Pyrazolo[4,3-]tetrazolo[1,5-][1,2,4]triazine Sulfonamides as an Important Scaffold for Anticancer Drug Discovery-In Vitro and In Silico Evaluation.

Pyrazolo[4,3-]tetrazolo[1,5-][1,2,4]triazine sulfonamides (-compounds) are a relatively new class of heterocyclic compounds that exhibit a wide variety of biological actions, including anticancer properties. Here, we used caspase enzyme activity assays, flow cytometry analysis of propidium iodide (PI)-stained cells, and a DNA laddering assay to investigate the mechanisms of cell death triggered by the -compounds (, , , and ). Due to inconsistent results in caspase activity assays, we have performed a bromodeoxyuridine (BrdU) incorporation assay, colony formation assay, and gene expression profiling. The compounds' cytotoxic and pro-oxidative properties were also assessed. Additionally, computational studies were performed to demonstrate the potential of the scaffold for future drug discovery endeavors. MM-compounds exhibited strong micromolar (0.06-0.35 µM) anti-proliferative and pro-oxidative activity in two cancer cell lines (BxPC-3 and PC-3). Activation of caspase 3/7 was observed following a 24-h treatment of BxPC-3 cells with IC concentrations of , and compounds. However, no DNA fragmentation characteristics for apoptosis were observed in the flow cytometry and DNA laddering analysis. Gene expression data indicated up-regulation of BCL10, GADD45A, RIPK2, TNF, TNFRSF10B, and TNFRSF1A (TNF-R1) following treatment of cells with the compound. Moreover, in silico studies indicated AKT2 kinase as the primary target of compounds. -compounds exhibit strong cytotoxic activity with pro-oxidative, pro-apoptotic, and possibly pro-necroptotic properties that could be employed for further drug discovery approaches.