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UBC Theses and Dissertations

Total synthesis of veratrum alkaloids Cable, John 1968-12-31

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THE TOTAL SYNTHESIS OF VERATRUM ALKALOIDS by John Cable B.Phm., U n i v e r s i t y o f Sydney, Sydney, A u s t r a l i a , 1963 M.Phm., U n i v e r s i t y of Sydney, Sydney, A u s t r a l i a , 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C olumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f 6tt/?l/H/ST%Lf The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date - i i - Abstract A synthetic approach to members of the Veratrum alkaloids and i t s application i n the synthesis of verarine i s described. The condensation of o p t i c a l l y active 3B-acetoxy-5a-etiojerv-12- en-17-one (76), a known compound available from the degradation of hecogenin acetate, with the lithium derivatives of various substituted 2-ethylpyridines i s outlined as a general scheme for synthesising the carbon skeleton" of members of the Veratrum alkaloids. Condensation with the lithium derivative of 2-ethy]-5-methy]pyridine (105) followed by acetylation of the product gave a mixture of four isomers possessing the verarine skeleton (106). The two major isomers designated "A" and "13" were separately converted to the ring D aromatic compounds (107) by heating with palladised charcoal and the products shown to be isomeric. Selective hydrogenation of the pyridine moiety in either ring D aromatised compound (107) gave a mixture of four isomers which contained the piperidine ring (108). These compounds were separated and then converted to the N-acetyl derivatives (110) v i a the 3-0,N-diacetyl derivatives (109). Degradation of veratramine (2) by a known procedure gave 3-0,N- diacetylverarine (117). Hydrogenation of the 5,6-double bond employing Adams catalyst i n acetic acid gave a 1:1 mixture of the 5a,6- and 58,6- dihydro compounds which were separated as the N-acetyl derivatives (120, 121). The N-acetyl derivative of one of the eight isomers obtained from hydrogenation of the isomeric aromatic compounds (107) has been i d e n t i f i e d as N-acetyl-5a,6-dihydroverarine (120). The conversion of th i s compound to verarine (3) was carried out on a quantity of material obtained from veratramine. Oxidation with Jones reagent led to N-acetyl-3-keto-5a,6- dihydroveiarine (111) which was converted to N-acetyl-A 4-3-keto-5,6-dihyd verarine (112). Treatment of this a,B-unsaturated ketone with isopropenyl acetate gave the enol acetate (113) which was converted to N-acetylverarine (114). Removal of N-acetyl group gave verarine (3) which was i d e n t i f i e d by comparison with an authentic sample. This completes i n a formal sense the t o t a l synthesis of verarine since hecogenin has been t o t a l l y synthesised. The t o t a l synthesis of racemic 3$-acetoxy-5a-etiojervan-17-one from 6-naphthol by other members of t h i s laboratory i s mentioned and i t s comparison with the natural (+) 38-acetoxy-5a-etiojervan-17-one i s noted. Table of Contents Page T i t l e page 1 Abstract Table of Contents i v L i s t of Figures v Acknowledgements v i i Introduction 1 Discussion 22 Experimental 69 Bibliography 94 -V- L i s t o f F i g u r e s F i g u r e P a g e 1 3 2 4 3 6 4 10 5 11 6 15, 16 7 18, 19 8 20 9 23 10 25 11 26 12 33 13 35 14 36 15 37 16 38 17 43 18 44 19 45 20 50 - v i - L i s t o f Figures (Cont'd) Figures Page 21 53 22 54 23 58 24 59 25 62 25(a) 62 26 64 27 65 28 66 - v i i - Acknowledgements I wish to express my thanks t o Pro f e s s o r J . P. Kutney f o r h i s c o n t i n u a l encouragement and e x c e l l e n t guidance throughout the course of my research. I am g r a t e f u l f o r s c h o l a r s h i p s from the MacMillan Bloedel and Powell R i v e r Co. and the N a t i o n a l Research C o u n c i l of Canada which I have r e c e i v e d during my study. F i n a n c i a l support of t h i s p r o j e c t by Smith M i l l e r and Patch Inc., The N a t i o n a l Research Council of Canada and the Medical Research Council of Canada i s g r e a t l y appreciated. In a d d i t i o n the r e c e i p t of generous g i f t s of hecogenin acetate from Smith, M i l l e r and Patch Inc. and Syntex S.A., Mexico i s Acknowledged. 1 INTRODUCTION during the past decade s i g n i f i c a n t contributions have been made regarding the chemistry and stereochemistry of several sub-groups among the Veratrum alkaloids. The general c l a s s i f i c a t i o n , Veratrum alkaloids includes those alkaloids from the t r i b e Veratreae belonging to the Liliaceae family and s p e c i f i c a l l y from the genera Veratrum, Zygadenus, Stenanthium and Schoenocaulon. Alkaloids of s i m i l a r structure have also been shown to occur i n the F r i t i l l a r i a genus of the L i l i a c e a e . Reviews on the various aspects of the chemistry of the Veratrum _• i 2 3 alkaloids have been written by Fieser and Fieser , Boit , Narayanan and 4 Kupchan . In addition, the occurrence of the alkaloids i n the plants of the Veratreae, and the implications of a l k a l o i d occurrence and structure to the taxonomy of the Veratreae have been reviewed^. The d i v i s i o n of the Veratrum alkaloids into the jerveratrum and cerveratrum groups as proposed by Feiser has been adopted by Kupchan in the most recent review. The jerveratrum alkamines contain only 1 to 3 atoms of oxygen and are found i n the unhydrolysed plant extracts, i n part as the free alkamines, and i n part, i n combination with one molecule of D-glucose as glucoalkaloids. The cerveratrum alkamines are highly hydroxylated and contain 7 to 9 atoms of oxygen. They usually occur e s t e r i f i e d with various acids but are i n some instances unconjugated although they do not occur as glycosides. Crude extracts from Veratrum and related plants have been used for various medicinal purposes since the middle ages, and t h e i r use i n the control of hypertension dates from a report by Baker** i n 1859. 2 T r e a t m e n t s i n t h e second h a l f o f t h e n i n e t e e n t h c e n t u r y employed crude e x t r a c t s w h i c h gave e r r a t i c r e s u l t s and t h e i r usage was d i s c o n t i n u e d . D u r i n g t h e l a t e 1930's p u r i f i e d a l k a l o i d a l p r e p a r a t i o n s became a v a i l a b l e and improved t e c h n i q u e s l e d t o t h e f i r s t c r y s t a l l i n e a l k a l o i d a l p r e p a r a t i o n , p r o t o v e r a t r i n e , w h i c h was shown t o be a p o w e r f u l h y p o t e n s i v e agent ' . 9 F o l l o w i n g p h a r m a c o l o g i c a l i n v e s t i g a t i o n by K r a y e r t h i s p r e p a r a t i o n was i n t r o d u c e d i n t o c l i n i c a l use i n t h e t r e a t m e n t o f c e r t a i n t y p e s o f h y p e r t e n s i o n ^ . One o f t h e l i m i t i n g f a c t o r s i n t h e use o f t h i s d r u g i s t h e narrow dosage range between h y p o t e n s i v e and e m e t i c e f f e c t s . The advent o f t h e s u p e r i o r h y p o t e n s i v e compound, r e s e r p i n e , has l e d t o t h e use o f t h e p r o t o v e r a t r i n e s b e i n g l a r g e l y d i s c a r d e d . The j e r v e r a t r u m a l k a m i n e r u b i j e r v i n e ( 1 ) , so named because o f i t s r e d c o l o u r w i t h s u l p h u r i c a c i d r a t h e r t h a n a s i m i l a r i t y t o j e r v i n e , may be r e g a r d e d as t h e s i m p l e s t o f t h e v e r a t r u m a l k a l o i d s from a s t r u c t u r a l p o i n t o f v i e w . I t p o s s e s s e s t h e normal C-27 s t e r o i d Skeleton ( e . g . c h o l e s t e r o l ) and t h e E and F r i n g s may f o r m a l l y be r e g a r d e d as h a v i n g been formed by f o l d i n g t h e normal c h o l e s t e r o l s i d e c h a i n around t h e n i t r o g e n atom. O t h e r j e r v e r a t r u m members v e r a t r a m i n e ( 2 ) , v e r a r i n e (3) and j e r v i n e (4) a r e c h a r a c t e r i s e d by t h e C-nor-D-^homo r i n g s k e l e t o n w h i c h may f o r m a l l y be r e g a r d e d as h a v i n g o r i g i n a t e d by m i g r a t i o n o f t h e C-13, C-14 bond o f a normal s t e r o i d to t h e C-12, C-14 p o s i t i o n . S i n c e i t was o u r aim to p r o v i d e a s y n t h e t i c e n t r y i n t o t h e C-nor,D-homo j e r v e r a t r u m a l k a l o i d s t h e s t r u c t u r a l e l u c i d a t i o n of v e r a t r a m i n e , v e r a r i n e and j e r v i n e w i l l now be r e v i e w e d . 3 b Figure I Fried and K l i n g s b e r g ^ proposed the term "jervane" be adopted to represent the carbon skeleton of jervine (4) and the term "etiojervane" to represent the parent t e t r a c y c l i c hydrocarbon. This proposal has been widely adopted and s i m p l i f i e s the nomenclature of these alkaloids. The systematic steroid nomenclature for the "etiojervane" portion i s 17-methyl-C-nor-D-homo-18-nor-5,12 androstane indicating the obvious advantages of the above proposal. The numbering scheme for these compounds i s as indicated for jervine (4) i n f i g 1. It has been proposed recently ( l l a ) that the term "cholojervane" be adopted for the 24 carbon skeleton which arises by cleavage of the C-24, C-25 bond. Both jervine (2) and veratramine (4) have been converted to the t r i a c e t y l derivative (5) and jervine has been interrelated with 12 • hecogenin (6) v i a compound 7. This suggested the 9a configuration for veratramine and further support for this assignment was obtained by conversion of hecogenin*^ and veratramine^ to compound 8. Additional Figure . 2 5 evidence for the 9a configuration was provided by Johnson^ i n a base catalysed e q u i l i b r a t i o n study of compound 9 formed from N-acetyl-11- ketoveratramine Q.0). Treatment of the diketone 9 with methanolic potassium hydroxide effected p a r t i a l conversion to a new compound. The n.m.r. spectrum of the diketone 9 showed a sharp signal attributed to the C-19 methyl at T 8.81 which was superimposable upon the corresponding signal i n the synthetic trans compound 12. The e q u i l i b r a t i o n mixture exhibited a new sharp signal at T8.45 which was superimposable on the corresponding C-19 methyl signal i n the synthetic c i s compound 11. This work established the B/C trans junction for N-acetyl, 11- ketoveratramine Q.0) which was related to veratramine by conversion to N-ethyl veratramine under conditions which allowed no epimerisation at C 9. The configuration of the piperidine moiety was f i r s t investigated by Sicher and T i c h y ^ by infrared comparison with two pairs of pipecolinol derivatives (13 and 14). They found that the c i s isomers exhibited two hydroxyl bands at 3u whilst the trans isomers showed a single hydroxyl band. They assumed that the ethyl group would occupy an equatorial position placing the hydroxyl group a x i a l and so allow hydrogen bonding to the nitrogen. Consequently the c i s isomers show a hydroxyl band due to the free hydroxyl as well as one due to the hydrogen bonded species. The trans isomers can exist with both groups equatorial and the infrared spectrum shows only the band-due to free hydroxyl. The infrared spectrum of veratramine shows only one hydroxyl band supporting a trans diequatorial arrangement for the alk y l side chain at C-22 and the C-23 hydroxyl. It 1 8 had been proposed that the C-26 methyl group belongs to the (S)-series and occupied the £5 position. From these considerations the configuration of veratramine was assigned as in 15. However, a recent investigation by Johnson has indicated that veratramine i s correctly represented by 16 i n which the substituents on the piperidine are a l l equatorial. Johnson synthesised 5a,6- dihydro-veratramine (17)and 5a,6-dihydro-23-iso-veratramine (18)without epimerising the C-22 a l k y l side chain. The iso compound must have the C-23 hydroxyl group i n an a x i a l orientation. This would be expected to cause a 12-13 cps s h i f t to lower f i e l d of the C-26 methyl signal i f th i s group i s also a x i a l l y oriented. The C-26 signal was shown to occur at T 9.18 i n both compounds 17 and 18 indicating that the C-26 methyl group i s equatorial. Since t h i s group occupies the 8 configuration and the substituents at C-22 and C-23 are trans and equatorial the correct assignment for veratramine i s represented by 16. E a r l i e r work 19 20 by Augustine and Masamune involving e q u i l i b r a t i o n studies on the keto amide of veratramine had supported the assignment of Sicher and T i c h y ^ . Masamune studied the-base catalyzed epimerisation of 3,N-7 diacetyl-23-dehydro-veratramine (19j which i n the o r i g i n a l postulate has one of the a l k y l groups on the piperidine moiety i n an a x i a l orientation. Treatment of 19 with meth.molic potassium hydroxide gave a predominance of the C-22-isomeric compound i n a r a t i o of 10:1. This resu l t was explained on the basis that epimerisation at th i s position would lead to the piperidine ring being able to assume the more stable conformation with both groups equatorial. 21 22 Paulson and Todt ' have shown that acylation of 2-methyl piperidine i s accompanied by conformational inversion of the ring (methyl group axial) to reli e v e s t e r i c interaction between the equatorial methyl group and amide carbonyl. 19 20 The recent n.m.r. study by Johnson supports t h i s inversion of the piperidine ring i n the N-benzoyl derivative of veratramine ( 2 0 ) . The C-23 proton appears as a r e l a t i v e l y sharp unresolved multiplet indicative of an a x i a l hydroxyl whilst the signal assigned to the C-26 methyl group now appears at T 9 . S 7. This downfield s h i f t of the C-26 methyl group indicates a 1,3 d i a x i a l relationship between the C-23 hydroxyl and the C-26 methyl groups.' It therefore seems very probable that the ketoamide 19 exists p r e f e r e n t i a l l y i n that conformation with the C-22 and C-25 substituents 8 both a x i a l . Epimerisation to the C-22 iso compound occurs readily since this compound can exist in the conformation with the C-22 a x i a l and she C-26 methyl equatorial. These studies indicate that veratramine i s correctly represented as 16 since the configuration at a l l centres except C-20 has been investigated. The configuration at C-20 i s assigned only on the basis of biogenetic analogy with other steroids. The configurations at C-22 and C-23 i n jervine have been interr e l a t e d with those of veratramine and therefore the tetrahydrofuran ring i n jervine must be attached to the piperidine ring in a trans manner. Degradation of jervine by K l i n s b e r g ^ gave 7 which had been synthesised 12 from hecogenin . The i n t e r r e l a t i o n with veratramine gave additional support for the 9a configuration whilst the configurations at a l l centres of 5, 6, 12-tetrahydrojervine (21) has been proposed by Wintersteiner 23 and Moore . In t h i s investigation the configurations at C-17 and C-20 were assigned on the basis of biogenetic analogy with other steroids rather than by investigation. Under fragmentation conditions N-methyl jervine gave 1, 5- 17 dimethyl-3-piperidone which was then reduced to 1,3-dimethylpiperidine A synthesis of 1-(3R)-dimethylpiperidine from D-(+) c i t r o n e l l a l was carried out and the product shown to be the antipode of the compound from N-methyljervine. Consequently the absolute configuration at C-25 i n jervine belongs to the (S)-series and the C-26 methyl group has the 8 orientation. The recently revised configurations of the piperidine ring i n veratramine also apply to jervine since the two compounds have been int e r r e l a t e d . In view of the 11-keto group i n jervine allowing for some 9 doubt tbout the actual configuration at C-9 i n jervine and i t s derivatives, Masamune has recently reexamined t h i s problem and has provided conclusive evidence for the 9a configuration v i a the conversion of veratramine and "Jervine-118-ol" to the 5a,6-dihydro derivative 22. At t h i s time the configurations at C-17 and C-20 were assigned only by biogenetic analogy and not by any direct evidence. A recent X-ray d i f f r a c t i o n determination on veratrobasine has revealed that t h i s compound i s probably jervin-118- 25 ol . This study reveals that the configuration which had been assigned 26 to C-17 i n jervine might be incorrect. Kupchan has investigated the relationship of jervine and veratrobasine and has shown that the l a t t e r compound i s indeed j e r v i n - l l B - o l . In the l i g h t of the X-ray work on veratrobasine the structure of jervine i s correctly represented by 23 in which the ether bridge i s B at both C-17 and C-23. Tomko who proposed that the compound was probably 23-desoxy veratramine 20 Masamune has investigated t h i s p o s s i b i l i t y by the removal of the 23- hydroxyl from veratramine and has shown that verarine i s i n fact 23 - desoxyveratramine. The reactions involved i n t h i s conversion w i l l be discussed l a t e r since they are pertinent to the work of the thesis. From th i s i n t e r r e l a t i o n the configurations assigned to veratramine must necessarily apply to verarine. The jerveratrum a l k a l o i d verarine (3) was f i r s t isolated by 27 HO 21 H 10 28 29 Two new veratrum a l k a l o i d s veralkamine (25) and veramine (26) have r e c e n t l y been i s o l a t e d and i n v e s t i g a t e d by Tomko and Sch r e i b e r . These compounds have been shown to possess the unusual 17B-methyl-18- nor-17-iso-cholestane carbon s k e l e t o n as i n d i c a t e d i n f i g . 