Synthesis and antileishmanial activity of new 1-aryl-1H-pyrazole-4- carboximidamides derivatives

Article abstract of DOI:10.1590/s0103-50532011000200022

Chemotherapy for leishmaniasis, diseases caused by protozoa of the genus Leishmania, remains inefficient in several treatments. So there is a need to search for new drugs. In this work, we have synthesized 1-aryl-1H-pyrazole-4- carboximidamides derivatives and evaluated antileishmanial activities in vitro, as well as cytotoxic effects. Structure-activity relationship (SAR) studies were carried out with all the compounds of the series. Compound 2 showed an activity profile that can be improved through medicinal chemistry strategies.

Full text of DOI:10.1590/s0103-50532011000200022

J. Braz. Chem. Soc., Vol. 22, No. 2, 352-358, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Short Report
S
Synthesis and Antileishmanial Activity of New 1-Aryl-1H-Pyrazole-4-
Carboximidamides Derivatives
Maurício S. dos Santos,a Adriana O. Gomes,a Alice M. R. Bernardino,*,a
Marcos C. de Souza,a Misbahul A. Khan,b Monique A. de Brito,c Helena C. Castro,d
Paula A. Abreu,d Carlos R. Rodrigues,e Rosa M. M. de Léo,f Leonor L. Leonf and
Marilene M. Canto-Cavalheirof
aPrograma de Pós-Graduação em Química Orgânica and dLABioMol, GCM - IB,
Universidade Federal Fluminense, Outeiro de São João Baptista, 24020-150 Niterói-RJ, Brazil
bChemistry Department, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan
cLaboratório de Química Medicinal Computacional, Faculdade de Farmácia,
Universidade Federal Fluminense, 24241-000 Niterói-RJ, Brazil
eFaculdade de Farmácia, ModMolQSAR, Universidade Federal do Rio de Janeiro,
24020-150 Rio de Janeiro-RJ, Brazil
fLaboratório de Bioquímica de Tripanosomatídeos, IOC, Fundação Oswaldo Cruz,
21040-900 Rio de Janeiro-RJ, Brazil
A quimioterapia para as leishmanioses, doenças causadas por protozoários do gênero
Leishmania, ainda permanece ineficiente em diversos tratamentos. Portanto, existe a necessidade
de pesquisa por novos fármacos. Nesse trabalho, foram sintetizados derivados 1-aril-1H-pirazol-
4-carboximidamidas, avaliadas as atividades leishmanicida e os efeitos citotóxicos in vitro, e
realizado um estudo de relação estrutura-atividade (REA) com a série de compostos. O composto
2 apresentou um perfil de atividade que pode ser melhorado através de estratégias de modificação
molecular da química medicinal.
Chemotherapy for leishmaniasis, diseases caused by protozoa of the genus Leishmania, remains
inefficient in several treatments. So there is a need to search for new drugs. In this work, we have
synthesized 1-aryl-1H-pyrazole-4-carboximidamides derivatives and evaluated antileishmanial
activities in vitro, as well as cytotoxic effects. Structure-activity relationship (SAR) studies were
carried out with all the compounds of the series. Compound 2 showed an activity profile that can
be improved through medicinal chemistry strategies.
Keywords: synthesis, 1-aryl-1H-pyrazole-4-carboximidamides, leishmaniasis
Introduction
manifestations occur in four major forms in humans
including: i) visceral leishmaniasis (VL) that is usually
fatal when untreated, ii) muco-cutaneous leishmaniasis
(MCL) that is a mutilating disease, iii) diffuse cutaneous
leishmaniasis (DCL), which is a long-lasting disease due
to a deficient cellular-mediated immune response and,
iv) cutaneous leishmaniasis (CL) that is disabling when
there are multiple lesions.3
The difficulty to control this parasitic disease remains a
serious problem mainly due to the diversity of mammalian
reservoirs (wild and domestic animals), species of vectors
and Leishmania species.4
Leishmaniasis is a group of vector-borne diseases
caused by species of the genus Leishmania that affects
about 12 million people in 88 countries in the world.
These life-threatening diseases are of medical, social and
economic importance in endemic areas, particularly in
subtropical and tropical regions. Leishmania parasites
exist in two forms: amastigote in the mammalian host and
a flagellated promastigote in the insect vector.1,2 Clinical
*e-mail: alicerolim@globo.com

Vol. 22, No. 2, 2011
dos Santos et al.
