New aryloxy-quinone derivatives with promising activity on Trypanosoma cruzi

Article abstract of DOI:10.1002/ardp.201900213

Continuing with a program to develop new quinone derivatives as biologically active compounds, we designed and synthesized a new series of aryloxy-quinones, which were evaluated in vitro against Trypanosoma cruzi in epimastigote form. Chemical modificatio

Full text of DOI:10.1002/ardp.201900213

Received: 24 July 2019
Revised: 7 October 2019
Accepted: 16 October 2019
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DOI: 10.1002/ardp.201900213
F U L L P A P E R
New aryloxy‐quinone derivatives with promising activity on
Trypanosoma cruzi
Christian Espinosa‐Bustos1
Margot Paulino5 Rodrigo Segura6
Mercedes González3 Ana M. Zarate7
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Karina Vázquez2
Jaime Pizarro6
Cristian O. Salas7
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Javier Varela3
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Hugo Cerecetto3,4
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Brenda Vera5
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1Departamento de Farmacia, Facultad de
Química y de Farmacia, Pontificia Universidad
Católica de Chile, Santiago, Chile
Abstract
Continuing with a program to develop new quinone derivatives as biologically active
compounds, we designed and synthesized a new series of aryloxy‐quinones, which
were evaluated in vitro against Trypanosoma cruzi in epimastigote form. Chemical
modifications in three specific moieties on the aryloxy‐quinone core were considered
for developing new anti‐T. cruzi agents. The majority of our new quinones showed
higher potency (IC50 values of <0.70 µM) than nifurtimox, a known pharmaceutical
used as a baseline drug (IC50 values of 7.00 µM); however, only two of them elicited
higher selectivity than nifurtimox against Vero cells. A structure–activity relationship
analysis provided information about the stereoelectronic features of these
compounds, which are responsible for an increase in trypanosomicidal activity. Using
2Campus de Ciencias Agropecuarias, Facultad
de Medicina Veterinaria y Zootecnia,
Universidad Autónoma de Nuevo León,
Nicolás de Los Garza, México
3Grupo de Química Medicinal, Laboratorio de
Química Orgánica, Facultad de Ciencias,
Universidad de la República, Montevideo,
Uruguay
4Área de Radiofarmacia, Centro de
Investigaciones Nucleares, Facultad de
Ciencias, Universidad de la República,
Montevideo, Uruguay
5Centro de Bioinformática Estructural,
DETEMA, Facultad de Química, Universidad
de la República, Montevideo, Uruguay
a
pharmacophore model, we mapped the substitution patterns of the five
pharmacophoric features of trypanosomicidal activity. We chose the Epc1 compounds
and found no relationship with the trypanosomicidal effects. These results provided
useful information about the structural characteristics for developing new aryloxy‐
quinones with higher potency against the protozoan parasite T. cruzi.
6Departamento de Química de los Materiales,
Facultad de Química y Biología, Universidad
de Santiago de Chile, Santiago, Chile
7Departamento de Química Orgánica,
Facultad de Química y de Farmacia, Pontificia
Universidad Católica de Chile, Santiago, Chile
K E Y W O R D S
Correspondence
aryloxy‐quinones, cyclic voltammetry, pharmacophoric model, Trypanosoma cruzi
Christian Espinosa‐Bustos and Cristian O.
Salas, Facultad de Química y de Farmacia,
Pontificia Universidad Católica de Chile,
Avenida Vicuña Mackenna 48604860, Macul,
7820436, Santiago, Chile.
Email: ccespino@uc.cl (C. E.‐B.) and
cosalas@uc.cl (C. O. S.)
Funding information
Fondo Nacional de Desarrollo Científico y
Tecnológico, Grant/Award Number: 1161816
and 11180292
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1
INTRODUCTION
economic burden. Parasitosis, such as Chagas disease (American
trypanosomiasis), affects more than eight million individuals within
Infectious diseases are responsible for mortality worldwide and
constitute a global health problem. They led to the death of 8.8
Latin America and contributes to approximately 10,000 annual
deaths, which is higher than the deaths caused by malaria in the
Americas. This parasite is responsible for 89% of the deaths caused
million persons in 2008.[1] Many such diseases cause
a huge
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by tropical clustered diseases in the Americas.[1] In seven of its endemic
countries, Chagas disease causes productivity loss of over US$ 1.2
billion/year.[2] Moreover, nowadays, this neglected disease is observed
in nonendemic areas, such as the United States of America and Europe,
due to international migration.[3] This disease is a chronic infection with
hidden symptoms triggered by Trypanosoma cruzi, which is mainly
transmitted to humans through hematophagous triatomine insects.[4]
The current treatment for Chagas disease includes only two drugs:
nifurtimox and benznidazole, both discovered more than 50 years ago.
These drugs are generally not useful during the chronic phase of the
disease, in addition to causing undesirable side effects.[5,6] For this
reason, the need for new drugs has sparked research efforts to find
new chemical entities with potential therapeutic utility.
