Synthesis of New Pyrazolo[3,4-d]pyrimidine Derivatives: NMR Spectroscopic Characterization, X-Ray, Hirshfeld Surface Analysis, DFT, Molecular Docking, and Antiproliferative Activity Investigations

Four new pyrazolo[3,4-<i>d</i>]pyrimidines (<b>P1</b>-<b>P4</b>) were successfully synthesized in good relative yields by reacting 3-methyl-1-phenyl-1H-pyrazolo[3,4-<i>d</i>]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 <b>P1</b>-<b>P4</b> structures were elucidated using NMR spectroscopy and X-ray diffraction. Intermolecular interactions in <b>P1</b>-<b>P4</b> 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 <b>P1</b>-<b>P4</b> 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 <b>P1</b> and <b>P2</b> 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-<i>d</i>]pyrimidines and DNA topoisomerase, a common target for various anticancer agents.