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The total synthesis of veratrum alkaloids Fortes, Carlos Camiza 1973

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c l THE TOTAL SYNTHESIS OF VERATRUM ALKALOIDS BY CARLOS C. FORTES Pharm. Chem., University of Brazil, Brazil, 1964 M.Sc, University of Brazil, Brazil, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1973 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 o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree 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 agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted 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 understood t h a t copying 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 allowed without my w r i t t e n p e r m i s s i o n . Department o f Chemistry 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 November 16 1973 - i i -ABSTRACT The condensation of a C-nor-D-homo s t e r o i d a l portion with the li t h i u m d e r i v a t i v e of various substituted pyridines i s outlined as a general method for the synthesis of Veratrun a l k a l o i d s . The a p p l i c a t i o n of t h i s approach to the synthesis of v e r t i c i n e (138), a representative of the a-cevanine bases i s described. Hecogenin acetate (180) was converted i n excel l e n t y i e l d to 33-acetoxy-13a-acetoxymethyl~18-nor-5a,12a-spirostan (240). Performic acid degradation of the s p i r o k e t a l side chain i n 240 gave 38-acetoxy-13et-acetox.ymethyl-18-nor-12a-pregnajervan-20-one (260). This ketone was coupled with 2-lithio-5-methyl pyridine to give a f t e r a c e t y l a t i o n 33,18-diacetoxy-20-hydroxy-20,23,24,25,26-N-hexadehydro-5a,136(H),17a(H)-vcratraiiine (277). The coupling product vras reduced with PtC^ i n acetic acid to 33,18-diacetoxy-5ct,133(H),17a(H)-veratranine (287). The f i n a l conversion to the hexacyclic a-cevane skeleton (90) was attempted by heating 287 at 130° for 72 hours i n triglyme. The above r e s u l t s open the p o s s i b i l i t y of the synthesis of several hypotensive Veratrum a l k a l o i d s and the access to several compounds not e a s i l y obtained from the natural products by degradation reactions. While some of the n a t u r a l l y occurring a l k a l o i d s are known to e x h i b i t hypotensive a c t i v i t y , i t i s not known whether a l t e r a t i o n s i n stereo-chemistry and structure w i l l cause enhancement or loss of such a c t i v i t y . - i i i -TABLE OF CONTENTS Page INTRODUCTION 1 1. Apocynaceae Bases 1.1 Conanine Bases ^ 1.2 Amino Pregnane Bases 0 1.3 D-.Homo Androstane Bases 0 1.4 Lactonic Bases ^ 1.5 Glyco Bases 8 2. Buxaceae Bases 2.1 Cyclobuxine Bases ^ 2.2 Buxenine Bases 1 0 3. Solanaceae Bases 3.1 Amino Spirotane Bases 3.2 Spirosolane Bases 3.3 Epimino Cholestane Bases 3.4 Solanidane Bases ^ 3.5 Solanocapsine L ^ 4. L i l i a c e a e Bases 4.1 Veratranine Bases i J 4.2 Jervanine Bases 27 4.3 Cevanine Bases 4.3.1 Jerveratrum Alkaloids 29 4.3.2 Ceveratrum A l k a l o i d s ^5 4.4 n-Methylene Amino Cholestane Bases 50 4.5 Summary of Discussion - i v -P a g e 5. Synthetic Proposal 51 5.1 Veratranine and Jervanine Bases 51 5.2 Cevanine Bases 55 DISCUSSION 57 6. F u n c t i o n a l i z a t i o n of C-18 i n C-nor-D-Homo Steroids. 6.1 Processes Described 57 6.2 Description of the Process U t i l i z e d 58 6.3 Modification of the Exocyclic O l e f i n Structure 61 7. Elimination of the Spiro Ketal System 86 7.1 Processes Described 86 7.2 Process U t i l i z e d 89 7.2.1 B a e y e r - V i l l i g e r Oxidation 89 7.2.2 Hydrolysis Studies 90 7.2.3 Conversion of the Side Products 112 8. Coupling of the Nitrogen Portion to C-Nor-D-Homo Steroid 114 8.1 Processes Described 114 8.2 Process U t i l i z e d 119 9. C y c l i z a t i o n of the Coupling Product 126 9.1 Process Described 126 9.2 Process U t i l i z e d 128 10. Conclusion 130 EXPERIMENTAL 138 BIBLIOGRAPHY 163 - v -LIST OF FIGURES Figure Page 1 Fundamental skeletons of the Apocynaceae Alkaloids. 2 2 Synthesis of the 5a-conanine skeleton 4 3 Synthesis of Latifoline and Conessine 5 4 Synthesis of Progesterone from Holamine 6 5 Synthesis of 5-dehydrodicytolucidamine 7 6 Synthesis of 5a-dihydrokibataline 7 7 Synthesis of Holantogenin and Anhydrohblantogenin . 9 8 Fundamental skeletons of the Buxus alkaloids 10 9 Synthesis of Cycloprotobuxine-C 11 10 Synthesis of a Buxenine base 11 11 Synthesis of the Buxenine skeleton 12 12 Synthesis of Buxidienine-F 12 13 Fundamental skeletons of the Solanum alkaloids .... 13 14 Synthesis of Pregnadienolone acetate from Solasodine 14 15 Synthesis of Jurubidine 15 16 Synthesis of Soladulcine and Tomatidine 16 17 Synthesis of Solafloridine 17 18 Synthesis of Solanidine 18 19 Synthesis of Solanocapsine 20 20 Fundamental skeletons of the Liliaceae alkaloids .. 21 21 Synthesis of 17-acetyl-5a-etiojervane-12,14,16-trien-3-ol 24 22 Synthesis of Veratramine 25 23 Synthesis of C-nor-D-homo steroids 26 24 Synthesis of Jervine 28 - v i -Figure Page 25 Mass s p e c t r a l fragmentation of Eduardine and Ed p e t i l i d i n e 36 26 Mass s p e c t r a l fragmentation of Korsine 39 27 Mass s p e c t r a l fragmentation of Ko r s e v e r i l i n e 41 28 Fundamental skeletons of the Ceveratrum a l k a l o i d s . 46 29 Carboxylic acids present i n the Ceveratrum esters.. 48 30 Synthetic scheme for Veratrum a l k a l o i d s 51 31 Synthesis of 5a,6-dihydroveratramine 53 32 Synthesis of 33-acetoxy-5a-etiojer-12(13)-en-17-one 54 33 Synthetic scheme for Cevanine bases 55 34 Degradation of 11-deoxo je r v i n e to an etiojervane d e r i v a t i v e f u n c t i o n a l i z e d at C-18 59 35 Synthesis of the C-nor-D-homo sapogenin fu n c t i o n a l i z e d at C-18 62 36 Hydroboration of the C-nor-D-homo sapogenin 64 37 Mass s p e c t r a l fragmentation of the s p i r o k e t a l system 66 38 N.M.R. spectrum of the 3 alcohol (209) 67 39 Mass spectrum of the 8 alcohol (209) 68 40 N.M.R. spectrum of the t e r t i a r y alcohol (211) 69 41 Mass spectrum of the t e r t i a r y alcohol (211) 70 42 Hydrogenation of the C-nor-D-homo sapogenin 73 43 Epoxidation of the C-nor-D-homo sapogenin 75 44 Osmic acid oxidation of the C-nor-D-homo sapogenin. 76 45 N.M.R. spectrum of the 3 aldehyde (238) 80 46 N.M.R. spectrum of the a aldehyde (239) 81 47 N.M.R. spectrum of the a alcohol (210) 84 48 N.M.R. spectrum of the a diacetate (240) 85 49 C l a s s i c a l elimination of the s p i r o k e t a l system .... 86 - v i i -Figure Page 50 C l a s s i c a l s p i r o k e t a l elimination applied to a C-nor-D-homo sapogenin 87 51 Degradation of the s p i r o k e t a l system v i a the Baeyer-V i l l i g e r r eaction 89 52 N.M.R. spectrum of the d i o l (252) 92 53 Mass spectrum of the d i o l (252) 93 54 N.M.R. spectrum of the tetraacetate (254) 95 55 Mass spectrum of the tetraacetate (254) 96 56 Compounds obtained i n the buffer hydrolysis 97 57 N.M.R. spectrum of the 6 diketone (257) 99 58 Mass spectrum of the 3 diketone (257) 100 59 N.M.R. spectrum of the diformate (258) 102 60 Mass spectrum of the diformate (258) 103 61 N.M.R. spectrum of the a,g-unsaturated ketone (256) 106 62 Mass spectrum of the a,g-unsaturated ketone (256) . 107 63 N.M.R. spectrum of the saturated ketone (260) 108 64 N.M.R. spectrum of the saturated ketone (260) 109 65 B a e y e r - V i l l i g e r oxidation mechanism 110 66 Conversion of the acetonide (270) to the saturated ketone (260) 115 67 N.M.R. spectrum of the diacetonide (270) 116 68 Mass spectrum of the diacetonide (270) 117 69 Coupling of the nitrogen portion i n the Solanum a l k a l o i d synthesis 118 70 High pressure l i q u i d chromatogram of the coupling product (277) 121 71 N.M.R. spectrum of the coupling product (277) 122 72 Mass spectrum of the coupling product (277) 123 - v i i i -Figure Page 73 N.M.R. spectra^ of the coupling products 124 74 Mass spectra of the coupling products 125 75 Synthesis of q u i n o l i z i d i n e d erivatives 127 76 Mass s p e c t r a l fragmentation of the p i p e r i d i n e s t e r o i d a l product (287) 129 77 N.M.R. spectrum of the p i p e r i d i n e product (287) ... 133 78 Mass spectrum of the p i p e r i d i n e product (287) 139 79 Mass sp e c t r a l fragmentation of deoxyverticirione . (288) 130 80 Mass spectrum of the c y c l i z a t i o n product 135 81 Mass spectrum of deoxyverticinone (288) 136 82 Mass spectrum of verti c i n o n e • 138 83 Proposal for the conversion of the a-cevanine base (288) to v e r t i c i n e (138) 131 84 Synthetic proposal for; the C-20 non-hydroxylated Veratrum a l k a l o i d s 132 - i x -GLOSSARY OF ABBREVIATIONS W.K. Wolff-Kishner reduction NCS N-chloro-succinimide BzCl Benzoyl chloride TsCl p-methyl benzene s u l f o n y l chloride NBS N-bromo succinimide NBA N-bromo acetamide DDQ Dichloro-dicyanoquinone TosNH-NH2 p-methyl benzene sulfbnyl_, hydrazine DMF Dimethyl formamide DMSO Dimethyl sulfoxide DCC Dicyclo hexyl carbodiimide - x -ACKNOWLEDGEMENTS I am indebted to Professor James P. Kutney for his guidance and continual encouragement throughout the course of my research. I would also like to acknowledge the many helpful suggestions from Dr. Alan F. Preston during this project. I am grateful to the National Research Council of Brazil for the award of a scholarship during the course of my studies. - 1 -INTRODUCTION The importance of steroidal compounds in l i f e processes has led to intense research into the chemistry of such compounds, particularly with regard to their synthesis. Although total synthesis s t i l l holds great possibilities,"'' i t appears that partial synthesis from plant natural products will remain the principal source of these substances. An important class of steroids is the steroidal alkaloids, several hundred of which have been isolated. These alkaloids may be conveniently divided into four main groups on the basis of their botanical source: 1. Apocynaceae Bases 2. Buxaceae Bases 3. Solanaceae Bases 4. Liliaceae Bases, While i t would be inappropriate in this thesis to discuss in detail a l l the extensive work involving the isolation, structure elucidation and syntheses of these natural products, a brief summary of the synthetic approaches developed in the above families will be presented. Also, in order to show how the present synthesis of Veratrum alkaloids has evolved, a discussion of the results involving structure elucidation in the Liliaceae group is presented since these - 2 -investigations have direct relevance to the main objective of our research. 1. Apocynaceae Bases: These bases are derived from five fundamental skeletons 1-5. Hi 4 5 R = amino sugar Figure 1. Fundamental skeletons of the Apocynaceae alkaloids. Conanine derivatives (1) are found in the genera Holarrhena, Funtumia and Malouetia. Simple amino pregnane derivatives (2) are found-' in Conopharyngia, Chonemorpha, and Dictyophleba. The latter genus also contains alkaloids derived from a 17a-methyl-g-homo androstane - 3 -skeleton (3). Basic s t e r o i d lactones derived from (4) were discovered i n P a r a v a l l i s and K i b a t a l i a . The representatives of c l a s s (5) were i s o l a t e d from Holarrhena spp. and constitute a new c l a s s of glyco-a l k a l o i d s i n which an amino sugar i s linked to t h i s s t e r o i d a l genin. The s t r u c t u r a l feature which imparts s p e c i a l i n t e r e s t to several of the above f a m i l i e s (1, 4 and 5) i s the f u n c t i o n a l i z a t i o n of C-18, a feature shared by the important hormone aldesterone (6). This s i m i l a r i t y has been u t i l i z e d i n the synthesis of aldosterone from 2 3 these a l k a l o i d s . ' 6 1.1 Conanine Bases Jeger's and Arigoni's groups collaborated i n the synthesis of the conanine skeletone from 3B-acetoxy-20-keto-5a-pregnane (7) as shown i n Figure 2. The most i n t e r e s t i n g step i n t h i s sequence involves r i n g closure to form the h e t e r o c y c l i c r i n g necessary for the a l k a l o i d system. Thus treatment of (10) with N-chlorosuccinimide gave the chloroamine which undergoes subsequent acid-catalyzed r i n g -closure generating 5a-conanine (11). The most important conanine a l k a l o i d i s conessine (21) of which several p a r t i a l and t o t a l syntheses have been published.^ Nagata et a l . reported a t o t a l synthesis of l a t i f o l i n e (20) which also - 4 -Figure 2. Synthesis of the 5a-Conanine skeleton . constitutes a formal synthesis of conessine since the intermediate (19) had been converted to this alkaloid previously.^ This synthesis (Figure:'3) started from the enone (12) and proceeded in a straight-forward manner to the tetracyclic intermediate 16 . One of the interesting steps in the synthesis involves the stereospecific introduction of the angular substituent at C-13 of the latter , this being achieved by addition of hydrogen cyanide catalyzed with triethylaluminum. Elaboration of the resultant cyano intermediate (17) via a reductive cyclization provided the natural system. 21 Figure 3. Synthesis of L a t i f o l i n e and Conessine. - 6 -1.2 Amino Pregnane Bases These bases are derived from pregnane with an amino substituent at either C-3 and/or C-20 and as might be expected several of these alkaloids have been synthesized from the corresponding pregnane derivatives. The importance of the amino pregnanes i s their role in the synthesis of steroidal hormones. For example, holamine (22) was transformed into progesterone (23), in 40% yield, by the action of g hypochlorite and aqueous a l k a l i . 22 23 Figure 4. Synthesis of Progesterone from Holamine. 1.3 D-Homo Androstane Bases These steroidal compounds are in general synthesized by ring expansion of the five membered ring D. The progesterone derivative (24) undergoes D-homo steroid rearrangement on treatment with base to give 183-hydroxy-18a-methyl-3,17-diketo-D-homo androst-4-ene (25) which was converted to the dienamine system in rings A and B and the latter was then reduced to yield 5-dehydrodicytolucidamine (26) as one of the products. - 7 -24 25 26 Figure 5. Synthesis of 5-dehydrodicytolucidamine. 1.4 Lactonic Bases Synthesis of the skeleton present in the paravallarine bases was accomplished by employing the cyanodione (27) previously obtained by Figure 6. Synthesis of 5a-dihydrokibataline. - 8 -6 XO Nagata i n the synthesis of conessine. ' Stepwise conversion of 27 by reduction and hydrolysis gave the intermediate lactone (28) which was subsequently elaborated to 5a-dihydrokibataline ( 3 0 ) . ^ Pa r a v a l l a r i n e bases have been considered as a l t e r n a t i v e precursors for aldesterone synthesis since such a l k a l o i d s are already oxygenated at C-18 and C-20. 1 2 1.5 Glyco Bases These bases found i n Holarrhena spp. have several c h a r a c t e r i s t i c features. In one ser i e s of compounds, the presence of a hydroxyl at C-14 i s noted (33) while i n another a c y c l i c k e t a l i s found (34). The l a t t e r system can also be formed when 33 i s treated under dehydration conditions and the anhydro'.rbase (34) i s reconvertible to the s t a r t i n g dihydroxy compound on addition of water (aqueous ethanol 13 or dioxan). This conversion has important synthetic implications since the synthesis of the g l y c o a l k a l o i d automatically implies the synthesis of the anhydro base. P a r t i a l syntheses of holantogenin (33) and anhydro holantogenin (34), the s t e r o i d a l moieties of the amino-glyco-steroids holantosines 14 A, B, C, and D, have been r e a l i z e d by two independent groups employing s i m i l a r pathways (see Figure 7 ) . ^ ' ^ The keto-acetate (31) was hydroxylated i n the 143-position by standard c l a s s i c a l methods."*"^ Saponification of the hydroxy-derivative (32) afforded holantogenin (33) , which was then dehydrated to anhydroholantogenin (34). - 9 -Figure 7. Synthesis of Holantogenin and anhydroholantogenin. 2. Buxaceae Bases Buxus a l k a l o i d s represent a new type of s t e r o i d a l k a l o i d which can be derived from structures 35 and 36. The two prototype skeletons, cyclo buxine (35) and buxenine (36) 18 have already been synthesized. In addition, i t should be noted that pregnane-type a l k a l o i d s such as those mentioned previously, can also be found i n some Buxaceae species. - 10 -^ YNR 2 R R 35 36 •NR. Figure 8. Fundamental skeletons of the Buxus alkaloids. 2.1 Cyclobuxine Bases Cycloprotobuxine-C (40), has been synthesized by a side chain degradation of cyclo artenol (37). Ozonolysis of the acetate and double Barbier-Wieland degradation gave the acid (38). Curtius degradation, followed by reduction of the resulting isocyanate, N-methylation and oxidation at C-3, gave the product (39), which was 19 readily transformed to the natural product via the oxime. 2.2 Buxenine Bases The buxenine skeleton has been synthesized by two interesting entries into the seven-membered ring B system. In the f i r s t of these, N-isobutyryl cyclobuxine-F (41) was treated with boron 20 trifluoride to produce the unsaturated seven-membered ring (42). In the other synthesis the steroidal compound (43) was subjected to a Wolff-Kishner reduction, giving an unexpected alpha-cleavage 21 to the product mixture of C-10 epimers (44). - 11 -Figure 9. Synthesis of Cycloprotobuxine-C. 2 Figure 10. Synthesis of a Buxenine base. - 12 -Figure 11. Synthesis of the Buxenine skeleton. Conversion of cyclobuxine into the buxenine structure has also 22 23 been achieved by Goutarel et a l . ' The Hot-alcohol resulting from mild reduction of cyclobuxidine-F (45) was treated with 30% sulfuric acid in dioxane; the product was identical with the natural base buxidienine-F (47). Figure 12. Synthesis of Buxidienine-F. - 13 -3. Solanaceae Bases Many Splanum species contain bases which are derived from five fundamental skeletons 48-52 . 