Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of Porcelia macrocarpa Is Associated with Alterations in Both Plasma Membrane Electric Potential and Mitochondrial Membrane Potential Emerson A. Oliveira,† Ivanildo A. Brito,‡ Marta L. Lima,§ Maiara Romanelli,§ José T. Moreira-Filho,⊥ Bruno J. Neves,⊥,∥ Carolina H. Andrade,⊥ Patricia Sartorelli,† Andre G. Tempone,§ Thais A. Costa-Silva,*,‡ and João Henrique G. Lago*,‡ Downloaded via UNIV AUTONOMA DE COAHUILA on May 2, 2019 at 21:25:55 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, São Paulo 09972-270, Brazil Center for Natural and Human Sciences, Federal University of ABC, São Paulo 09606-045, Brazil § Centre for Parasitology and Mycology, Instituto Adolfo Lutz, São Paulo 01246-000, Brazil ⊥ Faculty of Pharmacy, Federal University of Goias, Goias 74605-170, Brazil ∥ Laboratory of Cheminformatics, University Center of Anápolis, Goias 75083-515, Brazil ‡

S Supporting Information *

ABSTRACT: As part of a drug discovery program aimed at the identification of antiTrypanosoma cruzi metabolites from Brazilian flora, four acetogenins (1−4) were isolated from the seeds of Porcelia macrocarpa and were identified by NMR spectroscopy and HRESIMS. The new compounds 1 and 2 displayed activity against the trypomastigote (IC50 = 0.4 and 3.6 μM) and amastigote (IC50 = 23.0 and 27.7 μM) forms. The structurally related known compound 3 showed less potency to the amastigotes, with an IC50 value of 58 μM, while the known compound 4 was inactive. To evaluate the potential mechanisms for parasite death, parameters were evaluated by fluorometric assays: (i) plasma membrane permeability, (ii) plasma membrane electric potential (ΔΨp), (iii) reactive oxygen species production, and (iv) mitochondrial membrane potential (ΔΨm). The results obtained indicated that compounds 1 and 2 depolarize plasma membranes, affecting ΔΨp and ΔΨm and contributing to the observed cellular damage and disturbing the bioenergetic system. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were nonmutagenic, noncarcinogenic, nongenotoxic, and weak hERG blockers. Additionally, none of the isolated acetogenins 1−4 were predicted as pan-assay interference compounds.

■

RESULTS AND DISCUSSION Compounds 1−4 (Figure 1), isolated as solids, showed similar NMR spectra, suggesting them to be structurally related compounds. In the 13C and DEPT NMR spectra of compounds 1−3 were observed signals at δ 80.3 and 80.2, attributed to sp carbons C-11′ and C-12′, at δ 139.0 and 114.2 to sp2 carbons C-19′ and C-20′, and several methylene signals in the range δ 32−19 (C-1′/C-10′ and C13′/C-18′). This profile indicated the occurrence of related compounds containing an aliphatic side chain with one acetylenic system at C-11′ and a terminal double bond, as previously detected in acetogenins isolated from seeds of P. macrocarpa.9 These signals were absent in the 13C NMR spectrum of compound 4, suggesting the occurrence of a hydrogenated derivative. In the 1H NMR spectrum of compound 1 were observed, besides those assigned to the unsaturated side chain at δ 5.80 (ddt, J = 17.0, 10.0, and 6.7 Hz, H-19′), 4.95 (m, H-20′), 2.13

Porcelia macrocarpa (Warming) R. E. Fries (Annonaceae) is a species found in the Atlantic Forest and “Cerrado” regions of Brazil.1 Previous studies by our group have shown the antiTrypanosoma cruzi activity of acetylenic acids from the seeds and flowers from P. macrocarpa.2,3 T. cruzi protozoan parasites are responsible for Chagas disease, a neglected disease with eight million cases each year in North and South America.4 The available chemotherapy is unsatisfactory and is limited to two nitrogen-containing heterocyclic drugs, benznidazole and nifurtimox, 5 with severe adverse effects and reduced efficacy.6−8 In this context, the search for new chemotherapeutic alternatives for Chagas disease is crucial, and natural products are a promising approach to identify new lead compounds. In the present work, the antitrypanosomal activities of four acetogenins (1−4) isolated from the seeds of P. macrocarpa were evaluated, including two new compounds (1 and 2). Furthermore, studies concerning the mechanism of cellular death were performed on compounds 1 and 2, in addition to in silico evaluations of their ADMET properties. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 23, 2018

