Antileishmanial Effects of Acetylene Acetogenins from Seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae) and Semisynthetic Derivatives

As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11′-yn-19′-enyl)butanolide (1) and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11′-ynyl)butanolide (2), from the seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 μM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 μM (1a) and 23.1 μM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 μM, with no toxicity for NCTC mammalian cells (CC50 > 200 μM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.


Introduction
Leishmaniasis is a neglected tropical disease caused by the protozoan parasite Leishmania sp. Different forms of this disease can be found, and the visceral is responsible for the high lethality of this disease [1]. The treatment of leishmaniasis is limited and involves using toxic drugs such as antimonial derivatives, amphotericin B, and miltefosine. Based on this scenario, the search for new hit compounds is crucial, and natural products can provide inspiring molecules for drug discovery studies against this neglected tropical diseases [2,3]. Aiming to discover new natural products with antiprotozoal activity, differen studies with P. macrocarpa have previously been performed. Initially, the presence of ant trypanosomal fatty acid containing a diyn system was reported, but this compoun exhibited activity only against trypomastigote forms of Trypanosoma cruzi [5]. In a secon study, chemically related fatty acids containing enyn and diyn systems were isolated from flowers and exhibited anti-T. cruzi activity against trypomastigotes [6]. More recently, new acetylene acetogenins were obtained from seeds of P. macrocarpa with anti-T. cruzi activity especially against amastigote forms [7]. Additionally, these compounds induce alterations in the plasma membrane permeability and in the electric potential of th mitochondrial membrane as well as in the reactive oxygen species (ROS) levels of th parasite [6,7]. Despite the effect against T. cruzi, there is one unique study evaluating th effect of different acetogenins and fatty acid from P. macrocarpa against intracellular form of Leishmania (L.) infantum, which is the most relevant form of the parasite [8]. Aiming to discover new natural products with antiprotozoal activity, different studies with P. macrocarpa have previously been performed. Initially, the presence of antitrypanosomal fatty acid containing a diyn system was reported, but this compound exhibited activity only against trypomastigote forms of Trypanosoma cruzi [5]. In a second study, chemically related fatty acids containing enyn and diyn systems were isolated from flowers and exhibited anti-T. cruzi activity against trypomastigotes [6]. More recently, new acetylene acetogenins were obtained from seeds of P. macrocarpa with anti-T. cruzi activity, especially against amastigote forms [7]. Additionally, these compounds induced alterations in the plasma membrane permeability and in the electric potential of the mitochondrial membrane as well as in the reactive oxygen species (ROS) levels of the parasite [6,7]. Despite the effect against T. cruzi, there is one unique study evaluating the effect of different acetogenins and fatty acid from P. macrocarpa against intracellular forms of Leishmania (L.) infantum, which is the most relevant form of the parasite [8].
In continuation to our studies, in the present work, two acetylene acetogenins were isolated from seeds of P. macrocarpa and evaluated against amastigote forms of L. (L.) infantum. Additionally, these compounds were subjected to different modifications, including acetylation, and conversions to elimination and hydrogenation reactions products, to discover important check critical pharmacophoric groups in these chemically related compounds. Furthermore, considering that natural acetogenins were obtained in a mixture and showed higher potential against L. (L.) infantum than purified compounds, we also evaluated them as a mixture of natural products or as semisynthetic derivatives.
Aiming to establish relationships between the chemical structures and the antiparasitic activity, these compounds were subjected to a sequence of reactions including hydrogenation, acetylation, and elimination to afford compounds (2S,3R,4R)-3-   As previously reported [5][6][7], P. macrocarpa is an important source of natural antiprotozoal natural products including acetylenic fatty acids and acetogenins with anti-T. cruzi potential. Recently, our group reported the occurrence of different acetylene acetogenins and fatty acids with anti-Leishmania activity, especially against the intracellular amastigote forms [8].
