Design, Synthesis, In Vitro and In Silico Evaluation of Triazole–Benzimidazole Hybrid Derivatives as Potential Inhibitors of EGFR, EGFR-L858R, and EGFR-L858R/T790M

Epidermal growth factor receptor (EGFR) has become one of the most important molecular targets for the development of anticancer agents because of its critical role in regulating cell proliferation, differentiation, and survival. In the present study, a novel series of benzimidazole–triazole derivatives were designed by incorporating pharmacophoric features of clinically used EGFR inhibitors, synthesized, characterized, and evaluated for their anticancer potential. Cytotoxicity studies against the A549 lung cancer cell line identified 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-methylacetamide, 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-(hydroxymethyl)acetamide, 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-methoxy-N-methylacetamide, and 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-phenylacetamide as active derivatives, with compound 5a exhibiting the highest antiproliferative activity (IC50 = 6.014 ± 0.758 µM), and relatively low cytotoxicity toward NIH/3T3 cells. Based on these findings, 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-methylacetamide was further evaluated against EGFR and its clinically relevant mutant forms, EGFR-L858R and EGFR-L858R/T790M. Retrospective in silico ADME analysis indicated that the synthesized compounds generally possessed favorable drug-like properties, with 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-methylacetamide showing the most favorable overall drug-likeness profile among the series. Molecular docking and molecular dynamics studies revealed that compound 2-((4-Ethyl-5-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-4H-1,2,4-triazol-3-yl)thio)-N-methylacetamide formed stable interactions with key residues in the ATP-binding site of unmutated EGFR, supporting the in vitro inhibition results. Overall, synthesized benzimidazole–triazole derivatives may be promising precursor compounds for the development of novel EGFR inhibitors, particularly against unmutated EGFR cancer cells.