A

DOI: 10.1021/acs.jnatprod.8b00890 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

the presence of signals attributed to the C-5 methylene carbon. Additionally, a carbinol carbon peak was observed at δ 75.0 (CH, C-4), which, in association with the peak at δ 17.9 (CH3, C-5), suggested the presence of a methyl group at C-4 from the α,β-unsaturated lactone system due to the peaks at δ 177.1 (C-1 and C-3) and 100.9 (C-2). The 1H NMR spectrum of compound 2 showed a quartet at δ 4.80 (J = 6.6 Hz) and a doublet at δ 1.49 (J = 6.6 Hz), which were attributed to H-4 and H-5, respectively. This information, along with the HRESIMS, which showed a [M − H]− peak at m/z 387.2893, corresponded to a C25H40O3 molecular formula for compound 2 and allowed its characterization as 3-hydroxy4-methyl-2-(n-eicos-11′-yn-19′-enyl)but-2-enolide. Furthermore, the configuration of C-4 was determined as R, based on the comparison of the specific rotation value recorded for compound 2 with that reported to chemically related γlactones.11 Compounds 3 and 4 were identified respectively as the known acetogenins (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos11′-yn-19′-enyl)butanolide and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicosanyl)butanolide, respectively, by comparison of their NMR and MS data with those reported in the literature.9 Compounds 1 and 2 are new natural products and biosynthetically related to compound 3, previously isolated from seeds of P. macrocarpa.9 Compound 4 was described earlier9 as a hydrogenation product of compound 3, with this being its first occurrence as a natural product. From earlier reports, P. macrocarpa is a source of several natural bioactive products such as antifungal and cytotoxic alkaloids12 and antimicrobial terpenoids13 in addition to acetylenic fatty acids and triglycerides with anti-T. cruzi activity.2,3 The antitrypanosomal activity of compounds 1−4 was determined against cell-derived trypomastigotes and amastigotes of T. cruzi (Table 1). The results obtained demonstrated that compounds 2 and 3 induced no mammalian cytotoxicity at the highest tested concentration of 200 μM, while compound 1 displayed a CC50 value of 80 μM. Compounds 1 and 2 were effective against trypomastigotes of T. cruzi with IC50 values of 0.4 and 3.6 μM, respectively. The selectivity index (SI), given by the ratio between the mammalian toxicity and the activity against the trypomastigote form of T. cruzi, resulted in values of 200 and 55.6 for compounds 1 and 2, respectively. Intracellular amastigotes of T. cruzi were also susceptible to compounds 1 and 2 and displayed IC50 values of 23.0 and 27.7 μM, respectively, and SI values of 3.5 and 7.2. In addition, Figure 2 shows the treatment with compounds 1 and 2 in the intracellular amastigote form, making it possible to observe the integrity of the cells as well as the complete elimination of the parasites by the macrophages after treatment

Figure 1. Structures of compounds 1−4 isolated from the seeds of P. macrocarpa.

(t, J = 6.0 Hz, H-10′ and H-13′), 2.04 (q, J = 6.7 Hz, H-18′), and 1.26 (s, H-2′/H-9′ and H-14′/H-17′), additional coupled doublets at δ 5.26 and 5.12 (J = 2.9 Hz) attributed to the H-5 geminal hydrogens. The 13C and DEPT NMR spectra of compound 1 displayed four non-hydrogenated carbons at δ 173.1, 105.3, 162.3, and 149.8, and these were assigned respectively to C-1, C-2, C-3, and C-4 of a γ-lactone unity. An additional peak at δ 93.1 (CH2) was thus attributed to C-5, indicating the presence of an unsaturated carbonyl system, as reported for related compounds isolated from Lomatium dissectum (Apiaceae).10 The above information, when combined with the HRESIMS that showed a [M − H]− peak at m/z 385.2750, allowed the characterization of compound 1, with a molecular formula of C25H38O3, as 3hydroxy-4-methylene-2-(n-eicos-11′-yn-19′-enyl)but-2-enolide. The 13C and DEPT NMR spectra of compound 2 displayed a similar profile to those observed in compound 1 except for

Table 1. Anti-T. cruzi and Cytotoxicity Activities of Compounds 1−4 Isolated from the Seeds of P. macrocarpaa IC50 (μM)

CC50 (μM)

SI

compound

trypomastigotes

amastigotes

NCTC

trypomastigote

amastigote

1 2 3 4 stearolic acid stearic acid

0.4 ± 0.1 3.6 ± 0.9 7.8 ± 2.2 NA 27.6 ± 0.9 NA

23.0 ± 6.9 27.7 ± 4.6 58.3 ± 33.4 NA NA NA

80.0 ± 27.0 >200 >200

200.0 >55.6 >25.6

3.5 >7.2 >3.4

>200

>7.2

a

IC50, 50% inhibitory concentration; CC50, 50% cytotoxic concentration; SI, selectivity index in trypomastigote and amastigote forms; NA, not active. Standard drug benznidazole, IC50 5.5 and 18.7 μM against trypomastigotes and amastigotes, respectively. B



