Effectiveness of novel 5-(5-amino-1-aryl-1H-pyrazol-4-yl)-1H-tetrazole derivatives against promastigotes and amastigotes of leishmania amazonensis

Article abstract of DOI:10.1111/cbdd.12235

In this research, a series of substituted 5-(5-amino-1-aryl-1H-pyrazol-4- yl)-1H-tetrazoles were synthesized and evaluated for in vitro antileishmanial activity. Among the derivatives, examined compounds 3b and 3l exhibited promising activity against promastigotes and amastigotes forms of Leishmania amazonensis. The cytotoxicity of these compounds was evaluated on murine cells, giving access to the corresponding selectivity index (SI).

Full text of DOI:10.1111/cbdd.12235

Chem Biol Drug Des 2014; 83: 272–277
Research Article
Effectiveness of Novel 5-(5-amino-1-aryl-1H-pyrazol-
4-yl)-1H-tetrazole Derivatives Against Promastigotes
and Amastigotes of Leishmania amazonensis
1
1
~
insect vector, the female sandfly of the genus Lutzomyia in
the New World and Phlebotomus in the Old World, they
exist as extracellular motile promastigotes. In Brazil, Leish-
mania amazonensis is responsible for several clinical mani-
festations reported, which include cutaneous, mucosal,
diffuse cutaneous, and visceral leishmaniasis (4,5) and is
considered a species of epidemiologic importance. How-
ever, the chemotherapy for leishmaniasis is generally inef-
fective mainly due to the emergence of drug-resistant
strains and toxicity of the therapeutic agents. The pentava-
lent antimonials compounds are widely used as primary
therapy whereas other drugs such as amphotericin B,
pentamidine, paromomycin, and azoles derivatives have
been also employed (6). The azole antifungals ketoconaz-
ole, miconazole, and itraconazole have been used to treat
cutaneous leishmaniasis with variable success rates (7).
Allopurinol, a pyrazolopyrimidine, was described as a leish-
manicidal drug in the late 1970s (8) and is considered an
alternative to antimonials in some cases. In this context,
our research group has been devoted to the synthesis and
evaluation of antileishmanial activity of cyclic systems
containing nitrogen (9–11), including the compound
5-amino-1-(3,5-dichlorophenyl)-4-(4,5-dihydro-1H-imidazol-
2-yl)-1H-pyrazole, which was tested in vivo and exhibited
significant inhibition relative to an untreated control (12). In
addition, it has been reported that anti-inflammatory and
related biological activities have been improved by the
substitution of a 5-tetrazole group in place of carboxyl
function (13–15). Recently, 5-substituted tetrazoles have
been mentioned more and more frequently as non-classi-
cal isosteres of the carboxyl group. It has been also shown
that ionized tetrazoles are ten times more lipophilic than
the corresponding carboxylic acids, which in some cases
enables these compounds to penetrate the cell membrane
with greater ease (15). In this way, we have designed pyr-
azole–tetrazole hybrids by applying rational drug design
approach. Twelve 5-(5-amino-1-aryl-1H-pyrazol-4-yl)-1H-
tetrazoles (3a–l) were synthesized and evaluated against
Leishmania amazonensis.
Viviane dos Santos Faioes , Leonor L. Leon ,
Marilene M. Canto-Cavalheiro1, Eduardo
C. Torres-Santos1, Alice M. R. Bernardino2,
2
3,
ꢀ
Percilene F. Vegi and Maurıcio S. dos Santos *
1
ꢀ
ꢀ
ꢀ
Laboratorio de Bioquımica de Tripanosomatıdeos,
~
Janeiro, RJ 21040-900, Brazil
Instituto Oswaldo Cruz, Fundacßao Oswaldo Cruz, Rio de
2
^
ꢀ
Departamento de Quımica Organica, Programa de Pos-
ꢀ
~
ꢀ
ꢀ
Graduacßao em Quımica, Instituto de Quımica,
ꢀ
Universidade Federal Fluminense, Niteroi, RJ 24020-150,
Brazil
Instituto de Fısica e Quımica, Centro de Estudos e
3
ꢀ
ꢀ
~
Inovacßao em Materiais Biofuncionais Avancßados,
ꢀ
Universidade Federal de Itajuba, Itajuba, MG 37500-903,
ꢀ
Brazil
*Corresponding author: Maurıcio S. dos Santos,
mauriciounifei@yahoo.com.br
ꢀ
In this research, a series of substituted 5-(5-amino-1-
aryl-1H-pyrazol-4-yl)-1H-tetrazoles were synthesized
and evaluated for in vitro antileishmanial activity.