353
Chemotherapy for leishmaniasis is generally ineffective
mainly due to the emergence of drug-resistant strains
and toxicity of the therapeutics agents. The pentavalent
antimonials compounds are widely used as primary
therapy whereas other drugs such as amphotericin B,
pentamidine, paromomycin, azole derivatives and
glucantime are also used.5
In the present work, we prepared five new 1-aryl-1H-
pyrazole-4-carboximidamides derivatives 1-5 and evaluated
their leishmanicidal acitivity, cytotoxicity and theoretical
profiles (Scheme 1).
Results and Discussion
Pyrazoles are a class of heterocyclic compounds
that exhibit a broad spectrum of biological activities
such antiinflamatory, antimicrobial and antitumor.6
Consequently, a large number of synthetic routes
to pyrazoles have been reported and summarized in
some monographs and reviews.7,8 These reports have
been useful for biologists and chemists engaged in
the development of new drugs and/or synthetic routes.
Our group has synthesized pyrazole carbohydrazides
with anti-Leishmania in vitro9 and in vivo10 activity.
Simultaneously, substances containing the amidine group
and affecting large number of pathogens (i.e., Giardia
lamblia, Leishmania sp., Pneumocystis carinii, Candida
albicans, Aspargillus sp. and Trypanosoma sp.) have
been reported,11 as well as some reviews about synthetic
approaches.12 Pentamidine (Figure 1) is clinically used in
the treatment of pneumonia caused by the opportunistic
fungus, Pneumocystis jirovecii, early stage human
african tripanosomiasis (HAT) and when treatment with
pentavalent antimonials or amphotericin B has failed
against Leishmania.5,13
The key intermediates 1-aryl-1H-pyrazole-4-
carbonitriles 24-28 were obtained from two alternate
routes (methods A and B). In method A, 1-aryl-1H-
pyrazole-4-carbaldehydes 10-13 were synthesized through
aVilsmeyer-Haack reaction involving 1-aryl-1H-pyrazoles
6-9, dimethylformamide (DMF) and POCl3.14 The
aldehydes formed were converted to key intermediates from
the “one-pot reaction” with hydroxylamine and methanoic
acid.15 In method B, arylhydrazine hydrochlorides
14-18 reacted with ethoxymethylenemalononitrile in
ethanol and the resulting 5-amino-1-aryl-1H-pyrazole-4-
carbonitriles 19-23 were subjected to aprotic deamination
with t-butyl nitrite in tetrahydrofuran to generate the key
intermediates.16,17 Finally, the targets 1-aryl-1H-pyrazole-
4-carboximidamides derivatives 1-5 were obtained by
the reaction of the key intermediates 24-28 with gaseous
hydrochloric acid followed by treatment with ammonia.18
The compounds 1-5 were identified by proton nuclear
magnetic resonance (1H NMR), carbon nuclear magnetic
resonance (13C NMR), Fourier transform infrared (FTIR)
spectroscopies and elementary analysis.
Biological and cytotoxity assays
The effect of different concentrations (40, 80, 160 and
320µgmL-1)ofthe1-aryl-1H-pyrazole-4-carboximidamides
Figure 1. Structural formula of pentamidine.
Scheme 1.

354
Synthesis and Antileishmanial Activity
J. Braz. Chem. Soc.
derivatives against L. amazonensis promastigotes growth
inhibition was monitored microscopically at the end of the
exponential growth phase. Interestingly, an antiproliferative
effect was observed for compounds 2 and 3 (IC50 = 105
45 µmol L-1 or 279 12 µg mL-1 and 112 41 µmol L-1 or
259 12 µg mL-1 respectively) on L .amazonensis in contrast
to compounds 1 (IC50 > 1720 µmol L-1 or > 320 µg mL-1),
4 (IC50 > 1480 µmol L-1 or > 320 µg mL-1) and 5
(IC50 > 1250 µmol L-1 or > 320 µg mL-1) (Figure 2). Despite
the lower profile of 2 and 3 compared to pentamidine effect
(IC50 = 3.6 1.6 µmol L-1), their activity is still promising
since new substitutions may be performed to improve it.
