Design, synthesis and biological evaluation of hybrid bioisoster derivatives of N-acylhydrazone and furoxan groups with potential and selective anti-Trypanosoma cruzi activity

Article abstract of DOI:10.1016/j.ejmech.2014.05.077

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

Full text of DOI:10.1016/j.ejmech.2014.05.077

European Journal of Medicinal Chemistry 82 (2014) 418e425
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of hybrid bioisoster
derivatives of N-acylhydrazone and furoxan groups with potential and
selective anti-Trypanosoma cruzi activity
a
b
c
ꢀ
Ricardo Augusto Massarico Serafim , Jose Eduardo Gonçalves , Felipe Pereira de Souza ,
Ana Paula de Melo Loureiro c, Silvia Storpirtis b, Renata Krogh d,
Adriano Defini Andricopulo d, Luiz Carlos Dias e, Elizabeth Igne Ferreira a
,
*
a
ꢀ
ꢀ
~
LAPEN e Laboratorio de Planejamento e Síntese de Quimioterapicos Potencialmente Ativos contra Doenças Negligenciadas, Universidade de Sao Paulo
(USP), Faculdade de Ciencias Farmaceuticas, Departamento de Farmacia, Av. Prof. Lineu Prestes, 580, 05508-900 Sao Paulo, SP, Brazil
LPFCC e Laboratorio de Permeabilidade de Farmacos em Culturas Celulares, Universidade de Sao Paulo (USP), Faculdade de Ciencias Farmaceuticas,
^
^
ꢀ
~
b
ꢀ
ꢀ
~
^
^
ꢀ
~
Departamento de Farmacia, Sao Paulo, SP, Brazil
c
~
ꢀ
ꢀ
~
Universidade de Sao Paulo, Departamento de Analises Cl
LQMC e Laboratorio de Qu
sica de Sao Carlos, Sao Carlos, SP, Brazil
LQOS e Laboratorio de Qu
í
nicas e Toxicologicas, Sao Paulo, SP, Brazil
d
ꢀ
~
ꢀ
~
í
mica Medicinal e Computacional, Centro de Pesquisa e Inovaçao em Biodiversidade e Farmacos, Universidade de Sao Paulo
~
~
(USP), Instituto de F
í
e
ꢀ
^
ꢀ
í
mica Organica Sintetica, Universidade Estadual de Campinas (UNICAMP), Instituto de Qu
í
mica, Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the
objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO)
releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhy-
drazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms,
NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14)
both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the ar-
omatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were per-
formed for those most active compounds and both showed to be less cytotoxic than the reference drug,
benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability
and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a
possible candidate for additional studies.
Received 6 February 2014
Received in revised form
30 May 2014
Accepted 31 May 2014
Available online 3 June 2014
Keywords:
Anti-Trypanosoma cruzi compounds
Molecular hybrids
Bioisosters
Nitric oxide donor groups
N-acylhydrazone derivatives
Furoxan derivatives
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Despite the discovery of Chagas' disease occurred more than one
hundred years ago, chemotherapy still remains scarce and inef-
Chagas' disease, also named American trypanosomiasis, is an
anthropozoonosis common in Latin America and its transmission
occurs through the parasite Trypanosoma cruzi. It is one of the most
important neglected diseases, representing a serious public health
problem for the continent [1e3]. However, several cases have now
been reported in the United States and Canada, as well as in other
non-endemic countries such as Japan, Australia and Spain [3,4].
Currently, there are around 100 million people in 21 endemic
countries exposed to the infection, 8 million infected individuals
and beyond 41,000 new cases occur annually [2,3,5].
fective. Only two drugs have been used in its treatment: nifurtimox
and benznidazole, which present low rate of effectiveness in
chronic phase, as more than 80% of the patients treated at this stage
do not get parasitological cure. Thus it is very important to search
for new and better drugs for Chagas' disease chemotherapy [2,6,7].
Molecular modification represents a useful strategy in the
search for new biologically active compounds. By this process,
better pharmacokinetics and pharmacodynamics properties can be
achieved [8]. Molecular hybridization and bioisosterism [9,10] are
two of the most important molecular modification processes. They
have been widely used in the design of many therapeutic classes.
Furoxan, 1,2,5-oxadiazole N-oxide, has been a very important
scaffold in Medicinal Chemistry as nitric oxide (NO) donor. Many
furoxan derivatives have been reported in the literature as
* Corresponding author.
E-mail addresses: elizabeth.igne@gmail.com, hajudan@usp.br (E.I. Ferreira).
http://dx.doi.org/10.1016/j.ejmech.2014.05.077
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
419
Table 1
biologically interesting, especially as anti-T. cruzi compounds
Structure of synthesized compounds.
[11,12]. NO has been considered an important molecule with
cytotoxic and cytostatic activity to many parasites. Potential anti-
T. cruzi agents having NO releasing groups in its structure are
interesting alternative for obtaining new trypanomicide com-
pounds [11,13,14].
On the other hand, N-acylhydrazone group is considered a
privileged structure in Medicinal Chemistry, as it has a potential to
interact with different biological targets, including parasitic
cysteine proteases [15,16]. Cruzain is the main cysteine protease of
T. cruzi, being a specific target for designing new and selective
antichagasic agents [17,18].
Based on the foregoing considerations, we designed compounds
as depicted in Fig. 1, which are hybrids of N-acylhydrazone, po-
tential inhibitor of cruzain [15,16], and furoxan, a NO donor. This
dual mechanism of action would be important for achieving high
efficacy and selectivity, taken into account the N-acylhydrazone
part is an inhibitor of an enzyme found in the parasite but not in the
host. In addition, classic bioisosterism approach, especially through
retroisosterism, besides ring equivalents, and halides, would pro-
vide physicochemical properties changes, influencing not only re-
ceptor interaction but also pharmacokinetics profile.
For the aforementioned reasons, the objective of this study was
the design, synthesis, nitric oxide releasing study and biological
evaluation of fifteen (1e15) hybrid bioisoster compounds contain-
ing N-acylhydrazones and furoxan groups (Fig. 1). They have
different substituted and non-substituted aromatic rings, and
methyl or phenyl substituent on furoxan ring. This design allows
foreseeing the structureeactivity relationship (SAR) of the pro-
posed analogues.
Compounds
R1
R2
N-oxide position
1
2
3
1
1
1
4
5
6
1
1
1
7
8
1
1
Additionally, to evaluate the drug-like properties, permeability
in Caco-2 cells and cytotoxicity assay using tetrazolium salt (XTT)
were performed with the most active compounds.
