A novel series of thiazolopyrimidines and fused thiazolopyrimidines was designed and synthesized as topoisomerase II alpha inhibitors. All synthesized compounds were screened by the National Cancer Institute (NCI), Bethesda, USA for anticancer activity against 60 human cancer cell lines representing the following cancer types: leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancers. Compound 3a was found to be the most potent inhibitor on renal cell line (A-498) causing 83.03% inhibition (IC = 1.89 μM). DNA-flow cytometric analysis showed that compound 3a induce cell cycle arrest at G2/M phase leading to cell proliferation inhibition and apoptosis. Moreover, fused thiazolopyrimidines 3a showed potent topoisomerase II inhibitory activity (IC = 3.19 μM) when compared with reference compound doxorubicin (IC = 2.67 μM). Docking study of all the synthesized compounds showed that compound 3a interacts in a similar pattern to etoposide and stabilizing the topoisomerase cleavage complex (Top2-cc) that accounts for its high potency.Copyright © 2020 Elsevier Inc. All rights reserved.