Benzoic acid derivatives with trypanocidal activity: Enzymatic analysis and molecular docking studies toward trans-sialidase

Article abstract of DOI:10.3390/molecules22111863

Chagas, or American trypanosomiasis, remains an important public health problem in developing countries. In the last decade, trans-sialidase has become a pharmacological target for new anti-Chagas drugs. In this work, the aims were to design and find a new series of benzoic acid derivatives as trans-sialidase (TS) inhibitors and anti-trypanosomal agents. Three compounds (14, 18, and 19) sharing a para-aminobenzoic acid moiety showed more potent trypanocidal activity than the commercially available drugs nifurtimox and benznidazole in both strains: the lysis concentration of 50% of the population (LC₅₀) was <0.15 μM on the NINOA strain, and LC50 < 0.22 μM on the INC-5 strain. Additionally, compound 18 showed a moderate inhibition (47%) on the trans-sialidase enzyme and a binding model similar to DANA (pattern A).

Full text of DOI:10.3390/molecules22111863

molecules
Article
Benzoic Acid Derivatives with Trypanocidal Activity:
Enzymatic Analysis and Molecular Docking Studies
toward Trans-Sialidase
Muhammad Kashif 1 ID , Antonio Moreno-Herrera 1, Juan Carlos Villalobos-Rocha 2,
Benjamín Nogueda-Torres 3, Jaime Pérez-Villanueva 4, Karen Rodríguez-Villar 4,
José Luis Medina-Franco 5 ID , Peterson de Andrade 6, Ivone Carvalho 6 and Gildardo Rivera 1,
*
1
Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico
Nacional, Boulevard del Maestro, s/n, Esq. Elías Piña, Reynosa 88710, Mexico;
chkashif987@gmail.com (M.K.); amorher9@hotmail.com (A.M.-H.)
Laboratorio de Bioquímica Microbiana, Departamento de Microbiología, Escuela Nacional de Ciencias
2
Biológicas, Instituto Politécnico Nacional, Ciudad de México 11340, Mexico; jvillalobosr@ipn.mx
Departamento de Parasitología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
Ciudad de México 11340, Mexico; bnogueda@yahoo.com
Departamento de Sistemas Biológicos, División de Ciencias Biológicas y de la Salud, UAM-X,
Ciudad de México 04960, Mexico; jpvillanueva@correo.xoc.uam.mx (J.P.-V.);
qkarenrodv@hotmail.com (K.R.-V.)
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México,
México City 04510, Mexico; medinajl@unam.mx
3
4
5
6
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. Café s/n,
Ribeirão Preto SP 14040-930, Brazil; petersondeandrade32@gmail.com (P.d.A.); carronal@usp.br (I.C.)
Correspondence: gildardors@hotmail.com; Tel.: +52-899-924-3627; Fax: +52-899-924-3628
*
Received: 5 October 2017; Accepted: 24 October 2017; Published: 30 October 2017
Abstract: Chagas, or American trypanosomiasis, remains an important public health problem in
developing countries. In the last decade, trans-sialidase has become a pharmacological target for
new anti-Chagas drugs. In this work, the aims were to design and find a new series of benzoic acid
derivatives as trans-sialidase (TS) inhibitors and anti-trypanosomal agents. Three compounds (14, 18,
and 19) sharing a para-aminobenzoic acid moiety showed more potent trypanocidal activity than the
commercially available drugs nifurtimox and benznidazole in both strains: the lysis concentration of
50% of the population (LC50) was <0.15 µM on the NINOA strain, and LC50 < 0.22 µM on the INC-5
strain. Additionally, compound 18 showed a moderate inhibition (47%) on the trans-sialidase enzyme
and a binding model similar to DANA (pattern A).
Keywords: benzoic acid; Chagas disease; docking; inhibitors; trans-sialidase
1. Introduction
Chagas disease, or American trypanosomiasis, is a chronic disease caused by the kinetoplastid
protozoan parasite Trypanosoma cruzi. Although this disease is present mostly in the endemic poor
rural areas of America (from Southern California to Argentina), it is also becoming an important
health issue in metropolitan areas [
1
] and other non-endemic areas, such as North America [
2] and
Europe [ ], due to population migrations [5–
3
,
4
11]. In fact, the World Health Organization (WHO) has
estimated that Chagas disease is nowadays affecting about 7 to 8 million people worldwide [12] and
accounting for the death of over 12,500 patients per year [13,14].
Strategies to manage Chagas disease are based fundamentally on the regional elimination of
domestic vectors [15] and on the pharmacological treatment of patients. According to chemotherapy
Molecules 2017, 22, 1863; doi:10.3390/molecules22111863
www.mdpi.com/journal/molecules

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treatment, the nitroheterocyclic compounds benznidazole (N-benzyl-2-(2-nitro-1H-imidazole-1-
yl)acetamide, Bnz) and nifurtimox (3-methyl-N-[(5-nitrofuran-2-yl)methylidene]thiomorpholin-4-
amine-1,1-dioxide, Nfx), launched by Bayer in 1967 and Roche in 1972, respectively, are the only
two drugs currently used for the treatment of Chagas disease, although they are not effective
against its chronic form [16], diminish in efficacy the longer a person has been infected [12], and the
treatment is associated with severe toxic side-effects [17–22]. In order to address this major health
issue, new anti-Chagas drugs directed to different targets are being designed in order to modulate
relevant enzyme activities in biochemical pathways, such as cysteine proteases, trans-sialidase,
and trypanothione reductase [23].
Trypanosoma cruzi trans-sialidase (TcTS) is a potential target for Chagas disease chemotherapy.
The surface of invasive T. cruzi trypomastigotes is covered by numerous trans-sialidases, whose
main role is to acquire sialic acid units from mammalian host glycoconjugates and transfer them to
the parasite membrane generating α-2,3-sialylated-β-galactopyranose units and providing (i) direct
protection from recognition by the host’s immune system [24], and (ii) resistance to the complement
and immediate survival of trypomastigotes released to the bloodstream [25]. TcTS also aids in the
recognition of and attachment to host cells through active site-mediated binding to sialic acids and/or
β
-galactosyl residues on the surface of host cells [26
site [31,32].
The active site of TcTS contains several conserved microbial sialidase features showing a donor
site related to sialic acid interaction and an acceptor site associated with the -galactose molecule.
–30] or through domains distinct from the active
β
The donor site contains an arginine triad (Arg35, Arg245, and Arg314), which interacts with the
carboxylate group (Figure 1) of sialic acid, two residues for the stabilization of the transition state
(Tyr342 and Glu230), an aspartate (Asp59) essential for catalysis, and a hydrophobic pocket for
accommodating sialic acid’s N-acetyl group. The acceptor site contains the amino acids Tyr119 and
Trp312, which is crucial for the trans-glycosylation process, Asp59 (common to both sites), and Glu362,
which directly interacts with the β-galactose acceptor [14,33].
Figure 1. Sialic acid analogous 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (DANA) interaction
on active sites of Trypanosoma cruzi trans-sialidase (TcTS) enzyme (green line showing a conventional
hydrogen bonding interaction and red lines showing a hydrophobic interaction). The image was
produced with LigPlot+ software (v.1.4, European Bioinformatics Institute (EMBL-EBI), Hinxton,
Cambridge, UK).