The major pyrazolic compounds presented a cytotoxicity
profile better than pentamidine (Figure 2).
adenosine monophosphate) protein kinase (PKA)21 and
on nitric oxide production by promastigotes and axenic
amastigotes forms of L. amazonensis.22
C o n c e r n i n g t h e 1 - a r y l - 1 H - p y r a z o l e - 4 -
carboximidamides compounds, the pyrazoles nucleus have
displayed an impressive array of biological activities, among
which antiprotozoa, anti-malarial, anti-inflammatory,
immunomodulatory, nitric oxide inhibition, cytotoxic and
anti-cancer activities.23
Molecular modeling and Lipinski rule of five studies
The minimum energy conformations of 1-5 derivatives,
calculated by theAM1 semiempirical Hamiltonian,24 showed
that, as expected, all rings of these compounds are co-planar
except for 5 where the two chlorines lead to a rotation of the
ring (Figure 3). Despite of this conformational difference,
this structural feature did not contribute for a biological
activity for 5. Subsequently, a single-point energy ab initio
calculation was performed at the 6-311G* level in order
to derive electronic properties, such as highest occupied
molecularorbital(HOMO)andlowestunoccupiedmolecular
orbital (LUMO) energy values, volume, molecular dipole
moment (µ), and molecular electrostatic potential (MEP),
which could be related to the variation of the antileishmanial
activity of these compounds.25 The results showed that,
although the substitution pattern into the 1-aryl-1H-
pyrazole-4-carboximidamides structures lead to different
antileishmanial profiles, the molecular dipole moment,
volume and TPSA (total polar surface area) values did not
present any direct correlation with it, as shown in Table 1
and Figure 3.
Figure 2. Biological features of 1-aryl-1H-pyrazole-4-carboximidamides
compounds compared with pentamidine. Antiparasitic effect against
Leishmania amazonensis (left) and cytotoxicity profile against mice
peritoneal macrophages (right).
These biological results may suggest that the two
amidine groups of the pentamidine (Figure 1) are important
for binding in a non-intercalative way to the minor grove
regions of DNA (kDNA) in the Leishmania.19
Literature reports described monoamidine derivatives
with three hydrophobic phenyl groups with potential
effects against L. amazonensis.20 This effect may be
associated to an increase of lipophilicity of these
compounds, which facilitates the transport through
parasite membrane. In addition, it has been demonstrated
the inhibitory effect of these monoamidine derivatives on
the phosphorylating activity of cAMP-dependent (cyclic
Differently, HOMO and LUMO energy values
apparently seem to be related to these derivatives biological
profile since compounds 2 and 3 showed an antileishmanial
profile and also presented the lowest values for these orbital
energies (Table 1). This suggests a different reactivity for
these molecules compared to the other derivatives that
probably allowed their active profile.
As receptors recognize stereo-electronic effects and
not atom per se, studies of molecular electronic properties
Table 1. Theoretical evaluation of 1-aryl-1H-pyrazole-4-carboximidamides compounds
Compound
R
IC50 (mg mL-1
Energy (eV)
Dipole
(Debie)
Volume
(A3)
TPSA
Lipinski rule of five
)
HOMO
LUMO
2.60
Mw
HBA
HBD
clogP
-0.025
0.784
1
2
3
4
5
H
> 320
279
-8.38
-9.33
-9.20
-7.99
-8.97
4.17
2.88
3.59
5.19
5.50
215.37
240.04
246.52
246.64
250.98
67.7
67.7
113.5
76.9
67.7
186.22
265.11
231.22
216.24
255.11
4
4
7
5
4
3
3
3
3
5
Br
-0.79
0.85
NO2
OMe
2,6Cl
259
-0.066
0.032
> 320
> 320
2.78
2.34
1.658

Vol. 22, No. 2, 2011
dos Santos et al.
355
could be very effective in interpreting the electronic
structure in a comprehensive way.26 Therefore, the MEP
is a useful approach for understanding the electrostatic
contribution for the receptor-ligand binding process that
has been used in different reports for elucidating this issue.
In this work, the analysis of R position on MEPs generated
for these compounds revealed that the substitution lead to a
different electrostatic distribution depending on the added
group mainly on compound 2 (Figure 3). However this
MEP difference is not directly expressed in the biological
activity as expected.
were recorded as potassium bromide (KBr) pellets on
a Perkin-Elmer Model 1420 FT IR Spectrophotometer.
Microanalyses were performed on a Perkin-Elmer Model
2400 instrument and all values were within 0.4% of the
calculated compositions. Purity of the reaction products
were checked by means of thin layer chromatography
(TLC) using silica gel plates with fluorescent indicator and
hexane/ethyl acetate (1:1, v/v) as eluent, melting points, IR
and 1H NMR spectra.
