Acta Tropica 110 (2009) 7–14
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Biological activity of 1,2,3,4-tetrahydro--carboline-3-carboxamides against Trypanosoma cruzi Rodrigo Hinojosa Valdez a , Lilian Tatiani Düsman Tonin b , Tânia Ueda-Nakamura c , Benedito Prado Dias Filho a,c , José Andrés Morgado-Diaz d , Maria Helena Sarragiotto b , Celso Vataru Nakamura a,c,∗ a Programa de Pós-graduac¸ão em Microbiologia, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445, Km 380, Campus Universitário, CEP 86051-990 Londrina, Paraná, Brazil b Departamento de Química, Universidade Estadual de Maringá, Av. Colombo 5790, CEP 87020-900 Maringá, Paraná, Brazil c Departamento de Análises Clínicas, Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos, Bloco I-90 Sala 123 CCS, Universidade Estadual de Maringá, Av. Colombo 5790, CEP 87020-900 Maringá, Paraná, Brazil d Divisão de Biologia Celular, Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 6 June 2008 Received in revised form 4 October 2008 Accepted 12 November 2008 Available online 20 November 2008 Keywords: Trypanocidal -Carbolines Chemotherapy Electron microscopy
a b s t r a c t Several -carboline compounds were evaluated for in vitro trypanocidal activity against Trypanosoma cruzi and their potential toxic effects was also assessed. -Carboline derivative 4 showed good activity against epimastigote, trypomastigote, and amastigote forms of T. cruzi, with a dose-dependent inhibitory effect. It showed an IC50 of 14.9 M against the epimastigote form and an EC50 of 45 M and 33 M against trypomastigote and amastigote forms, respectively. Additionally, 4 was able to be active on mammalian cell–protozoan interaction, reducing the number of infected cells and the number of internalized parasites. The compound showed low cytotoxicity, with a selective index 31 times higher to the parasite than for mammalian cells. In human red-blood cells -Carboline 4 at 14.9 M not caused haemolysis. Observed at electron microscopy 4-treated epimastigotes showed abnormal swelling of the mitochondrion, a diffuse kinetoplast, and distortions of the parasite cell body. The present data support the potential effect of this class of compounds against T. cruzi and encourage further experiments in vitro to evaluate the action mechanism of this drug and also with in vivo models. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Chagas’ disease, also called American trypanosomiasis, is a parasitic disease caused by the kinetoplastid protozoan Trypanosoma (Schizotrypanum) cruzi. It is endemic from Mexico to Argentine, and afflicts 16–18 million people. Mortality rates range from 8% to 12% depending on the age and physiological state of the patient (WHO, 2002). The current treatment for this disease is very limited, and no successful vaccine has been developed (Maya et al., 2007). The available drugs for clinical treatment are the nitroderivatives Benznidazole and Nifurtimox, both unsatisfactory. These drugs have variable therapeutic effects, according to the geographical region, and require long-term treatment, besides frequently having toxic side effects.
