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UBC Theses and Dissertations

Synthetic and structural studies in natural products Hall, Judith Eleanor 1967

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The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF JUDITH ELEANOR HALL B.Sc.,M.Sc.3 U n i v e r s i t y of Alberta, Calgary . MONDAY, SEPTEMBER 11, at 3:30 p.m. IN ROOM 261, CHEMISTRY BUILDING COMMITTEE IN CHARGE Chairman: I. McT. Cowan . Dr. C A „ McDowell R.C. Thompson D„G„ McGreer G.H.N. Towers T. Money J.P. Kutney External Examiner: W. Klyne Department of Chemistry Westfield College Hampstead, London NW3 England Research Supervisor: J 0P, Kutney SYNTHETIC AND STRUCTURAL STUDIES IN NATURAL PRODUCTS ABSTRACT Part 1 describes the synthesis of r i n g A-oxygenated 6-aza steroids i n the cholestane s e r i e s . Methyl-5-oxo-5,7-seco-6-nor-3-cholesten-7~oate (89) was reacted with benzylamine to give N-benzyl-6-aza-2,4-cholestadien-7-one (88) . S e l e c t i v e hydrobora-ti o n of the 2,3-double bond of t h i s compound yiel d e d three alcohols which were i d e n t i f i e d as 3 -Ct, 3^ 3- and 2a.-hydroxy-N-benzyl-6-aza-4-chole-sten-7-one (91,92 and 90 r e s p e c t i v e l y ) . Oxidation of the f i r s t two with chromium t r i o x i d e i n either acetone or pyridine gave N-benzyl-6-aza-4-choles-ten-3,7-dione (93). In Part 2 the ORD curves of 6- and 11-aza steroids possessing lactam, enol lactam and amide functions are discussed. A l l the compounds studied exhibited p o s i t i v e Cotton e f f e c t s . An attempt i s made to in t e r p r e t the sign of the Cotton e f f e c t s observed for the 6-aza s t e r o i d lactams i n terms of the configuration at C^. An i n v e s t i g a t i o n of the spores of Equisetum  telmateia i s described i n Part 3. Equisporoside was i s o l a t e d from the methanol extracts and shown to be' i d e n t i c a l with the known flavonoid, gossy-p i t r i n (22). It was concluded that the structure of e q u i s e t o l i c a c i d which was i s o l a t e d from the ether extracts of the spores was H00C(CH o) o oC00H. Z Z o This work corrects the previous formulations sug-gested for these compounds by other workers. The a l k a l o i d content of the spores was found to be n e g l i g i b l e . GRADUATE STUDIES F i e l d of Study: Chemistry Topics i n Organic Chemistry Heterocyclic Compounds A l k a l o i d Chemistry Isoprenoid Compounds Recent Synthetic Methods i n Organic Chemistry Seminar i n Chemistry Physical Organic Chemistry Structure of Newer Natural Products D. McGreer L.D. H a l l F. McCapra F. McCapra J„P. Kutnev To Mone.j E. Piers A„ Rosentha R. Stewar*-To Monet PUBLICATIONS M.H. Benn and J 0 E . May, The Biosynthesis of Diterpenoid A l k a l o i d s , Experientia, 20, 252 (1964) M„H„ Benn and J.E„ May, Raney Nickel Desulphurisation of Catechol Thionocarbonate: .A New Synthesis of; Methyl'enedioxyBenzene, Chem, and Ihd., 499 (1964) J„P„ Kutney, G. Eigendorf, and J„E. May, Opt i c a l Rotatory Dispersion Studies on Aza Steroids, Chem, Commun., 59 (1966) J.Po Kutney, G. Eigendorf and J„E„ H a l l , Synthesis of Ring A-Oxygenated 6-Aza Steroids, Tetrahedron, 23, i n press (1967) SYNTHETIC AND STRUCTURAL STUDIES IN NATURAL PRODUCTS Part 1. Synthesis of Ring A-Oxygenated 6-Aza Steroids Part 2. ORD Studies of Lactam and Amide Chromophores Part 3. Investigation of the Spores of Equisetum telmateia by JUDITH ELEANOR HALL B.Sc., The Univ e r s i t y of Alberta, Calgary, 1962. M.Sc, The University of Alberta, Calgary, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Chemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1967. 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 t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 a nd S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r b y h.i.'S r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f 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 n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , C a n a d a i i ABSTRACT Part 1 describes the synthesis of r i n g A-oxygenated 6-aza steroids i n the cholestane s e r i e s . Methyl-5-oxo-5,7-seco-6-nor-3-cholesten-7-oate (89) was reacted with benzylamine to give N-benzyl-6-aza-2,4-cholestadien-7-one (88). S e l e c t i v e hydroboration of the 2,3-double bond of t h i s compound yiel d e d three alcohols which were i d e n t i f i e d as 3a- , 33-and 2a-hydroxy-N-benzyl-6-aza-4-cholesten-7-one (91, 92 and 90 r e s p e c t i v e l y ) . Oxidation of the f i r s t two with chromium t r i o x i d e i n e i t h e r acetone or pyridine gave N-benzyl-6-aza-4-cholesten-3,7-dione (93). In Part 2 the ORD curves of 6- and 11-aza steroids possessing lactam, enol lactam, and amide functions are discussed. A l l the compounds studied exhibited p o s i t i v e Cotton e f f e c t s . An attempt i s made to in t e r p r e t the sign of the Cotton e f f e c t s observed f o r the 6-aza s t e r o i d lactams i n terms of the configuration at C5. An i n v e s t i g a t i o n of the spores qf Equisetum telmateia i s described i n Part 3. Equisporoside was i s o l a t e d from the methanol extracts and shown to be i d e n t i c a l with the known flavonoid, g o s s y p i t r i n (22). It was concluded that the structure of e q u i s e t o l i c a c i d which was i s o l a t e d from the ether extracts of the spores was HOOC(CH2)28C00H. This work corrects the previous formulations suggested f o r these compounds by other workers. The a l k a l o i d content of the spores was found to be n e g l i g i b l e . TABLE OF CONTENTS Page Abstract . . . . . i i Table of Contents i i i L i s t of Figures . . . . . . . . i v L i s t of Tables . . . . . . . . . . . . . . . . . . . . . . v i i Acknowledgements . . . . v i i i PART 1 Introduction . . . . . . . . . 1 Discussion 28 Experimental . . . . 49 References • 62 PART 2 Introduction . . . . . . . . . . . . 67 Discussion • 81 Experimental 92 References . . . . . . . . . . . . . . . . . 93 PART 3 Introduction . . . . . . . . . . . . . 96 Discussion . . . . . . . . . . . . . . . 103 Experimental = .' 119 References . . . . 128 i v LIST OF FIGURES PART 1 Figures Page 1. Important Intermediates i n the Biosynthesis of Cholesterol 2 2. Synthesis of 1-Aza and 1-Aza-A-Homo Steroids . . . . . 5 3. Synthesis of 2- and 3^Aza Steroids 6 4. Synthesis of 3- and 4-Aza-ALHomo Steroids . 7 5. Synthesis of A-Homo-A-Azapregnanedione . 8 6. Synthesis of 5-Aza Steroids . 9 7. Synthesis of 6- and 7-Aza Steroids 10 8. Synthesis of 3-Aza-4-Hydroxyequilenin 10 j 9. Synthesis of 6-Aza Steroids v i a a Modified Curtius Reaction 11 10. 6-Aza Steroids 11 11. C y c l i z a t i o n of Keto Acids to 4-Aza Steroids . . . . . 12 12. C y c l i z a t i o n of Keto Esters to 6-Aza Steroids . . . . . 13 13. 11-Aza Steroids . . , . 14 14. 17-Azapregnane Derivatives 16 15. 173-Amino a.ndrostane Derivatives 17 16. 17a-Amino androstane Derivatives . . 17 17. Synthesis of 23-, 24- and 25-Azacholesterols 18 18. Side-Chain A z a c h o l e s t e r o l s 19 19. Total Synthesis of A-Nor-2,3-diaza-3-phenyl-oestra-l,5 (10), 8,14-tetraen-17-one . . . . . 21 20. To t a l Synthesis of d l - 6 - A z a e q u i l e n i n 21 21. Total Synthesis of dl-6-Aza e-quilenin and other 6-Aza-estrone Derivatives 22 22. Total Synthesis of 8-Azaestrone . . . . . . . . . . . . 23 V LIST OF FIGURES (cont'd) PART 1 Figures Page 23. Total Synthesis of 8-Azaestrone 23 24. Total Synthesis of 8- and 9-Aza Steroids 24 25. Synthesis of dl-8-Azaestrone Methyl Ether and Related Systems . . . . . . . . . . . . . 25 26. Synthesis of 13-Aza-18-Nor e.quilenin Methyl Ether . . 26 27. Synthesis of 13-Azaequilenin Derivatives 26 28. Total Synthesis of 8,13-Diaza-18-r)orestrone Methyl Ethers 27 29. Low F i e l d Region of NMR Spectra of Hydroboration Products 33 30. Mass Spectra 43 31. ORD Curves . . . . . . . . . . . . . . . . . . . . 47 PART 2 1. Synthesis of 6-0xa and 6-Aza Steroids . 71 2. ORD Curves of Compounds 37, 38 and 40 i n 6-Aza St e r o i d Series 83 3. ORD Curves of Compounds 44, 47 and 52 i n 6-Aza Steroid Series . . . . . . . . . . . . . 83 4. ORD Curves of Compounds 55, 57 and 59 i n 11-Aza Steroid Series . . . . . . . . . . . . . . . . 83 5. ORD Curve of Compound 41 Showing E f f e c t of Concentration 83 6. ORD of S t e r o i d Lactones . . . . . . . . . . . . . 87 PART 3 1. U l t r a v i o l e t Spectra of Equisporoside . 105 2. NMR Spectrum of Equisporoside . . . . . . 108 3. Paper Chromatography of the Sugar 110 v i LIST OF FIGURES (cont'd) PART 3 Figures Page 4. NMR Spectrum of Equisporol I l l 5. Mass Spectrum of E q u i s e t o l i c Acid . 115 6. Mass Spectrum of E q u i s e t o l i c Acid Methyl Ester . . 115 7. NMR Spectrum of E q u i s e t o l i c Acid Methyl Ester . . . 116 8. Other Constituents of Equisetum telmateia Methanol Extracts . . . . . . . . . 118 v i i LIST OF TABLES PART 1 Tables Page 1. V i n y l - A l l y l i c Spin Couplings 36 2. Coupling Constants f o r Benzylic Methylene Protons . 40 3. NMR Data (100 Mc/s) on Benzylic Protons 41 4. Mass Spectra of Hydroboration Products 57-61 PART 2 1. Chromophoric Derivatives . . . 69 2. Molecular Rotation Differences . . . . 70 3. Molecular Rotations of Oxa Steroids . 72 4. ORD of 6-Aza Steroid Derivatives . . . . . . . 82 5. ORD of 11-Aza Steroid Derivatives 89 PART 3 1. U l t r a v i o l e t Spectra of Equisporoside . . 104 2. U l t r a v i o l e t Spectra of Equisporol . . . . . . . . 104 3. Pentamethyl Ethers of Gossypetin . . . . . . . . 113 4. Rp Values of Sugars 125 ACKNOWLEDGEMENTS I wish to express my thanks to Pro f e s s o r J.P. Kutney f o r the opportunity o f working with him and f o r h i s guidance during the course of t h i s research. We are indebted to Pro f e s s o r T.A. Geissman f o r samples of g o s s y p i t r i n , gossypetin and gossypetin hexamethyl ether. PART I SY N T H E S I S OF RING ATOXYGENATED 6-AZA STEROIDS 1 INTRODUCTION In recent years there have been numerous investigations concerned with the e f f e c t of substituents attached to the normal s t e r o i d skeleton. These investigations led to the r e a l i z a t i o n that very dramatic a l t e r a t i o n s i n b i o l o g i c a l properties are encountered when substituents such as methyl, hydroxyl, and halogen, p a r t i c u l a r l y f l u o r i n e , are placed at s p e c i f i c 1-3 positions i n the molecule. A more s i g n i f i c a n t a l t e r a t i o n i n the structure and chemical nature of st e r o i d s , replacement of one or more carbon atoms with a nitrogen atom, i s known i n some cases to provide b i o l o g i c a l l y active substances. The object of t h i s research was to synthesize a r i n g B aza s t e r o i d which possessed the C-3 oxygen function l o s t during the early stages of previous syntheses. This was of i n t e r e s t as the A ^ k e t o moiety i s present i n most of the active s t e r o i d a l hormones. Recent b i o l o g i c a l tests with a number of A and B r i n g modified 20,25-diazacholesterol analogues showed that those possessing an oxygen function at C-3 were more active than those without. It therefore appears that hypocholesterolemic a c t i v i t y i s 4 associated with the l o c a l i z a t i o n of electrons near the C-3 atom. The a c t i v i t y of aza steroids i s due to t h e i r a b i l i t y to block the biosynthesis of cholesterol (Figure 1) apparently e i t h e r by i n h i b i t i n g the conversion of 3-hydroxy-3-methy.lglutaryl coenzyme A into mevalonic a c i d or by i n h i b i t i n g the reduction of desmosterol to c h o l e s t e r o l . Aza steroids may therefore be of c l i n i c a l value f o r the treatment of at h e r o s c l e r o s i s , a disease associated with abnormally high serum cho l e s t e r o l l e v e l s . A number of diaza steroids having the nitrogen atoms i n the side chain were found to be extremely potent i n h i b i t o r s of cholesterol biosynthesis i n animals. Further studies*' indicated that the monoaza st e r o i d s , 24- and 25-aza c h o l e s t e r o l , were the most active of those tested. 2 CH-. CH3COSCcA ^0 HQ SCoft COOH 3-hydroxy-3-methyl g l u t a r y l CoA HOHzC COOH mevalonic acid squalene lanosterol zymosterol HO desmosterol cho l e s t e r o l Figure 1. Important Intermediates i n the Biosynthesis of Cholesterol 3 This i s consistent with r e s u l t s of more recent tests with 20-aza-24-oxa- (1), 22-oxa-25-aza- (2), and 20-oxa-21-nor-25-az a-cholesterol (3), which i n d i -cated that 24- and 25-aza.cholesterols and r e l a t e d compounds are potent hypocholesterolemic agents regardless of whether C-20 and C-22 are present as carbon, oxygen, or nitrogen atoms. An absolute reduction of cholesterol and an almost constant t o t a l s t e r o l l e v e l was observed.^ Results of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p studies suggested that a receptor s i t e with 7 dimensions s p e c i f i c f o r cho l e s t e r o l i s involved and a non-steroidal 4 analogue (4) was found to be i n a c t i v e when tested on r a t s . 4 4 Subsequent c l i n i c a l s t u d i e s demonstrated that 22,25-diazacholestanol ( 5 ) ^ and 20,25-diaz a c h o l e s t e r o l ( 6 ) ^ ' ^ caused a s i g n i f i c a n t r e d u c t i o n i n serum c h o l e s t e r o l l e v e l s i n subjects w i t h hypocholesterolemia and coronary a t h e r o s c l e r o s i s . N-methyl-4-aza-36-methyl-5a-cholestane (7) and N,N-dimethyl-4-aza-3B-benzyl-5a-cholestane i o d i d e (8) a l s o i n h i b i t e d the re d u c t i o n of des-mosterol t o c h o l e s t e r o l . C e r t a i n aza s t e r o i d s , however, cause a marked increase i n c h o l e s t e r o l b i o s y n t h e s i s . The most a c t i v e o f these were 3a-N-ethanolaminocholestane (9) and N-phenyI-4-aza-5-cholestan-3-one (10) which are u s e f u l i n reducing s c l e r o t i c l e s i o n s i n l a b o r a t o r y animals without H 0 C H J . C M " ' 5 r e s o r t i n g to high levels of cholesterol i n the d i e t . A d e t a i l e d discussion 11 12 of the b i o l o g i c a l a c t i v i t y of aza steroids i s a v a i l a b l e . ' In addition to the properties already mentioned aza steroids have been reported to possess anabolic, a n t i - b a c t e r i a l , anti-fungal, hypotensive, coronary artery d i l a t i n g , CNS stimulant, CNS depressant, neuromuscular 13 blocking, anti-inflammatory, and androgenic a c t i v i t i e s . The introduction of a nitrogen atom into the s t e r o i d nucleus has a t t r a c t e d the attention of chemists f o r some time. As a r e s u l t i t has been introduced i n t o v i r t u a l l y every p o s i t i o n of the s t e r o i d nucleus. An excellent review of the l i t e r a t u r e up to 1962. describes .the various methods which have been used.*^ The more recent work w i l l be b r i e f l y discussed i n the following pages. The Beckmann rearrangement has been u t i l i z e d frequently i n these syntheses and a,recent review a r t i c l e on the uses of t h i s reaction and the Schmidt reaction has been p u b l i s h e d . ^ Beckmann rearrangement of the oximes of a 1-keto s t e r o i d (11) and the 1-keto-A-nor der i v a t i v e (14) y i e l d e d 1-aza-A-homoc h o l e s t e r o l (12) and 1-az SLCholesterol (15) r e s p e c t i v e l y . The 1,10-Figure 2. Synthesis of 1-Aza and 1-Aza-A-Homo Steroids. 6 seco-l-cyano compounds (13) and (16) were also obtained v i a abnormal rearrangement (Figure 2) . 1 6 The oxime of 5a-:cholestan-2-one (17) provided a mixture of 2- and 3-aza-A-homo lactams, (18) and (19), which were reduced to the corresponding 2- and 3-aza-A-homo-5ot-cholestanes, (20) and (21). (Figure 3). 1 (* On the other hand, Beckmann rearrangement of the oxime of A-nor-5a-cholestan-2-one (22) led to an inseparable.mixture of 2-aza-5a-cholestan-3-one (23) and 3-aza-5a-cholestan-2-one (24). They were separated Figure 3. Synthesis of 2- and 3-Aza Steroids. 7 as the dichloro derivatives which were characterized by t h e i r r e a c t i v i t y 17 with c o l l i d i n e (Figure 3). S i m i l a r l y , 5a-cholestan-3-one oxime (25) y i e l d e d an inseparable mixture of the 3- and 4 -azaTA-homo derivatives (26) 18 and (27). (Figure 4). The A^-S-keto s t e r o i d oxime (28), however, gave only the unsaturated lactam (29) which, a f t e r hydrogenation and l i t h i u m aluminum hydride reduction, y i e l d e d the 3-aza-A-homo s t e r o i d analogue (30), Figure 4). Figure 4. Synthesis of 3- and 4-Aza-A-Homo Steroids. Recently A-homo-4-aza-5a-pregnane-3,20-dione (31) was synthesized i n 93% y i e l d from the oxime of 5a-pregnane-3,20-dione (32) using benzene s u l f o n y l chloride, as the reagent. Standard Beckmann conditions, t h i o n y l chloride i n 21 dioxane, gave a 39% y i e l d of the same product. (Figure 5). C = 0 equivalent HON 32 31 Figure 5. Synthesis of A-Homo-A-Azapregnanedione. 22 23 The synthesis of 5-aza-A-nor-B-homocholestane (35) ' and 5-aza-24 25 A-no rc i i o l e s t a n e (38) ' v i a a Beckmann rearrangement of the keto-ester oximes (33) using phosphorous oxychloride, boron t r i f l u o r i d e , or phosphorous pentoxide i n toluene, and i t s a p p l i c a t i o n to the conversion of testosterone acetate (39) in t o A-nor-B.-homo-.5-aza.androstan-178-ol (40) have recently appeared i n the l i t e r a t u r e . (Figure 6). The Beckmann rearrangement has also been used to synthesize a 6-aza-B-homo s t e r o i d 27 28 29 (41) , and,7-aza-B-homo s t e r o i d (42), and a 7-aza s t e r o i d (43). (Figure 7). Other methods of synthesizing aza steroids have also been employed i n the past few years; The synthesis of a 3-azae q u i l e n i n d e r i v a t i v e (46) v i a an intramolecular conversion of 2-aminoequilenin-3,4-quinone (44) with per a c e t i c acid and subsequent decarboxylation of the acid (45) with copper 30 powder has been reported. (Figure 8). 