Synthesis and in vitro Evaluation of New Benzenesulfonamides as Antileishmanial Agents

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.


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. 2,3owever, 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. 4Another effective medicine is Vol.25, No. 5, 2014   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. 5,6Azole antifungals ketoconazole, miconazole and itraconazole have been used to treat cutaneous leishmaniasis with variable success rates. 7][10][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)-1Hpyrazoles, 12 1H-pyrazole-4-carbohydrazides, 13,14 1-aryl-1H-pyrazole-4-carboximidamides 15 and 4-(1H-pyrazol-1-yl)benzenesulfonamides. 16The sulfonamide group, in particular benzenesulfonamide, has proven importance in medicinal chemistry.A considerable number of sulfonamides are well known as antibacterial, 17 antiinflammatory 18 and anticancer 19 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.][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). 20,22We previously described the preparation of 2-(1H-pyrazol-3-yl)anilines (2) in mild conditions and excellent yields from 4-chloroquinolines (3). 224-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 NH 2 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 1 H nuclear magnetic resonance ( 1 H NMR), 13 C 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) pyrazole 22 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. 13Data from literature report that those different sensitivities occur among species and even in developmental growth stages. 24The 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. 25Metacyclogenesis consists in a life cycle differentiation process known to occur naturally in the developmental stage of Leishmania parasites. 26uring 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. 26. 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. 28Based in our data, we can suggest that the highest 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 IC 50 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. 16Thus, 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.Identified by 1 H NMR, 13 C NMR, FT-IR and HR-MS; b all compounds were recrystallized from ethanol; c isolated yields.

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 IC 50 , the compound is not hazardous to the host cells, afact that is a stimulus to continue our studies with this class of compounds.

Experimental
1 H and 13 C 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, CDCl 3 or DMSO-d 6 .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.

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 × 10 6 parasites mL -1 .Drugs were added to parasite Vol. 25, No. 5, 2014   cultures for screening, in a concentration range from 320 to 0.15 mg 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. 29Ketoconazole (Sigma-Aldrich) was used as reference drug.After 24 h of incubation at specific temperatures (26 o C for promastigotes and 32 o C 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 IC 50 /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 (10 5 IU L -1 ) and streptomycin sulfate (0.10 g L -1 ).The 2 × 10 6 cells per well were cultivated on microplate and incubated at 37 °C in a humidified 5% CO 2 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 IC 50 /24 h for axenic amastigotes of L. amazonensis and incubated for 48 h.After that, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) was added and after 2 h the reaction was interrupted with DMSO. 30he 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 COLLECT 31 and data reduction and unit cell refinement were achieved with the COLLECT 31 and DENZO 32 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. 33The ORTEP-3 for Windows 34 program was used in the preparation of the Figures.SHELXL-97 35 and PLATON 36 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.

Figure 1 .
Figure 1.Atom arrangement and atom numbering scheme for molecule 1e.Probability ellipsoids are drawn at the 50% level.Hydrogens on nitrogens are drawn as spheres of arbitrary radii.

Table 2 .
IC 50 (compound concentration required to kill parasites by 50% ± standard deviation) of pyrazolyl benzenesulphonamide compounds against L. amazonensis.All assays were performed three times, in triplicate