In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis

Vanessa Kaplum; Juliana Cogo; Diego Pereira Sangi; Tânia Ueda-Nakamura; Arlene Gonçalves Corrêa; Celso Vataru Nakamura

doi:10.1128/AAC.02582-15

. 2016 May 23;60(6):3433–3444. doi: 10.1128/AAC.02582-15

In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis

Vanessa Kaplum ^a, Juliana Cogo ^a, Diego Pereira Sangi ^b,^c, Tânia Ueda-Nakamura ^a, Arlene Gonçalves Corrêa ^c, Celso Vataru Nakamura ^a,^✉

PMCID: PMC4879393 PMID: 27001812

Abstract

Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis. The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (ΔΨm), mitochondrial superoxide anion (O₂·⁻) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ΔΨm, an increase in the O₂·⁻ concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of action of LSPN329 and LSPN331 and the decrease in lesion thickness in vivo brought about by LSPN329 and LSPN331 make them potential candidates for new drug development for the treatment of leishmaniasis.

INTRODUCTION

Neglected tropical diseases largely affect economically and socially marginalized populations in developing countries (1). Leishmaniasis is endemic in 98 countries and territories worldwide, with approximately 350 million people being at risk of infection and 12 million people currently being infected (2).

Leishmaniasis is caused by over 20 species of the protozoan Leishmania, which is divided into two developmental forms: promastigotes and amastigotes (3). Promastigotes are characterized by elongation and a free flagellum. They are transmitted during blood feeding by sandflies of the genus Lutzomyia in the New World and sandflies of the genus Phlebotomus in the Old World (4). Amastigotes are characterized by a rounded body with a short flagellum and are located inside the parasitophorous vacuoles of macrophages (5, 6).

The clinical manifestations of leishmaniasis can be classified primarily as cutaneous, mucocutaneous, and visceral (7). Cutaneous leishmaniasis predominates, with 1.5 million new cases occurring per year (2). Lesions appear as papules that may progress to nodules and even severe forms characterized by ulcerated lesions (8, 9). In Brazil, the most relevant species are Leishmania amazonensis, L. guyanensis, L. braziliensis, and L. chagasi, and approximately 30,000 cases were reported in 2010 alone (2, 10).

The first-line treatment of cutaneous leishmaniasis is pentavalent antimonials, represented by meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam) (11). Among the second-line drugs are miltefosine (the first antileishmanial drug that could be orally administered), pentamidine, and amphotericin B (the liposomal or free form), despite its high level of toxicity (12).

Quinoxaline derivatives have promising biological properties because of their privileged scaffold (13 –15). Several biological effects have been reported, including anticancer (14, 16, 17), antimicrobial (18 –22), antiviral (23), and anti-inflammatory and antioxidant (24, 25) effects.

The antiprotozoal activities of quinoxalines, especially their antitrypanosomatid activities, such as those of 1,4-di-N-oxide quinoxaline (26), 3-trifluoromethylquinoxaline N,N′-dioxides (27), and 3-aminoquinoxaline-2-carbonitrile 1,4-dioxides to antimony(III) (28), have been reported. The antileishmanial activities of the quinoxaline derivatives 4-alkapolynylpyrrolo[1,2-α]quinoxalines (29, 30) and 1,4-di-N-oxide quinoxaline (13, 26, 31, 32) have also been reported. We recently reported the activity of 2,3-disubstituted quinoxaline derivatives against Trypanosoma cruzi and L. amazonensis (33, 34).

The currently available therapeutic options for the treatment of cutaneous leishmaniasis have serious side effects, a long treatment duration, and variability in efficacy (35). The search for new therapeutic options is important, and quinoxaline derivatives are among the promising compounds. The aim of the present study was to investigate the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives, characterize the biochemical changes induced by these compounds in promastigotes, and evaluate their in vivo antileishmanial activities in a murine cutaneous leishmaniasis model.

MATERIALS AND METHODS

Chemicals.

The following chemicals, assay kits, and medium were used: actinomycin D, amphotericin B, antimycin A (AA), fetal bovine serum (FBS), carbonyl cyanide m-chlorophenylhydrazone (CCCP), the Cell Titer-Glo luminescent cell viability assay, dimethyl sulfoxide (DMSO), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate), rhodamine 123 (Rh 123), RPMI 1640 medium (Gibco Invitrogen, Grand Island, NY, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3,8-phenanthridinediamine-5-(6-triphenyl-phosphoniumhexyl)-5,6-dihydro-6-phenyl (MitoSOX Red kit), propidium iodide (PI), and a terminal deoxynucleotidyltransferase-mediated dUTP-bitoin nick end labeling (TUNEL) kit. All of the other reagents were of analytical grade. All synthetic compounds are known, were identified by the comparison of their spectroscopic data with those reported in the literature (34, 36 –39), and showed purity rates greater than 99% by gas chromatography.

Synthesis and preparation of quinoxalines LSPN329 and LSPN331.

The quinoxalines compounds 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) were synthesized as previously described by Cogo et al. (34). Stock solutions of LSPN329 and LSPN331 were aseptically prepared in DMSO (the concentration used in the experiments did not exceed 1%) and diluted in culture medium. The concentrations of LSPN329 and LSPN331 that were used in the assays were based on the concentration that inhibited cell growth by 50% (IC₅₀) and IC₉₀ values.

Animals.

Male BALB/c mice (weight, 20 to 25 g) between 4 and 6 weeks of age were obtained from the Universidade Estadual de Maringá (UEM; PR, Brazil). The animals were maintained at room temperature (22 ± 2°C) on a constant 12-h day/12-h night cycle. Food and water were given ad libitum. The Institutional Ethics Committee of the Universidade Estadual de Maringá approved all procedures adopted in this study (protocol 029/2014).

