Antileishmanial activity of amides from Piper amalago and synthetic analogs

Antileishmanial activity of amides from Piper amalago and synthetic analogs

Vanessa da Silva Carrara^I; Edézio Ferreira Cunha-Júnior^II; Eduardo Caio Torres-Santos^II; Arlene Gonçalves Corrêa^III; Júlia L. Monteiro^III; Izabel Galhardo Demarchi^IV; Maria Valdrinez Campana Lonardoni^IV; Diógenes Aparício Garcia Cortez^I,*

^IDepartamento de Farmácia, Universidade Estadual de Maringá, Brazil

^IILaboratório de Bioquímica de Tripanossomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Brazil

^IIIDepartamento de Química, Universidade Federal de São Carlos, Brazil

^IVDepartamento de Análises Clínicas, Universidade Estadual de Maringá, Brazil

ABSTRACT

Two natural amides isolated from the chloroform extract of Piper amalago L., Piperaceae, leaves, a hydrogenated derivative and seven synthetic analogs were tested against the promastigote and intracellular amastigote forms of Leishmania amazonensis. The antileishmanial activity was evaluated in terms of growth inhibitory concentration for 50% of protozoa (IC50). The cytotoxicity toward the J774A1 macrophages was evaluated in terms of the cytotoxic concentrations for 50% of macrophages (CC50). The ability to induce nitric oxide production was also investigated for all compounds. The saturated amide 7-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-1-heptanone was obtained by hydrogenation of the natural compound N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine. Synthetic amides were prepared by addition of the appropriate amine to the corresponding acyl chloride. The natural compound, N-[7-(3',4'-methylenedioxyphenyl)-2(E),4(E)-heptadienoyl]pyrrolidine, was the most active of all tested compounds against the promastigote and intracellular amastigote forms with IC50 values of 15 µM and 14.5 µM, respectively. None of the compounds modulated the production of nitric oxide.

Keywords: amides, antileishmanial activity, Piper amalago, synthetic analogs

Introduction

Leishmaniases are a group of diseases caused by several intracellular protozoan parasites belonging to the genus Leishmania. Studies report that around 350 million women, men and children are at the risk in 88 tropical and subtropical countries (Singh & Sivakumar, 2004). The therapy in most countries is still restricted to drugs, which are painful, toxic, have a high cost and are administrated by intravenous injection. New alkylphosphocholine derivatives with amides have shown antileishmanial activity (Obando et al., 2007). Therefore, all these facts reveal the urgency associated with need for the development of new cheaper and safer drugs. In this context, plants have been researched as sources of new compounds with potential activity and lower toxicity (Bero et al., 2011; Brenzan et al., 2012).

Species of the genus Piper L. (Piperaceae), such as, P. chaba, P. claussenianum, P. longum, P. sanguineispicum and P. tuberculatum, have shown antileishmanial activity. These plants have been studied chemically and the following compounds have been identified: propanoic acid, esters, lignans, terpenes and amides (Cabanillas et al., 2010; Ferreira et al., 2010; Marques et al., 2011; Ghosal et al., 2012; Naz et al., 2012).

Piper amalago L. has been used in folk medicine as an anti-inflammatory, analgesic, antipyretic, therapy for stomach problems, and vermifuge. The phytochemical composition of P. amalago L. roots consists mainly of sesquiterpenes, pyrrolidines and isobutylamides (Heckel, 1897; Achenbach et al., 1984; 1986; Domínguez & Alcorn, 1985; Domínguez et al., 1986). The supercritical extract of the leaves rich in amide 1 showed significant antileishmanial activity (Carrara et al., 2012).

The purpose of this study was to screen the amides isolated from the leaves of P. amalago, a hydrogenated derivative, and synthetic analogs and evaluate the activity of these compounds against L. amazonensis. The derivative and synthetic analogs were synthesized with the aim of investigating whether they could significantly improve the antileishmanial activity, as has already been demonstrated for their antifungal and insecticidal activity (Pagnocca et al., 2006; Sangwan et al., 2008; Castral et al., 2011). All compounds were analyzed in terms of the activity against the promastigote and amastigote forms of the protozoa, the cytotoxicity toward the macrophages, and the ability to induce nitric oxide production.