2. Results and discussion
9
2
2
2
2.1. Chemistry
10
11
Fifteen (1e15) hybrid bioisoster derivatives from N-acylhy-
drazones and furoxan groups (Fig. 1 and Table 1) were synthesized
in 3 steps (Figs. 2 and 3): a) esterification of different carboxylic
acids to the correspondent methyl ester and further formation of
hydrazide (IVeVI); b) cyclization of crotonaldehyde (VIII) or cin-
namic alcohol. The latter was then oxidized to the respective
12
2
13
14
15
2
2
2
aldehyde (X), and c) formation of final compounds from Schiff's
bases (1e15). The yields of the last step were considered reason-
able, ranging from 6% to 91%. All final compounds were charac-
terized by melting point, TLC, IR, 1H NMR, 13C NMR, HRMS and
elemental analyses. The compound 2 was used as reference for
diastereoisomeric analysis by NOE-diff experiment. Due to the
appearance of signs 8.49 ppm and 7.92 ppm (spectrum in the
Supporting Information) it can be concluded that the compound is
present in its E-diastereoisomer.
Fig. 1. General scaffold of hybrid bioisoster derivatives with potential dual mechanism
of action.

420
R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
Fig. 2. Synthetic route for obtaining derivatives with methyl substituent on furoxan ring. Reaction conditions: a) CH3OH, H2SO4/Reflux; N2H4 80% aq/60 ꢁC; b) NaNO2(aq), AcOH/
14 ꢁC, 1 h; c) C2H5OH, Hþ/reflux. * Other hydrazides were purchased from commercial suppliers.
2.2. Biological activity (T. cruzi amastigote forms)
In the halogenated compounds derived from methyl (R2) furo-
xan, the activity (5, IC50 ¼ 14.3 M, and 4, IC50 ¼ 27.43 M) was
higher than that observed for compounds derived from phenyl (R2)
M). Considering only the
difference in R2, it would be possible to deduce that bulky groups,
as phenyl group, in this position are unfavorable to the activity.
However, for benzyl substituents in R1, (3, and 15), the opposite was
m
m
Trypanocidal activity is presented in Table 2. The IC50 values
ranged from 2.88 to >100
showed similar trypanocidal activities (IC50 ¼ 2.88 and 2.90
respectively), although lower than the reference drug, benznida-
zole (Bzd, IC50 ¼ 1.50 M). These compounds contain a nitro group,
mM. The most active compounds (6, 14)
furoxan (10 and 11 both with IC50 ꢀ 100
m
mM,
m
which is considered a parasitophoric group, in para position of the
aromatic ring. The electron attractive property of nitro group
probably has a positive influence on biological activity. Further-
more, this group is able to interfere on the redox system of the
parasite, thus having a synergic effect on trypanocidal activity [19].
The orientation of the nitro group showed to be important, as when
it is in meta position (7, 12) the activity decreases considerably
observed: IC50 were ꢀ100
mM and 9.1 mM, respectively. It would be
possible that the overall lipophilicity displayed an important role in
the activity, since log P (calculated by MarvinView software,
Ref. [20]), for compound 3 is 0.36 while it is 2.27 for compound 15.
Notwithstanding, log
P (calculated by MarvinView software,
Ref. [20]) for the most active compounds (6 and 14) are 0.2 and 2.11,
respectively. Those data lead to deduce that the different behavior
of the compounds with methyl and phenyl is more related to the
differences in interaction with cruzain, one possible target for the
designed derivatives, than to the lipophilicity itself.
(IC50 ¼ 9.94 and 15.30
mM, respectively). The only structural dif-
ference between those molecules is the substituent (methyl or
phenyl) in the furoxan ring.
Fig. 3. Synthetic route for obtaining derivatives with phenyl substituent on furoxan ring. Reactions conditions: a) CH3OH, H2SO4/Reflux; N2H4 80% aq/60 ꢁC; b) NaNO2(aq), AcOH/
70 ꢁC, 1 h; CH2Cl2/r.t., 5 h c) C2H5OH, Hþ/reflux. * Other hydrazides were purchased from commercial suppliers.

R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
421
Table 2
2.4. Cruzain inhibition assay
Trypanocidal activity (amastigotes in vitro) and nitric oxide releasing potential (ni-
trite anion generation).
Submitted to cruzain inhibition assay, the most active com-
Compounds
Trypanocidal activity
(IC50 e M)
Nitric oxide releasing
pounds (6, 14) showed IC50 11.2
0.2
mM and 15.1
1.6 mM,
m
(IC50
e mM)
respectively, indicating that cruzain could be considered the bio-
logical target of those compounds. It is well known that cruzain is a
cysteine protease that displays many important activities in T. cruzi,
as invasion in the host cells, nutrition, differentiation of the parasite
besides interference in host immunological system mechanisms
[17]. Since this enzyme is not found in the human host, it has been
considered a good and selective target for trypanocidal agents.
Benznidazole
1
2
3
4
5
6
7
1.50 0.35
6.70 0.95
>100
ND
1220.0
210.2
898.4
101.8
56.0
>100
27.43 4.97
14.3 2.54
2.88 0.38
9.94 2.06
5.69 0.89
8.12 1.18
>100
>100
15.30 2.80
>100
242.0
52.0
8
9
124.0
867.0
4232.0
601.0
912.0
132.0
110.0
33.4
2.5. Drug-like properties
10
11
12
13
14
15
For the most active compounds (6, 14) additional studies were
performed to evaluate cell permeability and cytotoxicity in human
cell (Table 3).
2.90 0.32
9.1 2.11
2.5.1. Caco-2 cell permeability screening
ND e Not determined.
Human intestinal absorption of new drug candidates is
frequently estimated by measuring the permeation of the drug
across a monolayer of Caco-2 cells. Caco-2 cells are a human
epithelial colon carcinoma cell line, which provides a suitable
in vitro model for the evaluation of intestinal absorption of drugs
[22]. As the integrity and permeability of monolayers can vary
between experiments, control compounds as metoprolol and
amoxicilin, which are known to have high and low permeability,
respectively, and fluorescein, a paracellular marker, were also used
in the same experiment. Permeability studies were conducted in
the directions of influx (apical to basolateral A-B) towards the efflux
(basolateral to apical B-A). Caco-2 permeation studies were per-
formed because the comparison of apparent permeability co-
efficients (Papp) in the absorptive and secretory directions provides
an indication of the possible involvement of active transport
mechanisms, either absorptive or secretory. The transporter
Glycoprotein-P (P-gp) can be responsible for secreting drugs in the
blood-to-lumen, and B-A direction can function as a barrier to oral
absorption [23].
