Synthesis of quinone imine and sulphur-containing compounds with antitumor and trypanocidal activities: Redox and biological implications

Article abstract of DOI:10.1039/d0md00072h

Ortho-Quinones represent a special class of redox active compounds associated with a spectrum of pronounced biological activities, including selective cytotoxicity and antimicrobial actions. The modification of the quinone ring by simple nitrogen and sulphur substitutions leads to several new classes of compounds with their own, distinct redox behaviour and equally distinct activities against cancer cell lines and Trypanosoma cruzi. Some of the compounds investigated show activity against T. cruzi at concentrations of 24.3 and 65.6 μM with a selectivity index of around 1. These results demonstrate that simple chemical modifications on the ortho-quinone ring system, in particular, by heteroatoms such as nitrogen and sulphur, transform these simple redox molecules into powerful cytotoxic agents with considerable "potential", not only in synthesis and electrochemistry, but also, in a broader sense, in health sciences. This journal is

Full text of DOI:10.1039/d0md00072h

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RESEARCH ARTICLE
Synthesis of quinone imine and sulphur-
containing compounds with antitumor and
trypanocidal activities: redox and biological
implications†
Cite this: DOI: 10.1039/
d0md00072h
Renata G. Almeida, ‡a Wagner O. Valença,
‡
ab Luísa G. Rosa, a Carlos A. de Simone,c
e
Solange L. de Castro, d Juliana M. C. Barbosa, d Daniel P. Pinheiro,
Carlos R. K. Paier, e Guilherme G. C. de Carvalho,e Claudia Pessoa,
e
Marilia O. F. Goulart, f Ammar Kharma ag and Eufrânio N. da Silva Júnior
a
*
Ortho-Quinones represent a special class of redox active compounds associated with a spectrum of
pronounced biological activities, including selective cytotoxicity and antimicrobial actions. The modification
of the quinone ring by simple nitrogen and sulphur substitutions leads to several new classes of compounds
with their own, distinct redox behaviour and equally distinct activities against cancer cell lines and
Trypanosoma cruzi. Some of the compounds investigated show activity against T. cruzi at concentrations of
24.3 and 65.6 μM with a selectivity index of around 1. These results demonstrate that simple chemical
modifications on the ortho-quinone ring system, in particular, by heteroatoms such as nitrogen and sulphur,
transform these simple redox molecules into powerful cytotoxic agents with considerable “potential”, not
only in synthesis and electrochemistry, but also, in a broader sense, in health sciences.
Received 3rd March 2020,
Accepted 8th June 2020
DOI: 10.1039/d0md00072h
rsc.li/medchem
worldwide.1 At the same time, an increased global population
is also more susceptible to the outbreak and spread of
1. Introduction
The rapid expansion and simultaneous ageing of populations
around the globe represent a demographical change and a
challenge that, among many other issues, such as shortage of
nutrition and increase of pollution, is also associated with
higher incidence of new cases of cancer and related deaths.
The projection for the year 2030 estimates 27 million new
cases of cancer with 17 million cancer-related deaths
infectious diseases, such as Chagas disease. This disease is
caused by the protozoan Trypanosoma cruzi, which is
considered by the World Health Organization (WHO) as one
of the twenty neglected tropical diseases, affecting more than
5 million people worldwide. Its current chemotherapy is still
restricted to the nitroderivatives benznidazole and nifurtimox,
available for half a century, which present limited activity and
severe adverse effects.2–8 These demographical developments
and new ways of transmission, combined with the emergence
of drug resistance, require new and effective drugs for the
treatment of such diseases, i.e. agents which may be effective
as (cyto)toxins and also selective for their targets.
a Institute of Exact Sciences, Department of Chemistry, Federal University of Minas
Gerais, Belo Horizonte, 31270-901, MG, Brazil. E-mail: eufranio@ufmg.br
b Center for the Development of Chemical Technologies, State University of Mato
Grosso do Sul, Naviraí, 79950-000, MS, Brazil
Within this context, naphthoquinoidal compounds have
been studied widely.9–13 The derivatization of naphthoquinones
has been a subject of considerable interest among medicinal
c Department of Physics and Informatics, Institute of Physics, University of São
Paulo, São Carlos, 13560-160, SP, Brazil
d Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, 21045-900, RJ, Brazil
e Department of Physiology and Pharmacology, Federal University of Ceará,
Fortaleza, CE, 60430-270, Brazil
chemists and
a wide variety of natural and synthetic
naphthoquinones have already been reported as potent
trypanocidal and anticancer agents.14–17 Mechanistically, the
quinone core may undergo one-electron reduction under
aerobic conditions to form a semiquinone radical which will
redox cycle to release reactive oxygen species (ROS).10,18–20
Within the context of cancer, the cytotoxicity of quinones is
therefore mainly associated with the catalytic generation of ROS
and the alkylation of crucial proteins and nucleic acids, both
f Institute of Chemistry and Biotechnology, Federal University of Alagoas, CEP
57072-970, Maceió, AL, Brazil
g Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland, D-
66123 Saarbruecken, Germany
† Electronic supplementary information (ESI) available.
CCDC 1928261,
1928296, 1928326, 1928361, 1928382 and 1928407. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/d0md00072h
‡ These authors contributed equally to this work.
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processes, which provoke cell damage.14–16 Similar
considerations also apply to pathogenic microorganisms, some
of which are affected considerably by – similar – redox
modulating agents thanks to their weak(er) cellular antioxidant
defence systems. Indeed, the concept of catalytic “sensor/
effector” agents, i.e. “smart” redox molecules responding to
specific intracellular redox signatures and peculiarities, and
thereby combining considerable activity with selectivity, has
been evaluated for over a decade now, often with amazing
results.21–23
Whilst initial studies on such catalytic agents have employed
comparably simple redox agents, subsequent ones have
developed hybrid molecules with two or more redox sites
integrated into one molecule. The hybridization of a triazole
nucleus with β-lapachone, for instance, has already resulted in a
derivative with high antitumor activity (IC50 < 2 μM) for different
cancer lineages and, more recently, we have also reported the
antitumor activity of selenium-containing quinone-based
triazoles, which were inspired by earlier quinone–chalcogen
structures tested more than a decade ago (Scheme 1A).21,27–29
Quinone-based N-sulfonyl-1,2,3-triazoles were also prepared by
our group using click chemistry reactions (Scheme 1B).30 In this
For almost twenty years, our groups have been involved in
the synthesis and biological evaluation of naphthoquinones.24–26
Scheme 1 A schematic overview of the strategies employed in the preparation of bioactive compounds and transformation of ortho-quinones
into para-quinone imines and sulphur containing compounds.
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work, we employed as a strategy the insertion of the 1,2,3-
triazolic nucleus associated with a chalcogen atom, allowing the
formation of a redox active system. The compounds were
subsequently evaluated against eight types of cancer cell lines
and some derivatives exhibited potent antitumor activity.30
Interestingly, the application of such “smart” catalytic agents is
not limited to cancer research, and a pronounced – and selective
– cytotoxicity also affects protozoan pathogens, such as T. cruzi,
with some molecules exhibiting trypanocidal activity about two
or six times higher than that of the standard drug
benznidazole.31–35
Interestingly, the ortho-quinone redox centre found in
β-lapachone is prone to chemical modifications which impact
considerably the redox behaviour of these agents.10 Here, we
have investigated modifications with nitrogen and sulphur
substituents, as both heteroatoms are redox active and, besides
electronic effects, promise a major “transformation” of the
original redox system into considerably more active species
(Scheme 1C). We are now able to report the synthesis,
electrochemistry, anticancer and trypanocidal evaluation of
quinoidal derivatives containing alkynes and N-sulfonyl
triazoles and propose a distinct relationship between the
chemical structure and redox system, and their electrochemical
potentials and biological activities.
prepared arylamino naphthoquinones (2a–h) by the reaction
of 1 and the respective anilines following a procedure
published previously in the literature with minor
modifications.36–38 In general, the desired products were
obtained in moderate to excellent yields (Scheme 2). It should
be noted from the outset that these compounds now possess
more complicated redox cores spanning the original
ortho-quinone and the amine functionality in the
para-position.
