Design, synthesis and molecular docking studies of novel N-arylsulfonyl-benzimidazoles with anti Trypanosoma cruzi activity

Article abstract of DOI:10.1016/j.ejmech.2019.01.013

Currently, only two drugs (i.e. benznidazole (BZN) and nifurtimox (NFX)) have been approved for the treatment of Trypanosoma cruzi (Tc) infection, the etiological agent causing Chagas disease. Since both drugs exhibit severe side effects, patients frequently abandon therapy, resulting in an inefficient pharmacotherapeutic treatment. In this context, there is an urgent need to develop new, safer and optimised anti-Tc agents. In this report, we present the synthesis and biological activity of 11 novel and 3 already reported N-arylsulfonyl-benzimidazole derivatives (NBSBZD,1–14) currently in development as potential anti-Tc compounds. These compounds were designed as part of a library of synthetic arylsulfonyl heterocycle derivatives constructed from privileged structures exhibiting drug-like properties. Based on bioactivity assays against Tc, (in both the extracellular and intracellular forms), we observed that 10 compounds exhibited bioactivity against the epimastigote form, while six of them exhibited activity against the amastigote counterpart. Also, the compounds showed less cytotoxicity compared to the reference drug BZN as measured in Vero cell culture. In order to elucidate the potential mechanism of action, metabolite excretion profiles studies were performed, and complemented with molecular modeling studies performed over known Tc druggable targets. Consistency was observed between experimental and theoretical findings, with metabolic profiles showing that compounds 1, 2, 9, 12 and 14 interfered with the normal glycolysis cycle of Tc, while molecular modeling studies were able to establish a solid structure-activity relationship towards the inhibition of 6-phospho-1-fructokinase, a key enzyme involved in the parasite glycolytic cascade. Overall, the present study constitutes a multidisciplinary contribution to the development of new anti-Chagas compounds.

Full text of DOI:10.1016/j.ejmech.2019.01.013

European Journal of Medicinal Chemistry 165 (2019) 1e10
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and molecular docking studies of novel
N-arylsulfonyl-benzimidazoles with anti Trypanosoma cruzi activity
a
a
b
c
ꢀ
Gisele E. Miana , Sergio R. Ribone , Domingo M.A. Vera , Manuel Sanchez-Moreno ,
María R. Mazzieri a, Mario A. Quevedo a
Departamento de Ciencias Farmaceuticas, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Cordoba, Argentina
b Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar Del Plata, Mar Del Plata, Buenos Aires, Argentina
c Departamento de Parasitología, Facultad de Ciencias, Universidad de Granada, Granada, Spain
,
*
a
ꢀ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Currently, only two drugs (i.e. benznidazole (BZN) and nifurtimox (NFX)) have been approved for the
treatment of Trypanosoma cruzi (Tc) infection, the etiological agent causing Chagas disease. Since both
drugs exhibit severe side effects, patients frequently abandon therapy, resulting in an inefficient phar-
macotherapeutic treatment. In this context, there is an urgent need to develop new, safer and optimised
anti-Tc agents. In this report, we present the synthesis and biological activity of 11 novel and 3 already
reported N-arylsulfonyl-benzimidazole derivatives (NBSBZD,1e14) currently in development as potential
anti-Tc compounds. These compounds were designed as part of a library of synthetic arylsulfonyl het-
erocycle derivatives constructed from privileged structures exhibiting drug-like properties. Based on
bioactivity assays against Tc, (in both the extracellular and intracellular forms), we observed that 10
compounds exhibited bioactivity against the epimastigote form, while six of them exhibited activity
against the amastigote counterpart. Also, the compounds showed less cytotoxicity compared to the
reference drug BZN as measured in Vero cell culture. In order to elucidate the potential mechanism of
action, metabolite excretion profiles studies were performed, and complemented with molecular
modeling studies performed over known Tc druggable targets. Consistency was observed between
experimental and theoretical findings, with metabolic profiles showing that compounds 1, 2, 9, 12 and 14
interfered with the normal glycolysis cycle of Tc, while molecular modeling studies were able to establish
a solid structure-activity relationship towards the inhibition of 6-phospho-1-fructokinase, a key enzyme
involved in the parasite glycolytic cascade. Overall, the present study constitutes a multidisciplinary
contribution to the development of new anti-Chagas compounds.
Received 4 October 2018
Received in revised form
12 December 2018
Accepted 6 January 2019
Available online 7 January 2019
Keywords:
Chagas disease
Synthesis
Biological activity
N-Arylsulfonyl-benzimidazoles
Molecular modeling
Trypanosoma cruzi
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
25 million people [1].
To date, only two drugs have been approved to treat Tc infection,
Chagas disease (CD), or American trypanosomiasis, is caused by
infection with the protozoan parasite Trypanosoma cruzi (Tc). This
illness, originally observed in rural areas of the Americas in the 20th
century, changed its epidemiological pattern with progressive ur-
banisation and spread to other continents. Current estimates indi-
cate that around 7 million people are infected with Tc worldwide,
and mostly in the continental Latin American countries, with CD
remaining as one of the main public-health problems, causing more
than 7000 deaths per year and exposing the risk of infection to over
namely benznidazole (BZN) and nifurtimox (NFX). The former
constitutes the first-line treatment in most countries and exhibits
higher efficacy in the acute phase of CD compared to the chronic
stage [2,3]. Unfortunately, both drugs exhibit severe side effects,
including anorexia and weight loss, nausea and vomiting, nervous
excitation, insomnia, depression, convulsions, vertigo, headache,
sleepiness, myalgia, arthralgia, loss of balance, disorientation,
forgetfulness, paresthesia, adynamia, acoustic phenomena, pe-
ripheral neuropathies, gastralgia, mucosal oedema, hepatic intol-
erance and skin manifestations [4,5]. These effects lead to poor
patient adherence to treatment, resulting not only in therapeutic
failure, but also increased parasitic resistance [6]. In this context,
there is an urgent need for action with regard to the design and
* Corresponding author.
E-mail address: alfredoq@fcq.unc.edu.ar (M.A. Quevedo).
https://doi.org/10.1016/j.ejmech.2019.01.013
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
development of safe and efficient novel drug candidates to treat CD,
that are effective against the Tc forms associated with the acute and
chronic phases of the disease.
characterization and in vitro bioactivity against Tc is reported for the
novel series of NBSBZD. Also, to shed light on their potential
mechanism of action, bioactivity assays were complemented with
profiling the glucose metabolism of Tc parasites exposed to these
novel compounds. Finally, in order to study at the molecular level
the potential mechanism of antitrypanosomal activity, molecular
modeling techniques were used to describe the pharmacodynamic
behaviour of the studied compounds against druggable targets of Tc.
