic This  possi-  o f t h e des-N-  ( 1 ) i n A. d a s y c a r p o n A. D C .  7  and  21 A. v a r g a s i i A.DC. methyl  transferase  of c e l l - f r e e Part  The s t u d y o f t h i s m e t h y l  enzyme may b e a g o o d way t o i n i t i a t e  enzyme e x t r a c t s o f t h e A s p i d o s p e r m a I I Io f t h i s  thesis describes  an i n v e s t i g a t i o n  species.  a s y n t h e t i c study designed to  make a v a i l a b l e , f o r b i o s y n t h e t i c e v a l u a t i o n , derivatives  t r a n s f e r r e a c t i o n by t h e  appropriately  i n t h e preakuammicine- and s t e m m a d e n i n e - s e r i e s .  labelled  -93-  EXPERIMENTAL  For is  a d e s c r i p t i o n of general  r e f e r r e d to the e x p e r i m e n t a l R a d i o a c t i v i t y was  experimental  s e c t i o n of P a r t  Counter.  procedure were p r e v i o u s l y d e s c r i b e d  the  reader  I.  measured u s i n g a Nuclear-Chicago  a Mark I I M o d e l 6860 L i q u i d S c i n t i l a t i o n counting  information  and  The are  Mark I  or  d e t a i l s of  pertainant  the  to  this  section. The  A s p i d o s p e r m a p y r i c o l l u m p l a n t s and  t h e A.  australe plants  were grown f r o m seeds i n a Dept. of H o r t i c u l t u r e g r e e n h o u s e , U n i v e r s i t y of B r i t i s h  Columbia, under the  Dept. of Chemistry. vicinity  The  o f R i o de  Isolation The  and  was  later  de  Janeiro,  u s e d by  (15.8  g).  washed w i t h  the in  the  bath  and  a cooled  t o 10-12, the r e s u l t i n g  was  15%  d r i e d to g i v e a dark  (2x50 m l ) .  The  suspension  The  acidic  aqueous sodium h y d r o x i d e  suspension  Ondetti  and  then  filtered  e x t r a c t was  carefully  while  After raising  extracted with d i e t h y l ether  e x t r a c t i o n w i t h methylene c h l o r i d e (3x100 m l ) .  vicinity  cooled  ml)  s o l u t i o n (50%)  t e m p e r a t u r e down. was  (100 was  22  coloured  then suspended i n a  aqueous a c e t i c a c i d  f o r 1 hour.  to keep the  by  a u s t r a l e p l a n t s c o l l e c t e d i n the  T h i s m a t e r i a l was  petroleum ether  added p e r i o d i c a l l y  f o l l o w e d by  Gilbert  that published 21 i n our l a b o r a t o r i e s .  i n 1967,  (15 m l )  ice-water  made b a s i c w i t h was  B.  of  adapted from  e x t r a c t o f A.  Janeiro, Brazil  s o l u t i o n of methanol  and  Dr.  Salsbury  Brazil.  others  methanolic  i n an  P.  of A l k a l o i d s from Aspidosperma a u s t r a l e ( M u l l . Argov.) E x t r a c t  amorphous s o l i d  stirred  s e e d s w e r e c o l l e c t e d by  f o l l o w i n g p r o c e d u r e was  The of Rio  The  s u p e r v i s i o n of Dr.  These  the  ice pH  (3x100  organic  ml)  -94-  e x t r a c t s were combined, washed once w i t h w a t e r , and d r i e d o v e r sodium sulphate. (1.6  g).  (alumina,  Removal  anhydrous  o f the s o l v e n t i n vacuo y i e l d e d a brown  An e x a m i n a t i o n o f t h i s m a t e r i a l by  solid  t h i n - l a y e r chromatography  15% methanol i n e t h y l a c e t a t e ) r e v e a l e d  a l a r g e r number o f  components w h i c h s e p a r a t e d i n t o a " p o l a r " group and a " n o n - p o l a r " group.  Subsequent  column chromatography  ethylacetate/methanol  on a l u m i n a ( a c t . I l l ) u s i n g  an  g r a d i e n t y i e l d e d two f r a c t i o n s o f a l k a l o i d s w h i c h  were s e p a r a t e l y p a r t i t i o n e d . The  p o l a r f r a c t i o n s were r e - c h r o m a t o g r a p h e d on a l u m i n a  using a toluene/chloroform  gradient  This m a t e r i a l  recrystallized  ( 1 2 0 mg)  c u b i c c r y s t a l s , m.p. compound was  was  to o b t a i n almost pure u l e i n e ( 1 ) .  75-80° ( l i t . m.p.  76-80°)  The  to give c o l o u r l e s s  identity  chloroform/ethylacetate  (1)  18  gel (act.III).  Elution with  i d e n t i t y of this  o f i t s p h y s i c a l and 20  compound was  312-315° ( l i t . m.p.  data.  19  '  an a u t h e n t i c 22  a  continued (190  from methanol  u n a m b i g u o u s l y e s t a b l i s h e d by  spectroscopic data with  and w i t h p u b l i s h e d  and  g r a d i e n t y i e l d e d guatambuine  y e l l o w n e e d l e - l i k e c r y s t a l s , m.p.  as  s e v e r a l drops of  f r a c t i o n s c o n t a i n i n g o l i v a c i n e (4) w e r e r e c r y s t a l l i z e d  o l i v a c i n e . (4)  this  as w e l l  g r a d i e n t y i e l d e d o l i v a c i n e ( 4 ) ( 2 1 0 mg)  e l u t i o n u s i n g an e t h y l a c e t a t e / m e t h a n o l  give l i g h t  of  chromatographic separation  c o n t a i n i n g 5% m e t h a n o l a n d  p y r i d i n e and r e - c h r o m a t o g r a p h e d on s i l i c a  The  14  sample o f u l e i n e •  l e s s p o l a r f r a c t i o n s from the f i r s t  were d i s s o l v e d i n c h l o r o f o r m  The  from methanol  u n a m b i g u o u s l y e s t a b l i s h e d by c o m p a r i s o n o f i t s p h y s i c a l a n d  s p e c t r o s c o p i c d a t a w i t h an a u t h e n t i c 14 22 with published data. ' The  (act.Ill)  sample  mg) to  314-316). comparison of  -95-  The  guatambuine obtained  was  i d e n t i f i e d by  comparison w i t h  a  22 sample  obtained  by  partial  The  I s o l a t i o n of A l k a l o i d s from Aspidosperma Olivaceum E x t r a c t  vicinity solid  of Rio  (26  g).  procedure,  de  this  Olivaceum plants c o l l e c t e d i n  J a n e i r o , B r a z i l , was described  e x t r a c t was  methanol s o l u t i o n , s o l u t i o n was  e x t r a c t o f A.  I n t h e way  filtered  taken and  made b a s i c w i t h  d r i e d to g i v e an  i n the p r e v i o u s  up  i n 15%  g).  (150  g,  a  T h i s m a t e r i a l was act.III).  The  toluene/chloroform  could  r e a d i l y be  obtained  appropriate  methyl iodide Removal of  a slightly  extract  c h r o m a t o g r a p h e d on  alumina  from e l u t i o n  with  t o g i v e u l e i n e ( 1 ) ( 2 4 0 mg)  identified  (1 ml) the  coloured  was  by  comparison w i t h  and  d i s s o l v e d i n a s o l u t i o n of methanol l e t stand  s o l v e n t from the  from ethanol  283(sh),  290,  a t 5°  which  previously  gave c o l o r l e s s ,  302,  305(sh),  i n vacuo  R e c r y s t a l l i z a t i o n of  short needle-like crystals 205-206° ( l i t .  312(sh);  (3  ml)  overnight.  reaction mixture  crystalline material.  w h i c h w e r e d r i e d i n h i g h v a c u u m , m.p. A  alkaloidal  (23)  U l e i n e ( 1 ) (75 mg)  product  and  s o l u t i o n and  f r a c t i o n s obtained  g r a d i e n t were pooled  The  material.  Uleine methiodide  and  extraction  aqueous sodium h y d r o x i d e  crystallized  amorphous  washed w i t h p e t r o l e u m e t h e r .  subsequently  the  aqueous a c e t i c a c i d -  e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e to y i e l d e d an (3.0  in  21  l a b o r a t o r i e s by  methanolic  Grierson.  used i n another study  our  The  Dr.  s y n t h e s i s and  m.p.  yielded this (95  mg)  204-206°) ;  mass s p e c t r u m : 2 8 1 ( 1 0 0 ) ,  1 4  266(10),  -96-  222(22), 207(29), Anal.  205(29), 204(23), 194(9), 128(77),  Calculated f o r  C  H 1  9  2  5  N  2  I  :  c  >  5 5  -  8 8  5  127(44).  H,6.17; N , 6 . 8 6 .  Found:  C . 5 6 . 1 3 ; H,6.00; N , 6 . 6 9 .  l-Methyl-2-(3-dimethylaminoethyl)-3-ethylcarbazole Uleine methiodide ethanol  (3 m l ) , w a t e r  ( 1 9 ) ( 6 2 m g ) was d i s s o l v e d i n a s o l u t i o n o f  (2 m l ) a n d p o t a s s i u m h y d r o t i d e  m i x t u r e w a s r e f l u x e d f o r 1.5 h o u r s . i n vacuo  (25)  ( 7 5 mg) a n d t h e  Most o f t h e s o l v e n t was t h e n removed  a n d t h e r e s i d u e was p a r t i t i o n e d b e t w e e n w a t e r  and d i e t h y l e t h e r .  The  a q u e o u s l a y e r was e x t r a c t s e v e r a l t i m e s w i t h d i e t h y l e t h e r ( 5 0 m l )  and  t h e combined o r g a n i c  over anhydrous sodium  e x t r a c t s were washed once w i t h w a t e r  and d r i e d  sulphate.  Removal o f t h e s o l v e n t y i e l d e d a l i g h t brown g l a s s y m a t e r i a l c o u l d be c r y s t a l l i z e d a crystalline 262,  from d i e t h y l e t h e r / p e t r o l e u m  p r o d u c t , m.p.  114-116° ( . l i t . " m.p.  2 8 7 ( s h ) , 2 9 3 , 3 0 7 ; pmr s i g n a l s :  xl.9-2.3  ether  which  s o l u t i o n to give  14 115-116°) ; X ' . 2 3 0 , 2 4 8 , IT13X  (3H, m u l t i p l e t , a r - H ) ,  2.6-2.8 ( 2 H , m u l t i p l e t , a r - H ) , 7.1 ( 4 H , m u l t i p l e t , a r - C H - C H - N ) , 7.20 2  ( 2 H , q u a r t e t , a r - C H _ - C H ) , 7.50(3H, s i n g l e t , 2  N(CH ) ), 3  8.75(3H, t r i p l e t ,  2  222(29),  3  220(29),  Anal.  206(38),  ar-CH ),  2  3  204(38), 186(66),  Calculated for C  i g  H  2 4  N : 2  7.61(6K,  3  - C H C H ) ; mass s p e c t r u m :  2  280(86),  singlet,235(22),  171(100).  C , 8 1 . 3 8 ; H,8.63; N , 9 . 9 9 .  Found:  C . 8 1 . 1 0 ; H,8.59; N , 1 0 . 1 0 . T h i s m a t e r i a l was f o u n d of quatambuine o b t a i n e d  t o be i d e n t i c a l w i t h a d e g r a d a t i o n p r o d u c t  by Dr. G r i e r s o n  i n ourlaboratories.  l-Methyl-2-(B-trimethylaminoethyl)-3-ethylcarbazole The  carbazole  21  i o d i d e (26)  a m i n e ( 2 1 ) ( 4 0 mg) • was d i s s o l v e d i n a s o l u t i o n o f  -97-  methanol  (3 ml)  and m e t h y l i o d i d e ( 1 ml)  Removal o f the s o l v e n t material ethanol  ( 6 1 mg). and  i n vacuo  and  l e t stand  gave a s l i g h t l y  a t 5°  coloured  A p o r t i o n o f t h i s m a t e r i a l was  overnight.  crystalline  recrystallized  d r i e d i n h i g h v a c u u m o v e r n i g h t , m.p.  from  300-302° ( l i t .  m.p.  14 300-302°). Anal.  Calculated for C^H^N  I : C , 5 6 . 8 7 ; H,6.44; N , 6 . 6 3 .  F o u n d : C , 5 6 . 6 0 ; H,6.37; N,6.59.  H o f m a n n F r a g m e n t a t i o n o f Compound The _t-butanol The  carbazole (10 m l )  m e t h i o d i d e ( 2 2 ) ( 6 0 m g ) was  i n which  reaction vessel, a  potassium metal  two-knecked  a s o u r c e o f d r y n i t r o g e n gas and a collector  (26)  round  added t o f r e s h l y  ( 5 3 mg)  bottom  had  been d i s s o l v e d .  f l a s k , was  equipped  with  a g l a s s tube o u t l e t w h i c h  extended  into  charged w i t h methyl iodide-methanol s o l u t i o n .  s l o w s t r e a m o f n i t r o g e n g a s was  distilled  passed  through  I n t h i s way  a  the r e a c t i o n m i x t u r e to  carry with  i t any v o l a t i l e m a t e r i a l , formed  during  collecting  flask.  s t i r r e d i n an o i lb a t h s e t a t  The  80°  f o r 2 hours.  the  r e a c t i o n m i x t u r e was The  The  r e a c t i o n m i x t u r e was s y s t e m was allowed  r e a c t i o n m i x t u r e was  p a r t i t i o n e d between water several was  continuously  washed once w i t h w a t e r  and  Removal o f the s o l v e n t i n vacuo A t h i n - l a y e r chromatography  and  The  as  temperature.  e v a p o r a t e d jLn v a c u o  t i m e s w i t h d i e t h y l e t h e r (60 m l )  the  p u r g e d w i t h n i t r o g e n gas  t o c o o l t o room  and d i e t h y l e t h e r .  the r e a c t i o n , i n t o  and  the r e s i d u e  a q u e o u s l a y e r was  the combined o r g a n i c  d r i e d over anhydrous  sodium  was  washed  extract  sulphate.  y i e l d e d a brown amorphous m a t e r i a l  ( s i l i c a g e l , CHCl^) e x a m i n a t i o n o f t h i s  (12  mg).  product  -98-  revealed  one m a j o r component p l u s  Attempted p u r i f i c a t i o n  s e v e r a l more p o l a r m i n o r  o f the major product  components.  by p r e p a r a t i v e t h i n - l a y e r  chromatography y i e l d e d a s l i g h t l y y e l l o w g l a s s w h i c h - c r y s t a l l i z e d only poorly  from d i e t h y l e t h e r / p e t r o l e u m  Lack o f m a t e r i a l prevented A  228(sh),  multiplets, doublets, 3,  to purify  2  a r - C H ) , 8.75(3H, t r i p l e t , 3  this  J=24 a n d  J = 7 , a r - C H _ C H ) , 7.50 ( 3 H , s i n g l e t , 2  3  235(100),  3  220(90), 205(56), 204(39), 117(19), 107(20), 103(21), 102(26);  high  r e s o l u t i o n mass measurement: C a l c u l a t e d f o r C ^ H ^ N : 2 3 5 . 1 3 6 0 .  Found:  The  solvent  precipitate  i n t h e t r a p was e v a p o r a t e d  ( 1 9 mg) w h i c h w a s r e c r y s t a l l i z e d  n e e d l e - l i k e c r y s t a l s which were crushed m a t e r i a l was r e a d i l y  identified  the f o l l o w i n g data:  from methanol t o y i e l d  arid d r i e d i n h i g h  vacuum.  as tetramethylammonium i o d i d e  m.p. 230° ( l i t .  142(100), 127(94), 59(33), Anal.  long This  (31) based  m.p. 230°)  ; mass  spectrum:  57(76).  C a l c u l a t e d f o r C ^ H ^ N I : C , 2 3 . 8 9 ; H,6.02; N , 6 . 9 6 .  Found:  C , 2 3 . 9 6 ; H,6.10; N,7.07.  The  Isolation of Uleine  pyricollum The the  (1) from Feeding Experiments i n Aspidosperma  plants f o l l o w i n g p r o c e d u r e was u s e d t o o b t a i n u l e i n e  f e e d i n g e x p e r i m e n t s u s i n g A. p y r i c o l l u m p l a n t s .  o r w h o l e p l a n t s w e r e c u t up w i t h  235.1371.  i n vacuo t o give a c o l o u r l e s s  23 on  1 4  product,  of doublets,  J = 7 , - C E - C K _ ) ; mass s p e c t r u m : 2  67-68°).  4.4(1H, d o u b l e t o f  c i s ) , 6.7(1H, d o u b l e t  t r a n s ) , 7.20(2H, q u a r t e t ,  m.p.  3 0 6 ; pmr s i g n a l s : r l . 9 - 2 . 3 ( 3 H ,  2.6-2.8(2H, m u l t i p l e t s , a r - H ) ,  J=17 a n d 3,-CH=CH_ 2  solution (lit.  f u r t h e r attempts  235, 248, 265, 292(sh),  ar-H),  -CH=CH  ether  s c i s s o r s before  (1) from a l l  The r o o t  being  segments  masurated  ina  -99-  Waring blender filtered  w i t h methanol.  and r e - m a s c e r a t e d  became c o l o u r l e s s . i n vacuo. (10%)  This  (50%).  being  i n methanol  p u l p - l i k e m a t e r i a l was  (4 t i m e s ) u n t i l  The c o m b i n e d m e t h a n o l i c  the f i l t r a t e  e x t r a c t s were then  evaporated  r e s i d u e was t h e n s u s p e n d e d i n c o o l a q u e o u s a c e t i c a c i d  and f i l t e r e d .  before  The r e s u l t i n g  The f i l t r a t e  basified with  cooled  The e x t r a c t i o n o f t h i s  the crude a l k a l o i d a l  was w a s h e d t w i c e w i t h d i e t h y l e t h e r  extract.  aqueous sodium h y d r o x i d e  suspension  solution  w i t h methylene c h l o r i d e y i e l d e d  A t t h i s p o i n t , u l e i n e ( 2 0 mg) was a d d e d t o t h e  e x t r a c t and r e - i s o l a t e d by column c h r o m a t o g r a p h y on a l u m i n a ( u s u a l l y 25  g, a c t . I l l )  was a c h i e v e d  using a toluene/chloroform  by r e c r y s t a l l i z a t i o n  z a t i o n s were done b e f o r e in preparation  gradient.  from methanol.  Further  Usually  described  f o r counting  the r a d i o a c t i v i t y  previously i n Part I.  t h a t used by o t h e r s  The  studying  in i t .  In a l l the feeding  (0.5  m l , 1:4) w i t h  the root bark,  (1 ml) and counted  Precursors  This  were  similar  t o A. p y r i c o l l u m p l a n t s  e x p e r i m e n t s u s i n g r o o t segments  ( n o s . 1-4) t h e  was d i s s o l v e d i n a s o l u t i o n o f e t h a n o l  r o o t s which were c o n t a i n e d by  Samples between  T h i s p r o c e d u r e was  1 o r 2 drops of IN a c e t i c a c i d .  aided by s o n i c a t i o n .  overnight  t h e same p l a n t s y s t e m i n o u r l a b o r a t o r i e s .  Administration of Radioactive  radioactive precursor  recrystalli-  A t l e a s t "two r e c r y s t a l l i z a t i o n s  done between measurements o f r a d i o a c t i v i t y . to  two  t h e m a t e r i a l was d r i e d i n h i g h v a c u u m  0.150 a n d 0.250 mg i n w e i g h t w e r e d i s s o l v e d i n b e n z e n e as  purification  D i s s o l u t i o n could  s o l u t i o n was a p p l i e d d i r e c t l y i n test  tubes.  and w a t e r be  to freshly cut  After absorption  of the s o l u t i o n  the c o n t a i n e r which had the r a d i o a c t i v e s o l u t i o n i n i t  -100-  was  washed w i t h  and  applied  applied them  to  the  aliquots  the  roots.  root  test  This  throughout  the tube  end  of  the  the  i n which  w a s h s o l u t i o n was applied  the  of  the  solvent  washings with incubation  residual radioactivity  fed,  the  the  The  Table  the  similar data  had  of to  this  mixture  distilled  period  described  water  i n order  to  was  for radioactivity  found  that  was  5 and used.  described  pertaining  to  fed  t o be  (nos.  feeding  container,  the  were keep  pipette  been were a l l washed w i t h  radioactivity  experiments  w i c k method  a manner  and  two  root  time  then measured  the  In  to  feeding  t o be  2  ml)  Further  had  in  (0.5  moist. At  the  to  two  each  to  the  methanol.  i n case  plant.  In  and  a  correction  a l l cases  insignificant. 6)  i n which whole  This  plants  t e c h n i q u e was  i n Part  I.  feeding  experiment  are  were  employed  given  in  Tables  4.  4.  Exp.No.  A d d i t i o n a l data  Plant  System  associated  Plant weight(g.)  with  Table  Feeding Technique  2  Weight of precursor(mg)  Feeding Time(days)  37.0  hydroponic  7.01  5  14.1  hydroponic  9.56  6  27.2  hydroponic  2.46  5  plant  197  wick  8.94  90  plant  183  wick  0.03  91  1  tap  2  fibrous  3  tap  4  whole  5  whole  root root  root  -101-  The  I s o l a t i o n of Uleine  i n Aspidosperma The  australe  the feeding  to  that described  to o b t a i n u l e i n e  experiments using  A.  were added to the crude a l k a l o i d a l on a l u m i n a  The A d m i n i s t r a t i o n  of Radioactive  used.  w e r e u p - r o o t e d and E a c h p l a n t was dissolved  ( 2 0 mg)  e x t r a c t , and  Precursors  i n a t e s t tube.  The  and w a t e r  After absorption  the  similar  plants.  plants  the f o l l o w i n g plants  from the r o o t  ( 0 . 5 m l , 1:4)  s o l u t i o n was  w h i c h had  australe  applied  with  directly  o f the s o l u t i o n by  r o o t s were kept m o i s t d u r i n g  p e r i o d i c a d d i t i o n of a l i t t l e water  grow u n d e r f l u o r e s c e n t  lights.  The  system. was 1 or to the the  t h e r a d i o a c t i v e s o l u t i o n i n i t was  a l i q u o t s o f the s o l v e n t m i x t u r e which were a l s o The  mg)  radioactive precursor  root  system of each p l a n t .  t o A.  each experiment the  This  to the r o o t s .  from  t h e n i s o l a t e d by  this plant  2 drops of IN a c e t i c a c i d .  two  pyricollum  c a r e f u l l y brushed  i n a s o l u t i o n of ethanol  washed w i t h  A.  very  a n d o l i v a c i n e (20  experiments using  t h e s o i l was  r o o t b a r k , the c o n t a i n e r  o l i v a c i n e (4)  a u s t r a l e p l a n t s was  Immediately before  placed  Experiments  (act.III).  In a l l the feeding p r o c e d u r e was  (1) and  above f o r e x p e r i m e n t s u s i n g  s t u d y , however, b o t h u l e i n e  chromatography  (4) from F e e d i n g  plants  procedure used  all  In this  (1) and O l i v a c i n e  period  by  as t h e p l a n t s were a l l o w e d  to  data which  e x p e r i m e n t s i s summarized i n T a b l e s 3 and  5.  the f e e d i n g  applied  i s pertinent  to these  -102-  T a b l e 5.  Exp.No.  The  Additional data associated  Number o f plants  with  T o t a l wt of plants(g)  Table  3.  Weight o f precursor(mg)  Feeding Time(days)  1  3  21.0  2  4  28.1  8.2  3  3  24.5  1.21  6  4  3  22.7  1.80  6  5  4  24.0  0.28  5  6  4  27.9  1.12  5  Degradation of Uleine The  described  from methanol Experiment  (1) i s o l a t e d  tetramethylammonium  to constant  6 '  5  ( 1 ) I s o l a t e d f r o m F e e d i n g E x p e r i m e n t s 5 a n d 6.  radioactive uleine to y i e l d  .03  was d e g r a d e d  iodide  as p r e v i o u s l y  ( 2 9 ) w h i c h was  recrystallized  radioactivity.  5:  14 mg o f u l e i n e  ( 4 . 9 6 x 10  dpm/mmole) y i e l d e d 5 mg o f  tetramethyl-  6 ammonium i o d i d e Experiment  ( 4 . 8 0 x 10  dpm/mmole).  6:  12 mg o f u l e i n ammonium i o d i d e  ( 4 . 4 7 x 10  ( 4 . 3 8 x 10  dpm/mmole) y i e l d e d 4.1 mg o f  dpm/mmole).  tetramethyl-  -103-  BIBLIOGRAPHY 1.  E. W e n k e r t , J . Am. Chem. S o c , 84, 98 (1962).  2.  J.  J o u l e , H. M o n t e i r o , L . J . D u r k a m , B. G i l b e r t J.  3.  Chem. 