July 20, 1957
COMMUNICATIONS TO THE EDITOR
392 1
to the known 2a-acetoxy-4-androstene-3,17-dionePab m.p. 209-211'; [a]D +146.8' (CHCl3); X ~ ~ $ ' n o ' Sir : 239.5 mp (15,500). Extensive studies1 have shown that steroids can The structure of I was established by the conbe hydroxylated by microorganisms a t positions 6P, version of 1 acetate, m.p. 112.5-113.5'; [ab 7a, 7& 8- and/or 9-, 10, l l a , l l p , 14a, 15a, 150,16a, +191.7' (CHC13); 239.5 mp (16,500); 17a and 21. We now wish t o report the preparation (found: C, 72.71; H, 8.07); by means of dilute (I) ; m.p. methanolic sodium hydroxide to 1,4-androstadieneof la-hydroxy-4-androstene-3,17-dione 215-221'; [ a ] D +184' (CHC13); XEt$anol 240 mp 3,17-di0ne,~ m.p. and mixed m.p. 139-140'. Com(15,000); (found: C, 75.32; H, S.46); and 2P- parison of the infrared spectrum with that of an hydroxy-4-androstene-3,17-dione (11) ; m.p. 143- authentic sample confirmed identity. Oppenauer 145'; [ a ] D -36.8' (CHCls); AE~\hhPnol242 mp oxidation of I11 to 1,4-androstadiene-3,17-dione6 (14,200); (found: C, 75.80; H, 8.71); by the established the structure of 111. microbiological action of a species of Penicillium, The configuration of the 1-hydroxyl group in isolated from local soil, on 4-androstene-3,l'i-dione.both I and I11 was initially assigned on the basis of By subjecting dehydroepiandrosterone to the same molecular rotatory contributions.' The structure oxidative fermentation, we obtained, besides I, of I11 was definitively confirmed by its catalytic la-hydroxydehydroepiandrosterone (111) ; m.p. reduction to la,3p-dihydroxyandrostan-l7-one [ a ] 4-10.6' ~ (CHCls); (found: C, (IV), m.p. 202-203.5"; 275-277.5'; [.ID +88.2' (CHC13); 74.92; H, 9.21). The hydroxylated steroids were (found: C, 74.32; H, 9.74); followed by reduction prepared by the fermentation and extraction tech- of IV with sodium borohydride to 1aJ3P,17/3niques previously described,* and, in the case of the androstanetriol (V), m.p. 235-239' ; [a]D+20.2' hydroxylated androstenediones, were purified by (CHCI3); (found: C, 73.99; H, 10.47). This comchromatography on silica gel. 1a-Hydroxydehy- pound (V) was identical in all respects with that droepiandrosterone (111) was readily obtained by prepared by Benn, Colton, and Pappos by the redirect crystallization on concentration of the duction of 1aJ2a-oxidoandrostane-3,17-dione. methylene chloride extract. Reduction of I11 with sodium borohydride or 0 0 lithium aluminum hydride gave 5-androsteneIa,3P,17P-trioI (VI), m.p. 212-213'; [ a ] D -54.8' (CHC13); (found: C, 74.41; H, 10.22); triacetate, m.p. 179-181'; (found: C, 69.11; H, 8.24). Cautious acetylation of I11 produced 1a-hydroxy3P-acetoxy-5-androsten-17-one ,,(VII), m.p. 243244'; [ a ] -6.7' ~ (CHCI3); (found: C , . 72.58; H, 8.83); which was oxidized with chromium trioxide in pyridine to 3/3-acetoxy-5-androstene-l, 17dione (VIII), m.p. 156-157.5'; [ a ]$40.7' ~ (CHC13); (found: C, 73.27; H, 8.27). The reduction of VI11 with sodium borohydride yielded VI and 5-androstene-lp,3P,17P-triol(IX), m.p. 270-278" ; triacetate, m.p. 147.5-148.5' ; [ a ] -32.4' ~ (CHC13); (found: C, 69.11; H, 8.54). Compound I X HO and its triacetate were identical in all respects The structure of I1 was indicated by the con- (m.p., mixed m.p., and infrared) with the corretribution of the new hydroxyl t o the molecular sponding triol and triacetate obtained from Ruscorotation of the compound ( A M D -658),3a by a genin by Benn, Colton, and Pappo.8 positive "blue tetrazolium" test, and by the char(6) (a) H. H. Inhoffen, G.Ziihlsdorff and Huang-Minlon, Bar., 7SB, acteristic change of ultraviolet spectra with time (1940); (b) C. Djerassi and C. R. Scholz, J . Org. Chem., 13, 697 in 0.1 N methanolic potassium h y d r ~ x i d e . ~The 451 (1948). structure was established by converting I1 t o its (7) W. Schlegel and Ch. Tamm, H e h . C h i m . Acta, 40, 100 (1957), acetate, m.p. 157-158'; [ a ] -5.9' ~ (CHC13); and preceding papers. Amethanol max 243 mp (15,300); (found: C, 73.47, H ; (8) W. R. Benn, F. Colton and R. Pappo, THISJOURNAL, 7'9,3920 8.04) ; and epimerizing I1 acetate, by heating with (1957). AXD COMPASY R. M. DODSON anhydrous potassium acetate in glacial acetic acid,b G . D. SEARLE P. 0. Box 5110 ARTHURH.GOLDKAMP (1) For excellent reviews see: (a) A. Wettstein, Ergerienfia, XI, MICROBIOLOGICAL HYDROXYLATION OF Cle-STEROIDS AT POSITIONS C-1 AND C-2
465 (1955); (b) G. M. Shull, Trans. N . Y.Acad. Sci., 19, 147 (1958); (c) S.H.Eppstein, P. D. Meister, H. C. Murray and D. H. Peterson, Vitamins and Hormones, XIV, 359 (1956),Academic Press, Inc., New York, N. Y.; (d) J. Fried, R. W. Thoma, D. Perlman, J. E. H e n , and A. Barman, Recent Progr. Hormone Research, XI,149 (1955),Academic Press, Inc., New York, N. Y. (2) D. H. Peterson, H. C.Murray, S. H. Eppstein, L. M. Reineke. A. Weintraub, P. D. Meister and H. M. Leigh, THIS JOURNAL, 74, 5933 (1952). (3) (a) F. Sondheimcr, St. Kaufmann, J. Romo, H. Martinez and G.Rasenkranz, ibid., 7 5 , 4712 (1953); (b) G.Rosenkranz, 0. Mancera and F. Sondheimer, ibid., 77, 145 (1955). (4) A. S. Meyer, I. Org. Chem., 20, 1240 (1955). (5) R. 1,. Clarke, K. Dobriner, A. Mooradian and C. M. Martini, T H IJ ~ OTTRN~I..
7 7 , f i R 1 (19.55).
CHICAGO 80, ILLINOIS RECEIVED MAY2 5 , 1957
R. D. MUIR
MICROBIOLOGICAL TRANSFORMATION OF STEROIDS. Z@-HYDROXYLATION
Sir: It has become increasingly evident that enzymatic hydroxylations of steroid occurring in the mammalian tissues will inevitably find their counterparts in microbially-induced transformations of the same or similar substrates. Of the hydroxylation processes occurring in mammals the