∗ Corresponding author at: Universidade Estadual de Maringá, Departamento de Análises Clínicas, Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos, Bloco I-90 Sala 123 CCS, Avenida Colombo, 5790, BR-87020-900 Maringá, PR, Brazil. Tel.: +55 44 3261 4863; fax: +55 44 3261 4860. E-mail address:
[email protected] (C.V. Nakamura). 0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.11.008
They are only effective against the acute infection, during which most patients do not know that they are infected with T. cruzi. In addition, they have limited efficacy in the chronic stage (Coura and De castro, 2002). Furthermore, important differences in susceptibility to these drugs have been detected among different parasite strains isolated in various parts of the Americas (Rivas et al., 1999). A number of new drugs have been reported to be effective in vitro or in vivo against T. cruzi, but none has been found to be completely satisfactory, for either the treatment of Chagasic patients or for prophylaxis of blood to prevent infection via blood transfusion. Clearly, new drugs and new approaches for confronting these problems are necessary (Bernacchi et al., 2002). In this context, an exhaustive search for new synthetic and natural products for treatment of Chagas disease is ongoing (Croft et al., 2005). The carbolines, which are widespread in nature and have been isolated from fungi, higher plants, marine organisms, and mammals, have shown good activity against T. cruzi (Rivas et al., 1999). Natural and synthetic -carbolines and tetrahydro--carboline alkaloids are well-known compounds that possess several biological properties, such as anticonvulsive, ansiolytic, sedative, antimicrobial, antithrombotic, anti-HIV, antiproliferative, insecti-
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cidal, and parasiticidal (Ishida et al., 2001; Lin et al., 2002; Castro et al., 2003; Takasu et al., 2004; Cao et al., 2005; Costa et al., 2006). This class of compounds has been tested in trypanocidal assays, and some reports have shown the activity of -carbolines and tetrahydro--carbolines against T. cruzi (Calvin et al., 1987). In a previous work we demonstrated the activity of 1-(nitrophenyl)tetrahydro--carbolines against epimastigote forms of T. cruzi (Tonin et al., 2008). In the present study, a series of 12 1-substituted-1,2,3,4tetrahydro--carboline-3-carboxamides was synthesized and screened for antitrypanosomal activity against epimastigote form of T. cruzi. The effect of the most active compound N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-carboline-3-carboxamide (4) was evaluated against amastigote, and trypomastigote forms of T. cruzi also. 2. Materials and methods 2.1. Chemistry The aldehydes and amines used in this study were purchased from Sigma–Aldrich (Saint Louis, USA), Acros-Organics (Geel, Belgium) and Merck (Darmstadt, Germany). l-Tryptophan was purchased from Synth (São Paulo, Brazil) and trifluoroacetic acid (TFA) was obtained from Carlo Erba (Milan, Italy). The compounds were characterized by the mass spectra (EIMS) obtained on Shimadzu-CG/MS model QP 2000A spectrometer, NMR spectra data, recorded in a Varian spectrometer model Mercury plus BB 300 MHz, and (IR) spectra (KBr) recorded on a Bomem spectrophotometer, MB-100 model. 2.2. General procedure for synthesis of ˇ-carboline-3-carboxamides 4–15 The tetrahydro--carboline-3-carboxamides were prepared from l-tryptophan 1, through a Pictet-Spengler condensation (Bailey et al., 1987), of the l-tryptophan methyl ester 2 with a series of aromatic aldehydes, followed by the reaction of the methyl tetrahydro--carboline-3-carboxylate intermediates with different amines, according to the procedure previously reported (Coutts et al., 1984). The N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-carboline-3-carboxamide 4 was obtained from the reaction of the corresponding ester 3 with butylamine (Fig. 1). A mixture of l-tryptophan methyl ester 2 (0.5 mmol), 4dimethylaminobenzaldehyde (0.5 mmol) and trifluoroacetic acid (trace) in CH2 Cl2 (10 mL) was stirred at 0 ◦ C, over molecular sieves. After 24 h, an excess of TFA (1.0 mmol) was added, and the mixture was stirred at room temperature for 6 h, followed by evaporation of the solvent. Treatment of the crude product with 10% Na2 CO3 , extraction with EtOAc (3× 10 mL), drying of the organic layer under anhydrous Na2 SO4 , filtration and solvent evaporation
