Direct synthesis of 4-organylsulfenyl-7-chloro quinolines and their toxicological and pharmacological activities in Caenorhabditis elegans

Article abstract of DOI:10.1016/j.ejmech.2014.01.037

We describe herein our results on the synthesis and biological properties in Caenorhabditis elegans of a range of 4-organylsulfenyl-7-chloroquinolines. This class of compounds have been easily synthesized in high yields by direct reaction of 4,7-dichloroq

Full text of DOI:10.1016/j.ejmech.2014.01.037

European Journal of Medicinal Chemistry 75 (2014) 448e459
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Direct synthesis of 4-organylsulfenyl-7-chloro quinolines and their
toxicological and pharmacological activities in Caenorhabditis elegans
Willian G. Salgueiro a, Maurício C.D.F. Xavier b, Luis Fernando B. Duarte b,
Daniela F. Câmara a, Daiandra A. Fagundez a, Ana Thalita G. Soares a, Gelson Perin b,
**
,
a,
*
Diego Alves b , Daiana Silva Avila
a Grupo de Pesquisa em Bioquímica e Toxicologia em Caenorhabditis elegans (GBToxCE), Universidade Federal do Pampa-UNIPAMPA, BR 592,
Km 472, CEP 97500-970 Uruguaiana, RS, Brazil
b Laboratório de Síntese Orgânica Limpa e LASOL e CCQFA e Universidade Federal de Pelotas e UFPel, CEP 96010-900 Pelotas, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein our results on the synthesis and biological properties in Caenorhabditis elegans of a
range of 4-organylsulfenyl-7-chloroquinolines. This class of compounds have been easily synthesized in
high yields by direct reaction of 4,7-dichloroquinoline with organylthiols using DMSO as solvent at room
temperature under air atmosphere and tolerates a range of substituents in the organylsulfenyl moiety.
We have performed a toxicological and pharmacological screening of the synthesized 4-organylsulfenyl-
7-chloroquinolines in vivo in C. elegans acutely exposed to these compounds, under per se and stress
conditions. Hence, we determined the lethal dose 50% (LD50), in order to choose a nonlethal concen-
Received 30 August 2013
Received in revised form
15 January 2014
Accepted 17 January 2014
Available online 31 January 2014
Keywords:
Quinolines
tration (10 mM) and verified that at that concentration some of the compounds depicted protective action
against the induced damage inflicted by paraquat, a superoxide generator. Two compounds (3c and 3h)
reduced the toxicity inflicted by paraquat above survival, reproduction and longevity of the worms, at
least in part, by reducing the reactive oxygen species (ROS) generated by the toxicant exposure. Besides,
these compounds increased the quantities of superoxide dismutase (SOD-3::GFP) and catalase (CTL-
1,2,3::GFP), antioxidant enzymes. We concluded that the protective effects of the compounds observed in
this study might have been a hormetic response dependent of the transcriptional factor DAF-16/FOXO,
causing a non-lethal oxidative stress that protects against the subsequently damage induced by paraquat.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Sulfur
Antioxidants
Hormesis
Caenorhabditis elegans
1. Introduction
these, 91% contains nitrogen and 24% contains sulfur [4]. The syn-
thesis of new compounds that can treat different ROS-associated
It is well known that reactive oxygen species (ROS) such as su-
peroxide radical (O2ꢀꢁ), hydrogen peroxide (H2O2) and hydroxyl
radical (OHꢀꢁ) are capable of causing damage that increases
gradually with aging in many organisms, originating alterations in a
variety of molecules (lipid, protein, and DNA) from invertebrates to
humans [1,2]. The broad spectrum of severe diseases in which an
important role is played by reactive oxygen species, such as cancer,
Alzheimer and atherosclerosis [3] leads to the necessity of
searching for drugs that effectively neutralize/modulate ROS.
Actually, 85% of the drugs available in the market come from syn-
thetic origins and 62% of these are heterocyclic compounds. Among
pathologies is relevant and, to reach that, the insertion of
different substituents into a chemical structure that has described
pharmacological properties is a common synthesis practice. In this
context, quinolines have been described as a very successful
molecule in terms of pharmacological properties [5]. A number of
compounds based on the quinoline ring have been known since the
late 1800 s, such as quinidine (a class I antiarrhythmic agent),
chloroquine, hidroxychloroquine, primaquine (antimalarial drugs)
and quinine (antipyretic drug).
