UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on the synthesis and biosynthesis of indole alkaloids Lewis, Norman G. 1978

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STUDIES ON THE SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS BY NORMAN GEORGE LEWIS .Sc., U n i v e r s i t y o f S t r a t h c l y d e , Glasgow, (1973) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES Department o f C h e m i s t r y ' U n i v e r s i t y o f B r i t i s h Columbia We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y , 19 78 © Norman George L e w i s , 19 78 In presenting th is thes is in par t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permiss ion. Department of CHEMIST^ The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date ABSTRACT P a r t I o f t h i s t h e s i s d e s c r i b e s the more r e c e n t i n v e s t i g a t i o n s towards t h e e l u c i d a t i o n o f t h e b i o s y n t h e t i c pathways l e a d i n g t o the f o r m a t i o n of a c l a s s o f i n d o l e a l k a l o i d s found i n Aspidosperma v a r g a s i i . I n t h i s r e s p e c t , the i n v i v o r o l e o f t r y p t o p h a n (lb) and stemmadenine (63) were s t u d i e d but the i n c o r p o r a t i o n l e v e l s o b t a i n e d were not co n d u c i v e w i t h the a c t i v e i n t e r m e d i a c y o f e i t h e r ( l b ) o r (63) i n the b i o s y n t h e s i s o f the a l k a l o i d s u l e i n e (103), guatambuine (104) o r 9-methoxy-o l i v a c i n e (111). C o n d i t i o n s f o r the growth o f Aspidosperma a u s t r a l e ,  A. p y r i c o l l u m and A. v a r g a s i i t i s s u e c u l t u r e s a re a l s o r e p o r t e d . P a r t I I d i s c u s s e s the more r e c e n t s t u d i e s towards the s y n t h e -s i s o f stemmadenine (63) w i t h r a d i o a c t i v e l a b e l s a t the r e q u i r e d p o s i t i o n s i n the m o l e c u l e . The s t u d i e s i n i t i a l l y i n v o l v e d c o n v e r s i o n o f s t r y c h n i n e (5) t o 2yS, 16c(-cur-19-en-17-ol (143) by a p r e v i o u s l y d e s c r i b e d sequence o f r e a c t i o n s . C o n d i t i o n s f o r the e f f i c i e n t c o n v e r s i o n t o the known 2 / 3 - c u r - 1 9 - e n - l 7 - a l (145) were de v e l o p e d b u t subsequent c o n v e r s i o n t o stemmadenine (63) was n o t a c c o m p l i s h e d . The c o n v e r s i o n o f (143) t o des-carbomethoxystemmadenine (128) i s r e p o r t e d . F u r t h e r s t u d i e s towards the s y n t h e s i s o f stemmadenine (6 3) were i n i t i a t e d from methyl - 2 y 3 ,16<*-cur-19-en-17-oate (133). The e s t e r ( 1 3 3 ) , d e r i v e d from s t r y c h n i n e (5) i n o v e r a l l low y i e l d v i a W i e l a n d - G u m l i c h aldehyde (129) was an i m p o r t a n t i n t e r m e -d i a t e i n t h e s y n t h e s i s o f epistemmadenine (138). A more e f f i c i e n t s y n t h e s i s o f (133) was de v e l o p e d from W i e l a n d - G u m l i c h a l d o x i m e (130). E s t e r (133) was e f f i c i e n t l y c o n v e r t e d t o (-) akuammicine (64) by t r e a t m e n t w i t h l e a d t e t r a - a c e t a t e and t h e s e r e c e n t c o n d i t i o n s have been s u c c e s s f u l l y a p p l i e d i n t h e t o t a l s y n t h e s i s o f v i n d o l i n e ( 1 1 ) . Akuammicine (64) was con-v e r t e d t o deshydroxymethylstemmadenine (122). A t t e m p t s t o c o n v e r t (122) o r N -carbomethoxydeshydroxymethylstemmadenine (175) t o stemmadenine (63) were u n s u c c e s s f u l . These f a i l u r e s prompted a l k y l a t i o n s t u d i e s w i t h t h e model system, 1 - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (156) p r e p a r e d from t e t r a h y d r o c a r b a z o l e (155) v i a a t h r e e s t e p s y n t h e s i s . The N-carbomethoxy d e r i v a t i v e (170) o f (156) was t r e a t e d w i t h formaldehyde i n t h e p r e s e n c e o f p o t a s s i u m h y d r i d e and gave the r e q u i r e d 1 - c a r b o m e t h o x y - l - h y d r o x y m e t h y l -1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (157) i n good y i e l d . F u r t h e r a l k y l a t i o n s t u d i e s w i t h 18/ 3-carbomethoxycleavamine (72) and t h e c o r r e s p o n d i n g N - c a r b o m e t h o x y (180) and N - m e t h y l (183) d e r -a a i v a t i v e s were u n s u c c e s s f u l . Indeed, i t appears t h a t i n t r o -d u c t i o n o f t h e h y d r o x y m e t h y l group i n the more complex systems cannot be a c c o m p l i s h e d u s i n g t h i s s t r a t e g y . P a r t I I I o f t h i s t h e s i s i n v e s t i g a t e d the r o l e o f c a t h a r a n t h i : N ^ - o x i d e (205) as a p o s s i b l e p r e c u r s o r f o r the i n v i v o forma-t i o n o f t h e m e d i c i n a l l y i m p o r t a n t d i m e r i c a l k a l o i d v i n c r i s t i n e (201) i n C a t h a r a n t h u s r o s e u s . I n t h e s e s t u d i e s t h e c h e m i s t r y o f c a t h a r a n t h i n e (12) was a p p r o p r i a t e l y d e v e l o p e d i n o r d e r t h a t r a d i o a c t i v e l a b e l s a t (1) t h e a r o m a t i c p o s i t i o n s C-j^-C-j^ (2) C-19 (3) C-18 and (4) C-22 c o u l d be i n t r o d u c e d . (Ar 3H) c a t h a r a n t h i n e - N b - o x i d e (205) was a d m i n i s t e r e d t o i v C. r o s e u s and t h e a l k a l o i d v i n c r i s t i n e (201) i s o l a t e d by c o l d d i l u t i o n . The i n c o r p o r a t i o n l e v e l s o b t a i n e d do n o t g i v e s u b s t a n t i a l i n v i v o s u p p o r t f o r t h e i n t e r m e d i a c y o f (205) i n t h e b i o s y n t h e s i s o f (201). P a r t IV o f t h i s t h e s i s d i s c u s s e s the f o r m a t i o n o f i m p o r t a n t i n t e r m e d i a t e s i n t h e r e c e n t i n v e s t i g a t i o n s towards the s y n t h e -s i s o f the a n t i - t u m o u r a l k a l o i d s e l l i p t i c i n e (106) and o l i v a -c i n e (105) . I n t h i s r e s p e c t the s y n t h e s i s o f i n d o l - 2 - y l - l - ( 4 1 p y r i -d y l ) - e t h a n o l (239) was c a r r i e d o u t . H y d r o g e n o l y s i s o f (239) w i t h H 2/Pd/C a f f o r d e d i n d o l - 2 - y l - l - ( 4 * p y r i d y l ) - e t h a n e (240). T reatment o f (239) w i t h a c e t i c a c i d i n p y r i d i n e gave the r e q u i r e d i n d o l - 2 - y 1 - 1 - ( 4 * p y r i d y l ) - e t h e n e (241). W i t h t h e c h e m i s t r y d e v e l o p e d f o r t h e f o r m a t i o n o f d e r i v a t i v e s (239-241) f u r t h e r s t u d i e s f o r the i n t r o d u c t i o n o f t h e N'-methyl group and t h e C-3 s i d e c h a i n ((CH 3)- N CH 2) were e x e c u t e d t o g i v e d e r i v a t i v e s (246) and (247). The t e t r a h y d r o p y r i d i n e d e r i v a t i v e (248) was o b t a i n e d by sodium b o r o h y d r i d e r e d u c t i o n o f (246). The c y c l i s a t i o n o f (24 8) t o the p y r i d o c a r b a z o l e d e r i v a t i v e (235) was n o t a t t e m p t e d . However t h e c o n d i t i o n s n e c e s s a r y f o r t h e c y c l i s a t i o n have been r e p o r t e d f o r t h e s y n t h e s i s o f t h e c l o s e r e l a t e d a l k a l o i d e l l i p t i c i n e (106). F u r t h e r c y c l i s a t i o n s t u d i e s u s i n g t h e c o r r e s p o n d i n g d i h y d r o p y r i d i n e d e r i v a t i v e s o f (246) and (247) a r e c u r r e n t l y under i n v e s t i g a t i o n . V TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES v i LIST OF TABLES x ACKNOWLEDGEMENTS x i PART I INTRODUCTION 1 DISCUSSION 53 EXPERIMENTAL 61 PART II INTRODUCTION 1 DISCUSSION 66 EXPERIMENTAL 113 PART III INTRODUCTION 136 DISCUSSION 149 EXPERIMENTAL 170 PART IV INTRODUCTION 18 3 DISCUSSION 184 EXPERIMENTAL 193 REFERENCES 200 v i L i s t o f F i g u r e s F i g u r e 1 - Robinson p o s t u l a t e f o r b i o s y n t h e s i s of corynanthe and s t r y c h n o s s k e l e t a . F i g u r e 2 - Woodv/ard and Robinson p o s t u l a t e f o r the b i o s y n t h e s i s o f c o r y n a n t h e and s t r y c h n o s s k e l e t a . F i g u r e 3 - Some r e p r e s e n t a t i v e i n d o l e a l k a l o i d s commonly i s o -l a t e d i n b i o s y n t h e t i c i n v e s t i g a t i o n s . F i g u r e 4 - Thomas-Wenkert monoterpene p o s t u l a t e . F i g u r e 5 - Rearrangement o f monoterpene u n i t i n t o a s p i d o -sperma and i b o g a s k e l e t a . F i g u r e 6 - B i o s y n t h e t i c f o r m a t i o n of g e r a n i o l - p y r o p h o s p h a t e (32) F i g u r e 7 - B i o s y n t h e t i c f o r m a t i o n o f l o g a n i n (35). F i g u r e 8 - P o s t u l a t e d b i o s y n t h e t i c c o n v e r s i o n s f o r f o r m a t i o n of c a m p t o t h e c i n (56). F i g u r e 9 - P o s t u l a t e d b i o s y n t h e t i c f o r m a t i o n o f c o r y n a n t h e i n e aldehyde (57) and g e i s s o s c h i z i n e (5,8). F i g u r e 10 - Proposed b i o s y n t h e t i c o r i g i n s o f the s t r y c h n o s f a m i l y . F i g u r e 11 - Wenkert's proposed b i o s y n t h e t i c o r i g i n s o f the aspidosperma and i b o g a f a m i l i e s . F i g u r e 12 - S c o t t ' s m o d i f i c a t i o n of Wenkert's h y p o t h e s i s f o r the aspidosperma and i b o g a f a m i l i e s . F i g u r e 13 - Proposed mechanism f o r f o r m a t i o n o f n o r - f l u o r o -c u r a r i n e (82) . 3 F i g u r e 14 - I n c o r p o r a t i o n of (5 H) l o g a n i n (35) i n t o v i n d o -l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e (15). F i g u r e 15 - B i o s y n t h e s i s o f t r y p t o p h a n ( l b ) . F i g u r e 16 - The Wenkert p o s t u l a t e f o r the b i o s y n t h e s i s o f o l i v a c i n e (105), e l l i p t i c i n e (106) and u l e i n e (103). F i g u r e 17 - The D j e r a s s i p o s t u l a t e f o r the b i o s y n t h e s i s of a p p a r i c i n e (102). F i g u r e 18 - P o t i e r ' s i n v i t r o e x t r u s i o n o f C-2 u n i t o f fZ - e t h y l a m i n e s i d e c h a i n . v i i F i g u r e 19 - S c o t t ' s i n v i t r o c o n v e r s i o n o f stemmadenine (63) to v a l l e s a m i n e [ l 0 1 ) . F i g u r e 20 - P o t i e r ' s p o s t u l a t e d b i o s y n t h e s i s o f a p p a r i c i n e (102) from stemmadenine ( 6 3 ) . F i g u r e 21 - P o t i e r - J a n o t p o s t u l a t e f o r the b i o s y n t h e s i s o f a p p a r i c i n e (102), u l e i n e (103), guatambuine ( 1 0 4 ) , o l i v a c i n e (105), and e l l i p t i c i n e (106) . F i g u r e 22 - Kutney's a t t e m p t e d s y n t h e s i s o f 1 9 , 2 0 - d i h y d r o -stemmadenine (116). F i g u r e 23 - S n i e c k u s ' s y n t h e s i s o f a stemmadenine model. F i g u r e 24 - Kutney's a t t e m p t e d s y n t h e s i s o f stemmadenine ( 6 3 ) . F i g u r e 25 - Base c a t a l y s e d r i n g o p e n i n g o f descarbomethoxy-akuammicine (126). F i g u r e 26 - S y n t h e s i s o f 2,16-dihydroakuammicine (133). F i g u r e 27 - Kutney's s y n t h e s i s o f epi-stemmadenine (138). F i g u r e 28 - D e g r a d a t i o n o f s t r y c h n i n e (5) t o 2 ft , 16c(-cur-19-e n - 1 7 - o l (143). ' F i g u r e 29 - F o r m a t i o n o f W i e l a n d - G u m l i c h a l d e h y d e (129). F i g u r e 30 - Recent i n v e s t i g a t i o n s towards the s y n t h e s i s o f stemmadenine (63) - approach 1. F i g u r e 31 - M o d i f i e d Oppenauer o x i d a t i o n o f 2'8,16oC-cur-19-e n - 1 7 - o l (143). ' F i g u r e 32 - Pr o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f m e t h y l -c u r - 1 9 - e n e - f a m i l y . F i g u r e 33 - P o s t u l a t e d mass s p e c t r a l f r a g m e n t a t i o n o f a c y l a t e d d e r i v a t i v e (153). F i g u r e 34 - L e n z i ' s s y n t h e s i s o f 1 - c a r b o e t h o x y t e t r a h y d r o c a r -b a z o l e (156a). F i g u r e 35 - An a l t e r n a t i v e t o L e n z i ' s p o s t u l a t e . F i g u r e 36 - S y n t h e s i s o f 1 - b e n z o t r i a z o l e t e t r a h y d r o c a r b a z o l e (162) . F i g u r e 37 - F o r m a t i o n o f 1 - p y r i d i n i u m - l , 2 , 3 , 4 - t e t r a h y d r o -c a r b a z o l e bromide (163). F i g u r e 38 - S y n t h e s i s o f 1 - c a r b o m e t h o x y - l - c y a n o t e t r a h y d r o c a r -b a z o l e (168). F i g u r e 39 - S y n t h e s i s o f 1 - c a r b o m e t h o x y - l - h y d r o x y m e t h y l t e t -r a h y d r o c a r b a z o l e (157). v i i i F i g u r e 40 -F i g u r e 41 -F i g u r e 42 -F i g u r e 43 -F i g u r e 44 -F i g u r e 45 -F i g u r e 46 -F i g u r e 47 -F i g u r e 48 -F i g u r e 49 -F i g u r e 50 -F i g u r e 51 -F i g u r e 52 -F i g u r e 53 -F i g u r e 54 -F i g u r e 55 -F i g u r e 56 -F i g u r e 57 -F i g u r e 58 -Pro p o s e d mechanism f o r f o r m a t i o n o f 1-carbomethoxy-1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157). F u r t h e r i n v e s t i g a t i o n s towards the s y n t h e s i s o f stemmadenine (63) - approach 2. Proposed mass s p e c t r a l f r a g m e n t a t i o n o f N.-carbomethoxydeshydroxymethylstemmadenine (175). S t e r e o c h e m i c a l f e a t u r e s o f o x a z i n e s (138) and (187) . ProDosed r o u t e f o r t h e s y n t h e s i s o f stemmadenine (63) . S t r u c t u r a l p o s s i b i l i t i e s o b t a i n e d from c y c l i s a t i o n o f i n t e r m e d i a t e (195). Kutney's p r o p o s e d s y n t h e s i s o f stemmadenine ( 6 3 ) . A c i d c a t a l y s e d f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . S y n t h e s i s o f d i m e r i c a l k a l o i d s : c h l o r o i n d o l e n i n e approach. S y n t h e s i s o f d i m e r i c a l k a l o i d s : m o d i f i e d P o l o n o v -s k i a pproach. B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : enzyme a s s i s t e d f r a g m e n t a t i o n and c o u p l i n g . B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c i n d o l e n i n e approach. B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c " P o l o n o v s k i " c o u p l i n g . F o r m a t i o n o f 18 -/3 - carbomethoxycleavamine (72) from c a t h a r a n t h i n e (12) i n t r i f l u o r o a c e t i c a c i d . P r o p o s e d f o r m a t i o n o f 19 - o x o c a t h a r a n t h i n e (212). P r o p o s e d f o r m a t i o n o f 5 - oxo - 18/3 -carbomethoxy-c l e a v a m i n e (213). P r o p o sed mass s p e c t r a l f r a g m e n t a t i o n o f c a t h a r a n -t h i n e (12) . B u c h i ' s s y n t h e s i s o f v o a c a n g i n e (220). B u c h i ' s s y n t h e s i s o f (±) c a t h a r a n t h i n e ( 1 2 ) . i x F i g u r e 59 - A t t e m p t e d s y n t h e s i s o f " ' c c^CH^ c a t h a r a n t h i n e ( 1 2 ) . F i g u r e 60 - P r o p o s e d rearrangement o f c a t h a r a n t h i n e - -o x i d e (205). F i g u r e 61 - S y n t h e s i s o f I n d o l - 2 - y 1 - 1 - ( 4 * p y r i d y l ) - e t h a n o l (239). F i g u r e 62 - S y n t h e s i s o f i n t e r m e d i a t e s (246) and (247) . F i g u r e 63 - P o t i e r ' s s y n t h e s i s of e l l i p t i c i n e (106). F i g u r e 64 - S y n t h e s i s o f 5 - h y d r o x y - 2 , 6 , - d i m e t h y l - 6 H - p y r i d o -(4,3 b ) - c a r b a z o l e (257). F i g u r e 65 - P o s t u l a t e d mechanism f o r the f o r m a t i o n o f p y r i d o -c a r b a z o l e (257) from i n t e r m e d i a t e (256). F i g u r e 66 - S y n t h e s i s o f N - m e t h y l - 3 - e t h y l - 5 - b e n z y 1 - t e t r a h y d r o -p y r i d i n e (263). i X L i s t o f T a b l e s T a b l e 1 - F o r m a t i o n o f a l k a l o i d s from C. r o s e u s s e e d l i n g s . T a b l e 2 - F i n d i n g s o f t r y p t o p h a n ( l b ) i n c o r p o r a t i o n s i n t o a l k a l o i d s o f A. a u s t r a l e (A) and A. p y r i c o l l u m ( B ) . T a b l e 3 - Summary o f r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. p y r i c o l l u m . T a b l e 4 - Summary o f r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. a u s t r a l e . 3 T a b l e 5 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i r o o t c u t t i n g s . 3 T a b l e 6 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i whole p l a n t . 3 T a b l e 7 - R e s u l t s o f (Ar H) stemmadenine (63) a d m i n i s t r a t i o n t o A. v a r g a s i i . T a b l e 8 - C o m p o s i t i o n o f n u t r i e n t medium f o r s o l i d (agar) medium. 3 14 T a b l e 9 - I n c o r p o r a t i o n o f ( H, C) l o g a n i n (35) i n t o a l k a l o i d s o f C. r o s e u s . 1 2 T a b l e 10 - R e a c t i o n s o f c a t h a r a n t h i n e (12) w i t h H and H t r i f l u o r o a c e t i c a c i d . T a b l e 11 - Mass s p e c t r a l fragments o f c a t h a r a n t h i n e (12) -d e u t e r i u m e n r i c h m e n t e x p e r i m e n t s . 3 T a b l e 12 - I n c o r p o r a t i o n d a t a from a d m i n i s t r a t i o n o f (Ar H) c a t h a r a n t h i n e - - o x i d e (205) t o C. r o s e u s . ACKNOWLEDGEMENT S The a u t h o r w i s h e s t o thank Dr. James P. Kutney f o r h i s d i r e c t i o n , encouragement and op t i m i s m t h r o u g h o u t the cour s e o f these s t u d i e s . I am a l s o g r a t e f u l t o t h e v a r i o u s s t a f f members f o r h e l p f u l s u g g e s t i o n s , b u t , i n p a r t i c u l a r D r s . B.R. Worth and T. Honda r e c e i v e s p e c i a l c o n s i d e r a t i o n i n t h i s r e s p e c t . S i n c e r e g r a t i t u d e i s e x p r e s s e d t o my w i f e whose u n f a i l i n g s u p p o r t , a s s i s t a n c e , and u n d e r s t a n d i n g d u r i n g the co u r s e o f t h i s s t u d y were a c o n t r i b u t i o n beyond measure. R e c e i p t o f a S c i e n c e R e s e a r c h C o u n c i l (U.K.) p o s t g r a d u a t e s c h o l a r s h i p f o r 1974-1977 i s g r a t e f u l l y acknowledged as i s f u r t h e r f i n a n c i a l s u p p o r t from the N a t i o n a l I n s t i t u t e s o f H e a l t h (U.S.A.) and t h e c h e m i s t r y department (U.B.C.) f o r a t e a c h i n g a s s i s t a n t s h i p . INTRODUCTION - PARTS I & I I The e l u c i d a t i o n o f t h e major b i o s y n t h e t i c pathways i n i n d o l e a l k a l o i d b i o s y n t h e s i s has been t h e s u b j e c t o f much i n t e r e s t i n t h e l a s t decade. Many r e v i e w s have been p u b l i s h e d and w i l l be d i s -c u s s e d a t a p p r o p r i a t e j u n c t u r e s . 'However one r e c e n t comprehensive r e v i e w has p r o v i d e d i n v a l u a b l e a s s i s t a n c e . 1 B i o s y n t h e s i s i s an i n t r i g u i n g p r o c e s s and, c o n s e q u e n t l y , p r i o r • t o d i s c u s s i o n o f t h e h i s t o r i c a l a c c o u n t o f i n d o l e a l k a l o i d f o r m a t i o n some g e n e r a l comments on t h i s t o p i c a r e i n o r d e r . B i o s y n t h e t i c pathways a r e o f t e n i n f e r r e d by (1) a d m i n i s t e r i n g l a b e l l e d i n t e r m e d i a t e s and (2) showing t h a t t h e l a b e l appears i n t h e s u b s t a n c e r e g a r d e d as t h e end p r o d u c t o f the pathway. The p r e c u r s o r i s g e n e r a l l y l a b e l l e d w i t h "^C, 3H, 1 5 N , o r a c o m b i n a t i o n of t h e s e , and t h i s i s t h e n a d m i n i s t e r e d t o an i n t a c t p l a n t or p a r t o f a p l a n t ( r o o t c u l t u r e s , e x c i s e d l e a v e s o r t i s s u e c u l t u r e ) . Q u i t e o f t e n low i n c o r p o r a t i o n s a r e o b t a i n e d - t h e s e b e i n g r a t i o n a l i s e d as b e i n g due t o (1) low a l k a l o i d a l b i o s y n t h e s i s a t time o f p r e c u r s o r a d m i n i s t r a t i o n (2) n o n - p e r m e a b i l i t y o r (3) d e s t r u c t i o n o r m o d i f i c a t i o n o f the l a b e l l e d compound b e f o r e i t re a c h e s t h e b i o s y n t h e t i c s i t e . 2 C o r n f o r t h has c r i t i c a l l y a s s e s s e d t h e s u b j e c t i n a R o b e r t Robinson l e c t u r e "The l o g i c o f w o r k i n g w i t h enzymes". T h i s e x c e l l -e n t paper h e l p s d e f i n e c r i t e r i a r e q u i r e d f o r m e a n i n g f u l b i o s y n -t h e t i c p u r s u i t s . He argues t h a t w i t h r e s p e c t t o a d m i n i s t r a t i o n o f l a b e l l e d compounds i n o r d e r t o d e t e r m i n e p r e c u r s o r s o f the supposed -2-end product the p o s i t i v e r e s u l t s obtained may also be compatible with the precursor not being on the p r i n c i p a l pathway but, instead merely degradable or transformable to a true intermediate coming largely from a d i f f e r e n t precursor. Furthermore administration of l a b e l l e d precursors for the i d e n t i f i c a t i o n of possible intermediates i s cautioned since the "intermediates" i s o l a t e d may be detectable, not because they are intermediates, but because they are not. Indeed, the intermediates may only be slowly incorporated into the end-product, or, perhaps, not at a l l . Cornforth also points out that experiments revealing n e g l i g i b l e or no incorporation of the l a b e l from (1) the precursor into the suspected intermediate or (2) the intermediate into the product must be treated with caution since, on t h i s basis, mevalonic acid (26) could be shown not to be an intermediate i n the polyisoprenoid biosynthesis. 3 Leete has recently c r i t i c i s e d workers reporting low incor-porations as being s i g n i f i c a n t and yet f a i l i n g to determine the 4 location of the r a d i o a c t i v i t y i n the molecule and Barton has stated that low incorporations (<0.01%) are probably not meaningful. I t has also been shown, that, on occasion, plants may u t i l i z e "unnatural precursors" to y i e l d "unnatural products" e.g. conversion of 5 - f l u o r o n i c o t i n i c acid into 5 - f l u o r o n i c o t i n e . 5 ' ^ Consequently, plant enzymes may convert unnatural precursors into ones l y i n g d i r e c t l y on the biosynthetic route. Thus the determination of the major biosynthetic pathway might be d i f f i c u l t to prove by administration of precursors to plant -3-m a t e r i a l . Moreover, i n t h e i n s t a n c e o f p o s i t i v e i n c o r p o r a t i o n s , i t must be e s t a b l i s h e d t h a t i n t a c t i n c o r p o r a t i o n o f t h e p r e c u r s o r i n t o t h e a l k a l o i d has o c c u r r e d . T h i s i s g e n e r a l l y o b t a i n e d by f e e d i n g m u l t i p l e l a b e l l e d p r e -c u r s o r s , i s o l a t i n g t h e d e s i r e d a l k a l o i d and c r y s t a l l i s i n g i t t o c o n s t a n t a c t i v i t y . The r a t i o o f the l a b e l s from p r e c u r s o r t o a l k a l o i d s h o u l d be i n agreement w i t h t h a t e x p e c t e d . D e g r a d a t i o n o f t h e a l k a l o i d s h o u l d t h e n be c a r r i e d o u t t o d e t e r m i n e t h e e x a c t p o s i t i o n o f the l a b e l ( s ) . I t s h o u l d a l s o be n o t e d t h a t i n t h e i n d o l e a l k a l o i d f i e l d o n l y a s m a l l number o f p l a n t s have w a r r a n t e d i n v e s t i g a t i o n . Indeed, i n c o r p o r a t i o n o r l a c k o f i t , has been n o t e d t o d i f f e r i n d i f f e r -e n t p l a n t s - even a l t h o u g h they c o n t a i n the same a l k a l o i d s . ( D i s c u s s e d l a t e r i n t e x t . ) C l e a r l y , i n f o r m a t i o n from p l a n t e x p e r i m e n t s w i l l always be l i m i t e d , and i t i s hoped t h a t t h i s a r e a w i l l g i v e way t o i n v e s t i -g a t i o n i n , h o p e f u l l y , s i m p l e r systems e.g. p l a n t enzyme(s). The e v i d e n c e l e a d i n g t o and s u p p o r t i n g the c u r r e n t b i o s y n t h e -t i c p o s t u l a t e s i s now r e v i e w e d below. To a c c o u n t f o r t h e f o r m a t i o n o f ^ - c a r b o l i n e d e r i v e d 7 a l k a l o i d s , t y p i f i e d by ( 2 ) and ( 3 ) , Robinson s u g g e s t e d i n 1919 t h a t b i o s y n t h e t i c f o r m a t i o n o f t h e s e m o l e c u l e s o c c u r s by c o n d e n s a t i o n o f t r y p t a m i n e ( l a ) o r t r y p t o p h a n ( l b ) w i t h s i m p l e a l d e h y d e s i n a Mannich f a s h i o n as shown i n f i g u r e 1. - 4 -F i g u r e 1 - Robinson p o s t u l a t e f o r b i o s y n t h e s i s o f c o r y n a n t h e and s t r y c h n o s s k e l e t a . 8 9 As e x p e c t e d t r y p t o p h a n ( l b ) and t r y p t a m i n e ( l a ) have been shown t o be p r e c u r s o r s i n i n d o l e a l k a l o i d b i o s y n t h e s i s . The pr o v e d b i o s y n t h e t i c r o u t e t o t r y p t o p h a n ( l b ) u t i l i s i n g s h i k i m i c a c i d (6) and a n t h r a n i l i c a c i d (7) i s shown l a t e r i n f i g u r e 1 5 and d i s c u s s e d a t a more a p p r o p r i a t e p o i n t . I n a c c o r d w i t h Robinson's p o s t u l a t e s H a h n 1 0 gave f u r t h e r s u p p o r t by c o n d e n s i n g aldehydes w i t h t r y p t a m i n e ( l a ) t o g i v e t e t r a h y d r o c a r b o l i n e s . Barger and Hahn th e n p o s t u l a t e d t h a t i n b i o s y n t h e s i s o f i n d o l e a l k a l o i d s t h e aldehyde was t y r o s i n e (8) d e r i v e d . 1 ^ 1 1 T h i s 12 13 h y p o t h e s i s was l a t e r e l a b o r a t e d by Robinson and Woodward t o ac c o u n t f o r the f o r m a t i o n o f the cor y n a n t h e and s t r y c h n o s s k e l e t a . ( F i g u r e 2) Figure 2 - Woodward and Robinson postulate for the biosynthesis of corynanthe and strychnos skeleta. C H ^ C 0 2 C H VINDOLINE 11 AJMALICINE 15 DEHYDROASPIDOSPERMIDINE 10 CATHARANTHINE 12 RESERPININE 14 PERIVINE 16 F i g u r e 3 - Some r e p r e s e n t a t i v e i n d o l e a l k a l o i d s commonly i s o l a t e d i n b i o s y n t h e t i c i n v e s t i g a t i o n s . -7-ments, These t h e o r i e s were l a t e r i n v a l i d a t e d by f e e d i n g e x p e r i -14-20 and, i n t h i s r e s p e c t , i n d o l e a l k a l o i d s u s u a l l y examined i n b i o s y n t h e s i s a re shown i n f i g u r e 3. The numbering system 2 1 i s t h a t employed by Hesse. 22 A more u n l i k e l y monoterpenoid h y p o t h e s i s p r oposed by Thomas , 2 3 24 Wenkert , and Levy was n e x t c o n s i d e r e d . T h i s was based on the f a c t t h a t i n a l k a l o i d s i l l u s t r a t e d by c o r y n a n t h e i n e ( 1 7 ) , s t r y c h n i n e ( 5 ) , t a b e r s o n i n e (18) and c a t h a r a n t h i n e ( 1 2 ) , t h e r e a re t h r e e main t y p e s o f s k e l e t a p r e s e n t i n the n o n - t r y p t o p h a n ( l b ) p o r t i o n , i . e . (A, B and C.) C 0 2 C H 3 A 1 8 B C 0 2 C H 3 12 c - 8 -The occurrence i n nature of cyclopentane or secocyclopentane monoterpenoid g l y c o s i d e s ( 1 9 - 2 5 ) having ( 1 ) the same s t e r e o -chemistry i n the a p p r o p r i a t e p o s i t i o n as C - 1 5 of i n d o l e a l k a l o i d s and ( 2 ) a carbomethoxy group or corresponding d e r i v a t i v e at the c o r r e c t p o s i t i o n prompted t h e a b o v e a u t h o r s t o p o s t u l a t e b i o s y n t h e t i c o r o -cesses u t i l i s i n g such i n t e r m e d i a t e s . VERBENALIN 1 9 GENIPIN 2 0 ASPERULOSIDE 2 1 SWERTIAMARIN 2 2 BAKANKOSIN 2 3 ELENOLIDE 2 4 OLEUROPIN 2 5 2 2 Thomas suggested i n h i s hypothesis t h a t formation of s k e l e t o n A i n the corynanthe-strychnos s e r i e s was mevalonate ( 2 6 ) d e r i v e d as shown i n f i g u r e 4 . The cyclopentane u n i t so formed i s then c l e a v e d and r e a c t s w i t h tryptamine (la) g i v i n g entry i n t o t h i s area. -9-F i g u r e 4 - Thomas-Wenkert monoterpene p o s t u l a t e . F u r t h e r r earrangements were r e q u i r e d t o a c c o u n t f o r the s k e l e t a e x i s t i n g i n the aspidosperma (B) o r i b o g a a l k a l o i d s ( C ) . ( F i g u r e 5) F i g u r e 5 - Rearrangement o f monoterpene u n i t i n t o aspidosperma and i b o g a s k e l e t a . The h y p o t h e s i s was f i r s t t e s t e d by f e e d i n g sodium d l ( 2 ^ C ) mevalon-a t e (26) and ( 2 1 4 C ) m e v a l o n o l a c t o n e (27) t o C e p h a s l i s i p e c a c u a n h a and R a u w o l f i a s e r p e n t i n a w h i c h c o n t a i n a l k a l o i d s c e p h a e l i n e (2 8) and a j m a l i n e (9) r e s p e c t i v e l y . C e p h a e l i n e (28) i s an i s o q u i n o l i n e a l k a l o i d c o n t a i n i n g a d e o x y p h e n y l a l a n i n e r e s i d u e and p o s s e s s e s -10-the same C n , n u n i t as found i n the c o r y n a n t h e - s t r y c h n o s s k e l e t a . y - x u 19 33 N e g l i g i b l e l a b e l l i n g o c c u r r e d i n c e p h a e l i n e (28) ' o r a j m a l m e ( 9 ) 3 2 . 2 7 2 8 The p o s t u l a t e was however shown t o be c o r r e c t by f e e d i n g v a r i o u s l y l a b e l l e d forms o f mevalonate (26) , m e v a l o n o l a c t o n e ( 2 7 ) , g e r a n i o l (29) and n e r o l (30) t o a l t e r n a t i v e p l a n t s (Rhazya s t r i c t a , 34-39 C a t h a r a n t h u s r o s e u s , V i n c a major ). E x t e n s i v e d e g r a d a t i o n s t u d i e s o f i s o l a t e d a l k a l o i d s showed i n t a c t i n c o r p o r a t i o n o f t h e 40-46 p r e c u r s o r i n the manner d e s c r i b e d i n t h e h y p o t h e s i s 2 9 3 0 The s t e r e o c h e m i s t r y o f each o f the s t e p s has been e l u c i d a t e d t o t h e f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e 1 ( 3 2 ) , and, a l t h o u g h much of the s t e r e o c h e m i c a l s t u d y has been e x e c u t e d on l i v e r and y e a s t s y s -- l i -tems i t has been su g g e s t e d t h a t t he same r o u t e i s i n v o l v e d i n p l a n t s y s t e m s . 4 7 ( F i g u r e 6) H C p H H X fiH C H , C O S C o A 1 T. ^ W I i 1 A , c r A H O C C O S C o A H 0 2 C g S C ° A F i g u r e 6 - B i o s y n t h e t i c f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e (32). Thus i n the f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e (32) the p r o 4S hydrogens o f the m evalonates a r e c o m p l e t e l y l o s t . I t has been 4 8 49 d e monstrated i n t h e mould m y c e l i a n a m i d e and a t r i t e r p e n e t h a t t h e m e t h y l groups o f d i m e t h y l a l l y l - p y rophosphate (31) a r e n o t b i o s y n t h e t i c a l l y e q u i v a l e n t and i n g e r a n i o l - p y r o p h o s p h a t e (32) t h e C-10 was d e r i v e d from C-2 o f m e v a l o n i c a c i d (26) and C-8 and 9 from C-6 o f m e v a l o n i c a c i d ( 2 6 ) . F u r t h e r s i n c e g e r a n i o l (29) and n e r o l (30) were i n c o r p o r a t e d i n t o t h e t h r e e b a s i c s k e l e t a ( A , B and C) i t was thus o f g r e a t im-p o r t a n c e t o d e t e r m i n e a t what s t a g e t h e t e r p e n o i d r earrangements t o o k p l a c e . 50 14 I n C. i p e c a c u a n h a , (2 C) g e r a n i o l (29) was shown t o be a p r e c u r s o r o f i p e c o s i d e ( 3 3 ) 5 1 w h i c h t h e n l e d t o the s u g g e s t i o n t h a t l o g a n i n (35) c o u l d g i v e r i s e t o an a l d e h y d e (44) w h i c h on conden-s a t i o n w i t h t r y p t a m i n e ( l a ) gave the p r e c u r s o r f o r t h e d i v e r s i f i e d 52 m d o l i c s k e l e t a . The s t e r e o c h e m i s t r y a t C-5 o f i p e c o s i d e (33) 53 was o b t a i n e d by c o r r e l a t i o n w i t h ( - ) d i h y d r o p r o t o e m e t i n e (34) -13-T h i s p o s t u l a t e was q u i c k l y v e r i f i e d and l o g a n i n (35), d e r i v e d from mevalonate (ZBj^and g e r a n i o l ( 2 9 ) 1 , was i n c o r p o r a t e d i n t a c t into 5 2 a l k a l o i d s c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( 1 1 ) , s e r p e n t i n e ( 1 3 ) , 14 a j m a l i c i n e (15) and p e r i v i n e (16). S u r p r i s i n g l y (2 C) mevalonate 54 (26) f a i l e d t o l a b e l l o g a n i n (35) i n M. t r i f o l i a t a . F u r t h e r f e e d i n g e x p e r i m e n t s w i t h s i m i l a r systems e.g. v e r b e n a l i n (19) and 52 g e n i p i n (20) showed absence o f l a b e l i n the a l k a l o i d s . L o g a n i n (35) was then shown t o be a n a t u r a l p r o d u c t i n a v a r i e t y o f p l a n t systems and f u r t h e r e x p e r i m e n t s w i t h p r o 4R and 55 p r o 4S mevalonates extended the s t e r e o s p e c i f i c i t y t o l o g a n i n as shown i n f i g u r e 7. 3 2 3 5 F i g u r e 7 - B i o s y n t h e t i c f o r m a t i o n o f l o g a n i n ( 3 5 ) . F u r t h e r i n t e r m e d i a c y t o l o g a n i n (35) has been i n d i c a t e d by s p e c i f i c i n c o r p o r a t i o n o f 1 0 - h y d r o x y - g e r a n i o l ( 3 6 ) 5 6 , 10-hydroxy--14-5 6 5 V 5 8 n e r o l (37) , d e o x y l o g a n i n (38) and d e o x y l o g a n i c a c i d (39) i n t o l o g a n i n (35) and a l k a l o i d s v i n d o l i n e (11),' c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e ( 1 6 ) . 5 6 , 5 8 3 6 3 7 co 2R Cu O G I u 3 3 8 ; R = C H 3 3 9 , R = H The n e x t b i o s y n t h e t i c s t e p from l o g a n i n (35) was c l e a v a g e o f the c y c l o p e n t a n e r i n g , and i n t h i s r e s p e c t , i t s h o u l d be n o t e d t h a t i n a d d i t i o n t o t h e n a t u r a l l y o c c u r r i n g s e c o - i r i d o i d s (19-25) p r e -v i o u s l y mentioned, n a t u r a l p r o d u c t s s w e r o s i d e ( 4 0 ) , f o l i a m e n t h i n ( 4 1 ) , m e n t h i a f o l i n (42) and d i h y d r o f o l i a m e n t h i n (43) a l s o p o s s e s s a seco r i n g masked by l a c t o l f o r m a t i o n . 1 40 ? a = 41 , -42 , = O G I u 4 3 , 1= M i l d a l k a l i n e h y d r o l y s i s o f (40-42) a f f o r d e d s e c o l o g a n i n (44) wh i c h has been shown t o o c c u r i n C. ro s e u s and M. t r i f o l i a t a . 