Imidazolium salts as an alternative for anti-Leishmania drugs: Oxidative and immunomodulatory activities

In this study we explored the previously established leishmanicidal activity of a complementary set of 24 imidazolium salts (IS), 1-hexadecylimidazole (C₁₆Im) and 1-hexadecylpyridinium chloride (C₁₆PyrCl) against Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) infantum chagasi. Promastigotes of L. amazonensis and L. infantum chagasi were incubated with 0.1 to 100 μM of the compounds and eight of them demonstrated leishmanicidal activity after 48 h – C₁₀MImMeS (IC_{50 L. amazonensis} = 11.6), C₁₆MImPF₆(IC_{50 L. amazonensis} = 6.9), C₁₆MImBr (IC_{50 L. amazonensis} = 6), C₁₆M₂ImCl (IC_{50 L. amazonensis} = 4.1), C₁₆M₄ImCl (IC_{50 L. amazonensis} = 1.8), (C₁₀)₂MImCl (IC_{50 L. amazonensis} = 1.9), C₁₆Im (IC_{50 L. amazonensis} = 14.6), and C₁₆PyrCl (IC_{50 L. amazonensis} = 4).The effect of IS on reactive oxygen species production, mitochondrial membrane potential, membrane integrity and morphological alterations of promastigotes was determined, as well as on L. amazonensis-infected macrophages. Their cytotoxicity against macrophages and human erythrocytes was also evaluated. The IS C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl, and the compounds C₁₆Im and C₁₆PyrCl killed and inhibited the growth of promastigote forms of L. amazonensis and L. infantum chagasi in a concentration-dependent manner, contributing to a better understanding of the structure-activity relationship of IS against Leishmania. These IS induced ROS production, mitochondrial dysfunction, membrane disruption and morphological alterations in infective forms of L. amazonensis and killed intracellular amastigote forms in very low concentrations (IC_{50 amastigotes} ≤ 0.3), being potential drug candidates against L. amazonensis.

1 Introduction

Leishmaniasis are endemic in 102 countries or territories with around 1.3 million new cases and 20.000 to 30.000 deaths yearly (1). Brazil is one of the countries with the highest incidence of leishmaniasis (2). Leishmania (Leishmania) amazonensis is an important etiological agent of cutaneous, anergic diffuse cutaneous and less commonly mucocutaneous leishmaniasis, especially in South America (3, 4), and Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis, mainly in the Mediterranean Basin, Middle East, Central Asia and South America (4).

Currently, there is no vaccine for leishmaniasis, and their treatment is based on very toxic and poorly tolerated drugs, such as pentavalent antimonials and amphotericin B (5). Azole antifungals presented leishmanicidal activity against some Leishmania species (6). Moreover, various biological activities have been described for the cationic equivalent of imidazoles, imidazolium salts (IS), including antimicrobial (7), antifungal (8, 9), larvicidal (10), antitumoral, antioxidant and others (11). The IS present high structural variability and tunable interactions with biomolecules like phospholipids and proteins, especially because their amphiphilic nature (11), becoming potential candidates in medicinal chemistry (12). Recently, our group described for the first time the leishmanicidal activity of IS (13). Within the tested IS, 1-methyl-3-octadecylimidazolium chloride (C₁₈MImCl), 1-hexadecyl-3-methylimidazolium methanesulfonate (C₁₆MImMeS), 1-hexadecyl-3-methylimidazolium chloride (C₁₆MImCl), 1-decyl-3-methylimidazolium chloride (C₁₀MImCl) and 1-hexadecyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (C₁₆MImNTf₂) were identified as potent agents against both promastigote and amastigote forms of L. amazonensis. Here, we further explored this leishmanicidal action with a structurally complementary set of 24 IS, 1-hexadecylimidazole (C₁₆Im) and 1-hexadecylpyridinium chloride (C₁₆PyrCl) (Figure 1) to (i) investigate the structure-activity relationship of these molecules on promastigote and amastigote forms of L. amazonensis and promastigote forms of L. infantum chagasi, (ii) identify mortality pathways, and (iii) evaluate cytotoxicity.

FIGURE 1

Figure 1 Chemical structures of the imidazolium salts, C₁₆Im and C₁₆PyrCl screened for anti-Leishmania activity tested in this work, and imidazolium salts studied previously for anti-Leishmania activity (ref 13). The compounds were given sample codes that contain information on their chemical structures. For instance, for C₁₈MImCl: (1) C₁₈ = N-octadecyl substituent; (2) M = N-methyl substituent; (3) Im = imidazolium cation; and (4) Cl = chloride anion.

2 Materials and methods

2.1 Imidazolium salts and reference compounds

1,3-Didecyl-2-methylimidazolium chloride ((C₁₀)₂MImCl) (CJC China JIE Chemical), 1-hexadecylpyridinium chloride monohydrate (C₁₆PyrCl.H₂O) (Sigma-Aldrich), and 1-(2-hydroxyethyl)-3-methylimidazolium chloride (HOC₂MImCl) (IOLITEC) were purchased and used as received.

