In vitro and in silico evaluations of new aryloxy-1,4-naphthoquinones as anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1007/s00044-020-02512-9

In the search for new therapeutic alternatives for Chagas disease, a series of six aryloxy -naphthoquinone derivatives were synthesized and evaluated in vitro against Trypanosoma cruzi epimastigotes of the Tulahuén 2, INC-5, and NINOA strains. The compoun

Full text of DOI:10.1007/s00044-020-02512-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02512-9
ORIGINAL RESEARCH
In vitro and in silico evaluations of new aryloxy-1,4-
naphthoquinones as anti-Trypanosoma cruzi agents
Alejandra González1 Nohemí Becerra1 Muhammad Kashif2 Mercedes González3 Hugo Cerecetto3,4
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Elena Aguilera3 Benjamín Nogueda-Torres5 Karla F. Chacón-Vargas5,6 J. José Zarate-Ramos1
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Uziel Castillo-Velázquez1 Cristian O. Salas7 Gildardo Rivera2 Karina Vázquez
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Received: 23 December 2019 / Accepted: 16 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
In the search for new therapeutic alternatives for Chagas disease, a series of six aryloxy -naphthoquinone derivatives were
synthesized and evaluated in vitro against Trypanosoma cruzi epimastigotes of the Tulahuén 2, INC-5, and NINOA strains.
The compounds 3d and 4a showed better or similar trypanosomicidal activity than the reference drug nifurtimox. In
addition, 3d and 4a also elicited better trypanosomicidal activity than nifurtimox against T. cruzi bloodstream
trypomastigotes. On the other hand, 3b showed the highest selective indexes (SI values between 44 and 500, in the three
T. cruzi strains). Finally, molecular docking studies suggested that these compounds could be potential trypanothione
reductase inhibitors. Therefore, based on these new results, we validated that the aryloxy-naphthoquinone scaffold is
essential to obtain more selective cytotoxic and trypanosomicidal compounds.
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Keywords Aryloxy-naphthoquinones Chagas disease Trypanosoma cruzi Trypanothione reductase Cytotoxicity
Molecular docking.
Introduction
Pereira and Navarro 2013; De Souza 2014; Galaviz-Silva
et al. 2017). In Latin America, 25 million people live in risk
infection areas, while the Pan American Health Organiza-
tion reports 12,000 annual deaths related to Chagas disease
(CD). The incidence of the disease is high in rural areas
where environmental conditions favor the installation and
breeding of triatomine insects (Marques Pereira and
Navarro 2013).
American trypanosomiasis (AT) is recognized by the World
Health Organization (WHO) as one of the neglected tropical
diseases and a main cause of morbidity and mortality with 6
to 7 million people infected and ~21,000 deaths per year in
21 countries in Central and South America (Marques
AT is also known as CD, new world trypanosomiasis,
South American trypanosomiasis, and Chagas-Mazza dis-
ease. It was first described by Carlos Chagas in Brazil in
1909 and it is caused by a flagellated protozoan,
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02512-9) contains supplementary
material, which is available to authorized users.
* Gildardo Rivera
Montevideo, Uruguay
gildardors@hotmail.com
4
Área de Radiofarmacia, Centro de Investigaciones Nucleares,
Facultad de Ciencias, Universidad de la República,
Montevideo, Uruguay
* Karina Vázquez
karina.vazquezcn@uanl.edu.mx
5
Departamento de Parasitología, Escuela Nacional de Ciencias
1
Universidad Autónoma de Nuevo Leon, Facultad de Medicina
Veterinaria y Zootecnia, 66050 Escobedo, Nuevo Leon, México
Biológicas, Instituto Politécnico Nacional, 11340 Ciudad de
México, México
2
6
Laboratorio de Biotecnología Farmacéutica, Centro de
Facultad de Odontología, Universidad Cuauhtémoc Plantel
Biotecnología Genómica, Instituto Politécnico Nacional, 88710
Reynosa, Tamaulipas, México
Aguascalientes, 20196 Aguascalientes, Aguascalientes, México
7
Facultad de Química y de Farmacia, Pontificia Universidad
3
Grupo de Química Orgánica Medicinal, Instituto de Química
Católica de Chile, 702843 Santiago de Chile, Chile
Biológica, Facultad de Ciencias, Universidad de la República,

Medicinal Chemistry Research
Fig. 1 Compounds derived from
naphthoquinones (I and III) and
indolequinone (II) with
trypanosomicidal activity.
Trypanosoma cruzi (T. cruzi), a member of the Trypano-
somatidae family, Kinetoplastida order, which affects a
variety of mammals including humans (De Souza 2014;
Galaviz-Silva et al. 2017).
selectivity index (SI) that expresses the ratio of IC50 in L6
cells (rat skeletal myoblasts) and IC50 against epimastigotes
(SI = L6/IC50), expressed a low selectivity (SI < 5) which
led to future research to obtain molecules with less toxicity
(Bolognesi et al. 2008).
