Synthesis and evaluation of the anti parasitic activity of aromatic nitro compounds

Article abstract of DOI:10.1016/j.ejmech.2011.09.002

A series of nitroaromatic compounds was synthesized and evaluated as potential antileishmanial and trypanocidal agents. Five compounds exerted significant anti-leishmanial activity in vitro against promastigotes forms of Leishmania (L.) amazonensis, with

Full text of DOI:10.1016/j.ejmech.2011.09.002

European Journal of Medicinal Chemistry 46 (2011) 5443e5447
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of the anti parasitic activity of aromatic nitro
compounds
Marcela S. Lopesa, Renata C.C. de Souza Pietrab, Tatiane F. Borgatia, Carla F.D. Romeiroc,
Policarpo A.S. Júniord, Alvaro J. Romanhad, Ricardo J. Alvesa, Elaine M. Souza-Fagundesc,
,
Ana Paula S.M. Fernandesb, Renata B. de Oliveiraa
*
a Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais (UFMG), Avenida Antônio Carlos 6627, Belo Horizonte,
MG 31.270-901, Brazil
b Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas, Gerais (UFMG), Brazil
c Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas, Gerais (UFMG), Brazil
d Centro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, MG, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nitroaromatic compounds was synthesized and evaluated as potential antileishmanial and
Received 6 May 2011
Received in revised form
27 August 2011
Accepted 1 September 2011
Available online 8 September 2011
trypanocidal agents. Five compounds exerted significant anti-leishmanial activity in vitro against
promastigotes forms of Leishmania (L.) amazonensis, with IC50 in the range of 23e59 m
mol Lꢀ1, but none
were active against amastigotes intracellular forms of Trypanosoma cruzi. In vitro cytotoxicity on the
proliferation of human peripheral blood mononuclear cells (PBMC) stimulated with phytohemaglutinin
(PHA) was also evaluated. Two compounds, 6 and 7, were found to present a promising anti-leishmanial
activity with IC50 values of 59.5 and 50.6 mM, respectively, without affecting the lymphocyte proliferation
in PBMCs (selectivity index of 16.1 and 21.7, respectively), indicating low toxicity to human cells.
Keywords:
Nitro compounds
Antileishmanial activity
L. amazonensis
Trypanocidal
Ó 2011 Elsevier Masson SAS. All rights reserved.
T. cruzi
1. Introduction
causative Leishmania species of cutaneous leishmaniasis are Leish-
mania (Leishmania) amazonensis, L. (L.) major, L. (L.) tropica, L. (L.)
Parasitic trypanosomatids cause
diseases, including Chagas disease and the leishmaniasis.
a
number of important
ethiopica, L. (Viannia) braziliensis, and L. (L.) mexicana, while L. (L.)
donovani and L. (L.) infantum are the main etiologic agents of
visceral leishmaniasis [3]. Administration of pentavalent antimony
organic compounds remains as the first choice therapy for leish-
maniasis forms, followed by amphotericin B, pentamidine and
miltefosine. However, all currently available chemotherapeutic
agents have serious drawbacks, like severe side effects and paren-
teral administration for long time [3,4]. In this unfavorable scenario
the development of new drugs for the treatment of Chagas disease
and leishmaniasis is mandatory.
It’s well known that free-radical are generated in a futile reac-
tion cycle (or redox cycling) during the metabolism of nitro-
compounds and these reactive species can be correlated with the
pharmacological effects of these compounds [5,6]. Based on the
trypanocidal activity of the nitrocompounds [5] and on their
potential pharmacophoric effect, we decided to investigate the
efficacy of new nitro compounds against L. (L.) amazonensis pro-
mastigote forms and amastigote forms of T. cruzi. Furthermore, we
tested the effect of removing the nitro group of the aromatic system
Chagas disease is a potentially life-threatening illness caused by
the flagellate protozoan Trypanosoma cruzi. An estimated 10 million
people are infected worldwide, mostly in Latin America where
Chagas disease is endemic. More than 25 million people are at risk
of the disease. It is estimated that in 2008 Chagas disease has killed
more than 10,000 people [1]. Unfortunately, there is no ideal
treatment for the infection, especially for the chronic phase. In
Brazil, only benznidazole is available for the treatment of the
disease, although this drug presents toxic side effects and is active
only in the acute phase of illness [2].
