Rhodium-catalyzed C-H bond activation for the synthesis of quinonoid compounds: Significant Anti-Trypanosoma cruzi activities and electrochemical studies of functionalized quinones

Article abstract of DOI:10.1016/j.ejmech.2017.05.011

Thirty four halogen and selenium-containing quinones, synthesized by rhodium-catalyzed C-H bond activation and palladium-catalyzed cross-coupling reactions, were evaluated against bloodstream trypomastigotes of T.?cruzi. We have identified fifteen compounds with IC₅₀/24?h values of less than 2?μM. Electrochemical studies on A-ring functionalized naphthoquinones were also performed aiming to correlate redox properties with trypanocidal activity. For instance, (E)-5-styryl-1,4-naphthoquinone 59 and 5,8-diiodo-1,4-naphthoquinone 3, which are around fifty fold more active than the standard drug benznidazole, are potential derivatives for further investigation. These compounds represent powerful new agents useful in Chagas disease therapy.

Full text of DOI:10.1016/j.ejmech.2017.05.011

European Journal of Medicinal Chemistry 136 (2017) 406e419
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Rhodium-catalyzed C-H bond activation for the synthesis of quinonoid
compounds: Significant Anti-Trypanosoma cruzi activities and
electrochemical studies of functionalized quinones
Guilherme A.M. Jardim a, Thaissa L. Silva b, Marilia O.F. Goulart b, Carlos A. de Simone c,
d
d
d
e
~
Juliana M.C. Barbosa , Kelly Salomao , Solange L. de Castro , John F. Bower ,
a, *
^
Eufranio N. da Silva Júnior
a Institute of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil
b
ꢀ
Institute of Chemistry and Biotechnology, Federal University of Alagoas, CEP 57072-970, Maceio, AL, Brazil
c
~
~
Department of Physics and Informatics, Institute of Physics, University of Sao Paulo, Sao Carlos, 13560-160, SP, Brazil
d Laboratory of Cellular Biology, IOC, FIOCRUZ, Rio de Janeiro, RJ, 21045-900, Brazil
e School of Chemistry, University of Bristol, Bristol, BS8 1TS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty four halogen and selenium-containing quinones, synthesized by rhodium-catalyzed C-H bond
activation and palladium-catalyzed cross-coupling reactions, were evaluated against bloodstream try-
pomastigotes of T. cruzi. We have identified fifteen compounds with IC50/24 h values of less than 2 mM.
Electrochemical studies on A-ring functionalized naphthoquinones were also performed aiming to
correlate redox properties with trypanocidal activity. For instance, (E)-5-styryl-1,4-naphthoquinone 59
and 5,8-diiodo-1,4-naphthoquinone 3, which are around fifty fold more active than the standard drug
benznidazole, are potential derivatives for further investigation. These compounds represent powerful
new agents useful in Chagas disease therapy.
Received 4 February 2017
Received in revised form
1 May 2017
Accepted 2 May 2017
Available online 4 May 2017
Keywords:
C-H functionalization
Quinones
© 2017 Elsevier Masson SAS. All rights reserved.
Chagas disease
Trypanosoma cruzi
Electrochemistry
1. Introduction
chronic form, but after 10e30 years, the other third will develop a
symptomatic chronic disease with digestive and/or cardiac distur-
Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is clas-
sified as a neglected tropical disease by the World Health Organi-
zation. This disease has high morbidity and mortality rates, affects
5e7 million people and displays a limited response to therapy [1]. It
is transmitted to humans by triatomine vectors, blood transfusions,
oral and congenital routes and less commonly, by organ trans-
plantation, and laboratory accidents [2]. Chagas disease is charac-
terized by two clinical phases: a short, acute phase defined by
patent parasitemia and a long, progressive chronic phase. The acute
phase appears shortly after infection and is frequently asymptom-
atic. After 2e3 months, in most infected individuals, a host/parasite
balance is achieved, leading to the chronic phase. Approximately
two thirds of infected individuals remain in the indeterminate
bances [3]. Two current major concerns are outbreaks of acute
Chagas disease associated with the ingestion of contaminated food
[4], and the disease's emergence in non-endemic areas such as
North America and Europe, due to the immigration of infected in-
dividuals [5].
T. cruzi is a hemoflagellate protozoan and its life cycle involves
distinct forms during its passage through vertebrate and inverte-
brate hosts. The trypomastigote form ingested by the insect dif-
ferentiates into the proliferative epimastigote form, which, on
reaching the posterior intestine of the insect, differentiates into the
metacyclic form. The latter, following invasion of vertebrate host
cells, undergoes differentiation into the amastigote form, which,
after several reproductive cycles, transforms into the trypomasti-
gote form, responsible for the dissemination of the infection.
The current treatment for Chagas disease is restricted to two
nitroheterocyclic drugs, benznidazole (Bz) and nifurtimox (Nif),
* Corresponding author.
E-mail address: eufranio@ufmg.br (E.N. da Silva Júnior).
http://dx.doi.org/10.1016/j.ejmech.2017.05.011
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
407
and their efficacy varies according to the phase of the disease, with
their effectiveness decreasing with advancement of the infection
[6]. The major limitations of these drugs are their limited and
variable curative activity in the established chronic form of the
disease and their toxic effects, leading to treatment abandonment
in several instances [7]. These drawbacks justify the urgent need to
identify better drugs to treat chagasic patients. In this context, an
intensive research program has focused upon the search for alter-
native natural, semi-synthetic and synthetic lead compounds [8].
Over the last few years, our group has dedicated efforts to the
synthesis and identification of novel naphthoquinoidal compounds
with potent antiparasitic activity [9]. In this context, we have
modified the “redox centre” A- and C-rings of lapachones with the
aim of preparing new bioactive compounds [9c]. For instance, a
[12] described the synthesis and evaluation of A-ring modified
lapachones, which show activity against cancer cells (Scheme 1A);
this study is of direct relevance to the strategies outlined herein.
~
Salomao and co-workers have reported the trypanocidal activity
of C-2 and C-5 substituted naphthoquinones, as well as evidence for
their mode of action [13]. As shown in Scheme 1B, in assays per-
formed in the absence of blood and with incubation at 37 ꢀC several
compounds presented IC50 values of <2
instance, further assays with 5-acetoxy-1,4-naphthoquinone
(IC50 ¼ 0.16 M) revealed activity against the proliferative forms
mM against T. cruzi. For
m
of T. cruzi, intracellular amastigotes and epimastigotes. In experi-
ments with this latter parasite form, this compound led to mito-
chondrial swelling, vacuolization, and flagellar blebbing, as well as
a remarkable decrease in the mitochondrial membrane potential
and a discrete increase of ROS production. Such redox dependent
effects are likely reliant on the acetyl group facilitating quinone
reduction, as previously demonstrated by electrochemical analysis
[13b]. These results show that A-ring substituted naphthoquinones
can kill T. cruzi and highlight the importance of synthesizing and
evaluating new quinoidal compounds against parasites.
simple redox centre modification of b-lapachone led to a phenazine
derivative with trypanocidal activity about two and six times
higher than benznidazole and the original quinone, respectively
[10]. Intrinsically related to this strategy, we have demonstrated
that appendage of a triazole group to the C-ring of b-lapachone
affords a derivative four times more active against the parasite than
the naphthoquinoidal precursor [11]. Recently, Lim and co-workers
Recently, our group has embarked on a research program aimed
Scheme 1. Overview.

