Sept., 1950
BIOLOGICALLY ACTIVEN’-ALKYLSTREPTOMYCYLAMINES
3969
[CONTRIBUTION PROM THE FOOD RESEARCH LABORATORIES,LONGISLANDCITY, N. Y., AND THE RESEARCH LABORATORIES OF CHAS.PFIZERAND C o . , INC.]
Biologically Active N’-ALkylstreptornycylamines’ BY W. A . WINSTEN,C. I. JAROWSKI, F. X. MURPHYAND W. A . LAZIER Using the paper chromatographic technique, there has been demonstrated2 the presence of streptomycin derivatives having antibiotic activity in solutions of streptomycin and primary amines subjected to reductive alkylation. From the method of synthesis used, it was proposed that these derivatives are N’-substituted streptomycylamines. This paper describes the preparation, isolation, purification and proof of structure of streptomycylamine and several N’n-alkyl derivatives. The general reaction is represented by the equation St.CHO
Hz + RKHz + St.CHzSHR + HzO Pd-C
covered. It was necessary to heat a solution of I1 in normal sulfuric acid on a steam cone for one hour before hydrolytic cleavage occurred. Thus the insertion of a nitrogen atom in the streptose moiety of the streptomycin molecule strengthens the hemiacetal union between streptidine and streptobiosamine. Although this is not strictly analogous, i t recalls to mind the resistance shown by D-glucosaminides to similarly mild hydrolytic treatment.’ The only product isolated by this acid hydrolysis of I1 a t elevated temperatures was streptidine sulfate (111). The other fragment underwent extensive degradation. Nevertheless, the isolation of I11 shows that the alkylamino group in I1 must be attached to the streptobiosamine portion. Additional proof was provided by mercaptolysiss and chromatographic purification of the reaction mixture using acid-washed alumina. An amorphous product (IV) was obtained, the analysis of which conformed to the scheme
where St. equals streptomycin minus the aldehyde group. The compounds in Table I1 were prepared by hydrogenating a solution of the base and streDtomycin calcium chloride double salt3 in absdute methanol. Five per cent. palladium-on-carbon was used as the catalyst under 1000 p. s. i. of hydrogen a t 70-85’. The derivatives were purified by 0 I fractional crystalliza1 H-C-OH CHI 1 tion of their helian.2HCI + Streptithates. The fractions HO-C-H dine.2HCI I which were found to be devoid of streptomycin and dihydrostreptomyIV CHzOIf cin, as determined by The antibacterial activity of the N’-alkylstreppaper chromatograph;, were combined and recrystallized from methanol. The analytically toniycylamines against Escherichia coli and Bacilpure tetrahelianthates were converted to tetra- lus subtilis is listed in Table 11. Preliminary hydrochlorides by a method already d e ~ c r i b e d . ~studies indicate that the acute mouse toxicity for The general formula I was assigned to these all these compounds is higher than that of streptosubstituted streptomycylamines after a study of mycin. Replacement of the newly-formed hythe degradation of N‘-n-hexadecylstreptomycyl- droxyl group of dihydrostreptomycin by an amino amine tetrahydrochloride (11). When I1 was group lowers the antibacterial activity and raises treated with acid by procedures described for the the acute toxicity in mice. Potency reappears degradation of streptomycin into streptidine and when the ”-alkyl substituent is the n-decyl group. s t r e p t ~ b i o s a n i i n eonly , ~ ~ ~starting material was re- Insofar as E. coli potency is concerned, the most effective member of the series studied is N’-n(1) N’ designates the nitrogen atom introduced into the streptomycin molecule by catalytic hydrogenation in the presence of primary dodecylstreptomycylamine. This derivative is amines. even more effective than streptomycin against (2) (a) Winsten and Eigen, THISJ O U R N A L , 70, 3333 (1948). this organism. (b) Winsten, paper presented at the 114th meeting of the American Chemical Society in Washington, D. C., August 1948. The hydrochlorides listed in Table I1 are white, (3) Peck, Brink, Kuehl, Flynn, Walti and Folkers, ibid., 67, 1866 amorphous solids. All but the parent member of (1945). the series, streptomycylamine, may be extracted (4) Kuehl, Peck, Hoffhine, Craber and Folkers. ibid., 68, 1460 (1946). into n-butanol from aqueous solutions a t @H11.5. (5) Peck, Oraber, Walti, Peel, Hoffhine and Folkers, i b i d , 68, This property provides a convenient method for 29 (1946). I
f t i ) Carter, Clark, Dickman, Loo, Meek, Skell, Strong, Alberi, Bartz, Rinkley. Crooks,Hooper and Rebstock, Science. 108,53 (1946).
(7) Fried and Wintersteiner. THISJOURNAL, 68, 2096 (1946). (8) Kuehl, F l y n n , Brink and Folkers, i b i d . , 68, 2046 (1946).
