Design, Synthesis, and Trypanosomicidal Evaluation of Eugenol-Based Azole Hybrids: Discovery of an <i>In Vitro</i> Active and Selective Compound

Chagas disease, caused by <i>Trypanosoma cruzi</i>, remains a major public health concern in Latin America and beyond, with limited treatment options and high morbidity in chronic stages. Currently, benznidazole remains the first-line therapy for Chagas disease but exhibits considerable limitations, including dose-dependent toxicity and poor efficacy in long-term infections. In response to the pressing need for safer and more effective therapies, this study reports the design, synthesis, and biological evaluation of 17 novel eugenol-based hybrid compounds (<b>18</b>-<b>34</b>), strategically constructed by integrating pharmacophoric features from nitroaromatic trypanosomicidal agents, azole-based CYP51 inhibitors, and the phenylpropanoid core of eugenol. The synthetic sequence included key steps such as nitration, <i>O</i>-alkylation, epoxidation, nucleophilic epoxide opening, and CuAAC reactions to afford 1,2,4-triazole-, imidazole-, and 1,2,3-triazole-containing hybrids. All compounds were structurally characterized by IR, HRMS, and NMR spectroscopy. Biological assays were performed against both trypomastigote and amastigote forms of <i>T. cruzi</i> (Y strain), revealing compound-dependent antiparasitic profiles. Hybrid <b>29</b>, bearing benzyl and nitro substituents on an imidazole ring, emerged as the most active candidate (EC₅₀ = 26.5 μmol·L^-1) with a selectivity index exceeding 49. Enantioseparation by chiral HPLC enabled the isolation of <b>(+)-29</b> and <b>(-)-29</b> enantiomers, both of which showed comparable activity, indicating no significant stereoselectivity. Cytotoxicity assays in mammalian cell lines (Vero and H9c2) confirmed low toxicity for most hybrids (CC₅₀ > 1300 μmol·L^-1). Although the compounds were inactive against intracellular amastigotes, the observed trypomastigote-selective activityparticularly of nitrobenzylated derivatives suggests a non-CYP51-related mechanism of action. These findings highlight the value of phenylpropanoid-based molecular hybridization as a promising strategy for developing new antiparasitic agents against <i>T. cruzi</i>.