General procedure for the preparation of 1-aryl-1H-
pyrazole-4-carbaldehydes compounds 10-13
POCl3 (0.023 mol) was added to DMF (0.033 mol)
at 0 oC and the mixture was stirred for 15 min. After this
time 1-aryl-1H-pyrazoles 6-9 (0.003 mol) dissolved in
DMF were added dropwise with stirring. The reaction
mixture was then heated 3 h at 110 oC. The solution was
then poured slowly into 5 mL saturated sodium carbonate
aqueous solution and stirred 30 min. The organic layer
was diluted with ether, washed with saturated Na2CO3
aqueous solution, and dried with MgSO4 anhydrous.
Evaporation of the organic extract under reduced pressure
gave the corresponding products. The recrystallization
was made from ethanol/water. 10 (R = H): mp 84-85
Figure 3. Conformational and electronic properties of 1-aryl-1H-
pyrazole-4-carboximidamides derivatives. Tube (left) and CPK (middle)
representation of the most stable conformation are represented by atom
color. On the right, the molecular electrostatic potential energy isosurfaces
(MEP) is superimposed onto total electrons density of 0.002 e au-3. The
color code (see online) is in the range of -25 to +55 kcal mol-1.
o
oC, yield 73%; 11 (R = 4-Br): mp 124-126 C, yield
o
86%; 12 (R = 4-NO2): mp 150-151 C, yield 75%; 13
(R = 4-OCH3): mp 91-92 oC, yield 59%.
Since the compounds are considered for oral delivery, in
this work we submitted them to the analysis of Lipinski rule
of five,27 which indicates if a chemical compound could be
an orally active drug in humans. The rule states that most
“drug-like” molecules have clogP ≤ 5, molecular weight
(Mw) ≤ 500, PSA ≤ 140, and number of hydrogen bond
acceptors (HBA) ≤ 10 and donors (HBD) ≤ 5. Molecules
violating more than one of these rules may have problems
with bioavailability.27 Our results pointed all compounds
as fulfilling this rule and therefore with a good theoretical
biodisponibility (Table 1).
General procedure for the preparation of 5-amino-1-aryl-
1H-pyrazole-4-carbonitriles compounds 19-23
Arylhydrazine hydrochlorides 14-18 (0.01 mol) were
reacted with ethoxymethylenemalononitrile (0.01 mol)
and sodium acetate (0.02 mol) in ethanol (40 mL), under
reflux, during 40 min. Afterwards, the mixture was poured
in cold water and the precipitate formed was filtered out
and recrystallized from ethanol/water. The reactions were
accomplished by means of TLC using silica gel plate with
fluorescent indicator and hexane/ ethyl acetate (1:1) as
eluent. 19 (R=H): mp 135-136 oC, yield 90%; 20 (R=4-Br):
mp 168-169 oC, yield 86%; 21 (R=4-NO2): mp 220-221 oC,
Experimental
o
yield 68%; 22 (R=4-OCH3): mp 135-136 C, yield 75%;
Unless otherwise noted, all the reagents and solvents
were obtained from the market and used without further
purification. Melting points were obtained with a Fisher
23 (R=2,6-diCl): mp 190-191 oC, yield 78%.
General procedure for the preparation of 1-aryl-1H-
pyrazole-4-carbonitriles compounds 24-28
1
apparatus and were uncorrected. H NMR spectra were
recorded at room temperature on a Varian Unity plus
300 MHz employing tetramethylsilane as the internal
reference. The chemical shifts (d) are reported in ppm and
the coupling constants (J) in hertz. Infrared (IR) spectra
Method A
The reaction mixture of the aldehydes 10-13
(7.8 mmol L-1) with hidroxylamine and methanoic acid

356
Synthesis and Antileishmanial Activity
J. Braz. Chem. Soc.
(10 mL) was maintained under reflux form 6 h, until the
end of reaction was indicated by TLC. Then the reaction
mixture was poured in cold water and the precipitate formed
was filtered out washed with ethanol and recrystallized from
ethanol/water to afford crystals.The purity of the compounds
was checked by means of TLC using silica gel plate with
fluorescent indicator and hexane/ethyl acetate (1:1, v/v) as
eluent, melting point, IR spectra and 1H NMR. 24 (R = H):
mp 91-92 C, yield 85%; 25 (R = 4-Br): mp 198-199 C,
yield 80%; 26 (R = 4-NO2): mp 185-186 oC, yield 65%; 27
(R = 4-OCH3): mp 138-141 oC, yield 79%; 28 (R = 2,6-diCl):
not synthesized.
H, 5.28; N, 29.95. Calc. for C10H10N4: C, 64.50; H, 5.41;
N, 30.09%.
