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The synthesis of 11-aza pregnane derivatives Sood, Rattan Sagar 1967

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THE SYNTHESIS OF 11-AZA PREGNANE DERIVATIVES by RATTAN SAGAR SOOD B.Sc.(Hons.), The U n i v e r s i t y of Panjab - I n d i a , 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry V?e accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1957 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t t he L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e 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 by the Head o f my Department or by hils 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 not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f CfrfcA-y/sTRY The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 2 2- ^ . ABSTRACT The Beckmann rearrangement of the oxime of l l - o x o - 3 C< ,20-dihydroxy-C-nor pregnane (54) provided the corresponding lactam (56). O x i d a t i o n of the l a t t e r w i t h Jones reagent f u r n i s h e d the 3-keto-alcohol (57), which upon i t s conversion t o the acetate d e r i v a t i v e (58) fo l l o w e d by bromination and dehydrobromination y i e l d e d ll-aza-20-acetoxy-pregn-4-ene-3,12-dione (60). H y d r o l y s i s 4 of the l a t t e r to the ^> -3-keto a l c o h o l (62) f o l l o w e d by o x i d a t i o n f u r n i s h e d the d e s i r e d ll-aza-pregn-4-ene-3,12,20-t r i o n e (63). The s y n t h e t i c sequence provid e s the f i r s t examples of 11-aza s t e r o i d analogues i n which r i n g C i s 4 a s i x membered and r i n g A possesses the ^^-3-keto moiety. TABLE OF CONTENTS Page T i t l e Page - i A b s t r a c t i i T a b l e o f C o n t e n t s . i i i L i s t o f F i g u r e s i v Acknowledgements v I n t r o d u c t i o n 1 D i s c u s s i o n 21 E x p e r i m e n t a l 49 R e f e r e n c e s 59 i v LIST OF FIGURES F i g u r e Page 1 3 2 . 7 3 8 4 9 5 11 6 12 7 . 13 8 13 9 . 15 10 16 11 18 12 22 13 24 14 25 15 27 16 28 17 30 18 . 35 19 37 20 38 21 41 22 42 23 43 24 45 25 46 26 47 V ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e thanks and a p p r e c i a t i o n t o D r . James P . Kutney f o r h i s e x p e r t g u i d a n c e and e n c o u r a g e -ment d u r i n g the c o u r s e o f t h i s p r o j e c t and h i s c o n s t a n t h e l p i n the p r e p a r a t i o n o f t h i s t h e s i s . Thanks a r e a l s o due to Smi th K l i n e and F r e n c h L a b o r a t o r i e s , P h i l a d e l p h i a f o r f i n a n c i a l a s s i s t a n c e and a generous g i f t o f the oxime o f l l - o x o-3 o(,20Tdihydroxy-C - n o r p r e g n a n e . INTRODUCTION The f i e l d of s t e r o i d s has provided a host of i n t e r e s t i n g problems such as i s o l e i t i o n of s t e r o i d e n t i t i e s from n a t u r a l sources, t h e i r s t r u c t u r a l e l u c i d a t i o n , b i o c h e m i c a l s t u d i e s , p r e p a r a t i o n of many analogues of p o t e n t i a l m e d i c i -n a l i n t e r e s t and search of s u i t a b l e methods f o r i n d u s t r i a l p r oduction of s t e r o i d drugs. During t h i s unending process many problems of fundamental organic chemistry i n v o l v i n g r e a c t i o n mechanistic and stereochemical aspects have been s u c c e s s f u l l y solved and many new compounds of di v e r s e pharmacological a c t i v i t y and th e r e a p e u t i c i n t e r e s t have been discovered. Aza s t e r o i d s , that i s n i t r o g e n c o n t a i n i n g s t e r o i d s are r e c e i v i n g concerted a t t e n t i o n s of chemists and pharm-1 2 a c o l o g i s t s a l x k e . Alauddin and M a r t i n Smith ' and M a r t i n Smith and Sugrue ^ have reviewed the b i o l o g i c a l a c t i v i t y of s t e r o i d possessing n i t r o g e n atoms, both i n n a t u r a l and s y n t h e t i c o r i g i n . Out of numerous s y n t h e t i c aza s t e r o i d s * prepared, many have shown i n t e r e s t i n g b i o l o g i c a l a c t i v i t y . 4 Dorfman has reported some i n t e r e s t i n g f i n d i n g s i n t h i s 5 regard. A more d e t a i l e d d i s c u s s i o n by Doorenbos i n d i c a t e d t h a t aza, s t e r o i d s indeed provide a r i c h source of new drugs agai n s t c e r t a i n diseases. The a c t i v i t y of aza s t e r o i d s i s due to t h e i r a b i l i t y t o b l o c k the b i o s y n t h e s i s of c h o l e s t e r o l ( F i g . 1 ) a p p a r e n t l y e i t h e r by i n h i b i t i n g the conversion of 3-hydroxy-3-methyl-glutaryl coenzyme A i n t o mevalonic a c i d or by i n h i b i t i n g the r e d u c t i o n of desmosterol to c h o l e s t e r o l . Aza s t e r o i d s may be of c l i n i c a l value f o r the treatment of a t h e r o s c l e r o s i s , a disease a s s o c i a t e d w i t h abnormally high serum c h o l e s t e r o l l e v e l s . A number of diaza s t e r o i d s having n i t r o g e n atoms i n the s i d e c h a i n were found to be extremely potent i n h i b i t o r of c h o l e s t e r o l b i o s y n t h e s i s i n animals. Further s t u d i e s ^ i n d i c a t e d t h a t mono aza s t e r o i d s , 24 and 25-aza c h o l e s t e r o l were the most a c t i v e of these t e s t e d . T h i s i s c o n s i s t e n t w i t h the r e s u l t s of more recent t e s t s w i t h 2 0-aza-24-oxa ( 1 ) , 2 2-oxo-25-aza ( 2 ) and 2 0-oxo - 2 1-nor-25-aza (3) c h o l e s t e r o l , which i n d i c a t e d t h a t 24 and 2 5-aza c h o l e s t e r o l and r e l a t e d compounds are potent hypocholesterolemic agents r e g a r d l e s s of whether C 2 0 a n < 3 C 2 2 a r e P r e s e n t a s carbon, oxygen or n i t r o g e n atoms. An absolute r e d u c t i o n of c h o l e s t e r o l and an almost constant t o t a l s t e r o i d l e v e l was observed. ^ R e s u l t s of s t r u c t u r a l a c t i v i t y r e l a t i o n s h i p s t u d i e s suggest-ed that a r e c e p t o r s i t e w i t h dimensions s p e c i f i c f o r c h o l e s t e r o l i s i n v o l v e d ® and a non s t e r o i d a l analogue (4) 9 was found t o be i n a c t i v e when te s t e d on r a t s . Ao C H2.6H Coo H 3-hy droxy-3-methy1 g l u t a r y l CoA mevalonic a c i d squalene zymosterol desmosterol c h o l e s t e r o l F i g . 1. Important Intermediates i n the B i o s y n t h e s i s of C h o l e s t e r o l . 