Similarly, organic compounds with chalcogen atoms in their
structure, such as sulfur, are well known by their antioxidants
properties. Many studies have demonstrated efficacious treatment
with these type of compounds against disease models associated
with oxidative stress [6e10]. Their ROS-scavenging activity is
probably due to their high nucleophilicity. There are in the literature
some methodologies for the synthesis of sulfur-containing quino-
lines, however these methodologies have some disadvantages,
* Corresponding author. Tel./fax: þ55 55 34134321.
** Corresponding author. Tel.: þ55 53 32757357; fax: þ55 53 32757533.
E-mail addresses: diego.alves@ufpel.edu.br (D. Alves), avilads1@gmail.com
(D.S. Avila).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.037

W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
449
including long reaction times and harsh conditions [11e16,21].
2.1.1. General procedure for the synthesis of 7-chloro-4-
(organylsulfenyl)quinolines 3aeh
Consequently, the search for new and efficient methodologies for
the synthesis of novel sulfur-containing quinolines remains an
important challenge for synthetic organic chemistry.
To a round-bottomed flask containing 4,7-dichloroquinoline
(0.3 mmol) and DMSO (1.0 mL) was added the appropriated
organylthiol (0.3 mmol). The reaction mixture was stirred at room
temperature for the time indicated in Table 2. After that, the solu-
tion was cooled at room temperature, diluted with ethyl acetate
(15 mL), and washed with water (3 ꢃ 15 mL). The organic phase was
separated, dried over MgSO4, and concentrated under vacuum. The
obtained products were purified by flash chromatography on silica
gel using a mixture of ethyl acetate/hexane (20:80) as the eluent.
Some level of toxicity for these organochalcogens compounds
are related in the literature [7,17e19] and, for that reason, toxico-
logical screenings are needed in order the refine and reduce the
number of animals involved in this type of research. The nematode
Caenorhabditis elegans came as an alternative because of its easy
and inexpensive maintenance in the laboratory, based on a diet of
Escherichia coli. The entire life cycle, from an egg to an adult pro-
ducing more eggs, takes just 3.5 days at 20 ꢂC. Population growth is
fastest at 20 ꢂC, with brood sizes of w300 produced over approx-
imately a 4-day period [20]. Remarkably, the relatively straight-
forward generation of knockout strains for genes of interest and of
transgenic worms expressing green fluorescent tagged proteins
(GFP) are very useful for protein expression or localization studies.
Remarkably, it was the first multicellular organism to have its
genome completely sequenced, which has been found to have a
high level of conservation with the vertebrate genome [20e22].
This includes the antioxidant system.
2.1.1.17-Chloro-4-(phenylthio)quinoline (3a) [12]. Yield: 0.078
g
(96%); white solid; mp 73e75 ꢂC. 1H NMR (CDCl3, 200 MHz)
d
¼ 8.54 (d, J ¼ 5.0 Hz,1H), 8.29 (d, J ¼ 2.0 Hz,1H), 8.20 (d, J ¼ 8.9 Hz,
1H), 7.64e7.53 (m, 6H), 6.78 (d, J ¼ 5.0 Hz, 1H). 13C NMR (CDCl3,
50 MHz)
d
¼ 153.54, 147.63, 144.84, 137.47, 135.53, 130.54, 130.47,
128.44, 127.72, 126.72, 124.88, 124.04, 117.05. MS (relative intensity)
m/z: 273 (38), 271 (100), 235 (54), 204 (8), 135 (15), 127 (8), 109 (6),
77 (8).
