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

Article abstract of DOI:10.3390/molecules171112961

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.

Full text of DOI:10.3390/molecules171112961

Molecules 2012, 17, 12961-12973; doi:10.3390/molecules171112961
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
4-(1H-Pyrazol-1-yl) Benzenesulfonamide Derivatives:
Identifying New Active Antileishmanial Structures for
Use against a Neglected Disease
Roberta K. F. Marra 1, Alice M. R. Bernardino 1,*, Tathiane A. Proux 2, Karen S. Charret 3,
Marie-Luce F. Lira 2, Helena C. Castro 2,*, Alessandra M. T. Souza 4, Cesar D. Oliveira 1,
Júlio C. Borges 1, Carlos R. Rodrigues 4, Marilene M. Canto-Cavalheiro 3, Leonor L. Leon 3
and Veronica F. Amaral 2,*
1
Programa de Pós-graduação em Química, Instituto de Química, Universidade Federal Fluminense,
Outeiro de São João Baptista, Niterói, RJ, 24020-150, Brazil; E-Mails: katlenfusco@bol.com.br
(R.K.F.M.); posgraduacaouff@vm.uff.br (C.D.O.); juliusborges@yahoo.com.br (J.C.B.)
2
Programa de Pós-graduação em Ciências e Biotecnologia PPBI, Instituto de Biologia,
Universidade Federal Fluminense, Outeiro de São João Baptista, Niterói, RJ, 24020-150, Brazil;
E-Mails: tathimir@hotmail.com (T.A.P.); marie.biouff@gmail.com (M.-L.F.L.)
3
Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz,
Fundação Oswaldo Cruz, RJ, 21040-900, Brazil; E-Mails: karenbio@hotmail.com (K.S.C.);
mcantocavalheiro@hotmail.com (M.M.C.-C.); lleon@ioc.fiocruz.br (L.L.L.)
4
Faculdade de Farmácia, ModMolQSAR, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
RJ, 21941-590, Brazil; E-Mails: amtsouza2@yahoo.com.br (A.M.T.S.);
rangelrodrigues2003@yahoo.com.br (C.R.R.)
* Authors to whom correspondence should be addressed; E-Mails: gqolice@vm.uff.br (A.M.R.B.);
hcastrorangel@yahoo.com.br (H.C.C.); veronicadoamaral@gmail.com (V.F.A.);
Tel.: +55-21-2629-9954 (H.C.C.).
Received: 30 August 2012; in revised form: 8 October 2012 / Accepted: 12 October 2012 /
Published: 1 November 2012
Abstract: 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

Molecules 2012, 17
12962
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.
Keywords: pyrazoles; benzenesulfonamide; Leishmania; cytotoxicity; in silico evaluation
1. Introduction
Leishmaniasis is a parasitic disease with severe morbidity and mortality rates. According to the
World Health Organization, there are two million infected people and approximately 56,000 deaths
reported every year [1,2]. Ninety percent of the visceral leishmaniasis (VL) cases occur in Brazil,
India, Nepal, and Bangladesh [1,2]. Thus leishmaniasis is included in the WHO neglected diseases
group since the pharmaceutical industry has little commercial incentive to invest in new treatment options.
Leishmania spp. causes a broad spectrum of infectious diseases ranging from self-healing cutaneous
ulcerations to progressive and lethal visceral infection. L. amazonensis infection in humans can be
associated with a spectrum of disease manifestations, depending on the immune status of the host or
other external factors [3]. Currently the epidemiological pattern of Leishmania spp. [e.g., L. amazonensis
and L. infantum (L. chagasi syn.)] is changing, with a tendency to urbanization and geographic
expansion. Despite the high worldwide prevalence, few advances were made in the treatment of this
disease [4–9]. There are no vaccines for Leishmaniasis and vector control is complex [2,8].
Currently, the drugs used for leishmaniasis treatment present many disadvantages, including serious
clinical side effects such as nephrotoxicity, hepatotoxicity and cardiac arrhythmia, whereas the
emerging strain resistance to available drugs has also decreased the treatment options [7]. Resistance
to pentavalent antimonials is generating a problem in the treatment of visceral leishmaniasis in India
whereby naturally resistant parasites have higher virulence than susceptible L. donovani [9]. Some
additional drugs including pentamidine (aromatic diamine), amphotericin B (polyene antibiotic) and
miltefosine (alkyl phopholipid) were introduced as substitutes in chemotherapy but without complete
efficacy [4]. Currently the pentavalent antimonials are the first line treatment employed in Brazil,
followed by pentamidine and amphotericin B [10].
The leishmaniasis treatment restrictions and the resistant strains point to the urgent need for new
therapeutic options. Therefore, several reports regarding natural and synthetic new antileishmanial
compounds have been described [4,7], including some by our group [11–13]. Literature about the
pyrazole nucleus chemistry has reported a broad spectrum of pharmaceutical activities [14]. Similarly,
sulfonamides show different biological activity profiles, including antibacterial [15], anti-HIV [16],
anti-Trypanosome [17,18] and anti- Leishmania properties[17–20].
Recently, we reported new pyrazole carbohydrazide derivatives with in vitro antiparasitic activity
against L. amazonensis promastigotes and, to a lesser extent, L. braziliensis and L. infantum (chagasi syn.),
with no toxicity to murine macrophages [11]. The literature also described that mice experimentally
infected with L. amazonensis and treated with pyrazole carbohydrazide derivatives controlled the
evolution of both footpads cutaneous lesions and dissemination of parasites to draining lymph nodes [6].

