Novel acetophenonic-isoxazoline derivatives with potent anti-cancer and selective COX-2 inhibitory effects: Design, synthesis, and biological evaluation

Abstract

Overexpression of the COX-2 enzyme is a major factor in the development of various malignancies, including colorectal cancer. A new series of acetophenonic-isoxazoline derivatives was designed, synthesized, and evaluated for selective COX-2 inhibitory and anticancer effects. A synthetic route based on the 1,3-dipolar cycloaddition mechanism was employed for the regiospecific formation of the isoxazoline ring. The newly synthesized compounds were characterized by combustion analysis, IR, ¹H NMR, ¹³C NMR, and ESI-MS. Cytotoxicity was assessed using the MTT assay against HT-29 human cancer cells and HFF and HEK293 human normal cell lines. In addition, cell cycle analysis, apoptosis induction, and the expression levels of BAX, Bcl-2, caspase-3, PTGS2, and VEGF genes were evaluated in HT-29 cells treated with the most promising derivatives. The most probable binding modes were predicted by docking the 3D structures into the COX-2 binding site (PDB code: 1CX2), and the results showed hydrogen-bond formation with key residues. The fluoro-containing derivative 9b showed superior in vitro COX-2 selectivity (SI = 141.7), whereas compound 9f, bearing a 3,4-dimethoxyphenyl moiety, exhibited the highest COX-2 inhibition (IC₅₀ = 0.08 μM) and potent cytotoxicity against HT-29 cells (IC₅₀ = 7.34 ± 2.88 μM). The most potent compounds (9b, 9e, 9f, and 9 g) promoted apoptosis, with significantly increased mRNA expression of BAX and caspase-3 and decreased Bcl-2 expression. Collectively, these findings suggest that the newly designed acetophenonic-isoxazoline derivatives, as COX-2 inhibitors, may exert multitarget therapeutic effects against colorectal cancer.