(Figure 1) showed IC50 values of 1.72 and 9.02 µM against
epimastigote forms and a mortality percentage of 69% and 86%
on trypomastigote forms, respectively.[12]
Likewise, we have reported on the synthesis of new aryloxy‐
quinones, with several modifications on the naphthoquinone ring as
well as on the aryloxy moiety for in vitro evaluation against the T. cruzi
epimastigote form, and compounds VII and VIII (Figure 1) were the
most active, showing IC50 values of 20 and 40 nM, respectively, and
also presented low cytotoxicity against J‐774 nonneoplastic cells.[14]
Continuing with our program to develop new trypanosomicidal
agents with a higher potency,[13] herein, we report 15 new molecules
that combine two well‐known pharmacophoric patterns of bioactive
compounds (shown as red or blue dashes in Figure 1), with their
respective results on epimastigotes of T. cruzi. From the analysis of
the results discussed in the next sections, we validate the hypothesis
that the influence of the substitution pattern of the aryloxy moiety
or the replacement of the benzene ring by the heterocyclic ring is
structural features determining the biological activity of these
naphthoquinones derivatives. A pharmacophoric model was built to
confirm this observation. We also inform about the electrochemical
properties of all these compounds, which may be associated with a
potential action mechanism. Thus, these new results provide the
basis for the design of future anti‐T. cruzi agents.
Derivatives of natural quinones are considered privileged structures
for the development of compounds with antiparasitic activity,[7,8] such
as lapachol I and α‐lapachone II (Figure 1), which exhibit interesting
anti‐T. cruzi activity and have produced a framework for the creation
and elaboration of new derivatives.[9,10] Considering these antecedents,
Bolognesi et al.[11] synthesized a library of compounds derived from
2‐aryloxy‐1,4‐naphthoquinone. In this series, the 2‐phenoxy‐1,4‐
naphthoquinone III (Figure 1) was the most prominent compound, with
an IC50 of 1.70 µM on the amastigote form of T. cruzi.[11]
In recent years, our research group has synthesized several
derivatives of naphthoquinones with excellent trypanosomicidal
activity.[12–14] For example, in one in vitro study, phenoxy
derivatives of indole‐4,9‐dione were synthesized and evaluated
against T. cruzi epimastigotes, where compound IV (Figure 1) showed
high selectivity and remarkable inhibitory activity in the nanomolar
range (IC50 = 20 nM).[13] Subsequently, derivatives of 2‐phenylami-
nonaphtoquinones were synthesized, where compounds V and VI
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
We synthesized new aryloxy‐quinones by the nucleophilic substitu-
tion reaction of phenols with haloquinones in a basic medium at room
FIGURE 1 Chemical structures of lapachol (I), α‐lapachone (II), phenoxy‐ and phenylamino‐quinones (III–VIII) with trypanosomicidal
activities, used as templates to design the new series of aryloxy‐quinones as anti‐Trypanosoma cruzi agents

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temperature (Scheme 1).[14,15] 2‐Aryloxy‐naphtoquinones 3a–g were
attained in 72% to 92% yield, by the reaction of several phenols
2a–e with halo‐naphthoquinones 1a–c in dimethylformamide (DMF)
in the presence of potassium carbonate. The same procedure was
employed in the preparation of compounds 3h and 3i in 58%
and 44%, respectively, using naphthols 2f,g and 2,3‐dichloro‐1,4‐
naphtoquinone 1b. Compounds 6a and 6b were synthesized from
7‐bromoquinoline‐5,8‐dione 4, as reported previously by our
research group.[14] The reaction of haloquinone 4 with phenols 5a,b
gave derivatives 6a,b in good yields (85% for 6a and 63% for 6b).
Finally, the compounds 8a–d were prepared from the halobenzofur-
anquinones 7a,b[15] and the corresponding phenols 5a,b, with yields
among 36–62%. The target compounds’ chemical structures were
determined by the spectral properties they possess (infrared [IR],
SCHEME 1 Synthesis of aryloxy‐quinone derivatives. Reagents and conditions: (i) K2CO3, DMF (dimethylformamide), rt, 2–4 hr (36–96% yield)

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1H NMR [nuclear magnetic resonance], 13C NMR, and HRMS [high‐
activity but high toxicity on host mammalian cells will not have
therapeutic significance. For this reason, the cytotoxicity of these
derivatives on nonneoplastic monkey kidney cells (Vero) was
evaluated and their selectivity index (SI) was determined. In general,
most of the compounds showed high toxicity on Vero cells (Table 1),
even with SIs lower than nifurtimox. However, compounds 6a and 8d
showed greater selectivity for the parasite and better than
nifurtimox, with SIs of 19 and 9.4, respectively.
resolution mass spectrometry]; see Section
4 and Electronic
Supporting Information [ESI]).
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2.2
Trypanosomicidal effect and
structure–activity relationship
The in vitro trypanosomicidal activity of new 2‐aryloxynaphthoqui-
nones 3a–i, 7‐aryloxy‐quinolinquinones 6a,b, and 6‐aryloxy‐furanqui-
nones 8a–d was first tested against T. cruzi epimastigotes (Y strain,
Tc II). To determine the IC50 concentration (50% inhibitory
concentration) of each new derivative, a dose–response analysis
between 0.01 and 50 µM was performed. Nifurtimox (Bayer) was
utilized as the trypanosomicidal drug of reference. The new 15
aryloxy‐quinones showed potent effects on T. cruzi and more activity
than nifurtimox, which has an IC50 of 7.0 µM (Table 1). The
compound 8a was the sole exception with an IC50 value of >10 µM.
