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UBC Theses and Dissertations

Studies on the synthesis and biosynthesis of indole alkaloids Fuller, George Bohn 1974

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STUDIES ON THE SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS BY GEORGE BOHN FULLER B.A. (cum laude) , M a c a l e s t e r C o l l e g e , 1969 M.Sc, The U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , 19 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard /-) THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1974 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of th is thesis for f inanc ia l gain sha l l not be allowed without my written permission. Depa rtment The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT P a r t A of t h i s t h e s i s p r o v i d e s a resume1 of the s y n t h e s i s of v a r i o u s r a d i o a c t i v e l y l a b e l l e d forms of secodine C76) and p r o v i d e s an e v a l u a t i o n of these compounds, as w e l l as some r a d i o a c t i v e l y l a b e l l e d forms of tryptophan C25), as p r e c u r s o r s i n the B i o s y n t h e s i s of a p p a r i c i n e (81), u l e i n e C83), guatam-buine (90) , and o l i v a c i n e (88) i n Aspidosperma a u s t r a l e . Only a p p a r i c i n e (81) c o u l d be shown to i n c o r p o r a t e these p r e c u r s o r s to a s i g n i f i c a n t e x t e n t . Degradation of a p p a r i c i n e (81) from Aspidosperma p y r i c o l l u m p r o v i d e d evidence f o r the i n t a c t i n c o r p o r a t i o n of the secodine system. P a r t B d i s c u s s e s the s y n t h e s i s of 16-epi-stemmadenine (161), which p r o v i d e s an e n t r y i n t o the stemmadenine system with, r a d i o a c t i v e l a b e l s a t key p o s i t i o n s i n the molecule. The s y n t h e s i s i n v o l v e d the d e g r a d a t i o n of s t r y c h n i n e (29) to Wieland-Gumlich aldehyde (130) by a p r e v i o u s l y e s t a b l i s h e d sequence of r e a c t i o n s . I n i t i a l c o n v e r s i o n of Wieland-Gumlich aldehyde to n o r ^ f l u o r o c u r a r i n e (134) succeeded by a p r e v i o u s l y d e s c r i b e d r o u t e , although some study was necessary f o r determin-i n g the c o n d i t i o n s by which the Oppenauer o x i d a t i o n of 2B,16a-cur-19-en-17-ol (137) c o u l d s e l e c t i v e l y y i e l d e i t h e r 23,16a-cur-19-en-17-al (133) or n o r - f l u o r o c u r a r i n e (134). When n o r - f l u o r o -c u r a r i n e (134) c o u l d not be converted to the d e s i r e d stemmadenine system, Wieland^GxunlictL aldehyde was converted to methyl 18-hydroxy^2&,16a-cur-19-en-17^oate (156) by a p r e v i o u s l y e s t a b l i s h e d procedure. Conversion of t h i s compound to m e t h y l 2 6/, 16a-cur-19-en-17-^oate 0.571 was accomplished by s u c c e s s i v e treatment with, hydrogen bromide and z i n c i n a c e t i c a c i d . The e s t e r 157 was converted to i t s - N Ca I *s£ o rmy-1 d e r i v a t i v e 158 by r e a c t i o n w i t h methyl formate and sodium h y d r i d e . Treatment of t h i s product w i t h dry formaldehyde and sodium hy d r i d e i n dimethyl s u l f o x i d e l e d to the f o r m a t i o n of the unexpected but n e v e r t h e l e s s u s e f u l t e t r a h y d r o o x a z i n e d e r i v a t i v e 159. H y d r o l y s i s of the t e t r a h y d r o o x a z i n e moiety was accomplished w i t h methanolic hydrogen c h l o r i d e , r e s u l t i n g i n the i s o l a t i o n of 2g,16g-carbo-methoxy-cur-19-en-17-ol (160) . O x i d a t i o n of compound 160 w i t h l e a d t e t r a a c e t a t e f o l l o w e d immediately by treatment w i t h sodium borohydride i n methanolic a c e t i c a c i d p r o v i d e d 16-epi-stemmaden-i n e C161). Hydride r e d u c t i o n of the C-16 e s t e r f u n c t i o n i n 161 and a u t h e n t i c stemmadenine (6a) l e d to the same d i o l 175 thereby p r o v i d i n g the r e q u i r e d i n t e r r e l a t i o n s h i p between the s y n t h e t i c and n a t u r a l compounds. T h i s sequence a l s o e s t a b l i s h e d the p r e v i o u s l y unknown c o n f i g u r a t i o n of stemmadenine (6a) about C-16 and p r o v i d e d an obvious pathway f o r the s y n t h e s i s of stemmadenine v i a the s a t u r a t e d aldehyde 133. A l s o d i s c u s s e d i n P a r t B i s the l e a d t e t r a a c e t a t e o x i d a t i o n of the e s t e r 157 to akuammicine (66), r e p r e s e n t i n g the f i r s t t o t a l s y n t h e s i s of t h a t compound. P a r t C d i s c u s s e s the s y n t h e s i s of 16-epi-stemmadenine (161) l a b e l l e d w i t h t r i t i u m i n the aromatic r i n g . Simultaneous 3 administration of t h i s material and stemmadenine-Car- H) (6a) to separate p o r t i o n s of A., pyricolluro r o o t sections e s t a b l i s h e d t h a t , w h i l e the l a t t e r was incorporated i n t o a p p a r i c i n e (81), - i v -no i n c o r p o r a t i o n c o u l d be d e t e c t e d i n the. case of the former. y — TABLE OF CONTENTS Page T I T L E PAGE . . i ABSTRACT i i TABLE OF CONTENTS V L I S T OF FIGURES v i L I S T OF TABLES i x ACKNOWLEDGEMENTS x INTRODUCTION 1 DISCUSSION 35 PART A 35 PART B 57 PART C 10 7 EXPERIMENTAL 108 SECTION A 112 SECTION B 116 SECTION C 135 BIBLIOGRAPHY 136 - y i -LIST OF FIGURES F i g u r e Page 1 Some R e p r e s e n t a t i v e I n d o l e A l k a l o i d s 2 2 The B i o s y n t h e s i s of A n t h r a n i l i c A c i d C21) 5 3 The B i o s y n t h e s i s of Tryptophan (25) from A n t h r a n i l i c A c i d (21) 6 4 The Barger-Hahn-Robinson-Woodward P o s t u l a t e f o r Ind o l e A l k a l o i d B i o s y n t h e s i s 8 5a The Wenkert Prephenate P o s t u l a t e f o r Indole A l k a l o i d B i o s y n t h e s i s 9 5b The Thomas Monoterpene P o s t u l a t e f o r Indole A l k a l o i d B i o s y n t h e s i s 9 6a The Leete Acetate-Malonate Hypothesis f o r In d o l e A l k a l o i d B i o s y n t h e s i s 14 6b The Hendrickson P o l y k e t i d e M o d i f i c a t i o n 14 7 The E a r l y Stages of Indole A l k a l o i d B i o s y n t h e s i s as Proven by Experiment 16 8 The P o s t u l a t e d D e r i v a t i o n of Corynanthe A l k a l o i d s from V i n c o s i d e (54) 19 9 The Wenkert (A) and S c o t t (B) P o s t u l a t e s f o r the B i o s y n t h e s i s of Strychnos A l k a l o i d s 21 10 The Wenkert P o s t u l a t e f o r the B i o s y n t h e s i s of Iboga and Aspidosperma A l k a l o i d s 22 11 The P o s t u l a t e d D e r i v a t i o n of Aspidosperma and Iboga A l k a l o i d s from Intermediate 75 26 12 The Wenkert P o s t u l a t e f o r the B i o s y n t h e s i s of U l e i n e (83) ' 29 13 The D j e r a s s i P o s t u l a t e f o r the B i o s y n t h e s i s of A p p a r i c i n e (81) 29 14 The R e s u l t s of I n c o r p o r a t i o n of V a r i o u s P o s s i b l e Intermediates i n t o A p p a r i c i n e (81) and U l e i n e (83) 33 - y i i -F i g u r e Page 15 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 Non-tryptamine A l k a l o i d s 34 16 The Co n v e r s i o n of O l i v a c i n e (88) to Guatam-buine (90) 37 17 The O z o n o l y t i c Degradation of A p p a r i c i n e (81)... 39 18 The S y n t h e s i s o f Secodine-(Ar- 3H) and ( 1 4COOCH^) (76) 7. 40 19 The S y n t h e s i s of Secodine-(19- 3H) (76) 44 20 The S y n t h e s i s o f 1 - ( 3 1 - p y r i d y l ) - e t h a n e - ( 1 - 3 H ) (116) 45 21 The Proposed R e l a t i o n s h i p s of Secodine (76) to Stemmadenine (6) i n Indole A l k a l o i d B i o s y n t h e -s i s 49 22 The C o r r e l a t i o n of Akuammicine (66) and Stemm-adenine (6) v i a Preakuammicine (2) 58 23 The Attempted S y n t h e s i s of 19, 20-Dihydrostemm-adenine 60 24 The Degradation of S t r y c h n i n e (29) to Wieland-Gumlich Aldehyde 61 25 A Summary of Some Known Reactions i n the Wieland-Gumlich Aldehyde S e r i e s 64 2 6 The Boekelheide Mechanism f o r the Oppenauer O x i d a t i o n o f 133 to 134 66 27 The Routes Con s i d e r e d f o r the Conv e r s i o n of N o r - f l u o r o c u r a r i n e (134) to Stemmadenine ( 6 ) . . . . 68 2 8 The Nucl e a r Magnetic Resonance Spectrum of Nor-f l u o r o c u r a r i n e (134) 72 29 The R e a c t i o n of 16-epi-WGA w i t h HCN 73 30 The Rea 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 (134) w i t h Cyclohexylamine, P y r r o l i d i n e and Morpholine 75 31 The S y n t h e s i s o f 16-epi-stemmadenine (161) from WGA (130) 78 y i i i -F i g u r e Page 32 The Proposed Mass S p e c t r a l Fragmentation of t h e M e t h y l Cur-19-en-17-oate System 80 33 Two P o s s i b l e Routes to Stemmadenine (6) from Methyl 23,16a-cur-19-en-17-oate 0-57) 82 34 The P o s s i b l e Condensation of Formaldehyde w i t h Methyl 23 ,16a-cur-19-en-17-oate (157) 83 35 The N u c l e a r Magnetic Resonance Spectrum of the Carbomethoxy T e t r a h y d r o o x a z i n e 159 87 36 The R e v e r s i b l e Formation of the Model T e t r a h y d r o -oxazine (166) 86 37 The N u c l e a r Magnetic Resonance Spectrum of the Model T e t r a h y d r o o x a z i n e (166) 89 38 The Proposed Mechanism f o r the Formation of the Carbomethoxy T e t r a h y d r o o x a z i n e 159 93 39 The Edwards and Smith Mechanism f o r the Z i n c and S u l f u r i c A c i d Reduction of Akuammicine (66) 96 4 0 The Nuc l e a r Magnetic Resonance Spectrum of Akuammicine (66) 97 41 The Proposed Mechanism f o r the Formation of Indole E s t e r 141a 98 42 The N u c l e a r Magnetic Resonance Spectrum of the Indole E s t e r 141a 99 43 The N u c l e a r Magnetic Resonance Spectrum of the Indole E s t e r 141b 99 44 The Reduction of N a t u r a l and S y n t h e t i c Stemmaden-i n e Systems to the D i o l 175 103 4 5 The I n f r a r e d S p e c t r a of A u t h e n t i c and S y n t h e t i c D i o l 175 104 46 The N u c l e a r Magnetic Resonance S p e c t r a of A u t h e n t i c and S y n t h e t i c D i o l 175 105 47 The Proposed Route f o r the S y n t h e s i s of Stemm-adenine (6a) 106 - i x -LIST OF TABLES T a b l e Page 1 R e s u l t s of I n c o r p o r a t i o n of Secodine (76) i n t o A p p a r i c i n e (81) 47 2 S p e c i f i c A c t i v i t i e s A s s o c i a t e d w i t h the E x p e r i -ments i n T a b l e 1 48 3 S p e c i f i c A c t i v i t i e s A s s o c i a t e d w i t h the Ozonoly-t i c D e g r a d a t i o n of A p p a r i c i n e (81) i n Experiments 2 and 5 50 4 The V a r i o u s Compounds Fed to A. a u s t r a l e 52 5 I n c o r p o r a t i o n R e s u l t s A s s o c i a t e d w i t h T a b l e 3.... 53 6 E f f e c t s of D i f f e r e n t R e a c t i o n C o n d i t i o n s on the Oppenauer O x i d a t i o n of the A l c o h o l 137 66 7 Summary of R e s u l t s of the I n v e s t i g a t i o n s i n t o Anion Formation a t C-16 of the N-formyl e s t e r (158) 85 8 A Summary of P e r t i n e n t N u c l e a r Magnetic Reson-ance Chemical S h i f t s f o r Compounds 156-159 91 9 Comparison of NMR Data of N a t u r a l and S y n t h e t i c Stemmadenine Systems 102 10 The Stemmadenine Systems A d m i n i s t e r e d t o A. p y r i c o l l u m 106 11 I n c o r p o r a t i o n R e s u l t s A s s o c i a t e d w i t h T a b l e 10... 107 - X -ACKNOWLEDGEMENTS I would l i k e to express xny a p p r e c i a t i o n to P r o f e s s o r James P. Kutney f o r h i s guidance and encouragement, and the generous c o n t r i b u t i o n s of h i s time which were p r o v i d e d throughout the course o f t h i s work. I would a l s o l i k e to express my g r a t i t u d e t o my w i f e , whose u n f a i l i n g support, c o n f i d e n c e , and understanding d u r i n g the course o f t h i s study were a c o n t r i b u t i o n beyond measure. I am g r a t e f u l to The U n i v e r s i t y of B r i t i s h Columbia and to the N a t i o n a l Research C o u n c i l of Canada f o r the f i n a n c i a l support which they p r o v i d e d . INTRODUCTION To a chemist, one of the roost f a s c i n a t i n g aspects of nature i s the abundance of h i g h l y complex molecules found i n p l a n t systems. Even more f a s c i n a t i n g i s the f a c t t h a t these complex molecules are made by the p l a n t s from the m i n e r a l s a v a i l a b l e i n the s o i l , the gases a v a i l a b l e from the atmosphere, water and s u n l i g h t . Even the most s o p h i s t i c a t e d of l a b o r a t o r y procedures p a l e s i n comparison w i t h the s y n t h e t i c o p e r a t i o n s o c c u r r i n g d a i l y i n p l a n t systems. I t i s , t h e r e f o r e , of g r e a t importance to chemists to study p l a n t systems i n an e f f o r t t o d i s c o v e r how complex molecules are c o n s t r u c t e d i n v i v o i n o r d e r t h a t i n v i t r o t r a n s f o r m a t i o n s may become more e f f i c i e n t . T h i s t h e s i s concerns i t s e l f w i t h such a study, i n which an attempt i s made to b e t t e r understand the l a t e r stages of 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 t l e a s t 10-20% of a l l p l a n t s produce alkaloids,"'" and approximately 25% of these a l k a l o i d s c o n t a i n the i n d o l e , d i h y d r o -2 i n d o l e , i n d o l e n i n e , or a-m e t h y l e n e - i n d o l i n e system. Roughly 3 4 800 i n d o l e a l k a l o i d s are a l r e a d y known, ' and they can be con-v e n i e n t l y grouped i n t o f o u r main groups: corynanthe, s t r y c h n o s , aspidosperma and iboga. Examples of these groups are g e i s s o s c h i z i n e C l ), preakuammicine (2), v i n d o l i n e (3) and - 2 -( 7 ) F i g u r e 1. Some R e p r e s e n t a t i v e I n d o l e A l k a l o i d s . X - 3 -c a t h a r a n t h i n e C4), r e s p e c t i v e l y . D i f f e r e n c e s do occur w i t h i n v a r i o u s groups i n terms of s t e r e o c h e m i s t r y , f u n c t i o n a l i t y , e t c . One of the most i n t e r e s t i n g v a r i a t i o n s i n each group i s the e x i s t e n c e o f nine (or ten) membered r i n g d e r i v a t i v e s . Examples of these are p i c r a p h y l l i n e (5) (corynanthe), stemmadenine (6) ( s t r y c h n o s ) , v i n c a d i n e (7) (aspidosperma) and 16-carbomethoxycleavamine (8) ( i b o g a ) . D e s p i t e the d i f f e r e n c e s between and w i t h i n the f o u r main groups o f i n d o l e a l k a l o i d s , t h e r e e x i s t s an o v e r a l l u n i f y i n g s i m i l a r i t y . That i s , they can a l l be f o r m a l l y d e r i v e d from a tryptamine r e s i d u e j o i n e d w i t h a C^-C^Q u n i t . In a formal sense, the f o u r f a m i l i e s can be d e f i n e d by the arrangement of carbon atoms i n the c g ~ c ^ Q r e s i d u e . S t r u c t u r e (9) i n d i c a t e s the arrangement found i n both corynanthe and s t r y c h n o s a l k a l o i d s , w h i l e the arrangements f o r aspidosperma and iboga are i n d i c a t e d by s t r u c t u r e s (10) and (11) r e s p e c t i v e l y . (9 ) ( 10 ) ( 1 1 ) There i s g e n e r a l agreement t h a t the tryptamine segment i s 5—9 d e r i v e d from tryp t o p h a n , i n a c c o r d w i t h the p o s t u l a t e of P i c t e t 1 0 i n 1906. However, the o r i g i n of the C G - C 1 Q u n i t has - 4 -been the o b j e c t of s p e c u l a t i o n s i n c e the e a r l y t h i r t i e s when 11 12 Barger and Hahn p o s t u l a t e d t h a t the non-tryptamine p o r t i o n of yohimbine was t y r o s i n e - d e r i v e d . In the p a s t decade, the ready a v a i l a b i l i t y of r a d i o a c t i v e m a t e r i a l s f o r t r a c e r s t u d i e s has p r o v i d e d s u f f i c i e n t e x p e r i m e n t a l data to e l u c i d a t e the b i o s y n t h e t i c o r i g i n s of both the tryptamine r e s i d u e , and the Cg-C^n segment. The b i o s y n t h e s i s of tryptophan, as shown i n F i g u r e 2, has 13 14 been proven ' to occur v i a the s h i k i m a t e - c h o r i s m a t e pathway. Erythrose-4-phosphate (12) undergoes a l d o l c ondensation w i t h phosphoenolpyruvic a c i d (13) to form 3-deoxy-D-arabinoheptulo-s o n i c acid-7-phosphate (14), which undergoes c y c l i z a t i o n and o x i d a t i o n t o form 5-dehydroquinic a c i d (15). Loss of water r e s u l t s i n 5-dehydroshikimic a c i d (16) which i s then reduced by NADPH to s h i k i m i c a c i d (17) and p h o s p h o r y l a t e d to form s h i k i m i c acid-5-phosphate (19). Condensation w i t h phosphoenol-p y r u v i c a c i d i n i t i a l l y r e s u l t s i n 3 - e n o l p y r u v y l s h i k i m i c acid-5-phosphate (19) which then undergoes d e p h o s p h o r y l a t i o n to form c h o r i s m i c a c i d (20). C h o r i s m i c a c i d then a c q u i r e s the amide n i t r o g e n of glutamine to form a n t h r a n i l i c a c i d " ^ (21), 16 17 the major p r e c u r s o r to tryptophan. I t has been shown ' i n E. c o l i t h a t the c o n v e r s i o n of a n t h r a n i l i c a c i d t o tryptophan occurs as shown i n F i g u r e 3. Formation of N - ( 5 1 - p h o s p h o r i b o s y l ) -a n t h r a n i l i c a c i d (22) f o l l o w e d by o x i d a t i v e r i n g opening t o form e n o l - 1 - ( o - c a r b o x y l p h e n y l a m i n o - ) - l - d e s o x y r i b u l o s e - 5 - p h o s p h a t e (23) and subsequent c y c l i z a t i o n w i t h l o s s of carbon d i o x i d e r e s u l t s i n the f o r m a t i o n of i n d o l e - 3 - g l y c e r y l p h o s p h a t e (24). CHO HCOH H C O H COO COPOoH ChUOPOoH 1 3 ( 13 ) (12) HO ? 0 0 0 S-OH \ r l OH ( 15 ) COO ( 16 ) COO HO l\ H H OH ATP / ( -| 7 ) C H -H- - O - C * H 0 3 P 0 ' ^6HH (19 ) COO H ~i coo" -H 2PO 4 C H --o-c . '•. H COO H OH ( 20 ) HO^ PO i'-H 3 H OH (18 ) .COO glutamine >r > 'NH-( 21 ) F i g u r e 2. The B i o s y n t h e s i s of A n t h r a n i l i c A c i d (21). - 6 -( 21) C O O -> N H C H 9 0 P 0 . H HO OH ( 2 2 ) H 0 H H 0 0 c — C H H C H 2 O P 0 3 H ( 2 3 ) H 0 •C H -co H 0 •C H •CI-^OPO^H H +serxne -3-phospho-g l y c e r a l d e h y d e p y r i d o x a l phosphate COO ( 2U ) ( 2 5 ) F i g u r e 3. The B i o s y n t h e s i s of Tryptophan C25) from A n t h r a n i l i c A c i d (21). - 7 -Condensation o f 24 w i t h s e r i n e f o l l o w e d by l o s s of 3-phospho-g l y c e r a l d e h y d e completes the b i o s y n t h e s i s o f t r y p t o p h a n (25). Subsequent to the e l u c i d a t i o n of the m i c r o b i a l b i o s y n t h e s i s 18 of tryptophan, i t was shov/n t h a t the pathway shown i n F i g u r e s 2 and 3 i s a l s o v a l i d i n h i g h e r p l a n t systems. P r i o r to the f i r s t t r a c e r experiments concerned w i t h the o r i g i n of the cg~ c^o u n ;'- t' t h e r e were t h r e e main p o s t u l a t e s i n e x i s t e n c e . The o r i g i n a l Barger-Hahn p o s t u l a t e had been 19 20-22 e l a b o r a t e d by Robinson and Woodward to the p o i n t where i t c o u l d accommodate the f o r m a t i o n of a l k a l o i d s of the s t r y c h n o s and corynanthe, as w e l l as the o r i g i n a l yohimbe, a l k a l o i d s k e l e t o n s as shown i n F i g u r e 4. The combined h y p o t h e s i s i n v o l v e s the d e g r a d a t i o n of t y r o s i n e (26) to 3,4-dihydroxy-phenylacetaldehyde (27). Condensation of 27 w i t h tryptamine (28) c o u l d then occur a t the £ p o s i t i o n of the i n d o l e system t o y i e l d s t r y c h n i n e (29), or a t the a p o s i t i o n t o y i e l d 30, which c o n t a i n s an aromatic r i n g E. F i s s i o n of the aromatic r i n g E between the two h y d r o x y l groups r e s u l t s i n 31 which can combine w i t h two C^ u n i t s to y i e l d yohimbine (32), or be d i r e c t l y c o n v e r t e d to cinchonamine (33). 23 24 The second h y p o t h e s i s was suggested by Wenkert ' i n 1959. As shown i n F i g u r e 5a, Wenkert proposed t h a t s h i k i m i c a c i d (17) d e r i v e d from carbohydrate metabolism would combine w i t h p y r u v i c a c i d (34) to form prephenic a c i d (35). Rearrangement, h y d r a t i o n , r e t r o a l d o l cleavage and a d d i t i o n of formaldehyde r e s u l t s i n 36, the s o - c a l l e d seco-prephenate-formaldehyde (SPF) 2 6 u n i t . Wenkert commented upon the s i m i l a r i t i e s between the - 8 ~ F i g u r e 4. The Barger-Ilahn--Robinson-Woodward P o s t u l a t e f o r Indole A l k a l o i d B i o s y n t h e s i s . - 9 -COO H HO' Jr-OH \ H H OH ( 1 7 ) 0 • II H 3 C — C —COO ( 3 4 ) + O ^ / C O O ( 35 ) H "ooc-"OOC\^o ^o 0 ^ / C O O " 'ooc-o ^ c o o 4 e t r o -CHO a l d o l 'OOC OOC CHO ( 3 6 ) , OH F i g u r e 5a. The Wenkert Prephenate P o s t u l a t e f o r Indole A l k a l o i d \ B i o s y n t h e s i s . "00 c CH3C00 H0 -"00C-ooc' -OH ( 45 ) F i g u r e 5b. The Thomas Monoterpene P o s t u l a t e f o r 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 . - 10 -27-30 SPF u n i t and the r e c e n t l y d i s c o v e r e d c y c l o p e n t a n o i d monoterpene g l u c o s i d e s e x e m p l i f i e d by v e r b a n a l i n (37), g e n i p i n (38), aucubin (39) and a s p e r u l o s i d e (40) , and f e l t t h a t these monoterpenes c o u l d be d e r i v e d from the SPF u n i t (dotted l i n e i n F i g u r e 5), or they c o u l d be the r e a l p r e -c u r s o r s to the i n d o l e a l k a l o i d C 9 ~ c i o u n i t s - I n a nY event, b o t h the SPF u n i t and the monoterpenes possess the carbon s k e l e t o n shown i n s t r u c t u r e 9 t o be r e q u i s i t e f o r e n t r y i n t o the corynanthe-strychnos f a m i l y of i n d o l e a l k a l o i d s . .«0Glu C 0 0 C H - , ( 3 7 ) C O O C H . ( 3 8 ) TV U -25 The t h i r d p o s t u l a t e was proposed by Thomas who f e l t t h a t t h e c y c l o p e n t a n o i d monoterpenes c o n t a i n e d t h e s t r u c t u r a l f e a t u r e s p r e s e n t i n t h e Cg^C]_Q u n i t of the i n d o l e a l k a l o i d s , and t h a t t h e i r b i o s y n t h e s e s may be r e l a t e d to t h a t of the i n d o l e systems. In o t h e r words, the monoterpenes were themselves d e r i v e d from the acetate-mevalonate pathway (Figure 5b) independent of carbohydrate metabolism. T h i s o p t i o n was l a t e r 2 6 chosen by Wenkert i n 1962. S h o r t l y a f t e r the appearance of the Thomas-Wenkert 31 p o s t u l a t e , Leete began the f i r s t e x p e r i m e n t a l e v a l u a t i o n of 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 u s i n g r a d i o a c t i v e t r a c e r s t u d i e s . In o r d e r to t e s t the Barger-Hahn-Robinson-Woodward 14 p o s t u l a t e (Figure 4), Leete f e d t y r o s i n e - 2 - C (26) to R auwolfia s e r p e n t i n a and c o u l d not d e t e c t any r a d i o a c t i v i t y i n the a j m a l i n e (41) and r e s e r p i n e (42) which were i s o l a t e d . T h i s t e n t a t i v e r e f u t a t i o n of the Barger-Hahn-Robinson-Woodward 32 h y p o t h e s i s was subsequently confirmed by B a t t e r s b y i n h i s s t u d i e s on the i s o q u i n o l i n e a l k a l o i d s c e p h a e l i n e (43) and emetine (44). A lthough these a l k a l o i d s l a c k an i n d o l e n u c l e u s , they can be thought of as f o r m a l l y d e r i v e d from two t y r o s i n e m o l e c u l e s , and one C g u n i t which i s s t r u c t u r a l l y r e l a t e d to Wenkert's proposed SPF u n i t . 