Molecules 2012, 17
12963
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 [6,11–13].
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.
2. Results and Discussion
2.1. Chemistry
This work examined the functionalization of the core 1-phenylpyrazoles with a sulfonamide due to
the group’s antileishmanial profile previously described in the literature [11]. The 4-(4-bromo-5-
chloro-3-methyl-1H-pyrazol-1-yl)benzenesulfonyl chloride (1) derivative was obtained in good
yield by a regioselective electrophilic aromatic substitution reaction between the corresponding
1-phenylpyrazole derivative and chlorosulfonic acid. The compound 4-(5-chloro-3-methyl-1H-
pyrazol-1-yl)phenylamine (2) was prepared by nitration of the corresponding 1-phenylpyrazole
derivative, followed by reduction with iron powder and ammonium chloride.
The target compounds 3a–g could be easily prepared with these key intermediates in hand through
substitution reactions between the sulfonyl chloride moiety and amino intermediates [11,17]. Sulfonyl
chlorides are electrophilic reagents that react readily with primary and secondary amines, such as the
NH2 of the benzene ring as shown in Scheme 1.
Scheme 1. Synthesis of 4-(1H-pyrazol-1-yl)benzenesulfonamide derivatives 3a–g based on
the previous synthesis of 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles
(A) and 1-aryl-1H-pyrazole-4-carboximidamides (B).

Molecules 2012, 17
12964
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
1
characterized by spectroscopic techniques (IR, H-NMR and 13C-NMR) and elemental analysis.
The IR spectra of the compounds showed the presence of the characteristic bands for SO2 in the
1330–1630 cm−1 range. In addition, the 1H-NMR spectra of these compounds revealed the presence of
a singlet amino (NH) peak and multiplets due to the aromatic protons.
2.2. 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 [21–23]. Interestingly, 3b and 3e showed the best in vitro active
profile against the infective L. amazonensis promastigotes forms (IC50 = 0.070 mM and 0.072 mM,
respectively) as well as against L. infantum (L. chagasi syn.) (IC50 = 0.059 mM and 0.065 mM,
respectively) as shown in Table 1.
Table 1. Comparison of the antileishmanial (IC50) effect against Leishmania spp. and the
theoretical parameters evaluation of the molecular electronic properties of the new 4-(1H-
pyrazol-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).
Antileishmanial activity
Lipinski “rule of five”
(IC50 = mM) a,b
LUMO Dipole
Compound
(Ev)
(Debye)
Leishmania
infantum
Leishmania
amazonensis
S.I c
S.I c
MW clogP HBA HBD
3a
3b
3c
3d
3e
3f
0.228 ± 0.19
0.059 ± 0.01
0.123 ± 0.05
0.099 ± 0.08
0.065 ± 0.04
0.138 ± 0.11
0.149 ± 0.12
0.78
2.44
1.33
0.49
1.78
0.76
1.21
0.228 ± 0.33
0.070 ± 0.02
0.318 ± 0.59
0.075 ± 0.01
0.072 ± 0.05
0.153 ± 0.22
0.78
2.05
0.51
0.65
1.61
0.68
−1.61
−1.67
−1.63
−1.79
−1.77
−1.29
−1.18
4.61
4.53 426.72 3.27
4.80 440.75 3.58
3.69 505.62 3.97
3.75 461.17 3.88
4.84 347.83 2.57
5.24 361.85 2.88
4
4
4
4
4
4
1
1
1
1
1
1
3g
a
0.136 ± 0.054 1.33
b
Mean of IC50 (mM) ± S.D. for three determination; Pentamidine was used as control drug (IC50 = 0.062
and 0.021 mM; S.I = 0.87 and 2.57 respectively); c Selectivity index (SI): CC50 drug/IC50 drug.
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.
Our antileishmanial results with this new series are in agreement to the previous literature that
shows the sulfonamide functionality can display antiparasitic [17–20] as well as antibacterial [13,25]

