Synthesis and in vitro evaluation of new benzenesulfonamides as antileishmanial agents

SHORT REPORT

Synthesis and in vitro evaluation of new benzenesulfonamides as antileishmanial agents

Julio C. Borges^{I, *}; Adriana V. Carvalho^II; Alice M. R. Bernardino^I; César D. Oliveira^I; Luiz C. S. Pinheiro^I; Roberta K. F. Marra^I; Helena C. Castro^III; Solange M. S. V. Wardell^IV; James L. Wardell^{V, VI}; Veronica F. Amaral^VII; Marilene M. Canto-Cavalheiro^II; Leonor L. Leon^II; Marcelo Genestra^{II, †}

^IPrograma de Pós-Graduação em Química, Departamento de Química Orgânica Universidade Federal Fluminense, Outeiro de São João Baptista, s/n, 24020-141 Niterói-RJ, Brazil

^IILaboratório de Bioquímica de Tripanosomatídeos, IOC, Fundação Oswaldo Cruz, 21040-900 Rio de Janeiro-RJ, Brazil

^IIIPrograma de Pós-Graduação em Ciências e Biotecnologia e LABiEMol, GCM, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, 24020-141 Niterói-RJ, Brazil

^IVCHEMSOL, 1 Harcourt Road, Aberdeen, AB15 5NY, Scotland

^VInstituto de Tecnologia em Fármacos, Fundação Oswaldo Cruz, Farmanguinhos, 21041-250 Rio de Janeiro-RJ, Brazil

^VIChemistry Department, University of Aberdeen, Meston Walk, Old Aberdeen, AB243EU, Scotland

^VIIPrograma de Pós-Graduação em Ciências e Biotecnologia, Departamento de Imunobiologia, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de São João Baptista, s/n, 24020-141 Niterói-RJ, Brazil

ABSTRACT

This paper describes the synthesis and the antileishmanial activity of new pyrazolyl benzenesulfonamide derivatives. These were elucidated by spectrometric methods. Some compounds showed a significant in vitro activity against Leishmania amazonensis, highlighting the derivative 1e. These pyrazolyl benzenesulfonamide derivatives did not show any toxicity in murine macrophage.

Keywords: pyrazolyl benzenesulfonamides, synthesis, antileishmanial activity

RESUMO

Este artigo descreve a síntese e a atividade antileishmania de novos derivados pirazolil benzenossulfonamídicos. Estes foram elucidados por métodos espectrométricos. Alguns compostos mostraram uma atividade in vitro significativa contra Leishmania amazonensis, destacando-se o derivado 1e. Nenhum dos derivados pirazolil benzenossulfonamídicos mostraram qualquer toxicidade em macrófagos murinos.

Introduction

Leishmania species are the casual agents of several clinical manifestations called leishmaniasis, which are transmitted to human and others mammals by the bite of an infected female Phlebotomine sandfly. Depending on the causative species, it can be manifested as cutaneous, mucocutaneous, diffuse cutaneous, and visceral leishmaniasis.¹ More than 2 million new cases of leishmaniasis occur each year, with approximately 350 million persons at risk of infection.^2,3 However, the chemotherapy for leishmaniasis is generally ineffective mainly due to the emergence of drug-resistant strains and toxicity of the therapeutic agents. The pentavalent antimonials meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam) are the drugs of first choice since the 50s. These compounds are widely used as primary therapy whereas other drugs, such as amphotericin B, pentamidine and paromomycin have been also employed as second line drugs.⁴ Another effective medicine is miltefosine, an alkylphosphocholine derivative originally developed as an anticancer drug, which has been registered in India, Germany and Colombia for oral treatment of visceral leishmaniasis (VL). However, it is not indicated in pregnant women and shows severe gastrointestinal side effects.^5,6 Azole antifungals ketoconazole, miconazole and itraconazole have been used to treat cutaneous leishmaniasis with variable success rates.⁷

There is a great interest in the synthesis of pyrazole derivatives, since they show a wide range of pharmacological properties including antimicrobial, anti-inflammatory, analgesic, antipyretic and antileishmania.^8-11 Our research group reported the synthesis of various pyrazole derivatives that showed antileishmanial activity, among them we can highlight 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles,¹² 1H-pyrazole-4-carbohydrazides,^13,14 1-aryl-1H-pyrazole-4-carboximidamides¹⁵ and 4-(1H-pyrazol-1-yl)benzenesulfonamides.¹⁶ The sulfonamide group, in particular benzenesulfonamide, has proven importance in medicinal chemistry. A considerable number of sulfonamides are well known as antibacterial,¹⁷ anti-inflammatory¹⁸ and anticancer¹⁹ agents.

