Jan. 20, 1957
COMMUNICATIONS TO THE EDITOR
known t o become radioactive under the same experimental conditions when C14-acetate is the If the side-chain substitution carbon s o ~ r c e . ~ occurred a t the squalene stage, a radioactive hydrocarbon containing 31 carbon atoms and presumably having the same chromatographic properties as squalene would be a n expected intermediate. Since formate failed to contribute any radioactivity t o the hydrocarbon fraction, i t is likely that the one-carbon unit is attached to an already completed steroid structure. An inoculum of Fleischmann's baker's yeast was grown anaerobically for 48 hours.* The cells were harvested by centrifugation (yield 4.8 g., wet weight) and were suspended in 20 vol. of 0.1 iZf KHzPO4 containing 1% glucose, and aerated for 1.5 hours. The cells were again centrifuged, resuspended in 30 ml. of 0.03 M phosphate buffer containing 1% glucose and 2 mg. of CL4formate (60 pc.) and incubated in air a t 25' for 24 hours. The non-saponifiable fraction extracted from the cells contained 7.4 X lo5 c.p.m. (1.2y0 of the C14 added). Chromatography on deactivated (7% acetic acid) alumina yielded no significant radioactivity in the petroleum ether eluate (hydrocarbon fraction) and on elution with 14% benzene in petroleum ether, 6.6 X lo5 c.p.m. in the ergosterol fraction. Carrier ergosterol was added and the mixture crystallized three times from methanolchloroform t o yield material having 29 c.p.m./mg. This ergosterol was degraded according to Hanahan and Waki13 with the modification that the methyl isopropyl ketone was isolated as the semicarbazone before degradation to iodoform.
501
A STABLE PHENOXY RADICAL INERT TO OXYGEN
Sir : A very dark blue crystalline compound [11] has been isolated from oxidation of 3,3',5,5'tetra-tert-butyl-4,4'-dihydroxydiphenylmethane[I] R is H, by lead dioxide in ether or isooctane OH C(C&)8
(CHd&~)!,f~CHd3
CH
R-CH
1
1
I
A I II OH [I111
TABLE I
Compound 11 has m.p. 153'. Anal. Calcd. for C29H4102: C, 82.6; H, 9.7. Found: C, 82.8, 82.7; INCORPORATION O F CI4-??ORMATE INTO ERGOSTEROL AND H, 9.9, 9.9. This corresponds to the loss of three DEGRADATION PRODUCTS hydrogens from the st'drting phenol. C.pJ"m.g.5 C.p.m.b Compound I1 is a radical. Magnetic susceptiFound Ca1cd.c Found Ca1cd.c bility measurements indicate that there is one Ergosterol 29 .. 15 ... unpaired electron per molecule in the solid state. DNP-a,B-dimethyl-butyralThe electron magnetic resonance spectrum of I1 dehyded 40 41 34 35 is a singlet both in the solid state and in isooctane Methyl isopropyl ketone solution, "g" value of 2. The unique property of semicarbazone" 78 80 65 70 I1 is its unreactivity toward oxygen. No reaction Iodoform (G) .. .. 310' 420 occurs in the solid state after three months and none in isooctane after three days. The radical Infinitely thin samples. Infinitely thick samples of BaCOs. e Calcd. on the assumption that C Z is~ the only character decays slowly in solution but not through labeled carbon atom. M.p. 123-125', reported3 124-125'. reaction with oxygen. M.p. 112-114', reported 112O.9 f The liquid combustion The infrared spectrum indicates no OH stretching of iodoform has been reportedlo t o give BaCOa of lower and the only absorption between 3.5 and 6 . 4 ~ specific activity than expected. The low value (74% of calcd.) in the iodoform reported here may therefore be is an intense band a t 6 . 3 5 ~ . This band has been ascribed, a t least in part, to this analytical difficulty. reported by Cook and by Mueller to be present in the spectra of phenoxy radicals derived from Acknowledgment.-This work was supported oxidation of hindered phenols. lv2 The ultraviolet spectrum indicates that over a by a grant-in-aid from Eli Lilly and Company. concentration range variation of twenty-five (7) J . D. Johnston and K. Bloch, THIS JOURNAL, in press. Beer's law is obeyed; measurements were made on ( 8 ) H. P. Klein, J. Bacteriology, 69,620 (1955). a very strong band a t 420 mp, €molar 200,000. (9) G.Ciamician and .'k Silber. Bcr., 43,947 (1910). (10) G. Ehrensvard, Q.Reio, E. Saluste and R. Stjernholm, J. Biol. Compound I1 is reduced t o I11 with hydroquiChcm., 189, 93 (1951). none in ether; it is reduced to the parent phenol or to compound I11 by hydrogen and platinum. CONVERSE MEMOFSALLABORATORY HENRYDANIELSSON Compound I11 (m.p. 157-158', Anal. Calcd. for H ARVARD UNIVERSITY 0
CAMBRIDGE 38, MASSACHUSETTS
KONRAD BLOCH
RECEIVEDDECEMBER 17,1956
(1) C. C. Cook a n d B. E. Norcross, THIS JOURNAL, 78,3797 (1966). (2) 1. E.Mueller, K.Ley and W.Kiedaisch, Bcr., 88,1819 (1955).
