UBC Theses and Dissertations

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UBC Theses and Dissertations

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

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STUDIES RELATED TO THE SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS HARALD WILHELM HANSSEN M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES (Dept. o f C h e m i s t r y ) 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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA by i n M a r c h , 1978 H a r a l d W i l h e l m Hanssen, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requ i rement s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f CZL-/^2^?^c.,r^J The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date MTQV ¥ 7 2 - i i -ABSTRACT I n P a r t I , a m o d i f i e d s y n t h e s i s o f r a d i o - l a b e l l e d s e c o d i n e (68) and i t s 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 (7) i s d e s c r i b e d . I n a model s t u d y , f o r t h e s y n t h e s i s o f s i d e - c h a i n l a b e l l e d 2 3 - e t h y l p y r i d i n e ( 7 4 ) , [ 2 - H ] - ( 3 ' - p y r i d y l ) - e t h a n e was a c h i e v e d f r o m the c o r r e s p o n d i n g l y l a b e l l e d 3 - a c e t y l p y r i d i n e by d e s u l p h u r i z a t i o n o f 3 the i n t e r m e d i a t e t h i o k e t a l ( 9 3 ) . I n a second s t u d y , [ 1 - H ] - ( 3 ' - p y r i d y l ) -e t h a n e was s y n t h e s i z e d by t r e a t i n g 3 - a c e t y l p y r i d i n e w i t h sodium b o r o h y d r i d e -3 H. The r e s u l t i n g a l c o h o l (95) was a c e t y l a t e d , and h y d r o g e n o l y s i s a c h i e v e d the d e s i r e d p r o d u c t . 3 The e s t e r a l c o h o l (74) was c o u p l e d t o [ 1 - H ] - ( 3 ' - p y r i d y l ) - e t h a n e and t h e r e s u l t i n g p y r i d i n u m s a l t (90) was r e d u c e d t o the c o r r e s p o n d i n g p i p e r d e i n e e s t e r (80) i n a " o n e - p o t " s y n t h e s i s . The c o n v e r s i o n o f (80) 3 t o [19- H ] - s e c o d i n e was a c h i e v e d by a known p r o c e d u r e . 3 14 3 14 I n two e x p e r i m e n t s , [19- H, C 0 2 C H 3 ] - s e c o d i n e ( 6 8 ) ( H/ C r a t i o s = 3.00 and 1.54) was a d m i n i s t e r e d t o C a t h a r a n t h u s r o s e u s p l a n t s . The v i n d o l i n e (7) w h i c h was i s o l a t e d was shown to have been b i o s y n t h e s i z e d 3 14 f r o m the e n t i r e s e c o d i n e m o l e c u l e ( H/ C = 3.31 and 1.35 r e s p e c t i v e l y ) . I n P a r t I I , a d e g r a d a t i o n scheme d e s i g n e d t o a c h i e v e the i s o l a t i o n o f t h e N-methyl group o f u l e i n e (1) i s d e s c r i b e d as w e l l as p r e l i m i n a r y r e s u l t s f r o m an i n v e s t i g a t i o n i n t o t h e b i o s y n t h e s i s o f u l e i n e (1) and o l i v a c i n e ( 4 ) . V a r i o u s l y r a d i o - l a b e l l e d forms o f t r y p t o p h a n ( 1 5 ) , a n t h r a n i l i c a c i d and s e c o d i n e (18) were a d m i n i s t e r e d t o A s p i d o s p e r m a p y r i c o l l u m r o o t segments and w h ole p l a n t s . The u l e i n e (1) w h i c h was i s o l a t e d was found t o i i i be i n a c t i v e i n a l l experiments. V a r i o u s l y r a d i o - l a b e l l e d forms of t r y p t o p h a n (15), a n t h r a n i l i c 14 a c i d and s e c o d i n e (18) as w e l l as CH^-methionine (30) was a d m i n i s t e r e d to Aspidosperma a u s t r a l e p l a n t s . U l e i n e (1) and o l i v a c i n e (4) was i s o l a t e d . The o n l y i n c o r p o r a t i o n t h a t c o u l d be demonstrated was t h a t 14 o f CK^-methionine (30) i n t o u l e i n e (1) to the e x t e n t of 0.168% and 0.147%. The i s o l a t i o n of the N-methyl group from (1) showed t h a t i t c o n t a i n e d 97% and 98% o f the a c t i v i t y . In P a r t I I I , the attempted s y n t h e s i s o f compounds of the preakuammicine- and stemmadenine-series i s d e s c r i b e d . A new method f o r the C-18 d e o x y g e n a t i o n o f curan d e r i v a t i v e s u s i n g B i r c h r e d u c t i o n c o n d i t i o n s was a c h i e v e d . A l s o , a m o d i f i c a t i o n o f the Oppenauer o x i d a t i o n o f the c u r e n o l (36) to a c h i e v e improved y i e l d s of the aldehyde (37) and n o r - f l u o r o c u r a r i n e (39) was d e v e l o p e d . The i n t r o d u c t i o n o f a carbomethoxy group i n t o the C-16 p o s i t i o n of the c u r a n aldehyde d e r i v a t i v e s (44) and (50) u s i n g a base and m e t h y l c h l o r o f o r m a t e was u n s u c c e s s f u l . A l s o , the i n t r o d u c t i o n of c y a n i d e i n t o p o s i t i o n C-16 o f the i n d o l e a l c o h o l (52) o r i n d o l e a c e t a t e (57) v i a the 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 s was u n s u c c e s s f u l . The s y n t h e s i s o f p r o d u c t (60), which i s b e l i e v e d to be i d e n t i c a l w i t h preakuammicine aldehyde (7), was a c h i e v e d . T h i s m a t e r i a l c o u l d n o t be c o n v e r t e d i n t o akuammicine (5) o r stemmadenine ( 4 ) . Only the dehydrated i n d o l e n i n e (72) c o u l d be o b t a i n e d . The r i n g - o p e n i n g r e a c t i o n o f the c o r r e s p o n d i n g t h i o a c e t a l d e r i v a t i v e (73) y i e l d e d the d e c a r b o x y l a t e d i n d o l e t h i o a c e t a l s (75) and ( 7 6 ) . iv TABLE OF CONTENTS Page Ti t l e Page i Abstract i i Table of Contents iv List of Figures v Li s t of Tables v i i l Acknowledgements x Part I Introduction 2 Discussion 31 Experimental 44 Bibliography 59 Part II Introduction 67 Discussion 75 Experimental 93 Bibliography ^03 Part III Introduction 106 Discussion 112 Experimental 168 Bibliography 202 V LIST OF FIGURES PART I Figure Page 1. The Barger-Hahn Robinson-Woodward hypothesis for indole a l k a l o i d biosynthesis 7 2. The Wenkert prepheric a c i d hypothesis 9 3. The Thomas-Wenkert monoterpene hypothesis .' 10 4. Leete's modified acetate hypothesis 11 5. The monoterpene o r i g i n of the Cg-C^^ unit 12 6. Summary scheme of the e a r l y stages of a l k a l o i d biosynthesis as proven by experiment 17 7. The poss i b l e b i o g e n i s i s of the Corynanthe a l k a l o i d s as supported by experiment 20 8. Wenkert's hypothesis f o r the o r i g i n of the Strychnos family of a l k a l o i d s 21 9. The Scott hypothesis f o r the biosynthesis of the Strychnos a l k a l o i d s 23 10. The Wenkert hypothesis f o r the biosynthesis of the Aspidosperma and Iboga a l k a l o i d s 24 11. Hypothetical biosynthesis of the Aspidosperma and Iboga a l k a l o i d s from the Strychnos family v i a dehydrosecodine (64) 29 12. The synthesis of the chloro-indole. (73) 32 13. The Kutney synthesis of secodine (68) 33 14. The synthesis of ester a l c o h o l (84) 36 15. An alternate synthesis of piperdeine ester (80) .... 37 v i PART I I Fi g u r e Page 1. The Wenkert hypothesis f o r the b i o s y n t h e s i s of e l l i p t i c i n e ( 6 ) , u l e i n e (1) and o l i v a c i n e (4) 69 2. The D j e r a s s i - G i l b e r t r a t i o n a l f o r the b i o s y n t h e s i s of a p p a r i c i n e (2) 70 3. The P o t i e r - J a n o t p o s t u l a t e f o r the b i o s y n t h e s i s of u l e i n e ( 1 ) , a p p a r i c i n e (2) and the py r i d o c a r b a z o l e a l k a l o i d s 73 4. Expected p o s i t i o n s of r a d i o - l a b e l i n u l e i n e (1) and o l i v a c i n e (4) from a v a i l a b l e precursors as suggested by the P o t i e r - J a n o t hypothesis (Figure 3) -. 77 5. Degradation of u l e i n e (1) by means of Hofmann e l i m i n a t i o n r e a c t i o n s 83 PART I I I 1. The proposed b i o s y n t h e s i s of the Strychnos a l k a l o i d s from the Corynanthe f a m i l y 108 2. The i m p l i c a t i o n of the dehydrosecodine J stemmadenine b i o - e q u i l i b r i u m i n i n d o l e a l k a l o i d b i o s y n t h e s i s as supported by experiment 109 3. Some biomimetic conversions of stemmadenine (4) 110 4. Synthesis of 16-epi-stemmadenine (28) 116 5. The degradation of s t r y c h n i n e (29) to Wieland-Gumlich aldehyde (22) 117 6. Two methods f o r deoxygenation of C-18 of Wieland-Gumlich aldehyde (22) 119 7. A p o s s i b l e mechanism f o r the two-step r e d u c t i o n of Wieland-Gumlich aldehyde (22) to 23,16a-cur-19-ene-17-ol (36) 120 v i i Page 8. The o x i d a t i o n o f a l c o h o l (36) t o n o r -f l u o r o c u r a r i n e (39) 122 9. The s y n t h e s i s o f N - f o r m y l - 1 8 - d e o x y - W i e l a n d -G u m l i c h a l d e h y d e (44) . 127 10. A r a t i o n a l e f o r t h e f r a g m e n t a t i o n o f a l d e h y d e (44) i n the mass s p e c t r o m e t e r 129 11. A t t e m p t e d f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) and t h e p r o p o s e d p l a n f o r i t s c o n v e r s i o n t o p r e a k u a m m i c i n e and stemmadenine d e r i v a t i v e s 131 12. The s y n t h e s i s o f N-carbomethoxy-18-deoxy W i e l a n d -G u m l i c h a l d e h y d e (50) 137 13. The p l a n t o s y n t h e s i z e stemmadenine (4) v i a the c h l o r o i n d o l e n i n e o f d e s - c a r b o m e t h o x y stemmadenine (52) 143 14. The p l a n t o s y n t h e s i z e stemmadenine (4) v i a t h e c h l o r o i n d o l e n i n e a c e t a t e (58) 146 15. The n u c l e o p h i l i c r e a c t i o n s o f e n a m i n o c a r b o n y l systems 149 16. The p o s s i b l e r e a c t i o n s o f n o r - f l u o r o c u r a r i n e (38) w i t h m e t h y l c h l o r o f o r m a t e 150 17. The uv s p e c t r u m o f the p r o d u c t f r o m the r e a c t i o n o f n o r - f l u o r o c u r a r i n e (39) w i t h m e t h y l c h l o r o f o r m a t e . . . 152 18. The c a r b o n y l r e g i o n o f t h e i n f r a r e d s p e c t r u m o f p r o d u c t (60) 153 19. The pmr s p e c t r u m o f compound (60) 155 20. Some e x p e c t e d r e a c t i o n s o f p r e a k u a m m i c i n e d e r i v a t i v e s 157 21. A p o s s i b l e mechanism f o r t h e f o r m a t i o n o f compound (72) 159 22. R e a c t i o n s o f t h i o a c e t a l (73) 165 v i i i L I S T OF TABLES PART I T a b l e Page 3 14 1. The i n c o r p o r a t i o n o f [ 1 9 - H, C 0 2 C H . l -s e c o d i n e i n t o v i n d o l i n e (7) 43 2. A d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 1 58 PART I I 1 I n c o r p o r a t i o n s t u d i e s i n t o a p p a r i c i n e (2) i n A. p y r i c o l l u m p l a n t s 78 2. I n c o r p o r a t i o n s t u d i e s i n A. p y r i c o l l u m p l a n t s 86 3. I n c o r p o r a t i o n s t u d i e s i n A. a u s t r a l e p l a n t s 90 4. A d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 2 100 5. A d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 3 102 PART I I I 1. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) . . . . 134 2. F u r t h e r s t u d i e s on the f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) 135 3. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f N-carbomethoxy a l d e h y d e (50) 139 4. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a the c h l o r o i n d o l e n i n e o f i n d o l e (52) 144 5. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a t h e c h l o r o i n d o l e n i n e o f i n d o l e a c e t a t e (57) 147 6. R e a c t i o n s o f compound (60) w i t h NaBH^ 161 7. F u r t h e r s t u d i e s on the m e t a l h y d r i d e r e d u c t i o n o f compound (60) 161 8. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 1 186 i x T a b l e Page 9. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 2 187 10. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 5 0 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 3 190 11. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 4 194 12. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 5 195 X ACKNOWLEDGMENTS The a u t h o r i s g r a t e f u l t o P r o f . J . P. Kutney f o r g i v i n g him t h e o p p o r t u n i t y t o do r e s e a r c h i n s e v e r a l i n t e r e s t i n g a r e a s o f n a t u r a l p r o d u c t s c h e m i s t r y and a l s o f o r h i s encouragement and s u p e r v i s i o n . Many h e l p f u l d i s c u s s i o n s w i t h many h e l p f u l c o l l e a g u e s a r e g r a t e f u l l y a c k n o w l e d g e d . S p e c i a l t hanks a r e due t o D r s . R. Sood and J . Beck f o r i n t r o d u c i n g t h e a u t h o r t o t e c h n i q u e s i n b i o s y n t h e s i s . I a l s o t h a n k Anna Wong f o r t y p i n g t h e m a n u s c r i p t . x i To Brenda, who endured. - 1 -P A R T I Studies Related to the B i o s y n t h e s i s of V i n d o l i n e -2-INTRODUCTION Man's wonder and c u r i o s i t y a b o ut h i s n a t u r a l w o r l d i s i l l u s t r a t e d i n h i s e a r l i e s t r e c o r d s . Out o f c u r i o s i t y , and o f t e n n e c e s s i t y , h i s e a r l y a t t e m p t s a t u n d e r s t a n d i n g and u s i n g t h e m a t e r i a l s p r o v i d e d by n a t u r e was, i n f a c t , t h e s t u d y o f o r g a n i c compounds. From t h e s e b e g i n n i n g s t h e modern f i e l d o f o r g a n i c c h e m i s t r y e v o l v e d . Up u n t i l t h e 1 9 t h c e n t u r y , t h e s t u d y o f o r g a n i c c h e m i s t r y was e x c l u s i v e l y t h e s t u d y o f n a t u r a l p r o d u c t s . I n most c a s e s , o r g a n i c e x t r a c t s had been used as f o l k m e d i c i n e s , perfumes o r c o l o u r i n g m a t e r i a l s t h a t were o b t a i n e d f r o m p l a n t s o r m i c r o - o r g a n i s m s . The c l a s s i c a l s y s t e m a t i c s t u d y o f p u r i f i e d n a t u r a l compounds began w i t h t h e i d e n t i f i c a t i o n o f f u n c t i o n a l g r o u p s , d e l i n e a t i o n o f the c a r b o n s k e l e t o n and t h e n a t t e m p t e d s y n t h e s i s o f t h e compound t o c o n f i r m the p r o p o s e d m o l e c u l a r s t r u c t u r e . A b e w i l d e r i n g a r r a y o f m o l e c u l a r s t r u c t u r e s were d i s c o v e r e d . T e r p e n e s , s t e r o i d s , amino a c i d s , p e p t i d e s , n u c l e o t i d e s , s u g a r s and a l k a l o i d s were some c l a s s e s o f compounds w h i c h emerged and soon demanded t h e i r own s p e c i a l t i e s . JThese c l a s s d i v i s i o n s were o f t e n a r b i t r a r y and u n s a t i s f y i n g . C h e m i s t s and b o t a n i s t s began t o wonder about the i n t e r -r e l a t i o n s h i p s between t h e s e c l a s s e s and t h e o r i g i n s o f t h e move complex m o l e c u l e s . Were t h e r e any " b u i l d i n g b l o c k s " w h i c h n a t u r e used t o c o n -s t r u c t t h e m o l e c u l e s o f each c l a s s , o r were t h e r e " b u i l d i n g b l o c k s " common t o s e v e r a l c l a s s e s o f n a t u r a l p r o d u c t s ? - 3 -P e rhaps some o f the most e l e g a n t e a r l y s p e c u l a t i o n s on t h e b i o g e n e s i s o f n a t u r a l p r o d u c t s were t h o s e of S i r R o b e r t R o b i n s o n 1 , who i s now r e g a r d e d as the f a t h e r o f t h i s r e l a t i v e l y l a r g e a r e a o f s t u d y i n o r g a n i c c h e m i s t r y . The s t u d y o f the b i o g e n e s i s o f n a t u r a l compounds has made p o s s i b l e a r e - c l a s s i f i c a t i o n o f some c l a s s e s i n t o f a m i l i e s o f s t r u c t u r e s a c c o r d i n g t o t h e i r b i o g e n e t i c o r i g i n . F o r example, we now know t h a t t e r p e n e s r a n g i n g from the t e n c a r b o n (C-^Q) monoterpene t h r o u g h t o the t h i r t y c a r b o n (C^Q) t r i t e r p e n e s as w e l l as th e s t e r o i d s , have a l l been u n i v e r s a l l y b u i l t up from the f i v e c a r b o n (C,.) i s o p r e n e u n i t . The d e l i n e a t i o n o f b i o s y n t h e t i c pathways have o f t e n shown t h e way t o s i m p l e r and more d i r e c t l a b o r a t o r y s y n t h e s i s w h i c h have p r o v e n more e f f i c i e n t t h a n a pproaches t h a t have n o t t a k e n n a t u r a l r o u t e s i n t o c o n s i d e r a t i o n . One f a m i l y o f n a t u r a l p r o d u c t s w h i c h have p r e s e n t e d many o f the more c h a l l e n g i n g and s u b t l e c h e m i c a l problems i n s t r u c t u r a l e l u c i d a t i o n , and i n s y n t h e s i s , has been the i n d o l e a l k a l o i d s . These a r e compounds whose main s t r u c t u r a l f e a t u r e s a r e the i n d o l e (1) o r i n d o l i n e (2) n u c l e u s p l u s one b a s i c n i t r o g e n atom e l s e w h e r e i n the m o l e c u l e . (1) (2) -4-I n d o l e a l k a l o i d s have been f o u n d i n a l a r g e number o f t r o p i c a l and s u b - t r o p i c a l p l a n t f a m i l i e s s u c h as Apocynaceae, Loganaceae and R u b i a c e a e . The Apocynaceae p l a n t f a m i l y has been p a r t i c u l a r i l y p r o d u c t i v e i n p r o v i d i n g a l k a l o i d s o f m e d i c i n a l i m p o r t a n c e . Some examples a r e : (a) r e s e r p i n e (3) f r o m t h e genus R a u w o l f i a w h i c h i s w i d e l y used i n the t r e a t m e n t o f m e n t a l d i s o r d e r s ; (b) s t r y c h n i n e (4) f r o m v a r i o u s s t r y c h n o s s p e c i e s i s a c a r d i a c and c e n t r a l n e r v o u s s y s t e m s t i m u l a n t ; and ( c ) v i n b l a s t i n e ( 5 ) , a b i s - i n d o l e a l k a l o i d f r o m t h e genus C a t h a r a n t h u s w h i c h i s now e x t r e m e l y i m p o r t a n t i n c a n c e r chemotherapy. ( 7 ) - 5 -These a l k a l o i d s a r e r e p r e s e n t a t i v e o f t h e h i s t o r i c a l c l a s s i f i c a t i o n o f i n d o l e a l k a l o i d s i n t o t h e f o u r m a j o r groups w h i c h have been most a r d e n t l y s t u d i e d . The c a r b o n s k e l e t o n i l l u s t r a t e d by r e s p e r p i n e (3) i s r e f e r r e d t o as b e i n g o f the C o r y n a n t h e f a m i l y . S t r y c h n i n e (4) i s i l l u s t r a t i v e o f t h e S t r y c h n o s a l k a l o i d s . The b i s - i n d o l e (5) has as i t s " u p p e r " h a l f an I b o g a s k e l e t o n a 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 (6) w h i l e t h e " l o w e r " p a r t i s a n a t u r a l l y o c c u r r i n g monomeric a l k a l o i d c a l l e d v i n d o l i n e ( 7 ) . The l a t t e r i s r e p r e s e n t a t i v e o f t h e A s p i d o s p e r m a f a m i l y o f a l k a l o i d s . The f i r s t b i o g e n e t i c s p e c u l a t i o n s w h i c h a t t e m p t e d t o e s t a b l i s h some o r d e r i n the g r e a t v a r i e t y o f s t r u c t u r e s f o u n d i n t h e i n d o l e 2 a l k a l o i d a r e a were o f f e r e d by P i c t e t , i n 1906. S i n c e a l l t h e i n d o l i c s t r u c t u r e s e s t a b l i s h e d a t t h a t time had a 3 - a m i n o e t h y l group a t t a c h e d t o t h e 3 - p o s i t i o n o f t h e i n d o l e p o r t i o n , i t was l o g i c a l t o s p e c u l a t e t h a t t h e amino a c i d t r y p t o p h a n ( 8 , F i g u r e 1) was d e c a r b o x y l a t e d t o become t r y p t a m i n e ( 9 ) , and t h e l a t t e r was t h e n e l a b o r a t e d i n t o t h e C^g and C^Q i n d o l e a l k a l o i d s . One h a l f c e n t u r y p a s s e d b e f o r e t h i s s p e c u l a t i o n was g i v e n e x p e r i m e n t a l s u p p o r t . I n 1960, L e e t e showed t h a t t r y p t a p h a n , w h i c h had been s p e c i f i c a l l y l a b e l l e d w i t h r a d i o a c t i v e c a r b o n 14 i n t h e s i d e c h a i n , was i n c o r p o r a t e d i n t o t h r e e R a u w o l f i a 3 4 a l k a l o i d s : a j m a l i n e , r e s e r p i n e and s e r p e n t i n e ' . That l a b e l l e d c a r b o n atom was i s o l a t e d f r o m t h e s e a l k a l o i d s , t h u s l e a v i n g no doubt about s p e c i f i c 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 . These v e r y i m p o r t a n t r e s u l t s i n i t i a t e d a d e l u g e o f work i n the a r e a o f 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 . A l a r g e number o f r e v i e w s o f 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 have -6-appeared o v e r the y e a r s , the more r e c e n t b e i n g p a r t i c u l a r l y i n f o r m a t i v e ^ Two o f t h e s e r e v i e w s ^ '"^ have done an o u t -s t a n d i n g j o b o f p u t t i n g o r d e r to the t o t a l h i s t o r y o f work done i n t h i s a r e a . Another r e v i e w a t t h i s time cannot be j u s t i f i e d . However, i t w i l l be i n s t r u c t i v e i n the p r e s e n t work to summarize b r i e f l y the h i g h l i g h t s o f t h i s e x c i t i n g s t o r y i f o n l y t o p u t the work to be d e s c r i b e d l a t e r i n t o some p e r s p e c t i v e . W i t h the e s t a b l i s h m e n t of t r y p t o p h a n (8) as a fundamental b i o s y n t h e t i c p r e c u r s o r , the o r i g i n o f the o t h e r n i n e o r t e n c a r b o n atoms o f the complex a l k a l o i d s remained a g r e a t p u z z l e . In 1933, B a r g e r and Scholz''""'" thought t h a t a t y r o s i n e d e r i v e d a l dehyde (10, F i g u r e 1) condensed w i t h t r y p t a m i n e (9) and the p r o d u c t was then e l a b o r a t e d i n t o yohimbine (13) a r e p r e s e n t a t i v e of the then w e l l -12 known C o r y n a n t h e - t y p e . M o d i f i c a t i o n s of t h i s p o s t u l a t e by Hahn and l a t e r by W o o d w a r d " ^ ' a n d Robinson"' - a l l o w e d the h y p o t h e t i c a l b i o g e n e s i s of the S t r y c h n o s f a m i l y o f i n d o l e a l k a l o i d s from 3,4-d i h y d r o x y d e r i v a t i v e (14) p l u s a s i n g l e a c e t a t e u n i t to g i v e an i n d o l e n i n e ( 1 5 ) . I n h e r e n t i n t h i s scheme was the d a r i n g s p e c u l a t i o n o f t h e o x i d a t i o n and subsequent c l e a v a g e o f the p h e n o l i c system v i a i n t e r m e d i a t e (16) t o a c h i e v e the S t r y c h n o s c a r b o n s k e l e t o n ( 4 ) . The "Barger-Hahn-Robinson-Woodward" h y p o t h e s i s , i l l u s t r a t e d i n F i g u r e 1, remained u n c h a l l e n g e d f o r some time. 15 16 I n 1959, Wenkert and B r i n g i ' p o i n t e d out t h r e e major d e f i c i e n c i e s i n t h i s h y p o t h e s i s . F i r s t l y , a l l the y o h i m b i n e - l i k e a l k a l o i d s , c h a r a c t e r i z e d a t t h a t time, p o s s e s s e d the same a b s o l u t e (13) F i g u r e 1. The Barger-Hahn-Robinson-Woodward h y p o t h e s i s f 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 . - 8 -s t e r e o - c h e m i s t r y a t C-15, a f a c t w h i c h seemed u n l i k e l y i f r i n g E was i n d e e d a r o m a t i c a t t h a t l e v e l o f b i o s y n t h e s i s . S e c o n d l y , o n l y one compound o f t h i s t y p e p o s s e s s e d an a r o m a t i c r i n g E. And t h i r d l y , t h e i n t r o d u c t i o n o f a carbomethoxy group a t C-16 was m e c h a n i s t i c a l l y u n l i k e l y . They p r o p o s e d i n s t e a d t h a t t h e p r o g e n i t o r o f t h e C^-C^Q u n i t c o u l d be p r e p h e n i c a c i d ( 1 7 , F i g u r e 2 ) , a known p r o d u c t o f c a r b o h y d r a t e m e t a b o l i s m . The s t e r e o s p e c i f i c r e a r r a n g e m e n t o f t h e p r e p h e n a t e m o i e t y t o t h e s e c o - p r e p h e n a t e - f o r m a l d e h y d e u n i t (SPF,20) f i x e s t h e s t e r e o c h e m i s t r y a t C-15 i n t h e C o r y n a n t h e a l k a l o i d s and a l l o w s t h e C-16 carbomethoxy group t o be an i n a t e p a r t o f the p r o -g e n i t o r . T h i s scheme i s summarized i n F i g u r e 2. Not o n l y d i d t h e SPF u n i t a c c o u n t w e l l f o r t h e n o n - t r y p t a m i n e p a r t o f t h e y o h i m b i n e - l i k e a l k a l o i d s , b u t c o u l d a l s o be seen as p a r t o f t h e S t r y c h n o s a l k a l o i d s , and by a more complex mechanism i t c o u l d a l s o a c c o u n t f o r t h e C^-C^Q p a r t o f t h e Ib o g a and A s p i d o s p e r m a a l k a l o i d s . M o r e o v e r , t h i s s t r u c t u r e was s e e n i n a number o f monoterpenes, some o f w h i c h had been i s o l a t e d f r o m S t r y c h n o s s p e c i e s . The l a t t e r f e a t u r e l e d Thomas'*"^ t o p r o p o s e t h a t the C^-C^Q u n i t was i n f a c t d e r i v e d f r o m monoterpenes and had i t s o r i g i n i n t h e c o n d e n s a t i o n o f two m e v a l o n i c a c i d m o i e t i e s (21) t o g i v e a c y c l i c s t r u c t u r e ( 2 2 ) . T h i s s t r u c t u r e c o u l d t h e n be e l a b o r a t e d i n t o t h e c y c l o p e n t a n o i d monoterpenes and t h e C g - C ^ p r o g e n i t o r . About t h i s same t i m e two l a b o r a t o r i e s began t o c o n s i d e r an 18 19-21 a c e t a t e d e r i v e d C Q-C, n u n i t . S c h l i t t l e r and L e e t e s u g g e s t e d t h a t t h r e e a c e t a t e u n i t s condensed w i t h m a v a l o n i c a c i d (18) p l u s a o n e - c a r b o n - 9 -F i g u r e 2. The Wenkert p r e p h e n i c a c i d h y p o t h e s i s . ^ ' -10-u n i t t o g i v e a C^Q s t r u c t u r e w i t h t h e same c a r b o n s k e l e t o n as t h a t o f the SPF u n i t ( 2 0 ) . T h i s p r o p o s a l soon l e d t o some i m p o r t a n t i n v e s t i -g a t i o n s . I n t h e e a r l y 1960's, the r e s u l t s f r o m many r a d i o a c t i v e t r a c e r e x p e r i m e n t s began t o c a s t d o u b t s on a l l the h y p o t h e s i s c o n c e r n i n g the 20 21 o r i g i n o f t h e u b i q u i t o u s C^-C^Q u n i t . L e e t e ' , w o r k i n g w i t h 14 R a u w o l f i a S e r p e n t i n a f r o m the Apocynaceae f a m i l y , f e d [ 2 - C ] - t y r o s i n e , 14 14 14 [ 2 - C ] - a l a n i n e , [ 2 - C ] - m e v a l o n i c a c i d and sodium [ 1 - C ] - a c e t a t e , and i s o l a t e d a j m a l i n e (26) and r e s e r p i n e ( 3 ) , b o t h r e p r e s e n t i n g t h e C o r y n a n t h e f a m i l y . A l l o f t h e s e f e e d i n g s r e s u l t e d i n a n e g a t i v e i n c o r p o r a t i o n e x c e p t f o r t h e sodium a c e t a t e e x p e r i m e n t . One q u a r t e r o f t h e a c t i v i t y (22) F i g u r e 3. The Thomas-Wenkert monoterpene h y p o t h e s i s . - 1 1 -i s o l a t e d was f o u n d a t p o s i t i o n s C-3 and C-19 i n a j m a l i n e ( 2 6 ) . The Thomas-Wenkert h y p o t h e s i s r e q u i r e d a c t i v i t y a t C-14, C-19, C-21 and C-16. L e e t e t h e n m o d i f i e d t h e a c e t a t e h y p o t h e s i s t o i n c l u d e t h e c onden-s a t i o n o f t h r e e a c e t y l - c o e n z y m e - A u n i t s ( 2 3 ) , one malonyl-conenzyme-A u n i t (24) and one C ^ - u n i t t o g i v e s t r u c t u r e ( 2 5 ) , w h i c h i s s i m i l a r t o W e n k e rt's SPF u n i t (20) b u t b e t t e r e x p l a i n s t h e e x p e r i m e n t a l r e s u l t s ( F i g u r e 4 ) . (26) (25) F i g u r e 4. L e e t e ' s m o d i f i e d a c e t a t e h y p o t h e s i s . 22 23 Then i n 1965, S c o t t and h i s c o l l a b o r a t o r s ' r e p o r t e d the 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 m e v a l o n a t e ( 2 1 ) , the w e l l - k n o w n " b u i l d i n g b l o c k " i n t e r p e n e b i o s y n t h e s i s , i n t o t h e A s p i d o s p e r m a a l k a l o i d , v i n d o l i n e (7) b i o s y n t h e s i z e d i n C. r o s e u s . Soon th e r e s u l t s o f t r a c e r 24—28 e x p e r i m e n t s f r o m s e v e r a l l a b o r a t o r i e s e s t a b l i s h e d m e v a l o n a t e ( 2 l ) as a t r u e p r o g e n i t o r 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 . E q u a l l y e x c i t i n g was t h e q u i c k e s t a b l i s h m e n t of g e r a n i o l ( 2 7 ) , the i mmediate p r e c u r s o r t o 26 29 t h e m o n oterpenes, as a p r e c u r s o r i n s e v e r a l f a m i l i e s o f t h e s e a l k a l o i d s . ' ' F i g u r e 5 summarizes t h i s new h y p o t h e s i s as s u p p o r t e d by e x p e r i m e n t a l e v i d e n c e . The monoterpene h y p o t h e s i s was now on f i r m g r o u n d . F i g u r e 5. The monoterpene o r i g i n o f t h e C Q-C _ u n i t . - 1 3 -Th e n e x t s t e p i n the b i o s y n t h e s i s o f the C^-C^Q u n i t had to be .the i d e n t i f i c a t i o n o f the h y p o t h e t i c a l c y c l o p e n t a n o i d i n t e r m e d i a t e ( 2 8 ) . As mentioned e a r l i e r , t h e r e were a number of c y c l o p e n t a n o i d ( o r i r i d o i d ) mono t e r p e n e s known w h i c h had the c o r r e c t s t e r e o c h e m i s t r y and, i n some c a s e s , c o - e x i s t e d w i t h i n d o l e a l k a l o i d s i n s e v e r a l 32-34 p l a n t s p e c i e s . B a t t e r s b y and h i s c o - w o r k e r s f e d r a d i o - l a b e l l e d v e n b a n a l i n ( 3 1 ) , g e n i p i n ( 3 2 ) and l o g a n i n ( 3 3 ) t o C. r o s e u s and found 14 t h a t o n l y [CG*2 C H ^ ] - l o g a n i n ( 3 3 ) was i n c o r p o r a t e d i n t o t h r e e major f a m i l i e s o f a l k a l o i d s . T h i s f i n d i n g was q u i c k l y c o n f i r m e d by a S w i s s 35 3 group who were a b l e to i n c o r p o r a t e [C H ^ ] - l o g a n i n i n t o the same t h r e e a l k a l o i d s i n the same p l a n t s p e c i e s . The h i g h l e v e l s o f i n c o r p o r a t i o n a c h i e v e d i n t h e s e s t u d i e s l e f t no doubt as to the i m p o r t a n c e o f t h i s p r o g e n i t o r . Subsequent work i n t h i s a r e a d e m o n s t r a t e d t h e o c c u r r e n c e o f l o g a n i n ( 3 3 ) i n s e v e r a l a l k a l o i d c o n t a i n i n g p l a n t s p e c i e s and i t s b i o s y n t h e s i s from m e v a l o n a t e ( 2 1 ) and g e r a n i o l ( 2 7 ) was c o n f i r m e d . 3 6 " 3 8 The 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 o f i p e c o s i d e ( 3 4 ) , the f i r s t t e t r a --14-39 h y d r o i s o q u i n o l i n e monoterpene t o be d i s c o v e r e d , p r o v i d e d a c l u e t o t h e n e x t s t e p 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 . B a t t e r s b y s p e c u l a t e d (34) (35) t h a t i f h y d r o x y l o g a n i n e l i m i n a t e d t h e e l e m e n t s o f H^O w i t h 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 , t h e n one w o u l d have a m o i e t y w h i c h c o u l d 40 condense d i r e c t l y w i t h t r y p t a m i n e (9) t o g i v e i p e c o s i d e (34) . Such a s t r u c t u r e , c a l l e d s e c o l o g a n i n ( 3 5 ) , would c l o s e l y c o r r e s p o n d t o the c r u c i a l i n t e r m e d i a t e (29) i n t h e Thomas-Wenkert h y p o t h e s i s ( F i g u r e 5 ) . The i s o l a t i o n and s t r u c t u r a l e l u c i d a t i o n o f t h r e e new g l u c o s i d e s l e n t ^ 1 -^2 g r e a t c r e d i b i l i t y to t h i s i d e a . ' M e n t h i a f o l i n ( 3 6 a ) , f o l i a m e n t h i n (36a) (36b) 0 -15-(36b)and i t s d i h y d r o - d e r i v a t i v e c o n t a i n e d - t h e s k e l e t o n o f s e c o l o g a n i n (35) i n a masked l a c t o l f o r m . B a t t e r s b y and h i s c o - w o r k e r s were s u c c e s s f u l i n c l e a v i n g l o g a n i n (33) t o s e c o l o g a n i n (35) and f e e d i n g the l a t t e r as a d o u b l y -l a b e l l e d p r e c u r s o r t o C. r o s e u s p l a n t s . A h i g h l e v e l o f i n c o r p o r a t i o n i n t o s e r p e n t i n e , a j m a l i c i n e ( 3 0 ) , c a t h a r a n t h i n e (6) and p e r i v i n e was f o u n d . I n a d d i t i o n , a l a r g e s c a l e e x t r a c t o f t h i s p l a n t s p e c i e s r e v e a l e d t h e p r e s e n c e o f s e c o l o g a n i n (35) as a n a t u r a l p r o d u c t . Thus a l l the r e q u i r e m e n t s f o r t h i s compound t o be a t r u e p r e c u r s o r 43 44 t o t h e t h r e e f a m i l i e s o f i n d o l e a l k a l o i d s were met. ' The n e x t s t e p i n d e l i n e a t i n g t h i s b i o s y n t h e t i c pathway was t o l o o k f o r t h e p r o d u c t r e s u l t i n g f r o m the c o n d e n s a t i o n o f s e c o l o g a n i n (35) and the n i t r o g e n o u s m o i e t y t r y p t a m i n e ( 9 ) . An i n v i t r o e x p e r i m e n t 45 by the B a t t e r s b y group showed t h a t , a t pH 4.5, t r y p t a m i n e (9) condensed w i t h s e c o l o g a n i n (35) t o g i v e as a m a j o r p r o d u c t v i n c o s i d e (37) and i s o v i n c o s i d e (38) as the m i n o r p r o d u c t . I n an e l e g a n t s e r i e s o f e x p e r i m e n t s , r e p o r t e d by t h e s e w o r k e r s , b o t h o f t h e s e compounds were 4 6 4 7 r a d i o - l a b e l l e d and f e d them t o C. r o s e u s p l a n t s . ' They found t h a t -16-o n l y t h e 36(R) i s o m e r , v i n c o s i d e (37), c o u l d be i n c o r p o r a t e d i n t o the t h r e e i n d o l e a l k a l o i d f a m i l i e s . They were a l s o a b l e t o d e m o n s t r a t e t h a t b o t h i s o m e r s e x i s t e d as n a t u r a l p r o d u c t s i n C. r o s e u s . The p r o b l e m , however, was t h a t v i n c o s i d e (37) h a v i n g the 3B(R) c o n f i g u -r a t i o n was o f o p p o s i t e s t e r e o c h e m i s t r y p o s s e s s e d by t h e C o r y n a n t h e a l k a l o i d s , as r e p r e s e n t e d by a j m a l i c i n e (30). I t was s u g g e s t e d t h a t 48 C-3 underwent i n v e r s i o n w i t h r e t e n t i o n o f t h e h y d r o g e n . T h i s p o i n t 4 9-51 has been t h e s u b j e c t o f much c o n t r o v e r s y . A l a b o r a t o r y i n v e s t i -g a t i o n showed, i n f a c t , t h a t C-3 o f v i n c o s i d e d e r i v a t i v e s was e a s i l y 52-54 e p i m e r i z e d by an o x i d a t i v e - r e d u c t i v e mechanism. T h i s f i n d i n g l e n t g r e a t c r e d e n c e t o B a t t e r s b y ' s o r i g i n a l p r o p o s a l . More r e c e n t l y , a carbon-13 m a g n e t i c r e s o n a n c e (CMR) s t u d y ^ o f v a r i o u s s e c o l o g a n i n d e r i v a t i v e s and an X - r a y d i f f r a c t i o n study"' 6 on v i n c o s i d e (37) c o n f i r m e d t h e e a r l i e r a s s i g n e d s t e r e o c h e m i s t r y of t h e s e compounds. An i n t r i g u i n g r e s u l t made p u b l i c i n l a t e 1977 by a West German g r o u p " ^ has added more f u e l t o t h i s c o n t r o v e r s y . They r e p o r t e d t h a t i n a c e l l - f r e e enzyme e x t r a c t f r o m C. r o s e u s c e l l c u l t u r e s , o n l y t h e 3a(S) i s o v i n c o s i d e (38) c o u l d be i n c o r p o r a t e d i n t o the t h r e e i n d o l e a l k a l o i d f a m i l i e s f o u n d i n t h a t s p e c i e s . They a l s o r e p o r t e d t h a t t h e i r c e l l - f r e e p r e p a r a t i o n s c a t a l y z e d t h e f o r m a t i o n o f o n l y t h e 3a(S) i s o m e r and t h e f o r m a t i o n o f t h e 33(R) v i n c o s i d e (37) was n e v e r o b s e r v e d i n t h e f i v e Apocynaceae s p e c i e s Amsonia tabernaemontianum, R h a z i a  o r i e n t a l i s , R h a z i a s t r i c t a , V i n c a m i n o r and C. r o s e u s . T h i s i s v e r y c o n v i n c i n g e v i d e n c e f o r i s o v i n c o s i d e (38) b e i n g t h e o b l i g a t e p r e c u r s o r . T h i s compound i s now more commonly c a l l e d s t r i c t o s i d i n e , a name s u g g e s t e d by S m i t h who f i r s t i s o l a t e d i t f r o m R. s t r i c t a p l a n t s i n 1968. "^'"^ -17-The s t o r y o f 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 can a t t h i s p o i n t be d i v i d e d between t h e e a r l y s t a g e s w h i c h f o r m t h e c o n d e n s a t i o n p r o d u c t , s t r i c t o s i d i n e ( 3 8 ) , a r i s i n g f r o m monoterpene b i o g e n i s i s and amino a c i d b i o g e n i s i s , and the l a t e r s t a g e s i n w h i c h t h i s compound i s f u n c t i o n a l i z e d and r e a r r a n g e d i n t o the v a r i o u s a l k a l o i d f a m i l i e s . F i g u r e 6 summarizes t h e e a r l y s t a g e s as p r o v e n by e x p e r i m e n t . (38) F i g u r e 6. Summary scheme o f t h e e a r l y s t a g e s o f a l k a l o i d b i o s y n t h e s i s as p r o v e n by e x p e r i m e n t . -18-A number o f i n v e s t i g a t o r s soon began u n r a v e l l i n g the pathways •by w h i c h s t r i c t o s i d i n e (38) was c o n v e r t e d i n t o the v a r i o u s a l k a l o i d f a m i l i e s . I t s c o n v e r s i o n t o the C o r ynanthe s k e l e t o n c o u l d s i m p l y be a c h i e v e d by a l o s s o f a one c a r b o n u n i t and r i n g c l o s u r e t o f o r m r i n g D. Subsequent e l a b o r a t i o n o f t h i s s t r u c t u r e c o u l d r e s u l t i n the o t h e r members o f t h e Corynanthe f a m i l y . F i g u r e 7 summarizes some o f t h e s e p r o p o s a l s . . The n a t u r a l p r o d u c t c a thenamine ( 3 9 ) ^ appeared t o be t h e most immediate r e a r r a n g e m e n t p r o d u c t o f s t r i c t o s i d i n e (38) as i t was f ound t o a c c u m u l a t e i n c e l l - f r e e enzyme p r e p a r a t i o n s o f C. r o s e u s t h a t were d e p r i v e d o f NADPH ( t h e w i d e s p r e a d n a t u r a l r e d u c t i o . n - o x i d a t i o n system) ."^ H y d r o l y s i s and c l e a v a g e o f the e t h y l i d e n e group w i t h l o s s o f f o r m a l d e h y d e from t h e l a t t e r would a l l o w c o n d e n s a t i o n between the n o w - r e l e a s e d a l d e h y d e group and the b a s i c n i t r o g e n . Subsequent d e h y d r a t i o n o f the i n t e r m e d i a t e c a r b o n o l a m i n e would g i v e cathenamine ( 3 9 ) . R e d u c t i o n o f r i n g C g i v e s a j m a l i c i n e (30) and a t r i v i a l e l a b o r a t i o n of r i n g E would g i v e g e i s s o c h i z i n e (40) o r c o r y n a n t h e i n e a l d e h y d e (42a) and c o r y n a n t h e i n e ( 4 2 b ) . A l t e r -n a t i v e l y s t r u c t u r e (38) c o u l d be e l a b o r a t e d t o the immonium s p e c i e s and r e d u c t i o n o f t h e l a t t e r w o u l d g i v e g e i s s o c h i z i n e (40) and c y c l i z a t i o n o f r i n g E would r e s u l t i n a j m a l i c i n e ( 3 0 ) . 62 I n 1969, the B a t t e r s b y group showed t h a t g e i s s o c h i z i n e (40) was p r e s e n t i n C. r o s e u s and was a good p r e c u r s o r n o t o n l y o f the C o r y n a n t h e , -19-I boga and A s pidosperma a l k a l o i d s b u t a l s o o f the S t r y c h n o s f a m i l y r e p r e s e n t e d i n t h a t p l a n t by a k u a m i c i n e ( 4 5 ) . E a r l i e r w o r k ^ ' ^ i n d i c a t e d t h a t c o r y n a n t h e i n e a l d e h y d e (42a) was n o t a p r e c u r s o r o f a j m a l i c i n e (30) o r c a t h a r a n t h i n e (6) i n mature C. r o s e u s p l a n t s . However, s i g n i f i c a n t i n c o r p o r a t i o n was r e p o r t e d i n s e e d l i n g s 6 3 o f t h i s p l a n t . I n f a c t , subsequent work by S c o t t ' s group s u g g e s t e d t h a t t h e s e compounds a r e a l l a c t i v e l y i n v o l v e d i n b i o s y n t h e s i s i n C. r o s e u s s e e d l i n g s . ' ^ However, s i n c e t h i s group based t h e i r c o n -c l u s i o n s m a i n l y on e v i d e n c e from t h i n - l a y e r chromatography t h e d e t a i l s o f b i o s y n t h e s i s i n t h i s a r e a must r e m a i n an open q u e s t i o n . The e l u c i d a t i o n o f the b i o s y n t h e t i c e v e n t s w h i c h must t a k e p l a c e i n the t r a n s f o r m a t i o n o f g e i s s o c h i z i n e (40) t o akuammicine (45) and the o t h e r S t r y c h n o s a l k a l o i d s p r o v i d e d an i n t r i g u i n g c h a l l e n g e . One o f t h e o r i g i n a l p r o p o s a l s c o n c e r n i n g S t r y c h n o s b i o s y n t h e s i s was t h a t o f W e n k e r t . ^ ( F i g u r e 8 ) . The c r u c i a l s t e p i n t h i s scheme i s the a t t a c k a t the 3 - p o s i t i o n o f the i n d o l e by the a c t i v a t e d c a r b o n o f the a l d e h y d o - e s t e r ( p o s s i b l y by a r a d i c a l mechanism) to g i v e a p e n t a c y c l i c i n d o l e n i n e ( 4 1 ) . Subsequent r e a r r a n g e m e n t c o u l d r e s u l t i n t h e l i t t l e -known S t r y c h n o s a l k a l o i d stemmadenine ( 4 3 ) . A t r a n s a n n u l a r c y c l i z a t i o n o f t h e i n d o l e n i n e a l d e h y d e and l o s s o f t h e a l d e h y d e f u n c t i o n a l i t y , would g i v e akuammicine ( 4 5 ) . The n a t u r a l p r o d u c t s f o r m y l s t r y c t a m i n e ( 4 1 ) ^ 68 and i t s C-16 epimer w h i c h were i s o l a t e d r e c e n t l y f r o m Rhazya s t r i c t a add i n t e r e s t i n g s u p p o r t t o the Wenkert h y p o t h e s i s . -20-0H (30) (40) F i g u r e 7. The p o s s i b l e b i o g e n i s i s o f the Corynanthe a l k a l o i d s as s u p p o r t e d by e x p e r i m e n t . - 2 1 --22-6 y A second hypothesis was offered by Scott and Quereshi in 1969. They proposed that the hydroxyindolenine (46) could rearrange to an oxindole (47) which in turn could cyclize to preakuamicine aldehyde (44), a structure which possess the Strychnos skeleton. However, i t is interesting to note that this type of cyclization is a very d i f f i c u l t one to perforin in the laboratory. Compound (44) could then be elaborated to stemmadenine (43) via a ring-opening reaction of a type already known in the laboratory (this reaction w i l l be discussed in detail in Part III of this thesis). A great deal of evidence to support this hypothesis was provided by Scott's laboratory.^ He reported the isolation of the oxindole (47) and i t s incorporation into akuammicine (45) in C. roseus seedlings. Also reported was the isolation of the hydroxyindolenine (46) 69 and preakuammicine (48) . The co-occurrence of these structures lends great credence to the Scott hypothesis. More recently, Heimberger and Scott were able to incorporate geissoschizine (40) into Wieland-Gumlich aldehyde (49) and strychnine ( 4 ) . ^ (40) (4) (49) -23-OH (45) (48) F i g u r e 9. The S c o t t h y p o t h e s i s f o r t h e b i o s y n t h e s i s o f the S t r y c h o s a l k a l o i d s . -24-Wenkert's h y p o t h e s i s f o r the b i o s y n t h e s i s of t h e A s p i d o s p e r m a and I b o g a a l k a l o i d s i s i l l u s t r a t e d i n F i g u r e 10. He p r o p o s e d t h a t the S t r y c h n o s s k e l e t o n ( 5 0 ) , s i m i l a r t o s t r u c t u r e ( 4 2 ) ( F i g u r e 8) c o u l d be th e c r u c i a l p i v o t a l p r e c u r s o r t o the A s p i d o s p e r m a and I b o g a f a m i l i e s . (53) (52) (56) F i g u r e 10. The Wenkert h y p o t h e s i s f o r the b i o s y n t h e s i s o f t h e A s p i d o s p e r m a and Iboga a l k a l o i d s . - 2 5 -The i m p o r t a n t f e a t u r e i n t h i s scheme i s the t r a n s a n n u l a r c y c l i -z a t i o n u s e d t o c o n v e r t s t r u c t u r e (52) t o (53) and s t r u c t u r e (55) t o ( 5 6 ) . A g r e a t d e a l o f e f f o r t i n our l a b o r a t o r i e s was made to t e s t t h i s h y p o t h e s i s i n C. r o s e u s p l a n t s . D e s p i t e t h e s u c c e s s o f t h e c o r r e s -72-74 p o n d i n g l a b o r a t o r y t r a n s f o r m a t i o n , t h e i n v i v o s t u d y s u g g e s t e d t h a t t h i s was n o t a v i a b l e r o u t e . ? 