60 -15-4 4 , R » C H 3 4 4 A , - H The t e r p e n o i d n a t u r e o f s e c o l o g a n i n (44) was e s t a b l i s h e d i n C. r o s e u s and M. t r i f o l i a t a by t h e 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 ( 2 6 ) 6 0 a , and l o g a n i n ( 3 5 ) 6 0 b ' c . M e v a l o n a t e (26) was a l s o i n c o r p o r a t e d i n t o s e c o l o g a n i c a c i d 6 0a (44a) i n C. r o s e u s . S w e r t i a m a r i n (22) and s w e r o s i d e (40) i n S w e r t i a m o r i n j a p o n i c a were found t o be m e v a l o n o l a c t o n e (27) de-r i v e d 6 1 . S e c o l o g a n i n (44) was found t o l a b e l the a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e ( 1 6 ) 5 9 ' 6 0 b . F u r t h e r s w e r o s i d e (40) l a b e l l e d v i n d o l i n e (11) i n C. r o s e u s 6 2 3 ' * 3 and r e s e r p i n i n e (14) i n V. major ' . I p e c o s i d e ( 3 3 ) , d i s c u s s e d e a r l i e r , was t h e f i r s t example o f an i n t a c t s e c o - i r i d o i d u n i t i n a l k a l o i d s and l o g a n i n (35) and 6 3 51 g e r a n i o l (29) were shown t o be s p e c i f i c p r e c u r s o r s r e s p e c t i v e l y . ' However t h e f i r s t examples o f a s e c o - i r i d o i d u n i t i n combina-t i o n w i t h t r y p t o p h a n ( l b ) were n o t e d i n a l k a l o i d s c o r d i f o l i n e ( 4 5 ) , 6 4 a d i f o l i n e (46) and t h e i r c o r r e s p o n d i n g deoxy d e r i v a t i v e s (47,48). -16-S t r i c t o s i d i n e ( 4 9 ) , no s t e r e o c h e m i s t r y i m p l i e d , was then i s -o l a t e d from R. s t r i c t a . Thus t h e c r u c i a l i n t e r m e d i a t e between the i r i d o i d s and a l k a l o i d s was o b t a i n e d . S t r i c t o s i d i n e (49) was then shown t o be t r y p t o p h a n ( l b ) and l o g a n i n 6 8 (35) d e r i v e d . S i g n i f i c a n t l y the a u t h o r s mentioned f u r t h e r b i o - s y n t h e t i c p u r s u i t s were b e i n g i n i t i a t e d u s i n g (49) as a p r e c u r s o r . I n t h i s r e g a r d , Brown e t a l . o n l y o b t a i n e d low i n c o r p o r a t i o n o f 69 s t r i c t o s i d i n e (49) i n t o t h e i n d o l e a l k a l o i d s B a t t e r s b y then r e p o r t e d t h e p a r t i a l s y n t h e s i s o f i p e c o s i d e (33a i s o i p e c o s i d e (33b) and v i n c o s i d e ( 4 9 a ) / i s o v i n c o s i d e (49b) by conden-s a t i o n o f s e c o l o g a n i n (44) w i t h 3,4 d i h y d r o x y p h e n e t h y l a m i n e (50) and t r y p t a m i n e ( l a ) r e s p e c t i v e l y 7 ^ ' ^ . -17-5 0 The r e l a t i v e s t e r e o c h e m i s t r y about C-3 o f v i n c o s i d e ( 4 9 a ) / i s o v i n c o -s i d e (49b) b e i n g o b t a i n e d by c o r r e l a t i o n w i t h i p e c o s i d e ( 3 3 a ) / i s o -i p e c o s i d e (33b). V i n c o s i d e (49a) and i s o v i n c o s i d e (49b) were shown t o be 71 p r e s e n t i n C. r o s e u s . An a d m i x t u r e o f (49a,b) was f e d t o C. r o s e u s and a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e (16) were l a b e l l e d . ' ' F u r t h e r l a b e l l i n g e x p e r i m e n t s w i t h pure v i n c o s i d e (49a) and pure i s o v i n c o -s i d e (49b) showed s p e c i f i c i n c o r p o r a t i o n o f v i n c o s i d e (49a) w i t h 71 73 n e g l i g i b l e i n c o r p o r a t i o n b e i n g n o t e d u s i n g (49b). ' T h i s was t h e l o g i c a l r e s u l t s i n c e v i n c o s i d e (49a) p o s s e s s e s the same s t e r e o -c h e m i s t r y a t C-3 as t h a t o f t h e c o r y n a n t h e a l k a l o i d s . However t h i s r e s u l t i n i t i a t e d a c o n t r o v e r s y w h i c h a p p a r e n t l y has o n l y been r e s o l v e d v e r y r e c e n t l y . I n 1969, Smi t h p r o p o s e d t h a t t h e s t e r e o c h e m i s t r y o f v i n c o s i d e (49a) and i s o v i n c o s i d e (49b) s h o u l d be r e v e r s e d 7 4 due t o the s i m i -l a r s p e c t r a l q u a l i t i e s o f (49b) w i t h s t r i c t o s i d i n e (49) w h i c h t h e y had c o r r e l a t e d w i t h a n t i r h i n e (51) thus showing i t t o be the C-3 75 o(-H epimer. -18-X-ray i n v e s t i g a t i o n o f 0 ^ O - d i m e t h y l i p e c o s i d e ( 5 2 ) , now r e v i s e d the s t e r e o c h e m i s t r y a t C-5 from t o fi t h u s a u t o m a t i c a l l y r e v i s i n g t h e s t e r e o c h e m i s t r y a t C-3 t o v i n c o s i d e (49b) and i s o v i n c o s i d e (49a) i 76 r e s p e c t i v e l y . 5 2 C o n s e q u e n t l y d e s a c e t y l i p e c o s i d e (53) was p a r t i a l l y s y n t h e s i s e d and f e d i n l a b e l l e d form t o C. i p e c a c u a n h a . I t was found t o be i n c o r p o r a t e d i n t o i p e c o s i d e ( 3 3 ) , now (33b), c e p h a e l i n e (28) and 77 emetine (54) . 5 3 5 4 -19-F u r t h e r , l a b e l l i n g w i t h ( 5 % ) l o g a n i n (35) showed s p e c i f i c i n c o r p o r a t i o n o f l a b e l i n t o (33b), (28) and (53) - w i t h no l o s s o f t r i t i u m on i n v e r s i o n o f s t e r e o c h e m i s t r y . (Shown by d e g r a d a t i o n . ) (5^H, OMe^H) l o g a n i n (35) was t h e n f e d t o C. roseus and v i n -d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e (15) were shown t o 7 8 be s p e c i f i c a l l y l a b e l l e d . Thus as v i n c o s i d e ( 4 9 b ) and i p e c o s i d e (33b) had been d e t e r -mined t o be p r e c u r s o r s o b v i o u s l y i n c o r p o r a t i o n o c c u r r e d v i a an i n v e r s i o n and r e t e n t i o n p r o c e s s . Brown a l s o s u p p o r t e d the C-3 79 r e v i s i o n o f s t e r e o c h e m i s t r y . However i n the b i o s y n t h e s i s o f c a m p t o t h e c i n (56) i t was found 8 0 t h a t i n a d d i t i o n t o t h e e x p e c t e d i n t e r m e d i a c y o f mevalonate (26) , 80 8 0 t r y p t o p h a n ( l b ) and s e c o l o g a n i n (44) , i s o v i n c o s i d e (49a) was i n -80a c o r p o r a t e d t o the e x t e n t o f 2%. V i n c o s i d e (49b) was n o t i n c o r -, , 80a p o r a t e a . These h i g h i n c o r p o r a t i o n s a l l o w e d H u t c h i n s o n t o f e e d ( 5 1 3 C ) i s o v i n c o s i d e l a c t a m ( 5 5 ) , and, i t was t h u s d i s c o v e r e d t h a t C-5 o f c a m p t o t h e c i n (56) was e n r i c h e d . The p o s t u l a t e d scheme f o r b i o s y n -t h e s i s i s shown . ( F i g u r e 8) -20-F i g u r e 8 - P o s t u l a t e d b i o s y n t h e t i c c o n v e r s i o n s f o r f o r m a t i o n o f c a m p t o t h e c i n ( 5 6 ) . V e r i f i c a t i o n o f t h e s t r u c t u r e o f v i n c o s i d e (49b) was t h e n 81 o b t a i n e d by X-ray and was shown t o be C^-H^. Presumably i n l i g h t o f t h i s most s i g n i f i c a n t b i o s y n t h e t i c r e s u l t , Zenk, e n j o y i n g s u c c e s s w i t h c e l l s u s p e n s i o n c u l t u r e s from C. r o s e u s 8 2 i n c u b a t e d ( 2 1 4 C ) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) 8 3 w i t h a c e l l f r e e system from C. r o s e u s . A c h e m i c a l l y s y n t h e s i s e d - 2 1 -m i x t u r e o f n o n - r a d i o a c t i v e v i n c o s i d e (49b) and i s o v i n c o s i d e (49a) was th e n added. S e p a r a t i o n and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y r e v e a l e d t h a t a l l a c t i v i t y was p r e s e n t i n i s o v i n c o s i d e (49a) . S i n g l y and d o u b l y l a b e l l e d forms o f i s o v i n c o s i d e (49a) were the n f e d t o C. r o s e u s p l a n t s and a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , a j m a l i c i n e (15) and s e r p e n t i n e (13) were 8 3b l a b e l l e d . S i m i l a r e x p e r i m e n t s w i t h v i n c o s i d e (49b) showed no i n c o r p o r a t i o n i n t o t h e s e a l k a l o i d s . Thus, the (C^ c< e p i m e r ) , i s o v i n c o s i d e ( 4 9 a ) , now c a l l e d s t r i c t o s i d i n e , was i n c o r p o r a t e d i n t o the i n d o l e a l k a l o i d s as e x p e c t e d . I t i s n o t the purpose o f t h i s i n t r o d u c t i o n t o s p e c u l a t e on re a s o n s l e a d i n g t o t h e c o n t r o v e r s y . From t h e f e e d i n g e x p e r i m e n t s so f a r d e s c r i b e d , a few g e n e r a l p o i n t s were n o t e d : (1) i n c o r p o r a t i o n s were s u p p o r t e d by e i t h e r d e g r a d a t i o n and/or do u b l e l a b e l l i n g . I n c o r p o r a t i o n s were on a v e r a g e ~ 0 . 3 % a l t h o u g h i n some a l k a l o i d s e.g. a j m a l i c i n e ( 1 5 ) , p r e c u r s o r i n c o r p o r a t i o n was n o t a r e l i a b l e g u i d e t o t h e i m p o r t a n c e o f the p r e c u r s o r s . i n c o r p o r a t i o n m e v a l o n o l a c t o n e (27) 0.003 l o g a n i n (35) 0.26-0.46 s e c o l o g a n i n (44) 0.55 s t r i c t o s i d i n e (49a) 0.5 (2) v a r i o u s p l a n t systems may n o t i n c o r p o r a t e p r e c u r s o r s . (3) b l a n k e x p e r i m e n t s were n o t u s u a l l y r e p o r t e d b u t assuming t h a t - 2 2 -they were c a r r i e d o u t one thus o b t a i n s r e a s o n a b l y s i g n i f i c a n t r e -3 4 s u i t s - w i t h comments by L e e t e and B a r t o n b e i n g accomodated f u l l y . The i n t e r m e d i a c y beyond s t r i c t o s i d i n e (49a) has been the s u b j e c t o f many i n v e s t i g a t i o n s . Two p o s t u l a t e s have been f o r w a r d e d and 84 85 the f o l l o w i n g sequence o f f o r m a t i o n was s u g g e s t e d . ' c o r y n a n t h e —> s t r y c h n o s — • a s p i d o s p e r m a —• i b o g a C o n v e r s i o n o f s t r i c t o s i d i n e (49a) t o c o r y n a n t h e a l k a l o i d s , as shown by c o r y n a n t h e i n e aldehyde (57) o r g e i s s o s c h i z i n e ( 5 8 ) , would be n o n - e x c e p t i o n a l ( e n z y m a t i c a l l y \ ) . ( F i g u r e 9) F i g u r e 9 - P o s t u l a t e d b i o s y n t h e t i c f o r m a t i o n o f c o r y n a n t h e i n e a l d e h y d e (57) and g e i s s o s c h i z i n e ( 5 8 ) . - 2 3 -F u r t h e r c o n v e r s i o n o f the a l k a l o i d s (57) and (58) were i n v e s t i g a t e d , and, i n t h i s r e s p e c t , c o r y n a n t h e i n e aldehyde (57) was c o n s i d e r e d 8 6 not t o be an i n t e r m e d i a t e i n the b i o s y n t h e s i s o f v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and akuammicine ( 6 4 ) ? whereas g e i s s o s c h i z i n e (58) was e f f i c i e n t l y 8 7 i n c o r p o r a t e d i n t o t h e s e a l k a l o i d s S c o t t has, h o w e v e r , r e p o r t e d a s m a l l i n c o r p o r a t i o n o f c o r y n a n t h e i n e aldehyde (57) i n C. r o s e u s s e e d l i n g s 8 6 0 - b u t , as B a t t e r s b y s u g g e s t s , t h i s f a c t i s of l i t t l e s i g n i f i c a n c e as d e g r a d a t i o n and/or 8 7 m u l t i p l e l a b e l l i n g was not pursued t o ensure i n t a c t i n c o r p o r a -F u r t h e r s u p p o r t t o the i n t e r m e d i a c y o f g e i s s o s c h i z i n e (58) was 8 8 r e p o r t e d by i t s s u c c e s s f u l i n c o r p o r a t i o n i n t o c o r o n a r i d i n e (59) The p o s t u l a t e s ' f o r the f o r m a t i o n o f the s t r y c h n o s , aspidosperma, i b o g a b a s e s , as i n i t i a l l y s u g g e s t e d , are shown ( F i g . 10-12). Both s t r y c h n o s p o s t u l a t e s u t i l i z e g e i s s o s c h i z i n e ( 5 8 ) . ( F i g u r e 10) To account f o r t h e f o r m a t i o n o f the a s p i d o s p e r m a / i b o g a a l k a -l o i d s Wenkert proposed the f o l l o w i n g scheme 8^ wh i c h was the f i r s t s u g g e s t i o n t h a t a discrete C 7 - C 3 seco s p e c i e s was i n v o l v e d i n the b i o s y n t h e s i s o f t h e s e s k e l e t a . ( F i g u r e 11) t i o n . 59 84, 85 WENKERT'S HYPOTHESIS H C O X * C H O 3 Zt a;R, = C H O ; R 2 = C O 2 C H 3 b , R 2 = C 0 A C H 3 ; R , = C H O H 3 C 0 2 C H ^ O f t H O -0. -24-SCOTT'S HYPOTHESIS H X O C - C H O 3 2 I 8 3 H C 0 2 C CHO 61 7 H3C 02C 7 7 H C O C ' C H O 3 c. . 6 1 F i g u r e 10 - Pro p o s e d b i o s y n t h e t i c o r i g i n s o f the s t r y c h n o s f a m i l y . 1 F i g u r e 11 - Wenkert's p r o p o s e d b i o s y n t h e t i c o r i g i n s o f the aspidosperma and i b o g a f a m i l i e s . S l i g h t m o d i f i c a t i o n by S c o t t o f Wenkert's h y p o t h e s i s f o r the 85 a s p i d o s p e r m a / i b o g a s k e l e t a r e s u l t e d i n the f o l l o w i n g p o s t u l a t e ( F i g u r e 12) -26-F i g u r e 12 - S c o t t ' s m o d i f i c a t i o n o f Wenkert's h y p o t h e s i s f o r the aspidosperma and i b o g a f a m i l i e s . A s i m i l a r i n t e r m e d i a t e (73) has a l s o been p r o p o s e d by Levy C O £ C H 3 C 0 2 C H 3 7 3 7 4 I t i s o b v i o u s t h a t the i n t e r m e d i a c y o f stemmadenine (63) and an a c t i v a t e d p y r i d i n i u m s p e c i e s (65,70,73,74) are o f paramount im-p o r t a n c e i n the schemes o u t l i n e d . L e t us now c o n s i d e r t h e e v i d e n c e c o n c e r n i n g t h e s e schemes. -27-I n s u p p o r t o f Wenkert's s t r y c h n o s h y p o t h e s i s f o r m y 1 - s t r i c t a m i n e s 89 1 (60a,b) and p l e i o c a r p a m i n e (75) have been i s o l a t e d m R. s t r i c t a . Compound (76) was a l s o i s o l a t e d from Aspidosperma desmanthum 9 0 . U n f o r t u n a t e l y no b i o s y n t h e t i c e v a l u a t i o n o f t h i s d a t a has been f o r w a r d e d , and, c o n s e q u e n t l y , no comment can be made. I n v i t r o c o n v e r s i o n s o f c o r y n a n t h e —> s t r y c h n o s s k e l e t a have however been r e p o r t e d by S c o t t e.g. c o n v e r s i o n o f g e i s s o s c h i z i n e (58) i n t o dehydropreakuammicine (61) i n r e f l u x i n g a c e t i c a c i d . However t h e y i e l d s w h i c h were r e p o r t e d were poor and non-91 r e p r o d u c i b l e . 6 1 I n s u p p o r t o f S c o t t ' s s t r y c h n o s h y p o t h e s i s , o x i n d o l e a l k a l o i d s have been found i n M . p a r v i f o l i a and i n t h i s r e s p e c t a j m a l i c i n e (15) and 3 - i s o a j m a l i c i n e (78) were each i n c o r p o r a t e d i n t o the o x i n d o l e s m i t r a p h y l l i n e (79) and i s o m i t r a p h y l l i n e ( 80). -28-S c o t t has a l s o r e p o r t e d i s o l a t i o n o f t h e h y d r o x y i n d o l e n i n e o f g e i s s o s c h i z i n e (62) and g e i s s o s c h i z i n e o x i n d o l e (83, R=H) from C. 93 r o s e u s s e e d l i n g s . Subsequent i n c o r p o r a t i o n s o f g e i s s o s c h i z i n e o x i n d o l e (83, R=H), i n t o akuammicine (64) and v i n d o l i n e (11) were 9 3 a l s o r e p o r t e d . Treatment o f i n d o l e n i n e (81) w i t h p o t a s s i u m t - b u t o x i d e a f f o r d e d 9 4 n o r - f l u o r o c u r a r i n e (82) l e n d i n g an i n v i t r o s u p p o r t f o r S c o t t ' s s t r y c h n o s h y p o t h e s i s ( f i g u r e 13). A mechanism f o r t h i s c o n v e r s i o n was a l s o r e p o r t e d . F i g u r e 13 - P r o p osed mechanism f o r f o r m a t i o n o f n o r - f l u o r o c u r a r i n e ( 8 2 ) . Both hypotheses u t i l i s e stemmadenine (63) and i n t h i s r e s p e c t -29-(3 ±' 1C) t r y p t o p h a n ( l b ) was f e d t o C. r o s e u s and ( S " 1 ^) stemmadenine 95 3 (63) was o b t a i n e d by i s o t o p i c d i l u t i o n . F u r t h e r (OMe H) stemmadenine (63) was o b t a i n e d by t r i v i a l c o n v e r s i o n o f (63)-—» a c i d 9 5 ->(63) . F e e d i n g e x p e r i m e n t s w i t h t h e r e s u l t a n t d o u b l y l a b e l l e d 9 5 stemmadenine (63) showed r e a s o n a b l e i n c o r p o r a t i o n i n t o t a b e r s o n m e (1 8 ) , c a t h a r a n t h i n e (12) and v i n d o l i n e (11) w i t h no l o s s o f r a t i o o f l a b e l s on i n c o r p o r a t i o n thus g i v i n g f u r t h e r s u p p o r t f o r t h e i n t e r m e d i a c y o f an a c t i v a t e d p y r i d i n i u m s p e c i e s (e.g. 65,70,73,74). B a t t e r s b y gave f u r t h e r s u p p o r t t o the i n t e r m e d i a c y o f (1) stemmadenine (63) and (2) an a c t i v a t e d p y r i d i n e i n t e r m e d i a t e 3 7 8 (65,70,73,74) by f e e d i n g (5 H) l o g a n i n t o C. r o s e u s and i s o l a t i n g l a b e l l e d v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e ( 1 5 ) . A c c o r d i n g t o the h y p o t h e s i s t h e l a b e l would be found i n the f o l l o w i n g p o s i t i o n s i n the m o l e c u l e . ( F i g u r e 14) S p e c i f i c d e g r a -d a t i o n s showed l a b e l from f e e d i n g t o be l o c a t e d e x c l u s i v e l y a t Carbon (#) o f ( 1 1 ) , (12) and ( 1 5 ) . F u r t h e r e v i d e n c e f o r the f o r m a t i o n o f a seco i n t e r m e d i a t e such as t h e a c r y l i c e s t e r (74) was o b t a i n e d by i s o l a t i o n o f d i m e r i c i n d o l i c secamines (84) from R. s t r i c t a . Of p a r t i c u l a r s i g n i f i c a n c e was t h e p r e s e n c e o f monomeric s e c o d i n e s ( 8 5 ) , ( 8 6 ) , ( 8 7 ) . 9 7 H F i g u r e 14 - I n c o r p o r a t i o n o f (5 H) l o g a n i n (35) i n t o v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e ( 1 5 ) . -31-8 6 8 7 B a t t e r s b y a l s o e s t a b l i s h e d t h e n a t u r a l o c c u r r e n c e o f 16,17 d i h y d r o -9 8 s e c o d i n - 1 7 - o l (88) i n C. r o s e u s and Rhazya o r i e n t a l i s . I t s h o u l d be n o t e d t h a t no b i o s y n t h e t i c i n f o r m a t i o n f o r the forma-t i o n o f t h e s e compounds from s t r i c t o s i d i n e (49a), g e i s s o s c h i z i n e (58) o r stemmadenine (63) have been r e p o r t e d . I n v e s t i g a t i o n s aimed a t v e r i f i c a t i o n o f the l a t t e r s t a g e s o f b i o s y n t h e s i s were i n i t i a t e d i n o u r l a b o r a t o r i e s . I n t h i s r e s p e c t two pathv/ays were p u r s u e d . (1) i m p o r t a n c e o f 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 aspidosperma and i b o g a a l k a l o i d s (66-67,68-69). (2) i m p o r t a n c e o f d e h y d r o s e c o d i n e (74) i n subsequent c o n v e r s i o n s 8 8 -32-t o aspidosperma o r i b o g a a l k a l o i d s . (1) The 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 quebrachamine (89) and 1 8 y 3-carbomethoxycleavamine (72) t o the p e n t a c y c l i c analogues a s p i d o s p e r m i d i n e (90) and c a t h a r a n t h i n e (12) had been shown t o be 99 a f a c i l e i n v i t r o p r o c e s s and thus i t was o f i m p o r t a n c e t o d e t e r -. c . , . i • , • 100 mine i f t h e r e was any m v i v o a p p l i c a t i o n 72 12 U n f o r t u n a t e l y v e r y low i n c o r p o r a t i o n s were o b t a i n e d , w h i c h , on com-p a r i s o n w i t h the c o r r e s p o n d i n g b l a n k e x p e r i m e n t s n e g a t e d t h e i r i m p o r t a n c e . I t i s o f some i n t e r e s t t o note t h a t c l e a v a m i n e monomers (e.g. 72) do n o t appear t o o c c u r i n n a t u r e t h u s perhaps g i v i n g some ex-p l a n a t i o n t o the i n v i v o o b s e r v a t i o n . However s u p p o r t f o r the i n t e r -mediacy o f d e h y d r o s e c o d i n e (74) was c o n s i d e r e d , i n s p i t e o f t h e s e poor r e s u l t s , by the low i n c o r p o r a t i o n o f t a b e r s o n i n e (18) i n t o v i n d o l i n e (11) and c a t h a r a n t h i n e (12) i n C. r o s e u s p l a n t s 1 ^ 0 and - 3 3 -s e e d l i n g s . Thus r e v e r s a l of t h e t r a n s a n n u l a r c y c l i z a t i o n was p o s t u l a t e d - a p r o c e s s n o t found t o o c c u r i n o t h e r f e e d i n g ex-. 101 p e r i m e n t s However l a b e l l i n g o f t a b e r s o n i n e (18) i n t h e s e e x p e r i m e n t s o n l y u t i l i z e d t h e t r y p t a m i n e ( l a ) p o r t i o n and thus the r e s u l t s a r e o f l i m i t e d b i o s y n t h e t i c s i g n i f i c a n c e s i n c e double l a b e l l i n g e x p e r i -ments must be c a r r i e d o u t w i t h the l a b e l i n b o t h (1) t h e t r y p t a m i n e p o r t i o n ( l a ) and (2) t h e t e r p e n o i d p o r t i o n . (A,B,C) S u i t a b l e d e g r a d a t i o n would thus prove such a p r o c e s s o f r e a r r a n g e -ment . (2) Importance o f d e h y d r o s e c o d i n e (74) i n subsequent c o n v e r s i o n s U n f o r t u n a t e l y l a b e l l i n g e x p e r i m e n t s w i t h (74) c o u l d n o t be pur s u e d due t o t h e i n s t a b i l i t y o f such an i n t e r m e d i a t e and u n t i l c o n d i t i o n s a re met f o r the s t a b i l i z a t i o n o f t h i s compound no d a t a can be f o r w a r d e d f o r i t s i m p o r t a n c e . 3 However l a b e l l i n g e x p e r i m e n t s w i t h (Ar H) 1 6 , 1 7 - d i h y d r o s e c o d i n -1-7 i / o o v l 0 2 - 1 0 6 A / 0 1 \ 102-3,106-7 . , „. 1 7 - o l (88) and s e c o d i n e (91) ' i n C. r o s e u s and V i n c a minor were i n v e s t i g a t e d as i t was hoped t h a t they c o u l d produce a (74) type intermediate i n vivo. In the case of (Ar H) 16,17-dihydro--34-s e c o d i n - 1 7 - o l (88) i n c o r p o r a t i o n s were v e r y low (<0.001%) i n t o c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( 1 1 ) , a j m a l i c i n e (15) i n C. r o s e u s and v i n c a m i n e (92) and m i n o v i n e (93) i n V. minor . Subsequent ex-3 p e r i m e n t s w i t h (Ar H) s e c o d i n e (91) showed s i m i l a r i n c o r p o r a t i o n s i n a l l a l k a l o i d s e x c e p t v i n d o l i n e (11) t o w h i c h a 0.02% i n c o r p o r a t i o n was o b t a i n e d . F u r t h e r f e e d i n g e x p e r i m e n t s w i t h d o u b l y - l a b e l l e d s e c o d i n e ( A r 3 H , 1 4 C 0 2 C H 3 ) (91) showed t h a t i n c o r p o r a t i o n i n t o v i n d o l i n e (11) was accompanied by some l o s s o f t r i t i u m - t h i s a l s o b e i n g noted 8 7 by B a t t e r s b y ' s e x p e r i m e n t s w i t h o t h e r a r o m a t i c l a b e l l e d p r e c u r s o r s . 9 2 9 3 Subsequent e x p e r i m e n t s w i t h ("^CC^CH^,3,21°H) s e c o d i n e (91) showed 60% l o s s o f t r i t i u m on c o n v e r s i o n i n t o v i n d o l i n e (11) -a p r o c e s s s u g g e s t i n g t h e p o s s i b l e i n v o l v e m e n t o f a d e h y d r o s e c o d i n e (74) m o i e t y . However i t i s o b v i o u s t h a t t h e low i n c o r p o r a t i o n s , i f accom-p a n i e d by b l a n k e x p e r i m e n t s , do n o t g i v e t h e i n f e r e n c e o f a major b i o s y n t h e t i c pathway. Indeed i t was n o t e d t h a t n e i t h e r i n v e s t i -g a t i o n s i . e . e i t h e r (1) t r a n s a n n u l a r c y c l i z a t i o n o r (2) s e c o d i n e (91) i n v o l v e m e n t gave e n c o u r a g i n g r e s u l t s . I t i s hoped t h a t subse-quent f e e d i n g s o f a s t a b i l i s e d d e h y d r o s e c o d i n e (74) w i l l , i n the near f u t u r e , g i v e more s i g n i f i c a n t i n c o r p o r a t i o n s . -35-F i n a l l y i n s u p p o r t o f a c r y l i c e s t e r (74) i n v o l v e m e n t , S c o t t has r e p o r t e d a s e r i e s o f i n v i t r o e x p e r i m e n t s s u b s t a n t i a t i n g t h e s e 10 8 p r e d i c t i o n s . I t i s n o t t h e purpose o f t h i s i n t r o d u c t i o n t o d i s c u s s , i n d e t a i l , t h e i n v i v o t r a n s f o r m a t i o n s d e s c r i b e d . However i t s h o u l d be n o t e d t h a t t h e s e p a p e r s r e c e i v e d an u n h e r a l d e d r e -109 b u t t a l by S m i t h and P o i s s o n . I n r e s p o n s e , and d e f e n s e , S c o t t has p u b l i s h e d f u r t h e r work p a r t i a l l y s u b s t a n t i a t i n g h i s e a r l i e r c l a i m s 1 1 ^ ' 1 1 1 and from the e x p e r i m e n t a l s e c t i o n major s k e l e t a l con-v e r s i o n s u s i n g a c r y l i c e s t e r (74) as an i n t e r m e d i a t e were d e s c r i b e d . I n t h i s r e s p e c t some t r a n s f o r m a t i o n s were n o t e d i n 0.2% y i e l d from 6 mg:. o f s t a r t i n g m a t e r i a l ] C o n s e q u e n t l y , t h e c l o s e e x a m i n a t i o n of t h i s work by t h e i n t e r e s t e d r e a d e r i s s t r o n g l y s u g g e s t e d . From the work d e s c r i b e d e a r l i e r we have n o t e d p r e l i m i n a r y r e s u l t s s u g g e s t i n g t h e sequence c o r y n a n t h e — * s t r y c h n o s —> aspidosperma —•• i b o g a « • -, 4 . 1 0 . ^84,85 i s m e f f e c t as p r e v i o u s l y p o s t u l a t e d ' F u r t h e r s u p p o r t t o t h i s h y p o t h e s i s has been g i v e n by S c o t t 95 112 113 w i t h C. r o s e u s s e e d l i n g s . ' ' I n t h i s r e s p e c t f o r m a t i o n o f a l k a l o i d s i n a time domain was m o n i t o r e d . (Table 1) The a l k a l o i d s were i s o l a t e d and p a r t i a l l y c h a r a c t e r i s e d by (1) t h i n l a y e r chrom-a t o g r a p h y and (2) mass s p e c t r o m e t r y . Preakuammicine (77) was however a p p a r e n t l y i s o l a t e d and i n f o r -114 m a t i o n w i t h r e s p e c t t o i t s s t r u c t u r e was p r e s e n t e d . F u r t h e r s t u d i e s on i n t e r m e d i a c y i n C. r o s e u s s e e d l i n g s u t i l i s -i n g s h o r t term i n c u b a t i o n t e c h n i q u e s were then c a r r i e d o u t . 14 DL(2 C) t r y p t o p h a n ( l b ) was added t o the s e e d l i n g s and t h e r i s e and f a l l i n r a d i o a c t i v i t y o f o b s e r v e d i n t e r m e d i a t e s m o n i t o r e d by Table 1: F o r m a t i o n o f a l k a l o i d s from C. r o s e u s s e e d l i n g s . Time (hours) 0 26 28-32 28-40 42-48 50 72 100-160 168 A l k a l o i d I s o l a t e d none v i n c o s i d e (49b), c o r y n a n t h e i n e (17) v i n c o s i d e (49b), c o r y n a n t h e i n e a i d . ( 5 7 ) , g e i s s o s c h i z i n e (58) preakuammicine (77), c o r y n a n t h e i n e a i d . ( 5 7 ) , g e i s s o s c h i z i n e (58) akuammicine (64) stemmadenine (63) t a b e r s o n i n e (18) 11-methoxy t a b e r s o n i n e (18a) c a t h a r a n t h i n e (12) c o r o n a r i d i n e (59) c a t h a r a n t h i n e / v i n d o l i n e (12)/(11) Type cory n a n t h e c o r y n a n t h e c o r y n a n t h e -s t r y c h n o s s t r y c h n o s c o r y n a n t h e -s t r y c h n o s aspidosperma aspidosperma i b o g a i b o g a i b o g a / aspidosperma i I -37-a u t o r a d i o g r a p h y . The a l k a l o i d s were c h a r a c t e r i s e d by TLC b e h a v i o u r i n two d i m e n s i o n s . I t was n o t e d i n t h e s e e x p e r i m e n t s t h a t t r y p t o p h a n ( l b ) was q u i c k l y c o n v e r t e d t o a m e t a b o l i t e h a v i n g a r a p i d ' r a d i o - p r o f i l e ' and a c t i v i t y t h a t l a s t e d f o r o n l y 60 m i n u t e s . S c o t t s u g g e s t e d t h a t i t m i ght be ( A 2 0 , 2 1 ) c o r y n a n t h e i n e aldehyde (94) on t h e b a s i s o f i t s TLC b e h a v i o u r . ( H e v e r i f i e d t h i s p r o p o s a l i n a l a t e r communication 91 by TLC comparison of s y n t h e t i c (94) w i t h the ' r a d i o - p r o f i l e ' . ) He t h e n s t a t e s t h a t t h e f o l l o w i n g a l k a l o i d s are formed h a v i n g 115 dynamic r a d i o - p r o f i l e s i . e . t h e y were r a p i d l y m e t a b o l i z e d . They were (1) g e i s s o s c h i z i n e (58) (1.5-2.5 hours) (2) preakuammi-c i n e (77) (2-2.5 hours) and (3) t a b e r s o n i n e (18) (9 h o u r s ) . O ther a l k a l o i d s d e t e c t e d e.g. v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , a j m a l i c i n e ( 1 5 ) , c o r o n a r i d i n e (59) and l o c h n e r i c i n e (95) had ' s t a t i c ' p r o f i l e s s u g g e s t i n g t h e i r f o r m a t i o n t o be s h u n t - l i k e i n n a t u r e . U n f o r t u n a t e l y c h a r a c t e r i s a t i o n was o n l y by two d i m e n s i o n a l TLC and d e t e c t i o n by means of c e r i c s u l f a t e s p r a y . I n t h i s r e s -p e c t o n l y 4 a l k a l o i d s c o u l d be d e t e c t e d by t h i s s p r a y i n g p r o c e s s e.g. v i n d o l i n e ( 1 1 ) , akuammicine ( 6 4 ) , v i n e r v i n e (96) and an u n i d e n --38-t i f i e d a l k a l o i d , - t h e remainder n o t b e i n g d e t e c t e d by t h i s s e n s i t i v e r e a g e n t . 0 HO 9 5 9 6 However a r e a s c o r r e s p o n d i n g t o t h e Rf o f a l l o f t h e s e components were removed (by s c r a p i n g ) and s u b j e c t e d t o r a d i o a c t i v e c o u n t i n g . A ssignment o f s t r u c t u r e must thus be s p e c u l a t i v e s i n c e a s m a l l amount o f r a d i o a c t i v i t y i n t h e s e a r e a s c o u l d g i v e r i s e t o such ob-s e r v a t i o n s . T h i s i s n o t a t o t a l l y c r i t i c a l comment but i t has been n o t e d i n my e x p e r i m e n t s t h a t s u b s t a n t i a l l o s s o f a c t i v i t y i n chromatographed f r a c t i o n s o c c u r r e d f o l l o w i n g r e p e a t e d c r y s t a l l i s a -t i o n s t h u s s u g g e s t i n g s m a l l q u a n t i t i e s o f r a d i o - a c t i v e m a t e r i a l as i m p u r i t i e s . T h i s p u b l i c a t i o n 1 1 5 was r e p r i n t e d v e r b a t i m , i n i t s e n t i r i t y , i n Recent Advances i n P h y t o c h e m i s t r y 1 1 6 w i t h o n l y o m i s s i o n o f the e x p e r i m e n t a l s e c t i o n b e i n g n o t e d . Thus, so f a r , we have n o t e d o n l y p r e l i m i n a r y e v i d e n c e t o sup-p o r t the p o s t u l a t e d b i o s y n t h e t i c o r i g i n s 8 4 , 8 " ' beyond s t r i c t o s i d i n e ( 4 9a). I n t h i s r e s p e c t many e x p e r i m e n t s d e s c r i b e d have s e v e r e l i m i t a t i o n s on t h e i r b i o s y n t h e t i c s i g n i f i c a n c e i f r e c e n t comments 2 by C o r n f o r t h are t o be r e s p e c t e d . C o n s e q u e n t l y t h e advancement o f e n z y m o l o g i c a l t e c h n i q u e s f o r a l k a l o i d b i o s y n t h e s i s have been pu r s u e d i n C. r o s e u s . -39-I n t h i s r e s p e c t some s l i g h t p r o g r e s s was i n i t i a l l y made by 117 i s o l a t i o n o f an O-methyl t r a n s f e r a s e w h i c h c a t a l y s e d the c o n v e r -s i o n o f l o g a n i c (97) and s e c o l o g a n i c a c i d (44a) t o l o g a n i n (35) and s e c o l o g a n i n (44) r e s p e c t i v e l y . A c etone powders o f C. r o s e u s , i n c u b a t e d w i t h t r y p t a m i n e ( l a ) and s e c o l o g a n i n ( 4 4 ) , gave no a l k a l o i d a l biosynthesis. 1"'""' H An i m p o r t a n t b r e a k t h r o u g h a t t h e c e l l f r e e l e v e l was n o t e d when 118 S c o t t o b t a i n e d a m i x t u r e o f s o l u b l e enzymes from C. r o s e u s (1) s e e d l i n g s and (2) c a l l u s e s - w h i c h on i n c u b a t i o n w i t h ( 2 1 4 C ) t r y p -tamine ( l a ) and s e c o l o g a n i n (44) i n the p r e s e n c e o f NADPH, t h i o l s and t r i s b u f f e r , a f f o r d e d g e i s s o s c h i z i n e (58) and a j m a l i c i n e ( 1 5 ) . The a l k a l o i d s were c h a r a c t e r i s e d by two d i m e n s i o n a l TLC b u t i t was n o t e d t h a t g r e a t e r c o n v e r s i o n s o c c u r r e d w i t h t h e s o l u b l e en-zymes from t h e c a l l u s e s . However, more i m p o r t a n t l y , a l k a l o i d a l b i o s y n t h e s i s had been dem o n s t r a t e d a t the c e l l f r e e l e v e l . T h i s f i n d i n g was s u b s e q u e n t l y c o n f i r m e d by Zenk, who i n c u b a t e d 14 (2 C) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) w i t h a s u p e r n a t a n t enzyme e x t r a c t o f a h i g h b e a r i n g a l k a l o i d s t r a i n o f C. r o s e u s and 8 2 NADPH. B i o s y n t h e s e s o f a j m a l i c i n e ( 1 5 ) , 1 9 - e p i d j m a l i c i n e (98) and -40-t e t r a h y d r o a l s t o n i n e (99) were n o t e d . - the s t r u c t u r e s b e i n g v e r i f i e d by (1) TLC comparison and (2) i n t e r n a l d i l u t i o n and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . I n t h i s r e s p e c t i t was no t e d t h a t i n c o r p o r -118 a t i o n s were h i g h e r than t h o s e o f S c o t t by a f a c t o r o f 10. 9 8 119 F u r t h e r o b s e r v a t i o n s by Zenk showed t h a t i n c u b a t i o n , as d e s c r i b e d , b u t w i t h o u t NADPH o r NADH r e s u l t e d i n a c c u m u l a t i o n o f an i n t e r m e d i a t e (100), w h i c h has been c a l l e d cathenamine. I t s s t r u c t u r -120 a l p r o p e r t i e s have r e c e n t l y been p u b l i s h e d . Sodium b o r o h y d r i d e r e d u c t i o n o f cathenamine (100) a f f o r d e d t e t r a h y d r o a l s t o n i n e ( 9 9 ) . I t was a l s o n o t e d t h a t , i n the absence o f NADPH o r NADH, no (15 ) , (98) o r (99) formed. C o n s e q u e n t l y when cathenamine (100), p r e p a r e d i n h i g h s p e c i f i c a c t i v i t y , was i n c u b a t e d w i t h C. r o s e u s p r o t e i n , NADPH, and phosphate b u f f e r , ( 1 5 ) , (98) and (99) were ob-t a i n e d . C o n t r o l e x p e r i m e n t s w i t h (1) h e a t d e n a t u r e d p r o t e i n and (2) no NADPH showed no f o r m a t i o n o f ( 1 5 ) , (9 8) and (99) . H 3C0 2 c " 1 0 0 F u r t h e r cathenamine (100) has been found t o be a n a t u r a l p r o d u c t . A j m a l i c i n e (15) was a l s o i n c u b a t e d w i t h t h e enzyme m i x t u r e and i t was found not t o be c o n v e r t e d t o (9 8) o r (99) s u g g e s t i n g t h a t -41-s t e r e o c h e m i c a l c o n t r o l p r o b a b l y o c c u r s a t the cathenamine (100) s t a g e . S t r i c t o s i d i n e (49a), p r e v i o u s l y d i s c u s s e d , was p r e p a r e d by i n c u -b a t i o n o f ( 2 1 4 C ) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) w i t h the en-zyme p r e p a r a t i o n i n t h e p r e s e n c e o f D - ^ - g l u c o n o l a c t o n e (a g l u c o -8 3a s i d a s e i n h i b i t o r ) S t r i c t o s i d i n e (49a) was c h a r a c t e r i s e d by (1) TLC (2) M.S. (3) c o l d d i l u t i o n - and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . I n c u b a t i o n o f s t r i c t o s i d i n e (49a) w i t h t h e enzyme p r e p a r a t i o n , i n the absence o f NADPH, y i e l d e d cathenamine (100). I n the p r e s e n c e o f NADPH, a l k a l o i d s ( 1 5 ) , (98) and (99) were o b t a i n e d . Thus f o r m a t i o n o f t h e c o r y n a n t h e a l k a l o i d s have been demon-s t r a t e d a t the c e l l f r e e l e v e l . I t w i l l be o f immense i m p o r t a n c e i f i t i s found t h a t subsequent t r a n s f o r m a t i o n s t o the s t r y c h n o s , a spidosperma and i b o g a a l k a l o i d s can a l s o be o b t a i n e d u s i n g such systems. I n a d d i t i o n t o the a f o r e m e n t i o n e d c o r y n a n t h e , s t r y c h n o s , aspidosperma and i b o g a a l k a l o i d s i t s h o u l d be n o t e d t h a t t h e ab-n o r m a l l y b r i d g e d a l k a l o i d s v a l l e s a m i n e (101), a p p a r i c i n e (102), u l e i n e (103), guatambuine (104), o l i v a c i n e (105) and e l l i p t i c i n e (106) a r e c u r r e n t l y presumed t o be formed a t l a t e s t a g e s i n a l k a -121 l o i d a l b i o s y n t h e s i s . However as c u r r e n t p o s t u l a t e s have n o t y e t been c o n c l u s i v e l y s u b s t a n t i a t e d , an o v e r a l l d i s c u s s i o n o f t h e v a r i o u s p o s t u l a t e s i s b r i e f l y mentioned below. - 4 2 -f 0 4 1 0 5 1 0 6 2 3 In Wenkert's o r i g i n a l monoterpenoid hypothesis he considered the non-tryptamine (la) portion of the indole alkaloids to be derived from prephenic acid (107). This was converted to a seco-prephenate formaldehyde (SPF) unit (108) which combined with t r y p t -amine (la) to give the indole alkaloids previously mentioned. 1 0 7 1 0 8 Wenkert extended his hypothesis to include the formation of uleine (103), o l i v a c i n e (105) and e l l i p t i c i n e (106). In t h i s respect i t was postulated that the SPF unit (108) combined with - 4 3 C H O - O H • O H C H O P O ^ H 2 3 © co 2 I 0 C 0 P 0 3 H C H , O c o 2 -0 C H 0 H O ' O H O H N A D H C H ^ O P O ^ H H 0, A C 0 - H 2 0 C O _© 2. H O P O = 3 O H O H C O 0 © H O P O N 3 A T P £ H ? G l u t a m m e R 0 ' ' C O © O H C O . © / V ^ O H O H 7 N A D P H . C O . © • N 0 © C 0 9 O H © c o 2 N H C H 2 0 P 0 3 H ' 0 H O O H - C O ? 