MTHFD1/2-IN-1 is an orally active dual inhibitor of MTHFD1 and MTHFD2, exhibiting IC50 values of 0.26 μM and 0.031 μM for human MTHFD1 and MTHFD2, respectively. It disrupts one-carbon metabolism by inhibiting the dehydrogenase activity of MTHFD1 and both the dehydrogenase and cyclohydrolase activities of MTHFD2, thereby impairing nucleotide biosynthesis and redox homeostasis in cancer cells. This compound demonstrates good Caco-2 permeability and hepatic microsome metabolic stability. Furthermore, MTHFD1/2-IN-1 shows significant anti-leukemia activity, reducing the viability of various leukemia cells and inhibiting tumor growth in mouse models of acute myeloid leukemia (AML).