Antileishmanial Acetylene Fatty Acid and Acetogenins from Seeds of Porcelia macrocarpa

In the present work five acetylene derivatives (1-5), including three unknowns (1, 3 and 4), were isolated from seeds of Porcelia macrocarpa (Annonaceae). The structures of isolated compounds were determined as docos-13-yn-21-enoic acid (1), 3-hydroxy-4-methylene-2-(eicos-11’-yn19’-enyl)but-2-enolide (2), 3-hydroxy-4-methylene-2-(octadec-9’-yn-17’-enyl)but-2-enolide (3), 3-hydroxy-4-methylene-2-(hexadec-7’-yn-15’-enyl)but-2-enolide (4), and (2S,3R,4R)-3-hydroxy4-methyl-2-(eicos-11’-yn-19’-enyl)butanolide (5) by analysis of nuclear magnetic resonance (NMR) and electrospray ionization high-resolution mass spectrometry (ESI-HRMS) data. Moreover, all isolated compounds demonstrated selectivity towards intracellular amastigotes of Leishmania (L.) infantum, especially 2-4 with 50% inhibitory concentration (IC50) values of 9.2, 10.4 and 11.0 μM, respectively, indicating superior activity of that determined to positive control miltefosine (IC50 of 17.8 μM). Furthermore, these compounds showed higher selectivity index (SI) in comparison with miltefosine. Since related acetylene fatty acid 1 displayed reduced antiparasitic potential (IC50 of 48.5 μM), the obtained results suggested that the γ-lactone plays an important role in the antileishmanial activity. However, 2-4 exhibited cytotoxicity to mammalian NCTC cells (CC50 ca. 80 μM), which could be a result of the presence of a conjugated carbonyl system in the lactone ring, since 5, the only acetogenin that presents the saturated ring, lacked mammalian cytotoxicity (CC50 > 200 μM).


Introduction
Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, is common in different sites of tropical and subtropical regions of the world. 1,2 The chemotherapy for treatment of leishmaniasis consists of usage of toxic compounds discovered more than 50 years ago such as antimonial derivatives, amphotericin B, pentamidine, and miltefosine. 3,4 Therefore, the search for new compounds to treatment of leishmaniasis is crucial and natural products can be an interesting source of bioactive lead. 5 The history of drugs against leishmaniasis has a strong relationship to natural products. Amphotericin B, produced by the Streptomyces nodosus is a leading broad-spectrum antibiotic antifungal and have been used for about 50 years. 6 Paromomycin, also an anti-leishmanicidal drug, was originally isolated from the bacteria Streptomyces rimosus. 7 These data show the importance of natural products as a source of bioactive molecules.
Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae) has been found in the Atlantic Forest region of Brazil. 8 Different parts of this plant such as leaves, stem bark, and seeds have been chemically studied and shown to be composed of alkaloids, amides/lignanamides, flavonoids, steroids, trimethylamonium salts, amino acids, and acetylene acetogenins. [9][10][11][12][13][14] Additionally, the chemical composition of essential oils from its leaves was also investigated and Antileishmanial Acetylene Fatty Acid and Acetogenins from Seeds of Porcelia macrocarpa Ivanildo  the presence of antimicrobial terpenoids was detected. 15 In previous papers, [16][17][18] the occurrence of new acetylene acetogenins and fatty acids from seeds and flowers of P. macrocarpa with anti-T. cruzi activity was reported, including the determination of mechanism of parasite death, which involved alterations in the plasma membrane permeability and electric potential, in the mitochondrial membrane potential as well as in the production of reactive oxygen species (ROS). As a complementary segment of our studies, in the present work five biosynthetic related acetylene derivatives were isolated including one new fatty acid (1), two known (2 and 5) and two new related acetogenins (3 and 4) from seeds of P. macrocarpa. Additionally, the effects of 1-5 against intracellular (amastigote) forms of Leishmania (L.) infantum as well as the toxicity against mammalian cells (NCTC) were evaluated.

General experimental procedures
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (USA) Ultrashield 300 Avance III spectrometer, operating at 300 MHz to 1 H and at 75 MHz to 13 C nuclei, respectively. CDCl 3 (Aldrich, USA) and tetramethylsilane (TMS, Aldrich, USA) were used as the solvent and internal standard. Electrospray ionization high-resolution mass spectrometry (ESI-HRMS) analyses were performed on Bruker (USA) Daltonics micrOTOF-QII mass spectrometer equipped with an Apollo II electrospray ion source operating in negative and positive modes. Silica gel (230-400 mesh, Merck, USA) and Sephadex LH-20 (Amersham Biosciences, UK) were used for column chromatography (CC). For all extraction and chromatography procedures, analytical grade solvents were used (Labsynth Ltd., Brazil).

