New aryloxy-quinone derivatives as potential anti-Chagasic agents: synthesis, trypanosomicidal activity, electrochemical properties, pharmacophore elucidation and 3D-QSAR analysis

Article abstract of DOI:10.1039/c5ra10122k

A set of new aryloxy-quinones were synthesized and evaluated in vitro against the epimastigote form of Trypanosoma cruzi and their unspecific cytotoxicity was tested on murine macrophages J-774 cells. Most of these novel compounds were found to be much more potent and selective than the standard drug nifurtimox. Interestingly, 2-phenoxy-naphthoquinone 3b displayed a remarkable nanomolar inhibitory activity, IC₅₀ = 20 nM, and a high selectivity index, SI = 625. The E_pc1 was determined for the most interesting compounds and no correlation with the trypanosomicidal effect was found. Therefore, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure-trypanosomicidal activity relationship. The designed pharmacophore recognized the more potent and selective molecules, exhibiting five pharmacophoric features. A correlation coefficient R² of 0.99 of pIC₅₀ plotted against the predicted values indicated that the 3D-QSAR equation could be applied to further predictions of newly designed trypanosomicidal compounds.

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Gonzalez, M. Paulino and E. Birriel, RSC Adv., 2015, DOI: 10.1039/C5RA10122K.
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New aryloxy-quinone derivatives as potential Anti-Chagasic
agents: Synthesis, trypanosomicidal activity, electrochemical
properties, pharmacophore elucidation and 3D-QSAR analysis
Karina Vázquez, Christian Espinosa-Bustos, Jorge Soto-Delgado, Ricardo A. Tapia, Javier
Varela, Estefanía Birriel, Rodrigo Segura, Jaime Pizarro, Hugo Cerecetto, Mercedes
González, Margot Paulino and Cristian O. Salas

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New aryloxyꢀquinone derivatives as potential AntiꢀChagasic agents:
Synthesis, trypanosomicidal activity, electrochemical properties,
pharmacophore elucidation and 3DꢀQSAR analysis
a
a
b
a
Karina Vázquez , Christian EspinosaꢀBustos , Jorge SotoꢀDelgado , Ricardo A. Tapia , Javier Varela
c
c
d
d
c,e
c
,
Estefanía Birriel , Rodrigo Segura , Jaime Pizarro , Hugo Cerecetto , Mercedes González ,
f
a
Margot Paulino ,* and Cristian O. Salas ,*
a
Departamento de Química Orgánica, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago 6094411, Chile.
b
Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Quillota 980, Viña del Mar, Chile.
c
Grupo de Química Medicinal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo,
Uruguay.
d
Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 9170022 Chile.
e
Área de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Mataojo 2055, Montevideo,
Uruguay.
f
Centro de Bioinformática Estructural-DETEMA, Facultad de Química, Universidad de la República, C.C. 1157, Montevideo, Uruguay.
*Corresponding authors. eꢀmail address: margot@fq.edu.uy (M. Paulino) and cosalas@uc.cl (C.O.
Salas).
Abstract
A set of new aryloxyꢀquinones were synthesized and evaluated in vitro against the epimastigote form
of Trypanosoma cruzi and their unspecific cytotoxicity was tested on murine macrophages Jꢀ774 cells.
Most of these novel compounds were found to be extremely more potent and selective than the
standard drug nifurtimox. Interestingly, 2ꢀphenoxyꢀnaphthoquinone 3b displayed a remarkable
nanomolar inhibitory activity, IC = 20 nM, and a high selectivity index, SI = 625. The E was
5
0
pc1
determined for the most interesting compounds and no correlation with the trypanosomicidal effect was
found. Therefore, an in silico study was carried out to obtain a pharmacophoric model and quantitative
structureꢀtrypanosomicidal activity relationship. The designed pharmacophore recognized the more
2
potent and selective molecules, exhibiting five pharmacophoric features. A correlation coefficient R of
0
.99 of pIC50 plotted against the predicted values indicated that the 3DꢀQSAR equation could be
applied to further predictions of newly designed trypanosomicidal compounds.
1

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1
Introduction
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a neglected tropical
1
endemic disease in Central and South America. As a result of international migrations, Chagas disease
2
3
4
is also found now in nonꢀendemic countries such as in the United States, Europe and Australia. In
these countries where the vectors do not exist, the infection can be transmitted through donation of
5
infected blood or organs, and through vertical transmission from mother to child. The World Health
Organization (WHO) estimates that about 10 million people worldwide are currently infected, mainly
in endemic areas of Latin America. This disease represents an important health problem in Central and
South America and although mortality indexes have been decreased in the last few years, actually more
6
than 10.000 deaths are estimated to occur annually from Chagas disease. This disease has two
successive clinical phases: acute and chronic. There are two main drugs used for the treatment,
benznidazole and nifurtimox, developed more than 40 years ago, exhibit limited efficacy in the chronic
7
, 8
phase and both present severe toxic side effects. The need of more effective drugs has stimulated the
search for new compounds with potential clinical utility.
In view of the structural diversity and wide range of pharmacological activities exhibited by
compounds having a quinone scaffold, many natural and synthetic naphthoquinones have been tested
9
, 10
against T. cruzi parasites as possible antiꢀChagas agents.
Thus, considering the important antiꢀ
trypanosomal and leishmanicidal activity exhibited by lapachol (I, Fig. 1), Bolognesi and coꢀworkers
1
1
have synthesized a small library of 2ꢀaryloxyꢀ1,4ꢀnaphthoquinone derivatives. The most active
compound was 2ꢀphenoxyꢀ1,4ꢀnaphtoquinone II (Fig. 1), which showed an IC of 1.70 µM against the
5
0
1
1
amastigote forms of T. cruzi, but with low selectivity index (SI < 5).
In the search for new compounds with improved trypanosomicidal activity, we have been focused in
the synthesis of new aryloxyꢀindolequinones with different substitution patterns, which were tested for
their trypanosomicidal effect and selectivity in cultures of T. cruzi epimastigotes and macrophages,
respectively. Interestingly, 6ꢀphenoxyindolequinone III (Fig. 1), displayed excellent nanomolar
1
2
inhibitory activity, IC = 20 nM, and high selectivity index, SI = 625. Based on these results, we
5
0
recently carried out a study of 2ꢀarylaminonaphthoquinone and 5Hꢀbenzo[b]carbazoleꢀ6,11ꢀdione
derivatives. These compounds were assayed against T. cruzi epiꢀ and trypomastigotes and some of
1
3
them against cancer cells and normal fibroblasts. It was found that certain chemical modifications on
the naphthoquinone moiety increase the trypanosomicidal and cytotoxic effects. Several of these
compounds were more potent than the reference drug nifurtimox against both stages of the parasite and
2

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exhibited increased selectivity against T. cruzi in comparison with VERO cells, being the most active
ꢀarylaminonaphthoquinone IV (Fig. 1). On the other hand, the 2ꢀphenylꢀaminonaphthoquinone V
2
showed significant in vitro cytotoxicity against prostate and mammary cancer cells and high selectivity
in respect to fibroblasts. A preliminary analysis confirmed an earlier conclusion that the replacement of
a benzene ring by a pyridine ring condensed to the quinone core is an important modification to
1
4, 15
increase these activities.
The attempts to disclose a general structureꢀactivity relationships using
stereoelectronic and lipophilicity properties, point out that the presence of a chlorine atom at Cꢀ3 and a
highly lipophilic alkyl group or aromatic ring are newly observed elements with the aim of to design
13
more selective cytotoxic and trypanosomicidal compounds.
On the other hand, quantitative structureꢀactivity relationship (QSAR) studies have emerged as a useful
1
6, 17
tool to find relevant physicochemical descriptors for the design of new drugs,
as well as to get
some insight into the underlying mechanisms of action of the QSARꢀbased predicted activities.
Recently, QSAR analysis was applied to naphthoquinone derivatives and can be useful in the search for
1
8ꢀ20
new compounds with enhanced antiꢀtrypanosomal activity.
Fig. 1 Chemical structures of lapachol, phenoxyꢀ and phenylaminoꢀquinones with trypanosomicidal
activities and designed template for new antiꢀT. cruzi agents.
Herein, we designed a set of 27 new molecules based on the aryloxyꢀ quinone scaffold (Fig. 1), which
combine two pharmacophoric patterns of trypanosomicidal compounds (IIꢀV). These new compounds
could validate our hypothesis that chemical modifications on the naphthoquinone core or aromatic ring
would increase the trypanosomicidal effects and the selectivity as well. The in vitro trypanosomicidal
3