According to the WHO, in humans, AT can be treated
with benznidazole (Bnz) and nifurtimox (Nfx), which are
lethal against T. cruzi. These drugs are almost 100%
effective when they are administered in the acute phase of
the disease, which is the initial stage of infection. Never-
theless, their effectiveness diminishes as time elapses from
the start of infection. Besides, toxicity reactions and adverse
effects occur with Bnz and Nfx that on many occasions
cause abandonment of treatment (Heitmann et al. 2008). In
addition, both drugs are mutagenics (Melo and Ferreira
1990; Jurado and Pueyo 1995).
Recently, naphthoquinones were paid more consideration of
trypanosomicidal agents, in this sense Tapia et al. synthesized
and evaluated a series of new aryloxindol-4,9-diones against
the Y strain of T. cruzi epimastigotes. Compound II (Fig. 1)
presented the best IC50 with a value of 20 nM and a high SI =
625 (Selectivity index: expressed as the ratio of IC50 in J774
cells to IC50 in epimastigotes). A pharmacophoric study was
performed for this kind of molecules revealed that the presence
of an aromatic ring, hydrogen acceptor groups, and a hydro-
phobic ring are related with the biological activity (Tapia Apati
et al. 2014). Subsequently, a new series of aryloxy-quinones
were synthesized and evaluated against the Y strain of the T.
cruzi epimastigote stage and a murine macrophage cell line.
The obtained results showed that compound III displayed the
best trypanosomicidal activity and selectivity with an
IC50 = 20 nM and a SI = 625 (Fig. 1) (Vázquez et al. 2015).
In addition, it has been reported that naphthoquinone
derivatives are potential inhibitors of the flavoenzyme try-
panothione reductase of T. cruzi (TRTc), by enzymatic
assays and molecular docking. For example, it have been
reported that 3-(dibutylamino)-propylamine-1,4-naphtho-
quinone derivatives have a high inhibitory potential on
TRTc without inhibiting the homologous enzyme human
glutathione reductase, therefore the inhibition of TRTc is
specific. Likewise, they highlighted that the protonable
amino groups are critical for the recognition of TR (Salmon-
Chemin et al. 2000). However, naphthoquinone derivatives
could have more than one target for their trypanosomicidal
action, according to works carried out by Pieretti and col-
laborators, who suggested that naphthoquinones could carry
out their trypanosomicidal activity via multi-target
mechanism (Pieretti et al. 2013).
Currently, many research groups and nongovernmental
organizations are in search of various natural and/or synthetic
molecules as new alternatives for pharmacotherapy for AT.
Among these compounds, naphthoquinones stands out
because it presents diverse of biological properties, as in the
case of α and β-lapachone derivatives, which have
demonstrated activity against T. cruzi through various
mechanisms of action, such as the generation of reactive
oxygen species (Bombaca et al. 2019), and DNA and RNA
inhibition (Pinto and De Castro 2009). They also have
better activity against Candida albicans (Moraes et al.
2018). According to their origin, naphthoquinones are
classified as natural and synthetic. In the case of natural
naphthoquinones with trypanosomicidal activity against T.
cruzi epimastigotes, we find lapachol, β-lapachone, and α-
lapachone with IC50 values of 31.3, 0.21, and 24.7 µM,
respectively. The above naturally obtained naphthoquinone
derivatives were isolated from the bark of Bignoniaceae
trees (Salas et al. 2008). Synthetic naphthoquinones have
been reported as trypanosomicidal agents, for example, a
series of naphthoquinone derivatives were synthesized,
evaluated, and tested against Trypanosoma brucei rhode-
siense trypomastigotes, T. cruzi amastigotes, and Leishma-
nia donovani amastigotes by Bolognesi et al. in 2008. The
results obtained by this research group showed that com-
pound I (Fig. 1) had the best trypanosomicidal activity with
an IC50 of 1.26 µM against the amastigote stage of Tulahuén
C2C4 T. cruzi strain (Bolognesi et al. 2008). Furthermore,
compound I was more powerful than Bnz; nevertheless, the
Based on the aforementioned, in this work, the use of
naphthoquinone as a building block for the development of
new trypanosomicidal agents was considered. The naph-
thoquinone ring was functionalized according with three
criteria, to evaluate the effect of these modifications on the
trypanosomicidal effect. These modifications were: (a) the
substitution of the hydrogen atom on C-3 by a bromine, due

Medicinal Chemistry Research
Scheme 1 Synthesis of
compounds 3a–d and 4a, b.
