Mechanism Insight of Cell Death Signaling by Thymol Derivatives on Trypanosomatidae Protozoan Parasites

Leishmaniasis and Chagas disease are parasitic diseases considered to be among the most important neglected diseases, with implications for both developed and developing countries. Currently, there are no effective therapeutic treatments for these diseases due to challenges in drug administration, high toxicity, high costs, and drug resistance. In this study, a series of eleven thymol derivatives were designed, synthesized, and evaluated for their in vitro kinetoplastid activity against <i>Leishmania amazonensis</i> and <i>Trypanosoma cruzi</i>, as well as their cytotoxicity against a murine macrophage cell line. The most active compounds, thymol anysoate (<b>9</b>) and thymol picolinate (<b>10</b>), displayed the highest kinetoplastid activity with IC₅₀ values of 22.87 and 25.16 µM against <i>L. amazonensis</i> and <i>T. cruzi</i>, respectively. Notably, both compounds demonstrated an excellent selectivity index against the mammal cell line. Structure-activity relationship studies revealed that the ester group plays a crucial role in activity. The most promising derivatives, <b>9</b> and <b>10</b>, activate autophagy and apoptosis-like processes in the treated cells. Atomic force microscopy observations showed that derivative <b>9</b> induces the formation of cytoplasmic vacuoles, indicating an autophagic process, and drug-likeness analysis revealed that it meets all the pharmacokinetic criteria. Overall, these results highlight derivative <b>9</b> as a potential lead compound for the development of new drugs for the treatment of Trypanosomatidae infections and warrants further studies to elucidate the cell death cascade involved.