Parasites and cell culture.

Leishmania amazonensis (strain WHOM/BR/75/JOSEFA) promastigotes were maintained at 25°C in Warren's medium (brain heart infusion plus hemin and folic acid, pH 7.2) supplemented with 10% heat-inactivated FBS. J774-A1 macrophages were maintained at 37°C under a 5% CO₂ atmosphere in RPMI 1640 medium (pH 7.2) supplemented with 10% heat-inactivated FBS.

Activity against promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (1 × 10⁶ cells/ml) were treated with LSPN329 and LSPN331 (1, 10, 50, and 100 μM) for 72 h at 25°C. The positive controls were amphotericin B and miltefosine. After incubation, promastigotes were counted in a Neubauer hemocytometer. The IC₅₀ was determined by regression analysis of the data.

Activity against intracellular amastigotes of L. amazonensis.

Peritoneal macrophages from BALB/c mice were harvested by washing with cold phosphate-buffered saline (PBS) supplemented with 3% FBS, centrifuged, and resuspended in RPMI 1640 medium supplemented with 10% FBS (5 × 10⁵ cells/ml). The macrophages were then allowed to adhere to coverslips, which were placed in 24-well tissue culture plates for 2 h at 37°C under a 5% CO₂ atmosphere. Adherent macrophages were infected with metacyclic promastigotes (3.5 × 10⁶ cells/ml) for 4 h at 34°C. Noninteriorized parasites were then removed by washing, and the infected culture was incubated with different concentrations of LSPN329 and LSPN331 (5, 10, 20, and 25 μM) for 48 h at 34°C. After incubation, the coverslips were washed, fixed with methanol, and stained with Giemsa. The percentage of infected macrophages was determined by counting by light microscopy at least 200 cells in duplicate. The parasite load was assessed by use of the association index (the percentage of infected cells multiplied by the number of intracellular amastigotes per macrophage divided by the total number of macrophages), which was used to calculate the IC₅₀, determined by regression analysis of the data.

Cytotoxicity assay in macrophages.

J774-A1 macrophages (5 × 10⁵ cells/ml) were plated in RPMI 1640 medium supplemented with 10% FBS and incubated for 24 h at 37°C under a 5% CO₂ atmosphere. The macrophages were then treated with LSPN329 and LSPN331 (10, 100, 500, and 1,000 μM) and incubated for 48 h at 37°C under a 5% CO₂ atmosphere. The macrophages were then washed in PBS. MTT (50 μl, 2 mg/ml) was added, followed by incubation for 4 h at 37°C. Formazan crystals were solubilized in DMSO, and the absorbance at 492 nm was read in a microplate reader (BioTek Power Wave XS spectrophotometer). The concentration that inhibited the absorbance by 50% compared with that obtained with the negative control (the 50% cytotoxic concentration [CC₅₀]) was determined by regression analysis of the data.

Hemolytic assay.

Human blood (type A positive) was collected, defibrinated, and washed with a saline solution of glucose. Red blood cells at 3% in glycosylated saline were inoculated in 96-well plates with different concentrations of LSPN329 and LSPN31 (25 to 200 μM). The plates were incubated for 2 h at 37°C, and the absorbance of the supernatant was read at 540 nm. To calculate the percent hemolysis, 1% Triton X-100 was used as a positive control and 1% DMSO was used as a negative control. The Institutional Ethics Committee of the Universidade Estadual de Maringá approved all procedures adopted in this study (protocol 293/2006 COPEP-UEM).

Scanning electron microscopy (SEM) of promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (10⁶ cells/ml) were treated with the IC₅₀ of LSPN329 (5.3 μM) or LSPN331 (30.0 μM). After 48 h of incubation, the cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 25°C for 2 h. The promastigotes were then placed on glass coverslips that contained poly-l-lysine and incubated for 1 h. After incubation, the coverslips were dehydrated in an ethanol gradient, dried to the critical point in CO₂, coated with gold, and observed in a Shimadzu SS-550 scanning electron microscope.

Transmission electron microscopy of promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (10⁶ cells/ml) were treated with the IC₅₀ of LSPN329 (5.3 μM) or LSPN331 (30.0 μM). After 48 h of incubation, the cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 25°C for 2 h. The promastigotes were then postfixed with 1% osmium tetroxide and 0.8% potassium ferricyanide for 60 min at room temperature and protected from light. The cells were washed with 0.1 M sodium cacodylate buffer and dehydrated in increasing concentrations of acetone. The material was embedded in increasing concentrations of Epon resin and polymerized at 60°C for 72 h. Thin sections were contrasted with uranyl acetate and lead citrate and observed in a JEM 1400 JEOL transmission electron microscope.

Assay of mitochondrial membrane potential (ΔΨm) of promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (5 × 10⁶ cells/ml) were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). The positive control was CCCP (100 μM). After 24 h of incubation, the promastigotes were centrifuged, washed, resuspended in 0.9% saline solution, and then stained with the marker Rh 123 (13.1 μM) for 15 min. After resuspension, 10,000 events were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Rutherford, NJ, USA) and the data were analyzed using CellQuest software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA, USA). Alterations in Rh 123 fluorescence were quantified using an index of variation (IV), obtained from the equation (M_T − M_C)/M_C, in which M_T is the median fluorescence emitted by the treated parasites, and M_C is the median fluorescence for the untreated parasites (control group). Negative IV values correspond to depolarization of the mitochondrial membrane, and positive IV values correspond to hyperpolarization of the mitochondrial membrane.

