A series of novel pyrazolo[3,4-d]pyrimidines bearing benzenesulfonamide moiety 5a-f, 6 and 7 were synthesized. Cytotoxic screening was conducted against MCF-7 and HepG2. 6-(4-Methoxyphenyl)-4-oxopyrazolopyrimidine derivative 5e and 4-imino-6-oxopyrazolopyrimidine derivative 6 revealed potent cytotoxic activity with IC50 1.4 μM (MCF-7) and 0.4 μM (HepG2), respectively compared to that of doxorubicin, (IC50 = 1.02 μM and 0.9 μM, respectively). Compounds 5e and 6 were subjected to cell cycle analysis and apoptosis assay after 24 h and 48 h treatment. Compound 5e arrested cell at G1 phase, while 6 arrested cell at S and G2/M phases, respectively. The apoptotic effect of both compounds were evidenced by pre G1 apoptosis as its percentage increased by time (7.38%, 11.61%) and (13.92%, 16.71%), respectively. Apoptosis induction capability was confirmed by the effect on early and late apoptosis and augmentation of caspase-3 level. Furthermore, compound 6 inhibited CDK2 enzyme with IC50 = 0.19 μM and increased levels of its regulators, P21 and P27 by 10.06% and 8.5%, respectively. Moreover, a molecular docking study of compound 6 on CDK2 enzyme was adopted to explore binding interaction with amino acid residues of its active site.