Among the derivatives, examined compounds 3b and
3l exhibited promising activity against promastigotes
and amastigotes forms of Leishmania amazonensis.
The cytotoxicity of these compounds was evaluated
on murine cells, giving access to the corresponding
selectivity index (SI).
Key words: antileishmanial activity, Leishmania amazonensis,
tetrazole derivatives
Received 28 May 2013, revised 23 August 2013 and
accepted for publication 5 September 2013
Leishmaniasis, caused by protozoa of the genus Leish-
mania, remains as a significant health issue in large part
due to the lack of effective and affordable drugs and to
the increasing resistance against existing drugs. More than
2 million new cases of leishmaniasis occur each year, with
approximately 350 million persons at risk of infection (1,2).
All parasites of the genus Leishmania are obligate intracel-
lular parasites that infect cells of the mononuclear phago-
cyte lineage of their vertebrate hosts, in which they exist
as non-motile intracellular amastigotes (3). Within the
Methods and Materials
Chemistry
Unless otherwise noted, all the reagents and solvents
were obtained from the market and used without further
272
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12235

Tetrazoles Derivatives Evaluated Against Leishmania amazonensis
purification. Thin-layer chromatography (TLC) was per-
Measurement of antileishmanial activity in
formed on silica gel plates (60 F254). Melting points were
determined with a Fisatom 430 apparatus and were not
corrected. 1H NMR spectra were recorded at room tem-
perature on a Varian Unity plus 300 MHz employing tetra-
methylsilane as the internal reference. The chemical shifts
(d) are reported in ppm and the coupling constants (J) in
hertz. Infrared (IR) spectra were recorded in a Perkin–
Elmer Model 100 FT-IR spectrophotometer using attenu-
ated total reflection (ATR) accessory. Mass spectral data
were obtained using a Finingan MAT 711A.
intracellular amastigote
Murine macrophages were seeded in Lab-Tek chamber
slide (2.0 9 106 cells/mL) and allowed to adhere (about
1 h) at 37 °C, 5% CO2. After removal of non-adherent
macrophages with RPMI medium, cells were infected by
4 hours in the same environment with stationary phase
promastigotes at a parasite: macrophage ratio of 3:1. Sub-
sequently, non-phagocytized parasites were removed by
gentle washing, and infected macrophages were incu-
bated with tetrazole derivatives for an additional 72 h. The
slides were stained using an Instant Prov hematological
dye system (Newprov, Curitiba, Brazil) for microscopic
evaluation of amastigote burden. At least 100 macrophag-
es/well were analyzed, and IC50 was established, consid-
ering equal proportion of DMSO treated cells as control.
