Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Article abstract of DOI:10.1016/j.bmc.2008.10.085

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO₂, pIC₅₀ = 4.55 M) and 6l (X = F, Y = p-CN, pIC₅₀ = 4.27 M) as the most potent derivatives compared to crystal violet (pIC₅₀ = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising profile with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it.

Full text of DOI:10.1016/j.bmc.2008.10.085

Bioorganic & Medicinal Chemistry 17 (2009) 295–302
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular
modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-
acrylic acid benzylidene-carbohydrazide series
Maria A. F. Vera-DiVaio a, Antônio C. C. Freitas a, Helena C. Castro b, Sérgio de Albuquerque c,
Lucio M. Cabral d, Carlos R. Rodrigues d, Magaly G. Albuquerque e, Rita C. A. Martins f,
Maria G. M. O. Henriques g, Luiza R. S. Dias a,
*
a Laboratório de Química Medicinal (LQMed) - Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mário Viana 523, Santa Rosa, 24241-000 Niterói, RJ, Brazil
b Laboratório de Antibióticos, Bioquímica e Modelagem Molecular (LABioMol) - Instituto de Biologia, Universidade Federal Fluminense,
Outeiro de São João Baptista, 24020-150 Niterói, RJ, Brazil
c Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, 29020-159 São Paulo, SP, Brazil
d Laboratório de Modelagem Molecular e QSAR (ModMolQSAR) - Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
e Departamento de Química Orgânica - Instituto de Química, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brazil
f Curso de Farmácia, Centro de Ensino Superior do Pará, Av. Nazaré 630, Nazaré, 66035-170 Belém, PA, Brazil
g Departamento de Farmacologia Aplicada, Instituto de Tecnologia em Fármacos, Fundação Oswaldo Cruz (FIOCRUZ), Rua Sizenando Nabuco 100,
Manguinhos, 21041-250, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious
social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypan-
ocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series
of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO2,
pIC50 = 4.55 M) and 6l (X = F, Y = p-CN, pIC50 = 4.27 M) as the most potent derivatives compared to crystal
violet (pIC50 = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas
6l showed the most promising profile with low toxicity (0% of cell death). The best equation from the 4D-
QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical represen-
tation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen
bond acceptor Y-substituents increased it.
Received 19 August 2008
Revised 30 October 2008
Accepted 31 October 2008
Available online 5 November 2008
Keywords:
1-Phenyl-1H-pyrazol
Benzylidene-carbohydrazides
Trypanocidal evaluation
Chagas disease
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
duces blood micro-agglutination and presents a potential mutage-
nicity profile.5,6 In addition, it is not well-accepted by patients due
According to the World Health Organization (WHO), 16–18 mil-
lion people are infected with Trypanosoma cruzi, the causative
agent of Chagas disease.1 Since the first report from the Brazilian
physician Carlos Chagas, in 1909, the chemotherapy of Chagas dis-
ease is still unsatisfactory, based primarily in nitroimidazole (e.g.,
benznidazole) and nitrofuran (e.g., nifurtimox) drugs. These drugs
present poor efficacy in this disease chronic stage and frequent
highly toxic side effects.1–3
to the bluish color conferred on blood and tissues.5,6 Despite of
that, crystal violet remains as a reference drug for in vitro test.
As a part of a research program aiming at synthesis and phar-
macological evaluation of pyrazole derivatives as potential antich-
agasic agents more effective and safe for the treatment of Chagas
disease, in a previous work we reported work the synthesis of ac-
tive 1H-pyrazole derivatives and the importance of carbohydrazide
structural subunit for the trypanocidal activity.7a–d This moiety is
Therefore, Chagas disease treatment is still a challenge, mainly
in Brazil, where the only commercially available drug is benznidaz-
ole (RochaganÒ).4 Since 1984, the WHO has recommended the use
of crystal violet (gentian violet) in blood banks in endemic areas to
prevent the transfusion transmission. However, crystal violet in-
an isoster of a,b-unsaturated ketone group, which is a known phar-
macophoric group of inhibitors of cysteine proteases such as cruz-
ain, the main cysteine protease from T. cruzi.7e–h
Since literature describes several phenyl-pyrazole derivatives
active against parasites,8 herein we evaluated new derivatives of
the 1H-pyrazole system containing carbohydrazide moiety against
T. cruzi. Thus, in this work, we described the synthesis, in vitro try-
panocidal analysis, cytotoxicity assays, and molecular modeling
* Corresponding author. Tel.: +55 21 26299588; fax: +55 21 26299578.
E-mail address: ldias@vm.uff.br (L.R.S. Dias).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.085

296
M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
and SAR/QSAR studies of a new series of benzylidene-carbohydra-
pIC50 = 3.72 M) and 6j (X = H and Y = NMe2, pIC50 = 3.70 M) that
were equipotent to crystal violet (pIC50 = 3.77 M) (Table 1 and
Fig. 1). In contrast, 6c (X = H and Y = Cl, pIC50 = 2.07 M) and 6o
(X = F and Y = H, pIC50 = 2.11 M) were the least potent, whereas
6p (X = Br and Y = H) was inactive. The others remaining 21 com-
pounds and the intermediate 5 were of middle potency, showing
values spread over one logarithm unity (pIC50 = 2.5–3.5 M) (Table
1 and Fig. 2).
Overall, the X-substituent (Table 1) seems to depend on the Y-
substituent. Derivatives substituted in Y by H, OH, NMe2 and NO2
showed the best profiles, when X was non-substituted (X = H)
(derivatives 6a, 6e, 6j, and 6k). Whereas, when Y = CN, OMe, and
Br, the derivatives substituted at X position with any substituent
but hydrogen (X – H) showed the best profiles. These observations
suggest a dual behavior at X position. In relation to the remaining
Y-substituents (OEt, SMe, F and Cl), we were unable to perform a
proper analysis as there are not examples of derivatives with
X – H (Table 1).
