Conformational restriction of aryl thiosemicarbazones produces potent and selective anti-Trypanosoma cruzi compounds which induce apoptotic parasite death

Article abstract of DOI:10.1016/j.ejmech.2014.02.001

Chagas disease, caused by Trypanosoma cruzi, is a life-threatening infection leading to approximately 12,000 deaths per year. T. cruzi is susceptible to thiosemicarbazones, making this class of compounds appealing for drug development. Previously, the hom

Full text of DOI:10.1016/j.ejmech.2014.02.001

European Journal of Medicinal Chemistry 75 (2014) 467e478
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Conformational restriction of aryl thiosemicarbazones produces
potent and selective anti-Trypanosoma cruzi compounds which induce
apoptotic parasite death
,
b
Diogo Rodrigo Magalhaes Moreira a , Ana Daura Travassos de Oliveira ,
*
Paulo André Teixeira de Moraes Gomes b, Carlos Alberto de Simone c, Filipe Silva Villela d,
Rafaela Salgado Ferreira d, Aline Caroline da Silva e, Thiago André Ramos dos Santos e,
Maria Carolina Accioly Brelaz de Castro e, Valéria Rego Alves Pereira e,
Ana Cristina Lima Leite b
,
*
a Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, CEP 40296-710 Salvador, BA, Brazil
b Universidade Federal de Pernambuco, Departamento de Ciências Farmacêuticas, CEP 50740-520 Recife, PE, Brazil
c Universidade de São Paulo, Instituto de Física de São Carlos, CEP 13560-970 São Carlos, SP, Brazil
d Universidade Federal de Minas Gerais, Departamento de Bioquímica e Imunologia, CEP 31270-901 Belo Horizonte, MG, Brazil
e Fundação Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhães, CEP 50670-420 Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas disease, caused by Trypanosoma cruzi, is a life-threatening infection leading to approximately
12,000 deaths per year. T. cruzi is susceptible to thiosemicarbazones, making this class of compounds
appealing for drug development. Previously, the homologation of aryl thiosemicarbazones resulted in an
increase in anti-T. cruzi activity in comparison to aryl thiosemicarbazones without a spacer group. Here,
we report the structural planning, synthesis and anti-T. cruzi evaluation of new aryl thiosemicarbazones
(9aex), designed as more conformationally restricted compounds. By varying substituents attached to
the phenyl ring, substituents were observed to retain, enhance or greatly increase the anti-T. cruzi ac-
tivity, in comparison to the nonsubstituted derivative. In most cases, hydrophobic and bulky substituents,
such as bromo, biphenyl and phenoxyl groups, greatly increased antiparasitic activity. Specifically,
thiosemicarbazones were identified that inhibit the epimastigote proliferation and were toxic for
trypomastigotes without affecting mouse splenocytes viability. The most potent anti-T. cruzi thio-
semicarbazones were evaluated against cruzain. However, inhibition of this enzyme was not observed,
suggesting that the compounds work through another mechanism. In addition, examination of T. cruzi
cell death showed that these thiosemicarbazones induce apoptosis. In conclusion, the structural design
executed within the series of aryl thiosemicarbazones (9aex) led to the identification of new potent anti-
T. cruzi agents, such as compounds (9h) and (9r), which greatly inhibited epimastigote proliferation, and
demonstrated a toxicity for trypomastigotes, but not for splenocytes. Mechanistically, these compounds
do not inhibit the cruzain, but induce T. cruzi cell death by an apoptotic process.
Received 18 November 2013
Received in revised form
30 January 2014
Accepted 2 February 2014
Available online 3 February 2014
Keywords:
Chagas disease
Trypanosoma cruzi
Thiosemicarbazones
Hydrazones
Conformational restriction
Apoptosis
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
phase, but is less effective in patients that progressed to the chronic
phase [2,3]. Furthermore, benznidazole is less than ideal due to the
Chagas disease, caused by the flagellate protozoan Trypanosoma
cruzi, affects approximately 10 million people in Latin America [1].
Currently, the only drug in use is nitroheterocyclic benznidazole
(LAFEPE, Brazil), which is effective in curing the disease in the acute
fact that it causes severe side effects, leading to treatment inter-
ruption in a large number of patients [4]. In fact, Chagas disease is
considered to be ‘the most concerning’ infectious tropical disease in
Latin America [5]. Thus, there is a need to identify new pharma-
ceuticals for its treatment.
To address this need, a number of molecular targets for anti-
T. cruzi drugs have been investigated, increasing the quality of drug
identification for Chagas disease treatment. Examples of such
* Corresponding authors.
E-mail
addresses:
diogollucio@gmail.com,
diogollucio@yahoo.com.br
(D.R. Magalhaes Moreira), ana.leite@pq.cnpq.br (A.C.L. Leite).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.02.001

468
D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
Fig. 1. Structure of thiosemicarbazones (1), (2) and thiazolidinones (3) and (4) previously identified as anti-T. cruzi agents [40e43]. Red lines highlight the spacer groups. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
targets are the lanosterol 14
a
-demethylase [6], trypanothione
antiparasitic activity as well low selectivity indexes than benzni-
dazole. The same strategy was employed in thiosemicarbazone
bioisosters, leading to the identification of potent anti-T. cruzi
thiazolidinones, exemplified by compounds (3) and (4) (Fig. 1)
[42,43].
Recent studies have demonstrated examples of the homologa-
tion strategy for drug design by using spacer groups with some
degree of conformational restriction (i.e., high rotational energy
barrier) [44e48]. The preparation of more conformationally
restricted N-acylhydrazones, as well as thiosemicarbazones, has
produced compounds with enhanced antiparasitic activity [49,50].
Based on these findings, we hypothesized that applying this strat-
egy would increase the anti-T. cruzi activity for the compound (1)
chemical class. Therefore, we selected the alkoxyl group displayed
in Fig. 2 as the spacer group for aryl thiosemicarbazone homolo-
gation, which would increase molecular rigidity, as well as rapidly
enable synthesis of multiple derivatives.
Here, we used the alkoxyl spacer group to prepare a new series
of aryl thiosemicarbazones (9aex) (Fig. 2). A range of substituents
were attached to the phenyl ring to examine their role in terms of
electronic and steric contribution to the anti-T. cruzi activity. First,
we prepared 4-substituted phenyl derivatives, followed by prepa-
ration of derivatives with one substituent at position 3- or 2- in the
phenyl ring. Derivatives containing two substituents attached to
the phenyl ring were synthesized, as well as the replacement of a
phenyl by a naphthyl ring. The substituents were selected based on
the Hammett Rho values as well as the hydrophobic parameter Pi,
therefore, enabling to examine the substituent effect for the aryl
reactivity. Evaluation of the anti-T. cruzi and cruzain activity for the
compounds (9aex) led to the identification of a number of struc-
tureeactivity relationships, which ultimately resulted to the iden-
tification of new compounds equally or more potent than
benznidazole.
reductase [7], cruzain [8], trans-sialidase [9] and phosphatidylino-
sitol 3-kinase [10]. By using structure-based drug design, two
small-molecules were recently developed and are now considered
strong drug candidates: K11777, a vinyl sulfone peptide that in-
hibits cruzain [11], and VNI, an oxadiazole derivative inhibitor of
14-a-demethylase activity [12,13]. These compounds are likely to
enter into clinical trials; however, the chances of any drug being
approved in the clinical stage are very low. Additionally, with the
goal of avoiding the development of resistant T. cruzi strains, there
is a necessity for combined drug therapy [14,15]. Therefore, the
number of potential drug candidates must be enhanced. Given the
outcomes observed for K11777 and VNI as anti-T. cruzi agents, the
design of compounds to inhibit cruzain and the 14-
activity have received special attention [16,17].
a-demethylase
Cruzain, the major trypanosomal cysteine protease, is involved
during the parasite invasion, differentiation and proliferation in
host cells [18]. Specifically, it is responsible for degrading host cell
proteins, and therefore contributing to the outcome of the infection
[19]. Regarding the identification of cruzain inhibitors, most of the
efforts have been conducted through the investigation of peptides
and peptide-like compounds, such as ureas [20,21], hydrazones
[22e25], triazoles [26,27] and thiosemicarbazones [28e30]. Thio-
semicarbazones were originally developed as potential cathepsin-L
inhibitors, one of the principal proteases involved in cancer cells
[28]. However, based on the homology and similar biochemical
properties between cathepsin-L and cruzain, thiosemicarbazones
were investigated as a potential class of cruzain inhibitors. Later on,
aryl thiosemicarbazones were found to be a class of anti-T. cruzi
compounds that inhibits cruzain activity [29,30].