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W e a s t  Ed.,  -105-  PART I I I Studies Related  to the Synthesis  P r e a k u a m m i c i n e - and Type  Alkaloids  Stemmadenine  -106-  INTRODUCTION  A great  d e a l o f e l e g a n t w o r k has  the e a r l y stages  of indole a l k a l o i d  d e a l of work has  yet  t o be  later  stages  outline  In Parts by  the developments i n t h i s  described in  supported  but  stages  area.  of indole a l k a l o i d  i n which the l a t t e r  Aspidosperma skeletons speculation.  i s not  1  proposed  (1) u n d e r w e n t c y c l i z a t i o n  c l e a r and  t h a t the  ( r o u t e A)  s t r u c t u r e (2) w h i c h c o u l d  ring-opened would give and  l o s s of  Strychnos the  skeleton  little  the  (3).  rearrangement of oxindole  S c o t t and  has  to the  t o be  and  been the  that the i s the  be  first  Corynanthe, Iboga  for this  conversion. geissoschizine  two-step r e d u c t i o n of  the  penta-  to g i v e the  the  latter  (4), while ring-closure  i n akuammicine ( r o u t e B)  (5).  An  i n v o l v e d a known  d e r i v a t i v e o f g e i s s o s c h i z i n e (1) this  and  s u b j e c t o f much  rearrangement to g i v e  stemmadenine  C y c l i z a t i o n of  to  important  Strychnos,  Corynanthe a l k a l o i d  Quereshi  the h y d r o x y i n d o l i n e (6).  conjecture  hypothesis  A  thesis,  described  e a s i l y undergo bond f i s s i o n  known a l k a l o i d  by  this  w o r k was  biosynthesized  the aldehyde group would r e s u l t  alternative proposal  form the  I I of  biosynthesis.  i s converted  F i g u r e 1 s u m m a r i z e s two  W e n k e r t , i n 1965,  the  In t h i s P a r t , there w i l l  t h e m a j o r f a m i l i e s o f a l k a l o i d s t o be t h e way  great  understand  t h e a t t e m p t e d s y n t h e s i s o f compounds b e l i e v e d  the l a t e r  cyclic  I and  experimental  T h e r e i s much e v i d e n c e t o s u p p o r t of  to f u l l y  However, a  of  f a m i l i e s of indole a l k a l o i d s i n  of biosynthesis.  a number o f h y p o t h e s i s  biosynthesis.  done i n o r d e r  r e l a t i o n s h i p s between the v a r i o u s the  l e d t o a good u n d e r s t a n d i n g  s t r u c t u r e would give  the  to  -107-  i n d o l e n i n e preakuammicine aldehyde aldehyde group of the  the  has  not  preakuammicine  (8)  of  (8), a  the compound  a n a t u r a l product i n Catharanthus  c h a r a c t e r i z a t i o n data  on  this  compound  These i n v e s t i g a t o r s a l s o c l a i m to i n t o b o t h stemmadenine (4) and  biosynthesis  of other  major f a m i l i e s of  Strychnos family i s believed  (4).  Figure  experimental  have  akuammicine  evidence  (described  in detail  i s t h a t stemmadenine  with dehydrosecodine forms of  t o o c c u r by  e l a b o r a t i o n of  the A s p i d o s p e r m a ( 1 1 ) , the 4  o f a l k a l o i d s as w e l l as Stemmadenine f a m i l i e s of  i n Parts  (4)  (4)  i n t o the  I and  stemmadenine supported  II).  is in biological  (9) v i a i s o - s t e m m a d e n i n e  s e c o d i n e have b e e n shown i n o u r  smaller  i n d o l e a l k a l o i d s from  2 summarizes the b i o s y n t h e t i c schemes w h i c h a r e  current hypothesis  into  reduction  2  The the  However, f u l l  b e e n made a v a i l a b l e .  converted  A simple  l a t t e r would give preakuammicine  S c o t t group c l a i m to have found as  roseus seedlings.  (5).  (7).  (10).  3  t o be  The  equalibrium  Variously  laboratories  by  labelled  incorporated  I b o g a ^ ( 1 2 ) and- t h e H u n t a r i a ^ ( 1 3 ) f a m i l i e s anomolous s t r u c t u r e a p p a r i c i n e  i s also believed  to be  the  precursor  i n d o l e a l k a l o i d s w h i c h h a v e more d i v e r s e  to  (14).  7  '  8  three  carbon  9 skeletons.  These a r e  compounds o f  the  ellipticine,  o l i v a c i n e and  uleine  t o members o f  the  types. The  biomimetic  conversion  of  I b o g a and  A s p i d o s p e r m a f a m i l i e s has  S c o t t and  Quereshi  1 0  and  catharanthine  support  the  precursor  of  proposal the  claimed (12)  stemmadenine been the  subject  to have r e a r r a n g e d i n hot  (4)  this molecule  acetic acid.  that a dehydrosecodine-like  I b o g a and  o f much  Aspidosperma f a m i l i e s .  controversy. to  tabersonine  These r e s u l t s were s a i d m o l e c u l e m u s t be  a  to  pivotal  However, a c a r e f u l  -108-  F i g u r e 1.  The p r o p o s e d b i o s y n t h e s i s o f t h e S t r y c h n o s a l k a l o i d s from the Corynanthe f a m i l y .  Figure  2.  The i m p l i c a t i o n o f t h e d e h y d r o s e c o d i n e t s t e m m a d e n i n e b i o - e q u i l b r i u m i n i n d o l e a l k a l o i d b i o s y n t h e s i s as s u p p o r t e d by e x p e r i m e n t .  -110-  i n v e s t i g a t i o n o f t h i s work by t h e Smith  group f a i l e d  to confirm  these  results. Two o t h e r b i o m i m e t i c  conversions  o f stemmadenine  (4) have  been  12 claimed. resulted  Scott  i n a 20% y i e l d  been a c h i e v e d . mentation  reported  of the n a t u r a l product  This conversion  o f the N-oxide.  This r e a c t i o n i s very stemmadenine  that a r i n g - c o n t r a c t i o n r e a c t i o n which vallesamine  involved a modified Polonovski  However, no e x p e r i m e n t a l  similar  (15) had frag-  details are available.  to the p r e v i o u s l y proposed c o n v e r s i o n o f  (4) t o a l k a l o i d s p o s s e s s i n g  skeletons. A second r e a c t i o n o f stemmadenine  t h e more d i v e r s e  carbon  9  ( 4 ) w h i c h may h a v e  biological  13 s i g n f i c a n c e was r e p o r t e d b y S a n d o v a l achieved  et a l .  They, c l a i m e d  an o x i d a t i v e r i n g - c l o s u r e which y i e l d e d the product  to have condylocarpine  ( 1 6 ) , a member o f t h e A s p i d o s p e r m a f a m i l y w i t h w h i c h i t c o - o c c u r s plant  i n some  species.  COoCHo OH  C0 CH 2  OH  3  (17)  (15) F i g u r e 3.  Some b i o m i m e t i c  conversions  o f stemmadenine (4)  -111-  S t emma-d en i n e Steimnadenia  ( 4 ) was  Donnell-Smithii.  first 14  isolated,  i n 1958,  from  the f r u i t  Subsequent work r e v e a l e d the  of  novel  13 ring-opened C-16  was  Strychnos carbon  not determined.  stemmadenine  The  skeleton  , but  only reported attempt  (4) d e r i v a t i v e s i s t h a t by  m o d e l compound  the s t e r e o c h e m i s t r y a t  S n i e k u s ^ who  Corynanthe type  i n 1968.  be  and  (4) a p p e a r s  to c o n v e r t the  i n our  available  (20).  the l a t t e r A l l efforts  19,20-dihydro-  to achieve t h i s  cyclization  e v a l u a t e the c e n t r a l  to p l a y i n i n d o l e a l k a l o i d  r o l e which  I t would  (19)  stemma-  also  preakuammicine  the c h e m i s t r y of these  t o e v a l u a t e t h e i r r o l e i n the b i o s y n t h e s i s o f i n d o l e  (18)  failed.  biosynthesis various radio-  to have a v a i l a b l e d e r i v a t i v e s of the  (8) i n o r d e r t o i n v e s t i g a t e  the  to a c h i e v e the  f o r m s o f t h i s m o l e c u l e m u s t be made a v a i l a b l e .  ( 7 ) and also  p l a n was  i n order to f u l l y  of great i n t e r e s t  type  The  then to c y c l i z e  Clearly,  labelled  1 6  initiated  (18) v i a i t s c h l o r o i n d o l e n i n e t o s t r u c t u r e s o f  preakuammicine s e r i e s  denine  the  (17).  alkaloid  (19) and  synthesizing achieved  1  S y n t h e t i c w o r k i n t h e S t r y c h n o s a r e a was laboratories  at  compounds  alkaloids.  (20)  -112-  DISCUSSION  In order  to evaluate  effectively  the r o l e s played  by t h e  preakuammicine and t h e stemmadenine s e r i e s i n t h e b i o s y n t h e s i s o f indole alkaloids a synthesis This of  synthesis  o f t h e s e compounds h a d t o be d e v e l o p e d .  n o t o n l y h a d t o make a v a i l a b l e r e a s o n a b l e  quantities  t h e s e m a t e r i a l s b u t a l s o make p o s s i b l e t h e i n t r o d u c t i o n o f a r a d i o -  label  i n c e r t a i n positions i n the structures.  Also,  since  the stereo-  chemistry  a t p o s i t i o n C-16 i n t h e s e compounds was u n k n o w n i t w o u l d b e  desirable  to synthesize  both epimers o f each  compound.  T h e r e was a v a i l a b l e i n t h e l i t e r a t u r e d e s c r i p t i o n s o f a deal and  o f work b r i d g i n g  the degradation  the c o n s t i t u e n t s of calabash  preparation  from s e v e r a l species  curare.  biological  interest  activity  1 7  possessing  of these preparations  the basic  (29)  1 7  Curare i s an e x t r a c t  as arrow head p o i s o n s .  i n them l e d t o t h e i d e n t i f i c a t i o n  structures  of strychnine  o f t h e p l a n t genus S t r y c h n o s w h i c h was  used by the South American n a t i v e s high  chemistry  great  the chemical  and b o t a n i c a l  o f a l a r g e number o f  Strychnos skeleton  (21a)  Due t o t h e  (21a).  molecular  The s y s t e m o f  -113-  n o m e n c l a t u r e u s e d h e r e i s t h a t p r o p o s e d by the n u m b e r i n g s y s t e m u s e d by Since, readily  a v a i l a b l e from l i t e r a t u r e of  f r o m one  these  (21)  Bernauer et. a l .  compounds p o s s e s s i n g  possibility  as  of  Janot  we  C-7  and  C-15  of  s k e l e t o n were  considered  the  stemmadenine d e r i v a t i v e s  advantages i n using a curan d e r i v a t i v e  s t a r t i n g m a t e r i a l were t h r e e - f o l d .  a t p o s i t i o n s C-3,  1  the b a s i c Strychnos  preparations  The  L e Men' '" f o l l o w i n g  19  s y n t h e s i z i n g p r e a k u a m m i c i n e and structures.  and  Firstly,  the  t h e p i p e r d i n e r i n g was  known  fixed.  S e c o n d l y , known c h e m i s t r y  i n t h i s area  convert  the r i n g - c l o s e d Strychnos  s k e l e t o n to the ring-opened  system.  T h i r d l y , the a v a i l a b i l i t y  provided  an  in  opportunity  to i n t r o d u c e  t h e m o l e c u l e n e a r t h e end The  first  was  to introduce  C-16. (8)  As and  of  ( 4 ) was  to s y n t h e s i z e b o t h epimers to  biosynthetic the  stereochemistry  controlled of  the  face an  and  systetti o f  during  the  stereochemistry  unknown.  the  the  the  curan  type  Strychnos  stemmadenine s e r i e s  a t C-16  at  i n preakuammicine  I t w o u l d , t h e r e f o r e , be  experiments  stereochemistry  i n t r o d u c t i o n of  c u r a n s y s t e m (21b) c r e a t e d An  stemmadenine  advantageous  (a) a s c e r t a i n t h e n a t u r a l c o n f i g u r a t i o n , and  p r e d i c t e d because the  the molecule.  to  oxygenated carbon f u n c t i o n a l i t y  both epimers i n p l a n t feeding  importance of  d e r i v a t i v e s of  used  s y n t h e t i c sequence.  s t e r e o s e l e c t i v e l y an  stemmadenine  p o s s i b l y be  o f a scheme t o c o n v e r t  the  and  a radio-label i n various positions  ten-membered r i n g  mentioned e a r l i e r ,  (b) a d m i n i s t e r  the  requirement  s y s t e m to the d e s i r e d  of v a r i o u s  could  stereochemistry  examination  a t C-16.  the  C^  steric  of molecular  i n c o m i n g e l e c t r o p h i l e o r n u c l e o p h i l e a t C-16  the  I t was  that  u n i t a t C-16  stereochemistry  considerable  to e v a l u a t e  of  felt could  be  the p i p e r d i n e  part  crowding over the  m o d e l s l e f t no must r e a c t  doubt  from the  £-  that a-face.  -114-  Therefore,  by s e l e c t i n g  conversion  t o b o t h C-16 e p i m e r s  stemmadenine  the a p p r o p r i a t e  ( 4 ) systems  oxidation  level  o f the preakuammicine  would  be  of the C ^ - u n i t ,  ( 8 ) and  possible.  (21b)  The was  curan d e r i v a t i v e  that  became a v a i l a b l e  18-hydroxy-2g,16a-cur-19-ene-17-ol,also  to our  laboratories  known a s c a r a c u r i n e V I I  20 and W i e l a n d - G u m l i c h importance  alkaloids  after  from  strychnine  This  alkaloids  chemical routes.  to as Wieland-Gumlich  was made r e a d i l y  ( 2 9 ) , by a m o d i f i c a t i o n  compound, w h i c h  (29) thus l i n k i n g  v i a established  referred  simplicity,  (22).  i n the chemistry of the curan  synthesized of  aldehyde  aldehyde  available  the l a b o r a t o r i e s  o f Schmid  to  the a v a i l a b i l i t y  of strychnine  had a l s o  t h e s e two l a r g e This (WGA)  compound  of  pivotal  been families (22here-  i n the i n t e r e s t  of  from  of the o r i g i n a l  in  was  and K a r r e r  the degradation of s t r y c h n i n e 21 22 d e g r a d a t i v e work ' , developed 23 and p u b l i s h e d  ( 2 9 ) , and hence  WGA  i n 1969.  Due  (22) t h e l a t t e r  -115-  (22)  compound was  an  preakuammicine In  one  attractive ( 8  ),  area  intermediate  stemmadenine of  these  f o r the  ( 4 ) and  synthesis  of  their derivatives.  i n v e s t i g a t i o n s i n our l a b o r a t o r i e s ,  24 (Figure  4)  Fuller  was  16-epi-stemmadenine  successful i n achieving  (28)  from  the  intermediate  the  synthesis  (23) , the  of  latter  25 being  a v a i l a b l e f r o m WGA  step  in this  C^-unit this  was  s e q u e n c e was introduced  therefore  a  C^-unit  a  study  of  will The  form  part  (22)  excess  sodium e t h o x i d e  this  be  isolated using  of  an  aqueous  crucial  i n which  the  the  mechanism  of  involves anion  formation  at  similar  to a c h i e v e  5.  used  manner, t o  the  other  to c o n v e r t  Strychnine nitrite  introduce  epimer.  Such  salt/  chloride followed aldehyde  solution  of  (32)  (31)  (29)  was  strychnine  treated  (29)  with  to g i v e i s o - n i t r o s o s t r y c h n i n e  i t s hydrochloride  1-cyanoformyl-Wieland-Gumlich  (26)  Although  t h a t was 23  isoamyl  thionyl  to  The  following discussion.  scheme  as  (25)  possible, in a  the  and  procedure.  C-16.  i t apparently  i s shown i n F i g u r e  product  Treatment  conversion  position  of  degradation  w h i c h was  Smith's  oxidation level  t o WGA  of  at  i t should  higher  by  the  r e a c t i o n i s unknown,  C-16,  (30)  (22)  as  with  by the  A Beckman  rearrangement  hydrolysis yielded hydrochloride  steam p e r m i t t e d  salt.  hydrolysis  -116-  1.  HBr/HoAc  2.  Zn/HoAc  CO2CH3  CO2CH3  (23)  (24)  NaH HC0 CH 2  NaH CH 0 2  C r CO2CH3  (26)  CO2CH3  HCl  (25)  1. P b ( O A c ) ^ 2.  NaBH,  <; C02CH  3  OH (27)  Figure  4.  (28)  Synthesis  o f 16-epi-stemmadenine ( 2 8 ) .  3  -117-  (22)  Figure  5.  The d e g r a d a t i o n o f s t r y c h n i n e G u m l i c h a l d e h y d e (22)  (29)  to W i e l a n d -  -118of  the  c y a n o f o r m y l g r o u p t o g i v e WGA  35-40%.  T h i s m a t e r i a l had  identical with The  those  p h y s i c a l and  o f an  next step  i n an  C-18  overall yield  spectroscopic  a u t h e n t i c sample  i n the  the  (22)  s y n t h e s i s of  20  o f WGA  properties (22).  the d e s i r e d curan  oxygen f u n c t i o n a l i t y  of  was  the removal of  Two  methods f o r t h i s o b j e c t i v e were a l r e a d y a v a i l a b l e i n the  system  in structure  (22). literature.  22 The  first  method  hemi-acetal converted  involved  (22)  to  the  sodium borohydride  to Wieland-Gumlich d i o l  the  corresponding  allylic  (33).  The  bromide  (34)  s o l u t i o n of hydrogen bromide i n g l a c i a l ment o f of  the l a t t e r  r e d u c t i o n of l a t t e r was using  acetic acid.  i n a c e t i c a c i d gave the a c e t a t e  3%  the then  saturated  Zinc dust  (35).  treat-  Basic hydrolysis  t h i s m a t e r i a l r e s u l t e d i n the a l c o h o l d e r i v a t i v e , 2g,16a-cur-19-ene-  l7-ol  (36).  The  identity  of  its  spectroscopic data with  was  t h e UV  spectrum  (A  t h i s product  was  t h a t w h i c h was  confirmed  published.  by Of  comparison particular  243,297) s h o w i n g the p r e s e n c e o f an  of note  indoline  max c h r o m o p h o r e and as b e i n g  296 This  t h e mass s p e c t r u m w h i c h c o n f i r m e d  mass  technique  f o r d e o x y g e n a t i n g C-18 C-18  c o m p o u n d s c o u l d be  f o l l o w e d by duction of  synthesized  the anhydrous a l l y l i c treatment  with  t r i t i u m a t C-18  was  considered  a means  o f s t r u c t u r e (36)  the e t h y l i d i n e s i d e - c h a i n of preakuammicine  dissolving  weight  units.  of i n t r o d u c i n g a r a d i o - l a b e l i n t o  these  the m o l e c u l a r  from  bromide  (8) and  and  as  hence  stemmadenine  this starting material. (34)  in tritiated  z i n c dust  i n an  c o u l d be  accomplished.  into  (4) i f By  acetic acid  i n e r t atmosphere, the  intro-  -119-  A s e c o n d m e t h o d " " f o r d e o x y g e n a t i o n o f C-18 hydrogenolysis  of Wieland-Gumlich d i o l  i n aqueous a c i d i c medium and  afforded  The  catalytic  l a t t e r was  dissolved  treated with palladium-on-charcoal  under an a t m o s p h e r e o f h y d r o g e n g a s . C-18  (33).  involved  I n t h i s way  the a l c o h o l (36) i n h i g h y i e l d  and  catalyst  selective reduction purity.  (35)  Figure  6.  Two m e t h o d s f o r d e o x y g e n a t i o n o f C-18 aldehyde (22).  of Wieland-Gumlich  at  -120-  A t h i r d method w h i c h a c h i e v e d  t h i s deoxygenation  d u r i n g the c u r r e n t i n v e s t i g a t i o n i n a r e l a t e d described shortly.  WGA  1 , 2 - d i m e t h o x y e t h a n e and Excess  l i t h i u m m e t a l was  d i c a t i n g an e x c e s s c o l o u r f o r 15-30 monitoring) and  ( 2 2 ) was  added.  A b l u e c o l o u r was  quenched w i t h w a t e r .  and  which t h i s hydrogenolysis occurred b a s i c media promotes r i n g - o p e n i n g  blue  chromatographic  Subsequent work-up  i n good y i e l d  (75-82%).  provides a better overall  p r e v i o u s l y described procedures.  -78°.  soon achieved i n -  (as r e q u i r e d from t h i n - l a y e r  t h e r e a c t i o n was  be employing  After maintaining this  c h r o m a t o g r a p h y y i e l d e d t h e a l c o h o l (36)  t h e two  ammonia,  anhydrous ^ - b u t a n o l as c o - s o l v e n t s , a t  T h i s m e t h o d i s more c o n v e n i e n t than  discovered  study which w i l l  dissolved in liquid  of d i s s o l v e d metal.  minutes  was  yield  A p o s s i b l e mechanism  i s shown i n F i g u r e 7. of the hemiacetal  Probably  by the  t o g i v e s t r u c t u r e (22  (36) Figure 7.  A possible mechanism for the two-step reduction of WielandGumlich aldehyde (22) to 2g,16a-cur-19-ene-17-ol (36).  -121-  which then accepts two electrons followed by protonatlon to give the alcohol (36). To further explore the u t i l i t y  of this reduction, Wieland-  Gumlich d i o l (33) was subjected to the same experimental as just described.  conditions  I t was found that t h i s material could also be  e f f i c i e n t l y hydrogenolysed to give the alcohol (36). The reaction just described was discovered during the course of an i n v e s t i g a t i o n directed at developing a method f o r an e f f i c i e n t conversion of WGA  (22) to 18-deoxy Wieland-Gumlich aldehyde (37).  The  l a t t e r transformation was desirable i n view of the anticipated d i f f i c u l t i e s associated with the oxidation of the C-17 hydroxyl i n the curenol (36) to  (22)  (37)  27 the aldehyde (37).  F r i t z , Besh and Wieland  f i r s t reported that the  Woodward modification of the Oppenauer oxidation of (36), using potassium _t-butoxide and benzophenone, yielded only the unsaturated fluorocurarine (39).  aldehyde, nor-  Their r a t i o n a l f o r this unusual conversion was that  the saturated aldehyde (37) underwent f a c i l e a i r oxidation to give the  -122-  unsaturated conversion  system.  A later  study  b y t h e same g r o u p  c o u l d be a c h i e v e d  using  l i t h i u m _t-butoxide  s o l v e n t b u t no i n c r e a s e s  i n yield  of either  z a  showed in  that  this  nitrobenzene  ( 3 7 ) o r ( 3 9 ) was  reported.  28 I n 1964, B o e k e l h e i d e (39)  et. a l .  reported  of n o r - f l u o r o c u r a r i n e  from Oppenauer o x i d a t i o n o f (36) u s i n g p o t a s s i u m _t-butoxide  5 moles o f benzophenone as the h y d r i d e of  a 40% y i e l d  the s a t u r a t e d aldehyde  unsaturation  occurred  agent, rather  (39)  Only s m a l l q u a n t i t i e s  They p r o p o s e d  that  a,g-  t r a n s f e r to the o x i d i z i n g  than by a i r o x i d a t i o n , to g i v e  (36)  8.  (37) were o b t a i n e d .  by a second h y d r i d e  (38), which tautomerized  Figure  acceptor.  with  the i n d o l e n i n e  intermediate  to n o r - f l u o r o c u r a r i n e (39).  (37)  (38)  The o x i d a t i o n o f a l c o h o l ( 3 6 ) t o n o r - f l u o r o c u r a r i n e ( 3 9 ) .  -123-  Our aldehyde  first  attempts  to obtain  (37) i n v o l v e d v a r y i n g  Oppenauer o x i d a t i o n o f a l c o h o l a t room t e m p e r a t u r e r a t h e r  quantities of the saturated  the Boekelheide conditions (36).  f o r the  When t h e r e a c t i o n was p e r f o r m e d  than r e f l u x i n g benzene the 'conversion  required  4 hours to complete rather  quantity  o f o x i d i z i n g agent from 5 moles t o 2 moles prevented the  r e a c t i o n from going In  0.5 h o u r s .  Also, decreasing  t o c o m p l e t i o n a n d s t a r t i n g m a t e r i a l was  the  recovered.  t h e s e s t u d i e s , t h e p r o g r e s s o f t h e o x i d a t i o n was m o n i t o r e d b y  layer  chromatography  mixture at regular curarine  ( t i c ) a n d b y w o r k i n g up a l i q u o t s o f t h e r e a c t i o n  time i n t e r v a l s .  The i d e n t i f i c a t i o n o f n o r - f l u o r o -  ( 3 9 ) was made o n t h e b a s i s  of i t s ultraviolet  w h i c h showed a c h a r a c t e r i s t i c s t r o n g infrared cm . (39)  absorption  ( i r ) spectrum which e x h i b i t e d a carbonyl  was a s i n g l e t a b s o r p t i o n  proton of the unsaturated  (uv) spectrum  a t 3 6 3 nm a n d i t s absorption  Of s i g n i f i c a n c e i n t h e p r o t o n m a g n e t i c r e s o n a n c e  ±  thin-  a t 1640  (pmr) s p e c t r u m o f  a t x l . 0 w h i c h was a t t r i b u t e d t o t h e a l d e h y d i c  carbonyl  group.  O t h e r means o f o x i d i z i n g a l c o h o l  (36) t o the s a t u r a t e d  aldehyde  30 (37) was  were s t u d i e d . unsuccessful.  with while  alcohol  The R a t c l i f f e m o d i f i c a t i o n  of the C o l l i n ' s oxidation  S i x moles o f t h e p y r i d i n e - c h r o m a t e complex were  ( 3 6 ) f o r 1 h o u r , 6 h o u r s a n d 24 h o u r s a t r o o m  a s e c o n d s i m i l a r s t u d y was made a t 30-33°.  desired  r e a c t i o n p r o d u c t be d e t e c t e d  amounts o f t h e s t a r t i n g m a t e r i a l mixture.  by t i c .  could  organic  material.  temperature  A t no t i m e c o u l d t h e  I n a l l cases only  small  be i s o l a t e d f r o m t h e r e a c t i o n  The l a t t e r was w o r k e d up u s i n g  attempt t o recover  stirred  a variety of conditions  Besides using  aqueous  i n an  sodium  -124-  bicarbonate hydroxide  solution,  the r e a c t i o n m i x t u r e  s o l u t i o n i n one  attempt and  f o l l o w e d by n e u t r a l i z a t i o n i n another work-up p r o c e d u r e  was  washed w i t h IN sodium  IN h y d r o c h l o r i c a c i d attempt.  The  solution  iriations in  d i d not y i e l d b e t t e r r e c o v e r y of o r g a n i c m a t e r i a l .  A p o s s i b l e e x p l a n a t i o n f o r these r e s u l t s i s t h a t a chromium VI i o n could coordinate with group i n compound not be  the i n d o l i n e n i t r o g e n atom and  (36)  the  hydroxyl  to form a v e r y s t a b l e complex ( 4 0 ) , which would  i s o l a t e d under the c o n d i t i o n s used.  (40)  Related  to t h i s  problem  (41)  25 is  the o b s e r v a t i o n by  Smith  Wieland-Gumlich aldehyde at  t h a t the s i m i l a r s t r u c t u r e N - a c e t y l -  (41) c o u l d not be o x i d i z e d w i t h chromic  acid  100°. A third  study  i n v o l v e d attempts to o x i d i z e a l c o h o l (36)  using  30 as a r e a g e n t . When d i c y c l o h e x y l c a r b o d i m i d e 31 trioxide-pyridine were used as d e h y d r a t i n g agents no r e a c t i o n  anhydrous d i m e t h y l s u l p h o x i d e or sulphur products  c o u l d be d e t e c t e d and  a nearly quantitative y i e l d  of  starting  32 m a t e r i a l was  recovered.  However, when phosphorus p e n t o x i d e  a v e r y complex p r o d u c t m i x t u r e by  t i c and  pmr  was  obtained.  Examination  r e v e a l e d t h a t none of. the d e s i r e d aldehyde  was  of t h i s (37) was  used mixture present.  -125-  At (36)  a later  was i n i t i a t e d  of both  time,  (39).  solution with  of the o x i d a t i o n of a l c o h o l  and r e s u l t e d i n t h e a c h i e v e m e n f o f good y i e l d s  the saturated  curarine  a f o u r t h study  aldehyde  I t was f o u n d  (37) and t h e u n s a t u r a t e d  t h a t when a l c o h o l  nor-fluoro-  (36), i n a benzene  2 m o l e s o f b e n z o p h e n o n e a t 6 5 ° , was t r e a t e d w i t h  potassium hydride  the aldehyde  (37) c o u l d  T h i s m a t e r i a l was i d e n t i c a l w i t h amounts f r o m t h e p r e s e n t  study  be o b t a i n e d  i n 70-78%  that previously obtained  excess yield.  i n small  o f t h e Oppenauer o x i d a t i o n and a  similar  24 study  i n our l a b o r a t o r i e s .  Of p a r t i c u l a r  importance i n the c h a r a c t e r i -  z a t i o n o f t h i s p r o d u c t was i t s i n d o l i n e uv s p e c t r u m  (A  242, 2 9 8 ) , a max  carbonyl proton  absorption  absorbing  a t 1 7 1 5 cm  as a s i n g l e t  When a l c o h o l  1  i n i t s i r s p e c t r u m and an  a t x O . 2 3 i n i t s pmr  ( 3 6 ) was f i r s t  spectrum.  treated with  potassium hydride i n  a b e n z e n e s o l u t i o n a t 65°-70° a n d t h e n t r e a t e d w i t h b e n z e n e o v e r a 15 m i n u t e (39)  was o b t a i n e d .  c o u l d be d e t e c t e d  time p e r i o d  be  time both aldehydes  i fprecautions  procedures.  to exclude  aldehyde (37)  reaction mixture,  before  (37) and (39) c o u l d Good y i e l d s c o u l d  m o i s t u r e were e x e r c i s e d .  p r o c e d u r e s i t was f o u n d u s e f u l t o d i s t i l the  of nor-fluorocurarine  by t i c .  i n good y i e l d s by r e l i a b l e  obtained  a good y i e l d  4 moles o f n i t r o -  Only t r a c e q u a n t i t i e s of the saturated  Thus, f o r t h e f i r s t obtained  aldehydic  or nitrobenzene  only  I n both  about 10% o f the benzene  potassium hydride  be  from  were  added, t o e n s u r e t h a t any t r a c e s o f m o i s t u r e were removed. In order  to obtain anion  Wieland-Gumlich-aldehyde n i t r o g e n atom s i n c e  formation  a t p o s i t i o n C-16 o f 1 8 - d e o x y -  ( 3 7 ) i t w o u l d be n e c e s s a r y  t h e i n d o l i n e NH i s a l s o w e a k l y  to protect acidic.  the i n d o l i n e  -126-  Two  r o u t e s were developed  aldehyde  to o b t a i n N - f u n c t i o n a l i z e d d e r i v a t i v e s of  (37). I n the  first  route,  the c u r e n o l  ( 3 6 ) was  treated with  acetic-  29 formic anhydride i n v a c u o and  288,  289)  c h r o m o p h o r e was 1660  cm  a t 1720  cm  product  present.  assigned  two  The  pmr  product  compound.  t o an  0-formyl  spectrum of  to the p r o t o n ( 4 2 ) was  (^  had  i r spectrum which corresponded  The  l a t t e r was  o f an  a uv  at  absorption  group p r o v i d e d showed  further the  absorption of t l . 5 1  0-formyl  was  a singlet absorption  at  group.  spectrum s i m i l a r  to the  carbonyl absorption  (1660  to the p r e s e n c e o f an amide  a s i n g l e t absorption at tl.24  to  yield  starting cm  X  )  in  group.  i n the  spectrum. A m o l e c u l a r i o n w h i c h a p p e a r e d a t m/e 324 i n t h e mass of t h i s p r o d u c t a l l o w e d the a s s i g n m e n t of s t r u c t u r e ( 4 3 ) . The  m a x  indoline  r e f l u x e d i n m e t h a n o l f o r 30 m i n u t e s  the  by  N-  spectrum  t h i s product  A singlet  e x h i b i t e d o n l y one  confirmed  uv  a second c a r b o n y l  o f an N - f o r m y l g r o u p and  This product  (42) b u t  The  the  i r spectrum a carbonyl absorption  aldehydic protons.  attributed The  In the  amide g r o u p and  to the p r o t o n  x l . 9 9 was  obtained.  reagent  of  i n d i c a t e d t h a t an N - a c y l a t e d  attributed  evidence.  presence of  material  this  w h i c h was  X  supporting  a new  of  the  chromatography a high y i e l d  d e r i v a t i v e ( 4 2 ) was  i n d i c a t i n g an  X  f o r 30 m i n u t e s . A f t e r r e m o v a l o f  p u r i f i c a t i o n by  formyl-0-formyl 247,  a t 0°  pmr spectrum  s y n t h e s i s of N-formyl-18-deoxy-Wieland-Gumlich aldehyde  (44)  30 was  achieved  ment o f  the  by  Collins  o x i d a t i o n of N-formyl a l c o h o l (43).  l a t t e r w i t h 6 moles of chromic a n h y d r i d e - p y r i d i n e  45 m i n u t e s f o l l o w e d b y  The  treat-  complex f o r  a work-up w i t h s a t u r a t e d sodium b i c a r b o n a t e  solution  -127-  (44)  Figure  9.  The s y n t h e s i s o f aldehyde (44).  afforded  the d e s i r e d  (44) was  made o n  spectrum  showed  a  carbonyl  second  aldehyde  the b a s i s  i n 40%  of  i n addition absorption  N-formyl-18-deoxy-Wieland-Gumlich  yield.  The  the c h a r a c t e r i s t i c  to  the N - f o r m y l  a t 1720  cm  X  assignment  of  spectral data.  carbonyl  while close  absorption  structure The i r a t 1660 >  examination of  the  -128-  pmr s p e c t r u m and  of t h i s product  a singlet  a b s o r p t i o n a t xO.38 w i t h a t o t a l  aldehydic proton. provided of  presence  alter  10).  which  exhibited  multiplicity  indicated the  aldehyde  by t i c .  (44).  There  No a t t e m p t s  ( 4 4 ) was o b t a i n e d f r o m  a characteristic  The l o s s o f t h e a l d e h y d e  or ion (i) resulted  This  were  sequence.  for structure  i o n ( i ) a t m/e  f o r one  s i n c e t h e s t e r e o c h e m i s t r y a t C-16 w o u l d  the following reaction  would y i e l d ion  of the N-formyl  isomers  Further evidence  group.  r e g i o n of the spectrum  C-16 e p i m e r s  these  integral  a b s o r p t i o n a t x l . 1 4 and x l . 3 8  h o w e v e r , t o be o n l y one p r o d u c t  made t o s e p a r a t e  spectrum  singlet  f o r a proton o f an N-formyl  i n the low f i e l d  of both  appeared,  not  Similarly,  evidence  signals  r e v e a l e d a d o u b l e t a b s o r p t i o n a t T0.24  fragmentation pattern (Figure  group from  293.  i t s mass  the molecular  i o n (m/e 3 2 2 )  A complex f r a g m e n t a t i o n o f t h e m o l e c u l a r  i n a significant  i o n ( i i ) which  a p p e a r e d a t m/e 34  279. and  This fragmentation i s of particular  A second type  important  fragmentation  piperidine . aldehyde  yields  i n this  164 i n d i c a t i n g  series  an important  p o r t i o n of the molecule.  a p p e a r e d a t m/e  i n the Strychnos  series  diagnostic value f o r N^-functionalized derivatives.  fragmentation which  the p i p e r i d i n e  i s very characteristic  i o n ( i i i ) corresponding to  In this  the presence  i s the r e t r o - D i e l s - A l d e r -  example,  this  of the aldehyde  ion (iii)  group i n the  p o r t i o n of the s t r u c t u r e .  Other  means o f o x i d i z i n g  were e x p l o r e d i n order  a l c o h o l was t r e a t e d w i t h f r e s h l y  the N-formyl  alcohol  to increase the y i e l d . prepared  (43) t o t h e d e s i r e d I n one s t u d y , t h e  s i l v e r carbonate  on c e l i t e i n  '  +  H-  CHO  CHO  CO  CHO  (iii) m/e  •N  (ii) i/e 279  164  "f" CHO  Figure 10.  A r a t i o n a l e for the fragmentation of aldehyde in the mass spectrometer.  m /e 121  (44)  36  r e f l u x i n g benzene.  Although s i g n i f i c a n t quantities of the desired  product were obtained, the reaction always f a i l e d to go to completion and the desired product was d i f f i c u l t to i s o l a t e from minor components,  -130-  In another study,  the a l c o h o l (43)  was  t r e a t e d w i t h 2 moles of  pyrazole-chromate complex i n dichloromethane a c c o r d i n g  to a  dimethyl-  procedure  37 by  Corey.  T h i s o x i d a t i o n m e t h o d was  to  the aldehyde  hyde c o u l d in  order  (44)  o n l y be  was  apparent.  obtained  by  s u c c e s s f u l i n t h a t good  However, p u r e q u a n t i t i e s o f  repeated  the  alde-  chromatographic separations  to remove the d i m e t h y l p y r a z o l e .  desirable  conversion  This  to  t e d i o u s p r o c e d u r e made i t  to i n v e s t i g a t e another o x i d a t i o n r e a c t i o n .  The  N - f o r m y l a l c o h o l (43)  was  treated w i t h another  reagent  3 8 d e v e l o p e d by achieved  Corey  , p y r i d i n i u m chlorochromate.  the d e s i r e d  conversion  A second s y n t h e s i s of accomplished.  A f t e r the  p e r f e c t e d as formic (44)  conversion  to give  o f a l c o h o l (36) the  structure  W i t h the initiated  The  plan, illustrated having  Removal of  the  i n Figure  stereochemistry  1 1 , was  p r o t e c t i n g g r o u p and  in detail  In the  first  ( 4 4 ) was  the  the  to s y n t h e s i z e  C-16  the  subsequent o x i d a t i o n of  to stemmadeninine  ring-opened  was  acetic-  to f u r t h e r  preakuammicine s e r i e s . (.4)  by  The  study  position.  aldehydo-ester  shown f o r r e a s o n s p r e v i o u s l y  t h e n be  compound  served  (37)  aldehyde  f u n c t i o n a l i z e d curan system i n hand, a  lead to  discussed  acylated with  This" s y n t h e s i s  n i t r o g e n atom would  be  to aldehyde  to i n t r o d u c e a carbomethoxy group i n t o  the  later  (44).  desired  was  l a t t e r was  ( 4 4 ) was  the N-formyl-18-deoxy-Wieland-Gumlich  i n a comparable o v e r a l l y i e l d . the  approach  yield.  the N-formyl aldehyde  previously described,  anhydride  confirm  (45)  i n 78%  This  discussed. the  indoline  latter  could  a known r e a c t i o n w h i c h  will  later.  attempt to i n t r o d u c e  treated with  the d e s i r e d e s t e r  sodium h y d r i d e  functionality,  i n dimethoxyethane  and  -131-  F i g u r e 11.  A t t e m p t e d f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) and t h e p r o p o s e d p l a n f o r i t s c o n v e r s i o n t o p r e a k u a m m i c i n e and stemmadenine d e r i v a t i v e s .  -132-  m e t h y l c h l o r o f o r m a t e a t 80°. mixture after  A t i c examination of the reaction  1 hour revealed  polar material.  almost complete c o n v e r s i o n to a  T h i s p r o d u c t had a uv spectrum s i m i l a r  less  to starting  m a t e r i a l , b u t t h e i r s p e c t r u m showed a d d i t i o n a l a b s o r p t i o n i n t h e c a r b o n y l r e g i o n n e a r 1 6 8 0 cm which appeared carbomethoxy appeared  i n i t s pmr s p e c t r u m a t x 6 . 2 4 s h o w e d  group.  I n t h e mass s p e c t r u m o f  two m o l e c u l a r i o n p e a k s  suggested  a t m/e  these reaction conditions  t h e more b a s i c n i t r o g e n  absorption  the presence of a  this product there  446 a n d 448 i n a p a t t e r n  t h e p r e s e n c e o f an atom o f c h l o r i n e .  t h a t under with  A three-proton singlet  I t therefore  which  appeared  the methylchloroformate reacted  (N^) t o g i v e s t r u c t u r e  (47). This type 39  of r e a c t i o n has p r e v i o u s l y been observed i n s t r y c h n i n e c h e m i s t r y . A similar  r e a g e n t , c y a n o g e n b r o m i d e , was f o u n d t o c a u s e  five-membered  ring  Clearly,  of strychnine elevated  conversion to structure  fission  of the  to give s t r u c t u r e (48).  t e m p e r a t u r e s c o u l d n o t be u s e d  to achieve  ( 4 5 ) , t h e r e f o r e a s e l e c t i o n o f o t h e r more  p o w e r f u l bases were used a t lower temperatures.  Table 1 summarizes a  study i n which r e a c t i o n of the N-formyl aldehyde  ( 4 4 ) was a t t e m p t e d .  u s i n g e x c e s s sodium h y d r i d e t o f o r m t h e a n i o n f o l l o w e d by q u e n c h i n g which methylchloroformate.  I n each experiment  r e a c t i o n was f o l l o w e d b y t i c .  When d i m e t h y l f o r m a m i d e  s u l p h o x i d e were used, s m a l l a l i q u o t s and  examined  to determine  the progress of the or dimethyl-  o f t h e r e a c t i o n w e r e w o r k e d up  the progress of the reaction.  I n a l l cases  t h e r e a c t i o n s w e r e w o r k e d up a n d t h e p r o d u c t m i x t u r e was s e p a r a t e d a n d all  components were examined Starting material  spectroscopically. ( 4 4 ) was r e c o v e r e d i n e v e r y  experiment  shown e x c e p t E x p e r i m e n t 3 i n w h i c h c o m p l e t e d e c o m p o s i t i o n h a d o c c u r r e d .  -133-  In  Experiment  7 i n which  the e f f e c t i v e  base i s t h e d i m s y l a n i o n ,  some  d e f o r m y l a t i o n o f t h e s t a r t i n g m a t e r i a l h a d o c c u r r e d and s m a l l amounts of  aldehyde  (37) were i s o l a t e d .  since N-formyl  groups have been d e f o r m y l a t e d  Further attempts  to functionalize  (44) w i t h m e t h y l c h l o r o f o r m a t e 1-3 p o t a s s i u m plus  unexpected  under s i m i l a r c o n d i t i o n s .  t h e C-16 p o s i t i o n o f  a r e summarized i n Table  2.  In  aldehyde  Experiments  h y d r i d e d i s p e r s e d i n o i l was u s e d a n d s t a r t i n g m a t e r i a l  the deformylated  excess  T h i s o b s e r v a t i o n was n o t  aldehyde  ( 3 7 ) was r e c o v e r e d .  When a 2 m o l a r  o f p o t a s s i u m _t-butoxide (Exp.4-6) o r t h e "harpoon base"  d i i s o p r o p y l amine  (LDA) w e r e u s e d  to generate  (44) o n l y s t a r t i n g m a t e r i a l was r e c o v e r e d position  the anion o f  i n addition  lithium  aldehyde  t o t a r r y decom-  products. T h e r e a r e two s t e p s i n v o l v e d i n t h e n o r m a l f u n c t i o n a l i z a t i o n o f  a c a r b o n y l group. the second  The f i r s t  f o r m a t i o n a t t h e a-carbon and  i s the r e a c t i o n o f the anion w i t h the e l e c t r o p h i l i c  T h e r e f o r e , when s u c h asked  i s anion  a reaction  fails  the f i r s t  has t o be  i s whether o r n o t anion formation has occurred d u r i n g the r e a c t i o n  c o n d i t i o n s used. a parallel  In order  t o t r y t o o b t a i n some i n f o r m a t i o n a b o u t  s t u d y was made i n w h i c h  deuterium  o x i d e was u s e d  the r e a c t i o n i n s t e a d o f m e t h y l c h l o r o f o r m a t e . such a r e a c t i o n would have a deuterium a n i o n was f o r m e d .  the present  7 i n Table  The e x p e c t e d  this,  t o quench products  from  atom i n t h e p o s i t i o n i n w h i c h t h e  The p o s i t i o n o f a n d t h e e x t e n t o f d e u t e r a t i o n c o u l d  t h e n be a s c e r t a i n e d by e x a m i n a t i o n In  question which  reagent.  study,  o f t h e mass s p e c t r u m  of the product.  t h e r e a c t i o n c o n d i t i o n s f o r l i x p e r i m e n t s 2, 4, 5 a n d  1 were d u p l i c a t e d quenched w i t h D 0 2  and the recovered  starting  -134-  T a b l e 1.  Exp.  Reaction  l  a  6  2  a  2  3  a  2  4  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) u s i n g a n e x c e s s o f s o d i u m h y d r i d e and q u e n c h i n g w i t h m e t h y l c h l o r o f o r m a t e  a,b  Time(hrs)  Solvent  THF _ti_ •  DMF _n_  1  5  C  6  C  7  d  1  8  d  3  Temperature  _n_  4 0.5  e  (44) p l u s  decomposition  50° rt  Complete  50°  (44) p l u s  rt  _"_  decomposition decomposition  _"_  _n_  55° DMSO _n_  a.  10-Fold excess  b.  Granular rather  c.  1.5  d.  2 Moles  e.  Room  Moles  rt  Products  of sodium  of sodium  temperature.  (44) p l u s  rt  (44)  aldehyde(37)  hydride.  than o i l - d e s p e r s e d  of sodium  50°  hydride.  hydride.  sodium  h y d r i d e was  used.  -135-  T a b l e 2.  Exp.  1  F u r t h e r s t u d i e s on t h e f u n c t i o n a l i z a t i o n o f aldehyde (44)  Reaction Time(hrs)  3  Base  Solvent  Temperature  5 m o l e s KH  THF  r t  a  Products  (44) p l u s  2  1  —"—  —"—  60°  3  0.5  -"-  DMSO  35°  4  2  C,H, o o  55°  5  2  -"-  DMSO  r t  6  1  -"-  -"-  50°  (44)  7  0.5  2 m o l e s LDA  THF  -78°  (44)  8  1  -"-  -"-  0°  g  3  _ " _  _ " _  a.  Room  b.  R e f l u x e d f o r 30 m i n u t e s c h l o r o f ormate  2 m o l e s t-BuOk —  R  ^  b  "  aldehyde(37)  "  "  (44)  (44) p l u s  (44) p l u s II  decomposition  decomposition  II  temperature before cooling  t o 55° f o r a d d i t i o n o f m e t h y l -  II  -136-  material  ( 4 4 ) was  significant  e x a m i n e d by mass s p e c t r o m e t r y .  i n c o r p o r a t i o n o f d e u t e r i u m was  the r e a c t i o n c o n d i t i o n s were used  detected.  and  similar Due  A similar  h y d r o f u r a n and  9 i n Table  i n c o r p o r a t i o n of  a l s o done by L e w i s  laboratories  4 0  i t was  ( 3 6 ) i n F i g u r e 12 was  decided that  products.  