4. These compounds are unusual when compared with the known jerveratrum a l k a l o i d s and c l o s e l y resemble the spiroaminoketal a l k a l o i d s r s o l a s o d i n e and tomatidine. The above workers have f u r t h e r i n v e s t i g a t e d these types o f a l k a l o i d s 30 31 and the r e s u l t s w i l l soon appear i n p r i n t ' Figure 4 The cerveratrum a l k a l o i d s are h i g h l y oxygenated and g e n e r a l l y occur as e s t e r s . A l l have the cevan nucleus which i s c h a r a c t e r i s e d by the C-nor,D-homo nucleus with an a l t e r n a t e f o l d i n g o f the si d e chain around the n i t r o g e n atom. The commonly o c c u r r i n g alkamines are veracevine (27), germine (28), zygadenine(29), p r o t o v e r i n e (30) and sabine (31). ( F i g . 5) The p r o t o v e r i n e e s t e r s A and B (32 and 33) have been i n v e s t i g a t e d thoroughly. S t r u c t u r a l s t u d i e s on thes.e compounds culminated i n 1960 wi t h the. e l u c i d a t i o n o f the s t r u c t u r e and c o n f i g u r a t i o n o f the alkamine 32 pr o t o v e r i n e and the t e t r a e s t e r s p r o t o v e r a t r i n e A and p r o t o v e r a t r i n e B P r o t o v e r a t r i n e A i s a potent hypotensive agent with a narrow t h e r a p e u t i c dosage range w h i l s t p r o t o v e r a t r i n e B i s l e s s a c t i v e but the emetic s i d e Figure 5 e f f e c t ! are not as pronounced. S t r u c t u r a l l y the difference between these two compounds i s small and t h i s prompted Kupchan to examine a number of protoveratrine derivatives with the aim of improving the 33 therapeutic dosage range . This study led to a number of generalisations concerning structure a c t i v i t y relationships for the protoveratrines but no r a d i c a l improvement of the therapeutic dosage range. The assignment of configurations at C-8, C-13, C-16, C-20 and C-22 i n veracevine has been presented and a subsequent X-ray study of cevine hydroiodidehas provided conclusive evidence for the configurational 35 assignements of thirteen of the fourteen asymmetric centres whilst evidence has been advanced to support the 20-8 configuration. Alkaloids of related structure have been isolated from plants of the F r i t i l l a r i a genus of the Liliaceae family. The F r i t i l l a r i a a l k a l o i d v e r t i c i n e (54) was f i r s t i solated by Fukuda from £. v e r t i c i l l a t a W i l l d . var. thunbergii Baker. The same al k a l o i d was isolated by Chou 37 and Chen from F_. r o y l e i Hook and named peimine. The i d e n t i t y of the two compounds was established by direct comparison. S t r u c t u r a l l y v e r t i c i n e represents the simplest example of a compound having the cevane nucleus as found i n the cerveratrum alkaloids. A further a l k a l o i d F r i t i l l a r i n e was isola t e d i n the above studies and has since been characterised as verticinone 38 (35) by mixed melting point and infrared spectra comparison An extensive n.m.r. study of members of the cerveratrum group 39 has been carried out . The spectra of 37 alkaloids of t h i s group were recorded and data for methyl group signals examined. The study provides additional support for the configuration of the C-27 methyl group and the effect of forming the D ring orthoacetate upon the stereochemistry of the D ring was examined. Masamune has examined the n.m.r. spectra of 65 derivatives of the jerveratrum group of alkaloids. A l l the compounds contain the 22,27-imino- 17,23-oxidojervane ring skeleton (36) and the data from the spectra i s confined to the correlation of the chemical s h i f t s of the methyl groups with changes i n environment. In the n.m.r. spectra of steroids and triterpenoids the long range shielding effects of various substituents on the chemical s h i f t of the angular methyl protons have been shown to be additive. In order to examine both a d d i t i v i t y and shielding effects in the 22,27-imino-17, 23-oxidojervane skeleton,the compounds were divided into those derived from the A/B trans form (37) and those from the A/B 14 c i s (38). The contributions due to various functional groups were obtained by pairing compounds which d i f f e r only by the group;in question. Values calculated for the C-19 methyl protons using these contributions were in good agreement (within 0.02 t) with the observed chemical s h i f t s , indicating the "p r i n c i p l e of a d d i t i v i t y " holds s a t i s f a c t o r i l y for the 19-methyl protons of these C-nor-D-homosteroid alka l o i d s . 43 44 Masamune and Johnson have recently published the results of t h e i r independent investigations regarding the synthesis of some Veratrus alkaloids Masamune has succeeded i n synthesising veratramine and jervine whilst Johnson has synthesised veratramine. Both groups u t i l i s e d 17- acetyl-5a-etiojerva-12,14,16-triene-38-ol (8) as the source of the C-nor- D-homo skeleton followed by elaboration of the 17-acetyl side chain to provide the hetereocyclic portion to complete the jerveratrum skeleton. An outline of Masamunes' synthetic approach i s given in Fig. 6. In t h i s sequence compound 8 was obtained by degradation of hecogenin and the piperidine portion i s attached v i a an al k y l a t i o n of the pyrrolidine enamine 46 with 45. This leads d i r e c t l y to an isomeric mixture of 3,N- diacetyl-5a;6-dihydro-23-dehydro-veratramines (47) since the (S) configuration of the methyl group i s established i n 43. This isomeric mixture was compared with an authentic sample obtained from 5a,6- dihydroveratramine. The ether bridge between C-17 and C-23 was synthesised by a series of elegant steps including formation of the important compound 22,27-iminojervan-13(17)-ene-36,236-diol (49) which was i d e n t i f i e d by comparison with an authentic sample. Epoxidation of this compound gives 50 which undergoes cleavage, of the epoxide and concommitant attack of the C-23 hydroxyl to give the desired ether bridge. Dehydration with thionyl chloride gave 3-;0N-diacetyl-15 Figure 6 H CI Ao , l c 16 Figure 6 continued 17 l l - d e o x o - 5 a , 6 - d i h y d r o j e r v i n e ( 5 2 ) w h i c h was c o m p a r e d w i t h an a u t h e n t i c s a m p l e p r e p a r e d f r o m j e r v i n e i n a n u n a m b i g u o u s m a n n e r . T h e i n t r o d u c t i o n o f t h e 1 1 - k e t o g r o u p u n f o r t u n a t e l y p r o c e e d s i n a y i e l d o f 1% b u t t h e a u t h o r s w e r e a b l e t o i s o l a t e 5 3 a n d c o m p l e t e t h e s e q u e n c e t o j e r v i n e a s 26 s h o w n . I t s h o u l d b e n o t e d t h a t i n v i e w o f t h e r e c e n t p a p e r b y K u p c h a n t h e f o r m u l a e 5 1 - 5 6 a r e i n d i c a t e d w i t h t h e c o r r e c t s t e r e o c h e m i s t r y a t 45 C - 1 7 . S i n c e h e c o g e n i n h a s b e e n t o t a l l y s y n t h e s i s e d t h i s w o r k r e p r e s e n t s i n a f o r m a l s e n s e a t o t a l s y n t h e s i s o f v e r a t r a m i n e a n d j e r v i n e . T h e W. S . J o h n s o n g r o u p a l s o e m p l o y e d 1 7 - a c e t y l - 5 a - e t i o j e r v a - 1 2 , 1 4 , 1 6 - t r i e n - 3 B - o l ( 8 ) a s t h e s o u r c e o f t h e C - n o r - D - h o m o p o r t i o n b u t i n t h e i r a p p r o a c h t h i s c o m p o u n d was t o t a l l y s y n t h e s i s e d f r o m Haoemann ' s e s t e r ( 5 7 ) a s o u t l i n e d i n E i g . 7 r a t h e r t h a n o b t a i n e d f r o m t h e d e g r a d a t i o n o f h e c o g e n i n . T h e s y n t h e s i s i n v o l v e s t h e u s e o f a r e l a y c o m p o u n d f r o m v e r a t r a m i n e f o r t h e c o n v e r s i o n o f 66 t o '8 . T h i s same g r o u p o f w o r k e r s 46 h a s r e c e n t l y p u b l i s h e d a n a l t e r n a t e s y n t h e s i s o f c o m p o u n d 8 b y e x t e n s i o n 47 o f t h e h y d r o c h r y s e n e a p p r o a c h w h i c h t h e y h a d i n v e s t i g a t e d e a r l i e r F i g . 8 o u t l i n e s t h e s y n t h e s i s o f v e r a t r a m i n e f r o m 1 7 - a c e t y l - 5 a - e t i o j e r v a - 1 2 , 1 4 , 1 6 - t r i e n - 3 6 - o l i n w h i c h t h e p i p e r i d i n e r i n g i s b u i l t up f r o m t h e u n c y c l i z e d c o m p o u n d ( 7 2 ) t h r o u g h 73 a n d 74 t o y i e l d a m i x t u r e o f e p i m e r i c N - b e n z o y l 5 B , 6 - d i h y d r o - 3 , 2 3 - d i k e t o v e r a t r a m i n e s ( 7 5 ) w h i c h was c o m p a r e d w i t h an a u t h e n t i c s a m p l e p r e p a r e d f r o m v e r a t r a m i n e . I n t r o d u c t i o n 4 o f t h e 5-6 d o u b l e b o n d was a c h i e v e d v i a f o r m a t i o n o f t h e A - 3 - k e t o n e w h i c h was c o n v e r t e d t o t h e e n o l a c e t a t e . R e d u c t i o n o f t h i s c o m p o u n d w i t h s o d i u m b o r o h y d r i d e p r o v i d e d N - b e n z o y l v e r a t r a m i n e w h i c h was d e b e n z y l a t e d t o g i v e v e r a t r a m i n e . T h i s s y n t h e s i s r e p r e s e n t s a d i r e c t t o t a l s y n t h e s i s o f v e r a t r a m i n e w h i c h c a n b e e x t e n d e d t o j e r v i n e e m p l o y i n g p r o c e d u r e s d e v e l o p e d 66 65 Figure 7 Figure 7 continued 20 Figure 8 21 b y t h e M a s a m u n e g r o u p . D u r i n g t h e p e r i o d o f t i m e t h a t t h e s e t w o g r o u p s h a v e i n v e s t i g a t e d t h e t o t a l s y n t h e s i s o f t h e j e r v e r a t r u m a l k a l o i d s we h a v e a l s o d i r e c t e d o u r e f f o r t s , i n a s o m e w h a t d i f f e r e n t m a n n e r , t o w a r d s a t o t a l l y s y n t h e t i c e n t r y i n t o t h e v e r a t r u m a l k a l o i d s . T h e o v e r a l l p l a n was t o e m p l o y a m e t h o d w h i c h w o u l d a l l o w a g e n e r a l e n t r y i n t o t h e s e a l k a l o i d s s o t h a t s l i g h t m o d i f i c a t i o n s o f p r o c e d u r e w o u l d l e a d t o d i f f e r e n t a l k a l o i d s . T h i s t h e s i s r e p r e s e n t s t h e w o r k d o n e o n t h e l a t t e r s t a g e s o f t h i s a p p r o a c h w h i c h I w o u l d now l i k e t o p r e s e n t . 22 DISCUSSION During the past few years members of this laboratory have been act i v e l y engaged i n research designed to provide a t o t a l l y synthetic entry into the Veratrum alkaloids. The approach has been to consider the veratrum skeleton as consisting of an etiojervane portion and a substituted piperidine, coupled by the C-17, C-20 bond. The aim was to provide a t o t a l synthesis of these two molecules and then couple them i n such a manner as to provide the desired skeleton. The proposed sequence i s outlined i n f i g . 9 i n which 38-acetoxy-5a-etiojerv- 12-en-17-one(76) i s reacted with the lithium derivative of the appropriately substituted 2-ethylpyridine (77) to provide a isomeric mixture of compounds possessing the structure 78. Aromatisation of ring D would provide compounds of type 79 d i f f e r i n g only i n configuration at C-20. A selective reduction of the pyridine to the piperidine system generates new asymmetric centres arid would give r i s e to a mixture of isomers (80) with the number depending upon the nature of R'. I f a l l the possible isomers are obtained after the selective hydrogenation then one of these isomers should be i d e n t i c a l with the 5 ,6-dihydro a l k a l o i d for which the sequence i s designed. I f R' i s hydrogen then the isomeric 5a,6-dihydroverarines w i l l be obtained and i n the instance where R' i s hydroxyl the sequence leads to an isomeric mixture of the 5a,6-dihydroveratramines. The reason for choosing 3B-acetoxy-5a-etiojerv-12-en-17-one (76) as the source of the etiojervane portion stems from e a r l i e r research carried out i n our laboratory which has been directed towards the t o t a l 2 3 Figure 9 24 synthesis of t h i s compound, which i s readily available from the degradation of hecogenin by published procedures. It was f e l t that the quantity of 3 B-acetoxy-5 a-etiojerv-12-en-17-one wl.'ich would result from the t o t a l l y synthetic approach would be i n s u f f i c i e n t to complete the t o t a l synthesis of members of the veratrum alkaloids. Consequently we envisaged the use of 3B-acetoxy-5a-etiojerv-J2-en-l7-one 'Obtained from degrading hecogenin, as a relay compound^ and the t o t a l synthesis of t h i s compound, as providing the t o t a l l y synthetic approach to representatives of the veratrum alkaloids. 48 The procedure of W. F. Johns was employed i n the degradation of hecogenin as outlined i n f i g . 10. This sequence gives a reasonable y i e l d of the desired 3B-acetoxy-5cc-etiojerv-12-en-17-one although some twelve steps are involved i n the degradation. A feature of t h i s sequence which proved important i n our t o t a l l y synthetic approach i s that the desired a 6-unsaturated ketone (76) i s formed from the saturated ketone (87) by bromination and then dehydrobromination. A synthesis of any naturally occurring al k a l o i d from 3B-acetoxy- 5a-etiojerv-12-en-17-one obtained from hecogenin constitutes a formal t o t a l synthesis of that a l k a l o i d since hecogenin has been t o t a l l y synthesised 45 from isoandrosterone . A more notable achievement however i s the direct t o t a l synthesis which would result i f 38-acetoxy-5a-etiojerv-12-en-17- one (76) could be prepared by a t o t a l l y synthetic route. The t o t a l 49 synthesis of t h i s compound has recently been achieved i n our laboratory and the approach employed i s outlined i n f i g . 11. The synthesis of the d i o l aldehyde (94) has appeared i n p r i n t ^ and involves a ring contraction developed by W. S. Johnson i n an isomeric series of compounds***. Attempts to prepare 38-acetoxy-5a-etiojerv-12-en-i7-onei from 25 Figure 10 27 compound 98 v i a al k y l a t i o n of the enamine were unsuccessful. However alk y l a t i o n v i a enolate anion led to compound 99 which was shown to be st r u c t u r a l l y i d e n t i c a l with the saturated ketone 87 obtained i n the degradation of hecogenin. This compound provides the point of linkage between the use of a relay compound from hecogenin and the t o t a l l y synthetic approach to the veratrum alkaloids. The compound (99) prepared by the t o t a l l y synthetic approach was racemic and was not resolved, due to the small quantities available, whereas that obtained from hecogenin i s a single enantiomer and has a rotation of + 122°' To test the f e a s i b i l i t y of the sequence in f i g . 9 the reaction of compound (101) with 2-ethylpyridine was investigated. E a r l i e r work ( f i g . 