For instance, the sod-3 gene, which encodes the mitochondrial
superoxide dismutase, and ctl-1 and ctl-2, that encode the cytosolic
and peroxisomal catalase, posses 97, 97 and 98% of homology with
the human iso forms, respectively [23,24]. Such proteins can be
used as markers to access the redox state of C. elegans [25e28]. The
sod and ctl isoforms are, under oxidative stress response, regulated
by the FOXO homolog DAF-16, a transcription factor widely related
as essential for normal development, reproduction and lifespan in
C. elegans [29].
2.1.1.27-Chloro-4-(m-tolylthio)quinoline (3b). Yield: 0.074 g (87%);
white solid; mp 38e39 ꢂC. 1H NMR (CDCl3, 400 MHz)
d
¼ 8.55 (d,
J ¼ 4.8 Hz, 1H), 8.12 (d, J ¼ 8.9 Hz, 1H), 8.06 (d, J ¼ 2.0 Hz, 1H), 7.50
(dd, J ¼ 8.9, 2.0 Hz, 1H), 7.40e7.29 (m, 4H), 6.72 (d, J ¼ 4.8 Hz, 1H),
2.39 (s, 3H). 13C NMR (CDCl3, 100 MHz)
d
¼ 150.37; 149.30; 147.99;
140.11; 135.86; 135.59; 132.39; 130.71; 129.87; 128.73; 128.48;
127.27; 124.83; 124.26; 117.68; 21.24. MS (relative intensity) m/z:
287 (37), 285 (100), 270 (37), 251 (7), 249 (25), 236 (9), 135 (13), 111
(11), 91 (13), 77 (5).
We describe herein the direct synthesis of 4-organylsulfenyl-7-
chloro quinolines by reaction of 4,7-dichloroquinoline with organ-
ylthiols using DMSO as solvent at room temperature under air at-
mosphere. Additionally we investigated the toxicological and
pharmacological effects of the synthesized 4-organylsulfenyl-7-
chloro quinolines in C. elegans. We hypothesized that our thioquino-
lines could act by indirectly neutralizing oxygen reactive species by a
hormetic response induced by low ROS formation, which in turn up-
regulates SOD-3 and CTL-1,2,3 possibly via DAF-16 transcription fac-
tor, protecting the worms against the paraquat-induced oxidative
damage in the survival, reproduction and lifespan parameters.
2.1.1.37-Chloro-4-(4-methoxyphenylthio)quinoline
0.065 g (72%); white solid; mp 133e135 ꢂC. 1H NMR (CDCl3,
400 MHz)
¼ 8.52 (d, J ¼ 4.8 Hz, 1H), 8.13 (d, J ¼ 8.9 Hz, 1H), 8.05 (d,
(3c). Yield:
d
J ¼ 1.7 Hz, 1H), 7.53e7.50 (m, 3H), 7.02 (d, J ¼ 8.6 Hz, 2H), 6.63 (d,
J ¼ 4.8 Hz, 1H), 3.88 (s, 3H). 13C NMR (CDCl3, 100 MHz)
d
¼ 161.25;
150.45; 150.36; 148.00, 137.53; 135.60; 128.77; 127.21; 124.70;
124.09; 118.69; 116.86; 115.77; 55.44. MS (relative intensity) m/z:
303 (37), 301 (100), 270 (9), 266 (7), 257 (5), 251 (33), 235 (11), 223
(24), 139 (10).
2. Material and methods
Table 1
2.1. Chemistry
Optimization of reaction conditions in the synthesis of 7-chloro-4-(phenylsulfenyl)
quinoline 3a.a
The reactions were monitored by TLC carried out on Merck silica
gel (60 F254) by using UV light as visualizing agent and 5% vanillin in
10% H2SO4 and heat as developing agents. Baker silica gel (particle
size 0.040e0.063 mm) was used for flash chromatography. Proton
nuclear magnetic resonance spectra (1H NMR) were obtained at
300 MHz on a Varian Gemini NMR and at 400 or 200 MHz on
Bruker DPX 400 and DPX 200 spectrometers. Spectra were recor-
ded in CDCl3 solutions. Chemical shifts are reported in ppm,
referenced to tetramethylsilane (TMS) as the external reference.
Hydrogen coupling patterns are described as singlet (s), doublet (d),
double doublet (dd), double triplet (dt), double double doublet
(ddd), triplet (t), quintet (qui), sextet (sex), multiplet (m), and broad
singlet (bs). Coupling constants (J) are reported in Hertz. Carbon-13
nuclear magnetic resonance spectra (13C NMR) were obtained at
75 MHz on a Varian Gemini NMR and at 100 or 50 MHz on Bruker
DPX 400 and DPX 200 spectrometers respectively. Chemical shifts
are reported in ppm, referenced to the solvent peak of CDCl3. Low-
resolution mass spectra were obtained with a Shimadzu GC-MS-
QP2010 mass spectrometer.
