evaporated t o a small volume, and extracted with ethyl acet,ate. The extract was washed with water, sodium bicarbonate solution, water, dried over magnesium sulfate, and evaporated to 2.05 p. of a foam t h a t was chromatographed on Florisil (200 9.). N u tion with 5.2 l. of 12.5% acetone-87.5% Skellysolve B gave 0. g. of oils. Further elution with 2 1. each of 15% acetone-85 Skellylsolve and 20%;: acetone-80%, Skellyslolve I3 gave 0.90 of a gelatinous precipitate. Crystallization of the latter froiii methanol-water (1:2) gave 0.63 g. of (4), m.p. 206-208" dec. 'The analytical sample had m.p. 210-211' dec. froin rriethani~l water; A,, 241 mp ( B 16,100). Ar~al. Calcd. for C23H3,FOe.0.25H20:C, 64.69; H, 7. 4.45. Found: C, 64.54; H, 7.68; F, 4.41. 9a,156-Difluoro-11p,1701,21-trihydroxypregn-4-ene-3,20dione 21-Acetate (5).-N-Rromoacetaniide 10.38 9.) w m added to a solution of 0.7 g. (1.65 mmoles) of (4)in 15 ml. of pyridine. The misture was stirred under an at,rnosphere of nitrogen for 20 min. :tt) 25", cooled to 10' in a n ice bath, and sulfur dioxide X:LS i n t r o d u c d over the surface for 15 n i i ~ i . The ~ ~ temperature ri rapidly to 20", then dropped gradual1)- to 10". T h e react' mixture was diluted to 250 ml. with ice water yielding a white precipitate of the 9(11)-dehydro compound, amounting to 0.53 g. :tft,er filtration and drying in uacuo. The crude 9(1l)-olefiii (0.53 g. I was dissolved in :L mixture of 10 nil. of niet~hylene vhloride :tiit1 18 nil. of t-butyl :tlcnhol: perchloric. acid i 7 O i 1 I O . 16 nil.' in 1.25 nil. of Jvater and 0.25 g. of X-bronioacetamide i n 2 , 5 nil. of t-but>,l alcohol were added. The mixture \\-:is stirrtd lor I5 min. a t 25-30" :tnd 0.25 g. 01 sodiiini sulfite in 2.5 i i i l . of \v:iter \vas added. Concentration of the solution in ,'nciio :md dilution with 100 mi. uf ice water gave after filtration :iritl drying 0.55 g. of the bromohydrin which was dissolved i i i :wetone ( 1 5 m1.j and heated under reflux r i t h 0.6 g. of potassium :wet:tte for 24 hr. The mixture wits evaporated to dryness, t t i r , residue partitioned bet>n.eeriwater :md methylene chloride, and the orginic rxtract Ivashed with n:ater :tnd dried over magnesium sii1f:ttc.. I-:vaporation of solvent gave 0.486 g. of n yellon- fo:tni roniatographed on 50 g. of Florisil. Elution with acetone-Plcellvsolve R removed trace of materi:il. n with 2.2 1. of the same solvent mixture ga.ie crystalline 17~,21-dihydroxy-l5p-fluoro-9~,11epoxypregn-4-ene-3,2O-dione21-acetate, n1.p. 205--207". X solution of 0.35 g. of the epoxide in 3.5 nil. of niethylene chloride was cvoled t o -70" and added to a mixture of anhydrouu hydrogen fluoride (3.0 g . ) and tetrahjdrofuran ( 5 . 3 nil.) at, -20". The mist,ure was allowed t o stand 16 h r . at -20' and 1 hr. at, +5', then poured into a stirred ice-cold suspension of 21.6 g. of sotiillin kiicarbon:ite in 160 nil. of m;tter. Iktraction with eth>.l acetate yielded 0.386 g. of t: j.ellon- foam. '!ho crystallizations gave 110 mg. of ( , 5 ) , m.p. 21.1-214'
iicetone-Ykellysolve Uj gave 0.3% #. of t i ) . ~:r~-stallizatioii from acetone-Skell3-solve B and aqueous acetone gave :in :t 11:)1ytio:il sample, 1ii.p. 235-238'; A,,,, 243 nip ( e 15,200). .-I nu/. Calcd. for C23H29F06: F:4.52. b'ouurl: F', .I.>I
Interest in tlic relativc anabolic -androgenic acti\.iti(+' oi some of tlie preiwxisly unknowi steroid diinc~thylliydrazones prepared in our Laboratories lias suggehtd
the desirability of reporting the methods of preparatioii and physical characterization data for these material\. The properties of the steroid dimet1iylhydrazo1ic.s ai given in Table I. ,111 n ere prepared by reaction of tlw sterol in excess diiiipthylhydraziiie with a trace of ace ti(^ acid as a catalyst. The sterols are readily soluble iii dimethylhydrazine and the product precipitates 011 staiidirig or is precipitated by addition of water. 111 tlici absence of acetic acid to catalyze the reactioii, tlic qteroid is often recovered unchanged. X typical pwparation is described in tlie follon ing paragraph (5