11) had made available the anisole compound (100) which was converted to the unsaturated ketone (101) v i a Birch reduction and acid hydrolysis of the resulting enol ether. When 2-ethylpyridine i s added to a tetrahydrofuran solution of methyl lithium a deep red colour rapidly develops. The lithium derivative of the 2-ethylpyridine i s formed by the removal of a proton from the a position of the ethyl side chain. The resultant charge i n the carbanion can be extensively delocalised by resonance into the pyridine ri n g . Other carbanions where s i m i l a r delocalisation of the negative charge can occur (e.g. triphenylmethyl, and fluorenyl ) give r i s e to a deep red colour m solution. By analogy the deep red colour observed i n t h i s reaction i s 28 probably indicat i v e of the formation of the desired lithium s a l t . Addition of compound 101 to the red solution of 2-ethylpyridine and methyl lithium was continued u n t i l the colour faded and then the reaction quenched with water. Examination of the ether extract by t . l . c . indicated that one major compound had been formed. After p u r i f i c a t i o n by column chromatography t h i s substance, i n i t s u l t r a v i o l e t spectrum, showed peaks at 257, 262 and 270 ny corresponding to the pyridine chromophore whilst the mass spectrum exhibited a peak at m/e 395 corresp onding to the parent ion for compound 102. The absence of a carbonyl peak i n the infrared supported the 1,2 addition of the organolithium derivative to the.a^ 6-unsaturated carbonyl compound as opposed to any 1, 4 addition. This procedure was then extended to the reaction of the lithium s a l t of 2-ethylpyridine with 3 6-acetoxy-5 a-etiojerv-12-en-17-one (76). Column chromatography of the products from t h i s reaction primarily gave a material which appeared as one compound on t . l . c . The u l t r a v i o l e t spectrum showed peaks at 257, 262 and 269 mj ind i c a t i v e of the pyridine chromophore whilst the mass spectrum exhibited a peak at m/e 395 corresponding to the parent ion for compound 103. 29 The elemental analysis supported the assumption that the material obtained was the product of the desired coupling but the n.m.r. spectrum indicated that i t was probably a mixture of two isomers. The spectrum showed two doublets i n the region x 8.84 and these were tentatively assigned to the C-2.1 methyl group i n each of two compounds. These reactions indicated that the desired coupling between _3j|r_acetoxy-5a-etiojervrl2-en-17-one .._..(76) and 2-ethylpyridine was indeed possible and so our efforts were turned to considering substituted 2- ethylpyridines which would allow an entry to the skeleton of some of the naturally occurring veratrum alkaloids. The i n i t i a l aim was the synthesis of veratramine, which has as i t s heterocyclic portion, 2-ethyl-3-hydroxy-5-methylpiperidine. It was therefore necessary to prepare the corresponding 2-ethyl-3-hydroxy-5- methylpyridine for coupling with "^'B-^etoxy-5'a-etiojfty-li-en^J^one (76). This pyridine i s not readily available but was synthesised by Dr. G.V. Nair of our laboratory. Treatment of t h i s compound with methyl lithium would possibly lead to the formation of the lithium alkoxide which might hinder the formation of the carbanion at the a position on the adjacent 2-ethyl side chain. Of the protecting groups available we chose to prepare the benzyl ether since the benzyl group should be eas i l y removed during the subsequent c a t a l y t i c hydrogenation of the pyridine ring. The treatment of 2-ethyl-3-benzyloxy-5-methylpyridine with methyl lithium gave only a faint transient pink colouration rather than the deep red colour which had been observed with 2-ethylpyridine. The 30 product after addition of 3 g-acetoxy-5 a-etiojerv-12-en-17-one (76) showed no coupled compounds. Repeated attempts to achieve this coupling under varying conditions of temperature and time were unsuccessful. The only compounds which could be isolated in any quantity from the column chromatography of the reaction products were the a,8-unsaturated ketone 76 and the o r i g i n a l substituted pyridine. In an attempt to investigate the effect of the benzyl group on the formation of the desired carbanion, the methyl ether was prepared. A deep red colour slowly developed when 2-ethyl-3-methoxy-5-methylpyridine was refluxed with methyl lithium i n tetrahydrofuran. Compound 76 was added u n t i l the colour faded and the reaction worked up as usual. Examination of the ether extract by t . l . c . indicated one major compound besides recovered . a, 8-unsaturated ketone. This product was separated by preparative t . l . c . on s i l i c a gel and the re s u l t i n g material obtained c r y s t a l l i n e from ether. The u l t r a v i o l e t spectrum showed a peak at 284 m:y ind i c a t i v e of the pyridine chromophore whilst the mass spectrum exhibited a peak at m/e = 439 corresponding to the parent ion for compound 104. That the material obtained was a mixture of two compounds was indicated by the n.m.r. spectrum which exhibited two sharp singlets at 6.21 and 6.24 due to the presence of two methoxyl groups. Two doublets were also seen at 8.84 and 8.81 which were probably, due to the C-21 methyl group i n each 31 of the two compounds. A separation of these two compounds was not done at this time due to the r e l a t i v e l y small quantities available. Some preliminary investigations concerning the aromatisaticn of ring D were carried out on the mixtures of isomers obtained from the reaction of 38-aYetoxy-5a-et^^^^ with 2-ethylpyridine and 2-ethyl-3-methoxy-5-methylpyridine. Treatment of compound 103 with 10% Pd/C at 200° for 10 minutes gave a new compound which was separated by preparative t . l . c . The mass spectrum of t h i s compound ( f i g . 17) exhibited a peak at m/e 375 corresponding to the molecular ion for the ring D aromatised compound. The n.m.r. spectrum supported the conclusion that t h i s aromatisation had taken place as a new three proton singlet appeared at 7.85 and was assigned to the C-18 methyl group. A three proton singlet at 9.07 i n the product was due to the C-19 methyl group which appeared at 9.26 i n compound 100. This downfield s h i f t would be expected upon aromatisation of the D ring . Treatment of the mixture of isomers of structure 104 with Pd/C under the same conditions yielded a new compound which was separated from the product mixture by preparative t . l . c . The mass spectrum exhibited a peak at m/e 419 corresponding to the expected molecular ion for the ring D aromatised compound. The C-19 methyl resonance i n the n.m.r. spectrum of th i s compound now appeared at 9.09 compared with 9.22 i n compound 101 whilst a s i x proton singlet appeared at 7.75 corresponding to the C-18 and C-26 methyl groups. A shortage of the desired 2-ethyl-3-methoxy-5-methylpyridine at that time prevented any further investigation and c l a r i f i c a t i o n of the isomers encountered i n t h i s sequence. However t h i s work has since been further extended by Dr. G. V. Nair who has confirmed the existence 32 of the compounds as indicated above. The naturally occurring jerveratrum a l k a l o i d verarine has recently been confirmed to possess the 2-ethyl-5-methylpiperidine 20 molecule as the hetereocyclic portion . Since the pyridine precursor of t h i s compound should be readily available v i a the methylation of 2,5-lutidine our efforts were turned towards the synthesis of verarine whilst a larger quantity of the appropriate pyridine for the synthesis of veratramine was being prepared. The methylation of 2,5-lutidine was achieved by the addition of th i s compound to an ether solution of phenyl lithium followed by the addition of methyl iodide. The ether extract obtained after d i l u t i o n of the reaction mixture with water was reduced to a small volume i n vacuo and then the desired 2-ethyl-5-methyl pyridine was separated from the 2,5-lutidine by use of a spinning band d i s t i l l a t i o n column. The n.m.r. spectrum of the fra c t i o n d i s t i l l i n g at 62°/10 mm showed the replacement of^methyl sing l e t present i n the st a r t i n g material by a quartet at ^7.20 and a t r i p l e t at 8.80. The picrate derivative of this f r a c t i o n , with a melting point of 144°, was i n good agreement with that reported 53 i n the l i t e r a t u r e The reaction sequence for the synthesis of verarine i s shown in f i g . 12 with the i n i t i a l steps following the proposal of f i g . 9 whilst the introduction of the 5,6-double bond represents an application of 44 procedures employed by W. S. Johnson in his synthesis of veratramine Refluxing 2-ethyl-5-methyl pyridine with methyl lithium i n anhydrous tetrahydrofuran led to the rapid development of a deep red colouration i n the solution, 3 B-Acetoxy-5 a-etiojerv-12-en- 117-one (76) 33 Figure 12 v;as added u n t i l the colour faded and then the reaction was diluted with water. Examination of the ether extract by t . l . c . showed that two new compounds with s i m i l a r values had been formed but the separation of these two compounds was very d i f f i c u l t . In an attempt to simplify t h i s separation the compounds were converted to t h e i r 3-0 acetates by standing overnight i n the presence of pyridine-acetic anhydride (1:1).. The ether extract obtained after working up th i s mixture, deposited needle-like c r y s t a l s , m.pt. 191-192°C, when the solution was allowed to evaporate slowly at room temperature. Examination of these crystals by t . l . c , indicated that only one compound was present and th i s was designated compound "A". a The u l t r a v i o l e t spectrum of compound "A" showed peak at 270 m u with shoulders at 265 and 276 indicative of the pyridine chromophore, whilst the mass spectrum exhibited a peak at m/e 451 corresponding to the molecular ion for the desired compound 106. The n.m.r. spectrum i s reproduced i n f i g . 13 and i s i n accord with the proposed structure for compound A. A quartet occurring a t f 6 . 7 1 was assigned to the proton at C-20 and a decoupling experiment showed th i s signal to be coupled to a doublet at 8.79. This doublet was assigned to the C-21 methyl since the integral indicated three protons, whilst the C-19 methyl signal appeared as a sharp three-proton singlet at 7*9.21. The pyridine protons were readily discernible in the aromatic region of the spectrum with a one- proton doublet at i 2.82, J = 8 cps, corresponding to the C-23 H. The C-24 proton gave r i s e to two doublets at T 2.52 (J = 8 cps and J = 2 cps) since i t i s ortho coupled to C-23 H and meta to the C-25 H. The l a t t e r proton appeared as a broad singlet at T 1.67. A broad unresolved one proton signal at 3.98 which disappears upon addition of deuterium oxide 35 RELATIVE INTENSITY RELATIVE INTENSITY 92 Figure 15 -0 Figure 16 CO 39 was assigned to the hydroxyl proton at C-17. Elemental analysis confirmed the molecular formula of compound "A" as corresponding to the desired compound 106. Column chromatography of the mother, liquor from the c r y s t a l l i s a t i o n of compound "A" gave a second compound which had a larger value than compound "A" but the same colour reaction when sprayed with antimony pentachloride reagent. This compound gave rhomboid crystals m.pt. 189-190° from a small volume of ether and was designated compound "B". The u l t r a v i o l e t spectrum showed a maximum at 270 m u with shoulders at 265 and 276 m y ind i c a t i v e of the pyridine chromophore whilst the mass spectrum exhibited a peak at m/e 451 corresponding to the parent ion for structure 106. The overall mass spectrum of t h i s l a t t e r substance was v i r t u a l l y superimposable on that of compound "A" ( f i g . 14). Elemental analysis confirmed the isomeric nature of compounds "A" and "B". The n.m.r. spectrum of compound "B" i s reproduced i n f i g . 15 and shows the same signals as are present i n compound "AV with some differences i n chemical s h i f t . The largest differences are seen i n the positions of the C-18 and C-21 methyl groups. A three-proton singlet for the C-18 methyl appears at T 8.28 representing a s h i f t of 0.12 to lower f i e l d whilst the three- proton doublet corresponding to the C-21 methyl appears at 8.88 and corresponds to an up f i e l d s h i f t of 0.09 when compared with isomer "A". The hydroxyl proton now appears as a one-proton signal at T4.20 and i s not quite as broad as the corresponding signal in isomer "A". The pyridine protons occur at higher f i e l d with the one proton doublet due to the C-23 H at 2.92 whilst C-24 H gives r i s e to two doublets at T2.70. The broad signal corresponding to the C-27 proton appears at T 1.75. It can be seen from f i g . 12 that the coupling reaction creates two asymmetric centres and 40 and consequently there e x i s t s the p o s s i b i l i t y o f o b t a i n i n g four compounds possessing s t r u c t u r e 106. In an attempt to determine whether the other two compounds are formed i n t h i s r e a c t i o n , two o f the f r a c t i o n s , from the column chromatographic s e p a r a t i o n o f compound "B", were f u r t h e r i n v e s t i g a t e d . T . l . c . examination o f these consecutive f r a c t i o n s showed two compounds, which gave a s i m i l a r c o l o u r r e a c t i o n when sprayed with SbClj. as do compounds "A" and "B". However the values were not i d e n t i c a l w i t h the two compounds already c h a r a c t e r i s e d and these two a d d i t i o n a l compounds were designated "C" and "D". P r e p a r a t i v e t . l . c . y i e l d e d these compounds i n a pure s t a t e although n e i t h e r could be induced to c r y s t a l l i s e . Compound "C" was present i n the product mixture to the extent of 4% w h i l s t compound D only c o n s t i t u t e s about 1% o f the t o t a l product. The u l t r a v i o l e t spectrum of compound C showed a maximum at 270 mj with shoulders at 265 and 276/^ i n d i c a t i n g the p y r i d i n e chromophore w h i l s t the mass spectrum ( f i g . 14) e x h i b i t e d a peak at m/e 451 corresponding to the molecular i o n f o r s t r u c t u r e 106. Elemental a n a l y s i s confirmed that compound "C" was isomeric with "A" and "B" and the n.m.r. spectrum which i s reproduced i n f i g . 16 i s e n t i r e l y c o n s i s t e n t with t h i s c o n c l u s i o n . A three-proton singlet, at T 9.26 was assigned to the C-19 methyl w h i l s t the C-21 methyl appears as a three-proton doublet at 8.77. The quartet corresponding to the C-20 proton i s centred at T 6.90 and the C-18 methyl peak appears as a broad three-proton s i n g l e t at 8,43. The p y r i d i n e protons give r i s e to the usual p a t t e r n o f a doublet f o r the C-23 proton, two doublets f o r the C-24 proton and a broad s i n g l e t corresponding to the C-27 H i n the aromatic region o f the spectrum. A broad unresolved m u l t i p l e t at about T 5.8 which disappears on the a d d i t i o n of deuterium 41 oxide was assigned to the h y d r o x y l i c proton. The u l t r a v i o l e t spectrum o f compound "D" a l s o e x h i b i t e d the c h a r a c t e r i s t i c maximum at 270 m y with shoulders at 265 and 276 which was a s s o c i a t e d w i t h the presence o f the p y r i d i n e chromophore. A peak at m/e 451 corresponding to the parent i o n f o r compounds o f s t r u c t u r e 106 was again evident i n the mass spectrum. The n.m.r. o f compound "D" i n d i c a t e d that i t possessed the same f u n c t i o n a l i t i e s that are present i n the three compounds already i s o l a t e d from the r e a c t i o n . Elemental a n a l y s i s was not done on compound D due to the r e l a t i v e l y small amount a v a i l a b l e but the molecular formula was confirmed by high r e s o l u t i o n mass spectrometry. The f u r t h e r extension o f t h i s sequence to the r i n g D aromatised s e r i e s only i n v o l v e d the compounds "A" and "B" as the small amounts of II I! II 1 C and D which were a v a i l a b l e precluded any u s e f u l r e a c t i o n s . Compound "B" was ground w i t h 10% p a l l a d i s e d charcoal u n t i l the two substances were thoroughly mixed and then the powder was heated at 200°C f o r 10 minutes. The r e s u l t a n t s o l i d r esidue was washed s e v e r a l times w i t h chloroform to remove the organic m a t e r i a l . Examination o f the f i l t e r e d chloroform s o l u t i o n by t . l . c . i n d i c a t e d compound B had l a r g e l y been converted to a new compound possessing a s i m i l a r R^ value. Separation o f the r e a c t i o n mixture by p r e p a r a t i v e t . l . c . i n d i c a t e d t h i s new compound, which was designated "aromatic I I " , had been produced i n 80% y i e l d . The u l t r a v i o l e t spectrum showed no q u a l i t a t i v e d i f f e r e n c e from that o f compound B but the e x t i n c t i o n c o e f f i c i e n t at 270 m y increased from 4,130 i n the l a t t e r t o 5,400 i n t h i s compound. The mass spectrum e x h i b i t e d a peak at m/e 431 corresponding to the molecular i o n f o r a compound of s t r u c t u r e 107. The o v e r a l l mass spectrum ( f i g . 17) 42 shows a completely d i f f e r e n t p a t t e r n to that observed w i t h the s e r i e s o f compounds A to D. The n.m.r. spectrum reproduced i n f i g . 