afforded a residue which was purified on a chromatographic column (silica flash; hexane-EtOAc 20%) to give the compound 3 as a cis/trans mixture. A solution of methyl-1-(4-dimethylamino)phenyl-1,2,3,4tetrahydro--carboline-3-carboxylate 3 (1.0 mmol) and butylamine (5 mL) was refluxed for 24 h; then the excess amine was removed under vacuum. The residue was crystallized by using MeOH as a solvent, to give the compound 4 as a cis/trans mixture. The synthesized compound was characterized by spectral (1 H and 13 C NMR, MS and IR) data. The stereochemistry of cis and trans isomers was assigned on the basis of 13 C NMR data (Düsman et al., 2005). 2.3. Parasite The epimastigote form of T. cruzi Y strain was grown in Liver Infusion Tryptose (LIT) supplemented with 10% foetal calf serum (FCS, Gibco, Invitrogen Corporation, New York, USA) at 28 ◦ C for 96 h. Trypomastigote and amastigote forms were obtained by infection of LLCMK2 cell monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Invitrogen Corporation, New York, USA) at 37 ◦ C and 5% CO2 atmosphere. 2.4. Cell culture LLCMK2 (monkey kidney cells) were maintained in DMEM supplemented with 2 mM l-glutamine, 10% FCS, 50 mg/L gentamicin and buffered with sodium bicarbonate. 2.5. Antiproliferative activity of ˇ-carboline 4 on the epimastigote form The epimastigote form of T. cruzi in the logarithmic phase was used for this assay. The -carboline compounds were dissolved in dimethylsulfoxide (DMSO) and LIT medium to obtain concentrations of 3 M, 13 M, 26 M, 128 M and 256 M. The final concentration of DMSO did not exceed 1%. For each experiment, there was a growth control with and without DMSO. A cell density of 1 × 106 epimastigotes/mL was cultured in a 24well microplate to obtain a final volume of 1 mL. The cells were incubated at 28 ◦ C and their growth was determined by counting the parasites with a haemocytometer chamber every day for 7 days. The IC50 values (50% inhibition concentration) were determined using linear regression analysis from this inhibition percentage. Benznidazole-Rochagan® (Roche Pharmaceuticals, Rio de Janeiro, Brazil) was used as the reference drug. 2.6. Effect of ˇ-carboline 4 on the viability of the trypomastigote and amastigote forms The tissue-culture-derived parasite trypomastigote and amastigote forms were resuspended in Dulbecco’s modified
Fig. 1. General procedure for the synthesis and general chemical structure of N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro--carboline-3-carboxamide 4. Reagents and conditions: (a) H3 COH, H2 SO4 , reflux, 12 h; (b) 4-dimethylaminobenzaldehyde, CH2 Cl2 , molecular sieves, TFA (trace), 0 ◦ C, 24 h, TFA (2.0 mol equiv.), rt, 6 h; (c) butylamine, reflux, 24 h.
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Eagle medium supplemented with FCS containing 10% mouse blood in a concentration of 107 parasites/mL. In a 96-well microplate, 100 L of this suspension was added to the same volume of the drug diluted in DMSO and DMEM at twice the desired final concentration (8 M, 16 M, 32 M, 64 M, 128 M, 256 M, 513 M, 1026 M and 2051 M), and incubated for 24 h at 37 ◦ C. Considering the mobility of this form of parasite, which permit distinguishes his viability, we used the Pizzi-Brener method. For this an aliquot of 5 L of each sample were placed on slides plus coverslips and immediately counted in an optical microscopy (Brener, 1962), subsequently the EC50 (concentration which lysed 50% of the parasites) was calculated. In the case of amastigote forms where is impossible to distinguish the mobility of viable and unviable cells, the viability of free amastigotes was determined by counting in a haemocytometer chamber (Improved Double Neubauer) with a light microscope, after addition of 0.4% erythrosine B. Crystal violet (Inlab, São Paulo, Brazil) was used as the reference drug. The EC50 value was also evaluated. 2.7. Effect of ˇ-carboline 4 on the intracellular amastigote in the LLCMK2 cell line In the 24-well microplate containing glass coverslips, a 500 L aliquot of the LLCMK2 cells (2.5 × 105 cells/mL) was seeded in each well and incubated for 24 h at 37 ◦ C with 5% CO2 . Next, the LLCMK2 cell monolayer was infected with trypomastigotes (10:1) for 24 h, then washed with 0.01 M phosphate-buffered saline (PBS) pH 7.2, and fresh medium with and without the drug in different concentrations (16 M, 32 M, 64 M and 128 M) was added to the wells. The microplate was incubated for 96 h at 37 ◦ C with 5% CO2 atmosphere. The cells were fixed with methanol and stained with May-Grunwald-Giemsa (Gibco, Invitrogen Corporation, New York, USA) for 20 min, then the glass coverslips were permanently prepared with Entellan® (Merck, Darmstadt, Germany). The percentage