By considering the substituents at Y-position, derivatives 6k
(Y = p-NO2) and 6l (Y = p-CN), which contain highly electron-
withdrawing groups, presented the best biological profile, fol-
lowed by 6a (Y = H) and 6j (Y = p-NMe2, a highly electron-donat-
ing group), which were equipotent to crystal violet (Table 1).
Apparently, this is not a structural requirement, since others
derivatives containing NO2 (6v and 6z0), CN (6i, 6t and 6y) or
NMe2 (6m, 6q, 6r and 6x) groups in Y-position showed an inter-
mediary biological profile.
zides (6a–z0).
2. Chemistry
The synthesis of N-phenylpyrazole derivatives (6a–z0) is illus-
trated in Scheme 1. The 1-phenyl-1H-pyrazole (1) was obtained
according to the procedure described by Finar.9 Vilsmeier–Haack
formylation of 1 leads to 1-phenyl-1H-pyrazole-4-carboxyalde-
hyde (2) in 65% yield. Condensation of 2 with phenylacetic acid
by the Karminsky–Zamola method10 afforded the acid 3 in 80%
yield. By using these conditions, we obtained the (E) isomer of
the acid 3 in accord to Perkin, Cope, and Toullec condensations
methods,11 and to Zimmerman that previously emphasized this
reaction stereochemistry.12 The acid 3 was reacted with methanol
and sulfuric acid13 to generate the corresponding methyl ester 4, in
95% yield, which was reacted with hydrazine hydrate leading to
the carbohydrazide derivative 5, in 65% yield.14,15 The carbohyd-
razide 5 was treated with several aromatic aldehydes in an acid
catalysis in different reaction times, which generated the benzyli-
dene-carbohydrazides 6a–6z0 in different yields, depending on
the X- and Y-para-substituents of the phenylacetic acid (p-X-
PhCH2CO2H) and benzaldehyde (p-Y-PhCHO) reagents.16
3. Biology and SAR studies
The 28 compounds of the N-phenylpyrazole benzylidene-carbo-
hydrazide series (6a–6z0), including the intermediate 5, were
tested and showed some level of antiparasite activity, except for
The intermediate 5 was also tested to evaluate the impor-
tance of the benzylidene-carbohydrazide moiety of derivatives
6a–z0. The experimental data showed that 5 was less potent
(pIC50 = 2.55 M) than the correlated benzylidene-carbohydrazide
compound 6a (pIC50 = 3.72 M). These data pointed out an impor-
tant role for this substructure in the improvement of the anti-
parasitic profile, probably due to the enhancement of the
general lipophilic character of this series. This hypothesis is rein-
forced by the calculated LogP values (cLogP, Table 1) for the
6p that was inactive even at 500 lg/mL (Table 1 and Fig. 1). These
data reinforced N-phenylpyrazole as a potential system for anti-
parasite activity as suggested by the literature.7,8
Derivatives 6k (X = H and Y = NO2, pIC50 = 4.55 M) and 6l (X = F
and Y = CN, pIC50 = 4.27 M) were the most potent compounds, fol-
lowed by 6a (the non-substituted derivative, X = Y = H,
derivatives
(cLogP = 1.13).
6a–z0
(cLogP = 4.07–6.25)
compared
to
5
In this work, we tested the mice Balb/c cells viability, after drug
treatment, using the standard MTT colorimetric assay for evaluat-
ing the derivatives cytotoxicity profile. This test is based on the
reduction of the yellow-colored MMT tetrazolium salt to the pur-
ple-colored formazan by mitochondrial reductase enzymes in liv-
ing cells.17 Interestingly, the compounds with pIC50 higher than
3.50 M (most potent) and lower than 2.50 M (least potent) (Table
1 and Fig. 1) presented no significant cytotoxicity activity (cell
death <5%), except for compound 6k (ꢀ20% cell death). Com-
pounds with pIC50 between 2.50 and 3.50 M showed no specific
pattern of cytotoxicity profile, displaying high (6h and 6x with
ꢀ23% of cell death), medium (ꢀ10–15%), or low (0%) cytotoxicity
in the assays.
4. Molecular modeling and QSAR studies
The conformational analysis of the non-substituted compound
(6a), considering both the (E)- and (Z)-isomers of the benzyli-
dene-carbohydrazide moiety (N@C double bond), showed the
(E)-isomer of 6a as the most stable one, with the main chain
in a totally extended conformation (as drawn in Scheme 1).
Interestingly, others derivatives (6b–z0) presented the same
structural profile.
The calculated electronic properties such as HOMO, LUMO, and
GAP energy values of the 6a–6z0 derivatives and intermediate 5
(Table 1) were not correlated with the activity values. However,
the map of the three-dimensional isosurfaces of the MEP superim-
Scheme 1. The synthetic pathway of N0-benzylidene-carbohydrazide (6a–6z0).