18 was p a r t i c u l a r l y u s e f u l i n confirming that the d e s i r e d r i n g D aromatisation had occurred. The most s i g n i f i c a n t feature i s the appearance o f a second p a i r o f doublets i n the downfield region o f the spectrum corresponding to the expected AB system f o r the protons at C-15 and C-16. The one-proton quartet assigned t o the C-20 H i n compound B now appears at i 5.58 si n c e i t i s deshielded by the aromatic D r i n g and the p y r i d i n e ; w h i l s t the three-proton doublet due to the C-21 methyl has a l s o s u f f e r e d a downfield s h i f t and now appears at T 8.40. The broad three-proton s i n g l e t which corresponds to the C-18 methyl i n the s t a r t i n g m a t e r i a l has disappeared but a new sharp s i n g l e t appears at T 7.89 i n the product i n accord with the a r o m a t i s a t i o n o f r i n g D. One of the important features o f the n.m.r. spectrum i s that i n the aromatic region (2.75 - 3.1 T) a l l 8 l i n e s expected f o r the two AB systems present i n s t r u c t u r e 107 are c l e a r l y r e s o l v e d . The molecular formula was confirmed by high r e s o l u t i o n mass spectrometry ( c a l c : 431.2824 found 431.2809). Isomer A was s i m i l a r l y t r e a t e d with 10% Pd/C at 200°C f o r seven minutes and the residue washed with chloroform. Examination o f t h i s chloroform e x t r a c t by t . l . c . i n d i c a t e d that three major compounds had r e s u l t e d from the r e a c t i o n . A l l three compounds were obtained i n a pure s t a t e by p r e p a r a t i v e t . l . c . and two of these were i d e n t i f i e d as the a-B unsaturated ketone (76) and 2-ethyl-5-methypyridine. The t h i r d compound which was formed i n 25% y i e l d showed a colour r e a c t i o n w i t h SbCl^ s i m i l a r to that, obtained with the aromatic compound from isomer B. This t h i r d component was designated "aromatic I " and the mass spectrum e x h i b i t e d a peak at m/e 431 corresponding to the molecular ion f o r a compound of RELATIVE INTENSITY RELATIVE INTENSITY RELATIVE INTENSITY r r ~1 _ C 46 s t r u c t u r e 107. The o v e r a l l mass spectrum was very s i m i l a r to that of aromatic compound 'II ( f i g . 17) w h i l s t the n.m.r. spectrum shown i n f i g . 19 l e n t strong support to the presence o f an aromatic r i n g D i n the compound. The one-proton quartet due to the C-20 proton appears at T 5.61 with the three-proton doublet assigned to the C-21 methyl o c c u r r i n g at 8.41, The three-proton s i n g l e t at 8.40 assigned to t h t C-18 methyl i n compound A has now disappeared and a new s i n g l e t i n t e g r a t i n g f o r three protons appears at 7.90 i n accord w i t h the arom a t i s a t i o n o f r i n g D. The i n t e r e s t i n g d i f f e r e n c e if n -I i n the nature o f the n.m.r. s p e c t r a o f the aromatic compounds I and I I was noted i n the aromatic region (2.75 - 3.1 ). In I only seven o f the t h e o r e t i c a l e i g h t l i n e s were observed f o r the overlapping AB systems w h i l s t as mentioned above a l l eight l i n e s were c l e a r l y present i n compound I I . The molecular formula o f aromatic compound I was confirmed by high r e s o l u t i o n mass spectrometry as being the same as that o f aromatic I I . These two compounds must be isomers s i n c e the n.m.r. s p e c t r a i n d i c a t e they are not i d e n t i c a l . These compounds contain only one a d d i t i o n a l asymmetric centre (C-20) when compared w i t h 3B-acetoxy-5a-etiojerv-12-en-17rone. Since t h i s l a t t e r substance was obtained from hecogenin i t i s not racemic, and i n f a c t has a r o t a t i o n o f + 122°. The i n t r o d u c t i o n o f the asymmetric centre at C-20 would t h e r e f o r e be expectedto give r i s e to two diastereomers. This may w e l l represent the nature o f the d i f f e r e n c e between the two aromatic compounds obtained i n t h i s work. In view of the d i f f e r e n t extent o f r i n g D aro m a t i s a t i o n encountered w i t h isomers "A" and "B", the r e l a t i v e s t a b i l i t y o f these two compounds was f u r t h e r i n v e s t i g a t e d . The C-17, C-20 bond cleavage, which occurs w i t h isomer A under the c o n d i t i o n s f o r the D r i n g a r o m a t i s a t i o n , appears t o be very f a c i l e s i n c e t h i s cleavage a l s o occurs s l o w l y i n methanol or ethanol. The cleavage i s s l i g h t l y enhanced when the compound i s d i s s o l v e d i n 0.1 N methanolic potassium hydroxide. Isomer A i s s t a b l e i n dimethylformaraide or benzene but i s converted to 3B-acetoxy-5ct-etiojerv- 12-en-17-one and 2-ethyl-5-methylpyridine when d i s s o l v e d i n aqueous dimethylformamide c o n t a i n i n g potassium hydroxide (pH = 9). Isomer B i s s t a b l e under the above c o n d i t i o n s and shows no C-17, C-20 bond cleavage. Dehydration of the t e r t i a r y a l c o h o l appears to be the main process f o r both isomers A and B i n a c i d i c methanol s o l u t i o n . Thus two compounds o f s t r u c t u r e 107 had been prepared and the s e l e c t i v e r e d u c t i o n of the p y r i d i n e to a p i p e r i d i n e r i n g was i n v e s t i g a t e d . One of these aromatic compounds should lead to an isomeric mixture of 5ct,6-dihydroverarines w h i l s t the other would lead to a mixture of isomers o f s i m i l a r s t r u c t u r e but d i f f e r i n g i n c o n f i g u r a t i o n at C-20 from the n a t u r a l l y o c c u r r i n g a l k a l o i d v e r a r i n e . The s e l e c t i v e hydrogenation of 54 a p y r i d i n e r i n g i n the presence of a benzene r i n g i s reported t o occur smoothly when PtO^ i s used as the c a t a l y s t i n a c e t i c a c i d at room temperature and a hydrogen atmosphere at 45 p s i i s employed. These co n d i t i o n s were used i n our work i n the conversion o f the aromatic compounds, to the p i p e r i d i n e c o n t a i n i n g compounds of s t r u c t u r e 108. Aromatic compound I I was hydrogenated under these c o n d i t i o n s f o r three hours and the f i l t r a t e was reduced to a small volume by evaporation i n vacuo. The r e s u l t i n g mixture was d i l u t e d w i t h water, b a s i f i e d w i t h aqueous ammonia and then e x t r a c t e d w i t h chloroform. Examination of the product by t . l . c . i n d i c a t e d that four new compounds possessing very s i m i l a r R^ . values had been formed. These compounds which were numbered 1 to 4 i n order o f decreasing R,- values on s i l i c a g e l , were 4 8 separated by careful preparative t . l . c . These compounds were suspected to be isomers of 3-0-acetyl-5a,6-dihydroverarine. However preparation of a sample of th i s compound from verarine for comparison purposes i s d i f f i c u l t since acetylation of verarine yields N-acetylverarine or 3-0,N-diacetylverarine. None of the four compounds from the hydrogenation of aromatic compound II could be induced to c r y s t a l l i s e but i t was found from other investigations that N-acetyl-5a,6-dihydroverarine (110) c r y s t a l l i s e s readily from an ethereal solution. Consequently i t was decided to convert the four compounds obtained above to the N-acetyl derivatives (110), v i a acetylation to the 3-0,N-diacetates (109) followed by selective hydrolysis of the 3-acetoxyl group. The conversion to the respective diacetat.es was accomplished upon standing overnight with acetic anhydride:pyridine (1:1) at room temperature, whilst hydrolysis of the 3-acetoxyl group was achieved with 0.1 M potassium hydroxide in methanol. The other alternative open to us at th i s point, was the removal of the 3-acetate by saponification to give the 5a,6-dihydroverarine. This proposal was discarded i n favour of the approach outlined above for two reasons. F i r s t l y the amount of verarine available was r e l a t i v e l y 20 small and secondly Masamune has shown that veratramine, which i s readily available, can be converted to N-acetylverarine. Since the quantities of the isomeric compounds obtained from the hydrogenation were expected to be i n s u f f i c i e n t for the introduction of the 5,6-double bond which i s necessary to complete the synthesis of verarine, i t was desirable to compare the products with a cdmpound which was readily available. Once the comparison had been made then t h i s compound could be used for the further reactions involved in the synthesis of verarine. 49 The conversion of veratramine to N - a c e t y l v e r a r i n e as published 20 by Masavune i s o u t l i n e d i n f i g . 20. The procedure gave a reasonable y i e l d o f 3-0,N-diacetyl v e r a r i n e (117) although some d i f f i c u l t l y was experienced w i t h the t h i o k e t a l formation. The t h i o k e t a l d e r i v a t i v e (116) was obtained by d i s s o l v i n g the ketone (19) i n g l a c i a l a c e t i c a c i d and t r e a t i n g t h i s s o l u t i o n w i t h e t h a n e d i t h i o l and boron t r i f l u o r i d e etherate. This r e a c t i o n was not accompanied by the loss o f the 3-acetoxyl group as was the case when the compound was d i s s o l v e d i n methanol and then t r e a t e d w i t h e t h a n e d i t h i o l and hydrogen c h l o r i d e . Consequently the procedure f o r the conversion as repeated here l e d to 3-0,N-diacetylverarine r a t h e r than N - a c e t y l v e r a r i n e p r e v i o u s l y obtained by Masamune. The d i a c e t a t e was i d e n t i f i e d by t . l . c . comparison w i t h an a u t h e n t i c sample, melt i n g p o i n t , r o t a t i o n , mass spectrum and n.m.r., a l l 27 o f which were i n accord w i t h the p u b l i s h e d data . Subsequent h y d r o l y s i s of the 3-acetoxyl gave N - a c e t y l v e r a r i n e w i t h the same melti n g point as recorded i n the l i t e r a t u r e . Hydrogenation of the 5-6 double bond i n veratramine employing Adams c a t a l y s t i n a c e t i c a c i d had been reported to give mainly the 5a,6- d i h y d r o v e r a t r a m i n e ^ . By analogy i t was expected that the hydrogenation of the 5,6-double bond i n 3-0,N-diacetylverarine (117) using these c o n d i t i o n s would provide mainly the d e s i r e d 3-0,N-diacetyl-5a,6- d i h y d r o v e r a r i n e . This hydrogenation was c a r r i e d out and the compound was observed to consume approximately one mole of hydrogen a f t e r 4 hours. Examination of the product by t . l . c . employing both s i l i c a g e l and alumina under s e v e r a l solvent systems i n d i c a t e d that only one compound had been; formed. The n.m.r. spectrum i n d i c a t e d the unresolved one-proton m u l t i p l e t at x 4.5 present i n 3-0,N-diacetylverarine which had been assigned to the v i n y l i c proton, was no longer present. Thus the hydrogenation of the 50 Figure 20 51 double bond, had indeed occurred. However, a c l o s e r examination o f the spectrum p a r t i c u l a r l y i n the r e g i o n . T 8.5 - 9.5 i n d i c a t e d that both the 5a and the 58 compounds had been formed. The n.m.r. spectrum o f 3-0,N-diacetylverarine 117 e x h i b i t e d a three-proton doublet at T 9.02 which was a t t r i b u t e d to the C-26 methyl w h i l s t the C-19 methyl appeared as a three-proton s i n g l e t at 8.85. The n.m.r. spectrum o f the hydrogenation product e x h i b i t e d a sharp s i n g l e t at 8.92 and a doublet at 9.03. The u p f i e l d h a l f o f the doublet centred at 9.03 was however very intense and the i n t e g r a l o f t h i s doublet corresponded to 4.5 protons. From the recent e x c e l l e n t n.m.r. study o f 22,27-imino-17,23-oxidojervane d e r i v a t i v e s by Masamune^ one would expect the C-19 methyl s i g n a l f o r the 5a compound t o be at .a higher f i e l d p o s i t i o n than i n the 58 compound. This d i f f e r e n c e i s due to the s h i e l d i n g o f the C-19 methyl group by the r i n g A protons which are i n c l o s e p r o x i m i t y i n the 5a d e r i v a t i v e s but removed to a large extent i n the 58 41 d e r i v a t i v e s . This e f f e c t has been noted i n various s t e r o i d s where the stereochemistry o f the molecule i s f i x e d . The d i f f e r e n c e i n chemical s h i f t between the s i n g l e t at 8.92 and the u p f i e l d h a l f o f the doublet due to the C-26 methyl group was 13 cps and i t was i n f o r m a t i v e to compare t h i s d i f f e r e n c e w i t h the C-19 methyl resonances i n compounds 118 and 119. 52 The n.m.r. spectra of these two compounds have been studied by Masamune who found the C-19 methyl resonance i n compound 118 to occur at x 9.19 whilst in compound 119 the three-proton singlet i s observed at T 9.07 - a separation of 12 cps. It therefore appeared that the hydrogenation of 3-0, N-diacetyl verarine had given both the 5a and 58 compounds. In an attempt to obtain some separation of these two compounds and v e r i f y t h e i r existence the product was converted to the monoacetate by hydrolysis of the 3-acetoxyl group by means of 0.1M potassium hydroxide in methanol. A t . l . c . examination of the res u l t i n g N-acetyl-5,6-dihydroverarines showed c l e a r l y that ttoo compounds (120, 121) were present. The n.m.r. spectra of the two compounds after separation by preparative t . l . c . on s i l i c a g e l , supported the analysis of the C-19 methyl group positions outlined above, (see f i g s . 21, 22) Additional support for the 58 configuration corresponding to the compound which showed the C-19 methyl resonance at T 8.92 was gained from the presence of a r e l a t i v e l y narrow multiplet at x 5.97 assigned to the proton geminal to the C-3 hydroxyl supporting the equatorial position of t h i s proton which should result i n the conversion of ^  ^ to the 58,6 dihydro compound. The compound i n which the C-19 methyl resonance overlies the upfie l d h a l f of the C-26 methyl doublet exhibited a broad multiplet in the region T 6.3 - 6.5 which was assigned to t h i s proton and indicated i t s a x i a l position supporting the 5a configuration. It was v i t a l to establish firmly the identity of these two compounds whose molecular formulae had been v e r i f i e d by t h e i r mass spectra and elemental analysis. The O.R.D. curves of 3-keto steroids with the 5a 53 and 56 c o n f i g u r a t i o n has been e x t e n s i v e l y s t u d i e d and Masamune^ has recorded the O.R.D. curves o f compounds 122 and 123. These l a t t e r substances provide a very good analogy with the compounds under c o n s i d e r a t i o n . 122 123 Compound 120 e x h i b i t s a strong +ve Cotton e f f e c t curve with values o f [4>] + 2 7 4 0 ° and [6] SUgh - 1180° w h i l s t compound 121 e x h i b i t s L T J 304 m u L T J 265 my j a negative Cotton e f f e c t curve with values of [<i] ™ ? U g ^ + 265° and b L T J 304 m u ty] £ * k • 2340°. L^ J 265 m y The hydrogenation products which had t e n t a t i v e l y been assigned the 5a (120) and 56 (121) c o n f i g u r a t i o n s were converted t o the re s p e c t i v e 3-keto compounds (111 and 124) using Jones reagent as the o x i d i s i n g agent. The presence of the N-acetyl group tended to complicate the O.R.D. curves to some extent since i t had a strong absorption at 230 m y but the absorption at 300 m y was not s u f f i c i e n t to a f f e c t the curve i n t h i s r e g i o n . Compound 111 which had 56 been assigned the 5ot c o n f i g u r a t i o n on the b a s i s o f the n.m.r. a n a l y s i s o f i t s parent a l c o h o l , showed a stror.g p o s i t i v e Cotton e f f e c t curve w i t h a peak at 305 m u. The p o s i t i o n tnd i n t e n s i t y o f the trough at 270 rw was probably a f f e c t e d by the i n c r e a s i n g absorption due to the N- a c e t y l group. Compound 124 on the other hand showed only a very weak trough at 305 m y and the p o s i t i o n of the peak at 275 m u was probably i n f l u e n c e d by the N-acetyl group. In view o f these O.R.D. curves, however one can f i r m l y assign the 5a and 53 c o n f i g u r a t i o n s to the hydrogenation products as already noted above. appeared i n which the hydrogenation of veratramine employing Adams C a t a l y s t i n a c e t i c a c i d has been more c l o s e l y examined. S a i t o ^ had reported only the formation o f the 5a,6-dihydroveratramine but the recent work i n d i c a t e s t h a t a mixture o f 5a,6-dihydroveratramine (41%) and 58,6- dihydroveratramine (44%) i s produced. I t i s i n t e r e s t i n g to note that these workers d i s t i n g u i s h e d between the two compounds on the b a s i s o f the chemical s h i f t s o f the C-19 methyl resonances i n the n.m.r. spectra. d i h y d r o v e r a r i n e (120) the comparison o f t h i s compound with the N-acetyl d e r i v a t i v e s (110) of the four compounds obtained from the hydrogenation of aromatic compound I I was now undertaken. Only hydrogenation products I and 2 had a s i m i l a r value to N-acetyl-5a,6-dihydroverarine when compared by t . l . c . on s i l i c a g e l . Since t h i s work has been completed, a p u b l i c a t i o n has Having e s t a b l i s h e d the i d e n t i t y o f the d e s i r e d N-acetyl-5a,6- The m e l t i n g p o i n t s o f the four compounds were as f o l l o w s , No m.pt. 1 247 - 2 263 - 264 o 3 270 - 274 o 57 No 4 m.pt. 260 - 261 whereas the N-acetyl-5a,6-dihydroverarine prepared from veratramine had a m e l t i n g p o i n t o f 248-249°. A mixture o f compound 1 with the authe n t i c sample was observed to melt at 248-249° w h i l s t a mixture o f compound No. 2 and the au t h e n t i c compound had a melti n g p o i n t o f 230-240°. The i n f r a r e d s p e c t r a o f a l l f i v e compounds were determined i n chloroform and only t h a t o f compound No 1 and the au t h e n t i c sample were completely superimposable. The mass s p e c t r a o f a l l f i v e compounds are e s s e n t i a l l y the same e x h i b i t i n g a peak at m/e 437 corresponding to the molecular ion f o r N-acetyl-5a,6-dihydroverarine. The mass s p e c t r a o f N-acetyl-5a,6- d i h y d r o v e r a r i n e , compound "No 1" and compound "No 2" are reproduced i n f i g . 