COMMUNICATIONS TO THE EDITOR
3160
Yol.
so
COMMUNICATIONS T O T H E E D I T O R 16-METHYLATED STEROIDS. I. 16a-METHYLATED ANALOGS OF CORTISONE, A NEW GROUP OF ANTI-INFLAMMATORY STEROIDS
The 1Ga-methyl configuration is assigned t o the entering group on the basis of rotational data and precedents for a-side a t t a c k g Sir: Reaction of I with acetic anhydride-toluenesulDuring the nine years which have elapsed since fonic acidlo afforded a mixture of non-crystalline the discovery t h a t cortisone was effective in the enol acetates from which after oxidation with pertreatment of rheumatoid arthritis, intense efforts benzoic acid followed by an alkaline hydrolysis, have been expended toward modifying the struc- 16a-inethylpregnane-3 a , 17a-diol-3,20-dione (II ) , ture of this hormone in the hope of finding a related m.p. 135-187', [ a ] 60" (Anal. Found: C, 73.57; compound with superior therapeutic properties. H, 9.38) was obtained. Bromination of I1 a t (2-21 Noteworthy candidates have included All4 and was followed by conversion of the resulting bromide A1,4.Ganalogs, 2 a , b (2-9 halogenated derivatives,2c to 1Ga-methyl pregnane-3aj licu,21-triol-l1.20-dione C? and c6 methylated analogs,2dfeC-14*' and 21-acetate ( I I I ) , 1n.p. 182-1S&", [ a ] i j " (Anal. C-I 6'" hydroxylation products and compounds com- Found: C, 08.51; H, 5.41) by iiieans of potassium bining these furictioiis.?",',j," acetate-sodium iodide in boiling acetone." The It has been established that in addition to reduc- corresponding 3-ketone, 1Ga-methylpregnane-1Sa,tions in the A-ring, two pathways of metabolic in- 21-dio1-3,11,2O0-trione 2l-acetate, IV, n1.p. 235activation of cortisone or hydrocortisone involve 240" [ a ] 7G" (tetrahydrofuran), (Anal. Found: reduction a t C-203,4to an alcohol and scission of C, 69.00; 1-1,S.04) was obtained froin 111 by oxidathe side chain4,jto 17-keto compounds. tion with the chromic anhydride-pyridine coniOn the hypothesis t h a t the side chain could per- plex. l? Bromine in acetic acid was added to a soluhaps be stabilized against such catabolic inactiva- tion of I V in chloroform affording 4{-bronio-l(jcu-91. . tion by an appropriately placed but otherwise Iziethylpregnane-170(,'71-diol-3,1.1,20-trione chemically inert substituent, some analogs of corti- acetate (V), dec. 240-241 ', [ a ] SA" (tetrahydrosone containing a 1Gcu-methyl group were synthe- furan), (Anal. Found: C, 5'7.93; H, 6.70; Br, sized. 16.16) and the latter with semicarbazide (cf. ref. Introduction of the methyl group was accom- 10) was converted to 16a-1nethyl-l-preg~lene-l7a,plished by reaction of 16-pregnene-3a-ol-l1,20-21-diol-3,11,20-trionr 21 -acetate 3-semicarbazone dione acetate6 with methylmagnesium iodide7 t o (IT), dec. 225-228", Amax 2G9 mp,13 E 25,400 (Anal. give 16a-methylpregnane-3a-01-1 1,20-dione, I, m.p. Found: C, 63.56; H, 7.31; N, 9.07). 155-157", [ a ] 110',8 (Anal. Found: C, 76.26; Pyruvic acid treatment of 1 3 provided 1GaH, 10.04). The acetate of I had m a p . 