Materials and Methods

General experimental procedures

Compounds 1-3 were identified on a mass spectrometer (EI) QP DSQ II (Thermo Electron Corporation^®, USA). ¹H and ¹³C NMR were recorded on a Varian Gemini 2000 BB (300 and 75 MHz, respectively) (Varian^®, USA). Column chromatography was performed to purify the compounds, using silica gel 60 (70-230 and 230-430 mesh).

For the synthesis of compounds 4-10, unless otherwise noted, all commercially available reagents were purchased from Aldrich^® Chemical Co. Reagents and solvents were purified when necessary according to the usual procedures described in the literature. 1H and ¹³C NMR spectra were recorded on a Bruker Avance^® III spectrometer (400 and 100 MHz respectively). The IR spectra refer to films and were measured on a Bomem^® M102 spectrometer. Mass Spectra were recorded on a Shimadzu^® GCMS-QP5000. Analytical thin-layer chromatography was performed on a 0.25 µm film of silica gel containing fluorescent indicator UV₂₅₄ supported on an aluminum sheet (Sigma-Aldrich^®). Flash column chromatography was performed using silica gel (Kieselgel 60, 230-400 mesh, E. Merck^®). Gas chromatography was performed with a Shimadzu^® GC-17A with H₂ as carrier and using a DB-5 column. Melting points were performed in Microquimica^® MQAPF - 301. Reactions were irradiated in a focused microwave oven CEM Discover.

Plant material

Leaves of Piper amalago L., Piperaceae, were collected in the forestry garden belonging to "Dr. Luís Teixeira Mendes" in Maringá, Paraná, Brazil. The voucher specimen is deposited in the Herbarium of Universidade Estadual de Maringá (number HUEM 9885).

Extraction and isolation

The isolation of the compounds N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine (1) and N-[7-(3',4'-methylenedioxyphenyl)-2(E),4(E)-heptadienoyl]pyrrolidine (2) from the leaves of P. amalago and their spectral data were described in a previous study (Carrara et al., 2012).

Hydrogenation product of 1

Considering that compound 1 was the major compound isolated, it was chosen for preparing the derivative. A mixture of the methanol solution of compound 1 (36 mg) and 5% Pd/C (10 mg) was hydrogenated at 40 psi for 4 h and then filtered, washed with methanol (5 x 10 mL) and concentrated in a rotary evaporator to give compound 3 (22 mg) in a yield of 95.3% (Sangwan et al., 2008). This compound was identified by EIMS, ¹H and ¹³C NMR spectral data.

7-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-1-heptanone (3): ¹H NMR (300 MHz-CDCl₃) δ: 6.71 (d, J = 7.8 Hz, H5'; 1H); 6.66 (d, J = 1.8 Hz, H2'; 1H); 6.60 (dd, J = 1.4, 7.9 Hz, H6'; 1H); 5.90 (s, H1"'; 2H); 3.46 (t, J = 6.6 Hz, H1"; 2H); 3.39 (t, J = 6.6 Hz, H4"; 2H); 2.51 (t, J = 7.8 Hz, H7; 2H); 2.25 (t, J = 7.8 Hz, H2; 2H); 1.92-1.99 (m, H2"; 2H); 1.79-1.90 (m, H3"; 2H); 1.64 (t, J = 7.5 Hz, H3, 2H); 1.57 (t, J = 7.5 Hz, H6; 2H); 1.23-1.41 (m, H4, H5; 4H). ¹³C NMR (75 MHz-CDCl₃) δ: 24.55 (C3"); 25.01 (C3); 26.25 (C2"); 29.07 (C4); 29.47 (C5); 31.73 (C6); 34.93 (C2); 35.74 (C7); 45.83 (C1"); 46.84 (C4"); 100.82 (C1"'); 108.15 (C2'); 108.98 (C5'); 121.17 (C6'); 136.78 (C1'); 145.51 (C3'); 147.56 (C4'); 172.17 (C1). EIMS: m/z:304 (6); 303 (M⁺, 36); 168 (30); 147 (10); 126 (58); 135 (32); 113 (100); 112 (12); 72 (10); 71 (15); 70 (24); 55 (16).