The general binding mode for aryl thiosemicarbazones with
cruzain involves covalent binding of a thioamide group to Cys25
amino acid, as well as location of the aryl group within a deep
hydrophobic pocket [30,31]. Based on this model, a number of
molecular modifications have been investigated, including bis-thi-
osemicarbazones, heterocyclic-derived thiosemicarbazones, thio-
semicarbazones containing bioreductive groups and metallic
complexes [32e37]. This model suggests that aryl thio-
semicarbazones contain two different structural determinants and
therefore the presence of a spacer group (i.e., the homologation
strategy) [38,39] between the aryl group and thiosemicarbazone is
a feasible plan to enhance potency of the anti-T. cruzi and cruzain
activity.
2. Results
2.1. Synthesis
The synthetic procedures employed in aryl thiosemicarbazones
(9aex) preparation is shown in Scheme 1. First, the phenolic
compounds (5aex) reacted with 3-chloro-2-butanone (6) in basic
conditions at room temperature, similar to previously described
protocol [51]. After 12 h of reaction, b-ketoether (7aex) compounds
Our group has observed that the use of homologation strategy
produces thiosemicarbazones with enhanced anti-T. cruzi activity
in comparison to thiosemicarbazones without a spacer group.
Thiosemicarbazones (1) and (2) exhibited higher anti-T. cruzi ac-
tivity when compared to their respected analogs lacking a spacer
group [40,41]. As limitation, these compounds displayed lower
were obtained with yields varying from 66 to 99%. Compounds
(7aex) were then reacted with thiosemicarbazide (8) and catalytic
HCl in an ultrasound bath at room temperature [52]. After 2 h,
thiosemicarbazones (9aex) precipitated in the reaction mixture,
which were collected by simple filtration. All thiosemicarbazones
(9aex) were recrystallized and obtained at an acceptable purity

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469
Fig. 2. Conformational restriction of compound (1) leading to the design of (9a). Structure of the aryl groups employed during the aryl thiosemicarbazones (9aex). The red
highlights the spacer groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(>95%) in yields ranging from 36 to 98%. The structures were
determined by 1H and 13C NMR, DEPT, IR and high-resolution mass.
Two diastereoisomers can be produced from these thio-
semicarbazones (Fig. 3). However, when the 1H NMR spectra and
thin-layer chromatography were analyzed, only the formation of
one isomer was observed. These results are consistent with the
synthetic procedures, since the last reaction step was performed
under room temperature. To determine its configuration, single
crystals of the thiosemicarbazone (9a) were analyzed by X-ray
crystallography. As we can see from the Ortep-3 diagrams in Fig. 3,
the C(2) atom is antiperiplanar to N(2) atom, therefore the thio-
semicarbazone (9a) has an E-geometry in the solid state. The same
kind of assignment has been described in the literature [53]. In the
1H NMR spectra of all compounds (9aex), the chemical shift of the
methine proton C(2) appears at a narrow range, from 4.88 to
5.15 ppm. Based on these findings, it is reasonable to suggest that
thiosemicarbazones have an E-geometry. Due to the presence of
asymmetric C1 carbon, specific optical rotation was examined and
revealed that all analyzed thiosemicarbazones have small optical
rotation activity, suggesting that the enantiomeric excess in these
compounds is low.
2.2. Pharmacological evaluation
After structural characterization of aryl thiosemicarbazones
(9aex), the antiparasitic and host cell cytotoxicity was determined.
First, compounds were evaluated by their ability to inhibit the
epimastigote proliferation of T. cruzi Dm28 strain, as well as the
toxicity against trypomastigotes of Y strain. Results were respec-
tively expressed in terms of IC50 and CC50 values. Following this,
cytotoxicity was determined in splenocytes of BALB/c mouse and
results were expressed as the highest non-toxic concentration
(HNC) and given in
reference anti-T. cruzi drugs (Table 1). Benznidazole exhibited a
CC50 ¼ 6.2 M against trypomastigotes. The cutoff between active
mM. Benznidazole and nifurtimox were used as
m
and inactive compounds was determined using the CC50 ¼ 6.2
mM
against trypomastigotes, and were considered active anti-T. cruzi
agents [54].
Scheme 1. Synthesis of aryl thiosemicarbazones (9aex). Reagents and conditions: (A) K2CO3, KI, butanone, r.t., 12 h, yields from 66 to 99%. (B) HCl, EtOH, ultrasound bath for 2 h,
yields from 36 to 98%. Ac ¼ acetyl, iPr ¼ isopropyl, tBu ¼ tertbutyl.

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D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
Fig. 3. (A) Representations for the (9a) thiosemicarbazone diastereoisomers. (B) Ortep-3 diagrams with atom-labeling scheme of (9a) and its unit cell showing the formation of
centrosymmetric dimmers. Displacement ellipsoids are shown at the 50% probability level.
Regarding the activity against trypomastigotes, nonsubstituted
phenyl thiosemicarbazone (9a) was inactive. In comparison to (9a),
anti-T. cruzi activity was enhanced when an alkyl group was
attached to 4-position of the phenyl ring. The 4-ethylphenyl de-
rivative (9b) was 6-times more potent than (9a). When a 4-iso-
propyl (9c) or a 4-tertbutyl (9d) substituent is attached to the
phenyl ring, the resulting thiosemicarbazones were twice as potent
as benznidazole. The 4-methoxyphenyl derivative (9e) was inac-
tive, while its isomer (3-methoxyphenyl, 9f) was active. However,
the insertion of a 3-methoxyphenyl was not enough to produce a
compound more potent than benznidazole. In comparison to a
nonsubstituted phenyl thiosemicarbazone (9a), a 4-nitro group
(9g) enhanced the anti-T. cruzi activity. More remarkably, attaching
an acetamide group (9h) greatly increased the anti-T. cruzi activity.
In fact, the potency of (9h) against trypomastigotes was very similar
to that observed for nifurtimox and twice as potent as
benznidazole.
ring. In these conditions, 4-fluorophenyl (9i) and 4-chlorophenyl
(9j) derivatives were inactive. However, when the position of the
chloro atom was changed, the potency increased with the following
order: (2-Cl, 9l) > (3-Cl, 9k) > (4-Cl, 9j). Bromo atom attachment
greatly increases the activity, regardless of its position in the phenyl
ring. Likewise, attaching an iodo atom (9o) greatly increases the
activity in comparison to nonsubstituted phenyl thio-
semicarbazone (9a). Based on the results with bromo and iodo, we
evaluated compounds containing biphenyl groups. Placing
a
biphenyl greatly increased activity against trypomastigotes. When
we analyzed the CC50 values for compounds (40-phenyl, 9p), (30-
phenyl, 9q) and (20-phenyl, 9r), we observed that the position of the
biphenyl group did not affect potency. Interestingly, the 4-(phe-
noxy)phenyl derivative (9s) was very active against trypomasti-
gotes, exhibiting
a CC50 value in the same range of the
thiosemicarbazones containing biphenyl groups.
Next, we tested thiosemicarbazones containing a naphthyl, as
opposed to a phenyl ring. While the phenyl thiosemicarbazone (9a)
We evaluated the activity against trypomastigotes for thio-
semicarbazones containing halogen atoms attached to the phenyl
is inactive, the
a-naphthyl (9t) and the b-naphthyl (9u)

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471
Table 1
Anti-T. cruzi activity and host cell toxicity for thiosemicarbazones (9aex).
Compd.
Ar
T. cruzi
IC50
M) epimastigotesa
Cytotoxicity, HNC (m
M)c
(m
CC50 (m
M) trypomastigotesb
(9a)
(9b)
(9c)
(9d)
(9e)
(9f)
(9g)
(9h)
(9i)
C6H5
4-EtC6H4
26.1
11.6
5.9
4.6
27.6
6.8
176.9
71.5
86.4
13.5
45.8
10.3
20.1
5.8
4.9
4.5
2.2
2.2
ND
4.9
17.9
4.4
ND
27.3
4.6
1.5
1.4
26.8
6.2
5.9
1.4
23.2
22.8
5.5
1.5
3.9
4.5
1.1
1.3
1.3
1.1
1.3
1.5
5.6
4.4
3.5
11.9
6.2
2.7
210.6
18.8
17.9
17.0
93.5
4-iPrC6H4
4-tBuC6H4
4-MeOC6H4
3-MeOC6H4
4-NO2C6H4
4-(Ac)NHC6H4
4-FC6H4
4-ClC6H4
3-ClC6H4
2-ClC6H4
4-BrC6H4
3-BrC6H4
4-IC6H4
4-(C6H5)C6H4
3-(C6H5)C6H4
2-(C6H5)C6H4
4-(C6H5O)C6H4
37.4
88.5
>339.7 (350)d
>391.8
92.0
(9j)
(9k)
(9l)
36.8
36.8
31.6
31.6
13.7
15.9
31.9
(9m)
(9n)
(9o)
(9p)
(9q)
(9r)
(9s)
(9t)
(9u)
(9v)
(9w)
(9x)
Bdz
Nfx
31.9 (50)d
30.3
34.8
34.8
16.3
34.5
32.6
96.0
3.4
a
b
-Naphthyl
-Naphthyl
3,4-diClC6H3
3-Cl-4-FC6H3
2,3-diClC6H3
e
2.2
48.8
5.7
e
HNC ¼ highest non-cytotoxic concentration.