M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
297
Table 1
Experimental in vitro trypanocidal activity (IC50, mM and pIC50, M) and cytotoxicity (Tox,% of cell death) compared with calculated energies (eV) of HOMO, LUMO, and GAP
(ELUMO ꢁ EHOMO), and Lipinski’s parameters of lipophilicity (cLogP), molecular weight (MW, g/mol), number of hydrogen bond acceptor (HBA) and donor (HBD) groups of the N0-
phenylpyrazole benzylidene-carbohydrazide series (6a–6z0) and intermediate 5
Y
O
N
N
H
N
N
X
a
c
#
X
Y
IC50
pIC50
Tox
EHOMO
ELUMO
GAP
0.37
cLogP
MW
HBA
HBD
5
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
Br
Br
OH
OH
OMe
OMe
OMe
OMe
CF3
CF3
CF3
CF3
—
H
F
Cl
2.81
0.19
2.44
8.59
1.50
0.76
1.51
0.38
0.44
1.87
0.20
0.03
0.05
0.89
0.34
7.83
NAb
0.70
1.88
1.36
1.31
1.42
0.98
0.93
0.92
0.59
0.62
0.75
2.55
3.72
2.61
2.07
2.82
3.12
2.82
3.42
3.36
2.73
3.70
4.55
4.27
3.05
3.47
2.11
NA
3.15
2.73
2.87
2.88
2.85
3.01
3.03
3.04
3.23
3.21
3.13
0
4
0
0
ꢁ0.29
ꢁ7.96
ꢁ8.02
ꢁ8.03
ꢁ8.04
ꢁ7.78
ꢁ7.73
ꢁ7.72
ꢁ7.91
ꢁ8.15
ꢁ7.25
ꢁ8.19
ꢁ8.22
ꢁ7.30
ꢁ7.80
ꢁ8.03
ꢁ7.95
ꢁ7.17
ꢁ7.11
ꢁ7.86
ꢁ8.09
ꢁ7.75
ꢁ8.13
ꢁ8.17
ꢁ7.35
ꢁ8.32
ꢁ8.21
ꢁ8.35
0.08
2.07
2.01
1.91
1.88
2.14
2.15
2.16
1.96
1.39
2.23
0.81
1.33
2.15
2.07
1.99
1.83
1.97
2.11
1.96
1.41
2.17
0.82
1.92
1.97
1.26
1.68
0.71
1.13
4.55
4.71
5.22
5.36
4.07
4.60
4.98
4.98
4.30
4.65
4.51
4.47
4.81
4.77
4.71
5.36
5.46
4.17
4.07
4.36
4.12
4.56
5.41
5.54
5.20
6.25
5.40
304.35
392.46
410.45
426.90
471.35
408.46
422.48
436.51
438.55
417.47
435.53
437.45
435.46
453.52
440.47
410.45
471.35
514.42
451.53
408.46
447.49
438.48
467.48
501.38
503.52
485.46
539.35
505.45
5
5
5
5
5
6
6
6
5
6
6
8
6
6
6
5
5
6
7
6
7
7
9
6
6
6
5
8
3
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
2
1
1
1
1
1
1
6a
6b
6c
6d
6e
6f
6g
6h
6i
10.03
10.03
9.94
9.92
9.92
9.88
9.88
9.87
9.54
9.48
9.00
9.55
9.45
9.87
10.02
9.78
9.14
9.22
9.82
9.50
9.92
8.95
10.09
9.32
9.58
9.89
9.06
Br
14.35
12.32
0
OH
OMe
OEt
SMe
CN
NMe2
NO2
CN
0
22.8
0
6j
6k
6l
0
19.4
0
12.20
14.8
0
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
6z0
NMe2
OMe
H
H
0
0
NMe2
NMe2
H
12.94
9.64
14.35
12.04
11.22
0
CN
OH
NO2
Br
NMe2
CN
23.07
0
Br
NO2
0
0
a
b
c
Values (mM) are medium of triplicate assays.
Not active at 500 g/mL concentration.
Cell death values P5% means significant cytotoxicity.
l
posed onto the total electron density of the most representative
members (6a, 6b, 6k, 6l, 6o, 6p) of this series (Fig. 2) revealed
the derivatives 6k and 6l with high electronic density and high
LUMO coefficient. The derivatives 6k and 6l presented the lowest
electronic density at the N-benzylidene-carbohydrazide moiety
and the higher LUMO (blue region) electron density compared to
other compounds.
cidal activity, two with positive contribution, GC2 and GC4
(blue spheres), represented by unspecific atom types (all), and
two with negative contribution, GC1 and GC3 (red spheres),
represented by apolar atom types (ap). The frequency of occu-
pation of these GCs was given in Table 2 with the experimental
and
calculated
pIC50
,
and
the
residual
values
(pIC50Calc ꢁ pIC50Exp).
Herein, we performed quantitative structure-activity relation-
ship (QSAR) studies, which are mostly used to correlate property
parameters (e.g., biological response) with structural parameters.
Currently, with the advent of the molecular modeling tools, the
three-dimensional (3D) descriptors replaced the didactical phys-
ico-chemical and bidimensional descriptors.18 In those studies,
methods that incorporate molecular flexibility, such as the 4D-
QSAR method proposed by Hopfinger and co-workers,19 proved
advantageous because they allowed the identification of the con-
formation that maximizes the potency.
In this work, we obtained the best 4D-QSAR equation (Model
1) that was able to explain 85% of the activity variability (con-
ventional R2 = 0.85) with a high predictive feature (leave-one-
out cross-validated R2, Q2 = 0.79). It is important to emphasize
that this model cannot be interpreted in terms of ligand–recep-
tor interaction, as the current biological data are not related to
the intrinsic activity.
Model 1
pIC50 = 2.91 ꢁ 102.71 (GC1)(ap) + 18.15 (GC2)(all) ꢁ 7.20
(GC3)(ap) + 1.26 (GC4)(all)
N = 26 R2 = 0.85 SE = 0.23 Q2 = 0.79 F = 30.66
The GC1 (located behind the plane of phenyl ring A) was fre-
quently occupied when the relative orientation between ring A
and pyrazole ring was not co-planar, decreasing the potency.
The GC2 (located close to the ortho position of ring B) was fre-
quently occupied when the relative orientation between phenyl
ring B and pyrazole ring was almost parallel, allowing a ‘pi-
stacking’ type of interaction between them, increasing the po-
tency. The GC3 (located close to the X-substituent) was fre-
quently occupied when X was larger than H and F, pointing
that bulky substituents on this position decrease the potency.
Model 1 (graphically represented in Fig. 3 using derivative
6d) presented four grid cells (GC) correlated with the trypano-

298
M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
Figure 1. Plot of the experimental in vitro trypanocidal activity values (pIC50, M) of intermediate 5 and 6a–z0 derivatives, excluding 6p (inactive).
Figure 3. The four grid cells (GC) of the 4D-QSAR Model 1 represented with
derivative 6d (X = H and Y = Br).
phobic and hydrogen bond acceptor groups in this position in-
crease the potency. The only outlier compound, 6d, was
predicted more potent than its actual activity value (Table 2).