Entry
Solvent
Temperature (ꢂC)
Time (min)
Isolated yield 3a (%)
1
2
3
4
5
6
7
8
9
DMSO
DMSO
DMSO
DMSO
EtOH
Glycerol
PEG-400
MeCN
100
100
100
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
120
60
15
15
15
15
15
15
15
98
97
96
96
85
55
55
75
15
Toluene
a
Reactions are performed with 4,7-dichloroquinoline 1 (0.3 mmol), benzenethiol
2a (0.3 mmol) under air atmosphere.

450
W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
Table 2
Variability in the synthesis of 7-chloro-4-(arylsulfenyl)quinoline.a
Entry
RSH
Time
Product
Yield (%)b
1
15 min
96
2a
2b
3a
2
20 min
87
3b
3
1.5 h
72
3c
4
20 min
98
2d
3d
3e
5
20 min
80
2e
6
20 min
60
2f
3f
7
20 min
85
2g
3g

W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
451
Table 2 (continued )
Entry
RSH
Time
2 hc
Product
Yield (%)b
8
80
2h
3h
a
Reactions are performed with 4,7-dichloroquinoline 1 (0.3 mmol), organylthiol 2aeh (0.3 mmol), in DMSO (1 mL) at room temperature under air atmosphere.
Yields are given for isolated products.
Reaction was performed at 70 ꢂC.
b
c
2.1.1.47-Chloro-4-(p-tolylthio)quinoline (3d). [15] Yield: 0.084
g
(19), 251 (5), 207 (17), 160 (20), 147 (5), 135 (15), 129 (23), 127 (19),
116 (10), 115 (38), 77 (9).
(98%); white solid; mp 115e117 ꢂC. 1H NMR (CDCl3, 200 MHz)
d
¼ 8.52 (d, J ¼ 4.9 Hz, 1H), 8.17e8.10 (m, 2H), 7.56e7.46 (m, 3H),
7.31 (d, J ¼ 7.9 Hz, 2H), 6.69 (d, J ¼ 4.9 Hz, 1H), 2.44 (s, 3H). 13C NMR
2.2. Caenorhabditis elegans strains and maintenance of the worms
(CDCl3, 50 MHz)
d
¼ 150.89; 149.68; 147.18, 140.61; 136.01; 135.62;
131.01; 128.23; 127.50; 124.78; 124.66; 124.12; 117.09; 21.36. MS
(relative intensity) m/z: 287 (38), 285 (100), 272 (13), 270 (35), 250
(25), 236 (11), 235 (60), 207 (14), 135 (21), 127 (10),111 (10), 91 (28),
77 (10).
NGM plates seeded with live Escherichia coli OP50 bacteria were
used for growth and maintenance of the worms, as previously
described by Brenner [22]. The C. elegans strains Bristol N2 (wild
type), CF1553 (muls84), GA800 (wuls151), TJ356 (zls356), CF1038
(daf-16 [mu86]) were maintained and handled at 21 ꢂC. Synchro-
nous L1 population was obtained by isolating embryos from gravid
hermaphrodites using bleaching solution (1% NaOCl; 2.4% NaOH),
followed by floatation on a sucrose gradient to segregate eggs from
dissolved worms and bacterial debris, accordingly to standard
procedures previously described by Brenner [22].
2.1.1.57-chloro-4-(4-chlorophenylthio)quinoline (3e). Yield: 0.073 g
(80%); white solid; mp 151e152 ꢂC. 1H NMR (CDCl3, 200 MHz)
d
¼ 8.58 (d, J ¼ 4.8 Hz, 1H), 8.15e8.10 (m, 2H), 7.58e7.44 (m, 5H),
6.76 (d, J ¼ 4.8 Hz, 1H). 13C NMR (CDCl3, 50 MHz)
d
¼ 149.75;
149.28; 147.32; 136.49; 136.45; 136.25; 130.44; 128.34; 127.81;
127.30; 124.79; 124.22; 117.81. MS (relative intensity) m/z: 309 (13),
308 (13), 307 (65), 305 (100), 270 (52), 235 (99), 207 (19), 162 (12),
135 (44), 127 (21), 117 (18), 108 (22).
2.3. Per se, protection and reversion protocols of treatment
1500 L1 worms previously synchronized were exposed for
30 min to the compounds in liquid media containing 0.5% NaCl and
in absence of bacteria. L1 worms were chosen for these treatments
because some reasons: The exposure at the L1 larval stage offers the
possibility of to observe effects in the entire life of the worm and
the treatment could be done in completely absence of E. coli,
eliminating the possibility of any undesirable bacterial metabo-
lization of the compounds. At the end of treatment, the worms
were washed three times in NaCl to remove the compounds and
soon after placed on NGM recovery plates seeded with E. coli OP50
(this procedure was made at the same mode before all the assays).
