Design and Synthesis of New Multi-Target Coumarin Analogues Acting as Carbonic Anhydrases IX/XII and Topoisomerase II Inhibitors With Apoptotic Potential.

Through applying the hybridization technique, new coumarin derivatives (2-17) were prepared with substitution at coumarin C-3 utilizing various heterocyclic derivatives, aiming to afford multi-target carbonic anhydrases (CAs) IX/XII and topoisomerase II (Topo II) inhibitors with potent antiproliferative activity. Eight different cell lines were used to evaluate the growth inhibition percentages (GI%) of cancer cells determined by coumarin analogues 1-17. Analogues 16 and 17 had the most substantial cytotoxic effects, achieving mean GI% of 86.59% and 85.74%, respectively, exceeding doxorubicin (Dox), which demonstrated a mean GI% of 69.15. Furthermore, the CAs IX/XII and Topo II inhibitory potentials for compound 17 were evaluated according to the protein expression analysis in the MG-63 cancer cells. Analogue 17 downregulated the expression of CA IX, CA XII, and Topo II proteins by 0.43-, 0.51-, and 0.56-fold change, respectively, indicating frontier inhibitory potentials. Additionally, analogue 17 achieved upregulation of apoptotic proteins (Caspases 3, 7, and 9, and BAX by 1.52-, 3.10-, 1.67-, and 1.90-fold change, respectively). On the contrary, compound 17 induced downregulation of the antiapoptotic proteins BCL-2, MMP2, and MMP9 by 0.44-, 0.38-, and 0.45-fold change, respectively. Besides, compound 17 achieved an arrest at the G0-G1 phase of the cell cycle and elevated the cellular levels from 65.75% of the control cells to 92.54%. Finally, three molecular docking processes of the superior analogue 17 toward CA IX, CA XII, and Topo II targets were performed to investigate its molecular interactions.