Cationic Peptidomimetic Amphiphiles Having a N-Aryl- or N-Naphthyl-1,2,3-Triazole Core Structure Targeting Clostridioides (Clostridium) difficile: Synthesis, Antibacterial Evaluation, and an In Vivo C. difficile Infection Model

Abstract

Clostridioides (also known as Clostridium) difficile is a Gram-positive anaerobic, spore producing bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are inadequate, expensive, and limited, and thus inexpensive drug treatments for C. difficile infection (CDI) with improved efficacy and specificity are urgently needed. To improve the solubility of our cationic amphiphilic 1,1'-binaphthylpeptidomimetics developed earlier that showed promise in an in vivo murine CDI model we have synthesized related compounds with an N-arytriazole or N-naphthyltriazole moiety instead of the 1,1'-biphenyl or 1,1'-binaphthyl moiety. This modification was made to increase the polarity and thus water solubility of the overall peptidomimetics, while maintaining the aromatic character. The dicationic N-naphthyltriazole derivative 40 was identified as a C. difficile-selective antibacterial with MIC values of 8 µg/mL against C. difficile strains ATCC 700057 and 132 (both ribotype 027). This compound displayed increased water solubility and reduced hemolytic activity (32 µg/mL) in an in vitro hemolysis assay and reduced cytotoxicity (CC₅₀ 32 µg/mL against HEK293 cells) relative to lead compound 2. Compound 40 exhibited mild efficacy (with 80% survival observed after 24 h compared to the DMSO control of 40%) in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.

Keywords: Clostridioides (Clostridium) difficile; antibacterial; peptidomimetic; triazole.

Figure 1

Previously published cationic amphiphilic hydrophobic anchored peptidomimetic antimicrobial agents. MIC values against C. difficile in µg/mL. SR = solubility ratio relative to that of compound 1–see Ref [13].

Figure 2

Hydrophobic scaffold replacements of the binaphthyl moiety for the target peptidomimetics.

Scheme 1

Synthesis of N-aryltriazole monocationic peptidomimetics 10–16. i. HOBt (1.1 eq), EDCI.HCl (1.1 eq), Et₃N (1.0 eq), CH₂Cl₂, rt, 16 h. ii. R³C≡CH, CuSO₄.5H₂O (0.2 eq), Na.ascorbate (0.4 eq), t-BuOH:H₂O (4:1), rt, 16 h. iii. TFA/H₂O/DCM, rt, 16 h; then HCl in Et₂O.

Scheme 2

Synthesis of N-naphthyltriazole monocationic peptidomimetics 21–26. i. HOBt (1.1 eq), EDC.HCl (1.1 eq), Et₃N (1.0 eq), CH₂Cl₂, rt, 16 h. ii. R³C≡CH, CuSO₄.5H₂O (0.2 eq), Na.ascorbate (0.4 eq), t-BuOH:H₂O (4:1), rt, 16 h. iii. TFA/H₂O/DCM, rt, 16 h; then HCl in Et₂O.

Scheme 3

Synthesis of dicationic peptidomimetics 36–50-i. HOBt (1.1 eq), EDC.HCl (1.1 eq), Et₃N (1.0 eq), CH₂Cl₂, rt, 16 h. ii. R′C≡CH, CuSO₄.5H₂O (0.2 eq), Na.ascorbate (0.4 eq), t-BuOH:H₂O (4:1), rt, 16 h. iii. TFA/H₂O/DCM, rt, 16 h; then HCl in Et₂O.

Figure 3

C57BL/6J mice (n = 5 per group) were infected with 10⁵ spores of C. difficile strain M7404 (RT027). Six hours post-infection and then every 12 h thereafter, mice were administered either 10% DMSO (blue) or 2.5 mg (100 mg/kg in 10% DMSO) of compound 40 (red) by oral gavage. Mice were monitored daily for survival (a) and weight loss relative to day 0, which was the day of infection (b). Fecal spore load at 1-day post-infection was determined by plating (c). Data are presented as CFU/gram feces, with each point representing a single mouse. Mouse cages were scored on day 1 post-infection for appearance (d) and mice were individually assessed for fecal consistency (e) and physiological appearance (f). Data represent the mean ±S.E.M. and statistical significance was assessed using a log-rank (Mantel–Cox) test or one-way ANOVA with a post hoc Tukey’s multiple comparison test.