4 3 4 Subsequent c l i n i c a l s t u d i e s demonstrated t h a t 22,25-diaza c h o l e s t a n o l (5) ^ and 20,25-diaza c h o l e s t e r o l (6) 1 1 * I 2 ' I 3 causes 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-3 j3—methyl-5°< -cholestane (7) and N-N -dimethyl-4-aza-3 [Z—benzyl-5 -cholestane i o d i d e (8) a l s o i n h i b i t e d the r e d u c t i o n of desmosterol 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 of these were 3 -N-ethanolamino-cholestane (9) and N-phenyl-4-aza-5°<-cholestan-3-one (10) and they are u s e f u l i n reducing s c l e r o t i c t e n s i o n s i n l a b o r a t o r y animals without r e s o r t i n g t o a high l e v e l of c h o l e s t e r o l i n the d i e t . 4*- 1 0 In a d d i t i o n t o the p r o p e r t i e s a l r e a d y mentioned aza s t e r o i d s have been found to possess a n a b o l i c , a n t i -b a c t e r i a l , a n t i f u n g a l , hypotensive, coronary a r t e r y d i l a t i n g , CNS s t i m u l a n t , CNS depressant, neuromuscular b l o c k i n g , a n t i -14 flammatory and androgenic a c t i v i t i e s . The i n t r o d u c t i o n of a n i t r o g e n atom i n t o the s t e r o i d nucleus has a t t r a c t e d the a t t e n t i o n 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 tho* s t e r o i d a l nucleus. An e x c e l l e n t review of the l i t e r a t u r e up t o 1962 d e s c r i b e s the v a r i o u s methods t h a t have been used. ^ The more recent work w i l l be d i s cussed i n the f o l l o w i n g t a c t i o n of the t h e s i s . The Beckmann rearrangement has been u t i l i z e d f r e q u e n t l y i n these syntheses and a recent review a r t i c l e on the uses of t h i s r e a c t i o n and the Schmidt r e a c t i o n has been p u b l i s h e d . ^ Since the Beckmann rearrangement was used i n our own work a l s o , i t s a p p l i c a t i o n s i n the s y n t h e s i s of aza s t e r o i d s w i l l be discussed i n greater d e t a i l . The Beck-mann rearrangement of an oxime of a 1-keto s t e r o i d (11) and the 1-keto-A-nor d e r i v a t i v e (14), y i e l d e d 1-aza-A-horao-cholestan-2-one (12) and l-aza-cholestan-2-one (15) r e s p e c t i v e l y . The 1,10-seco-l-cyano compounds, (13) and (16) 17 were a l s o obtained v i a abnormal rearrangements ( F i g . 2 ) . The oxime of 5 <* -cholestan-2-one (17) provided a mixture of 2 and 3-aza-A-homo lactams (18) and (19) which were reduced t o the corresponding 2 and 3-aza-A-homo-5 -cholestanes (20) and (21) ( F i g . 3 ) . 1 7 On the other hand the Beckmann rearrangement of the oxime of A-nor-5 <*• -cholestan-2-one (22) l e d t o an inseparable mixture of 2-aza-5 <* -cholestan-3-one (23) and 3-aza-5 c< -cholestan-2-one (24). They were separated 7 CgHn 11 12 13 14 15 c s H n - f - r 1 16 F i g . 2. S y n t h e s i s of 1-Aza and 1-Aza-A-Homo S t e r o i d s as t h e i r d i c h o l o r o d e r i v a t i v e s which were c h a r a c t e r i s e d by t h e i r r e a c t i v i t y w i t h c o l l i d i n e ( F i g . 3 ) . *® S i m i l a r l y 5 cx -cholestan-3-one-oxime (25) f i e l d e d an inseparable ,-mixture of 3 and 4-aza-A-homo d e r i v a t i v e s (26) and (27) 19 4 ( F i g . 4 ) . The ^ - 3 - k e t o s t e r o i d oxime (28) however gave o n l y unsaturated lactam (29) which a f t e r hydrogenation and l i t h i u m aluminum hydride r e d u c t i o n y i e l d e d the 3-aza-" 20 21 A-homo s t e r o i d analogue ( 3 0 ) . ( F i g . 4) ' F i g . 3. Sy n t h e s i s of 2 and 3-Aza s t e r o i d s . 9 28 P. 29 F i g . 4. Synth e s i s of 3-and 4-Aza-A-Homo S t e r o i d s . The s y n t h e s i s of 5-aza-A-nor- B -horao-cholestan (33) 22,23 and-5-aza-A-nor-cholestan (36) 2 4 » 2 5 v i a a Beckmann rearrangement of the ke t o - e s t e r oxime (31) and (34), c y c l i -z a t i o n of the r e s u l t i n g lactams (32) and (35) using phosphorus o x y c h l o r i d e , boron t r i f l u o r i d e , or phosphorus pentaoxide i n toluene, and i t s a p p l i c a t i o n t o the conversion of te s t o s t e r o n e acetate (37) i n t o A-nor- & -homo-S-aza-androstan-17/?—ol(38) have r e c e n t l y appeared i n l i t e r a t u r e ( F i g . 5 ) . The Beckmann rearrangement has a l s o been used t o synthesize a 6 - a z a - S -27 28 homo ste.-roid (39), a 7-aza- B -homo s t e r o i d (40) and a 7-aza s t e r o i d (41) 2 9 ( F i g . 6) In past years many other methods have been employed i n the syntheses of aza s t e r o i d s . S i x aza s t e r o i d s have been s u c c e s s f u l l y prepared using the modified C u r t i u s r e a c t i o n ( F i g . 7 ) . 30,31 E l i m i n a t i o n of the C 3 oxygen f u n c t i o n was Overcome by reducing the intermediate iso-cyanate (42) w i t h l i t h i u m aluminum hydride t o y i e l d (44), or c a t a l y t i c a l l y to 32 y i e l d (45). To e x p l a i n these r e s u l t s an e q u i l i b r i u m between the isocyanate (42) and the lactone (43) was suggested. ( F i g . 8) I n t r o d u c t i o n of a n i t r o g e n atom i n t o the s t e r o i d nucleus has a l s o been accomplished by the c y c l i z a t i o n of intermediate k e t o - a c i d s w i t h amines. T h i s has been r e c e n t l y 33 34 a p p l i e d t o the s y n t h e s i s of 4-hydroxy, 4-amino, 4-alkylamino and 4,6 [% -dimethyl d e r i v a t i v e s of 4-aza s t e r o i d s . In our own l a b o r a t o r y t h i s method was a p p l i e d t o the s y n t h e s i s of 6-aza-cholestane ( 4 6 ) , 3 ^ 6-aza-androstane ( 4 7 ) 3 8 and 6-aza-pregnane ( 4 8 ) 3 9 d e r i v a t i v e s ( F i g . 9) and t h i s , along w i t h a s i m i l a r one c a r r i e d out independently 40 elsewhere provided the f i r s t general s y n t h e s i s of F i g . 5 . S y n t h e s i s of 5 - A z a S t e r o i d s . F i g . 6. Synthesis of 6-and 7-Aza Steroids. C ? r t n 13 F i g . 7 . Synthesis of 6-Aza s t e r o i d s v i a a M o d i f i e d C u r t i u s R e a c t i o n . F i g . 8. S y n t h e s i s of 6-Aza S t e r o i d s . 14 r i n g B -*~aza s t e r o i d s . T h i s work was subsequently extended t o the f i r s t s y n t h e s i s of the 11-aza s t e r o i d a l sapogenin (49), 4 1 and f i n a l l y t o 11-aza pregnane s e r i e s ( 5 0 ) . 4 2 (Fig.lO) Nitrogen atoms have a l s o been introduced i n t o the v a r i o u s p o s i t i o n s of the side chain of c h o l e s t e r o l and t h i s 43 subject has been reviewed r e c e n t l y . The syntheses d e s c r i b e d so f a r have i n v o l v e d a m o d i f i c a t i o n of the s t e r o i d nucleus. A number of t o t a l syntheses of aza s t e r o i d s have been r e p o r t e d i n the l a s t few years. T o t a l s y n t h e s i s of DL-8-aza-estrone methyl ether (51) and r e l a t e d s t e r o i d a l systems (52) has been accomplished. Separation of 14 and 14 j- isomers 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 ( F i g . 