Four new pyrazolo[3,4-d]pyrimidines (P1–P4) were successfully synthesized in good relative yields by reacting 3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ol with various alkylating agents (methyl iodide, propargyl bromide, and phenacyl bromide) at room temperature in DMF solvent, employing liquid–solid phase transfer catalysis. The P1–P4 structures were elucidated using NMR spectroscopy and X-ray diffraction. Intermolecular interactions in P1–P4 were analyzed via Hirshfeld surface analysis and 2D fingerprint plots. Geometrical parameters were accurately modeled by DFT calculations using the B3LYP hybrid functional combined with a 6–311++G(d,p) basis set. The antiproliferative activity of P1–P4 towards colorectal carcinoma (HCT 116), human hepatocellular carcinoma (HepG2), and human breast cancer (MCF-7) cell lines, along with one normal cell line (WI38) was investigated using the MTT assay and sunitinib as a reference. Compounds P1 and P2 exhibited antiproliferative activities comparable to the reference drug towards all tested cells, with an IC50 range of 22.7–40.75 µM. Both compounds also showed high selectivity indices and minimal cytotoxic effects on the normal cell line. Molecular docking revealed that the significant antiproliferative activity may attributed to the number and type of intermolecular hydrogen bonding established between pyrazolo[3,4-d]pyrimidines and DNA topoisomerase, a common target for various anticancer agents.