7 ' 7 8 I n t h e l a t t e r s t u d y , quebrachamine (57) and c l e a v a m i n e (58) b o t h l a b e l l e d w i t h t r i t i u m i n t h e a r o m a t i c r i n g , f a i l e d t o i n c o r p o r a t e i n t o c a t h a r a n t h i n e (6) and v i n d o l i n e ( 7 ) . (57) (58) (59) However, o u t o f t h i s work came the s u r p r i s i n g r e s u l t t h a t t h e A s p i d o s p e r m a a l k a l o i d t a b e r s o n i n e (59) was i n c o r p o r a t e d i n t o the more e l a b o r a t e A s p i d o s p e r m a a l k a l o i d v i n d o l i n e (7) and t h e I b o g a a l k a l o i d c a t h a r a n t h i n e ( 6 ) . The r e v e r s e p r o c e s s , t h e t r a n s f o r m a t i o n o f c a t h a r a n t h i n e (6) i n t o t a b a r s o n i n e (59) c o u l d n o t be d e m o n s t r a t e d . T h i s f a c t s t r o n g l y s u g g e s t s t h e b i o s y n t h e t i c sequence t o be 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 . S e q u e n t i a l f e e d i n g e x p e r i m e n t s by the S c o t t g r o u p 6 3 ' ^ u s i n g C. r o s e u s s e e d l i n g s p r o v i d e d e v i d e n c e f o r t h i s p r o p o s a l . A t t h i s p o i n t , one must c o n c l u d e t h a t the Wenkert h y p o t h e s i s i s i n c o r r e c t . -26-Another i m p o r t a n t o b s e r v a t i o n had been made i n our l a b o r a t o r y d u r i n g 7 7 14 the above d e s c r i b e d s t u d y . A d m i n i s t r a t i o n of [3- C ] - t r y p t o p h a n to V i n c a minor p l a n t s over v a r y i n g l e n g t h s of time and o b s e r v i n g the r a t i o s o f a c t i v i t y i n two " r i n g - o p e n " a l k a l o i d s v i n c a d i n e (60) and v i n c a m i n o r e i n e (61), and two " r i n g - c l o s e d " a l k a l o i d s v i n c a d i f f o r m i n e (62) and minovine (63) r e v e a l e d t h a t the r a t i o s were r e l a t i v e l y c o n s t a n t . (60) R = H (62) R =H (61) R = CH 3 (63) R = CH 3 T h i s o b s e r v a t i o n appeared to i n d i c a t e the e x i s t e n c e of a p i v o t a l i n t e r m e d i a t e which was c o n v e r t a b l e to the " r i n g - o p e n e d " and the " r i n g - c l o s e d " Aspidosperma a l k a l o i d s w i t h o u t g o i n g through a t r a n s -a n n u l a r c y c l i z a t i o n r e a c t i o n . R a t i o n a l i z a t i o n of e a r l i e r r e s u l t s from S c o t t ' s l a b o r a t o r y a l s o demanded a new b i o s y n t h e t i c i n t e r m e d i a t e . In v i v o and i n v i t r o e xperiments u s i n g the S t r y c h n o s a l k a l o i d stemmadenine ( 4 3 ) i n d i c a t e d the 80—82 l a t t e r was c o n v e r t e d i n t o b o t h Aspidosperma and Iboga a l k a l o i d s . The s t r u c t u r e f a v o u r e d f o r t h i s i n t e r e m d i a t e was the a c r y l i c e s t e r ( 6 4 ) , which was c l o s e l y r e l a t e d to the Wenkert i n t e r m e d i a t e s (51) and (54) but n e c e s s a r i l y o f a d i f f e r e n t o x i d a t i o n l e v e l . -27-(64) The i s o l a t i o n from Rhazya s p e c i e s a new f a m i l y o f d i m e r i c i n d o l e a l k a l o i d s , c a l l e d the secamines, r e l a t e d d i r e c t l y to the h y p o t h e t i c a l 83 8 >4 a c r y l i c e s t e r . ' L a t e r , t h r e e r e l a t e d monomeric a l k a l o i d s ( 6 5 ) , (66) and ( 6 7 ) , w h i c h were o f p a r t i c u l a r i m p o r t a n c e , were i s o l a t e d from -v, • 8 5 > 8 6 the same s p e c i e s . (65) (66) (67) 87 The Manchester group d i s c o v e r e d a n o t h e r group o f a l k a l o i d s , c a l l e d p r e s e c a m i n e s , which underwent a c i d c a t a l y s e d rearrangement to the p r e v i o u s l y c h a r a c t e r i z e d secamines. They a l s o found t h a t the p r e -secamines were D i e l s - A l d e r t y p e dimers of s e c o d i n e s p o s s e s s i n g an a -a c r y l i c e s t e r f u n c t i o n . The p o s s i b l e p r e s e n c e o f s e c o d i n e (68) and -28-C0 2 CH 3 C0 2 CH 3 (68) (69) i t s 1 5 , 2 0 - d i h y d r o d e r i v a t i v e (67) i n the p l a n t s t r o n g l y s u p p o r t s the h y p o t h e s i s t h a t d e h y d r o s e c o n d i n e (64) may be the p r i v o t a l p r e c u r s o r from which the v a r i o u s f a m i l i e s of i n d o l e a l k a l o i d s i s b i o s y n t h e s i z e d . T h i s c u r r e n t h y p o t h e s i s , i l l u s t r a t e d i n F i g u r e 11, s u g g e s t s t h a t the S t r y c h n o s a l k a l o i d stemmadenine (43) i s i n b i o l o g i c a l e q u i l i b r i u m w i t h d e h y d r o s e c o d i n e (64) v i a the i n t e r m e d i a t e iso-stemmadenine ( 7 0 ) . D e h y d r o s e c o d i n e (64) c o u l d undergo bond f o r m a t i o n between atoms, as shown, to g i v e the c a r b o n s k e l e t o n s of the Aspidosperma and Iboga t y p e s . The s y n t h e s i s and r a d i o l a b e l l i n g of d e h y d r o s e c o d i n e d e r i v a t i v e s was i m p e r a t i v e f o r the e v a l u a t i o n of t h i s new h y p o t h e s i s . To d a t e the s y n t h e s i s o f s t r u c t u r e (64) has n o t been p o s s i b l e as i t i s w e l l known t h a t d i h y d r o - p y r i d i n e m o i e t i e s (71) undergo v e r y f a c i l e o x i d a t i o n to the p y r i d i n i u m system (78) or r a p i d r e a r r a n g e m e n t s and p o l y m e r i z a t i o n : * T h i s b i o g e n e t i c numbering system was s u g g e s t e d by LeMen and T a y l o r . (71) R (72) -29-(60) (58) F i g u r e 11. H y p o t h e t i c a l b i o s y n t h e s i s o f the Aspidosperma and Iboga a l k a l o i d s from the S t r y c h n o s f a m i l y v i a d e h y d r o -s e c o d i n e . -30-Therefore, a more practical synthetic target was the tetrahydro-acrylic ester, secodine (68) and i t s hydrated derivative 16,17-dihydrosecodine-17-ol (69). These two compounds soon became available for biosynthetic investigation. 89 Battersby's group synthesized the hydroxy ester (69) and determined by isotopic dilution that i t was present in Rhazya brientalis plants and possibly present in (3. roseus seedlings. Both structures (68) 90 and (69) were synthesized in our laboratories by Drs. R. Sood and 91 J. Beck. In the next section of this thesis, this work and i t s biosynthetic implications w i l l be described. Part II of this thesis w i l l discuss investigations into two other families of indole alkaloids for which no biosynthetic evidence has been obtained. These two groups, the ulein-type and the pyridocarbazole-type of indole alkaloids are considered somewhat anomalous in that they do not possess the normal two-carbon bridge between the indole portion and the basic nitrogen atom. In the uleine-type structures this bridge i s absent, while in the pyridocarbazole alkaloids a three-carbon bridge i s found. Clearly, the evaluation of the biosynthesis of these compounds provides an interesting challenge. Part III of this thesis describes work directed towards the synthesis of the radio-labelled precursors preakuammicine aldehyde (44), preakuammicine (48), stemmadenine (43) and i t s iso-derivative (70). As previously described these structures are deeply implicated in the later stages of biosynthesis of a l l indole alkaloids and the possession of them in suitable radio-labelled form would be extremely desirable. - 3 1 -DISCUSSION As was r e v e a l e d i n t h e I n t r o d u c t i o n , the b i o s y n t h e s i s o f b o t h t h e Cg-C^ n u n i t f r o m t h e monoterpene pathway and t h e t r y p t a m i n e p o r t i o n o f t h e s t r u c t u r e s o f t h e f o u r l a r g e i n d o l e a l k a l o i d f a m i l i e s h as b e e n f i r m l y e s t a b l i s h e d . The i m m e d i a t e c h a l l e n g e , however, was the t e s t i n g o f t h e d e h y d r o s e c o d i n e h y p o t h e s i s ( F i g u r e 11) p u t f o r w a r d i n d e p e n d e n t l y by S c o t t and K u t n e y . As m e n t i o n e d e a r l i e r , 16,17-d i h y d r o s e c o d i n - 1 7 - o l (69) was s y n t h e s i z e d and shown t o p o s s i b l y be 89 p r e s e n t i n (3. r o s e u s p l a n t s by t h e B a t t e r s b y g roup. The s y n t h e s i s o f (69) and i t s d e h y d r a t i o n p r o d u c t s e c o d i n e (68) was a l s o a c c o m p l i s h e d i n 90 91 our l a b o r a t o r i e s . ' I n t h i s s e c t i o n t h e r e w i l l be d e s c r i b e d a m o d i f i e d s y n t h e s i s o f r a d i o - l a b e l l e d s e c o d i n e (68) and 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 A s p i d o s p e r m a a l k a l o i d s i n _C. r o s e u s p l a n t s . 90 A l r e a d y a v a i l a b l e i n our l a b o r a t o r i e s was the 2 - c a r b o e t h o x y i n d o l e d e r i v a t i v e (73) p r e p a r e d a c c o r d i n g t o the sequence shown i n F i g u r e 8. U s i n g t h i s compound as s t a r t i n g m a t e r i a l 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l (69) and s e c o d i n e (68) were s y n t h e s i z e d , by t h e sequence shown i n F i g u r e 13, to o b t a i n s u f f i c i e n t q u a n t i t i e s f o r r a d i o - l a b e l l i n g and p l a n t f e e d i n g e x p e r i m e n t s . The c h l o r o e t h y l i n d o l e (73) was c o u p l e d w i t h 3 - e t h y l p y r i d i n e (74) i n a s e a l e d t u b e a t 120° t o g i v e t h e p y r i d i n i u m s a l t (75) i n h i g h y i e l d . Sodium b o r o h y d r i d e r e d u c t i o n t o the t e t r a h y d r o p y r i d i n e d e r i v a t i v e (76) f o l l o w e d by l i t h i u m aluminum h y d r i d e r e d u c t i o n o f t h e e s t e r g roup gave the a l c o h o l ( 7 7 ) , a l s o i n good y i e l d . -32-(73) 9 0 F i g u r e 12. The S y n t h e s i s o f the c h l o r o - i n d o l e (73) The n e x t s t e p i n t h i s sequence was d e s i g n e d to l e n g t h e n the s i d e -c h a i n by one c a r b o n i n a manner which would a l l o w the i n t r o d u c t i o n o f c a r b o n 14 u s i n g an i n e x p e n s i v e and c o n v e n i e n t r e a g e n t . T h i s was a c c o m p l i s h e d by d i s p l a c i n g the c o r r e s p o n d i n g b e n z o a t e (78) w i t h c y a n i d e i o n to g i v e compound (79) . Subsequent h y d r o l y s i s of (79) w i t h 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 the c o r r e s p o n d i n g m e t h y l e s t e r ( 8 0 ) . When a r o m a t i c a l l y l a b e l l e d s e c o d i n e (68) was r e q u i r e d , t r i t i u m was i n t r o d u c e d a t t h i s s t a g e i n the s y n t h e s i s . The e s t e r (80) was s t i r r e d w i t h 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 ( p r e p a r e d from t r i f l u o r o a c e t i c 3 a n h y d r i d e and H^O) f o r 48 h o u r s . A f t e r chromatography and r e c r y s t a l l i --33-n (73) 'N ( 7 4 ) 0 C C C 1 OH (77) OCOC 6H 5 (78) CO2CH3 (81) NaBH, CO2CH3 (69) LiAlH, KCN HC02Et K a H (75) NaBH, -N N X 0 2 E t H L (76) -N (79) II ll N' H CN HC1/CK30H CO2CH3 (80) (68) CO2CH3 F i g u r e 13. The Kutney S y n t h e s i s o f s e c o d i n e ( 6 8 ) 9 0 ' 9 1 -34-z a t i o n t o c o n s t a n t r a d i o a c t i v i t y , a r o m a t i c a l l y l a b e l l e d (80) was o b t a i n a b l e i n 80% y i e l d . To c o m p l e t e t h e s e c o d i n e s k e l e t o n , 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 e n e group a to t h e e s t e r group was a c h i e v e d by t r e a t i n g t h e a n i o n o f (80) w i t h m e t h y l f o r m a t e and c a r e f u l l y r e d u c i n g t h e r e s u l t i n g p r o d u c t (81) w i t h sodium b o r o h y d r i d e . T h i s r e a c t i o n had t o be p e r f o r m e d a t -30° and m o n i t o r e d by t h i n - l a y e r c h r o m a t o g r a p h y . Good y i e l d s o f t h e e s t e r a l c o h o l (69) were o b t a i n e d o n l y when p r e c a u t i o n s a g a i n s t o v e r -r e d u c t i o n t o the c o r r e s p o n d i n g d i o l were e x e r c i s e d . D e h y d r a t i o n o f the a l c o h o l e s t e r (69) u s i n g sodium h y d r i d e gave th e somewhat u n s t a b l e s e c o d i n e ( 6 8 ) . I t was f o u n d t h a t i t was b e s t t o s t o r e t h e a l c o h o l (69) as a c r y s t a l l i n e m a t e r i a l and p r e p a r e t h e s e c o d i n e f o r f e e d i n g e x p e r i m e n t s i m m e d i a t e l y b e f o r e u s e . Thus, we were a b l e t o s t o r e [ a r - H ] - 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l and [ C 0 2 C H 3 ] - 1 6 , 1 7 - d i h y d r o -s e c o d i n - 1 7 - o l f o r c o n v e r s i o n to the c o r r e s p o n d i n g l y l a b e l l e d s e c o d i n e p r e c u r s o r ( 6 8 ) . 90 The i n i t i a l 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 s e new p r e c u r s o r s , w h i c h were done by Dr. Sood i n our l a b o r a t o r i e s , i n V i n c a m i n o r r e v e a l e d t h a t t h i s p l a n t s y s t e m c o u l d n o t u t i l i z e t h e a l c o h o l ( 6 9 ) . S e c o d i n e was, however, c o n v e r t e d i n t o v i n c a m i n e (82) and, m i n o v i n e (83) i n a l o w b u t d e f i n i t e i n c o r p o r a t i o n . I n C_. r o s e u s p l a n t s a l o w i n c o r p o r a t i o n o f C0 2CH^\ CH3 CO2CH3 (82) (83) 1 -35-o n l y s e c o d i n e (68) i n t o v i n d o l i n e (7) and c a t h a r a n t h i n e (6) c o u l d be o b t a i n e d . S t u d i e s w i t h d o u b l y - l a b e l l e d p r e c u r s o r s w i t h a known r a t i o o f r a d i o a c t i v i t y f r o m t r i t i u m i n t h e a r o m a t i c r i n g and carbon-14 i n the carbomethoxy group o f s e c o d i n e ( 6 8 ) , p r o v i d e d t h e i m p o r t a n t i n f o r m a t i o n t h a t a t l e a s t the i n d o l i c - e s t e r p a r t o f t h e m o l e c u l e was b e i n g u t i l i z e d 90 91 by the p l a n t . ' T h e r e f o r e , i t w o u l d be h i g h l y d e s i r a b l e t o a l s o have t h e s e c o d i n e m o l e c u l e r a d i o - l a b e l l e d i n t h e p i p e r i d e i n e p o r t i o n , as w e l l , t o c o n f i r m u t i l i z a t i o n o f t h e whole s t r u c t u r e . The s y n t h e s i s o f s e c o d i n e (68) l a b e l l e d i n t h i s way became t h e n e x t s t a g e i n t h e e v a l u a t i o n o f s e c o d i n e as a p r e c u r s o r common to the b i o s y n t h e s i s o f the m a j o r f a m i l i e s o f i n d o l e a l k a l o i d s . C l e a r l y , i t i s o f g r e a t a d v a n t a g e t o i n t r o d u c e r a d i o - l a b e l s n e a r t h e c o m p l e t i o n o f a l e n g t h y s y n t h e s i s o f a d e s i r e d p r e c u r s o r i n c o n -s i d e r a t i o n o f the expense o f the e x p e r i m e n t and o f the s a f e t y o f the w o r k e r s i n v o l v e d . I f a r a d i o - l a b e l i s i n t r o d u c e d e a r l y i n the se q u e n c e , a h i g h e r t o t a l a c t i v i t y must be h a n d l e d d u r i n g a g r e a t e r number o f m a n i p u l a t i o n s . Thus, i f one d e s i r e d i n t r o d u c t i o n o f a r a d i o - l a b e l i n t o t h e p i p e r d e i n e p o r t i o n o f s e c o d i n e (68) the f i r s t s y n t h e t i c r o u t e ( F i g u r e 13) w o u l d n o t be p a r t i c u l a r l y f a v o r a b l e . An a l t e r n a t e s y n t h e s i s o f s e c o d i n e (68) was r e q u i r e d . The b a s i c r e q u i r e m e n t i n a second s y n t h e t i c r o u t e t o s e c o d i n e (68) was t h a t i f the p i p e r d e i n e u n i t c a r r i e d a r a d i o - l a b e l i t s h o u l d be c o u p l e d t o the t r y p t a m i n e p o r t i o n by a s i m p l e p r o c e d u r e near the end o f t h e sequence. The a l c o h o l e s t e r (84) was a v a i l a b l e i n our l a b o r a t o r i e s by the sequence o u t l i n e d i n F i g u r e 14. Due t o the i n i t i a l d evelopment o f t h i s sequence by -36-92 W e s t c o t t , b u l k q u a n t i t i e s o f the a l c o h o l e s t e r (84) c o u l d be o b t a i n e d and i t was c h o s e n to be the s t a r t i n g m a t e r i a l i n t h e new s y n t h e s i s . The c o m m e r c i a l l y a v a i l a b l e i n d o l e e s t e r (85) was r e d u c e d to the c o r r e s p o n d i n g a l c o h o l (86) and c o n v e r t e d t o the b e n z o a t e e s t e r ( 8 7 ) . Subsequent i n t r o d u c t i o n o f c y a n i d e and m e t h a n o l y s i s o f the cyano group y i e l d e d the h o m o l ogated e s t e r (89) . T r e a t m e n t o f t h e l a t t e r w i t h e t h y l e n e o x i d e i n the p r e s e n c e o f s t a n n i c c h l o r i d e gave t h e a l c o h o l (84) i n good y i e l d and as a s t a b l e m a t e r i a l t h a t c o u l d be c o n v e n i e n t l y s t o r e d . L i A l H , H (85) (84) (86) (89) 0 C0C1 O H H C l C H 3 0 H O 2 C H 3 (87) F i g u r e 14. The S y n t h e s i s o f e s t e r a l c o h o l (84) 92 0 C 0 C 5 H 5 K C N (88) C N W i t h t h i s a l c o h o l i n hand s t u d i e s were i n i t i a t e d t o d e v e l o p methodology f o r the c o u p l i n g w i t h the 3 - e t h y l p y r i d i n e u n i t ( 7 4 ) . Our f i r s t c o n s i d e r -a t i o n was t o d i s p l a c e the h y d r o x y l w i t h c h l o r i d e i o n u s i n g m e t h a n o l i c h y d r o g e n 93 c h l o r i d e a c c o r d i n g to a p r o c e d u r e p u b l i s h e d by Wenkert. T h i s method met -37-w i t h o n l y p a r t i a l s u c c e s s as t h e p r o d u c t was c o n t a m i n a t e d w i t h s e v e r a l o t h e r components. Our a t t e n t i o n t u r n e d t o c o n s i d e r a t i o n o f t h e c o r r e s p o n d i n g p r i m a r y t o s y l a t e . I t i s w e l l known t h a t p r i m a r y t o s y l a t e s a r e good l e a v i n g groups i n n u c l e o p h i l i c d i s p l a c e m e n t r e a c t i o n s . Therefore,, i f t h e t o s y l a t e d e r i v a t i v e c o u l d be formed u s i n g 3 - e t h y l p y r i d i n e as the b a s e , the d e s i r e d p y r i d i n i u m s a l t d e r i v a t i v e w o u l d be t h e e x p e c t e d p r o d u c t . T h i s a p p r o a c h was i n d e e d s u c c e s s f u l . The a l c o h o l (84) was d i s s o l v e d i n m e t h y l e n e c h l o r i d e and c o o l e d t o 0° and f r e s h l y r e c r y s t a l l i z e d t o s y l c h l o r i d e was added. A f t e r two h o u r s t h i s m i x t u r e was s l o w l y warmed t o r e f l u x u n t i l t h i n l a y e r c h r o m a t o g r a p h y ( t i c ) i n d i c a t e d t h a t o n l y b a s e - l i n e m a t e r i a l was p r e s e n t . A f t e r r e m o v a l o f t h e s o l v e n t the r e s i d u e was d i s s o l v e d i n m e t h a n o l and t r e a t e d w i t h sodium b o r o h y d r i d e to g i v e the t e t r a h y d r o p y r i d i n e s t r u c t u r e ( 8 0 ) , w h i c h was i d e n t i c a l w i t h the c o r r e s p o n d i n g m a t e r i a l f r o m the f i r s t s y n t h e s i s o f s e c o d i n e shown i n F i g u r e 13. (84) (90) F i g u r e 15. An a l t e r n a t e s y n t h e s i s o f p i p e r d e i n e e s t e r ( 8 0 ) . T h i s sequence a l l o w e d u s , t h e n , t o c o u p l e the i n d o l i c p o r t i o n t o 3 - e t h y l p y r i d i n e and o b t a i n the r e d u c e d p r o d u c t (80) i n a " o n e - p o t " o p e r a t i o n , i n 21% y i e l d . S u b s e q u e n t l y , t h i s sequence was s t u d i e d by o t h e r s -38-94 i n o ur group and t h e y i e l d was r a i s e d t o 35%. The n e x t s t e p i n our i n v e s t i g a t i o n s r e q u i r e d a method f o r i n t r o d u c i n g a r a d i o - l a b e l i n t o t h e p i p e r i d e i n e m o i e t y o f s e c o d i n e ( 6 8 ) . T r i t i u m was c h o s e n o v e r c a r b o n 14 f o r t h i s p u r p o s e s i n c e i n t r o d u c t i o n o f the l a t t e r w o u l d r e q u i r e a more e l a b o r a t e s y n t h e s i s o f the 3 - e t h y l p y r i d i n e s k e l e t o n . I f t r i t i u m were t o be used i t was d e s i r a b l e , i n the f i r s t i n s t a n c e , t o i n t r o d u c e t h i s i s o t o p e i n t o t he e t h y l s i d e c h a i n r a t h e r t h a n i n t o t he p y r i d i n e r i n g b e c a u s e t h e l a t t e r u n dergoes e x t e n s i v e e l a b o r a t i o n d u r i n g t h e b i o s y n t h e s i s o f the v a r i o u s a l k a l o i d s k e l e t o n s and, t h e r e f o r e , a s i g n i f i c a n t l o s s o f t h e l a b e l w o u l d be e x p e c t e d . Our f i r s t c o n s i d e r a t i o n f o r t r i t i u m i n t r o d u c t i o n was t o exchange th e l a b i l e p r o t o n s i n the m e t h y l group o f 3 - a c e t y l p y r i d i n e (.91) and t h e n remove the oxygen f u n c t i o n under c o n d i t i o n s w h i c h w o u l d n o t a l l o w exchange b a c k to t h e o r i g i n a l i n a c t i v e s y s t e m . I n a model s t u d y u s i n g d e u t e r i u m i t 2 was f o u n d t h a t , i n a two-phase s y s t e m o f t e t r a h y d r o f u r a n and H^O c o n t a i n i n g sodium c a r b o n a t e , more t h a n 95% o f t h e m e t h y l p r o t o n s c o u l d be exchanged as measured by p r o t o n n u c l e a r m a g n e t i c r e s o n a n c e (p m r ) . T h i s d e t e r m i n a t i o n was made by i n t e g r a t i n g the s i g n a l a t T 7.4. W i t h t h i s d e u t e r a t e d m a t e r i a l i n hand we began a s t u d y d i r e c t e d a t t h e r e m o v a l o f the oxygen f u n c t i o n . Or*** (91) (92) -39-The c o n v e n t i o n a l p r o c e d u r e f o r d e o x y g e n a t i o n o f a l d e h y d e s and k e t o n e s w h i c h w o u l d p r o b a b l y n o t i n v o l v e a s i g n i f i c a n t d e g r e e o f e n o l i z a t i o n c o n c e r n e d t h e f o r m a t i o n o f t h e c o r r e s p o n d i n g e t h y l e n e t h i o k e t a l f o l l o w e d by d e s u l p h u r i z a t i o n w i t h Raney n i c k e l . T r e a t m e n t o f t h e d e u t e r a t e d k e t o n e (92) w i t h e t h a n e d i t h i o l and b o r o n t r i f l u o r i d e e t h e r a t e gave t h e t h i o k e t a l ( 9 3 ) . E x a m i n a t i o n o f the pmr s p e c t r u m o f t h i s p r o d u c t r e v e a l e d m u l t i p l e t s i g n a l s a t T 6 . 6 c o r r e s p o n d i n g t o t h e f o u r m e t h y l e n e p r o t o n s i n the t h i o k e t a l r i n g . I n t e g r a t i o n o f t h e s i g n a l a t T 7 . 9 r e v e a l e d t h a t o n l y 1.4% l o s s o f d e u t e r i u m f r o m the m e t h y l group had o c c u r r e d . D e s u l p h u r i z a t i o n o f t h e t h i o k e t a l (93) u s i n g Raney n i c k e l gave i n i t i a l l y a p o o r r e c o v e r y o f d e u t e r o - 3 - e t h y l p y r i d i n e ( 9 4 ) . I t was r e a s o n e d t h a t p e r h a p s t h e b a s i c n i t r o g e n o f the p y r i d i n e r i n g was c o o r d i n a t i n g w i t h the Raney n i c k e l i n such a way as t o r e t a r d i t s l i b e r a t i o n . When the r e a c t i o n m i x t u r e was worked up w i t h a 5% s o l u t i o n o f aqueous sodium h y d r o x i d e a good y i e l d o f the p r o d u c t was o b t a i n e d . When d e s u l p h u r i z a t i o n of t h e d e u t e r a t e d t h i o k e t a l (93) was a c h i e v e d , the pmr s p e c t r u m o f the d e u t e r a t e d 3 - e t h y l p y r i d i n e (94) showed t h a t a p p r o x i m a t e l y 2.1% l o s s o f l a b l e -40-had o c c u r r e d on t h e s u r f a c e o f t h e Raney n i c k e l . T h i s s m a l l l o s s c o u l d be e a s i l y t o l e r a t e d i n a s u b s e q u e n t t r i t i a t i o n p r o c e d u r e . Our s e c o n d c o n s i d e r a t i o n f o r i n t r o d u c i n g t r i t i u m i n t o t h e s i d e -c h a i n d 3 - e t h y l p y r i d i n e i n v o l v e d l a b e l l i n g t he m e t h y l e n e group. I n i t i a l s t u d i e s u s i n g sodium b o r o h y d r i d e showed t h a t the k e t o n e f u n c t i o n o f 3-a c e t y l p y r i d i n e c o u l d be r e d u c e d under a p r o t i c c o n d i t i o n s . I n t h i s manner, so d i u m b o r o t r i t i i d e was e f f i c i e n t l y u s e d t o i n t r o d u c e t r i t i u m i n t o t h e s i d e - c h a i n . A p r o t i c c o n d i t i o n s m i n i m i z e d l o s s o f l a b e l t h r o u g h exchange w i t h p r o t o n s o u r c e . The n e x t s t e p was the h y d r o g e n o l y s i s o f the a l c o h o l group i n the p r o d u c t o b t a i n e d f r o m s o d i u m b o r o h y d r i d e r e d u c t i o n o f (.91). Our p l a n i n v o l v e d c o n v e r t i n g t h e h y d r o x y l i n t o a b e t t e r l e a v i n g g roup. We a t t e m p t e d f i r s t t o make the c o r r e s p o n d i n g t o s y l a t e ( 9 6 ) , m e s y l a t e (97) and b e n z o a t e (98) (99) R = - C — C H 3 d e r i v a t i v e s , b u t i n each c a s e o n l y the s t a r t i n g a l c o h o l c o u l d be s e e n by t h i n l a y e r c h romatography o r r e c o v e r e d f r o m t h e r e a c t i o n m i x t u r e . The - 4 1 -c o r r e s p o n d i n g a c e t a t e (99) c o u l d , however, be o b t a i n e d i n h i g h y i e l d . The i d e n t i t y o f t h i s m a t e r i a l was e s t a b l i s h e d by i t s mass s p e c t r u m w h i c h e x h i b i t e d a m o l e c u l a r i o n peak a t m/e 165 and an i n f r a r e d s p e c t r u m w h i c h showed the p r e s e n c e o f a c a r b o n y l a b s o r p t i o n a t 1725 cm ^. F u r t h e r -more, the pmr s p e c t r u m o f t h i s p r o d u c t showed a t h r e e - p r o t o n s i n g l e t s i g n a l a t T 7 . 9 3 w h i c h was e x p e c t e d f o r t h e p r e s e n c e o f an a c e t a t e g r o u p . The s u b s e q u e n t h y d r o g e n o l y s i s o f t h e oxygen f u n c t i o n a l i t y was a c h i e v e d by t r e a t i n g t h e a c e t a t e (99) w i t h an atmosphere o f h y d r o g e n gas i n the p r e s e n c e o f p a l l a d i u m - o n - c h a r c o a l c a t a l y s t i n d i l u t e aqueous h y d r o c h l o r i c a c i d s o l u t i o n . I n t h i s way, 3 - e t h y l p y r i d i n e (74) was o b t a i n e d i n 65% o v e r a l l y i e l d . When t h i s sequence was used t o p r o d u c e r a d i o - l a b e l l e d m a t e r i a l , 3 s o dium b o r o h y d r i d e - H was a l l o w e d t o r e a c t w i t h the c a r b o n y l compound f o r an e x t e n d e d p e r i o d o f t i m e to e n s u r e a c o m p l e t e t r a n s f e r o f the t r i t i u m t o t h e s u b s t r a t e . The r e a c t i o n was t h e n c o m p l e t e d by t h e a d d i t i o n o f i n a c t i v e s o d i u m b o r o h y d r i d e . The o v e r a l l i n t r o d u c t i o n and r e t e n t i o n o f t h e r a d i o - l a b e l i n t h i s sequence as measured by the s p e c i f i c r a d i o a c t i v i t y o f 3 - e t h y l p y r i d i n e (74) was 87%. Of the two t r i t i u m l a b e l l e d forms of 3 - e t h y l p y r i d i n e (74) w h i c h were now a v a i l a b l e , the m e t h y l e n e l a b e l l e d d e r i v a t i v e was c h osen f o r c o u p l i n g t o t h e i n d o l e a l c o h o l ( 8 4 ) . The p r o d u c t o f t h i s r e a c t i o n was e l a b o r a t e d , as p r e v i o u s l y d e s c r i b e d , t o g i v e 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l (69) w i t h a s p e c i f i c r a d i o a c t i v i t y i n t h e p i p e r d e i n e s i d e - c h a i n o f 2.17x10"^ dpm p e r m i l l i m o l e . -42-W i t h s i d e - c h a i n l a b e l l e d s e c o d i n e (68) i n hand, p r o o f t h a t the e n t i r e p r e c u r s o r was b e i n g u t i l i z e d by t h e p l a n t s y s t e m to b i o s y n t h e s i z e t h e A s p i d o s p e r m a a l k a l o i d s c o u l d be o b t a i n e d . To f u r t h e r e v a l u a t e the b i o s y n t h e s i s o f t h i s p r e c u r s o r , d o u b l y - l a b e l l e d 3 14 [19- H, C C ^ C K ^ ] - s e c o d i n e h a v i n g a r a d i o a c t i v i t y r a t i o , o f t r i t i u m o v e r c a r -bon 14, e q u a l t o 3.00 was f e d t o a C_. r o s e u s p l a n t u s i n g the c o t t o n w i c k f e e d i n g t e c h n i q u e . A f t e r n i n e d a y s , t h e p l a n t was m a s c e r a t e d i n m e t h a n o l i n a W a r i n g b l e n d e r and v i n d o l i n e (7) was i s o l a t e d i n a manner s i m i l a r 90 91 to t h a t d e s c r i b e d by Sood and Beck. T h i s m a t e r i a l was r e c r y s t a l i z e d 91 and r e d u c e d w i t h l i t h i u m aluminum h y d r i d e to v i n d o l i n o l (100) . The l a t t e r was t h e n r e c r y s t a l i z e d to a c o n s t a n t r a d i o a c t i v i t y w i t h a r a t i o 3 14 H/ C e q u a l t o 3.31. I n v i e w o f the r e s u l t s o f many f e e d i n g e x p e r i m e n t s i n t h i s a r e a , t h i s r a t i o was c o n s i d e r e d t o be w i t h i n e x p e r i m e n t a l e r r o r . However, to v e r i f y t h i s r e s u l t , a second e x p e r i m e n t was c a r r i e d o u t . I n a second f e e d i n g e x p e r i m e n t u s i n g d o u b l y - l a b e l l e d s e c o d i n e (68) a r a d i o a c t i v i t y r a t i o o f 1.54 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 . I n a manner s i m i l a r t o t h a t d e s c r i b e d above, the i s o l a t e d v i n d o l i n e (7) was - 4 3 -c o n v e r t e d t o v i n d o l i n o l (100) and the l a t t e r was f o u n d t o have a c o n s t a n t r a t i o o f r a d i o a c t i v i t y e q u a l t o 1.35. These r e s u l t s a r e summarized i n T a b l e 1. The i m p o r t a n t c o n c l u s i o n t h a t can be drawn f r o m t h e r e s u l t s o f t h e s e two f e e d i n g e x p e r i m e n t s i s t h a t the e n t i r e s e c o d i n e m o l e c u l e (68) i s i n d e e d u t i l i z e d by t h e p l a n t s y s t e m f o r the b i o s y n t h e s i s o f t h e 95 A s p i d o s p e r m a a l k a l o i d v i n d o l i n e ( 7 ) . T a b l e 1. The i n c o r p o r a t i o n o f [19- H, C0„CH]-secodine i n t o v i n d o l i n e (7) Exp. R a t i o o f A c t i v i t y Fed ( 3 H / 1 4 C ) R a t i o o f A c t i v i t y I s o l a t e d 1 3.00 3.31 2 1.54 1.35 The work d e s c r i b e d i n t h i s s e c t i o n forms p a r t o f an o n g o i n g program, i n o u r l a b o r a t o r i e s , d e s i g n e d t o e v a l u a t e the i m p l i c a t i o n s o f the d e h y d r o s e c o d i n e h y p o t h e s i s i n the b i o s y n t h e s i s o f many f a m i l i e s o f i n d o l e a l k a l o i d s . C u r r e n t l y work i s p r o g r e s s i n g on the s y n t h e s i s o f a s t a b l e chromium complex o f d e h y d r o s e c o d i n e . A l s o i n the n e a r f u t u r e t h e s e p r e c u r s o r s w i l l be e v a l u a t e d i n c e l l - f r e e enzyme p r e p a r a t i o n s f r o m s e v e r a l p l a n t s p e c i e s . The n e x t p a r t o f t h i s t h e s i s d e s c r i b e s some p r e l i m i n a r y r e s u l t s f r o m an i n v e s t i g a t i o n i n t o the b i o s y n t h e s i s o f a l k a l o i d s o f the u l e i n e -and o l i v a c i n e - t y p e s . -44-EXP ERIMENTAL T h i n l a y e r c h r o m a t o g r a p h i c s t u d i e s were c a r r i e d o u t u s i n g e i t h e r M erck s i l i c a g e l o r Woelm n e u t r a l a l u m i n a as t h e a b s o r b e n t s . The c h r o m a t o p l a t e s , 0.3 mm i n t h i c k n e s s , were a i r d r i e d and a c t i v a t e d i n an oven a t 120°C f o r more t h a n t h r e e h o u r s . I n p r e p a r a t i v e s c a l e t h i n l a y e r c h romatography a t h i c k e r l a y e r o f 0.5 mm was u s e d . I n a l l c a s e s , e l e c t r o n i c p hosphor ( a b o u t 2% by w e i g h t ) was added t o t h e a b s o r b e n t as f l u o r e s c e n t i n d i c a t o r . The c h r o m a t o p l a t e s were d e v e l o p e d i n e i t h e r c h l o r o f o r m , o r a m i x t u r e o f c h l o r o f o r m p l u s 2% m e t h a n o l , and examined under a s h o r t and l o n g w a v e l e n g t h u l t r a v i o l e t s c a n n i n g lamp and v i s u a l i z e d w i t h a n t i m o n y p e n t a c h l o r i d e s p r a y o r i o d i n e v a p o r s . Column ch r o m a t o g r a p h y was p e r f o r m e d u s i n g Woelm s i l i c a g e l o r Woelm n e u t r a l a l u m i n a . The d i m e n s i o n s o f t h e columns were g e n e r a l l y m a i n t a i n e d a t the a c c e p t e d optimum r a t i o o f d i a m e t e r to h e i g h t as 1:10. T hroughout t h i s work, the s o l v e n t s were d i s t i l l e d b e f o r e u s e . Gas l i q u i d c h r o m a t o g r a p h y ( g l c ) was p e r f o r m e d on a V a r i a n model A-90-P i n s t r u m e n t , u s i n g h e l i u m as a c a r r i e r gas a t a f l o w r a t e o f 80-85 ml/min. F o r r o u t i n e a n a l y s i s a column 1/4 i n c h x 10 f e e t c o n s i s t e d o f 20% carbowax on 60/80 mesh Chromsorb W s u p p o r t p a c k i n g was u s e d . A l l work was done u s i n g a column t e m p e r a t u r e o f 175-205°. U l t r a v i o l e t s p e c t r a were r e c o r d e d i n m e t h a n o l on a C a r y 11 o r a C a r y 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 and t h e a b s o r p t i o n bands (A. ) a r e max r e c o r d e d i n nanometers (nm). I n f r a r e d s p e c t r a were o b t a i n e d on a P e r k i n -- 4 5 -E l m e r model 21 double-beam s p e c t r o p h o t o m e t e r . Samples were measured i n KBr p e l l e t s o r i n c h l o r o f o r m s o l u t i o n . The p o s i t i o n s o f a b s o r p t i o n maxima (v ) a r e q u o t e d i n wave numbers (cm . max P r o t o n m a g n e t i c r e s o n a n c e (p m r ) s p e c t r a were measured i n d e u t e r o c h l o r o f o r m a t room t e m p e r a t u r e . These were measured a t 60 MHz on a V a r i a n T-60 s p e c t r o m e t e r . Where a d d i t i o n a l r e s o l u t i o n o r d o u b l e r e s o n a n c e s t u d i e s were r e q u i r e d , the s p e c t r a were measured a t 100 MHz u s i n g a V a r i a n HA-100 i n s t r u m e n t . The p o s i t i o n s o f a l l p.m.r. a b s o r p t i o n s i g n a l s a r e g i v e n i n the T i e r s T s c a l e w i t h t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d a t T 10.00. F o r m u l t i p l e t s i g n a l s the x - v a l u e s g i v e n r e p r e s e n t the c e n t e r o f the s i g n a l . Mass s p e c t r a were measured on an A s s o c i a t e d E l e c t r i c a l I n d u s t r i e s MS-9 d o u b l e - f o c u s i n g mass s p e c t r o m e t e r o r an A t l a s CH-4 mass s p e c t r o -m e t e r . F r a g m e n t a t i o n d a t a i s g i v e n i n mass t o c h a r g e r a t i o s (m/e) f o l l o w e d by p e r c e n t r e l a t i v e abundance. H i g h r e s o l u t i o n measurements were a l s o d e t e r m i n e d on the MS-9 i n s t r u m e n t u s i n g s u i t a b l e s t a n d a r d s o f known m o l e c u l a r w e i g h t . R a d i o a c t i v i t y was measured u s i n g a N u c l e a r - C h i c a g o Mark I M o d e l 6860 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 . The measurement i n c o u n t s p e r m i n u t e (cpm) was c o n v e r t e d t o d i s i n t e g r a t i o n s p e r m i n u t e (dpm) by a p p l y i n g , f o r each sample, t h e c o u n t i n g e f f i c i e n c y d e t e r m i n e d by the 97 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 w h i c h u s e s a b a r i u m 133 gamma s o u r c e . The l i q u i d s c i n t i l l a t i o n s o l u t i o n c o n s i s t e d o f 2 , 5 - d i p h e n y l o x a z o l e (4 grams) and l , 4 - b i s [ 2 - ( 5 - p h e n y l o x a z o l y ) ] b e n z e n e (0.5 grams) d i s s o l v e d i n t o l u e n e (1 l i t r e ) . The b a c k g r o u n d r a d i a t i o n o f each c o u n t i n g v i a l -46-was d e t e r m i n e d b e f o r e i t was u s e d . A sample was d i s s o l v e d i n benzene o r m e t h a n o l (1 ml) i n t h e v i a l and s c i n t i l l a t o r s o l u t i o n (14 ml) was t h e n added. When d o u b l y l a b e l l e d samples were c o u n t e d t h e i n s t r u m e n t was r e c a l i b r a t e d and t h e o v e r l a p between t h e t r i t i u m and c a r b o n 14 measurements were o b t a i n e d f o r each sample. The C a t h e r a n t h u s r o s e u s p l a n t s used i n t h i s s t u d y were grown i n a Department o f H o r t i c u l t u r e g r e e n h o u s e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , under t h e s u p e r v i s i o n o f Dr. P. S a l s b u r y o f t h e Department o f C h e m i s t r y . M e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and a r e u n c o r r e c t e d . E l e m e n t a l a n a l y s e s were p e r f o r m e d by Mr. P. B o r d a o f t h e M i c r o a n a l y t i c a l L a b o r a t o r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . S y n t h e s i s o f S e c o d i n e (68) f r o m 2 - C a r b o e t h o x y ) - 3 - ( B - c h l o r o e t h y l ) - i n d o l e (73) The s y n t h e s i s o f s e c o d i n e (68) was p r e v i o u s l y worked o u t i n o u r 90 91 l a b o r a t o r i e s ' and has been d e s c r i b e d i n d e t a i l . T h e r e f o r e , o n l y q u a n t i t i e s used and y i e l d s o b t a i n e d , as w e l l as some m i n o r changes i n p r o c e d u r e w i l l be g i v e n f o r t h i s p a r t i c u l a r s equence. 90 N - [ B { 3 - ( 2 - C a r b o e t h o x y ) - i n d o l y l } - e t h y l ] - 3 " - e t h y l - p y r i d i n i u m c h l o r i d e (75) The c a r b o e t h o x y c h l o r o i n d o l e (73) (5g) was d i s s o l v e d i n 3 - e t h y l p y r i d i n e (74,16 ml) i n t h i c k - w a l l e d t u b e . A f t e r s e a l i n g , t h e tube was p l a c e d i n an o i l b a t h k e p t a t 120° f o r 24 h o u r s . The tube was t h e n c o o l e d , b r o k e n open and t h e c o n t e n t s were s t i r r e d i n d i e t h y l e t h e r f o r 2 h o u r s and f i l t e r e d t o y i e l d a w h i t e s o l i d w h i c h was d r i e d i n h i g h vacuum (6.3 g ) . -47-N - [ B - { 3 - ( 2 - C a r b o e t h o x y ) - i n d o l y l } - e t h y l ] - 3 ' - e t h y l - 3 " - p i p e r i d e i n e (76) The i n d o l e p y r i d i n i u m c h l o r i d e ( 7 5 ) ( 6 . 3 g) was d i s s o l v e d i n m e t h a n o l (400 ml) c o n t a i n i n g t r i e t h y l a m i n e (6 m l ) . Sodium b o r o h y d r i d e (20 g) was s l o w l y added a t room t e m p e r a t u r e w i t h v i g o r o u s s t i r r i n g . A f t e r 2.5 h o u r s most o f t h e s o l v e n t was removed i n vacuo b e f o r e w a t e r (100 ml) was added. The r e s u l t i n g s o l u t i o n was a c i d i f i e d w i t h 2N h y d r o c h l o r i c a c i d . A f t e r s t i r r i n g f o r 30 m i n u t e s the s o l u t i o n was b a s i f i e d w i t h s a t . s odium b i c a r b o n a t e s o l u t i o n and e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e . The combined o r g a n i c e x t r a c t s were washed w i t h w a t e r , d r i e d o v e r a nhydrous sodium s u l p h a t e and e v a p o r a t e d i n v acuo t o y i e l d an amorphous m a t e r i a l ( 7 6 ) ( 6 g ) . 90 N - [ g - { 3 - ( 2 - h y d r o x y m e t h y l e n e ) - i n d o l y l } - e t h y l ] - 3 ^ - e t h y l - 3 ^ - p i p e r i d e i n e (77) The p i p e r i d e i n e ( 7 6 ) ( 6 g ) f r o m t h e p r e v i o u s r e a c t i o n was d i s s o l v e d i n f r e s h l y d i s t i l l e d t e t r a h y d r o f u r a n (THF,70 ml) and was added o v e r 40 m i n u t e s t o a v i g o r o u s l y s t i r r i n g s u s p e n s i o n o f l i t h i u m aluminum h y d r i d e (8g) i n THF (300 ml) under a n i t r o g e n atmosphere. A f t e r t h e a d d i t i o n was c o m p l e t e , th e m i x t u r e was s l o w l y warmed t o r e f l u x f o r 2 h o u r s . A t t h e end o f t h i s t i m e t h e r e a c t i o n was c o o l e d and a d d i t i o n s o f w a t e r , 15% s odium h y d r o x i d e s o l u t i o n and w a t e r a g a i n d e s t r o y e d t h e e x c e s s r e a g e n t . The r e s u l t i n g s u s p e n s i o n was f i l t e r e d , e v a p o r a t e d and d r i e d i n h i g h vacuum. Column ch r o m a t o g r a p h y on a l u m i n a (150 g, a c t I I I ) p e r m i t t e d t h e s o l u t i o n o f t h e p i p e r i d e i n e a l c o h o l ( 7 7 ) ( 3 . 6 g ) u s i n g a b e n z e n e / c h l o r o f o r m g r a d i e n t . B e n z o y l a t i o n o f the p i p e r i d e i n e a l c o h o l (77) The m a t e r i a l f r o m t h e above r e a c t i o n (3.6 g) was d i s s o l v e d i n f r e s h l y -48-d r i e d and d i s t i l l e d p y r i d i n e (30 ml) a t 0°. B e n z o y l c h l o r i d e (11 ml) was added o v e r 15 m i n u t e s and r e s u l t i n g s o l u t i o n was s t i r r e d f o r a n o t h e r 3 h o u r s a t 0°. Water (30 ml) was added f o l l o w e d by 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 . A f t e r t h e e v o l u t i o n o f gas c e a s e d t h e m i x t u r e was e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e . The o r g a n i c e x t r a c t was washed r e p e a t e d l y w i t h w a t e r , d r i e d , e v a p o r a t e d i n vacuo and d r i e d i n h i g h vacuum o v e r n i g h t t o y i e l d a gummy, d a r k c o l o u r e d m a t e r i a l ( 7 8 ) ( 5 . 2 g ) . An a l t e r n a t e b e n z o y l a t i o n p r o c e d u r e w h i c h a v o i d s u s i n g 89 p y r i d i n e was l a t e r worked o u t . 90 N - [ g - { 3 - ( 2 - c y a n o m e t h y l e n e ) - i n d o l y l } - e t h y l ] - 3 " - e t h y l - 3 " - p i p e r i d e i r i e (79) The b e n z o a t e p i p e r i d e i n e ( 7 8 ) ( 5 . 2 g ) was d i s s o l v e d i n d i m e t h y l -formamide (135 ml) w i t h p o t a s s i u m c y a n i d e (.9.0 g) . T h i s m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 1 ho u r b e f o r e s l o w l y h e a t i n g t o 110°. A f t e r a b out 1 hou r the m i x t u r e was c o o l e d i n an i c e - w a t e r b a t h , d i l u t e d w i t h w a t e r (300 ml) and e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e . The o r g a n i c e x t r a c t was washed r e p e a t e d l y w i t h w a t e r , d r i e d o v e r anhydrous sodium s u l p h a t e and e v a p o r a t e d i n vacuo t o g i v e a t h i c k o i l . T h i s o i l was d r i e d i n h i g h vacuum to g i v e a c o l o u r e d s o l i d (3.7 g ) . Subsequent c h r o m a t o g r a p h y on a l u m i n a (150 g, a c t I I I ) y i e l d e d t h e n i t r i l e ( 7 9 ) ( 2 . 1 g ) . N - [ B - { 3 - ( 3 - C a r b o m e t h o x y - m e t h y l e n e ) - i n d o l y l } - e t h y l ] - 3 ' - e t h y 1 - 3 " - p i p e r i d e i n e wo) 9 0 The i n d o l e n i t r i l e ( 7 9 ) ( 2 . 1 g ) was d i s s o l v e d i n f r e s h l y d r i e d m e t h a n o l ( a b o u t 50 m l , d i s t i l l e d f r o m magnesium). Water (.0.5 ml) was added t o -49-c o n s t i t u t e 1% o f t h e s o l u t i o n and t h e m i x t u r e was c o o l e d i n an i c e -w a t e r b a t h . Hydrogen c h l o r i d e gas was b u b b l e d i n t o t he r e a c t i o n m i x t u r e t o a c h i e v e a s a t u r a t e d s o l u t i o n . T h i s m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 2.5 days and t h e n e v a p o r a t e d i n v a c u o . The r e s i d u e was p a r t i t i o n e d between s a t . sodium b i c a r b o n a t e s o l u t i o n and m e t h y l e n e c h l o r i d e . The o r g a n i c e x t r a c t was washed w i t h w a t e r , d r i e d o v e r a n h y d r o u s sodium s u l p h a t e and e v a p o r a t e d j i n vacuo t o g i v e an amorphous m a t e r i a l (1.8 g ) . T h i s p r o d u c t was chro m a t o g r a p h e d on a l u m i n a (100 g, a c t I I I ) t o y i e l d t h e carbomethoxy i n d o l e ( 8 0 ) ( 1 . 4 5 g) w h i c h c o u l d be r e c r y s t a l l i z e d f r o m m e t h y l e n e c h l o r i d e , m.p. 85-88°. 