COMMUNICATIONS TO THE EDITOR
502
Vol. 79
C29Hh202: C, 82.6; H I 10.0. Found: C, 82.4; H, 10.0) is oxidized to 11 by lead dioxide in ether. Compound I11 has an OH stretching band in the infrared a t 2.74 p and three intense bands between 6.1 and 6.5 p. Methanol adds to the quinone methide to yield the bisphenol I, R is OCH,, m.p. 160-161'. Anal. Calcd. for c30H46o3: c, 79.5; H. 10.2. Found: C, 79.3; H, 10.0, 10.3. Compound I1 is sensitive t o traces of strong acid in hydroxylic or hydrocarbon solvents. The radical is converted in methanol with a trace of toluenesulfonic acid to I, R is OCH3 and 3,3',5,5'tetra-tert-butyl-4,4'-diphenoquinone,m.p. 239240'. Base transforms I1 t o a different radical which reacts rapidly with oxygen. These reactions mill be described later. Compound I1 is being investigated as a standard for electron magnetic resonance measurements.
or its 21-acetate with Bacdlus sphaericzLs,Gleading directly to V, two forms (m.p. 235' (dec.); 22.5' (dec.); [Q]*'D +246' (dioxane); X ~ ~ ~222' l i-( (11,100), 255 (9,900), 296 (11,700); 2.79, 2.88 p (OH), 5.85 (11, 20 C=O), 6.06 ( 3 C=O), 6.13, 6.24, 6.31 (A1345); found: C, 70.83; H , 6.81). 6-Dehydrocortisol (IX) (m.p. 239-241'; [ a ] ? 5 ~ f177' (dioxane); 253 mp (24,900); Xz,U:O' 2.97 p (OH), 5.84 (20 C=O),6.11 (3 C=O), (5.20, 6.33 (A4e6); found: C, 69.86; H, S.06) was prepared from the 3-semicarbazone 21-acetate of 1'1115 by formation of the 3,20-bis-semicarbazone 21-acetate of VIII' (darkens %0', m.p,; >220'; 242 mp (14,500),301 (10,200); A&,":"' 2.89, 2.98, 3.05 p (OH, S H ) , 5.79 (ester C=O), 5.8G (11 C=-=O),5.99 (amide C=O), 6.35 ( A 4 G ) j7.99 (ester C-0-C); found: 5 , 16.46), reduction with EMERYVILLE RESEARCH CENTER sodium borohydride to the 3,20-bis-semicarbazone M. COPPINGER SHELLDEVELOPMENT COMPANY GALVIN of IX (darkens 260', m.p. > 320'; A,, 236 m p EMERYVILLE, CALIFORNIA (11,100), 298 (37.400); A",9'2.93, 3.06 p (OH, NH). RECEIVED DECEMBER 6, 1956 5.99 (amide C=O), 6.40 ( A 4 ~ 6 )found. ; N. 16.27). and cleavage with pyruvic acid and p-toluenesulfonic acid. Microbiological dehydrogenation of IX with B . sphaericzis gave 1,4,6-pregnatrieneSOME NEW ACTIVE CORTICOSTEROIDS 1lp,17a,2 l-triol-3,20-dione (VI) (in. p. 2330-24.1 '; [a]?'D +100' (dioxane); 222 my (11,6oo), Sir: 256 (9,600), 298 (12,200); X ~ 2 S 3~ , 2.93. ~ ;?.OOy ' The enhancement of the glucocorticoid' and (OH), 5.84 (20 C=-O\1, (i.09 (J C=O'i, 6.20, (i.27, antiarthritic2activities of prednisone (I) and prednisolone (11)4relative to cortisone (111) and cortisol 6.34 (A1.4,6);found' C, 70.25; €I, 7.26). The structure is supported by the preparation of (IV), led us t o introduce further unsaturation into l,-l.,6-androstatriene-3,1lj 1 7-trione (X) (m.p. 213 cortical hormones. To this end, we have prepared (dec.); [a]'jD +30g3 (acetone); 222 nip 6-dehydroprednisone (17) and B-dehydropredniso(11,300), 