Pregnenolone Dimethylhydrazone.-I'regnenolone I, 1 g I \\ . I \ heated with 5 ml of dimethylhydrazine containing 2 drops i ) i acetic acid until a clear solution n as obtained. Tlic soliitioii On dilution with 30 1111 \\-AS kept 12 hr a t room tc,mperaturcl. This was collwtcvl, O f water, :i mhite precipitate n as formed n :tshed with water, a n d recrj stall17c~lfroin nicthanol 1 ),it 'I c haracteriiing t h e product are given in Table I
The hiological actil ity of these compounds i5 ( i t interest in providing further examples of the ob3c.i 1 ;Ition that the oxygen atom of the three position in steroitfi is not essential to actiyity2 a i d of the biological activity of the dimetlzylliydrazoiies.~ Thew hydrazones m:~ytw regarded ab acyclic derivativrs of the steroidal pyi :tzoles2 4 sorne of nhicli have dioivri enliaiiced aliaholic. activity4 or antiinflammatory activity.? I11 .;talidaitl assay procedures in rats,l tlic dinietliyl2iydrazoii(~iI )I t h e five testosteroiic derixTative> (Tablo I) lin\*r >lion 11 :i considerahly reduced aiial.)olic-androgciiic activity :I< compared to the correspoiiding carhoiiyl compouiitlThe 19-nor c h i \ a t i w , l i o w ~ e r rt.taiiih , the diffcrciiti:it ion betu-eeu anabolic aiid aiicli ogeiiic acti\-itici c)kt\rhi,\ t t i with other 19-nor type-
.And. Chlcd. for C:23H30E1206: (1, 62.71; IT: G . S T , I*'% g . 6 3 . Found: C, 63.02; H, 6.85, F, 8.12. 9~,l5p-Difluoro-llp,l7a,21-trihydroxypregna-l,4-diene-3,20dione 21-Acetate i6).---Ahout 300 mg. of crude ( 5 ) in 40 nil. of t-lJIltyl alc~oholc~nt~airiing 0.4 ml. of acetic acid \vas heated under wflus \\-it11 150 nig. of selenium dioxide for 44 hr. ..in :adtlitiorial 150 mp. of seleniurn diosidc \vas added :tnd heating n-:ts c~)ntiiiuedfor 19 hr. The misturc \vas evaporated t o d r j w s s . taken up in 150 ml. of ethyl acetate, ant1 washed with cold aqueous hodiurn bimrbonate, aqueous ammonium polysulfide, dilute nninioriium hydroxide, xat,er, dilute Iiydrcicliloric :icitl, :md water. 1~:v:ipor:itionof t,he solvent gave 0.326 g. of x i orange foam t,h:it. Flurisil in methylene cliloridr. ne-O2..;i' 1 niethFlene chloride ..5(>; inetli~-leiiechloride gave d u c t . Tliree crystallizatitrns from :icetone--Skt~ilysolve13 pave 60 ing. of pure (61, 1ii.p. 232.Acknowledgment.-S H ' T\ ishes to acknon ledge a 2Y3" tfec.; An,.,, 23Xnip ( E 16,000). fellowship grant held during 1958-1960 under the SmithA l ~ Calcd. ~ d for C2rH28F,0,: (!, 63.00; H, fi.44; F, h . 6 7 ~ Mundt dct. The authors also \\ish to express thr.11 Pound: C, fi3.26; H, 6.24; F,8.62. 1 lp,l701,21-Trihydroxy-l~~-fluoropregna-1,4-diene-3,2O-dione appreciation for assistance in the> biological evaluatioiib 21-Acetate [7!.--A solution of 0.42 g. of (4)in t-butyl alcohol ( I ) C 11. I l c I < i n n t \ dtnd ii i'l\nc I' DL S'm Ezptl 0101l f t d 108, (50 nil.) cwiit:tining 0.5 mi. of acetic acid !vas dehydrogenated 273 (1961) \\.it11 :t total of 0.41 g. of seleniuni oxide :is in the previous ex( 2 ) R. Hirschmann t r i n b r i g , I' HuLhschacher J Ii I I I P ~( I i :impIe. Chromatography o n Florid ( 100 g . ; using linear celman, J Im Chem Soc 8 5 , 120 ( l ' l i gradient elution ( 2 1. each of 1: 9 acetone-Pkellj-solve B and 1:G C'ie\engel J W e d Pharwk Chcrn , 6 , (15) E. A. Drake, R . B. ]Coward, and -1, E.Fonkerr, L.S. Pat. 3.00.5,834 Oct. 2 4 , 1961).
1962), and references cited theieln (4) R 0 Clinton I? L Clarke, F I\ Sitonner, 4 J \latison. 11 i Jennings, and D K Philliun, J O r u C h e m , 27, 2800 (1962).
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 8.28 200 92 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 13.11 218 96 Cortisone (bis) M C21H36N20 75.85 10.91 8.43 75.08 10.6 8.69 180 87 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 6.23 158 73 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 CHESTERE. HOLMLUND, LOLTIS I. FELD~UAS, NEIL E. RIGLER, RALPHH. EVANS,JR., ROBERTH. B L A N KA, N D 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.
