in addition to undergoing reactions (3b), C6HbS. iuoiiodeutero-L-malate which is produced enzycnters into two termination processes : matically made it possible to determine whether the breaking of the methylene carbon-hydrogen bond CGHLS. (C&&CH. --f ( C G H ~ ) ~ C H S C(V) ~ H S(4a.j is the rate determining step in the enzymatic 2 CGH~S. --+ CsHsSSCsHs (VI) (4b) The infrared spectrum of residues after isolation reaction. The breaking of the methylene carbondeuterium bond in deutero-1,-malate must proceed of I11 was identical with that of a solution prepared a t approximately one-sixth the rate of that for the and VI.7 Evi- corresponding from an authentic sample of carbon-hydrogen bond.5 If the dence for the occurrence of reaction (3b) was found breaking of a carbon-hydrogen bond were rate in the formation of tetraphenylethane 111 in ‘727; limiting, the over-all rate of reaction would be yield by exposure of a dilute solution of diphenyl decreased by a factor of six in the dehydration of disulfide VI in diphenylmethane to a sun lamp. deutero-L-malate. Studies with S35 have shown that disulfides disWhen monodeutero-L-malate was dehydrated sociate into mercaptyl radicals on irradiation.8 enzymatically a t pH 8.0 in 0.005 M tris-(hydroxylilank experiments indicated that compounds IV, methyl)-aminomethane perchlorate buffer it was 1. and VI did not cause formation of I11 from 11, found that both the maximum initial velocity and nor did they adversely affect the isolation of I11 Michaelis constant were unchanged after a correcfrom II.9 tion for a small amount of fumarate in the malate ( 6 ) C. Fingi and V. Bellavita, G a m chim. i f a l . , 62, 699 (193?). preparation. Thus the breaking of the carbon-(7) T. Zinke and W. Frohneberg, Bsv., 43, 840 (1910). hydrogen bond is not involved in the rate-deter( 8 ) E. N. Guryanova and V. N. Vasileva, Zhui.. F i z . Khinz., 23, GO mining step. It seems likely from kinetic argu(1954). (9) This work was supported by a grant from the S a t i o n a l Science m e n t ~ ~that . ’ the dissociation of furnarate from the Foundation. enzyme is not the rate-determining step for the SCHOOL OF SCIENCE dehydration reaction at high L-malate concentraBRANDEIS UNIVERSITY SAUI, G. C O H E N tions. C1rr F1r.A M ’ A N G \\‘AI,THAM 64, hf ASSACMUSETTS The fact that the deuterium of monodeutero-1.RECEIVED JUSE 11, 1953 malate was removed in a step which is not rate limiting suggested that there should be a sterically CONCERNING THE STEREOSPECIFICITY OF THE specific exchange of this hydrogen atom proceeding FUMARASE REACTION AND THE DEMONSTRATION at a rate faster than the dehydration reaction. OF A NEW INTERMEDIATE’ Such a rapid exchange was indeed found to occur Sir: in an experiment in which L-malate was dehydrated When fumarase catalyzes the addition of water enzymatically in 99.5% D20to the extent of 0.04%. to fumarate only the L-isomer of malate is pro- The amount of deutero-L-malate which could duced.2 While the hydroxyl group is added have been formed by the reverse reaction from the stereospecifically, the question still arises as to the fumarate so produced and initially present in the stereospecificity of the addition of the hydrogen sample would be immeasurably small. However, atom. Vb7e have been able t o demonstrate that the dipotassium malate recovered had incorporated this process is absolutely stereospecific. Di- about 0.003 atom of deuterium per molecule. potassium fumarate was added to a reaction mix- This relatively rapid exchange demonstrates the ture containing crystalline fumarase, 0.038 M existence of an intermediate in which the hydrogen K2HP04, and 0.015 I, I.eigh, T H I J~ OUHNAI.. 1 4 , 5933 (195?).