1-(4’-Bromophenyl)-1H-pyrazole-4-carboximidamide (2)
o
mp 270-271 C, yield 66%; FT IR (KBr) nmax/cm-1
3400 to 3096, 1660; 1H NMR (DMSO, d in ppm): H3 9.23
(s), H5 10.00 (s), H2’ and H6’ 7.93 (d; 8.7 Hz), H3’ and H5’
7.21 (d; 9.0 Hz), NHNH2 3.48 (br); 13C NMR (DMSO, d
in ppm): C3 143.1, C4 114.2, C5 132.5, C1’ 145.8, C2’ and
C6’ 124.1, C3’ and C5’ 125.0, C4’ 142.5, C(NH)NH2 152.0;
MS: m/z 265.1107 (M+, 100%). Found: C, 45.21; H, 3.29;
N, 21.06; Br, 29.98. Calc. for C10H9N4Br: C, 45.30; H, 3.42;
N, 21.14; Br, 30.14%.
o
o
Method B
The reaction mixture of t-butyl nitrite (4 mL) with dry
THF (10 mL) was stirred and refluxed under 20 min. Then,
0.005 mol of 5-amino-1-aryl-1H-pyrazole-4-carbonitriles
19-23 was added. The mixture was stirred and refluxed
about 2 h. Afterwards, the mixture THF and t-butyl nitrite
was evaporated. The precipitate was recrystallized with
the mixture of ethanol/water. The purity of the compounds
was checked by means of TLC using silica gel plate with
fluorescent indicator and hexane/ethyl acetate (1:1, v/v) as
eluent, melting point, IR spectra and 1H NMR. 24 (R = H):
mp 90-91 oC, yield 82%; 25 (R = 4-Br): mp 198-200 oC,
1-(4’-Nitrophenyl)-1H-pyrazole-4-carboximidamide (3)
mp 257-258 oC, yield 48%; FT IR (KBr) nmax/cm-1 3400
to 3100, 1658; 1H NMR (DMSO, d in ppm): H3 8.53 (s),
H5 9.26 (s), 7.20-7.40 (m), NHNH2 3.86 (br); 13C NMR
(DMSO, d in ppm): C3 145.1, C4 117.5, C5 133.2, C1’147.7,
C2’and C6’126.0, C3’and C5’127.1, C4’146.5, C(NH)NH2
156.0; MS: m/z 231.2101 (M+, 100%). Found: C, 51.81; H,
3.79; N, 30.06; O, 14.34. Calc. for C10H9N5O2: C, 51.95;
H, 3.92; N, 30.29; O, 13.84%.
1-(4’-Methoxyphenyl)-1H-pyrazole-4-carboximidamide (4)
mp 239-240 oC, yield 65%; FT IR (KBr) nmax/cm-1 3211
to 3100, 1634; 1H NMR (DMSO, d in ppm): H3 8.06 (s),
H5 8.77 (s), H2’ and H6’ 7.74 (d; 9.0 Hz), H3’ and H5’ 7.06
(d; 8.7 Hz), NHNH2 4.62 (br), OCH3 3.88 (s); 13C NMR
(DMSO, d in ppm): C3 143.5, C4 92.6, C5 120.4, C1’131.2,
C2’and C6’121.1, C3’and C5’114.9, C4’149.8, C(NH)NH2
158.8, OCH3 55.7; MS: m/z 216.2193 (M+, 100%). Found:
C, 60.96; H, 5.49; N, 25.75; O, 7.80. Calc. for C11H12N4O:
C, 61.10; H, 5.59; N, 25.91; O, 7.40%.
o
yield 86%; 26 (R = 4-NO2): mp 187-188 C, yield
o
62%; 27 (R = 4-OCH3): mp 138-141 C, yield 82%; 28
(R = 2,6-diCl): mp 141-142 oC, yield 81%.
General procedure for the preparation of 1-aryl-1H-
pyrazole-4-carboximidamides compounds 1-5
A mixture of derivatives 24-28 (0.01 mol) and 20 mL
of dry ethanol was cooled at 0-5 ºC and saturated with
chloridric acid gas. The mixture was sealed and stirred at
room temperature for 5 days.After this, bubble ammonium
gas was added at mixture reaction and stirred for 7 days.
The solvent was evaporated and the crystals was purify with
ethanol/water. The purity of the compounds was checked
by means of TLC using silica gel plate with fluorescent
indicator and hexane/ethyl acetate (1:3) as eluent, melting
point, IR Spectra and 1H NMR.
