Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages

Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages

Accepted Manuscript Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitoc...

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Accepted Manuscript Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages Simone S. Grecco, Thais A. Costa-Silva, Gerold Jerz, Fernanda S. de Sousa, Vinicius S. Londero, Mariana K. Galuppo, Marta L. Lima, Bruno J. Neves, Carolina H. Andrade, Andre G. Tempone, João Henrique G. Lago PII:

S0009-2797(17)30349-6

DOI:

10.1016/j.cbi.2017.08.017

Reference:

CBI 8085

To appear in:

Chemico-Biological Interactions

Received Date: 30 March 2017 Revised Date:

13 July 2017

Accepted Date: 28 August 2017

Please cite this article as: S.S. Grecco, T.A. Costa-Silva, G. Jerz, F.S. de Sousa, V.S. Londero, M.K. Galuppo, M.L. Lima, B.J. Neves, C.H. Andrade, A.G. Tempone, Joã.Henrique.G. Lago, Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages, Chemico-Biological Interactions (2017), doi: 10.1016/ j.cbi.2017.08.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages Simone S. Grecco, Thais A. Costa-Silva, Gerold Jerz, Fernanda S. de Sousa, Vinicius da S. Londero, Mariana K. Galuppo, Marta L. Lima, Bruno J. Neves, Carolina H. Andrade, Andre G. Tempone, João Henrique G. Lago

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Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane

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and mitochondrial damages

Simone S. Grecco1,2,3, Thais A. Costa-Silva1, Gerold Jerz2, Fernanda S. de Sousa4,

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Vinicius S. Londero4, Mariana K. Galuppo5, Marta L. Lima5,6, Bruno J. Neves7,8,

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Carolina H. Andrade7, Andre G. Tempone5, João Henrique G. Lago1*

Center of Natural Sciences and Humanities, Federal University of ABC, Santo Andre, SP, 09210-180, Brazil.

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Institute of Food Chemistry, Technische Universität Braunschweig, Braunschweig,

Biotechnology and Innovation in Health Program, Anhanguera University of São Paulo, São Paulo, SP, 05145-200, Brazil.

Institute of Environmental, Chemical and Pharmaceutical Sciences,

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Federal University of São Paulo, Diadema, SP, 09972-270, Brazil. 5

Centre for Parasitology and Mycology, Instituto Adolfo Lutz, São Paulo, SP, 01246-902,

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38106, Germany.

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Brazil.

Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, 05403-000, Brazil.

LabMol, Laboratory for Molecular Modeling and Drug Design, Faculty of Pharmacy, Federal University of Goias, Goiânia, GO, 74605-170, Brazil. 8

Postgraduate Program in Society, Technology and Environment, UniEVANGELICA University Center, Anápolis, GO, 75083-515, Brazil.

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ACCEPTED MANUSCRIPT *corresponding author

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E-mail address: [email protected] (J.H.G.L.)

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ACCEPTED MANUSCRIPT Abstract

Chagas disease is a neglected tropical disease, caused by the protozoan parasite Trypanosoma cruzi, which affects more than eight million people in Tropical and Subtropical countries

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especially in Latin America. Current treatment is limited to nifurtimox and benznidazole, both with reduced effectiveness and high toxicity. In this work, the n-hexane extract from leaves of Nectandra leucantha (Lauraceae) displayed in vitro antitrypanosomal activity against T. cruzi.

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Using several chromatographic steps, four related neolignans were isolated and chemically characterized as dehydrodieugenol B (1), 1-(8-propenyl)-3-[3′-methoxy-1′-(8-propenyl)(2),

1-[(7S)-hydroxy-8-propenyl]-3-[3′-methoxy-1′-(8′-

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phenoxy]-4,5-dimethoxybenzene

propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (3), and 1-[(7S)-hydroxy-8-propenyl]-3[3′-methoxy-1′-(8′-propenyl)-phenoxy]-4,5-dimethoxybenzene (4). These compounds were tested against intracellular amastigotes and extracellular trypomastigotes of T. cruzi and for

