lack of growth. The yeast and fungus tube-dilutiori assays were performed by incorporating the compound and iiioculuni into sensitivity agar imtead of broth. Viiible inhibition of growth 011 the surface of the agar after 24 llr the of activity in these cases.
A&nowledgment.--The authors are grateful to Dr. David p. Jacobus of the jvalter Reed Institute of Research for providing the antimalarial screening results.
Synthesis and Activity of Some Nitro Steroids' ~ I A N F KE.E jD J TAND ~ ROBERT ~ ~ ~C. BOGUSLASKI Ucpar lrricnt of l'hui itiuLeulicu1 Chenizstry, School of Pharmacy, CTnzveiaity of Culifoi niu, Sun Fruncisco, CaliJorniu o4l&
Receieed September 26, 1967 The sytithesis of several 3- arid 20-1iitro-5a-androstane and -pregnciie derivatives was undertake11 by oxidation of the corresponding oximes. Improved conditions (irradiat,ioii and oxygenation) were developed for this technique. Biological evalimtioii of t'he final derivatives for anabolic aiid progestational activit'ies indicated that the replacement of a carbonyl oxygen by a nitro group in these compounds leads to weakly active or inactive products.
All steroid hormones except estradiol and testosterone possess a carbonyl group a t C-20 and all but estradiol have a keto group at C-3. I n work directed a t defining the function of this moiety in eliciting biological responses, we speculat'ed that a combination of high electron density and hydrogen-bond acceptance might be key factors in the importance of these ketones. I n the present work, we have examined this possibility by determining if a nitro group can be substituted in the region of a carbonyl function with retention of activit'y. Nitro st'eroids have been prepared by nitration of unsaturated steroids with nitric acid3&+ or nitrogen tetroxide,3dby condensation of steroidal aldehydes with ~ i i t r o m e t h a n e ,by ~ ~nitration of by oxidation of oximes with a ~ e r a c i d , ~from g reactions of steroids wit'h nitrosyl chloride, 3h and by displacement of alkyl ~iitrates,~' but these methods appeared too drastic or otherwise unsuitable for unsaturated steroids. httempts to displace steroidal 3-tosylates with sodium nitrite4 failed. Finally we employed and modified the mild oxidation of oximes5 which had been used for the preparation of 17-nitro steroids.6 Treatment of 1 with S-bromosuccinimide (KBS) in dioxane-water solution, followed by stirring and exposure to air for 4s hr arid final S a B H 4 reduction gave a mixture of the nitro compound 4 (27%) and androstane-3p,17P-diol (Scheme I). It was thus apparent that two competing reaction sequences occur during the KBS reaction: (a) formation of a yem-bromonitroso compound followed by air oxidation t o a gei,z-bromo(1) This investigation was supported in part by a PHS research grant (.\31-05016) f r o m t h e Kational Institute of Arthritis and RIetabolic Diseaces, U . S.Public Health Service. (2) 31. E. \Volff, TV. Ho, and 31. H o n j o l ~J, . X e d . Chem., 9, 682 (1966). ( 3 ) (a) . I. Windaus, Ber., 36, 3752 (1903): (b) A . IYindaus and C. IIrunken, 2.Phusiol. Chem., 140, 52 (1924); ( e ) J. 3Iauthner and TV. Suida, .IIo?iutsch., 24, 648 (1903); (d) C. dnagnostopoulos and L. F. Fieser. J . A m . Chem. Soc., 7 6 , 532 (1954); ( e ) A . Bowers and H. J. Ringold, t b d , 81, 3710 (1959); (f) 3. F. Bell, E. R. H. Jones, a n d G. D. Meakins, J . Chem. Soc., 2601 (1965); ( 9 ) C. €1. Robinson, L. Rlilewich, a n d P. Hofer, J . O r g . Chem., 31, 524 (1966); (h) W.A. Harrison, E. R. H. Jones, G. D. 3Ieakins. and P. A . Wilkinson, J . Chem. Soc., 3210 (1964); (i) R. Schaub and 31. J. \Veiss, U. 8. Patent 3,151,109 (Sept 29, 1964); Chem. Abstr., 61, 14754
(1964). (41 T. N . Korn1)lum. I T . I.arson, R . Illaclirvood, D. 3Ioolierry, E. Olivf+o, and G . Graham, J . Am. Chem. Soc., 78, 1497 (1956). ( 5 ) D. C. IWand and G. Criner, ibid., 7 6 , 4047 (1953). (6) A. G. Patchett. F. Hoffman, F. F. Giarrusso, H. Schwaillin, and G. E. Artli, J . Ore. Chem., 27, 3822 (1962).
SCHEME
1,R=OH 2, R=CH(B-OH)CHB
1
3
NaBH,
RO
4,3p, R = OH 5, R = CH(B-OH)CH, 6 , 3 &R = COCH,
9,20a 10,20f9
7,R=Ac;R'=Br 8, R = H; R = H
0 11
nitro compound, and (b) hydrolysis of the oxinie to the parent kctoric, arid subsequent reduction to the corrcsponding alcohol by the borohydride. It mas clear that b could be minimized by acceleratirig the steps in a. This ITas done by bubbling oxygen through the mixture rather than relying on atmospheric air, and by irradiating with ultraviolet light. We reasoned that the irradiation would generate bromine radicals, thus facilitating the bromination and, second, would convert molecular oxygen, a sluggish oxidizing agent, to atomic oxygen, a much better oxidizing agent. As a result of these modifications, the yield was increased to about 50%. The assignment of the configuration of the nitro group in 4 was based on the broad multiplet exhibited in the nmr spectrum of the 3a-proton; this is due t o axial-axial splittings and is compatible only with nri axial proton at C-3. In extending the method t o C-20 oximes, a complex mixture of epimeric C-20 nitro compounds and alcohols was obtained, as shown by glpc. Therefore, the interincdiate bromonitro compouiid 7 wab isolated and freed
of C-20 ketone by treatment lvith (;irard's re:tgeiit.
llcductioii and concomitant hydrolysib gavc 8. A4ttemptst o oxidize 8 by the O p p m a u c ~nwtliocl g:ir-v 011ly a complex mixture. Uw of chromic :wid in a c ~ toile at 0" guve the 3,G-dioiie 11, but at -TO' t h e clcl*ired 3-ketones were secured. These \vere iwnierizctl t u the coiijugated products 9 :md 10 with os:ilic nritl. 'l'hv structure of 11 was readily deduced from t h e iiiiir y e c t r u m . X sharp singlet at 6.2 ppin n-as producecl 1)) the C-4 proton, wliereas normally the resoiiaricc due to the. ('-4 proton in a 3-keto-14 steroid i- bro:tdeiied hi xllylic coupling to the protoil. :it C-6. The c*oiifiguri\tion of the nitro groups in 9, 10, and 11 ivti iiig L~iowiiIimr relationship>.5 111 tlie 20 ('-1s resorianee is shifted upfield xiid the C'-21 rcwii:iiicc is shifted downfield relative t ( J the psitioils of t h c pe:iki iti the spectra of the 206 isomers. Biological Testing.8- Compounds 4, 6, 9. a d 10 n ~ wevaluated in r.:isenberg-(:ordan-~Her~hl)crg~r) :tilt1 C'lnuberg-type tebts2 (T:Lblci I and 11). ('ompound 4