Article

action studies.14 In the present work, the observation of continuous plasma membrane integrity with the vital dye Sytox Green during a 120 min incubation indicated an absence of membrane disruption in the initial moments of the treatment (data not shown). Although the compounds caused no alterations in the permeability of the plasma membrane of T. cruzi parasites, the potential damage to the electric potential using the potential-sensitive fluorescent probe DiSBAC(3) was investigated. This probe enters depolarized cells, binding to the intracellular proteins and resulting in an increased fluorescence.15 A significant (p < 0.05) depolarization of the plasma membrane electric potential (ΔΨp) of trypomastigotes was observed after treatment with both compounds 1 and 2. The nonselective ionophoric peptide gramicidin was used as a positive control (p < 0.005) (Figure 3). Alteration of plasma membrane electric potential (ΔΨp) was also observed to the chemically related metabolite stearolic acid, isolated from the flowers of P. macrocarpa.3

Figure 2. Macrophages infected with T. cruzi and treated with compounds 1 (A) and 2 (B) at 100 μM and (C) untreated infected macrophages (control). Optical microscope images were obtained with a Nikon Eclipse E200, 1000× magnification.

at the highest tested concentration (100 μM). In the case of compound 3, a reduced potency was observed, with IC50 values of 7.8 and 58.3 μM to the trypomastigote and amastigote forms, respectively. Due to the occurrence of the related acetogenins 1−4, some conclusions between their structures and the anti-T. cruzi activity could be obtained. Hence, it was observed that the γlactone ring plays an important role in the detected antitrypanosomal potential, especially when comparing the IC50 values of compounds 1−4 with that determined for stearolic acid3 for the trypomastigote forms of T. cruzi (27.6 μM, Table 1). However, on comparing the IC50 values of compounds 1 and 2 with those obtained for compound 3, the effect against both forms of the parasite was intensified when a unsaturated conjugated system is present in the lactone ring, especially the C-2 and C-4 double bonds, as observed for compound 1. Otherwise, the presence of an exocyclic double bond (Δ4) causes an increase in toxicity to NCTC cells. Other important structural aspects are the presence of triple and double bonds at C-11′ and C-19′, on the side chain. As observed for compound 4, the absence of these unsaturated functionalities causes a reduction in the overall biological effects, and this compound showed similar effects to those observed for stearolic and stearic acids3 (Table 1). Therefore, the data obtained suggest that the presence of a conjugated unsaturated lactone system associated with the presence of triple and double bonds in the side chain appears to be essential for the biological activity observed for acetogenins 1 and 2 isolated from the seeds of P. macrocarpa. The most potent acetogenins against the intracellular parasites (compounds 1 and 2) were also the most selective ones in trypomastigotes and were selected for mechanism of

Figure 3. Effect of compounds 1 and 2 on the plasma membrane electric potential (ΔΨp) of trypomastigotes of T. cruzi. DiSBAC2 (3) dye (0.2 μM) fluorescence was measured with a flow cytometer (ATTUNE). Minimum (T. cruzi untreated, negative control (C−)) and maximum (gramicidin 0.5 μg/mL, positive control (C+)) alteration in the electric potential permeabilization was obtained. Fluorescence was quantified by calculating the mean percentages of untreated (0%) and gramicidin-treated (100%) parasites. **p < 0.005.

Considering that most reactive oxygen species (ROS) are generated inside the mitochondria and the excess may represent a harmful event, the ROS levels of T. cruzi after incubation with compounds 1 and 2 were studied. The present data demonstrated no alterations in ROS levels after 60 and 120 min of incubation (Figure 4). Although no alteration of ROS levels was observed, both compounds induced a rapid depolarization of the mitochondrial membrane potential of trypomastigotes, resulting in a reduction of the fluorescence intensity of the JC-1 probe, when compared to untreated controls (Figure 5). These data, obtained by flow cytometry, were also corroborated by an additional fluorescence microscopy study using a different probe, MitoTracker Red. A substantial reduction in the fluorescence levels of trypomastigotes treated with compounds 1 and 2 (Figure S1, Supporting Information) was observed clearly when compared with untreated parasites. As indicated in Figure S1, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control. Untreated parasites showed intense fluorescence levels in the mitochondria after labeling with C



Article

Figure 4. Effect of compounds 1 and 2 in the production of reactive oxygen species (ROS) in T. cruzi parasites. H2DCFDA dye (20 μM) fluorescence was measured using a fluorimetric microplate reader (FilterMax F5Multi-Mode microplate reader) with excitation and emission wavelengths of 485 and 520 nm, respectively. Minimum (T. cruzi untreated) is the negative control (C−) and maximum (azide, 10 mM) is the positive control (C+).

