COMMUNICATIONS TO THE EDITOR
Aug. 20, 1957
TABLE I FORMATE-CI4, METHIONINE-METHYL-C14AND SERINE-3-C" AS C-24 METHYL DONORS' Source of C"
HCOOH HCOOH HCOOH HCOOH HCOOH Methionine Methionine Methionine
Non-radioactive additions
...
Homocysteineb Homocysteine, folic acid Homocysteine, aminopterin Methionine
Non-saponifiable fraction Total S. A. counts Yield, Mg. X 10-8 X 1 0 - 8 %
3.7 3.5
2.3 4.3
8.6 15.2
6 10
4.0
6.4
25.7
17
4.2 3.8 3.9 5.2
0.8
3.5 2 0 . 4 0.3 37.2 25 31.5 22
0.1
... 9.5 Formate 6.1 Formate, homocysteine 3.3 2.5 8.5 6 Methionine Formate, hornocysteine, 3.7 6.2 20.3 14 aminopterin Methionine Aminopterin 1.9 34.4 65.4 44 Methionine Serine 3.6 28.4 102.1 68 Serine ... 3.5 4.7 16.6 11 a Each flask contained 4 ml. of homogenate made from 1 g. of dry yeast, 2 X iM A T P and 0.5 pc. of CI4 (1 mc./ mmole). Additions per flask (where indicated) : homocysteine, 5 mg.; folic acid, 1 mg.; aminopterin, 2 mg.; methionine, 15 mg.; HCOONa, 5 mg.; serine, 15 mg.
4555
without destroying its glucocorticoid activity, we have investigated the effect of l6a-hydroxylation on the activities of 2a-methyl steroids.2 Hydrolysis of 21-acetoxy-3,20-bis-ethylenedioxy5-pregnene-11/3,16a,17a-triol (I) in dilute acetic IVa, IVb, XIIIa, XIIIb,
X = H,R X = H, R X = F, R X = F, R
CH20R
= H = Ac
I c=o
=H = Ac
Ho\/\'/C.,R ---OH
I, x I
acid afforded 2 1-acetoxy-20-ethylenedioxy1lp, 16a,17a-triol-4-pregnen-3-one (11), m.p. 262-263', [ a I z 5 ~ 85' (CHCI3); (Anal. Found: C, 64.77; H, 8.03). Treatment of compound 11 with ethyl oxalate and sodium methoxide in t-butyl alcohol formed the sodium enolate of 20-ethylenedioxy-2ethoxyoxalyl- 11/3,16a,17a121 -tetrahydroxy-4-pregnen-3-one (111) as a pale yellow amorphous solid. Methylation of I11 with methyl iodide and potassium carbonate in acetone followed by removal of the ethoxyoxalyl group by sodium methoxide in methanol gave a glass. After removal of the 20ketal group with dilute ethanolic sulfuric acid, TABLE I1 partition chromatography4 yielded lip, 16a,17a,21TRITIUM:CARBON~~ RATIOSIN METHIONINE AND ERGOS- tetrahydroxy- 2 a -methyl- 4 -pregnene- 3,20 -dione (IVa) apparently with one molecule of acetone of TEROL Each e x p ~ m e n consisted t of 5 flasks, each containing crystallization, m.p. 201-203", Amax. 240-241 ml.c 4 ml. of yeast extract, 5 mg. of serine, 2 mg. of aminopterin, (E 16,600),5 [ c ~ ] * ~ D 145' (CHC13); ( A n d . 1 mg. of ATP, and 0.175 mg. of doubly labeled methionine. Found: C, 65.59; H, 8.39). Acetylation gave the Incubation time was 48 hr. ; radioactivities were determined on a Packard Tri-Carb Scintillation Counter. The values 16a,21-diacetate IVb, m.p. 253-254', Amax. 240241 mM (E 17,500), .E,",'. 3450, 1743, 1726 (shoulder), given are the averages of four samples. T : C " Ratio 1654, 1620 and 1238 cm.