1-Butyl-3-methylimidazolium methanesulfonate (C₄MImMeS) (14), 1-decyl-3-methylimidazolium methanesulfonate (C₁₀MImMeS) (15), 1-hexadecyl-3-methylimidazolium bromide (C₁₆MImBr) (16), 1-butyl-3-methylimidazolium tetrafluoroborate (C₄MImBF₄) (14), 1,10-bis(methylimidazolium-1-yl) decane bromide (MImC₁₀MImBr₂) (16), 1-triethylene glycol monomethyl ether-3-methylimidazolium hexafluorophosphate (C₇O₃MImPF₆) (15), 1-triethylene glycol monomethyl ether-3-methylimidazolium methanesulfonate (C₇O₃MImMeS) (15), 1-butyl-2,3-dimethylimidazolium methanesulfonate (C₄M₂ImMeS) (17), 1-hexadecylimidazole (C₁₆Im) (18), 1,3-dihexadecylimidazolium chloride ((C₁₆)₂ImCl) (19), 1-(3-hydroxypropyl)-3-methylimidazolium hexafluorophosphate (HOC₃MImPF₆) (20), 1-carboxymethyl-3-methylimidazolium chloride (HO₂CC₁MImCl) (21), 1,3-bis(carboxymethyl)imidazolium chloride ((HO₂CC₁)₂ImCl) (21), 1-carboxymethyl-3-methylimidazolium tetrafluoroborate (HO₂CC₁MImBF₄) (22), 1-carboxypropyl-3-methylimidazolium chloride (HO₂CC₃MImCl) (23), 1-hexadecyl-3-methylimidazolium hexafluorophosphate (C₁₆MImPF₆) (24), 1-hexadecyl-2,3,4,5-tetramethylimidazolium chloride (C₁₆M₄ImCl) (25), decyl methanesulfonate (C₁₀MeS) (15), hexadecyl methanesulfonate (C₁₆MeS) (26), butyl methanesulfonate (C₄MeS) (14), 1-hexadecyl-2-methylimidazole (C₁₆MIm) (27), and triethylene glycol monomethyl ether methanesulfonate (C₇O₃MeS) (15) were synthesized according to previously reported procedures. C₄MeS, C₁₀MeS, C₁₆MeS, C₁₆Im, C₁₆MIm, and C₇O₃MeS were used as source for the syntheses of C₄MImMeS, C₄M₂ImMeS, C₁₀MImMeS, (C₁₆)₂ImCl, C₇O₃MImMeS, 1,3-dihexadecylimidazolium methanesulfonate (C₁₆)₂ImMeS, 1,3-dihexadecyl-2-methylimidazolium chloride ((C₁₆)₂MImCl), and 1,3-dihexadecyl-2-methylimidazolium methanesulfonate ((C₁₆)₂MImMeS). Literature procedures were used as basis for the synthesis of 1-hexadecyl-2,3-dimethylimidazolium chloride (C₁₆M₂ImCl) (15), 1,5-bis(methylimidazolium-1-yl) pentane bromide (MImC₅MImBr₂) (16), 1,8-bis(methylimidazolium-1-yl) octane bromide (MImC₈MImBr₂) (16), (C₁₆)₂ImMeS (15), (C₁₆)₂MImCl (15) and (C₁₆)₂MImMeS (15), which are yet unpublished IS. See the Supporting Information for further details. Before use, the IS were dried under vacuum at 60°C for 5 h to remove residual water.

2.2 Parasite culture

Leishmania (L.) amazonensis (MHOM/BR/73/M2269 strain) and Leishmania (L.) infantum chagasi (MHOM/BR/74/PP75 strain) were cultured at 26 °C as previously described elsewhere (28). To avoid the loss of in vitro infectivity of promastigotes, samples of parasites were kept cryopreserved in liquid nitrogen soon after isolation of infected animals (BALB/c or hamsters, respectively). All experiments were performed using parasites with few passages of in vitro growth.

2.3 Anti-promastigote activity

The effect of IS, C₁₆Im and C₁₆PyrCl on L. amazonensis and L. infantum chagasi was evaluated as previously described (28). Promastigotes of L. amazonensis in the stationary phase were distributed in 96-well microplates (3 x 10⁶ cells/well) and incubated with M199 medium or 100 μM (screening concentration) of tested compound (IS, C₁₆Im or C₁₆PyrCl) for 44 h at 26 °C. Subsequently, 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich^®, St. Louis, MO, USA) was added and the incubations continued for an additional 4 h at 26 °C. The purple formazan product that was formed by the action of mitochondrial enzymes in living cells was solubilized by the addition of acidic isopropanol, and the absorbance at 570 nm was measured using a microplate reader spectrophotometer EZ Read 400 from Biochrom. The compounds that caused 100% of L. amazonensis mortality in the screening test (C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl, (C₁₀)₂MImCl, C₁₆Im and C₁₆PyrCl) were selected for additional tests against L. amazonensis and L. infantum chagasi at concentrations of 0.1 to 100 μM. The IS C₁₀MImCl, C₁₆MImCl and C₁₆MImMeS, tested against L. amazonensis in our previous study (13), were tested here against promastigotes of L. infantum chagasi. The 50% inhibitory concentration (IC₅₀) value for each compound was determined. The survival rate was calculated according to the formula: OD in treated group/OD of untreated group x 100. The 50% inhibitory concentration (IC₅₀) value for each compound was determined by nonlinear regression analysis using GraphPad Prism 5.03 software. The activity of each compound was compared with control samples incubated with culture medium (M199 medium for L. amazonensis and Schneider’s medium for L. infantum chagasi) containing less than 0.05% of polysorbate 80 (Sigma-Aldrich) or amphotericin B at 5 µM (100% mortality: Sigma-Aldrich) as reference antileishmanial drug.

