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 t h a t 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 t h a t 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 275-277.5'; [ a ] 4-10.6' ~ (CHCls); (found: C, (IV), m.p. 202-203.5"; [.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 t h a t 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 t h a t 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
reactions a t %CY', 2p2, 6 2 , 18-*and 19-* have not absorb in the region of 280 nip. Measurenient as yet been duplicated by incubations with micro- of the ultraviolet absorption in alkaline solution, organisms.4a according t o lleyer,j after heating a t 60' for 3 \Ye wish t o report that microbiological 2p- hours, afforded a curve which corresponded exactly hydroxylation of 4-pregnene-lia,21-diol-3,20-dione in location of niaxima with the highly characteris( I , Reichstein's Compound S) has now been tic curve from 2a-hydroxy-4-androstene-3,li-diachieved with the aid of several unidentified The rotation of the 2,21-diacetate of I1 difStreptomyces species isolated from soil (Schering fers markedly from that reported for +pregnenecollection numbers FC7-206, FC6-53S, DS 81-B). 2a, lia,21-trio1-3,20-dione 2,21-diacetate6 ( [cY]~'D Incubation of Compound S (600 mg.) in a peptone+122O (chloroform), m.p. 200-202', 213--21'T3 soybean meal-yeast extract-cerelose medium with (polymorphs)j. I n like fashion I1 contrasts with a 72-hour growth culture of Streptomyces sp. DS- the 4-pregnene-aa, 1ia,21-triol-3,20-dione, [ a ] % 81-B with rotary shaking a t 38' for 4S hours af130' (chloroform), m.p. 219-221'. Since 23forded, after chloroform extraction and chrornatog- hydroxyl reaches equilibrium with ?a-hydroxyl in raphy. 33 mg. of 4-pregnene-2P, lTa,21-triol-3,20- mildly alkaline solution,; it is obvious that the dione (11), m.p. 215-220' dec. Further recrystal- same alkaline ultraviolet spectrum must result lization from acetone-hexane raised the n1.p. t o for both configurations in a given pair of 2-hy22.5..j-22S0 dec. and gave I1 with the following dro~y-3-keto-A~-steroids.~ Hence, I1 must con243 m p tain a Bp-hydroxyl group. constants: [ a ] " ~-5s' (dioxanej,": ;A: ( E = 14,500),' : : A : 3.01 p (OH), 5.81 p (20The assignment is corroborated by the fact that carbonyl), 5.94 p (3-carbonyl) and (i.18 p ( A 4 ) ; the predicted shift in molecular rotation ( A L I three hydroxyl groups by integration of the OH %l)' based on t.he only 2P-hydroxy-3-keto-A4hand. Calcd. for C a H 3 0 0 ~ :C, 69.5s; H, 8.34. steroid known previously, 2,3-hydroxytestosterone Found: C, G9.Sl; H, 8.76. -4polymorphic modi- 2 , l 7-diacetate7 (2i3-hydroxytestosterorle itself' is fication of I1 exists which possesses an altered characterized incompletely) is in reasonable agree2.8'7 p (OH), 5.82 p (20- ment with the observed values of the shift for 21infrared spectrum, 'A':; carbonyl), 5.89 p and 6.02 p (3-carbonyl) and 6.20 p acetate of I "cs. 2,al-diacetate of I1 (Alf-300). (A4). The latter polymorph is converted to the This is especially noteworthy since the ?&hydroxyl former by recrystallization from acetone-hexane group contributes much more strongly to the and seeding with the former. The infrared spec- levorotation of 3-keto-Al-steroids than does any tra of the two forms differed in considerable detail other hydroxyl group. from the (8, l l a , IlP, 15a and 15@-hydroxydeIt is known that hydroxyl groups a t 2- or (i-iri rivatives of I. The 2,21-diacetate of 11, prepared 3-keto-Al-steroids can be removed reductively by with acetic anhydride and pyridine melts a t 218- mild treatment with zinc and acetic acid, affording 219', [ a I 2 ' D + g o (dioxane), xi:,"" 244 nip ( E = the parent steroid.' Treatment according t o this 16,200), ':A," 2.73, 2.83, 2.97 p (OH), 5.71 1.1 method converted I1 9,21-diacetate into I 21-ace(acetate carbonyl), 5 . 7 s p (20 carbonyl). 5.95 p tate, indistinguishable from an authentic saniplc (;