5. Calculated drug-likeness properties
The solubility ranking of chemical compounds may be esti-
mated using the Lipinski’s ‘rule-of-five’.20 The rule describes
molecular properties important for a drug pharmacokinetics in
the human body, including their absorption, distribution, metabo-
lism, and excretion (ADME). These parameters define the ‘drug-
likeness’ and predict a poor oral absorption or permeation when
the evaluated molecules present values higher than five H-bond
donors (HBD), 10 H-bond acceptors (HBA), and molecular weight
(MW) >500 Da and calculated LogP (cLogP) >5. Our results showed
that from the 28 compounds of the N-phenylpyrazole benzylidene-
carbohydrazide series (6a–6z0) evaluated, most of them (n = 19)
fulfilled the Lipinski’s ‘rule-of-five’ (Table 1), except for the com-
pounds with CF3 and Br substituents.
Figure 2. Comparison of Molecular Electrostatic Potential (MEP) energy isosurfaces
and LUMO orbital coefficients of 6a, 6b, 6k, 6l, 6o, and 6p.
6. Conclusion
The GC4 (located close to the Y-substituent) was frequently
occupied when Y was OMe, CN, and NMe2, pointing that hydro-
The molecular modeling and SAR results suggested that the
derivatives of N-phenylpyrazole benzylidene-carbohydrazide ser-

M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
299
Table 2
Experimental and calculated pIC50, frequency of occupation of the four grid cells (GC),
and residual values (pIC50Calc ꢁ pIC50Exp
7.2. 1-Phenyl-1H-pyrazole (1)
)
A mixture of phenylhydrazine (9.83 mL, 0.1 M), 1,1,3,3-tetra-
methoxypropane (16.46 mL, 0.1 M) and ethanol (100 mL) was stir-
red in a round flask. 5 mL of hydrochloric acid were added drop
wise. The reaction mixture was heated at reflux for 4 h. The reac-
tion was allowed to cool to room temperature, poured into water
and extracted with dichloromethane. The extracts were combined
and dried. Evaporation followed by distillation gave 1 as yellow oil
in 85% yield.
#
pIC50Exp
GC1
GC2
GC3
GC4
pIC50Calc
Residual
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6q
6r
3.72
2.61
2.06
2.82
3.12
2.82
3.42
3.36
2.73
3.70
4.55
4.27
3.05
3.47
2.11
3.15
2.73
2.87
2.88
2.85
3.01
3.03
3.04
3.23
3.21
3.13
0
0.036
0.033
0.044
0.034
0.020
0.033
0.020
0.028
0.042
0.027
0.083
0.043
0.027
0.015
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.563
2.378
2.167
3.424
3.272
2.734
3.319
3.212
3.025
3.439
4.416
3.926
3.441
3.539
2.037
3.000
2.852
2.949
2.849
2.805
2.909
2.902
2.956
3.427
3.243
3.157
ꢁ0.158
ꢁ0.232
0.107
0.604
0.152
ꢁ0.087
ꢁ0.101
ꢁ0.148
0.295
ꢁ0.261
ꢁ0.134
ꢁ0.344
0.391
0.011
0.015
0.001
0
0.011
0
0.002
0.008
0
0
0
0
0
0.282
0.037
0
0.139
0.032
0
0.188
0.033
0.284
0
0.048
0.033
0
7.3. 1-Phenyl-1H-pyrazole-4-carboxyaldehyde (2)
N,N-Dimethylformamide (25.6 mL, 0.33 M) was stirred in a
round flask in ice bath and POCl3 (21.6 mL, 0.23 M) was added
dropwise. Then N-phenylpyrazole was added (4.4 mL, 0.033 M) to
this cold mixture. The reaction was allowed to warm to room tem-
perature and then heated at reflux for 6 hours. The temperature
was kept at 95–100 °C. When the reaction was completed, the con-
tents were poured into crushed ice and weakly alkalinized with a
saturated solution of sodium carbonate. The solid was filtered off,
washed with water and recrystallized from ethanol giving the
formylated product in 65% yield; mp 84 °C (lit. 85 °C). 1 H NMR
(CDCl3) d: 10 (s, 1H, CHO), 8.5 (s, 1H, H3 pyrazolic) and 8.2 (s, 1H,
H5 pyrazolic), 7.3–7.5 (m, 5H, Ar). M (m/z): 172 (molecular ion).
0
0.069
0.008
0.002
0.002
0
0.007
0
ꢁ0.073
ꢁ0.150
0.122
0.013
0.017
0.003
0.030
0.017
0
0.028
0.002
0.097
0.043
0
0.001
0.017
0.015
0.004
0.011
6s
6t
0.079
0
0.075
0
0
ꢁ0.031
ꢁ0.045
ꢁ0.101
ꢁ0.128
ꢁ0.084
0.197
6u
6v
6w
6x
6y
6z
6z0
0.001
0
0
0
0
0.001
0.013
0.022
0.020
0.018
0.029
0.180
0
0
0
0
0.033
0.027
0
Grid cell legends: GC1 = (3;ꢁ7;ꢁ7) (ap); CC2 = (ꢁ1;ꢁ3;ꢁ1) (all); CC3 = (2;ꢁ6;3)
7.4. (E)-2-Phenyl-3-(1-phenyl-1H-pyrazol-4-yl) acrylic acid (3)
(ap); GC4 = (0;7;8) (all).
A mixture of the aldehyde (2) (0.172 g, 1 mM), phenylacetic acid
(0.136 g, 1 mM), triethylamine (1.0 mL) and acetic anhydride
(1.0 mL) was refluxed during 5 h. The reaction mixture was acidi-
fied with hydrochloric acid and extracted with diethyl ether. The
organic layer was washed with water and the product was ex-
tracted with a 10% solution of sodium carbonate. The aqueous layer
was acidified with acetic acid. The solid was filtered and washed
with a mixture of methylene chloride, hexane and ethyl acetate
ies (6a–6z0) may present more than one action mechanism, since
the most potent compounds 6k (X = H, Y = NO2, pIC50 = 4.55 M),
6l (X = F, Y = CN, pIC50 = 4.27 M) 6j (X = Y = H, pIC50 = 3.72 M) and
6a (X = H, Y = NMe2, pIC50 = 3.70 M) in the in vitro trypanocidal as-
says did not share the same stereo-electronic features to fit in just
one model of structure-activity relationship.
