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Studies on total synthesis of veratrum alkaloids Brookes, Roderick William 1969

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STUDIES ON TOTAL SYNTHESIS OF VERATRUM ALKALOIDS BY RODERICK W. BROOKES B. Eng., Royal Military College of Canada, Kingston, Ontario, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1969 In presenting th i s thes i s in pa r t i a l f u l f i lment of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of <C/-/Er>7/J"ir/2 / The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date ABSTRACT The i n i t i a l work toward a synthetic entry into the hexacyclic cevane nucleus (7) i s described, and the s p e c i f i c application of th i s work to the t o t a l synthesis of ve r t i c i n e (12) i s discussed. Hecogenin acetate (74) was converted to C-nor-D-homo-(25 R)-5a-spirost-13(18)-en-33-ol-3-acetate (79) v i a a known procedure, and hydroboration of the double bond gave the corresponding 133-hydroxymethyl compound (80(a)). The mass spectral fragmentation of the spiroketal system i s discussed. The stereochemistry at C-13 of the hydroborated compound (80(a)) was reversed by oxidation of the primary alcohol to the aldehyde, epimerization, and reduction. Acetylation gave the diacetate (85) which was used i n investigation of the spiroketal side chain degradation. The Baeyer-Villiger oxidation sequence developed by W.F. Johns was used i n the spiroketal degradation. 13ct-Acetoxymethyl-17-acetyl-18-nor-5a-etiojerv-16(17)-en-33-ol-3-acetate (110) was obtained with considerable d i f f i c u l t y i n low y i e l d from t h i s sequence, A model compound, 17-acetyl-5ct, 13B-etiojerv-16(17)-en-33-ol (111), was employed to te s t the f e a s i b i l i t y of attachment of a heterocyclic portion v i a a reaction developed by Schreiber and Adam. Reduction of the double bond and epimerization of the methyl ketone gave the ketone (120), which was then condensed with 2-lithio-5-methylpyridine. The structure of the condensation product was established by i t s n.m.r. and mass spectra. - i i i -TABLE OF CONTENTS Page Title Page i Abstract i i Table of Contents i i i List of Figures iv Acknowledgements vi Introduction 1 Discussion 18 Experimental 56 Bibliography 76 - iv -LIST OF FIGURES Figure Page 1 Examples of some Veratrum Alkaloids 2 2 Johnson's Synthesis of Veratramine - Part 1 ... 6 3 Johnson's Synthesis of Veratramine - Part 2 ... 7 4 Masamune's Synthesis of Jervine 9 5 Kutney's Synthesis of Veratramine and Verarine . 11 6 Synthesis of the Exocyclic Olefin (79) 19 7 N.m.r. Spectrum of Compound 80(a) 22 8 Mass Spectral Fragmentation of the Spiroketal System 24 9 Mass Spectral Dehydration of the M-114 Intermediate 26 10 Mass Spectra of Compounds 80(a) and 84 27 11 N.m.r. Spectrum of Compound 82 32 12 N.m.r. Spectrum of Compound 83 33 13 Mass Spectra of Compounds 82 and 83 34 14 N.m.r. Spectrum of Compound 84 35 15 Classical Degradation of the Spiroketal System . 37 16 Alternate Degradation of the Spiroketal System . 38 17 Mechanism of the Baeyer-Villiger Oxidation of Diacetate (94) 41 18 Side Chain Degradation of Diacetate (85) 42 - V -LIST OF FIGURES (Con't) Figure Page 19 N.m.r. Spectrum of Compound 110 45 20 N.m.r. Spectrum of Compound 111 46 21 Coupling of the Heterocyclic Unit in the Verarine Synthesis 47 22 Mass Spectrum of Compound 110 48 23 Coupling of the Heterocyclic Unit in the Solanum Alkaloid Synthesis 49 24 Mass Spectrum of Compound 121 53 25 N.m.r. Spectrum of Compound 121 54 26 Proposed Conclusion of the Synthesis of Verticine 55 vi -ACKNOWLEDGEMENTS I am deeply indebted to Professor James P. Kutney for his excellent guidance and continual encouragement throughout the course of my research. I would also like to acknowledge the many helpful suggestions from my colleagues during this project. I am grateful to the National Research Council of Canada for the award of an N.R.C. Bursary during the course of my studies. Financial support of this project by Smith, Miller, and Patch, Inc., The National Research Council of Canada and The Medical Research Council of Canada is greatly appreciated. The receipt of gifts of hecogenin acetate from Smith, Miller, and Patch, Inc., and Syntex S.A., Mexico is acknowledged. INTRODUCTION The Veratrum alkaloids are a group of ste r o i d a l alkaloids which occur i n the plants of the t r i b e Veratreae. This t r i b e belongs to the subfamily Melanthioideae of the family L i l i a c e a e , and the genera which have been studied are Veratrum, Zygadenus, Stenanthium, Amianthium, Melanthium, and F r i t i l l a r i a . Fieser* has proposed the d i v i s i o n of the Veratrum alkaloids into Jerveratrum and Ceveratrum groups. The Jerveratrum alkaloids contain only one to three atoms of oxygen, and are found i n the unhydrolysed plant extracts, either as free amines,, or as D-glucosides. Rubijervine (1), jervine (2), veratramine (3), and v e r t i c i n e (4) are cit e d as examples of the Jerveratrum a l k a l o i d s . Rubijervine i s one of the simpler Veratrum a l k a l o i d s , and possesses a normal st e r o i d skeleton. The l a t t e r three alkaloids possess the modified C-nor-D-homo st e r o i d skeleton. Tomko and Schreiber have recently i s o l a t e d two unusual Jerveratrum 2 3 al k a l o i d s , veralkamine (5) and veramine (6). They possess the 176-methyl-18-nor-l7-isocholestane skeleton, and closely resemble the spiroaminoketal alkaloids solanidine and tomatidine. The Ceveratrum alkaloids are highly oxygenated, usually containing seven to nine oxygen atoms, and are often heptacyclic. They frequently occur as esters of substituted benzoic, a c e t i c , and short chain a l i p h a t i c - 2 -Figure 1 : Examples of some Veratrum Alkaloids acids, but never occur as glycosides. They a l l contain the cevane nucleus (7), characterized by the C-nor-D-homo steroid skeleton and folding of the side chain around the nitrogen atom. Veracevine (8), protoverine A (9), and sabine (10) are cited as examples of the more common Ceveratrum alkaloids. The nomenclature of these alkaloids and their parent hydrocarbons has undergone a number of changes as new products were characterized and new families were recognized. The basic numbering for a normal C-27 steroid skeleton is as indicated for cholesterol (11). The numbering for - 4 -a C-nor-D-homo steroid skeleton is as indicated for verticine (12). 4 Fried and Klingsberg proposed that the term "jervane" be adopted to represent the carbon skeleton of jervine (2), They also proposed the term "etiojervane" to represent the parent tetracyclic hydrocarbon (13) whose systematic steroid nomenclature is 173-methyl-C-nor-D-homo-18-nor-5a,12a-androstane. The most recent review of nomenclature*' proposes the name (25 S)-5B-spirostan for the parent spiroketal skeleton (14). In both cases the stereochemistry as given in the diagrams is implied by the name, unless the name is otherwise modified. In this work, the spirostan nomenclature will be used for compounds containing an intact spiroketal side chain, and the etiojervane nomenclature will be used for the remaining structures. If a common name exists for a compound, the systematic nomenclature will be given once, but the common name will be used throughout for the sake of brevity. Much of the interest in the veratrum alkaloids has stemmed from their value for medicinal purposes. Crude extracts from Veratrum and related plants have been used in the treatment of fevers, as local counterirritants ft 7 in neuralgia, as cardiac toxins, and as insecticides » . The Jerveratrum - 5 -alkaloids show very l i t t l e hypotensive effect; the compounds mainly responsible for this effect are the esters of the Ceveratrum alkaloids. The first reported use of Veratrum in the control of hypertension dates g from the report of Baker in 1859. In the late 1930's, the first pure crystalline alkaloid, protoveratrine, was obtained, and was shown to be a 9 10 powerful hypotensive agent ' . After pharmacological investigation by Krayer 1 1, this compound was used in the treatment of certain types of hypertension. The limiting factor in the use of this drug is the narrow dosage range between hypotensive and emetic effects, and this prompted 12 Kupchan to examine a number of protoveratrine derivatives . He was able to make a number of generalizations concerning structure-activity relation-13 16 ships for the protoveratrines " , but l i t t l e improvement was made in widening the therapeutic dosage range. The use of the superior hypotensive compound reserpine has almost eliminated the use of the protoveratrines. 17 Kupchan has reviewed the occurrence of alkaloids in plants of the Veratreae, and the implication of alkaloid occurrence and structure to the taxonomy of the Veratreae. He has also summarized the relationships between structure and hypotensive activity of these alkaloids and some synthetic 18 derivatives Reviews on various aspects of the chemistry of Veratrum alkaloids 19 20 21 have been published by Fieser and Fieser , Boit , Narayanan , and 22 Kupchan . For the past decade, several groups have been working on the synthesis 23 of various members of the Veratrum alkaloids. W.S.. Johnson has 24 succeeded in synthesizing veratramine, while Masamune has synthesized Figure 2 : Johnson's synthesis of Veratramine - Part 1 - 7 -Figure 3 : Johnson's synthesis of Veratramine - Part 2 j c r v i n e . The W.S. Johnson group f i r s t synthesized the relay compound (25) from Hagemann's ester (15) i n a series of steps outlined i n f i g . 2. This 25 compound was also obtained by degradation of veratramine . This relay compound was converted i n a short series of steps to a second relay compound (28), and then compared to a t o t a l l y synthetic product derived from an extension of e a r l i e r work involving the hydrochrysene approach . Af t e r conversion of the methyl ketone (28) to the aldehyde (29) v i a the oxirane, the remaining nitrogen atom and carbon atoms were added v i a a Strecker reaction with L-t-butyl-3-methyl-4-aminobutyrate and potassium cyanide. Benzoylation gave the cyano ester (30).. Cyclization to the piperidine ring was accomplished by treatment of 30 with excess methyl-sulfinylcarbanion i n dimethylsulfoxide, and treatment with hydrogen chloride i n acetic acid gave the ketone (32). Saponification and oxidation gave the diketone (33) i n 6.1 % o v e r a l l y i e l d from the aldehyde (29), Reduction, benzoylation, and p a r t i a l hydrolysis afforded a dibenzoate with a hydroxyl at C-3. The corresponding 3-keto compound was converted 27 28 v i a established procedures ' into the 3B-hydroxy-5,6-dehydro der i v a t i v e , and hydrolysis of the two benzoate esters produced a sample of veratramine (34) i d e n t i c a l with the natural product. 