of infected cells and the number of intracellular parasites were estimated by observing 200 cells with a light microscope (Olympus CX31), and the survival index (product of the percentage of cells infected and the number of amastigotes per cell) and EC50 value (effective concentration) were determined. Benznidazole was used as reference drug. 2.8. Ultrastructural analysis 2.8.1. Transmission electron microscopy Epimastigote forms of T. cruzi, after treatment with 14.9 M (IC50 ) of -carboline 4, were harvested by centrifugation, washed in PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 1 h at 4 ◦ C. Next, the cells were post-fixed in a solution containing 1% OsO4 , 0.8% potassium ferrocyanide and 10 mM CaCl2 in 0.1 M cacodylate buffer at room temperature for 60 min, washed with 0.1 M cacodylate buffer, dehydrated in ethanol, and embedded in Epon® resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss EM900 transmission electron microscope. 2.8.2. Scanning electron microscopy Epimastigote and trypomastigote forms of T. cruzi treated with -carboline 4 at IC50 and EC50 values respectively were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at room temperature, washed three times in cacodylate buffer and adhered on poly-l-lysine coated coverslips. Next, the parasites were dehydrated in an ascending ethanol series, critical-point dried with CO2 , sputter-coated with gold,
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and observed in a Shimadzu SS-550 scanning electron microscope. 2.9. Cytotoxicity assay The LLCMK2 cells were seeded onto 96-well microplates at a concentration of 2.5 × 105 cells/mL and incubated for 24 h in DMEM supplemented with 10% FCS. The monolayer obtained was treated with different concentrations of -carboline (25 M, 128 M, 256 M and 1282 M). DMSO was used as a negative control, and Benznidazole was used as the reference drug. After incubation at 37 ◦ C with 5% CO2 for 96 h, cell viability was evaluated by the sulforhodamine B technique (Skehan et al., 1990). The absorbance was read at 530 nm in a microplate spectrophotometer (Biotek-Power Wave XS). Next, the CC50 of the drug (concentration of drug that lysed 50% of cells) was calculated. 2.10. Red blood cell lysis assay The potential haemolytic effect of -carboline was evaluated in this assay. A 4% suspension of fresh defibrinated human blood was prepared in sterile 5% glucose solution. One of several concentrations (3 M, 13 M, 26 M, 126 M, 256 M and 1282 M) of the -carboline compound was added to each test tube and gently mixed, and the tubes incubated at 37 ◦ C. After 1 h of incubation, the visual reading was made, and after 2 h the samples were centrifuged at 1000 × g for 10 min. The absorbance of the supernatant was determined at 540 nm for estimation of haemolysis. The results were expressed as percentage of haemolysis, by the equation Haemolysis: (%) = 100 − [(Ap − As)/(Ap − Ac) × 100]; where Ap, As and Ac are the absorbance of the positive control, test sample and negative control, respectively. Amphotericin B (Cristalia, São Paulo, Brazil) was used as the reference drug, Triton X-100 (Vetec, Rio de Janeiro, Brazil) was used as the positive control, and the cell suspension only was used as the negative control. 2.11. Statistical analysis Statistical analysis was done with the program GraphPad Prism 4 (GraphPad Software, San Diego, California, USA). Student’s t-test was applied and a p-value less than 0.05 was regarded as significant. The experiments were performed in triplicate, in at least three independent experiments. 3. Results 3.1. Effects of ˇ-carboline 4 on growth of the epimastigote form Some -carboline compounds showed good activity against the proliferation of epimastigotes, which is the form present in the reduviid vector. Comparison of the 50% inhibition concentration values (IC50 in M) for antitrypanosomal activity of 12 compounds synthesized showed that the compound bearing a 4dimethylaminophenyl and an N-butylcarboxamide group in the 1and 3-positions of the tetrahydro--carboline ring (compound 4, Fig. 1) showed the most activity, with IC50 of 14.9 M (Table 1). The presence of -carboline 4 in the culture of Y-strain epimastigotes caused progressive parasite injury, compared with the untreated cells, and a dose-dependent effect was observed. After 96 h of incubation with 256 M of the compound (the highest concentration tested), growth was completely arrested. The IC50 and the IC90 values of -carboline 4 were 14.9 ± 6.5 M and 76.9 ± 13.2 M, respectively (Fig. 2). IC50 to Benznidazole was 7.7 ± 2.9 M.
