UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies in the fields of steroids and alkaloids Cretney, Walter James 1968

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STUDIES IN THE FIELDS OF STEROIDS AND ALKALOIDS BY WALTER JAMES CRETNEY B.Sc. Honours, The U n i v e r s i t y of B r i t i s h Columbia, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o lumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the' Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Department o f A b s t r a c t In Part A of t h i s t h e s i s evidence i s presented concerning the l o c a t i o n and c o n f i g u r a t i o n of the bromine atom i n each of the two i s o m e r i c monobromo d e r i v a t i v e s (8a and 9a) of 5a,25R-spirostan (7, desoxytigogenin) and i n each of the two i s o m e r i c monobromo d e r i v a t i v e s (8b and 9b) of 33-acetoxy-5a,25R-s p i r o s t a n ( t i g o g e n i n acetate) prepared by the a c t i o n of bromine i n a c e t i c a c i on the parent compounds. From a study of the mass s p e c t r a and n u c l e a r magnetic resonance s p e c t r a obtained f o r the monobromotigogenins', i t was e s t a b l i s h e d t h a t the bromine atom was l o c a t e d at the C-23 s i t e . In a d d i t i o n , the c o n f i g u r a t i o n of the bromine atom i n each o f the compounds s t u d i e s was determined. In Part B of t h i s t h e s i s the syntheses of s e v e r a l d e r i v a t i v e s o f 43-dihydrocleavamine (116) having a s u b s t i t u e n t at the C-18 s i t e are d e s c r i b e d . The method employed an apparent SN^ 1 displacement of c h l o r i d e i o n from the a-methyleneindoline form (118, R=H) of the c h l o r o i n d o l e n i n e (113) of 46-dihydrocleavamine. The c h l o r o i n d o l e n i n e was prepared by the a c t i o n of t e r t -b u t y l h y p o c h l o r i t e on 48-dihydrocieavamine and was allowed to r e a c t w i t h s e v e r a l n u c l e o p h i l e s under a v a r i e t y of c o n d i t i o n s . Using s u i t a b l e condi-t i o n s 18a-methoxy-4g-dihydrocleavamine (140), 18g-methoxy-43-dihydrocleav-amine (141), 183~hydroxy-4B-dihydrocleavamine (142), and 183-cyano-43-dihydrocleavamine (143) were prepared. The l a s t compound was transformed i n t o 183-carbomethoxy-43-dihydrocleavamine (139) by unexceptional means. This t r a n s f o r m a t i o n provided a c r u c i a l l i n k i n the t o t a l syntheses of the Vinca a l k a l o i d , c o r o n a r i d i n e (45) and i t C-4 epimer dihydrocatharanthine (46) In Part B of t h i s t h e s i s are a l s o described the syntheses of dimeric compounds. The c h l o r o i n d o l e n i n e of 43-dihydrocleavamine was allowed to r e a c t w i t h d e a c e t y l v i n d o l i n e hydrazide (114) to give a dimer (115) . The c o u p l i n g of the two u n i t s was shown to have taken p l a c e between the C-18 s i t e of 48-dihydrocleavamine and the C-15 s i t e of d e a c e t y l v i n d o l i n e hydra-z i d e . The d i m e r i c Vinca a l k a l o i d s i s o l e u r o s i n e A (110) and vincaleuko-b l a s t i n e (as the methiodide s a l t , 109) are coupled i n the same manner. I s o l e u r o s i n e A and v i n c a l e u k o b l a s t i n e have i n common a carbomethoxy group at the C-18 s i t e o f the dihydrocleavamine p o r t i o n . The syntheses of two dimers (147.and 148) are described which a l s o have t h i s f e a t u r e . The syntheses were accomplished i n the manner of the previous coupling using the c h l o r o i n d o l e n i n e (117, R^COOMe) of 18B-carbomethoxy-43-dihydrocleavamine i n place of the c h l o r o i n d o l e n i n e of 4B-dihydrocleava;nine. In Part C of t h i s t h e s i s an e f f e c t i v e method f o r preparing t r i t i u m and deuterium l a b e l l e d i n d o l e a l k a l o i d s i s described. T r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d or t r i f l u o r o a c e t i c a c i d - d was used. A combination of the methods o f mass spectrometry and n u c l e a r magnetic resonance s p e c t r o -scopy was used to e s t a b l i s h that the deuterium atoms were lo c a t e d p r i m a r i l y i n the benzene p o r t i o n of deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydro-cleavamine (67) and 183-carbomethoxycieavamine (73). A l s o i n Part C of t h i s t h e s i s are described t r a c e r experiments of a 14 p r e l i m i n a r y nature i n Vinca rosea L. p l a n t s using [22- C]-18B-carbomethoxy-4B-dihydrocleavamine and [T-aromatic]-183-carbomethoxycleavamine and t r a c e r experiments i n Vinca minor L. p l a n t s using [T-aromatic]-vincadine (74) and [T-aromatic]-vincaminoreine (75). - i v -TABLE OF CONTENTS Pa^e TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES v LIST OF TABLES X 1 ACKNOWLEDGEMENTS X 1 1 PART A I. I n t r o d u c t i o n 2 I I . D i s c u s s i o n of Results 10 I I I . General Conclusions 23 IV. Experimental 27 References 29 PART B I. I n t r o d u c t i o n 3 2 I I . D i s c u s s i o n 76 I I I . Experimental 140 References 165 PART C I. I n t r o d u c t i o n 170 I I . D i s c u s s i o n 198 I I I . Experimental 227 References 241 LIST OF FIGURES PART A Figure Page 1 The Iso r e a c t i o n 5 2. Mass spectrum o f 23S-bromo-5a,25R-spirostan 9 3. Mass spectrum of 23R-bromo-5ct,25R-spirostan 10 4. High f i e l d r e g i o n o f nmr sp e c t r a at 60 Mcps 14 5. Low f i e l d r e g i o n of nmr s p e c t r a at 60 Mcps 19 6. Low f i e l d r e g i o n of nmr s p e c t r a at 60 Mcps 19 7. Low f i e l d r e g ion of nmr sp e c t r a at 100 Mcps 22 PART B 1. B i o s y n t h e t i c p o s t u l a t e f o r the pro d u c t i o n of Aspidosperma and Iboga skeletons 35 2. Transannular c y c l i z a t i o n g i v i n g the Aspidosperma s k e l e t o n . 36 3. Transannular c y c l i z a t i o n g i v i n g the Aspidosperma and Iboga skeletons 37 4. E q u i l i b r i a that would r e s u l t i n l o s s o f o p t i c a l p u r i t y .... 39 5. I n t e r c o n v e r s i o n of t u b i f o l i n e and c o n d i f o l i n e 40 6. A mechanism f o r t a u t o m e r i z a t i o n o f iminium f u n c t i o n s across n i t r o g e n 41 7. I n t e r c o n v e r s i o n o f veatchine and g a r r y i n e 41 8. Kutney's syntheses of dl-quebrachamine, dl-4ct-dihydrocleav-amine and dl-4B-dihydrocleavamine 46 9. Stork's s y n t h e s i s o f dl-1,2-dehydroaspidospermidine 48 - v i -PART B continued Figure Page 10. E q u i l i b r i u m w i t h stereochemical i m p l i c a t i o n s and s t e r e o -s p e c i f i c formation of dl-aspidospermine 49 11. Stork's s y n t h e s i s o f t r i c y c l i c keto amine 62 51 12. Ban's s y n t h e s i s of t r i c y c l i c keto amine 63 51 13. Kuehne's s y n t h e s i s of t r i c y c l i c keto amines 64 and 65 52 14. Harley-Mason 1s s y n t h e s i s of dl-aspidospermidine 52 15. Buchi's s y n t h e s i s of dl-ibogamine and dl-epiibogamine 53,54 16. Nagata's s y n t h e s i s o f dl-ibogamine 57 17. Reaction of bridged a z i r i d i n i u m ions w i t h n u c l e o p h i l e s .... 58 18. S a l l e y ' s s y n t h e s i s of dl-ibogamine 59,60 19. T a y l o r ' s hypothesis concerning an unusual r e a c t i o n of i n d o l e n i n e s 62 20. Reactions o f a hydroperoxyindoline 63 21. Reaction g i v i n g s u b s t i t u t i o n adjacent t o the a - p o s i t i o n of an i n d o l e 63 22. Reaction g i v i n g s u b s t i t u t i o n at the a - p o s i t i o n o f an i n d o l e 64 23. Buchi's s y n t h e s i s of voacangine 64 24. Kutney's r a t i o n a l i z a t i o n f o r the d e c a r b o x y l a t i o n of catharanthine 67 25. Dolby's r a t i o n a l i z a t i o n f o r the r e d u c t i o n of 2-indolecarb-i n o l d e r i v a t i v e s 68 26. I n t e r n a l r e t u r n w i t h i n an i o n p a i r 70 27. An unusual rearrangement o f the c h l o r o i n d o l e n i n e of ibogaine 71 - v i i -PART B continued Figure P c ge 28. Scheme envisaged f o r the p r e p a r a t i o n of 18 - s u b s t i t u t e d dihydrocleavamines 76 29. Mass spectrum of the c h l o r o i n d o l e n i n e of 48-dihydrocleav-^ amine 79 30. Nmr spectrum of the c h l o r o i n d o l e n i n e of 48-dihydrocleav-amine 81 31. Conceivable modes of r e a c t i o n of the c h l o r o i n d o l e n i n e of 43-dihydrocleavamine 82 32. Spiromidoether formation from a c h l o r o i n d o l e n i n e 83 33. An unusual S N 2 ' r e a c t i o n i n v o l v i n g n u c l e o p h i l i c a t t a c k on oxygen 85 34. Flow sheet showing the p a r t i a l s e p a r a t i o n of the products of the r e a c t i o n o f the c h l o r o i n d o l e n i n e 113 under the Buchi c o n d i t i o n s 87 35. Mass spectrum o f 18a-methoxy-48-dihydrocleavamine 92 36. Nmr spectrum o f 18a-methoxy-43-dihydrocleavamine 93 37. Mass spectrum of 18B-methoxy-4B-dihydrocleavamine 96 38. Nmr spectrum of 188-methoxy-48-dihydrocleavamine 97 39. Mass spectrum o f 18B-hydroxy-48-dihydrocleavamine 106 40. Nmr spectrum of 18B-hydroxy-46-dihydrocleavamine 107 41. Mass spectrum of 18B-cyano-48-dihydrocleavamine 109 42. Nmr spectrum o f 188-cyano-43-dihydrocleavamine 110 43. U l t r a v i o l e t spectrum of the dimer 115 117 44. Mass spectrum of the dimer 115 118 - v i i i -PART B continued Figure Page 45. Nmr spectrum o f the dimer 115 119 46. Nmr spectrum of 4B~dihydrocleavamine 121 47. Nmr spectrum o f d e a c e t y l v i n d o l i n e hydrazide 122 48. Nmr spectrum of the c h l o r o i n d o l e n i n e of 18B-carbomethoxy-4B -dihydrocleavamine 126 49. Mass spectrum o f the c h l o r o i n d o l e n i n e o f 18$-carbomethoxy-46-dihydrocleavamine 127 50. Mass spectrum o f the dimer 147 128 51. Nmr spectrum o f the dimer 147 130 52. Nmr spectrum o f 183-carbomethoxy-4B-dihydrocleavamine 131 53. Mass spectrum of the dimer 148 135 54. Nmr spectrum of the dimer 148 136 PART C 1. D e r i v a t i o n of Aspidosperma and Iboga types of backbone from the Corynanthe type of backbone 172 2. Barger-Hahn-Robinson-Woodward hypothesis 174 3. Pathway from s h i k i m i c a c i d to dihydroxyphenylpyruvic a c i d i n c o r r e c t l y i n v o l v i n g a hydrated prephenic a c i d intermediate 175 4. E s s e n t i a l features of hydrated prephenic a c i d hypothesis .. 176 5. Prephenic a c i d hypothesis 177 6. Acetate hypothesis f o r the pro d u c t i o n of a corynantheine-s t r y c h n i n e type of condensing u n i t 178 7. Examples of monoterpenic glucosides having corynantheine-s t r y c h n i n e type o f backbone 179 - i x -PART C continued Figure , Page 8. I n c o r p o i a t i o n of g e r a n i o l i n t o a l k a l o i d s r e p r e s e n t i n g the three s t r u c t u r a l types of i n d o l e a l k a l o i d s o f the tryptamine + C Q _ ^ Q type 182 9. Rearrangement i n backbone of g e r a n i o l to give m o d i f i c a t i o n 1. of the C N i n condensing u n i t i n v o l v i n g a cyclopentane monoterpene u n i t 183 10. I n c o r p o r a t i o n of loganin i n t o v a r i o u s i n d o l e a l k a l o i d s .... 185 11. Proposal f o r the pathway from mevalonate to i n d o l e a l k a l o i d s of the tryptamine + Cg ^ type 186 12. A cyclopentane cleavage r e a c t i o n of p o s s i b l e b i o s y n t h e t i c importance 187 13. P l a u s i b l e pathway t o ajmaline and r e l a t e d a l k a l o i d s 189 14. P l a u s i b l e pathway t o the Akuamma a l k a l o i d s 191 15. A l t e r n a t i v e pathway t o echitamine and r e l a t e d a l k a l o i d s ... 192 16. F i n a l stages i n Wenkert's proposal f o r the b i o s y n t h e s i s of a l k a l o i d s w i t h the Aspidosperma and Iboga types o f s k e l e t o n 193 17. P l a u s i b l e v a r i a t i o n on Wenkert's proposal f o r the biosynthe-s i s of a l k a l o i d s w i t h the Aspidosperma and Iboga types of sk e l e t o n 194 18. A p l a u s i b l e pathway from stemmadenine t o a l k a l o i d s w i t h the Aspidosperma and Iboga types o f s k e l e t o n 195 19. P l a u s i b l e pathway t o vincamine and r e l a t e d a l k a l o i d s and t o the a l k a l o i d v a l l e s a m i d i n e 196 20. Mass spectrum of unlabel l e d 18a-carbomethoxy-4et-dihydro-cleavamine 204 - x -PART C continued Figure Page 21. Mass spectrum o f deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine 205 22. P l a u s i b l e pathway g i v i n g a fragment with an m/e value of 210 207 23. Nmr spectrum of deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine 209 24. Nmr spectrum of u n l a b e l l e d 18a-carbomethoxy-4a-dihydrocleav-amine 210 25. Nmr spectrum of deuterium l a b e l l e d 186-carbomethoxycleavamine 214 26. Bag-on-leaf method of feeding 222 27. Apparatus f o r small s c a l e l a b e l l i n g experiments 230 - x i -LIST OF TABLES PART B Table Page 1 • : I l l 2 113 3 123 4 132 5 137 PART C 1 2 212 215 ACKNOWLEDGEMENTS I wish to express my s i n c e r e a p p r e c i a t i o n to Professo r J . P. Kutney f o r being an u n f a i l i n g source of advic e , encouragement, and i n s p i r a t i o n d u r i ng the course of t h i s research. I am a l s o g r a t e f u l f o r having r e c e i v e d a Na t i o n a l Research Council of Canada Studentship during my s t u d i e s . PART A STRUCTURE AND STEREOCHEMISTRY OF THE MONOBROMOTIGOGENINS I. I n t r o d u c t i o n The problem o f c o n c l u s i v e l y e s t a b l i s h i n g the s t r u c t u r e and stereo-chemistry o f bromosapogenins began almost three decades ago when Marker and Rohrmann* i n the course o f t h e i r study o f the s t e r o i d a l sapogenin s p i r o -k e t a l system found t h a t sarsasapogenin (1) y i e l d e d a monobromo d e r i v a t i v e on treatment w i t h bromine i n a c e t i c a c i d . Treatment o f t h i s monobromo 1 2 d e r i v a t i v e w i t h sodium i n a l c o h o l ' regenerated sarsasapogenin. Since the bromination occurred r e a d i l y , Marker r a t i o n a l i s e d that there must be a p o t e n t i a l carbonyl group i n sarsasapogenin and thus the bromination e x p e r i -ment provided a v a l u a b l e clue i n the eventual f o r m u l a t i o n of the s p i r o k e t a l system. At the time o f t h i s work, Marker considered that there were two 2 p o s s i b l e l o c a t i o n s f o r the bromine, at C-20 or at C-23. In a l a t e r study, chromic a c i d o x i d a t i o n o f bromosarsasapogenin acetate provided 30-acetoxy-16-oxo-53-pregnane 20S-carboxylic a c i d (2). On the b a s i s of having obtained t h i s compound, Marker was able to r u l e out the C-20 s i t e f o r the bromine l e a v i n g the C-23 s i t e as the probable s i t e f o r the bromine. Subsequently, other workers on the b a s i s of Marker's assignment have assumed t h a t other side-chain-brominated sapogenins have had the bromine at the C-23 s i t e . Furthermore, when i n the course o f these bromination s t u d i e s on s t e r o i d a l sapogenins, isomeric side-chain-brominated sapogenins were i s o l a t e d , they 3-5 were r a t h e r a r b i t r a r i l y assigned as 23a and 23b epimers. In f a i r n e s s to the i n t e r e s t e d workers, however, they were c a r e f u l to p o i n t out the r a t h e r - 3 -V H COOH MG 0 HO H AcO H 1 2 4 tenuous nature of t h e i r assignments. For i n s t a n c e , Wall and Jones i n r e p o r t i n g a study of the s i d e - c h a i n bromination of d i o s g e n i n and t i g o g e n i n i n which they chose the C-23 s i t e f o r the l o c a t i o n of the bromine were moved to r e c o r d t h a t "although t h i s assignment i s l o g i c a l , . . . , i t must be emphasized t h a t the C-23 assignment i s not based on a r i g i d s t r u c t u r e 3 proof." E a r l i e r M u e l l e r and Norton had reported t h a t they were unable to o x i d i z e 23a bromohecogenin acetate and o b t a i n a 12,.16-diketo-20-c a r b o x y l i c a c i d i n analogy w i t h Marker's reported o x i d a t i o n of bromosarsa-sapogenin acetate and hence could not o b t a i n d i r e c t evidence to exclude the C-20 s i t e as the l o c a t i o n of the bromine. To add f u r t h e r confusion to the side-chain-bromination s t o r y , Wall and Jones^ were s u c c e s s f u l i n preparing d e r i v a t i v e s of d i o s g e n i n and t i g o g e n i n acetate t h a t contained two r a t h e r than one bromine atom i n the s i d e chain i n analogy with the work o f others. In each case two products were obtained which had the d e s i r e d e x t r a bromine atom. The major product of these r e a c t i o n s was t e n t a t i v e l y assigned the 23,23-dibromo s t r u c t u r e , whereas the minor product was considered to have one bromine atom " l o c a t e d i n some p o s i t i o n other than C-23." By t h i s - 4 -time the choice of C-23 as the s i t e of s i d e - c h a i n bromination of stero.'dal sapogenins was indeed very a r b i t r a r y . F i n a l l y , work was cul m i n a t i n g i n an ex p l a n a t i o n of a seemingly u n r e l a t e d r e a c t i o n o f the s p i r o k e t a l s i d e chain. Accompanying t h i s e x p l a n a t i o n was the n e c e s s i t y to co n s i d e r C-25 f o r v a l i d chemical reasons as a p o s s i b l e s i t e o f bromination. I r o n i c a l l y , t h i s r e a c t i o n was f i r s t d escribed by Marker and Rohrmann i n the same paper^ i n which they described the s i d e - c h a i n bromination of sarsasapogenin. Marker and Rohrmann found t h a t sarsasapogenin on treatment w i t h r e f l u x i n g a l c o h o l i c h y d r o c h l o r i c a c i d was converted i n t o isosarsasapogenin, which was i d e n t i c a l w i t h the n a t u r a l l y o c c u r r i n g smilagenin. These authors assumed t h a t the two compounds d i f f e r e d only i n c o n f i g u r a t i o n at C-22. Much l a t e r , Scheer, 8 K o s t i c , and M o s e t t i g repeated experiments which were purported to e s t a b l i s h t h a t these two compounds (and other s i m i l a r p a i r s ) were C-22 epimers and found that degradation products of sarsasapogenin and smilagenin i n which the C-22 asymmetry had been destroyed were not the same as had been i n d i c a t e d by previous workers. In l i g h t of these r e s u l t s they c a r r i e d out f u r t h e r experiments and showed that sarsasapogenin and smilagenin d i f f e r e d i n c o n f i g u r a t i o n at C-25 although they were unable to e s t a b l i s h whether or not the compounds d i f f e r e d i n c o n f i g u r a t i o n at C-22. Working with another p a i r of compounds, neotigogenin and t i g o g e n i n , which are r e l a t e d i n the same 9 manner as sarsasapogenin and smil a g e n i n , Callow and Massey-Beresford were able t o destroy the asymmetry at C-25 without a f f e c t i n g the c o n f i g u r a t i o n at C-22 and thereby e s t a b l i s h that neotigogenin and t i g o g e n i n have the same c o n f i g u r a t i o n at C-22. The Iso r e a c t i o n was t h e r e f o r e shown to b r i n g about an e p i m e r i z a t i o n at C-25 without a l t e r i n g the r e s t o f the molecule. This e p i m e r i z a t i o n at f i r s t s i g h t was most unusual and seemingly i n e x p l i c a b l e s i n c e the p o s i t i o n a f f e c t e d was 3 to an ether l i n k a g e and under o r d i n a r y - 5 -circumstances would be c h e m i c a l l y i n e r t to r e f l u x i n g a l c o h o l i c h y d r o c h l o r i c a c i d . On the b a s i s of the f a c t that the e p i m e r i z a t i o n took p l a c e at a l l , c o n s i d e r a t i o n of the C-25 s i t e as a p o s s i b l e one f o r bromination would hav>^ to be made although the bromination r e a c t i o n s were c a r r i e d out under m i l d e r c o n d i t i o n s . In the l i g h t , however, o f the proposal put f o r t h by Woodward^ to e x p l a i n the Iso r e a c t i o n , i t became c l e a r that C-25 as a s i t e o f bromination must be s e r i o u s l y considered. Woodward suggested as shown i n Figure 1 that the i s o m e r i z a t i o n proceeds through a r e v e r s i b l e o x i d a t i o n -r e d u c t i o n mechanism i n which an intermediate C-26 aldehyde i s formed which Figure 1. The Iso r e a c t i o n permits e p i m e r i a t i o n of the C-25 p o s i t i o n v i a i t s enol form (6). In support of t h i s mechanism Woodward reported that a mixture of 25-epimeric aldehydes, which had been prepared by dichromate o x i d a t i o n of d i h y d r o t i g o g e n i n a c e t a t e , on treatment under the usual c o n d i t i o n s of the Iso r e a c t i o n gave a mixture from which t i g o g e n i n and neotigogenin could be i s o l a t e d . This showed that - 6 -the r e a c t i o n as proposed would at l e a s t proceed i n the d i r e c t i o n 5 —>- 3. That the r e a c t i o n could proceed i n the d i r e c t i o n 3 * 5 was demonstrated by D j e r a s s i and c o w o r k e r s ^ who found t h a t the aldehyde intermediate 5 could be trapped as i t s t h i o k e t a l d e r i v a t i v e when t i g o g e n i n acetate was t r e a t e d w i t h ethane d i t h i o l or propane d i t h i o l . The mechanism proposed by Woodward was, i n a d d i t i o n , supported by deuterium exchange experiments 12 c a r r i e d out by Callow and Massey-Beresford. I t was consequently q u i t e apparent that through the i n t e r m e d i a t i o n of the enol form 6 of the aldehyde 5 bromination could take place at C-25 and t h a t one or both of the monobromo-d e r i v a t i v e s of a s t e r o i d a l sapogenin could have the bromine atom l o c a t e d at C-25. When the study of the bromotigogenins reported here was undertaken, i t was apparent t h a t although the C-23 s i t e was s t i l l the most l i k e l y f o r the l o c a t i o n of the bromine atoms, a l l the p o s s i b l e p o s i t i o n s would have t o be considered and the methods used would have to d i s t i n g u i s h between a l l p o s s i b l e monobromo isomers i f c o n c l u s i v e assignments were t o be made. At the outset of the work i t seemed that the problem would lend i t s e l f n i c e l y to a mass spectrometric and nmr s p e c t r o s c o p i c study, i n p a r t i c u l a r the l a t t e r . 13 P r e v i o u s l y , Barton and coworkers " had made an extensive i n f r a r e d spectro-s c o p i c study of monobromosapogenins and were able to produce convincing evidence based on the p o s i t i o n of the C-Br sketching frequencies that the so c a l l e d 23a isomers had an e q u a t o r i a l bromine atom and the 23b isomers had an a x i a l bromine atom. U n f o r t u n a t e l y , the method was l i m i t e d i n t h a t i t could not e s t a b l i s h t h a t C-23 was the l o c a t i o n of the bromine atoms. In comparison i t was f e l t that an nmr s p e c t r o s c o p i c study coupled with a mass spectrometric study would lead to an unequivocal assignment of the l o c a t i o n as w e l l as the c o n f i g u r a t i o n o.c the bromine atoms i n monobrominated s t e r o i d a l sapogenins. The monobromo d e r i v a t i v e s of 5a, 2 5 R - s p i r o s t a n ^ (7, d e soxytigogenin)^ and 33-acetoxy-5aj25R-spirostan ( t i g o g e n i n acetate) were chosen f o r the purposes of the study. The study was a c o l l a b o r a t i v e e f f o r t w i t h Drs. G. N. P e t t i t and J . C. Knight at the U n i v e r s i t y of Maine, H H 7 Orono, Maine, U.S.A. Their work p a r a l l e l e d ours and they were able to supply us w i t h samples of both bromo-isomers of t i g o g e n i n acetate f o r study purposes as w e l l as samples of both bromo-isomers of desoxytigogenin f o r comparison w i t h those prepared i n our l a b o r a t o r y . I I . D i s c u s s i o n of R e s u l t s Bromination^ of desoxytigogenin gave two monobrominated products: one (8a) m e l t i n g at 193-194°, [a]*2 -643°, and another (9a), p r e v i o u s l y unreported, m e l t i n g at 215-217° to 225-226° (depending on r a t e of h e a t i n g ) , [ a ] p 2 -87.4°. Pure samples of each isomer could be c o n v e n i e n t l y obtained from the crude r e a c t i o n mixture by column chromatography on b a s i c (pH 8) s i l i c a g e l . Nevertheless, the two isomers had very s i m i l a r r e t e n t i o n times and t h e r e f o r e the bulk of the m a t e r i a l from any given chromatography s t i l l remained as a mixture. In Dr. P e t t i t ' s l a b o r a t o r y a much more tedious and time consuming f r a c t i o n a t i o n of the mixture by c r y s t a l l i z a t i o n had been c a r r i e d out. This method had e v e n t u a l l y brought about complete separation of the mixture. Elemental and mass s p e c t r a l (Figures 2 and 3) a n a l y s i s e s t a b l i s h e d that both isomers were products of monobromination. The fragmentation p a t t e r n s of both isomers were very s i m i l a r . Both e x h i b i t e d a molecular i o n at m/e 480 and of p a r t i c u l a r importance a fragment at m/e 331, which a l s o appeared i n the spectrum of desoxytigogenin. This p a r t i c u l a r fragment at m/e 331 has been a s s i g n e d ^ the s t r u c t u r e 10. The appearance of the fragment 10 i n both monobromodesoxytigogenins precluded C-20 as the l o c a t i o n of the bromine atom i n e i t h e r isomer. 4 Tigogenin acetate was brominated and gave two isomeric bromotigogenin ac e t a t e s : 8b, mp 206-208°, [ a ] 2 2 -61.9° and 9b, mp 201-202°, [ a ] 2 2 -57.0°, - 6 -100 80 60 40 20 55 71 69| 57 81 95 i.-LLL. 109 m.p. 225-226° [a]2-87.4° L JD 257 147 161 217 25827| 286 ll.. l!:L (III I I J. l,!lL ,iMu ,i!i„ „u mi! ill. L i 331 ' ' L_l L. 287 .4 i — _ i i i i i i 478||480(M+) 399 -I L__l I ' ' • 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 m/e Figu re 3. . Mass spectrum o f 23R-bromo-5a ,25R-spi ros tan - 11 -8: a, R=H; b, R=0Ac 9: a, R=H; b, R=OAc which were shown to be monobrominated products by elemental a n a l y s i s . 10 From a p r e l i m i n a r y nmr study"^ of s t e r o i d a l sapogenins of both the 25D (C-25 e q u a t o r i a l methyl group) and 25L (C-25 a x i a l methyl group) s e r i e s conducted i n our l a b o r a t o r i e s i t appeared that two regions of the nmr sp e c t r a o f the monobromotigogenins would be of p a r t i c u l a r value i n s o l v i n g the problem i n hand: ( a ) , the high f i e l d r e g i o n encompassing the C-methyl s i g n a l s and (b), the r e g i o n between 200 cps and 300 cps (at 60 Mcps) i n which the s i g n a l s of the protons at C-16, C-23 and C-26 (as w e l l as C-3 i n the monobromotigogenin acetates) occur. I t was found t o be u s e f u l to measure chemical s h i f t s and chemical s h i f t d i f f e r e n c e s i n cps. Chemical s h i f t s quoted are r e l a t i v e to the TMS s i g n a l set at 0 cps. The C-methyl r e g i o n at 60 Mcps The C-methyl r e g i o n of compounds 8a, 8b, 9a and 9b i s shown i n Figure 4. By analogy w i t h the assignments made f o r methyl protons i n the study mentioned above, i t was p o s s i b l e to make assignments w i t h confidence i n t h i s r e g i o n . In the sapogenins of the 25D s e r i e s , of which the compounds presented i n t h i s study are members, the protons of the C-25 methyl group were observed t o g i v e r i s e to a doublet of which one s i g n a l , g e n e r a l l y the only d i s c e r n a b l e member of the doublet, was found at highest f i e l d of a l l s i g n a l s . In 8a and 8b the higher f i e l d member of t h i s doublet was found at 44 cps and 45 cps, r e s p e c t i v e l y . The s i g n a l s from the angular methyl protons (C-18 and C-19) were observed to appear as two sharp spikes r i s i n g above the r e s t of the s i g n a l s and were o f t e n q u i t e c l o s e together. In 8a and 8b they were found at 47 and 51 cps, and 50 and 53 cps, r e s p e c t i v e l y . In a l l the s t e r o i d a l sapogenins of both the 25D and 25L s e r i e s examined i n the p r e l i m i n a r y study, the C-19 s i g n a l occurred at higher f i e l d than the C-18 s i g n a l . In the p a r t i c u l a r instance of the monobromotigogenins i t was d i f f i c u l t to a s s i g n w i t h c e r t a i n t y the C-18 and C-19 methyl resonances to p a r t i c u l a r spikes because the e f f e c t of the bromine atom was d i f f i c u l t to a s c e r t a i n . (This problem w i l l be discussed more f u l l y l a t e r . ) Like the doublet due to the C-27 methyl protons, the doublet due to the C-21 methyl group u s u a l l y was observed to show only one d i s c e r n a b l e s i g n a l at s l i g h t l y lower f i e l d than the s i g n a l s from the C-18 and C-19 methyl protons. In - 13 -the case of 8a, t h i s s i g n a l occurred at 37 cps and i n the case o f 8b, at 58 cps. These assignments were assumed t o describe only the twelve protons contained i n the four methyl groups found i n the molecules. That no s i g n a l from another proton i n a molecule had been confused f o r one of these twelve protons described was v e r i f i e d by the observation that the i n t e g r a l over t h i s r e g i o n corresponded t o e x a c t l y twelve protons. In the case o f compounds 9a and 9b there were some i n t e r e s t i n g features of the C-methyl r e g i o n which were germane t o the poi n t of the study, which was t o d e s c r i b e the l o c a t i o n and c o n f i g u r a t i o n o f the bromine atoms. In the s p e c t r a of both compounds, the C-21 methyl proton s i g n a l s were s h i f t e d appearing as a d i s t i n c t doublet (J = 7 cps) some 16 cps downfield from t h e i r p o s i t i o n i n 8a and 8b and were w e l l removed from the s i g n a l s due to the three other methyl groups. That t h i s s h i f t had taken p l a c e was supported by the i n t e g r a l of the s p e c t r a which showed that the doublet corresponded to 3 protons and the group of s i g n a l s assigned t o the other three angular methyl groups corresponded to nine protons. Confirmation f o r t h i s a s s i g n -ment was obtained when the nmr spectrum of 9a was run at 100 Mcps. The se p a r a t i o n i n the s i g n a l s assigned to the doublet from the C-21 methyl protons remained unchanged at 7 cps. I t i s i n t e r e s t i n g t o note that because the protons of the C-18 and C-19 methyl groups i n 9a occurred f o r t u i t o u s l y as a s i n g l e sharp spike at 47 cps, both s i g n a l s a t t r i b u t a b l e to the C-27 methyl doublet appeared, at 45 cps and 50 cps (J = 5 cps). In the case of 9b the s i t u a t i o n was more t y p i c a l and only the upper f i e l d member of the doublet a t t r i b u t a b l e to the C-27 methyl protons was seen to occur at 46 cps. As an a s i d e , the other member of t h i s doublet, by assuming the same co u p l i n g as i n the case of 9a, was expected at 51 cps and th e r e f o r e - 14 -would have c o i n c i d e d w i t h the methyl spike at 51 cps. Indeed, the s p i r e at 51 cps i n the spectrum o f 9b d i d seem i n o r d e n t l y l a r g e r than the one at 48 cps by comparison w i t h the same spikes i n the sp e c t r a of 8a and 8b. 53 51 . 4 7 50 Figure 4. High f i e l d r e g i o n of nmr s p e c t r a at 60 Mcps Co n s i d e r a t i o n of the f a c t s above gained by examination of the methyl r e g i o n o f the various s p e c t r a made i t p o s s i b l e to make a f a i r l y r e l i a b l e • assignment of l o c a t i o n and c o n f i g u r a t i o n of the bromine atom i n the four compounds s t u d i e d . For i n s t a n c e , C-20 and C-25 were r u l e d out as p o s s i b l e s i t e s f o r the l o c a t i o n of the bromine atom s i n c e i f the bromine atom had r e s i d e d at e i t h e r of these s i t e s the proton s i g n a l s of the methyl' group sha r i n g the same ca.rbon atom would have been s h i f t e d to lower f i e l d , and - 15 -would have appeared as a s i n g l e t . The s p e c t r a obtained showed no such f e a t u r e . Of the remaining s i t e s i n the s p i r o k e t a l s i d e c h a i n , C-23 seemed the most l o g i c a l s i n c e there appeared to be no chemical reason t o assume tha t bromination could occur at C-24 and d o u b t f u l reason to assume that bromination c o u l d occur at C-26. Examination of molecular models of the monobromotigogenins showed that an a x i a l C-23 bromine s u b s t i t u e n t would probably be i n a 1,3 d i a x i a l r e l a t i o n to the C-21 methyl group. F a i r l y 17-19 complete data have been compiled concerning the e f f e c t of v a r i o u s s u b s t i t u e n t s i n s t e r o i d systems on the C-18 and C-19 angular methyl protons. For example, the e f f e c t o f a bromine 1,3 to an angular methyl group has been determined f o r a 28-bromo and a 68-bromo s u b s t i t u e n t i n the 5a,14a-androstane system and found t o r e s u l t i n a downfield s h i f t i n the C-19 s i g n a l o f 14.0 cps and 15.0 cps, r e s p e c t i v e l y . The downfield s h i f t i n the proton resonance of the C-21 methyl group i n 9a and 9b compared to t h e i r parent compounds was found t o agree very w e l l and was about 16 cps i n each case. Since there was no noteworthy s h i f t i n the C-21 methyl resonance of compounds 8a and 8b, i t was t e n t a t i v e l y concluded that 9a and 9b had an a x i a l bromine at C-23 and- 8a and 8b had an e q u a t o r i a l bromine at C-23. Although not d i r e c t l y concerned w i t h the o b j e c t i v e of the nmr study, i t was an i n t e r e s t i n g e x c e r c i s e i n the case of the 23-monobromo epimers to attempt t o a s s i g n the C-18 and C-19 methyl proton resonances. In the spectrum of desoxytigogenin the C-18 methyl resonance occurred at 46 cps and the C-19 methyl resonance occurred at 48 cps. In the spectrum of t i g o g e n i n a c e t a t e , which- can be considered as the 33 - a c e t a t e d e r i v a t i v e o f desoxytigogenin, the C-18 methyl resonance occurred at 46 cps and the C-19 methyl resonance occurred at 50 cps. Since the C-19 methyl group i n the monobromotigogenins was seen to be f a r removed from the bromine s u b s t i t u e n t - It -i n the sapogenin s i d e c h a i n , the C-19 methyl resonance was expected t o occur i n n e a r l y the same p o s i t i o n as i t d i d i n ."he parent compound; that i s , at 48 cps i n the case o f 8a or 9a and at 50 cp^; i n the case of 8b or 9b. On t h i s b a s i s the l o g i c a l assignment of the C-19 methyl resonances r e q u i r e d that the spike at 47 cps i n the spectra of 8a and 9a and the spikes at 50 cps and 51 cps i n the s p e c t r a o f 8b and 9b, r e s p e c t i v e l y , be due to the C-19 methyl protons. The remaining unassigned spike i n each spectrum by d e f a u l t must have been due t o the C-18 methyl resonance. In the case of compounds 9a and 9b, i n which the bromine had been assigned the a x i a l c o n f i g u r a t i o n , the C-18 methyl resonances were assigned as the peaks at 47 cps and 48 cps, r e s p e c t i v e l y . Since as a general r u l e C-18 methyl resonances occur at higher f i e l d than C-19 methyl resonances, t h i s assignment was q u i t e u n exceptional. Regarding compounds 8a and 8b, however, the s i t u a t i o n was seen t o depart from the o r d i n a r y . The C-18 methyl resonances had to be assigned to the s i g n a l s at 51 cps and 53 cps, r e s p e c t i v e l y , and consequently at lower f i e l d than the C-19 methyl resonances. The e f f e c t of an e q u a t o r i a l bromine s u b s t i t u e n t at C-23 on the C-18 methyl resonances must have caused a downfield s h i f t o f 5 cps i n the case of 8a and of 7 cps i n the case of 8b. Since much had been deduced above on the b a s i s of very small chemical s h i f t d i f f e r e n c e s , an a l t e r n a t e assignment was considered. For the sake of argument i t was assumed th a t the C-18 methyl protons must always resonate at higher f i e l d than the C-19 methyl protons. The assignment f o r compounds 9a and 9b was unchanged, but i n the case of compounds 8a and 8b a reassignment was r e q u i r e d . The s i g n a l at 47 cps, f o r 8a had to be due t o the C-18 methyl protons and the one at 51 cps, to the C-19 methyl protons. This r e q u i r e d that an e q u a t o r i a l bromine s u b s t i t u e n t at C-23 must have r e s u l t e d i n a downfield s h i f t of 3 cps i n the p o s i t i o n of the C-19 methyl resonance and - 17 -only a downfield s h i f t o f 1 cps i n the C-18 methyl resonance. S i m i l a r l y , i n the case of 8b, an e q u a t o r i a l bromine s u b s t i t u e n t must have r e s u l t e d i n a downfield s h i f t o f 3 cps i n the p o s i t i o n of the C-19 methyl resonance to 53 cps and a downfield s h i f t i n the C-18 methyl resonance of 4 cps t o 50 cps. Examination of molecular models showed that the C-19 methyl group was about twice as f a r from the bromine s u b s t i t u e n t as the C-18 methyl group. Since long range magnetic a n i s o t r o p i c and d i p o l e e f f e c t s were known t o be i n v e r s e l y p r o p o r t i o n a l to the cube of the d i s t a n c e from a f u n c t i o n a l group, the e f f e c t of the bromine s u b s t i t u e n t on the C-19 methyl group was expected t o be roughly one-eigth (1/2 3) the e f f e c t on the C-18 methyl group. Thus, i t seemed th a t the a l t e r n a t i v e assignments above were untenable. I t was thus c l e a r that i n compounds 8a and 8b the bromine s u b s t i t u e n t must have had a l o c a t i o n and c o n f i g u r a t i o n that brought i t near enough to the C-18 methyl group t h a t the e f f e c t of bromine was to cause a downfield s h i f t i n the C-18 methyl resonance of o n e - t h i r d t o one-half the downfield s h i f t expected f o r a bromine atom i n a 1,3 d i a x i a l r e l a t i o n s h i p t o an angular methyl group. From an examination of molecular models i t appeared that aside from a C-20 bromine s u b s t i t u e n t , which had been r u l e d out on other grounds, only a C-23 e q u a t o r i a l bromine s u b s t i t u e n t would be near enough to the C-18 methyl group to have an e f f e c t on i t s resonance of the amount and d i r e c t i o n observed. In summary, the d i r e c t i o n and magnitude of the s h i f t i n the C-21 methyl resonance of 9a and 9b supported the assumption that these substances possess a C-23 a x i a l bromine atom. Likewise the s h i f t i n the C-18 methyl resonance of 8a and 8b supported the assumption that they possess a C-23 e q u a t o r i a l bromine atom. Further evidence of an unequivocal nature, which supported the s t r u c t u r a l and stereochemical assignments made above on the b a s i s of an a n a l y s i s of the C-methyl r e g i o n e x h i b i t e d by the monobromotigogenins, was - 18 -found through an analysis of the 200-300 cps region of t h e i r nmr spectra. The 200-300 cps region Examination of t h i s region (Figures 5 and 6) showed that each compound exhibited a broad set of signals a r i s i n g from the C-26 protons that approx-imated a doublet close to 200 cps. This feature was known to be character-i s t i c of sapogenins of the 25D series and i t s presence excluded on i t s own merits C-26 bromo, C-25 bromo and C-23 bromo-25L compounds from consideration as possible alternatives to the structural assignments made above. A more interesting feature of the low f i e l d region of the spectra was that the nmr spectra of compounds 8a and 8b on one hand and compound 9a and 9b on the other dif f e r e d markedly i n the region 230-300 cps. Comparison of t h i s region i n the spectra of the monobromotigogenins with the same region i n the parent compounds allowed assignment of the C-16 and C-3 proton resonances. Desoxy-tigogenin displayed a broad set of signals i n the region 250-275 cps attributed to the C-16 proton and tigogenin acetate displayed, i n addition, a broad set of signals which merged with the set from the C-16 proton on the low f i e l d side and was attributed to the C-3 proton. Comparison with the spectra of the parent compounds showed that the spectra of the monobromo derivatives exhibited an additional set of signals due to one proton on the u p f i e l d side of the signals from the C-16 proton. In the spectra of 8a and 8 b there were i n t h i s set four d i s t i n c t signals of nearly equal spacing and i n t e n s i t y which together resembled a doublet of doublets, whereas i n the spectra of 9a and 9b the set of signals resembled a not-too-well-resolved t r i p l e t . In the case of each monobromotigogenin t h i s set of signals must have arisen from a proton geminal to a bromine atom and the only sit e s i n the side chain where a bromine could be geminal to a proton are C-23, C-24 and - 19 -C-26. S i n c e C-24 and C-26 have been r u l e d o u t , t h e bromine atom must be a t 202 2 0 8 I F i g u r e 5. L o w - f i e l d r e g i o n o f nmr F i g u r e 6. Low f i e l d r e g i o n o f nmr s p e c t r a a t 60 Mcps s p e c t r a a t 60 Mcps C-23. On t h e b a s i s o f an a n a l y s i s o f t h e C-methyl r e g i o n t h e bromine atom i n compounds 8a and 8b had been t e n t a t i v e l y a s s i g n e d an e q u a t o r i a l c o n f i g u r -a t i o n and t h e bromine atom i n compounds 9a and 9b, an a x i a l c o n f i g u r a t i o n . I n t e r p r e t a t i o n o f t h e s e t o f s i g n a l s a r i s i n g f rom t h e C-23 p r o t o n p e r m i t t e d an u n e q u i v o c a l a s s i g n m e n t o f t h e o r i e n t a t i o n o f t h e bromine atom. E x a m i n a t i o n o f m o l e c u l a r models o f t h e monobromotigogenins i n d i c a t e d t h a t r i n g F s h o u l d e x i s t as an u n d i s t o r t e d c h a i r i n a l l c a s e s s i n c e t h e r e were no e x c e p t i o n a l i n t e r a c t i o n s a r i s i n g f rom t h e bromine s u b s t i t u e n t s i n e i t h e r t h e a x i a l o r e q u a t o r i a l c o n f i g u r a t i o n a t C-23. C o n s e q u e n t l y , i t - 20 -appeared t h a t the p a t t e r n o f the C-23 proton i n 8a and 8b should be i n t e r p r e t -able by c o n s i d e r a t i o n o f part s t r u c t u r e 11 and the p a t t e r n of the C-23 proton i n 9a and 9b by c o n s i d e r a t i o n of part s t r u c t u r e 12. 