Reagents and conditions:
K2CO3, DMF, rt, 6–7 h,
68–90%.
to it has been reported that this change could increase the
selectivity; (b) the substitutions of methoxy group or methyl
groups on aryloxy moiety, because in the previous studies
only electron-withdrawing groups (nitro or halogens) were
analyzed; and (c) a double substitution on C-2 and C-3 by
aryloxy moiety. In addition, to elucidate the possible
mechanism of action of this type of compounds, an in silico
molecular docking study was carried out on T. cruzi try-
panothione reductase (TcTR), a protein referred to as a
pharmacological target for naphthoquinone derivatives.
Table 1 Half-maximal inhibitory concentration on epimastigotes of T.
cruzi: Tulahuén 2, INC-5 and NINOA strains.
Compound
Tulahuén 2
IC50 (µM)
INC-5
NINOA
IC50 (µM)
IC50 (µM)
3a
3b
3c
0.37 0.01
0.20 0.02
0.31 0.03
0.45 0.009
0.49 0.03
0.79 0.08
7.7 0.5
2.60 0.13
1.56 0.12
4.30 0.14
1.09 0.10
1.20 0.12
10.23 0.14
1.30 0.23
4.42 0.16
2.27 0.13
6.43 0.17
1.69 0.12
1.30 0.11
15.51 0.20
1.14 0.34
3d
4a
4b
Nfx
Results and discussion
Chemistry
strains: Tulahuén 2, INC-5, and NINOA. The dose–response
assay used concentrations in a range of 1–50 μM to calculate
the half-maximal inhibitory concentration (IC50).
In Scheme 1 the synthetic step of compounds 3a–d and 4a,
b is shown. This synthesis followed the methodology
reported by Vázquez et al. 2015 with some modifications.
These aryloxy-naphthoquinones were obtained in moderate
to high yields (68–90%), and monosubstitution or double
substitution were controlled by the respective phenol molar
ratio. All compounds were purified by column chromato-
graphy and their structures were analyzed based on their
spectral properties (IR, UPLC-MS, 1H NMR, and 13C
NMR, see Supplementary section).
The results of the trypanosomicidal activity of aryloxy-
naphthoquinone derivatives on T. cruzi epimastigotes
(Tulahuén 2, INC-5, and NINOA strains) are shown in
Table 1. The results showed that all compounds displayed
better or equal trypanosomicidal activity than the reference
drug Nfx against Tulahuén 2 strain. The compound 3b
showed the best IC50 value (0.20 μM), which was 38 times
greater than Nfx. In a structure–activity relationship ana-
lysis, it was seen that compounds 3a and 3c, which had
methoxy groups in a phenyloxy-ring, did not increase their
trypanosomicidal activity with regard to 3b. On the other
hand, the presence of methyl groups in the aromatic ring
and the addition of the halogen bromine to the naphtho-
quinone ring (3d), reduced more than twofold the
Trypanosomicidal activity against the epimastigote
forms
The in vitro trypanosomicidal activity of the compounds was
initially tested against the epimastigote form of T. cruzi in three

Medicinal Chemistry Research
Table 2 Effect of the new
naphthoquinones on J774
murine macrophages and the
selectivity indexes.
Compound
J774
Tulahuén 2
Epimastigotes
SIb
NINOA
INC-5
IC50 (µM)
Epimastigotes
SIb
Epimastigotes
SIb
3a
3b
3c
55
1
2
149
500
84
12
44
4
21
64
6
100
26 0.6
43
51 0.8
60
316 23
3d
4a
4b
Nfx
2
96
25
39
4
39
43
6
104
76
1
41
277
243
aThe results are means of three independent experiments.
bSelectivity index: expressed as the ratio of IC50 on J774 cells to IC50 on epimastigotes.
trypanosomicidal effect. In the case of substitution of the
halogen atom for a second aromatic ring with methoxy (4a)
or methyl (4b) groups, biological activity did not improve.
According to the aforementioned, the presence of only
methyl groups in an aromatic ring (3b) was preferable to
maintain or improve the biological effect on Tulahuén
2 strain. Regarding the comparison of halogenated com-
pounds, it can be seen that compound 3c, which has a
bromine substituent and a methoxy group, had greater try-
panosomicidal activity than compound 3d with the sub-
stituents bromine and methyl groups.
In the case of the INC-5 strain, only three compounds
(3b, 3d, and 4a) displayed an activity similar to the refer-
ence drug, Nfx. The three left compounds presented low
activity. This means that the incorporation of a methoxy
substituent in the phenolic ring in compound 3a and 3c, and
the presence of a second aromatic ring with a methyl sub-
stituent, i.e., in 4b, did not favor the trypanosomicidal
activity of these molecules. The presence of a methyl group
improved the trypanosomicidal effect only in compounds
3b, 3d, and the presence of a second aromatic ring and a
methoxy group, enhanced the trypanosomicidal activity in
compound 4a. In the case of NINOA strain, as well as the
INC-5 strain, the compounds did not present better biolo-
gical activity than Nfx. Compound 4a showed the best
trypanosomicidal activity followed by compound 3d.
