New benzimidazolequinones as trypanosomicidal agents

Article abstract of DOI:10.1016/j.bioorg.2021.104823

Herein, the design and synthesis of new 2-phenyl(pyridinyl)benzimidazolequinones and their 5-phenoxy derivatives as potential anti-Trypanosoma cruzi agents are described. The compounds were evaluated in vitro against the epimastigotes and trypomastigote f

Full text of DOI:10.1016/j.bioorg.2021.104823

Bioorganic Chemistry 111 (2021) 104823
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New benzimidazolequinones as trypanosomicidal agents
a
a,
Claudia Lopez-Lira , Ricardo A. Tapia *, Alejandra Herrerab, Michel Lapierb, Juan D. Mayab,
´
Jorge Soto-Delgadoc *, Allen G. Oliverd, A. Graham Lappind, Eugenio Uriartee
,
a
´
Facultad de Química y de Farmacia, Pontificia Universidad Catolica de Chile, Santiago 6094411, Chile
b
´
Instituto de Ciencias Biomedicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile
c
´
˜
Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andres Bello, Vina del Mar 2531015, Chile
d Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, United States
e
´
Departamento de Química Organica, Facultad de Farmacia, Universidad Santiago de Compostela, 15782 Santiago de Compostela, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Herein, the design and synthesis of new 2-phenyl(pyridinyl)benzimidazolequinones and their 5-phenoxy de-
rivatives as potential anti-Trypanosoma cruzi agents are described. The compounds were evaluated in vitro against
the epimastigotes and trypomastigote forms of Trypanosoma cruzi. The replacing of a benzene moiety in the
naphthoquinone system by an imidazole enhanced the trypanosomicidal activity against Trypanosoma cruzi.
Three of the tested compounds (11a-c) showed potent trypanosomicidal activity and compound 11a, with IC50 of
Aryloxyquinones
Trypanosoma cruzi
Molecular docking
Trypanothione reductase
Old Yellow Enzyme
0.65 μM on the trypomastigote form of T. cruzi, proved to be 15 times more active than nifurtimox. Additionally,
molecular docking studies indicate that the quinone derivatives 11a-c could have a multitarget profile inter-
acting preferentially with trypanothione reductase and Old Yellow Enzyme.
1. Introduction
system by an imidazole. Furthermore, diverse biological activities dis-
played by benzimidazolequinone derivatives such as compound II
Chagas disease (CD), also called American trypanosomiasis, is caused
by the protozoan parasite Trypanosoma cruzi (T. cruzi). CD is endemic in
Central and South America; however, due to international migration, it
is also found in non-endemic countries such as in the United States,
Europe and Australia [1]. Currently, about 11 million people worldwide
are infected and more than 10.000 deaths are estimated to occur
annually from this disease [2]. Two main drugs are used for the treat-
ment of CD benznidazole and nifurtimox (Nfx), both exhibit limited
efficacy and present severe side effects [3]. Accordingly, there is a need
for developing more effective drugs for the treatment of this disease. The
trypanosomicidal activity exhibited by many natural occurring naph-
thoquinones [4] has stimulated the discovery and development of novel
naphthoquinoidal compounds as new agents useful in CD therapy [5].
Recent results suggest that 2-aryloxynaphthoquinones (I, Fig. 1) [6–8]
could be a good starting point to identify new potential molecular leads
for the development of more effective anti-chagasic agents. Taking into
account that the synthesis of bioisosteres has been a strategy in the
search for new compounds with improved biological activity [9,10], in
this study, we replaced the benzene moiety of the naphthoquinone
(Fig. 1), inhibitor of purine nucleoside phosphorylase of protozoan
Toxoplasma gondii [11], and compounds III and IV (Fig. 1), with cyto-
toxic activity against a variety of cancer cell lines [12,13] or potent
inhibitory activity against NAD(P)H:quinone oxidoreductase 1 (NQO1)
and indoleamine 2,3-dioxygenase (IDO) [14,15] have been described. In
previous studies on this type of benzimidazole-4,7-diones, the presence
of the quinone structure instead of the aromatic analog, the imidazole
ring instead of
a pirrol ring, or 2-aromatic ring substituted
benzimidazole-4,7-diones instead of aromatic ring fused analogues,
seems to be associated with an increase in its toxicity [13,16,17], which
may be due to different ways of interaction with DNA [18] or different
reductive potentials [17].
In addition, considering that the trypanosomicidal activity of 4,7-
benzimidazolediones has not been described, we report herein the
synthesis of new aryloxy heterocyclic quinone derivatives V (Fig. 1), to
evaluate their trypanosomicidal activity.
Currently, computer-aided drug discovery methods have played a
major role in the development of new bioactive compounds [19,20].
These strategies became essential to choose specific targets and design
´
´
* Corresponding authors at: Departamento de Química Organica, Facultad de Química y de Farmacia, Pontificia Universidad Catolica de Chile, Santiago 6094411,
´
˜
Chile (R. A. Tapia). Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andres Bello, Vina del Mar 2531015, Chile (J. Soto-Delgado).
E-mail addresses: rtapia@uc.cl (R.A. Tapia), jorge.soto@unab.cl (J. Soto-Delgado).
https://doi.org/10.1016/j.bioorg.2021.104823
Received 28 September 2020; Received in revised form 11 January 2021; Accepted 8 March 2021
Available online 12 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

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Bioorganic Chemistry 111 (2021) 104823
Fig. 1. Chemical structures of relevant biological active quinones (I-IV) and
target phenoxy benzimidazolequinones (V).
new molecules as potential inhibitors. In order to understand the
probable mechanism of action at the molecular level of the quinone
derivatives, an induced-fit docking methodology was used with the
purpose of identifying possible drug targets. Trypanothione reductase
(TcTR) and glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH)
have been usual targets to explore the biological activities of naph-
thoquinones, and it was proposed that these compounds could act using
a multi-target based mechanism [21,22]. However, in our recent studies
a general relationship between trypanosomicidal of aryloxy-quinones
and TcTR enzyme inhibition could not be established [8]. Interest-
ingly, recent studies indicated that T. cruzi Old Yellow Enzyme (TcOYE),
has a relevant role in the mechanism of action of benznidazole and Nfx,
as well as some quinone derivatives [23–25]. Accordingly, induced-fit
docking methodology was applied to investigate the probable binding
mode to TcOYE, and to shed light on the mechanisms of the trypano-
somicidal effects of the new aryloxy-quinone derivatives V.
Scheme 1. Reagents and conditions: (a) Sn, HCl, reflux, 2 h; (b) LaCl3⋅7H2O,
ArCHO, MeCN, rt, 24 h; (c) i: DMF, K2CO3, BTBAC, 100 ◦C, 1 h; ii: EtBr rt 18 h;
(d) THF, AgO, 6 N HNO3, 5 min.
phenylbenzimidazole 8a in a modest yield.
When the reaction was carried out under microwave irradiation at
140 ◦C for 15 min, phenylbenzimidazole 8a was obtained in 85% yield.
The same o-nitroaniline 7 was used for the synthesis of 2-pyridylbenzi-
midazoles applying Yang’s method [31] with minor modifications.
Thus, the reductive cyclization of o-nitroaniline 7 with the corre-
sponding pyridinecarboxaldehyde in the presence of Na2S2O4, yielded 2-
pyridylbenzimidazoles 8b-d in 65–78% yields.