The per se/protection/reversion protocols were assayed by counting
alive worms in the plates 24 h post-treatment. A LD50 curve was
2.1.1.67-Chloro-4-(2-chlorophenylthio)quinoline (3f). Yield: 0.055 g
(60%); white solid; mp 73e75 ꢂC. 1H NMR (CDCl3, 200 MHz)
d
¼ 8.60 (d, J ¼ 4.8 Hz, 1H), 8.16e8.09 (m, 2H), 7.59e7.26 (m, 5H),
6.74 (d, J ¼ 4.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
d
¼ 150.23;
148.22; 146.12; 138.46; 136.29; 135.90; 130.90; 130.74; 129.23;
128.80; 127.94; 127.66; 125.14; 124.77; 119.09. MS (relative in-
tensity) m/z: 309 (6), 307 (32), 304 (44), 272 (36), 270 (100), 236
(15), 235 (79), 207 (12), 162 (6), 135 (26), 127 (11), 117 (12), 108 (14).
2.1.1.77-Chloro-4-(4-fluorophenylthio)quinoline (3g). Yield: 0.074 g
(85%); white solid; mp 124e126 ꢂC. 1H NMR (CDCl3, 400 MHz)
d
¼ 8.56 (d, J ¼ 4.8 Hz,1H), 8.10 (d, J ¼ 8.9 Hz,1H), 8.06 (d, J ¼ 2.0 Hz,
obtained using concentrations that ranged from 1 mM to 1000 mM of
1H), 7.59e7.55 (m, 2H), 7.52 (dd, J ¼ 8.9, 2.0 Hz, 1H), 7.19 (t,
the compounds, and then, nonlethal doses were chosen. The pro-
tection or reversion of the induced oxidative damage were assayed
using paraquat (Gramoxone 200 Ò) as pro-oxidant agent at the
J ¼ 8.7 Hz, 2H), 6.66 (d, J ¼ 4.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
d
¼ 163.82 (d, J1 (CeF) ¼ 251.7 Hz); 150.38; 148.98; 148.09; 137.67
(d, J3 (CeF) ¼ 8.5 Hz); 135.77; 128.86; 127.43; 124.69; 124.15;
124.10 (d, J4 (CeF) ¼ 3.6 Hz); 117.44 (d, J2 (CeF) ¼ 22.1 Hz); 117.41.
MS (relative intensity) m/z: 292 (6), 291 (36), 290 (26), 289 (100),
254 (48), 222 (11), 207 (6), 162 (10), 135 (26), 127 (37), 57 (6).
concentration of 500
mM for another 30 min. The pre or post
exposure to the nonlethal concentrations of the compounds was
followed by a post or anticipated by a pre, respectively, exposure to
paraquat. Three additional washes were made before to proceed to
all the assays. All the experiments were made in duplicates and
repeated at least four times, as described by the Scheme 2.
2.1.1.87-Chloro-4-(naphthalen-2-ylthio)quinoline
0.077 g (80%); white solid; mp 66e67 ꢂC. 1H NMR (CDCl3, 400 MHz)
¼ 8.53 (d, J ¼ 4.8 Hz, 1H), 8.21 (d, J ¼ 8.9 Hz, 1H), 8.16 (s, 1H), 8.09
(3h). Yield:
d
2.4. Reproduction
(d, J ¼ 1.9 Hz, 1H), 7.96e7.86 (m, 3H), 7.63e7.54 (m, 4H), 6.77 (d,
J ¼ 4.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
d
¼ 150.46; 148.15;
In order to evaluate their reproduction, worms were individu-
ally transferred to new plates seeded with OP50 bacteria 48 h after
treatments. Brood size was measured by monitoring the worms
each 24 h during their fertile cycle. The mean eggs laid per day and
135.76; 135.42; 133.93; 133.43; 131.13; 129.95 (2C); 128.86; 127.90;
127.88; 127.60; 127.49; 127.06; 126.20; 124.96; 124.44; 118.16. MS
(relative intensity) m/z: 323 (36), 321(100), 288 (23), 286 (49), 254

452
W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
106. All experiments were made in duplicates and repeated at least
five times.