(CYP3A4, CYP2D6, CYP1A2, CYP2C19, and CYP2C9). However, all compounds tested showed unfavorable blood− brain barrier penetration (BBB+) and potential for hERG blocking. Compounds 1−4 were subjected to a similarity search and substructure filtering to identify aggregators and pan-assay interference compounds (PAINS), respectively. In our analysis, all compounds showed medium similarity with known aggregators and none contained PAINS substructures (Table S1, Supporting Information). The in silico multiparametric prediction indicated that none of the analyzed compounds contain PAINS substructures. Consequently, there is a low probability that their biological activities are artifacts caused by reactivity or colloidal aggregation. However, despite the detected anti-T. cruzi potential of acetogenins 1 and 2, these compounds display some structural aspects related to neurotoxic acetogenins found in several species of the genus Annona (Annonaceae).25,26

Figure 5. Effects of compounds 1 and 2 on the mitochondrial membrane potential of trypomastigotes of T. cruzi. JC-1 dye (0.2 μM) fluorescence was measured with a flow cytometer (ATTUNE). Minimum (T. cruzi untreated, negative control (C−)) and maximum (T. cruzi treated with CCCP, 100 μM, positive control (C+)) alteration in the mitochondrial membrane potential was obtained. Fluorescence was quantified by calculating the ratio between the channels BL2/BL1. ***p < 0.0005.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in dioxane or MeOH in a JASCO DIP-370 digital polarimeter (Na filter, λ = 588 nm). IR spectra were obtained as KBr pellets in a PerkinElmer infrared spectrometer model 1750. 1H (300 MHz) and 13C (75 MHz) NMR spectra were obtained on a Bruker Ultrashield Avance spectrometer using CDCl3 (Aldrich) and tetramethylsilane as solvent and internal standard, respectively. HRESIMS were measured on a Bruker Daltonics MicroTOF QII spectrometer. Silica gel (Merck, 230−400 mesh) and Sephadex LH20 (Aldrich) were used for column chromatography (CC). For all extraction and chromatography procedures, analytical grade solvents (Labsynth Ltd.) were used. Plant Material. Green fruits of P. macrocarpa were collected at the Instituto de Botânica de São Paulo (latitude 23°38′34.02″ S; longitude 46°37′17.68″ W) in November 2015. Plant material was identified by Profa. Dra. Maria Claudia M. Young, and a voucher specimen was compared with that deposited in the Herbarium of the Instituto de Botânica de São Paulo (IBT SMA, Sao Paulo, Brazil) under number SP76791. Extraction and Isolation. Seeds of P. macrocarpa were obtained from the green fruits immediately after the plant collection. After being dried and powdered, 479 g of seeds was obtained and extracted using n-hexane (4 × 800 mL). The combined organic solution was concentrated under reduced pressure to give 110 g of n-hexane extract and 2.7 g of a precipitate that was filtered. Part of the precipitated material (1.7 g) was chromatographed over a silica gel column (200 g, 40 × 4 cm) eluted with n-hexane containing increasing amounts of

MitoTracker Red, confirming the normal physiology of these mitochondria (Figure S1). Panels I represent images with a blue fluorescence, showing parasites labeled with the fluorescent probe DAPI, panels II represent images with red fluorescence, having parasites labeled with the fluorescent probe MitoTracker Red, and panels III represent the merged images. As previously reported,16−18 the growth inhibitory effects of different acetogenins on cancer cells have been associated with a mitochondrial imbalance, with consequent binding to the largest mitochondrial respiratory complex (complex I), named NADH-ubiquinone oxidoreductase. The data obtained in this work suggest that the depolarization of both the plasma membrane and mitochondrial membrane potential of T. cruzi may be the result of the inhibitory activity of acetogenins 1 and 2 isolated from P. macrocarpa. Additionally, compounds 1−4 were subjected to predictive assessment of ADME/Tox properties using AdmetSAR 2.0 and Pred-hERG 4.0 Web servers.19−24 According to data shown in Table S1 (Supporting Information), compounds 1−4 were predicted to be noncarcinogenic and nonmutagenic, with acceptable human intestinal absorption, low affinity for Pglycoprotein, low inhibitory promiscuity, and high metabolic stability within the main five cytochrome P450 isoforms D