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Table 1 General structure for -carbolines and its IC50 (M) values for 1-substituted-1,2,3,4-tetrahydro--carboline-3-carboxamides (4–15) against T. cruzi epimastigotes.
R1
R2
Isomer
IC50
4
cis + trans
5
trans
6
trans
79.6
7
cis + trans
33.9
8
cis
49.6
9
cis
>100
10
cis
>100
11
cis
>100
12
cis + trans
13
cis + trans
14
cis
15
trans
3.2. Effect of ˇ-carboline 4 on the viability of the trypomastigote and amastigote forms We evaluated the activity of -carboline 4 on the viability of the trypomastigote and amastigote forms. A lytic activity with a dosedependent trypanocidal effect was observed (Table 2). Against the trypomastigote form, in the presence of mouse blood, was observed a 50% effective concentration (EC50 ) of 45 ± 3.9 M, at 37 ◦ C after 24 h. In the amastigote form, the highest concentration of the compound tested was 256 M and showed a good lytic effect at 37 ◦ C. After 24 h of treatment, in the presence of mouse blood the EC50 was 33 ± 5.6 M (Table 2). The effect of the reference drug, crystal violet, against trypomastigote and amastigote forms showed EC50 of 12.8 ± 2.6 M and 6.7 ± 2.1 M, respectively.
14.9
>100
24.4
>100
28.1
>100
dependent trypanocidal effect (Fig. 3), leading to considerable reduction in both the percentage of infected cells and the mean of number of parasites per infected cells. After 96 h of incubation, the percentage of LLCMK2 cells with internalized parasites was higher for the control than for cells infected and treated with -carboline 4 (Fig. 3). At that time, the control showed a mean 31.9 amastigotes per cell and 72% of cells infected. Cells treated with 32 M showed a mean 4.9 internalized amastigotes, with 45% of cells infected. Treatment of the cells with 128 M resulted in only 11% infected cells and 2.1 parasites per cell. The effective concentration (EC50 ) against intracellular amastigotes was 20.2 ± 3.5 M, meanwhile the EC50 value for Benznidazole was 26.1 M. C4 also showed a lower value of SI50 at 17.5 M (survival index of 50%) when compared with Benznidazole with SI50 at 28.7 M. 3.4. Ultrastructural effects
3.3. Effect of ˇ-carboline 4 on the intracellular amastigote in the LLCMK2 cell line The treatment of LLCMK2 infected with amastigote forms showed that the compound had good activity, with a dose-
3.4.1. Transmission electron microscopy Electron microscopy analysis of epimastigote forms treated with 14.9 M (IC50 ) of -carboline 4, showed alterations of the typical morphology of the parasite, such as: swelling of the mitochondrion,
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Table 2 Biological effects of -carboline 4 on Trypanosoma cruzi (Y Strain) and over different mammalian cells.
