4-(1H-Pyrazol-1-yl) Benzenesulfonamide Derivatives: Identifying New Active Antileishmanial Structures for Use against a Neglected Disease

Leishmaniasis is a neglected disease responsible for about 56,000 deaths every year. Despite its importance, there are no effective, safe and proper treatments for leishmaniasis due to strain resistance and/or drug side-effects. In this work we report the synthesis, molecular modeling, cytotoxicity and the antileishmanial profile of a series of 4-(1H-pyrazol-1-yl)benzenesulfonamides. Our experimental data showed an active profile for some compounds against Leishmania infantum and Leishmania amazonensis. The profile of two compounds against L. infantum was similar to that of pentamidine, but with lower cytotoxicity. Molecular modeling evaluation indicated that changes in electronic regions, orientation as well as lipophilicity of the derivatives were areas to improve the interaction with the parasitic target. Overall the compounds represent feasible prototypes for designing new molecules against L. infantum and L. amazonensis.

In addition the evaluation of pyrazole carboximidamide derivatives also revealed potential in vitro activity against L. amazonensis [12].
A therapy using an anti-inflammatory drug with antileishmanial properties, lower toxicity, cost, side effects and patient compliance may be very advantageous [6]. Previous reports from our laboratory described the in vitro and in vivo activity of pyrazoles against Leishmania parasites [6,[11][12][13]. In this work we describe the synthesis of a new pyrazole family exploring this time the addition of a sulfonamide group. Thus we evaluated the activity of these 4-(1H-pyrazol-1-yl)benzenesulfonamide derivatives against Leishmania infantum and L. amazonensis and their cytotoxicity profile towards mammalian cells. We also performed a structure-activity relationship (SAR) evaluation of these derivatives using a molecular modeling approach.
These reactions were rapid with no side products, according to the experimental data. All the synthesized compounds were obtained as solids, purified by recrystallization from ethanol and characterized by spectroscopic techniques (IR, 1 H-NMR and 13 C-NMR) and elemental analysis. The IR spectra of the compounds showed the presence of the characteristic bands for SO 2 in the 1330-1630 cm −1 range. In addition, the 1 H-NMR spectra of these compounds revealed the presence of a singlet amino (NH) peak and multiplets due to the aromatic protons.

Biological Evaluation
In this work we evaluated the biological effect of 4-(1H-pyrazol-1-yl)benzenesulfonamides derivatives 3a-g against both the L. infantum (L. chagasi syn.) and L. amazonensis promastigote forms and compared with them with pentamidine, a reference drug used in leishmaniasis treatment [4] similar to other reports in the literature [21][22][23]. Interestingly, 3b and 3e showed the best in vitro active profile against the infective L. amazonensis promastigotes forms (IC 50 = 0.070 mM and 0.072 mM, respectively) as well as against L. infantum (L. chagasi syn.) (IC 50 = 0.059 mM and 0.065 mM, respectively) as shown in Table 1. Table 1. Comparison of the antileishmanial (IC 50 ) effect against Leishmania spp. and the theoretical parameters evaluation of the molecular electronic properties of the new 4-(1Hpyrazol-1-yl)benzenesulfonamide series 3a-e, including the lowest unoccupied molecular orbital (LUMO) energy (eV), dipole (Debye), and Lipinski "rule of five" (molecular weight -Mw, number of hydrogen bound donor -HBD, or acceptor -HBA groups and lipophilicity -cLogP). L. infantum (L. chagasi syn.) is a strain of epidemiological importance [24] and responsible for the American visceral clinic form, whereas L. amazonensis has been implicated in cutaneous, mucosal, visceral and diffuse clinic forms of leishmaniasis [3]. Our biological data pointed to the potential of compounds 3b and 3e as active pyrazole structures containing sulfonamide groups for treating infections caused by these two Leishmania strains.
Importantly, 3b showed an antileishmanial activity against both Leishmania species (IC 50 = 0.059 mM and 0.070 mM) and comparable to the effect of pentamidine on L. infantum (IC 50 = 0.062 mM) ( Table 1). These data suggest that this derivative has the most potential for activity against L. infantum strains as an alternative to pentamidine. This is also reinforced by the cytotoxic evaluation using murine peritoneal adherent cells that showed 3b (CC 50 = 0.144 mM) and 3e (CC 50 = 0.116 mM) with lower cytotoxicity than pentamidine (CC 50 = 0.054 mM) ( Figure 1). The selectivity index to L. infantum reinforced the fact that compound 3b (2.44) as better than pentamidine (0.87). Overall the in vitro results pointed to further explore these molecules in the in vivo tests as described for this and for other synthetic derivatives [25][26][27][28].