Molecules 2012, 17
12965
and anti-viral HIV activities [16]. In fact the addition of the sulfonamide together with the bromide
improved the antileishmanial activity of this series compared to our previous data from 5-amino-1-
aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles derivatives [12] (Scheme 1). The activity against
L. amazonensis of our new pyrazole series leads 3b and 3e, was slightly better than that of 1-aryl-1H-
pyrazole-4-carboximidamides derivatives (lowest IC50 = 0.105 mM) [12] but not than that of 5-amino-
1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (lowest IC50 = 0.015 mM). The antileishmanial
profile against L. infantum was also evaluated for 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-
1H-pyrazoles) [13]. Our results reveal this new series is active and better (3b and 3e IC50 = 0.059 mM
and 0.065 mM, respectively) compared to the non-active profiles of the 1-aryl-4-(4,5-dihydro-1H-
imidazol-2-yl)-1H-pyrazole derivatives (lowest IC50 > 0.500 mM).
Importantly, 3b showed an antileishmanial activity against both Leishmania species (IC50 = 0.059 mM
and 0.070 mM) and comparable to the effect of pentamidine on L. infantum (IC50 = 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 (CC50 = 0.144 mM) and 3e (CC50 = 0.116 mM) with lower
cytotoxicity than pentamidine (CC50 = 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 [25–28].
Figure 1. The experimental cytotoxicity (CC50) using murine adherent peritoneal cells and
the theoretical drugscore values of the new 4-(1H-pyrazol-1-yl)benzenesulfonamide series
(3a−g) compared with pentamidine (Ptm), a current antileishmanial drug on the market.
Higher values on both analyses suggest a good drug profile.
2.3. 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

Molecules 2012, 17
12966
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).
Figure 2. Theoretical structural analysis of the new 4-(1H-pyrazol-1-yl)benzenesulfonamide
series (3a–g) using a molecular modeling approach. Comparison of the highest occupied
molecular orbital (HOMO) distribution (A) and of the structural orientation through the
superposition (B) of the less (compounds 3a, 3f, 3g and 3c) and most active (3b–e)
compounds. The theoretical analysis allowed pointing derivatives electronic regions (A)
and orientation (B) that are probably related with a better interaction with the parasitic
target (white boxes).
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).

Molecules 2012, 17
12967
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.
3. Experimental
3.1. Chemistry
3.1.1. 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
1
(60 F-254 Merck) and the products visualized with ultraviolet lamp (254 nm). H- and 13C-NMR
spectra were determined in DMSO-d6 and CDCl3 solutions using a Varian spectrometer, operating at a
frequency of 500.0 MHz for proton and 125.70 MHz 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
1
(multiplet). All described products showed H- and 13C-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.

