Studies on the synthesis and biosynthesis of indole alkaloids

Lewis, Norman G.

doi:10.14288/1.0060870

UBC Theses and Dissertations

Featured Collection

UBC Theses and Dissertations

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Page Metadata

Item Metadata

Title	Studies on the synthesis and biosynthesis of indole alkaloids
Creator	Lewis, Norman G.
Publisher	University of British Columbia
Date Issued	1978
Description	Part I of this thesis describes the more recent investigations towards the elucidation of the biosynthetic pathways leading to the formation of a class of indole alkaloids found in Aspidosperma vargasii. In this respect, the in vivo role of tryptophan (lb) and stemmadenine (63) were studied but the incorporation levels obtained were not conducive with the active intermediacy of either (lb) or (63) in the biosynthesis of the alkaloids uleine (103), guatambuine (104) or 9-methoxy-olivacine (111). Conditions for the growth of Aspidosperma australe, A. pyricollum and A. vargasii tissue cultures are also reported. Part II discusses the more recent studies towards the synthesis of stemmadenine (63) with radioactive labels at the required positions in the molecule. The studies initially involved conversion of strychnine (5) to 2β, 16α-cur-19-en-17-ol (143) by a previously described sequence of reactions. Conditions for the efficient conversion to the known 2β-cur-19-en-l7-al (145) were developed but subsequent conversion to stemmadenine (63) was not accomplished. The conversion of (143) to des-carbomethoxystemmadenine (128) is reported. Further studies towards the synthesis of stemmadenine (6 3) were initiated from methyl-2β,16α-cur-19-en-17-oate (133). The ester (133), derived from strychnine (5) in overall low yield via Wieland-Gumlich aldehyde (129) was an important intermediate in the synthesis of epistemmadenine (138). A more efficient synthesis of (133) was developed from Wieland-Gumlich aldoxime (130). Ester (133) was efficiently converted to (-) akuammicine (64) by treatment with lead tetra-acetate and these recent conditions have been successfully applied in the total synthesis of vindoline (11). Akuammicine (64) was converted to deshydroxymethylstemmadenine (122). Attempts to convert (122) or Na-carbomethoxydeshydroxymethylstemmadenine (175) to stemmadenine (63) were unsuccessful. These failures prompted alkylation studies with the model system, 1-carbomethoxy-1,2,3,4-tetrahydrocarbazole (156) prepared from tetrahydrocarbazole (155) via a three step synthesis. The N-carbomethoxy derivative (170) of (156) was treated with formaldehyde in the presence of potassium hydride and gave the required 1-carbomethoxy-1-hydroxymethyl-1,2,3,4-tetrahydrocarbazole (157) in good yield. Further alkylation studies with 18β-carbomethoxycleavamine (72) and the corresponding Na-carbomethoxy (180) and Na-methyl (183) derivatives were unsuccessful. Indeed, it appears that introduction of the hydroxymethyl group in the more complex systems cannot be accomplished using this strategy. Part III of this thesis investigated the role of catharanthi: Nb-oxide (205) as a possible precursor for the in vivo formation of the medicinally important dimeric alkaloid vincristine (201) in Catharanthus roseus. In these studies the chemistry of catharanthine (12) was appropriately developed in order that radioactive labels at (1) the aromatic positions C₁₁-C₁₄ (2) C-19 (3) C-18 and (4) C-22 could be introduced. (Ar³H) catharanthine-Nb-oxide (205) was administered to C. roseus and the alkaloid vincristine (201) isolated by cold dilution. The incorporation levels obtained do not give substantial in vivo support for the intermediacy of (205) in the biosynthesis of (201). Part IV of this thesis discusses the formation of important intermediates in the recent investigations towards the synthesis of the anti-tumour alkaloids ellipticine (106) and olivacine (105) . In this respect the synthesis of indol-2-y1-1-(4' pyridyl)-ethanol (239) was carried out. Hydrogenolysis of (239) with H₂/Pd/C afforded indol-2-y1-1-(4' pyridyl)-ethane (240). Treatment of (239) with acetic acid in pyridine gave the required indol-2-y1-1-(4' pyridyl)-ethene (241). With the chemistry developed for the formation of derivatives (239-241) further studies for the introduction of the N'-methyl group and the C-3 side chain ((CH₃) ₂N CH₂) were executed to give derivatives (246) and (247). The tetrahydropyridine derivative (248) was obtained by sodium borohydride reduction of (246). The cyclisation of (24 8) to the pyridocarbazole derivative (235) was not attempted. However the conditions necessary for the cyclisation have been reported for the synthesis of the close related alkaloid ellipticine (106). Further cyclisation studies using the corresponding dihydropyridine derivatives of (246) and (247) are currently under investigation.
Subject	Indole alkaloids
Genre	Thesis/Dissertation
Type	Text
Language	eng
Date Available	2010-03-06
Provider	Vancouver : University of British Columbia Library
Rights	For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
DOI	10.14288/1.0060870
URI	http://hdl.handle.net/2429/21626
Degree	Doctor of Philosophy - PhD
Program	Chemistry
Affiliation	Science, Faculty of Chemistry, Department of
Degree Grantor	University of British Columbia
Campus	UBCV
Scholarly Level	Graduate
AggregatedSourceRepository	DSpace

Download

Media: 831-UBC_1978_A1 L49.pdf [ 8.8MB ]
Metadata: JSON: 831-1.0060870.json; JSON-LD: 831-1.0060870-ld.json; RDF/XML (Pretty): 831-1.0060870-rdf.xml; RDF/JSON: 831-1.0060870-rdf.json; Turtle: 831-1.0060870-turtle.txt; N-Triples: 831-1.0060870-rdf-ntriples.txt; Original Record: 831-1.0060870-source.json
Full Text: 831-1.0060870-fulltext.txt
Citation: 831-1.0060870.ris