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mammalian cytotoxicity. Neolignan 4 showed the higher selectivity index (SI) against trypomastigotes (> 5) and amastigotes (> 13) of T. cruzi. The investigation of the mechanism of action demonstrated that neolignan 4 caused substantial alteration of the plasma membrane

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permeability, together with mitochondrial dysfunctions in trypomastigote forms. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds

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were non-mutagenic, non-carcinogenic, non-genotoxic, weak hERG blockers, with acceptable volume of distribution (1.66 - 3.32 L/kg), and low rodent oral toxicity (LD50 810 - 2,200 mg/kg). Considering some clinical events of cerebral Chagas disease, the compounds also demonstrated favorable properties, such as blood-brain barrier penetration. Unfavorable properties were also predicted as high promiscuity for P450 isoforms, high plasma protein binding affinity (> 91%), and moderate-to-low oral bioavailability. Finally, none of the isolated neolignans was predicted as interference compounds (PAINS). Considering the

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promising chemical and biological properties of the isolated neolignans, these compounds could be used as starting points to develop new lead compounds for Chagas disease.

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permeability; mitochondrial dysfunctions; ADMET

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Key words: Nectandra leucantha; neolignans; Trypanosoma cruzi; plasma membrane

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ACCEPTED MANUSCRIPT 1. INTRODUCTION

Chagas disease, a protozoan disease caused by the parasite Trypanosoma cruzi, affect

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more than eight million people at Tropical and Subtropical countries especially in Latin America [1]. The currently therapeutic arsenal for the treatment of Chagas disease is very limited including nifurtimox and benznidazole, with severe side effects. Therefore, the search

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for new lead compounds, including those obtained from natural sources, is crucial.

Previous studies of our group performed with Nectandra leucantha afforded three

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neolignans with antileishmanial activity from twigs [2] as well as one eugenol dimer derivative with antitrypanosomal potential from leaves [3]. In continuation to this work, four additional neolignans (1 – 4) were isolated and chemically characterized from leaves extract of N. leucantha. These compounds were tested against intracellular amastigotes and

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extracellular trypomastigotes of T. cruzi and for mammalian cytotoxicity. By using flow cytometry and fluorimetric techniques, was investigated the cellular targets of the most selective compound, neolignan 4. Finally, an in silico study was performed to predict some

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pharmacokinetics and toxicity (ADMET) properties of the isolated neolignans, together with

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an analysis to exclude pan-assay interference compounds (PAINS).

2. RESULTS AND DISCUSSION

2.1 Chemical characterization of neolignans 1 – 4

The n-hexane crude extract from leaves of N. leucantha was analyzed by HPLC and four peaks were detected. Off-line HRESIMS analysis (Figure 1) indicated the occurrence of related neolignans due the quasi-molecular ion peaks [M + Na]+ at m/z 349.1423, 363.1585,

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365.1237 and 379.1506, corresponding to molecular formulas C20H22O4 (1), C21H24O4 (2), C20H22O5 (3), and C21H24O5 (4), respectively. As this extract displayed activity against trypomastigote forms of T. cruzi (100% of death at 300 µg/mL), it was subjected to several

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chromatographic steps to afford neolignans 1 – 4. H NMR spectra of neolignans 1 – 4 displayed signals at δ 6.27 – 6.90 (H-2, H-6, H-

2’, H-5’ and H-6’), 3.24 – 5.04 (H-7), 3.36 – 3.39 (H-7’) and δ 5.06 – 5.98 (H-8/H-8’ and H-

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9/H-9’). The 13C NMR spectra of compounds 1 – 4 displayed ten peaks assigned to aromatic ring at δ 105 – 153. Additional sp2 carbon atoms at δ 116 – 140 (C-8/C-8’ and C-9/C-9’)

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associated to the presence of methylene carbon atoms at approximately δ 40 (C-7/C-7’) in 1 and 2, indicated the presence of two allyl side chains. These evidences confirmed the occurrence of related compounds with similar structure of dehydrodieugenol B [4]. However, in case of 3 and 4, were observed additional signals at approximately δ 75.0 (CH), attributed

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to the carbinol carbon C-7. The spectroscopic data of isolated compounds were in good accordance with those previously reported in the literature [2, 5-7] allowing the identification of dehydrodieugenol B (1), 1-(8-propenyl)-3-[3′-methoxy-1′-(8-propenyl)-phenoxy]-4,5(2),

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dimethoxybenzene

1-[(7S)-hydroxy-8-propenyl]-3-[3′-methoxy-1′-(8′-propenyl)-

phenoxy]-4-hydroxy-5-methoxybenzene

(3),

and

1-[(7S)-hydroxy-8-propenyl]-3-[3′-

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methoxy-1′-(8′-propenyl)-phenoxy]-4,5-dimethoxybenzene (4), respectively (Figure 2).