6-Aza steroids have been success-31 32 f u l l y prepared using a modified Curtius reaction.. (Figure 9) ' Elimination of the C-3 oxygen function was overcome by reducing the i n t e r -mediate isocyanate (47) with l i t h i u m aluminum hydride to y i e l d (49), or 33 c a t a l y t i c a l l y to y i e l d (50). To explain these r e s u l t s an equilibrium between the isocyanate (47) and the lactone (48) was suggested. (Figure 10). 9 A-nor-B-hoanocholestane A-nor-B-hom oa.ndrostane-176-ol Figure 8. Synthesis of 3-Aza-4-Hydroxye q u i l e n i n . 12 Introduction of a nitrogen atom into, the s t e r o i d nucleus has also been accomplished by the c y c l i z a t i o n of intermediate keto acids with amines. 34 This has recently been applied to the synthesis of 4-hydroxy (51), 4-amino-(52), 3 5 4-alkylamino- ( 5 3 ) , 3 6 and 4,68-dimethyl- ( 5 4 ) 3 7 derivatives of 4-aza 38 steroids, as well as to other h e t e r o c y c l i c steroids (55). (Figure 11). Figure 11. C y c l i z a t i o n of Keto Acids to 4-Aza Steroids. 13 In our laboratory t h i s method was applied to the synthesis of 6-aza-39 40 41 cholestane (56), 6-aza a.ndrostane (57), and 6-az apregnane (58) derivatives (Figure 12) and t h i s , along with a s i m i l a r one c a r r i e d out 42 independently elsewhere, provided the f i r s t general synthesis of r i n g B aza s t e r o i d s . This work was subsequently extended to the synthesis of the Figure 12. C y c l i z a t i o n of Keto Esters to 6-Aza Steroids. 14 f i r s t l i - a z a s t e r o i d a l sapogenin ( 5 9 ) 4 3 and f i n a l l y to an 11-azapregnane deri v a t i v e ( 6 0 ) . 4 4 (Figure 13). AcO' 7.00 20 l-ioors A c O AcO ' Figure 13. 11-Aza Steroids. 15 Using the lactam (61) as s t a r t i n g material the 17-az a.pregnane d e r i -45 vative (62) was synthesized and with appropriate modifications, using the lactam (63) as s t a r t i n g material, 17-aza_progesterone (64) was also obtained. (Figure 14). The Leuckart-Wallach reductive amination of 17-oxoandrostane derivatives with a number of primary and secondary amines has been reported to be a convenient, general, s t e r e o s p e c i f i c method f o r the preparation of 47 176-aminoandrostane d e r i v a t i v e s . (Figure 15). . 17a-aminoandrostane derivatives have been prepared from the 176-alcohol tosylate e i t h e r by d i r e c t inversion with secondary amines or v i a the intermediate 17a-azide 48 and 17a-amine. (Figure 16). Nitrogen atoms have also been introduced into various positions of 49 the side-chain of cholesterol and t h i s subject has been reviewed recently. The b i o l o g i c a l a c t i v i t y of some of these derivatives has already been discussed and the syntheses w i l l now be b r i e f l y reviewed. The mono-aza analogues were synthesized as shown i n Figure 17. 5 S t a r t i n g from 38-hydroxypregn-5-en-20-one 3-tetrahydropyranyl ether (65), 22-oxa-25-azacholesterol (66) was obtained^ which d i f f e r e d from the 20-iso-22-oxa-25-azacholesterol recently s y n t h e s i z e d ^ i n the stereochemistry at C-20. (Figure 18). Syntheses of 20-oxa-21-nor-25-azacholestan-3B-ol (67) and 20-aza-24-oxa-chol e s t e r o l (68), as well as cholesterol analogues with the side chain at C-16 rather than at C-17 (69), were also reported.^ (Figure 18). The syntheses described so f a r have involved modifications of the s t e r o i d skeleton. A number of t o t a l syntheses of aza steroids have also been reported i n the l a s t few years, f o r example, the recent synthesis of A-nor-2,3-diaza s t e r o i d r i n g systems. (Figure 19)."^ The t o t a l synthesis of 6-aza steroids i n the estrogenic s e r i e s has been independently reported Figure 14. 17-Azapregnane D e r i v a t i v e s . 17 Figure 15. 178-Amino a.ndrostane Derivatives. Figure 16. 17a-Amino a.ndrostane Derivatives. 18 Figure 17. Synthesis of 23-, 24- and 25-Azacholesterols. 19 CHOH Na, EtOH NCH3 ^ 2, hydrolysis I, CHJNHJ. I. kN|Hz/l^.NH5 5» HO ( c H 5 ) i C H O C H 2 C O C I =^  (CHJVCHOCHJ.-C-O Figure 18. Side-Chain A z a c h o l e s t e r o l s . 20 by three groups of workers. One synthesis which l e d to dl-6-az a e q u i l e n i n 52 (71) from the ketone (70) i s outlined i n Figure 20. Another a t t r a c t i v e approach to 6-aza e q u i l e n i n (71) and other 6-azaestrone derivatives has 53 54 been independently reported. (Figure 21). ' Two syntheses of 8-aza-estrone which have been reported are out l i n e d i n Figures 22^ and 2 3 5 ^ ' 5 7 and a synthetic approach to both 8- and 9-aza steroids (74) and (75) res p e c t i v e l y i s shown i n Figure 2 4 . 5 8 ' 5 9 Very recently, a three-step t o t a l synthesis of DL-8-aza.estrone methyl ether (76) and r e l a t e d s t e r o i d systems (77) was accomplished i n 43% o v e r a l l y i e l d . Separation of the 14a and 146 isomer was accomplished by f r a c t i o n a l c r y s t a l l i z a t i o n . (Figure 2 5 ) . ^ App l i c a t i o n of th i s approach to the synthesis of the D-homo-8-aza s t e r o i d (78) was unsuccessful. The synthesis of 13-aza-18-norequilenin methyl ether (79) has been reported independently by two groups of workers. One synthetic sequence i s shown i n Figure 26 ^ and the other which also leads to 13-aza-18-nor-D-62 homo equilenin methyl ether (80) i n Figure**27 < The synthesis of j. derivatives of 8,13-diaza-18-norestrone methyl ether (81) and (82) s t a r t i n g with homoveratrylamine (83) and mescaline (84) re s p e c t i v e l y i s outlined i n 63 Figure 28. Both series of reactions proceeded i n good y i e l d . A review of the syntheses discussed reveals that very few lead to products which possess a non-aromatic A-ring, the true s t e r o i d skeleton and the C-3 oxygen function. The synthesis of 6-aza steroids by the c y c l i z a t i o n of an intermediate keto-ester with benzylamine as previously accomplished i n our laboratory has already been discussed. (Figure 12). The modifi-cation of t h i s work and the reintroduction of the oxygert function at C-3 i s described i n t h i s part of the t h e s i s . 21 Figure 19. Total Synthesis of A-Nor-2,3-diaza-3-phenyl-oestra-1,5 (10),8,14-tetraen-17-one. Figure 20. T o t a l Synthesis of d l - 6 - A z a e q u i l e n i n . 22 Figure 21. Total Synthesis of dl-6-Aza e q u i l e n i n and other 6-Aza-estrone Derivatives. Figure 22. Total Synthesis of 8-Azaestrone Figure 23. Total Synthesis of 8-Aza es t r o n e 24 Figure 24. T o t a l Synthesis of 8- and 9-Aza S t e r o i d s . 25 Figure 25. Synthesis of DL-8-Azaestrone Methyl Ether and Systems. 26 Figure 26. Synthesis of 13-Aza-18-Nore q u i l e n i n Methyl Ether 80 n = 2 Figure 27. Synthesis of 13-Azae q u i l e n i n Derivatives. Figure 28. T o t a l Synthesis of 8,13-Diaza-18-horoestrone Methyl Ethers. 28 DISCUSSION 39 40 42 In previous syntheses of 6-aza steroids of the cholestanei ' ' 39 40 41 androstane, ' and pregnane s e r i e s , the hetero atom was introduced by c y c l i z a t i o n of the appropriate seco k e t o e s t e r s with amines. (Figure 12). During the i s o l a t i o n of the keto e s t e r s , elimination of the oxygen function at C3 was normally observed. In cases where elimination was prevented by the exclusion of base from the work-up of the ozonolysis reaction, the sub-sequent reaction of the keto-ester with the amine resulted i n loss of the C 3 function. It was therefore necessary to investigate a possible modi-f i c a t i o n of the above sequence i n order to achieve a synthesis of r i n g A-oxygenated 6-aza s t e r o i d s . 42 Jacobs and Brownfield investigated re-introduction of an oxygen function at C3 of the enamine lactam (85). Using conditions s u i t a b l e f o r a l l y l i c bromination, (N-bromosuccinimide and bromine-carbon t e t r a c h l o r i d e ) H 85 a high y i e l d of the undesired v i n y l bromide (86) was obtained. I n i t i a l i n v e s t i g a t i o n s i n our laboratory were concerned with a method of introducing a functional group at C3 of the enol lactam (87). This p o s i t i o n did not exhibit normal a l l y l i c character and a l l attempts i n t h i s d i r e c t i o n met with f a i l u r e . The p o s s i b i l i t y of achieving a s e l e c t i v e reaction at the 2,3-double 29 30 bond i n the doubly unsaturated lactam (88) was then investigated. The 39 40 42 l a t t e r compound was prepared i n the usual manner ' ' when methyl-5-oxo-5,7-seco-6-nor-3-cholesten-7-oate (89) was reacted with r e f l u x i n g benzylamine. The u l t r a v i o l e t spectrum of the r e s u l t i n g N-benzyl-6-aza-2,4-cholestadien-7-one.(88) (^ m a x 297 my) was i n good agreement with the u l t r a v i o l e t spectrum 42 reported for the corresponding diene i n the -NH s e r i e s (A m a x 299 mu). The structure was conclusively established when c a t a l y t i c reduction provided. the known enol lactam ( 87). This reaction also indicated that a s e l e c t i v e reaction at the 2,3-double bond would be f e a s i b l e . An a t t r a c t i v e approach 64 was the hydroboration technique developed by H.C. Brown and co-workers. The hydroboration reaction has been extensively.investigated, and i t i s well established that i t provides,cis anti-Markownikoff addition 64 to unsymmetrical o l e f i n s . Consideration of the e l e c t r o n i c and s t e r i c factors whichnormally govern the course of the addition reveals several reaction products which might be a n t i c i p a t e d i n the hydroboration of the unsaturated lactam (88). Thus, the e l e c t r o n i c consideration which postulates that the d i r e c t i o n of addition i s c o n t r o l l e d p r i m a r i l y by the p o l a r i z a t i o n of the boron-hydrogen bond would predict that any formal p o s i t i v e charge which may; develop i n the intermediate would prefer to reside at C3, as i t s n e u t r a l i z a t i o n i s e a s i l y accommodated by, the adjacient 4,5-double bond. The subsequent conversion of t h i s intermediate (normally considered to be a four-64 centred t r a n s i t i o n state) to the f i n a l product would y i e l d the 2-hydroxy de r i v a t i v e . The s t e r i c a l l y less favoured B-approach of the reagent and the s t e r i c i n t e r a c t i o n s which would e x i s t between the 26-hydroxyl function and the angular methyl group at CIQ predict that a predominance of the 2a-hydroxyl der i v a t i v e (90) would,result. 31 91 92 90 On the other hand, the p o s s i b i l i t y of the reaction of 88 with the hydroborating reagent i n a manner which provides formation of a carbon-boron bond at C3 i s less c l e a r , both from an e l e c t r o n i c and s t e r i c viewpoint. The e l e c t r o n i c consideration provides no r e a l d i r e c t evidence, and molecular models reveal that the.approach of the hydroborating reagent at C3 i s f e a s i b l e from e i t h e r the a or 3 sides of the molecule, although there i s some s t e r i c preference f o r a approach. Therefore, i t was reasonable to expect that i f any 3-hydroxy analogues were formed both the 3a- and 38-hydroxy compounds would be i s o l a t e d (91 and 92 respectively) with the 3 isomer i n predominance. Indeed, the experimental r e s u l t s are i n reasonable agreement with the.above postulates. Hydroboration of the doubly unsaturated lactam (88) at 0° with diborane i n anhydrous diglyme, followed by treatment of the r e s u l t i n g organobbrane with.alkaline hydrogen peroxide, provided a white s o l i d reaction product, m.p. 133-136°. Thin layer chromatography (TLC) of t h i s product revealed that three components were a c t u a l l y present. Furthermore^ TLC i n d i c a t e d that separation into the pure compounds might prove d i f f i c u l t since the Rp values of these substances were very s i m i l a r . (A t y p i c a l TLC plate i s shown on the l e f t , below). The i n i t i a l separation of these 32 compounds by preparative t h i n layer chromatography was however, success-f u l l y accomplished and subsequent investigations showed that c a r e f u l and extensive column chromatography could be u t i l i z e d f o r the separation of larger q u a n t i t i e s . The least p o l ar compound, m.p. 151-153°, obtained c r y s t a l l i n e a f t e r the above separation, was of i n t e r e s t due to i t s desirable s p e c t r a l properties. The u l t r a -v i o l e t spectrum showed the c h a r a c t e r i s t i c ,enol lactam absorption (X 237 r * max my) i n d i c a t i n g that reaction at the 2,3-double bond had occurred. This was confirmed by the i n f r a r e d absorption bands at 1635 and 1670 cm - 1 which are c h a r a c t e r i s t i c of the enol lactam. That simple hydration of the diene system i n r i n g A had occurred was shown beyond doubt when the molecular formula was established as CssHi+gC^N by high r e s o l u t i o n mass spectrometry. The NMR spectra f o r a l l three compounds w i l l be discussed i n d e t a i l below. The second product i s o l a t e d was c r y s t a l l i z e d from aqueous methanol, m.p. 144-145.5°. It also exhibited the presence of an enol lactam system (X 237 my; v 1625 and 1670 cm - 1), and a simple hydration of the ITlcLX IT13.X 2,3-double bond was again evident from i t s molecular formula, 0 3 3 * ^ 9 0 ^ . The t h i r d and most polar compound i s o l a t e d from the hydroboration reaction was also a c r y s t a l l i n e substance, m.p. 176-178°, which again exhibited s p e c t r a l c h a r a c t e r i s t i c s i n agreement with the desired hydro-boration product. It also had the molecular formula C 3 3Hi tg02N. Solvent frorft —y 33 34 The NMR spectra obtained f o r the above three compounds w i l l now be discussed i n d e t a i l since a comparative analysis of the data allows s t r u c t u r a l assignments i n a l l three instances. The NMR studies were ca r r i e d out i n considerable d e t a i l at 60 and 100 Mc/s so that coupling constants and chemical s h i f t s were r e a d i l y determined. Figure 29 shows a reproduction of the relevant low f i e l d regions of the spectra of these compounds determined at 100 Mc/s. Examination of the s p l i t t i n g patterns reveals several c h a r a c t e r i s t i c features. These are: (a) the presence i n each instance of a p a i r of doublets (J = 16 c.p.s.), rather t y p i c a l of an AB system; (b) an o l e f i n i c proton absorption, the s p l i t t i n g pattern d i f f e r -ing i n each instance depending on the s u b s t i t u t i o n of the adjacent carbon; and (c) a broad m u l t i p l e t at higher f i e l d which i s r e a d i l y a t t r i b u t a b l e to a proton attached to the alcohol-bearing carbon atom. Analysis of sections A, B, and C i n Figure 29 can be made i n terms of structures 91, 90 and 92 respectively. I f 3a-hydroxy-N-benzyl-6-:aza-4-cholesten-7-one i s f i r s t considered i n terms of i t s conformational structure (91a), i t can be r e a d i l y shown that the presence of the 4,5-double bond transforms r i n g A into a h a l f -chair conformation, and the dihedral angles i n v o l v i n g substituents at C 2 and C3 are not s i g n i f i c a n t l y a l t e r e d from those encountered i n the simple cyclohexene s e r i e s . 35 Some d e t a i l e d studies of v i n y l - a l l y l i c proton spin couplings have been reported i n the l i t e r a t u r e and the observed values f o r the cyclohexene der i v a t i v e ( 9 4 ) , ^ D-glucal t r i a c e t a t e (95) shikimic a c i d (96), ^  and conduritol F (97) are l i s t e d i n Table 1. The o l e f i n i c proton s i g n a l at x 5.04 i n section A, Figure 29, i s a doublet. The magnitude of the coupling constant, J = 5.25 c.p.s., i s i n good agreement with the values o f • 3 g and J g 4 e observed f o r (94) and (97) r e s p e c t i v e l y , i n d i c a t i n g that the 3ct-hydroxyl function i s present. Further confirmation of t h i s assignment i s a v a i l a b l e from the width of the C3 proton s i g n a l . Since J ~ _ and J„ _ i n 91 are expected to be small (usually 2-5 c.p.s.; f o r example J 3 4 = 4.4 c.p.s. i n 97), and J , . i s shown to be approximately 5 c.p.s., the half-height width of the 0 , 4 C3 proton m u l t i p l e t should be of the order of 9-15 c.p.s. The observed value i s 10 c.p.s. On t h i s basis the least polar hydroboration product was 36 assigned structure 91. TABLE 1 Compound V i n y l - A l l y l i c Spin Couplings  J i n c.p.s. Reference 95 H 2,3a f2,3e 2,3a 2.5 5.0 3.2 65 66 J2,3e 4 - ° 1,6 J4,5 1.9 5.3 67 68 Section B i n Figure 29 represents the low f i e l d proton region of the NMR spectrum of the second hydroboration product (m.p. 144-145.5°). The o l e f i n i c proton s i g n a l i n t h i s case i s a quartet i n d i c a t i n g that the hydroxyl group must be at C 2 rather than at C 3 . The coupling constants observed f o r the substituted cyclohexene 9 4 ^ mentioned above were 3^ g a = 2.5 and J 2 3 e = 5.0. c.p.s. Our observed values of J 3a, 4 2 c.p.s. and 37 J j e 4 = 5.25 c.p.s. are i n agreement with these. I f the proton at C2 i s a x i a l i t should appear as a broad multiplet ,due to i t s spin coupling with the neighbouring protons at and C3 (J = 2-5 c.p.s. and J = 6-12 c . p . s . ) ^ ' 7 ^ Although no d i s t i n c t r e s o l u t i o n of the m u l t i p l e t could be achieved, i t was observed as a broad absorption with a half-height width of 25 c.p.s. and structure 90 was assigned. The most polar compound i s o l a t e d from the hydroboration reaction was also subjected to an extensive NMR study, and section C i n Figure 29 i l l u s t r a t e s the low f i e l d region of the spectrum. One of the s t r i k i n g differences noted i n t h i s spectrum i s the narrow doublet (J = 2 c.p.s.) observed f o r the o l e f i n i c proton. This s i t u a t i o n i s immediately reminiscent of tha small coupling constant, 3 a =2.5 c.p.s., observed i n the 65 68 spectrum of 94. In the case of conduritol F (97) the observed value of J, , was 1.9 c.p.s. Thus the proton at C3 was assigned the a x i a l p o s i t i o n . Further evidence to support,this assignment came from a 38 