Design of 4-(piperazin-1-yl)thieno[2,3-d]pyrimidine derivatives targeting the DFG-out conformation of VEGFR-2: Synthesis, biological evaluations, and molecular modeling approaches.

Abstract

Novel series of piperazinyl-thienopyrimidine acetamide derivatives (10a-l, 13a-b, and 16a) were rationally designed and synthesized as potential type II VEGFR-2 inhibitors based on the essential pharmacophoric features of sorafenib and the co-crystallized ligand in the VEGFR-2 crystal structure (PDB: 2OH4). The target compounds were prepared via a convergent synthetic route and confirmed by 1H NMR, 13C NMR, HRMS, and elemental analyses. In vitro VEGFR-2 kinase assays revealed inhibitory activity ranging from 0.056 to 1.346 μM, with compound 10d (4-methoxyphenyl) emerging as the most potent member (IC50 = 56 nM), essentially equipotent with sorafenib (IC50 = 59 nM). The most active derivatives showed significant antiproliferative activity against HepG2 and MCF-7 cancer cell lines, with 10d exhibiting IC50 values of 5.38 and 5.92 μM respectively, alongside markedly reduced cytotoxicity toward normal Vero cells (IC50 = 28.00 μM). Mechanistic studies confirmed that 10d suppressed VEGFR-2 phosphorylation and VEGF-A secretion in a dose-dependent manner, induced G2/M cell-cycle arrest, and triggered apoptosis (∼17-fold over control) via the intrinsic mitochondrial pathway, as validated by upregulation of Bax, Caspase-9, and Caspase-3, and downregulation of Bcl-2. Compound 10d further inhibited HepG2 cell migration at sub-cytotoxic concentrations. Molecular docking confirmed a type II binding mode with key interactions at Cys917, Glu883, and Asp1044 (ΔG = -9.85 kcal/mol), and 200 ns molecular dynamics simulations validated the stability of the lead complex with RMSD values below 2 Å, identifying 10d as a promising anticancer lead warranting further in vivo evaluation.