As part of this continuous study, the anti-L. (L.) infantum activity of two natural compounds (1 and 2) was performed against amastigotes forms of this parasite, whereas the toxicity of these compounds was determined using NCTC cells. The obtained results (Table 1) demonstrated that compounds 1 and 2 induced no mammalian cytotoxicity against NCTC cells to the highest tested concentration (200 µM). When tested against L. (L.) infantum amastigotes, compound 1 displayed activity with an IC 50 value of 29.9 µM, while compound 2 was inactive (IC 50 > 100 µM). This effect might be ascribed to the terminal double bond at C-19, which may play an important role in the activity. A similar effect was also observed for the hydrogenated derivative 3, which showed no activity (IC 50 > 100 µM). Considering the structure of γ-lactone unit and based on our previous studies describing the higher activity of conjugated natural acetogenins [7], the preparation of an α,β-unsaturated system was planned via acetylation of the hydroxyl group at C-3 followed by the elimination of acetic acid over alumina chromatographic column. This approach afforded acetyl (1a and 2a) and eliminated (1b and 2b) derivatives from acetogenins 1 and 2 as well as from hydrogenated products (3a and 3b). As a result, all derivatives prepared from compound 2 displayed no activity against amastigote forms of L.(L.) infantum. Considering the activity of acetogenin 1, with an IC 50 value of 29.9 µM, the acetylated compound 1a exhibited lower activity (IC 50 of 43.3 µM), while the eliminated product 1b showed a similar potential of natural product (IC 50 of 23.1 µM).
Finally, considering that compounds 1 and 2 were naturally obtained in a mixture of 2:1 and exhibited potent activity against amastigotes (IC 50 of 8.4 µM), reduced toxicity  Table 1, mixtures composed of 1:1 and 1:2, with IC 50 values of 13.6 and 19.4 µM, respectively, displayed lower potency than that at a 2:1 ratio. A similar effect was observed for the mixtures prepared with acetyl derivatives 1a and 2a, which showed an IC 50 value for the mixture 2:1 of 12 µM and a SI > 16.7. An interesting effect was observed for the eliminated derivatives 1b and 2b, since mixtures at 2:1, 1:1, and 1:2 displayed IC 50 values of 7.9, 10.5, and 18.2 µM, respectively, indicating the superior potential of that determined for standard drug miltefosine (IC 50 of 17.8 µM).
To evaluate the potential of bioactive compounds 1, 1a, and 1b as new prototypes for the development of drugs against L. (L.) infantum, an in-silico analysis using the SwissADME online platform was performed. Using this approach, it was possible to investigate different properties of the tested compounds such as physicochemical parameters, pharmacokinetics, and drug-likeness. As an initial evaluation, the bioavailability radar (Figure 3) of tested compounds demonstrated similar characteristics with adherence to some parameters such as unsaturation, polarity, and size. However, the presence of the C 20 side chain confers, to tested compounds, high lipophilicity and reduced solubilization in aqueous medium, as indicated by red lines outside of the physicochemical space in the bioavailability radar. To evaluate the potential of bioactive compounds 1, 1a, and 1b as new prototypes for the development of drugs against L. (L.) infantum, an in-silico analysis using the SwissADME online platform was performed. Using this approach, it was possible to investigate different properties of the tested compounds such as physicochemical parameters, pharmacokinetics, and drug-likeness. As an initial evaluation, the bioavailability radar (Figure 3) of tested compounds demonstrated similar characteristics with adherence to some parameters such as unsaturation, polarity, and size. However, the presence of the C20 side chain confers, to tested compounds, high lipophilicity and reduced solubilization in aqueous medium, as indicated by red lines outside of the physicochemical space in the bioavailability radar.
Based on the obtained data, bioactive compounds 1, 1a, and 1b exhibited similar results to all evaluated parameters, especially physicochemical parameters (fraction Csp 3 , number of rotatable bonds, H-bond acceptors, and H-bond donors), lipophilicity (log p), water solubility, and GI absorption. Furthermore, tested compounds showed partial adherence to Lipinski's rules-of-five (RO5), presenting one unique violation (log p > 4.15). The analysis of the binding to cytochrome-related isoenzymes CYP 450 indicated that all tested compounds could be considered not promiscuous molecules since they do not cause any inhibition of CYP2C1, CYP2D6, and CYP3A4 but only to CYP1A2 and CYP2CA. Finally, no alerts were evidenced for PAINS to tested compounds. Therefore, the obtained results suggested that the double bond and C-19 are crucial structural features associated with antileishmanial activity. However, the effect against the parasite is intensified when mixtures of natural compounds 1 and 2 are tested. As detected, the natural mixture of these compounds at 2:1 showed to be the best proportion of natural products, suggesting that the original mixture of acetogenins obtained from P. macrocarpa, easily obtained after simple chromatographic steps, could be considered an