The best equation from the 4D-QSAR analysis (Model 1) was
able to explain 85% of the activity variability. The Model 1 graphi-
cal representation showed that bulky X-substituents decrease the
potency whereas hydrophobic and hydrogen bond acceptor Y-sub-
stituents increase it.
The molecular modeling results showed 6k and 6l with the
highest orbital density, which may be involved in the trypanocidal
profile since both were more potent than crystal violet. Compound
6l showed to be the most potential lead compound from this series
due to its high biological activity and lower toxicity compared to
6k.
(1:1:1), giving the product in 80% yield; mp 265–267 °C. IR (m-
cmꢁ1) 3100–3500 (broad OH stretch), 1655 (CO); 1H NMR (dDMSO)
d: 6.7 (s, 1H, CH (E)), 7.75 (s, 1H, H3 pyrazole) and 8.3 (s, 1H, H5 pyr-
azole), 7.0–7.5 (m, 10H, Ar), 12 (s, 1H, COOH). The (Z) product pre-
sented the methylenic H in 6.4 (s, 1H, CH). 13C NMR (dDMSO) d:
170.9 (CO); M (m/z): 290 (molecular ion).
7.5. (E)-Methyl 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl) acrylate
(4)
The acrylic acid (3) (0.5 g, 1.72 mM) was heated at reflux with
methanol (30 mL) and sulfuric acid (0.2 mL) for a period of 5h,
and it was allowed to reach room temperature and poured into
ice water. The white precipitate was filtered, giving the methyl es-
7. Experimental
7.1. Chemistry
ter in 95% yield. IR (m
-cmꢁ1): 1700 (CO), 1200–1250 (CC=O), 1160
(OCC assym. stretch); M (m/z): 304 (molecular ion); 1H NMR
(dDMSO) d: 3.8 (s, 3H, CH3), 7.2 (s, 1H, CH), 7.8 (s, 1H, H5 pyrazole),
7.3–7.5 (m, 11H, Ar and H3 pyrazole).
All reactions were monitored by TLC performed on 2.0–6.0 cm
aluminum sheets precoated with silica gel 60 (HF-254, Merck) to
a thickness of 0.25 mm. The developed chromatograms were
viewed under ultraviolet light (254 265 nm). Hexane and ethyl-
acetate in the proportions of 7:3 and 1:1 were used as TLC eluent.
Melting points were determined through a Thomas Hoover appara-
tus and are uncorrected. The methanol, N,N-dimethylformamide
and POCl3 employed in the reactions were previously dried and
distilled. Triethylamine and acetic anhydride were only distilled.
Infrared (IR) spectra were recorded on a Perkin-Elmer 1710 spec-
trometer. Mass spectra were recorded on a VG Auto spec Q70 eV
spectrometer. The NMR spectra were determined in CDCl3 or
DMSO-d6 containing 1% TMS as an internal standard with a Varian
Gemini 200 spectrometer at 200 MHz.
7.6. 2-Phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylohydrazide (5)
The methyl ester (0.5 g, 2 mM) and ethanol (50 mL) were
warmed until complete dissolution. Hydrazine hydrate (3 mL)
was slowly added and the mixture was heated at reflux during
18 h. The reaction mixture was poured into ice water and the solid
filtered off giving the product in 65% yield; mp 133–136 °C. IR (m-
cmꢁ1): 3220 (NH), 1600 (NCO), 1500 (C=C); M (m/z): 304 (molecu-
lar ion); 1H NMR (CDCl3) d: 9.5 (s, 1H, NH), 8.6 (s, 1H, H5 pyrazole),
7.8 (s, 1H, H3 pyrazole), 7.2–7.8 (m, 13H, CH, Ar, NH2).

300
M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
7.7. General procedure for the preparation of N0-benzylidene-2-
phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-carbohydrazide (6)
Compound 6l: 41% yield; mp 154–157 °C; reaction time 10 h; IR
(
m
-cmꢁ1): 1656 (NCO, amide I), 1160 (C–F),1224 (C–O), 1394 (Ar-
F); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 9.2 (s, 1H, NH); 8.6 (s, 1H,
NCH), 8.2 (s, 1H, H5 pyrazole), 7.0–8.0 (m, 14H, H3 pyrazole and
Ar.); M (m/z): 13C NMR (CDCl3) d: 168 (C@O), 118 (CN), 162 (CF),
Compound 5 was heated (1 mM) until complete dissolution in
ethanol. The appropriate aldehyde (1 mM) was added with stirring.
The Hydrochloric acid (3 drops) was slowly added. The mixture
was heated at reflux during 18–22 h, depending on the aldehyde.
After the reaction was completed, the mixture was poured into
water. The solid was filtered off and recrystallized from chloroform
or ethanol/water giving the desired product in different yields
depending on the aldehyde (Table 1).
131 (Ca), 146 (Cb); M (m/z): 435 (molecular ion).
Compound 6m: 53% yield; mp 95–97 °C; time reaction 10 h; IR
(m
-cmꢁ1): 1596 (NCO, amide I), 3000 (NH, amide II), 1176 (C–F),
1365 (C–N); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 8.0 (s, 1H, NH),
8.8 (s, 1H, NCH), 8.0 (s, 1H, H3 pyrazole), 7.8 (s, 1H, H5 pyrazole),
2.9 (s, 3H, CH3), 3.0 (s, 3H, CH3), 6.8–8.2 (m, 13H, Ar); (CDCl3) d:
Compound 6a: 68% yield; mp 163–165 °C; reaction time: 8 h; IR
175 (C@O), 163 (C–F), 139 (NCH), 150 (C
62 (CH3); M (m/z): 453 (molecular ion).
a), 140 (Cb); 54 (CH3),
(m
-cmꢁ1): 1650 (NCO, amide I), 3200 (NH, amide II); 1H NMR
(CDCl3) d: 7,2 (s, 1H, CH), 7,8 (s, 1H, H5 pyrazole), 7,7 (s, 1H, H3 pyr-
azole), 7,3–7,7 (m, 16H, Ar. and NCH); M (m/z): 392 (molecular ion).