2 H H H 0 0 0 Q N-CH=44+CH O P C H H ^ 3 l b F i g u r e 15 - Bi o s y n t h e s i s of tryptophan ( l b ) . - 4 4 -0 1 0 3 1 0 6 F i g u r e 16 - The Wenkert P o s t u l a t e f o r the b i o s y n t h e s i s o f o l i v a c i n e (105), e l l i p t i c i n e (106) and u l e i n e (103). -45-g l y c o s y i i d e n e a n t h r a n i l i c a c i d (109), a p r e c u r s o r o f t r y p t o p h a n ( l b ) ( f i g u r e 15) t o then y i e l d (103), ( 1 0 5 ) , ( 1 0 6 ) a s shown i n f i g u r e 16. F u r t h e r f o l l o w i n g t h e s t r u c t u r a l e l u c i d a t i o n o f a p p a r i c i n e (102), D j e r a s s i and G i l b e r t p o s t u l a t e d t h a t i n t e r m e d i a t e (110) may 203 be x n v o l v e d i n a p p a r i c i n e (102) f o r m a t i o n . ( F i g u r e 17) O V A C C O 2 C H 3 no F i g u r e 17 - The D j e r a s s i P o s t u l a t e f o r t h e b i o s y n t h e s i s o f a p p a r i c i n e (102). Thus the p o s t u l a t e d b i o s y n t h e t i c schemes (16,17) f o r the f o r m a t i o n o f t h e s e a l k a l o i d s do n o t i n v o l v e t r y p t o p h a n ( l b ) . I n o r d e r t o t e s t 3 t h i s p r o p o s a l (Ar H) t r y p t o p h a n ( l b ) was a d m i n i s t e r e d t o A. p y r i c o l l u m , a p l a n t c o n t a i n i n g a p p a r i c i n e (102) and u l e i n e (103). I n c o r p o r a -122 t i o n s i n t o a p p a r i c i n e (102) were n o t e d i n d i c a t i n g t h a t t r y p t o -phan ( l b ) was b e i n g u t i l i z e d . C l e a r l y t h e s e r e s u l t s a r e incom-p a t i b l e w i t h t h e Wenkert and D j e r a s s i p r o p o s a l s . I t was p o s s i b l e , however, t h a t the p l a n t enzymes were c a p a b l e o f d e g r a d i n g t r y p t o -phan ( l b ) t o an a p p r o p r i a t e i n d o l e i n t e r m e d i a t e l a c k i n g t h e / ? - e t h y l -amino s i d e c h a i n w h i c h was the n i n v o l v e d i n the b i o s y n t h e s i s . -46-E x p e r i m e n t s were th e n d e s i g n e d t o de t e r m i n e whether one o r b o t h carbons o f the ^3-ethylamino s i d e c h a i n o f t r y p t o p h a n ( l b ) 3 14 3 14 were e x t r u d e d . C o n s e q u e n t l y (Ar H, 2 C) and (Ar H,3 C) t r y p t o -phan ( l b ) were f e d t o A. p y r i c o l l u m . I s o l a t i o n o f a p p a r i c i n e (102) showed e x t r u s i o n o f C-2 o f t r y p t o p h a n ( l b ) had o c c u r r e d (~9 7%) w h i l e 14 3 d e t e r m i n a t i o n o f the C/ H r a t i o i n the a l k a l o i d c l e a r l y r e -v e a l e d t h a t C-3 was r e t a i n e d . I n t h i s l a t t e r e x p e r i m e n t i t was n o t e d t h a t a l o s s o f 27% o f t r i t i u m o c c u r r e d from the a r o m a t i c r i n g . F u r t h e r a d m i n i s t r a t i o n o f (1) ( A r 3 H ) , ( 2 ) ( A r 3 H , 2 1 4 C ) , 3 14 (3) (Ar H, 3 C) t r y p t o p h a n ( l b ) t o A. a u s t r a l e y i e l d e d some i n -121 t e r e s t m g r e s u l t s . I n t h e s e e x p e r i m e n t s , a l k a l o i d s o l i v a c i n e (105), a p p a r i c i n e (102), u l e i n e (103) and guatambuine (104) were i s o l a t e d . W i t h r e s p e c t t o e x p e r i m e n t s (1) and ( 3 ) , o l i v a c i n e (105) c o u l d n o t be c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y and so no m e a n i n g f u l d i s c u s s i o n can be ensued on t h a t b a s i s . However e x p e r i m e n t ( 2 ) , c o n s t a n t a c t i v i t y , i n c o r p o r a t i o n (<0.001%), showed t h a t t h e 3 14 H/ C r a t i o had d o u b l e d on i n c o r p o r a t i o n s u g g e s t i n g l o s s o f 58% o f the l a b e l a t C-2 o f t r y p t o p h a n ( l b ) . I n e x p e r i m e n t s ( 1 ) , (2) and (3) where a p p a r i c i n e (102) was i s o l a t e d i t was n o t e d i n (1) t h a t s i g n i f i c a n t i n c o r p o r a t i o n i n t o (102) had o c c u r r e d , i.e. 0.208% . I n ( 2 ) , however, i n c o r p o r a t i o n l e v e l was •<0.0015% and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y c o u l d n o t be o b t a i n e d . Good i n c o r p o r a t i o n s were n o t e d though f o r e x p e r i m e n t (3) -47-(0.127%) a l t h o u g h a g a i n a l o s s o f 17% t r i t i u m o c c u r r e d . I n e x p e r i m e n t s (1) and (2) v/here u l e i n e (103) was i s o l a t e d i t was n o t e d t h a t i n (1) i n c o r p o r a t i o n was <0.001%. I n e x p e r i -ment (2) an i n c o r p o r a t i o n o f 0.001% was o b t a i n e d w i t h a 3H/^ 4C r a t i o a l m o s t i d e n t i c a l t o t h a t f e d . I n e x p e r i m e n t s (1) and (2) where guatambuine (104) was i s o -l a t e d , i n c o r p o r a t i o n s v/ere o f the o r d e r 0.002%. I n (2) l o s s o f 27% of the C-2 l a b e l from t r y p t o p h a n ( l b ) was n o t e d . These r e s u l t s a r e summarised i n t a b l e 2. T a b l e 2: F i n d i n g s o f t r y p t o p h a n ( l b ) i n c o r p o r a t i o n s i n t o a l k a l o i d s  o f A. a u s t r a l e (A) and A. p y r i c o l l u m ( B ) . Tryptophan ( l b ) A l k a l o i d s I s o l a t e d l a b e l f e d : A p p a r i c i n e U l e i n e Guatambuine O l i v a c i n e (102) (A) (103) (B) (104) (B) (105) (B) 1) A r 3 H , 2 1 4 C l o s s o f C-2 (%) 97 no l o s s 27 58 3 14 2) A r H,3 qC l o s s of H (%) 2 7 N/A N/A N/A N/A - n o t a p p l i c a b l e . 3 The p o s s i b l e i n v o l v e m e n t o f (Ar H) stemmadenine (63) was 105 t h e n p u r s u e d i n A. p y r i c o l l u m , and, a p p a r i c i n e (102), and u l e i n e (103), v/ere i s o l a t e d . S i g n i f i c a n t i n c o r p o r a t i o n i n t o (102) was n o t e d w i t h no a p p r e c i a b l e i n c o r p o r a t i o n i n t o (103) . F e e d i n g e x p e r i m e n t s were t h e n p u r s u e d w i t h v a l l e s a m i n e (101) due t o i t s s t r u c t u r a l s i m i l a r i t i e s w i t h stemmadenine ( 6 3 ) . Very low i n c o r p o r a t i o n i n t o (102) and (103) were, n o t e d . I n o r d e r t o e x p l a i n t h e s e r e s u l t s , i t was s u g g e s t e d t h a t e x t r u s i o n o f carbon -48-and m o d i f i c a t i o n t o the e x o c y c l i c methylene on c o n v e r s i o n from stemmadenine (63) t o a p p a r i c i n e (102) may have o c c u r r e d v i a a c o n c e r t e d p r o c e s s . 12 3 P o t i e r has r e c e n t l y r e p o r t e d an i n v i t r o f r a g m e n t a t i o n w h i c h may a c c o u n t f o r the ca r b o n e x t r u s i o n . ( F i g u r e 18) C H 3 H + <CF3C°!P C C E C C ^ H F h N H • H3 C\ / C H j / ^ N - O C O C R P h N ( C H j : 3 ' 2 H 4-N © H C C H . o ° F i g u r e 18 - P o t i e r ' s i n v i t r o e x t r u s i o n o f C-2 u n i t of /3 -ethylamine side chain . S c o t t has r e p o r t e d an i n v i t r o c o n v e r s i o n o f stemmadenine (63) i n t o v a l l e s a m i n e (101) a c e t a t e u t i l i s i n g the m o d i f i e d P o l o n o v s k i r e a c t i o n . ( F i g u r e 19) 123 P o t i e r had p r e v i o u s l y p o s t u l a t e d a s i m i l a r mechanism f o r t h e b i o s y n t h e s i s o f a p p a r i c i n e (102). ( F i g u r e 20) -49-F i g u r e 19 - S c o t t ' s i n v i t r o c o n v e r s i o n o f stemmadenine (63) t o v a l l e s a m i n e (101). 102 F i g u r e 2 0 - P o t i e r ' s p o s t u l a t e d b i o s y n t h e s i s o f a p p a r i c i n e (102) from stemmadenine ( 6 3 ) . -50-The p o s s i b l e i n v o l v e m e n t o f a d e h y d r o s e c o d i n e (74) type i n t e r -m e d i a t e v/as th e n e x p l o r e d and w h i l s t c o n d i t i o n s have n o t y e t been met f o r t h e s t a b i l i s a t i o n o f t h i s i n t e r m e d i a t e i t v/as f e l t t h a t l a b e l l i n g e x p e r i m e n t s w i t h 16,17 d e h y d r o s e c o d i n - 1 7 - o l (88) and 103-5 122 s e c o d i n e (91) might be i n f o r m a t i v e . ' L a b e l l i n g e x p e r i m e n t s w i t h (88) showed n e g l i g i b l e i n c o r p o r -a t i o n s i n t o a l k a l o i d s o f A. p y r i c o l l u m . L a b e l l i n g e x p e r i m e n t s w i t h v a r i o u s l a b e l l e d forms o f s e c o d i n e (91) were th e n i n i t i a t e d w i t h A. p y r i c o l l u m and A. a u s t r a l e 3 due t o s m a l l b u t h o p e f u l l y s i g n i f i c a n t i n c o r p o r a t i o n s of (Ar H) s e c o d i n e (91) i n t o (102). The r e s u l t s a re summarized i n t a b l e 3 and t a b l e 4. T a b l e 3: Summary o f r e s u l t s of l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. p y r i c o l l u m . S e c o d i n e (91) l a b e l f e d : 1) H, C r a t i o i n A p p a r i c i n e (102): T a b l e 4: Summary of r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. a u s t r a l e . S e c o d i n e (91) l a b e l : H, CO„CH_ r a t i o i n : A p p a r i c i n e (102) Guatambuine (104) O l i v a c i n e (105) l o s s o f 30% H l o s s o f 22% 3H * * * - not c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y -51-C o n s e q u e n t l y i t i s not p o s s i b l e t o draw any r i g i d c o n c l u s i o n s from th e s e r e s u l t s . 12 4 P o t i e r and J a n o t have r e c e n t l y extended t h e i r p o s t u l a t e t o encompass guatambuine (104), o l i v a c i n e ( 1 0 5 ) ^ e l l i p t i c i n e (106) and u l e i n e (103). ( F i g u r e 21) As n o t e d i n f i g u r e 20, the P o t i e r p o s t u l a t e f o r t h e f o r m a t i o n o f a p p a r i c i n e (102) i n v o l v e d a P o l o n o v s k i t y p e f r a g m e n t a t i o n f o l l o w e d by l o s s o f the carbomethoxy group o f stemmadenine (6 3 ) . T h i s p o s t u l a t e i s i n c o m p a t i b l e w i t h t h e s u g g e s t i v e e v i d e n c e t h a t the carbomethoxy group i s i n f a c t r e t a i n e d as shown i n the s e c o d i n e (91) f e e d i n g s . (Table 3) Moreover the g e n e r a l p o s t u l a t e f o r the b i o s y n t h e s e s o f u l e i n e (103), guatambuine (104), o l i v a c i n e (105) and e l l i p t i c i n e ( 1 0 6 ), are i n c o m p a t i b l e w i t h the i n c o r p o r a t i o n s n o t e d i n A. p y r i c o l l u m and A. a u s t r a l e . (Table 2) Thus i n o r d e r t o o b t a i n more d e f i n i t i v e d a t a the p l a n t 125 12 7 Aspidosperma v a r g a s i i , r i c h i n a l k a l o i d s a p p a r i c i n e (102) , 12 7 126 u l e i n e (103) , guatambuine (104) and 9 - m e t h o x y o l i v a c i n e 126 (111) , was n e x t c o n s i d e r e d . These i n v e s t i g a t i o n s w i t h c o r r e s p o n d i n g l a b e l l e d p r e c u r s o r s are d i s c u s s e d i n the n e x t s e c t i o n . See r e f . 199 f o r numberina svstem. 1 0 4 1 0 5 1 0 6 F i g u r e 21 - P o t i e r - J a n o t p o s t u l a t e f o r t h e b i o s y n t h e s i s of a p p a r i c i n e (102), u l e i n e (103), guatambuine (104), o l i v a -c i n e (105) and e l l i p t i c i n e (106) . -53-DPSCUSSION - PART I B i o s y n t h e s i s o f i n d o l e a l k a l o i d s l a c k i n g the normal t r y p t o p h a n  s i d e c h a i n : r o l e o f t r y p t o p h a n ( l b ) and stemmadenine (63) A. v a r g a s i i p l a n t s when mature are t r e e s s t a n d i n g up t o twenty metres i n h e i g h t and are i n d i g e n o u s t o r o c k y a r i d s l o p e s and t r a n s i t i o n f o r e s t i n V e n e z u e l a , and a d j a c e n t Colombia and Guiana. The p l a n t s r e q u i r e d f o r t h i s r e s e a r c h programme were f i v e d and 125(a) y e a r s o l d grown from s e e d s 1 2 5 ^ k i n d l y donated by Dr. B. G i l b e r t 3 The p r e c u r s o r s , c o m m e r c i a l l y a v a i l a b l e (Ar H) t r y p t o p h a n ( l b ) 3 and (Ar H) stemmadenine (63) , p r e p a r e d i n t h i s l a b o r a t o r y by the g e n e r a l a c i d c a t a l y s e d exchange of a r o m a t i c p r o t o n s (CF^C0 2T as t r i t i u m s o u r c e ) , were employed i n th e s e s t u d i e s . The r e s u l t i n g t r i t i a t e d p r e c u r s o r s were c r y s t a l l i s e d t o con-s t a n t a c t i v i t v and a d m i n i s t e r e d t o A. v a r g a s i i (whole p l a n t o r r o o t s e c t i o n s ) o v e r v a r y i n g time p e r i o d s . F o l l o w i n g t h e d u r a t i o n o f the p l a n t f e e d i n g , the p l a n t was removed, macerated, and e x t r a c t e d w i t h methanol. The a l k a l o i d s were s e p a r a t e d a c c o r d i n g t o a g e n e r a l p l a n t e x t r a c t i o n p r o c e d u r e 12 8 d e s c r i b e d by G i l b e r t . I n the a d m i n i s t r a t i o n o f t r y p t o p h a n ( l b ) t o A. v a r g a s i i r o o t c u t t i n g s the a l k a l o i d s p r e v i o u s l y mentioned (102-104, 111) were i s o l a t e d and c r y s t a l l i s e d r e p e a t e d l y t o remove i m p u r i t i e s . Re-c r y s t a l l i s a t i o n p r o c e d u r e s were r e p e a t e d u n t i l the f o l l o w i n g i n c o r p o r a t i o n l e v e l s were o b t a i n e d . (Table 5) S i m i l a r r e s u l t s 12 7 were r e c e n t l y n o t e d by D. G r i e r s o n i n stem f e e d i n g s . (Table 6) T a b l e 5 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i r o o t c u t t i n g s A c t i v i t y Fed (DPM) D u r a t i o n o f F e e d i n g (Days) A l k a l o i d I s o l a t e d S p e c i f i c A c t i v i t y I s o l a t e d (DPM/mmol) Weight I s o l a t e d (mg) A b s o l u t e I n c o r p o r a t i o n (%) S p e c i f i c I n c o r p o r a t i o n (%) 8.025x10 11 U l e i n e .(103) Guatambuine (10 4) 9-Methoxy-o l i v a c i n e (111) 0.00 1. 7388 x 10* 5.408 x 1 0 5 20 12 46 0.00 0.0026 0.0031 0.00 0.00069 0.00021 3 T a b l e 6 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i whole p l a n t 5.82 x l O 1 1 7 U l e i n e (103) 0.00 6 0.00 0.00 Guatambuine (104) 1.71 x 1 0 5 40 0.0018 0.000068 o n v a S n T u i l ) 3 ' 1 7 X 1 0 5 9 1 °- 0 0 4 9 0.000054 i I -55-C o u n t i n g was d i s c o n t i n u e d a t a l e v e l where b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . The g e n e r a l i t i e s a s s o c i a t e d w i t h the t r y p t o p h a n ( l b ) f e e d i n g s d i s c u s s e d so f a r a r e : (1) t h r e e d i f f e r e n t p l a n t s o u r c e s have been e x p l o i t e d (A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i ) (2) a d m i n i s t r a t i o n o f p r e c u r s o r had been c a r r i e d out a t v a r i o u s t i m e s o f t h e y e a r t o p l a n t s o f v a r y i n g s i z e s and ages. (3) p r e c u r s o r a d m i n i s t r a t i o n had employed s e v e r a l t e c h n i q u e s e.g. stem and r o o t f e e d i n g s . D i f f e r e n t s o l v e n t systems had been used t o a d m i n i s t e r the p r e c u r s o r , and, e f f i c i e n t i n c o r p o r a t i o n o f t r y p t o p h a n ( l b ) i n t o the p l a n t was n o t e d . (4) a f t e r i n i t i a l a d m i n i s t r a t i o n , e x p e r i m e n t s were o f v a r i e d d u r a t i o n (up t o 2 8 d a y s ) . (5) i n t a c t i n c o r p o r a t i o n i n t o any o f the a f o r e m e n t i o n e d 3 14 a l k a l o i d s was n o t demonstrated u s i n g e i t h e r (Ar H, 2 C) or ( A r 3 H , 3 1 4 C ) t r y p t o p h a n ( l b ) . ( I n c o r p o r a t i o n l e v e l s , l e s s than 0.001%). Thus a v a r i e t y o f e x p e r i m e n t s have been employed t o d e t e r m i n e 122 the r o l e o f t r y p t o p h a n ( l b ) . From the e a r l i e r s t u d i e s where c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y was o b t a i n e d i t i s o b v i o u s t h a t the r e s u l t s a re n o t i n agreement w i t h the P o t i e r - J a n o t 124 p o s t u l a t e . However as n o t e d i n some c a s e s , t h e p l a n t s (A. p y r i c o l l u m and A. a u s t r a l e ) d i d n o t always have s u f f i c i e n t q u a n t i t i e s o f a l k a l o i d s (102-105) t o o b t a i n c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . -56-I n the e x p e r i m e n t s w i t h A. v a r g a s i i , however, where the a l -k a l o i d s were i n s u f f i c i e n t q u a n t i t y , no e v i d e n c e c o u l d be found f o r the p o s i t i v e i n c o r p o r a t i o n o f t r y p t o p h a n ( l b ) thus c a s t i n g f u r -124 t h e r doubt on the p o s t u l a t e 3 The l e v e l s o f i n c o r p o r a t i o n on a d m i n i s t r a t i o n o f (Ar H) stemmadenine (63) as p r e c u r s o r t o the a l k a l o i d s (103), (104) and (111) o f A. v a r g a s i i a r e shown i n t a b l e 7 . A g a i n c o u n t i n g was d i s c o n t i n u e d a t a l e v e l where b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . The e x p e r i m e n t s des-c r i b e d d e m o n s t r a t e d t h a t stemmadenine (63), under the c o n d i t i o n s employed, was not u t i l i z e d as an a c t i v e b i o s y n t h e t i c i n t e r m e d i a t e i n the b i o s y n t h e s i s o f t h e s e a l k a l o i d s . I t i s o f t e n s a i d t h a t " g r e a t c a u t i o n " must be e x e r c i s e d i n i n t e r p r e t i n g r e s u l t s o b t a i n e d from low i n c o r p o r a t i o n s s i n c e t h e s e are o f t e n r a t i o n a l i s e d as due t o : 1) low a l k a l o i d a l b i o s y n t h e s i s a t the t i m e o f a d m i n i s t r a t i o n o f the p r e c u r s o r . 2) n o n - p e r m e a b i l i t y o f t h e b i o s y n t h e t i c s i t e by t h e p r e c u r s o r 3) m o d i f i c a t i o n o r d e s t r u c t i o n o f the p r e c u r s o r p r i o r t o r e a c h -i n g t h e b i o s y n t h e t i c s i t e . C o n s e q u e n t l y , i t i s o f t e n v e r y d i f f i c u l t t o e s t a b l i s h t h a t a s u g g e s t e d i n t e r m e d i a t e i s a t r u e b i o s y n t h e t i c p r e c u r s o r . I n c o n c l u s i o n , t h e s e r e s u l t s r e v e a l t h a t (1) t r y p t o p h a n ( l b ) and stemmadenine (63) d i d n o t appear t o be e f f i c i e n t l y i n v o l v e d as p r e c u r s o r s . (2) i f t r y p t o p h a n ( l b ) and stemmadenine (63) are i m p o r t a n t p r e c u r s o r s t o t h e s e a l k a l o i d s then c o n d i t i o n s must be T a b l e 7 - R e s u l t s of (Ar H) stemmadenine (63) a d m i n i s t r a t i o n t o A. v a r g a s i i . D u r a t i o n A c t i v i t y o f P l a n t Fed F e e d i n g (Net Wt.) (PPM) (Days) (gm) S p e c i f i c A c t i v i t y Weight A b s o l u t e S p e c i f i c A l k a l o i d I s o l a t e d I s o l a t e d I n c o r p o r a t i o n I n c o r p o r a t i o n I s o l a t e d (PPM/mmol) (mg) (%) (%) 6.29 x l O 10 130 U l e i n e (103) 9-MeO-O l i v a c i n e (111) 0.00 5.74 x 10 10 85 0.00 0 .0077 0.00 0.000091 3.447x10 10 13 120 U l e i n e (103) Guatambuine (104) 9-MeO-O l i v a c i n e (111) 0.00 2.479 x 10 1.5906 x 10 18 26 104 0.00 0.0019 0. 0048 0.00 0.000072 0.00046 8.48 xlO 10 28 150 U l e i n e (103) Guatambuine (104) 9-MeO-O l i v a c i n e (111) 1.86 x 10 6.819 x 10 5.386 x 10 28 20 70 0.0006 0.0016 0.0044 0.000022 0.00008 0.000064 i I -58-d e t e r m i n e d t o show t h e i r e f f e c t i v e use as p r e c u r s o r s . Conse-q u e n t l y i t was d e c i d e d t o grow t i s s u e c u l t u r e s o f A. p y r i c o l l u m and A. v a r g a s i i i n the hope t h a t more s i g n i f i c a n t i n f o r m a t i o n c o n c e r n i n g the b i o s y n t h e s i s o f a l k a l o i d s (102)-(10 6) and (111) c o u l d be o b t a i n e d . I f p r o p e r growth c o n d i t i o n s f o r such t i s s u e c u l t u r e s were e f f e c t e d , then s t u d y o f a l k a l o i d b i o s y n t h e s i s c o u l d be d e t e r m i n e d a t the c e l l - f r e e l e v e l . P e r m e a b i l i t y problems would h o p e f u l l y be d i m i n i s h e d and the r o l e o f t r y p t o p h a n ( l b ) and stemmadenine (63) c o u l d be r e - i n v e s t i g a t e d . A programme was thus i n i t i a t e d t o grow t i s s u e c u l t u r e s o f A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i . C o n s i d e r a b l e e f f o r t was e x p e n d e d 1 2 ^ * 3 t o t r y and d e t e r m i n e c o n d i t i o n s f o r t h e i r growth on e i t h e r s o l i d (agar) o r l i q u i d media and were i n i t i a l l y u n s u c c e s s -f u l i n b o t h media. R e c e n t l y c o n d i t i o n s f o r the v e r y s low growth o f t i s s u e c u l -t u r e s o f t h e s e p l a n t s have been o b t a i n e d u s i n g a medium w h i c h was a l s o s u c c e s s f u l f o r C. r o s e u s ( d i s c u s s e d l a t e r ) . I n a l l a t t e m p t s to" grow the c u l t u r e s , s m a l l s e c t i o n s ( s e v e r a l hundred) of stem, l e a f o r l e a f base were t a k e n and s t e r i l i s e d by p l a c i n g i n (1) e t h a n o l (2) 0.1% m e r c u r i c c h l o r i d e i n w a t e r (w/w) f o r 2 m i n u tes and (3) s t e r i l i s e d w a t e r . The s e c t i o n s were then p l a c e d on s o l i d agar medium i n an a t -129 tempt t o grow c a l l u s c u l t u r e s . I n the medium d e s c r i b e d , growth c o n d i t i o n s f o r e x t r e m e l y slow growth o f t h e s e c u l t u r e s have been o b t a i n e d . (Table 8) No c o n d i t i o n s f o r growth o f c u l t u r e s on l i q u i d media c o u l d be e f f e c t e d f o r A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i . -59-T a b l e 8 - C o m p o s i t i o n o f n u t r i e n t medium f o r s o l i d (agar) medium. KN0 3 MgSO 4-7H 20 N H 4 H 2 P 0 4 C a C l 2 - 2 H 2 0 F e S 0 4-7H 20 MnS0 4-H 20 I n o r g a n i c N u t r i e n t s mg/1 2,500 400 300 200 15 10 H 3 B O 3 ZnS0 4• KI CuS0 4< NaMoO 7H 20 5H 20 l-2H 20 C o C l 2 * 6 H 2 0 mg/1 5 1 1 0.2 0.1 0.1 O r g a n i c N u t r i e n t s I n o s i t o l N a 2 EDTA Thiamine-HC1 1,000 20 5 N i c o t i n i c A c i d P y r i d o x i n e HC1 5 0.5 Growth R e g u l a t o r s 2,4 D P-CPA 1 K i n e t i n 0.5 2.0 0.1 Sucrose Agar 30' P-CPA = P-chlorophenoxy a c e t i c a c i d 2 gm/1 -60-U n f o r t u n a t e l y <200 mg. o f c a l l u s c u l t u r e (dry w e i g h t ) were o b t a i n e d o v e r a s i x month growth p e r i o d and c o n s e q u e n t l y t h e s e c o u l d n o t be examined f o r t h e i r a l k a l o i d a l c o n t e n t . Thus, t o d a t e , the b i o s y n t h e s i s o f a l k a l o i d s (102,107,111) a t the c e l l - f r e e l e v e l has n o t been demonstrated. I f however the growth c o n d i t i o n s a r e c o r r e c t f o r a l k a l o i d b i o s y n t h e s i s , then the r o l e o f stemmadenine (63) and t r y p t o p h a n ( l b ) can be r e - e v a l u a t e d and, i f th e s e i n t e r m e d i a t e s are a g a i n n o t i n v o l v e d , t h e n a r e - a p p r a i s a l o f the b i o s y n t h e t i c t h e o r i e s f o r the f o r m a t i o n o f t h e s e a l k a l o i d s w i l l be n e c e s s a r y . F o l l o w i n g t h e e x p e r i m e n t a l , the n e x t s e c t i o n o f the t h e s i s c o n c e r n s i t s e l f w i t h the most r e c e n t i n v e s t i g a t i o n s d i r e c t e d towards t h e s y n t h e s e s o f a p p r o p r i a t e l y r a d i o - l a b e l l e d forms o f ' stemmadenine (63) f o r t h e s e f u t u r e i n v e s t i g a t i o n s . -61-EXP E RIMENTAL - PART I M e l t i n g p o i n t s were de t e r m i n e d on a K o f l e r b l o c k and are un-c o r r e c t e d . A l l u l t r a v i o l e t (UV.) s p e c t r a were r e c o r d e d i n methanol o r e t h a n o l u s i n g a Cary 15 r e c o r d i n g s p e c t r o p h o t o m e t e r . The i n f r a r e d (IR.) s p e c t r a were r e c o r d e d w i t h a P e r k i n - E l m e r Model 457 s p e c t r o p h o t o m e t e r e i t h e r i n c h l o r o f o r m s o l u t i o n ( c a v i t y c e l l s 0.1 mm.) o r as a n u j o l m u l l between sodium c h l o r i d e p l a t e s as i n d i c a t e d . A l l measurements were made i n cm ^ and c a l -i b r a t i o n o f the s n e c t r a was a c h i e v e d u s i n g the 1601 cm ^ abs o r p -t i o n band o f p o l y s t y r e n e . N u c l e a r m agnetic resonance s p e c t r a (N.M.R.) were o b t a i n e d i n d e u t e r o c h l o r o f o r m s o l u t i o n s ( u n l e s s o t h e r w i s e i n d i c a t e d ) a t 100 MHz on the V a r i a n HA-100, V a r i a n XL-100 o r B r u k e r TT-23 n u c l e a r magnetic resonance s p e c t r o m e t e r s . A l l N.M.R. s p e c t r a o b t a i n e d v i a t h e F o u r i e r T r a n s f o r m t e c h n i q u e (F.T.) w i l l be so noted and were o b t a i n e d w i t h the V a r i a n XL-100 o r B r u k e r TT-2 3 i n s t r u m e n t s . C h e m i c a l s h i f t s were g i v e n i n (ppm) w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as the i n t e r n a l s t a n d a r d . The m u l t i p l i c i t y , i n t e g r a t e d a r e a s , and p r o t o n assignments are g i v e n i n p a r e n t h e s e s . Mass s p e c t r a were d e t e r m i n e d on AEI MS-9, AEI MS-5 0 o r V a r i a n Mat CH-4B mass s p e c t r o m e t e r s , w i t h h i g h r e s o l u -t i o n mass s p e c t r a b e i n g d e t e r m i n e d w i t h the f i r s t two. Woelm n e u t r a l a l u m i n a and EM Reagents GF254 s i l i c a g e l were used f o r t h i n and o r e p a r a t i v e l a y e r chromatography. Woelm n e u t r a l a lumina (Ac t . I l l ) and Merck s i l i c a g e l ( A c t . 2-3) were used f o r column chromatography. A l l r e a c t i o n s d e s c r i b e d h e r e i n , u n l e s s o t h e r w i s e i n d i c a t e d , were performed under an atmosphere o f n i t r o g e n . E l e m e n t a l a n a l y s e s were c a r r i e d out by Mr. P. Borda, M i c r o a n a l y s t , - 6 2 -U n i v e r s i t y o f B r i t i s h C o lumbia. R a d i o a c t i v i t y was measured w i t h a N u c l e a r - C h i c a g o Mark I I l i q u i d s c i n t i l l a t i o n c o u n t e r i n counts p e r minute (cpm). The r a d i o a c t i v i t y o f the sample i n d i s i n t e g r a t i o n s p e r minute (dpm) was s u b s e q u e n t l y d e t e r m i n e d by the e x t e r n a l s t a n d a r d t e c h n i q u e u s i n g a b u i l t - i n Barium-133 s o u r c e o f gamma r a d i a t i o n . S c i n -t i l l a t i o n c o c k t a i l s used were e i t h e r a p r e p a r e d s o l u t i o n o f t o l u e n e and a N u c l e a r - C h i c a g o PPO-POPOP c o n c e n t r a t e o r a pre m i x e d s o l u t i o n o f N u c l e a r - C h i c a g o PCS c o c k t a i l . A s c i n t i l l a t i o n c o u n t i n g sam-p l e c o n s i s t e d o f a s o l u t i o n o f the sample d i s s o l v e d i n 1 m l . o f methanol and 14 ml. o f t h e a p p r o p r i a t e c o c k t a i l ( t o t a l volume 15 m l . ) . F o r each sample the background o f t h e v i a l was p r e -d e t e r m i n e d and s u b s e q u e n t l y s u b t r a c t e d from the measured dpm's f o r the sample i n subsequent c a l c u l a t i o n s . Each sample was counted f o r a time p e r i o d l o n g enough f o r the t o t a l c o u n t s f o r t h e sample, l e s s t h e t o t a l c o u n t s f o r t h e background t o exceed t e n thousand c o u n t s . The A. v a r g a s i i p l a n t s used i n t h i s s t u d y were grown i n the H o r t i c u l t u r e Department greenhouse, t h e U n i v e r s i t y o f B r i t i s h C o l umbia. I n t h i s s e c t i o n o n l y f u r t h e r c h a r a c t e r i s a t i o n d a t a f o r the a l k a l o i d s i s o l a t e d were g i v e n w i t h the e x c e p t i o n o f m e l t i n g p o i n t s . -63-A d m i n i s t r a t i o n o f (Ar^H) t r y p t o p h a n ( l b ) t o A. v a r g a s i i To A. v a r g a s i i r o o t c l i p p i n g s (36 g , wet w e i g h t ) w e r e added (Ar 3H) t r y p t o p h a n ( l b ) (3.21 mg , 2.50 x 1 0 1 1 dpm/mmol) dis-s o l v e d i n a w a t e r / a c e t i c a c i d s o l u t i o n (20:0.5 ml ). The r o o t s v/ere i n c u b a t e d w i t h t h i s s o l u t i o n f o r n i n e days a f t e r w h i c h they were removed and c o p i o u s l y washed w i t h w a t e r . The r o o t s were s i m u l t a n e o u s l y p u l p e d and e x t r a c t e d w i t h methanol - t h i s b e i n g e f f e c t e d by suspending the r o o t s i n methanol i n a b l e n d e r . The s o l i d p l a n t r e s i d u e was then removed by s u c t i o n f i l t r a t i o n . The r e s i d u e was the n r e - e x t r a c t e d t w i c e i n an i d e n t i c a l manner. The combined methanol e x t r a c t s were evapor-a t e d t o d r y n e s s . The methanol e x t r a c t was then suspended i n 10% h y d r o c h l o r i c a c i d (300 ml ) f o r 45 min ( i n an atmosphere o f n i t r o g e n ) . The aqueous e x t r a c t was the n e x t r a c t e d (1) w i t h p e t r o l e u m e t h e r (30-60°C) (3x300 ml ) and (2) c h l o r o f o r m (3x300 ml ). The c h l o r o f o r m e x t r a c t was d r i e d o v e r anhydrous sodium s u l -p h a t e , f i l t e r e d , and e v a p o r a t e d t o dryn e s s (0.1224 g ) . (Extract 1). The r e s u l t i n g a c i d l a y e r was then c o o l e d t o 5° and t r e a t e d s l o w l y w i t h c o n c e n t r a t e d ammonium h y d r o x i d e s o l u t i o n u n t i l pH 11. E x t r a c t i o n o f the b a s i c l a y e r w i t h c h l o r o f o r m gave, a f t e r d r y i n g , the c o r r e s p o n d i n g c h l o r o f o r m s o l u b l e s (0.390 g ) . (Extract 2). The c h l o r o f o r m e x t r a c t 1 was chromatographed on alumina (Act I I I ) u s i n g a c h l o r o f o r m / e t h y l a c e t a t e g r a d i e n t . A p p a r i c i n e (102), (16 mg , c o l o u r l e s s n e e d l e s , m.pt. 191°, l i f 1 . 2 7 1 9 2 - 4 0 ), u l e i n e (103) (28 mg , c o l o u r l e s s c r y s t a l s , m.pt. 75-100°, - 6 4 -lit."'79-100° , (±) guatambuine (104) (12 mg , c o l o u r l e s s cubes, 12 6 m.pt. 249° l i t . 250-52°), were thus o b t a i n e d . These a l k a l o i d s i s o l a t e d were f u r t h e r checked by comparison of t h e i r TLC, NMR, IR, UV and MS b e h a v i o u r with that of authentic samples. The c h l o r o f o r m e x t r a c t 2 was chromatographed on alumina (Act I I I ) u s i n g a c h l o r o f o r m / e t h y l a c e t a t e g r a d i e n t . The 9 - m e t h o x y - o l i v a c i n e (111) o b t a i n e d (46 mg , y e l l o w n e e d l e s , m.pt. 126 289-91° , l i t . 293-4°), was i d e n t i c a l i n a l l r e s p e c t s w i t h an a u t h e n t i c sample, (e.g. TLC, UV, I R ) . NMR: $ (CDC1 ) 100 MHz (FT): 8.72 ( s , l H , NH) , 8.40 (d, IH, J=6Hz, C-3H), 7.95 (m, IH, C-12 H) , 7.76 (d, 1H, J=6Hz, C-14 H), 7.70 (s , IH, C-19 H), 7.43 (d, IH, J=8Hz, C-9 H), 7.28 (d, IH, J=3 and 8Hz, C-10 H) , 4.00 ( s , 3H, OCH_3) , 3.15 (s, 3H, C-18 H_3) , 2.80 ( s , 3H, C-17 H ). M.S. m/e: 276 (M +)/ 261, 245. H.R.M.S.: C a l c - f o r C 1 8H 1 6N 20=276.1623 Found=276.1618 A d m i n i s t r a t i o n o f (Ar JH) stemmadenine (63) t o A. v a r g a s s i 3 10 (Ar H) stemmadenine (63) (3.64 x 10 dpm/m mol ) was d i s -s o l v e d i n methanol/water (1:4, 5 ml ) and a d m i n i s t e r e d t o A.  v a r g a s i i p l a n t s o v e r 7, 13 and 28 day p e r i o d s r e s p e c t i v e l y u s i n g t h e c o t t o n w i c k t e c h n i q u e . T h i s p r o c e d u r e r e q u i r e s b o r i n g a s m a l l h o l e i n the p l a n t -65-stem about 2 " above the s o i l . One end of a moist cotton wick was then placed i n the hole and the other end into a test-tube containing the precursor i n solution. In this way the precursor was introduced into the plant. The alkaloids were then i s o l a t e d i n a manner analagous to that already described. -66-DISCUS'SION ~ PART I I In view of comments made e a r l i e r i t i s o b v i o u s t h a t a stemmade-n i n e (63) type i n t e r m e d i a t e i s c o n s i d e r e d t o be o f paramount im-p o r t a n c e i n the a l k a l o i d b i o s y n t h e s i s so f a r d i s c u s s e d . Stemmadenine (63) was f i r s t i s o l a t e d from Stemmadenia d o n n e l l s m i t h i i (Rose) V7oodson i n 19 5 8 " ^ a n d was l a t e r shown t o po s s e s s 131 g r o s s s t r u c t u r e as shown . Only c o n f i g u r a t i o n s a t C-15 and 131a 13 ^  C-19 were determined u n t i l r e c e n t l y *. ( D i s c u s s e d l a t e r ) However, of the n o n - t r y p t o p h a n a l k a l o i d s (102-106, 111), 3 o n l y a p p a r i c i n e (10 2) gave p o s i t i v e i n c o r p o r a t i o n w i t h (Ar H) stemmadenine (63) p l a n t f e e d i n g e x p e r i m e n t s ? 5 F u r t h e r s i n c e o n l y t h e a r o m a t i c r i n g was l a b e l l e d the b i o s y n t h e t i c i n f o r m a t i o n from t h i s i n c o r p o r a t i o n was l i m i t e d . I n o r d e r t o demonstrate t h a t (63) i s a p r e c u r s o r o f the a l -k a l o i d s (102-106, 111) the f o l l o w i n g must be det e r m i n e d i n v i v o  (a) f a t e of carbon atoms 5 and 6 o f stemmadenine (6 3) - i n p a r -t i c u l a r was C-5 l o s t i n the f o r m a t i o n o f a p p a r i c i n e (102) as a l -ready sugges t e r r o r t r y p t o p h a n ( l b ) ? F u r t h e r , i f carbons 5 and 6 from stemmadenine (63) were m a i n t a i n e d i n (103-106, 111) t h e i r po-s i t i o n s must be de t e r m i n e d by d e g r a d a t i o n p r o c e d u r e s . -67-(b) f a t e o f carboinethoxy and hydroxymethy 1 groups of stemmadenine (63) -103-5,122 as suggested from the s e c o d i n e (91) f e e d i n g e x p e r i m e n t s , i n c o r p o r -a t i o n i n t o a p p a r i c i n e (102) o c c u r r e d w i t h the carbomethoxy group b e i n g r e t a i n e d . I t was o f c o n s i d e r a b l e i n t e r e s t t o determine whether a s i m i l a r p r o c e s s o c c u r r e d w i t h stemmadenine (63). (c) f a t e of C-18 m e t h y l group of stemmadenine (63) - assuming i n -c o r p o r a t i o n s c o u l d be o b t a i n e d , a p p r o p r i a t e d e g r a d a t i o n e x p e r i -ments t o show p o s i t i o n of the l a b e l must be pursued. (d) f a t e o f C-3 methylene o f stemmadenine (63) - a g a i n i n c o r p o r a -t i o n i n t o a l k a l o i d s must be s u p p o r t e d by d e g r a d a t i o n o f the i s o -l a t e d a l k a l o i d t o v e r i f y the p o s i t i o n o f the l a b e l . Thus, i n o r d e r t o m o n i t o r the f a t e o f t h e s e atoms, i t was n e c e s s a r y t o i n v e s t i g a t e p o s s i b l e s y n t h e s e s of (63) p o s s e s s i n g r a d i o a c t i v e l a b e l s a t (1) C,. and Cg (2) C-16 f u n c t i o n a l i t i e s (carbomethoxy and hydroxymethy1 groups) (3) C-18 and (4) C-3. I f t h e s e c o u l d be r e a l i s e d then subsequent f e e d i n g e x p e r i m e n t s and complementary d e g r a d a t i o n s t u d i e s would g i v e much i n f o r m a t i o n t o -wards t h e u n d e r s t a n d i n g o f t h e s e l a t e r b i o s y n t h e t i c r e a r r a n g e -ments (assuming r e a s o n a b l e i n c o r p o r a t i o n s ) . I t s h o u l d be noted t h a t i n any s y n t h e s i s o f a b i o s y n t h e t i -c a l l y i n t e r e s t i n g r a d i o l a b e l l e d p r e c u r s o r i t i s h i g h l y d e s i r a b l e t o l a b e l the m o l e c u l e a t a v e r y l a t e s t a g e i n the s y n t h e s i s , and, as w i l l be r e a l i s e d , t h i s p o i n t r e c e i v e d f u l l c o n s i d e r a t i o n i n the s t u d i e s d i s c u s s e d l a t e r . Stemmadenine ( 6 3 ) , r e c e i v e d s y n t h e t i c i n t e r e s t p r i o r t o t h e s e s t u d i e s and comment on t h e s e i n v e s t i g a t i o n s seems mandatory. -68-Ban a c c o m p l i s h e d the f o l l o w i n g t r a n s f o r m a t i o n (112-113) w h i c h was t h e r e b y c o n s i d e r e d a f e a s i b l e e n t r y i n t o the aspidosperma and s t r y c h n o s a l k a l o i d s . I t had a l s o been n o t e d t h a t t r e a t m e n t o f preakuammicine ( 7 7 ) , i s o l a t e d from C. r o s e u s , w i t h b o r o h y d r i d e i n methanol v i e l d e d 8 5 stemmadenine (63) and akuammicine (64 ) . 