With the quinoidal compounds 2a–h available, the
changed “redox core” was modified further, this time by
alkylation of one of the oxygen atoms. Notably, this kind of
alkylation not only simply “blocks” one of the oxygen atoms
of the ortho-quinone, it also transforms the electronic
structure of the entire ring system by engaging the nitrogen
in the para-position in the form of a distinctive para-quinone
imine redox core (Scheme 3).39,40 The consequences of this
apparently innocent O-alkylation for the redox and biological
activity will be discussed later. Eight alkyne derivatives 3a–h
were synthesized in moderate to excellent yields ranging from
48% to 99% according to Scheme 3 and in the presence of
DMF as a solvent, excess K2CO3 as a base, and propargyl
bromide.
Alkynes derived from ortho-naphthoquinones represent
important intermediates for the synthesis of 1,2,3-triazole
derivatives, and such molecules can be synthesized easily by
means of a 1,3-dipolar cycloaddition reaction between an
azide and alkyne using copperIJI) as a catalyst, in a process
known as a click chemistry reaction (Scheme 4). This type of
reaction is considered an important tool to synthesize hybrid
molecules with two or more redox centres and pronounced
2. Results and discussion
2.1. Chemistry
The first class of compounds with a nitrogen substituent at
the ring was prepared from commercially available 1,2-
naphthoquinone-4-sulfonic acid sodium salt (1). Initially, we
Scheme 2 Synthesis of arylamino ortho-naphthoquinones 2a–h.
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Scheme 3 Synthesis of alkynes derived from arylamino ortho-naphthoquinones 3a–h.
Scheme 4 Synthesis of N-sulfonyl-1,2,3-triazoles 4a–h.
biological activities.41 In this context, the synthesis of the
N-sulfonyl-1,2,3-triazoles was accomplished according to the
methodology described by Fokin et al., employing copperIJI)-
thiophene-2-carboxylate (CuTC) as a catalyst, as this catalyst
promises high yields and selective formation of the 1,4-
regioisomer of the product.42 The reaction was carried
employing the corresponding alkynes (3a–h) (Scheme 3) with
an excess of tosyl azide in toluene, as a solvent, and at room
temperature according to Scheme 4. In general, novel
compounds were obtained in moderate to excellent (50–95%)
yields.
Nitrogen is not the only nucleophile able to replace the
sulfonate group in compound 1. Sulphur has a similar
nucleophilic character and is also redox active. Indeed, recent
studies have shown that naphthoquinone compounds
substituted with chalcogen atoms often exhibit considerable
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biological activity.17,43 Whilst these studies have focused
primarily on para-quinones, compound 1 provides the basis
for a series of similar ortho-quinones. For this synthesis, a
methodology adapted from the literature36,37 yielded
compounds 5a–5d in low to moderate yields (30–40%)
(Scheme 5).
reversible and irreversible reduction and oxidation processes
associated with such molecules. CV was performed in mixed
medium, i.e. phosphate buffer + 30% methanol, on a glassy
carbon working electrode, with E vs. Ag/AgCl as a reference
electrode (SSE), at 200 mV s−1. In the presence of a protic
organic solvent (methanol), the reduction occurs in two
monoelectronic steps, different from the mechanism in
totally aqueous medium, where the reduction occurs through
the capture of 2e− + 2H+.
The cyclic voltammograms of selected compounds
belonging to class 1 (compounds 1 and 2), class 3 (compound
3a) and class 5 (compound 5b) were obtained and are shown
in Fig. S39 (see the ESI† file), together with the values for the
relevant reduction signals (Epc) and corresponding oxidation
(Epa) ones for various quinones, in Table 1. We have focused
here on the prime signals associated with the quinone redox
centre, as these waves are important to rationalize the
relevant biological activities (see below).
The structures of the novel compounds were determined
initially by 1H and 13C NMR, and were corroborated further
using electrospray ionization mass spectra. In the case of
compounds 3a, 3d, 3f, 3g, 3h and 5d, X-ray crystallographic
analysis with suitable crystals obtained by the slow
evaporation method was performed. Here, the bond lengths
and angles are in good agreement with the expected values
reported in the literature.44 The atoms of the
naphthoquinonic ring (C1–C10) of all structures are coplanar
and the largest deviation from the least-squares plane for
each one is: 0.077(3) Å for atom C1 in 3a; 0.044(3) Å for atom
C5 in 3d; 0.035(2) Å for atom C1 in 3f; 0.039(2) Å for atom C5
in 3g; 0.039(2) Å for atom C8 in 3h and 0.017(4) Å for atom C5
in 5d. The dihedral angles between the planes of the rings
(C1–C10) and (C11–C12) are: 48.5IJ3)° for 3a; 57.4IJ2)° for 3d;
86.9IJ3)° for 3g; 57.1IJ4)° for 3h; 61.01° for 3f and 86.9° for 5d.
ORTEP-3 diagrams of each molecule are shown in Fig. 1.
By analysis of the first reduction potential (Epc1), the ease
of reduction is the following: 5c > 5b > 1 ≈ 5d > 3e > 3h ≈
3b > 3a > 2a.
As anticipated, the redox behaviour changes significantly,
once the original ortho-quinone 1 is modified by a nitrogen
substituent in the para-position to yield compounds of class
2, due to the electron donating character of the amino group.
Interestingly, the subsequent O-alkylation converting 2 into 3
also – quite dramatically – changes the entire redox system,
since there is a direct modification on the redox system. This
significant change from an ortho-quinone to a para-quinone
imine is also observed electrochemically, and is associated
with a shift of the first reduction potentials to more negative
potentials. Generally, the potentials for the para-quinone
imine are 150 to 200 mV more negative, when compared to
2.2. Redox transformations
The process of substituting the sulfonate group in compound
1 for the nitrogen or sulphur in 2 and 5, respectively, and
also by O-alkylation when moving from 2 to 3 and 4, changes
the “redox core” of these molecules, as an additional redox
active element is added to the initial ortho-quinone system.
Some of the resulting redox systems therefore appear to differ
significantly from the original structures, and these
differences should also be reflected in the redox behaviour
and biological activities associated with them. As such, cyclic
voltammetry (CV) was employed to study redox changes, as it
represents a readily applicable, fast, informative and also
fairly reliable method to obtain initial information about
those for the ortho-quinones, pointing towards
electrophilic compound, being reduced at more negative
potentials. Modifying the compounds of class (less
a less
2
electrophilic) to yield class 3 brings an anodic shift of the
relevant potentials, however less intense, when compared to
Scheme 5 Synthesis of chalcogenic aryl ortho-quinones 5a–d.
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Fig. 1 ORTEP-3 projections of 3a, 3d, 3f, 3g, 3h and 5d, showing the atom-numbering and displacement ellipsoids at the 50% probability level.
the original compound 1, and introducing another factor
involved in the redox cycle with the quinone imines, once
electrochemically, and is associated with a shift of the
relevant potentials to more positive potentials. Generally, the
potentials for the chalcogenic quinone are 20 to 100 mV
more positive, when compared to those for the
ortho-quinone, pointing towards a more oxidizing species.
Hence, its influence on the overall redox behaviour of these
compounds is primarily due to electronic effects.
they are known to undergo
a
redox cycle through
aminophenols. Another important fact should be considered:
arylquinone imines can suffer from hydrolysis, to generate
back the original quinone,45 possibly behaving as a pro-
drug.40
In contrast, the substitution of the initial “redox core”
with a sulfur atom seems to change the original redox
properties to
a
quasi-reversible electron transfer with
2.3. Antiparasitic activity
potentials in the range of −168 mV up to −254 mV (Epc1) and
from −500 mV up to −574 mV (Epc2). A significant change
from an ortho-quinone to a chalcogenic quinone is observed
Based on the strategy of modifying and hence modulating
the initial “redox core” of the ortho-quinone, and the insights
obtained by electrochemistry, it was then interesting to see if
these modifications also translate into specific – changes in –
biological activities. Here, two distinct models indicative of
the activity of quinones have been selected, i.e. cancer cell
lines and T. cruzi (see section 1 Introduction).