The presence of a second aromatic ring and a methoxy
substituent improved the trypanosomicidal activity in
molecules against the INC-5 and NINOA strains; these
structural modifications, nonetheless, did not improve the
activity on Tulahuén 2 strain (Table 1).
study, it was observed that Mexican strains, which have
already been shown to belong to TcI2 (I or II) (Gómez-
Hernández et al. 2011), presented greater drug resistance than
strains of Brazilian origin, which have a TcII type lineage.
Cytotoxicity
Cytotoxic studies were performed on murine macrophages
J774.1 (Table 2). Compounds 3c and 3d were the most
toxic of the series, where 3c had the lowest IC50 against
macrophages. This behavior could be attributed to the
presence of bromine on the quinone ring and the methoxy
substituent in the aromatic ring. Interestingly, compound
3b, which does not have the mentioned constituents, was
the least cytotoxic against macrophages. The results showed
that all compounds present a greater cytotoxic effect than
Nfx in murine macrophages; however, the SI for all the
studied compounds showed a selectivity higher than four.
In the case of Tulahuén 2 strain, the SI results showed
greater selectivity than the reference drug, especially 3b.
Likewise, for the clinical strains (NINOA and INC-5), com-
pound 3b presented the best SI, followed by compounds 4a
and 3d. According to the SI values obtained for compounds
3d (SI = 96) and 4a (SI = 104) in Tulahuén 2 strain, we
suggest that these values are acceptable according to that
reported by other research groups being higher than the SI of
Nfx (Nwaka and Hudson 2006; Romanha et al. 2010; Da
Silva et al. 2014). The SI in NINOA strain was lower than
Nfx. The same behavior occurred with INC-5 strain. When it
was compared with the data obtained from the three strains, it
was seen that Tulahuén 2 strain showed the greatest sensi-
tivity to the studied compounds.
The strains INC-5 and NINOA are Mexican strains iso-
lated from patients and these belong to the TcI of T. cruzi and
Tulahuén 2 strain, belonging to the genotype TcVI, which is a
reference with high sensitivity to drugs that explains the
variability of the results. The previously aforementioned
results reported that there are differences in drug susceptibility
in different T. cruzi strains (León-Pérez et al. 2007). In this
Trypanosomicidal activity against trypomastigote
forms
After evaluating the trypanosomicidal effect of compounds
on epimastigotes, an assay was carried out with T. cruzi

Medicinal Chemistry Research
Table 3 Half-maximal lytic concentration of aryloxy derivatives 3d
and 4a on trypomastigotes of T. cruzi strains INC-5 and NINOA.
Compound
INC-5
NINOA
Strain (µM)
Strain (µM)
3d
18.99 11.45
17.94 9.03
20.03 11.33
16.11 8.57
21.35 7.45
20.06 3.91
4a
Nfx
blood trypomastigotes (INC-5 and NINOA strains). The
blood trypomastigote stage is the infective form in mam-
mals, mainly during the acute stage of the disease; therefore,
it is important to assess the compounds activities during this
stage. Only compounds 3d and 4a were considered to have
the best trypanosomicidal activity over the two strains
selected, with each one being representative of every eval-
uated series. Table 3 shows the LC50 results of 3d and 4a
with both strains. Compounds 3d and 4a showed lytic
activity against trypomastigotes on both strains having LC50
values similar to the reference drug Nfx. Animal experi-
ments were performed according to Norma Oficial Mex-
icana (NOM-062-ZOO-1999) published on August 22,
2009 entitled Technical specifications for the production,
care and use of laboratory animals.
Fig. 2 Interacting residues in the complex TcTR-quinacrine mustard
(PDBID: 1GXF).
Table 4 Binding energy of aryloxy-naphthoquinones in the active site
of TcTR by molecular docking.
Compound
Vina score (kcal/mol)
Quinacrine mustard
−5.7
−6.1
−6.7
−6.0
−6.3
−5.9
−6.5
Molecular docking analysis
3a
3b
3c
3d
4a
4b
Naphthoquinones have been reported as potential trypa-
nothione reductase (TR) inhibitors (Persch et al. 2014).
Therefore, our aryloxy naphthoquinone derivatives were
docked, using AutodockVina (vina), in order to assess their
affinity to TR (PDB ID: 1GXF) using quinacrine as the
template compound. Figure 2 highlights the structural
conformation of quinacrine mustard in the active site of
TcTR. Quinacrine mustard acts as a competitive inhibitor of
TcTR on the hydrophobic amino acid TRY-111, which is
part of the hydrophobic wall (formed by LEU-18, TRP-22,
TYR-111, and MET-114) and modulates the enzyme
activity of TcTR (Nwaka and Hudson 2006).