11 12 I t was apparent from the par t s t r u c t u r e 11 that the a x i a l proton would enter i n t o a x i a l - a x i a l s p i n - s p i n i n t e r a c t i o n w i t h H and ax i a l - e a u a t o : A. s p i n - s p i n i n t e r a c t i o n w i t h Hg. In 12 the e q u a t o r i a l proton H was expected t o enter i n t o a x i a l - e q u a t o r i a l s p i n - s p i n i n t e r a c t i o n with and e q u a t o r i a l -e q u a t o r i a l s p i n - s p i n i n t e r a c t i o n w i t h Hg. Since the c o u p l i n g constants and J g ^ i n 12 were expected to be equal or n e a r l y so, the expected p a t t e r n 20 21 f o r the proton was a t r i p l e t . ' In a d d i t i o n what i s probably more important, the h a l f - h e i g h t width (J + Jg^) was expected to be l e s s than 10 22-24 cps. The s i g n a l s from the C-23 proton i n 9a and 9b agreed q u i t e w e l l w i t h t h i s p r e d i c t e d p a t t e r n . The p a t t e r n o f the s i g n a l s was a t r i p l e t , although i t was not too w e l l defined at 60 Mcps, with h a l f - h e i g h t width of 7 cps. A more complicated case was a n t i c i p a t e d when the bromine s u b s t i t u e n t was e q u a t o r i a l as i n 11. In t h i s case the a x i a l proton was expected to enter i n t o a x i a l - a x i a l and a x i a l - e q u a t o r i a l c o u p l i n g with the v i c i n a l protons H^ and Hg. The p a t t e r n a r i s i n g from the proton H^, i f the r i g h t c o n d i t i o n s - 21 -p r e v a i l e d , ^ ( ' ^ could a l s o have been a t r i p l e t . N e v e r t h e l e s s , i t was r e a l i z e d t h a t the h a l f - h e i g h t width (or the d i f f e r e n c e between highest and lowest f i e l d s i g n a l s ) of the t r i p l e t must as i n the previous case be equal 22-24 to J + J g ^ . This sum would be expected to be g r e a t e r than 9 cps. The h a l f - h e i g h t width (7 cps) f o r the t r i p l e t i n 9a and 9b was too small to f i t i n t o t h i s s p e c i a l case. The p a t t e r n observed f o r the C-23 proton resonances i n 8a and 8b resembled a doublet of doub l e t s , a more t y p i c a l p a t t e r n f o r the "X" p o r t i o n of an ABX system such as that shown i n 11. The sum, J + J g ^ j w n i c n w a s about 17 cps, was a l s o more t y p i c a l of the case shown i n 11. Nmr s p e c t r a at 100 Mcps The nature o f the s p e c t r a obtained at 60 Mcps p o i n t e d out that i t would be worthwhile to o b t a i n s p e c t r a at 100 Mcps. A c c o r d i n g l y , desoxytigogenin and i t s monobromo d e r i v a t i v e s were submitted f o r nmr a n a l y s i s at 100 Mcps. At 100 Mcps i n both 8a and 9a the C-23 proton s i g n a l s were unm'i stake ably separated from the C-16 proton s i g n a l s (see Figure 7) and the i n t e g r a l provided c o n f i r m a t i o n o f the one proton nature of each group of s i g n a l s . In the spectrum of 9a the s p l i t t i n g s o f the C-23 proton were measurable and J . (apparent) was found to be about 11 cps and J R Y (apparent), about 6 cps AX D A i n c l o s e agreement with the values expected from the theory. . In the spectrum of 9a the C-23 proton resonances, which at 60 Mcps appeared to form a p o o r l y r e s o l v e d t r i p l e t , formed a c l e a r and unmistakeable t r i p l e t f o r which the f o l l o w i n g estimates, a l s o i n c l o s e agreement w i t h the theory, could be made: JAX + JBX * 3 - 5 C P 5 -From the nmr data alone, enough i n f o r m a t i o n was obtained to permit complete s t r u c t u r a l and stereochemical assignments to be made i n the case of - 22 -the four bromosapogenins s t u d i e d . S i m i l a r nmr data would be expected to provide enough i n f o r m a t i o n to allow v a l i d s t r u c t u r a l and stereochemical assignments to be made i n the case of other bromosapogenins. 415 8a 9a Figure 7. Low f i e l d r e g ion of nmr s p e c t r a at 100 Mcps In summary, desoxytigogenin has been found to a f f o r d as bromination, two monobromo d e r i v a t i v e s which are epimeric at C-23. The compound me l t i n g at 193-194° has been assigned the s t r u c t u r e 8a and the one m e l t i n g at 225-226°, the s t r u c t u r e 9a. S i m i l a r l y , the monobromotigogenin acetate isomer m e l t i n g at 206-208° has been assigned s t r u c t u r e 8b and the one m e l t i n g at 201-202°, the s t r u c t u r e 9b. I I I . General Conclusions Since the epimeric C-23 monobromotigogenins represented by f a r the bulk o f the compounds produced i n the bromination r e a c t i o n , i t seemed t o be worth c o n s i d e r i n g why the C-23 s i t e was p r e f e r r e d f o r bromination. I t was apparent that intermediate 4 (Figure 1), which was suggested as p l a y i n g a r o l e i n the Iso r e a c t i o n , must be i n v o l v e d i n the bromination r e a c t i o n as w e l l . Such an intermediate would be expected, by l o s s of a proton from the C-20 carbon atom, to form the enol ether 13 o r , by l o s s o f a proton from the C-23 carbon atom, t o form the enol ether 14. The enol ether could then have reacted w i t h bromine t o form a C-20 bromo d e r i v a t i v e i n the case of 13 or a C-23 bromo d e r i v a t i v e i n the case of 14. As a general r u l e , the a c i d c a t a l y s e d bromina-25 t i o n o f an unsymmetrical ketone, which resembles the present problem, has l e d to p r e f e r e n t i a l attack at the most s u b s t i t u t e d a carbon atom. Since 13 14 - 24 -t y p i c a l a c i d c a t a l y s i s (Br 2/H0Ac) was used i n the present case, at f i r s t s i g h t the expected products would have been C-20 epimeric bromotigogenini. Since only C-23 epimeric bromo d e r i v a t i v e s were obtained, i t was assumed that e i t h e r the enol ether 13 was formed very s l o w l y under the r e a c t i o n c o n d i t i o n s i n comparison with the r a t e s of formation and bromination of the enol ether 14 or, i f i t were formed at a competitive r a t e , i t d i d not react w i t h bromine at a c o m p e t i t i v e r a t e . I t so happened that the proposed enol ether 13 was known to be a p e r f e c t l y r e s p e c t a b l e compound, a member of the pseudosapogenin f a m i l y , which could be formed simply by treatment of desoxy-t i g o g e n i n or t i g o g e n i n w i t h a c e t i c anhydride (though under f a i r l y vigorous c o n d i t i o n s ) and base. Therefore there seemed to be no reason to deprecate 13 on the grounds that i t would be a very high energy and hence u n l i k e l y i n t e r m e d i a t e . As f a r as the r e a c t i v i t y o f an intermediate such as 13 was 3 concerned, M u e l l e r and Norton brominated pseudohecogenin under m i l d a c i d c a t a l y s i s and obtained no product corresponding t o e i t h e r of the bromo-hecogenins obtained v i a the a c i d c a t a l y s e d bromination of hecogenin a c e t a t e . 26 Furthermore, i t has been shown by s e v e r a l workers that pseudohecogenin could be o x i d i z e d by e i t h e r chromic a c i d or p e r a c i d . O x i d a t i o n of pseudo-hecogenin w i t h chromic a c i d l e d to a 3,12-dione-20-ol (15). Therefore, i t seemed l i k e l y t h a t i f the enol ether 13 was formed i n the bromination r e a c t i o n i t would r e a c t . The only p l a u s i b l e e x p l a n a t i o n of the r e s u l t s of the bromin-a t i o n r e a c t i o n seemed to be that 13 was formed very s l o w l y under the c o n d i t i o n s employed. Perhaps more d i r e c t evidence, which supported the p r o p o s i t i o n that 13 was not an intermediate, was that a pseudosapogenin under a c e t i c a c i d c a t a l y s i s e q u i l i b r a t e s not w i t h the parent sapogenin, but w i t h a compound, having a - 25 -27 s p i r o l c e t a l s i d e c h a i n , c a l l e d a cyclopseudosapogenin. The cyclopseudo-sapogenins were known t o be l e s s s t a b l e than the parent sapogenins having s e v e r a l more s t e r i c i n t e r a c t i o n s and t o be converted to the parent sapogenins under vigorous a c i d c a t a l y s i s (hot a l c o h o l i c h y d r o c h l o r i c a c i d ) . Thus, i t was f e l t t h a t under a c e t i c a c i d c a t a l y s i s the parent sapogenins could e q u i l i b r a t e only extremely s l o w l y w i t h t h e i r pseudoderivatives and, by i n f e r e n c e , 13 could not be an important intermediate i n the bromination r e a c t i o n s i n c e a c e t i c a c i d c a t a l y s i s was used. Examination of molecular models showed th a t the 3 face of the D r i n g i n the case of both the sapogenins and pseudosapogenins was extremely hindered by the C-18 methyl group. Hence, p r o t o n a t i o n of the pseudosapogenin double bond was considered to be much p r e f e r r e d on the a face g i v i n g r i s e to the cyclopseudosapogenin. The energy b a r r i e r to p r o t o n a t i o n of the double bond on the 3 face was considered to be much higher than the b a r r i e r to p r o t o n a t i o n on the a face and, t h e r e f o r e , i t seemed t o be reasonable t o assume th a t more vigorous c o n d i t i o n s than provided by a c e t i c a c i d would be r e q u i r e d to b r i n g about e q u i l i b r i u m i n a reasonable time of the pseudosapogenin w i t h i t s parent - 26 -sapogenin. By the same token i t was assumed that l o s s of the C-20 3 proton i n 4 would be expected t o take place at much slower r a t e i n a c e t i c a c i d than the rat e s of l o s s of e i t h e r of the C-23 protons and subsequent r e a c t i o n w i t h bromine of the enol ether (14) formed. In consequence of the forgoing reasoning the nature of the monobromosapogenins formed i n the a c e t i c a c i d -bromine bromination o f s t e r o i d a l sapogenins was considered t o be c o n t r o l l e d by k i n e t i c f a c t o r s . IV. Experimental Pet. ether r e f e r s t o a petroleum ether f r a c t i o n b o i l i n g at 40-50°. Mps were observed u s i n g a K o f l e r mp apparatus. Elemental microanalyses were performed i n the l a b o r a t o r i e s o f Dr. A. Bernhardt, Max Planck I n s t i t u t e , 28 Mulheim, Germany. Mass s p e c t r a l r e s u l t s ' were obtained u s i n g an Atlas-Mat Model CH4 mass spectrometer equipped w i t h a d i r e c t i n l e t system. I o n i z i n g energy of the mass spectrometer was maintained at 70 ev and the i o n i z i n g current at 50 ya. 29 The nmr s p e c t r a at 60 Mcps and 100 Mcps were recorded employing, r e s p e c t i v e l y , V a r i a n A s s o c i a t e s Models A-60 and HR-100 nmr spectrometers. The s p e c t r a were taken i n de u t e r i o c h l o r o f o r m s o l u t i o n s w i t h t e t r a m e t h y l -s i l a n e used as an i n t e r n a l standard. Values are given i n c y c l e s per second (cps) w i t h respect to the TMS s i g n a l set at 0 cps. I r s p e c t r a l data were provided by Dr. R. A. H i l l ( U n i v e r s i t y of Maine) and o p t i c a l r o t a t i o n (chloroform s o l u t i o n ) measurements by Drs. Weiler and Str a u s s , Oxford, England. 30 Bromination o f 5a,25R-spirostan (7) (by J.C.K.) A s o l u t i o n of B r 2 (2.9 g) i n g l a c i a l a c e t i c a c i d (36 ml) was added (over 30 min) t o a s o l u t i o n (maintained at 60°) composed of desoxytigogenin (6.0 g ) , ^  2 drops o f 4N HBr i n g l a c i a l a c e t i c a c i d , and g l a c i a l a c e t i c a c i d 31 (800 ml). A deep blue c o l o u r formed at once and i n t e n s i f i e d upon f u r t h e r - 28 -a d d i t i o n of B r 2 - The mixture of l i q u i d and c r y s t a l s (separated during bromination) was allowed to remain at room temp f o r ca. 24 h r . A f t e r f i l t r a t i o n the c r y s t a l l i n e product was separated i n t o two p r i n c i p a l com-ponents (3.7 g, mp 186-196° and 1.0 g, mp 211-222°) by f r a c t i o n a l r e c r y s t a l -l i z a t i o n from chloroform (Norit-A)-methanol. Repeated r e c r y s t a l l i z a t i o n of the lower m e l t i n g mixture from chloroform-pet. ether e v e n t u a l l y gave a pure speciment o f 23S-bromo-5ct, 25R-spirostan m e l t i n g at 193-194°; [ a ] p 2 -64.3° KRr -1 (c, 1.24), v 1008, 950, 918, 865 and 730 cm . (Found: C, 67.42; 1T13.X H, 8.75; Br, 16.91; mol wt, 480. C^H^BrOg r e q u i r e s : C, 67.60; H, 9.00; Br, 16.70%; mol wt 480.) S i m i l a r treatment of the higher m e l t i n g f r a c t i o n y i e l d e d an a n l y t i c a l sample of 9a m e l t i n g at 215-217° to 225-226° (depending on r a t e o f h e a t i n g ) ; [ a ] 2 2 -87.4° (c, 1.36); v 1015, 972, 943, 905, U IT13.X and 880 cm"1. (Found: C, 67.36; H, 8.84; Br, 17.01%; mol wt 480.) - 29 -References 1. R. E. Marker and E. Rohrmann, J . Amer. Chem. Soc. 61, 846 (1939). 2. R. E. Marker, D. L. Turner, A. C. Shabica and P. R. U l s h a f e r , J . Amer. Chem. Soc. 63, 1032 (1941). 3. G. P. M u e l l e r and L. L. Norton, J . Amer. Chem. Soc. 76, 749 (1954). 4. M. E. Wall and H. W. Jones, J . Amer. Chem. Soc. 79, 3222 (1957). 5. D. W. H. Dickson and J . E. Page, J . Chem. S o c , 447 (1956). 6. C. D j e r a s s i , H. Martinez and G. Rosenkranz, J . Org. Chem. 16, 303 (1951). 7. J . B. Z i e g l e r , W. E. Rosen and A. C. Shabica, J . Amer. Chem. Soc. 77, 1223 (1955). 8. I. Seheer, R. B. K o s t i c , and E. M o s e t t i g , J . Amer. Chem. Soc. 75, 4871 (1953); 77, 641 (1955). 9. R. K. Callow and P. N. Massey-Beresford, J . Chem. S o c , 4482 (1957). 10. R. B. Woodward, F. Sondheimer, and Y. Mazur, J . Amer. Chem. Soc. 80, 6693 (1958). 11. C. D j e r a s s i , 0. Halpern, G. R. P e t t i t , and G. H. Thomas, J . Org. Chem. 24, 1 (1959) . 12. R. K. Callow and P. N. Massey-Beresford, J . Chem. S o c , 2645 (1958). 13. D. H. R. Barton, J . E. Page, and C. W. Shoppee, J . Chem. S o c , 331 (1956). 14. (a) G. P. M u e l l e r and G. R. P e t t i t , E x p e r i e n t i a 18, 404 (1962). (b) G. R. P e t t i t , i b i d . 19, 124 (1963). (c) E. O'Donnell and M. F. C. Ladd, Chem. § Ind., 1984 (1963). 15. H. Bu d z i k i e w i c z , J . M. Wilson and C. D j e r a s s i , Monatsh. Chem. 93, 1033 (1962). 16. J . P. Kutney, S t e r o i d s 2, 225 (1963). 17. R. F. Zurcher, Helv. Chim. Acta 46, 2054 (1963). 18. N. S. Bhacca and D. H. W i l l i a m s , " A p p l i c a t i o n s of NMR Spectroscopy i n Organic Chemistry", (Holden-Day, Inc.) pp 19-24. 19. A. I. Cohen and S. Rock, S t e r o i d s 3, 243 (1964). - 30 -20. J . D. Roberts, "An I n t r o d u c t i o n to the A n a l y s i s of Spin-Spin S p l i t t i n g i n High-Resolution Nuclear Magnetic Resonance S p e c t r a , " Benjamin, New York, 1961, p. 76. 21. Reference 18, pp. 135-138. 22. Reference 18, p. 51. 23. L. M. Jackman, " A p p l i c a t i o n s o f Nuclear Magnetic Resonance Spectroscopy i n Organic Chemistry," Pergamon Press, 1959, pp. 84-86. 24. A. C. H u i t r i c , J . B. Carr, W. F. Irager and B. J . N i s t , Tetrahedron 19, 2145 (1963). 25. E. S. Gould, "Mechanism and S t r u c t u r e i n Organic Chemistry" H o l t , Rinehart and Winston, New York, 1959, p. 383. 26. L. F. F i e s e r and M. F i e s e r , " S t e r o i d s " , Reinhold, New York 1959, p. 828, 829. 27. Reference 26, pp. 825-828. 28. We wish to thank Professor C. D j e r a s s i , Chemistry Department, Stanford U n i v e r s i t y f o r running the mass s p e c t r a . 29. We wish to thank P r o f e s s o r W. A. Ayer, Chemistry Department, U n i v e r s i t y of A l b e r t a , f o r running the nmr s p e c t r a at 100 Mcps. 30. We wish to acknowledge the c o n t r i b u t i o n s of Dr. T. R. K a s t u r i during and e a r l y phase of t h i s study. 31. Cf., G. P. M u e l l e r , L. L. Norton, R. E. Stobaugh, L. T s a i and R. S. Winniford, J . Amer. Chem. Soc. 75, 4892 (1953). PART B SYNTHESIS OF C-18 SUBSTITUTED DIHYDROCLEAVAMINES \ I. I n t r o d u c t i o n In a general sense a l k a l o i d s are b a s i c n i t r o g e n c o n t a i n i n g compounds found i n p l a n t s . The number of known a l k a l o i d s has been estimated to be about 4000. 1 Many compounds which would appear to f i t t h i s d e s c r i p t i o n of an a l k a l o i d are not numbered among these. Compounds such as p u t r e s c i n e (1) and tryamine (2), which are b a s i c , n i t r o g e n c o n t a i n i n g and found i n p l a n t s , are not considered to be a l k a l o i d s and are o f t e n r e f e r r e d t o as N H A ( C H A N H a 1 p r o t o a l k a l o i d s s i n c e they o f t e n occur as s t r u c t u r a l u n i t s i n a l k a l o i d s . P r o t o a l k a l o i d s are derived from amino a c i d s . For i n s t a n c e , p u t r e s c i n e i s derived from o r n i t h i n e (3) and tyramine from t y r o s i n e (4) by decarboxyla-t i o n . COOH I N H 4 C H ( C H , ) 3 N H a 3 COOH C H a C H N H A 4 - 33 -With some compounds the d i s t i n c t i o n between p r o t o a l k a l o i d s and true a l k a l o i d s would appear to be somewhat hazy. I f p r o t o a l k a l o i d s are considered to 2 in c l u d e as w e l l as b i o g e n i c amines, d e r i v a t i v e s of b i o g e n i c amines, then compounds such as the toad p o i s e n , bufotenine (5) and the h a l l u c i n a t o r y p r i n c i p l e of "peyote" mescaline (6 ) , might b e t t e r be r e f e r r e d to as proto-a l k a l o i d s r a t h e r than as a l k a l o i d s . With few exceptions the " a l k a l o i d s " such N(Me)* ^ MeO NH. MeO as bufotenine which are found i n animals are of the p r o t o a l k a l o i d type. Two 3 noteworthy t r u e a l k a l o i d s which are found i n animals are samandarme (7) 4 and b a t r a c h o t o x i n i n A (8). Samandarine occurs with s e v e r a l r e l a t e d a l k a l o i d s 7 3 - 3A -as the s k i n poison o f two salamanders (S. maculosa L a u r e n t i and S_. A t r a L a u r e n t i ) of European h a b i t a t , whereas b a t r a c h o t o x i n i n A i s i s o l a t e d from the s k i n o f the Columbian arrow poison f r o g (Phyllobates a u r o t a e n i a ) . B a t r a c h o t o x i n i n A may be a secondary product formed from pseudobatrachotoxinin 4 by the a d d i t i o n o f water during i s o l a t i o n and p u r i f i c a t i o n . There are many a l k a l o i d s which do not f i t the d e s c r i p t i o n " b a s i c " . In these compounds the n i t r o g e n forms p a r t of an amide as i n c o l c h i c i n e (9) or i s quaternary as i n r h a z i d i n e (10) and p l e i o c a r p o l i n i n e (11) . Rhazidine i s a c t u a l l y a conjugate a c i d form of the base 12 but the base form i s 64 observed only i n s t r o n g l y b a s i c s o l u t i o n s or i n hydrocarbon s o l v e n t s . C O O M G 11 12 A very l a r g e f a m i l y of a l k a l o i d s , v h i c h number about 600, ' contains the i n d o l e system. The Aspidosperma, Vin--.a and Iboga a l k a l o i d s are members of t h i s l a r g e group. In t h i s part of t h i s •".hesis a general method f o r the s y n t h e s i s o f the Aspidosperma, Vinca and Iboga a l k a l o i d s i s discussed. This approach has been developed i n our l a b o r a t o r i e s . A key step i n t h i s method was a tr a n s a n n u l a r c y c l i z a t i o n which i s a l a b o r a t o r y analogy of a step pro-posed by Wenkert i n a p l a u s i b l e scheme f o r the b i o s y n t h e s i s of these a l k a l o i d s According to Wenkert's scheme nine-membered-ring i n t e r m e d i a t e s , such as 13 and 15, are b u l t up and undergo a transannular c y c l i z a t i o n t o give a l k a l o i d s possessing the Aspidosperma (e.g. 13 —>• 14) or the Iboga skeletons (e.g. 15 > 16) as shown i n Figure 1. Figure 1. B i o s y n t h e t i c p o s t u l a t e f o r the production of Aspidosperma and Iboga skeletons - 36 -Kutney and coworkers were the f i r s t to demonstrate t h e . f e a s i b i l i t y of such c y c l i z a t i o n s under l a b o r a t o r y c o n d i t i o n s . They were able to converv 8-10 dihydrocleavamine (17) which i s conveniently obtained v i a a simple degradation of c a t h a r a n t h i n e 1 1 i n two steps to 7-ethyl-5-desethylaspido-12 spermidine (18). O x i d a t i o n of dihydrocleavamine (Figure 2) provided the intermediate iminium i o n 19 which c y c l i z e d to the i n d o l e n i n e 20. The in d o l e n i n e without complete p u r i f i c a t i o n was then reduced w i t h l i t h i u m aluminum hydride t o provide the more s t a b l e 1,2-dihydro compound 18. Figure 2. Transannular c y c l i z a t i o n g i v i n g the Aspidosperma s k e l e t o n . 13 In the same manner these workers were able to convert carbomethoxydihydro-9 14 cleavamine ' (21), which l i k e dihydrocleavamine i s e a s i l y obtained by a simple degradation of cat h a r a n t h i n e , i n t o 7 - e t h y l - 5 - d e s e t h y l v i n c a d i f f o r m i n e (24), and c o r o n a r i d i n e , ^ which i s a known Iboga a l k a l o i d , and the C-4 epimer, dihydrocatharanthine ( 2 6 ) . 1 1 O x i d a t i o n of carbomethoxydihydrocleav-amine i n the N,— C-19 d i r e c t i o n l e d to the iminium i o n 22 which c y c l i z e d i n b - 37 -the r e a c t i o n medium to provide 24. A l t e r n a t e l y , o x i d a t i o n of carbomethoxy-dihydrocleavamine i n the —C-5 d i r e c t i o n l e d t o an iminium i o n , which by v i r t u e of e q u i l i b r a t i o n with the enamine 25 r e s u l t e d i n the mixture of epimeric iminium ions 23. One epimer c y c l i z e d to c o r o n a r i d i n e , the other, to d i h y d r o c a t h a r a n t i i i n e . The r e a c t i o n sequence proposed i s o u t l i n e d i n Figure 3 below. I t should be observed t h a t a t r a n s a n n u l a r c y c l i z a t i o n o f -t-N 21 v Hg(OAc), HOAc MeOO MeOOC 26 Hg(OAc),s HOAc C O O M G _ C O O M G 24 H M G O O C 25 Figure 3. Transannular c y c l i z a t i o n g i v i n g the Aspidosperma and Iboga skeletons - 38 -both types generated two new asymmetric centres and t h e r e f o r e each i n t e r -mediate iminium i o n above might have been expected to cyc.lize to a mixture of f o u r diastereomers (see s t r u c t u r e s 27 to 30 f o r Aspidosperma s k e l e t o n ) . 29 30 The f a c t the above mentioned experiments showed that each of the intermediate iminium ions c y c l i z e d to g i v e only one of the p o s s i b l e d i a s t e r i o m e r s i n 12 16 s u f f i c i e n t q u a n t i t y to i s o l a t e . This r e s u l t was not unexpected ' s i n c e i t was r e a d i l y apparent (from examination of molecular models) t h a t only one conformation of each iminium i o n would permit the r e a c t i n g centres to be c l o s e together and at the same time not be h i g h l y s t r a i n e d . I t was not, however, immediately apparent that i n each of the transannular c y c l i z a t i o n s discussed t h a t an o p t i c a l l y a c t i v e s t a r t i n g m a t e r i a l would lead to an o p t i c a l l y a c t i v e product. Both dihydrocleavamine and carbomethoxydihydro-cleavamine have a proton at C-2. I f 19 and 22 could e q u i l i b r a t e w i t h t h e i r enamine forms under the c o n d i t i o n s of the r e a c t i o n , racemic products would - 39 -be formed. A s i m i l a r e q u i l i b r a t i o n (23 25) was shown to occur. Moreover, t a u t o m e r i z a t i o n of the iminium double bond across n i t r o g e n might a l s o occur. Thus a combination of e q u i l i b r i a (Figure 4) could serve t o destroy the Aspiclospe rma skeleton on Figure 4. E q u i l i b r i a that would r e s u l t i n l o s s of o p t i c a l p u r i t y C-2 asymmetry independently of the d i r e c t i o n o f the i n i t i a l o x i d a t i o n and, t h e r e f o r e , destroy the o p t i c a l a c t i v i t y of the compounds w i t h the Iboga ske l e t o n as w e l l . There was precedence f o r t a u t o m e r i z a t i o n of an iminium i o n across n i t r o g e n 1 ^ However, i t appeared t h a t such a process was promoted by base and so would not l i k e l y be a f a c t o r i n the mercuric a c e t a t e - a c e t i c a c i d conversion of the dihydrocleavamines. For i n s t a n c e , 17 Schumann and Schmid found that c o n d i f o l i n e (31) was converted i n t o t u b i f o l i n e (32) under a v a r i e t y of n e u t r a l and b a s i c c o n d i t i o n s (e.g. neat at 117-120°, i n t e t r a l i n at 160-180°, i n t r i e t h y l a m i n e , i n ammonia-isopro-panol or i n potassium t - b u t o x i d e - t - b u t a n o l , but not under a c i d i c c o n d i t i o n s (2-7N h y d r o c h l o r i c a c i d at 100-125°). They proposed the scheme shown i n 18 Figure 5 to account f o r t h e i r r e s u l t s . Edwards and Singh found that both - 40 -Figure 5. I n t e r c o n v e r s i o n of t u b i f o i i n e and c o n d i f o l i n e a t i s i n e h y d r o c h l o r i d e (33) and i s o a t i s i n e h y d r o c h l o r i d e (34) gave the same compound on treatment w i t h a c e t i c anhydride or a c e t i c anhydride-pyridine 19 mixtures. P e l l i t i e r and Jacobs showed that the compound was a d i a c e t a t e h y d r o c h l o r i d e (35) of a t i s i n e and proposed a mechanism " i n v o l v i n g a concerted - 41 -a b s t r a c t i o n and r e - a d d i t i o n of a proton by acetate i o n and a c e t i c a c i d " as 20 shown i n Figure 6 below. In a c l o s e l y r e l a t e d case Weisner and Edwards Figure 6. A mechanism f o r t a u t o m e r i z a t i o n of iminium f u n c t i o n s across n i t r o g e n found that veatchine (36) was converted to g a r r y i n e (37) by hot a l k a l i and proposed that the quaternary forms of these compounds were i n e q u i l i b r i u m w i t h the y l i d i o n 38 under these c o n d i t i o n s as shown i n Figure 7. :0Ac H £CH HO OH Me 36 Me HO Me Me 38 37 Figure 7. In t e r c o n v e r s i o n o f veatchine and g a r r y i n e As f a r as the problem o f the r e l a t i v e c o n f i g u r a t i o n of the s y n t h e t i c Iboga compounds was concerned) the transannular c y c l i z a t i o n of the iminium ions 23 l e d to the n a t u r a l s k e l e t o n as proven by the i s o l a t i o n of cor o n a r i d i n e and dihydrocatharanthine. In the case of the pseudoaspidosperma compounds 16 21 no such d i r e c t comparison was p o s s i b l e . Kutney and coworkers ' and 22 independently Schmid and coworkers, however, s u c c e s s f u l l y c a r r i e d out a conversion i n the n a t u r a l s e r i e s . I t was found that (-)-quebrachamine (39) was transformed i n t o (+)-aspidospermidine (40) under s u i t a b l e c o n d i t i o n s . Thus, the transannular c y c l i z a t i o n l e d to the n a t u r a l diastereomer. Schmid suggested the absolute c o n f i g u r a t i o n of (+)-aspidospermine, based on o p t i c a l 23 s t u d i e s of a l a r g e number of r e l a t e d a l k a l o i d s , shown i n 40. I t can be seen t h a t the C-5 e t h y l group and C-19 proton are c i s i n r e l a t i o n s h i p to each other and th a t the C-19 proton bears a trans r e l a t i o n s h i p w i t h the C-10 C - l l b r i d g e . This r e l a t i v e c o n f i g u r a t i o n was e x a c t l y the one that would be expected to a r i s e on conformational grounds through c y c l i z a t i o n of the intermediate iminium i o n . The re d u c t i o n of 1,2 dehydroaspidospermidine, which was the immediate product a f t e r c y c l i z a t i o n , w i t h hydride was also completely s t e r e o s p e c i f i c s i n c e the n a t u r a l C-2 epimer was obtained. Since - 45 -there was no p o s s i b l e means by which tht C-5 c o n f i g u r a t i o n could be a l t e r e d during the conversion of (-) quebrachamine to (+)-aspidospermidine, o p t i c a l l y a c t i v e product was obtained as expected. As p o i n t e d out above, the s i t u a t i o n w i t h regard to the absolute c o n f i g u r a t i o n of the pseudo-aspidsoperma and Iboga compounds produced v i a the t r a n s a n n u l a r c y c l i z a t i o n was not unambiguous because the o p t i c a l a c t i v i t y of the C-2 carbon i n the dehydrocleavamine molecule could conceivably have been destroyed i n the process. Kutney and coworkers showed, however, that the asymmetry of the C-2 carbon atom was r e t a i n e d i n the transannular c y c l i z a t i o n r e a c t i o n . 7-Ethyl-5-desethylaspidospermidine obtained from 43-dihydrocleavamine (41) was e s t a b l i s h e d to have the absolute c o n f i g u r a t i o n as shown i n 42 by X-ray 41 42 21 d i f f r a c t i o n a n a l y s i s of i t s N -acetyl-N^-methiodide d e r i v a t i v e . The 24 25 c o n f i g u r a t i o n of the C-2 carbon atom ' i n 43-dihydrocleavamine, t h e r e f o r e , remained u n a l t e r e d d u r ing the course of the conversion r e a c t i o n . The con-f i g u r a t i o n of the C-19, C-12 and C-2 carbon atoms r e l a t i v e to the c o n f i g u r a -t i o n of the C-5 carbon atom of 7-ethyl-5-desethylaspidospermidine was e x a c t l y that expected from conformation c o n s i d e r a t i o n . In a d d i t i o n , the c o n f i g u r a t i o n of the C-4 carbon atom of 46-dihydrocleavamine was unaltered d uring the course of the r e a c t i o n . Consequently, of the e q u i l i b r a t i o n - 44 -r e a c t i o n s sitown i n Figure 4 only "C" i s important. Since the transannular c y c l i z a t i o n ox 43-dihydrocleavamine to 73-ethyl-5-desethylaspidospermidine took place i n a s t e r e o s p e c i f i c manner, i t fo l l o w e d that the transannular c y c l i z a t i o n o f carbomethoxydihydrocleavamine to provide 7 - e t h y l - 5 - d e s e t h y l -v i n c a d i f f o r m i n e , dmydrocatharanthine and c o r o n a r i d i n e was a l s o a s t e r e o -s p e c i f i c process. The absolute c o n f i g u r a t i o n o f c a t h a r a n t h i n e , as shown i n 43, ibogaine (44), c o r o n a r i d i n e (45) dihydrocatharanthine (46), ibogamine (47) and voacangine (48) fo l l o w e d d i r e c t l y from the above c y c l i z a t i o n when coupled w i t h the f a c t t h a t the r e l a t i v e c o n f i g u r a t i o n of ibogaine was a v a i l a b l e from the X-ray a n a l y s i s and v a r i o u s i n t e r c o n v e r s i o n s were , 10,27,28 known. Besides the i n t r i n s i c value of these r e s u l t s to the b i o s y n t h e t i c p o s t u l a t e s of Wenkert, they had a s p e c i a l value from a s y n t h e t i c p o i n t of view. The transannular c y c l i z a t i o n step was in c o r p o r a t e d i n t o a general t o t a l s y n t h e s i s of a number of a l k a l o i d s bearing the Aspidosperma or Iboga sk e l e t o n s . Since t h i s step was shown to be completely s t e r e o s p e c i f i c with the c o n f i g u r a t i o n of each new asymmetric centre being completely determined by the c o n f i g u r a t i o n at C-2 i n the nine-membered r i n g i n t e r m e d i a t e s , the - 45 -t o t a l s y n t h e s i s based on t h i s step was not beset w i t h stereochemical problems during the v a r i o u s stages o f the s y n t h e t i c sequence. Moreover, p a r a l l e l t o t a l syntheses of the dihydrocleavamine and quebrachamine systems were f e a s i b l e . Thus the t o t a l syntheses of a l k a l o i d s bearing the Aspidosperma s k e l e t o n on the one hand and the Iboga s k e l e t o n on the other complemented each other w i t h obvious advantages over s y n t h e t i c approaches that required a completely d i f f e r e n t method f o r each s k e l e t a l type. The important f e a t u r e s of the syntheses of dl-quebrachamine" (57) and d l - d i h y d r o c l e a v -30 amine (58) as c a r r i e d , out by Kutney and coworkers are shown i n Figure 8. In each s y n t h e s i s of appropriate s u c c i n a t e e s t e r (49 or 50), which was condensed w i t h tryptamine (51), was elaborated by separate and unexceptional means. I t was seen that i n t r o d u c t i o n of a carbomethoxy f u n c t i o n at C-18 i n dihydrocleavamine would provide a carbomethoxydehydrocleavamine and thereby complete the t o t a l syntheses of c o r o n a r i d i n e and dihydrocatharanthine. I n t r o d u c t i o n of a carbomethoxy f u n c t i o n at C-3 i n quebrachamine would provide - 46 -57, R ^ E t ; R2=H 58^=11; R 2=Et Figure 8. Kutney's syntheses of dl-quebracharaine, dl-4a-dihydrocleavamine and dl-48-dihydrocleavamine - 47 -31 a carbomethoxyquebrachamine. (+)-Vincadine (59) i s a n a t u r a l l y o c c u r r i n g Vinca a l k a l o i d as i s (-)-vincadifforamine (60). A s u c c e s s f u l transannular H / \ | MeOOC H 59 c y c l i z a t i o n of a carbomethoxyquebrachamine i n the manner used to give the 13 pseudo compounds p r e v i o u s l y would provide d l - v i n c a d i f f o r m i n e . Part of t h i s s e c t i o n i s concerned with the i n t r o d u c t i o n of a carbomethoxy group i n t o dihydrocleavamine. As mentioned above s e v e r a l syntheses have been elab o r a t e d f o r the p r e p a r a t i o n of a l k a l o i d s w i t h the Iboga and Aspidosperma s k e l e t o n s . Various syntheses c a r r i e d out i n other l a b o r a t o r i e s were p e c u l i a r to one of these s k e l e t a l types and are best discussed i n two separate groups. Three approaches to the formation of the a l k a l o i d aspidospermine,were p a r t i c u l a r l y i n t e r e s t i n g . Each of these approaches i n v o l v e d the e l a b o r a t i o n of a t r i -c y c l i c keto-amine (61) which would be expected to y i e l d the Aspidosperma ske l e t o n when exposed t o the F i s h e r i n d o l e r e a c t i o n as shown i n Figure 9. There are three asymmetric centres i n 61 and t h e r e f o r e f o u r diastereomer-i c a l l y r e l a t e d isomers are p o s s i b l e (62, 63, 64 and 65). In f a c t a l l f o u r 32 of these isomers have been synthesized. Stork and coworkers achieved the the f i r s t t o t a l s y n t h e s i s of dl-aspidospermine (66) and dl-quebrachamine. 33 T y syn hesized a r i c y c l i c keto amine shown l a t e r to have s t r u c t u r e 62,- 48 -Figure 9. Stork's s y n t h e s i s of dl-1,2-dehydroaspidospermidine submitted i t to F i s h e r i n d o l i z a t i o n w i t h o-methoxyphenylhydrazine and obtained dl-1,2-dehydrodeacetylaspidospermine (67). Reduction of 67 w i t h l i t h i u m aluminum hydride i n d i e t h y l ether introduced the C-2 proton stereo-s p e c i f i c a l l y and the l a t t e r product upon a c e t y l a t i o n provided d l - a s p i d o -spermine (66). Since the c o r r e c t stereochemistry was obtained i n the products even though the i n c o r r e c t stereochemistry was possessed by the t r i c y c l i c keto amine 62, e q u i l i b r a t i o n of the asymmetric centres at C-12 and C-19 must have occurred during the F i s h e r s y n t h e s i s . Stork proposed the - 49 -68 Figure 10. E q u i l i b r i u m w i t h stereochemical i m p l i c a t i o n s and s t e r e o s p e c i f i c formation of dl-aspidospermine e q u i l i b r i u m 67 ^± 68 (Figure 10) to account f o r the c o r r e c t stereochemistry o f the product- Stork and coworkers were able to extend t h e i r s y n t h e s i s to provide a t o t a l s y n t h e s i s of dl-quebrachamine by making use of t h i s e q u i l i b r i u m . A p p l i c a t i o n of the F i s h e r i n d o l e r e a c t i o n to the keto amine 62 w i t h phenylhydrazine provided d'1-1,2-dehydroaspidospermidine (69) which was converted to dl-quebrachamine by b r i n g i n g about the analogous e q u i l i b r i u m to 67 ^zf 68 i n methanol and s e l e c t i v e l y reducing the very r e a c t i v e iminium f u n c t i o n i n the t e t r a c y c l i c c a t i o n w i t h potassium borohydride. 33 Ban and coworkers i n the course of t h e i r s t u d i e s r e l a t e d the t o t a l s y n t h e s i s of aspidospermine produced a t r i c y c l i c keto amine which had the same planar s t r u c t u r e (61) as the one described by S t o r k , but d i f f e r e d i n chemical and p h y s i c a l p r o p e r t i e s . Comparison of both compounds allowed - 50 -these workers t o propose the s t r u c t u r e 63 f o r t h e i r compound and 62 f o r Stork's compound. The t r i c y c l i c keto amine 63 a l s o provided d l - a s p i d o -spermine when subjected to the same sequence of r e a c t i o n as mentioned above. The remaining two p o s s i b l e t r i c y c l i c keto amines 64 and 65 were prepared 34 by Kuehne and Sayha. Since the F i s h e r i n d o l e r e a c t i o n r e q u i r e d l o s s of the asymmetry at the s i t e a to the ketone i n the c o r r e c t c y c l i z a t i o n process, these two new isomers o f f e r e d no a d d i t i o n a l stereochemical problems. Stork's s y n t h e s i s of the t r i c y c l i c keto amine i s o u t l i n e d i n Figure 11, w h i l e those of Ban and Kuehne are given i n Figures 12 and 13, r e s p e c t i v e l y . Another i n t e r e s t i n g approach t o the s y n t h e s i s of the aspidosperma 35 s k e l e t o n has been c a r r i e d out by Harley-Mason and Kaplow. An e n t i r e l y s t e r e o s p e c i f i c s y n t h e s i s of dl-aspidospermidine was achieved by these workers and i s o u t l i n e d i n Figure 14. 3 6 Buchi and coworkers achieved the f i r s t t o t a l s y n t h e s i s of an Iboga a l k a l o i d . They were able to s y n t h e s i z e dl-ibogamine (70) and d l - e p i i b o g -amine (71) as shown i n Figure 15. The c y c l i z a t i o n rearrangement of 72 (73) to g i ve 74 (75) i s p a r t i c u l a r l y noteworthy i n view of some l a t e r d i s c u s s i o n i n t h i s t h e s i s . Buchi suggested t h a t c y c l i z a t i o n proceeds to give a compound (76) which has the d e s i r e d iboga s k e l e t o n but which s u f f e r s a 1,2 s h i f t of the amine (amide) n i t r o g e n to give a rearranged product 74 (75). Buchi favored a n o n - c l a s s i c a l carbonium i o n (78) as an intermediate i n the + OAc OAc 78(79) 51 H ,. .. MeOOC .... MeOOC 1-pyrrolidine lA \ 1.pyrrolidine 0' H 2.CH=CHC00Me H { | 2.CH,=CHC0Me 3.H 40,H0Ac H 0 H "3.H0AcA 0 HjN' OH OH 1.. \—J 2. N H * OH" 0 ILiAlH^ 2. H + 3. OH" C l C H X O C l K + tBuO" PhH 1. ° ^ 7 0 H 2. L i A l H * 62 Figure 11. Stork's s y n t h e s i s of t r i c y c l i c keto amine 62 0 Figure 12. Ban's synt h e s i s of t r i c y c l i c keto amine 63 - 52 -MeOOC N" I • MeOOC C 0 0 E t 0 Ph,P=CHMe N Pt/ H. CHMe CK=CHCOOMe LiAlH-y I P t / H ^ H ' * 6 4 2.aluminium ~ 6 5 isopropoxide Figure 13. Kuehne's sy n t h e s i s of t r i c y c l i c keto amines 64 and 65 CHOCOOMe 1.HC(0Me)3 (MeO)CH COOMe 2.0, 3. BH HOAc/efW % c l l - 4 0 Figure 14. Harley-Mason's s y n t h e s i s o f dl-aspidospermidine - 53 -Bz _ CL N a E H .0 NH, N -Bz u CHrCHCOMe NaBH* Bz 0 NaOCl,KOH, M e O H , H a O P •OH MeO H \ A N 1.6N H.SOz, ea.ioo0 ^ Bz N 0 6 pyr. 0 P d / H , HCl Me OAc H\+/|Cl H - ^ M \ tryptyl bromide ndole-3-acetyl / — \ ^ L p T s O H , / M HOAc 0 chloride) OAc N , H 0 72(73) H 2.Na^C0 3 S OAc Figure 15. Buchi's s y n t h e s i s of dl-ibogamine and dl-epiibogamine - 54 -Figure 15. Continued - 55 -1,2 s h i f t of n i t r o g e n . Although Buchi favored the n o n - c l a s s i c a l i o n , a c l a s s i c a l bridged a z i r i d i n i u m i o n could a l s o have been p o s t u l a t e d . In connection w i t h another approach to the s y n t h e s i s of the iboga 37 38 s k e l e t o n , Huffman and coworkers discovered an analogous rearrangement f o r which they p o s t u l a t e d a bridged a c y l a z i r i d i n i u m intermediate. They found, f o r i n s t a n c e , t h a t treatment of the i s o q u i n u c l i d o n e t o s y l a t e 80 (R = Ts) with r e f l u x i n g sodium a c e t a t e - a c e t i c a c i d gave a mixture of the corresponding i s o q u i n u c l i d o n e acetate 80 (R = Ac), and an is o m e r i c acetate 81 (R = Ac). S i m i l a r l y , the t o s y l a t e 81 (R = Ts) was converted to the same mixture under the same c o n d i t i o n s . Heating e i t h e r t o s y l a t e 80 (R = Ts) or t o s y l a t e 81 (R = Ts) to the me l t i n g p o i n t formed a mixture o f the two t o s y l a t e s i n a r a t i o 80 (R = Ts):81(R = Ts) of about 5:1. Huffman proposed t h a t these r e s u l t s were best explained i n terms of an inte r m e d i a t e a c y l a z i r i -dinium i o n (82). . 