Based on the above report and in the continuation of our studies on chemotherapy of leishmaniasis, herein we described synthesis of some new pyrazolyl benzenesulfonamic derivatives, as well as the evaluation of their antileishmanial activity and cytotoxicity.

Results and Discussion

The synthesis of sulfonamide derivatives (1) was performed by reaction between 2-(1H-pyrazol-3-yl)anilines (2) and corresponding sulfonyl chlorides. The sequence is outlined in Scheme 1. The 4-hydroxyquinolines (4) can be readily made via the Gould-Jacobs method.^20-22 The compounds (4) were easily chlorinated in refluxing phosphorus oxychloride at 120 ºC over a period of 24 h, affording the 4-chloroquinolines (3).^20,22 We previously described the preparation of 2-(1H-pyrazol-3-yl)anilines (2) in mild conditions and excellent yields from 4-chloroquinolines (3).²² 4-chloroquinolines react with an excess of hydrazine in diethyleneglycol initally at 90-100 ºC for one hour. Nucleophilic aromatic substitution of the chlorine atom in 3 by hydrazine occur in this temperature range to generate intermediate 4-hydrazinoquinolines, which on raising the temperature to 130-140 ºC, react further over a period of six hours to favor the rearrangement, affording the products 2.²² The preparation of new pyrazolyl benzenesulfonamide derivatives (1a-g) was performed through a substitution reaction between derivatives 2-(1H-pyrazol-3-yl)anilines (2) and excess of sulfonyl chloride. Benzenesulfonyl chlorides are electrophilic reagents that react readily with primary and secondary amines, such as NH of pyrazole and NH₂ of the benzene ring in 2.²³

All products were obtained with high purity levels and excellent yields, ranging from 91-82%, as shown in Table 1. These compounds were generally identified by ¹H nuclear magnetic resonance (¹H NMR), ¹³C NMR, Fourier transform infrared absorption (FT-IR) spectroscopies and high-resolution mass spectrometry (HR-MS). In particular, the structure of 1e was obtained by X-ray diffraction.

The atom arrangements and atom numbering scheme for molecule 1e are shown in Figure 1. The bond angles and bond lengths are all in the expected regions. The angle between the best planes through the pyrazole and aminoaryl rings in 1e is 20.53 (0.11), compared to that in the recently published 3-(2'-amino-3'-methylphenyl)pyrazole²² of 12º. A strong intramolecular hydrogen bond, N(3)–H(3)---N(1), is present in 1e, while a series of weak C–H---O intermolecular interactions generate chains and dimers.

In order to correlate the infectivity of parasite and susceptibility to drugs, sulfonamide derivatives were evaluated in different growth stages of Leishmania amazonensis. Also, the toxicity of those compounds to the host cell was evaluated and did not show any toxicity in murine macrophage at 320 µg mL^-1 of concentration.

Variations in the efficacy of drugs for the treatment of leishmaniasis are frequently a consequence of the immune status of the patient, the pharmacokinetic properties of the drug and the intrinsic differences in drug sensitivities of each Leishmania species.¹³ Data from literature report that those different sensitivities occur among species and even in developmental growth stages.²⁴ The life cycle of Leishmania spp. includes sequential development of invertebrate-stage promastigotes from a noninfective to an infective stage. Promastigotes obtained during the stationary phase of growth within the fly or culture is more infective for a vertebrate host than during their logarithmic phase. Thus, the putative metacyclics in the stationary phase showed increased infectivity compared with log phase cells.²⁵ Metacyclogenesis consists in a life cycle differentiation process known to occur naturally in the developmental stage of Leishmania parasites.²⁶ During this process, besides the structural modifications, several proteins with functions in protein synthesis, protein metabolism, protein folding and messenger RNA (mRNA) processing are down-regulated^25,27 while several other surface macromolecules are over expressed, such as a lipophosphoglycan (LPG) and a protease (GP63), which are related to the infective capacity of those forms.²⁶L. amazonensis reach the higher amount of metacyclic forms (about 73%) at the 3^rd day of growth, while at 24 h in culture none of those forms are detected, using lysis complement test.²⁸ Based in our data, we can suggest that the highest susceptibility of logarithmic forms would be associated with the lack of metacyclic forms, which are the infective ones.

