Novel pyrido[2,3-<i>b</i>][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-<i>b</i>][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-<i>b</i>][1,4]oxazine analogues were rationally designed and synthesized using the Suzuki cross-coupling reaction in a 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 (IC₅₀ values: 0.09, 0.89, and 1.10 μM, in the HCC827, NCI-H1975 and A-549 cell lines, respectively) equivalent 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 and 9.1% late) compared to control conditions (2.4% early and 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-<i>b</i>][1,4]oxazine derivatives as potential anticancer candidates worth further exploration for the development of targeted therapies against non-small cell lung cancer.