Full Text

STUDIES ON THE SYNTHESIS AND BIOSYNTHESIS OF INDOLE ALKALOIDS BY NORMAN GEORGE LEWIS .Sc., U n i v e r s i t y o f S t r a t h c l y d e , Glasgow, (1973) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES Department o f C h e m i s t r y ' U n i v e r s i t y o f B r i t i s h Columbia We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y , 19 78 © Norman George L e w i s , 19 78 In presenting th is thes is in par t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permiss ion. Department of CHEMIST^ The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date ABSTRACT P a r t I o f t h i s t h e s i s d e s c r i b e s the more r e c e n t i n v e s t i g a t i o n s towards t h e e l u c i d a t i o n o f t h e b i o s y n t h e t i c pathways l e a d i n g t o the f o r m a t i o n of a c l a s s o f i n d o l e a l k a l o i d s found i n Aspidosperma v a r g a s i i . I n t h i s r e s p e c t , the i n v i v o r o l e o f t r y p t o p h a n (lb) and stemmadenine (63) were s t u d i e d but the i n c o r p o r a t i o n l e v e l s o b t a i n e d were not co n d u c i v e w i t h the a c t i v e i n t e r m e d i a c y o f e i t h e r ( l b ) o r (63) i n the b i o s y n t h e s i s o f the a l k a l o i d s u l e i n e (103), guatambuine (104) o r 9-methoxy-o l i v a c i n e (111). C o n d i t i o n s f o r the growth o f Aspidosperma a u s t r a l e ,  A. p y r i c o l l u m and A. v a r g a s i i t i s s u e c u l t u r e s a re a l s o r e p o r t e d . P a r t I I d i s c u s s e s the more r e c e n t s t u d i e s towards the s y n t h e -s i s o f stemmadenine (63) w i t h r a d i o a c t i v e l a b e l s a t the r e q u i r e d p o s i t i o n s i n the m o l e c u l e . The s t u d i e s i n i t i a l l y i n v o l v e d c o n v e r s i o n o f s t r y c h n i n e (5) t o 2yS, 16c(-cur-19-en-17-ol (143) by a p r e v i o u s l y d e s c r i b e d sequence o f r e a c t i o n s . C o n d i t i o n s f o r the e f f i c i e n t c o n v e r s i o n t o the known 2 / 3 - c u r - 1 9 - e n - l 7 - a l (145) were de v e l o p e d b u t subsequent c o n v e r s i o n t o stemmadenine (63) was n o t a c c o m p l i s h e d . The c o n v e r s i o n o f (143) t o des-carbomethoxystemmadenine (128) i s r e p o r t e d . F u r t h e r s t u d i e s towards the s y n t h e s i s o f stemmadenine (6 3) were i n i t i a t e d from methyl - 2 y 3 ,16<*-cur-19-en-17-oate (133). The e s t e r ( 1 3 3 ) , d e r i v e d from s t r y c h n i n e (5) i n o v e r a l l low y i e l d v i a W i e l a n d - G u m l i c h aldehyde (129) was an i m p o r t a n t i n t e r m e -d i a t e i n t h e s y n t h e s i s o f epistemmadenine (138). A more e f f i c i e n t s y n t h e s i s o f (133) was de v e l o p e d from W i e l a n d - G u m l i c h a l d o x i m e (130). E s t e r (133) was e f f i c i e n t l y c o n v e r t e d t o (-) akuammicine (64) by t r e a t m e n t w i t h l e a d t e t r a - a c e t a t e and t h e s e r e c e n t c o n d i t i o n s have been s u c c e s s f u l l y a p p l i e d i n t h e t o t a l s y n t h e s i s o f v i n d o l i n e ( 1 1 ) . Akuammicine (64) was con-v e r t e d t o deshydroxymethylstemmadenine (122). A t t e m p t s t o c o n v e r t (122) o r N -carbomethoxydeshydroxymethylstemmadenine (175) t o stemmadenine (63) were u n s u c c e s s f u l . These f a i l u r e s prompted a l k y l a t i o n s t u d i e s w i t h t h e model system, 1 - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (156) p r e p a r e d from t e t r a h y d r o c a r b a z o l e (155) v i a a t h r e e s t e p s y n t h e s i s . The N-carbomethoxy d e r i v a t i v e (170) o f (156) was t r e a t e d w i t h formaldehyde i n t h e p r e s e n c e o f p o t a s s i u m h y d r i d e and gave the r e q u i r e d 1 - c a r b o m e t h o x y - l - h y d r o x y m e t h y l -1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (157) i n good y i e l d . F u r t h e r a l k y l a t i o n s t u d i e s w i t h 18/ 3-carbomethoxycleavamine (72) and t h e c o r r e s p o n d i n g N - c a r b o m e t h o x y (180) and N - m e t h y l (183) d e r -a a i v a t i v e s were u n s u c c e s s f u l . Indeed, i t appears t h a t i n t r o -d u c t i o n o f t h e h y d r o x y m e t h y l group i n the more complex systems cannot be a c c o m p l i s h e d u s i n g t h i s s t r a t e g y . P a r t I I I o f t h i s t h e s i s i n v e s t i g a t e d the r o l e o f c a t h a r a n t h i : N ^ - o x i d e (205) as a p o s s i b l e p r e c u r s o r f o r the i n v i v o forma-t i o n o f t h e m e d i c i n a l l y i m p o r t a n t d i m e r i c a l k a l o i d v i n c r i s t i n e (201) i n C a t h a r a n t h u s r o s e u s . I n t h e s e s t u d i e s t h e c h e m i s t r y o f c a t h a r a n t h i n e (12) was a p p r o p r i a t e l y d e v e l o p e d i n o r d e r t h a t r a d i o a c t i v e l a b e l s a t (1) t h e a r o m a t i c p o s i t i o n s C-j^-C-j^ (2) C-19 (3) C-18 and (4) C-22 c o u l d be i n t r o d u c e d . (Ar 3H) c a t h a r a n t h i n e - N b - o x i d e (205) was a d m i n i s t e r e d t o i v C. r o s e u s and t h e a l k a l o i d v i n c r i s t i n e (201) i s o l a t e d by c o l d d i l u t i o n . The i n c o r p o r a t i o n l e v e l s o b t a i n e d do n o t g i v e s u b s t a n t i a l i n v i v o s u p p o r t f o r t h e i n t e r m e d i a c y o f (205) i n t h e b i o s y n t h e s i s o f (201). P a r t IV o f t h i s t h e s i s d i s c u s s e s the f o r m a t i o n o f i m p o r t a n t i n t e r m e d i a t e s i n t h e r e c e n t i n v e s t i g a t i o n s towards the s y n t h e -s i s o f the a n t i - t u m o u r a l k a l o i d s e l l i p t i c i n e (106) and o l i v a -c i n e (105) . I n t h i s r e s p e c t the s y n t h e s i s o f i n d o l - 2 - y l - l - ( 4 1 p y r i -d y l ) - e t h a n o l (239) was c a r r i e d o u t . H y d r o g e n o l y s i s o f (239) w i t h H 2/Pd/C a f f o r d e d i n d o l - 2 - y l - l - ( 4 * p y r i d y l ) - e t h a n e (240). T reatment o f (239) w i t h a c e t i c a c i d i n p y r i d i n e gave the r e q u i r e d i n d o l - 2 - y 1 - 1 - ( 4 * p y r i d y l ) - e t h e n e (241). W i t h t h e c h e m i s t r y d e v e l o p e d f o r t h e f o r m a t i o n o f d e r i v a t i v e s (239-241) f u r t h e r s t u d i e s f o r the i n t r o d u c t i o n o f t h e N'-methyl group and t h e C-3 s i d e c h a i n ((CH 3)- N CH 2) were e x e c u t e d t o g i v e d e r i v a t i v e s (246) and (247). The t e t r a h y d r o p y r i d i n e d e r i v a t i v e (248) was o b t a i n e d by sodium b o r o h y d r i d e r e d u c t i o n o f (246). The c y c l i s a t i o n o f (24 8) t o the p y r i d o c a r b a z o l e d e r i v a t i v e (235) was n o t a t t e m p t e d . However t h e c o n d i t i o n s n e c e s s a r y f o r t h e c y c l i s a t i o n have been r e p o r t e d f o r t h e s y n t h e s i s o f t h e c l o s e r e l a t e d a l k a l o i d e l l i p t i c i n e (106). F u r t h e r c y c l i s a t i o n s t u d i e s u s i n g t h e c o r r e s p o n d i n g d i h y d r o p y r i d i n e d e r i v a t i v e s o f (246) and (247) a r e c u r r e n t l y under i n v e s t i g a t i o n . V TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES v i LIST OF TABLES x ACKNOWLEDGEMENTS x i PART I INTRODUCTION 1 DISCUSSION 53 EXPERIMENTAL 61 PART II INTRODUCTION 1 DISCUSSION 66 EXPERIMENTAL 113 PART III INTRODUCTION 136 DISCUSSION 149 EXPERIMENTAL 170 PART IV INTRODUCTION 18 3 DISCUSSION 184 EXPERIMENTAL 193 REFERENCES 200 v i L i s t o f F i g u r e s F i g u r e 1 - Robinson p o s t u l a t e f o r b i o s y n t h e s i s of corynanthe and s t r y c h n o s s k e l e t a . F i g u r e 2 - Woodv/ard and Robinson p o s t u l a t e f o r the b i o s y n t h e s i s o f c o r y n a n t h e and s t r y c h n o s s k e l e t a . F i g u r e 3 - Some r e p r e s e n t a t i v e i n d o l e a l k a l o i d s commonly i s o -l a t e d i n b i o s y n t h e t i c i n v e s t i g a t i o n s . F i g u r e 4 - Thomas-Wenkert monoterpene p o s t u l a t e . F i g u r e 5 - Rearrangement o f monoterpene u n i t i n t o a s p i d o -sperma and i b o g a s k e l e t a . F i g u r e 6 - B i o s y n t h e t i c f o r m a t i o n of g e r a n i o l - p y r o p h o s p h a t e (32) F i g u r e 7 - B i o s y n t h e t i c f o r m a t i o n o f l o g a n i n (35). F i g u r e 8 - P o s t u l a t e d b i o s y n t h e t i c c o n v e r s i o n s f o r f o r m a t i o n of c a m p t o t h e c i n (56). F i g u r e 9 - P o s t u l a t e d b i o s y n t h e t i c f o r m a t i o n o f c o r y n a n t h e i n e aldehyde (57) and g e i s s o s c h i z i n e (5,8). F i g u r e 10 - Proposed b i o s y n t h e t i c o r i g i n s o f the s t r y c h n o s f a m i l y . F i g u r e 11 - Wenkert's proposed b i o s y n t h e t i c o r i g i n s o f the aspidosperma and i b o g a f a m i l i e s . F i g u r e 12 - S c o t t ' s m o d i f i c a t i o n of Wenkert's h y p o t h e s i s f o r the aspidosperma and i b o g a f a m i l i e s . F i g u r e 13 - Proposed mechanism f o r f o r m a t i o n o f n o r - f l u o r o -c u r a r i n e (82) . 3 F i g u r e 14 - I n c o r p o r a t i o n of (5 H) l o g a n i n (35) i n t o v i n d o -l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e (15). F i g u r e 15 - B i o s y n t h e s i s o f t r y p t o p h a n ( l b ) . F i g u r e 16 - The Wenkert p o s t u l a t e f o r the b i o s y n t h e s i s o f o l i v a c i n e (105), e l l i p t i c i n e (106) and u l e i n e (103). F i g u r e 17 - The D j e r a s s i p o s t u l a t e f o r the b i o s y n t h e s i s of a p p a r i c i n e (102). F i g u r e 18 - P o t i e r ' s i n v i t r o e x t r u s i o n o f C-2 u n i t o f fZ - e t h y l a m i n e s i d e c h a i n . v i i F i g u r e 19 - S c o t t ' s i n v i t r o c o n v e r s i o n o f stemmadenine (63) to v a l l e s a m i n e [ l 0 1 ) . F i g u r e 20 - P o t i e r ' s p o s t u l a t e d b i o s y n t h e s i s o f a p p a r i c i n e (102) from stemmadenine ( 6 3 ) . F i g u r e 21 - P o t i e r - J a n o t p o s t u l a t e f o r the b i o s y n t h e s i s o f a p p a r i c i n e (102), u l e i n e (103), guatambuine ( 1 0 4 ) , o l i v a c i n e (105), and e l l i p t i c i n e (106) . F i g u r e 22 - Kutney's a t t e m p t e d s y n t h e s i s o f 1 9 , 2 0 - d i h y d r o -stemmadenine (116). F i g u r e 23 - S n i e c k u s ' s y n t h e s i s o f a stemmadenine model. F i g u r e 24 - Kutney's a t t e m p t e d s y n t h e s i s o f stemmadenine ( 6 3 ) . F i g u r e 25 - Base c a t a l y s e d r i n g o p e n i n g o f descarbomethoxy-akuammicine (126). F i g u r e 26 - S y n t h e s i s o f 2,16-dihydroakuammicine (133). F i g u r e 27 - Kutney's s y n t h e s i s o f epi-stemmadenine (138). F i g u r e 28 - D e g r a d a t i o n o f s t r y c h n i n e (5) t o 2 ft , 16c(-cur-19-e n - 1 7 - o l (143). ' F i g u r e 29 - F o r m a t i o n o f W i e l a n d - G u m l i c h a l d e h y d e (129). F i g u r e 30 - Recent i n v e s t i g a t i o n s towards the s y n t h e s i s o f stemmadenine (63) - approach 1. F i g u r e 31 - M o d i f i e d Oppenauer o x i d a t i o n o f 2'8,16oC-cur-19-e n - 1 7 - o l (143). ' F i g u r e 32 - Pr o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f m e t h y l -c u r - 1 9 - e n e - f a m i l y . F i g u r e 33 - P o s t u l a t e d mass s p e c t r a l f r a g m e n t a t i o n o f a c y l a t e d d e r i v a t i v e (153). F i g u r e 34 - L e n z i ' s s y n t h e s i s o f 1 - c a r b o e t h o x y t e t r a h y d r o c a r -b a z o l e (156a). F i g u r e 35 - An a l t e r n a t i v e t o L e n z i ' s p o s t u l a t e . F i g u r e 36 - S y n t h e s i s o f 1 - b e n z o t r i a z o l e t e t r a h y d r o c a r b a z o l e (162) . F i g u r e 37 - F o r m a t i o n o f 1 - p y r i d i n i u m - l , 2 , 3 , 4 - t e t r a h y d r o -c a r b a z o l e bromide (163). F i g u r e 38 - S y n t h e s i s o f 1 - c a r b o m e t h o x y - l - c y a n o t e t r a h y d r o c a r -b a z o l e (168). F i g u r e 39 - S y n t h e s i s o f 1 - c a r b o m e t h o x y - l - h y d r o x y m e t h y l t e t -r a h y d r o c a r b a z o l e (157). v i i i F i g u r e 40 -F i g u r e 41 -F i g u r e 42 -F i g u r e 43 -F i g u r e 44 -F i g u r e 45 -F i g u r e 46 -F i g u r e 47 -F i g u r e 48 -F i g u r e 49 -F i g u r e 50 -F i g u r e 51 -F i g u r e 52 -F i g u r e 53 -F i g u r e 54 -F i g u r e 55 -F i g u r e 56 -F i g u r e 57 -F i g u r e 58 -Pro p o s e d mechanism f o r f o r m a t i o n o f 1-carbomethoxy-1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157). F u r t h e r i n v e s t i g a t i o n s towards the s y n t h e s i s o f stemmadenine (63) - approach 2. Proposed mass s p e c t r a l f r a g m e n t a t i o n o f N.-carbomethoxydeshydroxymethylstemmadenine (175). S t e r e o c h e m i c a l f e a t u r e s o f o x a z i n e s (138) and (187) . ProDosed r o u t e f o r t h e s y n t h e s i s o f stemmadenine (63) . S t r u c t u r a l p o s s i b i l i t i e s o b t a i n e d from c y c l i s a t i o n o f i n t e r m e d i a t e (195). Kutney's p r o p o s e d s y n t h e s i s o f stemmadenine ( 6 3 ) . A c i d c a t a l y s e d f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . S y n t h e s i s o f d i m e r i c a l k a l o i d s : c h l o r o i n d o l e n i n e approach. S y n t h e s i s o f d i m e r i c a l k a l o i d s : m o d i f i e d P o l o n o v -s k i a pproach. B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : enzyme a s s i s t e d f r a g m e n t a t i o n and c o u p l i n g . B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c i n d o l e n i n e approach. B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c " P o l o n o v s k i " c o u p l i n g . F o r m a t i o n o f 18 -/3 - carbomethoxycleavamine (72) from c a t h a r a n t h i n e (12) i n t r i f l u o r o a c e t i c a c i d . P r o p o s e d f o r m a t i o n o f 19 - o x o c a t h a r a n t h i n e (212). P r o p o s e d f o r m a t i o n o f 5 - oxo - 18/3 -carbomethoxy-c l e a v a m i n e (213). P r o p o sed mass s p e c t r a l f r a g m e n t a t i o n o f c a t h a r a n -t h i n e (12) . B u c h i ' s s y n t h e s i s o f v o a c a n g i n e (220). B u c h i ' s s y n t h e s i s o f (±) c a t h a r a n t h i n e ( 1 2 ) . i x F i g u r e 59 - A t t e m p t e d s y n t h e s i s o f " ' c c^CH^ c a t h a r a n t h i n e ( 1 2 ) . F i g u r e 60 - P r o p o s e d rearrangement o f c a t h a r a n t h i n e - -o x i d e (205). F i g u r e 61 - S y n t h e s i s o f I n d o l - 2 - y 1 - 1 - ( 4 * p y r i d y l ) - e t h a n o l (239). F i g u r e 62 - S y n t h e s i s o f i n t e r m e d i a t e s (246) and (247) . F i g u r e 63 - P o t i e r ' s s y n t h e s i s of e l l i p t i c i n e (106). F i g u r e 64 - S y n t h e s i s o f 5 - h y d r o x y - 2 , 6 , - d i m e t h y l - 6 H - p y r i d o -(4,3 b ) - c a r b a z o l e (257). F i g u r e 65 - P o s t u l a t e d mechanism f o r the f o r m a t i o n o f p y r i d o -c a r b a z o l e (257) from i n t e r m e d i a t e (256). F i g u r e 66 - S y n t h e s i s o f N - m e t h y l - 3 - e t h y l - 5 - b e n z y 1 - t e t r a h y d r o -p y r i d i n e (263). i X L i s t o f T a b l e s T a b l e 1 - F o r m a t i o n o f a l k a l o i d s from C. r o s e u s s e e d l i n g s . T a b l e 2 - F i n d i n g s o f t r y p t o p h a n ( l b ) i n c o r p o r a t i o n s i n t o a l k a l o i d s o f A. a u s t r a l e (A) and A. p y r i c o l l u m ( B ) . T a b l e 3 - Summary o f r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. p y r i c o l l u m . T a b l e 4 - Summary o f r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. a u s t r a l e . 3 T a b l e 5 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i r o o t c u t t i n g s . 3 T a b l e 6 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i whole p l a n t . 3 T a b l e 7 - R e s u l t s o f (Ar H) stemmadenine (63) a d m i n i s t r a t i o n t o A. v a r g a s i i . T a b l e 8 - C o m p o s i t i o n o f n u t r i e n t medium f o r s o l i d (agar) medium. 3 14 T a b l e 9 - I n c o r p o r a t i o n o f ( H, C) l o g a n i n (35) i n t o a l k a l o i d s o f C. r o s e u s . 1 2 T a b l e 10 - R e a c t i o n s o f c a t h a r a n t h i n e (12) w i t h H and H t r i f l u o r o a c e t i c a c i d . T a b l e 11 - Mass s p e c t r a l fragments o f c a t h a r a n t h i n e (12) -d e u t e r i u m e n r i c h m e n t e x p e r i m e n t s . 3 T a b l e 12 - I n c o r p o r a t i o n d a t a from a d m i n i s t r a t i o n o f (Ar H) c a t h a r a n t h i n e - - o x i d e (205) t o C. r o s e u s . ACKNOWLEDGEMENT S The a u t h o r w i s h e s t o thank Dr. James P. Kutney f o r h i s d i r e c t i o n , encouragement and op t i m i s m t h r o u g h o u t the cour s e o f these s t u d i e s . I am a l s o g r a t e f u l t o t h e v a r i o u s s t a f f members f o r h e l p f u l s u g g e s t i o n s , b u t , i n p a r t i c u l a r D r s . B.R. Worth and T. Honda r e c e i v e s p e c i a l c o n s i d e r a t i o n i n t h i s r e s p e c t . S i n c e r e g r a t i t u d e i s e x p r e s s e d t o my w i f e whose u n f a i l i n g s u p p o r t , a s s i s t a n c e , and u n d e r s t a n d i n g d u r i n g the co u r s e o f t h i s s t u d y were a c o n t r i b u t i o n beyond measure. R e c e i p t o f a S c i e n c e R e s e a r c h C o u n c i l (U.K.) p o s t g r a d u a t e s c h o l a r s h i p f o r 1974-1977 i s g r a t e f u l l y acknowledged as i s f u r t h e r f i n a n c i a l s u p p o r t from the N a t i o n a l I n s t i t u t e s o f H e a l t h (U.S.A.) and t h e c h e m i s t r y department (U.B.C.) f o r a t e a c h i n g a s s i s t a n t s h i p . INTRODUCTION - PARTS I & I I The e l u c i d a t i o n o f t h e major b i o s y n t h e t i c pathways i n i n d o l e a l k a l o i d b i o s y n t h e s i s has been t h e s u b j e c t o f much i n t e r e s t i n t h e l a s t decade. Many r e v i e w s have been p u b l i s h e d and w i l l be d i s -c u s s e d a t a p p r o p r i a t e j u n c t u r e s . 'However one r e c e n t comprehensive r e v i e w has p r o v i d e d i n v a l u a b l e a s s i s t a n c e . 1 B i o s y n t h e s i s i s an i n t r i g u i n g p r o c e s s and, c o n s e q u e n t l y , p r i o r • t o d i s c u s s i o n o f t h e h i s t o r i c a l a c c o u n t o f i n d o l e a l k a l o i d f o r m a t i o n some g e n e r a l comments on t h i s t o p i c a r e i n o r d e r . B i o s y n t h e t i c pathways a r e o f t e n i n f e r r e d by (1) a d m i n i s t e r i n g l a b e l l e d i n t e r m e d i a t e s and (2) showing t h a t t h e l a b e l appears i n t h e s u b s t a n c e r e g a r d e d as t h e end p r o d u c t o f the pathway. The p r e c u r s o r i s g e n e r a l l y l a b e l l e d w i t h "^C, 3H, 1 5 N , o r a c o m b i n a t i o n of t h e s e , and t h i s i s t h e n a d m i n i s t e r e d t o an i n t a c t p l a n t or p a r t o f a p l a n t ( r o o t c u l t u r e s , e x c i s e d l e a v e s o r t i s s u e c u l t u r e ) . Q u i t e o f t e n low i n c o r p o r a t i o n s a r e o b t a i n e d - t h e s e b e i n g r a t i o n a l i s e d as b e i n g due t o (1) low a l k a l o i d a l b i o s y n t h e s i s a t time o f p r e c u r s o r a d m i n i s t r a t i o n (2) n o n - p e r m e a b i l i t y o r (3) d e s t r u c t i o n o r m o d i f i c a t i o n o f the l a b e l l e d compound b e f o r e i t re a c h e s t h e b i o s y n t h e t i c s i t e . 2 C o r n f o r t h has c r i t i c a l l y a s s e s s e d t h e s u b j e c t i n a R o b e r t Robinson l e c t u r e "The l o g i c o f w o r k i n g w i t h enzymes". T h i s e x c e l l -e n t paper h e l p s d e f i n e c r i t e r i a r e q u i r e d f o r m e a n i n g f u l b i o s y n -t h e t i c p u r s u i t s . He argues t h a t w i t h r e s p e c t t o a d m i n i s t r a t i o n o f l a b e l l e d compounds i n o r d e r t o d e t e r m i n e p r e c u r s o r s o f the supposed -2-end product the p o s i t i v e r e s u l t s obtained may also be compatible with the precursor not being on the p r i n c i p a l pathway but, instead merely degradable or transformable to a true intermediate coming largely from a d i f f e r e n t precursor. Furthermore administration of l a b e l l e d precursors for the i d e n t i f i c a t i o n of possible intermediates i s cautioned since the "intermediates" i s o l a t e d may be detectable, not because they are intermediates, but because they are not. Indeed, the intermediates may only be slowly incorporated into the end-product, or, perhaps, not at a l l . Cornforth also points out that experiments revealing n e g l i g i b l e or no incorporation of the l a b e l from (1) the precursor into the suspected intermediate or (2) the intermediate into the product must be treated with caution since, on t h i s basis, mevalonic acid (26) could be shown not to be an intermediate i n the polyisoprenoid biosynthesis. 3 Leete has recently c r i t i c i s e d workers reporting low incor-porations as being s i g n i f i c a n t and yet f a i l i n g to determine the 4 location of the r a d i o a c t i v i t y i n the molecule and Barton has stated that low incorporations (<0.01%) are probably not meaningful. I t has also been shown, that, on occasion, plants may u t i l i z e "unnatural precursors" to y i e l d "unnatural products" e.g. conversion of 5 - f l u o r o n i c o t i n i c acid into 5 - f l u o r o n i c o t i n e . 5 ' ^ Consequently, plant enzymes may convert unnatural precursors into ones l y i n g d i r e c t l y on the biosynthetic route. Thus the determination of the major biosynthetic pathway might be d i f f i c u l t to prove by administration of precursors to plant -3-m a t e r i a l . Moreover, i n t h e i n s t a n c e o f p o s i t i v e i n c o r p o r a t i o n s , i t must be e s t a b l i s h e d t h a t i n t a c t i n c o r p o r a t i o n o f t h e p r e c u r s o r i n t o t h e a l k a l o i d has o c c u r r e d . T h i s i s g e n e r a l l y o b t a i n e d by f e e d i n g m u l t i p l e l a b e l l e d p r e -c u r s o r s , i s o l a t i n g t h e d e s i r e d a l k a l o i d and c r y s t a l l i s i n g i t t o c o n s t a n t a c t i v i t y . The r a t i o o f the l a b e l s from p r e c u r s o r t o a l k a l o i d s h o u l d be i n agreement w i t h t h a t e x p e c t e d . D e g r a d a t i o n o f t h e a l k a l o i d s h o u l d t h e n be c a r r i e d o u t t o d e t e r m i n e t h e e x a c t p o s i t i o n o f the l a b e l ( s ) . I t s h o u l d a l s o be n o t e d t h a t i n t h e i n d o l e a l k a l o i d f i e l d o n l y a s m a l l number o f p l a n t s have w a r r a n t e d i n v e s t i g a t i o n . Indeed, i n c o r p o r a t i o n o r l a c k o f i t , has been n o t e d t o d i f f e r i n d i f f e r -e n t p l a n t s - even a l t h o u g h they c o n t a i n the same a l k a l o i d s . ( D i s c u s s e d l a t e r i n t e x t . ) C l e a r l y , i n f o r m a t i o n from p l a n t e x p e r i m e n t s w i l l always be l i m i t e d , and i t i s hoped t h a t t h i s a r e a w i l l g i v e way t o i n v e s t i -g a t i o n i n , h o p e f u l l y , s i m p l e r systems e.g. p l a n t enzyme(s). The e v i d e n c e l e a d i n g t o and s u p p o r t i n g the c u r r e n t b i o s y n t h e -t i c p o s t u l a t e s i s now r e v i e w e d below. To a c c o u n t f o r t h e f o r m a t i o n o f ^ - c a r b o l i n e d e r i v e d 7 a l k a l o i d s , t y p i f i e d by ( 2 ) and ( 3 ) , Robinson s u g g e s t e d i n 1919 t h a t b i o s y n t h e t i c f o r m a t i o n o f t h e s e m o l e c u l e s o c c u r s by c o n d e n s a t i o n o f t r y p t a m i n e ( l a ) o r t r y p t o p h a n ( l b ) w i t h s i m p l e a l d e h y d e s i n a Mannich f a s h i o n as shown i n f i g u r e 1. - 4 -F i g u r e 1 - Robinson p o s t u l a t e f o r b i o s y n t h e s i s o f c o r y n a n t h e and s t r y c h n o s s k e l e t a . 8 9 As e x p e c t e d t r y p t o p h a n ( l b ) and t r y p t a m i n e ( l a ) have been shown t o be p r e c u r s o r s i n i n d o l e a l k a l o i d b i o s y n t h e s i s . The pr o v e d b i o s y n t h e t i c r o u t e t o t r y p t o p h a n ( l b ) u t i l i s i n g s h i k i m i c a c i d (6) and a n t h r a n i l i c a c i d (7) i s shown l a t e r i n f i g u r e 1 5 and d i s c u s s e d a t a more a p p r o p r i a t e p o i n t . I n a c c o r d w i t h Robinson's p o s t u l a t e s H a h n 1 0 gave f u r t h e r s u p p o r t by c o n d e n s i n g aldehydes w i t h t r y p t a m i n e ( l a ) t o g i v e t e t r a h y d r o c a r b o l i n e s . Barger and Hahn th e n p o s t u l a t e d t h a t i n b i o s y n t h e s i s o f i n d o l e a l k a l o i d s t h e aldehyde was t y r o s i n e (8) d e r i v e d . 1 ^ 1 1 T h i s 12 13 h y p o t h e s i s was l a t e r e l a b o r a t e d by Robinson and Woodward t o ac c o u n t f o r the f o r m a t i o n o f the cor y n a n t h e and s t r y c h n o s s k e l e t a . ( F i g u r e 2) Figure 2 - Woodward and Robinson postulate for the biosynthesis of corynanthe and strychnos skeleta. C H ^ C 0 2 C H VINDOLINE 11 AJMALICINE 15 DEHYDROASPIDOSPERMIDINE 10 CATHARANTHINE 12 RESERPININE 14 PERIVINE 16 F i g u r e 3 - Some r e p r e s e n t a t i v e i n d o l e a l k a l o i d s commonly i s o l a t e d i n b i o s y n t h e t i c i n v e s t i g a t i o n s . -7-ments, These t h e o r i e s were l a t e r i n v a l i d a t e d by f e e d i n g e x p e r i -14-20 and, i n t h i s r e s p e c t , i n d o l e a l k a l o i d s u s u a l l y examined i n b i o s y n t h e s i s a re shown i n f i g u r e 3. The numbering system 2 1 i s t h a t employed by Hesse. 22 A more u n l i k e l y monoterpenoid h y p o t h e s i s p r oposed by Thomas , 2 3 24 Wenkert , and Levy was n e x t c o n s i d e r e d . T h i s was based on the f a c t t h a t i n a l k a l o i d s i l l u s t r a t e d by c o r y n a n t h e i n e ( 1 7 ) , s t r y c h n i n e ( 5 ) , t a b e r s o n i n e (18) and c a t h a r a n t h i n e ( 1 2 ) , t h e r e a re t h r e e main t y p e s o f s k e l e t a p r e s e n t i n the n o n - t r y p t o p h a n ( l b ) p o r t i o n , i . e . (A, B and C.) C 0 2 C H 3 A 1 8 B C 0 2 C H 3 12 c - 8 -The occurrence i n nature of cyclopentane or secocyclopentane monoterpenoid g l y c o s i d e s ( 1 9 - 2 5 ) having ( 1 ) the same s t e r e o -chemistry i n the a p p r o p r i a t e p o s i t i o n as C - 1 5 of i n d o l e a l k a l o i d s and ( 2 ) a carbomethoxy group or corresponding d e r i v a t i v e at the c o r r e c t p o s i t i o n prompted t h e a b o v e a u t h o r s t o p o s t u l a t e b i o s y n t h e t i c o r o -cesses u t i l i s i n g such i n t e r m e d i a t e s . VERBENALIN 1 9 GENIPIN 2 0 ASPERULOSIDE 2 1 SWERTIAMARIN 2 2 BAKANKOSIN 2 3 ELENOLIDE 2 4 OLEUROPIN 2 5 2 2 Thomas suggested i n h i s hypothesis t h a t formation of s k e l e t o n A i n the corynanthe-strychnos s e r i e s was mevalonate ( 2 6 ) d e r i v e d as shown i n f i g u r e 4 . The cyclopentane u n i t so formed i s then c l e a v e d and r e a c t s w i t h tryptamine (la) g i v i n g entry i n t o t h i s area. -9-F i g u r e 4 - Thomas-Wenkert monoterpene p o s t u l a t e . F u r t h e r r earrangements were r e q u i r e d t o a c c o u n t f o r the s k e l e t a e x i s t i n g i n the aspidosperma (B) o r i b o g a a l k a l o i d s ( C ) . ( F i g u r e 5) F i g u r e 5 - Rearrangement o f monoterpene u n i t i n t o aspidosperma and i b o g a s k e l e t a . The h y p o t h e s i s was f i r s t t e s t e d by f e e d i n g sodium d l ( 2 ^ C ) mevalon-a t e (26) and ( 2 1 4 C ) m e v a l o n o l a c t o n e (27) t o C e p h a s l i s i p e c a c u a n h a and R a u w o l f i a s e r p e n t i n a w h i c h c o n t a i n a l k a l o i d s c e p h a e l i n e (2 8) and a j m a l i n e (9) r e s p e c t i v e l y . C e p h a e l i n e (28) i s an i s o q u i n o l i n e a l k a l o i d c o n t a i n i n g a d e o x y p h e n y l a l a n i n e r e s i d u e and p o s s e s s e s -10-the same C n , n u n i t as found i n the c o r y n a n t h e - s t r y c h n o s s k e l e t a . y - x u 19 33 N e g l i g i b l e l a b e l l i n g o c c u r r e d i n c e p h a e l i n e (28) ' o r a j m a l m e ( 9 ) 3 2 . 2 7 2 8 The p o s t u l a t e was however shown t o be c o r r e c t by f e e d i n g v a r i o u s l y l a b e l l e d forms o f mevalonate (26) , m e v a l o n o l a c t o n e ( 2 7 ) , g e r a n i o l (29) and n e r o l (30) t o a l t e r n a t i v e p l a n t s (Rhazya s t r i c t a , 34-39 C a t h a r a n t h u s r o s e u s , V i n c a major ). E x t e n s i v e d e g r a d a t i o n s t u d i e s o f i s o l a t e d a l k a l o i d s showed i n t a c t i n c o r p o r a t i o n o f t h e 40-46 p r e c u r s o r i n the manner d e s c r i b e d i n t h e h y p o t h e s i s 2 9 3 0 The s t e r e o c h e m i s t r y o f each o f the s t e p s has been e l u c i d a t e d t o t h e f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e 1 ( 3 2 ) , and, a l t h o u g h much of the s t e r e o c h e m i c a l s t u d y has been e x e c u t e d on l i v e r and y e a s t s y s -- l i -tems i t has been su g g e s t e d t h a t t he same r o u t e i s i n v o l v e d i n p l a n t s y s t e m s . 4 7 ( F i g u r e 6) H C p H H X fiH C H , C O S C o A 1 T. ^ W I i 1 A , c r A H O C C O S C o A H 0 2 C g S C ° A F i g u r e 6 - B i o s y n t h e t i c f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e (32). Thus i n the f o r m a t i o n o f g e r a n i o l - p y r o p h o s p h a t e (32) the p r o 4S hydrogens o f the m evalonates a r e c o m p l e t e l y l o s t . I t has been 4 8 49 d e monstrated i n t h e mould m y c e l i a n a m i d e and a t r i t e r p e n e t h a t t h e m e t h y l groups o f d i m e t h y l a l l y l - p y rophosphate (31) a r e n o t b i o s y n t h e t i c a l l y e q u i v a l e n t and i n g e r a n i o l - p y r o p h o s p h a t e (32) t h e C-10 was d e r i v e d from C-2 o f m e v a l o n i c a c i d (26) and C-8 and 9 from C-6 o f m e v a l o n i c a c i d ( 2 6 ) . F u r t h e r s i n c e g e r a n i o l (29) and n e r o l (30) were i n c o r p o r a t e d i n t o t h e t h r e e b a s i c s k e l e t a ( A , B and C) i t was thus o f g r e a t im-p o r t a n c e t o d e t e r m i n e a t what s t a g e t h e t e r p e n o i d r earrangements t o o k p l a c e . 50 14 I n C. i p e c a c u a n h a , (2 C) g e r a n i o l (29) was shown t o be a p r e c u r s o r o f i p e c o s i d e ( 3 3 ) 5 1 w h i c h t h e n l e d t o the s u g g e s t i o n t h a t l o g a n i n (35) c o u l d g i v e r i s e t o an a l d e h y d e (44) w h i c h on conden-s a t i o n w i t h t r y p t a m i n e ( l a ) gave the p r e c u r s o r f o r t h e d i v e r s i f i e d 52 m d o l i c s k e l e t a . The s t e r e o c h e m i s t r y a t C-5 o f i p e c o s i d e (33) 53 was o b t a i n e d by c o r r e l a t i o n w i t h ( - ) d i h y d r o p r o t o e m e t i n e (34) -13-T h i s p o s t u l a t e was q u i c k l y v e r i f i e d and l o g a n i n (35), d e r i v e d from mevalonate (ZBj^and g e r a n i o l ( 2 9 ) 1 , was i n c o r p o r a t e d i n t a c t into 5 2 a l k a l o i d s c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( 1 1 ) , s e r p e n t i n e ( 1 3 ) , 14 a j m a l i c i n e (15) and p e r i v i n e (16). S u r p r i s i n g l y (2 C) mevalonate 54 (26) f a i l e d t o l a b e l l o g a n i n (35) i n M. t r i f o l i a t a . F u r t h e r f e e d i n g e x p e r i m e n t s w i t h s i m i l a r systems e.g. v e r b e n a l i n (19) and 52 g e n i p i n (20) showed absence o f l a b e l i n the a l k a l o i d s . L o g a n i n (35) was then shown t o be a n a t u r a l p r o d u c t i n a v a r i e t y o f p l a n t systems and f u r t h e r e x p e r i m e n t s w i t h p r o 4R and 55 p r o 4S mevalonates extended the s t e r e o s p e c i f i c i t y t o l o g a n i n as shown i n f i g u r e 7. 3 2 3 5 F i g u r e 7 - B i o s y n t h e t i c f o r m a t i o n o f l o g a n i n ( 3 5 ) . F u r t h e r i n t e r m e d i a c y t o l o g a n i n (35) has been i n d i c a t e d by s p e c i f i c i n c o r p o r a t i o n o f 1 0 - h y d r o x y - g e r a n i o l ( 3 6 ) 5 6 , 10-hydroxy--14-5 6 5 V 5 8 n e r o l (37) , d e o x y l o g a n i n (38) and d e o x y l o g a n i c a c i d (39) i n t o l o g a n i n (35) and a l k a l o i d s v i n d o l i n e (11),' c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e ( 1 6 ) . 5 6 , 5 8 3 6 3 7 co 2R Cu O G I u 3 3 8 ; R = C H 3 3 9 , R = H The n e x t b i o s y n t h e t i c s t e p from l o g a n i n (35) was c l e a v a g e o f the c y c l o p e n t a n e r i n g , and i n t h i s r e s p e c t , i t s h o u l d be n o t e d t h a t i n a d d i t i o n t o t h e n a t u r a l l y o c c u r r i n g s e c o - i r i d o i d s (19-25) p r e -v i o u s l y mentioned, n a t u r a l p r o d u c t s s w e r o s i d e ( 4 0 ) , f o l i a m e n t h i n ( 4 1 ) , m e n t h i a f o l i n (42) and d i h y d r o f o l i a m e n t h i n (43) a l s o p o s s e s s a seco r i n g masked by l a c t o l f o r m a t i o n . 1 40 ? a = 41 , -42 , = O G I u 4 3 , 1= M i l d a l k a l i n e h y d r o l y s i s o f (40-42) a f f o r d e d s e c o l o g a n i n (44) wh i c h has been shown t o o c c u r i n C. ro s e u s and M. t r i f o l i a t a . 60 -15-4 4 , R » C H 3 4 4 A , - H The t e r p e n o i d n a t u r e o f s e c o l o g a n i n (44) was e s t a b l i s h e d i n C. r o s e u s and M. t r i f o l i a t a by t h e s u c c e s s f u l i n c o r p o r a t i o n o f mevalonate ( 2 6 ) 6 0 a , and l o g a n i n ( 3 5 ) 6 0 b ' c . M e v a l o n a t e (26) was a l s o i n c o r p o r a t e d i n t o s e c o l o g a n i c a c i d 6 0a (44a) i n C. r o s e u s . S w e r t i a m a r i n (22) and s w e r o s i d e (40) i n S w e r t i a m o r i n j a p o n i c a were found t o be m e v a l o n o l a c t o n e (27) de-r i v e d 6 1 . S e c o l o g a n i n (44) was found t o l a b e l the a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e ( 1 6 ) 5 9 ' 6 0 b . F u r t h e r s w e r o s i d e (40) l a b e l l e d v i n d o l i n e (11) i n C. r o s e u s 6 2 3 ' * 3 and r e s e r p i n i n e (14) i n V. major ' . I p e c o s i d e ( 3 3 ) , d i s c u s s e d e a r l i e r , was t h e f i r s t example o f an i n t a c t s e c o - i r i d o i d u n i t i n a l k a l o i d s and l o g a n i n (35) and 6 3 51 g e r a n i o l (29) were shown t o be s p e c i f i c p r e c u r s o r s r e s p e c t i v e l y . ' However t h e f i r s t examples o f a s e c o - i r i d o i d u n i t i n combina-t i o n w i t h t r y p t o p h a n ( l b ) were n o t e d i n a l k a l o i d s c o r d i f o l i n e ( 4 5 ) , 6 4 a d i f o l i n e (46) and t h e i r c o r r e s p o n d i n g deoxy d e r i v a t i v e s (47,48). -16-S t r i c t o s i d i n e ( 4 9 ) , no s t e r e o c h e m i s t r y i m p l i e d , was then i s -o l a t e d from R. s t r i c t a . Thus t h e c r u c i a l i n t e r m e d i a t e between the i r i d o i d s and a l k a l o i d s was o b t a i n e d . S t r i c t o s i d i n e (49) was then shown t o be t r y p t o p h a n ( l b ) and l o g a n i n 6 8 (35) d e r i v e d . S i g n i f i c a n t l y the a u t h o r s mentioned f u r t h e r b i o - s y n t h e t i c p u r s u i t s were b e i n g i n i t i a t e d u s i n g (49) as a p r e c u r s o r . I n t h i s r e g a r d , Brown e t a l . o n l y o b t a i n e d low i n c o r p o r a t i o n o f 69 s t r i c t o s i d i n e (49) i n t o t h e i n d o l e a l k a l o i d s B a t t e r s b y then r e p o r t e d t h e p a r t i a l s y n t h e s i s o f i p e c o s i d e (33a i s o i p e c o s i d e (33b) and v i n c o s i d e ( 4 9 a ) / i s o v i n c o s i d e (49b) by conden-s a t i o n o f s e c o l o g a n i n (44) w i t h 3,4 d i h y d r o x y p h e n e t h y l a m i n e (50) and t r y p t a m i n e ( l a ) r e s p e c t i v e l y 7 ^ ' ^ . -17-5 0 The r e l a t i v e s t e r e o c h e m i s t r y about C-3 o f v i n c o s i d e ( 4 9 a ) / i s o v i n c o -s i d e (49b) b e i n g o b t a i n e d by c o r r e l a t i o n w i t h i p e c o s i d e ( 3 3 a ) / i s o -i p e c o s i d e (33b). V i n c o s i d e (49a) and i s o v i n c o s i d e (49b) were shown t o be 71 p r e s e n t i n C. r o s e u s . An a d m i x t u r e o f (49a,b) was f e d t o C. r o s e u s and a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and p e r i v i n e (16) were l a b e l l e d . ' ' F u r t h e r l a b e l l i n g e x p e r i m e n t s w i t h pure v i n c o s i d e (49a) and pure i s o v i n c o -s i d e (49b) showed s p e c i f i c i n c o r p o r a t i o n o f v i n c o s i d e (49a) w i t h 71 73 n e g l i g i b l e i n c o r p o r a t i o n b e i n g n o t e d u s i n g (49b). ' T h i s was t h e l o g i c a l r e s u l t s i n c e v i n c o s i d e (49a) p o s s e s s e s the same s t e r e o -c h e m i s t r y a t C-3 as t h a t o f t h e c o r y n a n t h e a l k a l o i d s . However t h i s r e s u l t i n i t i a t e d a c o n t r o v e r s y w h i c h a p p a r e n t l y has o n l y been r e s o l v e d v e r y r e c e n t l y . I n 1969, Smi t h p r o p o s e d t h a t t h e s t e r e o c h e m i s t r y o f v i n c o s i d e (49a) and i s o v i n c o s i d e (49b) s h o u l d be r e v e r s e d 7 4 due t o the s i m i -l a r s p e c t r a l q u a l i t i e s o f (49b) w i t h s t r i c t o s i d i n e (49) w h i c h t h e y had c o r r e l a t e d w i t h a n t i r h i n e (51) thus showing i t t o be the C-3 75 o(-H epimer. -18-X-ray i n v e s t i g a t i o n o f 0 ^ O - d i m e t h y l i p e c o s i d e ( 5 2 ) , now r e v i s e d the s t e r e o c h e m i s t r y a t C-5 from t o fi t h u s a u t o m a t i c a l l y r e v i s i n g t h e s t e r e o c h e m i s t r y a t C-3 t o v i n c o s i d e (49b) and i s o v i n c o s i d e (49a) i 76 r e s p e c t i v e l y . 5 2 C o n s e q u e n t l y d e s a c e t y l i p e c o s i d e (53) was p a r t i a l l y s y n t h e s i s e d and f e d i n l a b e l l e d form t o C. i p e c a c u a n h a . I t was found t o be i n c o r p o r a t e d i n t o i p e c o s i d e ( 3 3 ) , now (33b), c e p h a e l i n e (28) and 77 emetine (54) . 5 3 5 4 -19-F u r t h e r , l a b e l l i n g w i t h ( 5 % ) l o g a n i n (35) showed s p e c i f i c i n c o r p o r a t i o n o f l a b e l i n t o (33b), (28) and (53) - w i t h no l o s s o f t r i t i u m on i n v e r s i o n o f s t e r e o c h e m i s t r y . (Shown by d e g r a d a t i o n . ) (5^H, OMe^H) l o g a n i n (35) was t h e n f e d t o C. roseus and v i n -d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e (15) were shown t o 7 8 be s p e c i f i c a l l y l a b e l l e d . Thus as v i n c o s i d e ( 4 9 b ) and i p e c o s i d e (33b) had been d e t e r -mined t o be p r e c u r s o r s o b v i o u s l y i n c o r p o r a t i o n o c c u r r e d v i a an i n v e r s i o n and r e t e n t i o n p r o c e s s . Brown a l s o s u p p o r t e d the C-3 79 r e v i s i o n o f s t e r e o c h e m i s t r y . However i n the b i o s y n t h e s i s o f c a m p t o t h e c i n (56) i t was found 8 0 t h a t i n a d d i t i o n t o t h e e x p e c t e d i n t e r m e d i a c y o f mevalonate (26) , 80 8 0 t r y p t o p h a n ( l b ) and s e c o l o g a n i n (44) , i s o v i n c o s i d e (49a) was i n -80a c o r p o r a t e d t o the e x t e n t o f 2%. V i n c o s i d e (49b) was n o t i n c o r -, , 80a p o r a t e a . These h i g h i n c o r p o r a t i o n s a l l o w e d H u t c h i n s o n t o f e e d ( 5 1 3 C ) i s o v i n c o s i d e l a c t a m ( 5 5 ) , and, i t was t h u s d i s c o v e r e d t h a t C-5 o f c a m p t o t h e c i n (56) was e n r i c h e d . The p o s t u l a t e d scheme f o r b i o s y n -t h e s i s i s shown . ( F i g u r e 8) -20-F i g u r e 8 - P o s t u l a t e d b i o s y n t h e t i c c o n v e r s i o n s f o r f o r m a t i o n o f c a m p t o t h e c i n ( 5 6 ) . V e r i f i c a t i o n o f t h e s t r u c t u r e o f v i n c o s i d e (49b) was t h e n 81 o b t a i n e d by X-ray and was shown t o be C^-H^. Presumably i n l i g h t o f t h i s most s i g n i f i c a n t b i o s y n t h e t i c r e s u l t , Zenk, e n j o y i n g s u c c e s s w i t h c e l l s u s p e n s i o n c u l t u r e s from C. r o s e u s 8 2 i n c u b a t e d ( 2 1 4 C ) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) 8 3 w i t h a c e l l f r e e system from C. r o s e u s . A c h e m i c a l l y s y n t h e s i s e d - 2 1 -m i x t u r e o f n o n - r a d i o a c t i v e v i n c o s i d e (49b) and i s o v i n c o s i d e (49a) was th e n added. S e p a r a t i o n and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y r e v e a l e d t h a t a l l a c t i v i t y was p r e s e n t i n i s o v i n c o s i d e (49a) . S i n g l y and d o u b l y l a b e l l e d forms o f i s o v i n c o s i d e (49a) were the n f e d t o C. r o s e u s p l a n t s and a l k a l o i d s v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , a j m a l i c i n e (15) and s e r p e n t i n e (13) were 8 3b l a b e l l e d . S i m i l a r e x p e r i m e n t s w i t h v i n c o s i d e (49b) showed no i n c o r p o r a t i o n i n t o t h e s e a l k a l o i d s . Thus, the (C^ c< e p i m e r ) , i s o v i n c o s i d e ( 4 9 a ) , now c a l l e d s t r i c t o s i d i n e , was i n c o r p o r a t e d i n t o the i n d o l e a l k a l o i d s as e x p e c t e d . I t i s n o t the purpose o f t h i s i n t r o d u c t i o n t o s p e c u l a t e on re a s o n s l e a d i n g t o t h e c o n t r o v e r s y . From t h e f e e d i n g e x p e r i m e n t s so f a r d e s c r i b e d , a few g e n e r a l p o i n t s were n o t e d : (1) i n c o r p o r a t i o n s were s u p p o r t e d by e i t h e r d e g r a d a t i o n and/or do u b l e l a b e l l i n g . I n c o r p o r a t i o n s were on a v e r a g e ~ 0 . 3 % a l t h o u g h i n some a l k a l o i d s e.g. a j m a l i c i n e ( 1 5 ) , p r e c u r s o r i n c o r p o r a t i o n was n o t a r e l i a b l e g u i d e t o t h e i m p o r t a n c e o f the p r e c u r s o r s . i n c o r p o r a t i o n m e v a l o n o l a c t o n e (27) 0.003 l o g a n i n (35) 0.26-0.46 s e c o l o g a n i n (44) 0.55 s t r i c t o s i d i n e (49a) 0.5 (2) v a r i o u s p l a n t systems may n o t i n c o r p o r a t e p r e c u r s o r s . (3) b l a n k e x p e r i m e n t s were n o t u s u a l l y r e p o r t e d b u t assuming t h a t - 2 2 -they were c a r r i e d o u t one thus o b t a i n s r e a s o n a b l y s i g n i f i c a n t r e -3 4 s u i t s - w i t h comments by L e e t e and B a r t o n b e i n g accomodated f u l l y . The i n t e r m e d i a c y beyond s t r i c t o s i d i n e (49a) has been the s u b j e c t o f many i n v e s t i g a t i o n s . Two p o s t u l a t e s have been f o r w a r d e d and 84 85 the f o l l o w i n g sequence o f f o r m a t i o n was s u g g e s t e d . ' c o r y n a n t h e —> s t r y c h n o s — • a s p i d o s p e r m a —• i b o g a C o n v e r s i o n o f s t r i c t o s i d i n e (49a) t o c o r y n a n t h e a l k a l o i d s , as shown by c o r y n a n t h e i n e aldehyde (57) o r g e i s s o s c h i z i n e ( 5 8 ) , would be n o n - e x c e p t i o n a l ( e n z y m a t i c a l l y \ ) . ( F i g u r e 9) F i g u r e 9 - P o s t u l a t e d b i o s y n t h e t i c f o r m a t i o n o f c o r y n a n t h e i n e a l d e h y d e (57) and g e i s s o s c h i z i n e ( 5 8 ) . - 2 3 -F u r t h e r c o n v e r s i o n o f the a l k a l o i d s (57) and (58) were i n v e s t i g a t e d , and, i n t h i s r e s p e c t , c o r y n a n t h e i n e aldehyde (57) was c o n s i d e r e d 8 6 not t o be an i n t e r m e d i a t e i n the b i o s y n t h e s i s o f v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , s e r p e n t i n e ( 1 3 ) , a j m a l i c i n e (15) and akuammicine ( 6 4 ) ? whereas g e i s s o s c h i z i n e (58) was e f f i c i e n t l y 8 7 i n c o r p o r a t e d i n t o t h e s e a l k a l o i d s S c o t t has, h o w e v e r , r e p o r t e d a s m a l l i n c o r p o r a t i o n o f c o r y n a n t h e i n e aldehyde (57) i n C. r o s e u s s e e d l i n g s 8 6 0 - b u t , as B a t t e r s b y s u g g e s t s , t h i s f a c t i s of l i t t l e s i g n i f i c a n c e as d e g r a d a t i o n and/or 8 7 m u l t i p l e l a b e l l i n g was not pursued t o ensure i n t a c t i n c o r p o r a -F u r t h e r s u p p o r t t o the i n t e r m e d i a c y o f g e i s s o s c h i z i n e (58) was 8 8 r e p o r t e d by i t s s u c c e s s f u l i n c o r p o r a t i o n i n t o c o r o n a r i d i n e (59) The p o s t u l a t e s ' f o r the f o r m a t i o n o f the s t r y c h n o s , aspidosperma, i b o g a b a s e s , as i n i t i a l l y s u g g e s t e d , are shown ( F i g . 