3 90 [ A r - H ] - i n d o l e e s t e r (80) The r e c r y s t a l l i z e d and d r i e d i n d o l e e s t e r ( 8 0 ) ( 2 1 0 mg) was d i s s o l v e d 3 i n p r e v i o u s l y p r e p a r e d t r i f l u o r a c e t i c a n h y d r i d e - [ H] (1.2 g, 0.9 m i l l i c u r i e s / mmole) u s i n g a vacuum t r a n s f e r t e c h n i q u e . T h i s s o l u t i o n was a l l o w e d t o s t i r a t room t e m p e r a t u r e f o r 2.5 days under a n i t r o g e n atmosphere. A f t e r r e m o v a l o f t h e s o l v e n t , the r e s i d u e was p a r t i t i o n e d between a cone, ammonium h y d r o t i d e s o l u t i o n and m e t h y l e n e c h l o r i d e . The o r g a n i c e x t r a c t was washed s e v e r a l t i m e s w i t h w a t e r , d r i e d o v e r anhydrous sodium s u l p h a t e and evapo-r a t e d i n . v a c u o . Subsequent c h r o m a t o g r a p h y on a l u m i n a y i e l d e d the e s t e r (157 mg) w h i c h c o u l d be r e c r y s t a l l i z e d t o a c o n s t a n t s p e c i f i c a c t i v i t y o f 4.71 x 10^ dpm/mg. [ A r - H j - S e c o d i n e a l d e h y d e (81) The a r o m a t i c a l l y l a b e l l e d i n d o l e e s t e r (157 mg) was d i s s o l v e d i n d r y -50-benzene and add d r o p w i s e t o a s u s p e n s i o n o f o i l - f r e e s o d i u m h y d r i d e (160 mg o f 55% d i s p e r s i o n i n p a r a f f i n ) i n benzene ( 5 ml) and f r e s h l y d i s t i l l e d m e t h y l f o r m a t e (4 ml) under a n i t r o g e n atmosphere. The r e a c t i o n t e m p e r a t u r e was r a i s e d t o 35° and s t i r r i n g was c o n t i n u e d f o r 2 h o u r s . The r e a c t i o n m i x t u r e was c o o l e d i n an i c e - w a t e r b a t h and t r e a t e d w i t h c o l d w a t e r . Subsequent e x t r a c t w i t h m e t h y l e n e c h l o r i d e , d r y i n g o v e r anhydrous sodium s u l p h a t e and e v a p o r a t i o n i n vacuo y i e l d e d a l i g h t y e l l o w foam (180 mg) w h i c h was used d i r e c t l y i n the n e x t r e a c t i o n . 3 90 [ A r - H ] - 1 6 - 1 7 - D l h y d r o s e c o d l n - 1 7 - o l (69) The amorphous m a t e r i a l (150 mg) f r o m the above r e a c t i o n was t d i s s o l v e d i n m e t h a n o l (15 ml) c o o l e d t o -30° and t r e a t e d w i t h s o d i u m b o r o h y d r i d e (250 mg) o v e r 1 h o u r and s t i r r e d f o r an a d d i t i o n a l 30 m i n u t e s u n t i l t h i n - l a y e r c h r o m a t o g r a p h y i n d i c a t e d t h e absence o f s t a r t i n g m a t e r i a l . T h i s m i x t u r e was t h e n t r e a t e d w i t h 3 d r o p s o f 2N h y d r o c h l o r i c a c i d , d i l u t e d w i t h w a t e r and r e d u c e d i n volume i n v a c u o . Subsequent p a r t i t i o n i n g between s a t . sodium b i c a r b o n a t e s o l u t i o n and m e t h y l e n e c h l o r i d e gave an o r g a n i c e x t r a c t w h i c h was washed w i t h w a t e r and d r i e d o v e r an h y d r o u s sodium s u l f a t e . Removal o f the s o l v e n t _in vacuo y i e l d e d a s l i g h t l y c o l o u r e d foam (180 mg). Column chr o m a t o g r a p h y on a l u m i n a (10 g, a c t I I I ) y i e l d e d the s e c o d i n o l ( 6 9 ) ( 4 1 mg) f r o m a b e n z e n e / c h l o r o f o r m g r a d i e n t as a w h i t e foam. R e c r y s t a l l i z a t i o n o f t h i s m a t e r i a l f r o m d i c h l o r o m e t h a n e a f f o r d e d a s p e c i f i c a c t i v i t y o f 4.54 x io'' dpm/mg. - 5 1 -A l t e r n a t i v e S y n t h e s i s o f N - { g - [ 3 - ( 2 - C a r b o m e t h b x y m e t h y l ) - i r i d d l y l ] - t h y l } - 3 ' - p i p e r d e i n e (80) The e s t e r a l c o h o l ( 8 4 ) ( 1 9 0 mg) was d i s s o l v e d i n d i c h l o r o m e t h a n e (15 ml) and 3 - e t h y l p y r i d i n e ( 7 4 ) ( 0 . 5 m l ) a t 0° under an i n h e r t a t mosphere. To the c o o l r e a c t i o n m i x t u r e p - t o l u e n e s u l p h o n y l c h l o r i d e (600 mg) was added and s t i r r i n g was c o n t i n u e d o v e r n i g h t . T h i s r e a c t i o n m i x t u r e was t h e n warmed t o 95° w h i l e the d i c h l o r o m e t h a n e was d i s t i l l e d o f f . S t i r r i n g was c o n t i n u e d a t 95-98° f o r 8 h o u r s . A f t e r c o o l i n g , t h e e x c e s s 3 - e t h y l p y r i d i n e was removed i n h i g h vacuum and t h e r e s i d u e was d i s s o l v e d i n m e t h a n o l a t 0°. Sodium b o r o h y d r i d e ( l g ) was s l o w l y added o v e r 1 h o u r and s t i r r i n g a t 0° was c o n t i n u e d f o r a f u r t h e r 2 h o u r s . Two volumes o f w a t e r were t h e n added and a f t e r s t i r r i n g f o r 10 m i n u t e s t h i s m i x t u r e was e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e (125 m l ) . The o r g a n i c 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 and e v a p o r a t e d i n vacuo to g i v e a d a r k c o l o u r e d r e s i d u e (340 mg). Chromatography o f t h i s m a t e r i a l on a l u m i n a (15, a c t . I l l ) u s i n g a b e n z e n e - d i c h l o r o m e t h a n e s o l v e n t g r a d i e n t y i e l d the d e s i r e d i n d o l e e s t e r (60 mg). T h i s m a t e r i a l had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h o s e o f p r e v i o u s l y p r e p a r e d m a t e r i a l ( 8 0 ) . 1- (3 **-Pyr i d y l ) - 1 - t h i o - e t h y l e h e k e t a l - e thane 3 - A c e t y l p y r i d i n e ( 9 1 ) ( l g ) was d i s s o l v e d i n a m i x t u r e o f e t h a n e d i t h i o l (5 ml) and b o r o n t r i f l u o r i d e e t h e r a t e (1 ml) and s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e . A f t e r r e m o v a l o f t h e s o l v e n t i n h i g h vacuum, the r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous sodium b i c a r b o n a t e -52-s o l u t i o n and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t was washed t w i c e w i t h s a t . b i c a r b o n a t e s o l u t i o n and once w i t h w a t e r b e f o r e d r y i n g o v e r a n hydrous sodium s u l p h a t e . Most o f t h e s o l v e n t was t h e n e v a p o r a t e d i n vacuo and t h e p r o d u c t was c hromatographed on a l u m i n a (15 g, a c t . I l l ) t o y i e l d a c o l o u r l e s s o i l (1.55 g ) , pmr: 1.0, 1.45, 1.90, 2.78 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 6.6 ( m u l t i p l e t , 4H, -S-CT^CH^-S-), 7.85 ( s i n g l e t , 3H, -CH^); mass s p e c t r u m : M +197. D e s u l p h u r i z a t i o n o f l - ( 3 " - p y r i d y l ) - l - t h i o e t h y l e r i e k e t a l - e t h a n e The t h i o k e t a l ( l g ) was d i s s o l v e d i n a b s o l u t e e t h a n o l (50 ml) and r e f l u x e d w i t h f r e s h l y p r e p a r e d Raney n i c k e l (20 g) f o r 16 h o u r s . A f t e r c o o l i n g t h i s m i x t u r e was s t i r r e d w i t h 5% aqueous sodium h y d r o x i d e f o r 15 m i n u t e s and t h e i n o r g a n i c m a t e r i a l was f i l t e r e d o f f . The r e s i d u e was washed t w i c e w i t h e t h a n o l and t h e combined f i l t e r a t e s were e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e . A f t e r d r y i n g o v e r anhydrous sodium s u l p h a t e the s o l v e n t was removed i n v a c u o . A g l c e x a m i n a t i o n showed th e p r e s e n c e o f one component w h i c h had t h e same r e t e n t i o n time (6 m i n u t e s , 175°) as an a u t h e n t i c sample o f 3 - e t h y l p y r i d i n e ( 7 4 ) . The p r o d u c t was d i s t i l l e d t o y i e l d a c o l o u r l e s s l i q u i d whose s p e c t r a l p r o p e r t i e s were a l s o i d e n t i c a l w i t h an a u t h e n t i c sample o f ( 7 4 ) . [ 2 " - 2 H ] - 3 - A c e t y l p y r i d i n e (92) 3 - A c e t y l p y r i d i n e (2 g) was s t i r r e d w i t h d r y t e t r a h y d r o f u r a n (5 m l ) , d e u t e r i u m o x i d e (3 ml) and d r i e d sodium c a r b o n a t e CO.5 g) o v e r n i g h t i n a c l o s e d v e s s e l . The r e a c t i o n m i x t u r e was t h e n p a r t i t i o n e d and t h e o r g a n i c -53-l a y e r was d r i e d o v e r a nhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f t h e s o l v e n t i n h i g h vacuum, t h e p r o d u c t was examined by pmr: 7.40 ( m u l t i p l e t , -CHIJ^) i n t e g r a t e d t o show 95% exchange o f i s o t o p e s . 2 [ 2 ' - H ] - l - ( 3 " - P y r i d y l ) - l - t h i o e t h y l e n e k e t a l - e t h a n e (93) The d e u t e r a t e d m a t e r i a l (1.5 g) f r o m t h e above r e a c t i o n 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 t h i o k e t a l ( 9 3 ) ( 2 . 1 g) as p r e v i o u s l y d e s c r i b e d and examined by pmr: 7.9 ( m u l t i p l e t , -CHE^) i n t e g r a t e d t o show l o s s o f 1.4% f r o m s t a r t i n g m a t e r i a l . [ 2 - 2 H ] - ( 3 " - P y r i d y l ) - e t h a n e (94) The above d e u t e r a t e d t h i o k e t a l ( 9 3 ) ( 2 g ) was c o n v e r t e d to d e u t e r a t e d 3 - e t h y l p y r i d i n e ( 9 4 ) ( 0 . 8 6 g) by t h e Raney n i c k e l d e s u l p h u r i z a t i o n p r o -c e d u r e p r e v i o u s l y d e s c r i b e d . E x a m i n a t i o n o f the p r o d u c t by pmr: 8.8 ( m u l t i p l e t , -CHTJ^) r e v e a l e d by i n t e g r a t i o n a l o s s o f 3.4% o f l a b e l f r o m th e s t a r t i n g 3 - a c e t y l p y r i d i n e ( 9 2 ) . 1 - ( 3 " - P y r i d y l ) - e t h a n o l (.95) 3 - A c e t y l p y r i d i n e (91) (.3.5 g) was d i s s o l v e d i n a n hydrous d i m e t h o x y -e t h a n e (25 ml) and t r e a t e d w i t h sodium b o r o h y d r i d e (265mg)at 80° f o r 24 h o u r s . T h i s m i x t u r e was t h e n c o o l e d and s t i r r e d w i t h 0.1 N aqueous h y d r o c h l o r i c a c i d f o r 5 m i n u t e s and the r e s u l t i n g m i x t u r e was made b a s i c w i t h s a t u r a t e d s odium b i c a r b o n a t e s o l u t i o n and e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t s were washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . Removal o f the s o l v e n t i n vacuo y i e l d e d -54-a c o l o u r l e s s o i l w h i c h was chromatographed on a l u m i n a (25 g, a c t . I I I ) . T h i s p r o d u c t ( 9 5 ) ( 3 . 4 5 g, b.p. 105-120° a t 1.2 mm) had t h e f o l l o w i n g d a t a ; uv: s i m i l a r t o 3 - e t h y l p y r i d i n e : v 3250 (-0H); pmr: 1.56, 2.22, 2.78 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.0 ( b r o a d s i n g l e t , I H , -OH), 5.10 ( q u a r t e t , J=7, I H , - C H ( 0 H ) C H 3 ) , 8.55 ( d o u b l e t , J=7, 3H, - C H ( 0 H ) C H 3 ) ; mass s p e c t r u m : M + 123. 1 - E t h y l - ( 3 ' - p y r i d y l ) - a c e t a t e (99) The above a l c o h o l (2.8 g) was d i s s o l v e d i n a c e t i c a n h y d r i d e and h e a t e d on a steam b a t h f o r 45 m i n u t e s . The s o l v e n t was t h e n removed i n v a cuo and the r e s i d u e was p a r t i t i o n e d between 5% aqueous sodium b i c a r b o n a t e and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t was t h e n washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f t h e s o l v e n t t h e r e s i d u e (3.2 g) was r a p i d l y c hromatographed on a l u m i n a (20 g, a c t . I I ) t o g i v e a c o l o r l e s s o i l (3.0 g ) : v 1725 max ( a c e t a t e ) ; pmr: 1.5, 2.30, 2.51 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.05 ( q u a r t e t , J=7, I H , -CH(OAc)CH 3), 7.93 ( s i n g l e t , 3H, - 0 2 C C H 3 ) , 8.44 ( d o u b l e t , 3H, -CH(OAc)CH 3); mass s p e c t r u m : M +165. C a t a l y t i c H y d r o g e n o l y s i s o f A c e t a t e (99) The p y r i d a l a c e t a t e ( 9 9 ) ( 2 . 0 g) was d i s s o l v e d i n d i m e t h o x y e t h a n e and t r e a t e d b r i e f l y w i t h h y d r o g e n c h l o r i d e gas. The s o l v e n t was removed i n vacuo and the r e s i d u e was d i s s o l v e d i n w a t e r (30 m l ) . To t h i s s o l u t i o n , p a l l a d i u m - o n - c h a r c o a l c a t a l y s t ( 1 0%, 400 mg) was added and the r e s u l t i n g m i x t u r e was s t i r r e d a t room t e m p e r a t u r e i n an atmosphere o f h y d r o g e n gas. -55-A f t e r one e q u i v a l e n t of hydrogen had been consumed, the r e a c t i o n m i x t u r e was f i l t e r e d and made b a s i c w i t h s a t . aqueous sodium b i c a r b o n a t e s o l u t i o n and e x t r a c t e d w i t h d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t s were d r i e d o v e r anhydrous sodium s u l p h a t e b e f o r e the s o l v e n t was c a r e f u l l y removed by d i s t i l l a t i o n . The r e s i d u e was t h e n d i s t i l l e d to y i e l d 3 - e t h y l - p y r i d i n e (0.9 g) w h i c h was compared w i t h an a u t h e n t i c sample. [ l - 3 H ] - l - ( 3 " - P y r i d y l ) - e t h a n o l 3 - A c e t y l p y r i d i n e (4.5 g) was d i s s o l v e d i n anhydrous dimethoxy-ethane (30 ml) and t r e a t e d w i t h d r y sodium b o r o h y d r i d e (10 mg) a t 85° 3 f o r 2 h o u r s . Sodium b o r o h y d r i d e - K (40 mg, 200 mc) was added and the r e a c t i o n was c o n t i n u e d o v e r n i g h t . Dry sodium b o r o h y d r i d e (450 mg) was then added and s t i r r i n g was c o n t i n u e d u n t i l e x a m i n a t i o n by t i c on s i l i c a g e l ( c h l o r o f o r m ) i n d i c a t e d a complete r e a c t i o n . T h i s m i x t u r e was then worked up as p r e v i o u s l y d e s c r i b e d . 3 [1- H ] - l - E t h y l - ( 3 ' - p y r i d y l ) - a c e t a t e The t r i t i a t e d a l c o h o l from the above r e a c t i o n was d i s s o l v e d i n a c e t i c a n h y d r i d e (25 ml) a t 100°. T h i s r e a c t i o n was m o n i t o r e d by t i c and worked up as p r e v i o u s l y d e s c r i b e d . [ 1 - 3 H ] - 1 - ( 3 ' - P y r i d y l ) - e t h a n e The t r i t i a t e d p y r i d y l a c e t a t e from the above r e a c t i o n was c o n v e r t e d to i t s h y d r o c h l o r i d e s a l t , d i s s o l v e d i n water and t r e a t e d w i t h p a l l a d i u m - o n - c h a r c o a l (10%, 80 mg). A f t e r h y d r o g e n a l y s i s i n -56-an atmosphere o f h y d r o g e n . The r e a c t i o n m i x t u r e was worked up as 3 p r e v i o u s l y d e s c r i b e d t o y i e l d [ 1 - H ] - l - ( 3 ' - p y r i d y l ) - e t h a n e (2.6 g, 2 . 1 8 x l 0 8 dpm/mg). [ 1 9 - 3 H ] - N [ 6 { 3 - ( 2 - C a r b o m e t h o x y m e t h y l ) - i n d o l y l } - e t h y l ] - 3 ' - e t h y l - 3 ' - p i p e r d e i n e The e s t e r a l c o h o l ( 8 4 ) ( 2 0 0 mg) was d i s s o l v e d i n d r y d i c h l o r o -methane (15 m l ) , t r i t i a t e d e t h y l p y r i d i n e (0.5 ml) w i t h p - t o l u e n e s u l p h o n y l c h l o r i d e (650 mg) a t 0°. The r e a c t i o n was t h e n c a r r i e d o u t as p r e v i o u s l y d e s c r i b e d t o y i e l d t he t r i t i a t e d p i p e r d e i n e (80) (65 mg). 3 [19- H ] - S e c o d i n e - 1 7 - a l The t r i t i a t e d a l c o h o l ( 8 0 ) ( 6 5 g) was d i s s o l v e d i n benzene (10 m l , f r e s h l y d i s t i l l e d f r o m l i t h i u m a l u m i n i u m h y d r i d e ) and m e t h y l f o r m a t e (4 ml) and t r e a t e d w i t h sodium h y d r i d e (65 mg o f a 55% o i l s u s p e n s i o n ) . A f t e r 2.5 h o u r s t h e r e a c t i o n was worked up as p r e v i o u s l y d e s c r i b e d . [ 1 9 - 3 H ] - 1 6 , 1 7 - D i h y d r o s e c o d i n - 1 7 - o l The above p r o d u c t was d i s s o l v e d i n m e t h a n o l (10 ml) a t -35° and t r e a t e d w i t h sodium b a r o h y d r i d e (20 mg) o v e r 2 h o u r s a c c o r d i n g to t he p r o c e d u r e p r e v i o u s l y d e s c r i b e d . The p r o d u c t (30 mg) was r e c r y s t a l l i z e d f r o m d i c h l o r o m e t h a n e t o g i v e t r i t i a t e d s e c o d i n o l ( 6 . 5 6 x l Q 7 dpm/mg). -57-The Isolation of Vindoline (7) from Feeding Experiments in C. roseus P l a n t s 9 0 ' 9 1 Vindoline (7) was isolated from whole plants of C^. roseus by a procedure similar to that used by others in our laboratories and w i l l only be b r i e f l y summarized. The plant was up-rooted and cleaned of s o i l before i t was mascerated in methanol in a Waring blender. The resulting pulp was re-mascerated twice more with additional quantities of methanol. The combined methanolic extracts were then evaporated and the residue was partitioned between 10% aqueous acetic acid and benzene. This aqueous extract was made basic with saturated sodium bicarbonate solution and extracted with chloroform. The organic extract was then washed with water and dried over anhydrous sodium sulphate before the solvent was removed. Chromatography of the residue on alumina (act.Ill) yielded vindoline (7), using a benzene-chloroform solvent gradient. 3 14 90 91 The Administration of [19- H, CO^H^]-Secodine (68) to C. roseus ' In both of the following feeding experiments, the radioactive precursor was dissolved in d i s t i l l e d water (0.5 ml) with 2 drops of IN acetic acid. This solution was administered to the plant via cotton wick (~5 cm) which had been passed through the stem of the potted plant. After the solution had been taken up by the plant d i s t i l l e d water (0.5 ml) with 1 drop of IN acetic acid was added to the container and allowed to be taken up by the plant. After that, the plant was allowed to grow under a r t i f i c i a l lights at room temperature for a total of 9 days. -58-The data r e l e v a n t to the feeding experiments u s i n g C. roseus p l a n t s i s summarized i n Table 2. In both cases the v i n d o l i n e i s o l a t e d was converted to v i n d o l i n o l before r e c r y s t a l l i z a t i o n to constant r a d i o a c t i v i t y . Table 2. A d d i t i o n a l data a s s o c i a t e d w i t h Table 1 S p e c i f i c A c t i v i t y Fed 3H,dpm/mmole 1 4C,dpm/mmole a 3.48 x 10' 1.16 x 10' 7.61 x 10' 4.94 x 10' S p e c i f i c A c t i v i t y I s o l a t e d 3H,dpm/mmole ^c^pm/mmole 2.15 x 10" 6.50 x 10 3.67 x 10" 2.72 x 10" a. ["''^C^CH.j]-Secodinol was a v a i l a b l e from the o r i g i n a l s y n t h e s i s by Sood. V i n d o l i n o l ( 1 0 0 ) 9 1 , 9 8 R a d i o a c t i v e v i n d o l i n e (7) was converted to v i n d o l i n o l by the 91 procedure reported by Beck. V i n d o l i n e (7) was d i s s o l v e d i n dry te t r a h y d r o f u r a n and r e f l u x e d w i t h excess l i t h i u m aluminum hydride f o r 2 hours. The excess reducing reagent was destroyed w i t h s a t . sodium sulphate s o l u t i o n . E x t r a c t i o n w i t h methylene c h l o r i d e y i e l d e d the d e s i r e d product, which could be r e c r y s t a l l i z e d to constant r a d i o a c t i v i t y from d i e t h y l ether. -59-BIBLIOGRAPHY 1. R. Robinson, The S t r u c t u r a l Relation of Natural Products, Claredon Press, Oxford (1955). 2. A P i c t e t , Arch. Pharm., 224 389 (1906). 3. E. Leete, J. Am. Chem. S o c , 82, 6338 (1960). 4. E. Leete, Tetrahedron 14, 35 (1961). 5. A. R. Battersby, In Natural Substances Formed B i o l o g i c a l l y from Mevalonic Acid (ed. T. W. Goodwin), Academic Press, New York, P-157 (1970). 6. A. I. Scott, Acc. of Chem. Res., _3, 151 (1970. 7. A. R. Battersby, In Chemical Society S p e c i a l i s t P e r i o d i c a l Report, The A l k a l o i d s , 1, 31, The Chemical Society, London. (1971). 8. I. Kompis, M. Hesse and H. Schmid, L l o y d i a 34 (3), 269 (1971). 9 m J. P. Kutney, J. of He t e r o c y c l i c Chem., 9_, Supplementary Issue, S- 1 (1972). 10. G. A. C o r d e l l , L l o y d i a 37(2), 219 (1974). 11. G. Barger and C. Scholz, Helv. Chim. Acta., 16, 1343 (1933). 12. G. Hahn, H. Werner, L i e b i g ' s Ann. Chem., 520, 123 (1935). 13. R. B. Woodward, Nature 162, 155 (1948). 14. R. B. Woodward, Angew. Chem., 6§_, 13 (1956). 15. E. Wenkert and N. V. B r i n g i , J . Am. Chem. S o c , 81, 1474 (1959). 16. E. Wenkert, J . Am. Chem.'Soc, 84, 98 (1962). 17. R. Thomas, Tet. L e t t . , 544 (1961). 18. E. S h l i t t l e r and W. I. Taylor, E x p e r i e n t i a , 16, 224 (1960). 19. P. N. Edwards and E. Leete, Chem. and Ind., 1666 (1960). 20. E. Leete, S. Ghosal and P. N. Edwards, J . Am. Chem. S o c , 84, 1068 (1962). 21. E. Leete and S. Ghosal, Tet. L e t t . , 1179 (1962). -60-22. E. Leete, A. Almad and I. Kompis, J. Am. Chem. Soc. 87_, 4168 (1965) . 23. T. Money, I. G. Wright, F. McCapra and A. I. Scott, Proc. Nat. Acad. Sci., U.S.A. 5_3, 901 (1965). 24. F. McCapra, T. Money, A. I. Scott and I. G. Wright, Chem. Comm., 537 (1965). 25. H. Goeggel and D. Arigoni, Chem. Comm., 538 (1965). 26. A. R. Battersby, R. T. Brown, R. S. Kapil, A. 0. Plunkett and J. B. Taylor, Chem. Comm., 46 (1966). 27. E. S. Hall, F. McCapra, T. Money, K. Fukumoto, J. Hanson, B. Mootoo, G. T. Phil l i p s and A. I. Scott, Chem. Comm., 348 (1966) . 28. A. R. Battersby, R. T. Brown, R. S. Kapil, J. A. Knight, J. A. Martin and A. 0. Plunkett, Chem. Comm., 801 (1966). 29. A. R. Battersby, R. T. Brown, R. S. Kapil and A. 0. Plunkett, Chem. Comm., 810 (1966). 30. A. R. Battersby, R. T. Brown, J. A. Knight, J. A. Martin and A. 0. Plunkett, Chem. Comm. 346 (1966). 31. P. Loew, H. Goeggel and D. Arigoni, Chem. Comm. 347 (1966). 32. A. R. Battersby, R. T. Brown, R. S. Kapil, J. A. Martin and A. 0. Plunkett, Chem. Comm., 812 (1966). 33. A. R. Battersby, R. S. Kapil and R. Southgate, Chem. Comm., 131 (1968). 34. A. R. Battersby, R. S. Kapil, J. A. Martin and L. Mo, Chem. Comm., 133 (1968). 35. P. Loew and D. Arigonni, Chem. Comm., 137 (1968). 36. A. R. Battersby, E. S. Hall and R. Southgate, J. Chem. Soc. (C), 721 (1969). - 6 1 -37. C. J . C o s c i a , R. G u a r n a c c i a and Chem. Comm. 138 ( 1 9 6 8 ) . 38. S. B r e c h b u h l e r - B a d e r , C. J . C o s c i a , P. Loew, C. von S z e z e p a n s k i and D. A r i g o n i , Chem. Comm., 136 ( 1 9 6 8 ) . 39. M. M. J a n o t , P. P o r t i e r , P. F r a n c o i s , J . L e v i s a l l e s , A. R. B a t t e r s b y , B. G r e g o r y , H. Spenser and J . C. T u r n e r , Chem. Comm., 219 ( 1 9 6 7 ) . 40. A. R. B a t t e r s b y , P u r e A p p l . Chem., 14_, 117 ( 1 9 6 7 ) . 41. P. Leow, Ch. v o n S z e z e p a n s k i , C. J . C o s i c a and D. A r i g o n i , Chem. Comm., 1276 ( 1 9 6 8 ) . 42. A. R. B a t t e r s b y , A. R. B u r n e t t , G. D. Knowles and P. G. P e a r s o n s , Chem. Comm., 1277 ( 1 9 6 8 ) . 43. A. R. B a t t e r s b y , A. R. B u r n e t t , and P. G. P a r s o n s , Chem. Comm., 1280 ( 1 9 6 8 ) . 44. A. R. B a t t e r s b y , A. R. B u r n e t t and P. G. P a r s o n s , J . Chem. Soc. ( C ) , 1187 ( 1 9 6 9 ) . 45. A. R. B a t t e r s b y , A. R. B u r n e t t and P. G. P a r s o n s , Chem. Comm., 1282 ( 1 9 6 8 ) . 46. A. R. B a t t e r s b y , A. R. B u r n e t t , E. S. H a l l and P. G. P a r s o n s , Chem. Comm., 1582 (1 9 6 8 ) . 47. A. R. B a t t e r s b y , A. R. B u r n e t t and P. G. P a r s o n s , J . Chem. Soc. ( C ) , 1193 ( 1 9 6 9 ) . 48. 0. K e n n a r d , P. J . R o b e r t s , N. W. I s a a c s , F. H. A l l e n , W. D. S. M o t h e r w e l l , K. H. G i b s o n , and A. R. B a t t e r s b y , Chem. Comm., 899 ( 1 9 7 1 ) . 49. A. R. B a t t e r s b y and K. H. G i b s o n , Chem. Comm., 902 ( 1 9 7 1 ) . 50. K, T. D. De S i l v a , G. N. S m i t h , and K. E. H. Warren, Chem. Comm., 905 ( 1 9 7 1 ) . -62-51. W. P. B l a c k s t o c k , R. T. Brown and G. K. L e e , Chem. Comm., 910 ( 1 9 7 1 ) . 52. R. T. Brown and C. L. C h a p p i e , Chem. Comm., 740 ( 1 9 7 4 ) . 53. R. T. Brown, C. L. Ch a p p i e and R. P i a t t , Chem. Comm., 929 ( 1 9 7 4 ) . 54. R. T. Brown, C. L. Ch a p p i e and A. G. L a s h f o r d , Chem. Comm., 295 (1 9 7 5 ) . 55. A. H. H e c k e n d o r f , K. C. M a t t e s and C. R. H u t c h i n s o n , E. W. Hagaman and E. Wenkert, J . Org. Chem. 42^, 2045 ( 1 9 7 6 ) . 56. K. C. M a t t e s , C. R. H u t c h i n s o n , J . P. S p r i n g e r and J . C l a r d y , J . Am. Chem. Soc. 97, 6270 ( 1 9 7 5 ) . 57. J . S t o c k i g t and M. H. Zenk, P r i v a t e Communication t o P r o f . J . P. Ku t n e y . 58. G. N. S m i t h , Chem. Comm., 912 ( 1 9 6 8 ) . 59. R. T. Brown, G. N. S m i t h and K. S. J . S t a p l e f o r d , T e t . L e t . , 4349 ( 1 9 6 8 ) . 60. H. P. Husson, C. Kan-Fan, T. Sevenet e t J-P V i d a l , T e t . L e t . , 1889 ( 1 9 7 7 ) . 61. J . S t o c k i g t , J . T r e i m e r and M. Zenk, FEBS L e t t e r s , 70_, 267 ( 1 9 7 6 ) . 62. A. R. B a t t e r s b y and E. S. H a l l , Chem. Comm., 793 ( 1 9 6 9 ) . 63. A. A. Q u r e s h i and A. I . S c o t t , Chem. Comm., 948 ( 1 9 6 8 ) . 64. A. B a t t e r s b y , J . C. B y r n e , R. S. K a p i l , J . A. M a r t i n , T. G. Payne, D. A r i g o n i and P. Loew, Chem. Comm., 951 ( 1 9 6 8 ) . 65. A. I . S c o t t , P. C. C h e r r y and A. A. Q u r e s h i , J . Am. Chem. S o c , 91, 4932 ( 1 9 6 9 ) . 66. E. Wenkert and B. W i c k b e r g , J . Am. Chem. S o c , 87_, 1580 ( 1 9 6 5 ) . 67. G. A. C o r d e l l and G. F. S m i t h , J . Chem. S o c , - 6 3 -68. A. C h a t t e r j e e , A. B a n e r j i , P. Majumder and R. Majumder, B u l l . Chem. Soc. J a p a n , 49, 2000 ( 1 9 7 6 ) . 69. A. I . S c o t t and A. A. Q u r e s h i , J . Am. Chem. S o c , 5874 ( 1 9 6 9 ) . 70. S. I . H e i m b e r g e r and A. I . S c o t t , Chem. Comm. 217 ( 1 9 7 3 ) . 71. E. Wenkert, J . Am. Chem. S o c , 90_, 3891 ( 1 9 6 2 ) . 72. J . P. K u t n e y , E. P i e r s and R. T. Brown, J . Am. Chem. S o c , 92, 1700 ( 1 9 7 0 ) . 73. J . P. K u t n e y , W. J . C r e t n e y , J . R. H a d f i e l d , E. S. H a l l and V. R. N e l s o n , J . Am. Chem. S o c , 92, 1704 ( 1 9 7 0 ) . 74. J . P. K u t n e y , R. T. Brown, E. P i e r s and J . R. H a d f i e l d , J . Am. Chem. S o c , 92_, 1708 ( 1 9 7 0 ) . 75. J . P. K u t n e y , W. J . C r e t n e y , P. Le Quesne, B. McKague and E. P i e r s , J . Am. Chem. S o c , £ 2 , 1712 ( 1 9 7 0 ) . 76. J . P. K u t n e y , N. Abdurahman, C. G l e t s o s , P. LeQuesne, E. P i e r s and I . V l a t t a s , J . Am. Chem. S o c , 92^, 1727 ( 1 9 7 0 ) . 77. J . P. K u t n e y , C. E h r e t , V. R. N e l s o n , W. C. W i g f i e l d , J . Am. Chem. S o c , 90, 5929 ( 1 9 6 8 ) . 78. J . P. K u t n e y , W. J . C r e t n e y , J . R. H a d f i e l d , E. S. H a l l , V. R. N e l s o n , D. C. W i g f i e l d , J . Am. Chem. S o c , £0, 3566 ( 1 9 6 8 ) . 79. A. I . S c o t t , P. B. R e i g h a r d t , M. B. S l a y t o r and J . G. Sweeny, B i o o r g . Chem., 1, 157 ( 1 9 7 1 ) . 80. A. I . S c o t t and A. A. Q u e r e s h i , T e t r a h e d r o n 30, 2993 ( 1 9 7 4 ) . 81. A. I . S c o t t and C.'C. Wei, T e t r a h e d r o n 30, 3003 ( 1 9 7 4 ) . 82. A. I . S c o t t , P. C. C h e r r y and C. C. Wei, T e t r a h e d r o n , 30, 3013 (1974) . -64-83. D. A. Evans, G. F. S m i t h , G. N. S m i t h and K. S. J . S t a p l e f o r d , Chem. Comm., 859 ( 1 9 6 8 ) . 84. D. A. E v a n s , J . A. J o u l e and G. F. S m i t h , Phytochem. , 7_, 1429 (1 9 6 8 ) . 85. G. A. C o r d e l l , G. F. S m i t h and G. N. S m i t h , Chem. Comm., 189 ( 1 9 7 0 ) . 86. R. T. Brown, G. F. S m i t h , K. S. J . S t a p l e f o r d and D. A. T a y l o r , Chem. Comm., 190 ( 1 9 7 0 ) . 87. G. A. C o r d e l l , G. F. S m i t h and G. N. S m i t h , Chem. Comm., 191 (1 9 7 0 ) . 88. J . LeMen and W. I . T a y l o r , E x p e r e n t i a , 21, 508 ( 1 9 6 5 ) . 89. A. R. B a t t e r s b y and A. K. B h a t n a g e r , Chem. Comm., 193 ( 1 9 7 0 ) . 90. R. S. Sood; S t u d i e s R e l a t e d t o t h e 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 , Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a ( 1 9 7 0 ) . 91. J . F. Beck, S t u d i e s on t h e 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 , Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a ( 1 9 7 1 ) . 92. N. D. W e s t c o t t , S t u d i e s R e l a t e d t o : B a r k E x t r a c t s o f some F i r and Spruce S p e c i e s ; and S y n t h e s i s and 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 , Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a ( 1 9 7 0 ) . 93. E. Wenkert, K. Dave, C. Gnewuch, J . Am. Chem. Soc. , 90_, 5251 ( 1 9 6 8 ) . 94. N. J . E g g e r s , S t u d i e s on t h e 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 , Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a ( 1 9 7 3 ) . 95. J . P. K u t n e y , J . F. Beck, N. J . E g g e r s , H. W. Han s s e n , R. S. Sood, N. D. W e s t c o t t , J . Am. Chem. S o c , 93_, 7322 ( 1 9 7 1 ) . - 6 5 -96. J . P. K u t n e y , H e t e r o c y c l e s , ]_, 593 ( 1 9 7 7 ) . 97. Mark I L i q u i d S c i n t i l l a t i o n Systems I n s t r u c t i o n M a n u a l , N u c l e a r C h i c a g o C o r p o r a t i o n , Des P l a i n e s , I l l i n o i s ( 1 9 6 6 ) . 98. T. Money, I . G. W r i g h t , F. McCapra, E. S. H a l l , and A. I . S c o t t , J . Am. Chem. S o c , 90, 4144 ( 1 9 6 8 ) . -66-PART I I Studies Related to the B i o s y n t h e s i s of U l e i n e and O l i v a c i n e -67-INTRODUCTION I n P a r t I a g e n e r a l i n t r o d u c t i o n to i n d o l e a l k a l o i d b i o s y n t h e s i s was p r e s e n t e d . U n t i l v e r y r e c e n t l y , a l l the work i n t h i s a r e a had been d i r e c t e d toward t h e f o u r major f a m i l i e s o f i n d o l e a l k a l o i d s , namely t h e C o r y n a n t h e , t h e S t r y c h n o s , the A s p i d o s p e r m a and t h e Ib o g a s k e l e t o n s . T h e r e i s , however, an i n c r e a s i n g number o f i n d o l e a l k a l o i d s b e i n g f ound i n n a t u r e i n w h i c h t h e t r y p t a m i n e p o r t i o n o f the s k e l e t o n has been a l t e r e d . Members o f t h e f o u r m a j o r f a m i l i e s j u s t m e n t i o n e d p o s s e s s a tw o - c a r b o n b r i d g e between the i n d o l e n u c l e u s and the b a s i c n i t r o g e n atom (N, ) . A l k a l o i d s o f the u l e i n e - t y p e ( 1 ) , however, have no s u c h c a r b o n b r i d g e , w h i l e members o f the a p p a r i c i n e (2) and v a l l e s a m i n e (3) f a m i l y have a o n e - c a r b o n b r i d g e . On the o t h e r hand, a t h r e e - c a r b o n b r i d g e i s a p p a r e n t i n 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 f w h i c h the b e s t known examples a r e o l i v a c i n e ( 4 ) , guatambuine (5) and e l l i p t i c i n e ( 6 ) . (4) (5) (6) -68-The f i r s t q u e s t i o n t h a t had t o be a s k e d was: a r e t h e s e f a m i l i e s o f i n d o l e a l k a l o i d s a l s o d e r i v e d f r o m t r y p t o p h a n (15)? Wenkert's e a r l y h y p o t h e s i s was t h a t they were not."'" He s u g g e s t e d t h a t a p r e c u r s o r o f t r y p t o p h a n , g l y c o s y l e n e a n t h r a n i l i c a c i d (7) condensed w i t h t h e s e c o -p r e p h e n a t e - f o r m a l d e h y d e (SPF) u n i t ( 8 , see a l s o F i g u r e 2 i n P a r t I ) t o g i v e an i n t e r m e d i a t e (10) w h i c h w i t h t h e a d d i t i o n o f a m e t h y l a m i n e u n i t , o r i t s b i o l o g i c a l e q u i v a l e n t , c o u l d undergo the a p p r o p r i a t e c y c l i z a t i o n s t o g i v e e l l i p t i c i n e ( 6 ) , u l e i n e (1) and o l i v a c i n e (4) as i l l u s t r a t e d i n F i g u r e J.. S i n c e t h e p u b l i c a t i o n o f t h i s h y p o t h e s i s t h e SPF u n i t (8) has been s e t a s i d e i n f a v o u r o f t h e s t r u c t u r a l l y s i m i l a r s e c o - l o g a n i n m o i e t y ( 9 ) , the development o f w h i c h was d e s c r i b e d i n P a r t I . S i n c e t h e l a t t e r compound c o u l d behave i n a s i m i l a r f a s h i o n t o t h a t o f the SPF u n i t , the Wenkert h y p o t h e s i s r e m a i n s i n t a c t and w o r t h y o f f u r t h e r s t u d y . I n 1965, w i t h the s t r u c t u r e o f a p p a r i c i n e (2) i n hand D j e r a s s i 2 and G i l b e r t s u g g e s t e d t h a t Wenkert's i n t e r m e d i a t e (13) c o u l d a l s o c y c l i z e t o p r o v i d e the o n e - c a r b o n b r i d g e between the i n d o l e and p i p e r i d i n e u n i t s t o g i v e t h e a p p a r i c i n e s k e l e t o n . The f a c t t h a t u l e i n e (1) and a p p a r i c i n e (2) c o - o c c u r i n t h e same p l a n t s p e c i e s may s u g g e s t t h a t t h e s e 3 two s t r u c t u r e s have a common b i o s y n t h e t i c i n t e r m e d i a t e . A l s o , i t i s known i n b e r b e r i n e a l k a l o i d b i o s y n t h e s i s t h a t N-methyl groups can be p r e c u r s o r s t o m e t h y l e n e b r i d g e s . -69-F i g u r e 1. The Wenkert h y p o t h e s i s f o r the b i o s y n t h e s i s of e l l i p t i c i n e ( 6 ) , u l e i n e (1) and o l i v a c i n e ( 4 ) . -70-OHC C O f OHC C02" (13) (14) (2) F i g u r e 2. The D j e r a s s i - G i l b e r t r a t i o n a l f o r 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 ( 2 ) . The f i r s t 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 ^ o f t h e s e a l k a l o i d s by W i g f i e l d and N e l s o n i n our l a b o r a t o r i e s r e v e a l e d t h a t , i n A s p i d o s p e r m a p y r i c o l l u m p l a n t s , t r y p t o p h a n (15) l a b e l l e d i n t h e a r o m a t i c r i n g w i t h t r i t i u m and i n the C-3 p o s i t i o n w i t h Carbon 14, was i n c o r p o r a t e d i n t o a p p a r i c i n e (2) (See T a b l e 1 ) . However, no a c t i v i t y c o u l d e v e r be d e t e c t e d i n u l e i n e (1) w h i c h was a l s o i s o l a t e d i n t h e s e e x p e r i m e n t s . stemmadenine (16) was v e r y e f f i c i e n t l y i n c o r p o r a t e d i n t o a p p a r i c i n e (2) t o t h e e x t e n t o f 0.55%, w h i l e the more s i m i l a r s t r u c t u r e v a l l e s a m i n e (17) was i n c o r p o r a t e d i n t o a p p a r i c i n e i n o n l y 0.01%. (15) (2) A l s o o f i m p o r t a n c e was the d i s c o v e r y by our group t h a t These r e s u l t s s t r o n g l y s u g g e s t t h a t a s t e m m a d e n i n e - l i k e p r e c u r s o r i s i n v o l v e d i n the l a t e r s t a g e s o f b i o s y n t h e s i s w i t h t h e C-2 c a r b o n atom o f t h e o r i g i n a l t r y p t a m i n e b r i d g e b e i n g l o s t i n the l a t e r s t a g e s o f the pathway. A g a i n , no i n c o r p o r a t i o n i n t o u l e i n e (1) c o u l d be "OH " " (17) d e m o n s t r a t e d f r o m any o f t h e p r e c u r s o r s t r i e d . W i t h t h i s new i n s i g h t i n t o the o r i g i n o f the n i t r o g e n o u s p o r t i o n o f a p p a r i c i n e , Kim and E r i c k s o n m o d i f i e d an e a r l i e r m e c h a n i s t i c p r o p o s a l ^ w h i c h a l l o w e d the i n v o l v e m e n t o f t r y p t o p h a n (15) w i t h the e x t r u s i o n o f the two c a r b o n b r i d g e t o f o r m the u l e i n e s k e l e t o n s . T h e i r i n v i t r o s t u d i e s f a i l e d to s u p p o r t t h i s p r o p o s a l . The p r o p o s a l a l s o s u f f e r e d f r o m a major d e f i c i e n c y i n t h a t i t was s p e c i f i c f o r u l e i n e -72-and c o u l d n o t t a k e i n t o a c c o u n t t h e f o r m a t i o n o f a p p a r i c i n e ( 2 ) . Our e a r l y r e s u l t s c o n c e r n i n g the i n c o r p o r a t i o n o f stemmadenine and t h e i r own e x p e r i e n c e w i t h the 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 o f 3 N - o x i d e s s t i m u l a t e d a F r e n c h group t o p r o p o s e a b i o s y n t h e s i s o f a p p a r i c i n e (2) w h i c h i n c l u d e d e x t r u s i o n o f the C-2 c a r b o n f r o m t h e t w o - c a r b o n b r i d g e . T h i s i d e a was s u p p o r t e d by i n v i t r o s t u d i e s on t r y p t a m i n e d e r i v a t i v e s . F u r t h e r work i n o u r l a b o r a t o r i e s , by Beck and F u l l e r , u s i n g d o u b l y - l a b e l l e d s e c o d i n e (18) r e v e a l e d t h a t t h i s m a t e r i a l was i n c o r p o r a t e d i n t a c t i n t o a p p a r i c i n e (2) i n A. p y r i c o l l u m C0 2CH 3 (18) (2) CH 20 9 10 p l a n t s w i t h r e t e n t i o n o f the carbomethoxy group. ' The e a r l i e r F r e n c h h y p o t h e s i s had s u g g e s t e d a c o n c e r t e d l o s s o f t h i s group d u r i n g t h e f r a g m e n t a t i o n r e a c t i o n . S u b s e q u e n t l y , a m o d i f i e d h y p o t h e s i s was p u b l i s h e d 11 12 i n 1973 i n w h i c h l o s s o f t h e h y d r o x y m e t h y l e n e group was s u g g e s t e d . ' T h i s new h y p o t h e s i s was a s a t i s f y i n g one i n t h a t i t had u t i l i z e d a l l the e x p e r i m e n t a l e v i d e n c e t o d a t e and had a l l o w e d f o r the b i o s y n t h e s i s o f a l l t h e "anomolous" a l k a l o i d s a l o n g v e r y s i m i l a r pathways. T h i s s u g g e s t i o n i s i n a c c o r d w i t h t h e i r c o - e x i s t e n c e i n s e v e r a l p l a n t s p e c i e s . F i g u r e 3. The P o t i e r - J a n o t p o s t u l a t e f o r 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 (2)> u l e i n e (1) and 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 . -74-An i m p o r t a n t p o i n t t o r e c o g n i z e i s t h a t t h e s e b i o s y n t h e t i c pathways i n v o l v e stemmadenine ( 1 6 ) , t h e c r u c i a l p r e c u r s o r t o t h e I b o g a and A s p i d o s p e r m a a l k a l o i d s d i s c u s s e d i n P a r t I . 13 A r e c e n t s y n t h e s i s o f e l l i p t i c i n e (6) by t h e P o t i e r group p r o v i d e s an i n t e r e s t i n g s u p p o r t t o t h e i r h y p o t h e s i s . They a c h i e v e d t h e c y c l i z a t i o n o f s t r u c t u r e (21) v i a a P o l o n o v s k i f r a g m e n t a t i o n t o g i v e (22) i n h i g h y i e l d . T h i s r e a c t i o n i s p a r a l l e l t o t h e c o n v e r s i o n (19)->(20)->-(6) i n F i g u r e 3. S t r u c t u r e (22) was dehydrop.enated (21) (22) t o e l l i p t i c i n e ( 6 ) . To d a t e a l l a t t e m p t s t o i n c o r p o r a t e any p r e c u r s o r i n t o u l e i n e (1) have f a i l e d . I t was f e l t , due t o t h e i m p o r t a n c e o f t h i s p r o b l e m , t h a t some r e - i n v e s t i g a t i o n s and e x t e n s i o n o f the e a r l i e r s t u d i e s was n e c e s s a r y . The f o l l o w i n g s e c t i o n d e s c r i b e s f u r t h e r work d i r e c t e d toward t h e b i o -s y n t h e s i s o f u l e i n e (1) and o l i v a c i n e ( 4 ) . -75-DISCUSSION The f a c t t h a t t r y p t o p h a n ( 1 5 ) , stemmadenine (16) and s e c o d i n e (18) c o u l d a l l be i n c o r p o r a t e d i n t o a p p a r i c i n e (2) s t r o n g l y s u g g e s t s t h a t u l e i n e (1) c o u l d a l s o a r i s e f r o m a s i m i l a r b i o s y n t h e t i c pathway s i n c e t h e y c o - o c c u r i n s e v e r a l A s p i d o s p e r m a p l a n t s p e c i e s . As p r e -4 v i o u s l y m e n t i o n e d , work i n o u r l a b o r a t o r i e s showed t h a t t h e C-3 c a r b o n atom o f t r y p t o p h a n was r e t a i n e d d u r i n g b i o s y n t h e s i s t o f o r m th e u n u s u a l s i n g l e - c a r b o n b r i d g e between t h e i n d o l e n u c l e u s and N, i n b a p p a r i c i n e ( 2 ) . The f i r s t q u e s t i o n t h a t had t o be answered w i t h r e g a r d t o t h e b i o s y n t h e s i s o f u l e i n e (1) was: i s t h i s m o l e c u l e w h i c h d o e s n ' t p o s s e s s s u c h a b r i d g e a l s o d e r i v e d f r o m t r y p t o p h a n (15) o r i s i t d e r i v e d f r o m a p r o g e n i t o r o f t r y p t o p h a n as s u g g e s t e d by t h e Wenkert h y p o t h e s i s ^ ( F i g u r e 1 ) ? S e c o n d l y , i f t r y p t o p h a n i s i n v o l v e d , what i s the f a t e o f c a r b o n s - 2 and 3? I s t h e C-3 c a r b o n atom e x t r u d e d w h i l e t h e C-2 atom i s r e t a i n e d t o become t h e N-methyl group o f u l e i n e (1) as s u g g e s t e d by t h e P o t i e r - J a n o t h y p o t h e s i s ( F i g u r e 3 ) 1 1 ? And t h i r d l y , what r o l e does the d e h y d r o s e c o d i n e h y p o t h e s i s , as r e v e a l e d i n P a r t I , p l a y i n the b i o s y n t h e s i s o f u l e i n e ( 1 ) . S i m i l a r q u e s t i o n s may be a s k e d o f the b i o g e n e t i c o r i g i n o f t h e p y r i d o c a r b o z o l e d e r i v a t i v e o l i v a c i n e ( 4 ) , a member o f t h e "anomolous" f a m i l y o f a l k a l o i d s h a v i n g a t h r e e - c a r b o n b r i d g e between t h e i n d o l e 11 n u c l e u s and N^. The P o t i e r - J a n o t h y p o t h e s i s ( F i g u r e 3) s u g g e s t s t h a t b o t h u l e i n e (1) and o l i v a c i n e (4) a r e d e r i v e d f r o m the same i n t e r m e d i a t e , f r o m w h i c h t h e C-3 c a r b o n atom o r i g i n a l l y i n t r y p t o p h a n has been e x t r u d e d . The f o r m a t i o n o f the f u l l y a r o m a t i c s t r u c t u r e o f o l i v a c i n e (4) a l s o -76-r e q u i r e s l o s s o f t h e C-2 atom w h i c h had been i m p l i c a t e d as t h e N-methyl g r o u p . F i g u r e 4 summarizes t h e e x p e c t e d p o s i t i o n s o f r a d i o - l a b e l i n u l e i n e (1) and o l i v a c i n e (4) as w o u l d a r i s e f r o m t h e a p p r o p r i a t e l y l a b e l l e d p r e c u r s o r s t r y p t o p h a n ( 1 5 ) , stemmadenine (16) and s e c o d i n e ( 1 8 ) . We had a v a i l a b l e i n o u r l a b o r a t o r i e s two A s p i d o s p e r m a p l a n t s p e c i e s , A. p y r i c o l l u m and A. a u s t r a l e , i n w h i c h u l e i n e ( 1 ) , a p p a r i c i n e (2) and o l i v a c i n e (4) c o - o c c u r . T h i s p r o v i d e d us w i t h a u n i q u e o p p o r t u n i t y t o s t u d y the b i o s y n t h e s i s o f t h e s e u n u s u a l a l k a l o i d s . As m e n t i o n e d e a r l i e r t h e s t u d y o f a p p a r i c i n e (2) was begun f i r s t , i n our l a b o r a t o r i e s , i n A. p y r i c o l l u m p l a n t s . The b i o s y n t h e t i c e v i d e n c e o b t a i n e d i s summarized i n T a b l e 1. 4 E x p e r i m e n t s 1 and 2 i n T a b l e 1, r e p o r t e d by N e l s o n and W i g f i e l d , showed f o r t h e f i r s t t i m e t h a t t r y p t o p h a n i s i n d e e d a p r e c u r s o r o f a p p a r i c i n e (2) and t h a t i t i s t h e C-2 c a r b o n atom w h i c h i s e x t r u d e d a t some p o i n t i n the b i o s y n t h e s i s . The S t r y c h n o s a l k a l o i d stemmadenine (16) was f o u n d to be i n c o r p o r a t e d i n the s u r p r i s i n g l y h i g h l e v e l of 0.55%, w h i l e the more s t r u c t u r a l l y s i m i l a r v a l l e s a m i n e (3) was i n c o r p o r a t e d i n o n l y 0.01%. A l t h o u g h t h e i n c o r p o r a t i o n o f v a r i o u s l y l a b e l l e d s e c o d i n e (18) p r e c u r s o r s was g e n e r a l l y low ( e x p e r i m e n t s • 5 - 8 ) t h e r e c a n be l i t t l e d o u b t t h a t t h e p r o p o s e d stemmadenine J s e c o d i n e b i o - e q u i l i b r i u m i s o p e r a t i v e i n t h e b i o s y n t h e s i s o f t h i s m o l e c u l e . I n none o f t h e s e e x p e r i -ments c o u l d i n c o r p o r a t i o n i n t o u l e i n e (1) be d e m o n s t r a t e d . C l e a r l y , i t w o u l d be o f g r e a t i n t e r e s t t o expand t h e 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 o f u l e i n e (1) i n A. p y r i c o l l u m p l a n t s i n an a t t e m p t to u n d e r s t a n d t h e n e g a t i v e r e s u l t s o b t a i n e d t o d a t e , and t h e n e v a l u a t e p o s s i b l e b i o s y n t h e s i s o f t h i s m o l e c u l e i n a n o t h e r A s p i d o s p e r m a p l a n t s p e c i e s . -77-F i g u r e 4. E x p e c t e d p o s i t i o n s o f r a d i o - l a b e l i n u l e i n e (1) and o l i v a c i n e (4) from a v a i l a b l e p r e c u r s o r s as s u g g e s t e d by the P o t i e r - J a n o t h y p o t h e s i s ( F i g u r e 3 ) . -78-T a b l e 1 I n c o r p o r a t i o n s t u d i e s i n t o a p p a r i c i n e (2) i n A. p y r i c o l l u m p l a n t s E n t r y ( r e f ) P r e c u r s o r P e r c e n t R a t i o R a t i o i n c o r p o r a t i o n A c t i v i t y A c t i v i t y , , F e d ( H/^C) I s o l a t e d ( 3 H / C ) 1(4) [ A r - 3 H , 3 - 1 4 C ] - 1.1 1.5 t r y p t o p h a n ( 1 5 ) 2(4) [ A r - 3 H , 2 - 1 4 C ] - 1.0 <0.03 t r y p t o p h a n ( 1 5 ) 3 ( 5 ) [ A r - 3 H ] - s t e m m a d e n i n e ( 1 6 ) 0.55 3 4( 5 ) [ A r - H ] - v a l l e s a m i n e ( 3 ) 0.01 5( 9 ) [ 1 4 C 0 2 C H 3 ] - s e c o d i n e ( 1 8 ) .01 6( 9 ) [ A r - 3 H , 1 4 C 0 2 C H 3 ] - 0.014, 0.015 8.7 8.4 s e c o d i n e ( i s ) 7(9) [ 3 , 1 4 , 1 5 , 2 1 - 3 H , 1 4 C 0 2 C H 3 ] - 0.005, 0.009 4.2 2.2 s e c o d i n e ( 1 8 ) 8(10) [ 1 9 - 3 H , 1 4 C 0 2 C H 3 ] - .024 3.98 2.05 s e c o d i n e ( i s ) -79-I n A. a u s t r a l e p l a n t s , s i m i l a r p r e c u s o r s c o u l d be a d m i n i s t e r e d t o . i n v e s t i g a t e t h e i r p o s s i b l e p a r t i c i p a t i o n i n the b i o s y n t h e s i s o f u l e i n e (1) and the p y r i d o c a r b a z o l e a l k a l o i d o l i v a c i n e ( 4 ) . The b i o s y n t h e s i s o f t h e l a t t e r c l a s s o f a l k a l o i d s has n o t been i n v e s t i g a t e d b e f o r e . The r e m a i n i n g p a r t o f t h i s d i s c u s s i o n d e a l s w i t h the c u r r e n t i n v e s t i g a t i o n o f the b i o s y n t h e s i s of u l e i n e (1) and o l i v a c i n e ( 4 ) . The h y p o t h e t i c a l l y l a b e l l e d s t r u c t u r e of u l e i n e (1) i n F i g u r e 4 i l l u s t r a t e s t h a t an i n v e s t i g a t i o n i n t o the b i o s y n t h e s i s o f t h i s m o l e c u l e w o u l d be g r e a t l y enhanced i f a d e g r a d a t i o n scheme c o u l d be a d a p t e d t o p e r m i t the i s o l a t i o n o f some o f the c a r b o n atoms w h i c h a r e e x p e c t e d t o b e a r a r a d i o l a b e l . W i t h t h i s o b j e c t i v e i n mind, the f o l l o w i n g p l a n was implemented. Our f i r s t o b j e c t i v e was t o i s o l a t e the N-methyl group o f u l e i n e ( 1 ) . U l e i n e was f i r s t i s o l a t e d by Schmutz e t . a l . f r o m A. u l e i i n 1957 . The s t r u c t u r e p r o p o s e d a t t h a t time was, however, i n c o r r e c t and was r e p l a c e d by the now p r o v e n s t r u c t u r e p r o p o s e d by B u c h i two y e a r s l a t e r . X ^ The l a t t e r group a l s o succeeded i n the t o t a l s y n t h e s i s o f u l e i n e i n 1 9 7 1 . 