Molecular Modeling Data
In order to identify structural features important to this series' antileishmanial profile we performed a structure-activity relationship (SAR) evaluation using a molecular modeling approach. The derivatives 3D-structures were constructed using the Spartan 10 program and the molecular properties were calculated as described in the Supplementary Material. Several parameters were evaluated, including the highest energy occupied molecular orbital (HOMO) and lowest energy unoccupied molecular orbital (LUMO). They are known as frontier orbitals or interacting molecular orbitals and a pair that lies closest in energy of any pair of orbitals in two molecules that interact, which allows them to interact most strongly. Therefore, it can be detected a correlation between the biological activity HOMO and LUMO energy and/or distribution as they may be directly involved in the interaction with the target [29,30].
The overall analysis pointed the lowest LUMO energy and dipole moment as well as the highest theoretical lipophilicity (cLog P) of the most active compounds as structural features that may contribute to the biological activity in this series. These features are probably related to the ability of penetrating the biological membranes of the parasite and interact with the biological target (Table 1).
According to our theoretical structural analysis the absence of the aromatic substituent affected the antileishmanial activity (i.e., 3a), in agreement to the literature [23]. The analysis of the minimum energy conformations of these compound showed that compounds 3b−e, 3f and 3g are coplanar, but with different spatial orientation ( Figure 2). This is probably due to the retroisosterism of the sulfonamide, where the sulphur atom is linked directly to the aromatic ring in derivatives 3a-e, whereas for 3f and 3g, the nitrogen atom of the sulfonamide is connected to the ring ( Figure 2). This variation led to different orientations that should influence the biological activity in this series as the spatial complementarity is a requirement to interact with the biological target ( Figure 2). The stereo-electronic features evaluation suggests that the HOMO energy level (not shown) and molecular weight have no direct correlation with the observed activity (Table 1). However compounds 3b-e exhibit different HOMO distribution profiles, which probably orient the interactions with the parasitic target (Figure 2).
The analysis of the steric parameters pointed to the importance of the substitution on the aromatic ring for the antileishmanial activity. Apparently a bigger substituent such as bromine (e.g., 3d) may cause some steric hindrance at the molecular interaction level and slight HOMO distribution profile differences resulting in a low antileishmanial profile. Meanwhile, smaller (e.g., chlorine) or no substituents on the aromatic ring may properly interact as in 3e and 3b, respectively (Table 1 and Figure 2).
The 4-(1H-pyrazol-1-yl)benzenesulfonamide derivatives 3a-g were also submitted to an in silico pharmacokinetics properties evaluation. Since good absorption is necessary for oral administration, we analyzed these derivatives according to the rule-of-five developed by Lipinski co-workers (Table 1) [31]. The rule-of-five indicates the theoretical potential for a chemical compound to have good oral bioavailability. The rule states that the most "druglike" molecules present clogP ≤ 5, molecular weight (MW) ≤ 500, number of hydrogen bond acceptors ≤ 10 and donors ≤ 5. Molecules violating more than one of these rules may have bioavailability problems. Our results showed that all compounds of the 4-(1H-pyrazol-1-yl)benzenesulfonamide (3a-g) fulfilled the Lipinski "rule-of-five". Importantly, according to the theoretical analysis of the lipophilicity (clog P), the most active inhibitors of L. amazonensis and L. infantum were sufficiently hydrophobic for penetrating the biological membranes.
We also compared the drugscore values of these new derivatives with pentamidine, an antileishmanial drug currently in use in the market (Figure 1). The most active compounds showed drugscore values (3b and 3e = 0.48 and 0.46, respectively) similar to pentamidine (0.45) (Figure 1), revealing their potential profile similar to drugs current on the market. Far from establishing absolute results, the molecular modeling data obtained in this work using this series may help to design new benzenesulfonamide-related compounds more activity and/or safety against leishmaniasis.