10-12). Both s t r y c h n o s p o s t u l a t e s u t i l i z e g e i s s o s c h i z i n e ( 5 8 ) . ( F i g u r e 10) To account f o r t h e f o r m a t i o n o f the a s p i d o s p e r m a / i b o g a a l k a -l o i d s Wenkert proposed the f o l l o w i n g scheme 8^ wh i c h was the f i r s t s u g g e s t i o n t h a t a discrete C 7 - C 3 seco s p e c i e s was i n v o l v e d i n the b i o s y n t h e s i s o f t h e s e s k e l e t a . ( F i g u r e 11) t i o n . 59 84, 85 WENKERT'S HYPOTHESIS H C O X * C H O 3 Zt a;R, = C H O ; R 2 = C O 2 C H 3 b , R 2 = C 0 A C H 3 ; R , = C H O H 3 C 0 2 C H ^ O f t H O -0. -24-SCOTT'S HYPOTHESIS H X O C - C H O 3 2 I 8 3 H C 0 2 C CHO 61 7 H3C 02C 7 7 H C O C ' C H O 3 c. . 6 1 F i g u r e 10 - Pro p o s e d b i o s y n t h e t i c o r i g i n s o f the s t r y c h n o s f a m i l y . 1 F i g u r e 11 - Wenkert's p r o p o s e d b i o s y n t h e t i c o r i g i n s o f the aspidosperma and i b o g a f a m i l i e s . S l i g h t m o d i f i c a t i o n by S c o t t o f Wenkert's h y p o t h e s i s f o r the 85 a s p i d o s p e r m a / i b o g a s k e l e t a r e s u l t e d i n the f o l l o w i n g p o s t u l a t e ( F i g u r e 12) -26-F i g u r e 12 - S c o t t ' s m o d i f i c a t i o n o f Wenkert's h y p o t h e s i s f o r the aspidosperma and i b o g a f a m i l i e s . A s i m i l a r i n t e r m e d i a t e (73) has a l s o been p r o p o s e d by Levy C O £ C H 3 C 0 2 C H 3 7 3 7 4 I t i s o b v i o u s t h a t the i n t e r m e d i a c y o f stemmadenine (63) and an a c t i v a t e d p y r i d i n i u m s p e c i e s (65,70,73,74) are o f paramount im-p o r t a n c e i n the schemes o u t l i n e d . L e t us now c o n s i d e r t h e e v i d e n c e c o n c e r n i n g t h e s e schemes. -27-I n s u p p o r t o f Wenkert's s t r y c h n o s h y p o t h e s i s f o r m y 1 - s t r i c t a m i n e s 89 1 (60a,b) and p l e i o c a r p a m i n e (75) have been i s o l a t e d m R. s t r i c t a . Compound (76) was a l s o i s o l a t e d from Aspidosperma desmanthum 9 0 . U n f o r t u n a t e l y no b i o s y n t h e t i c e v a l u a t i o n o f t h i s d a t a has been f o r w a r d e d , and, c o n s e q u e n t l y , no comment can be made. I n v i t r o c o n v e r s i o n s o f c o r y n a n t h e —> s t r y c h n o s s k e l e t a have however been r e p o r t e d by S c o t t e.g. c o n v e r s i o n o f g e i s s o s c h i z i n e (58) i n t o dehydropreakuammicine (61) i n r e f l u x i n g a c e t i c a c i d . However t h e y i e l d s w h i c h were r e p o r t e d were poor and non-91 r e p r o d u c i b l e . 6 1 I n s u p p o r t o f S c o t t ' s s t r y c h n o s h y p o t h e s i s , o x i n d o l e a l k a l o i d s have been found i n M . p a r v i f o l i a and i n t h i s r e s p e c t a j m a l i c i n e (15) and 3 - i s o a j m a l i c i n e (78) were each i n c o r p o r a t e d i n t o the o x i n d o l e s m i t r a p h y l l i n e (79) and i s o m i t r a p h y l l i n e ( 80). -28-S c o t t has a l s o r e p o r t e d i s o l a t i o n o f t h e h y d r o x y i n d o l e n i n e o f g e i s s o s c h i z i n e (62) and g e i s s o s c h i z i n e o x i n d o l e (83, R=H) from C. 93 r o s e u s s e e d l i n g s . Subsequent i n c o r p o r a t i o n s o f g e i s s o s c h i z i n e o x i n d o l e (83, R=H), i n t o akuammicine (64) and v i n d o l i n e (11) were 9 3 a l s o r e p o r t e d . Treatment o f i n d o l e n i n e (81) w i t h p o t a s s i u m t - b u t o x i d e a f f o r d e d 9 4 n o r - f l u o r o c u r a r i n e (82) l e n d i n g an i n v i t r o s u p p o r t f o r S c o t t ' s s t r y c h n o s h y p o t h e s i s ( f i g u r e 13). A mechanism f o r t h i s c o n v e r s i o n was a l s o r e p o r t e d . F i g u r e 13 - P r o p osed mechanism f o r f o r m a t i o n o f n o r - f l u o r o c u r a r i n e ( 8 2 ) . Both hypotheses u t i l i s e stemmadenine (63) and i n t h i s r e s p e c t -29-(3 ±' 1C) t r y p t o p h a n ( l b ) was f e d t o C. r o s e u s and ( S " 1 ^) stemmadenine 95 3 (63) was o b t a i n e d by i s o t o p i c d i l u t i o n . F u r t h e r (OMe H) stemmadenine (63) was o b t a i n e d by t r i v i a l c o n v e r s i o n o f (63)-—» a c i d 9 5 ->(63) . F e e d i n g e x p e r i m e n t s w i t h t h e r e s u l t a n t d o u b l y l a b e l l e d 9 5 stemmadenine (63) showed r e a s o n a b l e i n c o r p o r a t i o n i n t o t a b e r s o n m e (1 8 ) , c a t h a r a n t h i n e (12) and v i n d o l i n e (11) w i t h no l o s s o f r a t i o o f l a b e l s on i n c o r p o r a t i o n thus g i v i n g f u r t h e r s u p p o r t f o r t h e i n t e r m e d i a c y o f an a c t i v a t e d p y r i d i n i u m s p e c i e s (e.g. 65,70,73,74). B a t t e r s b y gave f u r t h e r s u p p o r t t o the i n t e r m e d i a c y o f (1) stemmadenine (63) and (2) an a c t i v a t e d p y r i d i n e i n t e r m e d i a t e 3 7 8 (65,70,73,74) by f e e d i n g (5 H) l o g a n i n t o C. r o s e u s and i s o l a t i n g l a b e l l e d v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e ( 1 5 ) . A c c o r d i n g t o the h y p o t h e s i s t h e l a b e l would be found i n the f o l l o w i n g p o s i t i o n s i n the m o l e c u l e . ( F i g u r e 14) S p e c i f i c d e g r a -d a t i o n s showed l a b e l from f e e d i n g t o be l o c a t e d e x c l u s i v e l y a t Carbon (#) o f ( 1 1 ) , (12) and ( 1 5 ) . F u r t h e r e v i d e n c e f o r the f o r m a t i o n o f a seco i n t e r m e d i a t e such as t h e a c r y l i c e s t e r (74) was o b t a i n e d by i s o l a t i o n o f d i m e r i c i n d o l i c secamines (84) from R. s t r i c t a . Of p a r t i c u l a r s i g n i f i c a n c e was t h e p r e s e n c e o f monomeric s e c o d i n e s ( 8 5 ) , ( 8 6 ) , ( 8 7 ) . 9 7 H F i g u r e 14 - I n c o r p o r a t i o n o f (5 H) l o g a n i n (35) i n t o v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e (12) and a j m a l i c i n e ( 1 5 ) . -31-8 6 8 7 B a t t e r s b y a l s o e s t a b l i s h e d t h e n a t u r a l o c c u r r e n c e o f 16,17 d i h y d r o -9 8 s e c o d i n - 1 7 - o l (88) i n C. r o s e u s and Rhazya o r i e n t a l i s . I t s h o u l d be n o t e d t h a t no b i o s y n t h e t i c i n f o r m a t i o n f o r the forma-t i o n o f t h e s e compounds from s t r i c t o s i d i n e (49a), g e i s s o s c h i z i n e (58) o r stemmadenine (63) have been r e p o r t e d . I n v e s t i g a t i o n s aimed a t v e r i f i c a t i o n o f the l a t t e r s t a g e s o f b i o s y n t h e s i s were i n i t i a t e d i n o u r l a b o r a t o r i e s . I n t h i s r e s p e c t two pathv/ays were p u r s u e d . (1) i m p o r t a n c e o f t r a n s a n n u l a r c y c l i z a t i o n i n aspidosperma and i b o g a a l k a l o i d s (66-67,68-69). (2) i m p o r t a n c e o f d e h y d r o s e c o d i n e (74) i n subsequent c o n v e r s i o n s 8 8 -32-t o aspidosperma o r i b o g a a l k a l o i d s . (1) The t r a n s a n n u l a r c y c l i z a t i o n o f quebrachamine (89) and 1 8 y 3-carbomethoxycleavamine (72) t o the p e n t a c y c l i c analogues a s p i d o s p e r m i d i n e (90) and c a t h a r a n t h i n e (12) had been shown t o be 99 a f a c i l e i n v i t r o p r o c e s s and thus i t was o f i m p o r t a n c e t o d e t e r -. c . , . i • , • 100 mine i f t h e r e was any m v i v o a p p l i c a t i o n 72 12 U n f o r t u n a t e l y v e r y low i n c o r p o r a t i o n s were o b t a i n e d , w h i c h , on com-p a r i s o n w i t h the c o r r e s p o n d i n g b l a n k e x p e r i m e n t s n e g a t e d t h e i r i m p o r t a n c e . I t i s o f some i n t e r e s t t o note t h a t c l e a v a m i n e monomers (e.g. 72) do n o t appear t o o c c u r i n n a t u r e t h u s perhaps g i v i n g some ex-p l a n a t i o n t o the i n v i v o o b s e r v a t i o n . However s u p p o r t f o r the i n t e r -mediacy o f d e h y d r o s e c o d i n e (74) was c o n s i d e r e d , i n s p i t e o f t h e s e poor r e s u l t s , by the low i n c o r p o r a t i o n o f t a b e r s o n i n e (18) i n t o v i n d o l i n e (11) and c a t h a r a n t h i n e (12) i n C. r o s e u s p l a n t s 1 ^ 0 and - 3 3 -s e e d l i n g s . Thus r e v e r s a l of t h e t r a n s a n n u l a r c y c l i z a t i o n was p o s t u l a t e d - a p r o c e s s n o t found t o o c c u r i n o t h e r f e e d i n g ex-. 101 p e r i m e n t s However l a b e l l i n g o f t a b e r s o n i n e (18) i n t h e s e e x p e r i m e n t s o n l y u t i l i z e d t h e t r y p t a m i n e ( l a ) p o r t i o n and thus the r e s u l t s a r e o f l i m i t e d b i o s y n t h e t i c s i g n i f i c a n c e s i n c e double l a b e l l i n g e x p e r i -ments must be c a r r i e d o u t w i t h the l a b e l i n b o t h (1) t h e t r y p t a m i n e p o r t i o n ( l a ) and (2) t h e t e r p e n o i d p o r t i o n . (A,B,C) S u i t a b l e d e g r a d a t i o n would thus prove such a p r o c e s s o f r e a r r a n g e -ment . (2) Importance o f d e h y d r o s e c o d i n e (74) i n subsequent c o n v e r s i o n s U n f o r t u n a t e l y l a b e l l i n g e x p e r i m e n t s w i t h (74) c o u l d n o t be pur s u e d due t o t h e i n s t a b i l i t y o f such an i n t e r m e d i a t e and u n t i l c o n d i t i o n s a re met f o r the s t a b i l i z a t i o n o f t h i s compound no d a t a can be f o r w a r d e d f o r i t s i m p o r t a n c e . 3 However l a b e l l i n g e x p e r i m e n t s w i t h (Ar H) 1 6 , 1 7 - d i h y d r o s e c o d i n -1-7 i / o o v l 0 2 - 1 0 6 A / 0 1 \ 102-3,106-7 . , „. 1 7 - o l (88) and s e c o d i n e (91) ' i n C. r o s e u s and V i n c a minor were i n v e s t i g a t e d as i t was hoped t h a t they c o u l d produce a (74) type intermediate i n vivo. In the case of (Ar H) 16,17-dihydro--34-s e c o d i n - 1 7 - o l (88) i n c o r p o r a t i o n s were v e r y low (<0.001%) i n t o c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( 1 1 ) , a j m a l i c i n e (15) i n C. r o s e u s and v i n c a m i n e (92) and m i n o v i n e (93) i n V. minor . Subsequent ex-3 p e r i m e n t s w i t h (Ar H) s e c o d i n e (91) showed s i m i l a r i n c o r p o r a t i o n s i n a l l a l k a l o i d s e x c e p t v i n d o l i n e (11) t o w h i c h a 0.02% i n c o r p o r a t i o n was o b t a i n e d . F u r t h e r f e e d i n g e x p e r i m e n t s w i t h d o u b l y - l a b e l l e d s e c o d i n e ( A r 3 H , 1 4 C 0 2 C H 3 ) (91) showed t h a t i n c o r p o r a t i o n i n t o v i n d o l i n e (11) was accompanied by some l o s s o f t r i t i u m - t h i s a l s o b e i n g noted 8 7 by B a t t e r s b y ' s e x p e r i m e n t s w i t h o t h e r a r o m a t i c l a b e l l e d p r e c u r s o r s . 9 2 9 3 Subsequent e x p e r i m e n t s w i t h ("^CC^CH^,3,21°H) s e c o d i n e (91) showed 60% l o s s o f t r i t i u m on c o n v e r s i o n i n t o v i n d o l i n e (11) -a p r o c e s s s u g g e s t i n g t h e p o s s i b l e i n v o l v e m e n t o f a d e h y d r o s e c o d i n e (74) m o i e t y . However i t i s o b v i o u s t h a t t h e low i n c o r p o r a t i o n s , i f accom-p a n i e d by b l a n k e x p e r i m e n t s , do n o t g i v e t h e i n f e r e n c e o f a major b i o s y n t h e t i c pathway. Indeed i t was n o t e d t h a t n e i t h e r i n v e s t i -g a t i o n s i . e . e i t h e r (1) t r a n s a n n u l a r c y c l i z a t i o n o r (2) s e c o d i n e (91) i n v o l v e m e n t gave e n c o u r a g i n g r e s u l t s . I t i s hoped t h a t subse-quent f e e d i n g s o f a s t a b i l i s e d d e h y d r o s e c o d i n e (74) w i l l , i n the near f u t u r e , g i v e more s i g n i f i c a n t i n c o r p o r a t i o n s . -35-F i n a l l y i n s u p p o r t o f a c r y l i c e s t e r (74) i n v o l v e m e n t , S c o t t has r e p o r t e d a s e r i e s o f i n v i t r o e x p e r i m e n t s s u b s t a n t i a t i n g t h e s e 10 8 p r e d i c t i o n s . I t i s n o t t h e purpose o f t h i s i n t r o d u c t i o n t o d i s c u s s , i n d e t a i l , t h e i n v i v o t r a n s f o r m a t i o n s d e s c r i b e d . However i t s h o u l d be n o t e d t h a t t h e s e p a p e r s r e c e i v e d an u n h e r a l d e d r e -109 b u t t a l by S m i t h and P o i s s o n . I n r e s p o n s e , and d e f e n s e , S c o t t has p u b l i s h e d f u r t h e r work p a r t i a l l y s u b s t a n t i a t i n g h i s e a r l i e r c l a i m s 1 1 ^ ' 1 1 1 and from the e x p e r i m e n t a l s e c t i o n major s k e l e t a l con-v e r s i o n s u s i n g a c r y l i c e s t e r (74) as an i n t e r m e d i a t e were d e s c r i b e d . I n t h i s r e s p e c t some t r a n s f o r m a t i o n s were n o t e d i n 0.2% y i e l d from 6 mg:. o f s t a r t i n g m a t e r i a l ] C o n s e q u e n t l y , t h e c l o s e e x a m i n a t i o n of t h i s work by t h e i n t e r e s t e d r e a d e r i s s t r o n g l y s u g g e s t e d . From the work d e s c r i b e d e a r l i e r we have n o t e d p r e l i m i n a r y r e s u l t s s u g g e s t i n g t h e sequence c o r y n a n t h e — * s t r y c h n o s —> aspidosperma —•• i b o g a « • -, 4 . 1 0 . ^84,85 i s m e f f e c t as p r e v i o u s l y p o s t u l a t e d ' F u r t h e r s u p p o r t t o t h i s h y p o t h e s i s has been g i v e n by S c o t t 95 112 113 w i t h C. r o s e u s s e e d l i n g s . ' ' I n t h i s r e s p e c t f o r m a t i o n o f a l k a l o i d s i n a time domain was m o n i t o r e d . (Table 1) The a l k a l o i d s were i s o l a t e d and p a r t i a l l y c h a r a c t e r i s e d by (1) t h i n l a y e r chrom-a t o g r a p h y and (2) mass s p e c t r o m e t r y . Preakuammicine (77) was however a p p a r e n t l y i s o l a t e d and i n f o r -114 m a t i o n w i t h r e s p e c t t o i t s s t r u c t u r e was p r e s e n t e d . F u r t h e r s t u d i e s on i n t e r m e d i a c y i n C. r o s e u s s e e d l i n g s u t i l i s -i n g s h o r t term i n c u b a t i o n t e c h n i q u e s were then c a r r i e d o u t . 14 DL(2 C) t r y p t o p h a n ( l b ) was added t o the s e e d l i n g s and t h e r i s e and f a l l i n r a d i o a c t i v i t y o f o b s e r v e d i n t e r m e d i a t e s m o n i t o r e d by Table 1: F o r m a t i o n o f a l k a l o i d s from C. r o s e u s s e e d l i n g s . Time (hours) 0 26 28-32 28-40 42-48 50 72 100-160 168 A l k a l o i d I s o l a t e d none v i n c o s i d e (49b), c o r y n a n t h e i n e (17) v i n c o s i d e (49b), c o r y n a n t h e i n e a i d . ( 5 7 ) , g e i s s o s c h i z i n e (58) preakuammicine (77), c o r y n a n t h e i n e a i d . ( 5 7 ) , g e i s s o s c h i z i n e (58) akuammicine (64) stemmadenine (63) t a b e r s o n i n e (18) 11-methoxy t a b e r s o n i n e (18a) c a t h a r a n t h i n e (12) c o r o n a r i d i n e (59) c a t h a r a n t h i n e / v i n d o l i n e (12)/(11) Type cory n a n t h e c o r y n a n t h e c o r y n a n t h e -s t r y c h n o s s t r y c h n o s c o r y n a n t h e -s t r y c h n o s aspidosperma aspidosperma i b o g a i b o g a i b o g a / aspidosperma i I -37-a u t o r a d i o g r a p h y . The a l k a l o i d s were c h a r a c t e r i s e d by TLC b e h a v i o u r i n two d i m e n s i o n s . I t was n o t e d i n t h e s e e x p e r i m e n t s t h a t t r y p t o p h a n ( l b ) was q u i c k l y c o n v e r t e d t o a m e t a b o l i t e h a v i n g a r a p i d ' r a d i o - p r o f i l e ' and a c t i v i t y t h a t l a s t e d f o r o n l y 60 m i n u t e s . S c o t t s u g g e s t e d t h a t i t m i ght be ( A 2 0 , 2 1 ) c o r y n a n t h e i n e aldehyde (94) on t h e b a s i s o f i t s TLC b e h a v i o u r . ( H e v e r i f i e d t h i s p r o p o s a l i n a l a t e r communication 91 by TLC comparison of s y n t h e t i c (94) w i t h the ' r a d i o - p r o f i l e ' . ) He t h e n s t a t e s t h a t t h e f o l l o w i n g a l k a l o i d s are formed h a v i n g 115 dynamic r a d i o - p r o f i l e s i . e . t h e y were r a p i d l y m e t a b o l i z e d . They were (1) g e i s s o s c h i z i n e (58) (1.5-2.5 hours) (2) preakuammi-c i n e (77) (2-2.5 hours) and (3) t a b e r s o n i n e (18) (9 h o u r s ) . O ther a l k a l o i d s d e t e c t e d e.g. v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e ( 1 2 ) , a j m a l i c i n e ( 1 5 ) , c o r o n a r i d i n e (59) and l o c h n e r i c i n e (95) had ' s t a t i c ' p r o f i l e s s u g g e s t i n g t h e i r f o r m a t i o n t o be s h u n t - l i k e i n n a t u r e . U n f o r t u n a t e l y c h a r a c t e r i s a t i o n was o n l y by two d i m e n s i o n a l TLC and d e t e c t i o n by means of c e r i c s u l f a t e s p r a y . I n t h i s r e s -p e c t o n l y 4 a l k a l o i d s c o u l d be d e t e c t e d by t h i s s p r a y i n g p r o c e s s e.g. v i n d o l i n e ( 1 1 ) , akuammicine ( 6 4 ) , v i n e r v i n e (96) and an u n i d e n --38-t i f i e d a l k a l o i d , - t h e remainder n o t b e i n g d e t e c t e d by t h i s s e n s i t i v e r e a g e n t . 0 HO 9 5 9 6 However a r e a s c o r r e s p o n d i n g t o t h e Rf o f a l l o f t h e s e components were removed (by s c r a p i n g ) and s u b j e c t e d t o r a d i o a c t i v e c o u n t i n g . A ssignment o f s t r u c t u r e must thus be s p e c u l a t i v e s i n c e a s m a l l amount o f r a d i o a c t i v i t y i n t h e s e a r e a s c o u l d g i v e r i s e t o such ob-s e r v a t i o n s . T h i s i s n o t a t o t a l l y c r i t i c a l comment but i t has been n o t e d i n my e x p e r i m e n t s t h a t s u b s t a n t i a l l o s s o f a c t i v i t y i n chromatographed f r a c t i o n s o c c u r r e d f o l l o w i n g r e p e a t e d c r y s t a l l i s a -t i o n s t h u s s u g g e s t i n g s m a l l q u a n t i t i e s o f r a d i o - a c t i v e m a t e r i a l as i m p u r i t i e s . T h i s p u b l i c a t i o n 1 1 5 was r e p r i n t e d v e r b a t i m , i n i t s e n t i r i t y , i n Recent Advances i n P h y t o c h e m i s t r y 1 1 6 w i t h o n l y o m i s s i o n o f the e x p e r i m e n t a l s e c t i o n b e i n g n o t e d . Thus, so f a r , we have n o t e d o n l y p r e l i m i n a r y e v i d e n c e t o sup-p o r t the p o s t u l a t e d b i o s y n t h e t i c o r i g i n s 8 4 , 8 " ' beyond s t r i c t o s i d i n e ( 4 9a). I n t h i s r e s p e c t many e x p e r i m e n t s d e s c r i b e d have s e v e r e l i m i t a t i o n s on t h e i r b i o s y n t h e t i c s i g n i f i c a n c e i f r e c e n t comments 2 by C o r n f o r t h are t o be r e s p e c t e d . C o n s e q u e n t l y t h e advancement o f e n z y m o l o g i c a l t e c h n i q u e s f o r a l k a l o i d b i o s y n t h e s i s have been pu r s u e d i n C. r o s e u s . -39-I n t h i s r e s p e c t some s l i g h t p r o g r e s s was i n i t i a l l y made by 117 i s o l a t i o n o f an O-methyl t r a n s f e r a s e w h i c h c a t a l y s e d the c o n v e r -s i o n o f l o g a n i c (97) and s e c o l o g a n i c a c i d (44a) t o l o g a n i n (35) and s e c o l o g a n i n (44) r e s p e c t i v e l y . A c etone powders o f C. r o s e u s , i n c u b a t e d w i t h t r y p t a m i n e ( l a ) and s e c o l o g a n i n ( 4 4 ) , gave no a l k a l o i d a l biosynthesis. 1"'""' H An i m p o r t a n t b r e a k t h r o u g h a t t h e c e l l f r e e l e v e l was n o t e d when 118 S c o t t o b t a i n e d a m i x t u r e o f s o l u b l e enzymes from C. r o s e u s (1) s e e d l i n g s and (2) c a l l u s e s - w h i c h on i n c u b a t i o n w i t h ( 2 1 4 C ) t r y p -tamine ( l a ) and s e c o l o g a n i n (44) i n the p r e s e n c e o f NADPH, t h i o l s and t r i s b u f f e r , a f f o r d e d g e i s s o s c h i z i n e (58) and a j m a l i c i n e ( 1 5 ) . The a l k a l o i d s were c h a r a c t e r i s e d by two d i m e n s i o n a l TLC b u t i t was n o t e d t h a t g r e a t e r c o n v e r s i o n s o c c u r r e d w i t h t h e s o l u b l e en-zymes from t h e c a l l u s e s . However, more i m p o r t a n t l y , a l k a l o i d a l b i o s y n t h e s i s had been dem o n s t r a t e d a t the c e l l f r e e l e v e l . T h i s f i n d i n g was s u b s e q u e n t l y c o n f i r m e d by Zenk, who i n c u b a t e d 14 (2 C) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) w i t h a s u p e r n a t a n t enzyme e x t r a c t o f a h i g h b e a r i n g a l k a l o i d s t r a i n o f C. r o s e u s and 8 2 NADPH. B i o s y n t h e s e s o f a j m a l i c i n e ( 1 5 ) , 1 9 - e p i d j m a l i c i n e (98) and -40-t e t r a h y d r o a l s t o n i n e (99) were n o t e d . - the s t r u c t u r e s b e i n g v e r i f i e d by (1) TLC comparison and (2) i n t e r n a l d i l u t i o n and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . I n t h i s r e s p e c t i t was no t e d t h a t i n c o r p o r -118 a t i o n s were h i g h e r than t h o s e o f S c o t t by a f a c t o r o f 10. 9 8 119 F u r t h e r o b s e r v a t i o n s by Zenk showed t h a t i n c u b a t i o n , as d e s c r i b e d , b u t w i t h o u t NADPH o r NADH r e s u l t e d i n a c c u m u l a t i o n o f an i n t e r m e d i a t e (100), w h i c h has been c a l l e d cathenamine. I t s s t r u c t u r -120 a l p r o p e r t i e s have r e c e n t l y been p u b l i s h e d . Sodium b o r o h y d r i d e r e d u c t i o n o f cathenamine (100) a f f o r d e d t e t r a h y d r o a l s t o n i n e ( 9 9 ) . I t was a l s o n o t e d t h a t , i n the absence o f NADPH o r NADH, no (15 ) , (98) o r (99) formed. C o n s e q u e n t l y when cathenamine (100), p r e p a r e d i n h i g h s p e c i f i c a c t i v i t y , was i n c u b a t e d w i t h C. r o s e u s p r o t e i n , NADPH, and phosphate b u f f e r , ( 1 5 ) , (98) and (99) were ob-t a i n e d . C o n t r o l e x p e r i m e n t s w i t h (1) h e a t d e n a t u r e d p r o t e i n and (2) no NADPH showed no f o r m a t i o n o f ( 1 5 ) , (9 8) and (99) . H 3C0 2 c " 1 0 0 F u r t h e r cathenamine (100) has been found t o be a n a t u r a l p r o d u c t . A j m a l i c i n e (15) was a l s o i n c u b a t e d w i t h t h e enzyme m i x t u r e and i t was found not t o be c o n v e r t e d t o (9 8) o r (99) s u g g e s t i n g t h a t -41-s t e r e o c h e m i c a l c o n t r o l p r o b a b l y o c c u r s a t the cathenamine (100) s t a g e . S t r i c t o s i d i n e (49a), p r e v i o u s l y d i s c u s s e d , was p r e p a r e d by i n c u -b a t i o n o f ( 2 1 4 C ) t r y p t a m i n e ( l a ) and s e c o l o g a n i n (44) w i t h the en-zyme p r e p a r a t i o n i n t h e p r e s e n c e o f D - ^ - g l u c o n o l a c t o n e (a g l u c o -8 3a s i d a s e i n h i b i t o r ) S t r i c t o s i d i n e (49a) was c h a r a c t e r i s e d by (1) TLC (2) M.S. (3) c o l d d i l u t i o n - and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . I n c u b a t i o n o f s t r i c t o s i d i n e (49a) w i t h t h e enzyme p r e p a r a t i o n , i n the absence o f NADPH, y i e l d e d cathenamine (100). I n the p r e s e n c e o f NADPH, a l k a l o i d s ( 1 5 ) , (98) and (99) were o b t a i n e d . Thus f o r m a t i o n o f t h e c o r y n a n t h e a l k a l o i d s have been demon-s t r a t e d a t the c e l l f r e e l e v e l . I t w i l l be o f immense i m p o r t a n c e i f i t i s found t h a t subsequent t r a n s f o r m a t i o n s t o the s t r y c h n o s , a spidosperma and i b o g a a l k a l o i d s can a l s o be o b t a i n e d u s i n g such systems. I n a d d i t i o n t o the a f o r e m e n t i o n e d c o r y n a n t h e , s t r y c h n o s , aspidosperma and i b o g a a l k a l o i d s i t s h o u l d be n o t e d t h a t t h e ab-n o r m a l l y b r i d g e d a l k a l o i d s v a l l e s a m i n e (101), a p p a r i c i n e (102), u l e i n e (103), guatambuine (104), o l i v a c i n e (105) and e l l i p t i c i n e (106) a r e c u r r e n t l y presumed t o be formed a t l a t e s t a g e s i n a l k a -121 l o i d a l b i o s y n t h e s i s . However as c u r r e n t p o s t u l a t e s have n o t y e t been c o n c l u s i v e l y s u b s t a n t i a t e d , an o v e r a l l d i s c u s s i o n o f t h e v a r i o u s p o s t u l a t e s i s b r i e f l y mentioned below. - 4 2 -f 0 4 1 0 5 1 0 6 2 3 In Wenkert's o r i g i n a l monoterpenoid hypothesis he considered the non-tryptamine (la) portion of the indole alkaloids to be derived from prephenic acid (107). This was converted to a seco-prephenate formaldehyde (SPF) unit (108) which combined with t r y p t -amine (la) to give the indole alkaloids previously mentioned. 1 0 7 1 0 8 Wenkert extended his hypothesis to include the formation of uleine (103), o l i v a c i n e (105) and e l l i p t i c i n e (106). In t h i s respect i t was postulated that the SPF unit (108) combined with - 4 3 C H O - O H • O H C H O P O ^ H 2 3 © co 2 I 0 C 0 P 0 3 H C H , O c o 2 -0 C H 0 H O ' O H O H N A D H C H ^ O P O ^ H H 0, A C 0 - H 2 0 C O _© 2. H O P O = 3 O H O H C O 0 © H O P O N 3 A T P £ H ? G l u t a m m e R 0 ' ' C O © O H C O . © / V ^ O H O H 7 N A D P H . C O . © • N 0 © C 0 9 O H © c o 2 N H C H 2 0 P 0 3 H ' 0 H O O H - C O ? 2 H H H 0 0 0 Q N-CH=44+CH O P C H H ^ 3 l b F i g u r e 15 - Bi o s y n t h e s i s of tryptophan ( l b ) . - 4 4 -0 1 0 3 1 0 6 F i g u r e 16 - The Wenkert P o s t u l a t e f o r the b i o s y n t h e s i s o f o l i v a c i n e (105), e l l i p t i c i n e (106) and u l e i n e (103). -45-g l y c o s y i i d e n e a n t h r a n i l i c a c i d (109), a p r e c u r s o r o f t r y p t o p h a n ( l b ) ( f i g u r e 15) t o then y i e l d (103), ( 1 0 5 ) , ( 1 0 6 ) a s shown i n f i g u r e 16. F u r t h e r f o l l o w i n g t h e s t r u c t u r a l e l u c i d a t i o n o f a p p a r i c i n e (102), D j e r a s s i and G i l b e r t p o s t u l a t e d t h a t i n t e r m e d i a t e (110) may 203 be x n v o l v e d i n a p p a r i c i n e (102) f o r m a t i o n . ( F i g u r e 17) O V A C C O 2 C H 3 no F i g u r e 17 - The D j e r a s s i P o s t u l a t e f o r t h e b i o s y n t h e s i s o f a p p a r i c i n e (102). Thus the p o s t u l a t e d b i o s y n t h e t i c schemes (16,17) f o r the f o r m a t i o n o f t h e s e a l k a l o i d s do n o t i n v o l v e t r y p t o p h a n ( l b ) . I n o r d e r t o t e s t 3 t h i s p r o p o s a l (Ar H) t r y p t o p h a n ( l b ) was a d m i n i s t e r e d t o A. p y r i c o l l u m , a p l a n t c o n t a i n i n g a p p a r i c i n e (102) and u l e i n e (103). I n c o r p o r a -122 t i o n s i n t o a p p a r i c i n e (102) were n o t e d i n d i c a t i n g t h a t t r y p t o -phan ( l b ) was b e i n g u t i l i z e d . C l e a r l y t h e s e r e s u l t s a r e incom-p a t i b l e w i t h t h e Wenkert and D j e r a s s i p r o p o s a l s . I t was p o s s i b l e , however, t h a t the p l a n t enzymes were c a p a b l e o f d e g r a d i n g t r y p t o -phan ( l b ) t o an a p p r o p r i a t e i n d o l e i n t e r m e d i a t e l a c k i n g t h e / ? - e t h y l -amino s i d e c h a i n w h i c h was the n i n v o l v e d i n the b i o s y n t h e s i s . -46-E x p e r i m e n t s were th e n d e s i g n e d t o de t e r m i n e whether one o r b o t h carbons o f the ^3-ethylamino s i d e c h a i n o f t r y p t o p h a n ( l b ) 3 14 3 14 were e x t r u d e d . C o n s e q u e n t l y (Ar H, 2 C) and (Ar H,3 C) t r y p t o -phan ( l b ) were f e d t o A. p y r i c o l l u m . I s o l a t i o n o f a p p a r i c i n e (102) showed e x t r u s i o n o f C-2 o f t r y p t o p h a n ( l b ) had o c c u r r e d (~9 7%) w h i l e 14 3 d e t e r m i n a t i o n o f the C/ H r a t i o i n the a l k a l o i d c l e a r l y r e -v e a l e d t h a t C-3 was r e t a i n e d . I n t h i s l a t t e r e x p e r i m e n t i t was n o t e d t h a t a l o s s o f 27% o f t r i t i u m o c c u r r e d from the a r o m a t i c r i n g . F u r t h e r a d m i n i s t r a t i o n o f (1) ( A r 3 H ) , ( 2 ) ( A r 3 H , 2 1 4 C ) , 3 14 (3) (Ar H, 3 C) t r y p t o p h a n ( l b ) t o A. a u s t r a l e y i e l d e d some i n -121 t e r e s t m g r e s u l t s . I n t h e s e e x p e r i m e n t s , a l k a l o i d s o l i v a c i n e (105), a p p a r i c i n e (102), u l e i n e (103) and guatambuine (104) were i s o l a t e d . W i t h r e s p e c t t o e x p e r i m e n t s (1) and ( 3 ) , o l i v a c i n e (105) c o u l d n o t be c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y and so no m e a n i n g f u l d i s c u s s i o n can be ensued on t h a t b a s i s . However e x p e r i m e n t ( 2 ) , c o n s t a n t a c t i v i t y , i n c o r p o r a t i o n (<0.001%), showed t h a t t h e 3 14 H/ C r a t i o had d o u b l e d on i n c o r p o r a t i o n s u g g e s t i n g l o s s o f 58% o f the l a b e l a t C-2 o f t r y p t o p h a n ( l b ) . I n e x p e r i m e n t s ( 1 ) , (2) and (3) where a p p a r i c i n e (102) was i s o l a t e d i t was n o t e d i n (1) t h a t s i g n i f i c a n t i n c o r p o r a t i o n i n t o (102) had o c c u r r e d , i.e. 0.208% . I n ( 2 ) , however, i n c o r p o r a t i o n l e v e l was •<0.0015% and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y c o u l d n o t be o b t a i n e d . Good i n c o r p o r a t i o n s were n o t e d though f o r e x p e r i m e n t (3) -47-(0.127%) a l t h o u g h a g a i n a l o s s o f 17% t r i t i u m o c c u r r e d . I n e x p e r i m e n t s (1) and (2) v/here u l e i n e (103) was i s o l a t e d i t was n o t e d t h a t i n (1) i n c o r p o r a t i o n was <0.001%. I n e x p e r i -ment (2) an i n c o r p o r a t i o n o f 0.001% was o b t a i n e d w i t h a 3H/^ 4C r a t i o a l m o s t i d e n t i c a l t o t h a t f e d . I n e x p e r i m e n t s (1) and (2) where guatambuine (104) was i s o -l a t e d , i n c o r p o r a t i o n s v/ere o f the o r d e r 0.002%. I n (2) l o s s o f 27% of the C-2 l a b e l from t r y p t o p h a n ( l b ) was n o t e d . These r e s u l t s a r e summarised i n t a b l e 2. T a b l e 2: F i n d i n g s o f t r y p t o p h a n ( l b ) i n c o r p o r a t i o n s i n t o a l k a l o i d s  o f A. a u s t r a l e (A) and A. p y r i c o l l u m ( B ) . Tryptophan ( l b ) A l k a l o i d s I s o l a t e d l a b e l f e d : A p p a r i c i n e U l e i n e Guatambuine O l i v a c i n e (102) (A) (103) (B) (104) (B) (105) (B) 1) A r 3 H , 2 1 4 C l o s s o f C-2 (%) 97 no l o s s 27 58 3 14 2) A r H,3 qC l o s s of H (%) 2 7 N/A N/A N/A N/A - n o t a p p l i c a b l e . 3 The p o s s i b l e i n v o l v e m e n t o f (Ar H) stemmadenine (63) was 105 t h e n p u r s u e d i n A. p y r i c o l l u m , and, a p p a r i c i n e (102), and u l e i n e (103), v/ere i s o l a t e d . S i g n i f i c a n t i n c o r p o r a t i o n i n t o (102) was n o t e d w i t h no a p p r e c i a b l e i n c o r p o r a t i o n i n t o (103) . F e e d i n g e x p e r i m e n t s were t h e n p u r s u e d w i t h v a l l e s a m i n e (101) due t o i t s s t r u c t u r a l s i m i l a r i t i e s w i t h stemmadenine ( 6 3 ) . Very low i n c o r p o r a t i o n i n t o (102) and (103) were, n o t e d . I n o r d e r t o e x p l a i n t h e s e r e s u l t s , i t was s u g g e s t e d t h a t e x t r u s i o n o f carbon -48-and m o d i f i c a t i o n t o the e x o c y c l i c methylene on c o n v e r s i o n from stemmadenine (63) t o a p p a r i c i n e (102) may have o c c u r r e d v i a a c o n c e r t e d p r o c e s s . 12 3 P o t i e r has r e c e n t l y r e p o r t e d an i n v i t r o f r a g m e n t a t i o n w h i c h may a c c o u n t f o r the ca r b o n e x t r u s i o n . ( F i g u r e 18) C H 3 H + <CF3C°!P C C E C C ^ H F h N H • H3 C\ / C H j / ^ N - O C O C R P h N ( C H j : 3 ' 2 H 4-N © H C C H . o ° F i g u r e 18 - P o t i e r ' s i n v i t r o e x t r u s i o n o f C-2 u n i t of /3 -ethylamine side chain . S c o t t has r e p o r t e d an i n v i t r o c o n v e r s i o n o f stemmadenine (63) i n t o v a l l e s a m i n e (101) a c e t a t e u t i l i s i n g the m o d i f i e d P o l o n o v s k i r e a c t i o n . ( F i g u r e 19) 123 P o t i e r had p r e v i o u s l y p o s t u l a t e d a s i m i l a r mechanism f o r t h e b i o s y n t h e s i s o f a p p a r i c i n e (102). ( F i g u r e 20) -49-F i g u r e 19 - S c o t t ' s i n v i t r o c o n v e r s i o n o f stemmadenine (63) t o v a l l e s a m i n e (101). 102 F i g u r e 2 0 - P o t i e r ' s p o s t u l a t e d b i o s y n t h e s i s o f a p p a r i c i n e (102) from stemmadenine ( 6 3 ) . -50-The p o s s i b l e i n v o l v e m e n t o f a d e h y d r o s e c o d i n e (74) type i n t e r -m e d i a t e v/as th e n e x p l o r e d and w h i l s t c o n d i t i o n s have n o t y e t been met f o r t h e s t a b i l i s a t i o n o f t h i s i n t e r m e d i a t e i t v/as f e l t t h a t l a b e l l i n g e x p e r i m e n t s w i t h 16,17 d e h y d r o s e c o d i n - 1 7 - o l (88) and 103-5 122 s e c o d i n e (91) might be i n f o r m a t i v e . ' L a b e l l i n g e x p e r i m e n t s w i t h (88) showed n e g l i g i b l e i n c o r p o r -a t i o n s i n t o a l k a l o i d s o f A. p y r i c o l l u m . L a b e l l i n g e x p e r i m e n t s w i t h v a r i o u s l a b e l l e d forms o f s e c o d i n e (91) were th e n i n i t i a t e d w i t h A. p y r i c o l l u m and A. a u s t r a l e 3 due t o s m a l l b u t h o p e f u l l y s i g n i f i c a n t i n c o r p o r a t i o n s of (Ar H) s e c o d i n e (91) i n t o (102). The r e s u l t s a re summarized i n t a b l e 3 and t a b l e 4. T a b l e 3: Summary o f r e s u l t s of l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. p y r i c o l l u m . S e c o d i n e (91) l a b e l f e d : 1) H, C r a t i o i n A p p a r i c i n e (102): T a b l e 4: Summary of r e s u l t s o f l a b e l l i n g e x p e r i m e n t s w i t h s e c o d i n e (91) t o A. a u s t r a l e . S e c o d i n e (91) l a b e l : H, CO„CH_ r a t i o i n : A p p a r i c i n e (102) Guatambuine (104) O l i v a c i n e (105) l o s s o f 30% H l o s s o f 22% 3H * * * - not c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y -51-C o n s e q u e n t l y i t i s not p o s s i b l e t o draw any r i g i d c o n c l u s i o n s from th e s e r e s u l t s . 12 4 P o t i e r and J a n o t have r e c e n t l y extended t h e i r p o s t u l a t e t o encompass guatambuine (104), o l i v a c i n e ( 1 0 5 ) ^ e l l i p t i c i n e (106) and u l e i n e (103). ( F i g u r e 21) As n o t e d i n f i g u r e 20, the P o t i e r p o s t u l a t e f o r t h e f o r m a t i o n o f a p p a r i c i n e (102) i n v o l v e d a P o l o n o v s k i t y p e f r a g m e n t a t i o n f o l l o w e d by l o s s o f the carbomethoxy group o f stemmadenine (6 3 ) . T h i s p o s t u l a t e i s i n c o m p a t i b l e w i t h t h e s u g g e s t i v e e v i d e n c e t h a t the carbomethoxy group i s i n f a c t r e t a i n e d as shown i n the s e c o d i n e (91) f e e d i n g s . (Table 3) Moreover the g e n e r a l p o s t u l a t e f o r the b i o s y n t h e s e s o f u l e i n e (103), guatambuine (104), o l i v a c i n e (105) and e l l i p t i c i n e ( 1 0 6 ), are i n c o m p a t i b l e w i t h the i n c o r p o r a t i o n s n o t e d i n A. p y r i c o l l u m and A. a u s t r a l e . (Table 2) Thus i n o r d e r t o o b t a i n more d e f i n i t i v e d a t a the p l a n t 125 12 7 Aspidosperma v a r g a s i i , r i c h i n a l k a l o i d s a p p a r i c i n e (102) , 12 7 126 u l e i n e (103) , guatambuine (104) and 9 - m e t h o x y o l i v a c i n e 126 (111) , was n e x t c o n s i d e r e d . These i n v e s t i g a t i o n s w i t h c o r r e s p o n d i n g l a b e l l e d p r e c u r s o r s are d i s c u s s e d i n the n e x t s e c t i o n . See r e f . 199 f o r numberina svstem. 1 0 4 1 0 5 1 0 6 F i g u r e 21 - P o t i e r - J a n o t p o s t u l a t e f o r t h e b i o s y n t h e s i s of a p p a r i c i n e (102), u l e i n e (103), guatambuine (104), o l i v a -c i n e (105) and e l l i p t i c i n e (106) . -53-DPSCUSSION - PART I B i o s y n t h e s i s o f i n d o l e a l k a l o i d s l a c k i n g the normal t r y p t o p h a n  s i d e c h a i n : r o l e o f t r y p t o p h a n ( l b ) and stemmadenine (63) A. v a r g a s i i p l a n t s when mature are t r e e s s t a n d i n g up t o twenty metres i n h e i g h t and are i n d i g e n o u s t o r o c k y a r i d s l o p e s and t r a n s i t i o n f o r e s t i n V e n e z u e l a , and a d j a c e n t Colombia and Guiana. The p l a n t s r e q u i r e d f o r t h i s r e s e a r c h programme were f i v e d and 125(a) y e a r s o l d grown from s e e d s 1 2 5 ^ k i n d l y donated by Dr. B. G i l b e r t 3 The p r e c u r s o r s , c o m m e r c i a l l y a v a i l a b l e (Ar H) t r y p t o p h a n ( l b ) 3 and (Ar H) stemmadenine (63) , p r e p a r e d i n t h i s l a b o r a t o r y by the g e n e r a l a c i d c a t a l y s e d exchange of a r o m a t i c p r o t o n s (CF^C0 2T as t r i t i u m s o u r c e ) , were employed i n th e s e s t u d i e s . The r e s u l t i n g t r i t i a t e d p r e c u r s o r s were c r y s t a l l i s e d t o con-s t a n t a c t i v i t v and a d m i n i s t e r e d t o A. v a r g a s i i (whole p l a n t o r r o o t s e c t i o n s ) o v e r v a r y i n g time p e r i o d s . F o l l o w i n g t h e d u r a t i o n o f the p l a n t f e e d i n g , the p l a n t was removed, macerated, and e x t r a c t e d w i t h methanol. The a l k a l o i d s were s e p a r a t e d a c c o r d i n g t o a g e n e r a l p l a n t e x t r a c t i o n p r o c e d u r e 12 8 d e s c r i b e d by G i l b e r t . I n the a d m i n i s t r a t i o n o f t r y p t o p h a n ( l b ) t o A. v a r g a s i i r o o t c u t t i n g s the a l k a l o i d s p r e v i o u s l y mentioned (102-104, 111) were i s o l a t e d and c r y s t a l l i s e d r e p e a t e d l y t o remove i m p u r i t i e s . Re-c r y s t a l l i s a t i o n p r o c e d u r e s were r e p e a t e d u n t i l the f o l l o w i n g i n c o r p o r a t i o n l e v e l s were o b t a i n e d . (Table 5) S i m i l a r r e s u l t s 12 7 were r e c e n t l y n o t e d by D. G r i e r s o n i n stem f e e d i n g s . (Table 6) T a b l e 5 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i r o o t c u t t i n g s A c t i v i t y Fed (DPM) D u r a t i o n o f F e e d i n g (Days) A l k a l o i d I s o l a t e d S p e c i f i c A c t i v i t y I s o l a t e d (DPM/mmol) Weight I s o l a t e d (mg) A b s o l u t e I n c o r p o r a t i o n (%) S p e c i f i c I n c o r p o r a t i o n (%) 8.025x10 11 U l e i n e .(103) Guatambuine (10 4) 9-Methoxy-o l i v a c i n e (111) 0.00 1. 7388 x 10* 5.408 x 1 0 5 20 12 46 0.00 0.0026 0.0031 0.00 0.00069 0.00021 3 T a b l e 6 - R e s u l t s o f (Ar H) t r y p t o p h a n ( l b ) a d m i n i s t r a t i o n t o A. v a r g a s i i whole p l a n t 5.82 x l O 1 1 7 U l e i n e (103) 0.00 6 0.00 0.00 Guatambuine (104) 1.71 x 1 0 5 40 0.0018 0.000068 o n v a S n T u i l ) 3 ' 1 7 X 1 0 5 9 1 °- 0 0 4 9 0.000054 i I -55-C o u n t i n g was d i s c o n t i n u e d a t a l e v e l where b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . The g e n e r a l i t i e s a s s o c i a t e d w i t h the t r y p t o p h a n ( l b ) f e e d i n g s d i s c u s s e d so f a r a r e : (1) t h r e e d i f f e r e n t p l a n t s o u r c e s have been e x p l o i t e d (A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i ) (2) a d m i n i s t r a t i o n o f p r e c u r s o r had been c a r r i e d out a t v a r i o u s t i m e s o f t h e y e a r t o p l a n t s o f v a r y i n g s i z e s and ages. (3) p r e c u r s o r a d m i n i s t r a t i o n had employed s e v e r a l t e c h n i q u e s e.g. stem and r o o t f e e d i n g s . D i f f e r e n t s o l v e n t systems had been used t o a d m i n i s t e r the p r e c u r s o r , and, e f f i c i e n t i n c o r p o r a t i o n o f t r y p t o p h a n ( l b ) i n t o the p l a n t was n o t e d . (4) a f t e r i n i t i a l a d m i n i s t r a t i o n , e x p e r i m e n t s were o f v a r i e d d u r a t i o n (up t o 2 8 d a y s ) . (5) i n t a c t i n c o r p o r a t i o n i n t o any o f the a f o r e m e n t i o n e d 3 14 a l k a l o i d s was n o t demonstrated u s i n g e i t h e r (Ar H, 2 C) or ( A r 3 H , 3 1 4 C ) t r y p t o p h a n ( l b ) . ( I n c o r p o r a t i o n l e v e l s , l e s s than 0.001%). Thus a v a r i e t y o f e x p e r i m e n t s have been employed t o d e t e r m i n e 122 the r o l e o f t r y p t o p h a n ( l b ) . From the e a r l i e r s t u d i e s where c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y was o b t a i n e d i t i s o b v i o u s t h a t the r e s u l t s a re n o t i n agreement w i t h the P o t i e r - J a n o t 124 p o s t u l a t e . However as n o t e d i n some c a s e s , t h e p l a n t s (A. p y r i c o l l u m and A. a u s t r a l e ) d i d n o t always have s u f f i c i e n t q u a n t i t i e s o f a l k a l o i d s (102-105) t o o b t a i n c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y . -56-I n the e x p e r i m e n t s w i t h A. v a r g a s i i , however, where the a l -k a l o i d s were i n s u f f i c i e n t q u a n t i t y , no e v i d e n c e c o u l d be found f o r the p o s i t i v e i n c o r p o r a t i o n o f t r y p t o p h a n ( l b ) thus c a s t i n g f u r -124 t h e r doubt on the p o s t u l a t e 3 The l e v e l s o f i n c o r p o r a t i o n on a d m i n i s t r a t i o n o f (Ar H) stemmadenine (63) as p r e c u r s o r t o the a l k a l o i d s (103), (104) and (111) o f A. v a r g a s i i a r e shown i n t a b l e 7 . A g a i n c o u n t i n g was d i s c o n t i n u e d a t a l e v e l where b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . The e x p e r i m e n t s des-c r i b e d d e m o n s t r a t e d t h a t stemmadenine (63), under the c o n d i t i o n s employed, was not u t i l i z e d as an a c t i v e b i o s y n t h e t i c i n t e r m e d i a t e i n the b i o s y n t h e s i s o f t h e s e a l k a l o i d s . I t i s o f t e n s a i d t h a t " g r e a t c a u t i o n " must be e x e r c i s e d i n i n t e r p r e t i n g r e s u l t s o b t a i n e d from low i n c o r p o r a t i o n s s i n c e t h e s e are o f t e n r a t i o n a l i s e d as due t o : 1) low a l k a l o i d a l b i o s y n t h e s i s a t the t i m e o f a d m i n i s t r a t i o n o f the p r e c u r s o r . 2) n o n - p e r m e a b i l i t y o f t h e b i o s y n t h e t i c s i t e by t h e p r e c u r s o r 3) m o d i f i c a t i o n o r d e s t r u c t i o n o f the p r e c u r s o r p r i o r t o r e a c h -i n g t h e b i o s y n t h e t i c s i t e . C o n s e q u e n t l y , i t i s o f t e n v e r y d i f f i c u l t t o e s t a b l i s h t h a t a s u g g e s t e d i n t e r m e d i a t e i s a t r u e b i o s y n t h e t i c p r e c u r s o r . I n c o n c l u s i o n , t h e s e r e s u l t s r e v e a l t h a t (1) t r y p t o p h a n ( l b ) and stemmadenine (63) d i d n o t appear t o be e f f i c i e n t l y i n v o l v e d as p r e c u r s o r s . (2) i f t r y p t o p h a n ( l b ) and stemmadenine (63) are i m p o r t a n t p r e c u r s o r s t o t h e s e a l k a l o i d s then c o n d i t i o n s must be T a b l e 7 - R e s u l t s of (Ar H) stemmadenine (63) a d m i n i s t r a t i o n t o A. v a r g a s i i . D u r a t i o n A c t i v i t y o f P l a n t Fed F e e d i n g (Net Wt.) (PPM) (Days) (gm) S p e c i f i c A c t i v i t y Weight A b s o l u t e S p e c i f i c A l k a l o i d I s o l a t e d I s o l a t e d I n c o r p o r a t i o n I n c o r p o r a t i o n I s o l a t e d (PPM/mmol) (mg) (%) (%) 6.29 x l O 10 130 U l e i n e (103) 9-MeO-O l i v a c i n e (111) 0.00 5.74 x 10 10 85 0.00 0 .0077 0.00 0.000091 3.447x10 10 13 120 U l e i n e (103) Guatambuine (104) 9-MeO-O l i v a c i n e (111) 0.00 2.479 x 10 1.5906 x 10 18 26 104 0.00 0.0019 0. 