Design, Synthesis, and Evaluation of Hydroxamic Acid Derivatives as

Dec 3, 2013 - Today, there are approximately 8 million cases of Chagas disease in the southern cone of South America alone, and about 100 million peop...
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Design, Synthesis, and Evaluation of Hydroxamic Acid Derivatives as Promising Agents for the Management of Chagas Disease Giseli Capaci Rodrigues,†,‡,§ Daniel Ferreira Feijó,∥ Marcelo Torres Bozza,∥ Peiwen Pan,⊥ Daniela Vullo,# Seppo Parkkila,⊥ Claudiu T. Supuran,#,∇ Clemente Capasso,○ Alcino Palermo Aguiar,† and Alane Beatriz Vermelho*,‡,◆ †

Laboratório de Síntese Orgânica, Departamento de Química, Instituto Militar de Engenharia, IME, Rio de Janeiro, Brasil Laboratório Proteases de Microrganismos, Departamento de Microbiologia, Instituto de Microbiologia Paulo de Góes, IMPG, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil § Escola de Ciência e Tecnologia e Programa de Pós-Graduaçaõ em Ensino das Ciências, Universidade do Grande Rio, Unigranrio, Duque de Caxias, Rio de Janeiro, Brasil ∥ Laboratório de Inflamaçaõ e Imunidade, Departamento de Imunologia, Instituto de Microbiologia Paulo de Góes, IMPPG, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil ⊥ Institute of Biomedical Technology, Fimlab Ltd., School of Medicine and BioMediTech, University of Tampere and Tampere University Hospital, Medisiinarinkatu 3, 33520 Tampere, Finland # Laboratorio di Chimica Bioinorganica, Universita degli Studi di Firenze, Via della Lastruccia 3, Rm. 188, Polo Scientifico, 50019 Sesto Fiorentino, Florence, Italy ∇ Dipartimento NEIROFARBA, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy ○ Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131 Napoli, Italy ◆ Biotecnologia − BIOINOVAR: Unidade de Bioenergia, Biocatalise e Bioprodutos, Instituto de Microbiologia Paulo de Góes, IMPG, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, Brasil ‡

ABSTRACT: Today, there are approximately 8 million cases of Chagas disease in the southern cone of South America alone, and about 100 million people are living with the risk of becoming infected. The present pharmacotherapy is sometimes ineffective and has serious side effects. Here, we report a series of 4,5dihydroisoxazoles incorporating hydroxamate moieties, which act as effective inhibitors of the carbonic anhydrase (CA) from Trypanosoma cruzi (TcCA). One compound (5g) was evaluated in detail and shows promising features as an antitrypanosomal agent. Excellent values for the inhibition of growth for all three developmental forms of the parasite were observed at low concentrations of 5g (IC50 values from 7.0 to 100 000 >100 000

802 27 900 47 300 94 500 297 808 815 847 733 257 3810 >100 000 >100 000

428 263 267 189 94.1 71.3 39.8 365 615 182 141 >100 000 >100 000

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The data presented here show that we discovered a quite promising class of hydroxamates with anti-Chagas disease activity. We have proven that one of these compounds, 5g, may be considered as a promising antitrypanosomal drug and a prototype for research and development for new drugs. Our preliminary data demonstrated that 5g may target two proteins that are important for the parasite life cycle: α-carbonic anhydrase (TcCA) and the metalloproteinases. These metalloproteins are probably targeted by the hydroxamates described here, leading to a powerful growth inhibition in vivo and a substantial decrease of parasitemia in mice infected with T. cruzi. Hydroxamate 5g is more effective compared to the clinically used agent benznidazole, thus warranting further studies to understand in more detail its efficacy and mechanism of action at the molecular level. Moreover, we are planning to carry out new structural changes in the scaffold. Such changes include the insertion of other heterocyclic rings and modification of the aromatic ring substituent to obtain a more potent drug for the treatment of the Chagas disease.