2.2. Bioassays

Neolignans 1 – 4 were evaluated for its activity against trypomastigote and amastigote forms of T. cruzi by resazurin and macrophage infections assays, respectively [8, 9]. Except by the inactivity of neolignan 2, IC50 values ranging of 15.2 to 86.5 µM being the neolignan 4 the most active with anti-amastigote activity merely 2.7-fold lower than the standard drug

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benznidazole (Table 1). Differences at the molecular structures between these neolignans afforded considerable impact on parasite activity. For example, the replacement of the hydroxyl (neolignan 1) by methoxyl group at position C-4 in the eugenol aromatic ring

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(neolignan 2) causes the loss of the anti-T. cruzi activity that its potentially restored by the presence of hydroxyl group at the allyl side chain (neolignan 4). Hence, the parasite selectivity seems associated with structure requirements suggesting the hydroxyl group as

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essential to anti-T. cruzi activity of these neolignans. Worthy the mentioning, such structural differences not affected the absence of cytotoxicity in mammalian cells (Table 1). In line to

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our results, structure-activity relationships have been extensively associated with antiprotozoal activity of natural compounds and analogues [10-12]. Considering that neolignan 4 showed the higher activity against T. cruzi parasites in this study, the mechanism

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of action was investigated in the trypomastigote form.

2.3. Plasma membrane permeabilization

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The effect of neolignan 4 in the plasma membrane permeabilization was investigated using Sytox Green dye. A significant increase in the fluorescence levels was detected in the

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first 60 min incubation with neolignan 4 when compared to untreated trypomastigotes. After 360 min (6 hours) incubation, the fluorescence was similar that achieved in trypomastigotes treated with 0.5% Tx-100 detergent solution (Figure 2). Substantial plasma membrane permeabilization is necessary to permit the entrance of dyes such as Sytox Green due its relatively high molecular weight. Inside the cell it binds to intracellular nucleic acids producing fluorescence [13]. Thus, the neolignan 4 entered into trypomastigotes causing substantial of permeabilization of the plasma membrane.

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ACCEPTED MANUSCRIPT 2.4. Mitochondrial membrane potential and PI measurements

The effect of neolignan 4 in the mitochondrial membrane potential was investigated by

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flow cytometry using a combination of rhodamine 123 (Rd123) and propidium iodide (PI). Rd123 is a fluorescent cationic dye that accumulates into active mitochondria accordingly to the mitochondrial membrane potential (∆ψm), whereas PI behaves similar to Sytox Green

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sharing high molecular weight and fluorescence mechanism by intracellular nucleic acids binding [13, 14]. After 60 min of incubation, the Rd123 fluorescence considerably decreased

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in trypomastigotes treated with neolignan 4 when compared to untreated parasites (Figure 4A), with a concomitant increased of PI fluorescence levels (Figure 4B). Here, the increase of PI fluorescence corroborated the effect of plasma membrane permeabilization determined by Sytox Green and these cells were rolled out of RD123 measurements. On the other hand, non-

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permeabilized trypomastigotes treated with neolignan 4 (positive to Rd123 and negative to PI) showed a lower accumulation of Rd123, a similar effect found in trypomastigotes treated with FCCP (10 µM), a known mitochondrial uncoupler. Therefore, mitochondrial membrane

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potential and plasma membrane permeability were affected by neolignan 4. Both plasma membrane permeabilization and alteration of mitochondrial membrane