Recently, Hernandez et al. [21] synthesized hybrid derivatives
containing furoxan and N-acylhydrazone, showing reasonable ac-
tivity against T. cruzi. In that work, six families (A to G) of com-
pounds were synthesized varying the general structure and the
substituents. Some of the compounds in the family G, which gen-
eral structure is that following (Fig. 4), correspond to the retro-
isosters of derivatives 1, 4, and 6, synthesized herein. All these
compounds but 4, the brominated derivative, showed to be more
active than their retroisosters [16]. Comparing compound 6, one of
the most active, with its retroisoster [16], the former is about 12
times more active against amastigotes than the later. Although
compound 14, with almost the same activity as 6, does not have the
correspondent retroisoster in Hernandez et al. work [21], a hy-
pothesis can be advanced in that the change in the position of the
groups (N-acylhydrazone and furoxan) may interfere with the
binding to the biological target, increasing the activity of the
derivatives.
Papp for compounds evaluated are presented in Table 3. Those
values showed that although the compound 6 is less lipophilic than
compound 14 it has high permeability according to the Biophar-
maceutical Classification System (SCB) [24]. This indicates that
compound 6 does not have problems related to absorption from the
gastrointestinal tract provided that it can adequately solubilize in
the physiological fluids of this system. On the other hand, for the
most lipophilic compound 14, the Papp indicates low permeability.
The net flux ratio lower than 2.0 (6 ¼ 0.87 and 14 ¼ 0.63), ob-
tained for the compounds, indicated there is no significant trans-
port in the efflux direction, possibly mediated by P-gp [23,25].
2.3. Nitric oxide releasing potential
In order to explore a possible mechanism of action of those
compounds, related to the potential NO releasing, the fifteen
compounds synthesized were submitted to the nitrite formation
test (by-product of the nitric oxide degradation). The value of
released nitrite ranged from 33.0 to 4232.0
with the standard (Spermine NONOate, IC50 211200.0
m
M (Table 2). Compared
M), all
m
compounds displayed lower ability to release NO. The compound
with the highest NO releasing potential (10) showed to be inactive
(IC50 ꢀ 100
tential (15) showed an intermediate trypanocidal activity
M). It seems there is no relationship between NO
mM), and the compound with the lowest releasing po-
2.5.2. Cytotoxicity assay
Cytotoxicity assays were carried out using human hepatocellu-
lar carcinoma cell line (HepG2). In this study the mitochondrial
(IC50 ¼ 9.1
m
releasing potential and trypanocidal activity. Likewise, there is no
direct relationship between the molecular structure and nitric ox-
ide releasing ability. Although NO releasing properties could pro-
vide a synergism of action, this might not be the main mechanism
of action of these compounds.
Table 3
Permeability and cytotoxicity in vitro assays of the most active compounds.
Compounds
Permeability screening
Cytotoxicity assay
(IC50 e mM)
Selectivity
indexb
(ꢂ10ꢃ6, cm/s, A-B/B-A)a
Benznidazole
6
14
ND
28.1
80.9
45.5
18.7
28.09
15.69
43.17 7.60/37.63 4.45
0.82 0.09/0.52 0.03
ND e Not determined.
a
A-B/BA ¼ apical to basolateral/basolateral to apical.
b
Fig. 4. General scaffold of compounds of Hernandez et al. [21].
SI ¼ Cytotoxicity assay IC50/Trypanocidal activity IC50
.

422
R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
respiratory chain activity, through XTT tetrazolium salt assay, was
evaluated.
4.1.1. General procedure for the synthesis of intermediates
hydrazides (Figs. 2 and 3; IVeVI)
The results of the cytotoxicity assay are depicted in Table 3. Both
First, the carboxylic acids (10 mmol) in anhydrous etanol was
refluxed with acid catalysis (0.5 mL H2SO4) until the total con-
sumption of the starting material (monitored by TLC). Then, the
reaction medium was cooled to room temperature, and the second
part of the reaction, in situ, was added 10 mL of hydrazide hydrate
(80%) and the system was kept under vigorous stirring at 60 ꢁC for
about 4 h. After total consumption of the ester (monitored by TLC),
the reaction medium was cooled (in ice bath) and the precipitate
formed was filtered and washed with water and etanol. The hy-
drazides IV and V were obtained with yields of 74% and 86%,
respectively (spectrum in the Supporting Information - DMSO-d6 at
250 MHz (1H)). Hydrazide VI was directly used for next step
without isolation.
molecules were less cytotoxic (6, IC50
¼
80.9
mM and 14,
IC50 ¼ 45.5 M) than Bzd (IC50 ¼ 28.1
m
mM). It is interesting to
observe that compound 6 is almost three-fold less cytotoxic than
Bzd. The selectivity index (SI) corresponds to the ratio between
cytotoxicity IC50 and trypanocidal activity IC50. The SI of 14 (15.69)
was so lower than that of Bzd (18.7). Notwithstanding, the SI of 6
(28.09) was higher than the reference, leading to the consideration
that this compound is an interesting hit and will be submitted to
additional pre-clinical studies.
3. Conclusion
Fifteen hybrid bioisoster compounds containing N-acylhy-
drazone and furoxan groups were designed, synthesized and
evaluated for their potential of NO releasing, trypanocidal activity.
The most active compounds (6, 14) were submitted to perme-
ability, cytotoxicity and cruzain inhibition tests. SAR indicates the
mode of interaction of the overall structures with the target is
interfering more than the R2 substituent. Due to the nature of the
general scaffold, a dual mechanism of action, NO releasing and
cruzain inhibition, was expected. Compounds 6 and 14 presented
lower trypanocidal activities compared with the reference drug
(Bzd). The potential of NO releasing seems to have no direct cor-
relation with the activity, but a synergic effect could be present.
Cruzain assay showed both compounds can be inhibitors, being
derivative 6 the most active. Those compounds showed to be less
cytotoxic in human cells than Bzd, but 6 showed to be the most
promising, as it presents good permeability in Caco-2 cells and SI
higher than Bzd.
4.1.2. General procedure for synthesis of N-acylhydrazones (Figs. 2
and 3; 1e15)
The hydrazides were obtained by synthesis (4.1.1) or purchased
commercially. A mixture of the aldehyde (1.1 mmol; Figs. 2 and 3)
with corresponding hydrazide (1 mmol; Figs. 2 and 3) and, using
acid catalysis (about 5 drops of HCl) in anhydrous ethanol (50 mL)
as solvent, the reaction was kept under agitation and reflux until
the total consumption of the starting materials. Then, after cooling,
the resulting precipitate was filtered and purified by washing with
cold ethanol and water.
4.1.2.1. (E)-4-((2-(Furan-2-carbonyl)hydrazono)methyl)-3-methyl-
1,2,5-oxadiazole 2-oxide (1). Pale yellow solid; Yield 77%; mp
199e200 ꢁC. IR (neat) 3433, 1668, 1625, 1589, 1465, 1282, 1265,
1168, 1135, 1020, 736 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d: 12.37
(1H; s), 8.45 (1H; s), 7.98 (1H; s), 7.35 (1H; d; J ¼ 5 Hz), 6.72 (1H; t;
J ¼ 5 Hz), 2.36 (3H; s) ppm. 13C NMR (125 MHz, DMSO-d6)
d: 154.2,
Therefore, compound 6 can be considered a lead in this design.
