CHEMICAL STRUCTURE AND ANTIMICROBIAL ACTIVITY OF BIS-PHENOLS 273 3,3'-Melhylenebis(2,5,64richlorophenol), No. 66 IId. 3,3'-Methy/enebis(¾-bromo-2,5,6-trichlorophenol). Condensation of 4-bromo-2,3,6-trichlorophenol (105 g.) with paraformaldehyde was con- ducted as described for IA. Crystallization of the crude bis-phenol from 420 ml. of methanol and 190 ml. of water with addition of Nuchar yielded 76 g. of dark material, m.p. :204-212 ø. Recrystallization fi'om 300 ml. of petroleum naphtha plus 300 ml. of toluene, with Filtrol as decolorizing agent, and then from a mixture of 100 ml. of carbon tetrachloride and 250 ml. of ethylene dichloride (plus Filtrol) yielded 30 g. of a tan-colored product, m.p. 222-225 ø. HB. 3,3'-Me•hylenebis(2,$,6-trichlorophenol). Debromination of 30 g. of IIA was performed in the same manner as that of IA. The crude substance (19 g. m.p. 138-148 ø) was crystallized from 100 ml. of methanol and 50 ml. of water (plus Nuchar). The first fraction (5.8 g. m.p. 169-175 ø) was recrystallized from 40 ml. of toluene and then from 30 ml. of methanol. White, small crystals (2.7 g.) were obtained. 3,3'-3¾ethylenebis(¾,$,6-trichlorophenol), No. 67 IHd. 3,3'-Methylenebis(2-bromo-¾,5,6-trichlorophenol). 2-Bromo-4,5,6- trichlorophenol (60 g.) was converted into the bis-phenol as described for IA. The crude product (39 g.) was twice crystallized from methanol plus dioxane 27 g. of satisfactory material,. melting at 294-295 ø, was obtained. [liB. 3,3'-Methylenebis(¾,5,64richlorophenol). Treatment of the alka- line solution of IIIA with zinc yielded 16.6 g. of crude material which •vas first dissolved in boiling 80% v./v. aqueous methanol 1.5 g. remained insoluble. On cooling the flitrate, 9.1 g. (m.p. 211-214 ø) was obtained. Subsequent crystallization from 300 ml. of petroleum naphtha, from 160 ml. of toluene (plus Filtrol), and finally from 20 ml. of methanol yielded 2.3 g. of the pure substance. H. Microbiological Each of the compounds was dissolved in alcohol (specially denatured No. 30) to make a 1% stock solution. Twofold serial dilutions of the stock solu- tion were made in alcohol, and 0.2 mi. of each dilution level was added to a tube containing 20 mi. of 50 ø molten agar. The tube was swirled thoroughly to distribute the test compound and the contents were poured into a sterile plate. Hardened plates were surface-inoculated with an Accu-Drop dispenser (Scientific Products, Flushing, L. I., N.Y.). Agar employed for bacteria was Dextrose Tryprone Extract Agar (Difco Lab- oratories, Inc., Detroit, Mich.) Sabouraud's Dextrose Agar (Difco) was used for yeasts and molds. The American Type Culture Collection liquid

274 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS medium and Fluid Thioglycollate (Difco) broth for the Chlorella and anaerobic bacteria, respectively, were also employed. lnocula for the aerobic bacteria consisted of one drop (0.007 mi.) of a 1-100 distilled water dilution of a 24-hour, 35 ø, Tryptic Soy Broth (Difco) culture onto the surface of the agar plates. Inoculum for Chlorel/a vulgaris consisted of one drop of a 1-100 dilution of a 5-day, 25 ø culture to 10 ml. of broth containing the test compound. Anaerobic bacterial inocula consisted of one drop of a 1-100 dilution of a 72-hour, 35 ø broth culture to 15 ml. of broth containing the test compound. Yeast inoculum was one drop per plate of a 1-100 water dilution of a heavy cell suspension prepared by the addition of 10 ml. of distilled water to a 72-hour agar slant. Mold inocula were prepared by suspending the conidia from the surface growth of a 7-day slant in 20 ml. of distilled water with no further dilution. Incubation for aerobic bacterial plates was 35 ø for forty-eight hours, at the end of which the level completely inhibiting growth was recorded. Anaerobic tubes were incubated for fourteen days at 35 ø , after which bacteriostatic levels were recorded. Yeast results were noted after four days incubation at 30 ø and mold results after fourteen days at 30 ø . In- hibition of Chlorella was recorded at the end of thirty days incubation at 25 ø over two 30-inch fluorescent tubes at a distance of 20 inches. Because of inherent error in the procedure, tests were performed in triplicate on different days for aerobic bacteria. Tests with all other organisms (cf. Table II) were performed in duplicate on different days. The average inhibitory levels, calculated as the geometric mean of the values obtained, are reported in Table III. TABLE III--ANTIMIOROBIAL SPECTRA or METHYLENEBIS(TKICHLOKOPHENOL) ISOMEKS. (MINIMAL INHIBITORY LEVEL, /.ZG./ML.) Compound (by Number) Micro6rganism 1 2 3 40 65 66 67 42 S. aureus 0.93 0.61 0.39 10 2 S. epidermidis 0.93 0.78 0.23 12.5 2 B. subtilis 0.19 0.39 0.19 3.9 1 B. ammoniagenes 0.39 0.48 0.19 15.6 2 P. vulgaris 3.9 1.9 3.9 25 E. coli 25 * 12.5 * S. typhosa 40 * 10 * Ps. aeruginosa 25 * 50 * Ps. fiuorescens 0.23 (t.61 0.19 12.5 2 Sh. sonnei 40 * 9.9 * K. pneumoniae 50 * 15.6 * T. mentagrophytes 1.74 3 12 3.12 8.9 8 T. rubrum 4.4 3.12 8.9 8.9 3 M. audouini 3.12 1.74 3.12 3.12 4 C. albicans and P. ovale * * * * C1. tetani 0.19 0.39 0.09 1.56 0 C1. perfringens 0.78 0.39 0.55 1.74 0 C1. sporogenes 0.78 0.55 0.55 1.56 0 C. vulgaris 3.12 4.4 8.9 * .5 20 1.56 25 ß 4 20 1.56 25 .9 9.9 1.56 15.6 .4 40 1.56 31.5 * 63 10 63 .4 31.5 1.56 25 .9 6.25 4.4 6.25 _1 1.56 1.74 9.9 .4 3.12 8.9 4.4 .27 1.74 0.19 4.4 .55 6.25 0.39 6 25 .55 6.25 0.55 6.25 * 17.8 4.4 17.8 * Denotes growth at 100 •g./ml.