Table 1 Reduction potential values (Epc1 and Epc2) and corresponding
oxidation potentials (Epa1 and Epa2), representing three different classes of
compounds and their synthetic precursor (1)
The selection of these models was based on studies
previously described in the literature that demonstrate the
antitumor and trypanocidal activity of quinoidal compounds
containing the 1,2,3-triazole nucleus and the sulphur
atom.27,29,30 As discussed in the Introduction, our research
group demonstrated that quinone-based N-sulfonyl-1,2,3-
triazoles exhibited cytotoxicity against various tumour cell lines.
These compounds were not evaluated against T. cruzi, the
parasite that causes Chagas disease, but it is well documented
that active redox systems are potential candidates to present
1st wave
2nd wave
Compounds
Epc1
Epa1
Epc2
Epa2
1
−250 mV
−490 mV
−429 mV
−387 mV
−339 mV
−384 mV
−210 mV
−168 mV
−254 mV
−340 mV
−420 mV
−493 mV
−475 mV
−415 mV
−414 mV
−304 mV
−324 mV
−360 mV
−602 mV
−716 mV
−669 mV
−687 mV
−690 mV
−614 mV
−552 mV
−500 mV
−574 mV
−28 mV
2a
3a
3b
3e
3h
5b
5c
5d
−202 mV
−259 mV
−292 mV
−235 mV
−190 mV
−132 mV
−110 mV
−178 mV
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Table 2 Activity of the naphthoquinones on trypomastigote forms of T.
between 1.3 μM for compound 5a and 3.9 μM for compound
5d. The SI was also improved, with values above 10 observed
for virtually all compounds of class 5 under investigation.
When compared to the standard drug benznidazole, which
under these experimental conditions shows an IC50 of 9.7
μM, the quinones 5a–5d were even more active. Nonetheless,
the selectivity for the parasite is notoriously low for the
quinones when compared to benznidazole (SI > 413.2),
regardless of the assay conditions.
The sulphur-containing quinones 5a–5d also exhibited
some activity against macrophages with LC50 values between
36.9 and 81.4 μM. The quinones 5a–5d were, however, more
cytotoxic when compared to the standard drug benznidazole,
cruzia
Compounds
IC50/24 h (μM)
LC50/24 hb (μM)
SI
4a
4b
4c
4d
4e
4f
>500
>500
>500
>500
>500
>500
80.8 6.5
44.3 6.4
88.5 3.5
26.9 1.2
>100
>6.19
>11.29
>5.65
>18.59
nd
>100
nd
4g
4h
5a
5b
5c
5d
Bz
>500
>500
>100
nd
>12.31
1.52
0.92
1.03
40.6 2.5
36.9 2.6
81.4 3.7
67.3 6.9
41.5 0.2
>4000
24.3 7.9
88.7 10.1
65.6 10.2
112.0 10.6
103.6 0.6
0.37
>38.6
which under these experimental conditions shows LC50
>
4000 μM, and it is worth noting here the correlation between
the observed activity and the recorded electrochemical
potentials for the chosen compounds.
a
Mean SD of at least three independent experiments, 5% blood at
b
4 °C. Cytotoxicity assays were performed using primary cultures of
peritoneal macrophages obtained from Swiss Webster mice. nd: not
determined. Bz: benznidazole. SI = selectivity index, represented by
the ratio LC50/IC50
.
2.4. Cytotoxic activity
In the case of the cancer cell lines, cytotoxicity was evaluated
trypanocidal activity, since in general such activity may be
intrinsically related to the generation of ROS.
in
vitro
by
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay
representative of six cancer cell lines, i.e. colon cancer cells (HCT-
116), lung cancer cells (NCI-H460), prostate cancer cells (PC3),
human promyelocytic leukemia cells (HL-60), human
immortalized myelogenous leukemia cells (K-562) and multidrug
resistant leukemia cells (Lucena 1) (Table 4). The murine, non-
tumorigenic fibroblast immortalized cell line (L929) was
employed as a control to evaluate the selectivity index of the
compounds for tumour cells, once its IC50 is compared to the
corresponding values of neoplastic cell lines through selectivity
index calculation. Doxorubicin was employed as a positive control
and benchmark drug. The compounds were classified according
to their activity as highly active (IC50 < 2 μM), moderately active
(2 μM < IC50 < 10 μM), or inactive (IC50 > 10 μM).48
As expected and reflected in Table 4, the redox active
quinones exhibit significant cytotoxicity against a range of
cancer cell lines, with IC50 values for some compounds in the
low micromolar range. For instance, some alkynes of class 3
are moderately active and generally present a non-selective
cytotoxicity against the tumour cells evaluated. Compound 3d
is an exception, as it is particularly active and selective
against HL-60 cells, with an IC50 of 9.20 μM (Table 4) and a
Whilst none of the N-sulfonyl triazoles 4a–4h appears to
be particularly active against T. cruzi, with IC50 values higher
than 500 μM, the sulphur-containing quinones 5a–5d exhibit
rather promising IC50 values between 24.3 and 112.0 μM
(Table 2). It therefore appears that this particular
microorganism is sensitive towards the substituted
ortho-quinone series, and less so towards the quinone
imines. From the perspective of redox chemistry, this may be
explained by the fact that the ortho-quinones with their more
positive electrochemical potentials are also generally more
oxidizing and hence toxic, whilst the quinone imines, with
their somewhat more negative potentials, are less oxidizing.
Nonetheless, practical applications of such compounds may
be compromised by a wider toxicity, as the selectivity index
(SI) is rather low, with values ranging from 0.37 for
compound 5d to 1.52 for compound 5a.
It is already established in the literature that
naphthoquinones are inactivated in the presence of certain
components present in human blood.46,47 Compounds 5a–5d
were therefore also assayed in the absence of blood at 37 °C
(Table 3).46 These “blood-free” assays resulted in IC50 values
Table 3 Activity of the synthetic derivatives against bloodstream trypomastigotes of T. cruzi (Y strain) at 37 °C in the absence of blooda
Compounds
IC50/24 h (μM) at 37 °C, absence of blood
LC50/24 hb (μM) macrophages
SI at 37 °C, absence of blood
5a
5b
5c
5d
Bz
1.3 0.2
3.4 1.0
4.6 1.1
3.9 1.4
9.7 2.4
36.9 2.6
81.4 3.7
67.3 6.9
41.5 0.2
>4000
28.38
23.94
14.63
10.64
>413.2
a
b
Mean SD of at least three independent experiments, absence of blood at 37 °C. Cytotoxicity assays were performed using primary cultures
of peritoneal macrophages obtained from Swiss Webster mice. nd: not determined. Bz: benznidazole. SI = selectivity index, represented by the
ratio LC50/IC50
.