As part of a lead discovery research project ongoing in our
research group [7e10], a library of arylsulfonyl derivatives con-
taining bioactive heterocyclic compounds (BSHet) was designed
and prepared. The approach was based on fragment drug design
(FDD) strategies aimed to combine privileged structures (scaffolds)
in order to develop new bioactive compounds. This methodology
has demonstrated to be very successful in the context of other drug
discovery campaigns [11]. As part of our previous research efforts,
the BSHet library was subjected to high throughput screening (HTS)
assays over Leishmania donovani, Plasmodium falciparum and Tc at
the Head Parasite Chemotherapy, Swiss Tropical Institute, Basel,
Switzerland [7e10], with seven compounds exhibiting an IC50
against Tc below 6 mM, and three of them being identified as
promising lead compounds for further optimisation based on their
2. Results and discussion
2.1. Drug-like properties of NBSBZD
Prior to our synthetic efforts, all compounds were evaluated as
drug-like candidates by analysing their Lipinski parameters (Ro5)
using the Molinspiration and OSIRIS Property Explorer software
packages [23,24]. These analyses showed that all NBSBZD pre-
sented less than one violation of the rules corresponding to oral
bioactivity. In particular, although compounds 11 and 12 exceeded
the preferred number of halogens, they are classified as drug-like
molecules. These results suggest adequate bioavailability for all
derivatives. In addition, calculated LogP values predicted an
appropriate lipophilicity range (2.42e4.97), while the topological
surface area (TPSA) showed acceptable values (51.97e97.79 Å2).
Overall, both parameters are consistent with good membrane
permeability (Table S1) [25]. In order to gain information regarding
the potential toxicity and drug-likeness of the NBSBZD, OSIRIS
Property Explorer software was also used [24], indicating that the
COCH3 and 2,3,5,6-CH3 substituents on the BS scaffold elicited a
more negative impact on the calculated toxicities, as well as in
terms of the tumorigenic, irritant and reproductive effect than the
rest of the functional groups (Table S2). It is worth mentioning that
the fragments and the topology of the reference compounds (BZN
and NFX) presented higher potential toxicity than the NBSBZD
(Table S2). Finally, the drug-score and the drug-likeness calculated
by the software showed positive values in all cases. (Table S2).
Based on these preliminary studies, it was possible to conclude that
the designed library of NBSBZD merited synthetic effort due to their
drug like properties.
activity-toxicity profiles [10]. Furthermore,
a family of BS-
benzotriazole derivatives was evaluated using the epimastigote
and trypomastigote forms of Tc, resulting in good inhibition of their
growth [9].
In line with our previous research efforts, we herein report the
synthesis and biological activity evaluation against Tc of a serie of
BSHet, comprising 11 new and 3 already reported compounds,
containing a combination of benzimidazole (BZD) and substituted
benzenesulfonyl (BS) scaffold, which were termed N-arylsulfonyl-
benzimidazole (NBSBZD, 1e14, Fig. 1) derivatives. The BZD scaffold
is present in compounds exhibiting diverse pharmacological ac-
tivities [11], such as antiproliferative and antimicrobial properties,
including anti-HIV, antioxidant, cysticidal, antiprotozoal, anti-
helmintic, analgesic, antihypertensive, antiinflammatory, antipsy-
chotic, anticoagulant and antidiabetic activity [12e14]. In addition,
the BS scaffold is also present in chemical entities with diverse
bioactivities, such as antibacterial and antileprotic effects, mostly
derived from its well described PABA antagonism [15,16]. Also, the
BS constitutes a very promising privileged scaffold able to enhance
the bioactivity of other heterocycles, leading to analogues dis-
playing similar or improved biological activities compared to their
precursors [17e22].
In addition to the chemical synthesis, the 2D structural
2.2. Synthetic procedures
The synthesis of 1e14 was carried out by applying a synthetic
methodology previously developed in our research group [7e10].
Briefly, the synthesis of 1e14 was accomplished in one step by
condensation of BZD with the corresponding arylsulfonyl chloride
in methylene chloride under nitrogen atmosphere. All derivatives
were obtained in good yields (71e100% of isolated products). All
the compounds obtained constitutes novel chemical entities, with
the exception of 1, 4 and 13, which have been previously reported
by applying a different synthetic methodology [26,27].
Compounds 1e14 were fully characterized by means of HRMS,
IR, 1H NMR and 13C NMR spectra (Figs. S1eS14), with their corre-
sponding yields and melting points shown in Table S3.
2.3. Biological activity studies
2.3.1. In vitro activity assays
The series of NBSBZD was screened against the epimastigote
form of Tc by applying in vitro assays, with selected compounds
tested afterwards against the Tc amastigote form. [28,29] In addi-
tion, their unspecific cytotoxicity against Vero cells was also stud-
ied, with the corresponding values of biological activity,
cytotoxicity and selectivity indexes (SI) being summarized in
Table 1.
Fig. 1. General structure and synthesis of the studied NBSBZD (1-14). a) Reagents and
conditions: methylene chloride, anhydrous pyridine, 25 ꢀC.

G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
3
Table 1
In-vitro activity, toxicity and selectivity index (SI) determined for the BSBZD derivatives against the extracellular and intracellular forms of Trypanosoma cruzi (Tc).
Compounds
Activity IC50
(
m
M)a
Toxicity IC50
Vero Cells
(m
M)b
SIc
Epimastigote
Amastigote
Epimastigote
Amastigote
BZN
1
2
3
4
5
6
7
8
15,8
9.0
7.2
26.8
23.8
8.4
16.8
23.5
5.5
4.6
2.6
13.9
6.2
3.0
23.3
37.3
10.1
n.d
n.d
n.d
n.d
35.7
n.d
15.9
n.d
n.d
13.6
0.8
0.6
1765.6
468.1
656.4
107.6
986.9
122.5
1188.0
120.1
647.7
346.7
59.8
195.5 (244)
65.4 (82)
24.5 (31)
4.5 (6)
117.5 (147)
7.3 (9)
50.5 (63)
22.0 (27)
141.1 (176)
135.4 (169)
4.3 (5)
74.8 (93)
22.5 (28)
91.7 (115)
47.3 (78)
45.9 (76)
n.d
n.d
n.d
n.d
33.3 (55)
n.d
40.6 (67)
n.d
n.d
9
10
11
12
13
14
24.0
n.d
19.0
459.8
68.5
205.3
19.2 (32)
n.d
10.8 (18)
2.2
Results are averages of three separate determinations.
n.d: not determined.
a
IC50 ¼ the concentration required to give 50% inhibition, calculated by using PROBIT® with (1, 10, 25, 50 and 100
mM).
b
c
Measured towards Cell Vero after 72 h of culture.
Selectivity index ¼ IC50 Cell Vero/IC50 extracellular and intracellular form of parasite. In parenthesis: number of times that compound exceeded the SI reference drug (on
extracellular and intracellular forms of T. cruzi).
Compounds 1e14 as well as the standard drug BZN were
assayed at concentrations of 1, 10, 25 and 50 mM. All the activity
determinations were carried out at the Department of Parasitology,
particular, Tc consumes glucose at a high rate, thereby acidifying
the culture medium as a consequence of the incomplete oxidation
to acids [33]. Usually, the final products of glucose catabolism in Tc
are CO2, succinate, acetate, D-lactate, pyruvate, and L-alanine [34].
Succinate is important because its main role is to maintain the
glycosomal redox balance that facilitates the re-oxidation of NADH
produced in the glycolytic pathway. Succinic fermentation has the
advantage of requiring only half of the phosphoenolpyruvate pro-
duced to maintain the NADþ/NADH balance, while the remaining
pyruvate is converted inside the mitochondrion and the cytosol
into acetate, D-lactate or L-alanine, depending on the degradation
pathway [35,36].
