Dec. 20, 1957
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grams: one with MeOH-H20 (1 : 1) on Celite with benzene-ethyl acetate8 (9: 1) as mobile phase and the other with MeOH-H20 (7:3) on Celite with EtC1z9 as mobile phase. This procedure yielded 0.5 mg. of amorphous I which was homogeneous in the following paper chromatograms : ethylene glycol-EtC12:toluene (1 : 1) (RTHE = 1.0); forniamide-EtClz: toluene (1: 1) (RTHE= 0.70) ; t-butyl alcohol :H20:isooctane10 (RTHE = 0.80) ; and MeOH :HzO (I : l)-benzene" (RTHE = 0.85). I exhibited no absorption in the ultraviolet and a 1705 cm.-l absorption band (CHC13) in the infrared. H I 0 4oxidation of 0.13 mg.12of I yielded 0.014 mg. of HCHOLS(theor. = 0.011 mg.) and 0.11 mg.14 of a neutral product I1 which possessed absorption bands in the infrared (CS2) characteristic of hydroxyl (3580, 3400 cm.-l), y-lactone (1775 cm.-') and unconjugated carbonyl (1705 cm.-l) functions. (10) C. A. Coulson, Research, 10, 149 (1957). I n an alternate method for the isolation of 11, (11) D . S. Trifan, P. T . Huang and J. W. Herrick, Abstracts of Papers, 131st Meeting, Am. Chem. Sac., Miami, Florida, April 7-12, unhydrolyzed urine was treated with HI04 to ac1057, p. 5-S. complish the simultaneous oxidative cleavage of PLASTICS LABORATORY gl~curonidesl~ and a-ketols. The neutral etherPRIXCETON UNIVERSITY DANIELS. TRIFAN extractable fraction was then hydrolyzed with PRINCETON, NEW JERSEY J. L. WEIRMANN NaOH and the saponifiable fraction so obtained was BALLtSTIC RESEARCH LABORATORIES ABERDEEX PROVING GROUND, MD. LESTERP. KUHN lactonized with acid. The neutral lactone fracRECEIVED AUGUST13, 1957 tion was chromatographed on alumina and yielded 1116in the benzene-CH2Clz (I : 1) eluates. I n order to determine the number of acetylatable EVIDENCE FOR THE OCCURRENCE OF A hydroxyl groups per mole,2 the acetates of I and I1 METABOLITE O F ALDOSTERONE IN URINE' (I Ac, I1 Ac) were prepared from acetic anhydrideSir : l-C14 pyridine and chromatographed on paper This report describes the isolation of a new a-ke- and oninalumina until constant isotope content was to1 (I) from human urine which yielded on oxidation achieved. When desoxycorticosterone and T H E with HI04 a monohydroxy-y-lactone (11). The were treated with same acetylation mixture, evidence suggested that I is a tetrahydro deriva- they yielded acetatesthe with specific activities of 30 f tive of aldosterone2 with a primary hydroxyl group 1 X l o a c.p.m./~mole" for each acetyl group. I a t Cia. Neutral extracts of urine3 which had been hydro- Ac, purified by chromatography on paper (RDOCA" = 1.0) and on alumina (eluted with benzene) gave lyzed with /3-glucuronidase4were chromatographed 92,000 c.p.m./pniole17 and therefore was a triaceon paper and appropriate eluates which reduced Its spectrum (CS2) showed absorptate. blue tetrazolium were oxidized with HI04. The tion bandsinfrared a t 1750 and 1730 cm.-', characteristic neutral oxidation products were treated with hy- of 21-acetoxy-20-ketosteroids, l9 and gave no evidroxylamine to convert y-lactones to hydroxamic of a free hydroxyl group. acid derivatives which were quantitatively esti- dence I1 Ac was chromatographed on paper (XDOCA'~ = mated by measurement of radioactivity of the hydroxamic acid-Fej9 complexes after extraction into 0.52) and on alumina. Fractions of constant spen-butanol. Substance I, detected by this proce- cific activity were eluted with benzene :ligroin (4 : 1) (8) In this system I had t h e same Ry as 3a,17a,118,21-tetrahydroxydure, migrated with THE5 in CHC13-formamide6 paper chromatograms, and was separated from pregnane-20-one. (9) I n this system I had t h e same Rf a s T H E . T H E in the EtC12-formamide system, in which I (10) EzB system. W. R. Eberlein and A. 31. Bongiovanni, Arch. migrated with an R T H E= ~ 0.72 5 0.02. Biochem. Biophys., 69, 90 (1955). (11) Bssystem. I. E . Bush, Biochem. J . , 60, 370 (1951). Eluates from the EtClz-formamide paper chro(12) Determined by t h e blue tetrazolium test with THE a s the matograms were purified by two partition chromato- standard. R. 0. Recknagel and hl. Litteria, J . Lob. Clin. M e d . , 48,
ring produces a substantially stronger hydrogen bond than that of the benzene ring, An interesting contrast in the steric requirements of the xelectron systems of the two aromatic rings is provided by the spectrum of a-phenylethanol, the phenyl analog of I, which shows only the single stretching frequency of the free hydroxyl a t 3617 cm.-l. In both of these a-substituted compounds the hydroxyl hydrogen is only above the outer periphery of the ring and the observed difference under these geometric conditions may reflect the greater contribution of delocalization energy to the total energy of the hydrogen bondL0in the case of the ferrocene ring, consistent with other evidence of its unusually high a-electron 1ability.l' Derivatives of varying side chain lengths are presently being studied in this connection.