24 Masamune's approach ( f i g . 4) was to prepare 17-acetyl-5a-etio-jerva-12, 14, 16-triene-3B-ol-3-acetate (35) using a known degradation 29 s t a r t i n g from hecogenin acetate (36) . This compound was then converted to the corresponding bromo derivative (37) v i a the alcohol, and was then trea-ted with the p y r r o l i d i n e enamine of L-acetyl-3(S)-methyl-5-piperidone to y i e l d - 9 -d i r e c t l y an isomeric mixture of 3,N-diacetyl-5a,6-dihydro-23-dehydro-veratramines (38, 39). Compound 39, and the reduced compound (40) lead 30 31 32 d i r e c t l y to veratramine , 11-deoxojervine and verarine , The remainder of the synthesis involved the conversion of the intermediate (39) into the c y c l i c ether (43). A l l y l i c oxidation of the o l e f i n (44) gave i n poor y i e l d (1 %) the a,B-unsaturated ketone (45). Introduction of the 5,6-double bond proceeded, also i n poor y i e l d (2 % ) , to give a sample of jervine i d e n t i c a l to the natural material. Figure 4 : Masamune's synthesis of Jervine Figure 4'(continued) : Masamuners synthesis of Jervine - 11 -For some years now, our research group has been engaged i n the 33-35 development of a general synthetic route into the Veratrum alkaloids . Part of this work was recently concluded with the t o t a l synthesis of 36 verarine , and a further extension of this work to veratramine i s now 37 complete and i n p r i n t . In this approach (figure 5), 3-napthol (49) was converted to the methoxy ketone (50) which was then subjected to two Robinson annelation reactions. The f i r s t of these was with the Mannich base of ethyl v i n y l ketone, and the second with methyl v i n y l ketone, to produce the bridged ketol (53). On treatment with strong base, this compound rearranged to one of the important intermediates, the t e t r a c y c l i c ketone (54). Functionality was introduced into the C-ring by oxidation of the acetate (55) to form the keto-acetate (56). The C-nor-D-homo ste r o i d skeleton was then constructed v i a a series of steps leading to the dialdehyde (57) followed by an i n t e r n a l aldol condensation to y i e l d the hydroxy-aldehyde (58). Treatment with sodium acetate i n refluxing acetic acid gave the o l e f i n (59). A series of steps were required to construct the enone (60), which could then be alkylated with methyl iodide to give the relay compound (61). This relay compound was compared to an authentic sample which can be 38 39 obtained from degradation of hecogenin » . The remainder of the sequence was done with the o p t i c a l l y active material obtained from hecogenin degradation. The 12,13-double bond was reintroduced, and the heterocyclic portion attached v i a the attack of the anion of 2-ethyl-5-methylpyridine on the D-ring ketone of compound 62. The D-ring was aromatized by heating - 1 2 -compound 63 with 10 % palladium on charcoal, and the pyridine ring s e l e c t i v e l y reduced to y i e l d , a fter acetylation, 3,N-diacetyl-5a,6-dihydroverarine (64). The 5,6-double bond was then introduced by a known method^ to y i e l d verarine, i d e n t i c a l to the naturally occurring material, 37 Quite recently , the synthesis of veratramine was completed v i a the condensation of 2-ethyl-5-methyl-6-methoxypyridine with compound 62. Figure 5 : Kutney's synthesis of Veratramine and Verarine - 1 3 -Figure 5 (continued) : Kutney's synthesis of Veratramine and Verarine - 14 -Since this thesis i s concerned with developing a synthetic entry into the hexacyclic v e r t i c i n e skeleton, i t i s appropriate here to b r i e f l y discuss the work which has been done to date on v e r t i c i n e . Verticine was f i r s t i s o l a t e d by Fukuda 4* from F r i t i l l a r i a v e r t i c i l l a t a W i l l d . var thunbergii Baker. Ve r t i c i n e , and the corresponding 6-keto 42 compound, verticinone, were also i s o l a t e d by Chou and Chen from bulbs of F. r o y l e i (Liliaceae) after i t was noted that the pharmacological effects of the plant extracts resembled those of the alkaloids of 43 Veratrum- . Several groups of workers subsequently isolated these a l k a l o i d s 4 4 " 4 7 . Chou and Chen 4 2 showed that v e r t i c i n e and verticinone were interconvertable v i a the Beckmann oxidation method and sodium/alcohol reduction. They also showed the presence of two e a s i l y acylable hydroxyl 48 49 groups i n v e r t i c i n e . Dehydrogenation studies by Qui, Hwang, and Loh » showed that dehydrogenation of v e r t i c i n e gave l u t i d i n e (65) and two hydrocarbons, compound 66 and an unknown. The unknown was shown to be i d e n t i c a l to a dehydrogenation product of j e r v i n e 5 0 . At the same time, extensive experiments were done on the dehydrogenation of imperialine, 51 52 an a l k a l o i d of F. imperial i s * . From the product mixture, 2,5-lutidine (65) and the hydrocarbon (66), as well as the base veranthridine (67) were is o l a t e d . Veranthridine had also been is o l a t e d from the dehydrogenation products of cevine, an a l k a l o i d of Veratrum having the modified steroid skeleton (68). This indicated that imperialine probably also had t h i s skeleton, and i t was presumed that v e r t i c i n e and verticinone had a s i m i l a r s k e l e t a l structure. Morimoto and Kimata isolated verticine and i t s C-3 B-D-glycoside from F. thunbergii MIQ. They assumed that verticine had the fundamental skeleton (68), and therefore formulated the new glycoside as 69. 69 In a later paper , the same authors showed that, in verticine, there were no ketonic functions or double bonds, that the nitrogen was tertiary, and that there was one tertiary hydroxyl. The two secondary hydroxyls were oxidized, and the infrared indicated that both corresponding ketones were in six membered rings, Ito and co-workers"'"' also worked on the structure of verticine, correctly placing one secondary hydroxyl at C-3, and incorrectly placing the other at C-7. In a more recent paper 5^, they correctly analysed the - 16 -structure as given i n f i g . 1 (4). The 8 configuration of the C-3 hydroxyl was supported by ORD and i n f r a r e d measurements. The other secondary hydroxyl was placed at C-6, since v e r t i c i n d i o n e (70) was observed to undergo a e r i a l oxidation i n a l k a l i n e medium to form a 4-ene-3,6-dione system (71), A x = 375 my. The t e r t i a r y hydroxyl group was placed next to a methyl ^roup ( e i t h e r C-20 or C-25) since only one methyl group i n v e r t i c i n e appeared as a doublet i n the n.m.r. spectrum. I t wa? placed at C-20 by pK measurements and mass spectrometry, and was shown to be a x i a l by b a s i c i t y measurements and by i t s i n f r a r e d absorption. The stereochemistry of the C-27 methyl group was concluded to be a x i a l from chemical s h i f t s and coupling constants i n the n.m.r. spectrum. Stereochemistry at carbons 8, 9, 12, 14, 16, and 17 was assigned only by analogy with the s t e r o i d s e r i e s and from biogenetic considerations. The correctness of t h i s proposed structure 57 was very recently confirmed by an X-ray analysis While the structures of numerous a l k a l o i d s containing the cevane nucleus have now been completely e l u c i d a t e d , to date there have been no attempts to synthesize t h i s skeleton. I would l i k e to present i n the following - 1 7 -discussion our i n i t i a l e f f o r t s towards a synthetic entry into the cevane nucleus, and, more s p e c i f i c a l l y , towards the synthesis of v e r t i c i n e . DISCUSSION The successful synthetic approach employed i n our laboratory i n the synthesis of both veratramine and verarine has been to consider the veratrum skeleton as consisting of an etiojervane portion coupled to an appropriately substituted piperidine. An examination of the structure of verarine (72) emphasizes i t s marked s i m i l a r i t y to v e r t i c i n e (73), the most notable difference being the lack of a C-18 to nitrogen bond i n verarine. I t was thus clear that i f t h i s general synthetic approach were to be extended to v e r t i c i n e , the etiojervane portion would have to contain some type of C-18 f u n c t i o n a l i t y i n order to complete construction of the E r i n g . The f i r s t objective of this synthesis was thus to obtain an etiojervane system with a useful f u n c t i o n a l i t y at C-18. - 19 A perusal of the l i t e r a t u r e revealed that W.F. Johns had employed a modified procedure by which hecogenin acetate (74) was converted to an etiojervane system i n which C-18 was present as an exocyclic methylene (79). The d e t a i l s of this procedure are given i n f i g , 6, Figure 6 : Synthesis of the Exocyclic Olefin (79) - 20 -The i n i t i a l work on this sequence was done by Hirschmann et. a l . They observed that by forming the 12B-mesylate of rockogenin (77, R = succinate), t h i s s t e r o i d a l sapogenin could be induced to undergo s o l v o l y t i c rearrangement. The 13-14 bond was observed to s h i f t to the 12-14 p o s i t i o n r e s u l t i n g i n C-ring contraction and D-ring expansion. Loss of a proton from the C-18 methyl group gave an exocyclic methylene at C-13. I t was also noted by these workers that s o l v o l y s i s proceeded some 280 times more slowly with the 12a-mesylate. 