Derivative 3a is a particularly interesting compound because it
elicited the highest trypanosomicidal activity (IC50 value = 0.23 µM),
showing potency nearly 30 times that of nifurtimox.
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2.3
Electrochemical study
Electrochemical aspects are beneficial in redox‐molecules since they
can provide information about their mechanism of action.[17,18] Thus,
to determine a possible relationship between structure and activity
in the tested quinones, we used the cyclic voltammetry (CV)
technique to determine the oxidation and reduction potentials.
Figure 2 depicts the voltammetric response for the two most active
compounds 3a and 3h (for the other compounds, CV profiles are
available in the ESI). Every quinone showed quasi‐reversible
reduction behavior, which is a typical triad of quinone/semiqui-
none/hydroquinone in equilibrium, and overall CV profiles resembled
reports for other quinones.[10,19] Epc1 values were taken from each
compound’s CV profile (Table 2), which is frequently used for
biological activity comparisons. The comparison between the values
of reduction potential with the trypanosomicidal activity (Figure 3)
does not reveal a correlation, which indicates that the redox
properties of these quinones alone are not determinants for the
activity as we demonstrated in our previous work,[14] and therefore,
In terms of chemistry, considering the 2‐phenoxy‐1,4‐naphtho-
quinone moiety, the result of the chemical changes proposed in
Figure 1 can be analyzed. We can observe a slight structure–
trypanosomicidal activity relationship between these 15 derivatives.
First, the substitution of the hydrogen atom on C‐3 by a bromine
atom in the aryloxy‐quinone derivatives (3a, 3b, and 8b) slightly
diminishes or maintains the trypanosomicidal activity compared to
the nonhalogenated analogous (3f, 3g, and 8d, respectively, Table 1).
An exception to these behaviors is shown in 8a versus 8c, where the
halogenated quinone was the most active. On the contrary, when
comparing the chlorinated naphthoquinones, 3c–e, 3h and 3i, among
them, the trypanosomicidal effect could be related to the hydro-
phobicity of these compounds. For this reason, the value of clog P
was calculated (Table 1), which showed that the compounds 3h and
3i presented the highest lipophilicities, values of clog P > 5.0, and at
the same time, the best IC50 values among the chlorinated
compounds.
other factors must be considered to establish
a correlation
structure–activity.
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2.4
Consensus pharmacophore elucidation
As a first step to design a pharmacophore, a molecular flexible
alignment was performed. It is a stochastic search to find conforma-
tion of the set of molecules and, further, a three‐dimensional (3D)
spatial alignment. The first molecule, considered as the lead
compound (3a), was selected as the spatial reference to align the
rest of the set. As a result, we obtained 33 putative alignments,
ranked by a measure of the average deformation energy and the
percentage of similitude between the different aligned conforma-
tions. The aligned set with the best score (−65.01 kcal/mol) was then
used for building the consensus pharmacophore.
Finally, if we compare the effect of replacing the benzene ring
(in naphthoquinone core) by pyridine or a dihydrofuran ring, two
different observations can be made. Analysis of benzene replacement
by a furan ring revealed a reduction in the trypanosomicidal effect on
the epimastigotes form (8b vs. 3a and 8d vs. 3f), which was strongly
detected for 8a compared to 3b (IC50 values, >10 and 0.59 µM,
respectively). The quinolinone derivative 6b was slightly less active
than its carbocyclic analog 3a (IC50 values = 1.03 and 0.23 µM,
respectively), but quinolinquinone 6a was slightly more active than 3b.
Replacing the benzene with a pyridine ring is a framework worth
considering in future naphthoquinone core modifications. Our group’s
previous observations align with these findings that show the
importance of substituting nitrogen in the aromatic ring for the
activity of the related compounds.[12]
The consensus pharmacophore model related with in vitro
trypanosomicidal was elucidated separately for the three sets of
compounds: naphthoquinones, quinolinquinones, and furanquinones,
respectively. Six pharmacophoric agreement features were obtained
in the consensus: hydrophobicity (Hyd), aromaticity (Aro), metal
ligator (ML), a combined region of hydrogen acceptors with metal
ligation features (Acc&ML), hydrophobicity with aromaticity features
(Hyd/Aro), and aromaticity with hydrophobicity features (Aro/Hyd).
