New pyrazolo[1,5-a]pyrimidine derivatives as potential CDK2 inhibitors and apoptosis-driven antiproliferative agents

Five new series of pyrazolo[1,5-a]pyrimidine derivatives were synthesized as potential anti-cancer agents targeting the CDK2 enzyme. Their cytotoxic activity was evaluated against colon cancer (HCT-116) and breast cancer (MDA-MB-231) cell lines. Among the tested compounds, 7-(4-bromophenyl)-2-(methylthio)pyrazolo[1,5-a]pyrimidine-3‑carbonitrile (13 g), 7-(2,4-dichlorophenyl)-2-(methylthio)pyrazolo[1,5-a]pyrimidine-3‑carbonitrile (13j), 7-[1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-2-(methylthio)pyrazolo[1,5-a] pyrimidine-3‑carbonitrile (21c), and 7-(6-bromo-2-oxo-2H-chromen-3-yl)-2-(methylthio)pyrazolo[1,5-a] pyrimidine-3‑carbonitrile (26b) exhibited strong growth inhibition in HCT-116 cells, comparable to the reference drug roscovitine. Compounds 13 g and 21c were identified as the most potent candidates against HCT-116 cells, with IC₅₀ values of 0.45 μM and 0.09 μM, respectively, compared to roscovitine (IC₅₀ = 0.07 μM). Moreover, compounds 13 g, 13j, 21c, and 26b also displayed low toxicity toward normal WI-38 fibroblast cells, indicating superior selectivity. CDK2 inhibition assay for the most potent compounds 13 g, 13j, and 21c, demonstrated promising IC₅₀ values ranging from 18 to 150 nM, compared to roscovitine (IC₅₀ = 140 nM). Further biological evaluation revealed that compound 21c (IC₅₀ = 18 nM) triggered G1-phase cell cycle arrest and promoted more apoptosis than necrosis in HCT-116 cells (total apoptosis: 32.96 % compared to 0.63 % in the control). RT-PCR analysis indicated that apoptosis was mediated through both intrinsic and extrinsic pathways, accompanied by downregulation of the anti-apoptotic gene BCL-2 (0.7-fold change). Molecular docking studies supported these results, showing favorable interactions of compound 21c within the CDK2 active site. Overall, compound 21c emerges as a promising lead candidate for the development of selective CDK2 inhibitors exhibiting potent anti-cancer activity and low toxicity toward normal cells.