Bdz ¼ benznidazole.
Nfx ¼ nifurtimox.
ND ¼ not determined.
a
Determined 5 days after incubation with compounds, using Dm28c epimastigotes.
Determined 24 h after incubation with compounds, using Y strain trypomastigotes.
Cell viability of BALB/c mouse splenocytes determined 24 h after treatment.
In parenthesis, CC50 values against mouse splenocytes. IC50 ¼ inhibitory concentration for 50%. CC50 ¼ cytotoxic concentration for 50%. CC50 and IC50 values were calculated
b
c
d
using concentrations in triplicate and experiment was repeated, only values with a standard deviation < 10% mean were considered.
thiosemicarbazones were active. In addition to evaluating com-
pounds with a monosubstituted phenyl ring, we tested the thio-
semicarbazones containing two substituents attached to the phenyl
ring. While the 4-chlorophenyl (9j) was inactive, the 3,4-dichlor-
ophenyl derivative (9v) was active. The same was observed for the
3-chloro-4-fluorophenyl derivative (9w). However, attaching
chloro atoms at the positions 2- and 3- (9x) slightly decreased the
activity against trypomastigotes in comparison to the 3,4-dichlor-
ophenyl derivative (9v).
Having ascertained the antiparasitic activity for trypomasti-
gotes, we analyzed the antiproliferative activity against epi-
mastigotes. Excluding (9g) and (9h), most compounds which were
active against trypomastigotes were also able to inhibit epi-
mastigote proliferation. Regarding cytotoxicity in splenocytes,
some of the thiosemicarbazones that were active against T. cruzi
exhibited low cytotoxicity. For instance, compounds (9h), (9r), (9s)
Compounds (9a), (9h), (9k), (9m), (9n), (9o) and (9s) were screened
at 100 M, while compounds (9g), (9p) and (9t) were tested at
50 M, the maximum concentration at which they were soluble in
the assay buffer. However, cruzain inhibition by these compounds
was not observed (data not shown).
m
m
To understand the parasite death process caused by thio-
semicarbazones, untreated and treated trypomastigotes were
treated for 24 h of incubation and then double labeled with annexin
V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) [56].
For this assay, compounds (9h, 9r) and (9k) were selected because
they contain electron-donor and electron-withdrawing sub-
stituents, respectively, and assayed in concentrations equal to the
CC25 and CC100. The data were acquired and analyzed by flow
cytometry and results are shown in Fig. 4. In comparison to un-
treated parasites, treatment with thiosemicarbazones (9h, 9k and
9r) decreased parasite cell viability. Until 24 h of treatment with
compounds (9h) and (9k), only few parasite cells were stained
(data not shown) while in contrast, an increase in the percentage of
stained parasites was observed during thiosemicarbazone (9r)
treatment and this was concentration-dependent. Parasites treated
and (9t) had CC50 < 1.5
mM against trypomastigotes, while they
were non-toxic for splenocytes at concentrations up to 30
m
M.
Compounds (9c), (9d), (9o), (9p) and (9v) were the most cytotoxic
thiosemicarbazones for splenocytes, albeit less cytotoxic than
nifurtimox. We determined the selectivity index (CC50 splenocytes/
CC50 trypomastigotes) for compound (9h) and (9r). They displayed
indexes 250 and 45, respectively.
with compound (9r) at 1.1 mM presented 18.62% positively stained
cells, of which 7.8% were early apoptotic (annexin V), 6.2% were late
apoptotic (PI þ annexin V), while only 4.62% were necrotic (PI).
We investigated the inhibitory activity for thiosemicarbazones
(9aex) against cruzain. To this, we selected thiosemicarbazones
3. Discussion
with CC50 < 5.0 mM against trypomastigotes (i.e., derivatives 9g, 9h,
9k, 9m, 9n, 9o, 9p, 9s, 9t). For comparison reasons only, we
included the nonsubstituted phenyl derivative (9a). We measured
cruzain mediated enzymatic activity inhibition by using an assay
based on competition with the substrate Z-FR-AMC [55].
Currently, the only available treatment of Chagas disease is
benznidazole, therefore identification of new pharmaceuticals is
vital [4]. Aryl thiosemicarbazones were previously demonstrated to
act as strong anti-T. cruzi agents, targeting the trypanosomal

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D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
Fig. 4. (A) Thiosemicarbazone (9r)-based treatment causes parasite death through apoptosis induction. Trypomastigotes were treated with compound (9r) for 24 h. Parasites were
examined by flow cytometry with annexin V and PI staining. The percentage of cells in each quadrant represent the following: lower left, double negative; upper left, PI single
positive; lower right, annexin V single positive; upper right, PI and annexin V double positive. (B) Quantitative analysis of parasites positive only for annexin V. Values were taken
from six different readings of two independent experiments. (Cꢀ) ¼ negative control. *Compared to negative control (ANOVA one-way, p < 0.05).
protease cruzain [28e30]. It was recently discovered a sub-class of
these inhibitors, which contains a spacer group between aryl ring
and thiosemicarbazone, with an enhanced anti-T. cruzi activity in
comparison to aryl thiosemicarbazones without the spacer group.
Interestingly, the use of a spacer group not only enhanced the
thiosemicarbazone activity, but also thiazole and thiazolidinone
compound activity [40e43].
In the current study, we sought to use an alkoxyl spacer group to
design new aryl thiosemicarbazones. By using the alkoxyl spacer
group and varying a number of different substituents attached to
the phenyl ring, we were able to observe some structural de-
terminants involved in anti-T. cruzi activity. Specifically, sub-
stituents were observed to retain, enhance or greatly increase the
anti-T. cruzi activity in comparison to the nonsubstituted phenyl
thiosemicarbazone. For instance, the attachment of a 4-methoxyl,
4-fluoro or 4-chloro substituents to the phenyl ring led to de-
rivatives that retained activity in comparison to the nonsubstituted
thiosemicarbazone. However these compounds showed lower po-
tency than benznidazole.
Next, the effect of electron-donor or electron-withdrawing
properties of the substituents was examined here. In regard to
moderate electron-donor substituents, the 4-methoxyl derivative
was inactive, while the 4-phenoxyl and 4-acetamide were active.
Weak electron-donor substituents, such as alkyl and phenyl, also
produced active compounds. In regard to electron-withdrawing
substituents, strong deactivating substituents, such as the nitro
group, produced active compounds. However, bromo and iodo,
which are weak electron-withdrawing substituents, produced
active compounds and they were more potent than the nitro de-
rivative. This shows that the activity cannot be explained by the
substituent effect in terms of aryl reactivity. Considering that the
thiosemicarbazones containing halogens, biphenyl or phenoxyl
substituents exhibited the highest anti-T. cruzi activity, factors such
as hydrophobic properties, volume and polarisability (in the pres-
ence of halogens) could contribute to the observed activity [58]. An
exception to this general rule is the acetamide substituent, which
does not fit into these factors, but this substituent produced a
highly potent antiparasitic compound.
Next, we observed that bromo, biphenyl and phenoxyl at 4-
position are substituents that greatly increase the anti-T. cruzi ac-
tivity in comparison to the nonsubstituted phenyl thio-
semicarbazone. In agreement with activity observed for the
In general, the thiosemicarbazones were able to reduced epi-
mastigote proliferation with IC50 values similar to nifurtimox. In
addition, these compounds exhibited, in most cases, low cytotox-
icity for host cells in comparison to the anti-T. cruzi drugs, benz-
nidazole and nifurtimox. Specifically, compound (9h) and (9r)
displayed selectivity indexes comparable with some anti-T. cruzi
lead-compounds described in the literature [13,59,60]. Another
important comparison is that the antiparasitic activity and selec-
tivity observed for these molecules are higher than the thio-
biphenyl derivatives, the replacement of a phenyl group by a
a- or
b-naphthyl group also increased the activity. These results
demonstrate that hydrophobic substituents may be structural de-
terminants. However, we also observed that, while the derivative
containing an isopropyl, tertbutyl and iodo substituents greatly
increased the anti-T. cruzi activity, an unusual cytotoxicity for host
cells was observed.
semicarbazone previously investigated (1, CC50 ¼ 3.7
mM for
trypomastigotes and HNC < 1.0 M for splenocytes) [40e43].
m
An interesting structureeactivity relationship was observed by
comparing the 4-nitrophenyl and 4-acetamidephenyl derivatives.
Nitro is a well-known antiparasitic pharmacophoric group [57], but
here its attachment to the phenyl ring was not effective to produce
a compound as potent as benznidazole. On the other hand, the
insertion of an acetamide greatly increased the activity in com-
parison to the nonsubstituted phenyl thiosemicarbazone. In fact,
inserting an acetamide group produced one of the most potent
anti-T. cruzi thiosemicarbazones observed in this study.