408
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
at addressing unmet synthetic challenges related to quinone
modification. For instance, we have demonstrated that chiral
squaramide catalyzed reaction of lawsone with Morita-Baylis-
Hillman acetates of nitroalkenes leads to the asymmetric forma-
tion of pyranonaphthoquinones [14]. We have also developed C-H
activation-based processes that achieve challenging bond forma-
tions and enable late stage functionalization of complex systems
[15]. For example, we reported recently a method for the A-ring
functionalization of naphthoquinones that is reliant on a rhodium-
catalyzed C-5 selective C-H iodination reaction [16]. In this report,
we outline the C-H functionalization enabled synthesis of 30 qui-
noidal derivatives and their evaluation against T. cruzi. Four com-
pounds were also prepared by C-C bond forming reactions achieved
via palladium catalysis. Since the biological activity of quinones is
intrinsically related to the quinoidal redox centre, we also describe
the electrochemical behavior of nine representative compounds.
changed the regioselectivity of iodination to afford 5, where func-
tionalization had occurred ortho to the hydroxyl group (C7) rather
than at C5; this demonstrates that the carbonyl-directed pathway
can be overridden by strong donor substituents. Control experi-
ments showed that in the absence of the Rh-catalyst only traces of
product were observed. Use of 250 mol% NIS enabled subsequent
C5 iodination of 5 to provide 6 in 82% yield. Reaction of plumbagin 7
with 100 mol% NIS provided C7 iodination product 8 in 85% yield.
Juglone (4) and plumbagin (7) were reacted with iodomethane and
Ag2O in CHCl3 to afford 9 and 10 in 73% and 78% yield, respectively
[17]. Rh-catalyzed C-H iodination of these substrates gave 11 and 12
in excellent yields. To prepare compounds containing methoxy
groups with iodine in different A-ring position, derivatives 5 and 8
were also methylated to afford 13 and 14 in 91% and 92% yield,
respectively (Scheme 3).
Other substrates containing methoxy groups were obtained in
good yields by oxidation of tetralones 15 and 16 with 2-iodo-ben-
zoic acid and Oxone (substrates 17 and 18) [18]. When the C-H
iodination methodology was applied to these substrates derivatives
19 and 20 were generated in good to excellent yields. The regio-
selectivity observed for the iodination of 19 is suggestive of sec-
ondary coordination of the methoxy group to the Rh-center,
thereby facilitating functionalization at the most hindered A-ring
position (Scheme 4). Consistent with this assertion, iodination of
compound 21, in which the methoxy group of 19 has been replaced
by a methyl substituent, occurred selectively at the less hindered
position to generate 22 in 63% yield (Scheme 5).
2. Results and discussion
2.1. Chemistry
Naphthoquinone derivatives were the first class of compounds
targeted for preparation. A-ring modifications were performed
utilizing a carbonyl directed Rh-catalyzed C-H bond activation
protocol reported recently by our group [16]; this methodology
allowed C-5 selective iodination of several 1,4-naphthoquinones. In
the simplest case, using 1,4-naphthoquinone (1) as substrate, the
synthesis of mono-iodinated product 2 occurred in 69% yield,
accompanied by a 5% yield of bis-iodinated by-product 3 (Scheme
2). For the purpose of clarity, the numbering system shown in
Scheme 2 is used during the following discussion.
In order to investigate the effects of other electron donating
groups, iodination of 24, which contains an amino group, was
pursued. Consistent with hydroxy containing systems 4 and 7,
functionalization occurred at C-7 to afford 25 in 41% yield and with
high selectivity. On the other hand, iodination of 27, which contains
an electron withdrawing ester at C-8, was selective for C-5,
affording derivative 28 in 57% yield (Scheme 6). Note that Ivashkina
and co-workers [19] have described the synthesis of 25 using I2-
HIO3 but the product was obtained as mixture of C-5 and C-7
iodinated products.
Iodinated naphthoquinones containing electron donating and
withdrawing groups were selected for subsequent biological eval-
uation. The use of hydroxyl A-ring-substituted substrates (e.g. 4)
Electron deficient naphthoquinones were also used as sub-
strates, affording the targets in moderate to good yields. Interest-
ingly, unsymmetrical substrate 29 delivered 30 (59% yield) as the
sole product, indicating that the bromine substituent controls the
relative directing ability of the two carbonyl groups for the met-
alation event (Scheme 7).
We also sought to investigate the biological effects of
Scheme 2. Iodinated naphthoquinoidal derivatives 2 and 3.
Scheme 3. Iodinated A-ring substituted naphthoquinones 5, 6, 8 and 11e14.

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
409
Scheme 4. Preparation of iodinated derivatives 19 and 20 from tetralones.
degradation. For benzoquinone 47, which contains an electron-
donating methoxy-group, iodination occurred at the most elec-
tron rich position to afford 50 in 91% yield. Bis-iodination of bis-
methoxy system 48 was achieved in high yield by using an excess
of DIH to generate 51 in 76% yield (Scheme 10).
Systems possessing electron-withdrawing substituents proved
to be more challenging; for example, electron deficient substrate 52
reacted smoothly with DIH to provide 53 in 56% yield. Bromination
of 52 provided 54 in only 45% yield (Scheme 11).
Scheme 5. Synthesis of iodinated quinone 22.
As outlined in Scheme 12, application of the C-H functionali-
zation conditions to 1,4-naphthoquinone 1 provided derivatives 55
and 56 in excellent yields and high selectivity. The use of
[RhCptCl2]2 in combination with CuSO4 was required to achieve C-2
rather than C-5 selectivity. This aspect is notable because site-
selective C-H functionalization of naphthoquinones (C-2 versus C-
5) is achieved under catalyst control (cf. Scheme 2).
Because iodination of 1,4-naphthoquinone was readily achieved,
subsequent palladium-catalyzed C-C bond forming derivatizations
also became accessible (Scheme 13), thereby providing an avenue
for the synthesis of different classes of hybrid quinonoid-based
compounds. Under highly optimized conditions [21], Stille
coupling of 2 afforded 57 in 75% yield. Heck alkenylations [22] of 2
with ethyl acrylate and styrene proceeded smoothly to provide 58
and 59, respectively. Several Sonogashira cross-coupling protocols
were evaluated without success. To solve this problem, a previously
reported procedure [23] was employed, wherein reaction of 2 with
an alkynyl-copper(I) reagent provided Sonogashira-like product 60
in 64% yield.
substituents on other quinone-based systems. The evaluation of
heteroatom substituted benzoquinoidal compounds as potential
trypanocidal agents was particularly attractive because these can
be accessed directly by our reported C-H functionalization protocol
(Scheme 4) [20]. Rh-catalyzed reaction of 1,4-benzoquinone 35
with 1,3-diiodo-5,5-dimethylhydantoin (DIH) yielded derivative 36
in 95% yield. Exchanging the electrophile source to 1,3-dibromo-
5,5-dimethylhydantoin (DBH) afforded 37 in 82% yield. C-H phe-
nylselenation processes were also investigated. Reaction of 35 with
150 mol % N-(phenylseleno)phthalimide afforded a mixture of
target 38 and bis-functionalization product 39. However, it was
found that by using only 100 mol% N-(phenylseleno)phthalimide,
38 could be obtained in good yield and with high selectivity over 39
(4.5:1 selectivity). The latter compound could be accessed exclu-
sively in 74% yield by using an excess (250 mol %) of N-(phenyl-
seleno)phthalimide (Scheme 8).
Rh-catalyzed iodination of alkyl-substituted system 40 afforded
target 41 in 66% yield and good regioselectivity. Here, iodination
occurs at the site remote to the bulky tert-butyl group. A similar
result was obtained for bromination of 40 to deliver 42 in 57% yield
(Scheme 9).
The metal-catalyzed protocol was also applied to benzoquinone
46, containing two adjacent methyl groups, and this afforded 49 in
81% yield. Interestingly, attempted bromination of 46 lead to
The structures of novel compounds 5, 6, 8, 13, 14, 25, 49 and 59
were determined by 1H, 13C and 2D NMR analysis in combination
with either electrospray ionization or electronic impact mass
spectrometry. Additionally, compounds 5, 6 and 51 were analyzed
by single crystal X-ray diffraction, as summarized below.
Scheme 6. Iodinated compounds 25 and 28 prepared from 23 and 26.