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Table 5 Selectivity index for the most active compoundsa
Compounds
HCT116
NCI-H460
PC3
HL-60
K-562
Lucena I
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
1.2
2.9
1.6
1.8
1.6
1.2
1.5
1.0
3.8
2.9
4.9
3.5
2.0
1.9
1.0
1.7
2.2
1.3
1.8
1.0
1.6
3.3
2.0
1.9
1.9
1.3
1.0
1.0
2.9
2.6
2.2
2.7
1.6
1.9
1.0
0.7
0.7
0.8
1.0
1.0
2.5
3.6
1.8
1.0
2.1
1.7
1.0
24
1.0
1.0
6.7
9.8
1.0
5.2
1.0
2.7
0.7
0.8
1.0
1.0
2.0
4.4
2.8
3.6
2.8
1.9
2.0
1.1
3.1
3.3
3.3
2.6
2.8
4.2
2.6
2.0
4.6
6.6
6.6
3.6
1.0
1.0
1.5
2.4
2.4
1.2
1.4
1.0
1.7
1.0
2.9
2.6
2.0
3.3
1.5
1.9
1.8
1.8
2.2
2.5
1.5
2.8
2.4
1.9
1.9
2.2
1.6
1.9
5.6
5.4
12.3
3.0
6.1
5.3
5.6
1.7
1.8
2.1
2.8
1.0
a
Selectivity index, represented by the ratio of cytotoxicities between normal cells and different lines of cancer cells.
selectivity index (SI) superior to 3.6 (Table 5). Compound 3b
presents a broader cytotoxicity against NCI-H460 (IC50 = 9.45
μM), PC-3 (IC50 = 8.82 μM) and HL-60 (IC50 = 7.18 μM) cells
with a SI higher than 1.8. Generally, the members of class 4
exhibit a more selective cytotoxic activity against all the cell
lines, with IC50 values ranging from 1.44 to > 20 μM. For
example, 4d presents IC50 values between 2.03 and 7.70 μM
(Table 4), with a SI higher than 2.6 (Table 5), while 4f
presents an IC50 from 3.32 to 9.41 μM (Table 4) and a SI
higher than 1.9 (Table 5). Interestingly, the only difference
between these molecules is the deactivating halogen
substituent in the benzene ring, –F in 4d and –Br in 4f,
suggesting that the more electronegative the halogen
substituent, the more cytotoxic the compound. This pattern
is followed by 4g, which possesses an –I substituent and is a
Fig. 2 Effect of compounds 3e, 4f and 5a on ROS production in
less cytotoxic compound against the cell lines tested, except
for a particularly high selectivity for Lucena 1, i.e. IC50 of 2.90
μM and SI higher than 5.6 (Table 4).
K-562 cells determined by flow cytometry using CM-H2DCFDA, after 1
and
3 hours incubation. Menadione (MEN, 20 μM) and hydrogen
peroxide (H2O2, 150 μM) were used as positive controls. A total of
10 000 events were analyzed per sample. Data are expressed as mean
SEM from three independent experiments. *, p < 0.05 compared to
the negative control by ANOVA followed by Tukey's test, performed
using GraphPad Prism 8.
Notable differences can be observed in the SI values shown in
Table 5. The sulphur containing quinones seem to be fairly
selective against certain cancer cell lines, such as HL-60, as
observed in the case of compounds 5b and 5c. Indeed,
compounds of class 5 are particularly attractive as far as observed
activity and selectivity are considered, reflecting the potential of
the redox core in concert with the sulfur atom added to this
analogue. Compounds 5b and 5c present IC50 values of 4.14 μM
and 5.42 μM against HL-60, respectively, with an SI of 6.6.
In general, generation of intracellular ROS is a key
mechanism associated with the cytotoxic effects of quinones
in tumor cells. Thus, the redox status of treated K-562 cells
was monitored using the oxidation-sensitive fluorescent dye
CM-H2DCFDA after 1 and 3 hours of incubation. Fig. 2 shows
cell count at 3 hours. Compound 5a was the most potent in
inducing significant changes (p < 0.05) in ROS levels, at both
1 and 3 hours. In contrast, compound 4f generated lower
levels of ROS, but still significant (p < 0.05) at 3 hours. As
expected, menadione and hydrogen peroxide increased ROS
generation in treated cells.
3. Conclusions
that all three tested compounds induced
increase in intracellular ROS levels, reaching a higher ROS +
a
significant
Considered together, the modification of the ortho-quinone
lead 1 provides access to a wide range of different classes of
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compounds with distinctive “redox cores”, electrochemical
potentials, biological activities and SI values. Here, the
underlying mode(s) of action and also possible selectivity
need to be investigated, together with potential applications
against different human diseases. Whilst some of the
compounds synthesised as part of this study already exhibit a
combination of high activity and selectivity, these aspects
may need to be further improved. It remains to be shown, for
instance, against which cells or organisms these compounds
are especially active, and if there is some selectivity
associated with this activity, which may be exploited in
practice.
Fortunately, the synthetic chemistry associated with these
compounds is straightforward and provides wide
opportunities for structural modifications, from the initial
nucleophilic substitution and alkylation to the side chains
and prospects provided by click chemistry reactions.
until the total consumption of the starting material. After
completion of the reaction, the solvent was removed in vacuo
and the resulting residue was purified by column
chromatography over silica gel, using as the eluent a gradient
mixture of hexane/ethyl acetate with increasing polarity. In
some cases, ultrasound can be used to promote the reaction.
The analytical data for compounds 2a–h are in accordance
with those reported in the literature.36–38
General procedure for the synthesis of compounds 3a–h.
To a stirred solution of a type 2 compound (2.0 equiv.) in
DMF (4 mL), K2CO3 (250 mg, 0.18 mmol, 1.1 equiv.) was
added and the reaction mixture was stirred at room
temperature. After 10 min, propargyl bromide (80 mg, 0.7
mmol, 2.1 equiv.) was added dropwise and stirred under
ultrasound for 40 min. The solvent was evaporated in vacuo
and water (5 mL) was added to the crude reaction mixture.
The aqueous phase was extracted with ethyl acetate (3 × 10
mL) and the organic phases were combined and dried over
anhydrous sodium sulfate and concentrated under reduced
pressure. The crude residue was purified by silica gel column
chromatography by eluting with an increasing polarity
gradient mixture of hexane and ethyl acetate to afford the
respective alkynes (3a–h).
Similarly,
electrochemical
investigations
by
cyclic
voltammetry are fast and simple, and may provide initial
information on the redox behaviour and activities one may
expect.
4. Experimental section
(E)-4-(Phenylimino)-2-(prop-2-yn-1-yloxy)naphthalen-1IJ4H)-
one (3a). The product was obtained as an orange solid (99%
yield); mp 139–141 °C; 1H NMR (400 MHz, CDCl3): δ 8.52
(1H, d, J = 7.8 Hz), 8.24 (1H, d, J = 7.8 Hz), 7.74 (1H, t, J = 7.7
Hz), 7.67 (1H, t, J = 7.7 Hz), 7.43 (2H, t, J = 7.8 Hz), 7.21 (1H,
t, J = 7.4 Hz), 6.96 (2H, d, J = 7.4 Hz), 6.60 (1H, s), 4.61 (2H,
d, J = 2.2 Hz), 2.55 (1H, t, J = 2.2 Hz); 13C NMR (100 MHz,
CDCl3): δ 180.3, 154.8, 154.3, 150.5, 134.8, 133.5, 131.3, 131.3,
129.2, 126.7, 125.5, 125.0, 120.6, 104.6, 77.5, 76.6, 56.2; IR
vmax (cm−1, KBr): 3264, 3067, 2974, 2117, 1670, 1612, 1483,
1383, 1332, 1255, 1189, 1044, 1019, 949, 857, 769, 694, 648;
EI/HRMS (m/z) [M + H]+: 288.1023. Cald. for [C19H14NO2]+:
288.1024.
(E)-4-((4-Methoxyphenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3b). The product was obtained as a
red solid (97% yield); mp 149–150 °C; 1H NMR (400 MHz,
CDCl3): δ 8.51 (1H, d, J = 7.8 Hz), 8.22 (1H, d, J = 7.8 Hz), 7.73
(1H, t, J = 7.9 Hz), 7.65 (1H, t, J = 7.9 Hz), 6.98 (4H, s), 6.72
(1H, s), 4.65 (2H, d, J = 2.3 Hz), 3.78 (3H, s), 2.59 (1H, t, J =
2.3 Hz); 13C NMR (100 MHz, CDCl3): δ 180.4, 157.7, 154.3,
154.1, 143.6, 135.1, 133.4, 131.2, 131.0, 126.6, 125.3, 122.7,
114.6, 104.6, 77.5, 76.8, 56.2, 55.7; IR vmax (cm−1, KBr): 3264,
2930, 2843, 2128, 1675, 1597, 1497, 1461, 1354, 1320, 1260,
1243, 1201, 1044, 1022, 847, 777, 682, 653; EI/HRMS (m/z) [M
+ H]+: 318.1122. Cald. for [C20H16NO3]+: 318.1125.