Results of the molecular docking studies on the aryloxy
naphthoquinones are shown in Table 4. These six com-
pounds showed a higher affinity than quinacrine mustard.
Figure 3 shows the docking conformation of these results.
The 3a interacts with the four residues of the hydrophobic
wall, however unlike quinacrine mustard; TRY-111 forms a
hydrogen bond to 3a. This hydrogen bond is also present
with the 3c structure. This compound also interacts with
TRP-22 trough stacking, these interactions sometimes have
a big impact on affinity, as in 3b, the compound with the
highest affinity, nevertheless for 3c this is seen to be not
true. Compound 3c also form two hydrogen bonds with
TRY-111 and LEU-18. In addition, its boron atom interacts
with oxygen from ILE-339. For 4a, interactions are mainly
hydrophobic. These interactions occur with LEU-18, TYR-
111, TRP-22, ILE-339, ARG-472. In addition, a hydrogen
bond is formed with ARG-472.
On the other hand, interactions with 3b are diverse, this
compound has a π-stacking with TRP-22, establishes
hydrophobic interactions with LEU-18, ILE-339, TRY-111,
and TRP-22 and forms hydrogen bonds with GLU-19, ILE-
339, and TYR-111. The π-stacking with TRP-22 is also
present with 3d, as well as the hydrophobic interaction with
ILE-339. Along with those interactions, bromine atom of 3d
is interacting with SER-15 and two hydrogen bonds are
formed with TYR-111 and GLU-19. Finally, 4b establishes
hydrophobic interactions with LEU-18, VAL-59, ILE-107,
ILE-339, and LEU-399 and forms a hydrogen bond and
π-stacking interaction with TRY-111.
Based on these interactions, it seems that, just as qui-
nacrine mustard, all the six compounds interact with the

Medicinal Chemistry Research
Fig. 3 Docking conformations and their interactions. Aryloxy-naphthoquinones were docked using vina programme. The shown interactions were
calculated using protein ligand interaction profiler (PLIP).

Medicinal Chemistry Research
hydrophobic wall residues. This points out that additionally
to their importance acting as support for the correct orien-
tation of the natural subtract of TcTR, these amino acids
also contribute with an important set of potential interac-
tions for the binding of small molecules.
General procedure for preparation of novel, aryloxy
quinones (3a–3d to 4a–4b)
In a reaction flask, the appropriate phenol (1 mmol) and
K2CO3 (3 mmol) was dissolved in DMF (10 mL). The mix-
ture was stirred at room temperature for 20 min and the cor-
responding quinone (1 mmol) was added. The reaction
mixture was stirred for 6–8 h at room temperature and poured
in ice-cold water. The organic layer was extracted with ethyl
acetate (3 × 25 mL), washed with a saturated solution of
sodium sulfite and brine (3 × 25 mL). The obtained organic
layer was dried with sodium sulfate and concentrated under
vacuum. The product was purified by using column chro-
matography on silica gel and TLC. For compound 4a and 4b,
appropriate phenol was used (2 mmol).
Conclusions
In the present work, new aryloxy-naphthoquinone derivatives
were synthesized and evaluated in vitro against two form of T.
cruzi and in three strains of them. The trypanosomicidal
activity of these compounds be determined by the T. cruzi
strains and these results were compared with the reference
drug Nfx. The most promising naphthoquinones were 3d and
4a, due to their highest trypanosomicidal effect on INC-5 and
NINOA strains than Nfx. In addition, 3d and 4a had a similar
trypanosomicidal activity in a trypomastigotes ex vivo model
of the same strains. Even the cytotoxicity on J774-1 cells of
the compounds was higher than Nfx; 3b showed the best
selectivity behavior. The results of the molecular docking on
T. cruzi TR suggested that these compounds could have an
inhibitory effect as a mechanism of action. Therefore, this
study validates the aryloxy-naphthoquinone moiety as a pri-
vileged scaffold for the design of new molecules with try-
panosomicidal activity and selectivity.
