May 5, 1952
COMMUNICATIONS TO THE EDITOR
2383
in the pharmacology of the veratrum alkaloids3 and loylzygadenine with diazomethane gave veratroylin the clinical use of the hypotensive activity of the zygadenine, identical with an authentic sample by tertiary ester alkaloidsI4it appeared of importance map., mixed m.p. and infrared spectrum. The two ester alkaloids and zygadenine were to isolate and study the active principles of Zygaexamined by Professor 0. Krayer at Harvard denus venenosus. Heyl and co-workers5isolated the first crystalline Medical School for their circulatory action in the alkaloid in this series, zygadenine. It appeared anesthetized cat, their effect upon the failing heart unlikely, however, t h a t zygadenine is the most in the heart-lung preparation of the dog, and their important toxic agent in Zygadenus veneno~us.~~ effect upon the amphibian skeletal muscle. I n all The probable presence in Zygadenus venenosus of three types of experiments, the actions of zygaester alkaloids similar to the tertiary alkamine ester denine were similar to those of cevine, and the alkaloids of the veratrum series has been indicated actions of veratroylzygadenine and vanilloylzygadeby the pharmacodynamic properties of the alka- nine were similar, quantitatively and qualitatively, loidal We have now isolated two of the to the actions of the cevine ester, veratridine. ester alkaloids responsible for this activity. This work was supported by grants from IieThe chloroform-extractable alkaloids of Zyga- search Corporation and the National Institutes of denus zlenenosus (WATS)' were subjected to sim- Health, and the assistance of Eli Lilly and Complified 8-plate countercurrent distribution pat- pany and Riker Laboratories, Inc., in gathering and terned on the procedure of Fried, White and extracting Zygadenus alenenosus is gratefully ackW i n t e r ~ t e i n e r . ~Benzene ~ and phosphate buffer of nowledged. pH 7.1 were used as solvents. Veratroylzygadenine DEPARTMENT OF CHEMISTRY separated from a solution of the crude plate 8- HARVARD UNIVERSITY S.MORRIS KUPCHAV fraction in acetone; germine and zygadenine were CAMBRIDGE 38, MASSACHUSETTS C. V. DELIWALA obtained from the plate 0-fraction. RECEIVED MARCH 24, 1952 Veratroylzygadenine crystallized from absolute ethanol as rectangular prisms, m.p. 270-271" dec.; THE CARBON SKELETON OF [ a l Z o D -27" (C 2.08, Chf.); 262, 293 mp IDENTIFICATION OF o!-LIPOIC ACID (log E 4.13; 3.85). Anal. Calcd. C3~H51010N: Sir : C, 65.i3; H, 7.82; N, 2.13. Found: C, 65.90; It has been reported' recently that a-lipoic acid, H I 7.86; N, 2.09. Alkaline hydrolysis of veratroyl- a catalytic agent required for oxidative decarboxyzygadenine yielded veratric acid and a base iso- lation of pyruvic acid by certain lactic acid bacteria, meric with zygadenine, pseudozygadenine. This is a monocarboxylic acid, pKa 4.7, containing a base was also obtained by similar alkaline treat- disulfide linkage and possessing the empirical forment of zygadenine. Pseudozygadenine crystal- mula C8H1402S2. Infrared absorption spectrum lized from ethyl acetate-petroleum ether as needles, failed to show the presence of any carbon-carbon ~ (c 2.00, chf.). double bonds. These data indicate that a-lipoic m.p. 169-171" dec.; [ a I z 5-33" Anal. Calcd. C ~ T H ~ ~ O C,~65.69; N: H I 8.78; N, acid is a cyclic disulfide. The PKa value indicates 2.84. Found: C, 65.46, 65.79; H, 9.10, 8.69; N, that the sulfur atom is not attached to the carbon 2.95. Acetylation with acetic anhydride and pyri- atoms a- or P- to the carboxyl group. Comparison dine gave pseudozygadenine triacetate which crys- of the polarography of a-lipoic acid with that of tallized from ether as rhomboids, m.p. 235-236" several dithiols and cyclic and linear disulfides also dec.; [ ( u I z 3 ~-33" (c 1.89, chf.). Anal. Calcd. indicated its structure to be that of a cyclic difor C2,H4007i\T(COCH3)3: C, 63.95, HI 7.97; acetyl, sulfide. The presence of a six-membered ring in 20.84. Found: C, 64.13; HI 8.11; acetyl, 20.69. a-lipoic acid was suggested by the following obserThe filtrate obtained by removal of veratroyl- vation. The catalysis of hydrogen ion reduction a t zygadenine from the plate 8-fraction was lyophil- the dropping mercury electrode by the reduced ized and the residue was dissolved in chloroform form of a-lipoic acid resembled that of 1,4-dithiols and chromatographed on acid-washed alumina. more than 1,3- or lI5-dithiols. Vanilloylzygadenine crystallized from an ethanol Even with these limitations, the number of solution of the most difficultly eluted fractions as structures possessing the empirical formula of rods, m.p. 258-259" dec.; [aI2OD -27.5" (c 2.00, a-lipoic acid is considerable. It was therefore of chf.); 264, 294 mp (log t 4.07, 3.83). Anal. paramount importance to determine the nature of Calcd. C35H49010N;C, 65.30; H, 7.67; N, 2.18; the carbon skeleton. Assuming the cyclic disulfide 1 OCH3, 4.82. Found: C, 65.35; H, 7.93; N, 2.29; nature of the molecule, much information could be OCH3, 4.34. Alkaline hydrolysis yielded vanillic obtained by the desulfurization of the substance. acid and pseudozygadenine. Methylation of vanil- A 3-mg. sample was subjected to treatment with Raney nickel to remove the sulfur atoms.2 The (3) 0. Krayer and G. Acheson, Physiol. R e v . , 26, 383 (1946); G . L. product was isolated as the crystalline silver salt. hlaison, E. Gatz and J. W. Stutzman, J. Pharmacol. E x p l l . Therapy, ion, 74 (i~ji). A comparison of the X-ray powder diagrams of this (4) (a) E. Meilman and 0. Krayer, Circulation, 1, 204 (1950); material with that of silver n-caprylate3 revealed (b) J. Fried, H. L. White and 0. Wintersteiner, THISJOURNAL, 72, that the two samples were identical. The silver 4621 (1950). ( 5 ) (a) F. \V. Heyl, F, E. Hepner and S. K. Lay, ibid., 86, 258 (1913); (11) 1;. W. IIeyl and BI. E. Herr, ibid.,71, 1751 (1949). (I;) S. \-uRe nil