Then, the alkylation of compounds 8a-d with ethyl bromide, as
above, gave the N-ethyl derivatives 9a-d (62–74%). As a result of the
tautomerism of the imidazole moiety, the alkylation of benzimidazoles 8
could occur at N-1 or N-3. It has been reported that the alkylation of 5-
bromobenzimidazoles give the 1,5-isomer preferentially [32,33]. Here,
only the 1,5-isomer was isolated, and their structures were established
using 1H, 13C and 2D NMR techniques and further confirmed by single
crystal X-ray analysis of two representative compounds, 9a and 9b
(Fig. 2 and Table 1).
2. Results and discussion
2.1. Chemistry
Frequently, the synthesis of 2-arylbenzimidazoles involves conden-
sation of o-phenylenediamines with aromatic aldehydes, but many of
the reported methods require harsh reaction conditions and often a
demanding work up [26]. After trying some milder methods, the best
result was obtained using lanthanum chloride as catalyst [27]. The
starting 1,4-dimethoxy-2,3-dinitrobenzene (1), was obtained following
a literature method [28]. The reduction of 1 with tin granules and hy-
drochloric acid afforded 3,6-dimethoxybenzene-1,2-diamine (2) in
excellent yield. The later was converted into benzimidazole derivatives
3a (48%) and 3b (85%) by condensation with the corresponding pyri-
dine carboxaldehyde using lanthanum (III) chloride. Alkylation of
benzimidazole derivatives 3a-b by reaction with ethyl bromide in the
presence of potassium carbonate and benzyltributylamonium chloride
(BTBAC) gave the N-ethyl benzimidazole derivatives 4a-b in excellent
yields. Then, oxidation of the dimethoxy benzimidazole derivatives 4a-b
with silver (II) oxide and 6 N nitric acid provided benzimidazole-4,7-
diones 5a and 5b in 67% and 58% yields, respectively (Scheme 1).
Next, in order to obtain bromobenzimidazole-4,7-dione 10b, the
reaction of 5a with hydrobromic acid following a described methodol-
ogy [29] was attempted but gave unsatisfactory results. Therefore, the
synthesis of the target benzimidazole-4,7-diones was carried out as
shown in Scheme 2. First, the selective reduction of 1,4-dimethoxy-2,3-
dinitrobenzene (1) was achieved using hydrazine hydrate in the pres-
ence of the iron chloride and charcoal to give o-nitroaniline 6 in excel-
lent yield. Then, o-nitroaniline 6 was treated with N-bromosuccinimide
(NBS) in chloroform to afford compound 7 (70%, two steps) regiose-
lectively. The one pot solvent-free iron-catalysed redox condensation
[30] of o-nitroaniline 7 with benzylamine at 120 ◦C for 12 h afforded
Oxidative demethylation of benzimidazoles 9a-d with AgO/HNO3,
gave
bromoquinones
10a-d
in
good
yields.
Finally,
phenoxybenzimidazole-4,9-diones 11a-d were obtained in 50–58%
yield by nucleophilic substitution reaction [34] of 10a-d with phenol
and potassium carbonate in dimethylformamide (Scheme 2).
2.2. Biological evaluation
The benzimidazole-4,7-dione derivatives (compounds 5, 10 and 11)
were initially evaluated in vitro against the epimastigote form of T. cruzi,
Dm28c strain. The convenience of this assay as a preliminary test to
evaluate the in vitro trypanosomicidal activity has been recently
described [35]. The compounds were evaluated at different concentra-
tions (0.5–100 μM) to obtain a dose–response curve for calculation of
IC50 values, including Nfx as reference drug. The IC50 values for all
quinones on the epimastigote form of T. cruzi as well as the cytotoxicity
using mammalian Vero cells and the selectivity index (SI = IC50 Vero
cell/IC50 epimastigote form) are shown in Table 2. Interestingly, all
compounds were more actives than Nfx on epimastigotes form T. cruzi.
Comparing quinones 5a, 5b and 10a-d with their corresponding
phenoxybenzimidazole-4,7-diones 11a-d (IC50 = 1.42–4.07 μM) an in-
crease on the trypanosomicidal activity was observed. Only compounds
10b and 11b shown similar activity (Table 2).
Subsequently, the trypanosomicidal activity of benzimidazole-4,7-
dione derivatives was assayed in vitro against the trypomastigote infec-
tive form of T. cruzi (Dm28c strain). Firstly, the viability of parasites was
evaluated at a concentration of 5 μM. The phenoxyquinones 11 showed
2

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Bioorganic Chemistry 111 (2021) 104823
Scheme 2. Reagents and conditions: (a) i: Activated charcoal, FeCl3⋅6H2O, MeOH, 65 ◦C, 1 h; ii: H2NNH2; (b) i: CHCl3, NBS, ꢀ 5 ◦C, 5 h; ii: rt, 20 h; (c) PhCH2NH2,
FeCl3⋅6H2O, 140 ◦C, 15 min; (d) 1 M Na2S2O4, PyrCHO, EtOH, 70 ◦C, 10 h; (e) i: DMF, K2CO3, BTBAC, 100 ◦C, 1 h; ii: EtBr, rt, 18 h; (f) THF, AgO, 6 N HNO3, 5 min;
(g) i: PhOH, K2CO3, DMF, 25 ◦C, 1 h , ii: 10, 25 ◦C, 12 h.
Fig. 2. Crystal structures of benzimidazoles 9a (A) and 9b (B).
significant inhibition against trypomastigote forms of T. cruzi, with
compound 11a proved to be 15 times more active than Nfx. It is
important to highlight that the trypomastigote form is more relevant
from the clinical perspective, because is the parasite infective form.
Therefore, this result is very relevant within the study. Although the SI
of compounds 11a-c (SI = 10.6–11.5) are lower than Nfx (SI > 20), in
agreement to some authors, potential antichagasic agents must have
viability percentages in the range of 13.1% to 24.1% versus 85% for Nfx.
Then, quinones 11a-d and I were evaluated at 0.1–5 μM concentrations
to determine their IC50 values. Noteworthy, phenoxyquinones 11a-
d (IC50 = 0.65–1.39 M) exhibited higher activity on the trypomastigote
form of T. cruzi than on the epimastigote form of the parasite and
μ
3

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Bioorganic Chemistry 111 (2021) 104823
(GAPDH) and trypanothione reductase (TR) has been common drugs
targets to explore the trypanosomicidal activities of the naph-
thoquinones, proposing their multi-target biochemical pathway
[21,40].
Table 1
X-ray crystallographic data for compounds 9a and 9b.
Compound
9a
9b
Chemical formula
Formula weight
Crystal system, space
group
C
16H14BrCl3N2O2
C16H16BrN3O2
362.23
Recently, it has been described that the flavoprotein old yellow
enzyme of T. cruzi (TcOYE) is an oxidoreductase that has a relevant role
in the mechanism of action of some trypanasomicidal naphthoquinones
drugs [23]. The binding mode of β-lapachone, a trypanosomicidal agent,
and other naphthoquinones was investigated by molecular docking and
dynamics, suggesting that their binding to TcOYE are stabilized pre-
dominantly through interactions with the isoalloxazine ring of flavin
mononucleotide (FMN) prosthetic group and residues from the active-
site pocket [24]. Therefore, in this study the phenoxybenzimidazole-
4,9-diones and 2-phenoxy-1,4-naphthoquinone were used as ligands to
perform molecular docking with TcOYE as well as with TcGAPDH and
TcTR for comparison purposes.