2-(3,5-Dimethoxyphenoxy)-1,4-dihydronaphthalene-1,4-
dione (3a)
Yield: 85%, mp 126 °C, FT IR (υ cm−1): 3100 (CH sp2,
aromatic), 2842 (C-H stretch methoxy), 1677, (C=O). H
1
NMR (400 MHz, DMSO-d6) δ 3.76 (s, 6H, OCH3), 5.90
(s, 1H, H-3), 6.47 (s, 3H, H-2′, H-4′, and H-6′), 7.84–7.92 (m,
2H, H-6 and H-7), 7.93–7.99 (m, 1H, H-5), 8.04–8.11 (m,
1H, H-8). 13C NMR (101 MHz, DMSO-d6) δ 55.59 (OCH3),
98.25 (C-6′), 99.14 (C-3′ and C-4′), 113.60 (C-3), 125.62 (C-
5), 126.18 (C-8), 130.86 (C-8a), 131.44 (C-4a), 133.90 (C-6
or C-7), 134.63 (C-6 or C-7), 154.44 (C-1′), 159.83 (C-2),
161.52 (C-5′ and C-2′), 179.12 (C-1), 184.39 (C-4).
Experimental
Material and methods
Chemistry
2-(2,4-Dimethylphenoxy)-1,4-dihydronaphthalene-1,4-
dione (3b)
Yield: 90%, mp 120 °C, FT IR (υ cm−1): 2920 (CH sp2,
1
All reagents and solvents were purchased from Sigma-
Aldrich and were used without further purification.
Melting points were determined on Mel-Temp capillary
apparatus and are uncorrected. Solvent evaporation under
vacuum was carried out with a R-100. 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 Japan), with a
thickness of 0.25 nm. The developed chromatograms were
visualized under ultraviolet light at 254–265 nm. Infrared
spectra were recorded using OPUS_7.5.18 software with
PLATINUM-ATR Bruker Alpha FT-IR spectrometer.
NMR data were collected with Bruker Avance 500 spec-
trometer operating at 400 MHz (1H), and 126 MHz (13C).
1H proton NMR spectra were obtained in DMSO-d6, with
TMS as an internal standard. Chemical shifts are pre-
sented on the δ scale (ppm). Multiplicities are indicated as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet) or br (broad).
aromatic), 1680, (C=O). H NMR (400 MHz, DMSO-d6) δ
2.12 (s, 3H, Ar–CH3), 2.31 (s, 2H, Ar-CH3), 5.56 (s, 1H, H-
3), 7.06 (d, J = 8.2 Hz, 1H, H-6′), 7.14 (dd, J = 8, 1.6 Hz, 1H,
H-5′), 7.20 (d, J = 1.6 Hz, 1H, H-3′), 7.83–7.91
(m, 2H, H-6 and H-7), 7.93–7.96 (m, 1H, H-5), 8.05–8.12 (m,
1H, H-8).
13C NMR (101 MHz, DMSO-d6) δ 15.17 (Ar-CH3),
20.40 (Ar-CH3), 111.92 (C-3), 120.77 (C-6′), 125.60 (C-5),
126.19 (C-8), 128.35 (C-5′), 129.15 (C-4′), 130.96 (C-4a),
131.47 (C-8a), 132.35 (C-3′), 133.84 (C-2′), 134.60 (C-6 or
C-7), 135.90 (C-7 or C-6), 148.34 (C-1′), 159.65 (C-2),
179.08 (C-1), 184.25 (C-4).
2-Bromo-3-(3,5-dimethoxyphenoxy)-1,4-
dihydronaphthalene-1,4-dione (3c)
Yield: 87%, mp 133 °C, FT IR (υ cm−1): 3056 (CH sp2,
aromatic), 2839 (C–H stretch methoxy), 1679, (C=O). H
1
NMR (400 MHz, DMSO-d6) δ 3.71 (s, 6H, OCH3), 6.26

Medicinal Chemistry Research
(t, J = 2.1 Hz, 1H, H-4′), 6.36 (d, J = 2.1 Hz, 2H, H-2′ and
H-6′), 7.85–7.93 (m, 2H, H-6 and H-7), 7.96–8.01 (m, 1H, H-
8), 8.10 (m, 1H, H-5). 13C NMR (101 MHz, DMSO-d6) δ
55.50 (OCH3), 95.13 (C-3′ and C-6′), 95.38 (C-4′), 126.52
(C-8), 126.81 (C-5), 128.51 (C-2), 130.67 (C-4a), 131.42 (C-
8a), 134.28 (C-6 or C-7), 134.44 (C-6 or C-7), 154.63 (C-3),
157.77 (C-1′), 161.24 (C-2′ and C-5′), 177.30 (C-1),
178.41 (C-4).
C-1′′), 134.23 (C-6 and C-7), 145.91 (C-2 and C-3), 153.00
(C-2′ and C-2′′), 180.26 (C-1 and C-4).