11). ^ 4 A p p l i c a t i o n of t h i s approach t o the s y n t h e s i s of D-homo-8-aza s t e r o i d (53) was u n s u c c e s s f u l . S i m i l a r l y the t o t a l s y n t h e s i s of 13-aza-1 8 - n o r - e q u i l i n i n - m e t h y l ether has been re p o r t e d independently v 4 - : * 45,46 by two groups of workers. I t i s obvious t h a t the above methods provide a v a r i e t y of aza s t e r o i d s . To keep t h i s manuscript w i t h i n reasonable dimensions of length, i t i s not p o s s i b l e to d i s c u s s the r e l a t i v e merit of each of these methods. * t i s s u f f i c i e n t t o s t a t e t h a t most of these methods are s u i t a b l e f o r the syn-t h e s i s of r i n g A and D aza s t e r o i d s but very o f t e n cannot be F i g . 9. C y c l i z a t i o n of Keto-Esters t o 6-Aza S t e r o i d s . 16 used t o prepare aza s t e r o i d s possessing the heteroatom i n r i n g s B or C. In f a c t s e v e r a l years ago when research i n our l a b o r a t o r y was i n i t i a t e d i n t h i s area, there was no r i n g C aza s t e r o i d known i n which a normal s t e r o i d a l s keleton had been r e t a i n e d . I t was of i n t e r e s t t o develop a success-f u l sequence l e a d i n g to these novel substances. T h i s aim was f i n a l l y accomplished and e s s e n t i a l f e a t u r e s of t h i s approach have been o u t l i n e d i n F i g . 10. T h i s sequence as w e l l as a s i m i l a r one independently conceived by Rakhit 47 and Engel p r o v i d e d the f i r s t general synthesis of r i n g C aza s t e r o i d . Although t h i s approach o u t l i n e d i n F i g . 10 f o r the sy n t h e s i s of 11-aza s t e r o i d s i s very a t t r a c t i v e since degradation of the sapogenin side chain of 11-aza-hecogenin 48 acetate by a known procedure p r o v i d e s a d i r e c t entry i n t o r i n g C a z a - s t e r o i d s of the pregnane s e r i e s , i t s u f f e r s from the f o l l o w i n g s e r i o u s drawbacks. (a) F i r s t l y , the y i e l d of the key r e a c t i o n , namely the o z o n o l y s i s of 9, 11-dehydro-hecogenin acetate i s very poor. 8 9 (b) The r e d u c t i o n of double bond i n -11-aza-hecogenin acetate presented another d i f f i c u l t y . I t i s w e l l known from the normal s t e r o i d s e r i e s that t h i s p a r t i c u l a r double bond i s d i f f i c u l t to hydrogenate and the necessary c o n d i t i o n s * } i 52 53 ' F i g . 1 1 . S y n t h e s i s of DL-8-Aza Estrone Methyl Ether and R e l a t e d Systems. would destroy the s p i r o k e t a l system. T h i s d i f f i c u l t y , of course, was l a t e r e l i m i n a t e d by u t i l i z i n g a modified proce-42 dure. Although some evidence was provided t o i n d i c a t e t h a t during r e d u c t i o n the normal stereochemistry (8/3 ,9<K ) p e r s i s t s at these stereochemical c e n t r e s , c o n c l u s i v e evidence c o u l d not be obtained since c o r r e l a t i o n t o the con v e n t i o n a l s t e r o i d a l system was not d i r e c t l y f e a s i b l e . (c) Since the r e a c t i o n sequence o u t l i n e d i n F i g . 10 f o r the syntheses of 11-aza s t e r o i d s u t i l i z e s the A/B t r a n s s e r i e s , t h i s does not represent a conventient system t o introduce 4 the < i 3-3-keto moiety i n t o the r i n g A of the 11-aza-pregnane d e r i v a t i v e s . T h i s l a t t e r chromophore i s of i n t e r e s t as e s s e n t i a l l y a l l p h y s i o l o g i c a l l y potent s t e r i o d s i n the androgenic, p r o g e s t a t i o n a l and c o r t i c a l hormone s e r i e s 4 possess ^>-3-keto moiety. (d) Since i t i s necessary to degrade the sapogenin side c h a i n of 11-aza-hecogenin acetate t o get i n to the pregnane s e r i e s , t h i s makes the whole r e a c t i o n sequence extremely lengthy. Therefore i t was of i n t e r e s t t o develop a b e t t e r sequence f o r the sy n t h e s i s of 11-aza-pregnane d e r i v a t i v e s . T h i s aim was f i n a l l y accomplished and e s s e n t i a l f e a t u r e s of t h i s new approach are o u t l i n e d i n F i g . 12. T h i s work,in general provided the f i r s t examples of r i n g C-aza-pregnane derivatives which possess a ^ ^ - 3-keto moiety. r 21 DISCUSSION With an object i n mind to prepare aza s t e r o i d s An the pregnane s e r i e s w i t h the heteroatom i n the r i n g C an intermediate s u i t a b l e f o r t h i s purpose was the oxime of l l - o x o - 3 c< , 20-dihydroxy-C-nor pregnane (54). T h i s compound could be obtained i n reasonable q u a n t i t i e s through the k i n d co-operation of Smith K l i n e and French L a b o r a t o r i e s , P h i l a d e l p h i a , U.S .A. and i t s u t i l i z a t i o n was prompted by c e r t a i n advantageous f e a t u r e s . (1) I t c o n t a i n s a c i s A/B f u s i o n which would a l l o w us t o brominate the corresponding 3-ketone s e l e c t i v e l y a t the 4 p o s i t i o n . Subsequent dehydro bromination of l a t t e r would 4 introduce the d e s i r e d ^_ -keto moiety i n t o the r i n g A . T h i s l a t t e r chromophore was of i n t e r e s t since i t i s present i n most of the a c t i v e s t e r o i d hormones. (2) I t c o n t a i n s a l l the necessary f u n c t i o n a l groups which c o u l d a l l o w e n t r y i n t o the pregnane s e r i e s . Of s e v e r a l a t t r a c t i v e pathways p o s s i b l e the f o l l o w -i n g scheme was given s e r i o u s c o n s i d e r a t i o n ( F i g . 12). Our f i r s t c o n s i d e r a t i o n was t o convert the oxime of l l - o x o - 3 c< , 20-dihydroxy-C-nor pregnane (54) t o the corresponding lactam (56). The Beckmann rearrangement has been u t i l i z e d very f r e q u e n t l y f o r t h i s purpose and i t 6 2 6 3 F i g . 12. Synthesis of 11-Aza Pregnane D e r i v a t i v e s . 23 was s e l e c t e d i n t h i s i n s t a n c e . Treatment of oxime (54) w i t h t r i f l u o r o a c e t i c anhydride i n anhydrous methylene c h l o r i d e gave a white c r y s t a l l i n e residue (55) which without any f u r t h e r p u r i f i c a t i o n was s a p o n i f i e d w i t h aqueous sodium carbonate s o l u t i o n . The r e s u l t i n g product from t h i s r e a c t i o n was c r y s t a l l i s e d from acetone-hexane t o provide the d e s i r e d lactam (56), m.p. 237-238 C . The s t r u c t u r a l a s s i g n -ment of the l a t t e r compound was made on the b a s i s o f the f o l l o w i n g evidence. A strong band i n the I R spectrum -1 a t 1630 cm was r e a d i l y a t t r i b u t e d t o a lactam carbonyl w h i l e the presence of the - NH f u n c t i o n was confirmed by a sharp band a t 3380 cm and a s i g n a l a t T 4.12 (IH) i n the nuclear magnetic resonance (NMR) spectrum ( F i g . 