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potential of T. cruzi were also described for a related neolignan, isolated from leaves of Piper regnellii var. pallescens in trypomastigotes [15]. Besides, mitochondrial dysfunction has also been described for other trypanocidal compounds [16, 17]. In our work, the permeabilization of the plasma membrane seems to be an important effect of neolignan 4 in T. cruzi, a lethal mechanism that hinders a resistance by the parasite. As example, the amphotericin B, a standard leishmanicidal drug, disrupts the integrity of plasma membrane, and resistance tends to be species-dependent and uncommon [18]. Membranes of T. cruzi parasites are considerably different in lipid and protein composition from mammalian cells [19], which

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may explain the absence of cytotoxicity in mammalian NCTC cells, ensuring the selectivity found in this work (Table 1). Moreover, the antiparasitic activity of neolignans has also been

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associated to an immunomodulatory effect [20-22].

2.5. In silico prediction of ADMET properties and PAINS analysis

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It is well established that poor pharmacokinetics and toxicity (ADMET) properties represent one of the main reasons for the clinical failures of drug candidates [23]. Therefore, it

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is justifiable the use computational tools to predict ADMET properties at early stages of drug discovery process [24, 25]. In view of this advantage, an in silico investigation of ADMET properties using web services was performed with the studied neolignans 1 – 4. According to Table 2, the tested compounds were predicted as non-mutagenic, non-

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carcinogenic, non-genotoxic, weak hERG blockers, with acceptable volume of distribution (ranging from 1.66 to 3.32 L/kg), and low rodent oral toxicity (LD50 ranging from 810 to 2,200 mg/kg). Considering that Chagas disease could atypically cause a cerebral disease, with

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parasites also found in the brain [26], our analysis predicted that all compounds presented a favorable property, blood-brain barrier penetration. On the other hand, some unfavorable

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properties were also predicted, such as high promiscuity to cytochrome P450 isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4), high plasma protein binding affinity (> 91%), and moderate-to-low oral bioavailability. Additionally, neolignans 1 – 4 were subjected to a computational filter to identify

substructures present in pan-assay interference compounds (PAINS) [27]. This analysis has substantial value during screening of compounds because apparent activity of PAINS is typically caused by their reactivity rather than non-covalent binding, aggregate formation, and by nonspecific interactions with proteins of buffer in a high percentage of bioassays [28].

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None of the tested neolignans was predicted as PAINS. All compounds also showed low chemical similarity (Tanimoto coefficients < 0.22) to any compound inside in a dataset of

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12,600 likely aggregators [29], and therefore, they are unlikely to be aggregators or PAINS.

3. MATERIALS AND METHODS

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3.1. General

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For all extraction and chromatography procedures, analytical grade solvents (Labsynth Ltda, SP, Brazil) were used. HPLC fingerprint analysis were performed using an Ultimate 3000 system using a Luna C-18 column (250 × 4.6 mm, 5 µm) with a gradient from MeOH:H2O 1:1 (0 min) to MeOH 100% (35 min), flow rate 1.0 mL/min and detection at 210 nm. Bruker Daltonics MicroTOF QII spectrometer (Billerica, MA, USA) was used to

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measured HRESIMS spectra. 1H and 13C NMR spectra were recorded on a Bruker Daltonics Ultrashield 300 Avance III spectrometer (Billerica, MA, USA) at 300 and 75 MHz,

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respectively, using CDCl3 (TediaBrazil, RJ, Brazil) as solvent and TMS as internal standard.

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3.2. Plant material

Nectandra leucantha leaves were collected in the Atlantic Forest area of Cubatão city,

São Paulo State, Brazil in July 2012. The plant material was identified by Prof. MSc. Euder G. A. Martins and a voucher specimen (EM357) has been deposited in the Herbarium of Institute of Biosciences, University of São Paulo, SP, Brazil.

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ACCEPTED MANUSCRIPT 3.3. Extraction

N. leucantha leaves were dried, powdered (382 g) and exhaustively extracted using n-

obtained 10.3 g of hexane extract.