52 Figure 13. Fundamental skeletons of the Solanum alkaloids. The 3-amino spirostanes (48), the 22,26-epimino cholestanes (49), the spirosolanes (50), the solanidanes (51) and solanocapsine (52) (the single steroidal alkaloid; of this type), have been obtained from a number of Veratrum, Sabadilla, and Zygadenus species in which they occur as glycosides or i n the free state. A l l the fundamental 24 skeletons of the Solanum alkaloids have been synthesized. - 14 -Like the conanine and paravallarine alkaloids, the spirosolane alkaloids are easily degradable to steroidal hormones. Thus, solasodine 25 afforded pregnadienolone acetate in about 65% yield. This facile conversion has resulted in the Solanum alkaloids receiving increased attention as starting materials for the commercial synthesis of hormonal steroids. 53 54 55 Figure 14. Synthesis of Pregnadienolone acetate from Solasodine. 3.1 Amino Spirotane Bases Syntheses of the 3-amino spirostan (48) from the corresponding 26 27 sapogenins have been already described. ' An outline of this synthetic work i s given i n Figure 15. Neotigogenin (56) was oxidized to a ketone -and i t s 3-oxime reduced with sodium-alcohol to jurubidine (59). Consideration of both the above conversion and the total synthesis 28 of sapogenins by Sondheimer et a l . , shows that total synthesis of amino spirostan alkaloids has been acomplished. - 15 -Figure 15. Synthesis of Jurubidine. 3.2 Spirosolane Bases Spirosolanes have been synthesized from simple, synthetically 29 available pregnane derivatives. Thus, 38-a.cetoxy-5a-preg-16-en-20-one (60) was converted into 38,168-diacetoxy-5a-pregnan-20-one (61), which by reaction with 2-lithium-5-methyl pyridine afforded a mixture of epimeric pyridyl alcohols (.62). Reacetylation to the 3,16-diacetate and subsequent dehydration gave the conjugated 20-olefin (63). - 16 -Catalytic hydrogenation of 63 followed by alkaline hydrolysis gave as the major product an octahydro derivative identical with tetrahydrosolasodine A (65). Cyclization of the compound 65 via the corresponding N-chloro 30 derivative using sulfuric acid, or by U.V. irradiation in a strong 31 acid medium of the nitrosamine, afforded the natural spirosolane alkaloids soladulcine (66) and tomatidine (67) in high yields. H H 66 67 Figure 16. Synthesis of Soladulcine and Tomatidine. - 17 -3.3 22-26 Epimino Cholestane Bases Epimino cholestanes have been synthesized by a minor modification of the sequence of reactions leading to soladulcine and tomatidine. Thus 3B,16a-diacetoxy-5a-preghan-20-one (68) was converted ( as outlined in Figure 17) into the diacetate (70), the latter being isomeric with 63. Figure 17 . Synthesis of Solafloridine. - 18 -The N-chloro derivative of the diacetate (71) gave, after HC1-elimination, the alkaloid solafloridine (72), which did not cyclize to 33 34 a spiro amino ketal. ' The same diacetate was also oxidized and then reduced to yield the solanidane derivative (73). 3.4 Solanidane Bases A totally synthetic route leading directly to solanidane derivatives 35 has been achieved by Kassar e_t a l . In this study Michael addition of methyl-5-nitro-2S-methyl pentanoate (75) to cis-5,17(20)-pregnadien-38-20-one (74) gave the nitro esters (76) and (77). Subsequent reduction of 76 gave the amide (78) and the pyrrolidine derivative (79). The pyrrolidine ring in the latter was then reduced by means of sodium borohydride to the amide (78) which on further reduction yielded solanidine (80). Figure 18. Synthesis of Solanidine . - 19 -3.5 Solanocapsine 36 This unusual a l k a l o i d , i s o l a t e d from Solanum pseudo capsicum L., was proposed as having the structure of a 3B-amino-5a-steroid with an a-epimino-cyclo hemiketal moiety. 37 In the f i r s t synthesis published for t h i s a l k a l o i d the synthetic product was very s i m i l a r to, but not i d e n t i c a l with the natural product. The intermediates i n t h i s degradation were not i d e n t i c a l with the corresponding compounds obtained from the natural product. Recent chemical and N.M.R. inves t i g a t i o n s on derivatives of both s e r i e s have c l a r i f i e d these ambiguities. It was shown that solanocapsine possesses the e n e r g e t i c a l l y most favored structure as shown i n (52), with the trans f i s s i o n of rings E and F, thereby p l a c i n g the 25-Rrmethyl group i n the equatorial p o s i t i o n . 38 The s t a r t i n g material selected f or a second synthesis was the a l k a l o i d s o l a f l o r i d i n e (81) and the successful sequence i s shown i n Figure 19. 4. L i l i a c e a e Bases Many genera of t h i s family contain a l k a l o i d a l bases which are derived from f i v e fundamental skeletons .88'-,,92^  . 5a-Veratranine (88), 5a-jervanine (89), and 5a-cevanine (90) derivatives are obtained from a number of Schoenocaulon, Zygadenus, Stenanthium, F r i t i l l a r i a , P e t i l l i u m , and Veratrum species i n which they occur as glycosides or i n the free state. Alkamines with the fundamental structures (49 and 50) are also found i n t h i s family. - 20 -AcO 81 l)Ac 20,ZnCl 2,AcOH * / C H 2 ) M n 0 2 ' C H 2 C 1 2 3) NaBH. AcO Cr0 3,H 2S0 4 AcO KOH.MeOH O-C-Cl H- 83 H ! 1 J L°* > l)HBr,AcOH C i v * 2)NH2OH Figure 19. Synthesis of Solanocapsine. - 21 -88 90 91, R=glucose 89 92 Figure 20. Fundamental skeletons of the L i l i a c e a e a l k a l o i d s . Sewkorin'e (91) and veracintine (92) have been i s o l a t e d from 39 Korolkovia ( F r i t i l l a r i a ) sewerzsowii and Veratrum album subsp. 40 lobelianum res p e c t i v e l y and constitute the only representative of each group . Despite the number of genera mentioned, these bases have been referred to as Veratrum a l k a l o i d s . Reviews of the Chemistry of 41 42 43 Veratrum a l k a l o i d s have been written by F i e s e r , B o i t , Narayanan, 44 and Kupchan. D i v i s i o n of the Veratrum a l k a l o i d s into jerveratrum and ceveratrum groups as proposed by Fieser and Fieser i s now generally - 22 -accepted. The jerveratrum alkamines contain only 1 to 3 atoms of oxygen; they are found i n the unhydrolyzed plant extracts i n part as free a l k a l o i d s and p a r t l y i n combination with one molecule of g-glucose as glucoalkaloids. One of the most i n t e r e s t i n g features of these natural products i s the presence of a C-nor-D-homo s t e r o i d template. E0-93 Verarine R=H 94 Jervine 95 Veramarine Veratramine R=0H The ceveratrum a l k a l o i d s are.highly oxygenated and generally occur as esters. A l l the cevanine a l k a l o i d s are also characterized by the C-nor-D-homo nucleus. 97 Veracevine 4.1 Veratranirie Bases 45 Johnson reported the total synthesis of an appropriate C-nor-D-homo skeleton i n his synthesis of veratramine from 17-acetyl-5 -etiojervane-12,14,16-trien-3-ol (109). This compound was totally synthesized from Hagemann's ester (98) as outlined i n Figure 21. - 24 -108 Figure 21. Synthesis of 17-Acetyl-5a-^etl"o j ervane-12,14,16-trien-3-ol. The f i n a l conversion of compound (109) into veratramine (116) was achieved by the sequence shown in Figure 22. The piperidine ring is built up from the uncyclized compound (111) through .!110 and 112 to yield a mixture of epimeric-N-benzoyl-5,6-dihydro-3,23-diketoveratramines (113). The latter mixture was compared with an authentic sample prepared from veratramine. Introduction of the 5,6- double bond was 4 performed via standard steroid reactions (115 -*- A -3-ketone enol acetate -*• 116). - 25 -116 Figure 22. Synthesis of Veratramine. - 26 -46 Recently Fried reported an efficient approach to the veratranine bases u t i l i z i n g readily available fluorene derivatives. The synthetic pathway involves only seven steps from 117 (see Figure 23). Figure 23. Synthesis of C-nor-D-homo Steroids. - 27 -The cis stereochemistry between rings B and C in compounds (123a) and (123b) was determined using an interesting dehydrogenation reactioi with cells of Arthrobacter simplex. The remaining relative configura-tion was assigned by X-ray analysis. 4.2 Jervanine Bases Masamune employed 17-acetyl-5a-etiojerva-12,14,16-triene-3B-ol 47 (126) i n his synthesis of jervine (see Figure 24). In this approach an enamine derivative (128) is ut i l i z e d in attacking the heterocyclic unit to the appropriate side chain of the C-nor-D-homo steroidal template (127). The resulting product (130) is then elaborated to the f i n a l product. Perhaps the most interesting reactions in the later stages concern the formation of the furano system required for the jervine molecule. Thus Birch reduction of 131 with L i in ethylamine containing isopropyl alcohol produced a mixture of dihydro derivatives which were hydrogenated without further purification over platinum in acetic acid to give 132. The ether bridge between C-17 and C-23 was formed by a series of elegant steps. Acetylation followed by perbenzoic acid oxidation produced the 13,17-epoxide (133) which was reacted with potassium hydroxide in aqueous dimethyl sulfoxide to form 173,233-oxidojervane-33>13a-diol (134). Although the introduction of the 11-keto group in intermediate (135) proceeded in 1% yield, the authors were able to complete the sequence to jervine. The 3-D,N-diacetyl derivative of 134 underwent dehydration with thionyl chloride and pyridine to give 3-0,N-diacetyl-ll-deoxo-5a-* This compound was also utilized by Johnson in his synthesis of veratramine. - 28 -Figure 24. Synthesis of Jervine. - 29 -dihydrojervine (135). Oxidation with chromic anhydride gave the compound 136 from which jervine (137) was obtained in several steps in a yield of 2%. 4? 3 Cevanine Bases As mentioned before, the hexacyclic cevanine skeleton present in Liliaceae alkaloids has yet to be synthesized. Since this thesis is concerned with developing a synthetic entry into jerveratrum alkaloids incorporating this skeleton, i t i s appropriate here to discuss briefly the work which has thus far been done in this f i e l d . Special attention w i l l be given to the structure elucidation because in a number of instances the structural postulates are inconclusive and i t is hoped that our synthetic work should prove whether or not the assigned structures are correct. 4.3.1 Jerveratrum Bases -Several representatives of these alkaloids have been isolated from F r i t i l l a r i a , Petillium, and Veratrum genera. 23 6 Alkaloid Substituents in the Cevanine skeleton C-3 C-6 C - l l C-14 C-15 C-16 C-20 C-23 C:C References Verticine- 60H aOH - - - - ."• 80H - - 52,53 Verticinone BOH C:0 - - - - BOH - - 52 Veramarine OH - - - - OH OH - 54 Edpetilidine BOH BOH - - - - - - - 55 Eduardine BOH C:0 _ _ _ _ _ _ _ 55 Petilinine aOH aOH - - - - - - 64 Petilidine BOH aOH - - - - - - - 65 Korseveriline aOH BOH - aOH - - - - - 62 Korseveramine BOH aOH - aOH - - - - - 68 Korseveridine BOH - - BOH - - C8(14) 58 Korsine BOH - aOH - - - - OH C8( 9) 60 Korsinine aOH aOH - - - - - - C8( 9) 67 Korseverinine BOH BOH - - - - - - C8( 9) 69 Korseverine BOH C:0 - - - - - - C8( 9) 66 peimine; peiminine, f r i t i l l a r i n e , imperialine, seipimine and raddeaninei - 31 -Verticine, C2yH^^0^N (138), the most extensively investigated alkaloid of this group, was originally isolated from F r i t i l l a r i a 48 v e r t i c i l l a t a var. Thunbergii Baker. B O H 138 Verticine, as well as the corresponding 6-keto compound verticinone, was also isolated by Chou and Chen from the bulbs of F r i t i l l a r i a roley after i t was noted that the pharmacological effects of the plant 49 extracts resembled those of Veratrum alkaloids. 50 Dehydrogenation studies by Chu, Hwang and Loh showed that verticine gave lutidine (139) and two hydrocarbons, one of which was identified as compound 140. Extensive experiments were carried out concurrently on the dehydrogenation of verticinone, permitting the isolation of 2,5-lutidine, and the hydrocarbon (140) as well as the base veranthridine (141). The identity of the products in the dehydro-genation has indicated that these bases had a similar skeleton; and since the reduction of this alkaloid gave verticine, these two alkaloids were therefore interrelated. Morimoto and Kimata isolated verticine and i t s C-3-D-glycoside from F r i t i l l a r i a ^Thunbergii MIQ."^ They assumed that verticine had the cevanine skeleton and therefore formulated the new glycoside as (142). - 32 -Ito and coworkers'^ also investigated the structure of verticine.. They postulated that one hydroxyl group was in the 33-position from evidence derived from O.R.D. measurements on the keto derivative. The other hydroxyl group was; assigned to C-6, since verticinedione (143) was observed to undergo aerial oxidation in a l k a l i medium to form a 4-ene-3,6-dione system (144) (\ 375 nm). max The tertiary hydroxyl group was assigned to C-20 on the basis of mass spectrometry, and was suggested to be axial by basicity and I.R. absorption studies. The stereochemistry of the C-27 methyl group was assigned as axial from N.M.R. studies. Although the stereochemistry at carbons 8, 9, 12, 14, 16, and 17 was postulated only by analogy with the s t e r o i d series and from biogenetic considerations, the correctness of the proposed structure was l a t e r shown by an X-ray analysis . 53 Veramarine, C-H.-O.N (145), was i s o l a t e d from Veratrum album 27 43 3 54 145 It possesses three hydroxyl groups, one of them being t e r t i a r y since i t i s not acetylated with a c e t i c anhydride i n pyridine while chromic acid oxidation led to a diketone, the I.R. spectrum of which s t i l l i n dicated the presence of an hydroxyl group. On the basis of molecular r o t a t i o n differences the carbonyl groups were assigned to the C-3 and C-16 po s i t i o n s . - 34 -The N.M.R, spectrum of veramarine exhibits one secondary and two tertiary methyl groups, and one vinylic proton at 5.4 x, the secondary methyl group being attached at C-25 while the tertiary groups are at C-10 and C-20. On the basis of molecular rotation differences between veramarine and dihydroveramarine, the double bond i n the former compound was proposed to be located in the 5-position. Biogenetic considerations were used in proposing the f i n a l structure for veramarine. Although this structure proposal for veramarine i s plausible, further proof i s desirable. In 1968, Kupchan published a review of Veratrum alkaloids which included a l i s t of several alkamines who.se structures were either not 44 known or incompletely elucidated. Since then, Russian workers have published structural proposals for several members of this l i s t based mainly on mass spectrometry and biogenetic considerations. Although complete structural elucidation of these compounds has s t i l l to be achieved, the mass spectral evidence put forward w i l l be most useful in the characterization of synthetic cevane analogues. Edpetilidine, C^H^O^ (146) and Eduardine, C^H^O^ (147) were isolated from Petillium eduardii. A r e l a t i o n s h i p between edpetiiidiwe-and eduardine was established when i t was shown that the two compounds could be interconverted by oxidation and reduction. I.R. studies showed that e d p e t i l i d i n e contains hydroxyl groups and eduardine i s a k e t o l while the e l u c i d a t i o n of the cevane skeleton was based on mass s p e c t r a l and N.M.R. comparisons with v e r t i c i n o n e . ^ ' - ^ As shown below, verticinone r e a d i l y fragments to give peaks at m/e 155 and 156, the absence of such peaks i n d i c a t i n g that e d p e t i l i d i n e and eduardine do not possess a.hydroxyl group at C-20. Moreover, the formation of a m/e 218 peak analogous to the m/e 217 peak i n verticinone,. indicates that rings D, E, or F do not contain any oxygen functions. From N.M.R. studies of the chemical s h i f t s of the C-19 methyl protons, e d p e t i l i d i n e was assigned both 38- and 6B-hydroxyl groups, while eduardine was assigned a 38-hydroxyl group and a C-6 carbonyl group. - 36 -149 ' m/e 218 Figure 25. Mass s p e c t r a l fragmentation of Eduardine and E d p e t i l i d i n e . The values of the C-19 methyl signals i n the N.M.R. spectra of eduardine, e d p e t i l i d i n e and t h e i r d e r i v a t i v e s confirm that the linkage of the A, B, C, and D rings i s the same i n v e r t i c i n o n e . ^ From the p o s i t i o n of the signals and chemical s h i f t s i t was concluded that the C-21 methyl i s equatorial and the C-27 methyl i s a x i a l . - 37 -H 21 150 I n i t i a l chemical studies on koseveridine indicated that i t possessed two hydroxyl groups, neither of which was t e r t i a r y , while the prominence of the peaks m/e 155 and 156 i n the mass spectrum (see reduction studies showed that the molecule had a tetrasubstituted double bond, confirmation of this assignment coming from the N.M.R. spectrum which exhibited no o l e f i n i c protons. By a process of elimina-t i o n using N.M.R. and mass s p e c t r a l data i t was concluded that the double bond was located between C-8 and C-14. The N.M.R. spectrum of korseridione indicated a C-3 hydroxyl group with the second hydroxyl i n koseveridine being located at eith e r C - l l or C-15. The i n s t a b i l i t y of korseveridione i n chromic acid supported the p o s t u l a t i o n of C-15 as the s i t e f o r the second hydroxyl group. Detailed analysis of the N.M.R. spectrum led to assignment of the stereochemistry as shown i n 150. Figure 25 ) eliminated the p o s s i b i l i t y of a C-20 hydroxyl. 