**3. Results and Discussion**

The ethanolic extract of the leaves of *P. pseudoarboreum* was evaluated against promastigote forms of *L. amazonensis*, *L. braziliensis*, *L. guyanensis,* and *L. infantum*. The active EtOH crude extract was further fractionated by liquid–liquid partition to obtain DCM, EtOAc, and H2O fractions, which were assayed for their in vitro activity against the four *Leishmania* strains.

The DCM fraction showed an improved profile compared to the crude extract, displaying IC50 values ranging from 14.7 to 19.1 μg/mL for the four *Leishmania* strains assayed, whereas the EtOAc and H2O fractions showed to be inactive (IC50 > 50 μg/mL). Thus, DCM fraction was further fractionated to yield seven sub-fractions. Sub-fractions F1–F4 showed to be inactive (IC50 > 100 μM), whereas sub-fraction F5 showed some degree of activity on the four *Leishmania* strains (IC50 15.7–20.8 μg/mL) and F7 exhibited only slight potency on *L. amazonensis* and *L. brazilensis.* Moreover, the most active sub-fraction F6 exhibited higher potency than miltefosine, used as the reference drug (IC50 ranging from 2.2 to 3.4 μM vs. 17.7 to 30.7 μM), although showed a slightly low selectivity index taking J774 macrophages as reference mammalian cells (CC50 values ranging from 1.9 to 3.0 vs. 4.4 to 7.7) (Table 1).



*<sup>a</sup>* Fractions and compounds not included in the table were inactive (IC50 > 50 μg/mL and IC50 > 100 μM, respectively). *<sup>b</sup>* IC50: concentration able to inhibit 50% of parasites. The IC50 values of the ethanol extract (EtOH) and dichloromethane (DCM) and F5–F7 fractions are expressed as μg/mL ± standard deviation. The IC50 values of the compounds are expressed as μM ± standard deviation. *<sup>c</sup>* CC50 concentration able to inhibit 50% of murine macrophages. *<sup>d</sup>* SI: selectivity index (CC50/IC50). *<sup>e</sup>* M: miltefosine was used as a positive control.

Therefore, sub-fraction F6 was submitted to multiple chromatographic steps on silica gel and Sephadex LH-20 affording the known alkamides **1**–**6** (Figure 1). Their chemical structures were elucidated on the basis of their spectroscopic data (Supplementary Materials Figures S1–S6) and comparison with data reported in the literature. Thus, the isolated metabolites were identified as sintenpyridone (**1**) [18], (*E*)-demethoxypiplartine (**2**) [19], (*E*)-piplartine (also known as piperlongumine, **3**) [19], (*Z*)-piplartine (**4**) [20], 3,4-epoxy-8,9-dihydropiplartine (**5**) [21], and 10,11-dihydropiperine (**6**) [20].

**Figure 1.** Chemical structures of piperamides (**1**–**6**) isolated from *Piper pseudoarboreum.*

Alkamides **1**–**6** were tested by in vitro assays against the four strains of *Leishmania* promastigotes. The results indicated that alkamides **2** and **3** were 4.7 to 18-fold more potent (IC50 ranging from 1.6 to 3.8 μM) than miltefosine (IC50 ranging from 17.7 to 30.7 μM), and exhibited a selectivity index ranging from 3.0 to 6.6 for all *Leishmania* strains tested (Table 1). Recently, Araújo-Vilges and co-workers reported that (*E*)-piplartine was able to reduce the growth of *L. amazonensis* promastigotes (MHOM/BR/pH8) in a dose-dependent pattern, exhibiting an IC50 value of 179.0 μg/mL [22]. On the other hand, Capello et al. [23] reported that no antileishmanial activity on macrophages infected with *L. (L.) amazonensis* was found for (*E*)-piplartine at 50 μg/mL. We assume that such differences in potency depend to a great extent on the infecting *Leishmania* strain used in the assay and cell culture procedures.

