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VOl. 79
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, THISJOGRNAL, 79, 572
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~
(1957).
F
(2)
K. Bernauer
and I. Skudrzyk. J . p r a k f . Chcm.. 166, 310 (1940).
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I n an alternate preparation of this diamine, a (And. Calcd. for CzaHaO: C, 84.00; H, 12.00. solution containing ethyl p - c y a n o b ~ t y r a t e(12 ~ g. ; Found: C, 83.35; H, 12.11). The infrared specb.p. 66' a t 1 mm., n z 5 1.4195)) ~ dimethylamine trum indicated absorptions a t all the wave lengths (16 g.) and ethanol (100 ml.) was heated in a bomb believed to be characteristic of a A7-stenolIs and tube a t 105' for 5 days. The N,N-dimethyl-fi- the rate of color development with the Liebermanncyanobutyramide (7.25 g., b.p. 77-78' a t 5 mm;. Burchard reagent was similar to that of A7-stenols.I nZ5D 1.4620) so prepared was reduced with excess The behavior of the acetate of the new sterol (11), lithium aluminum hydride (10 g.) in ether (600 m.p. 93-94') to hydrogenation in glacial acetic acid over Adams catalyst (no hydrogen uptake) was l-Amino-4-dimethylamino-2-methylbutane ml.). and (3 g., b.p. 106-108' a t 100 mm.) was i ~ o l a t e d . ~also characteristic of AI- and AE(g)-stenols,8 Its dioxalate (m.p. 175-176') was the same as that resulted in isomerization. The reaction product, of the diamine derived from the pyridazinium bro- m.p. 76-77', absorbed one mole of hydrogen upon forced hydrogenation in the presence of HC1 a t mide (infrared spectra identical). The ion (RzN=NH+) is probably the interme- 60°9 to yield a saturated sterol acetate, m.p. 99diate that reacts with the isoprene rather than the 101'. The alcohol, m.p. 153-155') resulting from neutral species (RZN +=N-) obtained when the reac- the hydrolysis of the acetate failed to respond to the tion mixture is neutralized. The above pyridazin- Liebermann-Burchard reagent, and its infrared ium salt was obtained (84% yield) directly from spectrum indicated the absence of double bonds. the acidic reaction mixture. When an acid solu- On Kuhn-Roth C-methyl determination, lo I1 liber0.27 equivalents of acetic acid (calcd. tion from oxidation of 1,l-dimethylhydrazine and ated 3.48 concentrated base were added simultaneously to a as 4-methyl-A'-cholestenyl acetate) or 3.60 f 0.26 solution of isoprene in methanol a t such rates that (calcd. as 4,4-dimethyl-Ai-cholestenyl acetate). the reaction mixture remained just basic to phenol- Synthetic 4,4-dimethylcholesteryl acetate," cholphthalein, tetramethyltetrazene (84% yield) was esteryl acetate and A7-cholestenylacetate gave 3.60 obtained. f 0.31, 2.90 f 0.03, and 3.00 f 0.22 equivalents Evidence that this intermediate reacts with ani- of acetic acid, respectively. Thus I1 possesses line, phenol, styrene and vinyl n-butyl ether has approximately one more C-methyl group than the been gained from experiments in which acid solu- Cz7 sterol acetates (C-methyl = cu. 0.6 mole of acetions of i t were mixed with each of these substances tic acid). From chemical and biogenetic considera(1-2 hour reaction times), and little or no tetra- tions, either 4(a or p)-methyl-A7-cholesten-3P-ol or became attractive methyltetrazene was found by spectroscopic analy- 4,4-dimethyl-A7-cholesten-3p-ol sis after the reaction mixtures were