0048 0.00 0.000072 0.00046 8.48 xlO 10 28 150 U l e i n e (103) Guatambuine (104) 9-MeO-O l i v a c i n e (111) 1.86 x 10 6.819 x 10 5.386 x 10 28 20 70 0.0006 0.0016 0.0044 0.000022 0.00008 0.000064 i I -58-d e t e r m i n e d t o show t h e i r e f f e c t i v e use as p r e c u r s o r s . Conse-q u e n t l y i t was d e c i d e d t o grow t i s s u e c u l t u r e s o f A. p y r i c o l l u m and A. v a r g a s i i i n the hope t h a t more s i g n i f i c a n t i n f o r m a t i o n c o n c e r n i n g the b i o s y n t h e s i s o f a l k a l o i d s (102)-(10 6) and (111) c o u l d be o b t a i n e d . I f p r o p e r growth c o n d i t i o n s f o r such t i s s u e c u l t u r e s were e f f e c t e d , then s t u d y o f a l k a l o i d b i o s y n t h e s i s c o u l d be d e t e r m i n e d a t the c e l l - f r e e l e v e l . P e r m e a b i l i t y problems would h o p e f u l l y be d i m i n i s h e d and the r o l e o f t r y p t o p h a n ( l b ) and stemmadenine (63) c o u l d be r e - i n v e s t i g a t e d . A programme was thus i n i t i a t e d t o grow t i s s u e c u l t u r e s o f A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i . C o n s i d e r a b l e e f f o r t was e x p e n d e d 1 2 ^ * 3 t o t r y and d e t e r m i n e c o n d i t i o n s f o r t h e i r growth on e i t h e r s o l i d (agar) o r l i q u i d media and were i n i t i a l l y u n s u c c e s s -f u l i n b o t h media. R e c e n t l y c o n d i t i o n s f o r the v e r y s low growth o f t i s s u e c u l -t u r e s o f t h e s e p l a n t s have been o b t a i n e d u s i n g a medium w h i c h was a l s o s u c c e s s f u l f o r C. r o s e u s ( d i s c u s s e d l a t e r ) . I n a l l a t t e m p t s to" grow the c u l t u r e s , s m a l l s e c t i o n s ( s e v e r a l hundred) of stem, l e a f o r l e a f base were t a k e n and s t e r i l i s e d by p l a c i n g i n (1) e t h a n o l (2) 0.1% m e r c u r i c c h l o r i d e i n w a t e r (w/w) f o r 2 m i n u tes and (3) s t e r i l i s e d w a t e r . The s e c t i o n s were then p l a c e d on s o l i d agar medium i n an a t -129 tempt t o grow c a l l u s c u l t u r e s . I n the medium d e s c r i b e d , growth c o n d i t i o n s f o r e x t r e m e l y slow growth o f t h e s e c u l t u r e s have been o b t a i n e d . (Table 8) No c o n d i t i o n s f o r growth o f c u l t u r e s on l i q u i d media c o u l d be e f f e c t e d f o r A. a u s t r a l e , A. p y r i c o l l u m and A. v a r g a s i i . -59-T a b l e 8 - C o m p o s i t i o n o f n u t r i e n t medium f o r s o l i d (agar) medium. KN0 3 MgSO 4-7H 20 N H 4 H 2 P 0 4 C a C l 2 - 2 H 2 0 F e S 0 4-7H 20 MnS0 4-H 20 I n o r g a n i c N u t r i e n t s mg/1 2,500 400 300 200 15 10 H 3 B O 3 ZnS0 4• KI CuS0 4< NaMoO 7H 20 5H 20 l-2H 20 C o C l 2 * 6 H 2 0 mg/1 5 1 1 0.2 0.1 0.1 O r g a n i c N u t r i e n t s I n o s i t o l N a 2 EDTA Thiamine-HC1 1,000 20 5 N i c o t i n i c A c i d P y r i d o x i n e HC1 5 0.5 Growth R e g u l a t o r s 2,4 D P-CPA 1 K i n e t i n 0.5 2.0 0.1 Sucrose Agar 30' P-CPA = P-chlorophenoxy a c e t i c a c i d 2 gm/1 -60-U n f o r t u n a t e l y <200 mg. o f c a l l u s c u l t u r e (dry w e i g h t ) were o b t a i n e d o v e r a s i x month growth p e r i o d and c o n s e q u e n t l y t h e s e c o u l d n o t be examined f o r t h e i r a l k a l o i d a l c o n t e n t . Thus, t o d a t e , the b i o s y n t h e s i s o f a l k a l o i d s (102,107,111) a t the c e l l - f r e e l e v e l has n o t been demonstrated. I f however the growth c o n d i t i o n s a r e c o r r e c t f o r a l k a l o i d b i o s y n t h e s i s , then the r o l e o f stemmadenine (63) and t r y p t o p h a n ( l b ) can be r e - e v a l u a t e d and, i f th e s e i n t e r m e d i a t e s are a g a i n n o t i n v o l v e d , t h e n a r e - a p p r a i s a l o f the b i o s y n t h e t i c t h e o r i e s f o r the f o r m a t i o n o f t h e s e a l k a l o i d s w i l l be n e c e s s a r y . F o l l o w i n g t h e e x p e r i m e n t a l , the n e x t s e c t i o n o f the t h e s i s c o n c e r n s i t s e l f w i t h the most r e c e n t i n v e s t i g a t i o n s d i r e c t e d towards t h e s y n t h e s e s o f a p p r o p r i a t e l y r a d i o - l a b e l l e d forms o f ' stemmadenine (63) f o r t h e s e f u t u r e i n v e s t i g a t i o n s . -61-EXP E RIMENTAL - PART I M e l t i n g p o i n t s were de t e r m i n e d on a K o f l e r b l o c k and are un-c o r r e c t e d . A l l u l t r a v i o l e t (UV.) s p e c t r a were r e c o r d e d i n methanol o r e t h a n o l u s i n g a Cary 15 r e c o r d i n g s p e c t r o p h o t o m e t e r . The i n f r a r e d (IR.) s p e c t r a were r e c o r d e d w i t h a P e r k i n - E l m e r Model 457 s p e c t r o p h o t o m e t e r e i t h e r i n c h l o r o f o r m s o l u t i o n ( c a v i t y c e l l s 0.1 mm.) o r as a n u j o l m u l l between sodium c h l o r i d e p l a t e s as i n d i c a t e d . A l l measurements were made i n cm ^ and c a l -i b r a t i o n o f the s n e c t r a was a c h i e v e d u s i n g the 1601 cm ^ abs o r p -t i o n band o f p o l y s t y r e n e . N u c l e a r m agnetic resonance s p e c t r a (N.M.R.) were o b t a i n e d i n d e u t e r o c h l o r o f o r m s o l u t i o n s ( u n l e s s o t h e r w i s e i n d i c a t e d ) a t 100 MHz on the V a r i a n HA-100, V a r i a n XL-100 o r B r u k e r TT-23 n u c l e a r magnetic resonance s p e c t r o m e t e r s . A l l N.M.R. s p e c t r a o b t a i n e d v i a t h e F o u r i e r T r a n s f o r m t e c h n i q u e (F.T.) w i l l be so noted and were o b t a i n e d w i t h the V a r i a n XL-100 o r B r u k e r TT-2 3 i n s t r u m e n t s . C h e m i c a l s h i f t s were g i v e n i n (ppm) w i t h r e f e r e n c e t o t e t r a m e t h y l s i l a n e as the i n t e r n a l s t a n d a r d . The m u l t i p l i c i t y , i n t e g r a t e d a r e a s , and p r o t o n assignments are g i v e n i n p a r e n t h e s e s . Mass s p e c t r a were d e t e r m i n e d on AEI MS-9, AEI MS-5 0 o r V a r i a n Mat CH-4B mass s p e c t r o m e t e r s , w i t h h i g h r e s o l u -t i o n mass s p e c t r a b e i n g d e t e r m i n e d w i t h the f i r s t two. Woelm n e u t r a l a l u m i n a and EM Reagents GF254 s i l i c a g e l were used f o r t h i n and o r e p a r a t i v e l a y e r chromatography. Woelm n e u t r a l a lumina (Ac t . I l l ) and Merck s i l i c a g e l ( A c t . 2-3) were used f o r column chromatography. A l l r e a c t i o n s d e s c r i b e d h e r e i n , u n l e s s o t h e r w i s e i n d i c a t e d , were performed under an atmosphere o f n i t r o g e n . E l e m e n t a l a n a l y s e s were c a r r i e d out by Mr. P. Borda, M i c r o a n a l y s t , - 6 2 -U n i v e r s i t y o f B r i t i s h C o lumbia. R a d i o a c t i v i t y was measured w i t h a N u c l e a r - C h i c a g o Mark I I l i q u i d s c i n t i l l a t i o n c o u n t e r i n counts p e r minute (cpm). The r a d i o a c t i v i t y o f the sample i n d i s i n t e g r a t i o n s p e r minute (dpm) was s u b s e q u e n t l y d e t e r m i n e d by the e x t e r n a l s t a n d a r d t e c h n i q u e u s i n g a b u i l t - i n Barium-133 s o u r c e o f gamma r a d i a t i o n . S c i n -t i l l a t i o n c o c k t a i l s used were e i t h e r a p r e p a r e d s o l u t i o n o f t o l u e n e and a N u c l e a r - C h i c a g o PPO-POPOP c o n c e n t r a t e o r a pre m i x e d s o l u t i o n o f N u c l e a r - C h i c a g o PCS c o c k t a i l . A s c i n t i l l a t i o n c o u n t i n g sam-p l e c o n s i s t e d o f a s o l u t i o n o f the sample d i s s o l v e d i n 1 m l . o f methanol and 14 ml. o f t h e a p p r o p r i a t e c o c k t a i l ( t o t a l volume 15 m l . ) . F o r each sample the background o f t h e v i a l was p r e -d e t e r m i n e d and s u b s e q u e n t l y s u b t r a c t e d from the measured dpm's f o r the sample i n subsequent c a l c u l a t i o n s . Each sample was counted f o r a time p e r i o d l o n g enough f o r the t o t a l c o u n t s f o r t h e sample, l e s s t h e t o t a l c o u n t s f o r t h e background t o exceed t e n thousand c o u n t s . The A. v a r g a s i i p l a n t s used i n t h i s s t u d y were grown i n the H o r t i c u l t u r e Department greenhouse, t h e U n i v e r s i t y o f B r i t i s h C o l umbia. I n t h i s s e c t i o n o n l y f u r t h e r c h a r a c t e r i s a t i o n d a t a f o r the a l k a l o i d s i s o l a t e d were g i v e n w i t h the e x c e p t i o n o f m e l t i n g p o i n t s . -63-A d m i n i s t r a t i o n o f (Ar^H) t r y p t o p h a n ( l b ) t o A. v a r g a s i i To A. v a r g a s i i r o o t c l i p p i n g s (36 g , wet w e i g h t ) w e r e added (Ar 3H) t r y p t o p h a n ( l b ) (3.21 mg , 2.50 x 1 0 1 1 dpm/mmol) dis-s o l v e d i n a w a t e r / a c e t i c a c i d s o l u t i o n (20:0.5 ml ). The r o o t s v/ere i n c u b a t e d w i t h t h i s s o l u t i o n f o r n i n e days a f t e r w h i c h they were removed and c o p i o u s l y washed w i t h w a t e r . The r o o t s were s i m u l t a n e o u s l y p u l p e d and e x t r a c t e d w i t h methanol - t h i s b e i n g e f f e c t e d by suspending the r o o t s i n methanol i n a b l e n d e r . The s o l i d p l a n t r e s i d u e was then removed by s u c t i o n f i l t r a t i o n . The r e s i d u e was the n r e - e x t r a c t e d t w i c e i n an i d e n t i c a l manner. The combined methanol e x t r a c t s were evapor-a t e d t o d r y n e s s . The methanol e x t r a c t was then suspended i n 10% h y d r o c h l o r i c a c i d (300 ml ) f o r 45 min ( i n an atmosphere o f n i t r o g e n ) . The aqueous e x t r a c t was the n e x t r a c t e d (1) w i t h p e t r o l e u m e t h e r (30-60°C) (3x300 ml ) and (2) c h l o r o f o r m (3x300 ml ). The c h l o r o f o r m e x t r a c t was d r i e d o v e r anhydrous sodium s u l -p h a t e , f i l t e r e d , and e v a p o r a t e d t o dryn e s s (0.1224 g ) . (Extract 1). The r e s u l t i n g a c i d l a y e r was then c o o l e d t o 5° and t r e a t e d s l o w l y w i t h c o n c e n t r a t e d ammonium h y d r o x i d e s o l u t i o n u n t i l pH 11. E x t r a c t i o n o f the b a s i c l a y e r w i t h c h l o r o f o r m gave, a f t e r d r y i n g , the c o r r e s p o n d i n g c h l o r o f o r m s o l u b l e s (0.390 g ) . (Extract 2). The c h l o r o f o r m e x t r a c t 1 was chromatographed on alumina (Act I I I ) u s i n g a c h l o r o f o r m / e t h y l a c e t a t e g r a d i e n t . A p p a r i c i n e (102), (16 mg , c o l o u r l e s s n e e d l e s , m.pt. 191°, l i f 1 . 2 7 1 9 2 - 4 0 ), u l e i n e (103) (28 mg , c o l o u r l e s s c r y s t a l s , m.pt. 75-100°, - 6 4 -lit."'79-100° , (±) guatambuine (104) (12 mg , c o l o u r l e s s cubes, 12 6 m.pt. 249° l i t . 250-52°), were thus o b t a i n e d . These a l k a l o i d s i s o l a t e d were f u r t h e r checked by comparison of t h e i r TLC, NMR, IR, UV and MS b e h a v i o u r with that of authentic samples. The c h l o r o f o r m e x t r a c t 2 was chromatographed on alumina (Act I I I ) u s i n g a c h l o r o f o r m / e t h y l a c e t a t e g r a d i e n t . The 9 - m e t h o x y - o l i v a c i n e (111) o b t a i n e d (46 mg , y e l l o w n e e d l e s , m.pt. 126 289-91° , l i t . 293-4°), was i d e n t i c a l i n a l l r e s p e c t s w i t h an a u t h e n t i c sample, (e.g. TLC, UV, I R ) . NMR: $ (CDC1 ) 100 MHz (FT): 8.72 ( s , l H , NH) , 8.40 (d, IH, J=6Hz, C-3H), 7.95 (m, IH, C-12 H) , 7.76 (d, 1H, J=6Hz, C-14 H), 7.70 (s , IH, C-19 H), 7.43 (d, IH, J=8Hz, C-9 H), 7.28 (d, IH, J=3 and 8Hz, C-10 H) , 4.00 ( s , 3H, OCH_3) , 3.15 (s, 3H, C-18 H_3) , 2.80 ( s , 3H, C-17 H ). M.S. m/e: 276 (M +)/ 261, 245. H.R.M.S.: C a l c - f o r C 1 8H 1 6N 20=276.1623 Found=276.1618 A d m i n i s t r a t i o n o f (Ar JH) stemmadenine (63) t o A. v a r g a s s i 3 10 (Ar H) stemmadenine (63) (3.64 x 10 dpm/m mol ) was d i s -s o l v e d i n methanol/water (1:4, 5 ml ) and a d m i n i s t e r e d t o A.  v a r g a s i i p l a n t s o v e r 7, 13 and 28 day p e r i o d s r e s p e c t i v e l y u s i n g t h e c o t t o n w i c k t e c h n i q u e . T h i s p r o c e d u r e r e q u i r e s b o r i n g a s m a l l h o l e i n the p l a n t -65-stem about 2 " above the s o i l . One end of a moist cotton wick was then placed i n the hole and the other end into a test-tube containing the precursor i n solution. In this way the precursor was introduced into the plant. The alkaloids were then i s o l a t e d i n a manner analagous to that already described. -66-DISCUS'SION ~ PART I I In view of comments made e a r l i e r i t i s o b v i o u s t h a t a stemmade-n i n e (63) type i n t e r m e d i a t e i s c o n s i d e r e d t o be o f paramount im-p o r t a n c e i n the a l k a l o i d b i o s y n t h e s i s so f a r d i s c u s s e d . Stemmadenine (63) was f i r s t i s o l a t e d from Stemmadenia d o n n e l l s m i t h i i (Rose) V7oodson i n 19 5 8 " ^ a n d was l a t e r shown t o po s s e s s 131 g r o s s s t r u c t u r e as shown . Only c o n f i g u r a t i o n s a t C-15 and 131a 13 ^  C-19 were determined u n t i l r e c e n t l y *. ( D i s c u s s e d l a t e r ) However, of the n o n - t r y p t o p h a n a l k a l o i d s (102-106, 111), 3 o n l y a p p a r i c i n e (10 2) gave p o s i t i v e i n c o r p o r a t i o n w i t h (Ar H) stemmadenine (63) p l a n t f e e d i n g e x p e r i m e n t s ? 5 F u r t h e r s i n c e o n l y t h e a r o m a t i c r i n g was l a b e l l e d the b i o s y n t h e t i c i n f o r m a t i o n from t h i s i n c o r p o r a t i o n was l i m i t e d . I n o r d e r t o demonstrate t h a t (63) i s a p r e c u r s o r o f the a l -k a l o i d s (102-106, 111) the f o l l o w i n g must be det e r m i n e d i n v i v o  (a) f a t e of carbon atoms 5 and 6 o f stemmadenine (6 3) - i n p a r -t i c u l a r was C-5 l o s t i n the f o r m a t i o n o f a p p a r i c i n e (102) as a l -ready sugges t e r r o r t r y p t o p h a n ( l b ) ? F u r t h e r , i f carbons 5 and 6 from stemmadenine (63) were m a i n t a i n e d i n (103-106, 111) t h e i r po-s i t i o n s must be de t e r m i n e d by d e g r a d a t i o n p r o c e d u r e s . -67-(b) f a t e o f carboinethoxy and hydroxymethy 1 groups of stemmadenine (63) -103-5,122 as suggested from the s e c o d i n e (91) f e e d i n g e x p e r i m e n t s , i n c o r p o r -a t i o n i n t o a p p a r i c i n e (102) o c c u r r e d w i t h the carbomethoxy group b e i n g r e t a i n e d . I t was o f c o n s i d e r a b l e i n t e r e s t t o determine whether a s i m i l a r p r o c e s s o c c u r r e d w i t h stemmadenine (63). (c) f a t e of C-18 m e t h y l group of stemmadenine (63) - assuming i n -c o r p o r a t i o n s c o u l d be o b t a i n e d , a p p r o p r i a t e d e g r a d a t i o n e x p e r i -ments t o show p o s i t i o n of the l a b e l must be pursued. (d) f a t e o f C-3 methylene o f stemmadenine (63) - a g a i n i n c o r p o r a -t i o n i n t o a l k a l o i d s must be s u p p o r t e d by d e g r a d a t i o n o f the i s o -l a t e d a l k a l o i d t o v e r i f y the p o s i t i o n o f the l a b e l . Thus, i n o r d e r t o m o n i t o r the f a t e o f t h e s e atoms, i t was n e c e s s a r y t o i n v e s t i g a t e p o s s i b l e s y n t h e s e s of (63) p o s s e s s i n g r a d i o a c t i v e l a b e l s a t (1) C,. and Cg (2) C-16 f u n c t i o n a l i t i e s (carbomethoxy and hydroxymethy1 groups) (3) C-18 and (4) C-3. I f t h e s e c o u l d be r e a l i s e d then subsequent f e e d i n g e x p e r i m e n t s and complementary d e g r a d a t i o n s t u d i e s would g i v e much i n f o r m a t i o n t o -wards t h e u n d e r s t a n d i n g o f t h e s e l a t e r b i o s y n t h e t i c r e a r r a n g e -ments (assuming r e a s o n a b l e i n c o r p o r a t i o n s ) . I t s h o u l d be noted t h a t i n any s y n t h e s i s o f a b i o s y n t h e t i -c a l l y i n t e r e s t i n g r a d i o l a b e l l e d p r e c u r s o r i t i s h i g h l y d e s i r a b l e t o l a b e l the m o l e c u l e a t a v e r y l a t e s t a g e i n the s y n t h e s i s , and, as w i l l be r e a l i s e d , t h i s p o i n t r e c e i v e d f u l l c o n s i d e r a t i o n i n the s t u d i e s d i s c u s s e d l a t e r . Stemmadenine ( 6 3 ) , r e c e i v e d s y n t h e t i c i n t e r e s t p r i o r t o t h e s e s t u d i e s and comment on t h e s e i n v e s t i g a t i o n s seems mandatory. -68-Ban a c c o m p l i s h e d the f o l l o w i n g t r a n s f o r m a t i o n (112-113) w h i c h was t h e r e b y c o n s i d e r e d a f e a s i b l e e n t r y i n t o the aspidosperma and s t r y c h n o s a l k a l o i d s . I t had a l s o been n o t e d t h a t t r e a t m e n t o f preakuammicine ( 7 7 ) , i s o l a t e d from C. r o s e u s , w i t h b o r o h y d r i d e i n methanol v i e l d e d 8 5 stemmadenine (63) and akuammicine (64 ) . 7 7 A t t e m p t s t o s y n t h e s i s e 19,20-dihydrostemmadenine (116) i n t h i s 13 4 l a b o r a t o r y by c y c l i z a t i o n o f i n t e r m e d i a t e (115), o b t a i n e d from 18,19 d i h y d r o c o r y n a n t h e i n e aldehyde (114), i n a s i m i l a r manner were u n s u c c e s s f u l . ( F i g u r e 22) F a i l u r e o f t h i s c y c l i z a t i o n has 135 a l s o been r e p o r t e d by W i n t e r f e l d t From a b i o s y n t h e t i c s t a n d p o i n t t h i s approach was o f l i t t l e i n t e r e s t s i n c e i t c o u l d n o t r e a d i l y be adapted f o r our l a b e l l i n g p u r s u i t s . -69-H 1 1 6 F i g u r e 22 - Kutney's a t t e m p t e d s y n t h e s i s o f 19,20-dihydrostemmadenine (116). C o n s e q u e n t l y , s t r u c t u r a l s i m i l a r i t i e s between stemmadenine (63) and s t r y c h n i n e (5) were t a k e n i n t o a ccount and i t was hoped t h a t t h e s e c o u l d be e x p l o i t e d . T h i s approach was l i m i t e d i n the sense t h a t C,. and Cg o f (63) would be p r e f o r m e d and thus l a b e l l -i n g t h e s e p o s i t i o n s would be e x t r e m e l y d i f f i c u l t . - 7 0 -I n t h i s r e s p e c t a n o t h e r s y n t h e s i s o f a stemmadenine type m o l e c u l e (118) had r e c e n t l y been reported"*" 3^ and, h e r e , a d d i t i o n o f the C 5 - C 5 bond o c c u r r e d i n the f i n a l s t a g e s . T h i s approach would p r o b a b l y have t o be d r a s t i c a l l y m o d i f i e d t o y i e l d stemmadenine (63) and w h i l e o f i n t e r e s t was g i v e n no f u r t h e r c o n s i d e r a t i o n . ( F i g u r e 23) 1 1 8 F i g u r e 2 3 - S n i e c k u s ' s y n t h e s i s o f a stemmadenine model. -71-F i g u r e 24 - Kutney's a t t e m p t e d s y n t h e s i s o f stemmadenine ( 6 3 ) . -72-Recent s t u d i e s , i n t h i s l a b o r a t o r y towards the s y n t h e s i s 13 8 o f stemmadenine ( 6 3 ) , used n o r - f l u o r o - c u r a r i n e (119) , d e r i v e d from s t r y c h n i n e ( 5 ) . Each r o u t e a t t e m p t e d ( f i g u r e 24) had l i t e r a t u r e precedence i n s i m p l e r systems and d e s e r v e s b r i e f comment. Pathway ( a ) : c o n v e r s i o n of <xl ^ - u n s a t u r a t e d a l d e h y d e s t o 139 t h e i r c o r r e s p o n d i n g e s t e r s has been w e l l documented. C o n d i -t i o n s f o r t h i s t r a n s f o r m a t i o n t o akuammicine (64) c o u l d n o t be o b t a i n e d . Pathways '(b) and (c) : l i t e r a t u r e p r ecedences had e s t a b l i s h e d t h a t the i n d o l e (12 7) was o b t a i n e d from descarbomethoxyakuammi-140 c i n e (126) by t r e a t m e n t w i t h b o r o h y d r i d e i n me t h a n o l . ( F i g u r e 25) 1 2 6 1 2 7 F i g u r e 2 5 - Base c a t a l y s e d r i n g o p e n i n g o f descarbomethoxyakuammicine (126). I n t h i s r e s p e c t , t r e a t m e n t o f (119) w i t h sodium b o r o h y d r i d e i n 137 methanol gave an i n d o l i c compound s u g g e s t e d t o be (128). 1 2 8 -73-I t was hooed t h a t the r i n g o p e n i n g t o t h e i n d o l e c o u l d be e f f e c t e d k e e p i n g the ald e h y d e f u n c t i o n i n t a c t t o g i v e (124) by e i t h e r k e t a l i s a t i o n o r f o r m a t i o n o f the e n o l a c e t a t e p r i o r t o r i n g o p e n i n g ( f i g u r e 24, pathway b , c ) . The aldehyde (124) was r e q u i r e d s i n c e t h i s f u n c t i o n a l i t y i n l a t e r a l k y l a t i o n s t u d i e s w o u l d be o f g r e a t i m p o r t a n c e . C o n d i t i o n s , however, c o u l d n o t be found f o r the f o r m a t i o n o f e i t h e r the e n o l 137 a c e t a t e o r k e t a l and t h i s approach was d i s c o n t i n u e d . I t had a l s o been shown t h a t e n o l a t e a n i o n s are not r e d u c e d 141 by b o r o h y d r i d e ' and i t was c o n s i d e r e d t h a t t h i s o b s e r v a t i o n c o u l d be used t o o b t a i n (123) - however, t r e a t m e n t of e n o l a t e a n i o n (121) w i t h b o r o h y d r i d e gave no r e a c t i o n and pathway c was 137 abandoned. Pathway d w h i c h i n v o l v e d t r e a t m e n t o f p y r r o l i d i n e o r c y c l o -h e x y l d i e n a m i n e (125), w i t h m e t h y l c h l o r o f o r m a t e under e q u i l i b r i u m c o n d i t i o n s f a i l e d t o g i v e any p r o d u c t s o t h e r than s t a r t i n g ma-137 t e r i a l and was a l s o s u b s e q u e n t l y abandoned. A t t e n t i o n was t h e n f o c u s s e d onto the c o r r e s p o n d i n g e s t e r (132) s i n c e a r o u t e from W i e l a n d - G u m l i c h a l d e h y d e (129), p r e -142 v i o u s l y d e r i v e d from s t r y c h n i n e (5),had been r e p o r t e d i n 43% crud e y i e l d from oxime (130). E s t e r (132) v/as c o n s i d e r e d as an i m p o r t a n t i n t e r m e d i a t e f o r f u r t h e r s y n t h e t i c a t t e m p t s t o y i e l d stemmadenine ( 6 3 ) . Two l i t e r a t u r e p r e c e d e n t s ^ " 3 8 * 3 ' ± 4 3 s p e c i f i c a l l y a p p l i c a b l e t o t h e s t r y c h n o s system, were a v a i l a b l e f o r removal o f a l l y l i c h y d r o x y l g r o u o s . These were (1) h y d r o g e n o l y s i s 1 3 ^ w i t h H2/Pd/C -74-1 2 9 H 2 N O H - H C I l ) B a ( O H ) 2 ) H C I M e O H C 0 2 C H 3 Ac OyPyridine O C O C H -1 3 2 P d C 131 C 0 2 C H 3 1 3 4 + C 0 2 C H 3 1 3 3 F i g u r e 26 - S y n t h e s i s o f 2,16-dihydroakuammicine (133) and (2) t r e a t m e n t w i t h HBr/HOAc t o g i v e the bromide f o l l o w e d by 143 r e d u c t i v e removal w i t h Zn/HOAc. -75-Procedure (1) was r e p o r t e d 1 J ' to give ester (133) together with an undetermined quantity of dihydroester (134) as an im-purity which could not be removed. Procedure (2) was thus u t i l i z e d y i e l d i n g required ester i n 65% y i e l d . With ester (133) i n hand, attempts to y i e l d stemmadenine (63) were as follows: (Figure 27) Figure 27 - Kutney's synthesis of epi-stemmadenine (138). -76-F i g u r e 27, Pathway (a) F o r m a t i o n of akuammicine (64) was r e p o r t e d by t r e a t m e n t o f N - t o s y l d e r i v a t i v e (140) o f (133) w i t h p o t a s s i u m - t - b u t o x i d e i n 144 d i m e t h y l s u l f o x i d e ' f o r m a t i o n . No y i e l d s were g i v e n f o r t h i s t r a n s -6 4 C 0 2 C H 3 However i t had a l s o been n o t e d t h a t l e a d t e t r a a c e t a t e c o n v e r t e d 145 i n d o l i n e s d i r e c t l y t o t h e i r c o r r e s p o n d i n g m d o l e n i n e s . Con-s e q u e n t l y akuammicine (64) was formed u s i n g the l a t t e r method. F u r t h e r t r e a t m e n t o f (64) w i t h (1) a c e t i c a c i d and (2) sodium 132 137 b o r o h y d r i d e gave i n d o l e e s t e r (122). ' An a t t e m p t t o a l k y l a t e (122) t o g i v e stemmadenine (63) o r 137 i t s C-16 epimer was u n s u c c e s s f u l . F i g u r e 27, Pathway (b) f a r e d b e t t e r and was e x e c u t e d as shown t o g i v e a compound r e p o r t e d t o be (138) - b u t w h i c h was n o t stemmade-n i n e (63). U n f o r t u n a t e l y , the p u r i f i c a t i o n p r o c e s s e s employed were  not a b l e t o y i e l d (138) i n a form w h i c h a l l o w e d p o s i t i v e i d e n t i f i c a -t i o n . C o n s e q u e n t l y i t was r e p o r t e d t h a t (138) and (63) were r e d u c e d t o t h e d i o l (139) h Y t r e a t m e n t w i t h sodium b i s - ( m e t h o x y m e t h y l e n e -oxy ) -a l u m i n i u m h y d r i d e . Comparison o f the IR and NMR s p e c t r a r e -p o r t e d f o r t h e d i o l s (139) showed c l o s e s i m i l a r i t i e s and thus i t was -77-f o r w a r d e d t h a t (138) was the C-16 epimer o f (63) . J" J Z ' x J ' A s t e r e o c h e m i c a l argument was s u b s e q u e n t l y p u r s u e d i n w h i c h i t was s u g g e s t e d t h a t (138) had the f o l l o w i n g s t e r e o c h e m i s t r y thus g i v i n g stemmadenine (63) t h e a b s o l u t e c o n f i g u r a t i o n shown."'' ( D i s c u s s e d l a t e r ) 6 3 ' 3 8 As i s c l e a r l y n o t e d the i n v e s t i g a t i o n s d i s c u s s e d t h r o u g h o u t t h i s t h e s i s are p a r t o f l o n g range s y n t h e t i c and b i o s y n t h e t i c programmes and t h i s s e c t i o n now concerns i t s e l f w i t h the more r e c e n t a t t e m p t s t o s y n t h e s i z e stemmadenine (63) from s t r y c h n i n e ( 5 ) . C o n s e q u e n t l y i n i t i a l i n v e s t i g a t i o n s c o n s i d e r e d a c y l a t i o n o f a l d e -hyde (145), o r an a p p r o p r i a t e l y m o d i f i e d form o f (145), s i n c e i t was hoped t h a t t h i s would g i v e the o p p o s i t e s t e r e o c h e m i s t r y t o t h a t p r e v i o u s l y o b t a i n e d . I f t h i s c o u l d be a c c o m p l i s h e d , then r e d u c t i o n o f t h e al d e h y d e and s i m i l a r m a n i p u l a t i o n t o t h a t a l r e a d y ]32 13 7 d e s c r i b e d ' ' would y i e l d stemmadenine (63) . The c o n v e r s i o n o f s t r y c h n i n e (5) t o 2j5r 16 U. - c u r - 1 9 - e n - 1 7 - o l (143) as shown i n f i g u r e 28 i s w e l l documented. The i n i t i a l s t e p s t o W i e l a n d - G u m l i c h aldehyde (129) (WGA) were f i r s t r e p o r t e d i n 1932" 1" 4 6, improved i n 1 9 5 8 1 4 7 , and o p t i m i s e d i n 1 9 6 9 1 4 8 . As can be seen t r e a t m e n t o f s t r y c h n i n e (5) w i t h sodium e t h o x i d e -78-F i g u r e 2 8 - D e g r a d a t i o n o f s t r y c h n i n e (5) t o 2B, 1 6 o ( - c u r - l 9 - e n - 1 7 - o l (143) . 1 2 9 o = s I 1 4 4 C I F i g u r e 2 9 - F o r m a t i o n o f W i e l a n d - G u m l i c h aldehyde (12 9 ) . -79-and i s o a m y l n i t r i t e gave isonitrosostrychnine (141) which was i s -olated i n high o v e r a l l y i e l d as the corresponding hydrochloride s a l t . Treatment of this s a l t with thio n y l chloride followed by quenching i n ice gave a crude p r e c i p i t a t e of 1-cyanoformy1-WGA (144) which a f t e r r e c r y s t a l l i s a t i o n from methanol exhibited phy-14 8 s i c a l data previously reported. The compound (144) was then subjected to steam d i s t i l l a t i o n at pH 3.3-3.5 to give the required hemi-acetal WGA (129) and hydrogen cyanide. Treatment of WGA (12 9) v/ith sodium borohydride afforded 2j$, -147 16o(-cur-19-en-17,18-diol i n good y i e l d . This d i o l (142) can be converted to 2j$, ,16o(-cur-19-en-17-ol (143) i n either of two ways: (1) Bernauer had reported that treatment of d i o l (142) v/ith HBr/HOAc afforded the a l l y l i c bromide shown below. The bro-mide was then treated with zinc dust i n acetic acid to give the acetate of alcohol (143), which on hydrolysis with methanolic potassium hydroxide afforded required alcohol (143). 142 H B r H O A c p 1 4 3 (2) Hydrogenolysis using a spe c i a l palladium on charcoal catalyst prepared by Wieland" 1" 3 8^. Either method could thus be used for the introduction of a t r i t i u m l a b e l at this p o s i t i o n (C-18). Following the l a b e l l i n g , checks could be made to ensure that no random l a b e l l i n g had occurred elsev/here in the molecule (e.g. by ozonisation) . - 8 0 -Th e h y d r o g e n o l y s i s was the p r o c e d u r e o f c h o i c e i n t h e s t u d i e s employed s i n c e i t gave (143) i n 25-30% h i g h e r y i e l d . W i t h a l c o h o l (143) on hand, t h r e e pathways were c o n s i d e r e d t o g i v e stemmadenine (63) ( F i g u r e 3 0 ) : (1) f o r m a t i o n o f 1 - f o r m y l aldehyde (146), c o n d e n s a t i o n w i t h c y c l o h e x y l a m i n e t o g i v e (147), and t r e a t m e n t w i t h base and methy1-c h l o r o f o r m a t e t o y i e l d i n t e r m e d i a t e (148). F u r t h e r m i l d a c i d h y d r o l y s i s and b o r o h y d r i d e r e d u c t i o n , removal of f o r m y l group, would y i e l d t he r e q u i r e d (149), the e x p e c t e d C-16 epimer o f (137). (2) d i r e c t a c y l a t i o n of 1- f o r m y l aldehyde (146), r e d u c t i o n o f al d e h y d e t o a l c o h o l and removal of f o r m y l group t o y i e l d (149). (3) c o n v e r s i o n t o n o r - f l u o r o c u r a r i n e (119), r i n g - o p e n i n g and r e d u c t i o n t o a l c o h o l (128), o x i d a t i o n t o aldehyde (124), f o l l o w e d by a c y l a t i o n and r e d u c t i o n t o (63). The c o n v e r s i o n o f (143) t o (145) r e q u i r e s some comment s i n c e 137 d i f f i c u l t i e s had been e n c o u n t e r e d i n t h e s e l a b o r a t o r i e s and 138 e l s e w h e r e t o o b t a i n (145) i n good y i e l d . A t b e s t , t r e a t m e n t o f (143) under anhydrous, a n a e r o b i c c o n d i t i o n s w i t h p o t a s s i u m t - b u t o x i d e and benzophenone i n r e f l u x i n g benzene gave a m i x t u r e o f (145) and (119) i n 39 and 10% r e s p e c t i v e l y . More commonly, however, f u r t h e r o x i d a t i o n t o (119) was n o t e d as t h e major p r o d u c t . Replacement o f the base employed by sodium h y d r i d e y i e l d e d r e q u i r e d a l d e h y d e (145) i n 73% w i t h no measurable q u a n t i t y o f (119) b e i n g o b s e r v e d . Presumably t h i s i s formed v i a the f o l l o w i n g mechanism. ( F i g u r e 31) -81--82-( P h ) C = 0 1 4 5 C H O F i g u r e 31 - M o d i f i e d Oppenauer o x i d a t i o n o f ' 2y0,16od-cur-19-en-17-ol. As n o t e d the i n d o l i n e was p r o t e c t e d i n the n e x t s t e p by a d d i n g the f o r m y l group. T h i s group was chosen due t o the r e l a -t i v e ease w i t h w h i c h i t can be removed ( s t i r r i n g a t room temper-149 a t u r e w i t h sodium h y d r i d e i n THF ). The most e f f i c i e n t documented means of f o r m y l a t i o n o f d i h y d r o -indoles"'' 5^ was w i t h f o r m i c a c e t i c anhydride"'"5"'" and c o n s e q u e n t l y (146) was o b t a i n e d as e v i d e n c e d by absence o f NH a b s o r p t i o n i n the IR spectrum and the p r e s e n c e o f a two c a r b o n y l system a t 1730 c m - 1 and 1670 cm - 1. The U.V. spectrum (^max 287,278,250 nm) 142 was c h a r a c t e r i s t i c o f an N - a c y l i n d o l i n e . The NMR e x h i b i t e d the e x p e c t e d 3 p r o t o n d o u b l e t o f d o u b l e t s a t j l ' 5 2 (J=2 and 6 H z ) f o r =CHCH 3 and a 1 p r o t o n q u a r t e t f o r CHCH 3 a t 5.47 (J=6 H z ) . A more i n t e r e s t i n g f e a t u r e , however, were two s i n g l e t s a t £ 8.58, 8.83 f o r the p r o t o n (N-CHO), and t h r e e s i n g l e t s a t 6 9.58, 9.72, 9.76 f o r the p r o t o n (CHO). I n a d d i t i o n two d o u b l e t s a t cf 4.74, 4.88 were n o t e d f o r c-16 H. A s i m i l a r o b s e r v a t i o n had 152 been n o t e d w i t h N - f o r m y l d i h y d r o i n d o l e s (150) and (151). -83-I n b o t h c a s e s , the N-CHO p r o t o n had e x i s t e d as two s i n g l e t s and t h i s had been a s c r i b e d due t o f r e e r o t a t i o n h i n d r a n c e o f the N-CHO group. 0^0 I C H O 1 5 0 F u r t h e r s u p p o r t t o t h i s r a t i o n a l e was g i v e n by h e a t i n g t o 72-77° where c o l l a p s e t o a s i n g l e t was ob s e r v e d . These r e s u l t s a r e however a l s o c o m p a t i b l e w i t h " l o n e p a i r " i n v e r s i o n i s o m e r i s m . The mass spectrum of (146) a l s o showed major peaks a t m/e 322 (M +),279,172,164 & 109 w h i c h a r e i n agreement w i t h the 153 p r o p o s e d f r a g m e n t a t i o n s o f t h i s d i h y d r o i n d o l i n e system as shown i n f i g u r e 32. Cl e a v a g e , f o l l o w i n g r e t r o D i e l s - A l d e r r e a c t i o n , a t p o s i t i o n s marked , j$ and / gave o b s e r v e d i o n s b, c and d (R=R^"=CH0) a t m/e 16 4, 172, and 109. I n a d d i t i o n h y d r i d e t r a n s f e r and r u p t u r e of r i n g C f o l l o w e d by c l e a v a g e o f the C-15, 16 bond and rearrangement gave i o n e a t m/e 279. C o n v e r s i o n t o the i m i n e (147) ( f i g u r e 30, pathway 1) was r e a d i l y e f f e c t e d by h e a t i n g c y c l o h e x y l a m i n e and (146) t o g e t h e r i n r e f l u x i n g benzene u s i n g a m o d i f i e d Dean-Stark a p p a r a t u s ( i . e . a s o x h l e t c o n t a i n i n g anhydrous m o l e c u l a r s i e v e s ) - t h i s -84-F i g u r e 32 - P r o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f methyl-cur-19-ene f a m i l y . - 8 5 -b e i n g n o t e d by a one c a r b o n y l a b s o r p t i o n i n the IR a t 16 65 cm x , a mass spec, m/e 403 (M +) and the o t h e r peaks f o l l o w e d a s i m i -l a r f r a g m e n t a t i o n p r o c e s s t o t h a t g i v e n i n f i g u r e 32. The r e l e v a n t NMR d a t a f o r t h i s assignment showed a 1 p r o t o n s i n g l e t a t 5 9.02 (NCHO) , an AB q u a r t e t a t £8.69 (J=8 & 15 Hz) f o r CH=N and a <5l.91-l.37, CH2 en v e l o p e (cyclohexane) . The U.'V. remained v i r -t u a l l y unchanged (Amax 288, 282, 252 nm) as e x p e c t e d . H y d r o l y s i s w i t h h y d r o c h l o r i c a c i d (5%) gave (146) i n h i g h y i e l d . However a l l endeavours t o a c y l a t e (147) a t C-16 met w i t h f a i l u r e . These a t t e m p t s i n v o l v e d t r e a t m e n t o f the m o l e c u l e w i t h (1) base and (2) m e t h y l c h l o r o f o r m a t e . A v a r i e t y o f bases (e.g. t - b u t y l l i t h i u m , d i - i s o p r o p y l l i t h i u m amide, p o t a s s i u m and sodium h y d r i d e , e t c . ) , s o l v e n t s (e.g. THF, benzene, DMSO, HMPA) were em-p l o y e d a t v a r y i n g t e m p e r a t u r e s and e i t h e r s t a r t i n g m a t e r i a l was i s o l a t e d o r i n t h e case o f h i g h e r t e m p e r a t u r e s (>30°C) s e v e r e d e c o m p o s i t i o n r e s u l t e d . One p o s s i b l e r a t i o n a l e f o r t h i s f a i l u r e was t h e o b s e r v a t i o n t h a t f o r m a t i o n o f 1 4 7 1 5 2 1 4 8 i n t e r m e d i a t e (152) from (147) r e s u l t s i n an u n f a v o r a b l e s t e r i c i n t e r a c t i o n between the C-18 me t h y l group and the C-17 p r o t o n . Con-- 8 6 -sequently the approach was abandoned. Direct acylation (figure 30, pathway 2) was considered even although i t was appreciated that most conditions would favour 0-acylation. However i t was hoped that a small, but s i g n i f i c a n t quantity of C-acylation would occur. Again a var i e t y of condi-tions were employed, and, again none of the required acylated product could be obtained. It was, however, noted that (146), on treatment with sodium hy i n r e f l u x i n g benzene gave (153) i n very high y i e l d . Formation of (153) at elevated temperatures, can be consi-dered v i a i n i t i a l quaternary s a l t formation followed by nucleo-p h i l i c attach by chlorine. The UV was again c h a r a c t e r i s t i c of a N-formyl dihydroindole ( X f 4 287 , 278, 248 nm.). The IR showed CO absorption at 1668 and 168 8 cm respectively i n agreement with the proposed structure. The NMR spectrum exhi-bited three s i n g l e t s at £9.74, 9.67 and 9.5 for CHO, two singlets at<f 8.84, 8. 6 0 for NCHO, a one proton quartet at 6 5.52 for C-19 H, a doublet of doublets at 6 4.52 (J=10 and 16 Hz) for -87-C-16H, and a t h r e e p r o t o n s i n g l e t a t & 3.74 ( C O ^ C I - ^ ) . The mass spectrum showed an m/e 416 (M +) and e x h i b i t e d major peaks a t 210, 209, 208, 194, 180, 150, ( f i g u r e 12) w h i c h are 1 5 3 i n agreement w i t h the D j e r a s s i p r o p o s a l ( f i g u r e 33). The compound (15 3) was reduced t o the c o r r e s p o n d i n g a l c o -h o l (154) and f o l l o w i n g v e r i f i c a t i o n o f t h i s s t r u c t u r e the approach was d i s c o n t i n u e d . Pathway 3 ( f i g u r e 30) was then n e x t c o n s i d e r e d , and, i n t h i s r e s p e c t , t r e a t m e n t o f (143) w i t h e i t h e r p o t a s s i u m - t -b u t o x i d e o r p o t a s s i u m h y d r i d e i n benzene and n i t r o b e n z e n e gave n o r - f l u o r o c u r a r i n e (119) w h i c h was t h e n c o n v e r t e d t o the c o r r e s p o n d i n g i n d o l e a l c o h o l (128) by t r e a t m e n t w i t h b o r o h y d r i d e 137 i n m e t h a n o l . S e v e r a l a t t e m p t s t o o x i d i s e a l c o h o l (12 8) under a v a r i e t y o f c o n d i t i o n s met w i t h f a i l u r e . I n a l l cases a p l e t h o r a o f p r o d u c t s was o b t a i n e d on a t t e m p t i n g the c o n v e r s i o n and t h e approach was dropped. Thus t h e s e complementary s t u d i e s t o pathways p r e v i o u s l y 137 i n v e s t i g a t e d w i t h aldehydes (119) o r (146) a l s o e n j o y e d f a i l u r e and the u t i l i s a t i o n o f t h e s e s u b s t r a t e s f o r f u r t h e r s y n t h e t i c p u r s u i t s was abandoned. R e - c o n s i d e r a t i o n was t h e r e -f o r e g i v e n t o t h e c o r r e s p o n d i n g i n d o l e (122) s e r i e s , s i n c e i t c o n t a i n e d a more f l e x i b l e n i n e membered r i n g f o r w h i c h the D r e i d i n g model s u g g e s t e d a l k y l a t i o n c o u l d o c c u r t o g i v e ii " e i t h e r epimer - a l t h o u g h t h e °(-face appeared t o be f a v o u r e d f o r a t t a c k . m / e 18 0 F i g u r e 33 - P o s t u l a t e d mass s p e c t r a l f r a g m e n t a t i o n o f a c y l a t e d d e r i v a t i v e (153). - 8 9 -I n i t i a l a l k y l a t i o n o f (122) w i t h formaldehyde had r e c e i v e d 137 p r e l i m i n a r y i n v e s t i g a t i o n w i t h o u t s u c c e s s and t h i s w i l l be mentioned as i t p e r t a i n s t o the d i s c u s s i o n . C o n s e q u e n t l y , p r i o r t o embarking upon a l e n g t h y s y n t h e t i c r o u t e w h i c h p r o m i s e d f a i l u r e , i t was i n i t i a l l y d e c i d e d t o i n v e s -t i g a t e a l k y l a t i o n w i t h a model i n d o l e compound. The model system chosen was t e t r a h y d r o c a r b a z o l e (155) and i t was hoped t h a t c o n d i t i o n s c o u l d be found f o r the f o r m a t i o n o f e s t e r (156) w h i c h t h e n under s u i t a b l e m a n i p u l a t i o n would g i v e the c o r r e s p o n d i n g 1-carbomethoxy-1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157). A l i t e r a t u r e s u r v e y showed t h a t 1 - c a r b o e t h o x y t e t r a h y d r o c a r b a z o l e was p r o v e n by d e h y d r o g e n a t i o n t o the c o r r e s p o n d i n g c a r b a z o l e (156b) system and comparison w i t h an a u t h e n t i c sample. As can be seen from f i g u r e 34, (158) was c o n s i d e r e d t o be formed as an i n t e r m e d i a t e . However c y c l i z a t i o n o f (158) would be e x p e c t e d t o g i v e 4 - c a r b o e t h o x y t e t r a h y d r o c a r b a z o l e (159) and as o n l y m e l t i n g p o i n t , e l e m e n t a l a n a l y s i s and f o r m u l a were r e p o r t e d f o r (158) i t can a l s o be e n v i s a g e d t h a t t h e r e a c t i o n i n f a c t goes v i a an i n t e r m e d i a t e such as (160) - w h i c h would be more i n a c c o r d w i t h t h e d a t a r e p o r t e d . ( F i g u r e 35) (156a) was a v a i l a b l e 154,155 as shown i n f i g u r e 34. T h i s s t r u c t u r e 154 - 9 0 -Figure 34 - Lenzi's synthesis of 1-carboethoxytetrahydrocarbazole (156a Although th i s approach could be readi l y modified for the f o r -mation of (156), i t was considered that a more e f f i c i e n t synthesis d i r e c t l y from tetrahydrocarbazole (155) could be effected. Consequently attempts to introduce a one carbon function at C-l were investigated. I n i t i a l attempts to introduce a n i t r i l e function, v i a the corresponding chloroindolenine ( 1 6 1 ) x 5 6 met with -91-1 5 6 a 1 6 0 F i g u r e 35 - An a l t e r n a t i v e t o L e n z i ' s p o s t u l a t e . f a i l u r e s i n c e (161) r a p i d l y r e a c t e d w i t h the b e n z o t r i a z o l e formed i n the r e a c t i o n t o y i e l d 1 - b e n z o t r i a z o l e - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (162) ( f i g u r e 36) as e v i d e n c e d by the UV h a v i n g the c h a r a c t e r i s t i c 162 F i g u r e 36 - S y n t h e s i s o f 1 - b e n z o t r i a z o l e t e t r a h y d r o c a r b a z o l e (162). -92-i n d o l e chromophore ( J 291, 282, 276, 223). S u p p o r t f o r t h i s Kiel X s t r u c t u r e was a l s o g i v e n by t h e mass spectrum m/e 2 88 (M +) and the IR spectrum w h i c h had NH a b s o r p t i o n a t 3470 cm \ The NMR was a l s o i n d i c a t i v e h a v i n g a s i n g l e t a t i 8.4 (NH) and a one p r o t o n t r i p l e t a t $ 6.36-6.62 (J=5.5 Hz) C - l H . However t h i s p r o b lem was r e s o l v e d by t r e a t i n g 1 - p y r i d i n i u m -15 7 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e bromide (163) w i t h p o t a s s i u m c y -a n i d e i n a c e t o n i t r i l e . The crude p y r i d i n i u m bromide s a l t (163) was formed as a crude c r y s t a l l i n e mass by t r e a t m e n t o f t e t r a h y d r o c a r -b a z o l e (155) w i t h N-bromosuccinimide and b e n z o y l p e r o x i d e i n py-r i d i n e . The p r o p o s e d mechanism f o r the f o r m a t i o n o f the p y r i d i n i u m s a l t (163) i s shown i n f i g u r e 37. Treatment o f t h i s w i t h c y a n i d e gave r e q u i r e d 1 - c y a n o t e t r a h y d r o c a r b a z o l e (164). 155 163 1 6 4 The IR spectrum showed NH and CN a b s o r p t i o n a t 3470 and 2240 cm 1 and the UV spectrum was a g a i n c h a r a c t e r i s t i c o f an i n d o l e (/\ m a x 289 , 281, 273, 223 nm.) . The mass spectrum c o n t a i n e d the e x p e c t e d m/e 19 6 ( M + ) , and the NMR showed a one p r o t o n s i n g l e t a t S 8.12 (NH) and a one p r o t o n t r i p l e t a t S 4.15,(J=5.4 Hz) C - l H . A s m a l l amount (^10%) o f 1 - s u c c i n i m i d o t e t r a h y d r o c a r b a z o l e (165) was a l s o formed i n the r e a c t i o n presumably v i a a s i m i l a r -93-d i s p l a c e m e n t o f t h e p y r i d i n i u m s a l t (163) w i t h a s m a l l amount o f s u c c i n i m i d e . 165 I n t h i s c a s e , the IR spectrum showed NH and C=0 a b s o r p t i o n s a t 3470 and 1705 cm - x. The UV a g a i n showed the p r e s e n c e o f an i n d o l e chromophore ( A 288, 278, 223 nm) and t h e mass spectrum gave v ^max I I I r ? m/e 268 (M ). The NMR spectrum showed a one p r o t o n s i n g l e t a t £7.96 (NH), a one p r o t o n t r i p l e t a t £5.54,(J=7.0 Hz) ( C - l H ) , and a f o u r p r o t o n s i n g l e t a t 52. 72 (N- ( C O C H 2 ) 2 ) • P r i o r t o c o n v e r s i o n o f n i t r i l e (164) t o e s t e r (156) i t was d e c i d e d t o d e t e r m i n e c o n d i t i o n s f o r the C - l a c y l a t i o n o f (164). I n t h i s r e s p e c t , i t was r e a l i s e d t h a t a s u i t a b l e p r o t e c t i n g group f o r the i n d o l e hydrogen o f b o t h (164) and (156) would be r e q u i r e d s i n c e o t h e r w i s e a t t a c k w o u l d be a n t i c i p a t e d t o g i v e p r e f e r e n t i a l l y t h e N - a c y l / a l k y l p r o d u c t . A number o f p r o t e c t i n g groups were c o n s i d e r e d e.g. b e n z y l , m e t h y l , b e n z e n e s u l p h o n y l , carbomethoxy e t c . a n d , o b v i o u s l y , p r e -f e r e n c e was g i v e n t o a group w h i c h would h o p e f u l l y be s t a b l e t o the c o n d i t i o n s used and t h a t c o u l d a l s o r e a d i l y be added o r removed. I n t h i s r e s p e c t t h e carbomethoxy group seemed i d e a l s i n c e i t a p p a r e n t l y f u l f i l l e d a l l o f t h e s e r e q u i r e m e n t s . -94-F i g u r e 37 - F o r m a t i o n o f 1 - p y r i d i n i u m - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e bromide (153). C o n s e q u e n t l y N - c a r b o r n e t h o x y - l - c y a n o t e t r a h y d r o c a r b a z o l e (166) was formed by t r e a t m e n t o f (164) w i t h 1) p o t a s s i u m h y d r i d e and 2) m e t h y l c h l o r o f o r m a t e . ( F i g u r e 38) The UV spectrum was now t h a t o f an N - a c y l i n d o l e ( A m a x 292, 282 256 and 225 nm) and the IR showed CN and C=0 a b s o r p t i o n a t 2260 and 1745 cm 1 . The mass spectrum gave the e x p e c t e d m/e 254 (M +) and t h e NMR showed a one p r o t o n t r i p l e t a t & 4 . 