Aug. 20, 1955
COMMUNICATIONS TO THE EDITOR
cortisone acetate2 and hydrocortisone acetate3 from I has been described. In both of these schemes the 3-keto-A*-system was selectively reduced and reconstituted a t the end of the synthesis. We now report the synthesis of these and other members of the cortical family from I via "direct" chemical procedures which avoid this undesirable expedient. 11-Ketoprogesterone (11),obtained by oxidation of I , was treated with ethyl oxalate (1.1 mole) and sodium methoxide (1 mole) in benzene (2530'). The sodium salt of 21-ethoxyoxalyl-11ketoprogesterone (111) was precipitated in S590% yield by the addition of ether. Treatment of I11 with iodine in aqueous potassium iodide and subsequently with potassium acetate in methanol produced 11-dehydrocorticosterone acetate (IV) (m.p. 181-182.5') in approximately 40% yield. I n a similar way corticosterone acetate (VI) was prepared from 1ID-hydroxyprogesterone (V). 4 A solution of I11 in t-butyl alcohol and methanol, neutralized with acetic acid, was treated (0'') with bromine (2 moles) in the presence of sodium acetate. Subsequent addition of sodium methoxide in methanol gave methyl 3,11-diketo-4,17(20)[cis]-pregnadien-21-oate(VII)5 (60%), m.p. 212216", [ a I z 3 D +18g0 (acetone), iEmt,OxH 233 mp, E = 24,375. (Anal. Calcd. for Cz2Hzs04: C, 74.13; H, 7.92. Found: C, 74.40; H, 7.78). Methyl 3-keto-1la-hydroxy-4,17(20)-[cis]-pregnadien-21-oate (IX),6m.p. 205-210"; [ c x ] ~ ~ D 133' (acetone), HX:z: 239 mp, E = 22,425. (Anal. Calcd. for C22H3004: C, 73.75; H, 8.48. Found: C, 73.77; H I S.38), was prepared in a like manner from the salt of 21-ethoxyoxalyl-l1 a-hydroprogesterone (VIII).* The hydrolysis of VI1 with potassium hydroxide in aqueous methanol gave the corresponding acid (X), m.p. 252-254' (dec.), and 3,11-diketo-4,16pregnadien-21-oic acid (XI),m.p. 169-172" [methyl ester (XII), m.p. 114-115", [ ( r I z 3 D +179" (acetone), XgzH 239 mk, E = 15,450. Anal. Calcd. for C22H2804: C, 74.13; H I 7.92. Found: C, 74.34; H, 7.691. Esterification of X with diazomethane gave VII. However, sodium methoxide in anhydrous methanol converted VI1 to its 17,20trans isomer (XIII), m.p. 155-159', [ a I z 3+121" ~ (acetone), ",",A: 230 mp, E = 24,300. (Anad. Calcd. for CiH2804: C, 74.13; H, 7.92. Found: C, 73.93; H, 7.66). Treatment of VI1 with pyrrolidine and p-toluenesulfonic acid according to procedures described by Heyl and Herr,' resulted in the quantitative formation of the corresponding 3-enamine (XIV). An analytical sample melted a t 181-183"; - 110' (CHC13); hk:xHC'-MeoH 225 mp, E = 14,550;
+
(2) 0. Mancera, A. Zaffaroni, E. A. Rubin, F. Sondheimer. G. Rosenkranz and C. Djerassi, ibid., 74,3711 (1952). (3) R . H. Levin, B. J. Magerlein, A. V. McIntosh, A. R. Hanze. (1.S. Fonken, J. L. Thompson, A. M. Searcy, M. A. Scheri and E. S . Gutsell. ibid., 76,502 (1953). (4) J. A. Hogg and A. H. Nathan, U. S. Patent 2,683,724 (1954). ( 5 ) J. A . Hogg, P. F. Beal and F . H. Lincoln, U. S. Patent 2,707,184 (1955). (6) J. A. Hogg, P. F. Beal and F. H. Lincoln, U.S. Patent 2,6R5,900 (1954). (7) F. W.Heyl and M. E. Herr, THIS J O U R N A L , 76, 1918 (1953).