Detection of mitochondrion-derived superoxide anion (O₂·⁻) in promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (2 × 10⁷ cells/ml) were washed with Krebs-Henseleit (KH) solution buffer (pH 7.3; 15 mM NaHCO₃, 5 mM KCl, 120 mM NaCl, 0.7 mM Na₂HPO₄, 1.5 mM NaH₂PO₄) and then marked with 5 μM MITOSox Red for 10 min at 25°C. Marked promastigotes were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). The positive control was antimycin A (10 μM). The mitochondrion-derived O₂·⁻ concentration was measured at 0, 1, 2, 3, and 4 h. The fluorescence intensity was quantified using a fluorescence microplate reader (Victor X3; PerkinElmer) at excitation and emission wavelengths of 510 and 580 nm, respectively.

Determination of intracellular ATP concentration in promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (1 × 10⁶ cells/ml) were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). The positive control was CCCP (100 μM). After 24 h of incubation at 25°C, the promastigotes were centrifuged, washed, and resuspended in PBS. Equal volumes of CellTiter-Glo reagent (50 μl) and an aliquot of each sample (50 μl) were added to the wells of a white 96-well plate and mixed, and the plate was incubated for 10 min. The luminescence intensity was quantified using a luminescence microplate reader (Victor X3; PerkinElmer).

Determination of cell volume of promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (5 × 10⁶ cells/ml) were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). The positive control was actinomycin D (50 μM). After 24 h of incubation at 25°C, the promastigotes were centrifuged, washed, and resuspended in PBS. After resuspension, 10,000 events were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Rutherford, NJ, USA) and the data were analyzed using CellQuest software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA, USA).

Assay of phosphatidylserine externalization and cellular membrane integrity of promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (5 × 10⁶ cells/ml) were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). The positive control was CCCP (100 μM). After 24 h of incubation at 25°C, the promastigotes were centrifuged, washed, resuspended in binding buffer (140 mM NaCl, 5 mM CaCl₂, 10 mM HEPES-Na, pH 7.4), and then stained with 5 μl of the marker fluorescein isothiocyanate-conjugated annexin V for 15 min. Binding buffer and PI (3 μM) were then added. Afterward, 10,000 events were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Rutherford, NJ, USA) and the data were analyzed using CellQuest software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA, USA).

Nile red accumulation in promastigotes of L. amazonensis.

Promastigotes of L. amazonensis (5 × 10⁶ cells/ml) were treated with LSPN329 (1, 5, 10, and 20 μM) or LSPN331 (20, 40, 75, and 100 μM). After 24 h of incubation at 25°C, the promastigotes were centrifuged, washed, resuspended in PBS, and labeled with 10 μl of Nile red solution (1,000 μg/ml) for 30 min at 25°C. The fluorescence intensity was quantified using a fluorescence microplate reader (Victor X3; PerkinElmer) at excitation and emission wavelengths of 485 and 535 nm, respectively.

SEM of intracellular amastigotes of L. amazonensis.

Peritoneal macrophages that adhered to the surface of small glass coverslips were incubated with L. amazonensis promastigotes for 4 h at 34°C. For the first SEM evaluation, the samples were immediately treated with the IC₅₀ of LSPN329 (16.3 μM) or LSPN331 (19.6 μM). After 24 and 48 h of treatment, macrophages infected with intracellular amastigotes were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 12 h. The coverslips were then dehydrated in an ethanol gradient and dried to the critical point in CO₂. The slides were placed in an appropriate medium, and infected macrophages were fractured with adhesive tape, coated with gold, and observed in a Shimadzu SS-550 scanning electron microscope.

In vivo antileishmanial assay.

Male BALB/c mice were subcutaneously inoculated in the left hind footpad with 1 × 10⁷ infective promastigotes of L. amazonensis (late log phase of growth). After 8 weeks, the animals were randomly separated into 4 groups of 5 animals each, as follows: infected control mice, mice treated with 5 mg/kg of body weight/day of LSPN329 administered intralesionally, mice treated with 5 mg/kg/day of LSPN331 administered intralesionally, and mice treated with 20 mg/kg/day miltefosine administered orally. Treatment was administered daily for 3 weeks. Stock solutions of LSPN329 and LSPN331 were prepared daily in oil after previous solubilization in DMSO (final concentration, 0.1%). The volume injected in the left hind footpad was 20 μl daily. The infected control mice were administered the same number of vehicle injections (oil and DMSO). Lesion development was monitored weekly by measuring the thickness of the infected footpad with a dial caliper (150 mm; Digimess), and the thickness of the contralateral uninfected footpad was subtracted from that value.

Statistical analysis.

The data shown in the tables and the graphs in the figures are expressed as the means ± standard deviations (SDs) from at least three independent experiments. The data were analyzed using one- or two-way analysis of variance (ANOVA) followed by Dunnett's or Bonferroni's post hoc test. Values of P of ≤0.05 were considered statistically significant. The statistical analysis was performed using Prism (version 5) software (GraphPad, San Diego, CA, USA). The selectivity index (SI) was calculated as CC₅₀/IC₅₀, where CC₅₀ is the concentration that inhibited the absorbance by 50% compared with that obtained with the negative control, and IC₅₀ is the concentration that inhibits the cell growth of 50% of the promastigotes or intracellular amastigotes compared with that obtained with the negative control.

RESULTS

Synthesis of quinoxalines LSPN329 and LSPN331.

The quinoxalines 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) were synthesized as described in Fig. 1 (34) through the condensation of 1,2-diarylethanediones with o-phenylenediamine by employing ultrasound irradiation as the energy source (37). The diarylethanediones were prepared by Friedel-Crafts acylation followed by SeO₂ oxidation under microwave irradiation (36).

FIG 1 — Synthesis of 2,3-disubstituted quinoxalines LSPN329 and LSPN331. MW, microwave; EtOH, ethanol; AcOH, acetic acid; OMe, methanol; ))), the reaction was carried out under ultrasonication.