Against axenic amastigotes, only three compounds (1d, 1e and 1g) showed good activity superior to that obtained with ketoconazole. The difference encountered for IC₅₀ values between promastigotes and amastigotes would be explained by the differences in the culture conditions (temperature, pH and fetal calf serum concentration), resulting in low drug availability, since some amount of drug could be linked to the serum albumin. However, we cannot explain the good result obtained for compound 1d, assayed at the same conditions. Alternatively, the overall activity profile of the tested compounds 1 could be compared with the antileishmanial properties shown by previously described sulfonamides.¹⁶ Thus, we found that there is significant difference in the antipromastigote activity by addition of the sulfonamide group.

The most active sulfonamide compound against L. amazonensis promastigotes was the compound 1e. This compound was also effective against axenic amastigote, as well as compound 1d. Also, the derivatives 1d and 1e showed an activity about 50-52% higher against axenic amastigotes, when compared to ketoconazole. Furthermore, none of the compounds showed toxicity to murine macrophage at 320 µg mL^-1 of concentration.

Conclusions

Our results seem promising since pyrazolyl benzenesulfonamide derivative 1e showed to be active in vitro against L. amazonensis promastigotes and axenic amastigotes. Furthermore, the in vitro experiment with macrophage demonstrated that, in a concentration equivalent to IC₅₀, the compound is not hazardous to the host cells, afact that is a stimulus to continue our studies with this class of compounds.

Experimental

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained at 300.00 and 75.00 MHz, respectively, on a Varian Unityplus instrument equipped with a 5 mm probe, using tetramethylsilane as the internal standard. Chemical shifts (d) are reported in ppm and coupling constants (J) in Hz. Samples were dissolved in the solvents specified, CDCl₃ or DMSO-d₆. Fourier transform infrared (FT-IR) absorption spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrophotometer by reflectance in KBr. The experimental analysis of electrospray ionization high-resolution mass spectrometry (ESI/MS) were performed in a high-resolution Micromass apparatus, model Q-TOF. Melting points (m.p.) were determined with a Fisatom model 430 D apparatus.

Preparation of pyrazolyl benzenesulfonamides derivatives (1a-g): 0.9 mmol of 2, 7.0 mL of THF, 200 mg of triethylamine and 2.0 mmol of the sulfonyl chloride were stirred under reflux for 3 hours. Later, the solvent was evaporated and the mixture was dropped in a beaker with ice and water. The formed crystals were recrystallized from ethanol.

4-Methyl-N-(2-methyl-6-{1-[(4-methylphenyl)sulfonyl]-1H-pyrazol-3-yl}phenyl)benzenesulfonamide, 1a: R = 2-CH₃; R' = CH₃

m.p.: 149 ºC; FT-IR (KBr) ν_max/cm^-1: 3275.8, 1335.9, 1163.7, 1595.3, 1524.8, 1380.4, 1304.3, 1115.4, 1089.9, 1036.1; ¹H NMR (CDCl₃, 300.00 MHz): δ 5.87 (d, 1H, J 2.7 Hz, H4), 7.80 (d, 1H, J 2.7 Hz, H5), 7.02 (d, 1H, J 7.8 Hz, H4'), 7.15 (t, 1H, J 7.8 Hz, H5'), 7.30 (d, 1H, J 7.8 Hz, H6'), 8.00 (dd, 2H, J 8.4, 1.8 Hz, H2"and H6"), 6.71 (dd, 2H, J 8.4, 1.8 Hz, H3" and H5"), 7.46 (d, 2H, J 8.1 Hz, H2"' and H6"'), 6.78 (d, 2H, J 8.1 Hz, H3"' and H5"'), 2.66 (s, 3H, CH₃), 2.46 (s, 3H, CH₃), 2.26 (s, 3H, CH₃), 8.79 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 154.9 (C3), 106.6 (C4), 132.3 (C5), 132.4 (C1'), 139.6 (C2'), 127.5 (C3'), 126.9 (C4'), 126.3 (C5'), 128.3 (C6'), 146.5 (C1"), 130.3 (C2" and C6"), 126.7 (C3" and C5"), 133.2 (C4"), 142.6 (C1"'), 128.4 (C2"' and C6"'), 126.2 (C3"' and C5"'), 134.4 (C4"'), 21.6 (CH₃), 21.2 (CH₃), 19.7 (CH₃); ESI-HRMS (M + H⁺) calcd.: 482.5972; found: 482.1410.