1 6 The s t r u c t u r a l p r o o f o f t h i s m o l e c u l e was b a s e d on a c l a s s i c a l Hofmann d e g r a d a t i o n scheme w h i c h p e r m i t t e d the r e m o v a l o f the b a s i c n i t r o g e n f r o m the i n d o l e p o r t i o n . T h i s scheme, t h e n , c o u l d p r o b a b l y be adapted t o p e r m i t the i s o l a t i o n of the N-methyl group i n a manner w h i c h would be u s e f u l i n a 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 . / To o b t a i n b u l k q u a n t i t i e s o f u l e i n e f o r our d e g r a d a t i o n work and s ubsequent p l a n t f e e d i n g e x p e r i m e n t s we had a v a i l a b l e e t h a n o l i c e x t r a c t s o f A. a u s t r a l e p l a n t s c o l l e c t e d i n B r a z i l . The e x t r a c t s were d r i e d and t r e a t e d w i t h a 15% aqueous a c e t i c a c i d s o l u t i o n , f i l t e r e d t o remove -80-i n s o l u b l e m a t e r i a l , and the r e s u l t i n g s o l u t i o n was washed w i t h p e t r o l e u m e t h e r t o remove any r e m a i n i n g n o n - a l k a l o i d a l m a t e r i a l . T h i s a c i d i c s o l u t i o n was made b a s i c w i t h aqueous sodium h y d r o x i d e 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 t o y i e l d t h e a l k a l o i d c o n t a i n i n g f r a c t i o n . Subsequent c h r o m a t o g r a p h y on s i l i c a g e l y i e l d e d q u a n t i t i e s o f u l e i n e ( 1 ) , a p p a r i c i n e (2) and o l i v a c i n e (4) and s e v e r a l o t h e r _ 17,18 components. A s i m i l a r i s o l a t i o n scheme was used t o o b t a i n f u r t h e r q u a n t i t i e s o f u l e i n e (1) and o l i v a c i n e (4) f r o m A s p i d o s p e r m i a O l i v a c e u m p l a n t e x t r a c t s . Chromatagraphy o f t h e a l k a l o i d a l f r a c t i o n u s i n g d e a c t i v a t e d a l u m i n a as a d s o r b e n t p r o v i d e d an e a s i e r method f o r o b t a i n i n g u l e i n (1) as p u r e c r y s t a l l i n e m a t e r i a l . U l e i n e ( 1 , m.p.75-80°) was i d e n t i f i e d by i t s c h a r a c t e r i s t i c u l t r a v i o l e t s p e c t r u m ( ^ m a x 210, 307, 3 1 6 ) , mass s p e c t r u m ; and pmr s p e c t r u m . The l a t t e r e x h i b i t e d two s i n g l e t a b s o r p t i o n s o f T4.64 and 4.72 w h i c h a r e c h a r a c t e r i s t i c o f e x o c y c l i c m e t h y l e n e p r o t o n s . A l s o p r e s e n t was a s i n g l e t a t T 7 , ^ 0 w h i c h was a s s i g n e d t o the N-methyl group. The s p e c t r a l and p h y s i c a l d a t a were f o u n d t o be i d e n t i c a l w i t h p u b l i s h e d d a t a 1 ^ as 18 w e l l as w i t h an a u t h e n t i c sample o f u l e i n e ( 1 ) . O l i v a c i n e ( 4 , m.p.312-315°) was s i m i l a r i l y i d e n t i f i e d . I t s u l t r a v i o l e t s p e c t r u m (X 224, 338, 277, 288, 315, 329, 375) showed max the p r e s e n c e o f the f u l l y a r o m a t i c p y r i d o c a r b a z o l e s y s t e m . The pmr s p e c t r u m e x h i b i t e d b e s i d e s t h e s e v e n a r o m a t i c r i n g p r o t o n s , a s i n g l e t a t x6.86 w h i c h was a s s i g n e d t o t h e C - l m e t h y l group and a n o t h e r s i n g l e t a t T 7 . 2 1 was a t t r i b u t e d t o the C-5 m e t h y l group. The s p e c t r a l and - 8 1 -p h y s i c a l data"'"'' on t h i s compound were i d e n t i c a l w i t h p u b l i s h e d d a t a 24 and w i t h an a u t h e n t i c sample o f o l i v a c i n e ( 4 ) . The f i r s t d e g r a d a t i o n o f u l e i n e (1) t h a t we c o n s i d e r e d was one i n v o l v i n g two c l a s s i c a l Hofmann e l i m i n a t i o n r e a c t i o n s w h i c h w o u l d remove the b a s i c n i t r o g e n w i t h i t s m e t h y l group f r o m t h e r e s t o f t h e m o l e c u l e . U l e i n e (1) was c o n v e r t e d t o i t s q u a t e r n a r y s a l t w i t h m e t h y l i o d i d e t o g i v e a v e r y h i g h y i e l d o f t h e m e t h i o d i d e d e r i v a t i v e m.p. 205-206°. The mass s p e c t r u m o f t h i s compound e x h i b i t e d t h e p a r e n t peak (m/e 281) as t h e base peak w h i c h c o r r e s p o n d e d t o t h e a d d i t i o n o f one m e t h y l group t o t h e u l e i n e m o l e c u l e . The i d e n t i t y o f s t r u c t u r e (23) was c o n f i r m e d by e l e m e n t a l a n a l y s i s . U l e i n e m e t h i o d i d e (23) was r e f l u x e d w i t h p o t a s s i u m h y d r o x i d e i n aqueous e t h a n o l t o g i v e a s i n g l e p r o d u c t w h i c h had an u l t r a v i o l e t s p e c t r u m (A 2 3 5 ( s h ) , 248, 262, 287, 293, 307) c h a r a c t e r i s t i c o f a max c a r b a z o l e d e r i v a t i v e . The mass s p e c t r u m o f t h i s p r o d u c t showed a m o l e c u l a r p a r e n t peak a t m/e 280. The pmr s p e c t r u m , a p a r t f r o m two g r o u p s o f a r o m a t i c p r o t o n s i g n a l s , r e v e a l e d a q u a r t e t a t x7.10 and a m e t h y l t r i p l e t a t T 8 . 7 5 i n d i c a t i n g an e t h y l s i d e - c h a i n , s i m i l a r t o t h a t i n u l e i n e ( 1 ) . A l s o t h e r e a p p e a r e d a t h r e e p r o t o n s i n g l e t a t x7.50 w h i c h was a s s i g n e d t o a m e t h y l group a t t a c h e d t o an a r o m a t i c r i n g and a s i x - p r o t o n s i n g l e t a t T 7 . 6 1 w h i c h was a s s i g n e d t o two N-methyl g r o u p s . These d a t a a r e c o n s i s t e n t w i t h s t r u c t u r e ( 2 4 ) . U l e i n e m e t h i o d i d e (23) underwent t h e e x p e c t e d Hofmann f r a g m e n t a t i o n r e a c t i o n t o g i v e an i n t e r -m e d i a t e (24), w h i c h underwent f a c i l e a r o m a t i z a t i o n t o g i v e t h e c a r b a z o l e s y s t e m (25) i n 82% y i e l d . -82-The c a r b a z o l e d e r i v a t i v e (25) was r e a c t e d w i t h m e t h y l i o d i d e t o g i v e i t s c o r r e s p o n d i n g m e t h i o d i d e s a l t (22, m.p. 300-302°) w h i c h formed i n q u a n t i t a t i v e y i e l d . The n e x t s t e p i n t h e d e g r a d a t i o n was t o p e r f o r m a Hofmann e l i m i n a t i o n r e a c t i o n on t h e m e t h i o d i d e (26) t o remove t h e b a s i c n i t r o g e n f r o m the c a r b a z o l e p a r t o f t h e m o l e c u l e . I n i t i a l a t t e m p t s , c o n t r a r y t o a l i t e r a t u r e r e p o r t , f a i l e d t o a c h i e v e the d e s i r e d f r a g m e n t a t i o n when p o t a s s i u m h y d r o x i d e d i s s o l v e d i n e t h y l e n e g l y c o l -w a t e r s o l u t i o n a t 200°C, was employed. However, p o t a s s i u m _ t - b u t o x i d e i n f r e s h l y d r i e d ^ - b u t a n o l a c h i e v e d t h e e l i m i n a t i o n a t 80°. The p r o d u c t (27) f r o m t h i s r e a c t i o n had an u l t r a v i o l e t s p e c t r u m (A. 228, 248, 265, max 292, 306 nm) e x p e c t e d f o r a c a r b a z o l e s y s t e m . I t s mass s p e c t r u m showed a m o l e c u l a r i o n as t h e p a r e n t peak a t m/e 235 w i t h a f a c i l e l o s s o f two m e t h y l g r o u p s t o g i v e f r a g m e n t s a t m/e 220 and 205. The pmr s p e c t r u m r e v e a l e d a m u l t i p l e t c e n t e r e d a t x3.54 w h i c h was a s s i g n e d t o a v i n y l group bonded t o an a r o m a t i c r i n g . I n o r d e r f o r t h i s d e g r a d a t i o n scheme to be u s e f u l i n a 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 i t was n e c e s s a r y t o e v a l u a t e the sequence s t a r t i n g w i t h 15-20 m i l l i g r a m s (mg) o f u l e i n e ( 1 ) , t h i s b e i n g the e x p e c t e d q u a n t i t y o b t a i n a b l e f r o m p l a n t f e e d i n g e x p e r i m e n t s . I t was a l s o e s s e n t i a l t o be a b l e t o t r a p e f f i c i e n t l y the t r i m e t h y l a m i n e (28) w h i c h was l i b e r a t e d i n t h e l a s t r e a c t i o n o f t h i s s e q u ence. T h i s r e q u i r e m e n t was a c h i e v e d by a s l o w p a s s a g e o f d r y n i t r o g e n gas t h r o u g h t h e r e a c t i o n m i x t u r e i n t o a c o l l e c t o r c o n t a i n i n g a m e t h a n o l i c s o l u t i o n o f m e t h y l i o d i d e . C r y s t a l l i n e t etramethylammonium i o d i d e (29) was o b t a i n e d i n an o v e r a l l y i e l d o f 70-75%. -83-( 2 9 ) F i g u r e 5. D e g r a d a t i o n o f u l e i n e by means o f Hofmann e l i m i n a t i o n r e a c t i o n s . -84-I t was a l s o d e s i r a b l e t o e v a l u a t e t h i s d e g r a d a t i o n scheme u s i n g r a d i o a c t i v e m a t e r i a l b e f o r e p r o c e e d i n g w i t h t h e p l a n t f e e d i n g e x p e r i m e n t s i n o r d e r t o d i s c o v e r any f u r t h e r d i f f i c u l t i e s w h i c h m i g h t a r i s e . U l e i n e 14 ( l ) ( 2 0 m g ) was r e a c t e d w i t h C - m e t h y l i o d i d e and t h e r e s u l t i n g q u a t e r n a r y ammonium s a l t (19) was r e c r y s t a l l i z e d t o a c o n s t a n t s p e c i f i c r a d i o -a c t i v i t y o f 7.17x10"' d i s i n t e g r a t i o n s p e r m i n u t e (dpm) p e r m i l l i m o l e . D e g r a d a t i o n o f t h i s m a t e r i a l by t h e p r o c e d u r e d e s c r i b e d above y i e l d e d t e t r a m e t h y l ammonium i o d i d e (25) ( 7 2 % y i e l d ) , w h i c h was r e c r y s t a l l i z e d t o a c o n s t a n t r a d i o a c t i v i t y o f 6.95x10^ dpm p e r m i l l i m o l e . T h i s r e c o v e r y o f 97% o f t h e s p e c i f i c a c t i v i t y was c o n s i d e r e d w i t h i n t h e l i m i t s o f e x p e r i m e n t a l e r r o r . A 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 o f u l e i n e was now i n i t i a t e d . The f i r s t p l a n t s p e c i e s c h o s e n f o r t h i s s t u d y was A. p y r i c o l l u m . T h i s p l a n t i s a s l o w g r o w i n g s h r u b , i n d i g e n o u s t o S o u t h A m e r i c a , w h i c h s t o r e s u l e i n e i n t h e r o o t s . We f o u n d t h a t by r e m o v i n g some f i b r o u s r o o t s o r t h e t a p r o o t f o r u s e i n f e e d i n g e x p e r i m e n t s t h e r e m a i n i n g p l a n t c o u l d be m a i n t a i n e d i n a s a t i s f a c t o r y g r o w i n g s t a t e f o r s u b s e q u e n t e x p e r i m e n t s . I n o u r i n i t i a l f e e d i n g e x p e r i m e n t s u s i n g e i t h e r f i b r o u s o r t a p r o o t s t h e w a t e r s o l u b l i z e d p r e c u r s o r was a d m i n i s t e r e d t o p o l o g i c a l l y t o the s y s t e m . A t t h e end o f t h e f e e d i n g t i m e , t h e r o o t s were m a s u r a t e d i n m e t h a n o l and the n o r m a l a l k a l o i d e x t r a c t i o n p r o c e d u r e , as d e s c r i b e d e a r l i e r , was u s e d t o o b t a i n a c r u d e e x t r a c t . I n e a c h e x p e r i m e n t u l e i n e (l)(20mg) was added t o t h i s e x t r a c t t o a c t as a c a r r i e r f o r s m a l l amounts o f r a d i o a c t i v e u l e i n e w h i c h may have been p r e s e n t i n q u a n t i t i e s i n s u f f i c i e n t t o s t u d y d i r e c t l y . T h i s t e c h n i q u e o f r a d i o - d i l u t i o n a l l o w s one t o s t u d y -85-t h e t o t a l i n c o r p o r a t i o n i n t o a compound b u t n o t s p e c i f i c i n c o r p o r a t i o n ( d e f i n e d as r a d i o a c t i v i t y per^ m i l l i m o l e ) s i n c e t h e amount o f compound o b t a i n e d f r o m t h e p l a n t i s n e v e r known. T a b l e 2 summarizes t h e f e e d i n g e x p e r i m e n t s u s i n g A. p y r i c o l l u m . I n e x p e r i m e n t s 1 and 2, t r y p t o p h a n l a b e l l e d i n t h e a r o m a t i c r i n g w i t h t r i t i u m and w i t h carbon-14 i n t h e s i d e c h a i n (C-2) was a d m i n i s t e r e d h y d r o p o n i c a l l y t o t a p r o o t and f i b r o u s r o o t s , r e s p e c t i v e l y . A f t e r a t i m e p e r i o d o f 6 d a y s , u l e i n e (1) was i s o l a t e d and r e c r y s t a l l i z e d u n t i l an i n s i g n i f i c a n t amount o f r a d i o a c t i v i t y was d e t e c t e d . I t i s g e n e r a l l y a c c e p t e d i n the f i e l d o f b i o s y n t h e s i s t h a t i f a t o t a l i n c o r p o r a t i o n o f a p r e c u r s o r i n t o a t a r g e t m o l e c u l e i s b e l o w 0.001% t h e r e s u l t s s h o u l d be v i e w e d as b e i n g n e g a t i v e . I n t h e t h i r d e x p e r i m e n t i n t h i s p l a n t s p e c i e s , t h e a c e t a t e s a l t o f s e c o d i n e (18) l a b e l l e d w i t h c a r b o n 14 i n the carbomethoxy group was s i m i l a r i l y a d m i n i s t e r e d t o a t a p r o o t . A g a i n , i n c o r p o r a t i o n i n t o u l e i n e (1) c o u l d n o t be d e m o n s t r a t e d . The p r e p a r a t i o n o f t h i s p r e c u r s o r i s d e s c r i b e d i n P a r t I . A second s e r i e s o f f e e d i n g s t o A. p y r i c o l l u m were i n i t i a t e d to t e s t t h e p o s s i b i l i t y t h a t the p r o d u c t i o n o f u l e i n e (1) i n the p l a n t was a s l o w p r o c e s s and t h e r e f o r e t h e 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 (15) o r i t s p r e c u r s o r a n t h r a n i l i c a c i d t o a g r o w i n g p l a n t may r e s u l t i n some i n c o r p o r a t i o n i n t o u l e i n e . The r e s o u r c e s r e q u i r e d t o do a c o m p l e t e t i m e s t u d y o f i n c o r p o r a t i o n were n o t a v a i l a b l e a t t h a t t i m e . I n e x p e r i m e n t s 4 and 5, t r y p t o p h a n and a n t h r a n i l i c a c i d were a d m i n i s t e r e d i n v e r y d i l u t e a c e t i c a c i d s o l u t i o n s t o g r o w i n g p l a n t s u s i n g the w i c k f e e d i n g t e c h n i q u e . The r e s u l t s o f t h e s e l o n g term f e e d i n g e x p e r i m e n t s i n whole p l a n t s -86-T a b l e 2. I n c o r p o r a t i o n s t u d i e s i n A. p y r i c o l l u m p l a n t s Exp. No. P r e c u r s o r A c t i v i t y Fed(dpm) P e r c e n t i n c o r p o r a t i o n i n t o U l e i n e (1) [ A r - 3 H , 2 - 1 4 C ] 3 H - 1 . 1 3 x l 0 7 t r y p t o p h a n ( 1 5 ) a 1 / fC - 1 . 1 4 x l 0 8 < 0.001 [ A r - 3 H , 2 - 1 4 C ] - 3 H - 2 . 3 4 x l 0 8 t r y p t o p h a n ( 1 5 ) a 1 4 C - 7 . 6 2 x l 0 9 < 0.001 [ 1 4 C 0 2 C H 3 ] - b s e c o d i n e ( 1 8 ) 3.70x10' < 0.001 [ A r - 3 H , 2 - 1 4 C ] - 3 H - 3 . 8 9 x l 0 8 t r y p t o p h a n ( 1 5 ) 3 1 4 C - 4 . 9 2 x l 0 7 < 0.001 [ A r - H ] -a n t h r a n i l i c a c i d ' 2.22x10' < 0.001 a A v a i l a b l e f r o m New E n g l a n d N u c l e a r b A v a i l a b l e from t h e o r i g i n a l s y n t h e s i s by Sood i n our l a b o r a t o r i e s . -87-a p p e a r e d a l s o t o be n e g a t i v e . A d d i t i o n a l d a t a c o n c e r n i n g t h e f e e d i n g e x p e r i m e n t s i n A. p y r i c o l l u m a p p e a r i n T a b l e 4. A n o t h e r p l a n t s p e c i e s w h i c h had become a v a i l a b l e i n our l a b o r a t o r i e s was A s p i d o s p e r m a a u s t r a l e . Not o n l y d i d t h i s p l a n t b i o s y n t h e s i z e u l e i n e (1) b u t a l s o i t p r o d u c e d s e v e r a l 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 f w h i c h o l i v a c i n e (4) was o f p a r t i c u l a r i n t e r e s t . The p l a n t s used i n t h i s work were e i g h t e e n t o t w e n t y - f o u r months o l d and t w e n t y to t w e n t y - f i v e c e n t i m e t e r s t a l l . I n t h i s s e r i e s o f e x p e r i m e n t s , the p r e c u r s o r was a d m i n i s t e r e d d i r e c t l y t o the t a p r o o t o f the w h o l e p l a n t and the p l a n t was t h e n a l l o w e d to grow h y d r o p o n i c a l l y i n a t e s t tube d u r i n g a f e e d i n g time o f 6 d a y s . As b e f o r e t h e t a r g e t compounds u l e i n e (1) and o l i v a c i n e (4) were o b t a i n e d by d i l u t i o n o f the a l k a l o i d a l e x t r a c t s w i t h i n a c t i v e a l k a l o i d s and s u b s e q u e n t i s o l a t i o n . T a b l e 3 summarizes t h e r e s u l t s o f t h e s e e x p e r i m e n t s . E x p e r i m e n t s 1 and 2 show t h a t n e i t h e r a n t h r a n i l i c a c i d n o r t r y p t o p h a n c o u l d be i n c o r p o r a t e d s i g n i f i c a n t l y i n t o u l e i n e (1) o r o l i v a c i n e (4) under t h e s e e x p e r i m e n t a l c o n d i t i o n s . I n t h e hope t h a t a l a t e r s t a g e p r e c u r s o r , b e i n g c l o s e r t o the end o f t h e b i o s y n t h e t i c pathway, m i g h t be e l a b o r a t e d i n t o t h e s e a l k a l o i d s two f e e d i n g e x p e r i m e n t s u s i n g d o u b l y - l a b e l l e d s e c o d i n e (18) were p e r f o r m e d . The p r o c e d u r e f o r p r e p a r i n g s e c o d i n e (18) i m m e d i a t e l y p r i o r to i t s a d m i n i s t r a t i o n to the p l a n t was t h e same as t h a t d e s c r i b e d i n P a r t I . No i n c o r p o r a t i o n o f s e c o d i n e (18) i n t o u l e i n e (1) o r o l i v a c i n e (4) c o u l d be d e m o n s t r a t e d . A n o t h e r b i o g e n i s i s o f the N-methyl group o f u l e i n e (1) was c o n s i d e r e d . I t i s w e l l known t h a t the m ain s o u r c e o f m e t h y l groups i n b i o l o g i c a l s y stems i s t h e amino a c i d m e t h i o n i n e . M e t h i o n i n e (30) i n i t s a c t i v e f o r m as S--88-a d e n o s y l m e t h i o n i n e (31) c o u l d t r a n s f e r i t s S-methyl group to the b i o l o g i c a l e q u i v a l e n t o f d e s - N - m e t h y l u l e i n e (32). The l a t t e r (32) C H 3 - S - C H 2 C H 2 C H - C O - , H (30; C O f H - C - N H 2 C H 3 C H 2 H OH (31) c o u l d p o s s i b l y be on the b i o s y n t h e t i c r o u t e to u l e i n e (1) o r i t c o u l d be i n b i o l o g i c a l e q u i l i b r i u m w i t h the m e t h y l a t e d d e r i v a t i v e . (32) . (1) -89-T h e r e f o r e i t was d e c i d e d to l a b e l the " C ^ - p o o l " i n the p l a n t w i t h m e t h i o n i n e (30) l a b e l l e d w i t h c a r b o n 14 i n the S-methyl group. As shown by e x p e r i m e n t s 5 and 6 i n T a b l e 3, a h i g h i n c o r p o r a t i o n o f m e t h i o n i n e i n t o u l e i n e (1) was f o u n d . To a s c e r t a i n the p r e c i s e p o s i t i o n o f l a b e l i n the i s o l a t e d u l e i n e t h e p r e v i o u s l y d e s c r i b e d d e g r a d a t i o n scheme p e r m i t t e d t h e i s o l a t i o n o f 97% and 98% o f the s p e c i f i c r a d i o -a c t i v i t y o b t a i n e d as the tetramethylammonium i o d i d e s a l t ( 2 9 ) . The r e s u l t s of the 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 d e s c r i b e d i n t h i s s e c t i o n do n o t p r o v i d e any d i r e c t i n f o r m a t i o n r e l a t i n g to e i t h e r the Wenkert h y p o t h e s i s 1 o r the P o t i e r - J a n o t h y p o t h e s i s 1 1 c o n c e r n i n g the b i o s y n t h e s i s the u l e i n e and o l i v a c i n e t y p e of a l k a l o i d s . The i n c o r p o r a t i o n o f a n t h r a n i l i c a c i d i s e s s e n t i a l t o the Wenkert h y p o t h e s i s as i s the 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 (15) t o t h e P o t i e r - J a n o t h y p o t h e s i s . The l a t t e r would a l s o r e q u i r e the i n c o r p o r a t i o n o f s e c o d i n e (18) s i n c e i t has a l r e a d y been d e m o n s t r a t e d (as d e s c r i b e d i n P a r t I ) t h a t s e c o d i n e i s an a c t i v e p r e c u r s o r t o the Aspidosperma and Iboga t y p e of a l k a l o i d s by way o f t h e d e h y d r o s e c o d i n e (18) Z stemmadenine (16) e q u i l i b r i u m ( F i g u r e 4 ) . However, the f a c t t h a t none of t h e s e p r e c u r s o r s c o u l d be e l a b o r a t e d by A. p y r i c o l l u m o r A. a u s t r a l e i n t o more complex a l k a l o i d s under t h e c o n d i t i o n s d e s c r i b e d does n o t mean t h a t t h e s e compounds a r e n e c e s s a r i l y on the t r u e b i o s y n t h e t i c pathway. 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 n e g a t i v e r e s u l t s 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 due t o t h r e e problems i n h e r e n t t o t h e a d m i n i s t r a t i o n o f m a t e r i a l t o p l a n t s . The f i r s t p r o b l e m t h a t must be overcome i s one of t r a n s p o r t a t i o n t o the s i t e o f b i o s y n t h e s i s w i t h i n the p l a n t . We know t h a t the p l a n t has -90-T a b l e 3. I n c o r p o r a t i o n s t u d i e s i n A. a u s t r a l e p l a n t s Exp.No. P r e c u r s o r A c t i v i t y Fed(dpm) P e r c e n t I n c o r p o r a t i o n o l i v a c i n e ( 4 ) u l e i n e ( 1 ) [ A r - H ] - , a n t h r a n i l i c a c i d ' 2.22x10' < 0.001 < 0.001 [ A r - 3 H , 2 - 1 4 C ] -t r y p t o p h a n ( 1 5 ) ' -< 0.001 < 0.001 [ A r - 3 H , 1 4 C 0 2 C H 3 ] -s e c o d i n e ( 1 8 ) D 3 H - 2 . 4 8 x l 0 7 14 C - 7.44xlO c < 0.001 < 0.001 [19- 3H, 1 4 C 0 2 C H 3 ] -s e c o d i n e ( 1 8 ) c 1 4 c 1.19x10' 7 . 5 0 x l 0 6 < 0.001 < 0.001 [ 1 4 C H 3 ] -m e t h i o n i n e ( 3 0 ) a 2.22x10 8 < 0.001 0.168 [ 1 4 C H 3 ] -methionine(30 ) a 2.22x10 8 < 0.001 0.147 a. A v a i l a b l e from New England N u c l e a r b. [ " ^ C ^ C H ^ ] - s e c o d i n e was a v a i l a b l e from the o r i g i n a l s y n t h e s i s by Sood i n our l a b o r a t o r i e s o 3 c. S y n t h e s i s o f [ A r - H ] - s e c o d i n e and [19- H ] - s e c o d i n e i s d e s c r i b e d i n P a r t I - 9 1 -a b s o r b e d a l a r g e p e r c e n t o f t h e p r e c u r s o r b e c a u s e the r e s i d u a l r a d i o -a c t i v i t y i n t h e v e s s e l i s a l w a y s measured. But we d o n ' t know where i n t h e p l a n t t h e b i o s y n t h e s i s o f the a l k a l o i d s o c c u r s n o r do we know i f o u r p r e c u r s o r i s t r a n s p o r t e d t o where i t may be r e q u i r e d . The s e c o n d p r o b l e m i s one o f p e r m e a b i l i t y o f t h e c e l l w a l l s b e h i n d w h i c h b i o s y n t h e s i s t a k e s p l a c e . T h i s p r o b l e m i s b e l i e v e d t o be more s e r i o u s w i t h l a r g e r m o l e c u l e s s u c h as s e c o d i n e ( 1 8 ) . I t may be t h a t under n o r m a l c o n d i t i o n s t h e b i o s y n t h e t i c b u i l d i n g b l o c k s a r e a t t a c h e d t o c a r r i e r p r o t e i n m o l e c u l e s and u n a t t a c h e d p r e c u r s o r s may be t r e a t e d as f o r e i g n m a t e r i a l by t h e p l a n t s y s t e m s . The t h i r d p r o b l e m a r i s i n g i n a 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 i s t h e q u e s t i o n o f w h e t h e r o r n o t the p l a n t i s a c t i v e l y b i o s y n t h e s i z i n g t h e t a r g e t compounds d u r i n g t h e t i m e o f t h e e x p e r i m e n t . I t i s p o s s i b l e t h a t t h e s e compounds a r e b i o s y n t h e s i z e d v e r y s l o w l y and s t o r e d o r t h e y may be b i o -s y n t h e s i z e d o n l y d u r i n g c e r t a i n g r o w t h p e r i o d s i n the l i f e o f the p l a n t and a g a i n s i m p l y s t o r e d d u r i n g o t h e r t i m e s . I f b i o s y n t h e s i s i s o c c u r r i n g v e r y s l o w l y i t i s l i k e l y t h a t the p r e c u r s o r t h a t was f e d w o u l d undergo b i o d e g r a d a t i o n b e f o r e a s i g n i f i c a n t amount o f i t c o u l d be u s e d t o s y n t h e s i z e t h e a l k a l o i d s under i n v e s t i g a t i o n . I n o r d e r t o overcome some o f t h e s e p r o b l e m s i n h e r e n t i n t h e 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 s t o p l a n t s y s t e m s a g r e a t d e a l o f m a t e r i a l and work w o u l d be r e q u i r e d . One way a r o u n d t h e s e p r o b l e m s i s t o work w i t h c e l l - f r e e e x t r a c t s o f the enzyme systems o f the p l a n t s p e c i e s . Work i n t h i s a r e a w i l l be i n i t i a t e d i n our l a b o r a t o r i e s i n the n e a r f u t u r e . The f a c t t h a t u l e i n e (1) was e f f i c i e n t l y m e t h y l a t e d w i t h c a r b o n 14 l a b e l l e d m e t h i o n i n e (30) c a s t s g r a v e doubt on the P o t i e r - J a n o t h y p o t h e s i s -92-w h i c h r e q u i r e d t h e N-methyl group t o come f r o m C-2 c a r b o n o f t r y p t o p h a n ( 1 5 ) . I t i s t h e r e f o r e l o g i c a l t o s p e c u l a t e t h a t d e s - N - m e t h y l u l e i n e /32) i s an imme d i a t e p r e c u r s o r t o u l e i n e (1) and t h a t m e t h y l a t i o n o f t h e b a s i c n i t r o g e n atom o c c u r s as the l a s t s t e p i n t h e b i o s y n t h e s i s . T h i s p o s s i -b i l i t y i s s u p p o r t e d by the d i s c o v e r y o f t h e c o - e : : i s t e n c e o f t h e des-N-m e t h y l d e r i v a t i v e (32) w i t h u l e i n e (1) i n A. d a s y c a r p o n A. DC. 7 and 21 A. v a r g a s i i A.DC. The s t u d y o f t h i s m e t h y l t r a n s f e r r e a c t i o n by t h e m e t h y l t r a n s f e r a s e enzyme may be a good way t o i n i t i a t e an i n v e s t i g a t i o n o f c e l l - f r e e enzyme e x t r a c t s o f the A s p i d o s p e r m a s p e c i e s . P a r t I I I o f t h i s t h e s i s d e s c r i b e s a s y n t h e t i c s t u d y d e s i g n e d t o make a v a i l a b l e , f o r b i o s y n t h e t i c e v a l u a t i o n , a p p r o p r i a t e l y l a b e l l e d d e r i v a t i v e s i n t h e p r e a k u a m m i c i n e - and s t e m m a d e n i n e - s e r i e s . - 9 3 -EXPERIMENTAL F o r a d e s c r i p t i o n o f g e n e r a l e x p e r i m e n t a l i n f o r m a t i o n t h e r e a d e r i s r e f e r r e d t o t h e e x p e r i m e n t a l s e c t i o n o f P a r t I . R a d i o a c t i v i t y was measured u s i n g a N u c l e a r - C h i c a g o Mark I o r a Mark I I M o d e l 6860 L i q u i d S c i n t i l a t i o n C o u n t e r . The d e t a i l s o f t h e c o u n t i n g p r o c e d u r e were p r e v i o u s l y d e s c r i b e d and a r e p e r t a i n a n t t o t h i s s e c t i o n . The A s p i d o s p e r m a p y r i c o l l u m p l a n t s and t h e A. a u s t r a l e p l a n t s were grown f r o m seeds i n a Dept. o f H o r t i c u l t u r e g r e e n h o u s e , U n i v e r s i t y o f B r i t i s h C o l u m b i a , under t h e s u p e r v i s i o n o f Dr. P. S a l s b u r y o f t h e Dept. o f C h e m i s t r y . The seeds were c o l l e c t e d by Dr. B. G i l b e r t i n t h e v i c i n i t y o f R i o de J a n e i r o , B r a z i l . The I s o l a t i o n o f A l k a l o i d s f r o m A s p i d o s p e r m a a u s t r a l e ( M u l l . A r g o v . ) E x t r a c t 22 The f o l l o w i n g p r o c e d u r e was a d a p t e d f r o m t h a t p u b l i s h e d by O n d e t t i 21 and was l a t e r used by o t h e r s i n o u r l a b o r a t o r i e s . The m e t h a n o l i c e x t r a c t o f A. a u s t r a l e p l a n t s c o l l e c t e d i n t h e v i c i n i t y o f R i o de J a n e i r o , B r a z i l i n 1967, was d r i e d t o g i v e a d a r k c o l o u r e d amorphous s o l i d (15.8 g ) . T h i s m a t e r i a l was t h e n suspended i n a c o o l e d s o l u t i o n o f m e t h a n o l (15 ml) and 15% aqueous a c e t i c a c i d (100 ml) and s t i r r e d i n an i c e - w a t e r b a t h f o r 1 h o u r . The s u s p e n s i o n was t h e n f i l t e r e d and washed w i t h p e t r o l e u m e t h e r (2x50 m l ) . The a c i d i c e x t r a c t was c a r e f u l l y made b a s i c w i t h a c o o l e d aqueous sodium h y d r o x i d e s o l u t i o n (50%) w h i l e i c e was added p e r i o d i c a l l y t o keep t h e t e m p e r a t u r e down. A f t e r r a i s i n g t h e pH t o 10-12, t h e r e s u l t i n g s u s p e n s i o n was 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 (3x100 ml) f o l l o w e d by e x t r a c t i o n w i t h m e t h y l e n e c h l o r i d e (3x100 m l ) . These o r g a n i c -94-e x t r a c t s were combined, washed once w i t h w a t e r , and d r i e d o v e r a n h y d r o u s s o d i u m s u l p h a t e . Removal o f t h e s o l v e n t i n v a c u o y i e l d e d a brown s o l i d (1.6 g ) . An e x a m i n a t i o n o f t h i s m a t e r i a l by t h i n - l a y e r c h r o m a t o g r a p h y ( a l u m i n a , 15% m e t h a n o l i n e t h y l a c e t a t e ) r e v e a l e d a l a r g e r number o f components w h i c h s e p a r a t e d i n t o a " p o l a r " group and a " n o n - p o l a r " group. Subsequent column c h r o m a t o g r a p h y on a l u m i n a ( a c t . I l l ) u s i n g an e t h y l a c e t a t e / m e t h a n o l g r a d i e n t y i e l d e d two f r a c t i o n s o f a l k a l o i d s w h i c h were s e p a r a t e l y p a r t i t i o n e d . The p o l a r f r a c t i o n s were r e - c h r o m a t o g r a p h e d on a l u m i n a ( a c t . I l l ) u s i n g a t o l u e n e / c h l o r o f o r m g r a d i e n t t o o b t a i n a l m o s t p u r e u l e i n e ( 1 ) . T h i s m a t e r i a l (120 mg) was r e c r y s t a l l i z e d f r o m m e t h a n o l t o g i v e c o l o u r l e s s 14 c u b i c c r y s t a l s , m.p. 75-80° ( l i t . m.p. 76-80°) The i d e n t i t y o f t h i s compound was u n a m b i g u o u s l y e s t a b l i s h e d by c o m p a r i s o n o f i t s p h y s i c a l and 18 s p e c t r o s c o p i c d a t a w i t h an a u t h e n t i c sample o f u l e i n e (1) as w e l l as 14 22 • w i t h p u b l i s h e d d a t a . ' The l e s s p o l a r f r a c t i o n s f r o m the f i r s t c h r o m a t o g r a p h i c s e p a r a t i o n were d i s s o l v e d i n c h l o r o f o r m c o n t a i n i n g 5% m e t h a n o l and s e v e r a l d r o p s o f p y r i d i n e and r e - c h r o m a t o g r a p h e d on s i l i c a g e l ( a c t . I I I ) . E l u t i o n w i t h 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 y i e l d e d o l i v a c i n e (4)(210 mg) and c o n t i n u e d e l u t i o n u s i n g an e t h y l a c e t a t e / m e t h a n o l g r a d i e n t y i e l d e d guatambuine (190 mg) The f r a c t i o n s c o n t a i n i n g o l i v a c i n e (4) were r e c r y s t a l l i z e d f r o m m e t h a n o l t o g i v e l i g h t y e l l o w n e e d l e - l i k e c r y s t a l s , m.p. 312-315° ( l i t . m.p. 314-316). The i d e n t i t y o f t h i s compound was u n a m b i g u o u s l y e s t a b l i s h e d by c o m p a r i s o n o f i t s p h y s i c a l and s p e c t r o s c o p i c d a t a w i t h an a u t h e n t i c sample o f 20 19 22 o l i v a c i n e . (4) and w i t h p u b l i s h e d d a t a . ' -95-The guatambuine o b t a i n e d was i d e n t i f i e d by c o m p a r i s o n w i t h a 22 sample o b t a i n e d by p a r t i a l s y n t h e s i s and used i n a n o t h e r s t u d y i n 21 o u r l a b o r a t o r i e s by Dr. G r i e r s o n . The I s o l a t i o n o f A l k a l o i d s f r o m A s p i d o s p e r m a O l i v a c e u m E x t r a c t The m e t h a n o l i c e x t r a c t o f A. O l i v a c e u m p l a n t s c o l l e c t e d i n t h e v i c i n i t y o f R i o de J a n e i r o , B r a z i l , was d r i e d t o g i v e an amorphous s o l i d (26 g ) . I n the way d e s c r i b e d i n the p r e v i o u s e x t r a c t i o n p r o c e d u r e , t h i s e x t r a c t was t a k e n up i n 15% aqueous a c e t i c a c i d -m e t h a n o l s o l u t i o n , f i l t e r e d and washed w i t h p e t r o l e u m e t h e r . The s o l u t i o n was made b a s i c w i t h aqueous sodium h y d r o x i d e s o l u t i o n and e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e t o y i e l d e d an a l k a l o i d a l e x t r a c t (3.0 g ) . T h i s m a t e r i a l was s u b s e q u e n t l y c h r o m a t o g r a p h e d on a l u m i n a (150 g, a c t . I I I ) . The a p p r o p r i a t e f r a c t i o n s o b t a i n e d f r o m e l u t i o n w i t h a t o l u e n e / c h l o r o f o r m g r a d i e n t were p o o l e d to g i v e u l e i n e ( 1 ) ( 2 4 0 mg) w h i c h c o u l d r e a d i l y be c r y s t a l l i z e d and i d e n t i f i e d by c o m p a r i s o n w i t h p r e v i o u s l y o b t a i n e d m a t e r i a l . U l e i n e m e t h i o d i d e (23) U l e i n e (1) (75 mg) was d i s s o l v e d i n a s o l u t i o n o f m e t h a n o l (3 ml) and m e t h y l i o d i d e (1 ml) and l e t s t a n d a t 5° o v e r n i g h t . Removal o f the s o l v e n t f r o m the r e a c t i o n m i x t u r e i n v a c u o y i e l d e d a s l i g h t l y c o l o u r e d c r y s t a l l i n e m a t e r i a l . R e c r y s t a l l i z a t i o n o f t h i s p r o d u c t f r o m e t h a n o l gave c o l o r l e s s , s h o r t n e e d l e - l i k e c r y s t a l s (95 mg) w h i c h were d r i e d i n h i g h vacuum, m.p. 205-206° ( l i t . m.p. 2 0 4 - 2 0 6 ° ) 1 4 ; A 2 8 3 ( s h ) , 290, 302, 3 0 5 ( s h ) , 3 1 2 ( s h ) ; mass s p e c t r u m : 2 8 1 ( 1 0 0 ) , 2 6 6 ( 1 0 ) , -96-2 2 2 ( 2 2 ) , 2 0 7 ( 2 9 ) , 2 0 5 ( 2 9 ) , 2 0 4 ( 2 3 ) , 1 9 4 ( 9 ) , 1 2 8 ( 7 7 ) , 1 2 7 ( 4 4 ) . A n a l . C a l c u l a t e d f o r C 1 9 H 2 5 N 2 I : c > 5 5 - 8 8 5 H,6.17; N,6.86. Found: C.56.13; H,6.00; N,6.69. l - M e t h y l - 2 - ( 3 - d i m e t h y l a m i n o e t h y l ) - 3 - e t h y l c a r b a z o l e (25) U l e i n e m e t h i o d i d e (19)(62mg) was d i s s o l v e d i n a s o l u t i o n o f e t h a n o l (3 m l ) , w a t e r (2 ml) and p o t a s s i u m h y d r o t i d e (75 mg) and t h e m i x t u r e was r e f l u x e d f o r 1.5 h o u r s . Most o f t h e s o l v e n t was t h e n removed i n v a cuo and t h e r e s i d u e was p a r t i t i o n e d between w a t e r and d i e t h y l e t h e r . The aqueous l a y e r was e x t r a c t s e v e r a l t i m e s w i t h d i e t h y l e t h e r (50 ml) and t h e combined o r g a n i c e x t r a c t s were washed once w i t h w a t e r and d r i e d o v e r a nhydrous sodium s u l p h a t e . Removal o f t h e s o l v e n t y i e l d e d a l i g h t brown g l a s s y m a t e r i a l w h i c h c o u l d be c r y s t a l l i z e d from d i e t h y l e t h e r / p e t r o l e u m e t h e r s o l u t i o n t o g i v e 14 a c r y s t a l l i n e p r o d u c t , m.p. 114-116° ( . l i t . " m.p. 115-116°) ; X ' . 230, 248, I T13X 262, 2 8 7 ( s h ) , 293, 307; pmr s i g n a l s : x l . 9 - 2 . 3 (3H, m u l t i p l e t , a r - H ) , 2.6-2.8 (2H, m u l t i p l e t , a r - H ) , 7.1 (4H, m u l t i p l e t , a r - C H 2 - C H 2 - N ) , 7.20 (2H, q u a r t e t , ar-CH_ 2-CH 3) , 7.50(3H, s i n g l e t , a r - C H 3 ) , 7.61(6K, s i n g l e t , -N ( C H 3 ) 2 ) , 8.75(3H, t r i p l e t , - C H 2 C H 3 ) ; mass s p e c t r u m : 2 8 0 ( 8 6 ) , 2 3 5 ( 2 2 ) , 2 2 2 ( 2 9 ) , 2 2 0 ( 2 9 ) , 2 0 6 ( 3 8 ) , 2 0 4 ( 3 8 ) , 1 8 6 ( 6 6 ) , 1 7 1 ( 1 0 0 ) . A n a l . C a l c u l a t e d f o r C i g H 2 4 N 2 : C,81.38; H,8.63; N,9.99. Found: C.81.10; H,8.59; N,10.10. T h i s m a t e r i a l was found t o be i d e n t i c a l w i t h a d e g r a d a t i o n p r o d u c t 21 o f quatambuine o b t a i n e d by Dr. G r i e r s o n i n o u r l a b o r a t o r i e s . l - M e t h y l - 2 - ( B - t r i m e t h y l a m i n o e t h y l ) - 3 - e t h y l c a r b a z o l e i o d i d e (26) The c a r b a z o l e a m i n e ( 2 1 ) ( 4 0 mg) • was d i s s o l v e d i n a s o l u t i o n o f -97-m e t h a n o l (3 ml) and m e t h y l i o d i d e (1 ml) and l e t s t a n d a t 5° o v e r n i g h t . Removal o f t h e s o l v e n t i n vacuo gave a s l i g h t l y c o l o u r e d c r y s t a l l i n e m a t e r i a l (61 mg). A p o r t i o n o f t h i s m a t e r i a l was r e c r y s t a l l i z e d f r o m e t h a n o l and d r i e d i n h i g h vacuum o v e r n i g h t , m.p. 300-302° ( l i t . m.p. 14 300-302°). A n a l . C a l c u l a t e d f o r C ^ H ^ N I : C,56.87; H,6.44; N,6.63. Found: C,56.60; H,6.37; N,6.59. Hofmann F r a g m e n t a t i o n o f Compound (26) The c a r b a z o l e m e t h i o d i d e (22)(60mg) was added t o f r e s h l y d i s t i l l e d _ t - b u t a n o l (10 ml) i n w h i c h p o t a s s i u m m e t a l (53 mg) had been d i s s o l v e d . The r e a c t i o n v e s s e l , a two-knecked round b o t t o m f l a s k , was e q u i p p e d w i t h a s o u r c e o f d r y n i t r o g e n gas and a g l a s s tube o u t l e t w h i c h e x t e n d e d i n t o a c o l l e c t o r c h a r g e d w i t h m e t h y l i o d i d e - m e t h a n o l s o l u t i o n . I n t h i s way a s l o w s t r e a m o f n i t r o g e n gas was p a s s e d t h r o u g h the r e a c t i o n m i x t u r e t o c a r r y w i t h i t any v o l a t i l e m a t e r i a l , formed d u r i n g the r e a c t i o n , i n t o t h e c o l l e c t i n g f l a s k . The r e a c t i o n m i x t u r e was s t i r r e d i n an o i l b a t h s e t a t 80° f o r 2 h o u r s . The s y s t e m was c o n t i n u o u s l y p urged w i t h n i t r o g e n gas as t h e 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 c o o l t o room t e m p e r a t u r e . The r e a c t i o n m i x t u r e was e v a p o r a t e d jLn vacuo and t h e r e s i d u e was p a r t i t i o n e d between w a t e r and d i e t h y l e t h e r . The aqueous l a y e r was washed s e v e r a l t i m e s w i t h d i e t h y l e t h e r (60 ml) and the combined o r g a n i c e x t r a c t was washed once w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . Removal o f t h e s o l v e n t i n vacuo y i e l d e d a brown amorphous m a t e r i a l (12 mg). A t h i n - l a y e r c h r o m a t o g r a p h y ( s i l i c a g e l , CHCl^) e x a m i n a t i o n o f t h i s p r o d u c t -98-r e v e a l e d one m a j o r component p l u s s e v e r a l more p o l a r m i n o r components. A t t e m p t e d p u r i f i c a t i o n o f the m a j o r p r o d u c t by p r e p a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y y i e l d e d a s l i g h t l y y e l l o w g l a s s w h i c h - c r y s t a l l i z e d o n l y p o o r l y f r o m d i e t h y l e t h e r / p e t r o l e u m e t h e r s o l u t i o n ( l i t . m.p. 6 7 - 6 8 ° ) . 1 4 L a c k o f m a t e r i a l p r e v e n t e d f u r t h e r a t t e m p t s t o p u r i f y t h i s p r o d u c t , A 2 2 8 ( s h ) , 235, 248, 265, 2 9 2 ( s h ) , 306; pmr s i g n a l s : r l . 9 - 2 . 3 (3H, m u l t i p l e t s , a r - H ) , 2.6-2.8(2H, m u l t i p l e t s , a r - H ) , 4.4(1H, d o u b l e t o f d o u b l e t s , J=17 and 3,-CH=CH_2 c i s ) , 6.7(1H, d o u b l e t o f d o u b l e t s , J=24 and 3, -CH=CH 2 t r a n s ) , 7.20(2H, q u a r t e t , J=7, ar-CH_ 2CH 3), 7.50 (3H, s i n g l e t , a r - C H 3 ) , 8.75(3H, t r i p l e t , J=7, -CE 2-CK_ 3); mass s p e c t r u m : 2 3 5 ( 1 0 0 ) , 2 2 0 ( 9 0 ) , 2 0 5 ( 5 6 ) , 2 0 4 ( 3 9 ) , 1 1 7 ( 1 9 ) , 1 0 7 ( 2 0 ) , 1 0 3 ( 2 1 ) , 1 0 2 ( 2 6 ) ; h i g h r e s o l u t i o n mass measurement: C a l c u l a t e d f o r C ^ H ^ N : 2 3 5 . 1 3 6 0 . Found: 235.1371. The s o l v e n t i n t h e t r a p was e v a p o r a t e d i n vacuo t o g i v e a c o l o u r l e s s p r e c i p i t a t e (19 mg) w h i c h was r e c r y s t a l l i z e d f r o m m e t h a n o l t o y i e l d l o n g n e e d l e - l i k e c r y s t a l s w h i c h were c r u s h e d arid d r i e d i n h i g h vacuum. T h i s m a t e r i a l was r e a d i l y i d e n t i f i e d as tetramethylammonium i o d i d e (31) based 23 on t h e f o l l o w i n g d a t a : m.p. 230° ( l i t . m.p. 230°) ; mass s p e c t r u m : 1 4 2 ( 1 0 0 ) , 1 2 7 ( 9 4 ) , 5 9 ( 3 3 ) , 5 7 ( 7 6 ) . A n a l . C a l c u l a t e d f o r C ^ H ^ N I : C,23.89; H,6.02; N,6.96. Found: C,23.96; H,6.10; N,7.07. The I s o l a t i o n o f U l e i n e (1) f r o m F e e d i n g E x p e r i m e n t s i n A s p i d o s p e r m a  p y r i c o l l u m p l a n t s The f o l l o w i n g p r o c e d u r e was used t o o b t a i n u l e i n e (1) f r o m a l l t h e f e e d i n g e x p e r i m e n t s u s i n g A. p y r i c o l l u m p l a n t s . The r o o t segments o r w h ole p l a n t s were c u t up w i t h s c i s s o r s b e f o r e b e i n g m a s u r a t e d i n a -99-W a r i n g b l e n d e r w i t h m e t h a n o l . The r e s u l t i n g p u l p - l i k e m a t e r i a l was f i l t e r e d and r e - m a s c e r a t e d i n m e t h a n o l (4 t i m e s ) u n t i l t h e f i l t r a t e became c o l o u r l e s s . The combined m e t h a n o l i c e x t r a c t s were t h e n e v a p o r a t e d i n v a c u o . T h i s r e s i d u e was t h e n suspended i n c o o l aqueous a c e t i c a c i d (10%) and f i l t e r e d . The f i l t r a t e was washed t w i c e w i t h d i e t h y l e t h e r b e f o r e b e i n g b a s i f i e d w i t h c o o l e d aqueous sodium h y d r o x i d e s o l u t i o n ( 5 0 % ) . The e x t r a c t i o n o f t h i s s u s p e n s i o n w i t h m e t h y l e n e c h l o r i d e y i e l d e d t h e c r u d e a l k a l o i d a l e x t r a c t . A t t h i s p o i n t , u l e i n e (20 mg) was added t o t h e e x t r a c t and r e - i s o l a t e d by column c h r o m a t o g r a p h y on a l u m i n a ( u s u a l l y 25 g, a c t . I l l ) u s i n g a t o l u e n e / c h l o r o f o r m g r a d i e n t . F u r t h e r p u r i f i c a t i o n was a c h i e v e d by r e c r y s t a l l i z a t i o n f r o m m e t h a n o l . U s u a l l y two r e c r y s t a l l i -z a t i o n s were done b e f o r e the m a t e r i a l was d r i e d i n h i g h vacuum o v e r n i g h t i n p r e p a r a t i o n f o r c o u n t i n g t h e r a d i o a c t i v i t y i n i t . Samples between 0.150 and 0.250 mg i n w e i g h t were d i s s o l v e d i n benzene (1 ml) and c o u n t e d as d e s c r i b e d p r e v i o u s l y i n P a r t I . A t l e a s t "two r e c r y s t a l l i z a t i o n s were done between measurements o f r a d i o a c t i v i t y . T h i s p r o c e d u r e was s i m i l a r t o t h a t used by o t h e r s s t u d y i n g t h e same p l a n t s y s t e m i n o u r l a b o r a t o r i e s . The A d m i n i s t r a t i o n o f R a d i o a c t i v e P r e c u r s o r s to A. p y r i c o l l u m p l a n t s I n a l l t h e f e e d i n g e x p e r i m e n t s u s i n g r o o t segments ( n o s . 