59 Oxidation-- 38 -Korsine C^H^^O^ (151), was isolated from the bulbs of Korolkovia (Fritillaria) sewerzowii Rgl^ This base forms a triacetate, showing the presence of three acylable hydroxyl groups. Hydrogenation gives dihydrokorsine, C^ H^^ O^^ N while oxidation gives two ketones: korsinone, ^27^41^3^' a m * korsinedione, C2yH^g03N. Korsinone has a uv spectrum (X 245 and 300 nm) characteristic of an a,3-unsaturated ketone, max The fact that the molecule contains 27 carbon atoms, the mass spectral fragmentation being characteristic for a cevane type and the UV spectrum mentioned above a l l suggest the cevanine skeleton for korsine 50,56 151 The electron-impact induced fragmentation of korsine and korsine-dione is analogous to the fragmentation of verticinone and edpetilidine.' In the mass spectra of the latter two alkaloids, peaks at m/e 98, 111 and 112 appear in the low-mass region. In the spectrum of korsine,-peaks at 114, 127 and 128 are present; in that of korsinedione, peaks at 112, 125 and 127. Since the ions of korsine differ from the corresponding ions of verticinone and edpetilidine by 16 mass units, the hydroxyl group in korsine is most likely located in the heterocyclic ring F. If korsinedione had a carbonyl in positions 18 or 26, i t - 39 -would possess the.properties of an amide. Since there i s no absorption of an amide carbonyl group in the I.R. spectrum of korsinedione, the location of a hydroxyl at position C-18 or C-26 can be excluded. Biogenetic considerations were uti l i z e d to assign the hydroxyl group to the C-23 position. The absence of an olefinic proton in the N.M.R. spectra of korsine and i t s triacetate permits the assignment of the double bond between either C-8 and C-9, C-8 and C-14, C-12 and C-13, C-12 and C-14 ,C-13 and C-17, C-17 and C-20 or C-20 and C-22 . The latter two positions are excluded because of the absence in the N.M.R. spectrum of a singlet expected for the protons of a methyl group attached to an unsaturated Figure 26. Mass spectralfragmentation of Korsine. - 40 -carbon atom. If the double bond were present between either C-13 and C-17, C-12 and C-13, or C-12 and C-14, m/e 178 and 180 fragments could not be formed from korsine nor m/e 176 and 178 from korsinedione. If the double bond were located between C-8 and C-14, the resonance signal from the protons of the C-19 methyl groups of korsine should shift to a lower f i e l d after hydrogenation, i.e. deshielding should take place; this, however, i s not the case. The only possible position remaining for the double bond i s between C-8 and C-9. Hence, the second hydroxyl i s placed at C-7 or C - l l , since the U.V. spectrum of korsinone has an absorption maximum characteristic of a,6-unsaturated ketones. N.M.R. and I.R. considerations were ut i l i z e d to locate the remaining hydroxyl groups at C-3 and C - l l and to assign the stereo-chemistry presented for korsine. Korseveriline C^E^O^N (153), was isolated from Korolkbvia 62 ( F r i t i l l a r i a ) sewerzowii Rgl. It is a saturated tertiary base containing one tertiary and two secondary hydroxyl groups. The mass spectra of korseveriline indicated a cevanine skeleton with the formation of m/e 162, 164 and 178 fragments, produced from the heterocyclic parts of the molecule, indicating that the most probable position for the tertiary hydroxy group i s at C-14. Differences in the chemical shifts observed i n the N.M.R. spectra of korseriline and i t s oxidation product korseverilinedione (C^R^O^) were used to locate the secondary 63 hydroxyl groups in the a- and 3-positions at C-3 and C-6, respectively. By comparison with known chemical shifts of C-21 and C-27 protons, the a-configuration was assigned to these groups. - 41 -Figure 27. Mass spectralfragmentation of Kor s e v e r i l i n e . P e t i l i n i n e C2^H^^02N (154), has two secondary hydroxyl groups, as shown by the formation of a diacetate, and a diketone p e t i l i n i n e d i o n e . Evidence from U.V. absorption and from the mass and N.M.R. spectra indicates that p e t i l i n i n e has the cevanine skeleton. The formation of an m/e 111 ion indicated that t h i s a l k a l o i d does not possess a C-20 hydroxyl g r o u p . ^ N.M.R. and I.R. spectra confirmed the presence of 3a- and 68-hydroxyl group and established the stereochemistry at C-21 and C-27. From the magnitude of t h e i r -chemical shifts, the C-21 and C-27 methyl groups were assigned the a-configuration. Petilidine C2-H^ ,.02N (155), has two acylable hydroxyl groups and is oxidized with chromic acid to a diketone identical to petilininedione, suggesting a structural relationship between petilinine and petilidine.^ In the mass spectrum of petilidine, as i n that of petilinine, the main characteristic peaks are those at m/e 97, 98, 111, 112, 397, 400, and 415 (M+). Consequently, petilidine differs from petilinine only in the configuration of the hydroxyl group. From N.M.R. i t was established that in petilidine the 3-hydroxyl has the 8-configuration and the 6-hydroxyl i s i n the a-orientation. i 27 155 - 43 -Korseverine C-H.-O-N (156), was isolated from Korolkowia 27 41 2 — 66 ( F r i t i l l a r i a ) sewerzown. It was shown by I.R. spectra and chemical modifications to possess an alcohol, a ketone and a tetrasubstituted double bond. The cevanine skeleton of korseverine was established by U.V. and mass spectral considerations.^»56 T^ .M.R. studies on the parent alkaloid and i t s acetylated derivative located the hydroxyl at the 3g-position, the carbonyl at C-6, and the double bond i n the 8,9-position. Korsinine C„-,H/o0oN (157), was isolated from Korolkowia ( F r i t i l l a r i a ) 27 43 2 sewerzowii 67 157 - 44 -Chemical modifications coupled with I.R. and U.V. spectra established that korsinine possessed a double bond and two secondary hydroxyl groups. Mass and N.M.R. spectra were used to locate the double bond between C-8 and C-9, while the hydroxyls were assigned the 33- and 63-positions. Furthermore, i t was shown that the C-21 a C-27 methyl groups have a and 3 configurations respectively. Korseveramine C2 7H^0 3N (158) was isolated from Korolkowia 68 ( F r i t i l l a r i a ) sewerzowii. 158 I n i t i a l chemical studies on korseveramine established that the compound possessed two secondary hydroxyl groups and a tertiary one. N.M.R. studies established the secondary hydroxy groups at the 38-and 68-positions. The position of the tertiary hydroxy group was assigned as 14a by a process of elimination through comparison of the mass and N.M.R. spectra with those of other cevanine alkaloids. - 45 -Korseverinine C0-H,o0oN (159) was isolated from Korolkowia Korseverinine gave both a monoketone, korseverinone (160) and a dione, korseveriniriea dione (161) on oxidation with chromic acid. N.M.R. studies on the parent alkaloid and i t s two oxidation products led to the assignment of the hydroxyl groups to the 3a- and 6a-positions. The mass spectra of these compounds showed that the double bond was in the 8(9) position. 4.3.2 Ceveratrum Bases The structures of most of the hypotensive veratrum alkaloids have been elucidated during the late 1960's. These alkaloids are esters of 6 fundamental bases; zygadenine (162), zygadenilic acid 6-lactone (163), sabine (164), veracevine (165), germine (166) and protoverine (167). A l l known alkaloids of this type, except sabadine, are cyclic hemiacetals in which the 4- and 9-positions are joined by an oxygen bridge. They contain from six to eight hydroxyl groups; one to four - 46 -Figure 28. Fundamental skeletons of the Ceveratrum alkaloids. - 47 -* Structures of the Ceveratrum A l k a l o i d s . Compound Acyl Groups C-3 C-6 C-7 C-15 Esters of Zygadenine (162) Angeloyl zygadenine An -Ac e t y l zygadenine Ac " 3 ( 1 , 2 - m e t h y l butyryl)zygadenine MB Veratoyl zygadenine Ve - - -V a n i l l o y l zygadenine Va Ester of Zygadenilic acid 6-lactone (163) Angeloyl zygadenilic a c i d 6-lactone (a) - -Ester of Sabine (164) Sabadine Ac Esters of Veracevine (165) Cevacine Ac Cevadine An - - -Veratridine Ve - - -V a n i l l o y l veracevine Va — — — Esters of Germine (166) Germitetrine HA Ac MB Germitrine MB - Ac HM ne o-Ger mi t r ine Ac - Ac MB Germanitrine An - Ac MB Germerine MB - - HM Germedine Ac - - MB neo-Germidine - - Ac MB Gerbudine TMD - - MB neo-Germbudine EMD - - MB Protoveratridine MB - - -Germinitrine (b) - - — — Esters of Protoverine (167) Protoveratrine A HM Ac Ac MB Protoveratrine B TMD Ac Ac MB Escholerine An Ac Ac MB Desacetyl protoveratrine A HM Ac - MB Desacetyl protoveratrine B TMD Ac MB From page 217 Reference 44. (a) This ester has been characterized as the 16-angelate. (b) This a l k a l o i d has been reported to be a t r i e s t e r which y i e l d s a c e t i c acid, t i g l i c a c i d , and angelic acid upon hydro l y s i s . The p o s i t i o n of the a c y l groups have not been determined. - 48 -of which may be e s t e r i f i e d at p o s i t i o n s 3, 6, 7, and 15 with the following acids: Ceveratrum acids Acyl group Acetic Ac Erythro-2-methyl-2,3-dihydroxy b u t y r i c EDM cis-2-Methyl-2-butenoic (angelic) An (£)-2-Hydroxy-2-methyl b u t y r i c HM Erythro-2-hydroxy-2-methyl-3-acetoxy b u t y r i c HA («,)-2-Methyl b u t y r i c MB threo-2-Methyl-2,3-dihydroxy b u t y r i c TMD 4-Hydroxy-3-methoxy benzoic ( v a n i l l i c ) Va 3,4-Dimethoxy benzoic ( v e r a t r i c ) Ve Figure 29. Carboxylic acids present i n the Ceveratrum esters. A d e t a i l e d d e s c r i p t i o n of the s t r u c t u r a l studies developed for 44 these bases i s described i n Kupchan's review. Since the p u b l i c a t i o n of the l a s t review, only one other a l k a l o i d has been i s o l a t e d . ^ Germinaline C.-H-.-O.. „N (168), was i s o l a t e d from Veratrum lobelianum. J9 o l 1_ This alkaloid.revealed a band i n the I.R. spectrum at 3450 cm 1 due to a hydroxyl group and bands at 1738, 1250 cm 1 due to an ester group. The N.M.R. spectrum exhibited at x 8.21 a s i n g l e t (3 protons) i n d i c a t i v e of an acetate. - 49 -Basic hydrolysis of germinaline gave an amino alcohol, C^H^gOgN, which was identical to the hydrolysis product obtained from the known ceveratrum alkaloid germerine (169). Acetylation of germinaline gave a product identical to the one obtained under the same conditions from germerine. The two alkaloids, therefore, had the same nucleus (166) and differ only in the esterification of the hydroxyl groups. The above data also require, that germinaline, as well as germerine, must possess (£)-2-methyl butyric ester at C-3 and a (£)-2-methyl-2-hydroxy butyric ester at C-15. The acetate group could be present at either C-7 or C-16, the two remaining secondary alcohol.igroups in the germine nucleus. Its position was determined by a process of elimination, since germinaline i s different from the known alkaloid germitrine (170) in which the acetate is at C-7. The only position l e f t for the acetate group in germinaline was at C-16 and therefore i t s structure should be represented as (168). - 50 -4.4 n-Methylene Amino Cholestane Bases Sewkorine (91) and veracintine (92) represent two new types of biogenetically interesting alkaloids in which the side chain of the steroidal skeleton is cyclized, forming a 5- and 7-membered heterocycle. Since biogenetic considerations were used extensively in their structure elucidation work, syntheses of sewkorine and veracintine are required in order to prove their structure conclusively. To date, none have been published. 4.5 Summary of Discussion In the above discussion I have tried to provide a very brief summary of the extensive investigations which have been performed in this large area of natural products. In particular, emphasis has been placed on the members which have direct relevance to the synthetic problem to be discussed in the next section. It should be clear from the discussion that in many of the most recent studies., involving the structures of new cevanine bases, only tentative conclusions can be put forth since heavy reliance has been placed on spectroscopic data and unambiguous chemical correlations have not been done. I hope to reveal in the next section of this thesis how the synthetic investigations undertaken in our laboratory w i l l provide an opportunity to evaluate some of these structural proposals and hopefully to place them on a more rigorous basis. - 51 -5. Synthetic Proposal 5.1 Veratranihe and Jervanine Bases^ During the past few years members of this laboratory have been actively engaged in research designed to provide a totally synthetic entry into the Veratrum alkaloids with the 5a-veratranine and 5a-jervanine skeletons. The approach has been to consider the veratrum skeleton as consisting of two fundamental building blocks: (1) the C-nor-D-homo steroidal portion and, (2) a substituted piperidine. The aim has been to provide a total synthesis of these two molecules and then couple them in such a manner as to provide the desired skeleton, as represented in Figure 30. a : 5a-veratranine bases a + b: 5a-jervanine bases a + c: 5a-cevanine bases Figure 30. Synthetic scheme for Veratrum alkaloids. 71 72 The f i r s t two approaches have been completed with success. ' Coupling of 38-acetoxy-5a-etiojerv-12-en-17-one (171) with the lithium derivative of 2-ethyl-5-methoxy pyridine (172) gave the compound 173) which on treatment with 10% palladized charcoal is converted to the aromatic compound (174;. Reduction of the latter compound in 95% - 52 -ethanol containing 2% hydrochloric acid i n the presence of Adams' ca t a l y s t (PtC^) gave several products from which 5a ,6-dihydroveratramine (179) was i s o l a t e d i n 18% y i e l d . I t was expected that the reduction of the pyridine r i n g , under a c i d i c conditions, would lead d i r e c t l y to the desired system since the possible dihydropyridine intermediates 176 and 177 would react further i n the required manner (Figure 31). The t o t a l synthesis of 5a,6-dihydroveratramine was considered 45 since t h i s compound had already been converted to veratramine, 47 ... , 73 , . . 74 j e r v i n e , 11-deoxojervme, and veratrabasme. The s t e r o i d a l moiety desired to provide the etiojervane portion was 3g-acetoxy-5a-etiojerv-12(13)-en-17-one (171), a substance r e a d i l y a v a i l a b l e from the degradation of hecogenin. The procedure due to W.F. Johns was employed.^ Treatment of hecogenin (180) with t o s y l hydrazine i n a c e t i c a c i d gave the tosylhydrazone (181) which on r e f l u x with sodium hydroxide i n ethylene g l y c o l gave the endocyclic o l e f i n (182). An o v e r a l l y i e l d of 65% of t h i s o l e f i n from hecogenin was obtained. Reduction of 182 over palladium c a t a l y s t proceeded r e a d i l y to give a dihydro d e r i v a t i v e (183) i n which the side chain was l e f t i n t a c t . Degradation of the s p i r o k e t a l side chain i n a w e l l known manner gave the a,8-unsaturated ketone (184).' Degradation of the two carbon side chains i n the l a t t e r by use of the Beckmann rearrangement gave the saturated ketone (185). Monobromination, then dehydrobromination, led predominantly to the unsaturated ketone (171). 76 This compound was also prepared by a t o t a l l y synthetic route which involves a modification of the r i n g contraction process developed by W.S. Johnson from h i s hydrochrysene s y n t h e s i s . ^ - 53 -Figure 31. Synthesis of 5a,6-dihydroveratramine. - 54 -AcO 180 TdsNH-NEL TosNHN AcO 181 KOH (CH 2OH) 2 AcO 183 D(CH 3(CH 2) 6CO) 20 2) Cr0 3 3) t-BuOK 4) Ac 20/pyr. 1) H2/Pd 2) NH.;>0H 182 18 3) POCl 3/pyr 4) HC1 "AcO 184 185 D B r 2 2)MgO/DMF 171 Figure 32. Synthesis of 33-acetoxy-5a-etiojerv-12(13)-en-17-one. - 55 -5.2 Cevanine Bases The general synthetic approach followed i n the syntheses of veratranine and jervanine bases can be applied to cevanine bases, provided that the etiojervane portion contains some type of C-18 f u n c t i o n a l i t y i n order to permit the construction of the rin g E. S = substituent; R = protecting group Figure 33. Synthetic scheme for Cevanine bases. This thesis represents our e f f o r t s towards the synthesis of the etiojervane portion (186) and i t s conversion into the skeleton present i n the a l k a l o i d v e r t i c i n e (138). This base i s the only a l k a l o i d i n t h i s 53 series whose structure i s known unambiguously. With the appropriate modifications to th i s sequence , as mentioned l a t e r , i t should be possible to synthesize the more recently i s o l a t e d jerveratrum a l k a l o i d s with the cevanine skeleton -^5,64,65 w e r e presented b r i e f l y i n the previous section of th i s t h e s i s . - 56 -The most important aspect of this synthetic program w i l l be the access to several compounds not easily obtained from the natural products by degradation reactions. These compounds w i l l be submitted to biological screening since many of them have potential pharmaco-78 79 logical and insecticidal activity. This fact combined with the f i n a l structural proof of these alkaloids associate the pharmaceutical and academic interest and explain our own interests in the subject. - 57 -DISCUSSION 6. Functionalization of C-18 in C-nor-D-homo Steroids The i n i t i a l objective of our synthetic scheme was to obtain an etiojervane system with a useful functionality at C-18. In this respect our approach would parallel the synthesis of the conanine and paravallarine bases (see Section 1.1 and 1.4) which requires functionalization in a similar manner. 