Synthesis, antimicrobial, and cytotoxicity evaluation of selective growth inhibitors of Clostridioides difficile

Abstract

The World Health Organisation has estimated the cost of antimicrobial resistance (AMR) to the global economy to be $100,000,000,000,000 with AMR becoming the leading cause of death by 2050. Clostridioides difficile is a major healthcare-associated infection in the UK, Europe, the USA, and other developed countries. Strains of this microorganism that are less sensitive to the frontline therapies (metronidazole, vancomycin, and fidaxomicin) are emerging, causing great concern and negative prognosis for AMR. Developing new, potent, efficacious antimicrobial agents with a narrow spectrum of activity will provide alternative treatment options for C. difficile and AMR. Representative derivatives were prepared in good yield by Knoevenagel condensation of arylaldehydes with an active methylene substrate, in ethanol or water at reflux and without additional catalyst. Compounds were purified, characterised, and assayed against C. difficile NCTC 11204 to determine potency, and against Escherichia coli NCTC 35218 and Staphylococcus aureus NCTC 29213 to determine selectivity. The lead compound 5-[(5-Nitro-2-furyl)methylene]hexahydropyrimidine-2,4,6-trione (2.10) gave a minimum inhibitory concentration (MIC) value of 2 μg/mL against C. difficile NCTC 11204. Replacement of the nitro group by hydrogen 5-(2-furylmethylene)hexahydropyrimidine-2,4,6-trione (2.8) resulted in good selectivity. This inhibitory activity improved further, using isosteres. For example, the replacement of oxygen 2.10 by sulfur 5-[(5-Nitro-2-thienyl)methylene]hexahydropyrimidine-2,4,6-trione (2.16) gave a MIC value of 0.5 μg/mL against CD. Replacement of the nitro group in compound 2.16 by hydrogen 5-(thiophen-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione (2.20) resulted in the most potent compound in the series (MIC = 0.125 μg/mL) albeit with reduced selectivity. The active diene derivatives 5-[(E)-3-(2-Furyl)prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.9), 5-[(E)-3-(5-Nitro-2-furyl)prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.13), and 5-[(2-Methoxy-1-naphthyl)methylene]hexahydropyrimidine-2,4,6-trione (2.21) demonstrate scope for homologation in the inhibitors. Replacement of the five-membered aryl ring with benzene in compound 5-[(E)-3-(4-Methoxyphenyl) prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.5) gave excellent selectivity but with reduced growth inhibitory against C. difficile. Isosteric modification provided derivatives with improved potency and selectivity against C. difficile compared with S. aureus and E. coli. Six representatives of the lead compounds that were tested for their cytotoxic activity against normal mice liver cells and colorectal cancer cells, where compound 2.8 showed the lowest cytotoxic activity against both cell lines. Of the compounds prepared and evaluated, a subset provided promising development candidates for testing against clinical isolates from patients presenting with C. difficile infection, to examine effectiveness against ribotypes that are less sensitive for frontline therapies.