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3.4. HPLC and off line HRESIMS analysis

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hexane at room temperature. After evaporation of the solvent at reduced pressure, were

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Part of crude n-hexane extract from leaves of N. leucantha (5 mg) was dissolved in MeOH and filtered on a C18 Sep-Pak. Obtained sample was analyzed by HPLC and each detected peak was collected. After dried under reduced pressure each sample was dissolved in

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1 mL of MeOH and analyzed by HRESIMS.

3.5. Bioactivity-guided fractionation of n-hexane extract from leaves of N. leucantha

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Part of the crude n-hexane extract (9.9 g) was applied to a silica-gel column and eluted with a gradient mixture of EtOAc in n-hexane in gradient form. A total of 150 fractions (50

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mL each) were collected and combined into 12 groups (A – L) on the basis of similarities in TLC profiles. The activity against trypomastigote forms of T. cruzi was detected in groups C (1.05 g), F (1.52 g) and I (271 mg), which were then submitted to further purification procedures. Part of groups C (40 mg) and D (42 mg) were individually purified by prep. TLC (silica-gel, n-hexane:EtOAc 1:1) to yield 25 mg of 2 and 29 mg of 1, respectively. Part of group I (150 mg) was subjected to CC on silica-gel, eluted with gradient mixtures of EtOAc in n-hexane yielding 35 fractions which were pooled in three groups (I1 – I3). Individual

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purification by prep. TLC (silica-gel, n-hexane:EtOAc 1:1) of groups I2 (81 mg) and I3 (35 mg) yielded 29 mg of 3 and 15 mg of 4, respectively.

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3.6. In silico prediction of ADMET properties and PAINS analysis

Some physicochemical and ADMET properties were predicted using five different

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web services. The admetSAR server [30] was used to predict P-glycoprotein inhibition [31], carcinogenicity [32], and AMES mutagenicity [33]. The Online Chemical Database

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(OCHEM, https://ochem.eu/home/show.do) was used to predict inhibition of five cytochrome P450 isoforms while ACD/ILabs server (https://ilab.acdlabs.com/iLab2/) was used to predict genotoxicity, blood-brain barrier penetration, protein plasma binding, volume of distribution and oral bioavailability. The hERG inhibition was predicted using the Pred-hERG server [34,

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35] whereas the rodent oral toxicity (LD50) was predicted using PROTOX server [36]. Finally, the structures were imported into KNIME workspace v. 2.12.1 and filtered for identifying PAINS using a workflow provided by Saubern and co-workers [37]. In addition,

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to estimate the aggregation potential, we calculated the pairwise Tanimoto coefficient (using Morgan fingerprints [38-40] between each compound and a dataset of 12,600 likely

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aggregators [29].

3.7. Ethics Statement

Golden hamsters and BALB/c mice were obtained by the animal breeding facility at the Adolfo Lutz Institute-SP, Brazil. The animals were maintained in sterilized cages under a controlled environment and received water and food ad libitum. Animal procedures were performed with the approval of the Research Ethics Commission (project number CEUA

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IAL/Pasteur 02/2011), in agreement with the Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences.

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3.8. Parasites and mammalian cell maintenance

Trypomastigotes of T. cruzi (Y strain) were maintained in Rhesus monkey kidney cells

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(LLC-MK2-ATCC CCL 7), cultivated in RPMI-1640 medium supplemented with 2% FBS at 37°C in a 5% CO2-humidified incubator. Macrophages were collected from the peritoneal

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cavity of BALB/c mice by washing them with RPMI-1640 medium supplemented with 10% FBS and were maintained at 37°C in a 5% CO2-humidified incubator. The murine conjunctive cells (NCTC clone 929, ATCC) were maintained in RPMI-1640 supplemented with 10% FBS

3.9. Biological activity

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at 37°C in a humidified atmosphere containing 5% CO2.

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Crude n-hexane extract from leaves of N. leucantha and groups A – L from chromatographic fractionation were dissolved in MeOH at 300 µg/mL. Top concentrations for

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pure neolignans 1 – 4 were 100 µM to test anti-parasitic activity and 200 µM to test cytotoxic activity using 2-fold serially dilution over seven concentrations. Each point was tested in duplicate.

