XOTES
September, 1963
611
TABLEI STEROID D~METHYLHYDRAZOKES Yield,
%
Sterol"
M.P.,* OC.
SolventC
Formula
--C
---Calcd.--H
Analysesd-------------Found-N C
H
N
166 M Cz&&?0 77.04 10.68 7.81 76.92 10.74 7.92 90 Pregnenolone E C26H4Zx4 75.32 10.62 14.06 75.23 10.60 14.52 93 148 Progesterone (bis) M C21H34N2O i6.31 10.37 8.48 76.03 10.69 8.76 93 179 Testosterone hI C22H36XT20 76.69 10.53 8.13 76.60 10.63 8.31 190 96 17-a-Methyltestosterone ?*I C23&?j20 77.92 9.67 7.90 77.62 9.60 8.07 206 92 Ethisterone E C21H34820 i6.31 10.37 8.48 75.83 10.35 8.28 92 200 cis-Testosteronee E C20H28N20 76.88 9.03 8.97 77.12 9.07 8.88 174 60 Estrone h'f C2sHaaN40a 67.53 9.07 12.60 67.34 8.99 13.11 96 218 Cortisone (bis) M C21H36N20 75.85 10.91 8.43 75.08 10.6 8.69 87 180 Dihydrotestosterone hI C20H32N20 75.90 10.19 8.85 75.33 10.11 8.52 171 96 19-Nortestosterone C CZSH~BN~O~ 6.54 6.23 73 158 Methyl dehydrocholate'(mono) * 811 melting points are with decomposition. Solvent for recrystallization: hl, a Commercial materials were used as received. nq. methanol; E, aq. ethanol; C, cyclohexane. -4nalpses by hlicro Tech IJal)iratorieP,Sktrkie, Illinois. Methanol solution used i n preparation. f Prepared as described in ref. 5.
b y Dr. G. R. MrKinney of the Mead Johnson Company. (5)
W.Rorsche, Rer., 52, 1353 (1919).
The Microbiolgoical Preparation of 17-Deoxytriamcinolone CHESTER E. HOLMLUND, LOLTIS I. FELD~UAS, NEIL E. RIGLER, AND RALPHH. EVANS, JR., ROBERTH. BLANK, BARBARA E. XIELSOX Biochemical Research Section, Lederle Laboratories Division, American Cyanamid Company, Pearl Riaer, .\lex York Received March 11, 1,963
The recognition that glucocorticoid activity may be enhanced by the addition of certain functional groups to steroids has motivated the preparation of biologically active compounds that lack one or more of the structural features of the hydrocortisone molecule. An early example is provided by 9a-fluorocorticosterone acetate, which, though lacking the 17a-hydroxy group, displayed a glucocorticoid activity greater than that of hydrocortisone. However, this compound also has powerful sodium retaining properties. It is known that 16a-hydroxylation of 9a-fluorohydrocortisone and 9a-fluoroprednisolone yields derivatives that are lacking in sodium retention properties and yet retain considerable glucocorticoid activity. It was of interest to ascertain whether the analogous 17-deoxysteroids mould possess similar biological properties. Fermentation of 9a-fluorocorticosterone (I) with Streptomyces roseochromogenes (ATCC 3347) yielded a product that was identified as 9a-fluoro-llfi,lGa,21trihydroxypregn-4-ene-3,20-dione(11). This product was ultraviolet-absorbing and displayed a positive blue tetrazolium reaction, indicating the continued presence of a A4-3-ketone and the a-ketolic side chain, in agreement with infrared absorption data. Elemental analysis supported the presence of one additional hydroxyl group. Appearance of the typical 415 mp-absorbing chromogen after reaction of I1 with the Porter-Silber reagent3 strongly suggested the presence of a hydroxyl group a t C-16 or C-17. The rate of formation of the (1) J. Fried, J . E. Herz, E. F. Sabo, A . Borrnan, F. M. Singer, and P. Numerof, J. A m . Chem. Soc., 77,1068 (1956).