consideration of the C 3 proton s i g n a l . I f i t was a x i a l l y oriented, the electron coupled spin-spin i n t e r a c t i o n of t h i s proton with the two adjacent protons ( a x i a l and equatorial) at C 2, as well as with the Ci+ o l e f i n i c proton would predict a m u l t i p l e t with an approximate half-height width of 10-20 c.p.s. The experimental value observed was 20 c.p.s. The NMR data i s therefore consistent with the assignment of structure 92 to t h i s product. As predicted from s t e r i c considerations only the 2a-hydroxy alcohol was i s o l a t e d . A predominance of the 3a-isomer (91) was observed as expected. The r e l a t i v e amounts of 90, 91 and 92 i s o l a t e d were.5:2:1 re s p e c t i v e l y . To complete the discussion of the NMR data i t i s necessary to consider.the p a i r of doublets (J = 16 c.p.s) which i s a c h a r a c t e r i s t i c feature i n the NMR spectra of a l l the N-benzyl-6-aza-7-one s t e r o i d , d e r i -vatives synthesized irt our laboratory. These are t y p i c a l l y shown i n Figure 29 and are due to the methylene protons of the benzyl group. It has been recognized f o r some time that geminal protons and f l u o r i n e atoms attached to any dissymmetric moiety are magnetically non-equivalent and often couple with each other i n the NMR spectrum. The 71 72 e a r l i e r work on substituted ethanes (98) ' suggested that t h i s feature F R, H R 2 l l I I Ri- C - C - R 3 R i - C - C - R 3 I I I I F R i . H R k ^ 9 8 * was c h a r a c t e r i s t i c o f ; t h e methylene protons (or f l u o r i n e s ) when t h i s group was adjacent to an asymmetric center. Later studies revealed that t h i s 73 74 requirement was unnecessary. Recent publications from two laboratories ' have reported r e s u l t s on the magnetic non-equivalence of b e n z y l i c methylene protons i n various h e t e r o c y c l i c bases, while two other groups have presented 39 data i n the phthalimidine and imidazolidinone ser i e s which are even more d i r e c t l y pertinent to the present discussion. (Table 2). In the two l a t t e r instances, the N-benzyl moiety i s attached through the nitrogen atom to an asymmetric centre. In the present i n v e s t i g a t i o n a somewhat d i f f e r e n t system i s a v a i l a b l e as the compounds do not contain an asymmetric centre i n the immediate v i c i n i t y of the benzylic protons. The data f o r a v a r i e t y of N-benzyl-6-aza-7-one s t e r i o d analogues i s summarized i n Table 3. The difference between the chemical s h i f t s of these two protons i s possibly r a t i o n a l i z e d on the basis of reasonable assumptions about preferred conformations of these compounds. Molecular models reveal that minimum in t e r a c t i o n s between the phenyl group and the neighbouring atoms occur when t h i s group l i e s i n a plane approximately perpendicular to the plane of the lactam system. As the conformational structures 90a, 91a and 92a i n d i c a t e , t h i s s i t u a t i o n places the two geminal protons (H a and H D ) i n d i f f e r e n t environments. C l e a r l y Ha would be influenced by the lactam carbonyl group while Hb i s i n close proximity to the C4 o l e f i n i c linkage i n the r i n g A unsaturated 6-aza compounds. Since the r e l a t i v e e f f e c t s on these two protons are d i f f i c u l t to ascertain, i t i s not possible to make d e f i n i t e predictions as to which proton i s deshielded. The NMR data provided good evidence f o r the structures of the three hydroboration products. Chemical evidence to support,the proposed s t r u c t u r a l assignment was also obtained. The most d i r e c t confirmation of the suggested structures would be simple dehydration to the parent diene 88. Although dehydration of 90, 91 and 92 might be a reasonably f a c i l e process, i t would be expected that the rate of dehydration might d i f f e r somewhat. I t i s well-known that d i a x i a l elimination i s a preferred process, 40 TABLE 2 Coupling Constants f o r Benzylic Methylene Protons Compound J (c.p.s.) Reference n = 1 n = 0 14 13.2 73 * M C 6H 5 CH 3 15 75 CHzC,.Hs f«H>7 15 12^13 76 74 41 TABLE 3 Nuclear Magnetic Resonance Data (100 Mc/s) on Benzylic Protons Compound Proton 87 ( see 87a) a, b Line Positions (cps separation from TMS)  528,512 460,444 Chemical S h i f t x-scale  4.80 5.48 Chemical S h i f t Difference, ppm 0.68 90 (see 90a) 90b 91 (see 91a) 92 (see 92a) 93 (see 93a) a, b a, b a, b a, b a, b 522,506 460,444 522,506 465,449 512,496 469,453 520,504 456,440 516,500 484,468 4.86 5.48 4.86 5.43 4.96 5.39 4.88 5.52 4.92 5.24 0.62 0.57 0.43 0.64 0.32 42 and therefore i t was reasonable to assume that the 3a-hydroxyl group should eliminate more r e a d i l y than the isomeric 3g function. The i n s t a b i l i t y of the a x i a l isomer, 3<x-hydroxy-N-benzyl-6-aza-cholest T4-ene-7-one (91), was immediately apparent during i t s attempted p u r i f i c a t i o n . Column or t h i n - l a y e r chromatography of t h i s compound on alumina immediately led to p a r t i a l conversion to the diene (88). On standing, pure 91 had a tendency to dehydrate to the s t a r t i n g diene. On the other hand, the equatorial 3$-hydroxy d e r i v a t i v e (92) was considerably more stable and i t s p u r i f i c a t i o n was not quite so d i f f i c u l t . Dehydration occurred to a lesser extent and i t was possible to p u r i f y t h i s compound by chromatographic procedures with only a small loss due to conversion to the diene. the r e l a t i v e ease of dehydration was again evident i n the attempted a c e t y l a t i o n of 91 and 92. In both.instances, reaction with a c e t i c anhydride and pyridine provided the diene 88 as one of the major components. In each case some ac e t y l a t i o n had occurred as indicated by the appropriate carbonyl absorption in,the i n f r a r e d spectrum of the crude reaction mixture, but i s o l a t i o n of the pure compounds was rendered impossible by t h e i r constant tendency to revert to the diene. A difference i n the ease of dehydration was also noted i n the mass spectra of the isomeric 3-hydroxy compounds 91 and 92 (Figure 30, see also Table 4). The 3a-hydroxy isomer 91 lo s t water most,readily and i n both cases the M-18 fragment (m/e 473) formed the base peak. On the other hand, the base peak i n the mass spectrum of the 2-hydroxy d e r i v a t i v e (90) was the molecular ion (m/e 491) i n d i c a t i n g that loss of water does not occur so r e a d i l y i n t h i s case. 77 The mass spectra of aza steroids have been studied i n d e t a i l and 44 peaks corresponding to M-15, M-28 or 29, M-43 or 44, and, i n the case of cholesterol d e r i v a t i v e s , M-85 were c h a r a c t e r i s t i c of these compounds. The peak at m/e 91 due to the benzyl fragment (C7H 7 +) was common to a l l the spectra of N-benzyl derivatives and was also observed i n the spectra of the three hydroboration products, The peaks at m/e 476 (M-15), 463 (M-28) and 447 (M^44) i n the mass spectrum of 90 are c h a r a c t e r i s t i c and are probably due to the loss of CH 3, CO, and CHO + CH 3 r e s p e c t i v e l y . It i s i n t e r e s t i n g to note that the mass spectra of the two 3-hydroxy isomers 91 and 92 are quite d i f f e r e n t . The peaks at 458 (M-33), 406 (M-85), 373 (M-118), 358 (M-133) and 316 (M-175) i n the mass spectrum of 92 were r e l a t i v e l y intense. The f i r s t two are probably due to the loss of water plus CH 3, and CsH 1 3 of the cho l e s t e r o l side chain r e s p e c t i v e l y . The l a s t three peaks are 16 mass units greater than the corresponding M-28, M-43 and M-85 peaks observed at m/e 357, 342 and 300 i n the spectrum of 99 and are probably due to preliminary loss of the benzyl group followed by the 77 normal fragmentations observed f o r 6-aza s t e r o i d s . The peaks at m/e 458 and 406 also appeared i n the mass spectrum of 91. The r e l a t i v e l y strong peak at 338 probably corresponds to the one observed 16 mass units lower i n the spectrum of the enol lactam (87) which 77 was a t t r i b u t e d to fragmentation of the C and D rings: 45 C 8Mn HO H HO m/e 338 The remainder of the spectrum i s complex, and cannot be r e a d i l y i n t e r p r e t e d . The experimental r e s u l t s i n d i c a t e d that the 2-hydroxy d e r i v a t i v e (90) was considerably more s t a b l e than the other hydroboration products. Chromatographic p u r i f i c a t i o n could be achieved without d i f f i c u l t y , and r e a c t i o n of t h i s m a t e r i a l with a c e t i c anhydride and p y r i d i n e provided the corresponding acetate. This compound e x h i b i t e d the expected s p e c t r a l features i n the i n f r a r e d and u l t r a v i o l e t s p e c t r a (1740 cm - 1, X 237 my), r v max J ' and the molecular formula, C35H51O3N, was e s t a b l i s h e d by high r e s o l u t i o n mass spectrometry. S a p o n i f i c a t i o n of the acetate regenerated i n pa r t the s t a r t i n g m a t e r i a l 90, but provided i n a d d i t i o n a small amount of the diene (88). This demonstrated that dehydration i s a l s o f e a s i b l e i n t h i s system, but somewhat stronger c o n d i t i o n s are necessary than i n the case of the 3-hydroxy .de r i v a t i v e s 91 and 92. The f i n a l step i n the s y n t h e t i c sequence was the o x i d a t i o n of the a l c o h o l i c f u n c t i o n s to y i e l d the d e s i r e d A^-S-keto chromophore i n r i n g A. Several d i f f e r e n t experiments were c a r r i e d out and these are discussed i n succession. 46 Oxidation of 92 with chromium t r i o x i d e i n acetone (Jones reagent) or i n pyridine (Sarett reagent) yi e l d e d a new c r y s t a l l i n e compound, m.p. 173-174.5°, C33H|+702N, which exhibited s p e c t r a l properties i n accord with the 3-keto structure 93. Of p a r t i c u l a r note was the u l t r a v i o l e t spectrum EtOH which changed on the addition of a l k a l i to the s o l u t i o n (X 284 my; X 287 my 5 minutes a f t e r addition of a few drops of 0.1N NaOH; X 292 my p max a f t e r 30 minutes; and X 294 my a f t e r 3 hours). The bathochromic s h i f t max J (237 my — • 284 my) observed i n neutral s o l u t i o n and the spectral changes i n a l k a l i are i n excellent agreement with those reported f o r the H 0 i d e n t i c a l chromophore (100), (X 2 284 my; x 334 a f t e r 3 minutes i n max max 78 0.1N NaOH: X 293 my a f t e r 30 minutes). There was also a marked max J U H 100 a l t e r a t i o n i n the o p t i c a l rotatory dispersion (ORD) curve of the oxidation product compared to the t y p i c a l Cotton e f f e c t already known f o r the enol 79 lactam systems and observed f o r the three hydroboration products. (Figure 31). As the ORD curves of aza steroids w i l l be discussed i n Part 2 of t h i s thesis no further comment w i l l be made here. The o l e f i n i c proton signal i n the NMR spectrum was a sharp s i n g l e t at lower f i e l d than i n the spectra of the corresponding alcohols 91 and 92. These r e s u l t s established the structure of the oxidation products as 93. In a s i m i l a r manner, compound 91 was oxidized to 93. This provided conclusive evidence that the only difference i n the structures of 91 and 92 was the stereochemistry at C3, as had been indicated previously by the NMR 47 Fig. 31 ORD Curves 48 data. In subsequent experiments the t o t a l hydroboration mixture was subjected to oxidation and the desired A^-S-keto-S-aza s t e r o i d 93 was i s o l a t e d . In t h i s way better y i e l d s of 93 could be obtained. Since the appropriate unsaturated lactam system required f o r hydroboration i s also a v a i l a b l e i n the androstane and pregnane series t h i s synthesis of r i n g A-oxygenated steroids could be extended to these s e r i e s . 49 EXPERIMENTAL Melting points were determined on a K o f l e r block and are uncorrected. U l t r a v i o l e t spectra were measured i n methanol s o l u t i o n on a Cary 11 spectrophotometer, and i n f r a.red spectra were taken as KBr p e l l e t s on a Perkin-Elmer Model 21 spectrophotometer. Nuclear magnetic resonance (NMR) spectra w e r e recorded at 60 megacycles/sec. on a Varian A60 instrument and at 100 megacycles/sec. on a Varian HA100 instrument, using deuteriochloroform as solvent; the l i n e positions or centres of multiplets are given i n the Tier s T scale with reference to tetramethylsilane as the in t e r n a l standard; the m u l t i p l i c i t y , integrated areas and type of protons are indicated i n parentheses. Only the values obtained at 100 megacycles/ sec. are recorded below. The mass spectra were taken on an Atlas CH4 mass spectrometer, using the d i r e c t i n s e r t i o n technique, the electron energy being maintained at 70 eV. The high r e s o l u t i o n mass spectra f o r the determination of molecular formulae were obtained on an AEI MS9 mass spectrometer. The o p t i c a l rotatory dispersion (ORD) curves were taken i n methanol s o l u t i o n on a. JASCO UV/ORD/GD-5 spectropolarimeter. In a l l of our experiments, the th i n - l a y e r chromatography plates were prepared from neutral alumina (Woelm), to which 1% by weight of a fluorescent i n d i c a t o r ( E l e c t r o n i c phosphor, General E l e c t r i c Co.) was added. Antimony t r i c h l o r i d e i n g l a c i a l a c e t i c a c i d or 50% aqueous orthophosphoric acid were used as the spray reagents. In general, the plates were heated f o r 10 minutes at 100°C a f t e r spraying, during which time, the compounds are recognized as blue spots on the chromatoplates. The solvent systems u t i l i z e d benzene and chloroform, and these are indicated below i n parentheses. For column chromatography, neutral alumina (Woelm) was used i n a l l cases, and deactivation was done by the addition of water. The approximate 50 a c t i v i t y of the adsorbent u t i l i z e d i n s p e c i f i c experiments i s indicated below. N-Benzyl -6-aza-2 >4-cholestadien-7-one ( 8 8 ) Methyl 5-oxo-5,7-seco-6-nor-3-cholesten-7-oate ( 8 9 , 2.2 g.) was taken up i n benzylamine ( 5 ml.), and the mixture refluxed f o r 15 hours under an atmosphere of nitrogen. The cooled reaction mixture was treated with ether, and the l a t t e r was washed with d i l u t e aqueous hydrochloric acid to remove excess benzylamine. The separated ether s o l u t i o n was washed with 5% aqueous sodium hydroxide and water, and f i n a l l y , d r i e d over anhydrous magnesium s u l f a t e . Removal of the solvent i n vacuo, i n i t i a l l y on a steam bath, and f i n a l l y , at 2 0 0 - 2 2 0 ° / 0 . 1 mm., provided a yellow-brown glass ( 2 . 0 g.). Chromatography of t h i s reaction product on alumina ( 2 0 0 g., a c t i v i t y IV) y i e l d e d the desired lactam ( 8 8 , 1.2 g.) as a c o l o r l e s s viscous o i l . A l l attempts to obtain the product i n c r y s t a l l i n e form f a i l e d , although i t s p u r i t y was established by TLC (benzene); X (log e): 2 9 7 mu ( 3 . 7 3 ) ; v : 1 6 7 0 , 1 6 3 8 and 1575 cm" 1(diene lactam); NMR: 2.88 (multiplet, IT13.X 5H, aromatic), 4.24, 4 . 6 0 , 4 . 9 5 , 5.09 (multiplets, 5H, o l e f i n i c H and - N C H ? CK H Q . Calc. for C ^ H ^ N O : 4 7 3 . 3 6 5 ; found 4 7 3 . 3 6 5 . C a t a l y t i c Reduction of N-Benzyl-6-aza-2^4-cholestadien-7-one The lactam 88 ( 5 0 mg.) was dissolved i n ethanol ( 1 0 ml.) , and to t h i s s o l u t i o n , 1 0 % palladium on charcoal ( 3 0 mg.) was added. The reduction was allowed to proceed f o r 30 minutes, during which time, one mole of hydrogen had been absorbed. Removal of the c a t a l y s t and evaporation of the solvent y i e l d e d the reduction product ( 4 5 mg.), which was i d e n t i c a l i n every 39 41 respect (IR, mixed melting point, UV, TLC) with the known enol lactam 8 7 . ' 51 Hydroboration of N-Benzyl-6-aza-2,4-cholestadien-7-one The lactam (88, 1.2 g.) was dissolved i n anhydrous diglyme (24 ml.) and placed i n a small three-necked f l a s k . Diborane gas generated i n another vessel was then slowly passed into t h i s mixture kept at 0°C over a period of two hours. The reagent was prepared from sodium borohydride (630 mg.) i n diglyme (17 ml.) to which a s o l u t i o n of boron t r i f l u o r i d e - e t h e r a t e (2.8 ml.) i n diglyme (12 ml.) was slowly added over a period of two hours. The reaction mixture was immediately treated with 5% aqueous sodium hydroxide (45 ml.) and 30% aqueous hydrogen peroxide (12 ml.), and a f t e r allowing to stand f o r 5.minutes, i t was extracted with ether. The ether extract was thoroughly washed with water and 5% aqueous ferrous s u l f a t e s o l u t i o n . A f t e r drying over anhydrous magnesium s u l f a t e , and evaporation of the solvent, a s l i g h t l y yellow amorphous material (1.1 g.) was obtained. T r i t u r a t i o n of the l a t t e r with petroleum ether caused some separation of a white s o l i d (m.p. 133-136°). However, f o r the chromato-graphic separations mentioned below, the t o t a l crude product was always used. The crude reaction product was i n i t i a l l y shown by TLG to be a mixture of three components (chloroform, values of 0.49, 0.50 and 0.60). A d e t a i l e d i n v e s t i g a t i o n of the p o s s i b i l i t y of u t i l i z i n g preparative TLC as a method of separation was c a r r i e d out, and a t y p i c a l experiment i s described. The crude hydroboration mixture (120 mg.) was placed on an a i r -drie d chromatoplate (60 X 20 cm., 65 g. of adsorbent, 0.4 mm. approximate thickness), and the plate was developed i n chloroform. Removal of the zones and extraction with methanol-ether (1:1) provided a f r a c t i o n (10 mg.) subsequently shown to be 92, another f r a c t i o n (69 mg., 90), and a t h i r d 52 f r a c t i o n (22 mg., 91). The recovery (101 mg., 80%) was co n s i s t e n t l y good i n t h i s separation. For larger scale separations, c a r e f u l column chromatography could be u t i l i z e d . For t h i s purpose, alumina ( a c t i v i t y IV) was the most desirable adsorbent, and benzene was the e l u t i n g solvent. In a t y p i c a l separation, the crude hydroboration mixture (2.4 g. obtained i n another experiment) was chromatographed on alumina (200 g.)