-', [cx]*~D 92' (CHC13); Expt. Methionine Ergosterol (Anal. Found: C, 65.43; H, 7.75). Oxidation of 1 1.12 f 0.06 0.97 f 0.06 IVb with chromium trioxide-pyridine reagent6 2 1.12 f 0.06 1.02 f 0.02 gave 16a,2 1-diacetoxy-17a-hydroxy-2 oc-methyl-4methyl-T, was incubated with yeast homogenates pregnene-3,11,20-trione (V), m.p. 240.5-241.5', and the resulting ergosterol rigorously purified] the [ a I z 5f~ 129' (CHC13); (Anal. Found: C, 65.49; T:CI4 ratios of the substrate and product showed H I 7.30). Acetylation of 3,20-bis-ethylenedioxy-5-pregthat all three hydrogen atoms of the methyl group (VIa)' yielded the 16a1of methionine are transferred to ergosterol. Were nene-11/3,16a,l7a,21-tetrol the methyl group oxidized to the level of formal- 21-diacetate (VIb), m.p. 129-135°1s [cUIz5D dehyde, one third of the tritium would be lost, and 61.5' (CHC13); (Anal. Found: C, 62.49; H, 7.81). the T:C14ratio in ergosterol (Table 11) would have Treatment with phosphorus oxychloride in pyridine been 67%. Since the ratio is 86-91%, a t least some afforded 3,20-bis-ethylene-dioxy-l6a,21-diacetoxymethyl groups of methionine must have been 5 9( 11)-pregnadien-17a-01 (VII) m.p. 22 1-224', [ a I z 5~ 48" (CHClJ; (Anal. Found: C, 65.52; transferred intact to the carbon 24 of the sterol. H , 7.67). Hydrolysis of VI1 in dilute acetic acid WORCESTER FOUNDATION FOR EXPERIMENTAL BIOLOGY 17a-hy16a,21-diacetoxy-20-ethylenedioxySHREWSBURY, MASS. GEORGE J. ALEXANDERgave ERWIN SCHWENK droxy-4,9(1l)-pregnadien-3-one (VIII), m.p. 184.5RECEIVED JULY 2, 1957 1S6', [ a I z 5 5 ~ 0" (CHC13); (Anal. Found: C, 66.63; H, 7.65). The sodium enolate (IX) of the 2-ethoxyoxalyl
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~
16-HYDROXYLATED STEROIDS. V.' THE SYNTHESIS OF THE 16a-HYDROXY DERIVATIVES OF 2a-METHYL-STEROIDS
Sir: I n view of our previous Communication1 concerning the ability of the 16a-hydroxyl group to abolish the sodium retaining property of a steroid
(2) J. A. Hogg, F. H . Lincoln, R. W. Jackson and W. P. Schneider, rbid., 7 7 , 6401 (1955). (3) W.S.Allen and S. Bernstein, ibid., 78,3223 (1956). (4) R. Littell and S. Bernstein, ibid., 78,984 (1956). (5) T h e ultraviolet spectra were determined in absolute alcohol
solutions. (6) G. I. Poos, G. E. Arth, R. E. Beyler a n d L. H . Sarett, THIS JOURNAL, 7 6 , 422 (1953). (1) Paper IV,S. Bernstein, R. H. Lenhard, W. S. Allen, M. Heller, (7) W. S.Allen and S.Bernstein, ibid., 78,1909 (1956). R . Littell, S.M. Stolar, L. I. Feldman a n d R. H. Blank, THISJOURNAL, ( 8 ) This compound seemed t o be solvated and could not be brought 78,5693 (1956). to a better melt or analytical value.
COMMUNICATIONS TO THE EDITOR
4,j.S C i
i - ( ~ li!) .