2 ^ When t y r o s i n e - 2 - 1 4 C was f e d to C e p h a e l i s ipecacuanha p l a n t s , the a l k a l o i d s i s o l a t e d were shown to be a c t i v e o n l y a t p o s i t i o n C-3, w i t h no a c t i v i t y found i n the C - l p o s i t i o n as would have been p r e d i c t e d by the Barger-Hahn-Robinson-Woodward p o s t u l a t e as shown i n F i g u r e 4. T h i s e x p e r i -ment then p r o v i d e d c o n v i n c i n g evidence t h a t the cg~C^n, u n i t i - s not t y r o s i n e - d e r i v e d . - 12 -( U2 ) 31 Le e t e t e s t e d WenkertIs prephenic a c i d h y p o t h e s i s by f e e d -14 33 i n g a l a n i n e - 2 - C, a known p r e c u r s o r of p y r u v i c a c i d (34) , t o R. s e r p e n t i n a p l a n t s . The a j m a l i n e (41) i s o l a t e d was of low a c t i v i t y and w i t h o n l y 2% of i t s a c t i v i t y a t the p o s t u l a t e d p o s i t i o n . The Thomas-Wenkert monoterpene p o s t u l a t e was a l s o 14 t e s t e d . Mevalonic a c i d - 2 - C (45) was f e d to R. s e r p e n t i n a , and no a c t i v i t y c o u l d be d e t e c t e d i n the a j m a l i n e (41) which 14 was i s o l a t e d . However, when sodium a c e t a t e - 1 - C was f e d to the p l a n t system, a c t i v e a j m a l i n e (41) was i s o l a t e d i n which ~ 13 ^ 25% o f t h e a c t i v i t y was- l o c a t e d a t CT3 a n d C-r-14. T h e mono-^ t e r p e n e ' p o s t u l a t e r e q u i r e s - a c t i v i t y a t C-^,14, ( X L 9, C^21 a n d C<^16 o f a j m a l i n e ' C411. On t h e B a s i s o f t h e s e e x p e r i m e n t a l f i n d i n g s , L e e t e c h o s e 34 t o e x p a n d t h e S c h l i t t l e r ^ - T a y l o r a c e t a t e t h e o r y . A s shown i n F i g u r e 5 a , t h e L e e t e p r e c u r s o r 46 w h i c h c o r r e s p o n d s t o W e n k e r t ' s SPF u n i t i s b u i l t u p f r o m t h r e e a c e t y l - C o A u n i t s , a m a l o n y l - C o A 35 . . u n i t and f o r m a l d e h y d e . L e e t e was a b l e t o show p o s i t i v e i n c o r p o r a t i o n o f l a b e l l e d m a l o n i c a c i d i n t o a j m a l i n e (41) a n d r e s e r p i n e (42) a f t e r f e e d i n g e x p e r i m e n t s i n R. s e r p e n t i n a . H o w e v e r , t h e a l k a l o i d s p o s s e s s e d 74% o f t h e i r a c t i v i t y a t C-17 a n d n o n e a t C-18 a n d C-19. H e n d r i c k s o n s u b s e q u e n t l y 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 i n o r d e r t o i n c o r p o r a t e a p o l y k e t i d e -t y p e b i o g e n e s i s o f t h e L e e t e p r e c u r s o r . F i g u r e 6b shows t h e H e n d r i c k s o n m o d i f i c a t i o n a s t h e l i n e a r c o n d e n s a t i o n o f f i v e a c e t a t e u n i t s , f o l l o w e d b y l o s s o f t h e t e r m i n a l m e t h y l g r o u p f r o m t h e t e n c a r b o n c h a i n . F u r t h e r c o n d e n s a t i o n w i t h a C^ u n i t f o l l o w e d b y a l d o l c y c l i z a t i o n a n d t h e n a r e t r o - a l d o l c l e a v a g e y i e l d s 4 7 , w h i c h h a s t h e same c a r b o n s k e l e t o n a s L e e t e ' s p r e c u r s o r 46 a n d W e n k e r t ' s SPF u n i t ( 3 6 ) . B a t t e r s b y , w o r k i n g w i t h b o t h R. s e r p e n t i n a a n d C. i p e c a -32 37 c u a n h a ' was u n a b l e t o c o n f i r m t h e r e s u l t s o f L e e t e . H i s r e s u l t s showed random i n c o r p o r a t i o n o f l a b e l l e d s o d i u m a c e t a t e i n t h e C 9 ^ c ^ Q u n £ t ? °£ a x x t n - e a l k a l o i d s s t u d i e d . The e n d o f 38 t h e a c e t a t e h y p o t h e s i s was made o f f i c i a l when L e e t e y e r i f i e d B a t t e r s b y ' s w o r k a n d w i t h d r e w h i s p r e c u r s o r f r o m c o n s i d e r a t i o n . I n t h e same s t u d y , B a t t e r s b y a d m i n i s t e r e d l a b e l l e d s o d i u m - 14 ^ C o A S ^ 0 ^ HCHO 3 CH3COSC0A 0 OOCCH^COSCoA OOC C O S C o A ( 46 ) F i g u r e 6 a . The Leete Acetate-Malonate Hypothesis f o r Indole A l k a l o i d B i o s y n t h e s i s . 5 CH3COSC0A C o A S O C C o A S O C OHC OH C o A S O C CHO ( 47 ) F i g u r e 6D, The. Hendrickson Polyketi.de M o d i f i c a t i o n - 15 -f o r m a t e t o h i s p l a n t s y s t e m s and r e c o v e r e d t h e l a b e l o n l y a t t h e i n d o l i c N - m e t h y l o f a j m a l i n e (.41) and t h e a r o m a t i c O - m e t h y l -39 o f c e p h a e l m e ( 4 3 ) . T h i s f i n d i n g , l a t e r c o n f i r m e d by B a r t o n , i n d i c a t e d t h a t f o r m a t e was m e r e l y l a b e l l i n g t h e C-^  p o o l i n t h e p l a n t s y s t e m s and n o t p a r t i c i p a t i n g i n t h e b u i l d u p o f an SPF u n i t ( s e e F i g u r e 5 ) . More c o n v i n c i n g e v i d e n c e a g a i n s t t h e W e n k e r t p r o p o s a l o f a c a r b o h y d r a t e m e t a b o l i s m d e r i v e d p r e c u r s o r 40 was o b t a i n e d when i t was shown t h a t r a d i o a c t i v e s h i k i m i c a c i d (17) was i n c o r p o r a t e d o n l y i n t h e i n d o l e n u c l e u s o f t h e a l k a l o i d s i n v e s t i g a t e d . T h i s was t o be e x p e c t e d on t h e b a s i s o f t h e known b i o s y n t h e s i s o f t r y p t o p h a n a s shown e a r l i e r i n F i g u r e s 2 a n d 3. B a t t e r s b y was a b l e t o p r o v i d e t e n t a t i v e c o n f i r m a t i o n f o r t h e Thomas-Wenkert m o n o t e r p e n e p o s t u l a t e b y d e m o n s t r a t i n g low 14 i n c o r p o r a t i o n o f s o d i u m m e v a l o n a t e - 2 - C i n t o a j m a l i n e (41) a n d c e p h a e l i n e (43) i n R. s e r p e n t i n a and C. i p e c a c u a n h a . F u r t h e r c o n f i r m a t i o n was f o r t h c o m i n g f r o m t h e work o f Money 41 42 and S c o t t i n V i n c a r o s e a L i n n . , a n d t h e work o f A r i g o n i i n b o t h V. r o s e a L i n n , and V. maj o r L i n n . . More e x p e r i m e n t s a l o n g t h e s e l i n e s , c o u p l e d w i t h d e g r a d a t i v e d a t a , d e m o n s t r a t e d t h e i n t a c t i n c o r p o r a t i o n o f t h e m e v a l o n a t e u n i t a c c o r d i n g t o t h e p a t t e r n r e q u i r e d by t h e Thomas-Wenkert 41-46 h y p o t h e s i s ( F i g u r e 5 b ) . S u b s e q u e n t l y , i t was shown t h a t • n u v, a. I A O ,42,44,46-52 , , / * ov.\ 4 9,50 g e r a n i o l p y r o p h o s p h a t e (48a) ' ' and n e r o l (48b) c o u l d b o t h s e r v e as p r e c u r s o r s t o t h e Cg-C^Q u n i t . M o v i n g f u r t h e r a l o n g t h e pathway, i t was shown t h a t l o g a n i n ( 5 1 ) , now known t o be d e r i v e d f r o m m e v a l o n a t e (4 5) v i a g e r a n i o l (48a) 53-64 a n d / o r n e r o l ( 4 8 b ) , was i n c o r p o r a t e d i n t a c t i n t o t h e - 16 -( 53 ) Figure 7. The Early Stages o f Indole A l k a l o i d Biosynthesis as Proven by Experiment. 53 54 65 54 a l k a l o i d s o f V. r o s e a , ' ' R. s e r p e n t i n a , a n d C. i p e c a c -6 6 u a n h a . M o r e o v e r , l o g a n i n was f o u n d t o c o - o c c u r w i t h t h e 53 64 64 a l k a l o i d s o f V. r o s e a ' a n d S t r y c h n o s n u x v o m i c a . I t i s n o t e w o r t h y t h a t t h e s t e r e o c h e m i s t r y a t C-15 o f y o h i m b i n e (32) i s i d e n t i c a l t o t h e s t e r e o c h e m i s t r y a t t h e c o r r e s p o n d i n g p o s i t i o n o f l o g a n i n (51) . On e i t h e r s i d e o f l o g a n i n (51) o n t h e b i o s y n t h e t i c p a t h w a y a r e d e o x y l o g a n i n ( 5 0 ) , a n d s e c o l o g a n i n ( 5 2 ) . B o t h h a v e b e e n i s o l a t e d f r o m V. r o s e a , a n d h a v e b e e n shown t o be s p e c i f i c a l l y i n c o r p o r a t e d i n t o t h e a l k a l o i d s o f V. r o s e a . ^ " ^ F u r t h e r , t h e i n t e r m e d i a c y o f t h e h y d r o x y g e r a n i o l d e r i v a -t i v e (49) h a s b e e n d e m o n s t r a t e d i n V. r o s e a w i t h r a n d o m i z a t i o n 70 71 o f t h e l a b e l a t t h e p o s i t i o n s m a r k e d 2,6, ' i n d i c a t i n g t h a t o x i d a t i o n a t b o t h o f t h e s e c a r b o n atoms i s a n e c e s s a r y p a r t o f t h e s e q u e n c e t o d e o x y l o g a n i n ( 5 0 ) . W i t h t h e o r i g i n o f t h e Cg-C^Q u n i t o n a s o u n d e x p e r i m e n t a l f o o t i n g , t h e n e x t p r o b l e m was t o d e t e r m i n e how s e c o l o g a n i n (52) was u t i l i z e d b y t h e p l a n t s y s t e m i n t h e b i o s y n t h e s i s o f a n i n d o l e a l k a l o i d . S i m u l t a n e o u s l y a n d i n d e p e n d e n t l y , v i n c o s i d e (54) a n d 72 i s o v i n c o s i d e (55) w e r e i s o l a t e d f r o m V. r o s e a , a n d 73 74 75 s t r i c t o s i d i n e (55) f r o m R h a z y a s t r i c t a . X - r a y a n a l y s i s ' h a s p r o v e n t h e r e l a t i v e c o n f i g u r a t i o n s a b o u t C-3 t o be a s shown. I n a d d i t i o n , i t h a s b e e n shown t h a t v i n c o s i d e (54) i s s p e c i f i -c a l l y i n c o r p o r a t e d b y V. r o s e a p l a n t s i n t o a l l t h r e e t y p e s o f 72 76 77 i n d o l e a l k a l o i d s , ' ' a n d t h a t i t i s i t s e l f d e r i v e d f r o m 72 t r y p t o p h a n (25) a n d l o g a n i n ( 5 1 ) . I s o v i n c o s i d e (55) c o u l d - 18 -not be i n c o r p o r a t e d i n t o any of the a l k a l o i d s s t u d i e d . ( 5 4 ) . ( 55 ) These r e s u l t s i n d i c a t e the answer to the q u e s t i o n : are the d i f f e r e n t f a m i l i e s of i n d o l e a l k a l o i d s b i o s y n t h e s i z e d by d i f f e r e n t convergent pathways, or i s t h e r e a s i n g l e u n i f y i n g s e q u e n t i a l pathway, p o s s i b l y w i t h d i v e r g e n t branches? The f a c t t h a t v i n c o s i d e i s s p e c i f i c a l l y i n c o r p o r a t e d i n t o a l l t h r e e 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 n d i c a t e s t h a t the former i s probably not the case. Rather, i t appears t h a t v i n c o s i d e i s formed by a convergent pathway i n v o l v i n g s e p a r a t e b i o s y n t h e s i s of tryptophan (25) and s e c o l o g a n i n (52). The v i n c o s i d e (54) molecule must then undergo the a p p r o p r i a t e rearrangements to 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 . V i n c o s i d e (54) , then, serve s as a c o n v e n i e n t d i v i d i n g l i n e . The b i o s y n t h e s i s of v i n c o s i d e (54) i t s e l f may be thought of as the e a r l y stages of 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 , w h i l e the subsequent r e a r r a n g e -ments make up the l a t e r stages of 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 . I t can be seen t h a t the corynanthe f a m i l y of a l k a l o i d s can be d e r i v e d d i r e c t l y from v i n c o s i d e (54) i n a s t r a i g h t f o r w a r d manner i n v o l v i n g no rearrangements. Thus, F i g u r e 8 shows the d e r i v a t i o n of g e i s s o s c h i z i n e (1), corynantheine (57), corynan-^ 1 9 r F i g u r e 8. The P o s t u l a t e d D e r i v a t i o n of Corynanthe A l k a l o i d s from V i n c o s i d e ( 5 4 ) . - 20 -t h e i n e aldehyde (56). and a j m a l i c i n e C58) . Proceeding f u r t h e r along the pathway, Wenkert proposed 7 8 i n 1965 t h a t , on s t e r e o c h e m i c a l grounds, the s t r y c h n o s , iboga and aspidosperma a l k a l o i d s c o u l d be d e r i v e d from the corynanthe s k e l e t o n . He proposed a p o s s i b l e mechanism f o r the c o n v e r s i o n of the corynanthe s k e l e t o n to the s t r y c h n o s system, as shown 79 i n pathway A of F i g u r e 9. S c o t t proposed a d i f f e r e n t mechanism (Figure 9, pathway B) f o r the same c o n v e r s i o n , but so f a r e x p e r i m e n t a l data i s l a c k i n g f o r a d e c i s i o n to be made between the two pathways. 2 6 E a r l i e r , Wenkert had put f o r t h a p o s t u l a t e f o r the subsequent rearrangement of the s t r y c h n o s f a m i l y of a l k a l o i d s t o the iboga and aspidosperma systems. As shown i n F i g u r e 10, the iminium i n t e r m e d i a t e 60, which i s analogous to i n t e r m e d i a t e 59 i n F i g u r e 9A, undergoes r e t r o - M i c h a e l r e a c t i o n to form the p i v o t a l i n t e r m e d i a t e 61. Intermediate 61, a f t e 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 a c r y l i c a c i d 62 and subsequent r i n g c l o s u r e y i e l d s the iboga (63) s k e l e t o n . A l t e r n a t i v e l y , i n t e r m e d i a t e 61 can form the c o r r e s p o n d i n g p a r t i a l l y reduced i n t e r m e d i a t e 64 and undergo M i c h a e l a d d i t i o n to the a c r y l i c system f o l l o w e d by t r a n s a n n u l a r c y c l i z a t i o n to form the a s p i d o s -perma (65) r i n g system. The p o s t u l a t e d sequence of b i o s y n t h e s i s , t h a t i s , c o r y -nanthe -*• s t r y c h n o s aspidosperma -> iboga a l k a l o i d s , was g i v e n e x perimental support when i t was found t h a t g e i s s o s c h i z i n e (1) i s i n c o r p o r a t e d i n t a c t i n t o the s t r y c h n o s a l k a l o i d akuammicine 8 0*- 8 2 8 2 8 3 (66) i n V. r o s e a , and co-occurs w i t h i t . ' Furthermore - 21 -F i g u r e 9. The W e n k e r t (A) and S c o t t (B) P o s t u l a t e s f o r t h e B i o s y n t h e s i s of S t r y c h n o s A l k a l o i d s . coo' ( 6 3 ) ( 6 5 J F i g u r e 10. The Wenkert P o s t u l a t e f o r the B i o s y n t h e s i s of Iboga and Aspidosperma A l k a l o i d s . i t was shown t h a t the strychnos a l k a l o i d stemmadenine (6), analogous to Wenkert's i n t e r m e d i a t e 60, i s a c o n s t i t u e n t of V. r o s e a , ^  8 3 and i s i n c o r p o r a t e d i n t a c t 8 ^ * i n t o the iboga and aspidosperma a l k a l o i d s i n t h a t p l a n t system. (6) 80 82 F i n a l l y , S c o t t ' s work w i t h V. r o s e a s e e d l i n g s ' showed t h a t s e q u e n t i a l a n a l y s i s of the a l k a l o i d a l c ontent of germinated s e e d l i n g s r e v e a l s the appearance of corynanthe a l k a l o i d s f i r s t , f o l l o w e d by s t r y c h n o s , then aspidosperma, then iboga a l k a l o i d s . 14 These r e s u l t s were confirmed by f e e d i n g tryptophan-2- C (25) to germinated s e e d l i n g s of V. r o s e a and o b s e r v i n g the s e q u e n t i a l uptake of r a d i o a c t i v i t y . Once a g a i n , the sequence corynanthe -»• s t r y c h n o s -> aspidosperma -»- iboga was observed. Work i n our l a b o r a t o r i e s concerned i t s e l f w i t h the t r a n s -annular c y c l i z a t i o n r e q u i r e d to form the aspidosperma and iboga a l k a l o i d s a c c o r d i n g to Wenkert's p o s t u l a t e (Figure 10). T h i s 8 3—9 7 p r o c e s s was shown to be a f a c i l e one i n v i t r o . For example, o x i d a t i o n of quebrachamine (68) t o i t s N(b) iminium s a l t , f o l l o w e d by c y c l i z a t i o n and r e d u c t i o n , y i e l d s a s p i d o s p e r m i d i n e (69). S i m i l a r l y , 16-carbomethoxycleavamine (8) can be c o n v e r t e d to c a t h a r a n t h i n e (4). ( 8 ) ( A ) Whether or not t r a n s a n n u l a r c y c l i z a t i o n was a p p l i c a b l e i n v i v o , however, remained to be shown. I n i t i a l f e e d i n g experiments r e s u l t e d i n no i n c o r p o r a t i o n of r a d i o a c t i v e l y l a b e l l e d 16-carbomethoxycleavamine (8), 6,7-dehydrovincadine (70), quebrachamine (68) and v i n c a m i n o r e i n e (71) i n t o the 8 8 c o r r e s p o n d i n g aspidosperma or iboga s k e l e t o n s i n V. r o s e a . T h i s p r e l i m i n a r y i n d i c a t i o n of the n o n - u t i l i t y of t r a n s a n n u l a r c y c l i z a t i o n was confirmed by a d m i n i s t e r i n g tryptophan-3-^" 4C (25) to V. minor p l a n t s over v a r y i n g time i n t e r v a l s and o b s e r v i n g the r a t i o of a c t i v i t y i n the t e t r a c y c l i c a l k a l o i d s v i n c a d i n e (72) and v i n c a m i n o r e i n e (71) to t h a t of the penta-8 9 c y c l i c a l k a l o i d s v i n c a d i f f o r m i n e (73) and minoyine (74). T h i s r a t i o was found to be r e l a t i v e l y c o n s t a n t over a time p e r i o d extending from f o u r hours to f o u r t e e n days. The p o s s i b i l i t y t h a t the c o n s t a n t r a t i o might be due to an e q u i l i b r i u m between the t e t r a c y c l i c and p e n t a c y c l i c systems was e l i m i n a t e d when i t was shown t h a t l a b e l l e d minovine (74) t r a n s f e r s no a c t i v i t y to the t e t r a c y c l i c a l k a l o i d s a f t e r a one - 25 -week feeding period. COOCH3 ( 73 ) R = H ( l h ) R = C H 3 The above evidence appeared to indicate the existence of a p i v o t a l intermediate which could be converted i n vivo to the t e t r a c y c l i c and pentacyclic aspidosperma and iboga a l k a l o i d s each independently of the other, and without the necessity of a transannular c y c l i z a t i o n . Thus, the scheme shown i n Figure 2 6 11 appeared to be a v i a b l e a l t e r n a t i v e to the Wenkert postulate. I t i s noteworthy that the key intermediate, a c r y l i c ester 75 strongly resembles the a c r y l i c ester 62 contained i n Wenkert's proposal. Since the dihydropyridinium system contained i n intermediate 75, could be expected to be unstable and d i f f i c u l t to obtain s y n t h e t i c a l l y , i t was f e l t that the corresponding piperideine 90 76 named secodine by Smxth or the hydroxy ester 77 could be useful a l t e r n a t i v e s i n biosynthetic investigations. This view 91 received support when Battersby proposed, on the basis of i s o t o p i c d i l u t i o n experiments that 16,17-dihydrosecodin-17-ol - 26 ^ F i g u r e 11. The P o s t u l a t e d D e r i v a t i o n of Aspidosperma and Iboga A l k a l o i d s from Intermediate 75. - 27 -occurs n a t u r a l l y i n V. rosea and Rhazya o r i e n t a l i s . Subsequently, the i s o l a t i o n o f 16,17,15,20-tetrahydrosecodine (78), 16,17-dihydrosecodine (79) and 16,17,15,20-tetrahydro-s e c o d i n - 1 7 - o l (80) from p l a n t sources was r e p o r t e d 90,92 COOCH3 ( 7 8 ) C O O C H -( 7 9 ) COOCH3 ( 80 ) 3 16,17-Dihydrosecodin-17-ol-(ar- H) (77) was du l y synthe-93 94-97 95 98 s i z e d and a d m i n i s t e r e d t o V. r o s e a , V. minor, ' and 95-97 99 Aspidosperma p y r i c o l l u m . ' In each case, d e t e r i o r a t i o n o f the p l a n t s was observed, and no i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n the a l k a l o i d s i s o l a t e d c o u l d be d e t e c t e d . Thus i t was f e l t t h a t the t r a n s f o r m a t i o n o f the hydroxy e s t e r 77 t o the c o r r e s -ponding a c r y l i c e s t e r was prob a b l y not o c c u r r i n g i n the p l a n t systems under i n v e s t i g a t i o n s . Consequently, the necessary 3 d e h y d r a t i o n was performed i n v i t r o , and s e c o d i n e - ( a r - H) (76) fe d t o the same p l a n t systems. In t h i s case, low but d e f i n i t e i n c o r p o r a t i o n s of a c t i v i t y were observed i n the t h r e e a l k a l o i d a l f a m i l i e s . 9 3 " 9 5 ' 9 8 F u r t h e r s t u d i e s u s i n g v a r i o u s forms of doubly l a b e l l e d secodine and i n v o l v i n g d e g r a d a t i o n o f the a l k a l o i d s i s o l a t e d gave s t r o n g i n d i c a t i o n s t h a t the secodine s k e l e t o n i s i n c o r -- 28 -p o r a t e d i n t a c t i n t o v i n d o l i n e (3), a p p a r i c i n e (81) c a t h a r a n t h i n e (4), 1 0 0 ' 1 0 1 a n d vincamine ( 8 2 ) . 1 0 0 ( 8 3 ) The a l k a l o i d a p p a r i c i n e (81) as w e l l as the a l k a l o i d u l e i n e (83) r e p r e s e n t a n o v e l branch o f the i n d o l e a l k a l o i d f a m i l i e s . I t can be seen t h a t b o t h l a c k the u s u a l e t h y l e n e b r i d g e between the 8 p o s i t i o n on the i n d o l e n u c l e u s and the b - n i t r o g e n o f the a l k a l o i d , and possess o n l y a methylene b r i d g e a t t h a t p o i n t . Up to t h i s p o i n t , i t has been assumed t h a t tryptophan i s the p r e c u r s o r of the i n d o l e nucleus of a l l i n d o l e a l k a l o i d s . T h i s assumption appears to be open to q u e s t i o n i n the cases of a p p a r i c i n e (81), and u l e i n e (83). In f a c t , 2 6 Wenkert proposed i n 1962 t h a t the b i o s y n t h e t i c pathway f o r u l e i n e , as shown i n F i g u r e 12, i n v o l v e s g l y c o s y l i d e n e a n t h r a n i l i c a c i d (84), a p r e c u r s o r of tryptamine analogous to e n o l - 1 ( o -c a r b o x y l p h e n y l a m i n o - ) - l - d e s o x y r i b u l o s e - 5 - p h o s p h a t e (23). Condensation w i t h an a p p r o p r i a t e l y f u n c t i o n a l i z e d C-^ Q u n i t , a t t h a t time thought to be the SPF u n i t (36), f o l l o w e d by conden-s a t i o n w i t h methylamine o r i t s e q u i v a l e n t and c y c l i z a t i o n would H ( 81 ) F i g u r e 13. The D j e r a s s i P o s t u l a t e f o r the B i o s y n t h e s i s of A p p a r i c i n e (81) ^ 30. T V then y i e l d u l e i n e C831. 102 L a t e r , i n 1965, D j e r a s s i proposed t h a t Wenkert's i n t e r m e d i a t e 8 5 c o u l d a l s o serve as a p r e c u r s o r i n the b i o s y n t h e s i s of a p p a r i c i n e (81). I s o m e r i z a t i o n to the e x o c y c l i c iminium s p e c i e s 8 6 f o l l o w e d by c y c l i z a t i o n w i t h the i n d o l e nucleus as shown i n F i g u r e 13 would y i e l d a p p a r i c i n e (81). N e i t h e r one of the above p o s t u l a t e s allows f o r a p p a r i c i n e (81) or u l e i n e (83) to be d e r i v e d from tryptophan i t s e l f , and t h e r e f o r e , b o t h r e g a r d these a l k a l o i d s as products of a separate convergent b i o s y n t h e t i c pathway r a t h e r than as products of some branch along the d i v e r g e n t pathway, which has h e r e t o f o r e been shown to be the case. E x p e r i m e n t a l evidence c o n c e r n i n g the b i o s y n t h e s i s of a p p a r i c i n e (81) and u l e i n e (83) was f i n a l l y p r o v i d e d by our 3 l a b o r a t o r y when try p t o p h a n - (ar- H) (25) was a d m i n i s t e r e d to the A. p y r i c o l l u m p l a n t system. S i g n i f i c a n t i n c o r p o r a t i o n of 99 a c t i v i t y c o u l d be d e t e c t e d i n a p p a r i c i n e (81). Thus, i t appeared t h a t a p p a r i c i n e was probably tryptophan d e r i v e d , 102 c o n t r a r y to the p o s t u l a t e of D j e r a s s i . I f tryptophan i s the t r u e p r e c u r s o r of the i n d o l e p o r t i o n of a p p a r i c i n e (81), a t l e a s t one of the two carbons i n the tryptophan s i d e c h a i n must be extruded. To study t h i s p o i n t , 3 14 s tryptophan l a b e l l e d w i t h H. i n the aromatic r i n g and C i n e i t h e r C-2 or C-3 was f e d to A. p y r i c o l l u m . I t was found t h a t C-3 of tryptophan i s 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 (81) 3 14 w i t h r e t e n t i o n of the R/ C r a t i o . On the other hand, over 97% of the l a b e l a t C-2 was shown to be l o s t . ^ 31 ^ Stemmadenine (61, which, has a l r e a d y been shown to be an important i n t e r m e d i a t e i n the b i o s y n t h e s i s of the aspidosperma 8 0 and iboga a l k a l o i d s , was then c o n s i d e r e d as a p o s s i b l e i n t e r -mediate i n the l a t e r stages of a p p a r i c i n e b i o s y n t h e s i s because of i t s s i m i l a r i t y of s t r u c t u r e w i t h a p p a r i c i n e , and because i t has been found to co-occur w i t h a p p a r i c i n e i n the f r u i t s of - -i-i 103 A. p y r i c o l l u m . 104 A s e r i e s o f experiments ( F i g u r e 14) was run i n o r d e r t o p r o y i d e some i n s i g h t i n t o the mechanism by which C-2 of t r y p t o -phan i s extruded i n the b i o s y n t h e s i s of a p p a r i c i n e (81). Experiment 2 i n d i c a t e s t h a t stemmadenine i s a p r e c u r s o r to a p p a r i c i n e , and t h e r e f o r e , as experiment 4 a l s o suggests, t h a t e x t r u s i o n of C-2 of tryptophan o c c u r s w e l l a f t e r condensation of tryptophan w i t h s e c o l o g a n i n . Further,, experiment 3 suggests, but does not prove, t h a t the c o n v e r s i o n of the f u n c t i o n a l i t y a t C-16 o f stemmadenine t o the e x o c y c l i c methylene of a p p a r i c i n e may be a c o n c e r t e d p r o c e s s , a c c o u n t i n g f o r the low l e v e l of i n c o r p o r a t i o n o f v a l l e s a m i n e (87). A t t h i s p o i n t , a group of French w o r k e r s " ^ 5 put f o r t h a p o s t u l a t e f o r the b i o s y n t h e s i s of a p p a r i c i n e (81), which appears to be c o n s i s t e n t w i t h the above experimental evidence (Figure 15, 10 6 pathway A) . Some time l a t e r , t h i s same group p u b l i s h e d a communication p o s t u l a t i n g a u n i f i e d system 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 (81), u l e i n e (83), guatambuine (90), e l l i p t i c i n e (89)., and o l i v a c i n e (.88) , as shown i n F i g u r e 15, pathways A,B,C, D and E-, r e s p e c t i v e l y . However, no experimental evidence f o r t h i s u n i f i e d h y p o t h e s i s has y e t been p u b l i s h e d , and a p p a r i c i n e - 3 2 -remains t h e o n l y non-tryptophan type a l k a l o i d f o r which, b i o s y n t h e t i c i n f o r m a t i o n i s a y a i l a b l e . - 33 -% I n c o r p o r a t i o n C H . Experiment No. ( 81 ) ( 8 3 ) 1 DL-Try/ptophan- (Ar- H) 0.02 <0.0001 F i g u r e 14. The R e s u l t s of I n c o r p o r a t i o n of V a r i o u s P o s s i b l e Intermediates i n t o A p p a r i c i n e (81) and U l e i n e (83). ( 88 ) F i g u r e 15. 