The human mitochondrial enzyme, called methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), plays a key role in one-carbon metabolism in purine or thymidine biosynthesis. In mitochondrial folate carbon metabolism, MTHFD2 has attracted considerable attention as a potential target for cancer treatment. Its inducement is a higher expression in various human tumor types, but low or undetectable expression level in most adult tissues. In addition, MTHFD2 inhibitor is considered as potential cancer therapies with minimal side effects. Inhibition of MTHFD2 can inhibit the proliferation of cancer cells in vitro and reduce (but not eliminate) the growth of tumors in vivo. The expression of MTHFD2 is also related to the prognosis of breast cancer, liver cancer and acute myeloid leukemia (AML). Furthermore, the normal function of MTHFD2 may be in embryogenesis and the expression of MTHFD2L in normal adult tissues. DS18561882 is a highly potent, isozyme-selective MTHFD2 inhibitor with a good oral pharmacokinetic profile. DS18561882 is a highly potent, isozyme-selective methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) inhibitor with a good oral pharmacokinetic profile. Specifically, DS18561882 shows a cell-based activity with a GI value of 140 nM. Studies on substitution of tricyclic coumarin on the skeleton and regulation of molecular acidity showed modifications significantly improved efficiency and cell permeability. Besides, DS18561882 exhibited a high plasma concentration when administered orally. Moreover, it inhibits dose-dependently tumor growth at the doses of 30, 100, and 300 mg/kg, BID. Remarkably, the tumor growth was almost completely inhibited (TGI: 67%) at the dose of 300 mg/kg, BID. All in all, DS18561882 is a highly potent, isozyme-selective methylenetetrahydrofolate dehydrogenase 2 inhibitor with a good oral pharmacokinetic profile.