13). Furthermore the parent lactam system present i n (56) e x h i b i t e d a negative Cotton e f f e c t curve i n the o p t i c a l r o t a t o r y d i s p e r s i o n measurements ( F i g . 14). Elemental a n a l y s i s on the c r y s t a l l i n e compound supported the assigned formula and f i n a l c o n f i r m a t i o n was obtained from the mass + s p e c t r a l molecular weight d e t e r m i n a t i o n . ( M 335) I t was now necessary t o o x i d i s e the hydroxyl group a t i n (56) w h i l e l e a v i n g the C^Q -OH i n t a c t . Jones' 49 : reagent i s w e l l known as a m i l d oxidant and i t was s e l e c t -ed f o r t h i s purpose. Indeed when the d i o l (56) was t r e a t e d 24 25U 8 In o o •ft %0 ^1-rH O •H O o o o 0 ° — r o —t— o o —r-o 0 under these c o n d i t i o n s , the crude product upon chromatograph-i c p u r i f i c a t i o n a f f o r d e d the d e s i r e d k e t o - a l c o h o l (57), as a c r y s t a l l i n e substance, M.P. 242.5-244 C. The s p e c t r a l data provided strong support f o r the success of the r e a c t i o n and was i n complete agreement w i t h the p o s t u l a t e d s t r u c t u r e (57). For example a strong band at 1703 cm i n the IR spectrum was r e a d i l y a t t r i b u t e d t o the carbonyl group while a weak, broad band at 3240 cm was compatible w i t h the presence of the h y d r o x y l group. The NMR spectrum was p a r t i c u l a r l y i n s t r u c t i v e and i n accord w i t h the above experiment. Apart from r e t a i n i n g the C^Q hydroxyl s i g n a l a t f 3,14, i t showed the complete disappearance of protons i n the r e g i o n T 6.30-6.64 ( F i g . 15). The o p t i c a l r o t a t o r y d i s p e r s i o n curve of ll-aza-20-hydroxy-pregnan-3, 12-dione (57, F i g . 16) i n d i c a t e d both the c o n t r i b u t i o n of the s a t u r a t e d carbonyl group (trough at 307 mu and peak a t 279 mu) and the lactam carbonyl (trough at 237 mu). F i n a l l y elemental a n a l y s i s and a mass s p e c t r a l molecular weight determination proved the v a l i d i t y of s t r u c t u r e (57) beyond any doubt. In view of the subsequent r e a c t i o n s i t was consider worthwhile t o p r o t e c t the hydroxyl group i n (57) and thereby i t s conversion to the acetate d e r i v a t i v e was accomplished by r e a c t i o n w i t h p y r i d i n e and a c e t i c anhydride. The acetate F i g . 16 (58) was obtained d i r e c t l y af v.er chromatographic p u r i f i c a t i o n of the r e a c t i o n product on d e a c t i v a t e d alumina. The I R spectrum of the acetate showed complete absence of absorp--1 t i o n i n the h y d r o x y l r e g i o n w h i l e a new peak a t 1715 cm c o u l d be r e a d i l y a t t r i b u t e d t o the e s t e r c a r b o n y l group. NMR data, p a r t i c u l a r l y a sharp spike at "7*7.98 and a narrow q u a r t e t centred at T4.96 provided f u r t h e r c onfirm-a t o r y evidence f o r t h i s compound ( F i g . 17). Although the s p e c t r a l data and t h i n l a y e r chromatography (TLC) behavior i n d i c a t e d t h a t t h i s compound (58) was pure, we were unsuccess-f u l i n o b t a i n i n g t h i s i n c r y s t a l l i n e form. For a n a l y t i c a l purposes a s m a l l amount of t h i s m a t e r i a l was p u r i f i e d by p r e p a r a t i v e TLC and a pure sample was obtained as a white amorphous s o l i d . The mass spectrum of t h i s compound i n d i c a t e d a molecular i o n peak at 375 which was i n agree-ment w i t h the molecular formula, C „ H „ NO, . 22 33 4 Our next c o n s i d e r a t i o n was t o introduce a C. - C 4 5 double bond i n t o the keto-acetate (58). The procedure f o r t h i s purpose was chosen from the p u b l i s h e d l i t e r a t u r e on the 50 corresponding s t e r o i d analogues. Brommation of l l - a z a - 2 0 acetoxy-5 -pregnan-3,12-dione (58) i n b u f f e r e d a c e t i c a c i d s o l u t i o n at room temperature y i e l d e d a mixture of s e v e r a l products as i n d i c a t e d by TLC examinations of 31 the crude reaction mixture. The major product was expect-ed to be 4 p -bromo-ll-aza-20-acetoxy-5 J3> -pregnan-3,12-dione (59) i n accordance with the precedent a v a i l a b l e i n 51 the l i t e r a t u r e . In order to e s t a b l i s h t h i s more conclusive-52 l y one could normally use i n f r a - r e d spectroscopy. Jones observed that a single equatorial bromine atom increases the frequency of the carbonyl maximum by 15 - 17 cm i n the i n f r a - r e d region. Unfortunately a comparison of the i n f r a red spectra of 4 ji> -bromo-ll-aza-20-acetoxy-5 y 3 -pregnan-3,12-dione (59) with the corresponding saturated 3-ketone (58) was not possible since the i n t e r f e r i n g absorption due to actoxy group appears i n the expected region of bromo-ketone absorption. An additional d i f f i c u l t y arose when i t was r e a l i s e d that the crude bromo-ketone was not s u f f i c i e n t l y stable for p u r i f i c a t i o n . Therefore the bromination mixture was now subjected to dehydrobromination conditions. I t has been reported by many workers that the action of organic bases such as pyridine, c o l l i d i n e , sodium acetate etc., on 4-bromo-keto steroids generally produces the 4 53 54 55 -3-keto steroids i n very poor y i e l d s . ' It i s believed that one of the factors responsible for t h i s poor y i e l d i s the formation of two diastereoisomeric 4-bromo derivatives (4c<and 413 ) . Only one of these compounds (4 °< ) could 32 undergo f a c i l e t r a n s e l i m i n a t i o n of hydrogen bromide. Mattox and K e n d a l l ^ introduced a method f o r the conversion * 4 of 4-broroo-3-keto s t e r o i d s i n t o ---3-ketones through the intermediacy of a hydrazone d e r i v a t i v e which l o s t hydrogen bromide spontaneously. For example formation of a 2 , 4 - d i n i t r o phenyl-hydrazone d e r i v a t i v e and e l i m i n a t i o n of HBr proceeds i n e x c e l l e n t y i e l d s . Subsequent cleavage 4 . . of the resulting__.-3-hydrazone w i t h p y r u v i c a c i d provides 4 the desired__*-3-keto s t e r o i d s . A t h i r d method f o r the conversion of 4-halo-3-. 4 keto s t e r o i d s i n t o the corresponding __-3-ketones has been 5 7 developed by Holysz. T h i s procedure i n v o l v e s heating 4-halo-3-keto s t e r o i d s w i t h c e r t a i n m e t a l l i c h a l i d e s p a r t i c u l a r l y l i t h i u m c h l o r i d e , i n a amide sol v e n t such as dimethylformamide. Holysz has a l s o suggested a p l a u s i b l e mechanism f o r the e l i m i n a t i o n of hydrogen bromide i n 4-halo-3-keto s t e r o i d s where the C.