A series of novel pyrazolo[3,4-d]pyrimidine derivatives have designed and synthesized in synthetically useful yields. All the new synthesized compounds were biologically evaluated in vitro for their cytotoxic activities against a panel of three cancer cell lines namely, HepG-2, MCF-7, and HCT-116. The results of cytotoxic evaluation indicated that compound (9) exhibited the most prominent cytotoxic effect against all tested cell lines with IC₅₀ values ranging from (4.03- 6.18) µM comparable to that of doxorubicin as a control drug (IC₅₀ values of 8.17 and 9.27 µM). In particular, compounds (8,9,11) and (12) exhibited higher intercalative activity with IC₅₀ value of 30 µM than doxorubicin (31 µM).