Leishmaniasis is a neglected tropical disease caused by parasitic
protozoa of the genus Leishmania and threatens 350 million people
in 88 countries around the world. There are several different forms
of leishmaniasis. The most common are cutaneous and visceral. The
* Corresponding author. Tel.: þ55 31 34096395; fax: þ55 31 34096935.
E-mail address: renatabo@farmacia.ufmg.br (R.B. de Oliveira).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.002

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M.S. Lopes et al. / European Journal of Medicinal Chemistry 46 (2011) 5443e5447
to demonstrate the importance of this group to activity. Cytotox-
icity tests were performed on human peripheral blood mono-
nuclear cells (PBMC).
between 23 and 59
inactive in concentrations below 100
m
M. The other compounds were considered
m
M. Compounds 6 and 7
displayed low cytotoxicity to mammalian cells. In contrast,
compounds 3 and 5 exhibited toxicity to both cells (parasite and
mammalian cells).
All compounds tested showed low activity against amastigote
forms of T. cruzi, which indicates a specificity of these compounds
against L. (L.) amazonensis.
2. Results and discussion
The compounds 1e8 were synthesized from 4-(bromomethyl)
benzoic acid 1 [7], as shown in Scheme 1. The methyl ester 3 and
alcohol 4 were obtained by esterification and hydrolysis reaction,
respectively, of carboxylic acid 2, according to literature procedures
[8,9]. Synthesis of the amides 5, 6 and 7 was carried out by reaction
of the carboxylic acid 2 with ethanolamine and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDAC). The
displacement of the bromomethyl group in 1 and 2 by chloride ion
present in the reaction medium depends on the temperature.
When the reaction is performed at low temperature (0 ꢁC) the
bromomethyl group is maintained (amide 5). The synthesis of
dichloro derivative 7 involves in situ formation of the correspond-
ing methanesulfonate which is displaced by chloride ion. The
amide 8 was synthesized in the same way as 7.
According to described by Wakselman et al., o-nitrobenzyl
halides can generate the alkylating agents quinonimine methide
upon reduction to the corresponding hydroxylamino or amino
derivatives [11]. These reactive species could alkylate important
biomolecules of the parasite, as nucleic acids and proteins. As the
redox cycling of nitroaromatics generate reactive oxygen species,
these compounds may potentially have a dual mode of action,
through oxidative stress and/or alkylation, as previously proposed
by us [10]. So, the toxicity of compounds 1, 3 and 5 might be
associated to their intrinsic alkylating properties due to presence of
the benzyl bromine substituent, which is a good leaving group. The
benzyl chloride group is less reactive per se and consequently less
toxic (compounds 6, 7 and 8). The lower reactivity of the benzyl
chloride group is, most likely, a consequence of the higher strength
of the CeCl bond (81 kJ molꢀ1) than of CeBr (67 kJ molꢀ1). It is
clearly easier to break a CeBr bond than a CeCl bond.
Mammalian cells possess elaborate defense mechanisms to
detoxify radicals, what may explain the lower cytotoxicity of the
nitrocompounds, such as 6 and 7, to mammalian cells than to
parasite cells. In mammalian cells, low intracellular concentrations
of reactive oxygen species are maintained by the action of super-
oxide dismutase, glutathione peroxidase and glutathione reduc-
tase. Trypanosomatids contain superoxide dismutase, which
prevents accumulation of the superoxide anion radical. However,
the antioxidant enzymes catalase and glutathione peroxidase are
absent in parasite, resulting in a limited capacity to remove H2O2
and, consequently, higher susceptibility to oxidative stress.
The presence of nitro group and of a good leaving group at the
benzylic position seem to be important for antileishmanial activity,
since compounds 1 and 8, bearing no nitro substituent, and
compound 4, bearing no leaving group, were inactive.
Compounds 1e8 were assayed in vitro at 100
mM against
L. amazonensis and the results are expressed as percentage of inhi-
bition of parasite growth after treatment with each compound.
Invitrotrypanocidal effect of the compounds upon amastigote forms
of T. cruzi was also evaluated (Table 1). Trypanocidal activity of
compounds 1e3 against trypomastigotes of T. cruzi was previously
described byus [10] and all of them displayed IC50 values higher than
the highest concentration used (w500 mM). All compounds were
tested in vitro with human PBMC stimulated with phytohemaglu-
tinin (PHA) and proliferation evaluated by 3-(4,5-Dimethylthiazol-
2-yl)-2,5-Diphenyltetrazolium bromide (MTT) method (Table 1).
