Identification of N-(5-nitrothiazol-2-yl)-3-oxopyrazolidine-4-carboxamide derivative as a potent inhibitor of Clostridioides difficile

The continuous need to develop novel antibacterial agents remains a challenging predicament facing the drug development industry due to the major problem of bacterial resistance. In this study, we report the identification of a new N-(5-nitrothiazol-2-yl)-3-oxopyrazolidine-4-carboxamide derivative (RW18) as a potent inhibitor of Clostridioides difficile. C. difficile is a gram-positive, anaerobic spore-forming bacterium that is listed by the CDC as an urgent threat. It is the leading cause of nosocomial diarrhea ranging from mild diarrhea to lethal colitis, spreading worldwide with the lack of many treatment alternatives. Altogether, C. difficile is ranked as an alarming concern to the health system. RW18 was discovered during our synthetic efforts to discover MurA inhibitors targeting cell wall synthesis in bacteria. RW18 exhibited significant MurA inhibition, with IC₅₀ value of 9.8 μM as well as demonstrated nearly the same inhibitory activity against MurA C115D, the mutant version developed by resistant E. coli towards fosfomycin. Notably, RW18 displayed potent activity against several Clostridioides difficile clinical isolates with MIC values ranging between 0.125 and 1 μg/mL. It was found to possess bactericidal activity with MBC values 0.25 -1 μg/mL. When tested against the normal intestinal microbiota, RW18 showed a significantly reduced inhibition. In addition, when tested against Caco-2 cells, RW18 showed a high safety index, indicating that it should not show harmful effects to the colon cells if administered orally. Finally, the compound was highly stable in LB culture medium as well as with bacterial cell lysate. Overall, RW18 presents a promising lead compound against Clostridioides difficile.