Computationally guided design and in vitro validation of a benzanilide VEGFR-2 inhibitor with selective anticancer activity

Targeted inhibition of VEGFR-2 is a validated strategy for anti-angiogenic cancer therapy. In this study, we report the design, synthesis, and biological evaluation of ( Z )- N -(4-(1-((3-cyano-4,6-dimethyl-2-oxopyridin-1(2 H )-yl)imino)ethyl)phenyl)benzamide ( Z-CMPB ) as a novel VEGFR-2 inhibitor. The compound was rationally designed based on pharmacophoric features, optimized using density functional theory (DFT), and evaluated via molecular docking and 200 ns molecular dynamics simulations, which demonstrated stable binding at the VEGFR-2 active site and persistence of critical interactions. In silico ADMET and toxicity predictions indicated favorable oral absorption, limited CNS penetration, low carcinogenic and mutagenic risk, and high plasma protein binding, supporting the compound’s drug-like profile. Z-CMPB was synthesized and tested in vitro , showing potent enzymatic inhibition of VEGFR-2 (IC ₅₀ = 0.3 ± 0.008 µM). Western blot analysis in HepG-2 cells confirmed suppression of VEGFR-2 phosphorylation at Tyr1054/1059 (∼50% reduction), validating intracellular target engagement. Cytotoxicity assays revealed selective antiproliferative activity, with the greatest effect in HepG-2 cells (IC ₅₀ = 2.17 ± 0.6 µM) and minimal toxicity toward normal Vero cells (IC ₅₀ = 49.4 ± 7.6 µM), corresponding to a selectivity index of ∼22.8. Mechanistic studies indicated that Z-CMPB induces G0/G1 cell cycle arrest and apoptosis (total apoptotic fraction = 37.58%), consistent with VEGFR-2–dependent inhibition of proliferative signaling. Collectively, these findings highlight Z-CMPB as a promising lead compound with potent VEGFR-2 inhibitory activity, favorable selectivity, and a mechanistically supported anticancer profile, warranting further in vivo studies for hepatocellular carcinoma therapy.