Design, Synthesis, and Biological Evaluation of 5-(5-Iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2,4-diamine (AF-353) Derivatives as Novel DHFR Inhibitors against Staphylococcus aureus .

The high lethality of Staphylococcus aureus infections and the emergence of antibiotic resistance make the development of new antibiotics urgent. Our previous work identified a hit compound h1 ( AF-353 ) as a novel Mycobacterium tuberculosis ( Mtb ) dihydrofolate reductase (DHFR) inhibitor. Herein, we analyzed the antimicrobial profile of h1 and performed a comprehensive structure-activity relationship (SAR) assay based on h1 . The representative compound j9 exhibited potent antibacterial activity against S. aureus without cross-resistance to other antimicrobial classes. Multiple genetic and biochemical approaches showed that j9 directly binds to Sa DHFR, resulting in strong inhibition of its enzymatic activity (IC 50 = 0.97 nM). Additionally, j9 had an acceptable in vivo safety profile and oral bioavailability ( F = 40.7%) and also showed favorable efficacy in a mouse model of methicillin-resistant S. aureus (MRSA) skin infection. Collectively, these findings identified j9 as a novel Sa DHFR inhibitor with the potential to combat drug-resistant S. aureus infections.