General procedure for the preparation of 5-(5-
amino-1-aryl-1H-pyrazol-4-yl)-1H-tetrazoles (3a–l)
The key intermediates 5-amino-1-aryl-1H-pyrazole-4-car-
bonitriles (2a–l) were obtained from the reaction between
arylhydrazine hydrochlorides (1a–l) and ethoxymethylene-
malononitrile (0.01 mol), using sodium acetate (0.02 mol)
in ethanol (40 mL), under reflux, during 1 h. After, the mix-
ture 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 sil-
ica gel plate with fluorescent indicator and hexane/ethyl
acetate (1:1) as eluent (16).The 5-amino-1-aryl-1H-
pyrazole-4-carbonitriles (2a–l) (0.002 mol) were mixed
with sodium azide (0.004 mol), ammonium chloride
(0.004 mol), and DMF (15 mL). The reaction mixture was
heated at 130–135 °C, during 14 h. After, the mixture
was poured in cold water, and the precipitate formed was
filtered out and recrystallized from ethanol/water. The tar-
gets (3a–l) were characterized by Fourier transform infra-
red (FT-IR), Nuclear magnetic resonance (NMR), and
mass spectrometry (MS).
Cytotoxic assays on murine peritoneal
macrophages
Cytotoxicity was assessed on murine peritoneal macro-
phages by a colorimetric assay based on the mitochon-
drial reduction in MTT as described previously (17). The
cells were isolated from peritoneal cavity of BALB/c mice
with cold RPMI 1640 medium, supplemented with 10%
FCS. The 2 9 106 cells/well were cultivated on microplate
and incubated at 37 °C in a humidified 5% CO2 atmo-
sphere. After 2 h of incubation, no adherent cells were
then removed and the adhered macrophages were
washed with RPMI. Compounds were solubilized in DMSO
at concentrations ranging from 500 to 12.5 lM and added
to the cell culture for 72 h incubation at 37 °C and 5%
CO2. After that, culture supernatant was removed. The
macrophage viability was measured by adding MTT
(0.5 mg/mL in PBS, 200 lL/well), incubating plates for 2 h
at 37 °C, and the colored product formazan was solubi-
lized with DMSO. The results were read in spectrophotom-
eter with wavelength of 570 nm (17).
Antileishmanial activity assay
Promastigotes in late log phase in Schneider’s medium
supplemented with 10% fetal calf serum (FCS), 2 mM
L-glutamine, and antibiotics (100 U/mL penicillin and
100 lg/mL streptomycin) were incubated at an average
density of 107 parasites/mL in sterile 96-well plates with
various concentrations of compounds dissolved in DMSO
(final concentration <0.5% v/v). The assay was carried out
in triplicate. Appropriate controls containing DMSO, pent-
amidine (reference drug) were added to each set of experi-
ments. Parasite viability was assessed by a dye-reduction
assay employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) (17), and the absorbance mea-
sured in a spectrophotometer at 570 nm. Inhibitory con-
centration 50% (IC50) was defined as the concentration of
drug required to inhibit by 50% the metabolic activity of
Leishmania promastigotes compared with the control. IC50
/24 h was calculated by nonlinear regression analysis pro-
cessed on dose–response curves, using GRAPHPAD PRISM
5.0 (GraphPad Software Inc., La Jolla, CA, USA). IC50 val-
ues represent the mean value calculated from three inde-
pendent experiments.
Results and Discussion
Chemistry
The synthesis of the compounds 5-amino-1-aryl-1H-pyraz-
ole-4-carbonitriles (2a–l) and 5-(5-amino-1-aryl-1H-pyra-
zol-4-yl)-1H-tetrazole (3a–l) are summarized in Scheme 1.
The key intermediates 5-amino-1-aryl-1H-pyrazole-4-car-
bonitriles (2a–l) were obtained from the reaction between
arylhydrazine hydrochlorides (1a–l) and ethoxymethylene-
malononitrile, in ethanol. Previously, sodium acetate was
added to provide an acid–basic reaction with hydrochlo-
ride. The reaction occurs via a similar Michael addition
mechanism followed by cycloaddition. In the second step,
the compounds 5-(5-amino-1-aryl-1H-pyrazol-4-yl)-1H-
tetrazole (3a–l) were obtained via [3 + 2] cycloaddition by
the reaction of the compounds 2a–l with sodium azide
and ammonium chloride, in N, N-dimethylformamide
(DMF), according to similar methodology published by our
Chem Biol Drug Des 2014; 83: 272–277
273

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dos Santos Faioes et al.