The ability to inhibit the growth of the parasite without affecting
lymphocyte proliferation in PBMC is a good indication of selectivity
of the compound, a desirable property for a drug candidate.
The selectivity index (SI) was determined based on ratio of IC50
values measured on the mammalian cells (PBMC) and for the para-
site(leishmania) (IC50 for mammaliancell line/IC50 forparasite). This
ratio provides an indication of the selectivity of these compounds
against L. (L.) amazonensis promastigotes when compared to normal
cells.
The results revealed an interesting selectivity displayed by
compounds 6 and 7, which were much more active to promasti-
gotes of L. (L.) amazonensis line than to normal cells (selectivity
index higher than 15). The ability of compounds 6 and 7 to inhibit
specifically the growth of L. (L.) amazonensis and the high selectivity
index indicate that they may serve as promising lead compounds
for future drug development for the treatment of leishmaniasis.
The compounds 3, 5-7 were found to be active against pro-
mastigotes of L. (L.) amazonensis lines used, with IC50 values
H
N(CH2)2R1
O
OH
O
OH
O
iv or v
NO2
i
3. Conclusion
5 R1 = OH; R2 = Br
NO2
CH2R2
CH2Br
CH2Br
The nitrocompounds 6 and 7 evaluated in the present work can
be useful as lead molecules for the design of new analogues with
improved antileishmanial activity. The mechanism of action of
active compounds can be associated with the formation of reactive
species (free radical and reactive electrophile), after reduction of
aromatic nitro group. None of the tested compounds were active
against T. cruzi.
6 R1 = OH; R2 = Cl
7 R1 = R2 = Cl
2
1
vi
iii
ii
v
H
OCH3
O
O
OH
O
N(CH2)2OH
NO2
NO2
4. Experimental
CH2Br
CH2OH
CH2Cl
3
4
8
All melting points were determined on a Kofler Sybron appa-
ratus and are uncorrected. The IR spectra were recorded on a Shi-
madsu IR-408 spectrometer. The NMR spectra were recorded on
a Bruker AVANCE DRX200 instrument, using tetramethylsilane
Scheme 1. Reagents, conditions and yield: i ¼ fuming HNO3; 76%; ii ¼ methanol, 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC); 71%; iii ¼ Na2CO3, H2O,
acetone; 81%; iv ¼ ethanolamine, EDAC, N-hydroxysuccinimide (NHS), CH2Cl2, 0 ꢁC, 47%
(compound 5);
v
¼
ethanolamine, EDAC, NHS, CH2Cl2, room temperature, 35%
(TMS) as the internal standard. Chemical shifts are given in
d (ppm)
(compound 6), 30% (compound 9); vi ¼ Methanesulfonyl chloride (MsCl), triethylamine,
CH2Cl2, room temperature, 84%.
scale and J values are given in Hz. All reagents used were of

M.S. Lopes et al. / European Journal of Medicinal Chemistry 46 (2011) 5443e5447
5445
Table 1
In vitro effect of compounds on the growth of L. (L.) amazonensis, T. cruzi and on the proliferation of human lymphocytes stimulated with PHA.
O
R1
a
b
c
f
e
R2
d
R3
Compound
R1
R2
R3
Trypanocidal
Antileishmanial
PBMC Proliferation
SIc
activity IC50
(
m
M)a
activity IC50
(
m
M)a ꢃ SD
IC50
(m
M)b ꢃ SD
1
2
3
4
5
6
7
8
OH
OH
OCH3
OH
NH(CH2)2OH
NH(CH2)2OH
NH(CH2)2Cl
NH(CH2)2OH
e
H
Br
Br
Br
OH
Br
Cl
Cl
Cl
e
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
e
>100
>100
88.8 ꢃ 7
344.2 ꢃ 96d
44.1 ꢃ 13
ND
53.9 ꢃ 13
959.9 ꢃ 98d
1099 ꢃ 119d
329.9 ꢃ 69d
e
<0.9
<3.4
1.1
NO2
NO2
NO2
NO2
NO2
NO2
H
39.3 ꢃ 5
>100
e
23.1 ꢃ 0.5
59.5 ꢃ 6
50.6 ꢃ 6
>100
2.3
16.1
21.7
<3.3
e
Amphotericin Be
Benznidazolee
e
0.9 ꢃ 0.01
e
e
e
3.8 ꢃ 0.8
e
e
e
a
Half maximal inhibitory concentration (IC50) represents the concentration of drug able to inhibit by 50% the in vitro growth.
b
IC50 represents the concentration of drug able to inhibit by 50% the lymphocyte proliferation in Peripheral Blood Mononuclear Cell (PBMC) stimulated with phytohe-
maglutinin A.
c
Selectivity index (SI) is the ratio of IC50 on normal cell line to the IC50 on leishmania.