Synthetic procedures

Analogs 4-6

Compounds N-[3-(3',4'-methylenedioxyphenyl)-2-(E)-propenoyl]pyrrolidine (4), N-[3-(3',4'-methylenedioxyphenyl)-2-(E)-propenoyl]benzylamide (5) and N-[3-(3',4'-methylenedioxyphenyl)-2-(E)-propenoyl]piperidine (6) were prepared as described by Corrêa and workers (Pagnocca et al., 2006).

Analogs 7-10

Appropriate amounts of cinnamic acid (4.5 g; 23.4 mmol) and thionyl chloride (8 mL) were added to a 50 mL round-bottomed flask under nitrogen atmosphere, equipped with a magnetic stir bar and a condenser. The system was maintained at 50 ºC for 4 h, and anhydrous hexane (30 mL) was then added and the solvent was removed in a rotary evaporator. The reaction afforded the corresponding cinnamoyl chloride as a solid. This compound was diluted in anhydrous dichloromethane (36 mL) under nitrogen atmosphere and the appropriate amine (25.7 mmol) was added in order to obtain the corresponding amide. A saturated solution of sodium bicarbonate (3 mL) was added after 12 h, and the extraction was carried out with dichloromethane (3 x 3 mL). The organic phase was washed with distilled water (2 mL) followed by brine (2 mL), and dried with anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography (230-400 mesh) using hexane-ethyl acetate (1:2 v/v) as the eluent. Compounds 7-10 were obtained in 64, 93, 54 and 52% yield, respectively. The amides were analyzed through IR, EIMS, ¹H and ¹³C NMR spectral data and by comparison with data available in the literature (Castral et al., 2011).

N-[3-(2'-fluorophenyl)-2-(E)-propenoyl]piperidine (7): ¹H NMR (200 MHz-CDCl₃) δ: 1.54-1.75 (m, H2", H3", H4"; 6H); 3.58-3.67 (m, H1", H5"; 4H); 7.04 (d, J = 16 Hz, H2; 1H); 7.13-7.50 (m, H3', H5', H6'; 4H); 7.69 (d, J = 16 Hz, H4'; 1H). ¹³C NMR (100 MHz-CDCl3) δ: 24.67 (C3"); 25.62 (C2"); 26.76 (C4"); 43.37 (C1"); 47.05 (C5"); 116.00 (C3'); 116.22 (C2); 121.00 (C1'); 123.48 (C5'); 124.33 (C6'); 129.63 (C4'); 135.08 (C3); 160.01 (C2'); 165.34 (C1). EIMS: m/z:233(M⁺, 59), 149 (100), 138 (48), 121 (46), 101 (62), 84 (30), 75 (14). IR (v_max, film): 2937, 2854, 1645, 1610, 1487, 1440, 1274, 1218, 1137, 1018, 756 cm^-1.

N-[3-(4'-fluorophenyl)-2-(E)-propenoyl]piperidine (8): ¹H NMR (400 MHz-CDCl₃) δ: 1.63-1.86 (m, H2", H3", H4"; 6H); 3.57-3.61 (m, H1", H5"; 4H); 6.80 (d, J = 16 Hz, H2, 1H); 6.99-7.07 (m, H2', H6'; 2H); 7.44-7.54 (m, H3, H3', H5'; 3H). ¹³C NMR (100 MHz-CDCl3) δ: 25.55 (C3"); 26.88 (C2"); 27.92 (C4"); 44.65 (C1", C5"); 116.62, 116.91 (C2); 118.52, 118.55 (C3', C5'); 131.01, 131.12 (C2', C6'); 133.05, 133.10 (C1'); 142.55 (C3); 167.47, 163.34 (C4'); 166.64 (C1). EIMS: m/z:233(M+, 48), 149 (100), 138 (29), 121 (50), 101 (44), 84 (48). IR (v_max, film): 3041, 2935, 2850, 1647, 1598, 1510, 1442, 1280, 1222, 1162, 101, 993, 833, 511 cm-1.

