Synthesis, characterization and anticancer evaluation of different fused pyridopyrimidopyrimidines as dual EGFR/ACK1 inhibitors with c-Met activity in solid tumor cancer cell lines

Drug resistance in non-small cell lung cancer (NSCLC), frequently driven by EGFR mutations and compensatory bypass signaling via ACK1 and c-Met, remains a critical clinical challenge. Consequently, the design of multi-targeted agents capable of dual EGFR/ACK1 inhibition with c-Met activity represents a rational strategy to overcome these resistance mechanisms. Based on this hypothesis, a novel series of fused pyridopyrimidopyrimidine derivatives was designed and synthesized. The structures of the target compounds were confirmed using elemental analysis and spectral methods (IR, NMR, MS). The anticancer potential of the synthesized compounds was evaluated against a panel of solid tumor cell lines, with a focus on drug-resistant phenotypes. Among these, compound 11b and its analogue 7g demonstrated the strongest activity in drug-resistant H1975 cells, showing IC₅₀ values of 0.42 μM and 0.85 μM, respectively, and favorable selectivity indices. Mechanistic assays confirmed that 11b effectively suppressed the phosphorylation of EGFR, ACK1, and c-Met kinases. Furthermore, flow cytometric analysis indicated the induction of apoptosis, characterized by Annexin-V binding and Caspase-3 activation, alongside cell cycle modulation. Computational ADME-toxicity prediction indicated drug-like properties and low predicted toxicity risks. These findings position compound 11b , and to a lesser extent 7g , as strong candidates for further optimization as multitargeted kinase inhibitors against resistant forms of non-small-cell lung cancer. N-substituted and Fused different pyridopyrimidopyrimidines systems.