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The inhibitors reported for TcTS have complex chemical structures. DANA, (
1
, Figure 2), a potent
inhibitor of influenza neuraminidase, poorly inhibits TcTS with a reported Ki of 12.3 mM [34].
The DANA analogs have exhibited improved (Ki of 12.2 mM, IC50 of 5 mM and 10 M, respectively)
but still weak TcTS inhibition [35 36]. The 2,3-difluorosialic acid analogs, such as (Figure 2),
2–
4
µ
,
5
are mechanism-based TcTS inhibitors, which form a covalent bond with the hydroxyl group of Tyr342,
but only at high concentrations (mM range) [37]. Mimicking a sialic acid structure has been explored
in the search for potential TcTS inhibitors. The benzoic acid derivative compound
be highlighted, showing an IC50 of 0.54 mM. In the predicted docked conformation of
6
(Figure 2) [35] can
(Figure 2),
6
its carboxylate moiety provides the expected strong interaction with the arginine triad (residues 35, 245
and 314). The hydroxyl group can form hydrogen bonds with both Glu230 and Gln195, and the amide
NH group is hydrogen-bonded to Asp96, with the acetyl group located in the hydrophobic pocket.
However, the apparently good fit predicted for 6 did not translate into strong TcTS inhibition [35].
Figure 2. Structure of representative trans-sialidase inhibitors.
Compounds such as lactitol
analogues, which target the sialic acid acceptor site, were also weak inhibitors [38
TcTS inhibitor known, resulting from modifications in the GM3 ganglioside ( , Figure 2) and with IC50
values in the 10–100 M range [40], fills in both sialic acid acceptor and donor sites. This result shows
the importance of designing compounds occupying both binding sites. This can clearly be observed in
the docked conformation of compound (MuNANA assay screening at 1 mM: 87% inhibition and
7
(IC50 = 0.57 mM) (Figure 2) and other lactose (acceptor substrate)
,
39]. The most potent
8
µ
9
IC50 = 0.21 mM) (Figure 2), where the respective carboxylate group interacts well with the arginine
triad, and the amide function hydrogen bonds with Arg245 and Asp59, thus providing additional
stabilization of the triad. The thiophene ring is seen in the sialic acid acceptor binding site and the
benzene ring is positioned in the sialic acid site. The latter does not fill this site, thus the introduction
of functional groups could provide further interactions with key amino acid residues (e.g., Asp96,
and Gln195) [41].
In view of these previous works, and taking into account that inhibitor design against the
sialidases or trans-sialidases of trypanosomes is reduced, our approach involved the synthesis of
new non-sugar-based benzoic acid derivative inhibitors of TcTS that may lead to biological tools for

Molecules 2017, 22, 1863
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or potential leads in drug design. The fact that the negatively charged carboxylate group of sialic
acid represents the most important group able to interact with the sialic acid donor site through
strong interactions with the arginine triad led us to design simple structures containing this group.
Neres et al., 2007 synthesized meta- and para-substitutions of benzoic acid derivatives providing
4-acetylamino-3-hydroxymethylbenzoic acid (6, Figure 2) as the lead inhibitor, not containing the
synthetically complex sialic acid structure (IC50 = 0.54 mM). However, the weak inhibition prompted
us to synthesize new non-sugar-based benzoic acid derivative inhibitors of TcTS with better interaction
with both the sialic acid donor and acceptor sites of the TcTS enzyme. Our strategy was based on a
molecular simplification of these previous compounds, performing slight, but chemically sensitive
changes in the meta and para positions to the carboxylate group, and at the same time, we wondered
whether or not an ortho substituent to the carboxylate group in the aromatic ring could establish better
interaction with both the sialic acid donor and acceptor sites of the TcTS enzyme. In fact, docking
studies were performed to support the design of better TcTS inhibitors and showed that there is a
possibility of including an ortho substituent to the carboxylate group in the aromatic ring, which could
bind the acceptor substrate binding site, thus offering room for improvement [35]. In this context,
and following the interest in our research group to develop new therapeutic options for Chagas disease,
we designed new derivatives of benzoic acid with ortho-, meta-, and para-substitutions according to
preliminary docking studies and calculated physicochemical properties (Figure 3).
Figure 3.
(a) dry CH3CH2OH, conc H2SO4,
; (d) H2SO4, ; (e) p-ClC6H4COCl, Et3N (19), p-CH3OC6H4COCl, Et3N (20); (f) HCl, NaNO2,
and CH3COONa/NaN3 in H2O; (C) Compounds acquired from Sigma-Aldrich, Mexico.
(
A,
B) Compounds designed and synthesized in this work. Reagents and condition:
∆
; (b) CH3CO2H/(CH3CO)2O, ; (c) HNO3 and conc H2SO4,
∆
∆
∆

