2078
COMRIUNICATIONS T O T H E
EDITOR
Vol. s:!
(Anal. Found for CluHzoNnO.HC1:C, 69.59; H, 6.68; N, 8.62), m.p. 225-226", which was reduced catalytically to the 3-benzylphthalimidine, then reduced with lithium aluminum hydride to 11. 11 showed an interesting wide spectrum of pharmacological activity. It is a moderately active antihistaminic, a potent local anesthetic, has an antiinflammatory effect in the rat dextran edema test and shows a marked tranquilizing effect on mice, I n exploring variations in the structure of 11, compounds obtained by the replacement of the nitrogen of the isoindoline nucleus by carbon, ;.e., indenes, were investigated. Such analogs were prepared as follows. 2-(Dimethylaminoethyl) -indane-1-one, (111), b.p. 132" a t 1.7 mm. ( A n d . Found: C, 76.36; H , 8.53; N, 7.12), prepared by a modification of the method by Hoffmann, et al.,l for similar basic indanones, was treated with Grignard reagents or organolithium compounds. The intermediate amino alcohols dehydrated easily by warming in dilute aqueous acid to give the desired indenes. A large number of compounds were prepared by varying the different structural elements of such indenes. Reaction of I11 with the organolithium derivative generated by the action of phenyllithium on 2-ethylpyridine and dehydration gave dl-2- ( 1-[2-(2-dimethylarninoethyl)-3-indenyl]-ethyl] -pyridine I V (Anal. as the maleate, C21H22N2C2H404. Found: C, 69.81; H, 6.07; N, 7.16), m.p. 159-1Gl0, the most active antihistaminic of the series.2 Resolution of I V via the tartrate salts was accomplished. The activity resides chiefly in the levo rotating isomer. This substance as the maleate in the standard in vivo guinea pig assay is about four times as potent (oral EDso-31y=t3.5ylkg.) as dextrochloropheniramine maleate, the hitherto most active antihistamine reported4
B three-week assay involving quantitative iiieasurernent of the degree of hydropericardium produced in chicks on a test diet containing the imterials to be tested was used to guide the purification of the factor. The details of the biological studies will be reported elsewhere.3 The toxic tallow was refluxed in aqueous methanolic sodium hydroxide solution and the neutral, non-saponifiable fraction separated by extraction with ethylene dichloride to yield an active ( N X ) concentrate.4 This was dissolved in hexane and chromatographed on alumina (load,'adsorbent ratio 1:4). The bulk of the inert material passed through the column and the activity was eluted with 10% ether in hexane. The oily product (500 X) was separated from carbonyl-containing impurities by chromatography on Decalso (1: 100) using hexane as the developing and eluting solvent. The product (5,000x) was again chroinatographed on alumina (1 : 30) to give a 30,OOOX concentrate. Finally, this high potency preparation was chromatographed on a high-ratio alumina column ( I : 1000) using isooctane as the developing and eluting agent. The final chromatogram yielded partly crystalline peak fractions. These were triturated with isooctane to remove oily impurities and the residues were crystallized from a benzene-hexane mixture. After recrystallization to constant properties, the toxic fat factor was obtained as colorless needles which did not melt on the hot stage but sublimed slowly above 225 O, rapidly a t 243 '. I t was homogeneous when examined by reverse-phase chromatography on silanated paper. In 1 : 1 dimethylformamide-isooctane, a single spot vias observed a t IZf 0.8s. In iso6ctane solution, the factor was characterized by absorption maxima at 234 m p (El?m 1431) and 312 m p (E;%, I I i ) , and a shoulder a t 238 mp 11%). KO absorption maxima were observed iii the infrarccl (1) H. J. Schmid, A. Hunger and K. Hofimann, Hela. Chiin. Acta, below G.3 p , indicating the absence of carbonyl 39,607 (1956). (2) The generic name is dimethpyrindene. and hydroxyl groups. In the chick assay, this (3) These compounds were investigated pharmacologically by Drs. crystalline toxic factor produced