1-(2’,6’-Dichlorophenyl)-1H-pyrazole-4-carboximidamide
(5)
mp 222-223 oC, yield 60%; FT IR (KBr) nmax/cm-1 3400
to 3189, 1647; 1H NMR (DMSO, d in ppm): H3 8.28 (s), H5
9.05 (s), H3’ and H5’ 7.93 (d; 8.7 Hz), H4’ 7.55 (t; 8.7 Hz),
NHNH2 4.65 (br); 13C NMR (DMSO, d in ppm): C3 144.0,
C4 95.9, C5 132.1, C1’ 136.8, C2’ 112.4, C3’ and C5’ 118.3,
C4’ 128.3, C5’ 130.1, C6’ 112.4, C(NH)NH2 156.3; MS:
m/z 255.0925 (M+, 100%). Found: C, 46.94; H, 3.02; N,
21.88; Cl, 28.16. Calc. for C10H8N4Cl2: C, 47.09; H, 3.16;
N, 21.96; Cl, 27.79%.
1-Phenyl-1H-pyrazole-4-carboximidamide (1)
mp 220-221 oC, yield 67%; FT IR (KBr) nmax/cm-1 3303
to 3077, 1682; 1H NMR (DMSO, d in ppm): H3 8.63 (s), H5
9.64 (s), H2’ and H6’ 7.93 (d; 8.1 Hz), H3’ and H5’ 7.71 (t;
7.8 Hz), H4’ 7.56 (t; 7.8 Hz), NHNH2 4.08 (br); 13C NMR
(DMSO, d in ppm): C3 141.3, C4 113.3, C5 131.4, C1’138.7,
C2’and C6’119.3, C3’and C5’130.1, C4’128.0, C(NH)NH2
158.1; MS: m/z 186.2022 (M+, 100%). Found: C, 64.34;
Biological and cytotoxity assays
Leishmania amazonensis (MHOM/BR/LTB0016
o
strain) promastigotes were grown at 26 C in Schneider’

Vol. 22, No. 2, 2011
dos Santos et al.
357
Drosophila medium21 supplemented with 10% v/v heat-
inactivated foetal calf serum (FCS) at pH 7.2. Parasites were
harvested from the medium on day 4, when there was a high
percentage of infective forms (metacyclic promastigotes),
were counted in a Neubauer’ Chamber and adjusted to a
concentration of 4×06 parasites mL-1, for the drug assay.22,28
The assay was carried out in 96-well flat-bottom
microplate with a volume of 200 mL/well. The compounds
1-5 solubilized in dimethyl sulfoxide (DMSO) (the highest
concentration used was 1.6% v/v, not hazardous to the
parasite) were added to the culture, in a concentration
range from 320 from 80 mg mL-1.After 24 h incubation in a
temperature of 26 oC, the remaining parasites were counted
in a Neubauer’s chamber and compared with the controls
with DMSO, without the drugs and with the parasites
alone. All tests were done in triplicate and pentamidine
isethionate was used as reference drug. The IC50/24 h was
calculated by means of dose-response curves at a wider
range of concentrations, and the results were expressed
as the mean standard deviation determined from three
independent experiments.
The cytotoxicity effect of the derivatives 1-5 expressed
as cell viability was assayed on mice’s peritoneal
macrophages. The cells were isolated from peritoneal cavity
ofBalb/cmicewithcoldRPMI1640medium, supplemented
with 1 mmol L-1 L-glutamine, 1 mol L-1 HEPES, penicillin
G (105IUI-1), streptomycin sulfate(0.10 g L-1). The 2×105
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 then removed and the
adhered macrophages were washed twice with RPMI.
Compounds were added to the cell culture at the respective
EC50 /24 h for L. amazonensis and cells incubated for
24 h. Then, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide, MTT was added and after 2-4 h the
reaction was interrupted with DMSO. The results could be
read in spectrophotometer with wavelength of 570 nm.23,28,29
orbital coefficients distribution, molecular dipole
moment (µ), and molecular electrostatic potential (MEP)
were calculated. MEP isoenergy surface maps were
generated in the range from −25.0 (deepest red color) to
+30.0 (deepest blue color) kcal mol-1 and superimposed
onto a molecular surface of constant electron density of
0.002 e au-3. Each point of the three dimensional molecular
surface map expresses the electrostatic interaction energy
value evaluated with a probe atom of positive unitary charge
providing an indication of the overall molecular size and
location of attractive (negative) or repulsive (positive)
electrostatic potentials.