4437
274 mp, E = 21,825. (Anal. Calcd. for CZGHabN03: C, 76.24; HI 8.61; N, 3.42. Found: C, 76.34; H, 8.31; N, 3.64). The crude enamine (XIV) was reduced with lithium aluminum hydride in dibutyl ether, N-ethylmorpholine or etherbenzene to 3-pyrrolidyl-1lp,21-dihydroxy-3,5,17(20)- [cis]-pregnatriene (XV), m.p. 228-232" (dec.), [ L L ] ~ ~ -88" D (chloroform). (Anal. Calcd. for C25H37N02: C, 78.28; HI 9.72; N, 3.65. Found: C, 78.31; H, 9.82; N, 4.03). The 3-keto-A4system was regenerated by hydrolysis with alkali in methanol-water yielding 11P,21-dihydroxy-4,17(20)- [cis]-pregnadien-3-one(XVI),5m.p. 156-158", [ a ] 2 3 ~+12so (acetone), xE:~H 242.5 mp, E = 16,100. (Anal. Calcd. for C21H3003: C, 76.32; H, 9.15. Found: C, 76.04; H, 9.43). Acetylation of the total crude XVI yielded the 21-acetate (XVII)5 (70-76y0 over-all from VII), m.p. 190191", [ L L ] ~ ~+128" D (acetone), xE~H 243 mp, E = 15,750. (Anal. Calcd. for C23H3204: c, 74.16; H, 8.66. Found: C, 74.18; H, 8.45). Chromic acid oxidation of XVII gave 21-hydroxy-4,17(20)[cis]-pregnadien-3,1l-dione21-acetate (XVIII), m.p. 196-199", [a!Iz3D 145' (acetone). (Anal. Calcd. for C23H3004: C, 74.50; H, 8.22. Found: C, 74.37; H, 8.43). Alternatively, reaction of VI1 with ethylene glycol and p-toluenesulfonic acid5 afforded the 3-ketal (XIX), m.p. 188-190°, [ a I z 3 D +go, XzH: 225 mp, E = 13,525. (Anal. Calcd. for Cn4H3205: C, 71.94; H, 8.05. Found: C, 71.90; H, 7.95). Reduction of X I X with lithium aluminum hydride gave the 3-ethylene ketal (XX) of XVI, m.p. 185188", [ C X ] ~ ~+D4 O (acetone). (Anal. Calcd. for C23H3404: C, 73.76; H, 9.15. Found: C, 73.87; H, 9.22). Acid hydrolysis of XX yielded XVI. In a similar manner XI11 was converted to l l P , Z l dihydroxy - 4,17 - 4,17(20) - [trans]- pregnadien - 3one (XXI), m.p. 181-185", [ a ] 2 +120° 3~ (acetone). (Anal. Calcd. for Cp1H3003: C, 76.32; H, 9.15. Found: C, 76.60; H, 9.33). The acetate (XXII) ~D (aceof XXI melted a t 107-110"; [ L L ] ~ +113O tone). (Anal. Calcd. for C23H3204:C, 74.16; H, 8.66. Found: C, i4.16; H I 9.07). Evidence for the stereochemical assignments a t the 17,20-double bonds in XVII and X X I I and their precursors was provided by oxidation studies a t these positions. Hydroxylation of XVII with osmium tetroxide according to procedures similar to those described by Milass or Reichstein9 gave 1I@, 17a120a,21-tetrahydroxy-4-pregnen-3-one 21acetate (XXII), m.p. 228-229', [ a I 2 3 ~+89" (acetone). (Anal. Calcd. for C23H3406: C, 67.95; H, 8.43. Found: C, 68.33; H, 8.29). Upon saponification, X X I I I yielded the corresponding tetrol (XXIV), m.p. 256-260°, [ a I 2 3 ~ 65' (acetone). ( Anul. Calcd. for C21H3205-2H20: C , 63.00; H, 9.05. Found: C, 63.45; H, 9.32). Similarly, the trans-isomer10 (XXII) was converted to 11~,17a,2OP,21-tetrahydroxy-4 - pregnen- 3 -one
+
+
(8) N. A. Milas and S. Sussman, ibid., 68, 1302 (19%). (9) D. A. Prins and T. Reichstein. Heiu. Chirn. Acta, 26, 300 (1942). (10) It has been shown t h a t trans-17,ZO-steroid olefins yield 17m,208-
glycols: see Fieser and Fieser, "Natural Products Related t o Phenanthrene,'' Third Edition, Reinhold Puhlishing Corporation, New York, N. Y., 1949, pp, 410-119.