0 Bz v. A N H OR 0 N 80 81 OR 82 S h o r t l y a f t e r the p u b l i c a t i o n of the work o f the Buchi and Huffman groups, Nagata and coworkers de s c r i b e d the f i r s t s y n t h e s i s of bridged a z i r i d i n e 39 d e r i v a t i v e s and demonstrated the . a b i l i t y of t h i s approach to production of the' i s o q u i n u c l i d i n e p o s i t i o n o f the iboga s k e l e t o n by s y n t h e s i z i n g d e s e t h y l -40 ibogamine. Recently, these workers have pu b l i s h e d a s t e r e o s p e c i f i c t o t a l s yntheses^ 1 of dl-ibogamine and dl-epiibogamine. The racemic o l e f i n i c - 56 -amines 83 and 84 were synthesized by separate and r a t h e r lengthy procedures which w i l l not be presented here. The refaction sequence which l e d to d l -ibogamine i n the case of 83 and d l - e p i i b o g a u i n e i n the case of 84 i s shown i n Figure 16 f o r the s y n t h e s i s of the n a t u r a l l y o c c u r r i n g dl-ibogamine. The mechanism of the cleavage of the a z i r i d i n e system i s worth consider-i n g e s p e c i a l l y as i t r e l a t e s to the proposals of Buchi and Huffman. Nagata and coworkers found t h a t the cleavage r e a c t i o n of the bridged a z i r i d i n e s w i t h an a c y l a t i n g agent i n an a l k a l i n e medium took place without i n c o r p -o r a t i o n of the solvent anion. They proposed that intermediate formation of an i o n p a i r such as 87 took place w i t h cleavage by i o n - p a i r r e t u r n from the opposite s i d e of the n i t r o g e n atom. Q u a t e r n i z a t i o n of the a z i r i d i n e s with poorer e l e c t r o n withdrawing groups than an a c y l group lead to reasonably s t a b l e s a l t s . For i n s t a n c e , the a z i r i d i n e 88 reacted w i t h methyl i o d i d e at -50°C to give the methiodide 89 i n good y i e l d (Figure 17). The methiodide was r e a d i l y cleaved by a v a r i e t y of n u c l e o p h i l e s such as a c e t a t e , methoxide and cyanide. A mixture of the a p p r o p r i a t e l y s u b s t i t u t e d i s o q u i n u c l i d i n e 90 and i t s isomer 91 was obtained i n each case i n a r a t i o of about 2:1. From the examples discussed above i t . became apparent that the i s o q u i n u c l i dine system was i d e a l l y set up f o r n i t r o g e n p a r t i c i p a t i o n i n r e a c t i o n o c c u r r i n - 57 -H 83 ndoleacetyl-0 H 86 NaOMe, HOMe / Pb(OAc)^ — — > 1.0H 2.DMSO/A c ,0 •3.PhH,pTsOH .0 reflux N, H MeO H Li A id R-indolyUcetic anhydride, ^ acetone l4 85 pTsO ft MeO H H,/cat. 1 N J N H MeO H LiAlH^.EtaO reflux 70 Figure 16. Nagata's s y n t h e s i s of dl-ibogamine - 58 -Figure 17. Reaction of bridged a z i r i d i n i u m ions w i t h n u c l e o p h i l e s at v i c i n a l carbon atoms. This p a r t i c u l a r p r o p e r t y of the i s o q u i n u c l i d i n e system a l s o arose i n another r e a c t i o n of i n t e r e s t through i t s r e l a t i o n to the work described l a t e r i n t h i s part of t h i s t h e s i s . S h o r t l y before the Nagata group pu b l i s h e d t h e i r elegant s y n t h e s i s of dl-ibogamine and dl-epiibogamine, S a l l e y p u b l i s h e d a s t e r e o s p e c i f i c t o t a l 42 s y n t h e s i s of dl-ibogamine. S a l l e y ' s scheme i s o u t l i n e d i n Figure 18. In t h i s p a r t i c u l a r s y n t h e s i s the i n i t i a l stereochemical r e l a t i o n s h i p of the C-4, C-5, and C-18 protons i n ibogamine was e s t a b l i s h e d at the outset of the s y n t h e s i s . The D i e l s - A l d e r r e a c t i o n between quinone and 1,3-hexadiene 43 44 proceeded i n the endo f a s h i o n . Woodward and Hoffman have shown that - 59 -Diels-Alder 92 reduction OH OH H i n f h l ^ H + v o r.s.T NH.OH Figure 18. S a l l e y ' s s y n t h e s i s o f dl-ibogamine Figure 18. Continued - 61 -endo a d d i t i o n allows maximum mixing o f the highest occupied o r b i t a l of the one molecule with the lowest occupied o r b i t a l o f the other. Thus the e t h y l group i n 92 bor^ a c i s r e l a t i o n s h i p to the ene-dione r i n g . This r e l a t i o n -ship was necessary i n order t h a t the e v e n t u a l l y formed C-19—N-6 bridge would bear a c i s r e l a t i o n t o the same e t h y l group. A l s o important to the s y n t h e s i s was that the Beckman rearrangement of the oxime 93 proceeded t o give the lactam 94 r a t h e r than the isomeric lactam which would be formed by m i g r a t i o n of the secondary centre. Consequently, the oxime 93 must have had the t r a n s c o n f i g u r a t i o n as shown s i n c e the Beckman rearrangement i s know to proceed with p r e f e r e n t i a l m i g r a t i o n of the centre which bears an a n t i r e l a t i o n s h i p to the hydroxyl of the.oxime f u n c t i o n . S a l l y ' s syntheses of dl-ibogamine provided a p r e p a r a t i v e proof f o r the c i s r e l a t i o n s h i p of the e t h y l group to the C-19—N-6 bridge. Nagata's s y n t h e s i s which was a l s o s t e r e o s p e c i f i c , provided the same proof. As pointed out e a r l i e r the approach of Kutney and coworkers to the s y n t h e s i s of Iboga, Aspidosperma and Vinca a l k a l o i d s r e q u i r e d as an important step the i n t r o d u c t i o n of a carbomethoxy group i n t o the quebrachamine and 45 dihydrocleavamine systems. In 1962 Taylor proposed a general r e a c t i o n sequence (Figure 19) t o e x p l a i n some i n t e r e s t i n g and p u z z l i n g transformations present i n the l i t e r a t u r e . I t was apparent t h a t such a sequence, i f of a general nature, would provide the means of f u n c t i o n a l i z i n g the p o s i t i o n adjacent to the a - p o s i t i o n of an i n d o l e c o n t a i n i n g a l k a l o i d . At the time Taylor made t h i s p o s t u l a t e examples of such t r a n s f o r m a t i o n s were sparse. Nevertheless he was able to c i t e s e v e r a l examples from the previous l i t e r a -t ure which would f i t i n t o h i s general scheme. For i n s t a n c e , when 2 , 3 - d i e t h y l -3-hydroperoxyindolenir\e ( 9 5 ) ^ which had been prepared by a e r i a l o x i d a t i o n - 62 -X t X Y H Y Figure 19. Ta y l o r ' s hypothesis concerning an unusual r e a c t i o n o f in d o l e n i n e s o-propionaminopropiophenone (96) was obtained as the only product (Figure 20). On the other hand, when 95 was heated at 100° f o r 30 minutes i n the absence o f s o l v e n t , only a small amount of 96 was obtained and the main product (52% y i e l d ) was 2 - a c e t y l - 3 - e t h y l i n d o l e (97). Taylor proposed t h a t the r e a c t i o n could proceed v i a h i s mechanism (X=Y=00H). In a subsequent step the unstable secondary hydroperoxide would decompose to the ketone 97 w i t h l o s s of water. This p a r t i c u l a r example (and s e v e r a l others c i t e d ) i n v o l v e d an i n t e r n a l rearrangement. Another example of a r e a c t i o n which T a y l o r b e l i e v e d could f i t i n t o h i s scheme was the bromine-acetic a c i d bromination of 2,3-dimethylindole and subsequent base treatment o f the i n i t i a l product to f u r n i s h 2-hydroxymethyl-3-methylindole (Figure 19: X = of the corresponding i n d o l e was subjected t o r e f l u x i n g water f o r 20 minutes, Br, Y = OH). 47 0 + 96 (minor) Figure 20. Reactions o f a hydroperoxyindoline A more recent example of a r e a c t i o n (Figure 21) that l i k e l y proceeded according to Tay l o r ' s scheme was the conversion of t e t r a h y d r o c a r b a z o l e (98) i n t o the p y r i d i n i u m bromide 99 by the a c t i o n of N-bromosuccinimide i n the 48 presence of p y r i d i n e . As a matter of i n t e r e s t to some l a t e r d i s c u s s i o n workers i n the same l a b o r a t o r y had shown that under s i m i l a r c o n d i t i o n s i n -49 doles u n s u b s t i t u t e d at the a - p o s i t i o n gave a-pyridinium s a l t s as shown f o r 3-methylindole i n Figure 22. Figure 21. Reaction g i v i n g s u b s t i t u t i o n adjacent to the a - p o s i t i o n of an in d o l e - 64 -Figure 22. Reaction g i v i n g s u b s t i t u t i o n at the a - p o s i t i o n of an i n d o l e At the time that the i n t r o d u c t i o n of a carbomethoxy group i n t o the t e t r a c y c l i c dihydrocleavamines was a n t i c i p a t e d , there was a v a i l a b l e , however, what appeared to be an e x c e l l e n t and c l o s e analogy i n the t r a n s -formation of ibogaine (44) i n t o voacangine (48) by Buchi and M a n n i n g ^ (Figure 23). According to the general scheme by T a y l o r , the c h l o r o i n d o l e n i n e 44 48 (Me)3C0Cl KCNjMeOH, 'H*0,Et<0 1.K0H MeO. r 2.H*. C H 2 N a H N CN H Figure 23. Buchi's syn t h e s i s of voacangine - 65 -100 would be i n e q u i l i b r i u m w i t h i t s enamine form 101 which could r e a c t w i t h cyanide i o n v i a a S^' mechanism. ' Regeneration of the i n d o l e system would provide a d r i v i n g f o r c e f o r the r e a c t i o n . Buchi and Manning, however, favored the imine 102 as the i n t e r m e d i a t e . They a l s o suggested the non-c l a s s i c a l carbonium i o n 103 as a p l a u s i b l e a l t e r n a t i v e to the imine 102, but r e j e c t e d a c l a s s i c a l a z i r i d i n i u m intermediate on the grounds that, such an intermediate would be s e v e r e l y s t r a i n e d i n these cases and the n i t r o g e n 3 6 lone p a i r would not be a v a i l a b l e f o r displacement of the l e a v i n g group. Nevertheless at that time bridged a z i r i d i n i u m d e r i v a t i v e s were completely unknown, but i n view of the work of Nagata and coworkers c i t e d p r e v i o u s l y , the c l a s s i c a l i o n 104 should a l s o be considered as a p l a u s i b l e intermediate. 103 104 - 66 -Of the fou r p l a u s i b l e intermediates above, i t was apparent that e i t h e r 101 or 102 would have t o be a precursor to e i t h e r Of the other two p l a u s i b l e intermediates 103 and 104. There was some precedence f o r an intermediate l i k e 101. For example, dihydrocatharanthine and r e l a t e d Iboga b a s e s " ^ ' ^ ' ^ ^ decarboxylate r e a d i l y i n a c i d and are b e l i e v e d to proceed through the in d o l e n i n e 105 to g i v e i n i t i a l l y 106. Catharanthine i t s e l f does not decarboxy-l a t e under the same c o n d i t i o n s . Molecular models revealed that the equivalent intermediate to 106 i n the case of catharanthine would be extremely s t r a i n e d by comparison. Kutney and c o w o r k e r s ^ have p o s t u l a t e d t h a t the decarbo-methoxylation of catharanthine to descarbomethoxycatharanthine proceeded through s e v e r a l t e t r a c y c l i c ions as i n d i c a t e d i n Figure 24. 105 106 ' 2-Alkylidene-2H-indole intermediates such as 102 have been p o s t u l a t e d 54 i n the l i t h i u m aluminum hydride r e d u c t i o n o f 2 - i n d o l e c a r b i n o l d e r i v a t i v e s . Dolby p o s t u l a t e d t h a t generation of the 2 - a l k y l i d i n e - 2 H - i n d o l e intermediates proceeded v i a the i n d o l e anion. Thus, the l i t h i u m aluminum hydride was r e q u i r e d to act as a base i n the i n i t i a l step and as a reducing agent i n the f i n a l step (Figure 25). The a l t e r n a t i v e mechanism would be d i r e c t r e p l a c e -ment of the "X" group by hydride. Dolby and Booth demonstrated, however, that N-methylation e f f e c t i v e l y blocked the hydride r e d u c t i o n . They f e l t t h at t h i s r e s u l t supported t h e i r mechanism s i n c e i t seemed to them that i f - 67 -Figure 24. Kutney's r a t i o n a l i z a t i o n f o r the d e c a r b o x y l a t i o n of catharanthine Figure 25. Dolby's r a t i o n a l i z a t i o n f o r the r e d u c t i o n of 2 - i n d o l e c a r b i n o l d e r i v a t i v e s the r e a c t i o n were to proceed by d i r e c t displacement by hydride the N-H compounds should be l e s s r e a c t i v e to r e d u c t i o n than the N-methyl compounds because formation of the i n d o l e anion would s u r e l y have preceded r e d u c t i o n and the anion would be expected to be l e s s s u c e p t i b l e to n u c l e o p h i l i c attack. I t was apparent t h a t intermediates 101 and 102 could not be r u l e d out as the species that i n i t i a l l y react w i t h the incoming n u c l e o p h i l e , although they both appeared to represent q u i t e s t r a i n e d s t r u c t u r e s as i n d i c a t e d by molecular models and i n the case of 102 there would be d i s r u p t i o n of the - 69 -benzene resonance to some extent. I t was a t t r a c t i v e t o p o s t u l a t e that 101 or 102 was i n i t i a l l y formed, but s u f f e r e d immediate attack by the N-6 lone p a i r of e l e c t r o n s (which i n c i d e n t l y would a l s o f i t the T a y l o r scheme) to form e i t h e r the n o n - c l a s s i c a l i o n 103 or the c l a s s i c a l i o n 104. The c l a s s i c a l i o n 104 appeared to be the b e t t e r choice i n the l i g h t o f the work by Nagata and coworkers. They showed t h a t a l k y l a z i r i d i n i u m ions such as 89 are reasonably s t a b l e e n t i t i e s . I f 104 i s redrawn t o emphasize the s i m i l a r i t y i n t h e i r s t r u c t u r e s , i t i s seen that the i n d o l e p o r t i o n i n 104 forms a 6 membered r i n g along one of the C-N- bonds of the a z i r i d i n i u m r i n g . 89 104 When a molecular model of the i o n 104 was b u i l t the b r i d g i n g t r i p t y l f u n c t i o n d i d not appear t o b r i n g any a d d i t i o n a l s t r a i n to bear on the bridged a z i r i d i n i u m system. In f a c t 104 appeared t o be f a r l e s s s t r a i n e d than e i t h e r 101 or 102 and possess the f u l l i n d o l e resonance as w e l l . I t could have been argued t h a t although 104 appeared t o be a n i c e l o o k i n g i n t e r m e d i a t e , i t seemed h a r d l y necessary to p o s t u l a t e i t s i n c e e i t h e r 101 or 102 must be formed i n a p r i o r step and s i n c e the arguement has been to show th a t they are l e s s s t a b l e ( i . e . more r e a c t i v e ) why bother to p o s t u l a t e - 70 -.105 when 10:1 or 102 would serve as w e l l . The f a c t t h a t i n t e r n a l attack by the b a s i c n i t r o g e n would seem to be p o s s i b l e h a r d l y seemed t o represent evidence that i n t e r n a l a t t a c k would be p r e f e r r e d to e x t e r n a l attack by cyanide or another n u c l e o p h i l e . In t h i s regard one p i e c e of i n f o r m a t i o n was p a r t i c u l a r l y i n t e r e s t i n g . The conversion of 72 (73) i n t o 74 (75) i n 36 the Buchi s y n t h e s i s proceeded without i n c o r p o r a t i o n of an anion from the s o l v e n t . I t appeared t h e r e f o r e that the steps i n Figure 26 could o f f e r the 107 (108) Figure 26. I n t e r n a l r e t u r n w i t h i n an i o n p a i r only p l a u s i b l e e x p l a n a t i o n of t h i s r e s u l t . I f i n t e r m e d i a t e 76 (77) gave r i s e to the i o n p a i r 107 (108) then i n t e r n a l r e t u r n w i t h i n the p a i r would give 74 (75). Buchi and coworkers were able to i n c o r p o r a t e s o l v e n t anions only under more vigorous c o n d i t i o n s . I t thus appeared a f t e r c l o s e s c r u t i n y t h a t the s u c c e s s f u l s u b s t i t u t i o n of a cyanide i n t o the c h l o r o i n d o l e n i n e of ibogaine was not a good analogy f o r a s i m i l a r s u b s t i t u t i o n i n t o the c h l o r o i n d o l e n i n e s of dihydrocleavamine - 71 -and quebrachamine i f the formation of the a z i r i d i n i u m intermediate was an important f e a t u r e of the r e a c t i o n . On the other hand at t a c k by the n i t i o g e n ' s lone p a i r of e l e c t r o n s d i d appear t o be i n keeping w i t h the Taylor hypothesis. Buchi and Manning found that at elevated temperatures there was a s e r i o u s side r e a c t i o n a l s o i n v o l v i n g the lone p a i r of e l e c t r o n s on the i s o q u i n u c l i d i n e n i t r o g e n atom. These workers p o s t u l a t e d a rearrangement as shown i n Figure 27 t o account f o r the product obtained. I t was apparent that CI Figure 27. An unusual rearrangement of the c h l o r o i n d o l e n i n e of ibogaine i n the t e t r a c y c l i c s e r i e s the e l e c t r o n s of the n i t r o g e n atom could be i n v o l v e d i n cleavage of the C r i n g i n an analogous manner. Several other r e a c t i o n s of the c h l o r o i n d o l e n i n e system were a l s o considered to be a l s o p o s s i b l e and w i l l be discussed l a t e r . I t was apparent, t h e r e f o r e , that the i n t r o d u c t i o n of cyanide i n t o the c h l o r o i n d o l e n i n e of dihydrocleavamine i n the d e s i r e d manner would be d i f f i c u l t , i f p o s s i b l e at a l l , to achieve. Nevertheless, the approach was a t t r a c t i v e f o r s e v e r a l important reasons. I t r e q u i r e d only three steps, i n theory, t o transform r e a d i l y a v a i l a b l e dihydro 14 cleavamine i n t o a carbomethoxydihydrocleavamine. R a d i o a c t i v e ( C) cyanide could be used and the r a d i o a c t i v e carbomethoxydihydrocleavamines produced could be used i n b i o s y n t h e t i c s t u d i e s to t e s t Wenkert's hypotheses f o r the b i o s y n t h e s i s of the Iboga a l k a l o i d s . F u r t h e r , n u c l e o p h i l e s other than cyanide might be i n c o r p o r a t e d i n t o the 1 8 - p o s i t i o n of dihydrocleavamine. In connection w i t h the l a s t p o s s i b i l i t y an entry i n t o the d i m e r i c Vinca a l k a l o i d s might be r e a l i z e d . Probably the most, important member of t h i s c l a s s i s v i n c a l e u k o b l a s t i n e (VLB), an e f f e c t i v e a n t i c a n c e r agent. The s t r u c t u r e of v i n c a l e u k o b l a s t i n e has been determined by the X-ray method^ and i t s methiodide d e r i v a t i v e i s shown i n 109. An important f e a t u r e of the X-ray d i f f r a c t i o n a n a l y s i s was t h a t i t showed that the upper p o r t i o n of the dimer had a dihydrocleavamine s k e l e t o n r a t h e r than an Iboga s k e l e t o n as p r e v i o u s l y p o s t u l a t e d . 57 Recently, a new dimeric a l k a l o i d , c a l l e d i s o l e u r o s i n e A , has been reported and assigned the s t r u c t u r e 110. The i n t e r e s t i n g f e a t u r e about t h i s compound i s that the dihydrocleavamine p o r t i o n i s a carbomethoxydihydro - 73 -H i COOMe " N H M e O " " ^ COOMe 110 cleavamine and i t d i f f e r s from v i n c a l e u k o b l a s t i n e only i n t h a t i t does not possess the 4-hydroxyl f u n c t i o n . Several other dimeric Vinca a l k a l o i d s have a l s o been i s o l a t e d which a l s o d i f f e r from v i n c a l e u k o b l a s t i n e i n oxida-t i o n l e v e l or placement of the oxygen atom i n the p i p e r i d i n e r i n g of the dihydrocleavamine p o r t i o n of the molecule. The important f e a t u r e of a l l these compounds i s that they a l l possess the dihydrocleavamine s k e l e t o n . From a b i o s y n t h e t i c p o i n t o f view t h i s i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e , n e i t h e r dihydrocleavamine nor any monomeric d e r i v a t i v e s of i t have been i s o l a t e d from p l a n t sources to the present time. From a s y n t h e t i c as w e l l as a b i o s y n t h e t i c p o i n t o f view the dimers could be thought of as a r i s i n g through a coupling of a v i n d o l i n e molecule with an app r o p r i a t e carbomethoxy-dihydrocleavamine d e r i v a t i v e . Indeed, s i m i l a r d i m e r i z a t i o n s have been 28 c a r r i e d out i n the l a b o r a t o r y . Buchi and coworkers were able to c a r r y out an a c i d - c a t a t l y z e d condensation of voacangine with v o b a s i n o l (111) to produce voacamine (112), an important member of the group of dimeric - 74 -MeOOC H 112 Voacanga a l k a l o i d s . Vobosinol possesses a p o t e n t i a l carbonium i o n at the 3 - p o s i t i o n by v i r t u e of the hydroxyl group and would be expected to lead to d i m e r i z a t i o n with voacangine at one of t h i s compounds e l e c t r o n r i c h c e n t r e s , i . e . , at N-16, C-13, or C - l l . Indeed, under m i l d c o n d i t i o n s the condensation of voacangine with v o b a s i n o l y i e l d e d voacamine and a small amount of another known b i s i n d o l e , voacamidine, which arose by coupling between the C - l l s i t e of the voacangine molecule and the C-3 s i t e o f the vobasinol molecule. 57 Recently, Neuss, Gorman, and coworkers have re p o r t e d a s u c c e s s f u l a c i d c a t a l y s e d condensation between the c h l o r o i n d o l e n i n e of 46-dihydrocleav-amine (113) and d e a c e t y l v i n d o l i n e hydrazide (114) to g i v e a dimer (115) coupled i n the same f a s h i o n as v i n c a l e u k o b l a s t i n e . In t h i s p o r t i o n of t h i s t h e s i s are d e s c r i b e d p a r a l l e l experiments t h a t were c a r r i e d out i n our - 75 -114 l a b o r a t o r i e s . In p a r t i c u l a r a d e s c r i p t i o n o f the f i r s t s u c c e s s f u l e x p e r i -ments i n which dimers possessing a C-181. carbomethoxy group i s given. Because t h i s f u n c t i o n a l i t y i s present i n the n a t u r a l Vinca dimers these experiments w i l l l i k e l y represent an important step i n any subsequent s y n t h e s i s of these a l k a l o i d s i n our l a b o r a t o r i e s . I I . D i s c u s s i o n The scheme that was envisaged f o r the i n t r o d u c t i o n of a carbomethoxy group i n t o dihydrocleavamine i s shown i n Figure 28 (Y = CN, R = H ) ( c f Figure 19). Conversion of the 18-cyano-48-dehydrocleavamine (119; Y = CN, R = H) R Y R 119 118 Figure 28. Scheme envisaged f o r the p r e p a r a t i o n of 1 8 - s u b s t i t u t e d dihydro-cleavamines i n t o carbomethoxy-48-dehydrocleavamine was not expected to i n v o l v e any d i f f i c u l t i e s . No d i f f i c u l t i e s were expected i n the p r e p a r a t i o n of the c h l o r o i n d o l e n i n e 113 e i t h e r s i n c e the reagent t - b u t y l h y p o c h l o r i t e had been used s u c c e s s f u l l y i n s e v e r a l instances t o convert i n d o l e a l k a l o i d s i n t o - 77 -t h e i r corresponding chloroindolenin.es. ^ ' ^ ^ On the other hand i t was recognized that the sequence 117 -»- 118 119 might be d i f f i c u l t to achieve i n r e a l i t y . Treatment of 48-dihydrocleavamine (116; R = H) w i t h t - b u t y l h y p o c h l o r i t e gave a mixture of products with one component i n predominance. I t was found, however, that the y i e l d of the major product v a r i e d c o n s i d e r a b l y according to the procedure used i n the r e a c t i o n and work up. In a l l experiments the r e a c t i o n was c a r r i e d out by adding a standard carbon t e t r a c h l o r i d e s o l u t i o n of t - b u t y l h y p o c h l o r i t e to a c h i l l e d methylene c h l o r i d e s o l u t i o n of 48-dihydrocleavamine c o n t a i n i n g a c a t a l y t i c amount of t r i e t h y l a m i n e . I t was observed that as the t - b u t y l h y p o c h l o r i t e s o l u t i o n was added to the s o l u t i o n of the 48-dihydrocleavamine, the l a t t e r s o l u t i o n became s t e a d i l y more orange i n c o l o u r , u n t i l an equivalent amount of the oxident had been added. A g r e a t l y a c c e l e r a t e d deepening of the orange c o l o u r then occurred as excess oxident was added. The progress of the r e a c t i o n could c o n v e n i e n t l y be f o l l o w e d by a n a l y t i c a l t h i n - l a y e r chromatography (alumina, benzene or s i l i c a g e l , c h l o r o f o r m : e t h y l acetate 1:1). I t was observed t h a t the sudden increase i n c o l o r a t i o n of the r e a c t i o n medium was concomitant with the disappearance of 48-dihydrocleavamine and what appeared to be an i n c r e a s e i n m a t e r i a l s that were very p o l a r i n nature. Washing the r e a c t i o n s o l u t i o n w i t h water was found to be i n e f f e c t i v e i n removing the coloured and p o l a r i m p u r i t i e s . I t was d i s c o v e r e d , however, t h a t a c o l o u r l e s s sample of the major product from the o x i d a t i o n r e a c t i o n , which showed only one spot by t h i n - l a y e r chrom-atographic a n a l y s i s w i t h alumina and s i l i c a g e l absorbents and s e v e r a l systems, could be obtained by f i r s t mixing the r e a c t i o n s o l u t i o n with an equal volume of benzene and p e r c o l a t i n g the new s o l u t i o n at a r a p i d r a t e through a s h o r t , broad alumina (Woelm a c t i v i t y I I I ) column. Evaporation of - 78 -the s olvent from the eluant f i r s t i n a r o t a t o r y evaporator at reduced pressure and room temperature and f i n a l l y in_ vacuo at a pump provided a pure sample of the major r e a c t i o n product as a c o l o u r l e s s o i l . I t was found t h a t the best y i e l d s were obtained when the p e r c o l a t i o n step above was aided by the a p p l i c a t i o n of p o s i t i v e pressure to the top of the column i n order to maintain a steady stream of s o l u t i o n from the column. Use of the procedures described above r e s u l t e d i n c o n s i s t e n t y i e l d s of the major o x i d a t i o n product i n the range of 80 to 90 percent. Although t h i s m a t e r i a l was found to run as one compound i n a v a r i e t y of t h i n - l a y e r chromatography systems and so appeared to be a s i n g l e compound, i t could not be induced t o c r y s t a l l i z e . In f a c t a l l attempts to c r y s t a l l i z e the m a t e r i a l r e s u l t e d i n decomposition t o a g r e a t e r or l e s s e r degree. I t was r e a l i z e d t h a t the m a t e r i a l could be a mixture. In p a r t i c u l a r , i t seemed very probable that i t could be a mixture of the C-9 epimers of the d e s i r e d c h l o r o i n d o l e n i n e 113. The m a t e r i a l however, always appeared t o be a s i n g l e compound when i t was subjected to t h i n - l a y e r chromatographic or s p e c t r a l a n a l y s i s . The s p e c t r a l p r o p e r t i e s , of the m a t e r i a l were i n accord w i t h the f o r m u l a t i o n 113. The i n f r a r e d spectrum showed bands at 1600 cm * (m) and 1560 cm * ( s ) . This p a t t e r n i s a c h a r a c t e r i s t i c f e a t u r e of c h l o r o i n d o l e n i n e s ^ ' ^ ^®. The u l t r a v i o l e t spectrum and the nmr spectrum were a l s o i n keeping with the f o r m u l a t i o n 113. The low r e s o l u t i o n mass spectrum was p a r t i c u l a r l y inform-a t i v e and i s shown i n Figure 29. These were two molecular i o n peaks at 35 37 m/e 316 ( CI) and 318 ( CI) as would be expected. The base peak at m/e 281 corresponded to a l o s s of a c h l o r i n e atom from the molecular i o n . Other peaks t y p i c a l of the cleavamine s y s t e m ^ were a l s o present. The molecular formula of the m a t e r i a l was determined f o r the molecular i o n at m/e 316 and was found t o be C..H N CI, The m a t e r i a l was, t h e r e f o r e , - 80 -unquestionably a monochloro derivative of 4B-dihydrocleavamine. A l l the spectral data were i n accord therefore with the formulation of a chloroindol-enine 1.13 assuming no rearrangement of the cleavamine skeleton had taken place. That no rearrangement had occurred was established when 4g-dihydro-cleavamine was regenerated on treatment of the chlorinated material with lithium aluminum hydride i n diethyl ether. After the product of the chlorination reaction had been s a t i s f a c t o r i l y established as the desired chloroindolenine 113, the reaction of t h i s substance with cyanide ion was contemplated. It was recognized that a successful conversion of the chloroindolenine 113 into an 18-cyano-48-dihydrocleavamine (119; R = H, Y = CN) would depend, among other things, upon the equilibrium 117 * 118 being established under the reaction conditions employed. It was clear from the nmr spectrum (Figure 30) of the chloroindolenine, which showed no resonances that might correspond to the a-methylene-indoline form (118; R = H), that the indolenine form (117; R = H) in deuteriochloroform at least, was by far the predominant form. Neverthe-l e s s , the u l t r a v i o l e t spectrum of the chloroindolenine did show some pH dependency that could have been related to the desired imine-enamine tautomerization. In the absence of a more analogous sequence, the same conditions which had been used by Buchi and his coworkers to bring about the formation of 18-cyanoibogaine from the chloroindolenine of ibogaine were chosen for the study at hand. Thus, the chloroindolenine of 4B-dihydrocleav-amine was allowed to react with a complex mixture of potassium cyanide, methanol, diethyl ether and water for 48 hours at room temperature under a nitrogen atmosphere. These conditions were found by the Buchi group to be optimum for the formation of the desired product i n t h e i r sequence. In the Figure 30. Nmr spectrum of the c h l o r o i n d o l e n i n e o f 48-dihydrocleavamine - 82 -present cas^, however, a very complex mixture of products was obtained. Several m o d i f i c a t i o n s o f the r e a c t i o n c o n d i t i o n s were used i n an attempt to improve the r e a c t i o n , but no d i s c e r n a b l e improvement could be made. Although the r e s u l t s were very d i s c o u r a g i n g , they were not r e a l l y unexpected. I t was apparent that the c h l o r o i n d o l e n i n e had the p o t e n t i a l to undergo many transformations other than the d e s i r e d one. Several modes by which the c h l o r o i n d o l e n i n e might conceivably have reacted are i n d i c a t e d i n Figure 31. Reasonable arguements could be presented f o r the modes (a t o i ) o f r e a c t i o n f Figure 31. Conceivable modes of reaction- o f the c h l o r o i n d o l e n i n e of 43-dihydrocleavamine shown. For example r e a c t i o n mode " c " seemed t o have a high p r o b a b i l i t y of success. I t has been demonstrated that the di a s t e r e o m e r i c c h l o r o i n d o l e n i n e s 6 2 derived from yohimbine are converted to imidoethers on treatment w i t h a c i d 6 3 or base. 7-Chloro-7H-yohimbine (120) on treatment w i t h methanolic a l k a l i was found to be converted i n t o the corresponding s p i r o i m i d o e t h e r 121. The transformation was envisaged to proceed through a t t a c k of the methoxide i n a c i s manner to the c h l o r i n e atom a l l o w i n g the m i g r a t i n g centre to d i s p l a c e c h l o r i d e i o n by trans attack (Figure 32). Buchi and coworkers found that - 83 -F igu re 32. Sp i romidoe ther format ion from a c h l o r o i n d o l e n i n e the c h l o r o i n d o l e n i n e o f iboga ine d i d not undergo such a rearrangement . On the b a s i s o f t h i s r e s u l t they t e n t a t i v e l y proposed t ha t the ch lo r ine , atom was 6 - o r i e n t e d s i n c e o f the two t h e o r e t i c a l l y p o s s i b l e d i a s t e r i o m e r s the one w i t h the a c o n f i g u r a t i o n o f the c h l o r i n e atom would have l e d to an e x c e s s i v e l y crowded in t e rmed ia t e through " c i s " a t t a c k by methoxide i o n . In the case o f e i t h e r o f the two p o s s i b l e c h l o r o i n d o l e n i n e s o f 4 6 - d i h y d r o c l e a v -amine, which would have a more f l e x i b l e n a t u r e , there seemed to be no s t e r i c i n h i b i t i o n to fo rmat ion of an i n t e rmed ia t e w i th the c i s arrangement of the c h l o r i n e atom and a methoxyl (or hydroxyl) group. Spiroimidoether or s p i r o o x i n d o l e formation appeared t o be a favorable r e a c t i o n i n the case of a c h l o r o i n d o l e n i n e prepared from 4g-dihydro,-.leavamine. As f a r as r e a c t i o n mode "a" was concerned i t represented a simple SK^ displacement of c h l o r i d e i o n a l b e i t at a t e r t i a r y centre. A SN^ displacement i n v o l v i n g , perhaps, a 2-alkylidene-2H-indole intermediate was a l s o conceivable. I f the a t t a c k i n g n u c l e o p h i l e were water, the B-hydroxyindolenine 122 (Y = OH) would be formed which bears a s t r i k i n g resemblance to the conjugate base 64 form 12 of the Aspidosperma a l k a l o i d r h a z i d i n e (10). Rhazidine has been 64 shown to e x i s t i n i t s s a l t form i n a c i d i c or n e u t r a l a l c o h o l i c s o l u t i o n s . The conjugate base form was observed i n s t r o n g l y b a s i c a l c o h o l i c s o l u t i o n s or i n hydrocarbon sol v e n t s such as heptane. I t was tempting to consider t h a t compounds such as 122 (Y = OH, CI, CN, OMe) might a l s o be represented by the s t r u c t u r e s 123 (Y = OH, CI, CN, OMe) under the proper c o n d i t i o n s . 122 123 Reaction mode "b" was considered although i t represented a very unusual SN 2' r e a c t i o n . There was t h e o r e t i c a l j u s t i f i c a t i o n f o r such a r e a c t i o n ^ and a remote analogy which could be found i n the l i t e r a t u r e ^ i s shown i n Figure 33. - 85 -Figure 33. An unusual S^' r e a c t i o n i n v o l v i n g n u c l e o p h i l i c attack on oxygen Reaction mode "c" was expected to occur s i n c e the i m p l i e d r e a c t i o n was found to have been serious competing r e a c t i o n w i t h the d e s i r e d t r a n s f o r m a t i o n i n the conversion shown i n Figure 27. In the case of the c h l o r o i n d o l e n i n e of 4g-dihydrocleavamine i t was f e l t that t h i s mode would lead t o an i n t e r -mediate (124) which could undergo a v a r i e t y of r e a c t i o n s . cf 124 Reaction mode " f " had to be considered as a p o s s i b i l i t y . I t was f e l t t h at e l i m i n a t i o n of hydrogen c h l o r i d e i n the manner i n d i c a t e d would provide the a,8-unsaturated inline 125 which could undergo Michael a d d i t i o n of cyanide and regeneration o f the i n d o l e would provide a strong d r i v i n g f o r c e f o r the a d d i t i o n r e a c t i o n . The product, 8-cyano-4B-dihydrocleavamine (126) - 86 -•would be expected to be very d i f f i c u l t to d i s t i n g u i s h from the d e s i r e d l i cyano isomer. s<—\ <' N N \ 125 H 126 Reaction mode "g" was an i n t e r e s t i n g p o s s i b i l i t y s i n c e such a t r a n s -annular c y c l i z a t i o n r e a c t i o n would produce a quaternary s a l t (56) which was encountered as a key intermediate i n Kutney and coworkers' s y n t h e s i s of dihydrocleavamine (Figure 8). From a cursory examination by t h i n - l a y e r chromatography of the r e a c t i o n mixture from the r e a c t i o n of the c h l o r o i n d o l e n i n e of 4g-dihydrocleavamine under the Buchi c o n d i t i o n s , i t seemed that every conceivable r e a c t i o n and a few i n c o n c e i v a b l e ones as w e l l might have taken p l a c e . There were a great many products formed and no one product seemed t o be dominant. Chromatography of the r e a c t i o n mixture on Woelm alumina ( a c t i v i t y I I I ) accomplished a r e s o l u t i o n of the mixture i n t o three groups of products. E l u t i o n with benzene provided group "A", e l u t i o n w i t h chloroform-benzene provided group "B" and e l u t i o n w i t h s e v e r a l solvent systems i n c l u d i n g methanol-water provided group "C". Each group represented about o n e - t h i r d of the t o t a l r e a c t i o n mixture. A f t e r a cursory s p e c t r o s c o p i c examination, i t was decided that group A was the most l i k e l y group to conta i n the sought a f t e r 18-cyano-46-dihydrocleavamine. Although the compounds i n group A showed almost - 87 -i d e n t i c a l r a t e s of t r a n s p o r t on alumina "hromatoplates, there was a more d i s t i n c t d i f f e r e n c e i n the rates of t r a n s p o r t on s i l i c a g e l chromatoplates. Because of t h i s d i f f e r e n c e , group A was chromatographed on Woelm s i l i c a gel ( a c t i v i t y I I I ) . E l u t i o n w i t h benzene-chloroform (3:1) provided a s e r i e s of f r a c t i o n s c a l l e d group A^ which were shown to be a mixture of at l e a s t two compounds by a n a l y t i c a l t h i n - l a y e r chromatography on f e s h l y a c t i v a t e d s i l i c a gel p l a t e s . E l u t i o n with 2% t r i e t h y l a m i n e i n acetone provided a m a t e r i a l (Ag) which could be obtained i n c r y s t a l l i n e form from methanol. The chromato-graphic s e p a r a t i o n procedure i s summarized i n a flow sheet shown i n Figure 34. 113 (559mg) KCN.MeOH, Et-Ar-UO mixture(491mg) chromatography (alumina ) CH aCl a >Et aO, MeOH-Et.O PhH CHCL-PhH MeOH.MeOH-HjO group A m f c m g ) 9roup B(140mg) group C(l64mg) chromat. (silica gel) 71 PhH-CHCl, group A,(36mg) cmpA 3 ( 40mg ) Figure 34. Flow sheet showing the p a r t i a l s e paration of the products of the r e a c t i o n of the c h l o r c i n d o l e n i n e 113 under the Buchi c o n d i t i o n s - 88 -Group A ^ r e s i s t e d complete r e s o l u t i o n i n t o i t s component compounds. Neverthe-l e s s , a very s m a l l sample of the n i t r i l e c o n t a i n i n g component of group A^ was obtained by c a r r y i n g out s e v e r a l successive p u r i f i c a t i o n s on f r e s h l y a c t i v a t e d s i l i c a g e l p r e p a r a t i v e chromatoplates. The s p e c t r a l data which could be obtained f o r t h i s m a t e r i a l was c o n s i s t e n t w i t h i t being the sought f o r 18-cyano-43-dihydrocleavamine. I t e x h i b i t e d a t y p i c a l i n d o l e u l t r a v i o l e t spectrum. I t s i n f r a r e d spectrum d i s p l a y e d absorptions at 3300 cm ' and 2220 cm * which were c o n s i s t e n t with the compound having an i n d o l e NH group and a n i t r i l e group. The low r e s o l u t i o n mass spectrum of the m a t e r i a l d i s p l a y e d a molecular i o n peak at m/e 307 and a t y p i c a l cleavamine-type fragmentation p a t t e r n . High r e s o l u t i o n mass a n a l y s i s on the m/c 307 peak provided a molecular weight f o r the m a t e r i a l of 307.205 which corresponded to the formula C„ nH o rN e s t a b l i s h i n g that the m a t e r i a l indeed was a cyano-ZU z5 3 4B-dihydrocleavamine. A sample of group A^  which had been p u r i f i e d to the extent of about 90% i n the n i t r i l e c o n t a i n i n g component gave a nmr spectrum which was c o n s i s t e n t w i t h that which was expected f o r the d e s i r e d m a t e r i a l . Because i t proved t o be d i f f i c u l t to r e s o l v e group A i n t o i t s components i t was decided to proceed w i t h the mixture i n the next step of the syntheses; that i s , to convert the n i t r i l e to a methyl e s t e r . I t was hoped that p u r i f i c a t i o n o f the e s t e r would prove to be e a s i e r than p u r i f i c a t i o n of the n i t r i l e . .'