4-Methyl-N-(4-methyl-2-{1-[(4-methylphenyl)sulfonyl]-1H-pyrazol-3-yl}phenyl)benzenesulfonamide, 1b: R = 4-Me; R' = CH₃

m.p.: 163 ºC; FT-IR (KBr) ν_max/cm^-1: 3144.0, 1339.5, 1161.1, 1594.8, 1520.8, 1506.8, 1455.3, 1400.1, 1382.6, 1319.1; ¹H NMR (CDCl₃, 300.00 MHz): δ 6.50 (d, 1H, J 2.8 Hz, H4), 8.08 (d, 1H, J 2.8 Hz, H5), 7.58 (d, 1H, J 8.3 Hz, H3') 7.07 (dd, 1H, J 8.3, 2.0 Hz, H4'), 7.19 (d, 1H, J 2.0 Hz, H6'), 8.08 (dd, 1H, J 8.6, 1.8 Hz, H2" and H6"), 7.36 (dd, 2H, J 8.6, 1.8 Hz, H3" and H5"), 7.45 (dd, 2H, J 8.5, 1.8 Hz, H2"' and H6"'), 6.98 (dd, 2H, J 8.5, 1.8 Hz, H3"' and H5"'), 2.43 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 2.26 (s, 3H, CH₃), 10.11 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 155.3 (C3), 106.4 (C4), 131.1 (C5), 119.7 (C1'), 130.6 (C2'), 121.4 (C3'), 128.9 (C4'), 132.9 (C5'), 133.6 (C6'), 143.0 (C1"), 130.4 (C2" and C6"), 128.6 (C3" and C5"), 136.0 (C4"), 146.4 (C1"'), 128.9 (C2"' and C6"'), 126.9 (C3"' and C5"'), 21.6 (CH₃), 21.1 (CH₃), 20.5 (CH₃); ESI-HRMS (M + H⁺) calcd.: 482.5972; found: 482.0807.

N-(4-Chloro-2-{1-[(4-methylphenyl)sulfonyl]-1H-pyrazol-3-yl}phenyl)-4-methylbenzenesulfonamide, 1c: R = 4-Cl; R' = CH₃

m.p.: 144 ºC; FT-IR (KBr) ν_max/cm^-1: 3143.4, 1341.2, 1165.7, 1595.2, 1516.9, 1493.5, 1466.4, 1384.6, 1314.6, 1089.0; ¹H NMR (CDCl₃, 300.00 MHz): δ 6.52 (d, 1H, J 2.7 Hz, H4), 8.12 (d, 1H, J 2.7 Hz, H5), 7.65 (d, 1H, J 9.0 Hz, H3') 7.22 (dd, 1H, J 9.0, 2.4 Hz, H4'), 7.38 (d, 1H, J 2.4 Hz, H6'), 8.08 (d, 2H, J 8.1 Hz, H2" and H6"), 7.40 (d, 2H, J 8.1 Hz, H3" and H5"), 7.46 (d, 2H, J 8.1 Hz, H2"' and H6"'), 7.03 (d, 2H, J 8.1 Hz, H3"' and H5"'), 2.44 (s, 3H, CH₃), 2.29 (s, 3H, CH₃), 10.27 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 153.9 (C3), 106.4 (C4), 134.6 (C5), 122.4 (C1'), 135.8 (C2'), 126.9 (C3'), 129.2 (C4'), 121.2 (C5'), 131.4 (C6'), 146.7 (C1"), 131.4 (C2" and C6"), 130.5 (C3" and C5"), 116.6 (C4"), 143.4 (C1"'), 130.7 (C2"' and C6"'), 128.5 (C3"' and C5"'), 132.7 (C4"'), 21.6 (CH₃), 21.3 (CH₃); ESI-HRMS (M + H⁺) calcd.: 503.0154; found: 503.0162.

N-(4-Bromo-2-{1-[(4-methylphenyl)sulfonyl]-1H-pyrazol-3-yl}phenyl)-4-methylbenzenesulfonamide, 1d: R = 4-Br; R' = CH₃

