Dual topoisomerase IIα/EGFR-TK inhibitors: Synthesis, biological evaluation, and in silico studies of new thiadiazolo[3,2-a]pyrimidinone derivatives as potential anticancer agents

A new series of 2-(methylthio)-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one derivatives was synthesized and evaluated for in vitro Anticancer activity against six human Cancer cell lines; laryngeal carcinoma (Hep2), human prostate Cancer (PC3), cervical carcinoma (Hela), hepatocellular carcinoma (HepG-2), human colon Cancer (HCT-116), and mammary gland breast Cancer (MCF-7). The most active compounds against the selected Cancer cell lines, 3, 4, 6a and 6b were further screened for in vitro cytotoxicity against lung fibroblast (WI38) normal cells. The results revealed that they exhibited high SI values toward Hela and HepG-2 Cancer cells. Analysis of cell cycle manifested that compound 3 induced cell cycle arrest in Hela and HepG-2 at G0-G1 phase causing cell death principally via Apoptosis. Apoptosis induction by compound 3 was confirmed through elevated levels of Caspase-3 and Bax/Bcl-2 ratio in the screened Cancer cells. Moreover, the same compounds were further evaluated for inhibitory activity against EGFR-TK^WT and Topoisomerase IIα. The obtained results revealed that compound 6a displayed strong inhibitory activity against EGFR-TK^WT (IC₅₀ = 0.078 μM) that closely approximates the reference drug erlotinib and promising inhibitory activity against Topoisomerase IIα (IC₅₀ = 12.6 μM) surpassing that of etoposide. Compound 6a dual inhibitory activity on the expression of EGFR-TK^WT and topo IIα in HepG-2 cells was further evidenced through Western blot analysis. Furthermore, compound 3 demonstrated good inhibitory activity against EGFR-TK^WT (IC₅₀ = 0.122 μM) compared to erlotinib and superior inhibitory activity against Topoisomerase IIα (IC₅₀ = 16.8 μM) compared to etoposide. Additionally, compound 6a was evaluated for its inhibitory activity against EGFR^L858R and EGFR^T790M mutants, and it displayed promising activity against EGFR^T790M and moderate activity against EGFR^L858R (IC₅₀ = 0.088 and 0.189 μM, respectively) compared to erlotinib. Moreover, human epidermoid carcinoma (A431) and non-small cell lung Cancer (NSCLC) (H1975) cells were utilized to assess the in vitro cytotoxicity of compound 6a and it showed good activity over A431 and H1975 cells (IC₅₀ = 7.89 and 13.11 μM, respectively) compared to erlotinib. Molecular docking studies of 3 and 6a confirmed the high binding affinity of both compounds toward the active site of EGFR-TK^WT and Topoisomerase IIα. Finally, compound 6a was proved to adopt the exact fit into the active site of a double mutant EGFR^T790M/L858R. Molecular dynamics simulations of compound 6a were performed, and the results confirmed the stable binding interactions toward EGFR-TK^WT and Topoisomerase IIα active sites, supporting the consistency of the docking results.

Anticancer agents; Apoptosis; Cell cycle analysis; In silico studies; Thiadiazolo[3,2-a]pyrimidinones; Topoisomerase IIα/EGFR-TK inhibitors; Western blot analysis.