452.55
Monoclinic, P21/c
Monoclinic, P21/c
Temperature (K)
Wavelength
120(2) K
0.71073 Å
monoclinic
P21/c
120(2) K
0.71073 Å
monoclinic
P21/c
Crystal system
Space group
Unit cell dimensions
a, b, c (Å)
12.026(6), 15.567(6),
10.013(4)
10.2655(4),14.9077(6),
10.8243(4)
α
, β, γ (◦)
90, 111.494(4), 90
1744.1(13)
90, 116.4950(10), 90
1550.8(4)
Volume (Å3)
Z, Calculated density
Radiation type
Absorption
4, 1.723 g.cmꢀ 3
4, 1.623 g.cmꢀ 3
Mo K
α
Mo Kα
2.828 mmꢀ 1
2.784 mmꢀ 1
First, the IFD was carried out to study the interaction between
TcGAPDH and the new synthesized quinones. The TcGAPDH (PDBCode:
1K3T), has a binding site characterized principally by the catalytic
residue Cys166, together with Val255 and Asn335. These last two resi-
dues take importance due that probably are related to orientate correctly
the side chain in order to receive the incoming NAD+ cofactor [41]. In
this work, the superimposed images in the docking simulation of the
ligands is provided in supporting information (SI Figure S1) and best-
ranked pose for the most active quinone 11a at TcGAPDH active site
is shown in Fig. 3A. Compound 11a shows H-bond interactions between
oxygen atom of the quinone with NH fragment of Asn335 (2.1 Å), and
nitrogen of Cys166 (2.4 Å), and hydrophobic interactions between
phenyl rings with Arg249, Glu336, Arg12 and Ile13. This result is in
accord to the proposed mechanism that Cys166-thiol may react with 2-
phenoxy-1,4-naphthoquinones taking place a nucleophilic substitution
reaction with the phenolate anion displacement and formation of a co-
coefficient (μ)
F(000)
904
736
Crystal colour, habit
Crystal size
colourless, rod
colourless, block
0.199 × 0.169 × 0.140
mm3
0.246 × 0.053 × 0.052 mm3
θ range for data
collection
1.820 to 26.752◦
2.217 to 28.364◦
Index ranges
ꢀ 15 ≤ h ≤ 15, ꢀ 19 ≤ k ≤ 19,
ꢀ 12 ≤ l ≤ 12
ꢀ 12 ≤ h ≤ 13, ꢀ 19 ≤ k ≤
19, ꢀ 14 ≤ l ≤ 13
22,433
Reflections collected
Independent
20,762
3711 [Rint = 0.1381]
3703 [Rint = 0.0294]
reflections
Completeness to θ =
99.9%
100.0%
25.242◦
Data Collection
Absorption
Numerical
Numerical
correction
Max. and min.
transmission
0.9802, 0.6936
0.8142, 0.6917
–
valent C S bond [6]. Nevertheless, the binding affinity to TcGAPDH
Refinement method
Full-matrix least-squares on
Full-matrix least-squares
on F2
F2
predicted by IFD showed a low docking score respect to the other en-
zymes and there is no correlation with the trypanosomicidal activity of
the compounds. (Table 3).
Data / restraints /
parameters
3711 / 0 / 220
3703 / 0 / 202
Goodness-of-fit on F2
Final R indices [I >
1.079
1.026
In relation with the interactions of naphthoquinones with TcTR
(PDBCode: 1BZL), it has been proposed the Z-site of this enzyme as
binding site, which is characterized by a hydrophobic region defined by
Phe396, Pro 462, and Leu399 [42,43]. Heterocyclic quinones 11a-d and
I showed good affinity to the Z-site of TcTR, and the superimposed im-
ages in the docking simulation of the ligands is provided in supporting
information (SI Figure S2). The most active compound 11a showed the
best affinity to TcTR with a docking score of ꢀ 7.905 kcal/mol (Table 3).
For compound 11a favourable H-bond interactions are suggested be-
tween carbonyl groups of the quinone system with Lys62 (2.5 Å) and
Ser464 (2.2 Å), N-3 of imidazole scaffold also with Lys62 (2.1 Å) and the
oxygen of the phenoxy group with Asn433 (2.4 Å). In addition, hydro-
phobic interactions between phenyl ring with the residues Leu63,
Leu399 and Phe396 are proposed. It is important to point out that a
similar binding mode was described previously for naphthoquinones,
known by their non-competitive inhibition mechanism of TcTR [8].
Probably, the key H-bond interaction between N-3 and Lys62 may be the
R1 = 0.0692, wR2 = 0.1580
R1 = 0.0253, wR2 = 0.0550
2σ(I)]
R indices (all data)
Extinction
R1 = 0.1218, wR2 = 0.1742
R1 = 0.0356, wR2 = 0.0582
n/a
n/a
coefficient
Largest diff. peak
and hole
1.130, ꢀ 1.063 e–. Åꢀ 3
0.432, ꢀ 0.400 e–.Åꢀ 3
IC50 ≤ 10 μM and the SI (SI = IC50 Vero cells/IC50 trypomastigote forms)
must be higher than 10 [36]. Three of the tested compounds (11a-c),
meet both criteria and showed potent trypanosomicidal activity
(Table 2). In this context, the phenoxyquinones described here for the
first time can be considered as an important starting point for designing
more active compounds with lower cytotoxicity against normal cells.
Considering the structural modification, it worth noting that those
quinones having a fused imidazole system (11a-d) were most active
than the reference compound 2-phenoxy-1,4-naphthoquinone (I).
Regarding the C-2 substituent, the compounds showed the following
order of decreasing trypanosomicidal activity 11a (phenyl) > 11b (2-
pyridyl) > 11c (3-pyridyl) > 11d (4-pyridyl). Interestingly, similar re-
sults were described for the trypanosomicidal activity of 2-arylnaphthoi-
midazoles [37] and the antifungal activity of 6-arylamino-2-(pyridyl)-
4,7-benzimidazolediones [38].
reason that replacing phenyl group in I (IC50 = 2.09
scaffold led to the increase of trypanosomicidal activity of 11a (IC50
0.63 M). (Fig. 3B)
μM) by imidazole
=
μ
Next, the ligand-enzyme interactions of quinones with TcOYE
(PDBCode: 3ATZ) by IFD were investigated and the superimposed im-
ages of the ligands is provided in supporting information (SI Figure S3).
The docking prediction showed a ternary complex TcOYE/FMN/
quinone with the FMN prosthetic group close enough to quinone 11a
2.3. Molecular modelling studies
that an electron/proton transfer could occur and stabilized by
π-π
stacking interactions between FMN and the ligand (Fig. 3C). Favourable
H-bond interactions involving the carbonyl groups of the quinone sys-
tem with Tyr364 (1.9 Å), Asn198 (2.1 Å) and His195 (3.0 Å) together
To investigate the potential targets of quinones derivatives and un-
derstand the mechanism of action at the molecular level, we next
focused on target identification through induced-fit docking (IFD)
methodology [39]. Glyceraldehyde-3-phosphate dehydrogenase
with hydrophobic and
π-π stacking interactions that present the
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Bioorganic Chemistry 111 (2021) 104823
Table 2
In vitro activity, cytotoxicity and Selectivity Index (SI) of all quinones on epimastigote and trypomastigote forms of T. cruzi (Dm28c strain).
a
a
a
Compd.
5a
Structure
IC50
Epim.
(μ
M)
Viability (%)
IC50
(
μ
M)
IC50
(
μ
M)
Selectivity index
Trypom. (5 μM
)
Trypom.