Taking into account that the NBSBZD presented in this work
demonstrated good potential as new trypanocidal agents, and in
order to further study if the bioactivity of the studied compounds is
related to some extent of glucose metabolism modification, we
studied the 1H NMR spectra of the culture media corresponding to
the Tc epimastigote form after treatment with 1, 2, 9, 12 and 14. In
this way, the final excretion products were qualitatively and
quantitatively identified (Fig. S15), and compared to those obtained
from parasites maintained in drug-free medium (control) for 96 h.
The presence of acetate, succinate, D-lactate and L-alanine was
confirmed in the control experiments and, as expected, succinate
and acetate were the most abundant end products identified.
Remarkably, after treatment of the parasites with the selected
NBSBZD, the excretion profiles of catabolites was substantially
modified at the dosages assayed (Table 2), with each compound
producing a particular metabolite excretion profile.
ꢀ
University of Granada, by using the method reported by Sanchez
Moreno et al. [30e32] As shown in Table 1, 10 out of the 14 com-
pounds assayed exhibited a higher inhibitory potency than BZN
against the epimastigote form of Tc. Remarkably, derivatives 8, 9,10,
13 and 14, were between three and five-fold more active than BZN.
Clearly, the nature and position of the substituent on the BS group
markedly affected the trypanocidal activity. Indeed, p-NO2-NBSBZD
(10), showed a two-fold increase in IC50, relative to 8 and 9, both of
them bearing the nitro moiety in the ortho and meta positions,
respectively. In line with this observation, the p-CF3-NBSBZD (12)
exhibited twice the potency of the m-CF3 analogue 11, while a
fluorine atom on the para position of NBSBZD (5) also resulted in
higher potencies compared to compounds containing p-Cl or p-Br
substituents (6 and 7, respectively). The enhanced anti-Tc activity of
compounds 13 (p-C(CH3)3-NBSBZD) and 14 (2,3,5,6-(CH3)4-
NBSBZD) compared to 1, could be attributed to the more bulky
substituents of the former derivatives. On the other hand, com-
pounds 3, 4, and 7 were less potent than BZN, but notably they were
more selective for Tc (Table 1).
The activity against the intracellular parasite (amastigote form)
was determined for selected compounds and based on the most
promising ones after primary screening against epimastigote forms.
Therefore, compounds 1, 2, 7, 9, 12 and 14 were assayed at the
concentrations of 1, 10, 25 and 50 mM, with their IC50 values shown
in Table 1. Noteworthy, compounds 2, 9 and 14, exhibited higher
inhibitory potencies than the reference BZN, with 2 showing an
almost two-fold increase in the inhibitory activity against the
amastigote form of Tc and a 76-fold increase in the SI value.
Table 2
Variation percentages in the height of the peaks corresponding to catabolites
excreted by Tc epimastigotes in the presence of BSBZD derivatives at their IC25
compared to a control sample.
2.3.2. Determination of metabolites excretion
It is well-known that trypanosomatids are unable to completely
degrade glucose into CO2 under aerobic conditions. Consequently,
they excrete a considerable part of the hexose skeleton into the
medium as partially oxidised fragments in the form of fermented
metabolites. The nature and percentage of the partially oxidised
fragments is dependent on the metabolic pathway involved for
glucose biotransformation by each of the species considered [33]. In
Metabolite/Compound Succinate Pyruvate Acetate L-Alanine D-Lactate
1
2
ꢁ15,96% ꢁ13,41% 3,31%
28,33% 2,38% 12,30%
ꢁ3,24%
16,43%
25,52%
6,80%
9
12
14
ꢁ33,16% ꢁ30,20% ꢁ16,60% ꢁ22,73% ꢁ3,97%
ꢁ7,22%
ꢁ7,22%
10,63%
10,90%
11,76%
11,97%
9,92%
25,78%
26,43%
10,41%
(ꢁ) and (þ) ¼ 1H NMR peak intensity reduction and increase, respectively.

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G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
Based on the presented evidence, we hypothesised that the
includes one subcavity responsible for binding ATP, and a proximal
subcavity in which fructose 6-phosphate is bound prior to being
phosphorylated. The first one is located in the interface between
subdomains B and C of each monomer, while the fructose binding
site is formed at the interface between domains B and C, with and
additional contribution of a lysine residue (Lys347) belonging to a
second protein subunit. Also, it has been reported that significant
conformational changes are produced in the apo enzyme (aTc-PFK)
upon ATP binding [40], leading to a structurally rearranged catalytic
site in the holo protein (hTc-PFK). Although, the crystallographic
structure of TcePFK has not been solved yet, that of homologous
trypanosomatids, such as Trypanosoma brucei (Tb-PFK), has indeed
been obtained in both the apo and holo forms. A high degree of
homology in PFK has been reported in different species, with 77%
overall sequence identity and more than 90% sequence identity
within the region corresponding the enzyme active site [42,43]. In
this way, in the present study, we used available crystallographic
structures as templates for homology modeling techniques to
obtain three dimensional models of both aTc-PFK and hTc-PFK. (see
Materials and Methods for details).
biological activity of the series of NBSBZD is mediated via inter-
ference with one or more enzymes participating in the glycolytic
cycle. In order to further study this feature, molecular modeling
techniques were used to study their inhibitory potential over pre-
viously reported therapeutic targets of Tc, with particular emphasis
on those involved in the metabolism of glucose.
2.4. Virtual screening of Tc potential targets
2.4.1. Target search in databases
To initiate the screening of potential targets, the ChEMBL data-
base from the European Bioinformatics Institute was explored [37],
in order to compile available information regarding known Tc
therapeutic targets. Results were clustered by protein ID, UniProt
accession numbers [38], and the number of tested small molecule
inhibitors with reported IC50 values (Table 3).
As can be seen, a total of 14 possible targets were found, with
one of them corresponding to 6-phospho-1-fructokinase (PFK).
This enzyme catalyses the phosphorylation of fructose 6-phosphate
(F6P) to fructose 1,6-bisphosphate, an early step in the glycolytic
pathway in Tc [39,40]. Considering its crucial biological role, PFK,
has been previously identified as a promising and selective thera-
peutic target for the chemotherapy of diverse diseases caused by
several trypanosomatides, including Tc [41]. Of note, our metabolic
studies in the presence of the inhibitors (section 2.3.2) revealed
that they interfered with an early process in the glycolytic cascade,
which is consistent with the potential inhibition of PFK. Also, Bri-
macombeet et al. [42] previously reported a series of PFK inhibitors
that are indeed structurally related to the series of NBSBZD re-
ported in this work. The above mentioned aspects, led us to further
study the inhibition of FPK elicited by the reported NBSBZD as a
pathway for their observed anti-Tc activity.
2.4.2.1. Inhibitor-PFK model development for
a training set. A
training set of 32 PFK inhibitors reported in the ChEMBL database
(Figs. S16eS21) was used to develop a structure-activity relation-
ship model using aTc-PFK and hTc-PFK as the target enzymes. In this
way, molecular docking techniques were used to predict the
binding mode to the corresponding catalytic site for each com-
pound in the training set. Docked poses were scored using the
ChemGauss3 scoring function as implemented in the FRED docking
software [44], with the five lowest energy docked poses being
selected for further SAR model development.