Figure 7. Effect of 5g on T. cruzi peptidases. (A) Zymographic gel containing gelatin as substrate. Compound 5g was incubated with the gels for 12 h and with the epimastigote forms at different concentrations. The parasite cell (epimastigote) was lysed and applied to the electrophoresis test. (B) Western blot analysis of the cell extract of epimastigotes treated for 5 days with 5g at different concentrations using MMP-9 polyclonal antibodies. CTL, control (without treatment); E-64, cysteine peptidase inhibitor; DTT, cysteine peptidase activator; 1,10-PHE, metallopeptidase inhibitor; *, metallopeptidase; and **, mysteine peptidase.

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Chemicals. Media constituents, reagents used in electrophoresis, buffer components, and nitrocellulose membrane were obtained from Amersham Life Science (Little Chalfont, England). The metallo (1,10phenanthroline (1,10-PHE)) and cysteine (trans-epoxysuccinyl-Lleucylamido-(4-guanidino) butane [E-64]) peptidase inhibitors, dithiothreitol (DTT), and secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit Fc) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-MMP-9 polyclonal antibody was purchased from BD Pharmigen and Santa Cruz (Biotechnology), respectively. All other reagents were analytical grade. All reagents used in the synthesis of 4,5-dihydroisoxazole derivatives were obtained from Sigma-Aldrich. General Procedure for the Synthesis of Aldoxime. The formation of aldoximes occurred using 20 mmol of different aldehydes (1a−j) previously dissolved in 50 mL of ethanol and 60 mmol (4.17 g) NH2OH·HCl solubilized in 10 mL of distilled water.41 This reaction was maintained for 2 h at 27 °C under constant agitation. This was followed by extraction of the compound formed in the organic phase (20 mL of methylene chloride and 10 mL of distilled water). Anhydrous sodium sulfate was added, and the solution was filtered. Monitoring the reaction was carried out by GC, and the disappearance of the aldehyde peak retention and appearance of a new major peak for the product formed (product not isolated) was observed. General Procedure for the Synthesis of 4,5-Dihydroisoxazol. Each aldoxime obtained (2a−j, 20 mmol) was cooled to 15 °C in an ice bath, 20 mmol (2.02 g) of triethylamine was added, and 10 mmol (2.32 g) of cyanuric acid trichloride was then added carefully.26,34,35 The system remained under cooling for 15 min, and after removing the ice bath, the medium was maintained at 27 °C with constant stirring for 30 min to generate the nitrile oxide. Isocyanuric acid, a byproduct of the reaction, was removed at the end of the reaction by simple filtration. Then, the dipolarophiles were added to the solution, and after 24 hours, the 4,5-dihydroisoxazol derivatives were generated by 1,3-cycloaddiction reactions. The yields varied from 35 to 54%. General Procedure for the Synthesis of Hydroxamic Acid Derivatives. A solution of hydroxylamine hydrochloride (NH2OH· HCl; 0.2 mmol) in methanol (100 mL) was mixed with a solution of potassium hydroxide (KOH; 0.3 mmol) in methanol (50 mL) at 0 °C with stirring. The mixture was keep at 0 °C (15 min), and the precipitated potassium chloride removed. The filtrate was added to the respective 4,5-dihydroisoxazol derivative (4a−k; 0.1 mmol) and kept at reflux for 24 h. The respective generated hydroxamic acid derivatives (5a−k) were recrystallized from hot water and filtered.37,42 The yields varied from 40 to 99%.

a

KI values were determined from the CO2 hydration reaction catalyzed by these enzymes (Khalifah stopped-flow method).40

