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, 1H NMR, 13C 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 (IC50 = 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 IC50 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.