- Careful e l u t i o n with benzene yi e l d e d a t o t a l of 46 f r a c t i o n s (50 ml. each), which were then examined by TLC. Fractions 13-17 (230 mg.) were combined and contained mainly 91 and a trace of 90. Fractions 18-24 (630 mg.) were combined and contained mainly 92 and traces of 90. Each of these combined portions were rechromatographed ( r a t i o of adsorbent ."material of 100:1) to f i n a l l y y i e l d pure 91 (210 mg., 0.60), 90 (570 mg., R £ 0.50), and 92 (110 mg., R f 0.49). In each instance, the column chromatographic separation was complicated by decomposition of the compounds. It w i l l be noted that considerably less than h a l f (only 890 mg. from 2.2 g.) of the desired hydroboration products were recovered. Appreciable dehydration of both 91 and 92 to the lactam 88 was always observed, whereas 90 was stable and could be p u r i f i e d without d i f f i c u l t y . In general, the TLC technique was superior i n o v e r a l l separation and recovery, although obviously more tedious i n i t s a p p l i c a t i o n . The data obtained f o r the three hydroboration products i s as follows: 3g-Hydroxy-N-benzyl-6-aza-4-cholesten-7-one (92): m.p. 176-178° ( c r y s t a l l i z e d from ether-hexane), X (log e): 237 my (4.03); v : 3460 ITleLX IHctX (OH), 1667 and 1630 cm - 1(enol lactam); NMR: 2.90 (multiplet, 5H, aromatic), 53 4.88 (doublet, J = 16 c.p.s., 1H, H of -N-CH7CfiH^ group, see 92 a), 5.52 (doublet, J = 16 c.p.s., 1H, H of -N-CH 2C 6H 5 group, see 92a), 5.18 (doublet, J = 2 c.p.s., 1H, o l e f i n i c ) , 5.93 (broad m u l t i p l e t , 1H, ^CHOH); ORD: (Figure 3, C, 0.0999 mg./ml.), [<j,] 3 5 0 + 1472, [ $ ] 3 0 0 + 2945, [*] 263 + 17,700 (peak), [<f»]25o 0, [<f>]236 " 46,300 (trough), [<fr] 2 1 8 + 21,600. Calc. f o r C3 3H t t 9N0 2: 491.376. Found: 491.371. 3a-Hydroxy-N-benzyl-6-aza-4-cholesten-7-one (91): m.p. 151-153° ( c r y s t a l l i z e d from ether-hexane), X (log e): 237 my (4.01); v : 3510 nicix rns-X (OH), 1670 and 1635 cm"1 (enol lactam); NMR: 2.90 (multiplet, 5H, aromatic), 4.96 (doublet, J = 16 c.p.s., 1H, H of -N-CH?CfiHs group, see 91a), 5.39 (doublet, J = 16 c.p.s., 1H, H of -N-CH 2C 6H 5 group, see 91a), 5.04 (doublet, J = 5.25 c.p.s., 1H, o l e f i n i c ) , 5.89 (broad m u l t i p l e t , 1H, ^CHOH); ORD: (Figure 3, C, 0.0948 mg./ml.), [*] 3 5 0 + 1037, [<f>] 3 0 o + 3630, U h f i l - + 28,500 (peak), [ c f r h ^ 0, [<f>] 235 " 47,700 (trough), [ • ] 2 2 0 - 23,300. Calc. f o r C 3 3H^ 9N0 2: 491.376. Found: 491.371. 2a-Hydroxy-N-benzyl-6-aza-4-cholesten-7-one (90): m.p. 144-145.5° ( c r y s t a l l i z e d from aqueous methanol), X (log e): 237 my (4.01); v : 3460 (OH), 1670 and 1630 cm"1 (enol lactam); NMR: 2.90 (multiplet, 5H, aromatic), 4.86 (doublet, J = 16 c.p.s., 1H, H of -NCHyCfiHq group, see 90a), 5.48 (doublet, J = 16 c.p.s., 1H, H of -NCH 2C 6H 5 group, see 90a), 5.28 (quartet, J . = 2 c.p.s., J _ . = 5.25 c.p.s., 1H, o l e f i n i c ) , 6.17 (broad m u l t i p l e t , 1H, ^CHOH); ORD: (Figure 3, C, 0.0973 ing./ml.) , [<t>] 3 3 0 + 1010, [<f>] 3 0 0 +2020, [*]262 + 22,950 (peak), [ < t > ] 2 1 t 8 0, [ < f > ] 2 3 1 + - 51,000 (trough), [<>] 2i 6 +8080. Calc. f o r C 3 3H 1 + 9N0 2: 491.376. Found 491.373. Acetyl a t i o n of Hydroboration Products Attempts to obtain the acetate derivatives of 90, 91 and 92 were only p a r t i a l l y successful. The isomeric C 3-hydroxy compounds (91 and 92) 54 could not be s u c c e s s f u l l y acetylated (acetic anhydride, p y r i d i n e ) , since both compounds led to a mixture of the desired acetates ( i n f r a r e d data only) and the lactam 88. Further chromatographic p u r i f i c a t i o n of the a c e t y l a t i o n mixture merely provided f o r further conversion of the acetate to the lactam 88. The experiment was more successful i n the conversion of the 2-hydroxy compound (90) and i s described. The 2a-hydroxy-6-aza s t e r o i d (90, 25 mg.) was treated with pyridine (0.5 ml.) and a c e t i c anhydride (0.5 ml.), and the mixture was heated f o r 25 minutes at 70°C. The cooled mixture was poured onto i c e water, and the white p r e c i p i t a t e which formed was separated and dried (22 mg.) This material was r e c r y s t a l l i z e d from ether-methanol to y i e l d the pure acetate (10 mg.), m.p. 156-157.5°; X (log e): 237 my (4.08); v : 1740 and 1245 TH3.X HI 3.X (OAc), 1670 and 1643 cm - 1 (enol lactam); NMR: 2.90 (multiplet, 5H, aromatic), 4.86 (doublet, J = 16 c.p.s., IH, H of -NCH2C6H5 group, see 90b), 5.43 (doublet, J = 16 c.p. s., IH, H of -NCHgC^Hc; group, see 90b), 5.26 (quartet, J 3 g 4 = 2.4 c.p.s., J ^ a 4 = 5.8 c.p.s., IH, o l e f i n i c ) , 5.03 (broad m u l t i p l e t , IH, CHOAc), 8.05 ( s i n g l e t , 3H, CH 3C0). Calc. f o r C35H 5 1N0 3: 533.387. Found: 533.383. Saponification of 2q-Acetoxy-N-benzyl-6-aza-4-cholesten-7-one The above acetate (5 mg.) was taken up i n a mixture of ethanol (1 ml.) and IN aqueous potassium hydroxide (0.3 ml.), and the mixture allowed to stand f o r 10 minutes at 20°C. The reaction mixture was made a c i d i c by.the addition of 1M a c e t i c a c i d i n ethanol, and then evaporated to dryness. The residue was extracted with chloroform and t h i s extract was placed on a chromatoplate (chloroform). Two zones were eluted from the plate (methanol-ether 1:1). One of these was shown to be the 2a-hydroxy compound 55 90 (2 mg.), and the other was the lactam 88 (1 mg.). Oxidation of Hydroboration Products A s e r i e s of experiments were performed on the i s o l a t e d pure products, as well as on the crude hydroboration mixture. Chromium t r i o x i d e i n acetone (Jones reagent) and i n pyridine (Sarett reagent) gave i d e n t i c a l r e s u l t s , Several t y p i c a l experiments are described. The 3 -hydroxy-6-aza de r i v a t i v e (90, 22 mg.) was treated f o r 12 hours at room temperature with chromium t r i o x i d e (30 mg.) i n pyridine (1 ml.). A f t e r t h i s time, methanol (0.5 ml.) was added and the reaction mixture was evaporated to dryness. The residue was taken up i n chloroform, and the concentrated chloroform extract was placed d i r e c t l y on a t h i n -layer chromatoplate and separated (benzene-chloroform 1:1). The material which was eluted from the plate with ether-methanol was r e c r y s t a l l i z e d from ether-hexane to provide a pure sample of N-benzyl-6-aza-4-cholesten-3, 7-dione (93, 5 mg.), m.p. 173-174.5°; X (log e): 284 my i n neutral methanol s o l u t i o n (4.39): 2,87 my, a f t e r 5 minutes i n the presence of 0.1N sodium hydroxide (4.33); 292 my, a f t e r 30 minutes i n the presence of 0.1N sodium hydroxide (4.06); 294 my, a f t e r 3 hours i n contact with a l k a l i (4.32); 296 my, a f t e r 20 hours i n contact with a l k a l i (4.31); Vmax* 1 6 ^ 0 , 1 6 6 5 a n d 1 5 9 0 c m - 1 ( k e t o n e and lactam carbonyl); NMR: 2.88 (multiplet, 5H, aromatic), 4.92 (doublet, J = 16 c.p.s., IH, H of -NCHj>C6H5 group, see 93a), 5.24 (doublet, J = 16 c.p.s., IH, H of -NCH 2C 6H 5 group see 93a), 4.60 (s i n g l e t , IH, o l e f i n i c ) ; ORD: (Figure 3, C, 0.103 mg./ml.), [<fr]it00 + 9 5 0 » [*] 3^ 0 + 13,070 (peak), [<t>]32o 0, [<j>] 3 0 0 - 29,400 (trough), [<f>]270 -11,880, [<f>]260 - 12,360, [<f>]230 - 8070, [<f>]208 - 18,040. Calc. f o r C 3 3H 1 + 7N0 2: 489.361. Found: 489.362. 56 A s i m i l a r oxidation of the 3a-hydroxy-6-aza s t e r o i d (91, 20 mg.) provided 93 (4 mg.). The l a t t e r compound was shown to be i d e n t i c a l i n every respect (mixed melting point, i n f r a r e d , TLC) with the above oxidation product. The crude mixture (53 mg.) taken d i r e c t l y from the hydroboration reaction was oxidized with chromium t r i o x i d e (85 mg.) i n pyridine (2.2 ml.) f o r 12 hours at room temperature. Addition of methanol (2 ml.), and work-up of the reaction mixture as described above provided the crude oxidation product. The l a t t e r was again subjected to TLC separation, and the eluted material (13 mg.) was c r y s t a l l i z e d from ether-hexane to provide pure 93 (11 mg.). 57 TABLE 4 Mass Spectra of Hydroboration Products  Compound 91 m/e I m/e I m/e I m/e I m/e I m/e I 491 2 400 3 358 4 320 6 282 3 244 3 490 1 398 1 357 3 319 3 281 5 243 3 480 2 394 2 356 7 318 4 280 8 242 3 479 5 393 1 355 3 317 4 279 6 241 5 478 1 392 3 354 4 316 6 278 8 240 4 477 1 391 1 353 2 315 3 277 7 239 6 476 2 390 3 352 5 314 5 276 14 238 7 475 8 389 2 351 4 313 7 275 4 237 6 474 38 388 4 350 7 312 23 274 4 236 6 473 100 387 2 349 2 311 2 273 3 235 5 472 2 386 3 348 5 310 7 272 3 234 8 465 3 385 1 347 2 309 7 271 3 233 18 464 2 384 2 346 3 308 10 270 3 232 6 459 2 383 1 • 345 2 307 5 269 3 231 4 458 6 382 3 344 5 306 7 268. 5 230 2 450 2 381 1 343 6 305 4 267 6 229 3 446 1 380 3 342 21 304 6 266 9 228 2 445 1 379 2 341 2 303 6 265 11 227 5 437 1 378 4 340 6 302 39 264 12 226 7 436 1 377 2 339 16 301 4 263 8 225 30 434 1 376 3 338 58 300 4 262 6 224 6 432 1 375 1 337 5 299 5 261 6 223 5 431 1 374 3 336 7 298 3 260 4 222 7 430 373 2 335 3 297 2 259 8 221 22 426 1 372 4 334 6 296 4 258 3 220 10 425 1 371 2 333 3 295 8 257 4 219 6 423 1 370 5 332 5 294 16 256 3 218 4 422 1 369 1 331 6 293 4 255 4 217 5 420 368 2 330 6 292 8 254 6 216 2 418 1 367 1 329 2 291 4 253 7 215 3 416 1 366 4 328 4 290 4 252 8 214 1 414 1 365 3 327 2 289 3 251 13 213 4 408 3 364 5 326 3 288 4 ' 250 14 212 4 407 10 363 2 325 6 287 2 249 7 211 10 406 17 362 3 324 17 286 2 248 8 210 6 404 2 361 2 323 14 285 2 247 10 209 6 402 2 360 3 322 9 284 6 246 4 208 9 401 1 359 2 321 3 283 2 245 4 207 6 58 TABLE 4 (cont'd) Mass Spectra of Hydroboration Products  Compound 91 m/e I m/e I m/e I m/e I 206 6 168 8 130 20 92 40 205 5 167 8 129 31 91 50 204 3 166 8 128 10 90 2 203 8 165 12 127 13 89 7 202 21 164 5 126 13 87 7 201 5 163 15 125 30 86 5 200 3 162 5 124 16 85 43 199 7 161 14 123 30 84 31 198 6 160 4 122 13 83 52 197 7 159 10 121 28 82 28 196 7 158 4 120 12 81 34 195 6 157 8 119 23 80 10 194 9 156 4 118 20 79 25 193 24 .155 8 117 47 78 19 192 7 154 9 116 12 77 30 191 9 153 9 115 17 76 3.5 190 3 152 10 114 2 75 3 189 6 151 15 113 22 74 4 188 2 150 7 112 26 73 9 187 6 149 24 111 47 72 10 186 3 148 5 110 38 71 47 185 8 147 12 109 41 70 32 184 5 146 6 108 19 69 45 183 10 145 15 107 39 68 21 182 14 144 6 106 37 67 29 181 6 143 14 105 52 66 4 180 6 142 6 104 45 65 19 179 9 141 11 103 26 64 1 178 5 140 10 102 3 63 4 177 8 139 15 101 8 61 1 176 4 138 11 100 7 60 4 175 8 137 25 99 24 59 42 174 4 136 23 98 20 58 13 173 8 135 27 97 44 57 66 172 3 134 13 96 26 56 33 171 9 133 26 95 39 55 61 170 5 132 9 94 16 169 8 131 21 93 26 m/e I 54 53 52 51 50 9 14 5 20 11 59 Compound 90 m/e I m/e I m/e I m/e I m/e I m/e I 494 0.8 432 0.4 351 0.2 252 0.2 203 0.2 167 0.4 493 6.2 422 0.2 350 0.4 250 0.2 202 0.2 166 0.2 492 35.6 421 0.4 348 0.2 247. 0.2 201 0.2 165 0.4 491 100.0 420 0.4 347 0.2 246. 0.2 200 0.4 164 0.2 490 5.6 419 0.4 346 0.2 245 0.2 199 0.4 163 0.4 489 1.8 418 0.4 340 0.2 245 0.2 198 0.8 162 0.4 488 0.2 408 0.4 339 0.2 243 0.4 197 0.4 161 0.8 487 ° - i 407 0.6 338 0.4 242 0.4 196 0.4 160 0.4 479 0.3 406 0.8 337 0.2 241 0,2 • 195 0.2 159 0.4 478 0.2 405 0.4 336 0.6 240 0.2 194 0.2 158 0.3 477 1.2 404 0.6 334 0.4 239 0.2 193 0.2 157 0.4 476 2.8 402 0.2 310 0.2 238 0.2 192 0.2 156 0.2 475 1.2 401 0.4 308 0.4 237 0.2 191 0.2 155 0.2 474 2.8 400 0.2 302 0.4 236 0.4 190 0.2 154 0.2 473 6.4 392 0.2 297 0.2 229 0.2 189 0.2 153 0.2 472 0.4 391 0.2 296 0.2 228 0.5 188 0.2 152 0.4 465 0.2 390 1,2 286 0.2 227 0.4 187 0.4 151 0.3 464 1.8 388 0.4 285 0.2 226 0.8 186 0.4 150 0.4 463 5.2 387 0.2 284 0.2 225 0.4 185 0.4 149 1.0 462 1.2 386 0.4 279 0.2 224 Q.4 184 0.2 148 0.8 461 0.4 383 0.2 275 0.2 223 0.2 183 0.4 147 0.6 460 0,2 382 0.2 274 0.2 222 0.2 182 0.2 146 0.4 459 0.2 379 0.4 273 0.2 221 0.4 181 0.2 145 0.6 458 0.4 378 1.2 272 0.2 220 0.2 180 0.2 144 0.3 454 0.2 377 0.2 271 0.2 219 0.2 179 0.2 143 0.4 450 0.2 376 0.4 270 0.4 218 0.4 178 0.2 141 0.2 449 1.0 375 0.2 269 0.2 217 0.6 177 0.2 140 0.2 448 3.2 373 0.2 268 0.2 216 1.6 176 0.2 139 0.3 447 4.4 372 0.4 266 0.2 215 2.0 175 0.2 138 0.4 446 0.8 364 0.4 264 0.2 214 0,4 174 0.3 137 0.4 438 0.4 363 0.2 260 0.2_. 213 0.4 173 0.4 136 0.6 437 0.8 362 0.2 258 0.2 212 0.2 172 0.4 135 1.4 436 0.2 360 0.2 257 0.4 211 0,2 171 0.6 134 1.0 435 0.4 357 0.2 256 0.6 210 0.4 170 0.4 133 1.2 434 0.4 356 0.2 255 0.2 206 0.2 169 0.3 132 0,4 433 > 0.2 352 0.2 254 0.4 205 0.2 168 0.2 131 0.6 60 m/e I m/e I m/e Compoi I aid 92 m/e I m/e I m/e I 130 0.4 90 0.2 493 0.8 416 0.2 330 1.6 240 0.3 129 0.8 89 0.2 492 3.4 415 0.3 320 0.3 239 0.3 128 0.2 87 0.2 491 10.0 407 0.8 319 0.3 239 0.4 127 0.5 85 1.6 490 1.1 406 1.3 318 0.8 237 0.3 126 0.3 84 0.8 489 1.5 404 0.3 317 2.8 236 0.4 125 0.6 83 2.8 483 0.4 403 0.2 316 8.2 234 0.2 124 0.4 82 1.2 480 0.8 402 0.5 314 0.2 233 0.2 123 0.8 81 3.4 479 2.4 401 0.4 304 0.2 232 0.2 122 0.4 80 0.4 478 1.4 400 0.6 302 0.5 231 0.2 121 1.2 79 2.0 477 2.6 389 0.4 300 0.3 230 0.2 120 0.6 78 0.2 476 1.4 388 0.9 289 0.2 229 0.2 119 1.2 77 0.8 475 5.8 387 1.0 288 0.5 228 0.3 118 0.2 73 1.2 474 35.2 386 0.3 286 0.3 227 0.2 117 0.6 72 0.2 473 100.0 383 0.3 279 0.3 226 0.6 116 0.2 71 5.0 472 2.0 382 0.6 276 0.2 225 0.5 115 0.4 70 1.4 471 0.4 378 0.4 275 0.2 224 0.6 113 0.4 69 6.0 464 0.9 375 0.6 274 0.2 220 0.2 112 0.4 68 1.2 463 1.7 374 4.2 273 0.2 219 0.2 111 1.0 67 2.4 462 0.7 373 12.6 272 0.2 218 0.6 110 0.6 65 0.8 461 0.3 372 4.8 270 0.4 217 0.3 109 1.6 61 0.3 460 0.3 371 0.5 269 0.3 216 0.6 108 1.0 60= 0.8 459 1.6 370 0.2 268 0.4 215 1.1 107 2.0 59 0.3 4.58 4.0 366 0.2 264 0.2 214 0.4 106 1.6 58 0.4 457 0.3 362 0.2 261 0.2 213 0.3 105 1.6 57 8.0 448 0.7 361 0.5 260 0.6 212 0.3 104 0.4 56 1.6 447 1.1 360 1.6 258 0.3 211 0.2 103 0.2 55 6.6 446 0.5 359 1.0 256 0.2 210 0.5 101 0.2 54 0.4 445 0.7 358 3.4 254 0.3 205 0.2 99 0.6 53 0.6 444 0.2 356 0.2 253 0.2 204 0.2 98 0.8 437 Q.2 354 0.3 252 0.6 203 0.2 97 1.2 432 6.3 352 0.3 251 0.2 202 0.2 96 1.0 431 0.5 346 0.4 250 0.3 201 0.2 95 2.8 430 1.0 345 0.6 247 0.2 200 0.6 94 0.8 421 0.3 344 0.3 246 0.2 199 0.8 93 2.6 420 0.2 342 0.3 245 0.2 198 2.0 92 2.6 419 0.2 334 0.2 244 0.2 197 1.1 91 14.4 418 0.2 332 0.4 243 0.2 196 1.0 417 0.2 331 0.4 242 0.2 195 0.2 m/e I m/e I m/e I m/e I 194 0.3 156 0.4 117 1.0 70 2.4 193 0.2 155 0.2 116 0.2 69 4.6 192 0.2 153 0.3 115 0.4 68 1.6 191 0.3 152 0.3 113 0.6 67 3.3 190 0.3 151 0.9 112 1.0 66 0.5 189 0.3 150 3.4 111 2.0 65 1.4 188 0.4 149 2.0 . 110 2.2 63 0.2 187 0.4 148 0.8 109 2.4 61 0.5 186 0.4 147 1.1 108 1.8 60 0.8 185 0.4 146 0.9 107 3.4 59 1.0 184 0.4 145 0.9 106 2.2 58 1.0 183 0.4 144 0.6 105 2.4 57 6.8 182 1.4 143 0.5 104 0.7 56 4.6 181 0.2 142 0.3 103 0.2 55 6.3 180 0.2 141 0.2 99 0.5 54 0.5 179 0.2 140 0.2 98 0.9 53 1.3 178 0.2 139 0.4 97 2.6 52 0.2 177 0.4 138 0.3 96 1.8 51 0.7 176 0.4 137 0.6 95 4.8 50 0.3 175 0.6 136 0.7 94 1.6 174 0.7 135 1.9 93 4.4 173 0.5 134 1.6 92 3.6 172 0.6 133 1.7 91 10.4 171 0.9 132 0.8 90 0.3 170 0.6 131 1.1 89 0.4 169 0.2 130 0.6 86 2.2 168 0.2 129 0.7 85 1.6 167 0.7 128 0.3 84 1.4 166 0.2 127 0.2 83 3.0 165 0.6 126 0.4 82 1.8 164 1.7 125 1.0 81 4.0 163 0.7 124 1.5 80 0.8 162 0.7 123 1.2 79 3.0 161 1.1 122 1.8 78 0.5 160 1.0 121 2.0 77 1.8 159 0.6 120 1.6 73 0.4 158 0.5 119 2.0 72 0.2 157 0.5 118 .0.8 71 3.9 62 REFERENCES 1. 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V l a t t a s , Tetrahedron, i n press. 78. Y. Ban, Y. Sato, I. Inoue, M. Nagai (ne'e Seo) , T. O i s h i , M. Terashima, 0. Yonemitsu and Y. Kanaoka, Tetrahedron Letters, 2261 (1965). 79. J.P. Kutney, G. Eigendorf and J.E. May, Chem. Communs. 59 (1966). PART 2 ORD Studies of Lactam and Amide Chromophores 67 INTRODUCTION Opti c a l rotatory dispersion, the change i n o p t i c a l r o t a t i o n with wave-length, was discovered by Biot i n 1817 but i t s widespread ap p l i c a t i o n to s t r u c t u r a l , stereochemical and conformational problems did not begi n - u n t i l 1953. Optical rotatory dispersion (ORD) studies have been performed mainly with saturated and a,g-unsaturated ketones because the n -*• TT* t r a n s i t i o n occurs i n the region above 260 my to which studies have been r e s t r i c t e d u n t i l recently. Furthermore, ketones are one of the most common functional groups i n organic chemistry, e s p e c i a l l y i f one considers that alcohols are r e a d i l y oxidized to the corresponding ketones. For saturated ketones the octant r u l e * which permits the p r e d i c t i o n of the sign and, semiquantitatively, the i n t e n s i t y of the Cotton effect, has been formulated. This rule has been 2 3 extended to include a,3- and 6,y-unsaturated ketones. ' The combination of unequal absorption ( c i r c u l a r dichroism) and unequal v e l o c i t y of transmission ( o p t i c a l rotation) of l e f t and r i g h t c i r c u l a r l y p o l a r i z e d l i g h t i n the region i n which o p t i c a l l y active absorption bands are observed i s a phenomenon c a l l e d the Cotton e f f e c t . A p l o t of the molecular r o t a t i o n , [$], which i s proportional to the s p e c i f i c r o t a t i o n [a], against the wavelength, of the incident l i g h t gives a rotatory dispersion (ORD) curve. O p t i c a l l y active chromophores can be c l a s s i f i e d into two types: (a) the inherently dissymmetric chromophore, and (b) the inherently symmetric, but asymmetrically perturbed, chromophore. Examples of the f i r s t class are hexahelicene and twisted biphenyls. The molecular amplitudes of the ORD curves are generally quite high compared to those observed f o r 3 the second type, a t y p i c a l example of which i s the carbonyl group. Other chromophores are available which are o p t i c a l l y active and absorb i n a s p e c t r a l range convenient f o r i n v e s t i g a t i o n . These chromophores, 68 usually derivatives of s p e c i f i c functional groups, are l i s t e d i n Table 1 with the p o s i t i o n of t h e i r o p t i c a l l y active absorption bands. Other non-ketonic chromophores such as b i a r y l s , dienes, aporphines, disulphides, diselenides, t r i t h i o n e s , n i t r o compounds, azides, thiocyanates, ethylene t h i o k e t a l s , t h i o -acetates, polypeptides, proteins and n u c l e i c acids have also be studied. 