Click Chemistry-Facilitated Structural Diversification of Nitrothiazoles, Nitrofurans, and Nitropyrroles Enhances Antimicrobial Activity against Giardia lamblia

Abstract

Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.

Keywords: antimicrobial agents; drug screening.

FIG 1

Synthetic strategy. Azido derivatives of the nitroheterocycles, nitrothiazole (A), nitrofuran (B), and nitropyrrole (C) were synthesized following the depicted schemes. The azides were then reacted with one of ∼150 alkynes (see Table S1 in the supplemental material) utilizing the copper(I)-catalyzed azide alkyne cycloaddition (“click reaction”) to yield 1,4-substituted 1,2,3-triazoles. DMF, dimethylformamide; NaAsc, sodium ascorbate; MsCl, methanesulfonyl chloride; t-BuOH, tert-butyl alcohol; NEt₃, triethylamine; DCM, dichloromethane.

FIG 2

In vitro antigiardial activity of modified nitroheterocycles. (A to D) The activities of 442 newly synthesized nitroheterocyclic compounds were assayed against two metronidazole-sensitive (MzS) G. lamblia strains (713 and 106 [A and B]) and two metronidazole-resistant (MzR) G. lamblia strains (713M3 and WB-M2 [C and D]) in a 48-h growth and survival assay with ATP as a read-out. Panels A and C show activities separately against the indicated G. lamblia line, while panels B and D show the correlations between the two MzS strains and two MzR strains, respectively. Data are shown as means of the pEC50 values obtained in at least three independent experiments, with each point representing a single compound. Compounds are divided into groups with the same nitroheterocyclic cores: group G, nitrothiazoles; group H, nitrofurans; group I, nitropyrroles (see Fig. 1). The respective reference drugs are nitazoxanide (Ntz) (red circle), furazolidone (Fzd) (blue triangle), and metronidazole (Mz) (green square). The colored dotted lines in panels B and D show the activity of the respective reference compounds. The black dashed lines represent the assay sensitivity.

FIG 3

Cytotoxicity assessment of new nitroheterocycles in human cells. Cytotoxicity of the 442 new nitroheterocyclic compounds was tested against human HeLa epithelial cells in a 48-h growth and survival assay, using alamarBlue dye reduction as a read-out. Data are shown as means of at least three independent experiments, with each point representing a single compound. Panel A shows the results for all compounds, of which 52% displayed no measureable cytotoxicity at the highest tested compound concentration (50 μM, equivalent to a pCC50 of 4.3; assay limit), while the remaining compounds had measurable cytotoxicity (i.e., pCC50 of >4.3). Panel B depicts the selectivity index (ratio of CC₅₀ in HeLa cells versus EC₅₀ in G. lamblia strain 713) for the compounds with measurable cytotoxicity, while panel C shows the minimum predicted selectivity index for those compounds that did not have measurable cytotoxicity (the threshold pCC50 of 4.3 was used for the calculations, but the actual pCC50 could be significantly lower, so the real selectivity index would be higher). Data obtained in parallel assays for the benchmark compounds nitazoxanide (Ntz) (red circle), furazolidone (Fzd) (blue triangle), and metronidazole (Mz) (green square) are shown for comparison. As for the other compounds with undetectable cytotoxicity (i.e., pCC50 of <4.3) in our assay, only the minimum predicted selectivity index could be shown for the benchmark compounds.

FIG 4

In vivo activity of selected modified nitrothiazoles in murine giardiasis. (A) Structures of the six representative nitrothiazoles that were selected for further tests, resynthesized, and purified to >90%. (B) In vitro activity of the depicted compounds against G. lamblia strain GS/M (a human-pathogenic strain belonging to assemblage B) was determined in a 48-h growth and survival assay. Data are shown as individual pEC50 values of three independent experiments, with metronidazole (Mz) shown as a reference drug. Mean pEC50 values that were significantly different (P < 0.05) from those for metronidazole are indicated by an asterisk. (C) The six compounds and Mz were subsequently tested for in vivo efficacy in mice infected with the G. lamblia strain GS/M. Two days after infection, compounds were orally administered to mice in two daily doses of 10 mg/kg for a total of 5 doses over a 3-day period or the mice were given solvent alone as controls, and trophozoite numbers in the small intestine were determined. Data are shown as individual log₁₀-transformed counts with six to eight animals in each group. Horizontal lines depict the geometric mean for each group. The gray zone represents the 95% confidence interval of the counts in the controls.

FIG 5

SAR analysis of modified nitroheterocycles. The contributions of the azide cores (A) and alkynes (B and C) to the antigiardial activity of the resulting triazoles were analyzed. The nine azide cores used for the analysis are shown in panel A; six of these (A to F compounds) were reported previously (24), while the other three (G to I compounds) were synthesized in this study. The antigiardial activities of the triazoles generated by combining one of the nine azide cores with each of 83 identical alkynes are depicted in the left graph in panel A. Data are means of at least three independent experiments with G. lamblia strain 713. Each symbol represents one compound. The gray horizontal bars show the median values for the compound groups, and the dashed line depicts the assay sensitivity. Significance was evaluated by one-way ANOVA, followed by a post hoc Dunnett test with all compounds as a control. (B) The activity (pEC50) of each of 83 triazoles with the same azide core was normalized against the average activity of all compounds with that core. The normalized values were used to determine mean and SE for each alkyne and sorted from highest to lowest. Significance was evaluated by one-way ANOVA, followed by a post hoc Dunnett test with all compounds as a control. Structures are shown for the alkynes with significant contributions to the triazole activity. (C) All alkynes were subsequently analyzed by quantitative SAR using 1,666 chemical descriptors for correlation-based attribute subset evaluation to yield correlations between triazole activity and alkyne descriptors. The relationships with significant correlations are shown.