58 W.F. Johns found that, of a number of reductive methods attempted , Birch reduction gave the lowest y i e l d (2-3 %) of the undesired 12a-hydroxyl isomer. He further modified the Hirschmann procedure by selective formation of the C-3 monopivalate of rockogenin (76). Mesylation, s o l v o l y s i s with accompanying rearrangement, and acetylation gave the o l e f i n (79) i n some 45 % o v e r a l l y i e l d from hecogenin acetate. This procedure was 58 carried out i n our laboratory following the procedure of Johns i n order to obtain a quantity of the exocyclic o l e f i n (79) f o r further elaboration. In order to obtain a useful etiojervane intermediate f o r elaboration to v e r t i c i n e , the spiroketal system present i n the exocyclic o l e f i n (79) would have to be degraded. The two methods available f o r t h i s degradation » involve quite d r a s t i c conditions, therefore i t was f e l t that some alt e r a t i o n of the exocyclic double bond would be necessary i n order to preserve the f u n c t i o n a l i t y at C-18. Hydroboration of the o l e f i n (79) followed by acetylation of the r e s u l t i n g primary alcohol was f i r s t considered as a method of introducing a reasonably stable functional group at C-18. I t was found, however, that - 21 th i s procedure had been attempted by Huffman et. a l . on the C-3-tetrahydropyranyl ether of t h i s p a r t i c u l a r o l e f i n , and negative re s u l t s were reported. Although the successful hydroboration of a sapogenin has 62 been reported , the above workers were only able to obtain products i n which the spiroketal side chain had apparently been reduced. Since i t was not immediately apparent why this hydroboration should f a i l , the exocyclic o l e f i n (79) was subjected to hydroboration conditions. 63 No reaction at a l l could be observed when externally generated diborane was passed into a tetrahydrofuran (THF) solution of the o l e f i n (79). 64 However, when diborane was generated i n s i t u , a reaction was observed which was complete i n three hours at room temperature. F i r s t attempts at th i s reaction gave complex mixtures of products, but i t was found that by adding a two mole excess of commercially available diborane i n THF to a THF solution of the o l e f i n (79) and s t i r r i n g at room temperature under nitrogen f o r three hours, a reasonably clean reaction product was formed. Decomposition of the borate ester with hydrogen peroxide and base yielded a product which by thin layer chromatography ( t . l . c . ) appeared to be predominantly one quite polar compound. Alumina chromatography and r e c r y s t a l l i z a t i o n provided the necessary p u r i f i c a t i o n . A good ind i c a t i o n that hydroboration had occurred as expected was obtained from the nuclear magnetic resonance (n.m.r.) spectrum of the product (see f i g . 7). I t showed a 2-proton doublet at T 6.42 (CH9OH), and no .signal at T 5.22 where the v i n y l protons of the exocyclic o l e f i n (79) had absorbed. In addition, the acetate signal at T 8.01 and the a l l y l i c proton resonances at T 7.52 i n the n.m.r. spectrum of the s t a r t i n g o l e f i n (79) - 23 -were absent i n the n.m.r. spectrum of the product. The mass spectrum of the compound showed a parent peak at m/e 432, while the infrared spectrum showed a hydroxyl absorption, but no carbonyl absorption. On the basis of this data, i t was concluded that the double bond had undergone hydroboration to give a primary alcohol. The only remaining question as to the gross structure of the product was whether or not the spiroketal system was s t i l l i n t a c t . Jones et. a l . 6 5 have carried out a study of the infrared spectra of s t e r o i d a l sapogenins, and have found that the region between 875 and 1350 cm"* contains some 18 absorption bands associated with the spiroketal system. They also found that introduction of oxygen atoms into the s t e r o i d a l sapogenin skeleton diminishes the i n t e n s i t i e s of these bands, while rupture of the spiroketal system eliminates many of them. Examination of the infrared spectrum of the exocyclic o l e f i n (79) revealed the presence of 19 absorption bands between 875 and 1350 cm x. Many of these were, however, notably d i f f e r e n t from those given by Jones, but t h i s i s presumably due to the modified steroid skeleton. Comparison of the infrared spectrum of 79 with the one f o r the hydroboration product showed that most of the bands were s t i l l present i n roughly the same i n t e n s i t i e s , but many were s h i f t e d by up to 20 cm"*. Although i t appeared from t h i s spectrum that the spiroketal system was s t i l l i n t a c t , no d e f i n i t e conclusion could be drawn. The fact that the spiroketal side chain had not ruptured was shown conclusively by a detailed examination of the mass spectrum. D j e r a s s i ^ has summarized the fragmentation of the spiroketal system, and his results - 24 -are given in f i g . 8. Two important fragments occur in the low mass range, a prominent one at m/e 139 (B), and a lower intensity fragment at m/e 115 (A). The high mass range, however, is much more useful in that six fragments in this range are characteristic of spiroketal systems. The fragments resulting in peaks at M-59, M-69, M-72, M-114, M-129, and M-143 are also summarized in f i g . 8. B, m/e 139 Figure 8 : Mass Spectral Fragmentation of the Spiroketal System Figure 8 (continued) : Mass Spectral Fragmentation of the Spiroketal System -26 -In the mass spectrum of the hydroboration product, the base peak was the molecular ion, m/e 432 (see fig.10). There were prominant peaks at m/e 115 and m/e 139, as well as at M-59, M-69, and M-72. Peaks at M-114 and M-129 were notably absent, but peaks at M-132, M-143, and M-161 were present. The absence of fragments at M-114 and M-129, and the presence of a prominent fragment at M-132 can be explained by assuming that the M-114 fragment undergoes rapid dehydration (see f i g . 9) leading to the M-132 peak, and that loss of a methyl radical (see f i g . 8) is a much less favoured process from this M-114 intermediate. M-114 M-132 Figure 9 : Mass Spectral Dehydration of the M-114 Intermediate The peak at M-161 can also be explained by dehydration, in this case, of the M-143 fragment. The only remaining question with regard to the structure of the hydroboration product was the stereochemistry at C-13. Examination of the literature revealed that a number of reactions had been done on the exocyclic olefin (79), and most of these reactions occurred from the a face of the molecule to give a C-188-carbon atom. For example, 5 8 catalytic hydrogenation of the olefin gave the C-133-methyl compound. R E L A T I V E I N T E N S I T Y R E L A T I V E I N T E N S I T Y - 2 8 -Coxon et. a l . showed that treatment of this molecule with osmic acid gave the 13ct,18-diol, attack having again come from the a face. 6 8 Epoxidation gave a mixture of the a and 3 epoxides i n the r a t i o of 1 to 3 . On the basis of this data, i t was concluded that the C-13-hydroxy-methyl group was 3 (see 8 0(a)), and hence the stereochemistry was i n -correct f o r elaboration to v e r t i c i n e . In order to f i n a l l y prove or disprove t h i s stereochemical assignment, i t was decided to t r y to prepare 67 the C-13-aldehyde (81) described by Coxon I f our stereochemical assignment was correct, then oxidation under non-epimerizing conditions would produce the isomeric C-133-aldehyde. Examination of models indicated that this aldehyde should be e a s i l y epimerized by base to produce the C-13o(pseudo-equatorial)-aldehyde. I f the assignment was wrong, then the C-13a-aldehyde should be produced d i r e c t l y on oxidation. In the hydroboration reaction with the exocyclic o l e f i n (79), i t was found that i f a small amount of base instead of a molar amount was used i n the decomposition of the a l k y l borane intermediates, an alcohol could - 29 -be iso l a t e d i n poor y i e l d with the C-3-acetate s t i l l i ntact (80(b)). The structure of th i s acetate was confirmed by i t s n.m.r. and mass spectra, and conversion v i a base hydrolysis to the d i o l (80(a)). When this acetate (80(b)) was subjected to oxidation by CrO^ i n acetic acid, a very complex mixture of products was obtained, including a substantial amount of the compound bearing a C-13 carboxylic acid 69 function. Moffat oxidation of t h i s acetate, however, gave predominantly one compound which could be p u r i f i e d by chromatography and r e c r y s t a l l i z a -t i o n . The n.m.r. spectrum of this compound showed an aldehydic proton as a poorly resolved doublet at T 0.58. The n.m.r. signals reported by Coxon for the C-13a-aldehyde (81) were a doublet at T 0.21 for the aldehydic proton and a doublet f o r the C-13 proton at x 7.32. We reasoned, therefore, that we had the epimeric C-13B-aldehyde, and thus proceeded to t r y to epimerize the compound. Repeated attempts, however, even with KOH i n refl u x i n g methanol, gave back only s t a r t i n g material. We were unable to explain t h i s r e s u l t , since a l l of the spectral data indicated that we had the correct gross structure f o r the compound. Due to the d i f f i c u l t y i n obtaining the hydroboration product as the C-3-acetate, and due to the s l i g h t i n s t a b i l i t y of the corresponding aldehyde, i t was decided to investigate the oxidation of the d i o l (80(a)). When th i s d i o l was subjected to Moffat oxidation, a compound could be isolat e d i n high y i e l d which, a f t e r p u r i f i c a t i o n by alumina chromatography, showed i n the n.m.r. spectrum the presence of an aldehydic proton at x 0.58 . Repeated attempts to epimerize t h i s compound also f a i l e d . However, l a t e r examination of the n.m.r. spectrum - 30 -of one column f r a c t i o n of this compound revealed a small doublet at T 0.22, as well as a large signal at T 0.58 . This prompted us to examine the aldehydic region of the n.m.r. spectrum of the crude Moffat oxidation product, and we were surprized to f i n d a large doublet at T 0.22, and np_ signal at x 0.58 . I t was also found that by treating this new aldehyde with K2CO3 i n methanol at room temperature, the doublet at x 0.22 disappeared, and the signal at T 0.58 appeared. I t was thus concluded that Coxon's n.m.r. assignments were wrong, that the C-133-aldehyde (82) i s formed on oxidation of the d i o l (80(a)), and that t h i s new aldehyde w i l l completely epimerize on contact with either alumina or weak base. The aldehyde (82) was p u r i f i e d by repeated r e c r y s t a l l i z a t i o n s , while the aldehyde (83) was p u r i f i e d by chromatography and r e c r y s t a l l i z a t i o n . - 31 -Very pure samples could not be obtained, since both aldehydes tended to decompose s l i g h t l y on r e c r y s t a l l i z a t i o n . The n.m.r. spectra of these isomeric aldehydes are given i n f i g . 11 67 and f i g . 