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 Non-tryptamine A l k a l o i d s - 35 -DISCUSSION As was shown i n the i n t r o d u c t i o n , the b i o s y n t h e s i s of i n d o l e a l k a l o i d s of the c o r y n a n t h e - s t r y c h n o s , aspidosperma and iboga f a m i l i e s i s r e l a t i v e l y w e l l e s t a b l i s h e d e x p e r i m e n t a l l y . However, much l e s s i s known about those a l k a l o i d s which do not possess the u s u a l two carbon c h a i n between the 8 p o s i t i o n on the i n d o l e nucleus and the b a s i c n i t r o g e n . The work d e s c r i b e d i n P a r t A of t h i s t h e s i s concerns i t s e l f w i t h p r e l i m i n a r y s t u d i e s o f the b i o s y n t h e s i s of a p p a r i c i n e (81), o l i v a c i n e (88), u l e i n e (83) and guatambuine (90). T h i s work i n c l u d e s the i s o l a t i o n of samples of u l e i n e 107 (83) and o l i v a c i n e (88) from Aspidosperma olivaceum e x t r a c t s , ' the development of o z o n o l y t i c d e g r a d a t i o n procedures f o r a p p a r i c i n e (81) and u l e i n e (83) and the i n v e s t i g a t i o n of the p o s s i b l e i n t e r m e d i a c y of tryptophan (25) and secodine (76). P a r t B concerns i t s e l f w i t h the development of a s y n t h e s i s of stemmadenine (6) which i s s u i t a b l y l a b e l l e d f o r more d e t a i l e d . 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 than had been h i t h e r t o p o s s i b l e . P a r t C of t h i s t h e s i s d i s c u s s e s the r e s u l t s o b t a i n e d w i t h the l a b e l l e d stemmadenine system as a p r e c u r s o r i n Aspidosperma  pyrico1lum. - 36 -P a r t A The e x t r a c t i o n of samples of o l i v a c i n e (88) and u l e i n e (83) from A. oiivaceum M u l l . - A r g . e x t r a c t s was accomplished by f o l l o w i n g the procedure already- p u b l i s h e d by G i l b e r t and 108 D j e r a s s i . T h i s method c o n s i s t e d of t h o r o u g h l y mixing the e x t r a c t w i t h an aqueous s o l u t i o n of a c e t i c a c i d (10%), f i l t e r i n g the mixture through c e l i t e , and e x t r a c t i n g the f i l t r a t e w i t h hexane, benzene and f i n a l l y w i t h c h l o r o f o r m . The aqueous p o r t i o n , a f t e r b e i n g a d j u s t e d to pH 8, was e x t r a c t e d w i t h c h l o r o f o r m and a second e x t r a c t i o n f o l l o w e d a t pH 10. I t was found t h a t b o t h e x t r a c t s were s i m i l a r , and c o u l d be combined f o r f u r t h e r p u r i f i c a t i o n . Column chromatography through alumina ( a c t i v i t y I I I ) , f o l l o w e d by t h i n l a y e r chromatography on s i l i c a g e l a llowed pure o l i v a c i n e (88) and u l e i n e (83) to be o b t a i n e d . 109 I t has been shown t h a t o l i v a c i n e (88), when t r e a t e d w i t h methyl i o d i d e to y i e l d o l i v a c i n e methiodide (9) f o l l o w e d by c a t a l y t i c h y d r ogenation y i e l d s guatambuine (90), as shown i n F i g u r e 16. F u r t h e r , work i n our l a b o r a t o r y on the p l a n t 95 system A. p y r i c o l l u m made a p p a r i c i n e a v a i l a b l e i n 100 mg q u a n t i t i e s . Thus, samples of a l l the a l k a l o i d s of i n t e r e s t were r e l a t i v e l y e a s i l y a v a i l a b l e . The reason samples of the a l k a l o i d s to be s t u d i e d were r e q u i r e d i s t h r e e f o l d . F i r s t , they would p r o v i d e a method of comparison w i t h the a l k a l o i d s of A. a u s t r a l e to determine t h e i r presence or absence i n t h a t p l a n t system. Second, they were - 37 -needed as s t a r t i n g m a t e r i a l s i n d e g r a d a t i o n s t u d i e s . T h i r d , they c o u l d serve as c a r r i e r s i n i s o t o p i c d i l u t i o n s t u d i e s . That i s , samples of the i n a c t i v e a l k a l o i d s c o u l d be mixed w i t h the crude e x t r a c t s o b t a i n e d from a p l a n t which had been f e d r a d i o a c t i v e l y l a b e l l e d compounds. The a l k a l o i d s which had been added to the e x t r a c t c o u l d then be r e - i s o l a t e d , p u r i f i e d and checked f o r r a d i o a c t i v i t y . Thus, i n f o r m a t i o n c o u l d be o b t a i n e d on the b i o s y n t h e s i s of a l k a l o i d s p r e s e n t i n the p l a n t i n v ery s m a l l amounts without the n e c e s s i t y of c o n d u c t i n g l a r g e s c a l e p l a n t f e e d i n g s . ( 90 ) F i g u r e 16. The C onversion of O l i v a c i n e (99) to Guatambuine (90). W i t h the r e q u i r e d a l k a l o i d s i n hand, work was begun on 95 d e v e l o p i n g d e g r a d a t i v e procedures. Work i n our l a b o r a t o r y had a l r e a d y e s t a b l i s h e d t h a t secodine (ar- 3H) and ( 1 4COOCH 3) i s 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 A. p y r i c o l l u m . As w e l l , r v 38 -secodine (19— 3 H l was being made a v a i l a b l e as a precursor." 1" 0^ T h e r e f o r e , an o z o n o l y t i c d e g r a d a t i o n of a p p a r i c i n e , as shown i n F i g u r e 17 appeared to be the method of c h o i c e . O z o n o l y s i s of a p p a r i c i n e would c l e a v e the 15,16 and 18,19 double bonds and l i b e r a t e formaldehyde and acetaldehyde, r e s p e c t i v e l y , and without l o s s of the hydrogen a t p o s i t i o n 18. Steam d i s t i l l -a t i o n of the o z o n o l y s i s r e a c t i o n m i x t u r e i n t o a s a t u r a t e d s o l u t i o n of dimedone (92) r e s u l t e d i n the f o r m a t i o n of formaldehyde-bisdimedone (93) and acetaldehyde-bisdimedone (94). The acetaldehyde d e r i v a t i v e 94 c o u l d be s e l e c t i v e l y c y c l i z e d to the t r i c y c l i c d e r i v a t i v e 95, a l l o w i n g the mixture t o be e a s i l y s e p a r a b l e by t h i n l a y e r chromatography on s i l i c a g e l . A l t e r n a t i v e l y , the acetaldehyde t r i c y c l i c d e r i v a t i v e 95 c o u l d be s e l e c t i v e l y e x t r a c t e d from a b a s i c s o l u t i o n o f compounds 93 and 95. A c i d i f i c a t i o n o f the aqueous phase f o l l o w e d by e x t r a c t i o n would a f f o r d the b i c y c l i c formaldehyde derivative."'""'"'''' Thus, an easy d e g r a d a t i o n was a t hand to determine whether or not secodine (76) i s 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 . F u r t h e r , the i d e n t i c a l o z o n o l y s i s procedure c o u l d a l s o be a p p l i e d to u l e i n e (83). In t h i s case, o n l y the e x o c y c l i c methylene was c l e a v e d , and the workup was g r e a t l y s i m p l i f i e d . Steam d i s t i l l -a t i o n of the o z o n o l y s i s r e a c t i o n m i x t u r e i n t o a s a t u r a t e d aqueous s o l u t i o n of dimedone (92) r e s u l t e d i n f o r m a t i o n of formaldehyde-bisdimedone (93). E v a p o r a t i o n of the s o l v e n t under reduced p r e s s u r e f o l l o w e d by r e c r y s t a l l i z a t i o n from e t h a n o l y i e l d e d the pure c r y s t a l l i n e d e r i v a t i v e i n e x c e l l e n t o v e r a l l y i e l d . ( 9U ) ( 9 3 ) F i g u r e 17. The O z o n o l y t i c Degradation of A p p a r i c i n e (81). The c o n d i t i o n s f o r the s y n t h e s i s of secodine (76), as shown i n F i g u r e 18, had p r e v i o u s l y been determined i n our 93 95 l a b o r a t o r y ' . N e v e r t h e l e s s , xt was necessary to r e p e a t t h i s s y n t h e t i c sequence i n o r d e r 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 of r a d i o a c t i v e secodine f o r the b i o s y n t h e t i c s t u d i e s d e s c r i b e d i n t h i s t h e s i s . The c h l o r o e t h y l i n d o l e 96 was condensed w i t h an excess of 3 - e t h y l p y r i d i n e i n a s e a l e d tube at 120° f o r 24 hours. The r e s u l t i n g p y r i d i n i u m s a l t was p r e c i p i t a t e d by pouring the m i x t u r e i n t o a l a r g e excess of e t h y l e t h e r . Conversion of ( 76 ) F i g u r e 18. The S y n t h e s i s of Secodine-(Ar- 3H) and C 1 4COOCH.J (26). - 41 -the p y r i d i n i u m s a l t 97 to the c o r r e s p o n d i n g p i p e r i d e i n e 98 was accomplished w i t h sodium b o r o h y d r i d e i n methanol a t 0°. The e t h y l e s t e r 98 was then reduced w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g t e t r a h y d r o f u r a n to the c o r r e s p o n d i n g a l c o h o l 99, which was t r e a t e d w i t h benzoyl c h l o r i d e t o form the c o r r e s p o n d i n g benzoate 100. Column chromatography on alumina ( a c t i v i t y I I I ) a f f o r d e d the pure benzoate 100 i n about 70% o v e r a l l y i e l d from the s t a r t i n g c h l o r o e t h y l i n d o l e 96. Treatment of the benzoate 100 w i t h a f i v e f o l d excess of potassium cyanide a t 95° a f f o r d e d the n i t r i l e 101 i n y i e l d s which v a r i e d from 50% to 70%. F o r the purpose of i n t r o d u c i n g a r a d i o a c t i v e l a b e l i n t o the carbomethoxy group of secodine 14 (76), potassium c y a n i d e - C was used to form the n i t r i l e 101. In t h i s case, use of a f i v e f o l d excess o f potassium cyanide w i t h s u f f i c i e n t s p e c i f i c a c t i v i t y to be u s e f u l f o r b i o s y n t h e t i c purposes would be p r o h i b i t i v e l y expensive. T h i s problem was 14 circumvented i n the f o l l o w i n g manner. The potassium c y a n i d e - C to be used i n the experiment was d i l u t e d w i t h i n a c t i v e potassium cyanide t o one e q u i v a l e n t , and the r e a c t i o n i n i t i a t e d by adding the benzoate 100 d i s s o l v e d i n N,N-dimethylformamide (DMF) and r a i s i n g the temperature to 95°. A f t e r a s h o r t time a t t h i s temperature, f o u r more e q u i v a l e n t s of i n a c t i v e potassium cyanide were added to a l l o w the r e a c t i o n to proceed to completion. In t h i s way, a h i g h e r s p e c i f i c a c t i v i t y c o u l d be o b t a i n e d f o r the n i t r i l e than when the e n t i r e d i l u t i o n took p l a c e p r i o r to the s t a r t of the r e a c t i o n . The n i t r i l e 101 was subsequently c o n v e r t e d to the c o r r e s -- 42 -ponding methyl e s t e r 102 u s i n g the procedure developed by 113 Wenkert. The n i t r i l e was d i s s o l v e d i n methanol whxch c o n t a i n e d 1% water. S a t u r a t i o n of t h i s s o l u t i o n a t 0° w i t h hydrogen c h l o r i d e gas f o l l o w e d by a l l o w i n g the mixture to stand a t room temperature f o r 4 8 hours r e s u l t e d i n f o r m a t i o n of the methyl e s t e r 102 i n good y i e l d . A t t h i s p o i n t i n the s y n t h e t i c sequence, t r i t i u m c o u l d be i n t r o d u c e d i n t o the aromatic r i n g of the molecule by a l l o w i n g the e s t e r 102 to r e a c t w i t h t r i t i a t e d 3 t r i f l u o r o a c e t i c a c i d (CF^COO H) a t room temperature f o r 48 hours. The t r i t i a t e d methyl e s t e r 103 c o u l d be r e c o v e r e d from t h i s r e a c t i o n mixture i n about 80% y i e l d . F o r m y l a t i o n of the methyl e s t e r 102 o r 103, was accomplished by f o r m a t i o n of the e n o l a t e a n i o n w i t h sodium h y d r i d e i n benzene f o l l o w e d by a d d i t i o n of methyl formate to form the e n o l 104. The e n o l 104 was then s u b j e c t e d t o r e d u c t i o n w i t h sodium 93 95 b o r o h y d r i d e i n methanol. I t had p r e v i o u s l y been determined ' t h a t t h i s r e d u c t i o n r e q u i r e d low temperatures i n o r d e r to a v o i d f o r m a t i o n of the d i o l 105. T h i s r e a c t i o n was improved by lower-i n g the temperature to -4 5° and adding the sodium b o r o h y d r i d e i n very s m a l l amounts over the course of the r e a c t i o n . Although the improved method was. somewhat t e d i o u s , i n v o l v i n g r e g u l a r a t t e n t i o n over a twelve hour time p e r i o d , the u n d e s i r e d d i o l 105 c o u l d be almost t o t a l l y a voided, w i t h a consequent i n c r e a s e i n y i e l d of the d e s i r e d hydroxy e s t e r 77. The hydroxy e s t e r 77 c o u l d be e a s i l y r e c r y s t a l l i z e d , and was r e l a t i v e l y s t a b l e to long term s t o r a g e i n a vacuum d e s s i -c a t o r . Thus, when a p l a n t f e e d i n g was to be undertaken, o n l y - 43 -the amount of hydroxy e s t e r 77 needed was dehydrated f o r immediate a d m i n i s t r a t i o n of secodine (7 6) to the p l a n t system. T h i s d e h y d r a t i o n was accomplished i n the manner p r e v i o u s l y 93 95 r e p o r t e d . ' Thus, the hydroxy e s t e r was t r e a t e d w i t h sodium h y d r i d e i n benzene, then q u i c k l y passed through a s h o r t column of alumina ( a c t i v i t y I I I ) . I n t h i s manner, secodine (76) c o n t a i n i n g t r i t i u m i n the 14 aromatic r i n g and C i n the c a r b o n y l of the carbomethoxy group was o b t a i n e d . In o r d e r t o p r o v i d e more d e t a i l e d b i o s y n t h e t i c r e s u l t s , an a l t e r n a t i v e s y n t h e s i s o f secodine (76) was developed i n our l a b o r a t o r y and t h i s allowed t r i t i u m to be i n t r o d u c e d a t C-19.^^ T h i s s y n t h e s i s , shown i n F i g u r e 19, a l l o w s the i n t r o -d u c t i o n o f 3 - e t h y l p y r i d i n e a t a r e l a t i v e l y l a t e stage i n the s y n t h e t i c pathway, making t h i s r o u t e more economical than t h a t p r e v i o u s l y developed. The s y n t h e s i s summarized i n F i g u r e 19 has a l r e a d y been d i s c u s s e d elsewhere"*"^ and w i l l be d e a l t w i t h o n l y b r i e f l y here. Commercially a v a i l a b l e e t h y l i n d o l e - 2 - c a r b o x y l a t e (106) was reduced t o the c o r r e s p o n d i n g a l c o h o l 107 w i t h l i t h i u m aluminum h y d r i d e . Formation of the corres p o n d i n g benzoate 108 w i t h b e n z o y l c h l o r i d e f o l l o w e d by displacement w i t h potassium cyanide y i e l d e d the n i t r i l e 109. The n i t r i l e was t r e a t e d w i t h methanolic hydrogen c h l o r i d e to p r o v i d e m e t h y l - 2 - i n d o l y l a c e t a t e (110). R e a c t i o n o f the methyl e s t e r 110 w i t h e t h y l e n e oxide and s t a n n i c c h l o r i d e y i e l d e d the h y d r o x y e t h y l i n d o l e d e r i v a t i v e 111. T r e a t -ment of the h y d r o x y e t h y l i n d o l e 111 w i t h p _ - t o l u e n e s u l f o n y l c h l o r i d e f o l l o w e d by 1 - ( 3 ' - p y r i d y l ) - e t h a n e - ( 1 - H) (116) p r o v i d e d - 44 -F i g u r e 19, The S y n t h e s i s of Secodine- (19- 3H) t h e c o r r e s p o n d i n g p y r i d i n i u m s a l t 1 1 2 . R e d u c t i o n o f t h e p y r i d i n i u m s a l t 112 w i t h s o d i u m b o r o h y d r i d e y i e l d e d t h e d e s i r e d p i p e r i d e i n e 1 0 3 . The p i p e r i d e i n e t h u s o b t a i n e d c o u l d t h e n be c o n v e r t e d t o s e c o d i n e (76) i n t h e manner d e s c r i b e d i n F i g u r e 18. 3 The s y n t h e s i s o f 1 - ( 3 ' - p y r i d y l ) - e t h a n e - ( 1 - H) h a s a l s o b e e n d e s c r i b e d e l s e w h e r e 1 " ^ a n d w i l l b e o n l y b r i e f l y d e s c r i b e d h e r e . A s shown i n F i g u r e 2 0 , 3 - a c e t y l p y r i d i n e (113) was t r e a t e d 3 w i t h s o d i u m b o r o t r i t i i d e t o f o r m 1 - ( 3 1 p y r i d y l ) - e t h a n o l - ( 1 - H) ( 1 1 4 ) . T r e a t m e n t w i t h a c e t i c a n h y d r i d e t o f o r m t h e c o r r e s p o n d i n g a c e t a t e 115 f o l l o w e d b y c a t a l y t i c h y d r o g e n o l y s i s r e s u l t e d i n t h e 3 f o r m a t i o n o f t h e d e s i r e d 1 - ( 3 1 - p y r i d y l ) - e t h a n e - ( 1 - H) ( 1 1 6 ) . 0 3, . l i U H (113) (114) 3 H OAc 3 H H H Pd/C 2 ( 115 ) ( 1 1 6 ) F i g u r e 20. The S y n t h e s i s o f 1 - C 3 ' - p y r i d y l ) - E t h a n e - ' C l - H) C 1 1 6 ) . - 46 -With a l l the necessary compounds at hand, i t was now p o s s i b l e t o commence b i o s y n t h e t i c f e e d i n g experiments t o determine the p o s s i b l e i n t e r m e d i a c y of tryptophan and/or secodine i n the b i o s y n t h e s i s of a p p a r i c i n e (81), u l e i n e (83), o l i v a c i n e (88) and guatambuine (90). I t was d e c i d e d to c o n t i n u e the work which had a l r e a d y 95 been done on the b i o s y n t h e s i s of a p p a r i c i n e (81) i n 3 14 A. p y r i c o l l u m . To t h i s end, s e c o d i n e - ( 1 9 - H, COOCH^) 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 to r o o t c u t t i n g s of A. p y r i c o l l u m f o r f i v e days. I s o l a t i o n of a p p a r i c i n e by the procedure 95 p r e v i o u s l y d e s c r i b e d was f o l l o w e d by o z o n o l y t i c d e g r a d a t i o n and i s o l a t i o n of the dimedone d e r i v a t i v e s of acetaldehyde and formaldehyde. The r e s u l t s o f t h i s experiment, as w e l l as those 95 114 experiments p r e v i o u s l y d e s c r i b e d are shown i n T a b l e s 1 and 2. Experiment 1 p r o v i d e d an i n d i c a t i o n t h a t the i n d o l e n u c l e u s of secodine was being used by the p l a n t system i n the b i o s y n t h e s i s of a p p a r i c i n e (81). Furthermore, experiments 2 and 3 showed t h a t the i n d o l e nucleus and the carbomethoxy group on C-16 were being i n c o r p o r a t e d as an i n t a c t u n i t . S i n c e the o r i g i n a l P o t i e r p o s t u l a t e f o r the b i o s y n t h e s i s of a p p a r i c i n e " ^ 5 (Figure 15, pathway A) c a l l e d f o r the l o s s of the carbomethoxy group and r e t e n t i o n of C-17, a m o d i f i c a t i o n of the p o s t u l a t e seemed to be necessary. R e c o g n i t i o n of t h i s f a c t 106 was p r o v i d e d by the F r e n c h group i n a subsequent p u b l i c a t i o n . Experiment 4 p r o v i d e d the f i r s t i n d i c a t i o n t h a t the secodine m olecule as a whole was being 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 (81). I f the o v e r a l l r o l e of secodine 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 T a b l e 1.. R e s u l t s o f I n c o r p o r a t i o n o f Secodine (76) i n t o A p p a r i c i n e (81) Experiment Number Compound Fed % R a t i o o f A c t i v i t y R a t i o o f A c t i v i t y I n c o r p o r a t i o n „ ^ 3 T T ,14„ , . . . 3„ ,14, Fed H/^C I s o l a t e d H/ C I 9 5 3 Secodine-(Ar- H 0.01 295 Secodine- ( 1 4COOCH 3) 0.01 _ _ 3 9 5 Secodine-(Ar- 3H, 1 4COOCH 3) 0.015 8.7 8.4 4 9 5 Secodine-(3,14,15,21- 3H, 14 COOCH3) 0.009 4.2 2.2 5 Secodine-(19- 3H, 1 4COOCH 3) 0.024 3. 98 2.05 S p e c i f i c A c t i v i t i e s A s s o c i a t e d w i t h t h e E x p e r i m e n t s i n T a b l e 1. E x p e r i m e n t A c t i v i t y F e d S p e c i f i c A c t i v i t y F e d S p e c i f i c A c t i v i t y Number I s o l a t e d 3 H ^"4C 3 H dpm/mmol "*"4C dpm/mmol 3 H dpm/mmol "^4C dpm/mmol 1 2 . 5 7 x l 0 8 1 . 8 2 x l 0 1 0 7 . 6 8 x l 0 5 2 7 . 0 6 x l 0 6 1 . 1 9 x l 0 9 1 . 0 6 c l 0 4 3 l . l l x l O 8 1 . 2 8 x l 0 7 l . l O x l O 1 0 1 . 2 8 x l 0 9 1 . 6 x l 0 5 1 . 9 4 x l 0 4 4 4 . 4 2 x l 0 7 1 . 0 7 x l 0 7 9 . 8 2 x l 0 9 1 . 2 7 x l 0 9 3 . 0 3 x l 0 4 1 . 3 5 x l 0 4 5 6 . 2 x l 0 6 1 . 5 6 x l 0 6 1 . 1 8 x l 0 1 0 . 1 . 9 8 x l 0 9 2 . 1 3 x l 0 3 1 . 0 6 x l 0 3 ~ 49 -ChhOX COOCI-LV COOCH3 ( 75 ) aspidosperma and iboga a l k a l o i d s ( Figure 11) CH2OHC°OCH (6) CH?C COOCH3 ( 117 ) a p p a r i c i n e (81) u l e i n e (83) guatambuine (90) o l i v a c i n e (88) e l l i p t i c i n e (89) (Figure 15) F i g u r e 21. The Proposed R e l a t i o n s h i p of Secodine (76) to Stemmadenine (6) i n Indole A l k a l o i d B i o s y n t h e s i s . - 50 -i s as shown i n F i g u r e 21, then the o x i d a t i o n of secodine i n the iminium s p e c i e s 75 f o l l o w e d by c y c l i z a t i o n and rearrangement o f the r e s u l t i n g enamine double bond t o form stemmadenine 16) would be expected t o r e s u l t i n the observed lowering o f the 3H/^"4C r a t i o i n experiment 4. 95 With the data from experiments 1-4 a l r e a d y a v a i l a b l e , i t remained to p r o v i d e d e g r a d a t i v e evidence f o r the i n t a c t i n -c o r p o r a t i o n of secodine i n t o a p p a r i c i n e . The de g r a d a t i o n s which were c a r r i e d out f o r experiments 2 and 5 are shown i n Table 3. Sinc e the t r i t i u m a t the C-19 p o s i t i o n of secodine i n experiment 5 would be expected t o be p r e s e n t i n equal amounts i n both the R and S c o n f i g u r a t i o n s , i t might be expected t h a t s t e r e o s p e c i f i c enzymic c o n v e r s i o n o f the enamine 118 to stemmadenine (6) would r e s u l t i n the observed l o s s o f 50% of the t r i t i u m l a b e l r e l a t i v e to 1 4 C . Table 3. S p e c i f i c A c t i v i t i e s A s s o c i a t e d w i t h the O z o n o l y t i c Degradation o f A p p a r i c i n e (81) i n Experiments 2 and 5 (Table 1 ) . u ^ CH 20 i C H 3 C 0 ( 81 ) 4 95 1.06x10 dpm/mmol Cexpt.2) 1.06xl0 3 dpm/mmol (.expt. 5) ( 1 4C) 2.13x10 3 dpm/mmol Cexpt.5) C3H) 1.05x10 1. 04x10' 2.13x10" - 51 -Once the data f o r a p p a r i c i n e had been o b t a i n e d , i t was d e c i d e d to i n v e s t i g a t e the A. a u s t r a l e p l a n t system w i t h the hope o f o b t a i n i n g b i o s y n t h e t i c i n f o r m a t i o n on u l e i n e (83)., o l i v a c i n e (88) and guatambuine (90). The p r e c u r s o r s to be s t u d i e d were f e d h y d r o p o n i c a l l y as t h e i r a c e t a t e s a l t s t o whole A. a u s t r a l e p l a n t s f o r 5 days. The crude a l k a l o i d a l e x t r a c t was d i l u t e d w i t h i n a c t i v e samples of the d e s i r e d a l k a l o i d s , which were r e - i s o l a t e d by t h i n l a y e r chromatography and r e c r y s t a l l i z e d to c o n s t a n t a c t i v i t y . The r e s u l t s of these experiments are shown i n T a b l e s 4 and 5. Experiment 1 p r o v i d e d an i n d i c a t i o n t h a t o l i v a c i n e and guatambuine might be d e r i v e d from tr y p t o p h a n , but no a c t i v i t y c o u l d be d e t e c t e d i n the u l e i n e i s o l a t e d i n t h i s experiment. Experiments 3-5 i n v o l v i n g doubly l a b e l l e d tryptophan r e s u l t e d i n e i t h e r e r r a t i c r a t i o s or extremely low l e v e l s of r a d i o a c t i v i t y i n the a l k a l o i d s i s o l a t e d . I f the P o t i e r 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 these a l k a l o i d s ( F i g u r e 15) i s c o r r e c t , C-2 of tryptophan s h o u l d be l o s t i n the cases of a p p a r i c i n e and o l i -v a c i n e , and r e t a i n e d i n the cases of u l e i n e and guatambuine. Although the i n c o r p o r a t i o n s are extremely low, the r a t i o s of 3 14 H/ C a c t i v i t y i n experiments 3 and 5 do show an i n c r e a s e i n the case of o l i v a c i n e . The i n c r e a s e observed f o r a p p a r i c i n e had a l r e a d y been e s t a b l i s h e d i n the A. p y r i c o l l u m p l a n t system."^ As w e l l , i t can be seen from experiment 3 t h a t C-2 of t r y p t o p h a n appears to be r e t a i n e d i n the b i o s y n t h e s i s of u l e i n e . The case of guatambuine i s , however, ambiguous. Experiment 4 shows t h a t C-3 of tryptophan i s i n c o r p o r a t e d T a b l e 4. The Various Compounds Fed to A. a u s t r a l e Experiment Number Compound Fed Weight Wet P l a n t Fed mg Weight g 14 A c t i v i t y Fed C dpm H dpm R a t i o Fed 3 ,14 H/ C 1 Tryptophan-(Ar- 3H) 6. 01 14. 0 8.28xl0 7 2 Secodine-(Ar- 3H, 1 4COOCH 3) 3. 43 15. 7 3 . 0 x l 0 7 8 . 7 x l 0 7 2. 9 3 Tryptophan-(Ar- 3H,2- 1 4C) 7. 05 33. 7 6.25xl0 7 1.90xl0 8 3. 0 4 Tryptophan-(Ar- 3H,3- 1 4C) 9. 52 25. 0 8.45xl0 7 2 . 9 7 x l 0 8 3. 5 5 Tryptophan-(Ar- 3H,2- 1 4C) 17. 8 51. 0 1.33xl0 8 4.18xl0 8 3. 15 6 Secodine-(3,14,15,21- 3H, 3. 28 27. 0 1.0 9 x l 0 7 2 . 1 9 x l 0 7 2. 01 14 COOCH3) to T a b l e 5. I n c o r p o r a t i o n R e s u l t s A s s o c i a t e d w i t h T a b l e 3 Experiment A l k a l o i d S p e c i f i c A c t i v i t y % I n c o r p o r a t i o n R a t i o Number ( D i l u t i o n mg) I s o l a t e d (dpm/mmol) 1 4 C 3H 1 4 c 3H V 1 4 c 1 O l i v a c i n e (6.6) 2.01X10 7 * 241 A p p a r i c i n e (0) 2.0x10;: 208 U l e i n e (8.1) 5.19x10^ 001 — _ _ _ Guatambuine (8.0) 4.28x10 • 00534 2 O l i v a c i n e (4.9) 2. 