General
The chemicals were obtained from commercial supplies and used without purification, unless otherwise noted. Reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (60 F-254 Merck) and the products visualized with ultraviolet lamp (254 nm). 1 H-and 13 C-NMR spectra were determined in DMSO-d 6 and CDCl 3 solutions using a Varian spectrometer, operating at a frequency of 500.0 MH z for proton and 125.70 MH z for carbon. Peak positions are given in parts per million () from tetramethylsilane as internal standard, and coupling constant values (J) are given in Hz. Signal multiplicities are represented by: s (singlet), d (doublet), t (triplet), q (quadruplet) and m (multiplet). All described products showed 1 H-and 13 C-NMR spectra consistent with the assigned structures. Infrared (IR) spectra were obtained using spectrometer models 1420, 1600 FT-IR and Spectrum One FT-IR. Samples were examined as potassium bromide (KBr) disks. Melting points are uncorrected and were determined on a Fisatom (430-D) apparatus. All organic solutions were dried over anhydrous sodium sulfate and all organic solvents were removed under reduced pressure on a rotatory evaporator.

Animals
The BALB/c mice from Laboratory Animals Nucleus (UFF) were sacrificed to obtain peritoneal cells and for both infection and isolation of Leishmania. The protocol assays was approved by the Institutional Committee of the Center for Biological Evaluation and Care of Research Animals (CEUA-UFF).

In Vitro Cytotoxicity
BALB/c mice peritoneal cavity cells (4 × 10 5 cells/well) were incubated with the derivatives (10, 20, 40 and 80 µg/mL) in 96 wells plate for 24 h in cold 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 ) at 37 °C in a humidified 5% CO 2 atmosphere. After that, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT (Sigma) was added and the reaction was interrupted with DMSO after 2 h. The results were determined in 540 nm by using a MicroQuant spectrophotometer (Biotek-Instrument Inc., Winooski, VT, USA). All assays were repeated at least four times in quadruplicate. The cytotoxicity profile was expressed as CC 50 /24 h, the concentration of a compound that caused cytotoxicity compared to non-treated cultures [6]. Index of selectivity (IS) was defined as the ratio of the CC 50 value on the macrophage to the IC 50 value on the L. amazonensis or L. infantum strains (promastigotes).

Molecular Modeling Studies:
All molecular computations were performed using SPARTAN'08 (Wavefunction Inc. Irvine, CA, USA) as described elsewhere [20]. The theoretical studies of druglikeness and drugscore and ADMET were performed using Osiris Property Explorer (http://www.organic-chemistry.org/). Briefly the structures were optimized to a local minimum and the equilibrium geometry obtained in vacuum using RM1 semi-empirical methods. Subsequently, molecules were submitted to a single-point energy ab initio calculation, at the 6-31G* level, to calculate some stereoelectronic properties and perform the SAR studies. Thus, we calculated for all compounds best conformation the values of HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energies and density isosurface, molecular weight (MW), molecular surface area and volume, polar surface area (TPSA), dipole moment and lipophilicity using the same program.
The druglikeness value is calculated based on the occurrence frequency of each fragment is determined within the collection created by shredering 3300 traded drugs as well as 15,000 commercially available chemicals (Fluka Chemical Co., Buchs, Switzerland) yielding a complete list of all available fragments. In this case, positive values point out that the molecule contains predominantly the better fragments, wich are frequently present in commercial drugs but not in the non-druglike collection of fluka compounds. The drugscore combines druglikeness, clogP, logs, molecular weight and toxicity risks in one handy value that may be used to judge the drug potential of a compound.