2.8. Epifluorescence microscopy
For each slide, at least 30 treated worms were mounted on 2%
agarose pads and anaesthetized with few drops of levamisole
22.5
mM. Fluorescence observations were performed under epi-
Scheme 1. Synthesis promoted by base of sulfenyl quinoline 3a.
fluorescence microscopy; housed in air-conditioned rooms (20e
22 ꢂC) for image acquisitions and scoring the expression levels.
total brood size were quantified. All the experiments were made in
duplicates and repeated at least five times.
2.9. Statistics
All the graphics were generated with GraphPad Prism (Graph-
Pad Software Inc.). Dose-response curves were generated in order
to obtain the LD50 of each compound. To analyze the ROS measures
and lifespan, a Repeated Measures ANOVA analysis and the Tukey
post-hoc were applied. A one-way ANOVA was applied for all the
other assays, followed by the Tukey post-hoc. All the p values less
than 0.05 were considered statistically significant. All the values
expressed in percentage (%) were normalized assuming that control
values as 100%. In all figures bars represent the SEM.
2.5. Lifespan
48 h after the acute exposure described above, twenty worms
per concentration were individually transferred to new plates
seeded with E. coli OP50. The worms were monitored and trans-
ferred to new plates every day until the last one died. All the ex-
periments were made in duplicates and repeated at least three
times.
2.6. ROS measurement
3. Results
The ROS levels were measured in the L1 and L4 larval stage. For
L1 worms, right after the treatments, the animals washed to
3.1. Synthesis of sulfenyl quinolines
remove the compounds and then incubated for 1 h with 50 mM of
Initially, our studies were focused on the synthesis of 7-chloro-
4-(phenylsulfenyl)quinoline 3a. Recently, our group described the
synthesis of 4-arylselanyl-7-chloroquinolines by reaction of 4,7-
dichloroquinoline with diaryl diselenides using KOH as base,
DMSO as solvent at 100 ꢂC under air atmosphere [31]. Extending
this reaction condition for the reaction of 4,7-dichloroquinoline 1
with benzenethiol 2a, the desired product 3a was obtained in
excellent yield (Scheme 1).
Notably, when we performed this reaction in the absence of
KOH, the desired product 3a was obtained in similar yield after
120 min (Table 1, entry 1). The same results were also observed in
reactions decreasing the reaction time to 60 and 15 min (Table 1,
entries 2 and 3). When the reaction of quinoline 1 and benzenethiol
2a was performed at room temperature for 15 min, an excellent
result was achieved (Table 1, entry 4). We observed that the nature
of the solvent was critical for obtain the desired product 3a in high
yields and short reaction time (Table 1, entries 4e9). As shown in
Table 1, reactions employing different solvents, such as EtOH,
glycerol, PEG-400, MeCN and toluene, gave moderated or only a
traces of product 3a (Table 1, entries 4e9). Thus, analysis of the
results showed in Table 1 indicated that the best reaction condi-
tions were the use of 4,7-dichloroquinoline 1 (0.3 mmol), benze-
nethiol 2a (0.3 mmol) using DMSO as solvent at room temperature
under air atmosphere for 15 min.
H2DCF-DA; then, worms were washed with 0.5% NaCl until the
concentration of the dye decreased approximately a thousand
times (three dilutions of 1:10). Worms were transferred to micro-
plates and the fluorescence levels were measured for 30 min using
a plate reader CHAMELEONÔV Hidex Model 425-106, heated at
20 ꢂC. For L4 worms, worms were treated at the L1 stage, which
were deposited in NGM plates seeded with E. coli. 48 h after, the
NGM plates were washed with M9 buffer and the worms were
transferred to microtubes. The E. coli supernatant was washed until
exhaustion (three dilutions of 1:10). Then, worms were concen-
trated to 100
H2DCF-DA for 1 h, washed for dye removal and the ROS levels
measured by the same manner as L1 worms. The remaining 50 L of
mL with M9 buffer. 50 mL was incubated with 50 mM of
m
the sample was ultrasonicated for protein measurement, according
to Bradford [30].