In the Figure 4 are shown (for each set) the features identification
On the contrary, it is important to consider that in the search of
new anti‐T. cruzi agents, compounds that have trypanosomicidal

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TABLE 1 Effect upon culture growth of Trypanosoma cruzi of aryloxy‐quinones, respective calculated cLog P values, cytotoxicity on Vero cell
line, and selectivity index
a
a
Entry
3a
Structure
Epimastigotes IC50 (µM)
cLog Pb
Vero IC50 (µM)
SIc
3.4
0.23 0.05
3.69
0.79 0.07
3b
0.59 0.13
5.85
1.07 0.06
1.8
3c
3d
3e
0.53 0.12
0.54 0.14
0.33 0.12
3.96
3.89
4.78
0.54 0.10
0.66 0.14
0.75 0.16
1.0
1.2
2.3
3f
3g
3h
3i
0.41 0.14
0.63 0.12
0.32 0.11
0.33 0.11
4.42
6.59
5.14
5.12
0.62 0.11
0.82 0.08
0.18 0.08
0.13 0.04
1.5
1.3
0.6
0.4
6a
6b
0.43 0.15
1.03 0.14
4.11
1.94
8.16 0.15
1.15 0.10
19
1.1
(Continues)

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TABLE 1 (Continued)
a
a
Entry
8a
Structure
Epimastigotes IC50 (µM)
cLog Pb
Vero IC50 (µM)
SIc
>10
5.51
7.24 0.12
0.91 0.13
1.68 0.08
4.52 0.19
>50
–
8b
8c
0.49 0.13
0.58 0.14
0.48 0.15
7.00 0.30
3.34
6.24
4.07
–
1.9
2.9
9.4
7.1
8d
Nfx
Abbreviation: Nfx, nifurtimox.
aThe results are the means of three independent experiments.
bcLog P values were predicted by Molinspiration (http://www.molinspiration.com).
cSelectivity index: Expressed as the ratio of IC50 in Vero cells to IC50 in epimastigotes.
code, the percentage of molecules with a given feature, the number
of conformations aligned, the radius of each feature, and their nature.
For the naphthoquinones set, pharmacophoric features centered
in the middle of the atomic sets (centroids) were annotated: three
regions (F1–3) of hydrophobic and aromaticity characteristics (Aro,
Aro/Hyd, and Hyd/Aro) are marked as consensus in mainly all the
molecules (dark and light spheres). Two hydrogen acceptor and metal
ligation regions (F4–5) are detected in 78% of the molecules. In
summary, the aromaticity, hydrophobicity, and the hydrogen accep-
tor characteristics define the basic level (consensus) of activity in the
set of naphthoquinones. A metal ligation feature (F6) is annotated
centered in the oxygen of the aryloxy moiety. It is worth noticing that
the radius of pharmacophoric spheres is related with the spatial
coincidence of atoms and centroids, and due to the flexibility
of the aryloxy group (in comparison with the naphthoquinone ring),
the sphere radius of the aromatic feature centered in this group is
twice (1.2 Å) than that of Aro spheres (0.71 and 0.67 Å). In the case of
quinolinequinones, the sphere radius is smaller, given that they
are only two molecules aligned and the special coincidence is higher.
The consensus detected features are mainly the same as for the
FIGURE 2 Cyclic voltammograms of aryloxy‐quinones (1.0 mM). DMF/TBAP (0.1 M), glassy carbon electrode, cathodic direction, scan rate
50 mV/s. DMF, dimethylformamide; TBAP, tetrabutylammonium perchlorate

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TABLE 2 Epc values of aryloxy‐quinones
most of them presented better potencies (IC50 values <0.70 µM) than
the reference drug, nifurtimox (IC50 = 7.00 µM), currently used in
therapy against Chagas disease. Two aryloxy‐quinones elicited higher
selectivity than nifurtimox, against T. cruzi in comparison with Vero
cells. To determine the potential structure–activity relationship,
electrochemical studies were performed; however, it was not
Compound
Epc1 (V)
−0.702
−0.623
−0.684
−0.621
−0.718
−0.588
−0.688
−0.621
−0.460
−0.696
−0.720
−0.671
−0.710
−0.590
−0.726
Epc2 (V)
−1.429
−0.873
−1.434
−1.389
−1.478
−1.171
−1.010
−1.357
−1.270
−1.235
−1.501
−0.097
−1.389
−1.260
−1.284
Epc3 (V)
−1.551
−1.476
−1.661
−1.510
−1.689
−1.324
−1.227
−1.584
–
Epc4 (V)
Epc5 (V)
3a
3b
3c
3d
3e
3f
−1.810
–
–
–
–
–
–
–
possible to identify
a relationship between the compounds’
–
–
redox potentials and their IC50. Finally, through the construction of
a pharmacophore, key structural aspects to develop more active
derivatives of aryloxy‐quinones were identified.
−1.527
−1.818
3g
3h
3i
−1.412
−1.637
–
–
–
–
–
–
–
–
–
–
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4
EXPERIMENTAL
Chemistry
6a
6b
8a
8b
8c
8d
−1.452
−1.871
−1.372
−1.575
−1.520
−1.519
–
–
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4.1
−1.646
−1.875
–
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4.1.1
General
Melting points were determined on a Kofler Thermogerate apparatus
and were uncorrected. IR spectra were recorded on a JASCO FT/IR‐
400 spectrophotometer. NMR spectra were recorded, unless
otherwise specified, on a Bruker AM‐400 instrument using deutero-
chloroform, deuteroacetone, or deuterodimethylsulfoxide solutions
containing tetramethylsilane as an internal standard. Mass spectra
(MS) were obtained on an HP 5988 A mass spectrometer. HRMS–
ESI–MS experiments were carried out using a Thermo Scientific
Exactive Plus Orbitrap spectrometer with a constant nebulizer
temperature of 250°C. The experiments were carried out in positive
−1.923
naphthoquinones set (Aro, Hyd: features F1–3), Acc, and metal
ligation features (F4–6). Again, a metal ligation feature is annotated
with F7 and centered in the oxygen of aryloxy fragment. For the third
set (furanquinones), the Hyd features are much more abundant
(F2–F5), suggesting a tendency of a decrease in the IC50 related to
the increment of hydrophobicity. Overall, these results show very
clearly that the inhibition of parasitic growth is defined by the
aromaticity/hydrophobicity and hydrogen acceptor characteristics of
the studied set of quinones.