N
N
N
N
CN
H
NHNH2.HCl
N
N
NH2
NH2
N
N
i, ii
iii
Scheme 1: Reagents and
conditions: (i) sodium acetate,
ethanol, 0.5 h, reflux; (ii)
ethoxymethylenemalononitrile,
ethanol, 1 h, reflux; (iii) NaN3,
NH4Cl, DMF, 14–18 h, 120–
130 °C.
R
R
R
1a-l
2a-l
3a-l
R= H, 2-Cl, 3-Cl, 4-Cl, 2,4-diCl, 2,6-diCl, 3,4-diCl, 3,5-diCl, 2-F, 3-F, 4-F, 3-Br
research group recently (15). All these final compounds
3a–l are new, except 5-(5-amino-1-phenyl-1H-pyrazol-4-
yl)-1H-tetrazole (3a) which was obtained by Walter and
Becker (18).
(300 MHz, DMSO)
d
3.45 (br, NH), 6.57 (br, NH),
7.61–7.64 (1H, m), 7.67–7.72 (2H, m), 7.81 (1H, t,
J = 1.8 Hz), 8.05 (1H, s); 13C-NMR (75 MHz, DMSO) d
87.8, 122.3, 126.0, 129.2, 131.6, 135.6, 138.3, 139.5,
146.5, 149.6; TOF-MS (ES+): (C10H8ClN7) calcd [M + H]+
262.0608; found 262.0629.
The structure of compounds 5-(5-amino-1-aryl-1H-pyrazol-
1
4-yl)-1H-tetrazoles (3a–l) were confirmed by their FT-IR, H
NMR, 13C NMR, and mass spectrometry analysis. The FT-
IR spectrum showed the absence of the cyano group
(2209–2234 cmÀ1), absorption characteristic of the key
intermediates 5-amino-1-aryl-1H-pyrazole-4-carbonitriles
(2a–l), and the presence of a strong band at 1638–
1613 cmÀ1 characteristic of the C=N bond of the tetrazole
ring. In the 1H NMR spectrum of each of these tetrazoles
(3a–l), the NH/NH2 appeared at 3.40-4.95 and 6.34–
6.68 ppm. The hydrogen atom at C-3 position in the pyraz-
ole ring gave characteristic singlets at 8.00–8.06 ppm. The
hydrogens present in benzene ring were assigned accord-
ing to ortho and meta couplings. The 13C NMR spectrum
showed the C = N bond of the tetrazole ring at 149.5–
149.7 ppm, the C-3 at 138.1–138.4 ppm, and the C-5 at
146.3–147.9 ppm. 2D NMR techniques COSY and HSQC
were used to assign hydrogen and carbon atoms.
5-[5-amino-1-(4′-chlorophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3d)
yield 72%; mp 139–140 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3417, 3361, 3250, 3189, 1634, 1602; 1H-NMR
(300 MHz, DMSO) d 3.47 (br, NH), 6.53 (br, NH), 7.72
(2H, d, J = 9.0 Hz), 7.79 (2H, d, J = 9.0 Hz), 8.06 (1H, s);
13C-NMR (75 MHz, DMSO) d 88.2, 120.3, 126.3, 133.4,
136.0, 138.4, 146.3, 149.7. TOF-MS (ES+): (C10H8ClN7)
calcd [M + H]+ 262.0608; found 262.0625.
5-[5-amino-1-(2′,4′-dichlorophenyl)-1H-pyrazole-
4-yl]-1H-tetrazole (3e)
yield 64%; mp 155–157 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3447, 3359, 3245, 3108, 1630, 1610; 1H-NMR
(300 MHz, DMSO) d 3.45 (br, NH), 6.56 (br, NH), 7.67
(1H, dd, J = 9.0;2.4 Hz), 7.72 (1H, d, J = 9.0 Hz), 7.85
(1H, d, J = 2.4 Hz), 8.03 (1H, s); 13C-NMR (75 MHz,
DMSO) d 87.6, 128.2, 130.4, 131.5, 131.9, 132.4, 135.0,
138.1, 147.4, 149.6; TOF-MS (ES+): (C10H7Cl2N7) calcd
[M + H]+ 296.0218; found 296.0254.