Another set of interesting structureeactivity relationships were
observed by comparing the substituent position (i.e, ortho, meta,
para). It was evaluated one representative electron-donor substit-
uent, which was the biphenyl group, and one electron-withdrawing
substituent, the chloro. Variations in the position of the chloro atom
when attached to the phenyl group increased the anti-T. cruzi ac-
tivity. The position of biphenyl substituents within the phenyl ring
did not result in potency variations. This suggests the nature of the
substituent is important for the activity, but in same cases, sub-
stituent position along the phenyl ring is of less importance.
Therefore, indicating the structural design employed here for
compounds (9aex), which was based on the conformational re-
striction, was successful in producing compounds with enhanced
anti-trypanosomal activity. Regarding the mechanism of action,
most the literature points out that thiosemicarbazones inhibit the
major trypanosomal protease, cruzain. Moreover, few reports sug-
gest that this class of compounds also inhibits trypanothione
reductase [32]. In this study, the compounds did not exhibit cruzain
inhibition, thus, other mechanisms of action must be involved.
Regarding the mechanism of action of parasite cell death, we
observed thiosemicarbazone (9r) causes parasite death by an
apoptotic process, suggesting this compound has more effects on
cytoplasm and cell nucleus than affecting cell membrane.
4. Conclusions
Thiosemicarbazones were structurally designed by employing a
homologation strategy followed by conformational restriction. This
led to the synthesis and chemical characterization of compounds

D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
473
(9aex), which were evaluated as anti-T. cruzi and cruzain inhibitors.
The pharmacological evaluation led to the identification of thio-
semicarbazones (9h) and (9r) as potent and selective anti-T. cruzi
agents, as similarly observed in benznidazole-treated cells. Anti-
T. cruzi activity was observed to be dependent on the nature of the
employed substituent, but the substituent position in the phenyl
ring had less importance for activity. Mechanistically, these com-
pounds did not inhibit cruzain and were observed to induce para-
site cell death through an apoptotic process. The data argue that the
conformational restriction is a feasible strategy to modify the
structure of antiparasitic thiosemicarbazones.
with a sintered disc filter, washed with cold water, ethanol and then
dried over SiO2. The resulting powder was recrystallized in EtOH to
yield 9.7 g (89%) of colorless crystals.
5.3.1. 2-(3-Phenoxybutan-2-ylidene)thiosemicarbazide (9a)
Yield ¼ 89%. Mp. (ꢁC): 160e162. ½a 1D8
ꢂ
¼ ꢀ1.1 ꢃ1.1 (c 1.0, THF). IR
(KBr): 3390 (NH2), 3228 (NeH), 1598 (C]N) cmꢀ1
.
1H NMR
(400 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6 Hz, CH3), 1.82 (s, 3H, CH3),
4.97 (q, 1H, J 6 Hz, CHeO), 6.89 (d, 2H, J 6 Hz, ArH), 6.97 (d, 2H, J
6 Hz, ArH), 7.25 (t, 1H, J 8.1 Hz, ArH), 7.83 (s, 1H, NH2), 8.24 (s, 1H,
NH2), 10.18 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
d 11.7 (CH3),
5. Experimental section
19.5 (CH3), 76.7 (CHeO), 116.2 (Ar), 121.6 (CH, Ar), 130.1 (Ar), 152.5
(C]N), 157.9 (CeO), 179.8 (C]S). HRESIMS: 238.0575 [MþH]þ.
Anal. Calcd. for C11H15N3OS: C, 55.67; H, 6.37; N, 17.71. Found: C,
56.02; H, 6.60; N, 17.98. Crystallization from ethanol gave (9a) as
light white prisms suitable for X-ray analysis. Formula: C11H15N3OS.
5.1. General
Most the chemicals were purchased from SigmaeAldrich (St.
Louis, USA), Merck (Berlin, Germany) or Alfa-Aesar (Massachusetts,
USA). Reactions in ultrasound bath were performed in a Unique
EM-804 TGR instrument, with a frequency of 40 kHz and a nominal
power of 180 W, and without external heating. Precoated
aluminum sheets (silica gel 60 F254, Merck) were used for thin-
layer chromatography (TLC) and spots were visualized under UV
light. Elemental analysis was performed with a Carlo Erba instru-
ment model E-1110. IR spectra in KBr pellets were acquired at
Bruker FT-IR spectrophotometer. 1H and 13C NMR were recorded on
a UnityPlus 400 MHz and Bruker AMX-300 MHz spectrometer,
using DMSO-d6 as a solvent and trimethylsilane (TMS) as the in-
ternal standard. Splitting patterns were defined as; s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. Chemical shift values
were given in ppm. DEPT was employed to confirm the carbon
assignment. High-resolution electrospray ionization mass spectra
(HRESIMS) were acquired on a nanoUPLC-Xevo G2 Tof (Waters) in
the positive ionization mode. Optical rotations were recorded using
a JASCO Dichron model P-2000 polarimeter using tetrahydrofuran
as solvent. X-ray diffraction of compound (9a) was performed on an
ꢀ
Space group triclinic, P-1; Unit cell parameters: a ¼ 7.4240(4) A,
ꢀ
ꢀ
a
b ¼ 7.8120(3) A, c ¼ 11.2780(5) A;
¼ 101.737(3),
b
¼ 95.465(2),
g
¼ 105.084(3).
5.3.2. 2-(3-(4-Ethylphenoxy)butan-2-ylidene)thiosemicarbazide
(9b)
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 72%. M.p. (ꢁC): 87e89. ½a D18
¼ þ2.7 ꢃ1.3 (c 1.0, THF). IR (KBr):
ꢂ
3491 (NH2), 3192 (NH),1571 (C]N) cmꢀ1.1H NMR (400 MHz, DMSO-
d6): d 1.13 (t, 3H, J 7.6 Hz, CH3), 1.40 (d, 3H, J 6.4 Hz, CH3), 1.82 (s, 3H,
CH3), 2.51 (q, 2H, J 7.6 Hz, CH2), 4.92 (q,1H, J 6.4 Hz, CH), 6.88 (d, 2H, J
8.4 Hz, ArH), 7.07 (d, 2H, J 8.4 Hz, ArH), 7.82 (s, 1H, NH2), 8.23 (s, 1H,
NH2), 10.16 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d 10.9 (CH3),
15.7 (CH3),18.8 (CH3), 27.2 (CH2), 76.2 (CH),115.5 (CH, Ar),128.6 (CH,
Ar), 136.2 (Ar), 152.0 (CeO), 155.2 (C]N), 179.1 (C]S). HRESIMS:
266.1264 [M]þ. Anal. Calcd. for C13H19N3OS: C, 58.84; H, 7.22; N,
15.83. Found: C, 59.11; H, 6.70; N, 16.42.
Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on k-
goniostat) using graphite monochromated MoK
(0.71073 A), at room temperature.
a
radiation
5.3.3. 2-(3-(4-Isopropylphenoxy)butan-2-ylidene)
thiosemicarbazide (9c)
ꢀ
Recrystallization in ethanol afforded colorless crystals,
5.2. Synthesis of
phenoxybutan-2-one (7a)
b-ketoethers (7aex). Example for 3-
yield ¼ 55%. M.p. (ꢁC): 106e108. IR (KBr): 3492 (NH2), 3193 (NH),
1571 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.14 (d, 6H, J 6.4
Hz, CH3), 1.39 (d, 3H, J 6.4 Hz, CH3), 1.82 (s, 3H, CH3), 2.74e2.84 (m,
1H, CH), 4.91 (q, 1H, J 6.4 Hz, CHeO), 6.88 (d, 2H, J 8.8 Hz, ArH), 7.10
(d, 2H, J 8.8 Hz, ArH), 7.81 (s, 1H, NH2), 8.23 (s, 1H, NH2), 10.16 (s, 1H,
In a round bottom flask for 100 mL, phenol (5a, 5.0 g, 53 mmol)
was dissolved in 30 mL butanone and stirred at room temperature.
K2CO3 (11 g, 80 mmol) and KI (0.8 g, 5 mmol) were added to the
mixture, following by addition of 3-chloro-2-butanone (6, 5.6 g,
53 mmol). The reaction mixture was then maintained under stir-
ring for 12 h. After this time, the precipitate was removed under
filtration and rinsed with ethyl acetate, and the organic phase was
washed with water, and the with saturated KOH solution until the
complete removal of unreacted phenol. The combined organic
phases were dried over anhydrous Na2SO4, concentrated and then
dried under vacuum. The resulting product was used in the next
step without further purification.
NH). 13C NMR (100 MHz, DMSO-d6):
d 10.9 (CH3), 18.8 (CH3), 24.0
(CH3), 24.0 (CH3), 32.5 (CH), 76.1 (CHeO), 115.3 (CH, Ar), 127.1 (CH,
Ar), 140.7 (CH, Ar), 152.1 (CeO), 155.3 (C]N), 179.1 (C]S). HRE-
SIMS: 280.1516 [M]þ. Anal. Calcd. for C14H21N3OS: C, 60.18; H, 7.58;
N, 15.04. Found: C, 60.88; H, 6.54; N, 15.76.