a  It  dissolved  The  a t room t e m p e r a t u r e  T h i s m a t e r i a l was  i r spectrum "*") .  f o r 30 m i n u t e s  identical  o b t a i n e d when t h e i n d o l i n e a l c o h o l  tetra-  structure of this product (A  rapid  o f t h e m i x t u r e showed After  this  total  be  to the s i n g l e product which  ( 3 6 ) was  ( 4 9 ) was  product  i n a 2% m e t h a n o l i c  dissolved  t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e a t room t e m p e r a t u r e  I t s uv s p e c t r u m  was  in  p o t a s s i u m h y d r o x i d e s o l u t i o n o n l y t h e more p o l a r p r o d u c t c o u l d detected.  group  t i c examination of the product mixture r e v e a l e d  c a r b o n y l a b s o r p t i o n ( 1 7 3 0 - 1 6 6 0 cm  stirred  the N-formyl  t r e a t e d w i t h an e x c e s s o f m e t h y l c h l o r o f o r m a t e a  t h e p r e s e n c e o f two  indoline  2  deuterium  i n our  t h e p r o d u c t m i x t u r e i n some e x p e r i m e n t s .  A  The  no  Similarily,  7, 8 a n d  the i n d o l i n e n i t r o g e n atom as  reaction occurred.  was  case,  s t u d y s h o u l d be made on a n a l d e h y d e p o s s e s s i n g a n o t h e r  t h a t when t h e a l c o h o l  a broad  2, 5,  to the importance of t h i s problem,  complicating  found  s t u d y was  r e s u l t s were o b t a i n e d .  p r o t e c t i n g g r o u p on was  detected.  i n a d e u t e r a t i o n s t u d y . A g a i n , no  was  similar  i n Experiments  I n each  a s s i g n e d on  i n methanol f o r 15  and  minutes.  the f o l l o w i n g  2 4 1 , 2 7 8 , 287) showed t h e p r e s e n c e o f max . c h r o m o p h o r e s i m i l a r t o t h a t w h i c h was previously  was  data.  an N - a c y l a t e d  -137-  CH 0H 2  CO2CH3  (36)  (49)  CHO  (37)  F i g u r e 12.  obtained  The s y n t h e s i s o f N-carbbmethoxy-18-deoxy-WielandG u m l i c h a l d e h y d e (50)  f o r the N - f o r m y l a l c o h o l ( 4 2 ) .  a b s o r p t i o n a t 3575 cm  1  1685 cm  The pmr  1  was  found.  was  I n the i r spectrum, the  N-H  absent but a strong c a r b o n y l a b s o r p t i o n a t s p e c t r u m of t h i s m a t e r i a l e x h i b i t e d a  t h r e e - p r o t o n s i n g l e t a t T6.20 w h i c h was a s s i g n e d  t o t h e N-carbomethoxy  -138-  group.  Also  i n this  spectrum,  a t T 2 . 5 2 , w h i c h was a t t r i b u t e d indicative  the p o s i t i o n of a one-proton  t o t h e a r o m a t i c p r o t o n a t C-12, i s  o f an N - f u n c t i o n a l i z e d  indoline  system as i t has  f r o m x 2 . 8 i n t h e c o r r e s p o n d i n g N-H d e r i v a t i v e . of  this  structure  Further confirmation  354 a s w e l l  166 ( i o n v ) .  as prominent  The l a t t e r  f r a g m e n t s a t m/e  two i o n s r e p r e s e n t a  m e n t a t i o n p a t t e r n o f t h e s e a l k a l o i d s , w h i c h was d i s c u s s e d  Compound  shifted  ( 4 9 ) was o b t a i n e d f r o m i t s mass s p e c t r u m w h i c h  a m o l e c u l a r i o n a t m/e ( i o n i v ) a n d m/e  multiplet  ( 4 9 ) was o x i d i z e d  i n good y i e l d  exhibited 309 frag-  earlier.  to the aldehyde  (50) by  p y r i d i n i u m c h l o r o c h r o m a t e i n a manner s i m i l a r  to that d i s c u s s e d  i n the  N-formyl s e r i e s .  a uv s i m i l a r  T h i s p r o d u c t (50) e x h i b i t e d  the s t a r t i n g m a t e r i a l .  to that of  I t s i r s p e c t r u m showed a d d i t i o n a l c a r b o n y l  a b s o r p t i o n a t 1 7 2 0 cm . 1  The pmr s p e c t r u m i n d i c a t e d  the presence of  a n a l d e h y d i c p r o t o n a p p e a r i n g a s a d o u b l e t a b s o r p t i o n a t T0.40 The  p r e s e n c e o f t h e a l d e h y d e g r o u p was a l s o  l o s s o f 29 mass u n i t s spectrum.  from  (J=7Hz).  e v i d e n t by t h e s i g n i f i c a n t  t h e m o l e c u l a r i o n a t m/e  352 i n i t s m a s s  -139-  Further c o n f i r m a t i o n of s t r u c t u r e q u a n t i t i e s of aldehyde was  dissolved  to y i e l d  Gumlich  i n t e t r a h y d r o f u r a n and  aldehyde  out  methylchloroformate  p h y s i c a l and s p e c t r o s c o p i c  s e r i e s and  In this  a r e summarized  attempted  i n T a b l e 3.  f o l l o w e d by  s e r i e s , no  new  tarry decomposition  Exp.  Reaction Time(hr)  1  6  2  2  3  2  2 moles  4  4  1 mole  5  0.5  6  1  7  1  Base  Solvent  NaH  _n_  isolated  products.  KH KH LDA  Temperature  THF  40°  DMSO  53°  C  (50) p l u s  80°  DMF  50°  (50)  THF  0  (50)  20° THF  35°  (50)  Products  H  6 6  _n_ _n_  ( 5 0 ) was  of  f u n c t i o n a l i z a t i o n of N-carbomethoxy aldehyde  10 m o l e s  2 moles  methyl-  sepctroscopic examination  along w i t h v a r i o u s q u a n t i t i e s of C-16  with  experi-  p r o d u c t s were d e t e c t e d by t i c  t h e r e a c t i o n m i x t u r e o r by  Attempted  aldehyde  In each  quenching  Starting material  3.  of  to that i n the study d i s c u s s e d  the worked-up r e a c t i o n p r o d u c t .  Table  compound  N-carbomethoxy-18-deoxy-Wieland-  i n a manner s i m i l a r  m e n t a n i o n f o r m a t i o n was  of  latter  t o i n t r o d u c e t h e c a r b o m e t h o x y g r o u p a t C-16  the N-formyl  examination  o b t a i n e d when  (50).  (50) were c a r r i e d  c h l o r o f ormate .  later  The  treated with  T h i s p r o d u c t had  i d e n t i c a l w i t h those of  Attempts  for  ( 3 7 ) became a v a i l a b l e .  a single product.  properties  ( 5 0 ) was  _!!_  decomposition  -140-  In The  this  experimental  study  t h e mass s p e c t r a o f  zation  bilities  isolated  failure  a t C-16  a b a s e and  quenched w i t h  incorporation The  by  at  a l l under  of  position  be  Secondly,  c h l o r o f o r m a t e may  be  environment.  thirdly,  And  on  case,  product would not  the  two  as  6 and  oxide.  7 were  or o b t a i n  derivatives  (44)  Firstly,  anion  f o r m a t i o n may  given  be  this  second  and  be  t o be  (50)  Three not  with  possibe o c c u r r i n g environment  o c c u r r i n g , but  a sterically  possibility,  resonance  rapidly  no  functionali-  the v e r y h i n d e r e d  i n such  expected  i t would  to  f o r m a t i o n may  to r e a c t  of  revealed that  understood.  anion  duplicated  Examination  fully  due  made.  occurred.  formation  curan  oxygen atom v i a the  c o n d i t i o n s employed  2,  i s not  too b u l k y  occurring  the  the  c o n d i t i o n s employed  C-16.  had  to d e t e c t anion  treating  1,  deuterium  of deuterium  considered.  the  the  d e u t e r a t i o n s t u d y was  s t a r t i n g m a t e r i a l (50)  methylchloroformate  may  a parallel  c o n d i t i o n s of Experiments  and e a c h r e a c t i o n was  significant  also,  shown.  unfavourable  reaction  may  I f t h a t was  detected or  hydrolyse  methyl-  to  isolated  be the  under  starting  material. A different  approach  to the  functionalization  of p o s i t i o n  24 was  initiated.  I t was  d i s c o v e r e d i n our  laboratories  t h a t when  the  C-16  -141-  unsaturated  aldehyde, nor-fluorocurarine  borohydride  i n m e t h a n o l a t room t e m p e r a t u r e t h e r i n g - o p e n e d  alcohol  (52) could  be o b t a i n e d  i n 40% y i e l d .  o p e n i n g r e a c t i o n w h i c h was f i r s t s e r i e s o f compounds p r o b a b l y  ( 3 9 ) was t r e a t e d w i t h  This  sodium  indole  reductive  ring-  developed by Smith and Wrobel i n t h i s  p r o c e e d s v i a t h e seco-immonium d e r i v a t i v e  k? (51).  I n the present  study,  60-65% c o u l d be o b t a i n e d  i t was f o u n d  i fthis ring-opening  methanolic potassium hydroxide product  that an increased  y i e l d of  r e a c t i o n was d o n e i n 2%  s o l u t i o n a t 40°.  The p l a n  to convert  ( 5 2 ) t o s t e m m a d e n i n e i s shown i n F i g u r e 1 3 . A w e l l known r e a c t i o n o f i n d o l e a l k a l o i d s i s c h l o r o i n d o l e n i n e  formation.  This  r e a c t i o n i s accomplished by t r e a t i n g the i n d o l e  vative with  a source of p o s i t i v e c h l o r i n e ions  _t-butyl hypochlorite normally  unstable  Therefore, the  have been used  a few have ever been  reagent.  i s the introduction of a nucleophile  This  to i n t r o d u c e  type of r e a c t i o n has been w i d e l y various  nucleophiles  form  isolated.  and t r e a t e d  The common r e a c t i o n f o r w h i c h  c a s e p o s i t i o n C-16) v i a t h e t a u t o m e r i c  (54)).  The c h l o r o i n d o l e n i n e s a r e  t h e s e d e r i v a t i v e s a r e u s u a l l y made _ i n s i t u  appropriate  this  such as sodium h y p o c h l o r i t e ,  or N-chlorobenztriazole.  molecules and only  deri-  with  chloroindolenines  a to the indole ( i n  (represented  by s t r u c t u r e  used i n the Iboga a l k a l o i d s  such as c y a n i d e i o nand v i n d o l i n e .  However, t h i s r e a c t i o n appeared n o t t o have been used i n t h e S t r y c h n o s series. C-16  I t was f e l t  could  of molecular on  still  that  the stereochemistry  o f t h e C^ i n t r o d u c t i o n a t  be c o n t r o l l e d i n t h e r i n g - o p e n e d  models r e v e a l e d  t h e 8 - f a c e was p r e s e n t  that again  a great  i n these d e r i v a t i v e s .  series since  deal of s t e r i c  examination crowding  -142-  The  indole alcohol  J^-butylhypochlorite  ( 5 2 ) was  a t 0°  treated with  f o r 15 m i n u t e s .  reaction mixture revealed  the presence  absence of s t a r t i n g m a t e r i a l .  The  uv  A  1.1  equivalents  t i c examination  of  of very p o l a r m a t e r i a l spectrum  (X  of the  and  310) max  indicated  that  indolenine parative  chromophore.  Attempts  l a y e r chromatography  pure m a t e r i a l . ( 5 3 ) was  t h e i n d o l e c h r o m o p h o r e had  For  to i s o l a t e  representative.summary In each experiment,  of the s t u d y , the  treated with  of r e s u l t s  from  chloroindolenine  cyanide  this  then monitored  reaction  by  t i c and  decomposition  product  up any  spectroscopic  study of  t h e end  this  A  of  of the  a  s e r i e s of experiments  also  sufficient  was  uv  the  tarry  chromatoplate. apparent.  A prohibitively  to o b t a i n  4.  s e p a r a t e d by p i c .  a m o u n t s was  o f these components f o r c h a r a c t e r i z a t i o n . found  give  reaction  these complex m i x t u r e s  i n d o l e chromophores were p r e s e n t .  m a t e r i a l was  The  and  the b a s e - l i n e  r e a c t i o n would have been r e q u i r e d one  At  isolated  i n very small  to  c o n f i r m e d by  t h e m a i n c o m p o n e n t o f t h e m i x t u r e was  A l a r g e number o f p r o d u c t s However, a uv  failed  s t u d y i s shown i n T a b l e  spectroscopy.  r e m a i n i n g on  pre-  ion directly.  f o r m a t i o n was  t i m e , t h e p r o d u c t m i x t u r e was  In every experiment,  t h a t no  uv  chloro-  chloroindolenine  e x a m i n a t i o n b e f o r e a d d i t i o n o f the c y a n i d e r e a g e n t . was  to a  t h i s m a t e r i a l by  ( p i c ) , not unexpectedly,  the remainder  f o r m e d i n s i t u and  been converted  revealed large  scaled-  q u a n t i t i e s of  S i n c e no abandoned.  indolic  Figure  13.  The p l a n t o s y n t h e s i z e s t e m m a d e n i n e ( 4 ) v i a the c h l o r o i n d o l e n i n e o f des-carbomethoxy stemmadenine ( 5 2 ) .  -144-  T a b l e 4.  Exp.  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a t h e c h l o r o i n d o l e n i n e o f i n d o l e (52)  Nucleophile  Solvent  1  KCN  C H  2  -"-  3  Temperature  Time(hrs)  r  t  24  DMF  r  t  6  _"_  _"_  80°  0.5  4  ZnCN  CH C1  r  24  5  -"-  DMF  6  AgCN  CH C1  7  -"-  DMF  8  Bu.NCN  CH C1_  4  2  C 1  2  2  2  2  0  _"_  10  -"-  DMF  11  Et AlCN  CH C1  50°  1.5  r  48  t  r  t  1.5 .  48  2  35°  12  35°  5  r  5  t  12  36°  12  13  5°  5  a. Room  temperature  Decomposition  1 1  _"_  2  t  55°  9  2  2  Products  days  -145-  P e r h a p s one p o s i t i o n C-16 compound its of  was  (52).  acetate  due  therefore,  s t u d y the  to introduce  the h y d r o x y l  decided  the  treatment of  pyridine  (10%)  indole alcohol  a t 5°  overnight  p r o d u c t e x h i b i t e d a uv  spectrum  N-acyl indole  ir  s p e c t r u m w h i c h s h o w e d an an  (56).  amide c a r b o n y l  (52)  in  the h y d r o x y l  as  chloroindolenine  This  (^  with  acetic  pic.  S u r p r i s i n g l y , the  248,  m a x  282,  290)  acetate  carbonyl  n e a r 1640  at  an  N - a c e t a t e and  0-acetate group, r e s p e c t i v e l y .  of  the  an  x 7 . 7 9 and  s t r u c t u r a l assignment of  7.88  Two  singlet absorptions  i o n a t m/e  59 mass u n i t s w h i c h c o r r e s p o n d e d  i t was  the  d a t a on  the  to give  alcohol  yielded obtained  an  the  (52)  (57)  felt as  when t h i s r e a c t i o n was  not  that  both  i n i t s mass  a prominent acetate  loss  fragment.  r e a c t i o n suggested  that  known t o o c c u r  nitrogen under  this reaction could  tic.  Good y i e l d s w e r e The  product  be  Treatment  1 drop of p y r i d i n e a t  the major product. m o n i t o r e d by  indole  the major product.  a c e t i c a n h y d r i d e and  d e s i r e d m a t e r i a l as  1  confirmation  evident  t o a c y l a t e an  g r o u p , and  i t was  the mono-acetate with  cm  (57).  hydroxyl  acetylating conditions,  controlled  l o s s o f an  i t i s g e n e r a l l y more d i f f i c u l t  atom i n c o m p a r i s o n w i t h standard  the  to  three-proton  Further  with  the m i n o r component from t h i s  d e s i r e d mono-acetate  Since  to  380  a t 1725  presence of  t h i s p r o d u c t "(56) was  s p e c t r u m w h i c h showed a m o l e c u l a r  Preliminary  i n d i c a t e d the  major  s u p p o r t e d by i t s  absorption  cm  two  corresponding  s t r u c t u r a l a s s i g n m e n t was  absorption  anhydride  y i e l d e d a product mixture of  s e p a r a t e d by  an  of  into  this derivative.  be  of  cyanide  group  to p r o t e c t  f u n c t i o n a l i z a t i o n of  components w h i c h c o u l d  and  failure  to i n t e r f e r e n c e of  I t was,  and  The and  reason f o r the  (57)  -10°  -146-  exhibited  the normal  i n d o l e chromophore i n the uv s p e c t r u m  (A  224, max  283,  290) and a c a r b o n y l  absorption  i n the i r spectrum  ( 1 7 2 5 cm  w h i c h c o r r e s p o n d s to t h e p r e s e n c e o f an 0 - a c e t a t e group. proton xl.69  singlet absorption i n t h e pmr  spectrum provided  Figure  14.  a t x 7 . 8 9 a s w e l l a s a n N-H  s p e c t r u m , and a m o l e c u l a r  The p l a n t o s y n t h e s i z e s t e m m a d e n i n e chloroindolenine acetate (58).  )  three-  absorption  i o n a t m/e  c l e a r evidence f o r the assignment  A  X  at  338 i n t h e mass  of s t r u c t u r e (57).  ( 4 ) v i a the  -147-  With  the i n d o l e acetate  to i n -  cyanide  similar  t o t h a t made i n t h e f r e e h y d r o x y l s e r i e s p r e v i o u s l y d i s c u s s e d .  before  ( 5 7 ) was  t h e C-16 p o s i t i o n was i n i t i a t e d  designed  troduce  As  into  (57) i n hand, a study  c h l o r o i n d o l e n i n e f o r m a t i o n was c l e a r l y t r e a t e d w i t h a s l i g h t excess  methane o r d i m e t h y l f o r m a m i d e . which represent C-16  product  indolic material. was o b t a i n e d  i n Table  mixtures  into  by t i c and uv  I n each experiment,  a great deal of  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e of i n d o l e acetate (57).  Nucleophile  Solvent  decomposition of products.  Nor c o u l d any  v i a the chloroindolenine  Time(tirs)  Products  KCN  2  -"-  DMF  r t  8  3  II  II  68°  1  II  rt  24  II  4  Bu.NCN  C 1  2  Temperature  of  1  C H  2  study,  spectroscopy,  a l o n g w i t h a s m a l l amount o f a c o m p l e x m i x t u r e  Table  r  t  2  found  position  were examined f o r the presence  c o u l d any i n d o l i c m a t e r i a l be d e t e c t e d .  Exp.  compound  ( 5 8 ) . As i n t h e p r e v i o u s  A t no t i m e  5.  when  5 are the r e s u l t s  of the i n t r o d u c t i o n of cyanide  o f t h e r e a c t i o n was m o n i t o r e d  the i s o l a t e d  achieved  of _t-butyl hypochlorite i n dichloro-  Presented  i n the chloroindolenine acetate  the progress and  the study  i n a manner  Decomposition  0  -"-  -148-  p u r e m a t e r i a l be o b t a i n e d i n q u a n t i t i e s s u f f i c i e n t f o r characterization. With  the f a i l u r e  ( 5 2 ) o r compound  to introduce  cyanide  was a b a n d o n e d .  Similar  r e s u l t s were o b t a i n e d i n o u r l a b o r a t o r i e s by L e w i s  i n which  cyanide  (57) t h i s approach  i n t o e i t h e r compound  i n t r o d u c t i o n v i a the  chloroindolenine  of the i n d o l e  ester  40 ( 5 9 ) was  studied.  C l e a r l y a new a p p r o a c h  t o t h e f u n c t i o n a l i z a t i o n o f t h e C-16  p o s i t i o n of the curan skeleton d e r i v a t i v e which for  elaboration  (39). system.  ( 2 1 ) was r e q u i r e d .  curan  h a d become a v a i l a b l e a n d w h i c h was p o t e n t i a l l y u s e f u l t o t h e p r e a k u a m m i c i n e d e r i v a t i v e s was  T h i s s t r u c t u r e may b e r e g a r d e d The n u c l e o p h i l i c r e a c t i o n s  classified  Another  i n t o four  types  (Figure  nor-fluorocurarine  as h a v i n g a r i g i d  o f such 15).  conjugated  Reaction types  cis-enaminocarbonyl s y s t e m s may  be  I , I I and I I I  41 a r e w e l l known  i n such  s y s t e m s and a r e a p p l i c a b l e i n t h e c u r r e n t  study,  -149-  42 type VI r e a c t i o n s " " i n which  but  the 8-carbon i s n u c l e o p h i l i c  r e l e v a n t here because i n the curan d e r i v a t i v e substituted. systems  The f a c t o r s w h i c h  are not w e l l understood  interest. reaction  this position i s fully  g o v e r n r e a c t i o n a t 0, C a n d N i n s u c h and have been a s u b j e c t o f r e c e n t  The p r o d u c t s t h a t w o u l d  be e x p e c t e d  types i f n o r - f l u o r o c u r a r i n e  ( 3 9 ) was  from these  three  r e a c t e d w i t h the  e l e c t r o p h i l e , m e t h y l c h l o r o f o r m a t e , a r e shown i n F i g u r e 1 6 . was  i s not  The p l a n  t o enhance r e a c t i o n c o n d i t i o n s t o f a v o u r type I I r e a c t i o n .  C-functionalized (60).  I t was  product would  felt  that the type I N - s u b s t i t u t e d product  e a s i l y be d i s t i n g u i s h e d expected would  be o f t h e d e s i r e d p r e a k u a m i c i n e  series  (61) c o u l d  f r o m t h e l a t t e r by i t s u v s p e c t r u m w h i c h  t o be v e r y d i f f e r e n t .  be e x p e c t e d  The  The O - f u n c t i o n a l i z e d p r o d u c t  to h y d r o l y s e back to s t a r t i n g  was  (62)  material.  -c-  E  +  Type I  F i g u r e 15.  E  +  Type I I  E  +  Type I I I  The n u c l e o p h i l i c r e a c t i o n s o f e n a m i n o c a r b o n y l  systems.  An  examination of the l i t e r a t u r e  revealed  favoured C - f u n c t i o n a l i z a t i o n of enaminocarbonyl  that  two  systems.  factors Firstly,  -151-  Meyers e t . a l . 63)  . reported  aprotic solvents  that  i n the  c i s s e r i e s (see p a r t i a l  favoured C - a l k y l a t i o n of a v a r i e t y of  structure cyclic  enamino-ketones w h i c h were t r e a t e d w i t h m e t h y l i o d i d e , w h i l e trans  series (partial  s t r u c t u r e 64)  the  O - a l k y l a t i o n p r e d o m i n a t e d and  was 44  not  i n f l u e n c e d by  choice  of s o l v e n t .  S e c o n d l y , a s t u d y by  which involved  the  b o n d and  a c e t y l chloride revealed  in  using  a c y l a t i o n of a c y l i c  the p r e s e n c e o f a b a s e s u c h as  e n a m i n o - k e t o n e s h a v i n g an t h a t C - a c y l a t i o n was  p y r i d i n e or  s t u d y a l s o showed t h a t C - a c y l a t i o n was f a v o u r e d  When t h e  i n the  l i t h i u m s a l t of n o r - f l u o r o c u r a r i n e a s i n g l e p r o d u c t was  spectrum  ) of  o f an  233,  indolenine  similar  carpine  be  nanometers  s t r u c t u r e s s u c h as  (65)(X  would not  305  chromophore ( F i g u r e  t r a n s f e r b a n d a t 268 for  268,  max  280)  and  (nm)  This  cis series.  (39)  was  quenched  obtained.  The  t h i s p r o d u c t i n d i c a t e d the 17).  