23 and a l l show strong peaks at m/e 140 and 98. ''Budzkiewicz^ has examined the mass s p e c t r a o f veratramine, N-acetylveratramine and v e r a r i n e . Cleavage o f the b e n z y l i c C-20, C-22 bond i s p o s t u l a t e d to give r i s e to the strong peaks at m/e 114 and 98 i n veratramine and v e r a r i n e r e s p e c t i v e l y . This cleavage gives r i s e t o a strong peak at m/e 156 i n N-acetylveratramine w h i l s t a s l i g h t l y s m a l l e r peak at m/e 114 i s 0 a t t r i b u t e d to the m/e 156 fragment lessthe a c e t y l group. H + C H 3 C~- O 125 126 Therefore the peak at m/e 98 i n our mass s p e c t r a was assigned to s t r u c t u r e 125 w h i l s t the strong peak at m/e 140 was assigned to the N-acetyl d e r i v a t i v e (126). RELATIVE INTENSITY RELATIVE INTENSITY RELATIVE INTENSITY 8S 60 The n.m.r spectrum o f compound "No 1" was v i r t u a l l y i d e n t i c a l w i t h that o f the au t h e n t i c compound (see F i g . 21) although the r e s o l u t i o n was not good. ( F i g . 24) The n.m.r. spectrum o f compound "No 2" e x h i b i t e d a doublet at T 9.14 due to the C-26 or C-21 methyl group w h i l s t a sharp s i n g l e t assigned to the C-19 methyl group appeared at T 9.07. These f a c t o r s together c o n s t i t u t e a f i r m b a s i s f o r concluding that the N-acetyl d e r i v a t i v e o f the compound designated "No 1" obtained from the hydrogenation o f aromatic compound " I I " i s i d e n t i c a l with N- ac e t y l - 5 a , 6 - d i h y d r o v e r a r i n e . Aromatic compound I which was obtained i n 25% y i e l d from isomer A was subjected to hydrogenation under the same c o n d i t i o n s as ap p l i e d to aromatic compound I I . Examination o f the product by t . l . c . i n d i c a t e d t h a t four new compounds o f s i m i l a r values had been formed. For the sake of convenience these were numbered from 5 to 8 i n order o f decreasing R^ values on a t h i n - l a y e r chromatoplate. These compounds were separated by c a r e f u l p r e p a r a t i v e t . l . c . and then converted v i a the d i a c e t a t e s to the r e s p e c t i v e N-acetyl d e r i v a t i v e s . A t . l . c . comparison of the four N-acetates w i t h N-acetyl-5a,6-dihydroverarine i n d i c a t e d that only compounds No's. 5 and 6 had a s i m i l a r R^ . value, w h i l s t a l l showed the same co l o u r r e a c t i o n when sprayed with SbCl^. S u r p r i s i n g l y none of the N-acetyl d e r i v a t i v e s could be induced to c r y s t a l l i s e from ether i n co n t r a s t to the N-acetyl-5a,6-dihydroverarine and the N-acetates of compounds 1 - 4 which a l l c r y s t a l l i s e very r e a d i l y from t h i s s o l v e n t . The mass s p e c t r a o f the compounds 5 - 8 e x h i b i t e d a peak at m/e 437 corresponding to the molecular i o n f o r N-acetyl-5a,6-dihydroverarine as w e l l as prominent peaks at m/e 98 and 140. 61 Since compound No. 1 obtained i n these hydrogenation experiments has been shown to be i d e n t i c a l with N-acetyl-5a,6-dihydroverarine, the remaining steps i n the synthesis of verarine were carried out using a quantity of t h i s compound which had been prepared from veratramine as already discussed. To complete the synthesis i t was necessary to introduce the 5,6-double bond and remove the N-acetate function from N-acetyl-5a,6-dihydroverarine. The steps involved in achieving these goals are outlined i n f i g . 12 and are analogous to the reactions carried 44 out by W. S. Johnson i n the conversion of 23-0,N-dibenzoyl-Sa,6- dihydroveratramine to veratramine. 4 The formation of A -3-ketones from s t e r o i d a l 3-ketones has been extensively studied by various groups of workers and a thorough investigation of the application of t h i s reaction in the synthesis of 57 cortisone has been published by Evans et a l . The steps involved i n the introduction of the 4,5-double bond i n 4,5 -dihydro cortisone acetate 127 are outlined i n f i g . 25. Treatment of 127 with 2 moles of bromine in acetic acid led to the 2,2-dibromo compound (120) which readily rearranged to the 2,4-dibromo compound (129) i n the presence of hydrobromic acid. When the 2,4-dibromo compound i s refluxed with sodium iodide i n acetone the bromine atom at C-2 tends to undergo halogen exchange rather than dehydrobromination whilst the C-4 bromine shows no replacement by iodine but undergoes dehydrobromination, i f the refluxing i s prolonged, giving r i s e to the A^-2-iodo compound (131). Treatment of this compound with zinc i n acetic acid effects dehalogenation to give the desired cortisone acetate (132). Consequently N-acetyl-5a,6-dihydroverarine was oxidised with Jones reagent and a t . l . c . examination of the product was indicative of Figure 25 (a) an a l m o s t q u a n t i t a t i v e c o n v e r s i o n t o a new compound. The i n f r a r e d s p e c t r u m o f t h i s compound e x h i b i t e d a peak a t 5.89 u c o r r e s p o n d i n g t o a s a t u r a t e d k e t o n e . The O.R.D. c u r v e e x h i b i t e d as e x p e c t e d a s t r o n g + C o t t o n e f f e c t w h i l s t t h e n.m.r. s p e c t r u m f i g . 26 showed a t h r e e - p r o t o n s i n g l e t a t T 8.88 r e a d i l y a s s i g n e d t o t h e C-19 m e t h y l group. T h i s v a l u e r e p r e s e n t e d a s h i f t o f 0.18 T when compared w i t h t h e p a r e n t compound and was i n f a i r agreement w i t h t h e v a l u e o f 0.21 e x p e c t e d from t h e e x c e l l e n t 40 n.m.r. s t u d y o f Masamune me n t i o n e d p r e v i o u s l y . The 3 - k e t o - N - a c e t y l - 5 a , 6 - d i h y d r o v e r a r i n e (111) o b t a i n e d from t h e J o n e s o x i d a t i o n was t h e n s u b j e c t e d t o t h e c o n d i t i o n s as o u t l i n e d by Evans f o r t h e i n t r o d u c t i o n o f t h e 4,5-double bond. The r e a c t i o n m i x t u r e a f t e r t h e t r e a t m e n t w i t h z i n c d u s t i n a c e t i c a c i d was seen t o c o n t a i n one m a j o r compound and s e v e r a l m i n o r compounds when examined by t . l . c . The i n f r a r e d s p e c t r u m , o f t h e m a j o r compound, w h i c h was o b t a i n e d i n a p u r e s t a t e by p r e p a r a t i v e t . l . c , e x h i b i t e d a band a t 6.02 u w h i l s t t h e c a r b o n y l band p r e s e n t a t 5.89 u i n t h e 3-keto compound (111) had d i s a p p e a r e d . T h i s band a t 6.02 i n d i c a t e d t h a t t h e a , B - u n s a t u r a t e d c a r b o n y l system was p r o b a b l y p r e s e n t i n t h e m o l e c u l e . T h i s compound, w h i c h was o b t a i n e d i n 50% y i e l d from t h e 3 - k e t o - N - a c e t y l - 5 c t , 6 - d i h y d r o - v e r a r i n e , e x h i b i t e d a t h r e e - p r o t o n s i n g l e t a t T 8.72, a s s i g n e d t o t h e C-19 m e t h y l g r o u p , i n i t s n.m.r. s p e c t r u m ( f i g . 2 7 ) . T h i s m e t h y l r e s o n a n c e showed a d o w n f i e l d s h i f t o f 0.34 when compared w i t h N - a c e t y l - 5 c t , 6 - d i h y d r c v a r a r i n e and t h i s v a l u e c o r r e s p o n d e d c l o s e l y t o t h e -0.38 c o n t r i b u t i o n d u s t o a A ^ - 3 - k e t o i n t h e i m i n o j e r v a n e s e r i e s . I n a d d i t i o n t h e n.m.r, s p e c t r u m showed a o n e - p r o t o n s i n g l e t a t 4.22 w h i c h was i n t h e r e g i o n e x p e c t e d i n N - a c e t y l - A ^ - 3 - k e t o - 5 , 6 - d i h y d r o v e r a r i n e ( 1 1 2 ) . 58 4 Dauben* has s t u d i e d t h e c o n v e r s i o n o f A - c h o l e s t e n o n e (133) 65 66 67 to c h o l e s t e r o l (135) v i a the enol acetate (13if) as o u t l i n e d i n £13. 25(a) This procedure was a p p l i e d to N-acetyl-A 4-3-keto-5,6-dihydroverarine (112) wit h the aim of o b t a i n i n g N - a c e t y l v e r a r i n e . Compound (112) was r e f l u x e d w i t h isopropenyl acetate c o n t a i n i n g 1% s u l p h u r i c a c i d f o r one hour a f t e r which time t . l . c . i n d i c a t e d conversion to a new compound. The i n f r a r e d spectrum o f the product showed that the carbonyl at 6.02u i n the the s t a r t i n g m a t e r i a l was no longer present but a new band appeared at 5.75y which was very c h a r a c t e r i s t i c o f an enol acetate. This product was not p u r i f i e d but subjected to the sodium borohydride,reduction i n methanol under c o n d i t i o n s o u t l i n e d by Dauben. Examination o f the product from the sodium borohydride r e a c t i o n by t . l . c i n d i c a t e d that the major product was a compound o f i d e n t i c a l value and having the same co l o u r r e a c t i o n to SbCl^ as N - a c e t y l v e r a r i n e . The r e a c t i o n mixture was separated by p r e p a r a t i v e t . l . c . and the i d e n t i t y o f the major component with N - a c e t y l v e r a r i n e (114) was v e r i f i e d by comparison o f the i n f r a r e d , n.m.r.(Fig. 28jand mass s p e c t r a . The f i n a l step i n the conversion o f N-acetyl-5a,6-dihydroverarine t o v e r a r i n e i n v o l v e d the removal of the N-acetate group. N-acetylveratramine was used as a model compound i n the search f o r optimum c o n d i t i o n s f o r the h y d r o l y s i s o f such an N-acetate by potassium hydroxide i n aqueous d i m e t h y l s u l f o x i d e which had been reported by Masamune^. Some h y d r o l y s i s was encountered during these experiments but the procedure was f i n a l l y abandoned i n favour o f r e f l u x i n g w i t h 10% potassium hydroxide i n ethylene g l y c o l f o r 16 hours. This procedure gave a reasonable y i e l d o f veratramine wit h N-acetyl veratramine being the other major component i n the product mixture. When these c o n d i t i o n s were a p p l i e d t o N- a c e t y l v e r a r i n e (114) a compound o f s i m i l a r R f value and colour r e a c t i o n to that o f v e r a r i n e 68 was observed upon t . l . c . examination of the product. P r e p a r a t i v e t . l . c . i n d i c a t e d t h i s compound had been formed i n 60% y i e l d and i t s i d e n t i t y w i t h v e r a r i n e was e s t a b l i s h e d by i t s i n f r a r e d and mass sp e c t r a . The i n f r a r e d spectrum was superimposable upon that o f an a u t h e n t i c sample of 59 v e r a r i n e obtained from P r o f e s s o r Tomko *, w h i l s t the mass spectrum showed the c h a r a c t e r i s t i c m/e 98 and a more intense m-1 than m+ peak i n accord w i t h the l i t e r a t u r e . Compound No. 1 from the hydrogenation o f aromatic compound I I had been shown to be i d e n t i c a l w i t h N-acetyl-5a,6-dihydroverarine i n t h i s l a s t step represented the completion of the t o t a l s y n t h e s i s .of the n a t u r a l l y o c c u r r i n g a l k a l o i d v e r a r i n e from hecogenin. The r s y n t h e t i c approach c a r r i e d out by other woi'kers i n t h i s l a b o r a t o r y had provided racemic 38-acetoxy-r5a,etiojerv-17-one (99) which was not r e s o l v e d due t o the q u a n t i t y a v a i l a b l e whereas here (+) 38-acetoxy-5a- etiojerv-17-one (87) from hecogenin was u t i l i s e d i n the s y n t h e s i s o f v e r a r i n e . Very r e c e n t l y an account o f a s y n t h e t i c approach to 5,6-dihydro- v e r a r i n e has appeared i n the l i t e r a t u r e ^ and r e p o r t s problems s i m i l a r t o those of our work i n the s e p a r a t i o n of isomers. In t h i s recent p u b l i c a t i o n f o u r compounds possessing the d i h y d r o v e r a r i n e s k e l e t o n were obtained but the authors were unable to e s t a b l i s h the i d e n t i t y o f any of the compounds wi t h 5a,6-dihydroverarine. The approach in v o l v e d methods s i m i l a r to those employed by S c h r e i b e r ^ i n the s y n t h e s i s of the Solanum a l k a l o i d s . 69 •EXPERIMENTAL •Melting points were determined on a Kofler block and are uncorrected. U l t r a v i o l e t (U.V.) spectra were measured i n 95% ethanol on a Cary 11 recording spectrophotometer. Infrared (I.R.) spectra were obtained on a Perkin Elmer model 21 or 457 spectrophotometer. Nuclear magnetic resonance (n.m.r.) spectra were determined at 100 megacycles per second on a Varian Associates HA 100 spectrometer using deuteriochloroform solutions with tetramethylsilane as internal standard. The chemical s h i f t s are recorded i n the Tiers T scale. The types of protons, integrated area, m u l t i p l i c i t y and spin coupling constant J (in cps) are indicated i n parentheses. Double irjradiationexperiments were performed with a Hewlett Packard 200 CD o s c i l l a t o r and c a l i b r a t i o n was checked with an electronic frequency counter. Optical rotatory dispersion (O.R.D.) curves were taken i n chloroform solution on a Jasco UV/ORD/CD-5 spectropolarimeter. Specific rotations ([a] D) were determined i n chloroform solution at 20° on an; O.C. Rudolph and Sons No. 219 polarimeter using a one decimeter c e l l . Mass spectra were recorded on an Atlas CH-4 or Associated E l e c t r i c a l Industries MS-9 spectrometer, high resolution measurements being determined on the l a t t e r instrument. Unless otherwise specified s i l i c a gel G containing 1% electronic phosphor was used i n preparing thin-layer chromatoplates and Woelm neutral alumina, or Shawinigan alumina deactivated by addition of 3% of 10% aqueous acetic acid solution, was used for column chromatography. Elemental analyses were performed by Mr. P. Borda of the Microanalytical Laboratory, University of B r i t i s h Columbia. 70 Synthesis o f 3g-Acetoxy-5a-etiojerv-12(15)-en-17-one (76) Throughout the e n t i r e p r o j e c t t h i s compound occupied a p o s i t i o n o f prime importance s i n c e i t provided the e t i o j e r v a n e p o r t i o n of most of the compounds syn t h e s i s e d . I t i s not a commercially a v a i l a b l e compound 6 2 but was prepared from hecogenin acetate employing degradation procedures 48 w e l l documented by W. F. Johns . D e t a i l s o f the q u a n t i t i e s used and the y i e l d s obtained i n t h i s work, f o r the various steps are given below. Wherever m o d i f i c a t i o n s i n the experimental procedure of a d d i t i o n a l data was obtained, f o r c h a r a c t e r i s a t i o n o f the intermediates i n the degradation, t h i s i s i n c l u d e d . Hecogenin p-tpluenesulfonylhydrazone (82) A s o l u t i o n o f hecogenin acetate (400 g) i n g l a c i a l a c e t i c a c i d y i e l d e d hecogenin p-toluenesulfonylhydrazone (560 g) when t r e a t e d w i t h p- t o l u e n e s u l f o n y l h y d r a z i n e h y d r o c h l o r i d e as o u t l i n e d by Hirschmann et a l The product was not f u r t h e r p u r i f i e d but subjected to the next r e a c t i o n . 