Vero cell
Epim.b
Trypom.c
4.37 ± 0.45
5.62 ± 0.50
6.11 ± 0.52
4.08 ± 0.32
10.65 ± 0.80
6.49 ± 0.60
1.42 ± 0.07
4.04 ± 0.30
4.07 ± 0.27
3.73 ± 0.32
3.16 ± 0.28
21.05 ± 0.90
33.89
38.19
46.74
61.22
66.10
61.13
15.98
19.04
13.05
24.12
35.17
85.00
n.d.
6.52 ± 0.79
1.5
n.d.
5b
n.d.
10.43 ± 1.86
65.29 ± 2.81
>100
1.9
n.d.
n.d.
n.d.
n.d.
n.d.
10.6
11.5
10.6
5.5
10a
10b
10c
10d
11a
11b
11c
11d
I
n.d.
10.7
>25
8.1
n.d.
n.d.
85.68 ± 1.29
14.00 ± 0.76
6.89 ± 0.50
10.09 ± 0.70
10.20 ± 0.79
7.65 ± 0.66
6.25 ± 0.65
>200
n.d.
2.2
0.65 ± 0.10
0.88 ± 0.14
0.96 ± 0.13
1.39 ± 0.40
2.09 ± 0.12
10.00 ± 0.40
4.9
2.5
2.5
2.1
2.1
3.0
Nfx
>9.5
>20
n.d.: not determined.
a
Data are means of three independent experiments.
b
c
Selectivity index: expressed as the ratio of IC50 in Vero cells to IC50 in epimastigotes.
Selectivity index: expressed as the ratio of IC50 in Vero cells to IC50 in trypomastigotes.
Fig. 3. Proposed binding mode for quinone 11a at the active site of Glyceraldehyde-3-phosphate dehydrogenase (A), Trypanothione Reductase (B) and OLD Yellow
Enzyme (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
aromatic ring with residues Phe71 and Tyr200 are shown. This result
suggested that TcOYE could have an important role in the trypanoso-
micidal activity of quinones due to its reduction by the enzyme. Our
study has shown that this contribution is performed at an average
distance of 3.0 Å contributing with an important favourable interaction
to produce hydride transfer from the reduced FMN to quinone sub-
strates. It is worth noting that H-bond interactions with Tyr364, Asn198
and His195 are important for the ligand catalysis and were described for
5

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Bioorganic Chemistry 111 (2021) 104823
mixture was heated to reflux for 2 h, and the resulting white solid was
filtered. The solid was dissolved in water, the solution was made alkaline
(pH = 8) with 5% ammonium hydroxide and extracted with chloroform
(3x50 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated to give the crude product, which was purified
by flash column chromatography (chloroform/hexane, 1:1) to give 2
(3.5 g, 95%); mp 86–87 ◦C (Lit. [28] 86–87 ◦C).
Table 3
Binding affinity scores of quinones 11a-d and I according to the Glide score
(kcal/mol).
Compd.
TcGAPDH
TcTR
TcOYE
5a
ꢀ 5.46
ꢀ 5.00
ꢀ 5.00
ꢀ 4.93
ꢀ 5.05
ꢀ 6.01
ꢀ 5.55
ꢀ 6.19
ꢀ 6.06
ꢀ 6.17
ꢀ 5.69
ꢀ 5.76
ꢀ 5.47
ꢀ 5.68
ꢀ 5.57
ꢀ 5.32
ꢀ 5.21
ꢀ 7.91
ꢀ 6.14
ꢀ 6.29
ꢀ 6.88
ꢀ 6.95
ꢀ 6.77
ꢀ 6.17
ꢀ 6.76
ꢀ 7.52
ꢀ 6.17
ꢀ 6.61
ꢀ 9.40
ꢀ 7.68
ꢀ 7.62
ꢀ 8.06
ꢀ 8.37
5b
10a
10b
10c
10d
11a
11b
11c
11d
I
4.1.2. General procedure for preparing 4,7-dimethoxy-2-(pyridinyl)-1H-
benzo[d]imidazoles (3a-b)
A mixture of 3,6-dimethoxybenzene-1,2-diamine (1.68 g, 10 mmol)
(2), lanthanum (III) chloride heptahydrate (371.4 mg, 1.0 mmol) and
the corresponding pyridine carboxaldehyde (10 mmol) in acetonitrile
(60 mL) was stirred at room temperature for 24 h. After evaporation of
the solvent, the residue was dissolved in ethyl acetate, washed with
water, dried (MgSO4) and concentrated. The crude products were pu-
rified by column chromatography (ethyl acetate/hexane, 1:1).
known inhibitors of TcOYE [24].
In summary, these results suggest that the quinone derivatives could
have a multitarget profile and might preferentially interact with TcTR
and TcOYE which provides a molecular explanation of its anti-
Trypanosoma activity. Therefore, we conclude that this strategy based
on molecular docking over possible targets provides a helpful way for
the rational design of more potent inhibitors and the future synthesis of
highly potent trypanosomicidal compounds based on the phenox-
yquinones derivatives.
4.1.2.1. 4,7-Dimethoxy-2-(pyridin-2-yl)-1H-benzo[d]imidazole
(3a).
Colourless solid, mp 141–142 ◦C, yield 58%; IR (KBr, cmꢀ 1) 3430, 1615,
1600, 1540, 1460, 1270, 1100; 1H NMR (DMSO‑d6): 3.86 (s, 3H, OCH3),
4.01 (s, 3H, OCH3), 6.54 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H),
7.30 (dd, J = 1.2 and 4.9 Hz, 1H), 7.82 (dd, J = 1.2 and 7.8 Hz, 1H),
8.50–8.60 (m, 2H), 10.7 (s, 1H, NH); 13C NMR (DMSO‑d6): 55.7 (2C),
121.5 (2C), 124.5 (2C), 136.8 (2C), 148.1 (2C), 148.9 (2C), 149.3(2C).
HRMS (ESI+): [M+H]+ calcd. for C14H13N3O2, 256.1087; found,
256.1077.
3. Conclusions
In the search for new trypanosomicidal agents based on the replacing
of the benzene moiety of the naphthoquinone system by imidazole led to
phenoxy benzo[d]imidazole-4,7-diones as a promising class of multi-
target compounds against T. cruzi. The analysis of the trypanosomicidal
properties of these derivatives showed that phenoxyquinones 11a-
d were more active than the original naphthoquinone I, with IC50 values
4.1.2.2. 4,7-Dimethoxy-2-(pyridin-3-yl)-1H-benzo[d]imidazole
(3b).
Colourless solid, mp mp 218–220 ◦C (Lit. [44] 214–216 ◦C), yield 85%.
4.1.3. General procedure for synthesis of 1-ethyl-4,7-dimethoxy-2-
(pyridinyl)-1H-benzo[d]imidazoles (4a-b)
on the trypomastigote form of T. cruzi ranging from 0.65 to 1.39
most potent compound 11a was nearly 15 times more active against the
epimastigote form of the parasite (IC50 = 1.42 M) and>15 times more
active against the trypomastigote (IC50 of 0.65 M), although with a
μM. The
To a solution of the corresponding 2-aryl-4,7-dimethoxy-1H-benzo
[d]imidazole 3 (1.5 mmol) in DMF (2.0 mL) were added potassium
carbonate (235 mg, 1.7 mmol) and benzyltributylamonium chloride
(46.8 mg, 0.15 mmol) and the mixture was heated at 100 ◦C for 1 h.