4.1. Chemistry
Starting materials available from commercial suppliers were
employed as received, unless stated otherwise. All other
reagents requiring purification were purified by standard
laboratory techniques, according to methods published by
Perrin, Armarego and Perrin.49 Catalytic reactions were
performed under nitrogen or argon atmosphere. 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 a syringe through rubber septa.
Melting points of solid compounds were measured on a
Thomas Hoover melting point apparatus and are
uncorrected. Column chromatography was performed on
1
silica gel (SiliaFlash G60 Ultrapure 60–200 μm, 60 Å). H and
13C NMR spectra were recorded at room temperature on a
Bruker AVANCE DRX200 and DRX400, in the solvents
indicated. Chemical shifts (δ) are given in parts per million
(ppm) and coupling constants (J) are reported in Hertz (Hz).
MS analyses were performed on
a
LC-MS/ESI TOF
spectrometer (Model Xevo G2-XS QTof, Waters). The
compounds are named in accordance with IUPAC rules as
applied by ChemBioDraw Ultra (version 12.0).
General procedure for the synthesis of compounds 2a–h.
The synthesis of compounds 2a–h was performed according
to the methodology described in the literature by the Potter
(E)-2-(Prop-2-yn-1-yloxy)-4-(p-tolylimino)naphthalen-1IJ4H)-
one (3c). The product was obtained as an orange solid (95%
yield); mp 148–150 °C; 1H NMR (400 MHz, CDCl3): δ 8.50
(1H, d, J = 7.6 Hz), 8.21 (1H, d, J = 7.6 Hz), 7.71 (1H, t, J = 7.2
Hz), 7.64 (1H, t, J = 7.2 Hz), 7.22 (2H, d, J = 7.4 Hz), 6.88 (2H,
d, J = 7.4 Hz), 6.65 (1H, s), 4.62 (2H, s), 2.59 (1H, s), 2.40 (3H,
s); 13C NMR (100 MHz, CDCl3): δ 180.1, 154.3, 153.9, 147.7,
134.8, 134.6, 133.2, 131.1, 130.9, 129.6, 126.4, 125.2, 120.7,
and Chen groups with minor modifications.36,37 To
a
solution of sodium 3,4-dioxo-3,4-dihydronaphthalene-1-
sulfonate (1) (2.5 mmol) in EtOH : H2O 1 : 1 (75 mL), the
respective aniline (2.7 mmol) was added. The reaction
mixture was stirred at room temperature monitored by TLC
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104.4, 76.8, 76.6, 56.0, 21.0; IR vmax (cm−1, KBr): 3269, 2117,
1672, 1614, 1597, 1500, 1335, 1257, 1192, 1044, 1019, 772; EI/
HRMS (m/z) [M + H]+: 302.1170. Cald. for [C20H16NO2]+:
302.1176.
Hz), 7.70 (1H, t, J = 7.5 Hz), 7.01 (2H, d, J = 8.6 Hz), 6.44 (1H,
s), 4.62 (2H, d, J = 2.3 Hz), 2.65 (3H, s), 2.56 (1H, t, J = 2.3
Hz); 13C NMR (50 MHz, CDCl3): δ 197.0, 179.8, 155.1, 154.8,
154.5, 134.1, 133.6, 133.5, 131.4, 131.1, 129.7, 126.7, 125.6,
120.1, 104.0, 77.5, 76.1, 56.1, 26.5; IR vmax (cm−1, KBr): 3264,
2925, 2134, 1679, 1614, 1592, 1560, 1359, 1267, 1209, 1019,
726, 685; EI/HRMS (m/z) [M + H]+: 330.1122. Cald. for
[C21H16NO3]+: 330.1125.
General procedure for preparing the 1,2,3-triazole
derivatives. To a stirred solution of copperIJI) thiophene-2-
carboxylate (CuTC, 8 mg, 0.04 mmol, 0.08 equiv.) in toluene
(5 mL), the respective propargylated aminonaphthoquinones
(0.5 mmol, 1 equiv.) were added and the reaction mixture
was cooled in an ice–water bath. Subsequently, the sulfonyl
azide (0.5 mmol, 1 equiv.) was added dropwise. The reaction
mixture was then allowed to warm to room temperature and
stirred until the reaction was complete (as evidenced by
TLC). The reaction mixture was extracted with EtOAc (3 × 30
mL). The combined organic layers were dried over sodium
sulfate and filtered through Celite®. The eluent was
concentrated under vacuum and the product was purified by
silica gel column chromatography using eluents with an
increasing polarity gradient mixture of hexane and ethyl
acetate to afford compounds 4a–h.
(E)-4-((4-Fluorophenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3d). The product was obtained as an
1
orange solid (79% yield); mp 147–149 °C; H NMR (400 MHz,
CDCl3): δ 8.45 (1H, d, J = 7.7 Hz), 8.18 (1H, d, J = 7.7 Hz), 7.70
(1H, t, J = 7.4 Hz), 7.63 (1H, t, J = 7.4 Hz), 7.10 (2H, t, J = 8.6
Hz), 6.93–6.90 (2H, m), 6.56 (1H, s), 4.61 (2H, d, J = 2.1 Hz),
2.57 (1H, t, J = 2.1 Hz); 13C NMR (100 MHz, CDCl3): δ 180.1,
161.6, 159.2, 155.2, 154.3, 146.4, 134.6, 133.5, 131.3, 131.2,
126.6, 125.3, 122.2, 122.1, 116.1, 115.9, 104.1, 76.5, 56.2; IR
vmax (cm−1, KBr): 3296, 2990, 2123, 1667, 1624, 1597, 1497,
1339, 1264, 1221, 1204, 1184, 1044, 1019, 862, 772, 697, 648;
EI/HRMS (m/z) [M + H]+: 306.0919. Cald. for [C19H13FNO2]+:
306.0925.
(E)-4-((4-Chlorophenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3e). The product was obtained as an
1
orange solid (77% yield); mp 145–147 °C; H NMR (400 MHz,
CDCl3): δ 8.44 (1H, dd, J = 1.5 and 7.8 Hz), 8.19 (1H, dd, J =
1.5 and 7.8 Hz), 7.75–7.59 (2H, m), 7.36 (2H, d, J = 8.6 Hz),
6.89 (2H, d, J = 8.6 Hz), 6.51 (1H, s), 4.62 (2H, d, J = 2.4 Hz),
2.58 (1H, t, J = 2.4 Hz); 13C NMR (50 MHz, CDCl3): δ 180.0,
155.2, 154.4, 148.8, 134.5, 133.5, 131.4, 131.1, 130.4, 129.3,
126.7, 125.4, 122.0, 104.0, 77.6, 76.5, 56.2; IR vmax (cm−1,
KBr): 3275, 1670, 1614, 1483, 1337, 1252, 1192, 1044, 1019,
854, 772, 639; EI/HRMS (m/z) [M + H]+: 322.0629. Cald. for
[C19H13ClNO2]+: 322.0629.
