Novel Chalcone Derivatives as Anti- Leishmania infantum Agents with Potential Synergistic Activity and In Silico Insights

Abstract

Background: Visceral leishmaniasis (VL) is a neglected tropical disease with limited therapeutic options, often restricted by toxicity, high costs, and resistance. Chalcones are promising scaffolds for the development of antiparasitic agents. Objectives: This study aimed to synthesize novel acetamides derived from 4-hydroxychalcones and evaluate their antileishmanial activity, cytotoxicity, potential synergy with amphotericin B (AmB), and mechanisms of action through in silico analyses. Methods: Six chalcone-acetamides (3a-c, 4a-c) were synthesized and characterized by IR, NMR, and HRMS. In vitro activity against Leishmania infantum promastigotes and axenic amastigotes was assessed by colorimetric assays. Cytotoxicity was tested in human erythrocytes and PBMCs. Synergy with AmB was analyzed by the combination index. Molecular docking targeted parasite enzymes, and ADMET tools predicted pharmacokinetic and safety profiles. Results: Phenyl-substituted derivatives (3a-c) were inactive, while cyclohexyl-substituted analogs (4a-c) were active. Compound 4b displayed the strongest effect (IC₅₀: 7.02 μM for promastigotes, 3.4 μM for amastigotes), with low cytotoxicity and high Selectivity Indices. In combination with AmB, compound 4b reduced the effective dose (DRI: 2.87) and increased the therapeutic window. Docking revealed favorable interactions of compound 4b with deubiquitinase DUB16 and tryparedoxin peroxidase I, suggesting enzyme inhibition. ADMET predictions supported good absorption and low toxicity. Conclusions: Compound 4b demonstrated potent and selective antileishmanial activity, synergism with AmB, and predicted safety. These findings highlight chalcone derivative 4b as a promising lead for future preclinical development in VL therapy.

Keywords: acetamides; antileishmanial; flavonoids; synergy.

Scheme 1

Synthetic route to obtain 4-hydroxychalcone derivatives (2a–c, 3a–c, 4a–c). Reagents and reaction conditions: (i) 4-hydroxybenzaldehyde, H₂SO₄, EtOH, 0–25 °C, 24 h; (ii) K₂CO₃, DMF, 80 °C, 6 h.

Figure 1

In vitro antileishmanial activity of AmB, 4a, 4b, and 4c against L. infantum promastigotes (A) after 72 h treatment, and amastigotes (B) after 24 h treatment. The data represent the mean ± standard error of the mean (SEM) and were analyzed by one-way ANOVA, followed by the post hoc Tukey test. * p ≤ 0.05 and ns = non-significant.

Figure 2

In vitro cytotoxicity of AmB, 4a, and 4b against human PBMCs after 24 h-treatment. The data represent the mean ± standard error of the mean (SEM).

Figure 3

Three-dimensional representation of the molecular docking interactions between compound 4b (ligand in cyan) and the target enzymes. (A) shows the highest affinity pose of compound 4b in the binding site of the Leishmania Deubiquitinase, with the binding site surface and key residues highlighted. (B) displays the complex of compound 4b with the Leishmania Tryparedoxin Peroxidase I. In both images, the protein structures are represented as red ribbons, and the amino acid residues as sticks. ARG = arginine; ALA = alanine; GLU = glutamic acid; LEU = leucine; SER = serine.

Figure 4

SI = Selectivity Index. CC₅₀ = 50% Cytotoxic Concentration. IC₅₀ = 50% Inhibitory Concentration. DRI = dose reduction index.