410
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
Scheme 7. Iodinated naphthoquinoidal derivatives 30, 32 and 34.
Scheme 8. Iodinated derivatives 35 and 36 and chalcogenated 38 and 39.
Scheme 9. Synthesis of iodinated quinone 41 and brominated one 42 from 40.
Scheme 10. Iodinated quinoidal derivatives.
2.2. X-ray analysis
independent molecules in the asymmetric unit where two of them
are superimposed with occupational disorder on some atoms. For
clarity, only one molecule is shown in Fig. 1. The molecule is
Compound 5 crystallizes in space group P21/c with four

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
411
Scheme 11. Preparation of iodinated derivative 53 and brominated 54 from electron deficient compound 52.
Scheme 12. C-2 substituted naphthoquinones 55 and 56.
Styrene
Scheme 13. Derivatization of 2 via palladium catalyzed C-C bond formatting reactions.
perfectly planar (r.m.s. deviation ¼ 0.0029 Aꢀ for 14 non-hydrogen
atoms). In the packing of the crystal, five C-H—O type interactions
and three O-H—I type interactions link the molecules, forming
chains that extend along the [b] axis and parallel to (4 0 4) plane.
Compound 6 crystallizes in space group P21/n with two mole-
cules in the asymmetric unit. For clarity, only one molecule is
shown in Fig. 1. There are no significant conformational differences
between them. The molecule is almost planar (r.m.s.
deviation ¼ 0.0051 Aꢀ for I3 non-hydrogen atoms) except the I1 and
I2 atoms that are out of the mean plane of the rings with deviations
of ꢁ0.179 (3) and 0.137 (2) Aꢀ respectively. In the crystal, molecules
are linked by weak interactions C2-H2/O3 [d (H2/O3) ¼ 2.56 (2)
Å, \(C2-H2/O3) ¼ 125 (4)ꢀ], C18-H18/O2 [d (H18/O2) ¼ 2.42 (2)
Å, \(C18-H18/O2) ¼ 170 (3)ꢀ] and O-H—I [d (H/I) ¼ 3.53 (2)Å]
forming chains parallel to the (3 -3 -3) and (3 3 -3) planes.
Finally, compound 51 crystallizes in space group Pnma. The
molecule is symmetric where the asymmetric units are C1, C2, C3,
C4, C5 I1, O1, O2 and O3. The I1, O1 and O3 atoms deviate from the
mean plane of the ring by ꢁ0.154(2), ꢁ0.111(2) and ꢁ0.388 (3) Aꢀ,
respectively. The C2-C3-O2-C5 torsion angle of the methoxy group
Fig. 1. Projections ORTEP-3 of 5, 6 and 51, showing the atom-numbering and displacement ellipsoids at the 50% probability level.