.Mien a sample of group A was allowed to react w i t h anhydrous methanolic hydrogen c h l o r i d e and then with aqueous sodium carbonate, a complex mixture of compounds was obtained. F o r t u n a t e l y , a small amount of a u t h e n t i c 183-carbomethoxy-4S-dihydrocleavamine, obtained as a minor product i n the z i n c - a c e t i c a c i d r e d u c t i o n of catharanthine, was - a v a i l a b l e . Comparison of the r e a c t i o n mixture with the authentic sample by t h i n - l a y e r - 89 -chromatography i n d i c a t e d t h a t t h e compound was p r e s e n t i n t h e m i x t u r e . A c o m b i n a t i o n o f t h e methods o f column chromatography on a l u m i n a and p r e p a r a -t i v e t h i n - l a y e r chromatography on s i l i c a g e l p r o v i d e d a p u r e sample o f the compound b e l i e v e d t o be 1 8 g - c a r b o m e t h o x y - 4 3 - d i h y d r o c l e a v a m i n e . T h i s m a t e r i a l d i s p l a y e d t h e same r a t e s o f t r a n s p o r t and c o l o u r r e a c t i o n s as t h e a u t h e n t i c m a t e r i a l on a n a l y s i s by t h i n - l a y e r chromatography. T h i s m a t e r i a l a l s o e x h i b i t e d t h e same f r a g m e n t a t i o n p a t t e r n i n i t s mass s p e c t r u m as t h e a u t h e n t i c sample when b o t h compounds were r u n under t h e same c o n d i t i o n s . H i g h r e s o l u t i o n mass a n a l y s i s o f t h e m o l e c u l a r i o n peak a t m/e 340 p r o v i d e d a m o l e c u l a r w e i g h t f o r t h e m a t e r i a l o f 340.212 ( c a l c d mol wt o f 18 6-carbo-m e t h o x y - 4 3 - d i h y d r o c l e a v a m i n e , 340.215). A l t h o u g h t h e r e was no doubt t h a t t h e m e t h a n o l i c h y d r o g e n c h l o r i d e t r e a t -ment o f a sample o f group A had a c h i e v e d t h e d e s i r e d c o n v e r s i o n , t h e y i e l d o f 1 8 3 - c a r b o m e t h o x y - 4 8 - d i h y d r o c l e a v a m i n e was t e r r i b l e . S i n c e one o f t h e p r i n c i p l e r e a s o n s f o r d e v e l o p i n g a method f o r i n t r o d u c i n g a carbomethoxy group i n t o d i h y d r o c l e a v a m i n e v i a a cyano i n t e r m e d i a t e was t h a t p o t a s s i u m 14 14 c y a n i d e - C c o u l d be u s e d t o p r e p a r e C - l a b e l l e d c a r b o m e t h o x y d i h y d r o c l e a v -amine f o r t r a c e r e x p e r i m e n t s i n p l a n t s , i t was o f p a r t i c u l a r i m p o r t a n c e t o d e v e l o p as h i g h a y i e l d i n g sequence as p o s s i b l e . I t was c l e a r t h a t an a t t e m p t t o improve t h e method f o r making t h e 1 8 - c y a n o - 4 3 - d i h y d r o c l e a v a m i n e would i n v o l v e a c o n s i d e r a b l e e x p e n d i t u r e o f t i m e and e f f o r t w i t h no g u a r a n t e e o f s u c c e s s . T h e r e f o r e a t t e n t i o n was d i r e c t e d t o i m p r o v i n g t h e y i e l d o f 183-carbomethoxy-4 3 - d i h y d r o c l e a v a m i n e from t h e n i t r i l e . I t was f o u n d t h a t when a sample o f group A^ , w h i c h c o n t a i n e d r a d i o a c t i v e 1 8 - c y a n o - 4 3 - d i h y d r o c l e a v -14 amine from a p r e p a r a t i o n u t i l i z i n g p o t a s s i u m c y a n i d e - C, was c o n v e r t e d t o 1 8 8 - c a r b o m e t h o x y - 4 8 - d i h y d r o c l e a v a m i n e i n s u c h a manner t h a t a l l t h e r a d i o -a c t i v i t y p r e s e n t i n t h a t compound was scavenged w i t h i n a c t i v e compound, t h e - 90 -a c t i v i t y present as 183-carbomethoxy-4g-dihydrocleavamine represented only 5% of the a c t i v i t y present before the conversion. I t was decided, t h e r e f o r e , to attempt a b a s i c h y d r o l y s i s of the n i t r i l e . A c c o r d i n g l y , a sample of group A^ c o n t a i n i n g r a d i o a c t i v e n i t r i l e was allowed to r e a c t w i t h potassium hydroxide i n d i e t h y l e n e g l y c o l at 150° f o r 8 hours under a n i t r o g e n atmosphere. The c a r b o x y l i c a c i d formed was not i s o l a t e d , but was immediately converted i n t o i t s methyl e s t e r by treatment of the mixture of r e a c t a n t s with methanolic hydrogen c h l o r i d e and an e t h e r e a l s o l u t i o n of diazomethane. The 188-carbomethoxy-4g-dihydrocleavamine formed was estimated as before to con-t a i n not l e s s than 35% of the a c t i v i t y presented i n the sample of r a d i o a c t i v e group A^. Thus base h y d r o l y s i s o f the n i t r i l e f o l l o wed by e s t e r i f i c a t i o n w i t h diazomethane was shown to be a much b e t t e r method of producing 188-carbomethoxy-48-dihydrocleavamine from the 18-cyano-43-dihydrocleavamine component of group A^. Using t h i s method, enough s y n t h e t i c 188-carbomethoxy-48-dihydrocleavamine i n c r y s t a l l i n e form was obtained f o r the purposes of melt i n g p o i n t and i n f r a r e d comparisons with an a u t h e n t i c sample. The s y n t h e t i c and a u t h e n t i c samples had the same me l t i n g p o i n t , d i d not show depression of m e l t i n g p o i n t on admixture and d i s p l a y e d i d e n t i c a l i n f r a r e d s p e c t r a . The s y n t h e t i c carbomethoxydihydrocleavamine was thus i r r e f u t a b l y shown to be 183-carbomethoxy-4g-dihydrocleavamine and not another p o s s i b l e isomer such as an 8-carbomethoxy-48-dihydrocleavamine which would be formed from an 8-cyano-48-d-ihydrocleavamine (116). Compound A^ proved t o be a very i n t e r e s t i n g compound. Although the y i e l d (7%) of t h i s compound was f a i r l y s m a l l , i t was nevertheless a major component of the complex mixture of products obtained when the c h l o r o i n d o l e n i n e o f 43-dihydrocleavamine was allowed to react under the Buchi c o n d i t i o n s . The compound was found to d i s p l a y a t y p i c a l i n d o l e chromophore i n i t ' s - 91 -u l t r a v i o l e t spectrum. The i n f r a r e d spectrum showed a strong absorption band at 3280 cm 1 i n d i c a t i n g the presence of an i n d o l e N-H grouping and d i s p l a y i n g no bands i n the re g i o n 2500-1500 cm 1. Of p a r t i c u l a r note was a strong band at 1070 cm 1 which i n d i c a t e d the presence of a -C-O-Me grouping. The thought that the compound might be an 18-metl;oxy-48-dihydrocleavamine epimer (119, R = H, Y = OMe) was s e r i o u s l y e n t e r t a i n e d . The low r e s o l u t i o n mass spectrum (Figure 35) of t h i s compound supported t h i s conjecture. I t d i s p l a y e d a molecular i o n peak at m/e 312 and a fragmentation p a t t e r n which was a t y p i c a l cleavamine type. The molecular weight of the compound was found to be 312.220 ( c a l c d mol wt, 312.220) by high r e s o l u t i o n mass spectro-m e t r i c determination. The nmr spectrum (Figure 36) d i s p l a y e d a prominent s i g n a l at x 6.8 which corresponded to 3 protons i n the i n t e g r a l . This feat u r e v e r i f i e d the presence of a methoxyl f u n c t i o n i n the compound. The r e s t of the nmr spectrum was c o n s i s t e n t w i t h the compound being a methoxy-dihydrocleavamine. The compound was t e n t a t i v e l y proposed to be 18-methoxy-4B-dihydrocleavamine although on the ba s i s of the data C-8 could not be excluded as the s i t e of the methoxyl f u n c t i o n . Compelling nmr evidence to support the c l a i m that t h i s compound i s 18a-methoxy-4B-dihydrocleavamine w i l l be presented l a t e r . Some a t t e n t i o n was d i r e c t e d towards determining the nature of the compounds i n group B. This mixture was found to behave almost as one com-pound during a n a l y s i s by t h i n - l a y e r chromatography. The s p e c t r a l evidence, however, was not c o n s i s t e n t with i t being a s i n g l e compound. The u l t r a -v i o l e t spectrum seemed t o be made up of a s u p e r p o s i t i o n o f chromophores. The i n f r a r e d spectrum d i s p l a y e d bands which were i n d i c a t i v e of an ind o l e NH and a n i t r i l e , probably conjugated judging by the i n t e n s i t y of the absorption. R E L A T I V E A B U N D A N C E era c CD O CO CD -v) 00 CO o o o o O O o o o o S cn cn cn CD O r+ 4 O o o H i 124 00 P i 138 o o • X I -p* TO I o-H o o I—" (D I CD 187 182<M-130) S 1 o ho cn o X-00 o o 312(M ) - 36 -Figure 36. Nmr spectrum of 18a-methoxy-4g-dihydrocleavamine - 94 -The nmr spectrum d i s p l a y e d four sharp s i n g l e t s i n the region t 5.0-7.0 and two broad " s i n g l e t s " i n the r e g i o n x 1.0-2.0 i n which protons on i n d o l e n i t r o g e n o f t e n occur. A c a r e f u l chromatography of the mixture provided a p a r t i a l s e p a r a t i o n . The nmr spectrum of s e l e c t e d f r a c t i o n s showed there was a d e f i n i t e t r e n d i n the v a r i a t i o n of the i n t e n s i t y of s i g n a l s . I t was determined, t h e r e f o r e , that there were at l e a s t three compounds i n the mixture and because the r a t e s of t r a n s p o r t of the compounds were so n e a r l y the same, no attempt was made to f u r t h e r r e s o l v e the mixture. A cursory examination of group C by t h i n - l a y e r chromatographic a n a l y s i s showed that t h i s was a l s o a mixture which would not lend i t s e l f to easy r e s o l u t i o n i n t o i t s components. - I t was c l e a r from the r e s u l t s above th a t the 4g-dihydrocleavamine could be converted i n t o d e r i v a t i v e s possessing s u b s t i t u t i o n at the C-18 s i t e . N evertheless, the c o n d i t i o n s used were o b v i o u s l y not i d e a l . Enough 18-cyano-46-dihydrocleavamine could not be obtained f o r complete c h a r a c t e r i z a t i o n because of p u r i f i c a t i o n problems. In a d d i t i o n the y i e l d of t h i s m a t e r i a l was very poor. I t was f e l t t hat i f the r e a c t i o n 117 -> 124 was indeed a s e r i o u s side r e a c t i o n as i t had been shown to be by Buchi and h i s coworkers i n the p e n t a c y c l i c s e r i e s , then using c o n d i t i o n s which were designed to suppress t h i s u n d e s i r a b l e s i d e r e a c t i o n might lead to a b e t t e r y i e l d and a c l e a n e r r e a c t i o n . In view of t h i s , a c i d i c c o n d i t i o n s under which the b a s i c n i t r o g e n would be expected to be quaternary were chosen. An anhydrous methanolic hydrogen c h l o r i d e s o l u t i o n was added to a mixture of potassium cyanide and the c h l o r o i n d o l e n i n e of 48-dihydrocleavamine i n a f l a s k f i t t e d w i t h an e f f i c i e n t condenser and cooled to ice-water temperature. Escaping hydrogen cyanide gas was passed i n t o an aqueous potassium hydroxide s o l u t i o n . - 95 -The s o l u t i o n obtained was then heated t o r e f l u x temperature and r e f l u x e d f o r three hours. The reaction-product mixture obtained on work up was compared by t h i n - l a y e r chromatography w i t h the 18-cyano-4B-dihydrocleavamine and 18a-methoxy-48-dihydrocleavamine. There was no s i g n o f the former compound, but there was a major component o f the mixture which corresponded to the l a t t e r compound. That none o f the d e s i r e d n i t r i l e appeared to be formed was not unexpected because the c o n c e n t r a t i o n o f cyanide i o n would be expected to be very small under the a c i d i c c o n d i t i o n s employed. The crude r e a c t i o n mixture was chromatographed on alumina ( a c t i v i t y I I I ) . E l u t i o n w i t h petroleum ether (30-60)-benzene (3:1) provided a mixture of the compound which corresponded t o 18a-methoxy-4B-dihydrocleavamine and another compound which was s l i g h t l y l e s s p o l a r . The nmr spectrum o f the mixture i n d i c a t e d t h a t i t was.a mixture of both epimeric 18-methoxy-4B-dihydrocleavamines. On t h i s assumption the y i e l d of the methoxydihydrocleavamines was c a l c u l a t e d to be 39 percent. This r e s u l t represented a more than f i v e f o l d i n c r e a s e i n the y i e l d compared t o the previous r e a c t i o n . Chromatography of the mixture on s i l i c a g e l ( a c t i v i t y I I I ) provided a pure sample of each isomer. On the b a s i s of a comparison o f the s p e c t r a of these compounds, the l e s s p o l a r compound (10% y i e l d ) was considered to be 18B-methoxy-43-dihydrocleavamine and the more p o l a r compound (24% y i e l d ) was shown t o be 18a-methoxy-4B-dihydrocleavamine. The mass spectrum and the nmr spectrum o f the l e s s p o l a r methoxydihydrocleavamine are shown i n Figures 37 and 38, r e s p e c t i v e l y . The nmr s p e c t r a of the s u b s t i t u t e d dihydrocleavamines are discussed and the reasons f o r the stereochemical assignments are presented l a t e r on i n t h i s s e c t i o n . In view o f the higher y i e l d of the 18-methoxy-4B-dihydrocleavamines 96 O. o o CO o in cm o o £81 CD c cd > ni 0) i—i U O fn X (0£l-W)28l o in 8EI i X o •P CD ca oo CH o o o o CD cn IO o in o o o CO o o o co o in o o o CM CD U oo • H 3DNVQND8V 3 A U V 1 3 y - 98 -obtained i n the r e a c t i o n described above, i t was decided to attempt to prepare a d e r i v a t i v e o f 43-dihydro'cleavamine which possessed a good l e a v i n g group at C-18. The d i r e c t displacement of such a group w i t h cyanide i o n was a n t i c i p a t e d . I t was f e l t that a halogen would be a d e s i r a b l e l e a v i n g group. Consequently, an attempt to prepare an 18-iodo-4S-dihydrocleavamine was made. The h y d r o c h l o r i d e s a l t of the c h l o r o i n d o l e n i n e of 46-dihydrocleavamine was prepared. To the s a l t i n a small amount of anhydrous acetone was added a s o l u t i o n o f sodium i o d i d e i n anhydrous acetone. A dark pur p l e coloured s o l u t i o n formed i n s t a n t a n e o u s l y . The c o l o u r i n d i c a t e d the presence of i o d i n e . The s o l u t i o n was s t i r r e d f o r 3 hours under a dry, oxygen-free atmosphere at room temperature and then worked up. The i o d i n e c o l o u r was e f f e c t i v e l y removed by washing an e t h e r e a l s o l u t i o n of the r e a c t i o n mixture with an aqueous sodium t h i o s u l f a t e s o l u t i o n . I n v e s t i g a t i o n o f the crude r e a c t i o n mixture by a n a l y t i c a l t h i n - l a y e r chromatography showed the presence of 46-dihydrocleavamine and a mixture of compounds that were much too p o l a r by comparison to c o n t a i n the d e s i r e d iodo compounds. Chromatography on alumina ( a c t i v i t y I I I ) provided 46-dihydrocleavamine i n 22 percent y i e l d . 67 I t was r e c a l l e d that Dolby and G r i b b l e had reported a s i m i l a r r e d u c t i o n of a c h l o r o i n d o l e n i n e . They found that when the mixture of c h l o r o i n d o l e n i n e epimers shown as 127 was t r e a t e d w i t h potassium t-butoxide the t e t r a c y c l i c amine 128 was formed. They proposed that the r e a c t i o n took place by nucleo-CI - 99 -p h i l i c a t t a c k on c h l o r i n e . The same sort o f proposal could be made f o r the re d u c t i o n of the c h l o r o i n d o l e n i n e of 48-dihydrocleavamine by i o d i d e i o n The l i b e r a t i o n of i o d i n e on treatment of a h a l o i n d o l e n i n e w i t h i o d i d e has been p r e v i o u s l y reported. In 1933 Pla n t and Tomlinson reported t h a t 2-phenyl-3-methylindole on bromination gave a compound (which was presumably the 3-bromoindolenine) that l i b e r a t e d i o d i n e on treatment w i t h aqueous potassium i o d i d e . Because d i r e c t p r e p a r a t i o n of an 18-halo-48-dihydrocleavamine from the c h l o r o i n d o l e n i n e d i d not appear to be f e a s i b l e on the b a s i s of the above r e s u l t s , an attempt was made to prepare such a compound from the 18-methoxy-48-dihydrocleavamines. The boron t r i h a l i d e s have been used s u c c e s s f u l l y i n many instances to cleave e t h e r s ^ ' ^ to the corresponding a l k y l h a l i d e s w i t h the d i r e c t i o n of cleavage f a v o r i n g the d i r e c t i o n which would give r i s e to the most s t a b l e carbonium i o n . In a d d i t i o n , boron t r i b r o m i d e was used s u c c e s s f u l l y t o cleave benzyl ethers 53 and 54 i n the quebrachamine and dihydrocleavamine syntheses (Figure 8) developed i n our l a b o r a t o r i e s . In the present case treatment o f a mixture of 18-methoxy-48-dihydrocleavamines w i t h boron t r i c h l o r i d e produced a complex mixture of products and the method was abandoned. An attempt t o hydrolyse the 18-methoxy-48-dihydro-cleavamines w i t h aqueous h y d r o c h l o r i c a c i d to give the corresponding 18-hydroxy-48-dihydrocleavamines a l s o gave a complex mixture of products. In view of these r e s u l t s i t was apparent that the 18-methoxy-48-dihydrocleav-amines were a c i d s e n s i t i v e i n an u n d e s i r a b l e f a s h i o n . I t was f e l t at t h i s stage that d i r e c t formation of a 48-dihydrocleav-amine d e r i v a t i v e with a s u i t a b l e l e a v i n g group at the C-18 s i t e from the c h l o r o i n d o l e n i n e o f f e r e d the best chance of success. I t was decided t h a t a - 100 -C-18 acetoxy group might be a s u i t a b l e group. Although an acetoxy grcup under o r d i n a r y circumstances would not be a p a r t i c u l a r l y good l e a v i n g group, i t was f e l t t hat i t might be i n t h i s case and, i f not, there was good reason to b e l i e v e t h a t i t could be converted by; m i l d base h y d r o l y s i s i n t o a C-18 hydroxy group. The l a t t e r s u b s t i t u e n t might i n t u r n be converted 71 i n t o one of s e v e r a l good l e a v i n g groups. Dolby and Sakai found t h a t the acetoxylactam 129 gave the hydroxylactam 130 i n 52 percent y i e l d on standing f o r 2 hours at room temperature i n a water-methanol (1:1) s o l u t i o n c o n t a i n i n g 5% sodium hydroxide. The hydroxylactam 130 was converted i n q u a n t i t a t i v e y i e l d t o the acetoxylactam 129 when i t was t r e a t e d w i t h a sodium a c e t a t e - a c e t i c a c i d s o l u t i o n f o r t h i r t y minutes at room temperature. The c h l o r o i n d o l e n i n e of 46-dihydrocleavamine was t r e a t e d w i t h a 10% s o l u t i o n o f fused sodium acetate i n g l a c i a l a c e t i c a c i d . The r e a c t i o n was c a r r i e d out at 60°C i n an atmosphere of dry, oxygen-free n i t r o g e n and the progress of the r e a c t i o n was f o l l o w e d by t h i n - l a y e r chromatography (alumina, 3:1 benzene-ethylacetate; s i l i c a g e l , 2.5% trimethylamine i n e t h y l a c e t a t e ) . In both t h i n - l a y e r chromatography systems a r e a c t i o n product whose r a t e o f - 101 -tr a n s p o r t was s l i g h t l y l a r g e r than that of the c h l o r o i n d o l e n i n e appeared. Aside from " b a s e l i n e " m a t e r i a l there was no i n d i c a t i o n o f other products. A f t e r about 30 minutes the amount of t h i s r e a c t i o n product d i d not appear to in c r e a s e i n p r o p o r t i o n t o the amount of c h l o r o i n d o l e n i n e whereas the amount o f " b a s e l i n e " m a t e r i a l s t e a d i l y increased i n p r o p o r t i o n t o the c h l o r o i n d o l e n i n e . A f t e r two hours only " b a s e l i n e " m a t e r i a l remained. I n v e s t i g a t i o n by t h i n -l a y e r chromatography (alumina, 3:1 e t h y l acetate-ethanol) of the b a s e l i n e m a t e r i a l i n d i c a t e d that i t was composed of two compounds. Development o f a chromatoplate with antimony p e n t a c h l o r i d e revealed two overlapping spots t h a t were, s l i g h t l y d i f f e r e n t shades of green. Before the r e a c t i o n products obtained at 60° were i n v e s t i g a t e d f u r t h e r , the r e a c t i o n was repeated as before w i t h the exception that i t was c a r r i e d out at room temperature. At r o o m temperature the r e a c t i o n proceeded at a slower r a t e as expected, but there was no n o t i c e a b l e change i n the product d i s t r i b u t i o n . Since i t was apparent that the r e a c t i o n could not be c a r r i e d out without the formation o f the very p o l a r components even at room temperature, the nature of the p o l a r compounds was i n v e s t i g a t e d f u r t h e r . The behavior on t h i n - l a y e r chromato-graphy and s o l u b i l i t y p r o p e r t i e s o f the mixture of these p o l a r compounds suggested that they were s a l t s . In f a c t these p r o p e r t i e s were reminiscent tff those possessed by the quaternary ammonium mesylate s a l t s 55 and 56 which w e r e prepared i n the course of the synth e s i s of quebrachamine and dihydro-cleavamine (see Figure 8) c a r r i e d out i n our l a b o r a t o r i e s . The u l t r a v i o l e t spectrum of the mixture of these compounds was almost p r e c i s e l y the same as those obtained f o r the quaternary ammonium mesylate s a l t s . A r a t i o n a l e c o u l d be made f o r the formation of a quaternary ammonium s a l t . Such a s a l t c ould c o n c e i v a b l y be formed by reaction' mode "g" or by " i " (Y = OAc) fol l o w e d by "g" as i n d i c a t e d i n Figure 31. To t e s t the conjecture that the - 102 -p o l a r mixture might c o n s i s t o f the two e p i m e r i c a l l y r e l a t e d quaternary s a l t s 131 and 132, a small sample of i t was subjected to treatment w i t h OAc OAc l i t h i u m aluminum hydride i n r e f l u x i n g N-methylmorpholine This procedure had been shown i n our l a b o r a t o r y t c r e s u l t i n the r e d u c t i o n of the quaternary s a l t s i n the quebrachamine s e r i e s to give a f a i r y i e l d of que-brachamine. In the present case the procedure r e s u l t e d i n the regeneration of some 48-dihydrocleavamine. In view of t h i s r e s u l t the mixture was assumed t o c o n s i s t of 131 and 132, although polymeric systems such as the dimer 133 were conceivable by i n t e r m o l e c u l a r r a t h e r than i n t r a m o l e c u l a r attack of n i t r o g e n . In t h i s regard, however, a study of molecular models 133 - 103 -of the e p i m e r i c a l l y r e l a t e d 18-acetoxy-4B-dihydrocleavamines, which were considered to be the most l i k e l y intermediates i n the formation of the quaternary s a l t s by analogy with the r e a c t i o n of the c h l o r o i n d o l e n i n e of 43-dihydrocleavamine with methanol that was discussed e a r l i e r , showed that s u i t a b l e conformations f o r i n t r a m o l e c u l a r attack by n i t r o g e n could be e a s i l y achieved. I t was a]so observed that i f the r e a c t i o n were to proceed by a d i r e c t displacement mechanism, the conformation 134 i n the case of the 188-epimer would lead to the quaternary s a l t 131 and the conformation 135 i n the case of the 18a-epimer would lead to the quaternary s a l t 132. There was good evidence from the nmr s t u d i e s which w i l l be mentioned l a t e r t h a t i n the fOAc H ft 134 135 case o f the 188-acetoxy epimer the conformation shown i n 134 would be the p r e f e r r e d conformation o f the molecule. Several attempts were made to show that the 18-acetoxy epimers were formed i n the r e a c t i o n o f the c h l o r o i n d o l -enine of 43-dihydrocleavamine with sodium acetate i n acetic, a c i d . I t was f e l t that the m a t e r i a l which possessed a s l i g h t l y l a r g e r r a t e of t r a n s p o r t on t h i n - l a y e r chromatography than the c h l o r o i n d o l e n i n e might c o n s i s t of one or a mixture o f the 18-acetoxy-43-dihydrocleavamine. Although elaborate - 104 -steps were taken to secure a pure sample of t h i s m a t e r i a l no success was achieved. A crude mixture of the d e s i r e d m a t e r i a l w i t h unreacted c h l o r o -i n d o l e n i n e and the quaternary s a l t s could t e obtained by a l l o w i n g the r e a c t i o n to proceed f o r 30 minutes, then r a p i d l y quenching the r e a c t i o n i n a r a p i d l y s t i r r e d mixture of methylene c h l o r i d e and aqueous ammonium hydroxide cooled to ice-acetone temperature, separating the organic phase, d r y i n g with anhydrous sodium s u l f a t e , and removing the solvent at room temperature i n a r o t a r y evaporator. Chromatography on alumina r e s u l t e d i n complete l o s s of the d e s i r e d m a t e r i a l and the formation of a new m a t e r i a l . Chromatography on s i l i c a g e l was p r e f e r a b l e . E l u t i o n w i t h 2% t r i e t h y l a m i n e i n e t h y l acetate provided a m a t e r i a l which was i n i t i a l l y f r e e of the quaternary s a l t s but only p a r t i a l l y f r e e of the c h l o r o i n d o l e n i n e . Some of the m a t e r i a l encountered i n the attempt to chromatograph the mixture on alumina was a l s o formed. The longer the m a t e r i a l was on the column the more the new m a t e r i a l formed. Consequently, a c a r e f u l chromatography could not be c a r r i e d out. In a d d i t i o n quaterization of p u r i f i e d m a t e r i a l occurred even as i t stood at room tempera-t u r e . Nevertheless, the u l t r a v i o l e t , i n f r a r e d , and nmr s p e c t r a of the p a r t i a l l y p u r i f i e d m a t e r i a l suggested the presence of an 18-acetoxy-4B-dihydrocleavamine. Since i t was obvious that o b t a i n i n g a pure sample of an 18-acetoxy-4B-dihydrocleavamine would be exceedingly d i f f i c u l t i f not i m p o s s i b l e , a t t e n t i o n was d i r e c t e d to the m a t e r i a l which was seen to be formed when the crude r e a c t i o n product was chromatographed. This newly formed m a t e r i a l must have a r i s e n from the m a t e r i a l whose r a t e of t r a n s p o r t on t h i n - l a y e r chromatography was seen to be s l i g h t l y g r e a t e r than that of the c h l o r o i n d o l e n i n e s i n c e chromatography of a pure sample of the c h l o r o -i n d o l e n i n e on alumina d i d not give r i s e to any of i t . I t was f e l t t h e r e f o r e - 105 -t h a t the new m a t e r i a l might be a mixture o f the two 18-hydroxy-4$-dihyc 1ro-cleavamines. In f a c t t h i s m a t e r i a l was s i n g l e compound. A l l the.evidence i n c l u d i n g that provided by the mass spectrum (Figure 39) was c o n s i s t e n t wi'.h • t h i s compound being an C-18-hydroxy epimer and on the b a s i s o f the nmr spectrum (Figure 40) i t was proposed t o be 188-hydroxy-48-dihydrocleavamine. The nmr spectrum showed a broad s i g n a l at x 1.4 which was a t t r i b u t e d t o the OH proton and a p o o r l y defined m u l t i p l e t at x 4.78 which was a t t r i b u t e d to the C-18 proton. Since i n the s p e c t r a o f r e l a t e d compounds the C-18 proton resonances occurred as a p a i r of doublets or i n some cases, a doublet, the p o o r l y d e f i n e d nature o f the m u l t i p l e t i n the nmr spectrum suggested t h a t the C-18 proton was coupled t o the OH proton. Therefore the deute r i o c h l o r o f o r m s o l u t i o n was shaken with dueterium oxide and the nmr spectrum rerun. The s i g n a l s a t t r i b u t a b l e t o the OH and NH protons disappeared and i n a d d i t i o n the m u l t i p l e t at x 4.78 became a d i s t i n c t doublet i n appearance. These r e s u l t s were e n t i r e l y c o n s i s t e n t w i t h the compound being an 18-hydroxy--4g-dihydrocleavamine. The reasons f o r a s s i g n i n g the 8 - c o n f i g u r a t i o n to the hydroxyl group i n t h i s compound w i l l be discussed l a t e r . In view o f the problems encountered i n the attempted p r e p a r a t i o n of an 18-acetoxy-48-dihydrocleavamine, i t seemed that p r e p a r a t i o n of a compound with a s u i t a b l e l e a v i n g group at C-18 f o r displacement by cyanide and not by the b a s i c n i t r o g e n would be d i f f i c u l t to achieve. Instead of pursuing t h i s problem, i t was decided to attempt to use the mixture of quaternary s a l t s 131 and 132, which could be obtained i n 85% y i e l d , to prepare an 18-cyano-4g~dihdyrocleavamine. There was reason to b e l i e v e that t h i s approach would be s u c c e s s f u l . In our l a b o r a t o r y a f a i r l y complete study of the r e a c t i o n of the quaternary s a l t s i n the quebrachamine s e r i e s with cyanide 901 -- 107 -- 108 -had been worked out which gave the d e s i r e d n i t r i l e s i n reasonable y i e l d . 72 In a d d i t i o n , Harley-Mason and coworkers has reported some success i n a s i m i l a r r e a c t i o n i n the dihydrocleavamine s e r i e s . Samples of the mixture of quaternary s a l t s 131 and 132 were allowed to react w i t h cyanide under a v a r i e t y of c o n d i t i o n s . The r e a c t i o n s were monitored by t h i n - l a y e r chromatography using f o r comparison purposes a sample of the 18-cyano-46-dihydrocleavamine prepared p r e v i o u s l y . The best y i e l d o f the compound which corresponded to the 18-cyano-48-dihydrocleavamine was obtained when a s o l u t i o n of the mixture of quaternary s a l t s and potassium cyanide i n dry dimethylformamide was r e f l u x e d i n a dry, oxygen-free n i t r o g e n atmosphere f o r 100 minutes. In t h i s manner a 24% y i e l d o f . t h i s compound was obtained a f t e r chromatography of the crude r e a c t i o n product on s i l i c a g e l . The compound was obtained i n c r y s t a l l i n e form from methanol. The s p e c t r a l c h a r a c t e r i s t i c s of t h i s compound i n c l u d i n g those of the mass spectrum (Figure 41) were c o n s i s t e n t with i t being the same as the 18-cyano-48-dihydrocleavamine which had been prepared b e f o r e , but which could only be obtained i n pure form a f t e r an extensive chromatographic separation procedure and then only i n sub m i l l i g r a m amounts. On the basis o f i t s nmr spectrum (Figure 42) which w i l l be discussed l a t e r t h i s compound was pro-posed to be 186-cyano-4B-dihydrocleavamine. Using the b e t t e r of the procedures developed p r e v i o u s l y f o r the conversion of the n i t r i l e to the corresponding carbomethoxy d e r i v a t i v e , a sample of pure 18B-cyano-43-dihydro-cleavamine was converted i n t o 183-carbomet.hoxy-48-dihydrocleavamine i n 53% y i e l d . . The stereochemical assignments concerning the c o n f i g u r a t i o n of the C-18 f u n c t i o n i n the s e v e r a l d e r i v a t i v e s of 43-dihydrocleavamine discussed above were made on the b a s i s of a comparison of the nmr s p e c t r a of these compounds T1 OQ C H • 0) RELATIVE ABUNDANCE O CO O O O O O CD O O O O o in tt> o rt-o o o 00 TO I o v< CO o I I &• &• o o 1—• CD < fa 3 H * 3 CD o 139 177<M-130) o o cn O -o CO o o 307 (M+) - 601 -gure 42. Nmr spectrum of 18B-cyano-43-dihydrocleavamine - I l l -and s e v e r a l others which were a v a i l a b l e i n our l a b o r a t o r y . The s h i f t s of the p e r t i n e n t resonances are given i n Table 1. Comparison o f the resonances Table 1 Compound C H 2 - - C H 3 ( T ) C1E H ( T ) r e f . 18a-carbomethoxy-4a--dihydrocleavamine (136) 9 33 6. 13 9,14,73 188- " -4a- 11 (137) 9 09 4. 53 73 18a- " -48- " (138) 9 45 6. 12 73 188- " -48- " (139) 9 12 4. 98 73 18a-methoxy-48-dihydrocleavamine (140) 9 46 5. 50 188- " " " (141) 9 11 4 76 188-hydroxy-48- " (142) 9 17 4 77 188-cyano-48- " (143) 9 10 ' 4 58 Vincadine (144) 9 16 6. 20 31,75 Vincaminorine (145) 9 33 3 75 31,74 Vincaminoreine (146) 9 10 6. 10 31,74 137, Y-COOMe; R=H, R'=CH CH • R"=H, R"' =11 139, Y=COOMe; R=CH?CH ; R'=H; R"=H; R" 1 =H 141, Y=0Me, R=CI1 CH ; R'=H; R"=H; R" 1 =H 142, Y=0H; R=CH CH ; R'=H; R"=H; R"' =11 143, Y=CN; R=CH CH • R'=H; R"=H; R"' =H 145, Y=C00Me; R=H; R'=M; R"=CH CH,; R"'=Me ^ 136, Y=C00Me; R=H; R'=CH CH ; R"=H; R"' =H 138, Y=COOMe; R=CH CH ; R'=H; R"=H; R'"=H 140, Y=OMe; R=CH.CH • R'=H; R"=H; R"' =H 144, Y=COOMe; R=H; R'=H; R"=CH CH ; R'"=H 146, Y=C00Me; R=H; R'=H; R"=CH CH ; RM,=Me - 112 -showed th a t whenever both C-18 epimers c f a p a r t i c u l a r dihydrocleavamine or quebrachamine d e r i v a t i v e were a v a i l a b l e the resonances of the C-18 proton i n the epimer i n which the s u b s t i t u e n t was assigned the 8 - c o n f i g u r a t i o n occurred downfield from the resonances o f the C-18 proton i n the 18a-substi-t u t e d epimer. The p o s i t i o n o f the resonances of the C-18 proton i n the a-epimers was that which would be considered as normal f o r the C-18 d e r i v a t i v e i n q u e s t i o n . I t was apparent that there must be some e f f e c t that was p e c u l i a r to the 3-epimers which caused a chemical s h i f t d i f f e r e n c e between the C-18 proton resonances o f x 0.74 to x 2.35. The magnitude of t h i s d i f f e r e n c e seemed to.be e x t r a o r d i n a r i l y l a r g e , but could be explained i f the appropriate conformations f o r the members of the two s e r i e s (18a and 186) were assumed. In the compounds having the 186 c o n f i g u r a t i o n f o r the s u b s t i t u e n t i t was seen t h a t i n only one o f the p o s s i b l e conformations could the C-18 proton come i n very c l o s e p r o x i m i t y to the b a s i c n i t r o g e n atom. In f a c t i n molecular models the hydrogen and n i t r o g e n atoms were seen to be i n such c l o s e p r o x i m i t y t h a t i t was apparent that i n these molecules there was l i k e l y to be a strong i n t r a m o l e c u l a r van der Waals i n t e r a c t i o n . Because of the c l o s e p r o x i m i t y of the C-18 proton and the b a s i c n i t r o g e n of the p i p e r i d i n e moiety i n the conformation shown, i t followed that the C-18 proton would resonate at lower frequency. Mokry and Kompis who o r i g i n a l l y 76 observed t h i s e f f e c t i n the epimeric p a i r , vincaminoreine (146) and v i n c a -c a n i n o r i n e (145), a l s o showed that these two compounds e x h i b i t e d very slow 77 r a t e s o f methiodide formation which could only be r a t i o n a l i z e d on the b a s i s t h a t these compounds possessed a conformation as shown i n 145 and 146 i n which the lone p a i r o f e l e c t r o n s on the b a s i c n i t r o g e n atom i s s t e r i c a l l y s h i e l d e d by the i n d o l i c bridge. There was a l s o a d i f f e r e n c e observed i n the p o s i t i o n o f the methyl - 113 -proton resonances between the two groups of d e r i v a t i v e s . In the case of the dihydrocleavamine d e r i v a t i v e s the 183 epimers showed the resonances due to the methyl proton of the e t h y l s i d e chain at lower f i e l d than the resonances shown by the 18a-epimers. In the case of the quebrachamine d e r i v a t i v e s , the opposite r e l a t i o n s h i p was shown. Whatever the e f f e c t which r e s u l t e d i n the observed d i f f e r e n c e i t d i d not seem to be dependent on the c o n f i g u r a t i o n of the 4-ethyl group. Another s t r i k i n g d i f f e r e n c e between the 18a and 183 groups of epimers was that the nmr s p e c t r a of the former group showed i n a l l the cases, which we have s t u d i e d , a m u l t i p l e t which corresponded to one proton and resembled a doublet i n the region x 6.0-7.0. No such doublet was observed i n t h i s r e g i o n i n the 18g-epimers and presumably i n t h i s group the equivalent s i g n a l s occur under the methylene envelope. The p o s i t i o n of t h i s "doublet" i n the a-epimers i s given i n Table 2. The i n t e r e s t i n g f e a t u r e of t h i s d i f f e r e n c e Table 2 P o s i t i o n of Compound approx. doublet 18a-carbomethoxy-4a-dihydrocleavamine 6.54 18a- " -48- " 6.73 18a-methoxy-4 8- " 6.72 vincadine 6.77 vincaminoreine 6.89 ' between the 18a- and 18 8-epimers was that the d i f f e r e n c e appeared to be independent of the c o n f i g u r a t i o n and s i t e o f the e t h y l s i d e chain and a l s o appeared to be independent of the nature of the s u b s t i t u e n t at C-18. The' nmr spectra obtained f o r the methoxy, hydroxy, and cyano d e r i v a t i v e s prepared v i a the c h l o r o i n d o l e n i n e of 4 6-dihydrocleavamine were c o n s i s t e n t - 114 -w i t h the p o s i t i o n o f s u b s t i t u t i o n being at C-18 and not at C-8. Although only one C-18 epimer has been synthesized i n the case of the 18-cyano-4g-dihydrocleavamine and 18-hydroxy-4B-dihydrocleavamine, i t was c l e a r from the nmr data that the s u b s t i t u e n t i s B i n these compounds. Now that reasonably s a t i s f a c t o r y methods had been developed f o r i n t r o -ducing a s u b s t i t u e n t at C-18 of the dihydrocleavamine system, our a t t e n t i o n turned t o the s y n t h e s i s of dimeric systems. Aside from being a n a t u r a l extension to the t o t a l s y n t h e s i s of Vinca a l k a l o i d s developed i n our labora-t o r i e s , the approach, i f i t were s u c c e s s f u l , would provide a convenient method f o r s y n t h e s i z i n g an almost u n l i m i t e d number of p o t e n t i a l a n t i c a n c e r agents besides the w e l l known n a t u r a l l y o c c u r r i n g dimers such as v i n c a l e u k o b l a s t i n e which have a n t i c a n c e r a c t i v i t y . Dimers of s p e c i f i c design would be u s e f u l f o r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p studes. The s y n t h e s i s of the dimers 115, 147 and 148 are described and evidence i n support of the proposed s t r u c t u r e s are presented i n t h i s t h e s i s . Because i t was a n t i c i p a t e d that mass spectrometry would p l a y an important r o l e i n the c h a r a c t e r i z a t i o n of any dimers formed, an attempt to prepare the dimer 115 which possesses no carbomethoxy. groups, was made before the s y n t h e s i s of any other dimers was undertaken. I t had been shown p r e v i o u s l y that dimers such as v i n c a l e u k o b l a s t i n e which possess a carbomethoxyl group and have high molecular weight and low v o l a t i l i t y tend t o undergo thermal r e a c t i o n s i n the i n l e t system of a mass spectrometer which r e s u l t s i n "molecular i o n " peaks being observed which are m u l t i p l e s of 14 mass u n i t s l a r g e r than the t r u e 78 79 molecular weight. ' These spurious peaks have been a t t r i b u t e d to thermal decomposition products which a r i s e through i n t e r r n o l e c u l a r methyl t r a n s f e r from a carbomethoxyl group to n i t r o g e n f o l l o w e d by Hofmann e l i m i n a t i o n . - 115 -c=o I R 147,.R=NHNH2; R'=H; R"=COOMe 148, 6,7 dihydro; R=OMe, R'=OAc; R"=COOMe 151, R=OMe; R'=Ac; R"=H 152, R=OMe; R'=Ac; R"=C00Me 80 D e a c e t y l v i n d o l i n e hydrazide (114) was prepared from v i n d o l i n e (149) by r e f l u x i n g i t w i t h anhydrous hydrazine f o r 3 hours. The cou p l i n g r e a c t i o n o f the d e a c e t y l v i n d o l i n e hydrazide w i t h the c h l o r o i n d o l e n i n e of 46-dihydro-cleavamine was c a r r i e d out i n r e f l u x i n g anhydrous methanolic h y d r o c h l o r i c 81 a c i d (prepared from a c e t y l c h l o r i d e (2,5 ml) and anhydrous methanol (100 ml) ) under a n i t r o g e n atmosphere f o r 3 hours. In t h i s manner a 77% y i e l d of the dimer 115 was obtained. This dimer proved to be v i r t u a l l y i n s o l u b l e i n methanol and was obtained i n c r y s t a l l i n e form from the crude r e a c t i o n mixture simply by washing w i t h hot methanol. R e c r y s t a l l i z a t i o n from ethanol provided an a n a l y t i c a l sample o f the compound, mp 190-192 ( r a p i d - l i t -114, R=NHNH2; R'=H 149, R=OMe; R'=Ac 150, 6,7-dihydro; R=OMe; R'=Ac ra t e o f h e a t i n g ) . A l l the evidence was i n accord w i t h t h i s compound being a dimer. The u l t r a v i o l e t spectrum (Figure 43) di s p l a y e d i n superimposition the c h a r a c t e r i s t i c absorptions of both an in d o l e and a di h y d r o i n d o l e system. The low r e s o l u t i o n mass spectrum (Figure 44) of t h i s compound e s t a b l i s h e d beyond doubt i s dimeric nature. High r e s o l u t i o n mass a n a l y s i s provided the e m p i r i c a l formula C^H^O^N (found: mol wt, 694.421; c a l c d mol wt, 694.421). These r e s u l t s c l e a r l y were i n accord with the dimer being a product of coupling between d e a c e t y l v i n d o l i n e and 48-dihydrocleavamine. Cleavage of the dimer i n r e f l u x i n g 2N aqueous h y d r o c h l o r i c a c i d i n the presence of reducing agents ( t i n and stannous c h l o r i d e ) provided 48-dihydrocleavamine and d e a c e t y l v i n d o l i n e hydrazide, the i d e n t i t i e s of which were e s t a b l i s h e d i n the usual manner (mp, t i c , comparison i r ) . I t was thus c l e a r that the dimer contained d e a c e t y l v i n d o l i n e hydrazide and 48-dihydrocleavamine as i n t a c t u n i t s . The s i t e of attachment i n each u n i t thus remained to be determined. The nmr spectrum (Figure 45). of the dimer provided the remaining - 117 -- 8T'i " - 119 -- 120 -i n f o r m a t i o n t h a t was r e q u i r e d to e s t a b l i s h the s i t e s of attachment. In f a c t the important resonances a r i s i n g from both halves of the molecule were e a s i l y d i s t i n g u i s h a b l e from each other and .'"".he nmr spectrum i n i t s e l f , v i r t u a l l y c o n s t i t u t e d a proof of s t r u c t u r e . The nmr s p e c t r a of the monomers are shown i n Figure 46 and 47. The important s i g n a l s i n the nmr spectra of d e a c e t y l v i n d o l i n e hydrazide, 43-dihydrocleavamine and the dimer are compared i n Table 3. Deuterium exchange caused the s i g n a l s a t t r i b u t e d t o the N-16', C-4 hydroxyl and C-3 hydroxyl protons i n the nmr spectrum of the dimer to disappear and the i n t e g r a l showed that the three hydrazide protons must occur i n the region above 6x. Several important features of the dimer were seen from a comparison of the nmr resonances l i s t e d i n Table 3. The s i g n a l s a t t r i b u t e d t o the C-15 proton i n d e a c e t y l v i n d o l i n e hydrazide were absent i n spectrum of the dimer. In a d d i t i o n , the s i g n a l s a t t r i b u t e d to the C-14 and C-17 protons, which i n the d e a c e t y l v i n d o l i n e hydrazide occurred as doublets, were found as sharp s i n g l e t s i n the dimer. The absence of s i g n a l s a t t r i b u t a b l e to the C-15 proton and the s i n g l e t nature of the C-14 and C-17 proton resonances could only be r a t i o n a l i z e d i f the d e a c e t y l -v i n d o l i n e hydrazide p o r t i o n of the dimer was coupled at the C-15 s i t e . The appearance of a "doublet" at T 5.60 i n the dimer i s c o n s i s t e n t w i t h the 43-dihydrocleavamine p o r t i o n of the dimer being coupled at the C-18 or C-8 s i t e . Since i n no experiment had i t been shown that the C-8 s i t e i n the c h l o r o -i n d o l e n i n e o f 43-dihydrocleavamine was a c t i v a t e d toward a t t a c k by nu c l e o p h i l e s and since i t had been demonstrated i n s e v e r a l experiments that the C-18 s i t e was a c t i v a t e d i n t h i s manner, i t was v i r t u a l l y beyond doubt that C-18 was the s i t e at which the 43-dihydrocleavamine p o r t i o n of the dimer was coupled to the d e a c e t y l v i n d o l i n e hydrazide p o r t i o n of the dimer. - 122 -- 123 -Table 3 compound ! • • _ • 116(R=H) 115 114 ^ - ^ s i g n a l s proton (s)"*-^._ chemical s h i f t ( ), shape, no. of protons, c o u p l i n g , constant C-21 H 9.28,t,3 9.34,t,3 C-21'H 9.16,t,3 9.18,t,3 C-19 H 7. 34,s , 1 7.36,s,l N-1 CH 3 7.30,s,3 7.28,s,3 C-2 H 6.66,s,l 6.58,s,l C-16 OCH3 6.17,s,3 6.28,s,3 C-4 H 6.00,s,l 5.90,s,l C-18* under methylene envelope 5.60,"d",l J=10cps C-6 H 4.35,d,l J ^ l O c p s U.24,m,2 / C-7 H 4.17, p a i r of m's,l J 7 j 6 = 1 0 c p s C-17 H 3.97,s,l 3.98,d,l J 1 7 j l 5 - 2 . 3 c p s C-15 H" 3.79, p a i r of d ' s , l J 1 5 iy=2-3cVs> J 1 5 ' l 4 = 8 - 5 c P s C-14 H 3.35,s,l 3.19,d,l J 1 4 ^ 1 5 = 8 . 5 c p s C-11 1 H to C-H' H 2.45-3.07,m,4 2.45-3.07,m,4 * N-16* H 2.24,s,l 1.75?,s,l C-4 OH 2.05?,s,l | 1.8,broad "s",2 C-3 OH 0.57?,s,l - 124 -In view of the success obtained i n the r e a c t i o n o f the c h l o r o i n d o l e n i n e of 46-dihydrocleavamine with d e a c e t y l v i n d o l i n e hydrazide, i t became of immediate i n t e r e s t to see i f the same r e a c t i o n c o n d i t i o n s would b r i n g about r e a c t i o n between the c h l o r o i n d o l e n i n e of a carbomethoxydihydrocleav-amine and d e a c e t y l v i n d o l i n e hydrazide. I f the coupling proved to be p o s s i b l e i n t h i s case, a dimer would be obtained which would possess a carbomethoxy group at C-18'. The n a t u r a l l y o c c u r r i n g dimers which have a n t i c a n c e r a c t i v i t y possess a carbomethoxy group at the C-18' p o s i t i o n and i t i s p o s s i b l e that, the a c t i v i t y of these compounds i s dependent on such a f u n c t i o n -a l i t y being present at that s i t e . Before such a c o u p l i n g r e a c t i o n could be t r i e d i t was necessary to prepare the c h l o r o i n d o l e n i n e of carbomethoxydihydrocleavamine. I t was p o s s i b l e through a s e r i e s of r e a c t i o n s worked out i n our l a b o r a t o r i e s to prepare i n good y i e l d 186-carbomethoxy-4B-dihydrocleavamine. Treatment of t h i s compound with t - b u t y l h y p o c h l o r i t e y i e l d e d a c h l o r o i n d o l e n i n e i n almost q u a n t i t a t i v e y i e l d . This m a t e r i a l was obtained as an amorphous s o l i d which r e a d i l y became a gum on standing i n a i r . Like the c h l o r o i n d o l e n i n e of 46-dihydrocleavamine t h i s compound r e s i s t e d c r y s t a l l i z a t i o n , but behaved as a s i n g l e compound. I n v e s t i g a t i o n by t h i n - l a y e r chromatography and s p e c t r a l a n a l y s i s f a i l e d to show that the m a t e r i a l was not a s i n g l e compound. I t was f e l t i n the case o f t h i s compound t h a t . i t might p r e f e r to be i n the a-methylene i n d o l i n e form 118 (R=C00Me) r a t h e r than i n the in d o l e n i n e form 117 (R=C00Me) because a l k a l o i d s such as v i n c a d i f f o r m i n e (60) possess the a-methylene i n d o l i n e form i n which the double bond i s i n conjugation with the e s t e r group. Since i t i s the form 118 (P^COOCHj) which can undergo s u b s t i t u t i o n by an SN ? 1 mechanism, i t would have been encouraging i f the - 125 -compound possessed that form. The nmr spectrum (Figure 48), however, was s t r i c t l y i n accord w i t h the s t r u c t u r e 117 (R=C00CH^) f o r t h i s compound. The spectrum showed a "doublet" at x 5.53 which was a t t r i b u t e d to-a C-18 proton and d i d not show a s i g n a l that could be a t t r i b u t e d to a proton on the i n d o l e n i t r o g e n . ' In a d d i t i o n the i n f r a r e d spectrum revealed an uncon-jugated e s t e r a b s o r p t i o n at 1727 cm * and the c h a r a c t e r i s t i c absorptions at 1612 cm ^ and 1575 cm * observed i n t y p i c a l c h l o r o i n d o l e n i n e s . The mass spectrum (Figure 49) of the compound was i n accord w i t h i t being a monochloro d e r i v a t i v e of carbomethoxydihydrocleavamine with molecular i o n peaks at m/e 35 37 374 ( CI) and m/e 376 ( C I ) . Regeneration of 18B-carbomethoxy-4B-dihydro-cleavamine on r e d u c t i o n e s t a b l i s h e d that the c h l o r i n a t i o n had not brought about any backbone rearrangements. The c h l o r o i n d o l e n i n e prepared from 18B-carbomethoxy-4B-dihydrocleavamine and d e a c e t y l v i n d o l i n e hydrazide were allowed to react under the c o n d i t i o n s above. A complex mixture of products was obtained. The di m e r i c product was i d e n t i f i e d by a combination o f t h i n - l a y e r chromatography, u l t r a v i o l e t spectroscopy and mass spectrometry. I s o l a t i o n of the d i m e r i c product was c a r r i e d out by p r e p a r a t i v e t h i n - l a y e r chromatography ( s i l i c a g e l , 3:1 methanol-water). A 36.5% y i e l d of dimer 147 -as an amorphous powder was obtained i n t h i s manner. The u l t r a v i o l e t spectrum d i s p l a y e d maxima character-i s t i c of the i n d o l e and d i h y d r o i n d o l e chromophores. The i n f r a r e d spectrum was very s i m i l a r to that obtained f o r the dimer 115, but showed an a d d i t i o n a l -absorption a t t r i b u t a b l e to an e s t e r group at 1726 cm *. The low r e s o l u t i o n mass spectrum (Figure 50) e s t a b l i s h e d the dimeric nature of the compound. I t was noted that the mass spectrum of t h i s compound, which has a carbo-methoxy group, showed a .series of spurious peaks spaced by m u l t i p l e s of 14 - 126 -100 80 o < 60 o 40 20 iiiililiiiiiiiiaifc t Hi 1911 I n l r ! •I I t I I 1. J I r i -i i i i l I i 1 i so 100 150 200 250 m/e 300 350 400. 