The low solubility of these compounds in the assay medium prevented the determination of their IC50. Then, the values of IC50 were estimated by extrapolation of the
d
curve, by nonlinear regression analysis of data using Prism Graphpad software.
e
Positive control. SD: Standard Deviation.
analytical grade. Methyl 4-(bromomethyl)-3-nitrobenzoate 3 [8]
and 4-hydroxymethyl-3-nitrobenzoic acid 4 [9] were synthesized
according to previously reported procedures.
4.2. Synthesis of 4-(chloromethyl)-3-nitro-N-(2-hydroxyethyl)
benzamide 6
The synthesis of amide 6 was performed at room temperature
for 5 h from acid 2 using the same method described above. The
crude product was recrystallized from EtOAc (Yield: 35%). Mp
133e135 ꢁC; IR cmꢀ1: 3473e3154 (OH; NH); 1650 (C]O); 1538
(nitroaromatic (ArNO2), N]O assym.); 1332 (ArNO2, N]O sym.);
4.1. Synthesis of 4-(bromomethyl)-3-nitro-N-(2-hydroxyethyl)
benzamide 5
To a solution of acid 2 (1.15 mmol) in dichloromethane (6 mL)
were added N-hydroxysuccinimide (NHS, 1.85 mmol) and 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC,
1.85 mmol). The reaction mixture was stirred in ice bath for
20 min. The mixture was diluted with dichloromethane (60 mL)
and then extracted with saturated solution of NaCl (3 ꢂ 50 mL).
The organic layer was dried over anhydrous sodium sulfate,
filtered and the solvent was removed. Thus, the crude product
obtained (0.84 mmol) was transferred to a round-bottomed flask
and dichloromethane (5 mL) was added. To this mixture was
added dropwise a solution of ethanolamine (1.68 mmol) in
dichloromethane (5 mL), under stirring and ice-bath cooling. After
4 h, the reaction mixture was diluted with ethyl acetate (EtOAc)
(60 mL), washed with 0.1 mol/L HCl (3 ꢂ 30 mL), saturated solu-
tion of sodium bicarbonate (15 mL) and water (2 ꢂ 20 mL). The
organic portion was dried over anhydrous sodium sulfate, filtered
and the solvent was removed. The product 5 was obtained with
a yield of 47%. Mp 143e144 ꢁC; IR cmꢀ1: 3498e3144 (OH; NH);
1642 (C]O); 1527 (ArNO2, N]O assym.); 1327 (ArNO2, N]O
1056,1045 (CeO); 1H-NMR (200 MHz, DMSO-d6)
d: 8.83 (s broad,
1H, NH), 8.51 (s, 1H, H-b), 8.20 (d, 1H, Jf,e ¼ 7.6 Hz, H-f), 7.87 (d, 1H,
Je,f ¼ 7.6 Hz, H-e), 5.07 (s, 2H, eCH2Cl), 3.53e3.34 (m, 5H, eCH2NH,
eCH2OH and OH); 13C-NMR (50 MHz, DMSO-d6)
d: 163.93 (C]O),
147.81 (C-c), 136.01 (C-a), 134.54 (C-d), 132.49 (C-e and C-f), 123.95
(C-b), 59.59 (CH2OH), 42.50 (CH2NH), 42.39 (eCH2Cl).