415 mp-absorbing chromogen favored (3-16 as the hydroxylated p ~ s i t i o n . ~The ready formation of a diacetate and the separation of I1 from Sa-fluorohydrocortisone by paper chromatography further confirmed that the hydroxyl was located at C-16. Assignment of the configuration as 16a is based on the known capacity of Streptomyces roseochromogenes to 16a-hydroxylate steroids,6 and on optical rotation data (Table 1). TABLE I MOLECTJLAR ROTATION DATAOF SOMEC-16 HYDROXYLATED SWEROIDS AMD (I&-OH-H)
Steroid
RID
Solvent
Progesteronea 16a-Hydroxyprogesteronea 16,B-Hydroxyprogesteronea Sa-Fluorocorticosterone (I) 9~-Fluoro-lGa-hydroxycorticosterone (11) a See ref. 6.
+629O +519" +636" +675"
CHCl3 CHC13 CHC13 PIfeOH
- 110' +6"
+483"
MeOH
-192"
Fermentation of I1 with Nocardia corallina (ATCC 999) produced Sa-fluoro-1 lp,l6a,21-trihydroxypregna1,4-diene-3,20-dione (IV) which was isolated from the fermentation mash. Compound IV was ultraviolet absorbing, reduced blue tetrazolium, and gave a positive reaction with the Porter-Silber reagent. The infrared spectrum of IV is in accord with the assignment of a 1,4-diene-3-one system. The bathochromic shift observed in the ultraviolet in proceding from I1 to IV, together with the characteristic reaction of IV for a 1,4diene-3-one with isonicotinic acid hydrazide,' and phthalic acid p-phenylenediamines provide further support for the assigned structure. (2) S.Bernstein, R. H. Lenhard, W.6 . Allen, M. Heller, R. Littell, S. hI. Stolar, L. I. Feldman, and R. H. Blank, ibid.,78, 5693 (1956). (3) C. C. Porter and R. H. Silber, J . Bioi. Chem., 186, 201 (1950). (4) R. H. Blank, W. K. Hausmann, and C. E. Holmlund, Abstracts of Papers, 140th National Meeting of the American Chemical Society, September, 1961, p. 1P. ( 5 ) J. Fried, R. W. Thoma. D. Perlman, J. E. Herz, and 4. Borman, Recent Progr. Hormone Res.. 11, 149 (1955). (6) S.Bernstein, RI. Heller, and S. 11.Rtolar, J . Am. Chem. Soc., 77, 5327 (1955). (7) L. L. Smith and T. Foell, Anal. Chem., 31, 102 (1959). (8) Unreported results of R. H. Blank indicate that A'*'-3-ketosteroids produce a yellow color much more slowly than A'-3-ketosteroids when trested with the p-phenylenediamine-phthalic acid reagent [A. Bodenwky and J. Kollonitach, Nature, l?S, 729 (1955)l.
