Evaluation of Antibacterial Functionalized Dihydropyrimidine Photoaffinity Probes Toward Mechanism of Action Studies

Antibiotic-resistant bacteria are a global health concern, necessitating the development of antibiotics working through new or underutilized mechanisms. Functionalized amino dihydropyrimidines have previously demonstrated potential as antibacterial agents, but they had limited potency, and their biological mechanism was not understood. To further evaluate their potential, focused libraries were prepared and screened for bacterial growth inhibition, and these compounds provided additional insights into the structure–activity relationships, allowing for the preparation of compounds that inhibited all strains of Staphylococcus aureus with an MIC of 2 μg/mL. After eliminating the proposed mechanism of dihydrofolate reductase inhibition, trifluoromethyl diazirine photoaffinity probes were synthesized to investigate their mechanism, and these were tested to ensure the photolabile group did not impact the antibacterial activity. Finally, the compounds were screened for hemolysis and mammalian cytotoxicity. While they lacked nonspecific membrane rupturing activity, many of the compounds showed significant mammalian cytotoxicity, indicating further development will be required to render them selective for bacteria.