412
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
is ꢁ158.2 (2)ꢀ. In the crystal, the molecules are linked by I—O
(methoxy group) interactions [d ¼ 3.383 (2) Aꢀ] resulting in a
highly symmetrical arrangement that extends along the [a] axis.
An Ortep-3 diagram of each molecule is shown in Fig. 1 and
Table 1 lists the main crystallographic data for 5, 6 and 51. Packing
diagrams for all compounds are provided in the Supporting Infor-
mation File.
naphthoquinone 3 was obtained as a by-product (vide supra) in
poor yield (5%) and further optimization of its preparation will be
necessary. In general, the presence of iodine and electron donating
groups on the A-ring increases activity against T. cruzi and high-
lights the importance of our Rh-catalyzed C-H iodination meth-
odology. Compounds 22 (IC50
¼
0.49 mM, SI 24.7) and 25
(IC50 ¼ 0.56 M, SI 23.4), which combine either a methyl or amino
m
group with an iodine substituent, show important activity, being
around 20 times more active than benznidazole. As such, 22 and 25
are prototypes that warrant further study. Conversely, the presence
of electron withdrawing groups on A-ring and, in some cases, an
electron donating methyl group leads to decreased activity against
T. cruzi [34 (7.26/SI 3.8), 12 (7.57/SI 6.9) and 11 (4.06/SI 6.1)].
Certain benzoquinones also show interesting trypanocidal ac-
2.3. Biological studies
The potential of simple structural modifications for increasing
antiparasitic activity of naphthoquinones is shown in Scheme 6.
Juglone, a naturally occurring naphthoquinone, displays an IC50
/
24 h value of 6.51 M [13a]. We have previously reported that the
m
tivity, with an IC50 value of less than 2 mM determined for com-
addition of methyl or bromine groups to C-5 and/or C-7 leads to
new naphthoquinones that are 4.7 and 5.2 times more active
against T. cruzi than juglone, respectively. Remarkably, a 40 fold
increase in activity is observed for the O-acetoxy derivative of
juglone [13a] (Scheme 14).
Considering these previous results and recent reports in the
literature [24,25], we have accomplished modifications to the qui-
noidal system leading to A-, B-, or A- and B-ring substituted com-
pounds, and these were assayed against T. cruzi. Note that direct
functionalization by rhodium-catalyzed C-H bond activation allows
the efficient and fast preparation of potent quinoidal compounds,
as shown in Scheme 15.
pound 51. This compound is easily obtained from commercially
available benzoquinone in just one-step. C-2 functionalization may
be a good general strategy for preparing bioactive compounds,
since naphthoquinones substituted at C-2 with iodine (55
IC50 ¼ 1.79
m
M, SI 8.2) and selenium (56, IC50 ¼ 1.13
mM, SI 11.2)
were 5.4 and 8.5 times more active than benznidazole, respectively.
Finally, the most active compound (E)-5-styryl-1,4-
naphthoquinone 59 was prepared in two steps and is described
here for the first time. The synthesis of 59 requires C-5 iodinated
compound 2, which is prepared by Rh-catalyzed C-H functionali-
zation. The C-I bond then enables Pd-catalyzed alkenylation with
styrene to afford 59. This compound, with an IC50/24 h ¼ 0.19
mM, is
Compounds were evaluated against bloodstream forms of
T. cruzi (IC50/24 h) and mammalian cells (LC50/24 h), allowing the
determination of the selectivity index (SI) derived from the ratio of
LC50/IC50 (Table 2). From these 34 compounds, 22 are more active
fifty fold more active than benznidazole, with an SI of 62.5.
Although the studies described here are preliminary, it is worth
noting that the combination of a practical synthetic route, high
in vitro trypanocidal activity and low toxicity to mammalian cells,
makes 59 a very attractive hybrid quinone in the search for better
trypanocides.
than benznidazole, the reference drug (IC50/24 h ¼ 9.68 2.35
mM),
with 2, 3, 8, 13, 19, 20, 22, 25, 28, 30, 51, 55, 56, 57 and 59 exhibiting
IC50/24 h values lower than 2
62.5.
mM, with an SI of between 1.3 and
Compounds 2 and 3 show significant activity against the para-
site with IC50 values of 0.59 and 0.22 M, respectively, low toxicity
2.4. Electrochemistry aspects
m
for the mammalian cell, and an SI higher than 19. Diiodinated
Electrochemistry is a useful tool for the design, development
Table 1
Crystal data and structure refinement for compounds 5, 6 and 51.
Identification code
Compound 5
Compound 6
Compound 51
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C10H5IO3
315.5
293 (2) K
0.71073 Å
monoclinic
P21/c
C10H4I2O3
425.9
293 (2) K
0.71073 Å
monoclinic
P21/n
C8H6I2O4
419.9
293 (2) K
0.71073 Å
orthorhombic
Pnma
Unit cell dimensions
a ¼ 7.7911 (6) Å
b ¼ 27.0192 (4) Å
c ¼ 13.5802 (12) Å
2854.8 (9) Å3
4
a
b
g
¼ 90ꢀ
a ¼ 8.2431 (2) Å
b ¼ 15.1653 (4) Å
c ¼ 16.8525 (5) Å
2103.9 (2) Å3
8
a
b
g
¼ 90ꢀ
a ¼ 9.5066 (4) Å
b ¼ 17.3636 (8)Å
c ¼ 6.3177 (2) Å
1698.7 (2) Å3
4
a
b
g
¼ 90ꢀ
¼ 90ꢀ
¼ 90ꢀ
¼ 93.1 (5)ꢀ
¼ 90ꢀ
¼ 92.95 (2)ꢀ
¼ 90ꢀ
Volume
Z
Density (calculated)
Absorption coefficient
F (000)
2.17 Mg/m3
2.69 Mg/m3
5.9 mmꢁ1
1552
2.67 Mg/m3
6.01 mmꢁ1
768
3.6 mmꢁ1
1756
Theta range for data collection
Index ranges
1.7e27.9
1.8e27.1
2.3e27.9
ꢁ9ꢂh ꢂ 10,-35 ꢂ k ꢂ 35
ꢁ10 ꢂ h ꢂ 10, ꢁ19 ꢂ k ꢂ 19
ꢁ13 ꢂ l ꢂ 17
ꢁ21 ꢂ l ꢂ 21
ꢁ12 ꢂ h ꢂ 12, ꢁ22 ꢂ k ꢂ 22, ꢁ8 ꢂ l ꢂ 8
Collected
Reflections
25651
16620
none
8822
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
none
Full-matrix least-squares on F2
5975/0/448
none
Full-matrix least-squares on F2
3421/0/273
Full-matrix least-squares on F2
1241/0/71
1.34
1.004
1.21
R1 ¼ 0.074, wR2 ¼ 0.15
R1 ¼ 0.037, wR2 ¼ 0.062
R1 ¼ 0.014, wR2 ¼ 0.034
R1 ¼ 0.084, wR2 ¼ 0.152
R1 ¼ 0.064, wR2 ¼ 0.069
R1 ¼ 0.015, wR2 ¼ 0.034

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
413
Plumbagin
Scheme 14. Simple modifications in juglone leading to compounds with potent trypanocidal activity. *IC50/24 h values for the lytic activity on bloodstream trypomastigotes.
Scheme 15. Strategies for the synthesis of novel trypanocidal naphthoquinones and benzoquinones.
and characterization of redox-selective molecules and can provide
information on their mechanism of action [26]. Motivated by this
approach, we have successfully described the combined use of
electrochemistry and biological evaluation to provide insights
about the mechanism of action of 3-arylamino lapachone [27], as
well as selenium [28] and triazole substituted [29] quinones. The
ever-increasing interest in biologically active quinones has resulted
in new strategies to decrease toxicity, modulate activity, and direct
the compound to the target [30]. As such, it is pertinent to continue
to explore their synthesis, biological activities and redox mecha-
nisms through a combined theoretical and experimental approach.
Most quinones, including intermediary and reduced forms, exist
inside hydrophobic cell membranes. Dimethylformamide (DMF)
was chosen as an aprotic organic solvent for electrochemical
studies, because it can mimic the non-aqueous environment of the
membrane, where lipid peroxidation, one of the causes of mem-
brane fragmentation, normally occurs [24]. Non-aqueous solvents
provide better models of a membrane environment in which per-
oxidation processes take place, because both the superoxide anion
radical and its conjugated acid, the hydroperoxyl radical, are un-
stable in water and other protic solvents, owing to their fast
disproportionation [31a-c]. Additionally, in these conditions, single
electron transfer occurs, generating the active semiquinone
[26,31d].
We have studied the electrochemical properties of nine qui-
nones. These compounds were selected based on their trypanocidal
activity; both active and inactive variants have been studied to
enable an understanding of the main qualitative features associated
with activity.
As observed in Fig. 2, in general there are two reducible systems
(the quinone itself and the aryl iodide bond). The electrochemistry
of quinones [31e] and halogenated compounds, mainly aromatic
ones (ArX), has been thoroughly investigated [32] on several elec-
trodes. For quinones, in aprotic medium, general equations (1)e(3)
are expected and the quinone/semiquinone/hydroquinone (Q/SQꢃ-/
H2Q) triad is an important component of many redox systems in
biology [24,26].
For halides, bi-electronic uptake in aprotic solvents causes
rupture of the C-X bond, by either a concerted (Eq. (4)) or stepwise
mechanism (Eq. (5) and (6)), followed by reduction of the ensuing