66x10^ 1.04x10?, 0093* * 013 3.90 A p p a r i c i n e (0) 1. 36x10 2.75x10 0391 # 0274 2.04 U l e i n e (10.0) not„countable ~ <. 001 <. 001 Guatambuine . (10.0) 3. 35xlO D 7.55xlO b <. 001 <• 001 2.25 3 O l i v a c i n e (7.7) 2. 4 0 x l 0 J ? 1.72x10^ 0 0 0 5 ^ • 0011 7.18 A p p a r i c i n e (0) 6. 8 9 x l O J ? 1.06x10^ 0015 078* 154 U l e i n e (7.9) 7. 42x10;? ' 2.14x10^ m 0011 0011 2. 97 Guatambuine (9.2) 8. 24x10 3.34x10 • 0015 • 0022 4. 06 4 O l i v a c i n e (11.0) 1. 5 * 63x10:: 9.09x10^ 001 001 5. 57 A p p a r i c i n e (0) 9 .0x10 2.64xl0 a # 127 105 2. 9 0 U l e i n e (4.7) not countable <. 001 <. 001 Guatambuine (5.1) not countable <. 001 <• 001 5 . O l i v a c i n e (30.1) 1. 5* 5* 05x10 7.24x10 <. 001 <. 001 6.90 A p p a r i c i n e (0) (not counted) U l e i n e (8.2) not countable <. 001 <• 001 Guatambuine (10.1) not countable <. 001 <• 001 6 O l i v a c i n e (13) 8. 34x10^ 1.85X107. * 146 * • * 160 * 2.22 A p p a r i c i n e (0) 1. 67x10 4.17x10 # 00365 ^ 00508 2.50 U l e i n e (14) not countable <. 001 <. 001 Guatambuine (10) not countable <. 001 <. 001 * not c o n s t a n t a c t i v i t y 54 -without l o s s i n t o a p p a r i c i n e , as had been shown i n A. p y r i -104 c o l l u m . The P o t i e r p o s t u l a t e (Figure 15) p r e d i c t s t h a t C-3 of tryptophan should be l o s t i n the b i o s y n t h e s i s of o l i v a c i n e , guatambuine and u l e i n e . While no data c o u l d be o b t a i n e d f o r u l e i n e and guatambuine, o l i v a c i n e d i d show a r i s e 3 14 i n the H/ C r a t i o i n agreement w i t h th e o r y . Experiments 2 and 6, i n v o l v i n g secodine as the compound f e d y i e l d e d no d e f i n i t i v e d a t a . T h i s i s p a r t l y due to the low a c t i v i t i e s a s s o c i a t e d w i t h the i s o t o p i c d i l u t i o n technique r e q u i r e d to handle the s m a l l amounts o f the a l k a l o i d s b e i n g s t u d i e d . As w e l l , F i g u r e 21 shows t h a t i n the proposed pathway from secodine to stemmadenine, the b i o s y n t h e t i c pathway which has been e s t a b l i s h e d f o r the aspidosperma and iboga a l k a l o i d s must be r e v e r s e d . Although t h i s r e v e r s a l appears to take p l a c e i n the case of a p p a r i c i n e , i t does so w i t h r e l a t i v e l y poor e f f i c i e n c y , as can be seen i n T a b l e 1. Thus, the use of secodine as an i n t e r m e d i a t e 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 c o n c e r n i n g o l i v a c i n e , u l e i n e and guatambuine, can be shown to have f o u r main disadvantages. 0 F i r s t , secodine i s 115 r e l a t i v e l y u n s t a b l e . I t has been shown t h a t secodine under-goes a D i e l s - A l d e r d i m e r i z a t i o n to form presecamine (119). I t 95 has been e s t i m a t e d t h a t t h i s d i m e r i z a t i o n prevents roughl y 30% of secodine f e d t o the p l a n t from b e i n g taken up as the monomer. Second, secodine i t s e l f i s not a c t u a l l y on any p r o -posed b i o s y n t h e t i c pathway. Rather, a dehydrogenation must take p l a c e to form the iminium s p e c i e s 75, which i s thought to be a t r u e i n t e r m e d i a t e . ( 119 ) T h i r d , i n o r d e r t o be i n c o r p o r a t e d i n t o o l i v a c i n e , u l e i n e , a p p a r i c i n e and guatambuine, the iminium s p e c i e s 75 must proceed two steps backwards on the e s t a b l i s h e d aspidosperma-iboga b i o s y n t h e t i c pathway i n order to be i n s e r t e d i n t o the pathway l e a d i n g t o the d e s i r e d p r o d u c t . F o u r t h , i t can be seen from Table 1 t h a t secodine i s i n c o r p o r a t e d v e r y p o o r l y even i n a p p a r i c i n e . Whether t h i s i s due to a combination of the f i r s t t h r e e f a c t o r s , o r to some unknown f a c t o r , such as d i f f i c u l t y i n the t r a n s p o r t a t i o n o f secodine to the s i t e of b i o s y n t h e s i s , has not been determined. By way of c o n t r a s t , i t can be seen from F i g u r e 14 t h a t i n A. p y r i c o l l u m , stemmadenine (6) i s 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 about 50 times more e f f i c i e n t l y than s e c o d i n e . On f u r t h e r con-s i d e r a t i o n , i t can be seen t h a t stemmadenine would be 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 p l a n t f e e d i n g s i n v o l v i n g the "non-t r y ptamine" type a l k a l o i d s f o r t h r e e reasons. F i r s t , stemmadenine i s q u i t e s t a b l e and can be s t o r e d f o r long p e r i o d s of time as a c r y s t a l l i n e s o l i d w i t hout e x t e n s i v e decomposition. Second, stemmadenine i s d i r e c t l y on the b i o -s y n t h e t i c pathway proposed by P o t i e r (Figure 15), and need not be a l t e r e d by the p l a n t system i n order to be i n c o r p o r a t e d . T h i r d , stemmadenine has a l r e a d y been shown to be r e l a t i v e l y - 56 -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 . Thus, i t can be seen t h a t stemmadenine i s an.extremely a t t r a c t i v e compound f o r . 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 . However, the o n l y source o f stemmadenine up to t h i s p o i n t has been 116 e x t r a c t i o n from p l a n t sources which were u n a v a i l a b l e to our l a b o r a t o r y i n s i g n i f i c a n t amounts. Even i f stemmadenine c o u l d be o b t a i n e d , o n l y a l i m i t e d amount of b i o s y n t h e t i c i n f o r -mation c o u l d be o b t a i n e d , s i n c e o n l y the aromatic r i n g and the carbomethoxy methyl group c o u l d be l a b e l l e d w i t h t r i t i u m , and 14 o n l y the carbomethoxy methyl group c o u l d be l a b e l l e d w i t h C. As w e l l , the carbomethoxy methyl group i s expected to be l o s t i n the b i o s y n t h e s i s of a l l the "non-tryptamine" a l k a l o i d s . Thus, the most p r a c t i c a l course to take appeared to be to d e v i s e a s y n t h e s i s of stemmadenine which would a l l o w the molecule to be r a d i o a c t i v e l y l a b e l l e d i n such a way t h a t , w i t h the proper d e g r a d a t i v e procedure, i t c o u l d be determined whether or not the stemmadenine s k e l e t o n i s 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 , u l e i n e , o l i v a c i n e and guatambuine. T h i s s y n t h e s i s i s the s u b j e c t which i s d i s c u s s e d i n P a r t B. •r- 57 -P a r t B Stemmadenine C61 was f i r s t i s o l a t e d from Stemmadenia 116 d o n n e l T - s m i t h i i CRose) Woodson i n 1958 and was shown to 117 118 possess s t r u c t u r e 6 i n 19 62. ' At p r e s e n t , o n l y the a b s o l u t e c o n f i g u r a t i o n s a t C-15 and C-19 have been e s t a b -79 118 l i s h e d , .' w h i l e the a b s o l u t e c o n f i g u r a t i o n about C-16 remains t o be e s t a b l i s h e d . c ^ H C O O C H 3 (6) An i n t e r e s t i n g c o r r e l a t i o n between the s t r u c t u r e s o f 79 stemmadenine (6) and akuammicine (66) was p r o v i d e d by S c o t t . As shown i n F i g u r e 22, preakuammicine (2) was i s o l a t e d from s e e d l i n g s of V i n c a r o s e a and con v e r t e d i n t o a s e p a r a b l e mixture of akuammicine (66) and stemmadenine (6) by the a c t i o n of methanolic sodium b o r o h y d r i d e . As w e l l , l o s s of formaldehyde by preakuammicine to form e x c l u s i v e l y akuammicine was found t o occur upon treatment w i t h methanolic sodium methoxide. These r e s u l t s i n d i c a t e t h a t stemmadenine and akuammicine possess the same c o n f i g u r a t i o n a t both. C-15 and C-19. Moreover, the c o n y e r s i o n of preakuammicine to stemmadenine p r o v i d e s the b a s i s f o r a b i o g e n e t i c type s y n t h e s i s of stemmadenine. - 58 -F i g u r e 22. The C o r r e l a t i o n of Akuammicine (66) and Stemmadenine (6) v i a Preakuammicine (2). From a b i o g e n e t i c s t a n d p o i n t , t h e r e are two p o s s i b l e e n t r i e s i n t o the nine-membered r i n g system of stemmadenine. The r e a c t i o n of an i n d o l e n i n e such as preakuammicine w i t h sodium b o r o h y d r i d e . v i a the iminium 120 to form the i n d o l e system 6 119 120 (Figure 22, pathway a) has precedent i n the l i t e r a t u r e . ' As w e l l , the a - m e t h y l e n e - i n d o l i n e minovine (74) has been shown to r e a c t 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 a c e t i c a c i d t o 121 y i e l d v i n c a m i n o r e i n e (71). Thus, e i t h e r akuammicine or preakuammicine c o u l d serve as a t t r a c t i v e i n t e r m e d i a t e s i n a b i o g e n e t i c type s y n t h e s i s of stemmadenine. The use of akuammicine would r e q u i r e the i n t r o d u c t i o n of the hydroxymethylene group a t C-16 a f t e r f o r m a t i o n of the nine-membered r i n g . O r i g i n a l l y , work i n our l a b o r a t o r y was d i r e c t e d towards the f o r m a t i o n of 19,2 0-dihydropreakuammicine (121) as shown i n F i g u r e 122 23. Thus 18-,9-dihydrocorynantheine (122) was o x i d i z e d w i t h t e r t - b u t y l h y p o c h l o r i t e to y i e l d the 7 - c h l o r o i n d o l e n i n e 123 which- was then c o n v e r t e d to the r h y n c o p h y l l i n e e n o l e t h e r 12 4 w i t h sodium methoxide i n methanol. H y d r o l y s i s of 12 4 w i t h aqueous a c e t i c a c i d t o the c o r r e s p o n d i n g lactam 12 5 was f o l l o w e d by f u r t h e r h y d r o l y s i s t o y i e l d O-desmethylrhyncophy-l l i n e (126). The f r e e aldehyde was then p r o t e c t e d as the c o r r e s p o n d i n g e t h y l e n e a c e t a l 127 and t r e a t e d w i t h Meerwein's reagent to y i e l d O-desmethylrhyncophylline e t h y l e n o l e t h e r e t h y l e n e a c e t a l (128). A l t h o u g h the c y c l i z a t i o n of an e n o l a t e anion i n a system 123 such as compound 128 t o y i e l d the c o r r e s p o n d i n g i n d o l e n i n e 124 or a - m e t h y l e n e - i n d o l i n e has been shown to work i n some cases, r e p e a t e d attempts t o c y c l i z e e n o l e t h e r 128 to form the e t h y l e n e a c e t a l of the aldehyde c o r r e s p o n d i n g to 19,20-dihydropreakuammicine (12 9) were u n s u c c e s s f u l . T h i s r e s u l t was 125 confirmed by W i n t e r f e l d t and the e n o l e t h e r c y c l i z a t i o n approach was abandoned. T h i s t h e s i s concerns i t s e l f w i t h more r e c e n t e f f o r t s t o s y n t h e s i z e stemmadenine u s i n g commercially a v a i l a b l e s t r y c h n i n e (29) as s t a r t i n g m a t e r i a l , and proceeding by way of i t s degra-d a t i o n product Wieland-Gumlich aldehyde ( 1 3 0 ) . 1 2 6 ' 1 2 7 The d e g r a d a t i o n of s t r y c h n i n e to Wieland-Gumlich aldehyde 126 127 (WGA) has been known s i n c e 1932. ' The sequence was improved 12 8 by Anet and Robinson i n 1955, and f i n a l l y t h oroughly s t u d i e d 12 9 and o p t i m i z e d by Schmid and K a r r e r i n 1969. As shown i n F i g u r e 24, s t r y c h n i n e (29) i s t r e a t e d w i t h sodium e t h o x i d e i n the presence of isoamyl n i t r i t e , and the product c r y s t a l l i z e d as ( 121 ) F i g u r e 23. The Attempted S y n t h e s i s of 19,20-Dihydrostemmadenine (121) . - 61 -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 (131). R e a c t i o n of 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 w i t h t h i o n y l c h l o r i d e f o l l o w e d by quenching i n i c e y i e l d s N(a)-cyanoformyl-WGA h y d r o c h l o r i d e (132) as a c r y s t a l l i n e compound. Sodium carbonate h y d r o l y s i s and chromatographic p u r i f i c a t i o n of the r e s u l t a n t product p r o v i d e s c r y s t a l l i n e WGA. F u r t h e r p u r i f i c a t i o n of WGA thus o b t a i n e d can be c a r r i e d out by r e c r y s t a l l i z a t i o n i n e i t h e r c h l o r o f o r m or benzene. F i g u r e 24. The Degradation of S t r y c h n i n e (29) to Wieland-Gumlich Aldehyde (130). 130 Once WGA was o b t a i n e d , f o u r b a s i c s y n t h e t i c problems . remained. F i r s t , the C-18 hydroxy group must be removed i n such a way t h a t t r i t i u m can be i n t r o d u c e d a t C-18. Second, i n o r d e r f o r the r e q u i r e d nine-membered r i n g system to be formed, e i t h e r the 1,2- o r the 2,16-dehydro s p e c i e s must be o b t a i n e d . T h i r d , - 62 -X I 7 must e i t h e r remain as the. aldehyde or be f u r t h e r o x i d i z e d to the c o r r e s p o n d i n g methyl e s t e r . F o u r t h , a way must be found to i n t r o d u c e the remaining carbon atom a t C-16 i n r a d i o a c t i v e form. As an added c o m p l i c a t i o n , s i n c e the s t e r e o c h e m i s t r y about C-16 i n stemmadenine has not been e s t a b l i s h e d , a s u i t a b l e s y n t h e s i s must be a b l e to p r o v i d e e i t h e r or both c o n f i g u r a t i o n s a t C-16 i n the f i n a l p roduct. In r e s e a r c h i n g the v a s t amount of p u b l i s h e d l i t e r a t u r e c o n c erning s t r y c h n o s a l k a l o i d s , i t became immediately apparent t h a t a p p r o p r i a t e use of nomenclature would be a problem. Many a l k a l o i d s were d i s c o v e r e d and named ind e p e n d e n t l y , o n l y to be found i d e n t i c a l i n l a t e r y e a r s . S t i l l o t h e r s , f o r example 133 have been named a f t e r the p a r e n t dimer ( n o r - h e m i - d i h y d r o t o x i -f e r i n e ) or a f t e r a c l o s e l y r e l a t e d monomer (18-desoxy-Wieland-Gumlich a l d e h y d e ) , w h i l e an e q u a l l y c l o s e l y r e l a t e d monomer i s found as n o r - f l u o r o c u r a r i n e (134). (133 ) ( 1 3 4 ) ' In t h i s t h e s i s , the system o f nomenclature proposed by Janot 131 and Le Men as o u t l i n e d by Edwards and Smith w i l l be used. The curan system 135, w i t h no s t e r e o c h e m i s t r y s p e c i f i e d a t p o s i t i o n s 132 2, 16 and 20, i s numbered f o l l o w i n g Bernauer e t a l . Thus, — 63 — Wieland-Gumlich. aldehyde would become 18-hydroxy-2g , 16a-cur-19-ene-17-al h e m i a c e t a l . Due to t h e i r p i v o t a l nature i n the work d e s c r i b e d here, Wieland-Gumlich aldehyde (WGA), nor-f l u o r o c u r a r i n e (134) and akuammicine (66) w i l l be r e f e r r e d t o as such. A l l o t h e r compounds i n the curan system w i l l be named as d e s c r i b e d above. F i g u r e 25 r e p r e s e n t s a summary o f some of the known chemistry of WGA and i t s d e r i v a t i v e s which appeared to be most u s e f u l f o r our purposes. The r e a c t i o n of WGA w i t h sodium boro-h y d r i d e forms 23,16a-cur-19-en-17,18-diol ( 1 3 6 ) . 1 2 8 The d i o l 136 can then be c o n v e r t e d to 2 3,16a-cur-19-en-17-ol (137) i n 132 133 exther of two d i f f e r e n t ways. Bernauer e t a l ' were a b l e to prepare the a l l y l i c bromide 138 w i t h hydrogen bromide gas d i s s o l v e d i n a c e t i c a c i d . The a l l y l i c bromide 138 was then t r e a t e d , w i t h o u t p u r i f i c a t i o n , w i t h z i n c d u s t i n a c e t i c a c i d c o n t a i n i n g a s m a l l amount of methanol to i n c r e a s e the s o l u b i l i t y of the bromide. The r e s u l t a n t product was the d e s i r e d a c e t a t e 139 o b t a i n e d i n o v e r a l l 65% y i e l d . H y d r o l y s i s of the a c e t a t e 139 c o u l d then be accomplished i n e x c e l l e n t y i e l d w i t h methanolic potassium hydroxide. T h i s procedure c o u l d be e a s i l y m o d i f i e d to i n t r o d u c e a t r i t i u m atom a t C-18, thus s o l v i n g one of the b a s i c problems o u t l i n e d above. A l t e r n a t i v e l y , a l e s s t e d i o u s and h i g h e r y i e l d i n g (85%) 134 procedure was d e v i s e d by Wieland. Using a s p e c i a l l y prepared p a l l a d i u m on c h a r c o a l c a t a l y s t , the d i o l 136 c o u l d be s e l e c t i v e l y hydrogenolyzed i n an aqueous s o l u t i o n of a c e t i c a c i d and hydro-c h l o r i c a c i d to y i e l d the d e s i r e d a l c o h o l 137. T h i s procedure - 64 -F i g u r e 2 5. A Summary o f Some Known R e a c t i o n s i n t h e W i e l a n d -G u m l i c h A l d e h y d e S e r i e s . ~ 6 5 — was not c o n s i d e r e d f o r t r i t i a t i o n purposes f o r two reasons. F i r s t , t o conduct the h y d r o g e n o l y s i s w i t h t r i t i u m gas would be t e c h n i c a l l y more d i f f i c u l t than the a l t e r n a t i v e approach v i a the bromide 138. Second, when u s i n g t r i t i u m gas, t h e r e i s a danger of random h y d r o g e n - t r i t i u m exchange as i n the Wilzbach 135 . . l a b e l l i n g t e c h n i q u e . I f even a v e r y s m a l l amount of t r i t i u m were i n t r o d u c e d anywhere but C-18, the l a b e l l i n g procedure would be u s e l e s s i n b i o s y n t h e t i c experiments designed to demon-s t r a t e i n t a c t i n c o r p o r a t i o n of stemmadenine. In p r a c t i c e , the c a t a l y t i c h y d r o g e n o l y s i s approach was the method of c h o i c e when u n l a b e l l e d a l c o h o l 137 was d e s i r e d . As w e l l , the i d e n t i t y of the products o b t a i n e d by b o t h procedures served as an a d d i t i o n a l c o n f i r m a t i o n o f s t r u c t u r e . 136 In 1959, Wieland r e p o r t e d t h a t Oppenauer o x i d a t i o n of the a l c o h o l 137 w i t h potassium t e r t - b u t o x i d e and benzophenone i n benzene y i e l d e d 23,16a-cur-19-en-17-al (134) ( n o r - f l u o r o c u r a r i n e ) . 134 In a l a t e r p u b l i c a t i o n , Wieland r e p o r t e d t h a t the o x i d a t i o n c o u l d be made to proceed d i r e c t l y t o n o r - f l u o r o c u r a r i n e (134) by h e a t i n g the a l c o h o l 137 w i t h l i t h i u m t e r t - b u t o x i d e , benzophenone, 137 and n i t r o b e n z e n e a t 90° i n a s e a l e d tube. Boekelheide r e p o r t e d i n 1964 that'oppenauer o x i d a t i o n of the a l c o h o l 137 w i t h potassium t e r t - b u t o x i d e and benzophenone i n r e f l u x i n g benzene w i t h " r i g i d e x c l u s i o n of a i r " gave n o r - f l u o r o c u r a r i n e (134) as the major product, along w i t h s m a l l amounts of the s a t u r a t e d aldehyde 133. Boekelheide f e l t t h a t t h i s r e s u l t , t o g e t h e r w i t h the apparent s t a b i l i t y of the i s o l a t e d s a t u r a t e d aldehyde 133 to exposure to a i r , i n d i c a t e d t h a t f o r m a t i o n of the a - m e t h y l e n e - i n d o l i n e chromo-- 66 -phore of 134 was a d i r e c t r e s u l t of the Oppenauer r e a c t i o n c o n d i t i o n s , p r o b a b l y v i a the mechanism shown i n F i g u r e 26. \ (HO ) ( 134 ) F i g u r e 26. The Boekelheide Mechanism f o r the Oppenauer O x i d a t i o n of the Aldehyde 133 to N o r - f l u o r o c u r a r i n e (134). I n i t i a l Oppenauer o x i d a t i o n of the i n d o l i n e to form the i n d o l e n -inium s p e c i e s 140 i s f o l l o w e d by l o s s of a p r o t o n and r e a r r a n g e -ment to form the a - m e t h y l e n e - i n d o l i n e 134. I t was found t h a t , i n f a c t , e i t h e r the u n s a t u r a t e d aldehyde 134 or the s a t u r a t e d aldehyde 133, or a mixture of both, c o u l d be o b t a i n e d , depending on the s e v e r i t y of the c o n d i t i o n s used. The r e s u l t s of a s e r i e s of experiments concerning the Oppenauer o x i -d a t i o n of the a l c o h o l 137 are shown i n T a b l e 6. Table 6. E f f e c t s of D i f f e r e n t R e a c t i o n C o n d i t i o n s on the Oppenauer O x i d a t i o n of the A l c o h o l 137. Experiment Time % Y i e l d Number S o l v e n t Base Temperature (hrs.) (133) (134) 1 benzene t-BuOK r e f l u x 4 10 20 2 benzene t-BuOK room temp. .25 r e f l u x 1.25 39 10 3 t o l u e n e t-BuOLi 95° 72 10 47.5 4 t o l u e n e t-BuOK 90° * 3 0 41 * 2 e q u i v a l e n t s of n i t r o b e n z e n e were added a f t e r 3 hours and the r e a c t i o n was stopped 5 minutes l a t e r . ^ 67 -Focperiments 1 and 2 demonstrate t h a t , o t h e r f a c t o r s being e q u a l , the p r o p o r t i o n of u n s a t u r a t e d aldehyde 134 i n the r e a c t i o n mixture i n c r e a s e s as the r e a c t i o n time i n c r e a s e s . In an e f f o r t t o f o r c e the r e a c t i o n to completion, two methods were t r i e d . F i r s t , use o f l i t h i u m , r a t h e r than potassium, t e r t - b u t o x i d e w i t h t o l u e n e as s o l v e n t r e q u i r e d 72 h r s . f o r completion even a t 95° ( e x p e r i -ment 3 ) . As w e l l , t h e r e was s t i l l an a p p r e c i a b l e amount of s a t u r a t e d aldehyde 133 a f t e r the r e a c t i o n was stopped. However, when potassium t e r t - b u t o x i d e was used as the base i n t o l u e n e a t a temperature of 90° (experiment 4), no s t a r t i n g m a t e r i a l remained, and o n l y s a t u r a t e d aldehyde 133 c o u l d be d e t e c t e d by t h i n l a y e r chromatography. When two e q u i v a l e n t s of n i t r o b e n z e n e were added, immediate c o n v e r s i o n o f the s a t u r a t e d aldehyde 13 3 to the unsat-u r a t e d aldehyde 134 was observed, and no s a t u r a t e d aldehyde c o u l d be d e t e c t e d i n the product m i x t u r e . T h i s l a s t r e s u l t , when con-134 s i d e r e d a l o n g w i t h W i e l a n d 1 s method i n d i c a t e t h a t n i t r o b e n z e n e i s t a k i n g an a c t i v e p a r t i n the c o n v e r s i o n of 133 to 134. T h i s 138 i s r e m i n i s c e n t o f the Skraup q u i n o l i n e s y n t h e s i s where sodium ni t r o b e n z e n e s u l f o n a t e i s used as a dehydrogenating agent which promotes the f o r m a t i o n of an aromatic system. Once n o r - f l u o r o c u r a r i n e (134) was o b t a i n e d , t h e r e appeared to be two b a s i c pathways by which i t c o u l d be co n v e r t e d t o stemmadenine (6). As shown i n F i g u r e 27, pathway A, o x i d a t i o n of the aldehyde t o i t s cor r e s p o n d i n g e s t e r would y i e l d akuammi-c i n e (66), which c o u l d be co n v e r t e d to the i n d o l e 141 w i t h sodium borohydride i n a c e t i c a c i d . I n t r o d u c t i o n of the r e q u i r e d hydroxy-methylene group a t C-16 would complete the sequence. - 68 -F i g u r e 27, The Routes Considered f o r the Conversion of Nor-f l u o r o c u r a r i n e (134) to Stemmadenine (6). - 69 -S i n c e c o n y e r s i o n of n o r - f l u o r o c u r a r i n e (134) d i r e c t l y i n t o i t s i n d o l e d e r i v a t i v e with, sodium borohydride i n a c e t i c a c i d would l i k e l y be accompanied by r e d u c t i o n of the aldehyde to i t s c o r r e s p o n d i n g a l c o h o l , i t would be necessary t o p r o t e c t the aldehyde as shown i n pathway B. Two p o s s i b l e modes of p r o t e c t i o n of the aldehyde can be e n y i s i o n e d . F i r s t , the a - m e t h y l e n e - i n d o l i n e chromophore c o u l d be r e t a i n e d , as i n 142. An example of t h i s would be f o r m a t i o n of the e t h y l e n e a c e t a l d e r i v a t i v e 142 (X = OCH 2CH 20). Second, an i n d o l e n i n e e n o l d e r i v a t i v e c o u l d be formed, such as the e n o l a c e t a t e 143 (X = OAc). C o n v e r s i o n of e i t h e r o f these two compounds t o t h e i r r i n g opened i n d o l e d e r i v a t i v e s 144 and 145 f o l l o w e d by l o s s of the p r o t e c t i n g group would y i e l d the i n d o l e aldehyde 14 6. I n t r o -d u c t i o n of a carbomethoxy group a t C-16 f o l l o w e d by r e d u c t i o n of the aldehyde would y i e l d the d e s i r e d stemmadenine system. More-over, i f pathway A were shown to e x h i b i t a predominance of one isomer a t C-16 i n the f i n a l p r o d u c t , pathway B would be expected to y i e l d a predominance of the o t h e r isomer. B e f o r e d e s c r i b i n g the attempts to b r i n g the pathways d e s c r i b e d i n F i g u r e 26 to f r u i t i o n , i t would be worthwhile to d i s c u s s some of the more i n t e r e s t i n g p r o p e r t i e s of n o r - f l u o r o -c u r a r i n e (134). The N(b) m e t h o c h l o r i d e f l u o r o c u r a r i n e (134a) i s a n a t u r a l p r o d u c t f i r s t i s o l a t e d i n 1941 from a c a l a b a s h -c u r a r e p r e p a r a t i o n (used by South American Indians as an arrow 139 140 p o i s o n and packed i n calabashes or g o u r d s ) . ' L a t e r , i t was i d e n t i f i e d c h r o m a t o g r a p h i c a l l y i n e x t r a c t s from the bark of 141 Strychnos m i t s c h e r l i s c h i l . I t i s r e f e r r e d to i n the l i t e r a t u r e 7Q .