Design, synthesis, and anticancer evaluation of novel pyrazole–thiophene hybrid derivatives as multitarget inhibitors of wild EGFR, mutant (T790M) EGFR, and VEGFR-2

Abstract

A new series of pyrazole–thiophene hybrid derivatives was rationally designed, synthesized, and biologically evaluated for anticancer potential. Cytotoxicity screening toward MCF-7 and HepG2 cell lines identified 2 as the most potent analogue (IC₅₀ = 6.57 μM, MCF-7; 8.86 μM, HepG2), with activity comparable to the reference drugs doxorubicin, erlotinib, and sorafenib. Selectivity toward MCF-7 was observed for 8 (IC₅₀ = 8.08 μM), while 14 displayed moderate dual activity (IC₅₀ = 12.94 μM, MCF-7; 19.59 μM, HepG2). Enzyme inhibition assays revealed that 2 effectively suppressed wild-type EGFR (IC₅₀ = 16.25 μg mL⁻¹) and the clinically relevant T790M mutant (17.8 μg mL⁻¹), whereas 14 showed balanced dual inhibition (16.33 and 16.6 μg mL⁻¹, respectively). Notably, 8 emerged as the most active VEGFR-2 inhibitor (35.85 μg mL⁻¹), significantly outperforming 14 (112.36 μg mL⁻¹) and 2 (242.94 μg mL⁻¹). Mechanistic studies demonstrated that 14 brought MCF-7 cells in the G0/G1 phase to cell-cycle arrest (74.16% vs. 55.31% in control), increased the apoptotic population to 26.32%, and caused minimal necrosis (4.2%). Molecular docking supported these findings, revealing strong binding affinities and favorable interactions of 2, 14, and 8 with EGFR (wild-type and T790M mutant) and VEGFR-2, respectively. Taken together, these results highlight 2, 8, and 14 as promising pyrazole–thiophene multitargeted anticancer leads, offering potential for further optimization to overcome kinase-driven resistance in cancer therapy.