Synthesis, In Vitro Anticancer Evaluation, and Structure-Activity Relationships of Novel C4- and C7-Aryl-Substituted Pyrazolo[1,5-a][1,3,5]triazines

A series of pyrazolo [1,5-a][1,3,5]triazine derivatives was synthesized and characterized to assess their anticancer potential. Among the tested compounds, 2-(dichloromethyl)-7-(4-methylphenyl)-4-(pyridin-3-yl)pyrazole [1,5-a][1,3,5]triazine 4 and its analog 7 showed the highest activity. In contrast to the other derivatives, which displayed only weak growth-inhibitory effects, compounds 4 and 7 contain a 3-pyridyl substituent at the C4 position of the pyrazolotriazine scaffold. Both compounds demonstrated broad-spectrum antiproliferative activity in the NCI-60 human cancer cell line panel. Compound 4 exhibited GI₅₀ values ranging from 0.32 to 2.22 µM, with the highest potency against NSCLC, breast cancer, and leukemia cell lines. Compound 7 was even more potent, showing GI₅₀ values ranging from 0.03 to 3.04 µM and selective activity against renal cancer, breast cancer, and leukemia cell lines. In most cases, LC₅₀ values exceeded 100 µM, indicating low overall cytotoxicity despite strong growth inhibition. ADMET prediction suggested favorable drug-like properties, including high intestinal absorption, CNS permeability, weak P-gp substrate behavior, and probable P-gp inhibition. SAR analysis identified the 2-(dichloromethyl) group as a key determinant of activity, while the C4 substituent effect followed the order 3-pyridyl > 2-methylphenyl ≈ 2-fluorophenyl > phenyl ≈ 4-tert-butyl.