(E)-4-((4-Bromophenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3f). The product was obtained as a
red solid (72% yield); mp 156–158 °C; 1H NMR (400 MHz,
CDCl3): δ 8.48 (1H, d, J = 7.8 Hz), 8.23 (1H, d, J = 7.8 Hz), 7.75
(1H, t, J = 7.4 Hz), 7.68 (1H, t, J = 7.4 Hz), 7.54 (2H, d, J = 8.4
Hz), 6.85 (2H, d, J = 8.4 Hz), 6.54 (1H, s), 4.65 (2H, d, J = 2.1
Hz), 2.58 (1H, t, J = 2.1 Hz); 13C NMR (50 MHz, CDCl3): δ
180.1, 155.3, 154.6, 149.4, 134.6, 133.6, 132.3, 131.5, 131.3,
126.8, 125.5, 122.4, 118.2, 104.2, 77.6, 76.5, 56.3; IR vmax
(cm−1, KBr): 3275, 2117, 1667, 1612, 1478, 1337, 1255, 1192,
1046, 1024, 852, 772, 685, 641; EI/HRMS (m/z) [M + H]+:
366.0120. Cald. for [C19H13BrNO2]+: 366.0124.
(E)-4-(Phenylimino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)-
methoxy)naphthalen-1IJ4H)-one (4a). The product was
1
obtained as a red solid (60% yield); mp 153–155 °C; H NMR
(400 MHz, CDCl3): δ 8.48 (1H, d, J = 7.5 Hz), 8.22 (1H, s), 8.19
(1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 8.0 Hz), 7.72 (1H, t, J = 7.5
Hz), 7.65 (1H, t, J = 7.5 Hz), 7.43–7.37 (4H, m), 7.19 (1H, t, J =
7.1 Hz), 6.84 (2H, d, J = 8.0 Hz), 6.52 (1H, s), 5.02 (2H, s), 2.44
(3H, s); 13C NMR (100 MHz, CDCl3): δ 180.0, 154.6, 154.5,
150.2, 147.7, 141.8, 134.6, 133.4, 132.7, 131.1, 131.0, 130.5,
129.2, 129.2, 126.5, 125.3, 124.9, 123.2, 120.2, 120.1, 103.8,
61.7, 21.8; IR vmax (cm−1, KBr): 3132, 2922, 1668, 1607, 1392,
1254, 1193, 1014, 667, 587; EI/HRMS (m/z) [M
+
H]+:
485.1208. Cald. for [C26H21N4O4S]+: 485.1278.
(E)-4-((4-Methoxyphenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-
4-yl)methoxy)naphthalen-1IJ4H)-one (4b). The product was
1
obtained as a red solid (55% yield); mp 123–125 °C; H NMR
(400 MHz, CDCl3): δ 8.52 (1H, d, J = 7.8 Hz), 8.26 (1H, s), 8.22
(1H, d, J = 7.8 Hz), 8.02 (2H, d, J = 7.5 Hz), 7.74 (1H, t, J = 7.3
Hz), 7.66 (1H, t, J = 7.3 Hz), 7.41 (2H, d, J = 7.5 Hz), 6.98 (2H,
d, J = 7.2 Hz), 6.86 (2H, d, J = 7.2 Hz), 6.67 (1H, s), 5.09 (2H,
s), 3.89 (3H, s), 2.47 (3H, s); 13C NMR (100 MHz, CDCl3): δ
180.0, 157.5, 154.4, 154.0, 147.7, 143.3, 142.0, 134.8, 133.3,
132.8, 131.0, 130.9, 130.5, 128.9, 126.5, 125.2, 123.1, 122.2,
114.6, 104.0, 61.7, 55.5; IR vmax (cm−1, KBr): 3139, 2958, 2922,
1659, 1601, 1499, 1391, 1261, 1193, 1020, 670, 583; EI/HRMS
(m/z) [M + H]+: 515.1372. Cald. for [C27H23N4O5S]+: 515.1384.
(E)-4-(p-Tolylimino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)-
methoxy)naphthalen-1IJ4H)-one (4c). The product was
obtained as a yellow solid (50% yield); mp 157–159 °C; 1H
NMR (400 MHz, CDCl3): δ 8.52 (1H, d, J = 7.8 Hz), 8.23–8.21
(2H, m), 8.03 (2H, d, J = 8.2 Hz), 7.75 (1H, t, J = 7.6 Hz), 7.67
(1H, t, J = 7.6 Hz), 7.41 (2H, d, J = 8.2 Hz), 7.23 (2H, d, J = 8.0
(E)-4-((4-Iodophenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3g). The product was obtained as a
red solid (68% yield); mp 123–125 °C; 1H NMR (400 MHz,
CDCl3): δ 8.46 (1H, d, J = 7.8 Hz), 8.21 (1H, d, J = 7.8 Hz),
7.73–7.65 (4H, m), 6.72 (2H, d, J = 8.2 Hz), 6.52 (1H, s), 4.64
(2H, s), 2.59 (1H,s); 13C NMR (50 MHz, CDCl3): δ 179.8, 154.9,
154.2, 149.7, 137.9, 134.3, 133.3, 131.2, 130.9, 126.5, 125.2,
122.5, 103.8, 88.6, 77.4, 76.2, 56.1; IR vmax (cm−1, KBr): 3280,
1668, 1609, 1473, 1476, 1253, 1187, 1044, 1020, 770; EI/HRMS
(m/z) [M + H]+: 413.9989. Cald. for [C19H13INO2]+: 413.9985.
(E)-4-((4-Acetylphenyl)imino)-2-(prop-2-yn-1-yloxy)-
naphthalen-1IJ4H)-one (3h). The product was obtained as a
1
yellow solid (48% yield); mp 172–174 °C; H NMR (400 MHz,
CDCl3): δ 8.49 (1H, dd, J = 1.0 and 7.7 Hz), 8.25 (1H, dd, J =
1.0 and 7.7 Hz), 8.05 (2H, d, J = 8.6 Hz), 7.77 (1H, t, J = 7.5
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Hz), 6.78 (2H, d, J = 8.0 Hz), 6.59 (1H, s), 5.06 (2H, s), 2.47
(3H, s), 2.42 (3H, s); 13C NMR (100 MHz, CDCl3): δ; 180.2,
154.8, 154.6, 147.9, 142.3, 135.0, 135.0, 133.6, 133.1, 131.3,
131.2, 130.8, 130.1, 129.1, 126.7, 125.5, 123.3, 120.6, 104.1,
62.0, 22.1, 21.2; IR vmax (cm−1, KBr): 3132, 2917, 1665, 1597,
1396, 1260, 1197, 1015, 670, 587; EI/HRMS (m/z) [M + H]+:
499.1421. Cald. for [C27H23N4O4S]+: 499.1435.
(E)-4-((4-Fluorophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-
yl)methoxy)naphthalen-1IJ4H)-one (4d). The product was
obtained as a yellow solid (79% yield); mp 162–164 °C; 1H
NMR (400 MHz, CDCl3): δ 8.46 (1H, d, J = 7.6 Hz), 8.23 (1H,
s), 8.19 (1H, d, J = 7.6 Hz), 8.00 (2H, d, J = 7.8 Hz), 7.72 (1H,
t, J = 7.2 Hz), 7.65 (1H, t, J = 7.2 Hz), 7.39 (2H, d, J = 7.8 Hz),
7.11 (2H, t, J = 8.2 Hz), 6.83–6.81 (2H, m), 6.55 (1H, s), 5.06
(2H, s), 2.45 (3H, s); 13C NMR (100 MHz, CDCl3): δ 179.9,
155.0, 154.6, 147.7, 141.7, 134.5, 133.5, 132.6, 131.2, 131.0,
130.6, 128.9, 126.5, 125.3, 123.3, 121.8, 121.8, 116.2, 116.0,
61.5, 21.8; IR vmax (cm−1, KBr): 3127, 2925, 1670, 1607, 1495,
1388, 1260, 1197, 1015, 854, 670, 587; EI/HRMS (m/z) [M +
H]+: 503.1154. Cald. for [C26H20FN4O4S]+: 503.1184.
KBr): 3138, 2919, 1670, 1609, 1396, 1260, 1194, 1017, 668,
585; EI/HRMS (m/z) [M
+
H]+: 611.0246. Cald. for
[C26H20IN4O4S]+: 611.0244.