m.p.: 128 ºC; FT-IR (KBr) ν_max/cm^-1: 3143.2, 1340.3, 1164.0, 1594.6, 1517.7, 1492.5, 1386.7, 1314.7, 1088.6; ¹H NMR (CDCl₃, 300.00 MHz): δ 6.53 (d, 1H, J 2.7 Hz, H4), 8,13 (d, 1H, J 2.7 Hz, H5), 7.59 (d, 1H, J 8.7 Hz, H3'), 7.35 (dd, 1H, J 8.7, 2.4 Hz, H4'), 7.53 (d, 1H, J 2.4 Hz, H6'), 8.08 (d, 2H, J 8.4 Hz, H2" and H6"), 7.42 (d, 2H, J 8.4 Hz, H3" and H5"), 7.46 (d, 2H, J 8.1 Hz, H2"' and H6"'), 7.04 (d, 2H, J 8.1 Hz, H3"' and H5"'), 2.44 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 10.29 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 154.0 (C3), 106.3 (C4), 134.1 (C5), 121.0 (C1'), 135.8 (C2'), 122.3 (C3'), 129.2 (C4'), 127.8 (C5'), 131.4 (C6'), 143.6 (C1"), 130.5 (C2" and C6"), 129.1 (C3" and C5"), 132.7 (C4"), 146.7 (C1"'), 129.6 (C2"' and C6"'), 126.9 (C3"' and C5"'), 21.6 (CH₃), 21.3 (CH₃); ESI-HRMS (M + H⁺) calcd.: 547.4667; found: 547.4677.

N-{2-Methyl-6-[1-(phenylsulfonyl)-1H-pyrazol-3-yl]phenyl}benzenesulfonamide, 1e: R = 2-Me; R' = H

m.p.: 138 ºC; FT-IR (KBr) ν_max/cm^-1: 3146.5, 1344.9, 1170.9, 1584.2, 1524.1, 1481.3, 1449.2, 1427.6, 1386.1, 1092.2; ¹H NMR (CDCl₃, 300.00 MHz): δ 5.88 (d, 1H, J 3.0 Hz, H4), 7.80 (d, 1H, J 3.0 Hz, H5), 7.05-7.01 (m, 1H, H4'), 7.17 (t, 1H, J 7.8 Hz, H5'), 7.32 (d, 1H, J 7.8 Hz, H6'), 8.15-8.12 (m, 2H, H2" and H6"), 6.99-6.96 (m, 3H, H3", H4" and H5"), 7.76-7.66 (m, 2H, H2"' and H6"'), 6.84-6.80 (m, 3H, H3"', H4"' and H5"'), 2.67 (s, 3H, CH₃), 8.85 (s, 1H, CH₃); ¹³C NMR (CDCl₃, 75.00 MHz): δ 155.0 (C3), 106.9 (C4), 135.0 (C5), 127.9 (C1'), 139.6 (C2'), 132.4 (C3'), 126.7 (C4'), 126.3 (C5'), 128.2 (C6'), 137.2 (C1"), 131.8 (C2" and C6"), 129.7 (C3" and C5"), 127.0 (C4"), 136.3 (C1"'), 132.2 (C2"' and C6"'), 130.7 (C3"' and C5"'), 127.4 (C4"'), 19.7 (CH₃); ESI-HRMS (M + H⁺) calcd.: 454.5440; found: 454.0550.

N-{4-Chloro-2-[1-(phenylsulfonyl)-1H-pyrazol-3-yl]phenyl}benzenesulfonamide, 1f: R = 4-Cl; R' = H

m.p.: 154 ºC; FT-IR (KBr) ν_max/cm^-1: 3134.1, 1336.0, 1166.8, 1584.2, 1524.8, 1497.0, 1449.2, 1397.4, 1091.3; ¹H NMR (CDCl₃, 300.00 MHz): δ 6.51 (d, 1H, J 3.0 Hz, H4), 8.13 (d, 1H, J 3.0 Hz, H5), 7.85 (d, 1H, J 8.4 Hz, H3'), 7.24 (dd, 1H, J 8.4, 1.5 Hz, H4'), 7.41 (d, 1H, J 1.5 Hz, H6'), 8.22-8.18 (m, 2H, H2" and H6"), 7.50-7.44 (m, 3H, H3", H4" and H5"), 7.75-7.59 (m, 2H, H2"' and H6"'), 7.50-7.44 (m, 3H, H3"', H4"' and H5"'), 10.25 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 154.1 (C3), 106.6 (C4), 135.2 (C5), 121.2 (C1'), 138.7 (C2'), 122.8 (C3'), 129.9 (C4'), 126.8 (C5'), 132.6 (C6'), 135.8 (C1"), 132.6 (C2" and C6"), 131.4 (C3" and C5"), 127.9 (C4"), 136.7 (C1"'), 133.5 (C2"' and C6"'), 131.6 (C3"' and C5"'), 128.7 (C4"'); ESI-HRMS (M + H⁺) calcd.: 474.9622; found: 474.9761.