Molecules 2017, 22, 1863
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2. Results and Discussion
2.1. Synthesis
Benzoic acid derivatives (10
,
11,
13
,
21 and 23) [42
,
43] were purchased from Sigma-Aldrich.
The other compounds were synthesized through the reported procedure for esterification (14), nitration
17 and 18), hydrolysis (16), and condensation (15 19 20 22 and 24) [44 46] as detailed in the
(
,
,
,
–
Experimental Section. All compounds were obtained easily with good yields (60–90%), except
compounds 19 and 20 (20–40%).
2.2. Trypanocidal Activity
Initially, the lytic effect of the benzoic acid derivatives and reference drugs (Nfx and Bnz) on mouse
blood trypomastigotes was evaluated in vitro, using INC-5 and NINOA T. cruzi strains (Table 1) [47].
Furthermore, 4-acetamidobenzoic acid (24) previously assayed by Neres et al. [35] for TcTS inhibition
has been studied.
Table 1. Trypanocidal activity of benzoic acid derivatives on the NINOA and INC-5 strains.
R1
R2
R3
O
R4
Code
R1
R2
R3
R4
NINOA LC50 (µM)
INC-5 LC50 (µM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Nfx
Bzn
NH2
NHNH2
N3
NO2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
OH
OH
OH
0.52 ± 0.19
0.66 ± 0.39
0.60 ± 0.46
0.47 ± 0.16
0.10 ± 0.041
0.34 ± 0.18
1.37 ± 0.56
1.10 ± 0.58
0.02 ± 0.012
0.14 ± 0.08
0.61 ± 0.3
1.24 ± 1.0
0.58 ± 0.4
0.47 ± 0.35
0.46 ± 0.38
0.10 ± 0.047
0.21 ± 0.1
OH
OCH2CH3
OCH2CH3
OH
OH
OCH2CH3
OH
NHCOCH3
NH2
H
NO2
NO2
NO2
NO2
NO2
H
H
H
H
0.63 ± 0.3
NHCOCH3
NHCOCH3
NHCOC6H4-p-Cl
NHCOC6H4-OCH3
NH2
0.21 ± 0.1
0.22 ± 0.09
0.0008 ± 0.0001
0.43 ± 0.28
0.26 ± 0.09
1.28 ± 0.31
0.721 ± 0.42
0.878 ± 0.55
0.68 ± 0.17
0.62 ± 0.28
OH
OH
OH
OH
0.27 ± 0.10
1.28 ± 026
0.576 ± 0.32
1.39 ± 0.75
0.213 ± 0.08
0.292 ± 0.12
NHCOCH3
H
NHCOCH3
OH
LC50: lysis concentration of 50% of the population.
Biological activity showed that both reference compounds Nfx and Bnz have a similar lysis
concentration of 50% of the population (LC50) in each strain: 0.21–0.29 µM against the NINOA strain
and 0.62–0.68
the ethyl benzoate derivatives 14 (benzocaine, a known anesthetic agent), 15, and 18 displayed
significant trypanocidal activities in the range of 0.02–0.34 M against the NINOA strain and
0.10–0.22 M against the INC-5 strain, comparing to those of the corresponding carboxylic acid
precursors 10 24, and 17, respectively: 0.52–1.39 M and 0.21–1.24 M in the NINOA and INC-5
µM against the INC-5 strain. As shown in Table 1, amongst the tested compounds,
µ
µ
,
µ
µ
strains, respectively. Thus, the introduction of a hydrophobic group such as ethyl showed an increase
of activity. In fact, these compounds presented trypanocidal activity comparable to the reference Bzn
and Nfx against the NINOA strain and significantly higher against the INC-5 strain.

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Compound 19, showing significant trypanocidal activity against the NINOA strain (0.14
and the highest activity against the INC-5 strain (0.0008 M), became a lead structure of the benzoic
derivative series. The subsequent modification of this structure by replacing the chloride substituent
µM)
µ
by the acceptor methoxy group 20 did not improve the activity in both the NINOA (0.61
INC-5 (0.43 µM) strains.
µM) and
Compound 10 is a simplification of the structures synthesized by Neres et al. [35] displaying
moderate trypanocidal activity against the NINOA (0.52 M) and INC-5 (1.24 M) strains.
µ
µ
The structure-activity relationship (SAR) of these compounds showed that the replacement of the
para-amino group by nitro (13), hydrazine (11), or azide (12) groups showed comparable trypanocidal
activities against the NINOA strain (0.47–0.66
(0.46–0.58 M). On the other hand, the meta-nitration of compound 10 afforded compound 16 to
display a notably reduced activity against the NINOA strain (1.37 M), whereas it increased the
activity against the INC-5 strain (0.63 M). Alternatively, ortho-hydroxylation afforded compound
21 to display significant trypanocidal activity against the NINOA (0.27 M) and INC-5 (0.26 M)
µM) and an improvement against the INC-5 strain
µ
µ
µ
µ
µ
strains. Compound 21 presented a higher trypanocidal activity than references Bzn and Nfx. Thus,
the incorporation of a hydrophilic group as a hydroxyl in the ortho position of the para-amino benzoic
acid moiety may provide a center for H-bond interactions and it might be positively affecting the
biological activity. In fact, the elimination of the para-amino group generated compound 23 with lower
trypanocidal activities (0.57
in addition, the N-acetylation of this group (compound 22) significantly reduced the biological activity
(1.28 M in both strains). This was partially observed in compounds 24 and 17, which did not reflect an
improvement of the activity in relation to the para-aminobenzoic acid precursors 10 and 16 against the
NINOA strain (>1.0 M) but showed better trypanocidal activities against the INC-5 strain (0.87 and
0.21 M, respectively). It is remarkable that a different trypanocidal activities tendency was observed
in both strains. Finally, three compounds (14 18, and 19) showed more potent trypanocidal activity
than the commercially available drugs Nfx and Bnz in both strains: LC50 < 0.15 M in the NINOA
strain and LC50 < 0.22 M in the INC-5 strain, all of them sharing the modified para-aminobenzoic
µM and 0.72 µM against the NINOA and INC-5 strains, respectively) and
µ
µ
µ
,
µ
µ
ester moiety except 19. These structures can be used as references for current and future studies
for the synthesis of new anti-Chagas compounds. We have demonstrated that the incorporation of
ortho-hydroxyl groups in para-aminobenzoic acid derivatives successfully provided a more potent
compound (21). The esterification of the carboxylic acid (14, 15, and 18) generating a hydrophobic
moiety to increase the activity could be a good strategy in these compounds. However, the introduction
of a meta-nitro group did not initially generate a more active compound (16), although subsequent
structural modifications finally modulated the activity and improved it. Further assays are required to
understand the electronic and steric properties of meta and ortho substituents in the para-aminobenzoic
derivative structures.
2.3. TcTS Inhibition
TcTS inhibition screening results, including percentage inhibition values for a series of substituted
benzoic acid derivatives, are given in Table 2. The percentage inhibition at 1 mM concentration
is the average of at least three independent experiments. The enzymatic inhibition assay was
performed using a continuous fluorimetric method based on the TcTS-catalyzed hydrolysis of
2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MuNANA). As a control, the activities of
pyridoxal phosphate (Pyr) and compound 24
[35] were measured in the same concentrations of the
target compounds due to their respective moderate [48] and weak activities on TcTS.

