Novel Pyrido[2,3-b][1,4]oxazine-Based EGFR-TK Inhibitors: Rational Design and Synthesis of Potent and Selective Agents Targeting Resistance Mutations in Non-Small Cell Lung Cancer

Abstract

Several first-, second-, and third-generation EGFR-TKIs have proven effective as anti-cancer therapeutics. However, the rapid development of drug resistance and mutations continues to be a major challenge in EGFR-TKI therapy. Addressing both intrinsic and acquired resistance resulting from EGFR mutations requires further exploration and the identification of novel inhibitors. In this study, we identified a new class of pyrido[2,3-b][1,4]oxazine-based inhibitors that exhibited potent EGFR kinase inhibitory activity. These compounds demonstrated significant anti-proliferative effects against EGFR-mutated non-small cell lung cancer (NSCLC) cell lines, including HCC827 (EGFR exon 19 deletion), H1975 (EGFR L858R/T790M double mutation), and A549 (wild-type EGFR overexpression). These novel pyrido[2,3-b][1,4]oxazine analogues were rationally designed and synthesized using the Suzuki cross-coupling reaction in multi-step synthetic route. Anticancer evaluation of these derivatives using the MTT assay showed promising therapeutic potential. The most promising compounds were 7f, 7g, and 7h, with 7f showing potency equivalent (IC50 values: 0.09, 0.89, and 1.10 μM, in the HCC827, NCI-H1975 and A-549 cell lines, respectively) to clinically approved Osimertinib. Interestingly, these compounds are selectively cytotoxic against cancer cells while not harming normal BEAS-2B cells at doses over 61 μM. Mechanistic studies demonstrated that compound 7f acts as an EGFR-TK autophosphorylation inhibitor, causing significant apoptosis (33.7% early, 9.1% late) compared to control conditions (2.4% early, 1.8% late). Molecular docking showed that the compounds scored similar to Osimertinib, with the di-fluorophenyl group engaging the glycine-rich loop, pyridine substituents forming front pocket interactions, and essential hinge region interactions maintained, suggesting effective EGFR target engagement. These findings identify pyrido[2,3-b][1,4]oxazine derivatives as potential anticancer candidates worth further exploration for the development of targeted therapies against non-small cell lung cancer.