3.10. Anti-parasitic activity against trypomastigotes of T. cruzi

To determine the 50% inhibitory concentration (IC50) against T. cruzi, trypomastigotes were obtained from LLC-MK2 cultures, seeded at 1x106 cells/well in 96-well plates and

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incubated with neolignans 1 – 4 during 24 h at 37°C in a 5% CO2-humidified incubator. The viability was determined by determined by the resazurin assay (0.011% in PBS) [9].

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3.11. Cytotoxicity in mammalian cells

NCTC cells-clone L929 (6x104 cells/well) were seeded incubated with neolignans 1 –

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4 for 48 h at 37ºC in a 5% CO2 incubator and the cytotoxic concentration (CC50) was determined by MTT assay [8]. The optical density was determined in FilterMax F5

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(Molecular Devices) at 570 nm.

3.12. Anti-parasitic activity against amastigotes of T. cruzi

The activity against intracellular parasites was determined in infected macrophages.

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Macrophages obtained as previously described and seeded for 24 h at 1x105 cells/well in 16well slide chambers (Nunc). Trypomastigotes of T. cruzi obtained from LLC-MK2 cultures

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were added to the macrophages at a ratio of 1:5 (macrophage/parasite) for 4 hours at 37ºC in 5% CO2. Non-internalized parasites were removed by washing and cells incubated with

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neolignans 1 – 4 for 48 hours at 37ºC in 5% CO2. Benznidazol was used as the standard drug control. At the end of the assay, the cells were fixed in MeOH, stained with Giemsa and observed under a light microscope. The parasite burden was determined by the mean number of infected macrophages × mean number of amastigotes per macrophage out of 100 macrophages, infection index. At least 200 macrophages were counted per concentration tested.

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Late growth-phase trypomastigotes of T. cruzi (2x106/well) were washed and

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incubated in the dark with 1 µM Sytox Green probe (Molecular Probes) in HANKS' balanced salts solution (HBSS; Sigma-Aldrich) supplemented with 10 mM D-Glucose (HBSS+Glu) as described [13]. Neolignan 4 was added (t = 0 min) at IC50 (39 µM) and IC90 (106 µM) and

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fluorescence was measured every 1 hour for up to 6 hours. The maximum permeabilization was obtained with 0.5% Triton X-100. Fluorescence intensity was determined using a

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fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) with excitation and emission wavelengths of 485 and 520 nm, respectively. 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

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were tested in duplicate.

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3.14. Flow cytometry analysis

Late growth-phase trypomastigotes of T. cruzi (2x106/well) at late growth-phase were

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treated with the neolignan 4 at IC50 (39 µM) and IC90 (106 µM) in HBSS+Glu during 1 and 2 hours incubation at 26 °C. After the incubation, parasites were washed and co-stained with Rhodamine 123 (Rd123 0.3 µg/mL) and propidium iodate (PI 5 µg/mL) for 10 minutes under absence of light at 37°C in order to monitor the mitochondrial membrane potential (∆ψm) and cell viability, respectively. Flow cytometry was performed using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher) by analyzing 10,000 gated events using forward/side scatter (FSC/SSC), Rd123 fluorescence (BL1-A, filter 530/30 nm), PI fluorescence (BL2-A, filter 574/26 nm) and Attune Nxt® software included with the equipment. Unstained, Rd123-

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stained (untreated) and PI-stained (amphotericin B 1 µg/mL-treated) parasites were used to set background fluorescence and compensation. Promastigotes treated with FCCP (10 µM) and untreated were used to obtain maximal and minimal mitochondrial depolarization,

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respectively.

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3.15. Statistical analysis

The data obtained represent the mean and standard deviation of duplicate samples

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from two independent assays. IC50 and IC90 values were calculated using sigmoid dose– response curves in GraphPad Prism 5.0 software, and the 95% confidence intervals are included in parentheses. One-way ANOVA of variance with Tukey's Multiple Comparison

Acknowledgements

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Test was used for significance test (P value). All assays were repeated at least twice.

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This study is an activity within the Research Network Natural Products against Neglected Diseases (ResNetNPND: http://www.resnetnpnd.org/). This work was supported

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by grants from São Paulo State Research Foundation (FAPESP 2015/11936-2, 2015/50075-2, 2013/50228-8, and 2013/50228-8). We thank CAPES for scholarships from TACS, MKG and MLL. We also thank CNPq for the grant award to JHGL, AGT and CHA. We are grateful to ChemAxon for providing academic license of their software.