Molecules 2012, 17
12968
3.1.2. General Procedure for the Synthesis of 4-(1H-Pyrazol-1-yl)benzenesulfonamides 3a–g
The functionalized amine 2 (0.4 mmol) was added to a solution of the appropriate arylsulfonyl
chloride derivative (1.0 mmol) in THF (5 mL) containing triethylamine (0.5 mL). The reaction mixture
was stirred for about 2 hours at room temperature, the end of the reaction was observed by TLC. The
sulfonamide derivatives 3a–g were isolated by addition of base and subsequent acidification. The
products were recrystallized from ethanol, affording the sulfonamide derivatives in good yields [21].
4-(4-Bromo-5-chloro-3-methyl-1H-pyrazol-1-yl)benzenesulfonamide (3a). Brown powder, m.p. =
79–80 °C, Yield: 83%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499 (C=C/C=N), 1334 and
1
1162 (SO2). H-NMR (DMSO-d6): 2.4 (s, 3H, CH3, pyrazole), 8.0 (s, 1H, NH), 6.3 (s, 1H, H4
pyrazole), 7.7 (d, 2H, J = 8.8 Hz, H2' and H6' phenyl), 7.3 (d, 2H, J = 8.8 Hz, H3' and H5' phenyl).
13C-NMR: 12.3 (CH3), 133.5 (C3 pyrazole), 10.2 (C4 pyrazole), 144.2 (C5 pyrazole), 140.7 (C1'
phenyl), 139.4 (C4' phenyl), 121.4 (C2' and C6' phenyl), 130.9 (C3' and C5' phenyl). Anal. Calc. for
C10H9N3 (%): C 34.26; H 2.59; N 11.98. Found (%) C 34.18; H 2.41; N 11.75.
4-(4-Bromo-5-chloro-3-methyl-1H-pyrazol-1-yl)-N-phenylbenzenesulfonamide (3b). Brown powder,
m.p. = 97–100 °C, Yield: 79%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499 (C=C/C=N),
1
1334 and 1163 (SO2). H-NMR (DMSO-d6): 2.2 (s, 3H, CH3, pyrazole), 2.3 (s, 3H, CH3, phenyl), 7.7
(d, 2H, J = 8.8, H2' and H6' phenyl), 7.4 (d, 2H, J = 8.8 Hz, H3' and H5' phenyl), 7.6 (d, 2H, J = 8.8 Hz,
H2" and H6" phenyl), 7.2 (dd, 2H, J = 8.8 and 8.3 Hz, H3" and H5" phenyl), 6.2 (s, 1H, H4 pyrazole).
13C-NMR: 12.2 (CH3), 133.5 (C3 pyrazole), 100.2 (C4 pyrazole), 144.2 (C5 pyrazole), 140.4 (C1'
phenyl), 121.6 (C2' and C6' phenyl), 131.5 (C3' and C5'), 138.5 (C4' phenyl), 138.0 (C1" phenyl), 121.5
(C2" and C6" phenyl), 130.5 (C3" and C5"), 125.1 (C4" phenyl). Anal. Calc. for C16H13N3 (%): C 45.03; H
3.07; N 9.85. Found (%) C 44.09; H 3.01; N 9.76.
4-(4-Bromo-5-chloro-3-methyl-1H-pyrazol-1-yl)-N-(4methylphenyl)benzenesulfonamide (3c). Brown
powder, m.p. = 158–160 °C, Yield: 78%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499
1
(C=C/C=N), 1334 and 1162 (SO2). H-NMR (DMSO-d6): 2.4 (s, 3H, CH3), 7.6 (s, 1H, NH), 7.9 (dd,
13
2H, J = 8.8 Hz, H2' and H6' phenyl), 8.1 (dd, 2H, J = 8.8 Hz, H3' and H5' phenyl). C-NMR: 12.7
(CH3), 149.0 (C3 pyrazole), 96.3 (C4 pyrazole), 127.1 (C5 pyrazole), 143.9 (C1' phenyl), 140.1
(C4' phenyl), 124.8 (C2' and C6' phenyl), 127.1 (C3' and C5'). Anal. Calc. for C17H15N3 (%): C 46.33;
H 3.43; N 9.53. Found (%) C 46.27; H 3.34; N 9.41.
4-(4-Bromo-5-chloro-3-methyl-1H-pyrazol-1-yl)-N-(4-bromophenyl)benzenesulfonamide (3d). Brown
powder, m.p. = 142–144 °C, Yield: 74%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499
1
(C=C/C=N), 1334 and 1162 (SO2). H-NMR (DMSO-d6): 2.4 (s, 3H, CH3), 10.5 (s, 1H, NH), 7.9 (d,
2H, J = 8.3 Hz, H2' and H6' phenyl), 8.0 (d, 2H, J = 7.8 Hz, H3' and H5' phenyl), 7.2 (d, 2H, J = 7.8 Hz,
H2" and H6" phenyl), 8.0 (dd, 2H, J = 7.8 and 8.3 Hz, H3" and H5" phenyl), 7.2 (t, 1H, 7.8 and 8.3,
13
H4"). C-NMR: 12.8 (CH3), 149.2 (C3 pyrazole), 96.5 (C4 pyrazole), 126.7 (C5 pyrazole), 140.8 (C1'
phenyl), 124.7 (C2' and C6' phenyl), 129.3 (C3' and C5'), 139.1 (C4' phenyl), 137.4 (C1" phenyl), 120.5
(C2" and C6" phenyl), 128.0 (C3' and C5'), 124.5 (C4' phenyl). Anal. Calc. for C16H12N3 (%): C 38.01; H
2.39; N 8.31. Found (%) C 37.49; H 2.23; N 8.29.

