1H-NMR (DMSO-d6) (ppm) 12.08 (bs, 1H, NH), 8.10 (d, 1H, arom. 1H, arom. H, calculated [M?+?H]+ 280.0968; measured 280.0967. 6-[3-(4-Methoxyphenyl)-2-propenoyl]-3H-benzoxazol-2-one (3) Yield 58%. Mp: 210C213?C. 1H-NMR (DMSO-d6) (ppm) 8.08 (d, 1H, Tnfrsf10b arom. H, calculated [M?+?H]+ 296.0917; measured 296.0918. 6-[3-(4-Trifluoromethylphenyl)-2-propenoyl]-3H-benzoxazol-2-one (4) Yield 80%. Mp: 257C259?C. 1H-NMR (DMSO-d6) (ppm) 7.24 (d, 1H, arom. H, determined [M?+?H]+ 334.0686; measured 334.0687. 6-[3-(4-Isopropylphenyl)-2-propenoyl]-3H-benzoxazol-2-one (6) Yield 33%. Mp: 220C222?C. 1H-NMR (DMSO-d6) (ppm) 12.08 (bs, 1H, NH), 8.10 (d, 1H, arom. H, determined [M?+?H]+ 308.1281; measured 308.1286. Synthesis of the compounds 5, 7, and 8 To the mixture of 6-acetyl-2(determined [M?+?H]+ 282.0761; measured 282.0746. 6-[3-(4-Dimethylaminophenyl)-2-propenoyl]-3H-benzoxazol-2-one (7) Yield 48%. Mp: 248C250?C. 1H-NMR (DMSO-d6) (ppm) 12.02 (1?H, bs, NH), 8.00 (d,1H, Phenol-amido-C1-PEG3-N3 arom. H, determined [M?+?H]+ 309.1234; measured 309.1230. 6-[3-(4-Benzyloxyphenyl)-2-propenoyl]-3H-benzoxazol-2-one (8) Yield 63%. Mp: 243C245?C. 1H-NMR (DMSO-d6) (ppm) 8.07 (s, 1H, arom. H), 8.03 (dd, 1H, arom. H, determined [M?+?H]+ 372.1230; measured 372.1218. Biological activity Cytotoxicity test Materials The following chemicals and reagents were from the indicated companies: Dulbeccos altered Eagles medium (DMEM) from GIBCO BRL (Grand Island, NY); foetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin (DXR), and dimethyl sulphoxide (DMSO) from Wako Pure Chem. Ind. (Osaka, Japan); and tradition plastic dishes and plates (96-well) were purchased Phenol-amido-C1-PEG3-N3 from Becton Dickinson (Franklin Lakes, NJ). Cell tradition Human normal oral mesenchymal cells, gingival fibroblast (HGF), Phenol-amido-C1-PEG3-N3 and periodontal ligament fibroblast (HPLF) founded from your first premolar tooth extracted from the lower jaw of a 12-year-old woman47 and human being OSCC cell collection HSC-2 (derived from tongue), purchased from Riken Cell Lender (Tsukuba, Japan), were cultured at 37?C in DMEM supplemented with 10% heat-inactivated FBS, 100 models/ml penicillin G, and 100?g/ml streptomycin sulphate less than a humidified 5% CO2 atmosphere. HGF and HPLF cells at 10C18 populace doubling levels were used in this study. Assay for cytotoxic activity Cells were inoculated at 2.5??103 cells/0.1?ml inside a 96-microwell plate (Becton Dickinson Labware, Franklin Lakes, NJ). After 48?h, the medium was replaced with 0.1?ml of fresh medium containing different concentrations of solitary test compounds. Cells were incubated further for 48? h and the relative viable cell number was then determined by the MTT method22,48C54. All benzoxazolone derivatives were dissolved with DMSO in Phenol-amido-C1-PEG3-N3 the concentration of 40?mM and stored until use. Control cells were treated with the same amounts of DMSO (0.00156, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1.0%) and the cell damage induced by DMSO was subtracted from that induced by test agents. In brief, cells were stained with MTT reagent, dissolved with DMSO, and the absorbance of the MTT-stained cell lysate was measured at 560?nm, using a microplate reader (Infinite F 50R, TECAN, Kawasaki, Japan). Control cells were treated with the same amounts of DMSO and the cell damage induced by DMSO was subtracted from that induced by test agents. The concentration of compound that reduced the viable cell number by 50% (CC50) was identified from your dose-response curve and the mean value of CC50 for each cell type was determined from triplicate assays. Calculation of tumour specificity Tumour specificity (TS) was determined using the following equation: TS?=?Mean CC50 against three normal oral cell types (HGF, HPLF)/Mean CC50 against four OSCC cell lines (HSC-2). Since HGF cells were derived from gingival cells, the relative sensitivity of these cells was also compared (as mean CC50 against HGF/mean CC50 against HSC-2). Calculation of potency-selectivity manifestation Potency-selectivity manifestation (PSE) was determined by the following equation: PSE?=?Mean CC50 against two normal oral cell types/(CC50 against four OSCC cell lines)2100 (HGF, HPLF, HSC-2) and as.