Conflict of Interest The authors declare no conflict of interest.

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References [1] World Health Organization. http://www.who.int/chagas/en/ (accessed 17/02/2017).

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[2] T.A. Costa-Silva, S.S. Grecco, F.S. de Sousa, J.H.G. Lago; E.G. Martins, C.A. Terrazas, S. Varikuti, K.L. Owens, S.M. Beverley, A.R. Satoskar, A.G. Tempone, Immunomodulatory and antileishmanial activity of phenylpropanoid dimers isolated from Nectandra leucantha. J.

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Nat. Prod. 78 (2015) 653-657.

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Mesquita, M.K. Galuppo, A.G. Tempone, B.J. Neves, C.H. Andrade, R.L.O.R Cunha, M. Uemi, P. Sartorelli, J.H.G. Lago, Antitrypanosomal activity and evaluation of the mechanism of action of dehydrodieugenol isolated from Nectandra leucantha (Lauraceae) and its methylated derivative against Trypanosoma cruzi. Phytomedicine 15 (2017) 62-67.

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[4] A.M.P. Diaz, H.E. Gottlieb, O.R. Gottlieb, Dehydrodieugenols from Ocotea cymbarum. Phytochemistry 19 (1980) 681-682.

[5] M. Suarez, J. Bonilla, A.M.P. De Diaz, H. Achenbach, Dehydrodieugenols from

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Nectandra polita. Phytochemistry 22 (1983) 609-610. [6] A. Farias-Dias, An improved high yield synthesis of dehydrodieugenol. Phytochemistry.

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27 (1988) 3008-3009.

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Neolignans isolated from Nectandra leucantha induce apoptosis in melanoma cells by disturbance in mitochondrial integrity and redox homeostasis. Phytochemistry (2017), in press. [8] E.G. Pinto, D.C. Pimenta, M.M. Antoniazzi, C. Jared, A.G. Tempone, Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi. Exp. Parasitol. 135 (2013) 655-660.

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17

349.1423

150

175

200

225

250

275

300

327.1595 325

350

2

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1

375

363.1585

341.1753

175

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379.1323 250

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300

325

350

4

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325.1344

35 Rt (min)

m/z

150

3

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0

4

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3

2

375

m/z

379.1506

365.1237

200

225

250

275

300

325

350

375

m/z

150

175

200

225

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175

250

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339.1536 325

350

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Figure 1. HPLC analysis of crude n-hexane extract from leaves of N. leucantha and

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150

301.1368

HRESIMS of neolignans 1 – 4.

m/z

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ACCEPTED MANUSCRIPT R2 6 5

R1

3

O 4'

8' 9'

1'

OMe

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2'

1 - R1 = OH; R2 = H 2 - R1 = OMe; R2 = H 3 - R1 = R2 = OH

4 - R1 = OMe; R2 = OH

3' 7'

8

2

4

5' 6'

9

7 1

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MeO

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Figure 2. Chemical structures of neolignans 1 – 4.

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***

IC50 IC90 untreated Tx-100

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100 80 60

*

40

ns

20 0

0

60

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Fluorescence (% relative to Tx-100)

120

120 180 240 300 360 420

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Time (min)

Figure 3. Plasma membrane permeability of neolignan 4 on trypomastigotes of T. cruzi. The entrance of SYTOX® Green dye was monitored fluorimetrically up to 360 min. Untreated trypomastigotes and treated with Tx-100 were used to achieve minimal and maximal

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permeabilization, respectively. IC50 and IC90 values correspond to 39 µM and 106 µM, respectively. Ns: not significant; (∗∗∗) and (∗) indicate significant differences with the control < 0.05).

