New thiazole/coumarin hybrids as EGFR inhibitors with potential VEGFR-2 inhibition (design, synthesis, biological evaluation, and dynamic stimulation)

Two new series of coumarin-thiazole hybrids (6a-i and 9a-h) were designed and synthesized as EGFR/VEGFR-2 dual inhibitors. Hybrids 6a-i demonstrated remarkable antiproliferative activities on different NCI-60 cell lines in a single-dose assay. The antiproliferative activities of the newly synthesized hybrids were evaluated against a panel of three human cancer cell lines (HepG2, HCT-116, and MCF-7). Compounds 6a, 6h, 9a, and 9d were the four most active hybrids. Herein, compound 6a was the most active antiproliferative agent among the 6a-i hybrids, with IC₅₀ values ranging from 2.85 µM to 7.66 µM. At the same time, compound 9d was the most active antiproliferative agent among the 9a-h hybrids, with IC₅₀ values ranging from 3.79 µM to 9.80 µM. Furthermore, hybrids 6a, 6h, 9a, and 9d expressed low cytotoxicity for the normal fetal lung WI-38 cells. Consistent with the mechanistic study, the most potent compounds (6a, 6h, and 9d) displayed remarkable inhibitory effectiveness against EGFR and VEGFR-2. Moreover, the most potent hybrid (6a) caused the upregulation of Bax, cytochrome c, and caspase-3 levels and downregulation of Bcl-2 levels. Molecular docking study for compounds 6a, 6h, 9a, and 9d agreed with the mechanistic results. Molecular docking into the ATP-binding site of EGFR (PDB: 1M17 and re-docking RMSD = 1.43 Å) and VEGFR-2 (PDB: 4ASD and re-docking RMSD = 1.67 Å) confirmed strong binding affinities for 6a (ΔG = -10.32 and -9.98 kcal mol^-1, respectively) and 9d (ΔG = -10.23 and -9.87 kcal mol^-1, respectively), consistent with their enzymatic inhibition profiles. To validate the dynamic stability of binding, 100 ns all-atom molecular dynamics (MD) simulations were performed for 6a and 9d in complex with EGFR, alongside erlotinib and the apo receptor. Compound 6a was carried forward into the 100 ns molecular dynamics simulations of five systems, comprising the apo receptor, the erlotinib complex and the complexes of 6a, 9d, and 9a. Ligand binding compacted the kinase by roughly 2.3 Å in the radius of gyration relative to the apo receptor and reduced Cα fluctuation across the fold. The total protein-ligand interaction energy ranked the four ligands in an order consistent with the enzymatic assay, yielding values of -69.6, -55.6, -50.6 and -41.7 kcal mol^-1 for erlotinib, 6a, 9d and 9a against IC₅₀ values of 0.14, 0.27, 0.378, and 2.94 µM, respectively. Furthermore, every pairwise separation exceeded twice the block-averaged standard error. Compound 6a was identified as the lead compound of the series.