The  wavelength of  compares f a v o u r a b l y  preakuammicine  r h a z i n a l i n e (66)(X  e x p e c t e d f o r an N - f u n c t i o n a l i z e d  ( 8 max  )(^  N-H  favoured  triethylamine.  w i t h m e t h y l c h l o r o f o r m a t e a t 0° (X  Kozerski  m  266).  a  x  A 6  presence  the  with  uv  electron-  that  reported  2 263) .» p r e c o n d y l o S u c h a uv  d e r i v a t i v e (61).  spectrum  Although  -152-  J  I  l  220  Figure  no  17.  240  I  '  '  260  L  280  U  1  300  I  I  l i t e r a t u r e example of the chromophore i n s t r u c t u r e  320  340  i n which  ( 6 1 ) c o u l d be  t h e r e i s a d o u b l e bond a t o t h e i n d o l i n e  p r i n c i p a l band i n the uv s p e c t r u m a p p e a r s a t w a v e l e n g t h s  300 nm.  L  The u v s p e c t r u m o f t h e p r o d u c t f r o m t h e r e a c t i o n o f n o r f l u o r o c u r a r i n e (39) w i t h m e t h y l c h l o r o f o r m a t e .  i n a l l examples the  I  fluorocurarine  (39) a p p e a r e d  a t 363  nm.  nucleus  greater  T h i s band i n t h e uv s p e c t r u m o f the s t a r t i n g m a t e r i a l  found,  nor-  than  -153-  C H  3°A  Z^  0  N'  (8)  The  (66)  carbonyl region  three a b s o r p t i o n bands: was  i n t h e i r s p e c t r u m o f t h i s p r o d u c t showed  1720,  1670  and  cm -1 .  1640  a s s i g n e d t o a C-carbomethoxy group.  The  C-16  position  rotation  Due  t o t h e s t e r i c and  electronic  s u c h a n a l d e h y d e g r o u p may  so t h a t  two  rotomers would  environment  and  C a r b o n y l a b s o r p t i o n s o f d i f f e r e n t w a v e l e n g t h s may  F i g u r e 18.  The  18.00 I  i  i  i  cm  1  near  the  each c o n t r i b u t e a band i n the i r  i s i l l u s t r a t e d by  1  1640  have s u f f i c i e n t l y r e s t r i c t e d  This rationale  I  and  geminal to a carbo-  spectrum. (68).  cm -1  b a n d a t 1720  bands a t 1670  c o u l d be a d o u b l e t a b s o r p t i o n o f an a l d e h y d e g r o u p methoxy group.  The  '  the p a r t i a l s t r u c t u r e s be  (67)  expected  1600 1—  J  L  c a r b o n y l r e g i o n of the i n f r a r e d  spectrum of product  (60)  -154-  H  0  (67)  for  (68)  t h e a l i g n m e n t (67) and n o n - a l i g n m e n t  (68) o f the i r - o r b i t a l s o f  t h e two s y s t e m s . I n t h e pmr s p e c t r u m proton a b s o r p t i o n appeared paramagnetic  shift  ( F i g u r e 19) o f t h i s p r o d u c t a n a l d e h y d i c as a s i n g l e t  o f 0.6 ppm f r o m  spectrum of the s t a r t i n g m a t e r i a l  a t T0.02 w h i c h r e p r e s e n t s a  the corresponding absorption (39). A three-proton singlet  a b s o r p t i o n a t T6.08 c o n f i r m e d t h e p r e s e n c e o f a c a r b o m e t h o x y The  latter  chemical s h i f t  chemical s h i f t  compares f a v o u r a b l y w i t h  o f the carbomethoxy  precondylocarpine  group.  the corresponding  g r o u p s ' i n 'preakuammicine  45 (65) w h i c h w e r e x 6 . 1 4 a n d 6.15,  Of p a r t i c u l a r  i n the  (. 8 ) a n d  respectively.  i n t e r e s t was t h e r e m a r k a b l e r e s e m b l a n c e  t h e mass s p e c t r u m o f t h i s p r o d u c t a n d t h a t p u b l i s h e d  between  f o rprecondylo-  45 carpine  (65).  B o t h compounds f r a g m e n t  t o l o s e t h e carbomethoxy  from t h e m o l e c u l a r i o n t o g i v e a peak a t M -58 r a t h e r +  M -59. +  which  group  t h a n t h e more  usual  Due t o t h e l a c k o f i n f o r m a t i o n o n t h e c o m p l e x f r a g m e n t a t i o n s i n d o l e n i n e s appear  more s t r u c t u r a l spectrum of t h i s  t o undergo  assignment product.  i n t h e mass s p e c t r o m e t e r ,  c o u l d b e made a t t h i s  time based  little  o n t h e mass  F i g u r e 19.  The pmr s p e c t r u m o f compound  (60).  -156-  Based on t h e above d e s c r i b e d (60)  to this  product.  nor-fluorocurarine  (39) should  to pathway I I ( F i g u r e found  that  heating  this  as the s o l v e n t .  preparation signed  react with methylchloroformate  same r e a c t i o n c o u l d  of the aldehydo-ester  from s t e r i c  or just using  Due t o t h e s t e r i c  a t C-16 was a s -  crowding over the  should  I t was t h e l a t t e r  A  electrophile  argument upon w h i c h  o f 16-epi-stemmadenine (28) and hence stemmadenine 2  t o 65°,  the reagent  The s t e r e o c h e m i s t r y  ( 3 9 ) ( d i s c u s s e d . e a r l i e r ) an i n c o m i n g  chemistry  later  method f o r t h e  3-face o f the molecule be a - o r i e n t e d .  according  i n comparable y i e l d by  became t h e u s u a l (60).  considerations.  I t was  that  excess methylchloroformate  as a co-solvent,  The l a t t e r  used.  be a c h i e v e d  (39) w i t h  tetrahydrofuran  the s t r u c t u r e  made o n m o d e l s y s t e m s s u g g e s t e d  17) under t h e c o n d i t i o n s  nor-fluorocurarine  either with itself  Studies  d a t a we a s s i g n e d  the stereo( 4 ) was  4  assigned. Further  evidence f o r the assignment of s t r u c t u r e  a b o v e p r o d u c t was o b t a i n e d  from i t s s t a b i l i t y  i n b a s i c media.  material  ( 6 0 ) was f o u n d  solution  f o r more t h a n 6 h o u r s a t room t e m p e r a t u r e .  tends  to eliminate  the carbamate  When p r o d u c t  With of  type e a s i l y decarboxylate  hydroxide  observation as i t i s w e l l  under  milder  t o 65° i n 5% m e t h a n o l i c  c o m p l e t e h y d r o l y s i s -to t h e s t a r t i n g  (39), occurred  the aldehydo-ester  this material  This  (61) from c o n s i d e r a t i o n ,  ( 6 0 ) was h e a t e d  potassium hydroxide s o l u t i o n , nor-fluorocurarine  This  t o b e s t a b l e i n 5% m e t h a n o l i c p o t a s s i u m  known t h a t compounds o f t h i s conditions.  (60) t o t h e  material,  i n 2 hours. (60) i n hand, a study  to the preakuammicine  f o r the conversion  ( 8 ) and stemmadenine  ( 4) series  -157-  was  begun.  be d e s i r a b l e  Two  approaches were i n i t i a l l y  to simply  compare t h e l a t t e r w i t h  Figure  20.  considered.  Firstly,  r e d u c e t h e a l d e h y d e (60) t o t h e a l c o h o l a sample o f n a t u r a l  Some e x p e c t e d r e a c t i o n s  i t would (69) and  preakuammicine ( 8 ) .  of preakuammicine  derivatives.  -158-  Secondly,  t h e s y n t h e s i s o f s t e m m a d e n i n e s e r i e s may  be  by e i t h e r d i r e c t r e d u c t i v e r i n g - o p e n i n g o f p r e a k u a m m i c i n e series  (60) o r by  latter  c o n v e r s i o n was  b u t few  e x p e r i m e n t a l d e t a i l s were g i v e n .  have c o n v e r t e d preakuammicine Compound  ( 6 0 ) was  (8)  p r o d u c t was data.  The  These w o r k e r s  treated with recrystallized  i s o l a t e d by uv s p e c t r u m  p i c and (A  found  228,  o f one  also claim  indicated  to  ir> b a s i c m e d i a .  An  borohydride  examination  major product.  to have the f o l l o w i n g  292)  The  Quereshi  sodium  f o r 30 m i n u t e s .  t i c r e v e a l e d the presence  (69).  S c o t t and  to akuammicine ( 5 )  i n d r y m e t h a n o l a t room t e m p e r a t u r e t h i s r e a c t i o n by  aldehyde  r i n g - o p e n i n g of the c o r r e s p o n d i n g a l c o h o l c l a i m e d t o have been done by  achieved  of  This  spectral  the p o s s i b l e  presence  max o f an i n d o l e n i n e chromophore c o n j u g a t e d The  i r spectrum  molecule.  s h o w e d t h a t no  T h i s o b s e r v a t i o n was  the aldehyde  to a carbon-carbon  c o n f i r m e d by  p r o t o n a b s o r p t i o n and  of the p r o d u c t .  x3.89 and The  4.54  suggested  mass s p e c t r u m  the base peak.  t h e pmr  the m e t h y l  H o w e v e r , two the presence  spectrum.  a b s o r p t i o n of  best f i t s  19-en  (72).  r e v e a l e d a m o l e c u l a r i o n a t m/e  t h i s data i s that of  of the a l d e h y d o - e s t e r  C60)  the  t o be  Both carbopmr  singlet absorptions at  276  A h i g h r e s o l u t i o n mass m e a s u r e m e n t on  A p o s s i b l e mechanism by w h i c h treatment  one-proton  from  the  o f an e x o c y c l i c m e t h y l e n e  the m o l e c u l a r formula o f t h i s molecule which  bond.  c a r b o n y l groups were p r e s e n t i n the  methoxy g r o u p o f t h e s t a r t i n g m a t e r i a l (60) were a b s e n t spectrum  double  C^g^o^'  which  was  t h i s peak Th  e  group. also confirmed  structure  1,2-dehydro-16,17-dehydro-2g-cur-  compound  (72) c o u l d a r i s e  w i t h sodium borohydride  from  i s shown  -159-  in Figure  21.  The r e d u c t i o n o f a c a r b o m e t h o x y  aldehyde group under s i m i l a r see  group geminal t o an  c o n d i t i o n s i s w e l l known ( f o r e x a m p l e s  the synthesis of s e c o d i n o l described  i n Part  I and t h e r e d u c t i o n  of r h a z i n a l i n e (66) i n r e f .4 6 ) .  (72) Figure  21.  A p o s s i b l e m e c h a n i s m f o r t h e f o r m a t i o n o f compound ( 7 2 ) .  -160-  Th e p r o b l e m g r o u p i n compound achieve  now  (60)  became one to achieve  of e i t h e r reducing the  the preakuammicine s e r i e s o r  the r e d u c t i v e r i n g - o p e n i n g of  (60)  series, while preventing decarboxylation. two  possibilities  compound  (.60)  u n d e r v a r i o u s c o n d i t i o n s o f pH results  from  this  the main product A  study of  mixture (72).  temperature.  o f e a c h r e a c t i o n was  no  identifiable  results  from  8-10,  A t no  approach  c o u l d be  obtained.  and mass s p e c t r o s c o p y compounds.  by  uv and  The  failed  (5).  mass  stemmadenine  mixtures  were  (60)  In each experiment,  a  with complex  being the i n d o l e n i n e  detected. product  i n ammonia a n d  u n d e r t h e s e c o n d i t i o n s w o u l d be  d i h y d r o - d e r i v a t i v e or akuammicine  desired  7.  to the r e d u c t i o n of  treated b r i e f l y with lithium metal.  1-7  isolated.  obtained w i t h the major product  and  mixtures  borohydride  a s t u d y o f t h e r e d u c t i o n o f compound  dissolved  these  In Experiments  mixtures  components c o u l d be  a l d e h y d o - e s t e r was  expected  6.  v e r y complex product  The  be  to evaluate  of preakuammicine or  t i m e c o u l d i n d o l i c m a t e r i a l be  Another  stemmadenine  the deoxygenated i n d o l e n i n e (72).  h y d r i d e s a r e shown i n T a b l e  o f p r o d u c t s was  to  The r e p r e s e n t a t i v e  i n Table  t o show t h e p r e s e n c e  In Experiments  o b t a i n e d and  other metal  and  In order  t r e a t e d w i t h sodium  study are summarized  failed  derivatives.  The  was  t o g i v e the  the m i n o r components o f the p r o d u c t  spectroscopy  aldehyde  ( 6 0 ) was  considered.  dimethoxyethane a t  possible products  that  the a l c o h o l ( 6 9 ) , i t s  However, o n l y v e r y complex  C a r e f u l examination  of  -78° may  1,2product  t h e s e m a t e r i a l s by  to demonstrate the presence  o f any  of  the  uv  -161-  Table  6.  R e a c t i o n s o f Compound  Solvent  ( 6 0 ) w i t h NaBH.  Time(hr)  1  MeOH  0.5  2  MeOH  3  Temp.  Products  rt  (72)  2  -30°  (72)  MeOH + 1 0 % KOH  1  rt  4  MeOH + 1 0 % KOH  0.5  65°  5  MeOH + 1% HoAc  1  6  MeOH + 1 0 % H o A c  7  (72) p l u s m i n o r  "  _"_  rt  II  _n_  1  rt  II  _n_  MeOH + 5 0 % HoAc  1  rt  II  _n_  8  HoAc  0.5  rt  9  HoAc  0.3  90°  10  THF  .5  rt  Table  7.  products  Complex m i x t u r e  _n_  F u r t h e r s t u d i e s o n t h e m e t a l h y d r i d e r e d u c t i o n o f Compound ( 6 0 )  Reagent  Solvent  Temp.  Time  1  KBH. 4  MeOH  r t  1  2  -"-  MeOH+10%KOH  35°  0.5  3  NaCNBH  MeOH+10%HoAc  r t  1  4  -"-  HoAc  r t  1  5  Red-Al  THF  r t  0.2  3  Products (72) (72) p l u s c o m p l e x m i x t u r e (72) Complex m i x t u r e (72) p l u s complex m i x t u r e  -162-  The f a i l u r e  to o b t a i n from product  preakuammicine s e r i e s o r a ring-opened metal  hydride  into  shown i n F i g u r e 2 1 .  the chemistry  the aldehyde group i n o r d e r attempts  to ring-open  a catalytic  In order  reduction-  to gain further  to eliminate i t s participation  i n further  ( 6 0 ) was t r e a t e d w i t h e t h a n e  dithiol  amount o f b o r o n t r i f l o u r i d e e t h e r a t e i n d i c h l o r o -  T h i s m a t e r i a l was a s s i g n e d  o f t h e t h i o a c e t a l d e r i v a t i v e was  obtained.  s t r u c t u r e ( 7 3 ) on t h e b a s i s o f t h e f o l l o w i n g  The u v s p e c t r u m  (A  2 3 8 , 2 7 5 , 2 8 3 s h ) was v e r y max  to t h a t o f the s t a r t i n g m a t e r i a l .  similar  J  I n the i r spectrum, there  one c a r b o n y l a b s o r p t i o n b o n d n e a r 1710 cm a t 1560 cm \  to protect  the molecule.  methane a t 5 ° , a good y i e l d  spectral data.  to a f a c i l e  o f t h i s s t r u c t u r e i t was d e c i d e d  When t h e a l d e h y d o - e s t e r and  stemmadenine d e r i v a t i v e by  r e d u c i n g a g e n t s was a t t r i b u t e d  e l i m i n a t i o n process insight  (60) the a l c o h o l (69)of the  1  and an i n d o l e n i n e  appeared absorption  The pmr s p e c t r u m o f t h i s m a t e r i a l , e x h i b i t e d a 3 - p r o t o n  s i n g l e t a b s o r p t i o n a t x6.09 i n d i c a t i n g  (60)  the presence of the carbomethoxyl  S v /  (73)  -163-  group.  The  t h i o a c e t a l g r o u p was  e v i d e n t by  p r o t o n s i n g l e t a b s o r p t i o n a t T4.19 p r o t o n , and  the appearance of a  w h i c h was  attributed  a f o u r - p r o t o n m u l t i p l e t s i g n a l n e a r T6.68 was  the methylene p r o t o n s  of the t h i o a c e t a l group.  o f t h i s s t r u c t u r e was  g i v e n by  molecular  i o n a t m/e  the l a t t e r  426.  confirmed  A study derivative  sodium borohydride  to achieve  initiated.  When t h i s m a t e r i a l was  obtained.  supported  by  the  t h e a b s e n c e i n t h e pmr However, p r e s e n t  in this  The  revealed a molecular  i o n a t m/e  product  s t r u c t u r e (75)  assigned  c o u l d a t t h i s p o i n t n o t be  and  the  failure  The  carbomethoxy t h i o a c e t a l  370.  a  confirmed  methyl  one-proton  attributed  B a s e d on or  (76).  considered  t o the  C-17  the presence  these d a t a , The  this  (77) w h i c h would  of  reduction  stereochemistry at  C-lb  certainty. f o r the course  of this r e a c t i o n (74).  (73) c o u l d u n d e r g o d e c a r b o x y l a t i o n t o  Or, t h e r i n g - o p e n i n g r e a c t i o n may (74)  was  to d e t e c t the stemmadenine t h i o a c e t a l d e r i v a t i v e  the a - v i n y l i n d o l i n e  material  i r spectrum  mass s p e c t r u m o f t h i s m a t e r i a l  assigned with  p a t h w a y s m u s t be  The  This observation  s p e c t r u m was  a m u l t i p l e t a b s o r p t i o n n e a r x6.7  t h i o a c e t a l methylene protons.  Two  treated with  spectrum of the carbomethoxyl  a b s o r p t i o n a t x 4 . 8 2 ( J = 8 H z ) w h i c h was  was  thioacetal  i n r e f l u x i n g m e t h a n o l f o r 48 h o u r s a s m a l l q u a n t i t y  the absence o f c a r b o n y l a b s o r p t i o n s .  p r o t o n , and  on  o  indicated  doublet  a  C„_H„,N„0 S„. 23 26 2 2 2  r i n g - o p e n i n g of the  (A 2 2 8 , 2 8 4 , 2 9 1 ) m a t e r i a l was max  absorption.  to  Further confirmation  A h i g h r e s o l u t i o n mass m e a s u r e m e n t formula  C-17  assigned  i t s mass s p e c t r u m w h i c h showed  the m o l e c u l a r  designed  ( 7 3 ) was  of i n d o l i c  to the  one-  then undergo r i n g - o p e n i n g  have proceeded f i r s t  which could also decarboxylate  to  give product.  to g i v e the d e s i r e d  under these  reaction conditions.  -164-  H o w e v e r , a t no  time  during  intermediate products found  that this  refluxing  be  the course  detected  by  potassium  f r o m the carbomethoxy  I t was  sodium  by  t h i s m a t e r i a l to the i n d o l e t h i o a c e t a l  The  t h i s p a t h w a y c o u l d be  achieved,  the r e d u c t i v e r i n g - o p e n i n g The  indolenine  a b o v e r e s u l t s may (73)  o f compound indicate  i s base-catalysed  have caused d e c a r b o x y l a t i o n  Therefore,  i t was  decided  conditions  i n the presence  thioacetal  ( 7 3 ) was  2 h o u r s two  to attempt  this  (73)  to the  indolic  ions  anhydrous  sodium borohydride.  tic.  Subsequently,  f o l l o w i n g d a t a was uv  (74).  carbomethoxy  molar s o l u t i o n of  p i c and- t h e  the  or intermediate  The  were d e t e c t e d by  p o l a r m a t e r i a l e x h i b i t e d an  course  of h y d r o x y l  t r e a t e d w i t h anhydrous sodium  major products  (76)  unanswered.  c o n v e r s i o n under  of sodium methoxide.  the  Therefore,  remains  the presence  o f compound  t h e s e m a t e r i a l s were i s o l a t e d by less  but  (73)  or  t h a t the r i n g - o p e n i n g o f  d i s s o l v e d i n a 0.2  m e t h o x i d e i n m e t h a n o l and After  the q u e s t i o n as  of  subsequent  (75)  done i n r e f l u x i n g m e t n a n o l w i t h s o d i u m b o r o h y d r i d e .  since  The  hydroxide.  i t s synthesis  treatment  ring-opening of  may  potassium  achieved  10%  of  borohydride.  (77) w e r e r e q u i r e d a n d  ( 7 3 ) was  also  p r o b l e m some  l a t t e r w i t h hot  was  methanolic  and  any  i n 2 hours i n  approach to t h i s  thioacetal  thioacetal  reaction could  achieved  hydroxide  In connection w i t h another the a - v i n y l  this  t i c monitoring.  same c o n v e r s i o n c o u l d be  5% m e t h a n o l i c  q u a n t i t i e s of  of  spectrum  (A  obtained. 228,284,291)  max and  no  o f one to  c a r b o n y l a b s o r p t i o n i n the absorption  t h e pmr  the s p e c t r u m o f p r o d u c t  i r spectrum.  spectrum of (75)  or  (76)  Except  f o r the  t h i s m a t e r i a l was  very  previously obtained.  position similar A  one  -165-  F i g u r e 22  (75)  R  1  = H,  R  (76)  R  1  = < ], S  S  - Reactions of  epimer of  R  2  =  H  thioacetal  p r o t o n d o u b l e t appeared a t x5.32 which o f t h e o t h e r C-16  =  2  was  the ring-opened  (73).  assigned  t o t h e C-17  thioacetal  u n a m b i g u o u s a s s i g n m e n t o f t h e s t e r e o c h e m i s t r y a t C-16 tentative.  An  t h a t when t h e  examination  was  a-oriented (structure These r e s u l t s  of m o l e c u l a r models r e v e a l e d the  shielding  and  environment than  prevented  75)  The  point  possibility the  thioacetal  C-17 group  76).  s u b s e q u e n t w o r k on  s y n t h e s i s of stemmadenine d e r i v a t i v e s c o u l d n o t be  i f the  (76).  i s at this  group i s 3 - o r i e n t e d ( s t r u c t u r e  thioacetal  proton i s i n a less  (75) o r  proton  this  approach  made i t c l e a r  under b a s i c c o n d i t i o n s .  The  to a c h i e v e  the  that decarboxylation ring-opening  of  -166-  thioacetal acid  (73)  solvent.  was  then attempted under v a r i o u s  The  t h i o a c e t a l (73)  was  conditions  treated with  in glacial  a c e t i c a c i d a t r o o m t e m p e r a t u r e f o r one  and  f o r 15 m i n u t e s i n a s e c o n d s t u d y .  a t 90°  mixture  o f p r o d u c t s was  m a t e r i a l c o u l d be  obtained.  identified.  material.  A  third  methanol w i t h borohydride  failed  50%  results described ( 8 ) and  i n this Part  further investigation.  identical with  product  We  (7) has  (60).  may  f u t u r e be  Future One  the  isolated  as  work i n t h i s area  o b j e c t i v e i s the  achieve  At  other indolic  dissolved  in  sodium  this point  reduction  indicate that  this  of product  (69).  the  third  (60)  require a  been achieved  the  and  that (7)  of  great  synthesis latter  i s at  proposed b i o g e n e t i c  is  this  conversion  Strychnos f a m i l y of a l k a l o i d s , but  be  product. d i r e c t e d toward  three  objectives.  the aldehyde group i n p r o d u c t  p r e a k u a m m i c i n e d e r i v a t i v e (69)  conversion  synthesis  the  should of  the  b e l i e v e , however, t h a t  a natural  n a t u r a l l y o c c u r r i n g preakuammicine  The  the  treated with  (4) d e r i v a t i v e s w i l l  i n the  C o r y n a n t h e a l k a l o i d s to the  i n the  of  was  Preakuammicine aldehyde  point a hypothetical intermediate the  t h i o a c e t a l (73)  indolic material.  