derivative of V l I I (free steroid) was obtained as an pared3a-11and many of these substances exhibited amorphous solid in the manner described above, unusual biological activity. Treatment with methyl iodide and potassium carWe now wish to describe the synthesis oi a new bonate in acetone followed by reaction with sodium series of biologically active 19-nor compounds, methoxide in methanol and finally hydrolysis in namely, the 4,5-dihydroallo derivatives of nortestosdilute methanolic sulfuric acid yielded after parti- terone and 17a-alkyl substituted nortestosterones tion chromatography4 16~~,17a,21-trihydroxy-2a-as well as the corresponding 315,liP-diols. (Xa), 1n.p. methyl-4,9(ll)-pregnadiene-3-20-dione, While catalytic hydrogenation of Ia, Ib arid I C 203-207, [ a I 2 ' D 103' (CHCla); (Anal. Found: led to mixtures of the rings A/B cis and trans c o n C, 70.75; H , 8.29). Acetylation afforded the pounds, it was found that reduction of the unsat16a,21-diacetate (Xb), m.p. 221.5-224', [ a l Z 5 ~ urated ketones in ether-dioxane solution with lith104' (CHCl,); (Anal. Found: C, 68.10; H, 7.53). ium in liquid ammonia4 followed by ammoniuni ;Iddition of N-bromoacetamide and 10% per- chloride decomposition, furnished in excellent chloric acid to a solution of X b in dioxane gave the yield the dihydroallo derivatives : 19-norandrosbromohydrin X I as an amorphous solid, m.p. tan-17p-ol-3-one (IIa) (m.p. 130-132', [ a ] ~ 131-134' which could not be purified. Treatment 60°.5 Found for C18H2802:C, 78.34; H , 9.94); of X I with potassium acetate in acetone furnished 17cr-methyl-19-norandrostan-l7@-ol-3-one(LIb) the 9/3, llp-epoxide X I I , m.p. 222-223', [ c ~ ] ~ ~(m.p. D 145-146", [DID +35". Found for c19- 34' (CHC13); (Anal. Found: C, 65.57; H , 7.49). HzOO2: C, 78.49; H , 10.40); and 17cu-ethyl-19Hydrofluoric acid converted XI1 to 16a,21-diace- norandrostan-17p-ol-3-one (IIc) (m.p. 2 12-2 13O , toxy- 90 -fluor0 -11/3,17a- dihydroxy- 2cr -methyl- [ a ] D 4-33'. Found for C20H3202:C, 78.47; H , 4-pregnene-3,20-dione (XIIIb), m.p. 14O-20Oo8, 10.49). Reduction of the 17-vinyl (Id) and the Xmax 237-238 mp (E 16,300), 3420, 1740, 1732, 17-ethynyl (Ie) compounds by this technique resulted in saturation of the 4,5-double bonds only, 1723, 1660, 1627 (shoulder) and 1235 cm-'; (Anal. F, 3.87. Found: F, 4.29). The corre- furnishing, respectively, 17a-vinyl-19-norandros~ qonding 16a,21-diol XIIIa formed from X I I I b by tan-lip-ol-3-one (IId) (m.p. 192-193", [ a ]+47". C, 79.18; H , 10.05) and 1701potassium hydroxide hydrolysis melted a t 231- Found for C20H3002: (IIe) (111.p. 234' d., Amax 237-238 mp (E 15,100), v::: 3450, ethynyl-19-norandrostan-17~-01-3-one Found for C20H2802: e, 1720, 1660, and 1635 cm.-', [cY]'~D 115' (pyri- 932-223", [ a ] +~G o . dine); ( A d . Found: C, (54.30; H, 7.66; F, 4.57). 80.30; H, 9.52). T h a t the unsaturated sideBi~-assays.~-Preliminary assay (rat liver glyco- chains had withstood the reduction conditions was gen procedure) of l lp,l6a,lia,21-tetrahydroxy-demonstrated conclusively by the conversion of 2a-methyl-4-pregnene-3,20-dione (I1-a) indicated IIe to I I d by partial hydrogenation (palladium on definite activity (less than that of hydrocortisone) ; calcium carbonate-pyridine) and the derivation of the lBa,?l-diacetate IVb and the 16a,21-diacetoxy- I I c from either IId or TIe by reduction over pal11-one V were inactive. In the same assay, 9a- ladium-carbon