**1. Introduction**

Leishmaniases are neglected tropical diseases caused by the infection with *Leishmania* parasites, and are transmitted by the bite of a sand fly belonging to the genera *Lutzomyia* and *Phlebotomus*. Leishmaniases are endemic in large areas of the tropics, subtropics, and the Mediterranean basin, and are among the major neglected tropical diseases causing morbidity worldwide. Recently, it has broken out of its traditional boundaries and has been reported in new geographic locations with atypical disease manifestations involving novel parasite variants. Cutaneous leishmaniasis (CL) is endemic in more than 70 countries, with an estimated annual incidence of 1.5–2 million new

cases, and clinical manifestations ranging from small skin nodules to massive destruction of the mucous tissues. CL is mainly caused by *Leishmania major* in the Old World and by *L. amazonensis* and *L. braziliensis* in the New World, specifically in Brazil [1]. In spite of the high prevalence, and advances in the chemotherapy for leishmaniasis, the current available drugs, including pentavalent antimonials, amphotericin B, miltefosine, paromomycin, and pentamidine are compromised by the emergence of resistance, variable sensitivity between species, adverse side effects, requirements for long courses of administration, and high cost [2]. These drawbacks and the absence of vaccines underline the urgent need for searching alternative treatments with acceptable efficacy and safety profile.

Natural products are an important source of leishmanicidal drugs owing to their accessibility, structural diversity, low cost, and possible rapid biodegradation [3–5]. In South America, where resorting to medicinal plants represents a primary health care measure of the native population, several species of *Piper* genus are widely used as a remedy to relieve the symptoms of leishmaniasis disease. Thus, the leaves of *Piper aduncum*, *P. loretoanum,* and *P. hispidum* are used as poultices for healing wounds and to treat the symptoms of CL [6,7]. In addition, *Piper* species are used as culinary spices, and as a food preservative to control food spoilage and pathogenic microorganisms. In particular, *P. nigrum* (black pepper) is worldwide popular as a flavoring for food [8]. Phytochemical investigations of *Piper* species have reported numerous metabolites with ecological and medicinal properties, including amides, pyrones, lignanes, terpenes, and flavonoids [8]. Alkamides, also named piperamides, are characteristic bioactive constituents in *Piper* species [9]. In particular, (*E*)-piplartine, also called piperlongumine, is the major natural alkaloid from *P. longum* and *P. tuberculatum*, and in vitro and in vivo studies have demonstrated its promising pharmacological properties such as antioxidant, anxiolytic, anti-atherosclerosis, antidiabetic, and antiparasitic against neglected tropical diseases [10]. Moreover, (*E*)-piplartine is reported to kill a large variety of cancer cells while remaining nontoxic to normal cells, highlighting its therapeutic potential [11,12].

In previous investigations, we reported the isolation of an unprecedented chlorine-containing piperamide along with several known compounds and their antileihmanicidal activity from *Piper pseudoarboreum* [13]. In continuous research toward the discovery of natural occurring leihmanicidal agents, we report herein on the isolation and structure elucidation of six known alkamides from the leaves of *P. pseudoarboreum* Yunker through a bioassay-guided fractionation carried out against four promastigote strains of *Leishmania*. Compounds **2** and **3** were further evaluated on intracellular amastigotes of *L amazonensis* and *L. infantum*. (*E*)-piplartine (**3**) was selected to be assayed in an in vivo model for cutaneous leishmaniasis.

#### **2. Materials and Methods**

## *2.1. General Experimental Procedures*

The structure of the isolated compounds were elucidated using spectrometric and spectroscopic methods, and comparison with data previously reported. The Nuclear Magnetic Resonance (NMR) experiments were recorded on Bruker Avance 400 and 500 spectrometers (Bruker Co. Billerica, MA, USA); chemical shifts were referred to the residual solvent signal (CDCl3: δ<sup>H</sup> 7.26, δ<sup>C</sup> 77.36) (acetone *d*6: δ<sup>H</sup> 2.09, δ<sup>C</sup> 30.60 and 205.87), using trimethylsilane (TMS) as internal standard. Electron Impact Mass Spectrometry (EIMS) and High Resolution Electron Impact Mass Spectrometry (HREIMS) were recorded on a Micromass Autospec spectrometer (Micromass, Manchester, UK). Silica gel 60 (15–40 mm) and silica gel 60 F254 for column chromatography and Thin Layer Chromatography (TLC), respectively, were purchased from Panreac (Barcelona, Spain). Sephadex LH-20 was obtained from Pharmacia Biotech (Pharmacia, Uppsala, Sweden). Centrifugal planar chromatography was carried out in a Chromatotron instrument (model 7924T, Harrison Research Inc., Palo Alto, CA, USA) on manually coated silica gel 60 GF254 (Merck, Darmstadt, Germany) using 4-mm plates. The spots were visualized by UV light and heating silica gel plates sprayed with H2O-H2SO4-AcOH (1:4:20).