N-{4-Bromo-2-[1-(phenylsulfonyl)-1H-pyrazol-3-yl]phenyl}benzenesulfonamide, 1g: R = 4-Br; R' = H

m.p.: 156 ºC; FT-IR (KBr) ν_max/cm^-1: 3133.1, 1335.2, 1164.0, 1583.8, 1525.1, 1493.9, 1448.8, 1379.2, 1090.8; ¹H NMR (CDCl₃, 300.00 MHz): δ 6.52 (d, 1H, J 3.0 Hz, H4), 8.13 (d, 1H, J 3.0 Hz, H5), 7.60 (d, 1H, J 8.4 Hz, H3'), 7.40-7.37 (m, 1H, H4'), 7.53 (d, 1H, J 2.4 Hz, H6'), 8.22-8.19 (m, 2H, H2" and H6"), 7.52-7.48 (m, 3H, H3", H4" and H5"), 7.74-7.68 (m, 2H, H2"' and H6"'), 7.26-7.24 (m, 3H, H3"', H4"' and H5"'), 10.29 (s, 1H, NH); ¹³C NMR (CDCl₃, 75.00 MHz): δ 154.0 (C3), 106.6 (C4), 135.1 (C5), 116.9 (C1'), 138.5 (C2'), 122.8 (C3'), 129.8 (C4'), 121.4 (C5'), 132.6 (C6'), 134.4 (C1"), 132.6 (C2" and C6"), 130.8 (C3" and C5"), 126.8 (C4"), 135.8 (C1"'), 132.7 (C2"' and C6"'), 131.6 (C3"' and C5"'), 128.5 (C4"'); ESI-HRMS (M + H⁺) calcd.: 519.4135; found: 519.9426.

Biological and cytotoxity assays

Parasites

Promastigotes (early log/24 h and late log/72 h growth phases) of L. amazonensis (MHOM/BR/77/LTB0016 strain), were grown at 26 ºC in Schneider's medium at pH 7.2 supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 UI mL^-1 penicillin, 100 µg mL^-1 streptomycin and 1 mmol L^-1 L-glutamine. Promastigote metacyclogenesis rate from early to late logarithmic growth curve was evaluated through complement lysis test. L. amazonensis axenic amastigotes were cultured at 32 ºC in Schneider's insect medium pH 5.5 supplemented with 20% (v/v) FCS, 0.5 mmol L^-1 Hepes, 60 UI mL¹ and 60 µg mL¹ streptomycin.²⁸

Drug assay

Parasites (promastigotes and axenic amastigotes), after having been harvested from the medium were counted in Neubauer's chamber and adjusted to a concentration of 4 × 10⁶ parasites mL^-1. Drugs were added to parasite cultures for screening, in a concentration range from 320 to 0.15 µg mL^-1 solubilized in dimethyl sulfoxide (DMSO) and the final concentration of the solvent in the experiments never exceeding 1.6%, considered not hazardous for the parasite.²⁹ Ketoconazole (Sigma-Aldrich) was used as reference drug. After 24 h of incubation at specific temperatures (26 ºC for promastigotes and 32 ºC for axenic amastigotes), the number of surviving parasites was counted in Neubauer's chamber and the percentage of growth inhibition was calculated comparing to the controls (parasites without drug). The IC₅₀/24 h values were calculated by Origin 5.0 software (Microcal Software, Inc.) with a specific toolbox for estimating curves.

Cytotoxic assay

The cytotoxic effect of the pyrazolyl benzenesulfonamide compounds was assayed on mice peritoneal macrophages. The cells were isolated from peritoneal cavity of BALB/c mice with cold Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 1 mmol L^-1 L-glutamine, 1 mol L^-1 HEPES, penicillin G (10⁵ IU L^-1) and streptomycin sulfate (0.10 g L^-1). The 2 × 10⁶ cells per well were cultivated on microplate and incubated at 37 ºC in a humidified 5% CO₂ atmosphere. After 2 h of incubation, no adherent cells were removed and the adhered macrophages were washed twice with RPMI. Compounds were added to the cell culture at concentration of the respective IC₅₀/24 h for axenic amastigotes of L. amazonensis and incubated for 48 h. After that, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was added and after 2 h the reaction was interrupted with DMSO.³⁰ The results were read in a MicroQuant spectrophotometer (Biotek-Instrument Inc., Winooski, VT) at a wavelength of 570 nm.

Statistical analyses

Each experiment was done three to four times, in triplicate. Significance was determined using a non-paired Student t-test and Mann-Whitney analyses. Differences were considered to be significant when p < 0.05.

