NOTES
May, 1950
2297
cnts, prohbly silicates. The necessity of devising meth- methanol solution solids appeared, which after ods for the purification of the above products was elimicrystallization from aqueous methanol were toxic nated when it was lound that acetyl-oL-tryptophan could Recrystallization from water be hydrolyzed to oL-tryptophan in practically quantita- a t 5 nig./kg. tive yields by heating aqueous solutions 01 thr acetyl com- yielded rod-shaped crystals (see Fig. 1). They pound at 200' lor six 10 scvcn hours. The Idlowing pro- produced typical convulsions in rabbits a t a dose cedure was used lor the eotwcrsion of 111 lo V. I l l (1.5 of 1 - 1 3 mg./kg. and i n idice by intraperitoneal 9.) was heated with 21) ml. of s a t c r , cmlairwd in :LII tanThe material sealed bomb tube. at the refluxing temiwr;ttore lor 30 min.. injection a t about 40 mg./kg. the tube WIS scaled, the tube atid coutc111sheated at 200" decomposed slowly a t 220' and more rapidly a t for six to seven hours, the hyclrolysatc dccdorirecl with 240' without melting completely up to 350". Norite. evilpmntetl to dryness in iv~ctio, imd Ihr residue dried ovcr phosphoras pentoxide. 'Thr wsidw wits cxtracted with 15ml. 01 lloiliiigrlh:ii~oltogivriI.XXg.(84:;) of V , dec. p. 212-21i4". 'This prcxlwt wits rrrrystnllirrd from 3l17! aqueous cth:inol 10 g i w V, t l w . p. %i4-26,5". Anal. Cnlcd. for CIIHAN41: (??!.?): C. 59.5; H. 5.0; N, 12.6. Pound: C. 5!l,4; 11, %(I; S , 12.1;. A small amount of IV $viis rrrovrrccl from thr ethanol extract.
Acknowledgment.-The authors wish to express their indebtedness to Dr. A. EIek for the microanalyses given in this communication.
Studies on Nitrogen Trichloride Treated Prolamines. IV. Isolation of the Neurotoxic Principle BY L.REINER.F. MISANI,T. W. FAIR,P. WEISSAM) M.G. CORDAX0
In a recent publication Bentley, el d.,I announced the isolation of the toxic factor from nitrogen trichloride treated zein. We have also succeeded in isolating the toxic principle in a crystalline form and the purpose of this note is to report the procedure used and to describe the progress made in the characterization of this material. The treatment of zein and the initial steps of purification with cation exchanger (Duolite C,) were those reported previously.z For further purification the following procedure was adopted: The material (toxicity 0.5 g./kg.)' was hydrolyzed by refluxing with 5 N hydrochloric acid for four hours. After removal of the acid i t was extracted repeatedly with butanol from an aqueous solution Subsecontaining 5% trichloroacetic acid. quently, i t was precipitated a t an acid pH value with mercuric acetate, the mercury and hydrogen sulfide removed and the concentrated solution treated with picric acid. In some instances a second mercury precipitation was carried out after removal of the picric acid. This was followed by treatment with Duolite to bring the pH to 3.7. The material dried in wacuo possessed a toxicity of about 30 mg./kg. The solid was extracted with hot methanol. On concentrating and cooling the
Fig. 1
The Pauly and biuret tests were negative. The compound contained sulfur but no S H , -SC+ or chlorine. A bluish purple dye was formed with ninhydrin. Paper chromatograms (ascending) revealed a single spot with Rr values of 0.04, butanolacetic acid; 0.30, lutidin~ollidine-ethanol; 0.62, phenol. After refluxing with 2 N sodium hydroxide for twenty-four hours the reaction with ninhydrin was negative. Refluxing for twenty. four hours with 5 N hydrochloric acid yielded two additional spots and almost complete destruction of the toxicity. There was, however, no increase in the alpha amino nitrogen content after hydrolysis' and no liberation of ninhydrin-positive material after hydrolysis . of the dinitrophenyl derivative. Analysis by Dr. Richard Baltzly (Wellcome Research Laboratories) gave C, 33.31; H, 6.57; N, 15.65; S, 16.15.' A compound having the formula CsHlzNzOsS calculates for C, 33.32; H, 6.71; N, 15.55; S, 17.79; mol. wt., 180.2. Molecular weight determination by freezing point lowering of water gave the value of 187. Electrometric titration revealed only two ionizing groups between the pH values of 2.0 and 9.5 having the pK values of 2.5 and 8.4, respectively: thus only one of the nitrogens is present as a free amino group. The