414
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
Table 2
naphthoquinones analyzed have additional A-ring substituents
(-OH, -OCH3, and -CH3 groups) and two of them are bis-iodinated (3
and 6). All possess an aryl-iodide bond, except 55, which carries a
vinyl iodide. Data on the main reduction peaks, with an emphasis to
the first reduction step, Epc1, appear in Table 2.
Activity of synthetic derivatives against bloodstream trypomastigotes of T. cruzi (Y
strain) at 37 ꢀC, cytotoxicity to mammalian cells and Selectivity Index (SI).
Compd
IC50/24 h (
m
M)
LC50/24 h (
m
M)
SI
2
3
5
6
0.59 0.08
0.22 0.07
42.0 3.11
24.8 4.49
0.99 0.12
3.54 0.71
7.57 0.56
1.35 0.43
4.06 0.47
1.17 0.23
1.62 0.34
0.49 0.04
0.56 0.05
1.61 0.44
1.38 0.57
2.25 0.48
7.26 1.41
20.77 8.31
21.54 5.83
15.95 3.52
83.42 9.08
12.77 1.61
14.09 2.32
10.07 3.26
23.95 7.08
1.94 0.51
26.99 4.30
15.40 1.34
1.79 0.37
1.13 0.22
1.24 0.31
6.03 1.19
0.19 0.08
2.20 0.33
9.68 2.35
11.49 0.54
12.93 1.43
>100
19.5
28.8
>4.6
>5.1
15.8
3.8
6.9
9.8
6.1
12.1
7.5
24.7
23.4
8.7
8.8
5.3
3.8
4.1
1.6
Considering the ease of the reduction related to Epc1, the com-
pounds can be ranked as shown below. Less negative potentials
>100
indicate
higher
electrophilicity:
6
>
5
>
8
15.72 3.49
13.59 0.79
51.88 10.42
13.27 1.73
24.67 6.51
14.19 3.21
12.16 0.16
12.09 0.24
13.12 1.16
13.94 0.21
12.15 0.15
11.82 0.37
27.83 5.17
86.02 2.99
34.35 2.46
13.78 4.37
>100
55 > 3 > 2 > 22 > 20 > 19 > 11 (Fig. 2).
11
12
13
14
19
20
22
25
28
30
32
34
36
37
38
39
41
42
49
50
51
53
54
55
56
57
58
59
60
Bz
The CV profiles (Fig. 3) and structural similarities allow the
classification of the compounds into three main electrochemical
classes.
The first class contains only one compound, 55, and is important
as it allows comparison of the reduction of aromatic vs. vinylic C-I
bonds. Aromatic variants suffer much easier reduction (see Epc3),
while the ease of reduction of the quinone moiety is influenced by
the electron withdrawing halogen at C2, being reduced at more
positive potentials (ꢁ0.413 V) than similar C5-iodinated compound
2 (ꢁ0.535 V, see Table 3).
The second class comprises compounds with substituents that
can form hydrogen bond (5 and 6). This feature results in easier
reduction, presumably due to stabilization of the semiquinone (Eq.
(1)) through hydrogen-bonding. Reduction potentials for 5 and 6
are ꢁ0.216 V and ꢁ0.244 V, respectively; these values are much
more positive than for all the other quinones. The electrochemical
reduction of the C-I, leading to bond cleavage (Eqs. (4)e(7)) occurs
at highly negative potentials (Epc3, Table 2).
The third class of compounds, 2, 3, 22, 19, 11 and 20, has a
different electrochemical profile. The first waves for all of them are
related to the generation of the semiquinone (Eq. (1)). With the
exception of 2, 11 and 22, all of them present an enhanced current
at the level of the second reduction wave (Ipc2/Ipc1 > 1), with loss of
reversibility (Ipa/Ipc < 1). This is related to the reduction of the
semiquinone together with the reduction of the C-I bonding. In the
case of 3, where the current (Ipc2) is the highest, the cleavage of
both C-I bonds is expected [30] and observed.
0.86
>1.2
2.8
3.0
1.5
35.93 4.92
42.30 4.67
15.40 3.87
32.62 9.71
27.71 1.22
>100
1.4
1.3
>4.7
>6.5
8.2
11.2
10.2
2.1
62.5
5.7
>413.2
>100
14.72 2.68
12.67 0.70
12.67 0.10
12.65 0.26
11.87 0.43
12.49 0.23
>4000
radical (Eq. (7)) to an anion. The latter is usually protonated by
residual water to the respective C-H bond [30].
Comparison of the Epc1 values with trypanocidal activity re-
veals a trend, with the two more electrophilic quinones (5 and 6)
being less active than the others. As quinones are pro-drugs that
need to be activated, being so easily reduced, it is possible that 5
and 6 suffer reduction fast, before reaching the biological target,
thus leading, to inactivity.
ArX þ eꢁ/Arꢃ þ Xꢁ
ArX þ eꢁ#ArXꢃꢁ
ArXꢃꢁ/Arꢃ þ Xꢁ
Arꢃ þ eꢁ/Arꢁ
The electrochemistry of iodinated quinones where the halogen
is directly attached to the aromatic ring is less widely reported. In
the present case, the reduction process of nine iodinated 1,4-
naphthoquinones (1,4-NQ), 2, 3, 5, 6, 11, 19, 20, 22 and 55 was
investigated on glassy carbon electrode (GCE), in anhydrous DMF
with 0.1 M TBAPF6 as the supporting electrolyte and electro-
chemical data were compared to trypanocidal activity. A detailed
description of the voltammetric behavior of these novel com-
pounds is out of the scope of the present paper. Here we will only
consider some features relevant to the discussion of the correlation
of redox potentials with trypanocidal activity. Several of the iodo-
(4)
(5)
(6)
(7)
3. Conclusions
The present work shows how a complementary combination of
chemical, biological and electrochemical approaches can be used
for the identification of new lead compounds for the treatment of
Chagas disease. We have described potent trypanocidal naph-
thoquinones prepared in high efficiency by versatile synthetic
methods. Compound 59, which was prepared in just two steps, has
an IC50/24 h ¼ 0.19
mM (fifty fold more active than benznidazole)
and SI ¼ 62.5 and is thus an important starting point for the
development of a new drug against T. cruzi. In broader terms, the
present study provides an efficient framework to prepare quinoidal
compounds with potent activity against the etiological agent of
Chagas disease.
Fig. 2. Structures of the naphthoquinones ranked according to data obtained through electrochemical studies.

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
415
Fig. 3. Several inversion potentials in the CV of iodinated quinones 2, 3, 5, 6, 11, 19, 20 and 22 (1 mM) in DMF þ TBAPF6 (0.1 M), glassy carbon electrode, cathodic direction, potential
range: 0.5 V up to ꢁ2.5 V,
n
¼ 0.1 V sꢁ1
.