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activity against T. cruzi epimastigotes and the selectivity index for the set of 27 compounds was
evaluated. Additionally, considering that a possible mechanism of action is by reduction of the quinone
1
5, 21
system and production of freeꢀradical intermediates,
the electrochemical properties for most active
compounds were evaluated in order to find a probable correlation with their antiꢀT. cruzi activity.
Furthermore, an in silico strategy was applied to obtain a pharmacophoric model and 3DꢀQSAR
equation, to analyze the in vitro trypanosomicidal activity of the compounds under study.
2
2
Results and discussion
.1 Chemistry
A useful and direct approach for the synthesis of required aryloxyꢀquinones is through nucleophilic
1
1, 22
substitution reaction of phenols with haloquinones in basic medium at room temperature.
As shown
in Scheme 1, 2ꢀaryloxyꢀnaphthoquinones 3aꢀf (series I) were obtained in 40ꢀ97% through reaction of
phenols 2aꢀc with halonaphthoquinone 1a or 1b in dimethylformamide (DMF) in the presence of
potassium carbonate. Similarly, reaction of halonaphthoquinone 1a or 1b with naphthols 2d,e afforded
the corresponding naphthoquinones 3gꢀj in 53ꢀ95% yield.
Scheme 1. Synthesis of compounds of series I (3aꢀj). Reagents and conditions: (i) K CO , DMF, rt, 2ꢀ3
2
3
h.
The compounds belonging to series II were synthesized starting from bromoquinolinꢀ5,8ꢀdiones 6a and
b (Scheme 2). These quinones were obtained by bromination (Br /MeOH) of 8ꢀhydroxyquinoline 4a
6
2
or 8ꢀhydroxyꢀ2ꢀmethylquinoline 4b and subsequent oxidation of the corresponding dibromophenols
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2
3
5
a,b with nitric acid. The reaction of 6a and 6b with the phenols 2aꢀc or naphthols 2d,e furnished
regiospecifically the corresponding 7ꢀaryloxyꢀquinolineꢀ5,8ꢀdione derivatives 7aꢀi (24ꢀ92% yield).
Scheme 2. Synthesis of compounds of series II (7aꢀi). Reagents and conditions: (i) Br , MeOH, rt, 5
2
2
3
min; (ii) HNO , H SO , 0 °C, 30 min; (iii) K CO , 2aꢀe, DMF, rt, 3ꢀ5 h.
3
2
4
2
3
Scheme 3. Synthesis of compounds of series III (11aꢀi). Reagents and conditions: (i) Br , CHCl , rt,
2
3
1
.5 h; (ii) CrO , H O/AcOH, rt, 2 h; (iii) K CO , DMF, 2aꢀe, rt, 2 h.
3 2 2 3
5

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The required bromobenzofuranꢀ4,7ꢀdiones 10a,b (Scheme 3), were obtained using phenol 8 as
substrate. The bromination of 8 with two or four equivalents of Br in CHCl , followed by oxidation
2
3
14
with chromium trioxide, yielded the halobenzofuranquinones 10 a,b. The reaction of 10a,b and the
phenols 2aꢀe afforded compounds 11aꢀi (series III) in 23ꢀ83% yield. The chemical structures of the
1
13
target compounds were established on the basis of their spectral properties (IR, H NMR, C NMR and
HRMS, see experimental part and supplementary material).
2
.2 Trypanosomicidal effect
The in vitro trypanosomicidal activity of 2ꢀaryloxyꢀnaphthoquinones 3aꢀj (series I), 7ꢀaryloxyꢀ
quinolinquinones 7aꢀi (series II) and 6ꢀaryloxyꢀfuronaphthoquinones 11aꢀi (series III), was initially
2
4
tested against the epimastigote form of T. cruzi, Y strain (Tc II). For each derivative a doseꢀresponse
assay, between 0.01 and 50 ꢁM, was assayed to calculate the IC50 concentration (50% inhibitory
2
5, 26
concentration). Nifurtimox (Bayer) was used as the reference trypanosomicidal drug.
All aryloxyꢀ
quinones exhibited a potent trypanosomicidal activity and they were more active than the reference
drug nifurtimox, which had an IC of 7.0 ꢁM (Table 1). Among them, compounds 3aꢀb, 7f and 7h
5
0
displayed the most potent inhibitory activity with values of IC in the nanomolar order (IC < 90 nM
5
0
50
for epimastigotes).
ꢀPhenoxyꢀ1,4ꢀnaphthoquinone 3a (IC = 50 nM), was selected as a reference compound to evaluate
2
5
0
the effect of the chemical modifications for compounds of the series I. It was found that the
incorporation of a withdrawing group in the aryloxy moiety (nitro derivative 3b), enhanced the
trypanosomicidal effect and the compound with highest activity (IC = 20 nM) was obtained. On the
5
0
other hand, the electron donating methyl groups (3c) or αꢀ and βꢀnaphthyl rings (3g,i), weakly
decreased the trypanosomicidal effect compared to 3a, but these compounds were still more active than
nifurtimox (40 times). Regarding the replacement of the hydrogen atom at Cꢀ3 by bromine, compounds
3
dꢀf showed a decrease in trypanosomicidal effect compared with their unꢀhalogenated analogs;
whereas 2ꢀnaphthyloxyquinones 3h,j kept their antiꢀtrypanosoma activity. A similar behavior was
1
3
observed for the 2ꢀphenylaminoꢀ1,4ꢀnaphthoquinone derivatives previously studied.
For the compounds belonging to the series II (7aꢀi), the most active were 7f and 7h (IC = 40 and 90
5
0
nM respectively). In this case, the presence of the αꢀ or βꢀnaphthyl ring bound to the quinone core,
increased the trypanosomicidal effect in comparison to the reference compound 7a (IC = 110 nM), in
5
0
contrast to the results for the series I. The nitro derivative 7d was the less active of this series, but still
6

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it was more potent than nifurtimox (16 fold). The presence of methyl substituents on the phenyl ring of
a had no effect on the activity (7b). However, the trypanosomicidal activity of quinoline derivatives
7
with a methyl group at Cꢀ2 was lower than their Hꢀanalogues, showing a decrease up to four times (7f
vs 7g). Finally, analyzing the replacement of the benzene by a pyridine ring (series I vs II), an increase
in trypanosomicidal effect was observed on the epimastigote form (except 3a vs 7a). For instance,
compound 7f is almost four times more potent than its carbocyclic analog 3g, with IC values of 40
5
0
nM and 150 nM, respectively. These results are in agreement with previous findings from our group
that indicate the relevance of nitrogen substitution in the aromatic ring for activity of related
1
3ꢀ15
compounds.
The dihydrofurane derivatives of the series III (11aꢀi), were less active than the other series being most
of the IC values > 1.0 µM, except compound 11c which showed the lowest IC value (0.54 µM).
5
0
50
These results suggest that oxygen heterocyclic system fused to the quinone ring, decrease the
trypanosomicidal effect and that the halogen replacement at the quinone ring, give compounds with
less activity.
A preliminary analysis of several modifications on the naphthoquinone core showed a significant
improvement in the trypanosomicidal activity in compounds bearing nitro group substitution (3b) and
the replacement of a benzene ring by pyridine ring (7f and 7h), in comparison to the reference
compound 3a.
2
.3 Selectivity Studies
Compounds with trypanosomicidal activity do not have pharmacological relevance when display high
cytotoxicity on mammalian host cells and therefore exhibits low selectivity. For this reason,
compounds with the highest trypanosomicidal effect were studied for their unspecific cytotoxicity on
murine macrophages Jꢀ774 cells and the selective indexes (SI) were calculated as the ratio of the IC50
between epimastigotes and Jꢀ774 cells (Table 1). According to some authors in the search of new
strategies for the development of novel drugs for tropical disease, the selective index should be higher
2
7, 28
than 50.
concluded that most of them (19 compounds) exhibited a better selectivity than nifurtimox (SI value =
0). Interestingly, compounds 3a, 3b, 3d, 3e, 3f, 3j, 7a, 7b, 7f, 7g and 7h included in series I and II
Table 1 shows the IC values for the most compounds on Jꢀ774 cells and these results
50
4
showed a high selectivity index (SI values > 100). Among them, compound 3b deserved a special
importance, because exhibited the highest trypanosomicidal activity (IC50 value of 20 nM) and an
7

