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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 ,u (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)to 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 to 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 that 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 ,u (OH), 3.80 p (20-carbonyl), 6.0, ( i . l f i and that its synthesis can be accomplished in vitro' we 6.22 ,u (~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-l l$,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
Aug. 5 , 1955
COMMUNICATIONS TO THE EDITOR
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CHgOR this product was distributed in a 50-tube Craig all, glass countercurrent machine using the system ethyl c=o acetate-butanol-0.01 N HC1 (90: 10: 100) and the method of single withdrawal. The upper phases emerging between the 85th and 120th transfers were pooled. Evaporation of the solvents gave 177 mg. of the glucuronoside which was 95% pure as determined by the above method of analysis. In this experiment 47y0 of the administered tetrahydrocortisone was accounted for as the glucuronoside of this H purity. Since it was not possible to crystallize the purified glucuronoside, this and other similar lots I, R = COCH,, R' = CHa were treated with diazomethane followed by acetic 11, R = H, R ' = H OR anhydride and pyridine to give, after adsorption chromatography, needles from methanol which hydrocortisone-21-monoacetate with a-bromo. trimelted a t 208-209' uncor. [a]"D +39.3' (7.62 acetyl-glucuronic acid methyl ester in chloroform mg. in 2.40 ml. CHC13). Anal. Found: C, 60.00; in the presence of silver carbonate to give I, m.p. H, 7.01; CH3C0, 23.87. 209-212' (from methanol); [ffI3OD $37.8' f The purified glucuronoside was hydrolyzed by 1.5' (14.0 mg. in 1.00 ml. of CHC13). Anal. Calcd. beef liver 0-glucuronidase to give the calculated for C86H50016:C, 59.82; H, 6.92. Found: C, amount of tetrahydrocortisone as judged by the 59.75; H, 7.11. On admixture with the same above method of analysis. Similar hydrolysis of a derivative prepared from the glucuronoside isolated larger amount of the purified glucuronoside followed from urine the melting point was not depressed, and by acetylation and partition chromatography3 the infrared spectra of the two derivatives were of the neutral fraction showed that tetrahydro- identical. The structure of the naturally occurring cortisone diacetate was the only recoverable glucuronoside as deduced from the method of steroid, m.p. 232.5-233.5' uncor., and indistinguish- synthesis, optical rotation and hydrolysis with able from an authentic sample by mixed melting @-glucuronidasemust be that of 11, which may be point and infrared spectroscopy. Anal. Found : designated 3a-(P-~-glucuronoside) , 17a ,21-dihyC, 66.82; H, 8.00. droxypregnane-1l720-dione. An additional 10 to 15% of the administered This investigation was supported by research tetrahydrocortisone was accounted for a s a glu- grants A-272 and C-2210 from The National curonoside of p - c ~ r t o l o n e . ~The crude glucurono- Institute of Arthritis and Metabolic Diseases and side obtained from tubes 12 through 25 following The National Cancer Institute, of the National the countercurrent distribution was hydrolyzed Institutes of Health, Public Health Service, and an with @-glucuronidase. The recovered neutral frac- Institutional Grant to the College of Physicians tion was acetylated and chromatographed (3) and Surgeons by the American Cancer Society. to give 3 a ,20p,2 1- triacetoxy-17a-hydroxypregnane- The authors wish to thank Dr. Karl Pfister, Merck 11-one (p-cortolone triacetate) (4) m.p. 205-20G0 and Company, and Dr. Robert H. Levin of The uncor. Anal. Found: C, 65.81; H, 8.25. Through Upjohn Company for the tetrahydrocortisone used the courtesy of Dr. T. F. Gallagher its identity was in this work. established by mixed melting point with an authenDEPARTMENT OF EXPERIMENTAL MEDICINE tic sample and by infrared spectroscopy. JOHN J. SCHNEIDER JEFFERSON MEDICAL COLLEGE The synthesis of 3a-(triacetyl-P-~-glucuronosidePHILADELPHIA L. LEWBART 7 , PENNSYLVANIA MARVIN methyl ester)-2 1-acetoxy-17a-hydroxypregnane- DEPARTMENTS OF BIOCHEMISTRY AND 11,20-dione (I) was carried out by coupling tetra- OBSTETRICS A N D GYNECOLOGY (3) E. R. Katzenellenbogen, K. Dobriner and T. H. Kritchevsky, J . B i d . Chem., 207, 315 (1954). (4) D. K. Fukushima, N. S. Leeds, H. L. Bradlow, T. H. Kritchevsky, R I . B. S t o k e m a n d T.F Gallagher, ibid., 212, 449 (193.5).
OF PHYSICIANS AND SCRGEONS PAULLEVITAN COLLEGE COLUMBIA UNIVERSITY SEYMOUR LIEBERMAN SEW YORK32, XEWYORK RECEIVED J U N E 2 . 1955