(E)-4-((4-Acetylphenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-
yl)methoxy)naphthalen-1IJ4H)-one (4h). The product was
obtained as a yellow solid (87% yield); mp 174–176 °C; 1H
NMR (400 MHz, CDCl3): δ 8.46 (1H, d, J = 7.6 Hz), 8.22–8.20
(2H, m), 8.04–7.99 (4H, m), 7.76 (1H, t, J = 7.3 Hz), 7.68 (1H,
t, J = 7.3 Hz), 7.40 (2H, d, J = 8.1 Hz), 6.91 (2H, d, J = 8.1 Hz),
6.39 (1H, s), 5.03 (2H, s), 2.65 (3H, s), 2.46 (3H, s); 13C NMR
(100 MHz, CDCl3): δ 197.0, 179.7, 155.0, 154.6, 147.7, 141.6,
134.1, 133.7, 133.6, 132.7, 131.5, 131.1, 130.6, 129.8, 128.9,
126.6, 125.5, 123.2, 119.9, 103.6, 61.8, 29.7, 26.5, 21.8; IR vmax
(cm−1, KBr): 3133, 2919, 2859, 1670, 1594, 1393, 1269, 1194,
1019, 670, 590; EI/HRMS (m/z) [M + H]+: 527.1378. Cald. for
[C28H23N4O5S]+: 527.1384.
General procedure for the synthesis of compounds 5a–d.
The synthesis of compounds 5a–d was performed according
to the methodology described in the literature by the Potter
and Chen groups with minor modifications.36,37 To a 25 mL
round bottom flask, 5 mL of ethanol and 5 mL of water, and
then sodium 3,4-dioxo-3,4-dihydronaphthalene-1-sulfonate (1)
(1 mmol) and the corresponding thiophenol (2 mmol) were
added. The reaction mixture was stirred at room temperature
under ultrasound energy, and monitored by TLC until the
total consumption of the starting material. After completion
of the reaction, the organic phase was extracted with
dichloromethane and dried over sodium sulphate. The
solvent was removed under reduced pressure to afford the
(E)-4-((4-Chlorophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-
yl)methoxy)naphthalen-1IJ4H)-one (4e). The product was
1
obtained as an orange solid (95% yield); mp 155–157 °C; H
NMR (400 MHz, CDCl3): δ 8.47 (1H, d, J = 7.6 Hz), 8.25 (1H,
s), 8.21 (1H, d, J = 7.6 Hz), 8.03 (2H, d, J = 7.5 Hz), 7.75 (1H,
t, J = 7.2 Hz), 7.68 (1H, t, J = 7.2 Hz), 7.43–7.39 (4H, m), 6.82
(2H, d, J = 7.2 Hz), 6.53 (1H, s), 5.07 (2H, s), 2.47 (3H, s); 13C
NMR (100 MHz, CDCl3): δ 180.1, 155.3, 155.1, 148.9, 147.9,
142.0, 134.7, 133.8, 133.0, 131.6, 131.3, 130.8, 130.6, 129.7,
129.2, 126.9, 125.6, 123.5, 121.9, 61.7, 21.9; IR vmax (cm−1,
KBr): 3138, 2919, 1670, 1609, 1391, 1260, 1194, 1088, 1010,
852, 670, 583; EI/HRMS (m/z) [M + H]+: 519.0883. Cald. for
[C26H20ClN4O4S]+: 519.0888.
(E)-4-((4-Bromophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-
yl)methoxy)naphthalen-1IJ4H)-one (4f). The product was
obtained as a yellow solid (92% yield); mp 175–177 °C; 1H
NMR (400 MHz, CDCl3): δ 8.47 (1H, d, J = 7.7 Hz), 8.24 (1H,
s), 8.21 (1H, d, J = 7.7 Hz), 8.03 (2H, d, J = 8.4 Hz), 7.75 (1H,
t, J = 7.4 Hz), 7.68 (1H, t, J = 7.4 Hz), 7.54 (2H, d, J = 8.4 Hz),
7.42 (2H, d, J = 8.2 Hz), 6.76 (2H, d, J = 8.2 Hz), 6.52 (1H, s),
5.07 (2H, s), 2.48 (3H, s); 13C NMR (100 MHz, CDCl3): δ 179.8,
154.9, 154.8, 149.1, 147.7, 141.7, 134.4, 133.5, 132.7, 132.3,
131.3, 131.0, 130.6, 128.9, 126.6, 125.4, 123.2, 122.0, 118.1,
103.4, 61.7, 21.9; IR vmax (cm−1, KBr): 3127, 1670, 1609, 1393,
1260, 1192, 1015, 852, 774, 670, 587, 540; EI/HRMS (m/z) [M +
H]+: 563.0364. Cald. for [C26H20BrN4O4S]+: 563.0383.
(E)-4-((4-Iodophenyl)imino)-2-((1-tosyl-1H-1,2,3-triazol-4-yl)-
methoxy)naphthalen-1IJ4H)-one (4g). The product was
obtained as a yellow solid (89% yield); mp 173–175 °C; 1H
NMR (400 MHz, CDCl3): δ 8.45 (1H, d, J = 7.8 Hz), 8.21 (1H,
s), 8.19 (1H, d, J = 7.8 Hz), 8.01 (2H, d, J = 8.2 Hz), 7.75–7.69
(3H, m), 7.66 (1H, t, J = 7.4 Hz), 7.39 (2H, d, J = 8.2 Hz), 6.62
(2H, d, J = 8.3 Hz), 6.79 (1H, s), 5.05 (2H, s), 2.45 (3H, s); 13C
NMR (100 MHz, CDCl3): δ 179.8, 154.9, 149.7, 147.7, 141.7,
138.2, 134.4, 133.5, 132.7, 131.3, 131.0, 130.6, 128.9, 126.6,
125.4, 123.2, 122.3, 103.4, 88.8, 61.7, 21.9; IR vmax (cm−1,
crude
product,
which
was
purified
by
column
chromatography over silica gel, using as the eluent a gradient
mixture of hexane/ethyl acetate with increasing polarity. The
analytical data for compounds 5a are in accordance with
those reported in the literature.17b
4-(p-Tolylthio)naphthalene-1,2-dione (5b). The product
was obtained as an orange solid (40% yield); mp 205–206 °C;
1H NMR (200 MHz, CDCl3): δ 8.09 (1H, d, J = 7.4 Hz), 7.87
(1H, d, J = 7.6 Hz), 7.65 (t, 1H, J = 7.2 Hz), 7.52 (1H, t, J = 7.4
Hz), 7.37 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.0 Hz), 5.80 (s,
1H), 2.36 (s, 3H); 13C NMR (50 MHz, CDCl3): 179.5, 176.6,
161.9, 141.7, 135.8, 135.0, 133.4, 131.1, 130,3, 129.4, 125.0,
122.6, 121.1, 21.3; EI/MS (m/z) [M + H]+: 281.0. Cald. for
[C17H13O2S]+: 281.0.
4-(m-Tolylthio)naphthalene-1,2-dione (5c). The product
was obtained as an orange solid (32% yield); mp 210–212 °C;
1H NMR (400 MHz, CDCl3): δ 8.19 (1H, d, J = 7.4 Hz), 7.97
(1H, d, J = 7.6 Hz), 7.74 (1H, t, J = 7.2 Hz), 7.62 (1H, t, J = 7.4
Hz), 7.44–7.35 (m, 4H), 5.92 (s, 1H), 2.43 (s, 3H); 13C NMR
(100 MHz, CDCl3): 179.5, 176.6, 161.9, 140.6, 136.5, 135.0,
133.4, 131.1, 130,3, 129.4, 125.0, 122.6, 121.1, 21.3; EI/MS (m/
z) [M + H]+: 281.0. Cald. for [C17H13O2S]+: 281.0.
4-((4-Methoxyphenyl)thio)naphthalene-1,2-dione (5d). The
product was obtained as a red solid (30% yield); mp 215–216 °C;
1H NMR (400 MHz, CDCl3): δ 8.17 (1H, d, J = 7.6 Hz), 7.95 (1H,
d, J = 7.7 Hz), 7.73 (1H, t, J = 7.6 Hz), 7.60 (1H, t, J = 7.5 Hz), 7.48
(2H, d, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 5.88 (1H, s), 3.89 (3H,
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s); 13C NMR (100 MHz, CDCl3): 179.5, 176.6, 162.0, 137.5, 135.8,
135.0, 133.4, 131.1, 130,3, 129.4, 125.0, 122.6, 121.1, 55.6; EI/MS
(m/z) [M + H]+: 297.0. Cald. for [C17H13O3S]+: 297.0.