Crystallography

Data were obtained at 120(2) K with Mo-Ka radiation by means of the Bruker-Nonius 95mm CCD camera on k-goniostatr of the EPSRC crystallographic service, based at the University of Southampton. Data collection was carried out under the control of the program COLLECT³¹ and data reduction and unit cell refinement were achieved with the COLLECT³¹ and DENZO³² programs. Correction for absorption was achieved in each case by a semi-empirical method based upon the variation of equivalent reflections with the program SADABS 2007/2.³³ The ORTEP-3 for Windows³⁴ program was used in the preparation of the Figures. SHELXL-97³⁵ and PLATON³⁶ were used in the calculation of molecular geometry. The structures were solved by direct methods using SHELXL-97 and fully refined by means of the program SHELXL-97. Difference map peaks provided positions for the hydrogen atoms of the NH groups for which the coordinates, along with isotropic displacement parameters, were fully refined. All other hydrogen atoms were placed in calculated positions. Crystal data and structure refinement details are listed in Table 3.

Supplementary Information

Supplementary data (FT-IR, ¹H NMR, ¹³C NMR, HR-MS and crystallographic data) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Pró-Reitoria de Pesquisa, Pós-graduação e Inovação (PROPPi, UFF). The use of the National Crystallographic Service at Southampton, UK and the valuable assistance of the staff there are gratefully acknowledged.

Submitted: November 19, 2013

Published online: April 4, 2014

Supplementary Data

The supplementary data is available in pdf: [Supplementary data]