39.8−615 nM. The structure−activity relationship for the inhibition of TcCA with hydroxamate 5g is straightforward: the nature of the substituted aryl moiety in position C3 of the 4,5dihydro-isoxazol moiety strongly influences the CA inhibitory activity. Indeed, rather compact aromatic moieties (Ph, 2-, 3-, or 4-chlorophenyl) led to medium-potency TcCA inhibitors (compounds 5a−d, with KI’s in the range of 189−428 nM), whereas bulkier moieties, such as in 5e−g, led to better inhibitors (KI’s of 39.8 − 94.1 nM, Table 1). The presence of a methyl moiety in the 5 position (as in 5h−k) did not drastically influence the TcCA inhibitory properties of these compounds compared to corresponding nonmethylated derivatives 5a−d. Hydroxamates 5 (and carbohydrazides 6) were also assayed as inhibitors of the two dominate human (h) CA isoforms, hCA I and II (Table 1). Hydrazides 6 are ineffective as inhibitors of these enzymes, whereas the hydroxamates act as weak-tomedium-potency inhibitors of both hCA I and II. Indeed, against hCA I, inhibition constants in the range of 133 nM− 72.2 μM were obtained, whereas against hCA II, the inhibition constants were in the range of 257 nM−94.5 μM. Such data prove that the hydroxamates investigated here are effective protozoan CA inhibitors and ineffective as host (human) CA inhibitors, which is a favorable finding for the potential of these compounds as anti-Chagas disease agents. 304

dx.doi.org/10.1021/jm400902y | J. Med. Chem. 2014, 57, 298−308

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H), 1664 (CO), 1593 (CN). 1H NMR (DMSO-d6): 3.80 (d, H1, JH4a−H4b = 17.3 Hz, H-4a of isoxazoline ring), 3.40 (d, H1, 2JH4b−H4a = 17,3 Hz, H-4b of isoxazoline ring), 8.9 (s, H1, NH of hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for C11H11BrN2O3: C, 44.2; H, 3.7; N, 9.4. Found: C, 44.9; H, 3.8; N, 9.6. 3-(3-Fluorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5carboxamide (5j). Yield, 82%. mp 172−174 °C. IR (cm−1): 3244 (O− H), 1670 (CO), 1610 (CN). 1H NMR (DMSO-d6): 3.76 (d, H1, 2 JH4a−H4b = 17.6 Hz, H-4a of isoxazoline ring), 3.36 (d, H1, 2JH4b−H4a = 17.6 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for C11H11FN2O3: C, 55.5; H, 4.6; N, 11.8. Found: C, 55.9; H, 4.7; N, 12.2. 3-(2-Fluorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5carboxamide (5k). Yield, 41%. mp 100−102 °C. IR (cm−1): 3192 (O−H), 1667 (CO), 1601 (CN). 1H NMR (DMSO-d6): 3.78 (d, H1, 2JH4a−H4b = 17.5 Hz, H-4a of isoxazoline ring), 3.39 (d, H1, 2 JH4b−H4a = 17.5 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for C11H11FN2O3: C, 55.5; H, 4.6; N, 11.8. Found: C, 56.1; H, 4.9; N, 12.0. 3-(2-Ethoxyphenyl)-4,5-dihydroisoxazole-5-carbohydrazide (6e). Yield, 37%. mp 130−131 °C. IR (cm−1): 3282 and 3223 (NH2), 1676 (CO), 1589 (CN). 