Docking studies are in progress with the objective of under-
standing the interaction with the enzyme and driving further
work on lead optimization mainly to improve its pharmacody-
namics properties.
154.0, 146.5, 145.9, 136.1, 116.2, 112.3, 111.6, 9.12 ppm. Calculated for
C9H8N4O4: C, 45.77; H, 3.41; N, 23.72. Found: C, 45.88; H, 3.42; N,
23.79. HRMS (ESI þ TOF-MS) calculated for C9H8N4O4 [M þ Hþ]:
237.0624. Found: 237.0651.
4.1.2.2. (E)-4-((2-Benzoylhydrazono)methyl)-3-methyl-1,2,5-
oxadiazole 2-oxide (2). White solid; Yield 86%; mp 207e209 ꢁC. IR
(neat) 3406, 1645, 1608, 1575, 1488, 1460, 1379, 1334, 1288, 1147,
4. Experimental section
4.1. Chemistry
1033, 941, 800, 738, 705 cmꢃ1 1H NMR (250 MHz, DMSO-d6)
. d:
12.36 (1H; s), 8.48 (1H; s), 7.91 (2H; d; J ¼ 5 Hz), 7.59 (3H; m), 2.39
The progress of all reactions was monitored by Analytical thin-
layer chromatography, which was performed on silica-gel 60 GF
(5e40 mM thickness) plates. Visualization was accomplished with
(3H, s) ppm. 13C NMR (62.5 MHz, DMSO-d6)
d
: 163.9, 154.0.136.0,
132.5, 132.2, 128.5, 127.7, 111.5, 9.0 ppm. Calculated for C11H10N4O3:
C, 53.66; H, 4.09; N, 22.75. Found: C, 53.83; H, 4.11; N, 22.78. HRMS
(ESIþTOF-MS) calculated for C11H10N4O3 [MþHþ]: 247.0831. Found:
247.0828.
UV light and phosphomolybdic acid followed by heating. Melting
points were measured with an electrothermal melting-point
apparatus (Büchi, M-565 model) in open capillary tubes and are
uncorrected. For Infrared spectra, wavelengths of maximum
absorbance (max) are quoted in wavenumbers (cmꢃ1). 1H and
proton decoupled 13C NMR spectra were scanned on Bruker NMR
spectrometer in DMSO-d6 at 250 MHz (1H) and 62.5 MHz (13C),
400 MHz (1H) and 100 MHz (13C) or at 125 MHz (13C). The chemical
4.1.2.3. 3-Methyl-4-((2-(2-phenylacetyl)hydrazono)methyl)-1,2,5-
oxadiazole 2-oxide (3). (E:Z proportion ¼ 66:34). White solid; Yield
82%; mp 101e104 ꢁC. IR (neat) 3170, 1687, 1614, 1463, 1373, 1265,
1159, 1031, 802, 738, 703 cmꢃ1 1H NMR (250 MHz, DMSO-d6)
. d:
12.12/11.91 (1H; s), 8.28/8.06 (1H; s), 7.31e7.26 (5H; m), 3.96/3.60
shifts (d) are reported in ppm. Elemental analyses (C, H and N) were
(2H; s), 2.32/2.31 (3H; s) ppm. 13C NMR (62.5 MHz, DMSO-d6)
d:
performed on a PerkineElmer model 2400 analyzer, and the data
were within 0.4% of the theoretical values. High-resolution mass
spectrometry (HRMS) was performed using electrospray ionization
(ESI). The parent ions ([MþNaþ] or [MþHþ]) are quoted. Reagents
and solvents were purchased from commercial suppliers and used
as received.
172.7,167.2,154.0,153.7,136.0,134.8,131.7,129.3,128.3,128.2,126.7,
126.5,111.6,111.3, 41.1, 38.4, 9.2, 9.0 ppm. Calculated for C12H12N4O3:
C, 55.38; H, 4.65; N, 21.53. Found: C, 55.59; H, 4.66; N, 21.61. HRMS
(ESIþ TOF-MS) calculated for
C
12H12N4O3 [MþHþ]: 261.0988.
Found: 261.0966.
The furoxan rings (1,2,5-oxadiazole N-oxide) intermediates
containing methyl and phenyl substituents (VIII and X, respec-
tively) were synthesized according to a previously described
methodology [26,27].
4.1.2.4. (E)-4-((2-(4-bromobenzoyl)hydrazono)methyl)-3-methyl-
1,2,5-oxadiazole 2-oxide (4). Pale yellow solid; Yield 90%; mp
187e189 ꢁC. IR (neat) 3433, 1685, 1606, 1456, 1263, 1145, 734 cmꢃ1
.
1H NMR (250 MHz, DMSO-d6)
d: 12.39 (1H; s), 8.45 (1H; s), 7.85

R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
423
(2H; d; J ¼ 5 Hz), 7.77 (2H; d; J ¼ 5 Hz), 2.37 (3H; s) ppm. 13C NMR
1299, 1265, 1153, 860, 736 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
12.29 (1H; s), 8.37 (1H; s), 7.94e7.59 (9H; m) ppm. 13C NMR
(125 MHz, DMSO-d6) : 161.9, 155.9, 137.1, 133.4, 131.2, 131.2, 129.6,
129.0,128.7, 125.6,112.8 ppm. Calculated for C16H11ClN4O3: C, 56.07;
H, 3.23; N, 16.35. Found: C, 56.28; H, 3.24; N, 16.41. HRMS (ESIþ
TOF-MS) calculated for C16H11ClN4O3 [MþHþ]: 343.0598. Found:
343.0583.
d:
(62.5 MHz, DMSO-d6) d: 162.3, 153.8, 136.2, 132.2, 132.0, 129.6,
126.1, 111.5, 8.9 ppm. Calculated for C11H9BrN4O3: C, 40.64; H, 2.79;
N, 17.23. Found: C, 40.76; H, 2.80; N, 17.28. HRMS (ESI þ TOF-MS)
calculated for C11H9N4O3Br [MþHþ]: 324.9936. Found: 324.9959.
d
4.1.2.5. (E)-4-((2-(4-Chlorobenzoyl)hydrazono)methyl)-3-methyl-
1,2,5-oxadiazole 2-oxide (5). Pale yellow solid; Yield 91%; mp
165e168 ꢁC. IR (neat) 3413, 1622, 1593, 1490, 1332, 1265, 1147, 1099,
4.1.2.11. (E)-3-((2-(4-Bromobenzoyl)hydrazono)methyl)-4-phenyl-
1,2,5-oxadiazole 2-oxide (11). White solid; Yield 57%; mp
206e208 ꢁC. IR (neat) 3429, 3236, 1662, 1564, 1546, 1452, 1425,
1039, 856, 740, 678 cmꢃ1
.