(1) This work was supported by a grant (PHS-AlIO) from t h e National Institute of Arthritis and Metabolic Diseases of t h e National Institutes of Health, Education and Welfare. T h e capable technical assistance of Annabelle Long is gratefully acknowledged. ( 2 ) S. A. Simpson, J. F. Tait, A. Wettstein, R. Neher, J. v. Euw, 0. Schindler and T. Reichstein, Helv. Chim. Acta, 37, 1163 (1954). (3) Urine f r o m patients with edema a n d increased excretion of aldosterone was t h e best source of substance I. We are indebted t o Dr. Herbert C. Stoerk of t h e Merck Institute for Therapeutic Research for bioassays of aldosterone. (1) Ketodase, generously furnished by t h e Warner-Chilcott Laboratories. ( 5 ) Abbreviations: T H E = 3cr,17cr-21-trihydroxypregnane-l1,20dione. EtClt = 1,2-dichloroethane. ( 6 ) A. Zaffaroni and R . B. Burton, J . B i d . Chem., 193, 749 (1951). (7) RTHE, distance traveled by sample relative t o T H E on paper chromatograms scanned with blue tetrazolium.
463 (1956). (13) Determined colorimetrically with chromotropic acid. D. A. MacFadyen, J . Bioi. Chem., 168, 107 (1945). (14) Determined by lactone test (hydroxamic acid-FeS' complex). (15) C. F. Huebner, R. Lohmar, R. J. Dimler, S . Moore aud K . P. Link, J . B i d . Chem., 169, 503 (1945). (16) Samples of I1 obtained b y both methods of isolation were identical by infrared analysis of t h e free compounds and their acetates, and b y t h e RDOCAvalues of their acetates. (17) Counts per minute of C14 relative t o the density of formazan color formed from one pmole of desoxycorticosterone acetate (or T H E diacetate) in t h e blue tetrazolium test. (18) RDOCA,distance traveled by sample relative to desoxycorticosterone acetate on paper chromatograms in t h e methylcyclohexaneformamide system scanned by radioautography. (19) R. N. Jones, P. Humphries. F. Herling and K. Dobriner, THISJOURNAL, 74, 2820 (1932).
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and yielded 2.0 mg. of an amorphous solid. I t s infrared spectrum (Fig. 1) indicated the presence of
razene is detected by spectrographic analysis in such oxidized solutions; but when they are made alkaline, high yields of the tetrazenes are obtained; 100 r (2) quantitative yields of the 1,l-dialkylhydrazines are obtained upon reduction with stannous chloride, a reagent which does not reduce tetraalkyltetrazenes under the conditions used; and (3) the perchlorate salt of the intermediate, (CH3)2N= E 40 2 NH+C104-, has been isolated. The discovery of a 20 series of new organic reactions and additional evidence for the intermediate has resulted from a study -~ of its reactions with reactive organic substances. 3800 3000 1700 1200 1000 800 To a stirred solution of 1,l-dimethylhydrazine Frequency, cm.-'. (12 g., 0.20 mole) in hydrobromic acid (3.25 N,170 Fig. 1.-Infrared spectrum of monoacetoxy derivative of ml.) held a t ,'O a solution of bromine (32 g., 0.20 ylactone (I1 Ac) in carbon disulfide. mole) in hydrobromic acid (3.94 N , 200 nil.) was y-lactone (1775 cm.-l), acetoxy (1735 em.-') and added dropwise. Then, isoprene (40 g., 0.20 mole) unconjugated carbonyl (1706 cm.-l) groups and the was added and the mixture was stirred vigorously absence of a free hydroxyl group. The single ab- for two hours a t 0'. Unreacted isoprene was resorption band a t 1242 em.-' was characteristic of moved under vacuum (25 g. recovered), and the steroid acetates with axial conformations in ring remaining solution was made basic (pH 7.5) with concentrated sodium hydroxide solution. lA'orkA20 but did not distinguish between a 3a,5/3- and a up of an ether extract of this solution gave the di3P,5a-isomer. I1 Ac had a specific activity of 77,500 c.p.m./mg. which indicated an approximate methylhydrazone of tiglic aldehyde (3.0 g., b.p. molecular weight of 385 for a monoacetate.21 58-63' a t 25 mm., 12y0yield). I t s picrate (m.p Quantitative saponification of lactone and acetoxy 98') was prepared. Anal. Calcd. for CI3H17N6O7:C, 43.94; H, groups in I1 Ac confirmed that it was a lactone monoacetate; a sample which contained 1.07 f 4.82; N, 19.7. Found: C,44.35; H,4.94; N, 10.7. 