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A new series of N-(5-Mercapto-1, 3, 4-thiadiazol-2-yl)-2-phenylacetamide derivatives (3a-3j) were synthesized via an amidation reaction using EDC and HOBt in acetonitrile solvent at room temperature condition. Chemical structures were characterized by 1 H NMR, IR and MS spectroscopic methods and related melting points were also determined. The anticancer activity was evaluated using MTT procedure in-vitro. All compounds were tested against SKNMC (Neuroblastoma), HT-29 (Colon cancer) and PC3 (Prostate cancer) cell lines. According to the toxicological data, none of the synthesized derivatives exerted superior activity than doxorubicin as reference drug. Derivatives with Ortho chlorine (compound 3d), meta methoxy (compound 3h) and meta fluorine (compound 3b) substituents on the phenyl ring exhibited the best cytotoxic activity against SKNMC (IC 50= 4.5±0.035 µM), HT-29 (IC 50= 3.1±0.030 µM …