157-15SC, methyl-4-pregnene-1 7a,21-diol-3,11,20-trione2 1[CY] l l S o , ( A n d . Found: C, 74.08; H, 9.12). acetate (ITII), 1Ga-methylcortisone acetate, m.p. 207-210°, [ a ] 181', Xmax 238 mp, E 15,400 ( A d . (1) P. S. Hench, E. C. Kendall, C. H . Slocumb and H . F. Polley, Found: C, 69.11; H, 7.57). Dehydrogenation of Proc. S f a f M e e t . M a y o C l i n i c , 24, 181 (1949). VI1 b y means of selenium dioxide14 led t o 16a(2) (a) H . L. Herzog, A. Nobile, S. Tolksdorf, W. Charney, E. B. Hershberg, P. L. Perlman and M. M. Pechet, Science, 121, 176 (1955); methyl - 1,4 - pregnadiene - 17a,21 - diol - 3,11,20(b) D. Gould, E. L. Shapiro, 31.J. Gentles, E. B. Hetshberg, W. trione 81-acetate (VIII), m.p. 210-212", [ a ] Charney, A I . Gilmore, S. Tolksdorf, M. Eider, P. L. Perlman and 1SO" (6,0.00G),8A,, 23s mp, E 15,400 (Anal. Found : Vi. hl. Pechet, THISJ O U R N A L , 79, 502 (1957); (c) J. Fried and E. F. C, 69.42; H, 7.55) in satisfactory yield. Sodium Sabo. ibid , 75, 2273 (1953): J, Fried and E. F. Sabo, ibid., 76, 1455 ( l 9 5 i ) ; (d) J . A. Hogg, F. H . Lincoln,R. W.Jackson and X'. P. Schneider, borohydride effected reduction of the C-11 caribid., 77, 6401 (1955); (e) G. B. Spero, J. L. Thompson, B. J. hlagerbonyl functions of the 3,20-bis-~emicarbazones'~~'~ lein, A. R . Hanze, H. C. Murray, 0. K. Sebek a n d J. A. Hogg, i b i d . , of VI1 and VI11 whence, after hydrolysis a t C-3 78, 6213 (1956); (f) E. J. Agnello, B. L. Bloom a n d G. D. Laubach, and C-20 arid acetylation, 16a-methyl-4-pregneneibid., 77, 4684 (195.5); (g) W. S. Allen and S. Bernstein, ibid., 78, 1909 (1956); (h) S. Bernstein, R . H. Lenhard, W. S. Allen, 31. Heller, R . lip,17a,21-triol-3,20-dione 21-acetate (IX), 111.p. Littell, S. M. Stolar, L. I. Feldman and R. H . Blank, ibid., 78, 5691 , 3 1 0 - 2 1 2 O , [ a ] 1/16', Amax 242 nip e 16,900 (Anal. (1956): ( i ) J. E. Herz, J. Fried, P. Grabowich, E. F. Sabo, ibid., 78, Found: C, 69.03; H, 8.40) and lGa-methyl-1,44813 (1956); (j) G. B. Spero, J . L. Thompson, F. H. Lincoln, X'. P. pregnadiene-1 l,B,17a,21-triol-3,20-dione 2 1-acetate Schneider, J. A Hogg, ibid., 79, 1515 (1957); (k) S. Bernstein, hl. Heller. R.Littell, S . Stolar, R. Lenhard and W. Allen, ibid., 79, 4555 (X), m.p. 145-149°, A,, 242 m p , E 15,200, respec(1957). tively, were isolated.
+
+
+
+
+
+
+
+
+
( 3 ) E. Caspi, H. Levy and 0. M .Hechter, A r c h . Biochem. Riophys.. 45, 109 (1953). (-1) (a) D. K. Fukushima, Abstr. of 131st A.C.S. Meeting, Miami, 3937, p. 12-C; (b) E . bl. Glenn, R. 0. Stafford, S. C. Lyster and B. J. Bowman, E d o c r i n o l o g y , 61, 128 (1957) ( 5 ) S. Burstein, I(. Savard and K . I. Dorfman, ibid., 52, 418 (19.53). 73, 4765 (1951). (0) \V.R. X e s and H I>.J I a s o n , T H I S JOURXAL, ( 7 ) Cf.R.E. Marker nnd H . 31.Crooks, J r . , ibid.,64, 1280 (1942). (8) All rotations were taken in chloroform a t 25' unless otherwise noted, concn = 100 mg. per 10 ml., using the sodium-D line. We are indebted t o Dr. D. Williams for determining t h e rotational dispersion curve of VIII, t o Messrs. R. W. Walker for infrared spectra, R. N. Boos and associates fnr microanalyses and t o J. Wittick and associates for ultraviolet spectra.