2.7. Fluorescence quantification
The GFP expressing strains (CF1553 [muls84] and GA800
[wuls154]) were submitted to the acute exposure as above
described and transferred right after the end of the washes into
200 mL M9 buffer in a well of 96-well plate. Total GFP fluorescence
was measured using 485 nm excitation and 530 nm emission fil-
ters. Overall GFP fluorescence of GFP-expressing strains was
assayed using a plate reader CHAMELEONÔV Hidex Model 425-
After that, the variability of our methodology reacting 4,7-
dichloroquinoline 1 with other organylthiols 2beh was evaluated
Scheme 2. Schematic line of the treatments and assays.

W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
453
(Table 2). The results showed in Table 2 reveal that the reaction
worked well with a range of substituted arylsulfides containing
electron-donating (EDG) and electron-withdrawing groups (EWG),
affording good yields of the desired products (Table 2, entries 1e7).
We observed that steric effect had a little influence on the product
formation and reaction using 2-naphthylmercaptan gave the
desired product 3h in 80% yield, however at 70 ꢂC and in a longer
reaction time compared with other examples (Table 2; entry 8).
against the induced oxidative damage inflicted by paraquat
(500 M). We found that the compounds show their better po-
m
tential as protectors (Fig. 2A) by preventing the mortality of the
worms acutely exposed to paraquat. Worms treated only with
paraquat showed approximately 50% of mortality, whereas five
compounds showed reduced mortality levels around 34-26% when
pre-administered previous to paraquat. The reversion protocol was
not as efficient, as only 3h depicted a significant reduction on the
mortality induced by paraquat (Fig. 2B).
3.2. The compounds depicted different LD50 depending on the
substitutions of the thioquinoline ring
3.4. The compounds protect against paraquat-induced lifespan
toxicity
As showed in Fig. 1A, we found different LD50 values to the
compounds, indicating that the toxicity of the thioquinolines is
directly associated with the different substituents. In decrescent
Fig. 3A and B show that six compounds did not alter worms
lifespan. Compounds 3f and 3h increased the mean time of life
(p < 0.0001 compared with the control group). Paraquat caused
toxic effects, resulting in reduced lifespan of the exposed worms, as
demonstrated in Fig. 3C and D (p < 0.0001 when compared to the
control group). Three of the compounds (3c, 3f and 3h) were able to
protect the worms against the reduction in lifespan caused by
paraquat exposure (p < 0.0001 compared to paraquat group).
order of concentration: 3h (2500
m
M) > 3d (1700
M) > 3b (180 M) > 3a (120
M). The nonlethal concentrations were chosen
M).
M and no toxicity in the survival
m
M) > 3e
(520
(45
m
M) > 3c (230
m
m
mM) > 3f
m
M) > 3g (18 m
taking into account the lowest value of LD50 found (3ge18
The chosen concentration was 10
m
m
assay was verified at this concentration in all thioquinolines tested,
as showed in Fig. 1B.
3.3. Compounds protected against the induced oxidative damage
inflicted by paraquat
Searching for the best use of the compounds as antioxidant
agents, we choose to perform protection and reversion protocols
Fig. 2. Protection and reversion treatments with thioquinolines: A) Protection, B)
reversion. Data are expressed as mean ꢄ SEM. The control group was considered as
100%. *, ** and *** indicate, respectively, p < 0.05, p < 0.01 or p < 0.0001 compared to
the paraquat group.
Fig. 1. Per se effect of thioquinolines: A) Doseeresponse curve for LD50% (Lethal Dose
50%), B) non-lethal concentration of 10
mM on survival. Data are expressed as
mean ꢄ SEM.

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W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
Fig. 3. Longevity after per se or reversion treatment with thioquinolines: A and B) Per se C and D) Protection. Data are expressed as mean ꢄ SEM. Data were normalized to 100%. #
indicates significant difference (p < 0.05) compared to the control group. * indicates significant difference compared to the paraquat group.
3.5The compounds protect against the paraquat-induced
reproductive toxicity
3.7. Levels of mitochondrial superoxide dismutase (SOD-3) and
catalase (CTL-1, CTL-2, CTL-3) are increased in worms pre-treated
with the compounds
All the compounds did not shown any per se effects on repro-
duction, as observed by brood size assay (Fig. 4A). Fig. 4B shows
that the group treated only with paraquat depicted significant
reduction in the egg-laying (p < 0.0001 when compared to the
control group). 3d was able to partially protect the reduction in
brood size caused by paraquat (p < 0.01 compared to paraquat
group) whereas two other compounds (3c and 3h) fully protected
the worms, depicting number of eggs indistinguishable from con-
trol group (Fig. 4B).