6.1 Processes Described As compared with the normal steroids, l i t t l e success has been reported for the functionalization of C-18 methyl groups in the C-nor-D-homo series. Masamune's group has published some attempts in their goal of transforming Jerveratrum alkaloids into compounds containing the 80 Cevine nucleus. 11-Deoxp jervine (188) was hydrogenated to the 81 hexahydrojervine derivatives (189) and (190). Degradation of 189.' afforded an aldehyde (191) which was subsequently transformed into the keto alcohol (192). Acetylation of 192 followed by reduction gave the alcohol (193) which was then treated with iodine in cyclohexane containing lead tetraacetate and sodium carbonate, and this solution was irradiated for 30 minutes. Two products were formed, '.194, and - 58 -an unidentified compound. Compound 194, was oxidized to the corresponding y-lactone (195) with chromic acid in acetic acid in 19% yield. The treatment of 192 with either acid or base followed by acetylation converted i t into i t s C-17 epimer (196). Reduction and irradiation of the product (197) using conditions identical to those described above gave an amorphous compound whose structure was not determined. A Barton photolysis of this n i t r i t e prepared from 1193; was attempted in order to functionalize the C-18 methyl group. The reaction failed however, the starting material and ketone (192) being recovered. 6.2 Description of the Process Utilized As one can infer from the preceding discussion, the synthetic sequences previously described for the synthesis of the C-nor-D-homo alkaloids jervine and veratramine (Sections 4.1 and 4.2) and the functionalization of C-18 for etiojervane steroids give low yields. We needed a compound with such functionalization in reasonable amount as starting material for our synthetic project and consequently a totally different approach had to be developed. The scheme employed involved a modified procedure of the C-nor-D-homo rearrangement by which hecogenin acetate (200) was converted to a C-nor-D-homo spirostane system i n which C-18 was transformed into an exocyclic methylene (206). 82 83 The two groups ' who originally investigated these reactions - 59 -Figure 34. Degradation of 11-deoxo jervine to an etiojervane derivative functionalized at C-18. - 60 -isolated two rearranged olefinic compounds. One of the products was 17 18 identified as the A ' -exocyclic olefin (206). The other was thought 13 17 to be the A ' -isomer (198) unti l i t was demonstrated from chemical 12 13 and spectral evidence that i t was the A ' -endocyclic olefin (199). 84 Banford-Stevens decomposition of 12-tosylhydrazones always gave the endocyclic olefin as the major product, in yields varying between 30 and 70%. Buffered solvolysis of the 12-mesyloxy- or the tosyloxy-spirostane derivatives as described by Johns,^ afforded mainly the exocyclic olefin in high yields together with some endocyclic olefin. Rearrangement to give a similar mixture of C-nor-D-homo olefins 85 was also described in the diazotization of the 12-8-amino spirostane. 198 199 A detailed study of the conditions involved in this rearrangement 86 has been done by Coxon et a l . They found that when the 126-tosylate was decomposed by bases under aprotic conditions, the exocyclic olefin was formed almost exclusively. Applying these results to the sequence described by W.F. Johns, we obtained the desired olefin (206; R = (CH^^C-CO), i n almost quantitative yield, thereby providing 206 in an overall yield of 70-75% from hecogenin acetate. - 61 -The mesylate (205) had been obtained via a sequence in which hecogenin acetate was reduced by potassium and isopropanol to a mixture of 12a-(201) and 126-rockogenin (202). This mixture was treated with pivaloyl chloride in pyridine to promote selective formation of the corresponding C-3 monopivaloate. The resultant 3a-pivaloate-12a-hydroxy rockogenin (203) was insoluble i n pyridine and removed by f i l t r a t i o n . Treatment of (204) with mesyl chloride formed the corresponding 128-mesylate (205) with subsequent solvolysis using dry pyridine giving the exocyclic olefin (206; R = (CH^CCO). The cis configuration between rings C and D of the exocyclic olefin (206; R = CH^ CO) was assigned by Coxon on the basis of optical 87 rotatory dispersion. The 18-nor-17-ketone (207) obtained by degradation gave a negative Cotton curve (a = -29) which supported the a configuration for the C-13 hydrogen. 6.3 Modification of the Exocyclic Olefin Structure In order to obtain a useful etiojervane intermediate for elabora-tion into verticine, degradation of the spiroketal system present in the exocyclic olefin (206; R = (CH^)^CCO).was required. The methods which have been used for the degradation of the sapogenin side chain involve drastic conditions. In most cases the spiroketal system is ring opened in a strongly acidic medium and this would promote the rearrangement of the exocyclic olefin to the more stable endocyclic one. Consequently, the exocyclic olefin had to be altered in order to preserve the functionality at C-18. - 62 -Figure 35. Synthesis of the C-nor-D-homo sapogenin functionalized at C-18. Hydroboration of the olefin (206; R = (CH^CCO) followed by acetylation of the resulting primary alcohol was f i r s t considered as a method of introducing a reasonably stable group at C-18. It was - 63 -noted, however, that t h i s procedure had previously been attempted by 88 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 r e s u l t s were reported. Although the successful 89 hydroboration of a sapogenin has been reported, the above workers were able to obtain only products i n which the s p i r o k e t a l group had apparently been cleaved. Since i t was not clear why t h i s hydroboration should have f a i l e d , we submitted the exocyclic o l e f i n (206; R = (CH3>3CCO) to hydroboration. Our reaction also gave a complex mixture containing at l e a s t 8 compounds (thin layer chromatography) from which the desired product could be 90 i s o l a t e d i n only 10% y i e l d . Based on previous r e s u l t s i n which the corresponding acetate (206; R = CH3CO) was hydroborated with success, we decided to convert the pivaloate to the alcohol and then acetylate i t . Reduction with l i t h i u m aluminum hydride proceeded i n quantitative y i e l d , this method being used so as to avoid possible rearrangement of the exocyclic o l e f i n . The low y i e l d s of the a c e t y l a t i o n of (206; R = „H',' about" 50%) . . led us, however, to i n i t i a l l y attempt the hydroboration of the alcohol (208). It was found that by adding a two molar excess of commercially av a i l a b l e diborane i n tetrahydrofuran to a s o l u t i o n of the o l e f i n i n the same solvent, 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 mixture which was chromatographed on s i l i c a g e l . Two alcohols (209 and 210), corresponding to the anti-Markovnikoff addition of diborane to the exocyclic o l e f i n , were i s o l a t e d i n o v e r a l l 92% y i e l d . The other product (211), formed i n 4% y i e l d , i s the t e r t i a r y alcohol - 64 -which corresponds to the Markovnikoff addition of diborane to the starting material. 211 210 209 Figure 36. Hydroboration of the C-nor-D-homo sapogenin. Evidence which indicated that hydroboration had occurred as expected was obtained from the nuclear magnetic resonance, infrared and mass spectra of the products (see Figures 38-41 for N.M.R. and M.S. spectra). Compound ..,209, showed a two-proton doublet which-corresponds to the group (-G^OH) and compound (.211) showed a three-proton singlet at T 8.36 which corresponds to the group (-C(0H)CH3). No signal was observed at x 5.22 where the vinyl protons of the starting exocyclic - 65 -olefin had absorbed. The infrared spectra of the alcohols showed hydroxyl absorption; but no olefinic absorption at 1670 cm 1 . The mass spectra of the alcohols demonstrated that both have a molecular weight of 432, which corresponds to the molecular ion of the expected hydroboration product. On the basis of these data, i t was concluded that the double bond underwent hydroboration. One question remaining, however, was whether or not the spiroketal system was s t i l l intact. 91 Jones et a l . have studied the infrared spectra of steroidal sapogenins, and have found that the region between 875 and 1350 cm ^ contains some 18 absorption bands associated with the spiroketal system. They have found also that the introduction of oxygen atoms into the steroidal sapogenin skeleton diminishes the intensities of these bands, while rupture of the spiroketal system eliminates many of them. Examination of the infrared spectrum of the exocyclic olefin (206; R = Ac) revealed the presence of 19 absorption bands between 875 and 1350 cm \ Many of these were, however, notably different from those given by Jones, but this is presumably due to the modified steroid skeleton. Comparison of the I.R. spectrum of (206; R = Ac) with the spectra of the hydroboration products showed that most of the bands were s t i l l present in roughly the same intensities, but many were shifted by up to 20 cm \ Although i t appeared from these spectra that the spiroketal system was s t i l l intact, no definitive conclusion could be drawn. The fact that the spiroketal side chain had not been ruptured was shown conclusively by a detailed examination of the mass 92 spectra. Djerassi has summarized the fragmentation of the spiroketal system showing that two important fragments occur in the low mass range. - 66 -CH _ OH CH Gfii M-114 M-132 Figure 37. Mass spectral fragmentation of the spiroketal system. RELATIVE INTENSITY O.D 2 5 . 0 5 0 . 0 7 5 . 0 1 0 0 . 0 i i : i i • J09 • J J 5 -126 •J33 o m M O • O M Ln -o LH • o - 3 6 0 Ln o -Ln Ln • o - 4 J 4 - 4 3 2 cn o -o - 89 -- 71 -The high mass region, however, i s much more useful, with s i x character-i s t i c fragments at M-59, M-69, M-72, M-114, M-129, and M-143. In the mass spectrum of the primary alcohol (209), the base peak was the molecular ion, m/e 432. There were predominant 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 alcohol f i r s t undergoes rapid dehydration leading to the M-132 peak, and that loss of a methyl r a d i c a l i s a much less favoured process from t h i s M-114 intermediate. The peak at M-161 can also be explained by dehydration, i n t h i s case, of the M-143 fragment. The t e r t i a r y alcohol (211) has as i t s base peak : the molecular ion, m/e 432. There were also predominant peaks at m/e 414 (loss of water) and 115, 139, as well as M-59, M-69 and M-72 a l l associated with the s p i r o k e t a l system. Peaks at M-114 and M-129 were absent but again dehydration i s demonstrated by peaks corresponding to M-132 and M-161. The analysis of the mass spectra '--has therefore proven conclusively that the s p i r o k e t a l system of the hydroboration products (209,210 and 211) vwas s t i l l i n t a c t . The f i n a l question with regard to the structure of the hydroboration product was the stereochemistry at C-13. Examination of the l i t e r a t u r e revealed that there were three types of reactions documented for the exocyclic o l e f i n (206; R = CH^CO). The f i r s t example i s the c a t a l y t i c 93 hydrogenation with palladium-charcoal i n a c e t i c acid. The stereochemistry at C-13 was i n f e r r e d as 3 from the examination of the - 72 -optical rotatory dispersion curves of the two epimeric ketones (217 and 218) which had been derived from the a,6-unsaturated ketone (215) 94 via the Beckmann rearrangement followed by acid hydrolysis. The minor, thermodynamically less stable B-epimer (217) was correlated with the a,B-unsaturated ketone (215) through the 3,17-diketone (221), so that a l l these compounds would have the same configuration at C-13. The major ketone (218) showed an unusually strong positive Cotton effect (a = +216°) and the epimeric ketone (217) displayed a negative Cotton effect (a = -94°). Using as models the enantiomeric B-nor-coprostan-3-one (222) and (+)-cis-8-methylhydrindan-5-one (223), which exhibit negative Cotton effects, i t was concluded that the major ketone (218) 95 contains a 13a-methyl group. Further support for this assignment was provided by the demonstration that the 128-etiojervane derivative 96 (224) had the expected negative Cotton effect (a = -92°). Catalytic hydrogenation of the olefin (206; R = CH^CO) had therefore taken place with hydrogen attacking from the a face of the molecule to give the C-13 8 methyl group (214). 222 223 224 - 73 -221 217 218 Figure 42. Hydrogenation of the C-nor-D-homo sapogenin. - 74 -The second reaction involving the exocyclic o l e f i n (206; R = CH^CO) 94 was epoxidation with perbenzoic acid. I t was found that two epimeric epoxides, 6 (225) and a (226), were formed i n the r a t i o 3:1. The structure of the 8-epoxide has been established through the f i n d i n g that the l i t h i u m aluminum hydride reduction product (228, was i d e n t i c a l with the reduction product of one of the epoxides derived from the endocyclic o l e f i n (229). Since the alcohol (228) was obtained from both (206; R = CELjCO) and (229), i t must contain a hydroxyl at C-13 and the 12a-H configuration. Therefore, the alcohol (228) which was derived by trans attack of hydride ion on the epoxide (225), must have a 136-hydroxyl group. Consequently the epoxides (225) and (226) must have the 138,188- and 128,138-epoxy structures r e s p e c t i v e l y . Thus i n the case of epoxidation, the attack by perbenzoic acid has occurred predominantly from the 8 face of the molecule. This r e s u l t , although • 97 unexpected, has several precedents i n the l i t e r a t u r e . The t h i r d example i s the oxidation of the o l e f i n (206; R = CH3C0) 93 with osmium tetroxide. A c e t y l a t i o n of the derived d i o l (232) followed by treatment with t h i o n y l chloride-pyridine was shown to give a mixture containing the enol acetate (234). The formation of the exocyclic rather than the endocyclic enol acetate was regarded as e s t a b l i s h i n g the 13a-configuration of the t e r t i a r y hydroxyl group on the basis that the epimeric alcohols (228) and (227), under the same reaction conditions gave the endocyclic o l e f i n (229) and the exocyclic o l e f i n (206; R = CH^CO) re s p e c t i v e l y . In t h i s example the osmium tetroxide appears to have approached the exocyclic double bond from the a face. - 75 -Figure 43. Epoxidation of the C-nor-D-homo sapogenin. - 76 -Figure 44. Osmic acid oxidation of the C-nor-D-homo sapogenin. Examination of molecular models indicates that the D ring in the exocyclic olefin (206; R = CH^ CO) assumes a modified boat conformation (235) which i s imposed by the two adjacent cis-fused five-membered rings, and that the entire molecule is nearly planar 13(18) in shape. With regard to the A double bond, the a face appears to be more accessible than the 8 face since the latter is shielded by the 113,158 and 128-hydrogen atoms. - 77 -Since the addition of diborane has been found to occur in a cis 98 manner from the less hindered face of a double bond and on the basis of the previous examples, i t was speculated that the hydroboration occurred from the a face; the resulting primary alcohol being tentatively considered as having the 13-hydroxymethylene group in the 6 position. 93 Coxon described the conversion of the a-epoxide (226) by either boron trifluoride etherate or perchloric acid into an aldehyde to which he assigned the 13a-formyl structure (236) on the basis of I.R. and N.M.R. spectra. The compound (236) was not epimerized by base confirming the more stable a configuration of the formyl group. As the partial structure (237) shows, the substituent at C-13 occupies the pseudo-equatorial position in the boatlike ring D. - 78 -H H / \ l 3 A' y R CHO 18 237 Since verticine (138) has the C-18 in the a configuration, the possibility of epimerization of this center via the corresponding aldehyde was considered. At the same time comparison of the aldehyde obtained from the oxidation of the hydroboration product (209? with the previous results would permit some light to be shed upon our configurational assignment. When the diol (209) was subjected to 99 Moffatt oxidation, a compound was isolated by rapid chromatography on s i l i c a gel. The N.M.R. spectrum of this compound showed an aldehydic proton as a doublet (J = 5.5 Hz) at T 0.22 and a doublet of triplets with coupling constants of 5.5 and 7.0 Hz at x 7.32 (see Figure 45 for N.M.R. spectrum). - 79 -I t was evident that the s t r u c t u r a l assignment proposed by Coxon did not agree with ours, and consequently more d i r e c t evidence was necessary to c l a r i f y t h i s point. The i n f r a r e d spectra of the two aldehydes were very s i m i l a r and indicated that the s p i r o k e t a l system was s t i l l i n t a c t . The mass spectra 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). Oxidation of the second crop of c r y s t a l s obtained i n the r e c r y s t a l l i z a t i o n of the hydroboration product gave a material which exhibited two absorptions i n the N.M.R. spectrum, corresponding to formation of the two isomeric aldehydes (238 and 239). We therefore concluded that two unexpected r e s u l t s had occurred; f i r s t l y , i n the p u r i f i c a t i o n of the hydroboration product we had s e l e c t i v e l y c r y s t a l l i z e d out one of the isomeric alcohols (209) i n the presence of the other (210); secondly, the hydroboration of the o l e f i n (206; R = H) was not stereo-s e l e c t i v e as was the case with the 3-acetyl d e r i v a t i v e . This type of phenomenon may be accounted f o r presumably i n terms of "conformational . . ,. 