2.4 Evaluation of leishmania growth

Promastigote forms of L. amazonensis or L. infantum chagasi in the stationary growth phase were distributed in 12-well microplates at a density of 1 x 10⁵ mL^-1 in medium (control) (M199 medium for L. amazonensis and Schneider’s medium for L. infantum chagasi) or medium plus C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl, (C₁₀)₂MImCl, C₁₆Im or C₁₆PyrCl at the IC₅₀ found after 48 h of incubation, and the parasite growth at 26 °C was evaluated daily by motility and cell density using a hemocytometer (28).

2.5 Field emission scanning electron microscopy

To evaluate parasite ultrastructural alterations by scanning electron microscopy (SEM), promastigotes of L. amazonensis in the stationary phase were distributed in 96-well microplates (3 x 10⁶ cells/well) and incubated at 26 °C with M199, M199 containing IS at the IC₅₀ or amphotericin B at 0.4 µM (IC₅₀) for 6 h. The parasites were washed with phosphate buffered saline (PBS) and fixed with 25% glutaraldehyde in 0.2 M phosphate buffer pH 7.4. After being fixed three times with the same buffer, the parasites were adhered to glass slides previously coated with 0.1% aqueous poly-L-lysine for 30 min at room temperature. Subsequently, the parasites were washed three times with 0.2 M phosphate buffer pH 7.4 and post-fixed in a solution of 2% OsO₄ with 0.2 M phosphate buffer (1:1) for 45 min at room temperature. All samples were dehydrated in a graded series of acetone in water from 30% to 100%, critical point dried using CO₂, mounted on metal stubs, and coated with gold for observation in a field emission scanning electron microscope (Inspect F50, FEI) (29). In addition, the IS C₁₆MImMes and C₁₆MImCl, tested in our previous study (13), were also analyzed.

2.6 Evaluation of cell membrane integrity

L. amazonensis promastigotes (2 x 10⁵) in the logarithmic growth phase were distributed in 96-well microplates and either untreated, treated with IS (2-fold IC₅₀ for 48 h), hydrogen peroxide (H₂O₂) 2 mM or amphotericin B (0.4 μM) as positive control. After 48 h at 26°C, the parasites were harvested and washed twice with PBS, resuspended and incubated with 100 μL of propidium iodide (PI – Sigma–Aldrich) 50 μg/mL at 26°C for 15 min. Following, each sample was completed with 400 μL of PBS and a total of 20,000 events were acquired and analyzed on BD FACScalibur flow cytometer and CellQuest Pro software. PI fluorescence intensity was quantified as the percentage of the fluorescence compared with untreated promastigotes (30). Results were obtained from two independent experiments performed in triplicate.

2.7 Determination of the mitochondrial membrane potential (ΔΨm)

Mitochondrial membrane potential (ΔΨm) was quantified using the fluorescent dye rhodamine 123 (Rh 123, Sigma-Aldrich) (29). Briefly, promastigote forms of L. amazonensis (2 x 10⁵) in the logarithmic growth phase were incubated with M199 medium (6 h), medium plus IS (2-fold IC₅₀ – 6 h), amphotericin B (0.4 μM - 6 h) or H₂O₂ (2 mM - 1 h) in 96-well microplates, at 26°C. These tested concentrations and time were chosen based on previous results with other IS (13). Then, cells were washed in PBS and incubated with 500 μL of Rh 123 (1 µg/mL) for 10 min at 37 °C. After being washed, they were resuspended in 0.5 mL of PBS and the analysis was performed using a BD FACSCalibur (Becton–Dickinson^®, Rutherford, NJ, USA) flow cytometer and CellQuest^® Pro software (Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA), using the blue argon-ion 488 nm laser with the FL1 filter channel. A total of 30,000 events were acquired in the region that corresponded to the parasites. Histograms were build using the CellQuest Pro software (Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA).

2.8 Detection of reactive oxygen species production

ROS generation was quantified using the dye 2′,7′-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich) and flow cytometer analysis (13, 30, 31). Briefly, promastigote forms of L. amazonensis (2 x 10⁵/well) in the logarithmic growth phase were incubated with M199 medium (6 h), M199 plus IS (2-fold IC₅₀ - 6 h), amphotericin B (0.4 μM - 6 h) or H₂O₂ (2 mM – 1 h) in 96-well microplates, at 26°C. Then, cells were washed with PBS and incubated with 0.5 mL of 10 µM DCF-DA for 30 min at room temperature. After being washed with PBS, cells were resuspended in 0.5 mL PBS and analysed using a BD FACSCalibur (Becton–Dickinson, Rutherford, NJ, USA) flow cytometer and CellQuest Pro software (Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA) using the blue argon-ion 488 nm laser with the FL1 filter channel. A total of 30,000 events were acquired in the region that corresponded to living parasite.