Sincethecompoundsareconsideredfororaldelivery, they
were also submitted to the analysis of Lipinski rule of five,
which evaluate some properties of a compound that would
make it a likely orally active drug in humans. These structural
parameters were performed using Molispiration program.31
Conclusions
In this work we described a new set of 1-aryl-1H-
pyrazole-4-carboximidamide compounds synthesized
in good yields that presented an antileishmanial activity
profile. This series can be scaled up and easily produce new
analogues. Compound 2 (Br-substituted) also presented a
low cytotoxicity profile that pointed it as a lead compound
for further substitutions to improve its biological profile.All
compounds showed a good theoretical biodisponibility and
the molecular modelling evaluation showed that HOMO
and LUMO energies of 2 and 3 led to a different reactivity
profile that seem to be related to their antileishmanial
profile. The hydrophobic substituents in phenyl-pyrazolic
groups may be useful to investigate the contribution of this
structural unit on its bioactivity profile. Further experiments
are being carried out in order to define better chemical
structure and biological activity relationships.
Acknowledgments
Molecular modeling
We thank the Fundação deAmparo à Pesquisa do Estado
do Rio de Janeiro (FAPERJ), Programa de Desenvolvimento
Tecnológico em Insumos para Saúde (PDTIS), Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), Coordenação de Aperfeiçoamento de Pessoal
Docente (CAPES) and Universidade Federal Fluminense
(PROPP-UFF) for fellowships and financial support.
The molecular modeling study was performed using
SPARTAN’06 software package (Wavefunction Inc.
Irvine, CA, 2000).30 The minimum energy conformation
of the derivatives was obtained by the AM1 semiempirical
Hamiltonian. In order to better evaluate the electronic
properties of the AM1 minimum energy conformations,
they were submitted to a single-point energy ab initio
calculation at the 6-311G* level.
References
In order to perform structure-activity relationship
(SAR) studies, some electronic properties, such as
HOMO and LUMO energy values, HOMO and LUMO
1. Robert, J. M. H.; Sabourin, C.; Alvarez, N.; Piessard, S. R.; Le
Baut, G.; Le Pape, P.; Eur. J. Med. Chem. 2003, 38, 711.

358
Synthesis and Antileishmanial Activity
J. Braz. Chem. Soc.
2. Santos, D. O.; Coutinho, C. E. R.; Madeira, M. F.; Bottino,
13. Huang, T. L.; Eynde, J. J. V.; Mayence, A.; Collins, M. S.;
Cushion, M. T.; Rattendi, D.; Londono, I.; Mazumder, L.;
Bacchi, C. J.; Yarlett, N.; Bioorg. Med. Chem. Lett. 2009, 19,
5884.
C. G.; Vieira, R. T.; Nascimento, S. B.; Bernardino, A. M. R.;
Bourguignon, S. C.; Corte-Real, S.; Pinho, R. T.; Castro, H. C.;
Rodrigues, C. R.; Parasitol. Res. 2008, 103, 1.
3. Desjeux, P.; Comp. Immun. Microbiol. Infect. Dis. 2004, 27, 305.
4. Lainson, R.; Shaw, J. J.; The Leishmaniasis in Biology and
Medicine, Academic Press: London, 1987.
14. Finar, I. L.; Lord, G. H.; J. Chem. Soc. 1957, 3314.
15. Bekhit,A.A.; Fahmy, H. T.Y.; Rostom, S.A. F.; Baraka,A. M.;
Eur. J. Med. Chem. 2003, 38, 27.
5. Mayence, A.; Eynde, J. J. V.; LeCour, L.; Walker, L. A.;
Tekwani, B. L.; Huang, T. L.; Eur. J. Med. Chem. 2004, 39, 547;
Graebin, C. S.; Uchoa, F. D.; Bernardes, L. S. C.; Campo,V. L.;
Carvalho, I.; Eifler-Lima,V. L.; Anti-Infect. Agents Med. Chem.
2009, 8, 345; Cavalli,A.; Bolognesi, M. L.; J. Med. Chem. 2009,
52, 7339; Chappuis, F.; Sundar, S.; Hailu,A.; Ghalib, H.; Rijal,
S.; Peeling, R. W.; Alvar, J.; Boelaert, M.; Nat. Rev. Microbiol.
2007, 5, 873; Coler, R. N.; Reed, S. G.; Trends Parasitol. 2005,
21, 244.
16. Sakya, S. M.; Rast, B.; Tetrahedron Lett. 2003, 44, 7629.
17. Cadogan, J. I. G.; Molina, G. A.; J. Chem. Soc. Perkin Trans. I
1973, 541.
18. Tidwell, R. R.; Jones, S. K.; Geratz, J. D.; Ohemeng, K. A.;
Cory, M.; Hall, J. E.; J. Med. Chem. 1990, 33, 1252.
