MtTMPK-IN-3 (compound 25) is a potent Mycobacterium tuberculosis thymidylate kinase ( Mt TMPK) inhibitor with an IC 50 value of 0.12 μM. MtTMPK-IN-3 has inhibitory activity against Mtb H37Rv (MIC = 12.5 μM). MtTMPK-IN-3 exhibits certain cytotoxicity in human fibroblast cells MRC-5 (EC 50 = 12.5 μM). MtTMPK-IN-3 can be used for researching tuberculosis.