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Table 2. TcTS inhibition values of benzoic acid derivatives.
R1
R2
R3
O
R4
Code
R1
R2
R3
R4
% Inhib. at 1 mM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
NH2
NHNH2
N3
NO2
NH2
NHCOCH3
NH2
NHCOCH3
NHCOCH3
H
H
NO2
H
H
H
NO2
NO2
NO2
NO2
NO2
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
OH
OH
OH
30
61
40
43
1
7
77
66
OH
OCH2CH3
OCH2CH3
OH
OH
OCH2CH3
OH
47
NHCOC6H4-p-Cl
NHCOC6H4-p-OCH3
NH2
Not tested *
Not tested *
OH
OH
OH
OH
32
34
17
30
64
NHCOCH3
H
NHCOCH3
H
H
H
24
Pyr
OH
* Not tested due to low solubility. The standard deviation for each experiment was <5%.
Pyridoxal has been reported as a non-competitive TcTS inhibitor with a Ki = 7.3 mM [49] showing
a moderate inhibition (73% at 1.0 mM in the presence of MuNANA 0.1 mM). In this work, the inhibition
percentage of pyridoxal phosphate was 64%, similar to previous studies [50]. On the other hand,
contrary to results described by Neres et al. [35], the inhibition of compound 24 was 30% instead of 0%.
These differences could be mainly associated with the enzymatic assays’ particularities. Nonetheless,
this compound shows a weak inhibition, lower than pyridoxal phosphate.
According to Table 2, compounds 10–24 showed a variable influence on TcTS enzyme inhibition.
All of the substances displayed a lower inhibitory activity against TcTS than pyridoxal, excepting
compounds 11 and 17 which blocked 61 and 66% of the enzyme activity, respectively, with the same
inhibitory effect as that of pyridoxal, and compound 16 showed the best inhibition activity (77%).
Based on the inhibitory activity of these compounds, the para-amino-meta-nitrobenzoic acid core
may have a relevant role in TcTS inhibition. Accordingly, compound 18, obtained from the ethyl
esterification of compound 17, also showed moderate results (47%); however, esterification decreases
the activity. This behavior also can be observed in compounds 10 and 24, where the activity decreases
as compared with their ethyl ester analogs 14 and 15, respectively. The lack of the nitro substituent
reduced notably the inhibition activity of these compounds. Consequently, the ortho-hydroxyl benzoic
acid derivative compounds 21
–23 and the para-amino benzoic acid/benzoate derivative compounds
10 14 and 15 showed weak inhibition results in the range of 1–34%. However, the replacement of
,
the para-amine group of the benzoic acid by hydrazine (11), azide (12), or nitro (13) groups lead to
moderate results, with 61, 40, and 43% of inhibition, respectively. Again, the nitro group observed in
compound 13 plays an important role in the interaction with the TcTS active site. Compounds 19–20
were not tested due to a low solubility in DMSO 1.0% in the well.
The significant TcTS inhibition shown by compounds 11, 16 and 17 (>60% inhibition) may direct
the development of new derivatives as TcTS inhibitors, pointing out the necessity to maintain benzoic
acid/benzoate core and nitrogenous moieties (amine, N-acetyl, nitro, azide, or hydrazine) for an
efficient inhibition of this enzyme.

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In summary, we can observe that there is no correlation between trypanocidal activity and TcTS
inhibition. For example, of the benzoate derivatives 14 15 and 17 19 showing moderate trypanocidal
activities (0.10–0.028 M in the NINOA strain and 0.0008–0.22 M in the INC-5 strain), only compound
–
–
µ
µ
15 moderately blocked the TcTS enzyme as expected with 47%, whereas the other compounds
inhibited less than 7%. Nevertheless, the importance of para-amino carboxylic derivatives acting
as TcTS inhibitors or lysing trypomastigotes via other biological mechanisms against T. cruzi has
been demonstrated.
2.4. Molecular Docking
Docking studies were conducted for compounds 10–24 in order to obtain potential putative
interaction of these compounds on the active site of TcTS. The docking analysis was validated using the
crystal structure of TcTS against the reference inhibitor DANA [34]. The good superposition between
the DANA structures oriented with AutoDock Vina 5.6 as compared with the orientation in the crystal
structure (Protein Data Bank (PDB) accession number 1MS8) suggested that the chosen method is
appropriate (Figure 4). The potential DANA binding site was also predicted as a deep cavity including
a restricted space with two delimited regions, where the blue shift indicates a positive electrostatic
potential (carboxylate interaction) and the red shift a negative electrostatic potential (amide interaction)
(Figure 4).
Figure 4. Best scored binding mode for DANA obtained with AutoDock Vina (gray) and binding mode
for DANA in the original crystal structure (green). The TcTS surface is color-coded by the electrostatic
potential (blue shift showing positive electrostatic potential & red shift showing negative electrostatic
potential).
In this study, the proposed interaction modes of the benzoic and benzoate derivatives into the
active site of TcTS were determined as the highest-scored conformations (best-fit ligands), which
correspond to the structure with the most favorable free energy for binding in TcTS.
According to the results (Table S1, Supplementary Materials), two major binding patterns were
found, A and B (Figure 5A,B). Moreover, some conformations that did not have a particular binding
mode were grouped as C (Figure 5C). The first mode (A) is similar to that reported for DANA in
the crystal structure (Figure 5A). Although this binding mode was found to be the best result for
compounds 15, and 24, it does not correspond to the lowest energy conformation in most cases. It is
noteworthy that, binding mode A was found to be among the best nine conformations for most of the
ligands, having a good calculated affinity (Table S1). Similarly, molecules 12 and 16–19 showed their