3 4 These subjects have been reviewed recently ' and w i l l not be discussed further here. Compounds containing the carboxyl group show only p l a i n curves above 270 mu. The development of automatic recording spectropolarimeters capable of measuring o p t i c a l r o t a t i o n down to about 210 my has allowed examination of compounds containing the carboxyl chromophore i n the region of of t h e i r weak absorption band at 225 my. Cotton e f f e c t s have been observed 6 7 7 8 for the carboxyl and r e l a t e d groups i n acids, esters, lactones ' and 7 amides. Only the lactone chromophore w i l l be considered i n d e t a i l . It i s of p a r t i c u l a r i n t e r e s t because of i t s close r e l a t i o n s h i p to the lactam group. In connection with previous investigations i n the f i e l d of aza-9-11 steroids (Figures 12 and 13, Part 1) a series of compounds possessing lactam, enol lactam and amide functions were synthesized. These syntheses involved r i n g opening followed by c y c l i z a t i o n with an amine and reduction of the double bond. Thus the configuration at C 5 i n the 6-aza series i s 69 Functional Group -NH^ (and amino acids) -NH2 (and amino acids) ,^NH -NHCOR R-CH-COOH I NH 2 R-CH-COOH I NH2 RCH-COOH I NH2 RCH-COOH I NH2 R-CH-C02Ri NH2 -OH -OH -OH -COOH -COOH TABLE 1  Chromophoric Derivatives Chromophoric Derivative S -NHC-SR .CO CO ^NNO NO I -N-COR R-CHCOOH I NHC(=S)0C 2H 5 R-CH-COOH I NHC(=S)C 6H 5 R-CHCOOH I NHC(=S)CH 2C 6H 5 MHCHCOz'Ri /NR 2 -CONHC=S Absorption Maxima (my) 330 300 370 350-450 310 280 290, 380 NRR 270, 335 240-320 350 325-390 200-230 340 325-360 -C=C--C=C--C=C-s ^ s I I -C — c --c-,0 260 235,305,430 450, 550 70 unknown. It was hoped that ORD studies might provide the necessary information f o r the assignment of configuration to t h i s centre. In t h e i r paper on the synthesis of 6-oxa and 6-aza st e r o i d s , Jacobs 12 and Brownfield concluded that the products had the 5a configuration on the basis of molecular r o t a t i o n differences. (Figure 1). This difference (AMn) i s equal to the molecular r o t a t i o n of the lactone or lactam (Mp) minus the molecular r o t a t i o n of the parent keto acid (Mp 1)- The re s u l t s f o r compounds prepared by chemical conversions generally assumed to give the most thermodynamically stable products and thus ones of known stereo-chemistry are shown i n Table 2. Since the AMn value r e f l e c t s only the TABLE 2 Molecular Rotation Differences M D MD- AMn C-5 4-oxa-cholestan-3-one +313 +137.5 +175.5 a 4-oxa-178-hydroxy -17a-methyl +189 +51.2 +137.8 a androstan-3-one 4-aza-cholestan-3-one +170 +137.5 +32.5 a 4-aza-173-hydroxyandrostan- +96 -92.5 +188.5 a 3-one +71 +137.5 -66.4 6 4-oxa coprastan-3-one 1 -67 +376 -443 a 2 +81 +376 -295 a asymmetry about C5 the configurations of 1 and 2 were assigned on the basis of the observed sign of AM^. It was noted that C 5 i n the r i n g A lactones and lactams used f o r comparison may be v i s u a l i z e d as being epimeric with repsect to C 5 i n the B-ring lactone (1) and lactam (2) i f the l a t t e r compounds 71 Figure 1. Synthesis of 6-0xa and 6-Aza Steroids. are of the A/B trans configuration. Therefore i t was reasoned that i f 1 and 2 have t h i s configuration they would exhibit negative AMQ values as observed. I I Thes.e r e s u l t s were not i n accord with those found i n the 173-benzoyloxyandrostane seri e s i n which the lactone (5) and hemiacetal (6) 13 13 were i d e n t i f i e d . Repetition of Jacobs' and Brownfield's work by Atwater led to i d e n t i f i c a t i o n of the products as 7 and 8 i n the 6-oxa s e r i e s , rather than 4 and 1 respectively. It was also shown that i n both the cholestane and 17-keto series large negative rotations (AM^) were observed regardless of the C 5 configuration. (Table 3). The stereochemistry of these lactones was established when i t was found that the B a e y e r - V i l l i g e r oxidation of B-norcoprastan-6-one (11) gave the same lactone (8) that was obtained from the sodium borohydride reduction. Since t h i s oxidative 72 rearrangement i s known to occur with retention of configuration of the 14 migrating centre the A/B c i s structure could be assigned to 8. On th i s b a s i s , no d i r e c t evidence was therefore available on the stereochemistry of the 6-aza s e r i e s . 11 TABLE 3 Molecular Rotations of Oxa Steroids ketoester 56-lactone 5a-lactone AM„ AM u 6 a D Cholestane series (1, 8) +356 -68 -41 -423 -397 17-keto series (9,. 10)' +594 +58 +99 -536 -495 73 An e a r l i e r attempt to resolve t h i s question of stereochemistry based on the difference between the ORD curves of saturated-3-ketones^ i n the A/B c i s and A/B trans seri e s was unsuccessful. The ORD of the 5a-lactone (12) was v i r t u a l l y without a Cotton e f f e c t while that f o r Sg-lactone (13) was strongly p o s i t i v e . A p o s i t i v e Cotton e f f e c t was also observed f o r the 5a- compotincL (14). S i m i l a r r e s u l t s have been obtained by other workers and w i l l be discussed below. 13 14 Very few ORD studies of the lactam chromophore have been c a r r i e d out. During the course of our work the ORD curves of lactams (15) and (16) were r e p o r t e d . ^ P o s i t i v e Cotton e f f e c t s were observed f o r both compounds. 74 The N-substituted A - s t e r o i d enamine 17, and the c y c l i c enamine 18 were also 18 studied. A very weak p o s i t i v e Cotton e f f e c t was observed f o r 17 and a more intense negative one f o r 18. The unsaturated amide 19 also showed a 17 18 19 negative Cotton e f f e c t with the trough and peak at 250 mu ([<{>] =-30,000) and 235 my ([>] =+60,000) respectively. Lactones, which are c l o s e l y r e l a t e d to lactams, have however, been 7 16—20 22 quite extensively studied recently. ' ' The sector r u l e , a semi-t h e o r e t i c a l i n t e r p r e t a t i o n of the res u l t s comparable to the octant rule f o r 7 ketones, has been proposed. A series of seventy lactones representing nine of the twelve possible stereochemical types were.all found to obey t h i s r u l e . The lactone group may be considered to be planar according to X-ray 21 studies. To develop the sector rule i t i s assumed to a crude approximation that the two carbon-oxygen bonds are equivalent and that the plane b i s e c t i n g the carboxyl angle i s a symmetry plane. Each carbon-oxygen bond of the lactone group i s considered i n turn as a double bond and the signs of the contributions made by the atoms i n the f a r upper octants are all o c a t e d according to the ketone octant r u l e . These appear as 20 and 21 when viewed 75 from above i n p r o j e c t i o n on the plane of the lactone group. I f these two diagrams are superimposed (22A) the signs of the contributions i n some sectors cancel i n varying degrees while i n other sectors the contributions reinforce one another giving a p o s i t i v e contribution i n the back upper section E and a negative contribution i n the back upper sector B. Atoms near a sector boundary, f o r example i n sector F, near the E/F boundary, w i l l have a small but s i g n i f i c a n t contribution. The signs of the lactone sectors (22B) are the reverse of the signs used i n the ketone octant r u l e . 22A 22B i Since the lactone group l i e s i n a true symmetry plane the signs of the back lower sectors, that i s those below the plane of the lactone group, are n e c e s s a r i l y opposite to those of the back upper sectors. The signs of r o t a t i o n contributions i n the front sectors w i l l presumably be opposite to those of the back sectors but compounds with atoms i n near sectors have not yet been considered. In general the immediate neighbourhood of the lactone chromophore determines the sign of the Cotton e f f e c t and along any r a d i a l l i n e passing through the carboxyl carbon the quantitative e f f e c t of a given 76 substituent w i l l decrease with increasing distance from the chromophore. As a convention, when applying.the sector rule the hetero r i n g i s drawn to the l e f t of the formula with the upper angular substituent 8. In order to predict the sign of the Cotton e f f e c t i t i s necessary to consider two views of the molecule. These are (a) the view along the b i s e c t r i x of the O-C-0 angle i n the plane of the lactone group (23A, 24A, 25A), the usual octant p r o j e c t i o n and (b) the view of the molecule from above pro-jected onto the plane of the lactone r i n g (23B, 24B, 25B), the sector pr o j e c t i o n . 23 23A 23B For example, the sector rule predicts a p o s i t i v e Cotton e f f e c t f o r 3-oxo-4-oxa-5a-steroids (23) and a p o s i t i v e Cotton e f f e c t i s observed. Except f o r a few exceptions a l l the compounds considered had terminal lactone rings. Two of these exceptions were the 7-oxo-6-oxa-5B and -5a-steroi d s , (24) and (25) respectively. P o s i t i v e Cotton e f f e c t s were observed f o r both lactones. In these cases the sector projections (24B and 25B) are complicated by the fact that the lactone group i s i n the middle r i n g and no attempt has been made to in t e r p r e t the sign of the Cotton e f f e c t . 77 25 25A 25B The sector rule has also been successfully applied to a series of 17 19 bridged r i n g lactones representing eleven stereochemical types. *• For example, compounds of type 26 have strong p o s i t i v e Cotton e f f e c t s . This i s as would be expected from a consideration of the octant and sector pro-jections which show that the contributions of several pairs of atoms w i l l cancel and the remainder l i e i n the upper righ t and lower l e f t (atoms marked with a c i r c l e ) sectors. No exceptions to the rule were found f o r the lactones studied. 78 The fa c t that the lactone group (-C-CO-0-C) i s planar (as shown by 21a X-ray analysis) requires that a 6-lactone r i n g has e i t h e r the boat or h a l f - c h a i r conformation. An a l t e r n a t i v e i n t e r p r e t a t i o n of the sign of the Cotton e f f e c t of 6-lactones i n terms of the conformation of the lactone r i n g 22 has been suggested by.Wolf. In t h i s p u b l i c a t i o n a serie s of s t e r o i d lactones of known conformation were studied and those having e i t h e r the boat or h a l f - c h a i r conformation, (27) and (28) re s p e c t i v e l y , showed p o s i t i v e Cotton e f f e c t s while the enantiomers (29) and (30) showed negative Cotton e f f e c t s . Of p a r t i c u l a r relevance to t h i s discussion, may be c i t e d two examples, 6-oxa-56-cholestan-7-one (31) e x i s t i n g i n conformation 27 and possessing a p o s i t i v e Cotton e f f e c t while 6-oxa-5a-cholestan-3,7-dione (32) 7 13 e x i s t i n g i n conformation 28 also shows a p o s i t i v e Cotton e f f e c t . ' 29 30 79 The conformation of the saturated 6-lactone r i n g also influences the p o s i t i o n of the Cotton e f f e c t . Those compounds having the boat conformation showed the f i r s t extremum below 233 my while f o r those with the h a l f - c h a i r conformation i t was above 238 my. An early attempt to re l a t e the stereochemistry of a lactone to 23 the sign of i t s o p t i c a l r o t a t i o n was made by Hudson. In his well-known lactone rule he stated that i f the hydrogen atom at the alkoxy carbon, C*, i n 33 or 34 l i e s below the plane of the lactone r i n g the compound i s dextro-rotatory and, conversely, i f i t l i e s above i t w i l l have a negative r o t a t i o n . In the case of a complex lactone with many asymmetric centres, each centre contributes to the t o t a l r o t a t i o n of the molecule. In order to consider only that part due to the lactone formation i t i s necessary to subtract 3 3 34 35a R* = OH; R" = COOH b R' = OH; R" = COOMe c R' = OH; R" = CH20H d R' = H; R" = COOH the ro t a t i o n of a suitable reference compound (35a-d) containing a l l the same asymmetric centres as the parent compound. Hudson's o r i g i n a l rule was 80 based on 5- and 6-membered lactones of the sugar series and was l a t e r extended to many other lactones including s t e r o i d s , terpenes and other group 24 of natural products. opened reference compounds have now been compared by means of ORD curves instead of the monochromatic rotations at 589 my used by e a r l i e r workers. Lactones of general type 36 had negative difference curves ( o p t i c a l r o t a t i o n of lactone minus o p t i c a l r o t a t i o n of the reference compound) as predicted by Hudson's rule and also showed negative Cotton e f f e c t s . S i m i l a r l y compounds of type 33 and 34 showed p o s i t i v e difference curves and p o s i t i v e Cotton e f f e c t s . This agreement between the sign of the difference curve and the Cotton e f f e c t would be expected since both are measures of the r o t a t i o n contribution of the lactone group to the t o t a l r o t a t i o n of the molecule. The d i r e c t measurement of lactone Cotton e f f e c t s i s advantageous because i t eliminates the necessity of obtaining s u i t a b l e reference compounds. Hudson' rule i s l i m i t e d to lactones i n which the alkoxy-carbon i s asymmetric. ORD curves of carbohydrate lactones have been interpreted so f a r i n terms of the octant rule considering c h i e f l y the e f f e c t of a hydroxyl group a to the lactone carbonyl." The sector rule has as yet been concerne e n t i r e l y with the contributions of a l k y l and cyclohexane rings and cannot be extended to a-hydroxy or acetoxyl groups, or halogen atoms. The rotations of some representative lactones and t h e i r r i n g -20 H 36 81 DISCUSSION The ORD curves of a number of 6-aza and 11-aza s t e r o i d a l d e r i v a t i v e s were measured and the region between 380 and 200 my which i s of i n t e r e s t w i l l be discussed. For the sake of c l a r i t y the r e s u l t s f o r each s e r i e s w i l l be considered s e p a r a t e l y . The 6-Aza Se r i e s A s e r i e s of 6-aza s t e r o i d s i n which the b a s i c chromophore i s a lactam were s t u d i e d w i t h the hope of determining the c o n f i g u r a t i o n at C 5 . The lactam i s h e l d i n a more or less f i x e d conformation i n a r i n g and i s the r e f o r e a s u i t a b l e group with which to i n v e s t i g a t e the asymmetric environ-ment of i t s chromophore. Unfortunately there i s not a wide-range of compounds of known stereochemistry a v a i l a b l e as was the case with lactones and no equivalent of the s e c t o r r u l e f o r lactones e x i s t s at the moment f o r lactam systems. I t was t h e r e f o r e c l e a r at the outset that the r e s u l t s may not be e n t i r e l y c o n c l u s i v e but they would s t i l l be of some i n t e r e s t . The parent lactam system i n the cholestane (37) and androstane (38) s e r i e s e x h i b i t e d a p o s i t i v e Cotton e f f e c t with the peak i n the region between 250 and 260 my and the trough about 230 my. (Table 4, Figure 2). The R RJ_ 37 CgH^ H 38 OH H 39 C 8 H 1 7 -CH 2C 6H 5 40 OH -CH 2C 6H 5 41 -CH-CH3 -CH 2C 6H 5 OAc 82 TABLE 4 ORD of 6-Aza Ste r o i d Derivatives Compound Concentration i n mg/ml Amu F4>1 x 10 37 1.099 258 pk +9 232 t r -15 38 1.313 252 pk +9.5 228 t r -25 39 0.969 260 pk +3 230 t r -85 40 ' 1.206 246 pk +29.4 t r o f f scale 41 2.346 255 pk +198 t r o f f scale 1.173 255 pk +163.3 t r o f f scale 0.105 254 pk +26 230 t r -53.8 43 0.117 257 pk +215 228 t r -314 44 0.113 255 pk +118 228 t r -305 45 0.103 263 pk +241 233 t r -584 46 0.112 262 pk +567 234 t r -976 47 0.102 262 pk +253 235 t r -435 52 0.096 311 pk +118 260 t r -181 pk = peak; t r = trough 83 2 2 0 2 6 0 3 0 0 3 4 0 3 8 0 2 2 0 2 6 0 3 0 0 3 4 0 3 8 0 X (mju) X (m^.) F I G . 2 F I G . 3 RD Curves of Compounds 3 7 , 3 8 and 4 0 in R D Curves of Compounds 4 4 , 4 7 and 5 2 in 6 - A z a Stero id Ser ies . 6 - A z a S te r i od Ser ies . b X I—I T3. 2 2 0 RD Curves' of 5 9 in I I - A z a 2 6 0 3 0 0 3 4 0 3 8 0 \ (mjz) F I G . 4 Compounds 5 5 , 5 7 and S t e r o i d S e r i e s . 2 0 0 r 1 6 0 -1 2 0 -8 0 -4 0 -b o -X - 4 0 -1—1 •8. - 8 0 -t_J - 1 2 0 -- 1 6 0 -- 2 0 0 -- 2 4 0 • - 2 8 0 -- 3 2 0 --2.346 mgAnl 2 2 0 2 6 0 3 0 0 3 4 0 3 8 0 F I G . 5 RD Curve of Compound 41 Showing E f f e c t of Concent ra t ion . 84 appli c a t i o n of the lactone sector rule to these compounds may or may not be a v a l i d extension; however, f o r the sake of comparison an attempt was made to apply i t . F i r s t , the saturated lactam (15) was considered. The sector r u l e , (15A) and (15B), predicts a negative Cotton e f f e c t when i n fac t a weak v * 15 15A 15B p o s i t i v e one superimposed on a negative background was observed by Wolf. There i s some disagreement about the sign of the Cotton e f f e c t associated with the corresponding lactone (42). Wolf*^ observed a very weak p o s i t i v e curve superimposed on a strongly negative background. This was confirmed 25 by the p o s i t i v e c i r c u l a r dichroism curve. On the other hand, Klyne reported a negative Cotton e f f e c t i n agreement with the sector rule pre-d i c t i o n . In view of t h i s controversy and the fact that lactam (15) i s the only saturated one studied by other g r o u p s ^ i t i s not possible to draw any conclusions about the v a l i d i t y of extending the sector rule to lactams. S i m i l a r l y nothing can be sa i d about the use of the octant rule i n t h i s case 85 except to mention that i f i t i s applied to the lactam a negative Cotton e f f e c t i s predicted. It should be noted that the ORD curves of carbohydrate lactones 8 have been interpreted i n terms of the octant r u l e . I f the sector rule could be applied to lactams i t would be d i f f i c u l t to i n t e r p r e t the r e s u l t s f o r 6-aza steroids as the projections would be 7 s i m i l a r to those f o r lactones (24) and (25) which Klyne does not attempt to i n t e r p r e t . A further complication i s that both the A/B c i s (24) and A/B trans (25) lactones gave p o s i t i v e Cotton e f f e c t s , the l a t t e r of lower i n t e n s i t y . It i s reasonable on t h i s basis to suspect that the 6-aza steroids might give p o s i t i v e Cotton e f f e c t s regardless of the configuration at C 5. 