Article

SYTOX Green Assay for Cell Membrane Permeability. Trypomastigotes (2 × 106 /well) were incubated with 1 μM SYTOX Green (Molecular Probes) as described elsewhere.30 Compounds 1 and 2 were added at IC50 concentrations, and fluorescence was determined using a fluorimetric microplate reader (FilterMax F5-Molecular Devices) with excitation and emission wavelengths of 485 and 520 nm, respectively. The maximum permeabilization was obtained with 0.5% Triton X-100. The following internal controls were used in the evaluation: (i) the background fluorescence of the compound at the respective wavelengths; (ii) the possible interference of DMSO. Samples were tested in duplicate. Measurement of ROS. ROS levels were measured as previously described.31 Briefly, trypomastigotes of T. cruzi were incubated with compounds 1 and 2 (IC50 values) for 60 and 120 min. Then, 20 μM H2DCFDA was added, and the cells were incubated for 15 min at 37 °C. Fluorescence intensity was evaluated with excitation and emission wavelengths of 485 and 520 nm, respectively. Sodium azide (10 mM) was used as a positive control. The internal controls were similar to the SYTOX Green Assay. Assessment of Plasma Membrane Electric Potential (ΔΨp). Estimation of ΔΨp was monitored by measuring the increase in absorbance of bis(1,3-diethylthiobarbituric acid)trimethine oxonol [DiSBAC2(3)] (Invitrogen).14 Briefly, trypomastigotes of T. cruzi (2 × 106 /well) were treated with compounds 1 and 2 (IC50) at 37 °C for 60 min. Next, 0.2 μM DiSBAC2(3) was added and the samples were read with an Attune NxT flow cytometer (Thermo Fisher Scientific). Gramicidin D (0.5 μg/mL) (Sigma-Aldrich) was used as a positive control. Untreated parasites were used as negative controls. Assessment of Mitochondrial Membrane Potential (ΔΨm). Changes in ΔΨm were analyzed by flow cytometry using the cationic lipophilic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1,ThermoFisher), and the ratio between red/green fluorescence intensities (BL-2/BL-1; 590 nm/530 nm) was calculated.32 Trypomastigotes (2 × 106 /mL) treated for 2 h with compounds 1 and 2 (IC50 values) were incubated with JC-1 dye at a final concentration of 10 μM. For the positive control, 100 μM CCCP (Sigma) was used. Measurements were performed using an Attune NxT flow cytometer (ThermoFisher). For the fluorescence microscopy analysis, trypomastigotes of T. cruzi, after treatment with compounds 1 and 2, were costained with 10 μM DAPI (6,4′diamidino-2-phenylindole dihydrochloride) (Invitrogen, Molecular Probes) and 1 μM MitoTracker Red CM-H2XROS (Invitrogen, Molecular Probes) for 30 min at 37 °C in the dark. Thereafter, the samples were examined at 400× magnification. Merged images of blue (DAPI) and red (MitoTracker Red) images were obtained using the EVOS M5000 (Thermo, USA).33 In Silico ADMET Properties Calculation and PAINS Analysis. Some pharmacokinetics and toxicity (ADME/Tox) properties of compounds 1−4 were estimated through two different Web services. The potential for hERG inhibition was predicted using the PredhERG server (http://labmol.com.br/predherg).23,24 The AdmetSAR server (http://lmmd.ecust.edu.cn/admetsar2)22,34 was used to predict human intestinal absorption,35 human P-glycoprotein substrate,36 blood−brain barrier penetration,37 inhibition or substrate metabolism for the main cytochrome P450 isoforms,37 Ames mutagenicity,38 and carcinogenicity.39 The analysis of PAINS40,41 and aggregation potential22 were performed using KNIME workflows developed in our laboratory. Statistical Analysis. The mechanism of action assay data represent one representative assay of at least two independent assays in duplicate. The IC50 and CC50 values were calculated as described previously.42