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elevated selectivity in regard to its toxicity toward Jꢀ774 cells (SI value = 625). A second group of
compounds (3c, 3g, 3h, 3i, 7c, 7e, 7i and 11c) were slightly more selective than nifurtimox (SI values
among 70ꢀ90). Finally, a third group of compounds (11a, 11b, 11d, 11g, 11h, 11i and 7d) were less
selective than nifurtimox (SI < 20) and most of them belonged to compounds of the series III, which
showed the lowest trypanosomicidal activity.
A preliminary chemical structural analysis for the selectivity indicates that for those compounds of the
series I, the presence of the nitro group in the aryloxy moiety (3b and 3e) contributed to an increase of
both the trypanosomicidal effect and selectivity. The opposite effect was observed in the presence of
dimethylphenyl (3c) or naphthyl groups (3g and 3i), that increased the toxicity in mammals and
decreased selectivity.
In previous studies, our group had reported that the substitution of hydrogen by halogen at Cꢀ2 in the
1
3
quinone moiety increase the selectivity, nevertheless, for halogenated compounds (3dꢀf, 3h or 3j),
there is no a clear pattern. On the other hand, for those of the series II (regarding to 7a, SI = 200), the
presence of the βꢀnaphthyl group linked to the naphthoquinone provides an access to more selective
compounds (7f). In contrast, the replacement of the quinoline core by 2ꢀmethylquinoline in addition to
the aryloxy substitution by dimethylphenyl or αꢀnaphthyl ring, reduced significantly the selectivity in
all analogues.
There are several proposed mechanisms of action for the trypanosomicidal activity of quinone
derivatives that could be related with the selectivity shown in this study. One of them is the wellꢀknown
ability of quinones for generating reactive oxygen species (ROS) through redox cycle with molecular
10, 29ꢀ32
oxygen and consequently strong oxidative stress and cell death.
The biochemical difference
between the parasite and host cells to avoid the damage mediated by ROS, are considerable, meanwhile
3
3
mammalian cells defend themselves efficiently against free radicals in diverse ways, T. cruzi´s
3
4
defense mechanisms against oxidative stress are defective. On the other hand, it has been reported
that quinones are inhibitors of trypanothione reductase (TR), an enzyme that constitute part of the
3
5, 36
principal oxidativeꢀstress defense of the T. cruzi
and it is absent in the mammals. Studies on this
issue are currently developed in our laboratory.
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Table 1. Effect upon culture growth of T. cruzi and selectivity index vs Jꢀ774 cells, and the Epc1 of
aryloxyꢀquinones.
b
c
Entry
IC50
Jꢀ774
µM)
IC50 S.I.
Epc1 (V)
(
a
(µM)
3
3
3
3
3
3
3
3
3
3
7
7
7
7
7
7
7
7
a
b
c
d
e
f
0.05 ± 0.02
0.02 ± 0.01
0.17 ± 0.05
0.11 ± 0.04
0.11 ± 0.04
0.23 ± 0.06
0.15 ± 0.04
0.14 ± 0.05
0.17 ± 0.04
0.17 ± 0.06
0.11 ± 0.04
0.11 ± 0.04
0.14 ± 0.05
0.44 ± 0.09
0.23 ± 0.07
0.04 ± 0.02
0.15 ± 0.04
0.09 ± 0.04
<12.5
12.5
<12.5
18
<250
625
ꢀ0.610
ꢀ0.526
ꢀ0.610
ꢀ0.397
ꢀ0.369
ꢀ0.397
ꢀ0.633
ꢀ0.456
ꢀ0.624
ꢀ0.440
ꢀ0.517
ꢀ0.555
ꢀ0.566
ꢀ0.515
ꢀ0.608
ꢀ0.508
ꢀ0.536
ꢀ0.517
<73.5
163.6
418.2
169.6
<83.3
<89.3
<73.5
111.8
200
46
39
g
h
i
<12.5
<12.5
<12.5
19
j
a
b
c
d
e
f
22
18
163.6
89.3
12.5
12.5
<12.5
15
28.4
<54.3
375
g
h
16
106.7
<138.9
<12.5
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b
c
Entry
IC50
Jꢀ774
µM)
IC50 S.I.
Epc1 (V)
(
a
(µM)
7
1
1
1
1
1
1
1
1
1
i
0.17 ± 0.05
1.00 ± 0.02
2.30 ± 0.03
0.54 ± 0.13
5.00 ± 0.6
1.60 ± 0.2
1.80 ± 0.4
0.98 ± 0.17
7.00 ± 1.7
5.50 ± 0.4
7.00 ± 0.3
<12.5
21
<73.5
21
ꢀ0.527
ꢀ0.550
ꢀ0.456
1a
1b
1c
1d
1e
1f
1g
1h
1i
61
25.5
81.5
5.00
n.d.
d
44
n.d.
25
ꢀ0,347
n.d.
n.d.
n.d.
21
n.d.
n.d.
21.4
~ 3.60
~ 4.54
40
ꢀ0.508
ꢀ0.482
ꢀ0.465
ꢀ
~ 25
~ 25
316 ± 0.5
e
Nfx
a
The results are mean of three independent experiments.
b
c
d
e
Selectivity Index: expressed as the ratio of IC in Jꢀ774 cells to IC in epimastigotes.
5
0
50
ꢀ1
Values determinate at c = 1 x 10ꢀ3 mol Lꢀ1, in DMF/TBAP, 0.1 mol Lꢀ1, ν = 50 mV s .
n.d.: not determined.
5
µM was used for this assay.
2
.4 Pharmacophore elucidation
In order to generate a pharmacophore model (hypothesis), the compound with the highest
trypanosomicidal activity (3b, IC50 20 nM) was chosen as a structural template. Once the
tridimensional structure of the selected molecule 3b was created, the pharmacophore hypothesis was
3
7
generated using the polarꢀchargedꢀhydrophobic (PHC) scheme of MOE program. Several structural
particularities were identified: a quinonic ring, aromatic rings, nitro group, and an oxygen bridge with
1
0

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the aromatic ring. Thus, using 3b as template and with the PCH scheme, that combines polarity, charge
and hydrophobicity, different pharmacophoric features centered in the middle of the atomic sets
(centroids), with aromaticity (Aro), rings with π delocalization (Pir), positive charge (Cat), negative
charge (Ani), and hydrogen acceptor capacity (Acc) were defined (Fig. 2).
Fig. 2 a) Pharmacophore generated from 3b overlapped with a rod drawing of molecules; b)
Pharmacophoric features depicted in the pharmacophore of 3b. F1; F5 = Aro, Aromatic moiety; F2 =
Pir, Rings with pi delocalization; F3; F4; F9 = Acc, hydrogen bond acceptor; F6 = Cat, cationic; F7; F
=
Ani, anionic.
Once the five pharmacophoric features of 3b were identified, a combination of them, selecting as
essential Aromatic (Aro) and hydrogen acceptor (Acc) (Fig. 3) and taking any four of them each time,
the search algorithm reached to found just the molecules with selectivity index greater than 100. By
this way, a search was made over the quinone databases and all the eleven selective molecules were
found as hits: 3b, 3e, 7f, 7a, 3f, 7b, 3d, 3j, 3h, 7c and 7d. From them, all the selective molecules except
one (7g), were detected, having an 89% of recovering of true positives. Three molecules (3h, 7c and
7
d) are false positives that are; they are nonꢀselective being detected by the pharmacophore as
positives. To have a quantitative measurement of this result, it is useful to calculate the enrichment
factor R (Equation 1).
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1

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R = [number of selective hits (=8) / number of nonꢀselective hits (=17)] / [number of all selective (=9) /
number of all nonꢀselective (=19)] = 0.99 (1)
Fig. 3 Selective pharmacophore features and interatomic distances between the essential features Acc
F1 and F2) and Aro (F3).
(
2
.5 Electrochemical study
The cytotoxicity of quinones has been mainly attributed to two processes; redox cycling that generates
ROS or electrophilic arylation of critical cellular nucleophiles, in both cases the singleꢀelectroꢀ
reduction of the quinone ring is the first step. Accordingly, several attempts to establish some
3
8ꢀ41
correlation between the biological activity and redox potentials of quinones have been reported.
Cyclic voltammetry (CV) is commonly used to obtain electrochemical parameters for biological studies
with quinones, such as potentials of the oxidation (E ) and reduction (E ) peaks or E (for
pa
pc
redox
3
8ꢀ43
reversible systems).
Therefore, redox properties for the most active compounds of each series were
determinate by CV in a typical measurement.
Fig. 4 shows the cyclic voltammograms for the most active compounds of each series (for the rest
compounds, CV profiles are available in the supplementary information). All quinones exhibited quasiꢀ
reversible reduction behavior, which is a typical quinone/semiquinone/hydroquinone triad in
3
8, 39, 44, 45
equilibrium, and the overall CV profiles are similar to those reported for other quinones.
The Epc1 values were obtained from the CV profile of each compound (Table 1), which is most
commonly used to compare biological activities. For compounds from the series I, the relative
reduction potential is as follows: 3e > 3d ~ 3f > 3j > 3h > 3b > 3a ~ 3c > 3i > 3g. An interesting
characteristic that present the five compounds that are most easily reduced, is the presence of the
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bromine direct attachment of the quinone core, which is according to the electronꢀwithdrawing feature
of this halogen. The reduction trend for compounds of the series II was 7f > 7d > 7a ~ 7h > 7i > 7g >
7
b > 7c > 7e. The values for the Epc1 for these compounds were around ꢀ0.50 V, value which is smaller
than those having a halogen of the series I and confirm the effect of this substituent. However, the
replacement of the benzene ring by the pyridine ring facilitates the electron transfer due to the nitrogen
atom attached to the quinone moiety. On the other hand, the presence of an electronꢀdonating methyl
group (in either aromatic rings), increased the barrier to reduction (7i, 7g, 7b, 7c and 7e) except for 7d,
which could be related to the presence of nitro group in metaꢀposition of the phenyl moiety. The ease
of reduction in the series III, expressed by Epc1 (Table 1) was: 11d > 11b > 11i > 11h > 11g > 11a. All
of them display behavior similar to that observed for other benzofuranquinones. The first four
compounds of this series shares the presence of a bromine or nitro group as substituent, which confirm
the role of these substituents in the reduction process, diminishing the values of the Epc1.
Fig. 4 Cyclic voltammograms of quinones 3a, 3b, 7f and 11c (1.0 mM). DMF/TBAP (0.1 M), glassy
ꢀ1
carbon electrode, cathodic direction, scan rate 50 mV s .
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3