W. A. WINSTEN,C. I. JAROWSKI, F. X. MURPHYAND W. A. LAZIER
3 970
Vol. 72
TABLE I TETRAHELIANTHATES OF STREPTOMYCYLAMINE AND "-ALKYL DERIVATIVES Amine derivative
H
St.-CH2-d
\R
H n-Octyl n-Decyl n-Dodecyl n-Tetradecyl n-Hexadecyl
M. P . , C.
Formula
222-224 dec. 203-205 dec. 223-224 dec. 208-209 dec. 223 dec. 222-223 dec.
Carbon, 7c Calcd. Found
50.9 C;;H~OZOZ~ITZOS~ 51.1 C85H118023N2~S4~H20 52.7 52.6 C~~H122023NZOS4~3H20 52.2 52.3 C ~ ~ H ~ Z ~ ~ Z ~ N 5Z4O . 2 S ~ 54.5 55.2 CsiHi;oOz3NzoS4 54.6 55.0 C83H1340~3P\r~~S4 55.1
Hydrogen, % Calcd. Found
5.6 6.2 6.4 6.4 6.5 6.6
6.0 6.2 6.5 6.8 6.6 6.9
Nitrogen, '% Calcd. Found
15.5 14.4 14.0 14.2 14.0 13.8
15.4 13 8 13.3 14.7 14.4 13.9 I
TABLE I1 TETRAHYDROCHLORIDES OF STREPTOMYCYLAMINE AND "-ALKYL DERIVATIVES Amine derivative St.-CHs--NHR R=
H n-Octyl n-Decyl n-Dodecyl n-Tetradecyl n-Hexadecvl Streptomycin.3HCl
M. P., OC.
164-166 dec. 169-170 dec. 180-181 dec. 146 dec. 191-192 dec. 175-177 dec.
Formula
Carbon % Calcd. $ound
C Z ~ H ~ ~ O I I N E C ~3~4 . 6 35.0 C Z ~ H ~ Z O I ~ X ~ C ~4 ~ 0 . 5H Z 40.6 O C31H66011~8C14 42.8 42.9 C33HmOiiNaC14 44.1 44.5 Ca6Hdh1NsC14 45.4 4 5 . 6 2 C ! ~ ; H ~ ~ O I ~ N ~ C43.3 ~ ~ . ~ H4 3~.O
the separation of these "-alkyl derivatives from streptomycin and dihydrostreptomycin. I n addition to the compounds listed in the tables, the following amines were satisfactorily coupled with streptomycin under similar conditions : n-butylamine, cyclohexylamine, 2-amino-5-diethylaminopentane, ethanolamine, L-lysine, 2-aminoheptane, sulfanilamide, benzylamine, p-aminosalicylic acid, P-phenethylamine, 2-aminopyridine, 3-aminoquinoline, D-glucosamine, a-naphthylamine and 0-cyclohexylethylamine. Although these derivatives were not purified, their existence in predominant amount was proved by paper chromatography of the crude reaction mixtures. Experimental9 General Procedure for Reductive Alkylation.-Two hundred milliliters of a methanol solution containing 14.9 g. (0.01 mole) of the calcium chloride double salt of streptomycin and 9.4 g. (0.06 mole) of n-decylamine was reduced under 1000 p. s. i. of hydrogen a t 80-85 o in the presence of 5% Pd-C (5 g.). After six hours, heating was discontinued and the hydrogenation was continued for 18 hours. The catalyst was filtered off, washed with methanol and the combined filtrate and washings were poured into 10 volumes of absolute ether. The precipitate was filtered off, washed with ether and dissolved in distilled water. The solution was adjusted from PH 8 to 5.5 with hydrochloric acid, frozen and lyophilized. The crude residue weighed 13.2 g. It assayed as follows: maltol,10 0 -y/mg.; streptidine," 660 y/mg. The negative maltol test for crude N'-n-decylstreptomycylamine (VI) signifies that no streptomycin was present. Such a value would be given by dihydrostreptomycin or any N'-alkylstreptomycylamine. The streptidine assay, a colorimetric determination of guanidino groups, was carried out by a procedure developed in this laboratory. Preparation of He1ianthates.-The following description is typical of the procedure used in preparing helianthates. Crude VI (24.6 g., 0.028 mole) was dissolved in 100 ml. of (9) All melting points are uncorrected. (10) Boxer, Jelinek and Leghorn, J. B i d Chcm., 169, 153 (1947). (11) Monastero, method to be published.
Hydrogen % Calcd. Fohnd
6.3 7.5 7.6 7.8 8.1 8.1
6.7 7.5 7.4 8.0 8.1 8.1
Nitrogen, % ' Calcd. Found
15.4 13.0 12.9 12.4 12.1 10.8
14.7 13.3 12.9 12.5 12.2 10.8
Bioassay gamma/mg.
E. coli B. sublilis
465 238 635 1000 765 240 841
385