4.3. Synthesis of 4-(chloromethyl)-N-(2-hydroxyethyl)benzamide 8
The synthesis of amide 8 was performed at room temperature
for 24 h from acid 1 using the same method described above. The
crude product was recrystallized from EtOAc (Yield: 30%). Mp
116e118 ꢁC; IR cmꢀ1: 3508e3103 (OH; NH); 1635 (C]O); 1061,1037
(CeO); 1H-NMR (200 MHz, DMSO-d6)
d: 8.47 (s broad, 1H, NH), 7.85
(d, 2H, Jb,c ¼ 8.0 Hz, H-b), 7.51 (d, 2H, Jc,b ¼ 8.0 Hz, H-c), 4.80 (s, 2H,
-CH2Cl), 3.51 (m, 2H, -CH2OH), 3.31 (m, 2H, eCH2NH); 13C-NMR
(50 MHz, DMSO-d6) d: 165.94 (C]O), 140.54 (C-d), 134.44 (C-a),
128.69 (C-c), 127.60 (C-b), 59.75 (CH2OH), 45.50 (CH2Cl), 42.22
(CH2NH).
sym.); 1060 (CeO); 1H-NMR (200 MHz, DMSO-d6)
d: 8.76 (s broad,
1H, NH), 8.45 (s, 1H, H-b), 8.12 (d, 1H, Jf,e ¼ 7.8 Hz, H-f), 7.80 (d, 1H,
Je,f ¼ 7.8 Hz, H-e), 4.90 (s, 2H, -CH2Br), 3.51e3.29 (m, 5H, eCH2NH,
4.4. Synthesis of 4-(chloromethyl)-3-nitro-N-(2-chloroethyl)
benzamide 7
-CH2OH and OH); 13C-NMR (50 MHz, DMSO-d6)
d: 163.88 (C]O),
147.70 (C-c), 135.99 (C-a), 135.05 (C-d), 132.96 (C-e or C-f), 132.46
(C-f or C-e), 124.07 (C-b), 59.57 (CH2OH), 42.48 (CH2NH), 29.17
(eCH2Br).
Methanesulfonyl chloride (0.48 mmol) was added dropwise to
a solution of amide 6 (0.19 mmol) and triethylamine (0.48 mmol) in

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M.S. Lopes et al. / European Journal of Medicinal Chemistry 46 (2011) 5443e5447
dichloromethane (2 mL), under stirring in ice-bath. After 18 h at
room temperature, crushed ice was added to the flask and then
saturated sodium bicarbonate solution (2 mL) and water (5 mL)
were added. The mixture was extracted with EtOAc (3 ꢂ 15 mL),
and the combined extracts were washed with 0.1 mol/L HCl (30 mL)
and water until neutral pH, dried over anhydrous sodium sulfate,
filtered and the solvent was removed in rotavapor. The product 7
was obtained in 84% yield. Mp 92e97 ꢁC; IR cmꢀ1: 3312 (NH); 1649
(C]O); 1528 (ArNO2, N]O assym.); 1348 (ArNO2, N]O sym.); 1H-
compounds. Each compound was tested in triplicate. After 7 days
culture development, chlorophenol red -galactopyranoside at
b-D
100 mM and Nonidet P-40 at 0.1% were added to the plates and
incubated overnight at 37 ꢁC. The absorbance was measured at
570 nm in an automated micro plate reader. Benznidazole at its IC50
(1
m
g/mL ¼ 3.81
mM) was used as positive control. The results are
expressed as percentage of parasite growth inhibition [13].
4.8. Peripheral blood mononuclear cell (PBMC) assay
NMR (200 MHz, acetone-d6) d: 8.54e8.53 (m, 2H, NH, H-b), 8.25
(dd, 1H, Jf,b ¼ 1.7 Hz, Jf,e ¼ 8.0 Hz, H-f), 7.89 (d, 1H, Je,f ¼ 8.0 Hz, H-e),
The PBMC were prepared using the protocol previously
described by Gazzinelli et al [14]. Briefly, PBMC samples were ob-
tained through agreement with Minas Gerais Hematology and
Hemotherapy Center Foundation e HEMOMINAS (protocol nꢁ 105/
2004). The PBMC were obtained from healthy adult volunteers of
both sexes by centrifugation of heparinized venous blood over
a Ficoll cushion (SigmaeAldrich, St. Louis, MO). Mononuclear cells
were collected from the interphase after Ficoll separation and
washed three times in RPMI-1640 before further processing. All
cultures were carried out in RPMI-1640 medium SigmaeAldrich
(St. Louis, MO), supplemented with 5% (v/v) heat-inactivated,
5.09 (s, 2H, eCH2Cl), 3.78e3.75 (m, 4H, CH2OH and CH2NH); 13C-
NMR (50 MHz, acetone-d6) d: 165.13 (C]O), 148.98 (C-c), 136.70 (C-
a), 135.89 (C-d), 133.04 (C-e or C-f), 132.95 (C-e or C-f), 124.72 (C-b),
43.417e42.681 (CH2NH, and 2 ꢂ eCH2Cl).