in methanol solution. The A/B fluoro - llj3,16a;77a,21 - tetrahydroxy - 2a - ineth- allo configuration for compounds I1 which could y1-4-pregnene-3,20-dione(XIIIa) , and its di- be predicted on thermodynamic grounds,6 is acetate XIITb were found to be a t least two times firmly established by the rotatory dispersion curves7 of these dihydro compounds, the curves being viras active as hydrocortisone. In the rate electrolyte (sodium retention) assay, tually identical with that of androstan-l'ip-ol-3-one. Treatment of I I a through IIe with sodium boroIYa, IITb and V were inactive. The Sa-fluorocompounds X I I I a and X I I I b exhibited minor ac- hydride in aqueous dioxane gave the corresponding I I I a (m.p. 168tivity (much less than that of desoxycorticosterone). 19-norandrostan-3~,17@-diols: l T O " , [ a ] 4-37'". ~ Found for C ~ ~ H ~ O O ~ . ~ C ; ' H ~ O : (9) T h e assays were done hy L. Bortle, E. Hepder, J. Perrine, E. C, 7 2 . S S ; H, 10.94); ITIb (m.p. 174-176", [ a ] D Ross, a n d I. Ringler (Experimental Therapeutics Research Section of .too. Found for C19H3202,2C3H60:C, 73.76; these Laboratories). H, 11.12); IIIc (m.p. 181-183", [ a ] . +2". Found SEYMOUR BERNSTEIS for C20H3402: C, 78.20; H, 11.033); ITId (m.p. ORGASIC CHEMICAL RESEARCH SECTION MILTOSHELLER [DID 4-9'. Found for C20H3202: c, RESEARCH DIVISION R U D D Y LITTELL 167-169",
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+
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v::
+
XMERICAN CYANAMID COVPASY
PEARL RIVER,NEW YORK RECEIVED J U L Y
STEPHEN M. STOLAR ROBERT H. LENHARD (3) (a) L. Miramontes, G. Rosenkranz and C. Djerassi, T H I S WILLIAMS. ALLEN J O U R N A L , 73, 3540 (1951); 76, 4440 (1953); (h) .4. Sandoval, L. Miramontes, G. Rosenkranz, C. Djerassi a n d F. Sondheimer, ibid., 2 , 1957 75, 4117 (1953); (c) A . L. TVilds and S . A . Selson, i b i d . , 76, 53GF
STEROIDS. LXXXIX.' 19-NORDIHYDROTESTOSTERONE DERIVATIVES. A POTENT CLASS OF ANTI-ESTRO GENIC C 0MPO UND S.
Sir: Following Birch's2 synthesis of lg-nortestosterone (Ia) in 1940 a number of 19-nor analogs of the steroid hormones and metabolites have been pre(1) Paper l , X X X V I I l , J . Korno, . Knsmkranz and 1:. Srmrlheirnci. J O T , R N . I I . , 7 9 , in preqs, ( I q 5 7 ' . ( 1 ) A J . Tjircli .1. i I1i.111 \ ( I C , , 31,7 ( I ! 4 n I l 1
'1 H I S
I
(1953); (d) C. Djerassi, L. LIiramontes, G. Rosenkranz nnd I:. Sondheimer, i b i d . , 76, 4092 ( 1 9 5 4 ) ; (e) A . Zaffaroni, H. J . Rincolrl, C-. Rosenkranz, F. Sondheimer, C . €I. T h o m a s and C. Djeraisi, i b i L 76, 0 1 0 ( l Q 5 j ) ; ( f ) R.J. LIagerlein and J. A . IIogg, z h i J . , 79, 1508 (1!).;7), ( g ) 1'. R. Colton. I, S . Nysted, B. Rienel and A 79, 1123 ( 1 ! 4 5 i ) ; (h) F. R.Colton, U. S P a t e n t 2 , i (4) Cf,I;. Sondheimer, R . Yashin, G . Rosenkr TIIE J O U R N A L , 74, 2695 (lY.52). ( 3 ) All melting points are uncorrected and rutations were determined a t 20° in chloroform. T h a n k s are due M r . E. Denot for his able technical assistance a n d to Mr. E. Avila for rotations and spectra. (6) See D . H. R . Barton and C. € l , Rohinson, J . Chem. S O L . 30.15 , (lna?), and references cited t h e r e i n (7) We are gratefill t n I ' r o f F . v x C . I)jeras.i, W a y n e S t a t e Lriiivcr s i t y , f c r dctertniniLtioii ani1 coinparison of r n t a t o r v d i < i ) r r 4 n n ? . (8) i\nalytic:il S:LIII~IIY stililiiiivii i n hi;h \ A C ' I I I I I I ~