Plant material
Fresh seeds of P. macrocarpa were collected at Instituto de Botânica de São Paulo in November 2015 (registration code at SisGen A4123E4). As previously reported, 18 the identification of plant material was performed by Profa Dra Maria Claudia M. Young and a voucher specimen (SP76791) has been deposited in the Herbarium of the Instituto de Botânica (IBT SMA, São Paulo, Brazil).

Extraction and isolation
Fresh seeds of P. macrocarpa were dried at 40 °C during 72 h and the obtained plant material (479 g) was powdered and extracted (4 × 800 mL) using n-hexane. During evaporation of the solvent under reduced pressure was obtained a solid material (2.7 g) which was separated by filtration. Part of this material (1.7 g) was chromatographed over silica gel eluted with n-hexane containing increasing amounts of EtOAc (up to 100%) to afford eight fractions (I-VIII). Fraction I (248.3 mg) was subjected to fractionation over Sephadex LH-20 eluted with n-hexane:CH 2 Cl 2 (1:4) and CH 2 Cl 2 :acetone (3:2 and 1:4) to afford 30.8 mg of 1. Fractions III (1038.2 mg) and IV (146.7 mg) were found to be composed by pure 5 and 2, respectively. Fraction VIII (38.2 mg) was subjected to successive fractionation over Sephadex LH-20 eluted with n-hexane:CH 2 Cl 2 (1:4) to afford pure 3 (1.8 mg) and 4 (1.2 mg).  20 Macrophages were obtained from the peritoneal cavity of BALB/c mice by washing with Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal calf serum (FCS) and kept at 37 ºC in a 5% CO 2 -humidified incubator. 18 Murine fibroblast NCTC cells (clone L929 ATCC) were kept in cell culture flasks in M-199 medium supplemented with 10% fetal bovine serum (FBS) and 20 mg mL -1 gentamicin at 37 ºC in a 5% CO 2 -humidified incubator. To determine the IC 50 concentration against intracellular forms of L. (L.) infantum (amastigotes), macrophages collected from the peritoneal cavity of BALB/c mice were counted in a Neubauer chamber, seeded at 1 × 10 5 per well in a 16-well slide and kept in a 5% CO 2 -humidified incubator overnight. Posteriorly, amastigotes were collect from a previously infected hamster as described, 20 seeded at a ratio 1:10 (macrophages:amastigotes) and maintained at 37 °C in a 5% CO 2 -humidified incubator for 24 h. Subsequently, 1-5 were tested in a range concentration of 100 to 1.56 µM with infected macrophages for 96 h. Miltefosine was used as a standard drug. As the last step of the assay, the macrophages were fixed with MeOH, stained with Giemsa (Merck KGaA, Germany), and analyzed on a light microscope. The parasite burden was determined by the number of infected macrophages out of 200 cells.

Statistical analysis
The results were reported as the mean and standard deviation of duplicate samples from two or three independent assays. IC 50 and CC 50 values were calculated using sigmoid dose-response curves in Graph Pad Prism 5.0 software, 22 analysis of variance (ANOVA) for significance (p < 0.05).