stemmadenine  of preakuammicine aldehyde  of  study  complex  starting  t o show t h e p r e s e n c e o f  a c e t i c a c i d and  to g i v e  study  In both s t u d i e s a  spectroscopic  i n w h i c h the  and  h o u r i n one  abandoned.  preakuammicine d e a l of  10%  also failed  a p p r o a c h was The  1%,  study  borohydride  O n l y s m a l l amounts of  A uv  components from these r e a c t i o n s  sodium  in acetic  (8).  and  compare the  latter  Another o b j e c t i v e should  to stemmadenine  o b j e c t i v e that should  (4) d i r e c t l y be  included  (60)  to  with be  the  or v i a a l c o h o l in this investigation  -167-  is  t h e c o n v e r s i o n o f stemmadenine  M i g r a t i o n of the double  (4) t o iso-stemmadenine ( 1 0 ) .  b o n d f r o m p o s i t i o n C-19,20 t o p o s i t i o n C-20,21  has a l r e a d y been a c h i e v e d  i n the Strychnos  a r e a u s i n g Raney  The e v a l u a t i o n o f t h e b i o s y n t h e s i s a n d t h e b i o m i m e t i c iso-stemmadenine understanding  (10) w o u l d be o f g r e a t i m p o r t a n c e  of t h i s  nickel.  chemistry of  t o a more  f a s c i n a t i n g area of n a t u r a l products.  complete  -168-  EXPERIMENTAL  Thin  l a y e r chromatographic  u s i n g EM R e a g e n t s GF254 S i l i c a chromatoplates  ( t i c ) s t u d i e s were c a r r i e d o u t  g e l o r Woelm n e u t r a l a l u m i n a .  u s e d f o r a n a l y s i s w e r e 0.3 mm  u s e d f o r p r e p a r a t i v e s e p a r a t i o n s w e r e 1.0 mm electronic to  phosphor  the  i n t h i c k n e s s and thick.  ( a b o u t 2% b y w e i g h t ) was a d d e d  aidvisualization.  The c h r o m a t o p l a t e s  The those  I n a l l cases, to the absorbent  were d e v e l o p e d i n one o f  f o l l o w i n g solvent systems: eluent A c o n s i s t e d o f chloroform  1% m e t h a n o l , e l u e n t B c o n s i s t e d o f c h l o r o f o r m  plus  plus  2% m e t h a n o l , a n d  e l u e n t C c o n s i s t e d o f d i c h l o r o m e t h a n e p l u s 10% m e t h a n o l and 0.1% triethylamine.  The c h r o m a t o p l a t e s  wavelength u l t r a v i o l e t eerie sulphate  spray  scanning  or iodine  were examined under a l o n g and s h o r t  lamp and v i s u a l i z e d  alumina i n large scale preparations  to  height  a s 1:10.  Shawinigan n e u t r a l  a n d Woelm n e u t r a l a l u m i n a  s e p a r a t i o n s were r e q u i r e d .  were g e n e r a l l y maintained  a  vapors.  C o l u m n c h r o m a t o g r a p h y was p e r f o r m e d u s i n g  more e x a c t  further with  when  The d i m e n s i o n s o f t h e c o l u m n s  a t the accepted  optimum r a t i o  of diameter  Throughout t h i s work, a l l s o l v e n t s were  distilled  before use. Ultraviolet spectrophotometer. (nm).  s p e c t r a were recorded Absorption  values  i n m e t h a n o l o n a C a r y 15  ) a r e g i v e n i n nanometers max I n f r a r e d s p e c t r a were measured i n c h l o r o f o r m on a P e r k i n - E l m e r  m o d e l 137 d o u b l e beam i n s t r u m e n t , quoted i n wavenumbers  (cm "*") .  (A  and a b s o r p t i o n bands  (v ) are max  -169-  Proton magnetic resonance deuterochloroform  (pmr)  a t room t e m p e r a t u r e  o r a V a r i a n XL-100 s p e c t r o m e t e r .  s p e c t r a were r e c o r d e d  a t 100  The  l a t t e r was  s p e c t r a i n t h e F o u r i e r t r a n s f o r m mode. the T i e r ' s T s c a l e u s i n g a t TIO.OO.  Coupling  T-values g i v e n to m u l t i p l e t  ratios  (m/e)  signals refer  Fragmentation  f o l l o w e d by  percent  Elemental  Conversion The  has  data  of S t r y c h n i n e  t h e l a t t e r was  a v a i l a b l e no  mediates.  procedures  from those  on  (Hz). the  signal.  charge  High  the AEI-MS-902 o r an  resolution AEI-MS-50  on a K o f l e r b l o c k and  U n i v e r s i t y of B r i t i s h  (29)  21-23  P.  are  Borda  Columbia,  to Wieland-Gumlich Aldehyde  and  to Wieland-Gumlich  given here f o r these  u s e d a r e a p p r o p r i a t e l y r e f e r e n c e d and  of Vancouver.  (32) aldehyde  s i n c e an a u t h e n t i c sample  d a t a need be  20  of  interdiffer  published.  23-Isonitrosostrychnine Hydrochloride A  standard  i s g i v e n i n mass t o  a n a l y s e s w e r e p e r f o r m e d b y Mr.  been d e s c r i b e d i n d e t a i l  little  the i n t e r n a l  to the c e n t e r o f  c o n v e r s i o n o f s t r y c h n i n e (29)  The  s h i f t s are given i n  standards.  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  record  on a n A E I - M S - 9 0 2 o r a n A t l a s CH-43  M e l t i n g p o i n t s were determined uncorrected.  a l s o used to  r e l a t i v e abundance.  mass m e a s u r e m e n t s w e r e d e t e r m i n e d using suitable  o n a V a r i a n HA-100  (J-values)are given i n hertz  Mass s p e c t r a were r e c o r d e d mass s p e c t r o m e t e r .  Chemical  t e t r a m e t h y l s i l a n e as  constants  MH^  in  three-necked  (30)  round bottom f l a s k equipped  with a  thermometer  -170-  and  a dry  ice-acetone  (50g, d r i e d (98g,  ^2^5^  over  t o , and  maintained  dissolved hours.  n  The  (100 ml)  reduced  (100)  and  Norit  (3g)  u  t  filtration  of  again  a t 70°  occurred  d i s s o l v e d i n water  w a t e r and lization  dried  was  continued  allowed  being raised  (16g) for 4  to c o o l  and  To  this  brought to a b o i l  acetic  added  and  After  filter.  o b t a i n e d by  T h i s s o l u t i o n was  23  the above s a l t  a gray  stirred  (32) (40g)  previously cooled  30 m i n u t e s and  i t was  was  i n an  a t room t e m p e r a t u r e .  c a u t i o u s l y poured onto i c e , w i t h precipitate.  vacuum  from methanol,  Wieland-Gumlich Aldehyde Hydrochloride i n vacuo  acid  s o l u t i o n was  recrystallized  220°, d e c o m p ) .  water  iso-pentylnitrite.  (500), g l a c i a l  (40).  A s m a l l s a m p l e was  t h e s o l u t i o n was  after  was  o v e r n i g h t a c r y s t a l l i n e m a t e r i a l was  to y i e l d  While  i n vacuo t r e a t e d w i t h c o l d  s l o w l y a d d e d t o t h i o n y l c h l o r i d e (120 m l )  stirring,  added.  the r e a c t i o n mixture  to remove u n r e a c t e d  After drying thoroughly  4 hours,  Iso-pentylnitrite  the a d d i t i o n of sodium metal  evaporated  ( d e c o m p ) ( l i t . m.p.  ice-water bath.  (29)  overnight.  t h e m i x t u r e was  (43.5g).  N(a)-Cyanoformyl  (800 m l ) .  p r e s s u r e ) was  Stirring  cone, h y d r o c h l o r i c a c i d and  ethanol  e  during  with strychnine  d a r k b r o w n s o l u t i o n was  evaporated  s t a n d i n g a t 5°  218°  °l  (500 m l ) .  continued  b u l k was  S  temperature  s o l u t i o n was  and  D  charged  under reduced  the  resulting  The  a  a t , 70°  i n ethanol  s t i r r i n g was  m.p.  ^  freshly distilled  stirred mechanically  The  c o n d e n s e r was  Crystallization  then f i l t e r e d ,  After  vigorous  of t h i s m a t e r i a l  washed w i t h  i n vacuo to g i v e a h y d r o c h l o r i d e s a l t  (31g).  f r o m m e t h a n o l y i e l d e d c o l o u r l e s s m a t e r i a l , m.p.  220°  cold  Recrystal(decomp)  -171-  (lit.  m.p.  220°, decomp).  Wieland-Gumlich The two-necked was  crude s a l t  (32)  2 3  ( 2 9 g ) f r o m t h e a b o v e r e a c t i o n was a d d e d  f l a s k charged w i t h d i s t i l l e d  heated i n an o i lbath  rapidly of  Aldehyde  23  injected  into  (120°).  water  (400 m l ) .  A f t e r b o i l i n g began,  t h e b o i l i n g m i x t u r e f o r 1-2 h o u r s .  to a  This mixture s t e a m was The p r o g r e s s  t h e r e a c t i o n was m o n i t o r e d b y w o r k i n g up s m a l l a l i q u o t s w h i c h  examined  by t i c on a l u m i n a  (eluent B).  When t h e h y d r o l y s i s a p p e a r e d c o o l e d a n d made a l k a l i n e w i t h c o l d  t o be c o m p l e t e ,  (19g).  act.Ill)  t h e m i x t u r e was  ammonium h y d r o x i d e .  e x t r a c t i o n w i t h c h l o r o f o r m and e v a p o r a t i o n i n vacuo residue  were  T h i s m a t e r i a l was c h r o m a t o g r a p h e d  using benzene-ethylacetate gradient.  f r a c t i o n s as d e t e r m i n e d by t i c y i e l d e d  Repeated  yielded  a brown  on a l u m i n a  (500g,  Pooling of the appropriate  a colourless  crystalline  material  (17g) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h an authentic  sample  methanol-acetone 212.5-215°).  20  '' of Wieland-Gumlich aldehyde. v  yielded  Recrystalization  c o l o u r l e s s n e e d l e s , m.p.  212-215° ( l i t .  from m.p.  2 3  2g,16a -cur-19-en-17,18-diol Wieland-Gumlich  (33)  aldehyde  2 2  (5g)  was d i s s o l v e d  i n methanol  and  s o d i u m b o r o h y d r i d e ( 0 . 5 g ) was a d d e d o v e r 30 m i n u t e s  The  s o l u t i o n was s t i r r e d  f o ra further  (10 m l ) was a d d e d a n d s t i r r i n g  30 m i n u t e s  (50 m l )  a t room t e m p e r a t u r e .  a f t e r which  time water  c o n t i n u e d f o r a f u r t h e r 15 m i n u t e s .  This  -172-  s o l u t i o n was in  chloroform  t h e n e v a p o r a t e d i n v a c u o and  t h e r e s i d u e was  t a k e n up  c o n t a i n i n g 1 0 % m e t h a n o l and  chromatographed  on a l u m i n a  (lOOg, a c t . I I I ) .  Elution with ethylacetate yielded a material  ( 4 . 1 g ) w h i c h was crystals.  recrystallized  After drying  250-254° ( l i t . m.p.  from methanol-water  thoroughly  251-253) ;  i n vacuo, t h i s  X  2 2  (indoline);  4H,  aromatic OH),  J=2  and J=7;  4.27  pmr  compound h a d  243, 297; v max  3300 (0NH), 1600  to g i v e c o l o u r l e s s  3600  J=7,  (multiplets,  I H , C - 1 9 H ) , 5.88  2H, C - 1 8 H ) ; m a s s s p e c t r u m : M  +  2  312  (35)  Wieland-Gumlich g l y c o l hydrogen bromide  in glacial  f l a s k was  and  acetic  sealed  acid  ( 4 0 0 m l ) was  and  was  removed  t h e m i x t u r e was  was  t h e n removed  added  to a s o l u t i o n of  _ i n v a c u o and more  F r e s h l y a c t i v a t e d z i n c powder  shaken v i g o r o u s l y f o r 5 hours.  and washed w i t h a c e t i c a c i d and  These w a s h i n g s were combined w i t h i n vacuo.  Found:  The  a t room t e m p e r a t u r e f o r 2 d a y s .  added.  by f i l t r a t i o n  312•1837.  (100);  22  ( 3 3 , 3.6g)  l e t stand  o f t h e s o l v e n t was  added  :  ( 4 3 ) , 144  a c e t i c a c i d -(250 m l , 3% s a t u r a t e d ) .  Most  was  (multiplet,  ( 8 2 ) , 182  h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c . f o r ^^9^24^2°2 312.1841.  2g-16a-17-acetoxy-cur-19-en  (-0H),  max  s i g n a l s : 2.9-3.4  (triplet,  m.p.  the f i l t r a t e  then  glacial (50g) The  zinc  ethanol.  and r e d u c e d i n volume  The  r e s i d u e was  d i s s o l v e d i n d i l u t e ammonium h y d r o x i d e a n d  extracted with  chloroform,  d r i e d over sodium s u l p h a t e  and  evaporated to  * P o w d e r e d z i n c was a c t i v a t e d b y w a s h i n g t w i c e w i t h 5% a q u e o u s h y d r o c h l o r i c a c i d , f o l l o w e d by s u c c e s s i v e washings w i t h methanol and e t h e r , and t h e n d r i e d i n v a c u o .  -173-  g i v e a l i g h t b r o w n gum on a l u m i n a v  ( e l u e n t B)  1720  max  (1.2g).  E x a m i n a t i o n o f t h i s m a t e r i a l by t i c  r e v e a l e d t h e p r e s e n c e o f one  (acetate).  T h i s m a t e r i a l was  used  major  component,  as s u c h i n the  next  reaction.  2g,16a-Cur-19-en-17-ol The  (36)  2 2  a b o v e a c e t a t e ( l g ) was  hydroxide  (10%) f o r 1 hour.  refluxed  With cooling,  neutralized with dilute hydrochloric extracted with chloroform. w i t h anhydrous  sodium  amorphous m a t e r i a l  The  this reaction mixture  acid,  reduced  e v a p o r a t e d jLn v a c u o  chromatographed  on a l u m i n a  with a benzene-ethylacetate gradient yielded c o u l d be  177-178 ) 0  pmr  2 6  ;  signals:  crystallized  X' max  2.8-3.5  I H , C - 1 9 H ) , 8.50 M  +  243,  i n volume  was  and  were  dried  to g i v e a brown  mg).  T h i s m a t e r i a l was  which  potassium  combined o r g a n i c washings  s u l p h a t e and  (910  i n methanolic  from benzene, v  297,  max  (multiplets,  (multiplet,  J=7  (act.IV).  a p u r e compound  m.p.  and  h i g h r e s o l u t i o n mass measurement; C a l c .  m.p.  (quartet,  C - 1 8 C H ) ; mass 3  296(89), 281(17), 279(15), 265(12), 166(100),  mg)  1600(indoline);  a r o m a t i c - H ) , 4.58 3=2,  (850  172-175° ( l i t .  3575(N-H), 3350(0-H), 4H,  Elution  144(43),  J=7,  spectrum: 130(31);  f o r C^gH^^N^O: 2 9 6 . 1 8 8 7 , F o u n d :  296.1888.  Catalytic  Hydrogenolysis of Wieland-Gumlich  The acetic  acid  glycol  ( 3 0 0 mg)  (12 m l )  and  was  dissolved  hydrochloric acid  Glycol  (33)  i n a s o l u t i o n of water (0.2 m l ) .  (20 m l ) ,  After addition  of  -174-  palladium-charcoal  c a t a l y s t (10%,  75 mg) t h e r e a c t i o n m i x t u r e  f l u s h e d w i t h hydrogen and l e t s t i r  i na slightly  hydrogen atmosphere.  o f t h e r e a c t i o n was m o n i t o r e d b y  The p r o g r e s s  positive  was  pressure  volume o f gas u p - t a k e . The bath  reaction mixture  was t h e n f i l t e r e d ,  cooled  i n an  a n d made a l k a l i n e w i t h a q u e o u s p o t a s s i u m h y d r o x i d e  white  precipitate.  chloroform  The t o t a l m i x t u r e  (500 m l ) .  foam  a compound  resulting i n  was e x t r a c t e d r e p e a t e d l y  (285 mg).  and e v a p o r a t e d _in vacuo t o g i v e a  Chromatography o f t h i s product  23,16a-cur-19-en-17-ol  with  light  on a l u m i n a  ( 2 7 0 mg) w h i c h h a d p h y s i c a l a n d s p e c t r o s c o p i c  identical with  with  The o r g a n i c w a s h i n g s w e r e c o m b i n e d , d r i e d  anhydrous sodium sulphate yellow  ice-water  gave  properties  (36) which had p r e v i o u s l y been  obtained.  Birch Reduction The  o f Wieland-Gumlich Aldehyde (32)  aldehyde  and  d r y _t-butanol  dry  ice-acetone  stirring  bar.  ( l g ) was s u s p e n d e d i n d r y d i m e t h o x y e t h a n e  (0.5 ml) i n a round bottom f l a s k equipped w i t h  condenser, a source This mixture  (-78°) a n d t h e n f i l l e d  30 m i n u t e s .  a dark blue  c o l o u r was r e m o v e d ) .  allowed  t o warm a s t h e ammonia  c o l o u r c o u l d be  maintained  to destroy  The r e a c t i o n m i x t u r e  the excess was t h e n  evaporated.  r e s i d u e was e v a p o r a t e d  between water and c h l o r o f o r m .  bath  Freshly cut chips of  Ammonium c h l o r i d e was t h e n a d d e d  ( i . e . blue  The  i n a dry ice-acetone  w i t h ammonia ( 1 0 0 m l ) .  lithium  a  o f ammonia g a s a n d a m a g n e t i c  was c o o l e d  l i t h i u m m e t a l were added u n t i l for  (75 m l )  f u r t h e r i n vacuo before  The c o m b i n e d c h l o r o f o r m  partitioning  washings were  -175-  d r i e d w i t h anhydrous a light yellow  sodium sulphate  f o a m ( 9 1 5 mg).  and e v a p o r a t e d i n vacuo  T h i s m a t e r i a l was p a s s e d  to give  through a  column of a l u m i n a (20g, a c t . I l l ) w i t h a b e n z e n e - e t h y l a c e t a t e s o l v e n t gradient  to y i e l d  a compound  scopic properties identical had been p r e v i o u s l y  ( 8 9 0 mg) w h i c h h a d p h y s i c a l a n d with  26,16a-cur-19-en-17-ol  spectro-  (36) w h i c h  obtained.  B i r c h R e d u c t i o n o f W i e l a n d - G u m l i c h G l y c o l (33) The g l y c o l described  ( 1 5 0 mg) was h y d r o g e n o l y s e d u n d e r  above f o r W i e l a n d - G u m l i c h aldehyde  a compound  ( 1 2 1 mg) was o b t a i n e d  properties  identical  previously  obtained.  with  t h e same  (32). After  w h i c h had p h y s i c a l and  2B,16a-cur-19-en-17-ol  conditions  chromatography,  spectroscopic  (36) w h i c h had been  -176-  Oppenauer O x i d a t i o n o f 2B,16a-cur-19-en-17-ol  (36) by  Boekelheide's  Procedure The (100  a l c o h o l ( 3 6 ) (300  ml, freshly d i s t i l l e d  benzophenone  mg) was d i s s o l v e d i n r e f l u x i n g from l i t h i u m aluminum h y d r i d e )  ( 9 1 0 mg, s u b l i m e d ) .  Potassium _t-butoxide  added and t h e r e a c t i o n was s t i r r e d atmosphere. and  three  times  was p a r t i t i o n e d .  with dichloromethane  ( 5 6 0 mg) w a s  ( 1 0 0 m l ) was a d d e d  The a q u e o u s l a y e r was e x t r a c t e d (250 m l ) and t h e combined  e x t r a c t s were washed w i t h w a t e r b e f o r e  A f t e r r e m o v a l o f t h e s o l v e n t , t h e r e s i d u e was  on  (30 g, a c t . I l l )  gradient. product  ( 1 1 8 mg) a s a y e l l o w  1640  m.p.  foam.  0  2 6  ;  I H , CHO), 2.7 - 3.2  ( m u l t i p l e t s , 4H, a r o m a t i c - H ) ,  3  =CH-CH ); mass s p e c t r u m : M 3  +  (doublet  of doublets,  measurement: C a l c . f o r C ^ H ^ ^ O :  17-al (37).  (N-H),  4.68  J=7 a n d 2, 3H,  292 ( 6 3 ) , 2 6 3 ( 1 5 ) , 249 ( 2 1 ) , 2 4 3 ( 2 6 ) ,  ( 1 7 ) , 208 ( 1 5 ) , 194 ( 1 8 ) , 167 ( 2 2 ) , 1 2 1 ( 1 0 0 ) ;  A minor product  182-185°  pmr: -0.2 ( b r o a d s i n g l e t , I H , N - H ) , 0.70  ( q u a r t e t , J = 7 , I H , = C H - C H ) , 8.48  222  T h i s m a t e r i a l was i d e n t i f i e d a s  X 244 2 9 0 s h , 2 9 8 , 3 6 3 ; v 3300 max max  (CHO), 1605 ( i n d o l i n e ) ;  (singlet,  solvent  f r a c t i o n s y i e l d e d the major  ( 3 9 ) b a s e d o n t h e f o l l o w i n g d a t a : m.p.  185-186 )  sodium  chromatographed  a petroleum ether-benzene  Pooling of the appropriate  nor-fluorocurarine (lit.  using  organic  d r y i n g over anhydrous  sulphate. alumina  with  f o r 2 hours under an i n h e r t  A f t e r c o o l i n g i n an i c e - b a t h , w a t e r  the mixture  benzene  292.1575.  ( 1 4 mg) was l a t e r  Found:  identified  high  r e s o l u t i o n mass  292.1567. as 23,16a-cur-19-en-  -177-  Modified  Oppenauer O x i d a t i o n of  2g,16ct-cur-19-en-17-al The  (50 m l ,  Approximately  5 ml  added.  This mixture  potassium hydride  water bath, water  was  was  alumina  solvent gradient.  The  the  mg  added and  (8 g,  the  major product  f r a c t i o n s was  247,  (indoline);  pmr:  aromatic-H), 2,  0.24  4.60  J=7  and  3H,  164  ( 9 7 ) , 144 294.1731.  297;  (singlet,  others  Modified  was p a r t i t i o n e d . the  combined  d r i e d o v e r an h y d r o u s  IH,  mg)  obtained  by  pooling  3 4 5 0 ( N - H ) , 1720  IH,  CHO),  2.8  - 3.5  = C H - C H ) , 8.40 3  was  the based  (CHO),  1600  +  294  (multiplets, (doublet  ( 4 2 ) , 251  of  4H, doublets,  ( 1 6 ) , 199  (15),  for'^ig^22^2  294.1747. compared s p e c t r o s c o p i c a l l y w i t h  laboratory f o r another  Nor-fluorocurarine  sodium  ether-benzene  ( 1 0 0 ) , h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c .  Oppenauer O x i d a t i o n  The  A f t e r c o o l i n g i n an i c e -  using a petroleum  v  3  i n our  was  2 3 , 1 6 a - c u r - 1 9 - e n - 1 7 - a l (37)  =CH-CH ); mass s p e c t r u m : M  T h i s m a t e r i a l was by  reaction  max  ( q u a r t e t , J=7,  Found:  mg)  s o l v e n t i n vacuo, the r e s i d u e  (105  i d e n t i f i e d as  X  following data,  (270  o i l suspension)  the m i x t u r e  act.Ill)  max  0:  of a 22%  from the  e x t r a c t e d w i t h d i c h l o r o m e t h a n e and  c h r o m a t o g r a p h e d on  on  (200  A f t e r r e m o v a l of most of  appropriate  give  from l i t h i u m aluminum h y d r i d e ) .  e x t r a c t s w e r e washed w i t h w a t e r and  sulphate.  to  d i s s o l v e d w i t h benzophenone  r e f l u x e d f o r 1 hour.  (50 m l )  a q u e o u s l a y e r was  organic  was  of benzene were then d i s t i l l e d  before  The  mg)  freshly distilled  mixture  (36)  (37)  a l c o h o l (36)(145  i n benzene  2g, 1 6 a - c u r - 1 9 - e n - 1 7 - o l  of  study.  that  prepared  24  2g,16a-cur-19-eri-17-ol  (36)  to  give  (39)  a l c o h o l (36)(295  mg)  was  d i s s o l v e d i n benzene  (70 m l ,  freshly  -178-  dlstilled  from  lithium  were d i s t i l l e d  from  of  the o i l b a t h  of  a 22%  was  had  o v p r  15  The  and  (50 m l )  water  layer  The  combined  and  most  of  r e s i d u e was  material  was  and  s t i r r i n g was  added.  then  This  the  solvent  was  chromatographed solvent  physical  nor-fluorocurarine  and (39)  the  of  benzene  temperature  i n an  (0.45  inhert  ml)  was  continued for a  c o o l e d i n an  m i x t u r e was  removed on  mg)  i n vacuo.  alumina The  which  over  further  partitioned  (30  g,  The  act.Ill)  and  crystallized  spectroscopic  previously synthesized.  ml).  