2.9 Intramacrophage anti-amastigote activity

The anti-amastigote assay was performed as previously described (13). Macrophages RAW 264.7 cell line (ATCC) were cultured in DMEM plus 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) until reaching 90% confluence, when were distributed into 96-well microplates at a concentration of 2 x 10⁵ cells/well in the presence of DMEM. After 12-16 h at 37°C in a humidified atmosphere of 5% CO₂, non-adherent cells were removed, and adherent macrophages were infected with promastigotes of L. amazonensis (10 parasites/cell) in the stationary phase, and 4 h later were washed with PBS to remove parasites and incubated with DMEM (control), lipopolysaccharide (LPS) (10 ng/mL) plus interferon-γ (IFN-γ (1 ng/mL) as positive control, or IS, C₁₆Im or C₁₆PyrCl at concentrations of 0.1, 1 μM or 5 μM for 48 h. After, cells were incubated with 100 µL of 0.01% (w/v) SDS solution in serum-free M199 medium at 37°C for 20 min. Then, cells were supplemented with M199 30% FBS and cultured at 26°C for 7 days to determine the number of promastigotes recovered, once only viable amastigotes are capable to differentiate to motile promastigotes. The leishmanicidal activity of macrophages was assessed by determining the number of viable parasites (4 replicates) using a hemocytometer.

2.10 Macrophage viability and hemolytic assay

RAW cells were incubated or not with IS, C₁₆Im or C₁₆PyrCl (5 to 200 μM) for 48 h at 37 °C and in vitro cytotoxicity determined by MTT assay (30). Triton X-100 1% was used as positive control. The hemolytic assay was performed according to the procedure used by (13). Briefly, red blood cells solution 1% (v/v) obtained from healthy voluntary donors (n=3) (project approved by the research ethical committee from the Universidade Federal de Ciências da Saúde de Porto Alegre - CAAE 63282416.6.0000.5345) were distributed into 96-well microplate and incubated with different IS, C₁₆Im or C₁₆PyrCl (1 to 100 μM). The microplate was incubated at 37°C under agitation (90 rpm) for 60 min. Then, the suspension was centrifuged at 3,000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm using a spectrophotometer EZ Read 400 from Biochrom. Triton X-100 1% and PBS were used as positive and negative control, respectively. The 50% cytotoxicity concentration (CC₅₀) and 50% of red blood cell hemolysis (RBC₅₀) were determined by nonlinear regression analysis. The selectivity index (SI) (CC₅₀ or RBC₅₀/IC₅₀ against L. amazonensis) were determined for each compound.

2.11 Statistical analysis

The results are expressed as mean ± standard error of the mean (SEM) from five replicates in each three independent experiments. Kolmogorov-Smirnov test was applied to verify normality. Comparisons were performed by the One-way ANOVA test followed by Bonferroni’s post-test, Two-way ANOVA repeated measures test followed by Bonferroni’s post-test. Differences were considered statistically significant when p < 0.05. All statistical tests were performed using GraphPad Prism 6.01 software.

3 Results

3.1 Imidazolium salts

The results regarding the syntheses of IS are reported in the Supplementary materials, together with ¹H NMR spectra of the synthesized compounds, and ¹³C NMR and FTIR spectra for the novel IS.

3.2 Leishmanicidal activity of the compounds on promastigotes of L. amazonensis and L. infantum chagasi

Six of the 24 IS tested (Figure 1) were able to kill promastigotes of L. amazonensis in a concentration-dependent manner (Figure S38A: Leishmanicidal activity of imidazolium salts, C₁₆Im and C₁₆PyrCl (0.1 to 100 μM) on promastigote forms of L. amazonensis, determined at 48 h incubation using MTT assay). C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl at 5 µM caused a significant decrease in the viability after 48 h, resulting in 60, 37, 42, 85, 95 and 97% of mortality, respectively. The neutral imidazole C₁₆Im and the pyridinium salt C₁₆PyrCl at concentrations ≥ 10 µM and ≥ 5 µM caused significant parasite mortality, respectively (Figure S38A). The IC₅₀ values of C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl on promastigotes of L. amazonensis were calculated to be 11.6, 6.9, 6.0, 4.1, 1.8 and 1.9 µM, respectively (Table 1). Although C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl also presented leishmanicidal activity against L. infantum chagasi (Figure S38B: Leishmanicidal activity of imidazolium salts, C₁₆Im and C₁₆PyrCl (0.1 to 100 μM) on promastigote forms of L. infantum chagasi, determined at 48 h incubation using MTT assay), the IC₅₀ values were in general higher than against L. amazonensis (Table 1). In contrast, the IC₅₀ values for C₁₆Im and C₁₆PyrCl were lower in the case of L. infantum chagasi. To reinforce the higher activity of IS towards L. amazonensis we tested the effect of IS with the best activity against L. amazonensis in our previous study (13) in promastigotes of L. infantum chagasi. The IS C₁₀MImCl, C₁₆MImCl and C₁₆MImMeS presented lower IC₅₀ values for L. amazonensis (2.31, 1.23 and 0.75 µM, respectively) (13) when compared to those values found for L. infantum chagasi (IC₅₀ = 114.9 µM, 9.9 µM and 10.8 µM, respectively; data not shown). As expected, amphotericin B at 5 µM caused 100% mortality of L. amazonensis and L. infantum chagasi.

TABLE 1

Table 1 Leishmanicidal activity, macrophage toxicity and hemolytic effect of compounds.

3.3 Imidazolium salts, C₁₆Im and C₁₆PyrCl inhibit the growth of promastigotes of L. amazonensis and L. infantum chagasi

When promastigotes of L. amazonensis or L. infantum chagasi were treated with IS, C₁₆Im or C₁₆PyrCl at IC₅₀ concentrations, a complete inhibition of the parasites growth was observed (Figures 2A, B).