Fig. 2 presents the predicted binding mode obtained for the
most potent inhibitor within the training set, bound to both the
apoenzyme (Fig. 2a) and the holoenzyme (Fig. 2b). In the first case,
(aTc-PFK), we found that the inhibitor binds to a solvent accessible
cleft normally occupied by fructose 6-phosphate during the cata-
lytic cycle, establishing intermolecular interactions with residues
Arg173(A), Arg274(A), Glu325(A), Asp339(A), Lys345(A), Leu346(A),
Asp348(A), Val351(A), Glu352(A), Tyr375(A), Asp377(A), Tyr380(A)
and Lys374(D) (Fig. 2a). Among the key stabilizing interactions, two
hydrogen bond contacts with Arg274 and Lys374 anchors the in-
hibitor to the binding site. When binding to hTc-PFK was studied
(Fig. 2b), it was found that the inhibitor was positioned within a
buried cavity adjacent to the position of the ATP-Mg complex of the
holoenzyme, establishing intermolecular contacts with residues
Gly198(A), Asp199(A), Gln200(A), Asp231(A), Leu232(A),
Arg274(A), Asp275(A), Asn343(A) and Ala430(A) (Fig. 2b). When
docking assays were extended to the whole training set, homolo-
gous binding modes to those described above were found (de-
pictions not shown).
2.4.2. Development of a structure-activity relationship model for
the inhibition of PFK
Structurally, PFK exhibits a homotetrameric structure (chains A-
D) forming a dimer of dimers. In addition, each monomer chain
contains four well defined domains (domains A-D) [39,40], two of
which present a compact structure, namely domain B (residues
95e233, 386e409) and domain C (residues 234e385, 442e453),
while subdomains A and D present a less organized structure
[39,40]. The catalytic site involved in the phosphorylation of fruc-
tose 6-phosphate has been characterized in detail [39,40], and
Table 3
List of Trypanosoma cruzi drugabble targets retrieved from the ChEMBL (https://
www.ebi.ac.uk/chembl/) database (accession date 2/2018).
Target
UniProta
nb
From the above mentioned aspects, it is clear that the interac-
tion of PFK inhibitors with the target strongly depends on the state
of the enzyme (i.e. apo or holo enzyme). To further assess which
form of the enzyme may constitute the target of the training set, we
searched for a quantitative correlation between the interaction
energies of the inhibitors docked to the catalytic sites of aTc-PFK
and hTc-PFK and the reported inhibitory potencies (pIC50). These
interaction energies were calculated by applying the MMGBSA
method, and considered only the amino acid residues lying within a
5 Å radius from the ligand (Tables S4 and S5). As shown in Fig. 3, the
reported inhibitory potencies (pIC50) exhibited a close correlation
with the sum of the interaction energies found for aTc-PFK (Fig. 3a),
while no correlation between the interaction energy and the
inhibitory potency was found after docking the training set to hTc-
PFK (Fig. 3b). These observations strongly suggest that compounds
6-phospho-1-fructokinase
Bifunctionaldihydrofolatereductase-thymidylate synthase
Cruzipain
Cyclic nucleotide specific phosphodiesterase
Deoxyuridinetriphosphatase
Farnesyldiphosphate synthase
Farnesylsynthetase
Farnesyltransferase
Glyceraldehyde-3-phosphate dehydrogenase
Hexokinase
Q4E657
Q27793
P25779
Q53I59
Q4DI50
Q8WS26
Q964Q8
Q4CXF7
P22513
Q8ST54
Q7Z1V1
Q26966
P52270
P28593
42
7
750
51
e
47
20
e
39
94
e
Sterol 14-alpha demethylase
Trans-sialidase
Triosephosphateisomerase
Trypanothionereductase
19
9
282
a
Deposition code in the UniProt database (https://www.uniprot.org/).
b
Number of chemical entities assayed against the corresponding targets as
deposited in ChEMBL.

G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
5
Fig. 2. Intermolecular interactions established by the most active PFK inhibitor belonging to the training set (Fig. S21, TS32) with residues of: a) aTc-PFK and b) hTc-PFK.
Fig. 3. Correlation between the sum of energetic components (
interaction of the training set with a) aTc-PFK and b) hTc-PFK.
DGSUM) determined by molecular docking (Tables S4 and S5) and the reported biological activity (pIC50) for the
included in the training set inhibits the enzyme by binding to the
studied set of NBSBZD derivatives.
PFK catalytic site in the apo form.
3. Conclusions
2.4.3. Modeling of aTc-PFK inhibition elicited by NBSBZD
In order to study if the bioactivity observed for the series of
synthesized NBSBZD compounds is elicited by the inhibition of PFK,
and particularly through binding to aTc-PFK, the set of 14 de-
rivatives was subjected to molecular docking studies and energy
calculation protocols described above for the training set. As a
general overview of the overall binding mode of the serie of
NBSBZD and the previously reported compounds in the training set,
Fig. 4 presents a superimposition of the binding mode of the most
active inhibitor within the training set (TS32, thinner licorice) and
the most active NBSBZD derivative.
As before, the sum of interaction components was calculated
within a 5 Å radius from the ligand (Table S6), with the calculated
pIC50 values being obtained by applying the equation shown in
Fig. 3.a. As can be seen, a good correlation between pIC50calc and
pIC50exp was observed (Fig. 5), strongly suggesting that aTc-PFK
constitutes the molecular target mediating the anti Tc activity of the
A series of new 14 NBSBZD were synthesized and their struc-
tures were characterized. All compounds exhibited adequate drug-
like properties, with Lipinski parameters showing that they may
exhibit good membrane permeability and adequate oral bioavail-
ability. Also, screening of the synthesized compounds through the
OSIRIS database resulted in lower tumorigenic risk or irritant and
reproductive side-effects than the reported NBSBZD compared to
the reference compounds BZN and NFX.
In vitro assays were conducted in order to evaluate the anti-
trypanosomal activity and toxicity towards Vero cells. Com-
pounds 2, 9 and 14 displayed the highest inhibitory potencies
against the epimastigote and amastigote forms of Tc, with a marked
increase in their bioactivities compared to the reference compound
BZN. In addition, a significant increase in the SI was calculated for
all the reported compounds.
In order to study the mechanism of the anti-Tc activity of the

6
G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
Fig. 4. Superimposition of the binding modes of the most active inhibitor within the training set (TS32, thinner licorice) and the most active NBSBZD derivative (Fig. 1, Cpd. 14, wider
licorice) to a) aTc-PFK and b) hTc-PFK.