12. I t should be noted that the so-called "doublet" i n the n.m.r. spectrum of aldehyde (82) at T 7.32 i s i n fact a doublet of t r i p l e t s with J values of 5.5 and 6.0 Hz. This i s consistent with our proposed structure (82), but i s not consistent with Coxon's proposed structure. The dihederal angles between the proton at C-13 and those at both C-12 and C-17 are nearly 60°. The t r i p l e t due to the C-13 proton, which arises due to equal coupling with the two adjacent protons at C-12 and C-17, i s further s p l i t by the aldehydic proton. The resonance of t h i s C-13 proton i s not v i s i b l e i n the n.m.r. spectrum of the isomeric aldehyde (83), presumably due to an u p f i e l d s h i f t caused by shielding from the D-ring and from the a x i a l proton at C-14. The infrared spectra of these two aldehydes were very s i m i l a r , and indicated that the spiroketal system was s t i l l i n t a c t . The mass spectra (see f i g . 13) were almost i d e n t i c a l , showing parent peaks at m/e 428, as well as fragments at m/e 115, 139, 285(M-143), 356(M-72), 359(M-69), and 369(M-59). Since the diacetate (85) was the desired compound for elaboration to v e r t i c i n e , the keto-aldehyde (83) was reduced with sodium borohydride. The r e s u l t i n g d i o l (84) was e a s i l y p u r i f i e d , and i t s n.m.r. spectrum (see f i g . 14), when compared to that of the o r i g i n a l d i o l (80(a))(see f i g ' ?) » showed that the doublet at T 6.42 due to the C-18 protons had sh i f t e d to x 6.34 as a poorly resolved t r i p l e t . The C-21 methyl doublet Figure 11 : N.m.r. Spectrum of Compound 82 Figure 12 : N.m.r. Spectrum of Compound 83 R E L A T I V E I N T E N S I T Y R E L A T I V E I N T E N S I T Y 4 5 6 7 8 9 10 T Figure 14 : N.m.r. Spectrum of Compound 84 - 36 -CHO 0 HO 83 AcO had moved from T 9.00 to T 8.97, while the remaining signals of the spectrum were almost the same. The mass spectra of the isomeric diols (see f i g . 10) showed the same major fragments, the only difference being the relative intensities of these fragments. The corresponding diacetate derivative (85) was characterized by i t s n.m.r, and mass spectra. It i s interesting to note that in the mass spectrum, a significant fragment occurs at M-114, and a large fragment at M-174. The M-174 fragment arises from deacetylation of the M-114 intermediate, in an analogous manner to dehydration of the diol (80(a)) leading to an M-132 peak. With the characterization of the diacetate, our f i r s t objective had been accomplished, namely the synthesis of a C-nor-D-homo steroid skeleton with an acetoxymethyl functionality of the correct stereochemistry at C-13. The next problem was to develop a scheme to degrade the - 37 -spiroketal side chain while at the same time preserving the f u n c t i o n a l i t y at C-13. 70 The c l a s s i c a l method for degradation of a spiroketal system i s outlined i n f i g . 15 . The F-ring of the sapogenin (86) i s opened with CH* l J = a i 2 m 2 a d - m 2 - o - c { Q i 2 } , C H 6 3 Figure 15 : C l a s s i c a l Degradation of the Spiroketal System octanoic anhydride, and the re s u l t i n g o l e f i n (87) i s then treated with CrOj to produce the keto-ester (88). Base catalyzed elimination of the side chain gives the a,3-unsaturated ketone (89). However, i n a recent 71 paper , Johns stated that "....the c l a s s i c a l pseudoisomerization f a i l e d i n the 18 substituted C-nor-D-homo sapogenins.", but he did not elaborate further. In this paper, he presented an alternate approach to th i s degradation v i a a Baeyer-Villiger oxidation. The d e t a i l s of this sequence are given i n f i g . 16. Treatment of the sapogenin (90) with performic acid i n formic acid gives the diformate (91). Adsorption of this - 38 -89 Figure 16 : Alternate Degradation of the Spiroketal System diformate on alumina re s u l t s i n p r e f e r e n t i a l hydrolysis of the formate esters to give the d i o l (92). Jones oxidation of the l a t t e r followed by base catalyzed elimination of the side chain gives the a,g-unsaturated ketone (89), the ove r a l l y i e l d comparing favourably with that from the c l a s s i c a l sequence. Since Johns reported negative results with the c l a s s i c a l degradation procedure, the Baeyer-Villiger oxidation procedure was attempted with the diacetate (85). When th i s diacetate was treated with performic acid i n aqueous formic acid at 70-80°, extensive decomposition took place. Numerous attempts with t h i s procedure eventually revealed that the best - 3 9 -reaction conditions involved reaction of the diacetate ( 8 5 ) with aqueous performic acid at 4 0 ° f o r f i v e hours. Even with these conditions, some decomposition occurred, but i t was found that lower temperatures or shorter reaction times resulted i n recovery of a large amount of st a r t i n g material. Two major products were v i s i b l e by t . l . c , along with numerous minor products. The least polar of the two major compounds was isolated with some d i f f i c u l t y by s i l i c a gel chromatography as an impure, non-cryst-a l l i n e gum. The n.m.r. spectrum of t h i s material revealed the presence of two formate protons, one at T 2 . 0 8 , and the other at T 1 . 9 8 . I t also showed the presence of two acetate groups, but the remainder of the spectrum was of l i t t l e value. Since t h i s gumny material was impossible to p u r i f y , i t was d i f f i c u l t to obtain useful spectral data. The infrared spectrum yielded no useful information except to show that no hydroxyl groups were present. The mass spectrum showed no parent peak; the highest v i s i b l e peak was at m/e 5 5 8 . Although the structure of the diformate was uncertain at this point due to d i f f i c u l t y i n p u r i f i c a t i o n and poor spectral data, selective hydrolysis of the formate esters was attempted. Adsorption of the diformate on alumina of various pH's resulted i n quite complex mixtures. Repeated attempts at t h i s hydrolysis revealed the best procedure to be a short treatment with aqueous ^CO-j i n methanol, followed by immediate extraction with chloroform. Three major products were v i s i b l e by t . l . c . from t h i s reaction, a l l three materials being much more polar than the diformate. From work on a previous sequence using the C - 1 3 B-acetoxymethyl compound ( 9 4 ) , i t was found that three major products were formed on Baeyer-Villiger oxidation and selective hydrolysis. These three products - 40 -were the two expected Baeyer-Villiger products (101, 102) plus the compound i n which the ring-opened side chain had been hydrolysed (103). The proposed mechanistic pathway leading to these compounds i s given i n f i g . 17. These three products could be separated by s i l i c a gel chromatography and f r a c t i o n a l c r y s t a l l i z a t i o n . The hydrolysed compound (103) was quickly i d e n t i f i e d by i t s n.m.r. and mass spectra, but the isomeric d i o l s (101 and 102) gave almost i d e n t i c a l spectral data. The structures of these d i o l s were f i n a l l y established by subjecting both compounds (101 and 102) to Jones oxidation. Examination of the n.m.r. spectra of the two r e s u l t i n g keto-acids revealed that one of them had a methyl ketone resonance, while the other did not. * The d i o l (101) was now compared by t . l . c . with the product mixture from the hydrolysis of the formate esters i n the C-13ct-acetoxymethyl series. I t was found that one of the three components had a roughly comparable R£ value. This material was i s o l a t e d by chromatography as an impure gum which could not be induced to c r y s t a l l i z e from any of a number of solvent systems. An n.m.r. spectrum of the crude material showed the presence of two acetate groups, three methyl groups, a 2-proton doublet at T 6.58 f o r the C-26 protons, and no formate protons. The infrared spectrum showed the presence of hydroxyl groups, but the mass spectrum showed no parent peak, only a large fragment at M-18. Jones oxidation of t h i s material confirmed that i t was the correct Baeyer-Villiger product. Hie n.m.r. spectrum of the oxidized product showed a methyl ketone resonance at T 7.85, while the infrared spectrum of the material confirmed the presence of an a c i d i c function. AcOQl AcO AcOCH CH, R = CH2CH2CH-CH2OaiO AcOCH2 96 Acoau : r 0CH0 98 ACOCH UiO O-C-R II 0 99 AcOCH A c o a u 101 OH C-R' li 0 CH, R' = CH 2ffl 2ai-CH 20H AcOCH O-C-R' II 0 102 AcOCH II 103 Figure 17 : Mechanism of the Baeyer-Villiger Oxidation of Diacetate (94) - 42 -I t was thus assumed that the sequence 85 to 106 had been completed, and therefore, the f i n a l reaction i n the degradation was elimination of the side chain at C-16. Figure 18 : Side Chain Degradation of Diacetate (85) 72 During work on the isomeric C-133-acetoxymethyl sequence , the acid r e s u l t i n g from Jones oxidation of the d i o l (101) was e s t e r i f i e d with diazomethane for purposes of complete characterization. Attempted p u r i f i c a t i o n of this methyl ester by alumina chromatography, however, resulted i n elimination of the C-16 side chain to form the ct,8-unsaturated ketone (108) i n almost quantitative y i e l d . Since the base catalyzed side chain elimination i s only reported - 43 AcOClI AcOCH 0 0 o-c-ai-auai-cooMe AcO' AcO' 58,71 to go i n some 50 % y i e l d ' , i t was decided to attempt the elimination v i a contact with alumina i n the C-13a-acetoxymethyl series. Accordingly, the keto-acid (106) was e s t e r i f i e d with diazomethane, and passed through an alumina column. Only about 10 % conversion to the corresponding a,B-unsaturated ketone (110) was accomplished, however, and prolonged contact with alumina did not improve t h i s y i e l d . The difference i n r e a c t i v i t y on alumina of the two keto-esters can be e a s i l y explained from an examination of t h e i r molecular models. I t i s assumed that the f i r s t step i n the elimination reaction i s enolate anion formation to give an aluminate ester. Molecular models cl e a r l y show that when the C-13 acetoxymethyl group i s B(pseudo-axial), there i s no s t e r i c hindrance to enolate formation, and the reaction proceeds to completion. When the acetoxymethyl group i s a(pseudo-equatorial), Tl 3) 77 however, enolate formation results i n a large A v » ' s t r a i n being introduced between the C-21 methyl group and C-18. Thus, enolate formation i s much more d i f f i c u l t i n t h i s case, and the reaction proceeds only p a r t i a l l y to completion. The side chain was thus removed by refl u x i n g the keto-acid (106) in base. Work with the isomeric sequence had shown that the a,B-unsaturated ketone (108) as the C-3,C-18-diol was very unstable, but the diacetate (108) - 44 -was completely stable. Hence, the crude product from the side chain elimination was immediately acetylated. Alumina chromatography of this l a t t e r mixture gave a t . l . c . pure product which could not be induced to c r y s t a l l i z e from any of a number of solvent systems. The n.m.r. spectrum of this material was very informative (see f i g . 