13-Methyl-C-nor-D-homo-18-nor-5a,22a-spirost-12(13)-en-58-ol. (83) A s o l u t i o n o f hecogenin p-toluenesulfonylhydrazone (560 g) i n potassium hydroxide/ethylene g l y c o l was heated under n i t r o g e n as o u t l i n e d by W. F. Johns. The product was obtained c r y s t a l l i n e from ethanol/water as c o l o u r l e s s needles. M.pt. 108-111° ( l i t . m.pt. 108-118°). N.m.r. s i g n a l s : 9.28 ( s i n g l e t , 3H, C-19 CH ), 9.24 (doublet, J2S-26 = 6' 3H»' C-26 CH ), 8.91 (doublet, J 2 Q = 6, 3H, C-21 CH ) , 8.40 (broad s i n g l e t , 3H, C-18 CH^). Mass spectrum: M;W. 414; base peak m/e 300; main peaks, m/e 414, 263, 271. 71 13-Methyl-C-nor-D-homo-18-nor-5a,22a-spirostan-38-ol. Acetate 13-Methyl-C-nor-D-homo-18-nor-5a,22a-spirost-12(13)-en-38-ol Acetate (280 g) which had been prepared by treatment of the o l e f i n e - 3 3 - ol(83) w i t h p y r i d i n e - a c e t i c anhydride overnight was hydrogenated i n a c e t i c a c i d over 5% rhodium on alumina as o u t l i n e d by W. P. Johns. The product (84) was r e a d i l y obtained c r y s t a l l i n e from m e t h a n o l N e e d l e s m.pt. 168-173° (220 g) ( l i t . m.pt. 173-177°). N.m.r. s i g n a l s : 9.20 ( s i n g l e t , 3H, C-19 CH ) , 8.01 ( s i n g l e t , 3H, £H -fi-). Mass spectrum: M.W. 458; base peak; m/e 342. 17-Acetyl-5ct, 138-etioj erv-16-en-3B-ol (85) The hydrogenation product (110 g) was subjected to r i n g opening by o c t a n o i c anhydride, o x i d a t i o n w i t h chromic a c i d and e l i m i n a t i o n employing potassium hydroxide i n t - b u t a n o l , according to the procedures o u t l i n e d by W. F. Johns. Chromatography o f the product on alumina (2 Kg, 64 Act. I l l ) e l u t i n g with 20% chloroform i n benzene gave 17-acetyl-5a,138- et i o j e r v - 1 6 - e n - 3 B - o l (34 g) which was c r y s t a l l i s e d from acetone/petroleum ether. M.pt. 162-165° ( l i t . m.pt. 164-168°). N.m.r. s i g n a l s : 9.22 ( s i n g l e t 3H, C-19 CH 3), 9.19 (doublet, J = 7.0, 3H, C-18 CH ) , 7.0 ( q u i n t e t , IH, C-13H), 3.1 (two doublets, J = hJ 16 15 = 7 ' 5 , 1 H ' C ~ 1 6 ^ ' Conversion to 17-acetyl-5a-138-etiojerv-16-en-38-ol acetate was accomplished by d i s s o l v i n g the product i n a c e t i c anhydride/pyridine (1:1) and a l l o w i n g the s o l u t i o n to stand at 20° f o r 12 hours. R e c r y s t a l l i s a t i o n o f the product from acetone/petroleum ether gave needles|34.2 g) m.pt. 143-144°. 17-Acetyl-5a 138-etiojerv-16-cn-3B-ol-3-Acetate 20-0xime (86) A s o l u t i o n o f 17-acetyl-5a,138-etiojerv-16-en-38-ol acetate (33 g) 72 i n p y r i d i n e was t r e a t e d w i t h hydroxylamine h y d r o c h l o r i d e according to the procedure of IV. F. Johns. The product was obtained c r y s t a l l i n e from acetone as needles (33.5 g) -m.pt. 198-202° ( l i t . 205-210). N.m.r. s i g n a l s 9.18 ( s i n g l e t , 3H, C-19 CH ) 9.07 (doublet J = 7, 3H, C-18 CH ), 8.0 P N ( s i n g l e t , 6H, CH_3 -C- and CH_3 -C-) , 7.03 ( q u i n t e t , IH, C-13H), 3.82 (two doublets J 1 6 1 5 = J j 6 1 5 = 7, IH, C-16 H). Mass spectrum: M.W. 373; base peak m/e 358; main peaks m/e 373, 296, 255. 3B-Acetoxy-Sa-etioj ervan-17-one (87) A s o l u t i o n o f 17-acetyl-5a,13B-etiojerv-16-en~38-ol a c e t a t e , 20-oxime (33.0 g) i n p y r i d i n e (400 ml) was t r e a t e d w i t h phosphorous o x y c h l o r i d e f o l l o w i n g the procedure o f W. F. Johns. The product, obtained by f i l t r a t i o n a f t e r pouring the r e a c t i o n mixture i n t o aqueous h y d r o c h l o r i c a c i d at 60°, was chromatographed on alumina (700 g, act I I I ) . E l u t i o n w i t h 15% chloroform i n benzene gave pure 38-acetoxy-5a-etiojervan-17-one (24.8 g) which was c r y s t a l l i s e d from methanol/water g i v i n g c o l o u r l e s s needles. M.pt. 168-169° ( l i t 175-177°); [a] + 122° ( l i t + 123°); N.m.r. s i g n a l s : 9.21 ( s i n g l e t , 3H, C-19 CH,) , 9.05 (doublet J 1 4 > ., = 6, 3H, C-18 p , 6 1 o - J J CH 3), 8.06 ( s i n g l e t , 3H, Cti^ Ci-). Mass spectrum: M.W. 332; base peak, m/e 272; main peaks m/e 332, 257, 200. 3B-Acetoxyetiojerv-12(13)-en-17-one (76) Bromination of 3B-acetoxy-5a-etiojervan-17-one (87) using an equimolar q u a n t i t y of bromine followed by dehydrobromination as o u t l i n e d by W. F. Johns gave a mixture of compounds. Chromatography on alumina (400 g, act I I I ) e l u t i n g w i t h 15% chloroform/benzene gave 3B-acetoxyetio- jervan-17-one w h i l s t f u r t h e r e l u t i o n with the same so l v e n t provided 73 38-acer.oxyetiojerv-12(13)-en-17-one (76) m.pt. 160-162°. N.m.r. s i g n a l s : 9.18 ( s i n g l e t , 311, C-19 CH ) , 8.31 ( s i n g l e t , 3H, C 18 CM ) , 8.01 ( s i n g l e t , . P 3H, CH^ -L-). Mass spectrum: M.W. 330; base peak, m/e 330; main peaks, m/e 288, 272, 270, 255. i 18-Nor-C-homo-26-nor-22,27-iminojerva-12q(13),22,24,27-tetraene-3B,17-diol (1 An ether s o l u t i o n o f 1.88 M methyl l i t h i u m (1.0 ml) was added to anhydrous t e t r a h y d r o f u r a n (4 ml) i n a round bottom f l a s k which had been flame d r i e d and flushed with dry n i t r o g e n . A t e t r a h y d r o f u r a n s o l u t i o n o f 2.0 M 2 - e t h y l p y r i d i n e (1 ml) was added immediately and the mixture was r e f l u x e d under n i t r o g e n f o r t h i r t y minutes. During t h i s time the s o l u t i o n developed a deep red c o l o u r . A f t e r the p e r i o d o f r e f l u x the a,B-unsaturated ketone (101) i n the form of a f i n e powder was added slo w l y u n t i l the c o l o u r faded (120 mg) and the r e a c t i o n mixture r e f l u x e d f o r a f u r t h e r t h i r t y minutes w h i l s t the f l a s k was s t i l l under n i t r o g e n . Water was then added c a u t i o u s l y to destroy any a l k y l l i t h i u m remaining and the r e s u l t i n g mixture d i l u t e d w i t h ether (10 ml). The organic phase was separated and washed with water (4 x 10 m l ) , p r i o r to dr y i n g over anhydrous sodium s u l f a t e . Evaporation of the ether gave a l i g h t o i l (100 mg ) which appeared as.one major and s e v e r a l minor components when examined by t h i n l a y e r chromatography. The major component was obtained pure by p r e p a r a t i v e t h i n - l a y e r chromatography on Woelm n e u t r a l alumina (20 x 20 cm., 0.4 mm, 2% methanol i n chloroform). S p e c t r a l data i n d i c a t e d t h i s was the de s i r e d condensation product (102) although the n.m.r. i n d i c a t e s the m a t e r i a l i s probably a mixture of two isomers. I n f r a r e d (CHC1.,): 6.3, 6.4 ( p y r i d i n e ) , 3.1 p ( h y d r o x y l ) , U l t r a v i o l e t X max ( l o g e): 257 (3.55), 262 (3.58), 270 74 (3.45) m/i. N.m.r. s i g n a l s : 9.26 ( s i n g l e t , 3H, C-19 CH 3), 8.8, 8.75 (two overlapping doublets due to the presence o f two isomers i n the product, J = 7, 3H, CH 3 - £ - Fl), 6.53 (quartet J = 7, 1H, -CH-CH3) , 5.1 (broad s i n g l e t , 1H, C-13H), 2.0 3.0 ( m u l t i p l e t , 311, p y r i d i n e ) , 1.5 ( m u l t i p l e t , 1H, p y r i d i n e ) . 26-nor-22,27-iminojerva-12(13), 22,24,27-tetraene-3<3,17-diol. (103) An ether s o l u t i o n o f 1.88 M methyl l i t h i u m (1.0 ml) was added to anhydrous t e t r a h y d r o f u r a n i n a dry round bottom f l a s k under n i t r o g e n . 1 A s o l u t i o n o f 2 - e t h y l p y r i d i n e (1.0 ml o f 2.0 M) i n te t r a h y d r o f u r a n was added immediately and the mixture r e f l u x e d f o r 30 minutes during which time a deep red c o l o u r developed. A d d i t i o n o f 38-acetoxy-5a-etiojerv-12(13) en-17-one (76) i n the form o f a f i n e l y ground dry s o l i d was continued u n t i l the c o l o u r faded (90 mg added) and the mixture s t i r r e d f o r a f u r t h e r t h i r t y minutes, w h i l s t the f l a s k was s t i l l under n i t r o g e n . Water was added to quench the r e a c t i o n , and the mixture d i l u t e d w i t h ether (10 ml). The organic phase tvas separated and •: washed with water (4 x 10 ml) p r i o r to dr y i n g over anhydrous sodium s u l f a t e . Examination o f the et h e r e a l e x t r a c t by t h i n - l a y e r chromatography i n d i c a t e d one major component which appeared as a b r i g h t y ellow spot when the chromatoplate was developed w i t h 1:1 antimony pentachloride/carbon t e t r a c h l o r i d e (1:1) spray reagent. Column^chromatography on alumina (Shawinigan, act I I I , 25 g) e l u t i n g with benzene/chloroform (4e. 1) enabled p u r i f i c a t i o n o f t h i s component which was c r y s t a l l i s e d from benzene/ether as needles. (42 mg) M.pt. 204-206°. I n f r a r e d (CHCl^): 6.30, 6.41 y ( p y r i d i n e ) . U l t r a v i o l e t Xmax ( l o g e): 257 ( s h ) , 262.5 (3.58), 269 (sh) my." N.m.r. s i g n a l s : 9.26 ( s i n g l e t , 3H ? C-19 CH 3), 8.84 (doublet J 2 ] 2 Q = 7.0,. 311, C-21 CH ) , 8.26 ( s i n g l e t , 311, C-18 CH 3), 2.8 ( m u l t i p l e t , 2H, p y r i d i n e ) , 2.34 ( m u l t i p l e t , 1H, p y r i d i n e ) , 1.5 ( m u l t i p l e t , 1H, p y r i d i n e ) . Mass spectrum: M.W. 395; main peak:;: m/e 377, 362, 288. Found: C, 78.85; H, 9.50; N, 3.50. C a l d . f o r C.,H,_O.N: C, 78.94; H, 9.43; N, 3.54. 23-Methoxy-22,27-iminojerva-12(13),22,24,27-tetraene-3B,17-diol (104) An ether s o l u t i o n (1.0 ml) of 1.88 M methyl l i t h i u m was added to anhydrous tetrahydrofuran (5.0 ml) i n a dry round bottom f l a s k which had been fl u s h e d w i t h n i t r o g e n . A s o l u t i o n o f 1.9 M 2-ethyl-3-methoxy-5- me t h y l p y r i d i n e (1 ml) i n t e t r a h y d r o f u r a n was added immediately, and the mixture r e f l u x e d f o r one hour; during which time a deep red c o l o u r s l o w l y developed i n the s o l u t i o n . The s o l u t i o n was cooled and 38-acetoxy-5a- etiojerv-12(13)-en-17-one (76) was added as a f i n e powder u n t i l the c o l o u r faded (116 mg) and the mixture s t i r r e d f o r a f u r t h e r t h i r t y minutes. Water (5.0 ml) was added c a u t i o u s l y w h i l s t the mixture was s t i l l under n i t r o g e n and the r e s u l t i n g s o l u t i o n d i l u t e d with ether (10 ml). The organic phase was washed with water (4 x 10 ml) p r i o r to dr y i n g over anhydrous sodium s u l f a t e . Evaporation of the s o l v e n t i n vacuo gave a l i g h t o i l (120 mg) which appeared as p r i m a r i l y one compound when examined by t h i n - l a y e r chromatography (yellow spot when the chromatoplate i s developed w i t h antimony p e n t a c h l o r i d e spray reagent). Column chromatography on alumina (40 g, act I I I ) e l u t i n g w i t h benzene removed the unreacted p y r i d i n e w h i l s t the d e s i r e d compound was only p a r t i a l l y p u r i f i e d . P r e p a r a t i v e t h i n - l a y e r chromatography on s i l i c a gel (20 x 20 cm., 0.4 mm, 2% methanol i n chloroform p l a t e developed 2 x) gave a pure sample of 23-methoxy-22,27-irainojerva-12(13),22,24,27-tetraene-36,17-diol (104) as a l i g h t o i l which c r y s t a l l i s e d from ether. M.pt. 209-213°. I n f r a r e d 76 (CHC1 3); 6.3, 9.7 u. U l t r a v i o l e t , X max ( l o g e): 284.5 (3,86) my. N.m.r. s i g n a l s : 9.22 ( s i n g l e t , 3H, C19 CH ) 8.84, 8.81 (two overlapping doublets J 21_26 = 7 , 0 » 3 H ' C " 2 1 C H3?-' 6 , 2 4 » 6 , 2 1 ^ t w o s i n g l e t s > 3 H> -0CH 3), 3.10 ( s i n g l e t , IH, p y r i d i n e ) , 2.04 (broad s i n g l e t , IH, p y r i d i n e ) . Mass spectrum: M.W. 439; base peak m/e 406; main peaks m/e 421, 288. Found: 439.3076. Calcd. f o r C_oH.,0,N: 439,3086. 28 41 3 26-Nor-22,27-iminojerv-12(13),14(15),16,22,24,27-hey 9 /ne-3B-ol(103a) Condensation compound 103 (20 mg) was ground with 10% p a l l a d i s e d charcoal (10 mg) u n t i l a homogeneous powder was obtained. This powder was heated at 200° i n an i n e r t atmosphere f o r ten minutes. The r e s u l t i n g s o l i d r esidue was washed s e v e r a l times with chloroform and the combined washings examined by t h i n - l a y e r chromatography which i n d i c a t e d a s l i g h t l y more p o l a r compound had been formed. P r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.3 mm. 2% methanol i n chloroform, s i l i c a g e l G) afforded t h i s component as a c o l o u r l e s s o i l (12 mg) which could not be induced to c r y s t a l l i s e . The s p e c t r a l data i s i n accord with t h i s compound possessing s t r u c t u r e 103(a). I n f r a r e d , (CHC1 3): 6.3, 6.4, 6.65. Ultr a v i o l e t , X m a x ( l o g e) . 263 (3.67) my. N.m.r. s i g n a l s : 9.07 ( s i n g l e t , 3H, C-19 CH 3), 8.35 (doublet, ^2l-20 = 7 ' 3 H ' C " 2 1 C1V' 7 , 8 5 ( s i n S l e t > 3 H » C - 1 8 C H 3 ) • Mass spectrum: M.W. 375; base peak m/e 375; main peaks m/e 360, 346, 320, 288. 23-methoxy-22,27-iminojerv-12(13),14(15),15,22,24,27-hexaene-3B-ol (104a) Condensation compound 104 (25 mg) was ground with 10% p a l l a d i s e d charcoal u n t i l a homogeneous powder was obtained. This powder was heated at 200°C f o r ten minutes. The chloroform e x t r a c t obtained by washing the s o l i d residue s e v e r a l times with chloroform appeared to contain one major 77 new compound when examined by t h i n - l a y e r chromatography. This component was separated by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.4 mm, 2% MeOH/CHClj) as a l i g h t o i l (10 mg) which could not be induced to c r y s t a l l i s e . The s p e c t r a l p r o p e r t i e s are i n accord with t h i s product having s t r u c t u r e 104(a). I n f r a r e d (K B r ) : 6.29, 11.6 u. U l t r a v i o l e t , X max, ( l o g e ) ; 283 (4.03) m/t . N.m.r. s i g n a l s : 9.09 ( s i n g l e t , 3H, C-19 CH 3), 7.75 ( s i n g l e t , 6H, C-26 and C-18 CH 3), 6.33 ( s i n g l e t , 3H, -OCK^. Mass spectrum: M.W. 419; main peaks, m/e 419, 404, 390, 372 288. 2-ethyl-5-methylpyridine (105) An anhydrous ether s o l u t i o n (250 ml) o f bromobenzene (31.4 g, 0.2 mole) was added s l o w l y to a dry round bottom f l a s k which had been f l u s h e d w i t h n i t r o g e n and which contained l i t h i u m wire (2.8 g. 0.4 mole). When the l i t h i u m had l a r g e l y d i s s o l v e d an e t h e r e a l s o l u t i o n (100 ml) of 2 , 5 - l u t i d i n e (21 g, 0.2 mole) was added and the mixture r e f l u x e d f o r t h i r t y minutes, during which time a deep red co l o u r developed i n the s o l u t i o n . Methyl i o d i d e (14.2 g, 0.1 mole) i n ether (40 ml) was added s l o w l y to t h i s red s o l u t i o n over a p e r i o d o f f i f t e e n minutes and the mixture s t i r r e d f o r ten minutes. The excess l i t h i u m and phenyl l i t h i u m was removed by the c a r e f u l a d d i t i o n o f water (100 ml) to the r e a c t i o n mixture w h i l s t the f l a s k was s t i l l under n i t r o g e n . A f u r t h e r 200 ml of water was then added, the organic phase separated and washed with water (2 x 50 ml). The et h e r e a l s o l u t i o n was reduced i n volume to 40 ml by use o f a r o t a r y evaporator. This 40 ml was then c a r e f u l l y d i s t i l l e d at a pressure o f 10 mm, employing a spi n n i n g band column. The f i r s t f r a c t i o n , d i s t i l l i n g at 50-55° was shown by n.m.r. to be recovered 2 . 5 - l u t i d i n e w h i l s t that d i s t i l l i n g at 62-63° was shown to be 2- e t h y l -78 5-methylpyridine (105). I n f r a r e d ( f i l m ) : 6.3, 6.4, 6.8, 7.02 u. U l t r a v i o l e t , X max (log e): 265 sh (3.54), 269 (3.60), 276 (3.48) m u . N.m.r. s i g r . a l s ; 8.70 ( t r i p l e t , J = 7.5, 3H, CH.J-CH ), 7.72 ( s i n g l e t , 3H, CH 3-C), 7.20 (quartet = 7.5, 2H, -CH^ - CH ), 2.93 ( doublet, J 3 = 8.0, IH, C-3H), 2.59 (two doublets J 4 _ 3 = 8.0, J 4 = 2.0, H i , C-4 H), 1.63 (doublet ^ = 2.0, IH, C 6-H). P i c r a t e d e r i v a t i v e from actone, m.pt. 143-144° ( l i t . 144° 5 3 ) . 