100 transmission . 00 CO CM 0) T} I ca cu o § a w in -d-a) u 3 60 •H fin - 82 -93 The N.M.R, signals (at 60 MHz) reported by Coxon et a l . for the 13a-formyl compound (236) were a doublet (J = 7 Hz) at T 0.21 for the aldehydic proton and a doublet of triplets (J = 5.5 and 7.0 Hz) for the C-13 6 proton at T 7.32. It was not clear at this point which epimer of the aldehyde had been obtained, since on the one hand no information was available for the 133-formyl compound while on the other hand the possibility of epimerization during the reaction could not be ruled out. If our assignment of the configuration for the hydroboration product (209) was correct and no epimerization had taken place, then the aldehydic product obtained by Moffatt oxidation would have a 138-formyl group and would be the less stable epimer of the two possible products. In fact, i t was subsequently found that treatment with potassium carbonate in methanol at room temperature smoothly and completely converted the i n i t i a l Moffatt oxidation product into the epimeric aldehyde (see Figure 46 for N.M.R. spectrum). It was thus concluded that on Moffatt oxidation of the diol (209) the thermodynamically less stable epimer is formed and that this epimer is completely epimerized on contact with weak base. Furthermore, providing that the D-ring is in a boat-like conformation and that a - pseudoequatorial (a) substituent is more favourable than a pseudoaxial ( 6 ) one, i t is possible to assign the structures \238? and (239) to the less stable and the more stable epimers respectively. - 83 -The aldehyde mixture obtained i n the Moffatt oxidation was treated with potassium carbonate i n methanol at room temperature to give one product, the N.M.R. spectrum of which showed only one peak at T 0.59. This compound was reduced with sodium borohydride to the corresponding alcohol (210) whose N.M.R. spectrum contained a poorly resolved t r i p l e t at x 6.34 due to the C-18 protons and a doublet f o r the C-21 methyl at T 8.97, the remaining signals of the spectrum being almost i d e n t i c a l to those of the isomeric alcohol (209) (see Figure 47 f o r the N.M.R. spectrum). The mass spectra of the isomeric d i o l s (209 and 210) showed the same major fragments, the only difference being the r e l a t i v e i n t e n s i t i e s of these fragments. The alcohol (210) was f i n a l l y acetylated with a c e t i c anhydride i n p y r i d i n e to give the diacetate (240) (see Figure 48 for the N.M.R. spectrum). CEO !! : H CH 0H H I E : CEo0Ac H ! 239 210 240 The multi-step synthesis of the diacetate (240) was ca r r i e d out concurrently with Dr. Y. U e d a 1 1 2 , each of us beingi'responsible, f o r the. development of d i f f e r e n t stages. CD 10 Oi CO 00 o O O a t H to i 3 0) O {_ 01 rt S !3 cu l-i 3 60 •H JSCO 0 1 2 3 4 5 6 7 8 9 1 0 Figure 48. N.M.R. spectrum of the a-diacetate (240). - 86 -7. Elimination of the Spiroketal System 7.1 Processes Described More than three decades ago Marker"*"^ made the important discovery that sapogenins on being heated to 200° with acetic anhydride undergo fission of the spiroketal system, with introduction of a 20,22-double bond. Subsequent oxidation of this enol ether to a keto acid followed by elimination of the ester group affords a a,8-unsaturated ketone as indicated in the following scheme: OAC 241 242 243 244 Figure 49. Classical elimination of the spiroketal system. Several oxidation processes have been described; the most common being oxidation by chromic a c i d . " ^ 2 ' ^ ^ The ester group has been eliminated under both acidic and basic ... 104,105 condxtxons. In practice the sequence is performed without the isolation of the intermediates, and i t has provided the crucial process for the large-scale production of progesterone, sex hormones, and the cortical hormones from sapogenins - substances which are readily available 106,107 xn nature. - 87 -This degradation process has been utilized with success in C-nor-D-homo steroidal sapogenins.^ Spiroketal opening of the sapogenin (214) was successfully accomplished by a short treatment with octanoic anhydride or acetic anhydride ethyl amine hydrochloride. The product, the enol ether (245) was oxidized to the keto ester (246) with chromic acid and this in turn was cleaved to the unsaturated ketone (215) with either base or acidf r CH OR Figure 50. Classical spiroketal elimination applied to a C-nor-D-homo sapogenin. - 88 -This process when applied to the C-18 substituted C-nor-D-homo 108 sapogenins f a i l e d , probably because the formation of the enol ether i s hindered owing to the strong i n t e r a c t i o n between the C-21 methyl group and the C-18 acetoxymethylene group, as indicated i n the p a r t i a l structure below. R = -CH2-CH2-CH-CH20Ac One of the few a l t e r n a t i v e processes f o r degradation of the sp i r o k e t a l system of sapogenins v i a B a e y e r - V i l l i g e r reaction as , , , „ , 109,110 , , o r i g i n a l l y developed by Marker, was then considered as a sub s t i t u t e . It should be mentioned that t h i s work was done i n the non-substituted se r i e s and that the corresponding studies i n the C-18 substituted series:have not been published up to the present date. Treatment of the sapogenin (214) with performic acid at 70-80° gives the diformate (247). Adsorption of th i s diformate on alumina r e 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 (248). Jones oxidation of the l a t t e r followed by base catalyzed elimination of the side chain gives the a,6-unsaturated ketone (215) i n an o v e r a l l y i e l d comparable with that from the c l a s s i c a l sequence. - 89 -Figure 51. Degradation of the s p i r o k e t a l system v i a the Ba e y e r - V i l l i g e r reaction. 7.2 Process U t i l i z e d 7.2.1 B a y e r - V i l l i g e r Oxidation i 0 8 The information provided by Johns showing that Marker's c l a s s i c a l degradation did not work with C-18 substituted C-nor-D-homo st e r o i d s , and the f a i l u r e of some v a r i a t i o n s of the same method"*""^ to provide the desired product induced us to adopt the B a y e r - V i l l i g e r oxidation process as described i n Section 7.1. - 90 -I n i t i a l work on the degradation of the s p i r o k e t a l side chain of 112 the diacetate (240) gave disappointingly low y i e l d s . In order to improve these r e s u l t s , the reactions involved have been studied i n great d e t a i l . In the procedure ultimately used i n the B a e y e r - V i l l i g e r oxidation, the diacetate (240) was treated with performic acid generated i n s i t u at 41-44°for 4 hours to give a gummy material e x h i b i t i n g two major spots by t . l . c . along with more polar minor products. No s t a r t i n g material could be detected. A s i m i l a r r e s u l t was obtained when the oxidation was c a r r i e d out at room temperature for 36 hours. The N.M.R. spectrum of the crude product revealed the presence of two formyl protons, one at x 2.00 and the other at x 1.98. In addition, a doublet appeared at x 5.97 (J = 6.5 Hz) with corresponding disappearance of the m u l t i p l e t at x 5.55, assigned to the C-26 methylene protons. These r e s u l t s suggested that the s p i r o k e t a l had been opened and that C-26 c a r r i e d a formyloxy group. Since i t proved impossible to p u r i f y the reaction products t h e i r structures were uncertain. 7.2.2 Hydrolysis Studies Since the inception of t h i s p r oject, the s e l e c t i v e hydrolysis phase of the synthetic scheme has proved to be the most troublesome b a r r i e r to completion of the cevane skeleton. A c a r e f u l study of the several reactions used i n the s e l e c t i v e removal of the formate groups \wa"s=r considered. 113 Hydrolysis of esters i s catalyzed by acids or bases. For 114 preparative purposes t h i s hydrolysis has been done under basic conditions unless the compound i s base s e n s i t i v e . - 91 -116 Corey obtained a very favourable result i n performing the selective hydrolysis of the acetate group in the compound (249) using potassium carbonate in ethanol at room temperature for 3 hours . C0 2Et 249 Applying similar reaction conditions to the performic acid oxidation product we found that hydrolysis with potassium carbonate in methanol gave a multitude of products from which the diol (252) could be isolated in 21% yield. The configurations at C-26 and C-20 in this compound are not known (see Figures 52 and 53 for the N.M.R. and M.S. spectra). 300 200 100 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ • I l I l l l - l l ; | ; i . . 1 . I . . . . i • . . . 1 • . . . i . . . . I i I . . . . i . . . . I . . . . i . . . . I . . . . i . . . . 1 . . . . i Figure 52. N.M.R. spectrum of the d i o l (252). - £6 -- 94 -It was then concluded that potassium carbonate i n methanol i s too d r a s t i c f o r s e l e c t i v e removal of the formate ester groups. The pH of the system used for hydrolysis described was found to be 11. It was speculated that better s e l e c t i v i t y i n the formate hydrolysis may be achieved i f a less basic system i s employed. Ester hydrolysis i s a common occurrence i n alumina column chromatography and a modified chromatographic technique has been used to perform s e l e c t i v e formate ester hydrolysis."'""'"^ Hydrolysis under these conditions, however, resulted i n considerable losses by absorption of the material i n the column. A hydrolysis employing a buffer at pH 8.0 was then considered. The crude B a e y e r - V i l l i g e r oxidation product was treated with sodium b a r b i t a l methanolic buffer adjusted to pH 8.0 with hydrochloric acid. Hydrolysis was c a r r i e d out at 42-45° for 8 days with readjustment of the pH to 8.0 by the d a i l y addition of further buffer. Studies of the crude hydrolysis mixture showed that the d i o l (252) was present as one of the minor products. The predominant product was considered to correspond to the intermediate d i o l (251). Subsequent r e s u l t s showed that t h i s assignment was i n c o r r e c t . Nevertheless, the hydrolysis product was oxidized with Jones reagent to give a product from which the a c i d i c material was extracted with 5% sodium hydroxide s o l u t i o n . The basic extracts were a c i d i f i e d with hydrochloric acid and extracted with e t h y l ether. A f t e r solvent evaporation the product was submitted to side chain elimination conditions (sodium hydroxide i n jt-butanol under r e f l u x f o r 1 hour), the material thus obtained being subsequently treated with a c e t i c anhydride - 96 -- 97 -i n p y r i d i n e . The major i s o l a t e d product was the tetraacetate (254) (see Figures 54 and 55 for the N.M.R. and M.S. spectra) from which the desired a , 3-unsaturated ketone (256) could not be separated by column chromatography. R'=HC00CH„-CH-CH o-CH -CO-; R"=H0-CHo-CH-CHo-CH -CO-; R"'=H0oC-CH-CH -CH -C0-2 | 2 2 2 j 2 2 2 | 2 2 CH^ CH^ CH^ Figure 56. Compounds obtained i n the buffer h y d r o l y s i s . - 98 -The major product i n the neutral f r a c t i o n was the g-diketone (257) (see Figures 57 and 58 for the N.M.R. and M.S. spectra), formed by oxidation of the d i o l (252). The U.V. spectrum of th i s f r a c t i o n showed an absorption at 289 nm att r i b u t e d to the g-diketone (257) but no absorption at 236 nm demonstrating that the base treatment, at room temperature, did not promote eli m i n a t i o n of the ester side chain. The f a i l u r e i n obtaining the pure desired product (256) and the p o s s i b i l i t y of modifying the alumina hydrolysis conditions i n order to avoid the losses observed before led us to return to the alumina process. The crude performic acid oxidation product was s t i r r e d at room temperature with alumina Woelm i n methanol (pH = 8.0) for 6 hours. The alumina was then f i l t e r e d and extracted several times with chloroform to give a crude material which was chromatographed on s i l i c a gel ( a c t i v i t y II) using chloroform/ethyl acetate mixtures as eluent. Three major f r a c t i o n s were i s o l a t e d from t h i s separation. The l e a s t polar f r a c t i o n , c o n s t i t u t i n g 30% of the chromatographed material indicated i n the N.M.R. the presence of two formyl protons at x 2.00 and T 1.98 but no absorption at x 5.97 i n d i c a t i v e of the formyloxy group. On the basis of this data the structure (258) was assigned to the major c r y s t a l l i n e material of t h i s f r a c t i o n . In order to prove i f our assignment was correct the d i o l (252) obtained i n the 118 previous hydrolysis was treated with acetic-formic anhydride, i n pyridine, to give the diformate (258) i n good y i e l d (see Figures 59 and 60 for N.M.R. and M.S. spectra). This compound i s i d e n t i c a l i n a l l aspects to the material i s o l a t e d from the column chromatography. Its - 100 -2£t>-ID a .tn in o . a LD o o 2_S-BSE- a .in m OEE-662-raz-es .in (M m CM o o .o r_r-a .tn _or- a . a 1 • O'SZ 3AIidl__ .a 001 D'OS 0"0 - 101 -formation can be rationalized in terms of transesterification during the Baeyer-Villiger oxidation. This least polar fraction did not give any a,B-unsaturated ketone (256) when rehydrolyzed for 6 hours on alumina and submitted to the oxidation-side chain elimination-acetylation sequence. 252 258 Hydrolysis with potassium carbonate in methanol as described before, completely removed the formate groups. Oxidation with Jones reagent gave a mixture from which the B-diketone (257) was isolated in 50% yield. The transformation of this B-diketone into the desired ketone (260) w i l l be discussed later. The middle fraction.- i n the separation constituted 58% of the chromatographed material. Its N.M.R. spectrum showed two formyl signals at the original positions, namely at x 2.00 and x 1.98, the latter being considerably reduced. The C-26 formate has evidently been removed as indicated by the shift of the C-26 methylene doublet to x 6.54. in o cn o - eoi -- 104 -The i s o l a t i o n of t h i s monoformate f r a c t i o n as the major f r a c t i o n from the hydrolysis performed at pH = 8.00 explains why previous attempted preparations of the unsaturated ketone (256) gave, the tetraacetate (254) as the major product. Since previously, the hydrolysis products were not chromatographed the unhydrolyzed mono-formate (256) was c a r r i e d through to the f i n a l steps of the degradation to give the tetraacetate (254). This material was now treated again with alumina and submitted to Jones oxidation followed by base treatment and a c e t y l a t i o n to give the a,6-unsaturated ketone i n 30% y i e l d . The major side product was again the tetraacetate (254). The attempted separation of the a,8-unsaturated ketone from the tetraacetate f a i l e d completely. Thus the crude material was reduced over platinum oxide (Adam's catalyst) to give a mixture of ketones (259) and (260) which was then epimerized with boron t r i f l u o r i d e - e t h e r a t e to the most stable ketone (260). The separation of the saturated ketone (260) from the r e s i d u a l tetraacetate (254) has proved most d i f f i c u l t , and u n t i l very recently only small scale separation was possible. However, preparative separation can be now be achieved using modified column chromatography on f i n e mesh s i l i c a g e l under pressure. The transformation of the tetraacetate, (254),as the major side product separated at t h i s stage, i n t o the desired ketone (260) w i l l be discussed l a t e r . - 105 -The polar f r a c t i o n obtained constituted 12% of the crude reaction mixture. I t showed only very weak formyl peaks i n the N.M.R. spectrum. Direct oxidation with Jones reagent followed by side chain elimination and ac e t y l a t i o n gave a,$-unsaturated ketone (256) which was p r a c t i c a l l y free of tetraacetate (254). The a,8-unsaturated ketone was reduced and epimerized to 260 as described above and chromatographed using the new technique (see Figures 61-64 for the N.M.R. and M.S. spectra). Before we discuss our studies towards the elaboration of the side products, the 6-diketone (257) and the tetraacetate (254) to the desired ketone (260) some considerations with respect to the proposed mechanistic pathway f or the performic acid oxidation of the s p i r o k e t a l system present i n (240) are necessary. A summary of the mechanistic r a t i o n a l e which appears consistent with the r e s u l t s obtained thus f a r i s presented i n Figure 65. Protonation of one of the spir.dketa'l oxygen atoms p r e f e r e n t i a l l y opens the s i x membered r i n g F. The same p r e f e r e n t i a l cleavage has been observed i n Marker's c l a s s i c a l process. The exact explanation 0.0 Ul RELATIVE 25.0 l INTENSITY 50.0 I 75.0 100.0 _J o -o -109 o 143 a O -187 M o -o o ON -270 m LO a -CD -3)0 LO CD -327 344 •356 fe-CD o Ln CD " CD + 16 Ln-CD - LOT -Figure 64. Mass spectrum of the saturated ketone (260). - 110 -Figure 65. Baeyer-Villiger oxidation mechanism. - I l l -for t h i s preference i s not understood. Attack of the peracid at C-22 followed by rearrangement and l i b e r a t i o n of the formate ion r e s u l t s i n the formation of the dioxalane (263). The n u c l e o p h i l i c attack by formate anion at eit h e r C-16 or C-20, since t h i s intermediate i s symmetrical, would produce two isomeric intermediates (264 and 265). It seems from our r e s u l t s that there i s a p r e f e r e n t i a l attack at carbon C-20. The formation of these isomeric formates had considerable synthetic s i g n i f i c a n c e f o r us because i t could imply that 50% of the s t a r t i n g material would not have immediate u t i l i z a t i o n i n our synthesis. We also could not i s o l a t e or f i n d any evidence that these i n t e r -mediates are indeed involved i n the degradation. Since t h e i r structures are very c l o s e l y r e l a t e d one could argue that our analysis of the mixture f a i l e d to separate them. This p o s s i b i l i t y appears i n v a l i d because we never could i s o l a t e or detect the isomeric a,8-unsaturated ketone (266) or i t s reduction product (267) i n the f i n a l steps of the degradation. There appears to be no reason to doubt that 266 would be produced i f any s i g n i f i c a n t amount of the intermediate 265 was present i n the B a e y e r - V i l l i g e r product mixture. 