Regarding the influence of the substitution pattern in the alkamide scaffold on the leishmanicidal activity, it seems that α,β-unsaturated carbonyl groups in both, the acyl chain and the lactam ring are critical functionalities for the activity (**2**, **3**, and **4** vs. **1**, **5**, and **6**). Moreover, isomerization of the unsaturated acyl chain leads to slight changes in the activity (**3** vs. **4**). No straightforward conclusion can be drawn from the type of functional group on the aromatic ring.

Based on the in vitro results on promastigote forms, alkamides **2** and **3** were selected to be evaluated on intracellular amastigotes of *L. amazonensis* and *L. infantum.* The results revealed that compounds **2** and **3** exhibited some degree of activity, showing two-fold higher potency on *L. amazonesis* (IC50 9.1 and 8.2 μM, respectively) than on *L. infantum* (IC50 17.1 and 16.1 μM, respectively). Moreover, both compounds exhibited higher activity than miltefosine on both assayed *Leishmania* strains. Furthermore, these compounds were 5- to 6-fold more potent than miltefosine on *L. amazonensis* (Table 2).


**Table 2.** Leishmanicidal activity on amastigote forms of alkamides **2** and **3**.

*<sup>a</sup>* IC50: concentrations able to inhibit 50% of the parasites, and values are expressed as μM ± standard deviation (SD). *<sup>b</sup>* SI: selectivity index (CC50 of murine macrophages/IC50). *<sup>c</sup>* M: Miltefosine was used as a positive control.

Taking into consideration its potency and efficacy on *Leishmania* promastigote and amastigote forms, and although a poor selectivity index, (*E*)-piplartine was selected for in vivo assays to investigate its potential as a lead compound targeting CL since previous toxicological studies indicate a good safety profile in murine models [10]. Previous works report the in vitro evaluation of (*E*)-piplartine against *Leishmania* spp. promastigotes [22,24] and *L. amazonensis* intracellular amastigote forms [25] as well as an in vivo study against *L. donovani* in a hamster model of visceral leishmaniasis [24]. However, to our knowledge, in vivo studies on CL have not been reported.

The in vivo assay in BALB/c mice infected with *L. amazonensis* for CL was performed by treatment of three randomly separated mice groups (8 mice per group). Thirty five days after infection, the treated mice group with (*E*)-piplartine received in the foot lesions (intralesion) a dose of 25 mg/kg/day for 4 days, whereas the treated group with glucantime received by intraperitoneal route 25 mg/kg/day for 4 days. The lesion size footpad was measured four times before infection and treatment, after treatment and at the end of the experiment (days 0, 35, 50, and 100) (Figures 2 and 3). The individual lesion size was calculated from two measurements (differences between the left and the right footpad).

Moreover, with the aim to establish the visceralization of the chronic infection disease, all mice were sacrificed at the end of the experiment to determine parasite burden in spleen by culture on microtiter plates. This in vivo assay indicated that from the day of infection to the day before treatment with (*E*)-piplartine, the progress of the lesion size was similar in the three mice groups (day 35), whereas after the end of treatment (day 50), the mean progress of lesions within groups treated with (*E*)-piplartine and glucantime were reduced by around 35% with respect to the untreated group. At the end of the experiment, both treated groups showed more than 40% reduction in the lesion size and 55% in spleen parasite burden compared to untreated mice group. The intralesion (*E*)-piplartine treatment efficacy was comparable to the intraperitoneal glucantime treatment, with *p*-values of 0.800 and 0.832 for the lesion size and spleen parasite burden, respectively, and significantly higher than the untreated control group, with *p-*values of 0.045 and 0.027, respectively (Table 3).

**Figure 2.** Effect of the treatment with (*E*)-piplartine on chronic cutaneous leishmaniasis in BALB/c mice. (**A**) Graphic representation of footpad measurement: thickness (**left**) and width (**right**). Lesion size is expressed as the difference in size between the infected and contralateral non-infected footpads. (**B**) Representative images of a mouse infected with *Leishmania amazonensis*, at day 0 (**B1**) and at the end of experiment (**B2**).