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However, attempts to reveal an eventual structureꢀactivity relationship using values of Epc1 and pIC50
were unsuccessful for each series (Fig. 5). The lack of correlation between these parameters could
indicate that the first reduction potential of these aryloxyꢀquinones is not decisive in the
trypanosomicidal activity or although essential for cellular event, not always show a quantitative
3
9
correlation with this property. In fact, many other factors such as lipophilicity, membrane
permeability, etc., need critical consideration in order to rationalize the relationship of structural pattern
and trypanosomicidal activity. Considering the small correlation between de Epcl and the pIC , the
5
0
search for other tools to understand the trypanosomicidal activity were explored. With this goal in
mind, we performed the generation of 3DꢀQSAR models to correlate chemical structures of quinones
with their biological data to identify specific features that influence the observed trypanosomicidal
4
6ꢀ49
properties.
Fig. 5 Graphs correlating the values of Epc1 of series I, II and III of quinones with the trypanosomicidal
effect, represented by pIC .
50
2
.6 3DꢀQSAR
A 3DꢀQSARꢀcorrelation analysis was performed with a set of 28 aryloxyꢀquinones with potent
trypanosomicidal activity, selecting 19 and 9 compounds as the training and test sets, respectively.
Conformational sampling on each molecule by quenched molecular dynamics and alignment by an
atomꢀbased fashion was carried out using Open3DAlign. Then, derivation of 3DꢀQSAR models was
carried out by Partial LeastꢀSquares (PLS) analysis with Iterative Variable Elimination (IVEꢀPLS)
4
9
procedure by means of Open3DQSAR. The statistical results for 3DꢀQSAR modelling gave for
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2
2
internal crossꢀvalidation q
of 0.75 and q
of 0.78, with five components in all models. Figure 6a
LOO
LMO
shows the aligned molecules and Fig. 6b the correlations between experimental versus predicted values
2
of pIC . The correlation coefficients of our model was R = 0.99, and standard deviation of the error of
5
0
2
prediction (SDEP) of 0.324 for training set, as well as, the r
= 0.94 and SDEP of 0.115 for test set.
(pred)
These data indicate that a significant 3DꢀQSAR model was obtained.
Fig. 6 a) Atomꢀbyꢀatom superposition used for 3DꢀQSAR analysis. b) Scatter plot of the experimental
activities versus predicted activities for 3DꢀQSAR model: (●) training set predictions, (○) LOO crossꢀ
validated predictions, (●) testꢀset predictions.
For the 3DꢀQSAR modeling, the contour maps were created using the data from PLS analyses. These
maps help us to explain the structural features of the compounds that are contributing towards the antiꢀ
trypanosomicidal activity of the aryloxyꢀquinones. The steric contour map indicates the areas where the
bulky substituents are predicted to increase (green) or decrease (yellow) the activity. (Fig. 7 a,b)
Therefore, considering the yellow area around the aromatic moiety suggests that this ring should not
have bulky groups. Furthermore, in compounds with a 2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuran system and
a bromine atom at the quinone ring (11d, 11e, 11h and 11i), the halogen substituent shifts the aryloxy
group inducing an unfavorable interaction, and consequently decreases the biological activity (Fig. 7b).
The electrostatic map show red contour where high electron density (negative charge) would increase
the activity, and the blue contour displays areas with low electron density (partial positive charge) is
expected to increase the activity (Fig. 7 c,d). The quinone ring is thought to be critical for the biological
activity and the changes of its electron density might have caused an increment of the activity. The
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5