Infrared spectra were recorded on a Nicolet 5 SXC FT-IR. NMR ex-
periments were performed on a Bruker Advance II 400 Hz, ultra-
shield TM spectrometer at 400.16(1H) and 100.62 (13C) equipped
with an inverse multinuclear detection probe, digital resolution
capabilities and a variable temperature unit. Chemical shift values
are reported in ppm, while the multiplicities of the signals are
described using the following abbreviations s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, sex ¼ sextet, m ¼ multiplet, and
were acquired in CHCl3-d as solvent (referred to residual CHCl3 at
7.26 ppm for 1H and 77 ppm for 13C). Coupling constants (J) are in
Hz. High resolution mass spectroscopy experiments were acquired
using a Micromass Q-TOF micro hybrid quadrupole/orthogonal
high resolution time of flight MS equipped with a Micromass
capillary HPLC (Waters Corporation). The benzimidazole and all the
arylsulfonyl chlorides were purchased from Sigma-Aldrich and
Acros Organics. Pyridine used for synthetic procedures was distilled
prior to use and stored over pellets of NaOH. All other reagents and
solvents (p.a. grade) were used as purchased from Dorwill, Biopack,
Sintorgan and Merck.
Fig. 5. Correlation between the experimental biological activity (pIC50exp) and the
corresponding activity predicted based on the developed QSAR model for Cpds 1e14
(pIC50calc).
4.2. General synthetic procedure
A mixture of the benzimidazole (3 mmol), 1.5 mL of anhydrous
pyridine and methylene chloride (15 mL) was stirred at room
temperature for 1 h, after which the corresponding arylsulfonyl
chloride (3.2 mmol) was added. The mixture was stirred for 1 h and
the completion of the reaction was tested by TLC using hex-
ane:acetone (7:3) or chloroform:methanol:acetone (8:1:1) as the
mobile phase. The sulfonylation time was around 1 h, after which
the reaction was stopped by the addition of HCl 10% (30 mL). The
resulting immiscible phases were separated, with the organic
phase being extracted by sequentially adding HCl 10% (1 ꢂ 30 mL), a
saturated Na2CO3 solution (1 ꢂ 30 mL) and brine (1 ꢂ 30 mL). The
resulting organic phase was dried over anhydrous MgSO4, filtered
and concentrated to dryness under vacuum to isolate the reaction
products as solids. End-products were purified by crystallisation
from ethanol, n-hexane or mixtures of them.
reported molecules, a combination of metabolite excretion studies
and molecular modeling techniques was used. Both, the experi-
mental and theoretical approaches were consistent in that the
target of the assayed compounds is related to the glycolytic
pathway of the parasite. Specifically, molecular modeling studies
strongly supported the binding of the studied compounds to the
apo form of PFK as the molecular target of the observed biological
activity.
From the presented results, we can conclude that the series of
NBSBZD synthesized constitutes a significant potential as anti Tc
agents, representing a valuable contribution towards the further
design of antichagasic compounds based on the NBSBZD scaffold.
4. Experimental section
4.1. General considerations
4.2.1. Specific procedures and spectral data for compounds 1e7, 9,
11e16
Melting points (mp) were determined by using an electro-
thermal apparatus, by microcapillary methods and are uncorrected.
- 1-(Benzenesulfonyl)-benzimidazole(1). [26] White crystals
(0.55 g, 79%). mp. 99e100 ꢀC (from n-hexane). IR ( max/cm_1, KBr):
n

G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
7
3066
(
n
CH), 1376
(
n
SO2asim), 1171
(
n
SO2sim). 1HNMR (CDCl3,
C13H9Cl1N2O2S1Na: 314.9965, found: 314.9970 [MþNa].
- 1-(4-Bromo-benzenesulfonyl)-benzimidazole (7). White crys-
tals(0.57 g, 54%). mp. 129e130 ꢀC (from EtOH). IR( max/cm_1, KBr):
3086 ( CH), 1381 ( SO2asim), 1163 ( SO2sim), 746 (
CBr). 1HNMR
400.16 Hz): 8.39 (s,1H,H2); 8.00 (d, 2H, 8.6 Hz, H2’); 7.87 (d, 1H,
7.76 Hz, H8); 7.77 (d, 1H, 8.6 Hz, H5); 7.64 (t, 1H, 6.88 Hz, H4’); 7.53
(t, 1H, 8.64 Hz, H3’); 7.39 (m, 2H, H6eH7).13CNMR (CDCl3,
100.62 Hz; assigned using HSQC): 144.23 (Cq-4); 141.22 (CH-2);
137.70 (Cq-1’); 134.95 (CH-4’); 130.94 (Cq-9); 129.83 (CH-3’);
127.42 (CH-2’); 125.65 (CH-6); 124.84 (CH-7); 121.40 (CH-8); 112.63
(CH-5). HRMS (EI) m/z: calculated mass for C13H10N2O2S1:
258.1000, found: 259.0000 [MþH]þ [26].
n
n
n
n
n
(CDCl3, 400.16 Hz): 8.36 (s, 1H, H2); 7.85 (m, 3H, H3’-H8); 7.78 (d,
1H, 7 and 0.8 Hz, H5); 7.66 (dd, 2H, 8.9 and 2 Hz, H2’); 7.4 (m, 2H,
H6eH7). 13CNMR (CDCl3, 100.62 Hz; assigned using HSQC): 144.07
(Cq-9); 141.03 (CH-2); 136.5 (Cq-4’); 133.05 (CH-2’); 131.1 (Cq-4);
130.4 (Cq-1’); 128.6 (CH-3’); 125.95 (CH-7); 125.2 (CH-6); 121.32
(CH-8); 122.25 (CH-5). HRMS (EI) m/z: calculated mass for
C13H9Br1N2O2S1Na: 358.9460, found: 358.9464 [MþNa].
- 1-(4-Acetamide-benzenesulfonyl)-benzimidazole (2). White
crystals (0.58 g, 76%). mp. 186 ꢀC - decomposition (from EtOH).
IR(n
max/cm_1, KBr): 3244 (
nNH), 3037 (
n
CH), 1701 (
n
CO), 1377
- 1-(2-Nitro-benzenesulfonyl)-benzimidazole (8). Yellow crys-
(n
SO2asim), 1162 (
n
SO2sim). 1HNMR (CDCl3, 400.16 Hz): 8.37 (s, 1H,
tals (0.36 g, 72%). mp. 117e118 ꢀC (from EtOH). IR ( max/cm_1, KBr):
n
H2); 7.94 (dd, 2H,8 Hz, H2’); 7.85 (d, 1H, 6.6 Hz, H8); 7.76 (d, 1H,
6.56 Hz, H5); 7.67 (d, 2H, 8.2 Hz, H3’); 7.40 (sa, 1H, H10); 7.38 (m,
2H, H6 and H7); 2.05 (s, H,H12). 13CNMR (CDCl3, 100.62 Hz;
assigned using HSQC): 168.44 (C6’ ¼ O); 144.0 (Cq-9); 143.70 (Cq-
4’); 141.17 (Cq-2); 131.83 (Cq-1’); 130.72 (Cq-4); 128.78 (CH-2’);
125.63 (CH-7); 124.84 (CH-6); 121.09 (CH-8); 119.48 (CH-3’); 112.47
(CH-5); 24.74 (CH3-7’). HRMS (EI) m/z: calculated mass for
C15H13N3O3S1: 338.0570, found: 338.0653 [MþNa].