10 r-8 -< 4 < 2 -cr MeO Me I OH CONHNHj L Myni •0«)rrlBh£:^lpriIiJiii;.1.Titf!fIIlijp tiiJljIfhi^^^ffn^^NrniinllcridjTrrifJfi^Ifllhir.t,:!::^!!^^ I I I I I I 1 1 I I I I f I liiiiin::i:[i 2 4 S X 5 4 ikn:ii5!!li»«»iire:.,«,rJii;^!«."!i:llU»:5lab i M h i njlilili n i* nts'lii 1 1 1 1 1 1 1 1 nil febr J L_ 450 500 550 600 m/e 650 700 750 Figure 50. Mass spectrum o f the dimer 147 - 129 -mass u n i t s above the expected molecular i o n peak at m/e 752. High r e s o l u -t i o n mass a n a l y s i s provided the emperical formula C^H^N^O^ (found mol wt, 752.427; c a l c d mol wt, 752.426) f o r the m/e 752 peak. Cleavage of the dimer i n r e f l u x i n g 2N aqueous h y d r o c h l o r i c a c i d i n the presence of t i n and stannous c h l o r i d e , provided a carbomethoxydihydrocleavamine which was i d e n t i f i e d as 18$-carbomethoxy-4B-dihydrocleavamine by t h i n - l a y e r chromatography and comparison of i n f r a r e d s p e c t r a . D e a c e t y l v i n d o l i n e hydrazide could not e f f e c t i v e l y be separated from the h y d r o l y s i s products but i t s presence, was demonstrated by t h i n - l a y e r chromatography. The nmr spectrum (Figure 51) of the dimer proved t o be i n v a l u a b l e i n e s t a b l i s h i n g i t s s t r u c t u r e . The important nmr s i g n a l s o f the dimer and those of 18B-carbomethoxy-48-dihydro-cleavamine (Figure 52) and d e a c e t y l v i n d o l i n e hydrazide are given f o r compar-i s o n purposes i n Table 4. Deuterium exchange made i t p o s s i b l e to account f o r the two hydroxyl protons, the i n d o l e NH proton and the three hydrazide protons. L i k e the hydrazide protons of the dimer 115, the three hydrazide protons o f t h i s dimer were shown to be i n the region above x 6.0 by a comparison of the i n t e g r a l s obtained before and a f t e r the deuterium exchange. I t was noted t h a t no observable s i g n a l s disappeared i n the nmr spectrum above x 6.0 and the i n t e g r a l above that r e g i o n showed only a gradual change. Thus i t appeared that the hydrazide protons occurred as a broad band or s e v e r a l broad bands above T 6.0. The same phenomenon was observed i n d e a c e t y l v i n d o l i n e hydrazide i t s e l f except i n t h i s case the decrease i n the i n t e g r a l over the range above x 6.0 corresponded to 7 protons. This was c o n s i s t e n t w i t h the compound e x i s t i n g as a dihydrate with formula ^22^30^4^4* 80 2H^0 which was proposed f o r i t by the L i l l y group. I t i s apparent t h a t these are only two "blanks" i n the column f o r the dimer 147 i n Table 4. These blanks correspond to the C-18 proton of the - 130 -- 131 -- 132 -Table < compound 139 K-7 114 "v^~~--^_^ s i g n a l s proton (s J~~-~»^_ Chemical s h i f t ( x ) , shape, no. of protons, coupling constants C-21 H 9. 20 i t , 3 9.34,c,3 C-21' 9.12,t,3 9.08,t,3 C-19 H 7.18,s,l 7.36,s,l N-1 CH 3 7.33,s,3 7.28,s,3 C-2 H . 6.64,s,l 6.58,s,l C-16 OCH3 6.18,s,3 6.28,s,3 C-18' C O O C H 3 6.39,s,3 6.30,s,3 C-18' H 4.9S, ud",l C-4 H 6.01,s,l 5.90,s,l C-6 H 4°.28,d,l J 6 , 7 = 1 ° C P S 1 ^4.24,m,2 C-7 H 4.10 p a i r of m's,1 C-17 H 4.07,s,l 3.98,d,l J 1 7 J L 5 = 2 . 3 c p s C-15 H 3.79,pair of d's, 1 JIS ]^7~2.3cps J 1 5 ; i 4 = 8 . 5 c p s C-14 H 3.06,s,l 3.18,d,l J 1 4 ) 1 5 = 8 . 5 c p s C-11* H to C-14' H 2.5-3.1,m,4 2 . 5-3.1,m,1 N-16' H 1.37,s,l 1.84?,s,l C-4 OH 1.00?,s,l 1 C-3 OH 0.73?,s,l - 133 -carbomethoxydihydrocleavamine p o r t i o n and to the C-15 proton of the d e a c e t y l -v i n d o l i n e p o r t i o n of the dimer. Assuming that the C-8' and C-18' proton "doublets" which would have t o be present i f the two halves of the dimer were coupled through the C-15 and C-8' s i t e s are not s h i f t e d under the methylene envelope (a very u n l i k e l y occurrence), the two halves must be coupled through the C-18' and C-15 s i t e s . The s u c c e s s f u l p r e p a r a t i o n of the dimer 115 and 147 demonstrated the u t i l i t y o f the r e a c t i o n sequence used. Two other dimers 151 and 152, which are the v i n d o l i n e c o n t a i n i n g analogues of these dimers, have been success-f u l l y prepared by other workers i n our l a b o r a t o r i e s and work i s i n progress which w i l l lead to d i m e r i c systems which possess oxygen c o n t a i n i n g f u n c t i o n s i n the p i p e r i d i n e r i n g of the cleavamine p o r t i o n of the dimers. The p r e p a r a t i o n of carbomethoxyvelbanamine (153) i s being a c t i v e l y pursued at the present time. A s u c c e s s f u l d i m e r i z a t i o n of t h i s compound through i t s c h l o r o i n d o l e n i n e w i t h v i n d o l i n e would provide v i n c a l e u k o b l a s t i n e or i t s C-18 epimer or both. Since the t o t a l synthesis of 6,7 d i h y d r o v i n d o l i n e (150) was being a c t i v e l y pursued i n our l a b o r a t o r i e s and the syntheses of 183-carbomethoxy 43-dihydrocleavamine had been accomplished, i t was of i n t e r e s t to b r i n g C O O M G 153 - 134 -about a coupling of these two compounds. In a d d i t i o n , the dimer obtained would be i n t e r e s t i n g f o r the purpose of b i o l o g i c a l t e s t i n g . The c h l o r o -i n d o l e n i n e o f 18S-carbomethoxy-43-dihydrocleavaminenand 6,7-dihydrovindoline (150), which was obtained by c a t a l y t i c r e d u c t i o n of v i n d o l i n e , were allowed to react under the usual c o n d i t i o n s . The pure dimer 148 was obtained by column chromatography i n 42% y i e l d . The expected u l t r a v i o l e t and i n f r a r e d s p e c t r a were obtained f o r t h i s compound. High r e s o l u t i o n mass a n a l y s i s of the peak at m/e 796 provided the formula ^45H5o°8^4 (^ounc* m o 1 w t j 796.441; c a l c d mol wt, 796.441) f o r the compound which was i n accord with the struc> t u r e . Cleavage of the dimer under c o n d i t i o n s which were designed t o favor r e t e n t i o n o f the e s t e r f u n c t i o n s (1.5N methanolic h y d r o c h l o r i c a c i d , t i n and stannous c h l o r i d e ) provided a mixture of cleavage products. These were i d e n t i f i e d by the usual means as 18B-carbomethoxy-4B-dihydrocleavamine, 18«-carbomethoxy-4B-dihydrocleavamine and 6,7-dihydrovindoline. The low r e s o l u t i o n mass spectrum and the nmr spectrum of the dimer 148 are shown i n Figures 53 and 54, r e s p e c t i v e l y . The p e r t i n e n t s i g n a l s of the dimer 148 18B-carbomethoxy-4g-dihydrocleavamine, and 6,7-dihydrovindoline are compared i n Table 5. The nmr evidence was c o n s i s t e n t with the two monomeric u n i t s being coupled between t h e i r r e s p e c t i v e C-18' and C-15 s i t e s . The p o s i t i o n of the proton resonances a r i s i n g from the methoxyl f u n c t i o n s were assigned a f t e r a comparison of the nmr s p e c t r a of the f i v e dimers which had been prepared i n our l a b o r a t o r i e s . . The assignment of the C-15 methoxyl protons i s undoubtedly c o r r e c t , but the assignment f o r the protons of the two methyl e s t e r s might a c t u a l l y be the r e v e r s e . There has been no d i s c u s s i o n of the stereochemistry of the C-18' s i t e i n the s y n t h e t i c dimers to t h i s p o i n t . Two c o n f i g u r a t i o n s about the C-18' carbon atom were considered to be p o s s i b l e i n each of the dimers prepared. - SSI -Figure 54. Nmr spectrum of the dimer 148 - 137 -Table 5 compound 139 148 150 "*^~-~^signals proton (s) "-~-»^^_ Chemical s h i f t ( T ) , shape, no. of protons, coupling constants C-21 H 9.47,t,3 9.52,t,3 C-21 1 H 9.12,t,3 9.04,t,3 C-4 O C O C H 3 7.92,s,3 7.94,s,3 C-19 H 7.84,s,l 7.90,s,l N-1 C H 3 7.48,s,3 7.42,s,3 C-2 H 6.39,s,l 7.32,s,l C-18' COQCHj 6.39,s,3 6.28,s,3 C-3 C O O C H 3 6.24,s,3 6.27?,s,3 C-16 OCH^ •6.18, s, 3 6.24?,s,3 C-18* H 4.98,"d",l C-4 H 4.4.3,s,l 4.38,s,l C-17 H 4.04,s,l 3.98,d,l C-15 H 3.73,pair o f d ' s , l C-14 H 3.08,s,l ' 3.12,d,l C - l l ' to C-14; H 2.5-3.l,m,4 2.54-3.1,m,4 N-16" H 0.07?,s,l C-3 OH 1 1.00?,s,l 0.10,s,l When the d i m e r i z a t i o n r e a c t i o n s were considered i t was f e l t t hat each r e a c t i o n would provide two e p i m e r i c a l l y r e l a t e d dimers. In none of the f i v e r e a c t i o n s which have been c a r r i e d out to t h i s time has a second dimer been detected. I t would appear on the bases of t h i s r e s u l t that the coupling r e a c t i o n s were s t e r e o s p e c i f i c . In t h i s regard i t was noted t h a t both C-18 - 138 -epimers of 18-methoxy-4 6-dihydrocleavamine were obtained when the c h l o r o -i n d o l e n i n e o l 46-dihydrocleavamine was allowed to r e a c t i n 1.5% methanolic h y d r o c h l o r i c a c i d f o r three hours under a n i t r o g e n atmosphere i n the absence of a v i n d o l i n e d e r i v a t i v e . A mixture of the two epimers was obtained i n 35% o v e r a l l y i e l d . Before the mixture was separated i t was determined by nmr that the r a t i o ^ o f the epimers was about 3:1 w i t h the 18a-epimer predominating. Although i t would r e q u i r e a d d i t i o n a l work to show whether t h i s product r a t i o has any r e a l s i g n i f i c a n c e , i t would appear that attack on the B-face of the intermediate 118 (R=H) (or an e q u i v a l e n t intermediate) was p r e f e r r e d . In the d i m e r i z a t i o n r e a c t i o n where the a t t a c k i n g group would be a bulky v i n d o l i n e moiety any s t e r i c preference would be expected to be enhanced. In the case of d i m e r i z a t i o n r e a c t i o n , however, i t was r e a l i z e d there was a d i s t i n c t p o s s i b i l i t y that the 18-methoxy-4B-dihydrocleavamines 82 were interm e d i a t e s . Harly-Mason and coworkers ' have r e c e n t l y reported a s u c c e s s f u l d i m e r i z a t i o n with the 18-hydroxydihydrocleavamine mixture 154 and v i n d o l i n e i n methanolic h y d r o c h l o r i c a c i d . The 18-methoxy d e r i v a t i v e s would be expected to react i n the same manner as the 18-hydroxy d e r i v a t i v e s of dihydrocleavamine. Without there being a c l e a r p i c t u r e of the mechanism of the d i m e r i z a t i o n r e a c t i o n , i t would be r i s k y to t r y and p i c k out the most l i k e l y c o n f i g u r a t i o n of the groups attached to the C-18' carbon atoms. HO 154 - 139 -In regard tc t h i s stereochemical problem the s y n t h e t i c dimer 143 and the 57 n a t u r a l l y o c c u r r i n g i s o l e u r o s i n e B must e i t h e r be the same compound or C-18' epimers. A f a i r l y r o u t i n e i n t e r r e l a t i o n of the s y n t h e t i c dimers and the i s o l e u r o s i n e s w i t h v i n c a l e u k o b l a s t i n e whose absolute c o n f i g u r a t i o n i s known from X-ray d i f f r a c t i o n a n a l y s i s , would be expected t o be p o s s i b l e . Such an i n t e r r e l a t i o n would provide the answer t o the stereochemical problem of the s y n t h e t i c dimers. The c h l o r o i n d o l e n i n e s of 43-dihydrocleavamine and 183-carbomethoxy-43-dihydrocleavamine have been shown to be extremely v e r s i t i l e substances f o r the p r e p a r a t i o n of a v a r i e t y of new dihydrocleavamine d e r i v a t i v e s d i s p l a y -ing s u b s t i t u t i o n at the C-18 s i t e . Undoubtedly, the most important of these compounds from a s y n t h e t i c and b i o l o g i c a l p o i n t of view are the dimeric compounds. As a whole the work presented i n t h i s t h e s i s gives a d d i t i o n a l support to the p o s t u l a t e of T a y l o r shown i n Figure 19. In the l i g h t of the work presented i n t h i s t h e s i s i t became apparent t h a t given the r i g h t c o n d i t i o n s s u b s t i t u t i o n adjacent to the a - p o s i t i o n of.any i n d o l e c o n t a i n i n g m a t e r i a l could probably be achieved v i a the c h l o r o i n d o l e n i n e . ( I I I . Exper imenta l M e l t i n g p o i n t s were determined on a K o f l e r b l o c k and are uncor rec t ed . The u l t r a v i o l e t s p e c t r a were recorded w i t h the use o f methanol as so lven t (unless o therwise s p e c i f i e d ) on a Cary 11 or Cary 14 r e c o r d i n g spec t ro -photometer. I n f r a r ed ( i r ) spec t r a were recorded on a P e r k i n - E l m e r Model 21 spectrophotometer and potass ium bromide p e l l e t s were used un less o therwise s p e c i f i e d . Nuc lear magnetic resonance (nmr) spec t ra were recorded us ing deu t e r i och lo ro fo rm as s o l v e n t at 100 Mcps on a V a r i a n HA-100 instrument by Mr . R. Bur ton and the chemical s h i f t s are g i v e n i n the T i e r s x s c a l e . Mass s p e c t r a were recorded on an A t l a s CH-4 or an AEI MS-9 mass spectrometer and h igh r e s o l u t i o n molecu la r weight de te rmina t ions were performed by e i t h e r Mr. G. E igendor f or Mr . G. Brown. Combustion analyses were c a r r i e d out by Mr . P. Borda. Woelm n e u t r a l s i l i c a g e l and alumina or S i l i c a Ge l G and Alumina G (accord ing to S t a h l ) c o n t a i n i n g 5% by wt o f Genera l E l e c t r i c Ratma p - 1 , Type 118-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and p r e p a r a t i v e t h i n - l a y e r chromatography ( t i c ) . Woelm n e u t r a l alumina and s i l i c a g e l ( a c t i v i t y I I I ) were used f o r column chromatography. Chromato-p l a t e s were developed u s i n g 2:1 carbon t e t r a c h l o r i d e - a n t i m o n y pen t ach lo r i de spray reagent . S o l u t i o n s were d r i e d e x c l u s i v e l y w i t h anhyd sodium s u l f a t e . Some o f the work was performed i n c o l l a b o r a t i o n w i th Dr . P. Le Quesne ( P . L . Q . ) , Mr . J . Beck ( J . B . ) and Mr . F . Bylsma. ( F . B . ) . - 141 -188-carbomethoxycleavamine (3,4 dehydro-139) ( i n part by JB) In an e f f i c i e n t fume hood a three-necked, round bottom, 1 I f l a s k was f i t t e d w i t h a r e f l u x condenser and a r e l i a b l e mechanical s t i r r e r . A piece of rubber t u b i n g l e a d i n g t o the fume hood a i r duct was connected to the top of the condenser. A c e t i c a c i d (300 ml) was introduced i n t o the f l a s k and heated to about 100°C. Catharanthine h y d r o c h l o r i d e (11.02 g) and a f u r t h e r p o r t i o n o f a c e t i c a c i d (100 ml) was introduced i n t o the f l a s k w i t h r a p i d s t i r r i n g . Immediately a p o r t i o n of sodium borohydride was added and a d d i t i o n a l p o r t i o n s were added i n r a p i d succession as soon as the vigorous r e a c t i o n , which f o l l o w e d each a d d i t i o n , had subsided. I t was intended by using t h i s procedure that some borohydride would always be present i n the s o l u t i o n . The r a t e of a d d i t i o n o f sodium borohydride which was used was s u f f i c i e n t l y slow that u n c o n t r o l l a b l e r e f l u x i n g d i d not occur, but s u f f i c -i e n t l y r a p i d t h a t the r e a c t i o n temperature was maintained i n the region 90°C-105°C. In t h i s manner a t o t a l of 42 g of sodium borohydride was added over a p e r i o d of an hour. A f t e r the a d d i t i o n was complete, the r e a c t i o n mixture was immediately cooled i n an ice-water bath. The r e a c t i o n mixture which became extremely viscous on c o o l i n g , was t r e a t e d w i t h 9 N ammonium hydroxide (800 ml) and e x t r a c t e d w i t h methylene c h l o r i d e (three 400 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t was d r i e d and the solvent was removed with the a i d of a r o t a r y evaporator and a vacuum pump to y i e l d a white foam (10.85 g) . The foam was taken up i n hot methanol (20 ml) and almost immediately 4.4 g of 18 8-carbomethoxycleavamine separated from the s o l u t i o n having mp 121-123°C (authentic sample 122-123°). A f u r t h e r 1.3 g of m a t e r i a l , ' which was shown to be almost pure 183-carbomethoxycleavamine by t i c , was obtained by concentrating and c h i l l i n g the methanol s o l u t i o n . - 14: -18 B-carbomethoxy-4 g-dihydrocleavamine^ (139) 183-carbomethoxycleavamine (4.4 g) wis hydrogenated at room temperature and atmospheric pressure over Adam's c a t a l y s t (368 mg) i n e t h y l acetate (51 ml). The uptake of hydrogen ceased a f t e r 130 minutes. The r e a c t i o n mixture was f i l t e r e d through a c e l i t e pad and solvent was removed to y i e l d a foam which c r y s t a l l i z e d on t r i t u r a t i o n w i t h methanol. A f t e r washing with methanol, 4.1 g of 188-carbomethoxy-48-dihydrocleavamine was obtained having mp 143-145°C (auth sample 146-148°C). A f u r t h e r 0.3 g with mp 142-145°C was obtained from the methanol washings. 73 43-dihydrocleavamine (116, R-H) A s o l u t i o n of 188-carbomethoxy-48-dihydrocleavamine (3.0 g) i n 5 N h y d r o c h l o r i c a c i d (191 ml) was heated at about 90°C i n a water bath f o r seven hours under a n i t r o g e n atmosphere. The s o l u t i o n was cooled i n an ice-water bath, made s t r o n g l y b a s i c by the a d d i t i o n of 15 N ammonium hydroxide and ex t r a c t e d w i t h methylene c h l o r i d e (three 125 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t was d r i e d w i t h anhyd sodium s u l f a t e , the solvent was removed and the residue c r y s t a l l i z e d once from hot methanol to give 2.4 g of 4 3-dihydrocleavamine with mp 134-138° (auth sample a f t e r s e v e r a l r e c r y s t a l l i z a t i o n s , mp 136-138). Oxidation of 43-dihydrocleavamine with t e r t - b u t y l h y p o c h l o r i t e A s o l u t i o n of 0.050 M t e r t - b u t y l h y p o c h l o r i t e (7.1 ml, 0.36 mmol) i n carbon t e t r a c h l o r i d e was added over a pe r i o d of 30 minutes to a s o l u t i o n of 43-dihydrocleavamine (100 mg, 0.36 mmol) and t r i e t h y l a m i n e (0.07 ml) i n methylene c h l o r i d e (13.3 ml) cooled i n an ice-acetone bath. A f t e r the a d d i t i o n was complete, the s o l u t i o n was s t i r r e d f o r a f u r t h e r 15 minutes at - 143 -the temperature of the ice-acetone bath. The orange coloured s o l u t i o n was then d i l u t e d with an equal volume of benzene and r a p i d l y p e r c o l a t e d through a column of alumina (1.5 g). The bulk of the solvent was removed at room temperature i n a r o t a r y evaporator and then the l a s t t r a c e s were removed i n vacuo to provide the c h l o r o i n d o l e n i n e 113 as a pale y e l l o w o i l (101 mg) : xxsooctane 2 2_^ 2 6 0 ( b r o a d ) , 303(broad) my. (log e 4.31, 3.55, 3.42, max CHC1 —1 r e s p e c t i v e l y ) : v 3 2770 (Bohlmann band), 1600 and 1560 cm ( i n d o l e n i n e r J max O N ) ; nmr T 2.5-3.1 ( d i f f u s e , 4H, aromatic p r o t o n s ) , 8.79 (approx. q u a r t e t , 2H, CH 2-CH 3) , 9.14 ( t r i p l e t , 3H, CH^CH^) ; mass spectrum ( A t l a s ) m/e ( r e l i n t e n s i t y ) 318(72), 316(88), 281(100), 138(46.5), 124(38). Anal. Calcd f o r C i g H 2 5 N 2 C l : mol wt, 316.171. Found: mol wt, 316.172 (mass spectrometry). Lithium aluminium hydride r e d u c t i o n of the c h l o r o i n d o l e n i n e 113 L i t h i u m aluminum hydride powder (5 mg) was added s l o w l y w h i l e s t i r r i n g to a s o l u t i o n of the c h l o r o i n d o l e n i n e (23 mg) i n anhyd d i e t h y l ether (20 ml). A f t e r 15 minutes e t h y l acetate (saturated with water) was added u n t i l gas e v o l u t i o : i ceased. The mixture was then f i l t e r e d and the f i l t r a t e d r i e d and evaporated to give a gummy residue (21 mg) which was chromatographed on alumina (2 g). E l u t i o n with 1:1 petroleum ether (bp 50-60°)-benzene provided 12.5 mg of a c r y s t a l l i n e m a t e r i a l , which on r e c r y s t a l l i z a t i o n from methanol gave 4g-dihydrocleavamine (116, R=H), mp 136-138°, i d e n t i c a l w i t h an a u t h e n t i c sample as shown by mp and mmp, comparison i r and t i c (alumina benzene and s i l i c a g e l , chloroform). Reaction of the c h l o r o i n d o l e n i n e 113 with potassium cyanide A s o l u t i o n of the c h l o r o i n d o l e n i n e 113 (559 mg), potassium cyanide (1.22 g ) , methanol (15.5 m l ) , water (1.67 ml) and d i e t h y l ether (3.34 ml) - 144 -was s t i r r e d under a n i t r o g e n atmosphere f o r 48 hours at room temperature. An aqueous potassium carbonate s o l u t i o n (10%, 25 ml) was then added and the s o l u t i o n e x t r a c t e d w i t h methylene c h l o r i d e (three 25 ml p o r t i o n s ) . The combined e x t r a c t s were d r i e d and evaporated to give a g l a s s y residue (491 mg). This residue was chromatographed on alumina (50 g). E l u t i o n w i t h benzene provided 118 mg of a mixture of compounds c a l l e d group A. T i c (alumina, benzene): chromatoplates showed one major spot which was blue i n colour w i t h a pink f r i n g e at 0.5. T i c ( s i l i c a g e l , 1:1 c h l o r o f o r m - e t h y l a c e t a t e ) : chromatoplates showed two major spots: one pink i n colour at R^ 0.9 and the other grey-blue at R, 0.1. X 294, 285, 278 (sh) , 225 ( i n d i c a t i v e of i n d o l e • ' f max v CHC1 -1 chromophore); v ^ 3410 ( NH), 2220 ( n i t r i l e group) cm max Further e l u t i o n i n the above chromatography with methylene c h l o r i d e (v o l %, from 12 to 30) i n benzene provided 140 mg over 42 f r a c t i o n s of a mixture of compounds c a l l e d group B: T i c (alumina 3:1 benzene-ethyl a c e t a t e ) : chromatoplates of e a r l y f r a c t i o n s of mixture showed one green-brown spot at R^ 0.5 and chromatoplates of l a t e f r a c t i o n s of mixture showed one red-brown spot at R^ 0.5. T i c ( s i l i c a g e l , 1:1 chloroform-ethyl a c e t a t e ) : chromatoplates showed one brown spot at R,, 0.5; A _ 290, 282, 278, 273, x max 222 ( i n d i c a t i v e o f mixture of chromophores i n c l u d i n g an i n d o l e chromophore); CHC1 v 3 3400 ( NH), 2220 (very s t r o n g , i n d i c a t i n g a conjugated n i t r i l e max group) cm Nmr s p e c t r a of s e l e c t f r a c t i o n s i n d i c a t e d the s p e c t r a were composed of s i g n a l s from three compounds: one w i t h d i s t i n g u i s h i n g s i g n a l s at r(approx) 6.65 ( s i n g l e t ) , 5.41 ( s i n g l e t ) , 1.63 ( s i n g l e t , exchangeable p r o t o n ) ; another with d i s t i n g u i s h i n g s i g n a l s at x (approx) 6.35 ( s i n g l e t ) , 1.45 ( s i n g l e t , exchangeable p r o t o n ) ; the t h i r d w i t h d i s t i n g u i s h i n g s i g n a l x (approx) 6.6 ( s i n g l e t . Group A was chromatographed on s i l i c a g e l (10 g). E l u t i o n with 3:1 benzene-chloroform provided a s e r i e s of f r a c t i o n s which contained 36 mg of a mixture of compounds that was i n i t i a l l y thought to be one compound: t i c ( f r e s h l y a c t i v a t e d s i l i c a g e l , chloroform) chromatoplates showed two over-l a p p i n g spots at Rp 0.3. P r e p a r a t i v e t i c ( f r e s h l y a c t i v a t e d s i l i c a g e l , chloroform) provided a sample o f both components of the mixture each of v/hich contained about 10% of the other component as an i m p u r i t y . The p a r t i a l l y p u r i f i e d sample having the l a r g e r R^ value had ^ m 2.93, 240, 210 mu; v 3300 and 2220 (both very weak and probably from contaminent) max r 1613 and 1595 cm"1 ( i n d o l e n i n e C=N?); nmr x 9.12 ( t r i p l e t , 3H, CH2CH_3) , 5.98 ( s i n g l e t , exchangeable proton, 1H, OH or NH), 2.8-3.5 ( d i f f u s e , 4H, aromatic p r o t o n s ) . Further p u r i f i c a t i o n was attempted without success. The p a r t i a l l y p u r i f i e d sample having the smaller Rf value had v m a x 293.5, 284.5, 272 (sh), 225 mp; v 3300 ( NH) 2670 (Bohlmann band), 2220 TTlclX (CN) cm"1. Nmr: x 9.10 ( t r i p l e t , 3H, CH2CH_3), 4.58 (doublet, 1H, C-18 p r o t o n ) j 2.45-3.0 ( d i f f u s e , 4H, aromatic p r o t o n s ) , 1.47 ( s i n g l e t , 1H, NH). The sample was subjected to r i g o r o u s p u r i f i c a t i o n by p r e p a r a t i v e t i c . F r e s h l y a c t i v a t e d s i l i c a g e l p l a t e s (5 x 20 cm, 0.25 mm t h i c k n e s s ) 'were used and chloroform was used as the t r a n s p o r t i n g s o l v e n t . The d e s i r e d band was scraped o f f and e l u t e d with e t h y l acetate to provide a sample of 18B-cyano-48-dihydrocleavamine (143) (ca. 1 mg). Mass spectrum (MS-9) m/e 307 (molecular i o n ) , 138 and 124. Anal. Calcd f o r c 2 n H 2 5 N 3 : m o 1 w t> 3 0 7 • 2 0 5 • Found: mol wt, 307, 205 (mass spectrometry). Further e l u t i o n i n the above chromatography with 2% t r i e t h y l a m i n e i n •acetone provided 40 mg of 18cx-methoxy-43-dihydrocleavamine (140) as a glass which became c r y s t a l l i n e i n form on standing. R e c r y s t a l l i z a t i o n of t h i s - 146 -compound frcm methanol provided a sample with mp 126-127°: X r 292, 284, 277 (sh), 225 i.<y (log z 3.86, 3.90, 3.86, 4.49, r e s p e c t i v e l y ) ; v 3280 ITlclX ( N-H), 2780 (3ohlmann band), 1070 (C-O-Me) cm"1; nmr x 1.63 ( s i n g l e t , IH, N-H), 2.45-3.2 ( d i f f u s e , 4H, aromatic p r o t o n s ) , 5.47 ( p a i r of doublets, IH, C-18 p r o t o n s ) , 6.80 ( s i n g l e t , 3H, COOCH3), 9.46 ( t r i p l e t , 311, CH2CH ). Mass spectrum: m/e ( r e l i n t e n s i t y ) 312(58), 281(16), 280(15), 187(6), 182(49), 138(100), 124(29). Anal. Calcd f o r C o r iH o oN„0: mol wt, 312.220. Found: mol wt, 312.220 2v 2o 2 (mass spectrometry) . 18B-carbomethoxy~46-dihydrocleavamine (139) v i a treatment of group A with  anhyd methanolic h y d r o c h l o r i c a c i d ( i n part by P.L.Q.) A s o l u t i o n of group A (113 mg) i n anhyd satura t e d methanolic hydro-c h l o r i c a c i d (35 ml) was heated under r e f l u x f o r 30 minutes, l e t stand over-night at room temperature, and heated again under r e f l u x f o r 4 1/2 hours. A f t e r the r e a c t i o n s o l u t i o n had been evaporated almost to dryness, i t was p a r t i t i o n e d between d i e t h y l ether and an aqueous s o l u t i o n of sodium carbonate. The organic l a y e r was separated, washed with water and d r i e d . Removal of the solvent provided a gummy residue (92 mg) which was chromato-graphed on alumina (4 g). E l u t i o n with benzene provided a mixture (30 mg) which contained 183-carbomethoxy-4B-dihydrocleavamine as a major component. This mixture was rechromatographed on alumina (2.5 g ) . E l u t i o n w i t h benzene provided s e v e r a l f r a c t i o n s c o n t a i n i n g 18B-carbomethoxy-48-dihydro-cleavamine. One of these f r a c t i o n s (3.3 mg) showed only one spot when subjected to an i n v e s t i g a t i o n on alumina chromatoplates (3:1 benzene-chloro-form, 3:1 benzene-ethyl a c e t a t e ) . Further i n v e s t i g a t i o n by t i c ( s i l i c a g e l , 1:1 c h l o r o f o r m - e t h y l acetate) showed that i t was a mixture of s e v e r a l - 147 -compounds. P u r i f i c a t i o n by p r e p a r a t i v e t i c u s i n g a s i l i c a g e l p l a t e (5 x 20 cm, 0.25 mm t h i c k n e s s ) and the above solvent system provided 18 6-carbomethoxy-46-dihydrocleavamine (0.7 mg) as shown by comparison t i c and mass sp e c t r a ( v i r t u a l l y i d e n t i c a l fragmentation p a t t e r n as an authe n t i c sample when both s p e c t r a were seen under the same c o n d i t i o n s on the A t l a s instrument). Anal. Calcd f o r C o l H o o N „ 0 o : mol wt, 340.215. Found: mol wt, 340.212 Zi Zo Z Z (mass spectrometry). 14 [22- C]-18B-carbomethoxy-46-dihydrocleavamine (139) v i a methanolysis of group A Group A (5.1 mg, 1.4 x 10 ^ mCi), which had been obtained by the method described above, was d i s s o l v e d i n an anhyd saturated methanolic h y d r o c h l o r i c a c i d s o l u t i o n (1 ml) and heated under r e f l u x f o r one hour under a n i t r o g e n atmosphere. Then, the solvent was r o t a r y evaporated to y i e l d a g l a s s y r e s i d u e which was p a r t i t i o n e d between methylene c h l o r i d e (20 ml) and a sa t u r a t e d aqueous sodium bicarbonate s o l u t i o n (5 ml). The orga n i c l a y e r was d r i e d and r o t a r y evaporated to give a gummy residue (1.2 mg). One-half (0.6 mg) of t h i s m a t e r i a l was mixed w i t h i n a c t i v e 186-carbomethoxy-46-dihydrocleavamine (5.4 mg) and the mixture was c r y s t a l l i z e d to constant a c t i v i t y (148 dpm/mg). The r a d i o a c t i v i t y of the 186-carbomethoxy 43-dihydrocleavamine was estimated t o be 7.1 x 10 mCi. Only 5% of the r a d i o a c t i v i t y i n group A was present a f t e r r e a c t i o n as 188-carbomethoxy-43-dihydrocleavamine. - 14c' -18g-carbomethoxy-4g-dihydrocleavamine (139) v i a treatment of group A-^  with  potassim hydroxide and then diazomethane A sample of group A^ (10.0 mg) was d i s s o l v e d i n a 20% s o l u t i o n (0.1 ml) of potassium hydroxide i n d i e t h y l e n e g l y c o l and heated at 150°C f o r 8 1/2 hours under a n i t r o g e n atmosphere. The s o l u t i o n was then allowed to cool to room temperature and d i l u t e d w i t h methanol (0.2 ml). This methanolic s o l u t i o n was cooled i n an ice-water bath and t r e a t e d w i t h a saturated s o l u t i o n of hydrogen c h l o r i d e i n methanol u n t i l i t had become s l i g h t l y a c i d i c as shown by i n d i c a t o r paper. An e t h e r e a l s o l u t i o n (1 ml) of diazomethane (ca. 20 mg) was added immediately and the r e s u l t i n g mixture was allowed to stand i n an ice-water bath f o r 15 minutes. In the same manner as above the mixture was r e - a c i d i f i e d and t r e a t e d w i t h excess diazomethane two more times before the ether, methanol and excess diazomethane were removed with the a i d of a n i t r o g e n stream and a warm water bath. The r e s i d u e obtained was shaken with an aqueous 10% s o l u t i o n of potassium carbonate (1 ml) and e x t r a c t e d w i t h d i e t h y l ether (three 5 ml p o r t i o n s ) . A f t e r the e t h e r e a l e x t r a c t had been d r i e d , the ether was removed to provide a viscous m a t e r i a l (14.5 mg) c o n t a i n i n g d i e t h y l e n e g l y c o l which was chromatographed on alumina (2 g). E l u t i o n w i t h 4:1 petroleum ether (bp 30-60°)-benzene provided 18g-carbomethoxy-4g-dihydrocleavamine (3.1 mg) which upon r e c r y s t a l l i z a t i o n from methanol had a mp of 145-148° and was found to be i d e n t i c a l with an a u t h e n t i c sample as shown by mp, mmp, comparison i r and t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , 1:1 chloroform e t h y l a c e t a t e ) . 14 [22- C]-18g-carbomethoxy-4g-dihydrocleavamine (139) v i a potassium hydroxide and diazomethane treatment of group A^  -4 Group A (10.15 mg, 6.76 x 10 mCi) was used and the above procedure - 149 -repeated. The crude reaction product (47 mg) was diluted with inactive 188-carbomethoxy-4B-dihydrocleavamine (10.3 mg) and chromatographed on alumina (2 g). Elution with 4:1 petroleum ether (bp 30-60°)-benzene 14 provided [22- C]-18B-carbomethoxy-4B-dihydrocleavamine (13.5 mg, 2.3 x 10~^ mCi). The r a d i o a c t i v i t y represented 35% of that present i n group A^. The reaction of the chloroindolenine 113 i n a methanolic solution of hydrogen  chloride and potassium cyanide An anhydrous methanolic solution (19.5 ml) containing 1.5% hydrogen chloride was added slowly with s t i r r i n g to a mixture of the chloroindolenine 113 (229 mg) and potassium cyanide (378 mg) i n a fl a s k which had been f i t t e d with an e f f i c i e n t condenser and was cooled i n an ice-water bath. Escaping hydrogen cyanide gas was passed into an aqueous potassium hydroxide solution and the entire experiment was carried out i n an e f f i c i e n t fume hood. After the addition of the methanolic hydrogen chloride had been completed, the re s u l t i n g solution was refluxed for three hours under a nitrogen atmosphere. Then the reaction solution was cooled i n an ice-water bath and s o l i d sodium carbonate was added u n t i l the solution was neutral to indicator paper. The solution was dilu t e d with water (29.5 ml), made quite basic by the addition of sodium carbonate and extracted with methylene chloride (five 20 ml portions). The methylene chloride solution was dried and rotary evaporated to y i e l d a glassy residue (219 mg). The major portion (196 mg) of this residue was chromatographed on alumina (20 g). Elution with 3:1 petroleum ether (bp 30-60°)-benzene provided a mixture (79 mg) of two compounds. A portion (62 mg) of t h i s mixture was chromatographed on s i l i c a gel (6 g). Elution with 1:1 chloroform-ethyl acetate provided a compound (15 mg) which immediately c r y s t a l l i z e d on t r i t u r a t i o n with methanol. - l i iO -R e c r y s t a l l i z a t i o n of t h i s compound fr o y methanol-diethyl ether provided a sample, mp 175-178° of 183-methoxy-43-dinydrocleavamine. (141): A m 294, 286, 279(sh), 227 (log e 3.85, 3.91, 3.87, 4.48.. r e s p e c t i v e l y ) ; v 3250 (N-H) 2780 (Bohlmann band)1075 cm"1 (C-O-Me); nmr x 1.70 ( s i n g l e t , IH, NH), 2.44-3.06 ( d i f f u s e , 4H, aromatic p r o t o n s ) , 4.76 ( p a i r of d o u b l e t s , IH, C-18 p r o t o n ) , 6.86 ( s i n g l e t , 3H, C00CH 3), 9.11 ( t r i p l e t , 3H, CH CH ). Mass spectrum ( A t l a s ) m/e ( r e l i n t e n s i t y ) 312(85), 281(22), 280(11), 187(100),' 182(35), 138(97), 126(35), 124(40). Anal. Calcd f o r C o o I L o N o 0 : mol wt, 312.220. Found: mol wt, 312.221 Zo Zo Z (mass spectrometry). E l u t i o n i n the above chromatography with 2% t r i e t h y l a m i n e i n acetone provided a compound (37 mg) which sl o w l y c r y s t a l l i z e d on standing. R e c r y s t a l -l i z a t i o n of t h i s compound from methanol provided a sample w i t h mp 126-127°, which was i d e n t i c a l (mp, nmr, i r , t i c ) with a u t h e n t i c 18a-methoxy-43-dihydrocleavamine (140). An a l . Calc f o r C o nH o oN„0: C, 76.88; H, 9.03; N, 8.97. Found: C, ZO Zo Z 77.13; H, 9.28; N, 8.75 (combustion). The r e a c t i o n of the c h l o r o i n d o l e n i n e 113 i n a s o l u t i o n of sodium i o d i d e i n acetone - an attempt to prepare an 18-iodo-43-dihydrocleavamine The c h l o r o i n d o l e n i n e 113 (79 mg) was converted i n t o h y d r o c h l o r i d e s a l t by d i s s o l v i n g i t i n ether-methylene c h l o r i d e and passing hydrogen c h l o r i d e gas over the surface of the s o l u t i o n . Removal of the solvent gave the s a l t as a white powder. Acetone (1 m l ) , which had been d r i e d by p e r c o l a -t i o n through and storage over Linde 4A molecular s e i v e s , was added to the s a l t and then a sodium iodide-acetone s o l u t i o n (2 m l ) , which had been prepared by d i s s o l v i n g sodium i o d i d e (6.6 g) i n dry acetone (30 ml). - 151 -Immediately upon the l a t t e r a d d i t i o n the s o l u t i o n became dark purple-brown i n c o l o u r . The coloured s o l u t i o n was s t i i r e d at room temperature f o r three hours under a dry oxygen f r e e ( F i e s e r ' s s o l u t i o n ) n i t r o g e n atmosphere. Then, the purple-brown coloured s o l u t i o n was d i l u t e d w i t h d i e t h y l ether (25 ml) and the r e s u l t i n g s o l u t i o n was washed with an aqueous s o l u t i o n of sodium t h i o s u l f a t e u n t i l i t had become c o l o u r l e s s . The aqueous washings were made s t r o n g l y b a s i c by the a d d i t i o n of potassium carbonate and were extracted w i t h methylene c h l o r i d e (three 10 ml p o r t i o n s ) . The methylene c h l o r i d e s o l u t i o n and the ether s o l u t i o n were combined and d r i e d . Removal of the s o l v e n t provided a. gummy resi d u e (77 mg) which was chromatographed on alumina (8 g). E l u t i o n with 3:1 petroleum ether (bp 30-60°)-benzene afforded 15.4 mg of a product which gave c r y s t a l s , mp 136-138°, from methanol and proved to be i d e n t i c a l with 4 6-dihydrocleavamine (116, R=H)-as shown by mp and mmp, comparison i r and t i c . The r e a c t i o n o f 18-methoxy-4g-dihydrocleavamine with boron t r i c h l o r i d e -an attempt to prepare a.n 18-chloro-43-dihydrocleavamine Boron t r i c h l o r i d e gas was condensed i n a f l a s k which had been cooled i n a dry ice-acetone bath. A s o l u t i o n of a mixture (5 mg) of 18a-methoxy-48-dihydrocleavamine and 183-methoxy-48-dihydrocleavamine ( r a t i o of the former compound to the l a t t e r , 3:1) i n anhydrous methylene c h l o r i d e (1 ml) was added sl o w l y to the l i q u i d boron t r i c h l o r i d e . A f t e r the r e a c t i o n s o l u t i o n had stood at the dry-ice-acetone bath temperature f o r ten minutes under a dry n i t r o g e n atmosphere, the methylene c h l o r i d e and excess boron t r i c h l o r i d e . w a s removed on a r o t a r y evaporator. The r e s i d u e obtained was mixed thoroughly wi t h a mixture of an aqueous sodium bicarbonate s o l u t i o n (1 ml) (saturated) and d i e t h y l ether (10 ml). The ether phase was separated - 152 -and replaced with methylene c h l o r i d e (10 ml). Methylene c h l o r i d e was mixed thoroughly w i t h the mixture of aqueous sodium bicarbonate and undissolved r e s i d u e . Both the ether and methylene c h l o r i d e e x t r a c t s were d r i e d and evaporated s e p a r a t e l y to y i e l d 1.9 mg and 2.3 mg, r e s p e c t i v e l y , of a gummy resi d u e . I n v e s t i g a t i o n by t i c o f these residues i n each case f a i l e d to show tl i e presence of unreacted m a t e r i a l , but d i d show that each mixture was composed of a minimum of f i v e compounds. The r e a c t i o n o f 18-methoxy-4g-dihydrocleavamine with aqueous 1.5% h y d r o c h l o r i c  a c i d - an attempt to prepare a 18-hydroxy-4B-dihydrocleavamine A s o l u t i o n of a 3:1 mixture (5 mg) of 18a-methoxy-43-dihydrocleavamine t o 188-methoxy-43-dihydrocleavamine i n 1.5% h y d r o c h l o r i c a c i d (0.5 ml) was s t i r r e d under a n i t r o g e n atmosphere at room temperature while a l i q u o t s were taken p e r i o d i c a l l y and analysed by t i c (alumina, e t h y l a c e t a t e ) . I t appeared that the 18-methoxy-4g-dihydrocleavamines were being s l o w l y hydrolysed to a s e r i e s of more p o l a r compounds of which the amount o f " b a s e l i n e " m a t e r i a l increased p r o g r e s s i v e l y . Only a small amount of un-reacted m a t e r i a l remained as shown by t i c a f t e r 3 1/2 hours. The r e a c t i o n o f the c h l o r o i n d o l e n i n e 113 i n a s o l u t i o n of sodium acetate i n a c e t i c a c i d - the p r e p a r a t i o n of a mixture of the quaternary n i t r o g e n  acetate s a l t s 131 and 132. A s o l u t i o n of the c h l o r i n d o l e n i n e 113 (101 mg) i n a g l a c i a l a c e t i c a c i d s o l u t i o n (6.5 m l ) , which contained 10% fused sodium acetate by weight, was heated at 60° f o r two hours under a n i t r o g e n atmosphere. The r e a c t i o n s o l u t i o n was then poured i n t o a mixture of 15 N ammonium hydroxide (8 ml) and methylene c h l o r i d e (26 ml) w i t h r a p i d s t i r r i n g . The organic phase was separated and saved and the aqueous phase was made s t r o n g l y b a s i c by the - 153 -a d d i t i o n o l 15 N ammonium hydroxide, s a t u r a t e d w i t h ammonium acetate and ex t r a c t e d w i t h methylene c h l o r i d e (three 10 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t s were then combined and d r i e d . Removal of the solvent provided 93 mg of a white powder: t i c (alumina., 3:1 e t h y l acetate -ethanol) : chromatoplat.es showed two overlapping green coloured spots at R r 012; X 220, 270, 280, 289 (cf A 220, 270 (sh) , 280, 289 f o r mixture f max max 55 .and X 226, 273(sh), 282, 289 f o r mixture 56). max J The r e a c t i o n of the c h l o r o i n d o l e n i n e 113 i n a s o l u t i o n o f sodium acetate i n  a c e t i c a c i d - an attempt to o b t a i n an 18-acetoxy-4$-dihydrocleavamine  (134 and/or 135) A s o l u t i o n of the c h l o r o i n d o l e n i n e 113 (367.5 mg) i n a g l a c i a l a c e t i c a c i d s o l u t i o n (23.3 m l ) , which contained 10% fused sodium acetate by wt, was heated at 60°C f o r 30 minutes under a n i t r o g e n atmosphere. Then, the r e a c t i o n s o l u t i o n was immediately poured i n t o a r a p i d l y s t i r r e d mixture of 15 N ammonium hydroxide (30 ml) and methylene c h l o r i d e (93 ml) which was maintained at low temperature i n an ice-acetone bath. The organic phase was separated and saved. A f u r t h e r p o r t i o n of methylene c h l o r i d e (46.5 ml) was added to the aqueous phase and while the mixture was being r a p i d l y s t i r r e d at the temperature of the ice-acetone bath, the wealkly b a s i c aqueous phase was made s t r o n g l y b a s i c by the a d d i t i o n of 15 N ammonium hydroxide. The org a n i c phase was separated and combined w i t h the p r e v i o u s l y separated organic phase. The combined s o l u t i o n was d r i e d and r o t a r y evaporated to give a gummy resi d u e (416 mg) which was chromatographed almost immediately on s i l i c a g e l (20 g). E l u t i o n w i t h e t h y l acetate provided 67 mg of m a t e r i a l over s e v e r a l f r a c t i o n s . Each f r a c t i o n was shown to c o n t a i n the c h l o r o i n d o l e n i n e 113 and another compound(s) b e l i e v e d to be - 154' -an 18-acetoxy-4$-dihydrocleavamine. The s p e c t r a l p r o p e r t i e s of the purest f r a c t i o n s were determined: X 292(sh), 284, 278 (sh), 226 ( s l i g h t l y d i s t o r t e d i n d o l e spectrum); v C H C l 3 (PE 137), 3300 (i n d o l e NH), 1720 cm"1 rn9.x ( e s t e r O O); nmr T 1.72 ( s i n g l e t , NH), 2.5-3.2 ( d i f f u s e , aromatic p r o t o n s ) , 3.97 ( p a i r of doubl e t s , C-18 p r o t o n ) , 7.96 ( s i n g l e t , CH^COO), 9.15 ( t r i p l e t , CH CH3) ( s i g n a l s which could be a t t r i b u t e d to an acetoxy-43-dihydrocleavamine). Further e l u t i o n i n the above chromatography with 3% t r i e t h y l a m i n e i n e t h y l acetate gave a f u r t h e r 116 mg of m a t e r i a l c o n s i s t i n g mainly of the c h l o r o i n d o l e n i n e 113 and the compound(s) b e l i e v e d to be an 18-acetoxy-48-dihydrocleavamine. F i n a l l y , the column was washed with 5% a c e t i c a c i d i n methanol and 1:1 methanol-water. The washings were combined, r o t a r y evapor-ated to g i v e a residue which was t r e a t e d w i t h a saturated aqueous ammonium acetate s o l t u i o n (10 ml) c o n t a i n i n g ammonium hydroxide and e x t r a c t e d with methylene c h l o r i d e (three 25 ml p o r t i o n s ) . The combined e x t r a c t s were d r i e d and evaporated to provide 164 mg of a mixture of compounds which c o n s i s t e d mainly of the quaternary ammonium s a l t s 131 and 132 as shown by t i c . A mixture (65.5 mg) c o n t a i n i n g the a l l e g e d 18-acetoxy-4g-dihydrocleav-amine from the chromatography above was chromatographed on alumina (6 g ) . E l u t i o n w i t h 1:1 petroleum ether (bp 30-60°)-benzene gave 27 mg of a mixture which was shown by t i c to conta i n the c h l o r o i n d o l e n i n e 113 as the major component. E l u t i o n w i t h 1:1 benzene-ethyl acetate gave 14.3 mg of 183-hydroxy-43-dihydrocleavamine (142). R e c r y s t a l l i z a t i o n from methanol provided a sample, mp 202-205°: X 292, 284, 278(sh), 226 my (log e 3.86, 3.91, 3.87, 4.50, r e s p e c t i v e l y ) ; v 3200 (broad, NH and OH), 2700 cm"1 1113. X (Bohlmann band); nmr T 1.42 (broad s i n g l e t , IH, OH), 2.19 ( s i n g l e t , IH, NH) , 2.5-3.15 ( d i f f u s e , 4H, aromatic protons) 34 .77 (unresolved m u l t i p l e t becoming a doublet a f t e r treatment of nmr sample with D-0, 111, C-18 p r o t o n ) , 9.17 ( t r i p l e t , 3H, CH2CH ); mass s p e c t r i n ( A t l a s ) m/e ( r e l i n t e n s i t y ) 298(46), 281(13.5), 280(13.5), 173(11.5), 168(43), 138(100), 124(51). Anal. Calcd for•C._Ho,N„0: mol wt, 298.205. Found: mol wt, 29S.205. 