iriarked hydroW. Barrett and A. Plummer. pericardium a t a dose of 0.1 nig./kg. of diet, and (4) F. Roth and W. M . Govier, J . Phavin. and Ex$. Thcr., 124, 347 was assigned a potency of approxiiriately one (1'358). C. F. HUEBXEK million times that of the crude toxic fat. The sinall quantity (less than 1 nig.) o f crystalELLESD O N O G H U E RESEARCH DEPARTMEKT PATRICIA ~ ' E N K line toxic factor isolated to date has not permitted CIBA PHARMACEUTICAL PRODUCTS INC. E. S C R Y structural studies, even to the extent of elementary J . -1.XEI.SOS SUMMIT, E,J . analysis. The infrared absorption spectruiii is RECEIVED MARCH 3, 1960 notably lacking in structural implications, but docs suggest absence of functional groups containing oxygen. Until the last step in the isolation, it was THE ISOLATION AND CHARACTERIZATION OF THE not possible to correlate any chemical or physical CHICK EDEMA FACTOR property with the active factor; complete dependSir: ence on the chick assay was required. The agent responsible for an edema condition in Other groups studying the toxic principle have broiler type chicks has been traced' to certain lots used purificatioii methods similar to those outlined of feed grade animal fat. This toxic factor has here and have suggested that the factor might been further restricted2 to the non-saponifiable possess a steroidal5 or other polynuclear s t r ~ c t u r e . ~ ~ ~ portion of the tallow. We wish to report here the (3) W. H. Ott, el al., t o be published. isolation of the edema-producing substance in (4) Activities are expressed as multiples of the toxicity of the crystalline form and its preliminary characteriza- original tallow. ( 5 ) W. B. Brew, J . D. Dore. J. H. Benedict, G . C. Potter and E. tion. (1) Anon., Fccdslufs, 3 0 , No 14, 1 (1Q58). (2) S C. Schmittle, H. M. Edwards and D Morris, J . A m . Vel
M c d . A S S G C 131, ., 216 (1958).
Sipos, J . ASSGC. Ofic.Agr.. Cheviists, 41, no. 1, 120 (193'3) (6) L. Friedman, D. Firestone, W. Horwitz, D. Banes, 11. Anstead and G. Shue. ibid.,41, no. 1, 12'3 (195'3). (7) J. C. Wootton and J. C. Alexander, ibid., 42, no. 1, 1-11 (1'359).
April 20, 1960
COMMUNICATIONS TO THE EDITOR
2079
C = 0.90 in 6 N HCI; calcd. for CbHgN04; C, 40.82; H, 6.12; N, 9.52; 1 C-Me, 10.2; reported for a(L),P-methylaspartic acid,4 [a] D 13.3", C = 3.0 in 5N HCI). It was separable by paper chromatography from glutamic acid and a-methylaspartic but not from P-methylaspartic acid.j The identity of the isolated compound with a ( ~ ) , p methylaspartic acid was supported further by the infrared spectrum. The a,P-diaminobutyric acid eluted from the Celite column was isolated as the monohydrochloride monohydrate by preparative zone electrophoresis and crystallization from 0 . l N HCI, pyridine and acetone (Found: C, 27.33; H, 7.59; MERCKSHARP& DOHME ROBERT E. HARMAN N, 16.59; C1-, 20.16; NH2-N, 16.3; ninhydrin E. DAVIS COZ, 23.9; C-Me, 5.85; N-Me, 0; [Cy]"D +16.B0, RESEARCH LABORATORIES GEORGE WALTHER H. OTT C = 0.72 in 5NHC1; calcd. for C ~ H ~ ~ N ~ O ~ . H Z O . H C I : DIVISIONOF MERCK& Co., Isc. RAHWAY, NEW JERSEY NORMAN G. BRIKK C,27.83; H,7.54; N, 16.23; C1-,20.56; 2NH2-N, FREDERICK A . KUEHL,JR. 16.3; 1 ninhydrin COS,25.3; 1 C-Me and 1 N-Me, RECEIVED MARCH5, 1960 8.7). The substituted phenylurea was prepared (Found: C, 60.40, 60.49; H, 5.90, 5.59; N, ISOLATION AND IDENTIFICATION OF D-a-PIPECOLIC 16.05, 15.83; calcd. for C18H20N404: C, 60.7; ACID, a[L],B-METHYLASPARTIC ACID AND alp- H, 5 . 