Biological activity in vitro on epimastigotes
T. cruzi Tulahuén 2, NINOA, and INC-5strain epimasti-
gotes were grown at 28 °C in an axenic medium (BHI), as
previously described, complemented with 5% fetal bovine
serum. Epimastigotes from a 10-day-old culture (stationary
phase) were inoculated into 50 mL of fresh culture medium
to reach an initial concentration 1 × 106. Cell growth was
monitored by measuring the absorbance of the culture at
600 nm in an Elisa Epoch reader (Epoch 2 Microplate
Spectrophotometer (BioTek Instruments, Inc., Winooski,
VT, USA) every day. Before inoculation, the media was
supplemented with a given amount of the drug from a stock
solution in DMSO (25 mM). The final DMSO concentration
in the culture medium never exceeded 0.4% and the control
was run in the presence of 0.4% DMSO and in the absence
of drugs. The percentage of growth inhibition (% GI) and
IC50 values (50% inhibitory concentrations), and parasite
growth was followed in the absence (control), and in the
presence, of a range of concentrations of the corresponding
drug. On day 5, the absorbance of the culture was measured
and related to the control. The IC50 value was determined as
the concentration of drug needed to reduce the absorbance
ratio by 50%. The experiments of each compound were
carried out in triplicate and Nfx was used as reference
compound (Álvarez et al. 2014).
2-Bromo-3-(2,4-dimethylphenoxy)-1,4-dihydronaphthalene-
1,4-dione (3d)
Yield: 87%, mp 150 °C, FT IR (υ cm−1): 2917 (CH sp2,
aromatic), 1680, (C=O). 1H NMR (400 MHz, DMSO-d6) δ
2.23 (s, 3H, Ar-CH3), 2.31 (s, 3H, Ar-CH3), 6.84 (s, 2H,
H-5′ and H-6′), 7.09 (s, 1H, H-3′), 7.83–7.90 (m, 2H, H-6
and H-7), 7.92–7.97 (m, 1H, H-5), 8.10 (m, 1H, H-8). 13C
NMR (101 MHz, DMSO-d6) δ 15.84 (Ar-CH3), 20.16 (Ar-
CH3), 115.08 (C5′ or C-6′), 126.02 (C-3), 126.53 (C-5),
126.83 (C-8), 127.19 (C-6′ or C-5′), 130.55 (C-8a), 131.20
(C-4a), 131.70 (C-3′), 132.46 (C-1′), 134.32 (C-6 or C-7),
134.52 (C-7 or C-6), 152.76 (C-2′ and C-4’), 155.78 (C-2),
177.44 (C-4), 178.36 (C-1).
2,3-Bis (3,5-dimethoxyphenoxy)-1,4-dihydronaphthalene-
1,4-dione (4a)
Yield: 68%, mp 159 °C, FT IR (υ cm−1): 2945 (CH sp2,
1
aromatic), 2841 (C-H stretch methoxy), 1670, (C=O). H
NMR (400 MHz, DMSO-d6) δ 3.67 (s, 12H, OCH3), 6.18
(t, J = 2.1 Hz, 2H, H-4′ and H-4′′), 6.32 (d, J = 2.1 Hz, 4H,
H-3′, H-3′′, H-6′, and H-6′′), 7.84–7.92 (m, 2H, H-6, and
H-7), 8.01 (m, 2H, H-5, and H-8). 13C NMR (101 MHz,
DMSO-d6) δ 55.39 (OCH3), 95.19 (C-3′, C-3′′, C-6′ and
C-6′′), 95.24 (C-4′ and C-4′′), 125.95 (C-5 and C-8),
131.17 (C-4a and C-8a), 134.15 (C-6 and C-7), 145.86 (C-2
and C-3), 158.39 (C-1′ and C-1′′), 160.97 (C-2′, C-2′′, C-5′
and C-5′′), 180.05 (C-1 and C-4).
Ex vivo biological activity towards T. cruzi
trypomastigotes
CD1 female mice, 6–8 weeks old, were infected with T.
cruzi bloodstream trypomastigotes of INC-5 and NINOA
strains. The course of infection continued for 4–6 weeks. At
the peak of parasitemia, blood was obtained by cardiac
puncture using sodium heparin as an anticoagulant. The
blood was adjusted to 1 × 106 bloodstream trypomastigotes/
mL. In each well of the 96-well plate, 90 µL of infected
blood and 10 µL of the new aryloxy 1,4-naphthoquinone or
reference drug dilutions were seeded with each well
reaching a final volume of 100 µL. Each assay was per-
formed in triplicate. Initially, all compounds were tested at
50 µg/mL. Afterwards, the compounds with a lysis per-
centage >50 were tested at four concentrations, 50 µM,
20 µM, 10 µM, and 5 µM, to obtain a lysis concentration of
50% (LC50). As a negative control of lysis, wells with
untreated blood trypomastigotes were used, and as a posi-
tive control, wells with the reference drugs were used (Nfx
and Bnz). The microplates were incubated at 4 °C for 24 h.