1H NMR (DMSO-d6): 3.60 (dd, H1, 2 JH4a−H4b = 17.7 Hz, 3JH4a−H5 = 11.0 Hz, H-4a of isoxazoline ring), 3.50 (dd, H1, 2JH4b−H4a = 17.7 Hz, 3JH4b−H5 = 7.8 Hz, H-4b of isoxazoline ring), 4.98 (dd, H1, 3JH5−H4a = 11.0 Hz, 3JH5−H4b = 7.8 Hz, H-5 of isoxazoline ring), 9.43 (s, H1, NH of hydrazine), 4.35 (s, H2, NH2 of hydrazine). 3-(3-Ethoxyphenyl)-4,5-dihydroisoxazole-5-carbohydrazide (6f). Yield, 40%. mp 151−152 °C. IR (cm−1): 3300 and 3251 (NH2), 1683 (CO), 1563 (CN). 1H NMR (DMSO-d6): 3.55 (dd, H1, 2 JH4a−H4b = 17.4 Hz, 3JH4a−H5 = 11.0 Hz, H-4a of isoxazoline ring), 3.45 (dd, H1, 2JH4b−H4a = 17.4 Hz, 3JH4b−H5 = 7.3 Hz, H-4b of isoxazoline ring), 4.95 (dd, H1, 3JH5−H4a = 11.0 Hz, 3JH5−H4b = 7.3 Hz, H-5 of isoxazoline ring), 9.40 (s, H1, NH of hydrazine), 4.30 (s, H2, NH2 of hydrazine). Biological Assays. Mice. The animals used for the in vivo assay and to obtain macrophage cells were a susceptible mice BALB/c lineage from 5 to 8 weeks of age from the UFRJ vivarium. All animals were maintained at constant temperature (22 °C) and with light/dark cycles of 12 h. The care and handling of the animals were in accordance with the standards of the Ethics Committee of UFRJ. Cells, Parasite, and Growth Conditions. T. cruzi epimastigote forms (Y and Dm 28c strains) are part of our trypanosomatid culture collection and were cultivated in LIT (liver infusion tryptose) supplemented with yeast extract 0.5%, peptone 0.5%, KCl 2%, sucrose 2%, hemin 2 mg (w/v), and 10% fetal bovine serum at 28 °C. Cellular viability was assessed by motility using trypan blue cell dye exclusion.43 Bloodstream trypomastigote forms of T. cruzi (Y and Dm 28c strains) were maintained by blood passage in Balb/C mice every 7 days.44 Trypomastigotes were isolated from heparinized blood by low-speed centrifugation and collection of the parasites that swam out of the pellet. Experimental infection was performed by i.p. injection of 104 blood trypomastigotes. The total number of parasites/milliliter was determined by quantification in a Neubauer chamber. Intracellular amastigotes were obtained after infection with trypomastigote forms of macrophage cells extracted from Balb/C mice and THP-1 cells. The highly infected cells ruptured, releasing amastigote forms of T. cruzi after 3−5 days of infection.45 Human acute monocytic leukemia cells lines (THP-1) were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 μg/mL of streptomycin and 100 μg/mL of penicillin G). Macrophages were obtained by injecting thioglycollate46 (4%) i.p. into Balb/C mice, and peritoneal cells were collected by chilled RPMI washes 4 days after injection. Epimastigotes Extracts. Six-day-old cultured epimastigotes, 1 × 107 cells, at the log growth phase, were harvested by centrifugation (1500g, 15 min, 4 °C) and washed three times with phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.2). The pellet was resuspended in extraction lysis buffer (125 mM Tris, pH 6.8, 4%