1 y 26 2. Reaction of the mixture of the quaternary ammonium s a l t s 131 and 132 with  l i t h i u m aluminium hydride i n N-methylmorpholine A mixture (25 mg) of s a l t s 131 and 132 and l i t h i u m aluminium hydride (101 mg) were allowed to react i n r e f l u x i n g anhyd N-methylmorpholine (10 ml) under a dry, oxygen f r e e n i t r o g e n atmosphere. A l i q u o t s (ca. 0.5 ml) were taken p e r i o d i c a l l y . Each a l i q u o t was t r e a t e d with e t h y l acetate (saturated with water) u n t i l gas e v o l u t i o n ceased. The mixture was f i l t e r e d and the residue washed w i t h methylene c h l o r i d e (ca. 2 ml). The f i l t r a t e and wash-ings were combined, d r i e d , and evaporated with the a i d o f a n i t r o g e n stream and a hot water bath. The residue was examined by t i c (alumina, 3:1 benzene-chloroform and 3:1 e t h y l a c e t a t e - e t h a n o l , and s i l i c a g e l , chloroform). A f t e r one hour the presence of 4 8-dihydrocleavamine was detected. A f t e r 4 1/2 hours, the chromatoplates on development showed a major spot which corresponded i n c o l o u r and R^ value t o a spot from an au t h e n t i c sample of 48-dihydrocleavamine (116, R=H). 188-cyano-48-dihydrocleavamine (143) A mixture of the quaternary ammonium s a l t s 131 and 132 (73.3 mg) was allowed to react with potassium cyanide (56 mg)' i n r e f l u x i n g dimethylform-amide (9 ml) f o r 1 2/3 hours under a ni t r o g e n atmosphere. The solvent was then removed by d i s t i l l a t i o n at reduced pressure (50°C and 6 mm of Hg). The residue obtained was t r e a t e d w i t h 6 N ammonium hydroxide (1 ml) and the aqueous mixture was ext r a c t e d with methylene c h l o r i d e (three 5 ml p o r t i o n s ) . - 156 -The e x t r a c t was then d r i e d and the solvent removed to p r o v i d e a gummy . residue (64.1 mg). The r e s i d u e was then chromatographed on s i l i c a g e l (7 g). E l u t i o n w i t h 1:1 benzene-chloroform provided 15.7 mg of 186-cyano-43-dihydrocleavamine which gave c r y s t a l s from methanol w i t h mp 150-152°C: A 294, 285, 277, 226 mp (log e 3.86, 3.93, 3.89, 4.44, r e s p e c t i v e l y ) ; IftclX v 3300 (NH), 2760 (Bohlmann band), 2220 cm"1 (CN); nmr x 1.72 ( s i n g l e t , IT13-X IH, NH), 2.48-3.04 ( d i f f u s e , 4H, aromatic p r o t o n s ) , 4.58 ( p a i r of d o u b l e t s , IH, C-18 p r o t o n ) , 9.10 ( t r i p l e t , 3H, CH 2CH 3); mass spectrum (MS-9) m/e ( r e l i n t e n s i t y ) 307(32), 281(<3), 280(<3), 182(14), 177(77), 138(100), 124(75) . Anal. Calcd f o r C 2 0 H 2 5 N $ : mol wt, 307.205. Found: mol wt, 307.205 (mass spectrometry). 18B-carbomethoxy-43-dihydrocleavamine (139) from 18g-cyano-4g-dihydrocleav- amine (143) A s o l u t i o n of 188-cyano-43-dihydrocleavamine (5.21 mg) i n a s o l u t i o n (0.05 ml) of d i e t h y l e n e g l y c o l c o n t a i n i n g 20% KOH by wt was heated at 150°C f o r nine hours under a n i t r o g e n atmosphere. Then, the s o l u t i o n was allowed to c o o l to room temperature and d i l u t e d with methanol (0.1 ml). While t h i s new s o l u t i o n was kept c o o l i n an ice-water bath, a s a t u r a t e d s o l u t i o n of methanolic h y d r o c h l o r i c a c i d was added t o i t u n t i l i t had become s l i g h t l y a c i d i c to t e s t w i t h i n d i c a t o r paper. A s o l u t i o n (0.5 ml, approx cone 20 mg/ml) of diazomethane i n ether was immediately added and the r e s u l t i n g mixture was allowed t o stand i n an ice-water bath f o r 15 minutes. In the same manner as above the r e a c t i o n mixture was r e - a c i d i f i e d and t r e a t e d with excess diazomethane two more times before the ether, methanol and excess diazomethane were removed w i t h the a i d of a n i t r o g e n stream and a warm - 157 -water bath. The residue obtained was shaken w i t h an aqueous 10% potassium carbonate s o l u t i o n and e x t r a c t e d w i t h d i e t h y l ether. A f t e r the e t h e r e a l e x t r a c t had been d r i e d , the ether was removed to provide a v i s c o u s m a t e r i a l (23 mg) which was mainly d i e t h y l e n e g l y c o l . This m a t e r i a l was chromato-graphed on alumina (1 g). E l u t i o n w i t h 4:1 petroleum ether (bp 30-60°)-benzene provided 18B-carbomethoxy-43-dihydrocleavamine (2.97 mg). R e c r y s t a l -l i z a t i o n provided a sample, mp 146-148, which was i d e n t i c a l w i t h an a u t h e n t i c sample of 18B-carbomethoxy-48-dihydroc!eavamine as shown by mp,mmp, comparison i r and t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , 1:1 c h l o r o f o r m - e t h y l a c e t a t e ) . Oxidation of 188-carbomethoxy-4B-dihydrocleavamine (139) w i t h t e r t - b u t y l h y p o c h l o r i t e ( i n p a r t by FB § JB) A s o l u t i o n of 188-carbomethoxy-48-dihydrocleavamine (400 mg) and t r i -ethylamine (0.2 ml) i n methylene c h l o r i d e (40 ml) was cooled i n an i c e -water bath. While the s o l u t i o n was being s t i r r e d under a n i t r o g e n atmosphere a s o l u t i o n (250 ml, 0.05 M) of t e r t - b u t y l h y p o c h l o r i t e i n carbon t e t r a c h l o r i d e was added over a p e r i o d of 45 minutes.. Then the s o l u t i o n was washed with ice-water (two 30 ml p o r t i o n s ) , d r i e d and r o t a r y evaporated at room temp-erature t o give the c h l o r o i n d o l e n i n e 117 (R=C00Me) as an amorphous s o l i d (440 mg): x d l 0 x a n e 2 9 2 , 275, 227 my ( l o g e 3.44, 3.44, 4.30, r e s p e c t i v e l y ) ; 1113. X v ™ 1 3 2775 (Bohlmann band), 1727 (ester OO) , 1612 and 1575 cm"1 (indolenine O N ) ; nmr x 2.40-2.98 ( d i f f u s e , 4H, a r o m a t i c ) , 5.53 (doublet, 1H, C-18 p r o t o n ) , 6.41 ( s i n g l e t , 3H, COOCH^), 9.14 ( t r i p l e t , 3H, CH CH ); mass spectrum (MS-9) m/e ( r e l i n t e n s i t y ) 376(8), 374(22), 138(100), 124(85). Anal. Calcd f o r c 2 1 i l 2 7 N 2 ° 2 C 1 : m o 1 w t ' 3 7 4 - 1 7 6 - Found: mol wt, 374.174 (mass spectrometry). - 15cJ -C a t a l y t i c r e d u c t i o n of the chloroindolei.'ine 117 (R=C00Me) The c h l o r o i n d o l e n i n e 117 (R=COOMe)mg) was hydrogenated over Adam's c a t a l y s t (37 mg) i n e t h y l acetate (10 ml) a" room temperature and atmospheric pressure f o r one hour. Then, the r e a c t i o n mixture was f i l t e r e d through a c e l i t e pad and the pad was washed with methanol. The methanol and e t h y l acetate s o l u t i o n s were combined and evaporated. The m a t e r i a l obtained was d i s s o l v e d i n methylene c h l o r i d e and washed with a satura t e d aqueous potassium carbonate s o l u t i o n . The methylene c h l o r i d e s o l u t i o n was then d r i e d w i t h anhyd sodium s u l f a t e and evaporated t o give 19 mg of a m a t e r i a l , which on r e c r y s t a l l i z a t i o n from methanol gave 188-carbomethoxy-43-dihydrocleavamine, mp 146-148° i d e n t i c a l w i t h an aut h e n t i c sample as shown by mp and mmp, comparison i r and t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , 1:1 ch l o r o f o r m - e t h y l a c e t a t e ) . D e a c e t y l v i n d o l i n e hydrazide (114) About 8 ml of anhydrous hydrazine was d i s t i l l e d from sodium hydroxide i n t o a round bottom f l a s k c o n t a i n i n g v i n d o l i n e (1.03 g) . The mixture was r e f l u x e d under a dry n i t r o g e n atmosphere f o r three hours. (The foregoing steps were c a r r i e d out i n a fume hood behind a s h i e l d . ) Then the r e a c t i o n s o l u t i o n was allowed to cool to room temperature and methylene c h l o r i d e (30 ml) was added w i t h s t i r r i n g . Next water was added with s t i r r . i n g i n a dropwise f a s h i o n to the s o l u t i o n u n t i l i t separated i n t o two phases. The upper hydrazine hydrate phase was separated from the lower methylene c h l o r i d e phase and e x t r a c t e d with an a d d i t i o n a l amount of methylene c h l o r i d e . The methylene c h l o r i d e s o l u t i o n s were combined and shaken with water, which was added a drop at a time, u n t i l a f l u f f y white c r y s t a l l i n e m a t e r i a l (228 mg) was seen to suddenly separate. A f t e r t h i s m a t e r i a l had been - 159 -separated Ly f i l t r a t i o n , the methylene.chloride s o l u t i o n was washed with an a d d i t i o n a l s v a l l amount of water and d r i e d . Removal of the solvent gaA^e deacetylvindol."ne hydrazide as a white powder (687 mg) which gave c r y s t a l s O A from hot 95% ethanol w i t h mp 130-180° ( l i t . mp 130-180°): [ a ] 2 2 +17.3 (CHC1,) ( l i t . 8 0 [ a ] " +18.6); A 308, 248(sh), 213 my (log e3.71, 4.11, o u iHcix 4.66, r e s p e c t i v e l y ) , v 3380, 3260, 3170 (NH and OH), 2810 (Bohlmann ITtclX band), 1650 and 1615 cm"1 (CONHNH^); nmr T 1.80 (broad, 2H, C-4 OH and C-5 OH), 3.19 (doublet, IH, C-14 p r o t o n ) , 3.79 ( p a i r of d o u b l e t s , IH, C-15 p r o t o n ) , 3.98 (doublet, IH, C-17 p r o t o n ) , 5.90 ( s i n g l e t , IH, C-4 p r o t o n ) , 6.28 ( s i n g l e t , 3H, OCHj), 7.28 ( s i n g l e t , 3H, N-CH3), 9.34 ( t r i p l e t , 3H, CH^t^) . Anal. Calcd f o r C 2 2 H 3 0 ° 4 N 4 : m o 1 w t j 4 1 4 • 2 2 7 • Found: mol wt, 414.228 (mass spectrometry). The dimer 115 A s o l u t i o n of the c h l o r o i n d o l e n i n e 113 (601 mg) and d e a c e t y l v i n d o l i n e hydrazide (521.5 mg) i n an anhydrous methanolic 1.5% h y d r o c h l o r i c a c i d s o l u t i o n (52 ml) was r e f l u x e d under a dry n i t r o g e n atmosphere f o r three hours. A f t e r t h i s p e r i o d of time had passed and the s o l u t i o n had been allowed to cool to room t e m p e r a t u r e } i t was d i l u t e d w i t h water (78.5 ml) and made s l i g h t l y b a s i c by the a d d i t i o n of sodium carbonate. The b a s i c s o l u t i o n was e x t r a c t e d w i t h methylene c h l o r i d e (three 130 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t was d r i e d and the s o l v e n t was removed to y i e l d 985.5 mg of a powder. Some of t h i s powder (894.5 mg) was washed s e v e r a l times with hot methanol to provide 543.6 mg of small white c r y s t a l s . Concentration of the methanol washings provided a f u r t h e r 18.2 mg of the same c r y s t a l l i n e m a t e r i a l . This m a t e r i a l d i s p l a y e d r a t h e r unusual met l i n g p r o p e r t i e s . When the - 160 -temperature was r a i s e d slowly (ca. 2°/min) no me l t i n g point, could be observed but r a t h e r a p r o g r e s s i v e darkening begining at about 190°C. When the temperature was r a i s e d r a p i d l y (ca. 10°/min) the sample appeared to melt and immediately s o l i d i f y i n the range 189-194°C. R e c r y s t a l l i z a t i o n from 95% ethanol provided a sample which melted and s o l i d i f i e d i n the same manner i n the range 190-192°C: X 309(sh), 294, 285, 263, 224 (sh ) , m 3.x 215 mu (log e 3.85, 4.04, 4.05, 4.17, 4.60, 4.64 r e s p e c t i v e l y ) ; v 3400 1T13.X and 3280 (NH and OH), 1668 and 1616 cm"1 (C0NHNH2); nmr x 2.45-3.07 ( d i f f u s e , 4H, aromatic protons of cleavamine p o r t i o n ) , 3.35 ( s i n g l e t , 1H, C-14 proton of v i n d o l i n e p o r t i o n ) , 3.97 ( s i n g l e t , 1H, C-17 proton of v i n d o l i n e p o r t i o n ) , 5.60 (doublet, 1H, C-18 proton of cleavamine p o r t i o n , J=10cps), 9.18 ( t r i p l e t , 3H, CH2CH of cleavamine p o r t i o n ) , 9.40 ( t r i p l e t , 3H, CH2CH of the v i n d o l i n e p o r t i o n ) . Anal-Calcd f o r C, ..H^O.N • mol wt, 694.421.- Found: mol wt, 694.420. 41 54 4 6 Calcd f o r C 4 1 H 5 2 0 4 N 6 CM+"2) •' m o 1 w t> 692.405. Found: 692.403 (mass spectrometry). Cleavage of the dimer 115 A mixture of the dimer 115 (51 mg), t i n (205 mg), stannous c h l o r i d e d i hydrate (205 mg) and 2 N h y d r o c h l o r i c a c i d (10 ml) was r e f l u x e d f o r two hours under a n i t r o g e n atmosphere. A f t e r t h i s p e r i o d of time had passed and the mixture had been cooled to room temperature, a saturated aqueous s o l u t i o n of potassium carbonate was added u n t i l the mixture was b a s i c . The mixture was e x t r a c t e d with methylene c h l o r i d e ( f i v e 5 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t contained a f i n e white suspension which was e f f e c t i v e l y removed by c e n t r i f u g a t i o n . The methylene c h l o r i d e c e n t r i f u g a t e was d r i e d and r o t a r y evaporated to give 41 mg of a brown coloured residue which was - 161 -chromatographed on alumina (4 g). E l u t i o n with 1:1 petroleum ether (bp 30-60°)-benzene provided a m a t e r i a l (8 mg) which on c y r s t a l l i z a t i o n from methanol gave 43-dihydrocleavamine (116, R-H), mp 135-138, i d e n t i c a l with an aut h e n t i c sample as shown by mp, mmp, comparison i r and t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , chloroform). E l u t i o n w i t h methanol provided a m a t e r i a l (22 mg) which on c r y s t a l l i z a t i o n from ethanol-water and r e c r y s t a l l i z a t i o n from 95% ethanol provided a sample that was shown to be i d e n t i c a l w i t h d e a c e t y l v i n d o l i n e hydrazide by comparison i r and t i c ( s i l i c a g e l , 95% ethanol and alumina, 95% etha n o l ) . The dimer 147 ( i n p a r t by JB) A s o l u t i o n of d e a c e t y l v i n d o l i n e hydrazide (294 mg) and the chlo r o -i n d o l e n i n e 117 (R=C00Me)(386 mg) i n an anhydrous methanolic 1.5% h y d r o c h l o r i c a c i d s o l u t i o n (2.9 ml) was r e f l u x e d f o r three hours under a dry nit r o g e n atmosphere. A f t e r t h i s p e r i o d o f time had passed and the s o l u t i o n had been allowed to co o l to room temperature, the s o l u t i o n was d i l u t e d w i t h water (44 ml) and made s l i g h t l y b a s i c by the a d d i t i o n of sodium carbonate. The ba s i c s o l u t i o n was ex t r a c t e d with methylene c h l o r i d e (three 75 ml p o r t i o n s ) . The methylene c h l o r i d e e x t r a c t was d r i e d and the solvent removed to give a powdery m a t e r i a l (621 mg). Some of t h i s m a t e r i a l (409.5 mg) was subjected to s e p aration by p r e p a r a t i v e t i c . S i l i c a g e l (Woelm) p l a t e s (20 x 20 cm, 0.5 mm th i c k n e s s ) were used, with about 60 mg of m a t e r i a l being a p p l i e d to each p l a t e . A f t e r t r a n s p o r t w i t h 3:1 methanol-water, the d e s i r e d band was scraped o f f each p l a t e and e x t r a c t e d , f i r s t with methanol at room tempera-tu r e and then w i t h b o i l i n g methanol. The solvent was then removed and the residue was extracted with methylene c h l o r i d e . F i l t r a t i o n o f the methylene c h l o r i d e e x t r a c t s and removal of the solvent provided 118 mg of the dimer - 162 -147 as an amorphous s o l i d : X 313(sh), 296, 290, 269, 218 my (log e 3.88, IllciX 4.05, 4.06, 4.11, 4.69, r e s p e c t i v e l y ) ; v „ 3410 and 3290 (NH and OH), 1726 H i d X ( e s t e r C=0), 1663 and 1615 cm"1 (C0NHNH2); nmr x 2.48-3.10 ( d i f f u s e , 4H, aromatic protons on cleavamine p o r t i o n ) , 3.06 ( s i n g l e t , 1H, C-14 proton of v i n d o l i n e p o r t i o n ) , 4.07 ( s i n g l e t , 1H, C--17 proton on v i n d o l i n e p o r t i o n ) , 6.30 ( s i n g l e t , 3H, COOCH^ at C-18' of cleavamine p o r t i o n ) , 9.08 ( t r i p l e t , 3H, CH2CH_3 of cleavamine p o r t i o n ) , 9.20 ( t r i p l e t , 3H, CH2CH_^ of v i n d o l i n e p o r t i o n ) . Anal. Calcd f o r C y 1 7H r.0,N, : mol wt, 752.426. Found: mol wt, 752.427 43 56 6 6 (mass spectrometry). Cleavage of the dimer 147 (by JB) A mixture of the dimer 147 (13 mg), t i n (750 mg), stannous c h l o r i d e dihydrate (750 mg) and 2 N h y d r o c h l o r i c a c i d (25 ml) was r e f l u x e d f o r one hour. The mixture was then d i l u t e d with water, cooled and n e u t r a l i z e d with sodium bicarbonate. The s o l i d m a t e r i a l was separated by f i l t r a t i o n . Both the s o l i d and f i l t r a t e were e x t r a c t e d with chloroform. The chloroform e x t r a c t was d r i e d and the sol v e n t removed to give a res i d u e (9 mg). This residue was subjected to sep a r a t i o n by p r e p a r a t i v e t i c . A s i l i c a g e l (Woelm) p l a t e (5 x 20 cm, 0.5 mm) was used. A f t e r t r a n s p o r t with 1:1 e t h y l acetate-acetone, the d e s i r e d bands were scraped o f f and e x t r a c t e d with methanol. In t h i s manner 2 mg of a m a t e r i a l was obtained which was shown to be 18g-carbomethoxy-4B-dihydrocleavamine (139) by comparison ( t i c and i r ) with an authentic sample. Another 3 mg of a m a t e r i a l was obtained which was impure but di s p l a y e d the t i c p r o p e r t i e s of d e a c e t y l v i n d o l i n e hydrazide The dimer 148 ( i n p a r t by FB § JB) A s o l u t i o n of 6,7-dihydrovindoline (150) and the c h l o r o i n d o l e n i n e 117 - 163 -(R=COOMe) (.440 mg) i n anhydrous methanolic 1.5% h y d r o c h l o r i c a c i d (57 ml) was r e f l u x e d f o r three hour's under a dry n i t r o g e n atmosphere. The solvent was then removed i n a r o t a r y evaporator and the residue was d i s s o l v e d i n methylene c h l o r i d e (100 ml). Water (100 ml) was added and then potassium carbonate w i t h mixing u n t i l the mixture was b a s i c . The organic phase was then separated from the aqueous phase and the aqueous phase was extracted w i t h an a d d i t i o n a l q u a n t i t y of methylene c h l o r i d e (two 30 ml p o r t i o n s ) . A f t e r the methylene c h l o r i d e e x t r a c t s had been combined and d r i e d , the s o l -vent was removed to give a gl a s s y residue (694.4 mg). The re s i d u e was chromtographed on alumina (70 g). E l u t i o n with 4:1 benzene-diethyl ether provided 248.4 mg of the dimer 148 as an amorphous s o l i d : ^ 307(sh), 295, 287, 263, 217 my (log e 4.01, 4.14, 4.15, 4.19, 4.64, r e s p e c t i v e l y ) ; v n i c i x 3430 (NH and OH), 1735 cm"1 (ester C=0); nmr x 2.54-3.10 ( d i f f u s e , 4H, aromatic protons of cleavamine p o r t i o n ) , 3.08 ( s i n g l e t , IH, C-14 proton of v i n d o l i n e p o r t i o n ) , 4.04 ( s i n g l e t , IH, C-17 proton of v i n d o l i n e p o r t i o n ) , 6.28 ( s i n g l e t , 3H, C00CH_3 at C-18' on cleavamine p o r t i o n ) , 9.04 ( t r i p l e t , 3H, CHCH^ o f cleavamine p o r t i o n ) , 9.47 ( t r i p l e t , 3H, CH^CH^ of v i n d o l i n e p o r t i o n ) . Anal. Calcd f o r C./CH,_0oN. : mol wt, 796.441. Found: mol wt, 796.441 46 60 8 4 (mass spectrometry). Cleavage of the dimer 148 A mixture o f the dimer 148 (50.1 mg), t i n (100 mg), stannous c h l o r i d e d i hydrate (100 mg) and anhyd 1.5 N (6.5%) methanolic h y d r o c h l o r i c a c i d (10 ml) was r e f l u x e d f o r one hour under a n i t r o g e n atmosphere. Then, a f t e r the mixture had been cooled to room temperature, an aqueous 10% potassium carbonate s o l u t i o n (15 ml) was added and the r e s u l t i n g mixture was extr a c t e d - 1 6 4 -w i t h methylene c h l o r i d e ( f i v e 10 ml pori'ions) . The e x t r a c t was cen t r i f u g e d to p r e c i p i t a t e , the suspended white s o l i d and the c l e a r c e n t r i f u g a t e was d r i e d . Removal o f the solvent provided a g-immy residue (55 mg) . The residue was subjected to chromatography on alumina (5 g ) . E l u t i o n with 4:1 petroleum ether (bp 30-60°)-benzene provided 4.2 mg of a c r y s t a l l i n e compound. R e c r y s t a l l i z a t i o n of t h i s compound provided a sample w i t h mp 146-148°C which was i d e n t i c a l with an au t h e n t i c sample of 188-carbomethoxy-4g-dihydro-cleavamine (139) as shown by mp and mmp, comparison i r and t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , 1:1 chloroform-ethyl a c e t a t e ) . Further e l u t i o n i n the above chromatography with 4:1 petroleum ether (bp 30-60°)-benzene provided 8.0 mg of a m a t e r i a l which could not be induced to c r y s t a l l i z e but was shown to be i d e n t i c a l w i t h 18a-carbomethoxy-48-dihydrocleavamine (138) by comparison i r and t i c (above systems). Further e l u t i o n i n the above chromatography with 4:1 benzene-chloroform provided 25.6 mg of a foam which was shown to be i d e n t i c a l w i t h an authentic sample of 6,7-dihydrovindoline (150) by comparison i r and t i c ( s i l i c a g e l , e t h y l acetate and the above systems). - 165 -References 1. E. 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Chem. 30, 1550 (1965). 55. J . W. Moncrief and W. N. Lipscomb, J . Amer. Chem. Soc. 87, 4963 (1965). 56. N. Neuss, M. Gorman, H. E. Boaz, and N. J . Cone, J . Amer. Chem. Soc. 84, 1509 (1962). 57. N. Neuss, M. Gorman, N. J . Cone and L.L. Huckstep, Tetrahedron L e t t e r s , 783 (1968). 58. W. 0. Godfredsen and S. Vangeldal, Acta. Chem. Scand. 10, 1414 (1956). 59. N. Finch and W. I. T a y l o r , J . Amer. Chem. Soc. 84, 5871 (1962). 60. H. Zinnes and J . Shavel, J r . , J . Org. Chem. 31, 1765 (1966). 61. N. Gorman, N. Neuss and R. J . Cone, J . Amer. Chem. Soc. 87, 93 (1965). - 168 -62. J . Shavel, J r . , and H. Zinnes, J . Amer. Chem. Soc. 84, 1320 (1962); see a l s o r e f . 60. 63. N. Finch and W. I. Ta y l o r , J . Amer. Chem. Soc. 84, 3871 (1962); _84, 1318 (1962); see als o IV. I. T a y l o r , e t . a l . i b i d . 87, 2229 (1965). 64. S. Markey, K. Biemann and B. Witkop, Tetrahedron L e t t e r s , 157 (1967). 65. R. G. Pearson and J . Songstad, J . Amer. Chem. Soc. 89, 1827 (1967). 66. B. M i l l e r , J . Amer. Chem. Soc. 88, 1841 (1966). 67. L. J . Dolby and G. W. G r i b b l e , J . Org. Chem. 32, 1391 (1967). 68. S. G. P. Plant and M. L. Tomlinson, J . Chem. S o c , 955 (1933). 69. P. Povlock, Tetrahedron L e t t e r s , 4131 (1967). 70. R. D. Youssefyeh and Y Mazur, Chem. Ind. (London), 609 (1963). 71. L. J . Dolby and S. Sa k a i , Tetrahedron 23, 1 (1967). 72. J . Harley-Mason and Atta-ur-Raman, Chem. Commun., 208 (1967). 73. James P. Kutney, Walter J . Cretney, John H a d f i e l d , Ernest S. H a l l and Vern R. Nelson, p r e s e n t l y being submitted f o r p u b l i c a t i o n . 74. I s o l a t i o n of the compounds and nmr sp e c t r a at 100 Mcps by Vern Nelson. 75. Prepared by C. Gle t s o s . 76. J . Mokry and I. Kompis, L l o y d i a 27, 428 (1964). 77. J . Mokry, I. Kompis, M. Shamma and R. J . Sine, Chem. Ind. (London), 1988 (1964). 78. G. Buchi, R. E. Manning and S. A. Monti, J . Amer. Chem. Soc. 86, 4631 (1964). 79. P. Bommer, W. McMurray, and K. Biemann, J . Amer. Chem. Soc. 86, 1439 (1964); K. Biemann, L l o y d i a 27, 397 (1964). 80. N. Neuss, M. Gorman and N. J . Cone, L l o y d i a 27, 389 (1964). 81. L. F. F i e s e r and M. F i e s e r , "Reagents f o r Organic S y n t h e s i s " , John Wiley and Sons, Inc., 1967, p. 668. 82. J . Harley-Mason and Atta-ur-Raman, Chem. Commun., 1048 (1967). PART C STUDIES RELATED TO THE BIOSYNTHESIS OF INDOLE ALKALOIDS I. I n t r o d u c t i o n Since the beginning of t h i s decade, a considerable amount of e f f o r t by s e v e r a l groups of workers has been expended i n the d i r e c t i o n of s o l v i n g the mystery of the b i o s y n t h e s i s of the i n d o l e a l k a l o i d s . A f a i r l y complete b i o s y n t h e t i c scheme has emerged, although much of the scheme i s s t i l l s p e c u l a t i v e i n nature. .Tracer s t u d i e s have e s t a b l i s h e d many key steps i n the b i o s y n t h e s i s . Some key steps, which have yet to be confirmed by t r a c e r s t u d i e s , have been performed under l a b o r a t o r y c o n d i t i o n s and t h e r e i n derive support. Other key s t e p s , which have been proposed, have de r i v e d support from the i s o l a t i o n of new a l k a l o i d s or other n a t u r a l compounds whose s t r u c -tures c l o s e l y resemble proposed intermediates. In some cases a unique s t r u c t u r e , which i s possessed by a n a t u r a l compound, has led to new and more v i a b l e p o s t u l a t e s . In t h i s s e c t i o n of t h i s t h e s i s are d e s c r i b e d t r a c e r experiments which were undertaken i n an attempt to v e r i f y a key step proposed by Wenkert i n h i s scheme f o r the b i o s y n t h e s i s of a l k a l o i d s possessing the Iboga and Aspidosperma types of s k e l e t o n . 1 This key s t e p , which involved a transannular c y c l i z a t i o n , was supported by s e v e r a l s u c c e s s f u l l a b o r a t o r y 2-5 conversions performed by Kutney and coworkers , which were described i n some d e t a i l i n the i n t r o d u c t i o n to part B of t h i s t h e s i s . A l k a l o i d s possessing the Iboga and Aspidosperma types of skeleton are of the tryptamine + Cg ^^ u n i t s t r u c t u r a l type; that i s , they can be envisaged to a r i s e from the union of tryptamine and a u n i t that contains - 17.1 -6 1 9 or 10 carbon atoms. There are severa' hundred ' i n d o l e a l k a l o i d s which f i t t h i s d e s c r i p t i o n . Nevertheless, a clo s e look at the nature of the Cg_io u n i t i n these a l k a l o i d s r e v e a l s that there are only three b a s i c g m o d i f i c a t i o n s o f the carbon backbone. One m o d i f i c a t i o n (1) ( i n which the dotted l i n e represents the bond cleaved when the C I Q u n i t becomes the Cg u n i t ) of the Cg_ ng u n i t i s found i n a l k a l o i d s which are ex e m p l i f i e d as a group by corynantheine (2) and stry c h n i n e (3). The second m o d i f i c a t i o n (4) of the Cg.-^ Q u n i t i s found i n a l k a l o i d s e x e m p l i f i e d as a group by tabersonine (5). The t h i r d m o d i f i c a t i o n (6) of the Cg_^^ u n i t i s found i n a l k a l o i d s e x e m p l i f i e d as a group by co r o n a r i d i n e (7). I t can a l s o be seen that the two m o d i f i c a t i o n s 4 and 6 are r e l a t e d q u i t e simply to the mo d i f i c a -t i o n 1. M o d i f i c a t i o n s 4 and 6 could be envisaged as being derived from m o d i f i c a t i o n 1 at some stage i n the b i o s y n t h e s i s by a tr a n s f o r m a t i o n such as "a" i n the case of 4 and "b" i n the case of 6 as shown i n Figure 1. - 172 -Figure 1. D e r i v a t i o n of Aspidosperma and Iboga types 'of backbone from the Corynanthe type of backbone - 173 -The i n d o l e p o r t i o n of a l k a l o i d s of the tryptamine + Cg 1 Q type and, indeed, a l l o*:her i n d o l e a l k a l o i d s would l o g i c a l l y be d e r i v e d i n part from tryptophan (8) or i t s d e c a r b o x y l a t i o n product tryptamine (9). Tracer 9 s t u d i e s have borne out t h i s conjecture and i n a l l cases r e p o r t e d tryptophan has been shown t o be a d i r e c t p r e c u r s o r of i n d o l e a l k a l o i d s . As might be expected s e v e r a l hypotheses concerning the b i o s y n t h e s i s of the Cg_^Q p o r t i o n i n i n d o l e a l k a l o i d s were proposed. The e a r l i e s t scheme, which can be con-s i d e r e d as the Barger^^-Hahn1'''' ^ -Robinsons-Woodward 1^ ' ^  hypothesis a f t e r the workers who proposed or elaborated on the scheme, i n v o l v e d dihydroxy-phenylpyruvic a c i d (10) or an e q u i v a l e n t compound and two C^ u n i t s i n the production of the non-tryptamine p o r t i o n of a l k a l o i d s of the corynanthine-s t r y c h n i n e type. The e s s e n t i a l d e t a i l s o f t h i s scheme as i t emerged are shown i n Figure 2. Several f e a t u r e s of the scheme are worthy of note. I t was i m p l i e d t h a t the d i r e c t p r e c u r s o r s • o f the c o r y n a n t h i n e - s t r y c h n i n e group of a l k a l o i d s were aromatic i n t h e i r non-tryptamine p o r t i o n . Two C^ u n i t s were re q u i r e d at d i f f e r e n t stages i n the s y n t h e s i s . Since a l k a l o i d s of the corynanthine-strychnine type are c h a r a c t e r i z e d by constant stereochemist at C-15, a s t e r e o s p e c i f i c hydrogenation of the aromatic r i n g was r e q u i r e d . In 1959 1^' 1'' Wenkert proposed that the condensing u n i t with tryptamine might be derived from a d i r e c t precursor of the p h e n y l p y r u v i c a c i d of the - 1.74 -i^Ohh o^-condensaiion | g Q I - > + a H V° CH , 0 ^-condensation v N ^ H N 21 fS OH OH Robinson ring expansion to . tropolone with C, unit or Woodward cleavage + C, unit Figure 2. Barger-Hahn-Robinson-Woodward hypothesis - 175 e a r l i e r theory. At tha t time the sequence s h i k i m i c a c i d (11) -~y prephenic a c i d (12) —>• 4-hydroxyphenylpyruvic a c i d (13) —>• 3,4-dihydroxyphenyl-pyruvic" a c i d (10) (Figure 3) was thought t o in c l u d e a hydrated prephenic COOH X00H 11 14 CH aC0CO0H 10 COOH OH 13 Figure 3. Pathway from s h i k i m i c a c i d to dihydroxyphenylpyruvic a c i d i n c o r r e c t l y i n v o l v i n g a hydrated prephenic a c i d intermediate a c i d (14) as an intermediate. Wenkert proposed that t h i s intermediate gave r i s e to the condensing u n i t 15 a f t e r some s t e r e o s p e c i f i c rearrangements as shown i n Figure 4. The important f e a t u r e s of t h i s hypothesis were that the r i n g never became aromatic, the stereochemistry at C-15 was predetermined by the stereochemistry of the hydrated prephenic acid molecule and the carbomethoxy f u n c t i o n was r e t a i n e d so that only one C^ u n i t was required i n the synthesis of the a l k a l o i d s . When i t was shown 1**'^ that a hydrated - 176 -COOH i 0 r ^ V0 H [o] OH OH OH COOH COOH COOH 15 Figure 4. E s s e n t i a l features of hydrated prephenic a c i d hypothesis prephenic a c i d was not inv o l v e d i n the s h i k i m i c acid-prephenic a c i d t r a n s f o r m a t i o n , Wenkert 1 modified h i s scheme to u t i l i z e prephenic a c i d i t s e l f to give a condensing u n i t as shown i n Figure 5. Corynantheine could a r e l a t e d a l k a l o i d , yohimbine (18), by condensation of tryptamine with e i t h e r 16 or 17. This modified prephenic a c i d theory of Wenkert embodied the same features as the e a r l i e r one; that i s , i n p a r t i c u l a r , the establishment of the c o n f i g u r a t i o n at C-15 and the r e q u i r e d i n c l u s i o n of one C^  u n i t . Wenkert r e f e r r e d to 17 as the seco-prephenate-formaldehyde (SPF)unit. I t s r e l a t i o n s l i i p to 1 i s obvious. 8 Another scheme that had been proposed f o r the b i o s y n t h e s i s of the non-tryptamine p o r t i o n of the i n d o l e a l k a l o i d s i s i l l u s t r a t e d i n Figure 6. In t h i s scheme the condensing u n i t 19 was to have been b u i l t up by conden-s a t i o n of a pol y - 3 - k e t o e s t e r of s i x carbon atoms i n length with malonate and a C^ u n i t . This scheme rece i v e d some experimental s u p p o r t ^ 0 ^ which 23 was subsequently withdrawn. a r i s e , according to t h i s scheme, by condensation of tryptamine with 17 and - 177 -COOH CO COOH 0 Cl-UOH HOOC H 0 0 C C O O H ! C O C H . O H N ^ O tryptamine 0 C H 2 0 -> H" HOOC CHO 17 tryptamine H ri'" H MeOOC 18 OH Figure 5. Prephenic a c i d hypothesis - 178 -CoASOC CHiO CoASOC H CH aOH 0 HOOC H COSCoA HOOC COSCoA 19 Figure 6. Acetate hypothesis f o r the production o f a corynantheine-s t r y c h n i n e type of condensing u n i t Another hypothesis f o r the b i o s y n t h e s i s o f the non-tryptamine p o r t i o n of the i n d o l e a l k a l o i d s was proposed independently by Wenkert 1^'^>^ a n ( j 24 Thomas. The "Monoterpenoid hypothesis", as i t i s c a l l e d , was suggested to these people by the s t r i k i n g s t r u c t u r a l s i m i l a r i t i e s that s e v e r a l monoterpenic g l u c o s i d e s bore to the non-tryptamine p o r t i o n o f the corynantheine-strychnine type o f a l k a l o i d s and t o the h y p o t h e t i c a l SPF u n i t proposed by Wenkert. Some monoterpenic glucosides are shown i n Figure 7. The backbone o f each compound i s emphasized by a heavy l i n e to p o i n t out the s i m i l a r i t y of the backbone to the Cg ^ u n i t 1. I t was observed that the t e r p e n i c p o r t i o n of these compounds possessed the r e q u i s i t e number o f carbon atoms and i n the compounds, f o r which the absolute c o n f i g u r a t i o n at the s t a r r e d centres was known, the stereochemistry was the same as that found i n the m a j o r i t y o f i n d o l e a l k a l o i d s . In a d d i t i o n , the - 179 -.OGlu bakankosin JMe ^h>.0 n A . O ft MeOOC , swertiarnirin verbenalin (20) CH aOH * ; MeOOC genipin ?Me Cv3 *Jr X H O iridod ial Figure 7. Examples of monoterpenic g l u c o s i d e s having corynantheine-s t r y c h n i n e type of backbone necessary backbone was i n t a c t i n these compounds and they were seen to be simply r e l a t e d t o the h y p o t h e t i c a l condensing u n i t s 17 and 19. Cleavage (dashed l i n e s ) and r o t a t i o n (curved arrows) would give i n the case of each of the examples i n Figure 7 s t r u c t u r e s whose backbones are superimposable wit h the corynantheine-strychnine C n n u n i t (1). An important f e a t u r e of the monoterpenoid hypothesis not possessed by any other hypothesis was that a C-^  u n i t was not r e q u i r e d t o be i n v o l v e d i n the b i o s y n t h e s i s . The Barger-Hahn-Robinson-Woodward hypothesis, the Wenkert hydrated prephenic a c i d and prephenic a c i d hypotheses, and the T a y l o r acetate h y p o t h e s i s , a l l r e q u i r e d - 180 -that C-21 i n yohimbine, corynantheine and r e l a t e d a l k a l o i d s should come from the one-carbon pool i n p l a n t s . In order to d i s c o v e r which, i f indeed any, of the hypotheses most c l o s e l y resembled the b i o s y n t h e t i c pathway, t r a c e r experiments were c a r r i e d out by s e v e r a l groups of workers. U n f o r t u n a t e l y , the i n i t i a l r e s u l t s of the t r a c e r experiments l e d to confusion. Indeed f o r a time i t could be con-22 strued t h a t a l l the hypotheses had been r u l e d out. One group of workers 14 reporte d that when sodium formate- C was fed to Rauwolfia s e r p e n t i n a p l a n t s , C-21 of ajmaline (21) became l a b e l l e d (12% of a c t i v i t y ) . This was i n keep-ing w i t h a hypothesis that r e q u i r e d the i n c l u s i o n of a C, u n i t from the one-carbon pool of the p l a n t . Further experiments ' were reported which could be purported to disprove a l l the hypotheses except the T a y l o r acetate 14 hypothesis. In p a r t i c u l a r mevalonic acid-2- C (22) was found not to be incorp o r a t e d i n t o ajmaline as r e q u i r e d by the monoterpenoid hypothesis. 25 Other workers who c a r r i e d out feeding experiments i n Rauwolfia s e r p e n t i n a obtained r e s u l t s which were c o n s i s t e n t with a C^ u n i t not being in v o l v e d . i n the b i o s y n t h e s i s except i n t r i v i a l ways. As a r e s u l t of experiments that 25-29 were c a r r i e d out by s e v e r a l groups of workers, i t was apparent t h a t n e i t h e r the Barger-Hahn-Robinson-Woodward h y p o t h e s i s , nor the Wenkert OH 22 21 20,21 - 181 -hydrated prephenic a c i d and prephenic a c i d hypotheses, nor the Tay l o r acetate hypothesis described the b i o s y n t h e s i s of the i n d o l e a l k a l o i d s . In a d d i t i o n , the reported f a i l u r e to i n c o r p o r a t e mevalonic a c i d i n t o ajmaline as r e q u i r e d by the monoterpenoid hypothesis seemed t o i n d i c a t e that t h i s hypothesis was not tenable e i t h e r . One u n i f y i n g f e a t u r e o f the experimental work described above was th a t the r e s u l t s were negative i n nature; that i s , they appeared to disprove r a t h e r than prove. Nevertheless the evidence against the i n c l u s i o n of a fragment from the one-carbon pool was strong s i n c e i n c o r p o r a t i o n of sodium 14 formate- C was achieved, but the c r u c i a l carbon atom (C-21) was e s s e n t i a l l y 25 i n a c t i v e . For example, when Battersby and coworkers fed sodium formate-14 22 C to Rauwolfia s e r p e n t i n a p l a n t s i n r e p e t i t i o n of the e a r l i e r work, they found that i n the ajmaline i s o l a t e d the N-methyl group c a r r i e d not l e s s than 25% o f the a c t i v i t y and that the carbon atom, C-21, had l i t t l e or no a c t i v i t y . The evidence against the monoterpenoid h y p o t h e s i s , on the other 14 hand, was weak. In the attempt to show i n c o r p o r a t i o n of mevalonate- C i n t o ajmaline by Rauwolfia s e r p e n t i n a p l a n t s , the ajmaline that was i s o l a t e d was completely i n a c t i v e and,therefore, there was no guarantee that the l a b e l l e d mevalonic a c i d ever reached the s i t e of s y n t h e s i s o r , i f i t d i d , that i t reached the s i t e of synt h e s i s at a p e r i o d when s y n t h e s i s of ajmaline 30 31 was t a k i n g p l a c e . Other work ' has demonstrated that negative r e s u l t s should be i n t e r p r e t e d w i t h extreme care. 32 Scott and coworkers were the f i r s t to r e p o r t a s u c c e s s f u l i n c o r p o r a -t i o n o f mevalonate i n t o an a l k a l o i d o f the tryptamine + Cg type. Subse-33 — 36 quent p u b l i c a t i o n by s e v e r a l groups Of workers e s t a b l i s h e d that s p e c i f i c a l l y l a b e l l e d mevalonic a c i d was i n c o r p o r a t e d i n t o i n d o l e a l a k l o i d s i n a mariner which was completely c o n s i s t e n t w i t h the monoterpenoid hypothesis A d d i t i o n a l i r r e f u t a b l e evidence for the terpenoid o r i g i n of the Cg_^Q 37-40 condensing u n i t was found independently by four research groups. L a b e l l e d g e r a n i o l (23) was found to be incorporated as an i n t a c t u n i t i n t o v i n d o l i n e (24), catharanthine (25), and a j m a l i c i n e (26) i n Vinca rosea L. p l a n t s (Figure 8). Each of these three a l k a l o i d s are r e p r e s e n t a t i v e of one Figure 8. I n c o r p o r a t i o n of g e r a n i o l i n t o a l k a l o i d s r e p r e s e n t i n g the-, three s t r u c t u r a l types of i n d o l e a l k a l o i d s of the tryptamine + Cc,_^g typ - 183 -of the three types of tryptamine + Cg_-j.g a l k a l o i d s . The l o c a t i o n of the r a d i o a c t i v e l a b e l i n the a l k a l o i d s was i n a l l cases c o n s i s t e n t with the formation of ai. intermediate cyclopentane monoterpene u n i t as shown i n Figure 9 and the transformations shown i n Figure 1. Strong evidence f o r 1 Figure 9, Rearrangement i n backbone of g e r a n i o l t o give m o d i f i c a t i o n 1 of the Cg_iQ condensing u n i t i n v o l v i n g a cyclopentane monoterpene u n i t the intermediacy of cyclopentane intermediates i n the pathway was f i r s t 41 obtained by Battersby and coworkei-s who succeeded i n showing that logamn (27) which had been l a b e l l e d i n the e s t e r methyl, was in c o r p o r a t e d i n t o a j m a l i n e , v i n d o l i n e and catharanthine by Vinca rosea L. p l a n t s . These workers fed a v a r i e t y o f cyclopentane monoterpene compounds a l l of which were l a b e l l e d i n the e s t e r methyl group. They found t h a t there was no i n c o r p o r a t i o n of a c t i v i t y when r a d i o a c t i v e v e r b e n a l i n (20), dihydroverben-a l i n e (28), or monotropeine methyl e s t e r (29) were fed and t h e r e f o r e they f e l t that the good i n c o r p o r a t i o n of loganin obtained (ca. 1% i n t o v i n d o l i n e ) d i d not come about by simple t r a n s m e t h y l a t i o n . Recently, any p o s s i b i l i t y of ambiguity was removed when l o g a n i n , l a b e l l e d b i o s y n t h e t i c a l l y with 42 43 44 4 5 i n the 2 - p o s i t i o n ' and i n the 4 - p o s i t i o n , ' " was incorporated by Vinca rosea L. p l a n t s i n t o s e v e r a l a l k a l o i d s r e p r e s e n t a t i v e of the three - 184 -main s t r u c t u r a l types of i n d o l e a l k a l o i d s . In a d d i t i o n , a s u c c e s s f u l i n c o r p -o r a t i o n of [ 1 - % ] - l o g a n i n by Rauwolfia s e r p e n t i n a p l a n t s i n t o ajmaline was 43 a l s o r e p o r t e d , thereby e s t a b l i s h i n g l o g a n i n as a precursor of a Corynanthe type a l k a l o i d i n another p l a n t s p e c i e s . These r e s u l t s are summarized i n Figure 10. Several compounds are shown as m u l t i p l y l a b e l l e d e n t i t i e s , but i t should be understood that each l a b e l corresponds t o a separate experiment. 