Molecules 2017, 22, 1863
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best-scored binding in an alternative way, designated A2 (Table S1), which is a slight variation of the
binding mode A.
Figure 5. Best-scored conformations obtained for compounds 10
ligand DANA in the crystal structure of TcTS (green). Compounds with binding modes classified as
A are shown in pannel ( ) as yellow structures; those classified as B are shown in pannel ( ) as pink
structures; and those classified as C are shown in pannel (C) as orange structures.
–24 and their comparison with the
A
B
Figure 6A shows the best conformation for compound 24, which corresponds to binding mode A,
where the carboxylate group forms hydrogen bound interactions with Arg314 and Arg245 (donor site);

Molecules 2017, 22, 1863
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these interactions can be slightly different for each ligand in binding mode A. For the molecules in
binding mode A2, the nitro group adopts a similar orientation to the carboxylate group in DANA.
Figure 6. Best-scored conformations and interactions of selected compounds with TcTS. (
A
) compound
24 in binding mode A; (B) compound 16 in binding mode A2; (C,D) compounds 13 and 10 in binding
mode B, respectively.
Figure 6B shows compound 16 in binding mode A2. It is noteworthy that, whereas the nitro group
shows interactions with Arg314, Arg245, and Trp312, the carboxylate and amino groups have additional
interactions with Gln195, Trp120, and Asp59, respectively. It is worth noting that all nitro benzoic
acids and nitro benzoates are classified among the most active compounds in this series. Moreover,
compound 16 has a higher score value and good inhibition as compared with its non-nitrated analog
15. Therefore, the results suggest that the nitro group plays an important role in binding TcTS.
The binding mode B observed in this study differs from the DANA crystal structure (opposite
binding conformation), and has not been previously described (Figure 5B). Binding mode B
is associated with compounds 10, 11, 13, 14, 21 and 24 in their best-scored conformation;
other compounds that exhibit binding mode B also have good scores, but not the best. Arg93 and
Trp120 act as hydrogen donors to bind the carboxylate group at the position 1. These interactions can
also be found involving a nitro group as a hydrogen acceptor. A variation of binding mode B was
found where the amine group or derivatives at the position 4 form an interaction with Arg93 and
Asp59. Figure 6C,D shows examples of compounds in binding mode B.
The third binding mode (C) did not show a specific binding conformation, and it was different
from modes A and B. It is associated with compounds 20, 22 and 23; other compounds that exhibit
binding mode C have good scores, but not the best. These compounds do not involve the characteristic
interactions observed in the A and B modes or are binding on the exterior of the cavity (Figure 5C).