-bromine and C -hydrogen 4 5 atoms possess a c i s r e l a t i o n s h i p . Both of the a l t e r n a t i v e pathways described above are e q u a l l y a t t r a c t i v e but since b e t t e r y i e l d s were g e n e r a l l y c p r e p o r t e d w i t h the Holysz procedure, we a l s o s e l e c t e d t h i s method f o r our own purpose. Indeed when the crude brominated product was heated w i t h a mixture of l i t h i u m c h l o r i d e and ' • 33 l i t h i u m carbonate i n dimethylformamide, under a n i t r o g e n atmosphere f o r 10 hours, we obtained a white c r y s t a l l i n e product. This m a t e r i a l as expected was found t o be a mixture of s e v e r a l components as shown by TLC examination of the crude r e a c t i o n product. C a r e f u l chromatographic p u r i f i c a t i o n of t h i s m a t e r i a l on d e a c t i v a t e d alumina and then two subseq-uent c r y s t a l l i z a t i o n s from methanol a f f o r d e d the d e s i r e d 4 o ^ - 3 - k e t o n e (60), M.P. 112-115 C (35% o v e r a l l y i e l d from the corresponding saturated 3-ketone,58). I t was however r e a l i s e d at a l a t e r stage t h a t a b e t t e r p u r i f i c a t i o n c o u l d be accomplished by h y d r o l y s i n g the acetate (60) t o the corresponding a l c o h o l (62) which f o r t u n a t e l y happens to be the next step i n our sequence. I t must be emphasised here t h a t the purest m a t e r i a l which we could o b t a i n always i n d i c a t e d a t r a c e i m p u r i t y of some other m a t e r i a l running extremely c l o s e t o the major component on TLC. A l l attempts t o separate these two com-ponents were of no a v a i l and indeed the mixture behaved as a homogeneous compound i n a l l c r i t e r i a except on TLC. We wish here t o emphasise th a t t h i s m a t e r i a l (60) may represent a mixture of two isomers epimeric a t C 2 Q . This s p e c u l a t i o n was l a t e r confirmed when both these compounds gave a s i n g l e component on o x i d a t i o n of the subsequent a l c o h o l s (62). 34 The s p e c t r a l data of the p u r i f i e d acetate was c o n s i s t e n t w i t h the assigned s t r u c t u r e (60). The NMR spectrum o f the l a t t e r d i s p l a y e d a sharp s i n g l e t centred at T 4 . 1 2 due t o the o l e f i n i c proton a t ( F i g . 18) While the UV spectrum (Amax. 236 mu) was compatible w i t h the presence of the oi,j3 -unsaturate 1 d ketone. The success o f the r e a c t i o n was f u r t h e r apparent i n the IR spectrum which possessed an a d d i t i o n a l shoulder at 1665 cm as w e l l as the lactam absorp-t i o n a t 1657 cm. The oi ,y3 -unsaturated ketone chromophore present i n ll-aza-20-acetoxy-preg-4-ene-3,12-dione (60) e x h i b i t e d a m u l t i p l e Cotton e f f e c t i n the 300-380 mu r e g i o n o f the ORD curve ( F i g . 19). The negative c o t t o n e f f e c t a s s o c i a t e d w i t h the lactam group at 235 mu was completely quenched by the intense p o s i t i v e background r o t a t i o n a s s o c i a t e d w i t h the 17-77 t r a n s i t i o n of the -c = c - c = 0 group ( a l s o c a l l e d the K band) i n (60). The l a t t e r a b s o r p t i o n now appeared at 232 mu ( F i g . 2 0). Although t e c h n i c a l d i f f i c u l t i e s sometimes hamper f a c i l e e v a l u a t i o n of the TT 77 t r a n s i t i o n i n cX ~> y3 -unsaturated ketones because of the higher absorption c o e f f i c i e n t s charact-e r i s i n g t h i s band, recent improvements i n the instrumentation now p e r m i t e a s i e r access t o t h i s important s p e c t r a l r e g i o n . The ,K-band of a number of /3 -unsaturated ketones has 36 been i n v e s t i g a t e d w i t h a spectropolarimeter equipped w i t h 5 9 a Faradcy e f f e c t type a n a l y z e r . On our own instrument the K-band was r e s o l v e d o n l y s u f f i c i e n t l y t o a l l o w us t o draw a t e n t a t i v e c o n c l u s i o n . Further c o n f i r m a t i o n would be r e q u i r e d on an instrument equipped w i t h a Faraday e f f e c t type analyzer before any d e f i n i t e comments can be made. F i n a l l y the molecular formula assigned was supported by elemental a n a l y s i s and a mass s p e c t r a l molecular weight deter-+ mination (M 373). No se r i o u s attempts were made to i s o l a t e the v a r i o u s side products formed i n the above r e a c t i o n since there was a lar g e number of components present and only a small amount of m a t e r i a l was a v a i l a b l e t o us. Only a small amount of one a d d i t i o n a l compound (one spot on TLC) was i s o l a t e d during chromatography of the crude product and has been t e n t a t i v e -l y l y assigned t o be ^ -ll-aza-20-acetoxy-pregnen-3,12-dione (61), merely on the b a s i s of i t s a b s o r p t i o n s p e c t r a (^ max. 244 and 232 mu). However f u r t h e r work i s necessary before a d e f i n i t e s t r u c t u r e can be assigned. At t h i s stage i t was necessary t o hydrolyse the 4 ,__.-3 keto-acetate (60) t o the corresponding a l c o h o l (62). The h y d r o l y s i s was performed by r e f l u x i n g a methanolic s o l u t -i o n of (60) w i t h 5% aqueous sodium carbonate s o l u t i o n . The 37 39 i s o l a t e d product was p u r i f i e d by column chromatography on de a c t i v a t e d alumina and f u r t h e r by r e c r y s t a l l i z a t i o n from benzene v.o giv e the d e s i r e d compound, m.p. 223-225 C. TLC examination of t h i s m a t e r i a l again showed a t r a c e amount of soma other m a t e r i a l running Very c l o s e to the major component. T h i s we b e l i e v e as s t a t e d i n the l a s t experiment t o be a mixture of two isomers epimeric at ^2Q' I t : w a s p o s s i b l e t o r e s o l v e these two isomers i n t o two d i s t i n c t spots on TLC by running the TLC p l a t e s e v e r a l times i n e t h y l acetate and i t seemed l i k e l y t h a t these two isomers c o u l d be separated by p r e p a r a t i v e t h i n l a y e r chromatography. But no such attempt was made since both these were expected t o gi v e the same compound on subsequent o x i d a t i o n . S t r u c t u r a l assignment of the above compound was made on the b a s i s of the f o l l o w i n g evidence. The strong e s t e r a b s o r p t i o n i n the IR had disappeared and i n s t e a d a -1 weak and broad band at 3260 cm due t o the hyd r o x y l group was now e v i d e n t . The NMR provided f u r t h e r c o n f irmatory evidence f o r t h i s s t r u c t u r e . Apart from the expected disappearance of a sharp s i n g l e t at T 7.98 (O-C^-CH^) , the narrow q u a r t e t due t o proton at C20 had s h i f t e d u p f i e l d and was now l o c a t e d a t T 6 . 