N-[3-(4'-difluoromethoxyphenyl)-2-(E)-propenoyl]piperidine (9): ¹H NMR (200 MHz, CDCl₃) δ: 1.59-1.77 (m, H2", H3", H4"; 6H); 3.56-3.84 (m, H1", H5"; 4H); 6.51 (t, J = 16 Hz, CHF₂O; 1H); 6.83 (d, J = 16 Hz, H2; 1H); 7.09 (d, J = 4 Hz, H3', H5'; 2H); 7.50 (d, J = 4, H2', H6'; 2H); 7.59 (d, J = 8 Hz, H3, 1H). ¹³C NMR (100 MHz, CDCl₃): 24.61 (C3"); 26.73 (C4"); 43.34 (C1"); 47.01 (C5"); 113.07; 115.65 (C2) 117.96; 119.59 (C3',C5'); 129.12 (C1'); 140.74 (C3); 151.79 (CHF₂O); 165.07 (C1). EIMS m/z:281 (M⁺), 279, 197 (100), 169, 138, 119, 84. IR(v_max, film): 2920, 2856, 2362, 2345, 1647, 1604, 1514, 1446, 1243, 1216, 1116, 1037, 981, 823 cm^-1.

N-[3-(4'-trifluoromethoxyphenyl)-2-(E)-propenoyl]piperidine (10): Compound 10 was prepared as described by Corrêa and workers (Castral et al., 2011).

Determination of the antileishmanial activity

Parasites

A strain of Leishmania amazonensis (MHOM/BR/77/LTB0016) was used. Parasites isolated from infected mice were maintained as promastigotes through weekly passages in Schneider medium (Sigma^®) supplemented with 10% of fetal bovine serum, penicillin (100 UI/mL), and streptomycin (100 µg/mL) at 26 ºC.

Cells

J774A1 macrophages were cultured in RPMI medium (pH 7.2) supplemented with 10% of fetal bovine serum, and incubated at 37 ºC under an atmosphere of 5% CO₂. Macrophage cultures were maintained by passages every two or three days, according to ATCC.

Animals

Male BALB/c mice (25-30 g) were kept in a 12 h light/dark cycle in a temperature-controlled room with free access to water and food. The study reported in this manuscript was carried out in accordance with the Fiocruz Ethical Committee on Animal Use (CEUA-Fiocruz protocol number LW-7/10).

Stock solutions

Stock solutions of 10 mM of the compounds (1-10) were prepared in dimethyl sulfoxide (DMSO) (Sigma^®, Sant Louis, USA). Assay concentrations were prepared with culture medium used in the experiments, as indicated in each case.

Antileishmanial activity against promastigotes

The method used to evaluate the anti-promastigote activity was adapted from Denizot & Lang (1986). Promastigotes were adjusted to a concentration of 1 x 10⁶ cells/mL in Schneider medium (supplemented with 10% of fetal bovine serum, penicillin (100 UI/mL), and streptomycin (100 µg/mL). Parasites were incubated with the compounds (3.125-200 µM) (0.5% DMSO was used to dissolve the highest concentration of the samples) at 26 ºC for 72 h. Promastigotes in culture medium supplemented without test compounds, were used as the negative control. Pentamidine isotionate (0.3125-20 µM) was used as the reference. The antileishmanial activity was evaluated by adding 22 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] at 5 mg/mL (Sigma^®, Sant Louis, USA) to each well. After 2 h, 80 µL of DMSO was added and the optical density was determined at a wavelength of 570 nm in a microplate reader (µQuant Bio-Tek Instruments^®, Winooski). The assays were carried out in triplicate in 96-well plates (Costar^®, New York, USA). The inhibition percentage was estimated by the comparison with the negative control. Logarithm regression analysis was performed in order to obtain IC50 values (concentrations that inhibit the growth of promastigotes by 50%).

