April 5, 1954
NOTES
cule as oxirane oxygen. Details will be published a t some later date. EASTERN REGIONAL RESEARCH LABORAT0RYl3 PHILADELPHIA 18, PENNSYLVANIA
The Keto Acids of the Tulip (Tulifia gesneriana with Special Reference to the Keto Analog of y-Methyleneglutamic Acid
(13) One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Service, U. S.Department of Agriculture.
1!)50
BY G. H. N. TOWERS'AND F. C. STEWARD RECEIVED NOVEMBER 11, 1953
Our interest in the keto acids of plants, because of their importance in the understanding of nitrogen metabolism, has prompted the development of a 3-Indolecarboxaldehyde Thiosemicarbazone, a general method for their identification and quantiNew Antitubercular Compound' tative determination. This method, which will be BY I.OWEI,LE. WELLER,HAROLDM. SELLAND R. Y . published in due course (Towers, Thompson and GOTTSHALL Steward) depends upon the following procedures. RECEIVED JANUARY 11, 1954 a. The plant material is killed and the keto comThe inhibition of growth of Mycobacterium tuber- pounds fixed with an alcoholic solution of either 2,4culosis by many thiosemicarbazones has been re- dinitrophenylhydrazine or 1,l-diphenylhydrazine. b. The keto-acid hydrazones are separated from ported.2ab , 3-Indolecarboxaldehyde thiosemicarbazone has been shown to have high bacteriostatic the amino acids and neutral carbonyl compounds activity in vitro and t o suppress tuberculosis in by extraction into ethyl acetate and subsequent mice after injection of virulent tubercle bacilli. extraction of the ethyl acetate solution with dilute Because of these properties the synthesis of this sodium carbonate. c. Specific 2,4-dinitrophenylhydrazonesare recnew thiosemicarbazone is described. ognized by chromatography on paper. 3-Indolecarboxaldehyde Thiosemicarbazone.-Thiosemid. The hydrazones are converted to the correcarbazide 8.2 g. (0.09 mole), dissolved in 100 ml. of warm 30% acetic acid was added to a solution of 12.3 g. (0.085 sponding amino compounds by catalytic hydromole) of 3-indolecarboxaldehyde4.6 in 200 ml. of methanol genolysis. and the resulting mixture refluxed for 2 hours. After coole. The amino compounds so produced are recoging, the precipitate was collected by filtration, washed with cold water and air-dried. The crude product was purified nized by two-directional chromatography on paper by recrystallization from methanol to give light yellow crys- and treatment of the chromatograms with nintals of 3-indolecarboxaldehyde thiosemicarbazone, m .p. hydrin. Thus the keto-acids may be recognized 230-232 dec . by drawing upon the extensive information, which Anal. Calcd. for GOHIONIS:N, 25.67. Found: N, is now available, on the chromatography of the 25.48. amino compounds on paper and by utilizing the Biological Properties.-The following organisms were not sensitivity of the ninhydrin method for their inhibited by 100 micrograms of 3-indolecarboxaldehyde thiosemicarbazone: D~plococcuspneumoniae, Streptococcus detection. hemolyticus, Bacillus subtilis, Eschekhe'a coli, Pseudomonas The recent recognition of y-methyleneglutamine aerugznosa, Salmonella typhimurium, Micrococcus Pyagenes and of y-methyleneglutamic acid as constituents var. aureus, Klebsiella pneuwoniae, Proteus vulgaris, Shiof the peanut plant2 and the proof that these subgella dysenteriae, Corynebacterium diphtheriae, Aerobacter aerogenes, Mycobarterium phlei, -21. smegmatis and Myco- stances are identical with nitrogenous compounds bacterium 607. earlier recognized in the tulip plant3v4gives rise to M . tuberculosis var. horninis, strains H37Rv, H37R. and interesting possibilities. Ts1760 were inhibited bv 6.2, 0.8 and 3.2 micrograms of Therefore, in making the first extensive examinathe chemical, respectively. When injected intraperitoneally as a suspension, 50 mg. tion of the ketoacids of the tulip plant, special was lethal for 20-g. white mice while 25 mg. was tolerated. attention was paid to the recognition and identiFour-and-one-half mg. injected daily on seven successive days was tolerated and this dose was used for a preliminary fication of the keto analog of y-methyleneglutamic protection test. For this test, 1.0 mg. of M . tuberculosis, acid. Work has been done on the keto compounds H37R., was injected intravenously into eight mice and the present in the tissue of the resting tulip bulb (in following day, 4 5 mg of the 3-indolecarboxaldehyde was which the 7-methyleneglutamine was first recoginjected subcutaneously. Daily injections of the same dose of chemical were made for 17 days. Kineteen days nized) and also of the green foliage leaves in which after the last injection, seven of the eight treated mice were it is present in larger amount. still alive while of the 10 control mice infected a t the same The 2,4-dinitrophenylhydrazones from the tulip time, but which did not receive the chemical, only three bulb yielded on hydrogenolysis the fallowing amino survived. compounds: glycine, alanine, aspartic acid, gluThis investigation was supported in part by a re- tamic acid and valine. In addition to these known search grant E-227(c) from the National Institutes amino acids, hydrogenolysis also yielded amino of Health, U. S. Public Health Service. compounds whose identity still remains unknown. DEPARTMENT OF AGRICULTURAL CHEMISTRY Therefore, one could infer that the following keto MICHIGAN STATE COLLEGE, EASTLANSING, AND compounds, corresponding to this list of amino MICHIGAN DEPARTMENT OF HEALTH acids, w e present as constituents of the tulip plant: LANSING, MICHIGAN glyoxylic acid, pyruvic acid, oxaloacetic acid, a(1) Journal article No. 1515, Michigan Agricultural Experiment ketoglutaric acid and unknown keto compounds. Station. (2) (a) G. Domagk, R. Behnisch. F. Mietzsch and H. Schmidt, NqfuuwisSenschaflen, 93, 315 (1946); (h) G . Domagk, Schwefz. Z . Path. U. Baki., is, 575 (1949). ( 3 ) G.Domagk, Beilr. Klin. Tzcbevk., 102, 603 (1950). (4) F. T.Tyson and J. T . Shaw, THISJOURNAL, 74, 2273 (1952). ( 5 ) E. Campaigne and W. I,. Archer, ihid., 75, 984 (19%).
(1) Lalor Foundation, Pre-doctoral FelIow at Cornell University. (2) J. Done and L. Fowden, Btochem. J . , 49, Proc. XX (1951). (3) F C Stenard and J. F. Thompson, A n n . Rcu. Plant Phrsiol. 2, 233 (1950). (4) R . Zacharias, J. K . Pollard and F. C . Steward, THISJ O U R N A L , 76, 1961 (1954)