General Procedure for the Synthesis of Acyl Hydrazine Derivatives. A solution of hydrazine hydrate 80% (NH2NH2; 5 mmol, 0.223 mL) in methanol (25 mL) was mixed with a solution of 4,5-dihydroisoxazol derivative (4e−f; 0.8 mmol) in methanol (20 mL). The mixture remained at reflux for 24 h. The respective generated acyl hydrazine derivatives (6e and 6f) were recrystallized from water and filtered. The yields were 37 and 40%, respectively. 3-Phenyl-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5a). Yield, 50%. mp 148−150 °C. IR (cm−1): 3194 (O−H), 1639 (C O), 1601 (CN). 1H NMR (DMSO-d6): 3.67 (dd, H1, 2JH4a−H4b ∼ 17.0 Hz, 3JH4a−H5 ∼ 11.0 Hz, H4a of isoxazoline ring), 3.53 (dd, H1, 2 JH4b−H4a ∼ 17.0 Hz, 3JH4b−H5 ∼ 7.3 Hz, H-4b of isoxazoline ring), 5.00 (dd, H1, 3JH5−H4a ∼ 11.0 Hz, 3JH5−H4b ∼ 7.3 Hz, H-5 of isoxazoline ring), 9.0 (s, H1, NH of hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid). Anal. Calcd for C10H10N2O3: C, 58.2; H, 4.9; N, 13.6. Found: C, 57.6; H, 4.8; N, 13.6. 3-(2-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5b). Yield, 35%. mp 105−108 °C. IR (cm−1): 3130 (O−H), 1666 (CO), 1592 (CN). 1H NMR (DMSO-d6): 3.84 (m, H2, H4 of isoxazoline ring), 5.27 (dd, H1, 3JH5−H4a ∼ 9.8 Hz, 3JH5−H4b ∼ 7.2 Hz, H-5 of isoxazoline ring), 6.4 (s, H1, NH of hydroxamic acid), 9.4 (s, H1, OH of hydroxamic acid). Anal. Calcd for C10H9ClN2O3: C, 49.9; H, 3.8; N, 11.6. Found: C, 46.4; H, 3.7; N, 11.4. 3-(3-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5c). Yield, 65%. mp 151−153 °C. IR (cm−1): 3256 (O−H), 1683 (CO), 1562 (CN). 1H NMR (DMSO-d6): 3.66 (dd, H1, 2 JH4a−H4b ∼ 17.2 Hz, 3JH4a−H5 ∼ 10.8 Hz, H-4a of isoxazoline ring), 3.56 (dd, H1, 2JH4b−H4a ∼ 17.2 Hz, 3JH4b−H5 ∼ 7.8 Hz, H-4b of isoxazoline ring), 5.02 (dd, H1, 3JH5−H4a ∼ 10.8 Hz, 3JH5−H4b ∼ 7.8 Hz, H-5 of isoxazoline ring), 9.1 (s, H1, NH of hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid). Anal. Calcd for C10H9ClN2O3: C, 49.9; H, 3.8; N, 11.6. Found: C, 48.1; H, 3.9; N, 11.6. 3-(4-Chlorophenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5d). Yield, 40%. mp 140−143 °C. IR (cm−1): 3259 (O−H), 1680 (CO), 1596 (CN). 1H NMR (DMSO-d6): 3.64 (dd, H1, 2 JH4a−H4b ∼ 17.2 Hz, 3JH4a−H5 ∼ 11.2 Hz, H-4a of isoxazoline ring), 3.50 (dd, H1, 2JH4b−H4a ∼ 17.2 Hz, 3JH4b−H5 ∼ 7.7 Hz, H-4b of isoxazoline ring), 5.0 (dd, H1, 3JH5−H4a ∼ 11.2 Hz, 3JH5−H4b ∼ 7.7 Hz, H-5 of isoxazoline ring), 10.0 (s, H1, OH of hydroxamic acid). 3-(2-Ethoxyphenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5e). Yield, 50%. mp 171−174 °C. IR (cm−1): 3203 (O−H), 1672 (CO), 1600 (CN). 1H NMR (DMSO-d6): 3.68 (dd, H1, 2 JH4a−H4b = 17.5 Hz, 3JH4a−H5 = 10.6 Hz, H-4a of isoxazoline ring), 3.6 (dd, H1, 2JH4b−H4a = 17.5 Hz, 3JH4b−H5 = 7.9 Hz, H-4b of isoxazoline ring), 4.9 (dd, H1, 3JH5−H4a = 10.6 Hz, 3JH5−H4b = 7.9 Hz, H-5 of isoxazoline ring); 4.3 (s, H1, NH of hydroxamic acid), 9.4 (s, H1, OH of hydroxamic acid). 3-(4-Ethoxyphenyl)-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5f). Yield, 40%. mp 155−157 °C. IR (cm−1): 3205 (O−H), 1641 (CO), 1608 (CN). 1H NMR (DMSO-d6): 3.56 (m, H2, H4 of isoxazoline ring), 4.93 (m, H1, H-5 of isoxazoline ring), 9.0 (s, H1, NH of hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid). 3-[4-(Benzyloxy)phenyl]-N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (5g). Yield, 54%. mp 100−102 °C. IR (cm−1): 3211 (O− H), 1639 (CO), 1597 (CN). 1H NMR (DMSO-d6): 3.60 (m, H2, H-4 of isoxazoline ring), 4.95 (dd, H1, 3JH5−H4a = 10.6 Hz, 3 JH5−H4b = 7.5 Hz, H-5 of isoxazoline ring), 9.0 (s, H1, NH of hydroxamic acid), 11.0 (s, H1, OH of hydroxamic acid). Anal. Calcd for C17H16N2O4: C, 65.4; H, 5.1; N, 8.9. Found: C, 64.6; H, 5.1; N, 8.7. 3-(3-Chlorophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5carboxamide (5h). Yield, 96%. mp 168−170 °C. IR (cm−1): 3188 (O−H), 1666 (CO), 1598 (CN). 1H NMR (DMSO-d6): 3.76 (d, H1, 2JH4a−H4b = 17.4 Hz, H-4a of isoxazoline ring), 3.38 (d, H1, 2 JH4b−H4a = 17,4 Hz, H-4b of isoxazoline ring), 8.8 (s, H1, NH of hydroxamic acid), 10.9 (s, H1, OH of hydroxamic acid). Anal. Calcd for C11H11Cl N2O3: C, 51.9; H, 4.5; N, 11.0. Found: C, 52.1; H, 4.4; N, 11.3. 3-(3-Bromophenyl)-N-hydroxy-5-methyl-4,5-dihydroisoxazole-5carboxamide (5i). Yield, 99%. mp 176−178 °C. IR (cm−1): 3188 (O−