Formule
Fig. 2. Effects of -carboline 4 on the proliferation of epimastigotes of Trypanosoma cruzi. The parasites were cultured in LIT medium at 28 ◦ C as described in Section 2. Initial cell density was 1 × 106 epimastigotes/mL. Control, ; 3 M, ; 13 M, ; 26 M, 䊉; 128 M, ; 256 M, . Values are the means from three independent experiments.
diffuse kinetoplasts, electron lucent reservosomes, and appearance of vacuoles (Fig. 4). 3.4.2. Scanning electron microscopy Morphological alterations in the epimastigote and trypomastigote forms treated with -carboline 4 were visualised by scanning electron microscopy. In epimastigotes, the IC50 (14.9 M) caused distortions of the parasite cell body such as rounding and swelling of cell body, and a shortening of the flagellum (Fig. 5). In trypomastigote forms treated with EC50 (45 M), the alterations observed in the cell body were loss of the normal shape and a reduction in the size of the flagellum (Fig. 5).
IC50 EC50a EC50b EC50c CC50 HC50
14.9 ± 6.5 45 ± 3.9 33 ± 5.6 20.2 ± 3.5 462 ± 112.1 >1.282
The values (M) of IC50 represent the 50% inhibition concentration against epimastigote forms (28 ◦ C), EC50a the 50% effective concentration on trypomastigote forms (37 ◦ C), EC50b 50% effective concentration on amastigote forms (37 ◦ C), EC50c 50% effective concentration on mammalian cell–parasite interaction, CC50 refers to 50% citotoxical concentration on LLCMK2 (37 ◦ C) and HC50 refers to 50% haemolytic concentration of red blood cells (37 ◦ C).
3.5. Cytotoxic effect of ˇ-carboline 4 on LLCMK2 cells This assay evaluated the potential toxic effects of this drug on the LLCMK2 lineage, after 96 h of treatment. The cells treated with 256 M led to 38.7% cellular inhibition. With 128 M the cellular inhibition was 18.4%. The 50% cytotoxic concentration (CC50 ) was 462 ± 112.1 M (Table 2). The cytotoxic effects on LLCMK2 cells and activity against the parasite were compared by using the selectivity index (SI), ratio (CC50 for LLCMK2 /IC50 for parasite). The -carboline
Fig. 3. (a) Effect of -carboline 4 on the Trypanosoma cruzi–LLCMK2 cell interaction. LLCMK2 cells were infected with trypomastigote forms and treated with -carboline 4. SI (survival index, percent) was calculated by the equation (P2/P1) × 100, where P1 is the SI for the control and P2 is the SI for treated cells. SI was calculated by multiplying the percentage of LLCMK2 cells with internalized parasites and the mean number of internalized parasites per cell. The data represent the mean values from three independent experiments. (b) Light microscopy of intra-cellular amastigotes and their interaction with LLCMK2 cells treated with -carboline 4 for 96 h. (A) Untreated cells, (B) cells treated with 32 M and (C) cells treated with 128 M; Bars = 10 m.
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Fig. 4. Transmission electron microscopy of Trypanosoma cruzi epimastigotes (Y strain) treated with -carboline 4 for 96 h. (a) Control parasite showing characteristic organelles: the kinetoplast (K), nucleus (N), reservosome (R) and flagellar pocket (FP). (b–d) Epimastigotes treated with -carboline 4 at 14.9 M. (b) A diffuse kinetoplast (arrowhead), with a vacuole present in cytoplasm (V) and altered reservosomes (R). (c) An altered mitochondrion (arrow). (d) A large vacuole (V) and a coiled mitochondrion with interruption in mitochondrial membrane (star). Bars = 1 m.
compound was more selective against the parasite than the mammalian cells, with an SI of 31. 3.6. Haemolytic assay In this experiment we evaluated the toxicity for human redblood cells of -carboline 4 incubated at 37 ◦ C for 120 min. -Carboline 4 at 14.9 M (IC50 ) caused only 2.2% haemolysis, and at the highest concentration tested (1282 M) caused only 28% lysis, therefore, the HC50 (haemolytic concentration that causes 50% of lysis) was not calculated. Amphotericin B showed a strong haemolytic effect, with 70% haemolysis at 14.9 M. Triton X-100 used as a positive control was considered 100% lysis, Benznidazole showed low levels of haemolysis, and 1% DMSO did not cause lysis (Table 2). 4. Discussion In the investigation of new trypanocidal compounds, many sources have been chosen, including natural and synthetic products. Natural products or derivatives play an important role in the development of all types of drugs and some natural compounds or extracts have shown trypanocidal activity (Paveto et al., 2004; Mesquita et al., 2005; Luize et al., 2006a; Dantas et al., 2006; Izumi et al., 2008). In additional to the natural products, some synthetic compounds presented activity against T. cruzi (Garzoni et al., 2004; Adade et al., 2007; Menna-Barreto et al., 2005; Bisaggio et al., 2008).