22 I f one applies Wolf's theory that the sign of the Cotton e f f e c t i s determined by the conformation of the lactone r i n g p o s i t i v e Cotton e f f e c t s are predicted, i n both cases i n accord with observations. I f , how-ever, his r e s u l t s can be extended to lactams the p o s i t i o n of the f i r s t extremum of the ORD curves of the 6-aza steroids may be important. It has 21a been noted that i n view of the planar nature of the l a t t e r group, i t i s probably reasonable to assume that the lactam group i s also planar. On t h i s basis r i n g B would have the h a l f - c h a i r comformation (28) i f the configuration at C5 was a, and the boat conformation (27) i f C5 was 3- Wolf observed that the f i r s t extremum occurred above 238 mu when the lactone r i n g had the h a l f -22 chair conformation and below 233 my when i t was i n the boat conformation. The fa c t that our lactams showed the f i r s t extremum between,250 and 260 my might be some i n d i c a t i o n that these compounds have the 5a configuration. The i n t e n s i t i e s observed were low (Table 4) and i t i s of i n t e r e s t to note, 7 that Klyne observed weaker Cotton e f f e c t s f o r the 6-oxa-5a-lactone (25) n f o r the cor esponding 53-compound (24). 86 Although the r e s u l t s on the 6-aza steroids are suggestive, they c e r t a i n l y do not provide conclusive evidence f o r the configuration at C5 i n these compounds. In view of the problems associated with the i n t e r -p retation of the ORD curves of lactams and amides further r e s u l t s w i l l be presented without any attempt to explain the sign of the Cotton e f f e c t observed. The e f f e c t of s u b s t i t u t i o n on the nitrogen atom was investigated f o r the N-benzyl seri e s (39-41). In general, the e f f e c t of t h i s s u b s t i t u -t i o n was not appreciable. The sign of the Cotton e f f e c t remained unchanged but the i n t e n s i t y varied. (Table 4). In the series 39-41 these i n t e n s i t y differences must be due to the C-17 substituent. S i m i l a r r e s u l t s were obtained i n the enol lactam series (43-47). (Table 4). R R l 43 C 8 H 1 7 H 44 OH H 45 C 8 H 1 7 - C H 2 C 5 H 5 46 -CH-CH3 OAc -CH 2C 6H 5 47 OH -CH2CgH5 52 OH -CH 2 C 6 H 5 + addit i o n a l at C 2-C 3. double bond The various enol lactams (43-47) gave much more intense Cotton e f f e c t s than the corresponding saturated lactams (37-41) (Figures 2 and 3). The r e l a t i v e p o s i t i o n s of the peaks were s h i f t e d s l i g h t l y i f at a l l by the presence of the double bond. In the case of the parent lactams the Cotton it e f f e c t i s due to the weak n -+ TT t r a n s i t i o n of the carbonyl group which i s 87 not usually observed i n the u l t r a v i o l e t spectrums under routine measurements (above 220 mp) . On the other hand, the enol lactams showed an u l t r a v i o l e t absorption at 237 mp (log e about 4) which i s probably due to the IT •> TT* t r a n s i t i o n of the conjugated system, ^C=C-N-C=0. Si m i l a r u l t r a v i o l e t spectra have been observed f o r r e l a t e d systems, f o r example (48) and (49), N H C - C H 3 26 48 I 49 the former having X 240 mu (log e 3.8), the l a t t e r , Xm<sv 238 my (log e max max 3.9). A s i m i l a r e f f e c t was observed by Wolf 1^ i n comparing the lactone (50) and enol lactone (51) but i n contrast to our r e s u l t s the sign of the Cotton e f f e c t was reversed. (Figure 6). 0 ^ 0 51 Figure 6. ORD of Steroid Lactones 88 The presence of an addi t i o n a l double bond which extends the con-jugation of the unsaturated lactam would be expected to have an appreciable e f f e c t on the ORD curve. In the case of 17B-hydroxy-N-benzyl-6-aza-2,4-androstadien-7-one (52) the curve i s s h i f t e d to higher wavelength with the peak and trough at 311 my and 260 my re s p e c t i v e l y (Figure 3). From a model, the expected c h i r a l i t y of the diene i s that of a right-handed h e l i x (53) and;on t h i s basis a p o s i t i v e Cotton e f f e c t would be expected i f the O H 53 54 rules developed f o r the diene chromophore can be applied to t h i s compound. The analogous diene (54) exhibited a p o s i t i v e Cotton e f f e c t with the peak 27 at 300 my ([<J>] = 8700) normally a t t r i b u t e d to a TT -»- TT* t r a n s i t i o n . The 11-Aza Series The ORD study of aza steroids was extended to the 11-aza series with the study of the unsaturated lactam (55). I t was already known from previous HO 89 r e s u l t s , ' that t h i s chromophore with the double bond i n the same r i n g as the lactam system absorbs i n the u l t r a v i o l e t spectrum, at a higher wave-length (255 mu) than the corresponding enol lactams i n the 6-aza serie s (237 my). It was therefore expected that the ORD curve would s i m i l a r l y e x h i b it a bathochromic s h i f t . In accord with expectation the peak was observed at 285 my with the trough at 240 my. (Table 5, Figure 4). Unfortunately the corresponding saturated system was not available f o r study. TABLE 5 ORD of 11-Aza Steroid Derivatives Compound Concentration (mg/ml) Xmy [<t>] x 10~ 55 0.118 285 pk +36.2 240 t r -246 56 p l a i n negative dispersion curve 57 0.107 238 pk +117 215 t r -126 58 0.101 355 pk +79 297 t r +3.6 238 pk +378 212 t r -742 59 0.112 306 pk +71.8 270 t r -18.0 233 pk +82.7 212 t r -201 pk = peak; t r = trough 3 It i s well-known from work on polypeptides and proteins that the amide chromophore can be o p t i c a l l y a c t i v e . Thus the N-acetyl-11-aza derivatives (57), (58) and (59) would be expected to have anomalous 90 58 59 The parent 11-aza compound (56) showed a p l a i n negative curve. On the other hand, the N-acetyl de r i v a t i v e (57) showed an anomalous Cotton e f f e c t with the peak and trough at 238 my and 215 my r e s p e c t i v e l y , which could be a t t r i b u t e d to the amide n •*• TT* t r a n s i t i o n . (Figure 4). The corresponding compounds from the 11-aza pregnane seri e s (58) and (59) also exhibited s i m i l a r anomalous dispersion i n t h i s region i n addition to the expected Cotton e f f e c t s due to the n -»• TT* t r a n s i t i o n of the C z o ketone group. (Table 5). 2 It i s well known that changes i n concentration of some o p t i c a l l y active substances can a f f e c t the r o t a t i o n appreciably. This e f f e c t i s often already noticeable at the sodium D l i n e and may be.enhanced i n rotatory dispersion measurements. For example, with (+)-3-methylcyclo-hexanone the s p e c i f i c rotations i n methanol at the peak (307.5 my) were found to be 910°, 840° and 720°, corresponding to concentrations of 0.132, 2 0.103 and 0.029 g. per 100 cc. A s i m i l a r concentration dependence was observed i n the case of some of the aza steroids- studied. The data f o r the N-benzyl-6-aza d e r i v a t i v e (41) i s included in.Table 4. The molecular 91 rotations were found to be 19800, 16330, and 2600° corresponding to con-centrations of 2.346, 1.173 and 0.105 mg. per ml. r e s p e c t i v e l y and the curves are shown i n Figure 4. Observation of anomalous Cotton e f f e c t s associated with amide and lactam functions may allow the extension of ORD studies to stereochemical problems i n a l k a l o i d chemistry. Aromatic alk a l o i d s have been s u c c e s s f u l l y 3 28 studied by ORD recently. ' Other classes of alkaloids can often be r e a d i l y converted i n t o the N-acetate or N-benzoate derivatives and provided the asymmetric environment i s favourable ORD studies might y i e l d useful information. 92 EXPERIMENTAL Optical rotatory dispersion curves were measured i n methanol on a JASCO Model ORD/UV-5 Spectropolarimeter (1 = 0.05 dm; t = 20-25°; c = 1 mg/ml or 0.1 mg/ml). i 93 REFERENCES 1. W. Moffit,,R.B. Woodward, A. Moscowitz, W. Klyne, and C. Dj e r a s s i , J . Am. Chem. 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Vlattas and G.V. Rao, Can. J . Chem., 41, 958 (1963). 11. J.P. Kutney and I. V l a t t a s , Steroids, 4_, 595 (1964). 12. T.L. Jacobs and R.B. Brownfield, J . Am. Chem. Soc., 82, 4033 (1960). 13. N. Atwater, J . Am. Chem. Soc., 82, 3071 (1961). 14. R.B. Turner, J . Am. Chem. S o c , 72, 878 (1950); T.F. Gallagher and T.H.. Kritchevsky, J . Am. Chem. S o c , 72_, 882 (1950); K. Mislow and J. Brenner, J . Am. Chem. S o c , 75, 2318 (1953). 15. C. Djerassi and W. Clossen, J . Am. Chem. S o c , 78, 3761 (1956). 16. H. Wolf, Tetrahedron Le t t e r s , 1075 (1965). 17. C.G. DeGrazia, W. Klyne, P.M. Scopes, D.R. Sparrow and W.B. Whalley, J . Chem. S o c , 896 (1966) . 18. A. Yogev and Y. Mazur, Tetrahedron, 22, 1317 (1966). 19. J.P. Jennings, W. Klyne and P.M. Scopes, J . Chem. S o c , 7229 (1965). 20. W. Klyne, P.M. Scopes and A. Williams, J . Chem. Soc., 7237 (1965). 94 21. (a) A. Mc L. Mathieson, Tetrahedron Letters , 81 (1963); (b) K.K. Cheung, K.H. Overton and G.A. Sim, Chem. Comm., 634 (1965). 22. H. Wolf, Tetrahedron Letters, 5151 (1966). 23. C S . Hudson, J . Chem. S o c , 338 (1910); 61.1525 (1939). 24. W. Klyne, Chem. and Ind. 1198 (1954). 25. J.P. Jennings, W. Klyne and P.M. Scopes, Proceedings, 412 (1964). 26. A.I. Scott, Interpretation of the U l t r a v i o l e t Spectra of Natural Products, MacMillan, New York, 1964. 27. A. Moscowitz, E, Charney, U. Weiss and H. Z i f f e r , J . Am. Chem. S o c , 83, 4661 (1961); U. Weiss, H. Z i f f e r and E. Charney, Tetrahedron, 21, 3105 (1965); 3121 (1965); E. Charney, Tetrahedron, 21, 3127 (1965). 28. A.R. Battersby, I.R.C. Bick, W. Klyne, J.P. Jennings, P.M. Scopes and M.J. Vernengo, J . Chem. Soc., 2239 (1965). PART 3 INVESTIGATION OF THE SPORES OF EQUISETUM TELMATEIA F e r t i l e Branches' of Equisetum t e l m a t e i a Ehrh. 97 INTRODUCTION With the f o s s i l i z e d forms of dinosaurs and other p r e h i s t o r i c animals p e r f e c t l y preserved specimens of giant h o r s e - t a i l s are found. They grew i n dense forests a t t a i n i n g a height of s i x t y to ninety feet and a diameter of three feet and disappeared from the f o s s i l record 150 m i l l i o n years ago. Today there are about twenty-five species of descendants of these giants which range from a few inches to three or four feet i n height with the exception of E. giganteum. This species i s native to t r o p i c a l South America and has a stem one inch i n diameter and s i x t y feet t a l l , clambering somewhat l i k e a vine on other vegetation. Horsetails contain large amounts of s i l i c e o u s compounds and were used i n pioneer times f o r scouring pots and pans. They were c a l l e d scouring rushes. Equisetum species have been known to medicine f o r centuries. The p r i n c i p l e use of the herb i s as a d i u r e t i c but i t has also been recommended for haemoptysis, haemorrhoids, varicose ulcers and tuberculosis. The Indians b o i l e d i t i n water for a drink and used i t f o r horse medicine. A novel use f o r h o r s e t a i l s has been found by Dr. Hans Lundberg* who operates gold farms i n Indiana and I l l i n o i s . In areas containing gold,deposits small traces of the metal get into undergound streams. Horsetails soak up th i s water, gold traces and a l l , and because the gold i s foreign to t h e i r systems, the plants t r y to eject i t through t h e i r leaves. As a r e s u l t the gold appears i n t i n y capsules on the l e a f t i p s . In an.early experiment near Timmins, Ontario, Dr. Lundberg burned a ton of h o r s e t a i l s and extracted four ounces of gold. The h o r s e t a i l s have both f e r t i l e and s t e r i l e branches which ar i s e from a subterranean stem. The f e r t i l e branches appear early i n spring and, since they lack c h l o r o p h y l l , draw upon the underground stem f o r food. They 98 bear the spores i n cones at the top of the stems and are s h o r t - l i v e d . As soqn as the spores are disseminated multi-branched, green s t e r i l e branches appear. Hor s e t a i l s are of i n t e r e s t chemically because they have remained e s s e n t i a l l y unchanged since p r e h i s t o r i c times. The spores of Equisetum maximum Lam., also known as Equisetum telmateia Ehrh., were investigated 2 by a French chemist, Sosa nearly twenty years ago. As the structures he proposed were incomplete i t was of i n t e r e s t to re-examine the spores of t h i s species. When Sosa extracted the spores with ether he obtained a colourless substance which melted at 127.5°. It showed no u l t r a v i o l e t absorption and did not saponify on heating with potassium hydroxide. This compound was a c i d i c and he named i t e q u i s e t o l i c acid, assigned the empirical formula, C 3 7 H 7 2 O 5 J and suggested that i t was a monohydroxylated a l i p h a t i c d i a c i d . A f t e r the spores had been extracted with ether they were treated with ethanol. . A glycoside which he c a l l e d equisporoside separated from t h i s extract and on p u r i f i c a t i o n of t h i s substance i n water he found that with i t there was a second glycoside, equisporonoside. Equisporoside (1) was hydrolyzed with acid to one molecule of d-glucose and equisporol, C^H^Og. To determine the p o s i t i o n of the sugar, the glycoside was methylated and sub-sequently hydrolyzed to give penta-methyl equisporol (2). This compound melted at 250° and gave a negative f e r r i c chloride t e s t . Sosa therefore concluded that glucose was attached at the C3 p o s i t i o n . Equisporol was methylated and the r e s u l t i n g hexamethyl ether (3) submitted to a l k a l i n e degradation. The products were an acid i d e n t i f i e d as verat-ric acid (4) and an u n i d e n t i f i e d phenol, m.p. 112°. On t h i s basis the p a r t i a l structure 5 was proposed f o r equisporol. 99 OCH 3 oc\\3 1 R = Glucose 5 R = H 2 R = H 3 R = CH 3 Equisporonoside, which was not soluble i n hot water but soluble i n ethanol, melted at 293° and i t s acetate melted at 237°. On hydrolysis i t gave a sugar which Sosa thought to be glucose and equisporonol. The colour reactions of equisporonoside and equisporonol were very s i m i l a r to those of equisporoside-and equisporol r e s p e c t i v e l y . The spores which had already been treated with ether and ethanol were extracted with acetone f o r two days. A small amount of the pigment was extracted along with a very l i g h t colourless s o l i d which melted at 410°. It was very soluble i n organic solvents and did not give p o s i t i v e phenol or sapogenin reactions. F i n a l l y , Sosa extracted the spores with a mixture of equal amounts of methanol, benzene, and ethyl-acetate and obtained more equisporoside. The s t e r i l e green shoots of Equisetum.telmateia have been reported 3 to contain very small amounts of n i c o t i n e , a saponin probably i d e n t i c a l 4 5 with equisetonin from E. arvense, and 4.4 to 11.3% s i l i c i c acid. The f e r t i l e brown shoots were reported to contain dimethylsulphone Other species of Equisetum have also been investigated. Equisetum 3 6 7 arvense has been studied by several groups. Nicotine (6), ' ' 3-methoxy-100 pyridin e , traces of p a l u s t r i n , dimethyl sulphone, equisetonin, ' ' a c o n i t i c a c i d (7), o x a l i c acid and a l i p i d have been i s o l a t e d . The flavone mixture known as f l a v e q u i s e t i n * * was further investigated and equisetroside (kaempferol-7-diglucoside) (8), i s o q u e r c i t r i n (9) and luteolin-5-glucoside 12 (10) were i d e n t i f i e d . Manganese was found i n determinable amounts (6.5Tng.% 13 of the dry weight of the p l a n t s ) . The f e r t i l e brown shoots y i e l d e d a r t i c u l a t i n , C 2 1 H 2 2 O 3 . 2 H 2 0 , and i s o a r t i c u l a t i n . The corresponding aglycones, a r t i c u l a t i d i n and i s o a r t i c u l a t i d i n both showed anthraquinone-type r e a c t i o n s . 1 4 The s i l i c i c a c i d content was lower than i n the s t e r i l e shoots; 3.21% compared to 16-18%.5 B - s i t o s t e r o l has also been i s o l a t e d from t h i s 15 species. Very l i t t l e work has been done on Equisetum sylvaticum L. It 3 contains a small amount of ni c o t i n e and a saponin which exhibits d i f f e r e n t properties to those of equisetonin from E. arvense. 4 Apart from E. arvense the most work has been done on Equisetum 101 palustre. The c h i e f a l k a l o i d was i s o l a t e d i n 1936 and named p a l u s t r i n . ^ Further work was done i n 1953* 7 and some years l a t e r the p a r t i a l sturcture 18 11 was proposed. Nicotine, a xanthophyll, l u t e i n , and p a l u s t r i d i n were H N H } i _ N U II 11 0 17 also found. Equisetin and equisetonin i s o l a t e d by other workers are 19 i d e n t i c a l to p a l u s t r i n and p a l u s t r i d i n r e s p e c t i v e l y . A hydrocarbon, C2).Htf2,*^ thymine, dimethylsulphone,^ kaempferol 3-rhamnosylglucoside-7- -21 21 glucoside, and a p a r t l y characterized kaempferol-3,7-diglycoside have also been reported. Equisetum f l u v i a t i l e L. i s very va r i a b l e i n habit. The non-branched form i s known as E. limosum L. and the branched form as E. f l u v i a t i l e . 