267 - 112,-7.2.3 Conversion of the side products In order to obtain optimum yields i n the overall conversion of the spiroketal system (240) to the desired ketone (260), attempts to u t i l i z e the various side products i n the Baeyer-Villiger reaction were explored and the chemistry relevant to this objective i s now discussed. The tetraacetate (254) was reduced with lithium aluminum hydride to the tetrol (268) the mass spectrum of which does not show the molecular ion at m/e 352 but the peaks at m/e 334 and 316, correspond-ing to the loss of water, are quite intense. This tetrol was stirred in acetone with p-toluene sulfonic acid at room temperature i n order to form the acetonide (269). Although i t i s possible that the seven membered acetonide formation linking the C-18 and C-20 hydroxyl groups could occur, only the six membered ring through C-16 and C-20 was 119 isolated. The stereochemistry at C-16 and C-20 in 269 is unknown, however, this i s not important to the development of the scheme. The acetonide (269) was acetylated with acetic anhydride in pyridine to compound (270). The N.M.R. spectrum of this compound (see Figure 67) shows a singlet at x 7.71 for the acetonide methyl groups and a multiplet at x 5.98 for the C-18 methylene group. The molecular ion 271 - 113 -peak is not visible in the mass spectrum (see Figure 68) but the base peak at m/e 461, corresponding to the loss of a methyl group to give a fragment (271), indicates that the desired product was obtained. 270 269 Treatment of 270 with methanol and acid gave the diol (252) which was oxidized with Jones reagent to the diketone (257). These two latter compounds were identical in a l l respects with the compounds synthesized previously during the alumina hydrolysis studies. The diketone (257) was acetylated with acetic anhydride to a mixture of enol acetates (272 and 273). These two enol acetates could not be separated by column chromatography or high pressure liquid chromato-graphy. The N.M.R. spectrum of these enol acetates shows two singlets at T 8.06 and 8.10 attributed to the C-3 and C-18 acetates as well as - 114 -singlets at T 7.82 (C-21 CH3), 7.85 (C-16 enol ester); 7.68 (C-21 vinyl CH^), 7.76 (C-21 enol ester). The mass spectrum reveals fragments at m/e 474 (M+), 432 (M-42, loss of CH2C=0), 359 (M-115, loss of CH2OAc + CH2C=0), 299 (M-175, loss of CH2OAc + CH2C=0 + AcOH) a l l agreeing with the expected fragmentation for these enol acetates. Hydrogenolysis of the mixture on platinum oxide in methanol gave a mixture of ketones (259) and (260) which was epimerized to the most stable one (260) as described before. Final purification was obtained on s i l i c a gel column chromatography. In this manner the conversion of the tetra-acetate (254) to the saturated ketone (260) was performed in 5% overall yield. This recent development involving the conversion of the side products as described above has permitted us to double the overall yield of the desired saturated ketone (260) from 240. 8. Coupling of the Nitrogen Portion to C-Nor-D-homo Steroid We have considered the cevanine skeleton as consisting of two units, a steroidal portion,the synthesis of which we have described and a substituted piperidine (see Figure 33). This latter portion 72 76 has been synthesized in the previous approaches to Veratrum ' and 24 Solanum bases from a substituted pyridine which has been attached to the steroidal compound prior to the hydrogenation step. For the verticine synthesis the required compound would be 2-bromo-5-methyl pyridine, easily accessible from the commercially available 2-amino-5-methyl pyridine. - 115 -Figure 66. Conversion of the acetonide 270 to the saturated ketone 260. - 116 -Ln u r o cn CD • CD - LIT -- 118 -Of particular .relevance to the present discussion is the work of 120 Schreiber. His group has utilized similar reactions in the later stages of the pathway to Solanum alkaloids. Addition of 2-lithium-5-methyl pyridine (prepared from 2-bromo-5-methyl pyridine and phenyl lithium) to 3B-acetoxy-preg-5-en-20-one (274) gave two alcohols (275) and (276) which were separated by alumina column chromatography. 276 Figure 69. Coupling of the nitrogen portion i n the Solanum alkaloid synthesis. - 119 -8.2 Process Utilized The etiojervane derivative (260) (1 mole) was reacted with 2-lithio-5-methyl pyridine prepared in advance from 2-bromo-5-methyl pyridine (15 moles) and n-butyl lithium (13 moles). The crude product was extracted with dilute hydrochloric acid and the basic material acetylated with acetic anhydride-pyridine prior to chromatographic purification. The neutral material was acetylated and the unreacted saturated ketone (260) was recovered by column chromatography on s i l i c a gel. This reaction was described for the f i r s t time in Dr. Y. Ueda's • t i n - • thesis. z We have improved the yields and developed the purification procedure as described below. The basic material was f i r s t purified by column chromatography on basic alumina to remove the nonsteroidal compounds and then by column chromatography on fine mesh s i l i c a gel under pressure to yield the coupling product with the structural features shown in (277). The - 120 -mass spectrum of this material (see Figure 71) showed the base peak at m/e 136 corresponding to the expected fragment (278). A significant 278 peak at m/e 511 was attributed to the molecular ion of the coupling product and fragments with m/e 496 (M-15, loss of CH^), 451 (M-60, loss of AcOH) and 358 (M-153, loss of AcOH + picoline) were also present. The N.M.R. spectrum (see Figure 72) shows a singlet at T 8.55 for C-21 CH3 and a singlet at T 7.68 for C-27 CH.^ . A good indication of the success of the coupling reaction i s given by the aromatic peaks at T 2.96 (C-23 H) , 2.50 (C-24 H), and 1.65 (C-26 H). Owing to the creation of a new asymmetric center (C-20) during the reaction of the carbanion with 260, the product obtained should 120 be a mixture of two epimers (279 and 280). Schreiber obtained the separation of the analogous epimers (275) and (276) by neutral alumina column chromatography but in our case we could not detect separation. Subsequently the product (277) was separated by neutral alumina high pressure liquid chromatography (see Figure 70). - 121 -Figure 70. High liquid pressure chromatogram of the coupling mixture (277). N3 -P-Figure 73. N.M.R. spectra of the coupling products. to o ,1— r — i — i —r" | I I — , — r — ] — i — r — r -5 0-° 100.0 150.D 200.0 .250.0 300.0 350 0 . • M/E 400.0 450.0 500.0 50.0 100.0 9 9 150.0 200.0 250.0 300.0 • M/E 350.0 400.0 450.0 500.0 Figure 74. Mass spectra of the coupling products. - 126 -A low molecular weight pyridine derivative which remained as a trace impurity was removed from each of the separated epimers by repetition of the neutral alumina column chromatography. The N.M.R. and M.S. spectra (see Figures 73 and 74) as well as the R^  values on s i l i c a gel thin layer chromatography of the two epimers were practically identical; the only difference being their retention times on high pressure liquid chromatography. No attempt has been made to assign the stereochemistry at C-20 in the two separated epimers. 9. Cyclization of the Coupling Product 9.1 Process Described The cyclization of the intermediate (277) can be considered as the formation of a quinolizidine ring (281). 281 A large variety of methods are available for building up the quinolizidine ring from a piperidine or pyridine derivative. Most of them have in common the closing of the heterocyclic ring at the „ 121 nitrogen atom. A l l these methods are based ton the nucleophilic character of the amino group which causes displacement of various leaving groups. - 127 -Figure 75. Synthesis of Quinolizidine derivatives. - 128 -9.2 Process Utilized The nucleophilic displacement of the C-18 acetate by either the pyridine nitrogen or the corresponding reduced product was considered, the latter being the method of choice because of the greater nucleo-p h i l i c i t y of the piperidine nitrogen. Thus the coupling product (277) was reduced to the piperidine derivative (287). The N.M.R. spectrum of 287 (see Figure 77) showed no aromatic peaks, thereby indicating successful removal of the pyridine ring. A further indication that the reduction had proceeded was given by the mass specfrum (see Figure 78). The molecular ion at m/e 517 was accompanied by the presence of the expected peaks at m/e 516 (M-l), 502 (M-CH3), 457 (M-AcOH) and 498 (M-H-R^O). The most characteristic fragmentation was observed in the lower mass range where the heterocyclic ring fragmentation is present. The base peak (m/e 98) arises from the cleavage of the C^o"*"^ k° n t* as shown in Figure 76. 277 287 - 129 -Figure 76. Mass spectral fragmentation of the piperidine steroidal product (287). The piperidine product (287) was heated to 130° in triglyme for 72 hours. Since we had a very small amount of material and the difference between the N.M.R. spectrum of the cyclized and uncyclized product should not be very conclusive the course of the reaction was followed by mass spectrometry. The mass spectrum of this product (see Figure 80) was then compared with the mass spectrum of the uncyclized product (287) and deoxyverticinone (288) a degradation 122 product obtained from verticinone. The base peak of 288 (m/e 112) also arises from the heterocyclic portion by cleavage of the C-13,18 and C-20 bonds (see Figure 81). - 130 -i i 288 m/e 112(100) Figure 79. Mass spectral fragmentation of deoxyverticinone (288). In the cyclization product there were two base peaks, m/e 98 and m/e 112 and therefore we concluded that the cyclization has occurred to some extent. Our analysis was restricted to the base peaks because in deoxyverticinone spectrum the relative intensity of the fragments leading to the base peak m/e 112 are too small to be measured. No further work was possible because of the small amount of material involved. 10. Conclusions.-.-It i s obvious that the cyclization product requires further characterization but the indication of a positive result opens the door to the possibility of the synthesis of verticine (138) and related alkaloids. Therefore in Figure 83 a proposal is presented for the conversion of the synthetic cevane base (288) to this alkaloid. - 131 -Figure 83. Proposal for the conversion of the a-cevanine base (288) to verticine { Finally i t should be mentioned that the successful construction of the cevane skeleton has tremendous implications as i t can be applied to different series of Veratrum alkaloids. While some of the best known Veratrum alkaloids have a hydroxyl at C-20 there i s a large group of these alkaloids .which does not have this substitution pattern. Consequently minor modifications in our scheme should permit the access to these bases. In Figure 84 we present a proposal modifying the present synthesis in order to prepare C-20 non-hydroxylated bases. - 132 -| (147) R = 0 Eduardine (154) R=a-OH Petilidine (155) R=B-OH Petilinine Figure 84. Synthetic proposal for the C-20 non-hydroxylated Veratrum alkaloids. Figure 77.., N.M.R. spectra of the piperidine product (287). if i - 135 -L9t> -60b -89C-ese -ot>e-Loe-LBX-E S t -921-LE-ztr--es-= 0*001 . o CD o i n i n i n o 4J -s produc o (3 o O _ i n •r-t P7 clizat o :>> - ^UJ o _ o ^» cu of th o a o _ i n CM spectrui a CO o CO ni _ a (M •3 o . i n o o O co cu u 3 60 •H rn 0 'SL 0*0S O'SS A1ISN31NI _AI1H"1__ 0*0 RELATIVE INTENSITY D.D 25.0 50. Q 75.0 I I 1_ 100.0 •112 -119 :131 — I * ul -o o -154 -164 -180 o -o a ui • -139 :t-0 a --235 X. II f=-272 00 CO U l " -304 -329 -343 -359 T74~ -372 -401 391 -415 uV U l ul -o cn o-9ET " - L£l -- 138 -EXPERIMENTAL Melting points were determined on a Kofler block and are uncorrected. Ultraviolet (U.V.) spectra were recorded in methanol on a Cary 15 recording spectrophotometer. Infrared (I.R.) spectra were recorded on a Perkin Elmer model 457 spectrophotometer as potassium bromide pellets unless otherwise specified. Nuclear magnetic resonance (N.M.R.) spectra were determined at 60 MHz on a Varian T-60 spectrometer and at 100 MHz on either a Varian HA-100 spectrometer or a Varian XL-100 spectrometer using deuteriochloroform as solvent with tetramethyl silane as an internal standard. The chemical shifts are recorded in the Tiers T scale and the types of protons, integrated areas, multiplicities, spin coupling constants J (in Hz) are indicated in parentheses. Mass spectra were determined on an Atlas CH-4 spectrometer or an Associated El e c t r i c a l Industries MS-902 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. For column chromatography Shawinigan alumina or Woelm neutral or anionotropic alumina were used. The experimental described from pages 139 to 149 is also presented in Dr. Y. Ueda's t h e s i s 1 1 2 since this work was developed as a collaborative study. - 139 -Reduction of hecogenin acetate (38-acetbxy-(25R)-5a-spirostan-12-one)(200) Hecogenin acetate (54 g) was put i n t o a 2 - l i t r e 3-necked round bottom f l a s k equipped with a thermometer, a condenser, a s t i r r e r and a nitrogen i n l e t and anhydrous isopropanol (150 ml) and tetrahydrofuran (350 ml) were added. The s o l u t i o n was heated to r e f l u x (70°) and the heat supply removed. Potassium (42.5 g), which had been cut and kept under petroleum ether (b.p. 65-110°), was introduced i n small pieces during 30 minutes. Vigorous evolution of gas was observed. A f t e r the addi t i o n of potassium the heating mantle was turned on again to maintain a mild r e f l u x f o r 150 minutes. At the end of t h i s period 15 ml of isopropanol (15 ml) was added to the reaction mixture to destroy the excess of potassium. The mixture was cooled to room temperature and then poured with vigorous s t i r r i n g into i c e water (5.5 1.) containing a c e t i c a c i d (100- g) (pH of the s o l u t i o n = 6.0; Universal pH i n d i c a t o r paper). White c r y s t a l l i n e p r e c i p i t a t e s were formed immediately. The c r y s t a l l i n e mass was l e f t overnight at room temperature. The p r e c i p i t a t e s were c o l l e c t e d by suction f i l t r a t i o n and transferred i n t o a 2 l i t r e beaker to be digested during 10 minutes with water (1.5 X ). The product was f i l t e r e d , suction dried and f i n a l l y dried i n a vacuum oven at 80° overnight. The crude product weighed 49.20 g (99.5%). Chromatography on a c t i v i t y II alumina, e l u t i n g with chloroform-benzene (7:3), gave a pure sample of 126-rockogenin (5cx-spirostan-(25R)-38,126-diol) (202). R e c r y s t a l l i z a t i o n from petroleum ether gave white needles, m.p. 202-205° ( l i t . m.p. 209-211°). 8 2 - 140 -N.M.R. signals: 9.23 (singlet, 3H, C-19 CH3), 9.22 (doublet, J = 6, 3H, C-27 CH3), 8.97 (doublet, J = 6.5 Hz, 3H, C-21 CH3), ca. 6.6 (broad multiplet, 4H, C-3 CH + C^CH + C-26 CH2) , 5.59 (quartet, J = 7.5 Hz, IH, C-16 CH). I.R.: 3360, 1455, 1245, 1181, 1056, 981, 958, 921, 898, 864 cm Mass spectrum: M.W. 432; base peak at m/e 139; main peaks at m/e 432, 417, 402, 373, 360, 318, 303, 289, 248, 139, 115. Found: C, 74.'72; H, 10.08%, Calc. for C^H^O^: C, 74.95; H, 10.25%. No attempt was made at this stage to eliminate epirockogenin from the product mixture. Rockogenin 3-pivaloate (3g-pivaloyloxy-(25%)-5a-spirostan-12B-ol) (201) Freshly d i s t i l l e d pivaloyl chloride (28.8 g), was added at 0° to a stirred solution of crude rockogenin (85.94 g), dry pyridine (250 ml) and benzene (500 ml) i n a 1-litre round bottomed flask. The flask was l e f t at room temperature for 10 hours, then a small portion of water was added to destroy the excess of pivaloyl chloride. The precipitates of pyridine hydrochloride and epirockogenin-3-pivalate formed were collected by suction f i l t r a t i o n . The f i l t r a t e was transferred into a flask and most of the solvent removed j.n vacuo. The whole residue was dissolved in benzene (1 1.) and the solution was washed twice with half saturated aqueous sodium chloride containing hydrochloric acid to remove the remaining pyridine. The benzene solution was dried over sodium sulfate and the solvent evaporated to give rockogenin-3-pivaloate (3B-pivaloyloxy-(25R)-5a-spirostan-12B-ol) (204) (<7>3.30 g, 71%) as colourless needles. Recrystalliza-tion from acetone gave an analytical sample m.p. 250-252° ( l i t . m.p. 255-257°). 