416
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
Table 3
Major electrochemical parameters of the iodinated quinones (c ¼ 1 mmol Lꢁ1), using cyclic voltammetry, on GCE, in DMF/TBAPF6, 0.1 mol Lꢁ1
,
n
¼ 100 mV sꢁ1
.
Compound
Epc1 (V)
Epc2 (V)
Epc3 (V)
Epa’1 (V)
Epa1 (V)
Epa’2 (V)
Epa2 (V)
2
3
5
6
11
19
20
22
55
ꢁ0.535
ꢁ0.521
ꢁ0.244
ꢁ0.216
ꢁ0.625
ꢁ0.616
ꢁ0.593
ꢁ0.560
ꢁ0.413
ꢁ1.212
ꢁ1.514
ꢁ0.903
ꢁ0.912
ꢁ1.181
ꢁ1.312
ꢁ1.314
ꢁ1.223
ꢁ1.152
ꢁ2.198
ꢁ1.759
e
e
e
e
e
ꢁ0.470
ꢁ0.414
ꢁ0.148
ꢁ0.111
ꢁ0.550
ꢁ0.534
ꢁ0.514
ꢁ0.493
ꢁ0.330
e
e
e
e
ꢁ1.105
ꢁ1.129
ꢁ0.779
ꢁ0.741
ꢁ1.052
ꢁ1.175
ꢁ1.146
ꢁ1.119
ꢁ1.044
ꢁ2.057
ꢁ2.346
e
e
e
e
e
e
ꢁ2.242
ꢁ2.880
e
e
ꢁ0.517
ꢁ1.204
4. Experimental section
4.1. Chemistry
HMBC). Mass spectra were recorded using a Brüker Daltonics FT-
ICRMS Apex 4e 7.0T FT-MS (ESIþ mode) and Shimadzu GCMS
QP2010þ (EIþ mode). Infrared spectra were recorded on a Perkin
Elmer Spectrum One FTIR spectrometer as thin films or solids
compressed on a diamond plate. Data were processed employing
Bruker Data Analysis software version 4.0. Compounds were named
following IUPAC rules as applied by ChemBioDraw Ultra (version 12.0).
General microwave procedure for the CeH iodination of
naphthoquinoidal compounds. In a glovebox, an oven dried re-
sealable reaction tube was charged with the corresponding naph-
thoquinone (0.10 mmol), [RhCp*Cl2]2 (3.75 mol %, 2.3 mg), silver
bis(trifluoromethanesulfonyl)imide (20 mol %, 7.8 mg), N-iodo-
succinimide (100 mol %, 0.10 mmol, 22.5 mg) and anhydrous copper
acetate (100 mol %, 0.10 mmol, 18.2 mg). The tube was removed
from the glovebox and an inert atmosphere was maintained.
Anhydrous CH2Cl2 (1.0 mL) was added via syringe and the tube was
sealed. The mixture was irradiated in a CEM Discover microwave
apparatus in open flask mode (60 W) and the nitrogen flow was
adjusted to maintain a reaction temperature of 45 ꢀC. After cooling,
the reaction mixture was filtered through a pad of celite and
concentrated under reduced pressure. The residue was purified by
FCC, under the conditions noted [16].
4.1.1. General procedures
1,4-Naphthoquinone (1) and 1,4-benzoquinone (35) were pur-
chased from Alfa Aesar and Sigma Aldrich, purified via reduced
pressure sublimation using a cold finger sublimation apparatus
(50 ꢀC, 0.9 mbar) and stored in a glovebox to prevent contact with
moisture. All commercially available naphthoquinones, benzoqui-
nones (compounds 15, 16, 21, 29, 31, 33 and 40) and further com-
mercial chemicals were purchased from Sigma Aldrich, Alfa Aesar,
Strem Chemicals and Santa Cruz Biotechnology. [RhCptCl2]2 was
purchased from Sigma Aldrich.
Substrates 9 and 10, as well as derivatives 13 and 14 were ob-
tained by reaction with iodomethane and Ag2O in CHCl3 [17].
Substrates 17 and 18 obtained by oxidation of corresponding tet-
ralones with 2-iodobenzoic acid and Oxone [18]. Substrate 24 was
synthesized by nitration of 1,4-naphthoquinone followed by
reduction with SnCl2/FeCl3 [33]. Substrate 27 was synthesized
following our latest report [16]. 2,3-Dimethyl-1,4-benzoquinone
(46), 2-methoxy-1,4-benzoquinone (47) and 2,6-dimethoxy-1,4-
benzoquinone (48) were prepared by oxidation of the corre-
sponding hydroquinones (in case of 2,3-dimethyl-1,4-
benzoquinone, the hydroxyl groups of the hydroquinone were
methylated with dimethyl sulfate) with silica supported cerium
ammonium nitrate [34]. 2,3-Dichloro-1,4-benzoquinone (52) was
obtained by reaction of 1,4-benzoquinone (35) with SOCl2/H2SO4
followed by dehydrogenation with Ag2O [35]. All experimental
details for the synthesis of the compounds previously reported are
in the Supporting Information.
Starting materials available from commercial suppliers were
used as received, unless otherwise stated. All reagents requiring
purification were purified using standard laboratory techniques,
according to methods published by Perrin, Armarego, and Perrin
(Pergamon Press, 1966). Catalytic reactions were run under an at-
mosphere of dry nitrogen or argon; glassware, syringes and needles
were either flame dried immediately prior to use or placed in an
oven (200 ꢀC), for at least 2 h, and allowed to cool either in a
desiccator or under an atmosphere of nitrogen or argon; liquid
reagents, solutions or solvents were added via syringe through
rubber septa; solid reagents were added inside a glovebox. Melting
points were obtained on a Thomas Hoover apparatus and are un-
corrected. Column chromatography was performed on silica gel
General procedure for halogenation and phenylselenation of
benzoquinones. In a glovebox, an oven dried re-sealable tube was
charged with the corresponding benzoquinone (0.10 mmol),
[RhCptCl2]2
(3.75
mol%,
2.6
mg),
silver
bis(tri-
fluoromethanesulfonyl)imide (20 mol%, 7.8 mg), the electrophile
source (see below) and anhydrous copper sulfate (220 mol%,
0.22 mmol, 34.9 mg). The tube was removed from the glovebox and
an inert atmosphere was maintained. Anhydrous CH2Cl2 (1 mL) was
added via syringe and tube was sealed. The mixture was heated at
100 ꢀC for 18 h. After cooling, the mixture was filtered through a
pad of celite and purified by FCC, under the conditions noted [20].
4.1.2. 5-Hydroxy-6-iodo-1,4-naphthoquinone (5)
(SiliaFlash G60 UltraPure 60e200 mm, 60 Å). Infrared spectra were
Yield: 88%; m.p. (ꢀC) ¼ 179.8e180.1; red crystals. IR (solid, cmꢁ1
)
recorded on an FTIR Spectrometer IR Prestige-21-Shimadzu. 1H and
13C NMR were recorded at room temperature using a Bruker
AVANCE DRX200 and DRX400 MHz instrument, in the solvents
n
: 3021 (w), 1698 (s), 1270 (m), 830 (m); HRMS (EIþ): 299.9280
[M]þ. Cald. for [C10H5IO3]: 299.9283; 1H NMR (400 MHz, CDCl3)
12.73 (s, O-H), 8.15 (d, J ¼ 8.0 Hz, C7-H), 7.33 (d, J ¼ 8.0 Hz, C8-H),
6.96 (s, C2-H, C3-H); 13C NMR (100 MHz, CDCl3)
: 190.0 (C4), 183.8
d:
indicated. Chemical shifts (d) are given in parts per million (ppm)
and coupling constants (J) in Hertz (Hz). All assignments of NMR
d
(C1), 160.1 (C5), 146.1 (C7), 139.8 (C3), 138.0 (C2), 131.5 (C8a), 120.1
(C8), 114.2 (C4a), 94.9 (C6-I).
spectra were based on 2D NMR data (DEPT-135, COSY, HSQC and

