Experimental Approach: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and in women. It’s expected to cause about 53,200 deaths during 2020. An effective drug for therapy and prognosis after surgery still does not exist. Therefore, the search for new lead structures and chemical entities for the development of new effective anticancer agents is an increasingly important task in medicinal chemistry. This trend of global research includes work on the use of 1,2,4-triazine scaffold as a source for the design of biologically relevant molecules with well-known broad biological applications. A series of new pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine sulfonamides were designed, synthesized, and assessed as anticancer activity agents. Experimental Approach: The impact of two selected compounds, MM-128, and MM-129 (MMs), were evaluated against human colon cancer in in vitro and in zebrafish embryo xenograft model. Key Results: Our results show that the new synthesized compounds effectively inhibit cell survival and DNA synthesis in both DLD-1 and HT-29 cell lines. Their effectiveness is much higher as compared with the standard chemotherapy used for colorectal cancer, i.e. 5-fluorouracil. Flow cytometry analysis after annexin V-FITC and propidium iodide staining revealed that apoptosis was the main response of colorectal cancer cells to MMs treatment. We also found that MM-129 effectively inhibits tumor development in both DLD-1 and HT-29 zebrafish xenografts. Conclusion and Implications: New pyrazolo[4,3-e]tetrazolo[4,5-b][1,2,4]triazine sulfonamides may be new candidates for further evaluation as chemotherapeutic agents against colorectal cancer.