Targeting colorectal cancer with pyrazolo[4,3-e][1,2,4]triazine and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides: comprehensive in vitro evaluation

Abstract Colorectal cancer (CRC) remains a major global health challenge, ranking as the third most common malignancy and the second leading cause of cancer-related mortality worldwide. This study aimed to evaluate a novel series of pyrazolo[4,3- e ][1,2,4]triazine and pyrazolo[4,3- e ]tetrazolo[1,5- b ][1,2,4]triazine sulfonamides as potential anticancer agents, with a focus on their cytotoxic and mechanistic effects against CRC cell lines. Incorporation of the tetrazole ring significantly increased cytotoxic activity (up to 50-fold) and selectivity (3.6-9.2-fold) compared to non-tetrazole analogues (2.2–3.3). Pyrazolo-tetrazolo-triazine derivatives exhibited IC₅₀ values ranging from 0.12 to 1.1 µM and demonstrated enhanced solubility and consistent biological activity. Selected derivatives (MM134, MM136, MM137, MM139) induced marked DNA damage and inhibition of cell proliferation, with cell cycle analysis confirming apoptotic and necrotic features depending on the compound and exposure time. Electrochemical analyses confirmed direct interactions between MM compounds and double-stranded DNA. Notably, MM137 was the potent inducer of cell death in both HCT-116 and HT-29 3D spheroids at concentrations as low as 5 µM, comparable to the most pre-clinically advanced MM129 derivative. In conclusion, the study highlights pyrazolo[4,3- e ]tetrazolo[1,5- b ][1,2,4]triazine sulfonamides as promising anticancer candidates with potent cytotoxicity, favorable selectivity, and potential multimodal mechanisms of action.