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most active compound 3b received the influence of the NO group electronꢀwithdrawing, having a
2
positive delocalized polar region in the aromatic ring NO substituted. According to that, the
2
electrostatic contour map (Fig. 7c) of the aromatic ring in 3b has a positiveꢀcharge favored region. The
electrostatic contour maps indicated that the positive electrostatic interaction of the aromatic ring with a
possible receptor which could be increase the activity. In comparison, this interaction is lost in the less
active compound 11i (Fig. 7d).
Fig. 7 a) Steric contour map for most active compounds 3a, 3b and 7a. b) Steric contour map for least
active compounds 11d, 11h and 11i. c) Electrostatic contour map for most active compounds 3a, 3b
and 7a. d) Electrostatic contour map for least active compounds 11d, 11h and 11i.
3
Conclusions
We have synthesized 27 new aryloxyquinones considering the 2ꢀphenoxyꢀnaphthoquinone (3a) as
reference compound and were grouped in three series according to similar structural pattern: 2ꢀaryloxyꢀ
naphthoquinones 3aꢀj, 7ꢀaryloxyꢀquinolinquinones 7aꢀi and 6ꢀaryloxyꢀfuronaphthoquinones 11aꢀi. All
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compounds were evaluated as trypanosomicidal agents and tested for their unspecific cytotoxicity on
murine macrophages Jꢀ774 cells. Most of these compounds were more potent than the reference drug
nifurtimox vs epimastigote form and exhibited unprecedented selectivity against T. cruzi in comparison
with Jꢀ774 cells, notably the 2ꢀ(3ꢀnitrophenyloxy)ꢀnaphthoquinone 3b (IC = 20 nM and SI = 625).
5
0
Also, it was demonstrated that certain chemical modifications on the naphthoquinone moiety and on
the aryloxy group, increased significantly the trypanosomicidal effect and the selectivity of some
derivatives. Electrochemical studies were done in order to find out a possible structureꢀactivity
relationship and used in an attempt to understand this biological activity, but no correlation was found
between the E and the pIC . Because of that, in silico studies were done through the pharmacophoric
pcl
50
mapping and QSAR analyses. Eight out of nine molecules with the best IC and SI were chosen using
50
the selective designed pharmacophore, proving that this procedure is able to find active and selective
molecules with an enrichment factor of R = 0.99. A 3DꢀQSAR equation was obtained with a
2
coefficient of determination R of 0.99 and the contour maps indicated that the trypanosomicidal
activity was related to the steric and electronic features in the aryloxy moiety or ring fused to the
quinone system. Our current work is focused to understand in depth the selectivity and mechanism of
action of the most promissory compounds.
4
4
Experimental
.1 Materials and measurements
Melting points were determined on a Kofler Thermogerate apparatus and were uncorrected. Infrared
spectra were recorded on a JASCO FT/IRꢀ400 spectrophotometer. Nuclear magnetic resonance spectra
were recorded, unless otherwise specified, on a Bruker AMꢀ400 instrument using deuterochloroform
solutions containing tetramethylsilane as an internal standard. Mass spectra were obtained on a HP
5
988A mass spectrometer. HRMSꢀESIꢀMS experiments were done using a Thermo Scientific Exactive
Plus Orbitrap spectrometer with a constant nebulizer temperature of 250 °C. The experiments were
carried out in positive ion mode, with a scan range of m/z 300.00ꢀ1510.40 with resolution 140.000. The
samples were infused directly into the ESI source, via a syringe pump, at flow rates of 5 µL minꢀ1,
through the instrument’s injection valve. Thin layer chromatography (tlc) was performed using Merck
GFꢀ254 type 60 silica gel. Column chromatography was carried out using Merck type 9385 silica gel.
The purity of the compounds was determined by tlc and highꢀresolution mass spectrometry (HRMS).
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4
.2 Synthesis
General synthetic procedure of aryloxy quinones:
In a reaction flask, the suitable phenol (1 mmol) and K CO (3 mmol) was suspended in DMF (5 ml).
2
3
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 2ꢀ5 hours at room temperature. Later, the reaction was
poured into ice water and extracted with ethyl acetate (3 x 25 mL). The combined organic layers were
washed with a saturated solution of sodium sulfite (3 x 25 mL) and brine (3 x 25 mL). Finally, the
organic layer was dried with anhydrous sodium sulfate and concentrated under vacuum. The solid
residue was purified by column chromatography on silica gel. For compounds 3aꢀj, the eluent used was
dichloromethane and for 7aꢀi and 11aꢀi were a mixture of methanolꢀdichloromethane 1%.
11
1
2
ꢀphenoxynaphthaleneꢀ1,4ꢀdione (3a). Yellow solid, yield 75%, mp 102ꢀ103 ºC (lit. 101 ºC). H
NMR (400 MHz, CDCl ) δ ppm: 5.99 (s, 1H), 7.15 (d, J = 7,7 Hz, 2H), 7.34 (t, J = 7,5 Hz, 1H), 7.46 (t,
3
1
3
J = 7,9 Hz, 2H), 7.74ꢀ7.81 (m, 2H), 8.04ꢀ8.10 (m, 1H), 8.18ꢀ8.23 (m, 1H). C NMR (101 MHz,
CDCl ) δ ppm: 113.34, 121.07 (2C), 126.18, 126.60, 126.73, 130.40 (2C), 131.06, 131.91, 133.50,
3
ꢀ1
1
34.41, 152.67, 160.45, 179.86, 184.88. IR (KBr, cm ): 1613, 1672, 1591. HRMSꢀ
+
ESI for (C H O [M+H] ). Calcd: 251.0708 Found: 251.0699.
1
6
11
3
1
2
ꢀ(3ꢀnitrophenoxy)naphthaleneꢀ1,4ꢀdione (3b). Yellow solid, yield 80 %, mp 170ꢀ173ºC. H NMR
(
400 MHz, CDCl ) δ ppm: 6.05 (s, 1H), 7.53 (dd, J = 1.5, 8.2 Hz, 1H), 7.69 (t, J = 8.2 Hz, 1H), 7.74ꢀ
3
1
3
7
1
1
.85 (m, 2H), 8.00ꢀ8.12 (m, 2H), 8.16ꢀ8.25 (m, 2H). C NMR (101 MHz, CDCl ) δ ppm: 115.14,
3
16.53, 121.43, 126.53, 127.03, 127.23, 131.05, 131.34, 131.90, 134.00, 134.83, 149.54, 153.61,
ꢀ1
59.31, 179.36, 184.53. IR (KBr, cm ): 1620, 1654, 1528, 1472, 1178, 1032. HRMSꢀ
+
ESI for (C H NO [M+H] ). Calcd: 296.0559 Found: 296.0560.
1
6
9
5
1
2
ꢀ(2,5ꢀdimethylphenoxy)naphthaleneꢀ1,4ꢀdione (3c). Yellow oil, yield 63 %. H NMR (400 MHz,
CDCl ) δ ppm: 2.15 (s, 3H), 2.33 (s, 3H), 5.83 (s, 1H), 6.86 (s, 1H), 7.02 (d, J = 7.6 Hz, 1H), 7.17 (d, J
3
1
3
=
7.7 Hz, 1H), 7.74ꢀ7.78 (m, 2H), 8.02ꢀ8.09 (m, 1H),8.20 (dd, J = 6.1, 2.7 Hz, 1H). C NMR (101
MHz, CDCl ) δ ppm: 15.38, 20.98, 112.86, 121.62, 126.28, 126.67, 126.82, 127.62, 131.27, 131.80,
3
ꢀ
1
1
1
32.12, 133.54, 134.47, 138.07, 150.74, 159.85, 179.94, 185.14. IR (KBr, cm ): 1610, 1651, 1288,
+
258, 1222. HRMSꢀESI for (C H O [M+H] ). Calcd: 279.1021. Found: 279.1032.
1
8
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1
2
ꢀbromoꢀ3ꢀphenoxynaphthaleneꢀ1,4ꢀdione (3d). Yellow solid, yield 80 %, mp 160ꢀ162 °C. H NMR
(
400 MHz, CDCl ) δ ppm: 7.01 (d, J = 7.8 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 7.29ꢀ7.38 (m, 2H), 7.71ꢀ
3
1
3
7
1
.81 (m, 2H), 7.98ꢀ8.06 (m, 1H), 8.16ꢀ8.23 (m, 1H). C NMR (101 MHz, CDCl ) δ ppm: 116.69,
3
24.10, 127.35, 127.69, 129.88, 130.73, 131.17, 134.42, 134.56, 156.06, 156.41, 177.47, 178.48. IR
ꢀ1
+
(
KBr, cm ): 1613, 1672, 1206, 1245, 1159, 1021. HRMSꢀESI for (C H BrO [M+H] ). Calcd:
16 9 3
3
27.9735. Found: 329.1719.
ꢀbromoꢀ3ꢀ(3ꢀnitrophenoxy)naphthaleneꢀ1,4ꢀdione (3e). Yellow solid, yield 76%, mp 197ꢀ199 °C.
2
1
H NMR (400 MHz, CDCl ) δ ppm: 7.40 (ddd, J = 0.8, 2.5, 8.3 Hz, 1H), 7.55 (t, J = 8.2 Hz, 1H), 7.76ꢀ
3
1
3
7
1
1
.86 (m, 3H), 8.00ꢀ8.10 (m, 2H), 8.23ꢀ8.27 (m, 1H). C NMR (101 MHz, CDCl ) δ ppm: 111.93,
3
18.78, 122.78, 127.24, 127.75, 128.72, 130.28, 130.63, 131.28, 134.36, 134.59, 149.49, 155.25,
ꢀ1
56.52, 176.92, 177.76. IR (KBr, cm ): 1677, 1536, 1349, 1245, 1018. HRMSꢀ
+
ESI for (C H BrNO [M+H] ). Calcd: 373.9664. Found: 373.9648.
1
6
9
5
2
ꢀbromoꢀ3ꢀ(2,5ꢀdimethylphenoxy)naphthaleneꢀ1,4ꢀdione (3f). Orange solid, yield 97 %, mp 196ꢀ
1
1
98 °C. H NMR (400 MHz, CDCl ) δ ppm: 2.22 (s, 3H), 2.38 (s, 3H), 6.48 (s, 1H), 6.86 (d, J = 7.6
3
1
3
Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.68ꢀ7.81 (m, 2H), 7.98ꢀ8.09 (m, 1H), 8.18ꢀ8.27 (m, 1H). C NMR
(
101 MHz, CDCl ) δ ppm: 15.79, 20.90, 115.94, 124.68, 124.95, 125.11, 126.75, 127.26, 127.55,
3
ꢀ1
1
28.21, 131.30, 134.25, 134.43, 136.83, 154.82, 156.76, 177.54, 178.51. IR (KBr, cm ): 1679, 199,
+
+
1
244, 1098, 1002. HRMSꢀESI for (C H BrO [M+Na ] ). Calcd: 380.9925 Found: 381.2984.
1
8
13
3
1
2
ꢀ(naphthalenꢀ2ꢀyloxy)naphthaleneꢀ1,4ꢀdione (3g). Yellow solid, yield 95%, mp 163ꢀ164 °C. H
NMR (400 MHz, CDCl ) δ ppm: 6.01 (s, 1H), 7.27 (dd, J = 2.4, 8.8 Hz, 1H), 7.48ꢀ7.57 (m, 2H), 7.60
3
(d, J = 2.3 Hz, 1H), 7.75 (dd, J = 2.8, 5.5 Hz, 2H), 7.80ꢀ7.84 (m, 1H), 7.87ꢀ7.90 (m, 1H), 7.94 (d, J =
1
3
8
1
1
.9 Hz, 1H), 8.02ꢀ8.07 (m, 1H), 8.18ꢀ8.22 (m, 1H). C NMR (101 MHz, CDCl ) δ ppm: 113.73,
3
18.35, 120.11, 126.28, 126.37, 126.84, 127.18, 127.72, 128.02, 130.80, 131.18, 131.74, 132.03,
33.58, 134.07, 134.49, 150.29, 160.58, 179.95, 184.90. IR (KBr, cmꢀ1): 1629, 1671, 1242. HRMSꢀ
+
ESI for (C H O [M+H] ). Calcd: 301.0865. Found: 301.0865.
2
0
12
3
2
ꢀbromoꢀ3ꢀ(naphthaleneꢀ2ꢀyloxy)naphthaleneꢀ1,4ꢀdione (3h). Orange solid, yield 67 %, mp 166ꢀ
1
1
68 °C. H NMR (400 MHz, CDCl ) δ ppm: 7.27 – 7.22 (m, 1H), 7.33 (dd, J = 8.9, 2.5 Hz, 1H), 7.42
3
(m, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.74 (m, 2H), 7.81 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 8.01
13
(
dd, J = 7.4, 1.3 Hz, 1H), 8.24 – 8.20 (m, 1H). C NMR (101 MHz, CDCl ) δ ppm: 111.78, 117.98,
3
1
25.13, 126.91, 127.19, 127.35, 127.69, 127.89, 128.01, 130.21, 130.49, 130.67, 131.16, 133.92,
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ꢀ1
1
34.40, 134.54, 154.15, 156.16, 177.39, 178.49. IR (KBr, cm ): 1671, 1509, 1572, 1195, 1158.
+
+
HRMSꢀESI for (C H BrO [M+Na ] ). Calcd: 400.9789 Found: 400.9766.
2
0
11
3
1
2
ꢀ(naphthalenꢀ1ꢀyloxy)naphthaleneꢀ1,4ꢀdione (3i). Yellow solid, yield 53%, mp 156ꢀ158 °C. H
NMR (400 MHz, CDCl ) δ ppm: 5.82 (s, 1H), 7.27 (d, J = 7.3 Hz, 1H), 7.45ꢀ7.59 (m, 3H), 7.71 – 7.79
3
(m, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 8.03 (br, 1H),
1
3
8
1
1
3
.24 (br, 1H). C NMR (101 MHz, CDCl ) δ ppm: 113.78, 117.63, 121.24, 125.75, 126.05, 126.33,
3
26.89, 126.93, 127.12, 127.16, 128.36, 131.27, 132.10, 133.64, 134.53, 135.21, 148.64, 160.62,
ꢀ1
+
79.91, 185.00. IR (KBr, cm ): 1602, 1682, 1034, 1248. HRMSꢀESI for (C H O [M+H] ). Calcd:
2
0
12
3
01.0865 Found: 301.0859.
ꢀbromoꢀ3ꢀ(naphthalenꢀ1ꢀyloxy)naphthaleneꢀ1,4ꢀdione (3j). Red solid, yield 59%, mp 161ꢀ163 °C.
2
1
H NMR (400 MHz, CDCl ) δ ppm: 6.74 (d, J = 7.5 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.52–7.65 (m,
3
3
H), 7.67–7.79 (m, 2H), 7.86 (d, J = 6.9 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 8.20 (d, J = 8.3 Hz, 1H),
1
3
8
1
1
.34 (d, J = 7.5 Hz, 1H). C NMR (101 MHz, CDCl ) δ ppm: 109.60, 121.99 (2C), 124.15, 125.22,
3
26.48, 127.09, 127.37, 127.72, 127.81, 130.72 (2C), 131.21, 134.43, 134.57, 134.97, 152.56, 156.74,
ꢀ1
77.27,
178.51.
IR
(KBr,
cm ):
1682,
1601,
1570,
1247,
1221.
HRMSꢀ
+
+
ESI for (C H BrO [M+Na ] ). Calcd: 400.9789. Found: 400.9771.
2
0
11
3
1
7
ꢀphenoxyquinolineꢀ5,8ꢀdione (7a). Yellow solid, yield 47 %, mp 172ꢀ173 °C. H NMR (400 MHz,
CDCl ) δ ppm: 6.04 (s, 1H), 7.19–7.13 (m, 2H), 7.34 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 8.0 Hz, 2H), 7.72
3
13
(
dd, J = 4.7, 7.9 Hz, 1H), 8.41 (dd, J = 1.7, 7.9 Hz, 1H), 9.07 (dd, J = 1.7, 4.7 Hz, 1H). C NMR (101
MHz, CDCl ) δ ppm: 112.78, 115.50, 121.08, 126.95, 128.18, 129.06, 129.61, 130.58, 134.46, 147.00,
3
ꢀ1
1
52.52, 154.55, 161.10, 178.05, 183.73. IR (KBr, cm ): 3052, 3016, 1610, 1280, 1240, 1083. HRMSꢀ
+
ESI for (C H NO [M+H] ). Calcd: 252.0661. Found: 252.0654.
1
5
9
3
1
7
ꢀ(2,5ꢀdimethylphenoxy)quinolineꢀ5,8ꢀdione (7b). Orange solid, yield 63 %, mp 177ꢀ179 °C. H
NMR (400 MHz, CDCl ) δ ppm: 2.16 (s, 3H), 2.35 (s, 3H), 5.91 (s, 1H), 6.87 (s, 1H), 7.04 (d, J = 7.8
3
Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.72 (dd, J = 4.7, 7.9 Hz, 1H), 8.41 (dd, J = 1.7, 7.9 Hz, 1H), 9.07
1
3
(
dd, J = 1.7, 4.7 Hz, 1H). C NMR (101 MHz, CDCl ) δ ppm: 183.84, 178.01, 160.37, 154.47, 150.52,
3
1
47.04, 138.18, 134.44, 131.86, 129.10, 128.15, 127.83, 126.53, 121.45, 112.26, 20.95, 15.34. IR
ꢀ1
+
(
KBr, cm ): 1654, 1613, 1244, 1141. HRMSꢀESI for (C H NO [M+H] ). Calcd: 280.0974. Found:
17 13 3
2
80.0968.
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1
2
ꢀmethylꢀ7ꢀphenoxyquinolineꢀ5,8ꢀdione (7c). Yellow solid, yield 49%, mp 165ꢀ167. H NMR (400
MHz, CDCl ) δ ppm: 2.80 (s, 3H), 5.98 (s, 1H), 7.15 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 7.2 Hz, 1H), 7.48
3
1
3
(
t, J = 7.9 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H). C NMR (101 MHz, CDCl ) δ
3
ppm: 25.60, 112.88, 121.49 (2C), 127.17, 127.27, 128.44, 130.84 (2C), 134.85, 146.88, 152.98, 161.27,
ꢀ1
1
65.21, 178.63, 184.27. IR (KBr, cm ): 1640, 1614, 1582, 1289, 1226. HRMSꢀ
+
ESI for (C H NO [M+H] ) Calcd: 266.0817. Found: 266.0810.
1
6
11
3
1
2
ꢀmethylꢀ7ꢀ(3ꢀnitrophenoxy)quinolineꢀ5,8ꢀdione (7d). Yellow solid, yield 24 %, mp 197ꢀ198°C. H
NMR (400 MHz, CDCl ) δ ppm: 2.81 (s, 3H), 6.06 (s, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 8.1
3
13
Hz, 1H), 7.70 (t, J = 8.2 Hz, 1H), 8.05 (s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H). C
NMR (101 MHz, CDCl ) δ ppm: 183.52, 177.72, 165.38, 159.71, 153.38, 149.56, 146.44, 134.71,
3
ꢀ1
1
31.44, 128.41, 127.36, 126.94, 121.66, 116.65, 114.06, 25.37. IR (KBr, cm ): 1698, 1654, 1350,
+
1
258, 1163. HRMSꢀESI for (C H N O [M+H] ) Calcd: 311.0668. Found: 311.0663.
1
6
10
2
5
7
ꢀ(2,5ꢀdimethylphenoxy)ꢀ2ꢀmethylquinolineꢀ5,8ꢀdione (7e). Yellow solid, yield 92 %, mp 165ꢀ
1
1
66°C. H NMR (400 MHz, CDCl ) δ ppm: 2.15 (s, 3H), 2.34 (s, 3H), 2.80 (s, 3H), 5.85 (s, 1H), 6.86
3
(s, 1H), 7.03 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.0 Hz,
1
3
1
1
1
H). C NMR (101 MHz, CDCl ) δ ppm: 15.28, 20.90, 25.20, 111.95, 121.49, 126.51, 126.92, 127.68,
3
ꢀ1
28.05, 131.77, 134.47, 138.05, 146.53, 150.57, 160.11, 164.74, 178.21, 183.98. IR (KBr, cm ): 1652,
+
322, 1114, 1110. HRMSꢀESI for (C H NO [M+H] ) Calcd: 294.1130. Found: 294.1121.
1
8
15
3
1
7
ꢀ(naphthalenꢀ2ꢀyloxy)quinolineꢀ5,8ꢀdione (7f). Yellow solid, yield 42 %, mp 190ꢀ191 °C. H NMR
(
400 MHz, CDCl ) δ ppm: 6.07 (s, 1H), 7.27 (d, J = 9.3 Hz, 1H), 7.54 (m, 2H), 7.61 (s, 1H), 7.71 (dd, J
3
=
5.1, 7.2 Hz, 1H), 7.79ꢀ7.93 (m, 2H), 7.96 (d, J = 8.9 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 9.07 (d, J =
13
3
.7 Hz, 1H). C NMR (101 MHz, CDCl ) δ ppm: 113.03, 118.43, 119.92, 126.54, 127.31, 127.77,
3
1
28.06, 128.18, 129.07, 130.96, 131.84, 134.04, 134.45, 147.02, 150.02, 154.57, 161.16, 178.09,
ꢀ1
+
1
83.67. IR (KBr, cm ): 1649, 1222, 1003. HRMSꢀESI for (C H NO [M+H] ) Calcd: 301.0739.
19 11 3
Found: 302.0809.
2
ꢀmethylꢀ7ꢀ(naphthalenꢀ2ꢀyloxy)quinolineꢀ5,8ꢀdione (7g). Yellow solid, yield 33 %, mp 198ꢀ199 °C.
1
H NMR (400 MHz, CDCl ) δ ppm: 2.80 (s, 3H), 6.01 (s, 1H), 7.24ꢀ7.30 (m, 1H), 7.49ꢀ7.58 (m, 3H),
3
1
3
7
.60 (d, J = 1.7 Hz, 1H), 7.79ꢀ7.91 (m, 2H), 7.93 (t, J = 8.7 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H). C
NMR (101 MHz, CDCl ) δ ppm: 25.25, 112.76, 118.43, 120.02, 126.44, 126.92, 127.22, 127.72,
3
1
28.01, 128.11, 130.84, 131.77, 134.01, 134.49, 146.52, 150.11, 160.95, 164.85, 178.27, 183.83. IR
2
1