Compound 6b: 62% yield; mp 159–162 °C; reaction time 13 h; IR
Compound 6n: 64% yield; mp 117 °C; time reaction 10 h; 1602
(NCO, amide I), 3000 (NH, amide II), 1164 (Ar–F); 1H NMR (CDCl3)
d: 7.8 (s, 1H, H5 pyrazole), 7.7 (s, 1H, H3 pyrazole), 7.0 (s, 1H, CH),
3.1 (s, 3H, CH3), 6.7–7.4 (m, 13H, Ar), 8.4 (s, 1H, NCH), 8.0 (s, 1H,
NH); M (m/z): 440 (molecular ion).
(m
-cmꢁ1): 1540 (NCO, amide I), 3000 (NH, amide II), 1500 (CN),
3300 (OH); 1H NMR (CDCl3) d: 7,2 (s, 1H, CH), 9,2 (s, 1H, NH),
1,1(s, 1H, OH), 7,4–82 (m, 17H, Ar., H5 and H3 pyrazole and NCH);
M (m/z): 408 (molecular ion).
Compound 6o: 54% yield; mp 138 °C; time reaction 10 h; IR (m-
cmꢁ1): 1630 (NCO, amide I), 3020 (NH, amide II), 1155 (Ar–F); 1H
NMR (CDCl3) d: 7.4 (s, 1H, H5 pyrazole), 7.6(s, 1H, H3 pyrazole),
7.3 (s, 1H, NCH), 6.7–7.4 (m, 14H, Ar), 8.3(s, 1H, NCH), 7.9 (s, 1H,
NH); M (m/z): 410 (molecular ion).
Compound 6c: 65% yield; mp 180–184 °C; reaction time 11h; IR
(m
-cmꢁ1): 3000 (NH, amide II), 1500 (NCO, amide I); 1H NMR
(CDCl3) d: 7,2 (s, 1H, CH), 8,6 (s, 1H, NH), 7,8 (s, 1H, H5 pyrazole),
7,7 (s, 1H, H3 pyrazole), 6,8–78 (m, 15H, Ar. and NCH), 1,5 (s, 3H,
CH3); M (m/z): 422 (molecular ion).
Compound 6p: 65% yield; mp 145–147 °C; time reaction 10 h; IR
(m
-cmꢁ1): 1600 (NCO, amide I), 3000 (NH, amide II), 950 (Ar–Br);
Compound 6d: 65% yield; mp 187–190 °C; reaction time 11 h; IR
1H NMR (CDCl3) d: 7.6 (s, 1H, H5 pyrazole), 7.8(s, 1H, H3 pyrazole),
6.8–7.0 (m, 14H, Ar), 7.3 (s, 1H, CH), 10 (s, 1H, NH), 7.7 (s, 1H, NCH);
M (m/z): 471 (molecular ion), 472 (M+1).
(m
-cmꢁ1): 3050 (NH, amide II), 1600 (NCO, amide I); 1H NMR
(CDCl3) d: 7,25 (s, 1H, CH), 8,6 (s,1H, NH), 7,8 (s, 1H, H5 pyrazole),
7,7 (s, 1H, H3 pyrazole), 6,8–7,8 (m, 15H, Ar. and NCH) 1,4 (t, 3H,
CH3), 4,1 (q, 2H, CH2); M (m/z): 436 (molecular ion).
Compound 6q: 55% yield; mp 151–154 °C; time reaction 10 h; IR
(m
-cmꢁ1): 1600 (NCO, amide I), 2900 (NH, amide II), 1170 (Ar–Br),
Compound 6e: 52% yield; mp 202–203 °C; reaction time 15 h; IR
1370 (C–N); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 8.6 (s, 1H, NCH), 8.2
(s, 1H, H3 pyrazole), 6.9–7.2 (m, 14H, Ar, H5 pyrazole), 11.2 (s, 1H,
NH), 2.9 (s, 3H, CH3), 3.0 (s, 3H, CH3); M (m/z): 514 (molecular ion),
515 (M+1).
(m
-cmꢁ1): 3050 (NH, amide II), 1500 (NCO, amide I), 1060 (4-Br-
Ar); 1H NMR (CDCl3) d: 7,25 (s, 1H, CH), 8,6 (s, 1H, NH), 7,7 (s,
1H, H5 pyrazole), 7,6 (s, 1H, H3 pyrazole), 7.3–7.8 (m, 15H, Ar.
and NCH); M (m/z): 471 (molecular ion).
Compound 6r: 54% yield; mp 161–163 °C; time reaction 10 h; IR
Compound 6f: 68% yield; mp 190–194 °C; reaction time 10 h; IR
(m
-cmꢁ1): 1606 (NCO, amide I), 1361 and 2900 (OH), 1168 (N–C),
(m
-cmꢁ1): 1550 (NCO, amide I), 3040 (NH, amide II); 1H NMR
3150 (NH, amide II); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 8.6 (s,
1H, OH), 7.9 (s, 1H, H3 pyrazole), 7.7 (s, 1H, H5 pyrazole), 9.2 (s,
1H, NH), 6.8–9.0 (m, 14H, Ar and NCH); M (m/z): 451 (molecular
ion), 452 (M+1).
(CDCl3) d: 7,05 (s, 1H, CH), 9,4 (s, 1H, NH), 8,6 (s, 1H, NCH), 8,2 (s,
1H, H5 pyrazole), 8,05 (s, 1H, H3 pyrazole), 7,2–7,8 (m, 14H, Ar);
M (m/z): 417 (molecular ion).