7 7 A t t e m p t s t o s y n t h e s i s e 19,20-dihydrostemmadenine (116) i n t h i s 13 4 l a b o r a t o r y by c y c l i z a t i o n o f i n t e r m e d i a t e (115), o b t a i n e d from 18,19 d i h y d r o c o r y n a n t h e i n e aldehyde (114), i n a s i m i l a r manner were u n s u c c e s s f u l . ( F i g u r e 22) F a i l u r e o f t h i s c y c l i z a t i o n has 135 a l s o been r e p o r t e d by W i n t e r f e l d t From a b i o s y n t h e t i c s t a n d p o i n t t h i s approach was o f l i t t l e i n t e r e s t s i n c e i t c o u l d n o t r e a d i l y be adapted f o r our l a b e l l i n g p u r s u i t s . -69-H 1 1 6 F i g u r e 22 - Kutney's a t t e m p t e d s y n t h e s i s o f 19,20-dihydrostemmadenine (116). C o n s e q u e n t l y , s t r u c t u r a l s i m i l a r i t i e s between stemmadenine (63) and s t r y c h n i n e (5) were t a k e n i n t o a ccount and i t was hoped t h a t t h e s e c o u l d be e x p l o i t e d . T h i s approach was l i m i t e d i n the sense t h a t C,. and Cg o f (63) would be p r e f o r m e d and thus l a b e l l -i n g t h e s e p o s i t i o n s would be e x t r e m e l y d i f f i c u l t . - 7 0 -I n t h i s r e s p e c t a n o t h e r s y n t h e s i s o f a stemmadenine type m o l e c u l e (118) had r e c e n t l y been reported"*" 3^ and, h e r e , a d d i t i o n o f the C 5 - C 5 bond o c c u r r e d i n the f i n a l s t a g e s . T h i s approach would p r o b a b l y have t o be d r a s t i c a l l y m o d i f i e d t o y i e l d stemmadenine (63) and w h i l e o f i n t e r e s t was g i v e n no f u r t h e r c o n s i d e r a t i o n . ( F i g u r e 23) 1 1 8 F i g u r e 2 3 - S n i e c k u s ' s y n t h e s i s o f a stemmadenine model. -71-F i g u r e 24 - Kutney's a t t e m p t e d s y n t h e s i s o f stemmadenine ( 6 3 ) . -72-Recent s t u d i e s , i n t h i s l a b o r a t o r y towards the s y n t h e s i s 13 8 o f stemmadenine ( 6 3 ) , used n o r - f l u o r o - c u r a r i n e (119) , d e r i v e d from s t r y c h n i n e ( 5 ) . Each r o u t e a t t e m p t e d ( f i g u r e 24) had l i t e r a t u r e precedence i n s i m p l e r systems and d e s e r v e s b r i e f comment. Pathway ( a ) : c o n v e r s i o n of <xl ^ - u n s a t u r a t e d a l d e h y d e s t o 139 t h e i r c o r r e s p o n d i n g e s t e r s has been w e l l documented. C o n d i -t i o n s f o r t h i s t r a n s f o r m a t i o n t o akuammicine (64) c o u l d n o t be o b t a i n e d . Pathways '(b) and (c) : l i t e r a t u r e p r ecedences had e s t a b l i s h e d t h a t the i n d o l e (12 7) was o b t a i n e d from descarbomethoxyakuammi-140 c i n e (126) by t r e a t m e n t w i t h b o r o h y d r i d e i n me t h a n o l . ( F i g u r e 25) 1 2 6 1 2 7 F i g u r e 2 5 - Base c a t a l y s e d r i n g o p e n i n g o f descarbomethoxyakuammicine (126). I n t h i s r e s p e c t , t r e a t m e n t o f (119) w i t h sodium b o r o h y d r i d e i n 137 methanol gave an i n d o l i c compound s u g g e s t e d t o be (128). 1 2 8 -73-I t was hooed t h a t the r i n g o p e n i n g t o t h e i n d o l e c o u l d be e f f e c t e d k e e p i n g the ald e h y d e f u n c t i o n i n t a c t t o g i v e (124) by e i t h e r k e t a l i s a t i o n o r f o r m a t i o n o f the e n o l a c e t a t e p r i o r t o r i n g o p e n i n g ( f i g u r e 24, pathway b , c ) . The aldehyde (124) was r e q u i r e d s i n c e t h i s f u n c t i o n a l i t y i n l a t e r a l k y l a t i o n s t u d i e s w o u l d be o f g r e a t i m p o r t a n c e . C o n d i t i o n s , however, c o u l d n o t be found f o r the f o r m a t i o n o f e i t h e r the e n o l 137 a c e t a t e o r k e t a l and t h i s approach was d i s c o n t i n u e d . I t had a l s o been shown t h a t e n o l a t e a n i o n s are not r e d u c e d 141 by b o r o h y d r i d e ' and i t was c o n s i d e r e d t h a t t h i s o b s e r v a t i o n c o u l d be used t o o b t a i n (123) - however, t r e a t m e n t of e n o l a t e a n i o n (121) w i t h b o r o h y d r i d e gave no r e a c t i o n and pathway c was 137 abandoned. Pathway d w h i c h i n v o l v e d t r e a t m e n t o f p y r r o l i d i n e o r c y c l o -h e x y l d i e n a m i n e (125), w i t h m e t h y l c h l o r o f o r m a t e under e q u i l i b r i u m c o n d i t i o n s f a i l e d t o g i v e any p r o d u c t s o t h e r than s t a r t i n g ma-137 t e r i a l and was a l s o s u b s e q u e n t l y abandoned. A t t e n t i o n was t h e n f o c u s s e d onto the c o r r e s p o n d i n g e s t e r (132) s i n c e a r o u t e from W i e l a n d - G u m l i c h a l d e h y d e (129), p r e -142 v i o u s l y d e r i v e d from s t r y c h n i n e (5),had been r e p o r t e d i n 43% crud e y i e l d from oxime (130). E s t e r (132) v/as c o n s i d e r e d as an i m p o r t a n t i n t e r m e d i a t e f o r f u r t h e r s y n t h e t i c a t t e m p t s t o y i e l d stemmadenine ( 6 3 ) . Two l i t e r a t u r e p r e c e d e n t s ^ " 3 8 * 3 ' ± 4 3 s p e c i f i c a l l y a p p l i c a b l e t o t h e s t r y c h n o s system, were a v a i l a b l e f o r removal o f a l l y l i c h y d r o x y l g r o u o s . These were (1) h y d r o g e n o l y s i s 1 3 ^ w i t h H2/Pd/C -74-1 2 9 H 2 N O H - H C I l ) B a ( O H ) 2 ) H C I M e O H C 0 2 C H 3 Ac OyPyridine O C O C H -1 3 2 P d C 131 C 0 2 C H 3 1 3 4 + C 0 2 C H 3 1 3 3 F i g u r e 26 - S y n t h e s i s o f 2,16-dihydroakuammicine (133) and (2) t r e a t m e n t w i t h HBr/HOAc t o g i v e the bromide f o l l o w e d by 143 r e d u c t i v e removal w i t h Zn/HOAc. -75-Procedure (1) was r e p o r t e d 1 J ' to give ester (133) together with an undetermined quantity of dihydroester (134) as an im-purity which could not be removed. Procedure (2) was thus u t i l i z e d y i e l d i n g required ester i n 65% y i e l d . With ester (133) i n hand, attempts to y i e l d stemmadenine (63) were as follows: (Figure 27) Figure 27 - Kutney's synthesis of epi-stemmadenine (138). -76-F i g u r e 27, Pathway (a) F o r m a t i o n of akuammicine (64) was r e p o r t e d by t r e a t m e n t o f N - t o s y l d e r i v a t i v e (140) o f (133) w i t h p o t a s s i u m - t - b u t o x i d e i n 144 d i m e t h y l s u l f o x i d e ' f o r m a t i o n . No y i e l d s were g i v e n f o r t h i s t r a n s -6 4 C 0 2 C H 3 However i t had a l s o been n o t e d t h a t l e a d t e t r a a c e t a t e c o n v e r t e d 145 i n d o l i n e s d i r e c t l y t o t h e i r c o r r e s p o n d i n g m d o l e n i n e s . Con-s e q u e n t l y akuammicine (64) was formed u s i n g the l a t t e r method. F u r t h e r t r e a t m e n t o f (64) w i t h (1) a c e t i c a c i d and (2) sodium 132 137 b o r o h y d r i d e gave i n d o l e e s t e r (122). ' An a t t e m p t t o a l k y l a t e (122) t o g i v e stemmadenine (63) o r 137 i t s C-16 epimer was u n s u c c e s s f u l . F i g u r e 27, Pathway (b) f a r e d b e t t e r and was e x e c u t e d as shown t o g i v e a compound r e p o r t e d t o be (138) - b u t w h i c h was n o t stemmade-n i n e (63). U n f o r t u n a t e l y , the p u r i f i c a t i o n p r o c e s s e s employed were  not a b l e t o y i e l d (138) i n a form w h i c h a l l o w e d p o s i t i v e i d e n t i f i c a -t i o n . C o n s e q u e n t l y i t was r e p o r t e d t h a t (138) and (63) were r e d u c e d t o t h e d i o l (139) h Y t r e a t m e n t w i t h sodium b i s - ( m e t h o x y m e t h y l e n e -oxy ) -a l u m i n i u m h y d r i d e . Comparison o f the IR and NMR s p e c t r a r e -p o r t e d f o r t h e d i o l s (139) showed c l o s e s i m i l a r i t i e s and thus i t was -77-f o r w a r d e d t h a t (138) was the C-16 epimer o f (63) . J" J Z ' x J ' A s t e r e o c h e m i c a l argument was s u b s e q u e n t l y p u r s u e d i n w h i c h i t was s u g g e s t e d t h a t (138) had the f o l l o w i n g s t e r e o c h e m i s t r y thus g i v i n g stemmadenine (63) t h e a b s o l u t e c o n f i g u r a t i o n shown."'' ( D i s c u s s e d l a t e r ) 6 3 ' 3 8 As i s c l e a r l y n o t e d the i n v e s t i g a t i o n s d i s c u s s e d t h r o u g h o u t t h i s t h e s i s are p a r t o f l o n g range s y n t h e t i c and b i o s y n t h e t i c programmes and t h i s s e c t i o n now concerns i t s e l f w i t h the more r e c e n t a t t e m p t s t o s y n t h e s i z e stemmadenine (63) from s t r y c h n i n e ( 5 ) . C o n s e q u e n t l y i n i t i a l i n v e s t i g a t i o n s c o n s i d e r e d a c y l a t i o n o f a l d e -hyde (145), o r an a p p r o p r i a t e l y m o d i f i e d form o f (145), s i n c e i t was hoped t h a t t h i s would g i v e the o p p o s i t e s t e r e o c h e m i s t r y t o t h a t p r e v i o u s l y o b t a i n e d . I f t h i s c o u l d be a c c o m p l i s h e d , then r e d u c t i o n o f t h e al d e h y d e and s i m i l a r m a n i p u l a t i o n t o t h a t a l r e a d y ]32 13 7 d e s c r i b e d ' ' would y i e l d stemmadenine (63) . The c o n v e r s i o n o f s t r y c h n i n e (5) t o 2j5r 16 U. - c u r - 1 9 - e n - 1 7 - o l (143) as shown i n f i g u r e 28 i s w e l l documented. The i n i t i a l s t e p s t o W i e l a n d - G u m l i c h aldehyde (129) (WGA) were f i r s t r e p o r t e d i n 1932" 1" 4 6, improved i n 1 9 5 8 1 4 7 , and o p t i m i s e d i n 1 9 6 9 1 4 8 . As can be seen t r e a t m e n t o f s t r y c h n i n e (5) w i t h sodium e t h o x i d e -78-F i g u r e 2 8 - D e g r a d a t i o n o f s t r y c h n i n e (5) t o 2B, 1 6 o ( - c u r - l 9 - e n - 1 7 - o l (143) . 1 2 9 o = s I 1 4 4 C I F i g u r e 2 9 - F o r m a t i o n o f W i e l a n d - G u m l i c h aldehyde (12 9 ) . -79-and i s o a m y l n i t r i t e gave isonitrosostrychnine (141) which was i s -olated i n high o v e r a l l y i e l d as the corresponding hydrochloride s a l t . Treatment of this s a l t with thio n y l chloride followed by quenching i n ice gave a crude p r e c i p i t a t e of 1-cyanoformy1-WGA (144) which a f t e r r e c r y s t a l l i s a t i o n from methanol exhibited phy-14 8 s i c a l data previously reported. The compound (144) was then subjected to steam d i s t i l l a t i o n at pH 3.3-3.5 to give the required hemi-acetal WGA (129) and hydrogen cyanide. Treatment of WGA (12 9) v/ith sodium borohydride afforded 2j$, -147 16o(-cur-19-en-17,18-diol i n good y i e l d . This d i o l (142) can be converted to 2j$, ,16o(-cur-19-en-17-ol (143) i n either of two ways: (1) Bernauer had reported that treatment of d i o l (142) v/ith HBr/HOAc afforded the a l l y l i c bromide shown below. The bro-mide was then treated with zinc dust i n acetic acid to give the acetate of alcohol (143), which on hydrolysis with methanolic potassium hydroxide afforded required alcohol (143). 142 H B r H O A c p 1 4 3 (2) Hydrogenolysis using a spe c i a l palladium on charcoal catalyst prepared by Wieland" 1" 3 8^. Either method could thus be used for the introduction of a t r i t i u m l a b e l at this p o s i t i o n (C-18). Following the l a b e l l i n g , checks could be made to ensure that no random l a b e l l i n g had occurred elsev/here in the molecule (e.g. by ozonisation) . - 8 0 -Th e h y d r o g e n o l y s i s was the p r o c e d u r e o f c h o i c e i n t h e s t u d i e s employed s i n c e i t gave (143) i n 25-30% h i g h e r y i e l d . W i t h a l c o h o l (143) on hand, t h r e e pathways were c o n s i d e r e d t o g i v e stemmadenine (63) ( F i g u r e 3 0 ) : (1) f o r m a t i o n o f 1 - f o r m y l aldehyde (146), c o n d e n s a t i o n w i t h c y c l o h e x y l a m i n e t o g i v e (147), and t r e a t m e n t w i t h base and methy1-c h l o r o f o r m a t e t o y i e l d i n t e r m e d i a t e (148). F u r t h e r m i l d a c i d h y d r o l y s i s and b o r o h y d r i d e r e d u c t i o n , removal of f o r m y l group, would y i e l d t he r e q u i r e d (149), the e x p e c t e d C-16 epimer o f (137). (2) d i r e c t a c y l a t i o n of 1- f o r m y l aldehyde (146), r e d u c t i o n o f al d e h y d e t o a l c o h o l and removal of f o r m y l group t o y i e l d (149). (3) c o n v e r s i o n t o n o r - f l u o r o c u r a r i n e (119), r i n g - o p e n i n g and r e d u c t i o n t o a l c o h o l (128), o x i d a t i o n t o aldehyde (124), f o l l o w e d by a c y l a t i o n and r e d u c t i o n t o (63). The c o n v e r s i o n o f (143) t o (145) r e q u i r e s some comment s i n c e 137 d i f f i c u l t i e s had been e n c o u n t e r e d i n t h e s e l a b o r a t o r i e s and 138 e l s e w h e r e t o o b t a i n (145) i n good y i e l d . A t b e s t , t r e a t m e n t o f (143) under anhydrous, a n a e r o b i c c o n d i t i o n s w i t h p o t a s s i u m t - b u t o x i d e and benzophenone i n r e f l u x i n g benzene gave a m i x t u r e o f (145) and (119) i n 39 and 10% r e s p e c t i v e l y . More commonly, however, f u r t h e r o x i d a t i o n t o (119) was n o t e d as t h e major p r o d u c t . Replacement o f the base employed by sodium h y d r i d e y i e l d e d r e q u i r e d a l d e h y d e (145) i n 73% w i t h no measurable q u a n t i t y o f (119) b e i n g o b s e r v e d . Presumably t h i s i s formed v i a the f o l l o w i n g mechanism. ( F i g u r e 31) -81--82-( P h ) C = 0 1 4 5 C H O F i g u r e 31 - M o d i f i e d Oppenauer o x i d a t i o n o f ' 2y0,16od-cur-19-en-17-ol. As n o t e d the i n d o l i n e was p r o t e c t e d i n the n e x t s t e p by a d d i n g the f o r m y l group. T h i s group was chosen due t o the r e l a -t i v e ease w i t h w h i c h i t can be removed ( s t i r r i n g a t room temper-149 a t u r e w i t h sodium h y d r i d e i n THF ). The most e f f i c i e n t documented means of f o r m y l a t i o n o f d i h y d r o -indoles"'' 5^ was w i t h f o r m i c a c e t i c anhydride"'"5"'" and c o n s e q u e n t l y (146) was o b t a i n e d as e v i d e n c e d by absence o f NH a b s o r p t i o n i n the IR spectrum and the p r e s e n c e o f a two c a r b o n y l system a t 1730 c m - 1 and 1670 cm - 1. The U.V. spectrum (^max 287,278,250 nm) 142 was c h a r a c t e r i s t i c o f an N - a c y l i n d o l i n e . The NMR e x h i b i t e d the e x p e c t e d 3 p r o t o n d o u b l e t o f d o u b l e t s a t j l ' 5 2 (J=2 and 6 H z ) f o r =CHCH 3 and a 1 p r o t o n q u a r t e t f o r CHCH 3 a t 5.47 (J=6 H z ) . A more i n t e r e s t i n g f e a t u r e , however, were two s i n g l e t s a t £ 8.58, 8.83 f o r the p r o t o n (N-CHO), and t h r e e s i n g l e t s a t 6 9.58, 9.72, 9.76 f o r the p r o t o n (CHO). I n a d d i t i o n two d o u b l e t s a t cf 4.74, 4.88 were n o t e d f o r c-16 H. A s i m i l a r o b s e r v a t i o n had 152 been n o t e d w i t h N - f o r m y l d i h y d r o i n d o l e s (150) and (151). -83-I n b o t h c a s e s , the N-CHO p r o t o n had e x i s t e d as two s i n g l e t s and t h i s had been a s c r i b e d due t o f r e e r o t a t i o n h i n d r a n c e o f the N-CHO group. 0^0 I C H O 1 5 0 F u r t h e r s u p p o r t t o t h i s r a t i o n a l e was g i v e n by h e a t i n g t o 72-77° where c o l l a p s e t o a s i n g l e t was ob s e r v e d . These r e s u l t s a r e however a l s o c o m p a t i b l e w i t h " l o n e p a i r " i n v e r s i o n i s o m e r i s m . The mass spectrum of (146) a l s o showed major peaks a t m/e 322 (M +),279,172,164 & 109 w h i c h a r e i n agreement w i t h the 153 p r o p o s e d f r a g m e n t a t i o n s o f t h i s d i h y d r o i n d o l i n e system as shown i n f i g u r e 32. Cl e a v a g e , f o l l o w i n g r e t r o D i e l s - A l d e r r e a c t i o n , a t p o s i t i o n s marked , j$ and / gave o b s e r v e d i o n s b, c and d (R=R^"=CH0) a t m/e 16 4, 172, and 109. I n a d d i t i o n h y d r i d e t r a n s f e r and r u p t u r e of r i n g C f o l l o w e d by c l e a v a g e o f the C-15, 16 bond and rearrangement gave i o n e a t m/e 279. C o n v e r s i o n t o the i m i n e (147) ( f i g u r e 30, pathway 1) was r e a d i l y e f f e c t e d by h e a t i n g c y c l o h e x y l a m i n e and (146) t o g e t h e r i n r e f l u x i n g benzene u s i n g a m o d i f i e d Dean-Stark a p p a r a t u s ( i . e . a s o x h l e t c o n t a i n i n g anhydrous m o l e c u l a r s i e v e s ) - t h i s -84-F i g u r e 32 - P r o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f methyl-cur-19-ene f a m i l y . - 8 5 -b e i n g n o t e d by a one c a r b o n y l a b s o r p t i o n i n the IR a t 16 65 cm x , a mass spec, m/e 403 (M +) and the o t h e r peaks f o l l o w e d a s i m i -l a r f r a g m e n t a t i o n p r o c e s s t o t h a t g i v e n i n f i g u r e 32. The r e l e v a n t NMR d a t a f o r t h i s assignment showed a 1 p r o t o n s i n g l e t a t 5 9.02 (NCHO) , an AB q u a r t e t a t £8.69 (J=8 & 15 Hz) f o r CH=N and a <5l.91-l.37, CH2 en v e l o p e (cyclohexane) . The U.'V. remained v i r -t u a l l y unchanged (Amax 288, 282, 252 nm) as e x p e c t e d . H y d r o l y s i s w i t h h y d r o c h l o r i c a c i d (5%) gave (146) i n h i g h y i e l d . However a l l endeavours t o a c y l a t e (147) a t C-16 met w i t h f a i l u r e . These a t t e m p t s i n v o l v e d t r e a t m e n t o f the m o l e c u l e w i t h (1) base and (2) m e t h y l c h l o r o f o r m a t e . A v a r i e t y o f bases (e.g. t - b u t y l l i t h i u m , d i - i s o p r o p y l l i t h i u m amide, p o t a s s i u m and sodium h y d r i d e , e t c . ) , s o l v e n t s (e.g. THF, benzene, DMSO, HMPA) were em-p l o y e d a t v a r y i n g t e m p e r a t u r e s and e i t h e r s t a r t i n g m a t e r i a l was i s o l a t e d o r i n t h e case o f h i g h e r t e m p e r a t u r e s (>30°C) s e v e r e d e c o m p o s i t i o n r e s u l t e d . One p o s s i b l e r a t i o n a l e f o r t h i s f a i l u r e was t h e o b s e r v a t i o n t h a t f o r m a t i o n o f 1 4 7 1 5 2 1 4 8 i n t e r m e d i a t e (152) from (147) r e s u l t s i n an u n f a v o r a b l e s t e r i c i n t e r a c t i o n between the C-18 me t h y l group and the C-17 p r o t o n . Con-- 8 6 -sequently the approach was abandoned. Direct acylation (figure 30, pathway 2) was considered even although i t was appreciated that most conditions would favour 0-acylation. However i t was hoped that a small, but s i g n i f i c a n t quantity of C-acylation would occur. Again a var i e t y of condi-tions were employed, and, again none of the required acylated product could be obtained. It was, however, noted that (146), on treatment with sodium hy i n r e f l u x i n g benzene gave (153) i n very high y i e l d . Formation of (153) at elevated temperatures, can be consi-dered v i a i n i t i a l quaternary s a l t formation followed by nucleo-p h i l i c attach by chlorine. The UV was again c h a r a c t e r i s t i c of a N-formyl dihydroindole ( X f 4 287 , 278, 248 nm.). The IR showed CO absorption at 1668 and 168 8 cm respectively i n agreement with the proposed structure. The NMR spectrum exhi-bited three s i n g l e t s at £9.74, 9.67 and 9.5 for CHO, two singlets at<f 8.84, 8. 6 0 for NCHO, a one proton quartet at 6 5.52 for C-19 H, a doublet of doublets at 6 4.52 (J=10 and 16 Hz) for -87-C-16H, and a t h r e e p r o t o n s i n g l e t a t & 3.74 ( C O ^ C I - ^ ) . The mass spectrum showed an m/e 416 (M +) and e x h i b i t e d major peaks a t 210, 209, 208, 194, 180, 150, ( f i g u r e 12) w h i c h are 1 5 3 i n agreement w i t h the D j e r a s s i p r o p o s a l ( f i g u r e 33). The compound (15 3) was reduced t o the c o r r e s p o n d i n g a l c o -h o l (154) and f o l l o w i n g v e r i f i c a t i o n o f t h i s s t r u c t u r e the approach was d i s c o n t i n u e d . Pathway 3 ( f i g u r e 30) was then n e x t c o n s i d e r e d , and, i n t h i s r e s p e c t , t r e a t m e n t o f (143) w i t h e i t h e r p o t a s s i u m - t -b u t o x i d e o r p o t a s s i u m h y d r i d e i n benzene and n i t r o b e n z e n e gave n o r - f l u o r o c u r a r i n e (119) w h i c h was t h e n c o n v e r t e d t o the c o r r e s p o n d i n g i n d o l e a l c o h o l (128) by t r e a t m e n t w i t h b o r o h y d r i d e 137 i n m e t h a n o l . S e v e r a l a t t e m p t s t o o x i d i s e a l c o h o l (12 8) under a v a r i e t y o f c o n d i t i o n s met w i t h f a i l u r e . I n a l l cases a p l e t h o r a o f p r o d u c t s was o b t a i n e d on a t t e m p t i n g the c o n v e r s i o n and t h e approach was dropped. Thus t h e s e complementary s t u d i e s t o pathways p r e v i o u s l y 137 i n v e s t i g a t e d w i t h aldehydes (119) o r (146) a l s o e n j o y e d f a i l u r e and the u t i l i s a t i o n o f t h e s e s u b s t r a t e s f o r f u r t h e r s y n t h e t i c p u r s u i t s was abandoned. R e - c o n s i d e r a t i o n was t h e r e -f o r e g i v e n t o t h e c o r r e s p o n d i n g i n d o l e (122) s e r i e s , s i n c e i t c o n t a i n e d a more f l e x i b l e n i n e membered r i n g f o r w h i c h the D r e i d i n g model s u g g e s t e d a l k y l a t i o n c o u l d o c c u r t o g i v e ii " e i t h e r epimer - a l t h o u g h t h e °(-face appeared t o be f a v o u r e d f o r a t t a c k . m / e 18 0 F i g u r e 33 - P o s t u l a t e d mass s p e c t r a l f r a g m e n t a t i o n o f a c y l a t e d d e r i v a t i v e (153). - 8 9 -I n i t i a l a l k y l a t i o n o f (122) w i t h formaldehyde had r e c e i v e d 137 p r e l i m i n a r y i n v e s t i g a t i o n w i t h o u t s u c c e s s and t h i s w i l l be mentioned as i t p e r t a i n s t o the d i s c u s s i o n . C o n s e q u e n t l y , p r i o r t o embarking upon a l e n g t h y s y n t h e t i c r o u t e w h i c h p r o m i s e d f a i l u r e , i t was i n i t i a l l y d e c i d e d t o i n v e s -t i g a t e a l k y l a t i o n w i t h a model i n d o l e compound. The model system chosen was t e t r a h y d r o c a r b a z o l e (155) and i t was hoped t h a t c o n d i t i o n s c o u l d be found f o r the f o r m a t i o n o f e s t e r (156) w h i c h t h e n under s u i t a b l e m a n i p u l a t i o n would g i v e the c o r r e s p o n d i n g 1-carbomethoxy-1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157). A l i t e r a t u r e s u r v e y showed t h a t 1 - c a r b o e t h o x y t e t r a h y d r o c a r b a z o l e was p r o v e n by d e h y d r o g e n a t i o n t o the c o r r e s p o n d i n g c a r b a z o l e (156b) system and comparison w i t h an a u t h e n t i c sample. As can be seen from f i g u r e 34, (158) was c o n s i d e r e d t o be formed as an i n t e r m e d i a t e . However c y c l i z a t i o n o f (158) would be e x p e c t e d t o g i v e 4 - c a r b o e t h o x y t e t r a h y d r o c a r b a z o l e (159) and as o n l y m e l t i n g p o i n t , e l e m e n t a l a n a l y s i s and f o r m u l a were r e p o r t e d f o r (158) i t can a l s o be e n v i s a g e d t h a t t h e r e a c t i o n i n f a c t goes v i a an i n t e r m e d i a t e such as (160) - w h i c h would be more i n a c c o r d w i t h t h e d a t a r e p o r t e d . ( F i g u r e 35) (156a) was a v a i l a b l e 154,155 as shown i n f i g u r e 34. T h i s s t r u c t u r e 154 - 9 0 -Figure 34 - Lenzi's synthesis of 1-carboethoxytetrahydrocarbazole (156a Although th i s approach could be readi l y modified for the f o r -mation of (156), i t was considered that a more e f f i c i e n t synthesis d i r e c t l y from tetrahydrocarbazole (155) could be effected. Consequently attempts to introduce a one carbon function at C-l were investigated. I n i t i a l attempts to introduce a n i t r i l e function, v i a the corresponding chloroindolenine ( 1 6 1 ) x 5 6 met with -91-1 5 6 a 1 6 0 F i g u r e 35 - An a l t e r n a t i v e t o L e n z i ' s p o s t u l a t e . f a i l u r e s i n c e (161) r a p i d l y r e a c t e d w i t h the b e n z o t r i a z o l e formed i n the r e a c t i o n t o y i e l d 1 - b e n z o t r i a z o l e - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (162) ( f i g u r e 36) as e v i d e n c e d by the UV h a v i n g the c h a r a c t e r i s t i c 162 F i g u r e 36 - S y n t h e s i s o f 1 - b e n z o t r i a z o l e t e t r a h y d r o c a r b a z o l e (162). -92-i n d o l e chromophore ( J 291, 282, 276, 223). S u p p o r t f o r t h i s Kiel X s t r u c t u r e was a l s o g i v e n by t h e mass spectrum m/e 2 88 (M +) and the IR spectrum w h i c h had NH a b s o r p t i o n a t 3470 cm \ The NMR was a l s o i n d i c a t i v e h a v i n g a s i n g l e t a t i 8.4 (NH) and a one p r o t o n t r i p l e t a t $ 6.36-6.62 (J=5.5 Hz) C - l H . However t h i s p r o b lem was r e s o l v e d by t r e a t i n g 1 - p y r i d i n i u m -15 7 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e bromide (163) w i t h p o t a s s i u m c y -a n i d e i n a c e t o n i t r i l e . The crude p y r i d i n i u m bromide s a l t (163) was formed as a crude c r y s t a l l i n e mass by t r e a t m e n t o f t e t r a h y d r o c a r -b a z o l e (155) w i t h N-bromosuccinimide and b e n z o y l p e r o x i d e i n py-r i d i n e . The p r o p o s e d mechanism f o r the f o r m a t i o n o f the p y r i d i n i u m s a l t (163) i s shown i n f i g u r e 37. Treatment o f t h i s w i t h c y a n i d e gave r e q u i r e d 1 - c y a n o t e t r a h y d r o c a r b a z o l e (164). 155 163 1 6 4 The IR spectrum showed NH and CN a b s o r p t i o n a t 3470 and 2240 cm 1 and the UV spectrum was a g a i n c h a r a c t e r i s t i c o f an i n d o l e (/\ m a x 289 , 281, 273, 223 nm.) . The mass spectrum c o n t a i n e d the e x p e c t e d m/e 19 6 ( M + ) , and the NMR showed a one p r o t o n s i n g l e t a t S 8.12 (NH) and a one p r o t o n t r i p l e t a t S 4.15,(J=5.4 Hz) C - l H . A s m a l l amount (^10%) o f 1 - s u c c i n i m i d o t e t r a h y d r o c a r b a z o l e (165) was a l s o formed i n the r e a c t i o n presumably v i a a s i m i l a r -93-d i s p l a c e m e n t o f t h e p y r i d i n i u m s a l t (163) w i t h a s m a l l amount o f s u c c i n i m i d e . 165 I n t h i s c a s e , the IR spectrum showed NH and C=0 a b s o r p t i o n s a t 3470 and 1705 cm - x. The UV a g a i n showed the p r e s e n c e o f an i n d o l e chromophore ( A 288, 278, 223 nm) and t h e mass spectrum gave v ^max I I I r ? m/e 268 (M ). The NMR spectrum showed a one p r o t o n s i n g l e t a t £7.96 (NH), a one p r o t o n t r i p l e t a t £5.54,(J=7.0 Hz) ( C - l H ) , and a f o u r p r o t o n s i n g l e t a t 52. 72 (N- ( C O C H 2 ) 2 ) • P r i o r t o c o n v e r s i o n o f n i t r i l e (164) t o e s t e r (156) i t was d e c i d e d t o d e t e r m i n e c o n d i t i o n s f o r the C - l a c y l a t i o n o f (164). I n t h i s r e s p e c t , i t was r e a l i s e d t h a t a s u i t a b l e p r o t e c t i n g group f o r the i n d o l e hydrogen o f b o t h (164) and (156) would be r e q u i r e d s i n c e o t h e r w i s e a t t a c k w o u l d be a n t i c i p a t e d t o g i v e p r e f e r e n t i a l l y t h e N - a c y l / a l k y l p r o d u c t . A number o f p r o t e c t i n g groups were c o n s i d e r e d e.g. b e n z y l , m e t h y l , b e n z e n e s u l p h o n y l , carbomethoxy e t c . a n d , o b v i o u s l y , p r e -f e r e n c e was g i v e n t o a group w h i c h would h o p e f u l l y be s t a b l e t o the c o n d i t i o n s used and t h a t c o u l d a l s o r e a d i l y be added o r removed. I n t h i s r e s p e c t t h e carbomethoxy group seemed i d e a l s i n c e i t a p p a r e n t l y f u l f i l l e d a l l o f t h e s e r e q u i r e m e n t s . -94-F i g u r e 37 - F o r m a t i o n o f 1 - p y r i d i n i u m - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e bromide (153). C o n s e q u e n t l y N - c a r b o r n e t h o x y - l - c y a n o t e t r a h y d r o c a r b a z o l e (166) was formed by t r e a t m e n t o f (164) w i t h 1) p o t a s s i u m h y d r i d e and 2) m e t h y l c h l o r o f o r m a t e . ( F i g u r e 38) The UV spectrum was now t h a t o f an N - a c y l i n d o l e ( A m a x 292, 282 256 and 225 nm) and the IR showed CN and C=0 a b s o r p t i o n a t 2260 and 1745 cm 1 . The mass spectrum gave the e x p e c t e d m/e 254 (M +) and t h e NMR showed a one p r o t o n t r i p l e t a t & 4 . 5 , ( J = 4 H z ) , C - l H and a t h r e e p r o t o n s i n g l e t a t £4.09 (CO C H J 1 - c a r b o m e t h o x y - l - c y a n o - N - c a r b o m e t h o x y t e t r a h y d r o c a r b a z o l e (167) was then formed by t r e a t m e n t o f (166) w i t h p o t a s s i u m h y d r i d e and m e t h y l c h l o r o f o r m a t e ( f i g u r e 17). The UV and IR were s i m i l a r t o t h a t d e s c r i b e d above and the mass spectrum gave an m/e 312 ( M + ) . The NMR showed two t h r e e p r o t o n s i n g l e t s a t £3.98 (N-C0 2CH ) and £3.86 ( C - l C02CK_3_) r e s p e c t i v e l y . M i l d a l k a l i n e h y d r o l y s i s w i t h p o t a s s i u m h y d r o x i d e i n methanol a f f o r d e d 1 - c a r b o m e t h o x y - l - c y a n o - t e t r a h y d r o c a r b a z o l e (168). Thus, w i t h c o n d i t i o n s d e t e r m i n e d f o r a c y l a t i o n o f the a n i o n a t C - l of the n i t r i l e ( 1 6 6 ), i t was a n t i c i p a t e d t h a t a l k y l a t i o n o f the c o r r e s p o n d i n g e s t e r (156) s e r i e s i n a s i m i l a r manner would g i v e t h e r e q u i r e d p r o d u c t (157). C o n s e q u e n t l y t r e a t m e n t of (164) w i t h hydrogen c h l o r i d e i n methanol a t room t e m p e r a t u r e f o r 1.5 hours gave th e i n s o l u b l e p r o -d u c t (169) w h i c h on t r e a t m e n t w i t h aqueous m e t h a n o l i c h y d r o c h l o r i c a c i d gave th e r e q u i r e d e s t e r (156) ( f i g u r e 3 9 ) . 1) KB 2) C I C O , C H -96-The IR spectrum showed NH and CO a b s o r p t i o n a t 3470 and 17 30 cm and the UV gave the e x p e c t e d i n d o l e chromophore. The mass spectrum gave e x p e c t e d m/e 229 (M +) and the NMR showed a one p r o t o n s i n g l e t a t £8.34 (NH), a one p r o t o n m u l t i p l e t a t £3.94-3.88 ( C - l H) and a t h r e e p r o t o n s i n g l e t at«S3.80 (C0 2CH 3) . C o n v e r s i o n t o N - c a r b o m e t h o x y - l - c a r b o m e t h o x y t e t r a h y d r o c a r -b a z o l e (170) was e f f e c t e d i n good y i e l d by t r e a t m e n t o f (156) w i t h p o t a s s i u m h y d r i d e and m e t h y l c h l o r o f o r m a t e . ( F i g u r e 3 9 ) . The IR spectrum showed CO a b s o r p t i o n a t 1730 cm and the UV was c h a r a c t e r i s t i c o f an N - a c y 1 - i n d o l e ( A 292 , 278, 261, 227 nm) . J ^' max t i t The mass spectrum gave an m/e .2 87 (M +) and the NMR showed a one p r o t o n t r i p l e t a t £4.26 (J=5 Hz) f o r C - l H, and two t h r e e p r o t o n s i n g l e t s a t £3.98 (N-C02CH_3) and £3.74 (C0 2CH 3) r e s p e c t i v e l y . F u r t h e r t r e a t m e n t o f (170) w i t h p o t a s s i u m h y d r i d e i n t e t r a -h y d r o f u r a n f o r twenty minutes a t 0° . f o l l o w e d by q u e n c h i n g w i t h formaldehyde gave, f o l l o w i n g work-up, the r e q u i r e d 1 - c a r b o m e t h o x y - l -h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157) ( f i g u r e 39) as e v i d e n c e d by t h e IR spectrum w h i c h had OH, NH and CO a b s o r p t i o n a t 3620, 3460 and 1710 cm r e s p e c t i v e l y . The UV had an i n d o l e chromophore (/J 290, 282, 277, 224) and t h e mass spectrum gave an m/e 259 (M +) . The NMR showed a one p r o t o n s i n g l e t a t £8.64 (NH), a two p r o t o n AB q u a r t e t a t £3.99 (CH_20H) and a t h r e e p r o t o n s i n g l e t a t £ 3 . 74 (C0 2CH_ 3) . S i n c e l o s s o f the 'N-carbomethoxy group had n o t been n o t e d w i t h (170) under i d e n t i c a l work-up c o n d i t i o n s presumably t h i s l o s s can be r a t i o n a l i s e d as b e i n g due t o t h e f o l l o w i n g mechan-ism ( f i g u r e 4 0 ) . A l k y l a t i o n o f (170) gave i n t e r m e d i a t e ( i ) w h i c h -97-c y c l i s e s as shown t o g i v e ( i i ) . Base h y d r o l y s i s on work-up would be e x p e c t e d t o h y d r o l y s e ( i i i ) as shown t o g i v e (157). Thus w i t h c o n d i t i o n s f o r i n t r o d u c t i o n o f the h y d r o x y m e t h y l group a t C - l b e i n g e s t a b l i s h e d i t was now c o n s i d e r e d t h a t t h e s e , o r s i m i l a r c o n d i t i o n s , c o u l d be a p p l i e d t o e s t e r (122), t o g i v e e i t h e r stemmadenine (63) o r i t s epimer (138). P r i o r t o d i s c u s s i o n o f t h e s e i n v e s t i g a t i o n s i t had been mentioned t h a t o x i d a t i v e a t t e m p t s a t c o n v e r s i o n o f (128) t o t h e aldeh y d e (124) had not met w i t h s u c c e s s . I t was thus o f some i n t -e r e s t t o r e - i n v e s t i g a t e a s p e c t s o f t h i s o x i d a t i o n . C o n s e q u e n t l y e s t e r (15 6) was r e d u c e d w i t h l i t h i u m aluminium h y d r i d e t o g i v e 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (171) i n good y i e l d . 171 172 A t t e m p t e d o x i d a t i o n o f 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (171) t o t h e r e q u i r e d aldehyde (172) a g a i n f a i l e d under a few o x i d a t i v e / ^ 158a _ 158b , , a t t e m p t s (e.g. Oppenauer, C o l l i n s o r Corey p r o c e d u r e s ) as none of the p r o d u c t s o b t a i n e d p o s e s s e d s p e c t r a l c h a r a c t e r i s t i c s o f (172) and the approach was d i s c o n t i n u e d . I t was thus d e c i d e d t o i n v e s t i g a t e the a l k y l a t i o n o f deshydroxymethylstemmadenine (122). -98-1 5 7 , 7 0 F i g u r e 39 - S y n t h e s i s " o f 1-carbomethoxy -1 - h y d r o x y r a e t h y l t e t r a h y d r o c a r b a z o l e (157). F i g u r e 40 - P r o p o s e d mechanism f o r f o r m a t i o n o f 1 - c a r b o m e t h o x y - 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e ( 1 5 7) -99-The c o n v e r s i o n s o f s t r y c h n i n e (5) t o epi-stemmadenine (138) and deshydroxymethylstemmadenine (122) d i s c u s s e d p r e v i o u s l y o c c u r r e d , o v e r a l l , i n low y i e l d . C o n s e q u e n t l y i n a d d i t i o n t o t h e a l k y l a t i o n s t u d y t h a t was t o be i n v e s t i g a t e d i t was a l s o hoped t h a t s u i t a b l e m o d i f i c a t i o n s c o u l d be e f f e c t e d t o improve the s y n t h e -t i c u t i l i t y o f the approach. C o n v e r s i o n t o W i e l a n d - G u m l i c h aldoxime (130) was r e a d i l y 1 5 9 o b t a i n e d a c c o r d i n g t o t h e l i t e r a t u r e p r o c e d u r e and gave the e x p e c t e d l i t e r a t u r e v a l u e s f o r IR, UV, m.pt. The mass spectrum gave an m/e 325(M )and the NMR was i n agreement w i t h t h e s t r u c t u r e . I t was hoped t h a t oxime (130) c o u l d be d i r e c t l y c o n v e r t e d t o the e s t e r (132) v i a a c o n c o m i t a n t d e h y d r a t i o n and h y d r o l y s i s p r o c e s s t h u s e l i m i n a t i n g a number o f s t e p s . T h i s , u n f o r t u n a t e l y , was n o t a c h i e v e d b u t the p r o c e d u r e was m o d i f i e d by d e h y d r a t i n g t h e oxime w i t h sodium f o r m a t e / f o r m i c acid^° T h i s p r o c e d u r e d i d n o t g i v e the e x p e c t e d d i h y d r o i n d o l e n i t r i l e (174) b u t i n s t e a d t h e 1 - f o r m y l n i t r i l e (173) ( f i g u r e 41) as shown by t h e UV (A^ v 287,283,247 nm.), and the mass spectrum m/e 335 ( M + ) . The in 3.x IR showed CN and CO bonds a t 2260 and 1675 cm x r e s p e c t i v e l y and t h e NMR spectrum e x h i b i t e d two s i n g l e t s a t £ 8.9 and 8.76 (N-CHO) , a t r i p l e t a t S 5 . 