(1) Bcntley. MeDermott. Pact. Whilehend s a d M m o . N ~ Y I I , 184, 438 (1949). (2) Rrincr. Wciu. Mirani, Coda.ad Pair, Fedmalion Prar.. 8, (4) Determined h y Dr. J n m a B. Nliwa. Rutgm U n i v a i t y . 241 (1848). (5) The loa sulfur value m i 7 be due to s slight mntnmination hv (3) Toxicity v s i u a reported in this mmmunieation were obtained sulfur-free amino acids. Annlysis of II second hatch wss in agree. lo nbbitn, onily. unless stated otherwise. mcnt with the first. except that rvllur was higher. s. 17.81.
2298
NOTES
Vol. 72
minimum molecular weight calculated from the iieutralization equivalent was 175. The evidence presented suggests that the compound is not a peptide but a derivative of methionine. The chromatogr.ams of acid-hydrolyzed material apparently contradicts this view. We found however that methionine sulfoxide also yields two additional chromatographic spots after refluxing with 5 N hydrochloric acid. Only one of these is similar to one of those obtained from the toxic principle. I t is also significant that methionine in large doses (reversal ratio about 1:lOOO) prevented the development of seizures in rabbits. Acknowledgment is due to Dr. H. K. Parker and Mr. K. L. Fortmann for the treatment of the zein.
tained from the corresponding dihydrodiol. The 9,lO-diethyl and 9,lO-diphenyldihydrodiolderivatives of phenanthrene, however, undergo pinacol rearrangement, giving 9,g-diethyl and 9,9-diphenyl-10-phenanthrone. Stannous chloride also reduces a number of 9,IO-dialkyldihydroanthracenediols and 9,lO-dimethy1-9,lO-dihydroxy-g, 10dihydro-1,2-benzanthraceneto the corresponding hydrocarbons. The demethylation of 9,10-dimethyl-3,6-dimethoxyphenanthrene gives 9,10-dimethyl-3,6dihydroxyphenanthrene, a compound that is structurally related to 2,3-bis-(p-hydroxyphenyl)2 - b ~ t e n e . ~The phenanthrene was therefore tested for hormone activity but there was no evidence of estrogenic activity a t the high dose of 3740 y. RESEARCH LABORATORIES Acknowledgment.-The authors are greatly & TIERNAN PRODUCTS, INC. WALLACE RECEIVED OCTOBER 1, 1949 indebted to Dr. Edward D. Campbell, Head, BELLEVILLE, NEWJERSEY Department of Biochemical Research, The Lilly Research Laboratories, Indianapolis, for carrying out the biological assay of 3,6-dihydroxyThe Reduction of Some 9,10-Dihydroxy-9,109,lO-dimethylphenanthrene. We are also very dihydro Derivatives of Phenanthrene and An- grateful to the Canadian Cancer Society for thracene financial aid in support of this work. One of BY ROBERTW. RIMMER,~ ROBERTG. CHRISTIASSEN,?us (R. W. R.) is grateful to the National ReAND REUBEN B. SANDIN ROBERTK. BROWN search Council of Canada for financial support. Experimental5 The reduction of triarylcarbinols, by agents The 9,10-dihydro-9,10-diol~, with the exception of comsuch as vanadous and titanous chlorides, to give I, were prepared by published procedures. hexa-arylethanes has been reported by Conant and pound Reduction of Diols .-The general procedure was as c o - ~ o r k e r s .The ~ present report is a study of the follows: a mixture of 1.0 g. of diol in 25 ml. of glacial aceaction of stannous chloride on certain dihydrodiols tic acid, 10 g. of stannous chloride and 10 ml. of concenTABLE I ACTIONOF STANNOUS CHLORIDEON ~ , ~ ~ - D I H Y D R O X Y - ~ DERIVATIVES , ~ ~ - D IOF H YPHENANTHRENE DRO AND ANTHRACENE Compound used in reaction Substituted 9,lO-dihydrophenanthrene
Product isolated
Yield,
%
60
Q,lO-DiphenyI-Q,10-dihydroxyd
9, 10-Dimethylphenanthreneb 9,O-Diethyl-10-phenanthrone 9,9-Diphenyl-lO-phenanthrone
3,6-Dimethoxy-9,10-dimethyl-9,lO-dihydroxy
3,6-Dirnethoxy-Q,lO-dimethylphenanthrene'
50
9,10-Dimethyl-9,10-dihydroxy" Q,lO-DiethyI-9,lO-dihydroxy"
M., P..