After cooling to room temperature, was added bromoethane (52.3 mg,
1.62 mmol) and the suspension was stirred for 18 h. The reaction
mixture was concentrated under vacuum and the residue was purified by
column chromatography (ethyl acetate/hexane, 1:2).
μ
μ
much lower selectivity index against the tested Vero cells (SI = 10.6),
than the Nfx as reference compound. These results stimulate further in
vivo studies of these compounds and derivatives to definitely elucidate
its multitarget profile. The results of induced fit molecular docking
showed a better affinity of phenoxyquinones for TcOYE and TcTR, and
that the imidazole scaffold has an important effect in the ligand-enzyme
stabilization.
4.1.3.1. 1-Ethyl-4,7-dimethoxy-2-(pyridin-2-yl)-1H-benzo[d]imidazole
(4a). Colourless solid, mp 100–101 ◦C, yield 90%; IR (KBr, cmꢀ 1) 1600,
1530, 1480, 1440, 1270, 1210, 1120, 1090; 1H NMR (CDCl3): 1.40 (t, J
= 6.5 Hz, 3H, CH3) 3.85 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 5.00 (q, J =
6.5 Hz, 2H, CH2) 6.5–6.60 (m, 2H), 7.20–7.25 (m, 1H), 7.70–7.75 (m,
1H), 8.35–8.40 (m, 1H), 8.55–8.60 (m, 1H); 13C NMR (CDCl3): 17.0,
42.3, 55.8 (2C), 101.3, 103.7, 123.4, 125.3, 127.1, 134.9, 136.4, 141.9,
146.1, 148.4, 148.9, 150.7. HRMS (ESI+): [M+H]+ calcd. for
4. Experimental section
4.1. Chemistry
All chemicals were obtained from Merck or Sigma Aldrich and used
without purification. Melting points (mp) were measured on an Elec-
trothermal IA 9300 melting point apparatus and are uncorrected. 1H and
13C NMR spectra were recorded on a Bruker Advance 400 MHz spec-
trometer; chemical shifts (δ) are given in parts per million (ppm) and
coupling constants (J) in Hertz (Hz). All assignments of NMR spectra
were based on 2D NMR data (DEPT-135, COSY, HSQC and HMBC).
High-resolution mass spectral analysis was carried out on a Bruker
Impact II instrument. Infrared spectra (IR) were determined using an
IRAffinity-1 Shimadzu spectrophotometer. The 1,4-dimethoxy-2,3-dini-
trobenzene (1) starting material, was synthesized by nitration of 1,4-
dimethoxybenzene according to the method described in literature [28].
C16H17N3O2, 284.1400; found, 284.1388.
4.1.3.2. 1-Ethyl-4,7-dimethoxy-2-(pyridin-3-yl)-1H-benzo[d]imidazole
(4b). Colourless solid, mp 115–117 ◦C, yield 90%; IR (KBr, cmꢀ 1) 1635,
1527, 1473, 1430, 1378, 1270, 1108; 1H NMR (CDCl3): 1.36 (t, J = 7.3
Hz, 3H, CH3) 3.87 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.38 (q, J = 7.3 Hz,
2H, CH2) 6.52 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 8.5 Hz, 1H), 7.35–7.40
(m, 1H), 8.02 (d, J = 7.8 Hz, 1H), 8.66 (d, J = 4.8 Hz, 1H), 8.91 (s, 1H);
13C NMR (CDCl3): 17.2, 41.5, 55.8 (2C), 101.8, 103.6, 123.3, 126.2,
135.3, 137.2, 136.4, 141.5, 146.0, 149.9, 150.4. HRMS (ESI+): [M+H]+
calcd. for C16H17N3O2, 284.1400; found, 284.1391.
4.1.1. Synthesis of 3,6-dimethoxybenzene-1,2-diamine (2)
4.1.4. General procedure for synthesis of 1-ethyl-2-pyridinyl-1H-benzo[d]
imidazole-4,7-diones (5a-b)
To a stirred mixture of granular tin (15.1 g, 127.2 mmol) and 36%
hydrochloric acid (60 mL) was added 1 (5.0 g, 21.9 mmol). The reaction
To a stirred solution of appropriated 2-arilbenzimidazole 4 (0.2
6

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Bioorganic Chemistry 111 (2021) 104823
mmol) in THF (2.5 mL), were added silver (II) oxide (70 mg; 0.6 mmol)
and 6 N nitric acid (0.2 mL). After 5 min, the reaction mixture was
diluted with water (5 mL) and extracted with chloroform (3x15 mL) The
combined organic extracts were dried (MgSO4), concentrated in vacuo,
and the residue was purified by column chromatography (ethyl acetate/
hexane, 1:2).
mmol) was added to a stirred mixture of 4-bromo-3,6-dimetoxy-2-nitro-
aniline 7 (138.6 mg, 0.5 mmol) and the corresponding pyridine car-
boxaldehyde (0.5 mmol) in ethanol (2 mL), and the reaction mixture was
heated at 70 ◦C for 10 h. After cooling, he resulting mixture was made
alkaline with ammonium hydroxide and then extracted with ethyl ace-
tate (3x10 mL). The combined organic layers were dried (MgSO4) and
the solvent was removed under vacuum. The crude product was purified
by column chromatography on silica gel (ethyl acetate/hexane, 1:1).
4.1.4.1. 1-Ethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-4,7-dione (5a).
Yellow solid, mp 170–172 ◦C, yield 67%; IR (KBr, cmꢀ 1) 1682, 1652,
1591, 1485, 1410, 1395; 1H NMR (CDCl3): 1.48 (t, J = 7.0 Hz, 3H, CH3),
4.42 (q, J = 7.0 Hz, 2H, CH2), 6.66 (d, J = 10.3 Hz, 1H), 6.72 (d, J =
10.3 Hz, 1H), 7.40–7.50 (m, 1H), 8.05 (d, J = 7.6 Hz, 1H), 8.76 (d, J =
7.6 Hz, 1H), 8.91 (s, 1H); 13C NMR (CDCl3): 17.0, 42.3, 55.8 (2C), 101.3,
103.7, 123.4, 125.3, 127.1, 134.9, 136.4, 141.9, 146.1, 148.4, 148.9,
150.7. HRMS (ESI+): [M+H]+ calcd. for C14H12N3O2, 254.0930; found,
254.0928.
4.1.8.1. 5-Bromo-4,7-dimethoxy-2-(pyridin-2-yl)-1H-benzo[d]imidazole
(8b). Colourless solid, mp 141–142 ◦C, yield 65%; IR (KBr, cmꢀ 1) 3490,
3150, 1600, 1514, 1457, 1429, 1386, 1286; 1H NMR (DMSO‑d6): 3.81 (s,
3H, OCH3), 4.05 (s, 3H, OCH3), 6.77 (s, 1H), 7.40 (dd, 1H, J = 4.7 and
7.9 Hz), 7.87 (t, 1H, J = 7.9 Hz), 8.21 (d, 1H, J = 7.9 Hz), 8.61 (d, 1H, J
= 4.7 Hz), 13.40 (s, 1H, NH). 13C NMR (DMSO‑d6): 56.6, 61.4, 122.4,
125.2, 137.9 (4C), 148.7 (2C), 149.8 (2C), 150.9 (2C); HRMS (ESI+):
[M+H]+ calcd. for C14H13BrN3O2, 334.0191; found, 334.0181.
4.1.4.2. 1-Ethyl-2-(pyridin-3-yl)-1H-benzo[d]imidazole-4,7-dione (5b).
Yield 58%, yellow solid mp 150–151 ◦C (Lit. [13]147–148 ◦C).