3022 (
nCH), 1527 (nNO2asim), 1388 (nSO2asim), 1352 (nNO2sim), 1152
(n
SO2sim). 1H NMR (CDCl3, 400.16 Hz): 8.5 (s, 1H,H2); 8.30 (m, 1H,
9.5 y 3.3 Hz, H3'a); 7.8 (m, 3H,H4’-H2'b-H3'b); 7.7 (m, 2H, H5eH8);
7.42 (m, 2H, H7eH6). 13CNMR (CDCl3, 100.62 Hz; assigned using
HSQC): 148.1 (Cq-2'a); 143.9 (Cq-9); 142.2 (CH-2); 136.0 (CH-3'b);
134.5 (Cq-4); 132.7 (CH-4’); 131.3 (CH-3'a); 130.46 (Cq-1’); 126.5
(CH-7); 125.7 (CH-6); 125.0 (CH-2'b); 121.5 (CH-8); 112.2 (CH-5).
HRMS (EI) m/z: calculated mass for C13H9N3O4S1: 304.0387,
found: 304.0390 [MþH]þ, calculated mass for C13H9N3O4S1Na:
326.0206, found: 326.0208 [MþNa].
- 1-(4-acetyl-benzenesulfonyl)-benzimidazole (3). White crys-
tals (0.31 g, 92%). mp. 119e120 ꢀC (from EtOH/n-hexane). IR(
n
max/cm_1, KBr): 3039 (
nCH), 2967 (
n
CH3), 1684 (
n
C ¼ O), 1381
- 1-(3-Nitro-benzenesulfonyl)-benzimidazole (9). Yellow crys-
(n
SO2asim), 1163 (
n
SO2sim). 1H NMR (CDCl3, 400.16 Hz): 8.39 (s, 1H,
tals (0.63 g, 64%). mp.151e152 ꢀC (from EtOH). IR ( max/cm_1, KBr):
n
H2); 8.07 (quadruplet, 4H, 8.4 Hz, H3’-H2’); 7.87 (d, 1H, 7.68 Hz,
H8); 7.78 (d, 1H, 6.6 Hz, H5); 7.40 (m, 2H, H6eH7), 2.60 (s, 3H, H6’).
13C NMR (CDCl3, 100.62 Hz; assigned using HSQC): 196.00 (C]O),
144.07 (Cq-9); 141.55 (Cq-1’); 141.05 (CH-2); 130.64 (Cq-4); 129.40
(CH-2’); 127.54 (CH-3’); 125.93 (CH-6); 125.18 (CH-7); 121.34 (CH-
8); 112.39 (CH-5); 26.82 (CH3-6’). HRMS (EI) m/z: calculated mass
for C15H12N2O3S1: 301.0641, found: 301.0644 [MþH]þ, calculated
mass for C15H12N2O3S1Na: 323.0461, found: 323.0466 [MþNa].
- 1-(4-Methyl-benzenesulfonyl)-benzimidazole (4) [27]. White
3022 (
nCH), 1527 (nNO2asim), 1388 (nSO2asim), 1352 (nNO2sim), 1152
(n
SO2sim).1HNMR (CDCl3, 400.16 Hz): 8.9 (t, 1H,H2'a); 8.50 (ddd, 1H,
7.24, 2 and 1 Hz, H4’); 8.4 (s, 1H,H2); 8.3 (ddd, 1H, 6.93, 1.8 and 1 Hz,
H2'b); 7.92 (d,1H, 7.44 and 0.72 Hz, H8); 7.8 (m, 2H, H5eH3'b); 7.48
(td, 1H, 7.48 and 1.2 Hz, H7); 7.48 (td, 1H, 7.44 y 1.2 Hz,H6). 13CNMR
(CDCl3, 100.62 Hz; assigned using HSQC): 148.5 (Cq-3'a); 144.1 (Cq-
9); 140.84 (CH-2); 139.6 (Cq-1’); 132.36 (CH-2'b); 131.35 (CH-3'b);
130.46 (Cq-4); 129.17 (CH-4’); 126.28 (CH-7); 125.49 (CH-6); 122.52
(CH-2'a); 121.55 (CH-8); 112.26 (CH-5). HRMS (EI) m/z: calculated
mass for C13H9N3O4S1: 304.0387, found: 304,0390 [MþH]þ,
calculated mass for C13H9N3O4S1Na: 326.0206, found: 326.0208
[MþNa].
crystals (0.5 g, 86%). mp. 74e75 ꢀC (from EtOH/n-hexane). IR(
cm_1, KBr): 3054 (
CH), 2963, 2869 ( CH3), 1378 ( SO2asim), 1169
SO2sim). 1HNMR (CDCl3, 400.16 Hz): 8.38 (s, 1H, H2); 7.87 (m, 3H,
nmax/
n
n
n
(n
H2’-H8); 7.76 (d, 1H, 5.5 Hz, H5); 7.37 (m, 2H, H6eH7); 7.31 (d, 2H,
H3’). 13CNMR (CDCl3, 100.62 Hz; assigned using HSQC): 146.18 (Cq-
4’); 144.07 (Cq-9); 141.24 (CH-2); 134.64 (CH-1); 130.80 (CH-4);
127.37 (CH-2’); 125.54 (CH-6); 124.75 (CH-7); 121.08 (CH-8); 112.5
(CH-5); 21.65 (CH3-5’). MS m/z: mass found for C14H12N2O2S1:
272.00 [M]þ [27].
- 1-(4-Nitro-benzenesulfonyl)-benzimidazole (10). Yellow crys-
tals (0.73 g, 73%). mp. 153e154 ꢀC (from EtOH). IR( max/cm_1, KBr):
n
3026 (
nCH), 1533 (nNO2asim), 1388 (nSO2asim), 1351 (nNO2sim), 1172
(n
SO2sim). 1HNMR (CDCl3, 400.16 Hz): 8.36 (m, 2H, H3’-H2); 8.2 (dd,
2H, 9.3 and 2.4 Hz, H2’); 7.85 (d, 1H, 8 and 1.3 Hz, H8); 7.78 (d, 1H,
7.9 and 1.3 Hz, H5); 7.4 (m, 2H, H6eH7). 13CNMR (CDCl3, 100.62 Hz;
assigned using HSQC): 151.3 (Cq-4’); 144.3 (Cq-9); 143.22 (Cq-1’);
140.97 (CH-2); 130.6 (Cq-4); 128.5 (CH-2’); 126.6 (CH-7); 125.75
(CH-6); 124.9 (CH-3’); 121.7 (CH-8); 112.19 (CH-5). HRMS (EI) m/z:
calculated mass for C13H9N3O4S1Na: 326.0206, found: 326.0206
[MþNa].