Molecules 2017, 22, 1863
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Table S1 (Supplementary Materials) shows the binding mode distribution for the best nine hits
for each compound. It is noteworthy that some compounds exhibit two and even more binding
modes with similar scores; therefore, the biological effect observed can be associated with several
binding orientations.
3. Materials and Methods
3.1. Chemistry: General Procedure
Compounds 10 (4-aminobenzoic acid, A9878), 11 (4-hydrazinobenzoic acid, 246395), 13
(4-nitrobenzoic acid, 461091), 21 (4-amino 2-hydroxybenzoic acid, A79604), and 23 (2-hydroxybenzoic
acid, 247588) were purchased from Sigma-Aldrich, Mexico city, Mexico, and used without further
purification. In all compounds, synthesized melting points were determined on a Mel-Temp capillary
apparatus (Electrothermal, Staffordshire, UK) and are uncorrected. Infrared spectra were recorded
1
using a Bruker Alpha FT-IR spectrometer (AXS Inc., Madison, WI, USA). The H-NMR spectra
were obtained in CDCl3 or DMSO-d6 with Me4Si as an internal standard on a Bruker Avance-300
1
Spectrometer operating at 400 MHz for H-NMR (AXS Inc., Madison, WI, USA). The purity and
reactions were monitored by thin-layer chromatography (TLC) performed on silica gel plates
prepared with silica gel 60 (PF-245 with gypsum, Merck, Tokyo, Japan), of the thickness of 0.25 nm.
The developed chromatograms were visualized under ultraviolet light at 254–265 nm.
For 4-Azidobenzoic acid (12): 4-aminobenzoic acid (10) (1.5 g, 10.9 mmol) was dissolved in HCl (15 mL)
◦
at 0 C. The reaction mixture was stirred for 1 h, and 25 mL aqueous solution of NaNO2 (0.1 N)
was added into the reaction mixture dropwise. The product was precipitated by adding the reaction
mixture to the solution of CH3COONa (4.5 g, 61.3 mmol) and NaN3 (0.70 g, 10 mmol) in H2O (500 mL).
The product was obtained by filtration and the residue was recrystallized in EtOH, yielding (12) (73%).
1
IR (KBr): 2980 (CH); 2180 (N3); and 1680 and 1610 (C=O) cm−1. H-NMR (400 MHz, DMSO-d6)
δ ppm:
7.1 (d, 2H, C6H4); 7.8 (d, 2H, C6H4); 12.1 (s, H, COOH). Calculated analysis for C7H5N3O2: C, 51.54; H,
3.09; N, 25.76. Found: C, 51.10; H, 2.75; N, 25.35.
Ethyl 4-aminobenzoate (14): 4-aminobenzoic acid (10) (3 g, 21.8 mmol) was dissolved in anhydrous
ethanol (30 mL). Concentrated H2SO4 (1.0 mL) was added to the mixture and refluxed for 60 min.
The reaction mixture was allowed to cool to room temperature, and the mixture was poured into 40 mL
of ice water with continuous stirring. The mixture was neutralized by adding 15 mL of 10% Na2CO3.
The white color precipitate was obtained, which was separated by vacuum filtration. The precipitates
were washed with H2O, yielding (14) (90%). IR (KBr): 3410 and 3335 (NH2); 2984 (CH); 1678 and 1628
1
(C=O) cm−1. H-NMR (400 MHz, DMSO-d6)
δ ppm: 1.3 (t, 3H, OCH2CH3); 4.1 (s, 2H); 4.3 (m, 2H,
OCH2CH3); 7.1–7.2 (m, 2H, C6H4); 7.8–7.9 (m, 2H, C6H4). Calculated analysis for C9H11NO2: C, 65.44;
H, 6.71; N, 8.48. Found: C, 65.30; H, 6.45; N, 8.15.
Ethyl 4-acetamidobenzoate (15): Ethyl 4-aminobenzoate (14) (1.5 g, 9 mmol) was added to a mixture (1:1)
of acetic acid and acetic anhydride (20 mL), stirred, and the reaction mixture was refluxed for 15 min.
After completion of the reaction, the mixture was poured into ice-cooled water and a solid residue was
obtained after filtration. The crude was washed three times with 100 mL H2O to remove excess acid.
The crude was recrystallized in EtOH, yielding (15) (86%). IR (KBr): 3332 (NH); 2984 (CH); 1680 and
1
1596 (C=O) cm−1. H-NMR (400 MHz, DMSO-d6)
δ
ppm: 1.29 (t, 3H, OCH2CH3); 2.1 (s, 3H, CH3); 4.29
(m, 2H, OCH2CH3); 7.70–7.72 (m, 2H, C6H4); 7.88–7.90 (m, 2H, C6H4); 10.27 (s, 1H, NH). Calculated
analysis for C11H13NO3: C, 63.76; H, 6.32; N, 6.76. Found: C, 63.47; H, 6.05; N, 6.52.
For 4-Amino-3-nitrobenzoic acid (16): 4-acetamido-3-nitrobenzoic acid (17) (2 g, 8 mmol) was taken
in a reaction flask, H2SO4 (30 mL) was added dropwise with stirring for 15 min, and the mixture
was heated for 15 min at 100 ◦C. After the completion of the reaction, the mixture was poured into
ice-cooled water and a solid residue was obtained after filtration. The crude was washed three times
with 100 mL H2O to remove excess acid. The crude was recrystallized in EtOH, yielding (16) (90%).