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SDS, and 20% glycerol with or without 0.002% bromophenol blue) and stored on ice. Protein concentration was determined using the BioRad protein assay reagent according to the manufacture’s instructions using lysine as a standard; however, in this test, bromophenol blue was not added. Effect of the 4,5-Dihydroisoxazole Derivatives on the Growth Inhibition of T. cruzi Epimastigotes. 4,5-Dihydroisoxazole derivatives were incubated with T. cruzi epimastigote forms (Y and Dm 28 strains; 5 × 105 cells/mL) with 6 days of culture at several concentrations for 5 days. The cell count was performed daily until the fifth day of treatment in a Neubauer chamber and a quantitative colorimetric assay was performed using the oxidation−reduction indicator resazurin.39 Zymography Assay. T. cruzi epimastigote forms were incubated with 5g (4, 8, 16, 32, 64 μM) for 12 h under similar conditions as the T. cruzi epimastigotes growth inhibition assay, and the protein concentration of the epimastigotes extracts was determined. The cellular proteolytic activities were evaluated in 10% SDS-PAGE with 0.1% gelatin incorporated as substrate. The gels were loaded with 25 μL of cell extract (equivalent to 1 × 107 cells) per lane. After electrophoresis at a constant voltage of 170 V for 2 h at 4 °C, the gels were soaked for 1 h in 2.5% Triton X-100 under constant agitation. The gels were incubated for 48 h at 37 °C in 50 mM sodium phosphate buffer (pH 5.5). The effect of peptidase inhibitors (10 μM E-64 (a cysteine-peptidase inhibitor), 10 mM 1,10-phenanthroline (a nonspecific metallopeptidase inhibitor), and 2 mM DTT (a cysteinepeptidase activator)) on cellular proteolytic activities was also tested. Western Blot Analysis. T. cruzi epimastigote forms were incubated with 5g (4, 8, 16 μM) and 32 μM doxycycline (a nonspecific metallopeptidase inhibitor) for 5 days under similar conditions to the T. cruzi epimastigote growth inhibition assay, and the protein concentration of the epimastigotes extracts was determined. The treated or untreated cell extracts (30 μg) were electro-blotted onto a nitrocellulose membrane following separation on a 15% SDS-PAGE gel. The immunoblot was incubated for 2 h with blocking solution (5% milk in TBS) at room temperature followed by overnight incubation with various dilutions of the primary antibody (1:500 rabbit antiMMP-9 polyclonal antibody). The blots were washed three times with Tween-20 0.05%/Tris-buffered saline (T-TBS) and incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were again washed three times with T-TBS, and the proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Life Science). Cytotoxic Effect of 5g on T. cruzi Trypomastigotes. Trypomastigotes (1 × 106 cells/mL, Y and Dm 28c strains) were incubated for 48 h at 37 °C with 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum and 5g (1, 2, 4, 8, 16, and 32 μM). Cell counting was performed at 24 (data not shown) and 48 h in a Neubauer chamber. The tests were performed using a positive control without addition of the compound to be tested, a negative control, only the culture medium in the well, and a control with the highest concentration of DMSO obtained in the medium (0.02%). It should be noted that the assays were performed in duplicate and the results were based on the average of three different experiments. The determination of IC50 (half-maximal inhibitory concentration able to reduce trypomastigotes viability) was performed by linear-regression analysis from the percent inhibition values. In Vitro Assays for Parasite Burden. Peritoneal macrophages from Balb/c mice and THP-1 cells adhered on glass coverslips placed in the wells of 24-well plates were infected with T. cruzi trypomastigotes Y strain (3:1, parasite/macrophage) for 12 h at 37 °C in 5% CO2. The cultures were washed with RPMI 1640 to remove noninternalized trypomastigotes, treated with different concentrations of 5g compound (1, 2, 4, 8, 16, 32 μM), and incubated for 48 h. The cells were fixed with methanol and stained with Giemsa. To determine the parasitism, 300 cells were counted for the percentage of infected cells and the amount of intracellular amastigotes per infected cell. The tests were conducted in triplicate, and the results were based on the average of three different experiments. The highest