ion mode, with a scan range of m/z 300.00–1,510.40 with
a
resolution of 140.000. The samples were infused directly into the
ESI source, via a syringe pump, at low rates of 5 µl/min, through the
instrument’s injection valve. Thin‐layer chromatography (TLC) was
performed using Merck GF‐254 type 60 silica gel. Column chroma-
tography was carried out using Merck type 9385 silica gel. The purity
of the compounds was determined by TLC and HRMS. All the spectra
of aryloxy‐quinones synthesized are shown in the ESI.
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3
CONCLUSIONS
In this study, fifteen aryloxy‐quinones were synthesized. The in vitro
evaluation of these derivatives on T. cruzi epimastigotes showed that
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
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4.1.2
General procedure for the synthesis of
aryloxy‐quinones
In a reaction flask, the suitable phenol (1 mmol) and K2CO3 (3 mmol)
were suspended in DMF (5 ml). The mixture was stirred for 10 min,
and then the corresponding quinone (1 mmol) was added. After the
addition, the reaction mixture was stirred for 2–6 hr at room
temperature. Then, the solvent was removed under vacuum, and
the solid residue was purified by column chromatography on silica gel.
2‐(4‐Methyl‐3‐nitrophenoxy)naphthalene‐1,4‐dione (3a)
Yellow solid, yield 74%, mp 161–162°C. 1H NMR (400 MHz, CDCl3) δ
7.95–7.93 (m, 1H), 7.85–7.82 (m, 1H), 7.59–7.50 (m, 3H), 7.24 (d,
J = 8.4 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 5.78 (s, 1H), and 2.41 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 184.46, 179.32, 159.47, 151.05,
FIGURE 3 Graphs correlating the values of Epc1 of quinones with
the trypanosomicidal effect, represented by pIC50

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FIGURE 4 Pharmacophore features depicted graphically in the three‐dimensional space for each set of (a) naphthoquinones, (b)
quinolinquinones, and (c) furanquinones
149.71, 134.65, 134.64, 133.79, 131.97, 131.79, 130.94, 126.86,
126.35, 125.72, 117.51, 114.39, and 20.11. IR (KBr, cm−1): 1,676,
1,650, 1,523, 1,349, 1,230, 822, and 685. HRMS for (C17H11NO5
[M+H]+). Calcd: 310.0710. Found: 310.0710.
22.60, and 14.03. IR (KBr, cm−1): 3,057, 3,008, 1,653, 1,618, 1,504,
and 1,213. HRMS for (C22H23O4 [M+H]+). Calcd: 351.1591. Found:
351.1605.
2‐Chloro‐3‐phenoxynaphthalene‐1,4‐dione (3c)
2‐(4‐(Hexyloxy)phenoxy)naphthalene‐1,4‐dione (3b)
Orange solid, yield 72%, mp 117–119°C (lit. 142‐143°C).[20] 1H NMR
(400 MHz, CDCl3) δ 8.10 (d, J = 7.5 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H),
7.67 (p, J = 7.1 Hz, 2H), 7.23 (t, J = 7.8 Hz, 2H), 7.03 (t, J = 7.4 Hz, 1H),
and 6.91 (d, J = 8.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 178.43,
177.99, 156.42, 153.60, 134.55, 134.44, 133.78, 131.22, 130.66,
129.81 (2C), 127.38, 127.23, 124.06, and 116.61 (2C). IR (KBr, cm−1):
1,672, 1,455, 1,292, 1,266, 880, 758, and 723. HRMS for
(C16H10ClO3 [M+H]+). Calcd: 285.0313. Found: 285.0308.
Yellow solid, yield 90%, mp 56–58°C. 1H NMR (400 MHz, CDCl3) δ
8.44–8.33 (m, 1H), 8.31–8.21 (m, 1H), 8.00–7.91 (m, 2H), 7.24 (d,
J = 8.7 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 6.17 (s, 1H), 4.17 (t, J = 6.5 Hz,
2H), 2.06–1.95 (m, 2H), 1.71–1.66 (m, 2H), 1.62–1.50 (m, 4H), and
1.12 (t, J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 185.02, 180.06,
161.01, 157.43, 145.75, 134.39, 133.45, 132.00, 131.13, 126.75,
126.19, 121.88 (2C), 115.86 (2C), 113.07, 68.52, 31.57, 29.19, 25.70,

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2‐Chloro‐3‐(3‐nitrophenoxy)naphthalene‐1,4‐dione (3d)
7.82 (d, J = 8.2 Hz, 1H), 7.76–7.68 (m, 2H), 7.58–7.50 (m, 3H), 7.23
(t, J = 7.9 Hz, 1H), and 6.69 (d, J = 7.6 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 178.45, 177.79, 154.24, 152.58, 134.88, 134.56, 134.46,
133.98, 131.27, 130.67, 127.74, 127.42, 127.26, 127.02, 126.42,
125.26, 125.11, 124.11, 121.85, and 109.76. IR (KBr, cm−1): 1,679,
1,231, 1,198, 872, and 691. HRMS for (C20H12ClO3 [M+H]+). Calcd:
