Aralkylpyrimidinetriones as Growth Inhibitors of Clostridioides difficile

Abstract

There is a pressing clinical need for effective new antimicrobial agents against Clostridioides difficile; one of the UK’s leading nosocomial healthcare-associated infection pathogens. Strains of C. difficile have become less sensitive to the current frontline therapies metronidazole and vancomycin with growing resistance to many other antibiotics.1 We have prepared aralkylpyrimidinetriones (APTs), some of which are active against C. difficile but inactive against Escherichia coli and Staphylococcus aureus. Selective targeting of C. difficile would provide a significant advantage over current antibiotic treatments that also destroy various gut-colonising bacteria and exacerbate life-threatening C. difficile associated diarrhoea. Dihydroorotate dehydrogenase (DHODase) catalyses the reversible and rate-determining fourth step of de novo pyrimidine biosynthesis. Arylidene barbiturates are known to inhibit purified DHODase from the organism Clostridioides oroticum with the rate of inhibition being strongly dependant upon the electronic properties of the aryl substitutents.2,3 Indiscriminate reactivity towards nucleophiles, poor aqueous solubility and the risk of unwanted anxiolytic, hypnotic and sedative properties associated with uncharged CNS-active barbiturate drugs, render arylidene barbiturates poor candidates as potential anti-infective agents. We considered APTs to be surrogate substrates with potential as novel pro-drug inhibitors of DHODase that exploit the enzyme’s ability to create C=C bonds.3 Once bound at the DHODase active site, the benzylic C-C bond in the APT prodrug may conceivably be oxidised to give an exocyclic C=C bond, generating the reactive arylidene at the DHODase active site to which a proximal nucleophile can attach followed by deactivating protonation, leading to irreversible inhibition of the enzyme. Better aqueous solubility and closer resemblance to the enzyme’s natural substrate is predicted for the APT derivatives were ionization to generally occur at C5 (pKa = 3.9)4 under physiological conditions. We demonstrated success in the use of uncatalyzed Knoevenagel condensation between barbituric acid and various 2-aryl-substituted acrylaldehydes in ethanol (1 h, reflux) to give moderate to good yields of diene derivatives of barbituric acid from which APT analogues were readily prepared by reduction using sodium borohydride or palladium on charcoal. We achieved regiospecific reduction of the exocyclic double bond using sodium borohydride whereas reduction of both double bonds to give a saturated propyl linkage resulted from use of palladium on charcoal (1 h, sonication, 2-3 atm). Representative compounds in the arylidene and APT series were evaluated and shown to have C. difficile growth inhibitory properties but with reduced or no activity against E. coli and S. aureus.