FIGURE 2

Figure 2 Growth kinetics of L. amazonensis and L. infantum chagasi promastigotes treated with the different compounds. L. amazonensis (A) or L. infantum chagasi (B) promastigotes in the stationary phase (1 × 10⁵ cells/mL) were incubated with medium as control (M199 to L. amazonensis and Schneider’s medium to L. infantum chagasi) or medium plus compounds at IC₅₀ values (for 48 h incubation), at 26°C, and the growth was determined daily using a hemocytometer. Data are expressed as means ± SEM (n = 5) and are representative of three independent experiments. The Two-way ANOVA repeated measures test followed by the Bonferroni’s test were applied. ****p < 0.0001.

3.4 Imidazolium salts increase reactive oxygen species and induce mitochondrial membrane potential (ΔΨm) alterations in L. amazonensis

The treatment of promastigotes of L. amazonensis with IS at 2-fold IC₅₀ values led to a significant increase in ROS production at 6 h of incubation (Figures 3A, B). C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl caused significant ΔΨm depolarization (loss of Rh 123 fluorescence), while C₁₀MImMeS induced ΔΨm hyperpolarization (increase in Rh 123 fluorescence) (Figures 3C, D). Hydrogen peroxide at 2 mM or amphotericin B at 0.4 µM also induced significant elevation in ROS production and mitochondrial depolarization.

FIGURE 3

Figure 3 Determination of intracellular ROS (A, B) and mitochondrial membrane potential (ΔΨm) (C, D) of L. amazonensis promastigotes incubated with imidazolium salts. Promastigote forms in the logarithmic phase (2 x 10⁵ cells/well) were incubated or not with 2-fold IC₅₀ value of IS at 26°C for 6 h. M199 was used as negative control, and amphotericin B (0.4 µM) and H₂O₂ (2 mM) as positive controls. Data are expressed as means ± SEM and are representative of three independent experiments. The One-way ANOVA test followed by the Bonferroni’s test were applied. **p < 0.01, ***p < 0.001 and ****p < 0.0001 when compare with the medium group.

3.5 Imidazolium salts induce loss of plasma membrane integrity in L. amazonensis

When parasites were treated with IS at 2-fold IC₅₀ values for 48 h, a significant increase in propidium iodide labeling was observed for all IS tested, indicating that the IS interfere with membrane integrity causing cell death (Figures 4A, B). Besides, as illustrated in Figure 4C, incubation with C₁₆M₄ImCl at 1.8 µM (IC₅₀) for 48 h led to total lysis of the parasites.

FIGURE 4

Figure 4 Determination of membrane integrity of L. amazonensis promastigotes treated with imidazolium salts (A, B). Promastigote forms in the logarithmic phase (2 x 10⁵ cells/well) were incubated or not with 2-fold IC₅₀ value of IS for 48 h at 26°C. M199 was used as negative control, and amphotericin B (0.4 µM) or H₂O₂ (2 mM) as positive controls. Data are expressed as means ± SEM (n = 5) and are representative of three independent experiments. The One-way ANOVA test followed by the Bonferroni’s test were applied. ****p < 0.0001 when compare with the medium group. (C) L. amazonensis integrity after 48 h of exposure to C₁₆M₄ImCl at 1.8 μM or M199 (panoptic stain – 1000 × magnification).

3.6 Imidazolium salts cause morphological alterations on L. amazonensis promastigotes

As illustrated in Figure 5, when promastigotes were treated with IS at IC₅₀ values for 6 h, the parasites showed striking morphological alterations. While untreated parasites exhibited elongated shape, integral flagellum and membrane integrity (Figures 5A, B), promastigotes treated with amphotericin B at 0.4 µM (Figures 5C, D), as well as with C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl or (C₁₀)₂MImCl (Figures 5E–J) evidenced rounded shape, membrane damage, flagellum disruption or twisted, and cell aggregation. In addition, C₁₆MImMeS and C₁₆MImCl that killed L. amazonensis in our previous study (13) also caused similar morphological alterations in promastigotes of L. amazonensis (Figures 5K, L).

FIGURE 5

Figure 5 Scanning electron micrographs of L. amazonensis promastigotes treated or not with imidazolium salts at IC₅₀ values for 6 h. Untreated parasites showing the typical elongated shape (parasite body and anterior flagella) (A, B). Parasites treatment with amphotericin B at 0.4 µM (C, D), C₁₀MImMeS at 11.6 µM (E), C₁₆MImPF₆ at 6.9 µM (F), C₁₆MImBr at 6 µM (G), C₁₆M₂ImCl at 4.1 µM (H), C₁₆M₄ImCl at 1.8 µM (I), (C₁₀)₂MImCl at 1.9 µM (J), C₁₆MImMeS at 0.75 µM (K) and C₁₆MImCl at 1.23 µM (L). The images show alterations in promastigote body shape or disruption of plasma membrane (arrows), flagellum loss (asterisks), and aggregation of cells (hashes). Bars: (A, C) represent 20 µm, (B) and (D–L) represent 10 µm.

