A novel series of building blocks consisting of benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate have been synthesized as potential anticancer compounds. These compounds were prepared from 2-aminobenzothiazole, benzaldehyde and ethyl acetoacetate in ethylene glycol by catalysing with TBAHS to give benzo[4,5]thiazo[1,2-a]pyrimidine derivative 4 followed by the formation of amide by reaction with several secondary amines in good yields. The cytotoxicity of these compounds was evaluated against human cancer cell lines in vitro (A549, HeLa, MDA-MB-231 and MCF-7). Compound 5b exhibited promising cytotoxicity with IC₅₀ values of 0.58 and 1.58 μM specifically against human breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231, while compound 5a showed promising cytotoxicity against MDA-MB-231 (IC₅₀ value of 5.01 μM).