1H NMR (250 MHz, DMSO-d6)
d
: 12.41
(1H; s) 8.46 (1H; s), 7.95 (2H; d; J ¼ 7.5 Hz), 7.65 (2H; d; J ¼ 7.5 Hz),
2.38 (3H; s) ppm. 13C NMR (62.5 MHz, DMSO-d6)
: 162.3, 154.0,
137.2, 136.4, 131.2, 129.6, 128.7, 111.6, 9.1 ppm. Calculated for
d
1265, 1153, 736 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d
: 12.29 (1H; s),
8.36 (1H; s), 7.95e7.59 (9H; m) ppm. 13C NMR (100 MHz, DMSO-d6)
: 162.1, 156.0, 133.4, 131.6, 131.5, 131.3, 129.7, 129.0, 128.7, 126.1,
C
11H9ClN4O3: C, 47.07; H, 3.23; N, 19.96. Found: C, 47.25; H, 3.24; N,
d
19.95. HRMS (ESIþ TOF-MS) calculated for C11H9N4O3Cl [MþHþ]:
125.6, 112.8 ppm. Calculated for C16H11BrN4O3: C, 49.63; H, 2.86; N,
14.47. Found: C, 49.78; H, 2.87; N, 14.51. HRMS (ESIþ TOF-MS)
calculated for C16H11BrN4O3 [MþHþ]: 387.0093. Found: 387.0051.
281.0441. Found: 281.0460.
4.1.2.6. 3-Methyl-4-((2-(4-nitrobenzoyl)hydrazono)methyl)-1,2,5-
oxadiazole 2-oxide (6). (E:Z proportion ¼ 85:15). Yellow solid; Yield
72%; mp 99e102 ꢁC. IR (neat) 3423, 1654, 1602, 1541, 1352, 1325,
4.1.2.12. 3-((2-(3-Nitrobenzoyl)hydrazono)methyl)-4-phenyl-1,2,5-
oxadiazole 2-oxide (12). (E:Z proportion ¼ 80:20). White solid;
Yield 63%; mp 187e189 ꢁC. IR (neat) 3234, 3066, 1662, 1564, 1454,
1284, 1141, 1114, 1037, 709 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d:
12.62 (1H; s), 8.49 (1H; s), 8.40 (2H; d; J ¼ 7.5 Hz), 8.15 (2H; d;
1348, 1265, 738 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d
: 12.53/12.33
(1H; s), 8.74 (1H; s), 8.46e7.20 (9H; m) ppm. 13C NMR (125 MHz,
DMSO-d6) : 160.9, 156.0, 147.7, 134.2, 134.1, 133.8, 131.3, 130.4,
129.0, 128.7, 126.8, 125.6, 122.3, 112.8 ppm. Calculated for
J ¼ 7.5 Hz), 2.39 (3H, s) ppm. 13C NMR (62.5 MHz, DMSO-d6)
d:
163.2, 153.9, 136.9, 132.3, 132.2, 128.5, 127.5, 111.5, 8.9 ppm. Calcu-
lated for C11H9N5O5: C, 45.37; H, 3.11; N, 24.05. Found: C, 45.55; H,
3.51; N, 24.13. HRMS (ESIþTOF-MS) calculated for C11H9N5O5
[MþNaþ]: 314.0501. Found: 314.0513.
d
C
16H11N5O5: C, 54.39; H, 3.14; N, 19.82. Found: C, 54.61, H, 3.15; N,
19.89. HRMS (ESIþ TOF-MS) calculated for C16H11N5O5 [MþHþ]:
354.0838. Found: 354.0807.
4.1.2.7. 3-Methyl-4-((2-(3-nitrobenzoyl)hydrazono)methyl)-1,2,5-
oxadiazole 2-oxide (7). (E:Z proportion ¼ 81:19). White solid; Yield
67%; mp 94e96 ꢁC. IR (neat) 3446, 1656, 1625, 1533, 1355, 1328,
4.1.2.13. (E)-3-((2-Benzoylhydrazono)methyl)-4-phenyl-1,2,5-
oxadiazole 2-oxide (13). White solid; Yield 70%; mp 206e208 ꢁC. IR
(neat) 3442, 1652, 1596, 1564, 1357, 1282, 1155, 979, 736 cmꢃ1. 1H
1265, 1153, 1101, 1045, 736 cmꢃ1. 1H NMR (400 MHz, DMSO-d6)
d:
12.66/12.49 (1H; s), 8.75 (1H; s), 8.50e8.35 (3H; m), 7.87 (1H; t;
NMR (250 MHz, DMSO-d6)
d
: 12.25 (1H; s), 8.36 (1H; s), 7.96e7.58
: 163.0, 156.0, 133.0,
J ¼ 5 MHz), 2.39 (3H; s) ppm. 13C NMR (62.5 MHz, DMSO-d6)
d:
(10H; m) ppm. 13C NMR (125 MHz, DMSO-d6)
d
161.3, 153.9, 147.8, 137.2, 134.2, 133.8, 130.5, 126.9, 122.3, 111.6,
9.1 ppm. Calculated for C11H9N5O5: C, 45.37; H, 3.11; N, 24.05.
Found: C, 45.55; H, 3.12; N, 24.46. HRMS (ESIþ TOF-MS) calculated
for C11H9N5O5 [MþHþ]: 292.0682. Found: 292.0698.
132.5, 132.2, 131.2, 129.0, 128.7, 128.6, 127.7, 125.7, 112.8 ppm.
Calculated for C16H12N4O3: C, 62.33; H, 3.92; N, 18.17. Found: C,
62.55; H, 3.93; N, 18.21. HRMS (ESIþ TOF-MS) calculated for
C
16H12N4O3 [MþHþ]: 309.0988. Found: 309.0957.