General Experimental Procedures
Column chromatographic procedures were performed using silica gel 60 (Merck-Darmstadt, Germany) or Sephadex LH-20 (Sigma-Aldrich-St. Louis, MO, USA), whereas analytical TLC separations were conducted using silica gel F 254 (Merck-Darmstadt, Germany). HPLC analysis was performed using a Dionex Ultimate 3000 chromatograph with a UVD-DAD-170 V as the detector, using a Luna Phenomenex C18 column (particle and pore size of 5 µm and 120 Å)-10 × 250 mm to semipreparative and 4.6 × 250 mm to analytical modes. Analytical grade solvents and reagents were used for every chromatographic procedure (Labsynth Ltd.a, SP, Brazil). NMR spectra were recorded on a Varian INOVA 500 (Palo Alto, CA, USA) operating at 500 and 125 MHz for 1 H and 13 C nuclei, respectively. Spectra were recorded using CDCl 3 (Aldrich, St. Louis, MO, USA) as solvent and TMS as internal standard. ESI-HRMS spectra were recorded on a MicroTOF QII Bruker Daltonics (Billerica, MA, USA) spectrometer using positive or negative ionization modes.
3.6. Bioassay Procedures 3.6.1. Animals The animal breeding facility at the Instituto Adolfo Lutz, São Paulo, Brazil supplied the BALB/c mice and Golden hamsters (Mesocricetus auratus). The animals were maintained in sterilized cages under a controlled environment, receiving water and food ad libitum. Animal procedures were performed with the approval of the Research Ethics Commission, which is in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences.

Mammalian Cells
Macrophages were collected from the peritoneal cavity of female BALB/c mice by washing with RPMI-1640 without phenol red, supplemented with 10% FBS at 37 • C in a humidified atmosphere containing 5% CO 2 . NCTC (clone 929) cells were maintained in RPMI-1640 (without phenol red and supplemented with 10% FBS) at 37 • C in the same conditions of the peritoneal macrophages [14]. The 50% inhibitory concentrations (IC 50 ) against intracellular amastigotes for tested compounds were determined in infected macrophages. Peritoneal macrophages were obtained as described in the Section 3.6.3, and L. (L.) infantum amastigotes were obtained from spleens of infected hamsters by differential centrifugation. Peritoneal macrophages were seeded at 1 × 10 5 cells per well in Nunc™ 16-well slide chambers (Aldrich, St. Louis, MO, USA) for 24 h at 37 • C in a 5% CO 2 -humidified incubator. Next, amastigotes were isolated from a spleen of an infected hamster as described [13], counted, seeded at a 1:10 macrophages/amastigotes ratio, and incubated for 24 h. Non-internalized parasites were removed by washing twice with a culture medium. Then, the macrophages were incubated with compounds or standard drug (miltefosine) in a range of 100 to 0.78 µM for 96 h at 37 • C in a 5% CO 2 -humidified incubator, using miltefosine as a standard drug. At the end of the assay, the cells were fixed in methanol, stained with Giemsa, and observed under a digital light microscope (EVOS M5000, Thermo, Waltham, MA, USA) to determine the number of infected macrophages out of 400 cells [8].

Determination of the Cytotoxicity against Mammalian Cells
The 50% cytotoxic concentration (CC 50 ) was determined in NCTC clone 929 cells. NCTC cells were seeded at 6 × 10 4 cells/well in 96-well microplates and incubated with serial dilutions of the tested compounds or standard drug (miltefosine) to the highest concentration of 200 µM for 48 h at 37 • C in a 5% CO 2 -humidified incubator. An MTT assay determined the viability of the cells at 570 nm [15]. The selectivity index (SI) was determined considering the following equation: CC 50 NCTC cells/IC 50 parasites.