Antileishmanial activity and cytotoxicity.

The 2,3-diarylsubstituted quinoxalines LSPN329 and LSPN331 showed antileishmanial activity against the promastigote and amastigote forms of the parasite (Fig. 2). The activities against the promastigotes was dose dependent for both compounds, especially the activity of LSPN329, which had an IC₅₀ of 5.3 μM. Likewise, the activities of the two compounds against the intracellular amastigotes were dose dependent, with the IC₅₀s of LSPN329 and LSPN331 being 16.3 and 19.3 μM, respectively (Table 1).

FIG 2 — Scanning electron microscopy of promastigotes of *L. amazonensis* treated for 48 h. (A) The control group (untreated parasites), which showed a typical morphology with an elongated cell body and free flagellum; (B) parasites treated with the IC₅₀ of LSPN329 (5.3 μM); (C) parasites treated with the IC₅₀ of LSPN331 (30.0 μM), which showed morphological changes. Bars = 1 μm.

TABLE 1.

In vitro antileishmanial activities (IC₅₀s) of quinoxaline derivatives against intracellular amastigote and promastigote forms of L. amazonensis^a

Compound	CC₅₀ (μM) at 48 h	IC₅₀ (μM) for:		SI for intracellular amastigotes
Compound	CC₅₀ (μM) at 48 h	Promastigotes at 72 h	Intracellular amastigotes at 48 h	SI for intracellular amastigotes
LPSN329	203.0 ± 6.9	5.3 ± 0.7	16.3 ± 1.6	12.5
LPSN331	589.6 ± 7.8	30.0 ± 0.6	19.3 ± 4.7	30.6
Miltefosine	40.5 ± 1.7	18.5 ± 1.1	2.4 ± 0.1	16.9
Amphotericin B	3.7 ± 0.3	0.06 ± 0.00	0.42 ± 0.08	8.8

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IC₅₀, the concentration that inhibits the cell growth of 50% of the promastigotes or intracellular amastigotes compared with that of cells receiving the negative-control treatment; CC₅₀, the concentration that inhibits the absorbance by 50% compared with that for the negative control, as evaluated in J774-A1 macrophages; SI, selectivity index, which was calculated as CC₅₀/IC₅₀. The data are expressed as the means ± SDs from three experiments performed in duplicate.

A preliminary safety assessment of LSPN329 and LSPN331 was performed using a cytotoxicity assay with J774-A1 macrophages, for which CC₅₀s of 203.0 and 589.6 μM, respectively, were shown. Thus, the relationship between cytotoxicity and antileishmanial activity resulted in the SI. For promastigote forms, LSPN329 was 38.5 times more selective for the parasite than for macrophages. For intracellular amastigotes, LSPN331 had a better SI (30.6; Table 1). Another toxicity test was conducted to determine hemolytic activity, which was <0.5% for both compounds at the highest concentration tested (200 μM; data not shown).

Scanning and transmission electron microscopy of promastigotes of L. amazonensis.

Morphological changes were evaluated by SEM. The control group exhibited typical features of the parasite, with an elongated cell body and a free flagellum (Fig. 2A). In contrast, parasites that were treated with the IC₅₀ of LSPN329 (5.3 μM) and LSPN331 (30.0 μM) exhibited an altered shape, a size reduction, and a rounded cell body. Promastigotes that were treated with LSPN331 exhibited a wrinkled cell body (Fig. 2B and C).

Ultrastructural alterations in promastigotes treated with the IC₅₀ of LSPN329 (5.3 μM) included the presence of concentric membranes inside mitochondria, vesicles within the flagellar pocket, and lipid inclusions (Fig. 3B and C). Treatment with the IC₅₀ (30.0 μM) of LSPN331 revealed alterations in the mitochondria, such as intense mitochondrial swelling, concentric membrane structures inside mitochondria, vesicles within the flagellar pocket, and lipid inclusions (Fig. 3D and F). The control group exhibited no ultrastructural alterations.

FIG 3 — Transmission electron microscopy of promastigotes of *L. amazonensis* treated for 48 h. (A) The control group (untreated parasites) showed a normal ultrastructure of organelles. m, mitochondria; k, kinetoplast; n, nucleus; f, flagellum. (B, C) Treatment with the IC₅₀ of LSPN329 (5.3 μM) induced ultrastructural changes, including the presence of concentric membranes inside mitochondria (black arrow), lipid inclusions (white arrows), protrusions (white arrowhead), and vesicles (black arrowhead), inside the flagellar pocket. (D to F) Treatment with the IC₅₀ of LSPN331 (30.0 μM) induced ultrastructural changes, including mitochondrial swelling (black asterisks), concentric membranes within the mitochondria (black arrows), lipid bodies (white arrows), and vesicles (black arrowhead) inside the flagellar pocket. Bars = 0.5 μm (A) and 0.2 μm (B to F).

Assay of ΔΨm of promastigotes of L. amazonensis.

On the basis of the mitochondrial ultrastructural alterations in promastigotes treated with both compounds, we proceeded with the evaluation of the mitochondrial membrane potential (ΔΨm) by flow cytometry using the cationic marker Rh 123. Promastigotes that were treated with LSPN329 showed a dose-dependent increase in ΔΨm compared with that for promastigotes receiving the control treatment, with the increase of the greatest significance being for the highest concentration tested (20 μM; Fig. 4A to D; Table 2). Compound LSPN331 caused a significant increase in ΔΨm at concentrations of 75 and 100 μM (Fig. 4F to I; Table 2). Thus, the treatment of promastigotes with both compounds induced the hyperpolarization of the mitochondrial membrane. In contrast, the positive control, CCCP, induced the depolarization of the mitochondrial membrane (Fig. 4E and J; Table 2).