5 , ( J = 4 H z ) , C - l H and a t h r e e p r o t o n s i n g l e t a t £4.09 (CO C H J 1 - c a r b o m e t h o x y - l - c y a n o - N - c a r b o m e t h o x y t e t r a h y d r o c a r b a z o l e (167) was then formed by t r e a t m e n t o f (166) w i t h p o t a s s i u m h y d r i d e and m e t h y l c h l o r o f o r m a t e ( f i g u r e 17). The UV and IR were s i m i l a r t o t h a t d e s c r i b e d above and the mass spectrum gave an m/e 312 ( M + ) . The NMR showed two t h r e e p r o t o n s i n g l e t s a t £3.98 (N-C0 2CH ) and £3.86 ( C - l C02CK_3_) r e s p e c t i v e l y . M i l d a l k a l i n e h y d r o l y s i s w i t h p o t a s s i u m h y d r o x i d e i n methanol a f f o r d e d 1 - c a r b o m e t h o x y - l - c y a n o - t e t r a h y d r o c a r b a z o l e (168). Thus, w i t h c o n d i t i o n s d e t e r m i n e d f o r a c y l a t i o n o f the a n i o n a t C - l of the n i t r i l e ( 1 6 6 ), i t was a n t i c i p a t e d t h a t a l k y l a t i o n o f the c o r r e s p o n d i n g e s t e r (156) s e r i e s i n a s i m i l a r manner would g i v e t h e r e q u i r e d p r o d u c t (157). C o n s e q u e n t l y t r e a t m e n t of (164) w i t h hydrogen c h l o r i d e i n methanol a t room t e m p e r a t u r e f o r 1.5 hours gave th e i n s o l u b l e p r o -d u c t (169) w h i c h on t r e a t m e n t w i t h aqueous m e t h a n o l i c h y d r o c h l o r i c a c i d gave th e r e q u i r e d e s t e r (156) ( f i g u r e 3 9 ) . 1) KB 2) C I C O , C H -96-The IR spectrum showed NH and CO a b s o r p t i o n a t 3470 and 17 30 cm and the UV gave the e x p e c t e d i n d o l e chromophore. The mass spectrum gave e x p e c t e d m/e 229 (M +) and the NMR showed a one p r o t o n s i n g l e t a t £8.34 (NH), a one p r o t o n m u l t i p l e t a t £3.94-3.88 ( C - l H) and a t h r e e p r o t o n s i n g l e t at«S3.80 (C0 2CH 3) . C o n v e r s i o n t o N - c a r b o m e t h o x y - l - c a r b o m e t h o x y t e t r a h y d r o c a r -b a z o l e (170) was e f f e c t e d i n good y i e l d by t r e a t m e n t o f (156) w i t h p o t a s s i u m h y d r i d e and m e t h y l c h l o r o f o r m a t e . ( F i g u r e 3 9 ) . The IR spectrum showed CO a b s o r p t i o n a t 1730 cm and the UV was c h a r a c t e r i s t i c o f an N - a c y 1 - i n d o l e ( A 292 , 278, 261, 227 nm) . J ^' max t i t The mass spectrum gave an m/e .2 87 (M +) and the NMR showed a one p r o t o n t r i p l e t a t £4.26 (J=5 Hz) f o r C - l H, and two t h r e e p r o t o n s i n g l e t s a t £3.98 (N-C02CH_3) and £3.74 (C0 2CH 3) r e s p e c t i v e l y . F u r t h e r t r e a t m e n t o f (170) w i t h p o t a s s i u m h y d r i d e i n t e t r a -h y d r o f u r a n f o r twenty minutes a t 0° . f o l l o w e d by q u e n c h i n g w i t h formaldehyde gave, f o l l o w i n g work-up, the r e q u i r e d 1 - c a r b o m e t h o x y - l -h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (157) ( f i g u r e 39) as e v i d e n c e d by t h e IR spectrum w h i c h had OH, NH and CO a b s o r p t i o n a t 3620, 3460 and 1710 cm r e s p e c t i v e l y . The UV had an i n d o l e chromophore (/J 290, 282, 277, 224) and t h e mass spectrum gave an m/e 259 (M +) . The NMR showed a one p r o t o n s i n g l e t a t £8.64 (NH), a two p r o t o n AB q u a r t e t a t £3.99 (CH_20H) and a t h r e e p r o t o n s i n g l e t a t £ 3 . 74 (C0 2CH_ 3) . S i n c e l o s s o f the 'N-carbomethoxy group had n o t been n o t e d w i t h (170) under i d e n t i c a l work-up c o n d i t i o n s presumably t h i s l o s s can be r a t i o n a l i s e d as b e i n g due t o t h e f o l l o w i n g mechan-ism ( f i g u r e 4 0 ) . A l k y l a t i o n o f (170) gave i n t e r m e d i a t e ( i ) w h i c h -97-c y c l i s e s as shown t o g i v e ( i i ) . Base h y d r o l y s i s on work-up would be e x p e c t e d t o h y d r o l y s e ( i i i ) as shown t o g i v e (157). Thus w i t h c o n d i t i o n s f o r i n t r o d u c t i o n o f the h y d r o x y m e t h y l group a t C - l b e i n g e s t a b l i s h e d i t was now c o n s i d e r e d t h a t t h e s e , o r s i m i l a r c o n d i t i o n s , c o u l d be a p p l i e d t o e s t e r (122), t o g i v e e i t h e r stemmadenine (63) o r i t s epimer (138). P r i o r t o d i s c u s s i o n o f t h e s e i n v e s t i g a t i o n s i t had been mentioned t h a t o x i d a t i v e a t t e m p t s a t c o n v e r s i o n o f (128) t o t h e aldeh y d e (124) had not met w i t h s u c c e s s . I t was thus o f some i n t -e r e s t t o r e - i n v e s t i g a t e a s p e c t s o f t h i s o x i d a t i o n . C o n s e q u e n t l y e s t e r (15 6) was r e d u c e d w i t h l i t h i u m aluminium h y d r i d e t o g i v e 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (171) i n good y i e l d . 171 172 A t t e m p t e d o x i d a t i o n o f 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e (171) t o t h e r e q u i r e d aldehyde (172) a g a i n f a i l e d under a few o x i d a t i v e / ^ 158a _ 158b , , a t t e m p t s (e.g. Oppenauer, C o l l i n s o r Corey p r o c e d u r e s ) as none of the p r o d u c t s o b t a i n e d p o s e s s e d s p e c t r a l c h a r a c t e r i s t i c s o f (172) and the approach was d i s c o n t i n u e d . I t was thus d e c i d e d t o i n v e s t i g a t e the a l k y l a t i o n o f deshydroxymethylstemmadenine (122). -98-1 5 7 , 7 0 F i g u r e 39 - S y n t h e s i s " o f 1-carbomethoxy -1 - h y d r o x y r a e t h y l t e t r a h y d r o c a r b a z o l e (157). F i g u r e 40 - P r o p o s e d mechanism f o r f o r m a t i o n o f 1 - c a r b o m e t h o x y - 1 - h y d r o x y m e t h y l t e t r a h y d r o c a r b a z o l e ( 1 5 7) -99-The c o n v e r s i o n s o f s t r y c h n i n e (5) t o epi-stemmadenine (138) and deshydroxymethylstemmadenine (122) d i s c u s s e d p r e v i o u s l y o c c u r r e d , o v e r a l l , i n low y i e l d . C o n s e q u e n t l y i n a d d i t i o n t o t h e a l k y l a t i o n s t u d y t h a t was t o be i n v e s t i g a t e d i t was a l s o hoped t h a t s u i t a b l e m o d i f i c a t i o n s c o u l d be e f f e c t e d t o improve the s y n t h e -t i c u t i l i t y o f the approach. C o n v e r s i o n t o W i e l a n d - G u m l i c h aldoxime (130) was r e a d i l y 1 5 9 o b t a i n e d a c c o r d i n g t o t h e l i t e r a t u r e p r o c e d u r e and gave the e x p e c t e d l i t e r a t u r e v a l u e s f o r IR, UV, m.pt. The mass spectrum gave an m/e 325(M )and the NMR was i n agreement w i t h t h e s t r u c t u r e . I t was hoped t h a t oxime (130) c o u l d be d i r e c t l y c o n v e r t e d t o the e s t e r (132) v i a a c o n c o m i t a n t d e h y d r a t i o n and h y d r o l y s i s p r o c e s s t h u s e l i m i n a t i n g a number o f s t e p s . T h i s , u n f o r t u n a t e l y , was n o t a c h i e v e d b u t the p r o c e d u r e was m o d i f i e d by d e h y d r a t i n g t h e oxime w i t h sodium f o r m a t e / f o r m i c acid^° T h i s p r o c e d u r e d i d n o t g i v e the e x p e c t e d d i h y d r o i n d o l e n i t r i l e (174) b u t i n s t e a d t h e 1 - f o r m y l n i t r i l e (173) ( f i g u r e 41) as shown by t h e UV (A^ v 287,283,247 nm.), and the mass spectrum m/e 335 ( M + ) . The in 3.x IR showed CN and CO bonds a t 2260 and 1675 cm x r e s p e c t i v e l y and t h e NMR spectrum e x h i b i t e d two s i n g l e t s a t £ 8.9 and 8.76 (N-CHO) , a t r i p l e t a t S 5 . 590, (J=6 Hz) , C-19 H and a d o u b l e t a t £4.85, (J=10 Hz), C-2 H. Treatment o f 1 - f o r m y l n i t r i l e (173) w i t h b a r i u m h y d r o x i d e i n aqueous e t h a n o l gave the c o r r e s p o n d i n g n i t r i l e (174) i n good y i e l d ( f i g u r e 41) as shown by the e x p e c t e d UV s h i f t t o a d i h y d r o i n d o l e s p e c i e s ( A 298, 243 nm.) and mass spectrum m/e 307 (M +) . The IR -100-6 3 F i g u r e 41 - Further investigations towards the synthesis of stemmadenine (63) - approach 2. - 1 0 1 -showed NH and CN a b s o r p t i o n a t 3430 and 2230 cm "*" r e s p e c t i v e l y . The NMR was a l s o i n agreement w i t h the s t r u c t u r e . Treatment o f the n i t r i l e (174) w i t h a s o l u t i o n o f hydrogen c h l o r i d e i n r e f l u x i n g methanol a f f o r d e d e s t e r (132) h a v i n g the 142 same m.pt. and s i m i l a r UV and IR t o t h a t r e p o r t e d . However 137 the NMR r e p o r t e d was n o t o f s u f f i c i e n t q u a l i t y t o a l l o w p o s i -t i v e i d e n t i f i c a t i o n and was e x t r e m e l y d i f f e r e n t from t h a t o f the pure p r o d u c t o b t a i n e d i n t h e s e s t u d i e s . The c o r r e c t e d d a t a showed a t r i p l e t a t £5.7, (J=7 Hz),C-19 H, a s i n g l e t a t £4.74 (OH), a s i n g l e t a t £4.2 (NH) and a s i n g l e t a t £ 3.74 (C0 2CH 3) . Subsequent h y d r o g e n o l y s i s o f (132) t o (133) w i t h the W i e l a n d c a t a l y s t " * " 3 8 * 3 had p r e v i o u s l y been r e p o r t e d t o r e s u l t i n o v e r -r e d u c t i o n g i v i n g (134) as an i m p u r i t y w h i c h c o u l d n o t be removed. I t was a l s o n o t e d from the NMR o f (133) o b t a i n e d v i a the a l t e r n a t i v e h y d r o g e n o l y s i s o f the a l l y l i c bromide t h a t s a t i s f a c t o r y p u r i f i -c a t i o n had not been a c h i e v e d . However i n t h e s e r e c e n t s t u d i e s o v e r - r e d u c t i o n d i d not o c c u r and t h e r e q u i r e d e s t e r (133) was o b t a i n e d as pure c r y s t a l l i n e m a t e r i a l i n 88% y i e l d . The NMR o f (133) showed a q u a r t e t a t £5.48, (J=7 Hz)j C-19 H, a s i n g l e t a t £4.1 (NH) , a d o u b l e t a t ^4.05 (J=10 Hz), C-16H, a s i n g l e t a t £3.70 (C02CH_ 3) and a d o u b l e t o f d o u b l e t s a t Si.56,(J=2 and 7 Hz),C-18 CH . W i t h e s t e r (133) now i n hand, t h e n e x t s t e p i n v o l v e d o p t i m i s a t i o n o f the d e h y d r o g e n a t i o n r e a c t i o n w i t h l e a d t e t r a a c e -t a t e t o g i v e akuammicine (64). T h i s r e a c t i o n had been shown t o o c c u r i n 35% y i e l d by t r e a t i n g e s t e r (133) w i t h two e q u i v a l e n t s of l e a d -102-t e t r a a c e t a t e i n a s o l u t i o n o f benzene and a c e t i c a c i d f o r 1.75 nr. I n my s t u d i e s , t h i s r e a c t i o n f a r e d l e s s f a v o u r a b l y than r e p o r t e d b u t , i t was found t h a t t r e a t m e n t of (133) w i t h one e q u i v a l e n t of l e a d t e t r a a c e t a t e i n d r y benzene f o r 10 minutes gave akuammi-c i n e (64) i n 60% y i e l d . T h i s r e a c t i o n was found t o be e x t r e m e l y s e n s i t i v e t o minute v a r i a t i o n s i n the e x p e r i m e n t a l p r o c e d u r e . 21 Akuammicine (64) e x i s t s i n n a t u r e i n two forms (±) and 21 1 3 2 1 3 7 (-) b u t no C°ClD w a s r e p o r t e d i n the p r e v i o u s s t u d i e s . ' The p r o d u c t i n my s t u d i e s had a n e g a t i v e r o t a t i o n o f £af] = -672° and thus th e s y n t h e s i s o f (-) akuammicine (64) i s now complete. Treatment of (64) w i t h (1) a c e t i c a c i d and (2) sodium b o r o h y d r i d e gave e s t e r (122) i n 50% y i e l d . I t s h o u l d be noted t h a t t h e i n t e r m e d i a t e formed by t h e a c i d c a t a l y s e d r i n g o p e n i n g c o u l d be reduced w i t h sodium b o r o t r i t i i d e t o a l l o w i n t r o d u c t i o n of a r a d i o - l a b e l a t C-3. C 0 2 C H 3 C 0 2 C H 3 C 0 2 C H 3 6 4 122 The NMR o b t a i n e d f o r e s t e r (122) i n my s t u d i e s gave more 132 d e f i n i t i v e d a t a t o t h a t r e p o r t e d p r e v i o u s l y (see e x p e r i m e n t a l ) . Thus the c o n v e r s i o n o f s t r y c h n i n e (5) t o e s t e r (122) w i t h t h e s e m o d i f i c a t i o n s gave h i g h e r y i e l d s t h e r e b y i n c r e a s i n g somewhat the s y n t h e t i c u t i l i t y o f t h i s approach. Treatment of e s t e r (122) w i t h p o t a s s i u m h y d r i d e and m e t h y l -- 1 0 3 -c h l o r o f o r n a t e gave N -carbomethoxy-16-carboraethoxy-deshydroxy-cl methy1-stemmadenine (175) i n 59% y i e l d . E v i d e n c e f o r t h i s s t r u c t u r e came from the UV g i v i n g the e x p e c t e d N - a c y l i n d o l e chromophore {A 296, 285, 263, 228 nm). The mass spectrum gave an m/e max t i t r 382 ( M + ) , and t h e IR spectrum showed CO a b s o r p t i o n a t 1730 cm - 1. The NMR spectrum showed a q u a r t e t a t £5.43, (J=7 H z ) ; f o r C-19 H a s i n g l e t a t £ 4.0 f o r (N-C02CH_3) , a d o u b l e t a t £3.95, (J=3 Hz) f o r C-16 H, a s i n g l e t a t £3.65 (C0 2CH 3) and a d o u b l e t o f d o u b l e t s a t 3 1 . 8 3 , (J=2 and 7 H z ) , f o r C-18 CH . The d o u b l e t f o r t h e C-16 H was o f some s i g n i f i c a n c e s i n c e e s t e r (122) had a s i n g l e t f o r the C-16 H thus showing t h a t e p i m e r i z a t i o n had t a k e n p l a c e . T h i s had p r e -v i o u s l y been n o t e d on t r e a t i n g e s t e r (122) w i t h sodium h y d r i d e i n 137 d i m e t h y l s u l f o x i d e t o g i v e e p i m e r i c e s t e r (176). D r e i d i n g models and NMR d a t a s u g g e s t s t e r e o c h e m i s t r y a t C-16 i n (122), (175) and (176) t o be as shown. -104-D r e i d i n g models a l s o s uggest t h a t the N -carbomethoxy de-r i v a t i v e (175) was s t e r i c a l l y l e s s h i n d e r e d f o r a l k y l a t i o n a ttempts than the 1 -formyl e s t e r (135) p r e v i o u s l y s t u d i e d . C onsequently w i t h (175) on hand i t was now a p p r o p r i a t e t o ensure t h a t c o n d i -t i o n s were c o r r e c t f o r a n i o n f o r m a t i o n . I n t h i s r e s p e c t t r e a t m e n t o f (175) w i t h p o t a s s i u m h y d r i d e , i n an a n a l o g o u s manner t o t h a t used f o r (170), f o l l o w e d by quenching w i t h d e u t e r i u m o x i d e gave a p r o d u c t which was s u b j e c t e d t o a mass s p e c t r o m e t r i c i n v e s t i g a t i o n . + + E v a l u a t i o n of the r a t i o M /M +1 i n d i c a t e d about 40% d e u t e r a t i o n a t C-16. The mass s p e c t r a l f r a g m e n t a t i o n f o r (175) e x h i b i t e d m/e a t 382 (M +),323,194 and 123. I n the r e s u l t i n g d e u t e r a t e d p r o d u c t M + + l peaks 383,324 and 195 were e n r i c h e d s u g g e s t i n g the f r a g m e n t a t i o n t o be as shown ( f i g u r e 4 2 ) . m/<? 19 4 m / e 123 F i g u r e 42 - Proposed mass s p e c t r a l f r a g m e n t a t i o n o f N a-carbomethoxydeshydroxymethyl-stemmadenine (175) . S e v e r a l a t t e m p t s t o i n t r o d u c e a hydroxymethy1 group a t C-16 met w i t h f a i l u r e . M o d i f i c a t i o n o f the p r o c e d u r e by change o f -105-base (e.g. t - B u L i , NaH) , s o l v e n t (HMPA, DMSO), t e m p e r a t u r e , and time o f r e a c t i o n g e n e r a l l y l e d t o s e v e r e d e c o m p o s i t i o n . These r e s u l t s were most d i s a p p o i n t i n g and an a l t e r n a t i v e a l k y l a t i o n p r o c e d u r e was a t t e m p t e d . T h i s was based on t h e f a c t t h a t i n the s y n t h e s i s o f 16,17-dihydrosecodin-17-ol (88) the 87 f o l l o w i n g t r a n s f o r m a t i o n had been e f f e c t e d . '' 177 178 3 o Presumably formation of (178) occurs v i a i n i t i a l formylation of the indole nitrogen, to give, following further a l k y l a t i o n , (179) which on work-up loses the l a b i l e N-formyl group to give (178). 179 This reaction was attempted with (122) under a variety of conditions and led only to severe decomposition under the conditions used. Consequently another model system, derived from 18/3-carbo-16 5 methoxycleavamine (72), was considered. Treatment of (72) with potassium hydride and methylchloroform-161 ate gave Na-carbomethoxy-18/?-carbomethoxycleavamine (180), A l l attempts to alkylate this molecule with formalde-hyde at C-18 to give (182) met with f a i l u r e , even although 55% -106-d e u t e r i u m i n c o r p o r a t i o n a t C-18 was e f f e c t e d . S i m i l a r a t t e m p t s t o f o r m y l a t e (72) under a v a r i e t y o f c o n d i t i o n s t o g i v e (181) met w i t h f a i l u r e . C o n s e q u e n t l y i t was d e c i d e d t o change the i n d o l e p r o t e c t i n g group from the carbomethoxy group t o a m e t h y l group thus f u r t h e r d i m i n i s h i n g any s t e r i c f a c t o r s . T reatment o f (72) w i t h p o t a s s i u m h y d r i d e and m e t h y l i o d i d e i n t e t r a h y d r o f u r a n gave a m i x t u r e o f the r e q u i r e d N-methyl (183) and C-methyl (184) d e r i v a t i v e s as shown. T h i s r e a c t i o n was o f some i n t e r e s t s i n c e i t d e monstrated s t a b i l i s a t i o n o f the a n i o n a t C-9. I t had p r e v i o u s l y been n o t e d t h a t no a c y l a t i o n a t C-9 o c c u r r e d under the c o n d i t i o n s employed i n the f o r m a t i o n o f (180) whereas a l k y l a t i o n gave b o t h C- and N-a l k y l a t i o n . F u r t h e r , t r e a t m e n t o f t h e a n i o n from (72) w i t h b e n z y l bromide gave o n l y C-9 a l k y l a t e d p r o d u c t (185) i n h i g h y i e l d . -107-These r e s u l t s were most f r u s t r a t i n g s i n c e the r e a s o n s f o r f a i l u r e s were n o t o b v i o u s . I t i s now a p p r o p r i a t e t o d i s c u s s the p r o p o s e d s t e r e o c h e m i s t r y a t C-16 of epi-stemmadenine (138). I t had been proposed t h a t t r e a t m e n t o f 1 - f o r m y l e s t e r (135) ( f i g u r e 27) w i t h sodium h y d r i d e 132 137 and formaldehyde gave th e o x a z i n e (136) i n 38% y i e l d , ' and as t h e s t e r e o c h e m i s t r y was now f i x e d subsequent t r a n s f o r m a t i o n s w o u l d g i v e epi-stemmadenine (138) v / i t h the a b s o l u t e c o n f i g u r a t i o n shown (p. 77). T h i s was based on a s t e r e o c h e m i c a l argument i n w h i c h , under the e q u i l i b r i u m c o n d i t i o n s employed, a l k y l a t i o n w o uld be e x p e c t e d t o o c c u r from the"o(-face" o f (135) . i n f a c t a l --108-k y l a t i o n from the'fl -face',' w h i l s t l e s s f a v o u r a b l e , was a l s o f e a s i b l e . However the s u g g e s t e d r i n g c l o s u r e t o g i v e t h e o x a z i n e (136) p l a c e d b o t h r i n g s C and F ( f i g u r e 43) i n l e s s f a v o u r a b l e b o a t c o n f o r m a t i o n s . I t s h o u l d be n o t e d t h a t a l t e r n a t i v e s - f a c e " approach under t h e e q u i l i b r i u m c o n d i t i o n s t f o l l o w e d by v e r y f a c i l e r i n g c l o s u r e would y i e l d the o x a z i n e (187) w i t h r i n g s A-F i n more f a v o u r a b l e c h a i r c o n f o r m a t i o n s . 1 3 6 , 8 7 F i g u r e 43 - S t e r e o c h e m i c a l f e a t u r e s o f o x a z i n e s (138) : and (187). I t s h o u l d a l s o be noted t h a t f a c i l e f o r m a t i o n o f the o x a z i n e (188) from the a l c o h o l (143) had a l s o been o b s e r v e d on t r e a t m e n t w i t h 132 137 formaldehyde ' t h e r e b y g i v i n g f u r t h e r s u p p o r t f o r t h e s e comments. C o n s e q u e n t l y i t i s n o t p o s s i b l e t o draw any c o n c l u s i o n s w i t h r e s p e c t t o the a b s o l u t e c o n f i g u r a t i o n a t C-16. The s y n t h e s i s o f stemmadenine (63) however may be e f f e c t e d -109-by t r e a t i n g e s t e r (122) t o g i v e c h l o r o i n d o l e n i n e (189) w h i c h on f u r t h e r t r e a t m e n t w i t h c y a n i d e would be e x p e c t e d t o y i e l d t he c o r r e s p o n d i n g epimers (190 a,b). 1 9 0 a ; R = C N . R = C O p C b , R = C 0 2 C H . ^ ' = C A p p r o p r i a t e m a n i p u l a t i o n would y i e l d stemmadenine (63) o r i t s epimer (138). However i t s h o u l d be no t e d t h a t many o f thes e subsequent s t e p s would be a n t i c i p a t e d t o o c c u r i n low y i e l d s and c o n s e q u e n t l y t h i s approach was no t a t t e m p t e d . A n o t h e r approach would be t o t r e a t t h e n i t r i l e (191) as shown ( F i g u r e 44). I t i s t h e f e e l i n g o f t h i s a u t h o r t h a t t h i s a p proach, u s i n g t h i s s t r a t e g y , would be the most p r o m i s i n g . 162 A n o t h e r approach r e c e n t l y gave p r e l i m i n a r y i n f o r m a t i o n w h i c h may be adapted towards the s y n t h e s i s o f stemmadenine (63) ( f i g u r e 45)-By a p p r o p r i a t e m o d i f i c a t i o n o f s t e p s i n the s y n t h e s i s o f 1 6 , 1 7 - d i h y d r o s e c o d i n - 1 7 - o l (88 ) , i n t e r m e d i a t e (195) was ob-t a i n e d w h i c h on subsequent t r e a t m e n t as shown g i v e a compound whose s t r u c t u r e c o u l d not d e f i n i t i v e l y be a s s i g n e d . Three p o s s i b i l i t i e s were c o n s i d e r e d (196-198). I f the s t r u c t u r e was (19 6 ) , as s u g g e s t e d , t h e n i t was hoped by the author"*" t i i a t stemmadenine (63) c o u l d be formed by a p p r o p r i -a t e m o d i f i c a t i o n o f t h i s approach as shown ( f i g u r e 4 6 ) . (199 -200) -110-F i g u r e 44 - P r o p o s e d r o u t e f o r the s y n t h e s i s o f stemma-denine (63) . F i g u r e 45 - S t r u c t u r a l p o s s i b i l i t i e s o b t a i n e d from c y c l i z a t i o n o f i n t e r m e d i a t e (195). F i g u r e 46 - K u t n e y 1 s proposed s y n t h e s i s o f stemmadenine (63). I n t h i s r e s p e c t t r e a t m e n t o f d e r i v a t i v e (199) w i t h sodium b o r o h y d r i d e under c o n t r o l l e d c o n d i t i o n s s h o u l d g i v e the d i h y -d r o p y r i d i n e ( i ) . I f s u i t a b l e c o n d i t i o n s can be o b t a i n e d f o r the f o r m a t i o n of ( i i ) then r e a c t i o n as shown t o g i v e ( i i i ) would be e x p e c t e d . R e d u c t i o n t o (200) f o l l o w e d by d e h y d r a t i o n w ould h o p e f u l l y g i v e stemmadenine (63) o r i t s epiraer (138). However t h e s e m a n i p u l a t i o n s , w h i l s t under a t t e n t i o n , have not y e t been r e a l i s e d . Indeed u n t i l the s t r u c t u r e o f the p r o d u c t o b t a i n e d by r e a c t i o n o f (195) i s unambigously v e r i f i e d , ( f i g 45), no f u r t h e r i n v e s t i g a t i o n s u s i n g t h i s approach w i l l be c o n s i d e r e d . -112-I n c o n c l u s i o n , the att e m p t e d s y n t h e s e s o f stemmadenine (63) have, so f a r , been u n s u c c e s s f u l . I t i s t o be hoped t h a t one o f the proposed schemes w i l l a f f o r d ( 6 3 ) . F o l l o w i n g the e x p e r i m e n t a l s e c t i o n , p a r t I I , i n v e s t i g a -t i o n s towards the u n d e r s t a n d i n g o f the i n v i v o c o u p l i n g o f monomeric a l k a l o i d s c a t h a r a n t h i n e (12) and v i n d o l i n e (11) i n C. r o s e u s a r e p r e s e n t e d . -113-EXPERTMENTAL - PART I I G e n e r a l e x p e r i m e n t a l d e t a i l s were g i v e n i n E x p e r i m e n t a l , p a r t 1. P r o c e d u r e s , p u b l i s h e d or u n p u b l i s h e d by o t h e r s , a r e d e s c r i b e d when (1) d e t a i l e d e x p e r i m e n t a l v/as n o t r e l e a s e d (2) u n s a t i s f a c t o r y d a t a was p r e s e n t e d o r (3) p r o c e d u r e was m o d i f i e d . 14 8 I s o n i t r o s t r y c h n i n e h y d r o c h l o r i d e (141) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 218-220° (lit.m.pt.=220° w i t h d e c o m p o s i t i o n ) . Y i e l d = 7 9 % . NMR:£d-DMSO 100 MHz (FT) 8.12-7.96,7.46-7.02 (m,4H,arom.), 5.92 ( t , IH, J=6.4 Hz, C-19 H), 5.00 (d, IH, J=3Kz, C-17 H) M.S.- m/e: 36 3 (M +) , 346 ,318,144,130. High r e s o l u t i o n mass spectrum: C a l c - f o r (-21 H21^3 <^3~ 3^ 3 * 8 3 Found=363.1576. W i e l a n d - G u m l i c h aldehyde (129) ~L'*0 To i s o n i t r o s o s t r y c h n i n e h y d r o c h l o r i d e (141) (10 g , 25.13 mmol) was added t h i o n y l c h l o r i d e (20 m l , 0.278 mol) o v e r 5 min. F o l l o w i n g c o n t i n u o u s s t i r r i n g f o r 1 h r the c o n t e n t s were poured onto i c e (100 g) and s t i r r i n g was m a i n t a i n e d f o r an a d d i t i o n a l 4 h r . The r e s u l t i n g p r e c i p i t a t e o f 1-c y a n o f o r m y l WGA (144) was removed by s u c t i o n f i l t r a t i o n . To 1-cyanoformyl WGA (144) was added water (100 ml) and t h i s s u s p e n s i o n was then h e a t e d t o 109° (by a steam g e n e r a t o r and o i l bath) u n t i l a l l hydrogen c y a n i d e formed was removed (steam -114-d i s t i l l a t i o n ) . The r e s u l t i n g s u s p e n s i o n was c o o l e d (5 ) and b a s i f i e d w i t h M N a 2 C 0 3 t o pH 10. The r e s u l t i n g p r e c i p i t a t e was removed ( s u c t i o n f i l t r a t i o n ) , t a k e n up i n c h l o r o f o r m , and c r y s t a l l i n e WGA (129) was o b t a i n e d i n t h i s manner. The f i l t r a t e was e x h a u s t i v e l y e x t r a c t e d w i t h c h l o r o f o r m (10 x 150 ml) and the c h l o r o f o r m e x t r a c t s combined, d r i e d , and s t r i p p e d o f s o l v e n t . The c h l o r o f o r m s o l u b l e s were chromato-graphed on A l u m i n a (Act I I I ) , u s i n g c h l o r o f o r m as e l u a n t t o g i v e WGA (129) (4.1 g, 4 8 % ) . M.Pt.: 209-211° ( l i t . m.pt. 214-216°). NMR: £ (CDC1 3) 100 MHz (FT) 7.36-6.6 (m, 4H, arom ), 5.84 (m, IH, C-19 H),5.10 (d, IH, J=1.6 Hz, OCHO), 4.30, 4.17 (dd, IH, J=7 and 9 Hz, C-16 H). M.S. m/e: 310 ( M + ) , 180, 144, 130, 120, 118 H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2 2N 2O 2=310.1681 Found=310.1673 147 2 j 3 ,16o(-cur-19-en-17,18-diol (142) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 251° ( l i t . m.pt. 251°). Y i e l d = 81%. 2 y 3 ,16o(-cur-19-en-17-ol ( 1 4 3 ) 1 3 8 b P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . M.pt. 174-176° ( l i t . m.pt. 177-178°). Y i e l d = 90%. 2 y g-cur-19-en-17-al (145) Sodium h y d r i d e (0.465 g, 19.38 mmol) was o b t a i n e d by w a s h i n g a 50% o i l d i s p e r s i o n (0.93 g) w i t h dry benzene ( 3 x 2 m l ) . To t h i s was added a s o l u t i o n o f a l c o h o l (143) -115-(0.62 g, 2.1 mmol) and benzophenone (1.86 g, 10.22 mmol) i n dry benzene (40 m l ) . The s o l u t i o n was made up t o 60 ml w i t h d r y benzene and heated t o r e f l u x . A f t e r 6 hr the temperature was reduced t o 0° and h y d r o c h l o r i c a c i d (10%) added u n t i l pH 1. The aqueous l a y e r was s e p a r a t e d and b a s i f i e d a t 0° w i t h ammonium h y d r o x i d e u n t i l pH 9. The b a s i c s o l u t i o n was e x t r a c t e d w i t h e t h y l a c e t a t e (4 x 60 ml) and the e t h y l a c e t a t e e x t r a c t s combined, d r i e d and e v a p o r a t e d t o y i e l d a w h i t e foam. Chromatography on alumina (Act I I I ) u s i n g a b e n z e n e / e t h y l a c e t a t e g r a d i e n t a f f o r d e d the r e q u i r e d aldehyde (145) (0.45 g, 137 73%) p o s s e s s i n g s p e c t r o s c o p i c f e a t u r e s a l r e a d y r e p o r t e d . l - f o r m y l - 2 y f f - c u r - 1 9 - e n - 1 7 - a l (146) The r e a g e n t ( a c e t i c f o r m i c anhydride) was p r e -p a r e d by c o o l i n g r e d i s t i l l e d a c e t i c a n h y d r i d e (4 ml) t o 0°, and then s l o w l y adding r e d i s t i l l e d f o r m i c a c i d (2 m l ) . The temper-a t u r e was r a i s e d t o 50° f o r 15 min and then c o o l e d i m m e d i a t e l y o 151 to 07 The r e a g e n t (3.7 ml) was added t o a s o l u t i o n o f the a l d e -hyde (14 5) (443 mg, 1.51 mmol) a t 0°. The temp e r a t u r e was then r a i s e d t o 6° and s t i r r i n g m a i n t a i n e d f o r 10 min. The r e a c t i o n m i x t u r e was s t r i p p e d o f s o l v e n t and the r e s i d u e chromatographed on alumina (Act I I I ) u s i n g a b e n z e n e / e t h y l a c e t a t e g r a d i e n t t o g i v e t h e 1 - f o r m y l aldehyde (146) (280 mg, 58%) M.Pt.: 10 3° (MeOH) I.R.: [) C H C l 3 2940, 2890, 1730, 1670 cm" 1 U . V . : / i M e ° H 287 (3.39), 280 (3.46), 250 (3.88) nm. IHdX NMR: $ (CDC1,) 100 MHz (FT) 4.76, 9.72, 9.58 (3s, IH, C-17 H), -116-8.83, 8.58 (2s, IH, N-CHO), 8.04-7.9, 7.46-7.00 (m, 4H, arom.), 5,47 (q, IH, J=6 Hz, C-19 H), 4.81 (dd, IH, J=9 and 15 Hz, C-16 H) , 1.52 (dd, 3H, J=2 and 6 Hz, C-18 H). M.S. m/e: 322 (M+), 279, 172, 164, 144, 136, 109, 105. Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 Q H 2 2 N 2 0 2 = 3 2 2 . 1 6 8 0 . Found=322.1665. E l e m e n t a l a n a l y s i s : Found: C, 74.35, H, 6.95, N, 8.50 C a l c - C, 74.53, H, 6.83, N, 8.69. 1-formyl-2/3-cur-19 e n - 1 7 - c y c l o h e x y l i m i n e (14 7) To a s o l u t i o n o f (146) (100 mg, 0.31 mmol) i n an-hydrous benzene (40 ml) was added c y c l o h e x y l a m i n e (4 m l , 33.13 mmol) and the s o l u t i o n was r e f l u x e d o v e r 4 A° m o l e c u l a r s i e v e s f o r 2.5 - 3 h r i n a s o x h l e t condenser. The s o l v e n t was t h e n removed and t h e r e s u l t i n g r e s i d u e chromatographed on s i l i c a g e l t o g i v e as an o i l , the i m i n e (147) (82 mg, 6 5 % ) . I.R.: 0 C H C l 3 2920, 2850, 1665, cm - 1. U.V.: A E t ° H 288, 282, 252 nm. 1 max ' ' NMR: (CDC1 3) 100 MHz (FT) 9.02 ( s , l H , N CHO), 8.68 (q, IH, J=8 and 15 Hz, C-17 H), 7.97-7.83, 7.26-6.71 (m, 4H, arom.), 5.26 (q* IH, J=6 Hz, C-19 H) , 4.72 (dd, IH, . 7 = 3 and 8 Hz, C-16 H) , 1.91-1.37 (CH 2 e n v e l o p e , 11H, (CH_2) 5 CH) , 1.29 (dd, 3H, J=2 and 6 Hz, C-18 H) . M.S. m/e: 403 (M+), 320, 279, 172, 144. Hig h r e s o l u t i o n mass spectrum: C a l c - f o r C„,H 0_N_O=403.2623. 2 6 3 3 3 Found=403.2620. -117-A c y l a t e d d e r i v a t i v e (153)  Sodium h y d r i d e (25mg, 1.04 mmol) was o b t a i n e d by washing a 50% o i l d i s p e r s i o n ( 50 mg) w i t h anhydrous benzene (2 x 1 m l ) . To t h i s was added a s o l u t i o n o f 1-f o r m y l aldehyde (146) (50 mg, 0.155 mmol) i n anhydrous benzene (8 m l ) . M e t h y l c h l o r o f o r m a t e (2.44 g, 0.026 mol) was added and the mix-t u r e h e a t e d t o r e f l u x f o r 2 h r . The r e a c t i o n m i x t u r e was then c o o l e d and f i l t e r e d t h r o u g h a s h o r t a l u m i n a column (Act I I I ) u s i n g benzene as e l u a n t . Compound (15 3) was o b t a i n e d as an amorphous s o l i d on attemp t e d r e c r y s t a l l i s a t i o n ( 5 0 mg, 7 7 % ) . M.Pt.: 5 8° ( e t h a n o l , amorphous s o l i d ) I.R. : l J „ „ „ , 2960, 2930, 1732 (sh) , 1688 (sh) , 1668 cm - 1. L i l t 13 U.V.: A M e 0 H 287 (3.24), 277 (3.30), 248 (3.87) nm. IT13.X NMR: S( CDC1 3) 100 MHz 9.74, 9.67, 9.50 (3s, IH, C-17 H) , 8.84, 8.6 (2s, IH, N-CHO) , 8. 03-7. 88, 7. 88-7. 4 (m, 4H, arom.) , 5.52 (q, IH, J=6 Hz, C-19 H) 4.47, (dd, IH, J=10 and 16 Hz, C-16 H), 3.74 (S, 3H, N-C02CH_3) . M.S.-m/e: 418 (M+), 416 , 380, 279, 210, 209, 208, 194, 180, 166, 150, 134. High r e s o l u t i o n mass spectrum: C a l c — f o r C22 H25 N2°4 Cl=416.1501 Found= 416.1486 E l e m e n t a l a n a l y s i s : C 2 2 H 2 5 N 2 ° 4 " C 2 H 5 ° H ' F o u n d : C ' 62.41, H, 6.70, N, 6.33 C a l c - C, 62.27, H, 6.70, N, 6.05. N o r - f l u o r o c u r a r i n e (119) P r e p a r e d e x a c t l y a c c o r d i n g t o the l i t e r a t u r e p r o c e -d u r e : 1 3 8 1 3 M.pf. 184° ( l i t . m.pt. 184-5°). Y i e l d = 4 1 % -118-137 Deshydroxymethylstemmadenine (128) N o r - f l u o r o c u r a r i n e (119) (50 mg, 0.169 mmol) was d i s s o l v e d i n methanol (5 ml) and sodium b o r o h y d r i d e (55 mg, 1.45 mmol) added o v e r t e n min. A f t e r s t i r r i n g f o r an a d d i -t i o n a l 10 min the methanol was removed under reduced p r e s s u r e . The r e s u l t a n t o i l was t r e a t e d w i t h (1) d i c h l o r o m e t h a n e (5 ml) and (2) w a t e r (2 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were removed and the aqueous l a y e r f u r t h e r e x t r a c t e d w i t h d i c h l o r o m e t h a n e ( 3 x 5 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were combined, d r i e d , and s t r i p p e d o f s o l v e n t . Chromatography on s i l i c a g e l p r o v i d e d deshydroxymethylstemmadenine (128) (16 mg, 4 0 % ) . I.R.: \) ~ v n , 3350, 3250 (sh) , 2900, 1600 cm - 1 . U.V./{ 290 (3 . 7 4 ) , 283 (3. 78). M.Pt.: 86-88° (MeOH) NMR: ^ (CDC1 3) 100 MHz 9.22 ( s , IH, NH, exchangeable w i t h D 2 0 ) , 7.6-6.96 (m, 4H, arom.), 5.5 (q, IH, J=6 Hz, C-19 H), 4.92 (S, IH, OH, exchangeable w i t h D 2 0 ) , 4.22 (d, 2H, J=5 Hz, C-17 H ) , 1.66 (d, 3H, J=6 Hz, C-18 H). M.S. m/e: 296 ( M + ),193,168,144,123. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c^ gH 2 4N 20=296.1889. Found=296.1860. E l e m e n t a l a n a l y s i s : C a l c - f o r C l gH 2 4N 20-CH 3OH_ c 7 3 ± H 8.53, N, 8.53 Found C, 72.75, H, 8.20, N, 8.49. 16 3 1,2,3,4 t e t r a h y d r o c a r b a z o l e (155) P r e p a r e d e x a c t l y a c c o r d i n g t o p r o c e d u r e by Corson and Rogers. M.pt. 119° ( l i t . m.pt. 120°). Y i e l d = 7 5 % . -119-1 - B e n z o t r i a z o l e - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (162) N - C h l o r o b e n z o t r i a z o l e (504 mg, 3.28 mmol) i n dry d i c h l o r o m e t h a n e (4 ml) was added t o a s o l u t i o n of t e t r a -h y d r o c a r b a z o l e (155) (554 mg, 3.24 mmol) i n dry d i c h l o r o m e -thane (6 ml) a t 0°. A f t e r s t i r r i n g f o r 20 min the r e a c t i o n m i x t u r e was d i l u t e d w i t h d i c h l o r o m e t h a n e (50 ml) and th e n e x t r a c t e d w i t h (1) s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n (20 ml) and (2) water (20 m l ) . The d i c h l o r o m e t h a n e s o l u b l e s were then d r i e d , f i l t e r e d , and e v a p o r a t e d t o g i v e a w h i t e foam. Chromatography on al u m i n a (Act I I I ) f o l l o w e d by r e c r y s t a l l i -s a t i o n from methanol gave (162) (800 mg, 86%). M.Pt.: 205-209° (MeOH). U.V.: maS H 2 9 1 ( 4 - 1 0 ) ' 2 8 2 (4.19), 276 (4.20), 223 (4.62) nm. I.R.: d CHC1 3 3470, 3020, 2950, 1455 cm - 1. NMR: £(CDC1 3)100 MHz (FT) 8.40 ( s , IH, NH) , 8.00-7. 83 (m, IH, C-4 H), 7.77-7.55 (m, IH, C-7 1 H), 7.4-7.08 (m, 5H, arom. i n d o l e + C-5' H), 6.97-6.76 (m, IH, C-6' H) , 6.49 ( t , IH, J=6 Hz, C - l H) , 2.98 ( t , 211, J=5.5 Hz, C-4 U^) , 2.65 -2.31 (m, 2H, C-2 H 2 ) , 2.26-1.98 (m, 2H, C-3 H 2 ) . M.S. m/e: 288 ( M + ) , 170, 169, 168 High r e s o l u t i o n mass spectrum: Calc^. f o r C l 8H l 6N4=288.1375 Found=288.1374 E l e m e n t a l a n a l y s i s : Found C, 75.0, H, 5.40, N, 19.51 C a l c ! C, 75.0, H, 5.55, N, 19.44. -120-1 - C y a n o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) and 1 - s u c c i n i m i d o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (165) N-Bromosuccinimide (550 mg, 3.09 mmol) and d i -benzoyl peroxi de ( 1 mg) were q u i c k l y a i d e d t o a s o l u t i o n o f t e t r a h y d r o c a r b a z o l e (155) (500 mg, 2.93 mmol) i n dry p y r i d i n e (0.6 ml),and dry benzene (10 m l ) . A f t e r s t i r r i n g f o r 24 hr a t room temperature ( r . t . ) two l a y e r s were formed as d e s c r i b e d . 1 ^ The benzene l a y e r was decanted l e a v i n g a v i s c o u s r e d o i l w h i c h on d r y i n g under vacuo a f f o r d e d a crude c r y s t a l l i n e mass o f the r e q u i r e d p y r i d i n i u m s a l t (163) (1.2 g ) . P o t a s s i u m c y a n i d e (1.6 g, 24.62 mmol) was added t o the crude s a l t (163) f o l l o w e d by anhydrous a c e t o n i t r i l e (12 m l ) . S t i r r i n g was c o n t i n u e d f o r 12 hr and s a t u r a t e d sodium b i -c a r b o n a t e (15 ml) was then added. E x t r a c t i o n w i t h e t h y l a c e t a t e (4 x 50 ml) gave, a f t e r d r y i n g and removal o f s o l v e n t , a crude m i x t u r e o f (164) and (165) which on chromatography on s i l i c a a f f o r d e d 1-cyano-1,2,3,4 t e t r a h y d r o c a r b a z o l e (164) (426 mg, 74%) and 1 - s u c c i n i m i d o - l , 2 , 3 , 4 t e t r a h y d r o c a r b a z o l e (165) (150 mg, 19.2%). 1 - C y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) M.Pt.: 103-104° ( C H 2 C l 2 o r CC1 4) U.V.: J M e 0 H 288 (3. 7 5 ) , 278 (3. 84), 273 (3. 8 5 ) , 222 (4 . 51) nm. " max I.R.: l ^ C H C l 3 3470, 2960, 2870 ( s h ) , 2240, cm" 1 NMR: (CDC1 3) 100 MHz (FT) 8.16 ( s , IH, NH), 7.44-7.04 (m, 4H, arom.), 3.90 ( t , IH, J=6 Hz, C - l H), 2.71 ( t , 2H, J=6 Hz, C-4 H 2 ) , 2.34-1.66 (m, 4H, C-2 and C-3 H) . -121-M.S. m/e: 19 6(M+), 16 8. High r e s o l u t i o n mass spectrum: C a l c — f o r C-^3H^2N2=19 6 ' 1 (^00 Found=196.0977. E l e m e n t a l a n a l y s i s : Found C, 79.30, H, 6.26, N, 14.00 C a l c - C, 79.59, H, 6.12, N, 14.28. 1 - s u c c i n i m i d o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (165) M.Pt.: 220° (CHC1 3) U.V.: y\ ^ 0 H 288 (3. 8 3 ) , 278 ( 3 . 8 8 ) , 273 (3. 8 8 ) , 225 (4.50) nm. I.R.: l ^ C H C 1 3470 , 3015, 2950, 1705 cm. NMR: £(CDC1 3) 100 MHz (FT) 7.94 ( s , IH, NH) , 7.6-7.4 (m, IH, C - 8 H) , 7.32-7.04 (m, 3H, C-5-)7 H) , 5.54 ( t , IH, J=7 Hz, C - l H) , 2.92-2.72 (m, 2H, C-4 H) , 2.69 ( s , 4H, N(CH_2) 2), 2.60-1.90 (m, 4H, C-2 and C-3 H_2) . M.S. m/e: 268 (M +), 240, 170, 169, 168, 143. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C. -H, rN„0„=268.1212 l b ± o Z A Found=268.1205 E l e m e n t a l a n a l y s i s : Found C, 71.38, H, 5.81, N, 10.19 C a l c - C, 71.64, H, 5.97, N, 10.44. 9 - C a r b o m e t h o x y - l - c y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (166) 1 - C y a n o - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (164) (100 mg, 0.51 mmol) i n anhydrous t e t r a h y d r o f u r a n (3 ml) was added o v e r 3 min t o a s u s p e n s i o n o f p o t a s s i u m h y d r i d e (0.16 ml, 1.03 mmol) i n anhydrous t e t r a h y d r o f u r a n (10 ml) a t 0°. S t i r r i n g was c o n t i n u e d f o r 30 min and d r y , r e d i s t i l l e d m e t h y l c h l o r o f o r m a t e (0.1 m l , 1.3 mmol) was added. A f t e r an -122-a d d i t i o n a l 30 min a l u m i n a (Act I I I ) was s l o w l y added t o d e s t r o y the excess p o t a s s i u m h y d r i d e . The s u s p e n s i o n was d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a g l a s s s i n t e r f u n n e l . T h e s o l i d w a s w a s h e d w i t h e t h y l a c e t a t e (10 m l ) . T h e e t h y l a c e t a t e s o l u b l e s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h i c h was chromatographed on s i l i c a and gave 9-carbo-m e t h o x y - l - c y a n o - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (166) (90 mg, 70%). M.Pt.: 68-70° (CHC1 3). U.V.: / j ^ ° H 292 (3 . 23), 282 (3.16), 273 (3.17), 258 (3. 4 5 ) , 226 (3. 77)nm. I.R.: [) C H C i 3 2980, 2870 (sh) , 2260, 1730 cm - 1. NMR: (CDC1 3) 100 MHz (FT) 8.22-8.04 (m, IH, C-8 H ) , 7.56-7.24 (m, 3H, C-5 H ) , 4.50 ( t , IH, J=7 Hz, C - l H), 4.09 ( s , 3H, C0 2CH 3) , 2. 86-1.92 (m, 6H, C-2, 3 & 4 H_2) • M.S. m/e: 254 (M +),194,168,84. High r e s o l u t i o n mass spectrum: C a l c — f o r C^^H^ 4N 20 2=254. 1055 Found=254.1066 E l e m e n t a l a n a l y s i s : Found C, 70.86, H, 5.51, H, 11.02 C a l c - C, 70.61, H, 5.64, N, 10.5. N - C a r b o m e t h o x y - l - c y a n o - l - c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o - c a r b a z o l e (167) 9 - C a r b o m e t h o x y - 1 - c y a n o - t e t r a h y d r o c a r b a z o l e (16 6) (100 mg, 0.394 mmol) i n anhydrous t e t r a h y d r o f u r a n (3 ml) was added o v e r 3 min t o p o t a s s i u m h y d r i d e (0.33 m l , 2.045 mmol) i n anhydrous t e t r a h y d r o f u r a n a t 0°. The remainder o f t h e r e a c t i o n and the work-up p r o c e d u r e was i d e n t i c a l w i t h t h a t j u s t d e s c r i b e d f o r (166). -123-Chromatography on s i l i c a a f f o r d e d (167) (48 mg, 39%) and s t a r t i n g m a t e r i a l (166) (27 mg, 2 7 % ) . U.V.: /) 292 (3. 2 8 ) , 279 (3. 1 8 ) , 261 (3.48), 227 (3. 94) I.R.: \J CHC1 3 2960, 2860 (sh) , 2245, 1750-25 cm - 1 . NMR: (CDC1 3) 100 MHz (FT) 8. 24-8. 04 (m, IH, C-8 H) , 7.62-7.26 (m, 3H, arom. C-5-*7 H) , 4.05 ( s , 3H, N-C02CH_3) , 3.86 (s , 3H, C02CH_3) , 3.0-2.58 (m, 3H, C-4 H_2 and C-2 H) , 2.48-2.06 (m, 3H, C-3 H_2 and C-2 H) . M.S. m/e: 312 ( M + ),254. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c H N 0 =312.1110 17 16 2 4 Found=3i2.1102 1 - C a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (156) R e d i s t i l l e d a c e t y l c h l o r i d e (11.6 g, 0.148 mol) was s l o w l y added t o anhydrous methanol (13 ml, 0.32 mol) a t r . t . To t h i s was added a s o l u t i o n o f 1 - c y a n o - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (164) (200 mg, 1.02 mmol) i n anhydrous methanol (2 m l ) . A f t e r s t i r r i n g f o r 1.5 h r , the r e a c t i o n m i x t u r e was poured o n t o i c e (20 g) and s t i r r i n g was c o n t i n u e d f o r an a d d i -t i o n a l 30 min. The r e a c t i o n m i x t u r e was b a s i f i e d t o pH 10 w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n . E x t r a c t i o n w i t h d i c h l o r o m e t h a n e gave, a f t e r d r y i n g and removal o f s o l v e n t , a crude foam w h i c h when chromatographed on s i l i c a g e l a f f o r d e d e s t e r (156) (163 mg, 7 0 % ) . i M P O H U , V , : / i 1 max ' 2 9 1 ( 3 ' 8 1 ) ' 2 8 3 ( 3 - 8 8 ) , 277 (3.83), 224 (4.53) nm. I.R.: 0 CHC1 3 3470, 2960, 2860 ( s h ) , 1730 cm" 1. -124-NMR: S (CDC1 3) 100 MHz (FT) 8.34 ( s , IH, NH), 7.58-6.98 (m, 4H, arom.), 3.94-3.88 (m, IH, C - l H), 3.80 ( s , 3H, C 0 2 C H 3 ) , 2.86-2.64 ( t , 2H, J=5.5 Hz, C-4 H), 2.34-1.72 (m, 4H, C-2 and C-3, H). M.S. m/e: 229 ( M + ) , 170, 168. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C-^H^N02=229.1103 Found=229.1101. N-Carbome thoxy-T-carbomethoxy-1, 2 3 , 4 - t e t r a h y d r o c a r b a z o l e (17 0) P o t a s s i u m h y d r i d e (0.142 ml, 0.88 mmol) was q u i c k l y added t o a s o l u t i o n o f 1 - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (156) (100 mg, 0.44 mmol) i n anhydrous t e t r a -h y d r o f uran (2 m l ) . The r e a c t i o n m i x t u r e was s t i r r e d f o r 2 5 min and m e t h y l c h l o r o f o r m a t e (0.1 m l , 1.3 mmol) was added. A f t e r s t i r r i n g f o r an a d d i t i o n a l 20 min, a l u m i n a . (Act I I I ) was added t o d e s t r o y e x c e s s p o t a s s i u m h y d r i d e . The f l a s k c o n t e n t s were d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and p a s s e d t h r o u g h a g l a s s s i n t e r f u n n e l . The f u n n e l was washed w i t h e t h y l a c e t a t e (2 x 10 ml) and the e t h y l a c e t a t e s o l u b l e s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h i c h a f t e r chromatography on s i l i c a g e l a f f o r d e d r e q u i r e d N - c a r b o m e t h o x y - l - c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r -b a z o l e (170) (75 mg, 6 0 % ) . M.Pt.: 68° (CHC1 3). U.V.: y\ ( J J l x 1 1 ) 292 (3.58), 278 ( 3 . 6 6 ) , 252 (4.08), 259 (4.08), 227 (4.30) nm. I.R.: 0 CHC1 3 2960, 2860 ( s h ) , 1780 ( s h ) , 1730 cm - 1. NMR: £(CDC1 3) 100 MHz (FT) 8.22-8.06 (m, IH, C-8 H), 7.56-7.2 (m, 3H, C-5-7, H), 4.26 ( t , IH, J=5.4 Hz, C - l H), -125-3.98 ( s , 3H, N-C0 2CH 3) , 3.72 (s, 3H, C0 2CH_ 3), 2.84-2.60 (m, 2H, C-4 H), 2.32-2.06 (m, 2H, C-2 H ) , 2.02-1.76 (m, 2H, C-3 H) M.S. m/e: 287(M +),255,228,168. Hig h r e s o l u t i o n mass S D e c t r u m : C a l c — f o r C, ,H,_N0. = 287.1157 16 17 4 Found=287.1149. E l e m e n t a l a n a l y s i s : Found C, 66.97, H, 5.92, N, 4.89 C a l c - C, 66.89, H, 5.92, N, 4.87. 