Cytotoxicity evaluation

The cytotoxicity test used was an adaption of that described in Mendez et al. (2009). A continuous J774A1 macrophage lineage was used in order to analyze the cytotoxicity of all compounds. The macrophages (2 x 10⁶ cells/well) in RPMI culture medium at pH 7.2 (supplemented with 10% of fetal bovine serum) were incubated with compounds (50-400 µM) for 72 h at 37 ºC under 5% CO₂ in 96-well plates. Cells in culture medium plus DMSO (0.5%) were used as control of viability. The supernatant was removed and viable cells were quantified by adding 200 µL of MTT at 5 mg/mL in phosphate buffer saline (PBS). The supernatant was removed again after 2 h, and 100 µL DMSO were added to each well. The optical density was determined at a wavelength of 570 nm in the microplate reader. The tests were carried out in triplicate. The percentage of viable cells was calculated relative to the control cells. Logarithm regression analysis was performed in order to obtain CC50.

Antileishmanial activity against intracellular amastigotes

BALB/c mice macrophages were obtained by peritoneal lavage with 5 mL of cold RPMI medium (Sigma^®, Saint Loius, USA). The cell suspension (2 x 106 macrophages/mL) was applied in a Labtek chamber (Nunc^®, New York, USA) and incubated for 1 h at 37 ºC in 5% CO₂. The cultures were then washed with PBS at 37 oC for the removal of non-adherent cells. The remaining cells were incubated with the stationary phase of promastigotes of L. amazonensis at a ratio of 3:1 at 37 ºC in 5% CO₂. After 3 h, the chambers were washed to remove free parasites and incubated with the compounds (12.5-200 µM) at 37 ºC in 5% CO₂ for 72 h. Infected cells with DMSO (0.5%) were used as the negative control. Pentamidine isethionate (1.25-10 µM) was used as the reference. The cells were stained with the haematological system Instant Prov (New Prov^®, Curitiba, Brazil). The anti-amastigote activity was analyzed by microscopy counting at least 100 macrophages per sample. The experiments were performed twice in duplicate (Torres-Santos et al., 1999). Logarithm regression analysis was performed in order to obtain IC50 values (concentrations that inhibit the growth of amastigotes by 50%). Results were expressed as the ratio of infection (IF) using the following formula:

IF = (% infected cells x number of amastigotes)/total number of macrophages

Assay for nitric oxide production

The supernatants of infected macrophages were collected to quantify the secreted nitric oxide by determining the nitrite concentration using the Griess assay. Griess reagents (1% sulfanilamide/0.1% naphthylethylenediamine dihydrochloride/3% phosphoric acid) were added to the supernatant (1:1 v/v), and left to stand for 5 min at room temperature. The absorbance was determined at 470 nm in a microplate reader. The nitrite concentration was calculated from a standard curve of sodium nitrite (10 to 50 µM). The experiments were performed twice in duplicate (Roach et al., 1991; Ding et al., 1998).

Statistical analysis

The results obtained from two or three independent experiments were presented as the mean+SD. The data were evaluated by analysis of variance and the Student's t-test using GraphPad Prism 5.0 software (San Diego, CA, USA). Differences were considered significant when the p value was <0.05.

Results and discussion

Chemistry

The known amides 1 and 2 were isolated from the extract of Piper amalago L., Piperaceae, leaves, and their spectral data were compared with those available in the literature (Alécio et al., 1998, Jacobs et al., 1999). Compound 3 was obtained by hydrogenation of compound 1, the major compound in P. amalago, in order to improve the antileishmanial activity. The reduction of the double bound was confirmed by the ¹H NMR and ¹³C NMR data of the compound 3. The mass spectra of the compound 3 showed the molecular ion at m/z 303 (C₁₈H₂₅NO₃.⁺). In addition, the fragments at m/z 126 (C₇H₁₂NO⁺) and m/z 168 (C₁₀H₁₈NO⁺) indicated the saturated lateral chain of the amide.