590, (J=6 Hz) , C-19 H and a d o u b l e t a t £4.85, (J=10 Hz), C-2 H. Treatment o f 1 - f o r m y l n i t r i l e (173) w i t h b a r i u m h y d r o x i d e i n aqueous e t h a n o l gave the c o r r e s p o n d i n g n i t r i l e (174) i n good y i e l d ( f i g u r e 41) as shown by the e x p e c t e d UV s h i f t t o a d i h y d r o i n d o l e s p e c i e s ( A 298, 243 nm.) and mass spectrum m/e 307 (M +) . The IR -100-6 3 F i g u r e 41 - Further investigations towards the synthesis of stemmadenine (63) - approach 2. - 1 0 1 -showed NH and CN a b s o r p t i o n a t 3430 and 2230 cm "*" r e s p e c t i v e l y . The NMR was a l s o i n agreement w i t h the s t r u c t u r e . Treatment o f the n i t r i l e (174) w i t h a s o l u t i o n o f hydrogen c h l o r i d e i n r e f l u x i n g methanol a f f o r d e d e s t e r (132) h a v i n g the 142 same m.pt. and s i m i l a r UV and IR t o t h a t r e p o r t e d . However 137 the NMR r e p o r t e d was n o t o f s u f f i c i e n t q u a l i t y t o a l l o w p o s i -t i v e i d e n t i f i c a t i o n and was e x t r e m e l y d i f f e r e n t from t h a t o f the pure p r o d u c t o b t a i n e d i n t h e s e s t u d i e s . The c o r r e c t e d d a t a showed a t r i p l e t a t £5.7, (J=7 Hz),C-19 H, a s i n g l e t a t £4.74 (OH), a s i n g l e t a t £4.2 (NH) and a s i n g l e t a t £ 3.74 (C0 2CH 3) . Subsequent h y d r o g e n o l y s i s o f (132) t o (133) w i t h the W i e l a n d c a t a l y s t " * " 3 8 * 3 had p r e v i o u s l y been r e p o r t e d t o r e s u l t i n o v e r -r e d u c t i o n g i v i n g (134) as an i m p u r i t y w h i c h c o u l d n o t be removed. I t was a l s o n o t e d from the NMR o f (133) o b t a i n e d v i a the a l t e r n a t i v e h y d r o g e n o l y s i s o f the a l l y l i c bromide t h a t s a t i s f a c t o r y p u r i f i -c a t i o n had not been a c h i e v e d . However i n t h e s e r e c e n t s t u d i e s o v e r - r e d u c t i o n d i d not o c c u r and t h e r e q u i r e d e s t e r (133) was o b t a i n e d as pure c r y s t a l l i n e m a t e r i a l i n 88% y i e l d . The NMR o f (133) showed a q u a r t e t a t £5.48, (J=7 Hz)j C-19 H, a s i n g l e t a t £4.1 (NH) , a d o u b l e t a t ^4.05 (J=10 Hz), C-16H, a s i n g l e t a t £3.70 (C02CH_ 3) and a d o u b l e t o f d o u b l e t s a t Si.56,(J=2 and 7 Hz),C-18 CH . W i t h e s t e r (133) now i n hand, t h e n e x t s t e p i n v o l v e d o p t i m i s a t i o n o f the d e h y d r o g e n a t i o n r e a c t i o n w i t h l e a d t e t r a a c e -t a t e t o g i v e akuammicine (64). T h i s r e a c t i o n had been shown t o o c c u r i n 35% y i e l d by t r e a t i n g e s t e r (133) w i t h two e q u i v a l e n t s of l e a d -102-t e t r a a c e t a t e i n a s o l u t i o n o f benzene and a c e t i c a c i d f o r 1.75 nr. I n my s t u d i e s , t h i s r e a c t i o n f a r e d l e s s f a v o u r a b l y than r e p o r t e d b u t , i t was found t h a t t r e a t m e n t of (133) w i t h one e q u i v a l e n t of l e a d t e t r a a c e t a t e i n d r y benzene f o r 10 minutes gave akuammi-c i n e (64) i n 60% y i e l d . T h i s r e a c t i o n was found t o be e x t r e m e l y s e n s i t i v e t o minute v a r i a t i o n s i n the e x p e r i m e n t a l p r o c e d u r e . 21 Akuammicine (64) e x i s t s i n n a t u r e i n two forms (±) and 21 1 3 2 1 3 7 (-) b u t no C°ClD w a s r e p o r t e d i n the p r e v i o u s s t u d i e s . ' The p r o d u c t i n my s t u d i e s had a n e g a t i v e r o t a t i o n o f £af] = -672° and thus th e s y n t h e s i s o f (-) akuammicine (64) i s now complete. Treatment of (64) w i t h (1) a c e t i c a c i d and (2) sodium b o r o h y d r i d e gave e s t e r (122) i n 50% y i e l d . I t s h o u l d be noted t h a t t h e i n t e r m e d i a t e formed by t h e a c i d c a t a l y s e d r i n g o p e n i n g c o u l d be reduced w i t h sodium b o r o t r i t i i d e t o a l l o w i n t r o d u c t i o n of a r a d i o - l a b e l a t C-3. C 0 2 C H 3 C 0 2 C H 3 C 0 2 C H 3 6 4 122 The NMR o b t a i n e d f o r e s t e r (122) i n my s t u d i e s gave more 132 d e f i n i t i v e d a t a t o t h a t r e p o r t e d p r e v i o u s l y (see e x p e r i m e n t a l ) . Thus the c o n v e r s i o n o f s t r y c h n i n e (5) t o e s t e r (122) w i t h t h e s e m o d i f i c a t i o n s gave h i g h e r y i e l d s t h e r e b y i n c r e a s i n g somewhat the s y n t h e t i c u t i l i t y o f t h i s approach. Treatment of e s t e r (122) w i t h p o t a s s i u m h y d r i d e and m e t h y l -- 1 0 3 -c h l o r o f o r n a t e gave N -carbomethoxy-16-carboraethoxy-deshydroxy-cl methy1-stemmadenine (175) i n 59% y i e l d . E v i d e n c e f o r t h i s s t r u c t u r e came from the UV g i v i n g the e x p e c t e d N - a c y l i n d o l e chromophore {A 296, 285, 263, 228 nm). The mass spectrum gave an m/e max t i t r 382 ( M + ) , and t h e IR spectrum showed CO a b s o r p t i o n a t 1730 cm - 1. The NMR spectrum showed a q u a r t e t a t £5.43, (J=7 H z ) ; f o r C-19 H a s i n g l e t a t £ 4.0 f o r (N-C02CH_3) , a d o u b l e t a t £3.95, (J=3 Hz) f o r C-16 H, a s i n g l e t a t £3.65 (C0 2CH 3) and a d o u b l e t o f d o u b l e t s a t 3 1 . 8 3 , (J=2 and 7 H z ) , f o r C-18 CH . The d o u b l e t f o r t h e C-16 H was o f some s i g n i f i c a n c e s i n c e e s t e r (122) had a s i n g l e t f o r the C-16 H thus showing t h a t e p i m e r i z a t i o n had t a k e n p l a c e . T h i s had p r e -v i o u s l y been n o t e d on t r e a t i n g e s t e r (122) w i t h sodium h y d r i d e i n 137 d i m e t h y l s u l f o x i d e t o g i v e e p i m e r i c e s t e r (176). D r e i d i n g models and NMR d a t a s u g g e s t s t e r e o c h e m i s t r y a t C-16 i n (122), (175) and (176) t o be as shown. -104-D r e i d i n g models a l s o s uggest t h a t the N -carbomethoxy de-r i v a t i v e (175) was s t e r i c a l l y l e s s h i n d e r e d f o r a l k y l a t i o n a ttempts than the 1 -formyl e s t e r (135) p r e v i o u s l y s t u d i e d . C onsequently w i t h (175) on hand i t was now a p p r o p r i a t e t o ensure t h a t c o n d i -t i o n s were c o r r e c t f o r a n i o n f o r m a t i o n . I n t h i s r e s p e c t t r e a t m e n t o f (175) w i t h p o t a s s i u m h y d r i d e , i n an a n a l o g o u s manner t o t h a t used f o r (170), f o l l o w e d by quenching w i t h d e u t e r i u m o x i d e gave a p r o d u c t which was s u b j e c t e d t o a mass s p e c t r o m e t r i c i n v e s t i g a t i o n . + + E v a l u a t i o n of the r a t i o M /M +1 i n d i c a t e d about 40% d e u t e r a t i o n a t C-16. The mass s p e c t r a l f r a g m e n t a t i o n f o r (175) e x h i b i t e d m/e a t 382 (M +),323,194 and 123. I n the r e s u l t i n g d e u t e r a t e d p r o d u c t M + + l peaks 383,324 and 195 were e n r i c h e d s u g g e s t i n g the f r a g m e n t a t i o n t o be as shown ( f i g u r e 4 2 ) . m/<? 19 4 m / e 123 F i g u r e 42 - Proposed mass s p e c t r a l f r a g m e n t a t i o n o f N a-carbomethoxydeshydroxymethyl-stemmadenine (175) . S e v e r a l a t t e m p t s t o i n t r o d u c e a hydroxymethy1 group a t C-16 met w i t h f a i l u r e . M o d i f i c a t i o n o f the p r o c e d u r e by change o f -105-base (e.g. t - B u L i , NaH) , s o l v e n t (HMPA, DMSO), t e m p e r a t u r e , and time o f r e a c t i o n g e n e r a l l y l e d t o s e v e r e d e c o m p o s i t i o n . These r e s u l t s were most d i s a p p o i n t i n g and an a l t e r n a t i v e a l k y l a t i o n p r o c e d u r e was a t t e m p t e d . T h i s was based on t h e f a c t t h a t i n the s y n t h e s i s o f 16,17-dihydrosecodin-17-ol (88) the 87 f o l l o w i n g t r a n s f o r m a t i o n had been e f f e c t e d . '' 177 178 3 o Presumably formation of (178) occurs v i a i n i t i a l formylation of the indole nitrogen, to give, following further a l k y l a t i o n , (179) which on work-up loses the l a b i l e N-formyl group to give (178). 179 This reaction was attempted with (122) under a variety of conditions and led only to severe decomposition under the conditions used. Consequently another model system, derived from 18/3-carbo-16 5 methoxycleavamine (72), was considered. Treatment of (72) with potassium hydride and methylchloroform-161 ate gave Na-carbomethoxy-18/?-carbomethoxycleavamine (180), A l l attempts to alkylate this molecule with formalde-hyde at C-18 to give (182) met with f a i l u r e , even although 55% -106-d e u t e r i u m i n c o r p o r a t i o n a t C-18 was e f f e c t e d . S i m i l a r a t t e m p t s t o f o r m y l a t e (72) under a v a r i e t y o f c o n d i t i o n s t o g i v e (181) met w i t h f a i l u r e . C o n s e q u e n t l y i t was d e c i d e d t o change the i n d o l e p r o t e c t i n g group from the carbomethoxy group t o a m e t h y l group thus f u r t h e r d i m i n i s h i n g any s t e r i c f a c t o r s . T reatment o f (72) w i t h p o t a s s i u m h y d r i d e and m e t h y l i o d i d e i n t e t r a h y d r o f u r a n gave a m i x t u r e o f the r e q u i r e d N-methyl (183) and C-methyl (184) d e r i v a t i v e s as shown. T h i s r e a c t i o n was o f some i n t e r e s t s i n c e i t d e monstrated s t a b i l i s a t i o n o f the a n i o n a t C-9. I t had p r e v i o u s l y been n o t e d t h a t no a c y l a t i o n a t C-9 o c c u r r e d under the c o n d i t i o n s employed i n the f o r m a t i o n o f (180) whereas a l k y l a t i o n gave b o t h C- and N-a l k y l a t i o n . F u r t h e r , t r e a t m e n t o f t h e a n i o n from (72) w i t h b e n z y l bromide gave o n l y C-9 a l k y l a t e d p r o d u c t (185) i n h i g h y i e l d . -107-These r e s u l t s were most f r u s t r a t i n g s i n c e the r e a s o n s f o r f a i l u r e s were n o t o b v i o u s . I t i s now a p p r o p r i a t e t o d i s c u s s the p r o p o s e d s t e r e o c h e m i s t r y a t C-16 of epi-stemmadenine (138). I t had been proposed t h a t t r e a t m e n t o f 1 - f o r m y l e s t e r (135) ( f i g u r e 27) w i t h sodium h y d r i d e 132 137 and formaldehyde gave th e o x a z i n e (136) i n 38% y i e l d , ' and as t h e s t e r e o c h e m i s t r y was now f i x e d subsequent t r a n s f o r m a t i o n s w o u l d g i v e epi-stemmadenine (138) v / i t h the a b s o l u t e c o n f i g u r a t i o n shown (p. 77). T h i s was based on a s t e r e o c h e m i c a l argument i n w h i c h , under the e q u i l i b r i u m c o n d i t i o n s employed, a l k y l a t i o n w o uld be e x p e c t e d t o o c c u r from the"o(-face" o f (135) . i n f a c t a l --108-k y l a t i o n from the'fl -face',' w h i l s t l e s s f a v o u r a b l e , was a l s o f e a s i b l e . However the s u g g e s t e d r i n g c l o s u r e t o g i v e t h e o x a z i n e (136) p l a c e d b o t h r i n g s C and F ( f i g u r e 43) i n l e s s f a v o u r a b l e b o a t c o n f o r m a t i o n s . I t s h o u l d be n o t e d t h a t a l t e r n a t i v e s - f a c e " approach under t h e e q u i l i b r i u m c o n d i t i o n s t f o l l o w e d by v e r y f a c i l e r i n g c l o s u r e would y i e l d the o x a z i n e (187) w i t h r i n g s A-F i n more f a v o u r a b l e c h a i r c o n f o r m a t i o n s . 1 3 6 , 8 7 F i g u r e 43 - S t e r e o c h e m i c a l f e a t u r e s o f o x a z i n e s (138) : and (187). I t s h o u l d a l s o be noted t h a t f a c i l e f o r m a t i o n o f the o x a z i n e (188) from the a l c o h o l (143) had a l s o been o b s e r v e d on t r e a t m e n t w i t h 132 137 formaldehyde ' t h e r e b y g i v i n g f u r t h e r s u p p o r t f o r t h e s e comments. C o n s e q u e n t l y i t i s n o t p o s s i b l e t o draw any c o n c l u s i o n s w i t h r e s p e c t t o the a b s o l u t e c o n f i g u r a t i o n a t C-16. The s y n t h e s i s o f stemmadenine (63) however may be e f f e c t e d -109-by t r e a t i n g e s t e r (122) t o g i v e c h l o r o i n d o l e n i n e (189) w h i c h on f u r t h e r t r e a t m e n t w i t h c y a n i d e would be e x p e c t e d t o y i e l d t he c o r r e s p o n d i n g epimers (190 a,b). 1 9 0 a ; R = C N . R = C O p C b , R = C 0 2 C H . ^ ' = C A p p r o p r i a t e m a n i p u l a t i o n would y i e l d stemmadenine (63) o r i t s epimer (138). However i t s h o u l d be no t e d t h a t many o f thes e subsequent s t e p s would be a n t i c i p a t e d t o o c c u r i n low y i e l d s and c o n s e q u e n t l y t h i s approach was no t a t t e m p t e d . A n o t h e r approach would be t o t r e a t t h e n i t r i l e (191) as shown ( F i g u r e 44). I t i s t h e f e e l i n g o f t h i s a u t h o r t h a t t h i s a p proach, u s i n g t h i s s t r a t e g y , would be the most p r o m i s i n g . 162 A n o t h e r approach r e c e n t l y gave p r e l i m i n a r y i n f o r m a t i o n w h i c h may be adapted towards the s y n t h e s i s o f stemmadenine (63) ( f i g u r e 45)-By a p p r o p r i a t e m o d i f i c a t i o n o f s t e p s i n the s y n t h e s i s o f 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l (88 ) , i n t e r m e d i a t e (195) was ob-t a i n e d w h i c h on subsequent t r e a t m e n t as shown g i v e a compound whose s t r u c t u r e c o u l d not d e f i n i t i v e l y be a s s i g n e d . Three p o s s i b i l i t i e s were c o n s i d e r e d (196-198). I f the s t r u c t u r e was (19 6 ) , as s u g g e s t e d , t h e n i t was hoped by the author"*" t i i a t stemmadenine (63) c o u l d be formed by a p p r o p r i -a t e m o d i f i c a t i o n o f t h i s approach as shown ( f i g u r e 4 6 ) . (199 -200) -110-F i g u r e 44 - P r o p o s e d r o u t e f o r the s y n t h e s i s o f stemma-denine (63) . F i g u r e 45 - S t r u c t u r a l p o s s i b i l i t i e s o b t a i n e d from c y c l i z a t i o n o f i n t e r m e d i a t e (195). F i g u r e 46 - K u t n e y 1 s proposed s y n t h e s i s o f stemmadenine (63). I n t h i s r e s p e c t t r e a t m e n t o f d e r i v a t i v e (199) w i t h sodium b o r o h y d r i d e under c o n t r o l l e d c o n d i t i o n s s h o u l d g i v e the d i h y -d r o p y r i d i n e ( i ) . I f s u i t a b l e c o n d i t i o n s can be o b t a i n e d f o r the f o r m a t i o n of ( i i ) then r e a c t i o n as shown t o g i v e ( i i i ) would be e x p e c t e d . R e d u c t i o n t o (200) f o l l o w e d by d e h y d r a t i o n w ould h o p e f u l l y g i v e stemmadenine (63) o r i t s epiraer (138). However t h e s e m a n i p u l a t i o n s , w h i l s t under a t t e n t i o n , have not y e t been r e a l i s e d . Indeed u n t i l the s t r u c t u r e o f the p r o d u c t o b t a i n e d by r e a c t i o n o f (195) i s unambigously v e r i f i e d , ( f i g 45), no f u r t h e r i n v e s t i g a t i o n s u s i n g t h i s approach w i l l be c o n s i d e r e d . -112-I n c o n c l u s i o n , the att e m p t e d s y n t h e s e s o f stemmadenine (63) have, so f a r , been u n s u c c e s s f u l . I t i s t o be hoped t h a t one o f the proposed schemes w i l l a f f o r d ( 6 3 ) . F o l l o w i n g the e x p e r i m e n t a l s e c t i o n , p a r t I I , i n v e s t i g a -t i o n s towards the u n d e r s t a n d i n g o f the i n v i v o c o u p l i n g o f monomeric a l k a l o i d s c a t h a r a n t h i n e (12) and v i n d o l i n e (11) i n C. r o s e u s a r e p r e s e n t e d . -113-EXPERTMENTAL - PART I I G e n e r a l e x p e r i m e n t a l d e t a i l s were g i v e n i n E x p e r i m e n t a l , p a r t 1. P r o c e d u r e s , p u b l i s h e d or u n p u b l i s h e d by o t h e r s , a r e d e s c r i b e d when (1) d e t a i l e d e x p e r i m e n t a l v/as n o t r e l e a s e d (2) u n s a t i s f a c t o r y d a t a was p r e s e n t e d o r (3) p r o c e d u r e was m o d i f i e d . 14 8 I s o n i t r o s t r y c h n i n e h y d r o c h l o r i d e (141) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 218-220° (lit.m.pt.=220° w i t h d e c o m p o s i t i o n ) . Y i e l d = 7 9 % . NMR:£d-DMSO 100 MHz (FT) 8.12-7.96,7.46-7.02 (m,4H,arom.), 5.92 ( t , IH, J=6.4 Hz, C-19 H), 5.00 (d, IH, J=3Kz, C-17 H) M.S.- m/e: 36 3 (M +) , 346 ,318,144,130. High r e s o l u t i o n mass spectrum: C a l c - f o r (-21 H21^3 <^3~ 3^ 3 * 8 3 Found=363.1576. W i e l a n d - G u m l i c h aldehyde (129) ~L'*0 To i s o n i t r o s o s t r y c h n i n e h y d r o c h l o r i d e (141) (10 g , 25.13 mmol) was added t h i o n y l c h l o r i d e (20 m l , 0.278 mol) o v e r 5 min. F o l l o w i n g c o n t i n u o u s s t i r r i n g f o r 1 h r the c o n t e n t s were poured onto i c e (100 g) and s t i r r i n g was m a i n t a i n e d f o r an a d d i t i o n a l 4 h r . The r e s u l t i n g p r e c i p i t a t e o f 1-c y a n o f o r m y l WGA (144) was removed by s u c t i o n f i l t r a t i o n . To 1-cyanoformyl WGA (144) was added water (100 ml) and t h i s s u s p e n s i o n was then h e a t e d t o 109° (by a steam g e n e r a t o r and o i l bath) u n t i l a l l hydrogen c y a n i d e formed was removed (steam -114-d i s t i l l a t i o n ) . The r e s u l t i n g s u s p e n s i o n was c o o l e d (5 ) and b a s i f i e d w i t h M N a 2 C 0 3 t o pH 10. The r e s u l t i n g p r e c i p i t a t e was removed ( s u c t i o n f i l t r a t i o n ) , t a k e n up i n c h l o r o f o r m , and c r y s t a l l i n e WGA (129) was o b t a i n e d i n t h i s manner. The f i l t r a t e was e x h a u s t i v e l y e x t r a c t e d w i t h c h l o r o f o r m (10 x 150 ml) and the c h l o r o f o r m e x t r a c t s combined, d r i e d , and s t r i p p e d o f s o l v e n t . The c h l o r o f o r m s o l u b l e s were chromato-graphed on A l u m i n a (Act I I I ) , u s i n g c h l o r o f o r m as e l u a n t t o g i v e WGA (129) (4.1 g, 4 8 % ) . M.Pt.: 209-211° ( l i t . m.pt. 214-216°). NMR: £ (CDC1 3) 100 MHz (FT) 7.36-6.6 (m, 4H, arom ), 5.84 (m, IH, C-19 H),5.10 (d, IH, J=1.6 Hz, OCHO), 4.30, 4.17 (dd, IH, J=7 and 9 Hz, C-16 H). M.S. m/e: 310 ( M + ) , 180, 144, 130, 120, 118 H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2 2N 2O 2=310.1681 Found=310.1673 147 2 j 3 ,16o(-cur-19-en-17,18-diol (142) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 251° ( l i t . m.pt. 251°). Y i e l d = 81%. 2 y 3 ,16o(-cur-19-en-17-ol ( 1 4 3 ) 1 3 8 b P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 174-176° ( l i t . m.pt. 177-178°). Y i e l d = 90%. 2 y g-cur-19-en-17-al (145) Sodium h y d r i d e (0.465 g, 19.38 mmol) was o b t a i n e d by w a s h i n g a 50% o i l d i s p e r s i o n (0.93 g) w i t h dry benzene ( 3 x 2 m l ) . To t h i s was added a s o l u t i o n o f a l c o h o l (143) -115-(0.62 g, 2.1 mmol) and benzophenone (1.86 g, 10.22 mmol) i n dry benzene (40 m l ) . The s o l u t i o n was made up t o 60 ml w i t h d r y benzene and heated t o r e f l u x . A f t e r 6 hr the temperature was reduced t o 0° and h y d r o c h l o r i c a c i d (10%) added u n t i l pH 1. The aqueous l a y e r was s e p a r a t e d and b a s i f i e d a t 0° w i t h ammonium h y d r o x i d e u n t i l pH 9. 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 e t h y l a c e t a t e (4 x 60 ml) and the e t h y l a c e t a t e e x t r a c t s combined, d r i e d and e v a p o r a t e d t o y i e l d a w h i t e foam. Chromatography on alumina (Act I I I ) u s i n g a b e n z e n e / e t h y l a c e t a t e g r a d i e n t a f f o r d e d the r e q u i r e d aldehyde (145) (0.45 g, 137 73%) p o s s e s s i n g s p e c t r o s c o p i c f e a t u r e s a l r e a d y r e p o r t e d . l - f o r m y l - 2 y f f - c u r - 1 9 - e n - 1 7 - a l (146) The r e a g e n t ( a c e t i c f o r m i c anhydride) was p r e -p a r e d by c o o l i n g r e d i s t i l l e d a c e t i c a n h y d r i d e (4 ml) t o 0°, and then s l o w l y adding r e d i s t i l l e d f o r m i c a c i d (2 m l ) . The temper-a t u r e was r a i s e d t o 50° f o r 15 min and then c o o l e d i m m e d i a t e l y o 151 to 07 The r e a g e n t (3.7 ml) was added t o a s o l u t i o n o f the a l d e -hyde (14 5) (443 mg, 1.51 mmol) a t 0°. The temp e r a t u r e was then r a i s e d t o 6° and s t i r r i n g m a i n t a i n e d f o r 10 min. The r e a c t i o n m i x t u r e was s t r i p p e d o f s o l v e n t and the r e s i d u e chromatographed on alumina (Act I I I ) u s i n g a b e n z e n e / e t h y l a c e t a t e g r a d i e n t t o g i v e t h e 1 - f o r m y l aldehyde (146) (280 mg, 58%) M.Pt.: 10 3° (MeOH) I.R.: [) C H C l 3 2940, 2890, 1730, 1670 cm" 1 U . V . : / i M e ° H 287 (3.39), 280 (3.46), 250 (3.88) nm. IHdX NMR: $ (CDC1,) 100 MHz (FT) 4.76, 9.72, 9.58 (3s, IH, C-17 H), -116-8.83, 8.58 (2s, IH, N-CHO), 8.04-7.9, 7.46-7.00 (m, 4H, arom.), 5,47 (q, IH, J=6 Hz, C-19 H), 4.81 (dd, IH, J=9 and 15 Hz, C-16 H) , 1.52 (dd, 3H, J=2 and 6 Hz, C-18 H). M.S. m/e: 322 (M+), 279, 172, 164, 144, 136, 109, 105. Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 Q H 2 2 N 2 0 2 = 3 2 2 . 1 6 8 0 . Found=322.1665. E l e m e n t a l a n a l y s i s : Found: C, 74.35, H, 6.95, N, 8.50 C a l c - C, 74.53, H, 6.83, N, 8.69. 1-formyl-2/3-cur-19 e n - 1 7 - c y c l o h e x y l i m i n e (14 7) To a s o l u t i o n o f (146) (100 mg, 0.31 mmol) i n an-hydrous benzene (40 ml) was added c y c l o h e x y l a m i n e (4 m l , 33.13 mmol) and the s o l u t i o n was r e f l u x e d o v e r 4 A° m o l e c u l a r s i e v e s f o r 2.5 - 3 h r i n a s o x h l e t condenser. The s o l v e n t was t h e n removed and t h e r e s u l t i n g r e s i d u e chromatographed on s i l i c a g e l t o g i v e as an o i l , the i m i n e (147) (82 mg, 6 5 % ) . I.R.: 0 C H C l 3 2920, 2850, 1665, cm - 1. U.V.: A E t ° H 288, 282, 252 nm. 1 max ' ' NMR: (CDC1 3) 100 MHz (FT) 9.02 ( s , l H , N CHO), 8.68 (q, IH, J=8 and 15 Hz, C-17 H), 7.97-7.83, 7.26-6.71 (m, 4H, arom.), 5.26 (q* IH, J=6 Hz, C-19 H) , 4.72 (dd, IH, . 7 = 3 and 8 Hz, C-16 H) , 1.91-1.37 (CH 2 e n v e l o p e , 11H, (CH_2) 5 CH) , 1.29 (dd, 3H, J=2 and 6 Hz, C-18 H) . M.S. m/e: 403 (M+), 320, 279, 172, 144. Hig h r e s o l u t i o n mass spectrum: C a l c - f o r C„,H 0_N_O=403.2623. 2 6 3 3 3 Found=403.2620. -117-A c y l a t e d d e r i v a t i v e (153)  Sodium h y d r i d e (25mg, 1.04 mmol) was o b t a i n e d by washing a 50% o i l d i s p e r s i o n ( 50 mg) w i t h anhydrous benzene (2 x 1 m l ) . To t h i s was added a s o l u t i o n o f 1-f o r m y l aldehyde (146) (50 mg, 0.155 mmol) i n anhydrous benzene (8 m l ) . M e t h y l c h l o r o f o r m a t e (2.44 g, 0.026 mol) was added and the mix-t u r e h e a t e d t o r e f l u x f o r 2 h r . The r e a c t i o n m i x t u r e was then c o o l e d and f i l t e r e d t h r o u g h a s h o r t a l u m i n a column (Act I I I ) u s i n g benzene as e l u a n t . Compound (15 3) was o b t a i n e d as an amorphous s o l i d on attemp t e d r e c r y s t a l l i s a t i o n ( 5 0 mg, 7 7 % ) . M.Pt.: 5 8° ( e t h a n o l , amorphous s o l i d ) I.R. : l J „ „ „ , 2960, 2930, 1732 (sh) , 1688 (sh) , 1668 cm - 1. L i l t 13 U.V.: A M e 0 H 287 (3.24), 277 (3.30), 248 (3.87) nm. IT13.X NMR: S( CDC1 3) 100 MHz 9.74, 9.67, 9.50 (3s, IH, C-17 H) , 8.84, 8.6 (2s, IH, N-CHO) , 8. 03-7. 88, 7. 88-7. 4 (m, 4H, arom.) , 5.52 (q, IH, J=6 Hz, C-19 H) 4.47, (dd, IH, J=10 and 16 Hz, C-16 H), 3.74 (S, 3H, N-C02CH_3) . M.S.-m/e: 418 (M+), 416 , 380, 279, 210, 209, 208, 194, 180, 166, 150, 134. High r e s o l u t i o n mass spectrum: C a l c — f o r C22 H25 N2°4 Cl=416.1501 Found= 416.1486 E l e m e n t a l a n a l y s i s : C 2 2 H 2 5 N 2 ° 4 " C 2 H 5 ° H ' F o u n d : C ' 62.41, H, 6.70, N, 6.33 C a l c - C, 62.27, H, 6.70, N, 6.05. N o r - f l u o r o c u r a r i n e (119) P r e p a r e d e x a c t l y a c c o r d i n g t o the l i t e r a t u r e p r o c e -d u r e : 1 3 8 1 3 M.pf. 184° ( l i t . m.pt. 184-5°). Y i e l d = 4 1 % -118-137 Deshydroxymethylstemmadenine (128) N o r - f l u o r o c u r a r i n e (119) (50 mg, 0.169 mmol) was d i s s o l v e d i n methanol (5 ml) and sodium b o r o h y d r i d e (55 mg, 1.45 mmol) added o v e r t e n min. A f t e r s t i r r i n g f o r an a d d i -t i o n a l 10 min the methanol was removed under reduced p r e s s u r e . The r e s u l t a n t o i l was t r e a t e d w i t h (1) d i c h l o r o m e t h a n e (5 ml) and (2) w a t e r (2 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were removed and the aqueous l a y e r f u r t h e r e x t r a c t e d w i t h d i c h l o r o m e t h a n e ( 3 x 5 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were combined, d r i e d , and s t r i p p e d o f s o l v e n t . Chromatography on s i l i c a g e l p r o v i d e d deshydroxymethylstemmadenine (128) (16 mg, 4 0 % ) . I.R.: \) ~ v n , 3350, 3250 (sh) , 2900, 1600 cm - 1 . U.V./{ 290 (3 . 7 4 ) , 283 (3. 78). M.Pt.: 86-88° (MeOH) NMR: ^ (CDC1 3) 100 MHz 9.22 ( s , IH, NH, exchangeable w i t h D 2 0 ) , 7.6-6.96 (m, 4H, arom.), 5.5 (q, IH, J=6 Hz, C-19 H), 4.92 (S, IH, OH, exchangeable w i t h D 2 0 ) , 4.22 (d, 2H, J=5 Hz, C-17 H ) , 1.66 (d, 3H, J=6 Hz, C-18 H). M.S. m/e: 296 ( M + ),193,168,144,123. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c^ gH 2 4N 20=296.1889. Found=296.1860. E l e m e n t a l a n a l y s i s : C a l c - f o r C l gH 2 4N 20-CH 3OH_ c 7 3 ± H 8.53, N, 8.53 Found C, 72.75, H, 8.20, N, 8.49. 16 3 1,2,3,4 t e t r a h y d r o c a r b a z o l e (155) P r e p a r e d e x a c t l y a c c o r d i n g t o p r o c e d u r e by Corson and Rogers. M.pt. 119° ( l i t . m.pt. 120°). Y i e l d = 7 5 % . -119-1 - B e n z o t r i a z o l e - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (162) N - C h l o r o b e n z o t r i a z o l e (504 mg, 3.28 mmol) i n dry d i c h l o r o m e t h a n e (4 ml) was added t o a s o l u t i o n of t e t r a -h y d r o c a r b a z o l e (155) (554 mg, 3.24 mmol) i n dry d i c h l o r o m e -thane (6 ml) a t 0°. A f t e r s t i r r i n g f o r 20 min the r e a c t i o n m i x t u r e was d i l u t e d w i t h d i c h l o r o m e t h a n e (50 ml) and th e n e x t r a c t e d w i t h (1) s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n (20 ml) and (2) water (20 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were then d r i e d , f i l t e r e d , and e v a p o r a t e d t o g i v e a w h i t e foam. Chromatography on al u m i n a (Act I I I ) f o l l o w e d by r e c r y s t a l l i -s a t i o n from methanol gave (162) (800 mg, 86%). M.Pt.: 205-209° (MeOH). U.V.: maS H 2 9 1 ( 4 - 1 0 ) ' 2 8 2 (4.19), 276 (4.20), 223 (4.62) nm. I.R.: d CHC1 3 3470, 3020, 2950, 1455 cm - 1. NMR: £(CDC1 3)100 MHz (FT) 8.40 ( s , IH, NH) , 8.00-7. 83 (m, IH, C-4 H), 7.77-7.55 (m, IH, C-7 1 H), 7.4-7.08 (m, 5H, arom. i n d o l e + C-5' H), 6.97-6.76 (m, IH, C-6' H) , 6.49 ( t , IH, J=6 Hz, C - l H) , 2.98 ( t , 211, J=5.5 Hz, C-4 U^) , 2.65 -2.31 (m, 2H, C-2 H 2 ) , 2.26-1.98 (m, 2H, C-3 H 2 ) . M.S. m/e: 288 ( M + ) , 170, 169, 168 High r e s o l u t i o n mass spectrum: Calc^. f o r C l 8H l 6N4=288.1375 Found=288.1374 E l e m e n t a l a n a l y s i s : Found C, 75.0, H, 5.40, N, 19.51 C a l c ! C, 75.0, H, 5.55, N, 19.44. -120-1 - C y a n o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) and 1 - s u c c i n i m i d o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (165) N-Bromosuccinimide (550 mg, 3.09 mmol) and d i -benzoyl peroxi de ( 1 mg) were q u i c k l y a i d e d t o a s o l u t i o n o f t e t r a h y d r o c a r b a z o l e (155) (500 mg, 2.93 mmol) i n dry p y r i d i n e (0.6 ml),and dry benzene (10 m l ) . A f t e r s t i r r i n g f o r 24 hr a t room temperature ( r . t . ) two l a y e r s were formed as d e s c r i b e d . 1 ^ The benzene l a y e r was decanted l e a v i n g a v i s c o u s r e d o i l w h i c h on d r y i n g under vacuo a f f o r d e d a crude c r y s t a l l i n e mass o f the r e q u i r e d p y r i d i n i u m s a l t (163) (1.2 g ) . P o t a s s i u m c y a n i d e (1.6 g, 24.62 mmol) was added t o the crude s a l t (163) f o l l o w e d by anhydrous a c e t o n i t r i l e (12 m l ) . S t i r r i n g was c o n t i n u e d f o r 12 hr and s a t u r a t e d sodium b i -c a r b o n a t e (15 ml) was then added. E x t r a c t i o n w i t h e t h y l a c e t a t e (4 x 50 ml) gave, a f t e r d r y i n g and removal o f s o l v e n t , a crude m i x t u r e o f (164) and (165) which on chromatography on s i l i c a a f f o r d e d 1-cyano-1,2,3,4 t e t r a h y d r o c a r b a z o l e (164) (426 mg, 74%) and 1 - s u c c i n i m i d o - l , 2 , 3 , 4 t e t r a h y d r o c a r b a z o l e (165) (150 mg, 19.2%). 1 - C y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) M.Pt.: 103-104° ( C H 2 C l 2 o r CC1 4) U.V.: J M e 0 H 288 (3. 7 5 ) , 278 (3. 84), 273 (3. 8 5 ) , 222 (4 . 51) nm. " max I.R.: l ^ C H C l 3 3470, 2960, 2870 ( s h ) , 2240, cm" 1 NMR: (CDC1 3) 100 MHz (FT) 8.16 ( s , IH, NH), 7.44-7.04 (m, 4H, arom.), 3.90 ( t , IH, J=6 Hz, C - l H), 2.71 ( t , 2H, J=6 Hz, C-4 H 2 ) , 2.34-1.66 (m, 4H, C-2 and C-3 H) . -121-M.S. m/e: 19 6(M+), 16 8. High r e s o l u t i o n mass spectrum: C a l c — f o r C-^3H^2N2=19 6 ' 1 (^00 Found=196.0977. E l e m e n t a l a n a l y s i s : Found C, 79.30, H, 6.26, N, 14.00 C a l c - C, 79.59, H, 6.12, N, 14.28. 1 - s u c c i n i m i d o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (165) M.Pt.: 220° (CHC1 3) U.V.: y\ ^ 0 H 288 (3. 8 3 ) , 278 ( 3 . 8 8 ) , 273 (3. 8 8 ) , 225 (4.50) nm. I.R.: l ^ C H C 1 3470 , 3015, 2950, 1705 cm. NMR: £(CDC1 3) 100 MHz (FT) 7.94 ( s , IH, NH) , 7.6-7.4 (m, IH, C - 8 H) , 7.32-7.04 (m, 3H, C-5-)7 H) , 5.54 ( t , IH, J=7 Hz, C - l H) , 2.92-2.72 (m, 2H, C-4 H) , 2.69 ( s , 4H, N(CH_2) 2), 2.60-1.90 (m, 4H, C-2 and C-3 H_2) . M.S. m/e: 268 (M +), 240, 170, 169, 168, 143. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C. -H, rN„0„=268.1212 l b ± o Z A Found=268.1205 E l e m e n t a l a n a l y s i s : Found C, 71.38, H, 5.81, N, 10.19 C a l c - C, 71.64, H, 5.97, N, 10.44. 9 - C a r b o m e t h o x y - l - c y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (166) 1 - C y a n o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) (100 mg, 0.51 mmol) i n anhydrous t e t r a h y d r o f u r a n (3 ml) was added o v e r 3 min t o a s u s p e n s i o n o f p o t a s s i u m h y d r i d e (0.16 ml, 1.03 mmol) i n anhydrous t e t r a h y d r o f u r a n (10 ml) a t 0°. S t i r r i n g was c o n t i n u e d f o r 30 min and d r y , r e d i s t i l l e d m e t h y l c h l o r o f o r m a t e (0.1 m l , 1.3 mmol) was added. A f t e r an -122-a d d i t i o n a l 30 min a l u m i n a (Act I I I ) was s l o w l y added t o d e s t r o y the excess p o t a s s i u m h y d r i d e . The s u s p e n s i o n was d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a g l a s s s i n t e r f u n n e l . T h e s o l i d w a s w a s h e d w i t h e t h y l a c e t a t e (10 m l ) . T h e e t h y l a c e t a t e s o l u b l e s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h i c h was chromatographed on s i l i c a and gave 9-carbo-m e t h o x y - l - c y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (166) (90 mg, 70%). M.Pt.: 68-70° (CHC1 3). U.V.: / j ^ ° H 292 (3 . 23), 282 (3.16), 273 (3.17), 258 (3. 4 5 ) , 226 (3. 77)nm. I.R.: [) C H C i 3 2980, 2870 (sh) , 2260, 1730 cm - 1. NMR: (CDC1 3) 100 MHz (FT) 8.22-8.04 (m, IH, C-8 H ) , 7.56-7.24 (m, 3H, C-5 H ) , 4.50 ( t , IH, J=7 Hz, C - l H), 4.09 ( s , 3H, C0 2CH 3) , 2. 86-1.92 (m, 6H, C-2, 3 & 4 H_2) • M.S. m/e: 254 (M +),194,168,84. High r e s o l u t i o n mass spectrum: C a l c — f o r C^^H^ 4N 20 2=254. 1055 Found=254.1066 E l e m e n t a l a n a l y s i s : Found C, 70.86, H, 5.51, H, 11.02 C a l c - C, 70.61, H, 5.64, N, 10.5. N - C a r b o m e t h o x y - l - c y a n o - l - c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o - c a r b a z o l e (167) 9 - C a r b o m e t h o x y - 1 - c y a n o - t e t r a h y d r o c a r b a z o l e (16 6) (100 mg, 0.394 mmol) i n anhydrous t e t r a h y d r o f u r a n (3 ml) was added o v e r 3 min t o p o t a s s i u m h y d r i d e (0.33 m l , 2.045 mmol) i n anhydrous t e t r a h y d r o f u r a n a t 0°. The remainder o f t h e r e a c t i o n and the work-up p r o c e d u r e was i d e n t i c a l w i t h t h a t j u s t d e s c r i b e d f o r (166). -123-Chromatography on s i l i c a a f f o r d e d (167) (48 mg, 39%) and s t a r t i n g m a t e r i a l (166) (27 mg, 2 7 % ) . U.V.: /) 292 (3. 2 8 ) , 279 (3. 1 8 ) , 261 (3.48), 227 (3. 94) I.R.: \J CHC1 3 2960, 2860 (sh) , 2245, 1750-25 cm - 1 . NMR: (CDC1 3) 100 MHz (FT) 8. 24-8. 04 (m, IH, C-8 H) , 7.62-7.26 (m, 3H, arom. C-5-*7 H) , 4.05 ( s , 3H, N-C02CH_3) , 3.86 (s , 3H, C02CH_3) , 3.0-2.58 (m, 3H, C-4 H_2 and C-2 H) , 2.48-2.06 (m, 3H, C-3 H_2 and C-2 H) . M.S. m/e: 312 ( M + ),254. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c H N 0 =312.1110 17 16 2 4 Found=3i2.1102 1 - C a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (156) R e d i s t i l l e d a c e t y l c h l o r i d e (11.6 g, 0.148 mol) was s l o w l y added t o anhydrous methanol (13 ml, 0.32 mol) a t r . t . To t h i s was added a s o l u t i o n o f 1 - c y a n o - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (164) (200 mg, 1.02 mmol) i n anhydrous methanol (2 m l ) . A f t e r s t i r r i n g f o r 1.5 h r , the r e a c t i o n m i x t u r e was poured o n t o i c e (20 g) and s t i r r i n g was c o n t i n u e d f o r an a d d i -t i o n a l 30 min. The r e a c t i o n m i x t u r e was b a s i f i e d t o pH 10 w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n . E x t r a c t i o n w i t h d i c h l o r o m e t h a n e gave, a f t e r d r y i n g and removal o f s o l v e n t , a crude foam w h i c h when chromatographed on s i l i c a g e l a f f o r d e d e s t e r (156) (163 mg, 7 0 % ) . i M P O H U , V , : / i 1 max ' 2 9 1 ( 3 ' 8 1 ) ' 2 8 3 ( 3 - 8 8 ) , 277 (3.83), 224 (4.53) nm. I.R.: 0 CHC1 3 3470, 2960, 2860 ( s h ) , 1730 cm" 1. -124-NMR: S (CDC1 3) 100 MHz (FT) 8.34 ( s , IH, NH), 7.58-6.98 (m, 4H, arom.), 3.94-3.88 (m, IH, C - l H), 3.80 ( s , 3H, C 0 2 C H 3 ) , 2.86-2.64 ( t , 2H, J=5.5 Hz, C-4 H), 2.34-1.72 (m, 4H, C-2 and C-3, H). M.S. m/e: 229 ( M + ) , 170, 168. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C-^H^N02=229.1103 Found=229.1101. N-Carbome thoxy-T-carbomethoxy-1, 2 3 , 4 - t e t r a h y d r o c a r b a z o l e (17 0) P o t a s s i u m h y d r i d e (0.142 ml, 0.88 mmol) was q u i c k l y added t o a s o l u t i o n o f 1 - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (156) (100 mg, 0.44 mmol) i n anhydrous t e t r a -h y d r o f uran (2 m l ) . The r e a c t i o n m i x t u r e was s t i r r e d f o r 2 5 min and m e t h y l c h l o r o f o r m a t e (0.1 m l , 1.3 mmol) was added. A f t e r s t i r r i n g f o r an a d d i t i o n a l 20 min, a l u m i n a . (Act I I I ) was added t o d e s t r o y e x c e s s p o t a s s i u m h y d r i d e . The f l a s k c o n t e n t s were d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and p a s s e d t h r o u g h a g l a s s s i n t e r f u n n e l . The f u n n e l was washed w i t h e t h y l a c e t a t e (2 x 10 ml) and the e t h y l a c e t a t e s o l u b l e s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h i c h a f t e r chromatography on s i l i c a g e l a f f o r d e d r e q u i r e d N - c a r b o m e t h o x y - l - c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r -b a z o l e (170) (75 mg, 6 0 % ) . M.Pt.: 68° (CHC1 3). U.V.: y\ ( J J l x 1 1 ) 292 (3.58), 278 ( 3 . 6 6 ) , 252 (4.08), 259 (4.08), 227 (4.30) nm. I.R.: 0 CHC1 3 2960, 2860 ( s h ) , 1780 ( s h ) , 1730 cm - 1. NMR: £(CDC1 3) 100 MHz (FT) 8.22-8.06 (m, IH, C-8 H), 7.56-7.2 (m, 3H, C-5-7, H), 4.26 ( t , IH, J=5.4 Hz, C - l H), -125-3.98 ( s , 3H, N-C0 2CH 3) , 3.72 (s, 3H, C0 2CH_ 3), 2.84-2.60 (m, 2H, C-4 H), 2.32-2.06 (m, 2H, C-2 H ) , 2.02-1.76 (m, 2H, C-3 H) M.S. m/e: 287(M +),255,228,168. Hig h r e s o l u t i o n mass S D e c t r u m : C a l c — f o r C, ,H,_N0. = 287.1157 16 17 4 Found=287.1149. E l e m e n t a l a n a l y s i s : Found C, 66.97, H, 5.92, N, 4.89 C a l c - C, 66.89, H, 5.92, N, 4.87. 1-Carbomethoxy-1-hydroxymethy1-1,2,3,4-tetrahydrocarbazole (157) To 9 - c a r b o m e t h o x y - l - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (170) (40 mg, 0.14 mol) i n anhydrous t e t r a h y d r o -f u r a n (2 ml) a t 0° was added p o t a s s i u m h y d r i d e (0.1 ml, 0.615 mmol). S t i r r i n g was c o n t i n u e d f o r 20 min a f t e r w h i c h f o r m a l d e -hyde was d i s t i l l e d i n t o the r e a c t i o n m i x t u r e . The temperature was r a i s e d t o r . t . a n d s t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 30 m i n m A l u m i n a (Act I I I ) was added t o d e s t r o y excess p o t a s s i u m h y d r i d e . The f l a s k c o n t e n t s were d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a g l a s s s i n t e r f u n n e l . The s o l i d was washed w i t h e t h y l a c e t a t e (10 m l ) . The s o l v e n t was removed under vacuo and chromatography of the crude foam gave r e q u i r e d 1-carbomethoxy-l-hydroxymethy1-1,2,3,4 t e t r a h y d r o c a r -b a z o l e (157) (16 mg, 45%) and s t a r t i n g m a t e r i a l (11 mg, 30%). U.V.: A M e 0 H 290 (3.82), 282 ( 3 . 