RIP1/RIP3/MLKL activator 1 (Compound 6i) is a potent anti-glioma agent. RIP1/RIP3/MLKL activator 1 induces necroptosis through RIP1/RIP3/MLKL pathway. RIP1/RIP3/MLKL activator 1 exerts acceptable BBB permeability In Vitro RIP1/RIP3/MLKL activator 1 (Compound 6i) (96 h) shows antiproliferative activities in human glioma cell lines. RIP1/RIP3/MLKL activator 1 (0-4 µM, 0-72 h) exhibits remarkable antiproliferative activity for U251 cells in a time- and concentration-dependent manner. RIP1/RIP3/MLKL activator 1 (10 µM, 0-72 h) shows acceptable stability. RIP1/RIP3/MLKL activator 1 (0-2 µM, 24 h) effectively inhibits the migration of U251 cells. RIP1/RIP3/MLKL activator 1 induces necroptosis through RIP1/RIP3/MLKL pathway, and induces mitochondrial depolarization in U251 cells. RIP1/RIP3/MLKL activator 1 could not induce apoptosis in U251 cells. MCE has not independently confirmed the accuracy of these methods. They are for reference only. Cell Proliferation AssayCell Line: A172, LN229, U87, U251 and L02 cell lines Concentration: 0-4 µM for U251 cells Incubation Time: 96 h; 24, 48, and 72 h for U251 cells Result: Showed antiproliferative activity with IC 50 values of 3.03 ± 0.70, 1.78 ± 0.79, 1.22 ± 0.89, 0.94 ± 0.45, and 0.99 ± 0.46 µM against A172, LN229, U87, U251 and L02 cells, respectively. Time- and concentration-dependently inhibited the growth in U251 cells. Western Blot AnalysisCell Line: U251 Concentration: 0, 0.5, 1, 2, and 4 µM Incubation Time: 24 or 48 h Result: Concentration-dependently upregulated the expression of p-RIP1, RIP1, p-RIP3, RIP3, p-MLKL, and MLKL at 24 or 48 h. In Vivo RIP1/RIP3/MLKL activator 1 (Compound 6i) (2.50 ng/tail; i.v.; 48 h) inhibits U251 cell proliferation in vivo and exerts acceptable BBB permeability . MCE has not independently confirmed the accuracy of these methods. They are for reference only. Animal Model: Zebrafish wide-type AB strain; 200 CM-DiI labeled U251 cells were transplanted into yolk sac of each wild-type zebrafish embryos at 2 dpf (2 days postfertilization) Dosage: 2.50 ng/tail Administration: Microinjection; 48 h Result: Remarkably reduced the U251 xenografts fluorescence intensity. Form:Solid IC50& Target:RIP1, RIP3, MLKL