concentration of DMSO (0.02%) obtained in the assay was used as control for each experiment. In Vitro Macrophage Cytotoxicity. Two different cytotoxicity tests (LDH and MTT) were used to evaluate the biocompatibility of 5g. The LDH test measures only severe cell damage and enzyme release upon damage, whereas the MTT test measures the mitochondrial activity of the cells.47 In vitro cytotoxicity was determined in Balb/C macrophages treated with 5g for 24 (10−200 μM) and 48 (1−256 μM) h. The selectivity index (SI) was calculated for 5g by cytotoxicity in the macrophage cell assay (CC50 = 64 μM) and the macrophage parasite burden reduction assay (IC50 = 9.1 μM), both treated for 48 h. In Vivo Assay. Mice (Balb/C) were infected (i.p.) with 1 × 104 T. cruzi blood trypomastigotes (Y strain). The animals were divided into three groups, with each consisting of 7 mice: group 1, untreated (infected with T. cruzi but not treated); group 2, infected and treated with 5g (25 mg/kg); and group 3, infected and treated with benznidazole (100 mg/kg). The treatment was carried out for 7 consecutive days from the second to the ninth day postinfection by i.p. The blood parasite count was carried out from the sixth to the fourteenth day postinfection by the Pizzi−Brenner method.48,49 Carbonic Anhydrase Inhibition Assay. An Applied Photophysics stopped-flow instrument was used to assay CA-catalyzed CO2 hydration activity.40 Phenol red (at a concentration of 0.2 mM) was used as the indicator, working at an absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM Na2SO4 (for maintaining the ionic strength constant), and the initial rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 10−100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5−10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total rates observed. Stock solutions of inhibitor (0.1 mM) were prepared in distilled−deionized water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature or for 6 h at 4 °C prior to the assay to allow for the formation of the E-I complex. The inhibition constants were obtained by the nonlinear least-squares methods using PRISM 3, as reported earlier,11 and represent the mean from at least three different determinations. All CA isoforms were recombinant and obtained in-house as reported earlier.11

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*Phone: +55-21-25626743. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from Coordenaçaõ de ́ Superior (CAPES), Instituto Aperfeiçoamento Pessoal de Nivel Militar de Engenharia (IME), Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico (MCT/CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/ UFRJ), and Fundaçaõ Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) as well as by two EU grants (Metoxia and Dynano). We are grateful to Iêda Coleto Miguel de Castro and Silva Rocha de Souza for technical support and Marcelo Rosado Fantappié and Amanda Roberta Revoredo Vicentino for western blot assay support.

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ABBREVIATIONS USED CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor; gp63, glycoprotein of 63 kDa; hCA, human isoform of the carbonic anhydrase; MMP, matrix metalloproteinase; TcCA, 306

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Trypanosoma cruzi carbonic anhydrase; Tcgp63, Trypanosoma cruzi glycoprotein of 63 kDa

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