Fused thiophene - benzimidazole conjugates targeting EGFR: Design, synthesis, anticancer evaluation and their mechanistic insights

The development of potent epidermal growth factor receptor (EGFR)-targeted Anticancer agents continues to be a major priority in medicinal chemistry. In the present study, a series of rationally designed acetamide-linked fused thiophene-benzimidazole hybrids (3a-h, 4a-h, 5a-h, and 6a-i) were synthesized and comprehensively characterized using ¹H NMR, ¹³C NMR, and HRMS; compound 6d was further validated by single-crystal X-ray diffraction. The in vitro Anticancer potential of the synthesized derivatives was evaluated against PANC-1 (human pancreatic Cancer cells), A549 (adenocarcinomic human alveolar basal epithelial cells), and BEAS-2B (normal human bronchial epithelial cells) cell lines. Among them, compound 4d exhibited remarkable potency and selectivity toward PANC-1 cells (IC₅₀ = 0.067 ± 0.019 μM; BEAS-2B, IC₅₀ = 101.93 ± 2.21 μM, SI = 1521.34), whereas compound 6d demonstrated pronounced cytotoxicity against A549 cells (IC₅₀ = 0.82 ± 0.02 μM) with moderate selectivity (BEAS-2B, IC₅₀ = 19.06 ± 0.58 μM, SI = 23.24). Mechanistic investigations in A549 cells revealed that both 4d and 6d induced cell cycle arrest, Apoptosis, and alterations in the expression of markers of cell cycle regulators and Apoptosis. Furthermore, Western blot analysis revealed that both compounds significantly inhibited the phosphorylation of the EGFR (pEGFR) and also upregulated the expression of LC3B-II (a key marker of Autophagy) and cyclin-dependent kinase inhibitor p27, suggesting that activation of Autophagy and cell cycle arrest occurred, respectively, thereby inhibiting EGFR phosphorylation and activating downstream cellular responses similar to those of Erlotinib. In addition, the target prediction, followed by molecular docking results, showed strong binding affinities of both compounds toward the EGFR tyrosine kinase domain (PDB ID: 1M17, 2ITY, 2J5F). In silico ADME profiling further highlighted favourable pharmacokinetic properties, reinforcing the potential of 4d and 6d as promising lead candidates. Collectively, these results establish fused thiophene-benzimidazole hybrids as selective EGFR-targeted Anticancer agents with strong therapeutic promise.

Anticancer activity; Benzimidazole; EGFR; Lung cancer; Pancreatic cancer; Thiophene.