4.1.2.8. (E)-3-Methyl-4-((2-(2-(phenylamino)benzoyl)hydrazono)
methyl)-1,2,5-oxadiazole 2-oxide (8). The last step reaction was
carried out in situ (without hydrazide purification). Yellow solid;
Yield 6%; mp 180e182 ꢁC. IR (neat) 3313, 1674, 1589, 1450, 1380,
4.1.2.14. (E)-3-((2-(4-Nitrobenzoyl)hydrazono)methyl)-4-phenyl-
1,2,5-oxadiazole 2-oxide (14). Yellow solid; Yield 70%; mp
194e196 ꢁC. IR (neat) 3238, 3076, 1666, 1602, 1556, 1517, 1473, 1454,
1427, 1299, 1265, 1157, 867, 738 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
1265, 1218, 1170, 736 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d
: 12.33
(1H; s), 9.17 (1H; s), 8.40 (1H; s), 7.89 (1H; s), 7.41e6.89 (8H; m),
2.36 (3H; s) ppm. 13C NMR (125 MHz, DMSO-d6)
: 165.2, 153.8,
d
: 12.46 (1H; s), 8.36e7.24 (10H; m) ppm. 13C NMR (125 MHz,
DMSO-d6) d: 162.0, 156.5, 150.0, 136.6, 134.7, 131.8, 129.7, 129.5,
d
129.2, 126.1, 124.2 ppm. Calculated for C16H11N5O5: C, 54.39; 3.14;
19.82. Found: C, 54.60; H, 3.15; N, 19.89. HRMS (ESIþ TOF-MS)
calculated for C16H11N5O5 [MþHþ]: 354.0838. Found: 354.0860.
144.8, 141.0, 135.8, 132.8, 129.1, 122.0, 119.7, 118.1, 116.8, 115.3, 111.4,
69.6, 8.9 ppm. Calculated for C17H15N5O3: C, 60.53; H, 4.48; N, 20.76.
Found: C, 60.76; H, 4.49; N, 20.82. HRMS (ESIþ TOF-MS) calculated
for C17H15N5O3 [MþHþ]: 338.1253. Found: 338.1264.
4.1.2.15. 4-Phenyl-3-((2-(2-phenylacetyl)hydrazono)methyl)-1,2,5-
oxadiazole 2-oxide (15). (E:Z proportion ¼ 79:21). Yellow solid;
Yield 17%; mp 178e181 ꢁC. IR (neat) 3423, 1683, 1627, 1593, 1450,
4.1.2.9. (E)-3-((2-(Furan-2-carbonyl)hydrazono)methyl)-4-phenyl-
1,2,5-oxadiazole 2-oxide (9). White solid; Yield 74%; mp
199e201 ꢁC. IR (neat) 3139, 2912,1668, 1606,1467,1407,1338,1205,
1429, 1265, 738 cmꢃ1. 1H NMR (250 MHz, DMSO-d6)
d
: 11.99/11.73
(1H; s), 8.16/8.13 (1H; s), 7.85e6.96 (10H; m), 3.64/3.53 (2H; s) ppm.
13C NMR (125 MHz, DMSO-d6)
: 172.5, 155.9, 146.9, 134.8, 131.0,
1155, 1027, 912, 761 cmꢃ1
.
1H NMR (250 MHz, DMSO-d6)
d
: 12.19
(1H; s), 8.33 (1H; s), 7.94e7.57 (6H; m), 7.25 (1H; s), 6.65 (1H; s)
ppm. 13C NMR (125 MHz, DMSO-d6)
: 156.0, 153.9, 146.5, 145.9,
d
129.2, 129.0, 129.0, 128.7, 128.6, 128.2, 128.1, 126.4, 113.0, 40.0,
36.1 ppm. Calculated for C17H14N4O3: C, 63.35; H, 4.38; N, 17.38.
Found: C, 63.59; H, 4.39; N, 17.43. HRMS (ESI þ TOF-MS) calculated
for C17H14N4O3 [MþHþ]: 323.1144. Found: 323.1136.
d
133.0, 131.2, 128.9, 125.7, 120.1, 115.9, 112.7, 112.2 ppm. Calculated
for C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. Found: C, 56.56; H, 3.39;
N, 18.84. HRMS (ESIþ TOF-MS) calculated for C14H10N4O4 [MþHþ]:
299.0780. Found: 299.0806.
4.2. In vitro assay against T. cruzi amastigotes
4.1.2.10. (E)-3-((2-(4-Chlorobenzoyl)hydrazono)methyl)-4-phenyl-
1,2,5-oxadiazole 2-oxide (10). White solid; Yield 62%; mp
205e207 ꢁC. IR (neat) 3240, 3068, 1662, 1562, 1546, 1454, 1425,
Rat skeletal muscle myoblasts (L6 cells) were seeded in 96-well
microtiter plates at 2000 (cells/well)/100 mL RPMI 1640 medium
with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. After

424
R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain C2C4 con-
taining the -galactosidase (Lac Z) gene) was added. After 48 h, the
chromatographer. Chromatographic separations (50 mL samples)
b
were performed using a Synergil C18 column (150 mm 4.6 mm,
4 mm, Phenomenex®) at 35 ꢁC, a phosphate buffer 20 mM (pH 3.5)
and acetonitrile (50:50 v/v) as the mobile phase, a 1 mL/min flux,
and a UV detector at 254 nm. The apparent permeability co-
efficients (Papp, cm/s) were calculated according to the following
equation:
medium was removed from the wells and replaced by 100 mL of
fresh medium with or without a serial drug dilution. Seven 3-fold
dilutions were used covering a range from 0.123 to 90 mg/mL.
The plates were incubated at 37 ꢁC in 5% CO2 for 4 days. Then the
substrate CPRG/Nonidet (50 mL) was added to all wells. The color
reaction that developed during the following 2e6 h was read
photometrically at 540 nm. From the sigmoidal inhibition curve,
IC50 values were calculated. Bzn was used as the standard drug.
Papp ¼ ðVR ꢂ DQ=DtÞ=
where VR is the volume of the receiver compartment (basolateral
or apical), DQ/Dt is the linear appearance rate of the compound on
4.3. Nitric oxide releasing potential
the receiver chamber (in ng/cm3/s), A is the membrane surface area
(cm2) and C0 is the initial concentration in the donor compartment
(ng/cm3). This calculation requires that the sink conditions are
fulfilled; therefore, only receiver concentrations below 10% of the
donor concentration were employed in the calculations.
The analysis was performed using the apparatus Sievers® Nitric
Oxide Analyser (NOA 280) by indirect method. Compounds were
diluted in DMSO (1 mg in 200 mL) and analyzed in medium with
oxygen. The final result is the quantifying (IC50) of anion nitrite
(NOꢃ2 ) generation, a major byproduct of nitric oxide degradation.
Spermine NONOate was used as the standard drug. For more
technical details see the Refs. [28and29].
4.6. Cytotoxicity assay
The human hepatocellular carcinoma cell line HepG2 was kindly
provided by Professor Terezinha de Jesus Andreoli Pinto (University
4.4. Cruzain inhibition assay
~
of Sao Paulo, Brazil). The monolayer culture was maintained in
Cruzain truncated in the C-terminal extension was obtained
from Escherichia coli (strain M15 or DH5a containing the expression
plasmid) [30]. The substrate Z-Phe-Arg-AMC and all reagents for
buffer preparation were purchased from SigmaeAldrich. Stock so-
lutions of substrate and inhibitors candidates at 10 mM in neat
DMSO were stored at ꢃ20 ꢁC and at ꢃ4 ꢁC, respectively.
