GROWTH INHIBITION OF STRAIN H37 OF THE HUMAN TUBERCLE

Transcription

1 GROWTH INHIBITION OF STRAIN H37 OF THE HUMAN TUBERCLE BACILLUS BY 4-N-ALKYLRESORCINOLS IN THE DEPTH OF A LIQUID, SYNTHETIC, NONPROTEIN CULTURE MEDIUM Colorado Foundation for Research in Tuberculosis at Colorado College, Colorado Springs, Colorado Received for publication December 20, 1945 It was concluded after an earlier unreported investigation that 104 per cent of 4-n-hexylresorcinol in Long's liquid, synthetic, nonprotein culture medium (Long and Seibert, 1926) inhibited the depth of 10-3 mg (about 101 bacilli) of the H37 strain of the human tubercle bacillus. When either n-hexylic acid to which equivalent molecular amounts of NaOH had been added or resorcinol was placed in the same medium, was prevented by 10-1 and permitted by 10-2 per cent concentrations (Drea, 1944). It seemed desirable to investigate the antibacterial effects of other 4-n-alkylresorcinols. EXPERIMENTAL The bacteriological technique was the same as that used by the writer in 1944 for other similar studies. The 4-n-alkylresorcinols were crystalline solids. Dr. L. Earle Arnow (1945) reported that all had been purified by recrystallization, except the ethyl, propyl, and butyl compounds, which were prepared by distillation only. All of them were dissolved in sterile solutions of 30 per cent glycerol in distilled water and placed in a dark closet at room temperature. The following percentages of concentrations-2 and 2 X 10-1 of ethyl, propyl, butyl, and amyl; 10-1 of hexyl and heptyl; and 2 X 10-2 of octyl, decyl, undecyl, dodecyl, and tetradecyl resorcinols-were sterile at the end of 24 hours. Any clouded suspensions were made clear by warming them. The foregoing concentrations were then diluted with sterile distilled water to give 2 X 10-1, 2 X 10-2, etc., per cent concentrations, and from these 1-ml amounts were taken and dropped into 19 ml of Long's liquid synthetic culture medium to give 10-2, 10-3, etc., per cent concentrations of the substances to be tested for their antibacterial effects. Smaller gradations than decimal dilutions were not studied. A check was made for possible additive antibacterial effects of both hexylic and heptylic soaps separately on that of resorcinol. Equivalent molecular amounts of NaOH were added to the fatty acids (from Eastman Kodak Company). The three substances were prepared as sterile solutions in the same way as were the alkylresorcinols. The proper amounts of either fatty acid compound and resorcinol were placed in the culture medium to give the desired equivalent total weight of the corresponding alkylresorcinol. Plantings of 10-3 mg of well-dispersed H37 bacilli were made and incubated 507

2 ,508 at 37 C for at least 60 days. Evaporation of water was nil. Three separate experiments were made for each tested substance with the exception of the hexyl and heptyl resorcinols for which 5 and 4 tests, respectively, were made. The ph of the culture media was 7.2. RESUJLTS Seven of the 11 alkylresorcinols inhibited at 10-4 per cent concentrations. All of these had 6 or more carbon atoms in the alkyl chain. The hexylresorcinol permitted questionable visible in three out of five trials and no in the other two attempts. The heptylresorcinol allowed one small clump of bacilli to grow in one out of four trials, the other three permitting no. Ethyl, butyl, and amyl resorcinols with 2-, 4-, and 5-carbon atom side chains, respectively, inhibited at 10-3 per cent concentrations. The propyl resorcinol with a 3-carbon atom chain inhibited at 10-2 per cent concentrations. The mixture of either hexylate or heptylate with resorcinol permitted at 10-2 and inhibited at 10-1 per cent concentrations. Only -inhibiting powers for 60-day incubation periods are reported. It was established that incubation for some time longer than 60 days or transplanting to fresh Long's liquid medium at the end of 60 days permitted of previously invisible cultures or plantings in 10-4 per cent n-hexylresorcinol media. DISCUSSION The 4-n-alkylresorcinols are produced by condensing resorcinol with the proper fatty acids in the presence of ZnCl2, followed by reduction according to the method of Clemmensen (Suter, 1941). The alkyl chain C.H2.+1 is attached to the carbon atom in the 4-position of the resorcinol molecule as represented by: COH HC( CH HCjCOH C CnH2n+1 If n has the value of 6, 7, 8, 10, 11, 12, or 14, the -inhibiting amount is of order of 10-4 per cent. Growth is permitted by 10-5 per cent. The reaction between a fatty acid and NaOH in water may be represented by the reversible formula: CH3(CH2),_2COO-H + NaOH t CH8(CH2)n-2COO.Na + H20 Here, n must have the value of 14 or 16 for the 14- and 16-carbon atom chains of myristic and palmitic acids, respectively, in order to inhibit at 10-4 per cent under the same conditions (Drea, 1944). The hexylic and heptylic acids with 6- and 7-carbon chains, respectively, have considerably less antibacterial power. The substitution, however, of the hexyl