19), showing the presence of an a,B-unsaturated methyl ketone at x 7.72, two acetates at x 8.04, and a v i n y l proton as a quartet at x 2.91. The a l l y l i c C-13 proton appears at x 6.85 as a distorted t r i p l e t , and the C-18 protons appear at x 6,14 as an octet. The m u l t i p l i c i t y of this l a t t e r resonance i s due to hindered rotation around the C-13/C-18 bond, making the two C-18 protons non-equivalent and therefore geninally coupled. The n.m.r. spectrum of t h i s a,B-unsaturated ketone as the C-13B-methyl compound i s given i n f i g . 20 for comparison. It i s inte r e s t i n g to note that the C-13a-proton appears i n t h i s compound as a symmetrical quintet Figure 19 : N.m.r. Spectrum of Compound 110 Figure 20 : N.m.r. Spectrum of Compound 111 -47 -at x 6.96. The coupling with the C-18-methyl protons and with the C-13-proton i s thus the same. In the C-133-acetoxymethyl s e r i e s , the C-13-proton appears as a symmetrical quartet at x 6.72. The s i m i l a r couplings i n these two series indicate that the stereochemistry at C-13 must be the same, and therefore 3. This provided further proof of the C-13a stereochemistry i n 110. The i n f r a r e d , u l t r a v i o l e t , and mass spectra (see f i g . 22) were a l l i n accord with the assigned structure. Thus, the second objective of the synthesis was accomplished, namely, the construction of a suitable etiojervane portion to which a heterocyclic unit could be attached. In previous work i n our laboratory, a heterocyclic portion has been attached to an etiojervane skeleton v i a the synthetic route shown i n f i g . 21. 115 Figure 21 : Coupling of the Heterocyclic Unit i n the Verarine Synthesis - 4 8 -O r - in AJLISN31NI 3AI±V13d - 49 -The a,S-unsaturated ketone (111) was converted v i a the oxime (112) to the 17-keto compound (113), Reaction of the anion of 2-ethyl-5-methyl-pyridine with the a,B-unsaturated ketone (114) provided the condensation product (115). While the conditions for this reaction have now been f u l l y worked out, i t should be noted that i f t h i s approach were to be applied to the v e r t i c i n e synthesis, i t would introduce a hydroxyl at C-17 rather than at C-20 where i t i s required f o r v e r t i c i n e . 73 A better approach seemed to be the one used by Schreiber and Adam in t h e i r work on the solanum alka l o i d s . They reacted 33-acetoxypregn-5-en-20-one (116) with 2-lithio-5-methylpyridine to produce a mixture of the two condensation products (117 and 118) i n an overall y i e l d of 64 %. Figure 23 : Coupling of the Heterocyclic Unit in the Solanum Alk a l o i d Synthesis - 50 -I f this approach were applied to the v e r t i c i n e synthesis, i t would eliminate the necessity of going to the D-ring ketone v i a a Beckmann rearrangement, and would introduce a hydroxyl at the C-20 position where i t i s required for v e r t i c i n e . Since only small quantities of the a,S-unsaturated ketone (110) were available, i t was decided to f i r s t attempt t h i s reaction i n a model serie s . The a,3-unsaturated ketone (111) was chosen as the model, since "7 A a large quantity of this compound was available from e a r l i e r work . Ca t a l y t i c hydrogenation provided the saturated ketone (119), characterized by i t s mass spectrum (parent peak at m/e 318), infrared spectrum (saturated carbonyl at 1696 cm"*) and n.m.r. spectrum (saturated methyl ketone at T 7.90, no v i n y l or a l l y l i c protons). I l l 119 The chromatographically pure material, after r e c r y s t a l l i z a t i o n , s t i l l possessed a rather wide melting range, and further p u r i f i c a t i o n was attempted. However, repeated r e c r y s t a l l i z a t i o n s of this material succeeded only i n r a i s i n g the melting range a few degrees each time, but not narrowing the 5° range. I t was thus f e l t that t h i s material was a mixture of C-16 epimers which were c o - c r y s t a l l i z i n g . Thus, this material was refluxed i n methanolic KOH to t r y to epimerize the methyl ketone to the - 51 -more stable a configuration. The product appeared i d e n t i c a l to s t a r t i n g material by t . l . c , and n.m.r. and mass spectra, but r e c r y s t a l l i z a t i o n provided a sample with a much lower, constant, and sharp melting point. Moreover, the infrared spectrum showed s l i g h t differences i n the carbonyl and fi n g e r p r i n t regions of the spectra of these two materials. It was thus concluded that a mixture of isomers had been produced on c a t a l y t i c hydrogenation of 111, and that t h i s mixture could be converted wholly to the C-16a isomer (120) under epimerizing conditions. The stage was now set f o r the investigations involving the possible attachment of the heterocyclic u n i t . For t h i s purpose, 2-bromo-5-methyl-75 pyridine was prepared v i a a known reaction from 2-amino-5-methylpyridine Several attempts were made to condense 2-lithio-5-methylpyridine with the ketone (120). I t was found that the optimum procedure which resulted i n y i e l d s of 60 to 70 % of the condensation product (121) involved the reaction of a cold THF solution of the organolithio derivative (prepared from n-butyllithium and a ten-fold excess of 2-bromo-5-methylpyridine at -40 to -50°) with the ketone (120) under a completely i n e r t (helium) atmos-phere at -40 to -50°. The aqueous acid soluble products from the reaction were p u r i f i e d by s i l i c a gel chromatography, and two r e c r y s t a l l i z a t i o n s from petroleum ether provided a chromatographically pure product. The mass spectrum (see f i g . 24) indicated that condensation had indeed occurred. A parent peak at m/e 411 was v i s i b l e , and a large peak at m/e 393 indicated dehydration of the t e r t i a r y benzylic alcohol. Peaks at m/e 136 and m/e 274 indicated fragments from benzylic cleavage of the C-16/C-20 bond. The n.m.r. spectrum (see f i g . 25) showed the presence of three aromatic protons, and an aromatic methyl group at x 7.70. The two methyl groups on the s t e r o i d a l nucleus were present at T 9.22 and T 9.00. The fact that this material would readily c r y s t a l l i z e , and the fact that only one peak could be observed f o r the C-21 methyl group at T 8.54 seemed to indicate that only one C-20 isomer had been formed i n the condensation. It was impossible, however, to assign the stereochemistry at C-20. The u l t r a v i o l e t spectrum, infrared spectrum, and elemental analysis were a l l i n good agreement with the proposed structure (121). It thus appears that t h i s route w i l l be the most at t r a c t i v e i n elaboration of the a,3-unsaturated ketone (110) to v e r t i c i n e . Reduction of the double bond followed by base epimerization of the methyl ketone and simultaneous hydrolysis of the acetates w i l l lead to the d i o l (122). Attachment of the heterocycle followed by nucleophilic attack of the - 53 -• . i I I I I I I I I — I I I I I I I I I • ' • • _ . . . . i . . . . I . ... i . ... I • ... • •••• I • ••• i . . . . I I I I • . • . I • . • . i . • . • T . . , . i . , , , I 0 1 2 3 4 5 6 7 8 9 10 Figure 25 : N.m.r. Spectrum of Compound 121 - 55 -nitrogen on C-18 should give the hexacyclic system (123). Reduction the pyridine r i n g , and introduction of a 6a-hydroxyl w i l l then give Figure 26 : Proposed Conclusion of the Synthesis of Verticine EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected. Ultraviolet spectra were recorded on a Cary 11 Recording Spectrophotometer, and infrared spectra were recorded on a Perkin Elmer Model 21 Spectrometer as potassium bromide pellets unless otherwise noted. Nuclear magnetic resonance (n.m.r.) spectra were recorded at 100 MHz on a Varian HA-100 Spectrometer, using tetramethylsilane as an internal standard set at 10.0 units on the Tiers T scale. The types of protons, integrated areas, multiplicities, and spin coupling constants J (in Hz) are indicated in parentheses. Deuterochloroform was used as the solvent. Mass spectra were determined on an Atlas CH-4 Spectrometer or an Associated Electrical Industries MS-9 Spectrometer, high resolution measurements being determined on the latter instrument. Elemental analyses were performed by Mr. P. Borda of the Microanalytical Laboratory, University of British Columbia. Thin layer chromatoplates were prepared with silica gel G containing 2 % electronic phosphor, and were developed in benzene/ ethyl acetate mixtures. Woelm neutral si l i c a and Woelm neutral alumina were used for column chromatography unless otherwise noted. - 57 -Birch Reduction of Hecogenin Acetate (12-Keto-(25 R)-5a-spirostan-3g-ol- 3-acetate) (74) Ammonia (1500 ml) was run into a 2 - l i t r e , 3-necked round bottomed flask (RBF) f i t t e d with an acetone/dry ice condenser and cooled in an acetone/dry ice bath. A few pieces of sodium were added to dry the ammonia. The acetone/dry ice bath and condenser were removed, and the ammonia d i s t i l l e d over into a 5 - l i t r e , 3-necked RBF f i t t e d with an acetone/dry ice condenser and a dropping funnel, and s i t t i n g in an acetone/dry ice bath. Dry t-butanol (750 ml) was added, and the solution stirred with a magnetic s t i r r e r . Hecogenin acetate (74)(50.0 g) was dissolved in tetrahydrofuran (THF)(750 ml), and was added through the dropping funnel to the ammonia/t-butanol solution. Lithium wire (20 g) was then added, and the mixture stirred at -78° for 3 hours. During the course of the reaction, the colour changed from white to deep blue, and a thick, o i l y , brown layer appeared at the top of the mixture. After 3 hours, the acetone/dry ice bath and condenser were removed, and enough methanol was added to quench the reaction. The solution was then stirred at room temperature for several hours with a stream of nitrogen passing over i t in order to evaporate most of the ammonia. Water (2 l i t r e s ) was then added, and the solution was acidified with aqueous hydrochloric acid. The organic layer was separated, and the lower aqueous layer was extracted with chloroform. The combined organic layers were dried over sodium sulphate, and the solvents removed in vacuo to y i e l d a creamy coloured solid which, after drying in a vacuum oven overnight at 80°, afforded crude 12B-rockogenin ((25 R)-5ct-spirostan-3B,12&-diol) (75) (62.1 g). - 58 -Chromatography using Shawinigan activity II alumina (2800 g), eluting with 70 % chloroform/30 % benzene, gave pure 12B-rockogenin (75)(42.80 g, 93.4 % ) . A portion of this was recrystallized from ethanol/water to give 126-rocko-genin as white needles, m.p. 202 - 205° ( l i t . m.p.59 209 - 211°). N.m.r. signals: 9.23(doublet, J=7, 9H, C-18 CH3 • C-19 CH3 + C-27 CH 3), 8.99 (doublet, J=7, 3H, C-21 CH 3), 6.60(broad multiplet, 4H, C-3 CH + C-12 CH + C-26 CH 2), and 5.65(quartet, J=7, IH, C-16 CH). Infrared: 2.93, 3.43, 6.87, 9.45, and 13.11u. Mass spectrum: M.W. 432; base peak at m/e 139; main peaks at m/e 248, 300, 318, 360, 363, and 373. High resolution mass spectrum, found: 432.325426; calc. for ^ 21^i\/pV 432.323941. Elemental analysis, found: C 72.50, H 10.40; calc. for C^H^O^.Q^O^OH: C 72.80, H 10.45. 