Hit-to-Lead Studies of Pyrazinylpiperazines against Visceral Leishmaniasis: Pharmacokinetic Profile and In Vivo Efficacy of Potent Compounds against Leishmania infantum.

As part of the hit-to-lead optimization of antileishmanial pyrazinylpiperazines, the Absorption, Distribution, Metabolism, and Excretion (ADME) properties of the initial hit and five candidates for in vivo studies were assessed in vitro. These candidates, which displayed improved potency against Leishmania infantum (IC50 < 5 μM), were selected from an earlier structure-activity relationship study. Such assessment revealed that, except for the catechol derivative 6, all derivatives exhibited an improved overall ADME profile in comparison to the initial hit. The para-hydroxyl analog 2 stood out as the most promising candidate, being the second most potent compound in vitro against the parasite and showing far superior metabolic stability (more than twice as stable as the initial hit) in mouse liver microsomes, together with a reasonable gastrointestinal absorption and a lack of blood-brain barrier permeation. In vivo assessment of the antileishmanial efficacy of 2 in a BALB/c mice model of visceral leishmaniasis revealed a reduction in parasitemia by 91.1 and 90.0% in the spleen and liver, respectively, after a 10 day treatment of infected animals with a 100 mg/kg daily dose of 2, without any acute toxicity or death among mice treated with 2. Flow cytometry demonstrated that the antileishmanial activity of 2 is linked to a cytostatic effect, marked by cell cycle arrest in the G0/G1 phase and enhanced production of reactive oxygen species. Subsequent in silico studies suggested that the activity of the novel antileishmanial pyrazinylpiperazine lead 2 could be due to the inhibition of a nonspecific serine/threonine protein kinase in Leishmania infantum; however, in vitro inhibition assays failed to identify a target for 2 among a set of kinases and other proteins.