Design, synthesis, structure-activity relationship and mechanism of action studies of a series of 4-chloro-1-phthalazinyl hydrazones as a potent agent against Leishmania braziliensis.

With the aim to identify a potential drug candidate to treat cutaneous leishmaniasis, a series of 1-phthalazinyl hydrazones were synthesized and tested against Leishmania braziliensis parasite, one of the main responsible of this disease in the world. A structure-activity relationship permitted to identify two phthalazines containing nitroheterocyclic moiety 3l and 3m as promising new lead compounds. These compounds showed a significant antileishmanial activity against promastigote form of L. braziliensis, with EC50 values in sub-micromolar and nanomolar ranges. The phthalazine 3l also displayed a selective and excellent activity against the clinically relevant intracellular amastigotes form, with a EC50 value in sub-micromolar range (0.59 µM), without affecting the viability of the host cells. Oxidative stress was identified as the possible mode of action of the most active phthalazine. Considering their significant antileishmanial activity and ease synthesis, the phthalazine containing nitroheterocyclic represents a promising agent against Leishmania braziliensis for the rational design of new leads.

Nuevo método de evaluación del efecto antiparasitario en modelos in vitro e in vivo, a través de la visualización de Trypanosoma cruzi-GFP / New method of evaluation of the antiparasitic effect models in in vitro and in vivo, through the viewing of Trypanosoma cruzi-GFP

Los tratamientos de primera línea para la enfermedad de Chagas generan importantes efectos adversos que acentúan el deterioro de la salud en los pacientes. La necesidad de generar fármacos alternativos ha permitido desarrollar estudios donde se emplean parásitos capaces de expresar una proteína fluorescente, a fin de correlacionar fluorescencia con población de protozoarios. En este sentido, ideamos una metodología para el seguimiento de la proliferación de Trypanosoma cruzi-GFP (Green Fluorescent Protein) en modelos in vitro e in vivo, empleando el equipo iBox- UVP. Los ensayos in vitro se iniciaron con una curva de calibración usando concentraciones entre 5x105 y 5x107 parásitos/mL. Seguidamente, con una curva de proliferación evidenciamos a través de la fluorescencia la susceptibilidad de los parásitos frente a la droga comercial Benznidazol (IC50= 5,3±1,3 μM). En el ensayo in vivo se corroboró cualitativamente el efecto quimioterapéutico del Benznidazol (100 mg/kg/día) en ratones C57BL/6, partiendo de un inóculo de 2,5x105 parásitos, haciendo captura de imágenes de fluorescencia cada dos días a partir del día 1, e inicio del tratamiento por vía oral el sexto día. El coeficiente de correlación cercano a 1 obtenido en la curva de calibración habla de un método de cuantificación parasitario sencillo y robusto; también los ensayos en modelos in vitro e in vivo permitieron monitorear el efecto dosis-dependiente de Benznidazol sobre T. cruzi-GFP. En síntesis, elaboramos una metodología novedosa, rápida, no invasiva y que sigue en tiempo real la respuesta quimioterapéutica de drogas anti-T. cruzi.

The first-line treatments for Chagas disease generate significant adverse effects that accentuate the health deterioration in patients. The need to generate alternative drugs has led to the development of studies in which parasites will express a fluorescent protein, and correlate this expression with protozoan population. We devised a methodology for monitoring the proliferation of Trypanosoma cruzi- GFP (Green Fluorescent Protein) in models in vitro and in vivo, using the equipment iBox-UVP. In vitro assays were initiated with a calibration curve using concentrations between 5x105 and 5x107 parasites/mL. Subsequently, with a proliferation curve, through fluorescence we determined the susceptibility of the parasites against the commercial drug Benznidazol (IC50= 5,3±1,3 μM). In vivo assays corroborated qualitatively the chemotherapeutic effect of Benznidazol (100 mg/kg/day) in C57BL/6 mice, starting from an inoculum of 2.5x105 parasites, making capture of fluorescence imaging every two days from day 1, and starting oral treatment on the sixth day. The correlation coefficient close to 1 obtained in the calibration curve showed that this quantification method of parasites is simple and robust; assays in vitro and in vivo allowed monitoring dose-dependent effects of Benznidazol agains T. cruzi-GFP. We have produced an innovative, rapid, non-invasive method that monitors in real time the chemotherapeutic response of anti-T. cruzi drugs.

Anti-leishmanial evaluation of C2-aryl quinolines: mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis.

A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels-Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.