FIG 4 — ΔΨm of promastigotes of *L. amazonensis* treated with LSPN329 at concentrations of 1 μM (A), 5 μM (B), 10 μM (C), and 20 μM (D) and LSPN331 at concentrations of 20 μM (F), 40 μM (G), 75 μM (H), and 100 μM (I) for 24 h. The gray area corresponds to the control group (untreated cells). The positive control was CCCP (100 μM). (E and J) ΔΨm was assessed using the fluorescent marker Rh 123, which fluoresces when it accumulates in mitochondria. Histograms typical of those from at least three experiments performed in triplicate and conducted separately for LSPN329 and LSPN331. In all experiments, treatment with CCCP induced depolarization of the mitochondrial membrane.

TABLE 2.

ΔΨm of promastigotes of L. amazonensis treated with LSPN329 and LSPN331 for 24 h^a

Compound and control group or concn	IV^b
LSPN329
Control group	0
Positive control	−0.43 ± 0.09^c
1 μM	−0.24 ± 0.05
5 μM	0.10 ± 0.04
10 μM	0.10 ± 0.01
20 μM	0.33 ± 0.05^c
LSPN331
Control group	0
Positive control	−0.67 ± 0.05^c
20 μM	−0.01 ± 0.06
40 μM	0.50 ± 0.12
75 μM	0.75 ± 0.22^c
100 μM	1.49 ± 0.20^c

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The controls were untreated cells (control group) and cells treated with CCCP (positive control). The mitochondrial membrane potential (ΔΨm) was assessed using the fluorescent marker Rh 123, which fluoresces when it accumulates in mitochondria.

IV, index of variation, which is equal to (M_T − M_C)/M_C, where M_T is the median fluorescence emitted by the treated parasites and M_C is the median fluorescence for the untreated parasites (control group). The data represent the averages from three experiments.

The value is significantly different (P ≤ 0.05, one-way ANOVA followed by Dunnett's post hoc test) from that for the negative control (untreated cells).

Detection of mitochondrion-derived O₂·⁻ production in promastigotes of L. amazonensis.

Considering the ΔΨm results, the production of mitochondrion-derived superoxide anion (O₂·⁻) was evaluated in promastigotes treated with LSPN329 and LSPN331 using the MitoSOX Red reagent, which emits fluorescence when oxidized (oxMitoSOX). Compound LSPN329 induced an increase in the O₂·⁻ concentration at the higher concentrations of LSPN329 tested (Fig. 5A). In promastigotes treated with different concentrations of LSPN331, the O₂·⁻ concentration increased for the two highest concentrations of LSPN331 tested during the 4-h period of evaluation (Fig. 5B). The positive control, AA, also induced an increase in fluorescence.

FIG 5 — Production of mitochondrial O₂·⁻ in promastigotes of *L. amazonensis* treated with 1, 5, 10, and 20 μM LSPN329 (A) and 20, 40, 75, and 100 μM LSPN331 (B) for up to 4 h. The positive control was AA (10 μM). The amount of the mitochondrial O₂·⁻ was assessed using the fluorescent marker MitoSOX Red, which fluoresces when oxidized (oxMitoSOX). The bars represent the means ± SDs from three experiments performed in triplicate. a.u., absorbance units. *, a result significantly different (P ≤ 0.05, two-way ANOVA followed by the Bonferroni's *post hoc* test) from that for the control (untreated cells).

Determination of intracellular ATP concentration in promastigotes of L. amazonensis.

Given the effects of the compounds on ΔΨm and the increase in the O₂·⁻ concentration, the intracellular ATP concentration was evaluated in promastigotes treated with LSPN329 and LSPN331 using the CellTiter-Glo reagent. Promastigotes treated with LSPN329 showed no changes in intracellular ATP levels (Fig. 6A). On the other hand, promastigotes treated with different concentrations of LSPN331 showed a significant decrease in intracellular ATP levels in a dose-dependent manner (Fig. 6B). The positive control, CCCP, also induced a decrease in luminescence.

FIG 6 — Determination of intracellular ATP levels in promastigotes of *L. amazonensis* treated with 1, 5, 10, and 20 μM LSPN329 (A) and 20, 40, 75, and 100 μM LSPN331 (B) for 24 h. The positive control was CCCP (100 μM). Intracellular ATP levels were assessed using the luminescence CellTiter-Glo reagent. The bars represent the means ± SDs from three experiments performed in triplicate. A.U., absorbance units. *, a result significantly different (P ≤ 0.05, one-way ANOVA followed by Dunnett's *post hoc* test) from that for the control (untreated cells).

Determination of cell volume of promastigotes of L. amazonensis.

Mitochondrial alterations are suggestive of cell death by apoptosis and may cause changes in cell volume (40). Promastigotes treated with LSPN329 did not show a significant reduction of cell volume (Fig. 7A). Promastigotes treated with different concentrations of LSPN331 showed a significant reduction of cell volume for the two highest doses tested (Fig. 7B). The positive control, actinomycin D, reduced the cell volume.

FIG 7 — Cell volume of promastigotes of *L. amazonensis* treated with 1, 5, 10, and 20 μM LSPN329 (A) and 20, 40, 75, and 100 μM LSPN331 (B) for 24 h. The positive control was actinomycin D (Act. D; 50 μM). The bars represent the means ± SDs from three experiments performed in triplicate. *, a result significantly different (P ≤ 0.05, one-way ANOVA followed by Dunnett's *post hoc* test) from that for the control (untreated cells).

Phosphatidylserine externalization and cell membrane integrity in promastigotes of L. amazonensis.