Bloodstream trypomastigotes were quantified by the
2,3-Bis(2,4-dimethylphenoxy)-1,4-dihydronaphthalene-1,4-
dione (4b)
Yield: 65%, mp 117 °C, FT IR (υ cm−1): 2919 (CH sp2,
aromatic), 1667, (C=O). 1H NMR (400 MHz, DMSO-d6) δ
1.97 (s, 6H, Ar-CH3), 2.18 (s, 6H, Ar-CH3), 6.80 (dd, J = 8,
0.8 Hz, 2H, H-5′ and H-5′′), 6.89 (d, J = 8.2 Hz, 2H, H-6′
and H-6′′), 6.93 (d, J = 0.8 Hz, 2H, H-3′ and H-3′′), 7.88
(br s, 2H, H-6 and H-7), 8.00 (br s, 2H, H-5 and H-8). 13C
NMR (101 MHz, DMSO) δ 15.36 (Ar-CH3), 20.10 (Ar-
CH3), 115.08 (C-6′ and C-6′′), 125.85 (C-5 and C-8),
125.97 (C-4′ and C-4′′), 126.94 (C-5′ and C-5′′), 130.85
(C-4a and C-8a), 131.22 (C-3′ and C-3′′), 131.82 (C-1′ and

Medicinal Chemistry Research
Fig. 4 Aryloxy-naphthoquinone
structures.
Brener-Pizzi method, 5 µL of blood was placed on a slide
and covered with a coverslip 18 × 18 mm. The mobile
protozoa were counted in 15 fields at ×40 magnification
using an optical microscope. The percentage lysis was
determined by comparing the remaining trypomastigotes
against the negative control. LC50 was determined with the
Probit statistical tool (Chacón-Vargas et al. 2017).
Molecular docking
Molecular docking studies were performed using Auto-
DockVina 1.1.2 (Trott and Olson 2010). The crystal struc-
ture of TcTR bound to the inhibitor quinacrine mustard
(PDB ID: 1GXF) was obtained from the Protein Data Bank
(PDB). Next, the protein structure was extracted from the
crystal, missing side chains were repaired, and all hydro-
gens were added with the Dock Prep tool of UCSF Chimera
(Morris et al. 2009). Then, the script prepare_receptor4.py
from MGTools 1.5.6 (Morris et al. 2009) was used to add
AutoDock atom types and the charge to the prepared protein
structure. Six aryloxy-naphthoquinones (Fig. 4) were
docked in the coordinates: X = 17.733, Y = 13.499, and
Z = −1.197, according to the binding site of quinacrine
mustard in the crystal complex (PDB ID: 1GXF). Before
performing the docking, aryloxy-naphthoquinone structures
were drawn using MarvinSketch (MarvinSketch. 2014) and
were minimized with the Open Babel tool (O’Boyle et al.
2011). In addition, the MGTools script, prepare_ligand4.py
was employed to assign charge and atom types from
Autodock to each ligand structure.
Measurement of cell cytotoxicity
Cell cytotoxicity was determined with the 3-(4,5-dime-
thylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)
assay for the evaluation of mitochondrial function (Mos-
mann 1983). In a 96-well plate for cell culture, 5 × 104 of
the J774.1 cell line of murine macrophages (ATCC, USA)
was incubated at 37 °C with 4% CO2 for 24 h. Later, the
synthesized compounds were added at decreasing con-
centrations from 100 to 1.25 µM, with three independent
repetitions. Removing the previously used culture medium
and adding 5 µL of the dilutions of the compounds, the
incubation of the macrophages along with the compounds
was allowed for 24 h. After incubation, the treatment was
removed and washed three times with PBS to ensure the
evacuation of the compounds and dead cells. Then, 50 μL
(5 mg/mL) of MTT (Sigma-Aldrich) and 200 μL of DMEM
free of phenol red (Sigma-Aldrich) were added, leaving it to
act for 4 h at 37 °C; at that time, the MTT and DMEM were
removed and replaced by 200 μL DMSO (Sigma-Aldrich) to
dissolve the formazan crystals, incubating for 30 min. The
plate reading was at a wavelength of 570 nm in an Elisa
Epoch reader (Epoch 2 Microplate Spectrophotometer
(BioTek Instruments, Inc., Winooski, VT, USA)).
Acknowledgements We wish to express our gratitude to the Secre-
taría de Investigación y Posgrado del Instituto Politécnico Nacional
(SIP-20190295) 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 the “Programa de Estímulos al Desempeño de los Inves-
tigadores” (EDI-IPN). Karina Vázquez thanks to the Proyecto
“Programa para el Desarrollo Profesional Docente, para el Tipo
Superior (PRODEP) DSA/103.5/16/10510 for their financial support
and Professor Jose Carlos Espinoza Hicks of the Universidad
Autonoma de Chihuahua.

Medicinal Chemistry Research
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