G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
417
4.1.6. 6-Iodo-5-methoxy-2-methyl-1,4-naphthoquinone (14)
Yield: 92%; m.p. (ꢀC) ¼ 117.3e118.1; yellow powder. IR (solid,
cmꢁ1 : 2985 (w), 1689 (s), 1321 (m), 902 (m); HRMS (ESIþ):
) n
328.9656 [MþH]þ. Cald. for [C12H10IO3]: 328.9669; 1H NMR
(400 MHz, CDCl3)
d
: 8.19 (d, J ¼ 8.2 Hz, C7-H), 7.65 (d, J ¼ 8.2 Hz, C8-
H), 6.76 (s, C3-H), 3.91 (s, C10-H3), 2.17 (s, C9-H3). 13C NMR
(100 MHz, CDCl3) : 185.0 (C1), 183.3 (C4), 158.9 (C5), 146.3 (C2),
d
144.3 (C7), 136.9 (C3),134.4 (C8a), 124.4 (C8),124.3 (C4a), 103.8 (C6-
I), 61.8 (C10), 15.9 (C9).
4.1.3. 5-Hydroxy-6,8-diiodo1,4-naphthoquinone (6)
Yield: 82%; m.p. (ꢀC) ¼ 164.3e165.8; purple crystals. IR (solid,
cmꢁ1 : 3002 (w), 1613 (s), 1211 (m), 911 (m); HRMS (EIþ):
) n
425.8241 [M]þ. Cald. for [C10H4I2O3]: 425.8250; 1H NMR (500 MHz,
CDCl3)
d
: 13.54 (s, O-H), 8.80 (s, C7-H), 7.04 (d, J ¼ 10.2 Hz, C2-H),
6.96 (d, J ¼ 10.2 Hz, C3-H); 13C NMR (125 MHz, CDCl3)
d: 189.2 (C4),
182.0 (C1), 161.7 (C5), 158.5 (C7), 140.4 (C2), 136.5 (C3), 130.0 (C8a),
115.8 (C4a), 96.0 (C6-I), 83.0 (C8-I).
4.1.7. 5-Amino-6-iodo-1,4-naphthoquinone (25)
Yield: 41%; m.p. (ꢀC) ¼ 181.2e182.9, purple powder. IR (solid,
cmꢁ1 : 3078 (w), 1720 (s), 1380 (m), 923 (m); HRMS (EIþ):
) n
298.9433 [M]þ. Cald. for [C10H6INO2]: 298.9443; 1H NMR (400 MHz,
CDCl3)
d
: 8.03 (d, J ¼ 8.0 Hz, C7-H), 7.15 (d, J ¼ 8.0 Hz, C8-H), 6.90 (d,
J ¼ 10.3 Hz, C3-H), 6.86 (d, J ¼ 10.3 Hz, C2-H); 13C NMR (100 MHz,
CDCl3) d: 186.7 (C4), 185.0 (C1), 149.2 (C5), 144.8 (C7), 140.1 (C3),
137.2 (C2), 132.9 (C8a), 117.5 (C8), 111.9 (C4a), 95.1 (C6-I).
4.1.4. 5-Hydroxy-6-iodo-2-methyl-1,4-naphthoquinone (8)
Yield: 85%; m.p. (ꢀC) ¼ 125.1e125.9; red crystals. IR (solid, cmꢁ1
)
n
: 3048 (w), 1687 (s), 1198 (m), 854 (m); HRMS (ESIþ): 314.9431
[MþH]þ. Cald. for [C11H8IO3]: 314.9513; 1H NMR (400 MHz, CDCl3)
d
: 12.82 (s, O-H), 8.11 (d, J ¼ 7.9 Hz, C7-H), 7.35 (d, J ¼ 7.9 Hz, C8-H),
6.81 (s, C3-H), 2.19 (s, C9-H3). 13C NMR (100 MHz, CDCl3)
d: 189.9
(C4), 184.3 (C1), 159.9 (C5-OH), 150.0 (C2), 145.6 (C7), 134.8 (C3),
131.9 (C8a), 120.3 (C8), 114.4 (C4a), 94.4 (C6-I), 16.5 (C9).
4.1.8. 2,6-Diiodo-3,5-dimethoxy-1,4-benzoquinone (51)
Yield: 91%; m.p. (ꢀC) ¼ 160.2e160.9; red crystals. IR (solid, cmꢁ1
)
n
: 2947 (w), 1667 (s), 1266 (s), 924 (s); HRMS (EIþ): 261.9493 [M]þ.
Cald. for [C8H6I2O4]: 419.8355; 1H NMR (500 MHz, CDCl3)
d: 4.16 (s,
C7-H6); 13C NMR (125 MHz, CDCl3)
(C2, C6), 98.1 (C3, C5), 61.6 (C7).
d: 175.2 (C1), 171.2 (C4), 160.4
4.1.5. 6-Iodo-5-methoxy-1,4-naphthoquinone (13)
Yield: 91%; m.p. (ꢀC) ¼ 152.3e152.8; yellow powder. IR (solid,
cmꢁ1 : 3014 (w), 1699 (s), 1199 (m), 867 (m); HRMS (ESIþ):
) n
336.9329 [MþNa]þ. Cald. for [C11H7IO3Na]: 336.9332; 1H NMR
(400 MHz, CDCl3)
d
: 8.24 (d, J ¼ 8.2 Hz, C7-H), 7.65 (d, J ¼ 8.2 Hz, C8-
H), 6.95 (d, J ¼ 10.3 Hz, C2-H), 6.90 (d, J ¼ 10.3 Hz, C3-H), 3.93 (s, C9-
H3). 13C NMR (100 MHz, CDCl3)
d
: 184.4 (C1), 183.3 (C4), 159.2 (C5),
144.7 (C7),139.9 (C2),136.9 (C3),134.1 (C8a),124.2 (C8),124.1 (C4a),
104.2 (C6-I), 61.8 (C9).
4.1.9. (E)-5-Styrylnaphthalene-1,4-dione (59)
Yield: 51%; m.p.(ꢀC): 140.8e141.9; red solid. IR (solid, cmꢁ1
) n:
2956 (w), 1717 (s), 1632 (s), 1278 (m), 1100 (m), 790 (m); HRMS
(EIþ): 260.0822. Cald. for [C18H12O2]þ: 260.0837; 1H NMR
(500 MHz, CDCl3)
d
: 8.30 (d, J ¼ 16.1 Hz, C9-H), 8.10 (dt, J ¼ 7.6,
1.2 Hz, C8-H), 7.98 (dd, J ¼ 7.9, 0.8 Hz, C6-H), 7.72 (t, J ¼ 7.8 Hz, C7-
H), 7.62e7.60 (m, 2H), 7.43e7.37 (m, 2H), 7.35e7.29 (m, 1H), 7.06 (d,
J ¼ 16.2 Hz, C10-H), 6.95 (d, J ¼ 1.0 Hz, C2-H, C3-H). 13C NMR
(125 MHz, CDCl3) d: 187.0 (C4), 185.2 (C1), 140.5 (C2), 140.2 (C5),