46 Battersby has proposed a reasonable pathway from mevalonate to the i n d o l e a l k a l o i d s of the tryptamine + Cg_-^ Q type (Figure 11) . He was c a r e f u l to p o i n t out, however, that " s e v e r a l c l o s e l y s i m i l a r schemes could be w r i t t e n i n which the sequence of operations i s a l t e r e d . " For example, there has been p u b l i s h e d no evidence concerning p r e c i s e l y where i n the b i o s y n -t h e s i s the union of tryptamine and the Cg ^ u n i t comes about, although 46 Battersby i n d i c a t e d that there was some recent experimental evidence that i n d i c a t e d t h a t "the conversion of the corynantheine-strychnine Cg ^ u n i t i n t o the Aspidosperma and Iboga C g _ ^ u n i t s occurs a f t e r the i n t r o d u c t i o n of the n i t r o g e n . . . " Of s e v e r a l p o s s i b l e intermediates which could undergo cyclopentane - 185 -OH T •Menyanthes t r i f o l i a t a MeO M E C O O M G • MeOOC O H P ^ 27 I i N H MeOOC .N MeOOC Figure 10. In c o r p o r a t i o n of loganin i n t o v a r i o u s i n d o l e a l k a l o i d s - 186 -HCL /Me 1 . + COO Na OH .OH M G O O C MeOOC CH aR OGlu 27,R=H 30,R=OH > | I H MeOOC CHO citronellal CHO <~ ^ C H O i ridodia I ^•CHO CHO OGlu MeOOC OGlu 0 MeOOC 32 Figure 11. Proposal f o r the pathway from mevalonate t o - i n d o l e a l k a l o i d s of the tryptamine + C Q _ ^ type - J 87 -cleavage, hydroxyloganin (3D) i s p a r t i c u l a r l y a t t r a c t i v e . A cleavage 46 mechanism i s i n d i c a t e d i n Figure 12 (w).ere "X" could be a phosphate I f -H _ H > 6 MeOOCr * Figure 12. A cyclopentane cleavage r e a c t i o n of p o s s i b l e b i o s y n t h e t i c importance residue to provide a good l e a v i n g group.) 47 I t has been reported very r e c e n t l y that the monoterpenic g l u c o s i d e , sweroside (33), was incorpo r a t e d very e f f i c i e n t l y (11%) i n t o v i n d o l i n e by Vinca rosea L. p l a n t s . Sweroside bears a s t r i k i n g resemblance to the hypo-t h e t i c a l condensing u n i t 31, having the same stereochemistry at C-2 and C-7 and a p o t e n t i a l aldehyde f u n c t i o n at C-5. The i n c o r p o r a t i o n of sweroside i n t o v i n d o l i n e was more e f f i c i e n t than the i n c o r p o r a t i o n of loganin i n t o v i n d o l i n e and t h i s would tend to suggest that sweroside i s f u r t h e r along the b i o s y n t h e t i c pathway than loga n i n . In t h i s regard there was good evidence that l o g a n i c a c i d (34) i s a precursor of g e n t i o p i c r o s i d e (35) i n 48 Swertia c a r b o l i n i e n s i s p l a n t s and sweroside i s an extremely e f f i c i e n t p recursor ( i n c o r p o r a t i o n r a t i o 40%) of g e n t i o p r i c r o s i d e i n Gentiana scabra p l a n t s . There are a few a l k a l o i d s i n which the h y p o t h e t i c a l condensing u n i t 31 occurs as an i n t a c t or n e a r l y i n t a c t u n i t . One of these i s ipecoside (36) 49 I t has been shown that the monoterpenic p o r t i o n of ip e c o s i d e i s derived - 188 -*OGlu .^OGlu C^oeiu 33 34 35 50 from l o g a n i n . Another i s the i n d o l e a l k a l o i d c o r d i f o l i n e (37). In t h i s compound the sugar p o r t i o n was found to be monoacetylated. A t h i r d a l k a l o i d . COOH OGlu ' H MeOOC 36 M e O O C K ^ 0 H O T ^ O H OH 37 51 macrosalkine (38), whose s t r u c t u r e has been determined by X-ray d i f f r a c t i o n 52 a n a l y s i s , has a unique s t r u c t u r e i n which the h y p o t h e t i c a l condensing u n i t 31 i s d i s g u i s e d . I f macrosalhine i s w r i t t e n i n a d i s t o r t e d form, as i n 39, the r e l a t i o n s h i p between r i n g s D and E of macrosalhine and loganin i s r r n yfMe, | \ /» H"' - 189 -c l e a r l y revealed. The c o n f i g u r a t i o n s of the centres 15, 20 and 21 i n macrosalhine are the same as the c o n f i g u r a t i o n s or the corresponding centres i n l o g a n i n . 53 A p l a u s i b l e pathway by which an intermediate such as 32 of g e i s s o s -c h i z i n e (40) might be transformed i n t o ajmaline-type a l k a l o i d s i s shown i n Figure 13. O-H-0 21 N-H MeOOC H-0 polyneurididine(41) Figure 13. P l a u s i b l e pathway t o ajmaline and r e l a t e d a l k a l o i d s - 190 -Wenkert*^ has proposed a r a t h e r i n t e r e s t i n g pathway to Akuamma (eg. akuammicine (45)) and p l e i o c a r p a m i n e - l i k e bases. The unusual s t r u c t u r e of pleiocarpamine (44) suggested the pathway. Wenkert envisaged an o x i d a t i o n of a corynantheine-type intermediate such as g e i s s o s c h i z i n e t o give r i s e to a d i r a d i c a l c a t i o n (42) as the k.ey step. The pathway suggested by Wenkert i s shown i n Figure 14. Two a d d i t i o n a l k a l o i d s which might be der i v e d from intermediate 43 are pseudoakuammigine (46) and echitamine (47). I t should be emphasized t h a t the pathway i n Figure 14 i s pu r e l y s p e c u l a t i v e i n nature and i s meant only to provide a p l a u s i b l e b i o s y n t h e t i c sequence. A l t e r n a t i v e schemes are a l s o conceivable. For example, another p l a u s i b l e route*''' from g e i s s o s c h i z i n e to echitamine i s i l l u s t r a t e d i n Figure 15. The key r e a c t i o n of t h i s pathway i s an i n t r a m o l e c u l a r Michael condensation. In t h i s regard i t i s i n t e r e s t i n g to note that the 2-acyl i n d o l e system does not seem to be succeptable to n u c l e o p h i l i c a t t a c k at the carbon atom.^ Wenkert 1 has p o s t u l a t e d t h a t the a l k a l o i d s of the Iboga- and Aspido-sperma-type are r e l a t e d b i o s y n t h e t i c a l l y to the Akuamma a l k a l o i d s and has suggested the scheme shown i n Figure 16 to account f o r t h e i r b i o g e n e s i s . I t should be observed that whereas Wenkert has chosen to p o s t u l a t e the formation - 191 -Figure 14. P l a u s i b l e pathway to the Akuamma a l k a l o i d s - 192 -Figure 15. A l t e r n a t i v e pathway to echitamine and r e l a t e d a l k a l o i d s - 193 -52 53 Figure 16. F i n a l stages i n Wenkert's proposal f o r the b i o s y n t h e s i s of a l k a l o i d s with the Aspidosperma and Iboga types of s k e l e t o n - 194 -of bond "b" p r i o r to the formation of bond "a", i t i s e q u a l l y p l a u s i b l e that, bond "a" could be formed p r i o r to bond "b" as i n d i c a t e d i n Figure 17. Figure 17. P l a u s i b l e v a r i a t i o n on Wenkert's proposal f o r the b i o s y n t h e s i s of a l k a l o i d s with.the Aspidosperma and Iboga types of s k e l e t o n Very r e c e n t l y there has been some i n d i c a t i o n t h a t the Aspidosperma and 57 Iboga bases are d e r i v e d from an Akuamma-type int e r m e d i a t e . R a d i o a c t i v e stemmadenine (54) was incorporated i n t o catharanthine (25) and tabersonine (5). I f these r e s u l t s should be confirmed, then the determination of the HO H2C COOMe 54 - 195 -p r e c i s e b i o s y n t h e t i c pathway from the Akuamma sk e l e t o n t o the Aspidosperma and Iboga skeletons w i l l become even more i n t e r e s t i n g . A p l a u s i b l e v a r i a t i o n on the Wenkert scheme u t i l i z i n g stemmadenine i s o u t l i n e d i n Figure 18. The conversion of 55 i n t o tabersonine and catharanthine i s shown t o be ^ j < 54 N H 55 C O O M G HOyHp C O O M G / 25 56 Figure 18. A p l a u s i b l e pathway from stemmadenine to a l k a l o i d s with the Aspidosperma and Iboga types of s k e l e t o n - 196 -•completely concerted i n Figure 18 and i s a p l a u s i b l e a l t e r n a t i v e to a step-wise f o r m a t i o r o f bonds. The enamine form (56) of 55 would a l s o be a p l a u s i b l e intermediate and could react to give catharanthine or tabersonine i n e i t h e r a two step r e a c t i o n or a concerted r e a c t i o n ( D i e l s - A l d e r ? ) as shown. 54 iVenkert has extended h i s p r e v i o u s l y proposed b i o s y n t h e t i c pathway to the Aspidosperma-type a l k a l o i d s to i n c l u d e a l k a l o i d s such as vincamine (59) (Figure 19). The key step i n t h i s pathway i s the formation of the intermediate Figure 19, P l a u s i b l e pathway to vincamine and r e l a t e d a l k a l o i d s and to the a l k a l o i d v a l l e s a m i d i n e - 197 -58 by t r a n s a n n u l a r c y c l i z a t i o n i n 57 through c o u p l i n g of the iminium f u n c t i o n t o the a - p o s i t i o n of the i n d o l e . In t h i s regard i t i s i n t e r e s t i n g to note that the s t r u c t u r e of an a l k a l o i d c a l l e d v a l l e s a m i d i n e (60) has 58 r e c e n t l y been e s t a b l i s h e d by X-ray d i f f r a c t i o n a n a l y s i s . ' I I . D i s c u s s i o n THE PREPARATION OF LABELLED COMPOUNDS The demonstration i n our l a b o r a t o r i e s that the transformations 50 —>• 52 and 51 —> 53, which were proposed by Wenkert as key steps i n the b i o -s y n t h e s i s of the Aspidosperma and Iboga a l k a l o i d s , were c h e m i c a l l y f e a s i b l e and, moreover, proceeded i n the l a b o r a t o r y w i t h complete s t e r e o s p e c i f i c i t y s t i m u l a t e d a study to determine whether or not the transformations were of any s i g n i f i c a n c e i n l i v i n g Vinca p l a n t s . Several p o s s i b l e p r e c u r s o r s were a v a i l a b l e i n our l a b o r a t o r i e s , but i t was necessary t o prepare them i n r a d i o a c t i v e form before any t r a c e r experiments could be c a r r i e d out. A 14 method f o r preparing C - l a b e l l e d 188-carbomethoxy-43-dihydrocleavamine (61), a p l a u s i b l e precursor f o r Iboga a l k a l o i d s , had been worked out as part of the t o t a l s y n t h e s i s of that compound. At the time that t r a c e r experiments i n Vinca p l a n t s were being contemplated, however, 18B-carbomethoxy-48-dihydrocleavamine was a v a i l a b l e only v i a a poor y i e l d i n g sequence and i t was recognized that the l e v e l of a c t i v i t y o b t a i n a b l e might be too low f o r d e f i n i t i v e r e s u l t s . In a d d i t i o n , attempts to prepare a carbomethoxyquebrach-amine (62) as a p o s s i b l e precursor of v i n c a d i f f o r m i n e (63) and a carbomethoxy-4a-dihydrocleavamine (64), which possesses the same c o n f i g u r a t i o n of the 4-e t h y l group as c o r o n a r i d i n e (7), by the same method were u n s u c c e s s f u l . I t thus appeared that the method would be a p p l i c a b l e to the p r e p a r a t i o n of - 199 -18$-carbomei'".hoxy-43-dihydrocleavamine only. At any r a t e the method would o b v i o u s l y not be a p p l i c a b l e to the p r e p a r a t i o n of l a b e l l e d compounds which do not possess a carbomethoxy group, but which could be i n c o r p o r a t e d , i n theory at l e a s t , i n t o s e v e r a l Aspidosperma a l k a l o i d s . For these and other H H ; • MeOOC ^ 61 COOMe 62 N-H i MeOOC 64 63 COOMe reasons s e v e r a l other methods f o r preparing l a b e l l e d compounds were i n v e s t i -gated i n our l a b o r a t o r i e s i n order t h a t t h e i r a p p l i c a b i l i t y to the p r e p a r a t i o n of the d e s i r e d l a b e l l e d compounds might be assessed. Since a l l the p o t e n t i a l precursors which were to be used i n t h i s study had the i n d o l e moiety i n common, the task set t o t h i s worker was to i n v e s t i g a t e methods of exchanging the hydrogen atoms of the benzene p o r t i o n of the i n d o l e moiety with t r i t i u m atoms. Although no in f o r m a t i o n could be found i n the l i t e r a t u r e concerning t r i t i u m l a b e l l i n g of i n d o l e a l k a l o i d s by exchange o f the aromatic protons, there were, however, some examples of deuterium exchange.' Buchi and 59 coworkers were able to exchange the aromatic hydrogen atoms i n voacangine - 200 -(65) with deuterium atoms to a reasonable extent by t r e a t i n g the compound with a s o l u t i o n of deuterium oxide, methanol-0-d, and hydrogen c h l o r i d e . Voacangine, however, possesses a methoxyl group i n the aromatic r i n g and as a r e s u l t exchange would be expected to take place much more e a s i l y than the exchange i n compounds l i k e the carbomethoxydihydrocleavamines which possess 48 no such a c t i v a t i n g group. These same workers, however, a l s o showed th a t dregamine (66) could be l a b e l l e d i n t h i s manner. Dregamine was t r e a t e d MeO 65 MeOOC w i t h a 1:1 mixture o f methanol-O-d and deuterium oxide v/hich had been s a t u r -ated wi t h hydrogen c h l o r i d e gas. A f t e r the s o l u t i o n was heated under r e f l u x f o r 23 hours under a n i t r o g e n atmosphere, the dregamine was recovered i n 95% y i e l d and the aromatic hydrogen atoms, as shown by the nmr spectrum, had been replaced by deuterium atoms to the extent of about 50%. I t was f e l t that t h i s method could be modified to "give t r i t i u m l a b e l l e d compounds. The p r a c t i c a l problems that were a n t i c i p a t e d i n making such a change were formidable. T r i t i u m l a b e l l e d water could not be d i r e c t l y s u b s t i t u t e d i n the r e c i p e above because an unknown q u a n t i t y of t r i t i u m l a b e l l e d hydrogen c h l o r i d e gas would escape i n the i n i t i a l s a t u r a t i o n and subsequent r e f l u x steps. This problem would n e c e s s i t a t e the use of e f f i c i e n t t r a p s f o r - 201 -escaping gar. Also the extent of d i l u t i o n of the l a b e l might be s e r i o u s . Although i t was f e l t that these problems might be overcome, i t was a l s o f e l t t h a t there might be more s u i t a b l e c o n d i t i o n s f o r b r i n g i n g about the a c i d c a t a l y z e d exchange r e a c t i o n i n which a non gaseous a c i d was s u b s t i t u t e d f o r hydrogen c h l o r i d e . Onepossible a c i d t h a t came to mind immediately was a c e t i c a c i d . The carbomethoxydihydrocleavamines were known to r e t a i n t h e i r e s t e r f u n c t i o n s i n r e f l u x i n g g l a c i a l a c e t i c a c i d because these compounds are formed by the treatment of catharanthine with a z i n c - a c e t i c a c i d mixture. T r i t i u m l a b e l l e d g l a c i a l a c e t i c a c i d could be e a s i l y prepared from a c e t i c anhydride by h y d r o l y s i s of the anhydride w i t h t r i t i u m l a b e l l e d water. There were two p o s s i b l e drawbacks that came to mind concerning the use of t r i t i u m l a b e l l e d a c e t i c a c i d . One was that i t was a much weaker a c i d than hydro-c h l o r i c a c i d and consequently the r a t e of exchange might be too slow f o r the l a b e l l i n g method to be p r a c t i c a l . The other was that the l a b e l might become d i l u t e d through exchange of the a-hydrogen atoms of the a c e t i c a c i d with the a c i d i c hydrogen atom. Co n s i d e r a t i o n of these p o s s i b l e drawbacks le d to the b e l i e f t h a t the i d e a l a c i d f o r the exchange r e a c t i o n might be t r i f l u o r o a c e t i c a c i d . T r i f l u o r o a c e t i c a c i d appeared to possess s e v e r a l d e s i r a b l e p r o p e r i t e s . I t was known to be a f a i r l y strong a c i d (pKa 0.3) and to possess only one hydrogen atom, the a c i d i c one. The b o i l i n g p o i n t (75°C) was considered to be s u f f i c i e n t l y low that the l a b e l l e d a c i d could be e a s i l y removed from samples by d i s t i l l a t i o n at reduced pressure f o r subsequent use, but high enough that there would be l i t t l e danger from r a d i o a c t i v e vapours. F i n a l l y , t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d could be prepared e a s i l y from t r i f l u o r o a c e t i c anhydride because the h y d r o l y s i s of the anhydride was known to take place v i r t u a l l y i n s t a n t a n e o u s l y . - 202 -When i t was dec ided to t e s t the a b i l i t y o f t r i f l u o r o a c e t i c a c i d to promote exchange, a p l e n t i f u l supply of l ° > a - c a r b o m e t h o x y - 4 a - d i h y d r o c l e a v -amine was on hand i n our l a b o r a t o r i e s and consequent ly t h i s compound was chosen as the t e s t compound.. S ince i t seemed to be a d v i s a b l e to use f a i r l y v igorous c o n d i t i o n s , a s o l u t i o n o f 18a-carbomethoxy-4a-dihydrocleavamine i n t r i f l u o r o a c e t i c a c i d - t r i t i u m l a b e l l e d water ( s p e c i f i c a c t i v i t y , ca . 0.1 mCi / mmol) was heated at 50°C f o r four hours under a n i t r o g e n atmosphere. I n v e s t i -g a t i o n o f the crude product by t h i n - l a y e r chromatography showed only the presence o f 18a-carbomethoxy-4a-dihydrocleavamine (67) and i t s epimer 188-carbomethoxy-4a-dihydrocleavamine (68) . P r e p a r a t i v e t h i n - l a y e r chromato-graphy and r e c r y s t a l l i z a t i o n from methanol p rov ided a pure sample of H rcOOMe H ilj 67 18a-carbomet.hoxy-4a-dihydrocleavamine i n 72% y i e l d . The s p e c i f i c a c t i v i t y of the 18a-carbomethoxy-4a-dihydrocleavamine was found to be 0.103 mCi/mmol. These were p a r t i c u l a r l y encouraging r e s u l t s s i nce they meant tha t the method would l i k e l y be s u i t a b l e f o r l a b e l l i n g o ther p o t e n t i a l p r e c u r s o r s w i t h a h igh l e v e l o f a c t i v i t y wi thout fear o f ex t ens ive decompos i t ion . I t remained o f course to be shown tha t the p o s i t i o n o f the l a b e l was i n the aromatic system. There was l i t t l e doubt tha t the l a b e l was not at the s i t e of the i n d o l e n i t r o g e n atom s ince the c o n d i t i o n s used i n the work up would cause - 203 -an immediate exchange of any t r i t i u m atoms at t h a t s i t e f o r hydrogen atoms. There was the p o s s i b i l i t y , however, that the l a b e l could r e s i d e at the C-18 s i t e . In order to r e s o l v e the problem of the l o c a t i o n of the l a b e l , deuterated 18a-carbomethoxy-4a-dihydrocleavamine was prepared. T r i f l u o r o a c e t i c a c i d - d , which had been prepared by h y d r o l y z i n g t r i f l u o r o a c e t i c anhydride with an equivalent amount of deuterium oxide, was allowed to r e a c t w i t h 18a-carbo-methoxy-4a-dihydrocleavamine f o r 4 hours at 50°C under a n i t r o g e n atmosphere. Again t h i n - l a y e r chromatography showed that the r e a c t i o n mixture contained both C-18 epimers. P r e p a r a t i v e t h i n - l a y e r chromatography and c r y s t a l l i z a t i o n provided a pure sample of deuterated 18a-carbomethoxy-4a-dihydrocleavamine i n 67% y i e l d . An estimate of the average number of dueterium atoms and t h e i r l o c a t i o n i n the molecule was made with the a i d o f the nmr and mass spe c t r a of the compound i n i t s l a b e l l e d and u n l a b e l l e d forms. I t was q u i t e simple to d i s t i n g u i s h the fragments which corresponded to p o r t i o n s of the molecule which had become l a b e l l e d from those which had not by comparing the mass spectrum of the deuterium l a b e l l e d compound w i t h t h a t of the un-l a b e l l e d compound. Each spectrum was obtained under as n e a r l y i d e n t i c a l c o n d i t i o n s as p o s s i b l e . Since the deuterium l a b e l l e d compound was i n a c t u a l i t y a mixture of hydrogen-isotope compounds ( £ 2 1 ^ 2 8 n^n^2^2^ ' ^ d i s p l a y e d a composite mass spectrum. In the case of the spectrum of the u n l a b e l l e d compound (Figure 20) there were four peaks at m/e 338, 3.39, 341, and 342 t h a t were a s s o c i a t e d with the molecular ion peak at m/e 340. In the spectrum of the l a b e l l e d compound (Figure 21) there were a c l u s t e r of peaks s t r e t c h i n g to an m/e value of 346 (and perhaps to 347). Assxuning that the peak at 346 represented the M+2 peak of the most h i g h l y s u b s t i t u t e d molecular i o n , there could have been a mixture of hydrogen-isotope compounds LU O z < Q . Z m < LU > < UJ IX 100 90 80 70 60 •50 AO 30 20 10 0 '— 50 Figure 20. CM OD CD 1 n o CO o C N o CO NJ o Ill I I I i I I 1 i 100 150 250 200 m/e Mass spectrum of u n l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine 300 350 RELATIVE ABUNDANCE C/q c CD so to cn cn -0 00 CD O o o o o o o o o o O O o [ 2 fa w t/i in >d CD O r t o a o c u CD c r t CD H 124 138 cn o CD CD CU OO a i o l-i cr o 3 CD r t 5 ' O X-I s I CU H-X C u >-i O O t—• CD fa < 3 H* CD O O ,210 cn o CO o o I a CO cn» o - soz -- 206 -corresponding to the formulae: <"2l' 128^2 <^2' ^21^?7 D N2^2' ("2 1 ^ 26 D2^2^2' C 0 1H D N 0 and C 0 1H D N 0 i n the sample of l a b e l l e d carbomethoxydihydro-21 25 6 2 2 2± 2Q 2 2 cleavamine. Although a s e r i e s of simulataneous equations might have been set up from v/hich the r e l a t i v e abundance of each of the species C 0,H 0 0 D N„0 r r 21 28-n n 2 could t h e o r e t i c a l l y have been c a l c u l a t e d , to do so seemed to be i m p r a c t i c a l . Only the average number of deuterium atoms at each s i t e i n the molecule was of i n t e r e s t f o r our purposes and t h i s number was e a s i l y obtained w i t h a c e r t a i n degree of p r e c i s i o n from the nmr s p e c t r a . Some u s e f u l i n f o r m a t i o n was a v a i l a b l e from the mass s p e c t r a , however. A comparison of the spectrum of the u n l a b e l l e d compound with t h a t of the l a b e l l e d compound revealed that the r e l a t i v e abundance of the peaks i n the v i c i n i t y of the peaks at m/e 138 and m/e 124 were v i r t u a l l y the same i n both s p e c t r a . This was c o n s i s t e n t w i t h there being no dueterium c o n t a i n i n g fragments corresponding to ions 69 and 70 i n the deuterium l a b e l l e d compound. Hence, the protons i n these fragments were not exchangeable under the c o n d i t i o n s employed. I t was 69 70 observed that both s p e c t r a d i s p l a y e d a strong peak at m/e 210. The stren g t h of t h i s peak i n the mass spectrum of the l a b e l l e d compound meant that the fragment arose from a p o r t i o n of the molecule which was not e a s i l y l a b e l l e d . By comparison the peak at m/e 340 was almost non e x i s t e n t i n the spectrum - 207 -of the l a b e l l e d compound. Since most of the l a b e l was in- the i n d o l e system (as shown by nmr below), i t was apparent that t h i s fragment must not c o n t a i n the i n d o l e moiety. The ion 71 would d i s p l a y an m/e value of 210 and might a r i s e from 18a-carbomethoxy-4a-dihydrocleavamine as shown i n Figure 22. Figure 22. P l a u s i b l e pathway g i v i n g a fragment with an m/e value of 210 I t was a l s o observed that the peak at. m/e 211 i n the spectrum of the deuter-ated compound was s l i g h t l y l a r g e r by comparison with the same peak i n the spectrum o f the u n l a b e l l e d compound. This would be c o n s i s t e n t with there - 208 -being a small c o n t r i b u t i o n to the m/e 211 peak from the species 72. The nmr spectrum of the deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydro-cleavamine provided s u b s t a n t i a t i o n o f the i n f o r m a t i o n obtained from the mass spectrum. In a d d i t i o n , the s i t e s i n which deuterium was l o c a t e d and the amount of deuterium i n each s i t e could be determined by c a r e f u l i n t e g r a -t i o n . The spectrum of the deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydro-cleavamine i s shown i n Figure 23. The spectrum of the u n l a b e l l e d compound i s shown i n Figure 24. I t was immediately apparent even without the a i d of the i n t e g r a l that the aromatic p o r t i o n contained most of the deuterium atoms. The spectrum showed strong s i g n a l due to the proton on the i n d o l e n i t r o g e n atom and the proton at C-18. In a d d i t i o n , the p o r t i o n of the spectrum above T 6 was v i r t u a l l y superimposable with the same p o r t i o n of the spectrum of the u n l a b e l l e d compound. The r e g i o n of the aromatic protons, however, was r a d i c a l l y d i f f e r e n t . The m u l t i p l e t at about T 2.55 i n the spectrum o f the u n l a b e l l e d compound had c o l l a p s e d to a s i n g l e t at T 2.53 i n the spectrum of the l a b e l l e d compound. The other groups of m u l t i p l e t s appeared to be s i m p l i f i e d . These f a c t o r s were i n accord with deuterium being present i n the aromatic p o r t i o n of the molecule i n a s u b s t a n t i a l amount. The apparent Figure 23. Nmr spectrum of deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine Figure 24. Nmr spectrum of u n l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine - 211 -s i m p l i f i c a t i o n of the region would not be expected i f only a small number of molecules c o n t a i n i n g deuterium atoms were present. Comparison of the i n t e g r a l s i n the s p e c t r a o f the l a b e l l e d and u n l a b e l l e d compounds i n the region above x 7 d i d not r e v e a l any s i t e s that were suspected to have been l a b e l l e d to some extent were determined by assuming the i n t e g r a l of the s i g n a l from the protons of the methyl e s t e r to be e x a c t l y equivalent t o 3 protons. This assumption was s u b s t a n t i a t e d by c a r e f u l l y comparing the i n t e g r a l of the s i g n a l from the methyl e s t e r to the i n t e g r a l of the s i g n a l s from the methyl group of the e t h y l side chain. As near as could be deter-mined both i n t e g r a l s were the same. The i n t e g r a l over the aromatic r e g i o n , a f t e r c o r r e c t i o n f o r the presence of chloroform had been made, corresponded to 1.55 protons. This meant that 61.5% of the aromatic hydrogen atoms had been exchanged f o r deuterium atoms. The i n t e g r a l over the r e g i o n of the C-18 proton resonances corresponded to 0.89 t 0.03 protons. This meant that about 10% of the C-18 hydrogen atoms had been exchanged f o r deuterium atoms and t h i s r e s u l t agreed with the observation made from the mass spectrum concerning the C-18 s i t e . The i n t e g r a l over the i n d o l e NH corresponded to about 0.9 protons as w e l l , but t h i s i n t e g r a l i n the spectrum of the undeuter-ated compound a l s o corresponded to about 0.9 protons. The nmr evidence was c o n s i s t e n t w i t h the aromatic hydrogen atoms and the C-18 hydrogen atoms being the only atoms f o r which there was net exchange under the c o n d i t i o n s of the deuterium l a b e l l i n g experiment and a l s o t h e r e f o r e during the t r i t i u m l a b e l l i n experiment. Only about 4% of the l a b e l was at the C-18 s i t e , with the remainder of the l a b e l being i n . t h e aromatic s i t e s . Since the deuterium l a b e l l i n g - experiment proceeded very w e l l at 50°C, the r e a c t i o n was repeated at room temperature. In t h i s i n s t a n c e a s o l u t i o n of 18a-ca.rbomethoxy-4a-dihydrocleavamin3 i n t r i f l u o r o a c e t i c - d was presented to the nmr s p e c t r o s c o p i s t who ran the spectrum every so o f t e n when i t was convenient t o do so. The nmr s p e c t r a Obtained were of very good q u a l i t y . The number of deuterium atoms that had replaced aromatic hydrogen atoms over s e v e r a l d i f f e r e n t time i n t e r v a l s are given i n Table 1. The number of deuterium atoms was estimated by assuming that the i n t e g r a l over the proton resonances Table 1 No. of deuterium atoms Time elapsed (hrs.) 0.5 1 6 0.8 3 3 1.2 6 8 1.85 23 8 3.3 336 (approx.) of the methyl e s t e r corresponded to 3 protons. Even a f t e r two weeks the nmr spectrum revealed b a r e l y any signs of decomposition although the s o l u t i o n had become red-purple i n c o l o u r . I n v e s t i g a t i o n of the m a t e r i a l obtained a f t e r two weeks by t h i n - l a y e r chromatography showed the presence of 18a-carbomethoxy-4a-dihydrocleavamine, some 18B-carbomethoxy-4a-dihydrocleavamine and only a small amount of u n i d e n t i f i e d i m p u r i t i e s . Since 18B-carbomethoxycleavamine (73) seemed to be the most l i k e l y - 213 -precursor of the n a t u r a l l y o c c u r r i n g a l k a l o i d catharanthine (25), i t was of i n t e r e s t to see how t h i s compound was a f f e c t e d by t r i f l u o r o a c e t i c acid-d. I t was a n t i c i p a t e d that the presence of the o l e f i n i c double bond might make the compound s e n s i t i v e to a c i d . In f a c t t h i s conjecture-was borne out by the experiment. When the l a b e l l i n g r e a c t i o n was c a r r i e d out at 50°C, a complex mixture of compounds as shown by t h i n - l a y e r chromatography was obtained. Chromatography on alumina provided q u i t e pure 183-carbomethoxy-cleavamine i n 40% y i e l d , Further p u r i f i c a t i o n by c r y s t a l l i z a t i o n provided an a n a l y t i c a l l y pure sample. The nmr spectrum (Figure 25) revealed-that as i n the case of the 18a-carbomethoxy-4a-dihydrocleavamine the aromatic hydrogen atoms were exchanged t o the greatest extent. Because the s i g n a l s a r i s i n g from the c-18 and C-3 protons overlapped to some extent, i t was not p o s s i b l e to d i s t i n g u i s h between these two s i t e s . The i n t e g r a l over the aromatic r e g i o n , a f t e r c o r r e c t i n g f o r the presence of chloroform, corresponded to 1.85 protons and the i n t e g r a l over the r e g i o n i n which the C-18 and C-3 protons resonances occurred corresponded to 3.55 protons. These r e s u l t s meant that the deuterium atoms were d i s t r i b u t e d with 82.5% of the deuterium atoms being on the aromatic r i n g and 17.5% of them being i n one or both of the other two s i t e s at C-18 and C-3. •Although the y i e l d o f deuterium l a b e l l e d 188-carbomethoxycleavamine was poor when the l a b e l l i n g r e a c t i o n was done at 50°, s e v e r a l other Workers i n our l a b o r a t o r i e s have been able to ob t a i n n e a r l y q u a n t i t a t i v e y i e l d of the l a b e l l e d compound by c a r r y i n g out the r e a c t i o n at room temperature f o r periods longer than f o u r hours. Because the aromatic hydrogen atoms of 18a-carbomethoxy-4q-dihdyrocleav-amine and 183-carbomethoxycleavamine were exchanged f o r deuterium atoms - 215 -(and hence, t r i t i u m atoms) i n preference to the other hydrogen atoms i n these molecules, the l o c a t i o n of l a b e l i n r e l a t e d compounds, which were to be prepared f o r use i n t r a c e r experiments by treatment w i t h t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d , could be p r e d i c t e d . Since s e v e r a l of the compounds which were t o be used i n the t r a c e r experiments were a v a i l a b l e i n amounts of only a few m i l l i g r a m s , i t was not p r a c t i c a l t o l a b e l them with deuterium f o r the purposes of mass spectrometry and nmr spectroscopy and i t was imperative i n these cases that the p o s i t i o n of the l a b e l could be p r e d i c t e d w i t h c e r t a i n t y . On t h i s b a s i s 18a-carbomethoxy-4a-dihydrocleavamine, v i n c a d i n e (74) and vincaminoreine (75) were l a b e l l e d w i t h t r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d ( s p e c i f i c a c t i v i t y , 0.88 mCi/mmol), the l a t t e r two compounds f o r the purposes of t r a c e r experiment with Vinca minor L. p l a n t s . The method used was a m o d i f i c a t i o n of the method used to prepare deuterium l a b e l l e d 18a-c.arbomethoxy-4a-dihydrocleavamine. Whereas a 65:1 molar r a t i o was used i n the deuterium l a b e l l i n g experiments, a sm a l l e r r a t i o of t r i f l u o r o a c e t i c a c i d to the compound being l a b e l l e d was used i n the t r i t i u m l a b e l l i n g e x p e r i -ments, e n t i r e l y i n the i n t e r e s t of conserving t r i t i u m l a b e l l e d t r i f l u o r o -a c e t i c a c i d . The s p e c i f i c a c t i v i t i e s obtained f o r the three compounds are given i n Table 2. Table 2 Compound molar r a t i o of r e a c t a n t s s p e c i f i c a c t i v i t y (m Ci/mmol) 18a-carbomethoxy-4a-dihydrocleavamine vincaminoreine vincadine 8.6 9.3 18.0 0.205 0.282 0.548 - 216 -Other workers i n our l a b o r a t o r i e s have used t r i t i u m l a b e l l e d t r i f l u o r o -a c e t i c a c i d to prepare t r i t i u m l a b e l l e d samples of a great many i n d o l e c o n t a i n i n g compounds f o r t r a c e r experiments i n p l a n t s . As a r u l e the com-pounds were recovered v i r t u a l l y unchanged from the a c i d i c medium. When the l a b e l l i n g experiments described i n t h i s t h e s i s were being done, i t was r e a l i z e d t h a t i t would be economically more f e a s i b l e to recover the l a b e l l e d t r i f l u o r o a c e t i c a c i d f o r reuse. A simple vacuum system was constructed i n which the t r i t i u m labelled t r i f l u o r o a c e t i c a c i d c o u l d be d i s t i l l e d at reduced pressure to and from the compound which was t o be l a b e l l e d . In a t y p i c a l l a b e l l i n g experiment one v e s s e l c o n t a i n i n g the compound and another c o n t a i n i n g the a c i d was attached to the system. The t r i f l u o r o a c e t i c a c i d was then f r o z e n w i t h a l i q u i d n i t r o g e n bath and the system evacuated. The l i q u i d n i t r o g e n bath was then t r a n s f e r r e d to the v e s s e l c o n t a i n i n g the compound t o be l a b e l l e d and the t r i f l u o r o a c e t i c a c i d was d i s t i l l e d over. When a l l the t r i f l u o r o a c e t i c a c i d had been t r a n s f e r r e d n i t r o g e n was allowed i n t o the system and the l i q u i d n i t r o g e n bath was removed from the v e s s e l c o n t a i n i n g the compound to be l a b e l l e d and the fr o z e n a c i d . The r e a c t a n t s were allowed t o come t o room temperature. A f t e r the r e a c t i o n had proceeded - 217 -at room temperature f o r s e v e r a l hours ( u s u a l l y o v e r n i g h t ) , the excess v r i -f l u o r o a c e t i c a c i d was t r a n s f e r r e d from the l a b e l l e d compound usi n g the vacuum system. Since a large excess of a c i d was used, the d i l u t i o n of the l a b e l i n the r e a c t i o n was very small and the recovered t r i t i u m l a b e l l e d t r i f l u o r o -a c e t i c a c i d was s u i t a b l e f o r reuse. TRACER EXPERIMENTS USING VINCA PLANTS' 14 Experiment using [22 C]-18g-carbomethoxy-4g-dihyd:cocleavamine as t r a c e r Each of the four e p i m e r i c a l l y r e l a t e d carbomethoxydihydrocleavamines was considered as a p o s s i b l e p r e c u r s o r of the a l k a l o i d s bearing the Iboga s k e l e t o n . One of these epimers, 18g-carbomethoxy-4g-dihydrocleavamine, was a v a i l a b l e from 43-dihydrocleavamine by a s y n t h e t i c route i n which the carbonyl ] 4 carbon atom'of the e s t e r group came from the cyanide i o n . Therefore [22-' C]-18g-carbomethoxy-4g-dihydrocleavamine v/as a v a i l a b l e f o r the i n i t i a l t r a c e r experiment i n our l a b o r a t o r i e s to t e s t the p o s t u l a t e put forward by Wenkert f o r the b i o s y n t h e s i s o f a l k a l o i d s possessing the Iboga s k e l e t o n . Vinca rosea L. p l a n t s were used f o r t h i s experiment. The f o l i a g e on the p l a n t s was about two months o l d , whereas the root system was o l d e r . The p l a n t s ] 4 were not f l o w e r i n g when used. [22- C]-18g-carbomethoxy-4g-dihydrocleavamine (6.823 mg, 1.67 x 10 ^ mCi) w a s converted to i t s h y d r o c h l o r i d e s a l t and an aqueous s o l u t i o n of the s a l t was fed hydroponicaly to Vinca rosea L. p l a n t s . Each p l a n t was cut on the diagonal across i t s stem and the severed end was immediately placed i n . t h e s o l u t i o n of the l a b e l l e d compound. Each p l a n t was allowed to take up the s o l u t i o n of the l a b e l l e d compound and an a d d i t i o n a l volume of d i s t i l l e d water. When, a p a r t i c u l a r p l a n t refused to take up any l i q u i d or d i d so at a r a t e t h a t was no comparable w i t h the average r a t e , i t s - 218 -stem was recut. The b i t s of stem that were cut o f f were saved and i n c l u d e d i n the work ,ip. The p l a n t s were maintained f o r a p e r i o d of eight days by a d d i t i o n of d i s t i l l e d water when i t was necessary except towards the end of the p e r i o d when the p l a n t s were allowed to take i n a l l the water which was a v a i l a b l e to them. The p l a n t s were then immediately macerated i n the presence of a. mixture of benzene and 15 N ammonium hydroxide. The a l k a l o i d s that were present i n the benzene phase a f t e r t h i s treatment were e x t r a c t e d i n t o 2N h y d r o c h l o r i c a c i d . The a c i d i c s o l u t i o n was then made b a s i c and e x t r a c t e d w i t h benzene. The benzene e x t r a c t provided an a l k a l o i d c o n t a i n i n g mixture which was found to possess 30% o f the a c t i v i t y that had been fed. P r e p a r a t i v e t h i n - l a y e r chromatography (alumina, 3:1, benzene-chloroform) of the crude e x t r a c t on a 20x60 cm p l a t e , which was run i n the d i r e c t i o n of i t s longer measurement, provided a p a r t i a l s e p a r a t i o n of the mixture i n t o nine groups of a l k a l o i d s . T h i n - l a y e r chromatography confirmed the presence of 188-carbomethoxy-4g-dihydrocleavamine i n group 4 (av R^ 0.55), c o r o n a r i d i n e i n group 5 (av R^ 0.45), and catharanthine i n group 8 (av R^ 0.1), but d i h y d r o c a t h a r a n t h i n e , which i s not a known a l k a l o i d , could not be detected by t h i n - l a y e r chromatography i n group 7 (av R^ 0.2) i n which i t would have been present. The a c t i v i t y i n each of the groups of a l k a l o i d s was counted s e p a r a t e l y and the t o t a l a c t i v i t y obtained from the p l a t e corresponded t o 59% of t h a t which had been put on the p l a t e . The a c t i v i t y i n group 7, i n which dihydrocatharanthine might, have been present, accounted f o r only 0.2% of the t o t a l a c t i v i t y fed and a small amount of c o l d dihydrocatharanthine which-was added to the mixture represented by group 7 and r e i s o l a t e d by p r e p a r a t i v e t h i n - l a y e r cyromatography was found to show background a c t i v i t y only. Catharanthine and c o r o n a r i d i n e were p u r i f i e d by p r e p a r a t i v e thin-la.yer - 2x9 -chromatography u n t i l a n a l y t i c a l t h i n - l t y e r chromatography i n d i c a t e d that they were pure. At each stage i n the p u r i f i c a t i o n the a c t i v i t y associated w i t h these compounds diminished. When f u r t h e r p u r i f i c a t i o n by t h i n - l a y e r chromatography became i m p r a c t i c a l , the t o t a l a c t i v i t y a s s o c i a t e d with c o r o n a r i d i n e was 0.09% o f the fed a c t i v i t y and the t o t a l a c t i v i t y associated w i t h catharanthine was 0.02% of the fed a c t i v i t y . These r e s u l t s i n d i c a t e d 14 t h a t the l e v e l of i n c o r p o r a t i o n of [22- C]-18B-carbomethoxy-4B-dihydrocleav-amine, i f i t was a precursor of the Iboga a l k a l o i d s , was going t o be very low under the c o n d i t i o n s i n which t h i s t r a c e r experiment was run and helped s t i m u l a t e a study of d i f f e r e n t methods of l a b e l l i n g and feeding compounds. The above described feeding experiment was conducted i n c o l l a b o r a t i o n with Dr. Stan H a l l and he determined the l e v e l of a c t i v i t y a s s o c i a t e d with cath-aranthine. He was a l s o a c o l l a b o r a t o r i n two of the other t r a c e r e x p e r i -ments that w i l l be d e s c r i b e d i n t h i s t h e s i s and h i s c o n t r i b u t i o n s w i l l be s i g n i f i e d by the l e t t e r s "S.H." Experiment using [T-aromatic]-186-carbomethoxycleavamine as t r a c e r We f e l t t h a t 18g-carbomethoxydihydrocleavamine would be the most l i k e l y p r ecursor of catharanthine. The conversion r e q u i r e d t h a t the p l a n t be able to b r i n g about o x i d a t i v e c y c l i z a t i o n between the C-18 and C-5 atoms i n the carbomethoxydihydrocleavamine molecule. An analogous c y c l i z a t i o n had been shown to take place a f t e r mercuric acetate o x i d a t i o n of 18a-carbomethoxy-4a-dihydrocleavamine. Therefore, 18B-carbomethoxydihydrocleavamine (3.105 mg, -4 8.38 x 10 mCi) , which had been l a b e l l e d i n the aromatic system, was fed t o Vinca rosea L. p l a n t s i n the same manner as above. In t h i s case, however, the f o l i a g e on the p l a n t s was about 5 months o l d and the p l a n t s were worked up a f t e r 46 hours. The e x t r a c t i o n procedure was the same as described above - 22C -and the crude a l k a l o i d a l e x t r a c t contained 35% of the a c t i v i t y t hat had been fed. The crude e x t r a c t was chromatographed on alumina ( a c t i v i t y I I I ) . E l u t i o n w i t h 3:1 petroleum ether (30-60)-benzene provided [T-aromatic]-IS 8 -carbomethoxycleavamine. This m a t e r i a l was d i l u t e d with i n a c t i v e 18 0-carbomethoxycleavamine and the mixture c r y s t a l l i z e d to constant a c t i v i t y . About 11% of the fed a c t i v i t y was recovered i n the form o f unchanged 18 8-carbomethoxycleavamine. E l u t i o n w i t h 1:1 petroleum ether (30-60)-benzene provided catharanthine. A n a l y t i c a l t h i n - l a y e r chromatography of the cathar-anthine obtained showed that i t contained s e v e r a l i m p u r i t i e s . Consequently, the c a t h a r a n t h i i i e was d i l u t e d with a measured amount of i n a c t i v e catharanthine and chromatographed on alumina. E l u t i o n as before provided catharanthine which was seen to be pure by t h i n - l a y e r chromatography except f o r some i m p u r i t i e s which were observable only by t h e i r f l u o r e s c e n c e under u l t r a v i o l e t l i g h t . These i m p u r i t i e s were t r a n s p o r t e d at or n e a r l y at the same r a t e as catharanthine i n alumina t h i n - l a y e r chromatoplates but were seen to be tra n s p o r t e d i n the main at a much slower r a t e on s i l i c a g e l chromatoplates. A c c o r d i n g l y the catharanthine was chromatographed on s i l i c a g e l ( a c t i v i t y I I I ) . A t r i a l chromatography showed that a cons i d e r a b l e amount of catharan-t h i n e would be l o s t t o the column i f the chromatography was c a r r i e d out slowl y . Therefore the catharanthine was chromatographed q u i c k l y . The catharanthine obtained (80% recovery) was seen to be f r e e from most of the f l u o r e s c e n t i m p u r i t i e s . I t was then combined with an a d d i t i o n a l p o r t i o n of i n a c t i v e catharanthine and the mixture r e c r y s t a l l i z e d a t o t a l of f i v e times from methanol. A f t e r each c r y s t a l l i z a t i o n a sample was counted. The a c t i v i t y i n the catharanthine was seen to decrease w i t h each r e c r y s t a l l i z a -t i o n to a counting r a t e of only 2.2 dpm/mg. This corresponded to a - 221 -maximum t o t a l a c t i v i t y i n the i s o l a t e d catharanthine of 208 dpm which corresponded t o 0.011% o f the a c t i v i t y fed. Experiment u s i n g [T-aromatic]-vincaminoreine as t r a c e r Three alkalo.'ds which could conceivably be b i o s y n t h e s i z e d from v i n c a -minoreine (75) by Vinca minor L. p l a n t s are minovine (76), vincamine (59) and 1,2-dehydroaspidospermidine (77). A c c o r d i n g l y , an experiment was COOMe conducted to see whether these transformations were c a r r i e d out by Vinca  minor L. p l a n t s . A d i f f e r e n t method of feeding than t h a t described above was used. Since i t was f e l t that the s i t e of b i o s y n t h e s i s of the a l k a l o i d s might be i n the leaves of the Vinca p l a n t s , there was some concern that the negative r e s u l t s obtained i n the previous experiments could have been caused by f a i l u r e of the t r a c e r to be tr a n s p o r t e d to the leaves. For t h i s reason a method of feeding the leaves was sought. One method g e n e r a l l y used f o r feeding leaves d i r e c t l y i s to make a s l i t along the c e n t r a l v e i n of the l e a f from the apex towards the heel and bend the t r i a n g u l a r p e n i n s u l a that i s formed i n t o a v e s s e l c o n t a i n i n g a s o l u t i o n of the t r a c e r . In our e x p e r i -ments small t r i a n g u l a r bags, made from p l i a b l e p l a s t i c s h e e t i n g , were used - 2 2 2 -as the v e s s e l s . The placement of the bags i s i l l u s t r a t e d below i n Figure 2 6 plastic bag Figure 2 6 . Bag-on-leaf method of feeding Mature greenhouse grown Vinca minor L. p l a n t s were fed w i t h an aqueous s o l u t i o n of [T-aromatic]-vincaminoreine as the acetate s a l t by the bag-on-l e a f method. The t r i t i u m l a b e l l e d vincaminoreine ( 1 . 