8 8 ) , 277 (3.85), 224 (4.46) nm. " max I.R.: 0 CHC1 3 3620, 3460, 2950, 2850, 1710 cm - 1. NMR: <£(CDC13) 100 MHz (FT) 8.68 ( s , IH, NH) , 7. 64-7. 44 (m, IH, C-8 H) , 7.4-7.2 (m, 3H, C 5-7 H) , 3.99 (ABq, 2H, J=5.4 Hz, -126-CH 2OH, 3.74 ( s , 3H, C02CH_3) , 2.9-2.6 (m, 2H, C-4 H_ 2), 2.36-1.66 (m, 4H, C-2 and C-3 H ). M.S. m/e: 259(M +),228,200,197,168. Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5H 1 7N0 3=259.1208 Found=259.1199 1 - H y d r o x y m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (157) L i t h i u m a l u m i n i u m h y d r i d e (267 mg, 7.62 mmol) was added p o r t i o n w i s e o v e r 10 min t o a s o l u t i o n o f 1-c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (150 mg, 0.66 mmol) i n anhydrous t e t r a h y d r o f u r a n (20 ml) a t -5°. A f t e r 30 min a s a t u r a t e d s o l u t i o n o f sodium s u l p h a t e was added d r o p w i s e t o d e s t r o y e x c e s s l i t h i u m a l u m i n i u m h y d r i d e . The r e s u l t i n g s o l u t i o n was then e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 25 ml) and t h e e t h y l a c e t a t e e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e a foam w h i c h f o l l o w i n g chromato-graphy on s i l i c a g e l a f f o r d e d 1-hydroxymethy1-1,2,3,4-tetra-h y d r o c a r b a z o l e (157) (96 mg, 7 3 % ) . U.V.:/f J ^ ° H ; 288 (3. 76) , 283 (3.81), 274 (3. 7 8 ) , 225 (4.49) nm. I.R.: 0 C H C l 3 3622, 3460, 2940, 2860 cm" 1. NMR: £ (CDC1 3) 100 MHz (FT) 8.63 ( s , IH, NH), 7.62-6.90 (m, 4H, arom.), 4.07-3.52 (m ,2H, CH OH), 3.26-2.84 (m, IH, C - l H), 2.82-2.17 (m, 2H, C-4 H), 2.17-1.04 (m, 4H, C-2 and 3 H) . M.S. m/e: 201 (M +),170. H i g h r e s o l u t i o n mass spectrum: C a l c - f o r C^3H^,-NO=201.1154 Found=201.1148. E l e m e n t a l a n a l y s i s : Found C, 76.8, H, 7.34, N, 7.09 -127-C a l c - C, 7 7.6, H, 7.46, N, 6.96. Wi e l a n d - Gumlich Aldoxime (130) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c a -159 m.pt. 246° ( l i t . m.pt. 245°) Y i e l d = 8 5 % dure U l V ' : /I max" 2 9 5 ( 3 - 5 0 ) ' 2 4 2 ( 3- 9 1) n m -NMR: £ (CDC1 3) 100 MKz (FT) 7.34-6.58 ( m, 4H, arom.), 5.65 ( t , IH, J=7Hz, C-19H), 4.07 ( t , IH, J=7Hz, C-16 H ) , 3.84-3.50 (m, IH, C-2H). d Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C-\ ^ 2 3 1 ^ 3 0 2 = 3 2 5.1789 Found=325.1768. E l e m e n t a l a n a l y s i s : Found C, 69.86, H, 7.05, N, 12.82 C a l c - , C, 70.15, H, 7.07, N, 12.92 18-hydroxy-l-formyl-16-cyano-17-nor - 2 y 6 > , 16fl£-cur-19-ene (173) To oxime (130) (1 g, 3.076 mmol) was added f o r m i c a c i d (97-100%, 10 ml, 265.2 mmol) and anhydrous sodium formate ( 420 mg, 6.17 mmo1). T h i s m i x t u r e was h e a t e d t o r e f l u x f o r 1 h r . The temperature was then l o w e r e d t o -10° and c o n c e n t r a t e d ammonium h y d r o x i d e added to pH 10. The aqueous l a y e r was e x t r a c t e d w i t h c h l o r o f o r m ( 5 x 30 ml ) The c h l o r o f o r m e x t r a c t s were combined, d r i e d , and the s o l v e n t removed under reduced p r e s s u r e . The r e s u l t i n g gum was chromato-graphed on s i l i c a g e l t o g i v e (173) (720 mg, 7 0 % ) . M.Pt.: 240-242° (acetone) U.V. : /] MeOH 287 (3.78), 283 (3.81), 248 (4.34) nm. max -128-I.R.: C H C 1 3 3600, 2960, 2900 (sh) , 2260, 1675 cm" 1 NMR: S(CDC13) 100 MHz (FT) 8. 90, 8. 76 (2s, IH, N-CHO) , 8.1-8.03, 7.46-7.1 (m, 4H, arom.), 5.90 ( t , IH, J=7Hz, C-19H), 4.85 (d, IH, J=5Hz, C-2H). M.S. M/e: 335 (M +) 318, 304, 295, 177, 172, 144, 130. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C2oH21N3°2:=^~'" ^ 3 4 Found: 335.1614. E l e m e n t a l a n a l y s i s : Found C, 71.34, H, 6.44, N, 12.50 C a l c - C, 71.60, H, 6.26, N, 12.53 16-cyano-17-nor-2/?, 16 <t cur-19-ene (17 4) To t h e N- f o r m y l n i t r i l e (173) (100 mg, 0.298 mmol) was added e t h a n o l (2ml), w a t e r (4 ml) and barium h y d r o x i d e (380 mg, 2.22 mmol). The t e m p e r a t u r e was r a i s e d u n t i l r e f l u x began and t h i s was m a i n t a i n e d f o r 4 h r . The tem p e r a t u r e was then l o w e r e d t o r . t . and the s o l u t i o n d i l u t e d w i t h an e q u a l volume of d i c h l o r o m e t h a n e . R a p i d f i l t r a t i o n t h r o u g h a s h o r t column o f s i l i c a g e l a f f o r d e d the c rude n i t r i l e w h i c h was f u r t h e r p u r i f i e d by t h i n l a y e r chromatography on s i l i c a g e l t o g i v e (174) (72 mg, 7 8 % ) . . MeOH U.V.: /) m a x 298 (3 . 4 8 ) , 245 (3. 83) nm. I.R.: 0 CHC1 3 3420,3330,2930,2880,2230,1660,1600,750 cm" 1. NMR: & (CD 3OD ) 100 MHz (FT) 7.18-6.58 (m, 4H, arom.), 5.54 ( t , IH, J=7Hz, C-19 H), 4.14 (d, IH, J=9Hz, C-16 H). M.S. m/e: 307 ( M + ) , 267, 199, 177, 144, 130. High r e s o l u t i o n mass spectrum: C a l c - f o r C 1 9H 2iN 3O=307.1686 Found=307.1685 -129-Methyl-18-hydroxy- 2ft, 16 oj -cur-19-en-17-oate (132) A c e t y l c h l o r i d e (1.3 m l , 18.33 mmol) was s l o w l y added t o methanol (10 ml, 0.25 mol) a t r . t . To t h i s was added n i t r i l e (174) (95 mg, 0.31 mmol). -The temperature was r a i s e d u n t i l r e f l u x began and t h i s was c o n t i n u e d f o r 5 h r . Water (2 ml , 0. 11 mol) was then added and r e f l u x was c o n t i n u e d f o r 1 h r . The r e a c t i o n m i x t u r e was c o o l e d and e v a p o r a t e d t o d r y n e s s . To the r e s i d u e was added (1) e t h y l a c e t a t e (10 ml) and (2) 20% Na 2C03 (10 ml) . The aqueous l a y e r was f u r t h e r e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 10 ml) and the e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s t o g i v e , a f t e r chromatography on s i l i c a g e l and r e c r y s t a l l i s a t i o n , the r e q u i r e d e s t e r (132) (73 mg, 7 0 % ) . 142 E s t e r had same m.pt. and U.V. as t h a t r e p o r t e d . M.Pt.: 154-156° ( l i t 154-156°). u-v-: A m!x H 2 9 7 (3.52), 242 (3.84) nm. 1. R.rlJ CHC1 3 3610, 2920, 2960 , 1725 cm'1 NMR: £(CDC1 3) 8.28-7.58 (m, 4H, arom.), 5.67 ( t , IH, J=7Hz, C-19R), 4.74 ( s , IH, OH, exchangeable w i t h D 2 0 ) , 4.26 ( s , IK, NH, exchangeable w i t h D 20) , 3.74 ( s , 3H, C 0 2 CH_3) . M.S. m/e: 340 ( M + ) , 308, 267, 210, 178, 144, 130. d High r e s o l u t i o n mass spectrum: C a l c — f o r C 2 0 H 2 4 N 2 ° 3 = 3 ^ " 1 7 8 7 Found=340.1762 E l e m e n t a l a n a l y s i s , C a l c - f o r C 2 ( ) H 2 4 N 2 0 3 . CH 3OH: Found C, 68.0, H, 7.61, N, 7.80. Calc^- C, 67. 74, H, 7.52, N, 7.52 M e t h y l 2/2, 16 < cur-19-en-17-oate (133) 138b To the p a l l a d i u m on c h a r c o a l c a t a l y s t (10% Pd by w e i g h t , 169.2 mg c a t a l y s t ) , was added (1) h y d r o c h l o r i c a c i d - 1 3 0 -(33;5 m l , 0.18 m) (2) hydroxy e s t e r (132) (693 mg, 2.04 mmol) and (3) a c e t i c a c i d (20.32 m l ) . The m i x t u r e was then p l a c e d under an atmosphere o f hydrogen gas. A f t e r 20 h r , hydrogen (54.05 ml) had been t a k e n up and the c a t a l y s t was removed by s u c t i o n f i l t r a t i o n . The f i l t e r f u n n e l was washed w i t h methanol (150 m l ) . The r e s u l t -i n g s o l u t i o n was e v a p o r a t e d t o an o i l , c o o l e d t o 0°, and concen-trated NH^OH added u n t i l pH 12. The b a s i c l a y e r was e x t r a c t e d w i t h c h l o r o f o r m (5 x 20 ml) and the c h l o r o f o r m e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e a c o l o r l e s s foam which a f t e r chromatography on s i l i c a g e l and r e c r y s t a l l i s a t i o n a f f o r d e d r e q u i r e d e s t e r (133). (583 mg, 88%) M.Pt.: 118° (benzene) U.V.:/| max n 297 (3. 5 0 ) , 244 (3. 84) nm. I.R.:\) 3420, 2950, 2890 (sh) , 1725 c m T 1 NMR: i (CDC1 3) 100 MHz (FT): 7.30-6.52 (m, 4H, arom.), 5.49 (q, IH, J=7Hz, C-19H), 4.1 ( s , IH, exchangeable w i t h D 20, NH), 4.01 (d, IH, J=4.8Hz, C-16H) 3.70 ( s , 3H, C0 2CH 3 ), 1.56 (dd, 3H J=2 and 7Hz, C-18H_3) M.S. m/e: 324 ( M + ) , 251, 194, 144, 139, 130 High r e s o l u t i o n mass spectrum: C a l c - f o r c 2 0H 2 4N 2O 2=324.1837 Found=324.1810 E l e m e n t a l a n a l y s i s : Found C, 73.87, H, 7.40, N, 8.37 d C a l c - C, 74.07, H, 7.41, N, 8.64. Akuammicine (64) 0 . 0 6 7 mmol) i n dry benzene (4 ml) was q u i c k l y added l e a d t e t r a -a c e t a t e ( 3 0 mg, 0 . 0 6 7 mmol). A f t e r s t i r r i n g f o r 10 min, the (133) (22 mg, -131-r e a c t i o n m i x t u r e was d i l u t e d w i t h c h l o r o f o r m (4 ml) and then i m m e d i a t e l y poured t h r o u g h a g l a s s s i n t e r f u n n e l c o n t a i n i n g a l u m i n a (Act I I I , 0.6g). The s i n t e r f u n n e l was washed w i t h c h l o r o f o r m (30 m l ) , and the c h l o r o f o r m s o l u b l e s d r i e d , and e v a p o r a t e d t o g i v e a r e s i d u e w h i c h was chromatographed on s i l i c a g e l and c r y s t a l l i z e d from e t h y l a c e t a t e t o g i v e akuammicine (64) (13 mg, 60%) h a v i n g a l m o s t i d e n t i c a l c h a r a c t e r i s t i c s w i t h t h a t ^ , 132,137 r e p o r t e d . M.Pt.: 181° ( l i t m . p t . 180-181.5°) Akuammicine jV] D =-672 (CHC1 3) , C=0.1 ( l i t = - 700 ) PeshydroxymethyIstemmadenine (122) To a v e s s e l c o n t a i n i n g g l a c i a l a c e t i c a c i d , (3 m l , 52 mmol) a t r e f l u x was q u i c k l y added akuammicine (64) (68 mg, 0.21 mmol). R e f l u x was m a i n t a i n e d by t h e a d d i t i o n o f sodium b o r o h y d r i d e (500 mg, 13.16 mmol) o v e r f i f t e e n min. A f t e r a d d i -t i o n , t h e t e m p e r a t u r e was l o w e r e d t o 5° and t h e s o l u t i o n b a s i f i e d w i t h cone— ammonium h y d r o x i d e s o l u t i o n u n t i l pH 12. E x t r a c t i o n o f t h e aqueous phase w i t h d i c h l o r o m e t h a n e (3 x 20 ml) a f f o r d e d a f t e r d r y i n g and chromatography, r e q u i r e d e s t e r (122) (39 mg, 5 7 % ) . NMR: S (CDC1 3) (100 MHz) (FT) 9.19 ( s , IH, NH), 7.54-7.06 (m, 4H, arom.), 5.79, (q, IH, J=7.6Hz, C-19H), 4.35 ( s , IH, C-16H), 3.90 ( s , 3H, C 0 2 C H 3 ) , 1.77 (d, J=7.6Hz, 3H, C-18 H 3) E l e m e n t a l a n a l y s i s : C a l c - f o r C 2 0 H 2 4 N 2 ° 2 z z C ' 7 4 - 0 7 ' H ' 7.40, N 8.64 Found C, 73.83, H, 7.28, N, 8.46. -132-N - c arbome thoxy de shy droxyme thy I s temmadeh i n e (175) To e s t e r (122) (40 mg, 0.123 mmol) i n anhydrous TKF at 0° was q u i c k l y added p o t a s s i u m h y d r i d e (6.38 mg, 0.16 mmol). The s u s p e n s i o n was a l l o w e d t o s t i r f o r 10 min and m e t h y l c h l o r o -formate (0.04 m l , 0.58 mmol) was added. The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r f o r an a d d i t i o n a l 15 min a f t e r w h i c h a l u m i n a (Act I I I ) was s l o w l y added t o d e s t r o y the excess potassium h y d r i d e The m i x t u r e was then d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a s i n t e r f u n n e l - the f u n n e l b e i n g washed w i t h e t h y l a c e t a t e (10 m l ) . The s o l u t i o n was d r i e d and s t r i p p e d o f s o l v e n t t o y i e l d , a f t e r chromatography on s i l i c a g e l the r e q u i r e d N a -carbomethoxy e s t e r (175) (28 mg, 59%) Me OH U.V.:/| m a x 296 (3. 6 7 ) , 285 (3. 8 8 ) , 263 (4.22), 228 (4.34) nm. I.R.-.l) C H C 1 3 2980, 2900 (sh) , 1730 cm" 1 NMR: £(CDC1 3) 100 MHz (FT) 8.23-8.05, 7.59-7.14 (m, 4H, arom.), 5.44 (q, IH, J=7Hz, C-19H) , 4.00 ( s , 3H, N- C0 2CH_ 3), 3.95 (d, IH, J=3Hz, C-16H), 3.65 ( s , 3H, C 0 2 C H 3 ) , 1.85 (d, 3H, J=7Hz, C-18H 3). M.S. m/e: 382 ( M + ) , 323, 194, 123 Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 2 H 2 6 N 2 ° 4 = 3 8 2 ' 1 8 9 2 Found=382.1883. 1 8 / 3 - carbomethoxycleavamine (72) P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . 1 ^ Y i e l d = 7 9 % M.pt.=121° ( l i t m.pt.=122.24°) -133-N a-carbomethoxy-T8 j3 -carbomethoxycleavamine (180) 161 P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e Y i e l d = 6 7 % M.pt.=123° ( l i t m.pt.= 126°) N a-methyl-18 -carbomethoxycleavamine (18 3) and 9-methy 1-18/?--carbomethoxycleavamine i h d o l e h i n e (184) To e s t e r (72) (110 mg, 0.325 mmol) i n anhydrous THF a t 0° was added p o t a s s i u m h y d r i d e (26 mg, 0.65 mmol). S t i r r i n g was c o n t i n u e d f o r 30 min and m e t h y l i o d i d e (48 mg, 0.338 mmol) was added. S t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 3 0 min and a l u m i n a (Act I I I ) was added t o d e s t r o y t h e r e m a i n i n g p o t a s s i u m h y d r i d e . The m i x t u r e was d i l u t e d w i t h e t h y l a c e t a t e (20 ml) and then poured t h r o u g h a g l a s s s i n t e r f u n n e l . The s o l v e n t was removed i n vacuo t o g i v e an o i l w h i c h , f o l l o w i n g chromato-graphy on s i l i c a g e l , a f f o r d e d a 1:1 m i x t u r e o f N a-methy1-18/5-carbomethoxycleavamine (183) (40 mg, 35%) and 9-methyl-18^ / 5 -carbomethoxycleavamine i n d o l e n i n e (184) (40 mg, 35%) N^-methyl-18/?-carbomethoxycleavamine (183) l.R.-.l) CHC1., 3010 (sh) , 2930, 1725, 1475 cm - 1. V max 3 U.V.: A m l x H 2 9 3 (4.068),287 (4.099),279 ( 4 . 0 3 ) , 228 (4.78) nm. NMR: & (CDC1 3) 100 MHz (FT) 7.58-7.46 (m, IH, C-14 H), 7.32-6.98 (m, 3H, arom), 5.73 (d, IH, J=7Hz, C-18H), 5.39 (d, IH, J=3Hz, C-5 H), 3.62 ( s , 3H, N - C H 3 ) , 3.57 ( s , 3H, C 0 2 C H 3 ) , 3.15 ( s , 2H, C-5 H) , 2.06 (q, 2H, J=7.4 Hz, CH_2CH3) , 1.06 ( t , 3H, J=7.4 Hz, CH 2CH 3). -134-M.S. m/e: 352 ( M + ) , 337, 229, 136, 135, 124, 122 Hig h r e s o l u t i o n mass spectrum: C a l c — f o r 8 N 2 ° 2 = 3 ! ^ 2 • 2150 Found=352.2149 9-methyl - 1 8 y 0 -carbomethoxycleavamine (184) M.pt.= 9 0° I.R.: 0 m = v CHClo 2980, 1725, 1618 c m - 1 Illci X u - v - : A m!x H 2 6 2 ( 3 . 7 7 ) , 219 ( s h ) (4.24) nm. NMR: <£(CDC13) 100 MHz (FT) 7.72-7.55 . (m, IH, C-14 H) , 7.44-7.13 (m, 3H, arom), 5.55 (d, IH, J=6Hz, C-3H), 4.87 (d, IH, J = l l H z , C-18 H) , 3.61 ( s , 3H, C02CH_3) , 1.31 ( s , 3H, C-9 CH_ 3), 1.07 ( t , 3H, J=7.5 Hz, CH2CH_3) . M.S. m/e: 352 (M+.) , 337, 293, 228, 135, 122 Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 2H 2gN 20 2=352.2151 Found=352.2151 9-benzyl-18y(g -carbomethoxycTeavamine i n d o l e n i n e (185) To e s t e r (72) (100 mg, 0.30 mmol) i n anhydrous THF (5ml) a t 0° was added p o t a s s i u m h y d r i d e (23.5 mg, 0.59 mmol). S t i r r i n g was c o n t i n u e d f o r 30 min and b e n z y l bromide (52 mg, 0. 31 mmol) was added. S t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 30 min a t r . t . The tem p e r a t u r e was the n l o w e r e d t o 0° and al u m i n a (Act I I I ) added t o d e s t r o y the r e m a i n i n g p o t a s s i u m h y d r i d e . The m i x t u r e was d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and f i l t e r e d . The s o l v e n t was removed i n vacuo t o y i e l d an o i l w h i c h , f o l l o w i n g chromatography on s i l i c a g e l , a f f o r d e d (185) . 1. R.: [) CHC1. 2970, 1745, 1460, 700 cm" 1. Ilia. X o U.V.: A " S ° H 260 (3. 7 6 ) , 222 (sh) (4.78) nm. Ill 3 .X -135-NMR: & (CDC1 ) 100 MHz (FT) 7.69 (d, lH,J=7.8Hz, Ph CH), 7.41-6.86 (m, 9H, arom.), 6.39 (d, IH, J=7.8 Hz, Ph CH), 5.35 (d, IH, J=6Hz, C-3 H), 4.50 (d, IH, J=10 Hz, C-18 H), 3.58 ( s , 3H, C 0 2 C H 3 ) , 2.47 ( s , 2H, C-5H), 1.1 ( t , 3H, J=7.5Hz CH 2CH 3). M.S. m/e: 428, (M +),387, 337, 230, 214, 136, 135, 122 H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C 0 0 H N o0~=428.2471 / o 3 2 -^Found=428.2467 -136-INTRODUCTION - PART I I I E l u c i d a t i o n o f b i o s y n t h e t i c r o u t e s t o d i m e r i c a l k a l o i d s P o s t u l a t e s a c c o u n t i n g f o r the f o r m a t i o n of the monomeric a l k a -l o i d s have so f a r been d i s c u s s e d i n p r e v i o u s s e c t i o n s . F u r t h e r c o n s i d e r a t i o n s a re n e c e s s a r y t o a c c o u n t f o r the f o r m a t i o n o f the m e d i c i n a l l y i m p o r t a n t d i m e r i c a l k a l o i d s , v i n c r i s t i n e (VCR) (201) and v i n b l a s t i n e (VLB) (202) . I t i s i m m e d i a t e l y o b v i o u s t h a t the upper h a l f o f t h e m o l e c u l e c o r r e s p o n d s t o a " c l e a v e d " c a t h a r a n t h i n e (12) m o i e t y , whereas the lower h a l f c o r r e s p o n d s t o v i n d o l i n e (.11) . I n an a t t e m p t t o det e r m i n e the p r e c u r s o r r o l e i n the d i m e r i c a l k a l o i d s , S c o t t a d m i n i s t e r e d l a b e l l e d v i n d o l i n e (11) and 91 c a t h a r a n t h i n e (12) t o C. r o s e u s . I n c o r p o r a t i o n l e v e l s o b t a i n e d d i d n o t " s e t t l e the q u e s t i o n " b e i n g o f the o r d e r 0.05 and 0.005% r e s p e c t i v e l y . No d e t a i l s o f l a b e l l i n g v/ere g i v e n nor t h a t o f any d e g r a d a t i o n of the i s o l a t e d a l k a l o i d . H u t c h i n s o n l a t e r 1 ^ 4 a d m i n i s t e r e d (3,6 3H_) l o g a n i n ( 3 5 ) , -137-14 shown below, i n a d m i x t u r e w i t h CO^CH^ l o g a n i n (35) t o C. ro s e u s and i s o l a t e d VLB (202) , c a t h a r a n t h i n e (12) and v i n d o l i n e (11) . 3 14 The H/ C r a t i o f e d was 8.2. 3 5 The i n c o r p o r a t i o n d a t a o b t a i n e d i s shown i n t a b l e 9 T a b l e 9 - I n of C. r o s e u s 3 14 T a b l e 9 - I n c o r p o r a t i o n of ( H, C) l o g a n i n (35) i n t o a l k a l o i d s S p e c i f i c ? o r a t 3 (%) I s o l a t e d H 14 I n c o r p o r a t i o n 3 / C R a t i o V i n b l a s t i n e (202) 0.069 7.5 C a t h a r a n t h i n e (12) 0.39 8.7 V i n d o l i n e (11) 0.26 6.0 I t was s t a t e d t h a t a v e r a g i n g the 3H/ 1 4C r a t i o f o r c a t h a r a n t h i n e (12) and v i n d o l i n e (11) gave a paper r a t i o f o r VLB (202) o f 7.35 wh i c h was s i g n i f i c a n t l y c l o s e t o t h a t i s o l a t e d . The r a t i o s o f s p e c i f i c a c t i v i t y o f (35) t o (11) and (12) r e s p e c t i v e l y were c o n s i d e r e d t o m i r r o r the i n v e r s e m o lar r a t i o o f t h e i r p o o l s i z e s w h i c h l e d to the s u g g e s t i o n t h a t a s t e a d y -s t a t e was i n o p e r a t i o n where the r e l a t i v e r a t e s o f f o r m a t i o n o f (11) and (12) were the same. -138-I f t h e r e l a t i v e r a t e o f f o r m a t i o n o f VLB (202) was t h e same from f o r m a t i o n o f an i n t e r m e d i a t e between a c r y l i c e s t e r (70) and c a t h a r a n t h i n e ( 1 2 ) o r v i n d o l i n e ( 1 1 ) , o r c a t h a r a n t h i n e (12) o r v i n d o l i n e (11) them s e l v e s t h e n i t was argued t h a t the 3 14 H/ C r a t i o would be a n t i c i p a t e d t o f a l l between t h a t o f c a t h a r a n t h i n e (12) o r v i n d o l i n e ( 11). The H/ C r a t i o i s o l a t e d f o r (202) and (12) a r e w i t h i n e x p e r i -m e n t a l e r r o r b u t t h a t o b t a i n e d f o r v i n d o l i n e (11) shows a l o s s o f 27% t r i t i u m . No s c i e n t i f i c e x p l a n a t i o n was f o r w a r d e d f o r t h i s o b s e r v a t i o n . I n o r d e r t o a s c e r t a i n the p o s s i b l e means by w h i c h t h e s e d i m e r i c a l k a l o i d s m ight be formed i n v i v o we f o c u s s e d our a t t e n t i o n on a number o f i n v i t r o o b s e r v a t i o n s . 165 I n t h i s r e s p e c t , i t had been n o t e d t h a t c a t h a r a n t h i n e (12) on t r e a t m e n t w i t h r e f l u x i n g a c e t i c a c i d f o l l o w e d by sodium b o r o h y d r i d e gave, i n h i g h y i e l d , the c l e a v e d c a t h a r a n t h i n e system, 18^-carbomethoxycleavamine (72). ( F i g u r e 47) 7 0 3. 14 - 1 3 9 -C0 2C H 3 I 2 2 0 3 72 F i g u r e 47 - A c i d c a t a l y s e d f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . The c l e a v a m i n e s e r i e s o f compounds have found use i n r e c e n t 166 t i m e s i n the s y n t h e s e s o f d i m e r i c a l k a l o i d s , u n f o r t u n a t e l y , p o s s e s s i n g t h e " u n n a t u r a l " s t e r e o c h e m i s t r y a t c - 1 8 . T h i s approach u t i l i s e d i n i t i a l a t t a c k by p o s i t i v e c h l o r i n e t o g i v e an i n t e r m e d i a t e c h l o r o i n d o l e n i n e (204) w h i c h then r e a c t e d w i t h vindo-l i n e (11) i n a c i d media t o g i v e the c o r r e s p o n d i n g dimer. ( F i g u r e 48 ) C 0 2 C H 3 U N N A T U R A L D I M E R S F i g u r e 4 8 - S y n t h e s i s o f d i m e r i c a l k a l o i d s : c h l o r o i n d o l e n i n e approach, The problem, w i t h r e s p e c t t o s t e r e o c h e m i s t r y , has been -140-1 fc\ 1 r e s o l v e d v i a a m o d i f i e d P o l o n o v s k i r e a c t i o n ( F i g u r e 4 9 ) . I n t h i s c a s e , the N-oxide o f c a t h a r a n t h i n e (205) was t r e a t e d w i t h ( i ) t r i f l u o r o a c e t i c a n h y d r i d e a n d v i n d o l i n e (11) a n d ( i i ) sodium b o r o h y d r i d e . Dimers p o s s e s s i n g " n a t u r a l " s t e r e o c h e m i s t r y a t C-18 were p r e d o m i n a n t l y formed. The c o u p l i n g p r o c e s s can be viewed as e i t h e r c o n c e r t e d o r n o n - c o n c e r t e d . 2 0 5 C 0 2 C H 3 l - C O C F . , ^ / u ' ^ T ^ < < c o n c e r t e d C 0 2 C H 3 1) V i n d o l i n e 2) N a B H 4 D I M E R S 2 0 3 C0£H 1) V i n d o l i n e 2) N * B H 4 D I M E R 5 F i g u r e 49 - S y n t h e s i s o f d i m e r i c a l k a l o i d s m o d i f i e d P o l o n o v s k i approach. F u r t h e r e x a m i n a t i o n o f f i g u r e s 47-49 from a b i o s y n t h e t i c s t a n d p o i n t y i e l d e d the f o l l o w i n g i n f o r m a t i o n . (1) f i g u r e 47 can be c o n s i d e r e d b i o s y n t h e t i c a l l y as i n v o l v i n g an i n i t i a l e n z y m a t i c a t t a c k ( f o r example, E ^enzyme, see f i g u r e 50) f o l l o w e d by d i s p l a c e m e n t t o g i v e c h a r g e d i n t e r m e d i a t e (203) whic h t h e n c o u p l e s w i t h v i n d o l i n e (11) t o g i v e the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s . ^ 2 0 3 • D I M E R S F i g u r e 50 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : enzyme a s s i s t e d f r a g m e n t a t i o n and c o u p l i n g . + + The enzyme may be e n v i s a g e d t o " d e l i v e r " OH , OOH o r an e q u i v a l e n t f u n c t i o n a l i t y . I t s h o u l d be n o t e d t h a t o x i d a t i o n o f i n d o l e s t o t h e i r c o r r e s p o n d i n g hydroxy - o r h y d r o p e r o x y i n d o l e -16 8 n i n e s i s a w e l l known p r o c e s s , thus g i v i n g s u p p o r t t o t h i s h y p o t h e s i s . (2) f i g u r e 48 w a s i n v o l v e d w i t h t h e w e l l known c h l o r o i n d o l e n i n e x approach t o the s y n t h e s i s o f d i m e r i c a l k a l o i d s from c l e a v a m i n e systems. I n t h i s r e s p e c t , however, no c l e a v a m i n e monomers have been i s o l a t e d from C. r o s e u s , b u t t h i s does not p r e c l u d e an i n v i v o f r a g m e n t a t i o n o f c a t h a r a n t h i n e (12) t o an lSyJ-carbomethoxy c l e a v a m i n e - ( 7 2 ) - t y p e i n t e r m e d i a t e . C o n s e q u e n t l y a t t a c k on an i n t e r m e d i a t e o f type (72) by + + E =0H , OOH, o r an enzyme ( f i g u r e 51) would g i v e an i n t e r m e d i a t e analogous t o (204) w h i c h i n a s i m i l a r manner c o u l d c o u p l e w i t h v i n d o l i n e t o y i e l d ' n a t u r a l ' d i m e r i c a l k a l o i d s . I n t h i s way, i t w ould o c c u r r e d be e n v i s a g e d t h a t e n z y m a t i c c o n t r o l g i v i n g the c o r r e c t s t e r e o c h e m i s t r y . o f the c o u p l i n g F i g u r e 51 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c i n d o l e n i n e approach. (3) N - o x i d e s are f r e q u e n t l y found i n n a t u r e and, i n t h i s r e s p e c t , i n v o l v e m e n t o f an enzyme as shown c o u l d be p o s t u l a t e d t o y i e l d the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s i n v i v o . ( F i g u r e 52) o " o-E • D I M E R S 2 0 5 F i g u r e 52 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c " P o l o n o v s k c o u p l i n g . I t s h o u l d a l s o be n o t e d t h a t f i g u r e s 50-52 c o u l d a l s o o c c u r - 1 4 3 -v i a one e l e c t r o n p r o c e s s e s and w h i l s t t h i s had no s u b s t a n t i a l i n v i t r o s u p p o r t , i t must be g i v e n f u l l c o n s i d e r a t i o n i n the i n v i v o s t u d i e s . As the i n i t i a l s t u d i e s w i t h l a b e l l e d c a t h a r a n t h i n e (12) i n 91 C. r o s e u s had met w i t h l i t t l e s u c c e s s , f u l l c o n s i d e r a t i o n o f o t h e r p o s s i b l e b i o s y n t h e t i c i n t e r m e d i a t e s were made. I n t h i s r e s p e c t , however,the p o s s i b l e i n v o l v e m e n t o f hydroxy o r h y d r o -peroxy t y p e i n t e r m e d i a t e s o f c a t h a r a n t h i n e (12) o r 18/?-carbo-methoxycleavamine (72) were n o t pu r s u e d due t o t h e i r s u s p e c t e d i n s t a b i l i t y . I t was d e c i d e d i n s t e a d t o i n v e s t i g a t e the p o s s i b l e r o l e o f c a t h a r a n t h i n e - N ^ - o x i d e ( 2 0 5 ) as a p r e c u r s o r i n the d i m e r i c a l k a -l o i d b i o s y n t h e s i s o f v i n c r i s t i n e (201) . C a t h a r a n t h i n e - N g o x i d e (205) on s t a n d i n g a t room t e m p e r a t u r e f o r 169 p r o l o n g e d p e r i o d s r e a r r a n g e s t o g i v e (206). However, i t was f e l t t h a t t h i s s i t u a t i o n would n o t be o f g r e a t i m p o r t a n c e as t h e a d m i n i s t r a t i o n p e r i o d t o t h e p l a n t was t o be o f s h o r t d u r a t i o n . 2 0 6 A more i m p o r t a n t p o i n t was the e x p e c t e d low c o n c e n t r a t i o n o f v i n c r i s t i n e (201) i n C. r o s e u s (~1 x 1 0 - 4 % ) 1 7 ^ and, c o n s e q u e n t l y , i t was f e l t t h a t a p a r a l l e l programme s h o u l d be d e v e l o p e d t o g e n e r a t e C. roseus t i s s u e c u l t u r e s w h i c h would a c t i v e l y b i o s y n t h e --144-s i s e c a t h a r a n t h i n e (12), v i n d o l i n e (11) and the c o r r e s p o n d i n g d i -m e r i c a l k a l o i d s (201,202). 118 82—83 As mentioned p r e v i o u s l y , S c o t t and Zenk have made some p r o g r e s s i n t h i s a r e a b u t so f a r have been unable t o r e p o r t the b i o s y n t h e t i c f o r m a t i o n o f ( 1 1 ) , ( 1 2 ) , (201) o r (202) from t h e s e c u l t u r e s . However t h e r e has been c o n s i d e r a b l e s t i m u l a t i o n i n t h e p a s t f i f t e e n y e a r s t o f i n d growth c o n d i t i o n s f o r t i s s u e c u l t u r e s t h a t w o uld p r o v i d e a s o u r c e f o r t h e s e a l k a l o i d s . The p r o g r e s s i n t h i s a r e a i s d e p i c t e d h i s t o r i c a l l y as f o l l o w s . 171 I n i t i a l i n v e s t i g a t i o n by Carew e t a l . y i e l d e d C. r o s e u s c a l l u s t i s s u e c u l t u r e s r e p o r t e d l y p o s s e s s i n g a s i m i l a r a l k a l o i d a l p a t t e r n t o t h a t o f C. r o s e u s seeds. Four u n i d e n t i f i e d a l k a l o i d s were n o t e d . 172 F u r t h e r i n v e s t i g a t i o n s by Carew w i t h t h e s e c u l t u r e s s u g g e s t e d the p r e s e n c e o f numerous a l k a l o i d s . These, t o o , were u n i d e n t i f i e d a l t h o u g h one was c o n s i d e r e d t o be v i n d o l i n e ( 1 1 ) . 17 3 Boder e t a l . grew C. roseus crown g a l l t i s s u e c u l t u r e s and n o t e d t h e p r e s e n c e o f two a l k a l o i d a l components - one h a v i n g a s i m i l a r Rf t o t h a t o f v i n d o l i n e (11) . I n c u b a t i o n o f t h e c u l -t u r e s w i t h v i n d o l i n e (11) gave t h r e e m e t a b o l i t e s - one b e i n g p o s t u l a t e d t o be d e s a c e t y I v i n d o l i n e (207a). 174 F u r t h e r s t u d i e s w i t h C. r o s e u s stem and l e a f c a l l u s c u l t u r e s a l s o gave r i s e t o the s u g g e s t i o n t h a t v i n d o l i n e (11) and v i n d o l i n i n e (208) v/ere p r e s e n t •••145-2 0 7 a , R =H . 2 0 8 b , R = C 0 C H 3 , A - l c H 2 ) 2 175 G a r n i e r and M o r e l s t u d i e d the a l k a l o i d a l c o n t e n t of C. r o s e u s t i s s u e c u l t u r e s under a v a r i e t y o f c o n d i t i o n s . I n t h i s r e s p e c t i t was n o t e d t h a t the a l k a l o i d a l p r o d u c t i o n was i n c r e a s e d by a d d i t i o n o f t r y p t o p h a n (lb) and 2,4 D. Moreover the a l k a l o i d a l c o n t e n t o f the c u l t u r e s were n o t e d t o be d i f f e r e n t from t h a t o f the whole p l a n t e x t r a c t a l t h o u g h no f u r t h e r i d e n t i f i c a t i o n was made. I n t h i s p a r t i c u l a r i n v e s t i g a t i o n i t was a l s o found t h a t the r a t e o f c u l t u r e growth was i n v e r s e l y p r o p o r t i o n a l t o the a l k a -l o i d a l c o n t e n t . 176 Carew r e c e n t l y r e v i e w e d t h e o v e r a l l p r o g r e s s i n t h i s a r e a and, s i g n i f i c a n t l y , c i t e d t h a t P a t e r s o n had been u n a b l e t o de-m o n s t r a t e the o c c u r r e n c e o f c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e (11) o r the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s (201,202) i n C. r o s e u s c a l l u s c u l t u r e s . Carew then e l a b o r a t e d t h a t , " t h e s e o b s e r v a t i o n s c l e a r l y i n d i -c a t e t h a t d i f f e r e n c e s e x i s t between the s y n t h e t i c c a p a b i l i t y o f the whole p l a n t and t h a t o f i s o l a t e d c a l l u s t i s s u e " . However he f a i l e d t o e x p l a i n t h i s r e s u l t i n the l i g h t o f h i s 172 own communication d e a l i n g w i t h the p r e s e n c e o f v i n d o l i n e (11) i n s i m i l a r c a l l u s c u l t u r e s . R e c e n t l y m e t a b o l i c s t u d i e s o f v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e -146-(12) h y d r o c h l o r i d e and v i n b l a s t i n e s u l p h a t e (202) , by i n c u b a t i o n w i t h C. r o s e u s t i s s u e c u l t u r e s have a l s o been 177 s t u d i e d . I n t h i s r e s p e c t , v i n d o l i n e (11) was n o t e d t o be c o n v e r t e d t o d e s a c e t y l v i n d o l i n e (207a) and d i h y d r o v i n d o l i n e (207b). No m e t a b o l i t e s from c a t h a r a n t h i n e (12) were n o t e d . I n c u b a t i o n w i t h v i n d o l i n e (11) and c a t h a r a n t h i n e (12) gave o n l y " m e t a b o l i t e s " d e s a c e t y l v i n d o l i n e (207a) and d i h y d r o v i n d o l i n e (207b). I n c u b a t i o n w i t h v i n b l a s t i n e (202) gave t h r e e u n i d e n t i f i e d " m e t a b o l i t e s " - two h a v i n g m/e>750 and one h a v i n g an m/e 418. These s t u d i e s and c o n c l u s i o n s p r e v i o u s l y d i s c u s s e d have s e v e r e l i m i t a t i o n s . C h a r a c t e r i s a t i o n o f the a l k a l o i d s from 1962-77 have o n l y been by TLC e x a m i n a t i o n . On o c c a s i o n the p l a t e s were viewed under u l t r a v i o l e t l i g h t and s p r a y e d w i t h a p p r o p r i a t e r e a g e n t s . C o - s p o t t i n g w i t h r e f e r e n c e a l k a l o i d s was not pursued i n any o f t h e s e i n v e s t i g a t i o n s n o r was any s p e c t r a l i n f o r m a t i o n o b t a i n e d . C o n s e q u e n t l y t h e s e p u r s u i t s were, a t b e s t , p r e l i m i n a r y , and c h e m i c a l l y , u n s a t i s f a c t o r y . I n t h i s r e s p e c t , now c o n s i d e r the s t a t u s o f t i s s u e c u l t u r e s t u d i e s w i t h a n o t h e r p l a n t V. minor. I t was r e c e n t l y s t a t e d t h a t t i s s u e c u l t u r e s from t h i s s p e c i e s p o s s e s s the same a l k a l o i d a l 17 8 c o n t e n t as t h a t o f the whole p l a n t . I n p a r t i c u l a r , v i n c a m i n e ( 92) was shown t o be p r e s e n t by t h e methods i n d i c a t e d above. 179 F u r t h e r work i n t h i s a r e a by Koch and Kunesch was v e r y i l l u m i n a t i n g . They grew c u l t u r e s o f minor under s i m i l a r c o n d i --147-t i o n s and i n c u b a t e d them w i t h D L ( 2 ± 4 C ) t r y p t o p h a n ( l b ) . TLC comparison o f t h e e x t r a c t s o b t a i n e d showed t h a t no v i n c a m i n e ( 92) was p r e s e n t . C o l d d i l u t i o n o f the e x t r a c t w i t h n o n - l a b e l l e d v i n c a m i n e ( 92)^ f o l l o w e d by r e - i s o l a t i o n o f (92 ) , showed no r a d i o a c t i v i t y , f o l l o w i n g r e p e a t e d c r y s t a l l i s a t i o n , thus s t r o n g l y s u g g e s t i n g i t s absence i n the c u l t u r e . However a component w i t h s i m i l a r TLC b e h a v i o u r and r e a c t i o n w i t h s p r a y r e a g e n t t o t h a t 178 d e s c r i b e d f o r v i n c a m i n e ( 92) was i s o l a t e d and shown t o be the l i g n a n , l i r i o r e s i n o l - y ^ (209) . T h i s compound was c h a r a c t e r i s e d by e s t a b l i s h e d s p e c t r o s c o p i c means (NMR, UV, IR, MS). T h i s i s a f a i r d e m o n s t r a t i o n o f the f o l l i e s i n h e r e n t i n TLC comparison d a t a s i n c e a compound can o n l y be a s s i g n e d a s t r u c t u r e when s u f f i c i e n t s p e c t r a l d a t a i s o b t a i n e d . 