255 (9,900), 296 (11,900);' : : :A 3.7:: y lone (VI). Bromination of the 21-acetate of I with N- (17 C=O), 5.S-1 (11 c=O), G O A ( 3 C=O'i, bromosuccinimidej gave 6-bromo-l,4-pregnadiene-6.34 (A1*416); found. C, i i . O O ; H, G.C,7),both 17a,2l-dioI-3,11,20-trione 21-acetate (VII) (m.p. degradation of 5' with sodium bismuthate'" ' and from 1,4-androstadiene-X,11,20-trione1b by 185-188' (dec.); [ a I z 5+173' ~ (dioxane); " : : :A 245 mp ( E = 16,300); found: Br, 16.54). De- reaction with N-bromosuccininiidej to give 0(1n.p. I fiSo hydrobromination of VI1 in refluxing collidine bromo-l,4-androstadiene-5,1l-?O-trione ":::X 24:3 m u gave 1,4,6-pregnatriene-17~,2 l-di01-3,11,20-trione, (dec.); [Q]"D + l O i O (dioxane); (v) 21-acetate (m.p. 225228'; [aI2'D +265' (15,300); X ~ 5.74~ p (17 ~ C=Oj, ' 5.85 (11 C=Ol. (dioxane); XE2H 222 mp (11,400), 255 (10,300), 6.01 (3 C=O), 6.19, 6.24 ( A I i G ) ; found. Rr. 22.13), followed by dehydrobroiiiination with 297 (12,100); X ~ 2.96 ~ p (OH), ~ ' 5.76 (20 C=O, 21-OL\c), 5.85 (11 C=O), 6.02 (3 C=O), 6.21, collidine. Animal assay of V and 1-1 showed potencies a\ 6.31 (A1,4,G);found: C, 69.18; H I 6.73). The 21-acetate of V also was obtained by dibromination given in the table compared to cortisone [IT1 = 1 I. of the 21-acetate of I11 in acetic acid, followed by Eosinopenia9 r h y m u s Involutiong I i t e r (:lycoF;ent dehydrobromination with collidine. An alternate procedure was the microbiological V 1 2 1 3 2 dehydrogenation of 6-dehydrocortisone (VI1I)j VI 1 9 2 5 I.i
Xg;:"
(1) (a) H . L. Herzog, A . Nobile, S. Tolksdorf, W. Charney, E. B. Hershberg, P. L. Perlman and M. M. Pechet, Science, 121, 176 (1955); rh) H . I,. Herzog, C. C. Payne, hI. A . Jevnik, D. Gould, E. L . Shapiro, l? P. Oliveto and E. B . Hershberg, THISJ O U R N A L , 77, 4781 (19.55). ( 2 ) J. J. Bunim, M.h l . Pechet and A J . Ballet, J . A m . U e d . Assoc., 167, 3 1 1 (1955). n 13) Xeticorten, '& 1,4-pregnadiene-17a.?l-diol-3,11,20-trinne. ( 4 ) lfeticortelone, I ,4-gregnadiene-l lp,l7~,21-triol-3,20-dione. ( 3 ) Cf. 1'. R . M a t t o x , E. I,. Woroch, G. A . Fleischer a n d E . C. Kendall, J . B i d . C h e m . , 197, 2131 (1952). ( 0 ) Cf.W. Charney, D. Sutter, C . Federbush, hf. Gilmore, H . L . Herzog, M. J . Gentles, h l . E. Tully and E. B Hershberx, t o be published; T. H . S t o u d t , a '. J. RZcAleer, J . h l . Chemerda, M .A . Kozlowski, R . J. Kirschmann, V . I l a r l a t t and R. RIiller, Avcli. Biochem R i o g h y s . , 69, 301 (19%).
8
The electrolyte excretion pattern using V arid \TI was the same as with I and II.lu Preliminary ( 7 ) Cf.E. P. Oliveto, R . Rausser, Ile Rice, A l c i andra D. Stephenson, James A . T r u a n and Felix H . 1X''nrren. Lye appreciate t h e aid 01 Lawrencr Finckennr, Herbert Gerber and RIerl Steinberg in preparing quantities of V and V I for testing.