1-4) the r a d i o a c t i v e p r e c u r s o r was d i s s o l v e d i n a s o l u t i o n o f e t h a n o l and w a t e r (0.5 m l , 1:4) w i t h 1 o r 2 d r o p s o f I N a c e t i c a c i d . D i s s o l u t i o n c o u l d be a i d e d by s o n i c a t i o n . T h i s s o l u t i o n was a p p l i e d d i r e c t l y t o f r e s h l y c u t r o o t s w h i c h were c o n t a i n e d i n t e s t t u b e s . A f t e r a b s o r p t i o n o f the s o l u t i o n by t h e r o o t b a r k , the c o n t a i n e r w h i c h had the r a d i o a c t i v e s o l u t i o n i n i t -100-was washed w i t h two a l i q u o t s (0.5 ml) o f the s o l v e n t m i x t u r e d e s c r i b e d and a p p l i e d to the r o o t s . F u r t h e r washings w i t h d i s t i l l e d water were a p p l i e d to the r o o t t h r o u g h o u t the i n c u b a t i o n p e r i o d i n o r d e r to keep them m o i s t . At the end o f the f e e d i n g time t h i s c o n t a i n e r , the p i p e t t e and the t e s t tube i n which the r o o t had been were a l l washed w i t h m ethanol. T h i s wash s o l u t i o n was then measured f o r r a d i o a c t i v i t y i n c a s e a c o r r e c t i o n had to be a p p l i e d to the r a d i o a c t i v i t y f e d to the p l a n t . I n a l l c a s e s the r e s i d u a l r a d i o a c t i v i t y was found to be i n s i g n i f i c a n t . I n the two e x p e r i m e n t s (nos. 5 and 6) i n which whole p l a n t s were f e d , the w i c k method of f e e d i n g was u s e d. T h i s t e c h n i q u e was employed i n a manner s i m i l a r to t h a t d e s c r i b e d i n P a r t I. The d a t a p e r t a i n i n g to each f e e d i n g e x p e r i m e n t a r e g i v e n i n T a b l e s 2 and 4. T a b l e 4. A d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 2 Exp.No. P l a n t System P l a n t F e e d i n g Weight o f F e e d i n g w e i g h t ( g . ) Technique p r e c u r s o r ( m g ) Time(days) 1 tap r o o t 37.0 h y d r o p o n i c 7.01 5 2 f i b r o u s r o o t 14.1 h y d r o p o n i c 9.56 6 3 tap r o o t 27.2 h y d r o p o n i c 2.46 5 4 whole p l a n t 197 w i c k 8.94 90 5 whole p l a n t 183 w i c k 0.03 91 - 1 0 1 -The I s o l a t i o n o f U l e i n e (1) and O l i v a c i n e (4) from F e e d i n g E x p e r i m e n t s  i n A s p i d o s p e r m a a u s t r a l e p l a n t s The p r o c e d u r e used t o o b t a i n u l e i n e (1) and o l i v a c i n e (4) f r o m a l l t h e f e e d i n g e x p e r i m e n t s u s i n g A. a u s t r a l e p l a n t s was v e r y s i m i l a r t o t h a t d e s c r i b e d above f o r e x p e r i m e n t s u s i n g A. p y r i c o l l u m p l a n t s . I n t h i s s t u d y , however, b o t h u l e i n e (20 mg) and o l i v a c i n e (20 mg) were added t o t h e c r u d e a l k a l o i d a l e x t r a c t , and t h e n i s o l a t e d by c h r o m a t o g r a p h y on a l u m i n a ( a c t . I I I ) . The A d m i n i s t r a t i o n o f R a d i o a c t i v e P r e c u r s o r s t o A. a u s t r a l e p l a n t s I n a l l t h e f e e d i n g e x p e r i m e n t s u s i n g t h i s p l a n t t h e f o l l o w i n g p r o c e d u r e was u s e d . I m m e d i a t e l y b e f o r e each e x p e r i m e n t t h e p l a n t s were u p - r o o t e d and t h e s o i l was c a r e f u l l y b r u s h e d f r o m the r o o t s y s t e m . Each p l a n t was p l a c e d i n a t e s t t u b e . The r a d i o a c t i v e p r e c u r s o r was d i s s o l v e d i n a s o l u t i o n o f e t h a n o l and w a t e r (0.5 m l , 1:4) w i t h 1 o r 2 d r o p s o f IN a c e t i c a c i d . T h i s s o l u t i o n was a p p l i e d d i r e c t l y t o the r o o t s y s t e m o f each p l a n t . A f t e r a b s o r p t i o n o f the s o l u t i o n by t h e r o o t b a r k , the c o n t a i n e r w h i c h had the r a d i o a c t i v e s o l u t i o n i n i t was washed w i t h two a l i q u o t s o f the s o l v e n t m i x t u r e w h i c h were a l s o a p p l i e d t o t h e r o o t s . The r o o t s were k e p t m o i s t d u r i n g the f e e d i n g p e r i o d by t h e p e r i o d i c a d d i t i o n o f a l i t t l e w a t e r as t h e p l a n t s were a l l o w e d t o grow under f l u o r e s c e n t l i g h t s . The d a t a w h i c h i s p e r t i n e n t t o t h e s e e x p e r i m e n t s i s summarized i n T a b l e s 3 and 5. -102-T a b l e 5. A d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 3. Exp.No. Number o f T o t a l wt Weight o f F e e d i n g p l a n t s o f p l a n t s ( g ) p r e c u r s o r ( m g ) T i m e ( d a y s ) 1 3 21.0 .03 6 2 4 28.1 8.2 ' 5 3 3 24.5 1.21 6 4 3 22.7 1.80 6 5 4 24.0 0.28 5 6 4 27.9 1.12 5 The D e g r a d a t i o n o f U l e i n e (1) I s o l a t e d f r o m F e e d i n g E x p e r i m e n t s 5 and 6. The r a d i o a c t i v e u l e i n e (1) i s o l a t e d was degraded as p r e v i o u s l y d e s c r i b e d t o y i e l d tetramethylammonium i o d i d e (29) w h i c h was r e c r y s t a l l i z e d f r o m m e t h a n o l t o c o n s t a n t r a d i o a c t i v i t y . E x p e r i m e n t 5: 14 mg o f u l e i n e (4.96 x 10 dpm/mmole) y i e l d e d 5 mg o f t e t r a m e t h y l -6 ammonium i o d i d e (4.80 x 10 dpm/mmole). E x p e r i m e n t 6: 12 mg o f u l e i n (4.47 x 10 dpm/mmole) y i e l d e d 4.1 mg o f t e t r a m e t h y l -ammonium i o d i d e (4.38 x 10 dpm/mmole). -103-BIBLIOGRAPHY 1. E. W enkert, J . Am. Chem. S o c , 84, 98 (1962). 2. J . J o u l e , H. M o n t e i r o , L. J . Durkam, B. G i l b e r t and C. D j e r a s s i , J . Chem. S o c , 4773 (1965). 3. R. R. A r n d t , S. H. Brown, N. C. L i n g , P. R o l e r , C. D j e r a s s i , J . M. F e r r e i r a , B. G i l b e r t , E. C. M i r a n d a , S. E. F l o r e s , A. P. D u a r t e and E. D. C a r r a z z o n i , Phytochem. , 6_, 1652 (1967). 4. J . P. K u t n e y , V. R. N e l s o n and D. C. W i g f i e l d , J . Am. Chem. S o c , 91, 4278 (1969). 5. J . P. K u t n e y , V. R. N e l s o n and D. C. W i g f i e l d , J . Am. Chem. S o c , 91, 4279 (1969). 6. I . K. Kim and K. L. E r i c k s o n , T e t r a h e d r o n , 27, 3979 (1971). 7. J . A. J o u l e , M. O k a s h i , B. G i l b e r t and C. D j e r a s s i , T e t r a h e d r o n , 21, 1717 (1965). 8. A. A t r o n d , A. Cave, C. Kan-Fan, Y. L a n g l o i s and P. P o t i e r , Chem. Comm., 517 (1970). 9. J . P. K u t n e y , J . F. Beck and G. B. F u l l e r , H e t e r o c y c l e s , 1, 5 (1973). 10. G. B. F u l l e r , S t u d i e s on t h e S y n t h e s i s and 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 , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o l u m b i a n 974~). 11. P. P o t i e r and M. M. J a n o t , C R . Acad. S c i . P a r i s , 276, 1727 (1973). 12. J . P. K u t n e y , H e t e r o c y c l e s , _4, 429 (1976). 13. Y. L a n g l o i s , N. L a n g l o i s e t P. P o t i e r , T e t . L e t t . , 955 (1975). 14. J . Schmutz, F. H u n z i k e r and R. H i r t , H e l v . Chim. A c t a . , XL, 1189 (1957). 15. G. B u c h i and E. W. W a r n o f f , J . Am. Chem. S o c , 81, 4433 (1959). -104-!6. G. B u c h i , S. J . G o u l d and F. N a i f , J . Am. Chem. S o c , 93_, 2492 ( 1 9 7 1 ) . 17 • J . A. J o u l e , M. O s a s h i , B. G i l b e r t and C. D j e r a s s i , T e t r a h e d r o n , 21, 1717 ( 1 9 6 5 ) . 18. We a r e g r a t e f u l t o Dr. C. D j e r a s s i , S t a n f o r d U n i v e r s i t y , Dr. J . J o u l e , M a n c h e s t e r U n i v e r s i t y , Dr. J . Schmutz, R e s e a r c h I n s t i t u t e , A. W a r d e r e r , ' S . A., B e r n , and Dr. B. G i l b e r t , L a b o r a t o r i o de Q u i m i c a O r g a n i c a , F a c u l d a d e N a c i o n a l de F a r m a c i a , R i o de J a n e i o r f o r p r o v i d i n g samples o f t h e s e a l k a l o i d s . 19. G. B. M a r i n i - B e t t o l o and J . Schmutz, H e l v . Chim. A c t a . , 42, 2146 ( 1 9 5 9 ) . 20. We a r e g r a t e f u l t o Dr. D. H e n r y , S t a n f o r d R e s e a r c h I n s t i t u t e , Menlo P a r k , C a l i f o r n i a , f o r samples o f p y r i d o c a r b a z o l e a l k a l o i d s . 21. D a v i d S. G r i e r s o n , S t u d i e s on the B i o s y n t h e s i s , D e g r a d a t i o n and S y n t h e s i s o f O l i v a c i n e - E l l i p t i c i n e Type A l k a l o i d s , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o l u m b i a ( 1 9 7 0 ) . 22. M . A. O n d e t t i and V . D e u l o f e u , T e t r a h e d r o n , 15, 160 ( 1 9 6 1 ) . 23. CRC Handbook o f C h e m i s t r y and P h y s i c s , 5 4 t h e d i t i o n , R. C. Weast Ed., CRC P r e s s , C l e v e l a n d , O h i o . -105-PART I I I S t u d i e s R e l a t e d t o t h e S y n t h e s i s P r e a k u a m m i c i n e - and Stemmadenine Type A l k a l o i d s -106-INTRODUCTION A g r e a t d e a l o f e l e g a n t work has l e d t o a good u n d e r s t a n d i n g o f t h e e a r l y s t a g e s o f 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 . However, a g r e a t d e a l o f work has y e t t o be done i n o r d e r t o f u l l y u n d e r s t a n d t h e r e l a t i o n s h i p s between t h e v a r i o u s f a m i l i e s o f i n d o l e a l k a l o i d s i n t h e l a t e r s t a g e s o f b i o s y n t h e s i s . I n P a r t s I and I I o f t h i s t h e s i s , a number o f h y p o t h e s i s s u p p o r t e d by e x p e r i m e n t a l work was d e s c r i b e d t o o u t l i n e the d e v e l o p m e n t s i n t h i s a r e a . I n t h i s P a r t , t h e r e w i l l be d e s c r i b e d t h e a t t e m p t e d s y n t h e s i s o f compounds b e l i e v e d t o be i m p o r t a n t i n t h e l a t e r s t a g e s o f 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 . T h e r e i s much e v i d e n c e t o s u p p o r t t h e c o n j e c t u r e t h a t t h e f i r s t o f the m a j o r f a m i l i e s o f a l k a l o i d s t o be b i o s y n t h e s i z e d i s t h e C o r y n a n t h e , b u t t h e way i n w h i c h t h e l a t t e r i s c o n v e r t e d t o t h e S t r y c h n o s , I b o g a and A s p i d o s p e r m a s k e l e t o n s i s n o t c l e a r and has been the s u b j e c t o f much s p e c u l a t i o n . F i g u r e 1 summarizes two h y p o t h e s i s f o r t h i s c o n v e r s i o n . W e n k e r t 1 , i n 1965, p r o p o s e d t h a t t h e C o r y n a n t h e a l k a l o i d g e i s s o s c h i z i n e (1) underwent c y c l i z a t i o n ( r o u t e A) and r e a r r a n g e m e n t to g i v e t h e p e n t a -c y c l i c s t r u c t u r e (2) w h i c h c o u l d e a s i l y undergo bond f i s s i o n t o g i v e the r i n g - o p e n e d S t r y c h n o s s k e l e t o n ( 3 ) . A t w o - s t e p r e d u c t i o n o f t h e l a t t e r w o u l d g i v e t h e l i t t l e known a l k a l o i d stemmadenine ( 4 ) , w h i l e r i n g - c l o s u r e and l o s s o f t h e a l d e h y d e group w o u l d r e s u l t i n akuammicine ( 5 ) . An a l t e r n a t i v e p r o p o s a l by S c o t t and Q u e r e s h i ( r o u t e B) i n v o l v e d a known r e a r r a n g e m e n t o f the h y d r o x y i n d o l i n e d e r i v a t i v e o f g e i s s o s c h i z i n e (1) t o f o r m the o x i n d o l e ( 6 ) . C y c l i z a t i o n o f t h i s s t r u c t u r e w o u l d g i v e the -107-i n d o l e n i n e p r e a k u a m m i c i n e a l d e h y d e ( 7 ) . A s i m p l e r e d u c t i o n o f t h e a l d e h y d e group o f the l a t t e r w o u l d g i v e p r e a k u a m m i c i n e ( 8 ) , a compound t h e S c o t t group c l a i m t o have f o u n d a s a n a t u r a l p r o d u c t i n C a t h a r a n t h u s  r o s e u s s e e d l i n g s . However, f u l l c h a r a c t e r i z a t i o n d a t a on t h i s compound has n o t been made a v a i l a b l e . These i n v e s t i g a t o r s a l s o c l a i m t o have c o n v e r t e d p r e a k u a m m i c i n e (8) i n t o b o t h stemmadenine (4) and akuammicine ( 5 ) . 2 The b i o s y n t h e s i s o f o t h e r m a j o r f a m i l i e s o f i n d o l e a l k a l o i d s f r o m t h e S t r y c h n o s f a m i l y i s b e l i e v e d t o o c c u r by e l a b o r a t i o n o f stemmadenine ( 4 ) . F i g u r e 2 summarizes the b i o s y n t h e t i c schemes w h i c h a r e s u p p o r t e d by e x p e r i m e n t a l e v i d e n c e ( d e s c r i b e d i n d e t a i l i n P a r t s I and I I ) . The c u r r e n t h y p o t h e s i s i s t h a t stemmadenine (4) i s i n b i o l o g i c a l e q u a l i b r i u m w i t h d e h y d r o s e c o d i n e (9) v i a i s o - s t e m m a d e n i n e ( 1 0 ) . V a r i o u s l y l a b e l l e d 3 forms o f s e c o d i n e have been shown i n o u r l a b o r a t o r i e s t o be i n c o r p o r a t e d i n t o t h e A s p i d o s p e r m a 4 ( 1 1 ) , t h e I b o g a ^ ( 1 2 ) and- t h e H u n t a r i a ^ ( 1 3 ) f a m i l i e s 7 8 o f a l k a l o i d s as w e l l as i n t o t h e anomolous s t r u c t u r e a p p a r i c i n e ( 1 4 ) . ' Stemmadenine (4) i s a l s o b e l i e v e d to be the p r e c u r s o r t o t h r e e s m a l l e r f a m i l i e s o f i n d o l e a l k a l o i d s w h i c h have more d i v e r s e c a r b o n 9 s k e l e t o n s . These a r e compounds o f the e l l i p t i c i n e , o l i v a c i n e and u l e i n e t y p e s . The b i o m i m e t i c c o n v e r s i o n o f stemmadenine (4) t o members o f t h e I b o g a and A s p i d o s p e r m a f a m i l i e s has been t h e s u b j e c t o f much c o n t r o v e r s y . S c o t t and Q u e r e s h i 1 0 c l a i m e d t o have r e a r r a n g e d t h i s m o l e c u l e to t a b e r s o n i n e and c a t h a r a n t h i n e (12) i n h o t a c e t i c a c i d . These r e s u l t s were s a i d t o s u p p o r t the p r o p o s a l t h a t a d e h y d r o s e c o d i n e - l i k e m o l e c u l e must be a p i v o t a l p r e c u r s o r o f t h e I b o g a and A s p i d o s p e r m a f a m i l i e s . However, a c a r e f u l -108-F i g u r e 1. The p r o p o s e d b i o s y n t h e s i s o f t h e S t r y c h n o s a l k a l o i d s f r o m the C o r y n a n t h e f a m i l y . F i g u r e 2. The i m p l i c a t i o n o f t h e d e h y d r o s e c o d i n e t stemmadenine b i o - e q u i l b r i u m i n i n d o l e a l k a l o i d b i o s y n t h e s i s as s u p p o r t e d by e x p e r i m e n t . -110-i n v e s t i g a t i o n o f t h i s work by t h e S m i t h group f a i l e d t o c o n f i r m t h e s e r e s u l t s . Two o t h e r b i o m i m e t i c c o n v e r s i o n s o f stemmadenine (4) have been 12 c l a i m e d . S c o t t r e p o r t e d t h a t a r i n g - c o n t r a c t i o n r e a c t i o n w h i c h r e s u l t e d i n a 20% y i e l d o f the n a t u r a l p r o d u c t v a l l e s a m i n e (15) had been a c h i e v e d . T h i s c o n v e r s i o n i n v o l v e d a m o d i f i e d P o l o n o v s k i f r a g -m e n t a t i o n o f t h e N - o x i d e . However, no e x p e r i m e n t a l d e t a i l s a r e a v a i l a b l e . T h i s r e a c t i o n i s v e r y s i m i l a r t o the p r e v i o u s l y p r o p o s e d c o n v e r s i o n o f stemmadenine (4) t o a l k a l o i d s p o s s e s s i n g the more d i v e r s e c a r b o n s k e l e t o n s . 9 A second r e a c t i o n o f stemmadenine (4) w h i c h may have b i o l o g i c a l 13 s i g n f i c a n c e was r e p o r t e d by S a n d o v a l e t a l . They, c l a i m e d to have a c h i e v e d an o x i d a t i v e r i n g - c l o s u r e w h i c h y i e l d e d the p r o d u c t c o n d y l o c a r p i n e ( 1 6 ) , a member o f the A s p i d o s p e r m a f a m i l y w i t h w h i c h i t c o - o c c u r s i n some p l a n t s p e c i e s . C0 2 CH 3 OH COoCHo OH (15) (17) F i g u r e 3. Some b i o m i m e t i c c o n v e r s i o n s o f stemmadenine (4) -111-St emma-d en i n e (4) was f i r s t i s o l a t e d , i n 1958, from the f r u i t o f 14 Steimnadenia D o n n e l l - S m i t h i i . Subsequent work r e v e a l e d the n o v e l 13 r i n g - o p e n e d S t r y c h n o s c a r b o n s k e l e t o n , but the s t e r e o c h e m i s t r y a t C-16 was n o t d e t e r m i n e d . The o n l y r e p o r t e d a t t e m p t a t s y n t h e s i z i n g stemmadenine (4) d e r i v a t i v e s i s t h a t by S n i e k u s 1 ^ who a c h i e v e d the model compound ( 1 7 ) . S y n t h e t i c work i n the S t r y c h n o s a r e a was i n i t i a t e d i n our l a b o r a t o r i e s i n 1 9 6 8 . 1 6 The p l a n was t o c o n v e r t the a v a i l a b l e Corynanthe a l k a l o i d (18) v i a i t s c h l o r o i n d o l e n i n e t o s t r u c t u r e s o f the t y p e (19) and t h e n t o c y c l i z e the l a t t e r t o a c h i e v e the 1 9 , 2 0 - d i h y d r o -preakuammicine s e r i e s ( 2 0 ) . A l l e f f o r t s t o a c h i e v e t h i s c y c l i z a t i o n f a i l e d . C l e a r l y , i n o r d e r t o f u l l y e v a l u a t e the c e n t r a l r o l e w h i c h stemma-d e n i n e (4) a p p e a r s t o p l a y 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 v a r i o u s r a d i o -l a b e l l e d forms o f t h i s m o l e c u l e must be made a v a i l a b l e . I t would a l s o be o f g r e a t i n t e r e s t t o have a v a i l a b l e d e r i v a t i v e s o f the preakuammicine ty p e (7) and (8) i n o r d e r t o i n v e s t i g a t e the c h e m i s t r y o f t h e s e compounds and a l s o t o e v a l u a t e t h e i r r o l e i n the b i o s y n t h e s i s o f i n d o l e a l k a l o i d s . (18) (19) (20) -112-DISCUSSION I n o r d e r t o e v a l u a t e e f f e c t i v e l y t he r o l e s p l a y e d by t h e pre a k u a m m i c i n e and t h e stemmadenine s e r i e s i n t h e b i o s y n t h e s i s o f i n d o l e a l k a l o i d s a s y n t h e s i s o f t h e s e compounds had t o be d e v e l o p e d . T h i s s y n t h e s i s n o t o n l y had t o make a v a i l a b l e r e a s o n a b l e q u a n t i t i e s o f t h e s e m a t e r i a l s b u t a l s o make p o s s i b l e t h e i n t r o d u c t i o n o f a r a d i o -l a b e l i n c e r t a i n p o s i t i o n s i n t h e s t r u c t u r e s . A l s o , s i n c e t h e s t e r e o -c h e m i s t r y a t p o s i t i o n C-16 i n t h e s e compounds was unknown i t w o u l d be d e s i r a b l e t o s y n t h e s i z e b o t h e p i m e r s o f each compound. There was a v a i l a b l e i n the l i t e r a t u r e d e s c r i p t i o n s o f a g r e a t d e a l o f work b r i d g i n g the d e g r a d a t i o n c h e m i s t r y o f s t r y c h n i n e ( 2 9 ) 1 7 and the c o n s t i t u e n t s o f c a l a b a s h c u r a r e . 1 7 C u r a r e i s an e x t r a c t p r e p a r a t i o n f r o m s e v e r a l s p e c i e s o f the p l a n t genus S t r y c h n o s w h i c h was used by t h e S o u t h A m e r i c a n n a t i v e s as a r r o w head p o i s o n s . Due t o the h i g h b i o l o g i c a l a c t i v i t y o f t h e s e p r e p a r a t i o n s the c h e m i c a l and b o t a n i c a l i n t e r e s t i n them l e d t o the i d e n t i f i c a t i o n o f a l a r g e number o f m o l e c u l a r s t r u c t u r e s p o s s e s s i n g t h e b a s i c S t r y c h n o s s k e l e t o n ( 2 1 a ) . The s y s t e m o f (21a) -113-n o m e n c l a t u r e used h e r e i s t h a t p r o p o s e d by J a n o t and Le Men'1'" f o l l o w i n g 19 the numbering s y s t e m u s e d by B e r n a u e r e t . a l . S i n c e , compounds p o s s e s s i n g t h e b a s i c S t r y c h n o s s k e l e t o n were r e a d i l y a v a i l a b l e f r o m l i t e r a t u r e p r e p a r a t i o n s we c o n s i d e r e d t h e p o s s i b i l i t y o f s y n t h e s i z i n g p r e a k u a m m i c i n e and stemmadenine d e r i v a t i v e s f r o m one o f t h e s e s t r u c t u r e s . The a d v a n t a g e s i n u s i n g a c u r a n d e r i v a t i v e (21) as s t a r t i n g m a t e r i a l were t h r e e - f o l d . F i r s t l y , t h e s t e r e o c h e m i s t r y a t p o s i t i o n s C-3, C-7 and C-15 o f t h e p i p e r d i n e r i n g was known and f i x e d . S e c o n d l y , known c h e m i s t r y i n t h i s a r e a c o u l d p o s s i b l y be used t o c o n v e r t the r i n g - c l o s e d S t r y c h n o s s k e l e t o n t o t h e r i n g - o p e n e d stemmadenine s y s t e m . T h i r d l y , the a v a i l a b i l i t y o f v a r i o u s d e r i v a t i v e s o f the c u r a n t y p e p r o v i d e d an o p p o r t u n i t y t o i n t r o d u c e a r a d i o - l a b e l i n v a r i o u s p o s i t i o n s i n t h e m o l e c u l e n e a r t h e end o f t h e s y n t h e t i c sequence. The f i r s t r e q u i r e m e n t o f a scheme to c o n v e r t the S t r y c h n o s s y s t e m to the d e s i r e d ten-membered r i n g systetti o f the stemmadenine s e r i e s was t o i n t r o d u c e s t e r e o s e l e c t i v e l y an o x y g e n a t e d c a r b o n f u n c t i o n a l i t y a t C-16. As m e n t i o n e d e a r l i e r , the s t e r e o c h e m i s t r y a t C-16 i n p r e a k u a m m i c i n e ( 8 ) and stemmadenine ( 4 ) was unknown. I t w o u l d , t h e r e f o r e , be a dvantageous t o s y n t h e s i z e b o t h e p i m e r s to (a) a s c e r t a i n t h e n a t u r a l c o n f i g u r a t i o n , and (b) a d m i n i s t e r b o t h e p i m e r s i n p l a n t f e e d i n g e x p e r i m e n t s to e v a l u a t e t h e b i o s y n t h e t i c i m p o r t a n c e of t h e s t e r e o c h e m i s t r y a t C-16. I t was f e l t t h a t t h e s t e r e o c h e m i s t r y d u r i n g i n t r o d u c t i o n o f the C^ u n i t a t C-16 c o u l d be c o n t r o l l e d and p r e d i c t e d b e c a u s e t h e s t e r e o c h e m i s t r y o f t h e p i p e r d i n e p a r t o f the c u r a n s y s t e m (21b) c r e a t e d c o n s i d e r a b l e s t e r i c c r o w d i n g o v e r t h e £-f a c e t h e m o l e c u l e . An e x a m i n a t i o n o f m o l e c u l a r models l e f t no d oubt t h a t an i n c o m i n g e l e c t r o p h i l e o r n u c l e o p h i l e a t C-16 must r e a c t f r o m t h e a - f a c e . -114-T h e r e f o r e , by s e l e c t i n g the a p p r o p r i a t e o x i d a t i o n l e v e l of the C ^ - u n i t , c o n v e r s i o n to b o t h C-16 epimers o f the preakuammicine ( 8 ) and stemmadenine ( 4 ) systems would be p o s s i b l e . (21b) The c u r a n d e r i v a t i v e t h a t became a v a i l a b l e to our l a b o r a t o r i e s was 18-hydroxy-2g,16a-cur-19-ene-17-ol,also known as c a r a c u r i n e V I I 20 and Wieland-Gumlich aldehyde (22). T h i s compound, which was o f p i v o t a l importance i n the c h e m i s t r y o f the cu r a n a l k a l o i d s had a l s o been s y n t h e s i z e d from s t r y c h n i n e (29) thus l i n k i n g these two l a r g e f a m i l i e s o f a l k a l o i d s v i a e s t a b l i s h e d c h e m i c a l r o u t e s . T h i s compound ( 2 2 h e r e -a f t e r r e f e r r e d to as Wieland-Gumlich aldehyde (WGA) i n the i n t e r e s t of s i m p l i c i t y , was made r e a d i l y a v a i l a b l e from the d e g r a d a t i o n o f s t r y c h n i n e 21 22 (29) , by a m o d i f i c a t i o n o f the o r i g i n a l d e g r a d a t i v e work ' , developed 23 i n the l a b o r a t o r i e s o f Schmid and K a r r e r and p u b l i s h e d i n 1969. Due to the a v a i l a b i l i t y o f s t r y c h n i n e ( 2 9 ) , and hence WGA (22) the l a t t e r -115-(22) compound was an a t t r a c t i v e i n t e r m e d i a t e f o r the s y n t h e s i s o f preakuammicine ( 8 ) , stemmadenine ( 4 ) and t h e i r d e r i v a t i v e s . In one a r e a o f these i n v e s t i g a t i o n s i n our l a b o r a t o r i e s , 24 ( F i g u r e 4) F u l l e r was s u c c e s s f u l i n a c h i e v i n g the s y n t h e s i s o f 16-epi-stemmadenine (28) from the i n t e r m e d i a t e (23) , the l a t t e r 25 b e i n g a v a i l a b l e from WGA (22) by Smith's p r o c e d u r e . The c r u c i a l s t e p i n t h i s sequence was th e c o n v e r s i o n (25) t o (26) i n w h i c h the C ^ - u n i t was i n t r o d u c e d a t p o s i t i o n C-16. A l t h o u g h the mechanism of t h i s r e a c t i o n i s unknown, i t a p p a r e n t l y i n v o l v e s a n i o n f o r m a t i o n a t C-16, t h e r e f o r e i t s h o u l d be p o s s i b l e , i n a s i m i l a r manner, to i n t r o d u c e a C ^ - u n i t o f h i g h e r o x i d a t i o n l e v e l to a c h i e v e the o t h e r epimer. Such a s t u d y w i l l f orm p a r t o f the f o l l o w i n g d i s c u s s i o n . The d e g r a d a t i o n scheme t h a t was used to c o n v e r t s t r y c h n i n e (29) 23 to WGA (22) i s shown i n F i g u r e 5. S t r y c h n i n e (29) was t r e a t e d w i t h e x c e s s sodium e t h o x i d e and i s o a m y l n i t r i t e to g i v e i s o - n i t r o s o s t r y c h n i n e (30) which was i s o l a t e d as i t s h y d r o c h l o r i d e s a l t / A Beckman rearrangement of t h i s p r o d u c t u s i n g t h i o n y l c h l o r i d e f o l l o w e d by h y d r o l y s i s y i e l d e d 1 - c y a n o f o r m y l - W i e l a n d - G u m l i c h a l d e h y d e (32) as the h y d r o c h l o r i d e s a l t . Treatment of an aqueous s o l u t i o n o f (31) w i t h steam p e r m i t t e d h y d r o l y s i s -116-1. HBr/HoAc 2. Zn/HoAc CO2CH3 (23) (24) CO2CH3 NaH HC0 2CH 3 C r CO2CH3 (26) HCl NaH CH 20 CO2CH3 (25) <; C02CH3 OH 1. Pb(OAc)^ 2. NaBH, (27) (28) F i g u r e 4. S y n t h e s i s o f 16-epi-stemmadenine ( 2 8 ) . -117-(22) F i g u r e 5. The d e g r a d a t i o n o f s t r y c h n i n e (29) t o W i e l a n d -G u m l i c h a l d e h y d e (22) -118-o f t h e c y a n o f o r m y l group t o g i v e WGA (22) i n an o v e r a l l y i e l d o f 35-40%. T h i s m a t e r i a l had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s 20 i d e n t i c a l w i t h t h o s e o f an a u t h e n t i c sample o f WGA ( 2 2 ) . The n e x t s t e p i n t h e s y n t h e s i s o f t h e d e s i r e d c u r a n s y s t e m was the r e m o v a l o f t h e C-18 oxygen f u n c t i o n a l i t y i n s t r u c t u r e ( 2 2 ) . Two methods f o r t h i s o b j e c t i v e were a l r e a d y a v a i l a b l e i n t h e l i t e r a t u r e . 22 The f i r s t method i n v o l v e d the sodium b o r o h y d r i d e r e d u c t i o n o f t h e h e m i - a c e t a l (22) t o W i e l a n d - G u m l i c h d i o l ( 3 3 ) . The l a t t e r 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 a l l y l i c b r o m i d e (34) u s i n g 3% s a t u r a t e d s o l u t i o n o f h y d r o g e n bromide i n g l a c i a l a c e t i c a c i d . Z i n c d u s t t r e a t -ment o f t h e l a t t e r i n a c e t i c a c i d gave the a c e t a t e ( 3 5 ) . B a s i c h y d r o l y s i s o f t h i s m a t e r i a l r e s u l t e d i n the a l c o h o l d e r i v a t i v e , 2 g,16a-cur-19-ene-l 7 - o l ( 3 6 ) . The i d e n t i t y o f t h i s p r o d u c t was c o n f i r m e d by c o m p a r i s o n o f i t s s p e c t r o s c o p i c d a t a w i t h t h a t w h i c h was p u b l i s h e d . Of p a r t i c u l a r n o t e was t h e UV s p e c t r u m (A 243,297) showing the p r e s e n c e o f an i n d o l i n e max chromophore and t h e mass s p e c t r u m w h i c h c o n f i r m e d t h e m o l e c u l a r w e i g h t as b e i n g 296 mass u n i t s . T h i s t e c h n i q u e f o r d e o x y g e n a t i n g C-18 was c o n s i d e r e d as a means o f i n t r o d u c i n g a r a d i o - l a b e l i n t o C-18 o f s t r u c t u r e (36) and hence i n t o t h e e t h y l i d i n e s i d e - c h a i n o f p r e a k u a m m i c i n e (8) and stemmadenine (4) i f t h e s e compounds c o u l d be s y n t h e s i z e d f r o m t h i s s t a r t i n g m a t e r i a l . By d i s s o l v i n g the anhydrous a l l y l i c b r o m ide (34) i n t r i t i a t e d a c e t i c a c i d f o l l o w e d by t r e a t m e n t w i t h z i n c d u s t i n an i n e r t a t m o s p h e r e , t h e i n t r o -d u c t i o n o f t r i t i u m a t C-18 c o u l d be a c c o m p l i s h e d . -119-A s e c o n d method"" f o r d e o x y g e n a t i o n o f C-18 i n v o l v e d c a t a l y t i c h y d r o g e n o l y s i s o f W i e l a n d - G u m l i c h d i o l ( 3 3 ) . The l a t t e r was d i s s o l v e d i n aqueous a c i d i c medium and t r e a t e d w i t h p a l l a d i u m - o n - c h a r c o a l c a t a l y s t under an atmosphere o f h y d r o g e n gas. I n t h i s way s e l e c t i v e r e d u c t i o n a t C-18 a f f o r d e d the a l c o h o l (36) i n h i g h y i e l d and p u r i t y . (35) F i g u r e 6. Two methods f o r d e o x y g e n a t i o n o f C-18 o f W i e l a n d - G u m l i c h a l d e h y d e ( 2 2 ) . -120-A t h i r d method w h i c h a c h i e v e d t h i s d e o x y g e n a t i o n was d i s c o v e r e d d u r i n g t h e c u r r e n t i n v e s t i g a t i o n i n a r e l a t e d s t u d y w h i c h w i l l be d e s c r i b e d s h o r t l y . WGA (22) was d i s s o l v e d i n l i q u i d ammonia, e m p l o y i n g 1,2-dimethoxyethane and anhydrous ^ - b u t a n o l as c o - s o l v e n t s , a t -78°. E x c e s s l i t h i u m m e t a l was added. A b l u e c o l o u r was soon a c h i e v e d i n -d i c a t i n g an e x c e s s o f d i s s o l v e d m e t a l . A f t e r m a i n t a i n i n g t h i s b l u e c o l o u r f o r 15-30 m i n u t e s (as r e q u i r e d f r o m t h i n - l a y e r c h r o m a t o g r a p h i c m o n i t o r i n g ) t h e r e a c t i o n was quenched w i t h w a t e r . Subsequent work-up and chromatography y i e l d e d the a l c o h o l (36) i n good y i e l d ( 7 5 - 8 2 % ) . T h i s method i s more c o n v e n i e n t and p r o v i d e s a b e t t e r o v e r a l l y i e l d t h a n t h e two p r e v i o u s l y d e s c r i b e d p r o c e d u r e s . A p o s s i b l e mechanism by w h i c h t h i s h y d r o g e n o l y s i s o c c u r r e d i s shown i n F i g u r e 7. P r o b a b l y t h e b a s i c media promotes r i n g - o p e n i n g o f t h e h e m i a c e t a l t o g i v e s t r u c t u r e (22 (36) Figure 7. A possible mechanism for the two-step reduction of Wieland-Gumlich aldehyde (22) to 2g,16a-cur-19-ene-17-ol (36). -121-which then accepts two electrons followed by protonatlon to give the alcohol (36). To further explore the u t i l i t y of this reduction, Wieland-Gumlich d i o l (33) was subjected to the same experimental conditions as just described. It was found that this material could also be ef f i c i e n t l y hydrogenolysed to give the alcohol (36). The reaction just described was discovered during the course of an investigation directed at developing a method for an eff i c i e n t conversion of WGA (22) to 18-deoxy Wieland-Gumlich aldehyde (37). The latter transformation was desirable in view of the anticipated d i f f i c u l t i e s associated with the oxidation of the C-17 hydroxyl in the curenol (36) to (22) (37) 27 the aldehyde (37). F r i t z , Besh and Wieland f i r s t reported that the Woodward modification of the Oppenauer oxidation of (36), using potassium _t-butoxide and benzophenone, yielded only the unsaturated aldehyde, nor-fluorocurarine (39). Their rational for this unusual conversion was that the saturated aldehyde (37) underwent fa c i l e air oxidation to give the -122-u n s a t u r a t e d s y s t e m . A l a t e r s t u d y by t h e same g r o u p z a showed t h a t t h i s c o n v e r s i o n c o u l d be a c h i e v e d u s i n g l i t h i u m _ t - b u t o x i d e i n n i t r o b e n z e n e s o l v e n t b u t no i n c r e a s e s i n y i e l d o f e i t h e r (37) o r (39) was r e p o r t e d . 28 I n 1964, B o e k e l h e i d e e t . a l . r e p o r t e d a 40% y i e l d o f n o r - f l u o r o c u r a r i n e (39) f r o m Oppenauer o x i d a t i o n o f (36) u s i n g p o t a s s i u m _ t - b u t o x i d e w i t h 5 moles o f benzophenone as the h y d r i d e a c c e p t o r . O n l y s m a l l q u a n t i t i e s o f the s a t u r a t e d a l d e h y d e (37) were o b t a i n e d . They p r o p o s e d t h a t a , g -u n s a t u r a t i o n o c c u r r e d by a second h y d r i d e t r a n s f e r t o the o x i d i z i n g a g e n t , r a t h e r t h a n by a i r o x i d a t i o n , to g i v e the i n d o l e n i n e i n t e r m e d i a t e ( 3 8 ) , w h i c h t a u t o m e r i z e d t o n o r - f l u o r o c u r a r i n e ( 3 9 ) . (36) (37) (39) (38) F i g u r e 8. The o x i d a t i o n o f a l c o h o l (36) t o n o r - f l u o r o c u r a r i n e ( 3 9 ) . -123-Our f i r s t a t t e m p t s t o o b t a i n q u a n t i t i e s o f t h e s a t u r a t e d a l d e h y d e (37) i n v o l v e d v a r y i n g t h e B o e k e l h e i d e c o n d i t i o n s f o r t h e Oppenauer o x i d a t i o n o f a l c o h o l ( 3 6 ) . When t h e r e a c t i o n was p e r f o r m e d a t room t e m p e r a t u r e r a t h e r t h a n r e f l u x i n g benzene the ' c o n v e r s i o n r e q u i r e d 4 h o u r s t o c o m p l e t e r a t h e r 0.5 h o u r s . A l s o , d e c r e a s i n g t h e q u a n t i t y o f o x i d i z i n g a g e n t f r o m 5 moles t o 2 moles p r e v e n t e d the r e a c t i o n f r o m g o i n g to c o m p l e t i o n and s t a r t i n g m a t e r i a l was r e c o v e r e d . I n t h e s e s t u d i e s , t h e p r o g r e s s o f the o x i d a t i o n was m o n i t o r e d by t h i n -l a y e r c h r o m a t o g r a p h y ( t i c ) and by w o r k i n g up a l i q u o t s o f the r e a c t i o n m i x t u r e a t r e g u l a r time i n t e r v a l s . The i d e n t i f i c a t i o n o f n o r - f l u o r o -c u r a r i n e (39) was made on the b a s i s o f i t s u l t r a v i o l e t (uv) s p e c t r u m w h i c h showed a c h a r a c t e r i s t i c s t r o n g a b s o r p t i o n a t 363 nm and i t s i n f r a r e d ( i r ) s p e c t r u m w h i c h e x h i b i t e d a c a r b o n y l a b s o r p t i o n a t 1640 cm ± . Of s i g n i f i c a n c e i n the p r o t o n m a g n e t i c r e s o n a n c e (pmr) s p e c t r u m o f (39) was a s i n g l e t a b s o r p t i o n a t x l . 0 w h i c h was a t t r i b u t e d t o t h e a l d e h y d i c p r o t o n o f the u n s a t u r a t e d c a r b o n y l g r o u p . O t h e r means o f o x i d i z i n g a l c o h o l (36) t o the s a t u r a t e d a l d e h y d e 30 (37) were s t u d i e d . The R a t c l i f f e m o d i f i c a t i o n o f t h e C o l l i n ' s o x i d a t i o n was u n s u c c e s s f u l . S i x moles o f t h e p y r i d i n e - c h r o m a t e complex were s t i r r e d w i t h a l c o h o l (36) f o r 1 h o u r , 6 h o u r s and 24 h o u r s a t room t e m p e r a t u r e w h i l e a se c o n d s i m i l a r s t u d y was made a t 30-33°. A t no ti m e c o u l d t h e d e s i r e d r e a c t i o n p r o d u c t be d e t e c t e d by t i c . I n a l l c a s e s o n l y s m a l l amounts o f the s t a r t i n g m a t e r i a l c o u l d be i s o l a t e d f r o m the r e a c t i o n m i x t u r e . The l a t t e r was worked up u s i n g a v a r i e t y o f c o n d i t i o n s i n an a t t e m p t t o r e c o v e r o r g a n i c m a t e r i a l . B e s i d e s u s i n g aqueous sodium -124-bicarbonate s o l u t i o n , the r e a c t i o n mixture was washed with IN sodium hydroxide s o l u t i o n i n one attempt and IN hydrochloric acid s o l u t i o n followed by n e u t r a l i z a t i o n i n another attempt. The i r i a t i o n s i n work-up procedure did not y i e l d better recovery of organic m a t e r i a l . A p o s s i b l e explanation f o r these r e s u l t s i s that a chromium VI ion could coordinate with the i n d o l i n e nitrogen atom and the hydroxyl group i n compound (36) to form a very stable complex (40), which would not be i s o l a t e d under the conditions used. Related to t h i s problem (40) (41) 25 i s the observation by Smith that the s i m i l a r s tructure N-acetyl-Wieland-Gumlich aldehyde (41) could not be oxidized with chromic acid at 100°. A t h i r d study involved attempts to o x i d i z e alcohol (36) using 30 anhydrous dimethylsulphoxide as a reagent. When dicyclohexylcarbodimide 31 or sulphur t r i o x i d e - p y r i d i n e were used as dehydrating agents no r e a c t i o n products could be detected and a nearly q u a n t i t a t i v e y i e l d of s t a r t i n g 32 material was recovered. However, when phosphorus pentoxide was used a very complex product mixture was obtained. Examination of t h i s mixture by t i c and pmr revealed that none of. the desired aldehyde (37) was present. -125-A t a l a t e r t i m e , a f o u r t h s t u d y o f the o x i d a t i o n o f a l c o h o l (36) was i n i t i a t e d and r e s u l t e d i n the achievemenf o f good y i e l d s of b o t h the s a t u r a t e d a l d e h y d e (37) and the u n s a t u r a t e d n o r - f l u o r o -c u r a r i n e ( 3 9 ) . I t was found t h a t when a l c o h o l ( 3 6 ) , i n a benzene s o l u t i o n w i t h 2 moles o f benzophenone a t 65°, was t r e a t e d w i t h e x c e s s p o t a s s i u m h y d r i d e the a l d e h y d e (37) c o u l d be o b t a i n e d i n 70-78% y i e l d . T h i s m a t e r i a l was i d e n t i c a l w i t h t h a t p r e v i o u s l y o b t a i n e d i n s m a l l amounts f r o m the p r e s e n t s t u d y o f t h e Oppenauer o x i d a t i o n and a s i m i l a r 24 s t u d y i n our l a b o r a t o r i e s . Of p a r t i c u l a r i m p o r t a n c e i n the c h a r a c t e r i -z a t i o n o f t h i s p r o d u c t was i t s i n d o l i n e uv spec t r u m (A 242, 2 9 8 ) , a max c a r b o n y l a b s o r p t i o n a t 1715 cm 1 i n i t s i r s p e c t r u m and an a l d e h y d i c p r o t o n a b s o r b i n g as a s i n g l e t a t xO.23 i n i t s pmr sp e c t r u m . When a l c o h o l (36) was f i r s t t r e a t e d w i t h p o t a s s i u m h y d r i d e i n a benzene s o l u t i o n a t 65°-70° and then t r e a t e d w i t h 4 moles of n i t r o -benzene over a 15 minute time p e r i o d a good y i e l d o f n o r - f l u o r o c u r a r i n e (39) was o b t a i n e d . O nly t r a c e q u a n t i t i e s o f t h e s a t u r a t e d a l d e h y d e (37) c o u l d be d e t e c t e d by t i c . Thus, f o r the f i r s t t ime b o t h a l d e h y d e s (37) and (39) c o u l d be o b t a i n e d i n good y i e l d s by r e l i a b l e p r o c e d u r e s . Good y i e l d s c o u l d o n l y be o b t a i n e d i f p r e c a u t i o n s t o e x c l u d e m o i s t u r e were e x e r c i s e d . I n b o t h p r o c e d u r e s i t was found u s e f u l t o d i s t i l a bout 10% o f the benzene from the r e a c t i o n m i x t u r e , b e f o r e p o t a s s i u m h y d r i d e o r n i t r o b e n z e n e were added, t o e n s u r e t h a t any t r a c e s o f m o i s t u r e were removed. I n o r d e r t o o b t a i n a n i o n f o r m a t i o n a t p o s i t i o n C-16 of 18-deoxy-W i e l a n d - G u m l i c h - a l d e h y d e (37) i t would be n e c e s s a r y t o p r o t e c t the i n d o l i n e n i t r o g e n atom s i n c e the i n d o l i n e NH i s a l s o w e a k l y a c i d i c . -126-Two r o u t e s were d e v e l o p e d t o o b t a i n N - f u n c t i o n a l i z e d d e r i v a t i v e s o f a l d e h y d e ( 3 7 ) . I n t h e f i r s t r o u t e , the c u r e n o l (36) was t r e a t e d w i t h a c e t i c -29 f o r m i c a n h y d r i d e a t 0° f o r 30 m i n u t e s . A f t e r r e m o v a l o f the r e a g e n t i n v a cuo and p u r i f i c a t i o n by c h r o m a t o g r a p h y a h i g h y i e l d o f t h e N-f o r m y l - 0 - f o r m y l d e r i v a t i v e (42) was o b t a i n e d . The uv s p e c t r u m ( ^ m a x 247, 288, 289) o f t h i s p r o d u c t i n d i c a t e d t h a t an N - a c y l a t e d i n d o l i n e chromophore was p r e s e n t . I n t h e i r s p e c t r u m a c a r b o n y l a b s o r p t i o n a t 1660 cm X i n d i c a t i n g an amide group and a second c a r b o n y l a b s o r p t i o n a t 1720 cm X w h i c h was a t t r i b u t e d t o an 0 - f o r m y l group p r o v i d e d f u r t h e r s u p p o r t i n g e v i d e n c e . The pmr s p e c t r u m o f t h i s p r o d u c t showed the p r e s e n c e of two a l d e h y d i c p r o t o n s . A s i n g l e t a b s o r p t i o n o f t l . 5 1 was a s s i g n e d t o t h e p r o t o n o f an N - f o r m y l group and a s i n g l e t a b s o r p t i o n a t x l . 9 9 was a t t r i b u t e d t o the p r o t o n o f an 0 - f o r m y l g r o u p . The p r o d u c t (42) was r e f l u x e d i n m e t h a n o l f o r 30 m i n u t e s t o y i e l d a new compound. T h i s p r o d u c t had a uv s p e c t r u m s i m i l a r t o t h e s t a r t i n g m a t e r i a l (42) b u t e x h i b i t e d o n l y one c a r b o n y l a b s o r p t i o n (1660 cm X ) i n t h e i r s p e c t r u m w h i c h c o r r e s p o n d e d to t h e p r e s e n c e o f an amide g r o u p . The l a t t e r was c o n f i r m e d by a s i n g l e t a b s o r p t i o n a t t l . 2 4 i n t h e pmr s p e c t r u m . A m o l e c u l a r i o n w h i c h a p p e a r e d a t m/e 324 i n the mass s p e c t r u m o f t h i s p r o d u c t a l l o w e d the a s s i g n m e n t o f s t r u c t u r e ( 4 3 ) . The s y n t h e s i s o f N - f o r m y l - 1 8 - d e o x y - W i e l a n d - G u m l i c h a l d e h y d e (44) 30 was a c h i e v e d by C o l l i n s o x i d a t i o n o f N - f o r m y l a l c o h o l ( 4 3 ) . The t r e a t -ment o f t h e l a t t e r w i t h 6 moles o f c h r o m i c a n h y d r i d e - p y r i d i n e complex f o r 45 m i n u t e s f o l l o w e d by a work-up w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n -127-(44) F i g u r e 9. The s y n t h e s i s o f N-formyl-18-deoxy-Wieland-Gumlich a l d e h y d e ( 4 4 ) . a f f o r d e d the d e s i r e d a l d e h y d e i n 40% y i e l d . The assignment of s t r u c t u r e (44) was made on the b a s i s o f the c h a r a c t e r i s t i c s p e c t r a l d a t a . The i r spectrum showed i n a d d i t i o n to the N - f o r m y l c a r b o n y l a b s o r p t i o n a t 1660 > a second c a r b o n y l a b s o r p t i o n a t 1720 cm X w h i l e c l o s e e x a m i n a t i o n of the -128-pmr s p e c t r u m o f t h i s p r o d u c t r e v e a l e d a d o u b l e t a b s o r p t i o n a t T0.24 and a s i n g l e t a b s o r p t i o n a t xO.38 w i t h a t o t a l i n t e g r a l f o r one a l d e h y d i c p r o t o n . S i m i l a r l y , s i n g l e t a b s o r p t i o n a t x l . 1 4 and x l . 3 8 p r o v i d e d e v i d e n c e f o r a p r o t o n o f an N - f o r m y l g r o u p . T h i s m u l t i p l i c i t y o f s i g n a l s i n t h e low f i e l d r e g i o n o f t h e s p e c t r u m i n d i c a t e d t h e p r e s e n c e o f b o t h C-16 e p i m e r s o f the N - f o r m y l a l d e h y d e ( 4 4 ) . There a p p e a r e d , however, t o be o n l y one p r o d u c t by t i c . No a t t e m p t s were made to s e p a r a t e t h e s e i s o m e r s s i n c e t h e s t e r e o c h e m i s t r y a t C-16 w o u l d n o t a l t e r t h e f o l l o w i n g r e a c t i o n s e q uence. F u r t h e r e v i d e n c e f o r s t r u c t u r e (44) was o b t a i n e d f r o m i t s mass s p e c t r u m w h i c h e x h i b i t e d a c h a r a c t e r i s t i c f r a g m e n t a t i o n p a t t e r n ( F i g u r e 1 0 ) . The l o s s o f t h e a l d e h y d e group f r o m t h e m o l e c u l a r i o n (m/e 322) w o u l d y i e l d i o n ( i ) a t m/e 293. A complex f r a g m e n t a t i o n o f t h e m o l e c u l a r i o n o r i o n ( i ) r e s u l t e d i n a s i g n i f i c a n t i o n ( i i ) w h i c h a p p e a r e d a t m/e 34 279. T h i s f r a g m e n t a t i o n i s v e r y c h a r a c t e r i s t i c i n the S t r y c h n o s s e r i e s ' and i s o f p a r t i c u l a r d i a g n o s t i c v a l u e f o r N ^ - f u n c t i o n a l i z e d d e r i v a t i v e s . A s e c o n d i m p o r t a n t f r a g m e n t a t i o n i n t h i s s e r i e s i s t h e r e t r o - D i e l s - A l d e r -t y p e f r a g m e n t a t i o n w h i c h y i e l d s a n i m p o r t a n t i o n ( i i i ) c o r r e s p o n d i n g to the p i p e r i d i n e p o r t i o n o f the m o l e c u l e . I n t h i s example, t h i s i o n ( i i i ) a p p e a r e d a t m/e 164 i n d i c a t i n g the p r e s e n c e o f the a l d e h y d e group i n the p i p e r i d i n e p o r t i o n o f t h e s t r u c t u r e . . O t h e r means o f o x i d i z i n g t h e N - f o r m y l a l c o h o l (43) t o t h e d e s i r e d a l d e h y d e were e x p l o r e d i n o r d e r t o i n c r e a s e t h e y i e l d . I n one s t u d y , the a l c o h o l was t r e a t e d w i t h f r e s h l y p r e p a r e d s i l v e r c a r b o n a t e on c e l i t e i n H-CHO CHO ( i i i ) m/e 164 + •N CO CHO ( i i ) CHO i/e 279 "f" m /e 121 Figure 10. A rationale for the fragmentation of aldehyde (44) in the mass spectrometer. 