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DOI: 10.1039/C5RA10122K
ꢀ1
+
(
KBr, cm ): 1696, 1586, 1283, 1176. HRMSꢀESI for (C H NO [M+H] ) Calcd: 316.0974. Found:
20 13 3
3
16.0964.
ꢀ(naphthalenꢀ1ꢀyloxy)quinolineꢀ5,8ꢀdione (7h). Yellow solid, yield 24 %, mp 118ꢀ120°C. H NMR
1
7
(
400 MHz, CDCl ) δ ppm: 6.07 (s, 1H), 7.27 (dd, J = 1.6, 8.9 Hz, 1H), 7.54 (m, 2H), 7.61 (d, J = 1.7
3
Hz, 1H), 7.71 (dd, J = 4.7, 7.8 Hz, 1H), 7.81ꢀ7.85 (m, 1H), 7.88ꢀ7.92 (m, 1H), 7.96 (d, J = 8.9 Hz, 1H),
1
3
8
1
1
.39 (d, J = 7.9 Hz, 1H), 9.07 (d, J = 4.6 Hz, 1H). C NMR (101 MHz, CDCl ) δ ppm: 113.01, 118.43,
3
19.92, 126.54, 127.31, 127.76, 128.05, 128.19, 129.06, 130.96, 131.83, 134.03, 134.45, 147.01,
ꢀ1
49.99, 154.57, 161.16, 178.09, 183.67. IR (KBr, cm ): 1656, 1609, 1242. HRMSꢀ
ꢀ
ESI for (C H NO [M] ) Calcd: 300.0666. Found: 300.0656.
1
9
11
3
2
ꢀmethylꢀ7ꢀ(naphthalenꢀ1ꢀyloxy)quinolineꢀ5,8ꢀdione (7i). Yellow solid, yield 25 %, mp 199ꢀ200 °C.
1
H NMR (400 MHz, CDCl ) δ ppm: 2.18 (s, 3H), 6.43 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6
3
1
3
Hz, 1H), 7.69 – 7.75 (m, 3H), 7.98 (d, J = 7.2 Hz, 1H), 8.14 – 8.18 (m, 2H). C NMR (101 MHz,
CDCl ) δ ppm: 15.79, 115.93, 124.68, 124.94, 126.72, 127.26, 127.55, 128.21, 130.65, 131.16, 131.29,
3
ꢀ1
1
34.22, 134.40, 134.51, 136.79, 142.58, 154.81, 156.76, 177.54, 178.56. IR (KBr, cm ): 1653, 1615,
ꢀ
1
225. HRMSꢀESI for (C H NO [M] ) Calcd: 314.0823 Found: 314.0814.
2
0
13
3
1
2
,2ꢀdimethylꢀ6ꢀphenoxyꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11a). Orange oil, yield 47 %. H NMR
(
400 MHz, CDCl ) δ ppm: 1.56 (s, 6H), 2.92 (s, 2H), 5.47 (s, 1H), 7.08 (d, J = 7.8 Hz, 2H), 7.30 (d, J =
3
13
7
.4 Hz, 1H), 7.44 (t, J = 7.8 Hz, 2H). C NMR (101 MHz, CDCl ) δ ppm: 28.44 (2C), 39.63, 93.44,
3
1
07.71, 117.94, 121.06 (2C), 126.79, 130.45 (2C), 153.02, 158.61, 161.13, 178.42, 180.24. IR (KBr,
ꢀ1
+
cm ): 2923, 1653, 1249, 1207. HRMSꢀESI for (C H O [M+H] ) Calcd: 271.0970. Found: 271.0978.
1
6
14
4
2
,2ꢀdimethylꢀ6ꢀ(3ꢀnitrophenoxy)ꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11b). Orangeꢀred oil, yield 26
1
%
. H NMR (400 MHz, CDCl ) δ ppm: 1.58 (s, 6H), 2.94 (s, 2H), 5.55 (s, 1H), 7.48 (d, J = 9.0 Hz,
3
1
3
1
H), 7.67 (t, J = 8.2 Hz, 1H), 7.99 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H). C NMR (101 MHz, CDCl ) δ
3
ppm: 28.34 (2C), 39.46, 93.70, 109.18, 116.43, 118.32, 121.42, 127.15, 131.26, 149.41, 153.65,
ꢀ1
1
58.44, 159.65, 177.53, 179.55. IR (KBr, cm ): 1613, 1535, 1203. HRMSꢀ
+
ESI for (C H NO [M+H] ) Calcd: 316.0821. Found: 316.0792.
1
6
13
6
6
ꢀ(2,5ꢀdimethylphenoxy)ꢀ2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11c). Orangeꢀred oil,
1
yield 50 %. H NMR (400 MHz, CDCl ) δ ppm: 1.57 (s, 6H), 2.13 (s, 3H), 2.31 (s, 3H), 2.92 (s, 2H),
3
1
3
5
.34 (s, 5H), 6.79 (s, 1H), 6.99 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 7.7 Hz, 1H). C NMR (101 MHz,
CDCl ) δ ppm: 15.25, 20.85, 28.31 (2C), 39.47, 93.25, 107.03, 117.76, 121.31, 126.34, 127.52, 131.63,
3
2
2