3.7 Cytotoxicity of imidazolium salts on mammalian cells

In vitro cytotoxicity of imidazolium salts, C₁₆Im and C₁₆PyrCl (5 to 200 μM) towards macrophages, determined by MTT assay

In relation to the effects of IS on macrophages, a concentration-dependent cytotoxicity was found (Figure S39A: In vitro cytotoxicity of imidazolium salts, C₁₆Im and C₁₆PyrCl [5 to 200 μM] towards macrophages, determined by MTT assay). All IS at 10 µM kept more than 90% of the macrophages alive after 48 h of incubation, and C₁₀MImMeS maintained this condition up to 50 µM. Besides that, C₁₆M₄ImCl, (C₁₀)₂MImCl and C₁₀MImMeS at concentrations of 5.6-, 5.3- and 4.3-fold IC₅₀ for L. amazonensis kept 90, 94 and 89% of macrophages viability, respectively. C₁₆M₄ImCl and (C₁₀)₂MImCl presented the best selectivity indexes (10 and 8.6, respectively) (Table 1). Moreover, human erythrocytes incubated with IS at 10 µM demonstrated hemolysis up to 8% and, as well as for macrophages, C₁₀MImMeS was the less toxic IS, without significant hemolysis up to 40 µM (Figure S39B: In vitro cytotoxicity of imidazolium salts, C₁₆Im and C₁₆PyrCl (1 to 100 μM) towards human red blood cells, determined by hemolysis rates at 540 nm). C₁₆M₄ImCl and (C₁₀)₂MImCl also presented the best SI when considering hemolysis – 8.7 and 11, respectively (Table 1). The neutral imidazole C₁₆Im presented lower cytotoxicity against both macrophages and erythrocytes, however it presented low SI (Table 1). The pyridinium salt C₁₆PyrCl presented the higher toxicity and lowest SI among the tested compounds (Table 1).

3.8 Imidazolium salts reduce the intracellular survival of L. amazonensis on macrophages

As Leishmania can survive inside macrophages, we evaluate whether IS, C₁₆Im and C₁₆PyrCl stimulate the leishmanicidal activity of macrophages. Our data showed that all compounds at 1 µM were able to reduce amastigotes survival in 86% to 93% (Figure 6).

FIGURE 6

Figure 6 Intramacrophage anti-amastigote activity of compounds. RAW cells (2 × 10⁵ cells/well) were infected with promastigotes of L. amazonensis (10 parasites/cell) in the stationary phase (day 5 of culture) and incubated with DMEM (control), lipopolysaccharide (LPS; 10 ng/mL) plus interferon-γ (IFN-γ; 1 ng/mL) as positive control, or IS, C₁₆Im or C₁₆PyrCl at concentrations of 0.1 μM, 1 μM or 5 μM for 48 h. After, cells were lysed, supplemented with M199 30% FBS and cultured at 26°C for 7 days to determine the number of promastigotes recovered using a hemocytometer. Data are expressed as means ± SEM (n = 5). The One-way ANOVA test followed by the Bonferroni’s test were applied. *p < 0.05, ** p < 0.01, ***p < 0.001 and ****p < 0.0001 when compared with the medium group (100% parasite recovered).

4 Discussion

C₄MImMeS, C₁₀MImMeS, C₁₆MImBr, C₄MImBF₄, MImC₁₀MImBr₂, C₇O₃MImPF₆, C₇O₃MImMeS, C₄M₂ImMeS, (C₁₆)₂ImCl, HOC₃MImPF₆, HO₂CC₁MImCl, (HO₂CC₁)₂ImCl, HO₂CC₁MImBF₄, HO₂CC₃MImCl, C₁₆MImPF₆, C₁₆M₄ImCl, C₁₆M₂ImCl, MImC₅MImBr₂, MImC₈MImBr₂, (C₁₆)₂ImMeS, (C₁₆)₂MmCl, and (C₁₆)₂MImMeS were synthesized, which along with the commercially available (C₁₀)₂MImCl, and HOC₂MImCl constituted the group of 24 IS tested (Figure 1). The spectral data of C₄MImMeS (14), C₁₀MImMeS (15), C₁₆MImBr (16), C₄MImBF₄ (14), MImC₁₀MImBr₂ (16), C₇O₃MImPF₆ (15), C₇O₃MImMeS (15), C₄M₂ImMeS (17), C₁₆Im (18), (C₁₆)₂ImCl (19), HOC₃MImPF₆ (20), HO₂CC₁MImCl (21), (HO₂CC₁)₂ImCl (21), HO₂CC₁MImBF₄ (22), HO₂CC₃MImCl (23), C₁₆MImPF₆ (24), C₁₆M₄ImCl (25), C₁₀MeS (15), C₁₆MeS (26), C₄MeS (14), C₁₆MIm (27), and C₇O₃MeS (15) are in agreement with those previously reported. For the new IS C₁₆M₂ImCl, MImC₅MImBr₂, MImC₈MImBr₂, (C₁₆)₂ImMeS, (C₁₆)₂MmCl, and (C₁₆)₂MImMeS, the NMR, FTIR and MS data support their structures and purity.

To the best of our knowledge, this is the first study reporting the leishmanicidal and immunomodulatory activities for C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl (Figure 1) on two Leishmania species. Here, it was demonstrated that depending on the concentration, IS were able to kill infective forms of L. amazonensis and L. infantum chagasi (Figure S38).

