Development of Quinoline-Isoxazole Hybrids With Selective Cytotoxicity and EGFR Kinase Binding: In Vitro and In...

Fourteen new quinoline-isoxazole hybrid compounds were synthesized through click reaction with different substituent groups on the isoxazole group. The in vitro therapeutic activeness of the synthesized hybrids had been assessed versus diverse cell lines (MCF7, SKOV3, A549, LN229), using the standard tamoxifen as a reference. According to cell viability results, the most active molecules were found to be 4b against MCF7 cells (IC₅₀ = 12.94 μM) and compound 4f in the SKOV3 cells (IC₅₀ = 17.89 μM), as well as showing no cytotoxicity on the healthy human fibroblast cell line (MRC-5). These results showed us that the new compounds were selective for cancer cell lines. As an indicator of apoptosis, a significant decrease in the level of PARP protein was observed in the MCF7 cells for 4b and in the SKOV3 cells for 4f. After analyzing PARP levels by Western blot, apoptosis levels were also investigated by flow cytometry analysis. The mechanism of the 1,3-dipolar cycloaddition reaction between acetylene and benzaldoxime derivatives was investigated using time-dependent density functional theory (TDDFT). The calculated HOMO-LUMO energy differences and the changes in frontier orbital distributions were found to be consistent with the experimentally observed variations in reaction yield. The molecular docking investigation was conducted to evaluate the binding affinities, interactions, and binding modes of novel hybrid compounds with a target receptor, EGFR kinase (PDB ID: 4G5J). Results showed that compounds 4e and 4f presented favorable binding energies compared to the reference molecule Afatinib. Molecular dynamics simulations confirmed the stable binding of compound 4f with EGFR, with the complex reaching equilibrium after ~20 ns and maintaining an average RMSD of 0.25 ± 0.05 nm throughout the 100 ns trajectory. In silico ADME results also show that all synthesized molecules obey Lipinski's rules.