°C/5% CO2. After that time, the cells were centrifuged, the
medium containing CM-H2DCFDA was removed and the cells
were washed with PBS buffer. From this stage, the cells were
always protected from light. Fresh medium containing
compounds 3e, 4f and 5a was added and the cells were
incubated at the times of interest (1 and 3 hours). After the
incubation time, the cells were centrifuged, washed and re-
suspended in PBS containing propidium iodide (PI) to a final
concentration of 1 μg mL−1. Tubes were placed on ice and
immediately analyzed by flow cytometry. Living cells, which
are PI negative, were selected by gating. In those living cells,
the DCF fluorescence was recorded using excitation and
emission wavelengths of 490 and 525 nm, respectively.
Menadione (MEN, 20 μM) and hydrogen peroxide (H2O2, 150
μM) were used as positive controls. A total of 10 000 events
were analyzed per sample. Data are expressed as mean SEM
from three independent experiments.
4.2.4. Trypanocidal assay and selectivity index. Stock
solutions of the compounds were prepared in dimethyl
sulfoxide (DMSO), with the final concentration of the latter
in the experiments never exceeding 0.4%. Preliminary
experiments showed that DMSO has no deleterious effect on
the parasites when its concentration is up to 4%. T. cruzi
bloodstream trypomastigotes (Y strain) were obtained at the
peak of parasitaemia from infected albino mice, purified by
differential centrifugation and resuspended in RPMI to a
parasite concentration of 107 cells per mL in the presence of
10% mouse blood. This suspension (100 μL) was added in
the same volume of each compound previously prepared at
twice the desired final concentrations for 24 h at 4 °C. Cell
quantification was performed in a Neubauer chamber and
the trypanocidal activity was expressed as IC50/24 h,
corresponding to the concentration that leads to lysis of 50%
of the parasites. In order to determine the SI, cytotoxicity
assays were performed with primary cultures of peritoneal
macrophages obtained from Albino Swiss mice. For the
experiments, 2.5 × 104 cells in 200 μL of RPMI-1640 medium
(pH 7.2 plus 10% foetal bovine serum and 2 mM glutamine)
were added to each well of a 96-well microtitre plate and
incubated for 24 h at 37 °C. The treatment of the cultures
was performed in fresh supplemented medium (200 μL per
well) for 24 h at 37 °C. After this period, 110 μL of the
medium was discarded and 10 μL of PrestoBlue (Invitrogen)
was added to complete the final volume of 100 μL. Thus, the
plate was incubated for 2 h and the measurement was
performed at 560 and 590 nm, as recommended by the
manufacturer. The results were expressed as the difference in
the percentage 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.
4.2. Biological assays
4.2.1. Animals. All experiments dealing with animals were
performed in accordance with the Brazilian Law 11.794/2008
and regulations of the National Council of Animal
Experimentation Control under the license L038/2018 from
the Ethics Committee for Animal Use of the Oswaldo Cruz
Institute (CEUA/IOC).
4.2.2. Cytotoxicity assays. The compounds were tested for
cytotoxic activity in cell culture in vitro employing several
human cancer cell lines obtained from the National Cancer
Institute, NCI (Bethesda, MD). Cytotoxicity was investigated
against six cancer cell lines, i.e. HCT-116 (human colon
carcinoma cells), NCI-H460 (human lung cancer cells), PC3
(human prostate cells), HL-60 (human promyelocytic
leukemia cells), K-562 (myelogenous leukemia cell line) and
Lucena
1
(MDR-cell line). The murine fibroblast
immortalized cell line (L929) was used as control lineage. All
culture media were supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, 100 IU mL−1 penicillin, and 100 μg
mL−1 streptomycin at 37 °C with 5% CO2. In cytotoxicity
experiments, cells were plated in 96-well plates (0.1 × 106 cells
per well for leukaemia cells, 0.7 × 105 cells per well for HCT-
116, and 0.1 × 106 cells per well for PC3, L929 and NCI-
H460). All the compounds tested were dissolved in DMSO.
The final concentration of DMSO in the culture medium was
kept constant (0.1%, v/v). Doxorubicin (0.001–1.10 μM) served
as positive control, and negative control groups received the
same amount of vehicle (DMSO). The cell viability was
determined by reduction of the yellow dye 3-(4,5-dimethyl-2-
thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue
formazan product as described by Mosmann.50a At
completion of the incubation after 72 h, the plates were
centrifuged and the medium was replaced by fresh medium
(200 μL) containing 0.5 mg mL−1 MTT. Three hours later, the
MTT formazan product was dissolved in DMSO (150 μL) and
the absorbance was measured on
a multiplate reader
(SpectraCount, Packard, Ontario, Canada). The influence of
the compound on cell proliferation and survival was
quantified as the percentage of control absorbance of the
reduced dye at 550 nm. The results were obtained by
nonlinear regression for all the cell lines from three
independent experiments. All cell treatments were performed
with three replicates. All cells were mycoplasma-free.
4.2.3. Measurement of intracellular reactive oxygen species
levels. Intracellular reactive oxygen species (ROS)
accumulation was monitored using 5-(6)-chloromethyl-2′,7′-
dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which
is converted to the highly fluorescent dichlorofluorescein
(DCF) in the presence of intracellular ROS.50b K-562 cells
(myelogenous leukemia cell line) were pre-loaded with 10 μM
CM-H2DCFDA and incubated for 1 hour, in the dark, at 37
5. X-ray crystallography
X-ray diffraction data collection for the compounds was
performed on an Enraf-Nonius Kappa-CCD diffractometer (95
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mm CCD camera on a κ-goniostat) and XtaLAB Mini (ROW)
two-circle diffractometer employing graphite monochromated
MoK_radiation (0.71073 Å), at room temperature. Data
collection was carried out using the COLLECT software and
CrysAlisPro.51,52 Integration and scaling of the reflections,
and correction for Lorentz and polarization effects were
performed with the HKL DENZO-SCALEPACK and the
CrysAlisPro system of programs.53 The structure of the
compounds was solved by direct methods with SHELXS-97.54
The models were refined by full-matrix least squares on F2
using SHELXL-97.54 The program ORTEP-3 was used for
graphic representation and the program WINGX was used to
prepare materials for publication.55,56 All H atoms were
located by geometric considerations placed (C–H = 0.93–0.96
Å; N–H = 0.86 Å) and refined as riding with UisoIJH) =
1.5UeqIJC-methyl) or 1.2UeqIJother). Crystallographic data for
the structures were deposited in the Cambridge
Crystallographic Data Centre, with CCDC numbers 1928261
(3a), 1928296 (3d), 1928326 (3f), 1928361 (3g), 1928382 (3h)
and 1928407 (5d).
Development Center of the Federal University of Ceará for
technical support. AK also thanks the University of Saarland,
CAPES and DAAD for the PROBRAL exchange program that
provided financial support for his exchange period at the
Federal University of Minas Gerais. The authors are grateful to
Prof. Claus Jacob from the University of Saarland for his
excellent suggestions during the preparation of this manuscript.
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Acknowledgements
This research was funded by grants from CNPq (404466/2016-8
and PQ 305741/2017-9, PDS 110818/2016-4 and PQ SR 302613/
2013-9), Chamada Universal MCTI/CNPq No 01/2016, FAPEMIG
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Capes-Humboldt research fellowship for experienced
researchers (Proc. No. 88881.145517/2017-01), Return
Fellowship of the Alexander-von-Humboldt foundation and the
Royal Society of Chemistry for the research fund grant (R19-
9781). CAS would like to thank the Institute of Physics of the
University of São Paulo (São Carlos). The authors would like to
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