XWL-5-11

Topoisomerases are crucial enzymes that manage DNA topology and are essential for biological processes. Dysregulation of topoisomerases has been closely linked to the development and progress of cancer. As a result, topoisomerase inhibitors have emerged as potential anti-cancer drugs. In the current study fifteen hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives were designed and synthesized to investigate their topoisomerase II inhibitory activity. The antiproliferative activity of all the synthesized derivatives was evaluated across a panel of 60 cancer cell lines at the National Cancer Institute. Compound 8 was further investigated at 5 dose concentrations due to its remarkable growth inhibiting activity. Furthermore, the three compounds with significant cytotoxic activity 6c, 8, and 9a, were tested in an in vitro topoisomerase IIα inhibition assay compared to etoposide as reference drug. Compound 8 displayed the highest topoisomerase II inhibitory activity with an IC₅₀ of 41.67 ± 3.89 μM compared to etoposide (IC₅₀ = 99.86 ± 5.02 μM). In addition, the cell cycle analysis proved that compound 8 induced cell growth arrest at the G2/M phase in colon HT-29 cell line. Also, compound 8 significantly increased the total apoptotic ratio by 19.1-fold. This apoptotic activity was supported by an increase in the level of caspase-3 in colon HT-29 cell line by 8.49 and 8.63-fold for 6c and 8, respectively. Molecular docking was performed for compounds 6c, 8 and 9 to estimate their binding modes at the active site of human topoisomerase IIα enzyme and correlate it with their measured biological activity. In conclusion, compound 8 represents a promising Topoisomerase II inhibitor with potent antiproliferative and apoptotic effects, demanding further investigation as a potential anticancer therapy.

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.