4184
C O M h l U N I C A T I O N S TO T H E
symmetrically. The 7-norbornenyl cation may be represented by (VI). It reacts with solvent
EDITOR
Vol. 77
AI'4-pregnadiene-2l-ol-3,20-dione 2 1-acetate (VII)6 (m.p. 202-204', [ a ] " ~ 143' (chloroform), 152'
+
+
(ethanol) Xzi:hanol243 mp ( E = 15,800, Xzzp' 2.93 p (OH), 5.72 and 5.&0 p (20-carbonyl, 21-acetate interaction), 6.01, 6.16 and 6.23 p (A1s4-diene-3-one) ' S.06 p (C-0-C of acetate), found: C, 74.46; H, A 8.241, and 9a-fluoro-A's4-pregnadiene-1 I(?, 17a,21VI triol-3,20-dione (IX) [m.p. 265-269' dec., [ c ~ ] * ~ D stereospecifically; complete retention of con- +111" (ethanolj, X~~:'lano1 -39 3 inp ( E = 14,800), figuration was observed in the hydrolysis of the found: C, 64.22; H, '7.31. Calcd. for C21H2iOjF.dibromide (111)t o the alcohol, and in the acetolysis CH,O: C, (j4.37; H, 7.611. of 7-norbornenyl toluenesulfonate (V). In addition to the recently noted, enhanced DEPARTMENT OF CHEMISTRY glucocorticoid activity of the 21-acetate of IX' USIVERSITYOF CALIFORNIA S WINSTEIN we wish to report that IX and its 21-acetate Los AXGELES24, CALIFORNIA I\l SHATAVSKY possess intense mineralocorticoid action,8 oi the COSVERSE MEMORIAL LABORATORY C SORTON order of the parent fluorinated steroid, 9a-fluoro-4HARVARD U~IVERSITY R. B \\'OODWARD pregnene-1 1B,17a,%l-triol-3,20-dione." CAMBRIDGE, hlASSACHUSE rTS In subsequent reports we will describe in greater RECEIVED J U L Y 13, 1955 detail the chemistry and microbiology of these and related transformations, and the biochemical studies MICROBIOLOGICAL TRANSFORMATION OF STER- of the previously undescribed A'-unsaturated OIDS. I. A'~"DIENE-3-KETOSTEROIDS derivatives of the known natural and synthetic Sit,: steroid hormones. I t has become a problem of to (6) Ci.R. L. Clarke, K. Dobriner, .4. Slooradian and C . ?if. Martini, devise efficient techniques for the introduction of i b i d . , 77, 661 (1955). AI-unsaturation in cortisone (Ij3 and cortisol (11) (7) R . F. Hirschmann, R . Miller, K . E . R e y l e r , L. H . Sarett and since it has been shown that A',4-pregnadiene- 11. Tishler, ibid., 77, 3166 (185n). 11, R . Cook, Jr., and F. Elmadjian, J . diii. P h o i ~ i i i .As.roC.. 17a,21-diol-3,11,20-trione (111) and A1m4-pregna- Sci.(8)Ed.; X L I I , 329 (1953). diene-1lp, 17a,21-triol-3,20-dione (IV) are consider(9) J. Fried and E. F Saho, THISJ O U R N A L , 76, 1455 (1954). ably more potent anti-inflammatory agents than A . SOBILE the natural corticosteroids. We wish to report BIOLOGICAL RESEARCH LABORATORIES IV, CHARNEY that I may be converted t o 111 and I1 may be con- SCHERING CORPORATION P. L. PERLMAX H . L. HERZOG verted to I V by the action of Corynebacterium RESEARCH LABORATORIES C. C. PAYNE simplex (X.T.C.C. (5946). Either I or 11, dissolved CHEMICAL CORPORATION 11.E. TULLY in methanol, was added to shake flasks containing SCHERING 31.A . JEVNIK X. J. a 24-hour culture of C . simplex in a nutrient medium BLOOMFIELD, E . R.HERSHBERC of 0.155 Difco yeast extract buffered a t PH 7. RECEIVED JLrL'i 11, 1955 The mixture was shaken a t 28' for ;3-24 hours. Extraction of the resultant broth with chloroform, followed by evaporation to a residue and crys- ISOLATION FROM URINE AND SYNTHESIS OF TETRAHYDROCORTISONE GLUCURONOSIDE tallization from acetone, afforded excellent yields of I11 or IV, respectively. Compounds I11 and S i r : IV, obtained in this way, were identical in every It is generally agreed that 3a,l7cr,2l-trihydroiyrespect with samples prepared by purely chemical pregnane- 11,20-dione (tetrahydrocortisone) is the means. By similar microbiological procedures most abundant adrenocortical steroid metabolite we have also prepared A1,~-pregnadiene-l'7cr,21- excreted by man, and t h a t it is present in urine diol-3,20-dione (17) [m.p. 246-249' dec., [ a I z 5 D largely as a glucuronoside. Because of the general 76' (CHC13), hz::ht*hanol244 mp (t = 15,900),'?:A: interest in this conjugate and the recent evidence 3.03 p (OH), 3.80 p (20-carboiiyl), 6.0, ( i . l f i and that its synthesis can be accomplished in vitro' we 6.22 p (~14,-dietie-:3-orie)," found: C, 7:l..j(j; H, wish to report its recovery froin urine in a relativelj. 8.401, A1*-'-pregnadiene-ll$,2l -diol-3,20-dione (VI) pure state and the synthesis and characterization i m p . 2%7.%230.3° dec., [CYI'l'D +173O (methanol), of its tetraacetyl methyl ester. Eight 230-mg. doses of free tetrahydrocortisone 2-13 mp ( E = 1-1,300),?':A: 2.88 and 2.9i in aqueous alcohol were given orally to a man a t half p (OH), 535 p (?O-carbonyl), 6.07, 6.20 and 0.25 p (A1,4-diene-3-one), found: C, 73.49; H, S.12], hourly intervals. The urine which was collected during this period and the twelve-hour interval (I) J. J . Bunim, bl. SI.Pechet and A . J. Bollet, J . .4nz. .bled . ~ S S O L . , that followed was acidified and extracted with 1 6 7 , 311 (1955). butanol. The butanol extract was washed with (2) H . L. Herzog, A . Xohile, S. Tolksdorf, XY. Charney, E . BI Hershberg, P. L. Perlman and hf. M. Pechet, Science, 121, 176 (1955). water, neutralized with aqueous sodium carbonate ( 3 ) E. Vischer, C. hleystre and A . Wettstein, HeZu. Chim. Acta, and concentrated in vacuo. The crude product 38. 855 (1955), have reported t h e preparation of 111 and V by t h e action of Fusarium solani on cortisone and Reichstein's Compound S, which separated weighed 2.92 g. and contained 1.45 g. of the desired sodium glucuronosidate as respectively, and the preparation of VI and VI1 by t h e action of Caloneclria decora on corticosterone and desoxycorticosterone (followed determined by analysis based on the method of by acetylation in t h e latter case). Porter and Silber.' Four hundred milligrams of (4) H. L. Herzog. C. C Payne. hl. A . Jevnik. D Gould, E. I.. ~
d'--
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Shapiro, E. P. Oliveto and E. B. Hershherg, THISJ O U R N A I , i n press , . IV, Thnmn and A R l i n g s h e r p . > h i d ,7 5 , 5 7 G i '1 ( i j .J. I i r i ~ dR
(1) kl. 1. Isselhacher and 1. Axelrod, Tins J O U R N A L , 7 7 , 1070 flRbd) I ? ) C' c' P , l r f P r i n 4 li €7 . ? l l h r r I Erai C l w n : , 185. 201 1'130) f
