Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

Abstract

Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.

Keywords: antibiotics; infectious diseases; medicinal chemistry.

Fig. 1.

Synthesis of comprehensive new 5-NI library. (A) Structure of Mz. (B) Six different 5-NI cores (A−F) were synthesized with a “clickable” azide (N₃) functional group. (C) Scheme of click chemistry-facilitated synthesis of 5-NI triazole library.

Fig. 2.

Expanded antimicrobial activity range of structurally diverse 5-NI compounds. (A) The activities of 378 compounds were tested against the indicated protozoa and bacteria in 24- to 48-h growth assays using ATP levels or OD₆₀₀ as readouts. Each data point or number represents the mean EC₅₀ for one compound, with Mz shown in blue for comparison. (B) Examples of compounds with enhanced broad-spectrum or pathogen-selective activity (key values in bold). (C) Relationships between activities of individual compounds against the four target pathogens. Compounds that exceeded the activity of Mz (blue point) for both bacteria (black circles in light pink-shaded region, Left) were examined for their activities against the two protozoa (Right). The region containing compounds with superior activity against all four pathogens is shaded in salmon color. (D) The relationship between compound activities against two colonic bacteria (Upper). The orange-shaded region highlights compounds more active than Mz against the opportunistic pathogen, C. difficile, but less active than Mz against the commensal, B. fragilis. Assay sensitivity is depicted by the dashed green line. The table (Lower) lists examples of compounds that showed greater or lesser selectivity than Mz against C. difficile compared with B. fragilis.

Fig. 3.

New 5-NI compounds overcome Mz resistance. (A) Compounds were tested against MzR strains of G. lamblia (Left) and T. vaginalis (Right), with those more active than Mz (purple) highlighted by light pink boxes. Of these, compounds more active against both MzR lines than Mz against the respective MzS lines (light blue) are further highlighted by salmon-colored boxes. (B) Activities against wild-type and double mutant (ΔfrxA ΔrdxA) strains of H. pylori. Five compounds (red dots) had measurable activity against the mutant; all others were below the assay sensitivity (dashed green line, Upper). Examples of active compounds are listed in the table (Lower).

Fig. 4.

Predictive bioactivity landscape of 5-NI compounds. A structural space was generated by principal component analysis using activity data of the 378 compounds in the 5-NI library against the four target microbes. (A) The individual compounds were plotted in the resulting space with the top 10% most potent broad-spectrum compounds shown as red circles. (B) A structural space was constructed from the activity data against MzR G. lamblia. Activities of all compounds were plotted in the space (z axis), and a contour graph was generated using the indicated color scale. (C) A service vector machine model was constructed from the activity data of the 378 compounds against MzR Giardia (training set) and applied prospectively to a new set of 281 independently synthesized 5-NI compounds (test set). Correctly predicted compounds, as defined by coincidence of model prediction and assay-determined activity against the two MzR Giardia lines, are highlighted by background coloring (light pink and light blue) for predicted superior (Left) and inferior (Right) compounds. Each data point represents one compound. The light blue dots show Mz activity against the MzS parental lines for comparison.

Fig. 5.

Structure−activity relationships of 5-NI building blocks. The entire library of 659 5-NI compounds (composed of the 378 training and 281 test compounds) was examined for structure−activity relationships of the two building blocks, azido-5-NI and alkyne, used for the click chemistry-facilitated synthesis. (A) The influence of the azido-5-NI cores (A−F, Fig. 1B) on activity against Mz-sensitive (MzS, Left) and Mz-resistant (MzR, Right) Giardia, with average activities shown as red lines. (B) Data of all compounds (cpds) generated from cores A−C (to minimize core bias in the alkyne evaluation) in a structural space derived by principal component analysis. The top most active compounds against MzS and MzR Giardia are highlighted in light brown and red, respectively. Several regions with clustering of the most active compounds are boxed, and the corresponding alkynes used for compound synthesis are depicted.

Fig. 6.

In vivo efficacy of 5-NI compounds against giardiasis. (A) Adult C57BL/6 mice were orally infected with G. lamblia and given five 10 mg/kg oral doses of the indicated compounds over 3 d at 12-h intervals, and trophozoite numbers in the small intestine were determined. The dashed green line represents the assay sensitivity. Data are mean + SEM (n ≥ 5); *P < 0.05 vs. vehicle-treated controls. Compounds significantly more efficacious than Mz (light blue) are highlighted in red. (B) The relationships of in vivo bioactivity, in vitro activity (Left), aqueous solubility (Center), and measured serum drug concentrations (Right, green dashed line represents the assay sensitivity; Mz is shown in blue for comparison). (C) Examples for in vivo active compounds, along with their in vitro activities (Table in Lower Right) against MzS and MzR Giardia.