128-Rockogenin-3g-pivalate ((25 R)-5a-spirostan-3p,12B-diol-3-pivalate)(76) 128-Rockogenin (75)(62.0 g) was dried overnight in a vacuum oven at 80°. It was then dissolved in dry pyridine (1 l i t r e ) , and placed in a 2- l i t r e , 3-necked RBF f i t t e d with a drying tube, a thermometer, and a septum i n l e t . Pivaloyl chloride (18.15 g) was added with a syringe through the septum i n l e t , and the solution was then heated to 100° and stirred for 2 hours. The reaction mixture was allowed to cool, and benzene (2 lit r e s ) was added. The pyridine was removed by washing the organic layer with 1 N hydrochloric acid (3 x 500 ml), and the remainder of the organic solvent was removed in vacuo. The resulting solid was dried in a vacuum oven overnight at 80° to yield crude 12B-rockogenin-3B-pivalate (76) (74.80 g). An analytical sample was obtained by chromatography on activity II alumina, eluting with benzene. Recrystallization from acetone gave - 59 -pure 12B-rockogenin-3B-pivalate as white needles, m.p. 250 - 253° ( l i t . in.p. 5 8 255 - 257°). N.m.r. signals: 9.25(singlet, 3H, C-19 CH 3), 9.22 (doublet, J=5, 3H, C-27 CH 3), 9.15(singlet, 3H, C-18 CH 3), 8.97(doublet, J=6.5, 3H, C-21 CH 3), 8.85(singlet, 9H, C-3 pivalate protons), 6.76(doublet, J=4.5, IH, one C-26 CH 2), 6.65(doublet, J=5.5, IH, other C-26 CH 2), 6.58 (broad singlet, IH, C-12 CH), 5.62(quartet, J=7, IH, C-16 CH), and 5.38 (multiplet, IH, C-3 CH). Infrared: 2.86, 3.40, 5.87, 6.84, 7.73, 8.45, 9.45, 10.14, and 11.06p. Mass spectrum: M.W. 516; base peak at m/e 384; main peaks at m/e 498, 457, 447, 444, 402, 207, and 139. High resolution mass spectrum, found: 516.388734; calc. for C 3 2H 5 20 5: 516.382418. Elemental analysis, found: C 74.13, H 10.00; calc. for C 3 2H 5 20 5: C 74.37, H 10.14. Rockogenin-38-pivalate-123-mesylate ((25 R)-5a-spirostan-3B,12B-diol-3- pivalate-12-mesylate)(77) Crude 12g-rockogenin-3B-pivalate (76)(74.50 g) was dissolved in pyridine (500 ml) and placed in a 1-litre RBF f i t t e d with a thermometer, a septum in l e t , and a drying tube. The solution was cooled to 5° in an ice bath, and was then treated with freshly d i s t i l l e d methanesulphonyl chloride (46.5 ml). Evolution of a gas was noted. The solution was stirred with a magnetic s t i r r e r and allowed to warm slowly to room temperature over a period of 18 hours. The mesylate was then cooled to below 5° in an ice bath, and aqueous sodium bicarbonate (500 ml) was added. Heat was evolved, and the mesylate precipitated as a granular, light brown solid. It was f i l t e r e d off, dissolved in benzene, dried over magnesium sulphate, - 60 -and the benzene removed in vacuo to yield a brown solid (85.50 g). The mesylate (77) was further dried on a vacuum pump. Since i t was quite unstable to alumina and s i l i c a chromatography, and to recrystallization, i t was used for the next reaction without further purification. Infrared: 3.39, 5.84, 7.35, 8.51, 9.43, 10.16, 11.24, 11.92, and 13.16u. C-Nor-D-homo-18-nor-(25 R)-5ct-spirost-13(18)-en-3B-ol-3-acetate (79) Crude rockogenin-3B-pivalate-12B-mesylate (77)(85.5 g) was dissolved in a solution of potassium t-butoxide prepared by dissolving potassium (30 g) in _t-butanol (2.5 l i t r e s ) . The solution was refluxed for 18 hours under nitrogen in a 5 - l i t r e , 3-necked RBF f i t t e d with an a i r condenser and a nitrogen i n l e t . The solution was allowed to cool, and methanol (1200 ml), THF (600 ml), and water (120 ml) were added. The solution was refluxed for a further 2 hours under nitrogen, and the organic solvents were then removed in vacuo. Water was added periodically to aid in the removal of the t-butanol. The solid which precipitated was f i l t e r e d , washed with water, and dried in a vacuum oven overnight at 80°. A highly solvated, cream coloured solid (120 g) was obtained. This solid (78) was placed in a 1-litre RBF and refluxed for .5 hours with pure, dry acetic anhydride (500 ml). On cooling, a cream coloured solid precipitated out. This was f i l t e r e d , washed with glacial acetic acid to remove the coloured impurities, then washed with water, and f i n a l l y dried in vacuo. Workup by recrystallization from methanol/chloroform of the solid and a l l the mother liquors afforded the pure exocyclic olefin (79)(18.8 g) and a mixture of this olefin (79) and the A 1 2^ 1 3^ isomer (36.7 g). Two - 61 -recrystallizations from methanol/chloroform of a portion of the f i r s t crop of crystals provided the pure exocyclic olefin (79) as plates, m.p. 221 -225.5° ( l i t . m.p. 5 9 221 - 225.5°). N.m.r. signals: 9.14(doublet, J=6, 3H, C-27 CH 3), 9.11(singlet, 3H, C-19 CH 3), 8.94(doublet, J=6.5, 3H, C-21 QI 3), 8.02(singlet, 3H, -OCOCH3), 7.56(multiplet, 2H, C-12 CH + C-17 CH), 6.56(quartet, J=7, 2H, C-26 CH 2), 5.94(octet, Ji5g>i6 = 1 2» Ji5a-16 = 4 , 5» J 1 6_ 1 7=9.5, IH, C-16 CH), 5.32(multiplet, IH, C-3 CH), 5.22(broad singlet, 2H, C-18 Q l 2 ) . Infrared: 3.41, 5.78, 6.11, 6.87, 7.29, 8.00, 9.42, 10.12, and 11.24y. Mass spectrum: M.W. 456; base peak at m/e 456; main peaks at m/e 438, 414, 384, 342, 165, and 126. High resolution mass spectrum, found: 456.332017; calc. for C29 H44°4 : 456.323941. Elemental analysis, found: C 76.43, H 9.90; calc. for C 2 gH 4 40 4: C 76.27, H 9.71. Hydroboration of the Exocyclic Olefin (79) The exocyclic olefin (79)(3.00 g) was dissolved in pure, dry THF (120 ml), and placed in a 250 ml, 3-necked RBF f i t t e d with a dropping funnel, a condenser, and a nitrogen i n l e t . The apparatus was flame dried before use. Dry nitrogen was passed slowly over the solution during the reaction. Diborane in THF (1 M, 15 ml) was added dropwise through the dropping funnel over a period of .25 hours, and evolution of a gas was noted. The solution was stirred at room temperature for 3 hours. At this point, 10 % aqueous sodium hydroxide (50 ml) was added slowly, the solution heated to gentle reflux, and 30 % aqueous hydrogen peroxide (50 ml) added very slowly. The mixture was refluxed for .5 hours, then cooled, diluted with water and extracted with ether. The ether extract was dried over - 62 -sodium sulphate, and the solvent removed in vacuo to yield crude 138-hydroxymethyl-C-nor-D-homo-18-nor-(25 R)-5a-spirostan-38-ol (80(a)) (4.005 g). This material was chromatographed on activity II alumina, eluting with 75 % chloroform/25 % benzene, to yi e l d the pure diol (80(a))( 2.571 g). An analytical sample was prepared by recrystallization from methanol/water, m.p. 179 - 183°. N.m.r, signals (see f i g . 7): 9.24 (singlet, 3H, C-19 QI 3), 9.22(doublet, J=5, 3H, C-27 CH 3), 9.00(doublet, J=6, 311, C-21 CH 3), 8.02(singlet, 2H, C-3 OH + C-18 OH), 6.60(multiplet, 3H, C-3 QI + C-26 CH 2), 6.42(doublet, J=5, 2H, C-18 CH 2), and 5.95 (multiplet, IH, C-16 CH). Infrared: 2.90, 3.42, 6.87, 8.02, 8.42, 9.62, 10.28, 11.24, and 11.48y. Mass spectrum (see f i g . 10): M.W. 432; base peak at m/e 432; main peaks at m/e 373, 363, 360, 300, 270, 139, and 115. High resolution mass spectrum, found: 432.320025; calc. for C 27H 4 40 4: 432.523941. Elemental analysis, found: C 72.66, H 9.98; calc. for C27 H44 04* a i3 O I I : C 7 2 ' 5 0 » H 10\°3» By following the same procedure as above, except using only 2 ml of 10 % aqueous sodium hydroxide, i t was found that 138-acetoxymethyl-C-nor-D-homo-18-nor-(25 R)-5a-spirostan-38-ol-3-acetate (80(b))(750 mg) could be isolated from the chromatography in impure form. Several r e c r y s t a l l i z -ations from heavy petroleum ether afforded the pure acetate (80(b)), m.p. 184 - 186°. N.m.r. signals: 9.21(doublet, J=5.5, 3H, C-27 CH 3), 9.21(singlet, 3H, C-19 CH^), 8.99(doublet, J=6, 3H, C-21 CH 3), 8.01(singlet, 3H, -0C0CH3), 6.38(doublet, J=5, 2H, C-18 CH 2), 6.22(multiplet, 2H, C-26 CH 2), 5.92(multiplet, IH, C-16 QI), and 5.33(multiplet, IH, C-3 CH). Infrared: 2.92, 3.43, 5.82, 6.86, 7.28, 8.05, 9.71, 10.30, 11.20 and 13.21u. - 63 -Mass spectrum: M.W. 474; base peak m/e 105; main peaks at m/e 460, 456, 414, 402, 342, 300, 253, 139, and 115. High resolution mass spectrum, found: 474.334392; calc. f o r C 29 H46°5 : 474.334505. Elemental analysis, found: C 73.62, H 9.67; calc. for C 2 gH 4 60 5: C 73.38, H 9.77. 3-Keto-133-aldehydo-C-nor-D-homo-18-nor-(25 R)-5ct-spirostan (82) The pure d i o l (80(a))(3.6 g) was placed i n a 250 ml RBF, and dicyclohexylcarbodiimide (DCC)(14.64 g), dimethylsulfoxide (DMSO)(50 ml), benzene (75 ml), pyridine (2 ml), and t r i f l u o r o a c e t i c acid (1 ml) were added to i t . The reaction mixture was then s t i r r e d at room temperature f o r 36 hours. It was worked up by adding ether (250 ml) and o x a l i c acid (6.0 g) in methanol (50 ml), and s t i r r i n g at room temperature f o r 1 hour. The white c r y s t a l l i n e dicyclohexylurea which had precipitated was f i l t e r e d o f f , and the organic layer washed with water (3 x 200 ml) and with aqueous sodium bicarbonate (2 x 200 ml). The ether layer was dried over sodium sulphate, and the solvent removed i n vacuo to y i e l d a yellow s o l i d (4,266 g). An a n a l y t i c a l sample of the aldehyde (82) thus obtained was prepared by repeated r e c r y s t a l l i z a t i o n from methanol/water. I t was found that s l i g h t decomposition occurred on r e c r y s t a l l i z a t i o n . N.m.r. signals (see f i g . 