Design, synthesis, and anticancer evaluation of new Phenylpyrazolone derivatives: Dual JAK1/ JAK2 inhibition, apoptosis-inducing activity, and molecular modelling studies.

A series of novel phenylpyrazolone derivatives 3a-k were synthesized and evaluated as potential multitargeted kinase inhibitors against JAK1 and JAK2, aiming to develop effective anticancer agents. The structures of the new compounds were confirmed using IR, H NMR, C NMR, and mass spectrometry. Their antiproliferative activity was assessed in vitro across the NCI-60 human tumor cell line panel. Among them, compound 3i demonstrated notable cytotoxicity, with marked activity against HL-60 (leukemia), MCF-7 (breast), HT-29, and HCT-116 (colon) cell lines. Compound 3i exhibited potent activity toward HT-29 cells (IC = 1.02 μM) and minimal toxicity toward normal WI-38 fibroblasts, indicating high selectivity (SI of 19.5), highlights it as the most responsive and promising target for further mechanistic studies. Compound 3i exhibited notable inhibitory activity, with IC values of 14.73 ± 0.79 nM for JAK1 and 10.03 ± 0.81 nM for JAK2. Compound 3i caused induction of G2/M cell cycle arrest, and activation of intrinsic apoptosis via increased Bax and caspase 3/7 levels and reduced Bcl2 expression. Structure-activity relationships (SAR) revealed that di-substitution on both aniline and benzylidene rings with at least one meta hydrogen-bond acceptor (HBA) on each ring is essential for enhanced and broad cytotoxic activity. This conclusion was supported by 3D-QSAR pharmacophore modelling, DFT calculations, and molecular docking studies, which together provided detailed insight into the binding behavior, stability, and hinge-region interactions of the most active compound 3i within the ATP-binding sites of JAK1 and JAK2. Also, molecular dynamics simulations of compound 3i confirmed the stability and favorable conformational dynamics of the ligand-protein complexes over time. These findings highlight compound 3i as a promising lead for further development as a selective anticancer agent targeting JAK kinases.