Molecules 2017, 22, 1863
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1
IR (KBr): 3479 and 3362 (NH); 1622 (C=O); 772 (NO2) cm−1. H-NMR (400 MHz, DMSO-d6)
δ
ppm: 7.2
(s, 1H, C6H3NO2); 7.8 (s, 1H, C6H3NO2); 7.9 (s, 2H, NH2); 8.5 (s, 1H, C6H3NO2); 12.6 (s, 1H, COOH).
Calculated analysis for C7H6N2O4: C, 46.16; H, 3.32; N, 15.38. Found: C, 45.89; H, 3.11; N, 15.08.
For 4-Acetamido-3-nitrobenzoic acid (17): 4-acetamidobenzoic acid (24) (2.5 g, 14 mmol) was added
slowly to a mixture (1:1) of HNO3 and conc. H2SO4 (40 mL) with stirring for 10 min at 0 ◦C. After,
the mixture was stirred for 30 min at room temperature. The reaction mixture was poured into ice
water and neutralized by adding 15 mL of 5% Na2CO3. The product was obtained by filtration and
washed with an excess of H2O. The crude was purified by recrystallization with ethanol, yielding (17
)
1
(84%). IR (KBr): 3324 (NH); 2912 (CH); 1717 and 1672 (C=O); 773 (NO2) cm−1. H-NMR (400 MHz,
DMSO-d6)
δ ppm: 2.1 (s, 3H, CH3); 7.8 (s, 1H, C6H3NO2); 8.1 (s, 1H, C6H3NO2); 8.3 (s, 1H, C6H3NO2);
10.2 (s, 1H, NH); 12.6 (s, 1H, COOH). Calculated analysis for C9H8N2O5: C, 48.22; H, 3.60; N, 12.50.
Found: C, 47.90; H, 3.30; N, 12.20.
Ethyl 4-acetamido-3-nitrobenzoate (18): Ethyl 4-acetamidobenzoate (15) (0.65 g, 3 mmol) and a mixture
(1:1) of HNO3 and conc. H2SO4 (20 mL) were stirred and refluxed for 2 h. The reaction mixture was
poured into ice water and neutralized by adding 15 mL of 5% Na2CO3. The product was obtained by
filtration, washed with an excess of H2O, and the crude was purified by recrystallization with ethyl
acetate, yielding (18) (75%). IR (KBr): 3357 (NH); 2991 (CH); 1712 and 1620 (C=O); 771 (NO2) cm−1
.
1H-NMR (400 MHz, DMSO-d6)
δ
ppm: 1.33 (t, 3H, OCH2CH3); 2.12 (s, 3H, CH3); 4.31–4.37 (m. 2H,
OCH2CH3); 7.85 (d, 1H, C6H3NO2); 8.20 (d, 1H, C6H3NO2); 8.36 (s, 1H, C6H3NO2); 10.56 (s, 1H, NH).
Calculated analysis for C11H12N2O5: C, 52.38; H, 4.80; N, 11.11. Found: C, 52.10; H, 4.60; N, 10.80.
For 4-(4-Chlorobenzamido)-3-nitrobenzoic acid (19): 4-amino-3-nitrobenzoic acid (16) (0.60 g, 3 mmol),
4-chlorobenzoyl chloride (0.84 g, 4 mmol), and Et3N (1.0 mL) were dissolved in dry CH2Cl2 (40 mL)
and the reaction mixture was stirred for 48 h at room temperature. The mixture was filtered using a
vacuum and the residue was washed thrice with 200 (mL) of H2O to remove the acid (HCl) produced
during the reaction. The crude mixture was purified by column chromatography on silica gel in a
CH2Cl2/EtOAc (3:1), yielding (19) (40%). IR (KBr): 3370 (NH); 2850 (CH); 1782 and 1683 (C=O) cm−1
.
1H-NMR (400 MHz, DMSO-d6)
δ
ppm: 7.05 (d, 1H C6H3COOH); 7.5–7.6 (m, 2H, C6H4Cl); 7.83 (d, 1H,
C6H3COOH); 7.87–7.89 (m, 2H, C6H4Cl); 7.9 (s, 1H, NH); 8.5 (s, 1H C6H3COOH); 12.8 (s, 1H, COOH).
Calculated analysis for C14H9ClN2O5: C, 52.43; H, 2.83; N, 8.74. Found: C, 52.15; H, 2.56; N, 8.45.
For 4-(4-Methoxybenzamido)-3-nitrobenzoic acid (20): 4-amino-3-nitrobenzoic acid (16) (0.60 g, 3 mmol),
4-methoxybenzoyl chloride (0.90 g, 4 mmol), and Et3N (1.0 mL) were dissolved in dry CH2Cl2 (40 mL)
and the reaction mixture was stirred for 48 h at room temperature. The crude mixture was purified
by column chromatography on silica gel in n-hexane/EtOAc (7:3), yielding (20) (24%). IR (KBr): 3338
. δ ppm: 3.89 (s, 3H,
(NH); 2965 (CH); 1786 and 1683 (C=O) cm−1 1H-NMR (400 MHz, DMSO-d6)
CH3); 7.13–7.16 (m, 3H: 2H, C6H4OCH3 and 1H, C6H3COOH); 7.98 (d, 1H, C6H3COOH); 8.07 (d, 2H,
C6H4OCH3); 8.23 (s, 1H, NH); 8.67 (s, 1H, C6H3COOH); 12.8 (s, 1H, COOH). Calculated analysis for
C15H12N2O6: C, 56.96; H, 3.82; N, 8.86. Found: C, 56.65; H, 3.45; N, 8.50.
For 4-Acetamido-2-hydroxybenzoic acid (22): 4-amino 2-hydroxybenzoic acid (21) (1 g, 6.5 mmol) was
added to a mixture (1:1) of acetic acid and acetic anhydride (20 mL), stirred, and the reaction mixture
was refluxed for 15 min. After the completion of the reaction, the mixture was poured into ice-cooled
water and a solid residue was obtained after filtration. The crude was washed three times with 100 mL
H2O to remove excess acid. The crude was recrystallized in EtOH, yielding (22) (85%). IR (KBr):
1
3326 (NH); 2912 (CH); 1720 and 1680 (C=O) cm−1. H-NMR (400 MHz, DMSO-d6)
δ
ppm: 2.1 (s, 3H,
CH3); 7.6 (s, 1H, C6H3OH); 7.8 (s, C6H3OH); 8.1 (s, C6H3OH); 10.21 (s, 1H, NH); 12.6 (s, 1H, COOH).
Calculated analysis for C9H9NO4: C, 55.39; H, 4.65; N, 7.18. Found: C, 54.95; H, 4.10; N, 6.85.
For 4-Acetamidobenzoic acid (24): 4-aminobenzoic acid (10) (4 g, 29 mmol) was added to a mixture (1:1)
of acetic acid and acetic anhydride (20 mL), stirred, and the reaction mixture was refluxed for 15 min.
After the completion of the reaction, the mixture was poured into ice-cooled water and a solid residue