References

• 1. World Health Organization (WHO); Leishmaniasis: Epidemiology and Access to Medicines - An Update Based on the Outcomes of WHO Regional Meetings, Literature Review and Experts' Opinion; Geneva, 2012. Available at http://www.who.int/leishmaniasis/resources/leishmaniasis_epidemiology_access_to_medicine/en/, accessed in March, 2014.
• 2. Alvar, J.; Vélez, I. D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.; Jannin, J.; den Boer, M.; PLoS One 2012, 7, e35671.
• 3. Singh, N.; Mishra, B. B.; Bajpai, S.; Singh, R. K.; Tiwari, V. K.; Bioorg. Med. Chem. 2014, 22, 18.
• 4. Croft, S. L.; Olliaro, P.; Clin. Microbiol. Infect 2011, 17, 1478.
• 5. Dorlo, T. P. C.; Balasegaram, M.; Beijnen, J. H.; Vries, P. J.; J. Antimicrob. Chemother 2012, 67, 2576.
• 6. Pandley, K.; Singh, D.; Lal, C. S.; Das, V. N. R.; Das, P.; J. Postgrad. Med. 2013, 59, 306.
• 7. Macedo-Silva, S. T.; Urbina, J. A.; Souza, W.; Rodrigues, J. C.; PLoS One 2013, 8, e83247.
• 8. Gilman, A. G.; The Pharmacological Basis of Therapeutics, 11^th ed.; McGraw Hill: New York, USA, 2006.
• 9. Abdel-Wahab, B. F.; Abdel-Latif, E.; Mohamed, H. A.; Awad, G. E.; Eur. J. Med. Chem. 2012, 52, 263.
• 10. El-Hawash, S. A.; Badawey, E. A. M.; El-Ashmawey, I. M.; Eur. J. Med. Chem. 2006, 41, 155.
• 11. Sánchez-Moreno, M.; Gómez-Contreras, F.; Navarro, P.; Marín, C.; Ramírez-Macías, I.; Olmo, F.; Sanz, A. M.; Campayo, L.; Cano, C.; Yunta, M. J. R.; J. Antimicrob. Chemother. 2012, 67, 387.
• 12. Santos, M. S.; Oliveira, M. L. V.; Bernardino, A. M. R.; Léo, R. M.; Amaral, V. F.; Carvalho, F. T.; Leon, L. L.; Canto-Cavalheiro, M. M.; Bioorg. Med. Chem. Lett. 2011, 21, 7451.
• 13. Bernardino, A. M. R.; Gomes, A. O.; Charret, K. S.; Freitas, A. C. C.; Machado, G. M. C.; Canto-Cavalheiro, M. M.; Leon, L. L.; Amaral, V. F.; Eur. J. Med. Chem 2006, 41, 80.
• 14. Charret, K. S.; Lagrota-Cândido, J.; Carvalho-Pinto, C. E.; Hottz, C. F.; Lira, M. F.; Rodrigues, M. F.; Gomes, A. O.; Bernardino, A. M.; Canto-Cavalheiro, M. M.; Leon, L. L.; Amaral, V. F.; Exp. Parasitol 2013, 133, 201.
• 15. Santos, M. S.; Gomes, A. O.; Bernardino, A. M. R.; Souza, M. C.; Khan, M. A.; Brito, M. A.; Castro, H. C.; Abreu, P. A.; Rodrigues, C. R.; Léo, R. M. M.; Leon, L. L.; Canto-Cavalheiro, M. M.; J. Braz. Chem. Soc 2011, 22, 352.
• 16. Marra, R. K. F.; Bernardino, A. M. R.; Proux, T. A.; Charret, K. S.; Lira, M. F.; Castro, H. C.; Souza, A. M.; Oliveira, C. D.; Borges, J. C.; Rodrigues, C. R.; Canto-Cavalheiro, M. M.; Leon, L. L.; Amaral, V. F.; Molecules 2012, 17, 12961.
• 17. Kamal, A.; Swapna, P.; Shetti, R. V. C. R. N. C.; Shaik, A. B.; Rao, M. P. N.; Gupta, S.; Eur. J. Med. Chem 2013, 62, 661.
• 18. Chandna, N.; Kumar, S.; Kaushik, P.; Kaushik, D.; Roy, S. K.; Gupta, G. K.; Jachak, S. M.; Kapoor, J. K.; Sharma, P. K.; Bioorg. Med. Chem 2013, 21, 4581.
• 19. Luo, Y.; Li, Y.; Qiu, K.; Lu, X.; Fu, J.; Zhu, H.; Bioorg. Med. Chem 2011, 19, 6069.
• 20. Price, C. C.; Roberts, R. M.; J. Am. Chem. Soc 1946, 68, 1204.
• 21. Mitscher, L. A.; Chem. Rev. 2005, 105, 559.
• 22. Borges, J. C.; Oliveira, C. D.; Pinheiro, L. C. S.; Marra, R. K. F.; Khan, M. A.; Wardell, J. L.; Wardell, S. M. S. V.; Bernardino, A. M. R.; J. Braz. Chem. Soc. 2007, 18, 1571.
• 23. Shriner, R. L.; Fuson, R. C.; Cutin, D. Y.; Identificação Sistemática dos Compostos Orgânicos, 6Ş ed.; Guanabara Dois: Rio de Janeiro, Brasil, 1983.
• 24. Silva, E. F.; Canto-Cavalheiro, M. M.; Braz, V. R.; Cysne-Finkelstein, L.; Leon, L. L.; Echevarria, A.; Eur. J. Med. Chem. 2002, 37, 79.
• 25. Bates, P. A.; Tetley, L.; Exp. Parasitol. 1993, 76, 412.
• 26. Sacks, D. L.; Perkins, P. V.; Am. J. Trop. Med. Hyg. 1985, 34, 456.
• 27. Mojtahedi, Z.; Clos, J.; Kamali-Sarvestani, E.; Exp. Parasitol 2008, 119, 422.
• 28. Cysne-Finkelstein, L.; Temporal, R. M.; Alves, F. A.; Leon, L. L.; Exp. Parasitol 1998, 89, 58.
• 29. Canto-Cavalheiro, M. M.; Echevarria, A.; Araújo, C. A.; Bravo, M. F.; Santos, L. H.; Jansen, A. M.; Leon, L. L.; Microbios 1997, 90, 51.
• 30. Denzinot, F.; Lang, R.; J. Immunol. Methods 1986, 89, 271.
• 31. Hooft, R. W. W.; COLLECT; Nonius B. V.: Delft, The Netherlands, 1998.
• 32. Otwinowski, Z.; Minor, W. In Macromolecular Crystallography in Methods in Enzymology; Carter, C. W.; Sweet Jr., R. M., eds.; Academic Press: New York, USA, 1997, vol. 276, part A.
• 33. Sheldrick, G. M.; SADABS Version 2007/2; Bruker AXS Inc.: Madison, Wisconsin, USA, 2007.
• 34. Farrugia, L. J.; J. Appl. Crystallogr 1997, 30, 565.
• 35. Sheldrick, G. M.; Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112.
• 36. Spek, A. L.; J. Appl. Crystallogr. 2003, 36, 7.

Publication Dates

• Publication in this collection
  30 May 2014
• Date of issue
  May 2014

History

• Received
  19 Nov 2013
• Accepted
  04 Apr 2014