3 ALKYLRESORCINOLS ON TUBERCLE BACILLI 509, or heptyl chain in the 4-position of the weakly inhibiting resorcinol ring results in a molecule with considerably greater antibacterial power. The ethyl, propyl, butyl, and amyl resorcinols have less inhibiting power than the hexyl and heptyl resorcinols, but are also characterized by being more inhibiting than their side chains as fatty acids and soaps or resorcinol. The lesser antibacterial property of the propyl resorcinol as compared with that of the ethyl and the butyl compound may be due to the fact that none of the three compounds were recrystallized. It was indicated that the octyl and longer alkyl chain resorcinols tested were somewhat more antibacterial than the hexyl and heptyl resorcinols, but within TABLE 1 Effect of alkylresorcinols on the of tubercle bacilli 10-3 mg of well-dispersed H37 bacilli planted Incubation for 60 days at 37 C NUMIBER OF GROWTH-INHIBITING CONCENTRATION GROWTH- C ATOMS TYPE OP ALKYLBE- PERMITTING IN AIYL SORCINOL TESTED CONCENTRATION CHIN Number of tests Per cent PER CENT 2 Ethyl 2 out of out of 3 with very small amount of Propyl 3 out of 3 10' Butyl 3 out of Amyl 3 out of Hexyl 2 out of out of 5 with questionable Heptyl 3 out of out of 4 with very small amount of Octyl 3 out of ' 10 Decyl 3 out of Undecyl 3 out of Dodecyl 3 out of Tetradecyl 3 out of the limits of error of this investigation the longest chain compound, tetra-decylresorcinol, had the same order of antibacterial power as the 14-carbon atom chain of myristic acid. The antibacterial effect of organic compounds with long carbon chains continues to excite interest in tuberculosis research. Hook and Robinson (1944) are investigating synthesized derivatives of dialkylbutyric acids. Roberts (1945) is studying agaric acid and related compounds. Barry and McNally (1945) concluded from their investigation of dialkyl succinic acid derivatives that a-ethyl-a'-dodecyl succinic monomethyl ester inhibited of a bovine strain in vitro for 6 weeks at 1:400,000, and that the isomeric a-a'-di-n-heptyl acid ester inhibited for 6 weeks at 1:300,000. They

4 510 were of the opinion that it was indicated that the molecular weight of the acid, or, alternately, the total number of carbon atoms in the two substituents, must lie within a definite range to produce maximum tuberculocidal activity in the half-ester. Fatty acids with fewer carbon atoms may also be of importance, because, when condensed with weakly inhibiting molecules such as those of resorcinol, they acquire a much greater -preventing action in vitro on the H37 strain of the tubercle bacillus. The present writer, in 1944, suggested that the addition of a 6-carbon atom or longer alkyl chain at the 4-position of the greatly inhibiting sodium ethylmercurithiosalicylate molecule, with or without the substitution of some element such as Cd or Mn for the Hg atom, might be advantageous. It may be desirable to add the 2- to 5-carbon atom chains in the same way. SUMMARY Hexylic acid and heptylic acid, to which equivalent molecular amounts of NaOH have been added, prevent the of 10-3 mg (about 106 bacilli) of the H37 strain of the human tubercle bacillus in the depth of Long's liquid, synthetic, nonprotein culture medium when present in 10-1 per cent concentrations and permit in 10-2 per cent concentrations. Resorcinol under the same conditions has the same effect. The corresponding n-alkylresorcinols with the hexyl and heptyl chains substituted for the H atom in the 4-positions of the resorcinol molecules inhibit at 10-4 per cent and permit at 10- per cent concentrations. Octyl, decyl, undecyl, dodecyl, and tetradecyl chain resorcinols have the same order of -preventing power. The first four of these are more inhibiting than the corresponding fatty acids or resorcinol. Tetradecyl resorcinol has about the same -preventing effect as the corresponding myristic acid. Ethyl, propyl, butyl, and amyl resorcinols have less -inhibiting power than the longer chain compounds but are more inhibiting than their respective fatty acids or resorcinol. The addition of alkyl chains to the 4-position of the greatly inhibiting sodium ethylmercuri-thiosalicylate molecule, with or without the substitution of some other element for the Hg atom, may be of antibacterial importance in both in vitro and chemotherapeutic studies. Bactexicidal effects were not studied. ACKNOWLEDGMENT The writer expresses his appreciation for the donation of the alkylresorcinols to Dr. W. A. Feirer of the Medical Research Division of Sharp and Dohme, Inc. REFERENCES AiNow, L. E Personal communication. BARmY, V. C., AND MCNALLY, P. A Inhibitory action of dialkyl succinic acid derivatives on the in vitro of acid-fast bacteria. Nature, 156,

5 ALKYLRESORCINOLS ON TUBERCLE BACILLI 511 DR1EA, W. F Antibacterial effects of various organic substances upon the H37 strain of human tubercle bacilli in a simple synthetic medium. J. Bact., 48, HooK, W. H., AND ROBINSON, R Long-chain acids containing a quaternary carbon atom. J. Chem. Soc., April, LONG, E. R., AND SEIBERT, F. B A non-protein medium suitable for the production of tuberculin in large quantity. Am. Rev. Tuberc., 13, ROBERTS, J. C Aliphatic acids, fungi and Mycobacterium tuberculosis. Nature, 155, SUTER, C. M Relationships between the structures and bactericidal properties of phenols. Chem. Revs., 28,