Novel 2-Anilinoquinazoline derivatives as apoptosis-inducing agents in drug-resistant NSCLC cells: Integrating biological and computational evaluation.

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Despite widespread use, the limited durability of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) highlights the need for strategies that suppress tumour proliferation and restore apoptosis in resistant NSCLC. In this study, a series of eleven 2-anilinoquinazoline derivatives was synthesised to identify new scaffolds capable of restoring apoptotic signalling in drug-resistant NSCLC cells. Among these, JS04 emerged as the most potent derivative, exhibiting antiproliferative activity with IC₅₀ values of 8.11 ± 0.43 μM and 11.58 ± 1.68 μM against H1975 (EGFR-TKI resistant) and A549 (EGFR-independent) cell lines, respectively, outperforming gefitinib. Real-time impedance (xCELLigence) and flow cytometry analyses demonstrated that JS04 induced rapid, dose-dependent apoptosis, achieving 74% apoptotic cell death at 20 μM, substantially higher than erlotinib (13%). Proteome profiling confirmed activation of both intrinsic (downregulation of Bcl-2, Bcl-xL, and survivin) and extrinsic (upregulation of TRAIL R2/D5) apoptotic pathways, accompanied by G₂/M phase cell cycle arrest comparable to osimertinib. Zebrafish embryo assays revealed a favourable safety profile (LC₅₀ = 38.42 ± 3.96 μM). Computational studies, molecular docking and 100 ns molecular dynamics simulations indicated that JS04 forms stable hydrogen bonds with hinge region residues MET793 and GLN791, and exhibits strong binding affinity toward the EGFRL858R/T790M kinase. Collectively, these findings position JS04 as a promising quinazoline-based lead candidate that effectively triggers dual apoptotic mechanisms, selectivity toward H1975 cells, and demonstrates early indications of safety, warranting further biochemical validation, structure-activity optimisation, and in vivo efficacy evaluation.