The association between the increase in ΔΨm and the O₂·⁻ concentration can induce cell death by either apoptosis or necrosis (41). To identify such changes, promastigotes were treated with different concentrations of LSPN329 and LSPN331 for 24 h, double stained with annexin V and PI, and compared with promastigotes treated with the negative control. Flow cytometry revealed no significant increase in promastigotes labeled with annexin V in the upper and lower right quadrants after treatment with LSPN329 or LSPN331 (Fig. 8). Similarly, treatment with both compounds did not increase the number of promastigotes marked by PI in the right and upper left quadrants (Fig. 8). Treatment with the positive control, CCCP, showed an increase in the number of labeled cells in the right and left upper quadrants (Fig. 8F).

FIG 8 — Phosphatidylserine exposure on the cell membrane in promastigotes of *L. amazonensis* treated with LSPN329 at concentrations of 1 μM (B), 5 μM (C), 10 μM (D), and 20 μM (E) and LSPN331 at concentrations of 20 μM (G), 40 μM (H), 75 μM (I), and 100 μM (J) for 24 h. (A) Negative control (untreated parasites); (F) positive control treated with CCCP (100 μM). The percentage of annexin V-positive cells is shown in the upper and lower right quadrants. The percentage of PI-positive cells is shown in the upper right and left quadrants. Histograms typical of those from at least three independent experiments are shown.

Nile red accumulation in promastigotes of L. amazonensis.

Mitochondrial dysfunction can lead to a buildup of lipid inclusions in the cytoplasm, a characteristic find in cells in apoptosis (42). Transmission electron microscopy showed that promastigotes that were treated with the 2,3-diarylsubstituted quinoxalines exhibited an increase in the amount of lipid inclusions (Fig. 3C and F). Another way to determine the presence of lipid inclusions in promastigotes is by labeling them with Nile red. Promastigotes that were treated with LSPN329 showed a dose-dependent increase in Nile red accumulation compared with that for promastigotes receiving the control treatment, with a significant increase being seen for the highest concentrations tested (Fig. 9A). LSPN331 at concentrations of 75 and 100 μM caused a significant increase in lipid inclusions (Fig. 9B).

FIG 9 — Lipid inclusions in promastigotes of *L. amazonensis* treated with 1, 5, 10, and 20 μM LSPN329 (A) and 20, 40, 75, and 100 μM LSPN331 (B) for 24 h. Lipid inclusions were assessed using the fluorescent marker Nile red. The bars represent the means ± SDs from three experiments performed in triplicate. *, a result significantly different (P ≤ 0.05, one-way ANOVA followed by Dunnett's *post hoc* test) from that for the control (untreated cells).

Scanning electron microscopy of intracellular amastigotes of L. amazonensis.

After the fracture of peritoneal macrophages, intracellular amastigotes in the control group showed typical features, such as a rounded cell body (Fig. 10A and D). In contrast, parasites that were treated with the IC₅₀ of LSPN329 (16.3 μM) and LSPN331 (19.3 μM) for 24 h exhibited alterations in size and shape, wrinkling, and extravasation of cytoplasmic material (Fig. 10B and C). With 48 h of treatment, alterations in the shape of the cell body were observed, with the presence of residues within the parasitophorous vacuole being observed (Fig. 10E and F).

FIG 10 — Scanning electron microscopy of intracellular amastigotes of *L. amazonensis* in peritoneal macrophages treated for 24 h (A to C) and 48 h (D to F). (A, D) Control group (untreated parasites); (B, E) parasites treated with the IC₅₀ of LSPN329 (16.3 μM); (C, F) parasites treated with the IC₅₀ of LSPN331 (19.3 μM). The treatments caused morphological changes in intracellular amastigotes, especially in treatments with LSPN329 and LSPN331 for 48 h (white arrows). Bars = 1 μm.

In vivo antileishmanial assay.

The in vivo antileishmanial activity of LSPN329 and LSPN331 was evaluated in a murine cutaneous leishmaniasis model. BALB/c mice were infected in the footpad with L. amazonensis. Animals were treated intralesionally with 5 mg/kg/day of LSPN329 or LSPN331 every day for 3 weeks. Lesion development was monitored weekly by measuring the footpad thickness. Treatment with LSPN329 and LSPN331 resulted in a significant decrease in lesion thickness compared to that obtained with the control treatment (Fig. 11). Miltefosine administered orally (20 mg/kg/day) also decreased the lesion thickness compared to that obtained with the control treatment (Fig. 11).

FIG 11 — *In vivo* antileishmanial assay of LSPN329 and LSPN331. BALB/c mice were infected with 1 × 10⁷ infective promastigotes of *L. amazonensis* (late log phase of growth). The treatments included an infected control treatment, LSPN329 administered intralesionally (5 mg/kg/day), LSPN331 administered intralesionally (5 mg/kg/day), and miltefosine administered orally (20 mg/kg/day). The treatments were administered daily for 3 weeks, and lesion thickness (in millimeters) was monitored weekly. The data points represent the means ± SDs. The mean was the average difference between the contralateral uninfected footpad and the infected footpad (five mice per group). *, a result significantly different (P ≤ 0.05, two-way ANOVA followed by Bonferroni's *post hoc* test) from that for the infected control.

DISCUSSION

The 2,3-diarylsubstituted quinoxalines showed significant antiproliferative activity against promastigote and intracellular amastigote forms of L. amazonensis. In promastigotes, the ultrastructural changes and biochemical results suggest the occurrence of mitochondrial alterations associated with increases in lipid inclusions. Furthermore, evaluation of the in vivo antileishmanial activity resulted in a significant decrease of lesion thickness in a murine cutaneous leishmaniasis model.