EtOAc, to afford eight fractions (I−VIII). Fractions I (eluted with nhexane−EtOAc, 95:5, 248 mg) and VIII (eluted with n-hexane− EtOAc, 1:1, 38 mg) were composed by fatty materials, while fraction III (eluted with n-hexane−EtOAc, 8:2, 1038 mg) was found to be pure compound 3. Fractions V (eluted with n-hexane−EtOAc, 7:3, 51 mg) and VI (eluted with n-hexane−EtOAc, 6:4, 12 mg) were composed by mixtures of compounds 1 and 2 in different proportions. Compounds 1 and 2 were isolated in pure form from fractions IV (eluted with n-hexane−EtOAc, 7:3, 147 mg) and VII (eluted with nhexane−EtOAc, 6:4, 25 mg), respectively. Fraction II (eluted with nhexane−EtOAc, 9:1, 30 mg) was subjected to separation over a Sephadex LH-20 column (50 g, 20 × 2 cm) eluted with n-hexane− CH2Cl2 (1:4) and CH2Cl2−acetone (3:2 and 1:4) to afford 4 mg of compound 4. 3-Hydroxy-4-methylene-2-(n-eicos-11′-yn-19′-enyl)but-2-enolide (1): white, amorphous solid; IR (KBr) νmax 3138, 3006, 2927, 2858, 1777, 1626, 1270, 1063 cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.80 (1H, ddt, J = 17.0, 10.0, and 6.7 Hz, H-19′), 5.26 (1H, d, J = 2.9 Hz, H-5a), 5.12 (1H, d, J = 2.9 Hz, H-5b), 4.95 (2H, m, H-20′), 2.30 (1H, t, J = 7.0 Hz, H-1′a), 2.13 (4H, t, J = 6.0 Hz, H-10′ and H-13′), 2.04 (2H, q, J = 6.7 Hz, H-18′), 1.48 (1H, m, H-1′b), 1.26 (24H, s, H-2′ to H-9′ and H-14′ to H-17′); 13C NMR (CDCl3, 75 MHz) δ 173.1 (C-1), 162.6 (C-3), 149.8 (C-4), 139.0 (C-19′), 114.2 (C-20′), 105.3 (C-2), 93.1 (C-5), 80.3 (C-11′), 80.2 (C-12′), 33.7 (C-18′), 31.8 (C-1′), 29.4−28.6 (C-3′ to C-9′ and C-14′ to C-17′), 28.1 (C2′), 18.7 (C-10′ and C-13′); HRESIMS m/z 385.2750 [M − H]− (calcd for C25H37O3, 385.2748). (4R)-3-Hydroxy-4-methyl-2-(n-eicos-11′-yn-19′-enyl)but-2-enolide (2): white, amorphous solid; [α]25D −20.7 (c 0.20, dioxane); IR (KBr) νmax 3308, 2921, 2852, 1755, 1661, 1466, 1073 cm−1; 1H NMR (CDCl3, 300 MHz) δ 5.81 (1H, ddt, J = 17.0, 10.0, and 6.7 Hz, H19′), 4.97 (2H, m, H-20′), 4.80 (1H, q, J = 6.6 Hz, H-4), 2.18 (2H, t, J = 7.0 Hz, H-1′), 2.13 (4H, t, J = 6.0 Hz, H-10′ and H-13′), 2.04 (2H, q, J = 6.7 Hz, H-18′), 1.49 (3H, d, J = 6.6 Hz, H-5), 1.26 (24H, s, H-2’ to H-9′ and H-14′ to H-17′); 13C NMR (CDCl3, 75 MHz) δ 177.1 (C-1 and C-3), 100.9 (C-2), 139.1 (C-19′), 114.2 (C-20′), 80.3 (C-11′), 80.2 (C-12′), 75.0 (C-4), 33.7 (C-18′), 31.9 (C-1′), 29.4− 28.6 (C-3′ to C-9′ and C-14′ to C-17′), 28.1 (C-2′), 18.7 (C-10′ and C-13′), 17.9 (C-5); HRESIMS m/z 387.2893 [M − H]− (calcd for C25H39O3, 387.2899). Ethics Statement. Female BALB/c mice (20 g) were obtained from the animal breeding facility at the Adolfo Lutz Institute-SP, Brazil. The animals were maintained in sterilized cages under a controlled environment, receiving water and food ad libitum. All procedures performed were previously approved by the Animal Care and Use Committee from Instituto Adolfo Lutz, Secretary of Health of São Paulo State (Project Number CTC 02/2011), in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences. The Animal Care and Use Committee is coordinated by Dr. Raquel dos Anjos Fazioli. T. cruzi Parasites and Mammalian Cell Maintenance. The methods employed in T. cruzi parasites and mammalian cell maintenance were reported previously.3 Anti-T. cruzi Activity. The 50% inhibitory concentration (IC50) was determined against trypomastigotes (1 × 106 /well) in 96-well plates after incubation with compounds 1−4 for 24 h at 37 °C. Benznidazole was used as standard, and the viability was determined using a resazurin assay at 570 nm.27 For intracellular amastigotes, peritoneal macrophages (1 × 105 /well) were infected with trypomastigotes (ratio 10:1) and incubated with compounds 1−4 for 48 h at 37 °C, using benznidazole as standard. The IC50 and SI values were calculated by the following equations, respectively: number of amastigotes × number of infected macrophages/total number of macrophages and CC50 against NCTC cells/IC50 against amastigotes.28 Cytotoxicity against Mammalian Cells. NCTC cells-clone L929 (6 × 104 /well) were seeded and incubated with compounds 1− 4 for 48 h at 37 °C. The 50% cytotoxic concentration (CC50) was determined by an MTT assay29 at 570 nm.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00890. E