Molecules 2012, 17
12969
4-(4-Bromo-5-chloro-3-methyl-1H-pyrazol-1-yl)-N-(4-chlorophenyl)benzenesulfonamide (3e). Brown
powder, m.p. = −178–179 °C, Yield: 70%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499
(C=C/C=N), 1334 and 1162 (SO2). 1H-NMR (DMSO-d6): 2.4 (s, 3H, CH3, pyrazole), 2.6 (s, 3H, CH3,
phenyl) 10.7 (s, 1H, NH), 7.6 (d, 2H, J = 8.8 Hz, H2' and H6' phenyl), 8.0 (d, 2H, J = 8.8 Hz, H3' and
H5' phenyl), 7.2 (d, 2H, J = 8.8 Hz, H2" and H6" phenyl), 7.9 (dd, 2H, J = 8.8 and 8.3 Hz, H3" and
H5" phenyl). 13C-NMR: 12.7 or 12.6 (CH3 pyrazole or phenyl), 149.2 (C3 pyrazole), 96.8 (C4
pyrazole), 136.9 (C5 pyrazole), 148.2 (C1' phenyl), 132.2 (C2' and C6’ phenyl), 132.5 (C3' and C5').
140.9 (C4' phenyl), 138.7 (C1" phenyl), 128.0 (C2" and C6" phenyl), 137.9.0 (C3' and C5'). 124.7 (C4'
phenyl). Anal. Calc. for C16H12N3 (%): C 41.67; H 2.62; N 9.11. Found (%) C 41.55; H 2.57; N 9.03.
N-[4-(5-Chloro-3-methyl-1H-pyrazol-1-yl)phenyl]benzenesulfonamide (3f). Brown powder, m.p. =
169–170 °C, Yield: 70%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499 (C=C/C=N), 1334
and 1162 (SO2). 1H-NMR (DMSO-d6): 2.4 (s, 3H, CH3, pyrazole), 7.7 (s, 1H, H4 pyrazole), 10.7 (s, 1H,
NH), 7.4 (d, 2H, J = 8.8 Hz, H2' and H6' phenyl), 8.0 (d, 2H, J = 8.8 Hz, H3' and H5' phenyl), 7.2 (d,
2H, J = 8.8 Hz, H2" and H6" phenyl), 7.9 (dd, 2H, J = 8.8 and 8.3 Hz, H3" and H5" phenyl). 13C-NMR
(, ppm):12.6 (CH3 pyrazole), 149.0 (C3 pyrazole), 96.6 (C4 pyrazole), 128.6 (C5 pyrazole), 140.8 (C1'
phenyl), 124.4 (C2' and C6' phenyl), 127.9 (C3' and C5'). 138.7.9 (C4' phenyl), 136.3 (C1" phenyl), 121.9
(C2" and C6" phenyl), 129.0 (C3' and C5'). 124.9 (C4' phenyl). Anal. Calc. for C16H13N3 (%): C 45.03; H
3.07; N 9.85. Found (%) C 49.93; H 2.94; N 9.77.
N-[4-(5-Chloro-3-methyl-1H-pyrazol-1-yl)phenyl]-4-methylbenzenesulfonamide (3g). Brown powder,
m.p. = 215–217 °C, Yield: 73%, IR (cm−1): 3051 (CH), 2928 (CH), 1599, 1527 and 1499 (C=C/C=N),
1
1334 and 1162 (SO2). H-NMR (DMSO-d6): 2.3 (s, 3H, CH3 pyrazole), 7.7 (s, 1H, H4 pyrazole), 7.6
(d, 2H, J = 8.8, H2' and H6' phenyl), 7.8 (d, 2H, J = 8.8 Hz, H3' and H5' phenyl), 6.9 (d, 2H,
13
J = 8.8 Hz, H2" and H6" phenyl), 7.1 (dd, 2H, J = 8.8 and 8.3 Hz, H3" and H5" phenyl). C-NMR:
13.6 or 12.7 (CH3 pyrazol or phenyl), 126.1 (C3 pyrazole), 168.9 (C4 pyrazole), 148.5 (C5 pyrazole),
134.2 (C1' phenyl), 120.8 (C2' and C6' phenyl), 120.9 (C3' and C5'), 129.1 (C4' phenyl), 137.6 (C1"
phenyl), 127.9 (C2" and C6" phenyl), 129.6 (C3' and C5'). 143.3(C4" phenyl). Anal. Calc. for C17H15N3
(%): C 46.33; H 3.43; N 9.53. Found (%) C 46.17; H 3.33; N 9.41.
3.2. Pharmacology
3.2.1. In Vitro AntiLeishmanial Drug Assay
Parasites in metacyclic phase (4 × 106 parasites/mL) were incubated with the derivatives
(40–160 g/mL) solubilized in dimethyl sulphoxide (DMSO, Sigma Chemical Co., St. Louis, MO,
USA) at 26 °C for 24 h [20]. The results were expressed as IC50/24 h, the concentration of a compound
that caused a 50% reduction in survival/viability compared with non-treated culture. Pentamidine was
used as reference drug and all tests were carried out in triplicate. Promastigotes of L. amazonensis
(MHOM/BR/77/LTB0016 strain) and L. infantum (syn. chagasi) (MCAN/BR/97/P142 strain) were
grown in Schneider’s insect medium (Sigma) at 26 °C in pH 7.2 supplemented with 10–20% (v/v)
heat-inactivated fetal calf serum.