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ꢀ1
1
37.88, 150.91, 158.51, 160.28, 178.24, 180.21. IR (KBr, cm ): 1659, 1589, 1104. HRMSꢀ
+
ESI for (C H O [M+H] ) Calcd: 299.1283. Found: 299.1273.
1
8
18
4
5
1
7
9
ꢀbromoꢀ2,2ꢀdimethylꢀ6ꢀphenoxyꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11d). Red solid, yield 50% mp
1
40ꢀ142 °C. H NMR (400 MHz, CDCl ) δ ppm: 1.56 (s, 6H), 2.84 (s, 2H), 6.97 (d, J = 7.8 Hz, 2H),
3
1
3
.12 (t, J = 7.4 Hz, 1H), 7.28ꢀ7.36 (m, 2H), C NMR (101 MHz, CDCl ) δ ppm: 28.45 (2C), 39.5,
3
3.95, 116.85 (2C), 118.85, 120.31, 124.14, 129.90 (2C), 155.11, 156.66, 157.54, 173.37, 176.15. IR
ꢀ1
(
KBr, cm ): 1653, 1588, 1282. HRMSꢀESI for (C H BrO [M]) Calcd: 347.9997. Found: 348.0137.
16 13 4
5
5
0
1
1
1
ꢀbromoꢀ2,2ꢀdimethylꢀ6ꢀ(3ꢀnitrophenoxy)ꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11e). Red solid, yield
1
7 %, mp 177ꢀ179 °C. H NMR (400 MHz, CDCl ) δ ppm: 1.58 (s, 6H), 2.87 (s, 2H), 7.36 (ddd, J =
3
.9, 2.5, 8.3 Hz, 1H), 7.52 (t, J = 8.2 Hz, 1H), 7.76 (t, J = 2.3 Hz, 1H), 8.00 (ddd, J = 0.9, 2.1, 8.2 Hz,
1
3
H). C NMR (101 MHz, CDCl ) δ ppm: 28.46 (2C), 39.50, 94.34, 111.84, 118.98, 119.04, 121.38,
3
ꢀ1
23.24, 130.63, 149.26, 154.06, 156.75, 157.73, 172.82, 175.57. IR (KBr, cm ): 1693, 1647, 1577,
+
552, 1268, 1243. HRMSꢀESI for (C H BrNO [M+H] ) Calcd: 393.9926. Found: 393.9908.
1
6
12
6
5
ꢀbromoꢀ6ꢀ(2,5ꢀdimethylphenoxy)ꢀ2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11f). Orange
1
solid, yield 83%, mp 146ꢀ147 °C. H NMR (400 MHz, CDCl ) δ ppm: 1.56 (s, 6H), 2.26 (s, 3H), 2.33
3
13
(
s, 3H), 2.84 (s, 2H), 6.47 (s, 1H), 6.84 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H). C NMR (101
MHz, CDCl ) δ ppm: 15.84, 21.24, 28.31, 39.53, 93.74, 116.31, 118.51, 119.06, 121.41, 124.81,
3
ꢀ1
1
1
25.05, 131.34, 136.84, 155.13, 155.73, 157.48, 173.41, 176.23. IR (KBr, cm ): 1674, 1656, 1570,
+
207. HRMSꢀESI for (C H BrO [M+H] ) Calcd: 377.0388. Found: 379.0361.
1
8
17
4
2
2
1
,2ꢀdimethylꢀ6ꢀ(naphthalenꢀ2ꢀyloxy)ꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11g). Orangeꢀred oil, yield
1
5 %. H NMR (400 MHz, CDCl ) δ ppm: 1.56 (s, 6H), 2.94 (s, 2H), 5.51 (s, 1H), 7.20 (d, J = 8.9 Hz,
3
H), 7.52 (dd, J = 4.1, 7.4 Hz, 3H), 7.80 (d, J = 7.1 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H) 7.91 (d, J = 8.9
13
Hz, 1H). C NMR (101 MHz, CDCl ) δ ppm: 28.41 (2C), 39.61, 93.43, 107.98, 117.96, 118.28,
3
1
19.99, 126.44, 127.23, 127.74, 128.04, 130.75, 131.77, 134.02, 150.51, 158.62, 161.11, 178.39,
ꢀ1
+
1
80.13. IR (KBr, cm ): 1672, 1594, 1197. HRMSꢀESI for (C H O [M+H] ) Calcd: 321.1127.
20 16 4
Found: 321.1127.
5
ꢀbromoꢀ2,2ꢀdimethylꢀ6ꢀ(naphthaleneꢀ2ꢀyloxy)ꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11h). Red solid,
1
yield 47 %, mp 182ꢀ183°C. H NMR (400 MHz, CDCl ) δ ppm: 1.63 (s, 6H), 3.01 (s, 2H), 7.62 (d, J =
3
13
8
.9 Hz, 3H), 7.71 (t, J = 8.3 Hz, 1H), 7.85ꢀ7.97 (m, 2H), 9.50 (d, J = 8.4 Hz, 1H). C NMR (101 MHz,
CDCl ) δ ppm: 28.56 (2C), 40.20, 93.10, 112.51, 119.63, 121.31, 125.92, 126.80 127.95, 128.09,
3
2
3