5.3.4. 2-(3-(4-Tertbutylphenoxy)butan-2-ylidene)
thiosemicarbazide (9d)
Recrystallization in methanol afforded colorless crystals,
yield ¼ 71%. M.p. (ꢁC): 69e71. IR (KBr): 3408 (NH2), 3254 (NeH),
5.3. Synthesis of thiosemicarbazones (9aex). Example for
compound (9a)
1603 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6):
. d 1.23 (s, 9H,
CH3), 1.40 (d, 3H, J 6 Hz, CH3), 1.83 (s, 3H, CH3), 4.92 (q, 1H, J 6 Hz,
CH), 6.89 (d, 2H, J 9 Hz, ArH), 7.25 (d, 2H, J 9 Hz, ArH), 7.83 (s, 1H,
NH2), 8.25 (s, 1H, NH2), 10.17 (s, 1H, NH). 13C NMR and DEPT
In a round bottom flask for 50 mL, the b-ketoether (7a, 7.5 g,
46 mmol) was dissolved in 15 mL EtOH, following by the addition of
two drops HCl. The flask was placed in an ultrasound bath (40 kHz,
180 W) and under sonication, 4.2 g (46 mmol) of thiosemicarbazide
(8) was added in portions to the reaction. After 2 h, the mixture was
cooled at 0 ꢁC and the precipitate was filtered in a Büchner funnel
(75.5 MHz, DMSO-d6): d 16.4 (CH3), 24.3 (CH3), 36.7 (CH3), 38.5
(Cq), 81.5 (CHeO), 120.4 (CH, Ar), 131.5 (CH, Ar), 148.5 (C, Ar), 157.3
(CeO), 160.4 (C]N), 183.90 (C]S). HRESIMS: 293.9266 [M]þ. Anal.
Calcd. for C15H23N3OS: C, 61.40; H, 7.90; N,14.32. Found: C, 60.66; H,
6.88; N, 14.44.

474
D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
5.3.5. 2-(3-(4-Methoxyphenoxy)butan-2-ylidene)
thiosemicarbazide (9e)
HRESIMS: 256.0987 [MþH]þ. Anal. Calcd. for C11H14N3OSF: C, 51.75;
H, 5.53; N, 16.46. Found: C, 52.18; H, 5.09; N, 17.14.
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 92%. M.p. (ꢁC): 120e122. IR (KBr): 3393 (NH2), 3224 (NeH),
5.3.10. 2-(3-(4-Chlorophenoxy)butan-2-ylidene)thiosemicarbazide
(9j)
1600 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.38 (d, 3H, J 6.6
Hz, CH3), 1.82 (s, 3H, CH3), 3.68 (s, 3H, OCH3), 4.85 (q, 1H, J 6.6 Hz,
CHeO), 6.80 (d, 2H, J 9 Hz, ArH), 6.90 (d, 2H, J 9 Hz, ArH), 7.79 (s, 1H,
NH2), 8.23 (s, 1H, NH2), 10.16 (s, 1H, NH). 13C NMR (75.5 MHz,
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 59%. M.p. (ꢁC): 119e121. IR (KBr): 3400 and 3353 (NH2),
3246 (NeH), 1608 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6):
.
DMSO-d6):
d
10.9 (CH3), 18.8 (CH3), 55.2 (OCH3), 76.7 (CHeO), 114.4
d 1.42 (d, 3H, J 6.6 Hz, CH3), 1.82 (s, 3H, CH3), 4.96 (q, 1H, J 6.6 Hz,
(CH, Ar), 116.7 (CH, Ar), 151.0 (CeO), 152.1 (CeO), 153.5 (C]N), 179.1
(C]S). HRESIMS: 268.0632 [MþH]þ. Anal. Calcd. for C12H17N3O2S:
C, 53.91; H, 6.41; N, 15.72. Found: C, 54.50; H, 6.53; N, 15.95.
CHeO), 7.01 (d, 2H, J 6.6 Hz, ArH), 7.29 (d, 2H, J 6.6 Hz, ArH), 7.85 (s,
1H, NH2), 8.27 (s, 1H, NH2), 10.21 (s, 1H, NH). 13C NMR (75.5 MHz,
DMSO-d6):
d 11.0 (CH3), 18.6 (CH3), 76.5 (CHeO), 117.4 (CH, Ar),
124.6 (Ar), 129.2 (CH, Ar), 151.2 (C]N), 156.0 (CeO), 179.1 (C]S).
HRESIMS: 272.0127 [M]þ. Anal. Calcd. for C11H14N3OSCl: C, 48.61;
H, 5.19; N, 15.46. Found: C, 49.01; H, 5.12; N, 16.11.
5.3.6. 2-(3-(3-Methoxyphenoxy)butan-2-ylidene)
thiosemicarbazide (9f)
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 52%. M.p. (ꢁC): 110e112. IR (KBr): 3403 (NH2), 3255 (NeH),
5.3.11. 2-(3-(3-Chlorophenoxy)butan-2-ylidene)thiosemicarbazide
(9k)
1596 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d 1.40 (d, 3H, J 6.6
Hz, CH3), 1.82 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 4.94 (q, 1H, J 6.6 Hz,
CHeO), 6.48e6.56 (m, 3H, ArH), 7.14 (t,1H, J 7.8 Hz, ArH), 7.82 (s,1H,
NH2), 8.20 (s, 1H, NH2), 10.16 (s, 1H, NH). 13C NMR (100 MHz, DMSO-
Recrystallization in ethanol afforded brown crystals, yield ¼ 89%.
M.p. (ꢁC): 96e98. IR (KBr): 3323 (NH2), 3256 (NH),1603 (C]N) cmꢀ1
.
1H NMR (400 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6.6 Hz, CH3),1.83 (s, 3H,
d6):
d
10.9 (CH3), 18.7 (CH3), 54.9 (OCH3), 76.2 (CHeO), 101.6 (CH,
CH3), 5.00 (q, 1H, J 6.6 Hz, CHeO), 6.94e6.99 (m, 2H, Ar), 7.04e7.06
(m,1H, Ar), 7.27 (t,1H, J 8.1 Hz, Ar), 7.84 (s,1H, NH2), 8.23 (s,1H, NH2),
Ar), 106.6 (CH, Ar), 107.7 (CH, Ar), 129.9 (CH, Ar), 151.7 (C]N), 158.4
(CeO), 160.3 (CeO), 179.16 (C]S). HRESIMS: 268.1216 [MþH]þ.
Anal. Calcd. for C12H17N3O2S: C, 53.91; H, 6.41; N, 15.72. Found: C,
54.15; H, 5.96; N, 15.98.
10.18 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
d 11.0 (CH3), 18.5
(CH3), 76.6 (CHeO),114.5 (CH, Ar),115.7 (CH, Ar),120.9 (CH, Ar),130.8
(CH, Ar), 133.6 (CeCl), 150.9 (C]N), 158.1 (CeO), 179.2 (C]S).
HRESIMS: 272.0702 [MþH]þ. Anal. Calcd. for C11H14N3OSCl: C,
48.61; H, 5.19; N, 15.46. Found: C, 49.28; H, 5.13; N, 16.05.
5.3.7. 2-(3-(4-Nitrophenoxy)butan-2-ylidene)thiosemicarbazide
(9g)
Recrystallization in ethanol afforded bright yellowish crystals,
5.3.12. 2-(3-(2-Chlorophenoxy)butan-2-ylidene)thiosemicarbazide
(9l)
yield: 87%. M.p. (ꢁC): 160e162. IR (KBr): 3453 (NH2), 3291 (NeH),
1589 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d
1.47 (d, 3H, J 6.6
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 69%. M.p. (ꢁC): 139e141. IR (KBr): 3410 (NH2), 3240 (NH),
1595 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6): 1.46 (d, 3H, J 6.0
Hz, CH3), 1.85 (s, 3H, CH3), 5.06 (q, 1H, J 6.4 Hz, CHeO), 6.92e6.96
(m,1H, ArH), 7.21e7.26 (m, 2H, ArH), 7.41 (d, J 7.6 Hz,1H, ArH), 7.822
(s, 1H, NH2), 8.256 (s, 1H, NH2), 10.210 (s, 1H, NH). 13C NMR
Hz, CH3), 1.85 (s, 3H, CH3), 5.15 (q, 1H, J 6.6 Hz, CHeO), 7.20 (d, 2H, J
9.3 Hz, ArH), 7.82 (s, 1H, NH2), 8.16 (d, 2H, J 9.3 Hz, ArH), 8.26 (s, 1H,
NH2), 10.21 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
d 11.1 (CH3),
18.4 (CH3), 77.2 (CHeO), 116.0 (CH, Ar), 125.7 (CH, Ar), 141.0 (CeN,
Ar), 150.2 (C]N), 162.6 (CeO), 179.3 (C]S). HRESIMS: 283.0980
[MþH]þ. Anal. Calcd. for C11H14N4O3S: C, 46.80; H, 5.00; N, 19.85.