Results and Discussion
A sample of the precipitate formed during the evaporation of n-hexane extract from seeds of P. macrocarpa was subjected to column chromatography over silica gel and Sephadex LH-20 to afford 1-5 (Figure 1).
Compound 1 was isolated as an amorphous solid. 1 H NMR spectrum showed one coupled system at which, associated to the signals attributed to propargyl and allyl hydrogens at d 2.13-2.14 (t, J 6.0 Hz) and 2.04-2.05 (q, J 6.7 Hz) as well as to one intense broad singlet at d 1.25-1.26 (s), suggested the occurrence of an acetylene/ olefin side chain, as previously observed for acetogenins isolated from P. macrocarpa. 11,18 This was confirmed by the presence of two coupled doublets at d 5.25-5.26 and 5.12 (J 2.9 Hz) attributed to the geminal hydrogens H-5 of the lactone ring. In the 13 C NMR spectra of 2-4, besides the signals attributed to C-1 to C-5 of the lactone ring at (m/z 235.1336), confirmed the location of the triple bond at C-11', C-9' and C-7', respectively, to 2, 3 and 4. The chain extension between the lactone ring and the triple bond also was supported by the cleavages at C-5' (2-4), C-7' (2 and 3) and C-9' (2). Furthermore, these spectra exhibited a set of intense ions at approximately m/z 123 and 137, assigned to fragmentation between lactone ring and side chain, 26 as shown in Figure 3. Based on these evidences, structures of new acetogenins 3 and 4 were determined, respectively, as 3-hydroxy-4-methylene-2-(octadec-9'-yn-17'-enyl)but-2-enolide and 3-hydroxy-4-methylene-2-(hexadec-7'-yn-15'-enyl)but-2-enolide. Compound 5, previously isolated from P. macrocarpa was identified as (2S,3R,4R)-3-hydroxy-4-methyl-2-(eicos-11'-yn-19'-enyl) butanolide by comparison of NMR and ESI-HRMS data with those reported in the literature. 11 P. macrocarpa consists of a source of several natural antiprotozoal products, especially acetylene fatty acids and acetogenins which exhibited anti-T. cruzi potential. 17,18 However, no information concerning the antileishmanial activity of these related compounds is reported in the literature. Therefore, the anti-L. (L.) infantum activity of 1-5 was evaluated against amastigotes forms of this parasite  respectively. Otherwise, when tested against L. infantum amastigotes, isolated 1-5 displayed activity with IC 50 values of 48. 5, 9.2, 10.4, 11.0 and 29.9 µM, respectively, while miltefosine (positive control) showed an IC 50 value of 17.8 µM. The selectivity index (SI), given by ratio between the mammalian toxicity and the activity against the parasite, resulted in values of approximately 8 to all tested acetogenins. Among the tested compounds, it was observed a reduced anti-Leishmania potential for fatty acid 1 suggesting the presence of lactone ring is a crucial structural feature associated to antileishmanial activity. Based on this aspect and on the structures of lactones 2-5, some preliminary structure/activity relationships could be established. Initially, it was observed that the effect against the parasite is intensified when a conjugated double bond is present in the lactone ring, as observed for 2-4. Otherwise, as observed for T. cruzi, 18 this structural aspect causes an enhancement in the toxicity to NCTC cells. In a previous study, 18 acetogenins 2 and 5 showed activity against other member of the Trypanosomatidae family with IC 50 values of 23 and 58 µM against amastigotes of T. cruzi. In the present work, no difference in the IC 50 values was observed for these compounds, indicating that the extension of side chain did not affect their activity against L. (L.) infantum. Therefore, the obtained data suggest that the presence of a lactone associated to the unsaturated carbonyl system appears to be important for the biological activity for related metabolites 2-5 isolated from seeds of P. macrocarpa.
Natural acetylene derivatives, especially acetogenins, are found in several species of Annonaceae. 27 Considering the structures of these compounds, especially those which contains tetrahydrofuran (THF) moiety in the side chain, important structural factors were established. As previously  reported, 28,29 the presence of THF groups was favorable, but not essential, for the activity. On the other hand, the presence of long alkyl tail could be considered an essential structural requirement for the activity. 30 Another study 31 reported that the γ-lactone ring, a common structural unit among a large number of natural acetogenins, plays an important role in the anti-Leishmania potential, especially those which contains α,β-unsaturated system, but it is not crucial to the activity. Therefore, due to their important pharmacological potential, including antiprotozoal activity, 29 Annonaceae acetogenins have been considered an important source of new prototypes for development of new drugs against Leishmania (L.) infantum.

Conclusions
This work reports the isolation and chemical characterization of five biosynthetically related compounds from seeds of P. macrocarpa, with one being a fatty acid (1) and four acetogenins (2-4). The occurrence of acetylene acetogenins 2 and 5 was previously reported in P. macrocarpa but this is the first description of 1, 3 and 4. The anti-leishmanial activities of 1-5 indicated that acetogenins 2-4 showed superior potential than positive control miltefosine. However, acetogenin 5, which did not display an unsaturated carbonyl system in the lactone ring, displayed reduced potential. Fatty acid 1, which showed a similar acetylene/olefin side chain compared to 2 and 5, but with absence of lactone ring, exhibited reduced potential. Despite related compounds displayed anti-T. cruzi activity, this work describes for the first time their antileishmanial activity demonstrating the potential of these compounds in the search for new drug candidates for neglected diseases.

Supplementary Information
Supplementary data (NMR and ESI-HRMS spectra for 1-5) are available free of charge at http://jbcs.sbq.org. br as PDF file.