In the present study, the activities of LSPN329 and LSPN331 against L. amazonensis promastigotes resulted in IC₅₀s of 5.3 and 30.0 μM, respectively. For intracellular amastigotes, the SIs were 12.5 for LSPN329 and 30.6 for LSPN331. These are promising results, given that both compounds proved to be more selective for intracellular amastigotes than amphotericin B, one of the drugs of choice for the treatment of leishmaniasis (35). Previous studies reported the activities of quinoxaline derivatives against evolutionary forms of Leishmania spp., further attesting to the potential of these compounds (13, 30 –32). On the other hand, the in vitro effects of LSPN329 or LSPN331 in combination with miltefosine indicate that they have indifferent interactions in promastigotes and intracellular amastigotes of L. amazonensis (data not shown). The low cytotoxicity of quinoxaline derivatives for LLCMK₂ cells has previously been reported by our group, with the CC₅₀s being 471.8 μM for LSPN329 and 330.6 μM for LSPN331 (34). The data on the cytotoxicities of both compounds for J774-A1 macrophages and LLCMK₂ cells indicated that they had lower cytotoxicities than amphotericin B and miltefosine.

In addition to the evaluation of in vitro cytotoxicity, the evaluation of hemolytic activity is also important when the safety of specific compounds is investigated (33). Here the hemolytic activity was <0.5% for both LSPN329 and LSPN331 at the highest concentration of each compound tested in our experiments. Treatment with amphotericin B at concentrations that were approximately four times lower (50.8 μM) caused 50% hemolysis of erythrocytes (43).

The ultrastructural alterations observed in promastigotes treated with LSPN329 and LSPN331 were characterized by intense mitochondrial changes, mainly the presence of concentric membranes inside the organelle, loss of the mitochondrial matrix, and mitochondrial swelling. Considering that trypanosomatids have only one mitochondrion, such an organelle may be considered a potential therapeutic target (44). Several studies have reported that different classes of compounds (40, 45 –47), including quinoxaline derivatives (27), cause mitochondrial dysfunction in trypanosomatids.

In addition to ultrastructural changes, biochemical dysfunction, reflected by ΔΨm, which is an indicator of cellular damage, was observed (48). We also observed an increase in marking by Rh 123 in promastigotes treated with LSPN329 and LSPN331, indicating mitochondrial membrane hyperpolarization. Hyperpolarization is considered to be less common than mitochondrial depolarization and is best described for mammalian cells, arising mainly from dysfunctions in F_o/F₁ ATPase (48). A few studies have reported mitochondrial hyperpolarization in promastigote forms, such as with camptothecin treatment (49), rotenone treatment (50), conditional heat stress (19), and treatment with bispyridinium derivatives (51).

Reactive oxygen species are formed naturally in mitochondria, but mitochondrial hyperpolarization causes an increase in the level of O₂·⁻ production in mammalian cells (52). However, in promastigotes of Leishmania spp., the occurrence of hyperpolarization and the increase in the level of O₂·⁻ production may not be directly related (19, 50). Treatment with bispyridinium derivatives resulted in concomitant hyperpolarization and an increase in the O₂·⁻ concentration (51). Likewise, mitochondrial membrane hyperpolarization and a significant increase in the O₂·⁻ concentration was found in promastigotes treated with 2,3-diarylsubstituted quinoxalines. Other work has also presented information on the mitochondrial damage associated with the increase in the O₂·⁻ concentration only at the highest concentrations tested and concluded that mitochondrial dysfunction was the main cause of cell death (47, 53).

Intracellular ATP levels are susceptible to alterations when there is mitochondrial damage, such as changes in the mitochondrial membrane potential and an increase in the levels of reactive oxygen species (51, 54). Promastigotes treated with LSPN331 showed a significant decrease in intracellular ATP levels in a dose-dependent manner. However, promastigotes treated with LSPN329 showed no changes in intracellular ATP levels.

Ultrastructural changes in the mitochondria of the parasite, together with changes in ΔΨm (both depolarization and hyperpolarization), a decrease in intracellular ATP levels, and the accumulation of reactive oxygen species, are considered signs of apoptosis (55).

Thus, the signs of apoptotic cell death that were evaluated included cell volume, DNA fragmentation, and phosphatidylserine exposure on the cell membrane (56). The cell volume of promastigotes treated with LSPN329 was slightly reduced. However, promastigotes treated with LSPN331 showed a significant reduction in cell volume. An alteration of the cell volume was also revealed by SEM. Just as with 2,3-diarylsubstituted quinoxalines, the compound 3-chloro-7-methoxy-2-(methylsulfonyl)quinoxaline (LSPN337) caused changes in cell volume in epimastigotes of T. cruzi only at higher concentrations (33).

In the DNA fragmentation assay using TUNEL staining, no DNA fragmentation was found in promastigotes that were treated with LSPN329 or LSPN331 for 72 h (data not shown). To confirm the absence of type I programmed cell death or apoptosis, we evaluated the exposure of phosphatidylserine at the cell membrane using annexin V; however, the promastigotes were not marked. Promastigotes that were treated with the 2,3-diarylsubstituted quinoxalines showed no marking characteristic of apoptosis-like death.

The association between hyperpolarization and increased reactive oxygen species generation may also result in the initiation and occurrence of necrotic cell death (41). The lack of integrity of the cell membrane is one of the signs of cell death by necrosis (57).

Scanning electron microscopy of promastigotes treated with LSPN329 and LSPN331 showed alterations in shape, such as a size reduction and cell body rounding. However, no cell membrane alterations were observed. Thus, PI labeling was absent in treated cells, indicating that the cell membrane was intact after treatment. This was also seen with T. cruzi epimastigotes treated with LSPN337 (33).

