Structure-Activity Relationship Studies of Benzothiazole-Based FabK Inhibitors as New Anti-Clostridioides difficile Agents

Abstract

Clostridioides difficile Infections (CDIs) remain a significant clinical challenge due to recurrence, antimicrobial resistance, and microbiome disruption associated with current therapies. The enoyl-acyl carrier protein reductase II enzyme (FabK) is essential for fatty acid biosynthesis in C. difficile and represents an attractive narrow-spectrum antibacterial target. We previously reported that a phenylimidazole-based inhibitor with a p-bromophenyl tail group demonstrated selective inhibition of CdFabK and low micromolar antibacterial activity. In this work, we aim to further expand the structure–activity relationship (SAR) of the phenylimidazole CdFabK inhibitor series in an effort to identify new scaffolds and improve biochemical potency, while maintaining whole-cell antibacterial activity. An expanded N-(benzo[d]thiazol-2-yl)-2-(2-(phenylamino)thiazol-4-yl)acetamide chemical series was designed and synthesized. Compounds were assessed for CdFabK enzymatic inhibition and in vitro antibacterial activity against C. difficile. A series of benzothiazole derivatives were synthesized following the Hantzsch thiazole synthesis, ester hydrolysis, and amide coupling of substituted carboxylic acids with 2-aminobenzothiazole derivative. Biological evaluation revealed that a compound with 6-ethoxybenzothiazole head group and phenylaminothiazole tail moiety exhibited CdFabK inhibition value of IC₅₀ = 4.40 μM, with minimum inhibitory concentration (MIC) of 1.60 μg/mL against C. difficile. Another close structural analog with the 3-chlorophenylaminethiazole tail showed excellent anti-difficile activity (MIC = 0.4 μg/mL) and low micromolar CdFabK inhibition of IC₅₀ = 4.90 μM. Systematic SAR exploration of phenylimidazole- and benzothiazole-based inhibitors led to improved anti–C. difficile activity while preserving CdFabK inhibition. These findings further validate FabK as a promising narrow-spectrum antibacterial target and support continued optimization of this class of new therapeutic agents for C. difficile infection.