335.0469. Found: 335.0473.
Orange solid, yield 72%, mp 117–119°C. 1H NMR (400 MHz,
dimethyl sulfoxide [DMSO])
δ 8.12 (dd, J = 16.5, 8.5 Hz, 2H),
8.05–7.84 (m, 4H), 7.74 (t, J = 7.8 Hz, 1H), and 7.76–7.63 (m, 1H).
13C NMR (101 MHz, DMSO) δ 178.54, 178.10, 156.94, 152.18,
149.35, 135.09, 134.91, 134.53, 132.04, 131.60, 131.15, 127.10,
126.94, 123.68, 118.97, and 111.65. IR (KBr, cm−1): 1,672, 1,455,
1,292, 1,266, 880, 758. HRMS for (C16H9ClNO5 [M+H]+). Calcd:
330.0164. Found: 330.0159.
7‐(4‐(Hexyloxy)phenoxy)quinoline‐5,8‐dione (6a)
Orange solid, yield 85%, mp 95–97°C. 1H NMR (400 MHz, CDCl3) δ 9.25
(d, J = 4.6 Hz, 1H), 8.59 (d, J = 7.8 Hz, 1H), 7.90 (dd, J = 7.8, 4.7 Hz, 1H),
7.24 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 6.23 (s, 1H), 4.16
(t, J = 6.5 Hz, 2H), 2.04–1.94 (m, 2H), 1.71–1.65 (m, 2H), 1.61–1.51 (m,
4H), and 1.11 (t, J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 183.71,
178.13, 161.54, 157.58, 154.42, 146.96, 145.52, 134.35, 129.00, 128.05,
121.79 (2C), 115.92 (2C), 112.49, 68.52, 31.54, 29.16, 25.68, 22.58, and
14.02. IR (KBr, cm−1): 2,929, 2,863, 1,656, 1,501, 1,211, 1,197, 870, and
726. HRMS for (C21H22NO4 [M+H]+). Calcd: 352.1543. Found: 352.1538.
2‐Chloro‐3‐(2,5‐dimethylphenoxy)naphthalene‐1,4‐dione (3e)
Orange solid, yield 92%, mp 150–152°C. 1H NMR (400 MHz, CDCl3)
δ 8.31 (dd, J = 7.0, 1.0 Hz, 1H), 8.16–8.09 (m, 1H), 7.92–7.80 (m, 2H),
7.24 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.57 (s, 1H), 2.48
(s, 3H), and 2.32 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.97,
177.54, 154.31, 153.79, 136.25, 133.89, 133.74, 132.23, 130.72,
130.69, 130.08, 126.73, 126.63, 124.40, 124.12, 115.40, 20.38, and
15.20. IR (KBr, cm−1): 1,682, 1,244, 1,148, and 1,120. HRMS for
(C18H14ClO3 [M+H]+). Calcd: 313.0626. Found: 313.0624.
7‐(4‐Methyl‐3‐nitrophenoxy)quinoline‐5,8‐dione (6b)
2‐Bromo‐3‐(4‐methyl‐3‐nitrophenoxy)naphthalene‐1,4‐dione (3f)
Yellow solid, yield 81%, mp 175–176°C. 1H NMR (400 MHz, CDCl3) δ
8.14 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 7.1 Hz, 1H), 7.77–7.65 (m, 2H),
7.53 (d, J = 2.2 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.13 (dd, J = 8.5,
2.3 Hz, 1H), and 2.49 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 178.09,
177.11, 155.22, 154.38, 149.29, 134.82, 134.60, 134.02, 131.08,
130.45, 129.37, 128.62, 127.87, 127.39, 121.65, 112.81, and 20.05.
IR (KBr, cm−1): 1,677, 1,528, 1,227, 1,197, and 816. HRMS for
(C17H11BrNO5 [M+H]+). Calcd: 387.9815. Found: 387.9822.
Yellow solid, yield 63%, mp 195–197°C. 1H NMR (400 MHz, acetone)
δ 8.99 (dd, J = 4.6, 1.5 Hz, 1H), 8.35 (dd, J = 7.9, 1.5 Hz, 1H), 7.90 (d,
J = 2.4 Hz, 1H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H),
7.54 (dd, J = 8.4, 2.5 Hz, 1H), 6.17 (s, 1H), and 2.58 (s, 3H). 13C NMR
(101 MHz, acetone) δ 182.97, 176.35, 159.70, 153.64, 151.30, 146.81,
134.07, 133.12, 130.29, 128.53, 127.38 (2C), 124.75, 116.16, 113.86,
17.99. IR (KBr, cm−1): 1,703, 1,656, 1,522, 1,330, 1,210, 812, and 648.
HRMS for (C16H11N2O5 [M+H]+). Calcd: 311.0662. Found: 311.0661.