Molecules 2012, 17
3.2.2. Animals
12970
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).
3.2.3. In Vitro Cytotoxicity
BALB/c mice peritoneal cavity cells (4 × 105cells/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 (105 IU·L−1) and streptomycin sulfate (0.10 g·L−1) at 37 °C
in a humidified 5% CO2 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 CC50/24 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 CC50
value on the macrophage to the IC50 value on the L. amazonensis or L. infantum strains (promastigotes).
3.2.4. 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.

Molecules 2012, 17
12971
3.2.5. Statistical Analysis
Each experiment was done three to four times, in triplicate. Significance was determined using a
non-paired t Student test and Mann–Whitney analyses and p < 0.05.
4. Conclusions
In summary, a novel family of 4-(1H-pyrazol-1-yl)benzenesulfonamide derivatives 3a–g has been
synthesized and evaluated against Leishmania spp. The antileishmanial data showed a better active
profile for 3b–e on promastigote forms of L. infantum and L. amazonensis, close to that of the
reference drug pentamidine. The cytotoxic tests using murine adherent peritoneal cells pointed out that
3b and 3e had better IC50 values than pentamidine. 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. The biological and theoretical data reinforced the
potential of these molecules as an alternative option for testing against resistant Leishmania strains
and/or for further synthetic and biological exploration for the development of better antileishmanial drugs.
Acknowledgements
This work is supported by grants from CAPES, CNPq, FAPERJ, FIOCRUZ, PROPPI/UFF and
fellowships from CAPES, CNPq and PIBIC/UFF, Brazil.
Conflict of Interest
The authors declare no conflict of interest.
References
1. Alvar, J.; Vélez, I.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.; Jannin, J.; den Boer, M.; the
WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its
incidence. PLoS One 2012, 7, e35671.
2. World Health Organization (WHO). Organization Control of the Leishmaniases; World Health
Organization Technical Report 2010, Number 949; WHO: Geneva, Switzerland, 2010; pp. 1–186.
3. Leon, L.L.; Barral, A.; Machado, G.M.C.; Grimaldi, G., Jr. Antigenic differences among
Leishmania amazonensis isolates and their relationship clinical forms of the disease. Mem. Inst.
Oswaldo. Cruz 1992, 87, 229–234.
4. Croft, S.L.; Olliaro, P. Leishmaniasis chemotherapy-challenges and opportunities.
Clin. Microbiol. Infect. 2011, 17, 1478–1483.
5. Richard, J.V.; Werbovetz, K.A. New antileishmanial candidates and lead compounds.
Curr. Opin. Chem. Biol. 2010, 14, 447–455.
6. Charret, K.S.; Rodrigues, R.F.; Bernardino, A.M.; Gomes, A.O.; Carvalho, A.V.;
Canto-Cavalheiro, M.M.; Leon, L.; Amaral, V.F. Effect of the oral treatment with pyrazole
carbohydrazides derivatives in the murine infection by Leishmania amazonensis. Am. J. Trop.
Med. Hyg. 2009, 80, 568–573.