Ribeiro et al. (2004) described the trypanocidal effects of the natural alkaloid piperine and twelve synthetic derivatives, including compound 6, against epimastigote and amastigote forms of the protozoan parasite Trypanosoma cruzi, the causative agent of the Chagas' disease. In order to contribute to the structure-activity relationship study of this class of compounds, we decided to test amides containing a shorter side chain, different amines and substituents in the aromatic ring, including fluorine (Hou & Xu, 2001).

Although fluorine does not have the same valency as hydrogen, it is often considered an isostere of that atom, since it is virtually the same size. Replacement of a hydrogen atom with a fluorine atom will have little steric effect, but since the fluorine is strongly electronegative, the electronic effect may be dramatic. The use of fluorine as an isostere for hydrogen has been highly successful in the development of new drugs (Patrick, 1995).

Amides 4-10 were prepared as described in the literature by conversion of the appropriate cinnamic acid to the corresponding acyl chloride followed by the addition of the amine (Pagnocca et al., 2006; Castral et al., 2011). The synthetic compounds were characterized by ¹H NMR spectroscopy. The vinylic hydrogens of the trans double bond presented coupling constants in the range of 16 Hz (Ribeiro et al., 2004). Furthermore, the presence of shielded signal corresponding to ten hydrogens of the piperidinic ring confirmed the structures of the synthetic products 6-10. All ¹³C NMR spectra confirmed the presence of the amidic carbonyl, in the range of 160 ppm. For compounds 7-10 it was also observed heteronuclear coupling ¹³C-¹⁹F. The IR spectra displayed C=O stretching bands in the region of 1650 cm^-1, and thus confirmed the presence of amide carbonyl groups. From the mass spectra it was possible to obtain the mass of the molecular ion, besides the main fragmentation of amides to the complete characterization of each synthesized compound (Castral et al., 2011).

Antileishmanial activity of the amides against the promastigotes and intracellular amastigotes of L. amazonensis

Piperamides with significant antileishmanial activity are characterized by a phenyl group, with a side chain with at least one insaturation ending in a carbonyl group, and a piperidine, pyrrolidine or isobutyl groups containing nitrogen (Bodiwala et al., 2007; da Silva et al., 2009; Regasini et al., 2009). These compounds have been investigated by some researchers as a model for the development of new compounds with potential biological activities (Pagnocca et al., 2006; Sangwan et al., 2008; Castral et al., 2011).

Natural compounds 1 and 2, the hydrogenation product 3, and the synthetic analogs 4-10 were evaluated against the promastigote and intracellular amastigote forms of L. amazonensis. Promastigote forms were treated for 72 h with increasing concentrations of the compounds (3.125-200 µM). In order to evaluate the anti-amastigote activity, macrophages obtained from the peritoneal cavity of mice were infected with promastigotes and treated with the compounds (12.5-200 µM) for 72 h. Pentamidine isethionate was used as the reference and the IC50 values were 2.2 µM, and 1.5 µM against the promastigote and amastigote forms, respectively, with a selectivity index (SI) towards the latter of 46.67. The cytotoxicity was evaluated using J774A1 macrophages treated for 72 h with the compounds at concentrations of 50-400 µM. The selectivity index (SI) for the protozoan was calculated according to the following formula: CC50 for J774A1 macrophages/ IC50 for the intracellular amastigotes. The compound was considered to be more selective for the amastigote forms than for the macrophages when the SI value was greater than 1. All compounds were compared according to their antileishmanial activity, cytotoxicity and SI (Table 1). Natural compounds 1 and 2 showed the best antileishmanial activity with IC50 values of around 20 µM and 15 µM respectively, compound 2 being more active and selective than compound 1 with a SI ratio of 12.89. The isolated amides inhibited significantly both promastigote and intracellular amastigote forms. Inhibitory action of a compound for all parasite forms of Leishmania is more desirable, considering that promastigotes are the forms which enter to the human bloodstream and finally infect the macrophages, where there is the change to the amastigote forms (Genaro, 1998). The derivative 3 was less active than compounds 1 and 2, because it showed lesser antipromastigote activity, as indicated by the IC50 value of around 70 µM. All synthetic analogs showed lower antileishmanial activity than the natural compounds. Compound 5 showed growth inhibition of Leishmania forms with an IC50 value of around 25 µM. Compound 6 was the most selective compound for the amastigote forms, (SI ratio of 13.11) and showed low activity against promastigote forms. Compounds 4, 7, 8 and 9 were the least active compounds against all forms of Leishmania with IC50>100 µM. Compound 10 showed low antileishmanial activity with IC50>54 µM.