Molecules 2017, 22, 1863
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was obtained after filtration. The crude was washed three times with 100 mL H2O to remove excess
acid. The crude was recrystallized in MeOH, yielding (24) (90%). IR (KBr): 3340 (NH); 2539 (CH); 1700
1
and 1607 (C=O), cm−1. H-NMR (400 MHz, DMSO-d6)
δ
ppm: 2.08 (s, 3H, CH3); 7.68 (d, 2H, C6H4);
7.87 (d, 2H, C6H4); 10.24 (s, 1H, NH); 12.68 (s, 1H, COOH). Calculated analysis for C9H9NO3: C, 60.33;
H, 5.06; N, 7.82. Found: C, 59.80; H, 4.90; N, 7.52.
3.2. Biological Assays
3.2.1. Trypanocidal Activity
In vitro studies were carried out using two strains of trypomastigotes of Trypanosoma cruzi:
NINOA and INC-5. CD-1 Mice (18–20 g) were inoculated intraperitoneally with 1
×
106/mL of
blood trypomastigotes (0.2 mL). Blood was obtained by cardiac puncture of mice infected with
trypomastigotes at the peak of parasitemia, using heparin as an anticoagulant. Blood was treated with
isotonic saline (NaCl 0.85%) to adjust to a concentration of approximately 2
×
106 trypomastigotes/mL
and 195 L of blood and 5 L of treatment was placed in 96-well plates. Treatments consisted
µ
µ
of a negative control, containing dimethyl sulfoxide (DMSO 2.5%) and compounds derived from
benzoic acid, and nifurtimox and benznidazole dissolved in DMSO at the following concentrations:
200
50% of the population (LC50). LC50 values were determined using a Probit statistical analysis of the
dose-response, and the results are expressed as the mean standard deviation. Finally, crystal violet
(1 g/mL) for the lysis of the trypomastigotes and as witness wells containing 200 L of blood without
receiving any treatment were used. Each concentration was tested in triplicate.
µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, and 12.5 µg/mL, to get the lysis concentration of
±
µ
µ
Once the compounds derived from benzoic acids after being homogenized with the blood were
added, the plates were incubated at 4 ◦C for 24 h. After the incubation, the plates were kept at room
temperature for 30 min and then an aliquot of 5 mL of each well was taken, which was placed between
a slide and a coverslip, and viable trypomastigotes were counted using the method of Brener [51
]
supplemented with Pizzi. The results were later converted to micromolar units.
3.2.2. Enzymatic Inhibition Assays
Inhibition was assessed using the continuous fluorimetric assay described by Douglas and
co-workers [52]. The assay was performed in triplicate (and on three different days) in 96-well plates
containing phosphate buffer solution at pH 7.4 (25
and an inhibitor solution (25 L of a 4.0 mM solution). This mixture was incubated for 10 min at
26 ◦C followed by the addition of MuNANA (Km = 0.68 mM; 25
L of a 0.4 mM solution giving an
µL), a recombinant enzyme solution (25 µL),
µ
µ
assay concentration of 0.1 mM). The final concentration of the tested compounds was 1.0 mM, and the
positive control was pyridoxal phosphate. The fluorescence of the released product (Mu) was measured
after 10 min, with excitation and emission wavelengths of 360 and 460 nm, respectively, and the data
were analyzed with GraphPad Prism software version 4.0 (San Diego, CA, USA). Inhibition percentages
were calculated by the equation: % I = 100
the inhibitor, and V0 is the velocity in the absence of the inhibitor.
×
[1
−
(Vi/V0)], where Vi is the velocity in the presence of
3.2.3. Molecular Docking
Ligand preparation: Compounds 10
optimized using the universal force field (UFF) [53
–
24 were built in Maestro 9.1 and their geometry was
54]. Then, the ligands were exported to AutoDock
,
Tools 1.5.6 in .pdb format to generate .pdbqt files [50,55,56].
Protein preparation: The TcTS were obtained from the Protein Data Bank with the PDB accession
number 1MS8 [53]. The structure was prepared using Maestro 9.1 [56], first, the chain A was selected
and the ligands, solvent, and other molecules were removed. Missing side chains were added and
alternative side chains were defined using Maestro. Then, the .pdb structure was submitted to

Molecules 2017, 22, 1863
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minimization using the YASARA web server [57]. The optimized structure was exported to AutoDock
Tools 1.5.6 in .pdb format to generate a .pdbqt file.
Docking studies: AutoDock Vina 5.6 was used to predict the mode of interaction for each ligand
within the active site of TcTS [58]. A grid box of x, y, and z dimensions was set to 60, 60, 60 angstroms
centered to the DANA binding site in the original crystal structure. Each compound was set to run
100 dockings, and the best nine conformations were retrieved for the analysis. The results were
analyzed employing PyMOL v0.99 and UCSF Chimera 1.11rc [59].
4. Conclusions
In this work, the benzoic acid derivatives 14, 18 and 19 clearly showed more potent trypanocidal
activity than the commercially available drugs beznidazole and nifurtimox towards the NINOA and
INC-5 strains of T. cruzi. It is noteworthy that compound 18 showed nanomolar trypanocidal activity
against the NINOA strain (20 nM), whereas compounds 11 and 17 displayed similar TcTS inhibition to
pyridoxal and compound 16 showed the best inhibitory activity.
TcTS inhibition assays provided evidence that the p/m-nitrobenzoic acid cores (13
p-hydrazine benzoic acid ( ) are relevant for TcTS inhibition, whilst trypanocidal assays against the
NINOA and INC-5 strains showed higher anti-parasite activity for the p-aminobenzoate derivative
compounds (14 15) and the p-amino-o-hydroxylbenzoic acid compound 21 via alternative mechanisms.
The ethyl benzoate compounds 14 15, and 18 displayed higher trypanocidal activities than their
, 16–18) and the
2
–
,
precursors (10, 24, and 17, respectively) but a reduced inhibition of TcTS.
The respective docked structures of the compounds showed three different binding patterns
according to DANA crystal structure in the active site cavity. Model A is similar to DANA interaction
in the cavity, model B represents the opposite binding conformation, and model C is interactions
outside the cavity or that do not involve the characteristic interactions observed in the A and B modes.
The benzoic acid derivatives (10–24) evaluated in the present work for the treatment of
Chagas disease by the inhibition of TcTS or alternative biological mechanisms (trypanocidal activity)
reinforce the development of more effective candidates of this disease. Therefore, we suggest ethyl
4-acetamido-3-nitrobenzoate 18 as a prototype for the development of more effective TcTS inhibitors
against Chagas disease, which shows a moderate inhibition (47%), a binding model similar to DANA
(pattern A), and significant trypanocidal activity (LC50 values of 0.02 and 0.22
and INC-5 strains, respectively).
µM against the NINOA
Supplementary Materials: The following are available online. Table S1: Binding modes and docking scores for
compounds 10–24.
Acknowledgments: Muhammad Kashif thanks the Consejo Nacional de Ciencia y Tecnologia (CONACyT) for
the scholarship (No. 590887/715369). We wish to express our gratitude to the CONACyT (Proyecto Apoyado
por el Fondo Sectorial de Investigación para la Educación, CB-2014-01, 241615) for their financial support.
Gildardo Rivera and Benjamín Nogueda-Torres hold a scholarship from the “Comisión de Operación y Fomento
de Actividades Académicas” (COFAA-IPN) and “Programa de Estímulos al Desempeño de los Investigadores”
(EDI-IPN).
Author Contributions: B.N.-T., A.M.-H. and G.R. conceived and designed the experiments; M.K., B.N.-T. and
J.C.V.-R. evaluated the biological activity against T. cruzi; J.P.-V., K.R.-V. and J.L.M-F. carried out the in silico
analysis; P.d.A. and I.C. evaluated the compounds toward the TcTS enzyme; and M.K., A.M.-H. and G.R. wrote
the paper. All authors read and approved the final manuscript.
Conflicts of Interest: All participants declare no conflict of interest.
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Sample Availability: Samples of the compounds 10–24 are available from the authors.
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