■

Article

(16) Akatsuka, A.; Kojima, N.; Okamura, M.; Dan, S.; Yamori, T. Pharmacol. Res. Perspect. 2016, 4, No. e00246. (17) Nakanishi, S.; Yamamoto, S.; Murai, M.; Miyoshi, H. Biochim. Biophys. Acta, Bioenerg. 2011, 1807, 1170−1176. (18) Konno, H.; Hiura, N.; Makabe, H.; Abe, M.; Miyoshi, H. Bioorg. Med. Chem. Lett. 2004, 14, 629−632. (19) Van de Waterbeemd, H.; Gifford, E. Nat. Rev. Drug Discovery 2013, 2, 192−204. (20) Waring, M. J.; Arrowsmith, J.; Leach, A. R.; Leeson, P. D.; Mandrell, S.; Owen, R. M.; Pairaudeau, G.; Pennie, W. D.; Pickett, S. D.; Wang, J.; Wallace, O.; Weir, A. Nat. Rev. Drug Discovery 2015, 14, 475−486. (21) Cheng, F.; Li, W.; Zhou, Y.; Shen, J.; Wu, Z.; Liu, G.; Lee, P. W.; Tang, Y. J. Chem. Inf. Model. 2012, 52, 3099−3105. (22) Braga, R. C.; Alves, V. M.; Silva, M. F.; Muratov, E.; Fourches, D.; Tropsha, A.; Andrade, C. H. Curr. Top. Med. Chem. 2014, 14, 1399−1415. (23) Braga, R. C. Mol. Inf. 2015, 34, 698−701. (24) Segall, M. D.; Barber, C. Drug Discovery Today 2014, 19, 688− 693. (25) Le Ven, J.; Schmitz-Afonso, I.; Lewin, G.; Brunelle, A.; Touboul, D.; Champy, P. J. Agric. Food Chem. 2014, 62, 8696−8704. (26) Escobar-Khondiker, M.; Höllerhage, M.; Muriel, M. P.; Champy, P.; Bach, A.; Depienne, C.; Respondek, G.; Yamada, E. S.; Lannuzel, A.; Yagi, T.; Hirsch, E. C.; Oertel, W. H.; Jacob, R.; Michel, P. P.; Ruberg, M.; Höglinger, G. U. J. Neurosci. 2007, 27, 7827−7837. (27) Reimão, J. Q.; Migotto, A. E.; Kossuga, M. H.; Berlinck, R. G. S.; Tempone, A. G. Parasitol. Res. 2008, 103, 1445−1450. (28) Mangoni, M. L.; Saugar, J. M.; Dellisanti, M.; Barra, D.; Simmaco, M.; Rivas, L. J. Biol. Chem. 2005, 280, 984−990. (29) Tempone, A. G.; Oliveira, C. M.; Berlinck, R. G. S. Planta Med. 2011, 77, 572−585. (30) Ribeiro, G. A.; Cunha-Júnior, E. F.; Pinheiro, R. O.; Silva, S. A.; Canto-Cavalheiro, M. M.; da Silva, A. J.; Costa, P. R.; Netto, C. D.; Melo, R. C.; Almeida-Amaral, E. E.; Torres-Santos, E. C. J. Antimicrob. Chemother. 2013, 68, 789−799. (31) Keil, V. C.; Funke, F.; Zeug, A.; Schild, D.; Müller, M. Pfluegers Arch. 2011, 462, 693−708. (32) Rea, A.; Tempone, A. G.; Pinto, E. G.; Mesquita, J. T.; Rodrigues, E.; Silva, L. G.; Sartorelli, P.; Lago, J. H. G. PLoS Neglected Trop. Dis. 2013, 7, No. e2556. (33) Yang, H.; Lou, C.; Sun, L.; Li, J.; Cai, Y.; Wang, Z.; Li, W.; Liu, G.; Tang, Y. Bioinformatics 2019, 35, 1067−1069. (34) Shen, J.; Cheng, F.; Xu, Y.; Li, W.; Tang, Y. J. Chem. Inf. Model. 2010, 50, 103−1041. (35) Li, D.; Chen, L.; Li, Y.; Tian, S.; Sun, H.; Hou, T. Mol. Pharmaceutics 2014, 11, 716−726. (36) Cheng, F.; Yu, Y.; Shen, J.; Yang, L.; Li, W.; Liu, G.; Lee, P. W.; Tang, Y. J. Chem. Inf. Model. 2011, 51, 996−1011. (37) Hansen, K.; Mika, S.; Schroeter, T.; Sutter, A.; Laak, A.; StegerHartmann, T.; Heinrich, N.; Müller, K. R. J. Chem. Inf. Model. 2009, 49, 2077−2081. (38) Lagunin, A.; Filimonov, D.; Zakharov, A.; Xie, W.; Huang, Y.; Zhu, F.; Shen, T.; Yao, J.; Poroikov, V. QSAR Comb. Sci. 2009, 28, 806−810. (39) Baell, J.; Walters, M. A. Nature 2014, 513, 481−483. (40) Baell, J. B.; Holloway, G. A. J. Med. Chem. 2010, 53, 2719− 2740. (41) Irwin, J. J.; Duan, D.; Torosyan, H.; Doak, A. K.; Ziebart, K. T.; Sterling, T.; Tumanian, G.; Shoichet, B. K. J. Med. Chem. 2015, 58, 7076−7087. (42) Conserva, G. A. A.; Costa-Silva, T. A.; Amaral, M.; Antar, G. M.; Neves, B. J.; Andrade, C. H.; Tempone, A. G.; Lago, J. H. G. Phytomedicine 2019, 54, 302−307.