4.5. In vitro assay with promastigotes forms of L. amazonensis
Promastigotes of L. (L.) amazonensis (IFLA/BR/1967/PH-8), were
maintained at 23 ꢁC in Schneider’s Drosophila medium (GIBCOÔ)
supplemented with 20% heat-inactivated fetal calf serum (FCS), pH
7.2.
pooled AB (GIBCO/BRL, Grand Island, NY) sera and 2 mM
mine. An antibiotic/antimycotic solution containing 1000 U/mL
penicillin, 1000 g/mL streptomycin and 25 g/mL fungisone
L-gluta-
4.6. MTT assay
m
m
(GIBCO/BRL, Grand Island, NY) was added to control fungal and
bacterial contamination.
To evaluate the leishmanicidal activity of nitroaromatic
compounds, an in vitro assay was performed in two steps: screening
and dose-response. This assay involves the conversion of a tetrazo-
lium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), into a colored, insoluble formazan product, the
amount of which depends on the number of viable cells present.
Briefly, promastigotes of L. amazonensis were plated in 96-well-flat-
bottom microplates at a plating density of 1 ꢂ107 parasites/well. For
4.9. Analysis of cell viability
PBMC were cultured in 96 well plates at densities 200,000 cells/
well in a final volume of 200 mL/well. The plates were pre-incubated
in a 5% CO2/95% air-humidified atmosphere at 37 ꢁC for 24 h to
allow adaptation of cells prior to the addition of the test
compounds. All compounds were dissolved in dimethyl sulfoxide
(DMSO), prior to dilution. The toxicity was determinated over
screening assay, parasites were exposed to 100
mM of each
compound, in triplicate. After 48 h of incubation, 10
mL of MTT
(10 mg/mL) was added to each well and the plates were further
incubated for 4 h. The enzymatic reaction was then stopped by
addition of 100 mL of 50% isopropanole10% sodium dodecyl sulfate
a range of concentrations (1e100 mM). All cell cultures were incu-
bated in a 5% CO2/95% air-humidified atmosphere at 37 ꢁC for 48 h.
Cell viability was estimated by MTT assay [15]. Results were
normalized to DMSO control (0.01%) and expressed as percent
inhibition of cell viability. Interactions of compounds and media
were estimated on the basis of the variations between drug-
containing medium and drug-free medium to control for false-
positive or false-negative results. The data were analyzed using
Prism 5.0 (GraphPad Software Inc.).
solution. The optical density at 570 nm was measured using an
ELISA plate reader (BioSource, Inc., EUA). For dose-response assay,
parasites were exposed to six points of dilution, in duplicate, of
those compounds that showed percent growth inhibition greater
than 50% in screening assay. The addition of MTT and all next steps
were the same as described previously. A curve was obtained and
the IC50 determined. Three independent experiments, in duplicate,
were performed to determine the leishmanicidal activity. Data were
analyzed using MiniTabÒ.
4.10. Statistical analysis
All results were expressed as the mean ꢃ standard deviation
(SD) of three independent experiments performed, at least, in
triplicate. These data were analyzed using Student’s t-test for
paired comparisons. Statistical significance was determined as
P < 0.05.
4.7. In vitro assay amastigotes intracellular forms of T. cruzi
In vitro assay with T. cruzi amastigote and trypomastigote forms
was performed according to protocols established by Buckner et al.
[12] with modifications. Briefly, parasites and culture procedures:
T. cruzi (Tulahuen strain) expressing the Escherichia coli
sidase gene were grown on monolayer of mouse L929 fibroblasts.
Cultures assayed for -galactosidase activity were grown in RPMI
b-galacto-
Acknowledgments
b
The authors are grateful to Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), for financial
support.
1640 medium (pH 7.2e7.4) without phenol red (Gibco BRL) plus 10%
fetal bovine serum and glutamine. Ninety-six-well tissue culture
micro plates were seeded with L929 fibroblasts at 4.0 ꢂ 103 per well
in 80 m b-galactosi-
L and incubated overnight at 37 ꢁC and 5% CO2.
dase-expressing trypomastigotes were then added at 4.0 ꢂ 104 per
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200 L of fresh medium. After 48 h, the medium was discarded
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