In a previous study, we demonstrated that IS are able to kill both promastigote and amastigote forms of L. amazonensis (13). Here, we investigated a structurally complementary set of mono- (N-substituent = methyl; N’-substituent = n-butyl, n-decyl, n-hexadecyl, 2-hydroxyethyl, methylcarboxylic acid, propylcarboxylic acid, methoxycarbonylmethyl, triethylene glycol monomethyl ether; C₂-substituent = H, methyl; C₄-substituent = H, methyl; C₅-substituent = H, methyl – N,N’-substituents = n-decyl, n-hexadecyl, methylcarboxylic acid; C₂-substituent = H, methyl) and dicationic (N-substituent = methyl; alkane bridge = pentane, octane, decane) IS with different anions, as well as the neutral C₁₆Im and the pyridinium salt C₁₆PyrCl (Figure 1) against L. amazonensis and L. infantum chagasi. This provided the perspective to compare the chemical structures and extend the comprehension about activity of this class of compounds against Leishmania. Significant differences in leishmancidal activity of IS against L. amazonensis were observed (Table 1). (C₁₀)₂MImCl and C₁₆M₄ImCl presented the highest activity (lowest IC₅₀ values): (C₁₀)₂MImCl = C₁₆M₄ImCl > C₁₆M₂ImCl > C₁₆MImBr > C₁₆MImPF₆ > C₁₀MImMes. Corroborating with scientific literature (10, 11, 13), the monocationic methylimidazolium salts with longer N-alkyl chain-lengths are closely related to higher leishmanicidal activities. In this sense, we point out that C₁₆MImMeS showed the higher leishmanicidal activity (IC₅₀ = 0.75 µM) (13) compared to C₁₀MImMeS (IC₅₀ = 11.6 µM) and C₄MImMeS (without activity at 100 µM) tested here. On the other hand, shorter N-alkyl chain-lengths afford lower lipophilicity to the molecule and consequently less cytotoxic effects (32), as demonstrated by C₁₀MImMeS which showed lower toxicity towards macrophages (CC₅₀ = 76.9 µM) and erythrocytes (RBC₅₀ > 100 µM) when compared to C₁₆MImMeS (CC₅₀ = 11.5 µM and RBC₅₀ = 36.9 µM) (13).

The N,N’dialkyl substituted IS with n-decyl and n-hexadecyl groups (Figure 1) showed that, independent of the anions present in the n-hexadecyl-based IS (MeS, Cl or Br), only (C₁₀)₂MImCl presented leishmanicidal activity (IC₅₀ = 1.9 μM), evidencing a higher leishmanicidal effect than C₁₀MImCl – tested in the previous study of our group (IC₅₀ = 2.31 µM) (13). Another study demonstrated that (C₁₀)₂MImCl also inhibits bacterial (Escherichia coli and Staphylococcus aureus) and fungal (Candida albicans and Candida tropicalis) growth in vitro (33), and the addition of two hydrophobic alkyl chains to the imidazolium ring also contributed greatly to improve the activity of IS against tumor-derived cell lines (34). This reaffirms the potential balance between hydrophilicity and lipophilicity, which is essential for the antimicrobial activity of IS (7).

The introduction of methyl groups on the C₂-, C₄- and C₅-positions of the imidazolium ring influences the L. amazonensis mortality (Table 1). When comparing the activities of C₁₆M₄ImCl (IC₅₀ = 1.8 µM) and C₁₆M₂ImCl (IC₅₀ = 4.1 µM), the additional two methyl groups reduced 2.3-fold the IC₅₀ value for L. amazonensis mortality. However, C₁₆MImCl presented the lowest IC₅₀ value among these three compounds (IC₅₀ = 1.23 µM) (13). This H-methyl substituent alteration in the imidazolium ring does not seem to interfere to the same extend on L. infantum chagasi mortality, according to the IS C₁₆M₄ImCl (IC₅₀ = 14.9 µM) and C₁₆M₂ImCl (IC₅₀ = 15.4 µM).

From results of this study and our previous work (13), it is possible to compare the effect of different IS anions on L. amazonensis mortality. The IS C₁₆MImMeS (IC₅₀ = 0.75 µM), C₁₆MImCl (IC₅₀ = 1.23 µM) and C₁₆MImNTf₂ (IC₅₀ = 1.64 µM) demonstrated higher capacities to kill L. amazonensis than C₁₆MImBr (IC₅₀ = 6.0 µM) and C₁₆MImPF₆ (IC₅₀ = 6.9 µM), reaffirming the hypothesis that the IS anion also has an important but secondary effect on activity against Leishmania (13). For the C₁₆MIm-based IS, the established order of anion-dependent inhibition was MeS > Cl > NTf₂ > Br > PF₆. As observed for L. amazonensis mortality with C₁₆MImBr and C₁₆MImPF₆, Br and PF₆ anions did not trigger differences in L. infantum chagasi mortality – C₁₆MImPF₆ (IC₅₀ = 26.3 µM) and C₁₆MImBr (IC₅₀ = 29.7 µM). A decrease in anion volume has been related to an increasing fungal mortality, evidencing the IS with MeS and chloride anions as the most promising antifungals (9). This was also observed for anti-leishmanial activity of the IS.

Considering the highest leishmanial activity achieved with C₁₆MImMeS, we tested the effect of its neutral imidazole equivalent C₁₆Im on promastigote forms of Leishmania. This study showed that C₁₆Im was not so effective to kill L. amazonensis as C₁₆MImMeS. Furthermore, this compound presented lower toxicity than all other tested compounds, except C₁₀MImMeS (Table 1). In contrast, C₁₆Im showed a higher activity than the IS tested in this work against L. infantum chagasi.