19. Bray, P. G.; Barret, M. P.; Ward, S. A.; Koning, H. P.; Trends
Parasitol. 2003, 19, 232.
20. Canto-Cavalheiro, M. M.; Echevarria, A.; Araújo, C. A. C.;
Bravo, M. F.; Santos, L. H. S.; Jansen, A. M.; Leon, L. L.;
Microbios 1997, 90, 51.
6. Fancelli, D.; Moll, J.;Varasi, M.; Bravo, R.;Artico, R.; Berta, D.;
Bindi, S.; Cameron, A.; Candiani, I.; Cappella, P.; Carpinelli, P.;
Croci,W.; Forte, B.; Giorgini, M. L.; Klapwijk, J.; Marsiglio,A.;
Pesenti, E.; Rocchetti, M.; Roletto, F.; Severino, D.; Soncini, C.;
Storici, P.; Tonani, R.; Zugnoni, P.; Vianello, P.; J. Med. Chem.
2006, 49, 7247; Bekhit,A.A.;Ashour, H. M.A.; Ghany,Y. S.A.;
Bekhit, A. E. A.; Baraka, A.; Eur. J. Med. Chem. 2008, 43, 456.
7. 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; Bonacorso, H. G.;
Oliveira, M. R.; Costa, M. P.; Silva, L. B.; Zanatta, N.; Martins,
M. A. B.; Flores, A. F. C.; J. Braz. Chem. Soc. 2005, 16, 868.
8. Makino, K.; Kim, H. S.; Kurasawa, Y.; J. Heterocycl. Chem.
1998, 35, 489.
21. Genestra, M. J.; Echevarria, A.; Cysne-Finkelstein, L.; Silva-
Gonçalves,A. J.; Leon, L. L.; Arzneimittel-Forshung Drug Res.
2001, 51, 920.
22. Temporal, R. M.; Cysne-Finkelstein, L.; Echevarria, A.; Silva-
Gonçalves, A. J.; Leon, L. L.; Genestra M. J.; J. Enzyme Inhib.
Med. Chem. 2005, 20, 13.
23. Bhat, B. A.; Dhar, K. L.; Puri, S. C.; Saxena, A. K.;
Shanmugavel, L. M.; Qazi, G. N.; Bioorg. Med. Chem. Lett.
2005, 15, 3177.
24. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P.;
J. Am. Chem. Soc. 1985, 107, 3902.
25. Leach,A. R.; Molecular Modeling: Principles and applications,
2nd ed., Pearson Education: London, 1996.
9. Bernardino, A. M.; Gomes, A. O.; Charret, K. S.; Freitas, A.
C.; Machado, G. M.; Canto-Cavalheiro, M. M.; Leon, L. L.;
Amaral, V. F.; Eur. J. Med. Chem. 2006, 41, 80.
26. Kubinyi, H.; Pharm. Acta Helv. 1995, 69, 259.
27. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.;
Adv. Drug Deliv. Rev. 2001, 46, 3.
10. Charret, K. S.; Rodrigues, R. F.; Bernardino, A. M. R.; Gomes,
A. O.; Carvalho, A. V.; Canto-Cavalheiro, M. M.; Leon, L. L.;
Amaral, V. F.; Am. J. Trop. Med. Hyg. 2009, 80, 568.
11. Croft, S. L.; Coombs, G. H.; Trends Parasitol. 2003, 19, 502;
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; Bell, C. A.;
Hall, J. E.; Kyle, D. E.; Antimicrob. Agents Chemother. 1990, 34,
1381; Dardonville, C.; Brun, R.; J. Med. Chem. 2004, 47, 2296;
Kramp, K. L.; DeWitt, K.; Flora, J. W.; Muddiman, D. C.; Slunt,
K. M.; Houston, T. A.; Tetrahedron Lett. 2005, 46, 695.
12. Santos, M. S.; Bernardino,A. M. R.; Souza, M. C.; Quim. Nova
2006, 29, 1301.
28. Cysne-Finkelstein, L.;Aguiar-Alves, F.; Temporal, R. M.; Leon,
L. L.; Exp. Parasitol. 1998, 89, 58.
29. Hendricks, L. D.; Wood, D. E.; Hajduk, M. E.; Parasitology
1978, 76, 309.
30. SPARTAN’06 software package. Wavefunction Inc. Irvine, CA,
2000.
31. Molinspiration Property Calculation Service, www.
molinspiration.com.
Submitted: April 28, 2010
Published online: September 21, 2010