Discovery of 1,2,3,4-Tetrahydrochromeno[3,4- <i>c</i> ]pyridin-5-one Derivatives as Novel MTHFD Inhibitors for the Treatment of Acute Myeloid Leukemia

One-carbon metabolism is essential for nucleotide biosynthesis and redox homeostasis, and its key enzymes, MTHFD2 and MTHFD1, are aberrantly activated in diverse cancers, particularly acute myeloid leukemia (AML). Herein, we solved the X-ray crystallography of the reported MTHFD inhibitor DS18561882 bound to MTHFD2 and performed systematic structure-activity optimization, leading to the identification of a highly selective MTHFD2 inhibitor (compound <b>31</b>) and a dual MTHFD1/2 inhibitor (compound <b>34</b>). Cocrystal structural analysis revealed that subtle modifications of the aminosulfonamide motif dictate isoform selectivity by reshaping hydrogen bond and hydrophobic networks within the MTHFDs active site. Notably, the dual inhibitor <b>34</b> exhibited superior antiproliferative activities across AML cell lines and induced marked tumor regression in MOLM-13 xenograft models with minimal toxicity, outperforming the reference compound DS18561882. Our findings establish rational design principles for isoform-selective and dual MTHFD1/2 inhibitors and highlight a combined MTHFD1/2 blockade as a promising therapeutic strategy for AML.