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 l l e y e r , j after heating a t 60' for 3 \Ye wish t o report t h a t 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 t h a t 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- t h e 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 t h a t 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 conconstants: [ a ] " ~-5s' (dioxanej,": ;A: 243 m p tain a Bp-hydroxyl group. ( E = 14,500),' : : A : 3.01 p (OH), 5.81 p (20The assignment is corroborated by the fact t h a t 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 t h a t 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 (;
