Nucleoside Analogue with Thymidine Nucleobase Inhibits <i>Leishmania infantum</i> and Depolarizes the Plasma Membrane Potential In Vitro

Visceral leishmaniasis, caused by the protozoan parasites <i>Leishmania infantum</i> or <i>L. donovani</i>, remains a lethal neglected tropical disease without effective therapy. Current drugs available are toxic, leading to severe side effects, treatment abandonment, and resistance, underscoring the urgent need for new therapeutic options. As <i>Leishmania</i> spp<i>.</i> are auxotrophic for purines, they rely on the purine salvage pathway for their nucleotide biosynthesis. Consequently, nucleoside analogues (NAs) represent a promising class of compounds for the design of antiparasitic agents. This study aimed to investigate the in vitro antileishmanial activity of NA compounds and the cellular alterations induced by treatment. Twelve NA compounds were screened for antileishmanial activity against promastigote and amastigote forms of <i>L. infantum</i> and cytotoxic effects in mammalian cells. Among all compounds evaluated, six of them demonstrated antipromastigote activity, exhibiting 50% effective concentration (EC₅₀) values ranging from 9.29 to 76.74 μM. As for amastigote activity, two compounds, <b>5</b> and <b>10</b>, were effective, with values of EC₅₀ of 8.02 and 31.95 μM, respectively. Only compound <b>1</b> maintained cellular viability at the maximum concentration tested (>200 μM). The selective index for the derivatives investigated ranges from 0.5 to 4.2. Compound <b>5</b>, a tritylated thymidine NA, the most active, was further subjected to analysis of cellular alterations using fluorescent-based approaches. This analogue demonstrated a lack of cytotoxicity against murine peritoneal macrophages up to 50 μM and nonhemolytic activity up to 100 μM. When applied at EC₅₀, it did not cause damage to plasma membrane permeability, the integrity of the genetic material, acidocalcisomes, intracellular Ca²⁺, and ROS levels of treated <i>Leishmania</i> parasites. However, it caused depolarization of the plasma membrane potential, leading to cell death. Further studies are also necessary to understand the enzymatic action of this most active compound, and optimization is required to develop more effective and safer antileishmanial lead compounds. In conclusion, compound <b>5</b> might be a suitable candidate for the development of antileishmanial agents.