418
G.A.M. Jardim et al. / European Journal of Medicinal Chemistry 136 (2017) 406e419
137.1 (C8a),136.9 (C3),133.5 (C10),133.5 (C6),133.3 (C7),128.8 (C13,
C15), 128.3 (C10, C14), 127.9 (C4a), 127.7 (C9), 127.1 (C12, C16), 126.5
(C8).
(Invitrogen) was added to complete the final volume of 100 mL.
Thus, the plate was incubated for 2 h and the measurement was
performed at 560 and 590 nm, as recommended by the manufac-
turer. The results were expressed as the difference in the percent-
age of reduction between treated and untreated cells being the
LC50 value, corresponding to the concentration that leads to
damage of 50% of the mammalian cells [28].
4.2. Crystallographic data
X-ray diffraction data collection for three compounds were
performed on an Enraf-Nonius Kappa-CCD diffractometer (95 mm
CCD camera on
MoK_radiation (0.71073 Å), at room temperature. Data collection
was carried out using the COLLECT software [36] up to 50ꢀ in 2
k-goniostat) using graphite monochromated
4.6. Electrochemical studies
q
.
Cyclic voltammetry (CV) experiments were performed with a
conventional three electrode cell in an Autolab PGSTAT-30 poten-
tiostat (Echo Chemie, Utrecht, the Netherlands) coupled to a PC
microcomputer, using GPES 4.9 software. The working electrode
was a glassy carbon (GC) BAS (d ¼ 3 mm), the counter electrode was
a Pt wire and the reference electrode an AgjAgCl, Clꢁ (saturated), all
contained in a one-compartment electrochemical cell with a
volumetric capacity of 5 mL. The GC electrode was cleaned up by
polishing with alumina on a polishing felt (BAS polishing kit). The
solvent used in aprotic media studies was extra dry N,N-
dimethylformamide (99.8%) acquired from Acros Organics. In CV
experiments, the scan rate varied from 10 to 1000 mV sꢁ1. Elec-
trochemical reduction and oxidation were performed in aprotic
media (DMF þ TBAPF6 0.1 mol Lꢁ1) at room temperature (25 2 ꢀC).
Each compound (1 ꢄ 10ꢁ3 mol Lꢁ1) was added to the supporting
electrolyte and the solution was deoxygenated with argon before
the measurements by cyclic voltammetry, in different potential
intervals.
Integration and scaling of the reflections, correction for Lorentz and
polarization effects were performed with the HKL DENZO-
SCALEPACK system of programs [37]. The structure of the com-
pounds was solved by direct methods with SHELXS-97 [38]. The
models were refined by full-matrix least squares on F2 using the
SHELXL-97 [39]. The program ORTEP-3 [40] was used for graphic
representation and the program WINGX [41] to prepare materials
for publication. All H atoms were located by geometric consider-
ations (C-H ¼ 0.93e0.97; O-H ¼ 0.82 Å) and refined as riding with
Uiso(H) ¼ 1.5Ueq (C-methyl) or 1.2Ueq (other). Crystallographic
data for the structures were deposited in the Cambridge Crystal-
lographic Data Centre, with numbers CCDC 1526294, 1526293 and
1526364.
4.3. Animals
Albino Swiss mice were employed for the trypanocidal and
ꢀ
cytotoxicity assays, in accordance to the guidelines of the Colegio
~
Brasileiro de Experimentaçao Animal (COBEA), and these were
Notes
performed under biosafety conditions. All animal experimentation
procedures were approved by the Comissao de Etica em
ꢀ
~
The authors declare no competing financial interest.
~
Experimentaçao Animal (CEUA/Fiocruz), license LW 16/13.
Acknowledgements
4.4. Trypanocidal assay
This research was funded by grants from CNPq (PVE 401193/
2014-4, PQ 305385/2014-3, Universal/CNPq 474797/2013-9 and PQ
SR Proc. 302613/2013-7), Chamada Universal MCTI/CNPq Nꢀ 01/
The experiments were performed with the Y strain of T. cruzi
[42]. Stock solutions of the compounds were prepared in dime-
thylsulfoxide (DMSO), with the final concentration of the latter in
the experiments never exceeding 0.1%. Preliminary experiments
showed that at concentrations of up to 0.5%, DMSO has no delete-
rious effect on the parasites [43]. Bloodstream trypomastigotes
were obtained from infected Albino Swiss mice at the peak of
parasitemia by differential centrifugation. The parasites were
resuspended to a concentration of 10 ꢄ 106 cells/mL in DMES
~
2016, Fundaçao Oswaldo Cruz, FAPERJ (CNE Proc. E-26/203.083/
2016), FAPEMIG (Edital 01/2014 APQ-02478-14 and Programa
Pesquisador Mineiro, PPM-X), FAPEAL, CAPES/PROCAD and INCT-
ꢀ
Catalise. J.F. Bower is indebted to the Royal Society for a University
Research Fellowship. E.N. da Silva Júnior also thanks the Royal So-
ciety of Chemistry for a JWT Jones Travelling Fellowship and CAPES/
DAAD for exchange PROBRAL program (99999.008126/2015-01).
Prof. CAS would like to thank the Institute of Physics of University of
medium. This suspension (100 mL) was added to the same volume
of each of the compounds, which had been previously prepared at
twice the desired final concentrations. The incubation was per-
formed in 96-well microplates (Nunc Inc., Rochester, USA) at 37 ꢀC
for 24 h. Benznidazole (Lafepe, Brazil), the standard drug for
treatment of chagasic patients, was used as control. Cell counts
were performed in a Neubauer chamber, and the activity of the
compounds corresponding to the concentration that led to 50%
lysis of the parasites was expressed as the IC50/24 h.
~
~
Sao Paulo (Sao Carlos), for kindly allowing the use of the KappaCCD
diffractometer.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.011.
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