In the combination livcr glycogen and tliyrnus involution assay^,^ 17-droxytt~iamcirioloi~e (11') \\ a\. respectively, 3.3 (%yoconfidence limits: 2.7 4.0) a i i t l 1.4 (1.2-1.6) times as active as hydrocortisone. C'onipounds I1 and I V caused excretion of sodium, pots.sium, and water in saline-loaded adrenolectoiiiized malr rats.I0 Thus, it appears that the preserirc, of a l(icuhydroxyl group is capable of reversing the sodiuni retaining property of the Sa-fluorine atom e ~ w in i 1 Tdeoxysteroids. The presence of a lea-hydroxyl group in 9a-H-17-deoxysteroids also alters sodium nietaholisni by either decreasing sodium retentioiill or effrctirig sodium Other substituents at C-1 (i liavc also been reported to reduce the soc3iiirn-i.ctaiiiii~~ pinperty of a C)a-fluoro-l7-tieo.;y~tt~t~oi~l I
HO I
CHLOH I C=O t
CHzOR I
HO
I
I
c=o I
S. roseochromogenes
T
(ATCC 3347)
O'
J
II,R=H 111,R = COCH3
om
N. corallina
(ATCC 999)
YH20R
/
w
0
IV,R = H V, R = COCH, Experimental General.-Melting points were determined on a Fisher-Johns block and are corrected. Infrared spertra were determined in KBr disks with a Perkin-Elmer spectrophotometer (Model 21). Ultraviolet data mere determined in methanol solution with a Cary recording spectrophotometer. Polarimetric data were ohtained in methanol sdution in a 1-dm. semimicro tube. All evaporations were carried out in vacuo. The solvent system employed for paper chromatography consisted of benzene-acetic acid-p-dioxane-water in the volume ratio 4: 1: 1:2. 9~-Fluoro-llp,l6~,2l-trihydroxypregn-4-ene-3,2O-dione (II).-Fifty 500-ml. erlenmeyer flasks, each containing 100 ml. of medium A,14were inoculated with 1 ml. of a 72 hr.-old inoculum of Streptomyces roseochromogenes (ATCC 3347). The flasks were placed on a reciprocating shaker (120 strokes/min.) a t 28" for 16 hr. At this time 20 mg. of 9a-fluoro-1 lp,21-dihydroxypregn-4ene-3,20-dione (I), dissolved in 1 nil. of methanol, waa added til (9) I. Ringler, S. X a u e r , a n d E. Heydpr, €'roc. SOC.Ezptl. B i d . .\fed., 107,451 (1961). (10) Compound I1 was tested a t 500 pg., a n d compound I V in graded doses from 50-1600 pg., in the manner reported b y I. Ringler. L. Bortle, E . Heyder, A . Monteforte, J. Perrine. and E. Ross, ibid.. 102, 628 (1959). (11) 16a-Hydroxy-11-deoxycorticosteronewas found t o be inactive in the odium retention assay [H. Hirschmann, F. B. Hirschmann. and G . L. Farrel, J . A m . Chem. Soc., 76, 4862 (1953)l. (12) 3p,5a.6p,16p-20a-Pentahydroxypregnsnehas been classed as a n aldosterone inhibitor [T. Nakao, K. Hiraga, T. Saito, a n d Y. Muragawa, Jikeikai M e d . J . , 6, 1 (19.59)I], a n d 3~.16a-dihydroxyallopregnan-20-one has been reported t o act under certain biological conditions as a sodium-excreting factor [R. Neher, P. Desaulles, E. Vischer, P. Wieland. a n d A . Wettstein. I l e h . Chim. Acta, 41, 1667 (195811. (13) A. R. Hoffman, H . hI. Kissmann, and M. J. Weiss, J . M e d . Pharm. Chem., 6, 962 (1962). (14) Medium A consisted of soybean oil meal, 0.22%; corn steep liquor, 0.3%; yeast extract, 0.25%; glucose, 1%; ammonium diacid phosphate, 0.3%; a n d calcium carbonate. 0.25%. The p A was adjusted to 7.0 with sodium hydroxide.
211
10,700
IO
1,i,500
1 .;, 000 1 :i, 100
SO
1.5,600
IO,500
1601,Zl -Diacetoxy-S~-fluoro-llB-hydroxypregn-4-ene-3,20dione (III).-TI (49 mg.) vas dissnlrcd i n 0.85 ml. of pyridine, and 0.4 ml. of acetic anhydride was added. The reaction mixture was allowed to stand at room teriiperature for abciiit 18 hr., it^ which time a crude product was separztted aft,er the addition i ) f 7 ml. of witer. 1iecryst:tllizstiori from wetone and c:trbon tetraiahloride yielded 23 mg. of pur(. 111, n1.p. 161.5~-l62.