3B-Acetoxy-22,27-iminojerva-12(13),22,24,27-tetraene-17-ol (106) A 2.05 M e t h e r e a l s o l u t i o n of methyl l i t h i u m (25 ml, 0.0512 moles) was added to a flame d r i e d round bottom f l a s k c o n t a i n i n g anhydrous te t r a h y d r o f u r a n (40 ml) under n i t r o g e n . A mixture of anhydrous tetrahydrofuran (10 ml) and 2-ethyl-5-methylpyridine (6 g, 0.05 moles) was added immediately and the r e a c t i o n mixture r e f l u x e d f o r one and a. h a l f hours. During t h i s p e r i o d a deep red c o l o u r developed i n the s o l u t i o n . A f a i n t pink colour p e r s i s t e d a f t e r 38-acetoxy-5a-etiojerv-12-en-17-one (76) (2.0 g. 0.006 moles) was added as a f i n e powder to the cooled s o l u t i o n , and the mixture s t i r r e d f o r ten minutes. Water (50 ml) was added c a u t i o u s l y to the r e a c t i o n mixture w h i l s t s t i l l under n i t r o g e n , followed by ether (50 ml) p r i o r to s e p a r a t i o n o f the organic and aqueous phases. The organic phase was washed w i t h water (3 x 25 ml) and then d r i e d over anhydrous sodium s u l f a t e . Removal of the s o l v e n t i n vacuo gave a l i g h t o i l (2.3 g) which appeared to c o n s i s t of two new components when examined by t h i n - l a y e r chromatography. Attempts to o b t a i n these compounds i n a pure s t a t e were uns u c c e s s f u l . However a small s c a l e i n v e s t i g a t i o n i n d i c a t e d that the compounds could be separated more r e a d i l y as the C-3 acetates r a t h e r than as the C-3 a l c o h o l s which were formed i n the r e a c t i o n . Conversion to the C-3 acetates was achieved by d i s s o l v i n g the 79 crude product from the r e a c t i o n i n a c e t i c anhydride-pyridine (1:1, 20 ml) and a l l o w i n g the s o l u t i o n to stand at room temperature f o r twelve hcurs. The s o l u t i o n was then poured onto crushed i c e and the r e s u l t a n t f l o e c u l a n t s o l i d e x t r a c t e d i n t o ether. The combined e t h e r e a l e x t r a c t s were washed wi t h s a t u r a t e d aqueous sodium hydrogen carbonate s o l u t i o n and then s e v e r a l times w i t h water before d r y i n g over anhydrous sodium s u l f a t e . The volume o f the e t h e r e a l s o l u t i o n was reduced i n vacuo to 20 ml and then allowed to stand whereupon c o l o u r l e s s needles were deposited. M.pt. 187-189° (370 mg). Examination o f the c r y s t a l s by t h i n - l a y e r chromatography i n d i c a t e d only one compound was present and r e c r y s t a l l i s a t i o n from ether gave an a n a l y t i c a l sample, m.pt. 189-190°. This compound was designated "A" and the p h y s i c a l and s p e c t r a l data are i n accord w i t h s t r u c t u r e 106. I n f r a r e d , (CHCl^): 5.83 (OAc) 6.25, 6.37 ( p y r i d i n e ) u. U l t r a v i o l e t , Xmax ( l o g e): 270 (3.61) rru . [ a ] D + 83° (c. 124). N.m.r. s i g n a l s : 9.21 ( s i n g l e t , 3H, C-19 CH 3), 8.79 (doublet, J 2 1 _ 2 Q = 7-°> c " 2 1 C H 3 ) 8.40 ( s i n g l e t , 3H, C-18 CH 3), 8.01 ( s i n g l e t , 3H, 0-C-CH ),7.70 ( s i n g l e t , 311, C-26 CH ) , 6.71 (quartet J^Q 2\= 7 ,^> C-20 H) 3.89 (broad m u l t i p l e t - disappears on D 20 a d d i t i o n , 1H, -OH),2.82 (doublet J 2 3 u = 8, 1H, C-23 H) 52.52 (two doublets, J 24-23 = 8 J 2 4 - 2 7 = 2* 1 H » C-24'>: H ) ' 1 , 6 7 ( d o " b l e t J 27_24 = 2 > 1H, C-27 H). Mass spectrum: M.W. 451; base peak m/e 418; main peaks m/e 433, 330, 147, 121. Found: C,76.92;H, 8.95; 0, 10.46; N, 3.21. C a l c d . f o r C29 M41°3 N : C ' 7 7 ' 1 2 ' H ' 9 - 1 5 ' °» 1 0- 6 3> N» 3 - 1 0 - Examination by t h i n - l a y e r chromatography o f the mother l i q u o u r s from the c r y s t a l l i s a t i o n o f compound A i n d i c a t e d the presence o f a f u r t h e r compound of s i m i l a r c o l o u r r e a c t i o n when sprayed with antimony p e n t a c h l o r i d e - carbon t e t r a c h l o r i d e reagent. These mother l i q u o u r s were combined and chromatographed on alumina (60 g. act I I I ) . E l u t i o n w i t h benzene y i e l d e d , 80 unreacted 2-ethyl-5-methylpyridine (105) i n i t i a l l y , w h i l s t f u r t h e r e l u t i o n provided an a d d i t i o n a l compound (350 mg) which was obtained c r y s t a l l i n e from a small volume o f ether as prisms, m.pt. 190-192°. R e c r y s t a l l i s a t i o n from acetone-petroleum ether gave an a n a l y t i c a l sample (250 mg) m.pt. 190- 192°. This compound was designated compound "B" and was s l i g h t l y l e s s p o l a r than compound "A" on a s i l i c a g e l chromatoplate developed w i t h 2% methanol i n chloroform. I n f r a r e d , (CHC1 3): 5.83 (OAc), 6.25, 6.37 ( p y r i d i n e ) u . U l t r a v i o l e t , X max ( l o g e): 265 sh (3.57), 270 (3.61), 276 sh (3.51) my. [ a ] D - 126° (c, 1.26). N.m.r. s i g n a l s : 9.23 ( s i n g l e t , 3H, C-19 CH ), 8.88 doublet, J 2 Q _ 2 1 = 7.0 3H, C-21 CH ^ 8 . 2 8 ( s i n g l e t , 3H, C-18 CH ), 8.02 ( s i n g l e t , 3H, CH_ -C-), 7.72 ( s i n g l e t , 311, C-26 CH ) , 6.71 (quartet ^20 21 = 7*^' '0 4.20 (broad s i g n a l , disappears on D^ O a d d i t i o n , IH; -OH), 2.92 ,(doublet, J 2 3 _ 2 4 = 8.'0, IH, C-23 H) , 2.70 (two doublets J24-23 = 8 ' 0 ' J24-27 = 2 , ° ' 1 H» C " 2 4 H ) 5 1 , 7 5 ( d o u b l e t J27~24» 1 H » C " 2 7 H ) ' Mass spectrum: M.W. 451; base peak m/e 418, main peaks; m/e 433, 330 147, 121. Found: C, 77.19; H, 9.21; N, 3.19. Cald. f o r C H ON;- C, 77.12; H, 9.15; N, 3.10. E l u t i o n w i t h 20% ether i n benzene gave a d d i t i o n a l compound A (150 mg) w h i l s t two f r a c t i o n s appeared to co n t a i n two f u r t h e r compounds o f s i m i l a r r e a c t i o n to the spray reagent when examined by t h i n - l a y e r chromatography. Separation by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.3 mm, 1% methanol i n chloroform, -chromatoplate developed twice) y i e l d e d 35 mg and 8 mg o f these compounds which were designated "C" and "D" r e s p e c t i v e l y . Compound "C" could not be induced to c r y s t a l l i s e and an a n a l y t i c a l sample was prepared by sublimation at 190° / 0.1 mm which gave a c l e a r g l a s s . The s p e c t r a l data was c o n s i s t e n t w i t h t h i s compound being isomeric w i t h "A" and "B". I n f r a r e d (KBr): 5 . 8 (-OAc), 6.25, 6.39 (p y r i d i n e ) y . U l t r a v i o l e t , X max ( l o g e) : 270 (3.60) my. N.m.r. s i g n a l s : 81 9.26 ( s i n g l e t , 3H, C-19 CH ), 8.77 (doublet, J = 7.0, 3H, C-21 CH ) , 8.43 (broad s i n g l e t , 3H, C-18 CH ), C.01 ( s i n g l e t , 3H, CH-C-), 7.73 ( s i n g l e t , 3H, C-26 CH ), 6.90 (quartet J 20-21 = 7 , ° * 1 H * C " 2 0 H ) > 2 - 9 7 (doublet, J23-24 = 8 - 0 , 1 H » C - 2 3 H ) > 2 - 6 5 ( t w o doublets, J 2 4 _ 2 3 = 8.0, J 2 4 _ 2 7 = 2.0, 1H,C-24H), 1.72 (doublet J 2 ? 2 4 =2.0, 1H, C-27H). Mass spectrum: M.W. 451jbase peak m/e 120; main peaks m/e 423, 418, 330. Found: C, 77.05; H, 9.33, C a l c d . f o r C 2 gH 0 N: C, 77.12, H, 9.15. Compound D could not be induced to c r y s t a l l i s e but the n.m.r. and mass spectrum i n d i c a t e s t h i s compound i s probably isomeric w i t h the three c h a r a c t e r i s e d above. I n f r a r e d (CHC1 ): 5.83 (OAc), 6.25, 6.37 (p y r i d i n e ) y U l t r a v i o l e t X max (l o g e): 2 70 (3,62) my. N.m.r. s i g n a l s 9.21 ( s i n g l e t , 3H, C-19 CH 3), 8.63 (doublet J 2 Q = 7.0, 3H, C-21 CH ), 8.27 ( s i n g l e t , 3H, C-18 CH,), 8.01 ( s i n g l e t , 3H, CH C-), 7.74 ( s i n g l e t , 3H, C-26 CH ), 6.89 (quartet J 20-21 = 7 ' ° ' 1 H ' C " 2 ° H') ' 3 , 0 8 ( d o u b l e t J 2 3 24 = 8 , ° ' 1 H ' C-23H), 2.62 (two doublets J 2 4 ' = 8.0, J 2 4 2 y = 2.0, 1H, C-24H), 1.70 (doublet J 2 7 _ 2 4 =2.0, 1H, C-27H). Mass spectrum: M.W. 451; base peak,m/e 121, main peaks, m/e 433, 418. 3B-Acetoxy-22,27-iminojerv-12(13),14(15),16,22,24,27-hexa.gne. (107) "Aromatic I." Compound "A" (118 mg) was thoroughly ground with 10% p a l l a d i s e d charcoal (30 mg) u n t i l a homogeneous powder was obtained. This powder was heated at 200° f o r seven minutes and the r e s u l t a n t s o l i d r esidue washed s e v e r a l times w i t h chloroform. T h i n - l a y e r chromatography o f the chloroform e x t r a c t i n d i c a t e d the product contained three major components and these were separated by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.4 mm, 2% methanol i n chloroform). One of these three 82 components was shown to be 2-ethyl-5-methylpyridine (105) by n.m.r. The band which e x h i b i t e d a strong fluorescence when the chromatoplate was viewed under u l t r a v i o l e t l i g h t was e x t r a c t e d and y i e l d e d a l i g h t o i l which c r y s t a l l i s e d from acetone-petroleum ether as needles m.pt. 167°. This compound was i d e n t i f i e d as 38-acetoxy-5a-etiojerv-12(13)-en-17-one (76) by i t s u l t r a v i o l e t spectrum and by mixed m e l t i n g p o i n t with an a u t h e n t i c sample. The t h i r d compound which appeared as a l i g h t orange spot when the chromatoplate was sprayed with antimony pentachloride-carbon t e t r a c h l o r i d e reagent was e x t r a c t e d to y i e l d a l i g h t o i l which could not be induced to c r y s t a l l i s e . An a n a l y t i c a l sample of t h i s compound which was designated "Aromatic I " was obtained as a c l e a r g l a s s a f t e r s u b l i m a t i o n at 185° / 0.1 mm. S p e c t r a l data i n d i c a t e d t h i s compound possessed s t r u c t u r e 107. I n f r a r e d (KBr): 5.82, 6.26, 6.39 y. U l t r a v i o l e t , X max ( l o g e): 265 ( s h ) , 269 (3.73) 277 (3.62) nu . [a] + 13° ( c , 0.84). N.m.r. s i g n a l s : 9.10 ( s i n g l e t , 311, C-19 CH 3) , 8.41 (doublet J 2 ] 2 Q = 7.0, 3H, C-21 CH 3), 8.03 ( s i n g l e t , 3H, CH_3 C-) , 7.90 ( s i n g l e t , 3H, C-18 CH 3) , 7.80 ( s i n g l e t , 311, C-26 CH 3) , 5.61 (quartet J 2 0 2 i = 7-°» 1 H » C-20H), 3.22 - 3.0 (two doublets overlapping J= 8, 211, C-15 or C-16 H and C-23 or C-24 H), 2.94 (doublet J = 8, 1H, C-15 or C-16 11), 2.72 (two doublets J 2 4 _ 2 3 = 8, J ^ 4 2 ? = 2, 111, C-24 H), 1.7 (doublet, J 2 ? 2 4 = 2, 1H, C-27 H). Mass spectrum: M.W. 431; base peak, m/e 431; main peaks m/e 416, 402. Found: 431.2809. Calcd. f o r C 2 gH 0 N : 431.2824. 3B-Acetoxy-22,27-iminojerv-12(13),14,(15,16,22,24,27-hexaene. (107) "Aromatic I I " Compound B (80 mg) was ground with 10% p a l l a d i s e d charcoal (20 mg) u n t i l a homogeneous powder was formed. This powder was heated 83 at 200 f o r seven minutes and the s o l i d residue washed s e v e r a l times with chloroform. T h i n - l a y e r chromatographic examination o f the chlorofoiTii e x t r a c t i n d i c a t e d the major component was a new compound which appeared as a l i g h t orange spot when the chromatoplate was sprayed w i t h antimony pentachloride/carbon t e t r a c h l o r i d e reagent. This compound was separated from the r e a c t i o n mixture by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.4 mm, 1% methanol i n chloroform - developed twice) as a l i g h t o i l (60 mg) which could not be induced to c r y s t a l l i s e . An a n a l y t i c a l sample o f the compound, which was designated "Aromatic. I I " , was obtained as a c l e a r g l a s s a f t e r s u b l i m a t i o n at 185° / 0.01 mm. I n f r a r e d (KBr): 5.82, 6.26, 6.39 y. U l t r a v i o l e t , X max ( l o g e): 265 sh, 269 (3.73), 277 (3.62), my. N.m.r. s i g n a l s : . 9.10 ( s i n g l e t , 311, C-19 CH ) , 8.40 (doublet 3 n Q = 7 , 3H, C-21 CH ) , 8.02 ( s i n g l e t , 3H, CH-C-), 7.89 ( s i n g l e t , 3H, C-18 CH ) , 7.80 ( s i n g l e t , 3H, C-26 CH ) , 5.58. (quartet J 2 Q _ 2 1 = 7, IH, C-20 H), 3.14 (doublet J 2 ^ _ 2 4 = 8, III, C-23 H), 3.09 (doublet, J = 8, IH, C-15 H or C-16 H), 2.91 (doublet, J J 6 = 8, IH, C-15 H or C-16 H), 2.75 (two doublets, J2A 2 3 = 8, J 2 4 2 ? = 2, IH, C-24 H) , 1.73 (doublet J ^ 24 = 2' 1 H ' C ~ 2 7 H-) ' M a S S s P e c t r u m : M- W- b a s e peak, 431, main peaks m/e 416, 402. Found: 431.2809. Ca l c d . f o r C29 H37°2 N : 4 3 1 * 2 8 2 4 ' N-acetyl-22,27-iminojerva-12(13),14(15),16-triene-3B-ol. (110) Isomers 1-4 Aromatic compound I I (50 mg) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (10 ml) and the s o l u t i o n hydrogenated at 20° and 45 p . s . i . over Adams c a t a l y s t (PtO^, 20 mg) f o r three hours. The mixture was f i l t e r e d to remove the c a t a l y s t which was c a r e f u l l y washed-with a d d i t i o n a l a c e t i c a c i d (5 ml). The combined f i l t r a t e s were reduced i n volume to 2 ml 84 i n vacuo, then d i l u t e d w i t h water (20 ml) and made b a s i c w i t h ammonia s o l u t i o n . The r e s u l t a n t suspension was e x t r a c t e d w i t h methylene c h l o r i d e (3 x 10 ml) p r i o r to d r y i n g over anhydrous sodium s u l f a t e . T h i n - l a y t r chromatography revealed the presence of four compounds of s i m i l a r value and c o l o u r r e a c t i o n w i t h various spray reagents. These compounds were designated 1-4 i n order o f decreasing R^ value, ( s i l i c a g e l G, 5% methanol i n chloroform) and were separated by c a r e f u l p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.3 mm, 1% methanol i n c h l o r o f o r m j p l a t e s developed three t i m e s ) . The bands corresponding to the four compounds were d e l i n e a t e d by i n s p e c t i o n o f the developed p l a t e s under u l t r a v i o l e t l i g h t (chromatovue, C-3). E x t r a c t i o n of the various bands and removal of the s o l v e n t (methanol / chloroform - 1:1) gave each of the compounds as a c l e a r o i l . None of the compounds could be induced to c r y s t a l l i s e . A l l four compounds were s e p a r a t e l y converted v i a the 3-0, N- d i a c e t a t e s (109) to the N-acetyl d e r i v a t i v e s ( s i n c e a sample of N - a c e t y l - 5a,6-dihydroverarine was a v a i l a b l e f o r comparison) as o u t l i n e d f o r one o f the isomers below. Compound No. 2 was d i s s o l v e d i n p y r i d i n e - a c e t i c anhydride (2 ml, 1:1) and allowed to stand f o r twelve hours at 20°. The s o l u t i o n was then poured i n t o i c e water (5 ml) and the r e s u l t i n g suspension e x t r a c t e d w i t h methylene c h l o r i d e ( 3 x 3 ml). The combined e x t r a c t s were washed w i t h s a t u r a t e d aqueous sodium hydrogen carbonate (10 ml) and then with water (2 x. 5 m l ) , p r i o r to d r y i n g over anhydrous sodium s u l f a t e . Removal o f the methylene c h l o r i d e i n vacuo gave a l i g h t o i l which c r y s t a l l i s e d from ether (the" d i a c e t a t e s of compounds 1, 3 and 4 were not obtained c r y s t a l l i n e ) as needles m.pt. 203-205°. I n f r a r e d (CHCl^) 5.80 (OAc), 6.19 (NAc) y . U l t r a v i o l e t : X max ( l o g e) : 268 (2.7) 277 (2.7) my. 85 The d i a c e t a t e (20 mg) war r e f l u x e d with 0.1 M potassium hydroxide i n methanol (5 ml) f o r one hour, the s o l u t i o n cooled and d i l u t e d w i t h water (20 ml), the aqiieois suspension was e x t r a c t e d with methylene c h l o r i d e ( 3 x 5 ml) and the combined e x t r a c t s washed with water ( 2 x 5 ml) p r i o r to d r y i n g over anhydrous sodium s u l f a t e . Evaporation o f the s o l v e n t i n vacuo gave a l i g h t o i l which was r e a d i l y c r y s t a l l i s e d from anhydrous ether. ( A l l four N-acetyl d e r i v a t i v e s were obtained c r y s t a l l i n e v i a t h i s procedure). The N-acetyl d e r i v a t i v e s of. compounds 1 and 2 had an R^ . value comparable w i t h that of N-acetyl-5oc,6-dihydroverarine w h i l s t those of the N-acetyl d e r i v a t i v e s of compounds 3 and 4 were s l i g h t l y s m a l l e r . A l l compounds showed s i m i l a r c o l o u r r e a c t i o n s when the chromatoplate was sprayed w i t h various reagents. The N-acetyl d e r i v a t i v e s were c h a r a c t e r i s e d as f o l l o w s . Compound No. 1 N-acetate; prisms m.pt. 249-250°, (6 mg). I n f r a r e d (CHCl^): 6.20, 9.70 u. U l t r a v i o l e t , X max ( l o g e) : 268 (2.68), 277 (2.66) iru . [.a] + 36° (c, 0.605). N.m.r. s i g n a l s : 9.07 ( s i n g l e t , C-19 CH ), 8.19 R ( s i n g l e t , 3H, CH 3 C-N), 7.74 ( s i n g l e t , 311, C-18 CH ), 3.15 (doublet J fi = IH, C-15H or C-16 H) , 2.96 (doublet J 1 5 _ 1 6 = 8, IH, C-15H or C-16 11). Mass spectrum: M.W. 437; base peak m/e 140; main peaks m/e 297, 98. Found: 437.325. Ca l c d . f o r C__H._O.N: 437.329. 29 43 2 Compound No. 2 N-acetate; prisms m.pt. 263-264° (]7 mg).Infrared (CHC1„): 6.19, 9.70 y. U l t r a v i o l e t X max (log c ) : 268 (2.65), 277 (2.61) nu . [ a ] D + 26°, ( c , 1.71). Mass spectrum: M.W. 437; base peak m/e 140; main peaks, m/e, 298, 297, 98. Compound No. 3 N-acetate; r o s e t t e s m.pt. 270-274°. (5 mg) I n f r a r e d (C11C1-): 6.20, 9.70 u. U l t r a v i o l e t X max: 268.277 my. Mass spectrum: 86 M.W. 437; base peak, m/e 140; main peaks m/e 297 ;98. Compound No. 4 N-acetate; long needles m.pt. 260-261° ^ (10 mg), I n f r a r e d (CHC1 3) : 6.20, 9.70, 9.80 L I . U l t r a v i o l e t , X max: 268/ 277 my. Mass spectrum: M.W. 437; base peak, m/e 140; main peaks, m/e 298, 297, 98. N-acetyl-22,27-iminojerva-12(13),14(15),16-triene-3 R-ol. (110) Isomers 5-8 Aromatic compound I (50 .Tig.) 