StagX1, an isoquinolinone compound, selectively targets CES1-positive Ewing sarcoma cells as a potential therapeutic agent.

StagX1 {ethyl 2-[[2-[2-[(2,3-dihydro-1,4-benzodioxin-6-yl)amino]-2-oxoethyl]-1,2-dihydro-1-oxo-5-isoquinolinyl]oxy]propanoate} is a derivative of isoquinolinone, possessing an ethyl propionate. StagX1 exhibits growth-inhibitory activity in multiple Ewing sarcoma cell lines. To advance StagX1 as a potential lead, we conducted experiments to examine its metabolism and stability in tissue culture media, plasma, liver microsomes, cells and mice. Our studies demonstrate that StagX1 is metabolically unstable and undergoes rapid hydrolysis to its corresponding acid metabolite (StagX1-acid) through cleavage of the ethyl ester group. We identified carboxylesterase 1 (CES1) as the primary enzyme responsible for this conversion. Notably, cells expressing CES1 are sensitive to StagX1, whereas CES1-deficient cells show minimal response, indicating that metabolic activation is required for its activity. In contrast, StagX1-acid is metabolically stable. These findings suggest that StagX1 functions as a prodrug that is enzymatically converted to its active metabolite, StagX1-acid, within cells. This metabolic conversion likely underlies its mechanism of action and contributes to its selective anticancer activity in Ewing sarcoma. Our findings provide insight into the metabolism of StagX1 and the role of CES1 in mediating its effects and demonstrate that StagX1 is a promising compound with growth inhibitory effects in CES1 positive Ewing sarcoma cells.