Furthermore some drugs used for the treatment of diseases caused by other microorganisms, such as fungal diseases, have been tested against T. cruzi (Urbina et al., 2000). Although -carbolines are widespread in nature and have been isolated from many sources, only a few reports on their trypanocidal activity have appeared. Some earlier papers have reported antimicrobial, antiproliferative, insecticidal, and parasiticidal activity of -carbolines (Cao et al., 2005). The results obtained in the present study showed that treatment of epimastigotes with chemically modified -carboline 4 for 7 days resulted in dose-dependent growth inhibition. In the first 24 h the compound already showed a significant inhibitory effect at 256 M (p < 0.05). After 96 h, concentrations above 26 M showed significant antiproliferative activity compared to the growth of the control (p < 0.01), and with 256 M growth was completely arrested. Transmission electron microscopy analysis of epimastigotes treated with IC50 of -carboline 4 demonstrated swelling of the mitochondrion and the presence of vacuoles and a diffuse kinetoplast, while the cytoplasmic membrane remained preserved. These alterations were also found in T. cruzi treated with other synthetic compounds such as risedronate, in which mitochondrial swelling was among the most prominent ultrastructural alterations seen in both epimastigote and amastigote forms (Garzoni et al., 2004). Additionally, scanning electron microscopy study showed that epimastigotes treated with -carboline 4 had some distortions on their surface, such as rounding and swelling of the cell body, and a reduction in the size of the flagellum compared to control cells. However, the plasma
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Fig. 5. Scanning electron microscopy of Trypanosoma cruzi epimastigotes (a–c) and trypomastigotes (d–f) treated with -carboline 4. Control parasites (a and d) showing the typical elongated body with terminal flagellum. (b and c) Epimastigotes treated with 14.9 M for 96 h, showing a rounded cell body and small flagellum. (e and f) Trypomastigotes treated with 45 M for 24 h, showing distortion in cell body, reduction in size, and probable loss of intracellular material (e). (a–c) Bar = 2 m. (d–f) Bar = 1 m.
membrane and subpellicular microtubules seemed to be preserved. These alterations are similar to those reported previously, such as those caused by essential oil of Syzugium aromaticum (Dantas et al., 2006) and eupomatenoid-5 isolated from Piper regnellii (Luize et al., 2006b). The -carboline compound also showed activity against the trypomastigote form, it caused heavy damage and extensive lysing. Concentrations above 16 M showed significant activity compared to the control test (p < 00.1). When observed in SEM, the cell surface was altered and the cells had a short flagellum. In free amastigotes the situation was similar and all concentrations showed significant activity compared with the control (p < 0.01). Against the clinically
important form (the intracellular amastigote) there was a huge reduction in the number of infected cells as well as in the number of intracellular amastigotes. The drug was able to penetrate into the host cell and act on the confined parasite, with no significant deleterious effects on the host cells. The EC50 value was 20.2 ± 3.5 M, a minor index than presented by Benznidazole which was 26.1 M, similar to presented by previous reports (Saraiva et al., 2007). Additionally, the SI50 of C4 (17.5 M), was also lower than presented by Benznidazole (28.7 M). Cytotoxicity assays demonstrated that -carboline 4 was 31 times more toxic to the parasite than to the LLCMK2 lineage. In the haemolytic assay, -carboline 4 showed only 28% haemolysis
