Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8-12.2 mu M. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin-resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli Delta tolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 mu M respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 mu g/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 mu g/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

Automated summary

The synthesis of novel compounds targeting the cytoplasmic steps of peptidoglycan synthesis as potential antibacterial agents was reported. Some of these compounds showed potent inhibition of MurA enzyme and exhibited antibacterial activity against Clostridioides difficile strains. The inhibition of MurA enzyme was found to be responsible for the observed effect on bacterial growth.