(9) T. F. Gallagher and T. H. Kritchevsky, ibid.,72, 882 (1950). (10) Cf.T. H. Kritchevsky, D. L. Carmaise and T. F. Gallagher. $bid ,74, 483 (1952). (11) Cf.ref. 10 and G . Rosenkranz, J. Pataki, S t . Kaufmann, J . Berlin and C. Djerassi, ibid., 72, 4081 (1950). (12) G. I. Poos, G. E . Arth, R. E . Beyler and L. H . Sarett, ibid. 75, 422 (1953). (13) Ultraviolet spectra are of methanolic solutions of the eompounds. (14) Cf.Ch. Meystre, H. Frey, IV,Voser and A . I V e t t i t e ~ n Ili,ln. , Chirn. A d a , 39, 734 (1956). (15) N. L. Wendler, Huang-Minlon and h l . Tishler, T H I S JOIJRNAI., 78, 3818 (1951). (16) C/. R. E. Jonejand S A. Robinson, J . Oug. C h e m . , 21,586 (1956).
June 20, 1958
3161
COMMUNICATIONS TO THE EDITOR
T h e biological activities of the compounds herein described are compared with those of other anti-inflammatory steroids in an accompanying Communication.l7 (17) G. E. Arth, J, Fried, D. B. R . Johnston, D. R. Hoff, L. H . Starett, R. H. Silber, H. C . Stoerk, C. A Winter, THISJ O U R N A L , 80, 3161 (1958).
have shown that only 0.2 p M of C T P is required t o give maximal activity. I t seems probable t h a t the extra enzyme required in the absence of added C T P was nucleoside diphosphate kinase, whose function was t o replenish the small amount of C T P which was present in the enzyme preparations.
TABLE I GLES E. ARTH FUNDAMENTAL RESEARCH DAVIDB. R . JOHNSTOS REQUIREMEKT O F CTP FOR SYNTHESIS OF 4'-PHOSPHOJOHNFRIED MERCK SHARP& DOHME PANTOTHENYLCYSTEINE LABORATORIES DIVISION WILLIAMW.SPOONCER The reaction mixture contained: 0.08 p M 4'-phosphoDALER. HOFF MERCK& Co., I s c . RAHWAY, NEWJERSEY LEWISH. SARETT pantothenic acid, 5 piW ATP, 10 p M MgClz, 80 p M tris(hydroxymethy1)-aminomethane buffer a t pH i.4, 10 RECEIVED MAY12, 1958 p M cysteine, 700 y of purified coupling enzyme and additions as shown below in a total volume of 2 ml. Incubation was for 3 hr. a t 37", followed by heating for 5 min. at 100" REQUIREMENT OF CYTIDINE TRIPHOSPHATE FOR and centrifugation to separate denatured protein. The THE BIOSYNTHESIS OF PHOSPHOPANTETHEINE' supernatant solutions were analyzed as described in the text. Sir:
T h e enzymes, present in cell-free extracts of Proteais morgnnii, which catalyze reactions 1 , 2 and 3
+
pantothenic acid ATP2 + 4'-phosphopantothenic acid ADP P , (1) 4'-phosphopantothenic acid cysteine C T P --+ 4'-phosphopantothenylcysteine C D P 3 P , (2) 4'-phosphopantothenylcysteine + 4'-phosphopantetheine+ COz (3)
+
Addi Lion
Xone ATP, ATP, ATP, CTP,
+ + + + +
have been named, respectively : (1) pantothenic acid kinase4m6; (2) phosphopantothenic acid-cysteine coupling enzyme5; and (3) phosphopantothenylcysteine decarboxylase. A crude extract, prepared from cells ruptured in a Hughes press, was treated with ammonium sulfate and calcium phosphate gel t o yield a preparation of the coupling enzyme which was purified 20-fold and was free of the other two enzymes. T h e substrates for the reaction catalyzed by the purified coupling enzyme were found t o be 4'phosphopantothenic acid and cysteine. The amount of product formed, 4/-phosphopantothenylcysteine, was determined by measurement of the disappearance of 4'-phosphopantothenic acid by determining the