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at 1282 M (a concentration 72 times higher than the IC50 for epimastigotes). Although the mechanism of action of -carboline compounds is unknown, recent reports have documented their significant antiproliferative activity in tumoral cells, and there is evidence that these compounds can intercalate DNA. It can be envisaged that the -carboline ring system, having a planar aromatic structure, could stack in the base pairs of DNA, and such intercalation could contribute to the biological activity (Boursereau and Coldham, 2004). Other reports have shown that a high concentration of -carboline was able to induce apoptosis in vitro (Hans et al., 2005). Furthermore, inhibition of the respiratory chain seems to be an important action mechanism of the majority of the -carbolines studied in the Tulahuen strain of T. cruzi (Rivas et al., 1999). The present data show the potential effect of the -carboline derivative (4), against the three forms of T. cruzi. This supports further screening of new analogs, and more in vitro and in vivo studies of this drug. Such studies are necessary to increase understanding of the mode of action of this drug and the possibility that it can be used alone or in combination with other drugs for treatment of Chagas’ disease in the future. Acknowledgements This study was supported through grants from DECIT/SCTIE/MS and MCT by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Programa de Núcleos de Excelência (PRONEX/Fundac¸ão Araucária), and Programa de Pós-graduac¸ão em Microbiologia da Universidade Estadual de Londrina. References Adade, C.M., Figueiredo, R.C.B.Q., De Castro, S.L., Soares, M.J., 2007. Effect of l-leucine methyl ester on growth and ultrastructure of Trypanosoma cruzi. Acta Trop. 101, 69–79. Bailey, P.D., Hollinshead, S.P., McLay, N.R., 1987. Exceptional stereochemical control in the Pictet–Spengler reaction. Tetrahedron Lett. 28, 5177–5180. Bernacchi, A.S., De Cazzulo, B.F., Castro, A.H., Cazzulo, J.J., 2002. Trypanocidal action of 2,4-dichloro-6-phenylphenoxyethyl diethylamine (Lilly 18947) on Trypanosoma cruzi. Acta Pharmacol. Sin. 23, 399–404. Bisaggio, D.F.R., Adade, C.M., Souto-Padrón, T., 2008. In vitro effects of Suramin on Trypanosoma cruzi. Int. J. Antimicrob. Agents 31, 282–286. Boursereau, Y., Coldham, I., 2004. Synthesis and biological studies of l-amino carbolines. Bioorg. Med. Chem. Lett. 14, 5841–5844. Brener, Z., 1962. Therapeutic activity criterion of cure on mice experimentally infected with Trypanosoma cruzi. Rev. Inst. Med. Trop. São Paulo 4, 386–396. Calvin, J., Krassner, S., Rodriguez, E., 1987. Plant derived alkaloids active against Trypanosoma cruzi. J. Ethnopharmacol. 19, 89–94. Cao, R., Chen, H., Peng, W., Ma, Y., Hou, X., Guan, H., 2005. Design, synthesis and in vivo antitumor activities of novel -carboline derivatives. Eur. J. Med. Chem. 40, 991–1001. Castro, A.C., Dang, L.C., Soucy, F., Grenier, L., Mazdiyasni, H., Hottelet, M., Parent, L., Pien, C., Palombella, V., Adams, J., 2003. Novel IKK inhibitors: -carbolines. Bioorg. Med. Chem. Lett. 13, 2419–2422. Coura, J.R., De castro, S.L., 2002. A critical review on Chagas disease chemotherapy. Mem. Inst. Oswaldo Cruz 97, 3–24. Coutts, R.T., Micetich, R.G., Baker, G.B., 1984. Some 3-carboxamides of -carboline and tetrahydro -carboline. Heterocycles 22, 122–131. Costa, E.V., Pinheiro, M.L.B., Xavier, C.M., Silva, J.R.A., Amaral, A.C.F., Souza, A.D.L., Barison, A., Campos, F.R., Ferreira, A.G., Machado, G.M.C., Leon, L.L.P., 2006. A pyrimidine--carboline and other alkaloids from Annnona foetida with antileishmanial activity. J. Nat. Prod. 69, 292–294.
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