~ b o t h . .as. C~£luorocurarine a n d as C ^ . c u r a r i n e I I I , a n d t h e f r e e 140 b a s e a s n o r - C - f l u o r o c u r a r i n e a n d n o r - C - c u r a r i n e I I I . The u l t r a v i o l e t s p e c t r u m o f n o r - f l u o r o c u r a r i n e (134) i s m o s t u n u s u a l , h a v i n g a v e r y i n t e n s e a b s o r p t i o n a t 363 nm. I t was p o s t u l a t e d d u r i n g t h e w o r k o n s t r u c t u r e e l u c i d a t i o n o f t h e n a t u r a l p r o d u c t t h a t t h i s a b s o r p t i o n was due t o an e q u i l i b r i u m 142 b e t w e e n t h e two t a u t o m e r s 134 a n d 1 4 7 , w h i c h w o u l d p r o v i d e f o r c o n j u g a t i o n w i t h t h e a r o m a t i c r i n g a n d i n c r e a s e d s t a b i l i z a t i o n o f t h e s y s t e m a s a w h o l e . . T h i s i s i n m a r k e d c o n t r a s t w i t h a k u a m m i c i n e (6.6). w h i c h w o u l d n o t t a u t o m e r i z e a s r e a d i l y , a n d d i s p l a y s a n e q u a l l y s t r o n g a b s o r p t i o n , b u t a t 326 nm. F u r t h e r m o r e , t h e a b s o r p t i o n b a n d f o r t h e c a r b o n y l i n t h e I R s p e c t r u m o f 134 —T a p p e a r s a t 1650 cm , w h i c h i s u n u s u a l l y l o w f o r a n a , g - u n s a t u r a -143 t e d a l d e h y d e . The NMR s p e c t r u m o f 134 ( F i g u r e 28) i s p a r t i c u l a r l y w o r t h y o f n o t e due t o i t s e x c e p t i o n a l r e s o l u t i o n o f t h e c o m p l e x s p l i t t i n g p a t t e r n o f t h e p r o t o n s o n t h e n o n - i n d o l i n e c a r b o n a t o m s . T h e . p r e d o m i n a n t f e a t u r e s a r e a 1 p r o t o n s i n g l e t a t 9.396 (CHO), a 1 p r o t o n q u a r t e t ( J - 7 Hz) a t 5.436 (~CHCH 3) a n d a .3 p r o t o n d o u b l e t CJ - 7Hz) c o n t a i n i n g f u r t h e r f i n e s p l i t t i n g s a t 1.696 (=CH-CH 3) . ^ 71 -With, n o r - f l u o r o c u r a r i n e (134) i n hand, i t was p o s s i b l e to i n v e s t i g a t e the pathways shown i n F i g u r e 27. I t was de c i d e d to attempt to o x i d i z e n o r - f l u o r o c u r a r i n e to an un s a t u r a t e d e s t e r 144 usxng the method developed by Corey. Treatment o f nor-f l u o r o c u r a r i n e (134) w i t h sodium cyanide i n a c i d i c methanol w i t h 145 a twenty f o l d excess o f a c t i v a t e d manganese d i o x i d e r e s u l t e d i n a complex mixture o f seven compounds, none of which d i s p l a y e d the UV a b s o r p t i o n a t 326 nm, c h a r a c t e r i s t i c of akuammicine (66). When the same r e a c t i o n was attempted i n n e u t r a l methanol, 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 along w i t h three compounds which once a g a i n l a c k e d the c h a r a c t e r i s t i c UV a b s o r p t i o n a t 32 6 nm. When the o x i d a t i o n was attempted w i t h s i l v e r (II) oxide i n t e t r a h y d r o f u r a n and water i n the presence o f sodium cyanide, f o l l o w e d by treatment w i t h diazomethane, o n l y decomposition of the s t a r t i n g m a t e r i a l was observed. A v a r i a t i o n o f the above sodium cya n i d e a s s i s t e d o x i d a t i o n s was a l s o attempted. I n s t e a d o f methanol, d i e t h y l a m i n e can be used as s o l v e n t . The o x i d a t i o n then proceeds to the c o r r e s p o n d i n g 146 a,3-unsaturated amide. When n o r - f l u o r o c u r a r i n e was t r e a t e d w i t h an excess o f a c t i v e manganese d i o x i d e and sodium cyanide i n d i e t h y l a m i n e , no products having the expected s p e c t r a l p r o p e r t i e s of the d e s i r e d p r o d u c t c o u l d be found i n the r e a c t i o n m i x t u re. A p o s s i b l e r a t i o n a l i z a t i o n f o r the f a i l u r e of the o x i d a t i o n s i n v o l v i n g sodium cyanide can be found i n the work of Schmid e t 129 a l . As shown i n F i g u r e 29, the cyanohydrin of 16-epi-WGA (148) under b a s i c aqueous c o n d i t i o n s c y c l i z e s and h y d r o l y z e s to the a-hydroxy lactam 149. F i g u r e 28. The Nuclear Magnetic Resonance Spectrum o f N o r - f l u o r o c u r a r i n e (134). F i g u r e 29. The R e a c t i o n of 16-epi-WGA w i t h HCN. In the case of n o r - f l u o r o c u r a r i n e (134) the s i t u a t i o n i s made even more complex by i t s known p r o p e n s i t y to deformylate 147 under a c i d c o n d i t i o n s , forming the co r r e s p o n d i n g desformyl i n d o l e n i n e . At t h i s p o i n t , i t was de c i d e d to abandon attempts to o x i d i z e n o r - f l u o r o c u r a r i n e and t r y to make use o f some of i t s unique p r o p e r t i e s . S p e c i f i c a l l y , i t was deci d e d to t r y to form some d e r i v a t i v e o f the aldehyde moiety which would a l l o w i t to s u r v i v e the c o n d i t i o n s r e q u i r e d f o r fo r m a t i o n of the n i n e -membered r i n g i n compounds 144 and 145, as shown i n F i g u r e 27, pathway B. The e a s i e s t d e r i v a t i v e to prepare was f e l t to be the e n o l a t e - 148 anion (143, X = 0 ). I t has been shown t h a t e n o l a t e anions of aldehydes are not reduced by sodium b o r o h y d r i d e . S t i l l another advantage of e n o l a t e s i s t h a t r e g e n e r a t i o n o f the aldehyde r e q u i r e s simple r e p r o t o n a t i o n . Thus n o r - f l u o r o c u r a r i n e - 74 -(134) was. t r e a t e d with, a suspension of sodium h y d r i d e i n t e t r a h y d r o f u r a n . A l t h o u g h the v i g o r o u s e v o l u t i o n of bubbles i n d i c a t e d t h a t the e n o l a t e had been formed, no r e a c t i o n o c c u r r e d when sodium b o r o h y d r i d e was added, even a f t e r h e a t i n g to 50°. S i m i l a r l y , when n o r - f l u o r o c u r a r i n e was t r e a t e d w i t h l i t h i u m t e r t - b u t o x i d e i n t e r t - b u t a n o l f o l l o w e d by sodium b o r o h y d r i d e , 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 unchanged a f t e r s t i r r i n g f o r 36 hours a t 35°. However, when methanolic sodium methoxide was used, f o r m a t i o n of the nine-membered r i n g was observed along w i t h r e d u c t i o n o f the aldehyde to form descarbomethoxy stemmadenine The s t r u c t u r e o f compound 150 was apparent from the d i s a p p e a r -ance of the c a r b o n y l a b s o r p t i o n i n the IR spectrum, the presence of two exchangeable protons i n the NMR spectrum, the presence of an i n d o l e a b s o r p t i o n p a t t e r n i n the UV spectrum and a c o n s i s t e n t m o l e c u l a r i o n and f r a g m e n t a t i o n p a t t e r n i n the mass spectrum. Thus, although the metal e n o l a t e of n o r - f l u o r o c u r a r i n e c o u l d be shown to p r o t e c t the aldehyde from b o r o h y d r i d e r e d u c t i o n w i t h the use of a s u f f i c i e n t l y s t r o n g base, the use of such a b a s i c medium appeared to p r e c l u d e the f o r m a t i o n o f the d e s i r e d nine-membered r i n g . F u r t h e r attempts to form e i t h e r the e n o l a c e t a t e or the (150) . C H 2 0 H (15 0 ) - 75 -e n o l e t h e r of n o r - f l u o r o c u r a r i n e were not met w i t h s u c c e s s . 149 I t has been shown i n model s t u d i e s t h a t compounds c o n t a i n i n g an aldehyde conjugated w i t h an a - m e t h y l e n e - i n d o l i n e can form the c o r r e s p o n d i n g dienamine w i t h r e l a t i v e ease. Thus, r e f l u x i n g n o r - f l u o r o c u r a r i n e i n benzene c o n t a i n i n g c y c l o h e x y l -amine and a c a t a l y t i c amount of a c i d w i t h a z e o t r o p i c removal of water r e s u l t e d i n f o r m a t i o n of a b r i g h t y e l l o w product 151. Compound 151 showed the expected bathochromic s h i f t i n the UV spectrum, having a s t r o n g a b s o r p t i o n a t 387 nm. As w e l l , the NMR spectrum c o n t a i n e d no a l d e h y d i c p r o t o n s i g n a l s , although a new s i n g l e t was observed a t 7.40 6 (NCH = C). No N-H a b s o r p t i o n s appeared i n the IR spectrum and an a,3-unsaturated imine absorp-t i o n was found a t 1630 cm ^. F i n a l l y , the mass spectrum showed the expected m o l e c u l a r i o n (m/e = 373). ( 153 ) F i g u r e 30... The R e a c t i o n s of Nor-f l u o r o c u r a r i n e (134) w i t h Cyclohexylamine, P y r r o l i d i n e , and Morpholine. - 76 -While r e a c t i o n of n o r - f l u o r o c u r a r i n e w i t h morpholine under s i m i l a r c o n d i t i o n s l e d o n l y to r e c o v e r y of s t a r t i n g m a t e r i a l along w i t h some decomposition p r o d u c t s , i t was found t h a t r e a c t i o n w i t h p y r r o l i d i n e o c c u r r e d w i t h r e l a t i v e ease. In f a c t , i t was found t h a t f o r m a t i o n of the p y r r o l i d i n e condensation product o c c u r r e d w i t h o u t a c i d c a t a l y s i s merely by i n v o l v i n g a S o x h l e t e x t r a c t o r c o n t a i n i n g m o l e c u l a r s i e v e s w i t h a r e f l u x i n g benzene s o l u t i o n of n o r - f l u o r o c u r a r i n e and p y r r o l i d i n e . While the UV spectrum of the r e a c t i o n mixture e v e n t u a l l y d i s p l a y e d the expected s h i f t t o 387 nm, attempts t o p u r i f y the p r o d u c t r e s u l t e d i n a hypsochromic s h i f t t o 372 nm, i n d i c a t i n g p a r t i a l h y d r o l y s i s of the enamine. T h i s r e s u l t was confirmed by the NMR spectrum which showed the presence of an a l d e h y d i c p r o t o n , and by the mass spectrum which showed a prominent peak a t m/e = 292 c o r r e s p o n d i n g to n o r - f l u o r o c u r a r i n e , as w e l l as a p a r e n t peak a t m/e = 345 c o r r e s p o n d i n g to the p y r r o l i d i n e enamine 152. I t was hoped t h a t , w i t h the p y r r o l i d i n e enamine 152, r e a c t i o n w i t h methyl c h l o r o f o r m a t e under e q u i l i b r i u m c o n d i t i o n s would r e s u l t i n the i n t r o d u c t i o n o f a carbomethoxy group a t C-16. A f t e r h y d r o l y s i s of the r e s u l t a n t p y r r o l i d i n i u m moiety, preaku-ammicine aldehyde (153) would be formed. However, a l l attempts to accomplish t h i s t r a n s f o r m a t i o n were u n s u c c e s s f u l . A t t h i s p o i n t i t was obvious t h a t the c o m p l i c a t e d f u n c t i o n a l i t y of nor-f l u o r o c u r a r i n e was not amenable to s t r a i g h t f o r w a r d chemical m a n i p u l a t i o n , and i t was d e c i d e d to abandon t h i s approach. During the course of t h e i r s t r u c t u r e e l u c i d a t i o n of 131 akuammrcine (66), Edwards and Smith d e s c r i b e d the c o n v e r s i o n of Wieland-Gumliclx aldoxime (154) to methyl 18-hydroxy-2g, 16a-cur-19-en-17-oate (156). S i n c e the i n d i r e c t o x i d a t i o n o f WGA to the c o r r e s p o n d i n g methyl e s t e r was one of the b a s i c r e q u i r e -ments f o r the s y n t h e s i s of the stemmadenine system, i t was dec i d e d to i n t e g r a t e t h i s sequence i n t o the o v e r a l l scheme shown i n F i g u r e 31. I t was found t h a t Wieland-Gumlich aldoxime c o u l d c o n v e n i e n t l y be prepared by a l l o w i n g WGA to s t i r o v e r n i g h t a t room temperature w i t h a l a r g e excess o f hydroxylamine h y d r o c h l o r i d e . The c r y s t a l -127 l i n e p roduct was o b t a i n e d i n pure form, m.p. 242-244° (d) ( l i t . m.p. 245 d e c ) , i n a f i n a l y i e l d o f 80-90%. In a d d i t i o n , the IR, UV, NMR and mass s p e c t r a l data a l l were c o n s i s t e n t w i t h s t r u c t u r e 154. 131 F o l l o w i n g the procedure o f Edwards and Smith, the Wieland-Gumlich aldoxime was dehydrated w i t h a c e t i c anhydride i n p y r i d i n e 131 to form the n i t r i l e d i a c e t a t e 155 which d i s p l a y e d the r e p o r t e d a b s o r p t i o n s a t 2260, 1748 and 1660 c m - 1 i n the IR spectrum. The UV spectrum C^ m a x 285, 277, 248 nm) was c o n s i s t e n t w i t h the expect-ed N - a c y l i n d o l i n e chromophore, and the NMR spectrum d i s p l a y e d two sharp methyl s i n g l e t s a t 2.01 and 2.106. Treatment of the n i t r i l e d i a c e t a t e 155 w i t h barium hydroxide i n r e f l u x i n g 35% e t h a n o l f o r 16 hours, f o l l o w e d by i s o l a t i o n o f the crude product and treatment, w i t h o u t p u r i f i c a t i o n , w i t h methanolic 5% h y d r o c h l o r i c a c i d f o r 5 hours y i e l d e d methyl 18-hydroxy-2g,16a-cur-19-en-17-oate (156) which d i s p l a y e d a m e l t i n g p o i n t (153-154°) i n agreement w i t h t h a t r e p o r t e d i n the l i t e r a -131 t u r e (152-154.5°). The IR spectrum was i n good agreement ( 160 ) ( 161 ) Figure 31. The S y n t h e s i s of 16-epi-SteiTimadenine (161) from WGA C130). - 79 -with, that reported by Edwards and Smith./ haying absorption bands at 3615, 3510 and 1730 cm"1, and the UV spectrum ( A m a x 297, 244 nm) was t y p i c a l of the expected indoline chromophore. The NMR spectrum contained two 1 proton s i n g l e t s at 3.40 and 4.226 which disappeared on exchange with D 20, a 3 proton s i n g l e t (-COOCH^) at 3.686 , and a 1 proton t r i p l e t (=CH-CH2-OH) at 5.606. The mass spectrum contained the expected molecular ion at m/e = 340. With the desired hydroxy-ester 156 i n hand, the next problem was to hydrogenolyze the hydroxy group at C-18. The method of 132 133 Bernauer et a l ' whxch has been previously described i n the conversion of the d i o l 136 to the alcohol 137 was attempted on the hydroxy ester 156, r e s u l t i n g i n a 65% y i e l d of the desired methyl 2g,16a-cur-19-en-17-oate (157). The IR spectrum contained peaks at 3420, 2980, 1730 and 1605 cm~x, i n d i c a t i n g that the methyl ester moiety had remained i n t a c t . the UV spectrum (A 297, 245 nm) was again c h a r a c t e r i s t i c of the expected indoline chromophore. the NMR spectrum contained a 3 proton doublet of doublets at 1.586 (J = 2 arid 7 Hz) (=CH-CH_3) , a 3 proton s i n g l e t at 3. 706 (COOCH3) , and a 1 proton quartet (J = 7Hz) at 5.486 (=CH-CH 3). As w e l l , the-molecular ion and fragmentation pattern was 150 found to be i d e n t i c a l to that reported i n the l i t e r a t u r e . As shown i n Figure 32, retro-Diels-Alder opening of ring C to form ion 162 simultaneously r e l i e v e s the s t r a i n of the fused pentacyclic system and provides for aromatization of the indoline nucleus. Cleavage at the positions marked a, 8 and y lead to the observed ions a, b, c and d CR=R'=H) at m/e =130, 194, 144 and 139. In addition, transfer of a hydride from C-14 to C-16, - 80 -during rupture of ring C forms ion 163, which, undergoes cleavage of the C-15, C-16 bond followed by rearrangement of the-double bonds to form the f u l l y aromatic ion e at m/e = 2 51 (R=H). a 3 y Figure 32. The Proposed JSlass Spectral Fragmentation .Reactions of the Methyl Cur-19-en-17-oate System. 131 Sxnce xt has been reported that the a l l y l i c hydroxyl group of the hydroxy ester 156 could not be removed by treatment with zinc i n . a c e t i c acid, the above r e s u l t was most g r a t i f y i n g . - 81 -However, when the a l t e r n a t i v e method of c a t a l y t i c hydrogenoly-134 s i s v i a W i e l a n d 1 s procedure was attempted, the mass spectrum of the product i n d i c a t e d s m a l l , but s i g n i f i c a n t , amounts of methyl 28,16a-curan-17-oate (156a) . Although t h i s s i d e product was not e v i d e n t i n the NMR spectrum, i t c o u l d not be t o l e r a t e d due to the importance of mass s p e c t r a l data i n the a n a l y s i s of p r o j e c t e d i n t e r m e d i a t e s i n the s y n t h e t i c pathway, and i t was t h e r e f o r e d e c i d e d to use o n l y the b r o m i n a t i o n - h y d r o g e n o l y s i s approach even f o r the p r e p a r a t i o n of u n l a b e l l e d m a t e r i a l . A t t h i s p o i n t , two c r u c i a l problems remained to be s o l v e d i n o r d e r to complete the s y n t h e s i s . F i r s t , i t would be e s s e n t i a l to determine the c o n d i t i o n s n ecessary f o r the i n t r o d u c t i o n of the hydroxymethylene group a t C-16. Second, a procedure would have to be d e v i s e d f o r the c o n v e r s i o n of the i n d o l i n e moiety to the c o r r e s p o n d i n g i n d o l e system w i t h cleavage of r i n g C a t the C-3, C-7 bond. As shown i n F i g u r e 33, two a l t e r n a t i v e pathways can be v i s u a l i z e d f o r the completion of the s y n t h e t i c sequence from methyl 28,16a-cur-19-en-17-oate (157), depending on the o r d e r i n which the above two o p e r a t i o n s are c a r r i e d out. Rather than c o n c e n t r a t e s o l e l y ' o n pathway A or pathway B of F i g u r e 33, i t seemed to be expedient to i n i t i a l l y develop methods f o r the c o n v e r s i o n o f compound 157 to the hydroxy e s t e r 160, and then to s o l v e the problem of the i n t r o d u c t i o n of the 2,16 double bond to form the u n s a t u r a t e d e s t e r 66 (akuammicine). S i n c e the ester . 157 was r e l a t i v e l y r e a d i l y a v a i l a b l e , t h i s would mean t h a t the two most d i f f i c u l t s y n t h e t i c problems c o u l d be i n v e s t i g a t e d w i t h a minimum e x p e n d i t u r e of e f f o r t and m a t e r i a l . - 82 -(6) F i g u r e 33. Two P o s s i b l e Routes to Stemmadenine (6) from Methyl 23 ,16ct-cur-19-en-17-oate (.157) . - 83 -( 165 ) F i g u r e 34-. The P o s s i b l e Condensation of Formaldehyde w i t h Methyl 28,16a-cur-19-en-17-oate (157). In d e a l i n g with, the f i r s t problem, i . e . i n t r o d u c t i o n of the hydroxymethylene group a t C-16, i t was f e l t t h a t treatment of the e s t e r 157 d i r e c t l y w i t h formaldehyde i n the presence of base c o u l d e a s i l y r e s u l t i n i n i t i a l c ondensation of formaldehyde w i t h the i n d o l i n e n i t r o g e n as shown i n F i g u r e 34. Subsequent conden-s a t i o n of the anion of the c a r b i n o l a m i n e 164 thus formed w i t h the e s t e r c a r b o n y l would r e s u l t i n the u n d e s i r e d t e t r a h y d r o oxazinone 165. Thus, i t was d e c i d e d to p r e c l u d e such a r e a c t i o n pathway by p r o t e c t i n g the i n d o l i n e n i t r o g e n . For t h i s purpose, the N(a) formyl d e r i v a t i v e 158 was chosen f o r the f o l l o w i n g reasons. F i r s t , i t appeared e a s i l y a c c e s s i b l e by condensation of 157 w i t h methyl formate. Second, i t would condense s e l e c -t i v e l y w i t h the secondary i n d o l i n e n i t r o g e n , s i n c e a t t a c k a t the t e r t i a r y n i t r o g e n would be much more r e a d i l y r e v e r s i b l e . T h i r d , i t c o u l d be e a s i l y removed by r e f l u x i n g w i t h sodium h y d r i d e i n - 84 -151 t e t r a h y d r o f u r a n , a procedure which would not cause any u n d e s i r a b l e r e a c t i o n s elsewhere i n the molecule. To t h i s end, the e s t e r 157 was t r e a t e d w i t h methyl formate and sodium h y d r i d e i n r e f l u x i n g benzene, and the d e s i r e d product methyl 1-formyl-2 ( 3,16a-cur-19-en-17-oate (158) was o b t a i n e d . The IR spectrum showed an absence o f NH a b s o r p t i o n , but d i d show the presence of two c a r b o n y l groups having a b s o r p t i o n s a t 1730 and 1668 cm" 1. The UV spectrum U 287, 278, 250 nm) was c h a r a c t e r i s t i c o f the d e s i r e d N - a c y l i n d o l i n e chromophore ( c f . compound 155). The NMR spectrum c o n t a i n e d a 1 p r o t o n s i n g l e t a t 8.716 (N-CHO) , a 1 pr o t o n q u a r t e t (J = 7 Hz) a t 5.386 (=CH-CH 3 ) , a 3 p r o t o n s i n g l e t a t 3.686 (COOCH^), and a 3 proton d o u b l e t o f d o u b l e t s (J = 2 and 7 Hz) a t 1. 536 (=CH-CH_3) . The mass spectrum c o n t a i n e d a m o l e c u l a r i o n a t m/e = 352, and a fragme n t a t i o n p a t t e r n c o n s i s t e n t w i t h t h a t shown i n F i g u r e 32. S p e c i f i c a l l y , i o n s a, b, c, d and e (R = CHO, R' = H) c o u l d be seen a t m/e = 158, 194, 172, 139 and 279, r e s p e c t i v e l y . I n i t i a l l y , i t had been hoped t h a t the r e a c t i o n c o n d i t i o n s employed f o r i n t r o d u c i n g the N-formyl p r o t e c t i n g group might s i m u l t a n e o u s l y r e s u l t i n the i n t r o d u c t i o n o f an aldehyde func-t i o n a l group on C-16 v i a the condensation of the e n o l a t e anion w i t h methyl formate. That t h i s d i d not oc c u r , and i n f a c t c o u l d not be induced to occur even by means of employing h i g h e r temperatures and lo n g e r r e a c t i o n times, seemed to i n d i c a t e t h a t an i n v e s t i g a t i o n o f the c o n d i t i o n s necessary to form a r e a c t i v e anion a t C-16 was i n o r d e r . In o r d e r to i n v e s t i g a t e anion f o r m a t i o n a t C-16, the N-formyl - 85 -e s t e r 158 was allowed to r e a c t w i t h a . v a r i e t y of b a s e / s o l v e n t combinations and then quenched w i t h V^O. The compounds r e -covered were s u b m i t t e d f o r mass s p e c t r a l a n a l y s i s . E f f i c i e n t i n c o r p o r a t i o n of deuterium a t C-16, i n d i c a t i n g anion f o r m a t i o n , c o u l d then be determined by a decrease i n peak h e i g h t s a t m/e = 352, 194 and 139 (ions M +, b and d, R = CHO, R« = H) w i t h a concomitant i n c r e a s e i n peak h e i g h t s a t m/e = 353, 195 and 140 (R = CHO, R' = D). The r e s u l t s o f t h i s i n v e s t i g a t i o n are summarized i n Tabl e 7. Table 7. Summary of R e s u l t s o f the I n v e s t i g a t i o n i n t o Anion Formation a t C-16 of the N-Formyl E s t e r 158. Experiment Number S o l v e n t Base Tempera-t u r e Time Hrs. % D I n c o r - ^ p o r a t i o n 1 THF 153 L i N U - P r ) ^ J -78° . 45 0 2 THF NaH r e f l u x 2.5 0 3 DMSO : NaH 22° .40 >95 4 HMPA NaH 22° .40 >95 5 THF t-BuOK 22° .40 40 6 DMSO t-BuOK 22° .40 * * 7 HMPA t-BuOK 22° .40 ** Approximate, based .on r e l a t i v e peak h e i g h t s i n mass s p e c t r a ** Severe decomposition of s t a r t i n g m a t e r i a l o c c u r r e d . I t can be seen from T a b l e 7 t h a t sodium h y d r i d e i n e i t h e r d i m e t h y l s u l f o x i d e o r hexamethyl phosphoramide (experiments 3 and 4) o f f e r e d the most hope of su c c e s s . Of the two s o l v e n t s , dimethyl s u l f o x i d e was chosen because i t appeared to be more e a s i l y removed ^ 86 ^ from the r e a c t i o n m i x t u r e , and r e s u l t e d i n b e t t e r r e c o v e r y o% m a t e r i a l . A c c o r d i n g l y , when t h e N-formyl e s t e r 158 was allowed to r e a c t w i t h d r y formaldehyde with, sodium h y d r i d e i n d i m e t h y l s u l f o x i d e a t room temperature, a product was formed which appeared to be l e s s p o l a r than the s t a r t i n g m a t e r i a l by t h i n l a y e r chromatography. The TR spectrum of the product showed no N-H bands, and o n l y one c a r b o n y l band a t 1730 cm 1 , w h i l e the UV spectrum CA 299, 246 nml i n d i c a t e d an i n d o l i n e chromophore. *• max *• The NMR spectrum (Figure 35) shows a 1 pr o t o n q u a r t e t (J = 7 Hz) at 5.466 C=CH-CH3), a t y p i c a l AB p a t t e r n (J = 1 0 Hz) a t 5.18 and 4.716 (N-CH 2-0) , a 1 p r o t o n s i n g l e t a t 4.946 (N~CH- C C R 3 ) 2 ) , a 3 pr o t o n s i n g l e t a t 3.706 (COOCH^), and a 3 p r o t o n d o u b l e t of do u b l e t s (J = 2 and 7 Hz) a t 1.576 C^CH-CH-). The h i g h r e s o -( l o s s o f CH 20). These data are a l l i n a c c o r d w i t h the unexpec-t e d , but n e v e r t h e l e s s u s e f u l carbomethoxy t e t r a h y d r o o x a z i n e s t r u c t u r e 159. In o r d e r to p r o v i d e f u r t h e r evidence f o r the proposed s t r u c t u r e 159, i t was d e c i d e d t o s y n t h e s i z e the analogous descarbomethoxy compound 166 v i a an independent r o u t e . F i g u r e 36'. The R e v e r s i b l e Formation of the Model T e t r a h y d r o -0"22 H26°3 N2 = 366.1943) w i t h a major fragment a t .m/e 366.1815 (137 ) ( 166 ) o x a z i n e (166). F i g u r e 35. The Nuclear Magnetic Resonance Spectrum of the Carbomethoxy T e t r a h y d r o o x a z i n e 15 88 r As shown i n Figure. 