36 refluxing benzene. Although significant quantities of the desired product were obtained, the reaction always failed to go to completion and the desired product was d i f f i c u l t to isolate from minor components, -130-I n a n o t h e r s t u d y , the a l c o h o l (43) was t r e a t e d w i t h 2 moles o f d i m e t h y l -p y r a z o l e - c h r o m a t e complex i n d i c h l o r o m e t h a n e a c c o r d i n g t o a p r o c e d u r e 37 by C o r e y . T h i s o x i d a t i o n method was s u c c e s s f u l i n t h a t good c o n v e r s i o n to t h e a l d e h y d e (44) was a p p a r e n t . However, p u r e q u a n t i t i e s o f the a l d e -hyde c o u l d o n l y be o b t a i n e d by r e p e a t e d c h r o m a t o g r a p h i c s e p a r a t i o n s t o i n o r d e r t o remove the d i m e t h y l p y r a z o l e . T h i s t e d i o u s p r o c e d u r e made i t d e s i r a b l e t o i n v e s t i g a t e a n o t h e r o x i d a t i o n r e a c t i o n . The N - f o r m y l a l c o h o l (43) was t r e a t e d w i t h a n o t h e r r e a g e n t 3 8 d e v e l o p e d by C o r e y , p y r i d i n i u m c h l o r o c h r o m a t e . T h i s a p p r o a c h a c h i e v e d the d e s i r e d c o n v e r s i o n i n 78% y i e l d . A s e c o n d s y n t h e s i s of t h e N - f o r m y l a l d e h y d e (44) was l a t e r a c c o m p l i s h e d . A f t e r the c o n v e r s i o n o f a l c o h o l (36) t o a l d e h y d e (37) was p e r f e c t e d as p r e v i o u s l y d e s c r i b e d , the l a t t e r was a c y l a t e d w i t h a c e t i c -f o r m i c a n h y d r i d e t o g i v e t h e N - f o r m y l - 1 8 - d e o x y - W i e l a n d - G u m l i c h a l d e h y d e (44) i n a c o m p a r a b l e o v e r a l l y i e l d . This" s y n t h e s i s s e r v e d t o f u r t h e r c o n f i r m t h e s t r u c t u r e ( 4 4 ) . W i t h the d e s i r e d f u n c t i o n a l i z e d c u r a n s y s t e m i n hand, a s t u d y was i n i t i a t e d t o i n t r o d u c e a carbomethoxy group i n t o t h e C-16 p o s i t i o n . The p l a n , i l l u s t r a t e d i n F i g u r e 11, was t o s y n t h e s i z e t h e a l d e h y d o - e s t e r (45) h a v i n g t h e s t e r e o c h e m i s t r y shown f o r r e a s o n s p r e v i o u s l y d i s c u s s e d . Removal o f t h e p r o t e c t i n g group and s u b s e q u e n t o x i d a t i o n o f t h e i n d o l i n e n i t r o g e n atom would l e a d t o the p r e a k u a m m i c i n e s e r i e s . The l a t t e r c o u l d t h e n be r i n g - o p e n e d to stemmadeninine (.4) by a known r e a c t i o n w h i c h w i l l be d i s c u s s e d i n d e t a i l l a t e r . I n t h e f i r s t a t t e m p t t o i n t r o d u c e the d e s i r e d e s t e r f u n c t i o n a l i t y , compound (44) was t r e a t e d w i t h sodium h y d r i d e i n d i m e t h o x y e t h a n e and - 1 3 1 -F i g u r e 11. A t t e m p t e d f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) and t h e p r o p o s e d p l a n f o r i t s c o n v e r s i o n t o preakuammicine and stemmadenine d e r i v a t i v e s . -132-m e t h y l c h l o r o f o r m a t e a t 80°. A t i c e x a m i n a t i o n o f t h e r e a c t i o n m i x t u r e a f t e r 1 h o u r r e v e a l e d a l m o s t c o m p l e t e c o n v e r s i o n t o a l e s s p o l a r m a t e r i a l . T h i s p r o d u c t had a uv s p e c t r u m s i m i l a r t o s t a r t i n g m a t e r i a l , b u t t h e i r s p e c t r u m showed a d d i t i o n a l a b s o r p t i o n i n t h e c a r b o n y l r e g i o n n e a r 1680 cm A t h r e e - p r o t o n s i n g l e t a b s o r p t i o n w h i c h a p p e a r e d i n i t s pmr s p e c t r u m a t x6.24 showed t h e p r e s e n c e o f a carbomethoxy g r o u p . I n the mass s p e c t r u m o f t h i s p r o d u c t t h e r e a p p e a r e d two m o l e c u l a r i o n peaks a t m/e 446 and 448 i n a p a t t e r n w h i c h s u g g e s t e d t h e p r e s e n c e o f an atom o f c h l o r i n e . I t t h e r e f o r e a p p e a r e d t h a t under t h e s e r e a c t i o n c o n d i t i o n s t h e m e t h y l c h l o r o f o r m a t e r e a c t e d w i t h t h e more b a s i c n i t r o g e n (N^) t o g i v e s t r u c t u r e ( 4 7 ) . T h i s t y p e 39 o f r e a c t i o n has p r e v i o u s l y been o b s e r v e d i n s t r y c h n i n e c h e m i s t r y . A s i m i l a r r e a g e n t , cyanogen b r o m i d e , was f o u n d t o cause f i s s i o n o f t h e five-membered r i n g o f s t r y c h n i n e t o g i v e s t r u c t u r e ( 4 8 ) . C l e a r l y , e l e v a t e d t e m p e r a t u r e s c o u l d n o t be used t o a c h i e v e c o n v e r s i o n t o s t r u c t u r e ( 4 5 ) , t h e r e f o r e a s e l e c t i o n o f o t h e r more p o w e r f u l b a s e s were u s e d a t l o w e r t e m p e r a t u r e s . T a b l e 1 summarizes a s t u d y i n w h i c h r e a c t i o n o f t h e N - f o r m y l a l d e h y d e (44) was a t t e m p t e d . u s i n g e x c e s s s o d i u m h y d r i d e t o f o r m t h e a n i o n f o l l o w e d by q u e n c h i n g w h i c h m e t h y l c h l o r o f o r m a t e . I n e a c h e x p e r i m e n t t h e p r o g r e s s o f the r e a c t i o n was f o l l o w e d by t i c . When d i m e t h y l f o r m a m i d e o r d i m e t h y l -s u l p h o x i d e were u s e d , s m a l l a l i q u o t s o f the r e a c t i o n were worked up and examined t o d e t e r m i n e t h e p r o g r e s s o f t h e r e a c t i o n . I n a l l c a s e s the r e a c t i o n s were worked up and the p r o d u c t m i x t u r e was s e p a r a t e d and a l l components were examined s p e c t r o s c o p i c a l l y . S t a r t i n g m a t e r i a l (44) was r e c o v e r e d i n e v e r y e x p e r i m e n t shown e x c e p t E x p e r i m e n t 3 i n w h i c h c o m p l e t e d e c o m p o s i t i o n had o c c u r r e d . -133-I n E x p e r i m e n t 7 i n w h i c h t h e e f f e c t i v e base i s t h e d i m s y l a n i o n , some d e f o r m y l a t i o n o f t h e s t a r t i n g m a t e r i a l had o c c u r r e d and s m a l l amounts o f a l d e h y d e (37) were i s o l a t e d . T h i s o b s e r v a t i o n was n o t u n e x p e c t e d s i n c e N - f o r m y l g r o u p s have been d e f o r m y l a t e d under s i m i l a r c o n d i t i o n s . F u r t h e r a t t e m p t s t o f u n c t i o n a l i z e t h e C-16 p o s i t i o n o f a l d e h y d e (44) w i t h m e t h y l c h l o r o f o r m a t e a r e summarized i n T a b l e 2. I n E x p e r i m e n t s 1-3 p o t a s s i u m h y d r i d e d i s p e r s e d i n o i l was used and s t a r t i n g m a t e r i a l p l u s t h e d e f o r m y l a t e d a l d e h y d e (37) was r e c o v e r e d . When a 2 m o l a r e x c e s s o f p o t a s s i u m _ t - b u t o x i d e (Exp.4-6) o r t h e "harpoon b a s e " l i t h i u m d i i s o p r o p y l amine (LDA) were used t o g e n e r a t e the a n i o n o f a l d e h y d e (44) o n l y s t a r t i n g m a t e r i a l was r e c o v e r e d i n a d d i t i o n t o t a r r y decom-p o s i t i o n p r o d u c t s . T h e r e a r e two s t e p s i n v o l v e d i n t h e n o r m a l f u n c t i o n a l i z a t i o n o f a c a r b o n y l group. The f i r s t i s a n i o n f o r m a t i o n a t the a - c a r b o n and the s e c o n d i s the r e a c t i o n o f the a n i o n w i t h t h e e l e c t r o p h i l i c r e a g e n t . T h e r e f o r e , when s u c h a r e a c t i o n f a i l s t he f i r s t q u e s t i o n w h i c h has t o be a s k e d i s w h e t h e r o r n o t a n i o n f o r m a t i o n has o c c u r r e d d u r i n g t h e r e a c t i o n c o n d i t i o n s u s e d . I n o r d e r t o t r y t o o b t a i n some i n f o r m a t i o n about t h i s , a p a r a l l e l s t u d y was made i n w h i c h d e u t e r i u m o x i d e was used t o quench th e r e a c t i o n i n s t e a d o f m e t h y l c h l o r o f o r m a t e . The e x p e c t e d p r o d u c t s f r o m s u c h a r e a c t i o n w o u l d have a d e u t e r i u m atom i n t h e p o s i t i o n i n w h i c h t h e a n i o n was formed. The p o s i t i o n o f and t h e e x t e n t o f d e u t e r a t i o n c o u l d t h e n be a s c e r t a i n e d by e x a m i n a t i o n o f t h e mass s p e c t r u m o f the p r o d u c t . I n t he p r e s e n t s t u d y , the r e a c t i o n c o n d i t i o n s f o r l i x p e r i m e n t s 2, 4, 5 and 7 i n T a b l e 1 were d u p l i c a t e d quenched w i t h D 20 and t h e r e c o v e r e d s t a r t i n g -134-T a b l e 1. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) u s i n g an e x c e s s o f sodium h y d r i d e and q u e n c h i n g w i t h m e t h y l c h l o r o f o r m a t e Exp. R e a c t i o n T i m e ( h r s ) S o l v e n t Temperature P r o d u c t s l a 6 THF r t e (44) p l u s d e c o m p o s i t i o n 2 a 2 _ti_ 50° 3 a 2 • DMF r t C omplete d e c o m p o s i t i o n 4 a , b 1 _n_ 50° (44) p l u s d e c o m p o s i t i o n 5 C 4 _n_ r t _"_ _"_ _n_ 6 C 0.5 55° 7 d 1 DMSO 50° (44) p l u s a l d e h y d e ( 3 7 ) 8 d 3 _n_ r t (44) a. 1 0 - F o l d e x c e s s o f sodium h y d r i d e . b. G r a n u l a r r a t h e r t h a n o i l - d e s p e r s e d sodium h y d r i d e was u s e d . c. 1.5 M o l e s o f s o d i u m h y d r i d e . d. 2 M o l e s o f sodium h y d r i d e . e. Room t e m p e r a t u r e . -135-T a b l e 2. F u r t h e r s t u d i e s on t h e f u n c t i o n a l i z a t i o n o f a l d e h y d e (44) Exp. R e a c t i o n T i m e ( h r s ) Base S o l v e n t Temper- P r o d u c t s a t u r e 1 3 5 moles KH THF r t a (44) p l u s a l d e h y d e ( 3 7 ) 2 1 —"— —"— 60° " " " 3 0.5 -"- DMSO 35° 4 2 2 moles t-BuOk C,H, 5 5 ° b (44) — o o 5 2 -"- DMSO r t (44) p l u s d e c o m p o s i t i o n 6 1 -"- -"- 50° (44) 7 0.5 2 moles LDA THF -78° (44) 8 1 -"- -"- 0° (44) p l u s d e c o m p o s i t i o n g 3 _ " _ _ " _ R ^ I I I I I I a. Room t e m p e r a t u r e b. R e f l u x e d f o r 30 m i n u t e s b e f o r e c o o l i n g t o 55° f o r a d d i t i o n o f m e t h y l -c h l o r o f ormate -136-m a t e r i a l (44) was examined by mass s p e c t r o m e t r y . I n each c a s e , no s i g n i f i c a n t i n c o r p o r a t i o n o f d e u t e r i u m was d e t e c t e d . S i m i l a r i l y , t h e r e a c t i o n c o n d i t i o n s i n E x p e r i m e n t s 2, 5, 7, 8 and 9 i n T a b l e 2 were u s e d i n a d e u t e r a t i o n s t u d y . A g a i n , no i n c o r p o r a t i o n o f d e u t e r i u m was d e t e c t e d . A s i m i l a r s t u d y was a l s o done by L e w i s i n o u r l a b o r a t o r i e s and s i m i l a r r e s u l t s were o b t a i n e d . 4 0 Due t o t h e i m p o r t a n c e o f t h i s p r o b l e m , i t was d e c i d e d t h a t a s i m i l a r s t u d y s h o u l d be made on an a l d e h y d e p o s s e s s i n g a n o t h e r p r o t e c t i n g group on t h e i n d o l i n e n i t r o g e n atom as the N - f o r m y l group was c o m p l i c a t i n g t h e p r o d u c t m i x t u r e i n some e x p e r i m e n t s . I t was f o u n d t h a t when the a l c o h o l (36) i n F i g u r e 12 was d i s s o l v e d i n t e t r a -h y d r o f u r a n and t r e a t e d w i t h an e x c e s s o f m e t h y l c h l o r o f o r m a t e a r a p i d r e a c t i o n o c c u r r e d . A t i c e x a m i n a t i o n o f t h e p r o d u c t m i x t u r e r e v e a l e d the p r e s e n c e o f two p r o d u c t s . The i r s p e c t r u m o f the m i x t u r e showed a b r o a d c a r b o n y l a b s o r p t i o n (1730-1660 cm "*") . A f t e r t h i s t o t a l p r o d u c t was s t i r r e d a t room t e m p e r a t u r e f o r 30 m i n u t e s i n a 2% m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n o n l y t h e more p o l a r p r o d u c t c o u l d be d e t e c t e d . T h i s m a t e r i a l was i d e n t i c a l t o the s i n g l e p r o d u c t w h i c h was o b t a i n e d when t h e i n d o l i n e a l c o h o l (36) was d i s s o l v e d i n m e t h a n o l and t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e a t room t e m p e r a t u r e f o r 15 m i n u t e s . The s t r u c t u r e o f t h i s p r o d u c t (49) was a s s i g n e d on the f o l l o w i n g d a t a . I t s uv s p e c t r u m (A 241, 278, 287) showed the p r e s e n c e o f an N - a c y l a t e d max . i n d o l i n e chromophore s i m i l a r t o t h a t w h i c h was p r e v i o u s l y -137-CH20H CO2CH3 (36) (49) CHO (37) Figure 12. The s y n t h e s i s of N-carbbmethoxy-18-deoxy-Wieland-Gumlich aldehyde (50) obtained f o r the N-formyl a l c o h o l (42). In the i r spectrum, the N-H a b s o r p t i o n at 3575 cm 1 was absent but a strong carbonyl a b s o r p t i o n at 1685 cm 1 was found. The pmr spectrum of t h i s m a t e r i a l e x h i b i t e d a three-proton s i n g l e t a t T6.20 which was assigned to the N-carbomethoxy -138-group. A l s o i n t h i s s p e c t r u m , t h e p o s i t i o n o f a o n e - p r o t o n m u l t i p l e t a t T2.52, w h i c h was a t t r i b u t e d t o t h e a r o m a t i c p r o t o n a t C-12, i s i n d i c a t i v e o f an N - f u n c t i o n a l i z e d i n d o l i n e s y s t e m as i t has s h i f t e d f r o m x2.8 i n the c o r r e s p o n d i n g N-H d e r i v a t i v e . F u r t h e r c o n f i r m a t i o n o f t h i s s t r u c t u r e (49) was o b t a i n e d f r o m i t s mass s p e c t r u m w h i c h e x h i b i t e d a m o l e c u l a r i o n a t m/e 354 as w e l l as p r o m i n e n t f r a g m e n t s a t m/e 309 ( i o n i v ) and m/e 166 ( i o n v ) . The l a t t e r two i o n s r e p r e s e n t a f r a g -m e n t a t i o n p a t t e r n o f t h e s e a l k a l o i d s , w h i c h was d i s c u s s e d e a r l i e r . Compound (49) was o x i d i z e d i n good y i e l d t o the a l d e h y d e (50) by p y r i d i n i u m c h l o r o c h r o m a t e i n a manner s i m i l a r to t h a t d i s c u s s e d i n the N - f o r m y l s e r i e s . T h i s p r o d u c t (50) e x h i b i t e d a uv s i m i l a r t o t h a t o f th e s t a r t i n g m a t e r i a l . I t s i r s p e c t r u m showed a d d i t i o n a l c a r b o n y l a b s o r p t i o n a t 1720 cm 1 . The pmr s p e c t r u m i n d i c a t e d t h e p r e s e n c e o f an a l d e h y d i c p r o t o n a p p e a r i n g as a d o u b l e t a b s o r p t i o n a t T0.40 (J=7Hz). The p r e s e n c e o f the a l d e h y d e group was a l s o e v i d e n t by the s i g n i f i c a n t l o s s o f 29 mass u n i t s from the m o l e c u l a r i o n a t m/e 352 i n i t s mass s p e c t r u m . -139-F u r t h e r c o n f i r m a t i o n o f s t r u c t u r e (50) was l a t e r o b t a i n e d when q u a n t i t i e s o f a l d e h y d e (37) became a v a i l a b l e . The l a t t e r compound was d i s s o l v e d i n t e t r a h y d r o f u r a n and t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e to y i e l d a s i n g l e p r o d u c t . T h i s p r o d u c t had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h o s e o f N - c a r b o m e t h o x y - 1 8 - d e o x y - W i e l a n d -G u m l i c h a l d e h y d e ( 5 0 ) . A t t e m p t s t o i n t r o d u c e t h e carbomethoxy group a t C-16 o f a l d e h y d e (50) were c a r r i e d o u t i n a manner s i m i l a r t o t h a t i n t h e s t u d y d i s c u s s e d f o r t h e N - f o r m y l s e r i e s and a r e summarized i n T a b l e 3. I n e a c h e x p e r i -ment a n i o n f o r m a t i o n was a t t e m p t e d f o l l o w e d by q u e n c h i n g w i t h m e t h y l -c h l o r o f ormate . I n t h i s s e r i e s , no new p r o d u c t s were d e t e c t e d by t i c e x a m i n a t i o n of t h e r e a c t i o n m i x t u r e o r by s e p c t r o s c o p i c e x a m i n a t i o n o f the worked-up r e a c t i o n p r o d u c t . S t a r t i n g m a t e r i a l (50) was i s o l a t e d a l o n g w i t h v a r i o u s q u a n t i t i e s o f t a r r y d e c o m p o s i t i o n p r o d u c t s . T a b l e 3. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f N-carbomethoxy a l d e h y d e (50) Exp. R e a c t i o n T i m e ( h r ) Base S o l v e n t Temperature 1 6 10 m o l es NaH THF 40° (50) 2 2 _n_ DMSO 53° 3 2 2 moles KH C 6 H 6 80° 4 4 1 mole KH DMF 50° (50) 5 0.5 2 moles LDA THF 0 (50) 6 1 _n_ 20° _!!_ 7 1 _n_ THF 35° P r o d u c t s p l u s d e c o m p o s i t i o n -140-In t h i s study a l s o , a p a r a l l e l d e u t e r a t i o n study was made. The e x p e r i m e n t a l c o n d i t i o n s of Experiments 1, 2, 6 and 7 were d u p l i c a t e d and each r e a c t i o n was quenched w i t h d e u t e r i u m o x i d e . E x a m i n a t i o n of the mass s p e c t r a of i s o l a t e d s t a r t i n g m a t e r i a l (50) r e v e a l e d t h a t no s i g n i f i c a n t i n c o r p o r a t i o n o f d e u t e r i u m had o c c u r r e d . The f a i l u r e to d e t e c t a n i o n f o r m a t i o n or o b t a i n f u n c t i o n a l i -z a t i o n a t C-16 by t r e a t i n g the two c u r a n d e r i v a t i v e s (44) and (50) w i t h a base and m e t h y l c h l o r o f o r m a t e i s not f u l l y u n d e r s t o o d . Three p o s s i -b i l i t i e s may be c o n s i d e r e d . F i r s t l y , a n i o n f o r m a t i o n may not be o c c u r r i n g a t a l l under the c o n d i t i o n s employed due to the v e r y h i n d e r e d environment of p o s i t i o n C-16. Secondly, a n i o n f o r m a t i o n may be o c c u r r i n g , but m e t h y l -c h l o r o f ormate may be too b u l k y to r e a c t i n such a s t e r i c a l l y u n f a v o u r a b l e environment. And t h i r d l y , g i v e n t h i s second p o s s i b i l i t y , r e a c t i o n may be o c c u r r i n g on the oxygen atom v i a the resonance shown. I f t h a t was the c a s e , the p r o d u c t would n o t be expected to be d e t e c t e d or i s o l a t e d under the c o n d i t i o n s employed as i t would r a p i d l y h y d r o l y s e to s t a r t i n g m a t e r i a l . A d i f f e r e n t approach to the f u n c t i o n a l i z a t i o n o f p o s i t i o n C-16 24 was i n i t i a t e d . I t was d i s c o v e r e d i n our l a b o r a t o r i e s t h a t when the - 1 4 1 -u n s a t u r a t e d a l d e h y d e , n o r - f l u o r o c u r a r i n e (39) was t r e a t e d w i t h s o d i u m b o r o h y d r i d e i n m e t h a n o l a t room t e m p e r a t u r e the r i n g - o p e n e d i n d o l e a l c o h o l (52) c o u l d be o b t a i n e d i n 40% y i e l d . T h i s r e d u c t i v e r i n g -o p e n i n g r e a c t i o n w h i c h was f i r s t d e v e l o p e d by S m i t h and W r o b e l i n t h i s s e r i e s o f compounds p r o b a b l y p r o c e e d s v i a t h e seco-immonium d e r i v a t i v e k? ( 5 1 ) . I n t h e p r e s e n t s t u d y , i t was fo u n d t h a t an i n c r e a s e d y i e l d o f 60-65% c o u l d be o b t a i n e d i f t h i s r i n g - o p e n i n g r e a c t i o n was done i n 2% m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n a t 40°. The p l a n t o c o n v e r t p r o d u c t (52) t o stemmadenine i s shown i n F i g u r e 13. A w e l l known r e a c t i o n o f i n d o l e a l k a l o i d s i s c h l o r o i n d o l e n i n e f o r m a t i o n . T h i s r e a c t i o n i s a c c o m p l i s h e d by t r e a t i n g t he i n d o l e d e r i -v a t i v e w i t h a s o u r c e o f p o s i t i v e c h l o r i n e i o n s s u c h as so d i u m h y p o c h l o r i t e , _ t - b u t y l h y p o c h l o r i t e o r N - c h l o r o b e n z t r i a z o l e . The c h l o r o i n d o l e n i n e s a r e n o r m a l l y u n s t a b l e m o l e c u l e s and o n l y a few have e v e r been i s o l a t e d . T h e r e f o r e , t h e s e d e r i v a t i v e s a r e u s u a l l y made _in s i t u and t r e a t e d w i t h t h e a p p r o p r i a t e r e a g e n t . The common r e a c t i o n f o r w h i c h c h l o r o i n d o l e n i n e s have been used i s t h e i n t r o d u c t i o n o f a n u c l e o p h i l e a t o t h e i n d o l e ( i n t h i s c a s e p o s i t i o n C-16) v i a t h e t a u t o m e r i c f o r m ( r e p r e s e n t e d by s t r u c t u r e ( 5 4 ) ) . T h i s t y p e o f r e a c t i o n has been w i d e l y used i n the Iboga a l k a l o i d s t o i n t r o d u c e v a r i o u s n u c l e o p h i l e s s u c h as c y a n i d e i o n and v i n d o l i n e . However, t h i s r e a c t i o n a p p e a r e d n o t t o have b e e n u s e d i n t h e S t r y c h n o s s e r i e s . I t was f e l t t h a t t he s t e r e o c h e m i s t r y o f the C^ i n t r o d u c t i o n a t C-16 c o u l d s t i l l be c o n t r o l l e d i n the r i n g - o p e n e d s e r i e s s i n c e e x a m i n a t i o n o f m o l e c u l a r models r e v e a l e d t h a t a g a i n a g r e a t d e a l o f s t e r i c c r o w d i n g on t h e 8 - f a c e was p r e s e n t i n t h e s e d e r i v a t i v e s . -142-The i n d o l e a l c o h o l (52) was t r e a t e d w i t h 1.1 e q u i v a l e n t s o f J ^ - b u t y l h y p o c h l o r i t e a t 0° f o r 15 m i n u t e s . A t i c e x a m i n a t i o n o f t h e r e a c t i o n m i x t u r e r e v e a l e d t h e p r e s e n c e o f v e r y p o l a r m a t e r i a l and a b s e n c e o f s t a r t i n g m a t e r i a l . The uv s p e c t r u m (X 310) max i n d i c a t e d t h a t the i n d o l e chromophore had been c o n v e r t e d t o a c h l o r o -i n d o l e n i n e chromophore. A t t e m p t s t o i s o l a t e t h i s m a t e r i a l by p r e -p a r a t i v e l a y e r c h r o m a t o g r a p h y ( p i c ) , n o t u n e x p e c t e d l y , f a i l e d t o g i v e p u r e m a t e r i a l . F o r t h e r e m a i n d e r o f t h e s t u d y , t h e c h l o r o i n d o l e n i n e (53) was formed i n s i t u and t r e a t e d w i t h c y a n i d e i o n d i r e c t l y . A r e p r e s e n t a t i v e . s u m m a r y o f r e s u l t s f r o m t h i s s t u d y i s shown i n T a b l e 4. I n e ach e x p e r i m e n t , c h l o r o i n d o l e n i n e f o r m a t i o n was c o n f i r m e d by uv e x a m i n a t i o n b e f o r e a d d i t i o n o f the c y a n i d e r e a g e n t . The r e a c t i o n was t h e n m o n i t o r e d by t i c and uv s p e c t r o s c o p y . A t t h e end o f t h e r e a c t i o n t i m e , t h e p r o d u c t m i x t u r e was i s o l a t e d and s e p a r a t e d by p i c . I n e v e r y e x p e r i m e n t , the main component o f t h e m i x t u r e was a t a r r y d e c o m p o s i t i o n p r o d u c t r e m a i n i n g on t h e b a s e - l i n e o f t h e c h r o m a t o p l a t e . A l a r g e number o f p r o d u c t s i n v e r y s m a l l amounts was a l s o a p p a r e n t . However, a uv s p e c t r o s c o p i c s t u d y o f t h e s e complex m i x t u r e s r e v e a l e d t h a t no i n d o l e chromophores were p r e s e n t . A p r o h i b i t i v e l y l a r g e s c a l e d -up r e a c t i o n w o u l d have been r e q u i r e d t o o b t a i n s u f f i c i e n t q u a n t i t i e s o f any one o f t h e s e components f o r c h a r a c t e r i z a t i o n . S i n c e no i n d o l i c m a t e r i a l was found t h i s s e r i e s o f e x p e r i m e n t s was abandoned. F i g u r e 13. The p l a n t o s y n t h e s i z e stemmadenine ( 4 ) v i a the c h l o r o i n d o l e n i n e o f d e s - c a r b o m e t h o x y stemmadenine ( 5 2 ) . -144-T a b l e 4. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a t h e c h l o r o i n d o l e n i n e o f i n d o l e (52) Exp. N u c l e o p h i l e S o l v e n t Temperature T i m e ( h r s ) P r o d u c t s 1 KCN C H 2 C 1 2 2 -"- DMF 3 _"_ _"_ 4 ZnCN C H 2 C 1 2 5 -"- DMF 6 AgCN C H 2 C 1 2 7 -"- DMF 8 Bu.NCN CH 0C1_ 4 1 1 9 _"_ _"_ 10 -"- DMF 11 E t 2 A l C N C H 2 C 1 2 12 13 r t 24 D e c o m p o s i t i o n r t 6 80° 0.5 r t 24 50° 1.5 r t 48 55° 1.5 r t . 48 35° 12 35° 5 r t 5 36° 12 5° 5 days a. Room t e m p e r a t u r e -145-P e r h a p s one r e a s o n f o r t h e f a i l u r e t o i n t r o d u c e c y a n i d e i n t o p o s i t i o n C-16 was due t o i n t e r f e r e n c e o f t h e h y d r o x y l group i n compound ( 5 2 ) . I t was, t h e r e f o r e , d e c i d e d t o p r o t e c t t h e h y d r o x y l as i t s a c e t a t e and s t u d y t h e f u n c t i o n a l i z a t i o n o f t h e c h l o r o i n d o l e n i n e o f t h i s d e r i v a t i v e . The t r e a t m e n t o f i n d o l e a l c o h o l (52) w i t h a c e t i c a n h y d r i d e and p y r i d i n e (10%) a t 5° o v e r n i g h t y i e l d e d a p r o d u c t m i x t u r e o f two components w h i c h c o u l d be s e p a r a t e d by p i c . S u r p r i s i n g l y , t h e m a j o r p r o d u c t e x h i b i t e d a uv s p e c t r u m ( ^ m a x 248, 282, 290) c o r r e s p o n d i n g to an N - a c y l i n d o l e ( 5 6 ) . T h i s s t r u c t u r a l a s s i g n m e n t was s u p p o r t e d by i t s i r s p e c t r u m w h i c h showed an a c e t a t e c a r b o n y l a b s o r p t i o n a t 1725 cm 1 and an amide c a r b o n y l a b s o r p t i o n n e a r 1640 cm Two t h r e e - p r o t o n s i n g l e t a b s o r p t i o n s a t x7.79 and 7.88 i n d i c a t e d t h e p r e s e n c e o f b o t h an N - a c e t a t e and an 0 - a c e t a t e g r o u p , r e s p e c t i v e l y . F u r t h e r c o n f i r m a t i o n o f t h e s t r u c t u r a l a s s i g n m e n t o f t h i s p r o d u c t "(56) was e v i d e n t i n i t s mass s p e c t r u m w h i c h showed a m o l e c u l a r i o n a t m/e 380 w i t h a p r o m i n e n t l o s s o f 59 mass u n i t s w h i c h c o r r e s p o n d e d to the l o s s o f an a c e t a t e f r a g m e n t . P r e l i m i n a r y d a t a on the m i n o r component f r o m t h i s r e a c t i o n s u g g e s t e d t h a t i t was t h e d e s i r e d m o n o - a c e t a t e ( 5 7 ) . S i n c e i t i s g e n e r a l l y more d i f f i c u l t t o a c y l a t e an i n d o l e n i t r o g e n atom i n c o m p a r i s o n w i t h an h y d r o x y l g r o u p , and n o t known t o o c c u r u n d e r s t a n d a r d a c e t y l a t i n g c o n d i t i o n s , i t was f e l t t h a t t h i s r e a c t i o n c o u l d be c o n t r o l l e d t o g i v e t h e m o n o - a c e t a t e (57) as t h e m a j o r p r o d u c t . T r e a t m e n t o f t h e a l c o h o l (52) w i t h a c e t i c a n h y d r i d e and 1 d r o p o f p y r i d i n e a t -10° y i e l d e d t h e d e s i r e d m a t e r i a l as t h e m a j o r p r o d u c t . Good y i e l d s were o b t a i n e d when t h i s r e a c t i o n was m o n i t o r e d by t i c . The p r o d u c t (57) -146-e x h i b i t e d t h e n o r m a l i n d o l e chromophore i n the uv s p e c t r u m (A 224, max 283, 290) and a c a r b o n y l a b s o r p t i o n i n the i r s p e c t r u m (1725 cm X ) w h i c h c o r r e s p o n d s t o t h e p r e s e n c e o f an 0 - a c e t a t e group. A t h r e e -p r o t o n s i n g l e t a b s o r p t i o n a t x7.89 as w e l l as an N-H a b s o r p t i o n a t x l . 6 9 i n t h e pmr s p e c t r u m , and a m o l e c u l a r i o n a t m/e 338 i n t h e mass s p e c t r u m p r o v i d e d c l e a r e v i d e n c e f o r t h e a s s i g n m e n t o f s t r u c t u r e ( 5 7 ) . F i g u r e 14. The p l a n t o s y n t h e s i z e stemmadenine ( 4 ) v i a t h e c h l o r o i n d o l e n i n e a c e t a t e ( 5 8 ) . -147-W i t h t h e i n d o l e a c e t a t e (57) i n hand, a s t u d y d e s i g n e d t o i n -t r o d u c e c y a n i d e i n t o t h e C-16 p o s i t i o n was i n i t i a t e d i n a manner s i m i l a r t o t h a t made i n t h e f r e e h y d r o x y l s e r i e s p r e v i o u s l y d i s c u s s e d . As b e f o r e c h l o r o i n d o l e n i n e f o r m a t i o n was c l e a r l y a c h i e v e d when compound (57) was t r e a t e d w i t h a s l i g h t e x c e s s o f _ t - b u t y l h y p o c h l o r i t e i n d i c h l o r o -methane o r d i m e t h y l f o r m a m i d e . P r e s e n t e d i n T a b l e 5 a r e t h e r e s u l t s f o u n d w h i c h r e p r e s e n t t h e s t u d y o f t h e i n t r o d u c t i o n o f c y a n i d e i n t o p o s i t i o n C-16 i n t h e c h l o r o i n d o l e n i n e a c e t a t e ( 5 8 ) . As i n the p r e v i o u s s t u d y , t h e p r o g r e s s o f the r e a c t i o n was m o n i t o r e d by t i c and uv s p e c t r o s c o p y , and t h e i s o l a t e d p r o d u c t m i x t u r e s were examined f o r t h e p r e s e n c e o f i n d o l i c m a t e r i a l . I n each e x p e r i m e n t , a g r e a t d e a l o f d e c o m p o s i t i o n was o b t a i n e d a l o n g w i t h a s m a l l amount o f a complex m i x t u r e o f p r o d u c t s . A t no t i m e c o u l d any i n d o l i c m a t e r i a l be d e t e c t e d . Nor c o u l d any T a b l e 5. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e v i a t h e c h l o r o i n d o l e n i n e o f i n d o l e a c e t a t e ( 5 7 ) . Exp. N u c l e o p h i l e S o l v e n t Temperature T i m e ( t i r s ) P r o d u c t s 1 KCN C H 2 C 1 2 r t 2 0 D e c o m p o s i t i o n 2 -"- DMF r t 8 -"-3 II I I 68° 1 I I 4 Bu.NCN r t 24 I I -148-p u r e m a t e r i a l be o b t a i n e d i n q u a n t i t i e s s u f f i c i e n t f o r c h a r a c t e r i z a t i o n . W i t h t h e f a i l u r e t o i n t r o d u c e c y a n i d e i n t o e i t h e r compound (52) o r compound (57) t h i s a p p r o a c h was abandoned. S i m i l a r r e s u l t s were o b t a i n e d i n o u r l a b o r a t o r i e s by L e w i s i n w h i c h c y a n i d e i n t r o d u c t i o n v i a t h e c h l o r o i n d o l e n i n e o f the i n d o l e e s t e r 40 (59) was s t u d i e d . C l e a r l y a new a p p r o a c h t o the f u n c t i o n a l i z a t i o n o f the C-16 p o s i t i o n o f the c u r a n s k e l e t o n (21) was r e q u i r e d . A n o t h e r c u r a n d e r i v a t i v e w h i c h had become a v a i l a b l e and w h i c h was p o t e n t i a l l y u s e f u l f o r e l a b o r a t i o n t o the preakuammicine d e r i v a t i v e s was n o r - f l u o r o c u r a r i n e ( 3 9 ) . T h i s s t r u c t u r e may be r e g a r d e d as h a v i n g a r i g i d c i s - e n a m i n o c a r b o n y l s y s t e m . The n u c l e o p h i l i c r e a c t i o n s o f s u c h c o n j u g a t e d systems may be c l a s s i f i e d i n t o f o u r t y p e s ( F i g u r e 1 5 ) . R e a c t i o n t y p e s I , I I and I I I 41 a r e w e l l known i n s u c h systems and a r e a p p l i c a b l e i n t h e c u r r e n t s t u d y , -149-42 b u t t y p e V I r e a c t i o n s " " i n w h i c h the 8-carbon i s n u c l e o p h i l i c i s n o t r e l e v a n t h e r e because i n the c u r a n d e r i v a t i v e t h i s p o s i t i o n i s f u l l y s u b s t i t u t e d . The f a c t o r s w h i c h g o v e r n r e a c t i o n a t 0, C and N i n s u c h systems a r e n o t w e l l u n d e r s t o o d and have been a s u b j e c t o f r e c e n t i n t e r e s t . The p r o d u c t s t h a t would be e x p e c t e d from t h e s e t h r e e r e a c t i o n t y p e s i f n o r - f l u o r o c u r a r i n e (39) was r e a c t e d w i t h the e l e c t r o p h i l e , m e t h y l c h l o r o f o r m a t e , a r e shown i n F i g u r e 16. The p l a n was t o enhance r e a c t i o n c o n d i t i o n s t o f a v o u r t y p e I I r e a c t i o n . The C - f u n c t i o n a l i z e d p r o d u c t would be o f the d e s i r e d p r e a k u a m i c i n e s e r i e s ( 6 0 ) . I t was f e l t t h a t the t y p e I N - s u b s t i t u t e d p r o d u c t (61) c o u l d e a s i l y be d i s t i n g u i s h e d f r o m the l a t t e r by i t s uv s p e c t r u m w h i c h was e x p e c t e d to be v e r y d i f f e r e n t . The O - f u n c t i o n a l i z e d p r o d u c t (62) would be e x p e c t e d t o h y d r o l y s e back to s t a r t i n g m a t e r i a l . - c -E + E + E + Type I Type I I Type I I I F i g u r e 15. The n u c l e o p h i l i c r e a c t i o n s o f e n a m i n o c a r b o n y l s y s t e m s . An e x a m i n a t i o n o f t h e l i t e r a t u r e r e v e a l e d t h a t two f a c t o r s f a v o u r e d C - f u n c t i o n a l i z a t i o n o f e n a m i n o c a r b o n y l s y s t e m s . F i r s t l y , -151-Meyers e t . a l . . r e p o r t e d t h a t i n the c i s s e r i e s (see p a r t i a l s t r u c t u r e 63) a p r o t i c s o l v e n t s f a v o u r e d C - a l k y l a t i o n o f a v a r i e t y o f c y c l i c enamino-ketones w h i c h were t r e a t e d w i t h m e t h y l i o d i d e , w h i l e the t r a n s s e r i e s ( p a r t i a l s t r u c t u r e 64) O - a l k y l a t i o n p r e d o m i n a t e d and was 44 n o t i n f l u e n c e d by c h o i c e of s o l v e n t . S e c o n d l y , a s t u d y by K o z e r s k i w h i c h i n v o l v e d the a c y l a t i o n o f a c y l i c enamino-ketones h a v i n g an N-H bond and u s i n g a c e t y l c h l o r i d e r e v e a l e d t h a t C - a c y l a t i o n was f a v o u r e d i n the p r e s e n c e o f a base such as p y r i d i n e o r t r i e t h y l a m i n e . T h i s s t u d y a l s o showed t h a t C - a c y l a t i o n was f a v o u r e d i n the c i s s e r i e s . When the l i t h i u m s a l t o f n o r - f l u o r o c u r a r i n e (39) was quenched w i t h m e t h y l c h l o r o f o r m a t e a t 0° a s i n g l e p r o d u c t was o b t a i n e d . The uv s p e c t r u m (X 233, 268, 305 ) o f t h i s p r o d u c t i n d i c a t e d the p r e s e n c e o f an i n d o l e n i n e chromophore ( F i g u r e 1 7 ) . The w a v e l e n g t h o f the e l e c t r o n -t r a n s f e r band a t 268 nanometers (nm) compares f a v o u r a b l y w i t h t h a t r e p o r t e d 2 f o r s i m i l a r s t r u c t u r e s such as preakuammicine ( 8 ) ( ^ m a x 263) .» p r e c o n d y l o -c a r p i n e ( 6 5 ) ( X 280) and r h a z i n a l i n e (66)(X 2 6 6 ) . A 6 Such a uv s p e c t r u m max max w ould n o t be e x p e c t e d f o r an N - f u n c t i o n a l i z e d d e r i v a t i v e ( 6 1 ) . A l t h o u g h -152-J I l I I ' ' L U 1 I I L 220 240 260 280 300 320 340 F i g u r e 17. The uv s p e c t r u m o f the p r o d u c t f r o m the r e a c t i o n o f n o r -f l u o r o c u r a r i n e (39) w i t h m e t h y l c h l o r o f o r m a t e . no l i t e r a t u r e example o f t h e chromophore i n s t r u c t u r e (61) c o u l d be f o u n d , i n a l l examples i n w h i c h t h e r e i s a d o u b l e bond a t o t h e i n d o l i n e n u c l e u s the p r i n c i p a l band i n the uv s p e c t r u m a p p e a r s a t w a v e l e n g t h s g r e a t e r t h a n 300 nm. T h i s band i n the uv s p e c t r u m o f the s t a r t i n g m a t e r i a l n o r -f l u o r o c u r a r i n e (39) a p p e a r e d a t 363 nm. -153-(8) C H 3 ° A Z^ 0 N' (66) The c a r b o n y l r e g i o n i n the i r s p e c t r u m o f t h i s p r o d u c t showed -1 -1 t h r e e a b s o r p t i o n b a n d s : 1720, 1670 and 1640 cm . The band a t 1720 cm was a s s i g n e d t o a C-carbomethoxy group. The bands a t 1670 and 1640 cm 1 c o u l d be a d o u b l e t a b s o r p t i o n o f an a l d e h y d e group g e m i n a l t o a c a r b o -methoxy g r o u p . Due t o the s t e r i c and e l e c t r o n i c e n v i r o n m e n t n e a r t h e C-16 p o s i t i o n s u c h an a l d e h y d e group may have s u f f i c i e n t l y r e s t r i c t e d r o t a t i o n so t h a t two r o t o m e r s w o u l d e a c h c o n t r i b u t e a band i n t h e i r s p e c t r u m . T h i s r a t i o n a l e i s i l l u s t r a t e d by t h e p a r t i a l s t r u c t u r e s (67) and ( 6 8 ) . C a r b o n y l a b s o r p t i o n s o f d i f f e r e n t w a v e l e n g t h s may be e x p e c t e d 18.00 1600 I 1 I ' 1— i i i J L F i g u r e 18. The c a r b o n y l r e g i o n o f t h e i n f r a r e d s p e c t r u m o f p r o d u c t (60) -154-0 H (67) (68) f o r t h e a l i g n m e n t (67) and n o n - a l i g n m e n t (68) o f the i r - o r b i t a l s o f t h e two s y s t e m s . I n t h e pmr s p e c t r u m ( F i g u r e 19) o f t h i s p r o d u c t an a l d e h y d i c p r o t o n a b s o r p t i o n a p p e a r e d as a s i n g l e t a t T0.02 w h i c h r e p r e s e n t s a p a r a m a g n e t i c s h i f t o f 0.6 ppm f r o m the c o r r e s p o n d i n g a b s o r p t i o n i n the s p e c t r u m o f the s t a r t i n g m a t e r i a l ( 3 9 ) . A t h r e e - p r o t o n s i n g l e t a b s o r p t i o n a t T6.08 c o n f i r m e d the p r e s e n c e o f a carbomethoxy g r o u p . The l a t t e r c h e m i c a l s h i f t compares f a v o u r a b l y w i t h the c o r r e s p o n d i n g c h e m i c a l s h i f t o f the carbomethoxy g r o u p s ' i n 'preakuammicine (. 8 ) and 45 p r e c o n d y l o c a r p i n e (65) w h i c h were x6.14 and 6.15, r e s p e c t i v e l y . Of p a r t i c u l a r i n t e r e s t was t h e r e m a r k a b l e r e s e m b l a n c e between the mass s p e c t r u m o f t h i s p r o d u c t and t h a t p u b l i s h e d f o r p r e c o n d y l o -45 c a r p i n e ( 6 5 ) . B o t h compounds f r a g m e n t t o l o s e t h e carbomethoxy group f r o m t h e m o l e c u l a r i o n t o g i v e a peak a t M +-58 r a t h e r t h a n t h e more u s u a l M +-59. Due t o the l a c k o f i n f o r m a t i o n on the complex f r a g m e n t a t i o n s w h i c h i n d o l e n i n e s appear t o undergo i n the mass s p e c t r o m e t e r , l i t t l e more s t r u c t u r a l a s s i g n m e n t c o u l d be made a t t h i s t i m e based on t h e mass s p e c t r u m o f t h i s p r o d u c t . F i g u r e 19. The pmr s p e c t r u m of compound ( 6 0 ) . -156-Based on t h e above d e s c r i b e d d a t a we a s s i g n e d t he s t r u c t u r e (60) t o t h i s p r o d u c t . S t u d i e s made on model systems s u g g e s t e d t h a t n o r - f l u o r o c u r a r i n e (39) s h o u l d r e a c t w i t h m e t h y l c h l o r o f o r m a t e a c c o r d i n g t o pathway I I ( F i g u r e 17) under t h e c o n d i t i o n s u s e d . I t was l a t e r f o u n d t h a t t h i s same r e a c t i o n c o u l d be a c h i e v e d i n co m p a r a b l e y i e l d by h e a t i n g n o r - f l u o r o c u r a r i n e (39) w i t h e x c e s s m e t h y l c h l o r o f o r m a t e t o 65°, e i t h e r w i t h t e t r a h y d r o f u r a n as a c o - s o l v e n t , o r j u s t u s i n g t h e r e a g e n t i t s e l f as the s o l v e n t . The l a t t e r became the u s u a l method f o r the p r e p a r a t i o n o f t h e a l d e h y d o - e s t e r ( 6 0 ) . The s t e r e o c h e m i s t r y a t C-16 was a s -s i g n e d f r o m s t e r i c c o n s i d e r a t i o n s . Due t o t h e s t e r i c c r o w d i n g o v e r t h e 3 - f a c e o f t h e m o l e c u l e ( 3 9 ) ( d i s c u s s e d . e a r l i e r ) an i n c o m i n g e l e c t r o p h i l e s h o u l d be a - o r i e n t e d . I t was t h e l a t t e r argument upon w h i c h the s t e r e o -c h e m i s t r y o f 16-epi-stemmadenine (28) and hence stemmadenine ( 4 ) was A 2 4 a s s i g n e d . F u r t h e r e v i d e n c e f o r t h e a s s i g n m e n t o f s t r u c t u r e (60) t o t h e above p r o d u c t was o b t a i n e d f r o m i t s s t a b i l i t y i n b a s i c m e d i a . T h i s m a t e r i a l (60) was f o u n d t o be s t a b l e i n 5% m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n f o r more t h a n 6 h o u r s a t room t e m p e r a t u r e . T h i s o b s e r v a t i o n t e n d s t o e l i m i n a t e t he carbamate (61) f r o m c o n s i d e r a t i o n , as i t i s w e l l known t h a t compounds o f t h i s t y p e e a s i l y d e c a r b o x y l a t e under m i l d e r c o n d i t i o n s . When p r o d u c t (60) was h e a t e d to 65° i n 5% m e t h a n o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n , c o m p l e t e h y d r o l y s i s -to t he s t a r t i n g m a t e r i a l , n o r - f l u o r o c u r a r i n e ( 3 9 ) , o c c u r r e d i n 2 h o u r s . W i t h the a l d e h y d o - e s t e r (60) i n hand, a s t u d y f o r the c o n v e r s i o n o f t h i s m a t e r i a l t o the preak u a m m i c i n e ( 8 ) and stemmadenine ( 4 ) s e r i e s -157-was begun. Two a p p r o a c h e s were i n i t i a l l y c o n s i d e r e d . F i r s t l y , i t w o u l d be d e s i r a b l e t o s i m p l y r e d u c e t h e a l d e h y d e (60) t o t h e a l c o h o l (69) and compare t h e l a t t e r w i t h a sample o f n a t u r a l p reakuammicine ( 8 ) . F i g u r e 20. Some e x p e c t e d r e a c t i o n s o f preakuammicine d e r i v a t i v e s . -158-S e c o n d l y , t h e s y n t h e s i s o f stemmadenine s e r i e s may be a c h i e v e d by e i t h e r d i r e c t r e d u c t i v e r i n g - o p e n i n g o f p r e a k u a m m i c i n e a l d e h y d e s e r i e s (60) o r by r i n g - o p e n i n g o f t h e c o r r e s p o n d i n g a l c o h o l ( 6 9 ) . The l a t t e r c o n v e r s i o n was c l a i m e d t o have been done by S c o t t and Q u e r e s h i b u t few e x p e r i m e n t a l d e t a i l s were g i v e n . These w o r k e r s a l s o c l a i m t o have c o n v e r t e d p r e a k u a m m i c i n e ( 8 ) t o akuammicine ( 5 ) ir> b a s i c m e d i a . Compound (60) was t r e a t e d w i t h r e c r y s t a l l i z e d sodium b o r o h y d r i d e i n d r y m e t h a n o l a t room t e m p e r a t u r e f o r 30 m i n u t e s . An e x a m i n a t i o n o f t h i s r e a c t i o n by t i c r e v e a l e d t h e p r e s e n c e o f one m a j o r p r o d u c t . T h i s p r o d u c t was i s o l a t e d by p i c and found t o have the f o l l o w i n g s p e c t r a l d a t a . The uv s p e c t r u m (A 228, 292) i n d i c a t e d the p o s s i b l e p r e s e n c e max o f an i n d o l e n i n e chromophore c o n j u g a t e d t o a c a r b o n - c a r b o n d o u b l e bond. The i r s p e c t r u m showed t h a t no c a r b o n y l g r o u p s were p r e s e n t i n t h e m o l e c u l e . T h i s o b s e r v a t i o n was c o n f i r m e d by the pmr s p e c t r u m . B o t h the a l d e h y d e p r o t o n a b s o r p t i o n and the m e t h y l a b s o r p t i o n o f t h e c a r b o -methoxy group o f t h e s t a r t i n g m a t e r i a l (60) were a b s e n t f r o m t h e pmr s p e c t r u m o f t h e p r o d u c t . However, two o n e - p r o t o n s i n g l e t a b s o r p t i o n s a t x3.89 and 4.54 s u g g e s t e d the p r e s e n c e o f an e x o c y c l i c m e t h y l e n e g r o u p . The mass s p e c t r u m r e v e a l e d a m o l e c u l a r i o n a t m/e 276 w h i c h was a l s o the base peak. A h i g h r e s o l u t i o n mass measurement on t h i s peak c o n f i r m e d t h e m o l e c u l a r f o r m u l a o f t h i s m o l e c u l e t o be C ^ g ^ o ^ ' T h e s t r u c t u r e w h i c h b e s t f i t s t h i s d a t a i s t h a t o f 1 ,2-dehydro-16,17-dehydro-2g-cur-19-en ( 7 2 ) . A p o s s i b l e mechanism by w h i c h compound (72) c o u l d a r i s e f r o m t r e a t m e n t o f the a l d e h y d o - e s t e r C60) w i t h sodium b o r o h y d r i d e i s shown -159-i n F i g u r e 21. The r e d u c t i o n o f a carbomethoxy group g e m i n a l t o an a l d e h y d e group under s i m i l a r c o n d i t i o n s i s w e l l known ( f o r examples see the s y n t h e s i s o f s e c o d i n o l d e s c r i b e d i n P a r t I and t h e r e d u c t i o n o f r h a z i n a l i n e (66) i n r e f . 