Found: C, 47.63; H, 4.88; N, 19.20.
(75.5 MHz, DMSO-d6):
d 10.9 (CH3), 18.5 (CH3), 77.3 (CHeO), 115.8
(CH, Ar), 121.9 (CH, Ar), 122.0 (Ar), 128.1 (CH, Ar), 130.0 (CH, Ar),
150.8 (CeO), 152.5 (C]N), 179.2 (C]S). HRESIMS: 272.0702
[MþH]þ. Anal. Calcd. for C11H14N3OSCl: C, 48.61; H, 5.19; N, 15.46.
Found: C, 48.58; H, 5.18; N, 15.78.
5.3.8. N-(4-(((3-(thiosemicarbazide)butan-2-ylidene)oxy)phenyl)
acetamide (9h)
Recrystallization in ethanol afforded pale yellow crystals,
yield ¼ 89%. M.p. (ꢁC): 177e179. ½a D18
ꢂ
¼ ꢀ2.4 ꢃ 5.2 (c 1.0, THF). IR
5.3.13. 2-(3-(4-Bromophenoxy)butan-2-ylidene)thiosemicarbazide
(9m)
(KBr): 3406 (NH2), 3270 (NeH), 1658 (C]N) cmꢀ1
.
1H NMR
(300 MHz, DMSO-d6):
d
1.39 (d, 3H, J 6.3 Hz, CH3), 1.82 (s, 3H, CH3),
Recrystallization in ethanol afforded yellowish crystals,
1.99 (s, 3H, CH3), 4.89 (q, 1H, J 6.3 Hz, CHeO), 6.90 (d, 2H, J 8.7 Hz,
ArH), 7.43 (d, 2H, J 8.7 Hz, ArH), 7.78 (s, 1H, NH2), 8.19 (s, 1H, NH2),
9.77 (s,1H, NHCO),10.11 (s,1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
yield ¼ 70%. M.p. (ꢁC): 141e143. IR (KBr): 3393 (NH2), 3224 (NeH),
1600 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6.6
Hz, CH3), 1.81 (s, 3H, CH3), 4.95 (q, 1H, J 6.6 Hz, CH), 6.95 (d, 1H, J
9 Hz, ArH), 7.40 (d, 1H, J 9 Hz, ArH), 7.83 (s, 1H, NH2), 8.26 (s, 1H,
d
11.0 (CH3), 18.4 (CH3), 77.1 (CHeO), 116.5 (CH, Ar), 117.7 (CH, Ar),
122.9 (Ar), 130.9 (CH, Ar), 131.55 (Ar), 150.5 (C]N), 156.7 (CeO),
NH2), 10.20 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d 11.0 (CH3),
179.3 (S]C). HRESIMS: 295.0880 [MþH]þ. Anal. Calcd. for
18.6 (CH3), 76.5 (CHeO), 112.44 (Ar), 117.9 (CH, Ar), 132.1 (CH, Ar),
151.2 (C]N), 156.5 (CeO), 179.1 (C]S). HRESIMS: 315.9171
[MþH]þ. Anal. Calcd. for C11H14N3OSBr: C, 41.78; H, 4.46; N, 13.29.
Found: C, 41.63; H, 4.52; N, 13.23.
C
13H18N4O2S: C, 53.04; H, 6.16; N, 19.03. Found: C, 53.29; H, 5.60; N,
19.20.
5.3.9. 2-(3-(4-Fluorophenoxy)butan-2-ylidene)thiosemicarbazide
(9i)
5.3.14. 2-(3-(3-Bromophenoxy)butan-2-ylidene)thiosemicarbazide
(9n)
Recrystallization in ethanol afforded colorless crystals,
yield ¼ 83%. M.p. (ꢁC): 148e150. IR (KBr): 3427 (NH2), 3265 (NH),
1605 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6): 1.40 (d, 3H, J 6.8
Hz, CH3),1.82 (s, 3H, CH3), 4.91 (q,1H, J 6.4 Hz, CHeO), 6.97e7.10 (m,
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 36%. M.p. (ꢁC): 121e123. IR (KBr): 3393 (NH2), 3224 (NeH),
1600 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6.3
4H, ArH), 7.81 (s, 1H, NH2), 8.24 (s, 1H, NH2), 10.17 (s, 1H, NH). 13
C
Hz, CH3), 1.82 (s, 3H, CH3), 5.00 (q, 1H, J 6.3 Hz, CHeO), 7.00 (d, 1H, J
8.4 Hz, ArH), 7.11 (d, 1H, J 8.4 Hz, ArH), 7.18 (s, 1H, ArH), 7.21 (t, 1H, J
NMR (100 MHz, DMSO-d6):
d 10.9 (CH3), 18.7 (CH3), 76.8 (CHeO),
115.6 (CH, Ar), 115.9 (CH, Ar), 116.9 (CH, Ar), 117.0 (CH, Ar), 151.5 (Ce
O), 153.5 (C]N), 155.4 (CeF, Ar), 157.8 (CeF, Ar), 179.1 (C]S).
8.1 Hz, ArH), 7.86 (s,1H, NH2), 8.27 (s,1H, NH2),10.22 (s,1H, NH). 13
C
NMR (75.5 MHz, DMSO-d6):
d 11.0 (CH3), 18.5 (CH3), 76.6 (CHeO),

D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
475
114.9 (CH, Ar), 118.6 (CH, Ar), 121.9 (Ar), 123.9 (CH, Ar), 131.2 (CH,
5.3.19. 2-(3-(4-Phenoxyphenoxy)butan-2-ylidene)
thiosemicarbazide (9s)
Ar), 150.9 (C]N), 158.2 (CeO), 179.2 (C]S). HRESIMS: 315.962
[MþH]þ. Anal. Calcd. for C11H14N3OSBr: C, 41.78; H, 4.46; N, 13.29.
Found: C, 41.85; H, 4.12; N, 13.39.
Recrystallization in ethanol afforded yellow crystals,
yield ¼ 71%. M.p. (ꢁC): 111e112. IR (KBr): 3423 (NH2), 3238 (NeH),
1593 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6):
. d 1.42 (d, 3H, J
6 Hz, CH3), 1.86 (s, 3H, CH3), 4.93 (q, 1H, J 6 Hz, CHeO), 6.90e6.95
(m, 4H, ArH), 7.00e7.05 (m, 3H, ArH), 7.32e7.38 (m, 2H, ArH), 7.81
(s, 1H, NH2), 8.26 (s, 1H, NH2), 10.20 (s, 1H, NH). 13C NMR (75.5 MHz,
5.3.15. 2-(3-(4-Iodophenoxy)butan-2-ylidene)thiosemicarbazide
(9o)
Recrystallization in ethanol afforded yellowish crystals,
DMSO-d6): d 11.8 (CH3),19.5 (CH3), 77.3 (CHeO),117.6 (CH, Ar),118.2
yield ¼ 83%. M.p. (ꢁC): 154e156. IR (KBr): 3347 (NH2), 3243 (NH),
(CH, Ar), 121.0 (CH, Ar), 123.4 (CH, Ar), 130.6 (CH, Ar), 133.6 (CH, Ar),
150.5 (CeO, Ar), 152.5 (C]N), 154.2 (CeO, Ar), 158.3 (CeO), 179.8
(C]S). HRESIMS: 330.0697 [MþH]þ. Anal. Calcd. for C17H19N3O2S:
C, 61.98; H, 5.81; N, 12.76. Found: C, 61.83; H, 5.90; N, 12.95.
1579 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6.4
Hz, CH3), 1.81 (s, 3H, CH3), 4.94 (q, 1H, J 6.4 Hz, CHeO), 6.83 (d, 2H, J
8.4 Hz, CH, ArH), 7.55 (d, 2H, J 8.4 Hz, 2CH, ArH), 7.81 (s, 1H, NH2),
8.24 (s, 1H, NH2), 10.18 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d
11.0 (CH3), 18.6 (CH3), 76.3 (CHeO), 83.8 (Ar), 118.4 (CH, Ar), 137.9
5.3.20. 2-(3-(Naphthalen-1-yloxy)butan-2-ylidene)
thiosemicarbazide (9t)
(CH, Ar), 151.2 (C]N), 157.1 (CeO), 179.1 (C]S). HRESIMS: 364.0118
[M]þ. Anal. Calcd. for C11H14N3OSI: C, 36.37; H, 3.89; N,11.57. Found:
C, 36.45; H, 3.65; N, 11.65.