36 f 2g, 16a^cur^l9-en-17-ol (1371 previously prepared by hydrogenolysis of Wieland—Gumlich. d i o l (136) (Figure 25) was allowed to react with paraformaldehyde i n methanol i n the presence of anhydrous sodium s u l f a t e at room temperature. This procedure, analogous to that previously 153—155 described i n the l i t e r a t u r e for geissoschizoline (167) gave i n good y i e l d the desired tetrahydrooxazine 166. The IR (167) spectrum showed the expected disappearance of the 0-H and N-H bands at 3150 and 3395 cm 1 , while the uv spectrum (A 297, *• max 249 nm) indicated the expected indoline chromophore. The NMR spectrum (Figure 37) contains a 1 proton quartet (J = 7 Hz) at 5 .416 (=CH-CH3), a t y p i c a l AB pattern (J = 11 Hz) at 5 . 2 3 and 4 .676 (N-CH2-0), and a 3 proton doublet (J = 7 Hz) at 1 .506 (=CH-CH_3) . The high r e s o l u t i o n mass spectrum indicates a mole-cular ion at m/e = 308 .1932 ( C 2 o H 2 4 O N2 r e ( 3 u i r e 308 .1887) with a major fragment occurring at m/e = 278 .1782 (loss of CH 2Q). The most s t r i k i n g s i m i l a r i t y i n the NMR spectra shown i n Figures 35 and 37 i s the presence of the two downfield doublets caused by the protons on the i s o l a t e d carbon of the tetrahydro-oxazine system. Because of the r i g i d i t y of the hexacyclic system, the a and g protons are held i n d i f f e r e n t magnetic environments and hence have d i f f e r e n t chemical s h i f t s as well as character-F i g u r e 37. The Nuclear Magnetic Resonance Spectrum of the Model T e t r a h y d r o o x a z i n e 166. - 90 -i s t i c s p l i t t i n g p a t t e r n s which, r e v e a l s p i n c o u p l i n g between them. The presence of the 1 p r o t o n s i n g l e t f o r the 2(3 p r o t o n a t such low f i e l d i n F i g u r e 36 can be e x p l a i n e d by the d i f f e r e n c e s i n c o n f i g u r a t i o n a t C-16. In the case of the carbomethoxy t e t r a h y d r o o x a z i n e 159, m o l e c u l a r models r e v e a l t h a t the e t h e r l i n k a g e o f the t e t r a h y d r o o x a z i n e system i s p r o j e c t e d i n t o the a p l a n e of the molecule, thus p r o j e c t i n g the lone p a i r o f e l e c t r o n s on the i n d o l i n e n i t r o g e n i n t o the |3 c o n f i g u r a t i o n , w i t h no f a c i l e i n v e r s i o n p o s s i b l e . T h i s s i t u a t i o n i n t u r n p l a c e s the 2 6 p r o t o n i n t o v e r y c l o s e p r o x i m i t y to the l o n e p a i r o f e l e c t r o n s , r e s u l t i n g i n the observed d o w n f i e l d s h i f t . On the o t h e r hand, i n the model t e t r a h y d r o o x a z i n e 166, e x a c t l y the o p p o s i t e i s the case, w i t h the e t h e r l i n k a g e of the t e t r a h y d r o -o x a z i n e p r o j e c t e d i n t o the 8 plane of the molecule, and no down-f i e l d s h i f t i s observed. That the carbomethoxy t e t r a h y d r o o x a z i n e does i n f a c t possess the c o n f i g u r a t i o n shown a t C-16 can be f u r t h e r e s t a b l i s h e d by 131 NMR evidence. I t has been shown by Edwards and Smith t h a t the hydroxy e s t e r 156 possesses the 3-carbomethoxy c o n f i g u r a t i o n a t C-16 and i s n o t ' e p i m e r i z a b l e . T h i s i s understandable, s i n c e w i t h r i n g C i n a c h a i r conformation, the 3 carbomethoxy group would be i n a f a v o r a b l e e q u a t o r i a l o r i e n t a t i o n . I f e p i m e r i z a t i o n were t o o c c u r a t C-16, t h i s would f o r c e the carbomethoxy group i n t o an extremely u n f a v o u r a b l e a x i a l p o s i t i o n . With the s t e r e o -c h e m i s t r y o f the hydroxy e s t e r 156 e s t a b l i s h e d , Table 8 c o r r e l a t e s the chemical s h i f t data of t h a t compound w i t h the data f o r the e s t e r 157, the N-formyl e s t e r 158, and the carbomethoxy t e t r a -hydrooxazine 159. Table 8. A Summary of Pertinent Nuclear Magnetic Resonance Chemical S h i f t s for Compounds 156-159 Chemical S h i f t s 6 (ppm) - 92 -The data shown i n Table 8 o f f e r s convincing evidence that, i n proceeding from the hydroxy ester 156 to the carbomethoxy tetrahydrooxazine 159, the configuration of the carbomethoxy group remains unchanged, i . e . 8. In addition, studies of the molecular model of the N-formyl ester 158 c l e a r l y indicate that approach of an e l e c t r o p h i l e from the a face would be far more favorable than from the quite hindered B face. With the structure of the carbomethoxy tetrahydrooxazine 159 established, i t would be i n t e r e s t i n g to speculate on the mechanism of i t s formation. As shown i n Figure 38, condensation of the enolate anion of the N-formyl ester 158 with formaldehyde would y i e l d an intermediate 168 which could then c y c l i z e r e v e r s i b l y to the intermediate 169. Although the equilibrium involving r i n g opening of 169 to the formate ester 17 0 would not necessarily favor the formate ester, the anion formed at the indoline nitrogen could e a s i l y condense with another molecule of formaldehyde to form 171 followed by an i r r e v e r s i b l e c y c l i z a t i o n involving d i s -placement of formate to y i e l d the observed carbomethoxy tetrahydro-oxazine 159. Once the carbomethoxy tetrahydrooxazine 159 had been obtained, the next problem was to hydrolyze the tetrahydrooxazine moiety without a f f e c t i n g the methyl ester. To t h i s end, the model compound 166 was used as a source of material for study. When the model tetrahydrooxazine was treated with 10% hydrochloric acid i n methanol at room temperature for 5 hours, quantitative hydrolysis of the tetrahydrooxazine occurred. This could e a s i l y be shown by comparison of the spectral data of the product of those - 93 -a l r e a d y a v a i l a b l e f o r the a l c o h o l 137. (171) F i g u r e 38. The Proposed Mechanism f o r the Formation of the Carbomethoxy Tetrahydrooxazine 159. S i m i l a r l y , when the carbomethoxy t e t r a h y d r o o x a z i n e 159 was t r e a t e d w i t h r e f l u x i n g 10% methanolic h y d r o c h l o r i c a c i d , the hydroxy e s t e r 160 was o b t a i n e d i n good y i e l d . The IR spectrum showed a b s o r p t i o n bands a t 3350, 2950 and 1725 cm 1 and the UV spectrum (^ m a x 296, 244 nm) was c o n s i s t e n t w i t h a d i h y d r o -i n d o l e chromophore. The NMR spectrum c o n t a i n e d a 3 pr o t o n q u a r t e t (J = 7 Hz) a t 5.546 (=CH-CH 3), a 1 pr o t o n s i n g l e t a t 3.736 C00CH 3), and a 3 p r o t o n d o u b l e t of d o u b l e t s (J = 2 and 7 Hz) a t 1.646. That the s i n g l e t f o r the 28 pr o t o n has o n l y s h i f t e d u p f i e l d 0.2 8 ppm may be c o n s i d e r e d t o be due - 94 -to hydrogen bonding between the p r o t o n on the i n d o l i n e n i t r o g e n and t h e oxygen of the a l c o h o l i c f u n c t i o n . T h i s would have the e f f e c t of m a i n t a i n i n g the c l o s e p r o x i m i t y between the 23 p r o t o n and the 3 l o n e p a i r of e l e c t r o n s on the i n d o l i n e n i t r o g e n . F i n a l l y , the mass spectrum e x h i b i t e d the expected m o l e c u l a r i o n a t m/e = 354. Having accomplished one of the twQ remaining s y n t h e t i c problems, i t remained to d e v i s e a method f o r the o x i d a t i o n of the i n d o l i n e system to e i t h e r an i n d o l e n i n e or an a-methylene-i n d o l i n e system, which c o u l d then be e a s i l y c onverted to the d e s i r e d i n d o l e system. As p r e v i o u s l y s t a t e d , i t was d e c i d e d to c o n c e n t r a t e the i n i t i a l i n v e s t i g a t i o n on the problem of c o n v e r t i n g the e s t e r 157 to the c o r r e s p o n d i n g a,3 u n s a t u r a t e d e s t e r 66 (akuammicine) . I t has been shown 1 5*' t h a t l e a d t e t r a -a c e t a t e i s capable o f o x i d i z i n g an i n d o l i n e system to the c o r r e s p o n d i n g i n d o l e n i n e w i thout s e r i o u s s i d e r e a c t i o n s i n v o l v i n g the b a s i c n i t r o g e n (N(b)) of the a l k a l o i d a l system. T h e r e f o r e , l e a d t e t r a a c e t a t e was the reagent of c h o i c e f o r the i n v e s t i g a t i o n a t hand. The i n i t i a l i n v e s t i g a t i o n c o n s i s t e d of adding a benzene s o l u t i o n of l e a d t e t r a a c e t a t e dropwise to a benzene s o l u t i o n of the e s t e r 157 w i t h f r e q u e n t m o n i t o r i n g of the p r o g r e s s o f the r e a c t i o n . T h i s was accomplished i n two ways. F i r s t , an a l i q u o t would be taken from the r e a c t i o n mixture, the s o l v e n t evaporated to dryness and a UV spectrum taken of the r e s i d u e . Second, a s m a l l a l i q u o t would be p l a c e d on a s i l i c a g e l t h i n l a y e r chroma-tography p l a t e and e l u t e d w i t h 20% methanol i n benzene. The - 95 -presence o f akuammicine i n the r e a c t i o n m i x t u r e c o u l d then e a s i l y be determined by the appearance of the c h a r a c t e r i s t i c a b s o r p t i o n a t 325 nm i n the UV spectrum, and the c h a r a c -t e r i s t i c b l u e c o l o r which appears when an a- m e t h y l e n e - i n d o l i n e 140 i s sprayed w i t h e e r i e s u l f a t e s o l u t i o n . In t h i s way, i t c o u l d be seen t h a t the r e a c t i o n was i n f a c t p r o c e e d i n g as d e s i r e d , and t h a t the f o l l o w i n g f a c t o r s were important. I t was found t h a t optimum r e s u l t s c o u l d be o b t a i n e d when the r e a c t i o n was c a r r i e d out i n a d i l u t e benzene s o l u t i o n c o n t a i n i n g 1-2 e q u i v a l e n t s of a c e t i c a c i d . F u r t h e r , i t was found t h a t 2 e q u i v a l e n t s o f l e a d t e t r a a c e t a t e were r e q u i r e d f o r optimum y i e l d s , and must be added v e r y s l o w l y as a benzene s o l u t i o n . Chromatographic p u r i f i c a t i o n o f the r e s u l t a n t product mixture and c r y s t a l l i z a t i o n p r o v i d e d akuammicine (66). The IR, UV and mass s p e c t r a l data were i d e n t i c a l to t h a t a l r e a d y d e s c r i b e d i n 150 157 the l i t e r a t u r e . ' F u r t h e r , i t can be seen t h a t the NMR spectrum ( F i g u r e 40) i s remarkably s i m i l a r to t h a t o f n o r - f l u o r o -c u r a r i n e ( F i g u r e 28) w i t h the e x c e p t i o n of the 3 pro t o n s i n g l e t a t 3.82 (COOCH^) and the l a c k o f an a l d e h y d i c p r o t o n i n the former. 158 S i n c e s t r y c h n i n e (29) has been t o t a l l y s y n t h e s i z e d , t h i s then r e p r e s e n t s the f i r s t t o t a l s y n t h e s i s o f akuammicine. With both major s y n t h e t i c problems s o l v e d , the completion of the s y n t h e s i s o f the stemmadenine system i n v o l v e d comparing the r e l a t i v e m e r i t s of pathway A o r pathway B shown i n F i g u r e 33. Pathway B was the f i r s t to be attempted. Treatment of akuammicine w i t h an excess of sodium b o r o h y d r i d e 121 i n r e f l u x i n g a c e t i c a c i d l e d to the d e s i r e d i n d o l e e s t e r 141a NaH. -> ( U l a ) COOCH 3 ( U 1 b ) and a s m a l l amount of the epimer 141b. That the major product would be the a carbomethoxy epimer would be expected from the 131 r e s u l t s o f Edwards and Smith who demonstrated t h a t r e d u c t i o n of the 2,16 double bond of akuammicine by z i n c and me t h a n o l i c s u l f u r i c a c i d r e s u l t s i n methyl 23,16B-cur-19-en-17-oate (172). As shown i n F i g u r e 39, they proposed t h a t the f i r s t s t e p would be p r o t o n a t i o n of C-16 of akuammicine to form the iminium i o n 173 COOCH3 " COOCH3 " COOCH ( 6 6 ) ( 1 7 3 ) M 7 2 ) F i g u r e 39. The Edwards and Smith Mechanism f o r the Zinc and S u l f u r i c A c i d Reduction o f Akuammicine (66). P r o t o n a t i o n from the 3 f a c e would p l a c e the carbomethoxy group i n the l e s s s t r a i n e d e q u a t o r i a l p o s i t i o n , w i t h r i n g C having a boat conformation. S i m i l a r l y , the iminium i o n 17 3 i n r e f l u x i n g a c e t i c a c i d would be expected to undergo opening o f r i n g C as shown i n F i g u r e 41. In the presence of sodium boro-h y d r i d e , the r e s u l t a n t N(b) iminium i o n 173 would be reduced to the c o r r e s p o n d i n g amine 141. The IR spectrum of the a-carbo— Hi F i g u r e 40. The Nuclear Magnetic Resonance Spectrum of Akuammicine (66). 98 -methoxy compound had a b s o r p t i o n bands a t .3450, 2930 and 1725 cm*"1 and the UV spectrum (A 290, 283, 225 nm) was c o n s i s t e n t w i t h the d e s i r e d i n d o l e chromophore. ( 173 ) (174) ( U 1 a ) F i g u r e 41. The Proposed Mechanism f o r the Formation of Indole E s t e r 141a. The NMR spectrum ( F i g u r e 42) c o n t a i n e d a 1 pro t o n s i n g l e t a t 9.086 ( i n d o l e N-H), a 1 pro t o n q u a r t e t (J - 7 Hz) a t 5.606 (=CH-CH 3), a 1 pr o t o n s i n g l e t a t 4.306 (indole-CH-COOCH 3), a 3 pro t o n s i n g l e t a t 3.886 (COOCH_3) , and a 3 pr o t o n d o u b l e t (J = 7 Hz) a t 1.776 . (=CH-CH 3). The mass spectrum showed a m o l e c u l a r i o n a t m/e = 324, w i t h a base peak a t m/e = 123. When the a-carbomethoxy compound was r e f l u x e d i n benzene w i t h sodium h y d r i d e , c l e a n c o n v e r s i o n t o the 8-carbomethoxy epimer o c c u r r e d . the 8-carbomethoxy compound 141b had a b s o r p t i o n bands i n the IR spectrum a t 3460, 2940 and 1725 cm - 1. The UV spectrum was i d e n t i c a l to t h a t o b t a i n e d f o r the epimer 141a, as was the mass spectrum. However, the NMR spectrum ( F i g u r e 43) was markedly d i f f e r e n t , c o n t a i n i n g a 1 pro t o n s i n g l e t a t 8.86 ( i n d o l e N-H), a 1 pro t o n q u a r t e t (J = 7 Hz) a t 4.466 (=CH-CH 3), a 1 pro t o n d o u b l e t (J = 4 Hz) a t 4.206 (indole-CH-COOCH 3) a 3 proton s i n g l e t a t 3.766 COOCH3) and a 3 pro t o n d o u b l e t (J = 7 Hz) a t 1.586 (=CH-CH3) . At t h i s p o i n t , two f a c t o r s l e d to the abandonment of 7 6 5 4 3 2 1 <5 Cppm) The Nuclear Magnetic Resonance Spectrum of the Indole E s t e r 141a. 9 8 7 6 5 4 3 2 1 0 6 (ppm) Figure 43. The Nuclear Magnetic Resonance Spectrum of the Indole Ester 141b. - 101 -pathway B as shown i n F i g u r e 33. F i r s t , the r e d u c t i y e r i n g opening gave, a t b e s t , a 30-40% y i e l d of the d e s i r e d i n d o l e e s t e r 141. T h i s , coupled w i t h the low y i e l d (35%) of akuammicine from the e s t e r 157 made pathway B u n f a v o r a b l e from the s t a n d p o i n t of f i n a l y i e l d . Second, a l l attempts t o i n t r o -duce the r e q u i r e d hydroxymethylene group a t C-16 met w i t h f a i l u r e . Thus, the o n l y p o s s i b i l i t y of success through pathway B l a y i n p r o t e c t i n g the i n d o l e n i t r o g e n p r i o r to f o r m a t i o n o f the anion a t C-16, making the f i n a l y i e l d ( i f any) of the stemmadenine system p r o h i b i t i v e l y low. Thus, i t was d e c i d e d t o attempt t o complete the s y n t h e s i s o f the stemmadenine system v i a pathway A ( F i g u r e 33). A c c o r d i n g l y , the hydroxy e s t e r 160 was t r e a t e d w i t h l e a d t e t r a a c e t a t e under the same c o n d i t i o n s used t o s y n t h e s i z e akuammicine. The r e a c t i o n was monitored v i a t h i n l a y e r chroma-tography, and the immediate appearance of a l e s s p o l a r product was observed. A f t e r 2 e q u i v a l e n t s of l e a d t e t r a a c e t a t e were added, the r e a c t i o n was t e r m i n a t e d by r a p i d l y f i l t e r i n g the crude mixture through a column of alumina (12% water) w i t h methylene c h l o r i d e as e l u e n t . S i n c e the r e a c t i o n product was expected to 79 be the preakuammicine system (2) which i s known to be u n s t a b l e , no attempt was made to i s o l a t e or p u r i f y the product o b t a i n e d . The crude o x i d a t i o n p r o d u c t was d i s s o l v e d d i r e c t l y i n 50% methanolic a c e t i c a c i d and an excess of sodium b o r o h y d r i d e was added t o reduce the a n t i c i p a t e d iminium i o n formed d u r i n g the r i n g opening o f the preakuammicine system (see F i g u r e s 33 and 42). P u r i f i c a t i o n o f the product thus formed was achieved by s i l i c a - 102 T. g e l p r e p a r a t i y e l a y e r chromatography p l a t e s . The pure product had a b s o r p t i o n bands i n the IR spectrum a t 3580, 3420, 2920 and -1725 cm""1. The UV spectrum CA 291, 284, 225 nm) was c o n s i s -r max ' ' t e n t w i t h the d e s i r e d i n d o l e chromophore. The NMR spectrum c o n t a i n e d s i g n a l s f o r two exchangeable protons a t 10.14 and 2.666 ( i n d o l e N-H and CH2-OH, r e s p e c t i v e l y ) , a 1 p r o t o n q u a r t e t (J = 7 Hz) a t 5.426 C=CH-CH3), a 2 pro t o n s i n g l e t a t 4.366 C-CHj-OH), a 3 pro t o n s i n g l e t a t 3.886 (COOCH^), and a 3 pr o t o n d o u b l e t CJ = 7 Hz) a t 1.526 C=CH-CH3) . The mass spectrum e x h i b i t e d the expected m o l e c u l a r i o n a t m/e = 354, w i t h fragments a t M +-17, 18 and 30 c o r r e s p o n d i n g t o l o s s o f hydroxide, water and formaldehyde i n a c c o r d w i t h the fragme n t a t i o n p a t t e r n a l r e a d y d e s c r i b e d f o r 117 stemmadenine. That the compound o b t a i n e d was not i n f a c t stemmadenine can be e a s i l y seen from the NMR evidence i n Table 9 T h i s comparison of the a s s i g n e d chemical s h i f t data p u b l i s h e d f o r 118a stemmadenine w i t h t h a t d e s c r i b e d above- c l e a r l y i n d i c a t e s t h a t the n a t u r a l s t e r e o c h e m i s t r y about C-16 i n the stemmadenine system has not been o b t a i n e d . T a b l e 9. Comparison o f NMR Data oh N a t u r a l and S y n t h e t i c Stemmadenine Systems. Compound Indole N-H =CH-CH3 CH2~OH COOCH_3 =CH-CH N a t u r a l 9.4 S y n t h e t i c 10.14 5.4 4.38 3.79 1.7 5.4 4.36 3.88 1.5 - 10.3 -In order to prove t h a t the s y n t h e t i c m a t e r i a l was indeed 16-epi-stemmadenine (161), i t was deci d e d to reduce b o t h the 159 n a t u r a l and s y n t h e t i c m a t e r i a l to the c o r r e s p o n d i n g d i o l as shown i n F i g u r e 44. The two products thus o b t a i n e d should then be i d e n t i c a l i n every r e s p e c t . ( 6a ) (175) ( 161 ) F i g u r e 44. The Reduction of N a t u r a l and S y n t h e t i c Stemmadenine Systems to the D i o l 175. A c c o r d i n g l y , b o t h the n a t u r a l and s y n t h e t i c stemmadenine compounds were 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 excess of sodium bis(methoxy^ethylenoxy) aluminum h y d r i d e (commercially a v a i l a b l e benzene s o l u t i o n ) . The r e a c t i o n was r a p i d and l e d to e s s e n t i a l l y o n l y one component i n both cases. A f t e r p u r i f i c a t i o n v i a p r e p a r a t i v e l a y e r chromatography on s i l i c a g e l , the two pr o d u c t s were compared and found to be i d e n t i c a l , p o s s e s s i n g superimposable IR (Figure 45) and NMR ( F i g u r e 46) s p e c t r a . S i n c e the s y n t h e t i c 16-epi-stemmadenine has known s t e r e o -c h e m i s t r y about C-16 e s t a b l i s h e d e a r l i e r on i n the s y n t h e s i s , i t now becomes p o s s i b l e to a s s i g n the n a t u r a l s t e r e o c h e m i s t r y about C-16 i n stemmadenine. Thus, the s t e r e o c h e m i s t r y shown i n F i g u r e 47 f o r stemmadenine (6a) i s pr o b a b l y c o r r e c t . 1 F i g u r e 45. The I n f r a r e d S p e c t r a of S y n t h e t i c CA) and A u t h e n t i c (B) D i o l 175 i n c h l o r o f o r m . - 105 -i g u r e 46. The N u c l e a r Magnetic Resonance Spectrum of S y n t h e t i c (A), and A u t h e n t i c (B) D i o l 175. ^ 1Q6 «-Although, t h e s y n t h e s i s of the n a t u r a l s t e r e o c h e m i s t r y stemmadenine system w i l l not De d e s c r i b e d i n t h i s t h e s i s , i t would be i n t e r e s t i n g to s p e c u l a t e as to how i t might be o b t a i n e d . As shown i n F i g u r e 47, a l i k e l y s t a r t i n g p o i n t would be the s a t u r a t e d aldehyde 133, the s y n t h e s i s of which has a l r e a d y been 137 d e s c r i b e d . Proceeding i n a manner s t r i c t l y analogous to t h a t o u t l i n e d i n F i g u r e 31, p r o t e c t i o n of the i n d o l i n e n i t r o g e n as i t s formamide 176 f o l l o w e d by r e a c t i o n w i t h methyl c h l o r o f o r m a t e i n base might be expected to y i e l d the N-formyl aldehyde e s t e r 177 which c o u l d be deformylated w i t h sodium h y d r i d e i n t e t r a h y d r o -f u r a n to y i e l d the aldehyde e s t e r 178. O x i d a t i o n w i t h l e a d t e t r a a c e t a t e f o l l o w e d by r e d u c t i v e r i n g opening would then y i e l d the d e s i r e d stemmadenine (6a). < 6a ) (178) F i g u r e 47. The Proposed £oute f o r the S y n t h e s i s of Stemmadenine (6a) P a r t c ^ 107 ^ Although, the o r i g i n a l g o a l of the s y n t h e s i s of stemmadenine having the n a t u r a l c o n f i g u r a t i o n about C-16 was not met, i t was n e v e r t h e l e s s of i n t e r e s t to determine the u s e f u l n e s s of s y n t h e t i c 16-epi-stemmadenine as a b i o s y n t h e t i c p r e c u r s o r . To t h a t end, a sample o f 16-epi-stemmadenine (161) was l a b e l l e d w i t h t r i t i u m i n the aromatic r i n g by exchange 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 121 a c i d , and the r a d i o a c t i v e compound a d m i n i s t e r e d to r o o t c u t t i n g s of s i x year o l d Aspidosperma p y r i c o l l u m p l a n t s . At the same time, a s i m i l a r l y l a b e l l e d r a d i o a c t i v e sample of a u t h e n t i c stemmadenine"''"'"^ was a l s o a d m i n i s t e r e d to a se p a r a t e p o r t i o n of r o o t s e c t i o n s . In t h i s way, the e f f i c i e n c y o f i n c o r p o r a t i o n of the C-16 epimer c o u l d be d i r e c t l y compared w i t h the n a t u r a l isomer. The r e s u l t s o f t h i s study are shown i n Tables 10 and 11. Tab l e 10. The Stemmadenine Systems A d m i n i s t e r e d to A. p y r i c o l l u m Experiment Compound Fed Weight Wet P l a n t A c t i v i t y Number Fed mg Weight g Fed dpm 3 1 stemmadenine-(Ar- H) 1.3 2 16-epi-stemmadenine- 1.4 (Ar- 3H 49 3.29x10 29 2 . 