82 — 83 I n t h i s r e s p e c t Zenk must r e c e i v e c r e d i t f o r b e i n g t he f i r s t t o have broken the b a r r i e r hampering s c i e n t i f i c p r o g r e s s i n c u l t u r e s o f C. rose u s ( i . e . by u s i n g s p e c t r o s c o p i c p r o c e d u r e s ) . O C H 3 O C H 3 2 0 9 -148-I t can o n l y be hoped t h a t o t h e r s , who f e e l t h e y can c o n t r i b u t e i n t h i s a r e a , w i l l a l s o s t r i v e t o meet t h e s e s t a n d a r d s . I n v e s t i g a t i o n towards growth r e q u i r e m e n t s o f C. r o s e u s t i s s u e c u l t u r e s f o r the f o r m a t i o n o f a l k a l o i d s c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( l l ) , v i n c r i s t i n e (201) and v i n b l a s t i n e (202) were p u r -sued s i n c e t h e r e was no c o n c r e t e e v i d e n c e f o r t h e i r f o r m a t i o n i n ,.. ^ 125b,180 the l i t e r a t u r e . I f t h e s e c o u l d be d e v e l o p e d t h e n i n v e s t i g a t i o n s a t t h e c e l l -f r e e l e v e l c o u l d be i n s t i g a t e d p a r t i c u l a r l y w i t h r e s p e c t t o (1) d i m e r i c a l k a l o i d b i o s y n t h e s i s and (2) l a t e r s t a g e s o f c a t h a r a n -t h i n e (12) and v i n d o l i n e (11) b i o s y n t h e s i s . The f o l l o w i n g d i s c u s s i o n p r e s e n t s our s t u d i e s as t h e y r e l a t e t o t h i s o v e r a l l a r e a . I n the f i r s t p a r t o f the d i s c u s s i o n the e x p e r i m e n t s d e s c r i b e d p r o v i d e an e f f i c i e n t approach t o v a r i o u s l y l a b e l l e d forms o f c a t h a r a n t h i n e (12) and i t s d e r i v a t i v e s w h i l e the l a t t e r p a r t i l l u s t r a t e s the i n c o r p o r a t i o n s t u d i e s w i t h such p r e c u r s o r s i n p l a n t s . Some p r e l i m i n a r y e x p e r i m e n t s w i t h t i s s u e c u l t u r e s are a l s o i n d i c a t e d . -149-DISCUSSION - PART I I I A l t h o u g h c a t h a r a n t h i n e - N ^ - o x i d e (205) had n o t been i s o l a t e d from C. r o s e u s i t was c o n s i d e r e d t o be the most l i k e l y p r e c u r s o r f o r the i n v i v o c o u p l i n g w i t h v i n d o l i n e ( 1 1 ) . C o n s e q u e n t l y from a b i o s y n t h e t i c s t a n d p o i n t t h e s t r a t e g y was thus t o s y n t h e s i s e l a b e l l e d forms o f c a t h a r a n t h i n e - N ^ - o x i d e (205) and a d m i n i s t e r t h e s e t o t h e C. r o s e u s p l a n t o r t i s s u e c u l t u r e . F o l l o w i n g t h e e x t r a c t i o n p r o c e d u r e , the a l k a l o i d v i n c r i s t i n e (201), would be i s o l a t e d and c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y . P r o v i d i n g p o s i t i v e i n c o r p o r a t i o n was d e m o n s t r a t e d , t h e d i -m e r i c a l k a l o i d v i n c r i s t i n e (201), would then be s u b j e c t e d t o r e d u c t i v e c l e a v a g e t o g i v e velbanamine (210) and c l e a v a m i n e (211) 181 as the i n d o l e d e r i v a t i v e s . I n t h i s r e s p e c t l o c a l i s a t i o n o f t h e a c t i v i t y i n t h e i n d o l i c p o r t i o n o f the m o l e c u l e would be d e t e r m i n e d e i t h e r by c o u n t i n g and/or f u r t h e r d e g r a d a t i o n . The l a b e l l e d forms o f catharanthine-Nj-,-oxide (205) w h i c h were c o n s i d e r e d are shown. They were: (1) g e n e r a l p r o t o n exchange on the a r o m a t i c r i n g by t r i t i u m . (2) r e p l a c e m e n t o f hydrogens a t t a c h e d t o C-19 by t r i t i u m . (3) r e p l a c e m e n t o f carbon i n e s t e r f u n c t i o n by 1 4 C . I n t h i s r e s p e c t , e i t h e r carbon c o u l d be r e p l a c e d s i n c e O-methyl groups 8 7 i n v a r i o u s f e e d i n g e x p e r i m e n t s had n o t been l o s t . However p r e f e r e n c e was t o have the l a b e l a t t a c h e d t o C-18 removing the p o s s i b i l i t y o f any O-methyl l o s s . - 1 5 0 -2 0 5 Double l a b e l l i n g e x p e r i m e n t s c o u l d be p u r s u e d u t i l i s i n g ( 1 ) and ( 2 ) , ( 1 ) and ( 3 ) , and ( 2 ) and ( 3 ) . I n t h i s manner, the p r e -c u r s o r would be l a b e l l e d n o t o n l y on t h e t r y p t a m i n e ( l a ) p o r -t i o n b u t a l s o on t h e n o n - t r y p t a m i n e p a r t . 2 2 ( 1 ) The p r e p a r a t i o n o f (Ar H) c a t h a r a n t h i n e ( 1 2 ) and (Ar H) c a t h -a r a n t h i n e - N ^ - o x i d e ( 2 0 5 ) w a s a t t e m p t e d by a l l o w i n g ( 1 2 ) and ( 2 0 5 ) 2 t o s t a n d a t room t e m p e r a t u r e i n ( H) t r i f l u o r o a c e t i c a c i d f o r 4 8 h o u r s . I t was n o t e d , h o w e v e r , t h a t no c a t h a r a n t h i n e ( 1 2 ) nor c a t h a r a n t h i n e - N j - j - o x i d e ( 2 0 5 ) remained a f t e r t h i s t i me and i n b o t h c a s e s a p l e t h o r a o f p r o d u c t s formed. F u r t h e r e x p e r i m e n t s w i t h c a t h a r a n t h i n e ( 1 2 ) w i t h t h i s r e a g e n t o v e r 9 6 hours a t - 3 0 ° gave 6 7 % r e c o v e r y o f s t a r t i n g m a t e r i a l b u t < 1% D i n c o r p o r a t i o n . (Table 1 0 ) I t had been, however, n o t e d t h a t a s m a l l q u a n t i t y o f lS^-carbomethoxyclea.vamine ( 7 2 ) was formed i n the f i r s t e x p e r i m e n t w h i c h s u g g e s t e d t h a t f r a g m e n t a -t i o n as shown i n f i g u r e 5 3 had o c c u r r e d . T h i s can be r a t i o n a l -i s e d as p r o c e e d i n g v i a t h e c h a r g e d s p e c i e s ( 2 0 3 ) w h i c h , i n the absence o f h y d r i d e i o n , d i s p r o p o r t i o n a t e d t o g i v e a p y r i d i n i u m -151-7 2 F i g u r e 53 - F o r m a t i o n o f 18-^-carbomethoxycleavamine (72) from c a t h a r a n t h i n e (12) i n t r i f l u o r o a c e t i c a c i d . s p e c i e s and IBp -carbomethoxycleavamine ( 7 2 ) . C o n s e q u e n t l y , o t h e r systems were c o n s i d e r e d f o r the d e u t e r a t i o n s t u d i e s . However t h e s e had t o be r e a d i l y c o n v e r t i b l e t o c a t h a r a n t h i n e -N ^-oxide (205). I n i t i a l l y 1 9 - o x o - c a t h a r a n t h i n e (212) was con-s i d e r e d s i n c e the a b i l i t y t o fragment as i n d i c a t e d i n f i g u r e 53 s h o u l d be d i m i n i s h e d by t h e e l e c t r o n - w i t h d r a w i n g c a p a b i l i t y of the c a r b o n y l group o f t h e l a c t a m . -152-2 1 0 212 211 T h i s l a c t a m (212) was p r e p a r e d a c c o r d i n g t o a p r o c e d u r e 182 u t i l i s e d by B a t t e r s b y e t a l . i n v o l v i n g t r e a t m e n t o f c a t h a r -a n t h i n e (12) w i t h ^/NaHCO^ a t room t e m p e r a t u r e . M e c h a n i s t i -c a l l y t h i s r e a c t i o n can be c o n s i d e r e d t o o c c u r as shown i n F i g u r e 54. T h i s r e a c t i o n was found t o be t e m p e r a t u r e s e n s i t i v e and a t 0° y i e l d e d 5-oxo-18^-carbomethoxycleavamine (213) i n a p p r o x i m a t e l y 25% y i e l d . I t s f o r m a t i o n i s e n v i s a g e d as i n f i g u r e 55. The compound, 5-oxo-18^-carbomethoxycleavamine (213) gave the — 1 18 3 e x p e c t e d (+)15 cm. f r e q u e n c y s h i f t i n the i n f r a r e d f o r an o(,jl-unsaturated l a c t a m . M e c h a n i s t i c a l l y the f o r m a t i o n o f p r o d u c t s (212) and (213) can be c o n s i d e r e d v i a i n i t i a l q u a t e r n a r y s a l t f o r m a t i o n w i t h i o d i n e ( i ) . The f o r m a t i o n o f (212) t h e n i n v o l v e d a b s t r a c t i o n o f hydrogen from C-19 t o g i v e ( i i ) w h i c h t h e n r e a c t e d w i t h hy-d r o x i d e t o g i v e ( i i i ) . S i m i l a r complex f o r m a t i o n ( i v ) , f o l l o w e d by l o s s o f HI gave (v) w h i c h r e a d i l y l o s t a p r o t o n t o g i v e r e q u i r e d (212). The -153-C0 ZCH 3 2 1 2 F i g u r e 54 - P r o p o s e d f o r m a t i o n o f 1 9 - o x o c a t h a r a n t h i n e (212). F i g u r e 55 - P r o p osed f o r m a t i o n o f S - oxo-lS^-carbomethoxycleavamine (213). f o r m a t i o n o f (213) was c o n s i d e r e d t o i n v o k e l o s s o f I v i a a P o l o n o v s k i t y p e r e a c t i o n t o g i v e (203) . F u r t h e r r e a c t i o n w i t h h y d r o x i d e i o n gave ( v i ) w h i c h i n a s i m i l a r manner t o t h a t de--155-s c r i b e d above y i e l d e d ( v i i i ) . R e d u c t i o n o f t h i s t o (213) was c o n s i d e r e d t o be v i a a d i s p r o p o r t i o n a t i o n r e a c t i o n analogous t o t h a t p r e v i o u s l y d e s c r i b e d ( f i g u r e 53) f o r 18/5-carbome thoxy -c l e a v a m i n e (72) v i a i n t e r m e d i a t e (203). Treatment o f 19-oxo-c a t h a r a n t h i n e (212) w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room temp-e r a t u r e f o r 48 hours gave a r e c o v e r y o f <5% a l t h o u g h d e u t e r i u m i n c o r p o r a t i o n was ~ 3 5 % (based on f o u r exchangeable a r o m a t i c p r o -tons ) . D e u t e r a t i o n s t u d i e s were c o n s e q u e n t l y i n i t i a t e d on N -c a r b o m e t h o x y - 1 9 - o x o - c a t h a r a n t h i n e (215) to determine the e x t e n t of d e u t e r i u m i n c o r p o r a t i o n . 184 N - c a r b o m e t h o x y c a t h a r a n t h i n e (214) was p r e p a r e d by t r e a t i n g c a t h a r a n t h i n e (12) w i t h p o t a s s i u m h y d r i d e f o l l o w e d by m e t h y l c h l o r o f o r m a t e . The N a ~ c a r b o m e t h o x y c a t h a r a n t h i n e (214) 2 so formed was t r e a t e d w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room te m p e r a t u r e f o r 60 hours and r e - i s o l a t e d (50% r e c o v e r y ) b u t w i t h no measurable d e u t e r i u m i n c o r p o r a t i o n b e i n g n o t e d . 184 N a - c a r b o m e t h o x y - 1 9 - o x o c a t h a r a n t h i n e (215) was p r e p a r e d by t r e a t m e n t of N a - c a r b o m e t h o x y c a t h a r a n t h i n e (214) w i t h ^/NaHCO^. The N a - c a r b o m e t h o x y - 1 9 - o x o c a t h a r a n t h i n e (215) formed was 2 t r e a t e d w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room temperature f o r 96 hours and r e c o v e r e d i n 63% y i e l d b u t a g a i n w i t h no measurable d e u t e r i u m i n c o r p o r a t i o n . -156-0 H C O C C 0 2 C H 3 C 0 2 C H 3 214 215 Further experiments with catharanthine (12) at reduced temperatures were employed over various time periods and i n th i s respect deuterium incorporation was obtained (see table 10) although, competition from the fragmentation process was s t i l l major product. Highest deuterium incorporation and recovery was obtained in experiment 6. Mass spectral fragmentation of catharanthine correct, deuterium incorporation would a f f e c t peaks at m/e 16 8, 170, 214, and 229 but not at m/e 121 and 135. Table 11 shows the res u l t s i n this respect and i t was noted that peaks 121/122/123 and 135/136 had ra t i o s which were not influenced by the deuterium incorporation. I t should also be noted that the NMR spectrum of deuterated (12) was i d e n t i c a l with the exception that s i m p l i f i c a t i o n of the aromatic region was noted showing the deuterium to be located exclusively i n the aromatic r i n g . important giving 18/3-carbomethoxy cleavamine (72) as the other (12), as postulated 165a , i s shown i n figure 56. If t h i s was -157-N - C H 2 m/e 170 m / e 168 m / e 135 F i g u r e 56 - P r o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . T a b l e 10 - R e a c t i o n s o f c a t h a r a n t h i n e (12) w i t h H and H t r i f l u o r o a c e t i c a c i d . Cath (12) R e l a t i v e r a t i o s o f w t - o f S o l v e n t Temp. t ( h ) Recovery (%) M/M+l E x p t . (12) (mgs) CF 3C0 2H CF. 3C0 2D (x°) (y) Cath (.12) 18 CMC (72) r a t i o M/M+l , M+l/M+2 , M+2/M+3 Volume (ml) 1 30 0. 07 - - 5 24 87 - 4 1 - -2 - 0.14 - 5 72 41 31 0. 89 1 0.92 0. 31 3 0.07 - -15 72 49 - - - - -4 0.07 - -30 96 70 - - - - -5a - 0.07 20 24 18 41 0.95 1 0.85 0.13 5b " 0.07 - 20 24 17 40 - - - -6 " - 0.14 -10 72 55 - 0. 77 0.91 1 0.25 7 - 0.14 -30 96 67 - 4 1 - -8 100 1 - -5 -10 48 50 27 — 1 _ _ I I—1 Ul 00 I -159-The f o r m a t i o n o f 18y# -carbomethoxycleavamine (72) i n th e s e e x p e r i m e n t s was a l m o s t c e r t a i n l y v i a c h a r g e d i n t e r m e d i a t e (203) d i s c u s s e d i n f i g u r e 53 w h i c h was a l s o the p o s t u l a t e d i n t e r m e d i a t e i n the s y n t h e s i s o f the d i m e r i c a l k a l o i d s ( f i g u r e 48-49). To i n v e s t i g a t e t h i s p o s t u l a t e c a t h a r a n t h i n e (12) and v i n d o l i n e (11) were 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 a t (-5)-(-10°) f o r 5 days. Normal b o r o h y d r i d e r e d u c t i o n and work up y i e l d e d 18/3 -carbornethoxy-c l e a v a m i n e ( 7 2 ) , c a t h a r a n t h i n e (12) and v i n d o l i n e ( 1 1 ) . No d i m e r i c a l k a l o i d s were n o t e d thus c a s t i n g doubt on the use o f t h i s s p e c i e s as an i n t e r m e d i a t e i n the c o u p l i n g p r o c e s s . (2) 19-oxocatha.ranthine (212) was p r e p a r e d as p r e v i o u s l y men-t i o n e d . Treatment o f t h i s compound w i t h d i b o r a n e gave c a t h a r a n -185 t h i n e (12) i n 33% y i e l d . C o n s e q u e n t l y t h e employ o f t r i t i a t e d d i b o r a n e would i n t r o d u c e the l a b e l a t the C-19 p o s i t i o n o f c a t h a r a n t h i n e (12). (3) T h i s l e f t t h e pr o b l e m o f d e t e r m i n i n g c o n d i t i o n s f o r l a -b e l l i n g t he carbons o f the e s t e r group o f c a t h a r a n t h i n e (12) a n d j i n t h i s r e s p e c t , c a t h a r a n t h i n e (12) was h y d r o l y s e d t o c a t h a r -186 a n t h i n i c a c i d (216) by base. The r e a c t i o n m i x t u r e was then a c i d i f i e d w i t h a methanol/hydrogen c h l o r i d e s o l u t i o n t o pH 6. Ex-ce s s e t h e r e a l diazomethane was added u n t i l pH 7.5-8. C a t h a r a n -t h i n e (12) was thus r e g e n e r a t e d . T a b l e 11 - Mass s p e c t r a l fragments o f c a t h a r a n t h i n e (12) - d e u t e r i u m e n r i c h m e n t e x p e r i m e n t s R a t i o s o f Peak h e i g h t o f : 121 122 123 135 136 168 169 170 171 214 215 229 230 336 337 1 15.2 : 20 2.2 60 7 40 12 20 : 4.2 20 : 4.2 60 : 11.2 20 4 2 15. 3 : 20 2.4 60 8.2 40 39 .4 20 16.8 20 26.5 60 51 20 22 5 16. 0 20 2.5 60 7.3 40 19 20 11 20 • 12 60 12.4 20 5 6 15. 2 20 : 2.4 60 8 40 : 39.2 20 : 13.4 20 : 27 60 56 20 26 7 15.0 20 : 2.5 60 : 8.2 40 : 11 20 : 4.8 20 : 5.5 60 : 12.4 20 : 5 I H cn o I -161-Thus c o n d i t i o n s f o r l a b e l l i n g t h e e s t e r m e t h y l group were e s t a b l i s h e d . However, as s t a t e d e a r l i e r , i t had been t h e i n t e n -t i o n t o l a b e l the o t h e r c a r b o n atom and l i t e r a t u r e p recedence s t i m u l a t e d t h o u g h t i n t h i s d i r e c t i o n as f o l l o w s : 187 (a) i n t h e s y n t h e s i s o f v o a c a n g i n e (220) from i b o g a i n e (217), c h l o r o i n d o l e n i n e (218) r e a c t e d w i t h c y a n i d e t o g i v e (219) w h i c h on (1) base h y d r o l y s i s and (2) t r e a t m e n t w i t h diazomethane y i e l d e d v o a c a n g i n e (220) ( F i g u r e 57) i n 49% c r u d e y i e l d . t - B u O C I 217 2 1 8 J-tP C 0 2 C H 3 2 2 0 F i g u r e 57 - B u c h i 1 s s y n t h e s i s o f v o a c a n g i n e (220). -162-I n a s i m i l a r , b u t more a p p r o p r i a t e , s y n t h e s i s o f (+) 188 c a t h a r a n t h i n e ( 1 2 ) , s y n t h e t i c h y d r o x y n i t r i l e (221) was c o n v e r t e d t o i t s c o r r e s p o n d i n g c h l o r o i n d o l e n i n e (222) w h i c h r e a c t e d w i t h c y a n i d e t o g i v e cyano d e r i v a t i v e (223). Treatment o f (223) w i t h c o n c e n t r a t e d s u l p h u r i c a c i d gave a crude m i x t u r e o f the n i t r i l e (224) and amide (225) w h i c h on subsequent base h y d r o l y s i s and t r e a t m e n t w i t h diazomethane y i e l d e d (+) c a t h a r a n t h i n e (12) ( f i g u r e 5 8 ) . C o n v e r s i o n o f hydroxy n i t r i l e (223) t o c a t h a r a n t h i n e (12) was r e p o r t e d i n 28% y i e l d . C o n s e q u e n t l y a s y n t h e s i s u s i n g t h i s s t r a t e g y was n e x t con-s i d e r e d , and, i n t h i s r e s p e c t , d e s c a r b o m e t h o x y c a t h a r a n t h i n e (226) was employed as s t a r t i n g m a t e r i a l . I t was hoped t h a t t r e a t m e n t o f t h e c o r r e s p o n d i n g c h l o r o i n d o l e n i n e (227) w i t h c y a n i d e would a f f o r d the n i t r i l e ( 224). H y d r o l y s i s o f t h i s d e r i v a t i v e would h o p e f u l l y r e g e n e r a t e c a t h a r a n t h i n e (12) o r i t s a c i d (216) ( f i g u r e 5 9 ) . The c h l o r o i n d o l e n i n e f o r m a t i o n was f i r s t i n v e s t i g a t e d w i t h 189 c a t h a r a n t h i n e (12) u s i n g a p r o c e d u r e by Lichman i n o r d e r t h a t c o n d i t i o n s f o r i s o l a t i o n c o u l d be d e t e r m i n e d . F o l l o w i n g chromatography, y i e l d s o f 85% f o r (228) were o b t a i n e d . I n a s i m i l a r manner, the c h l o r o i n d o l e n i n e o f descarbomethoxy-c a t h a r a n t h i n e (227) was formed i n 87% y i e l d on t r e a t m e n t o f (226) w i t h N - c h l o r o b e n z o t r i a z o l e . -163-2 2 8 Treatment o f c h l o r o i n d o l e n i n e (227) w i t h p o t a s s i u m c y a n i d e i n d i m e t h y l a c e t a m i d e a f f o r d e d t h e n i t r i l e (224) i n y i e l d s com-p a r a b l e t o t h a t p u b l i s h e d . 1 ^ 7 1 8 8 A c i d c a t a l y s e d h y d r o l y s i s i n methanol a t room and e l e v a t e d t e m p e r a t u r e s f a i l e d t o g i v e any r e a c t i o n i n w h i c h c a t h a r a n t h i n e (12) was formed and, i n one c a s e , the r e a c t i o n was c o n t i n u e d u n t i l a l l s t a r t i n g m a t e r i a l had decomposed. None o f t h e p r o d u c t s n o t e d c o n t a i n e d a c a r b o n y l group. T h i s o b s e r v a t i o n had a l s o been n o t e d i n t h e a t t e m p t e d h y d r o l y s i s o f (219) t o v o a c a n g i n e (220) w i t h hydrogen c h l o r i d e i n methanol and was presumably due t o the f a c t t h a t (219) c o n t a i n s a v e r y h i n d e r e d t e r t i a r y n i t r i l e . Base c a t a l y s e d h y d r o l y s i s w i t h p o t a s s i u m h y d r o x i d e i n d i -e t h y l e n e g l y c o l was t h e n p u r s u e d a t 80, 100, 120, 140, 155 and 160° o v e r p e r i o d s o f 5-16 h o u r s . A c i d i f i c a t i o n a t 0° f o l l o w e d by t r e a t m e n t w i t h diazomethane gave, i n a l l c a s e s , a m u l t i t u d e o f p r o d u c t s b u t none c o r r e s p o n d i n g t o c a t h a r a n t h i n e (12) e x c e p t i n the case o f 155° where a t r a c e o f c a t h a r a n t h i n e (12) may have formed as shown by TLC and mass s p e c t r a l d a t a . Treatment o f t h e n i t r i l e (224) w i t h c o n c e n t r a t e d s u l p h u r i c -164 -165-a c i d , as i n d i c a t e d i n f i g u r e 58 p r o v i d e d an i n f r a r e d s p e c -trum ( t a k e n on the crude m i x t u r e ) p o s s e s s i n g n i t r i l e (2235 cm x ) and amide (1665 cm x ) bands. Subsequent h y d r o l y s i s o f t h i s crude m i x t u r e w i t h p o t a s s i u m h y d r o x i d e i n d i e t h y l e n e g l y c o l , f o l l o w e d by e s t e r i f i c a t i o n as d e s c r i b e d , y i e l d e d no c a t h a r a n t h i n e ( 1 2 ) . F u r t h e r t r e a t m e n t o f c a t h a r a n t h i n i c a c i d (216) w i t h p o t a s s -ium h y d r o x i d e i n d i e t h y l e n e g l y c o l a t 155° f o r 11 hours f o l l o w e d by a c i d i f i c a t i o n and e s t e r i f i c a t i o n a t 0° gave c a t h a r a n t h i n e (12) i n 9% y i e l d . C o n s e q u e n t l y , due t o t h e d i f f i c u l t i e s e n c o u n t e r e d i n t h e s e p r e l i m i n a r y i n v e s t i g a t i o n s , t h i s p a r t i c u l a r avenue o f r e s e a r c h was abandoned as i t was d e c i d e d t h a t t h e m e t h y l d e r i v a t i v e o f c a t h a r a n t h i n e (12) would be employed i n s t e a d . Thus, w i t h t h e a b i l i t y t o l a b e l c a t h a r a n t h i n e (12) as des-c r i b e d , i t was o f some i n t e r e s t t o o b t a i n p r e l i m i n a r y d a t a w i t h 3 (Ar H) c a t h a r a n t h i n e - N ^ - o x i d e (205). C a t h a r a n t h i n e - N b - o x i d e (205) was p r e p a r e d from c a t h a r a n t h i n e (12) by t r e a t m e n t w i t h m e t a c h l o r o p e r b e n z o i c a c i d t o g i v e an i s o l a b l e p ure p r o d u c t . P r e v i o u s a t t e m p t s t o i s o l a t e t h i s com-190 pound i n pure form had met w i t h f a i l u r e , s i n c e i t had been n o t e d t h a t on s t a n d i n g f o r p r o l o n g e d p e r i o d s a t room t e m p e r a t u r e a r e a r r a n g e d p r o d u c t (206) was formed ( f i g u r e 6 0 ) . However, - o x i d e (205) was s t a b l e on s t o r a g e a t 0°. 3 3 (Ar H) c a t h a r a n t h i n e (12) was p r e p a r e d by exchange w i t h ( H) t r i f l u o r o a c e t i c a c i d - t h i s r e a g e n t b e i n g p r e p a r e d by a b u l b - t o -3 b u l b d i s t i l l a t i o n o f t r i f l u o r o a c e t i c a n h y d r i d e i n t o ( H) w a t e r . -166-2 0 5 2 0 6 F i g u r e 60 - P r o p osed rearrangement o f c a t h a r a n t h i n e - N r - o x i d e (205) . b 3 The (Ar H) c a t h a r a n t h i n e (12) o b t a i n e d was c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y and c o n v e r t e d t o the c o r r e s p o n d i n g N^-oxide 3 (205). F o l l o w i n g p u r i f i c a t i o n t e c h n i q u e s , (Ar H) c a t h a r a n t h m e -Nj-,-oxide (205) was a d m i n i s t e r e d t o C. r o s e u s o v e r p e r i o d s o f 3-28 days. To e n s u r e t h a t the p r e c u r s o r was c a t h a r a n t h i n e - N g O x i d e (205) and n o t r e a r r a n g e d p r o d u c t (206) two checks were c a r r i e d out (1) TLC comparison w i t h a sample p r e v i o u s l y s y n t h e s i s e d , a n d 191 (2) r e d u c t i o n w i t h z i n c and a c e t i c a c i d t o r e g e n e r a t e c a t h -a r a n t h i n e (12) . F o l l o w i n g the f e e d i n g p e r i o d , t h e p l a n t was macerated and m ethanol s o l u b l e s removed i n a manner analagous t o t h a t a l r e a d y d e s c r i b e d . I s o l a t i o n o f the crude a l k a l o i d a l f r a c t i o n c o n t a i n i n g VLB (201) and VCR ( 2 0 2 ) , as e v i d e n c e d by TLC c o m p a r i s o n d a t a , 192 was e x e c u t e d u s i n g a g r a d i e n t pH e x t r a c t i o n p r o c e d u r e . I n a c t i v e v i n c r i s t i n e (201) was t h e n added t o t h e e x t r a c t , r e - i s o l a t e d , and c r y s t a l l i s e d t o g i v e d a t a shown i n t a b l e 13. T a b l e 13 - I n c o r p o r a t i o n d a t a from a d m i n i s t r a t i o n o f (Ar H) c a t h a r a n t h i n e - N b ~ o x i d e (205) t o r o s e u s . A c t i v i t y a d m i n i s t e r e d (cpm/mmole) D u r a t i o n o f f e e d i n g (days) S p e c i f i c A c t i v i t y I s o l a t e d Wt. o f VCR (108) i s o l a t e d (mgs) S p e c i f i c A b s o l u t e I n c o r p o r a t i o n I n c o r p o r a t i o n 1.184 x 10" 1.184 x 10-1.184 x 10-1.184 x 10" 1. 594 x 10-3 6 12 21 28 2.26 x 10 1.70 x 10 2.64 x 10 2.03 x 10' 2.75 x 10 15 14 19 16 22 0.0044 0.0031 0.0065 0.0042 0.0078 0.00035 0. 00026 0. 00041 0.00032 0.00043 i i—• l -168-C o u n t i n g was d i s c o n t i n u e d when b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . F u r t h e r s t u d i e s were t h e r e f o r e n o t p u r s u e d on the i n t a c t p l a n t , a n d , i n s t e a d , work was i n i t i a t e d on C. r o s e u s t i s s u e c u l t u r e s t o d e t e r m i n e i f i t was p o s s i b l e t o f i n d c o n d i t i o n s f o r the f o r m a t i o n o f t h e a l k a l o i d s ( 1 1 ) , ( 1 2 ) , (201) and (202). C o n s e q u e n t l y t i s s u e c u l t u r e s o f C. r o s e u s grown under a v a r i e t y o f c o n d i t i o n s were i n v e s t i g a t e d f o r t h e i r a l k a l o i d a l c o n t e n t . The c u l t u r e s were grown a t the U n i v e r s i t i e s o f B r i t i s h Columbia (UBC) and L e i c e s t e r (England) r e s p e c t i v e l y . The c a l l u s c u l t u r e s a t U.B.C. were h a r v e s t e d a f t e r 3 weeks-4 months growth p e r i o d s . These were removed from t h e s o l i d agar medium ( t a b l e 8) and f r e e z e d r i e d . The c u l t u r e s from t h e U n i v e r s i t y of L e i c e s t e r were p a r t o f 180 a c o l l a b o r a t i v e p r o j e c t . C o n d i t i o n s f o r t h e i r growth have no t y e t been r e l e a s e d b u t f r e e z e d r i e d samples were g i v e n f o r our i n v e s t i g a t i o n s . A l l c u l t u r e s were e x t r a c t e d w i t h m ethanol t o g i v e , a f t e r r e m o v a l o f s o l v e n t , the crude " a l k a l o i d a l " e x t r a c t . The e x t r a c t was t h e n t r e a t e d w i t h ' V HC1 t o remove any a l k a l o i d s as t h e i r h y d r o c h l o r i d e s a l t s . The r e s u l t i n g a c i d i c s o l u b l e s were e x t r a c t e d w i t h benzene. o The aqueous l a y e r was t h e n c o o l e d t o 2 and b a s i f i e d w i t h ammonium h y d r o x i d e t o pH 7. The c h l o r o f o r m s o l u b l e s were t h e n removed. F u r t h e r t r e a t m e n t o f the aqueous l a y e r t o pH 10 was then c a r r i e d o u t and t h e c h l o r o f o r m s o l u b l e s a g a i n removed. -169-Chromatography o f the r e s i d u e s o b t a i n e d from the pH 1,7 and 10 e x t r a c t i o n s were found t o be d e v o i d o f the r e q u i r e d a l k a l o i d s ( 1 1 ) , ( 1 2 ) , (201) o r (202). Indeed no a l k a l o i d s were noted i n any o f the c u l t u r e s i n v e s t i g a t e d . F u r t h e r s t u d i e s w i t h t i s s u e c u l t u r e s ( s o l i d and l i q u i d ) a r e c u r r e n t l y under i n v e s t i g a t i o n as p a r t o f a l o n g term p r o j e c t t o o b t a i n c o n t r o l l e d c o n d i t i o n s f o r a l k a l o i d a l growth i n these l a b o r a t o r i e s . T h i s , and attempts t o i s o l a t e the enzymes from C. r o s e u s , are c u r r e n t l y b e i n g s t u d i e d by P r o f e s s o r S t u a r t . When c o r r e c t growth c o n d i t i o n s a re de t e r m i n e d the r o l e o f c a t h a r -a n t h i n e - N - o x i d e (205) can be r e i n v e s t i g a t e d . F o l l o w i n g the e x p e r i m e n t a l s e c t i o n , s y n t h e t i c s t u d i e s towards the s y n t h e s i s of a l k a l o i d s e l l i p t i c i n e (106) and o l e -v a c i n e (105) are r e p o r t e d . -170-E X P E R I M E N T A L - PART I I I A description of the general experimental information was given in Experimental, parts I and I I . As before procedures, published or unpublished by others, are described in f u l l when (1) detailed experimental was not released (2) unsatisfactory data was presented or (3) procedure was modified. 182 19-0xocatharanthine (212) and 5-oxo-18yg carbomethoxy- cleavamine (213) Sodium bicarbonate (65 mg, 0.774 m.mol) i n d i s -t i l l e d water (1 ml) was added to catharanthine (12) (45 mg, 0.134 m.mol) i n tetrahydrofuran (THF) (1.2ml) at room tem-perature. Iodine (59 mg, 0.232 m.mol) i n THF (1 ml) was added over a oeriod of 8 min and afte r an additional 30 min, the solution was dil u t e d with water (20 ml) and extracted with chloroform (4x15 ml). The chloroform layers were combined and washed with (1) 2 x 10 ml 0.1 M sodium thiosulphate (2) 10 ml water (3) 10 ml 2 N H 2S0 4 and (4) 10 ml H 20. Chromatography of the dried chloroform extract on s i l i c a gel gave 19-oxocatharanthine (212) (10.4 mg, 22%) and the lactam (213) (2.9 mg, 6 %) . 19-0xocatharanthine (212) was compared with an authentic 184 sample prepared v i a a longer pathway and was i d e n t i c a l i n a l l respects. 19-Oxocatharanthine (212) I.R.: 0 _„_. 3460,3000,2980-2960,2880,1725,1670(sh),1655,1520, U l L l 3 -171-1275-50 cm U.V.: A m J ° H 222, 276 (3.919), 283 (3.953), 292 (3.896) nm. NMR: c f (CDC1 3) 100 MHz (FT) 8.02 ( s , IH, N-H) , 7.51-7.34 (m, IH, arom.), 7.22-7.05 (m, 3H, arom.), 6.12 (dd, IH, J=2 and 4 Hz, C-3 H), 5.05 (d, IH, J=2 Hz, C-5 H), 4.26-3.94 (complex m, IH, C-2 H) , 3.64 ( s , 3H, C0 2CH_ 3) , 3.42-3.08 (complex m, 4H, C 7, C 8, U2)f 2.58 (dd, IH, J=2 and 12 Hz, C - l H), 2.34-2.14 (complex m, 3H, CH 2CH 3 + C 1 H) , 1.05 ( t , 3H, J=7 Hz, CH2CH_3) . M.S. m/e: 350 (M +) 228, 227, 195, 168, 167, 154. H i g h r e s o l u t i o n mass spectrum: c a l c ^ f o r C2iH22 N2°3 = 350•1630 Found=350.1627. 5-Oxo - 1 8 y g-carbomethoxycleavamlne (213) and 1 9 - o x o c a t h a r a n t h i n e (212) C a t h a r a n t h i n e (12) (90 mg, 0.268 mmol) i n THF (2.6 ml) was added t o a s o l u t i o n o f sodium b i c a r b o n a t e (130 mg, 1.548 mmol) i n d i s t i l l e d w a t e r (2 m l ) . The t e m p e r a t u r e was low-e r e d t o 0° and i o d i n e (108 mg, 0.425 mmol) i n THF (2 ml) was added o v e r a t h r e e h r p e r i o d . The s o l u t i o n was d i l u t e d w i t h w a t e r (20 ml) and e x t r a c t e d w i t h methylene c h l o r i d e ( 3x15 m l ) . The methylene c h l o r i d e l a y e r s were combined and washed w i t h (1) 2 x 10 ml 0.1 M sodium t h i o s u l p h a t e (2) 2 x 10 ml 2 N H 2 S 0 4 (3) 2 x 10 ml w a t e r . Chromatography o f the methylene c h l o r i d e s o l u b l e s on s i l i c a g e l y i e l d e d the l a c t a m (213) (20.2 mg, 20%) and the l a c t a m (212) (6 mg, 6%). 5 - 0 x 0 - 1 8 ^ carbomethoxycleavamine (213) I.R.: 0 CHC1 3 3460, 2960-40, 2870-50, 1745 ( s h ) , 1720, 1675, 1460 c m - 1 . -172-U.V.: X E t 0 H 223 ( 4 . 4 2 ) , 276 ( 3 . 8 1 ) , 283 ( 3 . 8 3 ) , 291 (3.77) nm. ITlclX NMR: 100 MHz £(CDC1 3) (FT) 8.20 ( s , IH, N-H), 7.64-7.10 (com-p l e x m, 4H, arom.) 6.55 (d, IH, J=8 Hz, C-3 H), 3.80 (s, 3H, C 0 2 C H 3 ) , 2.18 (q, 2H, J=7 Hz, C H 2 C H 3 ) , 1.00 ( t , 3H, J=7 Hz, C H 2CH 3). M.S. m/e: 352 ( M + ) , 336, 293, 291, 264, 263, 229, 228, 227, 214. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2^H 2 4N 20 3=352.1787 Found=352.1787. ( 2H) T r i f l u o r o a c e t i c a c i d ( 2H) Water (99.95%D) (0.2 m l , 10 mmol) was added t o r e d i s t i l l e d t r i f l u o r o a c e t i c a n h y d r i d e (2.75 m l , 10.5 mmol) a t -78°. The t e m p e r a t u r e was a l l o w e d t o r i s e s l o w l y t o room temp-2 e r a t u r e o v e r 15 min g i v i n g ( H) t r i f l u o r o a c e t i c a c i d . ( A r 2 H ) - 1 9 - O x o c a t h a r a n t h i n e (212) - G e n e r a l d e u t e r a t i o n p r o c e d u r e 1 9 - O x o c a t h a r a n t h i n e (212) (20 mg, 0.0568 mmol) was d i s s o l v e d i n ( 2H) t r i f l u o r o a c e t i c a c i d (1 m l , 13.35 mmol) a t room t e m p e r a t u r e . A f t e r 60 h r , t h e t e m p e r a t u r e was c o o l e d t o 0° and s a t u r a t e d sodium b i c a r b o n a t e added t o pH 9. E x t r a c t i o n w i t h methylene c h l o r i d e ( 3 x 5 ml) gave, a f t e r d r y i n g and chroma-2 t o g r a p h y on s i l i c a g e l , (Ar H) 1 9 - O x o c a t h a r a n t h i n e (212) (1 mg, 5%) . "I p A N ^ - C a r b o m e t h o x y c a t h a r a n t h i n e (214) P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e and had a l m o s t i d e n t i c a l p r o p e r t i e s w i t h t h a t r e p o r t e d . ( Y i e l d = 7 7 % , L i t . y i e l d = 8 3 % ) . -173-Attempted deuteration of UR-Carbometho'xycatharanthine (214) (2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol) was added to N a-carbomethoxycatharanthine (214) (30 mg, 0.076 mmol). Following incubation for (1) 24 (2) 60 hr , N -carbomethoxycatharanthine (214) was i s o l a t e d (according to gen-e r a l procedure described) i n (1) 70 and (2) 50% y i e l d respectively. N.M.R. and M.S. showed no measurable deuterium incorporation. 184 N a-Carbomethoxy-19-oxocatharanthine (215) Prepared according to l i t e r a t u r e procedure and had almost i d e n t i c a l properties with that reported by the second procedure quoted. Attempted deuteration of N a-carbomethoxy-19-oxocatharanthine (215) (2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol) was added to N a-Carbomethoxy-19-oxocatharanthine (215) (30 mg, 0.074 mmol). After incubation for 96 hr, i s o l a t i o n , according to general procedure described, gave (215) (19.5 mg, 65% recovery). N.M.R. and M.S. showed no measurable deuterium incorporation. Catharanthine (12) and 18 /3-carbomethoxycleavamine (72) (XH) or( 2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol), was added to catharanthine (12) (30 mg, 0.089 mmol) at x°C as described i n Table 10. After y hours, saturated sodium bicarbonate solution was added to pH 9. The mixture was extracted with methylene chloride ( 4 x 4 ml) which, a f t e r drying and chroma-tography on s i l i c a gel yielded catharanthine (12) and 18 j6 -car-borne thoxycleavamine (72). -174-(Ar 1!!) C a t h a r a n t h i n e (12) had i d e n t i c a l p r o p e r t i e s t o t h a t of s t a r t i n g m a t e r i a l . (Ar^-H) 1 8 / 3 - c a r b o m e t h o x y c l e a v a m i n e (72) was i d e n t i c a l t o an a u t h e n t i c sample e.g. TLC, NMR, UV, IR, M.S., m.pt. (118-120° , l i t . m.pt. = 121-23° ). At t e m p t e d c o u p l i n g between c a t h a r a n t h i n e (12) and v i n d o l i n e (11) C a t h a r a n t h i n e (12) (100 mg, 0.3 mmol) and v i n d o -l i n e (11) (120 mg, 0.27 mmol) were 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 (1 m l , 13.28 mmol) a t -78°. The te m p e r a t u r e was r a i s e d t o -5-fr-10°. A f t e r s i x days, t h e te m p e r a t u r e was a g a i n r e d u c e d t o -78° and sodium b o r o h y d r i d e i n methanol was added u n t i l pH 8. Water (5 ml) was added and the aqueous l a y e r e x t r a c t e d w i t h methy-l e n e c h l o r i d e ( 4 x 5 m l ) . Chromatography o f the methylene c h l o r i d e s o l u b l e s gave (a) 18^0 -carbomethoxycleavamine (72) (82 mg,82%)(b) c a t h a r a n t h i n e (12) ( 5 mg, 5 %) and (c) v i n d o l i n e (116 mg, 9 6 % ) . 157 C a t h a r a n t h i n i c a c i d (216) C a t h a r a n t h i n e (12) (200 mg, 0.595 mmol) was added t o a s o l u t i o n o f aqueous sodium h y d r o x i d e (30 mg, 12% w/w) and e t h a n o l (30 m l ) . R e f l u x was m a i n t a i n e d f o r 3.5 h r a f t e r w h i c h the t e m p e r a t u r e was re d u c e d t o 0°. A m b e r l i t e a c i d i c r e s i n IR-124 was added u n t i l s o l u t i o n pH ~ 7 . The r e s i n was f i l t e r e d , and washed w i t h d i c h l o r o m e t h a n e . The s o l v e n t s were e v a p o r a t e d t o d r y n e s s and the r e s u l t a n t s o l i d t r i t u r a t e d w i t h c h l o r o f o r m . Chromatography on s i l i c a g e l y i e l d e d c a t h a r a n t h i n i c a c i d (216) (153 mg, 80%) m.pt. = 189°. -175-I.R.: 0 KBr 3400, 3020 (sh), 2940, 2620-2240, 1700 (sh),1600 cm max U.V.:/) m a x H 224 (4.495), 275 (3.823), 282 (3.852), 291 (3.788) nm. NMR: S (CDC13) 100 MHz (FT) 7.47-6. 93 (m, 411, arom.), 6.17-5.97 (d, 111, J=5 Hz, C-3 H) , 4.49 (s, IH, C-5 H) , 1.19-0.95 ( t , 3H, J=7Hz, CH 2CH 3). M.S. m/e: 322 (M +), 278, 135, 122, 121. High r e s o l u t i o n mass spectrum: C a l c — f o r C 2 Q H ^02=322.1681 Found=322.1670. Elemental a n a l y s i s f o r C20 H22 W2°2" ^ * 5 H 2 0 : F o u n d c ' 72.73, H, 6.90, N, 8.43 C a l c - , C, 72.50, H, 6.95, N, 8.64 Catharanthine (12) C a t h a r a n t h i n i c a c i d (216) (20 mg, 0.062 mmol) was d i s s o l v e d i n 20% KOH i n d i e t h y l e n e g l y c o l (0.4 m l ) . The mixture was then cooled to 0° and a c i d i f i e d w i t h 1.5% HC1 i n methanol u n t i l pH 6.5. An excess e t h e r e a l s o l u t i o n of d i a z o -methane was added u n t i l s l i g h t l y a l k a l i n e . S t i r r i n g was c o n t i n -ued f o r 30 min and s a t u r a t e d sodium bicarbonate (5 ml) was added. E x t r a c t i o n of the r e s u l t i n g mixture w i t h dichloromethane ( 4 x 5 ml) y i e l d e d , a f t e r chromatography on s i l i c a g e l , catharan-t h i n e (12) (17 mg, 82%) i d e n t i c a l i n a l l respects w i t h an a u t h e n t i c sample. 165a,186 Descarbomethoxycatharanthine (226) Catharanthine (12) (810 mg, 2.41 mmol) was d i s s o l v e d i n a s o l u t i o n of aqueous sodium hydroxide (40 ml, 12% w/w) and eth-anol (40 ml). The s o l u t i o n was heated at r e f l u x f o r 3.5 hr. Concen-t r a t e d h y d r o c h l o r i c a c i d was slowly added, (the temperature being _ i -176-m a i n t a i n e d a t a p p r o x i m a t e l y 65° d u r i n g a d d i t i o n ) , u n t i l pH 1. The r e a c t i o n m i x t u r e was c o o l e d t o r . t . and e x t r a c t e d w i t h c h l o r o f o r m (4 x 40 m l ) . The c h l o r o f o r m e x t r a c t s were com-b i n e d , d r i e d , and chromatographed on s i l i c a g e l y i e l d i n g des-c a r b o m e t h o x y c a t h a r a n t h i n e (226) (480 mg, 7 2 % ) . m.pt.: 92° ( l i t m . p t . 91-92°) 1 6 5 a I.R.: 0 CHC1 3 3480, 2940, 2890, 1470 cm" 1. U.V.: y\ E t 0 H 226, 275 (3,809), 283 (3.869) , 290 (3.848) nm. NMR: £(CDC1 3) 100 MHz (FT) 7.74 (s , IH, NH) , 7.58-7.4 (m, IH, arom.), 7.34-7.00 (m, 3H, arom.),5.90 (dd, IH, J=2 and 6 Hz,C-3 H), 3.54 (s, IH, C-5 H) , 2. 42-2.08 (complex m, 2H, J=8 Hz, CH_2CH3) , 2.08-1.82 (complex m, IH, C - l H), 1.78-1.52 (m, IH, C - l H ) , 1.08 ( t , 3H, J=8 Hz, C H 2CH 3). M.S. m/e: 278 ( M + ) , 171, 170, 135, 122. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2 2N 2=278.1782 Found=278.1774. E l e m e n t a l A n a l y s i s : Found:C, 81.79, H, 8.10, N 9.97. C a l c -C, 82.01, H, 7.91, N, 10.07. 