4 6 ) . (72) F i g u r e 21. A p o s s i b l e mechanism f o r t h e f o r m a t i o n o f compound ( 7 2 ) . -160-Th e p r o b l e m now became one o f e i t h e r r e d u c i n g t h e a l d e h y d e group i n compound (60) t o a c h i e v e the p reakuammicine s e r i e s o r t o a c h i e v e t h e r e d u c t i v e r i n g - o p e n i n g o f (60) t o g i v e the stemmadenine s e r i e s , w h i l e p r e v e n t i n g d e c a r b o x y l a t i o n . I n o r d e r t o e v a l u a t e t h e s e two p o s s i b i l i t i e s compound (.60) was t r e a t e d w i t h sodium b o r o h y d r i d e under v a r i o u s c o n d i t i o n s o f pH and t e m p e r a t u r e . The r e p r e s e n t a t i v e r e s u l t s f r o m t h i s s t u d y a r e summarized i n T a b l e 6. I n E x p e r i m e n t s 1-7 t h e main p r o d u c t o f e a c h r e a c t i o n was t h e d e o x y g e n a t e d i n d o l e n i n e ( 7 2 ) . A s t u d y o f t h e m i n o r components o f the p r o d u c t m i x t u r e s by uv and mass s p e c t r o s c o p y f a i l e d t o show the p r e s e n c e o f p r e a k u a m m i c i n e o r stemmadenine d e r i v a t i v e s . I n E x p e r i m e n t s 8-10, v e r y complex p r o d u c t m i x t u r e s were o b t a i n e d and no i d e n t i f i a b l e components c o u l d be i s o l a t e d . The r e s u l t s f r o m a s t u d y o f t h e r e d u c t i o n o f compound (60) w i t h o t h e r m e t a l h y d r i d e s a r e shown i n T a b l e 7. I n each e x p e r i m e n t , a complex m i x t u r e o f p r o d u c t s was o b t a i n e d w i t h t h e m a j o r p r o d u c t b e i n g t h e i n d o l e n i n e ( 7 2 ) . A t no t i m e c o u l d i n d o l i c m a t e r i a l be d e t e c t e d . A n o t h e r a p p r o a c h to the r e d u c t i o n o f p r o d u c t (60) was c o n s i d e r e d . The a l d e h y d o - e s t e r was d i s s o l v e d i n ammonia and d i m e t h o x y e t h a n e a t -78° and t r e a t e d b r i e f l y w i t h l i t h i u m m e t a l . The p o s s i b l e p r o d u c t s t h a t may be e x p e c t e d under t h e s e c o n d i t i o n s w o u l d be t h e a l c o h o l ( 6 9 ) , i t s 1,2-d i h y d r o - d e r i v a t i v e o r akuammicine ( 5 ) . However, o n l y v e r y complex p r o d u c t m i x t u r e s c o u l d be o b t a i n e d . C a r e f u l e x a m i n a t i o n o f t h e s e m a t e r i a l s by uv and mass s p e c t r o s c o p y f a i l e d t o d e m o n s t r a t e t h e p r e s e n c e o f any o f t h e d e s i r e d compounds. - 1 6 1 -T a b l e 6. R e a c t i o n s o f Compound (60) w i t h NaBH. S o l v e n t T i m e ( h r ) Temp. P r o d u c t s 1 MeOH 0.5 r t (72) 2 MeOH 2 -30° (72) 3 MeOH + 10% KOH 1 r t (72) p l u s m i n o r p r o d u c t s 4 MeOH + 10% KOH 0.5 65° " _"_ 5 MeOH + 1% HoAc 1 r t II _n_ 6 MeOH + 10% HoAc 1 r t II _n_ 7 MeOH + 50% HoAc 1 r t II _n_ 8 HoAc 0.5 r t Complex m i x t u r e 9 HoAc 0.3 90° 10 THF .5 r t _n_ T a b l e 7. F u r t h e r s t u d i e s on the m e t a l h y d r i d e r e d u c t i o n o f Compound (60) Reagent S o l v e n t Temp. Time P r o d u c t s 1 KBH. MeOH r t 1 4 2 -"- MeOH+10%KOH 35° 0.5 3 NaCNBH 3 MeOH+10%HoAc r t 1 4 -"- HoAc r t 1 5 R e d - A l THF r t 0.2 (72) (72) p l u s complex m i x t u r e (72) Complex m i x t u r e (72) p l u s complex m i x t u r e -162-The f a i l u r e t o o b t a i n from p r o d u c t (60) the a l c o h o l ( 6 9 ) o f the preakuammicine s e r i e s o r a r i n g - o p e n e d stemmadenine d e r i v a t i v e by m e t a l h y d r i d e r e d u c i n g a g e n t s was a t t r i b u t e d t o a f a c i l e r e d u c t i o n -e l i m i n a t i o n p r o c e s s shown i n F i g u r e 21. I n o r d e r t o g a i n f u r t h e r i n s i g h t i n t o the c h e m i s t r y o f t h i s s t r u c t u r e i t was d e c i d e d t o p r o t e c t the a l d e h y d e group i n o r d e r t o e l i m i n a t e i t s p a r t i c i p a t i o n i n f u r t h e r a t t e m p t s t o r i n g - o p e n the m o l e c u l e . When the a l d e h y d o - e s t e r (60) was t r e a t e d w i t h ethane d i t h i o l and a c a t a l y t i c amount o f b o r o n t r i f l o u r i d e e t h e r a t e i n d i c h l o r o -methane a t 5°, a good y i e l d o f the t h i o a c e t a l d e r i v a t i v e was o b t a i n e d . T h i s m a t e r i a l was a s s i g n e d s t r u c t u r e (73) on t h e b a s i s o f the f o l l o w i n g s p e c t r a l d a t a . The uv s p e c t r u m (A 238,275,283sh) was v e r y s i m i l a r max J t o t h a t o f the s t a r t i n g m a t e r i a l . I n t h e i r s p e c t r u m , t h e r e appeared one c a r b o n y l a b s o r p t i o n bond n e a r 1710 cm 1 and an i n d o l e n i n e a b s o r p t i o n a t 1560 cm \ The pmr s p e c t r u m o f t h i s m a t e r i a l , e x h i b i t e d a 3 - p r o t o n s i n g l e t a b s o r p t i o n a t x6.09 i n d i c a t i n g the p r e s e n c e o f the c a r b o m e t h o x y l (60) S v / (73) -163-group. The t h i o a c e t a l group was e v i d e n t by t h e appearance o f a one-p r o t o n s i n g l e t a b s o r p t i o n a t T4.19 w h i c h was a t t r i b u t e d t o the C-17 p r o t o n , and a f o u r - p r o t o n m u l t i p l e t s i g n a l n e a r T6.68 was a s s i g n e d t o the m ethylene p r o t o n s o f the t h i o a c e t a l group. F u r t h e r c o n f i r m a t i o n o f t h i s s t r u c t u r e was g i v e n by i t s mass s p e c t r u m w h i c h showed a m o l e c u l a r i o n a t m/e 426. A h i g h r e s o l u t i o n mass measurement on the l a t t e r c o n f i r m e d the m o l e c u l a r f o r m u l a C„_H„,N„0 oS„. 23 26 2 2 2 A s t u d y d e s i g n e d t o a c h i e v e r i n g - o p e n i n g o f the t h i o a c e t a l d e r i v a t i v e (73) was i n i t i a t e d . When t h i s m a t e r i a l was t r e a t e d w i t h sodium b o r o h y d r i d e i n r e f l u x i n g m e t h anol f o r 48 h ours a s m a l l q u a n t i t y o f i n d o l i c ( A 228,284,291) m a t e r i a l was o b t a i n e d . The i r s p e c t r u m max i n d i c a t e d the absence o f c a r b o n y l a b s o r p t i o n s . T h i s o b s e r v a t i o n was s u p p o r t e d by the absence i n the pmr s p e c t r u m o f the c a r b o m e t h o x y l m e t h y l a b s o r p t i o n . However, p r e s e n t i n t h i s s p e c t r u m was a o n e - p r o t o n d o u b l e t a b s o r p t i o n a t x4.82 (J=8Hz) w h i c h was a t t r i b u t e d t o the C-17 p r o t o n , and a m u l t i p l e t a b s o r p t i o n n e a r x6.7 c o n f i r m e d the p r e s e n c e o f the t h i o a c e t a l m e t h y l e n e p r o t o n s . The mass s p e c t r u m o f t h i s m a t e r i a l r e v e a l e d a m o l e c u l a r i o n a t m/e 370. Based on t h e s e d a t a , t h i s r e d u c t i o n p r o d u c t was a s s i g n e d s t r u c t u r e (75) o r ( 7 6 ) . The s t e r e o c h e m i s t r y a t C - l b c o u l d a t t h i s p o i n t n o t be a s s i g n e d w i t h c e r t a i n t y . Two pathways must be c o n s i d e r e d f o r t h e c o u r s e o f t h i s r e a c t i o n and the f a i l u r e t o d e t e c t the stemmadenine t h i o a c e t a l d e r i v a t i v e ( 7 4 ) . The carbomethoxy t h i o a c e t a l (73) c o u l d undergo d e c a r b o x y l a t i o n t o g i v e the a - v i n y l i n d o l i n e (77) w h i c h would t h e n undergo r i n g - o p e n i n g t o p r o d u c t . Or, the r i n g - o p e n i n g r e a c t i o n may have p r o c e e d e d f i r s t t o g i v e the d e s i r e d m a t e r i a l (74) w h i c h c o u l d a l s o d e c a r b o x y l a t e under t h e s e r e a c t i o n c o n d i t i o n s . -164-However, a t no t i m e d u r i n g the c o u r s e o f t h i s r e a c t i o n c o u l d any i n t e r m e d i a t e p r o d u c t s be d e t e c t e d by t i c m o n i t o r i n g . I t was a l s o f o u n d t h a t t h i s same c o n v e r s i o n c o u l d be a c h i e v e d i n 2 h o u r s i n r e f l u x i n g 5% m e t h a n o l i c p o t a s s i u m h y d r o x i d e and sodium b o r o h y d r i d e . I n c o n n e c t i o n w i t h a n o t h e r a p p r o a c h to t h i s p r o b l e m some q u a n t i t i e s o f t h e a - v i n y l t h i o a c e t a l (77) were r e q u i r e d and i t s s y n t h e s i s f r o m the carbomethoxy t h i o a c e t a l (73) was a c h i e v e d by t r e a t m e n t o f the l a t t e r w i t h h o t 10% m e t h a n o l i c p o t a s s i u m h y d r o x i d e . The s u b s e q u e n t r i n g - o p e n i n g o f t h i s m a t e r i a l t o t h e i n d o l e t h i o a c e t a l (75) o r (76) was done i n r e f l u x i n g m e t n a n o l w i t h s o d i u m b o r o h y d r i d e . T h e r e f o r e , s i n c e t h i s pathway c o u l d be a c h i e v e d , t h e q u e s t i o n as to the c o u r s e o f the r e d u c t i v e r i n g - o p e n i n g o f compound (73) r e m a i n s unanswered. The above r e s u l t s may i n d i c a t e t h a t the r i n g - o p e n i n g o f t h e i n d o l e n i n e (73) i s b a s e - c a t a l y s e d b u t t h e p r e s e n c e o f h y d r o x y l i o n s may have c a u s e d d e c a r b o x y l a t i o n o f compound (73) o r i n t e r m e d i a t e ( 7 4 ) . T h e r e f o r e , i t was d e c i d e d t o a t t e m p t t h i s c o n v e r s i o n under anhydrous c o n d i t i o n s i n the p r e s e n c e o f sodium m e t h o x i d e . The carbomethoxy t h i o a c e t a l (73) was d i s s o l v e d i n a 0.2 m o l a r s o l u t i o n o f sodium m e t h o x i d e i n m e t h a n o l and t r e a t e d w i t h a n hydrous sodium b o r o h y d r i d e . A f t e r 2 h o u r s two m a j o r p r o d u c t s were d e t e c t e d by t i c . S u b s e q u e n t l y , t h e s e m a t e r i a l s were i s o l a t e d by p i c and- the f o l l o w i n g d a t a was o b t a i n e d . The l e s s p o l a r m a t e r i a l e x h i b i t e d an i n d o l i c uv s p e c t r u m (A 228,284,291) max and no c a r b o n y l a b s o r p t i o n i n t h e i r s p e c t r u m . E x c e p t f o r t h e p o s i t i o n o f one a b s o r p t i o n t h e pmr s p e c t r u m o f t h i s m a t e r i a l was v e r y s i m i l a r t o the s p e c t r u m o f p r o d u c t (75) o r (76) p r e v i o u s l y o b t a i n e d . A one -165-(75) R 1 = H, R 2 = (76) R 1 = < S S], R 2 = H F i g u r e 22 - R e a c t i o n s o f t h i o a c e t a l ( 7 3 ) . p r o t o n d o u b l e t a p p e a r e d a t x5.32 w h i c h was a s s i g n e d t o t h e C-17 p r o t o n o f t h e o t h e r C-16 e pimer o f t h e r i n g - o p e n e d t h i o a c e t a l (75) o r ( 7 6 ) . The unambiguous a s s i g n m e n t o f t h e s t e r e o c h e m i s t r y a t C-16 i s a t t h i s p o i n t t e n t a t i v e . An e x a m i n a t i o n o f m o l e c u l a r models r e v e a l e d t h e p o s s i b i l i t y t h a t when the t h i o a c e t a l group i s 3 - o r i e n t e d ( s t r u c t u r e 75) t h e C-17 p r o t o n i s i n a l e s s s h i e l d i n g e n v i r o n m e n t t h a n i f t h e t h i o a c e t a l group was a - o r i e n t e d ( s t r u c t u r e 7 6 ) . These r e s u l t s and s u b s e q u e n t work on t h i s a p p r o a c h t o a c h i e v e the s y n t h e s i s o f stemmadenine d e r i v a t i v e s made i t c l e a r t h a t d e c a r b o x y l a t i o n c o u l d n o t be p r e v e n t e d under b a s i c c o n d i t i o n s . The r i n g - o p e n i n g o f -166-t h i o a c e t a l (73) was t h e n a t t e m p t e d u n d e r v a r i o u s c o n d i t i o n s i n a c e t i c a c i d s o l v e n t . The t h i o a c e t a l (73) was t r e a t e d w i t h s odium b o r o h y d r i d e i n g l a c i a l a c e t i c a c i d a t room t e m p e r a t u r e f o r one h o u r i n one s t u d y and a t 90° f o r 15 m i n u t e s i n a s econd s t u d y . I n b o t h s t u d i e s a complex m i x t u r e o f p r o d u c t s was o b t a i n e d . O n l y s m a l l amounts o f s t a r t i n g m a t e r i a l c o u l d be i d e n t i f i e d . A uv s p e c t r o s c o p i c s t u d y o f t h e o t h e r components f r o m t h e s e r e a c t i o n s f a i l e d t o show th e p r e s e n c e o f i n d o l i c m a t e r i a l . A t h i r d s t u d y i n w h i c h the t h i o a c e t a l (73) was d i s s o l v e d i n m e t h a n o l w i t h 1%, 10% and 50% a c e t i c a c i d and t r e a t e d w i t h sodium b o r o h y d r i d e a l s o f a i l e d t o g i v e i n d o l i c m a t e r i a l . A t t h i s p o i n t t h i s a p p r o a c h was abandoned. The r e s u l t s d e s c r i b e d i n t h i s P a r t i n d i c a t e t h a t the s y n t h e s i s o f p r e a k u a m m i c i n e (8) and stemmadenine (4) d e r i v a t i v e s w i l l r e q u i r e a g r e a t d e a l o f f u r t h e r i n v e s t i g a t i o n . We b e l i e v e , however, t h a t the s y n t h e s i s o f p r e a k u a m m i c i n e a l d e h y d e (7) has been a c h i e v e d and t h a t the l a t t e r i s i d e n t i c a l w i t h p r o d u c t ( 6 0 ) . P r e a k u a m m i c i n e a l d e h y d e (7) i s a t t h i s p o i n t a h y p o t h e t i c a l i n t e r m e d i a t e i n the p r o p o s e d b i o g e n e t i c c o n v e r s 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 t o the S t r y c h n o s f a m i l y o f a l k a l o i d s , b u t may i n the f u t u r e be i s o l a t e d as a n a t u r a l p r o d u c t . F u t u r e work i n t h i s a r e a s h o u l d be d i r e c t e d toward t h r e e o b j e c t i v e s . One o b j e c t i v e i s the r e d u c t i o n of t h e a l d e h y d e group i n p r o d u c t (60) t o a c h i e v e the p reakuammicine d e r i v a t i v e (69) and compare the l a t t e r w i t h n a t u r a l l y o c c u r r i n g preakuammicine ( 8 ) . A n o t h e r o b j e c t i v e s h o u l d be the c o n v e r s i o n o f p r o d u c t (60) t o stemmadenine (4) d i r e c t l y o r v i a a l c o h o l ( 6 9 ) . The the t h i r d o b j e c t i v e t h a t s h o u l d be i n c l u d e d i n t h i s i n v e s t i g a t i o n -167-i s t he c o n v e r s i o n o f stemmadenine (4) t o iso-stemmadenine ( 1 0 ) . M i g r a t i o n o f the d o u b l e bond from p o s i t i o n C-19,20 t o p o s i t i o n C-20,21 has a l r e a d y been a c h i e v e d i n the S t r y c h n o s a r e a u s i n g Raney n i c k e l . The e v a l u a t i o n o f the b i o s y n t h e s i s and the b i o m i m e t i c c h e m i s t r y o f iso-stemmadenine (10) would be o f g r e a t i m p o r t a n c e t o a more complete u n d e r s t a n d i n g o f t h i s f a s c i n a t i n g a r e a o f n a t u r a l p r o d u c t s . -168-EXPERIMENTAL T h i n l a y e r c h r o m a t o g r a p h i c ( t i c ) s t u d i e s were c a r r i e d o u t u s i n g EM R e a g e n t s GF254 S i l i c a g e l o r Woelm n e u t r a l a l u m i n a . The c h r o m a t o p l a t e s used f o r a n a l y s i s were 0.3 mm i n t h i c k n e s s and t h o s e u sed f o r p r e p a r a t i v e s e p a r a t i o n s were 1.0 mm t h i c k . I n a l l c a s e s , e l e c t r o n i c p hosphor (about 2% by w e i g h t ) was added t o t h e a b s o r b e n t t o a i d v i s u a l i z a t i o n . The c h r o m a t o p l a t e s were d e v e l o p e d i n one o f the f o l l o w i n g s o l v e n t s y s t e m s : e l u e n t A c o n s i s t e d o f c h l o r o f o r m p l u s 1% m e t h a n o l , e l u e n t B c o n s i s t e d o f c h l o r o f o r m p l u s 2% m e t h a n o l , and e l u e n t C c o n s i s t e d o f d i c h l o r o m e t h a n e p l u s 10% m e t h a n o l and 0.1% t r i e t h y l a m i n e . The c h r o m a t o p l a t e s were examined under a l o n g and s h o r t w a v e l e n g t h u l t r a v i o l e t s c a n n i n g lamp and v i s u a l i z e d f u r t h e r w i t h a e e r i e s u l p h a t e s p r a y o r i o d i n e v a p o r s . Column chromatography was p e r f o r m e d u s i n g S h a w i n i g a n n e u t r a l a l u m i n a i n l a r g e s c a l e p r e p a r a t i o n s and Woelm n e u t r a l a l u m i n a when more e x a c t s e p a r a t i o n s were r e q u i r e d . The d i m e n s i o n s o f t h e columns were g e n e r a l l y m a i n t a i n e d a t the a c c e p t e d optimum r a t i o o f d i a m e t e r t o h e i g h t as 1:10. Throughout t h i s work, a l l s o l v e n t s were d i s t i l l e d b e f o r e u s e . U l t r a v i o l e t s p e c t r a were r e c o r d e d i n m e t h a n o l on a Car y 15 s p e c t r o p h o t o m e t e r . A b s o r p t i o n v a l u e s (A ) a r e g i v e n i n nanometers max -(nm). I n f r a r e d s p e c t r a were measured i n c h l o r o f o r m on a P e r k i n - E l m e r model 137 d o u b l e beam i n s t r u m e n t , and a b s o r p t i o n bands ( v ) a r e max quoted i n wavenumbers (cm "*") . -169-P r o t o n m a g n e t i c r e s o n a n c e (pmr) s p e c t r a were r e c o r d e d i n d e u t e r o c h l o r o f o r m a t room t e m p e r a t u r e a t 100 MH^ on a V a r i a n HA-100 o r a V a r i a n XL-100 s p e c t r o m e t e r . The l a t t e r was a l s o used t o r e c o r d s p e c t r a i n the F o u r i e r t r a n s f o r m mode. C h e m i c a l s h i f t s a r e g i v e n i n the T i e r ' s T s c a l e u s i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d a t TIO.OO. C o u p l i n g c o n s t a n t s ( J - v a l u e s ) a r e g i v e n i n h e r t z ( H z ) . T - v a l u e s g i v e n t o m u l t i p l e t s i g n a l s r e f e r t o the c e n t e r o f t h e s i g n a l . Mass s p e c t r a were r e c o r d e d on an AEI-MS-902 o r an A t l a s CH-43 mass s p e c t r o m e t e r . F r a g m e n t a t i o n d a t a i s g i v e n i n mass to c h a r g e r a t i o s (m/e) f o l l o w e d by p e r c e n t r e l a t i v e abundance. H i g h r e s o l u t i o n mass measurements were d e t e r m i n e d on the AEI-MS-902 o r an AEI-MS-50 u s i n g s u i t a b l e s t a n d a r d s . M e l t i n g p o i n t s were d e t e r m i n e d on a K o f l e r b l o c k and a r e u n c o r r e c t e d . E l e m e n t a l a n a l y s e s were p e r f o r m e d by Mr. P. B o r d a o f the M i c r o a n a l y t i c a l L a b o r a t o r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . The C o n v e r s i o n o f S t r y c h n i n e (29) t o W i e l a n d - G u m l i c h A l d e h y d e (32) The c o n v e r s i o n o f s t r y c h n i n e (29) t o W i e l a n d - G u m l i c h a l d e h y d e 21-23 20 has been d e s c r i b e d i n d e t a i l and s i n c e an a u t h e n t i c sample o f t h e l a t t e r was a v a i l a b l e no d a t a need be g i v e n h e r e f o r t h e s e i n t e r -m e d i a t e s . The p r o c e d u r e s used a r e a p p r o p r i a t e l y r e f e r e n c e d and d i f f e r l i t t l e f r o m t h o s e p u b l i s h e d . 2 3 - 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 (30) A t h r e e - n e c k e d r o u n d b o t t o m f l a s k e q u i p p e d w i t h a thermometer -170-and a d r y i c e - a c e t o n e c o n d e n s e r was c h a r g e d w i t h s t r y c h n i n e (29) ( 5 0 g , d r i e d o v e r ^2^5^ ^ n a D S ° l u t e e t h a n o l (800 m l ) . I s o - p e n t y l n i t r i t e (98g, f r e s h l y d i s t i l l e d u nder r e d u c e d p r e s s u r e ) was added. W h i l e b e i n g s t i r r e d m e c h a n i c a l l y t h e t e m p e r a t u r e o f t h e r e a c t i o n m i x t u r e was r a i s e d t o , and m a i n t a i n e d a t , 70° d u r i n g t h e a d d i t i o n o f sodium m e t a l (16g) d i s s o l v e d i n e t h a n o l (500 m l ) . S t i r r i n g a t 70° was c o n t i n u e d f o r 4 h o u r s . The r e s u l t i n g d a r k brown s o l u t i o n was a l l o w e d t o c o o l and s t i r r i n g was c o n t i n u e d o v e r n i g h t . The s o l u t i o n was e v a p o r a t e d i n v a c u o t r e a t e d w i t h c o l d w a t e r (100 ml) and e v a p o r a t e d a g a i n to remove u n r e a c t e d i s o - p e n t y l n i t r i t e . The r e d u c e d b u l k was d i s s o l v e d i n w a t e r ( 5 0 0 ) , g l a c i a l a c e t i c a c i d (100) and cone, h y d r o c h l o r i c a c i d ( 4 0 ) . To t h i s s o l u t i o n was added N o r i t (3g) and the m i x t u r e was b r o u g h t to a b o i l and f i l t e r . A f t e r s t a n d i n g a t 5° o v e r n i g h t a c r y s t a l l i n e m a t e r i a l was o b t a i n e d by vacuum f i l t r a t i o n ( 4 3 . 5 g ) . A s m a l l sample was r e c r y s t a l l i z e d f r o m m e t h a n o l , 23 m.p. 218° ( d e c o m p ) ( l i t . m.p. 220°, decomp). N ( a ) - C y a n o f o r m y l W i e l a n d - G u m l i c h A l d e h y d e H y d r o c h l o r i d e (32) A f t e r d r y i n g t h o r o u g h l y i n v acuo the above s a l t (40g) was s l o w l y added t o t h i o n y l c h l o r i d e (120 ml) p r e v i o u s l y c o o l e d i n an i c e - w a t e r b a t h . T h i s s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e . A f t e r 4 h o u r s , t h e s o l u t i o n was c a u t i o u s l y p o u r e d o n t o i c e , w i t h v i g o r o u s s t i r r i n g , t o y i e l d a g r a y p r e c i p i t a t e . C r y s t a l l i z a t i o n o f t h i s m a t e r i a l o c c u r r e d a f t e r 30 m i n u t e s and i t was t h e n f i l t e r e d , washed w i t h c o l d w a t e r and d r i e d i n vacuo to g i v e a h y d r o c h l o r i d e s a l t ( 3 1 g ) . R e c r y s t a l -l i z a t i o n f r o m m e t h a n o l y i e l d e d c o l o u r l e s s m a t e r i a l , m.p. 220° (decomp) - 1 7 1 -23 ( l i t . m.p. 220°, decomp). W i e l a n d - G u m l i c h A l d e h y d e ( 3 2 ) 2 3 The c r u d e s a l t (29g) f r o m the above r e a c t i o n was added t o a two-necked f l a s k c h a r g e d w i t h d i s t i l l e d w a t e r (400 m l ) . T h i s m i x t u r e was h e a t e d i n an o i l b a t h (120°). A f t e r b o i l i n g began, steam was r a p i d l y i n j e c t e d i n t o t he b o i l i n g m i x t u r e f o r 1-2 h o u r s . The p r o g r e s s o f the r e a c t i o n was m o n i t o r e d by w o r k i n g up s m a l l a l i q u o t s w h i c h were examined by t i c on a l u m i n a ( e l u e n t B ) . When t h e h y d r o l y s i s a p p e a r e d t o be c o m p l e t e , the m i x t u r e was c o o l e d and made a l k a l i n e w i t h c o l d ammonium h y d r o x i d e . R e p e a t e d e x t r a c t i o n w i t h c h l o r o f o r m and e v a p o r a t i o n i n vacuo y i e l d e d a brown r e s i d u e ( 1 9 g ) . T h i s m a t e r i a l was chrom a t o g r a p h e d on a l u m i n a (500g, a c t . I l l ) u s i n g 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 . P o o l i n g o f t h e a p p r o p r i a t e f r a c t i o n s as d e t e r m i n e d by t i c y i e l d e d a c o l o u r l e s s c r y s t a l l i n e m a t e r i a l (17g) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h an 20 v'' a u t h e n t i c sample o f W i e l a n d - G u m l i c h a l d e h y d e . R e c r y s t a l i z a t i o n f r o m m e t h a n o l - a c e t o n e y i e l d e d c o l o u r l e s s n e e d l e s , m.p. 212-215° ( l i t . m.p. 212.5-215°). 2 3 2g,16a - c u r - 1 9 - e n - 1 7 , 1 8 - d i o l ( 3 3 ) 2 2 W i e l a n d - G u m l i c h a l d e h y d e (5g) was d i s s o l v e d i n m e t h a n o l (50 ml) and sodium b o r o h y d r i d e (0.5g) was added o v e r 30 m i n u t e s a t room t e m p e r a t u r e . The s o l u t i o n was s t i r r e d f o r a f u r t h e r 30 m i n u t e s a f t e r w h i c h t i m e w a t e r (10 ml) was added and s t i r r i n g c o n t i n u e d f o r a f u r t h e r 15 m i n u t e s . T h i s -172-s o l u t i o n was t h e n e v a p o r a t e d i n vacuo and t h e r e s i d u e was t a k e n up i n c h l o r o f o r m c o n t a i n i n g 10% m e t h a n o l and chromatographed on a l u m i n a (lOOg, a c t . I I I ) . E l u t i o n w i t h e t h y l a c e t a t e y i e l d e d a m a t e r i a l (4.1g) w h i c h was r e c r y s t a l l i z e d f r o m m e t h a n o l - w a t e r t o g i v e c o l o u r l e s s c r y s t a l s . A f t e r d r y i n g t h o r o u g h l y i n v a c u o , t h i s compound had m.p. 250-254° ( l i t . m.p. 2 5 1 - 2 5 3 ) 2 2 ; X 243, 297; v 3600 (-0H), max max 3300 (0NH), 1600 ( i n d o l i n e ) ; pmr s i g n a l s : 2.9-3.4 ( m u l t i p l e t s , 4H, a r o m a t i c O H ) , 4.27 ( t r i p l e t , J=7, I H , C-19H), 5.88 ( m u l t i p l e t , J=2 and J=7; 2H, C - 1 8 H 2 ) ; mass s p e c t r u m : M + 312 ( 8 2 ) , 182 ( 4 3 ) , 144 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r ^^9^24^2°2 : 312•1837. Found: 312.1841. 22 2 g - 1 6 a - 1 7 - a c e t o x y - c u r - 1 9 - e n (35) W i e l a n d - G u m l i c h g l y c o l ( 3 3 , 3.6g) was added t o a s o l u t i o n o f h y d r o g e n b r o m i d e i n g l a c i a l a c e t i c a c i d -(250 m l , 3% s a t u r a t e d ) . The f l a s k was s e a l e d and l e t s t a n d a t room t e m p e r a t u r e f o r 2 d a y s . Most o f t h e s o l v e n t was t h e n removed _in vacuo and more g l a c i a l a c e t i c a c i d (400 ml) was added. F r e s h l y a c t i v a t e d z i n c powder (50g) was added and t h e m i x t u r e was shaken v i g o r o u s l y f o r 5 h o u r s . The z i n c was removed by f i l t r a t i o n and washed w i t h a c e t i c a c i d and t h e n e t h a n o l . These w a s h i n g s were combined w i t h t h e f i l t r a t e and r e d u c e d i n volume i n v a c u o . The r e s i d u e was d i s s o l v e d i n d i l u t e ammonium h y d r o x i d e and e x t r a c t e d w i t h c h l o r o f o r m , d r i e d o v e r sodium s u l p h a t e and e v a p o r a t e d t o * Powdered z i n c was a c t i v a t e d by w a s h i n g t w i c e w i t h 5% aqueous h y d r o c h l o r i c a c i d , f o l l o w e d by s u c c e s s i v e w a s h i n g s w i t h m e t h a n o l and e t h e r , and t h e n d r i e d i n v a c u o . -173-g i v e a l i g h t brown gum ( 1 . 2 g ) . E x a m i n a t i o n o f t h i s m a t e r i a l by t i c on a l u m i n a ( e l u e n t B) r e v e a l e d t h e p r e s e n c e o f one m a j o r component, v 1720 ( a c e t a t e ) . T h i s m a t e r i a l was used as s u c h i n the n e x t max r e a c t i o n . 2 g , 1 6 a - C u r - 1 9 - e n - 1 7 - o l ( 3 6 ) 2 2 The above a c e t a t e ( l g ) was r e f l u x e d i n m e t h a n o l i c p o t a s s i u m h y d r o x i d e (10%) f o r 1 h o u r . W i t h c o o l i n g , t h i s r e a c t i o n m i x t u r e was n e u t r a l i z e d w i t h d i l u t e h y d r o c h l o r i c a c i d , r e d u c e d i n volume and e x t r a c t e d w i t h c h l o r o f o r m . The combined o r g a n i c w a s h i n g s were d r i e d w i t h a nhydrous sodium s u l p h a t e and e v a p o r a t e d jLn vacuo t o g i v e a brown amorphous m a t e r i a l (910 mg). T h i s m a t e r i a l was c h r o m a t o g r a p h e d on a l u m i n a ( a c t . I V ) . E l u t i o n w i t h 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 y i e l d e d a p u r e compound (850 mg) w h i c h c o u l d be c r y s t a l l i z e d f r o m benzene, m.p. 172-175° ( l i t . m.p. 1 7 7 - 1 7 8 0 ) 2 6 ; X' 243, 297, v 3575(N-H), 3350 ( 0 - H ) , 1 6 0 0 ( i n d o l i n e ) ; max max pmr s i g n a l s : 2.8-3.5 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.58 ( q u a r t e t , J=7, I H , C-19H), 8.50 ( m u l t i p l e t , J=7 and 3=2, C-18CH 3); mass s p e c t r u m : M + 2 9 6 ( 8 9 ) , 2 8 1 ( 1 7 ) , 2 7 9 ( 1 5 ) , 2 6 5 ( 1 2 ) , 1 6 6 ( 1 0 0 ) , 1 4 4 ( 4 3 ) , 1 3 0 ( 3 1 ) ; h i g h r e s o l u t i o n mass measurement; C a l c . f o r C^gH^^N^O: 296.1887, Found: 296.1888. C a t a l y t i c H y d r o g e n o l y s i s o f W i e l a n d - G u m l i c h G l y c o l (33) The g l y c o l (300 mg) was d i s s o l v e d i n a s o l u t i o n o f w a t e r (20 m l ) , a c e t i c a c i d (12 ml) and h y d r o c h l o r i c a c i d (0.2 m l ) . A f t e r a d d i t i o n o f -174-p a l l a d i u m - c h a r c o a l c a t a l y s t ( 1 0 % , 75 mg) t h e r e a c t i o n m i x t u r e was f l u s h e d w i t h h y d r o g e n and l e t s t i r i n a s l i g h t l y p o s i t i v e p r e s s u r e h y d r o g e n a t m o s p h e r e . The p r o g r e s s o f the r e a c t i o n was m o n i t o r e d by volume o f gas u p - t a k e . The r e a c t i o n m i x t u r e was t h e n f i l t e r e d , c o o l e d i n an i c e - w a t e r b a t h and made a l k a l i n e w i t h aqueous p o t a s s i u m h y d r o x i d e r e s u l t i n g i n w h i t e p r e c i p i t a t e . The t o t a l m i x t u r e was e x t r a c t e d r e p e a t e d l y w i t h c h l o r o f o r m (500 m l ) . The o r g a n i c w a s h i n g s were combined, d r i e d w i t h a n h ydrous sodium s u l p h a t e and e v a p o r a t e d _in vacuo t o g i v e a l i g h t y e l l o w foam (285 mg). Chromatography o f t h i s p r o d u c t on a l u m i n a gave a compound (270 mg) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h 2 3 , 1 6 a - c u r - 1 9 - e n - 1 7 - o l (36) w h i c h had p r e v i o u s l y been o b t a i n e d . B i r c h R e d u c t i o n o f W i e l a n d - G u m l i c h A l d e h y d e (32) The a l d e h y d e ( l g ) was suspended i n d r y d i m e t h o x y e t h a n e (75 ml) and d r y _ t - b u t a n o l (0.5 ml) i n a round b o t t o m f l a s k e q u i p p e d w i t h a d r y i c e - a c e t o n e c o n d e n s e r , a s o u r c e o f ammonia gas and a m a g n e t i c s t i r r i n g b a r . T h i s m i x t u r e was c o o l e d i n a d r y i c e - a c e t o n e b a t h (-78°) and t h e n f i l l e d w i t h ammonia (100 m l ) . F r e s h l y c u t c h i p s o f l i t h i u m m e t a l were added u n t i l a d a r k b l u e c o l o u r c o u l d be m a i n t a i n e d f o r 30 m i n u t e s . Ammonium c h l o r i d e was t h e n added t o d e s t r o y the e x c e s s l i t h i u m ( i . e . b l u e c o l o u r was removed). The r e a c t i o n m i x t u r e was t h e n a l l o w e d to warm as the ammonia e v a p o r a t e d . The r e s i d u e was e v a p o r a t e d f u r t h e r i n vacuo b e f o r e p a r t i t i o n i n g between w a t e r and c h l o r o f o r m . The combined c h l o r o f o r m w a s h i n g s were -175-d r i e d w i t h a n h y d r o u s s o d i u m s u l p h a t e and e v a p o r a t e d i n v a c u o t o g i v e a l i g h t y e l l o w foam (915 mg). T h i s m a t e r i a l was p a s s e d t h r o u g h a column o f a l u m i n a (20g, a c t . I l l ) w i t h a b e n z e n e - e t h y l a c e t a t e s o l v e n t g r a d i e n t t o y i e l d a compound (890 mg) w h i c h had p h y s i c a l and s p e c t r o -s c o p i c p r o p e r t i e s i d e n t i c a l w i t h 2 6 , 1 6 a - c u r - 1 9 - e n - 1 7 - o l (36) w h i c h had been p r e v i o u s l y o b t a i n e d . B i r c h R e d u c t i o n o f W i e l a n d - G u m l i c h G l y c o l (33) The g l y c o l (150 mg) was h y d r o g e n o l y s e d under t h e same c o n d i t i o n s d e s c r i b e d above f o r W i e l a n d - G u m l i c h a l d e h y d e ( 3 2 ) . A f t e r c h r o m a t o g r a p h y , a compound (121 mg) was o b t a i n e d w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 - o l (36) w h i c h had been p r e v i o u s l y o b t a i n e d . -176-Oppenauer O x i d a t i o n o f 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 - o l (36) by B o e k e l h e i d e ' s  P r o c e d u r e The a l c o h o l (36) (300 mg) was d i s s o l v e d i n r e f l u x i n g benzene (100 m l , f r e s h l y d i s t i l l e d f r o m l i t h i u m aluminum h y d r i d e ) w i t h benzophenone (910 mg, s u b l i m e d ) . P o t a s s i u m _ t - b u t o x i d e (560 mg) was added and t h e r e a c t i o n was s t i r r e d f o r 2 h o u r s under an i n h e r t a t mosphere. A f t e r c o o l i n g i n an i c e - b a t h , w a t e r (100 ml) was added and t h e m i x t u r e was p a r t i t i o n e d . The aqueous l a y e r was e x t r a c t e d t h r e e t i m e s w i t h d i c h l o r o m e t h a n e (250 ml) and t h e combined o r g a n i c e x t r a c t s were washed w i t h w a t e r b e f o r e d r y i n g o v e r a nhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f the s o l v e n t , t h e r e s i d u e was chromatographed on a l u m i n a (30 g, a c t . I l l ) u s i n g a p e t r o l e u m e t h e r - b e n z e n e s o l v e n t g r a d i e n t . P o o l i n g o f t h e a p p r o p r i a t e f r a c t i o n s y i e l d e d t h e m a j o r p r o d u c t (118 mg) as a y e l l o w foam. T h i s m a t e r i a l was i d e n t i f i e d as n o r - f l u o r o c u r a r i n e (39) bas e d on t h e f o l l o w i n g d a t a : m.p. 182-185° ( l i t . m.p. 1 8 5 - 1 8 6 0 ) 2 6 ; X 244 290 s h , 298, 363; v 3300 (N-H), max max 1640 (CHO), 1605 ( i n d o l i n e ) ; pmr: -0.2 ( b r o a d s i n g l e t , I H , N-H), 0.70 ( s i n g l e t , I H , CHO), 2.7 - 3.2 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.68 ( q u a r t e t , J=7, I H , =CH-CH 3), 8.48 ( d o u b l e t o f d o u b l e t s , J=7 and 2, 3H, =CH-CH 3); mass s p e c t r u m : M + 292 ( 6 3 ) , 263 ( 1 5 ) , 249 ( 2 1 ) , 243 ( 2 6 ) , 222 ( 1 7 ) , 208 ( 1 5 ) , 194 ( 1 8 ) , 167 ( 2 2 ) , 121 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C ^ H ^ ^ O : 292.1575. Found: 292.1567. A m i n o r p r o d u c t (14 mg) was l a t e r i d e n t i f i e d as 23,16a-cur-19-en-1 7 - a l ( 3 7 ) . -177-M o d i f i e d Oppenauer O x i d a t i o n o f 2g, 1 6 a - c u r - 1 9 - e n - 1 7 - o l (36) t o g i v e  2 g , 1 6 c t - c u r - 1 9 - e n - 1 7 - a l (37) The a l c o h o l ( 3 6 ) ( 1 4 5 mg) was d i s s o l v e d w i t h benzophenone (270 mg) i n benzene (50 m l , f r e s h l y d i s t i l l e d f r o m l i t h i u m aluminum h y d r i d e ) . A p p r o x i m a t e l y 5 ml o f benzene were t h e n d i s t i l l e d f r o m t h e r e a c t i o n m i x t u r e b e f o r e p o t a s s i u m h y d r i d e (200 mg o f a 22% o i l s u s p e n s i o n ) was added. T h i s m i x t u r e was r e f l u x e d f o r 1 h o u r . A f t e r c o o l i n g i n an i c e -w a t e r b a t h , w a t e r (50 ml) was added and t h e m i x t u r e was p a r t i t i o n e d . The aqueous 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 and t h e combined o r g a n i c e x t r a c t s were washed w i t h w a t e r and d r i e d o v e r an h y d r o u s sodium s u l p h a t e . A f t e r r e m o v a l o f most o f t h e s o l v e n t i n v a c u o , t h e r e s i d u e was c h romatographed on a l u m i n a (8 g, a c t . I l l ) u s i n g a p e t r o l e u m e t h e r - b e n z e n e s o l v e n t g r a d i e n t . The m a j o r p r o d u c t (105 mg) o b t a i n e d by p o o l i n g the a p p r o p r i a t e f r a c t i o n s was i d e n t i f i e d a s 2 3 , 1 6 a - c u r - 1 9 - e n - 1 7 - a l (37) b a s e d on t h e f o l l o w i n g d a t a , X 247, 297; v 3450 (N-H), 1720 (CHO), 1600 max max ( i n d o l i n e ) ; pmr: 0.24 ( s i n g l e t , I H , CHO), 2.8 - 3.5 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.60 ( q u a r t e t , J=7, I H , =CH-CH 3), 8.40 ( d o u b l e t o f d o u b l e t s , J=7 and 2, 3H, =CH-CH 3); mass s p e c t r u m : M + 294 ( 4 2 ) , 251 ( 1 6 ) , 199 ( 1 5 ) , 164 ( 9 7 ) , 144 ( 1 0 0 ) , h i g h r e s o l u t i o n mass measurement: C a l c . f o r ' ^ i g ^ 2 2 ^ 2 0: 294.1731. Found: 294.1747. T h i s m a t e r i a l was compared s p e c t r o s c o p i c a l l y w i t h t h a t p r e p a r e d 24 by o t h e r s i n o u r l a b o r a t o r y f o r a n o t h e r s t u d y . M o d i f i e d Oppenauer O x i d a t i o n o f 2 g , 1 6 a - c u r - 1 9 - e r i - 1 7 - o l (36) t o g i v e  N o r - f l u o r o c u r a r i n e (39) The a l c o h o l ( 3 6 ) ( 2 9 5 mg) was d i s s o l v e d i n benzene (70 m l , f r e s h l y -178-d l s t i l l e d from l i t h i u m aluminum h y d r i d e ) and about 7 ml o f benzene were d i s t i l l e d from the r e a c t i o n m i x t u r e . A f t e r the temperature of the o i l b a t h had been lower e d to 60-65°, p o t a s s i u m h y d r i d e (350 mg o f a 22% o i l s u s p e n s i o n ) was added. S t i r r i n g i n an i n h e r t atmosphere was c o n t i n u e d f o r 10 m i n u t e s . N i t r o b e n z e n e (0.45 ml) was added dr o p w i s e o v p r 15 minutes and s t i r r i n g was c o n t i n u e d f o r a f u r t h e r 15 m i n u t e s . The r e a c t i o n v e s s e l was then c o o l e d i n an i c e - w a t e r b a t h and water (50 ml) was added. T h i s m i x t u r e was p a r t i t i o n e d and the aqueous l a y e r was e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e (250 m l ) . The combined o r g a n i c e x t r a c t s were d r i e d o v e r anhydrous sodium s u l p h a t e and most o f the s o l v e n t was removed i n vacuo. The h i g h l y c o l o u r e d r e s i d u e was chromatographed on a l u m i n a (30 g, a c t . I l l ) u s i n g a p e t r o l e u m e t h e r - b e n z e n e s o l v e n t g r a d i e n t . The a p p r o p r i a t e f r a c t i o n s were p o o l e d to y i e l d y e l l o w foam (179 mg) which c r y s t a l l i z e d from benzene. T h i s m a t e r i a l had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h those o f n o r - f l u o r o c u r a r i n e (39) p r e v i o u s l y s y n t h e s i z e d . -179-l-Formyl-2B-16a-cur-19-en-17-0-formate (42) The indoline alcohol (36)(900 mg) was dissolved in acetic-29 formic anhydride (10 ml) and stirred at room temperature for 15 minutes. The solvent was then removed in vacuo at ambient temperature. The residue was chromatographed on alumina (20 g, a c t . I l l ) using benzene as the eluent to yiel d , after removal of solvent, a light yellow foam (940 mg), A 247, 282, 287; v 1720 (0-CHO), 1660 max max (N-CHO), 1600 (indoline), pmr: 1.51 (singlet, IH, N -CHO), 2.00 (singlet, IH, 0-CH0), 4.4 (quartet, J=7, IH, =CH-CH3) 8.40 (doublet of doublets, J=7 and 2, 3H, =CH-CH3); mass spectrum: M + 352 (81), 337(37) 324 (49), 279 (100), 144 (81). l-Formyl-2g-16a-cur-19-en-17-ol (43) The diformate (42)(940 mg) was refluxed in methanol (15 ml) for 30 minutes. After the removal of the solvent in_ vacuo, a t i c examination (alumina, eluent A) revealed the presence of a single component. This material was chromatographed on alumina (20 g, a c t . I l l ) to yield a light yellow foam (780 mg), A 247, 282, 287, v 1660 (N-CHO), max max 1600 (indoline); pmr: 1.24 (singlet, IH, N-CHO), 4.48 (quartet, J=7, IH, =CH-CH3), 8.34 (doublet of doublets, J=7 and 2, 3H, =CH-CH3); mass spectrum: M + 324 (77), 309 (49), 293 (31), 279 (100); high resolution mass measurement: Calc. for C_ nH 0.N„0_ 324.1837. Found: 324.1826. 20 24 2 2 Oxidation of N-formyl Alcohol (43) with Collin's Reagent 20 Collin's reagent was prepared in situ in dichloromethane (30 ml, freshly d i s t i l l e d from using chromium trioxide (200 mg, -180-d r i e d o v e r n i g h t i n h i g h vacuum a t 100°) and p y r i d i n e (0.4 m l , d i s t i l l e d f r o m KOH) a t 0°. To t h i s s o l u t i o n was added t h e a l c o h o l ( 4 3 ) ( 1 0 0 mg). A f t e r s t i r r i n g f o r 45 m i n u t e s , s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n (30 ml) was added and t h e r e s u l t i n g m i x t u r e was s t i r r e d f o r a f u r t h e r 30 m i n u t e s b e f o r e p a r t i t i o n i n g . A f t e r r e p e a t e d e x t r a c t i o n o f t h e aqueous l a y e r w i t h d i c h l o r o m e t h a n e , t h e o r g a n i c e x t r a c t was washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . The d a r k c o l o u r e d r e s i d u e o b t a i n e d a f t e r r e m o v a l o f t h e s o l v e n t i n v a c u o was chroma t o g r a p h e d on a l u m i n a (5 g, a c t . I I I ) . The a p p r o p r i a t e benzene f r a c t i o n s were combined t o y i e l d a l i g h t y e l l o w amorphous m a t e r i a l (39 mg), w h i c h was a s s i g n e d s t r u c t u r e (44) base d on the f o l l o w i n g d a t a : X 242, 282, 288; v 1720 (C-CH0), 1665 (N -CHO), 1590 ( i n d o l i n e ) ; max max — pmr: 0.22 ( d o u b l e t , J=4, 0.5H, C-CHO), 0.37 ( s i n g l e t , 0.5H, C-CHO), 1.14 ( s i n g l e t , 0.5H, N-CH0) 1.40 ( s i n g l e t , 0.5H, N-CHO), 2.0 ( m u l t i p l e t , I H , C-12N), 4.46 ( m u l t i p l e t , I H , =CH-CH 3), 5.18 ( m u l t i p l e t , I H , C-16H), 8.45 ( m u l t i p l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 322 ( 4 1 ) , 293 ( 5 0 ) , 279 ( 7 4 ) , 180 ( 4 4 ) , 164 (100) h i g h r e s o l u t i o n mass measurement: C a l c . f o r C 2 Q H 2 2 N 2 0 2 322.1680. Found: 322.1678. O x i d a t i o n o f N - f o r m y l A l c o h o l (43) w i t h S i l v e r C a r b o n a t e S i l v e r c a r b o n a t e on c e l i t e was p r e p a r e d a c c o r d i n g t o F e t i z o n e ' s 36 p r o c e d u r e . T h i s r e a g e n t (0.4 mmoles) was r e f l u x e d w i t h t h e N - f o r m y l a l c o h o l (43) (96 mg, 0.3 mmoles) i n benzene o v e r n i g h t . A f t e r f i l t r a t i o n and r e m o v a l o f t h e s o l v e n t t h e r e s i d u e was examined by t i c ( a l u m i n a , e l u e n t A) and fo u n d t o c o n t a i n a m i x t u r e o f components. T h i s m i x t u r e (95 mg) was chromatographed on a l u m i n a (6.5 g, a c t . I l l ) u s i n g a benzene-- 1 8 1 -e t h y l a c e t a t e s o l v e n t g r a d i e n t . P o o l i n g o f t h e a p p r o p r i a t e c h r o m a t o g r a p h i c f r a c t i o n s y i e l d e d a l i g h t y e l l o w amorphous m a t e r i a l (24 mg), w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h o s e o f t h e N - f o r m y l a l d e h y d e (44) w h i c h had p r e v i o u s l y been c h a r a c t e r i z e d . A second component (10 mg) was i d e n t i f i e d as s t a r t i n g m a t e r i a l ( 4 3 ) . O x i d a t i o n o f N - f o r m y l A l c o h o l (43) w i t h DMS0-S0 o 3. -5 The a l c o h o l ( 4 3 ) ( 1 0 0 mg) was d i s s o l v e d i n d r y d i m e t h y l s u l p h o x i d e o (1 m l , f r e s h l y d i s t i l l e d f r o m 4A m o l e c u l a r s i e v e s ) and t r e a t e d w i t h p y r i d i n e - s u l p h u r t r i o x i d e complex (300 mg) and t r i e t h y l a m i n e ( 0 . 1 m l ) . A f t e r s t i r r i n g a t room t e m p e r a t u r e f o r 1 h o u r , 20 volumes o f w a t e r were added t o t h e r e a c t i o n m i x t u r e b e f o r e i t was e x t r a c t e d w i t h benzene. The o r g a n i c w a s h i n g s were d r i e d o v e r anhydro'us sodium s u l p h a t e and e v a p o r a t e d t o y i e l d a c o l o u r l e s s amorphous m a t e r i a l . Chromatography o f t h i s m a t e r i a l y i e l d e d an amorphous compound (18 mg) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h e N - f o r m y l a l d e h y d e (44) 3. p r e v i o u s l y c h a r a c t e r i s e d . A second component (62 mg) f r o m t h i s p r o d u c t m i x t u r e had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h s t a r t i n g m a t e r i a l ( 4 3 ) . O x i d a t i o n o f N ^ - f o r m y l A l c o h o l (43) w i t h 3 , 5 - D i m e t h y l p y r a z o l e - c h r o m i u m  T r i o x i d e Complex The a l c o h o l ( 4 3 ) ( 9 0 mg, 0.3 mmoles) was added t o a s t i r r e d s o l u t i o n v. -182-o f 3 , 5 - d i m e t h y l p y r a z o l e (60 mg, 0.6 mmoles) and c a r e f u l l y d r i e d chromium t r i o x i d e (65 mg) i n d i c h l o r o m e t h a n e (25 m l , f r e s h l y d i s t i l l e d f r o m ^2^5^ * '^ i e r e s u l t i n g brown s u s p e n s i o n was s t i r r e d a t room t e m p e r a t u r e f o r 1 h o u r . S a t u r a t e d aqueous sodium b i c a r b o n a t e was added t o t h e m i x t u r e and t h e l a t t 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 (100 m l ) . The o r g a n i c e x t r a c t was washed w i t h w a t e r and d r i e d o v e r a n hydrous s o d i u m s u l p h a t e and e v a p o r a t e d i n v a c u o . The r e s u l t i n g r e s i d u e was examined by t i c ( a l u m i n a , e l u e n t A) and f o u n d t o c o n t a i n t h r e e m a j o r components w h i c h a p p e a r e d to be d i m e t h y l p y r a z o l e , s t a r t i n g m a t e r i a l and t h e d e s i r e d a l d e h y d e . Subsequent c h r o m a t o g r a p h y on a l u m i n a and s i l i c a g e l y i e l d e d o n l y samples o f p u r e m a t e r i a l s w h i c h had s p e c t r o -s c o p i c p r o p e r t i e s i d e n t i c a l w i t h the N - f o r m y l a l d e h y d e (44) and t h e s t a r t i n g m a t e r i a l ( 4 3 ) . O x i d a t i o n o f N ^ - f o r m y l A l c o h o l (43) w i t h P y r i d i n i u m C h l o r o c h r o m a t e The N - f o r m y l a l c o h o l ( 4 3 ) ( 3 0 5 mg) was d i s s o l v e d i n d i c h l o r o -cL methane (60 m l , f r e s h l y d i s t i l l e d f r o m a n c * t r e a t e a w i t h p y r i d i n i u m c h l o r o c h r o m a t e (480 mg) w h i c h had been p r e p a r e d a c c o r d i n g to C o r e y ' s 38 p r o c e d u r e . T h i s c o l o u r e d s u s p e n s i o n was s t i r r e d a t room t e m p e r a t u r e f o r 2 h o u r s , a f t e r w h i c h t i m e , an e q u a l volume o f s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n was added and t h e m i x t u r e was s t i r r e d f o r a f u r t h e r 15 m i n u t e s . T h i s m i x t u r e was t h e n e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e (.250 ml) . The o r g a n i c e x t r a c t was w i t h aqueous sodium b i c a r b o n a t e s o l u t i o n and once w i t h w a t e r b e f o r e d r y i n g o v e r anhydrous sodium s u l p h a t e . -183-A f t e r r e m o v a l o f t h e s o l v e n t , t h e d a r k c o l o u r e d r e s i d u e was c h r o m a t o -g r a p h e d on a l u m i n a (10 g, a c t . I I I ) . F r a c t i o n s e l u t e d w i t h benzene y i e l d e d t h e d e s i r e d a l d e h y d e (218 mg) as a l i g h t y e l l o w foam. The p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s o f t h i s m a t e r i a l was i d e n t i c a l t o t h e N - f o r m y l a l d e h y d e (44) w h i c h had been p r e v i o u s l y c h a r a c t e r i z e d . 