3 7 as a broad unresolved m u l t i p l e t which i n t e g r a t e d f o r one proton ( F i g . 21). In the o p t i c a l r o t a t o r y d i s p e r s i o n measurement, the parent °^^p> - unsaturated ketone i n 11-aza-20-hydroxy-preg-4-ene-3,12-dior.e (62) e x h i b i t e d a m u l t i p l e Cotton e f f e c t curve i n the r e g i o r 300-380 mu ( F i g . 22), s i m i l a r t o one shown by l l - a z a - 2 0 acetoxy-preg> i—4-ene-3,12 -dione (60). The negative Cotton e f f e c t a s s o c i a t e d w i t h the lactam chromophore at 23 5 mu was again marked by the i n t e n s e p o s i t i v e bak ground r o t a t i o n of the K-band of the conjugated c a r b o n y l chromophore appearing a t 227 mu ( F i g . 23). E x c e l l e n t elemental a n a l y s i s on the c r y s t a l l i n e compound supported the formula assigned and f i n a l l y a .mass + s p e c t r a l molecular weight determination (M 331) proved the v a l i d i t y o f the s t r u c t u r e beyond any doubt. Our l a s t c o n s i d e r a t i o n was t o o x i d i s e the hydroxyl 4 group i n the 2\-3-keto-alcohol (62). Among the v a r i e t y of methods a v a i l a b l e f o r t h i s purpose, the procedure employed 60 by Krubiner and O l i v e t o was s e l e c t e d because of the success w i t h which i t has been employed on the corresponding s t e r o i d analogues. The o x i d a t i o n of the a l c o h o l (62) proceeded smoothly i n dimethylformamide using chromiumtrioxide as o x i d a n t . The crude product a f t e r chromatographic p u r i f i c a t i o n on alumina provided the t i t l e compound, ll-aza-pregn-4-ene-3,12,20-trione (63) which r e c r y s t a l l i z e d from benzene-o petroleum ether as needles, m.p. 220-222 C. The s p e c t r a l 41 42 43 CN fa _ 44 data on t h i s compound provided strong support f o r the success of the r e a c t i o n and was i n complete agreement w i t h the expected s t r u c t u r e (63). The IR spectrum showed the complete absence of h y d r o x y l group w h i l e an a d d i t i o n a l carbonyl peak -1 a t 1688 cm c o u l d be r e a d i l y a t t r i b u t e d t o a carbonyl group a t ^20' T ^ e N M R spectrum provided f u r t h e r support f o r the p o s t u l a t e d f o r m u l a t i o n . I t showed a sharp peak centred a t *T?7.60, a t t r i b u t e d t o the methyl group. Due t o i t s c l o s e p r o x i m i t y t o the carbonyl f u n c t i o n , the proton at C ^ had moved s u f f i c i e n t l y downfield now to be recognized as a broad unresolved t r i p l e t at 7*6.88 ( F i g . 24). Indeed comparison of these s i g n a l s w i t h the NMR spectrum of the 4 ^-pregnen-3,20-dione, whxch has the same D r i n g shows e x c e l l e n t agreement. The o p t i c a l r o t a t o r y d i s p e r s i o n spectrum of (63) was very c h a r a c t e r i s t i c s i n c e c o n t r i b u t i o n of both the s a t u r a t e d keto group (strong p o s i t i v e Cotton e f f e c t w i t h 4 peak a t 309 mu and the trough a t 266 mu) and the .^-3-keto moiety ( m u l t i p l e Cotton e f f e c t i n 340-380 mu region) were very c l e a r l y d i s c e r n e d ( F i g . 25). R e p e t i t i o n of the ORD measurement at much lower c o n c e n t r a t i o n again r e v e a l e d t h a t the strong negative Cotton e f f e c t a s s o c i a t e d w i t h the lactam chromophore had been o b l i t e r a t e d by extremely intense . . 4 p o s i t i v e background r o t a t i o n of t h e ^ - 3 - k e t o group, which 4 6 47 CP en O so o O O CP cvl A O o CVJ o o CU o CP VO •H fa 48 occured at 229 mu. (Fig. 26). F i n a l l y s a t i s f a c t o r y elemental a n a l y s i s on the c r y s t a l l i n e compound provided f i n a l support for the assigned formula. I n conclusion t h i s study provided the f i r s t successful synthesis of 11-aza pregnane derivatives which 4 possess a 3-keto moiety and with a six-membered C r i n g . The synthetic work described i s well substantiated with spectral and a n a l y t i c a l data. 49 EXPERIMENTAL M e l t i n g p o i n t s were determined on a K o f l e r b l o c k 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 r o t a t i o n s were taken i n chloroform s o l u t i o n . The i n f r a - r e d s p e c t r a were taken i n chloroform s o l u t i o n (unless otherwise stated) on a Perkin-Elmer Model 21 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were determined a t 100 mega c y c l e s / s e c . on a V a r i a n HA 100 instrument, using d e u t e r i o -chloroform as s o l v e n t ; the l i n e p o s i t i o n s or centre of m u l t i p l e t s are given in'"piers T s c a l e with reference t o t e t r a methyl s i 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 , i n t e g r a t e d areas and type of protons are i n d i c a t e d i n parentheses. The mass spectra were taken on an A t l a s CH4 mass spectrometer, using the d i r e c t i n s e r t i o n technique. The o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) curves were taken i n methanol or e l s e i n dioxane s o l u t i o n on a Jasco UV/ORD/CD-5 spe c t r o p o l a r i m e t e r . In a l l our experiments s i l i c a G e l G and Woelm alumina were used f o r t h i n layer chromatography. The alumina used f o r column chromatography was Shawinigan grade, approx-imate a c t i v i t y I I I (deactivated w i t h 3 y/a of 10 T» aqueous a c e t i c a c i d ) . Analyses were performed by Mr. P. Borda, the M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia. 50 Synthesis of l l - a z a - 3 oc. ,20-dihydroxy-5 p> —preqnan-12-one (56) A s o l u t i o n of the oxime of l l - o x o - 3 c* ,20-dihydroxy-C-nor pregnane (54,1.80 gm.) i n methylene c h l o r i d e (70 ml.) was t r e a t e d dropwise w i t h t r i f l u o r o a c e t i c anhydride (9.05 gm.) i n methylene c h l o r i d e (10 m l . ) . Reaction was allowed to proceed at i c e bath temperature f o r 15 minutes and at 9 25 C f o r 30 minutes. Evaporation of the s o l v e n t i n vacuo o gave a white c r y s t a l l i n e s o l i d (55), m.p. 124-126 C. Without f u r t h e r p u r i f i c a t i o n , t h i s s o l i d was d i s s o l v e d i n methanol (75 ml.) and t r e a t e d w i t h 5 ^ aqueous sodium carbonate s o l u t i o n (15 m l . ) . The s a p o n i f i e d s o l u t i o n was r e f l u x e d f o r two hours. I t was than cooled and concentrated i n vacuo t o about 25 ml. The concentrate was d i l u t e d w i t h water (50 ml*) and the r e s u l t i n g white p r e c i p i t a t e was e x t r a c t e d i n to methylene c h l o r i d e . The organic l a y e r was washed s e v e r a l times w i t h c o l d water and f i n a l l y d r i e d over anhydrous magnesium sulphate. Removal of the dryin g agent and evaporation of the solvent i n vacuo provided a c r y s t a l l i n e r e s i d u e (56). I t was r e c r y s t a l l i s e d from acetone-hexane and two crops were c o l l e c t e d . F i r s t crop (707 mg.), o o m.p. 237-238 C; second crop (330 mg.) m.p. 230-238 C; L^JT) + 25 (C 1.0). I n f r a - r e d : 3360 (-NH), 3240 (OH) -1 and 163 0 cm (lactam c a r b o n y l ) ; NMR: 2.98 ( s i n g l e t , IH, -OH), 51 4.12 ( s i n g l e t , IH,-C-N-H),6.30-6.64 (multiplet,4H,H-C 2 0+C 3-H o + C -H + C -OH); ORD: ( F i g . 