7 ; N, 15.7). Of all the structures theoretically DIAMINOBUTYRIC ACID FROM THE POLYPEPTIDE possible, only a,P-diaminobutyric and a,P-diaminoANTIBIOTIC ASPARTOCIN isobutyric acids met all the requirements. The Sir: The antibiotic Aspartocin produced by the micro- latter compound was ruled out on the basis that organisms Streptomyces griseus var. spiralis and deamination of the natural product with nitrous Streptomyces violaceus was isolated recently and acid, then periodate oxidation, yielded not formalcharacterized as an acidic fatty acid containing dehyde but acetaldehyde as shown by paper polypeptide similar to but different from Ampho- chromatography of the 2,4-dinitrophenylhydrazones. The postulated structure, cu,p-diaminomycin.' Glycine, L-aspartic acid, L-proline and butyric acid, was proved by synthesis! The natuL-valine were reported to have been isolated in not be separated crystalline form from a hydrolysate. We now wish ral and synthetic compounds could The infrared curves by paper chromatography. to report on the isolation in crystalline form of the other three ninhydrin-positive components and showed no differences. (4) H. A. Barker, R. D. Smyth. E. J. Wawszkiewicz, M. N. Lee and their identification as D-a-pipecolic acid, a [L] ,Wilson, Arch. Biochem. Biophys., 7 8 , 468 (1958). p-methylaspartic acid and a,p-diaminobutyric acid. K.(M. 5 ) We are indebted to Dr. H. A. Barker, University of California, Chromatography on Celite with the system 1- for these samples. butanol, 1-propanol, 0.1 N HCl ( 1 0 : l : l O by vol(6) C. Kolbe, J . p r a k l . Chem., [ 2 ] 25, 369 (1882); C. Neuberg, ume) gave a band containing pipecolic and p- Biockem. Z . , 1 , 282 (1906). J. H. MARTIN methylaspartic acids as well as a band of a,p- LEDERLELABORATORIES RESEARCH SECTION diaminobutyric acid. The former mixture was BIOCHEMICAL CYANAMID COMPANY resolved by ion exchange chromatography on AMERICAN PEARL RIVER,KEWYORK W. K. HAUSMAXN Dowex 50 X 8 using 1N HC1 as eluent. AnalytiRECEIVED MARCH7 , 1960 cally pure pipecolic acid monohydrochloride was obtained by recrystallization from ethanolic ether (Found: C, 43.09; H, 7.20; N, 8.13; C1-, 21.37; GLYCEROL : ISOLATION, IDENTIFICATION AND INCORPORATION FACTOR ACTIVITY FOR B. C-Me, none; N-Me, none; calcd. for C6HllNOZMEGA THERI U M HC1: C, 43.38; H, 7.25; N, 8.46; C1-, 21.15). Optical rotation measurements on the natural Sir: ~ C = 3.861 in H20) compound ( [ a I z 5+15.5", The purification of a component of bacterial indicated i t to have mainly the D-configuration cells and its demonstrated activity in the incorpora(reported2 for the L-form: [cx]'~D -24.6", C = tion of amino acids has been described.' We wish 1.087 in HzO). The isolated compound was sepa- to report here studies on a further purified sample rated by paper Chromatography from p- and y - of this factor which have led to its identification pipecolic acids but not from a-pipecolic acid. as glycerol. After racemization3 matching infrared curves were Gale and Folkes' noted that bacterial cells lose obtained for the isolated material and authentic their ability to incorporate amino acids into proDL-a-pipecolic acid. tein when disrupted and deprived of their nucleic The methylaspartic acid was recrystallized from acid. Upon addition of nucleic acid from the aqueous ethanol (Found: C, 40.75; H, 6.41; same or some other sources, the system was stimuN, 9.61; C-Me, 7.1; N-Me, 0 ; [ ( r I z 5 ~ +12.2', lated to resume incorporation of amino acids. Us(1) A. J. Shay, J. Adam, J. H. Martin, W. K. Hausmann. P. Shu ing sonically disrupted and depleted Staphylococcus and N. Bohonos, "Antibiotics Annual 1959/60," in press. aureus cells for measurement of activity, they
Since their preparations, however, were not pure, these structural interpretations must be regarded with reservations. We hope to be able to prepare more pure material shortly for further characterization of this most unusual compound. \Ye are indebted to Robert W. Walker for the spectral studies. We acknowledge the generosity of B. H. Barrows of the Hales and Hunter Co. and K'. B. Brew of the Ralston Purina Co. for supplying us with the fats used in these studies and for helpful discussions of the problem; and we also wish to thank L. Friedman and his associates of the Food and Drug Administration for useful discussions.
+
( 2 ) N. Grobbelaar, R. M. Zacharius and F. C. Steward, THIS 76, 2912 (1954). (1) E. F. Gale and J. P. Folkes, Biochem. J . , 69, 661 (1955); 69, 611 (3) M . Bergmann and L. Zervas, Biochem. Z., 203,280 (1928). (1958).
JOURNAL,