**Figure 3.** Effectiveness of (*E*)-piplartine in the treatment of chronic cutaneous leishmaniasis. Lesion size was measured four times (day 0, day 35, day 50, and day 100) and expressed as a mean of the group in mm. At end of the experiment, lesion size of (*E*)-piplartine vs. glucantime treated mice groups did not show statistically significant differences with *p-*value > 0.800; untreated control group vs. (*E*)-piplartine and glucantime showed significant differences with *p-*value < 0.000 \*.


**Table 3.** Efficacy of (*E*)-piplartine in the control of the visceralization in chronic Cutaneous Leishmaniasis.

*<sup>a</sup>* Number of parasites per gram in spleen measured on day 100 of infection. *<sup>b</sup>* SEM: Standard error of the mean.

Thus, the results indicated that (*E*)-piplartine was effective in the in vivo assay (Figures 2 and 3, and Table 3) as evidenced by a significant reduction in the lesion size footpad after infection, and in the spleen parasite burden at the end of the experiment (day 100 post-infection). These findings, together with previous safety [22,25] and pharmacokinetic studies [10,26], provide additional experimental evidence of the potential of (*E*)-piplartine as a promising leishmanicidal lead compound.

In this study, the intralesional route for (*E*)-piplartine was chosen in order to develop a prospective formulation for topical administration. This administration route is an attractive alternative for CL, offering significant advantages over systemic therapy with: fewer adverse side effects, easy administration, and low costs [27]. This later point is relevant as in regions with limited resources there are no dispensaries or qualified personnel for intramuscular or intravenous drug administration [28]. In addition, topical formulations can penetrate over the skin to diminish disease progression at the beginning of the infection [29].

Although the mechanism of action of (*E*)-piplartine has not been established on *Leishmania* parasites, previous studies performed on cancer cell lines [12,30,31] have demonstrated that this alkamide is able to inhibit the proliferative process by activation of mitochondrial apoptosis pathways and induction of reactive oxygen species. Considering these studies, the effect of (*E*)-piplartine on *Leishmania* parasites could also be related to the activation of apoptotic events. Moreover, further studies should be undertaken in order to determine the leishmanicidal mechanism of action of this promising lead compound.

#### **4. Conclusions**

The results reported herein reinforce the efficacy of (*E*)-piplartine against neglected tropical diseases caused by *Leishmania* spp., and deserve future investigations for further lead optimization with desired drug-likeness properties for the treatment of leishmaniasis. Furthermore, (*E*)-piplartine is a natural alkamide occurring in several species in the widely distributed *Piper* genus, and therefore, the in vivo studies results support and may validate the traditional uses of some *Piper* species by the indigenous people to treat the symptoms of cutaneous leishmaniasis.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/9/1250/s1, Figures S1–S6: 1H and 13C NMR spectra of compounds **1**–**6**.

**Author Contributions:** Conceptualization, I.A.J. and F.B.-F.; methodology, I.A.J., M.A.D.-A., and J.C.T.; investigation, J.C.T. and P.B.-R.; data curation, M.A.D.-A. and N.F.; writing—the original draft preparation; N.F. and I.A.J.; writing—reviewed and editing, I.L.B.; funding acquisition, I.L.B. and F.B.-F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by RTI2018-094356-B-C21 Spanish MINECO project, cofunded by the European Regional Development Fund (FEDER) and PCI-Iberoamerica A/030160/10 and AP/039767/11 projects from Spanish MAEC-AECID. J.C.T. and P.B.-R. are grateful to Spanish MAEC-AECID for their fellowships.

**Acknowledgments:** The authors would like to thank the botanist Juan Celedonio Ruiz Macedo, Amazonense Herbarium of the Universidad Nacional de la Amazonia Peruana, Iquitos, Perú, for the identification of the plant material.

**Conflicts of Interest:** The authors declare no conflict of interest.