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( <0.0009 and

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B A ∆Ψ m

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100 80 60

ns

40 20

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90

IC

50

IC

FC CP

un tre at ed

0

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Fluorescence (% relative to untreated)

** 120

Figure 4. Alteration of mitochondrial membrane potential of neolignan 4 in trypomastigotes

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of T. cruzi. (A) Changes of Rd123 fluorescence determined by flow cytometry and reported as percentage relative to untreated parasites. Maximal and minimal fluorescence of Rd123 were achieved by non-treatment and treatment with FCCP (10 µM), respectively. IC50 and IC90

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values correspond to 39 µM and 106 µM, respectively. Ns: not significant; (∗∗)

< 0.0033.

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(B) Flow cytometry graphs showing in y axis Rd123 fluorescence and x axis PI fluorescence.

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Table 1. Anti-T. cruzi (trypomastigote and amastigote forms) and mammalian cytotoxicity (NCTC cells) of neolignans 1 – 4.

3

4

trypomastigote

86.5

38.6

(70.7 – 105.9)

(26.7 – 55.8)

NA

>100

33.6

59.7

(26.2 – 43.1)

(27.8 – 131.0)

15.2

39.2

(9.7 – 23.7)

(28.8 – 53.3)

NCTC >200

SI

amastigote trypomastigote

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2

amastigote

CC50 / µM (95% CI)

> 2.3

> 5.2

ND

> 2.0

>200

> 5.9

> 3.3

>200

> 13.1

> 5.1

>200

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1

IC50 / µM (95% CI)

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compounds

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IC50 – 50% inhibitory concentration, CC50 – 50% cytotoxic concentration, ND – not determined, NA – not active, SI – selectivity index, 95% CI – 95% confidence interval. Standard drug benznidazole - IC50 5.5 and 18.7 µM to trypomastigotes and amastigotes of T. cruzi, respectively.

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ACCEPTED MANUSCRIPT Table 2. Predicted ADMET properties and PAINS analysis of neolignans 1 - 4. Property

1

2

3

4

Inhibitor

Inhibitor

Inhibitor

Inhibitor

AMES Toxicitya

Non-mutagenic

Non-mutagenic

Non-mutagenic

Non-mutagenic

Carcinogenicitya

Non-carcinogenic

Non-carcinogenic

Non-carcinogenic

Non-carcinogenic

CYP1A2b

Inhibitor

Inhibitor

Non-inhibitor

Inhibitor

CYP2C19b

Inhibitor

Inhibitor

Inhibitor

Inhibitor

CYP2C9b

Inhibitor

Inhibitor

CYP2D6b

Inhibitor

Inhibitor

CYP3A4b

Inhibitor

hERGc

Weak blocker

P-glycoprotein

1930mg/kg (LD50) d Genotoxicitye Blood-brain barrier penetratione

bindinge Volume of

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Oral bioavailabilitye PAINSf

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Inhibitor

Inhibitor

Inhibitor

Inhibitor

Weak blocker

Non-blocker

Weak blocker

1930mg/kg

810mg/kg

2200mg/kg

Non-genotoxic

Non-genotoxic

Non-genotoxic

Penetrate

Penetrate

Penetrate

93.7%

96.4%

91.39%

98%

2.36 L/kg

1.66 L/kg

3.32 L/kg

2.14 L/kg

distributione

Inhibitor

Penetrate

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Plasma protein

Inhibitor

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Non-genotoxic

Inhibitor

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Rodent oral Toxicity

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inhibitiona

between 30%

less than 30%

between 30% less than 30%

and 70% Not

Not

and 70% Not

Not

Predictions based on: aadmetSAR [30], bOCHEM (https://ochem.eu/home/show.do), cPred-hERG [34, 35], dPROTOX [36], eACD/I-Labs (https://ilab.acdlabs.com/iLab2/), fKNIME RDKit workflow for identifying PAINS [37].

ACCEPTED MANUSCRIPT Highlights

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► Neolignans 1 – 4 were isolated from leaves of Nectandra leucantha (Lauraceae) ► Neolignans 1 – 4 displayed activity against T. cruzi and reduced cytotoxicity. ► Neolignan 4 showed selectivity against trypomastigotes (> 5) and amastigotes (> 13). ► Neolignan 4 alters plasma membrane permeability and mitochondrial dysfunction. ► None of the isolated neolignans was predicted as interference compounds (PAINS).