4.1.8.2. 5-Bromo-4,7-dimethoxy-2-(pyridin-3-yl)-1H-benzo[d]imidazole
(8c). Yellow solid, mp 240–242 ◦C, yield 74%; IR (KBr, cmꢀ 1) 3450,
1604, 1507, 1475, 1453, 1416, 1386, 1280, 1H NMR (DMSO‑d6): 3.91 (s,
3H, OCH3), 4.14 (s, 3H, OCH3), 6.88 (s, 1H), 7.49–7.54 (m, 1H), 8.52 (d,
J = 7.5 Hz, 1H), 8.62 (d, J = 4.0 Hz, 1H), 9.36 (s, 1H), 13.42 (s, 1H, NH);
13C NMR (DMSO‑d6): 56.7, 61.3, 105.4, 108.1, 124.5, 126.3 (2C), 127.2,
134.8, 137.9, 142.5, 148.5, 149.1, 151.2; HRMS (ESI+): [M+H]+ calcd.
for C14H13BrN3O2, 334.0191; found, 334.0183.
4.1.5. Synthesis of 3,6-dimethoxy-2-nitroaniline (6)
A mixture of 1,4-dimethoxy-2,3-dinitrobenzene (4.56 g, 20 mmol)
(1), activated charcoal (0.8 g), iron (III) chloride hexahydrate (324.4
mg, 1.2 mmol) in methanol (25 mL), was stirred at 65 ◦C for 1 h. Then,
hydrazine monohydrate (3.84 mL, 79 mmol) was added drop wise and
stirred at 65 ◦C for 1 h. The reaction was allowed to cool to room tem-
perature and the solids were filtered and washed with methanol. The
filtrate was dried (MgSO4), concentrated and the residue was purified by
column chromatography (ethyl acetate/hexane, 1:1) to give 6 as an
orange solid (3.76 g, 95%), mp 68–69 ◦C (lit. [28] 67–68 ◦C).
4.1.8.3. 5-Bromo-4,7-dimethoxy-2-(pyridin-4-yl)-1H-benzo[d]imidazole
(8d). Yellow solid, mp 190–192 ◦C, yield 78%; IR (KBr, cmꢀ 1) 3450,
3260, 1600, 1510, 1450, 1390, 1275; 1H NMR (DMSO‑d6): 3.86 (s, 3H,
OCH3), 4.01 (s, 3H, OCH3), 6.82 (s, 1H), 8.08 (d, J = 5.0 Hz, 2H), 8.64
(d, J = 5.0 Hz, 2H), 13.47 (s, 1H, NH); 13C NMR (DMSO‑d6): 56.5, 61.3,
108.0, 121.1, 137.1 (4C), 148.8 (4C), 150.8 (2C); HRMS (ESI+):
[M+H]+ calcd. for C14H13BrN3O2, 334.0191; found, 334.0182.
4.1.6. Synthesis of 4-bromo-3,6-dimetoxy-2-nitroaniline (7)
A solution of 6 (260 mg; 1.31 mmol) in chloroform (5 mL) was cooled
to ꢀ 5 ◦C, and a solution of N-bromosuccinimide (230 mg, 1.31 mmol) in
acetonitrile (2 mL) was added dropwise. The mixture was stirred at
ꢀ 5 ◦C for 5 h, and then was further stirred for 20 h at room temperature.
After evaporation of the solvents, the residue was purified by column
chromatography (chloroform/hexane, 1:4) to afford 7 as an orange solid
(255 mg, 74%), mp 131–132 ◦C; IR (cmꢀ 1) 3510, 3390,1617, 1595,
1523, 1344; 1H NMR (CDCl3): 3.84 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
5.34 (s, 2H, NH2), 6.90 (s, 1H); 13C NMR (CDCl3): 56.6, 62.7, 102.5,
115.3, 116.3, 134.5, 144.2. 145.3; HRMS (ESI+): [M + Na]+ calcd for
C8H9N2NaO4, 298.9643; found, 298.9634.
4.1.9. Synthesis of 2-(aryl)-5-bromo-1-ethyl-4,7-dimethoxy-1H-benzo[d]
imidazoles (9a-d)
The general procedure described for the preparation of 4a-b (2.1.3.)
from 3a-b was utilized to synthesize compounds 9a from 8a and 9b-
d from 8b-d.
4.1.9.1. 5-Bromo-1-ethyl-4,7-dimethoxy-2-phenyl-1H-benzo[d]imidazole
(9a). White solid, mp 157–158 ◦C, yield 70%; IR (KBr, cmꢀ 1) 1610,
1510, 1475, 1460, 1410, 1290, 1240, 1140, 1085; 1H NMR (CDCl3):
1.37 (t, J = 7.0 Hz, 3H, CH3), 3.92 (s, 3H, OCH3), 4.21 (s, 3H, OCH3),
4.36 (q, J = 7.0 Hz, 2H, CH2), 6.81 (s, 1H), 7.48–7.51 (m, 3H), 7.65–7.69
(m, 2H). 13C NMR (CDCl3): 17.2, 41.5, 56.1, 61.5, 106.3, 107.6, 125.9
4.1.7. Synthesis of 5-bromo-4,7-dimethoxy-2-phenyl-1H-benzo[d]
imidazole (8a)
A mixture of 7 (138.6 mg, 0.5 mmol), benzylamine (251 mg, 1.5
mmol), and iron (III) chloride hexahydrate (6.8 mg, 0.025 mmol) in a
10-mL pressure tube was stirred at 140 ◦C for 15 min using an Anton
Paar Monowave 300 Microwave Synthesis Reactor (Anton Paar GmbH,
Graz, Austria). After cooling, the reaction mixture was diluted with ethyl
acetate, then filtered over a pad of Celite, and washed with ethyl acetate.
The filtrates were dried (MgSO4), evaporated and the residue was pu-
rified by flash chromatography on silica gel (ethyl acetate/hexane, 1:1)
to afford compound 8a (141.6 mg, 85%) as a colourless solid, mp
157–159 ◦C; IR (KBr, cmꢀ 1) 3500, 3430, 1614, 1514, 1486, 1457, 1386,
1300; 1H NMR (DMSO‑d6): 3.88 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.84
(2C), 128.7 (2C), 129.6, 129.8, 130.3, 137.9, 142.8, 142.9, 153.5 ppm.
+
HRMS (ESI+): [M+H]
calcd. for C17H18BrN2O2, 361.0552; found,
361.0549.
4.1.9.2. 5-Bromo-1-ethyl-4,7-dimethoxy-2-(pyridin-2-yl)-1H-benzo[d]
imidazole (9b). White solid, mp 114–115 ◦C, yield 74%; IR (KBr, cmꢀ 1
)
1625, 1590, 1500, 1430, 1390, 1290, 1125; 1H NMR (CDCl3): 1.44 (t, J
= 7.0 Hz, 3H, CH3), 3.93 (s, 3H, OCH3), 4.25 (s, 3H, OCH3), 4.50 (q, J =
7.0 Hz, 2H, CH2), 6.80 (s, 1H), 7.30–7.35 (m, 1H), 7.81 (t, J = 7.8 Hz,
1H), 8.34 (d, J = 7.8 Hz, 1H), 8.67 (d, J = 5.0 Hz, 1H); 13C NMR (CDCl3):
17.0, 42.3, 56.2, 61.5, 105.9, 108.0, 123.8, 125.3, 126.9, 136.7, 137.5,
142.8, 143.3, 148.7, 149.6, 150.4; HRMS (ESI+): HRMS (ESI+): [M+H]+
calcd. for C16H17BrN3O2, 362.0504; found, 362.0505.
