2.5.2. Cells

J774 murine macrophages were grown and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, penicillin G (100 U/mL), and streptomycin (100 μg/mL) at 37 ◦C and 5% CO2 air atmosphere.

### 2.5.3. Animals

Male BALB/c mice of 20–25 g body weigh were purchased from Harlan Interfauna Ibérica (Barcelona, Spain). All rodents were housed in plastic cages in a 12 h dark–light cycle under controlled temperature (25 ◦C) and humidity (70%) conditions. During the study, animals had unrestricted access to food and water.

#### 2.5.4. In Vitro Promastigotes Susceptibility Assay

In vitro antileishmanial assay was performed using a method described elsewhere [14]. Briefly, promastigotes were grown in vitro in a Schneider's insect medium supplemented with 20% heat-inactivated FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 26 ◦C in 25 mL in tissue culture flasks, and were cultured in 96-well plastic plates (2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> parasites/well). Compounds dissolved in dimethylsulfoxide 1% (DMSO) at the suitable concentration to be tested in serial dilutions (a first screening using 100 μg/mL, and then 100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 μg/mL) to get a final volume of 200 μL were added to each well. After an incubation of 48 h at 26 ◦C, 20 μL of 2.5 mM resazurin solution was added. Plates were then analyzed by fluorescence emission (535ex–590em nm) using a fluorometer Infinite 200 (Tecan i-Control, Tecan Group Ltd, Männedorf, Switzerland). All tests were carried out in triplicate, and miltefosine was used as the reference drug. The antileishmanial activity of each compound was estimated by calculating the GI% (percentage of growth inhibition) and then the IC50 value (concentration of the compound that produced a 50% reduction in parasites).

#### 2.5.5. Cytotoxicity Assay

The cytotoxicity assay of the tested compounds was performed according to a previously described method [14]. Briefly, J774 macrophages (5 <sup>×</sup> 104 cells/well) were placed in 96-well flat-bottom plates with 100 μL of RPMI-1640 medium, and allowed to attach at 37 ◦C and 5% CO2 for 2 h. Afterwards, 100 μL of RPMI-1640 medium containing the test compound in varying concentrations (100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μg/mL) were added to the cells and incubated for another 48 h. Growth controls and signal-to-noise were included. Following the aforementioned incubation time, 20 μL of 2.5 mM resazurin solution in PBS was added, and the plates were placed again in the incubator for another 3 h to evaluate cell viability. The ability of cells to reduce resazurin was determined by fluorometry as in the promastigote assay. Each concentration was assayed in triplicate. Cytotoxicity was expressed as the 50% reduction of cell viability of treated culture cells with respect to untreated culture (CC50).

#### 2.5.6. In Vitro Amastigote Assay

The effectiveness against intracellular amastigotes was evaluated using a fluorometric method described elsewhere [15]. Briefly, macrophages (5 <sup>×</sup> 104 cells) and stationary *Leishmania* promastigotes in a ratio of 1:10 (macrophage/parasite) were seeded in each well of a microtiter plate, suspended in 200 μL of culture medium and incubated at 33 ◦C and 5% CO2 for 24 h. After this incubation time, the temperature was increased up to 37 ◦C for another 24 h. Cells were washed with medium several times in order to remove free non-infective promastigotes, and the supernatant was replaced by 200 μL/well of culture medium containing two-fold serial dilutions of the test compounds (ranging from 5 to 0.038 μg/mL) and the reference drug (ranging from 50 to 0.38 μg/mL). The culture medium was removed carefully to be replaced by 200 μL/well of the lysis solution (RPMI-1640 with 0.048% HEPES and 0.006% sodium dodecyl sulfate (SDS)) and incubated at room temperature for 20 min. Thereafter, the plates were centrifuged at 3500× *g* for 5 min and the lysis solution was replaced by 200 μL/well of Schneider's insect medium. The culture plates were incubated at 26 ◦C for another 3 days for the transformation of viable promastigotes into amastigotes. Afterwards, 20 μL/well of 2.5 mM resazurin was added and incubated for 3 h. Plates were analyzed by fluorescence emission,

and IC50 was determined as described above. All tests were carried out in triplicate. Miltefosine was used as reference drug and was evaluated at the same conditions.