Molecules 2012, 17
12972
7. Kobets, T.; Grekov, I.; Lipoldova, M. Leishmaniasis: Prevention, parasite detection and treatment.
Curr. Med. Chem. 2012, 19, 1443–1474.
8. Singh, B.; Sundar, S. Leishmaniasis: Vaccine candidates and perspectives. Vaccine 2012, 30,
3834–3842.
9. Vanaerschot, M.; Doncker, S.; Rijal, S.; Maes, L.; Dujardin, J.-C.; Decuypere, S. Antimonial
resistence in Leishmania donovani is associated with increased in vivo parasite burden. PLoS One
2011, 6, e23120.
10. Ministry of Health of Brazil. 2008. Casos confirmados de leishmaniose visceral Segundo UF de
residências, Brasil grandes regiões
e
unidades federadas. Available online:
http://portal.saude.gov.br/arquivos/pdf/casos_lv.pdf (accessed on 31 October 2011).
11. Bernardino, A.M.R.; Gomes, A.O.; Charret, K.S.; Machado, G.M.; Canto-Cavalheiro, M.M.;
Leon, L.L.; Amaral, V.F. Synthesis and leishmanial activities of 1-(4-X-phenyl)-N'-[(4-Y-
phenyl)methyllene]-1H-pyrazole-4-carbohydrazides. Eur. J. Med. Chem. 2006, 41, 80–87.
12. 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.; et al. Synthesis and antileishmanial
activity of new 1-aryl-1H-pyrazole-4-carboximidamides derivatives. J. Braz. Chem. Soc. 2011,
22, 352–358.
13. 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. Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-
dihydro-1H-imidazol-2-yl)-1H-pyrazole derivatives. Bioorg. Med. Chem. Lett. 2011, 21, 7451–7454.
14. Fustero, S.; Sánchez-Reselló, M.; Barrio, P.; Simón-Fuentes, A. From 2000 to id 2010: A fruitful
decade for the synthesis of pyrazoles. Chem. Rev. 2011, 111, 6984–7034.
15. Patel, N.B.; Patel, J.N.; Lilakar, J.D. Sulfonamides of 2-[(2,6-dichlorophenyl)amino]phenyl
acetoxyacetic acid and their antibacterial studies. Acta Pol. Pharm. 2010, 67, 351–359.
16. Loh, B.; Vozzolo, L.; Mok, B.; Lee, C.C.; Fitzmaurice, R.J.; Caddick, S.; Fassati, A. Inhibition of
HIV-1 replication by isoxazolidine and isoxazole sulfonamides. Chem. Biol. Drug Des. 2010, 75,
461–474.
17. Silva, L.E.; Joussef, A.C.; Pacheco, L.K.; Silva, D.G.; Steindel, M.; Rebelo, R.A. Synthesis and in vitro
evaluation of leishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides.
Bioorg. Med. Chem. 2007, 15, 7553–7560.
18. Bilbao-Ramos, P.; Galiana-Roselló, C.; Dea-Ayuela, M.A.; González-Alvarez, M.; Veja, C.;
Rolón, M.; Pérez-Serrano, J.; Bolás-Fernández, F.; González-Rosende, M.E. Nuclease activity
and ultrastructural effects of new sulfonamides with anti-leishmanial and trypanocidal activities.
Parasitol. Int. 2012, 61, 604–613.
19. Dea-Ayuela, M.A.; Castillo, E.; Gonzalez-Alvarez, M.; Veja, C.; Rolón, M.; Bolás-Fernández, F.;
Borrás, J.; González-Rosende, M.E. In vivo and in vitro anti-leishmanial activities of 4-nitro-N-
pyrimidin and N-pyrazin-2-ylbenzenesulfonamides, and N2-(4-nitrophenyl)-N1-Propylglycinamide.
Bioorg. Med. Chem. 2009, 17, 7449–7456.
20. Souza, A.M.T.; Castro, H.C.; Brito, M.A.; Andrighetti-Fröhner, C.R.; Magalhães, U.;
Oliveira, K.N.; Gaspar-Silva, D.; Pacheco, L.K.; Joussef, A.C.; Steindel, M.; et al. Leishmania
amazonensis growth inhibitors: Biological and theoretical features of sulfonamide
4-methoxychalcone derivatives. J. Curr. Microbiol. 2009, 59, 374–379.