COM.MUNIC.ITIONS TO THE EDITOR
4438
Vol. 77
(XXV, Reichstein's Substance E"), m.p. 1301 3 2 O , identified as the 20,21-diacetate XXVI, m.p. 230-231.5"; [cx]'~D 168" (acetone). [Keported" m p . 229-230'; [ a l 2 ' D f163" (acetone)]. (Anal. Calcd. for C26H3607: C, 66.94; H, 8.09. Found: C, 66.69; H, 8.07). That XXIV is the 30a-epimer of Reichstein's Substance E was established by the oxidation of its 21-acetate (XXIII) with manganese dioxide to hydrocortisone acetate (XXVII), m.p. 317-2?O', [a]"D +139" (dioxane), XgzH 243 mM, E = 13,900. Xlso, the oxidation of XXIII with chromium trioxide in pyridine yielded cortisone acetate (XXVIII), 1n.p. 244 .'48", identified by its infrared spectrum and paper chromatogram. The oxidative hydroxylation of XVII in t-butyl alcohol-pyridine with phenyl iodosoacetate in the presence of catalytic amounts of osmium tetroxide afforded hydrocortisone acetate (XXirII) in 65%, yie1d.l' In like manner XXII and XVIII were converted to hydrocortisone acetate (XXT'TI) and cortisone acetate (XXVIII), respectively. The methods described h a w also been applied to progesterone and other ?@ketosteroids. The authors are indebted to X.Koniiig, K. 11.. Kratz, C. D. n'ickman and IT. K. Shellinan for technical assistance, to Dr. J . L. Johnson, Mrs. G. S. Fonken and J. E. Stafford for infrared and ultraviolet absorption studies, to Alr. N7.-1. Struck and associates for microanalyses and to Mr. L. A I . Reineke and associates for paper chromatographic analyses.
moles of diethyl oxalate and sodium niethoxide i n t-butyl alcohol. The yellow disodium salt of I precipitated at once. This slurry was neutralized with acetic acid-methanol and treated with three moles of bromine in the presence of sodium acetate. Subsequent treatment with sodium methoxide in methanol gave a 58.3y0yield of methyl 2-bromo~~,ll-cliketo-4,17(20r-[cis]-pregnadien-2l-oate (I1j , [ c Y ] ' ~ D$-20!)3(chloroform); (-4ncrL. n ~ p160-162'; . Calcd. for C22H27Br0.,:Br, 18.35. F(;untl: I!r, 18.4tii. Debromination of a sample of I1 with zinc and acetic acid yielded inethyl S,l t-diixto-$,IT(20i-[cis]-pregnadien-2-oate (III).l Ikhydrohalogenation of 111 with collidine or with lithium chloride in dimethyl fornianiide4 produced methyl :1,11 - diketo - 1,4,17(?0) - [cis]- pregnatrien-21 -oute (IVi ( i O C / b yield!, 11i.p. .'40--243"; [ c u ] : " ~ +210" (chloroform'); (Anal. Calcd. for C?."ZGO.I: C , 74.55; H , 7.39. Found: C, 74.30; H, 7.14 . Conversion of 11: to the pyrrolidyl enamine followd by reduction with lithium aluminum hydride ant1 removal of the enamine, as previously described, afforded 1 1p,? 1-dihydroxy-1,4,17(?0)- [cis]-pregn:itrier-3-one (\TI, 11i.p. I 74.--175'., [a]"ju i-110' ichloroformj ; (A7zuZ. Calcd. for C~~1-12~0:~: C, 7tj.78; H, h.59. Found: C, '76.98;H, 8 . 7 ; < , . The conversion of IT' to V also can bc efi'ectetl b!. blocking the %keto grouping as the ethylene bc.t:ii instead of the enamine. Acetylation o f 1- gi\-c.s quantitatively the 21-acetate (\TI), 11i.p.220.-222.3" ; [ a ] ~f122' (chloroform). (Anud. Calcd. f o r C2;IHaa04: C, 74.33;H, S.16. Found: C, 7-&.