2.5.7. In Vivo Experiments

BALB/c mice were infected subcutaneously at the left hand-foot with 1 <sup>×</sup> 107 promastigotes of *L. amazonensis* on day 0. Right hind paw was used as a negative control (no infection). Thirty five days after infection, chronic cutaneous leishmaniasis was developed, and animals were randomly divided into three groups (*n* = 8/group): animals treated with (*E*)-piplartine received in the foot lesions (intralesion) doses of 25 mg/kg/day for 4 days in a 15 μL volume of phosphate saline dilution/propylene glycol (9:1), a group treated with glucantime receiving 25 mg/kg/day for 4 days by intraperitoneal route, and the control group. The measurement of cutaneous lesion was monitored at 0, 35, 50, and 100 days post-infection, using a Vernier calliper to measure footpad size. The number of viable *L. amazonensis* parasites in the spleen of the different groups of mice was estimated using the limiting dilution assay method at the end of the experiment (day 100 post-infection) [16]. Mice were sacrificed, and the spleen were aseptically removed, weighed, and homogenized in Schneider's medium supplemented with 10% FBS. Briefly, serial dilutions were prepared and distributed to 96-well microtiter plates under sterile conditions, and incubated at 26 ◦C. On day 7 post-incubation, wells were analyzed using an inverted microscope. The number of parasites per milligram of tissue was determined based on the tissue weight and the parasite load from the culture dilutions [17].

## 2.5.8. Ethical Consideration

All animals were handled according to the European Union legislation Directive 2010/63/EU and Spanish law Real Decreto 53/2013 on the protection of animals used for scientific purposes. The experimental protocols involving the use of animals were approved by the local ethical committee of the University Complutense of Madrid (CEXAN170415) http://147.96.70.122/Web/Actas/CEXAN170415.pdf.

#### 2.5.9. Statistical Analysis

For in vitro assays, the antileishmanial activity (IC50) and cytotoxic activity (CC50) of compounds were analyzed by Probit test, using SPSS v20.0 software. All results were expressed as means ± standard error of the mean (S.E.M). For in vivo assays, results were analyzed by Shapiro-Wilk's normality test, and then by one-way ANOVA with Tukey's HSD post-hoc test. Significant differences were considered at *p-*value < 0.05, using SPSS v20.0 and Microsoft Excel 2010 software.
