356 JOURNAL O1• THE SOCIETY O1• COSMETIC CHEMISTS EXPERIMENTAL Syntheses 3'-Trifiuoromethylsalicylanilide.--A slurry of 11.05 g. (0.08 mole) of salicylic acid and 12.89 g. (0.08 mole) of 3-trifluoromethylaniline in 200 ml. of chlorobenzene was refluxed for three hours with 4 ml. of phosphorus trichloride and was filtered hot. The solution was re- frigerated overnight. The resulting precipitate was collected on a filter, washed with a 5% solution of hydrochloric acid, air-dried, and finally recrystallized from ethanol after decolorization with activated charcoal. A 45% yield of product was obtained (Table I). 2'-Chloro-5'-trifiuoromethylsalicylanilide was obtained in a similar manner in 40% yield. •'-Bromo-2-rnercaptobenzanilide was prepared in a similar manner in 50% yield. 3- (•-Bromophenyl) -6-bromo-1,3-benzoxazine-2,•-dione.--4, 5- Dibromo- salicylanilide (1) was condensed with ethyl chloroformate or phosgene following the procedure of Stenseth (2). The product recrystallized from toluene was obtained in 36% yield. 6 ,8-Dibromo-3- ( 3-trifiuoromethylphenyl)-1,3-benzoxazine-2,•-dione was obtained in a similar manner in 60% yield. 3- (3- Trifiuoromethylphenyl)-l,3-benzoxazine-2,•-dione was obtained in a similar manner in 61% yield. 3-(2-Chloro-3-trifiuoromethylphenyl)-1,3-benzoxazine-2,•-dione was similarly obtained in 43% yield. 3-(•-Bromophenyl)-l,3-benzothiazine-2,•-dione was obtained in a similar manner. The product was recrystallized from a tetrahydro- furan-acetone-water mixture. The yield was 47%. A ntibacterial Studies There are many tests for the determination of antibacterial activity, but there is no single test which is infallible. Due to the differences in solubility in agar of the compounds to be tested it was thought that the reliability of the results from an agar plate method would be question- able. Thus, the bacteriostatic activity of the compounds against Staphylococcus aureus and Alkaligines fecalis in vitro was determined by a modified minimum inhibitory concentration method (MIC). A 1:1000 stock solution of the compound to be tested was prepared by dissolving 100 mg. of the compound in a minimum quantity of either alcohol or acetone. The solution was then transferred with

CYCLIC SALICYLANILIDE(3 357 aseptic technique to a previously sterilized 100 ml. volumetric flask containing Brain Heart Infusion (BHI) broth. Serial dilutions in screw-capped test tubes were made from this stock solution. To each of the dilutions of a given compound was then added 0.1 ml. of a 24-hour broth culture of the organism to be tested. The turbidity of the broth TABLE I Salicylanilide and Cyclic Salicylanilides Calcd, %b Found, Compound Formula M.p. øC a C H C H 3-Trifiuoromethyl- salicylanilidc C14H10FaNO.o 195-6 59.78 3.58 59.93 3.59 2 •-Chloro-5 •-trifiuoro- methylsalicylanilide C•4H9C1F•NO2 197-8 53.23 2.87 53.44 3.06 4'-Bromo-2-mercapto- benzanilide ClaH10BrNOS 146-8 50.66 3.27 50.57 3.65 3-(4-Bromophenyl)-6- bromo-l,3-benzoxa- zine-2,4-dione C•4H7Br2NO• 141 42.35 1.78 42.15 2.35 6,8-Dibromo-3- (3-tri- fiuoromcthylphcnyl)- 1,3-bcnzoxazinc-2,4- dione Ci•H6Br2FaNOa 233-5 38.74 1.30 38.74 1.50 3-(3-Trifluoromcthyl- phcnyl)-l,3-bcn- zoxazine-2,4-dione C•HsF•NO• 196 58.64 2.62 59.08 2.82 3- (2-Chloro-3-trifiuoro- mcthylphcnyl )- 1,3- bcnzoxazinc-2 dione ClsH.7C1FaNOa 135-6 52.72 2.07 52.95 2.67 3-(4-Bromophenyl)-l,3- bcnzothiazinc-2,4- dionc C14HsBrNO•S 250-2 50.32 2.39 51.30 3.24 a All melting points were taken on a Fisher-Jones rodting point apparatus and are un- corrected. b Carbon-hydrogen analyses were conducted by the Weiler and Strauss Microanalytical Laboratory, Oxford, England. solutions was determined with the aid of a Welch Densichron. The densitometer was chosen over visual observation for purposes of ac- curacy, especially when end points were questionable. The broth dilutions were then allowed to stand for 24 hours at 37 øC. A control consisting of 0.1 ml. of a 24-hour broth culture in BHI broth was also prepared and subjected to the same conditions as the compounds to be tested. At the end of the 24-hour period the tubes were again observed