## *2.2. Chemicals and Reagents*

All solvents used were of analytical grade and purchased from Panreac (Barcelona, Spain). (*E*)-Piplartine, Scheneider's insect medium, RPMI-1640, fetal bovine serum (FBS), 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES), resazurin sodium salt, and sodium dodecyl sulphate (Sigma-Aldrich, St Louis, MO, USA), L-glutamine (Avantor Performance Material Inc., PA, USA), trypsin (Merck, Darmstadt, Germany), penicillin Penilevel® 100.000 U.I. (ERN laboratories, Barcelona, Spain), streptomycin sulphate (Reig Jofré laboratories, Barcelona, Spain), and Glucantime® (Merial Laboratories, Barcelona, Spain).

### *2.3. Plant Material*

Leaves of *Piper pseudoarboreum* Yunck. were collected in November 2009 at Iquitos, Maynas Province, Department of Loreto, Perú. The plant material was identified by the botanist Juan Celedonio Ruiz Macedo, and a voucher specimen (AMZ 11114) was deposited at the Amazonense Herbarium of the Universidad Nacional de la Amazonia Peruana, Iquitos, Perú.

## *2.4. Extraction, Bioassay-Guided Fractionation and Isolation*

The dried leaves of *P. pseudoarboreum* (200.3 g) were powdered and extracted in a Soxhlet apparatus with 5 L of 96% ethanol. The solvent was evaporated to give 42.9 g (21.4%) of extract. The ethanolic extract (EtOH) was partitioned into dichlorometane (DCM), ethyl acetate (EtOAc), and water (H2O). After removing the organic solvents under reduced pressure, the DCM (9.2 g, 4.6%) and EtOAc (1.2 g, 0.6%) fractions were obtained, whereas the aqueous-soluble extract was lyophilized providing the H2O fraction (8.9 g, 4.5%). The most active organic fraction (DCM, 9.2 g) was chromatographed over silica gel column eluting with mixtures of hexanes-EtOAc (10:0 to 0:10, 1 L each one) to obtain seven sub-fractions (F1–F7). The most active fraction, F6 (1.5 g), was subjected to column chromatography over Sephadex LH-20 by isocratic elution (MeOH-CHCl3, 1:1) to afford fifteen sub-fractions, which were combined based on their TLC profiles (F6A to F6F). Preliminary nuclear magnetic resonance (NMR) studies revealed that sub-fraction F6B was rich in aromatic alkamides, and were further investigated. Thus, F6B (448.1 mg) was chromatographed by centrifugal planar chromatography on 4-mm silica gel plates, using mixtures of hexanes-EtOAc (60:40 to 50:40) as eluent to give eleven sub-fractions (F6B1 to F6B11). Sub-fraction F6B2 (21.5 mg) was further purified on silica gel by preparative TLC (3 × development, hexanes-2-propanol, 8:2) to give compounds **1** (1.7 mg) and **5** (1.4 mg). Purification of sub-fraction F6B4 (18.3 mg) by preparative TLC (2 × development, CH2Cl2-Et2O, 95:5) yielded compounds **3** (11.4 mg) and **4** (2.2 mg), whereas sub-fraction F6B7 (23.8 mg) gave compounds **2** (19.8 mg) and **6** (0.9 mg) after purification by preparative TLC (2 × development, hexanes-2-propanol, 8:2). The compounds were identified by NMR spectroscopy and comparison with data reported in the literature.

#### *2.5. Biological Studies*

#### 2.5.1. Parasites

Autochthonous isolates of *Leishmania infantum* (MCAN/ES/92/BCN83) were obtained from an asymptomatic dog from the Priorat region (Catalunya, Spain), and kindly provided by Prof. Montserrat Portús (University of Barcelona). *L. braziliensis* (2903), *L. amazonensis* (MHOM/Br/79/Maria) and *L. guyanensis* (141/93) were kindly given by Prof. Alfredo Toraño (Instituto de Salud Carlos III, Madrid).