7 5 - 141 -N.M.R. signals: 9.25 (singlet, 3H, C-18 CR"3), 9.21 (doublet, J - 5.5 Hz, 3H, C-27 CH3), 9.14 (singlet, 3H, C<rl9 CH3), 8.97 (doublet, J = 6.5, 3H, C-21 CH3), 8.85 (singlet, 9H, C-3 pivalate protons), 6.5-6.7 (multiplet, 3H, C-26 CH2 + C-12 CH), 5.59 (quartet, J = 7, IH, C-16 CH), 5.35 (multiplet, IH, C-3 CH). I.R.: 3505, 1710, 1292, 1246, 1182, 1057, 984, 959, 923, 903, 866 cm"1. Mass spectrum: M.W. 516; base peak at m/e 139; main peaks at m/e 516, 498, 457, 447, 444, 429, 402, 384, 373, 332, 139, 115, 109. Found: C, 74.22; H, 10.03%. Calc. for C32H_20_: C, 74.37; H, 10.14%. The precipitates formed during the reaction were washed with water and the residue was dried in vacuo to yield epirockogenin 3-pivaloate (203) (10.85 g, 14%). An analytical sample was obtained by 75 '' recrystallization from methylene chloride, m.p. 299-299.5° ( l i t . m.p. 297-299°). Found: C, 74.44; H, 10.28%. Calc. for C32H_20_: C, 74.37; H, 10.14%. Rockogenin 12-methanesulfonate 3-pivalate (12B-mesyloxy-38-pivaloyloxy- (25R)-5ct-spirostan) (205) Crystalline rockogenin 3-pivalate (204) (66.90 g) was dissolved in dry pyridine (500 ml) in a 2-liter 3-necked round bottomed flask equipped with a s t i r r e r , a'dropping funnel and a Drierite tube. The solution was chilled below 5° and freshly d i s t i l l e d mesyl chloride (40 ml) was added dropwise. The reaction mixture was allowed to stand at room temperature for 20 hr, then the flask was immersed in an ice-salt bath and 10% aqueous sodium bicarbonate (350 ml) was added slowly so that the temperature did not exceed 5°. During this operation evolution of a gas was observed and an oily layer, which f i r s t appeared - 142 -on the walls of the flask, turned to a granular solid. Ice water (700 ml) was added and the mixture was stirred for 1 hr below 5°. The solid was f i l t e r e d off, washed with water, digested with ice water, fi l t e r e d again and suction dried over 3 hr, then dried in a vacuum oven overnight with temperature slowly rising to 50°. The grayish granular crude product (74.68 g, 97.0%) obtained appeared homogeneous by t . l . c . and was immediately used for the next reaction without further purification. N.M.R. signals: 8.83 (singlet, 9H, C-3 pivalate protons), 6.99 (singlet, 3H, -OS02CH3). I.R.: 1715, 1358, 1283, 1242, 1170, 1058, 982, 930, 908, 888, 838 cm"1. Mass spectrum: M.W. 594; base peak at m/e 384; main peaks at m/e 492, 477, 384, 355, 282, 253, 126, 115. 3g-Pivaloyloxy-C-nor-D-homo(25R)-5a,12q-spirost-13(18)-en (206, R = '(1CH3)3CC0) The methanesulfonate derivative (205) (43.6 g) twas dissolved i n dry pyridine (350 ml) i n a 1-liter round bottomed flask equipped with ' a condenser and a nitrogen inlet. The solution was refluxed for 14 hr, and pyridine was removed in vacuo. Benzene (600 ml) and ethyl acetate (600 ml) were added and the resultant solution was washed successively with water, dilute hydrochloric acid, 5% aqueous sodium bicarbonate andlxthen with water. The organic layer was dried over sodium sulfate and the solvents were removed in vacuo to yield a solid (37.0 g). Crystallization from petroleum ether (b.p. 65-73°) afforded exocyclic olefin pivalate (206, R = (CH^CCO) (30.2 g, 82.5%), m.p. 193.5-194.5°. N.M.R. signals: 9.21 (doublet, J = 6, 3H, C-27 CH 3), 9.20 (singlet, 3H, C-19 CH3), 8.92 (doublet, J = 6.5, 3H, C-21 CH3), 8.85 (singlet, 9H, C-3 pivalate protons), ca. 7.50 (multiplet, 2H, C-12 CH + C-17 CH), ca. 6.54 - 143 -(multiplet, 2H, C-26 CH2), 5.90 (octet, IH, C-16 CH), 5.31 (multiplet, IH, C-3 CH), 5.17 (broad singlet, 2H, C-18 -CH.), I.R.: 1728, 1719, 1644, 1285, 1247, 1171, 1061, 983, 902, 885, 868 cm"1. Mass spectrum: M.W. 498; base peak at m/e 165; main peaks at m/e 498, 480, 456, 439, 429, 426, 411, 397, 384, 355, 253, 165, 139, 126, 115, 105. Found: C, 76.92; H, 10.08%. Calc. for Cg-H -Cy. C, 77.06; H, 10.11%. C-Nor-D-homo-(25R)-5a, 12ct-spirost-13(18)-en-38-ol (208) The exocyclic olefin pivalate (206, R = (CH^CCO) (13.76 g) was dissolved in dry ether (300 ml) in a 1-liter round bottomed flask, and lithium aluminum hydride (1.00 g) was added slowly. The reaction mixture was stirred for 2 hr at room temperature, and ethyl acetate (5 ml) in ether (400 ml) was added. Celite (10 g), water (3 ml) and anhydrous sodium sulfate (30 g) were introduced consecutively and the mixture was stirred for 30 min before the solid was fi l t e r e d off. Removal of solvents from the f i l t r a t e followed by drying in a vacuum oven at 50° gave crude C-nor-D-homo-(25R)-5a,12a-spirost-13(18)-en-36-ol (208) (11.63 g). An analytical sample was prepared by recrystallization from ethyl acetate, m.p. 184-186°. N.M.R. signals: 9.22 (singlet, 3H, C-19 CH3), 9.21 (doublet, J = 6, 3H, C-27 CH3), 8.93 (doublet, J = 6.5, 3H, C-21 CH3), ca. 7.55 (multiplet, 2H, C-12 CH + C-17 CH), ca. 6.55 (multiplet, 3H, C-3 CH + C-26 CH 2), 5.93 (octet, IH, C-16 CH), 5.21 (broad singlet, 2H, C-18 =CH2). I.R.: 3475, 3360, 1619, 1247, 1053, 983, 903, 884, 866 cm-1. Mass spectrum: M.W. 414; base peak at m/e 126; main peaks at m/e 414, 396, 372, 355, 345, 342, 327, 300, 285, 271, 165, 139, 126, 115, 105. Found: C, 78.30; H, 10.11%. Calc. for C 2_H 4 20 3: C, 78.21; H, 10.21%. - 144 -Hydroboration of 3B-hydroxy-C-nor-D-homo-(25R)-5a,12g-spirost-13(18)-en  (208) The exocyclic o l e f i n (208) (8.02 g) was dissolved i n dry THF (150 ml) i n a 500 ml 3-necked round bottom f l a s k equipped with a s t i r r e r , a condenser and a nitrogen i n l e t . Diborane i n THF (1 M, 40 ml) was added over a 30 minute period, and the so l u t i o n was s t i r r e d at room temperature f o r 3 hour. Aqueous sodium hydroxide (10%, 50 ml) and aqueous hydrogen peroxide (30%, 40 ml) were added and the mixture was s t i r r e d overnight. Removal of the solvents i n vacuo, n e u t r a l i z a t i o n with d i l u t e hydrochloric acid followed by ether extraction, gave a crude product (7.83 g) which was chromatographed on s i l i c a g e l , a c t i v i t y I I , 150 g. El u t i o n with petroleum ether (b.p. 65-110°)-ethyl acetate (2:1) yielded the pure d i o l s (209) and (210) (7.20 g, 92%) and^the t e r t i a r y alcohol (211) (0.30 g, 4J%). 136-Hydroxymethyl-C-nor-D-homo-18-nor-(25R)-5a,12a-spirostan -3B-ol (209); m.p. 182-184° (pet. ether-ethyl acetate). N.M.R. signals (see Figure 38): 9.22 ( s i n g l e t , 3H, C-19 CHg), 9.20 (doublet, J = 6.0, 3H, C-27 CHg), 8.98 (doublet, J = 6.0, 3H, C-21 CH 3), 8.26 (s i n g l e t s , 2H, C-3 OH + -CH 20H), 6.4 (multiplet, IH, C-3 CH), 6.35 (doublet, J = 5.5, 2H, -CH^OH), 5.87 (distorted quartet, IH, C-16 CH), 5.58 (multiplet, 2H, C-26 CH 2), I.R.: 3385, 1241, 1029, 1017, 985, 907, 900, 863 cm - 1. Mass spectrum (see Figure 39): M.W. 432; base peak at m/e 115; main peaks at m/e 432, 402, 373, 363, 360, 345, 318, 300, 288, 145, 139, 126, 115, 107. High r e s o l u t i o n mass spectrum; Found: 432.3200; Calc. f o r C ^ H ^ O ^ 432.3239. Found: C, 74.98; H, 10.26%. Calc. f o r C^H^O^: C, 74.95; H, 10.25%. C-nor-D-homo-18-nor-(25R)-5a,12a-spirostane-36,13£;-diol (211), m.p. 170-172? (pet. ether-ethyl acetate). N.M.R. signals (see Figure 40): 9.24 (s i n g l e t , 3H, C-19 CH„), i J - 145 -9.20 (singlet, 3H, C-27 CHg), 8.90 (doublet, 3H, C-21 CH 3), 8.4 (singlet, 3H, C-18 CH3), 6.5 (multiplet, 3H, C-3 CH + C-26 CH2), 5.8 (multiplet, IH, C-16 CH). I.R.: 3385, 1241, 1029, 1017, 985, 900, 863 cm"1. Mass spectrum (see Figure 41): M.W. 432; base peak at 432; main peaks at m/e 414, 389, 373, 363, 360, 211, 149, 115, 108. High resolution mass spectrum, found 432.3248; calc. for C__H,,0,: 432.3239. Found: 27 44 4 C, 75.08; H, 10.05%. Calc. for C YL £ : C, 75.00; H, 10.05%. 3-Keto-13B-aldehydo-C-nor-D-homo-18-nor-(25R)-5a-spirostan (238) The pure diol (209) (3.6 g) was placed in a 250 ml round bottom flask, and dicyclohexylcarbodiimide (14.64), dimethyl sulfoxide (50 ml), benzene (75 ml), pyridine (2 ml), and trifluoroacetic acid (1 ml) were added to i t . The reaction mixture was stirred at room temperature for 36 hours, then worked up by adding ether (250 ml) and oxalic acid (6.0 g) in isopropyl alcohol (50 ml), and stirring at room temperature for 1 hour. The white crystalline dicyclohexylurea which had precipitated was f i l t e r e d off and the organic layer was 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 in vacuo to yield a yellow solid (4.2 g). An analytical sample was obtained as an amorphous solid by chromatography on s i l i c a gel. N.M.R. signals (see Figure 45): 9.21 (doublet, J = 6, C-27 CH3), 9.08 (singlet, 3H, C-21 CH3), 9.03 (doublet, J = 6.0, C-27 CH3), 7.33 (doublet of tri p l e t s , J12-13 a n d J13-17 = 5 , 5 » J13-18 = 6 ' 1 H ' C ~ 1 3 G H ^ ' 6 , 6 0 ( m u l t i P l e t » 2 H » C-26 CH2), 5.95 (octet, J 1 5 _ 1 6 = 12, J 1 5 _ 1 6 = 4.5, J 1 6 _ 1 7 = 9-5, IH, C-16 CH), and 0.22 (doublet, J = 6, IH, C-18 CHO). I.R.: 2720, 1720, - 146 -980, 920, 900, 867 cm . Mass spectrum: M.W. 428; base peak at m/e 149; main peaks at m/e 428, 369, 359, 356, 341, 314, 285, 256, 206, 149, 135, 126, 115. High resolution mass spectrum, found 428.2953; calc. for C 2_H 4 00 4: 428,2926. 13a-Formyl-C-nor-D-homo-18-nor-(25R)-5aT,12q-spirostan-3-one (239) The crude aldehyde (238) (13.26 g) in methanol (400 ml) was treated with anhydrous potassium carbonate (3.00 g) for 1.5 hr at room temperature. Most of the solvent was removed in vacuo and.iwater was added to the residue. Extraction with methylene chloride gave, after drying over sodium sulfate, the crude epimeric aldehyde (239) (12.69 g). Part of the crude mixture was chromatographed on s i l i c a gel to give an analytical sample. N.M.R. signals (see Figure 46): 9.22 (doublet, J = 6.0, 3H, C-27 CH3), 9.10 (doublet, J = 5.0, 3H, C-21 CH3), 9.08 (singlet, 3H, C-19 CH3), 6.58 (multiplet, 2H, C-26'CH2), 5.98 (octet, IH, C-16 CH), 0.57 (distorted doublet, J = 4, IH, -CHO). Mass spectrum: M.W. 428; base peak at m/e 115; main peaks at m/e 428, 369, 359, 356, 341, 314, 285, 257, 206, 149, 135, 126, 115. High resolution mass spectrum, found: 428.2933; calc. for Co_H..0.: 428.2926. 27 40 4 13a-Hydroxymethyl-C-nor-D-homo-18-nor-(25R)-5a,12a-spirostan-3B-ol (210) The crude aldehyde (239) (12.00 g) was dissolved in methanol (300 ml) and the solution was treated with sodium borohydride (2.00 g) at 0°. After 3 hr of sti r r i n g most of the solvent was removed in vacuo, and the products were extracted from the acidified aqueous layer with chloroform. Drying the organic layer over sodium sulfate followed by evaporation of the solvent gave the crude diol (210) (12.36 g). An - 147 -analytical sample was obtained by crystallization from ethyl acetate-petroleum ether (b.p. 65-73°), m.p. 243.5-245°. N.M.R. signals (see Figure 47): 9.25 (singlet, 3H, C-19 CH 3), 9.21 (doublet, J = 6.0, 3H, C-27 CH3), 8.90 (doublet, J = 6.5, 3H, C-21 CI^), 6.55 (multiplet, 2H, C-26 CH 2), 6.4 (multiplet, IH, C-3 CH), 6.25 (doublet, J = 4.5, CH^OH), 5.95 (octet, IH, C-16 CH). I.R.: 3400, 1247, 1058, 1027, 981, 922, 900, 867 cm Mass spectrum: M.W. 432; base peak at m/e 115; main peaks at m/e 432, 402, 373, 363, 360, 345, 318, 300, 288, 145, 139, 126, 115, 105. Found: C, 74.79; H, 10.38%. Calc. for C^H^O^ C, 74.95; H, 10.25%. 38-Acetoxy-13a-acetoxymethyl-C-nor-D-homo-18-nor-(25R)-5a,12a-spirostan  (240) The diol (210) (5.00 g) was treated with pyridine (30 ml) and acetic anhydride (30 ml) overnight at room temperature. The reaction mixture was then chilled i n an ice bath, and water (200 ml) was added. The mixture was stirred for 1 hr, extracted with ether and worked up to give the crude diacetate (240) (6.09 g) which was chromatographed on alumina (neutral, activity I). Elution with petroleum ether (b.p. 65-73°) gave the pure diacetate (5.63 g) as an amorphous solid. This material resisted crystallization. N.M.R. signals (see Figure 48): 9.23 (singlet, 3H, C-19 CH3), 9.21 (doublet, J = 6.0, 3H, C-27 CH3), 8.98 (doublet, J = 6.0, 3H, C-21 CH3), 8.01 (singlet, 3H, -0C0CH3), 7.99 (singlet, 3H, -0C0CH3), 6.55 (multiplet, 2H, C-26 CH.), 5.95 (multiplet, IH, C-16 CH), 5.85 (doublet, J = 2.0, -CH20Ac), 5.28 (multiplet, IH, C-3 CH). I.R.: 1735, 1385, 1366, 1243, 1057, 1028, 981, - 148 -920, 902, 864 cm~±. Mass spectrum: M.W. 516; base peak at m/e 105; main peaks at m/e 516, 486, 357, 447, 444, 402, 387, 384, 342, 327, 264, 253, 145, 129, 126, 115, 105. High resolution mass spectrum, found: 516.3486; calc. for C,,H/o0£: 516.3451. 31 48 6 Baeyer-Villiger oxidation of 3B-acetoxy-13a-acetoxymethyl-C-nor-D-homo-18- nor-(25R)-5cx,12ct-spirostan (240)  Method BV-I The diacetate (240) (4.90 g) was dissolved in formic acid (98-100%, 100 ml) containing water (5 ml). The solution was heated to 40° and aqueous hydrogen peroxide (30%, 20 ml) was added dropwise in 20 min, while the temperature was maintained at 41-44°. Three additional portions of aqueous hydrogen peroxide (30%, 5 ml) were introduced at hourly intervals. The reaction mixture was then diluted with water (1 l i t r e ) and extracted with methylene chloride. The organic layer was washed with aqueous sodium bicarbonate and with water, then dried and concentrated to give a clear glass (5.13 g) as product. Method BV-II Aqueous hydrogen peroxide (17%, 35 ml) was added dropwise to a solution of the diacetate (232) (10.62 g) in chilled formic acid (98-100%, 200 ml) at ice bath temperature; the ice bath was removed and the mixture was stirred at room temperature for 24 hour. Additional aqueous hydrogen peroxide (30%, 5 ml) was then introduced and sti r r i n g was continued^ for a 12 hour period. The reaction mixture was worked up as in Method I to give a clear glass (10.82 g). N.M.R. signals: - 149 -9.18 (singlet, 3H, C-19 CHg), 7.98 and 7.97 (two singlets, two -OCOCHg groups), 5.97 (doublet, J = 6.5, C-26 CH2), 2.00 and 1.92 (two singlets, two -OCHO groups). Hydrolysis of the Baeyer-Villiger oxidation product  Method I The crude performic acid oxidation mixture (5.13 g) was dissolved in methanol (100 ml) and placed in a 100 ml separatory funnel. Saturated aqueous potassium carbonate (5 ml) was added to the solution and the mixture was shaken for 1.5 min. The reaction mixture was promptly transferred to a 2-liter separatory funnel containing dilute hydrochloric acid (0.1 N, 500 ml), saturated aqueous sodium chloride (500 ml) and methylene chloride (400 ml). The organic layer was washed with aqueous sodium bicarbonate and with water, then dried over sodium sulfate. Evaporation of the solvent in vacuo gave the hydrolysis product as a clear glass (4.83 g). This material was chromatographed on alumina (neutral, activity II). Elution with benzene-ethyl acetate (1:2) gave the di o l (252) (875 mg, 21% based on the diacetate (240)) as crystalline solid. An analytical sample was obtained by recrystalliza-tion from benzene-methylene chloride, m.p. 211-212°. N.M.R. signals (see Figure 52): 9.22 (singlet, 3H, C-19 CH3), 8.71 (doublet, J = 6.5, 3H, C-21 CH3), 8.03 (singlet, 3H, -OCOCRy, 7.99 (singlet, 3H, -OCOCH^, 7.55 (singlet, 2H, two-OH groups; disappeared with D 20), 5.90 (multiplet, IH, C-20 CH), 5.82 (broad singlet, 2H, C-18 CH 2), 5.52 (broad singlet, IH, C-16 CH), 5.35 (multiplet, IH, C-3 CH). I.R.: 3240, 1733, 1378, 1365, 1025 cm "*". Mass spectrum (see Figure 53): M.W. 436; base peak at m/e 107; main peaks at m/e 400, 358, 340, 314, 273, 260, 254, 187, 147, - 150 -145, 107, 105. Found: C, 68.61; H, 9.19%. Calc. for C^H^Og: C, 68.77; H, 9.24%. Method II The crude performic acid oxidation mixture (1.164 g), obtained by the room temperature reaction, was dissolved in methanol (100 ml), a barbital buffer solution (0.05 M, pH 8.20, 10 ml) was added and the mixture carefully titrated with 0.2 M hydrochloric acid to pH 8.0. The solution was stirred at 42-45° for a period of 8 days readjusting the pH to 8.00 every 24 hr. The solvent was removed in vacuo and the products extracted with ethyl ether. These extracts were quickly washed with cold 1 N sodium hydroxide, then water, and f i n a l l y dried over sodium sulfate. Evaporation of the solvent gave a white foam (1.075 g). N.M.R. signals: 9.18 (singlet, 3H, C-19 CHg), 7.98 and 7.95 (two singlets, two -0C0CH3), 6.55 (doublet, J = 5.5, C-26 CH 2), 2.00 and 1.92 (two singlets, two -0CH0 groups). Method III The crude diformate (4.00 g) was dissolved in methanol (250 ml), and activity I neutral alumina pH 8.00 (200 g) was added. The reaction mixture was stirred at room temperature for 6 hours then worked up by addition of chloroform and f i l t r a t i o n , a yellow o i l being obtained (3.15 g). This material was chromatographed on activity II s i l i c a gel, eluting with chloroform ethyl acetate mixtures, to yield three major fractions: - 151 -1) least polar: (0.943 g; 30%) N.M.R. signals: 9.18 (singlet, 3H, C-19 CHg), 7.98 and 7.95 (two singlets, two -OCOCHg), 4.18 (broad singlet, 2H, C-18 CH 2), 4.65 (multiplet, IH, C-3 CH), 2i00 and 1.92 (two singlets, two -0CH0 groups). This material was dissolved in a separatory funnel and methanol (10 ml) and saturated aqueous potassium carbonate solution (1 ml) were added. After 3 minutes of shaking the mixture was extracted with methylene chloride. The methylene chloride layer was washed with water and dried over sodium sulfate. Evaporation of the solvent gave a crystalline material which was purified by column chromatography on s i l i c a gel to give the diol (2523) (0.470 g). 