14, C, 0.1225 mg./ml., 9 ~~~• 3 methanol). [4>] 5 0 0 + 534, \_4>] 3 Q Q + 534, i<P] 2 Q Q + 267, OJ 2 7 0 0,[4>]25Q - 2135, e4>] 2 3 5 " 5339 (trough),[ <f>] ^ 0, j^cb ] + 4806, [4^] + 8057. Found: C, 71.43; H, 10.09; 2 2 2 2 ^1 N , 4.22. C a l c . f o r C 2 0H 3-NO 3: C, 71.59; H, 9.83; N, 4.17. Syn t h e s i s of ll-aza-20-hydroxy-5 ^ — p r e q n a n - 3 , 12-dione (57). An i c e c o l d s o l u t i o n of the d i o l (56, 2.0 gm., 0.0060 moles) was t r e a t e d w i t h 4N Jones reagent (6.30 ml., 0.0120 moles) The r e a c t i o n was stopped a f t e r 5 minutes and immediately t r e a t e d w i t h i c e c o l d water t o destroy the excess of reagent. Methylene c h l o r i d e was added and the organic l a y e r was separated, washed w i t h water, aqueous sodium bicarbonate and again w i t h water. A f t e r d r y i n g over anhydrous magnesium sulphate, the s o l v e n t was removed i n vacuo to provide a crude mixture (1.80 gm), which was subjected to chromatography on alumina (90 gm.). E l u t i o n w i t h benzene-ethyl ether (1:1) y i e l d e d the d e s i r e d k e t o - a l c o h o l (57,1.50 gm.) as a c r y s t a l l i n e substance, • 23 .p. 242.5-244 C (from benzene) ;[or ]-> + 38 (c 1.0). m I n f r a - r e d : 3370 (-NH), 3240 (OH), 1703 (_3 carbonyl) and -1 1632 cm (lactam c a r b o n y l ) ; NMR: 3.40 ( s i n g l e t , IH, C 2 Q-0H), 3.54 ( s i n g l e t , IH -C-N-H), 6.32-6.60 ( m u l t i p l e t , 2H, 52 C- n -H + C - H); ORD: ( F i g . 16, C, 1.125 mg./ml., methanol), M 6 o o + 1 1 8 • C*] 4 0o + 3 2 6 - ' W 3 3 0 + 2 8 9 ' ^ ] 3 2 o + 5 9 ' [4>] 2 7 9 + 1 1 8 4 ( P e a k ) » [ > ] 2 6 o + 2 9 6 ; < F i g * 1 6 ' c ' 0 , 1 3 1 mg./ml. methanol), (> ] 4 5 0 0, [ 4> ] 4 0 Q + 508, [ 4> ] 3 3 q + 1271, M 310 + 5 0 8 ' f > ] 3 0 7 + 2 5 4 (trough). [ 4 » ] 2 9 0 + 1525, (4>J 279 + 2 0 3 3 < P e a k ) ' [4>]251 °'l<t>]246 ~ 1 4 2 3 ' M 237 " 4067 (trough), ["ct ] 2 3 Q 0,[4>] 2 2 6 + 7116. Found: C, 72.22; H, 9.55; N, 4.31. C a l c . f o r C 2 Q H 3 1 0 3 N : C, 72.03; H, 9.37; N, 4.20. Synthes i s of l l - a z a - 2 0-acetoxv-5 f> — preqnan-3 ,12-dione (58) The k e t o - a l c o h o l (57, 1.50 gm.) was d i s s o l v e d i n p y r i d i n e (75 ml.) and t r e a t e d w i t h a c e t i c anhydride (20 ml.). The mixture was allowed t o stand a t room temperature f o r four days. I t was then t r e a t e d c a u t i o u s l y w i t h water and e x t r a c t e d w i t h methylene c h l o r i d e . The orga n i c l a y e r was washed s e v e r a l times w i t h water, then w i t h 5 tf* aqueous sodium bicarbonate s o l u t i o n and again w i t h water. F i l t r a t i o n and removal of the solvent i n vacuo gave an o i l y product (1.79 gm.). T h i s m a t e r i a l was chromatographed on alumina (85 gm.). E l u t i o n w i t h benzene-ethyl ether (8:2) y i e l d e d the d e s i r e d acetate (58,1.260 gm.), as a white amorphous s o l i d . Although t h i n layer chromatography (TLC) behavior of t h i s m a t e r i a l i n d i c a t e d t h a t i t was pure, we were unsuccess-53 f u l i n o b t a i n i n g t h i s compound i n c r y s t a l l i n e form. For a n a l y t i c a l purposes a small amount (100 mg.) was p u r i f i e d by p r e p a r a t i v e TLC (Woelm alumina, ethyl-acetate-benzene (1:1) as developing medium, Rf *• 0.57) and a pure sample (80 mg.) was obtained as a white amorphous s o l i d . I n f r a - r e d (KBr): 3225 (-NH), no absorption i n the hydr o x y l r e g i o n , 1715 (ester carbonyl ), 1705 (C 3 c a r b o n y l ) , 1665 (lactam _ i carbonyl) and 1250 cm (acetate band); NMR: 4.81 ( s i n g l e t , IH, -C-N-H), 4.94 ( m u l t i p l e t , IH, H-C 2 0 -oAc), 6.63 o (doublet, J=10 Cps, IH, C--H.),7.98 ( s i n g l e t , 3H, -0-C-CH-) ; o Mass spectrum: MW 375; C a l c . f o r C 2 2 H 3 3 ° 4 N : 3 7 5 * Bromination of ll-aza-20-acetoxy-5 p> -preqnan-3,12-dione To a s o l u t i o n of the keto-acetate (58,1.124 gm., 3 m i l l i m o l e s ) i n g l a c i a l a c e t i c a c i d (20 ml.) was added a 30 °f» s o l u t i o n of hydrogen bromide i n a c e t i c a c i d (0.2 m l . ) . To the r e s u l t i n g pale y e l l o w s o l u t i o n was added dropwise a s o l u t i o n of bromine (480 mg., 3 m i l l i m o l e s ) and sodium acetate (246 mg., 3 m i l l i m o i e s ) i n g l a c i a l a c e t i c a c i d (5 ml . ) . The a d d i t i o n r e q u i r e d 30 minutes. Bromine absorption was slow a t the s t a r t and then proceeded more r a p i d l y . When a l l the bromine had been consumed, the r e a c t i o n mixture was sl o w l y d i l u t e d w i t h water (10 ml.) and the p r e c i p i t a t e d s o l i d was e x t r a c t e d i n t o methylene c h l o r i d e . The organic l a y e r 54 was washed s e v e r a l times w i t h water, aqueous sodium bicarbonate s o l u t i o n and again w i t h water. A f t e r d r y i n g over anhydrous magnesium sulphate, the s o l v e n t was removed i n vacuo t o provide the crude brominated product (1.140 gm.). TLC of t h i s m a t e r i a l ( s i l i c a g e l G, e t h y l acetate) showed one major (59, R^ = 0.45) and s e v e r a l small spots. This m a t e r i a l was not s u f f i c i e n t l y s t a b l e t o permit any p u r i f i -c a t i o n . Therefore the bromination mixture was now subjected t o dehydrobomination c o n d i t i o n s . S ynthesis of ll-aza-20-acetoxy-pregn-4-ene-3,12-dione (60). A s o l u t i o n of crude brominated mixture (59.1.140 gm.), l i t h i u m c h l o r i d e (130 mg.) and l i t h i u m carbonate (684 mg.) i n dimethylformamide (20 ml.) was heated a t 100 C under n i t r o g e n f o r 10 hours. D i l u t i o n of the r e a c t i o n mixture w i t h water (5 ml.) r e s u l t e d i n p r e c i p