Compound 6g: 58% yield; mp 160–163 °C; reaction time 10h; IR
Compound 6s: 56% yield; mp 158–160 °C; time reaction 10 h; IR
(
m
-cmꢁ1): 3050 (NH, amide II), 1600 (NCO, amide I); 1H NMR
(m
-cmꢁ1): 1590 (NCO, amide I), 1370 and 2950 (OH), 3150 (NH,
(CDCl3) d: 6.7 (s, 1H, CH), 8.6 (s, 1H, NH), 8.1 (s, 1H, H5 pyrazole),
7.8 (s, 1H, H3 pyrazole), 7.2–7.8 (m, 15H, Ar. and NCH), 3.0 (s, 6H,
CH3); M (m/z): 435 (molecular ion).
amide II); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 8.6 (s, 1H, OH), 7.9
(s, 1H, H3 pyrazole), 7.7 (s, 1H, H5 pyrazole), 9.0 (s, 1H, NH), 5.6
(s, 1H, OH), 9.8 (s, 1H, NCH), 7.0–7.7 (m, 14H, Ar); M (m/z): 408
(molecular ion).
Compound 6h: 70% yield; mp 154–157 °C; reaction time 10 h;
1H NMR (CDCl3) d: 2.5 (s, 3H, CH3), 6.8 (s, 1H, CH), 10.8 (s, 1H,
NH), 8.3 (s, 1H, H5 pyrazole), 8.2 (s, 1H, H3 pyrazole), 7–8 (m,
Compound 6t: 63% yield; mp 96–99 °C; time reaction 10 h; IR (m-
cmꢁ1): 1644 (NCO, amide I), 2219 (AR-CN), 2335 (NH, amide II); 1H
NMR (CDCl3) d: 6.8 (s, 1H, CH), 3.8 (s, 3H, CH3), 11 (s, 1H, NH), 8.4
(s, 1H, NCH), 7.0–7.7 (m, 13H, Ar); M (m/z): 457 (molecular ion).
Compound 6u: 62% yield; mp 100–101 °C; time reaction 10 h; IR
14H, Ar); 13C NMR (CDCl3) d: 140.5 (C@O), 136 (C
a), 140.2 (Cb),
14 (CH3); 439 (molecular ion).
Compound 6i: 65% yield; mp 210–211 °C; reaction time 9 h; IR
(m
-cmꢁ1): 3140 (NH, amide II), 1654 (NCO, amide I), 2220 (Ar–
(m
-cmꢁ1): 1602 (NCO, amide I), 1245 (Ar–OH), 2500 (NH, amide II);
CN); 1H NMR (CDCl3) d: 7 (s, 1H, CH), 8.1(s, 1H, H5 pyrazole), 8.0
(s, 1H, H3 pyrazole), 7.2–7.9 (m, 15H, Ar. and NCH), 417 (molecular
ion).
1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 3.8 (s, 3H, CH3), 10 (s, 1H, OH),
10.5 (s, 1H, NH), 8.2 (s, 1H, NCH), 8.4 (s, 1H, H3 pyrazole), 7.0–7.8
(m, 14H, Ar, H5 pyrazole); M (m/z): 438 (molecular ion).
Compound 6j: 65% yield; mp 125–128 °C; reaction time 9 h; IR
Compound 6v: 70% yield; mp 198–201 °C; time reaction 10h; IR
(m
-cmꢁ1): 3050 (NH, amide II), 1600 (NCO, amide I), 1360 (N–C);
(m
-cmꢁ1): 1602 (NCO, amide I), 1681 (N@O), 1342 (NO2), 3299 (NH,
1H NMR (CDCl3) d: 3.0 (s, 6H, (CH3)2), 7 (s, 1H, CH), 8.2 (s, 1H,
NH), 8.0 (s, 1H, H5 pyrazole), 7.7 (s, 1H, H3 pyrazole), 7.2–7.6 (m,
15H, Ar. and NCH); 435 (molecular ion).
amide II); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 3.8 (s, 3H, CH3), 11 (s,
1H, NH), 7.4–7.8 (m, 16H, Ar, H3 and H5 pyrazole, NCH); M (m/z):
467 (molecular ion).
Compound 6k: 80% yield; mp 208–209 °C; reaction time 3 h; IR
Compound 6w: 68% yield; mp 153–156 °C; time reaction 10 h;
((m
-cmꢁ1): 3200 (NH, amide II), 1680 (NCO, amide I), 1500, 1330
IR (m
-cmꢁ1): 1644 (NCO, amide I), 3174 (NH, amide II); 1078 (Ar–
(NO2); 1H NMR (CDCl3) d: 6.7 (s, 1H, CH), 11.2 (s, 1H, NH), 8.4 (s,
Br); 1H NMR (CDCl3) d: 6.8 (s, 1H, CH), 3.8 (s, 3H, CH3), 8.8 (s, 1H,
NCH), 10.8 (s, 1H, NH), 8.2 (s, 1H, H3 pyrazole), 7.0–8.0 (m, 14H,
Ar and H5 pyrazole); M (m/z): 501 (molecular ion).
1H, H5 pyrazole), 7.0–8.0 (m, 16H, H3 pyrazole, Ar. and NCH); 13C
NMR (CDCl3) d: 163 (C@O), 136 (Ca), 140.3 (Cb); M (m/z): 437
(molecular ion).

M. A. F. Vera-DiVaio et al. / Bioorg. Med. Chem. 17 (2009) 295–302
301
Compound 6x: 49% yield; mp 166 °C; time reaction 10 h; IR (
m
-
force field available in Spartan’04. The resultant conformers were
fully geometry optimized using the AM1 semi-empirical Hamilto-
nian (Spartan’04). All the conformers of the non-substituted deriv-
ative (6a) were submitted to a single-point energy calculation
cmꢁ1): 1602 (NCO, amide I), 3000 (NH, amide II); 1326 (Ar–CF3);
1H NMR (dDMSO) d: 6.4 (s, 1H, CH), 8.2 (s, 1H, NCH), 11.2 (s, 1H,
NH), 8.0 (s, 1H, H3 pyrazole), 7.4–8.0 (m, 14H, Ar and H5 pyrazole),
3.0 (s, 3H, CH3), 3,4 (s, 3H, CH3); M (m/z): 503 (molecular ion).