Recrystallization in ethanol afforded white crystals,
yield ¼ 98%. M.p. (ꢁC): 158e160. IR (KBr): 3408 (NH2), 3228 (NeH),
1597 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.56 (d, 3H, J 6.8
5.3.16. 2-(3-([1,10-Biphenyl]-4-yloxy)butan-2-ylidene)
thiosemicarbazide (9p)
Hz, CH3), 1.86 (s, 3H, CH3), 5.18 (q, 1H, J 6.4 Hz, CHeO), 7.05 (d, 1H, J
8 Hz, ArH), 7.36 (t, 1H, J 7.6 Hz, ArH), 7.45e7.53 (m, 3H, ArH), 7.84e
7.86 (m, 1H, ArH), 7.88 (s, 1H, NH2), 8.19e8.22 (m, 1H, ArH), 8.26 (s,
Recrystallization in ethanol afforded white crystals,
yield ¼ 91%. M.p. (ꢁC): 164e166. IR (KBr): 3404 (NH2), 3236 (NeH),
1H, NH2), 10.20 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d 10.4
1592 (C]N) cmꢀ1
.
1H NMR (400 MHz, DMSO-d6):
d
1.46 (d, 3H, J
(CH3), 18.3 (CH3), 76.0 (CHeO), 106.6 (CH, Ar), 119.8 (CH, Ar), 121.0
(CH, Ar),124.7 (CH, Ar), 124.9 (CH, Ar), 125.6 (CH, Ar), 125.9 (CH, Ar),
127.0 (CH, Ar), 133.6 (CH, Ar), 151.3 (C]N), 152.0 (CeO, Ar), 179.7
(C]S). HRESIMS: 288.1283 [MþH]þ. Anal. Calcd. for C15H17N3OS: C,
62.69; H, 5.96; N, 14.62. Found: C, 62.63; H, 6.07; N, 14.92.
6 Hz, CH3), 1.87 (s, 3H, CH3), 5.04 (q, 1H, J 6 Hz, CHeO), 7.09 (d, 2H, J
9 Hz, ArH), 7.20e7.47 (m, 1H, ArH), 7.49e7.73 (m, 2H, ArH), 7.78e
7.99 (m, 4H, ArH), 7.89 (s, 1H, NH2), 8.30 (s, 1H, NH2), 10.24 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6):
d 11.7 (CH3), 19.5 (CH3), 81.7
(CHeO), 116.7 (CH, Ar), 126.9 (CH, Ar), 128.4 (CH, Ar), 129.1 (CH, Ar),
129.5 (CH, Ar), 133.6 (CH, Ar), 140.4 (C, Ar), 152.4 (C]N), 157.6 (Ce
O), 179.8 (C]S). HRESIMS: 314.1336 [MþH]þ. Anal. Calcd. for
5.3.21. 2-(3-(Naphthalen-2-yloxy)butan-2-ylidene)
thiosemicarbazide (9u)
C
17H19N3OS: C, 65.15; H, 6.11; N, 13.41. Found: C, 64.93; H, 5.77; N,
Recrystallization in ethanol afforded white crystals,
13.70.
yield ¼ 89%. M.p. (ꢁC): 150e152. IR (KBr): 3341 (NH2), 3270 (NeH),
1614 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d 1.49 (d, 3H, J 6.4
Hz, CH3), 1.85 (s, 3H, CH3), 5.13 (q, 1H, J 6.3 Hz, CHeO), 7.19 (d, 1H, J
9.2 Hz, ArH), 7.19 (d, 1H, J 9.2 Hz, ArH), 7.33 (t, 1H, J 7.6 Hz, ArH),
7.41e7.46 (m, 2H, ArH), 7.74 (d, 1H, J 8.4 Hz, ArH), 7.80 (d, 1H, J
5.3.17. 2-(3-([1,10-Biphenyl]-3-yloxy)butan-2-ylidene)
thiosemicarbazide (9q)
Recrystallization in ethanol afforded white crystals,
8.8 Hz, ArH), 7.98 (s,1H, NH2), 8.27 (s,1H, NH2),10.20 (s,1H, NH). 13
C
yield ¼ 79%. M.p. (ꢁC): 138e140. IR (KBr): 3437 (NH2), 3324 (NeH),
NMR (100 MHz, DMSO-d6):
d 10.9 (CH3), 18.6 (CH3), 76.2 (CHeO),
1586 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d 1.45 (d, 3H, J 8.8
108.9 (CH, Ar), 118.9 (CH, Ar), 123.7 (CH, Ar), 126.3 (CH, Ar), 126.7
(CH, Ar), 127.4 (CH, Ar), 128.5 (CH, Ar), 129.3 (CH, Ar), 134.0 (CH, Ar),
151.6 (C]N), 154.9 (CeO, Ar), 179.2 (C]S). HRESIMS: 288.1283
[MþH]þ. Anal. Calcd. for C15H17N3OS: C, 62.69; H, 5.96; N, 14.62.
Found: C, 62.78; H, 6.01; N, 14.97.
Hz, CH3), 1.85 (s, 3H, CH3), 5.09 (q, 1H, J 8.8 Hz, CHeO), 6.96 (d, 1H,
ArH), 7.20e7.26 (m, 2H, ArH), 7.31e7.37 (m, 1H, ArH), 7.45 (t, 2H,
ArH), 7.62 (d, 2H, ArH), 7.90 (s, 1H, NH2), 8.26 (s, 1H, NH2), 10.20 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6):
d 11.0 (CH3), 18.8 (CH3),
76.4 (CHeO), 113.9 (CH, Ar), 114.8 (CH, Ar), 119.4 (CH, Ar), 126.7 (CH,
Ar), 127.6 (CH, Ar), 128.9 (CH, Ar), 130.0 (CH, Ar), 139.8 (C, Ar), 141.6
(C, Ar), 151.7 (C]N), 157.8 (CeO), 179.3 (C]S). HRESIMS: 314.1422
[MþH]þ. Anal. Calcd. for C17H19N3OS: C, 65.15; H, 6.11; N, 13.41.
Found: C, 65.17; H, 6.11; N, 13.86.
5.3.22. 2-(3-(3,4-Dichlorophenoxy)butan-2-ylidene)
thiosemicarbazide (9v)
Recrystallization in ethanol afforded white crystals,
yield ¼ 74%. M.p. (ꢁC): 154e156. IR (KBr): 3420 (NH2), 3259 (NeH),
1593 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.42 (d, J 6.6 Hz,
5.3.18. 2-(3-([1,10-Biphenyl]-2-yloxy)butan-2-ylidene)
thiosemicarbazide (9r)
3H, CH3), 1.82 (s, 3H, CH3), 5.00 (q, 1H, J 6.6 Hz, CHeO), 7.00 (dd, 1H,
J 9.0 Hz, 4J 3.0 Hz, ArH), 7.27 (d, 1H, 4J 3.0 Hz, ArH), 7.47 (d, 1H, J
9.0 Hz, ArH), 7.85 (broad s, 1H, NH2), 8.24 (broad s, 1H, NH2), 10.19
Recrystallization in ethanol afforded white crystals,
yield ¼ 83%. M.p. (ꢁC): 179e181. IR (KBr): 3416 (NH2), 3226 (NeH),
(s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
d 11.0 (CH3), 18.4 (CH3),
1596 (C]N) cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d
1.32 (d, 3H, J 6.0
77.1 (CHeO), 116.5 (CH, Ar), 117.7 (CH, Ar), 122.9 (CeCl, Ar), 130.9
(CH, Ar), 131.5 (Ar), 150.5 (C]N), 156.7 (CeO), 179.3 (C]S). HRE-
SIMS: 306.0320 [MþH]þ. Anal. Calcd. for C11H13N3OSCl2: C, 43.15;
H, 4.28; N, 13.72. Found: C, 43.39; H, 4.09; N, 13.69.
Hz, CH3), 1.76 (s, 3H, CH3), 4.97 (q, 1H, J 6.4 Hz, CHeO), 7.02 (t, 1H, J
7.6 Hz, ArH), 7.14 (d, 2H, J 8.4 Hz, ArH), 7.25e7.34 (m, 3H, ArH), 7.41
(t, 2H, J 7.2 Hz, ArH), 7.49 (d, 2H, J 7.2 Hz, ArH), 7.73 (s, 1H, NH2), 8.22
(s, 1H, NH2), 10.16 (s, 1H, NH). 13C NMR and DEPT (100 MHz, DMSO-
d6):
d
11.7 (CH3), 19.0 (CH3), 77.2 (CHeO), 114.8 (CH, Ar), 121.8 (CH,
5.3.23. 2-(3-(3-Chloro-4-fluorophenoxy)butan-2-ylidene)
thiosemicarbazide (9w)
Ar), 127.3 (CH, Ar), 128.4 (CH, Ar), 129.2 (CH, Ar), 129.7 (CH, Ar),
130.9 (C, Ar), 131.1 (CH, Ar), 138.6 (C, Ar), 152.2 (CeO), 154.4 (C]N),
179.6 (C]S). HRESIMS: 314.1336 [MþH]þ. Anal. Calcd. for
Recrystallization in ethanol afforded yellowish crystals,
yield ¼ 91%. M.p. (ꢁC): 134e136. IR (KBr): 3423 (NH2), 3236 (NH),
C
17H19N3OS: C, 65.15; H, 6.11; N, 13.41. Found: C, 65.70; H, 5.44; N,
1579 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.41 (d, 3H, J 6.4
13.67.