sulphate  coloured  were  from benzene.  properties  the  (250  using a  appropriate fractions  15  bath  sodium  highly  mg  added  ice-water  anhydrous  (350  atmosphere  repeatedly with dichloromethane  gradient. (179  7 ml  potassium hydride  Stirring  Nitrobenzene  v e s s e l was  extracted  y e l l o w foam had  minutes.  reaction was  added.  about  After  to 60-65°,  o r g a n i c e x t r a c t s were d r i e d  ether-benzene yield  lowered  minutes  minutes.  of  been  o i l s u s p e n s i o n ) was  dropwise  to  the r e a c t i o n m i x t u r e .  c o n t i n u e d f o r 10  aqueous  aluminum h y d r i d e ) and  identical  petroleum pooled This  with  those  -179-  l-Formyl-2B-16a-cur-19-en-17-0-formate  (42)  The indoline alcohol (36)(900 mg) was dissolved i n a c e t i c 29 formic anhydride minutes.  (10 ml) and s t i r r e d at room temperature f o r 15  The solvent was  then removed i n vacuo at ambient  The residue was chromatographed  temperature.  on alumina (20 g, a c t . I l l ) using  benzene as the eluent to y i e l d , a f t e r removal of solvent, a l i g h t 247, 282, 287; v  yellow foam (940 mg), A max (N-CHO), 1600  ( i n d o l i n e ) , pmr: 1.51  ( s i n g l e t , IH, 0-CH0), 4.4  1720  (0-CHO), 1660  max ( s i n g l e t , IH, N -CHO), 2.00  (quartet, J=7, IH, =CH-CH ) 8.40 3  of doublets, J=7 and 2, 3H, =CH-CH ); mass spectrum: M  +  3  324  (doublet  352 (81), 337(37)  (49), 279 (100), 144 (81).  l-Formyl-2g-16a-cur-19-en-17-ol  (43)  The diformate (42)(940 mg) was refluxed i n methanol 30 minutes.  (15 ml) for  After the removal of the solvent in_ vacuo, a t i c examination  (alumina, eluent A) revealed the presence of a single component. material was chromatographed  on alumina (20 g, a c t . I l l ) to y i e l d a 247, 282, 287, v  l i g h t yellow foam (780 mg), A max 1600  (indoline); pmr: 1.24  IH, =CH-CH ), 8.34 3  spectrum: M  +  This  1660  (N-CHO),  max  ( s i n g l e t , IH, N-CHO), 4.48  (quartet, J=7,  (doublet of doublets, J=7 and 2, 3H, =CH-CH ); mass 3  324 (77), 309 (49), 293 (31), 279 (100); high r e s o l u t i o n  mass measurement: Calc. for C_ H .N„0_ 324.1837. 20 24 2 2 n  0  Oxidation of N-formyl Alcohol (43) with C o l l i n ' s  Found: 324.1826.  Reagent  20 C o l l i n ' s reagent was prepared i n s i t u (30 ml, f r e s h l y d i s t i l l e d from  i n dichloromethane  using chromium trioxide (200 mg,  -180-  dried  overnight  i n h i g h v a c u u m a t 100°) a n d p y r i d i n e ( 0 . 4 m l , d i s t i l l e d  f r o m KOH) a t 0 ° . After stirring solution  To t h i s s o l u t i o n was a d d e d t h e a l c o h o l ( 4 3 ) ( 1 0 0 m g ) .  f o r 45 m i n u t e s , s a t u r a t e d a q u e o u s s o d i u m  (30 m l ) was added and t h e r e s u l t i n g m i x t u r e  a f u r t h e r 30 m i n u t e s b e f o r e of  partitioning.  t h e aqueous l a y e r w i t h d i c h l o r o m e t h a n e ,  bicarbonate  was s t i r r e d f o r  A f t e r repeated the organic  extraction  e x t r a c t was  washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . coloured  residue obtained  a f t e r r e m o v a l o f t h e s o l v e n t i n v a c u o was  chromatographed on a l u m i n a f r a c t i o n s were combined  The d a r k  (5 g, a c t . I I I ) .  to y i e l d  (39 m g ) , w h i c h was a s s i g n e d  The a p p r o p r i a t e  benzene  a l i g h t y e l l o w amorphous m a t e r i a l  s t r u c t u r e (44) based on t h e f o l l o w i n g d a t a :  X 242, 282, 288; v 1 7 2 0 ( C - C H 0 ) , 1 6 6 5 ( N -CHO), 1 5 9 0 ( i n d o l i n e ) ; max max — pmr:  0.22 ( d o u b l e t ,  1.14  (singlet,  IH,  (multiplet,  I H , = C H - C H ) , 5.18 ( m u l t i p l e t , 3  3H, = C H - C H ) ; mass s p e c t r u m : M 3  ( 7 4 ) , 1 8 0 ( 4 4 ) , 164 ( 1 0 0 ) h i g h  for C  2 Q  H  2 2  Oxidation  N 0 2  2  322.1680.  Found:  of N-formyl Alcohol  Silver  0.5H, C-CHO),  0.5H, N-CH0) 1.40 ( s i n g l e t , 0.5H, N-CHO), 2.0  C - 1 2 N ) , 4.46 ( m u l t i p l e t ,  8.45 279  J = 4 , 0.5H, C-CHO), 0.37 ( s i n g l e t ,  +  (multiplet, I H , C-16H),  322 ( 4 1 ) , 293 ( 5 0 ) ,  r e s o l u t i o n mass measurement:  Calc.  322.1678.  (43) w i t h  Silver  c a r b o n a t e o n c e l i t e was p r e p a r e d  Carbonate according  to Fetizone's  36 procedure. alcohol and  This reagent  ( 0 . 4 m m o l e s ) was r e f l u x e d w i t h  ( 4 3 ) ( 9 6 mg, 0.3 m m o l e s ) i n b e n z e n e o v e r n i g h t .  removal of the solvent  e l u e n t A) and f o u n d  the N-formyl  After  t h e r e s i d u e was examined b y t i c  to contain a mixture  (95 mg) was c h r o m a t o g r a p h e d o n a l u m i n a  o f components.  (6.5 g, a c t . I l l )  filtration  (alumina,  This  mixture  u s i n g a benzene-  -181-  ethyl acetate  solvent  gradient.  Pooling  of the  appropriate  chromatographic f r a c t i o n s y i e l d e d a l i g h t yellow (24 m g ) , with  w h i c h had p h y s i c a l and  spectroscopic  those of the N-formyl aldehyde  characterized. material  A second  component  amorphous  properties  (44) w h i c h had ( 1 0 mg)  was  material  identical  p r e v i o u s l y been  identified  as  starting  (43).  Oxidation  of N - f o r m y l Alcohol  (43) w i t h  DMS0-S0  The  alcohol  ( 4 3 ) ( 1 0 0 mg)  was  o  -5  3.  d i s s o l v e d i n dry dimethyl  sulphoxide  o  (1 m l , f r e s h l y  distilled  pyridine-sulphur After  stirring  trioxide  f r o m 4A m o l e c u l a r complex  organic  ( 3 0 0 mg)  and  a t room t e m p e r a t u r e f o r 1 h o u r ,  added t o the r e a c t i o n m i x t u r e The  sieves)  before  i t was  and  treated  t r i e t h y l a m i n e (0.1 m l ) . 20 v o l u m e s  of water  extracted with  w a s h i n g s w e r e d r i e d o v e r anhydro'us  with  sodium  were  benzene.  sulphate  and  e v a p o r a t e d t o y i e l d a c o l o u r l e s s amorphous m a t e r i a l .  Chromatography  of  w h i c h had  and  t h i s m a t e r i a l y i e l d e d a n a m o r p h o u s compound spectroscopic  properties identical with  (18 mg)  the N -formyl  physical  aldehyde  (44)  3.  previously mixture  characterised.  had  starting  component  p h y s i c a l and s p e c t r o s c o p i c  (62 mg)  from t h i s  properties identical  product  with  material (43).  Oxidation Trioxide  A second  of N^-formyl A l c o h o l  (43) w i t h  3,5-Dimethylpyrazole-chromium  Complex  The  alcohol  ( 4 3 ) ( 9 0 mg,  0.3 v.  m m o l e s ) was  added t o a s t i r r e d s o l u t i o n  -182-  of 3,5-dimethylpyrazole chromium t r i o x i d e f r o m ^2^5^ *  '^  ie  (60 mg,  (65 mg) r  e  s  u  0.6  i n dichloromethane  lting  brown s u s p e n s i o n  temperature f o r 1 hour.  Saturated  added to the m i x t u r e  the  (100  ml).  The  and  organic  examined by  l a t t e r was  and  and  the d e s i r e d aldehyde.  Oxidation The methane  N -formyl  freshly (480  room  i n vacuo.  e l u e n t A)  dichloromethane  t o be  dried  The  and  was  over  resulting  found  to  contain  dimethylpyrazole,  starting  S u b s e q u e n t c h r o m a t o g r a p h y on  the N - f o r m y l  (43)  aldehyde  with Pyridinium  a l c o h o l (43)(305  cL  chlorochromate  at  (44)  and  alumina spectrothe  (43).  of N^-formyl A l c o h o l  (60 m l ,  stirred  distilled  s a m p l e s o f p u r e m a t e r i a l s w h i c h had  scopic properties identical with starting material  was  dried  freshly  extracted with  evaporated  t i c (alumina,  s i l i c a gel yielded only  (25 m l ,  washed w i t h w a t e r and  t h r e e major components w h i c h appeared m a t e r i a l and  carefully  aqueous sodium b i c a r b o n a t e  e x t r a c t was  anhydrous sodium sulphate r e s i d u e was  mmoles) and  mg)  mg)  was  Chlorochromate  dissolved i n dichloro-  distilled  from  w h i c h had  been prepared  a n c  *  t  r  e  a  t  e  a  with  according  to  pyridinium Corey's  38 procedure. for  This  f u r t h e r 15  ml) .  The  s o l u t i o n and  s o l u t i o n was  an  equal  was  stirred  a t room  temperature  volume of s a t u r a t e d  added and  the m i x t u r e  was  aqueous  stirred  for  minutes.  This mixture (.250  suspension  2 hours, a f t e r which time,  sodium bicarbonate a  coloured  was  organic  then e x t r a c t e d repeatedly e x t r a c t was  once w i t h w a t e r b e f o r e  with  w i t h aqueous sodium  dichloromethane bicarbonate  d r y i n g over anhydrous sodium  sulphate.  -183-  After  removal of the s o l v e n t , the dark coloured  graphed  o n a l u m i n a ( 1 0 g, a c t . I I I ) .  yielded  the d e s i r e d aldehyde  ( 2 1 8 mg)  r e s i d u e was  chromato-  Fractions eluted with as a l i g h t  yellow  benzene  foam.  p h y s i c a l a n d s p e c t r o s c o p i c p r o p e r t i e s o f t h i s m a t e r i a l was to  the N - f o r m y l  aldehyde  The  identical  (44) w h i c h had been p r e v i o u s l y c h a r a c t e r i z e d .  3.  l-Formyl-2g,16ct-cur-19-en-17-al 18-Deoxy-Wieland-Gumlich tetrahydrofuran  (12 m l )  and  t h e n added and  aldehyde  ( 3 7 ) (47 mg)  Saturated  r e s u l t i n g m i x t u r e was  residue, obtained silica  after  g e l ( e l u e n t C)  p h y s i c a l and  partioned.  aldehyde  dissolved i n  d r i e d over anhydrous  to y i e l d  sodium  an a m o r p h o u s ' m a t e r i a l  bicarbonate  A f t e r washing  organic  r e m o v a l o f t h e s o l v e n t , was  spectroscopic  was  aqueous sodium  aqueous l a y e r w i t h d i c h l o r o m e t h a n e , the combined w e r e washed w i t h w a t e r and  (37)  t r e a t e d w i t h a c e t i c - f o r m i c a n h y d r i d e (0.5  a t room t e m p e r a t u r e f o r 1 h o u r . was  (44) From A l d e h y d e  extracts  sulphate.  purified ( 3 7 mg)  p r o p e r t i e s were i d e n t i c a l w i t h  (44) w h i c h had b e e n p r e v i o u s l y c h a r a c t e r i z e d , and  synthesized  by o t h e r s  i n our l a b o r a t o r i e s by a s i m i l a r  the  The  b y p i c on whose  those of later  route.  40  the  ml)  -184-  Attempted  C-16 F u n c t i o n a l i z a t i o n o f l - F o r m y l - 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 a l ( 5 0 )  The C-16  data relevant  to the study of the f u n c t i o n a l i z a t i o n of the  p o s i t i o n o f the N-formyl aldehyde  (44) u s i n g sodium h y d r i d e and  m e t h y l c h l o r o f o r m a t e i s s u m m a r i z e d i n T a b l e 8, a n d d a t a r e l e v a n t similar  study u s i n g o t h e r bases In  i s s u m m a r i z e d i n T a b l e 9.  a l l experiments i n Table 8 and 9 t h e f o l l o w i n g g e n e r a l  procedures were used. were conducted  A l l g l a s s w a r e was f l a m e - d r i e d and a l l r e a c t i o n s  i n an atmosphere o f d r y n i t r o g e n g a s .  1-8 o f T a b l e 8 a n d 1-3 o f T a b l e 9, t h e a l d e h y d e the  to a  appropriate solvent a t the required  a d d e d a n d t h e m i x t u r e was s t i r r e d  I n Experiments  ( 4 4 ) was d i s s o l v e d i n  temperature.  f o r the required  The b a s e was t h e n  time p e r i o d ,  after o  which  excess methyl  c h l o r o f o r m a t e (0.2-0.3  ml, d i s t i l l e d  f r o m 4A m o l e c u l a r  s i e v e s ) was added. S t i r r i n g was c o n t i n u e d f o r 10-20 m i n u t e s b e f o r e i c e w a t e r was a d d e d . and  The r e a c t i o n m i x t u r e was t h e n p a r t i t i o n e d b e t w e e n  dichloromethane.  The o r g a n i c e x t r a c t s w e r e w a s h e d w i t h w a t e r a n d  dried  over anhydrous  vacuo  to give the r e a c t i o n product.  dimethyl if,  formamide  water  sodium  s u l p h a t e b e f o r e t h e s o l v e n t was r e m o v e d i n  (DMF) w e r e u s e d  a t t h e end o f t h e r e a c t i o n  When d i m e t h y l s u l p h o x i d e (DMSO) a n d as s o l v e n t s  t h e work-up was s i m p l e r  time, o n l y a few drops o f water  were  a d d e d a n d m o s t o f t h e s o l v e n t was r e m o v e d i n h i g h v a c u u m b e f o r e  partitioning  between w a t e r and d i c h l o r o m e t h a n e . In  Experiments  p r o c e d u r e was e m p l o y e d .  4-6 o f T a b l e 9, a v a r i a t i o n  i n t h e above d e s c r i b e d  P o t a s s i u m _ t - b u t o x i d e was f i r s t  transferred to  the  r e a c t i o n v e s s e l from a p r e v i o u s l y prepared s t o c k s o l u t i o n  i n benzene.  The  b e n z e n e was t h e n r e m o v e d i n h i g h v a c u u m a n d t h e w e i g h t o f t h e r e a g e n t  -185-  was  determined.  The  a p p r o p r i a t e s o l v e n t was  the a d d i t i o n of the aldehyde In Experiments amine  (LDA)  was  first  d i i s o p r o p y l amine  7-9  o f T a b l e 9,  ( 4 4 ) was  a d d e d and  In every experiment, chromatography  on a l u m i n a  spectroscopically. unreacted  quantities  (HMPA).  After  15 m i n u t e s ,  of  t h e r e a c t i o n p r o d u c t was and  the employed.  s e p a r a t e d by  the components were  examined  I n e v e r y e x p e r i m e n t , e x c e p t number 3 i n T a b l e  aldehyde  1-3  ( 4 4 ) , was  o f T a b l e 9,  (37) were i s o l a t e d  copic comparison w i t h m a t e r i a l p r e v i o u s l y ments, q u a n t i t i e s  diisopropyl  t h e a b o v e d e s c r i b e d p r o c e d u r e was  (act.Ill)  7 o f T a b l e 8 and  Wieland-Gumlich  lithium  from potassium h y d r o x i d e ) , n - b u t y l  s t a r t i n g m a t e r i a l , aldehyde  Experiments  the base  p r e p a r e d a t 0° u s i n g e q u a l m o l a r  (distilled  by  (44).  l i t h i u m and h e x a m e t h y l p h o s p h o r a m i d e aldehyde  then added, f o l l o w e d  of very p o l a r  recovered.  samples and  of  t a r - l i k e m a t e r i a l was  In  18-deoxy-  identified  synthesized.  8,  by  spectros-  In a l l experi-  obtained.  -186-  T a b l e 8.  Exp.  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 1.  Aldehyde(44) (mg,mmoles)  Sodium H y d r i d e (mg,mmoles) 3  Solvent (ml)  Temperature  c  Time (hrs)  1  48  9 0.15  36  , 1.5  THF(30)  2  50  9 0.16  39  , 1.6  THF(25)  50°  2  3  82  9 0.25  60  , 2.5  DMF(20)  rt  2  4  50  9 0.16  40° , 1.7  DMF(20)  50°  1  5  85  9 0.26  10  , 0.4  DMF(30)  rt  4  6  85  9 0.26  10  , 0.4  DMF(20)  55°  0  7  48  9 0.15  7  , 0.3  DMSO(20)  50°  1  8  50  9 0.16  7  , 0.3  DMSO(20)  rt  3  a.  C a l c u l a t e d based  b.  G r a n u l a r sodium  c.  Room  temperature  on 55% o i l s u s p e n s i o n hydride  rt  6  -187-  Table  Exp.  9.  A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 2.  Aldehyde(44) (mg,mmoles)  Base (mg,mmoles)  Solvent (ml)  1  50  9 0.15  KH(32,0.8)  a  THF(30)  rt  2  45  9 0.14  KH(28,0.7)  a  THF(30)  60°  1  3  65  9 0.20  KH(40,1.0)  a  DMSO(20)  35°  0.5  4  38  9 0.12  _t-BuOK(28,0. 2 5 )  C H (50)  5  5  50 J> 0.15  t-BuOK(34,0. 30)  DMSO(25)  rt  2  6  85  9 0.26  t-BuOK(62,0. 55)  DMSO(25)  50°  1  7  46  9 0.14  LDA(0.28)  b  THF(20)  -78°  0.5  8  1 3 2 , 0.41  LDA(0.80)  b  THF(45)  0°  1  9  68  LDA(0.42)  b  THF(20)  rt  3  9 0.21  6  Temperature  5  C  o d  6  a.  C a l c u l a t e d based on 22% o i l s u s p e n s i o n  b.  P l u s 1 e q u i v a l e n t o f HMPA  c.  Room t e m p e r a t u r e  d.  R e f l u x e d f o r 30 m i n u t e s b e f o r e c o o l i n g of methyl chloroformate.  Reaction Time(hrs)  3  2  t o 55° f o r t h e a d d i t i o n  -188-  l-Carbomethoxy-2B,16a-cur-19-en-17-ol The and  alcohol  was  dissolved  t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e (0.2 ml)  temperature and  ( 3 6 ) ( 1 0 0 mg)  (49)  f o r 15 m i n u t e s .  t h e r e s i d u e was  b i c a r b o n a t e and were d r i e d material  and  The  s o l v e n t was  evaporated  The  2  3  (multiplet,  241,  (indoline);  278,  3H,  pmr:  287;  2  measurement: C a l c .  2.52  and  1685  (multiplet,  257(21),  IH,  (multiplets 3H,  J=7,  2  3  8.33  3  M  354(86),  +  166(85); high r e s o l u t i o n  f o r C H „ , 0 N „ : 354.1942 Found: 21 z o 3 2. o  freshly distilled  1H,=CH-CH ),  N-C0 CH ),  3  202(63),  o 1  C-12H),  2, = C H - C H ) ; m a s s s p e c t r u m :  N-carbomethoxy a l c o h o l  methane (100 m l ,  sodium  washings  3 3 5 0 (OH),  (singlet,  l-Carbomethoxy-2B, 16ct-cur-19-en-17-al The  t h e n removed i n vacuo  v  a r o m a t i c - H ) , 450  2H,-CH -0H) 6.20  309(100),  a t room  max  ( d o u b l e t o f d o u b l e t s , J=7 323(21),  ml)  i n vacuo to g i v e a l i g h t y e l l o w amorphous  X  2.65-3.0 ( m u l t i p l e t s , 5.68  let stir  combined o r g a n i c  max ( N - C 0 C H ) , 1600  and  (10  p a r t i t i o n e d between s a t u r a t e d aqueous  dichloromethane.  (110 mg),  i n methanol  mass  354.1916.  (50)  ( 4 9 ) ( 4 5 0 mg) from ^^^)  was a n c  '  dissolved  i n dichloro-  treated with pyridinium 38  chlorochromate  ( 7 5 0 mg)  procedure.  The  temperature  f o r 2.5  saturated  resulting  stirred  and  been prepared a c c o r d i n g to  c o l o u r e d s u s p e n s i o n was  stirred  Corey's  a t room  t i m e , an e q u a l v o l u m e o f  b i c a r b o n a t e s o l u t i o n was  f o r a f u r t h e r 15  T h i s m i x t u r e was (300 m l )  had  hours, a f t e r which  aqueous sodium  m i x t u r e was  which  a d d e d and  the  minutes.  extracted repeatedly with  dichloromethane  the combined o r g a n i c e x t r a c t s were washed w i t h  water  -189-  before  being  of  s o l v e n t , t h e r e s i d u e was  the  d r i e d over anhydrous sodium sulphate.  act I I I ) using a benzene-ethyl desired X  max  pmr:  a l d e h y d e was  242,  282,  288  ; v  0.40  (doublet,  2.6-3.0 ( m u l t i p l e t s , 6.23 3H,  (singlet,  3H,  -CHO), 2.38  N - C 0 C H ) , 8.45 2  3  Found:  +  4.45  ( q u a r t e t , J=7,  r e s o l u t i o n mass measurement: C a l c .  1H, J=7  for  C  =CH-CH ), 3  and  2,  202(47),  2 i 2 4 ^ 3 2 ' ' H  N  352.1737.  (50)  18-Deoxy-Wieland-Gumlich aldehyde tetrahydrofuran stirring  (10 m l )  and  e x t r a c t was being  evaporated i n vacuo.  w h i c h had  pic (silica p h y s i c a l and  with previously synthesized aldehyde  (50).  N  mg)  was  (37) dissolved in  methylchloroformate.  hours the  solvent  p a r t i t i o n e d between  s o l u t i o n and  washed w i t h w a t e r and  chromatographed by (35 mg)  treated with excess  t h e r e s i d u e was  aqueous sodium b i c a r b o n a t e  From A l d e h y d e  (37)(55  a t r o o m t e m p e r a t u r e f o r 1.5  r e m o v e d i n v a c u o and  before  C-12H),  352(58), 323(22), 309(78),  l-Carbomethoxy-2B,16a-cur-19-en-17-al  After  mg), (indoline);  IH,  of doublets,  g,  The  foam (325  (multiplet,  (doublet  3  =CH-CH ); mass s p e c t r u m : M  164(100); high  a light yellow  aromatic-H),  (20  solvent gradient.  o  IH,  3H,  as  removal  alumina  1 7 2 0 ( C H 0 ) , 1 6 9 0 ( N - C 0 C H _ ) 1600 z j  max  J=4,  c h r o m a t o g r a p h e d on acetate  obtained  After  dichloromethane.  saturated The  d r i e d over anhydrous sodium The  amorphous p r o d u c t  g e l , e l u e n t C)  to y i e l d  spectroscopic properties  a  was  organic sulphate  ( 4 1 mg)  was  product identical  -carbomethoxy-18-deoxy-Wieland-Gumlich  -190-  Attempted 17-al  C-16 C a r b o x y l a t i o n o f l - C a r b o m e t h o x y - 2 g  (50) The  data relevant  t h e C-16 p o s i t i o n in  ,16ot-cur-19-en-  to the study of the f u n c t i o n a l i z a t i o n  o f the N-carbomethoxy aldehyde  of  (50) i s summarized  T a b l e 1 0 . I n t h i s s t u d y , t h e same g e n e r a l p r o c e d u r e s d e s c r i b e d  above f o r t h e p r e v i o u s s t u d y o f t h e N - f o r m y l employed f o r s i m i l a r  aldehyde  experiments.  A t no t i m e c o u l d any p r o d u c t s o t h e r t h a n  the s t a r t i n g  (50) o r v e r y p o l a r d e c o m p o s i t i o n m a t e r i a l be d e t e c t e d after  chromatography  (44) were  on a l u m i n a  material  spectroscopically  (act.III).  T a b l e 1 0 . A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 5 0 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 3.  Exp.  Aldehyde(50) (mg,mmoles)  Base (mg,mmoles)  1  68 , 0.19  NaH(50,2.0)  a  THF(30)  40°  6  2  75  , 0.21  NaH(50,2.0)  a  DMS0(20)  53°  2  3  45  , 0.13  KH(10,0.25)  b  C H (60)  80°  2  4  50 , 0.14  KH(6,0.15)  DMF(20)  50°  4  5  50 , 0.14  LDA(0.28)  C  THF(20)  0°  0.5  6  46  , 0.13  LDA(0.25)  C  THF(25)  rt  1  7  35  , 0.10  LDA(0.20)  C  THF(15)  35°  1  b  Solvent (ml)  6  6  a.  C a l c u l a t e d based  on 55% o i l s u s p e n s i o n  b.  Calculated  on 22% o i l s u s p e n s i o n  c.  P l u s 1 e q u i v a l e n t o f HMPA.  based  Temperature  Time (hours)  -191-  Des-carbomethoxy-stemmadenine Nor-fluorocurarine potassium hydroxide  a d d e d o v e r a 0.5  a  1.5  further  ( 3 9 ) ( 6 6 0 mg)  (35 m l ,  w h i c h was  (52)  2% ) a t 40° hour  was and  period.  