Anti- Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives

Abstract

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC₅₀ of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC₅₀ in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Keywords: CYP51; Chagas disease; Trypanosoma cruzi; azole; heterocycle; imidazole; nitroreductase; triazole.

Figure 1

Design of azolic derivatives 5–10.

Scheme 1

Synthesis of azolic derivatives 5–10.

Figure 2

Comparison of the anti-T. cruzi activities among the 5–10 series of compounds.

Figure 3

Metabolization rate of nitroazole derivatives by the enzyme TcNTR. Higher values of K_obs obtained indicate a higher consumption rate of this compound at a given concentration compared to the other derivatives.

Figure 4

Superposition of the cocrystal fluconazole (TPF) and redocked pose. Three-dimensional (3D) representation of the TPF in the binding site of the 14α-demethylase structure (CYP51). The cyan color represents the cocrystal TPF pose (carbon atoms), and the redocked pose of TPF (carbon atoms) is preserved in magenta. Carbons of the heme group are represented in green. The other atoms were colored according to PyMOL defaults (v2.5.0, https://pymol.org, accessed on 18 April 2023) [43]. The root mean square deviation (RMSD) value between the TPF crystal coordinates and predicted coordinates is also shown.

Figure 5

Two-dimensional (2D) diagrams of protein–ligand interactions in the active sites of the 14α-demethylase structure (CYP51) (PDBid.: 2WUZ) [41] using the Maestro (Schrödinger) program [44]. The interactions of cocrystallized (A) and docked (B) poses of fluconazole (TPF) with CYP51 residues and heme groups. 2D docking interactions of Compounds 5–10 poses (C–H) with 14α-demethylase residues and heme groups. Protein–ligand interactions are represented with arrows/lines between ligand atoms and protein residues: H-bonds are represented by solid pink arrows; π–π stacking interactions are shown in green lines; π–cation interactions are represented by red lines; metal coordination is shown in purple line; the salt bridge is represented in red-to-blue gradient lines. The colors in residue circles indicate the residue type: (acidic, red; basic, purple; hydrophobic, green; polar, blue; glycines, light green; metal atoms, dark gray; other, light gray). Ligand atoms that are exposed to solvent are marked with gray spheres. The protein “pocket” is displayed with a line around the ligand, colored with the color of the nearest protein residue.

Figure 6

Distance (in Å) of the heme Fe²⁺ to the proximal aromatic nitrogen (N.ar) of the nitrotriazole and imidazole rings in cocrystallized (A) and docked (B) poses of fluconazole (TPF) and Compounds 5–10 (C–H) are shown in A through H. Furthermore, better docking energies are also shown (in kcal/mol). Heme carbons are represented in green, cocrystallized TPF carbons are shown in cyan and redocked TPF carbons are in magenta. Carbons in derivatives 5–10 are shown in yellow, pink, gray, purple–blue, orange, and green–cyan, respectively. The other atoms are colored according to PyMOL [43] defaults.