Compound 6y: 68% yield; mp 187–200 °C; time reaction 10 h; IR
*
using the ab initio 6-31G basis set (Spartan’04).
In order to evaluate the electronic properties, the most stable
conformer of each derivative was submitted to a single-point en-
(m
-cmꢁ1): 1670 (NCO, amide I), 2958 (NH, amide II); 2225 (Ar–CN),
1112 (C–N), 1326 (Ar–CF3); 1H NMR (CDCl3) d: 7.4 (s, 1H, CH), 8.3
(s, 1H, NCH), 8.2 (s, 1H, H3 pyrazole), 8.0 (s, 1H, H5 pyrazole), 11.8
(s, 1H, NH), 7.4–7.9 (m, 13H, Ar); M (m/z): 485 (molecular ion).
ergy calculation using the ab initio 6-31G basis set (Spartan’04).
*
The molecular electrostatic potential (MEP) maps, the HOMO
(Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccu-
pied Molecular Orbital) energies and orbital coefficients, and the
GAP (ELUMO ꢁ EHOMO) energies were calculated in Spartan’04. The
Compound 6z: 70% yield; mp 90–91 °C; time reaction 10 h; IR (m-
cmꢁ1): 1500 (NCO, amide I), 2931 (NH, amide II), 1326 (Ar- CF3),
1068 (Ar–Br); 1H NMR (CDCl3) d: 7.6 (s, 1H, CH), 8.9 (s, 1H, NCH),
11.6 (s, 1H, NH), 8.1 (s, 1H, H3 pyrazole), 7.9 (s, 1H, H5 pyrazole),
7.4–7.7 (m, 13H, Ar); M (m/z): 539 (molecular ion).
MEP isoenergy contours were generated in
a range from
ꢁ45.0 kcal/mol (red) to +25.0 kcal/mol (blue) and superimposed
onto a surface of constant electron density of 0.002 e/au3. HOMO
and LUMO isosurfaces were calculated at 0.0032 au. The constants
employed in these isosurfaces calculations are default parameters
in the program used.
The lipophilic descriptor, cLogP (octanol–water partition coeffi-
cient), was predicted using the Ghose–Crippen default method
available in Spartan’04.22 The parameters for drug-likeness were
evaluated according to the Lipinski’s ‘rule-of-five’,20 using the Mol-
inspiration WebME Editor 1.16. [http://www.molinspiration.com].
The 4D-QSAR methodology was applied in this work as de-
scribed elsewhere19 using the 4D-QSAR program23 and grid cell
size of 1 Å. The models were developed using the 6a–z0 series,
excluding 6p, the inactive derivative. The intermediate 5 was not
included into the biological data set because it does not contain
the benzylidene-carbohydrazide moiety. The biological activity
values of the 26 compounds, reported as the minimum concentra-
Compound 6z0: 68% yield; mp 174–177 °C; time reaction 10 h; IR
(m
-cmꢁ1): 1668 (NCO, amide I), 1394 (NO2), 1336 (Ar–CF3), 2962
(NH, amide II); 1H NMR (CDCl3) d: 8.6 (s, 1H, NCH), 11.8 (s, 1H,
NH), 8.2(s, 1H, H3 pyrazole), 8.1 (s, 1H, H5 pyrazole), 7.2–8.0 (m,
14H, CH and Ar); M (m/z): 505 (molecular ion).
7.8. Pharmacology
The bioassays were carried out using infected Swiss albino mice
blood collected by cardiac puncture at the peak of parasitic infec-
tion (7th day of infection for Y strain of T. cruzi). The infected blood
was diluted with healthy mice blood to achieve a concentration of
2.106 forms/mL. Stock solutions of the compounds were prepared
by dissolving in dimethylsulfoxide (DMSO). The activity profile of
the pure derivatives was evaluated at 100, 250, and 500
The bioassays were performed in triplicate on titration microplates
(96 wells), which contained 400 L of mixture/well. The plates
were incubated at 4 °C and the number of parasites was counted
lg/mL.
tion (lg/mL) capable of inhibiting 50% (IC50) of the T. cruzi growing
l
in cultured cells, were converted into molar units, and then in the
corresponding negative logarithm values (pIC50). The larger the
pIC50 value, the more potent the compound. The three atoms of
the alignment step were C1–N1–N2 from the acyl-hydrazone
framework as suggested in a previous work.24
21
after 24 h as described by Brener
Infected blood with the same
volume of DMSO was used as positive control. The activity re-
sponse for each substance was expressed as the percentage of
reduction of the number of parasites (lysis) and the corresponding
IC50 values were calculated using the program GraphPad Prims
v.3.0. Crystal violet was used as the reference drug.
Acknowledgements
We are grateful to ‘Central Analítica do Núcleo de Pesquisas de
Produtos Naturais’ (NPPN, UFRJ, Brazil) for the Mass and NMR
spectra and ‘Instituto de Química’ (UFF, Brazil) for the IR spectra.
We thank to the Brazilian governmental agencies (FAPERJ, CAPES,
and CNPq) and UFF for the financial support of this research and
HCC fellowship. We thank to Prof. Dr. Anton J. Hopfinger who
kindly supplied the 4D-QSAR program for academic use.
7.8.1. Cytotoxicity assays
The assays were performed with cells of the peritoneal cavity of
male Balb/c mice (18–25 g) from FIOCRUZ (Fundação Oswaldo
Cruz—Brazil). Briefly, the cells were washed with 5 mL of sterile
RPMI and centrifuged for 10 min at 1500 rpm. The pellet was
resuspended in 1 mL of RPMI containing 10% fetal bovine serum
and antibiotics. An aliquot of 10 lL was diluted in Trypan blue
for counting living cells. After that 2 ꢂ 105 cells/well were distrib-
uted on a 96 wells plate and maintained in 5% of CO2 at 37 °C for
30 min. Then the cells were incubated with the derivatives
References and notes
1. World Health Organization (WHO). Infectious Diseases, Chagas disease
(American trypanosomisis), 2005. Available from: [http://www.who.int/tdr/
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