189 C h l o r o i n d o l e n i n e from c a t h a r a n t h i n e (228) N - C h l o r o b e n z o t r i a z o l e (17.2 mg, 0.112 mmol) i n dr y benzene (1.2 ml) was added d r o p w i s e t o a s o l u t i o n o f c a t h a r a n -t h i n e (12) (30 mg, 0.089 mmol) i n d r y benzene (1.5 m l ) . A f t e r 30 min , the s o l u t i o n was e x t r a c t e d w i t h 10% sodium b i c a r b o n a t e s o l u t i o n ( 2 x 3 m l ) . The b a s i c e x t r a c t was backwashed w i t h ben-zene ( 4 x 4 m l ) . The combined benzene s o l u b l e s were chromato-graphed on s i l i c a g e l y i e l d i n g (228) (28 mg, 8 5 % ) . -177-I.R.: 0 CHC1 2878, 1737, 1551 cm X" max 3 U.V. : ^l°E 276, 228 nm. NMR: S(CDC1 3) 100 MKz (FT) 7 .56-7 .42 (m, IH, arom.), 7.28-6.86 (m, 3H, arom.), 5.78 (dd, IH, J=2 and 7 Hz, C-3 H) , 4.9 (d, IH, J=2 Hz, C-5 H), 4.96-3.68 ( f i v e l i n e m u l t i p l e t , I H ) , 3.42 ( d t , IH, J=3 and 16 H z ) , 2.80 ( d t , IH, J=3 and 16 H z ) , 2.62 ( d t , 2H, J=3 and 8 Hz , C 7 & C 8H 2) , 3.32 ( s , IH, C0 2CH_ 3), 2.40-1.98 (m, 2H, CH CH ) , 1.82-1.54 (m, 2H, C1 B_2) , 1.04 ( t , 3H, J=7.5 Hz, CH 2CH 3) . M.S. m/e: 372 ( M + ) , 371, 370 ( M + ) , 369, 335, 228, 121. d 35 Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 ^ H 2 3 C1N 20 2= 370.1447. Found=370.1456. C h l o r o i n d o l e n i n e from d e s c a r b o m e t h o x y c a t h a r a n t h i n e (22 7) N - C h l o r o b e n z o t r i a z o l e (44.4 mg, 0.289 mmol) i n dry benzene (2 ml) was added dropwise t o d e s c a r b o m e t h o x y c a t h a r a n t h i n e (226) (80 mg, 0.288 mmol) i n benzene (3 m l ) . A f t e r 30 m i n , the s o l u t i o n was e x t r a c t e d w i t h 10% sodium b i c a b o n a t e s o l u t i o n ( 2 x 4 m l ) . The r e s u l t i n g aqueous e x t r a c t was backwashed w i t h benzene ( 4 x 4 m l ) . The benzene s o l u b l e s were chromatographed on s i l i c a g e l y i e l d i n g c h l o r o i n d o l e n i n e (227) (78 mg, 87%). I.R.: 0 CHC1 3460 (sh) , 2960, 2880, 1520 cm" 1 U.V.: A S a x " 3 2 7 6 < 3 ' 8 8 ) ' 227 ( 4. 42) NMR: £(CDC1 3) l o o MHz (FT) 7.66 (m, IH, arom.), 7.32-6.86 (m, 3H, arom.), 5.70 (d, IH, J=7 Hz, C 3 H), 4.4 ( s , IH, C-5 H), 2.36-1.96 (complex m u l t i p l e t , 2H, CH 2CH 3, 0.98 ( t , 3H, J=7 Hz, -178-CH 2CH 3) M.S. m/e: 314 ( M + ) , 312 ( M + ) , 278, 277, 170, 135, 121. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2^N 2Cl=312.1427. Found=312.1410. 18-De s c a r b o m e t h o x y - 1 8 - c y a n o c a t h a r a n t h i n e (224) P o t a s s i u m c y a n i d e (104 mg, 1.6 mmol) was added t o a s o l u t i o n o f c h l o r o i n d o l e n i n e (227) (50 mg, 0.159 mmol) i n anhydrous d i m e t h y l acetamide (3 m l ) . A f t e r 30 h r a t room t e m p e r a t u r e , s a t u r a t e d sodium b i c a r b o n a t e (13 ml) was added. E x t r a c t i o n w i t h methylene c h l o r i d e (5 x 15 m l ) , f o l l o w e d by d r y i n g and removal o f a l l s o l v e n t i n vacuo gave a gum w h i c h was chromatographed on s i l i c a g e l y i e l d i n g n i t r i l e (224) (11 mg, 2 3 % ) . 1. R.: [)„ (CHC1.J 3740, 2940, 2880, 2230, 1450 cm" 1. u - v - : / J m a x H 2 2 2 ( 4- 4 7>' 2 7 5 ( 3 . 8 6 ) , 280 (3 . 8 9 ) , 288 (3.86) nm. NMR: £ (CDC1 3) 100 MHz (FT) 7.88 ( s , IH, NH), 7.58-7.46 (m, IH, arom.), 7.44-7.10 (m, 3H, arom.), 5.94 (dd, J=2 and 6 Hz, C-3 H), 2. 82-1. 88 (complex e n v e l o p e , 2H, CH_2CH3) , 1.08 (3H, J=7 Hz, CH 2CH 3). M.S. m/e: 303 ( M + ) , 160, 122. Hi g h r e s o l u t i o n mass spectrum; C a l c — f o r C 2gH 2^N 3=303.1735. Found=303.1731. C a t h a r a n t h i n e (12) To c a t h a r a n t h i n i c a c i d (216) (36 mg, 0.11 mmol) was added 20% KOH i n d i e t h y l e n e g l y c o l (0.75 ml) and t h e tempera-t u r e was r a i s e d t o 155° . A f t e r 11 h r the te m p e r a t u r e was lo w e r e d t o 0° and 1.5% HC1 i n methanol added u n t i l pH 6.5. The -179-s o l u t i o n was t r e a t e d w i t h e x c e s s e t h e r e a l diazomethane u n t i l s l i g h t -l y a l k a l i n e . S t i r r i n g was c o n t i n u e d f o r 30 min and s a t u r a t e d sodium b i c a r b o n a t e (10 ml) was added u n t i l pH 9. E x t r a c t i o n o f the r e s u l t i n g s o l u t i o n w i t h methylene c h l o r i d e (5 x 10 ml) gave, a f t e r d r y i n g and chromatography on s i l i c a g e l , c a t h a r a n t h i n e (12) (3.6 mg, 10%) . C a t h a r a n t h i n e - N h - o x i d e (205) M e t a - c h l o r o p e r b e n z o i c a c i d (157 mg, 0.91 mmol) was added t o a s o l u t i o n o f c a t h a r a n t h i n e (12) (300 mg, 0.893 mmol) i n methylene c h l o r i d e a t 0°. A f t e r 10 min' t h e s o l v e n t was removed by vacuum t r a n s f e r . The r e s i d u e was chromatographed on a l u m i n a (Act I I I ) y i e l d i n g N b - o x i d e (205) (220 mg, 7 0 % ) . I.R.:^max 3460, 2960, 1730, 1740 c m - 1 . U.V.: y\ E a ° H 291 (3. 8 ) , 283 ( 3 . 9 ) , 2. 75 ( s h , 3. 8 ) , 224 ( 4 . 5 ) . NMR: 5(CDC1 3) 100 MHz (FT) 8.37 (b. s. , IH, NH) , 7.62-7.42 (m, IH, arom.), 7.4-7.05 (m, 3H, arom.), 6.14 (d, J=5.6 Hz, H), 4.72 ( s , IH, C 5 E), 3.74 ( s , 3H, C 0 2 C H 3 ) , 2.65-2.05 ( e n v e l o p e , 2H, CH 2CH^) , 1.12 ( t , J=7.5 Hz, 3H, CH2CH_3) . M.S. m/e: 352 (M +) 336, 254, 248, 222, 218, 144, 135, 130. C a t h a r a n t h i n e (12) A c t i v a t e d z i n c d u s t (40 mg, 0.61 mmol) was added t o a s o l u t i o n o f c a t h a r a n t h i n e - N D - o x i d e (205) (40 mg, 0.1136 mmol) i n a c e t i c a c i d (1.5 ml) a t 20° . A f t e r 30 min, the z i n c d u s t was removed by f i l t r a t i o n and the s o l u t i o n b a s i f i e d w i t h s a t u r a t e d sodium b i c a r b o n a t e a t 0° . E x t r a c t i o n w i t h d i c h l o r o -methane ( 4 x 5 m l ) , gave a f t e r d r y i n g and chromatography on s i l i c a -180-g e l , c a t h a r a n t h i n e (12) (25 mg, 66%) i d e n t i c a l i n a l l r e s p e c t s t o an a u t h e n t i c sample. 3 (Ar H) C a t h a r a n t h i n e (12) T r i t i a t e d t r i f l u o r o a c e t i c a c i d was p r e p a r e d by -2 d i s t i l l a t i o n o f t r i f l u o r o a c e t i c a n h y d r i d e (2.71 m l , 1.86 x 10 mol) 3 -3 xnto ( H) w a t e r (0.3 ml, 1.86 x 10 mol, 500 m C i ) u s i n g a vacuum 3 t r a n s f e r system. The r e s u l t i n g ( H) t 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 t o a f l a s k c o n t a i n i n g c a t h a r a n t h i n e (12) (220 mg, 0.65 mmol) a t -78° by vacuum d i s t i l l a t i o n . The r e s u l t i n g a c i d i c s o l u -t i o n was warmed t o a p p r o x i m a t e l y -5° , t h i s b e i n g m a i n t a i n e d f o r 48 h r . Exces s ( H) t r i f l u o r o a c e t i c a c i d was then removed by vacuum t r a n s f e r . M e t h a n o l ( 3 x 2 ml) was added t o the r e s u l t i n g r e s i d u e and removed by vacuum t r a n s f e r t o g e t h e r w i t h any exchange-a b l e t r i t i u m . S a t u r a t e d aqueous sodium b i c a r b o n a t e was added a t 0 ° u n t i l ph 8 . 5 . E x t r a c t i o n w i t h d i c h l o r o m e t h a n e f o l l o w e d by d r y i n g i n the u s u a l manner gave a crude foam w h i c h was c r y s t a l l i s e d w i t h methanol t o y i e l d (Ar 3H) c a t h a r a n t h i n e (12) 118 mg, 5 4 % ) . Chromatography on s i l i c a g e l (twice) and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y y i e l d e d (Ar^H) c a t h a r a n t h i n e (12) (6.389 x 10 dpm/mmol). 3 (Ar H) C a t h a r a n t h i n e - N b - o x i d e (205) M e t a c h l o r o p e r b e n z o i c a c i d (34 mg, 0.197 mmol) i n 3 methylene c h l o r i d e (1 ml) was added t o a s o l u t i o n o f (Ar H) c a t h a r -a n t h i n e (12) (60 mg, 0.178 mmol) i n methylene c h l o r i d e (4 ml) a t 0°. S t i r r i n g was c o n t i n u e d f o r 15 min and the s o l u t i o n d i l u t e d w i t h e t h y l a c e t a t e (25 m l ) . The o r g a n i c l a y e r was then -181-e x t r a c t e d w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n ( 2 x 5 ml) . D r y i n g and e v a p o r a t i o n o f s o l v e n t i n the u s u a l manner a f f o r d e d a w h i t e foam w h i c h was chromatographed w i t h v a r i o u s s o l v e n t systems on s i l i c a g e l u n t i l c o n s t a n t a c t i v i t y t o g i v e (Ar 3H) c a t h a r a n t h i n e - N b - o x i d e (205) (15 mg, 2 5 % ) . (6.485 x 1 0 9 dpm/mmole). 3 A d m i n i s t r a t i o n o f (Ar H) C a t h a r a n t h i n e - N b - o x i d e (205) t o  C. r o s e u s 3 (Ar H) c a t h a r a n t h i n e - N b - o x i d e (205) (1.594 mg, 9 6.4 85 x 10 dpm/mmol ) was a d m i n i s t e r e d t o a C. r o s e u s p l a n t (68 g wet w e i g h t ) i n a methanol/water s o l u t i o n (4 m l , 1:1) by the c o t t o n w i c k method. A f t e r 28 days t h e p l a n t was macerated and methanol s o l u b l e s removed. The e x t r a c t c o r r e s p o n d o n g t o v i n c r i s t i n e (10 8) was 156 i s o l a t e d a c c o r d i n g t o Svoboda's g r a d i e n t pH t e c h n i q u e . I n t h i s way, the methanol s o l u b l e s were e v a p o r a t e d g i v i n g a r e s i -due w h i c h was suspended i n 2% t a r t a r i c a c i d s o l u t i o n (350 m l ) . A f t e r a g i t a t i o n f o r 45 min, t h e a c i d i c s o l u t i o n was e x t r a c t e d w i t h hexane (3 x 250 m l ) . F u r t h e r e x t r a c t i o n o f t h e a c i d s o l u t i o n w i t h benzene (4 x 250 ml) gave benzene s o l u b l e s and aqueous e x t r a c t ( 1 ) . The benzene s o l u b l e s were d r i e d and e v a p o r a t e d and the r e s i d u e was d i s s o l v e d i n 2% t a r t a r i c a c i d s o l u t i o n (150 m l ) . The a c i d i c l a y e r was e x t r a c t e d w i t h d i c h l o r o m e t h a n e t o g i v e e x t r a c t B ( a f t e r removal o f t h e s o l v e n t i n the u s u a l manner.) The r e -s u l t i n g aqueous l a y e r (2) was then b a s i f i e d w i t h 5% ammonium hy-d r o x i d e s o l u t i o n a t 5° u n t i l pH 8.5. E x t r a c t i o n w i t h methylene -182-c h l o r i d e (3 x 150 ml) gave, a f t e r d r y i n g , e x t r a c t C. (The aqueous l a y e r was then d i s c a r d e d ) . The o r i g i n a l aqueous e x t r a c t (1) was s i m i l a r i l y b a s i f i e d t o pH 8.5 and removal o f methylene c h l o r i d e s o l u b l e s (3 x 200 ml) gave, a f t e r d r y i n g , e x t r a c t A. ( o r g a n i c s o l u b l e s ) E x t r a c t C was d i l u t e d w i t h v i n c r i s t i n e (201) (20 mg) and t h i s was r e - i s o l a t e d a f t e r t h i n l a y e r chromatography (X2). The v i n c r i s t i n e (201) was t h e n c r y s t a l l i s e d t o l e v e l s shown i n t a b l e 12. ( I t s h o u l d be n o t e d t h a t , i n t h i s e x p e r i m e n t , e x t r a c t (A) and (B) were a l s o d i l u t e d w i t h VCR - w i t h no s i g n i f i c a n t a c t i v i t y b e i n g noted.) V i n c r i s t i n e ( 2 0 1 ) , r e i s o l a t e d , was checked by TLC, NMR, IR, M.S. and m.pt. w i t h an a u t h e n t i c sample. INTRODUCTION - PART IV S t u d i e s r e l a t e d t o the s y n t h e s e s o f a n t i - t u m o u r p y r i d o - c a r b a - z o l e a l k a l o i d s The p y r i d o - c a r b a z o l e a l k a l o i d s o l i v a c i n e (105), guatambuine (104) and e l l i p t i c i n e (106), d i s c u s s e d p r e v i o u s l y from a b i o s y n t h e t i c s t a n d p o i n t , have a l s o r e c e i v e d c o n s i d e r a b l e s y n t h e t i c a t t e n t i o n 193 and s e v e r a l approaches g i v i n g o l i v a c i n e (105) and e l l i p t i c i n e 194 (106) have been r e p o r t e d . The a n t i - t u m o u r and a n t i - l e u k a e m i c a c t i v i t y of' t h e s e a l k a l o i d s a c c o u n t f o r t h i s c o n s i d e r a b l e 195 s y n t h e t i c i n t e r e s t . The more r e c e n t s t u d i e s p r e s e n t e d were d i r e c t e d towards the s y n t h e s i s o f the d i h y d r o p y r i d i n e (229-231) and t e t r a h y d r o p y r i d i n e (232-234) i n t e r m e d i a t e s . A p p l i c a t i o n o f t h e i r c h e m i s t r y s h o u l d p e r m i t t h e s y n t h e s i s o f the p y r i d o c a r b a z o l e (23 5) system. 2 3 1 2 3 4 DISCUSSION - PART IV I n v e s t i g a t i o n s towards the s y n t h e s e s o f i n t e r m e d i a t e s ( 2 2 9 - 2 3 4 ) used the key i n t e r m e d i a t e ( 2 3 9 ) o b t a i n e d by base h y d r o l y s i s o f 19 6 the r e p o r t e d N - b e n z e n e s u l p h o n y l d e r i v a t i v e ( 2 3 8 ) as shown i n f i g 6 1 . 2 3 9 2 3 8 F i g 6 1 : S y n t h e s i s o f I n d o l - 2 - y 1 - 1 - ( 4 ' p y r l d y l ) - e t h a n o l . ( 2 3 9 ) E v i d e n c e f o r s t r u c t u r e ( 2 3 9 ) was i n d i c a t e d by the U.V. h a v i n g the e x p e c t e d chromophore (A max 2 8 3 , 2 7 4 , 2 5 6 , 2 5 3 , 2 1 1 nm) and the I.R. had NH and OH a b s o r p t i o n a t 3 4 0 0 and 3 2 9 0 cm x . r e s p e c -t i v e l y . The mass spectrum gave an m/e 2 3 8 (M ) and the NMR was i n t o t a l agreement w i t h the s t r u c t u r e (see e x p e r i m e n t a l ) . I n o r d e r t o s y n t h e s i s e d e r i v a t i v e s ( 2 2 9 - 2 3 4 ) i t was d e c i d e d t o f i r s t d e v e l o p the c h e m i s t r y f o r the f o r m a t i o n o f the c o r r e s -p o n d i n g deshydroxy ( 2 4 0 ) and d e h y d r a t e d ( 2 4 1 ) d e r i v a t i v e s o f the a l c o h o l ( 2 3 9 ). I n t h i s r e s p e c t , h y d r o g e n a t i o n o f ( 2 3 9 ) u s i n g a Pd/C c a t a l y s t a f f o r d e d the r e q u i r e d deshydroxv d e r i -v a t i v e ( 2 4 0 ) i n good y i e l d . -185-Th e s t r u c t u r e (240) was i n d i c a t e d by the u.V. ( A max 288, 278, 268, 263, 256, 217 nm) and the I.R. spectrum d i s p l a y e d NH a b s o r p t i o n a t 34 80 cm \ The mass spectrum gave an m/e 222 (M +) and the NMR gave the e x p e c t e d q u a r t e t a t £4.20 (J=7Hz) f o r CHCH_ and a d o u b l e t a t S 1-68 (J=7Hz) f o r CHCH,.. 2 4 0 2 3 9 2 4 1 D e h y d r a t i o n o f (239) w i t h a c e t i c a n h y d r i d e i n p y r i d i n e gave t h e d e r i v a t i v e (241) i n good y i e l d . E v i d e n c e f o r t h i s s t r u c t u r e came m a i n l y from t h e mass spectrum h a v i n g an m/e 220 (M ) and the NMR w h i c h e x h i b i t e d two s i n g l e t s a t S 5.83, 5.42 f o r C=CH_2. However no f u r t h e r s t u d i e s w i t h (241) were i n i t i a t e d due t o i t s n o t e d i n s t a b i l i t y . I n t h i s r e s p e c t i t was c o n s i d e r e d t h a t a p p r o p r i a t e d e h y d r a t i o n o f t h e hydroxy s e r i e s c o u l d be pur s u e d a t a l a t e r s t a g e i n the s y n t h e s i s . W i t h d e r i v a t i v e s (239) and (240) on hand, s t u d i e s toward t h e i n t r o d u c t i o n o f the c o r r e s p o n d i n g N'-methyl group and (CH^) 2 N C H 2 s^ <^ e c h a i n a t C-3, were i n i t i a t e d . I n t r o d u c t i o n o f the m e t h y l group was r e a d i l y o b t a i n e d by t r e a t m e n t o f (239) and (240) w i t h m e t h y l i o d i d e ( f i g 62) t o g i v e m e t h i o d i d e s a l t s (242) and (243). E v i d e n c e f o r the m e t h i o d i d e s a l t (242) came p r i n c i p a l l y from t h e U.V. (A max 2 87,277,262,257, & 217 nm) and the NMR whi c h e x h i b i t e d a s i n g l e t a t £ 4.26 f o r N-CH-.. The remainder o f the -186-spectrum was i n t o t a l agreement w i t h the s t r u c t u r e . F u r t h e r s u p p o r t f o r the s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n o f (242) t o g i v e the t e t r a h y d r o p y r i d i n e d e r i v a t i v e (244) ( f i g 6 2 ) . The U.V. o f t h i s compound d i s p l a y e d the c h a r a c t e r i s t i c i n d o l e chromophore ( A max 288, 279, 270 and 218 nm) and t h e I.R. s p e c -trum gave OH and NH a b s o r p t i o n a t 3480 and 3320 c m - 1 r e s p e c -t i v e l y . The mass spectrum gave an m/e 256 (M ) and the NMR e x h i b i t e d a m u l t i p l e t a t £ 5.65 f o r C-3'H, a d o u b l e t a t S 2.82 f o r C-2'H , a m u l t i p l e t a t S 2.32 f o r C-6'H~, a s i n g l e t a t S 2.16 —2 f o r N-CH 3, and a m u l t i p l e t a t S 2.00 f o r C-5'H_2. E v i d e n c e f o r t h e m e t h i o d i d e s a l t (243) came p r i n c i p a l l y from the U.V. spectrum ( A max 285, 275, 263, 257, 217 nm) and t h e NMR w h i c h e x h i b i t e d a s i n g l e t a t <S 4.29 f o r N-CH^. Once a g a i n f u r t h e r s u p p o r t f o r t h i s s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n t o t h e c o r r e s p o n d i n g t e t r a h y d r o p y r i d i n e s p e c i e s (245) ( f i g 6 2 ) . The n e x t s t e p i n v o l v e d i n t r o d u c t i o n o f the ( C H 3 ) 2 N C H 2 s i d e c h a i n a t C-3. T h i s t r a n s f o r m a t i o n o c c u r r e d i n h i g h y i e l d t o g i v e (246) by t r e a t m e n t o f m e t h i o d i d e s a l t (242) w i t h HCHO/(CH 3) 2NH i n g l a c i a l a c e t i c a c i d ( f i g 6 2 ) . E v i d e n c e f o r t h i s s t r u c t u r e p r i n c i p a l l y came from the NMR w h i c h was i n t o t a l agreement w i t h t h e p r o p o s e d s t r u c t u r e h a v i n g a m u l t i p l e t a t S 3.49 f o r CH_2N and a s i n g l e t a t £ 2.29 f o r N(CH_ 3) 2 i n a d d i t i o n t o t h e e x p e c t e d s i g n a l s . The U.V. and I.R. were a l s o i n agreement w i t h t h i s s t r u c t u r e . -187-2 4 7 F i g u r e 62: S y n t h e s i s o f i n t e r m e d i a t e s (246) and (247). i F u r t h e r v e r i f i c a t i o n o f t h i s s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n t o g i v e the t e t r a h y d r o p y r i d i n e (248). E v i d e n c e f o r t h i s s t r u c t u r e was o b t a i n e d from the U.V. ( A max 288, 279,273 & 217 nm) and the I.R. w h i c h showed NH and OH a b s o r p t i o n a t 3485 and 3280 cm 1 r e s p e c t i v e l y . The mass spectrum gave an m/e 313 (M +) and t h e NMR was i n t o t a l agreement w i t h the s t r u c t u r e (see e x p e r i m e n t a l ) . -188-Th e formation of the corresponding analogous d e r i v a t i v e (247) 197 has a l s o been c a r r i e d out i n more recent s t u d i e s . These p r e l i m i n a r y r e s u l t s c o n s t i t u t e the progress made 198 towards the syntheses of the pyrido- c a r b a z o l e a l k a l o i d s . 197 202 Completion of the remaining s y n t h e t i c steps i s c u r r e n t l y underway. ' However i t i s of some i n t e r e s t to speculate on the p o s s i b l e pathways f o r completion of the syntheses. In t h i s r e s p e c t two recent syntheses of py r i d o - c a r b a z o l e s have r e c e n t l y been pub-l i s h e d - both of which had s i m i l a r i t i e s to the ' t e t r a h y d r o p y r i d i n e approach'. Consequently treatment of the N-benzenesulphonyl d e r i v a t i v e (250) w i t h KQBu i n DMSO aff o r d e d an E/Z mixture of dienamines (251) which was then allowed to undergo the appro-p r i a t e Mannich r e a c t i o n and c y c l i z a t i o n to give N-methyltetra-hydro e l l i p t i c i n e (252) i n 2.2 % . y i e l d . Treatment of (252) w i t h Pd/C affo r d e d e l l i p t i c i n e ( 1 0 6 ) . 1 9 9 ( f i g 63) Figure 63: P o t i e r ' s s y n t h e s i s of e l l i p t i c i n e (106) -189-The second approach i s shown i n f i g 64 and a s i m i l a r c y c l i z a t i o n o c c u r r e d t o g i v e p y r i d o c a r b a z o l e (257) from i n t e r m e d i a t e ( 2 5 6 ) . 2 0 0 T h i s a c i d c a t a l y s e d c y c l i z a t i o n p r o b a b l y o c c u r r e d v i a the p o s t u l a t e d mechanism ( f i g 6 5 ) . 257 F i g 65: P o s t u l a t e d mechanism f o r the f o r m a t i o n o f p y r i d o c a r b a z o l e (257) from i n t e r m e d i a t e (256). -190-A l t h o u g h t h i s c y c l i s a t i o n has n o t y e t been a t t e m p t e d l i t e r a t u r e p r e c e d e n c e s u g g e s t s t h a t t h e f i n a l t r a n s f o r m a t i o n t o g i v e the r e q u i r e d c y c l i s a t i o n p r o d u c t (235) w i l l be s u c c e s s f u l . I t i s t o be hoped, however, t h a t a p p r o p r i a t e m o d i f i c a t i o n o f t h i s t r a n s f o r m a t i o n can be e f f e c t e d t o a l l o w f o r m a t i o n o f the r e q u i r e d c y c l i z e d p r o d u c t (235) i n a much g r e a t e r y i e l d t han the 2% r e p o r t e d i n the s y n t h e s i s o f e l l i p t i c i n e (106) ( f i g 63). 201 A more p r o m i s i n g a p p r o a c h , however, c o n c e r n s the u t i l i s a t i o n o f d i h y d r o p y r i d i n e i n t e r m e d i a t e s . E x t e n s i v e s t u d i e s i n t h e s e l a b o r a t o r i e s have r e a l i s e d c o n d i t i o n s f o r t h e s t a b i l i s a t i o n o f h i g h l y r e a c t i v e d i h y d r o p y r i d i n e i n t e r m e d i a t e s as t h e i r c o r r e s -p o n d i n g chromium t r i c a r b o n y l complexes. I n t h i s r e s p e c t the s t a b l e d i h y d r o p y r i d i n e complex (258), shown i n f i g 66, was t r e a t e d w i t h p y r i d i n e t o r e g e n e r a t e the c o r r e s p o n d i n g d i h y d r o -p y r i d i n e (259) i n s i t u . Treatment o f (259) w i t h b e n z y l bromide and sodium b o r o h y d r i d e a f f o r d e d a m i x t u r e o f b e n z y l a t e d i n t e r -m e d i a t e s (260) and (261). D i r e c t d e b e n z y l a t i o n o f (261) and (260) was r e a d i l y e x e c u t e d u s i n g n - p r o p y l mercaptan, l i t h i u m h y d r i d e and hexamethylphosphoramide t o g i v e r e q u i r e d N-methyl-3 - e t h y l - 5 - b e n z y l - l , 2 , 5 , 6 t e t r a h y d r o p y r i d i n e (263) and N-methyl-3 - e t h y l - l , 2 , 5 , 6 t e t r a h y d r o p y r i d i n e (262). -191-2 5 8 2 5 9 2 6 2 2 6 3 F i g 66: S y n t h e s i s o f N - m e t h y l - 3 - e t h y l - 5 b e n z y l - t e t r a h y d r o p y r i -d i n e (263) However a more e f f i c i e n t method f o r the f o r m a t i o n o f (263) was n o t e d on t r e a t m e n t of N - m e t h y 1 - 3 - e t h y l p y r i d i n i u m i o d i d e (264) w i t h sodium b o r o h y d r i d e t o g i v e the r e q u i r e d d i h y d r o p y r i d i n e (259) T h i s , when t r e a t e d w i t h a 10 x excess o f b e n z y l bromide gave, f o l l o w i n g d e b e n z y l a t i o n a t n i t r o g e n , the t e t r a h y d r o p y r i d i n e (263) i n an o v e r a l l y i e l d o f 48%. e C H 3 C H 3 C H 3 2 6 4 2 5 9 2 6 3 Thus the a p p r o p r i a t e c h e m i s t r y has been de v e l o p e d t o a l l o w i n v e s t i g a t i o n o f the c y c l i s a t i o n r e a c t i o n s i n b o t h the t e t r a h y d r o -p y r i d i n e and d i h y d r o p y r i d i n e s e r i e s . -192-Refinement o f the t e t r a h y d r o p y r i d i n e c y c l i s a t i o n i s 197 c u r r e n t l y under s t u d y . U t i l i s a t i o n o f e i t h e r t h e " f r e e " d i h y d r o p y r i d i n e i n t e r -m e d i a t e s (229) o r (230) o r t h e i r s t a b i l i s e d chromium t r i c a r b o n y l Complexes (264) o r (265) i s a l s o under i n v e s t i g a t i o n and c o n d i t i o n s f o r t h e e f f i c i e n t c y c l i s a t i o n t o the r e q u i r e d p r o d u c t s (266) o r (267) ar e b e i n g e f f e c t e d . 1 9 7 ' 2 0 2 When t h e s e c o n d i t i o n s are d e t e r m i n e d a p p r o p r i a t e m o d i f i -c a t i o n s f o r the s y n t h e s e s o f o l i v a c i n e (105) and e l l i p t i c i n e (106) w i l l be i n t r o d u c e d . 2 4 7 2 6 5 2 6 7 -193-EXPERIMENTAL - PART I V G e n e r a l e x p e r i m e n t a l d e t a i l s were g i v e n i n p a r t I and I I . 196 1 - B e n z e n e s u l p h o n y l i n d o l e (23 7) P o t a s s i u m h y d r i d e (3.6 m l , 0.09 mol) was added t o a s o l u t i o n o f anhydrous THF (75 ml) and HMPA (15 ml) a t 0°. To t h i s was added i n d o l e (236) (10 g, 0.085 m o l ) , dissolved in anhydrous THF (30 m l ) , over 30 min. The suspen-s i o n was a l l o w e d t o s t i r f o r 15 min and dry r e d i s t i l l e d benzene-s u l p h o n y l c h l o r i d e (16.5 g, 0.088 mol) was added d r o p w i s e . The te m p e r a t u r e was r a i s e d t o r . t . and s t i r r i n g t hen m a i n t a i n e d f o r 3 h r . Alumina (Act I I I ) was added to d e s t r o y the r e m a i n i n g p o t a s s i u m h y d r i d e . Water (50 ml) was added and the r e s u l t i n g m i x t u r e poured t h r o u g h a g l a s s s i n t e r f u n n e l and then e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 100 m l ) . The e t h e r e a l e x t r a c t s were combined and backwashed w i t h w a t e r (2 x 75 m l ) , d r i e d , and e v a p o r a t e d t o g i v e a p r e c i p i t a t e w h i c h on r e c r y s t a l l i s a t i o n from m e t h y l e n e c h l o r i d e - hexane a f f o r d e d the r e q u i r e d (237) (19.5 g, 88%) i d e n t i c a l i n a l l r e s p e c t s t o t h a t r e p o r t e d (m.pt.= 78°, l i t m.pt. 77.5-79°) 1- ( 1 - B e n z e n e s u l p h o n y l i n d o l - 2 - y l ) - 1 - ( 4 - p y r i d y l ) - e t h a n o l (238) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e -19 6 dure. M.pt. 228-229° ( l i t m.pt. 229-230°) I n d o l - 2 - y l - l - ( 4 p y r i d y l ) e t h a n o l - ( 2 39) To a methanol-2M sodium h y d r o x i d e s o l u t i o n (500 ml, 5:1) was added (238)(3 g, 7.96 mmol). The s o l u t i o n -194-was then h e a t e d t o r e f l u x f o r 4 h r , c o o l e d , and e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 200 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o y i e l d a crude s o l i d w h i c h was r e c r y s -t a l l i s e d from c h l o r o f o r m t o g i v e (239) ( 1.36 g, 72%). M.pt 218-221° ( l i t m.pt.=221°) I.E.: 0 KBr 3400, 3290, 3050, 3030, 2920, 1600, 1455, 1430, 1295, 835, 795, 750, 700,cm - 1 I MeOH U . V . : / 1 M A X 283 (3.86), 274 (3.92), 256 (3. 9 5 ) , 253 (3 .92), 211 (4.53) nm. NMR: £ (d-DMSO) 100 MHz (FT) 10.92 (s, IH, NH), 8.49 (d, 2H, J=6Hz, C-2', C - 6 \ H ) , 7.46 (d, 2H, J=6Hz, C-3', C - 5 \ H_ 2), 7.40-6.80 (m, 4H, arom.), 6.31 (d, IH, J=2Hz, C-3H), 6.18 ( s , IH, OH, ex-changeable w i t h D-,0) , 1.88 ( s , 3H, CH_ 3). M.S. m/e: 238 ( M + ) , 220, 205, 160. High r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5 H 4 N 2 0 = 238.1106 Found= 238.1123 E l e m e n t a l a n a l y s i s C a l c C, 75.63, H, 5.88, N, 11.76 Found, C, 75.55, H, 5.70, N, 11.95 I n d o l - 2 - y l - l - ( 4 p y r i d y l ) - e t h a n e (240) The a l c o h o l (239) (105 mg, 0.44 mmol) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d and t o t h i s was added a Pd/C (10% w/w) c a t a l y s t (30 mg). The r e s u l t i n g s u s p e n s i o n was p l a c e d under an atmosphere o f hydrogen f o r 72 h r . The c a t a l y s t was then removed ( s u c t i o n f i l t r a t i o n ) , c o o l e d t o 0°, and concentrated ammonium h y d r o x i d e added t o ph 9. The aqueous l a y e r was the n e x t r a c t e d w i t h c h l o r o f o r m (3 x 25 m l ) . The c h l o r o f o r m e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h ich was chromato--195-graphed on s i l i c a g e l to a f f o r d r e q u i r e d (240) ( 52 mg, 54 %) and s t a r t i n g m a t e r i a l ( 1 3 mg, 13 %) M.pt.: 12 3-2 4° I.R.: 0 C H C 1 3 3480, 2980, 1605, 1460, 1420, 1305, 915 cm"1. I Me OH U * V ' : ^max 2 8 8 < 3 ' 8 3 ) ' 2 7 8 (3. 88), 268 (3.87), 263 (3. 90), 256 (3.87), 217 (4.52) nm. NMR: S (CDC13) 100 MHz (FT) 8.43 (b.s.,3H, NH and C-2', C-61 H 2 ) , 7.67 - 7.49 (m, IH, C-7H) , 7.33-6. 95 (m, 5H, arom & C-3', C-5 1 , (d, IH, J=2Hz, C-3H), 4.20 (q, IH, J=7Hz, CHCH ) 1.68 (d, 3H, J=7Hz, CH 3). M.S. m/e: 222, (M+) , 207, 144. High r e s o l u t i o n mass spectrum: C a l c - f o r C-, r-H N = 222.1157 Found= 222.1157. Elemental a n a l y s i s : C a l c - f o r c i 5 H i 4 N 2 ' C ' 81.08, H, 6.30, N, 12.61 Found, C, 80.74, H, 6.18, N, 12.72 In d o l - 2 - y l - l - ( 4 - p y r i d y l m e t h i o d i d e ) - e t h a n e . ( 2 4 3 ) Excess methyl i o d i d e (1 ml) was added t o a s o l u t i o n of compound (240) (210 mg, 0.94 mmol) d i s s o l v e d i n anhydrous e t h y l acetate. The s o l u t i o n was allowed t o s t i r o v e r n i g h t at r . t . a f t e r which a f l o c c u l e n t p r e c i p a t e had accumulated. This was removed ( s u c t i o n f i l t r a t i o n ) and r e c r y s t a l l i s a t i o n from methanol gave the r e q u i r e d methiodide s a l t (243) (313 mg, 91 %) M.pt.: 127° (MeOH) I.R.:^ CHC1 3 3190,3150,2980,1660,1450,1422,1294,848,790,745,540 cm - 1. U . V . : y\ J J | ° H 2 85 (3. 69 ) sh, 275(3. 88) sh, 263(4. 05) sh, 257(4. 07), 217 (4. 73) nm. NMR: S (d-DMSO) 100 MHz (FT) 8.92 (d, 211, J=6.8 Hz, C-2 ' , C-6 1 ) , 8.04 (d, 2H, J=6.8Hz,C-3',C-5" ,H2) , 7. 58-6.84 (m, 411, arom.), -196-6.36 ( s , 1H, C-3H), 4.70 (q, 1H, J=7Hz, CHCH^, 4.29 ( s , 3H, N-CH_3) 1.73 (d, 2H, J=7Hz, CHCH_3) . E l e m e n t a l a n a l y s i s : C a l c - f o r C l g H N 2 I , C, 52 . 76 , H, 4.70, N, 7.69, Found, C, 52.95, H, 4.71, N, 7.57 I n d o l - 2 - y l - l - ( 4 - p y r i d y l ) - e t h e n e (241) The a l c o h o l (239) (50 mg, 0.48 mmol) was added t o a s o l u t i o n of a c e t i c a n h y d r i d e (2 ml) and p y r i d i n e (0.5 m l ) . The temperature was r a i s e d t o 80° f o r 3 hr and then a l l o w e d t o O ri s t a n d o v e r n i g h t a t 50 . F u r t h e r h e a t i n g a t 80 f o r 1 hr completed the r e a c t i o n . The r e a c t i o n m i x t u r e was c o o l e d and d i l u t e d w i t h chloroform (20 m l ) . The s o l u t i o n was washed w i t h w ater (2 x 10 m l ) . The c h l o r o -form s o l u b l e s were d r i e d and e v a p o r a t e d t o g i v e an o i l 'which f o l l o w i n g chromatography on s i l i c a g e l a f f o r d e d t h e r e q u i r e d ethene (241) (37 mg, 80%). I.R.:' \) CHC1 3 3480, 3010, 2960, 2930, 1600 cm" 1, i MeOH U . V . : / j m a x 288(3.85, 278(3.92), 268(3.92), 263(3.93), 217(1.09) nm. NMR: £ (CDC1.J 100 MHz (FT) 88.56 (m,2H, C-2', C-6' H ) , 7.33-6.89 (m, 6H, arom H_4 & C' 3, C' 5 H ), 6.38 (d, IH, J=2Hz, C-3H) , 5.64, 5.40 ( 2S, 2H, C H 9 ) . . M.S. m/e: 220 ( M + ) , 78. High r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5 H 1 2 N 2 = 220.1000 Found= 220.0987 I n d o 1 - 2 - y 1 - 1 - ( 4 - p y r 1 d y l m e t h l o d i de)-e t h a n o l (242) The a l c o h o l (239) (390 mg, 1.64 mmol) was added t o a s o l u t i o n o f m e t h y l i o d i d e ( 2 ml) and anhydrous e t h y l -197-a c e t a t e (30 ml) . The s u s p e n s i o n was h e a t e d t o 50° f o r 1 h r t o e f f e c t s o l u t i o n . The t e m p e r a t u r e v/as t h e n l o w e r e d t o r . t . and s t i r r i n g was m a i n t a i n e d o v e r n i g h t . The r e s u l t i n g p r e c i p i t a t e was removed by s u c t i o n f i l t r a t i o n t o a f f o r d an amorphous powder (554 mg, 8 9 % ) . I.R.: 0 KBr 3285, 3030, 2930, 1640, 1460, 755, 700 cm" 1. U . V . : A m i ° H 287 (3.63)sh,277 (3.85)sh,262 (4.06)sh,257 (4.07) ,217 (4. 70) NMR: & (d-DMSO) 100 MHz (FT) 8.93 (d, 2H, J=6.8Hz, C-2', C-6', H 2 ) , 8.17 (d, 2H, J=6.8Hz, C-3 ' , C-5 ' H_2) , 7 . 56-6 . 82 (m, 4H, arom.), 6.72 ( s , IH, OH, e x c h a n g e a b l e w i t h D O ) , 6.44 ( s , IH, C-3H), 4.26 (s , 3H, N-CH 3), 1.98 ( s , 3H, CH(OH)CH ). I n d o l - 2 - y 1 - 1 - ( N ' - m e t h y I t e t r a h y d r o p y r i d y 1 ) - e t h a n o l (244) The m e t h i o d i d e s a l t (242) (200 mg, 0.526 mmol) was d i s s o l v e d i n methanol (10 ml) and sodium b o r o h y d r i d e (184 mg, 5.26 mmol) v/as added. The m i x t u r e was a l l o w e d t o s t i r f o r 0. 5 h r and the s o l v e n t v/as t h e n removed i n vacuo. Water (10 ml) was t h e n added and the aqueous phase e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 50 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s . The o i l formed was t h e n chromato-graphed on s i l i c a g e l t o g i v e the r e q u i r e d t e t r a h y d r o p y r i d i n e (244) (145 mg, 88%) . M . D t . : 184-185° (CHC1 ) 1. R.: ^ CHC1 3 3480, 3320, 3020, 2990, 2950, 2800, 1665, 1460, 1375, 1295, cm" 1. -198-u-v- : A m a f 2 8 8 ( 3 . 7 4 ) , 278 (3. 8 6 ) , 270 (3.87), 217 (4.54) nm. NMR: £ (d-DMSO) 100 MHz (FT) 10.59 ( s , IH, NH), 7.46-7.20 (m, 2H, arom), 7.04-6.74 (m, 2H, arom), 6.16 (d, IH, J=2Hz, C-3 H) , 5.65 ( t , IH, J-4Hz, C-3* H), 2.82 (d, 2H, J=4Hz, C-2' H 2 ) , 2.32 (m, 2H, C-5' H_2) , 2.16 ( s , 3H, N-CH_3), 2.00 (m, 2H, C-6 1 H 2 ) , 1.6 0 ( s , 3H, C H 3 ) . M.S. m/e: 256 ( M + ) . High r e s o l u t i o n mass spectrum: C a l c — f o r C^^20^2o= 256.1575 Found= 256.1570 E l e m e n t a l a n a l y s i s , C a l c - f o r C 1 6 H 2 ( ) N 2 0 , C, 75.0, H, 7.81, N, 10.94 Found, C, 74.86, H, 7.72, N, 10.83. P y r i d i n i u m . s a l t (246) To an aqueous d i m e t h y l a m i n e s o l u t i o n (25% w/w, 250 mg) a t 0° was added (1) g l a c i a l a c e t i c a c i d (500 mg, 8.33 mmol) and (2) and aqueous formaldehyde s o l u t i o n (37% w/w, 105 mg, 1.3 mmol). T h i s m i x t u r e was the n added t o t h e p y r i d i n i u m s a l t (242) (541 mg, 1.42 mmol) and the r e s u l t i n g s u s p e n s i o n v/armed t o 45° and s t i r r e d o v e r n i g h t . The r e a c t i o n m i x t u r e was c o o l e d t o 0° and n e u t r a l i s e d w i t h 2M sodium h y d r o x i d e s o l u t i o n . F o l l o w i n g f r e e z e - d r y i n g the s o l i d o b t a i n e d was r e c r y s t a l l i s e d from e t h a n o l -hexane t o g i v e r e q u i r e d p y r i d i n i u m s a l t (246) (518 mg, 8 4 % ) . M.pt. 238-242° = 0 I.R.: \S m ^ v KBr 3420, 3230, 3020, 2980, 2930, 1635, 1455, 750, 70 0 cm" max -1 „ I MeOH u - v - : / i m a x 283 ( 3 . 7 3 ) , 275 (3 . 8 7 ) , 260(3.95), 217(4.70) nm. NMR: £ (d-DMSO) 100 MHz (FT) 9.2 ( s , IH, NH), 8.91 (d, 2H, -199-J=6.8Hz,C-2',C-6' H ) , 8.11 (d, 2H, J=6.8Hz, C-3*, C-5 1 H_ 2), 7.65-6.97 (m, 4H, arom), 4.32 ( s , 3H, N-CH_3) , 3.49 ( m, 2H, C H 2 N ( C H 3 ) 2 ) , 2.29 ( s , 6H, N ( C H 3 ) 2 ) f 2 . 0 1 ( s , 3H, CH_ 3). E l e m e n t a l a n a l y s i s : C a l c - f o r C l g H 2 4 N I , C, 52.18, H, 5.53, N, 9.61 Found C, 52.24, H, 5.63, N, 9.40 T e t r a h y d r o p y r i d i n e (24 8) The Mannich d e r i v a t i v e (246) (200 mg, 0.46 mmol) was d i s s o l v e d i n methanol (10 ml) and sodium b o r o h y d r i d e ( 160 mg, 4.6 mmol) added o v e r 10 min. The m i x t u r e was a l l o w e d t o s t i r f o r 0.5 h r and t h e s o l v e n t was removed i n vacuo. Water (10 ml) was t h e n added and t h e aqueous phase e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 25 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s . The amorphous powder was the n chromato-graphed on s i l i c a g e l t o g i v e the r e q u i r e d t e t r a h y d r o p y r i d i n e (248) (126 mg, 8 8 % ) . I.R.: 0 CHC1 3 3485, 2990, 2950, 2780, 1610, 1465, 1010 cm" 1. 1 MeOH U . V . : / ) m a v 288 (3.86, 279 (3. 9 2 ) , 273 (3.90, 2.7 (456) nm. NMR: & (CDC1 3) 100 MHz (FT) 8.39 ( s , IH, NH), 7.60-7.02 (m, 4H, arom), 5.92 (b.m, IH, C-3'H), 3.65 (d, 2H, J=2Hz, CH„N(CH ) ) , 2.42 ( s , 3H, N-CH 3) , 2.27 ( s , 6H, N- ( C H _ 3 ) 2 ) , 1.72 ( s , 3H, CH_ 3). M.S. m/e: 313 ( M + ) , 268, 250, 208, 172, 138. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c i 9 H 2 7 N 3 0 = : 313.2154 Found= 313.2158. - 2 0 0 -REFERENCES 1. G.A. C o r d e l l , L l o y d i a , 37_, 2 1 9 , ( 1 9 7 4 ) . 2 . J.W. C o r n f o r t h , Quart. Rev., 1 , ( 1 9 7 3 ) . 3 . E. Leete, The Chemical S o c i e t y , S p e c i a l i s t P e r i o d i c a l Reports, B i o s y n t h e s i s , 3., 1 1 3 , ( 1 9 7 5 ) • 4. D.H.R. Barton, Fourth I n t e r n a t i o n a l Symposium on the B i o -sy n t h e s i s and Physiology of A l k a l o i d s , H a l l e , D.D.R., ( 1 9 6 9 ) . 5 . G.B. Bodem, E. Leete, J.M.F. Manuel, Phytochemistry, 1 0 , 2 6 8 7 , ( 1 9 7 D . 6 . H. Rapoport, M.L. Rueppel, J . Amer. Chem. S o c , 9 3 , 7 0 2 1 , ( 1 9 7 D • 7 . 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