Spectroscopic data of compounds 1 and 2, Table S1, and Figure S1 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: (+5511) 49960001. Fax: (+5511) 49963166. E-mail: [email protected]. *Tel: (+5511) 49960001. Fax: (+5511) 49963166. E-mail: [email protected]. ORCID

Carolina H. Andrade: 0000-0003-0101-1492 João Henrique G. Lago: 0000-0002-1193-8374 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful for the financial support and fellowships provided by CAPES (Coordenaçaõ de Aperfeiçoá mento de Pessoal de Nivel Superior), CNPq (Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico), and Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2018/07885-1 and 2018/10279-6). This publication is part of the activities of the Research Network Natural Products against Neglected Diseases (ResNetNPND): http:// www.uni-muenster.de/ResNetNPND/. J.H.G.L., A.G.T., C.H.A., and P.S. thank CNPq for the scientific awards.

■

REFERENCES

(1) Murray, N. A. Syst. Bot. Monogr. 1993, 40, 8−121. (2) Santos, A. L.; Cavalheiro, A. J.; Tempone, A. G.; Correa, D. S.; Alexandre, T. R.; Quintiliano, N. F.; Rodrigues-Oliveira, A. F.; Oliveira-Silva, D.; Martins, R. C.C.; Lago, J. H. G. Molecules 2015, 20, 8168−8180. (3) Londero, V. S.; Costa-Silva, T.; Gomes, K. S.; Ferreira, D. D.; Mesquita, J. T.; Tempone, A. G.; Young, M. C. M.; Jerz, G.; Lago, J. H. G. Bioorg. Chem. 2018, 78, 307−311. (4) Barrett, M. P.; Croft, S. L. Br. Med. Bull. 2012, 104, 175−196. (5) Bestetti, R. B.; Cardinalli-Neto, A. Int. J. Cardiol. 2011, 147, 172−173. (6) Croft, S. L.; Barrett, M. P.; Urbina, J. A. Trends Parasitol. 2005, 21, 508−512. (7) Romanha, A. J.; Castro, S. L.; Soeiro, M. N.; Lannes-Vieira, J.; Ribeiro, I.; Talvani, A.; Bourdin, B.; Blum, B.; Olivieri, B.; Zani, C.; Spadafora, C.; Chiari, E.; Chatelain, E.; Chaves, G.; Calzada, J. E.; Bustamante, J. M.; Freitas-Junior, L. H.; Romero, L. I.; Bahia, M. T.; Lotrowska, M.; Soares, M.; Andrade, S. G.; Armstrong, T.; Degrave, W.; Andrade, Z. A. Mem. Inst. Oswaldo Cruz. 2010, 105, 233−238. (8) Morillo, C. A.; Marin-Neto, J. A.; Avezum, A.; Sosa-Estani, S.; Rassi, A., Jr.; Rosas, F.; Villena, E.; Quiroz, R.; Bonilla, R.; Britto, C.; Guhl, F.; Velazquez, E.; Bonilla, L.; Meeks, B.; Rao-Melacini, P.; Pogue, J.; Mattos, A.; Lazdins, J.; Rassi, A.; Connolly, S. J.; Yusuf, S. N. Engl. J. Med. 2015, 373, 1295−1306. (9) Chaves, M. H.; Roque, N. F. Phytochemistry 1997, 44, 523−528. (10) Bradford, C. V.; John, H. C. Tetrahedron 1986, 42, 1117−1122. (11) Zhao, Y.; Guo, Y.; Zhang, W. Helv. Chim. Acta 2005, 88, 349− 355. (12) Lago, J. H. G.; Chaves, M. H.; Ayres, M. C.; Agripino, D. G.; Young, M. C. M. Planta Med. 2007, 73, 292−295. (13) Silva, E. B.; Soares, M. G.; Mariane, B.; Vallim, M. A.; Pascon, R. C.; Sartorelli, P.; Lago, J. H. G. Molecules 2013, 18, 13574−13587. (14) Menna-Barreto, R. F.; Salomão, K.; Dantas, A. P.; Santa-Rita, R. M.; Soares, M. J.; Barbosa, H. S.; de Castro, S. L. Micron 2009, 40, 157−168. (15) Luque-Ortega, J. R.; Rivas, L. Methods Mol. Biol. 2010, 618, 393−420. F


Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of

Recommend Documents