The results obtained for the compound 4 were not statistically different from those of the negative control (p>0.05) in the anti-promastigote experiment, according to the Student t-test. The rest of the results for the anti-promastigote and anti-amastigote assays were significantly different to those of the control group (p<0.05), according to the Student's t-test.

Further experiments will be necessary to clarify the structure-activity relationships of the amides. However, it was possible to observe some tendencies:

1. The double bonds in the side chain having a (E,E) configuration in compound 2, led to a slight increasing in the antileishmanial activity;

2. The substituents of the compounds 7-10 with deactivating electron-withdrawing groups (e.g., fluorine, difluoromethoxy, trifluoromethoxy) have a negative effect on the activity, which was shown by their lesser activity than compounds 1-3, 5 and 6. This observation is in agreement with the results reported for the antileishmanial activity of imidothiocarbamates and imidoselenocarbamates described by Moreno et al. (2011).

The effects of the compounds were evaluated in relation to nitric oxide production, in order to determine whether anti-amastigote activity resulted from activation of this antileishmanial mechanism. None of the compounds promoted significant nitric oxide production, suggesting a direct and selective action of the compounds on the intracellular amastigotes. Moreover, all amides were less cytotoxic than pentamidine isothionate.

In summary, natural compounds 1 and 2 showed higher antileishmanial activity than the derivative and the synthetic analogs, with the compound 2 being the most active. Moreover, this compound was more selective for the amastigotes than macrophages and may act directly on the parasite, demonstrating a significant potential for the treatment of the cutaneous leishmaniasis (McConville & Handman, 2007). Compound 2 showed an interesting action and low cytotoxicity compared with other alkaloids with antileishmanial activity, especially pyrrolidine amides recognized for the growth inhibition of the intracellular amastigotes forms reported in the literature (Bodiwala et al., 2007; Mishra et al., 2009; Ghosal et al., 2012). These data could be important to further studies using natural compounds from P. amalago L. leaves in order to establish their mechanism of action. The present study showed the antileishmanial activity of the major compounds of P. amalago L., which might become useful for the development of a new medicine to the benefit of people afflicted by leishmaniasis.

Acknowledgment

The authors are grateful to the Brazilian governmental agencies CNPq, CAPES, FAPESP and Fundação Araucária for providing a research grant and fellowships.

Authors' contributions

VSC contributed in collecting plant sample and identification, confection of herbarium, running the laboratory work, analysis of the data and drafted the paper. EFCJ and ECTS contributed to the biological studies. AGC and JLM contributed in synthesizing the analogs. EFCJ, ECTS, AGC, JLM, IGD and MVCL contributed to critical reading of the manuscript. DAGC designed the study, supervised the laboratory work and contributed to critical reading of the manuscript. All the authors have read the final manuscript and approved the submission.

Received 10 Dec 2012

Accepted 7 Feb 2013

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Publication Dates

• Publication in this collection
  05 Mar 2013
• Date of issue
  June 2013

History

• Received
  10 Dec 2012
• Accepted
  07 Feb 2013