22 23 A c o n i t i c aci d was i s o l a t e d as i t s magnesium,calcium,and sodium s a l t s . ' 4 A saponin with the same properties as equisetonin was also found. From 17.5 kg. of dry,Equisetum hiemale L. small amounts of dimethyl-sulphone, 12 mg. of a reactive acid, a water soluble substance, small amounts of n i c o t i n e , and f e r r u l i c (12) and c a f f e i c acids (13) were i s o l a t e d . ^ P a l u s t r i n was not found. 12 R = CH 3 13 R = H Two flavonoids, apigenin (14) and l u t e o l i n (15) were i d e n t i f i e d i n 24 the methanol extracts of Equisetum ramosissimum Desf. and n i c o t i n e has been 102 i d e n t i f i e d i n Equisetum debile 25 O H OH 0 It i s obvious that very l i t t l e i s known about the chemistry of Equisetum and much work remains to be done. 103 DISCUSSION  Equisporoside Equisporoside which had been assigned the p a r t i a l structure (1) by 2 Sosa was i s o l a t e d from the methanol extracts of the spores. A small amount was p u r i f i e d by paper chromatography and t h i s was used f o r preliminary s p e c t r a l studies. (Table 1). Quantitative u l t r a v i o l e t and v i s i b l e spectra were done l a t e r using a sample of equisporoside which had been c r y s t a l l i z e d from aqueous a c e t i c acid. (Figure 1). U l t r a v i o l e t and v i s i b l e spectra of flavonoid compounds have been 26-29 extensively studied and can be used to determine various s t r u c t u r a l features of these compounds. The spectrum of equisporoside, * m a x 388,348 mu (Band I) and A 277, 261 mu (Band II) immediately suggests several points fflcLX about the structure of equisporoside. Flavones (16) and flavonols (17) generally exhibit high i n t e n s i t y absorptions i n the 320-380 my (Band I) and 240-270 my (Band II) regions. The p o s i t i o n and i n t e n s i t y of each of these bands varies with the r e l a t i v e resonance contributions of the benzoyl (18), cinnamoyl (19), and pyrone (20) groupings to the t o t a l resonance of the flavone molecule. Although.these groupings undoubtedly i n t e r a c t , the spectra of substituted flavones and flavonols i n neutral and a l k a l i n e solutions suggest that Band I i s associated c h i e f l y with the cinnamoyl grouping (19) and Band II with absorption i n the TABLE 1 U l t r a v i o l e t Spectra of Equisporoside Reagent Added Band I X (mu) maxv 1 Band II Ethanol s o l u t i o n Aluminum chloride Sodium Acetate Boric Acid-Sodium Acetate Sodium Ethoxide 388 455 394 404 390 348 383 351 277 261 275 280 266 266 274 250 TABLE 2 Reagent Added Ethanol s o l u t i o n Aluminum chloride Sodium Acetate Boric Acid-Sodium Acetate Sodium Ethoxide U l t r a v i o l e t Spectra of Equisporol Band I a. 384 450 b 341 380 328 412 357 387 (low in t e n s i t y ) X (my) max Band II 276 287 263 285 287 b 263 249 274 105 Figure 1. U l t r a v i o l e t Spectra of Equisporoside. 106 benzoyl grouping (18). Thus, the introduction of electron donating groups such as hydroxyl into r i n g B increase i t s r e l a t i v e resonance c o n t r i -bution and consequently produce considerable bathochromic s h i f t s of Band I. Introduction of hydroxyl or methoxyl groups into the A r i n g , on the other hand, p r i m a r i l y increases the resonance contribution of t h i s r i n g and tends to increase the wavelength and i n t e n s i t y of Band I I . The p o s i t i o n of Band II at 261 and 277 my suggested that equis-poroside was a gossypetin (21) d e r i v a t i v e f o r which ^ m a x 250-260 my and 30 270-280 my have been reported. The p o s i t i o n of Band I, 348 and 388 my OH indicated that i t was probably a flavonol rather than a flavone; the former us u a l l y have Band I between 340 and 380 my and the l a t t e r between 320 and 31 350 my. Band II of flavones and flavonols which have only a 4'-substituent 107 i n the B-ring has a s i n g l e , well-defined peak. For flavones and flavonols which have hydroxyl or methoxyl substituents i n both the 3'- and 4'-positions Band II shows two d e f i n i t e peaks or one peak and a pronounced i n f l e c t i o n . When three substituents are present i n the B-ring, Band II 32 has only a s i n g l e peak. Thus, the fact that Band II i n the spectrum of equisporoside has a peak and a pronounced i n f l e c t i o n indicates immediately 2 the presence of 3'- and 4'- substituents i n r i n g B as suggested by Sosa. The value of s p e c t r a l data i n the i d e n t i f i c a t i o n and s t r u c t u r a l analysis of flavonoid compounds i s increased considerably by the addition of c e r t a i n reagents which produce s h i f t s i n the maxima i n accordance with the location of various functional groups i n the molecule. Addition of 27 aluminum chloride caused a bathochromic s h i f t of 67 my i n Band l a (Table 1, Figure 1) which i s r e l i a b l e evidence f o r the presence of a free 27 33 3-hydroxy group. ' Sodium acetate i s s u f f i c i e n t l y b a s i c to ionize hydroxyl groups located at positions 7, 3, and 4* of the flavone nucleus. HydroxyIs located elsewhere are unaffected. Ionization of.3- and 4'-hydroxyl functions produces bathochromic s h i f t s of Band I but does not a f f e c t the p o s i t i o n of Band II. Since Band II i s associated mainly with absorption i n the A r i n g , however, i o n i z a t i o n of a 7-hydroxyl group r e s u l t s i n a pro-26 33 nounced (8-20 my) bathochromic s h i f t i n t h i s band. ' Addition of sodium 26 acetate r e s u l t e d i n a s h i f t of 6 my i n Band l a i n d i c a t i n g again the presence of 3- and 4'-hydroxyl groups. Band II remained e s s e n t i a l l y unchanged and t h i s provided i n i t i a l evidence f o r the attachment of the sugar at the 7-position. The presence of one sugar i n equisporoside was c l e a r l y shown i n the NMR spectrum (100 Mc/s of the t r i m e t h y l s i l y l ether i (Figure 2). In the presence of sodium acetate, b o r i c acid chelates with phenolic Figure 2. NMR Spectrum of Equisporoside. o 00 109 28 compounds containing o_-dihydroxyl groups. Addition of these reagents caused a bathochromic s h i f t of 16 my i n the p o s i t i o n of Band l a i n good agreement with the 15-20 my s h i f t reported f o r flavones and flavonols having £-dihydroxyl groups. The spectra of compounds which do not contain 34 such a group are not appreciably affected. The gossypetin system i s one of the few exceptions which decomposes i n sodium ethoxide regardless of whether the hydroxyl at C 3 i s protected. In t h i s case, therefore, a l k a l i i n s t a b i l i t y which i s usually c h a r a c t e r i s t i c of 3,4'-dihydroxyflavonols 2^'* 5 4 cannot be used as evidence f o r the presence of these substituents. Thus, the s p e c t r a l evidence i s i n agreement with equisporoside being a gossypetin d e r i v a t i v e . Addition of p_-benzoquinone to an ethanol sol u t i o n of :equisporoside gave a p o s i t i v e gossypetone reaction (red-brown 35 p r e c i p i t a t e ) i n d i c a t i n g the presence of para-hydroxyl groups. Equisporo-side was subsequently i d e n t i f i e d as g o s s y p i t r i n (22), the 7-glucoside of gossypetin. Evidence f o r t h i s assignment w i l l be given below. 22 Hydrolysis of equisporoside with d i l u t e aqueous acid gave the corresponding aglycone and a sugar which was i d e n t i f i e d as glucose by paper chromatography i n three d i f f e r e n t solvent systems. A t y p i c a l paper which was developed with ethyl acetate-pyridine-water, 8:2:1, i s shown i n Figure 3. 110 i o l v e . Go. I cud dlucose Unknown £ G i f t l a c t o s e +• ; U.M k n o w n glucose + jf. Unkhown Figure 3. Paper Chromatography of the Sugar. 2 The aglycone which Sosa named equisporol was c r y s t a l l i z e d from aqueous a c e t i c acid, m.p. 302-304° (decomposition). The u l t r a v i o l e t spectrum, X m a x 384, 341, 276 and 263 my agreed with that reported f o r gossypetin: X m a x 386, 341, 278 and 262 my. The NMR spectrum (100 Mc/s, Figure 4) showed the expected s p l i t t i n g pattern f o r the r i n g B aromatic protons, plus a s i n g l e t which integrated f o r one proton, i n d i c a t i n g the presence of only one unsubstituted p o s i t i o n on the A-ring. There were no methoxyl protons. The derivatives of equisporol also corresponded to those of gossypetin. The melting point of the hexacetate, 226-230°, agreed with 35 36 that reported f o r gossypetin hexaacetate. ' The melting point of equis-porol hexamethyl ether was undepressed on mixing with a sample of gossypetin hexamethyl ether. This sample was shown to be impure by t h i n layer chromatography (alumina, chloroform) and the i n f r a r e d spectrum was not quite as well resolved as that of equisporol hexamethyl ether; however, the two were e s s e n t i a l l y superimposable. The u l t r a v i o l e t spectrum, X 351 7 Figure 4. NMR Spectrum of E q u i s p o r o l . 112 36 272 . and 252 my was the same as that reported f o r hexamethyl gossypetin. F i n a l l y , comparison of equisporol with gossypetin showed the mixed melting point was undepressed and the i n f r a r e d spectra were super-imposable. Equisporol was therefore i d e n t i f i e d as gossypetin (21). The remaining question was the p o s i t i o n of attachment of the sugar residue. U l t r a v i o l e t studies of equisporoside had already indicated that t h i s was l i k e l y at the C7 p o s i t i o n . In order to d e f i n i t e l y e s t a b l i s h the p o s i t i o n equisporoside was methylated and the product hydrolyzed with d i l u t e sulphuric a c i d to y i e l d the pentamethyl ether, m.p. 249-252°, the acetate of which melted at 166-168°. The melting points of various penta-methyl ethers of gossypetin and the corresponding acetates are l i s t e d i n Table 3. From t h i s i t can be seen that equisporol pentamethyl ether must be 7-hydroxy-3,3', 4', 5,8-pentamethoxy flavone. The u l t r a v i o l e t spectrum, 36 *max ' 272 and 349 my agreed with that reported by Geissman, X 251 . 270 and 351 my, f o r t h i s compound. Addition of sodium max ' r ethoxide caused the expected bathochromic s h i f t i n Band II to 282 my (29 my with a 34% decrease i n i n t e n s i t y ) due to i o n i z a t i o n of the 7-hydroxyl group. The u l t r a v i o l e t spectrum and t h i s s h i f t were i n agreement with those 36 observed by Geissman f o r 7-hydroxy-3,3',4',5,8-pentamethoxyflavone; however the p o s i t i o n of Band I (387 my) i n the presence of sodium ethoxide 36 d i f f e r e d from the 368 my reported by Geissman. As the aglyconehad been i d e n t i f i e d as gossypetin and the p o s i t i o n of attachment of the sugar found to be at the 7-hydroxyl i t followed that equisporoside must be the 7-glucoside of gossypetin which i s c a l l e d g o s s y p i t r i n . Comparison of equisporoside with a sample of g o s s y p i t r i n (22) confirmed t h i s proposal. The i n f r a r e d spectra were superimposable and 2 the mixed melting point was undepressed. The structure proposed by Sosa 113 (1) i s therefore i n c o r r e c t . TABLE 3 Pentamethyl Ethers of Gossypetin A c e t ate. m.p. m.p. 7-hydroxy-3,5,8,3', 4'-pentamethox y f l a v o n e 3 6 250-251° 164-168° 8-hydroxy-3,5,7,3', 4'-pentamethox y f l a v o n e 3 7 196-198° 215-216° 38 3-hydroxy-5,7,8,3',4'-pentamethox yf l a v o n e 228-230° 207-208° 39 5-hydroxy-3,7,8,3 1, 4J -pentamethox y. f 1avone 166-168° Equisporol pentamethyl ether 249-252° 166-168° E q u i s e t o l i c Acid E q u i s e t o l i c acid was i s o l a t e d from the ether extracts of the spores, m.p. 127-129°. The i n f r a r e d spectrum showed the presence of a hydroxyl (2623 and 959 cm-1) and a carbonyl group (1693 cm" 1). I t possessed no u l t r a v i o l e t absorption i n the region above.220 my. High r e s o l u t i o n mass spectrometry established the molecular formula to be 0 3 ( ^ 5 3 0 1 + rather than 2 C37H72O5 as proposed by Sosa. It w i l l be noted from the mass spectrum (Figure 5) p a r t i c u l a r l y i n the region below m/e 378, that a regular fragmentation pattern i n which the fragments d i f f e r from each other by 14 mass un i t s , i s observed. This r e s u l t suggested immediately that e q u i s e t o l i c a c i d may possess a long hydrocarbon-like chain with carboxylic acid groups attached to both ends. On t h i s basis structure (23) was an a t t r a c t i v e p o s s i b i l i t y . 114 An attempt to saponify e q u i s e t o l i c acid with potassium hydroxide ROOC(CH2)28COOR 23 R = H 24 R = CH 3 resulted i n recovery of the unchanged acid. Due to the i n s o l u b i l i t y of e q u i s e t o l i c acid an attempt to determine i t s equivalent weight by t i t r a t i o n was unsuccessful. Acetylation of e q u i s e t o l i c a c i d using the procedure reported by 2 Sosa to give the monoacetate yi e l d e d a product, m.p. 119-123°. (Sosa reports m.p. 119°). However further i n v e s t i g a t i o n of t h i s product indicated that i n fac t no ac e t y l a t i o n had occurred. The i n f r a r e d spectra of e q u i s e t o l i c acid and the l a t t e r compound were superimposable. It was therefore c l e a r that Sosa's "acetate" was probably impure s t a r t i n g material. Methylation of e q u i s e t o l i c acid with diazomethane gave a methyl ester, m.p. 84-86°. The carbonyl absorption was now observed at 1748 cm - 1 i n the in f r a r e d spectrum. The NMR spectrum (Figure 7) showed a peak due to the methoxyl protons at T 6.4, a t r i p l e t at x 7.7 a t t r i b u t a b l e to the methylene protons adjacent to the carbonyl group, a broad.multiplet at T 8.39 which i s probably due to the neighbouring methylene protons, and an intense s i n g l e t at T 8.7- representing the remainder of the methylene protons i n the molecule. Since the previous r e s u l t s had already established the molecular formula, C 3 0 H 5 8 O 4 f o r the acid, i t i s cl e a r that the ester must possess e i t h e r the molecular formula, C ^ i ^ c O ^ (monoester, m/e 496) or C3 2Hg 20t t (diester, m/e 510). Mass spectrometry on the above compound indica t e d that the d i e s t e r formulation was correct. Confirmatory evidence i s a v a i l a b l e from the NMR spectrum i n which the integrated areas are as 100 80 60 --40 -20 -llllil lllbllbl H 3 C 0 0 C ( C H 2 ) 2 e C 0 0 C H 3 2 4 IbinWlndbblinn>tlftl(tn.ltl<ltli]d...[li,ili(ih.iJQJiiJ>L.I iLi: kn^nlitiliti b.tH, !ltbL.». Ii. nuui,L, dllX Ji il a 11 ili> IllJll III III ll|»H Illlli O 10 o ca L.jL'ii ilillil J Ii 50 100 150 200 250 m/e 300 350 400 450 Figure 6. Mass Spectrum of Equisetolic Ac id Methyl Ester 500 100 so -60 40 20 xillllllllijillUIIIUlll li.llUllllllllillilIlli,lll JIM lllll.hU! !ilii.lili H 0 0 C ( C H 2 ) 2 8 COOH 2 3 illlli»iillli,,,,llilil,llL,..iillll,..tiii 1,1 LIJUII I 1 „ J U . J i L I L , ,n J I I I J . J1L11 .1 .1,1,1 1 ,,...l,Jii, I1.1.I1.M1 1. gs 1 2 li.,i,.L..!.ll + s ao 50 100 150 200 250 m/e 300 350 400 450 Figure 5. Mass Spectrum of Equisetolic Ac id 1 500 H3COOC(CH2)28COOCH3 2.4 10 Figure 7. NMR Spectrum o f E q u i s e t o l i c A c i d Methyl E s t e r . 117 follows: OCH3 (6 H); -CH^ -C- (4 H);-CH^-C^-C- (4 H) and-CH2- (48 H). 0 0 It i s now concluded that e q u i s e t o l i c acid has the structure 23 and i t s methyl ester i s 24. The same acid has been i s o l a t e d from the spores of Equisetum arvense 41 and i s being studied by Bonnett and co-workers. Other Constituents An attempt to i s o l a t e a l k a l o i d s from the methanol extracts y i e l d e d only an i n s i g n i f i c a n t amount of material. This was not unexpected i n view of the very low a l k a l o i d content reported i n the l i t e r a t u r e f o r other Equisetum species. 40 . , Towers and co-workers extracted Equisetum telmateia spores with water and obtained large amounts of sucrose. This has not been i d e n t i f i e d yet i n our;extracts and i t i s probably i n the aqueous layers. These aqueous iayers from which equisporoside separated contained more equisporoside and several other components as shown by paper chromato-graphy i n butanol-acetic acid-water, 4:1:5. (Figure 8). Some separation was achieved on a polyamide column but no pure.substances were i s o l a t e d . The f r a c t i o n s a l l gave p o s i t i v e f e r r i c chloride t e s t s and undoubtedly contained phenolic compounds. The u l t r a v i o l e t spectra of various mixtures were not very informative. Investigation of the other constituents of Equisetum telmateia spores i s continuing i n our laboratory and should prove very i n t e r e s t i n g . 118 A - Methanol e x t r a c t a f t e r a d d i t i o n of ether and removal of equisporoside (Procedure A, Experimental). B - Aqueous e x t r a c t s o f methanol-petrol l a y e r a f t e r separation of equisporoside (Procedure B, Experimental). Spot Colour V i s i b l e U l t r a v i o 1 brown.' yellow 2 brown yellow 3 yellow blue 4 yellow brown 5 pale yellow blue 6 pale yellow yellow 7 pale yellow blue 8 pale yellow blue 9 pale yellow blue 10 green brown 11 green red 4 = equisporoside F i g u r e 1 8. Other Constituents of Equisetum t e l m a t e i a Methanol E x t r a c t s . 