-1-(4-Fluoro-benzenesulfonyl)-benzimidazole (5). White crys-
tals (0.53 g, 64%). mp. 124e125 ꢀC (from EtOH/n-hexane). IR(
n
max/cm_1, KBr): 3071 (
nCH), 1377 (nSO2asim), 1159 (nSO2sim), 1086
n
CF. 1HNMR (CDCl3, 400.16 Hz): 8.37 (s, 1H, H2); 8.03 (ddd, 2H, 7
and 2 Hz, JHF(orto) ¼ 4.9, H2’); 7.85 (d, 1H, 6.76 and 1.36 Hz, H8); 7.78
(d, 1H, 7.16 and 1.88 Hz, H5); 7.40 (m, 2H, H6eH7); 7.20 (td, 2H, 8.72
and 2 Hz, H3’). 13CNMR (CDCl3, 100.62 Hz; assigned using HSQC):
166.28 (Cq-4’, 1JCF ¼ 260 Hz); 144.06 (Cq-9); 141.08 (CH-2); 133.38
(Cq-1’); 130.63 (Cq-4); 130.21 (CH-2’); 125.82 (CH-7); 125.05 (CH-
6); 121.28 (CH-8); 117.26 (CH-3’); 112.36 (CH-5). HRMS (EI) m/z:
calculated mass for C13H9F1N2O2S1Na: 299.0261, found: 299.0272
[MþNa].
-
1-(3-trifluoromethyl-benzenesulfonyl)-benzimidazole (11).
White crystals (0.44 g, 87%). mp. 83e84 ꢀC (from EtOH). IR(
max/
cm_1, KBr): 3025 (
CH), 1332 ( SO2asim), 1165 ( SO2sim), 1133
CF3asim), 1099
CF. 1H NMR(CDCl3, 400.16 Hz): 8.39 (s, 1H, H2);
n
n
n
n
(n
n
8.28 (s, 1H, H2'a); 8.17 (d, 1H, 8.32 Hz, H2'b); 7.87 (m, 2H, H4’-H8);
7.79 (d, 1H, 7.0 Hz, H5); 7.70 (t,1H, 5.32 Hz, H3'b); 7.44 (t, 1H,
7.04 Hz, H7), 7.39 (t, 1H, 7.04 Hz, H6). 13CNMR (CDCl3, 100.62 Hz;
assigned using HSQC): 144.09 (Cq-9); 140.96 (CH-2); 138.85 (Cq-
1’); 131.45 (CH-4’, 3JCF ¼ 3.35 Hz); 130.7 (CH-3'b); 130.58 (Cq-4);
130.26 (CH-2'b-Cq-3'a 2JCF ¼ 40.2 Hz); 126.13 (CH-7); 125.26 (CH-
6); 124.28 (CH-2'a 3JCF ¼ 3.86 Hz- Cq-5’); 121.49 (CH-8); 112.20
(CH-5); 30.59 (Cq-6’). HRMS (EI) m/z: calculated mass for
C14H9F3N2O2S1: 327.0410, found: 327.0415 [MþH]þ.
- 1-(4-Chloro-benzenesulfonyl)-benzimidazole (6). [27] White
crystals (0.62 g, 63%). mp. 113e114 ꢀC (from EtOH). IR (
n
max/cm_1,
KBr): 3058 (nCH), 1384 (nSO2asim), 1171 (nSO2sim), 761 (nCCl).
1HNMR (CDCl3, 400.16 Hz): 8.37 (s, 1H, H2); 7.93 (dd, 2H, 9.3 and
1.3 Hz, H2’); 7.84 (d, 1H, 7.96 and 1.36 Hz, H8); 7.78 (d, 1H, 7.92 and
1.36 Hz, H5); 7.50 (dd, 2H, 9.3 and 1.3 Hz, H3’); 7.4 (m, 2H,
H6eH7).13CNMR (CDCl3, 100.62 Hz; assigned using HSQC): 144.04
(Cq-9); 141.81 (Cq-4’); 141.1 (CH-2); 135.93 (CH-1); 130.6 (Cq-4);
130.15 (CH-2’); 128.6 (CH-3’); 125.85 (CH-7); 125.1 (CH-6); 121.3
(CH-8); 122.36 (CH-5). HRMS (EI) m/z: calculated mass for
-
1-(4-trifluoromethyl-benzenesulfonyl)-benzimidazole (12).
White crystals(0.51 g, 83%). mp. 99e100 ꢀC (from EtOH). IR(
max/
cm_1, KBr): 3046 (
CH), 1321 ( SO2 asim), 1175 ( SO2sim), 1135
CF3asim), 1090
CF. 1H NMR(CDCl3, 400.16 Hz): 8.39 (s, 1H, H2);
n
n
n
n
(n
n

8
G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
8.14 (d, 2H, 7.5 Hz, H2’); 7.87 (d, 1H, 9.8 y 3.44 Hz, H8); 7.81 (d, 3H,
H3’-H5); 7.41 (m, 2H, H7eH6). 13C NMR (CDCl3, 100.62 Hz; assigned
using HSQC): 144.62 (Cq-9); 141.09 (CH-2); 136.25 (Cq-4’,
2JCF ¼ 33.20 Hz); 130.96 (Cq-4); 128.1 (Cq-1’,CH-2’); 126.56 (CH-3’,
Cq-5’, 3JCF ¼ 4.0 Hz); 126.07 (CH-7); 125.46 (CH-6); 121.5 (CH-8);
112.48 (CH-5). HRMS (EI) m/z: calculated mass for
C14H9F3N2O2S1: 327.0410, found: 327.0422 [MþH]þ.
the culture medium to reach final concentrations of 1, 10, 25 and
50 M, measuring the effect of each compound against Tc epi-
m
mastigotes during 72 h using a Neubauer haemocytometer. The
anti-Chagas effect is expressed as the IC50, which was calculated
using the PROBIT software and corresponded to the concentration
required to produce 50% growth inhibition. The corresponding re-
sults are expressed as the average of three separate experiments.
- 1-(4-t-butyl-benzenesulfonyl)-benzimidazole (13). [27] White
crystals (0.5 g,80%). mp. 116e117 ꢀC (from EtOH/n-hexane). IR
4.3.4. In vitro activity: amastigotes assay (intracellular forms)
Vero cells were grown in RPMI medium (Gibco) supplemented
with 10% inactivated foetal bovine serum and were kept in a hu-
midified atmosphere of 95% air and 5% CO2 at 37 ꢀC. Cells were
seeded at a density of 1 ꢂ 104 cells/well in 24-well microplates
(Nunc) with rounded cover slips on the bottom and cultured for 2
days. Afterwards the cells were infected in vitro with metacyclic
forms of Tc at a ratio of 10:1 during 24 h [35]. The non-
phagocytosed parasites were removed by washing, and then the
assayed compounds were added to reach final concentrations of 1,
(
n
max/cm_1, KBr): 3054 (
n
CH), 2963, 2869 (
nCH3), 1378 (nSO2asim),
1169 (
n
SO2sim). 1HNMR(CDCl3, 400.16 Hz): 8.39 (s, 1H, H2); 7.92 (m,
3H, H2’-H8); 7.78 (d, 1H, 7.2 and 0.72 Hz, H5); 7.52 (dd, 2H, H3’);
7.41 (t, 1H, 7.3 and 1 Hz, H7); 7.37 (t, 1H, 7.76 and 1.64 Hz, H6); 1.28
(s, 9H, H6’).13C NMR (CDCl3, 100,62 Hz; assigned using HSQC):
159.26 (Cq-4’); 144.28 (Cq-9); 141.34 (CH-2); 134.7 (CH-1); 130.84
(CH-4); 127.44 (CH-2’); 126.51 (CH-3’); 125.28 (CH-7); 124.7 (CH-
6); 121.61 (CH-8); 112.61 (CH-5); 35.53 (Cq-5’); 30.59 (CH3-6’). MS
m/z: found C17H19N2O2S1:314.00 [M]þ [27].