Molecules 2012, 17
12973
21. Souza, T.M.; Morais-Braga, M.F.; Saraiva, A.A.; Rolón, M.; Veja, C.; de Arias, A.R.; Costa, J.G.;
Menezes, I.R.; Coutinho, H.D. Evaluation of the anti-Leishmania activity of ethanol extract and
fractions of the leaves from Pityrogramma calomelanos (L.) link. Nat. Prod. Res. 2012, 8,
doi:10.1080/14786419.2012.686911.
22. Machado, M.; Pires, P.; Dinis, A.M.; Santos-Rosa, M.; Alves, V.; Salgueiro, L.; Cavaleiro, C.;
Sousa, M.C. Monoterpenic aldehydes as potential anti-Leishmania agents: Activity of Cymbopogon
citratus and citral on L. infantum, L. tropica and L. major. Exp. Parasitol. 2012, 130, 223–231.
23. De Carvalho, G.S.; Machado, P.A.; de Paula, D.T.; Coimbra, E.S.; da Silva, A.D. Synthesis,
Cytotoxicity, and Antileishmanial Activity of N,N'-Disubistituted Ethylenediamine and
Imidazolidine Derivatives. Sci. World. J. 2010, 10, 1723–1730.
24. Silveira, F.T.; Lainson, R.; Crescente, J.A.; de Souza, A.A.; Campos, M.B.; Gomes, C.M.;
Laurenti, M.D.; Corbett, C.E. A prospective study on the dynamics of the clinical and
immunological evolution of human Leishmania (L.) infantum chagasi infection in the Brazilian.
Amazon region. Trans. R. Soc. Trop. Med. Hyg. 2010, 104, 529–535.
25. Free Patent On Line. Sulfonamide-substituted fused 7-membered ring compounds, their use as a
medicament, and pharmaceutical preparations comprising them. Available online:
http://www.freepatentsonline.com/6908947.html (accessed on 15 January 2006).
26. Andrighetti-Frohner, C.R.; de Oliveira, K.N.; Gaspar-Silva, D.; Pacheco, L.K.; Joussef, A.C.;
Steindel, M.; Simões, C.M.; de Souza, A.M.; Magalhaes, U.O.; Afonso, I.F.; et al. Synthesis,
biological evaluation and SAR of sulfonamide 4-methoxychalcone derivatives with potential
antileishmanial activity. Eur. J. Med. Chem. 2009, 44, 755–763.
27. Parise-Filho, R.; Pasqualoto, K.F.; Magri, F.M.; Ferreira, A.K.; da Silva, B.A.; Damião, M.C.;
Tavares, M.T.; Azevedo, R.A.; Auada, A.V.; Polli, M.C.; et al. Dillapiole as Antileishmanial
Agent: Discovery, Cytotoxic Activity and Preliminary SAR Studies of Dillapiole Analogues.
Arch. Pharm. (Weinheim) 2012, 19, doi:10.1002/ardp.201200212.
28. Canto-Cavalheiro, M.M.; Echevarria, A.; Araujo, C.A.; Bravo, M.F.; Santos, L.H.; Jansen, A.M.;
Leon, L.L. The potential effects of new synthetic drugs against Leishmania amazonensis and
Trypanosoma cruzi. Microbios 1997, 90, 51–60.
29. Schetz, J.A. Structure-Activity Relationships. xPharm: The Comprehensive Pharmacology
Reference 2007, 1–12.
30. Lewis, D.F.V. Frontier orbitals in chemical and biological activity: Quantitative relationships and
mechanistic implications. Drug Metab. Rev. 1999, 31, 755–816.
31. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P. Experimental and computacional
approaches to estimate solubility and permeability in drug discovery and development settings.
J. Adv. Drug Deliv. Rev. 2001, 46, 3–26.
Sample Availability: Not available.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativICommons.org/licenses/by/3.0/).