(s, 1H), 7.44–7.49 (m, 3H), 7.60–7.70 (m, 2H), 13.47 (s, 1H, NH); 13
C
NMR (DMSO‑d6): 56.5, 61.1, 105.2, 107.8, 124.3 (2C), 126.1 (2C),
126.9, 134.5, 137.7, 142.3, 148.3, 148.9, 150.9; HRMS (ESI+): [M+H]+
calcd for C15H14BrN2O2, 333.0239; found, 333.0227.
4.1.9.3. 5-Bromo-1-ethyl-4,7-dimethoxy-2-(pyridin-3-yl)-1H-benzo[d]
4.1.8. General procedure for synthesis of 5-bromo-4,7-dimethoxy-2-
(pyridinyl)-1H-benzo[d]imidazoles (8b-d)
imidazole (9c). White solid, mp 78–80 ◦C, yield 63%; IR (KBr, cmꢀ 1
)
1615, 1520, 1470, 1440, 1390, 1290; 1H NMR (CDCl3): 1.39 (t, J = 7.0
Hz, 3H, CH3), 3.92 (s, 3H, OCH3), 4.18 (s, 3H, OCH3), 4.37 (q, J = 7.0
Freshly prepared 1 M aqueous sodium dithionite (261.2 mg, 1.5
7

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Bioorganic Chemistry 111 (2021) 104823
Hz, 2H, CH2), 6.81 (s, 1H), 7.42–7.47 (m, 1H), 8.04 (t, J = 5.0 Hz, 1H),
8.73 (d, J = 5.0 Hz, 1H), 8.92 (s, 1H); 13C NMR (CDCl3): 17.3, 41.8, 56.2,
61.6, 106.7, 108.1, 123.6, 126.1, 126.7, 137.3, 138.1, 142.9, 143.0,
149.9, 150.3, 150.8; HRMS (ESI+): [M+H] + calcd. for C16H17BrN3O2,
362.0504; found, 362.0495.
hexane, 1:2).
4.1.11.1. 1-Ethyl-5-phenoxy-2-phenyl-1H-benzo[d]imidazole-4,7-dione
(11a). Orange solid, mp 190–192 ◦C, yield 50%; IR (KBr, cmꢀ 1) 1710,
1660, 1615, 1600, 1540, 1430, 1380, 1320, 1200; 1H NMR (CDCl3):
1.43 (t, J = 7.0 Hz, 3H, CH3); 4.40 (q, J = 7.0 Hz, 2H, CH2); 5.58 (s, 1H);
7.14 (d, J = 7.4 Hz, 2H); 7.31 (t, J = 7.4 Hz, 1H); 7.46 (t, J = 7.4 Hz, 2H);
7.43–7.70 (m, 5H); 13C NMR (CDCl3): 16.2, 41.8, 110.4 (2C), 121.2,
126.7, 128.6 (2C). 129.0 (4C), 129.4 (2C), 130.5, 130.6, 140.6, 153.2,
160.1, 175.2, 178.5. HRMS-ESI: [M+H]+ calcd for C21H17N2O3,
345.1239; found, 345.1237.
4.1.9.4. 5-Bromo-1-ethyl-4,7-dimethoxy-2-(pyridin-4-yl)-1H-benzo[d]
imidazole (9d). White solid, mp 160–161 ◦C, yield 62%; IR (KBr, cmꢀ 1
)
1615, 1510, 1455, 1420, 1385, 1295, 1120; 1H NMR (CDCl3): 1.40 (t, J
= 7.0 Hz, 3H, CH3), 3.92 (s, 3H, OCH3), 4.18 (s, 3H, OCH3), 4.40 (q, J =
7.0 Hz, 2H, CH2), 6.81 (s, 1H), 7.61 (d, J = 5.0 Hz, 2H), 8.76 (d, J = 5.0
Hz, 2H), 13C NMR (CDCl3): 17.2, 41.7, 56.2, 61.5, 106.7, 108.3, 123.7
(2C), 126.2 (4C). 137.9, 142.9, 150.3 (2C). HRMS (ESI+): [M+H]+
calcd. for C16H17BrN3O2, 362.0504; found, 362.0496.
4.1.11.2. 1-Ethyl-5-phenoxy-2-(pyridin-2-yl)-1H-benzo[d]imidazol-4,7-
dione (11b). Red solid, mp 177–178 ◦C, yield 55%; IR (KBr, cmꢀ 1) 1710,
1650, 1610, 1595, 1535, 1480, 1420, 1390, 1205; 1H NMR (CDCl3):
1.45 (t, J = 6.9 Hz, 3H, CH3), 5.04 (q, J = 6.9 Hz, 2H, CH2), 5.57 (s, 1H),
7.10–7.38 (m, 4H), 7.44 (t, J = 7.8 Hz, 2H), 7.83 (t, J = 7.8 Hz, 1H), 8.38
(d, J = 7.2 Hz, 1H), 8.65 (d, J = 4.3 Hz, 1H); 13C NMR (CDCl3): 16.4,
43.4, 111.2 (2C), 121.5 (C), 124.8, 125.6, 126.9, 130.8 (2C), 132.5,
137.4, 140.4, 149.2, 149.4, 150.6, 153.5, 160.3, 175.6, 178.7; HRMS-
ESI: [M+H]+ calcd for C20H16N3O3, 346.1192; found, 346.1187.
4.1.10. Synthesis of 2-aryl-5-bromo-1-ethyl-1H-benzo[d]imidazole-4,7-
diones (10a-d)
The general procedure described for the preparation of 5a-b from 4a-
b (2.1.4.) was utilized to synthesize compounds 10a-d from 9a-d.
4.1.10.1. 5-Bromo-1-ethyl-2-phenyl-1H-benzo[d]imidazole-4,7-dione
(10a.). Orange solid, mp 178–179.5 ◦C, yield 86%; IR (KBr, cmꢀ 1
)
1695, 1650, 1590, 1480, 1430, 1380, 1225, 1190; 1H NMR (CDCl3):
1.49 (t, J = 7.1 Hz, 3H, CH3), 4.44 (q, J = 7.1 Hz, 2H, CH2), 7.22 (s, 1H),
7.55–7.75 (m, 5H); 13C NMR (CDCl3): 16.4, 42.2, 128.4 (2C), 129.3
(2C), 129.6 (2C), 131.1, 131.4, 137.8, 138.4, 154.6, 173.5, 175.8; HRMS
(ESI+): [M+H]+ calcd. for C15H12BrN2O2, 332.0060; found, 332.0038.