DMEM supplemented with 10% FCS, 10 U/mL of penicillin, 0.1 mg/
mL of streptomycin sulfate, and 1.2 mg/mL of sodium bicarbonate.
Cells were incubated at 37 ꢁC in atmosphere containing 5% CO2.
All the assays were performed with cells plated out at a density
of 5 ꢂ 104 cells/well in 96-well culture plates 24 h prior to the
beginning of the treatment. The culture medium was then replaced
by new complete ones containing concentrations of 3, 12, 24, 48 or
The highly purified enzyme (0.64 nM), in 50 mM sodium
phosphate, 100 mM sodium chloride, 5 mM EDTA, pH 6.5, con-
taining 5 mM DTT, was incubated with the N-acylhydrazone de-
rivatives for 5 min at room temperature followed by the addition of
the fluorogenic substrate Z-Phe-Arg-AMC [31,32]. Fluorescence was
monitored on a Wallac 1420-042 PerkinElmer spectrofluorometer
and measurements were done using 355 nm as the excitation
wavelength and 460 nm as the emission wavelength. Cruzain ac-
tivity was measured as an increase of fluorescence intensity by
cleavage of the substrate Z-Phe-Arg-AMC and release of
aminocoumarin.
96 mM of each molecules dissolved in dimethylsulfoxide (DMSO)
(final concentration of DMSO in the culture medium 0.44% v/v).
Control cell incubations were done with complete culture medium.
Cell exposure occurred for the following 24 h.
To perform the Tetrazolium Salt Assay (XTT) a Xenometrix kit
was used. At the end of the treatment, cells were carefully washed
twice with 200
culture medium containing 5% of SFB (200
pre-formulated 50 XTT mixed
m
L of phosphate buffered saline (PBS), and a fresh
L/well) was added. A
reagent (XTT
m
mL
reagent:buffer ¼ 100:1) was then added to each well and the cul-
ture plate was incubated for 1 h at 37 ꢁC, 5% CO2. Light absorbance
values (OD ¼ OD 450 nmeOD 690 nm) were read at a test wave-
length of 450 nm and a reference wavelength of 690 nm. Optical
density readings were normalized to control cells (percentage of
the control values).
The IC50 values were calculated using nonlinear regression
analysis employing the Sigma-Plot enzyme kinetics module and it
was based on percentage of enzyme activity in the presence of
inhibitor, relatively to the activity in the absence of inhibitor.
4.5. Caco-2 cell permeability screening
Submission declaration
Caco-2 cells were cultured in 12-well TranswellW insert filters
for 21 days to reach confluence and differentiation at a seeding
density of 5 ꢂ 104 cells/cm2. The integrity of the monolayer was
examined by measuring the transepithelial electrical resistance
(TEER) with an epithelial voltammeter (Millicell-ERSW, Millipore
Corporation, Bedford, USA) and running standard assays using
fluorescein, metoprolol and amoxicillin as paracellular and trans-
cellular flux markers, respectively, and the results were compared
with literature data [33]. Only cell monolayers with a TEER above
The work has not been published and it is not under consider-
ation for publishing elsewhere.
Contribution of the authors
Ricardo Augusto Massarico Serafim e synthesis and analysis of
the compounds, text elaboration.
300U
ꢂ cm2 were used for the transport assays. The transport ex-
Jose Eduardo Gonçalves e permeability assays.
ꢀ
periments were initiated with the addition in the apical compart-
ments of the Transwell® plates of 10 mM compound in Hanks
balanced salt solution (HBSS) buffer at pHs 7.4, while the baso-
lateral buffer was set at pH 7.4.
Felipe Pereira de Souza e cytotoxicity assays.
Ana Paula de Melo Loureiro e cytotoxicity assays supervisor.
Silvia Storpirtis e permeability assays supervisor.
Renata Krogh e cruzain inhibition tests, T. cruzi tests.
Adriano Defini Andricopulo e supervisor of cruzain inhibition
tests and T. cruzi tests.
The plates were kept under stirring in an orbital shaker at 37 ꢁC
(25 rpm). Samples were collected from the basolateral and apical
side at 30, 60, 90, 120 and 180 min. Quantitative determinations of
compounds were performed by HPLC on a Shimadzu LC-10A
Luiz Carlos Dias e synthesis and analysis of the compounds
supervisor.

R.A. Massarico Serafim et al. / European Journal of Medicinal Chemistry 82 (2014) 418e425
425
complexesas potential drugs for Chagas's disease: enzyme inhibition and
in vitro/in vivo trypanocidal activity, Br. J. Pharmacol. 160 (2010) 260e269.
[14] L.R. Brunet, Nitric oxide in parasitic infections, Int. Immunopharmacol. 1
(2001) 1457e1467.
Elizabeth Igne Ferreira e general supervisor, PhD student R. A.
M. Serafim supervisor, overall manuscript review, correspondence
author.
[15] N.C. Romeiro, G. Aguirre, P. Hernandez, M. Gomzalez, H. Cerecetto, I. Aldana,
S. Perez-Silanes, A. Monge, E.J. Barreiro, L.M. Lima, Synthesis, trypanocidal
activity and docking studies of novel quinaxoline-N-acylhydrazones, designed
as cruzain inhibitors candidates, Bioorg. Med. Chem. 17 (2009) 641e652.
[16] D.R.M. Moreira, A.C.L. Leite, R.R. Santos, M.B.P. Soares, Approaches for the
development of new anti-Trypanosoma cruzi agents, Curr. Drugs Targets 10
(2009) 212e231.
Conflict of interest
All authors declare there is no conflict of interest at all.
[17] M.E. McGrath, A.E. Eakin, J.C. Engel, J.H. McKerrow, C.S. Craik, R.J. Fletterick,
The crystal structure of cruzain: a therapeutic target for Chagas' disease,
J. Mol. Biol. 247 (1995) 251e259.
[18] W.A.S. Judice, M.H.S. Cezari, A.P.C.A. Lima, J. Scharfstein, J.R. Chagas,
I.L.S. Tersariol, M.A. Juliano, L. Juliano, Comparison of the specificity, stability
and individual rate constants with respective activation parameters for the
peptidase activity of cruzipain and its recombinant form, cruzain, from Try-
panosoma cruzi, Eur. J. Biochem. 268 (2001) 6578e6586.
[19] S.R. Wilkinson, C. Bot, J.M. Kelly, B.S. Hall, Trypanocidal activity of nitro-
aromatic prodrugs: current treatments and future perspectives, Curr. Med.
Chem. 11 (2011) 2072e2084.
[20] MarvinView 5.4.0. e Free License, ChemAxon Ltd, 1998e2010.