made basic. plausible structures for I. A7-Cholesten-3P-olwas The products here formed and further possible re- subjected to Oppenauer oxidation to give A7-choactions are under investigation. lesten-3-one, m.p. 143-145'. This ketone upon treatment with potassium, t-butyl alcohol, and (3) J. Bredt and J. Kallen, A n n . , 293, 351 (1896). methyl iodide, gave a mixture which was reduced (4) L. K. Amundsen and L. S. Nelson, THIS JOURNAL,73, 242 (1951). with LiA41H4. Purification of the resulting alcohols GEORGE HERBERT JONES LABORATORY through the digitonides12 followed by silicic acid UNIVERSITY OF CHICAGO W. H. URRY chromatography of the @-sterolsafforded two main CHICAGO 37, ILLINOIS compounds of m.p. 122-124' and m.p. 146-147', INORGANICCHEMISTRY BRANCH CHEMISTRY DIVISION HOWARD W. KRUSE respectively. The former compound was identical U. S. NAVAL ORDNANCETESTSTATION to A7-cholesten-3p-ol, and the latter sterol (less CHINA LAKE,CALIFORNIA WILLIAMR. MCBRIDR polar) was identical to I (Anal. Calcd. for CW RECEIVED ~'OVEMBER 2, 1957 C, 84.00; H, 12.00. Found: C, 83.22; H, 12.01) by m.m.p. and infrared spectrum, Evidence has been obtained which tentatively rules out 4,4ISOLATION AND SYNTHESIS OF A NEW STEROL dimethyl-A7-cholesten-3p-01.Future publication of FROM RAT FECES112 the data will be made. The possibility of the 2(a Sir: or p)-methyl isomers cannot as yet be eliminated, I n previous reports, 3.4 the presence of a new sterol but is unlikely on biological grounds. Others13*14 (I) in rat feces was announced. Separation from have reported the monoalkylation of A4-3-onesterthe less polar coprostanyl p-phenyl azobenzoate oids a t position 4 using conditions similar to ours. was effected by chromatography of the mixed es- Since these conditions favor the more stable equaters on silicic acid:Celite (2:1)5 columns using pe- torial configuration, it appears likely that I is 4atroleum ether (b.p., 90-95'). Saponification of (I) methyl-A7-cholesten-3p-01. The origin of I sugp-phenyl azobenzoate, and crystallization of the en- gests the possibility that this sterol arises from the suing sterol (I) from absolute ethanol yielded thin D. R. Idler, S. W. Nicksic, D. R. Johnson, V. W. Meloche, needles; m.p. 146-147', [ a I z 5 D 0.00 in chloroform H .(6) A. Schuette and C. A. Baumann, THIS JOWRNAL, 7 6 , 1712 (1953) (1) This work was supported by a research grant (H-2458 C, CS) from the National Institutes of Health, U. S. Public Health Services. (2) Some of the properties of this sterol were studied in collaboration with D. L. Coleman and C. A. Baumann, Department of Biochemistry, University of Wisconsin. (3) W. W. Wells, D. L. Coleman and C. A. Baumann, Arch. Biochcm. Biophys., S7, 437 (1955). ( 4 ) D. L. Coleman, W. W. Wells and C. A. Baumann, ibid., 60, 412 (1956). (5) D. R. Idler and C. A. Baumann, J . Bid. Chcm., 195,623 (1952).
(7) D. R . Idler and C. A . Baumann. J . B i d . Chcm., 203, 380 (1953). (8) H.Wieland and W. Benend, A n n . Chcm., SS4, 1 (1943). (9) A. Windaus and G. Zhhlsdorff, A n n . , S36, 204 (1938). (IO) E. Weisenberger, Mikrochcm. Microchim. Acla, 3 3 , 51 (1947). (11) R. B. Woodward, A. A. Patchett, D. H. R . Barton, D.A. Ives and R . B. Kelly, J . Chcm. Soc., 1131 (1957). (12) W. Bergmann, J . B i d . Chcm., 132, 471 (1940). (13) F.Sondheimer and Y.Mazur, THIS JOURNAL, 79, 2806 (1957). (14) N. W. Atwater, ibid., 79, 5315 (1957).
