Proline racemases (PRAC), catalyzing the L-proline and D-proline interconversion, are essential factors in eucaryotic pathogenes such as Trypanosoma cruzi, Trypanosoma vivax and Clostridioides difficile. If the discovery of irreversible inhibitors of Trypanosoma cruzi PRAC (TcPRAC) led to innovative therapy of the Chagas disease, no inhibitors of CdPRAC have been discovered to date. However, Clostridioides difficile, due to an increased incidence in recent years, is considered as a major cause of health threat.

In this work, we have taken into account the similarity between TcPRAC and CdPRAC enzymes to design new inhibitors of CdPRAC.

Starting from (E) 4-oxopent-2-enoic acid TcPRAC irreversible inhibitors, we synthesized 4-aryl substituted analogues and evaluated their CdPRAC enzymatic inhibition against eleven strains of Clostridioides difficile. This study resulted in promising candidates and allowed for identification of (E)-4-(3-bromothiophen-2-yl)-4-oxobut-2-enoic acid 20 that was chosen for complementary in vivo studies and did not reveal in vivo toxicity.