5-[5-amino-1-(2′-chlorophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3b)
yield 57%; mp 134–135 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1
: 3437, 3331, 3230, 3164, 3112, 1633, 1610;
1H-NMR (300 MHz, DMSO) d 3.46 (br NH), 6.34 (br, NH),
7.65–7.73 (3H, m), 7.83 (1H, d, J = 8.7 Hz), 8.00 (1H, s);
13C-NMR (75 MHz, DMSO) d 86.7, 128.3, 130.3, 130.4,
131.7, 131.9, 135.0, 138.1, 147.8, 149.6; TOF-MS (ES+):
(C10H8ClN7) calcd [M + H]+ 262.0608; found 262.0628.
5-[5-amino-1-(2′,6′-dichlorophenyl)-1H-pyrazole-
4-yl]-1H-tetrazole (3f)
yield 62%; mp 233–234 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3410, 3283, 3154, 3113, 1628, 1609; 1H-NMR
(300 MHz, DMSO)
d 3.48 (br, NH), 6.45 (br, NH),
5-[5-amino-1-(3′-chlorophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3c)
yield 74%; mp 164–165 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3473, 3425, 3296, 3111, 1635, 1595; 1H-NMR
7.62–7.67 (3H, m); 8.04 (1H, s); 13C-NMR (75 MHz,
DMSO) d 87.6, 127.8, 128.8, 133.5, 136.8, 138.2, 147.9,
149.5; TOF-MS (ES+): (C10H7Cl2N7) calcd [M + H]+
296.0218; found 296.0252.
274
Chem Biol Drug Des 2014; 83: 272–277

Tetrazoles Derivatives Evaluated Against Leishmania amazonensis
5-[5-amino-1-(3′,4′-dichlorophenyl)-1H-pyrazole-
5-[5-amino-1-(3′-fluorophenyl)-1H-pyrazole-4-yl]-
4-yl]-1H-tetrazole (3g)
1H-tetrazole (3j)
yield 67%; mp 160–162 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3415, 3277, 3128, 3110, 1629, 1605; 1H-NMR
(300 MHz, DMSO) d 3.46 (br, NH), 6.53 (br, NH), 7.53
(1H, dd, J = 8.7;2.4 Hz), 7.68 (1H, d, J = 8.7 Hz), 7.77
(1H, d, J = 2.4 Hz), 8.06 (1H, s); 13C-NMR (75 MHz,
DMSO) d 87.9, 128.5, 130.4, 131.2, 131.9, 132.8, 135.3,
138.2, 146.3, 149.5; TOF-MS (ES+): (C10H7Cl2N7) calcd
[M + H]+ 296.0218; found 296.0258.
yield 74%; mp 179–180 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3361, 3260, 3174, 3085, 1629, 1599; 1H-NMR
(300 MHz, DMSO)
d 4.40 (br, NH), 6.56 (br, NH),
7.35–7.41 (1H, m), 7.58-7.63 (2H, m), 7.67–7.75 (1H, m),
8.05 (1H, s); 13C-NMR (75 MHz, DMSO) d 88.2, 110.5,
114.3, 119.6, 131.6, 138.3, 139.8, 146.8, 149.7, 160.1;
TOF-MS (ES+): (C10H8FN7) calcd [M + H]+ 246.0903;
found 246.0948.