4.1.11.3. 1-Ethyl-5-phenoxy-2-(pyridin-3-yl)-1H-benzo[d]imidazol-4,7-
dione (11c). Yellow solid, mp 158–159 ◦C, yield 55%; IR (KBr, cmꢀ 1):
1700, 1660, 1612, 1580, 1520, 1495, 1400, 1390, 1330, 1225, 1195; 1H
NMR (CDCl3): 1.46 (t, J = 7.1 Hz, 3H, CH3), 4.41 (q, J = 7.1 Hz, 2H,
CH2), 5.60 (s, 1H), 7.13 (d, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1H),
7.43–7.49 (m, 3H), 8.07 (d, J = 7.4 Hz, 1H), 8.78 (d, J = 7.4 Hz, 1H),
8.93 (s, 1H); 13C NMR (CDCl3): 17.2, 42.9, 111.3 (2C), 122.0, 124.7,
4.1.10.2. 5-Bromo-1-ethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazol-4,7-
dione (10b). Orange solid, mp 175–177 ◦C, yield 82%; IR (KBr, cmꢀ 1
)
125.9, 127.6, 131.4, 132.6, 138.1, 141.6, 150.3, 151.9, 152.4, 153.9,
1690, 1660, 1580, 1530, 1470, 1420, 1380, 1270; 1H NMR (CDCl3):
1.55 (t, J = 6.9 Hz, 3H, CH3), 5.12 (q, J = 6.9 Hz, 2H, CH2), 7.33 (s, 1H),
7.45 (t, J = 7.7 Hz, 1H), 7.91 (t, J = 7.7 Hz, 1H), 8.44 (d, J = 7.7 Hz, 1H),
8.74 (d, J = 7.7 Hz, 1H); 13C NMR (CDCl3): 16.4, 43.6, 125.0, 125.8,
132.2, 137.5, 138.4, 138.4, 141.0, 149.1, 150.8, 149.3, 173.7, 176.0;
HRMS (ESI+): [M+H]+ calcd for C14H11BrN3O2, 332.0035; found,
332.0034.
161.0, 175.9, 179.1; HRMS-ESI: [M+H]
calcd for C20H16N3O3,
+
346.1192; found, 346.1188.
4.1.11.4. 1-Ethyl-5-phenoxy-2-(pyridin-4-yl)-1H-benzo[d]imidazole-4,7-
dione (11d). Yellow solid, mp 176–177 ◦C, yield 58%; IR (KBr, cmꢀ 1
)
1645, 1615, 1390, 1355, 1310, 1200; 1H NMR (CDCl3): 1.48 (t, J = 6.8
Hz, 3H, CH3), 4.45 (q, J = 6.8 Hz, 2H, CH2), 5.61 (s, 1H), 7.09–7.34 (m,
3H), 7.46 (t, J = 7.3 Hz, 2H), 7.63 (d, J = 4.0 Hz, 2H), 8.82 (d, J = 4.0,
2H); 13C NMR (CDCl3): 16.6, 42.4, 151.2 (2C), 141.0, 136.5, 132.3,
130.9 (2C), 127.1, 123.6 (2C), 121.5, 110.9 (2C); HRMS-ESI: [M+H]+
calcd for C20H16N3O3, 346.1192; found, 346.1188.
4.1.10.3. 5-Bromo-1-ethyl-2-(pyridin-3-yl)-1H-benzo[d]imidazol-4,7-
dione (10c). Orange solid, mp 174–176 ◦C, yield 65%; IR (KBr, cmꢀ 1
)
1690, 1660, 1580, 1540, 1470, 1405, 1295; 1H NMR (CDCl3): 1.50 (t, J
= 7.1 Hz, 3H, CH3), 4.44 (q, J = 7.1 Hz, 2H, CH2), 7.22 (s, 1H), 7,52 (t, J
= 7.7 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 8.87 (d, J = 7.7 Hz, 2H); RMN
13C (100 MHz; CDCl3): 16.5, 42.4, 124.2, 124.9, 131.7, 137.4, 137.9,
138.5, 141.5, 149.6, 151.5, 151.9, 173.3, 175.7; HRMS (ESI+): [M+H]+
calcd for C14H11BrN3O2, 332.0035; found, 332.0034.
4.2. Crystal structure determination
X-ray data for 9a and 9b were collected on a Bruker AXS APEX3
diffractometer using a combination of ω
- and φ-scans of 0.5◦. Data were
corrected for absorption and polarization effects and analyzed for space
group determination [45]. The structures were solved by dual-space
methods and expanded routinely [46]. The models were refined by
full-matrix least-squares analysis of F2 against all reflections [47]. All
non-hydrogen atoms were refined with anisotropic atomic displacement
parameters. Unless otherwise noted, hydrogen atoms were included in
calculated positions. Atomic displacement parameters for the hydrogens
were tied to the equivalent isotropic displacement parameter of the
atom to which they are bonded (Uiso(H) = 1.5Ueq(C) for methyl,
1.2Ueq(C) for all others). The crystal data, data collection and structure
refinement details of compounds 9a and 9b are tabulated in Table 1.
4.1.10.4. 5-Bromo-1-ethyl-2-(pyridin-4-yl)-1H-benzo[d]imidazol-4,7-
dione (10d). Yellow solid, mp 185–186 ◦C, yield 76%; IR (KBr, cmꢀ 1):
1700, 1650, 1595, 1570, 1530, 1490, 1430, 1420, 1280; 1H NMR
(CDCl3): 1.49 (t, J = 7.0 Hz, 3H, CH3), 4.45 (q, J = 7.0 Hz, 2H, CH2),
7.21 (s, 1H), 7.60 (d, J = 4.3 Hz, 2H), 8.81 (d, J = 4.3 Hz, 2H) ppm; RMN
13C (100 MHz; CDCl3):16.2, 42.2, 123.3 (2C), 131.6, 135.9, 137.7,
138.5, 141.2 (2C), 150.7, 173.0, 175.5; HRMS (ESI+): [M+H]+ calcd for
C
14H11BrN3O2, 332.0035; found, 332.0040.
4.1.11. General procedure for preparing 2-aryl-1-ethyl-5-phenoxy-1H-
benzo[d]imidazole-4,7-diones (11a-d)
A mixture of phenol (9.41 mg, 0.1 mmol) and K2CO3 (41.5 mg, 0.3
mmol) in DMF (0.5 mL) was stirred for 1 h at 25 ◦C. Then, the corre-
sponding bromoquinone 10 (0.1 mmol) was added and the reaction
mixture was stirred at 25 ◦C for 12 h. The reaction mixture was diluted
with water (5 mL) and extracted with chloroform (3x15 mL). The
combined organic extracts were dried (MgSO4), concentrated in vacuo,
and the residue was purified by column chromatography (ethyl acetate/
4.3. Biological evaluation
4.3.1. In vitro anti-T. Cruzi activity on epimastigotes.
T. cruzi epimastigotes, Dm28c strain, were grown at 28 ◦C in Di-
amond’s monophasic medium supplemented with 4 µM hemin and 5%
(v/v) foetal bovine serum as described earlier [48]. The compounds
were tested at a starting concentration of 0.5–100 µM and were added to
8

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C. Lopez-Lira et al.
Bioorganic Chemistry 111 (2021) 104823
suspensions of 3⋅106 parasites/mL and incubated at 28 ◦C for 24 h. After
this period, trypanosomicidal activity was measured using MTT [3–(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [49].
MTT was added to a final concentration of 0.5 mg/mL with phenazine
methosulfate (0.22 mg/mL) and incubated at 37 ◦C for 4 h. The parasites
were solubilized in 10% sodium dodecyl sulfate-0.01 M HCl and incu-
bated overnight. Formazan crystal formation was measured at 570 nm
using an ELISA spectrophotometer (Labsystems Multiskan MS, Finlan-
dia). A full dose–response curve was constructed for all compounds to
determine the concentration inhibiting 50% of parasite growth (IC50
value). The IC50 values were obtained using non-linear dose response
curve-fitting analysis (log of concentration vs percentage of viable cells)
via Graph Pad Prism 5 software. Reported values are means of at least
three independent experiments.
packages. C.L.L. thanks CONICYT for a doctoral fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104823.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgements
This work was supported by FONDECYT-CHILE Grant 11150988 and
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