11): 9.21(doublet, J=6, C-27 CH 3), 9.08(singlet, 3H, C-19 CH 3), 9.03(doublet, J=6, 3H, C-21 CHj), 7.33(doublet of t r i p l e t s , J 1 2 _ 1 3 and J13-17 = 5 , 5» J13-18 = 6» 1 H» C " 1 3 Q i^» 6 ' 6 ° ( r a u l t i P l e t » 2 H» c " 2 6 CH 2), 5.95 (octet, •J 1 5 e_ 1 6=12> J J 5 !6 = 4' 5» J16-17 = 9* 5> 1 H» C " 1 6 m ) » ^ 0.22(doublet, J=6, IH, C-18 CHO). Infrared: 3.42, 5.88, 6.86, 8.00, 9.34, 10.12, 11.05, and 11 . 4 4 p . Mass spectrum (see f i g . 13): M.W. 428; base peak at m/e 149; - 64 -main peaks at m/e 410, 400, 369, 359, 356, 341, 314, 285, 256, 206, 126, and 115. High resolution mass spectrum, found: 428.295336; calc. f o r Co7HJ,„0 : 428.292643. Zl 40 4 3-Keto-13a-aldehydo-C-nor-D-homo-18-nor-(25 R)-5g-spirostan (83) The crude aldehyde (82)(3.572 g) was dissolved i n methanol (100 ml), and potassium carbonate (1.5 g) was added. The mixture was s t i r r e d at room temperature f o r 1 hour, and most of the methanol was then removed in vacuo. Dilut i o n with water and extraction with chloroform gave, after drying the organic layer over sodium sulphate and removal of the solvent i n vacuo, the crude aldehyde (83)(3.583 g). An a n a l y t i c a l sample was prepared by chromatography on a c t i v i t y II alumina, eluting with benzene, and by r e c r y s t a l l i z a t i o n of this material from methanol/water. S l i g h t decomposition occurred on r e c r y s t a l l i z a t i o n . N.m.r. signals (see f i g . 12): 9.22(doublet, J=6, 3H, C-27 CH 3), 9.10(doublet, J=5, 3H, C-21 CH 3), 9.08 ( s i n g l e t , 3H, C-19 CH 3), 6.58(multiplet, 2H, C-26 CH 2), 5.98(octet, J15e-16 = 1 2» J15a-16 = 4 , 5» J16-17 = 9 , 5» 1 H» C " 1 6 Q I ) ' a n d ° . 5 7 ( d i s t o r t e d doublet, J=4, IH, C-18 CHO). Infrared: 3.42, 5.86, 6.84, 8.00, 9.39, 10.08, 11.03, and 11.41u. Mass spectrum (see f i g . 13): M.W. 428; base peak at m/e 115; main peaks at m/e 400, 369, 359, 356, 341, 328, 313, 300, 285, 257, 206, 149, and 139. High resolution mass spectrum, found: 428.293266; calc. for ^ W^^x 428.292643. 13ot-Hydroxymethyl-C-nor-D-homo-18-nor-(25 R)-5ot-spirostan-33-ol (84) Sodium borohydride (.475 g) was added to a solution of the crude aldehyde (83)(3.583 g) i n methanol (100 ml). The reaction mixture was then - 65 -s t i r r e d at room temperature for 2 hours. Most of the methanol was removed i n vacuo, and the solution was then dilut e d with water and extracted with chloroform to y i e l d the crude d i o l (84)(3.270 g). An a n a l y t i c a l sample was prepared by chromatography on a c t i v i t y II alumina, eluting with chloroform; the re s u l t i n g material was r e c r y s t a l l i z e d from methanol/ water to y i e l d the pure d i o l (84), m.p. 240.5 - 242°, N.m.r. signals (see f i g . 14): 9.33(singlet, 3H, C-19 CH-), 9.27(doublet, J=6, 3H, C-27 CH 3), 8.97(doublet, J=6, 3H, C-21 CH 3), 6.62(multiplet, 2H, C-26 CH 2), 6.34 (broad s i n g l e t , 2H, C-18 C l l 2 ) , and 6.04(octet, J ^ . ^ 1 2 . J i s a _ i 6 = 4 , 5 » J16-17 = 9' 5' 1 H» C " 1 6 I n £ r a r e d : 2« 9 5> 3' 4 2» 6' 8 6» 8« 0 2» 9' 4 4» 10.19, 11.08, and 11.50u. Mass spectrum (see f i g . 13): M.W. 432; base peak at m/e 115; main peaks at m/e 402, 373, 363, 360, 345, 300, 288, 145, and 139. High resolution mass spectrum, found: 432.324148; calc. f o r C 2yH 4 40 4: 432.323941. Elemental analysis, found: C 73.57, H 10.21; c a l c . f o r C 2 7H 4 40 4: C 74.96, H 10.25. 13q-Aldehydo-C-nor-D-homo-18-nor-(25 R)-5ot-spirostan-3g-ol-3-acetate (81) The acetate (80(b))(213 mg) was placed i n a 50 ml RBF, and DCC (826 mg), DMSO (2.5 ml), benzene (5 ml), pyridine (0.1 ml) and t r i f l u o r o a c e t i c acid (0.05 ml) were added. The flask was stoppered, and the reaction mixture s t i r r e d at room temperature for 36 hours. Ether (20 ml) was then added, followed by oxa l i c acid (0.35 g) i n methanol (10 ml), and the reaction mixture was s t i r r e d f o r a further 1 hour. The white c r y s t a l l i n e dicyclo-hexylurea which had precipitated was f i l t e r e d o f f . The organic layer was then washed with water (3 x 20 ml) and then with aqueous sodium bicarbonate - 66 (3 x 20 ml). The ether layer was then dried over sodiumjsulphate, and the ether removed i n vacuo to y i e l d a white s o l i d (209 mg). This material, without p u r i f i c a t i o n , was treated with potassium carbonate (500 mg) in methanol (20 ml) and water (5 ml), and s t i r r e d at room temperature f o r 1 hour. The product was isolated by removal of most of the methanol i n vacuo, d i l u t i o n with water, and extraction with chloroform. The crude product so obtained was treated immediately with pyridine (5 ml) and acetic anhydride (5 ml), and was l e f t overnight at room temperature. Benzene (25 ml) was then added, and the solution washed with aqueous 1 N hydrochloric acid (3 x 20 ml), aqueous sodium bicarbonate (3 x 20 ml), and water (2 x 20 ml). Removal of the solvent i n vacuo yielded the crude aldehydo-acetate (81)(224 mg). This material was chromatographed on a c t i v i t y II alumina, el u t i n g with benzene, to y i e l d the pure aldehyde (81) (143 mg). R e c r y s t a l l i z a t i o n of this material from heavy petroleum ether gave white needles, m.p. 155 - 160°. T.l.c. indicated that s l i g h t decomposition had occurred. N.m.r. signals: 9.25(singlet, 3H, C-19 CH 3), 9.22(doublet, J=6, 3H, C-27 CH 3), 9.11(doublet, J=6.5, 3H, C-21 CH 3), 8.02(singlet, 3H, -0C0CH3), 6.60(multiplet, 2H, C-26 CH 2), 5.99(octet, J15B-16 = 1 2» J15a-16 = 4' 5' J16-17 = 9 , 5» 1 H» C " 1 6 Q ° » 5 - 3 8 ( m u l t i P l e t » 1 H» C-3 CH), and 0.59(distorted doublet, J=4, IH, C-18 CHO). Infrared: 3.38, 3.69, 5.81, 6.83, 7.31, 8.02, 8.75, 9.72, 10.21, and 11.15y. Mass spectrum: M.W. 472; base peak at m/e 115; main peaks at m/e 444, 442, 413, 403, 400, 385, 358, 325, 300, 173, 149, 135, and 126. High resolution mass spectrum,, found: 472.315640; calc. f o r C 2 gH 4 40 5: 472.318855. - 67 -This material could, before acetylation, be converted to the d i o l (84) by sodium borohydride reduction using exactly the same procedure as for the reduction of the keto-aldehyde (83). The d i o l s were shown to be i d e n t i c a l by t . l . c . and melting point. 13a-Acetoxynethyl-C-nor-D-homo-18-nor-(25 R)-5a-spirostan-3B-ol-3-acetate (85) The crude d i o l (84)(5.100 g) was placed i n a 100 ml RBF, and acetic anhydride (25 ml) and pyridine (25 ml) were added. The solution was allowed to stand overnight, and was then d i l u t e d with benzene (200 ml). The solution was washed with 1 N hydrochloric acid (3 x 100 ml), aqueous sodium bicarbonate (3 x 100 ml) and water (2 x 100 ml). The solvent was then removed i n vacuo to y i e l d a yellow o i l (3.420 g). Chromatography on a c t i v i t y II alumina, e l u t i n g with 60 % benzene/40 % heavy petroleum ether, gave the pure diacetate (85)(2.570 g) as a clear glass. This material could not be induced to c r y s t a l l i z e from any of a number of solvent systems. N.m.r. signals: 9.25(singlet, 3H, C-19 CH3) , 9.22(doublet, J=7, 3H, C-27 C i y , 9.00(doublet, J=6, 3H, C-21 dl-j), 8.05(singlet, 3H, -OCOCH3), 8.01(singlet, 311, -0C0CH3), 6.60(multiplet, 2H, C-26 CH 2), 6.00(octet, J 1 C„ w = 12, J l r ,^=4.5, J , , ,.,=9.5, III, C-16 CH), 5.91(distorted doublet, log-lo * 15a-16 16-1/ J=2, 2H, C-18 CH 2), 5.33(multiplet, IH, C-3 CH). Infrared (chloroform s o l u t i o n ) : 3.42, 5.82, 6.87, 7.20, 7.31, 7.99, 9.51, 9.82, and 11.12p. Mass spectrum: M.W. 516; base peak at m/e 114; main peaks at m/e 484, 457, 447, 444, 402, 387, 342, 327, 224, 157, 139, and 115. High resolution mass spectrum, found: 516.348576; calc. f o r C,,H.oO{-• 516.345084. - 68 -Baeyer-Villiger Oxidation of the Diacetate (85) The pure diacetate (85)(5.3 g) was placed i n a 250 ml RBF, and formic acid (100 ml) and water (20 ml) were added. The solution was heated to 40°, and 30 % aqueous hydrogen peroxide (20 ml) was added dropwise. The solution was s t i r r e d at 40° f o r 4 hours, with three additional portions of 30 % aqueous hydrogen peroxide (5 ml) being added at hourly i n t e r v a l s . The mixture was then d i l u t e d with water (1 l i t r e ) , and the product isol a t e d by extraction with benzene. The solvent was removed i n vacuo to y i e l d a yellow o i l (5.910 g). Chromatography of a portion of t h i s material on a c t i v i t y II s i l i c a g e l , e l u t i n g with 60 % chloroform/40 % benzene, gave the p a r t i a l l y p u r i f i e d diformate (104) as a clear glass. No further p u r i f i c a t i o n could be accomplished by chromatography, and the material could not be induced to c r y s t a l l i z e . N.m.r. signals: 9.20(singlet, 311, C-19 CH 3), 9.04(doublet, J=6, 3H, C-27 Q l 3 ) , 8.70(doublet, J=6, 3H, C-21 CH 3), 8.03(singlet, 3H, -0C0CH3), 8.01(singlet, 311, -0C0Q13), 6.02(doublet, J=6, 211, C-26 CH 2), 5.90 (multiplet, 211, C-18 CH 2), 5.33(multiplet, IH, C-3 Ql), 2.08(singlet, IH, one -0CH0), 1.98(singlet, IH, other -0CH0). Infrared (chloroform s o l u t i o n ) : 3.41, 5.83, 6.87, 7.23, 7.97, 9.75, and l l . O l y . Mass spectrum: no parent v i s i b l e ; highest peak at m/e 558; base peak at m/e 105; main peaks at m/e 543, 530, 515, 501, 484, 433, 387, 359, 357, 340, 280, 187, and 115. Hydrolysis of the Diformate (104) The crude diformate (104)(5.40 g) was dissolved i n methanol (250 ml), - 69 -and the solution was placed i n a 500 ml RBF. A c t i v i t y I neutral alumina (200 g) was added, and the mixture s t i r r e d at room temperature for 6 hours. The product was isolated by addition of chloroform and f i l t r a t i o n , a yellow o i l being obtained (3.983 g). This material was chromatographed on a c t i v i t y II s i l i c a g e l , el u t i n g with chloroform/ethyl acetate mixtures, to y i e l d the previous s t a r t i n g diacetate (85)(400 mg), a mixture of the diformate (104) and two monoformates (2.50 g), and the r e l a t i v e l y pure d i o l (105)(825 mg). This material, however, could not be p u r i f i e d further by chromatography, and could not be induced to c r y s t a l l i z e . N.m.r. signals: 9.21(singlet, 3H, C-19 CH 3), 9.09(doublet, J=6, 3H, C-27 Ql^), 8.78(doublet, J=6, 3H, C-21 d l 3 ) , 8.02(singlet, 3H, one -0C0CH3), 7.97(singlet, 3H, other -0C0CH3), 6.58(doublet, J=6, 2H, C-26 CH 2), 5.76(broad s i n g l e t , 2H, C-18 CH 2), 5.34(multiplet, IH, C-3 CH). Infrared (chloroform so l u t i o n ) : 2.91, 3.42, 5.33, 6.87, 7.22, 8.09, and 9.69y. Mass spectrum: no parent v i s i b l e , highest peak at m/e 532; base peak at m/e 314; main peaks at m/e 532, 518, 502, 489, 473, 460, 418, 404, 400, 359, 340, 314, and 115. Jones Oxidation of the Diol (105) The e s s e n t i a l l y pure d i o l (105)(733 mg) was dissolved i n pure acetone (25 ml). The solution was placed i n a 50 ml RBF, and cooled to 5° i n an ice bath. Jones reagent (1.3 ml) was then added dropwise with s t i r r i n g over a period of 15 minutes. The reaction was quenched with methanol and dilut e d with water, and the crude product (745 mg) - 70 -was isolated by extraction with chloroform. The material was non-c r y s t a l l i n e , and could not be p u r i f i e d by chromatography; hence, spectral data was obtained on the crude material. N.m.r. signals: 9.20(singlet, 3H, C-19 CH 3), 8.02(singlet, 3H, one -OCOCH3), 8 , 0 0 ( s i n g l e t , 3H, other -OCOCII3). Infrared (chloroform s o l u t i o n ) : 2.75 - 3.70 (-C00H), 3.43, 5.82, 6.37, 7.28, 8.08, 9.12, and 9.72y. Mass spectrum: no parent v i s i b l e , highest peak at m/e 530; base peak at m/e 361; main peaks at m/e 416, 378, 347, 320, 296, 283, 197, 135, and 100. 