+jaq; H, S.3lb. In 65ch yield5 TI is convcrted 1))(11) T. Reichstein and J . von Euw, H l l z . C'hifif. A c t m , 2 4 , 2 4 7 E phenyl iodosoacetate in the presence of osmiuiii (1941). tetroxide i o 1 1~,17cu,21 -trih~drosy-l,4--pregii~~i(12) K. Lliescher and J . Schmidlin, i b d . , 33, 18-10 (1950), have described the use of hydrogen peroxide and osmium tetroxide for ene-3,20-dione-2 1-acetate iV11), ? i11.p. ?40-24.'3 : the oxidative hydroxylation of t h e steroidal 17,20-doubIe b o n d . This [ c u ] 2 4 ~+ I I C(dioxane;; ~ " '!::A 2-12 i n p ; E 13,225; procedure in the present studies gave hydrocortisone acetntr i n (L4jzui, Calcd. for C23H:;"OO: C , 6S.K;; 13, 7 . 2 ! . 35-40C'c yield when applied t o XVTI. ; H, 7.32,. Saponification (if \'I1 ?a,? 1-trihydroxy-1 ,-L-pregnadieneRESEARCH LABORATORIES 1,' (90',() yield), 111.1). 232-236' ; I:. H. Lrzcotx THECPJOHN COXPANY 102' (dioxane) ; (dm l . Calcd. for C O ~ H ~ ~ ( ~ ~ : KALAXAZOO, & I I C H I G A S 11.. P. SCHNEIDQR[ U]D U.J. MACERLEISC, 69.97;H, 7.83. Found: C, 60.79; 13, 7.SSl. -4. K.HASZE Oxidation of VI1 with N-bronioacetaniide in f-butyl I t . 11.. JACK SO,^ alcohol-pyridine formed 1i , 2 1-dihydrosy-I ,.i-pregK E C J ~ I V EJ UDL Y ls5,l!hX (q-' c d , I, nadiene-3,l I ,20-triorie-?l-acetate iTX) !,ield!, m p . 230k238" (dec.); [ m ] D lh5' (cliosanel; (.4ncilI. Calcd. for CaaHas05:C, 68.98; 1-1,7.05. THE ADRENAL HORMONES AND RELATED COM- Found: C , W.9>; 13, 6.65). Hydrolysis oi IX POUNDS. 11. SYNTHESIS OF I-DEHYDRO n-ith potassium bicarbonate i i i quecrus inethanol ANALOGSL gave 1ia,21-diliydroxv-l,-~-pregnadienc-:',,11 ,?O-triSi?: one (X)? (91 ' i yield), n1.p. 330.;5---2X2.33; [ o i ] ? ' : ~ ) In the previous paper of this series we described t 169" (tiiosane!; ( i l i i a L . CnIccl. for C'21Hd)a. the preparation of 2 1-ethoxyoxalyl-ll -ketoprog- C , 7 0 . 3 7 ; H, i . 3 1 . Fount1 : C ,70.(;4; H,7 . 2 0 . . csterone and its conversion to certain of the adrenal Pehydration of \'TI with thionyl chloriclr pyridine hormones. 11-e now report the preparation of ?,?Itli-(ethoxyoxalyl'i-l 1-ketoprogcsterone ( I ) and its conversion to 9a-fluoro-1-dehydrohydrocortisonc acetate via 1-dehydrohydrocortisorle acetate.? I 1 -Ketoprogesterone, prepared by the method of Peterson, Murray, e/ ~ z l . was , ~ treated with two 4' K .P. Holysz, LEiLfi., 7 5 , 1433 (19383.
+
+
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(1) Preceding paper in this berieb, T H I S J O U R N A L , 77, $436 (1955). (2) H. L . Herzog, A. Nobile, S. Tolksdorf, W. Charney, E. B. Hershberg, P . L. Perlman and M. &I. Pechet, Science, 121, 176 (1935). (3) D. H . Peterson, H. C. Murray, S. H . Eppstein, L. R I . Reineke. A . Weintraub, P . L). Slei,ter and H. R f . I . e i g h 'I H I S I U I ? K N A I . , 76, 8933 (1052)
'
i! K. Iliescher and J . Schmidlin, Hcla. chim o c t o , 33, IS40 (I%.>O have described t h e oxidative hydroxylation o l t h e steroidal 17 9 0 In this double bond wirh hydrogen peruiide o ; l d osiuiiini tetmY:ir!e work I-drhydrohydrocorliione acet.ite IYLS produced I:, 'I:?'; y i e l r l lroiu V I L y their p ~ u r r d u r r I'rulunged wurktip u i t i i resulted iu apprecidble ti! d r u l y 3 i a tu tlir t i e r ~ i i i - v l i i i l