1-Carbomethoxy-1-hydroxymethy1-1,2,3,4-tetrahydrocarbazole (157) To 9 - c a r b o m e t h o x y - l - c a r b o m e t h o x y - l , 2 , 3 , 4 - t e t r a -h y d r o c a r b a z o l e (170) (40 mg, 0.14 mol) i n anhydrous t e t r a h y d r o -f u r a n (2 ml) a t 0° was added p o t a s s i u m h y d r i d e (0.1 ml, 0.615 mmol). S t i r r i n g was c o n t i n u e d f o r 20 min a f t e r w h i c h f o r m a l d e -hyde was d i s t i l l e d i n t o the r e a c t i o n m i x t u r e . The temperature was r a i s e d t o r . t . a n d s t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 30 m i n m A l u m i n a (Act I I I ) was added t o d e s t r o y excess p o t a s s i u m h y d r i d e . The f l a s k c o n t e n t s were d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a g l a s s s i n t e r f u n n e l . The s o l i d was washed w i t h e t h y l a c e t a t e (10 m l ) . The s o l v e n t was removed under vacuo and chromatography of the crude foam gave r e q u i r e d 1-carbomethoxy-l-hydroxymethy1-1,2,3,4 t e t r a h y d r o c a r -b a z o l e (157) (16 mg, 45%) and s t a r t i n g m a t e r i a l (11 mg, 30%). U.V.: A M e 0 H 290 (3.82), 282 ( 3 . 8 8 ) , 277 (3.85), 224 (4.46) nm. " max I.R.: 0 CHC1 3 3620, 3460, 2950, 2850, 1710 cm - 1. NMR: <£(CDC13) 100 MHz (FT) 8.68 ( s , IH, NH) , 7. 64-7. 44 (m, IH, C-8 H) , 7.4-7.2 (m, 3H, C 5-7 H) , 3.99 (ABq, 2H, J=5.4 Hz, -126-CH 2OH, 3.74 ( s , 3H, C02CH_3) , 2.9-2.6 (m, 2H, C-4 H_ 2), 2.36-1.66 (m, 4H, C-2 and C-3 H ). M.S. m/e: 259(M +),228,200,197,168. Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5H 1 7N0 3=259.1208 Found=259.1199 1 - H y d r o x y m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (157) L i t h i u m a l u m i n i u m h y d r i d e (267 mg, 7.62 mmol) was added p o r t i o n w i s e o v e r 10 min t o a s o l u t i o n o f 1-c a r b o m e t h o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o c a r b a z o l e (150 mg, 0.66 mmol) i n anhydrous t e t r a h y d r o f u r a n (20 ml) a t -5°. A f t e r 30 min a s a t u r a t e d s o l u t i o n o f sodium s u l p h a t e was added d r o p w i s e t o d e s t r o y e x c e s s l i t h i u m a l u m i n i u m h y d r i d e . The r e s u l t i n g s o l u t i o n was then e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 25 ml) and t h e e t h y l a c e t a t e e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e a foam w h i c h f o l l o w i n g chromato-graphy on s i l i c a g e l a f f o r d e d 1-hydroxymethy1-1,2,3,4-tetra-h y d r o c a r b a z o l e (157) (96 mg, 7 3 % ) . U.V.:/f J ^ ° H ; 288 (3. 76) , 283 (3.81), 274 (3. 7 8 ) , 225 (4.49) nm. I.R.: 0 C H C l 3 3622, 3460, 2940, 2860 cm" 1. NMR: £ (CDC1 3) 100 MHz (FT) 8.63 ( s , IH, NH), 7.62-6.90 (m, 4H, arom.), 4.07-3.52 (m ,2H, CH OH), 3.26-2.84 (m, IH, C - l H), 2.82-2.17 (m, 2H, C-4 H), 2.17-1.04 (m, 4H, C-2 and 3 H) . M.S. m/e: 201 (M +),170. H i g h r e s o l u t i o n mass spectrum: C a l c - f o r C^3H^,-NO=201.1154 Found=201.1148. E l e m e n t a l a n a l y s i s : Found C, 76.8, H, 7.34, N, 7.09 -127-C a l c - C, 7 7.6, H, 7.46, N, 6.96. Wi e l a n d - Gumlich Aldoxime (130) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c a -159 m.pt. 246° ( l i t . m.pt. 245°) Y i e l d = 8 5 % dure U l V ' : /I max" 2 9 5 ( 3 - 5 0 ) ' 2 4 2 ( 3- 9 1) n m -NMR: £ (CDC1 3) 100 MKz (FT) 7.34-6.58 ( m, 4H, arom.), 5.65 ( t , IH, J=7Hz, C-19H), 4.07 ( t , IH, J=7Hz, C-16 H ) , 3.84-3.50 (m, IH, C-2H). d Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C-\ ^ 2 3 1 ^ 3 0 2 = 3 2 5.1789 Found=325.1768. E l e m e n t a l a n a l y s i s : Found C, 69.86, H, 7.05, N, 12.82 C a l c - , C, 70.15, H, 7.07, N, 12.92 18-hydroxy-l-formyl-16-cyano-17-nor - 2 y 6 > , 16fl£-cur-19-ene (173) To oxime (130) (1 g, 3.076 mmol) was added f o r m i c a c i d (97-100%, 10 ml, 265.2 mmol) and anhydrous sodium formate ( 420 mg, 6.17 mmo1). T h i s m i x t u r e was h e a t e d t o r e f l u x f o r 1 h r . The temperature was then l o w e r e d t o -10° and c o n c e n t r a t e d ammonium h y d r o x i d e added to pH 10. The aqueous l a y e r was e x t r a c t e d w i t h c h l o r o f o r m ( 5 x 30 ml ) The c h l o r o f o r m e x t r a c t s were combined, d r i e d , and the s o l v e n t removed under reduced p r e s s u r e . The r e s u l t i n g gum was chromato-graphed on s i l i c a g e l t o g i v e (173) (720 mg, 7 0 % ) . M.Pt.: 240-242° (acetone) U.V. : /] MeOH 287 (3.78), 283 (3.81), 248 (4.34) nm. max -128-I.R.: C H C 1 3 3600, 2960, 2900 (sh) , 2260, 1675 cm" 1 NMR: S(CDC13) 100 MHz (FT) 8. 90, 8. 76 (2s, IH, N-CHO) , 8.1-8.03, 7.46-7.1 (m, 4H, arom.), 5.90 ( t , IH, J=7Hz, C-19H), 4.85 (d, IH, J=5Hz, C-2H). M.S. M/e: 335 (M +) 318, 304, 295, 177, 172, 144, 130. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C2oH21N3°2:=^~'" ^ 3 4 Found: 335.1614. E l e m e n t a l a n a l y s i s : Found C, 71.34, H, 6.44, N, 12.50 C a l c - C, 71.60, H, 6.26, N, 12.53 16-cyano-17-nor-2/?, 16 <t cur-19-ene (17 4) To t h e N- f o r m y l n i t r i l e (173) (100 mg, 0.298 mmol) was added e t h a n o l (2ml), w a t e r (4 ml) and barium h y d r o x i d e (380 mg, 2.22 mmol). The t e m p e r a t u r e was r a i s e d u n t i l r e f l u x began and t h i s was m a i n t a i n e d f o r 4 h r . The tem p e r a t u r e was then l o w e r e d t o r . t . and the s o l u t i o n d i l u t e d w i t h an e q u a l volume of d i c h l o r o m e t h a n e . R a p i d f i l t r a t i o n t h r o u g h a s h o r t column o f s i l i c a g e l a f f o r d e d the c rude n i t r i l e w h i c h was f u r t h e r p u r i f i e d by t h i n l a y e r chromatography on s i l i c a g e l t o g i v e (174) (72 mg, 7 8 % ) . . MeOH U.V.: /) m a x 298 (3 . 4 8 ) , 245 (3. 83) nm. I.R.: 0 CHC1 3 3420,3330,2930,2880,2230,1660,1600,750 cm" 1. NMR: & (CD 3OD ) 100 MHz (FT) 7.18-6.58 (m, 4H, arom.), 5.54 ( t , IH, J=7Hz, C-19 H), 4.14 (d, IH, J=9Hz, C-16 H). M.S. m/e: 307 ( M + ) , 267, 199, 177, 144, 130. High r e s o l u t i o n mass spectrum: C a l c - f o r C 1 9H 2iN 3O=307.1686 Found=307.1685 -129-Methyl-18-hydroxy- 2ft, 16 oj -cur-19-en-17-oate (132) A c e t y l c h l o r i d e (1.3 m l , 18.33 mmol) was s l o w l y added t o methanol (10 ml, 0.25 mol) a t r . t . To t h i s was added n i t r i l e (174) (95 mg, 0.31 mmol). -The temperature was r a i s e d u n t i l r e f l u x began and t h i s was c o n t i n u e d f o r 5 h r . Water (2 ml , 0. 11 mol) was then added and r e f l u x was c o n t i n u e d f o r 1 h r . The r e a c t i o n m i x t u r e was c o o l e d and e v a p o r a t e d t o d r y n e s s . To the r e s i d u e was added (1) e t h y l a c e t a t e (10 ml) and (2) 20% Na 2C03 (10 ml) . The aqueous l a y e r was f u r t h e r e x t r a c t e d w i t h e t h y l a c e t a t e (3 x 10 ml) and the e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s t o g i v e , a f t e r chromatography on s i l i c a g e l and r e c r y s t a l l i s a t i o n , the r e q u i r e d e s t e r (132) (73 mg, 7 0 % ) . 142 E s t e r had same m.pt. and U.V. as t h a t r e p o r t e d . M.Pt.: 154-156° ( l i t 154-156°). u-v-: A m!x H 2 9 7 (3.52), 242 (3.84) nm. 1. R.rlJ CHC1 3 3610, 2920, 2960 , 1725 cm'1 NMR: £(CDC1 3) 8.28-7.58 (m, 4H, arom.), 5.67 ( t , IH, J=7Hz, C-19R), 4.74 ( s , IH, OH, exchangeable w i t h D 2 0 ) , 4.26 ( s , IK, NH, exchangeable w i t h D 20) , 3.74 ( s , 3H, C 0 2 CH_3) . M.S. m/e: 340 ( M + ) , 308, 267, 210, 178, 144, 130. d High r e s o l u t i o n mass spectrum: C a l c — f o r C 2 0 H 2 4 N 2 ° 3 = 3 ^ " 1 7 8 7 Found=340.1762 E l e m e n t a l a n a l y s i s , C a l c - f o r C 2 ( ) H 2 4 N 2 0 3 . CH 3OH: Found C, 68.0, H, 7.61, N, 7.80. Calc^- C, 67. 74, H, 7.52, N, 7.52 M e t h y l 2/2, 16 < cur-19-en-17-oate (133) 138b To the p a l l a d i u m on c h a r c o a l c a t a l y s t (10% Pd by w e i g h t , 169.2 mg c a t a l y s t ) , was added (1) h y d r o c h l o r i c a c i d - 1 3 0 -(33;5 m l , 0.18 m) (2) hydroxy e s t e r (132) (693 mg, 2.04 mmol) and (3) a c e t i c a c i d (20.32 m l ) . The m i x t u r e was then p l a c e d under an atmosphere o f hydrogen gas. A f t e r 20 h r , hydrogen (54.05 ml) had been t a k e n up and the c a t a l y s t was removed by s u c t i o n f i l t r a t i o n . The f i l t e r f u n n e l was washed w i t h methanol (150 m l ) . The r e s u l t -i n g s o l u t i o n was e v a p o r a t e d t o an o i l , c o o l e d t o 0°, and concen-trated NH^OH added u n t i l pH 12. The b a s i c l a y e r was e x t r a c t e d w i t h c h l o r o f o r m (5 x 20 ml) and the c h l o r o f o r m e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e a c o l o r l e s s foam which a f t e r chromatography on s i l i c a g e l and r e c r y s t a l l i s a t i o n a f f o r d e d r e q u i r e d e s t e r (133). (583 mg, 88%) M.Pt.: 118° (benzene) U.V.:/| max n 297 (3. 5 0 ) , 244 (3. 84) nm. I.R.:\) 3420, 2950, 2890 (sh) , 1725 c m T 1 NMR: i (CDC1 3) 100 MHz (FT): 7.30-6.52 (m, 4H, arom.), 5.49 (q, IH, J=7Hz, C-19H), 4.1 ( s , IH, exchangeable w i t h D 20, NH), 4.01 (d, IH, J=4.8Hz, C-16H) 3.70 ( s , 3H, C0 2CH 3 ), 1.56 (dd, 3H J=2 and 7Hz, C-18H_3) M.S. m/e: 324 ( M + ) , 251, 194, 144, 139, 130 High r e s o l u t i o n mass spectrum: C a l c - f o r c 2 0H 2 4N 2O 2=324.1837 Found=324.1810 E l e m e n t a l a n a l y s i s : Found C, 73.87, H, 7.40, N, 8.37 d C a l c - C, 74.07, H, 7.41, N, 8.64. Akuammicine (64) 0 . 0 6 7 mmol) i n dry benzene (4 ml) was q u i c k l y added l e a d t e t r a -a c e t a t e ( 3 0 mg, 0 . 0 6 7 mmol). A f t e r s t i r r i n g f o r 10 min, the (133) (22 mg, -131-r e a c t i o n m i x t u r e was d i l u t e d w i t h c h l o r o f o r m (4 ml) and then i m m e d i a t e l y poured t h r o u g h a g l a s s s i n t e r f u n n e l c o n t a i n i n g a l u m i n a (Act I I I , 0.6g). The s i n t e r f u n n e l was washed w i t h c h l o r o f o r m (30 m l ) , and the c h l o r o f o r m s o l u b l e s d r i e d , and e v a p o r a t e d t o g i v e a r e s i d u e w h i c h was chromatographed on s i l i c a g e l and c r y s t a l l i z e d from e t h y l a c e t a t e t o g i v e akuammicine (64) (13 mg, 60%) h a v i n g a l m o s t i d e n t i c a l c h a r a c t e r i s t i c s w i t h t h a t ^ , 132,137 r e p o r t e d . M.Pt.: 181° ( l i t m . p t . 180-181.5°) Akuammicine jV] D =-672 (CHC1 3) , C=0.1 ( l i t = - 700 ) PeshydroxymethyIstemmadenine (122) To a v e s s e l c o n t a i n i n g g l a c i a l a c e t i c a c i d , (3 m l , 52 mmol) a t r e f l u x was q u i c k l y added akuammicine (64) (68 mg, 0.21 mmol). R e f l u x was m a i n t a i n e d by t h e a d d i t i o n o f sodium b o r o h y d r i d e (500 mg, 13.16 mmol) o v e r f i f t e e n min. A f t e r a d d i -t i o n , t h e t e m p e r a t u r e was l o w e r e d t o 5° and t h e s o l u t i o n b a s i f i e d w i t h cone— ammonium h y d r o x i d e s o l u t i o n u n t i l pH 12. E x t r a c t i o n o f t h e aqueous phase w i t h d i c h l o r o m e t h a n e (3 x 20 ml) a f f o r d e d a f t e r d r y i n g and chromatography, r e q u i r e d e s t e r (122) (39 mg, 5 7 % ) . NMR: S (CDC1 3) (100 MHz) (FT) 9.19 ( s , IH, NH), 7.54-7.06 (m, 4H, arom.), 5.79, (q, IH, J=7.6Hz, C-19H), 4.35 ( s , IH, C-16H), 3.90 ( s , 3H, C 0 2 C H 3 ) , 1.77 (d, J=7.6Hz, 3H, C-18 H 3) E l e m e n t a l a n a l y s i s : C a l c - f o r C 2 0 H 2 4 N 2 ° 2 z z C ' 7 4 - 0 7 ' H ' 7.40, N 8.64 Found C, 73.83, H, 7.28, N, 8.46. -132-N - c arbome thoxy de shy droxyme thy I s temmadeh i n e (175) To e s t e r (122) (40 mg, 0.123 mmol) i n anhydrous TKF at 0° was q u i c k l y added p o t a s s i u m h y d r i d e (6.38 mg, 0.16 mmol). The s u s p e n s i o n was a l l o w e d t o s t i r f o r 10 min and m e t h y l c h l o r o -formate (0.04 m l , 0.58 mmol) was added. The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r f o r an a d d i t i o n a l 15 min a f t e r w h i c h a l u m i n a (Act I I I ) was s l o w l y added t o d e s t r o y the excess potassium h y d r i d e The m i x t u r e was then d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and passed t h r o u g h a s i n t e r f u n n e l - the f u n n e l b e i n g washed w i t h e t h y l a c e t a t e (10 m l ) . The s o l u t i o n was d r i e d and s t r i p p e d o f s o l v e n t t o y i e l d , a f t e r chromatography on s i l i c a g e l the r e q u i r e d N a -carbomethoxy e s t e r (175) (28 mg, 59%) Me OH U.V.:/| m a x 296 (3. 6 7 ) , 285 (3. 8 8 ) , 263 (4.22), 228 (4.34) nm. I.R.-.l) C H C 1 3 2980, 2900 (sh) , 1730 cm" 1 NMR: £(CDC1 3) 100 MHz (FT) 8.23-8.05, 7.59-7.14 (m, 4H, arom.), 5.44 (q, IH, J=7Hz, C-19H) , 4.00 ( s , 3H, N- C0 2CH_ 3), 3.95 (d, IH, J=3Hz, C-16H), 3.65 ( s , 3H, C 0 2 C H 3 ) , 1.85 (d, 3H, J=7Hz, C-18H 3). M.S. m/e: 382 ( M + ) , 323, 194, 123 Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 2 H 2 6 N 2 ° 4 = 3 8 2 ' 1 8 9 2 Found=382.1883. 1 8 / 3 - carbomethoxycleavamine (72) P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e . 1 ^ Y i e l d = 7 9 % M.pt.=121° ( l i t m.pt.=122.24°) -133-N a-carbomethoxy-T8 j3 -carbomethoxycleavamine (180) 161 P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e Y i e l d = 6 7 % M.pt.=123° ( l i t m.pt.= 126°) N a-methyl-18 -carbomethoxycleavamine (18 3) and 9-methy 1-18/?--carbomethoxycleavamine i h d o l e h i n e (184) To e s t e r (72) (110 mg, 0.325 mmol) i n anhydrous THF a t 0° was added p o t a s s i u m h y d r i d e (26 mg, 0.65 mmol). S t i r r i n g was c o n t i n u e d f o r 30 min and m e t h y l i o d i d e (48 mg, 0.338 mmol) was added. S t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 3 0 min and a l u m i n a (Act I I I ) was added t o d e s t r o y t h e r e m a i n i n g p o t a s s i u m h y d r i d e . The m i x t u r e was d i l u t e d w i t h e t h y l a c e t a t e (20 ml) and then poured t h r o u g h a g l a s s s i n t e r f u n n e l . The s o l v e n t was removed i n vacuo t o g i v e an o i l w h i c h , f o l l o w i n g chromato-graphy on s i l i c a g e l , a f f o r d e d a 1:1 m i x t u r e o f N a-methy1-18/5-carbomethoxycleavamine (183) (40 mg, 35%) and 9-methyl-18^ / 5 -carbomethoxycleavamine i n d o l e n i n e (184) (40 mg, 35%) N^-methyl-18/?-carbomethoxycleavamine (183) l.R.-.l) CHC1., 3010 (sh) , 2930, 1725, 1475 cm - 1. V max 3 U.V.: A m l x H 2 9 3 (4.068),287 (4.099),279 ( 4 . 0 3 ) , 228 (4.78) nm. NMR: & (CDC1 3) 100 MHz (FT) 7.58-7.46 (m, IH, C-14 H), 7.32-6.98 (m, 3H, arom), 5.73 (d, IH, J=7Hz, C-18H), 5.39 (d, IH, J=3Hz, C-5 H), 3.62 ( s , 3H, N - C H 3 ) , 3.57 ( s , 3H, C 0 2 C H 3 ) , 3.15 ( s , 2H, C-5 H) , 2.06 (q, 2H, J=7.4 Hz, CH_2CH3) , 1.06 ( t , 3H, J=7.4 Hz, CH 2CH 3). -134-M.S. m/e: 352 ( M + ) , 337, 229, 136, 135, 124, 122 Hig h r e s o l u t i o n mass spectrum: C a l c — f o r 8 N 2 ° 2 = 3 ! ^ 2 • 2150 Found=352.2149 9-methyl - 1 8 y 0 -carbomethoxycleavamine (184) M.pt.= 9 0° I.R.: 0 m = v CHClo 2980, 1725, 1618 c m - 1 Illci X u - v - : A m!x H 2 6 2 ( 3 . 7 7 ) , 219 ( s h ) (4.24) nm. NMR: <£(CDC13) 100 MHz (FT) 7.72-7.55 . (m, IH, C-14 H) , 7.44-7.13 (m, 3H, arom), 5.55 (d, IH, J=6Hz, C-3H), 4.87 (d, IH, J = l l H z , C-18 H) , 3.61 ( s , 3H, C02CH_3) , 1.31 ( s , 3H, C-9 CH_ 3), 1.07 ( t , 3H, J=7.5 Hz, CH2CH_3) . M.S. m/e: 352 (M+.) , 337, 293, 228, 135, 122 Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 2H 2gN 20 2=352.2151 Found=352.2151 9-benzyl-18y(g -carbomethoxycTeavamine i n d o l e n i n e (185) To e s t e r (72) (100 mg, 0.30 mmol) i n anhydrous THF (5ml) a t 0° was added p o t a s s i u m h y d r i d e (23.5 mg, 0.59 mmol). S t i r r i n g was c o n t i n u e d f o r 30 min and b e n z y l bromide (52 mg, 0. 31 mmol) was added. S t i r r i n g was c o n t i n u e d f o r an a d d i t i o n a l 30 min a t r . t . The tem p e r a t u r e was the n l o w e r e d t o 0° and al u m i n a (Act I I I ) added t o d e s t r o y the r e m a i n i n g p o t a s s i u m h y d r i d e . The m i x t u r e was d i l u t e d w i t h e t h y l a c e t a t e (10 ml) and f i l t e r e d . The s o l v e n t was removed i n vacuo t o y i e l d an o i l w h i c h , f o l l o w i n g chromatography on s i l i c a g e l , a f f o r d e d (185) . 1. R.: [) CHC1. 2970, 1745, 1460, 700 cm" 1. Ilia. X o U.V.: A " S ° H 260 (3. 7 6 ) , 222 (sh) (4.78) nm. Ill 3 .X -135-NMR: & (CDC1 ) 100 MHz (FT) 7.69 (d, lH,J=7.8Hz, Ph CH), 7.41-6.86 (m, 9H, arom.), 6.39 (d, IH, J=7.8 Hz, Ph CH), 5.35 (d, IH, J=6Hz, C-3 H), 4.50 (d, IH, J=10 Hz, C-18 H), 3.58 ( s , 3H, C 0 2 C H 3 ) , 2.47 ( s , 2H, C-5H), 1.1 ( t , 3H, J=7.5Hz CH 2CH 3). M.S. m/e: 428, (M +),387, 337, 230, 214, 136, 135, 122 H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C 0 0 H N o0~=428.2471 / o 3 2 -^Found=428.2467 -136-INTRODUCTION - PART I I I E l u c i d a t i o n o f b i o s y n t h e t i c r o u t e s t o d i m e r i c a l k a l o i d s P o s t u l a t e s a c c o u n t i n g f o r the f o r m a t i o n of the monomeric a l k a -l o i d s have so f a r been d i s c u s s e d i n p r e v i o u s s e c t i o n s . F u r t h e r c o n s i d e r a t i o n s a re n e c e s s a r y t o a c c o u n t f o r the f o r m a t i o n o f the m e d i c i n a l l y i m p o r t a n t d i m e r i c a l k a l o i d s , v i n c r i s t i n e (VCR) (201) and v i n b l a s t i n e (VLB) (202) . I t i s i m m e d i a t e l y o b v i o u s t h a t the upper h a l f o f t h e m o l e c u l e c o r r e s p o n d s t o a " c l e a v e d " c a t h a r a n t h i n e (12) m o i e t y , whereas the lower h a l f c o r r e s p o n d s t o v i n d o l i n e (.11) . I n an a t t e m p t t o det e r m i n e the p r e c u r s o r r o l e i n the d i m e r i c a l k a l o i d s , S c o t t a d m i n i s t e r e d l a b e l l e d v i n d o l i n e (11) and 91 c a t h a r a n t h i n e (12) t o C. r o s e u s . I n c o r p o r a t i o n l e v e l s o b t a i n e d d i d n o t " s e t t l e the q u e s t i o n " b e i n g o f the o r d e r 0.05 and 0.005% r e s p e c t i v e l y . No d e t a i l s o f l a b e l l i n g v/ere g i v e n nor t h a t o f any d e g r a d a t i o n of the i s o l a t e d a l k a l o i d . H u t c h i n s o n l a t e r 1 ^ 4 a d m i n i s t e r e d (3,6 3H_) l o g a n i n ( 3 5 ) , -137-14 shown below, i n a d m i x t u r e w i t h CO^CH^ l o g a n i n (35) t o C. ro s e u s and i s o l a t e d VLB (202) , c a t h a r a n t h i n e (12) and v i n d o l i n e (11) . 3 14 The H/ C r a t i o f e d was 8.2. 3 5 The i n c o r p o r a t i o n d a t a o b t a i n e d i s shown i n t a b l e 9 T a b l e 9 - I n of C. r o s e u s 3 14 T a b l e 9 - I n c o r p o r a t i o n of ( H, C) l o g a n i n (35) i n t o a l k a l o i d s S p e c i f i c ? o r a t 3 (%) I s o l a t e d H 14 I n c o r p o r a t i o n 3 / C R a t i o V i n b l a s t i n e (202) 0.069 7.5 C a t h a r a n t h i n e (12) 0.39 8.7 V i n d o l i n e (11) 0.26 6.0 I t was s t a t e d t h a t a v e r a g i n g the 3H/ 1 4C r a t i o f o r c a t h a r a n t h i n e (12) and v i n d o l i n e (11) gave a paper r a t i o f o r VLB (202) o f 7.35 wh i c h was s i g n i f i c a n t l y c l o s e t o t h a t i s o l a t e d . The r a t i o s o f s p e c i f i c a c t i v i t y o f (35) t o (11) and (12) r e s p e c t i v e l y were c o n s i d e r e d t o m i r r o r the i n v e r s e m o lar r a t i o o f t h e i r p o o l s i z e s w h i c h l e d to the s u g g e s t i o n t h a t a s t e a d y -s t a t e was i n o p e r a t i o n where the r e l a t i v e r a t e s o f f o r m a t i o n o f (11) and (12) were the same. -138-I f t h e r e l a t i v e r a t e o f f o r m a t i o n o f VLB (202) was t h e same from f o r m a t i o n o f an i n t e r m e d i a t e between a c r y l i c e s t e r (70) and c a t h a r a n t h i n e ( 1 2 ) o r v i n d o l i n e ( 1 1 ) , o r c a t h a r a n t h i n e (12) o r v i n d o l i n e (11) them s e l v e s t h e n i t was argued t h a t the 3 14 H/ C r a t i o would be a n t i c i p a t e d t o f a l l between t h a t o f c a t h a r a n t h i n e (12) o r v i n d o l i n e ( 11). The H/ C r a t i o i s o l a t e d f o r (202) and (12) a r e w i t h i n e x p e r i -m e n t a l e r r o r b u t t h a t o b t a i n e d f o r v i n d o l i n e (11) shows a l o s s o f 27% t r i t i u m . No s c i e n t i f i c e x p l a n a t i o n was f o r w a r d e d f o r t h i s o b s e r v a t i o n . I n o r d e r t o a s c e r t a i n the p o s s i b l e means by w h i c h t h e s e d i m e r i c a l k a l o i d s m ight be formed i n v i v o we f o c u s s e d our a t t e n t i o n on a number o f i n v i t r o o b s e r v a t i o n s . 165 I n t h i s r e s p e c t , i t had been n o t e d t h a t c a t h a r a n t h i n e (12) on t r e a t m e n t w i t h r e f l u x i n g a c e t i c a c i d f o l l o w e d by sodium b o r o h y d r i d e gave, i n h i g h y i e l d , the c l e a v e d c a t h a r a n t h i n e system, 18^-carbomethoxycleavamine (72). ( F i g u r e 47) 7 0 3. 14 - 1 3 9 -C0 2C H 3 I 2 2 0 3 72 F i g u r e 47 - A c i d c a t a l y s e d f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . The c l e a v a m i n e s e r i e s o f compounds have found use i n r e c e n t 166 t i m e s i n the s y n t h e s e s o f d i m e r i c a l k a l o i d s , u n f o r t u n a t e l y , p o s s e s s i n g t h e " u n n a t u r a l " s t e r e o c h e m i s t r y a t c - 1 8 . T h i s approach u t i l i s e d i n i t i a l a t t a c k by p o s i t i v e c h l o r i n e t o g i v e an i n t e r m e d i a t e c h l o r o i n d o l e n i n e (204) w h i c h then r e a c t e d w i t h vindo-l i n e (11) i n a c i d media t o g i v e the c o r r e s p o n d i n g dimer. ( F i g u r e 48 ) C 0 2 C H 3 U N N A T U R A L D I M E R S F i g u r e 4 8 - S y n t h e s i s o f d i m e r i c a l k a l o i d s : c h l o r o i n d o l e n i n e approach, The problem, w i t h r e s p e c t t o s t e r e o c h e m i s t r y , has been -140-1 fc\ 1 r e s o l v e d v i a a m o d i f i e d P o l o n o v s k i r e a c t i o n ( F i g u r e 4 9 ) . I n t h i s c a s e , the N-oxide o f c a t h a r a n t h i n e (205) was t r e a t e d w i t h ( i ) t r i f l u o r o a c e t i c a n h y d r i d e a n d v i n d o l i n e (11) a n d ( i i ) sodium b o r o h y d r i d e . Dimers p o s s e s s i n g " n a t u r a l " s t e r e o c h e m i s t r y a t C-18 were p r e d o m i n a n t l y formed. The c o u p l i n g p r o c e s s can be viewed as e i t h e r c o n c e r t e d o r n o n - c o n c e r t e d . 2 0 5 C 0 2 C H 3 l - C O C F . , ^ / u ' ^ T ^ < < c o n c e r t e d C 0 2 C H 3 1) V i n d o l i n e 2) N a B H 4 D I M E R S 2 0 3 C0£H 1) V i n d o l i n e 2) N * B H 4 D I M E R 5 F i g u r e 49 - S y n t h e s i s o f d i m e r i c a l k a l o i d s m o d i f i e d P o l o n o v s k i approach. F u r t h e r e x a m i n a t i o n o f f i g u r e s 47-49 from a b i o s y n t h e t i c s t a n d p o i n t y i e l d e d the f o l l o w i n g i n f o r m a t i o n . (1) f i g u r e 47 can be c o n s i d e r e d b i o s y n t h e t i c a l l y as i n v o l v i n g an i n i t i a l e n z y m a t i c a t t a c k ( f o r example, E ^enzyme, see f i g u r e 50) f o l l o w e d by d i s p l a c e m e n t t o g i v e c h a r g e d i n t e r m e d i a t e (203) whic h t h e n c o u p l e s w i t h v i n d o l i n e (11) t o g i v e the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s . ^ 2 0 3 • D I M E R S F i g u r e 50 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : enzyme a s s i s t e d f r a g m e n t a t i o n and c o u p l i n g . + + The enzyme may be e n v i s a g e d t o " d e l i v e r " OH , OOH o r an e q u i v a l e n t f u n c t i o n a l i t y . I t s h o u l d be n o t e d t h a t o x i d a t i o n o f i n d o l e s t o t h e i r c o r r e s p o n d i n g hydroxy - o r h y d r o p e r o x y i n d o l e -16 8 n i n e s i s a w e l l known p r o c e s s , thus g i v i n g s u p p o r t t o t h i s h y p o t h e s i s . (2) f i g u r e 48 w a s i n v o l v e d w i t h t h e w e l l known c h l o r o i n d o l e n i n e x approach t o the s y n t h e s i s o f d i m e r i c a l k a l o i d s from c l e a v a m i n e systems. I n t h i s r e s p e c t , however, no c l e a v a m i n e monomers have been i s o l a t e d from C. r o s e u s , b u t t h i s does not p r e c l u d e an i n v i v o f r a g m e n t a t i o n o f c a t h a r a n t h i n e (12) t o an lSyJ-carbomethoxy c l e a v a m i n e - ( 7 2 ) - t y p e i n t e r m e d i a t e . C o n s e q u e n t l y a t t a c k on an i n t e r m e d i a t e o f type (72) by + + E =0H , OOH, o r an enzyme ( f i g u r e 51) would g i v e an i n t e r m e d i a t e analogous t o (204) w h i c h i n a s i m i l a r manner c o u l d c o u p l e w i t h v i n d o l i n e t o y i e l d ' n a t u r a l ' d i m e r i c a l k a l o i d s . I n t h i s way, i t w ould o c c u r r e d be e n v i s a g e d t h a t e n z y m a t i c c o n t r o l g i v i n g the c o r r e c t s t e r e o c h e m i s t r y . o f the c o u p l i n g F i g u r e 51 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c i n d o l e n i n e approach. (3) N - o x i d e s are f r e q u e n t l y found i n n a t u r e and, i n t h i s r e s p e c t , i n v o l v e m e n t o f an enzyme as shown c o u l d be p o s t u l a t e d t o y i e l d the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s i n v i v o . ( F i g u r e 52) o " o-E • D I M E R S 2 0 5 F i g u r e 52 - B i o s y n t h e s i s o f d i m e r i c a l k a l o i d s : e n z y m a t i c " P o l o n o v s k c o u p l i n g . I t s h o u l d a l s o be n o t e d t h a t f i g u r e s 50-52 c o u l d a l s o o c c u r - 1 4 3 -v i a one e l e c t r o n p r o c e s s e s and w h i l s t t h i s had no s u b s t a n t i a l i n v i t r o s u p p o r t , i t must be g i v e n f u l l c o n s i d e r a t i o n i n the i n v i v o s t u d i e s . As the i n i t i a l s t u d i e s w i t h l a b e l l e d c a t h a r a n t h i n e (12) i n 91 C. r o s e u s had met w i t h l i t t l e s u c c e s s , f u l l c o n s i d e r a t i o n o f o t h e r p o s s i b l e b i o s y n t h e t i c i n t e r m e d i a t e s were made. I n t h i s r e s p e c t , however,the p o s s i b l e i n v o l v e m e n t o f hydroxy o r h y d r o -peroxy t y p e i n t e r m e d i a t e s o f c a t h a r a n t h i n e (12) o r 18/?-carbo-methoxycleavamine (72) were n o t pu r s u e d due t o t h e i r s u s p e c t e d i n s t a b i l i t y . I t was d e c i d e d i n s t e a d t o i n v e s t i g a t e the p o s s i b l e r o l e o f c a t h a r a n t h i n e - N ^ - o x i d e ( 2 0 5 ) as a p r e c u r s o r i n the d i m e r i c a l k a -l o i d b i o s y n t h e s i s o f v i n c r i s t i n e (201) . C a t h a r a n t h i n e - N g o x i d e (205) on s t a n d i n g a t room t e m p e r a t u r e f o r 169 p r o l o n g e d p e r i o d s r e a r r a n g e s t o g i v e (206). However, i t was f e l t t h a t t h i s s i t u a t i o n would n o t be o f g r e a t i m p o r t a n c e as t h e a d m i n i s t r a t i o n p e r i o d t o t h e p l a n t was t o be o f s h o r t d u r a t i o n . 2 0 6 A more i m p o r t a n t p o i n t was the e x p e c t e d low c o n c e n t r a t i o n o f v i n c r i s t i n e (201) i n C. r o s e u s (~1 x 1 0 - 4 % ) 1 7 ^ and, c o n s e q u e n t l y , i t was f e l t t h a t a p a r a l l e l programme s h o u l d be d e v e l o p e d t o g e n e r a t e C. roseus t i s s u e c u l t u r e s w h i c h would a c t i v e l y b i o s y n t h e --144-s i s e c a t h a r a n t h i n e (12), v i n d o l i n e (11) and the c o r r e s p o n d i n g d i -m e r i c a l k a l o i d s (201,202). 118 82—83 As mentioned p r e v i o u s l y , S c o t t and Zenk have made some p r o g r e s s i n t h i s a r e a b u t so f a r have been unable t o r e p o r t the b i o s y n t h e t i c f o r m a t i o n o f ( 1 1 ) , ( 1 2 ) , (201) o r (202) from t h e s e c u l t u r e s . However t h e r e has been c o n s i d e r a b l e s t i m u l a t i o n i n t h e p a s t f i f t e e n y e a r s t o f i n d growth c o n d i t i o n s f o r t i s s u e c u l t u r e s t h a t w o uld p r o v i d e a s o u r c e f o r t h e s e a l k a l o i d s . The p r o g r e s s i n t h i s a r e a i s d e p i c t e d h i s t o r i c a l l y as f o l l o w s . 171 I n i t i a l i n v e s t i g a t i o n by Carew e t a l . y i e l d e d C. r o s e u s c a l l u s t i s s u e c u l t u r e s r e p o r t e d l y p o s s e s s i n g a s i m i l a r a l k a l o i d a l p a t t e r n t o t h a t o f C. r o s e u s seeds. Four u n i d e n t i f i e d a l k a l o i d s were n o t e d . 172 F u r t h e r i n v e s t i g a t i o n s by Carew w i t h t h e s e c u l t u r e s s u g g e s t e d the p r e s e n c e o f numerous a l k a l o i d s . These, t o o , were u n i d e n t i f i e d a l t h o u g h one was c o n s i d e r e d t o be v i n d o l i n e ( 1 1 ) . 17 3 Boder e t a l . grew C. roseus crown g a l l t i s s u e c u l t u r e s and n o t e d t h e p r e s e n c e o f two a l k a l o i d a l components - one h a v i n g a s i m i l a r Rf t o t h a t o f v i n d o l i n e (11) . I n c u b a t i o n o f t h e c u l -t u r e s w i t h v i n d o l i n e (11) gave t h r e e m e t a b o l i t e s - one b e i n g p o s t u l a t e d t o be d e s a c e t y I v i n d o l i n e (207a). 174 F u r t h e r s t u d i e s w i t h C. r o s e u s stem and l e a f c a l l u s c u l t u r e s a l s o gave r i s e t o the s u g g e s t i o n t h a t v i n d o l i n e (11) and v i n d o l i n i n e (208) v/ere p r e s e n t •••145-2 0 7 a , R =H . 2 0 8 b , R = C 0 C H 3 , A - l c H 2 ) 2 175 G a r n i e r and M o r e l s t u d i e d the a l k a l o i d a l c o n t e n t of C. r o s e u s t i s s u e c u l t u r e s under a v a r i e t y o f c o n d i t i o n s . I n t h i s r e s p e c t i t was n o t e d t h a t the a l k a l o i d a l p r o d u c t i o n was i n c r e a s e d by a d d i t i o n o f t r y p t o p h a n (lb) and 2,4 D. Moreover the a l k a l o i d a l c o n t e n t o f the c u l t u r e s were n o t e d t o be d i f f e r e n t from t h a t o f the whole p l a n t e x t r a c t a l t h o u g h no f u r t h e r i d e n t i f i c a t i o n was made. I n t h i s p a r t i c u l a r i n v e s t i g a t i o n i t was a l s o found t h a t the r a t e o f c u l t u r e growth was i n v e r s e l y p r o p o r t i o n a l t o the a l k a -l o i d a l c o n t e n t . 176 Carew r e c e n t l y r e v i e w e d t h e o v e r a l l p r o g r e s s i n t h i s a r e a and, s i g n i f i c a n t l y , c i t e d t h a t P a t e r s o n had been u n a b l e t o de-m o n s t r a t e the o c c u r r e n c e o f c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e (11) o r the c o r r e s p o n d i n g d i m e r i c a l k a l o i d s (201,202) i n C. r o s e u s c a l l u s c u l t u r e s . Carew then e l a b o r a t e d t h a t , " t h e s e o b s e r v a t i o n s c l e a r l y i n d i -c a t e t h a t d i f f e r e n c e s e x i s t between the s y n t h e t i c c a p a b i l i t y o f the whole p l a n t and t h a t o f i s o l a t e d c a l l u s t i s s u e " . However he f a i l e d t o e x p l a i n t h i s r e s u l t i n the l i g h t o f h i s 172 own communication d e a l i n g w i t h the p r e s e n c e o f v i n d o l i n e (11) i n s i m i l a r c a l l u s c u l t u r e s . R e c e n t l y m e t a b o l i c s t u d i e s o f v i n d o l i n e ( 1 1 ) , c a t h a r a n t h i n e -146-(12) h y d r o c h l o r i d e and v i n b l a s t i n e s u l p h a t e (202) , by i n c u b a t i o n w i t h C. r o s e u s t i s s u e c u l t u r e s have a l s o been 177 s t u d i e d . I n t h i s r e s p e c t , v i n d o l i n e (11) was n o t e d t o be c o n v e r t e d t o d e s a c e t y l v i n d o l i n e (207a) and d i h y d r o v i n d o l i n e (207b). No m e t a b o l i t e s from c a t h a r a n t h i n e (12) were n o t e d . I n c u b a t i o n w i t h v i n d o l i n e (11) and c a t h a r a n t h i n e (12) gave o n l y " m e t a b o l i t e s " d e s a c e t y l v i n d o l i n e (207a) and d i h y d r o v i n d o l i n e (207b). I n c u b a t i o n w i t h v i n b l a s t i n e (202) gave t h r e e u n i d e n t i f i e d " m e t a b o l i t e s " - two h a v i n g m/e>750 and one h a v i n g an m/e 418. These s t u d i e s and c o n c l u s i o n s p r e v i o u s l y d i s c u s s e d have s e v e r e l i m i t a t i o n s . C h a r a c t e r i s a t i o n o f the a l k a l o i d s from 1962-77 have o n l y been by TLC e x a m i n a t i o n . On o c c a s i o n the p l a t e s were viewed under u l t r a v i o l e t l i g h t and s p r a y e d w i t h a p p r o p r i a t e r e a g e n t s . C o - s p o t t i n g w i t h r e f e r e n c e a l k a l o i d s was not pursued i n any o f t h e s e i n v e s t i g a t i o n s n o r was any s p e c t r a l i n f o r m a t i o n o b t a i n e d . C o n s e q u e n t l y t h e s e p u r s u i t s were, a t b e s t , p r e l i m i n a r y , and c h e m i c a l l y , u n s a t i s f a c t o r y . I n t h i s r e s p e c t , now c o n s i d e r the s t a t u s o f t i s s u e c u l t u r e s t u d i e s w i t h a n o t h e r p l a n t V. minor. I t was r e c e n t l y s t a t e d t h a t t i s s u e c u l t u r e s from t h i s s p e c i e s p o s s e s s the same a l k a l o i d a l 17 8 c o n t e n t as t h a t o f the whole p l a n t . I n p a r t i c u l a r , v i n c a m i n e ( 92) was shown t o be p r e s e n t by t h e methods i n d i c a t e d above. 179 F u r t h e r work i n t h i s a r e a by Koch and Kunesch was v e r y i l l u m i n a t i n g . They grew c u l t u r e s o f minor under s i m i l a r c o n d i --147-t i o n s and i n c u b a t e d them w i t h D L ( 2 ± 4 C ) t r y p t o p h a n ( l b ) . TLC comparison o f t h e e x t r a c t s o b t a i n e d showed t h a t no v i n c a m i n e ( 92) was p r e s e n t . C o l d d i l u t i o n o f the e x t r a c t w i t h n o n - l a b e l l e d v i n c a m i n e ( 92)^ f o l l o w e d by r e - i s o l a t i o n o f (92 ) , showed no r a d i o a c t i v i t y , f o l l o w i n g r e p e a t e d c r y s t a l l i s a t i o n , thus s t r o n g l y s u g g e s t i n g i t s absence i n the c u l t u r e . However a component w i t h s i m i l a r TLC b e h a v i o u r and r e a c t i o n w i t h s p r a y r e a g e n t t o t h a t 178 d e s c r i b e d f o r v i n c a m i n e ( 92) was i s o l a t e d and shown t o be the l i g n a n , l i r i o r e s i n o l - y ^ (209) . T h i s compound was c h a r a c t e r i s e d by e s t a b l i s h e d s p e c t r o s c o p i c means (NMR, UV, IR, MS). T h i s i s a f a i r d e m o n s t r a t i o n o f the f o l l i e s i n h e r e n t i n TLC comparison d a t a s i n c e a compound can o n l y be a s s i g n e d a s t r u c t u r e when s u f f i c i e n t s p e c t r a l d a t a i s o b t a i n e d . 82 — 83 I n t h i s r e s p e c t Zenk must r e c e i v e c r e d i t f o r b e i n g t he f i r s t t o have broken the b a r r i e r hampering s c i e n t i f i c p r o g r e s s i n c u l t u r e s o f C. rose u s ( i . e . by u s i n g s p e c t r o s c o p i c p r o c e d u r e s ) . O C H 3 O C H 3 2 0 9 -148-I t can o n l y be hoped t h a t o t h e r s , who f e e l t h e y can c o n t r i b u t e i n t h i s a r e a , w i l l a l s o s t r i v e t o meet t h e s e s t a n d a r d s . I n v e s t i g a t i o n towards growth r e q u i r e m e n t s o f C. r o s e u s t i s s u e c u l t u r e s f o r the f o r m a t i o n o f a l k a l o i d s c a t h a r a n t h i n e ( 1 2 ) , v i n d o l i n e ( l l ) , v i n c r i s t i n e (201) and v i n b l a s t i n e (202) were p u r -sued s i n c e t h e r e was no c o n c r e t e e v i d e n c e f o r t h e i r f o r m a t i o n i n ,.. ^ 125b,180 the l i t e r a t u r e . I f t h e s e c o u l d be d e v e l o p e d t h e n i n v e s t i g a t i o n s a t t h e c e l l -f r e e l e v e l c o u l d be i n s t i g a t e d p a r t i c u l a r l y w i t h r e s p e c t t o (1) d i m e r i c a l k a l o i d b i o s y n t h e s i s and (2) l a t e r s t a g e s o f c a t h a r a n -t h i n e (12) and v i n d o l i n e (11) b i o s y n t h e s i s . The f o l l o w i n g d i s c u s s i o n p r e s e n t s our s t u d i e s as t h e y r e l a t e t o t h i s o v e r a l l a r e a . I n the f i r s t p a r t o f the d i s c u s s i o n the e x p e r i m e n t s d e s c r i b e d p r o v i d e an e f f i c i e n t approach t o v a r i o u s l y l a b e l l e d forms o f c a t h a r a n t h i n e (12) and i t s d e r i v a t i v e s w h i l e the l a t t e r p a r t i l l u s t r a t e s the i n c o r p o r a t i o n s t u d i e s w i t h such p r e c u r s o r s i n p l a n t s . Some p r e l i m i n a r y e x p e r i m e n t s w i t h t i s s u e c u l t u r e s are a l s o i n d i c a t e d . -149-DISCUSSION - PART I I I A l t h o u g h c a t h a r a n t h i n e - N ^ - o x i d e (205) had n o t been i s o l a t e d from C. r o s e u s i t was c o n s i d e r e d t o be the most l i k e l y p r e c u r s o r f o r the i n v i v o c o u p l i n g w i t h v i n d o l i n e ( 1 1 ) . C o n s e q u e n t l y from a b i o s y n t h e t i c s t a n d p o i n t t h e s t r a t e g y was thus t o s y n t h e s i s e l a b e l l e d forms o f c a t h a r a n t h i n e - N ^ - o x i d e (205) and a d m i n i s t e r t h e s e t o t h e C. r o s e u s p l a n t o r t i s s u e c u l t u r e . F o l l o w i n g t h e e x t r a c t i o n p r o c e d u r e , the a l k a l o i d v i n c r i s t i n e (201), would be i s o l a t e d and c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y . P r o v i d i n g p o s i t i v e i n c o r p o r a t i o n was d e m o n s t r a t e d , t h e d i -m e r i c a l k a l o i d v i n c r i s t i n e (201), would then be s u b j e c t e d t o r e d u c t i v e c l e a v a g e t o g i v e velbanamine (210) and c l e a v a m i n e (211) 181 as the i n d o l e d e r i v a t i v e s . I n t h i s r e s p e c t l o c a l i s a t i o n o f t h e a c t i v i t y i n t h e i n d o l i c p o r t i o n o f the m o l e c u l e would be d e t e r m i n e d e i t h e r by c o u n t i n g and/or f u r t h e r d e g r a d a t i o n . The l a b e l l e d forms o f catharanthine-Nj-,-oxide (205) w h i c h were c o n s i d e r e d are shown. They were: (1) g e n e r a l p r o t o n exchange on the a r o m a t i c r i n g by t r i t i u m . (2) r e p l a c e m e n t o f hydrogens a t t a c h e d t o C-19 by t r i t i u m . (3) r e p l a c e m e n t o f carbon i n e s t e r f u n c t i o n by 1 4 C . I n t h i s r e s p e c t , e i t h e r carbon c o u l d be r e p l a c e d s i n c e O-methyl groups 8 7 i n v a r i o u s f e e d i n g e x p e r i m e n t s had n o t been l o s t . However p r e f e r e n c e was t o have the l a b e l a t t a c h e d t o C-18 removing the p o s s i b i l i t y o f any O-methyl l o s s . - 1 5 0 -2 0 5 Double l a b e l l i n g e x p e r i m e n t s c o u l d be p u r s u e d u t i l i s i n g ( 1 ) and ( 2 ) , ( 1 ) and ( 3 ) , and ( 2 ) and ( 3 ) . I n t h i s manner, the p r e -c u r s o r would be l a b e l l e d n o t o n l y on t h e t r y p t a m i n e ( l a ) p o r -t i o n b u t a l s o on t h e n o n - t r y p t a m i n e p a r t . 2 2 ( 1 ) The p r e p a r a t i o n o f (Ar H) c a t h a r a n t h i n e ( 1 2 ) and (Ar H) c a t h -a r a n t h i n e - N ^ - o x i d e ( 2 0 5 ) w a s a t t e m p t e d by a l l o w i n g ( 1 2 ) and ( 2 0 5 ) 2 t o s t a n d a t room t e m p e r a t u r e i n ( H) t r i f l u o r o a c e t i c a c i d f o r 4 8 h o u r s . I t was n o t e d , h o w e v e r , t h a t no c a t h a r a n t h i n e ( 1 2 ) nor c a t h a r a n t h i n e - N j - j - o x i d e ( 2 0 5 ) remained a f t e r t h i s t i me and i n b o t h c a s e s a p l e t h o r a o f p r o d u c t s formed. F u r t h e r e x p e r i m e n t s w i t h c a t h a r a n t h i n e ( 1 2 ) w i t h t h i s r e a g e n t o v e r 9 6 hours a t - 3 0 ° gave 6 7 % r e c o v e r y o f s t a r t i n g m a t e r i a l b u t < 1% D i n c o r p o r a t i o n . (Table 1 0 ) I t had been, however, n o t e d t h a t a s m a l l q u a n t i t y o f lS^-carbomethoxyclea.vamine ( 7 2 ) was formed i n the f i r s t e x p e r i m e n t w h i c h s u g g e s t e d t h a t f r a g m e n t a -t i o n as shown i n f i g u r e 5 3 had o c c u r r e d . T h i s can be r a t i o n a l -i s e d as p r o c e e d i n g v i a t h e c h a r g e d s p e c i e s ( 2 0 3 ) w h i c h , i n the absence o f h y d r i d e i o n , d i s p r o p o r t i o n a t e d t o g i v e a p y r i d i n i u m -151-7 2 F i g u r e 53 - F o r m a t i o n o f 18-^-carbomethoxycleavamine (72) from c a t h a r a n t h i n e (12) i n t r i f l u o r o a c e t i c a c i d . s p e c i e s and IBp -carbomethoxycleavamine ( 7 2 ) . C o n s e q u e n t l y , o t h e r systems were c o n s i d e r e d f o r the d e u t e r a t i o n s t u d i e s . However t h e s e had t o be r e a d i l y c o n v e r t i b l e t o c a t h a r a n t h i n e -N ^-oxide (205). I n i t i a l l y 1 9 - o x o - c a t h a r a n t h i n e (212) was con-s i d e r e d s i n c e the a b i l i t y t o fragment as i n d i c a t e d i n f i g u r e 53 s h o u l d be d i m i n i s h e d by t h e e l e c t r o n - w i t h d r a w i n g c a p a b i l i t y of the c a r b o n y l group o f t h e l a c t a m . -152-2 1 0 212 211 T h i s l a c t a m (212) was p r e p a r e d a c c o r d i n g t o a p r o c e d u r e 182 u t i l i s e d by B a t t e r s b y e t a l . i n v o l v i n g t r e a t m e n t o f c a t h a r -a n t h i n e (12) w i t h ^/NaHCO^ a t room t e m p e r a t u r e . M e c h a n i s t i -c a l l y t h i s r e a c t i o n can be c o n s i d e r e d t o o c c u r as shown i n F i g u r e 54. T h i s r e a c t i o n was found t o be t e m p e r a t u r e s e n s i t i v e and a t 0° y i e l d e d 5-oxo-18^-carbomethoxycleavamine (213) i n a p p r o x i m a t e l y 25% y i e l d . I t s f o r m a t i o n i s e n v i s a g e d as i n f i g u r e 55. The compound, 5-oxo-18^-carbomethoxycleavamine (213) gave the — 1 18 3 e x p e c t e d (+)15 cm. f r e q u e n c y s h i f t i n the i n f r a r e d f o r an o(,jl-unsaturated l a c t a m . M e c h a n i s t i c a l l y the f o r m a t i o n o f p r o d u c t s (212) and (213) can be c o n s i d e r e d v i a i n i t i a l q u a t e r n a r y s a l t f o r m a t i o n w i t h i o d i n e ( i ) . The f o r m a t i o n o f (212) t h e n i n v o l v e d a b s t r a c t i o n o f hydrogen from C-19 t o g i v e ( i i ) w h i c h t h e n r e a c t e d w i t h hy-d r o x i d e t o g i v e ( i i i ) . S i m i l a r complex f o r m a t i o n ( i v ) , f o l l o w e d by l o s s o f HI gave (v) w h i c h r e a d i l y l o s t a p r o t o n t o g i v e r e q u i r e d (212). The -153-C0 ZCH 3 2 1 2 F i g u r e 54 - P r o p o s e d f o r m a t i o n o f 1 9 - o x o c a t h a r a n t h i n e (212). F i g u r e 55 - P r o p osed f o r m a t i o n o f S - oxo-lS^-carbomethoxycleavamine (213). f o r m a t i o n o f (213) was c o n s i d e r e d t o i n v o k e l o s s o f I v i a a P o l o n o v s k i t y p e r e a c t i o n t o g i v e (203) . F u r t h e r r e a c t i o n w i t h h y d r o x i d e i o n gave ( v i ) w h i c h i n a s i m i l a r manner t o t h a t de--155-s c r i b e d above y i e l d e d ( v i i i ) . R e d u c t i o n o f t h i s t o (213) was c o n s i d e r e d t o be v i a a d i s p r o p o r t i o n a t i o n r e a c t i o n analogous t o t h a t p r e v i o u s l y d e s c r i b e d ( f i g u r e 53) f o r 18/5-carbome thoxy -c l e a v a m i n e (72) v i a i n t e r m e d i a t e (203). Treatment o f 19-oxo-c a t h a r a n t h i n e (212) w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room temp-e r a t u r e f o r 48 hours gave a r e c o v e r y o f <5% a l t h o u g h d e u t e r i u m i n c o r p o r a t i o n was ~ 3 5 % (based on f o u r exchangeable a r o m a t i c p r o -tons ) . D e u t e r a t i o n s t u d i e s were c o n s e q u e n t l y i n i t i a t e d on N -c a r b o m e t h o x y - 1 9 - o x o - c a t h a r a n t h i n e (215) to determine the e x t e n t of d e u t e r i u m i n c o r p o r a t i o n . 184 N - c a r b o m e t h o x y c a t h a r a n t h i n e (214) was p r e p a r e d by t r e a t i n g c a t h a r a n t h i n e (12) w i t h p o t a s s i u m h y d r i d e f o l l o w e d by m e t h y l c h l o r o f o r m a t e . The N a ~ c a r b o m e t h o x y c a t h a r a n t h i n e (214) 2 so formed was t r e a t e d w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room te m p e r a t u r e f o r 60 hours and r e - i s o l a t e d (50% r e c o v e r y ) b u t w i t h no measurable d e u t e r i u m i n c o r p o r a t i o n b e i n g n o t e d . 