3. l - F o r m y l - 2 g , 1 6 c t - c u r - 1 9 - e n - 1 7 - a l (44) From A l d e h y d e (37) 1 8 - D e o x y - W i e l a n d - G u m l i c h a l d e h y d e (37) (47 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (12 ml) and t r e a t e d w i t h a c e t i c - f o r m i c a n h y d r i d e (0.5 ml) a t room t e m p e r a t u r e f o r 1 h o u r . S a t u r a t e d aqueous sodium b i c a r b o n a t e was t h e n added and r e s u l t i n g m i x t u r e was p a r t i o n e d . A f t e r w a s h i n g the aqueous l a y e r w i t h d i c h l o r o m e t h a n e , t h e combined o r g a n i c e x t r a c t s were washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e . The r e s i d u e , o b t a i n e d a f t e r r e m o v a l o f t h e s o l v e n t , was p u r i f i e d by p i c on s i l i c a g e l ( e l u e n t C) t o y i e l d an a m o r p h o u s ' m a t e r i a l (37 mg) whose p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s were i d e n t i c a l w i t h t h o s e o f t h e a l d e h y d e (44) w h i c h had been p r e v i o u s l y c h a r a c t e r i z e d , and l a t e r 40 s y n t h e s i z e d by o t h e r s i n o u r l a b o r a t o r i e s by a s i m i l a r r o u t e . -184-A t t e m p t e d C-16 F u n c t i o n a l i z a t i o n o f l - F o r m y l - 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 a l (50) The d a t a r e l e v a n t t o t h e s t u d y o f the f u n c t i o n a l i z a t i o n o f t h e C-16 p o s i t i o n o f t h e N - f o r m y l a l d e h y d e (44) u s i n g s o d 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 i s summarized i n T a b l e 8, and d a t a r e l e v a n t t o a s i m i l a r s t u d y u s i n g o t h e r b a s e s i s summarized i n T a b l e 9. I n a l l e x p e r i m e n t s i n T a b l e 8 and 9 t h e f o l l o w i n g g e n e r a l p r o c e d u r e s were u s e d . A l l g l a s s w a r e was f l a m e - d r i e d and a l l r e a c t i o n s were c o n d u c t e d i n an atmosphere o f d r y n i t r o g e n g a s . I n E x p e r i m e n t s 1-8 o f T a b l e 8 and 1-3 o f T a b l e 9, t h e a l d e h y d e (44) was d i s s o l v e d i n the a p p r o p r i a t e s o l v e n t a t the r e q u i r e d t e m p e r a t u r e . The base was t h e n added and t h e m i x t u r e was s t i r r e d f o r t h e r e q u i r e d t i m e p e r i o d , a f t e r o w h i c h e x c e s s m e t h y l c h l o r o f o r m a t e (0.2-0.3 m l , d i s t i l l e d f r o m 4A m o l e c u l a r s i e v e s ) was added. S t i r r i n g was c o n t i n u e d f o r 10-20 m i n u t e s b e f o r e i c e -w a t e r was added. The r e a c t i o n m i x t u r e was t h e n p a r t i t i o n e d between w a t e r and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t s were washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e b e f o r e t h e s o l v e n t was removed i n vacuo t o g i v e the r e a c t i o n p r o d u c t . When d i m e t h y l s u l p h o x i d e (DMSO) and d i m e t h y l formamide (DMF) were used as s o l v e n t s the work-up was s i m p l e r i f , a t the end o f the r e a c t i o n t i m e , o n l y a few d r o p s o f w a t e r were added and most o f the s o l v e n t was removed i n h i g h vacuum b e f o r e p a r t i t i o n i n g between w a t e r and d i c h l o r o m e t h a n e . I n E x p e r i m e n t s 4-6 o f T a b l e 9, a v a r i a t i o n i n t h e above d e s c r i b e d p r o c e d u r e was employed. P o t a s s i u m _ t - b u t o x i d e was f i r s t t r a n s f e r r e d t o the r e a c t i o n v e s s e l f r o m a p r e v i o u s l y p r e p a r e d s t o c k s o l u t i o n i n benzene. The benzene was the n removed i n h i g h vacuum and t h e w e i g h t o f t h e r e a g e n t -185-was d e t e r m i n e d . The a p p r o p r i a t e s o l v e n t was t h e n added, f o l l o w e d by t h e a d d i t i o n o f t h e a l d e h y d e ( 4 4 ) . I n E x p e r i m e n t s 7-9 o f T a b l e 9, t h e b a s e l i t h i u m d i i s o p r o p y l amine (LDA) was f i r s t p r e p a r e d a t 0° u s i n g e q u a l m o l a r q u a n t i t i e s o f d i i s o p r o p y l amine ( d i s t i l l e d f r o m p o t a s s i u m h y d r o x i d e ) , n - b u t y l l i t h i u m and h e x a m e t h y l p h o s p h o r a m i d e (HMPA). A f t e r 15 m i n u t e s , t h e a l d e h y d e (44) was added and t h e above d e s c r i b e d p r o c e d u r e was employed. I n e v e r y e x p e r i m e n t , t h e r e a c t i o n p r o d u c t was s e p a r a t e d by c h r o m a t o g r a p h y on a l u m i n a ( a c t . I l l ) and t h e components were examined s p e c t r o s c o p i c a l l y . I n e v e r y e x p e r i m e n t , e x c e p t number 3 i n T a b l e 8, u n r e a c t e d s t a r t i n g m a t e r i a l , a l d e h y d e ( 4 4 ) , was r e c o v e r e d . I n E x p e r i m e n t s 7 o f T a b l e 8 and 1-3 o f T a b l e 9, samples o f 18-deoxy-W i e l a n d - G u m l i c h a l d e h y d e (37) were i s o l a t e d and i d e n t i f i e d by s p e c t r o s -c o p i c c o m p a r i s o n w i t h m a t e r i a l p r e v i o u s l y s y n t h e s i z e d . I n a l l e x p e r i -ments, q u a n t i t i e s o f v e r y p o l a r t a r - l i k e m a t e r i a l was o b t a i n e d . -186-T a b l e 8. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 1. Exp. A l d e h y d e ( 4 4 ) Sodium H y d r i d e S o l v e n t Temperature Time (mg,mmoles) (mg,mmoles) 3 (ml) ( h r s ) 1 48 9 0.15 36 , 1.5 THF(30) c r t 6 2 50 9 0.16 39 , 1.6 THF(25) 50° 2 3 82 9 0.25 60 , 2.5 DMF(20) r t 2 4 50 9 0.16 40° , 1.7 DMF(20) 50° 1 5 85 9 0.26 10 , 0.4 DMF(30) r t 4 6 85 9 0.26 10 , 0.4 DMF(20) 55° 0 7 48 9 0.15 7 , 0.3 DMSO(20) 50° 1 8 50 9 0.16 7 , 0.3 DMSO(20) r t 3 a. C a l c u l a t e d b a s e d on 55% o i l s u s p e n s i o n b. G r a n u l a r sodium h y d r i d e c. Room t e m p e r a t u r e -187-T a b l e 9. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 4 4 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 2. Exp. A l d e h y d e ( 4 4 ) (mg,mmoles) Base (mg,mmoles) S o l v e n t (ml) Temperature R e a c t i o n T i m e ( h r s ) 1 50 9 0.15 K H ( 3 2 , 0 . 8 ) a THF(30) r t C 3 2 45 9 0.14 K H ( 2 8 , 0 . 7 ) a THF(30) 60° 1 3 65 9 0.20 K H ( 4 0 , 1 . 0 ) a DMSO(20) 35° 0.5 4 38 9 0.12 _t-BuOK(28,0. 25) C 6 H 6 ( 5 0 ) 5 5 o d 2 5 50 J> 0.15 t-BuOK(34,0. 30) DMSO(25) r t 2 6 85 9 0.26 t-BuOK(62,0. 55) DMSO(25) 50° 1 7 46 9 0.14 L D A ( 0 . 2 8 ) b THF(20) -78° 0.5 8 132, 0.41 L D A ( 0 . 8 0 ) b THF(45) 0° 1 9 68 9 0.21 L D A ( 0 . 4 2 ) b THF(20) r t 3 a. C a l c u l a t e d b a s e d on 22% o i l s u s p e n s i o n b. P l u s 1 e q u i v a l e n t o f HMPA c. Room t e m p e r a t u r e d. R e f l u x e d f o r 30 m i n u t e s b e f o r e c o o l i n g t o 55° f o r t h e a d d i t i o n o f m e t h y l c h l o r o f o r m a t e . -188-l - C a r b o m e t h o x y - 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 - o l (49) The a l c o h o l ( 3 6 ) ( 1 0 0 mg) was d i s s o l v e d i n m e t h a n o l (10 ml) and t r e a t e d w i t h m e t h y l c h l o r o f o r m a t e (0.2 ml) and l e t s t i r a t room t e m p e r a t u r e f o r 15 m i n u t e s . The s o l v e n t was t h e n removed i n vacuo and t h e r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous s o d i u m b i c a r b o n a t e and d i c h l o r o m e t h a n e . The combined o r g a n i c w a s h i n g s were d r i e d and e v a p o r a t e d i n vacuo to g i v e a l i g h t y e l l o w amorphous m a t e r i a l (110 mg), X 241, 278, 287; v 3350 (OH), 1685 max max ( N - C 0 2 C H 3 ) , 1600 ( i n d o l i n e ) ; pmr: 2.52 ( m u l t i p l e t , I H , C-12H), 2.65-3.0 ( m u l t i p l e t s , 3H, a r o m a t i c - H ) , 450 ( m u l t i p l e t s J=7, 1H,=CH-CH 3), 5.68 ( m u l t i p l e t , 2H,-CH 2-0H) 6.20 ( s i n g l e t , 3H, N - C 0 2 C H 3 ) , 8.33 ( d o u b l e t o f d o u b l e t s , J=7 and 2, =CH-CH 3); mass s p e c t r u m : M + 3 5 4 ( 8 6 ) , 3 2 3 ( 2 1 ) , 3 0 9 ( 1 0 0 ) , 2 5 7 ( 2 1 ) , 2 0 2 ( 6 3 ) , 1 6 6 ( 8 5 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C o 1 H „ , 0 o N „ : 354.1942 Found: 354.1916. 21 zo 3 2. l-Carbomethoxy-2B, 1 6 c t - c u r - 1 9 - e n - 1 7 - a l (50) The N-carbomethoxy a l c o h o l ( 4 9 ) ( 4 5 0 mg) was d i s s o l v e d i n d i c h l o r o -methane (100 m l , f r e s h l y d i s t i l l e d f r o m ^ ^ ^ ) a n c ' t r e a t e d w i t h p y r i d i n i u m 38 c h l o r o c h r o m a t e (750 mg) w h i c h had been p r e p a r e d a c c o r d i n g t o C o r e y ' s p r o c e d u r e . The r e s u l t i n g c o l o u r e d s u s p e n s i o n was s t i r r e d a t room t e m p e r a t u r e f o r 2.5 h o u r s , a f t e r w h i c h t i m e , an e q u a l volume o f s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n was added and t h e m i x t u r e was s t i r r e d f o r a f u r t h e r 15 m i n u t e s . T h i s m i x t u r e was e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e (300 ml) and the combined o r g a n i c e x t r a c t s were washed w i t h w a t e r -189-b e f o r e b e i n g d r i e d o v e r a nhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f t h e s o l v e n t , the r e s i d u e was c hromatographed on a l u m i n a (20 g, a c t 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 s o l v e n t g r a d i e n t . The d e s i r e d a l d e h y d e was o b t a i n e d as a l i g h t y e l l o w foam (325 mg), X 242, 282, 288 ; v 1720(CH0), 1690(N-C0 oCH_) 1600 ( i n d o l i n e ) ; max max z j pmr: 0.40 ( d o u b l e t , J=4, I H , -CHO), 2.38 ( m u l t i p l e t , I H , C-12H), 2.6-3.0 ( m u l t i p l e t s , 3H, a r o m a t i c - H ) , 4.45 ( q u a r t e t , J=7, 1H, =CH-CH 3), 6.23 ( s i n g l e t , 3H, N - C 0 2 C H 3 ) , 8.45 ( d o u b l e t o f d o u b l e t s , J=7 and 2, 3H, =CH-CH 3); mass s p e c t r u m : M + 3 5 2 ( 5 8 ) , 3 2 3 ( 2 2 ) , 3 0 9 ( 7 8 ) , 2 0 2 ( 4 7 ) , 1 6 4 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C 2 i H 2 4 ^ 3 N 2 ' ' Found: 352.1737. l - C a r b o m e t h o x y - 2 B , 1 6 a - c u r - 1 9 - e n - 1 7 - a l (50) From A l d e h y d e (37) 1 8 - D e o x y - W i e l a n d - G u m l i c h a l d e h y d e ( 3 7 ) ( 5 5 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (10 ml) and t r e a t e d w i t h e x c e s s m e t h y l c h l o r o f o r m a t e . A f t e r s t i r r i n g a t room t e m p e r a t u r e f o r 1.5 h o u r s t h e s o l v e n t was removed i n vacuo and t h e r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t was washed w i t h w a t e r and d r i e d o v e r anhydrous sodium s u l p h a t e b e f o r e b e i n g e v a p o r a t e d i n v a c u o . The amorphous p r o d u c t (41 mg) was c h r o m a t o g r a p h e d by p i c ( s i l i c a g e l , e l u e n t C) t o y i e l d a p r o d u c t (35 mg) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h p r e v i o u s l y s y n t h e s i z e d N - c a r b o m e t h o x y - 1 8 - d e o x y - W i e l a n d - G u m l i c h a l d e h y d e ( 5 0 ) . -190-A t t e m p t e d C-16 C a r b o x y l a t i o n o f l-Carbo m e t h o x y - 2 g ,16ot-cur-19-en- 1 7 - a l (50) The d a t a r e l e v a n t t o t h e s t u d y o f t h e f u n c t i o n a l i z a t i o n o f th e C-16 p o s i t i o n o f the N-carbomethoxy a l d e h y d e (50) i s summarized i n T a b l e 10. I n t h i s s t u d y , t h e same g e n e r a l p r o c e d u r e s d e s c r i b e d above f o r t h e p r e v i o u s s t u d y o f t h e N - f o r m y l a l d e h y d e (44) were employed f o r s i m i l a r e x p e r i m e n t s . A t no t i m e c o u l d any p r o d u c t s o t h e r t h a n the s t a r t i n g m a t e r i a l (50) o r v e r y p o l a r d e c o m p o s i t i o n m a t e r i a l be d e t e c t e d s p e c t r o s c o p i c a l l y a f t e r c h r o m a t o g r a p h y on a l u m i n a ( a c t . I I I ) . T a b l e 10. A t t e m p t e d C-16 f u n c t i o n a l i z a t i o n o f a l d e h y d e ( 5 0 ) : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 3. Exp. A l d e h y d e ( 5 0 ) (mg,mmoles) Base (mg,mmoles) S o l v e n t (ml) Temperature Time ( h o u r s ) 1 68 , 0.19 N a H ( 5 0 , 2 . 0 ) a THF(30) 40° 6 2 75 , 0.21 N a H ( 5 0 , 2 . 0 ) a DMS0(20) 53° 2 3 45 , 0.13 K H ( 1 0 , 0 . 2 5 ) b C 6 H 6 ( 6 0 ) 80° 2 4 50 , 0.14 K H ( 6 , 0 . 1 5 ) b DMF(20) 50° 4 5 50 , 0.14 L D A ( 0 . 2 8 ) C THF(20) 0° 0.5 6 46 , 0.13 L D A ( 0 . 2 5 ) C THF(25) r t 1 7 35 , 0.10 L D A ( 0 . 2 0 ) C THF(15) 35° 1 a. C a l c u l a t e d b a s e d on 55% o i l s u s p e n s i o n b. C a l c u l a t e d based on 22% o i l s u s p e n s i o n c. P l u s 1 e q u i v a l e n t o f HMPA. -191-Des-carbomethoxy-stemmadenine (52) N o r - f l u o r o c u r a r i n e ( 3 9 ) ( 6 6 0 mg) was d i s s o l v e d i n m e t h a n o l i c p o t a s s i u m h y d r o x i d e (35 m l , 2% ) a t 40° and t r e a t e d w i t h NaBH^ (0.5 g) w h i c h was added o v e r a 0.5 h o u r p e r i o d . S t i r r i n g was c o n t i n u e d f o r a f u r t h e r 1.5 h o u r s b e f o r e the m i x t u r e was r e d u c e d i n volume i n vacuo and p a r t i t i o n e d between w a t e r and d i c h l o r o m e t h a n e . The aqueous l a y e r was r e p e a t e d l y e x t r a c t e d w i t h d i c h l o r o m e t h a n e and t h e combined e x t r a c t s were washed w i t h w a t e r b e f o r e d r y i n g o v e r anhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f t h e s o l v e n t , the r e s i d u e (470 mg) was chromatographed on a l u m i n a (15 g, a c t . I V ) u s i n g a b e n z e n e - e t h y l a c e t a t e s o l v e n t g r a d i e n t . A f t e r p o o l i n g o f t h e a p p r o p r i a t e f r a c t i o n a l i g h t y e l l o w amorphous m a t e r i a l (395 mg) was o b t a i n e d : X 226, 282, 288 ; v 3350 (N-H), max max 3250 ( 0 - H ) ; pmr: 1.0 ( b r o a d s i n g l e t , I H , N-H) 2.4-3.0 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.5 ( q u a r t e t , J=7, I H , =CH-CH 3), 5.72 ( d o u b l e t , J=5, 2H, -CH 2-0H), 6.15 ( m u l t i p l e t , I H , C-16H), 8.3 ( m u l t i p l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 2 9 6 ( 5 7 ) , 2 6 5 ( 1 2 ) , 2 5 1 ( 1 5 ) , 1 6 6 ( 6 1 ) , 1 4 4 ( 8 6 ) , 1 3 0 ( 3 2 ) , 1 2 3 ( 1 0 0 ) , 1 2 2 ( 5 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C 1 Q H o / N „ 0 : 296.1887. Found: 296.1887. 19 24 I 1 , 1 7 - D i a c e t y l - d e s - c a r b o m e t h o x y - s t e m m a d e n i n e (56) Des-carbomethoxy-stemmadenine ( 5 3 ) ( 6 0 mg) was d i s s o l v e d i n a c e t i c a n h y d r i d e (5 ml) and p y r i d i n e (0.5 ml) and l e t s t a n d a t 5° o v e r n i g h t . The s o l v e n t was t h e n removed i n vacuo and t h e r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous so d i u m b i c a r b o n a t e s o l u t i o n and d i c h l o r o m e t h a n e . The aqueous l a y e r was e x t r a c t e d f u r t h e r and t h e o r g a n i c e x t r a c t s were washed -192-w i t h w a t e r b e f o r e d r y i n g o v e r anhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f most o f t h e s o l v e n t t h i s m a t e r i a l was s e p a r a t e d by p i c on s i l i c a g e l ( e l u e n t C) t o y i e l d two amorphous m a t e r i a l s . The m a j o r p r o d u c t (45 mg) was a s s i g n e d s t r u c t u r e (56) b a s e d on t h e f o l l o w i n g d a t a : X 248, 282, 290 ; v 1725 ( O - A c ) , 1640 ( N - A c ) , pmr: 2.4-2.9 max max ( m u l t i p l e t s , 4H, a r o m a t i c - N ) , 4.47 ( q u a r t e t , J=7, I H , =CH-CH 3), 5.3 ( m u l t i p l e t , 2H, - C H 2 - 0 A c ) , 7. ( s i n g l e t , 3H, N - C ( 0 ) C H 3 ) , 7.89 ( s i n g l e t , 3H, - 0 2 C C H 3 ) , 8.26 ( m u l t i p l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 3 8 0 ( 2 7 ) , 3 2 1 ( 2 1 ) , 2 9 3 ( 5 3 ) , 2 0 8 ( 1 6 ) , 1 8 6 ( 1 7 ) , 1 4 4 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C „ o H _ o N o 0 o : 380.2100. Found: 380.2108. 1 7 - A c e t y l - d e s - c a r b o m e t h o x y - s t e m m a d e n i n e (57) Des-carbomethoxy-stemmadenine ( 5 2 ) ( 1 9 0 mg) was d i s s o l v e d i n a c e t i c a n h y d r i d e (5 ml) w i t h 1 drop o f p y r i d i n e and l e t s t a n d a t -10° f o r 6 h o u r s . The s o l v e n t was t h e n removed i n vacuo and the r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous sodium b i c a r b o n a t e and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t was washed w i t h w a t e r and d r i e d o v e r anhydrous s o d i u m s u l p h a t e . A f t e r r e m o v a l o f the s o l v e n t , t h e r e s i d u e was c a r e f u l l y c h romatographed on a l u m i n a (10 g, a c t . I l l ) u s i n g a b e n z e n e - e t h y l a c e t a t e s o l v e n t g r a d i e n t . The l e s s p o l a r f r a c t i o n s y i e l d e d an amorphous m a t e r i a l (6 mg) w h i c h was i d e n t i f i e d as t h e d i a c e t a t e ( 5 6 ) . The m a j o r p r o d u c t was i d e n t i f i e d as 1 7 - a c e t y l - d e s - c a r b o m e t h o x y -stemmadenine ( 5 7 ) ( 1 7 6 mg) b a s e d t h e f o l l o w i n g d a t a : X 224, 282, 288 ; max v 1730 (-OAc); pmr: 1.69 ( b r o a d s i n g l e t , I H , N-H), 2.4-2.9 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 4.49 ( q u a r t e t , J=7, I H , =CH-CH 3) 5.3 ( m u l t i p l e t , 2H, - C H 2 _ O A C ) , 7.89 ( s i n g l e t , 3H, - 0 2 C C H 3 ) > 8 - 2 6 ( m u l t i p l e t , 3H,=CH-CH 3); -193-mass s p e c t r u m : M + 3 3 8 ( 9 ) , 2 9 3 ( 7 ) , 2 7 9 ( 1 4 ) , 1 7 0 ( 2 5 ) , 1 4 4 ( 3 9 ) , 1 2 3 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C 2 1 H 2 6 ° 2 N 2 : 3 3 8 ' 1 9 7 6 ' F o u n d : 338.1972. A t t e m p t e d C-16 C y a n i d e I n t r o d u c t i o n i n A l c o h o l (52) v i a C h l o r o i n d o l e n i n e ( 5 3 ) The d a t a r e l e v a n t t o the s t u d y o f t h e i n t r o d u c t i o n o f a cyano group i n t o the C-16 p o s i t i o n o f i n d o l e a l c o h o l (52) v i a t h e c h l o r o i n d o l e n i n e (53) i s summarized i n T a b l e 11. I n a l l e x p e r i m e n t s i n t h i s s t u d y , t h e f o l l o w i n g g e n e r a l p r o c e d u r e was employed. The i n d o l e a l c o h o l (52) was d i s s o l v e d i n d i c h l o r o m e t h a n e ( f r e s h l y d i s t i l l e d f r o m ^ 0 ^ ) a t ®°' _ t - B u t y l h y p o c h l o r i t e (1.1 e q u i v a l e n t s f r o m a 0.05 M s t o c k s o l u t i o n i n C C l ^ ) was added and t h e r e a c t i o n m i x t u r e was s t i r r e d f o r 15 m i n u t e s . The r e a c t i o n was m o n i t o r e d by t i c on a l u m i n a ( e l u e n t B) and uv s p e c t r o s c o p y . When the uv chromophore o f t h e s t a r t i n g m a t e r i a l was a b s e n t and the c h r a c t e r i s t i c i n d o l e n i n e chromophore (X max 260, 310) was p r e s e n t , the n u c l e o p h i l e was added and the m i x t u r e was s t i r r e d a t t h e r e q u i r e d t e m p e r a t u r e . However, when even d i m e t h y l f o r m a m i d e (DMF) was used as s o l v e n t the d i c h l o r o m e t h a n e was removed i n v a c u o a f t e r 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 . A l l e x p e r i m e n t s were m o n i t o r e d by t i c on a l u m i n a and by uv s p e c t r o -s c o p y . A t t h e end o f the r e a c t i o n t i m e , the m i x t u r e was p a r t i t i o n e d between d i c h l o r o m e t h a n e and w a t e r . The o r g a n i c e x t r a c t s were d r i e d o v e r anhydrous sodium s u l p h a t e b e f o r e t h e s o l v e n t was removed i n v a c u o . The r e s i d u e s were p a r t i t i o n e d by p i c on s i l i c a g e l ( e l u e n t C ) . I n e v e r y e x p e r i m e n t , a v e r y complex m i x t u r e o f p r o d u c t s were o b t a i n e d . The m a j o r p r o d u c t s were d a r k c o l o u r e d t a r - l i k e m a t e r i a l w h i c h r e m a i n e d n e a r -194-t h e b a s e - l i n e o f t h e c h r o m a t o p l a t e . A t no t i m e c o u l d new i n d o l i c m a t e r i a l be d e t e c t e d by uv o r mass s p e c t r o s c o p y . T a b l e 11. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 4. Exp I n d o l e ( 5 2 ) a N u c l e o p h i l e S o l v e n t T e m p e r a t u r e Time (mg,mmoles) (mg,mmoles) (ml) ( h r s ) 1 48, 0.16 KCN(52, 0.8) C H 2 C 1 2 ( 1 5 ) r t C 24 2 35, 0.12 KCN(58, 0.9) DMF(10) r t 6 3 50, 0.17 KCN(98, 1.5) DMF(15) 80° 0.5 4 35, 0.12 Z n ( C N ) 2 ( 1 4 0 , 1.2) C H 2 C 1 2 ( 2 0 ) r t 24 5 36, 0.12 Z n ( C N ) 2 ( 7 0 , 0.6) DMF(15) 50° 1.5 6 50, 0.17 AgCN(228, 1.7) C H 2 C 1 2 ( 3 0 ) r t 48 7 40, 0.14 AgCN(200, 1,5) DMF(10) 55° 1.5 8 54, 0.18 Bu 4NCN(91, 0.9) C H 2 C 1 2 ( 2 5 ) r t 48 9 35, 0.12 Bu 4NCN(120,1.2) C E 2 C 1 2 ( 2 5 ) 35° 12 10 35, 0.12 Bu 4NCN(120,1.2) DMF(10) 35° 5 11 48, 0.16 E t 2 A l C N ( 1 . 6 ) b C H 2 C 1 2 ( 2 5 ) r t 5 12 37, 0.13 E t 2 A l C N ( 1 . 3 ) C H 2 C 1 2 ( 2 5 ) 36° 12 13 62, 0.21 E t A1CN(2.1) C H 2 C 1 2 ( 2 5 ) 5° 5 days a. S t i r r e d w i t h 1.1 e q u i v a l e n t s o f _t-BuOCl a t 0° f o r 15 m i n u t e s b e f o r e a d d i t i o n o f n u c l e o p h i l e . b. A v a i l a b l e f r o m A l f a P r o d u c t s . c. Room t e m p e r a t u r e . -195-A t t e m p t e d C-16 C y a n i d e I n t r o d u c t i o n i n A c e t a t e (57) v i a C h l o r o i n - d o l e n i n e (58) The d a t a r e l e v a n t t o t h e s t u d y o f t h e i n t r o d u c t i o n o f a cyano group i n t o t h e C-16 p o s i t i o n o f the i n d o l e a c e t a t e v i a t h e c h l o r o -i n d o l e n i n e (58) i s summarized i n T a b l e 12. I n a l l e x p e r i m e n t s , t h e same p r o c e d u r e as t h a t d e s c r i b e d above f o r t h e s t u d y i n v o l v i n g a l c o h o l (52) was employed. A t no t i m e i n t h i s s t u d y , c o u l d i n d o l i c m a t e r i a l be d e t e c t e d by uv o r mass s p e c t r o s c o p y . T a b l e 12. A t t e m p t e d C-16 i n t r o d u c t i o n o f c y a n i d e : a d d i t i o n a l d a t a a s s o c i a t e d w i t h T a b l e 5. Exp. I n d o l e ( 5 7 ) a (mg,mmoles) N u c l e o p h i l e (mg,mmoles) S o l v e n t (ml) T emperature Time ( h r s ) 1 45 , 0.13 KCN(85,1.3) C H 2 C 1 2 ( 3 0 ) r t b 20 2 35 , 0.10 KCN(65,1.0) DMF(20) r t 8 3 28 , 0.08 KCN(52,0.8) DMF(15) 68° 1 4 34 , 0.10 Bu 4NCN(50,0. 5) C H 2 C 1 2 ( 3 0 ) r t 24 5 26 , 0.08 Bu 4NCN(49,0. 5) DMF(20) r t 8 6 30 , 0.09 Bu ANCN(91,0. 9) DMF(20) 75° 1 a. S t i r r e d w i t h 1.1 e q u i v a l e n t s o f _t-Bu0Cl a t 0° f o r 15 m i n u t e s b e f o r e a d d i t i o n o f n u c l e o p h i l e b. Room t e m p e r a t u r e -196-R e a c t i o n o f N o r - f l u o r o c u r a r i n e (39) w i t h Me t h y l c h l o r o f o r m a t e N o r - f l u o r o c u r a r i n e ( 3 9 ) ( 1 2 0 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (2 m l , f r e s h l y d i s t i l l e d f r o m l i t h i u m aluminum h y d r i d e ) . T h i s s o l u t i o n was added to a p r e v i o u s l y p r e p a r e d s o l u t i o n o f l i t h i u m d i i s o p r o p y l a m i n e-hexamethylphosphoramide complex ( 1 : 1 , 2 e q u i v a l e n t s ) i n t e t r a -h y d r o f u r a n (6 ml) a t 0°. A f t e r s t i r r i n g f o r 10 m i n u t e s , m e t h y l c h l o r o -f o r m a t e (0.2 ml) was added and s t i r r i n g was c o n t i n u e d a t 0° f o r 15 m i n u t e s . C o l d w a t e r (10 ml) was added and t h e m i x t u r e was p a r t i t i o n e d . The aqueous 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 and t h e combined o r g a n i c e x t r a c t s were washed t h r e e t i m e s w i t h w a t e r b e f o r e d r y i n g o v e r anhydrous sodium s u l p h a t e . A f t e r r e m o v a l o f the s o l u e n t , t h e r e s i d u e was c hromatographed on a l u m i n a (8 g, a c t . I l l ) u s i n g benzene. The a p p r o p r i a t e f r a c t i o n s were combined to g i v e a s i n g l e p r o d u c t : X 233, 268, 305 ( s h ) ; v 1720 max max ( -C0 2CH 3), 1670 and 1640 (-CH0), 1600 ( i n d o l e n i n e ) pmr: 0.02 ( s i n g l e t , I H , -CHO), 2.3 ( m u l t i p l e t , I H , C-12H), 2.6-3.0 ( m u l t i p l e t s , 3H, a r o m a t i c - K ) , 4.56 ( q u a r t e t , J=7, =CH-CH 3), 6.08 ( s i n g l e t , 3H, - C 0 2 C H 3 ) , 8.34 ( m u l t i p l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 350 ( 1 0 0 ) , 321 ( 3 3 ) , 307 ( 1 7 ) , 292 ( 2 0 ) , 121 ( 5 7 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C 2 ^ H 2 2 0 3 N 2 : 3 5 0 . 1 6 2 9 Found: 350.1650. A l t e r n a t e S y n t h e s i s o f A l d e h y d o - e s t e r (60) N o r - f l u o r o c u r a r i n e ( 3 9 ) ( 1 8 0 mg) was suspended i n m e t h y l c h l o r o -o f o r m a t e (5 m l , d i s t i l l e d f r o m 4A m o l e c u l a r s i e v e s ) and s t i r r e d v i g o r o u s l y a t 60-65° under a n i t r o g e n gas a tmosphere. A f t e r 20 m i n u t e s , the s o l v e n t was removed i n vacuo and t h e r e s i d u e was p a r t i t i o n e d between s a t u r a t e d -197-aqueous sodium b i c a r b o n a t e s o l u t i o n and d i c h l o r o m e t h a n e . The o r g a n i c e x t r a c t was washed once w i t h w a t e r and t h e n d r i e d o v e r a nhydrous s o d i u m s u l p h a t e . A f t e r r e m o v a l o f t h e s o l v e n t , t h e r e s i d u e was c h r o m a t o -graphed on a l u m i n a (10 g, a c t . I l l ) t o g i v e two compounds. The m a j o r compound (152 mg) had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h e a l d e h y d o - e s t e r (50) w h i c h had p r e v i o u s l y been c h a r a c t e r i z e d . The s econd component (12 mg) was i d e n t i f i e d as the s t a r t i n g m a t e r i a l n o r -f l u o r o c u r a r i n e (39) . A l k a l i n e H y d r o l y s i s o f A l d e h y d o - e s t e r (60) The a l d e h y d e - e s t e r ( 6 0 ) ( 3 5 mg) was d i s s o l v e d i n m e t h a n o l i c p o t a s s i u m h y d r o x i d e (5 m l , 5%) and h e a t e d t o 65° f o r 2 h o u r s . C o l d w a t e r (20 ml) was added and t h e r e s u l t i n g m i x t u r e 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 . The o r g a n i c e x t r a c t s were combined, d r i e d o v e r anhydrous sodium s u l p h a t e and e v a p o r a t e d i n vacuo t o g i v e an amorphous m a t e r i a l (23 mg). T h i s m a t e r i a l was c h r o m a t o g r a p h e d on a l u m i n a ( a c t . I l l ) t o g i v e a l i g h t y e l l o w foam w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h t h o s e o f n o r - f l u o r o c u r a r i n e (39) w h i c h had been p r e v i o u s l y s y n t h e s i z e d . 1,2-Dehydro-16,17-dehydro-2g-cur-19-en (72) .. The a l d e h y d o - e s t e r ( 6 0 ) ( 5 0 mg) was d i s s o l v e d i n m e t h a n o l (15 m l , d i s t i l l e d f r o m magnesium) and t r e a t e d w i t h sodium b o r o h y d r i d e (20 mg, r e c r y s t a l l i z e d f r o m i s o - p r o p y l a m i n e ) . The r e a c t i o n was s t i r r e d a t room t e m p e r a t u r e f o r 30 m i n u t e s a f t e r w h i c h t i m e most o f t h e s o l v e n t was -198-removed ir± vacuo. The r e s i d u e was d i s s o l v e d i n w a t e r and e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e (150 m l ) . The o r g a n i c e x t r a c t was washed once w i t h w a t e r and d r i e d over anhydrous sodium s u l p h a t e . The r e s i d u e , a f t e r e v a p o r a t i o n o f t h e s o l v e n t , was chromatographed by p i c on s i l i c a g e l ( e l u e n t C) t o y i e l d a l i g h t y e l l o w amorphous m a t e r i a l (26 mg): A m a x 228, 292; pmr: 2.28-2.8 ( m u l t i p l e t s , 4H, a r o m a t i c - H ) , 3.89 ( s i n g l e t , I H , C-17H), 4.36 ( q u a r t e t , J=7, I H , =CH-CH 3), 4.54 ( s i n g l e t , I H , C-17H), 8.16 ( d o u b l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 276 ( 1 0 0 ) , 232 ( 3 9 ) , 1 9 4 ( 4 5 ) , 170 ( 3 2 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C ^ ^ Q ^ : 276.1621. Found: 276.1618. T h i s i n d o l i n e (72) was i s o l a t e d i n a v a r i e t y o f e x p e r i m e n t s i n w h i c h c o n v e r s i o n o f p r o d u c t (60) t o the preakuammicine - and stemmadenine-s e r i e s was a t t e m p t e d . These r e s u l t s a r e summarized i n T a b l e s 6 and 7. I n most e x p e r i m e n t s , o n l y samples of (72) were i s o l a t e d f o r i d e n t i f i c a t i o n s i n c e the p r o d u c t m i x t u r e s were v e r y complex. I n a l l e x p e r i m e n t s i n T a b l e s 6 and 7, t h e a l d e h y d o - e s t e r ( 6 0 ) ( 4 0 - 1 1 0 mg) was d i s s o l v e d i n t h e s o l v e n t i n d i c a t e d a t the r e q u i r e d t e m p e r a t u r e and t r e a t e d w i t h e x c e s s r e d u c i n g a g e n t , w i t h the e x c e p t i o n o f Exp. 9 i n T a b l e 6. I n t h e l a t t e r , compound (60) was added t o a s o l u t i o n o f NaBH^ i n g l a c i a l a c e t i c a c i d a t 90°, f o l l o w e d by a d d i t i o n o f more r e a g e n t . I n a l l c a s e s , t h e b a s i c r e a c t i o n s were worked up as d e s c r i b e d above. Those p e r f o r m e d under a c i d i c c o n d i t i o n s were f i r s t made b a s i c w i t h aqueous sodium b i c a r b o n a t e s o l u t i o n . -199-T h i o a c e t a l F o r m a t i o n o f A l d e h y d e - e s t e r (60) The a l d e h y d o - e s t e r ( 6 0 ) ( 2 0 0 mg) was d i s s o l v e d i n d i c h l o r o m e t h a n e (15 m l ) , e t h a n e d i t h i o l (1 ml) and 2 d r o p s o f b o r o n t r i f l u o r i d e e t h e r a t e . T h i s m i x t u r e was l e t s t a n d a t 5° o v e r n i g h t . The s o l v e n t was t h e n removed i n v a c u o and t h e r e s i d u e was p a r t i t i o n e d between s a t u r a t e d aqueous sodium b i c a r b o n a t e s o l u t i o n and c h l o r o f o r m . A f t e r s e v e r a l e x t r a c t i o n s w i t h c h l o r o f o r m , the o r g a n i c e x t r a c t s were combined and d r i e d o v e r a n hydrous sodium s u l p h a t e . The s o l v e n t was t h e n removed and the r e s i d u e was chromato-graphed on a l u m i n a (5 g, a c t . I l l ) u s i n g a b e n z e n e - m e t h y l e n e c h l o r i d e s o l v e n t g r a d i e n t . P o o l i n g o f t h e a p p r o p r i a t e f r a c t i o n s y i e l d e d a l i g h t y e l l o w amorphous m a t e r i a l (215 mg): A 238, 275, 283 ( s h ) ; v 1710 (-C0 oCH n): max max 2 3 pmr: 2.26 ( m u l t i p l e t , I H , C-12H) 2.65-2.8 ( m u l t i p l e t s , 3H, a r o m a t i c - H ) , 4.19 ( s i n g l e t , I H , C-17H), 4.42 ( q u a r t e t , 3=1, I H , =CH-CH 3) 6.09 ( s i n g l e t , 3H, - C 0 2 C H 3 ) , 6.69 ( m u l t i p l e t , 4H, -S-CH 2-CH 2-S- ) , 8.23 ( d o u b l e t , 3H, =CH-CH 3); mass s p e c t r u m : M + 426 ( 4 1 ) , 376 ( 1 1 ) , 355 ( 1 4 ) , 167 ( 1 0 0 ) , 121 ( 5 8 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r C „ o H „ , N o 0 o S o : 426.1436 Found: 426.1427. A t t e m p t e d R i n g - O p e n i n g o f T h i o a c e t a l (73) The carbomethoxy t h i o a c e t a l ( 7 3 ) ( 8 2 mg) was d i s s o l v e d i n a m e t h a n o l i sodium m e t h o x i d e s o l u t i o n (20,0.2N) and r e f l u x e d under an i n h e r t atmospher f o r 2.5 h o u r s . The r e a c t i o n m i x t u r e was t h e n c o o l e d t o 0° and i c e - w a t e r was added. T h i s m i x t u r e was e x t r a c t e d r e p e a t e d l y w i t h d i c h l o r o m e t h a n e and t h e o r g a n i c e x t r a c t was washed w i t h w a t e r b e f o r e d r y i n g o v e r anhydrous sodium s u l p h a t e . E v a p o r a t i o n o f t h e s o l v e n t y i e l d e d a r e s i d u e (76 mg). -200-E x a m i n a t i o n o f t h i s m a t e r i a l by t i c on s i l i c a g e l ( e l u e n t C) r e v e a l e d the p r e s e n c e o f two major p r o d u c t s , w h i c h were s u b s e q u e n t l y i s o l a t e d by p i c . The l e s s p o l a r m a t e r i a l (75) had the f o l l o w i n g s p e c t r a l d a t a : X 228, 284, 291; v 3300 (-N-H); pmr: 2.37 ( m u l t i p l e t , I H , C-12H) max max r — 2.65-2.8 ( m u l t i p l e t s , 3H, a r o m a t i c - H ) , 4.14 ( d o u b l e t , J=8, I H , C-17H), 4.44 ( q u a r t e t , J=7, I H , =CH-CH 3), 6.71 ( m u l t i p l e t , 4H, -S-CH 2-CH 2-S- ) , 8.24 ( d o u b l e t , 3H, =CH-CH 3); mass sp e c t r u m : M + 370 ( 3 0 ) , 309 ( 1 6 ) , 123 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r ^21^26^2^2 ;370.1537» Found: 370.1543. The second component ( 7 6 ) ( 1 4 mg) had the f o l l o w i n g d a t a : ^ m a x 228, 284, 290; v 3300 (N-H); pmr: 2.37 ( m u l t i p l e t , I H , C-12H), 2.65-2.8 max — ( m u l t i p l e t s , 3H, a r o m a t i c - H ) , 4.45 ( q u a r t e t , J=7, I H , =CH-CH 3 > 5.32 ( d o u b l e t , J=8, I H , C-17H), 6.74 ( m u l t i p l e t , 4H, -S-CH 2-CH 2-S-), 8.25 ( d o u b l e t , 3H, =CH-CH 3); mass spectrum: M + 370 ( 2 8 ) , 309 ( 1 5 ) , 123 ( 1 0 0 ) ; h i g h r e s o l u t i o n mass measurement: C a l c . f o r ^21^26^2^2 : ^70.1537, Found: 370.1511. I n a second e x p e r i m e n t , t h e i n d o l e n i n e t h i o a c e t a l (73) (47 mg) was r e f l u x e d i n m e t h a n o l i c p o t a s s i u m h y d r o x i d e (.10%, 10 ml) i n the p r e s e n c e o f sodium b o r o h y d r i d e (20 mg) f o r 2 h o u r s . Most o f the s o l v e n t was r e -moved jLn vacuo and t h e r e s i d u e was d i l u t e d w i t h w a t e r b e f o r e i t 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 . The o r g a n i c e x t r a c t was washed w i t h w a t e r , d r i e d o v e r anhydrous sodium s u l p h a t e and e v a p o r a t e d i n vacuo to g i v e an amorphous m a t e r i a l . . T h i s m a t e r i a l was chromatographed on s i l i c a g e l ( e l u e n t C) t o y i e l d a s l i g h t l y y e l l o w gum (18 mg) w h i c h had p h y s i c a l and s p e c t r o s c o p i c p r o p e r t i e s i d e n t i c a l w i t h compound ( 7 6 ) , p r e v i o u s l y i s o l a t e d . -201-I n a t h i r d s t u d y , the t h i o a c e t a l ( 7 3 ) ( 3 8 mg) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (5 ml) and t r e a t e d w i t h sodium b o r o h y d r i d e (40 mg) a t room t e m p e r a t u r e f o r 1 h o u r . The r e a c t i o n m i x t u r e was then d i l u t e d w i t h w a t e r , made b a s i c w i t h sodium b i c a r b o n a t e and 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 . The o r g a n i c e x t r a c t was washed w i t h w a t e r , d r i e d o v e r anhydrous sodium s u l p h a t e and e v a p o r a t e d i r i vacuo to y i e l d an amorphous m a t e r i a l . T h i s m a t e r i a l was chromatographed by p i c on s i l i c a g e l ( e l u e n t C) t o y i e l d the s t a r t i n g m a t e r i a l (73) (17 mg) and a t a r - l i k e m a t e r i a l f r o m the b a s e - l i n e o f the c h r o m a t o p l a t e . The t h i o a c e t a l (73) (45 mg) was d i s s o l v e d g l a c i a l a c e t i c a c i d (1 ml) and added t o a s o l u t i o n o f sodium b o r o h y d r i d e i n g l a c i a l a c e t i c a c i d (15 ml) a t 90°. Sodium b o r o h y d r i d e was added f r e q u e n t l y o v e r 10 m i n u t e s and the r e a c t i o n was a l l o w e d t o s t i r f o r a f u r t h e r 5 m i n u t e s . The r e a c t i o n was t h e n worked up as d e s c r i b e d above. E x a m i n a t i o n o f the p r o d u c t m i x t u r e by t i c r e v e a l e d a complex m i x t u r e o f components. Subsequent chromatography and a uv s p e c t r o s c o p i c s t u d y showed t h a t no i n d o l i c m a t e r i a l was p r e s e n t . -202-BIBLIOGRAPHY 1. E. Wenkert and B. W i c k b e r g , J . Am. Chem. S o c , 87_, 1580 (1965). 2. A I S c o t t and A.A. Q u e r e s h i , J . Am. Chem. S o c , 91, 5874 ( 1 9 6 9 ) . 3. J . P. Ku t n e y , H e t e r o c y c l e s , 4-, 169 (1976). 4. J . P. Ku t n e y , J . F. Beck, N. J . E g g e r s , H. W. Hanssen, R. S. Sood, N. D. W e s t c o t t , J . Am. Chem. S o c , 93, 7322 (1971). 5. J . F. Beck, S t u d i e s on the 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 , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o lumbia ( 1 9 7 1 ) . 6. N. J . E g g e r s , S t u d i e s on the 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 , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o lumbia ( 1 9 7 3 ) . 7. J . P. Kut n e y , V. R. N e l s o n and D. C. W i g f i e l d , J . Am. Chem. S o c , 91, 4279 ( 1 9 6 9 ) . 8. J . P. Kut n e y , J . F. Beck and G. B. F u l l e r , H e t e r o c y c l e s , 1, 5 ( 1 9 7 3 ) . 9. P. P o t i e r and M. M. J a n o t , C. R. Acad. S c i . P a r i s , 276, 1727 (1 9 7 3 ) . 10. A. I . S c o t t and A. A. Q u e r e s h i , Chem. Comm., 1945 (1968). 11. R. T. Brown, J . H i l l , G. F. Smith and K. S. S t a p l e f o r d , Chem. Comm., 1475 (1 9 6 9 ) . 12. A. I . S c o t t , B i o o r g . Chem., 3_, 398 ( 1 9 7 4 ) . 13. A. S a n d o v a l , F. W a l l s , J . S h o o l e v y , J . W i l s o n , H. B u d z i k i e w i e z and C. D j e r a s s i , T e t . L e t . , 409 (1 9 6 2 ) . 14. F. W a l l s , 0. C o l l e r a and A. S a n d o v a l , T e t r a h e d r o n , 2_, 173 ( 1 9 5 8 ) . 15. A. Wu and V. S n i e k u s , T e t . L e t . , 2057 (1975). 16. G. P o u l t o n and M. H e a r n , R e s e a r c h R e p o r t , U n i v . o f B r i t i s h C o lumbia ( 1 9 7 0 ) . -203-17. The A l k a l o i d s , R.H.F. Manske, e d . Academic P r e s s , New Y o r k . 18. M. M. J a n o t and J . LeMen^ Communication t o S m i t h , r e f . 25. 19. K. B e r n a u e r , F. B e r l a g e , W. v o n P h i l i p s b o r n , H. Schmid and P. K a r r e r , H e l v . Chim. A c t a . , 4 1 2 2 9 3 ( 1 9 5 8 ) . 20. We a r e g r a t e f u l f o r a g i f t o f W i e l a n d - G u m l i c h a l d e h y d e f r o m P r o f . H. Schmid, U n i v e r s i t y o f Z u r i c h , Z u r i c h , S w i t z e r l a n d . 21. H. W i e l a n d and K. K a z i r o , L i e b i g s Ann. 506, 60 ( 1 9 3 3 ) . 22. F. Anet and S i r R. R o b i n s o n , J . Chem. S o c , 2253 ( 1 9 5 5 ) . 23. J . R. Hymon, H. Schmid, P. K a r r e r , A. B o i l e r , H. E l s , P. F a h r n i and A. F i i r s t , H e l v . Chim. A c t a , 52_, 1564 ( 1 9 6 9 ) . 24. G. B. F u l l e r , S t u d i e s on the S y n t h e s i s and 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 , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o l u m b i a ( 1 9 7 4 ) . 25. P. N. Edwards and G. F. S m i t h , J . Chem. S o c , 152, ( 1 9 6 1 ) . 26. H. F r i t z , E. B e s c h and T. W i e l a n d , L i e b i g s Ann. 663, 150 ( 1 9 6 3 ) . 27. H. F r i t z , E. B e s c h and Th. W i e l a n d , Ang..Chem., _71t 126 ( 1 9 5 9 ) . 28. M. G r d i n i c , D. A. N e l s o n and B o e k e l h e i d e , J . Am. Chem. S o c , 86, 3357 ( 1 9 6 4 ) . 29. L. F. F i e s e r and M. F i e s e r , Reagents f o r O r g a n i c S y n t h e s i s , John W i l e y and Sons, I n c . New Y o r k , p.4. 30. R. R a t c l i f f e and R. R o d e h o r s t , J . Org. Chem., 3_5, 4000 ( 1 9 6 9 ) . 31. K. E. P f i t z n e r and J . G. M o f f a t t , J . Am. Chem. S o c , 85_, 3027 ( 1 9 6 3 ) ; 87, 5661 ( 1 9 6 5 ) ; 87_, 5670 ( 1 9 6 5 ) . 32. J . R. P a r i k h and W. von E. D o e r i n g , J . Am. Chem. S o c , J39, 5505 ( 1 9 6 7 ) . 33. K. Onodera, S. H i r a n o , N. K i s h i m u r a , F. Masuda, T. Y a j i m a and N. M i y a z a k i , J . Ong. Chem., 31, 1291 ( 1 9 6 6 ) . -204-34. H. B u d z i k l e w i c z , J . M. W i l s o n , C. D j e r a s s i , J . L e v y , J . LeMen and M-M. J a n o t , T e t r a h e d r o n , 1_9, 1265 ( 1 9 6 9 ) . 35. H. M i i l l e r , M. He s s e , P. Waser, H. Schmid and P. K a r r e r , H e l v . Chim. A c t a . , 48, 320 ( 1 9 6 5 ) . 36. M. F e t i z o n e and M. G o l f i e r , Compt. r e n d . , 267, 900 ( 1 9 6 8 ) . M. F i e s e r and L. F i e s e r , Reagents f o r O r g a n i c S y n t h e s i s , 2, 363 ( 1 9 6 9 ) . 37. E. J . Corey and G. W. F l e e t , T e t . L e t . , 4499 ( 1 9 7 3 ) . 38. E. J . Co r e y and J . W i l l i a m s Suggs, T e t . L e t . , 2647 ( 1 9 7 5 ) . 39. H. G. B o i t , Chem. B e r . , 86, 133 ( 1 9 5 3 ) . 40. N. G. L e w i s , Ph.D. T h e s i s , U n i v . o f B r i t i s h C o l u m b i a ( 1 9 7 8 ) . 4 1 . G. H. G a i t i n Enamines: S y n t h e s i s , S t r u c t u r e and R e a c t i o n s . E l e c t r o p h i l i c S u b s t i t u t i o n s and A d d i t i o n s t o Enamines, A. G. Cook, Ed., M a r c e l D e k k e r , New Y o r k ( 1 9 6 9 ) . 42. M. Y o s h i m o t o , N. I s h i d a and T. H i r a o k a , ' T e t . L e t . , 39 ( 1 9 7 3 ) . 43. A. I . M e y e r s , A. H. R e i n e , and R. G a u l t , J . Org. Chem., 34, 698 ( 1 9 6 9 ) . 44. L. K o z e r s k i , T e t r a h e d r o n , 32, 1299 ( 1 9 7 6 ) . 45. A. W a l s e r and C. D j e r a s s i , H e l v . Chim. A c t a . , 48^, 391 ( 1 9 6 5 ) . 46. A. C h a t t e r j e e , A. B a n e r j i , P. Majumder and R. Majumder, B u l l . Chem. Soc. J a p a n , £9, 2000 ( 1 9 7 6 ) . 47. A. F. S m i t h and J . T. W r o b e l , J . Chem. S o c , 792 ( 1 9 6 0 ) . 48. C. H. S c h l a t t e r , E. Walden, K. Schmid, W. M a i e r and D. G r o g e r , H e l v . Chim. A c t a . , 52, 776 ( 1 9 6 9 ) . PUBLICATIONS James P. K u t n e y , J o h n C a b l e , W i l l i a m A.F. G l a d s t o n e , H a r a l d W. Hanssen, G. V i j a y N a i r , Edward J . T o r u p k a and W i l l i a m D.C. Warnock. Can. J . Chem. 53, 1796 (1971) S y n t h e t i c S t u d i e s i n t h e V e r a t r u m A l k a l o i d S e r i e s I I . The t o t a l S y n t h e s i s o f V e r a r i n e , V e r a t r a m i n e , J e r v i n e and V e r a t r o b a s i n e . James P. K u t n e y , J o h n F. Beck, N i g e l J . E g g e r s , H a r a l d W. Hanssen, R a t t a n S. Sood, N e i l D. W e s t c o t t . J . Amer. Chem. Soc. 93_, 7322 (1971) S t u d i e s on 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 . V I I . The L a t e r S t a g e s o f A s p i d o s p e r m a A l k a l o i d B i o s y n t h e s i s . James P. Ku t n e y and H a r a l d W. Hanssen. P h y t o c h e m i s t r y 10, 3298 (1971) 5,6,7 - T r i m e t h o x y f l a v o n e and 5,6,7,8-T e t r a m e t h o x y f l a v o n e From Z e y h e r a T u b e r c u l o s a . James P. K u t n e y , H a r a l d W. Hanssen and G. V i j a y N a i r . T e t r a h e d r o n 27, 3323 (1971) S t u d i e s on Some E l e c t r o p h i l i c S u b s t i t u t i o n R e a c t i o n s i n t h e F u r a n S e r i e s . James P. K u t n e y , J o h n C a b l e , W i l l i a m A.F. G l a d s t o n e , H a r a l d W. Hanss en, Edward J . T o r u p k a , W i l l i a m D.C. Warnock. J . Amer. Chem. Soc. 90, 5332 (1968) The T o t a l s y n t h e s i s o f V e r a t r u m A l k a l o i d s . I . V e r a r i n e . 

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