Transmission electron microscopy showed that promastigotes treated with LSPN329 and LSPN331 exhibited an increase in the amount of lipid inclusions. This was confirmed by Nile red staining, in which a dose-dependent increase in the amount of lipid inclusions was observed, in addition to the random distribution of droplets throughout the cytoplasm of the cells. The increase in lipid inclusions may be related to changes in phospholipids and sterols in promastigotes of L. amazonensis (58). Lipid inclusions in the cytoplasm can be caused by mitochondrial dysfunction (42).

Despite mitochondrial damage and alterations, neither DNA fragmentation nor phosphatidylserine on the cell membrane was observed, thus ruling out the possibility of cell death by apoptosis. Given the cell membrane integrity, cell death was also not caused by necrosis. Thus, the treatment of L. amazonensis promastigotes with LSPN329 and LSPN331 caused a multifactorial response that culminated in cellular collapse, characterized by intense mitochondrial changes. Similar results were observed with the treatment of promastigotes with the iron chelator 2,2-dipyridyl and 4-amino bispyridinium derivatives (51, 59).

Scanning electron microscopy studies of amastigotes of Leishmania spp. have generally evaluated parasites that are external to the host cell (60, 61). In the present study, morphological changes were evaluated in intracellular amastigotes that were present in parasitophorous vacuoles of peritoneal macrophages. The changes were characterized by wrinkling, the extravasation of cytoplasmic material, and alterations in the size and shape of the cell body. Transmission electron microscopy will be performed to corroborate the changes observed by SEM.

On the basis of the in vitro selectivity of the 2,3-diarylsubstituted quinoxaline derivatives, especially in intracellular amastigotes, the in vivo activity was evaluated in a murine cutaneous leishmaniasis model. BALB/c mice treated intralesionally with LSPN329 or LSPN331 showed significant decreases in lesion thickness compared to that for the control mice. Oral miltefosine also decreased the thickness of the lesion in the footpad.

Although the mechanism of action of 2,3-diarylsubstituted quinoxalines in promastigotes of L. amazonensis has not been fully elucidated, intense mitochondrial changes were observed, together with an increase in lipid inclusions that resulted in a multifactorial response that may be responsible for cellular collapse.

The considerable selectivity of 2,3-diarylsubstituted quinoxalines for intracellular amastigotes of L. amazonensis in vitro and the significant decrease in lesion thickness in a murine cutaneous leishmaniasis model suggest that such compounds may be candidate therapeutic agents for leishmaniasis.

Conclusion.

The 2,3-diarylsubstituted quinoxalines were selective for promastigotes and intracellular amastigotes of L. amazonensis. For promastigote forms, the multifactorial response that was responsible for cellular collapse was based on intense mitochondrial alterations. Because of their in vitro antiprotozoal activity, the significant decrease in lesion thickness in a murine cutaneous leishmaniasis model, and the mitochondrial mechanism of action, such compounds may be candidates for therapeutic drug development for the treatment of leishmaniasis.

ACKNOWLEDGMENTS

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Capacitação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP grants 13/50680-8 and 13/07600-3), GlaxoSmithKline Trust in Science Project, and Programa de Pós-Graduação em Ciências Farmacêuticas.

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PERMALINK

In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis

Vanessa Kaplum

Juliana Cogo

Diego Pereira Sangi

Tânia Ueda-Nakamura

Arlene Gonçalves Corrêa

Celso Vataru Nakamura

Abstract

INTRODUCTION

MATERIALS AND METHODS

Chemicals.

Synthesis and preparation of quinoxalines LSPN329 and LSPN331.

Animals.

Parasites and cell culture.

Activity against promastigotes of L. amazonensis.

Activity against intracellular amastigotes of L. amazonensis.

Cytotoxicity assay in macrophages.

Hemolytic assay.

Scanning electron microscopy (SEM) of promastigotes of L. amazonensis.

Transmission electron microscopy of promastigotes of L. amazonensis.

Assay of mitochondrial membrane potential (ΔΨm) of promastigotes of L. amazonensis.

Detection of mitochondrion-derived superoxide anion (O2·−) in promastigotes of L. amazonensis.

Determination of intracellular ATP concentration in promastigotes of L. amazonensis.

Determination of cell volume of promastigotes of L. amazonensis.

Assay of phosphatidylserine externalization and cellular membrane integrity of promastigotes of L. amazonensis.

Nile red accumulation in promastigotes of L. amazonensis.

SEM of intracellular amastigotes of L. amazonensis.

In vivo antileishmanial assay.

Statistical analysis.

RESULTS

Synthesis of quinoxalines LSPN329 and LSPN331.

FIG 1.

Antileishmanial activity and cytotoxicity.

FIG 2.

TABLE 1.

Scanning and transmission electron microscopy of promastigotes of L. amazonensis.

FIG 3.

Assay of ΔΨm of promastigotes of L. amazonensis.

FIG 4.

TABLE 2.

Detection of mitochondrion-derived O2·− production in promastigotes of L. amazonensis.

FIG 5.

Determination of intracellular ATP concentration in promastigotes of L. amazonensis.

FIG 6.

Determination of cell volume of promastigotes of L. amazonensis.

FIG 7.

Phosphatidylserine externalization and cell membrane integrity in promastigotes of L. amazonensis.

FIG 8.

Nile red accumulation in promastigotes of L. amazonensis.

FIG 9.

Scanning electron microscopy of intracellular amastigotes of L. amazonensis.

FIG 10.

In vivo antileishmanial assay.

FIG 11.

DISCUSSION

Conclusion.

ACKNOWLEDGMENTS

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Detection of mitochondrion-derived superoxide anion (O₂·⁻) in promastigotes of L. amazonensis.

Detection of mitochondrion-derived O₂·⁻ production in promastigotes of L. amazonensis.