184 N a - c a r b o m e t h o x y - 1 9 - o x o c a t h a r a n t h i n e (215) was p r e p a r e d by t r e a t m e n t of N a - c a r b o m e t h o x y c a t h a r a n t h i n e (214) w i t h ^/NaHCO^. The N a - c a r b o m e t h o x y - 1 9 - o x o c a t h a r a n t h i n e (215) formed was 2 t r e a t e d w i t h ( H) t r i f l u o r o a c e t i c a c i d a t room temperature f o r 96 hours and r e c o v e r e d i n 63% y i e l d b u t a g a i n w i t h no measurable d e u t e r i u m i n c o r p o r a t i o n . -156-0 H C O C C 0 2 C H 3 C 0 2 C H 3 214 215 Further experiments with catharanthine (12) at reduced temperatures were employed over various time periods and i n th i s respect deuterium incorporation was obtained (see table 10) although, competition from the fragmentation process was s t i l l major product. Highest deuterium incorporation and recovery was obtained in experiment 6. Mass spectral fragmentation of catharanthine correct, deuterium incorporation would a f f e c t peaks at m/e 16 8, 170, 214, and 229 but not at m/e 121 and 135. Table 11 shows the res u l t s i n this respect and i t was noted that peaks 121/122/123 and 135/136 had ra t i o s which were not influenced by the deuterium incorporation. I t should also be noted that the NMR spectrum of deuterated (12) was i d e n t i c a l with the exception that s i m p l i f i c a t i o n of the aromatic region was noted showing the deuterium to be located exclusively i n the aromatic r i n g . important giving 18/3-carbomethoxy cleavamine (72) as the other (12), as postulated 165a , i s shown i n figure 56. If t h i s was -157-N - C H 2 m/e 170 m / e 168 m / e 135 F i g u r e 56 - P r o p o s e d mass s p e c t r a l f r a g m e n t a t i o n o f c a t h a r a n t h i n e ( 1 2 ) . T a b l e 10 - R e a c t i o n s o f c a t h a r a n t h i n e (12) w i t h H and H t r i f l u o r o a c e t i c a c i d . Cath (12) R e l a t i v e r a t i o s o f w t - o f S o l v e n t Temp. t ( h ) Recovery (%) M/M+l E x p t . (12) (mgs) CF 3C0 2H CF. 3C0 2D (x°) (y) Cath (.12) 18 CMC (72) r a t i o M/M+l , M+l/M+2 , M+2/M+3 Volume (ml) 1 30 0. 07 - - 5 24 87 - 4 1 - -2 - 0.14 - 5 72 41 31 0. 89 1 0.92 0. 31 3 0.07 - -15 72 49 - - - - -4 0.07 - -30 96 70 - - - - -5a - 0.07 20 24 18 41 0.95 1 0.85 0.13 5b " 0.07 - 20 24 17 40 - - - -6 " - 0.14 -10 72 55 - 0. 77 0.91 1 0.25 7 - 0.14 -30 96 67 - 4 1 - -8 100 1 - -5 -10 48 50 27 — 1 _ _ I I—1 Ul 00 I -159-The f o r m a t i o n o f 18y# -carbomethoxycleavamine (72) i n th e s e e x p e r i m e n t s was a l m o s t c e r t a i n l y v i a c h a r g e d i n t e r m e d i a t e (203) d i s c u s s e d i n f i g u r e 53 w h i c h was a l s o the p o s t u l a t e d i n t e r m e d i a t e i n the s y n t h e s i s o f the d i m e r i c a l k a l o i d s ( f i g u r e 48-49). To i n v e s t i g a t e t h i s p o s t u l a t e c a t h a r a n t h i n e (12) and v i n d o l i n e (11) were d i s s o l v e d i n t r i f l u o r o a c e t i c a c i d a t (-5)-(-10°) f o r 5 days. Normal b o r o h y d r i d e r e d u c t i o n and work up y i e l d e d 18/3 -carbornethoxy-c l e a v a m i n e ( 7 2 ) , c a t h a r a n t h i n e (12) and v i n d o l i n e ( 1 1 ) . No d i m e r i c a l k a l o i d s were n o t e d thus c a s t i n g doubt on the use o f t h i s s p e c i e s as an i n t e r m e d i a t e i n the c o u p l i n g p r o c e s s . (2) 19-oxocatha.ranthine (212) was p r e p a r e d as p r e v i o u s l y men-t i o n e d . Treatment o f t h i s compound w i t h d i b o r a n e gave c a t h a r a n -185 t h i n e (12) i n 33% y i e l d . C o n s e q u e n t l y t h e employ o f t r i t i a t e d d i b o r a n e would i n t r o d u c e the l a b e l a t the C-19 p o s i t i o n o f c a t h a r a n t h i n e (12). (3) T h i s l e f t t h e pr o b l e m o f d e t e r m i n i n g c o n d i t i o n s f o r l a -b e l l i n g t he carbons o f the e s t e r group o f c a t h a r a n t h i n e (12) a n d j i n t h i s r e s p e c t , c a t h a r a n t h i n e (12) was h y d r o l y s e d t o c a t h a r -186 a n t h i n i c a c i d (216) by base. The r e a c t i o n m i x t u r e was then a c i d i f i e d w i t h a methanol/hydrogen c h l o r i d e s o l u t i o n t o pH 6. Ex-ce s s e t h e r e a l diazomethane was added u n t i l pH 7.5-8. C a t h a r a n -t h i n e (12) was thus r e g e n e r a t e d . T a b l e 11 - Mass s p e c t r a l fragments o f c a t h a r a n t h i n e (12) - d e u t e r i u m e n r i c h m e n t e x p e r i m e n t s R a t i o s o f Peak h e i g h t o f : 121 122 123 135 136 168 169 170 171 214 215 229 230 336 337 1 15.2 : 20 2.2 60 7 40 12 20 : 4.2 20 : 4.2 60 : 11.2 20 4 2 15. 3 : 20 2.4 60 8.2 40 39 .4 20 16.8 20 26.5 60 51 20 22 5 16. 0 20 2.5 60 7.3 40 19 20 11 20 • 12 60 12.4 20 5 6 15. 2 20 : 2.4 60 8 40 : 39.2 20 : 13.4 20 : 27 60 56 20 26 7 15.0 20 : 2.5 60 : 8.2 40 : 11 20 : 4.8 20 : 5.5 60 : 12.4 20 : 5 I H cn o I -161-Thus c o n d i t i o n s f o r l a b e l l i n g t h e e s t e r m e t h y l group were e s t a b l i s h e d . However, as s t a t e d e a r l i e r , i t had been t h e i n t e n -t i o n t o l a b e l the o t h e r c a r b o n atom and l i t e r a t u r e p recedence s t i m u l a t e d t h o u g h t i n t h i s d i r e c t i o n as f o l l o w s : 187 (a) i n t h e s y n t h e s i s o f v o a c a n g i n e (220) from i b o g a i n e (217), c h l o r o i n d o l e n i n e (218) r e a c t e d w i t h c y a n i d e t o g i v e (219) w h i c h on (1) base h y d r o l y s i s and (2) t r e a t m e n t w i t h diazomethane y i e l d e d v o a c a n g i n e (220) ( F i g u r e 57) i n 49% c r u d e y i e l d . t - B u O C I 217 2 1 8 J-tP C 0 2 C H 3 2 2 0 F i g u r e 57 - B u c h i 1 s s y n t h e s i s o f v o a c a n g i n e (220). -162-I n a s i m i l a r , b u t more a p p r o p r i a t e , s y n t h e s i s o f (+) 188 c a t h a r a n t h i n e ( 1 2 ) , s y n t h e t i c h y d r o x y n i t r i l e (221) was c o n v e r t e d t o i t s c o r r e s p o n d i n g c h l o r o i n d o l e n i n e (222) w h i c h r e a c t e d w i t h c y a n i d e t o g i v e cyano d e r i v a t i v e (223). Treatment o f (223) w i t h c o n c e n t r a t e d s u l p h u r i c a c i d gave a crude m i x t u r e o f the n i t r i l e (224) and amide (225) w h i c h on subsequent base h y d r o l y s i s and t r e a t m e n t w i t h diazomethane y i e l d e d (+) c a t h a r a n t h i n e (12) ( f i g u r e 5 8 ) . C o n v e r s i o n o f hydroxy n i t r i l e (223) t o c a t h a r a n t h i n e (12) was r e p o r t e d i n 28% y i e l d . C o n s e q u e n t l y a s y n t h e s i s u s i n g t h i s s t r a t e g y was n e x t con-s i d e r e d , and, i n t h i s r e s p e c t , d e s c a r b o m e t h o x y c a t h a r a n t h i n e (226) was employed as s t a r t i n g m a t e r i a l . I t was hoped t h a t t r e a t m e n t o f t h e c o r r e s p o n d i n g c h l o r o i n d o l e n i n e (227) w i t h c y a n i d e would a f f o r d the n i t r i l e ( 224). H y d r o l y s i s o f t h i s d e r i v a t i v e would h o p e f u l l y r e g e n e r a t e c a t h a r a n t h i n e (12) o r i t s a c i d (216) ( f i g u r e 5 9 ) . The c h l o r o i n d o l e n i n e f o r m a t i o n was f i r s t i n v e s t i g a t e d w i t h 189 c a t h a r a n t h i n e (12) u s i n g a p r o c e d u r e by Lichman i n o r d e r t h a t c o n d i t i o n s f o r i s o l a t i o n c o u l d be d e t e r m i n e d . F o l l o w i n g chromatography, y i e l d s o f 85% f o r (228) were o b t a i n e d . I n a s i m i l a r manner, the c h l o r o i n d o l e n i n e o f descarbomethoxy-c a t h a r a n t h i n e (227) was formed i n 87% y i e l d on t r e a t m e n t o f (226) w i t h N - c h l o r o b e n z o t r i a z o l e . -163-2 2 8 Treatment o f c h l o r o i n d o l e n i n e (227) w i t h p o t a s s i u m c y a n i d e i n d i m e t h y l a c e t a m i d e a f f o r d e d t h e n i t r i l e (224) i n y i e l d s com-p a r a b l e t o t h a t p u b l i s h e d . 1 ^ 7 1 8 8 A c i d c a t a l y s e d h y d r o l y s i s i n methanol a t room and e l e v a t e d t e m p e r a t u r e s f a i l e d t o g i v e any r e a c t i o n i n w h i c h c a t h a r a n t h i n e (12) was formed and, i n one c a s e , the r e a c t i o n was c o n t i n u e d u n t i l a l l s t a r t i n g m a t e r i a l had decomposed. None o f t h e p r o d u c t s n o t e d c o n t a i n e d a c a r b o n y l group. T h i s o b s e r v a t i o n had a l s o been n o t e d i n t h e a t t e m p t e d h y d r o l y s i s o f (219) t o v o a c a n g i n e (220) w i t h hydrogen c h l o r i d e i n methanol and was presumably due t o the f a c t t h a t (219) c o n t a i n s a v e r y h i n d e r e d t e r t i a r y n i t r i l e . Base c a t a l y s e d h y d r o l y s i s w i t h p o t a s s i u m h y d r o x i d e i n d i -e t h y l e n e g l y c o l was t h e n p u r s u e d a t 80, 100, 120, 140, 155 and 160° o v e r p e r i o d s o f 5-16 h o u r s . A c i d i f i c a t i o n a t 0° f o l l o w e d by t r e a t m e n t w i t h diazomethane gave, i n a l l c a s e s , a m u l t i t u d e o f p r o d u c t s b u t none c o r r e s p o n d i n g t o c a t h a r a n t h i n e (12) e x c e p t i n the case o f 155° where a t r a c e o f c a t h a r a n t h i n e (12) may have formed as shown by TLC and mass s p e c t r a l d a t a . Treatment o f t h e n i t r i l e (224) w i t h c o n c e n t r a t e d s u l p h u r i c -164 -165-a c i d , as i n d i c a t e d i n f i g u r e 58 p r o v i d e d an i n f r a r e d s p e c -trum ( t a k e n on the crude m i x t u r e ) p o s s e s s i n g n i t r i l e (2235 cm x ) and amide (1665 cm x ) bands. Subsequent h y d r o l y s i s o f t h i s crude m i x t u r e w i t h p o t a s s i u m h y d r o x i d e i n d i e t h y l e n e g l y c o l , f o l l o w e d by e s t e r i f i c a t i o n as d e s c r i b e d , y i e l d e d no c a t h a r a n t h i n e ( 1 2 ) . F u r t h e r t r e a t m e n t o f c a t h a r a n t h i n i c a c i d (216) w i t h p o t a s s -ium h y d r o x i d e i n d i e t h y l e n e g l y c o l a t 155° f o r 11 hours f o l l o w e d by a c i d i f i c a t i o n and e s t e r i f i c a t i o n a t 0° gave c a t h a r a n t h i n e (12) i n 9% y i e l d . C o n s e q u e n t l y , due t o t h e d i f f i c u l t i e s e n c o u n t e r e d i n t h e s e p r e l i m i n a r y i n v e s t i g a t i o n s , t h i s p a r t i c u l a r avenue o f r e s e a r c h was abandoned as i t was d e c i d e d t h a t t h e m e t h y l d e r i v a t i v e o f c a t h a r a n t h i n e (12) would be employed i n s t e a d . Thus, w i t h t h e a b i l i t y t o l a b e l c a t h a r a n t h i n e (12) as des-c r i b e d , i t was o f some i n t e r e s t t o o b t a i n p r e l i m i n a r y d a t a w i t h 3 (Ar H) c a t h a r a n t h i n e - N ^ - o x i d e (205). C a t h a r a n t h i n e - N b - o x i d e (205) was p r e p a r e d from c a t h a r a n t h i n e (12) by t r e a t m e n t w i t h m e t a c h l o r o p e r b e n z o i c a c i d t o g i v e an i s o l a b l e p ure p r o d u c t . P r e v i o u s a t t e m p t s t o i s o l a t e t h i s com-190 pound i n pure form had met w i t h f a i l u r e , s i n c e i t had been n o t e d t h a t on s t a n d i n g f o r p r o l o n g e d p e r i o d s a t room t e m p e r a t u r e a r e a r r a n g e d p r o d u c t (206) was formed ( f i g u r e 6 0 ) . However, - o x i d e (205) was s t a b l e on s t o r a g e a t 0°. 3 3 (Ar H) c a t h a r a n t h i n e (12) was p r e p a r e d by exchange w i t h ( H) t r i f l u o r o a c e t i c a c i d - t h i s r e a g e n t b e i n g p r e p a r e d by a b u l b - t o -3 b u l b d i s t i l l a t i o n o f t r i f l u o r o a c e t i c a n h y d r i d e i n t o ( H) w a t e r . -166-2 0 5 2 0 6 F i g u r e 60 - P r o p osed rearrangement o f c a t h a r a n t h i n e - N r - o x i d e (205) . b 3 The (Ar H) c a t h a r a n t h i n e (12) o b t a i n e d was c r y s t a l l i s e d t o c o n s t a n t a c t i v i t y and c o n v e r t e d t o the c o r r e s p o n d i n g N^-oxide 3 (205). F o l l o w i n g p u r i f i c a t i o n t e c h n i q u e s , (Ar H) c a t h a r a n t h m e -Nj-,-oxide (205) was a d m i n i s t e r e d t o C. r o s e u s o v e r p e r i o d s o f 3-28 days. To e n s u r e t h a t the p r e c u r s o r was c a t h a r a n t h i n e - N g O x i d e (205) and n o t r e a r r a n g e d p r o d u c t (206) two checks were c a r r i e d out (1) TLC comparison w i t h a sample p r e v i o u s l y s y n t h e s i s e d , a n d 191 (2) r e d u c t i o n w i t h z i n c and a c e t i c a c i d t o r e g e n e r a t e c a t h -a r a n t h i n e (12) . F o l l o w i n g the f e e d i n g p e r i o d , t h e p l a n t was macerated and m ethanol s o l u b l e s removed i n a manner analagous t o t h a t a l r e a d y d e s c r i b e d . I s o l a t i o n o f the crude a l k a l o i d a l f r a c t i o n c o n t a i n i n g VLB (201) and VCR ( 2 0 2 ) , as e v i d e n c e d by TLC c o m p a r i s o n d a t a , 192 was e x e c u t e d u s i n g a g r a d i e n t pH e x t r a c t i o n p r o c e d u r e . I n a c t i v e v i n c r i s t i n e (201) was t h e n added t o t h e e x t r a c t , r e - i s o l a t e d , and c r y s t a l l i s e d t o g i v e d a t a shown i n t a b l e 13. T a b l e 13 - I n c o r p o r a t i o n d a t a from a d m i n i s t r a t i o n o f (Ar H) c a t h a r a n t h i n e - N b ~ o x i d e (205) t o r o s e u s . A c t i v i t y a d m i n i s t e r e d (cpm/mmole) D u r a t i o n o f f e e d i n g (days) S p e c i f i c A c t i v i t y I s o l a t e d Wt. o f VCR (108) i s o l a t e d (mgs) S p e c i f i c A b s o l u t e I n c o r p o r a t i o n I n c o r p o r a t i o n 1.184 x 10" 1.184 x 10-1.184 x 10-1.184 x 10" 1. 594 x 10-3 6 12 21 28 2.26 x 10 1.70 x 10 2.64 x 10 2.03 x 10' 2.75 x 10 15 14 19 16 22 0.0044 0.0031 0.0065 0.0042 0.0078 0.00035 0. 00026 0. 00041 0.00032 0.00043 i i—• l -168-C o u n t i n g was d i s c o n t i n u e d when b i o s y n t h e t i c s i g n i f i c a n c e was c o n s i d e r e d n e g l i g i b l e . F u r t h e r s t u d i e s were t h e r e f o r e n o t p u r s u e d on the i n t a c t p l a n t , a n d , i n s t e a d , work was i n i t i a t e d on C. r o s e u s t i s s u e c u l t u r e s t o d e t e r m i n e i f i t was p o s s i b l e t o f i n d c o n d i t i o n s f o r the f o r m a t i o n o f t h e a l k a l o i d s ( 1 1 ) , ( 1 2 ) , (201) and (202). C o n s e q u e n t l y t i s s u e c u l t u r e s o f C. r o s e u s grown under a v a r i e t y o f c o n d i t i o n s were i n v e s t i g a t e d f o r t h e i r a l k a l o i d a l c o n t e n t . The c u l t u r e s were grown a t the U n i v e r s i t i e s o f B r i t i s h Columbia (UBC) and L e i c e s t e r (England) r e s p e c t i v e l y . The c a l l u s c u l t u r e s a t U.B.C. were h a r v e s t e d a f t e r 3 weeks-4 months growth p e r i o d s . These were removed from t h e s o l i d agar medium ( t a b l e 8) and f r e e z e d r i e d . The c u l t u r e s from t h e U n i v e r s i t y of L e i c e s t e r were p a r t o f 180 a c o l l a b o r a t i v e p r o j e c t . C o n d i t i o n s f o r t h e i r growth have no t y e t been r e l e a s e d b u t f r e e z e d r i e d samples were g i v e n f o r our i n v e s t i g a t i o n s . A l l c u l t u r e s were e x t r a c t e d w i t h m ethanol t o g i v e , a f t e r r e m o v a l o f s o l v e n t , the crude " a l k a l o i d a l " e x t r a c t . The e x t r a c t was t h e n t r e a t e d w i t h ' V HC1 t o remove any a l k a l o i d s as t h e i r h y d r o c h l o r i d e s a l t s . The r e s u l t i n g a c i d i c s o l u b l e s were e x t r a c t e d w i t h benzene. o The aqueous l a y e r was t h e n c o o l e d t o 2 and b a s i f i e d w i t h ammonium h y d r o x i d e t o pH 7. The c h l o r o f o r m s o l u b l e s were t h e n removed. F u r t h e r t r e a t m e n t o f the aqueous l a y e r t o pH 10 was then c a r r i e d o u t and t h e c h l o r o f o r m s o l u b l e s a g a i n removed. -169-Chromatography o f the r e s i d u e s o b t a i n e d from the pH 1,7 and 10 e x t r a c t i o n s were found t o be d e v o i d o f the r e q u i r e d a l k a l o i d s ( 1 1 ) , ( 1 2 ) , (201) o r (202). Indeed no a l k a l o i d s were noted i n any o f the c u l t u r e s i n v e s t i g a t e d . F u r t h e r s t u d i e s w i t h t i s s u e c u l t u r e s ( s o l i d and l i q u i d ) a r e c u r r e n t l y under i n v e s t i g a t i o n as p a r t o f a l o n g term p r o j e c t t o o b t a i n c o n t r o l l e d c o n d i t i o n s f o r a l k a l o i d a l growth i n these l a b o r a t o r i e s . T h i s , and attempts t o i s o l a t e the enzymes from C. r o s e u s , are c u r r e n t l y b e i n g s t u d i e d by P r o f e s s o r S t u a r t . When c o r r e c t growth c o n d i t i o n s a re de t e r m i n e d the r o l e o f c a t h a r -a n t h i n e - N - o x i d e (205) can be r e i n v e s t i g a t e d . F o l l o w i n g the e x p e r i m e n t a l s e c t i o n , s y n t h e t i c s t u d i e s towards the s y n t h e s i s of a l k a l o i d s e l l i p t i c i n e (106) and o l e -v a c i n e (105) are r e p o r t e d . -170-E X P E R I M E N T A L - PART I I I A description of the general experimental information was given in Experimental, parts I and I I . As before procedures, published or unpublished by others, are described in f u l l when (1) detailed experimental was not released (2) unsatisfactory data was presented or (3) procedure was modified. 182 19-0xocatharanthine (212) and 5-oxo-18yg carbomethoxy- cleavamine (213) Sodium bicarbonate (65 mg, 0.774 m.mol) i n d i s -t i l l e d water (1 ml) was added to catharanthine (12) (45 mg, 0.134 m.mol) i n tetrahydrofuran (THF) (1.2ml) at room tem-perature. Iodine (59 mg, 0.232 m.mol) i n THF (1 ml) was added over a oeriod of 8 min and afte r an additional 30 min, the solution was dil u t e d with water (20 ml) and extracted with chloroform (4x15 ml). The chloroform layers were combined and washed with (1) 2 x 10 ml 0.1 M sodium thiosulphate (2) 10 ml water (3) 10 ml 2 N H 2S0 4 and (4) 10 ml H 20. Chromatography of the dried chloroform extract on s i l i c a gel gave 19-oxocatharanthine (212) (10.4 mg, 22%) and the lactam (213) (2.9 mg, 6 %) . 19-0xocatharanthine (212) was compared with an authentic 184 sample prepared v i a a longer pathway and was i d e n t i c a l i n a l l respects. 19-Oxocatharanthine (212) I.R.: 0 _„_. 3460,3000,2980-2960,2880,1725,1670(sh),1655,1520, U l L l 3 -171-1275-50 cm U.V.: A m J ° H 222, 276 (3.919), 283 (3.953), 292 (3.896) nm. NMR: c f (CDC1 3) 100 MHz (FT) 8.02 ( s , IH, N-H) , 7.51-7.34 (m, IH, arom.), 7.22-7.05 (m, 3H, arom.), 6.12 (dd, IH, J=2 and 4 Hz, C-3 H), 5.05 (d, IH, J=2 Hz, C-5 H), 4.26-3.94 (complex m, IH, C-2 H) , 3.64 ( s , 3H, C0 2CH_ 3) , 3.42-3.08 (complex m, 4H, C 7, C 8, U2)f 2.58 (dd, IH, J=2 and 12 Hz, C - l H), 2.34-2.14 (complex m, 3H, CH 2CH 3 + C 1 H) , 1.05 ( t , 3H, J=7 Hz, CH2CH_3) . M.S. m/e: 350 (M +) 228, 227, 195, 168, 167, 154. H i g h r e s o l u t i o n mass spectrum: c a l c ^ f o r C2iH22 N2°3 = 350•1630 Found=350.1627. 5-Oxo - 1 8 y g-carbomethoxycleavamlne (213) and 1 9 - o x o c a t h a r a n t h i n e (212) C a t h a r a n t h i n e (12) (90 mg, 0.268 mmol) i n THF (2.6 ml) was added t o a s o l u t i o n o f sodium b i c a r b o n a t e (130 mg, 1.548 mmol) i n d i s t i l l e d w a t e r (2 m l ) . The t e m p e r a t u r e was low-e r e d t o 0° and i o d i n e (108 mg, 0.425 mmol) i n THF (2 ml) was added o v e r a t h r e e h r p e r i o d . The s o l u t i o n was d i l u t e d w i t h w a t e r (20 ml) and e x t r a c t e d w i t h methylene c h l o r i d e ( 3x15 m l ) . The methylene c h l o r i d e l a y e r s were combined and washed w i t h (1) 2 x 10 ml 0.1 M sodium t h i o s u l p h a t e (2) 2 x 10 ml 2 N H 2 S 0 4 (3) 2 x 10 ml w a t e r . Chromatography o f the methylene c h l o r i d e s o l u b l e s on s i l i c a g e l y i e l d e d the l a c t a m (213) (20.2 mg, 20%) and the l a c t a m (212) (6 mg, 6%). 5 - 0 x 0 - 1 8 ^ carbomethoxycleavamine (213) I.R.: 0 CHC1 3 3460, 2960-40, 2870-50, 1745 ( s h ) , 1720, 1675, 1460 c m - 1 . -172-U.V.: X E t 0 H 223 ( 4 . 4 2 ) , 276 ( 3 . 8 1 ) , 283 ( 3 . 8 3 ) , 291 (3.77) nm. ITlclX NMR: 100 MHz £(CDC1 3) (FT) 8.20 ( s , IH, N-H), 7.64-7.10 (com-p l e x m, 4H, arom.) 6.55 (d, IH, J=8 Hz, C-3 H), 3.80 (s, 3H, C 0 2 C H 3 ) , 2.18 (q, 2H, J=7 Hz, C H 2 C H 3 ) , 1.00 ( t , 3H, J=7 Hz, C H 2CH 3). M.S. m/e: 352 ( M + ) , 336, 293, 291, 264, 263, 229, 228, 227, 214. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C 2^H 2 4N 20 3=352.1787 Found=352.1787. ( 2H) T r i f l u o r o a c e t i c a c i d ( 2H) Water (99.95%D) (0.2 m l , 10 mmol) was added t o r e d i s t i l l e d t r i f l u o r o a c e t i c a n h y d r i d e (2.75 m l , 10.5 mmol) a t -78°. The t e m p e r a t u r e was a l l o w e d t o r i s e s l o w l y t o room temp-2 e r a t u r e o v e r 15 min g i v i n g ( H) t r i f l u o r o a c e t i c a c i d . ( A r 2 H ) - 1 9 - O x o c a t h a r a n t h i n e (212) - G e n e r a l d e u t e r a t i o n p r o c e d u r e 1 9 - O x o c a t h a r a n t h i n e (212) (20 mg, 0.0568 mmol) was d i s s o l v e d i n ( 2H) t r i f l u o r o a c e t i c a c i d (1 m l , 13.35 mmol) a t room t e m p e r a t u r e . A f t e r 60 h r , t h e t e m p e r a t u r e was c o o l e d t o 0° and s a t u r a t e d sodium b i c a r b o n a t e added t o pH 9. E x t r a c t i o n w i t h methylene c h l o r i d e ( 3 x 5 ml) gave, a f t e r d r y i n g and chroma-2 t o g r a p h y on s i l i c a g e l , (Ar H) 1 9 - O x o c a t h a r a n t h i n e (212) (1 mg, 5%) . "I p A N ^ - C a r b o m e t h o x y c a t h a r a n t h i n e (214) P r e p a r e d a c c o r d i n g t o l i t e r a t u r e p r o c e d u r e and had a l m o s t i d e n t i c a l p r o p e r t i e s w i t h t h a t r e p o r t e d . ( Y i e l d = 7 7 % , L i t . y i e l d = 8 3 % ) . -173-Attempted deuteration of UR-Carbometho'xycatharanthine (214) (2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol) was added to N a-carbomethoxycatharanthine (214) (30 mg, 0.076 mmol). Following incubation for (1) 24 (2) 60 hr , N -carbomethoxycatharanthine (214) was i s o l a t e d (according to gen-e r a l procedure described) i n (1) 70 and (2) 50% y i e l d respectively. N.M.R. and M.S. showed no measurable deuterium incorporation. 184 N a-Carbomethoxy-19-oxocatharanthine (215) Prepared according to l i t e r a t u r e procedure and had almost i d e n t i c a l properties with that reported by the second procedure quoted. Attempted deuteration of N a-carbomethoxy-19-oxocatharanthine (215) (2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol) was added to N a-Carbomethoxy-19-oxocatharanthine (215) (30 mg, 0.074 mmol). After incubation for 96 hr, i s o l a t i o n , according to general procedure described, gave (215) (19.5 mg, 65% recovery). N.M.R. and M.S. showed no measurable deuterium incorporation. Catharanthine (12) and 18 /3-carbomethoxycleavamine (72) (XH) or( 2H) T r i f l u o r o a c e t i c acid (0.07 ml, 0.93 mmol), was added to catharanthine (12) (30 mg, 0.089 mmol) at x°C as described i n Table 10. After y hours, saturated sodium bicarbonate solution was added to pH 9. The mixture was extracted with methylene chloride ( 4 x 4 ml) which, a f t e r drying and chroma-tography on s i l i c a gel yielded catharanthine (12) and 18 j6 -car-borne thoxycleavamine (72). -174-(Ar 1!!) C a t h a r a n t h i n e (12) had i d e n t i c a l p r o p e r t i e s t o t h a t of s t a r t i n g m a t e r i a l . (Ar^-H) 1 8 / 3 - c a r b o m e t h o x y c l e a v a m i n e (72) was i d e n t i c a l t o an a u t h e n t i c sample e.g. TLC, NMR, UV, IR, M.S., m.pt. (118-120° , l i t . m.pt. = 121-23° ). At t e m p t e d c o u p l i n g between c a t h a r a n t h i n e (12) and v i n d o l i n e (11) C a t h a r a n t h i n e (12) (100 mg, 0.3 mmol) and v i n d o -l i n e (11) (120 mg, 0.27 mmol) were d i s s o l v e d i n t r i f l u o r o a c e t i c a c i d (1 m l , 13.28 mmol) a t -78°. The te m p e r a t u r e was r a i s e d t o -5-fr-10°. A f t e r s i x days, t h e te m p e r a t u r e was a g a i n r e d u c e d t o -78° and sodium b o r o h y d r i d e i n methanol was added u n t i l pH 8. Water (5 ml) was added and the aqueous l a y e r e x t r a c t e d w i t h methy-l e n e c h l o r i d e ( 4 x 5 m l ) . Chromatography o f the methylene c h l o r i d e s o l u b l e s gave (a) 18^0 -carbomethoxycleavamine (72) (82 mg,82%)(b) c a t h a r a n t h i n e (12) ( 5 mg, 5 %) and (c) v i n d o l i n e (116 mg, 9 6 % ) . 157 C a t h a r a n t h i n i c a c i d (216) C a t h a r a n t h i n e (12) (200 mg, 0.595 mmol) was added t o a s o l u t i o n o f aqueous sodium h y d r o x i d e (30 mg, 12% w/w) and e t h a n o l (30 m l ) . R e f l u x was m a i n t a i n e d f o r 3.5 h r a f t e r w h i c h the t e m p e r a t u r e was re d u c e d t o 0°. A m b e r l i t e a c i d i c r e s i n IR-124 was added u n t i l s o l u t i o n pH ~ 7 . The r e s i n was f i l t e r e d , and washed w i t h d i c h l o r o m e t h a n e . The s o l v e n t s were e v a p o r a t e d t o d r y n e s s and the r e s u l t a n t s o l i d t r i t u r a t e d w i t h c h l o r o f o r m . Chromatography on s i l i c a g e l y i e l d e d c a t h a r a n t h i n i c a c i d (216) (153 mg, 80%) m.pt. = 189°. -175-I.R.: 0 KBr 3400, 3020 (sh), 2940, 2620-2240, 1700 (sh),1600 cm max U.V.:/) m a x H 224 (4.495), 275 (3.823), 282 (3.852), 291 (3.788) nm. NMR: S (CDC13) 100 MHz (FT) 7.47-6. 93 (m, 411, arom.), 6.17-5.97 (d, 111, J=5 Hz, C-3 H) , 4.49 (s, IH, C-5 H) , 1.19-0.95 ( t , 3H, J=7Hz, CH 2CH 3). M.S. m/e: 322 (M +), 278, 135, 122, 121. High r e s o l u t i o n mass spectrum: C a l c — f o r C 2 Q H ^02=322.1681 Found=322.1670. Elemental a n a l y s i s f o r C20 H22 W2°2" ^ * 5 H 2 0 : F o u n d c ' 72.73, H, 6.90, N, 8.43 C a l c - , C, 72.50, H, 6.95, N, 8.64 Catharanthine (12) C a t h a r a n t h i n i c a c i d (216) (20 mg, 0.062 mmol) was d i s s o l v e d i n 20% KOH i n d i e t h y l e n e g l y c o l (0.4 m l ) . The mixture was then cooled to 0° and a c i d i f i e d w i t h 1.5% HC1 i n methanol u n t i l pH 6.5. An excess e t h e r e a l s o l u t i o n of d i a z o -methane was added u n t i l s l i g h t l y a l k a l i n e . S t i r r i n g was c o n t i n -ued f o r 30 min and s a t u r a t e d sodium bicarbonate (5 ml) was added. E x t r a c t i o n of the r e s u l t i n g mixture w i t h dichloromethane ( 4 x 5 ml) y i e l d e d , a f t e r chromatography on s i l i c a g e l , catharan-t h i n e (12) (17 mg, 82%) i d e n t i c a l i n a l l respects w i t h an a u t h e n t i c sample. 165a,186 Descarbomethoxycatharanthine (226) Catharanthine (12) (810 mg, 2.41 mmol) was d i s s o l v e d i n a s o l u t i o n of aqueous sodium hydroxide (40 ml, 12% w/w) and eth-anol (40 ml). The s o l u t i o n was heated at r e f l u x f o r 3.5 hr. Concen-t r a t e d h y d r o c h l o r i c a c i d was slowly added, (the temperature being _ i -176-m a i n t a i n e d a t a p p r o x i m a t e l y 65° d u r i n g a d d i t i o n ) , u n t i l pH 1. The r e a c t i o n m i x t u r e was c o o l e d t o r . t . and e x t r a c t e d w i t h c h l o r o f o r m (4 x 40 m l ) . The c h l o r o f o r m e x t r a c t s were com-b i n e d , d r i e d , and chromatographed on s i l i c a g e l y i e l d i n g des-c a r b o m e t h o x y c a t h a r a n t h i n e (226) (480 mg, 7 2 % ) . m.pt.: 92° ( l i t m . p t . 91-92°) 1 6 5 a I.R.: 0 CHC1 3 3480, 2940, 2890, 1470 cm" 1. U.V.: y\ E t 0 H 226, 275 (3,809), 283 (3.869) , 290 (3.848) nm. NMR: £(CDC1 3) 100 MHz (FT) 7.74 (s , IH, NH) , 7.58-7.4 (m, IH, arom.), 7.34-7.00 (m, 3H, arom.),5.90 (dd, IH, J=2 and 6 Hz,C-3 H), 3.54 (s, IH, C-5 H) , 2. 42-2.08 (complex m, 2H, J=8 Hz, CH_2CH3) , 2.08-1.82 (complex m, IH, C - l H), 1.78-1.52 (m, IH, C - l H ) , 1.08 ( t , 3H, J=8 Hz, C H 2CH 3). M.S. m/e: 278 ( M + ) , 171, 170, 135, 122. H i g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2 2N 2=278.1782 Found=278.1774. E l e m e n t a l A n a l y s i s : Found:C, 81.79, H, 8.10, N 9.97. C a l c -C, 82.01, H, 7.91, N, 10.07. 189 C h l o r o i n d o l e n i n e from c a t h a r a n t h i n e (228) N - C h l o r o b e n z o t r i a z o l e (17.2 mg, 0.112 mmol) i n dr y benzene (1.2 ml) was added d r o p w i s e t o a s o l u t i o n o f c a t h a r a n -t h i n e (12) (30 mg, 0.089 mmol) i n d r y benzene (1.5 m l ) . A f t e r 30 min , the s o l u t i o n was e x t r a c t e d w i t h 10% sodium b i c a r b o n a t e s o l u t i o n ( 2 x 3 m l ) . The b a s i c e x t r a c t was backwashed w i t h ben-zene ( 4 x 4 m l ) . The combined benzene s o l u b l e s were chromato-graphed on s i l i c a g e l y i e l d i n g (228) (28 mg, 8 5 % ) . -177-I.R.: 0 CHC1 2878, 1737, 1551 cm X" max 3 U.V. : ^l°E 276, 228 nm. NMR: S(CDC1 3) 100 MKz (FT) 7 .56-7 .42 (m, IH, arom.), 7.28-6.86 (m, 3H, arom.), 5.78 (dd, IH, J=2 and 7 Hz, C-3 H) , 4.9 (d, IH, J=2 Hz, C-5 H), 4.96-3.68 ( f i v e l i n e m u l t i p l e t , I H ) , 3.42 ( d t , IH, J=3 and 16 H z ) , 2.80 ( d t , IH, J=3 and 16 H z ) , 2.62 ( d t , 2H, J=3 and 8 Hz , C 7 & C 8H 2) , 3.32 ( s , IH, C0 2CH_ 3), 2.40-1.98 (m, 2H, CH CH ) , 1.82-1.54 (m, 2H, C1 B_2) , 1.04 ( t , 3H, J=7.5 Hz, CH 2CH 3) . M.S. m/e: 372 ( M + ) , 371, 370 ( M + ) , 369, 335, 228, 121. d 35 Hig h r e s o l u t i o n mass spectrum: C a l c — f o r C 2 ^ H 2 3 C1N 20 2= 370.1447. Found=370.1456. C h l o r o i n d o l e n i n e from d e s c a r b o m e t h o x y c a t h a r a n t h i n e (22 7) N - C h l o r o b e n z o t r i a z o l e (44.4 mg, 0.289 mmol) i n dry benzene (2 ml) was added dropwise t o d e s c a r b o m e t h o x y c a t h a r a n t h i n e (226) (80 mg, 0.288 mmol) i n benzene (3 m l ) . A f t e r 30 m i n , the s o l u t i o n was e x t r a c t e d w i t h 10% sodium b i c a b o n a t e s o l u t i o n ( 2 x 4 m l ) . The r e s u l t i n g aqueous e x t r a c t was backwashed w i t h benzene ( 4 x 4 m l ) . The benzene s o l u b l e s were chromatographed on s i l i c a g e l y i e l d i n g c h l o r o i n d o l e n i n e (227) (78 mg, 87%). I.R.: 0 CHC1 3460 (sh) , 2960, 2880, 1520 cm" 1 U.V.: A S a x " 3 2 7 6 < 3 ' 8 8 ) ' 227 ( 4. 42) NMR: £(CDC1 3) l o o MHz (FT) 7.66 (m, IH, arom.), 7.32-6.86 (m, 3H, arom.), 5.70 (d, IH, J=7 Hz, C 3 H), 4.4 ( s , IH, C-5 H), 2.36-1.96 (complex m u l t i p l e t , 2H, CH 2CH 3, 0.98 ( t , 3H, J=7 Hz, -178-CH 2CH 3) M.S. m/e: 314 ( M + ) , 312 ( M + ) , 278, 277, 170, 135, 121. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r C^gH 2^N 2Cl=312.1427. Found=312.1410. 18-De s c a r b o m e t h o x y - 1 8 - c y a n o c a t h a r a n t h i n e (224) P o t a s s i u m c y a n i d e (104 mg, 1.6 mmol) was added t o a s o l u t i o n o f c h l o r o i n d o l e n i n e (227) (50 mg, 0.159 mmol) i n anhydrous d i m e t h y l acetamide (3 m l ) . A f t e r 30 h r a t room t e m p e r a t u r e , s a t u r a t e d sodium b i c a r b o n a t e (13 ml) was added. E x t r a c t i o n w i t h methylene c h l o r i d e (5 x 15 m l ) , f o l l o w e d by d r y i n g and removal o f a l l s o l v e n t i n vacuo gave a gum w h i c h was chromatographed on s i l i c a g e l y i e l d i n g n i t r i l e (224) (11 mg, 2 3 % ) . 1. R.: [)„ (CHC1.J 3740, 2940, 2880, 2230, 1450 cm" 1. u - v - : / J m a x H 2 2 2 ( 4- 4 7>' 2 7 5 ( 3 . 8 6 ) , 280 (3 . 8 9 ) , 288 (3.86) nm. NMR: £ (CDC1 3) 100 MHz (FT) 7.88 ( s , IH, NH), 7.58-7.46 (m, IH, arom.), 7.44-7.10 (m, 3H, arom.), 5.94 (dd, J=2 and 6 Hz, C-3 H), 2. 82-1. 88 (complex e n v e l o p e , 2H, CH_2CH3) , 1.08 (3H, J=7 Hz, CH 2CH 3). M.S. m/e: 303 ( M + ) , 160, 122. Hi g h r e s o l u t i o n mass spectrum; C a l c — f o r C 2gH 2^N 3=303.1735. Found=303.1731. C a t h a r a n t h i n e (12) To c a t h a r a n t h i n i c a c i d (216) (36 mg, 0.11 mmol) was added 20% KOH i n d i e t h y l e n e g l y c o l (0.75 ml) and t h e tempera-t u r e was r a i s e d t o 155° . A f t e r 11 h r the te m p e r a t u r e was lo w e r e d t o 0° and 1.5% HC1 i n methanol added u n t i l pH 6.5. The -179-s o l u t i o n was t r e a t e d w i t h e x c e s s e t h e r e a l diazomethane u n t i l s l i g h t -l y a l k a l i n e . S t i r r i n g was c o n t i n u e d f o r 30 min and s a t u r a t e d sodium b i c a r b o n a t e (10 ml) was added u n t i l pH 9. E x t r a c t i o n o f the r e s u l t i n g s o l u t i o n w i t h methylene c h l o r i d e (5 x 10 ml) gave, a f t e r d r y i n g and chromatography on s i l i c a g e l , c a t h a r a n t h i n e (12) (3.6 mg, 10%) . C a t h a r a n t h i n e - N h - o x i d e (205) M e t a - c h l o r o p e r b e n z o i c a c i d (157 mg, 0.91 mmol) was added t o a s o l u t i o n o f c a t h a r a n t h i n e (12) (300 mg, 0.893 mmol) i n methylene c h l o r i d e a t 0°. A f t e r 10 min' t h e s o l v e n t was removed by vacuum t r a n s f e r . The r e s i d u e was chromatographed on a l u m i n a (Act I I I ) y i e l d i n g N b - o x i d e (205) (220 mg, 7 0 % ) . I.R.:^max 3460, 2960, 1730, 1740 c m - 1 . U.V.: y\ E a ° H 291 (3. 8 ) , 283 ( 3 . 9 ) , 2. 75 ( s h , 3. 8 ) , 224 ( 4 . 5 ) . NMR: 5(CDC1 3) 100 MHz (FT) 8.37 (b. s. , IH, NH) , 7.62-7.42 (m, IH, arom.), 7.4-7.05 (m, 3H, arom.), 6.14 (d, J=5.6 Hz, H), 4.72 ( s , IH, C 5 E), 3.74 ( s , 3H, C 0 2 C H 3 ) , 2.65-2.05 ( e n v e l o p e , 2H, CH 2CH^) , 1.12 ( t , J=7.5 Hz, 3H, CH2CH_3) . M.S. m/e: 352 (M +) 336, 254, 248, 222, 218, 144, 135, 130. C a t h a r a n t h i n e (12) A c t i v a t e d z i n c d u s t (40 mg, 0.61 mmol) was added t o a s o l u t i o n o f c a t h a r a n t h i n e - N D - o x i d e (205) (40 mg, 0.1136 mmol) i n a c e t i c a c i d (1.5 ml) a t 20° . A f t e r 30 min, the z i n c d u s t was removed by f i l t r a t i o n and the s o l u t i o n b a s i f i e d w i t h s a t u r a t e d sodium b i c a r b o n a t e a t 0° . E x t r a c t i o n w i t h d i c h l o r o -methane ( 4 x 5 m l ) , gave a f t e r d r y i n g and chromatography on s i l i c a -180-g e l , c a t h a r a n t h i n e (12) (25 mg, 66%) i d e n t i c a l i n a l l r e s p e c t s t o an a u t h e n t i c sample. 3 (Ar H) C a t h a r a n t h i n e (12) T r i t i a t e d t r i f l u o r o a c e t i c a c i d was p r e p a r e d by -2 d i s t i l l a t i o n o f t r i f l u o r o a c e t i c a n h y d r i d e (2.71 m l , 1.86 x 10 mol) 3 -3 xnto ( H) w a t e r (0.3 ml, 1.86 x 10 mol, 500 m C i ) u s i n g a vacuum 3 t r a n s f e r system. The r e s u l t i n g ( H) t r i f l u o r o a c e t i c a c i d was t r a n s f e r r e d t o a f l a s k c o n t a i n i n g c a t h a r a n t h i n e (12) (220 mg, 0.65 mmol) a t -78° by vacuum d i s t i l l a t i o n . The r e s u l t i n g a c i d i c s o l u -t i o n was warmed t o a p p r o x i m a t e l y -5° , t h i s b e i n g m a i n t a i n e d f o r 48 h r . Exces s ( H) t r i f l u o r o a c e t i c a c i d was then removed by vacuum t r a n s f e r . M e t h a n o l ( 3 x 2 ml) was added t o the r e s u l t i n g r e s i d u e and removed by vacuum t r a n s f e r t o g e t h e r w i t h any exchange-a b l e t r i t i u m . S a t u r a t e d aqueous sodium b i c a r b o n a t e was added a t 0 ° u n t i l ph 8 . 5 . E x t r a c t i o n w i t h d i c h l o r o m e t h a n e f o l l o w e d by d r y i n g i n the u s u a l manner gave a crude foam w h i c h was c r y s t a l l i s e d w i t h methanol t o y i e l d (Ar 3H) c a t h a r a n t h i n e (12) 118 mg, 5 4 % ) . Chromatography on s i l i c a g e l (twice) and c r y s t a l l i s a t i o n t o c o n s t a n t a c t i v i t y y i e l d e d (Ar^H) c a t h a r a n t h i n e (12) (6.389 x 10 dpm/mmol). 3 (Ar H) C a t h a r a n t h i n e - N b - o x i d e (205) M e t a c h l o r o p e r b e n z o i c a c i d (34 mg, 0.197 mmol) i n 3 methylene c h l o r i d e (1 ml) was added t o a s o l u t i o n o f (Ar H) c a t h a r -a n t h i n e (12) (60 mg, 0.178 mmol) i n methylene c h l o r i d e (4 ml) a t 0°. S t i r r i n g was c o n t i n u e d f o r 15 min and the s o l u t i o n d i l u t e d w i t h e t h y l a c e t a t e (25 m l ) . The o r g a n i c l a y e r was then -181-e x t r a c t e d w i t h s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n ( 2 x 5 ml) . D r y i n g and e v a p o r a t i o n o f s o l v e n t i n the u s u a l manner a f f o r d e d a w h i t e foam w h i c h was chromatographed w i t h v a r i o u s s o l v e n t systems on s i l i c a g e l u n t i l c o n s t a n t a c t i v i t y t o g i v e (Ar 3H) c a t h a r a n t h i n e - N b - o x i d e (205) (15 mg, 2 5 % ) . (6.485 x 1 0 9 dpm/mmole). 3 A d m i n i s t r a t i o n o f (Ar H) C a t h a r a n t h i n e - N b - o x i d e (205) t o  C. r o s e u s 3 (Ar H) c a t h a r a n t h i n e - N b - o x i d e (205) (1.594 mg, 9 6.4 85 x 10 dpm/mmol ) was a d m i n i s t e r e d t o a C. r o s e u s p l a n t (68 g wet w e i g h t ) i n a methanol/water s o l u t i o n (4 m l , 1:1) by the c o t t o n w i c k method. A f t e r 28 days t h e p l a n t was macerated and methanol s o l u b l e s removed. The e x t r a c t c o r r e s p o n d o n g t o v i n c r i s t i n e (10 8) was 156 i s o l a t e d a c c o r d i n g t o Svoboda's g r a d i e n t pH t e c h n i q u e . I n t h i s way, the methanol s o l u b l e s were e v a p o r a t e d g i v i n g a r e s i -due w h i c h was suspended i n 2% t a r t a r i c a c i d s o l u t i o n (350 m l ) . A f t e r a g i t a t i o n f o r 45 min, t h e a c i d i c s o l u t i o n was e x t r a c t e d w i t h hexane (3 x 250 m l ) . F u r t h e r e x t r a c t i o n o f t h e a c i d s o l u t i o n w i t h benzene (4 x 250 ml) gave benzene s o l u b l e s and aqueous e x t r a c t ( 1 ) . The benzene s o l u b l e s were d r i e d and e v a p o r a t e d and the r e s i d u e was d i s s o l v e d i n 2% t a r t a r i c a c i d s o l u t i o n (150 m l ) . The a c i d i c l a y e r was e x t r a c t e d w i t h d i c h l o r o m e t h a n e t o g i v e e x t r a c t B ( a f t e r removal o f t h e s o l v e n t i n the u s u a l manner.) The r e -s u l t i n g aqueous l a y e r (2) was then b a s i f i e d w i t h 5% ammonium hy-d r o x i d e s o l u t i o n a t 5° u n t i l pH 8.5. E x t r a c t i o n w i t h methylene -182-c h l o r i d e (3 x 150 ml) gave, a f t e r d r y i n g , e x t r a c t C. (The aqueous l a y e r was then d i s c a r d e d ) . The o r i g i n a l aqueous e x t r a c t (1) was s i m i l a r i l y b a s i f i e d t o pH 8.5 and removal o f methylene c h l o r i d e s o l u b l e s (3 x 200 ml) gave, a f t e r d r y i n g , e x t r a c t A. ( o r g a n i c s o l u b l e s ) E x t r a c t C was d i l u t e d w i t h v i n c r i s t i n e (201) (20 mg) and t h i s was r e - i s o l a t e d a f t e r t h i n l a y e r chromatography (X2). The v i n c r i s t i n e (201) was t h e n c r y s t a l l i s e d t o l e v e l s shown i n t a b l e 12. ( I t s h o u l d be n o t e d t h a t , i n t h i s e x p e r i m e n t , e x t r a c t (A) and (B) were a l s o d i l u t e d w i t h VCR - w i t h no s i g n i f i c a n t a c t i v i t y b e i n g noted.) V i n c r i s t i n e ( 2 0 1 ) , r e i s o l a t e d , was checked by TLC, NMR, IR, M.S. and m.pt. w i t h an a u t h e n t i c sample. INTRODUCTION - PART IV S t u d i e s r e l a t e d t o the s y n t h e s e s o f a n t i - t u m o u r p y r i d o - c a r b a - z o l e a l k a l o i d s The p y r i d o - c a r b a z o l e a l k a l o i d s o l i v a c i n e (105), guatambuine (104) and e l l i p t i c i n e (106), d i s c u s s e d p r e v i o u s l y from a b i o s y n t h e t i c s t a n d p o i n t , have a l s o r e c e i v e d c o n s i d e r a b l e s y n t h e t i c a t t e n t i o n 193 and s e v e r a l approaches g i v i n g o l i v a c i n e (105) and e l l i p t i c i n e 194 (106) have been r e p o r t e d . The a n t i - t u m o u r and a n t i - l e u k a e m i c a c t i v i t y of' t h e s e a l k a l o i d s a c c o u n t f o r t h i s c o n s i d e r a b l e 195 s y n t h e t i c i n t e r e s t . The more r e c e n t s t u d i e s p r e s e n t e d were d i r e c t e d towards the s y n t h e s i s o f the d i h y d r o p y r i d i n e (229-231) and t e t r a h y d r o p y r i d i n e (232-234) i n t e r m e d i a t e s . A p p l i c a t i o n o f t h e i r c h e m i s t r y s h o u l d p e r m i t t h e s y n t h e s i s o f the p y r i d o c a r b a z o l e (23 5) system. 2 3 1 2 3 4 DISCUSSION - PART IV I n v e s t i g a t i o n s towards the s y n t h e s e s o f i n t e r m e d i a t e s ( 2 2 9 - 2 3 4 ) used the key i n t e r m e d i a t e ( 2 3 9 ) o b t a i n e d by base h y d r o l y s i s o f 19 6 the r e p o r t e d N - b e n z e n e s u l p h o n y l d e r i v a t i v e ( 2 3 8 ) as shown i n f i g 6 1 . 2 3 9 2 3 8 F i g 6 1 : S y n t h e s i s o f I n d o l - 2 - y 1 - 1 - ( 4 ' p y r l d y l ) - e t h a n o l . ( 2 3 9 ) E v i d e n c e f o r s t r u c t u r e ( 2 3 9 ) was i n d i c a t e d by the U.V. h a v i n g the e x p e c t e d chromophore (A max 2 8 3 , 2 7 4 , 2 5 6 , 2 5 3 , 2 1 1 nm) and the I.R. had NH and OH a b s o r p t i o n a t 3 4 0 0 and 3 2 9 0 cm x . r e s p e c -t i v e l y . The mass spectrum gave an m/e 2 3 8 (M ) and the NMR was i n t o t a l agreement w i t h the s t r u c t u r e (see e x p e r i m e n t a l ) . I n o r d e r t o s y n t h e s i s e d e r i v a t i v e s ( 2 2 9 - 2 3 4 ) i t was d e c i d e d t o f i r s t d e v e l o p the c h e m i s t r y f o r the f o r m a t i o n o f the c o r r e s -p o n d i n g deshydroxy ( 2 4 0 ) and d e h y d r a t e d ( 2 4 1 ) d e r i v a t i v e s o f the a l c o h o l ( 2 3 9 ). I n t h i s r e s p e c t , h y d r o g e n a t i o n o f ( 2 3 9 ) u s i n g a Pd/C c a t a l y s t a f f o r d e d the r e q u i r e d deshydroxv d e r i -v a t i v e ( 2 4 0 ) i n good y i e l d . -185-Th e s t r u c t u r e (240) was i n d i c a t e d by the u.V. ( A max 288, 278, 268, 263, 256, 217 nm) and the I.R. spectrum d i s p l a y e d NH a b s o r p t i o n a t 34 80 cm \ The mass spectrum gave an m/e 222 (M +) and the NMR gave the e x p e c t e d q u a r t e t a t £4.20 (J=7Hz) f o r CHCH_ and a d o u b l e t a t S 1-68 (J=7Hz) f o r CHCH,.. 2 4 0 2 3 9 2 4 1 D e h y d r a t i o n o f (239) w i t h a c e t i c a n h y d r i d e i n p y r i d i n e gave t h e d e r i v a t i v e (241) i n good y i e l d . E v i d e n c e f o r t h i s s t r u c t u r e came m a i n l y from t h e mass spectrum h a v i n g an m/e 220 (M ) and the NMR w h i c h e x h i b i t e d two s i n g l e t s a t S 5.83, 5.42 f o r C=CH_2. However no f u r t h e r s t u d i e s w i t h (241) were i n i t i a t e d due t o i t s n o t e d i n s t a b i l i t y . I n t h i s r e s p e c t i t was c o n s i d e r e d t h a t a p p r o p r i a t e d e h y d r a t i o n o f t h e hydroxy s e r i e s c o u l d be pur s u e d a t a l a t e r s t a g e i n the s y n t h e s i s . W i t h d e r i v a t i v e s (239) and (240) on hand, s t u d i e s toward t h e i n t r o d u c t i o n o f the c o r r e s p o n d i n g N'-methyl group and (CH^) 2 N C H 2 s^ <^ e c h a i n a t C-3, were i n i t i a t e d . I n t r o d u c t i o n o f the m e t h y l group was r e a d i l y o b t a i n e d by t r e a t m e n t o f (239) and (240) w i t h m e t h y l i o d i d e ( f i g 62) t o g i v e m e t h i o d i d e s a l t s (242) and (243). E v i d e n c e f o r the m e t h i o d i d e s a l t (242) came p r i n c i p a l l y from t h e U.V. (A max 2 87,277,262,257, & 217 nm) and the NMR whi c h e x h i b i t e d a s i n g l e t a t £ 4.26 f o r N-CH-.. The remainder o f the -186-spectrum was i n t o t a l agreement w i t h the s t r u c t u r e . F u r t h e r s u p p o r t f o r the s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n o f (242) t o g i v e the t e t r a h y d r o p y r i d i n e d e r i v a t i v e (244) ( f i g 6 2 ) . The U.V. o f t h i s compound d i s p l a y e d the c h a r a c t e r i s t i c i n d o l e chromophore ( A max 288, 279, 270 and 218 nm) and t h e I.R. s p e c -trum gave OH and NH a b s o r p t i o n a t 3480 and 3320 c m - 1 r e s p e c -t i v e l y . The mass spectrum gave an m/e 256 (M ) and the NMR e x h i b i t e d a m u l t i p l e t a t £ 5.65 f o r C-3'H, a d o u b l e t a t S 2.82 f o r C-2'H , a m u l t i p l e t a t S 2.32 f o r C-6'H~, a s i n g l e t a t S 2.16 —2 f o r N-CH 3, and a m u l t i p l e t a t S 2.00 f o r C-5'H_2. E v i d e n c e f o r t h e m e t h i o d i d e s a l t (243) came p r i n c i p a l l y from the U.V. spectrum ( A max 285, 275, 263, 257, 217 nm) and t h e NMR w h i c h e x h i b i t e d a s i n g l e t a t <S 4.29 f o r N-CH^. Once a g a i n f u r t h e r s u p p o r t f o r t h i s s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n t o t h e c o r r e s p o n d i n g t e t r a h y d r o p y r i d i n e s p e c i e s (245) ( f i g 6 2 ) . The n e x t s t e p i n v o l v e d i n t r o d u c t i o n o f the ( C H 3 ) 2 N C H 2 s i d e c h a i n a t C-3. T h i s t r a n s f o r m a t i o n o c c u r r e d i n h i g h y i e l d t o g i v e (246) by t r e a t m e n t o f m e t h i o d i d e s a l t (242) w i t h HCHO/(CH 3) 2NH i n g l a c i a l a c e t i c a c i d ( f i g 6 2 ) . E v i d e n c e f o r t h i s s t r u c t u r e p r i n c i p a l l y came from the NMR w h i c h was i n t o t a l agreement w i t h t h e p r o p o s e d s t r u c t u r e h a v i n g a m u l t i p l e t a t S 3.49 f o r CH_2N and a s i n g l e t a t £ 2.29 f o r N(CH_ 3) 2 i n a d d i t i o n t o t h e e x p e c t e d s i g n a l s . The U.V. and I.R. were a l s o i n agreement w i t h t h i s s t r u c t u r e . -187-2 4 7 F i g u r e 62: S y n t h e s i s o f i n t e r m e d i a t e s (246) and (247). i F u r t h e r v e r i f i c a t i o n o f t h i s s t r u c t u r e was o b t a i n e d by b o r o h y d r i d e r e d u c t i o n t o g i v e the t e t r a h y d r o p y r i d i n e (248). E v i d e n c e f o r t h i s s t r u c t u r e was o b t a i n e d from the U.V. ( A max 288, 279,273 & 217 nm) and the I.R. w h i c h showed NH and OH a b s o r p t i o n a t 3485 and 3280 cm 1 r e s p e c t i v e l y . The mass spectrum gave an m/e 313 (M +) and t h e NMR was i n t o t a l agreement w i t h the s t r u c t u r e (see e x p e r i m e n t a l ) . -188-Th e formation of the corresponding analogous d e r i v a t i v e (247) 197 has a l s o been c a r r i e d out i n more recent s t u d i e s . These p r e l i m i n a r y r e s u l t s c o n s t i t u t e the progress made 198 towards the syntheses of the pyrido- c a r b a z o l e a l k a l o i d s . 