2) middle fraction (1.82 g; 58%) N.M.R. signals: 9.22 (singlet, 3H, C-19 CH3), 8.71 (doublet, J = 6.5, 3H, C-21 CH3), 8.03 (singlet, 3H, -OCOCHg), 7.99 (singlet, 3H, -0C0CH3), 6.57 (doublet, J = 4, 2H, C-26 CH 2), 5.94 (broad singlet, 2H, C-18 CH2), 5.35 (multiplet, IH, C-3 CH), 2.00 and 1.98 (two formates, the*; latter considerably reduced). This material was rehydrolyzed on alumina and submitted to Jones oxidation followed by base treatment and acetylation to give the a, 6-unsaturated ketone (256) (30%), with the tetraacetate (254) as the major side product. A l l purification attempts at this stage failed so the mixture was reduced over platinum and the product treated with boron trifluoride etherate in benzene solution. The separation of the saturated ketone (260) from the tetraacetate (254) was achieved using modified column chromatography on fine mesh s i l i c a gel under pressure (5 psi). - 152 -3) polar fraction (0.41 g; 12%) N.M.R. signals: 9.22 (singlet, 3H, C-19 CH.), 8.71 (doublet, J = 6.5, 3H, C-21 CH3), 8.03 (singlet, 3H, -OCOCHg), 7.99 (singlet, 3H, -0C0CH3), 6.57 (doublet, J = 4, 2H, C-26), 5.94 (broad singlet, 2H, C-18 CH2), 5.35 (multiplet, IH, C-3 CH). No formate groups. This material was submitted to the remaining steps of the spiro ketal system to give the a,8-unsaturated ketone (256) free of the tetraacetate (254). Formylation of the diol (252) The diol (252) was treated with acetic-formic anhydride (6 ml) and pyridine (4 ml) at 50° for 3 hr. Benzene (150 ml) was added and the solution washed with hydrochloric acid, water, a saturated solution of sodium bicarbonate and then dried over sodium sulfate. Evaporation of the solvent i n vacuo gave the crude product as a crystalline solid (121 mg). An analytical sample of the diformate (258) was obtained by recrystallization from heptane-benzene, m.p. 167-169°. N.M.R. signals (see Figure 59): 9.21 (singlet, 3H, C-19 CHg), 8.70 (doublet, J = 6.5, 3H, C-21 CH 3), 8.02 and 8.00 (two singlets, two -OCOCHg), 6.01 and 6.82 (AB quartet, J = 12, 2H, C-18 CH2), 5.35 (multiplet, IH, C-3 CH), 4.80 (quartet, J = 6, IH, C-20 H), 4.44 (broad singlet, IH, C-16 CH), 2.07 and 2.00 (two singlets, two -0CH0 groups). I.R1: 1736, 1726, 1720, 1711, 1248, 1208-1176 cm 1 (four bands). Mass spectrum (see Figure 60): M.W. 492 (M+ not v i s i b l e ) ; base peak at m/e 340; main peaks at m/e 432, 386, 340, 326, 280, 265, 187, 147, 145, 143, 131, 119, 107, 105. Found: C, 65.76; H, 8.30%. C a l c for C 0 7H / nO a: C, 65.83; H, 8.19%. - 153 -Jones oxidation of the diol (251) The essentially pure diol (251) (733 mg) was dissolved i n pure acetone (25 ml). The solution was placed in a 50 ml round bottom flask, and cooled to 5°in an ice bath. Jones reagent (1.3 ml) was then added dropwise with sti r r i n g over a period of 15 minutes . The reaction was quenched with methanol and diluted with water , and the crude product (745 mg) was isolated by extraction with chloroform . Theimaterial was non-crystalline, and could not be purified by chromatography; hence, spectral data was obtained on the crude material. N.M.R. signals: 9.20 (singlet, 3H, C-19 CHg), 8.02(singlet, 3H, -OCOCHg), 8.00 (singlet, 3H, -0COCH3). I.R.(chloroform solution): 3600-3200, 1730, 1450, 1360, 1230 cm """ . Mass spectrum: no parent vi 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, 100. j.3d-Acetoxymethyl-17-acetyl-18-nor-5a-etiojerv-16(17)-en-3B-ol-3- acetate (256) The crude keto-acid (248) (644 mg) was placed in a 100 ml, 3-necked round bottom flask fitted with a condenser and a nitrogen inlet , and t-butanol (50 ml) and 5% aqueous potassium hydroxide (10 ml) were added. The solution was refluxed for 1 hour under nitrogen, then cooled and the t-butanol was removed in vacuo. The resultant solution was diluted with water and extracted with ether, the ether layer being dried over sodium sulfate. The ether was removed in vacuo and the product treated with pyridine (25 ml) and acetic anhydride (25 ml) overnight. Benzene (100 ml) was added, and the organic layer was washed - 154 -with 1 N hydrochloric acid, saturated aqueous sodium bicarbonate and water. The solvents were removed in vacuo to yield the crude ct,8-unsaturated ketone (249) (490 mg). This material was chromatographed on activity II alumina, eluting with benzene-chloroform, to yield the pure, non-crystalline a,B-unsaturated ketone (256). N.M.R. signals: 9.34 (singlet, 3H, C-19 CE^), 8.04 (singlet, 6H, two acetates), 7.70 (singlet, 3H, C-21 CH 3), 6.12 (distorted t r i p l e t , J = 7.5, IH, C-13 CH), 6.07 (octet, 2H, -CH20Ac), 5.33 (multiplet, IH, C-3 CH), 2.84 (quartet, J = 3 and 7, IH, C-16 CH). I.R.: 1720, 1670, 1450, 1370, 1250, 1030 cm"1. U.V. spectrum X 233 nm (e 6000). Mass spectrum: M.W. 416; base max peak at m/e 356; main peaks at m/e 416, 388, 372, 370, 256, 343, 327, 296, 202, 187, 149, 135, 107, 105. High resolution mass spectra, found: 416.2569; calc. for C„_H_,0_: 416.2563. Z J Jo J Reduction of the q,B-unsaturated ketone (256) The a,B-unsaturated ketone (256) (200 mg) was dissolved in ethanol (25 ml) and added to a previously reduced suspension of Adams' catalyst (50 mg) in ethanol (5 ml) under hydrogen. After 1 hour the catalyst was f i l t e r e d off using celite and the solvent evaporated under vacuum to yield a mixture of epimeric ketones (259) and (260). This mixture was chromatographed on s i l i c a gel eluting with ether-petroleum ether to give: 3B-Acetoxy-13q-acetoxymethyl-18-nor-12q,17q-pregnajervan-20-one (259) (7 mg), m.p. 145-147° (petroleum ether). N.M.R. signals: 9.18 (singlet, 3H, C-19 CH 3), 8.08 (singlet, 6H, two -0C0CH3), 7.94 (singlet, 3H, C-21 CH 3), 7.15 (triplet, J = 5, IH, - 155 -C-13 CH), 5.92 (doublet, J = 7, 2H, C-18 CH2), 5.40 (multiplet, IH, C-3 CH). Mass spectrum: M.W. 418; base peak at m/e 43, other peaks at m/e 374, 358, 300, 298, 255, 149, 135, 107. Found: C, 71.54; H, 9.30%. Calc. for C^H^O.: C, 71.74; H, 9.15%. 3B-Acetoxy-13cx-acetoxymethyl-18-nor-12a,178-pregnajervan-20-one (260) (25 mg) as a glass; [a]^'2 -44° (c 1.47, CHClg). N.M.R. signals: 9.17 (singlet, 3H, C-19 CHg), 8.10 (singlet, 6H, two -0C0CH3), 7.87 (singlet, 3H, C-21 CHg) , 6.31 (multiplet, 3H, C-13 CH), 5.95 (doublet, J = 7, 2H, C-18 CH 2), 5.40 (multiplet, IH, C-3 CH). Mass spectrum: M.W. 418; base peak at m/e 43; other peaks at m/e 374, 358, 300, 298, 255, 149, 141, 135, and 107. The mass spectra of the two isomers were virtually identical. High resolution mass spectrum: Found: 418.2718; calc. for C.-Hoo0_: 418.2702. - 156 -Isomerization of the Saturated Ketone (259) The mixture of the saturated ketones (259) and (260) (42 mg) was dissolved in dry benzene (2 ml) and borontrifluoride etherate (0.15 ml) was added dropwise. The reaction was allowed to remain at room temperature for 15 hours, then diluted with ether (50 ml), washed with saturated bicarbonate solution (10 ml), water, dried over sodium sulfate and evaporated jln vacuo to give as the sole product the 178-isomer (260) (40 mg). Reduction of the Tetraacetate (254) Lithium aluminum hydride (0.475 g) was slowly added to a stirred solution of the tetraacetate (254) (0.675 g) in dry ethyl ether (45 ml) under nitrogen. The mixture was stirred overnight, then the excess of lithium aluminum hydride was destroyed by the careful addition of water. Dilute hydrochloric acid was added to the suspension obtained and this mixture was extracted with ethyl ether. The ether extract was dried and evaporated under vacuo to give the tetrol (268) (0.458 g) as a white solid. N.M.R. signals (CD3COCD3): 9.22 (singlet, 3H, C-19 CH 3), 8.73 (doublet, J = 5.0 Hz, 3H, C-21 CH 3), 7.25 (broad singlet, 4 -OH groups), 6.46 (broad singlet, 2H, C-18 CH_20H). Mass spectrum: M.W. 352 (not v i s i b l e ) ; base peak at m/e 107; main peaks at m/e 334, 317, 316, 299, 272, 251, Acetonide Formation from 13B-Hydroxymethyl-C-nor-D-homo-18-nor-pregnaj ervan-3B, 16E,, 21g-triol (268) - 157 -A s o l u t i o n of the t e t r o l (268) (0.132 g) and p-toluene s u l f o n i c a c i d (10 mg) i n acetone (25 ml) was s t i r r e d f o r 18 hr at room temperature and then d i l u t e d with water. The mixture was extracted with e t h y l ether, and the ether extract was washed with 5% aqueous sodium hydroxide and brine, then dried and concentrated to give a yellow o i l . The crude acetonide (269) was dissolved i n p y r i d i n e -a c e t i c anhydride (2:1) (35 ml) and s t i r r e d f o r 5 hr at 20°. The mixture was d i l u t e d with water, extracted with ether, and the ether extract was washed with d i l u t e hydrochloric acid then dried and concentrated to give a pale yellow o i l which was chromatographed on s i l i c a g e l . E l u t i o n with pet. ether-ethyl acetate (10:1) gave the diacetoxy acetonide (270) (0.116 g) as an o i l . N.M.R. signals (see Figure 67): 9.26 ( s i n g l e t , 3H, C-19 CH 3), 8.82 (doublet, J = 5.5 Hz, 3H, C-21 CH 3), 8.02 and 7.99 (two s i n g l e t s , two -0C0CH3 groups), 7.71 (s i n g l e t , 6H, acetonide CH 3) , 5.98 (multiplet, 2H, C-18 CH 2), 6.30 and 5.94 (two mu l t i p l e t s , C-20 and C-16 CH), 5.30 (multiplet, C-3 H). Mass spectrum (see Figure 68): M.W. 476 (not v i s i b l e ) ; base peak at m/e 461; main peaks at 462,. 358, 341, 314, 281, 254, 241, 187, 159, 147, 129, 119, 110, 107. Found: C, 70.31; H, 9.42%; Calc. f o r C 2 8 H 4 6 ° 6 : C ' 7 0 , 5 1 ; H ' 9 - 6 2 % -Hydrolysis of the Acetonide (270) i A s o l u t i o n of the diacetoxy acetonide (270) (84 mg) and p-toluene s u l f o n i c a c i d i n methanol (25 ml) was s t i r r e d at 50° for 1 hr, then cooled and d i l u t e d with brine s o l u t i o n . The mixture was extracted with ethyl ether and the ether extract was dried and concentrated to give the d i o l (252) (72 mg) as a c r y s t a l l i n e s o l i d which was r e c r y s t a l l i z e d - 158 -from benzene-methylene chloride, m.p. 211-212°. This material was identical in a l l respects to the diol obtained in the potassium carbonate hydrolysis of the Baeyer-Villiger product. Oxidation of the Diol (252) Jones reagent (0.6 ml) was added dropwise to a stirred solution of the diol (252) (0.115 g) i n tetrahydrofuran (30 ml) at 20°. The mixture was stirred for 15 min and methanol was added to destroy the excess of reagent. The solution was concentrated to a gum and ethyl ether added. The ether extract was washed with water, dried over sodium sulfate and evaporated in vacuo to yield the 3B-acetoxy-13ct-acetoxymethyl-C-nor-D-homo-18-nor-pregnajervan-16,20-dione (257) (0.107g) as a colourless o i l . N.M.R. signals (see Figure 57): 9.32 (singlet, 3H, C-19 CH-j), 8.00 and 8.04 (two singlets, two -OCOCHg), 7.90 (singlet, 3H, C-21 CH3), 6.04 (multiplet, 2H, C-18 CH2), 5.4 (multiplet, IH, C-2 CH). I.R.: 1720, 1260 cm"1. U.V. (0.1 N KOH-CHo0H) : 289 nm (11.520). Mass spectrum (see Figure 58): M.W. 3 max 432; base peak at m/e 359; main peaks at m/e 372, 360, 330, 300, 299, 281, 252, 199, 187, 185, 171, 161, 159, 157, 151, 149, 143, 137, 126, 121, 119, 107. High resolution mass spectrum: Found: 432.2556, Calc". for CocHo,0.: 432.2511. 25 36 6 Enol Acetate Formation from B-Diketone (257) A solution of the diketone (257) (0.176 g) and acetic anhydride in pyridine (10 ml) was stirred for 18 hr at 20° then poured into ice water. The mixture was extracted with ethyl ether and the ether extract was washed with dilute hydrochloric acid, dried and concentrated - 159 -to a gum which was chromatographed on s i l i c a gel. Elution with pet. ether (b.p. 65-110°)-ethyl acetate (6:1) gave the enol acetates (271 and 272) as a colourless o i l . N.M.R. signals: 9.24 (singlet, 3H, C-19 CH3), 8.06 and 8.10 (two singlets, two -OCOCHg), 7.85 (singlet, 3H, C-16 enol acetate), 7.82 (singlet, 3H, C-21 CR^), 7.76 (singlet, 3H, C-20 enol acetate), 7.68 (singlet, 3H, C-21 vinyl CHg), 6.00 (multiplet, 2H, C-18 CH2), 5.4 (multiplet, IH, C-3 CH). I.R.: 3010, 1730, 1630, 1250, 1150 cm"1. U.V.: X 240 nm. Mass spectrum: max M.W. 474; base peak at m/e 359; main peaks at m/e 432, 416, 401, 372, 356, 315, 314, 300, 299, 149, 147, 107. High resolution mass spectrum: Found: 474.2723; Calc. for Co_Ho_0o: 474.2617. LI 51 o Hydrogenolysis of the Enol Acetates (271 and 272) A solution of the enol acetates (271 and 272) (0.210 g) and platinum oxide (35 mg) in ethanol (30 ml) was stirred under hydrogen for four days and then fi l t e r e d . The f i l t r a t e was concentrated to a colourless o i l (0.180 g) which was chromatographed on s i l i c a gel. Elution with pet. ether-ethyl acetate gave the saturated ketones 259 (11 mg) and .260) (98 mg) as crystalline solids identical in a l l respects to the saturated ketones obtained previously in the reduction of the a,B-unsaturated ketone (256). 3B,18-Diacetoxy-20-hydroxy-22,23,24,25,26-N-hexadehydro-5ct,13B(H),17a(H)- veratranine (277) Dry ether (5 ml) was placed in a flame-dried 25 ml 3-necked round bottomed flask equipped with a s t i r r e r , a thermometer, and a condenser topped with a .,argon in l e t . The flask was cooled to -60° with an - 160 -acetone-dry ice bath. Addition of 2-bromo-5-methy-pyridine (1.45 g,, 15 equivalents) in ether (10 ml) and tetrahydrofuran (20 ml) followed by n-butyllithium (1.8 M in hexane, -7.8' ml, 13 equivalents) resulted in a deep red solution when the temperature was allowed to rise to -40° in 45 min. 3B-Acetoxy-13a-acetoxymethyl-18-nor-12a-pregnajervan-20-one (260) (234 mg) i n tetrahydrofuran (10ml) was then added i n 8 min and the mixture was stirred at -40° for 3 min before dilute hydrochloric acid (2 N, 50 ml) was added to quench the reaction. The aqueous layer was shaken with petroleum ether (65-73°)-methylene chloride (4:1, 50 ml) and the organic layer was washed with dilute hydrochloric acid (2 N,25 ml). The combined aqueous layers were neutralized with ammonium hydroxide and extracted with methylene chloride to give the basic reaction products (462 mg) which were acetylated with acetic anhydride-pyridine at room temperature overnight. The reaction mixture was worked up by extracting the products with methylene chloride to yield a yellow glass (462 mg). This material was chromatographed on alumina (anisotropic, activity II, 10.5 g) to remove the non-steroidal material. The f i n a l purification was achieved by column chromatography on fine mesh s i l i c a gel under pressure (5 psi) (54 mg). N.M.R. signals: 9.21 (singlet, 3H, C-19 CHg), 8.55 (singlet, 3H, C-21 CHg), 8.11 and 8.01 (two singlets, 6H, two -OCOCHg), 7.68 (singlet, 3H, C-27 CHg), 6.33 (octet, 2H, C-18 CH2), 5.28 (multiplet, IH, C-3 CH), 2.96 (doublet, J = 8, IH, C-23 CH), 2.50 (doublet of doublets, J = 8 and 2.5 Hz, IH, C-24 CH), 1.65 (broad singlet, IH, C-26 CH). U.V.: 267 nm (e = 3700). I.R.: 3400, 1725, 1603, 1581, 1381, 1367, 1260, 1026, 831 cm"1. Mass spectrum: M.W. 511; base peak at m/e 136; main peaks at m/e 511, - 161 -496, 493, 451, 358, 255, 137, 107. High resolution mass spectrum: Found: 511.3294; Calc. for C_.H.CN0C: 511.3298. 31 45 5 In a second experiment the coupling product (277) obtained according to the above procedure s t i l l had the presence of a pyridine impurity. This material was then submitted to high pressure liquid chromatography (Waters, ALC 100) in order to separate the two epimers at C-20 and remove the undesirable impurity. The preparative work was done according to the following conditions: Column neutral alumina Size 3/8" x 4 feet Solvent n-heptane-dioxane 15% Pressure 50 psi Flow 3 ml/min Chart speed 10 min/in Recorder R/10 Retention time coupling product A - 19 min coupling product B - 32 min The N.M.R. spectra of the two products indicated that the impurity was s t i l l present in both. In addition the analytical high pressure liquid chromatography (as above, column size 1.8" x 24", pressure: 1100 psi) indicated that complete separation had not been obtained., In order to separate the pyridine impurity from both epimers chromato-graphy columns on alumina Woelm, activity II, eluting with pet. ether (b.p. 60-110°) and ethyl acetate (2:1) were attempted. The fractions obtained were analyzed with high liquid pressure chromatography and N.M.R. which showed that the epimers were separated and the impurity - 162 -had been removed. For the N.M.R. and Mass spectra of the coupling products A and B see Figures 70 and 71 respectively. Reduction of the Coupling Product (277) A solution of the coupling product (21 mg) in glacial acetic acid (2 ml) was added to platinum oxide (Adam's catalyst) (15 mg) in acetic acid (5 ml), previously prehydrogenated and stirred under hydrogen overnight. The catalyst was removed by f i l t r a t i o n and most of the acetic acid removed under vacuo. Water was added and the acetic acid neutralized with ammonia. The mixture was extracted with ethyl ether and the ether extract dried over sodium sulfate. Evaporation of the ether under vacuo gave the desired reduced product 3B,18-diacetoxy-20-hydroxy-5a,138(H), 17a(H)-veratranine as one spot on t . l . c . (18 mg). N.M.R. signals: 9.20 (singlet, 3H, CT19 CHg), 8.98 (doublet, 3H, C-21 CH3), 8.92 (singlet, 3H, C-27 CH3), 8.00 and 7.96 (two singlets, 6H, two -0C0CH3 groups), 7.40 (singlet, 2H, C-22 CH(a) and C-26 CH(a)), 7.12 (singlet, 2H, C-22 CH(e) and C-26CCH(e)), 5.76 (multiplet, 2H, C-18 CH2), 5.28 (multiplet, IH, C-3 CH). I.R.: 3440, 1730, 1380, 1260 cm "*". Mass spectrum: M.W. 517; base peak at m/e 98; maintpeaks at m/e 517, 502, 498, 457, 406, 368, 350, 301, 265, 251, 199, 185, 173, 159, 142, 126, 112. Cyclization attempt The piperidine derivative (277) (8 mg) was heated in triglyme at 130 for 72 hr. The solvent was removed in vacuo and the mixture chromato-graphed on s i l i c a gel eluting with pet. ether:ethyl acetate (2:1) to give a steroidal material (2 mg) from which the mass spectrum was taken (see Figure 80). - 163 -1-. L. Velluz, J. Vails, and G. Nomine, Angew Chem. Int. Ed., 4_, 181 (1965). 2. L. Labler and F. Sorm, Coll. Czech. Chem. Comm., 27, 276 (1966). 3. R. Bengelmans, H.O. Husson, and J. LeMen, Bull. Soc. Chim. France, 136 (1964). 4. P. Bushchacher, J. Kalvoda, D. Arigoni, and 0. Jeger, J. Amer. Chem.8 Soc., 80, 2905 (1958). 5. V. Cerny,and F. Sorm, i n "The Alkaloids", Ed. by. R.H. Manske, Vol. IX, page 358, Academic Press, N.Y. (1962). 6. W. Nagata, T. Teresawa and T. Aoki, Tetrahedron Letters, 865, 869 (1963). 7. W.S_' Johnson, V.J. Bauer and R.W. Frank, Tetrahedron Letters, 72 (1961). 8. R. Goutarel, Bull. Chim. Soc. France, 1665 (1964). 9. M.M. Janot, C. 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