Hz, CH3), 1.82 (s, 3H, CH3), 4.96 (q, 1H, J 6.4 Hz, CHeO), 6.97e7.01

476
D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
(m, 1H, ArH), 7.20e7.22 (m, 1H, ArH), 7.28 (t, 1H, J 9.2 Hz, ArH), 7.88
was calculated using nonlinear regression on Prism 4.0 GraphPad
software. Benznidazole and nifurtimox were used as the reference
drugs.
(s, 1H, NH2), 8.26 (s, 1H, NH2), 10.21 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): d 11.0 (CH3),18.5 (CH3), 77.1 (CHeO),116.0 (CH, Ar),117.0
(CH, Ar), 117.4 (CH, Ar),119.7 (Ar), 150.7 (CeF, Ar),153.1 (CeO),153.8
(C]N), 179.2 (C]S). HRESIMS: 290.0551 [MþH]þ. Anal. Calcd. for
5.7. Anti-T. cruzi activity (trypomastigotes)
C
11H13N3OSFCl: C, 45.60; H, 4.52; N, 14.50. Found: C, 45.35; H, 4.49;
N, 14.42.
Metacyclic trypomastigotes were collected from the superna-
tant of infected LLC-MK2 cells and then seeded at 4 ꢄ 105 cells/well
in RPMI-1640 medium. All compounds were dissolved in DMSO
and then diluted in RPMI-1640 medium in a serial dilution (1.23,
5.3.24. 2-(3-(2,3-Dichlorophenoxy)butan-2-ylidene)
thiosemicarbazide (9x)
Recrystallization in ethanol afforded yellowish crystals,
3.70, 11.11, 33.33 and 100 mg/mL) and added to respective wells, in
yield ¼ 55%. M.p. (ꢁC): 132e134. IR (KBr): 3423 (NH2), 3236 (NH),
triplicate. The final DMSO concentration was 1%. Plate was incu-
bated for 24 h at 37 ꢁC and 5% of CO2. Aliquots of each well were
collected, and the number of viable parasites was counted in a
Neubauer chamber. The percentage of inhibition was calculated in
relation to untreated cultures. Cytotoxic concentration for 50%
(CC50) calculation was also carried out using nonlinear regression
with Prism 4.0 GraphPad software. Benznidazole and nifurtimox
were used as the reference drugs.
1507 (C]N) cmꢀ1. 1H NMR (300 MHz, DMSO-d6):
d 1.47 (d, 3H, J 6.0
Hz, CH3),1.85 (s, 3H, CH3), 5.09 (q,1H, J 6.0 Hz, CHeO), 7.18e7.27 (m,
3H, ArH), 7.84 (s, 1H, NH2), 8.28 (s, 1H, NH2), 10.23 (s, 1H, NH). 13
NMR (100 MHz, DMSO-d6): 10.9 (CH3), 18.5 (CH3), 78.0 (CHeO),
C
d
114.1 (CH, Ar), 120.6 (CH, Ar), 122.5 (Ar), 128.3 (CH, Ar), 132.3 (Ar),
150.33 (CeO, Ar), 154.0 (C]N), 179.2 (C]S). HRESIMS: 306.0235
[MþH]þ. Anal. Calcd. for C11H13N3OSCl2: C, 43.15; H, 4.28; N, 13.72.
Found: C, 42.61; H, 3.99; N, 13.62.
5.8. Inhibition of catalytic activity of cruzain
5.4. Cells
Recombinant cruzain was gently provided by Allison Doak and
Dr. Brian Shoichet, from the University of California San Francisco.
Cruzain activity was measured as previously described, by moni-
toring the cleavage of the fluorogenic substrate Z-Phe-Arg-amino-
methylcoumarin (Z-FR-AMC) in a Synergy 2 (Biotek), from the
Center of Flow Cytometry and Fluorimetry at the Biochemistry and
Immunology Department (UFMG), using filters of 340 nm for
excitation and 440 nm for emission. Assays were performed in
BALB/c mouse, housed in the Centro de Pesquisas Aggeu Mag-
alhaes (Recife, Brazil), were used to collect splenocytes accordingly
to a previously reported protocol [61]. T. cruzi Dm28c epi-
mastigotes, cloned derived from Dm28 strain (TcI) [62], were
maintained at 26 ꢁC in LIT (Liver Infusion Tryptose) medium sup-
plemented with 10% fetal bovine serum (FBS) (Life, Carlsbad, USA),
1% hemin (SigmaeAldrich, St. Louis, USA), 1% R9 medium (Sigmae
Aldrich, St. Louis, USA), and 50
m
g/mL gentamycin (Novafarma,
sodium acetate 0.1 M pH 5.5 and in the presence of 1 mM
captoethanol and 0.01% Triton X-100. The final concentration of
cruzain was 0.5 nM, and the substrate concentration was 2.5
(Km ¼ 1.0 M). Compounds (9a, 9h, 9k, 9m, 9n, 9o and 9s) were
screened at 100 M, while (9g, 9p and 9t) were tested at 50 M.
b-mer-
Anápolis, Brazil). Y strain (TcII) trypomastigotes were obtained
from the supernatant of infected LLC-MK2 cells and were main-
tained in RPMI-1640 medium (SigmaeAldrich, St. Louis, USA)
m
M
m
supplemented with 10% FBS, and 50
m
g/mL gentamycin at 37 ꢁC and
m
m
5% CO2. Experiments were carried out in accordance with the rec-
ommendations of ethical issues guidelines and were approved by
the local Animal Ethics Committee (number 0266/05).
Screening was performed in two conditions, without pre-
incubation with the enzyme and after a 10-min pre-incubation
with enzyme. All compounds were assayed in two independent
experiments, each performed in triplicates. Assays were followed
for 5 min, and activity was calculated based on a DMSO control.
5.5. Host cell cytotoxicity
BALB/c mouse splenocytes were seeded at 5 ꢄ 106 cells/well in
96-well plate. Compounds were dissolved in DMSO and then
diluted in RPMI-1640 medium in a serial dilution (1.23, 3.7, 33.33
5.9. Flow cytometry analysis
Trypomastigotes (4 ꢄ 105 cells/mL) were resuspended in RPMI-
and 100
final DMSO concentration was 1%. The plate was incubated for 24 h
at 37 ꢁC and 5% CO2 containing 1.0 Ci of 3H-thymidine (Perkine
mg/mL) and added to respective wells, in triplicate. The
1640 medium and treated with compound (9r) (0.25 and 1.1 mM)
for 24 h at 37 ꢁC with 5% CO2. Parasites were labeled with propi-
dium iodide (PI) and annexin V using the annexin V-FITC apoptosis
detection kit (Ebioscience, San Diego, USA) according to the
manufacturer instructions. Experiment was performed using a BD
Calibur flow cytometer (San Jose, USA) by acquiring 50,000 events
at least, and data were analyzed by BD CellQuest software (San Jose,
USA). Two independent experiments, in triplicate, were performed.
m
Elmer, Waltham, MA). Cells were harvested and then transferred to
a liquid scintillation counter (WALLAC 1209, Rackbeta Pharmacia,
Stockholm, Sweden) and the percent of 3H-thymidine incorpora-
tion was determined. The highest non-cytotoxic concentration
(HNC) was determined for each compound. For determining the
CC50 values, five different concentrations were used.
Acknowledgments
5.6. Anti-T. cruzi activity (epimastigotes)
This work was funded by CNPq (grant 471461/2011-3 to A.C.L.L.
and 477435/2012-2 to R.S.F.) and FACEPE. A.C.L.L. and C.A.S. and
V.R.A.P. are recipients of a CNPq fellowship, A.D.T.O. held a FACEPE
scholarship and D.R.M.M. holds a FAPESB post-doctoral scholarship.
Authors are thankful to the CPqGM Proteomic facility for deter-
mining mass spectral, Instituto de Física de São Carlos for allowing
theuse of the diffractometerand Centrode Citometria eFluorimetria
da UFMG/FAPEMIG for allowing the use of the fluorimeter. We are
very thankful to Allison Doak and Dr. Brian Shoichet (University of
California San Francisco) for providing recombinant cruzain.
Epimastigotes (Dm28c) in LIT media were counted in a hemo-
cytometer and then seeded at 106 cells/well into a 96-well plate.
Compounds were dissolved in DMSO and then diluted in LIT me-
dium in a serial dilution (1.23, 3.70, 11.11, 33.33 and 100 mg/mL) and
added to respective wells, in triplicate. The final DMSO concen-
tration in the plate was 1%. Plate was incubated for 5 days at 26 ꢁC,
aliquots of each well were collected, and the number of viable
parasites were counted in a Neubauer chamber and compared to
untreated parasite culture. Inhibitory concentration for 50% (IC50
)

D.R. Magalhaes Moreira et al. / European Journal of Medicinal Chemistry 75 (2014) 467e478
477
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