197 202 Completion of the remaining s y n t h e t i c steps i s c u r r e n t l y underway. ' However i t i s of some i n t e r e s t to speculate on the p o s s i b l e pathways f o r completion of the syntheses. In t h i s r e s p e c t two recent syntheses of py r i d o - c a r b a z o l e s have r e c e n t l y been pub-l i s h e d - both of which had s i m i l a r i t i e s to the ' t e t r a h y d r o p y r i d i n e approach'. Consequently treatment of the N-benzenesulphonyl d e r i v a t i v e (250) w i t h KQBu i n DMSO aff o r d e d an E/Z mixture of dienamines (251) which was then allowed to undergo the appro-p r i a t e Mannich r e a c t i o n and c y c l i z a t i o n to give N-methyltetra-hydro e l l i p t i c i n e (252) i n 2.2 % . y i e l d . Treatment of (252) w i t h Pd/C affo r d e d e l l i p t i c i n e ( 1 0 6 ) . 1 9 9 ( f i g 63) Figure 63: P o t i e r ' s s y n t h e s i s of e l l i p t i c i n e (106) -189-The second approach i s shown i n f i g 64 and a s i m i l a r c y c l i z a t i o n o c c u r r e d t o g i v e p y r i d o c a r b a z o l e (257) from i n t e r m e d i a t e ( 2 5 6 ) . 2 0 0 T h i s a c i d c a t a l y s e d c y c l i z a t i o n p r o b a b l y o c c u r r e d v i a the p o s t u l a t e d mechanism ( f i g 6 5 ) . 257 F i g 65: P o s t u l a t e d mechanism f o r the f o r m a t i o n o f p y r i d o c a r b a z o l e (257) from i n t e r m e d i a t e (256). -190-A l t h o u g h t h i s c y c l i s a t i o n has n o t y e t been a t t e m p t e d l i t e r a t u r e p r e c e d e n c e s u g g e s t s t h a t t h e f i n a l t r a n s f o r m a t i o n t o g i v e the r e q u i r e d c y c l i s a t i o n p r o d u c t (235) w i l l be s u c c e s s f u l . I t i s t o be hoped, however, t h a t a p p r o p r i a t e m o d i f i c a t i o n o f t h i s t r a n s f o r m a t i o n can be e f f e c t e d t o a l l o w f o r m a t i o n o f the r e q u i r e d c y c l i z e d p r o d u c t (235) i n a much g r e a t e r y i e l d t han the 2% r e p o r t e d i n the s y n t h e s i s o f e l l i p t i c i n e (106) ( f i g 63). 201 A more p r o m i s i n g a p p r o a c h , however, c o n c e r n s the u t i l i s a t i o n o f d i h y d r o p y r i d i n e i n t e r m e d i a t e s . E x t e n s i v e s t u d i e s i n t h e s e l a b o r a t o r i e s have r e a l i s e d c o n d i t i o n s f o r t h e s t a b i l i s a t i o n o f h i g h l y r e a c t i v e d i h y d r o p y r i d i n e i n t e r m e d i a t e s as t h e i r c o r r e s -p o n d i n g chromium t r i c a r b o n y l complexes. I n t h i s r e s p e c t the s t a b l e d i h y d r o p y r i d i n e complex (258), shown i n f i g 66, was t r e a t e d w i t h p y r i d i n e t o r e g e n e r a t e the c o r r e s p o n d i n g d i h y d r o -p y r i d i n e (259) i n s i t u . Treatment o f (259) w i t h b e n z y l bromide and sodium b o r o h y d r i d e a f f o r d e d a m i x t u r e o f b e n z y l a t e d i n t e r -m e d i a t e s (260) and (261). D i r e c t d e b e n z y l a t i o n o f (261) and (260) was r e a d i l y e x e c u t e d u s i n g n - p r o p y l mercaptan, l i t h i u m h y d r i d e and hexamethylphosphoramide t o g i v e r e q u i r e d N-methyl-3 - e t h y l - 5 - b e n z y l - l , 2 , 5 , 6 t e t r a h y d r o p y r i d i n e (263) and N-methyl-3 - e t h y l - l , 2 , 5 , 6 t e t r a h y d r o p y r i d i n e (262). -191-2 5 8 2 5 9 2 6 2 2 6 3 F i g 66: S y n t h e s i s o f N - m e t h y l - 3 - e t h y l - 5 b e n z y l - t e t r a h y d r o p y r i -d i n e (263) However a more e f f i c i e n t method f o r the f o r m a t i o n o f (263) was n o t e d on t r e a t m e n t of N - m e t h y 1 - 3 - e t h y l p y r i d i n i u m i o d i d e (264) w i t h sodium b o r o h y d r i d e t o g i v e the r e q u i r e d d i h y d r o p y r i d i n e (259) T h i s , when t r e a t e d w i t h a 10 x excess o f b e n z y l bromide gave, f o l l o w i n g d e b e n z y l a t i o n a t n i t r o g e n , the t e t r a h y d r o p y r i d i n e (263) i n an o v e r a l l y i e l d o f 48%. e C H 3 C H 3 C H 3 2 6 4 2 5 9 2 6 3 Thus the a p p r o p r i a t e c h e m i s t r y has been de v e l o p e d t o a l l o w i n v e s t i g a t i o n o f the c y c l i s a t i o n r e a c t i o n s i n b o t h the t e t r a h y d r o -p y r i d i n e and d i h y d r o p y r i d i n e s e r i e s . -192-Refinement o f the t e t r a h y d r o p y r i d i n e c y c l i s a t i o n i s 197 c u r r e n t l y under s t u d y . U t i l i s a t i o n o f e i t h e r t h e " f r e e " d i h y d r o p y r i d i n e i n t e r -m e d i a t e s (229) o r (230) o r t h e i r s t a b i l i s e d chromium t r i c a r b o n y l Complexes (264) o r (265) i s a l s o under i n v e s t i g a t i o n and c o n d i t i o n s f o r t h e e f f i c i e n t c y c l i s a t i o n t o the r e q u i r e d p r o d u c t s (266) o r (267) ar e b e i n g e f f e c t e d . 1 9 7 ' 2 0 2 When t h e s e c o n d i t i o n s are d e t e r m i n e d a p p r o p r i a t e m o d i f i -c a t i o n s f o r the s y n t h e s e s o f o l i v a c i n e (105) and e l l i p t i c i n e (106) w i l l be i n t r o d u c e d . 2 4 7 2 6 5 2 6 7 -193-EXPERIMENTAL - PART I V G e n e r a l e x p e r i m e n t a l d e t a i l s were g i v e n i n p a r t I and I I . 196 1 - B e n z e n e s u l p h o n y l i n d o l e (23 7) P o t a s s i u m h y d r i d e (3.6 m l , 0.09 mol) was added t o a s o l u t i o n o f anhydrous THF (75 ml) and HMPA (15 ml) a t 0°. To t h i s was added i n d o l e (236) (10 g, 0.085 m o l ) , dissolved in anhydrous THF (30 m l ) , over 30 min. The suspen-s i o n was a l l o w e d t o s t i r f o r 15 min and dry r e d i s t i l l e d benzene-s u l p h o n y l c h l o r i d e (16.5 g, 0.088 mol) was added d r o p w i s e . The te m p e r a t u r e was r a i s e d t o r . t . and s t i r r i n g t hen m a i n t a i n e d f o r 3 h r . Alumina (Act I I I ) was added to d e s t r o y the r e m a i n i n g p o t a s s i u m h y d r i d e . Water (50 ml) was added and the r e s u l t i n g m i x t u r e poured t h r o u g h a g l a s s s i n t e r f u n n e l and then e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 100 m l ) . The e t h e r e a l e x t r a c t s were combined and backwashed w i t h w a t e r (2 x 75 m l ) , d r i e d , and e v a p o r a t e d t o g i v e a p r e c i p i t a t e w h i c h on r e c r y s t a l l i s a t i o n from m e t h y l e n e c h l o r i d e - hexane a f f o r d e d the r e q u i r e d (237) (19.5 g, 88%) i d e n t i c a l i n a l l r e s p e c t s t o t h a t r e p o r t e d (m.pt.= 78°, l i t m.pt. 77.5-79°) 1- ( 1 - B e n z e n e s u l p h o n y l i n d o l - 2 - y l ) - 1 - ( 4 - p y r i d y l ) - e t h a n o l (238) P r e p a r e d e x a c t l y a c c o r d i n g t o l i t e r a t u r e p r o c e -19 6 dure. M.pt. 228-229° ( l i t m.pt. 229-230°) I n d o l - 2 - y l - l - ( 4 p y r i d y l ) e t h a n o l - ( 2 39) To a methanol-2M sodium h y d r o x i d e s o l u t i o n (500 ml, 5:1) was added (238)(3 g, 7.96 mmol). The s o l u t i o n -194-was then h e a t e d t o r e f l u x f o r 4 h r , c o o l e d , and e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 200 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o y i e l d a crude s o l i d w h i c h was r e c r y s -t a l l i s e d from c h l o r o f o r m t o g i v e (239) ( 1.36 g, 72%). M.pt 218-221° ( l i t m.pt.=221°) I.E.: 0 KBr 3400, 3290, 3050, 3030, 2920, 1600, 1455, 1430, 1295, 835, 795, 750, 700,cm - 1 I MeOH U . V . : / 1 M A X 283 (3.86), 274 (3.92), 256 (3. 9 5 ) , 253 (3 .92), 211 (4.53) nm. NMR: £ (d-DMSO) 100 MHz (FT) 10.92 (s, IH, NH), 8.49 (d, 2H, J=6Hz, C-2', C - 6 \ H ) , 7.46 (d, 2H, J=6Hz, C-3', C - 5 \ H_ 2), 7.40-6.80 (m, 4H, arom.), 6.31 (d, IH, J=2Hz, C-3H), 6.18 ( s , IH, OH, ex-changeable w i t h D-,0) , 1.88 ( s , 3H, CH_ 3). M.S. m/e: 238 ( M + ) , 220, 205, 160. High r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5 H 4 N 2 0 = 238.1106 Found= 238.1123 E l e m e n t a l a n a l y s i s C a l c C, 75.63, H, 5.88, N, 11.76 Found, C, 75.55, H, 5.70, N, 11.95 I n d o l - 2 - y l - l - ( 4 p y r i d y l ) - e t h a n e (240) The a l c o h o l (239) (105 mg, 0.44 mmol) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d and t o t h i s was added a Pd/C (10% w/w) c a t a l y s t (30 mg). The r e s u l t i n g s u s p e n s i o n was p l a c e d under an atmosphere o f hydrogen f o r 72 h r . The c a t a l y s t was then removed ( s u c t i o n f i l t r a t i o n ) , c o o l e d t o 0°, and concentrated ammonium h y d r o x i d e added t o ph 9. The aqueous l a y e r was the n e x t r a c t e d w i t h c h l o r o f o r m (3 x 25 m l ) . The c h l o r o f o r m e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o g i v e an o i l w h ich was chromato--195-graphed on s i l i c a g e l to a f f o r d r e q u i r e d (240) ( 52 mg, 54 %) and s t a r t i n g m a t e r i a l ( 1 3 mg, 13 %) M.pt.: 12 3-2 4° I.R.: 0 C H C 1 3 3480, 2980, 1605, 1460, 1420, 1305, 915 cm"1. I Me OH U * V ' : ^max 2 8 8 < 3 ' 8 3 ) ' 2 7 8 (3. 88), 268 (3.87), 263 (3. 90), 256 (3.87), 217 (4.52) nm. NMR: S (CDC13) 100 MHz (FT) 8.43 (b.s.,3H, NH and C-2', C-61 H 2 ) , 7.67 - 7.49 (m, IH, C-7H) , 7.33-6. 95 (m, 5H, arom & C-3', C-5 1 , (d, IH, J=2Hz, C-3H), 4.20 (q, IH, J=7Hz, CHCH ) 1.68 (d, 3H, J=7Hz, CH 3). M.S. m/e: 222, (M+) , 207, 144. High r e s o l u t i o n mass spectrum: C a l c - f o r C-, r-H N = 222.1157 Found= 222.1157. Elemental a n a l y s i s : C a l c - f o r c i 5 H i 4 N 2 ' C ' 81.08, H, 6.30, N, 12.61 Found, C, 80.74, H, 6.18, N, 12.72 In d o l - 2 - y l - l - ( 4 - p y r i d y l m e t h i o d i d e ) - e t h a n e . ( 2 4 3 ) Excess methyl i o d i d e (1 ml) was added t o a s o l u t i o n of compound (240) (210 mg, 0.94 mmol) d i s s o l v e d i n anhydrous e t h y l acetate. The s o l u t i o n was allowed t o s t i r o v e r n i g h t at r . t . a f t e r which a f l o c c u l e n t p r e c i p a t e had accumulated. This was removed ( s u c t i o n f i l t r a t i o n ) and r e c r y s t a l l i s a t i o n from methanol gave the r e q u i r e d methiodide s a l t (243) (313 mg, 91 %) M.pt.: 127° (MeOH) I.R.:^ CHC1 3 3190,3150,2980,1660,1450,1422,1294,848,790,745,540 cm - 1. U . V . : y\ J J | ° H 2 85 (3. 69 ) sh, 275(3. 88) sh, 263(4. 05) sh, 257(4. 07), 217 (4. 73) nm. NMR: S (d-DMSO) 100 MHz (FT) 8.92 (d, 211, J=6.8 Hz, C-2 ' , C-6 1 ) , 8.04 (d, 2H, J=6.8Hz,C-3',C-5" ,H2) , 7. 58-6.84 (m, 411, arom.), -196-6.36 ( s , 1H, C-3H), 4.70 (q, 1H, J=7Hz, CHCH^, 4.29 ( s , 3H, N-CH_3) 1.73 (d, 2H, J=7Hz, CHCH_3) . E l e m e n t a l a n a l y s i s : C a l c - f o r C l g H N 2 I , C, 52 . 76 , H, 4.70, N, 7.69, Found, C, 52.95, H, 4.71, N, 7.57 I n d o l - 2 - y l - l - ( 4 - p y r i d y l ) - e t h e n e (241) The a l c o h o l (239) (50 mg, 0.48 mmol) was added t o a s o l u t i o n of a c e t i c a n h y d r i d e (2 ml) and p y r i d i n e (0.5 m l ) . The temperature was r a i s e d t o 80° f o r 3 hr and then a l l o w e d t o O ri s t a n d o v e r n i g h t a t 50 . F u r t h e r h e a t i n g a t 80 f o r 1 hr completed the r e a c t i o n . The r e a c t i o n m i x t u r e was c o o l e d and d i l u t e d w i t h chloroform (20 m l ) . The s o l u t i o n was washed w i t h w ater (2 x 10 m l ) . The c h l o r o -form s o l u b l e s were d r i e d and e v a p o r a t e d t o g i v e an o i l 'which f o l l o w i n g chromatography on s i l i c a g e l a f f o r d e d t h e r e q u i r e d ethene (241) (37 mg, 80%). I.R.:' \) CHC1 3 3480, 3010, 2960, 2930, 1600 cm" 1, i MeOH U . V . : / j m a x 288(3.85, 278(3.92), 268(3.92), 263(3.93), 217(1.09) nm. NMR: £ (CDC1.J 100 MHz (FT) 88.56 (m,2H, C-2', C-6' H ) , 7.33-6.89 (m, 6H, arom H_4 & C' 3, C' 5 H ), 6.38 (d, IH, J=2Hz, C-3H) , 5.64, 5.40 ( 2S, 2H, C H 9 ) . . M.S. m/e: 220 ( M + ) , 78. High r e s o l u t i o n mass spectrum: C a l c — f o r C 1 5 H 1 2 N 2 = 220.1000 Found= 220.0987 I n d o 1 - 2 - y 1 - 1 - ( 4 - p y r 1 d y l m e t h l o d i de)-e t h a n o l (242) The a l c o h o l (239) (390 mg, 1.64 mmol) was added t o a s o l u t i o n o f m e t h y l i o d i d e ( 2 ml) and anhydrous e t h y l -197-a c e t a t e (30 ml) . The s u s p e n s i o n was h e a t e d t o 50° f o r 1 h r t o e f f e c t s o l u t i o n . The t e m p e r a t u r e v/as t h e n l o w e r e d t o r . t . and s t i r r i n g was m a i n t a i n e d o v e r n i g h t . The r e s u l t i n g p r e c i p i t a t e was removed by s u c t i o n f i l t r a t i o n t o a f f o r d an amorphous powder (554 mg, 8 9 % ) . I.R.: 0 KBr 3285, 3030, 2930, 1640, 1460, 755, 700 cm" 1. U . V . : A m i ° H 287 (3.63)sh,277 (3.85)sh,262 (4.06)sh,257 (4.07) ,217 (4. 70) NMR: & (d-DMSO) 100 MHz (FT) 8.93 (d, 2H, J=6.8Hz, C-2', C-6', H 2 ) , 8.17 (d, 2H, J=6.8Hz, C-3 ' , C-5 ' H_2) , 7 . 56-6 . 82 (m, 4H, arom.), 6.72 ( s , IH, OH, e x c h a n g e a b l e w i t h D O ) , 6.44 ( s , IH, C-3H), 4.26 (s , 3H, N-CH 3), 1.98 ( s , 3H, CH(OH)CH ). I n d o l - 2 - y 1 - 1 - ( N ' - m e t h y I t e t r a h y d r o p y r i d y 1 ) - e t h a n o l (244) The m e t h i o d i d e s a l t (242) (200 mg, 0.526 mmol) was d i s s o l v e d i n methanol (10 ml) and sodium b o r o h y d r i d e (184 mg, 5.26 mmol) v/as added. The m i x t u r e was a l l o w e d t o s t i r f o r 0. 5 h r and the s o l v e n t v/as t h e n removed i n vacuo. Water (10 ml) was t h e n added and the aqueous phase e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 50 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s . The o i l formed was t h e n chromato-graphed on s i l i c a g e l t o g i v e the r e q u i r e d t e t r a h y d r o p y r i d i n e (244) (145 mg, 88%) . M . D t . : 184-185° (CHC1 ) 1. R.: ^ CHC1 3 3480, 3320, 3020, 2990, 2950, 2800, 1665, 1460, 1375, 1295, cm" 1. -198-u-v- : A m a f 2 8 8 ( 3 . 7 4 ) , 278 (3. 8 6 ) , 270 (3.87), 217 (4.54) nm. NMR: £ (d-DMSO) 100 MHz (FT) 10.59 ( s , IH, NH), 7.46-7.20 (m, 2H, arom), 7.04-6.74 (m, 2H, arom), 6.16 (d, IH, J=2Hz, C-3 H) , 5.65 ( t , IH, J-4Hz, C-3* H), 2.82 (d, 2H, J=4Hz, C-2' H 2 ) , 2.32 (m, 2H, C-5' H_2) , 2.16 ( s , 3H, N-CH_3), 2.00 (m, 2H, C-6 1 H 2 ) , 1.6 0 ( s , 3H, C H 3 ) . M.S. m/e: 256 ( M + ) . High r e s o l u t i o n mass spectrum: C a l c — f o r C^^20^2o= 256.1575 Found= 256.1570 E l e m e n t a l a n a l y s i s , C a l c - f o r C 1 6 H 2 ( ) N 2 0 , C, 75.0, H, 7.81, N, 10.94 Found, C, 74.86, H, 7.72, N, 10.83. P y r i d i n i u m . s a l t (246) To an aqueous d i m e t h y l a m i n e s o l u t i o n (25% w/w, 250 mg) a t 0° was added (1) g l a c i a l a c e t i c a c i d (500 mg, 8.33 mmol) and (2) and aqueous formaldehyde s o l u t i o n (37% w/w, 105 mg, 1.3 mmol). T h i s m i x t u r e was the n added t o t h e p y r i d i n i u m s a l t (242) (541 mg, 1.42 mmol) and the r e s u l t i n g s u s p e n s i o n v/armed t o 45° and s t i r r e d o v e r n i g h t . The r e a c t i o n m i x t u r e was c o o l e d t o 0° and n e u t r a l i s e d w i t h 2M sodium h y d r o x i d e s o l u t i o n . F o l l o w i n g f r e e z e - d r y i n g the s o l i d o b t a i n e d was r e c r y s t a l l i s e d from e t h a n o l -hexane t o g i v e r e q u i r e d p y r i d i n i u m s a l t (246) (518 mg, 8 4 % ) . M.pt. 238-242° = 0 I.R.: \S m ^ v KBr 3420, 3230, 3020, 2980, 2930, 1635, 1455, 750, 70 0 cm" max -1 „ I MeOH u - v - : / i m a x 283 ( 3 . 7 3 ) , 275 (3 . 8 7 ) , 260(3.95), 217(4.70) nm. NMR: £ (d-DMSO) 100 MHz (FT) 9.2 ( s , IH, NH), 8.91 (d, 2H, -199-J=6.8Hz,C-2',C-6' H ) , 8.11 (d, 2H, J=6.8Hz, C-3*, C-5 1 H_ 2), 7.65-6.97 (m, 4H, arom), 4.32 ( s , 3H, N-CH_3) , 3.49 ( m, 2H, C H 2 N ( C H 3 ) 2 ) , 2.29 ( s , 6H, N ( C H 3 ) 2 ) f 2 . 0 1 ( s , 3H, CH_ 3). E l e m e n t a l a n a l y s i s : C a l c - f o r C l g H 2 4 N I , C, 52.18, H, 5.53, N, 9.61 Found C, 52.24, H, 5.63, N, 9.40 T e t r a h y d r o p y r i d i n e (24 8) The Mannich d e r i v a t i v e (246) (200 mg, 0.46 mmol) was d i s s o l v e d i n methanol (10 ml) and sodium b o r o h y d r i d e ( 160 mg, 4.6 mmol) added o v e r 10 min. The m i x t u r e was a l l o w e d t o s t i r f o r 0.5 h r and t h e s o l v e n t was removed i n vacuo. Water (10 ml) was t h e n added and t h e aqueous phase e x t r a c t e d w i t h d i e t h y l e t h e r (3 x 25 m l ) . The e t h e r e a l e x t r a c t s were combined, d r i e d , and e v a p o r a t e d t o d r y n e s s . The amorphous powder was the n chromato-graphed on s i l i c a g e l t o g i v e the r e q u i r e d t e t r a h y d r o p y r i d i n e (248) (126 mg, 8 8 % ) . I.R.: 0 CHC1 3 3485, 2990, 2950, 2780, 1610, 1465, 1010 cm" 1. 1 MeOH U . V . : / ) m a v 288 (3.86, 279 (3. 9 2 ) , 273 (3.90, 2.7 (456) nm. NMR: & (CDC1 3) 100 MHz (FT) 8.39 ( s , IH, NH), 7.60-7.02 (m, 4H, arom), 5.92 (b.m, IH, C-3'H), 3.65 (d, 2H, J=2Hz, CH„N(CH ) ) , 2.42 ( s , 3H, N-CH 3) , 2.27 ( s , 6H, N- ( C H _ 3 ) 2 ) , 1.72 ( s , 3H, CH_ 3). M.S. m/e: 313 ( M + ) , 268, 250, 208, 172, 138. Hi g h r e s o l u t i o n mass spectrum: C a l c — f o r c i 9 H 2 7 N 3 0 = : 313.2154 Found= 313.2158. - 2 0 0 -REFERENCES 1. G.A. C o r d e l l , L l o y d i a , 37_, 2 1 9 , ( 1 9 7 4 ) . 2 . J.W. C o r n f o r t h , Quart. Rev., 1 , ( 1 9 7 3 ) . 3 . E. Leete, The Chemical S o c i e t y , S p e c i a l i s t P e r i o d i c a l Reports, B i o s y n t h e s i s , 3., 1 1 3 , ( 1 9 7 5 ) • 4. D.H.R. Barton, Fourth I n t e r n a t i o n a l Symposium on the B i o -sy n t h e s i s and Physiology of A l k a l o i d s , H a l l e , D.D.R., ( 1 9 6 9 ) . 5 . G.B. Bodem, E. Leete, J.M.F. Manuel, Phytochemistry, 1 0 , 2 6 8 7 , ( 1 9 7 D . 6 . H. Rapoport, M.L. Rueppel, J . Amer. Chem. S o c , 9 3 , 7 0 2 1 , ( 1 9 7 D • 7 . W.H. P e r k i n , R. Robinson, J . Chem. S o c , 1 1 5 , 9 3 3 , ( 1 9 1 9 ) . 8 a . E. Leete, Chem & Ind.,(London), 6 9 2 , ( i 9 6 0 ) . b. E. Leete, J . Amer. Chem. S o c , 8_2, 6 3 3 8 , ( i 9 6 0 ) . c. E. Leete, Tetrahedron, 14, 3 5 , ( 1 9 6 1 ) . 9a. W.J. Cretney, J.R. H a d f i e l d , E.S. H a l l , J.P. Kutney, V.R. Nelson, D.R. W i g f i e l d , J.Amer. Chem. S o c , 90 3 5 6 6 , ( 1 9 6 8 ) . ~ b. A.R. Bat t e r s b y , A.R. Burnett, P.G. Parsons, Chem.Comm. ,1282,(1968) c. A.R. Battersby, A.R. Burnett, P.G. Parsons, J.Chem.Soc. (C), 1 1 9 3 , ( 1 9 6 9 ) . 1 0 . G. Hahn, H. Ludwig, Chem. Ber., 6]_, 2 0 3 1 , ( 1 9 3 * 0 . 1 1 . G. Barger, C. Scholz, Helv. Chim. Acta, 1 6 , 1 3 4 3 , ( 1 9 3 3 ) . 1 2 . R. Robinson, The S t r u c t u r a l R e l a t i o n of N a t u r a l Products, Clarendon Press, Oxford, ( 1 9 5 5 ) . 1 3 a . R.B. Woodward, Angew. Chem., 6 8 , 1 3 , ( 1 9 5 6 ) . b. R.B. Woodward, Nature, 1 6 2 , 1 5 5 , ( 1 9 4 8 ) . c R.B. Turner, R.B. Woodward, The A l k a l o i d s , 3 , R.F. Manske, ed., Acad. Press, N.Y., ( 1 9 5 3 ) . 14. A.R. Battersby, R. Binks, W. L a u r i e , G.V. Pa r r y , & B.R. Webster, Proc. Chem. S o c , 3 6 9 , ( 1 9 6 3 ) . - 2 0 1 -D. A r i g o n i , H. Goeggel, E x p e r i e n t i a , 21, 3 6 9 , ( 1 9 6 5 ) . D. Groger, K. Mothes, & K. S t o l l e , Chem. & I n d . (London), 2 0 6 5 , ( 1 9 6 5 ) . D.H.R. B a r t o n , G.W. K i r b y , R.H. P r a g e r , and E.M. W i l s o n , J . Chem. S o c , 3990, ( 1 9 6 5 ) . D. Gro g e r , K. Mothes, K. S t o l l e , Z. N a t u r f o r s c h . B. 2 1 , 2 0 6 , ( 1 9 6 8 ) . —-A.R. B a t t e r s b y , R. B i n k s , W. L a u r i e , G.V. P a r r y , B.R. Webster, J . C h e n . S o c , 7 4 5 9 , ( 1 9 6 5 ) . A. Ahmad, I . Kompis & E. L e e t e , J . Amer. Chem. S o c , 8j_, 4 l 6 8 , (1965) M. Hesse, I n d o l a l k a l o i d e i n T a b e l l e n , V o l I , I I e d . S p r i n g e r -v e r l a g B e r l i n , H e i d e l b e r g & New Y o r k , ( 1 9 6 4 ) , ( 1 9 6 8 ) . R. Thomas, T e t r a h e d r o n L e t t e r s , 5 4 4 , ( 1 9 6 1 ) . E. Wenkert, J . Amer. Chem. S o c , 84_, 9 8 , ( 1 9 6 2 ) . J . L evy, Apocynaceous i n d o l e a l k a l o i d s , D o c t o r a l T h e s i s , U . o f P a r i s , ( 1 9 6 2 ) . G.H. B u c h i & R.E. Manning, T e t r a h e d r o n L e t t e r s , 2 6 , 5 , ( I 9 6 0 ) . G. H. B u c h i & R.E. Manning, T e t r a h e d r o n , 1049, ( 1 9 6 2 ) . C. D j e r a s s i et a l , J . Org. Chem., 26, 1 1 9 2 , ( 1 9 6 1 ) . J . Grimshaw, Chem. & I n d . (London), 4 0 3 , ( 1 9 6 l ) . T. Kubota & Y. To m i t a , T e t r a h e d r o n L e t t e r s , 5_, 1 7 6 , ( 1 9 6 1 ) . D. A. P r i n s & U. R.enner, E x p e r i e n t i a , 3/7, 106, ( 1 9 6 1 ) . H. C. Beyerman, J.S. Bontekoe, V. G e r r i s , P . J . Oud, J.E. R i b b e r s , P.J. Van Ree and W.L.C. V e e r , Rec. T r a v . Chim. Pays-Bas 7 6 : 8 3 9 , ( 1 9 5 7 ) . H.C. Beyerman, L.A. Van D i j c k , J . L e v i s a l l e s , A. M a t e r a , and W.L.C. V e e r , B u l l . Soc. Chim. P r . : 1 8 1 2 , ( 1 9 6 1 ) . G. O r i e n t ! , L. P a n i z z i , and M.L. S c a r p a t i , Gazz. Chim. I t a l . 90: 1449, ( I 9 6 0 ) . P.N. Edwards, S. G h o s a l , E. L e e t e , J.Amer.Chem.Soc., 84, 1 0 6 8 , ( 1 9 6 2 ) . A.R. B a t t e r s b y , G.V. P a r r y , T e t r a h e d r o n L e t t e r s 7 8 7 , ( 1 9 6 4 ) . A.R. B a t t e r s b y , R.T. Brown, R.S. K a p i l , A.O. P l u n k e t t , G.B. T a y l o r , Chem. Comm., 46, ( 1 9 6 6 ) . D. A r i g o n i , H. Goeg g e l , Chem. Comm., 5 3 8 , ( 1 9 6 5 ) . -202-3 6 . N.R. F a r n s w o r t h , L l o y d i a , 24_, 1 0 5 , ( 1 9 6 1 ) . 37. W.T. S t e a r n , L l o y d i a , 29,196,(1966). 38. F.McCapra, T. M o n e y , A . I . S c o t t , I.G. W r i g h t , P r o c . N a t. Acad. S c i . U.S., 5 3 , 901,(1965). 39. F.McCapra, T. Money, A . I . S c o t t , I.G. W r i g h t , Chem. Comm., 537, (1965). 40. A.R. B a t t e r s b y , R.T. Brown, R.S. K a p i l , J.A. K n i g h t , J.A. M a r t i n , A.D. P l u n k e t t , Chem. Comm., 810, 888,(1966). 41. F. McCapra, E.S. H a l l , T. Money, A . I . S c o t t , I.G. W r i g h t , J.Amer.Chem.Soc ., 9 0 , >\l>i>\, C 1 9 6 8 ) . 42. D. G r o g e r , W. M a i e r , G. S c h l a t t e r , M. Schmid, G.E. Waldner, H e l v . Chim. A c t a , 5_2, 776,(1969). 43. A.R. B a t t e r s b y , R.T. Brown, J.A. K n i g h t , J.A. M a r t i n & A.D. P l u n k e t t , Chem. Comm., 346,(1966). 44. D. A r i g o n i , H. Goeggel, P. Loew, Chem. Comm., 347,(1966). 45. F. McCapra, K. Fukumoto, E.S. H a l l , J.R. Hanson, T. Money, B.S. Mootoo, G.T. P h i l i p p s , A . I . S c o t t , Chem. Comm., 348,(1966). 46. E. L e e t e , S. Ueda, T e t r a h e d r o n L e t t e r s , 4 9 1 5 , ( 1 9 6 6 ) . 47. G. P o p j a k , N a t u r a l S u b s t a n c e s Formed B i o l o g i c a l l y from M e v a l o n i c A c i d , l e d . T.w. Gooctwm; , A c a d . PreSS, N.Y. P. 1 7 , ( 1 9 7 0 ) . 48a. A . J . B i r c h , J.M. Ko c o r , N. Sheppard, J . Winter,J.Chem.Soc. 1502, (1962 ). b. A . J . B i r c h . R.J. E n g l i s h , R.A. Massy-Westrop, H. S m i t h , J . Chem. S o c , 3 6 9 , ( 1 9 5 8 ) . 49. D. A r i g o n i , E x p e r i e n t i a , 14_, 153,(1958). 50. A.R. B a t t e r s b y , P. F r a n c o i s , B. Gre g o r y , M.M. J a n o t , I . L e v i s a l l e s , P. P o t i e r , H. Spe n s e r , J . L . T u r n e r , Chem. Comm., 219, (1967). 51. A.R. B a t t e r s b y , B. Gr e g o r y , Chem. Comm., 134,(1968). 52. A.R. B a t t e r s b y , R.T. Brown, A.S. K a p i l , J.A. M a r t i n & A.O. P l u n k e t t , Chem. Comm., 812, 890,(1966). 53. A.R. B a t t e r s b y , Pure A p p l . Chem., 14, 1117,(1967). -203-54. D. A r i g o n i , S. B r e c h b u l e r , C.J. C o s c i a , P. Loew, Ch. Von. Szczepanski,Chem. Comm., 136,(1968). 55. L. B o t t a , C.J. C o s c i a , R. G u a r n a c c i a , A r c h . Biochem. B i o p h y s . , 904,( 1971 ). 56a. D. A r i g o n i , S. E s c h e r and P. Loew, Chem. Comm., 823,(1970) . b. A.R. B a t t e r s b y , S.H. Brown, and T.G. Payne, Chem. Comm., 827,(1970). 57. A.R. B a t t e r s b y , A.R. B u r n e t t , and P.G. P a r s o n s , Chem. Comm., 826,(1970). 58a. Y. A o k i , H. Inouye, Y. Takedo, and S. Ueda,Tetrahedron L e t t e r s , 2351,(1969). b. Y. A o k i , H. Inouye, Y. Takedo, & S. Ueda, Chem.&. Pharm. B u l l . (Tokyo), 20, 1287, (1972). 59. A.R. B a t t e r s b y , A . R . B u r n e t t , & P.G. P a r s o n s , Chem. Comm., 1280, (1968). 60a. C.J. C o s c i a and R. G u a r n a c c i a , J . A m e r . G h e m . S o c . , 9 3 , 6 3 2 0 , ( 1 9 7 1 ) • b. A.R. B a t t e r s b y , A.R. B u r n e t t & P. G. P a r s o n s , Chem. Comm., 1282, (1968). c. A.R.. B a t t e r s b y , A.R. B u r n e t t , & P.G. P a r s o n s , J . Chem. Soc ., (C), 1187, (1969). 61a. H. Inouye, Y. Nakamura, & S. Ueda,Tetrahedron L e t t e r s , 3 2 2 1 , ( 1 9 6 7 ) b. H. Inouye, Y. Nakamura, & S. Ueda, Chem.& Pharm. B u l l . (Tokyo), 18, 2043,(1970). 62a. H. Inouye, Y. Takeda, & S. Ueda,Tetrahedron L e t t e r s , 3 4 5 3 , ( 1 9 6 8 ) . b. H. Inouye, Y. Takeda, & S. Ueda, Chem.& P h a r m . B u l l . ( T o k y o ) , 19 , 587, (1971). c. H. Inouye, Y. Takeda, & S. Ueda, T e t r a h e d r o n L e t t e r s , 4 0 7 , ( 1 9 6 9 ) . 63. A.R. B a t t e r s b y , & B. Gre g o r y , Chem. Comm., 134,(1968), 64. R.T. Brown and L.R. Row, Chem. Comm., 453,(1967). 65. R.T. Brown, K.V.J. Rao, P.V.S. Rao, and L.R. Row, Chem. Comm., 350,(1968). 66. L. M e r l i n i & G. N a s i n i , Gazz. Chim. I t a l . , 98, 974,(1968). -204-67. G.N. Smith, Chem. Comm., 912,(1968). 68. R.T. Brown, G.N. Smith, & K.S.J. Stapleford, Tetrahedron Letters, 4349, (1968). 69. R.T. Brown, G.N. Smith, & K.S.J. Stapleford, 1968, Unpub-lis h e d results mentioned i n reference L p. 236, l a s t paragraph. 70. A.R. Battersby, A.R. Burnett, P.G. Parsons, Chem. Comm., 1280, (1968). 71. A.R. Battersby, A.R. Burnett, & P.G. Parsons,J.Chem.Soc.,(C), 1193, (1969). 72. A.R. Battersby, & E.S. H a l l , Chem. Comm., 793,(1969). 73. A.R. Battersby, A.R. Burnett, E.S. H a l l , & P.G. Parsons, Chem. Comm., 1582,(1968). 74. G.N. Smith, Symposium on the chemistry of the al k a l o i d s , Manchester, (19 69). 75. K . T . D . Desilva, G.N. Smith, & K.E.H. Warren, Chem. Comm., 905, (1971). 76. F . J . A l l e n , A.R. Battersby, K.H. Gibson, N.W. Isaacs, O. Kennard, W.D.S. Motherwell, P.J. Roberts, Chem. Comm., 899, (1971). 77. A.R. Battersby & R.J. Parry, Chem. Comm. , 901, (1971). 78. A.R. Battersby & K .H . Gibson, Chem. Comm., 902,(1971). 79. W.P. Blackstock, R.T. Brown and G.K. Lee, Chem. Comm., 910, (1971). 80a. P.E. Daddona, E. Hagaman, A.H. Heckendorf, C.R. Hutchinson and E. Wenkert, J.Amer. Chem. S o c , 96,5609, (1974). • Correction i b i d . 97_, 1988, (1975). b. H. Rapoport, Unpublished work referred to i n 80a. 81. J. Clardy, C.R. Hutchinson, K.C. Mattes, & J.P. Springer, J. Amer. Chem. S o c , 97_, :6270, (1975). 82. J . Stockigt, J . Treimer, & M.H. Zenk, Febs. Lett., 70, number 1, p. 267,(1976). 83a. J . Stockigt & M.H. Zenk, Febs. Lett., 79_, number 2, p. 233, (1977). b. J. Stockigt & M.H. Zenk, Chem. Comm., 646 ,(1977). -205-E. Wenkert and B. Wickberg, J . Amer. Chem. S o c , 8_7, 1580 (1965) . A.I. Scott & A.A. Querishi, J. Amer. Chem. S o c , 91, 5874 (1969). D. Arigoni, A.R. Battersby, J.C. Byrne, R.S. K a p i l , P. Loew, J.A. Martin, T.G. Payne, Chem. Comm., 951, (1968). A.R. Battersby, Chimia (Switz) , 2_2, 213,(1968). A.A. Querishi & A.I. Scott, Chem. Comm., 948,(1968). A.R. Battersby, E.S. H a l l , Chem. Comm., 793,(1969). N. Langlois, & P. Potier , C.R. Acad. S c i . , Paris , Ser. C, 2^ 73, 994, (1971). Z.Z. Dabrowski, G.F. Smith & G.N. Smith, I.U.P.A.C., Intern. Symp. Chem. Nat. Prod. (London). Abstracts, p . 425, (1968). J.R. Althaus, Ph.D. Thesis, M. I. T. , (1970). A.I. Scott, Bioorg. Chem. 3, 398,(1974). P.J. Houghton & E.J. Shellard, Planta Med.,21, 16,(1972). P.J. Houghton, K. Sarpong, & E.J. Shellard, J . Pharm. Pharmacol., 23, 244S,(1971). A.I. Scott, Accounts Chem. Res. 3_, 151,(1970). M-M. Janot, J.Le Men, L. O l i v i e r , J . Poisson, & J-L. Pousset, Compt. Rend.., 261, 5538,(1965). H. Budzikiewicz, C. Djerassi, M-M. Janot, J.Le Men, J . Levy, and L. O l i v i e r , B u l l . Soc. Chim. Fr., 868,(1965). A.A. Qureshi, and A.I. Scott, Chem. Comm., 948,(1968). D.A. Evans, G.F. Smith, G.N. Smith, and K.S.J. Stapleford, Chem. Comm., 859,(1968). D.A. Evans, J.A. Joule, and G.F. Smith, Phytochemistry, 7_, 1429,(1968). G.A. Cor d e l l , G.F. Smith, & G.N. Smith, Chem. Comm., 193,(1970) R.T. Brown, G.F. Smith, K.S.J. Stapleford, and D.A. Taylor, Chem. Comm., 190,(1970). A.R. Battersby, and A.K. Bhatnager, Chem. Comm., 189,(1970)»-- 2 0 6 -9 9 a . R.T. Brown, J.P. Kutney, & E. P i e r s , J.Amer.Chem.Soc. , 9 2 , 1 7 0 0 , ( 1 9 7 0 ) . b. W.J. Cretney, J.R. H a d f i e l d , E.S. H a l l , J.P. Kutney, & V.R. Nelson, J . Amer. Chem. Soc . , 9_2, 1 7 0 4 , ( 1 9 7 0 ) . c. R.T. Brown, J.R. H a d f i e l d , J.P. Kutney, & E. P i e r s , J.Amer.Chem.Soc., 9 2 , 1 7 0 8 , ( 1 9 7 0 ) . d. W.J. Cretney, J.P. Kutney, P. LeQuesne, B. McKaque, E. P i e r s , J.Amer.Chem.Soc., £ 2 , 1 7 1 2 , ( 1 9 7 0 ) . e. N. Abduraham, C. Gl e t s o s , J.P. Kutney, P. LeQuesne, E.' P i e r s , & I . V l a t t a s , J.Amer.Chem.Soc., 9 2 , 1 7 2 7 , ( 1 9 7 0 ) . 1 0 0 . W.J. Cretney, J.R. H a d f i e l d , E.S. H a l l , J.P. Kutney, V.R. Nelson, & D.C W i g f i e l d , J.Amer .Chem. S o c ,90_, 3 5 6 6 , ( 1 9 6 8 ) . 1 0 1 . C. Ehret, J.P., Kutney, V.R. Nelson, & W.C. W i g f i e l d , J.Amer.Chem.Soc., 9 0 , 5 9 2 9 , ( 1 9 6 8 ) . 1 0 2 . J.P. Beck, N.J. Eggers, H.W. Hanssen, J.P. Kutney, R.S. Sood, & N.D. Westcott,J.Amer.Chem.Soc. , 9 3 , 7 3 2 2 , ( 1 9 7 1 ) . 1 0 3 . J.F. Beck, Ph.D. Thes i s , U.B.C., ( 1 9 7 1 ) . 104. C. Ehret, J.P. Kutney, G. P u l t o n , R.S. Sood, & N.D. Westcott, Bioorg. Chem., 1 , 194, ( 1 9 7 1 ) . 1 0 5 . J.P. Kutney, J . H e t e r o c y c l i c Chem., 9., Supplementary Issue, S - l , ( 1 9 7 2 ) . 1 0 6 . J.F. Beck, J.P. Kutney, V.R. Nelson, R.S. Sood,J.Amer.Chem. S o c , 9 3 , 2 5 5 , ( 1 9 7 1 ) . 1 0 7 . R.S. Sood, Ph.D. Thes i s , U.B.C., ( 1 9 7 0 ) . 1 0 8 . A.A. Quereshi and A.I. S c o t t , Chem. Comm., 9 4 5 , 9 4 7 , ( 1 9 6 8 ) . 1 0 9 a . R.T. Brown, J.S. H i l l , N. Kunesch, M. Muquet, J . Poisson, G.F. Smith, K.S.J. S t a p l e f o r d , Chem. Comm., 1475, ( 1 9 6 9 ) . b. R.T. Brown, J.S. H i l l , G.F. Smith, K.S.J. S t a p l e f o r d , Tetrahedron, 2 7 , 5 2 1 7 , ( 1 9 7 1 ) . c. N. Kunesch, M. Muquet, and J . Poisson, Tetrahedron, 2 8 , 1 3 6 3 , ( 1 9 7 2 ) . 1 1 0 a . A.I. S c o t t , and C C . Wei, J.Amer.Chem.Soc., 9 £ , 8 2 6 3 , ( 1 9 7 2 ) . b. A.I. S c o t t , and C C . Wei, J.Amer .Chem. Soc. , 9_4, 8 2 6 4 , ( 1 9 7 2 ) . c. A.I. Scott and C C Wei, J .Amer. Chem. Soc., 94_> 8 2 6 6 , ( 1 9 7 2 ) . - 2 0 7 -d. A.I. S c o t t , J . Amer. Chem. Soc., 9_4, 8 2 6 2 , ( 1 9 7 2 ) . . 1 1 1 a . A.A. Quereshl, & A.I. S c o t t , Tetrahedron, 3_0, 2 9 9 3 , ( 1 9 7 4 ) . b. A.I. Scott and C C . Wei, Tetrahedron, 3 0 , 3 0 0 3 , ( 1 9 7 4 ) . c. P.C. Cherry, A.I. S c o t t , & C C . Wei, Tetrahedron, 3 0 , 3 0 1 3 , ( 1 9 7 4 ) . 1 1 2 . P.C. Cherry, A.A. Quereshi, and A.I. S c o t t , J.Amer.Chem.Soc., 9 1 , 4 9 3 2 , ( 1 9 6 9 ) . 1 1 3 . A.I. S c o t t , Accounts Chem. Res., 3 , 1 5 1 , ( .1970) . 114. A.A. Quereshi and A.I. S c o t t , J.Amer.Chem.Soc., 9 1 , 5 8 7 4 , ( 1 9 6 9 ) . 1 1 5 . P.B. Rei c h a r d t , A.I. S c o t t , M.B. S l a y t o r and J.G. Sweeny, Bioorg. Chem., 1 , 1 5 7 , ( 1 9 7 1 ) . 1 1 6 . P.B. Rei c h a r d t , A.I. S c o t t , M.B. S l a y t o r , & J.G. Sweeny, Recent Advances i n Phytochemistry, 6_, 1 1 7 , ( 1 9 7 3 ) . 1 1 7 a . C.J. Cos c i a , R. Guarnaccia, and K.M. Madyastha, Fed. Proc. Fed. Am. Soc. Exp. B i o l . , 3 0 , 1 4 7 2 , ( 1 9 7 1 ) . b. C.J. C o s c i a , R. Guarnaccia, & K.M. Madyastha, Febs. L e t t . , 14, 1 7 5 , ( 1 9 7 1 ) . c. C.J. C o s c i a , & R. Guarnaccia, J . Amer. Chem. Soc . , 9 3 . , 6 3 2 0 , ( 1 9 7 1 ) . 1 1 8 . S.L. Lee, A.I. Scott,J.Amer.Chem.Soc., 9 7 , 6 9 0 6 , ( 1 9 7 5 ) . 1 1 9 . H.P. Husson, C. Kan-Fan, J . S t o c k i g t , and M.H. Zenk, Chem. Comm., 1 6 4 , ( 1 9 7 7 ) . 1 2 0 . H.P. Husson, C Kan-Fan, T. Sevenet, and J.P. V i d a l , Tetrahedron L e t t e r s , 1 8 8 9 , ( 1 9 7 7 ) . 1 2 1 . J.P. Kutney, Heterocycles, 4_, 4 2 9 , ( 1 9 7 6 ) and references t h e r e i n . 1 2 2 . J.P. Kutney, V.R. Nelson, D.C Wigfield,J.Amer.Chem.Soc. , 9 1 , 4 2 7 9 , ( 1 9 6 9 ) . 1 2 3 . A. Ahond, A. Cone, CKan-Fan, Y. L a n g l o i s , P. P o t i e r , Chem. Comm., 5 1 7 , ( 1 9 7 0 ) . 124. P. P o t i e r , M. Janot, C.R. Acad. S c i . , P a r i s , 2 7 6 , 1 9 2 7 , ( 1 9 7 3 ) • 1 2 5 a . Seeds k i n d l y donated by Dr. B. G i l b e r t , Research Centre f o r Nat u r a l Products, F a c u l t y of Pharmacy, Rio de J a n e i r o , B r a z i l . -208-b. grown by P. S a l i s b u r y , M y c o l o g i s t , U.B.C., t o whom s i n c e r e g r a t i t u d e i s extended. 126. R.H. B u r n e l l , D.D. Casa, Canad. J . Chem., 4j5, 8 9 , ( 1 9 6 7 ) . 127. D. G r i e r s o n , Ph.D. T h e s i s , U n i v . o f B.C., (1975). 128. E.C. B l o s s e y , K.S. Brown, J r . , J . C a m p e l l o , E.P. C a r r a z z o n i , C. D j e r a s s i , A.P. D u a r t e , S.E. F l o r e s , B. G i l b e r t , Y. Nakagawa, R.J. O w e l l e n , T e t r a h e d r o n , 2_1, 1141,(1965). 129. A.C. H i l d e b r a n d t & R.U. Schenk, Canad. J . B o t . , 5 0 , 1 9 9 , ( 1 9 7 2 ) . 130. 0. C o l l e r a , A. S a n d o v a l , & F. W a l l s , T e t r a h e d r o n , 2_, 173, ( 1958). 131a. H. B u d z i k i e w i c z , C. D j e r a s s i , A. S a n d o v a l L, J.N. S h o o l e r y , F. W a l l s , J.M. W i l s o n , T e t r a h e d r o n L e t t e r s , 4 0 9 , ( 1 9 6 2 ) . b. K. Biemann, A.L. B u r l i n g a m e , D. S t a u f f a c h e r , T e t r a h e d r o n L e t t e r s , 5 2 7 , ( 1 9 6 2 ) . c. H. Schmid, D. Schumann, H e l v . Chim. A c t a . , 4_6, 1996,(1963), d. M.M. J a n o t , Pure A p p l . Chem., 6, 635,(1963). 132. G.B. F u l l e r & J.P. Kutney, H e t e r o c y c l e s , 3, 197,(1975). 133. Y. Ban, T. O i s h i , M. Magai, T e t r a h e d r o n L e t t e r s , 4 9 1 , ( 1 9 6 8 ) . 134. M. H e a r n e , u n p u b l i s h e d r e s u l t s , U n i v . o f B.C.,(1969). 135. A . J . G a s k e l l , H.E. Radung, E. W i n t e r f e l d t , T e t r a h e d r o n , 26, 5353, (1970). 136. V. S n i e c k u s & A. Wu,Tetrahedron L e t t e r s , 2 0 5 7 , (1975). 137. G.B. F u l l e r , Ph.D. T h e s i s , U n i v . o f B.C.,(1974). 138a. E. Bes c h , H. F r i t z , T. W i e l a n d , Angew. Chem., 7_1, 126, (1959). b. E. Bes c h , H. F r i t z , T. W i e l a n d , Ann, 663, 150,(1963). c. V. B o e k e l h e i d e , M. G r d i n i c , D.A. N e l s o n , J.Amer.Chem.Soc., 8 6 , 3 3 5 7 , ( 1 9 6 4 ) . 139a. E . J . Corey, B.E. Ganem,N.W. Gilman,J.Amer.Chem.Sec.,9 0 , 5 6 1 6 , ( 1 9 6 8 ; b. N.W. G i l m a n , Chem. Comm., 733,(1971). 140a. G.F. S m i t h , J.T. W r o b e l , J.Chem.Soc, 7 9 2 , ( I 9 6 0 ) . b. K. Biemann, G. S p i t e l l e r , J.Amer.Chem.Soc . ,84_, 4 5 7 8 , (1962). -209-141. D.H.R. Barton, R.H. Hesse, M.M. Pechet, C. Wi l t s h i r e , Chem. Comm., 1017,(1972). 142 . P.N. Edwards, G.F. Smith, J.Chem.Soc, 152, (1961). 143a. F. Berlage, K. Bernauer, P. Karrer, W. von Philipsborn, H. Schmid, Helv. Chim. Acta., 4_1, 2293 ,(1958). b. K. Bernauer, H. Schmid, (to Hoffman La Roche, Inc.,) U.S. Patent 3, 073, 832 (CI 260=293), Jan. 15, 1963., C.A. , 58, 9160 6, (1963). 144. M.M. Janot, J. Levy, J . Le Men, L. O l i v i e r , C.R. Acad. Sc., Par i s , C, 26L2, 1169,(1966). 145. M.F. B a r t l e t t , B.F. Lambert, & W.I. Taylor, J.Amer.Chem.Soc., 8 8 , 729, (1964). 146a. W. Gumlich, H. Wieland, Liebigs Ann. Chem., 494, 191,(1932). b. K. Kaziro, H. Wieland, Liebigs Ann. Chem., 506, 60,(1933). 147. F.A.L. Anet, S i r R. Robinson, J.Chem.Soc., 2253, (1955). 148. A. B o i l e r , H. E l s , P. Fabrui, A. Furst, J.R. Hymon, P. Karrer, H. Schmid, Helv. Chim. Acta., 5_2, 1564,(1969). 149. H.J. Berwin, T.G. Parsons, J.C. Powers, R. Seidner, J. Org, Chem., 31, 2623, (1966). 150. E. B i t t n e r , E. Dezseri, J . Eles, G. Fekete, K. Jovanovics, G. Richter, K. Szasz, G.R. Vegyeszeti, Ger. Offen. , 2, 259, 388 Chem. Ab., 81, 82, 369m, (1974). 151. L.F. Fieser & M. Fieser, Reagents for Org. Synth., p. 4, (John Wiley & Sons,) (1967 ). 152. J.C. Braekman, C. Hootele, B u l l . Soc. Chim. Belg., 7_8 (5/6) , 271, (1969). 153. H.Budzikiewicz, C. Djerassi, M.M. Janot, J . Levy, J . L e M e n , . J.M. Wilson, Tetrahedron, 19, 1265,(1963). 154. M. J u l i a & J . Lenzi, B u l l . Soc. Chim. Fr., 2262,(1962). - 2 1 0 -1 5 5 . C.A. Hartke, R.J. Owellen, J.Org.Chem., 4 l ( l ) , 1 0 2 , ( 1 9 7 6 ) . 1 5 6 . R.J. Owellen, J.Org.Chem., 3 9 ( 1 ) , 6 9 , ( 1 9 7 4 ) . 1 5 7 . T. Kobayoshi, H. Sakakibara, Tetrahedron, 2 2 , 2475, ( 1 9 6 6 ) . 1 5 8 a . E.J. Corey, J.W. Suggs, Tetrahedron L e t t e r s , 2 6 4 7 , ( 1 9 7 5 ) . b. R. R a t c l i f f e & R. Rodehurst,J.Org.Chem., 3 5 , 4000 ( 1 9 7 0 ) . 1 5 9 . K. K a z l r o , H. Wieland, Ann., 5 0 6 , 6 0 , ( 1 9 3 3 ) . 1 6 0 . E. Van Es, J.Chem. S o c . , 1564 , ( 1965). 1 6 1 . J . B a l s e v i c h , G.H. Bokelman, T. Hibino, T. Honda, I . I t o h , J.P. Kutney & A.H. Ratcliffe,Canad.J.Chem.(in p r e s s ) . 1 6 2 . P. Singh, unpublished observations, Univ. of B.C. ( 1 9 7 3 - 6 ) . 1 6 3 . B. Corson, H. Rogers, Org. Syn. C o l l . , ±3 884, ( 1 9 7 2 ) . 1 6 4 . P.E. Daddona, C.R. Hutchinson,J.Amer.Chem.Soc. , 9 6 , 6 8 0 6 , ( 1 9 7 4 ) . 1 6 5 a . N.J. Cone, M. Gorman & N. Neuss,J.Amer.Chem.Soc., 8 7 , 9 3 , ( 1 9 6 5 ) • b. R.T. Brown, J.P. Kutney & E. Piers,Canad.J.Chem., 4 3 , 1 5 4 5 , ( 1 9 6 5 ) . c. W.J. Cretney, J.R. H a d f i e l d , E.S. H a l l , J.P. Kutney & V.R. Nelson, J . Amer .Chem. Soc ., 92_, 1 7 0 4 , ( 1 9 7 0 ) . 166. J . Beck,- P. Bylsma, J . Cook, W.J. Cretney, K. F u j i , R. Imhof, J.P. Kutney, & A.M. Treasurywala, Helv. Chim. Acta., 5 8 , 1 6 9 0 , ( 1 9 7 5 ) . 1 6 7 a . N. L a n g l o i s , Y. La n g l o i s & P. P o t i e r , Chem. Comm., 6 7 0 , ( 1 9 7 5 ) . b. J.P. Kutney, A.H. R a t c l i f f e , A.M. Treasurywala & S. Wunderly, Heterocycles, 3 , 6 3 9 , ( 1 9 7 5 ) . 1 6 8 a . J.B. P a t r i c k & B. Wit kop,J. Amer .Chem. Soc . ,7_2_, 6 3 3 , ( 1 9 5 0 ) . b. T. Hino, T. Matsuura, M. Nakagawa & I . S a i t o , Acc.Chem.Res., 346, ( 1 9 7 7 ) . 169. R.Z. Andriamialosa, A. C h i a r o n i , F. G u e r i t t e , N. L a n g l o i s , Y. L a n g l o i s , P. P o t i e r & C. Riche, Tetrahedron, 3 2 , 9 4 5 , ( 1 9 7 6 ) . 1 7 0 . G.H. Svoboda, L l o y d i a , 2_4(1), 1 7 3 , ( .1961) . 1 7 1 . P.B. Babcock, D.P. Carew, L l o y d i a , 2 5 ( 4 ) , 2 0 9 , ( 1 9 6 2 ) . 1 7 2 . D.P. Carew, A.H. H a r r i s , H.B. Nylund, L l o y d i a , 2 7 ( 4 ) , 3 2 2 , C 1 9 6 4 ) . -211-17 3. G.H. Boder, M. Gorman, I.S. Johnson, P.J. SimDSon, Lloydia, 27(4), 328, (1964). 174. D. Groger, K. Mothes, I.Richter, K. S t o l l e , Naturwissen-schaften, 306, (1965). 175. J . Garnier & G. Morel, Physiol. Veg., 10 (4), 617, (1972). 176. D.P. Carew, 'The Catharanthus Al k a l o i d s ' , ed. N. Farns-worth (1975). 177. D.P. Carew, R.J. Krueger, Phytochemistry, 1 6 , l 4 6 l , ( 1 9 7 7 ) . 178. Y. Demarly, V. Petard, Ann. Amm. Plantes., 2_2 (4) , 361, (1972). 179. M. Koch, G. Kunesch, Phytochemistry, l4_, 1 3 8 5 , ( 1 9 7 5 ) . 180. C. roseus tissue cultures were grown at the Univ. of Leicester (Botany dept.) by Prof. H.E. Street. 181a. K. Biemann. G. Buchi, N.J. Cone, M. Gorman, W. Hargrove, R.E. Manning &'N. Neuss,J.Amer.Chem.Soc. , 8 6,1440,(1964). b. H.E. Boaz, N.J. Cone, M. Gorman & N. Neuss,J.Amer.Chem.Soc., 84, 1509, 1962. 182. A.R. Battersby & G.H. Gibson, Chem. Comm., 902, (1971). 183. R.T. Conley, Infrared Spectroscopy, p. 165, copyright A l l y n & Bacon (1966) . 184. G.H. Bokelman, M. Ichikawa, E. Jahngen, A.V. Joshua, J.P. Kutney, P.H. Liao & B.R. Worth,Canad.J. Chem., 55(18), 3227, (1977). 185. E. Jahngen, unpublished observations, Univ. of B.C., 1976. 186. Preliminary i n v e s t i g a t i o n towards the formation of t h i s compound had been reported by E. Jahngen, Univ. of B.C., 1976. 187. G. Buchi & R.E. Manning,J.Amer.Chem.Soc . ,88, 2 5 3 2 , ( 1 9 6 6 ) . 188. G. Buchi, P. Kulsa, K, Ogasawara, R.L. Rosati,J.Amer.Chem.Soc., 92, 999, 1970. 189. A.V. Lichman, J.Chem.Soc.,(C), ,2540, (1971). 190. A.M. Treasurywala, Ph.D. Thesis, Univ. of B.C., (1973). -212-191. In the research report of I. Itoh conditions for the conversion of N^-carbomethoxycleavamine-N^-oxide to N a-carbomethoxycleavamine were described. 192. G.H. Svoboda, Lloydia,' 27 (4) , 299, 1964 (and references therein). 193a. J. Schmutz & H. Wittwer, Helv. Chim. Acta, 43, 793 (1960). b. K.G. Dave & E. Wenkert,J.Amer.Chem.Soc.,84,94, ( 1 9 6 2 ) . c. E.M. Acton, O.P. Crews, L. Goodman, & C.W. Mosher, J . Medicin.Chem., 9, 237, (1966). d. D.S. Grierson & J.P. Kutney, Heterocycles, 3_, 171, (1975) . e. K. Fukumoto, Y. Ichikawa, T. Kametani & T. Suzuki, Heterocycles, 3_, 401, (1975). f. K. Fukumoto, Y. Ichikawa, T. Kametani & T. Suzuki,J.Chem. S o c , Perkin 1, 2102, (1975). g. R. Besselievre & H.P. Husson, Tetrahedron Letters,2 2 _ , 1 8 7 3 , (1976) . 194a. F.A. Hochstein, G.A. Iacobucci & R.B. Woodward, J.Amer. Chem.Soc,,^1, 4434, (1959). b. P.A. Cranwell & J.E. Saxton, J.Chem.Soc., 3 4 8 2 , ( 1 9 6 6 ) . c. T.R. Govindachari, S. Rajapa & V. Sudarsanan,Indian. J. Chem., 1, 247, (1963). d. R.N. S t i l l w e l l , Ph.D. Thesis, Harvard University, (1964.) e. L.K. Dalton, S. Demerac, B.C. Elmes, J.W. Loder, J.M. Swan & T. Tetei,Austral. J.Chem. , 20_, 2715,(1967.) f. K.N. Kilminster & M. Sainsbury,J.Chem.Soc , Perkin I, 2 2 6 4 . (1972). g. A. Ahond, F. Le G o f f i c & A. Gouyette, Tetrahedron, 29, 3357,(1973.) h. N.M. Hasan & A.P. Kozikowski, J.Org.Chem.,4_2 , 2039, (1977.) 195a. A. Ahond, A. Cave, T.A. Connors, L.K. Dalton, N. Dat-Xuong, M. Hayat, F. Le G o f f i c , A. Gouyette, M.M. Janot, C. Kan-Fan, G. Mathe, J . Le Men, J . Miet, J . Poisson, P. Potier, T. Sevenet, Biomedicine, 21, 101, (1974). -213-b. E.M. Acta, O.P. Crews, L. Goodman & C.W. Mosher, J. Medicin.Chem., 9_, 237, (1966). c. N. Dat-Xuong, C. Goss, C. P a o l e t t i , J.B. Le Pecq, Compt. Rend, 2 77, 2289, (1973). 196. H.F. Russell & R.J. Sundberg, J . Org. Chem. , 3 _ 8 , 3324, (1973). 197. This work i s under invest i g a t i o n by M.B. Monteiro, Univ. of B. C., (1977). 19 8. Preliminary investigations towards the formation of thi s class of compounds was reported by P.R. Jamieson, Univ. of B. C., (1976). 199. R. Besselievre, C. Thai, H.P. Husson & P. Potier, Chem. Comm., 90, (1975). 200. J.A. Joule & S.J. Martinez, Chem. Comm., 818, (1976). 201. R. Greenhouse, J.P. Kutney & V.E. Ridaura, J.Amer.Chem.Soc., 96, 7364, (1974). 202. This i s currently being investigated by Y.H. So, Univ. of B. C. 203. C. Djerassi, L.J. Durham, B. G i l b e r t , H. Monteiro, J.A. Joule, J.Chem.Soc, 4773, (1965).

Cite

Citation Scheme:

Citations by CSL (citeproc-js)

Usage Statistics

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.

Include Metadata Specify width in pixels (leave blank for auto-width):

                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>

Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:

https://iiif.library.ubc.ca/presentation/dsp.831.1-0060870/manifest

Open Collections

UBC Theses and Dissertations

UBC Theses and Dissertations

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

Page Metadata

Item Metadata

Download

Full Text

Cite

Usage Statistics

Embed

Comment

Related Items

Open Collections

UBC Theses and Dissertations

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

Page Metadata

Item Metadata

Download

Full Text

Cite

Usage Statistics

Share

Embed

Comment

Related Items