[21] P. Hernandez, R. Rojas, R.H. Gilman, M. Sauvain, L.M. Lima, E.J. Barreiro,
M. Gonzalez, H. Cerecetto, Hybrid furoxanyl N-acylhydrazone derivatives as
hits for the development of neglected diseases drug candidates, Eur. J. Med.
Chem. 59 (2013) 64e74.
Acknowledgments
The authors thank CAPES for R.A.M. Serafim scholarship and
CNPq for E.I. Ferreira, L.C. Dias and A. D. Andricopulo fellowship,
and FAPESP, CNPq, and GlaxoSmithKline, for financial support. We
also thank Prof. Roberto Parise Filho (FCF/USP) for assistance in
preparing the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.077.
ꢀ
ꢀ
[22] S. Storpirtis, J.E. Gonçalves, C. Chiann, M.N. Gai, Biofarmacotecnica-serie
^
^
Ciencias Farmaceuticas, Guanab. Koogan (2009) 204e211.
[23] J. Souza, L.Z. Benet, Y. Huang, S. Storpirtis, Comparison of bidirectional
Lamavudine and Zidovudine transport using MDCK, MDCK-MDR1, and Caco-2
cell monolayers, J. Pharm. Sci. 98 (2009) 4413e4419.
References
[1] J.R. Coura, J.C.P. Dias, Epidemiology, control and surveillance of Chagas disease
e 100 years after its discovery, Mem. Inst. Oswaldo Cruz 104 (2009) 31e40.
[2] R.J. Anis, R. Anis, J.A. Marin-Neto, Chagas disease, Lancet 375 (2010)
1388e1402.
[3] World Health Organization, WHO (2013). http://www.who.int/mediacentre/
factsheets/fs340/en/index.html.
[24] H.V. Chavda, C.N. Patel, I.S. Anand, Biopharmaceutical classification system,
Syst. Rev. Pharm. 1 (2010) 62e69.
^
[25] P.C. Huber, C.H. Maruiama, W.P. Almeida, Glicoproteína-P, resistencia a múl-
~
tiplas drogas (MDR) e relaçao estrutura-atividade de moduladores, Quim.
Nova 33 (2010) 2148e2154.
[4] J. Gascon, C. Bern, M.J. Pinazo, Chagas disease in Spain, the United States and
other non-endemic countries, Acta Trop. 115 (2010) 22e27.
[5] M.C.P. Nunes, W. Dones, C.A. Montillo, J.J. Encina, A.L. Ribeiro, Chagas disease:
an overview of clinical and epidemiological aspects, J. A. Coll. Cardiol. 62
(2013) 767e776.
[26] R. Fruttero, B. Ferrarotti, A. Serafino, D. Stiloa, A. Gasco, Unsummetrically
substituted furoxans, Part 11, methylfuroxancarbaldehydes, J. Heterocycl.
Chem. 26 (1989) 1345e1347.
[27] A.M. Gasco, R. Fruttero, G. Sorba, A. Gasco, Unsymmetrically substituted
furoxans, Part XIII, phenylfuroxancarbaldehydes and related compounds,
Liebigs Ann. Chem. (1991) 1211e1213.
[6] L.C. Dias, M.A. Dessoy, J.N.S. Silva, O.H. Thiemann, G. Oliva, A.D. Andricopulo,
Quimioterapia da doença de Chagas: estado da arte
e perspectivas no
[28] R.S. Braman, S.A. Hendrix, Nanogram nitrite and nitrate determination in
enviromental and biological materials by vanadium(III) reduction with
chemiluminescence detection, Anal. Chem. 61 (1989) 2715e2718.
[29] Castegnaro, et al., Food Addit. Contam. 4 (1978) 37e43.
[30] A.E. Eakin, A.A. Mills, G. Harth, J.H. McKerrow, C.S. Craik, The sequence, or-
ganization, and expression of the major cysteine protease (cruzain) from
Trypanosoma cruzi, J. Biol. Chem. 267 (1992) 7411e7420.
ꢀ
desenvolvimento de novos farmacos, Quim. Nova 32 (2009) 2444e2457.
[7] R.M. Alves, R.P. Thomaz, E.A. Almeida, J.S. Wanderley, M.E. Guariento, Chagas
disease and egeing: the coexistence of other chronic disease with Chagas
disease in elderly patients, Rev. Soc. Bras. Med. Trop. 42 (2009) 622e628.
~
[8] E.J. Barreiro, C.A.M. Fraga, Química Medicinal: as bases moleculares da açao
ꢀ
dos farmacos, second ed., Artmed, Porto Alegre, 2008, pp. 271e277, 343e345.
[9] L.M. Lima, E.J. Barreiro, Bioisosterism: a useful strategy for molecular modi-
fication and drug design, Curr. Med. Chem. 12 (2005) 23e49.
[10] C. Viegas-Junior, A. Danuello, V.S. Bolzani, E.J. Barreiro, C.A.M. Fraga, Molecular
hybridization: a useful tool in the design of new drug prototypes, Curr. Med.
Chem. 14 (2007) 1829e1852.
[31] R. Li, G.L. Kenyon, F.E. Cohen, X. Chen, B. Gong, J.N. Dominguez, E. Davidson,
G. Kurzban, R.E. Miller, E.O. Nuzum, P.J. Rosenthal, J.H. McKerrow, In vitro
antimalarial activity of chacones and their derivatives, J. Med. Chem. 38
(1995) 5031e5037.
[32] D.M. Borchardt, A. Mascarello, L.D. Chiaradia, R.J. Nunes, G. Oliva, R.A. Yunes,
A.D. Andricopulo, Biochemical evaluation of a series of synthetic chalcone and
hydrazide derivatives as novel inhibitors of cruzain from Trypanosoma cruzi,
J. Braz. Chem. Soc. 21 (2010) 142e150.
[11] R.A.M. Serafim, M.C. Primi, G.H.G. Trossini, E.I. Ferreira, Nitric oxide: state of
the art in drug design, Curr. Med. Chem. 19 (2012) 385e405.
ꢀ
[12] H. Cerecetto, M. Gonzalez, Anti-T. cruzi agents: our experience in the evalu-
ation of more than five hundred compounds, Mini-Rev. Med. Chem. 8 (2008)
1355e1383.
[13] J.J.N. Silva, P.M.M. Guedes, A. Zottis, T.L. Balliano, F.O.N. Silva, L.G.F. Lopes,
J. Ellena, G. Oliva, A.D. Andricopulo, D.W. Franco, J.S. Silva, Novel ruthenium
[33] J.E. Gonçalves, M.B. Fernandes, C. Chiann, M.N. Gai, J. Souza, S. Storpirtis, Effect
of pH, mucin and bovine serum on rifampicin permeability through Caco-2
cells, Biopharm. Drug. Dispos. 33 (2012) 316e323.