0.03 pmole of C1*-acetateconsumed 1.9 f 0.1 peg. The oil product obtained by the acid hydrolysis NaOH per ,umole of acetate. 1.800 mg. consumed of the hydrazone gave the known 2,4-dinitrophenyl9.78 peg. of NaOH to give a saponification equiva- hydrazone of tiglic aldehyde (map. 215-216'; m.p. lent of 154 (theor. for a lactone monoacetate, Cz0- of mixture with authentic sample2 215-216'). When this hydrolysis mixture was made alkaline H2704CH3CO = 374/2 = 187). These observations are consistent with the formu- and distilled, 1,l-dimethylhydrazine (picrate, m.p. lation of I as a CalOs-pregnane derivative possess- 150') was obtained. ing three hydroxyl groups a t Ca, Cl8 and Czl, one Water was removed from the original reaction carbonyl group a t Czoand another probably at GI. mixture in an evaporator, and extraction of the reA definitive characterization and assignment of maining salts with hot propanol-2 gave sodium structure, however, must await the isolation of bromide (162.8 g.). Evaporation of the propanol-2 larger amounts of I. Direct evidence that I is a solution gave a salt, presumed to be 1,l'Ptrimethylmetabolite of aldosterone has been obtained by the tetrahydro-A4-pyridazinium bromide (32.7 g., 7970 isolation of tritium-labeled I from urine after the yield, m.p., after recrystallization from propanol-2, administration of tritium-labeled aldosterone to a 151-1 53') . human subject. Anal. Calcd. for C7HI6N2Br:C, 40.6; H, 7.3; (20) R. N. Jones and F. Herling, TEISJOURNAL. 78, 1152 (1956). N, 13.5. Found: C,40.3; H, 7.6; N, 13.4. (21) 30 =k 1 X 103 counts per mlnute/pmole - 77,500 counts per This product (34 g., 0.165 mole) was hydrominute/mg = 385 =k 15 pg /#mole nf acetate genated over Adams catalyst in ethanol (7.4 1. S.C. (22) Public Health Servlce Research Fellow of the National Instit u t e of Arthrltls and Metaholx Dlseases, 1954-1956, American Heart of Hz absorbed; theory for 2 moles per mole of salt, Association Research Fellow, 1956-1957 7.4 1.). When the gummy salt that remained after DEPARTMENTS O F BIOCHEMISTRY, MEDICINE the ethanol was evaporated was treated with concenAND OBSTETRICS AND GYNECOLOGY trated sodium hydroxide solution, 1-amino-4-diCOLLEGE OF PHYSICIANS AND SURGEOXS methylamino-2-methylbutane separated. I t gave COLUMBIA UNIVERSITY STANLEY ULICK~* "mixed melting I~EW YORK32, N. Y. SEYMOUR LIEBERMANa dioxalate salt (m.p. 175-176'; point" with an authentic sample, no depression). RECEIVED OCTOBER14, 1957 Anal. Calcd. for CllH22N208: C, 42.6; H, 7.2; N,9.1. Found: C,42.4; H,7.1; N, 9.0. NOVEL ORGANIC REACTIONS O F THE INTERI n another hydrogenation experiment in which MEDIATE FROM THE TWO-ELECTRON OXIDATION OF 1,I-DIALKYLHYDRAZINES IN ACID hydrochloric acid was added to the ethanol solution after the hydrogenation, the mixed hydrobroSir : mide-hydrochloride salt of the diamine (m.p. 202Evidence has been presented' for the formation of an ionic intermediate (R*N=NH+) of surprising 203') precipitated as the ethanol was removed. Anal. Calcd. for C,HzoNzBrC1: C, 33.95; H, stability in the two-electron oxidation of 1,l-dialkylhydrazines in acid solution : (1) no tetraalkyltet- 8.14; N, 11.31; Ag equiv., 123.8. Found: C, 34.1; H , 8.3; N, 11.1; Agequiv., 124. (1) W. R. McBrlde and H W Kruse, THISJ O G R N A L79, , 572
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(1957).
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(2)
K. Bernauer
and I. Skudrzyk. J . p r a k f . Chcm.. 166, 310 (1940).