reduced  and  p a r t i t i o n e d between water  was  repeatedly extracted with dichloromethane  were washed w i t h w a t e r A f t e r removal on a l u m i n a After  ( 3 9 5 mg)  was  obtained: X  1.0  a r o m a t i c - H ) , 4.5  ( q u a r t e t , J=7,  (multiplet,  mass s p e c t r u m :  M  2  123(100), H  +  226,  max  (broad s i n g l e t ,  - C H - 0 H ) , 6.15  1 Q  The  and  aqueous  layer  the combined sodium was  extracts  sulphate.  chromatographed  (15 g, a c t . I V ) u s i n g a b e n z e n e - e t h y l a c e t a t e s o l v e n t g r a d i e n t .  3 2 5 0 ( 0 - H ) ; pmr:  19  continued f o r  p o o l i n g o f t h e a p p r o p r i a t e f r a c t i o n a l i g h t y e l l o w amorphous  material  C  dichloromethane.  t h e r e s i d u e ( 4 7 0 mg)  g)  i n volume i n vacuo  b e f o r e d r y i n g over anhydrous  of the s o l v e n t ,  i n methanolic  t r e a t e d w i t h NaBH^ ( 0 . 5  S t i r r i n g was  h o u r s b e f o r e t h e m i x t u r e was and  dissolved  282,  I H , N-H)  288  max  3  N„0: 24 I  (multiplet,  296(57), 265(12), 251(15), 166(61),  296.1887.  Found:  3H,  Des-carbomethoxy-stemmadenine  s o l v e n t was  (5 m l )  and  pyridine  a q u e o u s l a y e r was  and  =CH-CH ); 3  144(86),  130(32),  for  further  was  t h e r e s i d u e was  and  dissolved  i n acetic  l e t s t a n d a t 5° o v e r n i g h t .  b i c a r b o n a t e s o l u t i o n and  extracted  2H,  (56)  ( 5 3 ) ( 6 0 mg)  (0.5 ml)  t h e n removed i n v a c u o and  s a t u r a t e d aqueous sodium  4H,  296.1887.  1,17-Diacetyl-des-carbomethoxy-stemmadenine  anhydride  (N-H),  ( d o u b l e t , J=5,  1 2 2 ( 5 0 ) ; h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c .  o /  3350  2.4-3.0 ( m u l t i p l e t s ,  I H , = C H - C H ) , 5.72  I H , C - 1 6 H ) , 8.3  ; v  partitioned  dichloromethane.  The  between The  t h e o r g a n i c e x t r a c t s were washed  -192-  w i t h water  b e f o r e d r y i n g over anhydrous  o f most o f  t h e s o l v e n t t h i s m a t e r i a l was  gel  ( e l u e n t C)  (45 mg) X  was  248,  max  282,  M  +  4H,  3H,  380(27),  ; v  290  (multiplet,  (singlet,  two  max  1725  321(21),  293(53),  208(16),  r e s o l u t i o n mass m e a s u r e m e n t : C a l c . Found:  silica  major  product data:  2.4-2.9  =CH-CH ), 3  N-C(0)CH ),  spectrum:  3  o  o  144(100);  o  7.89  3  =CH-CH ); mass  for C„ H_ N 0 :  removal  high  380.2100.  o  380.2108.  anhydride  for  6 hours.  was  partitioned  over anhydrous r e s i d u e was  (5 m l )  The  (57)  w i t h 1 drop  s o l v e n t was  ( 5 2 ) ( 1 9 0 mg)  o f p y r i d i n e and  t h e n removed  The  sodium  carefully  o r g a n i c e x t r a c t was sulphate.  After  chromatographed  removal  The  major  stemmadenine  p r o d u c t was  ( 5 7 ) ( 1 7 6 mg)  The  w h i c h was  identified  based  of  on a l u m i n a  (6 mg)  dissolved  as  -10°  the r e s i d u e  bicarbonate  and and  the s o l v e n t ,  dried the  (10 g, a c t . I l l )  less  in  l e t stand at  washed w i t h w a t e r  a benzene-ethyl acetate solvent gradient. an amorphous m a t e r i a l  was  i n v a c u o and  between s a t u r a t e d aqueous sodium  dichloromethane.  (56).  IH,  3H,  186(17),  Des-carbomethoxy-stemmadenine  yielded  J=7,  3H,  17-Acetyl-des-carbomethoxy-stemmadenine  acetic  p i c on  ( N - A c ) , pmr:  (singlet,  (multiplet,  3  The  After  the f o l l o w i n g  (quartet,  2  - 0 C C H ) , 8.26  on  ( O - A c ) , 1640  - C H - 0 A c ) , 7.  2  s e p a r a t e d by  (56) based  a r o m a t i c - N ) , 4.47 2H,  sulphate.  amorphous m a t e r i a l s .  assigned structure  (multiplets, 5.3  to y i e l d  sodium  polar  identified  using  fractions as  the  diacetate  17-acetyl-des-carbomethoxy-  the f o l l o w i n g  data: X  224,  282,  288  ;  max v  1730  4H,  (-OAc); pmr:  a r o m a t i c - H ) , 4.49  - C H _ O A C ) , 7.89 2  1.69  (broad s i n g l e t ,  ( q u a r t e t , J=7,  (singlet,  3H,  I H , = C H - C H ) 5.3 3  -0 CCH )> 2  I H , N-H),  3  8  -  2 6  2.4-2.9  (multiplets,  (multiplet,  (multiplet,  2H,  3H,=CH-CH ); 3  -193-  mass s p e c t r u m : M  338(9), 293(7), 279(14), 170(25), 144(39),  +  h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c . f o r 1 2 6 ° 2 2 C  H  N  : 3 3 8  2  '  123(100); 1  9  7  6  '  F  o  u  n  d  :  338.1972. Attempted  C-16 C y a n i d e  The into is  Introduction  data relevant  i n Alcohol  (52) v i a C h l o r o i n d o l e n i n e ( 5 3 )  t o the s t u d y o f t h e i n t r o d u c t i o n o f a cyano  t h e C-16 p o s i t i o n o f i n d o l e a l c o h o l  group  (52) v i a t h e c h l o r o i n d o l e n i n e (53)  summarized i n Table 11. In a l l experiments  was e m p l o y e d .  i n this  study, the following  The i n d o l e a l c o h o l  (freshly d i s t i l l e d  from ^ 0 ^ )  a  t  ( 5 2 ) was d i s s o l v e d  ®°'  general procedure  i n dichloromethane  _t-Butyl hypochlorite  (1.1 e q u i v a l e n t s  f r o m a 0.05 M s t o c k s o l u t i o n  i n C C l ^ ) was a d d e d a n d t h e r e a c t i o n m i x t u r e  was s t i r r e d  The r e a c t i o n was m o n i t o r e d b y t i c o n a l u m i n a  f o r 15 m i n u t e s .  ( e l u e n t B) a n d u v s p e c t r o s c o p y .  When t h e u v c h r o m o p h o r e o f t h e s t a r t i n g  m a t e r i a l was a b s e n t a n d t h e c h r a c t e r i s t i c  i n d o l e n i n e chromophore  (X max  260,  3 1 0 ) was p r e s e n t , t h e n u c l e o p h i l e was a d d e d a n d t h e m i x t u r e was  stirred  a t the required  (DMF) was u s e d  temperature.  as s o l v e n t  H o w e v e r , when e v e n  dimethylformamide  t h e d i c h l o r o m e t h a n e was r e m o v e d i n v a c u o  after  chloroindolenine formation. All scopy.  e x p e r i m e n t s were m o n i t o r e d by t i c on a l u m i n a and by uv  A t t h e end o f t h e r e a c t i o n  between d i c h l o r o m e t h a n e and w a t e r . anhydrous  sodium  t i m e , t h e m i x t u r e was  spectro-  partitioned  The o r g a n i c e x t r a c t s w e r e d r i e d  s u l p h a t e b e f o r e t h e s o l v e n t was r e m o v e d i n v a c u o .  r e s i d u e s were p a r t i t i o n e d by p i c on s i l i c a  over The  g e l (eluent C).  I n e v e r y e x p e r i m e n t , a v e r y complex m i x t u r e of p r o d u c t s were o b t a i n e d . The  major  p r o d u c t s were d a r k c o l o u r e d t a r - l i k e m a t e r i a l which  remained  near  -194-  the b a s e - l i n e o f the chromatoplate.  A t no t i m e  m a t e r i a l b e d e t e c t e d b y u v o r mass  Table  11.  c o u l d new  indolic  spectroscopy.  A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l a s s o c i a t e d w i t h T a b l e 4.  data  Exp  Indole(52) (mg,mmoles)  Nucleophile (mg,mmoles)  Solvent (ml)  1  48,  0.16  KCN(52,  0.8)  CH C1 (15)  rt  2  35,  0.12  KCN(58,  0.9)  DMF(10)  rt  6  3  50,  0.17  KCN(98,  1.5)  DMF(15)  80°  0.5  4  35,  0.12  Zn(CN) (140,  CH C1 (20)  rt  24  5  36,  0.12  Zn(CN) (70,  DMF(15)  50°  1.5  6  50,  0.17  AgCN(228,  1.7)  CH C1 (30)  rt  48  7  40,  0.14  AgCN(200,  1,5)  DMF(10)  55°  1.5  8  54,  0.18  Bu NCN(91,  0.9)  CH C1 (25)  rt  48  9  35,  0.12  Bu NCN(120,1.2)  CE C1 (25)  35°  12  10  35,  0.12  Bu NCN(120,1.2)  DMF(10)  35°  5  11  48,  0.16  Et AlCN(1.6)  CH C1 (25)  rt  5  12  37,  0.13  Et AlCN(1.3)  CH C1 (25)  36°  12  13  62,  0.21  Et  CH C1 (25)  5°  5 days  a  2  1.2)  2  0.6)  2  4  4  4  b  2  2  A1CN(2.1)  a.  S t i r r e d w i t h 1.1 e q u i v a l e n t s o f _t-BuOCl addition of nucleophile.  b.  Available  c.  Room  from A l f a  temperature.  Products.  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  Temperature  C  a t 0° f o r 15 m i n u t e s  Time (hrs) 24  before  -195-  A t t e m p t e d C-16 dolenine  Cyanide I n t r o d u c t i o n  i n Acetate  (57) v i a C h l o r o i n -  (58)  The d a t a r e l e v a n t  to the study o f the i n t r o d u c t i o n o f a cyano  group i n t o  t h e C-16 p o s i t i o n o f t h e i n d o l e a c e t a t e  indolenine  (58) i s summarized i n Table 12.  v i a the c h l o r o -  I n a l l e x p e r i m e n t s , t h e same p r o c e d u r e a s t h a t d e s c r i b e d for  the study i n v o l v i n g a l c o h o l  ( 5 2 ) was  A t no t i m e i n t h i s s t u d y , c o u l d uv  o r mass  T a b l e 12.  Exp.  above  employed.  i n d o l i c m a t e r i a l be d e t e c t e d  by  spectroscopy. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a s s o c i a t e d w i t h T a b l e 5.  a d d i t i o n a l data  Indole(57) (mg,mmoles)  Nucleophile (mg,mmoles)  Solvent (ml)  1  45 , 0.13  KCN(85,1.3)  CH C1 (30)  rt  2  35  , 0.10  KCN(65,1.0)  DMF(20)  rt  8  3  28 , 0.08  KCN(52,0.8)  DMF(15)  68°  1  4  34  , 0.10  B u N C N ( 5 0 , 0 . 5) C H C 1 ( 3 0 )  rt  24  5  26  , 0.08  B u N C N ( 4 9 , 0 . 5) D M F ( 2 0 )  rt  8  6  30 , 0.09  B u N C N ( 9 1 , 0 . 9) DMF(20)  75°  1  a  4  2  2  Temperature  2  2  4  A  a.  S t i r r e d w i t h 1.1 e q u i v a l e n t s o f _ t - B u 0 C l a t 0° f o r before addition of nucleophile  b.  Room  temperature  b  15 m i n u t e s  Time (hrs) 20  -196-  Reaction  of Nor-fluorocurarine Nor-fluorocurarine  (2 m l , was  freshly  distilled  ( 3 9 ) w i t h Me t h y l c h l o r o f o r m a t e  ( 3 9 ) ( 1 2 0 mg)  formate  (0.2  Cold water l a y e r was  ml)  a t 0°. was  (10 m l )  2 equivalents)  f o r 10 m i n u t e s ,  s t i r r i n g was  a d d e d and  This s o l u t i o n  continued  in  tetra-  methylchloro-  a t 0°  f o r 15  the mixture  was  partitioned.  e x t r a c t e d w i t h d i c h l o r o m e t h a n e and  the  combined o r g a n i c  were washed t h r e e  times  with water before  sulphate.  A f t e r removal of  on  (8 g,  alumina  combined  (1:1,  After stirring  added and  was  tetrahydrofuran  s o l u t i o n of l i t h i u m d i i s o p r o p y l  amine-hexamethylphosphoramide complex (6 m l )  dissolved i n  from l i t h i u m aluminum h y d r i d e ) .  added to a p r e v i o u s l y p r e p a r e d  hydrofuran  was  act.Ill)  the  soluent,  d r y i n g over anhydrous t h e r e s i d u e was  u s i n g benzene.  to g i v e a s i n g l e p r o d u c t :  X  The  The 233,  2  -CHO), 2.3 4.56  and  3  1640  (multiplet,  ( q u a r t e t , J=7,  (-CH0), 1600  IH,  1720  3  3H,  = C H - C H ) ; mass s p e c t r u m : M  ( i n d o l e n i n e ) pmr:  121  (57); high  (singlet,  350  +  3  Found:  max 0.02  C - 1 2 H ) , 2.6-3.0 ( m u l t i p l e t s ,  = C H - C H ) , 6.08  sodium  f r a c t i o n s were  max ( - C 0 C H ) , 1670  extracts  (sh); v  305  aqueous  chromatographed  appropriate 268,  minutes.  (100),  3H,  321  3H,  2  IH,  aromatic-K),  - C 0 C H ) , 8.34  (multiplet,  3  ( 3 3 ) , 307  r e s o l u t i o n mass measurement: C a l c .  (singlet,  ( 1 7 ) , 292  (20),  for C ^H 0 N :350.1629 2  2 2  3  2  350.1650.  Alternate  Synthesis  of Aldehydo-ester  Nor-fluorocurarine  (60)  ( 3 9 ) ( 1 8 0 mg)  was  suspended i n  methylchloro-  o  formate  (5 m l ,  distilled  f r o m 4A m o l e c u l a r  a t 60-65° u n d e r a n i t r o g e n gas was  removed i n v a c u o and  atmosphere.  t h e r e s i d u e was  s i e v e s ) and  stirred  vigorously  A f t e r 20 m i n u t e s , t h e  p a r t i t i o n e d between  solvent  saturated  -197-  aqueous sodium b i c a r b o n a t e e x t r a c t was  washed once w i t h w a t e r and  sodium sulphate. g r a p h e d on compound  s o l u t i o n and  A f t e r removal of  alumina  (152  mg)  (10 g, had  the a l d e h y d o - e s t e r second component fluorocurarine  act.Ill)  p h y s i c a l and  (50) (12 mg)  w h i c h had was  the  aldehyde-ester  (20 m l )  was  dichloromethane. sodium sulphate (23 mg).  (5 m l ,  added and The  two  organic  anhydrous  r e s i d u e was  compounds.  spectroscopic  chromato-  The  major  properties identical  p r e v i o u s l y been c h a r a c t e r i z e d .  identified  as  the  with  The  starting material  nor-  and  give a l i g h t yellow those  (60)  (60)(35  mg)  5%)  heated  and  the  organic  evaporated  T h i s m a t e r i a l was  identical with  s o l v e n t , the  The  (39) .  potassium hydroxide water  then d r i e d over  to g i v e  A l k a l i n e H y d r o l y s i s of Aldehydo-ester The  dichloromethane.  was  dissolved in t o 65°  methanolic  f o r 2 hours.  r e s u l t i n g mixture  was  Cold  extracted  with  e x t r a c t s were combined, d r i e d over i n v a c u o t o g i v e an  c h r o m a t o g r a p h e d on  foam w h i c h had  p h y s i c a l and  anhydrous  amorphous m a t e r i a l  alumina  (act.Ill)  spectroscopic  o f n o r - f l u o r o c u r a r i n e (39) w h i c h had  to  properties  been p r e v i o u s l y  synthesized.  1,2-Dehydro-16,17-dehydro-2g-cur-19-en .. The distilled  aldehydo-ester  (60)(50  f r o m magnesium) and  recrystallized  mg)  (72) was  d i s s o l v e d i n methanol  t r e a t e d w i t h sodium borohydride  from i s o - p r o p y l amine).  The  r e a c t i o n was  r o o m t e m p e r a t u r e f o r 30 m i n u t e s a f t e r w h i c h t i m e m o s t o f  (20  stirred  (15  ml,  mg, at  the s o l v e n t  was  -198-  r e m o v e d ir±  vacuo.  The  r e s i d u e was  repeatedly with dichloromethane w a s h e d o n c e w i t h w a t e r and The by  material  silica  gel  (26 mg):  A  aromatic-H),  3.89  = C H - C H ) , 4.54 3  spectrum: M  +  (150 m l ) .  228,  to y i e l d  292;  pmr:  232  mass m e a s u r e m e n t : C a l c . f o r C ^ ^ Q ^ : T h i s i n d o l i n e ( 7 2 ) was which conversion s e r i e s was  isolated  of product  attempted.  I n most e x p e r i m e n t s , s i n c e the product  (60)  mixtures  were v e r y  6.  In the  latter,  cases,  4H, IH,  = C H - C H ) ; mass 3  (32); high r e s o l u t i o n Found: 276.1618.  i n a v a r i e t y of experiments i n stemmadenine-  summarized i n Tables  (72) w e r e i s o l a t e d  for  7,  agent, w i t h  (60)(40-110  the r e q u i r e d temperature the e x c e p t i o n  o f Exp.  and  9 i n Table  a d d e d t o a s o l u t i o n o f NaBH^ i n  f o l l o w e d by a d d i t i o n o f m o r e r e a g e n t . as d e s c r i b e d  above.  p e r f o r m e d u n d e r a c i d i c c o n d i t i o n s w e r e f i r s t made b a s i c w i t h solution.  7.  identification  the aldehydo-ester  t h e b a s i c r e a c t i o n s w e r e w o r k e d up  sodium bicarbonate  6 and  complex.  6 and  compound ( 6 0 ) was  g l a c i a l a c e t i c a c i d a t 90°, all  276.1621.  d i s s o l v e d i n the s o l v e n t i n d i c a t e d a t  treated w i t h excess reducing  amorphous  t o t h e p r e a k u a m m i c i n e - and  samples of  In a l l experiments i n Tables was  ( d o u b l e t , 3H,  These r e s u l t s a r e only  chromatographed  ( q u a r t e t , J=7,  ( 3 9 ) , 1 9 4 ( 4 5 ) , 170  was  sulphate.  2.28-2.8 ( m u l t i p l e t s ,  ( s i n g l e t , I H , C - 1 7 H ) , 4.36  (100),  extracted  extract  a l i g h t yellow  ( s i n g l e t , I H , C - 1 7 H ) , 8.16 276  organic  o f t h e s o l v e n t , was  ( e l u e n t C)  max  The  d r i e d over anhydrous sodium  residue, after evaporation p i c on  d i s s o l v e d i n w a t e r and  In Those  aqueous  mg)  -199-  T h i o a c e t a l Formation The (15 m l ) ,  of Aldehyde-ester  aldehydo-ester  (60)(200  ethane d i t h i o l  (1 m l )  This mixture  was  l e t stand  i n v a c u o and  t h e r e s i d u e was  bicarbonate  s o l u t i o n and  chloroform,  the o r g a n i c  sodium s u l p h a t e .  The  mg)  and  a t 5°  was  dissolved i n  The  s o l v e n t was  then  g r a p h e d on  alumina  gradient.  P o o l i n g of the a p p r o p r i a t e  act.Ill)  A  d r i e d over  r e m o v e d and  2.26  4.19  (singlet,  3H,  (multiplet, IH,  - C 0 C H ) , 6.69 2  fractions yielded a light 275,  283  (sh); v  I H , C-12H) 2.65-2.8 ( m u l t i p l e t s ,  = C H - C H ) ; mass s p e c t r u m : M  +  3  121  ( q u a r t e t , 3=1, 4H,  426  anhydrous chromato-  yellow  1710  (-C0 CH ): 2 3 o  max  C - 1 7 H ) , 4.42  IH,  3H,  =CH-CH ) 6.09  2  ( 4 1 ) , 376  (singlet,  3  - S - C H - C H - S - ) , 8.23  (doublet,  2  ( 1 1 ) , 355  n  aromatic-H),  ( 1 4 ) , 167  3H,  (100),  ( 5 8 ) ; h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c . f o r C „ H „ , N 0 S : o  Found:  sodium  u s i n g a benzene-methylene c h l o r i d e s o l v e n t  238,  (multiplet,  3  removed  t h e r e s i d u e was  max pmr:  then  After several extractions with  e x t r a c t s w e r e c o m b i n e d and  mg):  etherate.  p a r t i t i o n e d between s a t u r a t e d aqueous  chloroform.  (5 g,  dichloromethane  2 drops of boron t r i f l u o r i d e  overnight.  s o l v e n t was  a m o r p h o u s m a t e r i a l (215  (60)  o  o  o  426.1436  426.1427.  Attempted Ring-Opening of T h i o a c e t a l The  carbomethoxy t h i o a c e t a l  sodium methoxide s o l u t i o n for  2.5  hours.  was  added.  the o r g a n i c  The  e x t r a c t was  sodium s u l p h a t e .  (73)(82  (20,0.2N) and  reaction mixture  This mixture  (73)  was  was  mg)  was  r e f l u x e d under an then  cooled  t o 0°  methanoli  i n h e r t atmospher and  ice-water  extracted repeatedly with dichloromethane  washed w i t h w a t e r b e f o r e  Evaporation  dissolved i n a  of  d r y i n g over  anhydrous  the s o l v e n t y i e l d e d a r e s i d u e  (76  mg).  and  -200-  E x a m i n a t i o n o f t h i s m a t e r i a l by t h e p r e s e n c e o f two by X  pic.  max  The  228,  major products,  silica  284,  v  291;  4.44  ( q u a r t e t , J=7,  8.24  ( d o u b l e t , 3H, (100); high  had  the  3300 (-N-H); pmr:  max  gel  ( e l u e n t C)  w h i c h were subsequently  l e s s p o l a r m a t e r i a l (75)  aromatic-H),  4.14  I H , = C H - C H ) , 6.71  ( d o u b l e t , J=8,  = C H - C H ) ; mass s p e c t r u m : M  isolated  ( m u l t i p l e t , IH,  ( m u l t i p l e t , 4H,  3  revealed  f o l l o w i n g s p e c t r a l data:  2.37  r  2.65-2.8 ( m u l t i p l e t s , 3H,  123  t i c on  370  +  3  IH,  C-12H) — C-17H),  -S-CH -CH -S2  ),  2  ( 3 0 ) , 309  (16),  r e s o l u t i o n mass m e a s u r e m e n t : C a l c . f o r ^21^26^2^2 370.1537» ;  Found: 370.1543. The 284,  290;  s e c o n d component ( 7 6 ) ( 1 4 v  max  3300 ( N - H ) ; pmr:  ( m u l t i p l e t s , 3H, ( d o u b l e t , J=8, ( d o u b l e t , 3H,  aromatic-H),  IH,  C - 1 7 H ) , 6.74  mg)  2.37  4.45  had  the  following data:  ^  228,  m a x  ( m u l t i p l e t , I H , C - 1 2 H ) , 2.65-2.8 — ( q u a r t e t , J=7,  ( m u l t i p l e t , 4H,  = C H - C H ) ; mass s p e c t r u m : M  +  3  370  I H , =CH-CH  3>  5.32  -S-CH -CH -S-), 2  8.25  2  ( 2 8 ) , 309  h i g h r e s o l u t i o n mass m e a s u r e m e n t : C a l c . f o r ^ 2 1 ^ 2 6 ^ 2 ^ 2  :  ( 1 5 ) , 123  (100);  ^70.1537, Found:  370.1511. I n a second experiment, the refluxed  i n methanolic  of sodium borohydride moved jLn v a c u o and  i n d o l e n i n e t h i o a c e t a l (73) (47 mg)  potassium hydroxide (20 mg)  f o r 2 hours. Most of  t h e r e s i d u e was  extracted w i t h dichloromethane.  (.10%, 10 m l )  The  organic  e x t r a c t was and  g i v e an a m o r p h o u s m a t e r i a l . . T h i s m a t e r i a l was a s l i g h t l y yellow  gum  presence  t h e s o l v e n t was  d i l u t e d w i t h water before  water, d r i e d over anhydrous sodium sulphate  to y i e l d  i n the  was  i t  washed  re-  was with  evaporated i n vacuo c h r o m a t o g r a p h e d on  gel  ( e l u e n t C)  (18 mg)  and  s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h compound  w h i c h had  to  silica  physical  (76), previously  isolated.  -201-  In glacial at  a third acetic  study, the t h i o a c e t a l  acid  room t e m p e r a t u r e  (5 m l )  and  ( 7 3 ) ( 3 8 mg)  f o r 1 hour.  The  The  r e a c t i o n m i x t u r e was  o r g a n i c e x t r a c t was  o v e r a n h y d r o u s s o d i u m s u l p h a t e and amorphous m a t e r i a l . gel  ( e l u e n t C)  m a t e r i a l from The (1 m l ) acid  and  and  ( 7 3 ) (45 mg)  was  extracted with dried  a t 90°.  ( 7 3 ) (17 mg)  and  a  r e a c t i o n was  dissolved  allowed to s t i r  t h e n w o r k e d up  the p r o d u c t m i x t u r e by  glacial  acetic  i n d o l i c m a t e r i a l was  present.  tar-like  acid acetic  added f r e q u e n t l y over for a further 5  as d e s c r i b e d above.  10  minutes.  Examination  t i c r e v e a l e d a complex m i x t u r e of  S u b s e q u e n t c h r o m a t o g r a p h y and  silica  chromatoplate.  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