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 (10 ml) and the s o l u t i o n shaken w i t h Adams c a t a l y s t (PtO^, 20 mg) i n an atmosphere of hydrogen at 45 p . s . i . and 20° f o r a p e r i o d of three hours. The mixture was f i l t e r e d to remove the c a t a l y s t which was washed w i t h a f u r t h e r 10 ml of g l a c i a l a c e t i c a c i d and the f i l t r a t e s combined. The volume o f the a c e t i c a c i d was reduced to 2 ml.by use o f a r o t a r y evaporator p r i o r to d i l u t i o n w i t h water (20 ml). The aqueous a c i d s o l u t i o n was made b a s i c w i t h d i l u t e ammonia and the r e s u l t i n g suspension e x t r a c t e d s e v e r a l times w i t h methylene c h l o r i d e (3 x 10 ml) p r i o r to d r y i n g over anhydrous sodium s u l f a t e . T h i n - l a y e r chromatographic i n v e s t i g a t i o n o f the l i g h t o i l (45 mg) obtained by removal of the methylene c h l o r i d e i n d i c a t e d the presence o f four compounds, of s i m i l a r R^ . value and c o l o u r r e a c t i o n to the spray reagent. These compounds were numbered 5-8 i n order of decreasing R^ . values and were separated by c a r e f u l p r e p a r a t i v e t h i n - l a y e r chromatography ( s i l i c a g e l G. 20 x 20 cm., 0.3 mm, 1% methanol i n chloroform, p l a t e s developed three, t i m e s ) . The compounds were obtained as c o l o u r l e s s o i l s which could not be c r y s t a l l i s e d . Conversion of these compounds v i a the 3-0,N-diacetates to the N-acetyl d e r i v a t i v e s was accomplished as o u t l i n e d above f o r the 3B-acetoxy-22,27-iminojerva-12(l3), 14(15),16-triene isomers No. 1-4 obtained from Aromatic compound I I . None of the N-acetyl d e r i v a t i v e s o f compounds 5-8 could be induced to 87 c r y s t a l l i s e although compounds 5 ai.d 6 had comparable R^ . values with N- a c e t y l - 5 , 6 - d i h y d r o v e r a r i n e . These compounds were not f u l l y c h a r a c t e r i s e d but t h e i r s p e c t r a l p r o p e r t i e s were i n accord w i t h s t r u c t u r e 110. Compound No. 5 N-acetate: I n f r a r e d (KB:r): 6.19, 9.28 p. U l t r a v i o l e t X max: 267, 276 mu. Mass spectrum: M.W. 437; base peak, m/e 140; main peaks 298, 297, 98. S p e c t r a l p r o p e r t i e s f o r the N-acetyl d e r i v a t i v e of compounds 6T8 are v i r t u a l l y the same as f o r Nb. 5-acetate and no a d d i t i o n a l data was obtained f o r any of these compounds. Hydrogenation of 3-0,N-diacetylverarine (117) The 3 - 0 , N - d i a c e t y l v e r a r i n e u t i l i s e d i n t h i s r e a c t i o n was 20 prepared from veratramine and had the f o l l o w i n g p h y s i c a l constants. M.pt. 189-190° ( l i t . m.pt. 189-190°), [a] -23° - 2° (ethanol). A s o l u t i o n of 3 0, N - d i a c e t y l v e r a r i n e (190 mg) i n g l a c i a l a c e t i c a c i d (5 ml) was s t i r r e d with Adams c a t a l y s t (Pt02, 60 mg) i n an atmosphere of hydrogen at 20°. A f t e r 14 hours 9.1 ml of hydrogen had been consumed and the mixture was f i l t e r e d to remove the c a t a l y s t which was washed w i t h a f u r t h e r 10 ml of a c e t i c a c i d . The combined f i l t r a t e s were reduced i n volume (to 2 ml) i n vacuo and d i l u t e d w i t h water (30 ml) to give a white p r e c i p i t a t e which was taken up i n ether. The e t h e r e a l phase was washed with water (3 x 10 ml) p r i o r to d r y i n g over anhydrous sodium s u l f a t e . T h i n - l a y e r chromatographic examination on both s i l i c a g e l and alumina using a v a r i e t y o f s o l v e n t s i n d i c a t e d that a s i n g l e new compound had been formed. Removal o f the ether i n vacuo gave an o i l which could not be induced to c r y s t a l l i s e . A small s c a l e i n v e s t i g a t i o n revealed that a f t e r s e l e c t i v e h y d r o l y s i s of the 3-0-acet.ate the product appeared as two 88 components of s i m i l a r R^ value-on a s i l i c a gel chromatopiate. Consequently 3-0,N-diacetyl-S,6-dihydroverarine (170 mg) was r e f l u x e d with 0.1.N methanolic potassium hydroxide (5 ml) f o r one hour, the s o l u t i o n cooled, and then d i l u t e d w i t h water (20 ml). The r e s u l t i n g aqueous suspension was e x t r a c t e d w i t h methylene c h l o r i d e (3 x 10 ml) and the combined e x t r a c t s washed with water (3 x 10 ml) p r i o r to d r y i n g over anhydrous sodium s u l f a t e . Removal o f the solvent i n vacuo gave a l i g h t o i l which on t r i t u r a t i o n with ether gave c r y s t a l s . M.pt. 190-225°. T h i n - l a y e r chromatography showed the c r y s t a l l i n e product to c o n s i s t of two compounds which were separated by p r e p a r a t i v e t h i n - l a y e r chromatography on s i l i c a g e l . (20 x 20 cm., 0.4 mm, 2% methanol, i n chloroform). The l e a s t p o l a r compound (75 mg) was t e n t a t i v e l y assigned the 56 c o n f i g u r a t i o n on the b a s i s of i t s n.m.r. spectrum. R e c r y s t a l l i s a t i o n from ether gave an a n a l y t i c a l sample of N-acetyl-56,6-dihydroverarine (121) m.pt. 198-199°. I n f r a r e d (CHC1 ): 6.21 (-NC-CH) y. U l t r a v i o l e t , X max ( l o g e): 267 (2.74), 276 (2.74) mu. N.m.r. s i g n a l s : 9.02 (doublet J 2 6 2 5 = 7, 3H, C-26 CH ), 8.94 ( s i n g l e t , 3H,C-19 CH j , 8.82 (doublet J 2 Q = 8 , 3H, C-21 CH ), 8.14 ( s i n g l e t , 3H, CH_3 C-N), 7.71 ( s i n g l e t , 3H, C-18 CH ), 6.45 (doublet of quartets J 2 0 21 = ' 8 ' J 2 0 22 = 1 0 ' 1 H ' C ~ 2 0 5 - 9 7 ( n a r r o w m u l t i p l e t , 111, H-C-0- Ac), 3.12 (doublet J = 8, 111, C-15 H or C-16 H) , 2.96 (doublet J15 16 = 8 ' C " 1 5 o r C-1 6 H ) • Mass spectrum: M.W. 437; base peaks, m/e 140; main peaks, m/e 297, 98. Found: C, 79.51; H, 10.02; N, 3.25, Calcd. C29 H43°2 N : C ' 7 9 ' 5 8 ' H» 9 ' 9 0 ' N» 3 > 2 ° - The more p o l a r compound was c r y s t a l l i s e d from ether as prisms m.pt. 249-250°. This compound was t e n t a t i v e l y assigned the "5a" c o n f i g u r a t i o n on the b a s i s of i t s n.m.r. spectrum, and an a n a l y t i c a l sample of N-acetyl-Sa,6-dihydroverarine (120) was prepared by sublimation 89 at 220° / 0.01 mm. as prisms m.pt. 249-250°. I n f r a r e d (KBr): 6.22 ft (N-CCH ), 12.23 y. U l t r a v i o l e t , X max ( l o g . e ) : 267 (2.69), 276 (2.68) u p . [ a ] D + 41° (c, 0.961). N.m.r. s i g n a l s : 9.06, 9.02 (a s i n g l e t and fl a doublet, 6H, C-19 and C-26 CH groups), 8.19 ( s i n g l e t , 3H, CH_3 C-N) ', 7.72 ( s i n g l e t , 3H, C-18 CH 3), 3.13 (doublet, J J 5 _ 1 6 = 8, lH^C-15 or C-16, H), 2.99 (doublet J 1 6 = 8, 1H, C-15 or C-16 H). Mass spectrum: M.W. 437; base peak m/e 140; main peaks m/e 437, 394, 297. Found: C. 79.32; H, 9.99. Calc d . f o r C^H^C^N: C, 79.58; H, 9.90. N-acetyl-3-keto-5a,6-dihydroverarine ( T i l ) A s o l u t i o n o f N-acetyl-5a,6-dihydroverarine (120 ^ 212 mg) i n acetone (75ml) was t r e a t e d w i t h Jones r e a g e n t ^ 0 . 3 ml) and allowed to stand at 20° f o r two minutes. Isopropanol (2 ml) was added to remove the excess oxidant and the mixture d i l u t e d w i t h water (600 ml). The aqueous acetone s o l u t i o n was e x t r a c t e d with methylene c h l o r i d e (3 x 30 ml) and the combined e x t r a c t s washed with water (3 x 30 ml) p r i o r t o drying over anhydrous sodium s u l f a t e . Evaporation o f the methylene c h l o r i d e gave a c o l o u r l e s s o i l (191 mg) which could not be c r y s t a l l i s e d but appeared homogeneous when examined by t h i n - l a y e r chromatography. An a n a l y t i c a l sample was obtained as a c l e a r g l a s s a f t e r s u b l i m a t i o n at 185° / 0.01 mm. fl I n f r a r e d (KBr): 5.89 (saturated c a r b o n y l ) , 6.14 (N-C-CH ) y. U l t r a v i o l e t , X max ( l o g e): 267 (2.73), 276 (2.71) my. [a] + 66° (c, 1.15). O.R.D. [•] P " f • 1090°, [*] * ™ u 8 h + 115°, [*] • 3910°. N.m.r. L 1 305 m/t ' L J 270 m,« ' L J 230 m/v s i g n a l s : 9.03 (doublet J 2 & 2 5 = 7.0, 3H, C-26 CH ), 8.88 ( s i n g l e t , 311, C-19 'CH ), 6.45 (doublet o f quartets J =7.5, J 2 0 _ 2 2 = 9.0, 1H, C-20 H). 3.13 (doublet J = 8, 111, C-15 H or C-16 H) , 2.95 (doublet" J 1 5 _ 1 6 = 8» 111, C-15 H or C-16 H) . Mass spectrum: M.W. 435; base peak m/e 140; main peaks m/e 98, 295, 392. Found: 435,312. Calcd. for c 29 I ,4i°2 N : 435.313. 4 N-Acetyl - A -3-keto-5,6-dihydroverarine (.*. 12) A solution of N-acetyl-3-keto-5aj5-dihydroverarine (162 mg, 0.372 m. mole) i n acetic acid (6 ml) containing hydrogen bromide (60 mg, 0.744 m. moles) was placed i n a dry round bottom flask. Bromine (130 mg 0.8 m. moles) i n acetic acid (5 ml) was added slowly and the mixture s t i r r e d at 20° for twenty minutes and then diluted with water (25 ml). The res u l t i n g heavy white precipitate was taken up i n methylene chloride and the extract washed with water (3 x 10 ml) p r i o r to drying over anhydrous sodium sulfate. Removal of the solvent in vacuo gave a colourless gum (225 mg) which was shown by thin-layer chromatography to consist of one major compound. This crude product was immediately 57 subjected to the conditions of Evans for introduction of the 4,5-doubl bond as outlined below. Bromine (0.26 ml) was added to acetone (7.5 ml) at 0° and the solution s t i r r e d u n t i l the colour disappeared. Sodium carbonate (0.7 g) was then added and the mixture, s t i r r e d for a further t h i r t y minutes, and then the mixture was f i l t e r e d d i r e c t l y into hot acetone containing sodium iodide (7.0 g). This solution was refluxed for f i f t e e n minutes during which time a precipitate of sodium bromide was formed. A portion (6 ml) of the supernatant of t h i s hot solution was a d d e d to a round bottom flask containing 2,4-dibromo ketone (220 mg) a n d the solution refluxed for two and a h a l f hours. During this time more sodium bromide precipitated, indicative of the exchange of the 2-bromine for an iodine atome. Oxalic acid (220 mg) was added as a fine 91 powder and the s o l u t i o n r e f l u x e d f o r a f u r t h e r hour before c o o l i n g . The r e a c t i o n mixture was d i l u t e d with e t h y l acetate (20 ml) and f i l t e r e d . This f i l t r a t e was washed with water (2 x 10 ml), sat u r a t e d aqueous sodium hydrogen carbonate (2 x 10 ml) and water (2 x 10 ml). The et h y l acetate was d e c o l o u r i s e d by adding z i n c dust (600 mg) and a c e t i c a c i d (0.2 ml) and then shaking. When the s o l u t i o n had a t t a i n e d a l i g h t yellow colour the z i n c dust was removed by f i l t r a t i o n and the e t h y l acetate f i l t r a t e washed w i t h water (2 x 10 ml), sodium hydrogen carbonate s o l u t i o n (2 x 10 ml) and water (2 x 10 ml) p r i o r to dr y i n g over anhydrous sodium s u l f a t e . Removal o f the e t h y l acetate i n vacuo gave a l i g h t y ellow gum (158 mg) which was examined by t h i n - l a y e r chromatography. The spot on the chromatoplate corresponding to the major product e x h i b i t e d an intense fluorescence under u l t r a v i o l e t l i g h t , and appeared purple when the p l a t e was sprayed with antimony pentachloride-carbon t e t r a c h l o r i d e reagent. P r e p a r a t i v e t h i n - l a y e r chromatography on s i l i c a g e l (20 x 60 cm, 0.4 mm developed twice w i t h 1% methanol i n chloroform) gave t h i s compound as a l i g h t gum (110 mg. 50% y i e l d ) which was not c r y s t a l l i s e d . An a n a l y t i c a l sample was prepared by s u b l i m a t i o n at 180° / 0.01 mm which gave a l i g h t y e llow g l a s s . I n f r a r e d (KBr): 6.03 (a,8-unsaturated c a r b o n y l ) , 6.14 (N- Ac) u. [cx] D + 74° (c, 1.26). N.m.r. s i g n a l s : 9.02 (doublet J 26-25 = 1 > 3H, C-26 CH ) , 3.72 ( s i n g l e t , 3H, C-19 CHJ , 8.18 ( s i n g l e t , 3H, CH_3 C-N) , 6.45 (doublet o f quarte t s J2Q = 7.5, J 2 0 _ 2 2 =-10, 1H, C-20 II), 4.22 ( s i n g l e t , 1H, C-4 H), 3.10 (doublet J 1 5 _ 1 6 = 8, 1H, C-15 or C-16 H), 2.94 (doublet J 1 5 = 8, 1H, C-15 or C-16 H). Mass spectrum: M.W. 433 • base peak, m/e 140; main peaks, m/e 433, 390, 293, 98. Found: 433.2974, Calcd. f o r C 2 9 H 3 9 0 2 N : 433.2980.. 92 N - a c e t y l v e r a r i n e (114) 4 A s o l u t i o n o f N-acetyl-A -3-keto-;5,6-dihydroverarine (100 mg) i n i s o p r o p e n y l acetate (3 ml) c o n t a i n i n g 0.5% s u l f u r i c a c i d was r e f l u x e d f o r one hour. Anhydrous sodium acetate (60 mg) was then added and most of the isopropenyl acetate removed by use of a r o t a r y evaporator. The residue was d i l u t e d w i t h methylene c h l o r i d e (10 ml) and then f i l t e r e d . T h i n - l a y e r chromatography o f the f i l t r a t e i n d i c a t e d the complete absence of (112) and the formation of a l e s s p o l a r compound. Evaporation o f the methylene, c h l o r i d e gave an o i l (100 mg) the i n f r a r e d spectrum of which e x h i b i t e d a carbonyl band at 5.75 ui i n d i c a t i v e of an enol acetate (113). This o i l was d i s s o l v e d i n a mixture of methanol (10 ml) and ether (3 ml) and heated to r e f l u x . During the r e f l u x i n g a methanolic s o l u t i o n o f sodium borohydride (150 mg i n 5 ml) was added s l o w l y over a p e r i o d o f f i f t e e n minutes. The s o l u t i o n was r e f l u x e d f o r a f u r t h e r one hour and then concentrated h y d r o c h l o r i c a c i d (1 ml) was added and the r e f l u x i n g continued w i t h s t i r r i n g f o r t h i r t y minutes. During t h i s f i n a l p e r i o d vigorous s t i r r i n g was necessary to overcome severe bumping. The r e a c t i o n mixture was cooled and then d i l u t e d w i t h ether (50 m l ) c The e t h e r e a l s o l u t i o n was washed with water and then d r i e d over anhydrous sodium s u l f a t e . Removal of the solvent gave a l i g h t o i l (70 mg) which was shown to co n t a i n a compound of i d e n t i c a l R^ value to that of N-acetyl v e r a r i n e . Separation by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.3 mm, 2% methanol i n chloroform) and e x t r a c t i o n of the band corresponding to t h i s product y i e l d e d an o i l (30 mg). This compound was shown to be i d e n t i c a l with N - a c e t y l v e r a r i n e by t h i n - l a y e r chromatography, superimposable i n f r a r e d and n.m.r. sp e c t r a . I n f r a r e d (CHC1,).:. 6.19 93 P (NC-CH3) y. U l t r a v i o l e t , X max ( l o g e): 268 (2.76), 277 (2.65) my. N.m.r. s i g n a l s : 9.03 (doublet 3^ 2J.' = 7, 311, C-26 CH ) , 8,87 ( s i n g l - t , 3H, C-19 CH ) , 8.18 ( s i n g l e t , 3H, CH C-N-), 4.56 ( m u l t i p l e t , IH, C-6 H), 3.12 (doublet J 1 5 _ 1 6 = 8, IH, C-15 H or C-16 H), 2.94 (doublet "^ 15 16 = 8* ^-15 " o r C-16 H) . Mass spectrum: M.W. 435; base peak m/e 140; main peaks m/e 98, 295. Verar i n e (3) A s o l u t i o n o f N - a c e t y l v e r a r i n e (20 mg) i n ethylene g l y c o l (5 ml) co n t a i n i n g 10% potassium hydroxide was r e f l u x e d f o r twelve hours. The r e a c t i o n mixture was cooled, d i l u t e d w i t h water (20 ml) and ext r a c t e d w i t h methylene c h l o r i d e ( 3 x 5 ml). Examination of the e x t r a c t by t h i n - l a y e r chromatography i n d i c a t e d the presence o f N - a c e t y l v e r a r i n e and a more p o l a r compound which had the same value and colour r e a c t i o n to the spray reagent, as an au t h e n t i c sample o f v e r a r i n e . The more p o l a r compound was separated by p r e p a r a t i v e t h i n - l a y e r chromatography (20 x 20 cm., 0.3 mm 5% methanol i n chloroform) as a l i g h t o i l (8 mg) which c r y s t a l l i s e d from ether. This compound was shown to be i d e n t i c a l w i t h v e r a r i n e by t h i n - l a y e r chromatography, i n f r a r e d and n.m.r. comparison. I n f r a r e d (CHCl^): 9.57 y. U l t r a v i o l e t , X max ( l o g e) : 267 (2.74) 276 (2.73) my. N.m.r. s i g n a l s : 9.15 (doublet J 2 5 = 6, 311, C-26 CH ) 8.88 ( s i n g l e t , C-19 CH^), 7.73 ( s i n g l e t 3H, C-18 CH ) , 4.55 (broad doublet, IH, C-6 Ii) , 3.02 ( s i n g l e t , 2H, C-15 II and C-16 H). Mass spectrum: M.W 393; base peak m/e 98, main peaks, m/e 392, 376,295, 284, 256. High r e s o l u t i o n on m/e (m+ 392. Found: 392.2922. Calcd. f o r Co,H__0 N: 392.2953. 94 BIBLIOGRAPHY L. L. F. F e i s e r and M. F e i s e r . " S t e r o i d s " , pp. 867-895, Reinhold P u b l i s h i n g Corp., New York, 1959. 2. H. G. B o i t . "Ergebnisse der Alkaloid-Chemie b i s I960" pp 798-832, Akademie-Verlag, B e r l i n 1961. 3. C. R. Narayanan i n "Progress i n the Chemistry of Organic Natural Products" (L. Zeichmeister ed.) 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