5-[5-amino-1-(3′,5′-dichlorophenyl)-1H-pyrazole-
4-yl]-1H-tetrazole (3h)
5-[5-amino-1-(4′-fluorophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3k)
yield 66%; mp 210–211 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3466, 3427, 3336, 3297, 3113, 1634, 1608;
1H-NMR (300 MHz, DMSO) d 3.46 (br, NH), 6.68 (br,
NH), 7.79 (1H, t, J = 1.8 Hz), 7.82 (2H, d, J = 1.8 Hz),
8.06 (1H, s); 13C-NMR (75 MHz, DMSO) d 87.9, 128.0,
130.5, 133.7, 135.7, 138.2, 146.3, 149.5; TOF-MS
(ES+): (C10H7Cl2N7) calcd [M + H]+ 296.0218; found
296.0254.
yield 62%; mp 172–173 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3468, 3320, 3215, 3106, 1613, 1603; 1H-NMR
(300 MHz, DMSO) d 3.40 (br, NH), 6.43 (br, NH), 7.51
(2H, t, J = 9.0 Hz), 7.75 (2H, dd, J = 9.0;4.8 Hz), 8.01
(1H, s); 13C-NMR (75 MHz, DMSO)
d 88,0, 116.3,
126.3, 134.2, 138.1, 146.8, 149.6, 159.8; TOF-MS
(ES+): (C10H8FN7) calcd [M + H]+ 246.0903; found
246.0944.
5-[5-amino-1-(2′-fluorophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3i)
5-[5-amino-1-(3′-bromophenyl)-1H-pyrazole-4-yl]-
1H-tetrazole (3l)
yield 47%; mp 120–121 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1: 3436, 3321, 3164, 3117, 1638, 1614; 1H-NMR
yield 68%; mp 175–176 °C; FT-IR (ATR-diamond/ZnSe)
cmÀ1
: 3468, 3422, 3301, 3200, 3108, 1631, 1610;
(300 MHz, DMSO)
7.47–7.62 (2H, m), 7.66-7.73 (2H, m), 8.04 (1H, s);
13C-NMR (75 MHz, DMSO)
87.0, 117.0, 125.5,
d
4.95 (br, NH), 6.43 (br, NH),
1H-NMR (300 MHz, DMSO) d 3.45 (br, NH), 6.58 (br, NH),
7.63 (1H, t, J = 8.1 Hz), 7.76 (2H, t, J = 8.4 Hz), 7.93
(1H, t, J = 1.8 Hz), 8.05 (1H, s); 13C-NMR (75 MHz,
DMSO) d 88.2, 122.0, 122.2, 126.0, 130.2, 133.7, 138.4,
139.6, 146.3, 149.5; TOF-MS (ES+): (C10H8BrN7) calcd
[M + H]+ 306.0103; found 306.0135.
d
125.7, 129.6, 131.7, 138.5, 147.9, 149.7, 155.8; TOF-
MS (ES+): (C10H8FN7) calcd [M + H]+ 246.0903; found
246.0942.
a
Table 1: IC50 (lM) values of 5-(5-amino-1-aryl-1H-pyrazol-4-yl)-1H-tetrazoles (3a–l) on promastigotes and amastigotes of Leishmania
spp. and the cytoxicity in murine macrophages
Promastigote (IC50/lM)
Leishmania amazonensis
Amastigote intracellular (IC50/lM)
Leishmania amazonensis
Mø peritoneal murine
(LD50/lM)
Selectivity index (SI) (LD50/
IC50 in amastigote intracellular)
Compound
3a (H)
3b (2-Cl)
3c (3-Cl)
3d (4-Cl)
3e (2,4-diCl)
3f (2,6-diCl)
3g (3,4-diCl)
3h (3,5-diCl)
3i (2-F)
3j (3-F)
3k (4-F)
3l (3-Br)
Pentamidine
388 Æ 6.0
75.8 Æ 7.2
379 Æ 3.1
>800
>800
>800
130.5 Æ 0.0
46.5 Æ 4.8
ND
ND
ND
ND
ND
ND
>100
>500
ND
3.98
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.41
4.46
185.2 Æ 5.7
>500
ND
ND
ND
ND
>800
195.8 Æ 18.8
91.4 Æ 0.1
144.8 Æ 0.0
102.6 Æ 13.1
78.5 Æ 3.4
13.0 Æ 0.1
192.2 Æ 13.0
93.9 Æ 4.7
>500
ND
97.0 Æ 2.8
106.6 Æ 7.45
1.9 Æ 0.121
>250
500 Æ 0.0
8.49 Æ 1.25
aIC50 values were obtained from the drug concentration-response curve, and the results were expressed as the mean Æ standard devia-
tion determined from three independent experiments.
