Design, Synthesis, In Vitro and In Silico Biological Evaluation of New Pyridine-2,5-Dicarboxylates Esters Bearing Natural Source Fragments as Anti-Trypanosomatid Agents

<b>Background</b>: Chagas disease and leishmaniasis remain public health concerns. Despite the existence of approved medications for the treatment of these diseases, most patients discontinue treatment due to long drug regimens and/or the severe side effects of these drugs. This leads to treatment failure and potential future drug resistance. Therefore, the search for new molecules with trypanocidal activity, low cytotoxicity, and high selectivity is essential to address this challenge. <b>Methods</b>: In this work, three series (a, b, and c) of pyridine-2,5-dicarboxylate esters were synthesized using different β-keto-esters bearing naturally occurring fragments and 1,2,3-triazine-1-oxides via the inverse electron demand Diels-Alder (IEDDA) reaction. The structural elucidation of the compounds was performed using NMR (¹H and ¹³C) and HRMS, and the crystal structure of compound <b>6a</b> was also obtained. Furthermore, a biological assay was performed for all synthesized and characterized compounds to determine their cytotoxicity against <i>Trypanosoma cruzi</i>, <i>Leishmania mexicana</i>, and the J774.2 macrophage cell line. Finally, the in silico determination of their pharmacokinetic and toxicological properties was performed using the SwissADME and ProTox 3.0 platforms. <b>Results</b>: Compounds <b>3a</b>, <b>4a</b>, <b>5a</b>, <b>4b</b>, and <b>8c</b> had the highest anti-<i>Trypanosoma cruzi</i> activity against both strains (IC₅₀ ≤ 56.68 µM). Compounds <b>8b</b>, <b>10a</b>, <b>9b</b>, and <b>12b</b> had considerable leishmanicidal activity against <i>Leishmania mexicana</i> against both strains (IC₅₀ ≤ 161.53 µM). Furthermore, in silico prediction of ADMET properties suggest that these pyridine compounds possess good pharmacokinetic profile. The results are also consistent with low in vitro cytotoxicity and high selectivity. <b>Conclusions</b>: The synthesized pyridine-2,5-dicarboxylate esters have promising activity against <i>Trypanosoma cruzi</i> and <i>Leishmania mexicana</i>, with low cytotoxicity and good drug-like properties, suggesting that these compounds are potential candidates for further evaluation as new treatments for Chagas disease and leishmaniasis.