5,6-Fused heterocycle cholate derivatives as spore germination inhibitors of Clostridioides difficile

Clostridioides difficile is an anaerobic bacterium that is responsible for most antibiotic-associated cases of diarrhea. Clostridioides difficile infection (CDI) begins with the ingestion of an environmentally stable spore that germinates within the GI tract if the concentration and composition of bile salts, ions, and amino acids are favorable. We have shown that the bile salt analog N-phenyl-cholan-24-amide (1) can inhibit spore germination and prevent CDI in animal models of infection. Unfortunately, 1, while stable in the gut of antibiotic-treated animals, is rapidly degraded by normal gastrointestinal microbiota to release cholate and aniline. This instability prevents its use as a prophylactic for CDI. To generate more stable bile salt analogs, we removed the amide by either reducing it to an amine or converting it into 5,6-fused heterocycles. We found that reduction of the amide to an amine resulted in a significant loss of activity, but cyclization to benzimidazole (6a), benzothiazole (6b), or benzoxazole (6c) gave anti-germinants with good IC₅₀ values. Changes in the sterane group to chenodeoxycholate and deoxycholate gave active compounds only for the benzimidazole series. Mice treated for 7 days with 6a and 6b showed no signs of toxicity up to 300 mg/kg/day. Compound 6b, but not 6a, was able to prevent CDI in a murine model of CDI when given at a dose of 50 mg/kg for three days. Examination of 6b showed that it was stable for 96 h in the presence of feces taken from healthy mice validating the hypothesis that replacing the amide with a bioisostere could increase the stability of the compound.