Compounds containing cationic pyridinium heterocycles have concerned great awareness as prospective chemotherapeutic agents, such as anticancer, analgesic, antimicrobial and antiviral (35). Based on this, the effect of C₁₆PyrCl on Leishmania mortality was tested, and this compound showed being effective against L. amazonensis (IC₅₀ = 4.0 µM) (Figure S38A and Table 1), but being more toxic toward macrophages than IS (Figure S39A and Table 1). In our previous study we tested the structurally related IS C₁₆MImCl, which showed higher leishmanicidal activity and lower macrophage and red blood cell cytotoxicity than C₁₆PyrCl (13). C₁₆PyrCl also demonstrated high capacity to kill L. infantum chagasi (IC₅₀ = 2.6 µM) (Figure S38B). Although this compound is commonly used as antiseptic (mouthwash), its high toxicity against macrophages turns it into a weak candidate for drug development.

To elucidate possible mechanisms of action of IS on Leishmania parasites, membrane and mitochondrial dysfunctions, ROS generation and morphological changes in response to IS were investigated. In Leishmania, mitochondria are a drug target (36), since this unique organelle is the main site of ROS production, and excessive ROS may lead to oxidative stress, which may be one of the major contributors to cell death by IS (37, 38). In addition, the maintenance of an appropriate mitochondrial membrane potential (ΔΨm) is essential for Leishmania survival (36, 37). Here, we demonstrate that IS with leishmanicidal activity caused an increase in ROS production (Figures 4A, B) and ΔΨm alterations (Figures 34C, D) in L. amazonensis. C₁₀MImMeS triggered mitochondrial membrane hyperpolarization, while C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl caused depolarization of mitochondrial membrane. Both hyperpolarization and depolarization can result in promastigotes death (29), and depolarization can be preceded by a transient hyperpolarization as the last attempt by cells to avoid death (39). Moreover, all six IS presenting leishmanicidal activity caused plasma membrane alterations (Figures 4A, B), indicating cell death involving necrosis (40).

In this regard, the scanning electron microscopy analyses revealed that the treatment with IS induced swelling and overall rounding, cell aggregation, membrane rupture, besides lower parasite length and flagellum damage (Figure 5), corroborating with evidences of the action of IS on L. amazonensis (13). In addition to the biochemical alterations, IS inhibited the in vitro growth of promastigotes forms of L. amazonensis (Figure 2A) and L. infantum chagasi (Figure 2B). This effect could be related to the ergosterol availability. Similarly to fungi, ergosterol in Leishmania is essential for the organization of membrane domains, cell growth and survival. In a previous study it was demonstrated that C₁₆MImCl reduced the total ergosterol content of clinical isolates of yeasts, suggesting that IS inhibit the ergosterol biosynthesis (8). Although C₁₆MImCl and other IS presented potent leishmanicidal activity, the activity of enzyme involved in ergosterol synthesis (lanosterol 14α-demethylase) or ergosterol content were not evaluated in this study.

IS, C₁₆Im and C₁₆PyrCl at 1 µM reduced the amastigote survival of L. amazonensis in about 90% (Figure 6), with an IC₅₀ between 0.2 to 0.3 and higher SI (31.5 to 275) than those calculated for promastigotes, indicating a higher selectivity for amastigotes. These data are in agreement with our previous data showing the better selectivity of IS for amastigotes (13).

For the first time, the IS C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl, and (C₁₀)₂MImCl have been identified as potent agents against L. amazonensis. These compounds induced ROS generation, membrane, and mitochondrial dysfunction in parasites. The results reaffirm the hypothesis that IS with longer N-alkyl chain-lengths are related with higher biological activities, and on a secondary basis IS with MeS and chloride anions are the most promising antileishmanial agents. As a limitation of this study, we mentioned that we did not test the effects of these IS on antimony-resistant Leishmania spp. lines, or their in vivo effects.

5 Conclusions

The IS C₁₀MImMeS, C₁₆MImPF₆, C₁₆MImBr, C₁₆M₂ImCl, C₁₆M₄ImCl and (C₁₀)₂MImCl, and the compounds C₁₆Im and C₁₆PyrCl killed and inhibited the growth of promastigote forms of L. amazonensis and L. infantum chagasi in a concentration-dependent manner. Moreover, induced ROS production, mitochondrial dysfunction, membrane disruption and morphological alterations in infective forms of L. amazonensis and were able to kill intracellular amastigote forms in a very low concentration, being worthy of further in vivo studies as potential antileishmanial drugs.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Author contributions

Conceptualization PR, HS, FB. Methodology: FB, TF, MS, PF, VS, DP, JK, ES. Software: TF, FB, LJ. Validation: FB, TF. Formal analysis: FB, LJ. Investigation: FB, ED, TF. Resources: PR, HS, MM. Data curation: FB, PR. Writing—original draft preparation: KN-L, FB, PR, TF. Writing—review and editing: MM. PR, HS. Visualization: AP, LJ, MM, ED. Supervision: PR, HS, MM. Project administration: PR, HS. Funding acquisition: PR, HS, MM. All authors contributed to the article and approved the submitted version.

Funding

This project was funded, in part, with support from the Fundação de Amparo à Pesquisa do Estado de Rio Grande do Sul (FAPERGS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) Finance Code 001, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This project was also funded, in part, with support from the Federal University of Health Sciences of Porto Alegre. TF is recipient of a PNPD/CAPES fellowship. MM, AP, HS and PR are Research Career Awardees of the CNPq.

Acknowledgments

The authors were most grateful for the financial support by the Brazilian’s agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Rio Grande do Sul (FAPERGS), Federal University of Pará and MM, AP, HS and PR thanks for the fellowship from CNPq.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.1096312/full#supplementary-material

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