Design, synthesis, biological evaluation, and mechanistic characterization of novel dual VEGFR2/CRAF-directed kinase inhibitors with potent antitumor activity.

A series of novel type II kinase inhibitors based on a 4-phenylthiazol-2-amine scaffold were designed and synthesized to overcome limitations of VEGFR2-targeted therapy, including acquired resistance and RAF pathway reactivation. Through structure-guided optimization, compound 5j was identified as a promising lead, exhibiting potent inhibitory activity against VEGFR2 (IC50 = 12.22 nM) and CRAF (IC50 = 38.04 nM) in cell-free kinase assays. In vitro biological evaluations demonstrated that 5j effectively suppressed proliferation in multiple cancer cell lines. In H1299 cells, 5j significantly inhibited cell migration and clonogenic growth, induced G0/G1 cell-cycle arrest, apoptosis, andreactive oxygen species (ROS) accumulation in a concentration-dependent manner. Transcriptomic profiling indicated targeted modulation of RTK/RAF-related oncogenic signaling pathways, including interferon-stimulated gene responses and attenuation of KRAS-, EGFR-, and MEK-associated signatures. Consistent with dual VEGFR2/CRAF inhibition, Western blot analysis confirmed suppression of the downstream Raf-MEK-ERK pathway, as evidenced by decreased phosphorylation of MEK and ERK. Molecular docking and 100 ns molecular dynamics simulations supported stable binding of 5j to the ATP-binding site and allosteric pocket of both targets, consistent with a Type II inhibition mode. These findings identify 5j as a promising dual VEGFR2/CRAF-directed lead compound for further optimization.