6‐(4‐(Hexyloxy)phenoxy)‐2,2‐dimethyl‐2,3‐dihydrobenzofuran‐4,7‐
dione (8a)
2‐Bromo‐3‐(4‐(hexyloxy)phenoxy)naphthalene‐1,4‐dione (3g)
Red solid, yield 92%, mp 89–90°C. 1H NMR (400 MHz, CDCl3) δ
8.49–8.38 (m, 1H), 8.30–8.24 (m, 1H), 8.07–7.98 (m, 2H), 7.22 (d,
J = 8.9 Hz, 2H), 7.10 (d, J = 8.9 Hz, 2H), 4.17 (t, J = 6.5 Hz, 2H),
2.09–1.98 (m, 2H), 1.76–1.69 (m, 2H), 1.65–1.55 (m, 4H), and 1.17 (t,
J = 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 178.50, 177.63, 156.37,
155.74, 150.15, 134.43, 134.28, 131.05, 130.70, 128.21, 127.54,
127.23, 117.81 (2C), 115.33 (2C), 68.46, 31.59, 29.27, 25.73, 22.61,
and 14.06. IR (KBr, cm−1): 3,008, 2,872, 1,673, 1,503, and 1,206, 702.
HRMS for (C22H22BrO4 [M+H]+). Calcd: 429.0696. Found: 429.0702.
Brown solid, yield 36%, mp 62–64°C. 1H NMR (200 MHz, CDCl3) δ
7.00–6.66 (m, 5H), 3.91 (t, J = 6.5 Hz, 2H), 2.83 (s, 2H), 1.74 (dd,
J = 13.6, 6.2 Hz, 2H), 1.55 (s, 6H), 1.51–1.29 (m, 6H), and 0.90 (t,
J = 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 176.23, 173.32, 157.28,
155.72, 155.27, 150.27, 118.55, 117.90, 117.23 (2C), 115.27 (2C),
93.72, 68.45, 39.40, 31.55, 29.22, 28.27 (2C), 25.69, 22.57, and
14.00. IR (KBr, cm−1): 2,954, 1,671, 1,657, 1,475, and 1,239. HRMS
for (C22H27O5 [M]−). Calcd: 369.1707. Found: 369.3250.
2,2‐Dimethyl‐6‐(4‐methyl‐3‐nitrophenoxy)‐2,3‐dihydrobenzofuran‐
4,7‐dione (8b)
2‐Chloro‐3‐(naphthalen‐2‐yloxy)naphthalene‐1,4‐dione (3h)[21]
Orange solid, yield 88%, mp 158–159°C.[21] 1H NMR (400 MHz,
CDCl3) δ 8.21 (d, J = 7.4 Hz, 1H), 8.02 (d, J = 7.2 Hz, 1H), 7.86–7.73 (m,
5H), 7.66 (d, J = 7.9 Hz, 1H), 7.47–7.35 (m, 2H), and 7.31 (dd, J = 8.9,
1.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 178.46, 177.94, 154.22,
153.70, 134.57, 134.47, 133.97, 133.91, 131.27, 130.67, 130.50,
130.18, 127.88, 127.42, 127.28, 127.18, 126.91, 125.14, 117.93, and
111.79. IR (KBr, cm−1): 1,678, 1,231, 1,199, 870, and 691. HRMS for
(C20H12ClO3 [M+H]+). Calcd: 335.0469. Found: 355.0471.
Orange solid, yield 52%, mp 52–54°C. 1H NMR (400 MHz, CDCl3) δ
7.56 (d, J = 2.2 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.08 (dd, J = 7.4, 3.1 Hz,
1H), 5.32 (s, 1H), 2.73 (s, 2H), 2.43 (s, 3H), and 1.37 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 179.57, 177.59, 159.90, 158.42, 151.18, 149.65,
134.57, 132.06, 125.62, 118.15, 117.44, 108.56, 93.60, 39.43, 28.30
(2C), and 20.10. IR (KBr, cm−1): 2,971, 1,672, 1,641, 1,530, and 1,268.
HRMS for (C17H16NO6 [M+H]+). Calcd: 330.0972. Found: 330.0967.
5‐Bromo‐6‐(4‐(hexyloxy)phenoxy)‐2,2‐dimethyl‐2,3‐dihydrobenzo-
furan‐4,7‐dione (8c)
2‐Chloro‐3‐(naphthalen‐1‐yloxy)naphthalene‐1,4‐dione (3i)[21]
Red solid, yield 44%, mp 162–163°C.[21] 1H NMR (400 MHz, CDCl3) δ
8.29 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H),
Brown solid, yield 62%, mp 98–100°C. 1H NMR (400 MHz, CDCl3) δ
6.88 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.9 Hz, 2H), 3.87 (t, J = 6.5 Hz, 2H),

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ESPINOSA‐BUSTOS ET AL.