i t a t i o n of the crude product. T h i s white p r e c i p i t a t e was e x t r a c t e d i n t o methylene c h l o r i d e . The organic layer, was washed s e v e r a l times w i t h water, d r i e d over anhydrous magnesium sulphate and f i n a l l y concentrated i n vacuo t o provide a crude mixture (1.050 gm.), which was subjected t o chromatography on alumina (60 gm.). T h i s chromatographic separation allowed the i s o l a t i o n of two major components. The f i r s t compound was obtained i n the i n i t i a l f r a c t i o n s upon e l u t i o n w i t h benzene-ethyl ether (8:2), as a c r y s t a l l i n e s o l i d (61,20 mg.). T h i s m a t e r i a l has been 1,4 t e n t a t i v e l y assigned t o be -ll-aza-20-acetoxy-pregnan-3,12-dione (61) merely on the b a s i s of i t s a b s o r p t i o n s p e c t r a ; U l t r a v i o l e t : max.244 and 232 mu. However f u r t h e r work i s r e q u i r e d before a d e f i n i t e s t r u c t u r e can be assigned. Further e l u t i o n w i t h benzene-ethyl ether (8:2) 4 provided the d e s i r e d _-*-3-keto-acetate (60,490 mg.), which upon two subsequent c r y s t a l l i z a t i o n s from methanol provided o 2 3 a pure sample, m.p. 112-115 C; ]j> + 143 (C 0.50). I n f r a - r e d : 1665-1657 cm (overlapping a b s o r p t i o n of conjugated and lactam c a r b o n y l s ) ; NMR: 4.24 ( s i n g l e t , IH, o l e f i n i c ) , 4.73 ( s i n g l e t , IH, -C-N-H), 4.90 (quartet, IH, o H-C 2 0 -OAC), 7.22 (doublet, J =10 cps, IH, C g-H), 7.98 ( s i n g l e t , 3H, CH- -C-0); U l t r a v i o l e t : / ^ max. 236 mu ( l o g £ 4 . 1 6 ) , ORD: ( F i g . 19, C, 0.4025 mg/ml., dioxane), [<j>] ^ Q Q + 185, < C4>]450 + 8 3 4 ' M.385 + 1 2 0 5 ' C 4 > ] 3 7 ] t + 649, (>J ^ + 742, W 355 + 3 7 1 ' ^ 3 3 4 1 + 1 1 5 8 ' ^ ^ 331-326 + 2 7 8 1 < s h o u l d e r > ' t<p] + 3986 ( i n f l e c t i o n ) , 0 7 - + 6163; ( F i g . 20, C, 316 -^270 0.020125 mg./ml., dioxane), ] + 1 8 0 0 , ] 0 C- A + 6500, O ] 238 + 3 5 2 5 0 ' ^ ] 2 3 1 + 4 9 1 2 0 ( P e a k ) ' M 226 + 3 5 2 7 5 ' J 220 + 1 4 8 4 0 ' O j 214 °« F o l ;> n d : C, 70.58; H, 8.10; N, 3.99. C a l c . f o r C 22 H31°4 N: C, 70.74; H, 8.09; N, 3.84. Synthesis of ll-aza-20-hydroxy-preqn-4-ene-3,12-dione (62). 4 A methanolic s o l u t i o n (20 ml.) of __-3-keto-acetate 56 (60, 240 mg.) was t r e a t e d w i t h 5 ^ aqueous sodium carbonate s o l u t i o n (5 ml.). S u f f i c i e n t water was added to d i s s o l v e the suspended sodium carbonate. The s o l u t i o n was g e n t l y r e f l u x e d o v e r n i g h t . The r e a c t i o n mixture was then cooled and concentrated i n vacuo t o a volume of 10 ml. Water (10 ml.) was added and the r e s u l t i n g white gummy p r e c i p i t a t e was e x t r a c t e d i n t o methylene c h l o r i d e . The organic l a y e r was washed s e v e r a l times w i t h water and f i n a l l y d r i e d over anhydrous magnesium sulphate. Removal of the s o l v e n t i n vacuo provided a crude mixture (220 mg.) which was subjected to chromatography on alumina (12 gm.). E l u t i o n w i t h benzene-e t h y l ether (1:1) y i e l d e d an amorphous s o l i d (180 mg.), which on r e c r y s t a l l i z a t i o n from benzene gave an a n a l y t i c a l 0 23 sample of the a l c o h o l (62), m.p. 223-225 ; [« ~}p + 114 ( C 0.50). I n f r a - r e d : 3260 (OH), no e s t e r a b s o r p t i o n , 1660 (conjugated carbonyl) and 1635 cm (lactam c a r b o n y l ) ; NMR: 3.44 ( s i n g l e t , 2H, C 2 Q - OH + C-N-H), 7.11 (doublet, IH, o C g-H) ; u l t r a v i o l e t : " X max. 236 mu (log£.4.20); ORD: ( F i g . 22, C , 0.460 mg./ml. dioxane) ,_[<f>] 6 0 Q + 144, O J 5 0 0 + 3 2 4 ' ^ ^ 4 0 0 •+ 7 2 0 , O J 3 7 1 + 283,[<pJ 3 6 3 + 4 3 2 , O J 3 5 6 -180[<p] 3 4 6 + 720, 341 + 5 6 1 ' 327-322 + 2 2 0 9 ( s h o u M e r ) # 1 318-315 + 3 2 0 9 ( i n f l e c t i o n ) ; ( F i g . 23, C , 0.0230 mg./ml., dioxane), \ W350 0, ltf33Q + 1 4 4 0 , C < p ] 3 ] L 0 + 4 3 8 0 . [ 4 ? ] 2 8 ( ) + 7190, M 250 + 1 2 9 1 5 ' ^ 2 3 2 + 2 1 6 5 5 ' [ < f > ] 227 + 4 1 ? 2 ° ' [<p] + 28510, [4>] 2 i4 + 5 6 4 0 « F o u r * » d : c > 7 2 . 5 1 ; H, 9 . 0 0 ; N, 4 .311 C a l c . f o r C 2 0 H 2 9 N O 3 : C ' 7 2 - 4 7 " H , 8 . 7 4 ; N , 4 . 2 2 . S y h t h e s i s o f l l - a z a - p r e g n - 4 - e n e - 3 , 1 2 , 2 0 - t r i o n e (63) . 4 T o a s o l u t i o n o f ^ - 3 - k e t o - a l c o h o l (61, 100 mg.) i n d r y d imethy l formamide (10 m l . ) was added chromium t r i o x i d e (100 mg.) f o l l o w e d by d imethy l formamide (0.8 m l . ) c o n t a i n i n g c o n c e n t r a t e d s u l p h u r i c a c i d (0.04 m l . ) . A f t e r 4 h o u r s a t room t e m p e r a t u r e , the r e a c t i o n m i x t u r e was p a r t i t i o n e d between water and methylene c h l o r i d e . The o r g a n i c l a y e r was washed s e v e r a l t i m e s w i t h w a t e r , d r i e d over anhydrous magnesium s u l p h a t e and e v a p o r a t e d i n vacuo to p r o v i d e a c r y s t a l l i n e r e s i d u e (95 m g . ) . T h i s m a t e r i a l was d i s s o l v e d i n a s m a l l amount o f benzene and chromatographed on a l u m i n a (10 g m . ) . E l u t i o n w i t h b e n z e n e - e t h y l e t h e r (9:1) p r o v i d e d a c r y s t a l l i n e s o l i d (38 m g . ) , w h i c h upon r e c r y s t a l l i z a t i o n from b e n z e n e - p e t . e t h e r gave an a n a l y t i c a l sample o f the d e s i r e d compound (63) , m .p . 220-222 C . I n f r a r e d : no a b s o r p t i o n i n the h y d r o x y l r e g i o n , 1688 (C 2 Q c a r b o n y l ) -1 and 1660-1655 cm ( o v e r l a p p i n g a b s o r p t i o n o f c o n j u g a t e d and l a c t a m c a r b o n y I s ) ; NMR: 3.87 ( s i n g l e t , I H , - C - N - H ) , 4 .23 o ( s i n g l e t , I H , o l e f i n i c ) , 6.88 ( u n r e s o l v e d t r i p l e t , I H , C ^ - H ) , 58 7.15 (doublet, IH, C g -U) , 7.62 ( s i n g l e t , 3H, C^-CH*, ), 8.77 ( s i n g l e t , 3H, C^ g angular methyl group), 8.96 ( s i n g l e t , 3H, C ^ Q angular methyl group); u l t r a v i o l e t : max. 236 mu (log£4.25); ORD: ( F i g . 25, C, 0.340 mg./ml., dioxane), M60Q + 290, C<p] 4 5 0 + 870, O J 3 8 2 + 1548, O 7 3 7 0 1161, C4>] 3 6 1 + 1451 ,C<p] 3 5 6 + 1161, C<P1 3 4 6 _ 3 4 0 + 2564 (shoulder), O ] 3 1 Q + 9045 (peak), L<t>]266 + 97 (trough); ( F i g . 26, C, 0.0170 mg./ml., dioxane), O J 3 5 0 0, C4>] 3 4 Q + 1915; CP] 3 3 Q + 7725, 1 ^ 3 1 0 + 4 6 2 0'' ^ 2 9 6 + 3 4 8 4 ' ' ^ 2 8 6 °' ^ 2 5 0 + 3 8 7 0 ' ^ 2 4 6 9 6 9 0 ' ^ 2 4 0 2 2 2 0 ^ 2 2 9 + 4 4 5 2 0 ( P e a k ) ' ^ 2 1 8 1 1 6 0 1 ' [$>] = 0. 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