119 EXPERIMENTAL Melting points were.determined on,a Kofler block,and are uncorrected. U l t r a v i o l e t and v i s i b l e spectra were measured i n ethanol on Cary 14 and Cary 11 spectrophotometers. Infrared spectra unless otherwise reported were taken as KBr p e l l e t s on a Perkin-Elmer Model 21 spectrophotometer. Nuclear magnetic resonance spectra were recorded at 60 megacycles per second on a Varian A 60 instrument and at 100 megacycles per second on a V a r i a n HA 100 instrument using the solvents indicated; the values obtained at 100 mega-cycles per second are reported here. The l i n e positions or centres of multiplets are given i n the Tiers x scale with reference to tetramethyl-si l a n e as the i n t e r n a l standard. The m u l t i p l i c i t y , integrated areas and type of protons,are indicated i n parentheses. The mass spectra were recorded with an Atlas CH4 mass spectrometer using the d i r e c t i n s e r t i o n technique, the electron energy being maintained at 70 ev. High r e s o l u t i o n mass spectra f o r the determination of molecular formulae were obtained using an AEI MS9 mass spectrometer. The analyses were performed by,the microanalytical laboratory, University of B r i t i s h Columbia. C o l l e c t i o n of Spores The s t r o b i l i (cones) of Equisetum telmateia were c o l l e c t e d i n the Spring ( A p r i l 14-18, 1966) i n Vancouver and Squamish, B.C. Aft e r allowing the s t r o b i l i to dry f o r 3 or 4 days at room temperature, the spores (4.407 kg.) were shaken out. I s o l a t i o n of E q u i s e t o l i c Acid The spores (4.407 kg.) were extracted (Soxhlet) with ether f o r approximately eight hours ( u n t i l the extracts were c o l o u r l e s s ) . Upon con-, centration of the combined ether extracts.a white s o l i d (8 g.) separated, 120 which a f t e r one c r y s t a l l i z a t i o n from ethyl acetate melted at 127-129°; v J 1 max (nujol): 2623 and 959 (OH), 1693 cm - 1; no u l t r a v i o l e t absorption. Calc. .for C 3 0H 5 8O l +: C, 74.70; H, 12.02; 0, 13.28. Found: C, 75.16; H, 12.20; 0, 13.10. Calc. f o r C^o^O,,: 482.433. Found: 482.437. 2 Acetylation of E q u i s e t o l i c Acid E q u i s e t o l i c a c i d (97.9 mg.) i n anhydrous pyridine (3.6 ml.) and a c e t i c anhydride (1.2 ml.) was heated on a steam bath for 1 hour. The mixture was poured into ice-water and the product f i l t e r e d (90 mg.), m.p. * 119-123°. The i n f r a r e d spectrum of t h i s product showed no esier caroonyl absorption and merely indicated that i t was impure e q u i s e t o l i c acid. Sub-sequent comparison by i n f r a r e d showed these compounds to be i d e n t i c a l , Methylation of E q u i s e t o l i c Acid E q u i s e t o l i c a c i d (519.4 mg.) was dissolved i n a mixture of hot ether (250 ml), benzene (250 ml) and methanol (480 ml) and t h i s s o l u t i o n was allowed to cool slowly to 0°. An ethanolic-ether s o l u t i o n of diazo-methane (3.0 g., 0.71 moles) was poured into t h i s s o l u t i o n and i t was l e f t to stand overnight. Concentration of the s o l u t i o n caused the product to separate. It was c r y s t a l l i z e d from chloroform-methanol (465 mg.), m.p. 84-86°; v (chloroform): 1748 cm"1 (carbonyl); no u l t r a v i o l e t spectrum; max NMR (deuteriochloroform): 6.4 ( s i n g l e t , 6 H, - 0 C H 3 ) , 7.7 ( t r i p l e t , J = 6 c.p.s., 4 H, RCH 2 CH2COOH), 8.4 (broad multiplet 4 H, R C H 2 C H 2 C O O H ) , 8.7 ( s i n g l e t , 48 H, methylene protons). Mass spectrum: (Figure 6). I s o l a t i o n of Equisporoside The spores which had already been extracted with ether were submitted e i t h e r to procedure A or B below: A. The spores (721 g.) were extracted with methanol (Soxhlet) f o r four days, the extracts concentrated to a small volume and ether added. A yellow gum 121 which separated was removed from the s o l u t i o n by decantation, and then dissolved i n water. The f i l t e r e d s o l u t i o n was allowed to stand, during which time a l i g h t brown s o l i d (2; 49 g.) separated. B. The spores (750 g.) were extracted (Soxhlet) f o r ten hours with methanol and,the extract evaporated nearly to dryness. The residue was taken up i n petroleum ether and extracted with water. A f t e r concentration of the com-bined aqueous layers, yellow-brown needles (2.17 g.) separated. Extraction of the spores with methanol for a further ten hours followed by the same work-up gave a further 1.10 g. of yellow-brown needles. Paper chromatography using Whatman No.3 paper and 5% aqueous ac e t i c acid or butanol-acetic acid-water (4:1:5) showed that the yellow-brown s o l i d s were almost,pure but the aqueous solutions from which they separated were complex mixtures of up to s i x components. In the former solvent, equisporoside scarcely moved from the baseline while i n the l a t t e r i t had and Rp value of 0.43. P u r i f i c a t i o n of Equisporoside Equisporoside (4.1 mg.) i n a minimum amount of methanol was spotted on Whatman No.3 paper and developed with butanol-acetic acid-water (4:1:5). Equisporoside (Rp 0.43) and the minor, f a s t e r running impurity (Rp 0.62) appeared as brown bands under u l t r a v i o l e t l i g h t . The band corresponding to equisporoside was cut out,and the material was removed by allowing the paper to remain i n contact'with methanol-water (1:1, 100 ml) f o r 2 hours. The r e s u l t i n g s o l u t i o n was f i l t e r e d and solvent removed to give pure equis-poroside (2.6 mg.). E l u t i o n of the minor component gave 0.3 mg. A det a i l e d i n v e s t i g a t i o n of the u l t r a v i o l e t spectra of equisporoside was c a r r i e d out and.the r e s u l t s were as follows: A E t 0 H (log e): 388 (4.21), 348 (shoulder), fflcLX '277 (shoulder), 261 mp (4.30); a f t e r adding 1 drop of a 2% ethanolic aluminum 122 chloride s o l u t i o n A (log e ) : 455 (4.24), 383 (shoulder), 275 my (4.41); nicix a f t e r addition of excess fused sodium acetate to both.the sample and solvent c e l l s X • (log e ) : 394 (4.12), 351 (shoulder), 280 (shoulder), 266 my m3.x (4.38); 20 minutes a f t e r addition of saturated ethanolic b o r i c acid s o l u t i o n (2 ml.) to the ethanol s o l u t i o n of equisporoside (2 ml.), d i l u t i o n to 10 ml. and addition of excess anhydrous sodium acetate A (log e ) : 404 J max v • J (4.29), 266 (4.36), 250 my (shoulder); a f t e r addition of an 0.03 sodium ethoxide so l u t i o n (2 ml.) to the ethanol s o l u t i o n of equisporoside (2 ml.) and d i l u t i o n to 10 ml. X (log e): 390 very broad peak (3.95), 274 (4.75). Gossypitrin X (log e): 388 (4.20), 350, ;278, 262 my (4.33). 3 6 m 3.x When p_-benzoquinone was added to, an ethanol s o l u t i o n of equisporoside, a dark red-brown colour developed and a p r e c i p i t a t e formed;indicating a . 3 5 p o s i t i v e gossypetone reaction. Equisporoside was dark orange i n aqueous sodium hydroxide turning to brown on standing. In concentrated sulphuric acid i t was a very intense 42 yellow, i n d i c a t i n g i t was probably a f l a v o n o l . 43 The colour reactions on paper also indicated i t was a f l a v o n o l . With no reagent i n v i s i b l e l i g h t i t appeared yellow and under u l t r a v i o l e t l i g h t , brown. A f t e r being exposed to ammonia vapour i t turned a b r i g h t e r yellow i n v i s i b l e l i g h t and a l i g h t brown i n u l t r a v i o l e t l i g h t . Larger amounts of equisporoside were p u r i f i e d by column chromato-graphy on Woelm polyamide-celite (8:2). Equisporoside was not very soluble i n water so a mixture.of methanol-water was used. In a t y p i c a l experiment, equisporoside (788 mg.) was dissolved i n methanol-water (1:1, 40 ml.) and applied to a column of polyamide-celite (10 g.) which had been packed with water. E l u t i o n with methanol-water (1:1) y i e l d e d equisporoside (608 mg., 77% recovery). Equisporoside c r y s t a l l i z e d as. small yellow needles from 123 aqueous a c e t i c acid. A f t e r drying at 85° under vacuum f o r 12 hours, i t melted at 202-204°. An authentic sample of g o s s y p i t r i n (obtained from Geissman), melted at 199-201°; mixed m.p. 199-201°. v (KBr): 3400, 2900 J ' •. max ^ ' (shoulder), 1650, 1605, 1557 and 1510 cm"1; superimposable on that of g o s s y p i t r i n . Preparation of T r i m e t h y l s i l y l Ether of Equisporoside f o r NMR Study Equisporoside (40 mg.) was dissolved i n anhydrous pyridine (3 ml.) and hexamethyl d i s i l a z a n e . ( [ ( C H 3 ) 3 S i ] 2 N H , 0.5 ml) and trimethylchlorosilane (0.5 ml) was added. The solvent and excess reagents were immediately removed under high vacuum and the dry residue was extracted with carbon t e t r a c h l o r i d e . The c l e a r s o l u t i o n obtained b y . f i l t e r i n g o f f the s a l t s was concentrated to a s u i t a b l e volume (0.4 ml) and used d i r e c t l y f o r the NMR measurement. NMR s i g n a l s : 2.34 (multiplet, 1.8 H, H2» and H6>)> 3 - 2 0 (doublet, 1 H, H 5'), 3.73 ( s i n g l e t , with shoulder, 1 H, H 6), 5.2 (broad doublet, J = 6 cps., 1H, anomefic proton of the sugar), 6.20-6.70 (multiplet, 6H, sugar protons), 9.62-9.90(silyl ethers). Hydrolysis of Equisporoside Equisporoside (66.7 mg.) i n methanol (20 ml) and 2N sulphuric acid (20 ml) was refluxed f o r 4 hours, cooled and the s o l u t i o n extracted with ethyl acetate. Evaporation of the combined ethylacetate extracts y i e l d e d the-aglycone, equisporol, (43 mg.). It c r y s t a l l i z e d from aqueous a c e t i c acid as yellow needles, m.p. 301-304° (dec., Kofler preheated to 290°). There was no depression of the melting point when mixed with authentic gossypetin. Equisporol had an Rp value of 0.51 on paper chromatography using butanol-acetic acid-water (4:1:5) as e l u t i n g solvent. Dropwise addition of methanolic potassium hydroxide, to a methanol s o l u t i o n of equisporol 124 caused the o r i g i n a l l y yellow s o l u t i o n to change i n i t i a l l y to blue and then to green. In a c i d i c s o l u t i o n the colour was bright red. v m a x (KBr): 3360, 3270, 1648, 1620 (1602, shoulder), 1573, 1513 cm"1.; superimposable on that of authentic gossypetin; A E t 0 H (log e ) : 384 (4.08), 341 (4.01), 276 (4.21), nicLX 263 my (4.22); immediately a f t e r addition of one drop of a 2% ethanolic aluminum chloride s o l u t i o n X (log e): 450 (3.97), 380 (3.96), 287 my IT13.X (4.26); a f t e r addition of excess fused sodium acetate to both the sample and solvent c e l l s X (log e) ; 328 (4.15), 249 my (4.29); 20 minutes a f t e r IT13.X the addition of saturated ethanolic b o r i c acid s o l u t i o n (2 ml.) to the ethanol s o l u t i o n of equisporol (2 ml.), d i l u t i o n to 10 ml. and addition of excess anhydrous sodium acetate X (log e ) : 412 (3.92), 357 (4.03), 285 (shoulder), 274 my (4.30); a f t e r addition of an 0.03 M sodium ethoxide s o l u t i o n (2 ml.) to the ethanol s o l u t i o n of equisporol (2 ml.) and d i l u t i o n to 10.ml. A (log e): about 387 (broad peak), 287 my (4.26); Gossypetin A (log e): 386 (4.15), 341 (shoulder), 278 (4.23), 262 my (4.26); 3 6 IHclX • NMR (acetone - d 6 ) : 0.93; 1.61, and 1.82 (broad s i n g l e t s each in t e g r a t i n g f o r 1 H, presumably due to three of the hydroxyl protons), 2.19 (doublet, J = 2 c.p.s., 1 H, H 2'), 2.27 (quartet, J = 8.5 c.p.s. and 2 c.p.s., H6' ) , 3.10 (doublet, J = 8.5 c.p.s., H 5'), 3.74 (s i n g l e t , 1 H, H 5). The aqueous layer was ne u t r a l i z e d with barium carbonate, the bulk of the s a l t s removed b y , f i l t r a t i o n and the so l u t i o n passed through a series of columns containing Amberlite IR-120 H C P . medium porosity, strongly a c i d i c cation exchange r e s i n (100 ml.) which had been regenerated with 2N HC1 and backwashed with water u n t i l n e u t r a l , Duolite A-4 anion exchange r e s i n (100 ml.) which had been regenerated with 2N NaOH and backwashed with water u n t i l n e u t r a l , and Amberlite IR-120 H C P . r e s i n (20 ml.). Evaporation of the water at 40°C gave a cl e a r gum (22.09 mg.) which was i d e n t i f i e d as 125 glucose by paper chromatography i n three d i f f e r e n t solvent systems. (See Table 4). The papers were developed by immersing them twice i n s i l v e r nitrate-aqueous acetone ( s i l v e r n i t r a t e (1 g.) i n acetone,(100 ml.) to which just enough water was added to obtain a c l e a r s o l u t i o n ) , once i n ethanolic sodium hydroxide s o l u t i o n u n t i l the spots developed and f i n a l l y i n aqueous sodium thiosulphate s o l u t i o n . In each instance the papers were allowed to dry,between immersions. TABLE 4' R Values of Sugars Solvent Hours developed galactose glucose t unknown sucrose galactose + unknown glucose + unknown formic acid-a c e t i c a'cid-water-ethyl-acetate 1:3:4:18 65 • 14 16.2 16.1 8.3 14.6 16.5 15.8 ethylacetate-pyridine-water i 8:2:1 68 ' 7.3 9.6 9.6 3.6 9.9 7.9 9.6 butanol-ethanol-water 3:1:1 68 6V8 8.0 7.8 4.7 8.2 7.2 8.0 Equisporol Hexamethyl Ether Equisporol (108.2 mg.) was dissolved i n anhydrous acetone (40 ml.) ; anhydrous potassium carbonate (1.2 g.) and dimethyl sulphate (0.5 ml) added, and the mixture refluxed f o r a t o t a l of 36 hours. During the r e f l u x period two addi t i o n a l portions of potassium carbonate and dimethyl sulphate were 126 added. The dark s o l u t i o n was poured into water and extracted with ether, dried (MgSQ4) and the ether removed to give a brown o i l (200 mg) which was chromatographed on alumina (Woelm, neu t r a l , a c t i v i t y IV) (20 g.). E l u t i o n with chloroform yi e l d e d a pale yellow s o l i d (36.8 mgOjin.p. 152-153°. This l a t t e r substance was washed with a small amount of methanol to give a white s o l i d , m.p. 165-168°, which a f t e r r e c r y s t a l l i z a t i o n from methanol-chloro-form melted at 170-171.5°; gossypetin hexamethyl ether (obtained from Geissman ), 166-168°; mixed m.p. 166-169°. v (KBr): 2900, 1620, 1595, J > f m a x v J > 1570 (shoulder), 1510 cm - 1; superimposable on that of gossypetin hexamethyl ether, A E t 0 H (log E ) : 351 (4.32), 272 (4.33), 252 mp (4.35); Gossypetin hexamethyl ether X (log e): 351 (4.34), 273 (4.33), 252 my (4.34). Calc. fflcLX f o r C 2 1 H 2 2 0 8 : 402.131. Found: 402.130. Equisporol Hexaacetate Equisporol (19.73 mg.) was refluxed with a c e t i c anhydride (0.12 ml) and pyridine (2 drops) f o r two hours, poured into water, extracted with ether and the solvent removed. The residue gave a negative f e r r i c chloride t e s t . C r y s t a l l i z a t i o n of the crude product from a c e t i c anhydride-methanol afforded a very poor y i e l d (1 mg.) of the hexaacetate. During the melting point determination, t h i s compound f i r s t s i n t e r e d at 190°, again at 216° 35 and f i n a l l y melted at 226-230°. Gossypetin hexaacetate, si n t e r s at 190°, again at 210° and f i n a l l y melts at 226-228°. Equisporol Pentamethyl Ether Equisporoside (310 mg.) and anhydrous acetone (25 ml.) were placed i n a 50 ml. two-necked f l a s k f i t t e d with a condenser and dropping funnel. The so l u t i o n was s t i r r e d f o r a few minutes, anhydrous potassium carbonate (6 g.) added and the apparatus flushed with nitrogen. A f t e r heating to 127 r e f l u x , dimethyl sulphate (5 ml) was added dropwise over a period of 2.5 hours. Four hours a f t e r the addition of dimethyl sulphate was begun an aliquot gave a p o s i t i v e (olive green) f e r r i c chloride t e s t . A f t e r 8 hours the test was negative. The yellow s o l u t i o n was f i l t e r e d , evaporated to dryness and taken up i n ethanol (15 ml.) and 2N sulphuric acid (24 ml.). A f t e r r e f l u x i n g f o r 2 hours the s o l u t i o n was cooled, extracted with chloro-form and the combined extracts washed with water. Removal of the chloroform l e f t a yellow residue which c r y s t a l l i z e d as pale yellow needles (64.2 mg.) from! ethanol-chloroform, m.p. 249-252°. This compound gave a negative f e r r i c chloride t e s t . A ^ " (log e) : 253 (4.14), 272 (4.18), 349 my (4.13); a f t e r addition of a 0.03 M sodium ethoxide s o l u t i o n (2 ml.) to an ethanol s o l u t i o n of the pentamethyl ether of equisporol (2 ml.) and d i l u t i o n to 10 ml. A (log E ) : 282 (4.43), 387 my (3.96). Calc. f o r C 2 0 H 2 0 O 8 : 388.116; Found: 388.117. 7-hydroxy-3,3 1,4',5,8-pentamethoxyflavone, m.p. 250-251°. EtOH. x 7 0 2 a f t e r addition of sodium ethoxide A : 368, 280 my. max max Pentamethyl e q u i s p o r o l (12.91 mg.) was dissolved i n a c e t i c anhydride (0.12 ml.) and pyridine (5 drops) and allowed to stand overnight at room temperature. Water was added and the s o l u t i o n extracted with ether. The ether was evaporated and the product c r y s t a l l i z e d as yellow needles from ethyl acetate, m.p. 159-161°. Three further r e c r y s t a l l i z a t i o n s provided 6 mg. of a pure substance, m.p. 166-168°. Calc. f o r C 2 2 H 2 2 0 9 : 430.126. 36 Found: 430.128. 7-acetoxy-3,3 1,4 1,5,8-pentamethoxyflavone, m.p. 164-168°. 128 REFERENCES 1. McLean's Magazine, Jan. 1967, p.48. 2. A. Sosa, B u l l . Ste. Chim. B i o l . , 31, 57 (1949). 3. J.D. P h i l l i p s o n and C M e l v i l l e , J . Pharm. Pharmacol., 12_, 506 (1960). 4. R. Casparis and K. Haas, Pharm. Acta Helv., 6_, 181 (1931). 5. F. Gaudard, Pharm. Acta Helv., 4_, 157 (1929). 6. P. Karrer, CH. Eugster and D.K. 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The Chemistry of Flavonoid Compounds, Ed. T.A. Geissman, MacMillan, New York, 1962, p. 122. 34. The Chemistry of Flavonoid Compounds, Ed. T.A. Geissman, MacMillan, New York, 1962, p. 127. 35. A.G. Perkin, Chem. Society Transactions, 650 (1913). 36. T.A. Geissman and C. Steelink, J . Org. Chem., 22, 946 (1957). 37. S. Rajagopalan, R.R. Rao, K.V. Rao and T.R. Seshadri, Proc. Indian Acad. S c i . , 29A, 9 (1949); £ ^ . , 4 3 , 5398a (1949). 38. K.V. Rao and T.R. Seshadri, Proc. Indian Acad. S c i . , 24A, 375 (1946); CA. , 41, 2735d (1947) . 39. R.M. Horowitz, J . Am. Chem. S o c , 79, 6561 (1957). 40. G.H.N. Towers, pri v a t e communication. 41. R. Bonnett et a l . , p rivate communication. 42. The Chemistry of Flavonoid Compounds, Ed. T.A. Geissman, MacMillan, New York, 1962, p.72. 43. The Chemistry of Flavonoid Compounds, Ed. T.A. Geissman, MacMillan, New York, 1962, p. 51. 44. T.J. Mabry, J . Kagan and H. Rb'ster, "NMR Analysis of Flavonoids", The University of Texas P u b l i c a t i o n , Number 6418, September 15, 1964, Austin, Texas. 

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