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2.79 (s, 2H), 1.78–1.65 (m, 2H), 1.52 (s, 6H), 1.46–1.36 (m, 2H), 1.30
(d, J = 6.5 Hz, 4H), and 0.87 (t, J = 6.0 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 176.24, 173.33, 157.29, 155.73, 155.29, 150.29, 119.28,
118.59, 117.92 (2C), 115.29 (2C), 93.75, 68.46, 39.41, 31.57, 29.25,
28.30 (2C), 25.71, 22.60, and 14.04. IR (KBr, cm−1): 2,937, 1,671,
1,657, 1,475, and 1,239. HRMS for (C22H26BrO5 [M+H]+). Calcd:
449.0958. Found: 449.0965.
solubilized with 10% sodium dodecyl sulphate in 0.1 mM HCl and
incubated overnight at 37°C. A formazan formation was measured at
570 nm in a multiwell reader (StatFax 4200; Awareness Technology,
Inc., Palm City, FL).
|
4.4
Electrochemical studies
The cyclic voltammetry experiments were carried out with a CH
Instruments model 620C potentiostat, and the scan rate was 50 mV/s.
A three‐electrode system consisting of a working glassy carbon
electrode, 3‐mm diameter, an Ag/AgCl reference electrode (3 M KCl),
and a platinum wire auxiliary electrode was used. The glassy carbon
electrode was cleaned up by polishing with alumina on a polishing felt
(BAS polishing kit). All experiments were conducted at room
temperature (25 2°C) and purging with nitrogen. The compounds
(1.0 mM) were dissolved in 0.1 M tetrabutylammonium perchlorate in
DMF.
5‐Bromo‐2,2‐dimethyl‐6‐(4‐methyl‐3‐nitrophenoxy)‐2,3‐dihydroben-
zofuran‐4,7‐dione (8d)
Red solid, yield 46%, mp 177–178°C. 1H NMR (400 MHz, CDCl3) δ
7.57 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.19 (dd, J = 8.4,
2.5 Hz, 1H), 2.86 (s, 2H), 2.57 (s, 3H), and 1.58 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 175.53, 172.77, 157.51, 154.51, 154.05, 149.17,
133.94, 129.31, 121.80, 120.89, 118.76, 112.78, 94.14, 39.32, 28.30
(2C), and 20.00. IR (KBr, cm−1): 2,971, 1,689, 1,661, 1,590, 1,522,
1,363, and 1,207. HRMS for (C17H15BrNO6 [M+H]+). Calcd:
408.0077. Found: 408.0084.
|
4.5
Pharmacophore
|
4.2
Trypanosomicidal effect
After structural flexible alignment of all quinone 3D structures, the
pharmacophoric model generated using the MOE program[25] was
Y strain T. cruzi epimastigotes (discrete typing unit Tc II) were
grown at 28°C in an axenic milieu (BHI‐tryptose) as previously
described,[22,23] complemented with 5% fetal bovine serum. Epimas-
tigotes from a 10‐day‐old culture (stationary phase) were inoculated
into 50 ml of fresh culture milieu to reach an initial concentration of
1 × 106 cells/ml. Cell growth was monitored by measuring the
absorbance of the culture at 600 nm every day. Before inoculation,
the milieu was supplemented with a given amount of the drug from a
stock solution in DMSO (25 mM). The final DMSO concentration in
the culture milieu 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, parasite growth was followed in the
absence (control) and in the presence of a range of concentrations
of the corresponding compound. On day 5, the absorbance of the
culture was measured and related to the control. The IC50 value was
taken as the concentration of drug needed to reduce the absorbance
ratio to 50%.[22,23]
elucidated using
a polar‐charged hydrophobic (PCH) scheme.
Different pharmacophoric features centered in the middle of the
atomic sets (centroids), with aromaticity (Aro), rings with delocaliza-
tion (Pir), positive charge (Cat), negative charge (Ani), and hydrogen
acceptor capacity (Acc) H‐bond were available and depending on the
structural set under study, some of them will be elucidated.
A consensus set of features was generated and associated to the
biological activity (IC50).
ACKNOWLEDGMENT
C. S. and C. E.‐B. gratefully acknowledge the financial support from
FONDECYT (Research Grant Nos. 1161816 and 1180292).
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
|
ORCID
4.3
Cytotoxicity assays
Christian Espinosa‐Bustos
http://orcid.org/0000-0001-6038-8439
Cytotoxicity assays were performed by using the 3‐(4,5‐dimethylthia-
zol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction method
as described previously.[24] Briefly, Vero cell were plated in a flat‐
bottom 96‐wells plate at 1 × 104 cells per well density. Test
compound solutions were diluted from freshly made stock solutions
in DMSO with water. DMSO was used as a negative control. Then,
the cells were incubated with these compounds at different
concentrations (from 0.05 to 50 μM) in 200 μl of 10% fetal bovine
serum‐RPMI (Rosewell Park Memorial Institute) culture medium at
37°C for 72 hr. Ten microliters of MTT was added at a final
concentration of 0.5 mg/ml, incubated at 37°C for 4 hr, and then
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How to cite this article: Espinosa‐Bustos C, Vázquez K, Varela J,
et al. New aryloxy‐quinone derivatives with promising activity
on Trypanosoma cruzi. Arch Pharm Chem Life Sci. 2019;
e1900213. https://doi.org/10.1002/ardp.201900213
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