8 5 x l 0 1 0 - 108 T T a b l e 11, I n c o r p o r a t i o n R e s u l t s A s s o c i a t e d with. T a b l e 10. Experiment Compound I s o l a t e d S p e c i f i c A c t i v i t y % I n c o r p o r a t i o n Number Cwt, mg) (dpm/mmol) 1 a p p a r i c i n e (37) 3.52xl0 6- 0.109 2 a p p a r i c i n e (15) , <0.0001 The data shown i n Tables 10 and 11 c l e a r l y i n d i c a t e t h a t w h i l e a u t h e n t i c stemmadenine, as expected from the r e s u l t s shown i n F i g u r e 14 i s 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 to a s i g n i f i c a n t e x t e n t , the C-16 epimer i s not. A lthough r e s u l t s i n b i o s y n t h e t i c e x p e r i -ments must always be i n t e r p r e t e d w i t h g r e a t c a r e , i t may be the case t h a t e i t h e r the c o n v e r s i o n of the stemmadenine system to the i n d o l e n i n e 117 o r the subsequent l o s s o f formaldehyde as shown i n F i g u r e 15 (pathway A) or b o t h i s an e n z y m a t i c a l l y c o n t r o l l e d process f o r which the n a t u r a l s t e r e o c h e m i s t r y about C-16 i s v i t a l . T h i s o f course assumes t h a t the P o t i e r p o s t u l a t e i s c o r r e c t , something which has not y e t been proven. In any case, the r e s u l t s shown i n T a b l e s 10 and 11 i n d i c a t e , but do not prove', t h a t the s t e r e o c h e m i s t r y about C-16 of stemmadenine i s an important f a c t o r i n i t s r o l e as p r e c u r s o r i n the p l a n t system. F u r t h e r i n f o r m a t i o n on t h a t p o i n t await the s u c c e s s f u l completion of the s y n t h e s i s of stemmadenine, a p r o j e c t c u r r e n t l y underway i n our l a b o r a t o r y . - 109 -EXPERIMENTAL M e l t i n g p o i n t s were determined on a K o f l e r b l o c k and are u n c o r r e c t e d . The u l t r a v i o l e t (UV) s p e c t r a were r e c o r d e d i n methanol u s i n g a Cary 15 r e c o r d i n g spectrometer. The i n f r a r e d (IR) s p e c t r a were r e c o r d e d w i t h a Per k i n - E l m e r Model 457 spectrometer i n c h l o r o f o r m s o l u t i o n w i t h a c e l l path o f 0.2 mm , u s i n g a matched r e f e r e n c e c e l l f i l l e d w i t h c h l o r o f o r m (unless otherwise noted). C a l i b r a t i o n was achieved vising the -16-01 • cm ~ a b s o r p t i o n band of p o l y s t y r e n e . N u c l e a r magnetic resonance s p e c t r a (NMR) were o b t a i n e d w i t h d e u t e r i o c h l o r o f o r m s o l u t i o n s (unless otherwise i n d i c a t e d ) a t 100 MHz on a V a r i a n HA-100 or a V a r i a n XL-100 n u c l e a r magnetic resonance spectrometer. A l l NMR s p e c t r a o b t a i n e d v i a the F o u r i e r Transform technique (FT) w i l l be so noted and were o b t a i n e d w i t h the V a r i a n XL-100 instrument. Chemical s h i f t s are g i v e n i n 6(ppm) w i t h r e f e r e n c e to t e t r a m e t h y l s i l a n e as the i n t e r n a l s t andard. The m u l t i p l i -c i t y , i n t e g r a t e d peak areas, and p r o t o n assignments are g i v e n i n parentheses. Mass s p e c t r a were determined on an AEI-MS-902 or an A t l a s CH-4B mass spectrometer,with h i g h r e s o l u t i o n mass s p e c t r a determined by the former. Woelm n e u t r a l alumina and EM Reagents GF254 s i l i c a g e l \i?ere used f o r t h i n and p r e p a r a t i v e - 110 l a y e r chromatography. Woelm n e u t r a l alumina ( a c t i v i t y I I I ) was used f o r column chromatography i n s e c t i o n A. In s e c t i o n s B and C, Woelm n e u t r a l alumina (12% water) was used f o r column chromatography. In s e c t i o n B, two s o l v e n t systems were used f o r the development of t h i n and p r e p a r a t i v e l a y e r chromatography p l a t e s . System A c o n s i s t e d o f 20% t r i e t h y l a m i n e i n c h l o r o f o r m , and system B c o n s i s t e d of 20% methanol i n benzene. R a d i o a c t i v i t y was measured w i t h N uclear-Chicago Mark 1 or Mark XI l i q u i d s c i n t i l l a t i o n c ounters i n counts per minute (cpm). The r a d i o a c t i v i t y o f a sample i n d i s i n t e g r a t i o n s per minute (dpm) was subsequently determined u s i n g the e x t e r n a l s t a n d a r d t e c h n i -que 1 6^' 1*'' 1' u s i n g a b u i l t - i n Barium-133 source of gamma r a d i a t i o n . A t y p i c a l s u pply o f the l i q u i d s c i n t i l l a t o r s o l u t i o n which was used was made up of the f o l l o w i n g components: t o l u e n e (1 l i t e r ) , 2,5-diphenyloxazole (4 g) and 1,4 - b i s I 2 -(5-phenyloxazoly)Jbenzene (.0.05 g) . In p r a c t i c e a sample was d i s s o l v e d i n benzene (1 ml) or i n methanol (1 ml) i n a co u n t i n g v i a l . The volume was then made up t o 15 ml w i t h the above s c i n t i l l a t o r s o l u t i o n . F o r each sample counted the background a c t i v i t y was determined f o r the co u n t i n g v i a l t o be used by f i l l i n g the v i a l w i t h the a p p r o p r i a t e s o l v e n t and s c i n t i l l a t o r s o l u t i o n and coun t i n g - t h e background a c t i v i t y i n cpm. The v i a l was then emptied, r e f i l l e d w i t h the sample to be counted and the s o l v e n t / s c i n t i l l a t o r s o l u t i o n mixture and i t s a c t i v i t y determined. The d i f f e r e n c e i n a c t i y i t y (cpm) be-tween the sample and the p r e v i o u s l y determined background was then used f o r subsequent c a l c u l a t i o n s . Each v i a l was counted f o r a time p e r i o d long enough f o r the t o t a l counts f o r the sample, l e s s the - I l l -t o t a l counts f o r the background, to exceed one thousand counts. The A. p y r i c o l l u m and A. a u s t r a l e p l a n t s used i n t h i s study were grown i n the H o r t i c u l t u r e Department greenhouse, the U n i v e r s i t y o f B r i t i s h Columbia. - 112 -S e c t i o n A Degradation of a p p a r i c i n e (81) A s o l u t i o n of a p p a r i c i n e C81) CIO.1 mg) i n a c e t i c a c i d CIO ml) was t r e a t e d w i t h ozone gas u n t i l a b l u e c o l o r appeared (15 min)^ Water (10 ml) was then added, and the m i x t u r e allowed to stand f o r 30 min. The mixture was then steam d i s t i l l e d and the d i s -t i l l a t e (30 ml) c o l l e c t e d . The d i s t i l l a t e was then t r e a t e d w i t h a s a t u r a t e d s o l u t i o n o f dimedone (92) i n water (10 ml) and allowed v to stand a t room temperature f o r 16 h r . G l a c i a l a c e t i c a c i d (1 ml) was then added and the mixture r e f l u x e d f o r 6 h r . The s o l v e n t was then evaporated under reduced p r e s s u r e and the r e s i d u e p u r i f i e d v i a s i l i c a g e l p r e p a r a t i v e l a y e r chromatography p l a t e s developed w i t h a s o l u t i o n o f 2 0% e t h y l a c e t a t e i n c h l o r o f o r m . Formaldehyde bisdimedone (93) was o b t a i n e d (0.5 mg, 5%) as white c r y s t a l s which c o u l d be r e c r y s t a l l i z e d from e t h a n o l , m.p. 190-192° 112 ( l i t . m.p. 191 -191 .5° ) , and which was i d e n t i c a l w i t h an a u t h e n t i c sample of formaldehyde bisdimedone (93) by t h i n l a y e r chromatography and mixed m e l t i n g p o i n t comparison. C y c l i z e d acetaldehyde bisdime-done (95) was o b t a i n e d (0.8 mg, 8%) as white c r y s t a l s which c o u l d be p u r i f i e d by r e c r y s t a l l i z a t i o n from e t h a n o l , m.p. 175-176° 112 ( l i t . m.p. 176-177°) , and which was i d e n t i c a l w i t h an a u t h e n t i c sample by t h i n l a y e r chromatography and mixed m e l t i n g p o i n t compari-son. The a d m i n i s t r a t i o n of l a b e l l e d s ecodine (76) to A. p y r i c o l l u m The p a r t i c u l a r l a b e l l e d secodine (76) to be a d m i n i s t e r e d was 113 -prepared from 16,17-dihydrosecodin-17-ol C 7 7 ) as p r e v i o u s l y 93 95 d e s c r i b e d . ' The p a l e y e l l o w gum was then d i s s o l v e d i n et h a n o l CO.5 ml} and to t h i s s o l u t i o n was added 0.1 N a c e t i c a c i d CO.5 ml) and d i s t i l l e d water C1.0 m l ) . T h i s s o l u t i o n was ad m i n i s t e r e d to r o o t c u t t i n g s ( u s u a l l y 30-6 0 g) of 2-3 year o l d A. p y r i c o l l u m p l a n t s i n a l a r g e t e s t tube f o r 5 days. During t h a t time, the r o o t c u t t i n g s were kept m o i s t by p e r i o d i c a d d i t i o n s of d i s t i l l e d water. The e x t r a c t i o n o f a p p a r i c i n e (81) from A. p y r i c o l l u m . The r o o t c u t t i n g s t o which l a b e l l e d secodine (76) had been a d m i n i s t e r e d were mascerated w i t h methanol i n a Waring b l e n d e r , f i l t e r e d , and washed w i t h methanol. The s o l v e n t was evaporated under reduced p r e s s u r e and the r e s i d u e taken up i n 2 N h y d r o c h l o r i c a c i d (150 m l ) . T h i s m i x t u r e was f i r s t e x t r a c t e d w i t h benzene ( 3 x 75 m l ) , then made b a s i c w i t h 15 N ammonium hydroxide and e x t r a c t e d w i t h c h l o r o f o r m (3 x 10 0 m l ) . The combined c h l o r o f o r m e x t r a c t s were d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness under reduced p r e s s u r e . The r e s i d u e thus o b t a i n e d was p u r i f i e d v i a p r e p a r a t i v e l a y e r chromatography on s i l i c a g e l p l a t e s developed w i t h 30% e t h y l a c e t a t e i n e t h a n o l , a f f o r d i n g pure a p p a r i c i n e (81), which c o u l d be r e c r y s t a l l i z e d from acetone. The a p p a r i c i n e thus o b t a i n e d was degraded by o z o n o l y s i s as d e s c r i b e d above. F u r t h e r data p e r t a i n i n g to experiment 5, Tables 1 and 2 are as f o l l o w s . Weight of seco-d i n e C76) f e d : 2.1 mg; wet p l a n t weight: 64 g; weight a p p a r i c i n e i s o l a t e d : 33.2 mg; weight formaldehyde bisdimedone i s o l a t e d - 114 -a f t e r d e g r a d a t i o n : 2.1 mg; weight acetaldehyde bisdimedone d e r i v a t i v e i s o l a t e d a f t e r d e g r a d a t i o n o f a p p a r i c i n e : 4 mg. A d m i n i s t r a t i o n o f l a b e l l e d compounds t o A. a u s t r a l e The compound to be f e d was d i s s o l v e d i n e t h a n o l (5-10 d r o p s ) . To t h i s s o l u t i o n was added 0.1 N a c e t i c a c i d (5 drops) and d i s t i l l e d water (0.5 m l ) . T h i s s o l u t i o n was a d m i n i s t e r e d hydro-p o n i c a l l y t o whole 1-2 year o l d A. a u s t r a l e p l a n t s whose r o o t systems were c o n t a i n e d i n t e s t tubes. A b s o r p t i o n of the s o l u t i o n by the p l a n t s was observed i n 2-3 h r , a f t e r which the r o o t s were kept moist w i t h a d d i t i o n a l q u a n t i t i e s of d i s t i l l e d water f o r the 5 day f e e d i n g p e r i o d . E x t r a c t i o n o f a l k a l o i d s from A. a u s t r a l e The A. a u s t r a l e p l a n t s were mascerated i n a Waring b l e n d e r w i t h methanol, f i l t e r e d , and remascerated u n t i l the f i l t r a t e was c o l o r l e s s . The s o l v e n t was evaporated under reduced p r e s s u r e and the r e s i d u e taken up i n 2 N h y d r o c h l o r i c a c i d (150 m l ) . T h i s mixture was f i r s t e x t r a c t e d w i t h benzene (3 x 75 m l ) , then made b a s i c w i t h 15 N ammonium hydroxide and e x t r a c t e d w i t h c h l o r o -form (3 x 100 ml). The combined c h l o r o f o r m e x t r a c t s were d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and evaporated to dryness under reduced p r e s s u r e . The r e s i d u e was d i l u t e d w i t h a s m a l l amount of i n a c t i v e o l i v a c i n e (88) (4-13 mg) and chromatographed on a p r e p a r a t i v e l a y e r s i l i c a g e l p l a t e developed w i t h a s o l u t i o n of 30% e t h a n o l i n e t h y l a c e t a t e , y i e l d i n g pure o l i v a c i n e , which c o u l d be f u r t h e r p u r i f i e d by r e c r y s t a l l i z a t i o n i n a mixture o f T~ 115 -c h l o r o f o r m and methanol. The remainder of t h e p r e p a r a t i v e l a y e r p l a t e was e x t r a c t e d w i t h methanol and the s o l v e n t remoyed under reduced p r e s s u r e . The r e s u l t i n g r e s i d u e was then chromatograph-ed on a p r e p a r a t i v e l a y e r alumina p l a t e developed w i t h a s o l u t i o n of 10% methanol i n benzene, which a f f o r d e d pure a p p a r i c i n e (81) which c o u l d then be r e c r y s t a l l i z e d from acetone. The remainder of the p l a t e was e x t r a c t e d as b e f o r e , and the r e s i d u e d i l u t e d w i t h i n a c t i v e u l e i n e C83) and guatambuine C90) C8-14 mg). T h i s m i x t u r e was then chromatographed on a p r e p a r a t i v e l a y e r s i l i c a g e l p l a t e developed w i t h 50% a c e t i c a c i d i n e t h y l a c e t a t e , y i e l d -i n g the a c e t a t e s a l t s o f u l e i n e (83) and guatambuine (90). These s a l t s were made b a s i c w i t h 15 N ammonium hydroxide and e x t r a c t e d w i t h methylene c h l o r i d e . The methylene c h l o r i d e e x t r a c t s were then d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated t o dryness under reduced p r e s s u r e , y i e l d i n g u l e i n e (83) and guatambuine (90) as the f r e e bases. U l e i n e was r e c r y s t a l l i z e d from methanol and guatambuine was r e c r y s t a l l i z e d from a mixture of methanol and c h l o r o f o r m . The data o b t a i n e d from the a d m i n i s t r a t i o n of v a r i o u s l a b e l l e d compounds t o A. a u s t r a l e can be found i n Tables 4 and 5. r- 116 <r S e c t i o n B Degradation of s t r y c h n i n e C2 9) to 23,16q-cur-19-en-17-ol (137) The d e g r a d a t i o n of s t r y c h n i n e to Wieland-Gumlich aldehyde has been e x h a u s t i v e l y s t u d i e d and d e s c r i b e d i n g r e a t d e t a i l by 129 Schmid and K a r r e r . As w e l l , the subsequent t r a n s f o r m a t i o n of Wieland-Gumlich aldehyde to the a l c o h o l 137 has been d e s c r i b e d 132 133 b o t h v i a the a l l y l i c bromide 138, ' and v i a the d i r e c t hydro-134 g e n o l y s i s of the d i o l 126. D e t a i l s of 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 i n t h i s work are g i v e n below. Wherever m o d i f i -c a t i o n s i n the e x perimental procedure were employed or a d d i t i o n a l data were o b t a i n e d f o r c h a r a c t e r i z a t i o n o f the i n t e r m e d i a t e s i n the d e g r a d a t i o n , t h i s i s i n c l u d e d . As w e l l , t h i s p r a c t i c e w i l l be f o l l o w e d wherever e l s e a p a r t i c u l a r r e a c t i o n or sequence of r e a c t i o n s has been p r e v i o u s l y d e s c r i b e d . 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 (.131) A suspension of s t r y c h n i n e (134 g) i n dry e t h a n o l (800 ml) was t r e a t e d w i t h isoamyl n i t r i t e (211 g) and sodium e t h o x i d e (37.2 g sodium i n 1 1 e t h a n o l ) . . I s o l a t i o n of the product by c r y s t a l l i z a t i o n from aqueous h y d r o c h l o r i c a c i d y i e l d e d the d e s i r e d h y d r o c h l o r i d e 131 )129 g, 8 0 % ) . Wieland-Gumlich aldehyde (1301 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 (11 g) was t r e a t e d w i t h t h i o n y l c h l o r i d e (20 ml) f o l l o w e d by quenching i n i c e (100 g ) . - 117 -F i l t r a t i o n o f the aqueous s l u r r y f o l l o w e d by treatment w i t h steam a t pH 3-3.5 (methyl orange i n d i c a t o r ) y i e l d e d the d e s i r e d Wieland-Gumlich aldehyde (130) (5 g, 58%) as c o l o r l e s s c r y s t a l s which c o u l d be r e c r y s t a l l i z e d from c h l o r o f o r m or benzene m.p. 213-215° ( l i t . 1 2 9 m.p. 214-216). NMR s i g n a l s : 6.6-7.2 (2 m u l t i p l e t s , 4H, aromatic C-H), 5.82 (broad s i n g l e t , IH, C-19 H), 5.0 ( s i n g l e t , IH, C-17 H) . 2 8,16a-cur-19-en-17,18-diol (136) Wieland-Gumlich aldehyde (5 g) was t r e a t e d w i t h sodium boro-133 h y d r i d e (1.25 g) i n methanol (200 ml) as o u t l i n e d by Bernauer t o y i e l d the d e s i r e d d i o l 136 as c o l o r l e s s c r y s t a l s (4.1 g, 82%) 12 which c o u l d be r e c r y s t a l l i z e d from methanol m.p. 250-251° ( l i t . m.p. 251°). NMR s i g n a l s (CD^OD): 6.6-7.2 (2 m u l t i p l e t s , 4H, aro-matic C-H), 5.72 ( t r i p l e t , J = 7 Hz, IH, C-19 H), 4.13 (doublet of d o u b l e t s , J = 7 and 2 Hz, 2H, C-18 H_2) . Mass spectrum: M +, m/e = 312; main peaks = 294, 281, 182, 144 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C i n H „ . N „ 0 o : 312.1837. Found: 312.1784. 2g-16a-17-acetoxy-18-bromo-cur-19-en (138) The d i o l 136 (2.3 g) 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 (300 ml), and t r e a t e d w i t h a s a t u r a t e d (at 0°) s o l u t i o n of hydrogen bromide i n a c e t i c a c i d (6 ml) as o u t l i n e d by Bernauer. The a l l y l i c bromide thus o b t a i n e d was used i n the next r e a c t i o n w ithout i s o l a t i o n or p u r i f i c a t i o n . ^ 118 -2g,16q-17-acetoxy~cur-19-en (139! The crude a l l y l i c bromide o b t a i n e d i n the p r e c e d i n g 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 c i d C200 ml) and t r e a t e d w i t h z i n c powder (15 g) a c c o r d i n g to the method d e s c r i b e d by Bernauer. The crude product thus o b t a i n e d was p u r i f i e d by e l u t i o n w i t h benzene through a column of alumina, y i e l d i n g a c o l o r l e s s o i l (1.5 g, 63%). NMR s i g n a l s : 6.6-7.2 (2 m u l t i p l e t s , 4H, aromatic C-H), 5.58 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 2.04 ( s i n g l e t , 3H, OOCCH 3), 1.59 (doublet of d o u b l e t s , J = 7 and 2 Hz, 3H, C-18 H^). Mass spectrum: M +, m/e = 338; main peaks: 279, 144 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 2 i H 2 6 N 2 ° 2 : 338.1993. Found: 338.1978. 2g,16a-cur-19-en-17-ol (137) The a c e t a t e 139 (1.5 g) o b t a i n e d i n the p r e c e d i n g r e a c t i o n was t r e a t e d w i t h IN m e t h a n o l i c potassium hydroxide (75 ml) a t room temperature f o r 30 min. The s o l v e n t was evaporated under reduced p r e s s u r e , and the r e s i d u e t r e a t e d w i t h water (50 ml) and methylene c h l o r i d e (50 m l ) . E x t r a c t i o n of t h i s mixture w i t h methylene c h l o r i d e (3 x 50 ml) f o l l o w e d by d r y i n g of the combined e x t r a c t s over anhydrous sodium s u l f a t e , f i l t r a t i o n , and evapora-t i o n of the s o l v e n t under reduced p r e s s u r e y i e l d e d the crude a l c o h o l . P u r i f i c a t i o n was e f f e c t e d by e l u t i o n through a column of alumina w i t h benzene having an e t h e r g r a d i e n t , r e s u l t i n g i n i s o l a t i o n of the pure a l c o h o l 137 as c o l o r l e s s c r y s t a l s (1.0 g, 77%) which c o u l d be f u r t h e r p u r i f i e d by r e c r y s t a l l i z a t i o n from benzene m.p. 168-171° ( l i t . 1 3 4 m.p. 170-174°). NMR s i g n a l s : 6.5-7.2 (2 m u l t i p l e t s , 4H., aromatic C-H.)., 5.47 ( q u a r t e t , J - 7 Hz, IK, C-18 tt), 4.70 Cbroad s i n g l e t , IH, OH), 1.58 (doublet of . d o u b l e t s , J = 7 and 2 Hz, 3H, C-19 H^). Mass spectrum: M +, m/e = 296, main peaks: m/e = 294, 279, 266, 166 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C ] _ g H 2 4 N 2 0 : 296.1887. Found: 296.1883. C a t a l y t i c h y d r o g e n o l y s i s of 23,16a-cur-19-en-17,18-diol (136) The p r e v i o u s l y o b t a i n e d d i o l 136 (3.5 g) was d i s s o l v e d i n a mixture of water (175 m l ) , a c e t i c a c i d (105 ml), and c o n c e n t r a t e d h y d r o c h l o r i c a c i d (1.75 m l ) . P a l l a d i u m on c h a r c o a l (10%, 543 mg) was added, and the mixture hydrogenolyzed as o u t l i n e d by Wieland, y i e l d i n g the d e s i r e d a l c o h o l 137 (3.0 g, 91%) which c o u l d be r e c r y s t a l l i z e d d i r e c t l y from the crude product w i t h o u t the necess i t y o f chromatographic p u r i f i c a t i o n . The a l c o h o l thus o b t a i n e d was i d e n t i c a l i n every r e s p e c t w i t h t h a t d e s c r i b e d above. Oppenauer o x i d a t i o n of 23,16a-cur-19-en-17-ol (137); experiment 1 A s o l u t i o n of the a l c o h o l 137 (50 mg) i n benzene (10 ml) was t r e a t e d w i t h benzophenone (150 mg) and potassium t e r t - b u t o x i d e (84 mg) i n benzene (1.7 ml) a c c o r d i n g to the procedure o u t l i n e d 137 by Boekelheide. The crude r e a c t i o n product thus o b t a i n e d was p u r i f i e d v i a p r e p a r a t i v e l a y e r chromatography on s i l i c a g e l ( s o l v e n t system A ) , r e s u l t i n g i n the i s o l a t i o n of n o r - f l u o r o -c u r a r i n e (134) (10 mg, 20%), and 28-cur-19-en-17-al (133) (5 mg, 10%), which were c h a r a c t e r i z e d as f o l l o w s : N o r - f l u o r o c u r a r i n e m.p. 180-185° ( l i t . 1 3 4 m.p. 185-186°). NMR s i g n a l s : 9.39 12 0 -C s i n g l e t , I E , CHOI, 6.8-7.4 ( m u l t i p l e t , 4E, a r o m a t i c C-R) , 5.43 ( q u a r t e t , J - 7 H z , 1H, C-19 H) , 1.60 ( d o u b l e t w i t h f u r t h e r f i n e s p l i t t i n g , J = 7 H z , 3K, C-18 H 3 ) . Mass s p e c t r u m : M , m/e = 2 9 2 ; m a i n p e a k s : 2 6 3 , 2 4 9 , 1 6 7 , 121 ( b a s e p e a k ) . H i g h r e s o l u -t i o n mass s p e c t r u m : c a l c . f o r C ^ g H ^ ^ O : 2 9 2 . 1 5 7 5 . F o u n d : 2 9 2 . 1 5 5 0 . 133 S a t u r a t e d a l d e h y d e 133 I R b a n d s : 3 4 0 0 , 2 9 2 0 , 1 7 1 5 , 1 6 0 5 . UV a b s o r p t i o n s : A 2 9 8 , 2 4 5 ; l o g e 3.40, 3.83. NMR s i g n a l s : 9.78 r max / / ^ / v ( s i n g l e t , I H , CHO), 6.5-7.2 (2 m u l t i p l e t s , 4H, a r o m a t i c C-H), 5.46 ( q u a r t e t , J = 7 H z , I H , C-19 H ) , 1.60 ( d o u b l e t o f d o u b l e t s , J = 7 a n d 2 H z , 3H, C-18 H_3) . M a s s s p e c t r u m : M + , m/e = 294; m a i n p e a k s : 2 5 1 , 2 1 8 , 164 ( b a s e p e a k ) , 1 4 5 . H i g h r e s o l u t i o n mass s p e c t r u m : c a l c . f o r C 1 9 H 2 2 N 2 0 : 2 9 4 . 1 7 3 1 . F o u n d : 2 9 4 . 1 7 6 2 . O p p e n a u e r o x i d a t i o n o f 2 g , 1 6 o t - c u r - 1 9 - e n - 1 7 - o l ( 1 3 7 ) ; e x p e r i m e n t 2 To a s o l u t i o n o f t h e a l c o h o l 137 (300 mg) i n d r y b e n z e n e (50 ml) was a d d e d b e n z o p h e n o n e (900 mg) a n d a s a t u r a t e d s o l u t i o n o f p o t a s s i u m t e r t - b u t o x i d e i n b e n z e n e (10 m l ) . The 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 15 m i n , a n d a n o t h e r p o r t i o n o f t h e s a t u r a t e d b u t o x i d e s o l u t i o n (5 ml) was a d d e d a n d t h e m i x t u r e h e a t e d t o r e f l u x . A f t e r 15 m i n r e f l u x i n g , a f u r t h e r p o r t i o n (5 m l ) o f t h e b u t o x i d e s o l u t i o n was a d d e d , a n d t h e r e a c t i o n a l l o w e d t o c o n t i n u e 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 ( s i l i c a g e l , s o l v e n t s y s t e m A) i n d i c a t e d t h a t a l l s t a r t i n g m a t e r i a l h a d b e e n c onsumed ( t o t a l r e a c t i o n t i m e : 1.5 h r ) . The r e a c t i o n was t h e n q u e n c h e d w i t h w a t e r (25 m l ) a n d e x t r a c t e d w i t h b e n z e n e . The b e n z e n e e x t r a c t s w e r e c o m b i n e d , 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 f a t e , f i l t e r e d , a n d t h e s o l v e n t e v a p o r a t e d u n d e r r e d u c e d - 121 -p r e s s u r e . The. r e s i d u e thus o b t a i n e d was p u r i f i e d by e l u t i o n through a column of alumina with, benzene having an e t h e r g r a d i e n t , y i e l d i n g the s a t u r a t e d aldehyde 133 (115 mg, 39%) and a s m a l l amount of n o r - f l u o r o c u r a r i n e (134) (30 mg, 10%). Oppenauer o x i d a t i o n of 28,16a-cur-19-en-17-ol (137); experiment 3 To a mixture of benzene (10 ml) and tert-butan.ol (10 ml) was added l i t h i u m metal (1.48 g ) , and the mixture allowed to s t i r , a t 40° u n t i l a l l the metal had been consumed. The excess s o l v e n t was then evaporated under reduced p r e s s u r e , and a s o l u t i o n of the a l c o h o l 137 (600 mg) and benzophenone (3.6 g) i n t o l u e n e (150 ml) was added to the r e a c t i o n f l a s k . The mixture was a l l o w e d to s t i r a t 90° f o r 48 h r . A t t h i s p o i n t , potassium t e r t - b u t o x i d e (2.0 g) was added t o the r e a c t i o n m ixture, f o l l o w e d by s t i r r i n g a t 95° f o r 24 h r . E x t r a c t i o n of the crude r e a c t i o n product and p u r i f i c a t i o n as d e s c r i b e d above y i e l d e d n o r - f l u o r o c u r a r i n e (134) (280 mg, 47.5%) as w e l l as a s m a l l amount of the s a t u r a t e d aldehyde 133 (60 mg, 10%). Oppenauer o x i d a t i o n of 2g,16a-cur-19-en-17-ol (137); experiment 4 To a s o l u t i o n of the a l c o h o l 137 (1.75 g) i n t o l u e n e (450 ml) was added benzophenone (21.8 g ) , and the mixture heated to 90°. Potassium t e r t - b u t o x i d e (7.34 g) was then added and the m i x t u r e s t i r r e d a t 90° f o r 3 h r . Nitrobenzene (20 ml) was then added, and an i n s t a n t a n e o u s c o l o r change from dark orange to dark green was noted. E x t r a c t i o n of the crude r e a c t i o n product and p u r i f i -c a t i o n as d e s c r i b e d above y i e l d e d n o r - f l u o r o c u r a r i n e (709 mg, 41%). ^ 122 -No s a t u r a t e d aldehyde 133 c o u l d be d e t e c t e d In the r e a c t i o n m i x t u re. Des-carbomethoxy stemmadenine (150) N o r - f l u o r o c u r a r i n e (134) (50 mg) was d i s s o l v e d i n methanol (5 m l ) , and sodium b o r o h y d r i d e (55 mg) added i n t h r e e equal p o r t i o n s a t 15 min i n t e r v a l s a t room temperature. A f t e r a t o t a l r e a c t i o n time of 4 5 min, the s o l v e n t was removed under reduced p r e s s u r e , and the r e s i d u e t r e a t e d w i t h a m i x t u r e of water (5 ml) and methylene c h l o r i d e (5 m l ) . E x t r a c t i o n of t h i s mixture w i t h methylene c h l o r i d e (3 x 5 ml) was f o l l o w e d by d r y i n g the combined methylene c h l o r i d e e x t r a c t s over anhydrous sodium s u l f a t e . A f t e r f i l t r a t i o n , the s o l v e n t was evaporated under reduced p r e s s u r e , and the r e s i d u e p u r i f i e d by e l u t i o n through a column of alumina w i t h benzene having an e t h e r g r a d i e n t . The i n d o l e a l c o h o l 150 (24 mg, 48%) was o b t a i n e d as a c o l o r l e s s o i l which c o u l d not be induced to c r y s t a l l i z e . IR bands: 3350, 3250 (sh), 2900, 1460. UV a b s o r p t i o n s : A 290, 283 nm; l o g e 3.74, 3.78. NMR s i g n a l s : c max 3 ^ 9.08 ( s i n g l e t , IH, i n d o l e N-H), 7.0-7.6 ( m u l t i p l e t , 4H, aromatic C-H), 5.47 ( q u a r t e t , J = 7 Hz, IH, C-19 H) , 4.19 (doublet, J = 5 Hz, 2H, CH 2OH), 4.00 (doublet, J = 5 Hz, IH, C-16 H), 1.66 (doublet, J = 7 Hz, 3H, C-18 H_3) . Mass spectrum: M +, m/e =• 296; main peaks: 166, 123 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 1 9 H 2 4 N 2 0 : 296.1887. Found: 296.1906. N o r - f l u o r o c u r a r i n e - c y c l o h e x y l a m i n e enamine (151) N o r - f l u o r o c u r a r i n e (50 mg) was r e f l u x e d w i t h cyclohexylamine (2 ml) and p _ - t o l u e n e s u l f o n i c a c i d (5 mg) i n benzene (15 ml) w i t h - 123 -a z e o t r o p i c remoyal of water f o r 4 hr. The r e a c t i o n m i x t u r e was then cooled and shaken q u i c k l y w i t h a s a t u r a t e d aqueous s o l u t i o n of sodium carbonate CIO ml). The benzene l a y e r was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced pressure. The residue thus obtained was p u r i f i e d v i a p r e p a r a t i v e l a y e r chromatography ( s i l i c a g e l , s o l v e n t system B), r e s u l t i n g i n i s o l a t i o n of the d e s i r e d enamine 151 as a b r i g h t y e l l o w o i l (23 mg, 36%). IR bands: 2925, 1630. UV a b s o r p t i o n s : ^ m a x 387, 247 nm: l o g e 4.28, 3.91. NMR s i g n a l s : 6.9-7.6 C m u l t i p l e t , 5H, aromatic C-H and NCH=C), 5.44 (quartet, J = 7 Hz, IH, C-19 H), 4.06 ( s i n g l e t , IH, N-H), 1.64 (doublet, J = 7 Hz, 3H, C-18 H^). Mass spectrum: M , m/e = 373. N o r - f l u o r o c u r a r i n e - p y r r o l i d i n e enamine (152) N o r - f l u o r o c u r a r i n e (134) (50 mg) was r e f l u x e d w i t h p y r r o l i -dine (2 ml) i n benzene (10 ml) w i t h a z e o t r o p i c removal of water f o r 27 hr. At t h i s time, the UV spectrum of the crude r e a c t i o n mixture i n d i c a t e d the expected s h i f t to A 387, and the s o l v e n t ^ max ' evaporated under reduced pressure. However, any attempt to p u r i f y the crude product (65 mg) r e s u l t e d i n p a r t i a l h y d r o l y s i s of the enamine, as shown by the reappearance of a s i n g l e t at 9.39 i n the NMR spectrum, t h e r e f o r e , f u l l c h a r a c t e r i z a t i o n of the p y r r o l i d i n e enamine 152 was not achieved. Wieland-Gumlich aldoxime C154) Wieland-Gumlich aldehyde C130) (5.0 g) was dissolved i n pyridine (75 ml), and hydroxylamine hydrochloride (5.55 g) added. The m i x t u r e was allowed to s t i r a t room temperature f o r 16 h r . The s o l v e n t was then evaporated under reduced p r e s s u r e , and the r e s i d u e d i s s o l v e d i n water (150 m l ) . Concentrated ammonium hydroxide was then added dropwise w i t h v i g o r o u s s t i r r i n g , r e s u l t i n g i n the p r e c i p i t a t i o n of the d e s i r e d oxime as white c r y s t a l s . When the pH of the aqueous suspension reached 10, a d d i t i o n was stopped, and the mixture f i l t e r e d and washed w i t h water. The r e s i d u e was then r e c r y s t a l l i z e d from methanol, y i e l d i n g the d e s i r e d oxime 154 (4.75 g, 91%) m.p. 243°(d) ( l i t . 1 2 7 m.p. 245° ( d ) ) . IR a b s o r p t i o n s (KBr): 3350, 2900, 1600. NMR s i g n a l s : 6.6-7.4 ( m u l t i p l e t , 4H, aromatic C-H), 5.67 ( t r i p l e t J = 7 Hz, IH, C-19 H) . Mass spectrum: M +, m/e = 325; main peaks: 308, 267, 144 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 1 9 H 2 3 N 3 ° 2 : 325.1789. Found: 325.1817. 18-acetoxy-l-aoetyl-16-cyano-17-nor-2(B, 16a-cur-19-ene (155) Wieland-Gumlich aldoxime (4.75 g) was t r e a t e d w i t h a c e t i c anhydride (50 ml) and p y r i d i n e (8.25 ml) a c c o r d i n g to the procedure p r e v i o u s l y d e s c r i b e d by S m i t h . 1 3 1 The d e s i r e d n i t r i l e 155 was o b t a i n e d as a l i g h t brown o i l (5.35 g, 93%). NMR s i g n a l s 7.0-7.3 ( m u l t i p l e t , 4H, aromatic C-H), 5.64 ( t r i p l e t , J = 7 Hz, IH, C-19 H), 2.10 ( s i n g l e t , 3H, CHACON), 2.00 ( s i n g l e t , 3H, CH^COO). Mass spectrum: M , m/e = 391; main peaks; m/e = 351, 331, 219, 144 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 2 3 R 2 5 N 3 ° 3 : 2 9 1 - 1 8 9 5 - Found: 391.1801. - 125 -M e t h y l 18-hydroxy-2g , 16q-cur-19-en-17-oate (156) The n i t r i l e 155 (5.35 g). was t r e a t e d w i t h barium hydroxide (21.0 g) i n e t h a n o l (110 ml) and water (220 ral) e x a c t l y a c c o r d -131 m g to the procedure o u t l i n e d by Smith. The crude r e a c t i o n product was then t r e a t e d w i t h 5% m e t h a n o l i c hydrogen c h l o r i d e (330 ml) a c c o r d i n g to Smith's procedure, r e s u l t i n g i n the i s o l a t i o n of the d e s i r e d hydroxy e s t e r 156 as a c o l o r l e s s c r y s t a l l i n e s o l i d (2.5 g, 53.5%) which c o u l d be f u r t h e r p u r i f i e d 131 by r e c r y s t a l l i z a t i o n from methanol m.p. 153-154° ( l i t . m.p. 154-156). NMR signals-: 6.6-7.2 ( m u l t i p l e t , 4H, aromatic C-H), 5.62 ( t r i p l e t , J = 7 Hz, IH, C-19 H), 4.22 (disappears a f t e r a d d i t i o n of D 20) ( s i n g l e t , IH, O-H) , 3.68 ( s i n g l e t , 3H, COOCH_3) , 3.39 (disappears a f t e r a d d i t i o n of D 20) ( s i n g l e t , IH, N-H). Mass spectrum: M +, m/e = 340; main peaks: 308, 267, 178 (base peak), 144. High r e s o l u t i o n mass spectrum: c a l c . f o r C^H^N-jO^: 340.1786. Found: 340.1745. Methyl-2g,16ct-cur-19-en-17-oate (157) The hydroxy e s t e r 156 (200 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 (60 ml), and a s a t u r a t e d (at 0°) s o l u t i o n of hydrogen bromide i n a c e t i c a c i d (1.8 ml) was added. The mixture was allowed to s t i r a t room temperature i n the dark f o r 4 8 hr, a f t e r which the s o l v e n t was evaporated under reduced p r e s s u r e . The crude p r o d u c t thus o b t a i n e d 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 (100 ml) and methanol (10 m l ) , and powdered z i n c (10 g) was added. A f t e r s t i r r i n g a t room temperature f o r 3 h r , the mixture was f i l t e r e d , the r e s i d u e washed w i t h methanol and the f i l t r a t e evaporated under reduced p r e s s u r e . The r e s i d u e was taken up i n e t h y l a c e t a t e (50 m i l and a s a t u r a t e d aqueous s o l u t i o n of sodium carbonate (50 m l ) . The mixture was e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 50 m l ) , and the combined o r g a n i c e x t r a c t s d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated under reduced p r e s s u r e . The r e s i d u e was p u r i f i e d on a column of alumina w i t h e l u t i o n by benzene, r e s u l t i n g i n the d e s i r e d e s t e r 157 as a c o l o r l e s s o i l (130 mg, 65%) which c o u l d not be induced t o c r y s t a l l i z e . IR bands: 3420, 2950, 1730, 1605, 1480, 1440. UV a b s o r p t i o n s : Amax 2 9 7 ' 2 4 4 ; l o g e 3 - 4 7 ' 3 - 8 1 - N M R s i g n a l s : 6.5-7.2 ( m u l t i p l e t , 4H, aromatic C-H), 5.48 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 3.70 ( s i n g l e t , 3H, COOCH_3) , 1.58 (doublet of d o u b l e t s , J = 7 and 2 Hz, 3H, C-18 H^). Mass spectrum: M , m/e = 324; main peaks: m/e = 251, 194 (base p e a k ) r 144, 139 and 130. High r e s o l u t i o n mass spectrum: c a l c f o r C 2 Q H 2 4 N 2 0 2 : 324.1837. Found: 324.1766. Methyl-l-formyl-26,16a-cur-19-en-17-oate (158) The e s t e r 157 (250 mg) was d i s s o l v e d i n benzene (2 ml) and added t o sodium h y d r i d e (100 mg) (obtained by washing a 50% o i l d i s p e r s i o n (200 mg) w i t h benzene ( 3 x 1 m l ) ) , f o l l o w e d by the a d d i t i o n of methyl formate (3 ml). The mixture was heated t o r e f l u x w i t h s t i r r i n g under an atmosphere o f n i t r o g e n gas f o r 40 min. The excess sodium h y d r i d e was then d e s t r o y e d by the drop-wise a d d i t i o n o f g l a c i a l a c e t i c a c i d u n t i l e b u l l i t i o n was no lon g e r observed. The m i x t u r e was then e l u t e d through a column of alumina w i t h benzene. The f i r s t f r a c t i o n (25 ml) y i e l d e d pure N-formyl e s t e r 158 (178 mg), and the succeeding f o u r 127 -f r a c t i o n s C10Q ml) y i e l d e d l e s s pure m a t e r i a l which, was p u r i f i e d v i a p r e p a r a t i v e l a y e r chromatography on s i l i c a g e l ( s o l v e n t system B ), the combined processes y i e l d i n g a white c r y s t a l l i n e s o l i d (216 mg, 80%) m.p. 162-170°. IR bands: 2960, 1730, 1668, 1600. UV a b s o r p t i o n s : X 287, 278, 250; ' ' ' r max ' ' l o g e 3.44, 3.46, 3.98. NMR s i g n a l s : 8.71 ( s i n g l e t , . I H , NCHO), 5.38 ( q u a r t e t , J = 7 Hz, IH, C-19 H) , 3.68 ( s i n g l e t , 3H, COOCH_3) , 1.53 (doublet of d o u b l e t s , J = 7 and 2 Hz, C-18 H ) Mass spectrum: H+, m/e = 352; main peaks: 279, 194, 172 (base peak), 144. High r e s o l u t i o n mass spectrum: c a l c . f o r C 2 i H 2 4 N 2 ° 3 : 352.1785; Found: 352.1829. Carbomethoxy t e t r a h y d r o o x a z i n e 159 Paraformaldehyde (400 mg) was mixed w i t h phosphorous pen-t o x i d e (400 mg) and heated under vacuum (1 mm). The formaldehyde which was r e l e a s e d was condensed as a s l u r r y by p a s s i n g the vapor i n t o a f l a s k c o o l e d by l i q u i d n i t r o g e n . T h i s s l u r r y was then s i m i l a r l y d i s t i l l e d i n t o a f l a s k c o n t a i n i n g sodium h y d r i d e (25 mg, o b t a i n e d by washing a 50% o i l d i s p e r s i o n (50 mg) w i t h benzene ( 3 x 1 ml)) and d i m e t h y l s u l f o x i d e (2 m l ) . A f t e r the d i s t i l l a t i o n was complete, the r e a c t i o n f l a s k was warmed to room temperature and the N-formyl e s t e r 158 (50 mg) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e (1 ml) and added to the r e a c t i o n m i x t u r e . A f t e r s t i r r i n g a t room temperature f o r 4 hr, the r e a c t i o n was quenched by adding g l a c i a l a c e t i c a c i d u n t i l e b u l l i t i o n was no lo n g e r observed. The mixture was then made b a s i c w i t h a - . 128 -s a t u r a t e d aqueous s o l u t i o n of sodium carbonate (10 ml) and e x t r a c t e d with, e t h y l a c e t a t e (4 :x 15 ml}. The e t h y l a c e t a t e e x t r a c t s were then combined and washed w i t h water (-4 x 10 ml) , d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and the s o l v e n t evaporated under reduced p r e s s u r e . P u r i f i c a t i o n was a c h i e v e d by e l u t i n g the crude r e a c t i o n product through a column of alumina w i t h benzene as e l u e n t . The d e s i r e d p r o d u c t was o b t a i n e d as a c o l o r l e s s o i l (40 mg, 78%). IR bands: 2960, 1730, 1602. UV a b s o r p t i o n s : A m a x 299, 246; l o g e 3.51, 3.97. NMR (FT) s i g n a l s ( F i g u r e 36): 6.6-7.2 (2 m u l t i p l e t s , 4H, aromatic C-H), 5.46 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 5.18 (doublet, J = 10 Hz, IH, N-CHH-O), 4.71 (doublet, J = 10 Hz, IH, N-CHH-O), 4.94 ( s i n g l e t , IH, C-2 H) , 3.70 ( s i n g l e t , 3H, COOCH_3) , 1.57 (doublet o f d o u b l e t s , J = 7 and 2 Hz, 3H, C-18 H). Mass spectrum: M +, m/e = 366; main peaks: 336, 280, 279, 250, 206, 143 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 2 2 H 2 6 N 2 ° 3 : 3 6 6 - 1 9 4 3 - Found: 366.1905. Model t e t r a h y d r o o x a z i n e 166 The a l c o h o l 137 (500 mg) was d i s s o l v e d i n methanol (15 m l ) , and paraformaldehyde (500 mg) and anhydrous sodium s u l f a t e (1.0 g) were added. The mixture was allowed t o s t i r a t room temperature f o r 18 hr, then f i l t e r e d and the s o l v e n t evaporated under reduced p r e s s u r e . The r e s i d u e was taken up i n a s a t u r a t e d aqueous s o l u -t i o n of sodium carbonate (25 ml) and methylene c h l o r i d e (25 m l ) . The m ixture was e x t r a c t e d w i t h methylene c h l o r i d e (3 x 25 ml) and the o r g a n i c e x t r a c t s combined, d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced p r e s s u r e . The - 129 -r e s i d u e was p u r i f i e d by e l u t i o n through, a column of alumina w i t h methylene c h l o r i d e as e l u e n t , y i e l d i n g the d e s i r e d t e t r a h y d r o -o x a z i n e 166 as a c o l o r l e s s o i l (520 mg, 100%). IR bands: 2940, 2870, 1601.4. UV a b s o r p t i o n s : \ 297, 249; l o g e 3.52, 3.98. c max NMR s i g n a l s ( F i g u r e 37}: 6.76-7.3 (2 m u l t i p l e t s , 4H, aromatic C-H), 5.41 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 5.25 (doublet, J = 11 Hz, IH, N-CHH-O), 4.67 (doublet, J = 11 Hz, IH, N=CHH-0), 1.50 (doublet w i t h some f i n e s p l i t t i n g s , J = 7 Hz, 3H, C-18 H^). Mass spectrum: M +, m/e = 308; main peaks: 278, 149, 148, 144, 143 (base peak). High r e s o l u t i o n mass spectrum: c a l c . f o r C 2 0 H 2 4 N 2 O : 3 0 8 - 1 8 8 7 - Found: 308.1932. H y d r o l y s i s of model t e t r a h y d r o o x a z i n e 166 The t e t r a h y d r o o x a z i n e 166 (80 mg) was d i s s o l v e d i n 10% methanolic hydrogen c h l o r i d e (10 ml) and s t i r r e d a t room tempera-t u r e f o r 8 hr. The methanol was then evaporated under reduced p r e s s u r e and the r e s i d u e taken up i n a s a t u r a t e d aqueous s o l u t i o n of sodium carbonate (5 ml) and e x t r a c t e d w i t h methylene c h l o r i d e ( 3 x 5 m l ) . The combined methylene c h l o r i d e e x t r a c t s were d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced p r e s s u r e . The a l c o h o l 137 was o b t a i n e d as c o l o r -l e s s c r y s t a l s (77 mg, 100%) which were i d e n t i c a l w i t h a p r e v i o u s l y prepared s t a n d a r d sample i n every r e s p e c t . 2g,16g-carbomethoxy-cur-19-en-17-ol (16 0) The carbomethoxy t e t r a h y d r o o x a z i n e 159 (71 mg) was d i s s o l v e d i n 10% m e t h a n o l i c hydrogen c h l o r i d e (15 ml) and the s o l u t i o n r> 130 -r e f l u x e d f o r 2,5 hx. I s o l a t i o n of the product as. d e s c r i b e d aboye f o r the a l c o h o l 137 f o l l o w e d by; e l u t i o n through a s h o r t column of aiumijia 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 hydroxy e s t e r 160 as a c o l o r l e s s o i l (63.5 mg, 92%). IR bands: 3360, 2960, 1728, 1605. W a b s o r p t i o n s : ^ m a x 296, 244: l o g e 3.38, 3.76. NMR s i g n a l s (FT): 6.7-7.2 ( m u l t i p l e t , 4H, aromatic C-H), 5.54 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 4.66 ( s i n g l e t , IH, C-2 H) , 3.73 ( s i n g l e t , 3H, COOCH_3) , 1.64 (doublet of d o u b l e t s , J = 7 and 2 Hz, 3H, C-18 H_3) . Mass spectrum: M +, m/e = 352; main peaks: 324, 251, 224, 199, 144 (base peak). High r e s o l u -t i o n mass spectrum: c a l c . f o r C 2 1 H 2 6 N 2 ° 3 : 354.1942. Found: 354.1915. Akuammicine (66) . < The e s t e r 157 (200 mg) was d i s s o l v e d i n benzene (25 m l ) , and a c e t i c a c i d (35 p i ) was added. A s o l u t i o n of l e a d t e t r a -a c e t a t e (550 mg) i n benzene (40 ml) was added dropwise w i t h s t i r r i n g a t room temperature over a p e r i o d of 1.75 h r . The r e a c t i o n mixture was allowed t o s t i r f o r an a d d i t i o n a l 15 min, a f t e r which a 1 M aqueous sodium carbonate s o l u t i o n (10 ml) was added, and e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 10 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 over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced p r e s s u r e . The r e s i d u e was then p u r i f i e d by e l u t i o n through a column of alumina with, benzene, y i e l d i n g the d e s i r e d akuammicine (661 a s c o l o r l e s s c r y s t a l s which c o u l d be r e c r y s t a l l i z e d from e t h y l a c e t a t e (70 mg, 35%) m.p. 178-180° ( l i t . 1 5 8 m.p. 180-181.5°). - 131 -IR b a n d s ; 3495, 2960, 1670, 1601. UV a b s o r p t i o n s ; A 326, 296, 226; l o g e 4.17, 4.03, 4.05. NMR s i g n a l s (FT) ( F i g u r e 4 0 ) : 9.01 ( s i n g l e t , IH, N-H), 6.8-7.3 ( m u l t i p l e t , 4H, a r o m a t i c C-H), 5.34 ( q u a r t e t , J = 7 Hz, IH, C-19 H ) , 3.82 ( s i n g l e t , 3H, COOCH 3), 1.62 (.doublet, J = 7 Hz, 3H, C-18 H_3) . Mass s p e c t r u m : M +, m/e = . 322; m a i n p e a k s : 121 ( b a s e p e a k ) . H i g h r e s o l u t i o n mass s p e c t r u m : c a l c . f o r C 2 0 H 2 2 N 2 O 2 : 322.1680. F o u n d : 322.1657. D e s - h y d r o x y m e t h y l e n e stemmadenine (141a) To a s o l u t i o n o f a kuammicine (66) (94 mg) i n r e f l u x i n g g l a c i a l a c e t i c a c i d (10 ml) was a d d e d s o d i u m b o r o h y d r i d e (1 g) as r a p i d l y as p o s s i b l e . The s o l u t i o n was a l l o w e d t o s t i r w i t h o u t h e a t i n g f o r 15 m i n , t h e n c o o l e d i n an i c e b a t h . The m i x t u r e was s l o w l y made b a s i c w i t h 7 N ammonium h y d r o x i d e a n d t h e n e x t r a c t e d w i t h e t h y l a c e t a t e ( 3 x 5 m l ) . The e t h y l a c e t a t e e x t r a c t s were c o m b i n e d , 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 f a t e , f i l t e r e d , and t h e s o l v e n t e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e . The r e s i d u e was p u r i f i e d 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 on s i l i c a g e l ( s o l v e n t s y s t e m B ) , y i e l d i n g t h e d e s i r e d i n d o l e e s t e r 141a a s a c o l o r l e s s o i l (50 mg, 5 0 % ) . IR b a n d s : 3450, 2930, 1720, 1670 (w), 1601 (w). UV a b s o r p t i o n s : A 290, 283, 225; l o g e 3.73, HI 3 . X 3.74, 4.37. NMR s i g n a l s (FT) ( F i g u r e 4 2 ) : 9.08 ( s i n g l e t , IH, i n d o l e N-H), 7.1-7.6 ( m u l t i p l e t , 4H, a r o m a t i c C-H), 5.60 ( q u a r t e t , J = 7 Hz, IH, C-19 H ) , 4.30 ( s i n g l e t , IH, C-16 H ) , 3.88 ( s i n g l e t , 3H, COOCH 3, 1.77 ( d o u b l e t , J = 7 Hz, 3H, C-18 H_3) . Mass s p e c t r u m : M , m/e ~ 324; m a i n p e a k s : 194, 123 (base p e a k ) . H i g h r e s o l u t i o n mass s p e c t r u m : c a l c . f o r C o r . H _ . N „ 0 „ : 324.1837. F o u n d : 324.1857. 20 24 2 2 - 132 -E p i m e r i c I n d o l e e s t e r 141b A s o l u t i o n of i n d o l e e s t e r 141a (10 mg) i n benzene (1 ml) was added to sodium h y d r i d e (10 mg, o b t a i n e d by washing a 50% o i l d i s p e r s i o n (20 mg) w i t h benzene), and methyl formate (1 ml) was d i s t i l l e d (over phosphorous pentoxide) d i r e c t l y onto the m i x t u r e . The r e a c t i o n m i x t u r e was heated to r e f l u x f o r 2.5 hr, then c o o l e d and passed through a s h o r t column of alumina, f o l l o w e d by a d d i t i o n a l benzene (50 m l ) . The s o l v e n t was evapor-at e d under reduced p r e s s u r e , and the r e s i d u e p u r i f i e d on a s i l i c a g e l p r e p a r a t i v e l a y e r chromatography p l a t e ( s o l v e n t system B) y i e l d i n g the e p i m e r i c i n d o l e e s t e r 141b as a c o l o r l e s s o i l (3.6 mg, 36%). IR bands: 3460, 2940, 1720, 1670 (w), 1601 (w). UV a b s o r p t i o n s : ^ m a x 290, 283, 225; l o g e 3.73, 3.74, 4.37. NMR s i g n a l s (FT): 8.8 ( s i n g l e t , IH, i n d o l e N-H), 7.0-7.5 ( m u l t i p l e t , 4H, aromatic C-H), 4.64 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 4.20 (doublet, J = 4 Hz, IH, C-16 H) , 3.76 ( s i n g l e t , 3H, COOCH-j) , 1.58 (doublet, J = 7 Hz, 3H, C-18 H_3) . Mass spectrum: M +, m/e = 324; main peaks: 194, 123 (base peak). 16-epi-stemmadenine (161) To a s o l u t i o n of the hydroxy e s t e r 160 (27 mg) i n benzene (1 ml) was added a c e t i c a c i d (10 u1). A s o l u t i o n of l e a d t e t r a -a c e t a t e (67 mg) i n benzene (1 ml) was then added dropwise w i t h s t i r r i n g over a p e r i o d of 30 min. The r e a c t i o n was allowed to s t i r f o r 15 min, then was e l u t e d through a s m a l l column of alumina w i t h methylene c h l o r i d e (25 ml). The s o l v e n t was evaporated under reduced p r e s s u r e , and the r e s i d u e d i s s o l v e d i n 133 r-a 50%. s o l u t i o n of methanol i n a c e t i c a c i d (.4 m l ) . Sodium bo r o h y d r i d e CO.5 g) was then added to the s t i r r e d s o l u t i o n as r a p i d l y as p o s s i b l e , and the r e a c t i o n a llowed t o s t i r f o r 15 min. The mixture was then c o o l e d i n an i c e bath, made b a s i c w i t h 7 N ammonium hydro x i d e , and e x t r a c t e d w i t h methylene c h l o r i d e (3 x 5 m l ) . The combined methylene c h l o r i d e e x t r a c t s were d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced p r e s s u r e . P u r i f i c a t i o n o f the r e s i d u e by p r e p a r a t i v e l a y e r chromatography on s i l i c a g e l Csolvent system B) y i e l d e d 16-epi-stemmadenine (161) (7.0 mg, 26%) as a c o l o r l e s s o i l which c o u l d not be induced to c r y s t a l l -i z e . IR bands: 3580, 3420, 2920, 1725, 1605 (w). UV absorp-t i o n s : A 291, 284, 225; l o g e 3.68, 3.69, 4.33. NMR s i g n a l s max J (FT): 10.14 ( s i n g l e t , IH, i n d o l e N-H), 7.0-7.7 ( m u l t i p l e t , 4H, aromatic C-H), 5.42 ( q u a r t e t , J = 7 Hz, IH, C-19 H), 4.36 ( s i n g l e t , 2H, CH 2OH), 3.88 ( s i n g l e t , 3H, COOCH_3) , 1.52 (doublet, J = 7 Hz, 3H, C-18 H 3 ) . Mass spectrum: M +, m/e = 354; main peaks: 336, 324, 123 (base peak). High r e s o l u t i o n mass spectrum; c a l c . f o r C 2 1 H 2 6 N 2 ° 3 : 3 5 4 - 1 9 4 2 - Found: 354.1926. Stemmadenine d i o l (175) . 159 To a s t i r r e d s o l u t i o n of a u t h e n t i c stemmadenine (10 mg) i n t e t r a h y d r o f u r a n (11 ml) was added excess sodium b i s (methoxy-methylenoxy) aluminum h y d r i d e as the commercially a v a i l a b l e 70% benzene s o l u t i o n CRed-Al) CO.2 m l ) . The r e a c t i o n was allowed t o s t i r a t room temperature f o r 30 min, then made b a s i c w i t h 5 N ammonium hydroxide and e x t r a c t e d w i t h methylene c h l o r i d e (3 x TS 134 -5 m i l . The methylene ch l o r i d e extracts were combined, dried over anhydrous sodium s u l f a t e , f i l t e r e d , and the solvent evaporated under reduced pressure. The residue was p u r i f i e d by preparative layer chromatography on s i l i c a gel (solvent system B), y i e l d i n g the desired d i o l 175 (7 mg, 76%) as a c o l o r l e s s o i l which could not be induced to c r y s t a l l i z e . In an i d e n t i c a l manner, the synthetic 16-epi-stemmadenine (161) was reduced to d i o l 17 5. The two products were found to be i d e n t i c a l i n every respect, each providing the data given below. IR bands: 3400, 2920, 1470. UV absorptions: A 290, c • max 283, 266; log e 3.73, 3.74, 4.41. NMR s i g n a l s : 9.68 (singlet, IH, indole N-H), 7.0-7.6 (multiplet, 4H, aromatic C-H), 5.40 (quartet, J = 7 Hz, IH, C-19 H), 4.0-4.5 (multiplet, 4H, 2CCH_2OH), 1.64 (doublet, J = 7 Hz, 3H, C-18 H_3) . Mass spectrum: M+, m/e = 326; main peaks: 324, 276, 197, 123 (base peak). High resolution mass spectrum: c a l c . for C„ r tH„-N o0 o: 326.1994. 20 26 2 2 Found: 326.1983. f - 135 -S e c t i o n C . 16-epl— stemmadenine (Ar- Hi (161) T r i t i a t e d t r i f l u o r o a c e t i c acid -^- 1- (1.57 g, 1 Ci) was combined with. 16-epi-stemmadenine (18 mg) by means of a vacuum t r a n s f e r system. The a c i d s o l u t i o n was maintained under a dry n i t r o g e n atmosphere a t room temperature f o r 48 h r . The t r i t i a t e d t r i f l u o r o a c e t i c a c i d was then removed v i a the vacuum t r a n s f e r system, and the r e s i d u e taken up i n 7 N ammonium hydroxide and e x t r a c t e d w i t h methylene c h l o r i d e (3 x 5 ml). The methylene c h l o r i d e e x t r a c t s were combined, d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and the s o l v e n t evaporated under reduced p r e s s u r e . 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