C.
144' 64 198 138
Substituted 9,lO-dihydroanthracene
50 17&-179 35 96 9,lO-Diethylanthracene 25 145 9,10-Diethyl-Q,10-dihydroxy' 9,10-Dimethyl-1,2-benzanthracene 25 122-123 9,1O-Dirnethyl-9,lO-dihydroxy-1,2-ben~ The picrate melted a t 192". Bradsher and Amore, THISJOURNAL, Zincke and Tropp, Ann., 362, 242 (1908). 66, 1280 (1944), report 193-194". Anal. Calcd. for CzzHnOiN3: C, 60.7; H, 3.9. Found: C , 61.2; H, 4.2. Bradsher Werner and Grob, Ber., 37, 28870 (1904). e Anal. Calcd. for ClsHlsOz: C, and Amore, ib&, report 142.5-143". 81.2; H, 6.8. Found: C, 81.1; H, 6.8. The picrate melted a t 186-187 . Anal. Calcd. for C ~ ~ H Z I O C, ~ N58.2; ~: Bachmann and BradH, 4.3. Found: C, 58.3; H, 4.5. f Bachmann and Chemerda, J . Urg. Chem., 4, 583 (1939). bury, ibid., 2, 175 (1938).
9,10-Dimethyl-9,10-dihydroxy' 2,9,1O-Trimethyl-9,1O-dihydroxy'
9, IO-Dimethylanthracene
2,9,10-Trimethyianthracene
of phenanthrene and anthracene. 9,lO-Dimethyldihydrophenanthrenediol is reduced to 9,lO-dimethylphenanthrene by stannous chloride, while 9,10-dimethyl-3,6-dimethoxyphenanthreneis ob(1) Present address: -Department of Chemistry, Purdue University, Lafayette, Indiana. (2) Present address: Department of Chemistry, University of Wisconsin, Madison, Wisconsin. (3) Conant and Sloan, THIS JOURNAL, 47, 572 (1925); Conant, Small and Taylor, ibrd., 47, 1959 (1925); Conant and Bigelow. ibid., 63, 676 (1931).
trated hydrochloric acid was refluxed for one hour. The cooled solution was poured into 500 ml. of water and after standing overnight, the hydrocarbon was filtered, washed with water and dried. The picrate was made and crystallized from alcohol. The pure hydrocarbon was regenerated from the picrate by treatment with ammonia. (4) Dodds, Goldberg, Lawson and Robinson, Nafurc, 141, 247 (1938); Dodds, Goldberg, Lawson and Robinson, Proc. Roy. Sor. ( L o n d o n ) , B127, 140 (1939) ; Dodds, Goldberg, Grunfeld, Lawson, Saffer and Robinson, ibid., B132, 83 (1944). (5) All melting points are uncorrected.