13a-Acetoxymethyl-l7-acety1-18-nor-5a-etiojerv-16(17)-en-3S-ol-5- acetate (110) The crude keto-acid (106)(664 rag) was placed i n a 100 ml, 3-necked RBF f i t t e d with a condenser and a nitrogen i n l e t , and t^butanol (50 ml) and 5 % aqueous potassium hydroxide (10 ml) were added. The solution was refluxed f o r 1 hour under nitrogen, cooled, and the t-butanol removed i n vacuo. The resultant solution was dilu t e d with water and extracted with ether, the ether layer being dried over sodium sulphate. The ether was then removed i n vacuo to y i e l d the crude ct,B-unsaturated ketone (109)(480 mg). Since previous work had shown that this compound was l i k e l y to be very unstable, i t was not i s o l a t e d , but was immediately converted to the corresponding diacetate (110). The crude d i o l (109)(480 mg) was treated with pyridine (25 ml) and acetic anhydride (25 ml), and was allowed to stand overnight. Benzene (100 ml) was then added, and the organic layer was washed with 1 N hydrochloric - 71 -acid (3 x 50 ml), saturated aqueous sodium bicarbonate (3 x 50 ml), and water (2 x 50 ml). The solvents were then removed i n vacuo to y i e l d the crude diacetate (110)(490 mg). This material was chromato-graphed on a c t i v i t y II alumina, eluting with 25 % chloroform/75 % benzene, to y i e l d the pure, non-crystal line diacetate (110)(265 mg). N.m.r. signals (see f i g . 19): 9.34(singlet, 3H, C-19 CH 3), 8.04 ( s i n g l e t , 6H, two -OCOCH3), 7.72(singlet, 3H, C-21 C H 3 ) , 6.85 (distorted t r i p l e t , J=6, IH, C-13 CH) , 6.12(distorted octet, 2H, C-18 CH 2), 5.34(multiplet, IH, C-3 CH), and 2.91(quartet, JisQ-\e=3> J l 5 a -=7, IH, C-16 Ql). Infrared (chloroform s o l u t i o n ) : 3.42, 5.82, 6.01, 6.89, 7.31, 7.98, and 9.73p. U l t r a v i o l e t : Xmax=231my(e=6000). Mass spectrum (see f i g . 22): M.W. 416; base peak at m/e 356; main peaks at m/e 388, 372, 370, 343, 327, 296, 202, 187, 149, 135, 107, and 105. High resolution mass spectrum, found: 416.256870; calc. for C - r H ^ O • 416.256258. <-i> 00 5 17-Acetyl-5a >138-etiojervan-5B-ol (120) Pure 17-acetyl-5a,13B-etiojerv-16(17)-en-56-ol (111)(250 mg) was dissolved i n ethanol (100 ml) i n a 250 ml R B F , and palladium on charcoal (30 mg) was added. The solution was s t i r r e d under hydrogen at room temperature for 5 hours u n t i l the uptake of hydrogen had ceased. The catalyst was then f i l t e r e d o f f , and the solvent removed i n vacuo to y i e l d a white c r y s t a l l i n e s o l i d (249 mg). Chromatography on a c t i v i t y II alumina, el u t i n g with 15 % chloroform/85 % benzene, afforded a chromatographically pure material (226 mg). Two - 72 -r e c r y s t a l l i z a t i o n s from petroleum ether/chloroform gave white needles, m.p. 148 - 153°. Further r e c r y s t a l l i z a t i o n s did not narrow this melting range. N.m.r. signals: 9.22(singlet, 311, C-19 CH^), 9.21 (doublet, J=6, 3H, C-18 CH 3), 7.90(singlet, 3H, C-21 CH 3), and 6.41(septet, J=5, IH, C-3 CH). Infrared: 2.83, 3.44, 5.89, 6.92, 7.22, 7.37, 8.16, 8.39, and 9.42. Mass spectrum: M.W. 318; base peak at m/e 55; main peaks at m/e 303, 300, 257, 233, 205, 187, 175, 147, 107, and 85. High resolution mass spectrum, found: 318.255023; calc. f o r C2i H34°2 : 518.255866. Elemental analysis, found: C 79.36, H 10.61; calc. f o r C 2 1H 3 40 2: C 79.19, H 10.76. 17a-Acetyl-5a,13g-etiojervan-3B-ol (121) The ketone (120)(110 mg) was dissolved i n methanol (20 ml), and potassium hydroxide (1 g) was added. The mixture was refluxed f o r 1 hour, and was then cooled and dil u t e d with water. Chloroform extraction gave a white s o l i d (109 mg) which appeared i d e n t i c a l to st a r t i n g material by t . l . c . R e c r y s t a l l i z a t i o n from petroleum ether/ chloroform gave white needles, m.p, 143.5 - 145.5°. Infrared: 2.87, 3.44, 5.92, 6.88, 7.29, 7.68, 7.86, 8.50, 8.84, 9.36, and 9.58y. Other spectral data f o r this compound was i d e n t i c a l with that from the st a r t i n g material (120). 2-Bromo-5-methylpyridine 2-Amino-5-methylpyridine (1.40 g) was placed i n a 100 ml, 3-necked RBF and cooled to below 0° i n an i c e / s a l t bath. To this was added 48 % hydrobromic acid (7,25 ml) with s t i r r i n g . Bromine (2.00 ml) was added - 73 -dropwise, and an orange pr e c i p i t a t e appeared. A solution of sodium n i t r i t e (2.23 g) i n water (4 ml) was added dropwise, and evolution of a gas was noted. The mixture was s t i r r e d f o r a further 10 minutes below 0°, and then sodium hydroxide (5 g) i n water (15 ml) was added dropwise. S t i r r i n g was continued u n t i l the deep red layer at the bottom of the flask disappeared. The solution was then extracted with ether, and the solvent removed i n vacuo to y i e l d a yellow s o l i d (2.15 g). This material was decolourized with Darco, and r e c r y s t a l l i z e d from heavy petroleum ether to y i e l d pure 2-bromo-5-methylpyridine (1.68 g) as white plates, m.p. 46.5 - 47.5°. N.m.r. signals: 7.72(singlet, 3H, methyl group), 2.66(doublet, J=1.5, 211, C-3 + C-4 protons), and 1.82 (broad s i n g l e t , IH, C-6 proton). Infrared: 3.00, 3.37, 6.31, 6.39, 6.89, 7.33, 8.22, 8.88, 9.21, 9.73, and 12.21y. U l t r a v i o l e t : X m a x = 272 my. Mass spectrum: M.W. 172; base peak at m/e 92; main peaks at m/e 173, 171, 145, 143, 119, 117, 65, and 39. High resolution mass spectrum, found: 172.965971; calc. f o r C^H^BrN (Br = 81): 172.966444. Elemental analysis, found: C 42.30, H 3.45, N 8.40; calc. f o r C 6H 6BrN: C 42.10, H 3.50, N 8.20. 22,27-Imino-13S-jerva-22 t24,27-triene-3S t20-diol (122) A 50 ml, 3-necked RBF f i t t e d with a 10 ml dropping funnel, a small condenser with a drying tube, and a helium i n l e t was thoroughly flame dried before use. A l l materials used i n this reaction were rigorously dried and transferred using septums and syringes i n order to ensure completely anhydrous conditions. THF (6 ml) and n-butyl-- 74 -lithium (0.9 ml, 1 M i n hexane) were placed i n the RBF and cooled to -40 to -50° i n an acetone/dry ice bath. Dry helium was used as a protecting gas. 2-Bromo-5-methylpyridine (360 mg) i n THF (8 ml) was added dropwise over a period of 10 minutes, and the solution turned deep red i n colour. After 5 minutes, the ketone (121)(70 mg) in THF (8 ml) was added dropwise over a period of 5 minutes, and the solution was allowed to s t i r at -40 to -50° for 4 hours. The cooling bath was then removed, and the solution allowed to warm to room temperature over a period of 3 hours, during which time the colour of the reaction mixture changed from deep red to l i g h t yellow. The reaction was quenched with aqueous ammonium chloride, and the organic material was extracted with methylene chloride (150 ml). Removal of the solvent i n vacuo gave the crude product (214 mg). The material was redissolved i n methylene chloride, and the aqueous acid soluble material was extracted with 1 N hydrochloric acid. Of the material (26 mg) remaining i n the organic layer, 20 mg was s t a r t i n g material. The aqueous acid layer was cooled to 5°, and made basic with aqueous sodiun hydroxide. Extraction of this basic layer with methylene chloride gave, af t e r removal of the solvent i n vacuo, a mixture of simple pyridines and the desired consensation product (122)(185 mg). Chromatography of th i s material on a c t i v i t y II s i l i c a g e l , el u t i n g with 25 % benzene/75 % chloroform, gave what appeared by t . l . c . to be one ste r o i d a l condensation product plus two minor impurities (55 mg). Two r e c r y s t a l l i z a t i o n s from heavy petroluem ether gave the pure condensation product (122), m.p. 153 - 162°. - 75 -N.m.r. signals (see f i g . 25): 9.22(singlet, 3H, C-19 CH 3), 9.00 (doublet, J=6.5, 3H, C-18 QIJ, 8.54(singlet, 3H, C-21 Q^ 3), 7.70 (s i n g l e t , 3H, C-26 CH 3), 6.43(multiplet, IH, C-3 Oi), 2.83(doublet, J=8, IH, C-23 CH), 2.51(doublet, J=8, IH, C-24 CH), and 1.71(broad s i n g l e t , IH, C-27 CH). Infrared: 2.96, 3.04, 3.41, 3.47, 6.22, 6.36, 6.72, 6.89, 7.17, 8.71, 9.64, and 11.90u. U l t r a v i o l e t : X m a x = 268 (e = 3630). Mass spectrum (see f i g . 24): M.W. 411; base peak at m/e 136; main peaks at m/e 410, 409, 393, 378, 316, 274, 258, 219, 118, and 107. High resolution mass spectrum, found: 411.310478; calc. f o r C 2 7H 4 10 2N: 411.313713. BIBLIOGRAPHY 1. S.M. Kupchan and A.W. By in "The Alka l o i d s " (R.H.F. Manske ed.) v o l . X, pp. 193 - 280, Academic Press, New York, 1968. 2. J . Tomko, A. Vassova, G. Adam, K. Schreiber, and E. Hohne, Tetrahedron Letters, 3907 (1967). 3. G. Adam, K. Schreiber, J. Tomko, Z. Voticky, and A. Vassova, Tetrahedron Letters, 2815 (1968). 4. J. Fried and A. Klingsberg, J . Am. Chem. S o c , 75, 4929 (1953). 5. "Revised Tentative Rules f o r Nomenclature of Steroids", Steroids, 13, 277 (1969). 6. L. S. Goodman and A. 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