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ꢀ1
1
1
28.20, 129.08, 131.59, 132.05, 150.68, 156.04, 156.92, 169.03, 180.24. IR (KBr, cm ): 1681, 1647,
+
341. HRMSꢀESI for (C H BrO [M+H] ) Calcd: 399.0232. Found: 399.2946
2
0
15
4
5
ꢀbromoꢀ2,2ꢀdimethylꢀ6ꢀ(naphthaleneꢀ1ꢀyloxy)ꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (11i). Orange
1
solid, yield 42%, mp 179ꢀ180 °C. H NMR (400 MHz, CDCl ) δ: 1.61 (s, 6H), 2.98 (s, 2H), 7.26 (br,
3
1
8
1
H), 7.61 – 7.71 (m, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H),
13
.43 (d, J = 7.8 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.40, 39.64, 93.06, 119.26, 119.60, 121.07,
3
21.15, 127.29 (2C), 127.59 (2C), 127.94 (2C), 128.70 (2C), 133.73, 157.87, 168.96 178.49, 180.85.
ꢀ1
+
IR (KBr, cm ): 1793, 1650, 1370. HRMSꢀESI for (C H BrO [M+H] ) Calcd: 399.0232. Found:
2
0
15
4
3
99.1966.
4
,5,6ꢀtribromoꢀ2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuranꢀ7ꢀol (9b). To a solution of 2,3ꢀdihydroꢀ2,2ꢀ
dimethylꢀ7ꢀbenzofuranol (1 mmol) in chloroform (30 mL), under magnetic stirring at room
temperature, was added dropwise a solution of bromine (5 mmol) in chloroform (15 mL) . The mixture
was stirred for 1.5 hour and then was washed with sodium thiosulfate solution (3 x 15 mL) and water (3
x 15 mL). The organic extract was dried over anhydrous sodium sulfate filtered and concentrated. The
crude was purified on silica gel column using dichloromethane as eluent. White solid, yield 57%, mp
1
13
1
20 ꢀ121 °C. H NMR (400 MHz, CDCl ) δ ppm: 1.53 (s, 6H), 3.07 (s,1H), 5.43 (s, 2H). C NMR
3
(
101 MHz, CDCl ) δ ppm: 28.40 (2C), 46.46, 89.51, 112.40, 112.85, 116.10, 130.07, 138.14, 145.10.
3
5
,6ꢀdibromoꢀ2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuranꢀ4,7ꢀdione (10b). A solution of chromium oxide
(2 mmol) in water (4 mL) was added dropwise at room temperature to a solution containing the
compound 9b (0.5 mmol) in 9 ml of a mixture of glacial acetic acid/water (3.5/1). The mixture was
stirred for two hours and then water (20 mL) was added and extracted with CH Cl . The organic phase
2
2
was washed with sodium bicarbonate solution 10% until neutral, dried with anhydrous sodium sulfate
filtered and concentrated. The crude was purified on silica gel column using dichloromethane as eluent.
1
Red solid, yield 63%, mp 108ꢀ110 °C. H NMR (400 MHz, CDCl ) δ ppm: 1.57 (s, 6H), 2.94 (s, 2H).
3
1
3
C NMR (101 MHz, CDCl ) δ ppm: 28.23, 40.09, 46.47, 93.76, 120.27, 135.25, 141.46, 156.97,
3
ꢀ1
+
1
70.58, 174.36. IR (KBr, cm ): 1639, 1684, 1177, 762. HRMSꢀESI for (C H Br O [M+H] ) Calcd:
10 8 2 3
3
34.8918. Found: 336.8887.
4
.3 Biology
T. cruzi Y strain epimastigotes were grown at 28 °C in an axenic medium (BHIꢀtryptose) as previously
2
5, 26
described,
complemented with 5% foetal bovine serum. Epimastigotes from a 10ꢀdayꢀold culture
2
4

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DOI: 10.1039/C5RA10122K
(stationary phase) were inoculated into 50 mL of fresh culture medium to reach an initial concentration
6
of 1×10 cells/mL. Cell growth was monitored by measuring the absorbance of the culture at 600 nm
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 inhibition (%GI) and IC values, 50% inhibitory concentrations, parasite growth
5
0
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.
2
5,
The IC value was taken as the concentration of drug needed to reduce the absorbance ratio to 50%.
5
0
2
6
Jꢀ774 murine macrophageꢀlike cells (ATCC, USA) were maintained by passage in Dulbecco’s
modified Eagle’s medium (DMEM) containing 4 mM Lꢀglutamine, and supplemented with 10% heat
inactivated foetal calf serum and 1% of antibiotics (10.000 U/mL penicillin and 10.000 U/mL
5
streptomycin). Jꢀ774 cells were seeded (1×10 cells/well) in 96 well microplates with 200 mL of RPMI
1
640 medium supplemented with 20% heatꢀinactivated foetal calf serum. Cells were allowed to attach
for 48 h in a humidified 5% CO /95% air atmosphere at 37 °C and, then, exposed to compounds (100.0
2
to 400.0 ꢀM) for 48 h. Afterward, cell viability was assessed by measuring the mitochondrialꢀ
dependent reduction of MTT (Sigma) to formazan. For that purpose, MTT was added to cells to a final
concentration 0.4 mg/mL and cells were incubated at 37 °C for 3 h. After removing the media,
formazan crystals were dissolved in DMSO (0.18 mL), and the absorbance at 595 nm was read using a
microplate spectrophotometer. Results are expressed as IC (compound concentration that reduce 50%
5
0
25
control absorbance at 595 nm). Every IC is the average of three independent experiments. The
5
0
selectivity index, SI, was expressed as the ratio between IC in macrophages and IC in T. cruzi (Y
5
0
50
strain).
4
.4 Pharmacophore
The 3Dꢀstructure of the pharmacophore hypothesis was carried out using “Molecular Operating
Environment (MOE) software version 2013ꢀ2014”, Chemical Computing Group INC., 1010 Sherbrook
Street West, Suite 910, Montreal, H3A 2R7, Canada.
4
.5 3DꢀQSAR
The data set was split into a 19 compounds as training set (2/3 of the compounds) and 9 compounds as
test set (1/3 of the compounds). Alignments were carried out in an atomꢀbyꢀatom based on a
2
5

Page 27 of 30
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DOI: 10.1039/C5RA10122K
5
0
Conformational sampling by quenched molecular dynamics was carried out using Open3Daling on
each structure (MMFF94 forceꢀfield, GB/SA implicit solvent model, 1000 5 ps molecular dynamics
runs at 1000 K followed by energy minimization), keeping the most stable conformations in a 8
–
1
kcal/mol range from the global minimum; this energy strain threshold was previously shown to
include a percentage of experimentally found bioactive. The aligned ligand ensembles were enclosed
in a grid box exceeding the largest molecule by 5 Å in each direction; a 1 Å step size was preferred to
the original coarser 2 Å mesh to reduce the dependency from gridꢀtoꢀmolecule reciprocal orientations.
Steric and electrostatic MIFs were computed with Open3DQSAR using MMFF94 van der Waals
3
parameters and charges. The probe was constituted by a sp carbon atom bearing a unit positive charge.
Training set MIF data were pre filtered an energy cutꢀoff was set at (30 kcal/mol; variables having a
standard deviation below 2.0 were discarded; block unscaled weighting was applied to steric and
electrostatic fields to give them the same importance in the PLS model. pIC values corresponding to
5
0
the individual molecules were correlated with MIF data for each alignment using PLS regression; the
optimal number of principal components (PC) to be extracted was chosen with the same criterion
5
1
adopted by Tosco et al., namely the one giving rise to the best leaveꢀoneꢀout crossꢀvalidation
2
performance, expressed as q LOO. Finally, the predictive power of each PLS model was evaluated
2
against the external test set and expressed both as r
prediction (SDEP).
and as standard deviation of the error of
pred
4
.6 Electrochemical studies
The cyclic voltammetry experiments were carried out with a CH Instruments (USA) model 620C
ꢀ1
potentiostat, the scan rate was 50 mVs . A threeꢀelectrode 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. 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 ºC)
and purging with nitrogen. The compounds (1.0 mM) were dissolved in 0.1 M tetrabutylammonium
perchlorate (TBAP) in dimethylformamide (DMF).
Acknowledgements
We are grateful to Fondecyt (Grants 1110749 and 1120128), KV thanks to PROMEPꢀMéxico (Grant
1
03.5 /10/5345), CSICꢀUdelaR (Proyecto Grupos Nº 661) of Uruguay, MP is grateful to the
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PEDECIBAꢀUDelaR Master in Bioinformatics. JV and EB thank ANII (Uruguay) for their
scholarships. JSD acknowledges support from PostꢀDoctoral fellow Fondecyt (Grant 3130359).
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