Chem Biol Drug Des 2014; 83: 272–277
275

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dos Santos Faioes et al.
~
Biological activity
Coordenacßao de Aperfeicßoamento de Pessoal Docente
In this work, we evaluated the synthesis, cytotoxicity and
in vitro leishmanicidal activity of twelve 5-(5-amino-1-aryl-
1H-pyrazol-4-yl)-1H-tetrazole (3a–l) derivatives. Table 1
displays IC50 values of these derivatives against L. ama-
zonensis promastigotes and intracellular amastigotes
forms. Cytotoxic activity (LC50) was related to anti-amasti-
(CAPES), Universidade Federal Fluminense (PROPP-UFF),
~
Centro de Estudos e Inovacßao em Materiais Biofuncionais
ꢀ
Avancßados/UNIFEI, and Rede Mineira de Quımica (RMQ)
for fellowships and financial support.
gote activity(IC50
)
for determining their corresponding
References
selectivity index (SI = LC50/IC50). Among the compounds
tested 3a–l, four compounds 3b (R = 2-Cl), 3i (R = 2-F),
3k (R = 4-F), and 3l (R = 3-Br) presented interesting anti-
promastigote activity (IC50 75.83, 91.41, 102.58, and
78.53 lM, respectively), with greater selectivity index for
bromine derivative 3l (SI = 6.41). We carried out amasti-
gote assays with these four active compounds to evaluate
its ability for reducing the parasite load in infected host
cells. The results showed that derivative 3b is able to
reduce significantly the number of intracellular parasites
with IC50 = 46 lM (Table 1), while compounds 3a, 3k, and
3l displayed an average activity (IC50 of 130.5, 97.0,
and 106.6 lM, respectively). Concerning compound 3i
exhibited significant cytotoxicity on macrophages cells
(LC50 = 93.92 lM), the presence of the 2-fluoro in the aryl
group seems to play a more important role for the cytotox-
icity profile. In a previous report (9), we synthesized and
evaluated the in vitro leishmanicidal activity of 5-amino-1-
aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles. Thus,
considering the similarity of imidazole and tetrazole rings,
this study could compare the leishmanicidal activity of new
tetrazole derivatives. However, a close examination of the
similar compounds imidazole and tetrazole groups
revealed that their leishmanicidal activity is strongly bound
to the nature and position of the substituent on the
phenyl group (Scheme 1). Compound with no struc-
tural modification (R = H) compared with tetrazole (3a),
IC50 = 388 Æ 6.0 lM (Table 1), or imidazole rings,
IC50 = 365Æ3.5 lM showed similar activity (11).
ꢀ
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This study achieved the synthesis of a novel series of tetraz-
ole ring bearing arylpyrazol derivatives. This study con-
firmed this new class of compounds as innovative
antileishmanial agents. However, through a preliminary
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~
ꢁ
We thank the Fundacßao de Amparo a Pesquisa do Estado
do Rio de Janeiro (FAPERJ), Conselho Nacional de
ꢀ
ꢀ
Desenvolvimento Cientıfico e Tecnologico (CNPq), Progra-
ꢀ
ꢁ
ꢀ
ma Estrategico de Apoio a Pesquisa em Saude (PAPES),
276
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Tetrazoles Derivatives Evaluated Against Leishmania amazonensis
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