Integrated In Silico and In Vitro Discovery of a VEGFR-2-Targeted Anticancer Benzanilide Lead

Abstract

The development of selective vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors remains a promising strategy for anticancer therapy targeting tumor angiogenesis. In the study at hand, a novel benzanilide-based derivative, ACEB-Cl, was rationally designed by assimilating fundamental pharmacophoric characteristics essential for VEGFR-2 inhibition. ACEB-Cl was evaluated using a comprehensive approach that combined computational modeling, synthesis, and in vitro biological assessment. Density functional theory (DFT) calculations revealed a favorable electronic profile, with distinct nucleophilic and electrophilic regions that could support potential interactions within the VEGFR-2 binding pocket. Docking and molecular dynamics (MD) simulations suggested stable binding of ACEB-Cl within the ATP-binding pocket, supported by strong hydrophobic interactions and favorable binding free energy (ΔG = −63.21 kcal/mol). Following synthesis, ACEB-Cl confirmed potent anti-VEGFR-2 activity in an ELISA-based assay (IC50 = 0.086 0.002 M), showing higher potency than sorafenib. Western blot analysis further confirmed significant downregulation of phosphorylated VEGFR-2 in MDA-MB-231 cells. In vitro cytotoxicity studies revealed selective anticancer activity, with reduced toxicity toward normal Vero cells and favorable selectivity indices. Mechanistically, ACEB-Cl triggered G0/G1 phase arrest and significantly increased apoptotic cell death, highlighting its ability to suppress cancer cell proliferation. Additionally, ADMET and toxicity estimations indicated a balanced pharmacokinetic and safety profile, with improved solubility, acceptable absorption, and reduced toxicity risks compared to sorafenib. Overall, this integrated study pinpointed ACEB-Cl as a promising VEGFR-2targeted anticancer lead compound with strong inhibitory potency, and a clearly defined mechanism of action, supporting its progression for further improvement.