5 0 7 mg, 1 . 2 x 1 0 mCi) as the acetate s a l t was d i s s o l v e d i n d i s t i l l e d water. The aqueous s o l u t i o n was d i v i d e d i n t o two p o r t i o n s . One p o r t i o n ( 1 5 % , - 1 . 4 5 x 1 0 ^ mCi) was saved as a blank and the other p o r t i o n ( 8 5 % , 1 . 0 5 x 1 0 mCi) was used to f i l l the bags. A f t e r the feeding was complete the a c t i v i t y remaining i n the bags was determined and found to be 2 1 % of that put i n them. The amount of a c t i v i t y as [T-aromatic]-vincaminoreine that was taken i n t o the p l a n t was - 4 determined by d i f f e r e n c e t o be 8 . 2 5 x 1 0 mCi. Caution was taken to assure t h a t as l i t t l e time as p o s s i b l e was wasted from the time a l e a f was cut u n t i l a bag was put i n place and f i l l e d w i t h some of the s o l u t i o n of the t r a c e r . The bags were never allowed to be sucked to dryness. D i s t i l l e d water was i n j e c t e d i n t o the bags as r e q u i r e d . The p l a n t s were worked up a f t e r four days. The a l k a l o i d s were ex t r a c t e d u s i n g the procedure above except that methanol was used i n place of the benzene - 1 5 N ammonium hydroxide mixture and methylene c h l o r i d e was used i n pla c e of benzene i n the back e x t r a c t i o n - 223 -step. The t o t a l a c t i v i t y recovered i n the crude a l k a l o i d a l e x t r a c t was 46.5% of the a c t i v i t y fed.. The crude e x t r a c t was chromatographed on alumina ( a c t i v i t y I I I ) . E l u t i o n w i t h 1:1 petroleum ether (30-60) provided v i n c a -minoreine i n the i n i t i a l f r a c t i o i i s and minovine i n the l a t e r f r a c t i o n s with some overlap i n the middle f r a c t i o n s . E l t u i o n w i t h 3:1 benzene-petroleum ether (50-60) provided a mixture of compounds i n which 1,2-dehydroaspido-spermidine was a major component. E l u t i o n w i t h an a d d i t i o n a l q u a n t i t y of t h i s s olvent mixture provided vincamine. The vincamine was r e c r y s t a l l i z e d from methanol and i t s counting r a t e determined. The percent of the a c t i v i t y which had been fed that was a s s o c i a t e d w i t h the vincamine when f u r t h e r p u r i f i c a t i o n was i m p r a c t i c a l was 0.0021. The mixture of compounds that contained 1,2-dehydroaspidospermidine was reduced w i t h l i t h i u m aluminum hydride i n ether. The crude r e d u c t i o n product was chromatographed on alumina. E l u t i o n w i t h 3:1 benzene-petroleum ether (30-60) provided p a r t i a l l y pure aspidospermidine. Further p u r i f i c a t i o n o f the aspidospeimiidine, f i r s t as the f r e e base and then as the h y d r o c h l o r i d e s a l t brought the l e v e l of a c t i v i t y that was a s s o c i a t e d w i t h the aspidospermi-dine down to 0.008% of the a c t i v i t y which had been f e d . The minovine was p u r i f i e d and i t s r a t e of i n c o r p o r a t i o n was found t o be 0.7%(SH). When the blank was worked up, i t was found t h a t the vincaminoreine had been converted to minovine i n 0.3%- y i e l d (SH). This behavior was a l s o observed i n the case of quebrachamine and i t was e s t a b l i s h e d t h a t quebrach-amine was converted i n t o 1,2-dehydroaspidsopermidine simply by standing i n a i r . In one experiment (SH), quebrachamine was allowed to stand i n s o l u t i o n f o r 10 days, and then t r e a t e d with l i t h i u m aluminum hydride. The y i e l d of aspidospermidine was shown to be 3.4%. - 224 -Experiment using [T-aromatic]-vincadine as t r a c e r A l l the t r a c e r experiments described above and others conducted i n our l a b o r a t o r i e s at the same time by other workers gave negative r e s u l t s or questionable p o s i t i v e r e s u l t s . I t was f e l t at t h i s stage that i t would be d i f f i c u l t i n the case of the conversion of a precursor to an aspidosperma s k e l e t o n by transannular c y c l i z a t i o n to e s t a b l i s h whether the c y c l i z a t i o n was brought about by enzymic o x i d a t i o n i n the p l a n t or by a e r i a l o x i d a t i o n . Nevertheless, i t was hoped t h a t an a c t u a l i n c o r p o r a t i o n of a t r a c e r by the p l a n t could be demonstrated so that the question of whether the t r a c e r s were a c t u a l l y being t r a n s p o r t e d i n t o a s i t e of syntheses i n the p l a n t s could be answered. I f l a b e l l e d v incadine (74) were to be incorporated i n t o minovine (76) by the Vinca minor L. p l a n t s , t h i s question could be answered i n the a f f i r m a t i v e s i n c e only the p l a n t could b r i n g about an N-methylation. Vincadine was a l s o of i n t e r e s t as a probable precursor of vincamine. The conversion could be envisaged as being brought about by o x i d a t i o n of v i n c a -dine at the C-3 and C-19 s i t e s f o l l owed by a rearrangement such as that shown i n Figure 19. [T-aromatic]-vincadine was fed to mature Vinca minor L. p l a n t s as be-for e by the bag-on-leaf method. A s o l u t i o n of [T-aromatic]-vincadine -3 (1.133 mg, 1.79 x 10 mCi) was used. A f t e r the feeding was over, the a c t i v i t y remaining i n the bags was determined to be 31% of that contained i n the s o l u t i o n used. The t o t a l a c t i v i t y which had gone i n t o the leaves was -3 determined by d i f f e r e n c e to be 1.23 x 10 mCi and t h i s amount was considered to be the a c t i v i t y fed. The crude a l k a l o i d a l e x t r a c t was found to c o n t a i n 4 3% of the a c t i v i t y which had been fed to the p l a n t s . P u r i f i c a t i o n of the a l k a l o i d s of i n t e r e s t was c a r r i e d out i n the same fa s h i o n as i n the experiment - 225 -above. The maximum t o t a l a c t i v i t y which c o u l d have been present as the i s o l a t e d 1,2-iehydroaspidospermidine was found to be 0.003% o f the a c t i v i t y which had been fed t o the p l a n t . P u r i f i c a t i o n of minovine u n t i l i t was impossible to p u r i f y i t f u r t h e r brought the l e v e l of the maximum t o t a l a c t i v i t y which could have been present as i s o l a t e d minovine down t o 0.001%. In t h i s experiment the vincamine d i s p l a y e d a r a t e of i n c o r p o r a t i o n , but the blank experiment a l s o showed a r a t e of i n c o r p o r a t i o n that was comparable. Because no attempt had been made t o obta i n a r a d i o c h e m i c a l l y pure sample of [T-aromatic]-vincadine f o r the feeding experiment, i t was suspected that the vincadine which was l a b e l l e d contained a small amount o f vincamine which co u l d not be detected as an im p u r i t y by t h i n - l a y e r chromatography. When i n a c t i v e vincamine was added t o the t r i t i u m l a b e l l e d v i n c a d i n e and r e i s o -l a t e d i t was found t o have become r a d i o a c t i v e (SH). R e p e t i t i o n (SH) of the feeding experiment using p u r i f i e d t r i t i u m l a b e l l e d v i n c a d i n e showed that there was no i n c o r p o r a t i o n of vi n c a d i n e i n t o vincamine by Vinca rosea L. p l a n t s which could be detected i n our experiments. In summary, none of the experiments recounted i n t h i s t h e s i s were of a p o s i t i v e nature. The a c t i v i t i e s given f o r the most of the compounds s i g n i f y the degree to which the d i f f e r e n t samples co u l d be p u r i f i e d before f u r t h e r p u r i f i c a t i o n was no longer p o s s i b l e or p r a c t i c a l . In cases where a d e f i n i t e s p e c i f i c a c t i v i t y could be determined f o r a compound an e x p l a n a t i o n which d i d not i n v o l v e the p l a n t was p o s s i b l e . I t appeared on the b a s i s of the r e s u l t s obtained t h a t the transannular c y c l i z a t i o n as envisaged was not a step- i n the b i o s y n t h e s i s of the Iboga and Aspidosperma a l k a l o i d s . I t must be emphasized t h a t the above experiments were r e a l l y of a p r e l i m i n a r y nature to simply i n v e s t i g a t e whether high l e v e l s of i n c o r p o r a t i o n could be e a s i l y - 226 -achieved by the use of the above-mentioned l a r g e p r e c u r s o r s . C l e a r l y these r e s u l t s were not c o n c l u s i v e and more d e f i n i t i v e experiments were e s s e n t i a l . Due to lack of time I was unable to conduct such experiments but other workers i n t h i s l a b o r a t o r y have now done t h i s work. A recent communication on a l l of these r e s u l t s has now appeared. I t i s concluded that the t r a n s -annular c y c l i z a t i o n r e a c t i o n i s not of b i o s y n t h e t i c s i g n i f i c a n c e at l e a s t i n Vinca p l a n t s . I I I . Experimental M e l t i n g p o i n t s were determined on a K o f l e r block and are uncorrected. Nuclear magnetic resonance (nmr) s p e c t r a were recorded using d e u t e r i o c h l o r o -form as solvent on a Varian HA-100 instrument by Mr. R. Burton and the chemical s h i f t s are given i n the T i e r s T s c a l e . Mass s p e c t r a l were recorded on an A t l a s CH-4 mass spectrometer and high r e s o l u t i o n molecular weight determinations were performed by e i t h e r Mr. G. Eigendorf or Mr. G. Brown on an AEI MS-9 mass spectrometer. Woelm n e u t r a l s i l i c a g e l and alumina without binder or S i l i c a Gel G and Alumina G (according to S t a h l ) c o n t a i n i n g 5% by wt of General E l e c t r i c Ratma p-1. Type 118-2-7 e l e c t r o n i c phosphor were used f o r a n a l y t i c a l and p r e p a r a t i v e t h i n - l a y e r chromatography ( t i c ) . Chromatoplates were developed w i t h 2:1 carbon t e t r a c h l o r i d e - a n t i m o n y penta-c h l o r i d e spray reagent. Woelm n e u t r a l s i l i c a g e l and alumina ( a c t i v i t y I I I ) were used f o r column chromatography. S o l u t i o n s were d r i e d u s i n g anhyd sodium s u l f a t e . R a d i o a c t i v i t y was measured with a Nuclear-Chicago Mark 1 Model 6860 L i q u i d S c i n t i l l a t i o n counter i n counts per minute (cpm). The r a d i o a c t i v i t y of a sample i n d i s i n t e g r a t i o n s per minute (dpm) was c a l c u l a t e d using the counting e f f i c i e n c y which was determined f o r each sample by the e x t e r n a l standard t e c h n i q u e ^ 1 u t i l i z i n g the b u i l t - i n barium-133 gamma source. The standard d e v i a t i o n s i n the r a d i o a c t i v i t y of samples which are quoted 6 ^  ' were c a l c u l a t e d ~ so that the p r o b a b i l i t y of a number being w i t h i n the p r e s c r i b e d l i m i t s was 68%. The r a d i o a c t i v i t y of a l k a l o i d s as the f r e e - 22cJ -base was determined us i n g a s c i n t i l l a t e r s o l u t i o n made up according to the r e c i p e : toluene 1 I 2,5-diphenyloxazole (PPO) 4 g l , 4 - b i s [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] benzene (POPOP) 0.05 g The r a d i o a c t i v i t y of the s a l t s o f a l k a l o i d s and the r a d i o a c t i v i t y of crude e x t r a c t s were determined using a s c i n t i l l a t o r s o l u t i o n made up according to the r e c i p e : toluene 0.385 I dioxane 0.385 % methanol 0.230 I napthalene 80 g PPO 5 g POPOP 0.0625 g In p r a c t i c e a sample of an a l k a l o i d as the fr e e base was d i s s o l v e d i n 1 ml of benzene i n a counting v i a l using heat i f necessary and then the volume was made up to 15 ml with s c i n t i l l a t o r s o l u t i o n . In the case of the s a l t of an a l k a l o i d the sample was d i s s o l v e d i n i t i a l l y i n 1 ml of methanol. T r i t i u m l a b e l l e d 18a-carbomethoxy-4ct-dihydrocleavamine (67) 18a-carbomethoxy-4a-dihydrocleavamine (20.5 mg) was d i s s o l v e d i n t r i -f l u o r o a c e t i c a c i d (0.65 ml). Approx. 1 mCi of t r i t i u m l a b e l l e d water (0.01 ml) was added and the s o l u t i o n was heated at 50°C f o r four hours under a n i t r o g e n atmosphere. The s o l u t i o n was then added s l o w l y with s t i r r i n g to a saturate d sodium bicarbonate s o l u t i o n (5 ml) which was cooled i n an i c e -water bath. When the a d d i t i o n was complete, the s o l u t i o n was made f a i r l y s t r o n g l y b a s i c by the a d d i t i o n of sodium carbonate and e x t r a c t e d with methylene c h l o r i d e . The methylene c h l o r i d e e x t r a c t was d r i e d and the solvent was removed to y i e l d a residue which was comprised of -18a-carbo-methoxy-48-dihydrocleava.mine and a small amount of 188-carbomethoxy-43-dihydro-cleavamine as shown by t i c . P r e p a r a t i v e t i c ( s i l i c a g e l , 20 x 20 cm p l a t e , - 229 -0.5 t h i c k n e s s , chloroform as t r a n s p o r t i n g s o l v e n t , e x t r a c t i o n with e t h y l acetate) and c r y s t a l l i z a t i o n from methanol provided 14.8 mg of t r i t i u m l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine mp 171-172.5°C, w i t h a s p e c i f i c a c t i v i t y of 0.103 mCi/mmol. Deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine (67) 18a-carbomethoxy-43-dihydrocleavamine (50.0 mg) was d i s s o l v e d i n t r i -f l u o r o a c e t i c acid-d (0.7 ml) which had been prepared by h y d r o l y s i n g t r i -f l u o r o a c e t i c anhydride with deuterium oxide. The s o l u t i o n was heated at 50°C f o r four hours under a n i t r o g e n atmosphere. The s o l u t i o n was then added slo w l y w i t h s t i r r i n g to a sa t u r a t e d sodium bicarbonate s o l u t i o n (10 ml) which was maintained at the temperature of an ice-water bath. . A l i t t l e sodium carbonate was added to the s o l u t i o n to make i t d e f i n i t e l y b a s i c and the s o l u t i o n was e x t r a c t e d w i t h methylene c h l o r i d e (three 10 ml p o r t i o n s ) . A f t e r the e x t r a c t had been d r i e d , the solvent was removed to y i e l d a gummy residue (61 mg). C r y s t a l l i z a t i o n of the res i d u e y i e l d e d 17.4 mg o f deuterium l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine. The mother l i q u o r s from the above c r y s t a l l i z a t i o n were subjected to se p a r a t i o n by pr e p a r a t i v e t i c ( s i l i c a g e l , 20 x 20 cm p l a t e , 0.5 mm t h i c k n e s s , chloroform as t r a n s p o r t i n g s o l v e n t , e x t r a c t i o n w i t h e t h y l acetate) and i n t h i s manner an a d d i t i o n a l q u a n t i t y of deuterium l a b e l l e d 18a-carbcmethoxy-4a-dihydro-cleavamine (16.0 mg) and some deuterium l a b e l l e d 18g-carbomethoxy-4a-dihydro-cleavamine (68) (10.5 mg) as shown by t i c comparison w i t h an a u t h e n t i c sample were obtained. Deuterium l a b e l l e d I8B-carbomethoxycleavamine (73) Some 188-carbomethoxycleavamine (152.3 mg) was d i s s o l v e d i n t r i f l u o r o -a c e t i c a c i d - d and the s o l u t i o n was heated f o r four hours at 50°C under a - 230 -ni t r o g e n atmosphere. The same work up as given i n the previous experiment obtained (180.4 mg). This r e s i d u e was chromatographed on alumina (18 g). E l u t i o n with benzene provided 61.2 mg of deuterium l a b e l l e d 18 3-carbomethoxy-cleavamine, which showed some minor i m p u r i t i e s on a n a l y s i s by t i c ( s i l i c a g e l , c hloroform). A combination of p r e p a r a t i v e t i c ( s i l i c a g e l , chloroform, e x t r a c t i o n w i t h e t h y l acetate) and c r y s t a l l i z a t i o n from methanol and then methanol-water provided 16.1 mg of 183-carbomethoxycleavamine with mp 119-123 °C. T r i t i u m l a b e l l e d 18a-carbomethoxy-4a-dihydrocleavamine (67) - a small s c a l e  experiment A sample of 18a-carbomethoxy-4a-dihydrocleavamine (10.7 mg) was weighed i n a pie c e of gl a s s tubing which was sealed at one end. T r i t i u m l a b e l l e d t r i f l u o r o a c e t i c a c i d (20 y l , 0.88 mCi/mmol) was added and the gl a s s tubing was attached to a gl a s s "T" j o i n t as shown i n Figure 27. A f t e r the s o l u t i o n w i t h the appropriate adjustment of q u a n t i t i e s was used and a gummy residue dry NA sleeve /copper wire dry ice-acetone bath ^CF 3 C00H Figure 27. Apparatus f o r small s c a l e l a b e l l i n g experiments - 231 -had been'heated at 50°C f o r four hours under a n i t r o g e n atmosphere, i t was added to a cooled aqueous 10% sodium bicarbonate s o l u t i o n (0.5 ml)- with s t i r r i n g and the b a s i c s o l u t i o n obtained was ex t r a c t e d w i t h methylene c h l o r i d e (three 1 ml p o r t i o n s ) . A f t e r the methylene c h l o r i d e e x t r a c t had been d r i e d , the solvent was removed with the a i d of a n i t r o g e n stream and a warm water bath and a vacuum pump to y i e l d a foam (9.5 mg). C r y s t a l l i z a t i o n from methanol to constant a c t i v i t y gave a sample of 18a-carbomethoxy-4a-dihydrocleavamine w i t h s p e c i f i c a c t i v i t y of 0.205 mCi/mmol. T r i t i u m l a b e l l e d vincaminoreine (75) The experiment was c a r r i e d out as described above u s i n g vincaminoreine (10.29 mg) and l a b e l l e d t r i f l u o r o a c e t i c a c i d (20 y l , 0.88 mCi/mmol). A f t e r the work up had been completed, l a b e l l e d vincaminoreine (9.10 mg, 0.282 mCi/mmol), which showed only one spot on a n a l y s i s by t i c (alumina, benzene and s i l i c a g e l , 1:1 e t h y l a c e t a t e - c h l o r o f o r m ) , was obtained. T r i t i u m l a b e l l e d v i n c a d i n e (74) The experiment was c a r r i e d out as described above usi n g vincadine (5.14 mg) and l a b e l l e d t r i f l u o r o a c e t i c a c i d (20 ml, 0.88 mCi/mmol). A f t e r the work up, which i n c l u d e d r e c o v e r i n g the vincadine from a large volume of methanol to ensure that only an i n s i g n i f i c a n t percentage of the l a b e l would remain on the i n d o l e n i t r o g e n atom, had been completed,labelled vincadine (5.00 mg, 0.548 mCi/mmol), which appeared to be chemically pure as shown by t i c , was obtained. ] 4 Tracer experiment i n Vinca rosea L. p l a n t s using'[22- C]-183-carbomethoxy-48-dihydrocleava.mine (61) A sample of [22-^C]-188-carbomethoxy-46-dihydroc.leava.mine (6.823 mg, - 232 -1.67 x 10 mCi/mmol) was d i s s o l v e d i n d i e t h y l ether and p r e c i p i t a t e d as i t s h y d r o c h l o r i d e s a l t by blowing hydrogen c h l o r i d e vapours over the s o l u t i o n . A f t e r the d i e t h y l ether and excess a c i d had been removed by evaporation, the s a l t was d i s s o l v e d i n water (10 ml) and the aqueous s o l u t i o n was d i v i d e d among ten 5 ml t e s t tubes. Ten Vinca rosea L. p l a n t s with about two month o l d f o l i a g e and an o l d root system were cut on an angle through the stem. Each p l a n t as i t was severed from i t s root i n t h i s f a s h i o n was immediately placed i n one of the t e s t tubes c o n t a i n i n g a s o l u t i o n of the t r a c e r . A f t e r each p l a n t had been p e r m i t t e d t o take up the s o l u t i o n of t r a c e r u n t i l there was j u s t enough s o l u t i o n to keep the cut end moist, the volume was made up to 1 ml w i t h d i s t i l l e d water. A f t e r t h i s procedure was repeated s e v e r a l times to ensure that n e a r l y a l l the t r a c e r had been taken up by the p l a n t , the t e s t tubes were f i l l e d t o the top with d i s t i l l e d water and more water was added when i t was r e q u i r e d over a p e r i o d of e i g h t days. During t h i s p e r i o d of time between 10 ml and 30 ml of l i q u i d was taken up by each p l a n t and l i g h t was s u p p l i e d by a bank of neon tubes. A f t e r e i g h t days the p l a n t s (67.3 g) were chopped up with a p a i r of s c i s s o r s and macerated i n the presence o f 10:1 benzene - 15 N ammonium hydroxide (500 ml) i n a Waring blender. The green mash was f i l t e r e d through a c e l i t e pad i n a Buchner fu n n e l . The marc was separated from the c e l i t e pad and macerated again with 10:1 benzene - 15 N ammonium hydroxide (250 ml) and f i l t e r e d as before. The f i l t r a t e s were combined and the organic phase was separated from the aqueous phase. A f t e r the bulk of the solvent had been removed i n a r o t a r y evaporator, benzene (150 ml) was added and the s o l u t i o n extacted w i t h 2 N h y d r o c h l o r i c a c i d ( f i v e 50 ml p o r t i o n s ) . A f t e r the a c i d e x t r a c t had been washed with benzene ( f i v e 50 ml p o r t i o n s ) , an a d d i t i o n a l q u a n t i t y (100 ml) of benzene was added to i t and i t was made s t r o n g l y b a s i c by the a d d i t i o n of 15 N ammonium hydroxide. The organic phase was drawn o f f and the aqueous phase was extracted with a further-q u a n t i t y (three 50 ml p o r t i o n s ) of benzene. The aqueous phase was then sa t u r a t e d with potassium carbonate and again extracted w i t h benzene (two 50 ml p o r t i o n s ) . The benzene e x t r a c t s were combined, washed with water (three 50 ml p o r t i o n s ) and s a l t b r i n e (one 50 ml p o r t i o n ) , and.dried. Removal of the solvent provided 139.0 mg of a gummy r e s i d u e . The r a d i o -a c t i v i t y (3.44 x 10 mCi) of t h i s residue represented 30% of the t o t a l a c t i v i t y f ed. Most of the crude a l k a l o i d a l e x t r a c t .(133.2 mg) was subjected to a sep a r a t i o n by p r e p a r a t i v e t i c . An alumina (Woelm) p l a t e (20 x 60 cm, 0.5 mm thi c k n e s s ) was used and the crude e x t r a c t was ap p l i e d i n a narrow band at one end of the p l a t e . A f t e r being t r a n s p o r t e d i n the d i r e c t i o n o f the longer measurement with 3:1 benzene-chloroform, the a l k a l o i d s were located as dark bands by observation of the p l a t e under u l t r a v i o l e t l i g h t . The p l a t e was d i v i d e d i n t o nine s e c t i o n s , from b a s e l i n e to solvent f r o n t with the bands corresponding to 18B-carbomethoxy-48-dihydrocleavamine, c o r o n a r i d i n e , dihydrocatharanthine and catharanthine c o i n c i d i n g with a d i f f e r e n t s e c t i o n . Each s e c t i o n was scraped o f f the p l a t e and. e l u t e d w i t h methanol. Removal of the solvent gave nine groups of a l k a l o i d s . The a c t i v i t y i n each group was determined and the t o t a l r a d i o a c t i v i t y was 1.95 x 10 mCi (59% of that placed on the p l a t e ) . A t t e n t i o n was d i r e c t e d towards i n v e s t i g a t i n g groups 4(48% of recovered a c t i v i t y , av. 0.55), 5 (25% of recovered a c t i v i t y , av. R^ 0.45) and S (1.5% of recovered a c t i v i t y , av. R^ 0.1), which c o n s i s t e d mainly of 183-carbomethoxy-43-dihydrocleavamine, coronaridine and catharanthine, r e s p e c t i v e l y , as shown by comparison t i c using s e v e r a l systems, and group 7 (1.2% of recovered a c t i v i t y , av R f 0.2) i n which - 234 -dihydrocatharanthine would occur, i f i t were present. Group 5 was again subjected to p r e p a r a t i v e t i c on an alumina (Woelm) p l a t e (5 x 20 cm, 0.25 mm t h i c k n e s s ) . A f t e r being t r a n s p o r t e d with 3:1 benzene-chloroform, the bands corresponding to 183-carboamethoxy-43-dihydrocleavamine, c o r o n a r i d i n e and the i n t e r v a l between these were scraped o f f the p l a t e and e l u t e d with methanol. Removal of the solvent provided three residues which corresponded to 37.5%, 9.3% and 36.0%, r e s p e c t i v e l y , of the a c t i v i t y i n group 5. The residue which appeared to co n t a i n only c o r o n a r i d i n e as shown by comparison t i c using alumina p l a t e s was shown to be composed of two compounds on s i l i c a g e l p l a t e s . T i c ( s i l i c a g e l , 3:1 chlo r o f o r m - e t h y l a c e t a t e ) : chromatoplate showed a green spot of R^ 0.7, which corresponded i n R£ and c o l o u r r e a c t i o n to c o r o n a r i d i n e , and a pink spot at R^ 0.55. The mixture was then r e s o l v e d by p r e p a r a t i v e t i c using the above system. At t h i s stage i t was no longer p r a c t i c a l to attempt a f u r t h e r p u r i f i c a t i o n and i t was determined that the r a d i o a c t i v i t y of the c o r o n a r i d i n e was 242 dpm (0.09% of the r a d i o a c t i v i t y that was fed) whereas the r a d i o -a c t i v i t y of the unknown m a t e r i a l was 2000 dpm (0.8% of the r a d i o a c t i v i t y MeOH that was f e d ) . The s p e c t r a l data of t h i s unknown m a t e r i a l : A 327, 297, • max 228; mass spectrum m/e ( r e l i n t e n s i t y ) 338(35), 124(100) i n d i c a t e d that i t might be 7 8 - e t h y l - 5 - d e s e t h y l v i n c a d i f f o r m i n e (7S-ethyl-5-desethyl-63). Anal. C a l c d f o r CniH_^K' 0 • mol wt, 338.199. Found: mol wt, 338.197 21 26 6 2 (mass spectrometry). Group 7 was d i l u t e d w i t h a small amount of au t h e n t i c dihydrocatharanthine and subjected to p r e p a r a t i v e t i c . The r e i s o l a t e d dihydrocatharanthine a f t e r p r e p a r a t i v e t i c (alumina, 3:1 benzene-chloroform and s i l i c a g e l , 1:1 chloroform e t h y l acetate) was found to have no r a d i o a c t i v i t y . The catharanthine component i n group 8 was subjected to p u r i f i c a t i o n by - 235 -t i c (by SH) and when p u r i f i c a t i o n was found to be no longer p r a c t i c a l , the r a d i o a c t i v i t y of the catharanthine was 75 dpm (0.02% of the a c t i v i t y which had been f e d ) . Tracer Experiment i n Vinca rosea L. p l a n t s using [T-aromatic]-188-carbo-methoxycleavamine (73) A sample of [T-aromatic] -18 6-carbomethoxyc.leavamine (3.105 mg; 8.38 x -4 10 mCi) was fed as i t s h y d r o c h l o r i d e s a l t i n the same manner as was described i n the preceding experiment. Ten Vinca rosea L. p l a n t s which had f i v e month o l d f o l i a g e and were not f l o w e r i n g at the time of the experiment were used. The p l a n t s were worked up a f t e r 46 hours during which time each had taken up an average of 13 ml of s o l u t i o n . The p l a n t s (124.2 g) were worked up i n the same f a s h i o n as was described i n the previous experiment and i n t h i s case 213.1 mg of a crude -4 a l k a l o i d a l e x t r a c t was obtained, the r a d i o a c t i v i t y of which was 2.9 x 10 mCi (35% of the r a d i o a c t i v i t y which had been f e d ) . About 1% of the r a d i o -a c t i v i t y fed was recovered i n the t e s t tubes a f t e r the fee d i n g . Most of the crude a l k a l o i d a l e x t r a c t (212 mg) was chromatographed on alumina (20 g ) . E l u t i o n w i t h 3:1 to 2:1 petroleum ether (bp 30-60)-benzene provided crude [T-aromatic]-183-carbomethoxycleavamine. D i l u t i o n w i t h i n a c t i v e 186-carbomethoxycleavamine and r e c r y s t a l l i z a t i o n to constant a c t i v i t y from methanol showed that 11% of the a c t i v i t y which had been fed or 31% of the a c t i v i t y which was recovered i n the crude a l k a l o i d a l e x t r a c t was due to unchanged 186~carbomethoxycleavamine. E l u t i o n w i t h 1:1 petroleum ether (bp 30-60)-benzene provided 25.6 mg of crude catharanthine which was seen to conta i n s e v e r a l minor i m p u r i t i e s by t i c (alumina, 3:1 benzene-chloroform) The crude catharanthine was combined w i t h i n a c t i v e c a t h a ranthine t o give - 236 -53.2 mg of which 46.6 mg was chromatographed on alumina (5 g ) . E l u t i o n w i t h 1:1 petroleum ether (bp 30-60)-benzene provided 39.3 mg of catharanthine. which showed only f l u o r e s c e n t ( u l t r a v i o l e t l i g h t ) i m p u r i t i e s when subjected to t h i n - l a y e r chromatographic a n a l y s i s using the above mentioned system. The catharanthine from t h i s chromatography was chromatographed on s i l i c a g e l (4 g) at a very r a p i d r a t e (almost a p e r c o l a t i o n ) . Most; of the f l u o r -escent i m p u r i t i e s were e l u t e d w i t h 10% d i e t h y l ether benzene. E l u t i o n w i t h 15% to 50% d i e t h y l ether benzene provided 31.4 mg of catharanthine shown to be f r e e of much of the f l u o r e s c e n t i m p u r i t y . This m a t e r i a l was then d i l u t e d with i n a c t i v e catharanthine to give 56.6 mg of catharanthine which was r e c r y s t a l l i z e d f i v e times from methanol to provide a sample with a counting r a t e of 2.2 t 0.3 dpm/mg. The r a d i o a c t i v i t y of the catharanthine was 208 + 24 dpm (0.011 + 0.001% of the r a d i o a c t i v i t y t h a t was f e d ) . Tracer experiment i n Vinca minor L. p l a n t s using [T-aromatic]-vincaminoreine (75) Vinca minor L. p l a n t s , which were mature and greenhouse grown, were fed by the bag-on-leaf method w i t h [T-aromatic]-vincaminoreine as the acetate s a l t . The acetate s a l t was prepared from a sample of [T-atomatic]-vinca-minoreine (1.507 mg, 1.20 x 10 mCi) and d i s s o l v e d i n d i s t i l l e d water. The aqueous s o l u t i o n was d i v i d e d i n t o two p o r t i o n s , the l a r g e r p o r t i o n (85%, 1.05 x 10 mCi) of which was fed to the p l a n t s and the s m a l l e r p o r t i o n -4 (15%, 1.45 x 10 mCi) of which was put aside as a blank. The p e r i o d of time used i n feeding the p l a n t s was four days and du r i n g t h i s p e r i o d of -4 time the p l a n t s took i n 79% of the r a d i o a c t i v i t y or 8.25 x 10 mCi. The method used i n the i s o l a t i o n of the a l k a l o i d s ' from the p l a n t s was s i m i l a r to the method used i n the experiments using Vinca rosea L. p l a n t s described i n the preceding experiments. The only d i f f e r e n c e s were th a t methanol was used i n place of the benzene ammonium hydroxide mixture i n the maceration step and t h a t methylene c h l o r i d e was used i n the f i n a l , e x t r a c t : o n step. Using the modified procedure 111.1 mg of crude a l k a l o i d a l e x t r a c t was obtained from about 35 g of p l a n t m a t e r i a l . I t should be emphasized that the e n t i r e p l a n t was e x t r a c t e d - r o o t s , stems, and leaves. The r a d i o --4 a c t i v i t y recovered i n the crude a l k a l o i d a l was 3.38 x 10 mCi (46.5% of the a c t i v i t y which was determined to have been taken i n by the p l a n t s ) . Most of the crude a l k a l o i d a l e x t r a c t (110 mg) was chromatographed on alumina (10 g). E l u t i o n . w i t h 1:1 petroleum ether (bp 30-60)-benzene provided unchanged vincaminoreine i n the e a r l y f r a c t i o n s and minovine i n the l a t e f r a c t i o n s w i t h some overlap of the two i n the middle f r a c t i o n s . E l u t i o n w i t h 3:1 benzene-petroleum ether (bp 30-60) provided 1,2-dehydroaspidospermi-dine i n a mixture with other unknown a l k a l o i d s as shown by t i c . F urther e l u t i o n w i t h 3:1 benzene-petroleum ether (bp 30-60) provided a mixture (4.65 mg) which was almost e n t i r e l y composed of vincamine. C r y s t a l l i z a t i o n o f t h i s mixture from methanol provided 3.30 mg of vincamine with a count-i n g r a t e o f 8.5 t 0.9 dpm/mg. Assuming 4.64 mg to be the wt of vincamine th a t had been i s o l a t e d , the percent of the r a d i o a c t i v i t y fed t h a t was s t i l l a s s o c i a t e d w i t h the vincamine was 0.0021 t 0.0002. The mother l i q u o r s from the vincamine c r y s t a l l i z a t i o n , which were shown by t i c to c o n t a i n some 1,2-dehydroaspidospermidine, and the appropriate f r a c t i o n s from the chromatography were combined t o give 7.8 mg of a mixture which was ta-ken up i n d i e t h y l ether (3 ml) and mixed with a s o l u t i o n o f l i t h i u m aluminum hydride (30 mg) i n d i e t h y l ether (3 ml). The r e a c t i o n s o l u t i o n was s t i r r e d overnight at room temperature under a n i t r o g e n atmosphere. - 238 -A f t e r the excess l i t h i u m aluminum hydride had been destroyed by the a d d i t i o n of wet e t h y l a c e t a t e , the i n o r g a n i c m a t e r i a l was removed by f i l t r a t i o n and the f i l t r a t e was d r i e d w i t h anhyd sodium s u l f a t e . Removal of the solvent provided a gummy m a t e r i a l (7.3 mg) which was chromatographed on alumina (1 g). E l u t i o n w i t h 3:1 benzene-petroleum ether (bp 30-60) provided 0.58 mg of nea r l y pure aspidospermidine. The aspidospermidine was d i l u t e d t o 7.55 mg wit h i n a c t i v e aspidospermidine and c r y s t a l l i z e d from methanol to give 5.28 mg with a counting r a t e of 57.6 dpm/mg corresponding to an as s o c i a t e d l e v e l of a c t i v i t y of 0.023%. The aspidospermidine (4.24 mg) was then d i l u t e d again to 16.95 mg wi t h an i n a c t i v e sample, r e c r y s t a l l i z e d once from methanol and converted to the h y d r o c h l o r i d e s a l t . The s a l t was washed s e v e r a l times w i t h hot acetone and d r i e d vacuo overnight to provide a sample with a counting r a t e of 3.95 + 0.45 dpm/mg. The r a d i o a c t i v i t y of the aspido-spermidine was, t h e r e f o r e , 154 ± 17 dpm (0.0083 i " 0.0009% o f the r a d i o -a c t i v i t y which was f e d ) . The minovine (76) c o n t a i n i n g f r a c t i o n s (3.47 mg) were combined and d i l u t e d to 21.97 mg with i n a c t i v e minovine. Through three c r y s t a l l i z a t i o n s the l e v e l of a c t i v i t y remained constant. The t h i r d c r y s t a l l i z a t i o n gave a sample with a counting r a t e of 615 dpm/mg. The r a d i o a c t i v i t y of the i s o l a t e d minovine was 6.0 x 10 ^  mCi (apparent i n c o r p o r a t i o n 0.7%) (by SH). The work up of the blank, which had stood f o r ten days i n an open t e s t tube, was accomplished by r e v e r s i n g the pi-1 w i t h ammonium hydroxide and e x t r a c t i n g w i t h methylene c h l o r i d e . The e x t r a c t e d m a t e r i a l (40% of the a c t i v i t y ) was mixed with 2 mg of i n a c t i v e minovine and chromatographed on alumina. E l u t i o n w i t h 1:1 petroleum ether - benzene provided 0.7 mg of minovine which was d i l u t e d to 22.0 mg with i n a c t i v e minovine and r e c y s t a l l i z e d three times. The counting r a t e was seen to r i s e s l i g h t l y but not - 239 -s i g n i f i c a n t l y to give a f i n a l counting r a t e of 66 dpm/mg which corresponded to an apparent i n t r a c o n v e r s i o n o f vincaminoreine to minovine of 0.3% (by SH). Tracer experiment i n Vinca minor L. p l a n t s using [T-aromatic]-vincadine (74) Vinca minor I . , p l a n t s , which were mature and greenhouse grown, were fed by the bag-on-leaf method w i t h [T-aromatic]-vincadine (1.133 mg, 1.79 x -3 10 mCi) as i t s acetate s a l t . A f t e r seven days had passed, the r a d i o -_3 a c t i v i t y taken i n by the p l a n t was 1.23 x 10 mCi (69%). The a l k a l o i d s were i s o l a t e d i n the same manner th a t was used i n the previous experiment and 26 g of p l a n t m a t e r i a l afforded 89.0 mg of crude a l k a l o i d a l e x t r a c t , -4 the r a d i o a c t i v i t y of which was 5.45 x 10 mCi (43% of the r a d i o a c t i v i t y taken i n by the p l a n t s ) . The crude a l k a l o i d a l e x t r a c t (87.8 mg) was chroma-tographed i n the same f a s h i o n as i n the previous experiment. In t h i s experiment the f r a c t i o n s which contained vincamine weighed 4.26 mg and c r y s t a l l i z a t i o n , from methanol provided 1.36 mg of vincamine (59) w i t h a counting r a t e of 1173 dpm/mg. Some of t h i s sample of vincamine (0.83 mg) was then d i l u t e d to 10.17 mg with i n a c t i v e vincamine ( d i l u t i o n f a c t o r , 1225) and c r y s t a l l i z e d twice from methanol t o give a sample with a counting r a t e of 89.8 +3.7 dpm/mg (89.8 ± 2.7 dpm/mg x 12.25 = 1100 ± 33 dpm/mg). This r e s u l t meant that the apparent r a t e of i n c o r p o r a t i o n was between the l i m i t s 0.054% and 0.17% depending on whether 1.36 mg or 4.26 mg i s taken to be the amount of vincamine obtained fronvuthe p l a n t . The blank was worked up i n the f o l l o w i n g f a s h i o n . A f t e r i n a c t i v e vincamine (2.0 mg) had been added to the blank, i t was made b a s i c w i t h ammonium hydroxide and e x t r a c t e d w i t h methylene c h l o r i d e . The crude e x t r a c t which contained 80% of the a c t i v i t y was chromatographed on alumina. E l u t i o n w i t h 2:1 petroleum ether (bp 30-60)-benzene was c a r r i e d out u n t i l a l l the - 240 -a c t i v i t y .as v i n c a d i n e had been eluted as i n d i c a t e d by counting s e l e c t e d f r a c t i o n s . E l u t i o n with 3:1 benzene-petroleum ether then provided 1.85 .ng of vincamine which was d i l u t e d t o 11.65 mg with i n a c t i v e vincamine (59) and c r y s t a l l i z e d twice from methanol. The f i r s t c r y s t a l l i z a t i o n gave 7.88 mg wi t h counting r a t e of 94 t 4 dpm/mg and the second, 6.02 mg with a counting r a t e of 88.2 i 1.7 dpm/mg. The r a d i o a c t i v i t y of the vincamine was t h e r e f o r e 1100 + 21 dpm (0.068 ± 0.001% o f the r a d i o a c t i v i t y of the bl a n k ) . The 1,2-dehydroaspidospermidine _(77) was converted i n t o aspidospermi-dine i n the same f a s h i o n t h a t had been employed i n the case of the previous experiment. The p a r t i a l l y p u r i f i e d aspidsopermidine (0.34 mg) obtained from the column chromatography was d i l u t e d to 10.27 mg with i n a c t i v e aspido-spermidine and r e c r y s t a l l i z e d from methanol to a f f o r d 4.4 mg of c r y s t a l l i n e aspidsopermidine w i t h a counting r a t e of 9.25 ± 1.6 dpm/mg. The r a d i o -a c t i v i t y o f the aspidospermidine was t h e r e f o r e 9 5 + 1 3 dpm (0.0034 t 0.0006% of the r a d i o a c t i v i t y which had been taken up by the p l a n t s ) . The minovine c o n t a i n i n g f r a c t i o n s (ca. 1 mg) were combined and d i l u t e d to 18.3 mg with i n a c t i v e minovine. 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