amount of pantothenic acid which could be regenerated b y treatment with phosphatase. For this purpose, pantothenic acid assays were performed with Saccharomyces carlsbergensis 422Ek6 Unexpectedly, it was found t h a t the purified coupling enzyme did not function unless a small amount of crude extract was also included in the reaction mixture. The activating factor in the crude extract was heat labile and appeared to be an enzyme. Of a large number of compounds which were tested only one, C T P , was able to replace tbe requirement for this extra enzyme. The activating effects of the crude extract and C T P are shown in Table I. Other nucleoside di- and triphosphates were inactive. Additional experiments (1) T h i s investigation was supported by National Science Foundation Grant G4580. (2) A T P , A D P , C T P and C D P are adenosine and cytidine triand diphosphates. (3) Fragmentary evidence only indicates t h a t C D P and inorganic phosphate (P,) are products of this reaction. (4) G. B. Ward, G. M. Brown and E. E. Snell, J . Bid. Chem., 213, 869 (1955). (5) G. M . Brown, Federation PYOC., 11, 197 (1958). (6) L. Atkin, W. L. Williams, A. $, Shultz and C. N. Frey, I n d . Eng. Chem.. Anal. E d . , 16, 67 (1941).
4'-Phosphopantothenylcysteine formed, #,If x 10%
511111
5phl 5wM
4-crude extract 4- boiled extract
5pM
1.91 1.25 4.38 I .91 6.92
Incubation of the purified enzyme with cysteine and C T P gave no detectable cytidine-containing, sulfur-containing compound. Thus it seems likely that the C T P requirement in the reaction is for the activation of the carboxyl group of 4'-phosphopantothenic acid in a manner similar t o the way A T P functions in the synthesis of pantothenic acid from pantoic acid and P-alanine.' (7) W. K. Maas, Federation Pvoc., 15, 305 (1950).
DIVISIONOF BIOCHEMISTRY GENEM. BROWN O F BIOLOGY DEPARTMENT MASSACHUSETTS INSTITUTE OF TECHNOLOGY CAMBRIDGE, MASSACHUSETTS RECEIVED M A Y2, 1958 16-METHYLATED STEROIDS. 11. 16a-METHYL ANALOGS OF CORTISONE, A NEW GROUP OF ANTI-INFLAMMATORY STEROIDS. ~cY-HALO DERIVATIVES
Sir: Syntheses of the acetates of 16a-methylated analogs of cortisone, hydrocortisone and their 1,2unsaturated derivatives, I and 11, respectively, are reported in an accompanying communication. The enchanced activity and freedom from salt retention characteristic of this group of compounds prompted extension to 9-halogenated analogs. A dimethylformamide-pyridine solution of 16amethylhydrocortisone acetate (111) was treated with methanesulfonyl chloride,2 affording 1 6 0 ~ methyl-4,9(11)-pregnadiene- 17a,2 1-diol-3,20-dione 21-acetate (IV), m.p. 205-208" Xmax 239 mp, E 17,300, [ a ] 93°.3 (Anal. Found: C, 71.96; H, 8.30) which was coiiverted to 16a-methyl-9a-
+
(1) G. E Arth, D. B. R . Johnston, J . Fried, W. W. Spooncer, D. R . Hoff and L. H. Sarett, THISJ O U R N A L , 80, 3180 (1958). (2) Modification of unpublished procedure of E. hl. Chamberlain and J. M. Chemerda: cf. J. Fried, K . Florey, E. Sabo, J. H e n , A. Restivo, A. Borman and F . Singer, ibid., 77, 4181 (1955). (3) Ultraviolet spectra are of methanolic solutions of the compounds. Rotations were determined in chloroform a t 25'. concn. = 100 mg./lO ml. using the sodium-D line, unless otherwise noted.
