Convenient synthesis, characterization, evaluation and molecular docking of some new fused pyrazolo[3,4-d]pyrimidine derivatives and 3-methyl-8 phenylpyrazolo[3′,4':4,5]pyrimido[6,1-c][1,2,4]triazines against HeLa cancer cells

Utility of the precursors 3-aryl-4-imino-1-phenylpyrazolo[3,4-d]pyrimidin-5-amines 4a-f and 3-aryl-4-hydrazineyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines 5a-f in the synthesis of pyrazolo[3,4-d]pyrimidin-4-yl)hydrazono derivatives 10a-l via reaction of the hydrazines 5a-f with each of pyruvic acid or ethyl pyruvate 9. Refluxing of the hydrazone derivatives 10a-l in N,N-dimethylformamide for 90 h afforded the corresponding 3-methyl-8-phenylpyrazolo-[3′,4':4,5]pyrimido[6,1-c][1,2,4]triazine derivatives 11a-f. The cytotoxic results showed that the compound 11a was the most effective in suppressing the growth of HeLa cancer cells when compared to all other prepared compounds, with the most effective IC₅₀ value (3.46 ± 0.59 μg/mL) and no cytotoxic effects on normal human lung cells (WI-38). Moreover, the toxicity of the compound 11a against HeLa cells was confirmed by a significant increase in LDH levels in treated HeLa cells compared to untreated ones. Using annexin V/PI, treated HeLa cells with this IC₅₀ value of compound 11a displayed a considerable increase in early and late apoptotic cells in comparison to control cells. Additionally, apoptosis induction in the compound 11a-treated cells was mediated through increased reactive oxygen species (ROS) production. Moreover, the compound 11a markedly decreased the levels of antioxidant enzymes, including GSH, CAT, and SOD, in treated HeLa cells relative to untreated cells. Finally, the compound 11a markedly raised the expression levels of cleaved caspase-3, which is the initiator of apoptosis. Overall, these findings indicate that the compound 11a triggers significant cytotoxicity in HeLa cell cancer cells in a dose-dependent manner, primarily via ROS-mediated cell death, possibly via the mitochondrial pathway. So, compound 11a can be used as a promising treatment option for HeLa cancer in humans. In addition, in silico modelling, including, bioavailability, ADMET analysis, molecular docking and molecular dynamics simulation was conducted to evaluate the drug-likeness of the novel compounds (10a-11f). Compound 11a exhibited a promising binding affinity and stability against the receptors affirming its ability to act as antitumor and antioxidant ligand.