Design, synthesis, anticancer evaluation, biological screening, and computational study of novel 6,7-dimethoxyquinazoline derivatives as VEGFR-2 inhibitors and apoptotic inducers

Novel quinazoline analogs were designed, synthesized, and evaluated for their activities as potential Anticancer agents. The compounds bearing a 4-substituted-6,7-dimethoxyquinazoline scaffold underwent in vitro cytotoxicity screening against 60 Cancer cell lines under the National Cancer Institute (NCI) protocol, for all the novel synthesized compounds at 10 μM concentration. Notably, the initial single-concentration screening revealed that compounds 9a-d exhibited the most potent growth inhibition percentage (GI%) on most of the screened nine panels of Cancer, with a mean range of (50.23-189.98 %). The most potent compounds, 9a-c, continued to be tested at concentrations of 0.01, 0.1, 1, 10, and 100 μM to identify the GI₅₀, TGI, and LC₅₀ values and evaluate the compounds' efficacy and selectivity. Compound 9a generally demonstrated broad-spectrum potent growth inhibitory activity (GI₅₀) reaching to nanomolar level (GI₅₀ = 10 nM) against leukemia CCRF-CEM and colon COLO-205 cells. Compound 9b exhibited a leukemia subpanel midpoint inhibitory value (MIDb) of 0.56 μM, with a selectivity ratio (SR) of 9.15 against leukemia cell lines. Additionally, 9b induced Apoptosis and caused cell cycle arrest at the G1 phase in leukemia CCRF-CEM cells. The RT-PCR results showed that 9b increased the levels of proapoptotic mediators, Bax, Caspase-3, and p53, with values of 8.91, 6.15, and 4.95 fold change, respectively, in addition to the decreased level of anti-apoptotic protein Bcl-2 to 0.372 fold change in the treated CCRF-CEM leukemia cells. Compound 9b was docked to VEGFR-2 (ΔG = -14.1 kcal/mol), suggesting strong interactions within the VEGFR2 active site, comparable to the reference ligand sorafenib (ΔG = -14.8 kcal/mol). The molecular dynamic (MD) simulation (200ns) confirmed the docking results by demonstrating that compound 9b retained stable interactions inside the VEGFR-2 active site throughout the trajectory. Moreover, the MTT viability test for compounds 9a, 9b, and 9c demonstrated less cytotoxicity against normal fibroblast cells (WI38), revealing enhanced safety profiles with IC₅₀ values of 28.04, 219.79, and 43.77 μM, respectively, compared to Sorafenib (IC₅₀ = 26 μM). Enzyme inhibition assays revealed that compounds 9a-c effectively inhibited EGFR and VEGFR-2, confirming the multi-targeting potential of this series of compounds.

Angiogenesis; Apoptosis; EGFR; Fruquintinib; Multi-kinase inhibition; Quinazoline; VEGFR-2; Vandetanib.