- 1-(2, 3, 5, 6-tetramethyl-benzenesulfonyl)-benzimidazole (14).
10, 25 and 50 mM. The resulting Vero cells were incubated for
White crystals(0.49 g, 82%). mp. 130e131 ꢀC (from EtOH). IR (
n
max/
CH3), 1603 (aromatic substi-
CH3), 1357 ( SO2asim), 1158 ( SO2sim).
72 h at 37 ꢀC in a 5% CO2 atmosphere. Drug activity was determined
on the basis of number of amastigotes in treated and untreated
cultures in methanol-fixed and Giemsa-stained preparations. The
number of amastigotes was determined by analyzing 200 host cells
distributed in randomly chosen microscopic fields. The anti-Chagas
effect is expressed as the IC50. The results presented in Table 1 are
averages of three separate experiments.
cm_1, KBr): 3089 (
tution 1,2,3,5,6), 1463,1432 (
nCH), 2973,2918(n
d
n
n
1H NMR (CDCl3, 400.16 Hz): 8.40 (s, 1H, H2); 7.78 (d, 1H, 7.96 and
0.8 Hz, H8); 7.32 (td, 1H, 7.28 and 1.2 Hz, H7); 7.24 (m, 2H, H4’-H6);
7.16 (d, 1H, 7.44 y 0.7 Hz, H5); 2.47 (s, 6H, H6’); 2.23 (s, 6H, H5’). 13C
NMR(CDCl3, 100.62 Hz; assigned using HSQC): 143.63 (Cq-9); 141.3
(CH-2); 137.84 (Cq-1’-4’); 136.8 (CH-3’); 136.34 (CH-2’); 131.07 (Cq-
4); 125.02 (CH-6); 124.37 (CH-7); 120.9 (CH-8); 111.9 (CH-5), 21.0
(CH3-6’),17,5 (CH3-5’). HRMS (EI) m/z: calculated mass for
C17H18N2O2S1Na: 337.0981, found: 337.0994 [MþNa].
4.3.5. Metabolites excretion study
Cultures of Tc epimastigotes (initial concentration of
5 ꢂ 105 cells/mL) received IC25 concentrations of the compounds
under study (except for control cultures). After incubation during
96 h at 28 ꢀC, the cells were centrifuged at 400 g for 10 min. The
supernatants were collected to determine the excreted metabolites
using 1H NMR. Chemical shifts were expressed in parts per million
4.3. Biological evaluation
4.3.1. Parasite strain culture
Epimastigotes of Tc SN3 strain (IRHOD/CO/2008/SN3) isolated
from domestic Rhodnius prolixus from Guajira (Colombia) [33],
were cultured in vitro in trypanosomes liquid medium (TLM) with
10% inactivated foetal bovine serum, and were kept in an air at-
mosphere at 28 ꢀC in Roux flasks (Corning, USA) with a surface area
of 75 cm2, according to a previously described methodology [34].
(ppm, d scale), using sodium 2,2-dimethyl-2-silapentane-5-
sulfonate as the reference signal. The chemical displacements
used to identify the respective metabolites were consistent with
those previously described [30].
4.4. Molecular modeling studies
4.3.2. Cell culture and cytotoxicity tests
4.4.1. Development of TcePFK homology models
Vero cells (EACC number 84113001) originally obtained from
monkey kidney were grown in RPMI medium (Gibco), supple-
mented with 10% inactivated of foetal bovine serum in a humidified
95% air, 5% CO2 atmosphere at 37 ꢀC for two days. The cytotoxicity
test for Vero cells was performed according to a previously
described methodology [34]. After 72 h of treatment, cell viability
was determined by flow cytometry. Thus, 100 mL/well of a propi-
dium iodide solution (100 mg/mL) was added and incubated for
10 min at 28 ꢀC in darkness. Afterwards, 100 mL/well of fluorescein
diacetate (100 ng/mL) was added and incubated under the same
conditions. Finally, the cells were recovered by centrifugation at
400g for 10 min, with the pellet being washed with phosphate
buffered saline (PBS). Flow cytometry analysis was performed with
a FACS Vantage flow cytometer (Becton Dickinson). The percentage
of viability was calculated by comparison with the corresponding
control cultures. The IC50 was calculated using the software PROBIT.
The homology modeling of TcPFK was based on the crystallo-
graphic structure of TbPFK [39,40]. The three-dimensional structure
was constructed using Modeller v9.16 software [45], with the
sequence corresponding to TcPFK being downloaded from the
UniProt database [38]. Then both PFK sequences were aligned and
compared, reaching 76.7% sequence identities, in agreement with
previous reports [42,43].
Considering that the target can adopt two different conforma-
tions, and that the corresponding binding site for the training set
under study was unknown, two 3D TcPFK homology models were
generated using different crystallographic structures from TbPFK as
3D templates: 1) the apoenzyme form (PDB code: 2HIG) [39] and 2)
the holoenzyme form containing the ATP-Mg complex bound to the
active site (PDB code: 3F5M) [40]. In order to model the entire
active site, the homology model was constructed based on chain A
and B. Five 3D homology models were generated for every form of
the PFK, with the best model being chosen on the basis of their
discrete optimised protein energy (DOPE) score as calculated by
Modeller v9.16. The model exhibiting the lowest DOPE value was
used for further analysis.
4.3.3. In vitro activity: epimastigote assay (extracellular forms)
The assay was performed following a previously reported pro-
cedure [34]. Briefly, assayed compounds and the reference drug
benznidazole (BZN) were dissolved in a PEG:EtOH mixture (7:3),
which was been previously found to be non-toxic and lacking any
inhibitory effects on parasite growth. Compounds were added to
The resulting aTcPFK and hTcPFK structures were further refined
using molecular dynamics simulations. The Amber16 software
package was used [46], applying molecular mechanics parameters

G.E. Miana et al. / European Journal of Medicinal Chemistry 165 (2019) 1e10
9
corresponding to the Amber ff14SB force field [47]. The para-
the NVIDIA GPU Grant Program.
meterised systems were submitted to a standard minimisation
protocol, applying 5000 steps of computation (2500 steps of
steepest descent and 25000 conjugate gradient searches). The
resulting structures were used as structural templates for molec-
ular docking studies.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.01.013.
4.4.2. Molecular docking procedures
References
In order to elucidate the molecular target responsible of the
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initiated against diverse known targets of Tc. Among them, cruzi-
pain was initially screened using the AutoDock software; however a
lack of correlation between the corresponding binding modes and
the observed anti-Tc activity was found.
In a next stage, PFK was screened as potential target involved in
the anti-Tc activity. In this case, docking procedures were per-
formed using software packages developed by OpenEye Scientific
Software [48], and consisted of three sequential stages: 1) the
ligand conformer library generation stage, which was conducted
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software [44,51,52]. In this case, the ChemGauss3 scoring function
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lowest energy docked poses were selected for model development.
Stage 3 involved a three-dimensional visualisation and intermo-
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