High levels of ROS can lead to an oxidative stress state. In a
healthy organism, some enzymes play a detoxifying role. The sod-3
gene encodes the major isoform of mitochondrial superoxide dis-
mutase, an important antioxidant enzyme that catalyzes the dis-
mutation of superoxide to hydrogen peroxide. Thereafter, H2O2 is
converted to molecular oxygen and water by catalase, other anti-
oxidant enzyme. We found that the treatment with 3c, 3e and 3h
compounds were able to significantly increase the levels of both
SOD-3 and CTL-1,2 and 3, isoforms, whereas 3d was capable to
induce only SOD-3 expression and 3g only shows ability to up-
regulate the CTL isoforms, as showed in Fig. 6A and B, respectively.
3.6. The ROS levels in worms were altered in the per se and
protection protocols
To determine whether the ROS formation caused by paraquat
could be prevented by the compounds, we used H2DCF-DA, which
is de-esterified to H2DCF and then oxidized to the DCF fluorophore
in the presence of free radicals. Fig. 5A shows that all compounds
caused increase in ROS formation in L1 worms; nevertheless, in
these same worms, 48 after the treatment (Fig. 5B), ROS levels were
indistinguishable (3c) or lower (3b and 3d-3h) than the levels
found in the controls. 3a compound-treated group remained with
higher ROS levels even 48 h after the treatment. Paraquat- treat-
ment and compounds/paraquat treatment depicted increased ROS
levels in L4 worms when compared to control (p < 0.0001, Fig. 5C);
however, two groups of treated worms (3c and 3h) showed
significantly reduced levels of ROS when compared to paraquat-
exposed worms (p < 0.0001).
3.8. The protective effect of the compounds is dependent of DAF-16,
a homolog of the human transcription factor FOXO
Searching for a putative molecular target of the thioquinolines,
we investigated the subcellular localization of DAF-16. The tran-
scription factor DAF-16 is the worm homolog of human FOXO. This
transcriptional factor belongs to the insulin/IGF-like signaling,
which is the central determinant of the endocrine control of stress
response, aging, fat metabolism, fertility, and diapause in C. elegans.
In the worms pre-treated with the compounds, we observed a
significant change in DAF-16 localization for 3c, 3d and 3feh
compounds, inducing its nuclear migration and thus, its activation
(p < 0.0001, Fig. 7A).

W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
455
Fig. 5. ROS levels measured with H2DCF-DA probe in per se and protection treatment
with thioquinolines: A) Per se in N2 L1 animals, B) Per se in N2 L4 (48 after the
treatment) animals C) Compounds/paraquat in L4 (48 h after the treatment) animals.
Fig. 4. Brood size in per se and reversion treatment with thioquinolines: A) Per se, B)
Reversion. Data are expressed as mean ꢄ SEM. # indicates significant difference
(p < 0.05) compared the control group. * indicates significant difference compared to
the paraquat group (p < 0.05).
Arbitrary Units of Fluorescence/
mg of protein. Data are expressed as mean ꢄ SEM. *, **
or *** indicate difference when compared to paraquat group. # indicates difference
when compared to control group.
The compounds caused mortality per se in daf-16 mutants
(Fig. 7B). We also found that the protection against the oxidative
damage conferred by the pre-treatment with the thioquinolines in
the survival assay was completely lost in absence of the daf-16 gene,
as depicted in Fig. 7C. The elevated ROS formation induced by the
compounds per se was significantly higher in the daf-16 mutant
animals than the observed in N2 worms (Fig. 7D and Table 3). Be-
sides, 48 h after, these ROS levels remained significantly elevated
but without any difference in comparison to N2 animals, as showed
in Fig. 7E and Table 3. The post-treatment with paraquat in L4
animals significantly induced higher ROS formation in daf-16 ani-
mals than in N2 (Fig. 7F. and Table 3). Besides, none of the com-
pounds were able to prevent the induced ROS formation in the daf-
16 mutants (Fig. 7E).
compounds were able to protect against the oxidative damage
inflicted by paraquat in several parameters and demonstrated that
this ability is possibly due to a hormetic response against a small
oxidative damage induced by the thioquinolines. This response
could be, at least in part, mediated by ROS formation, which in turn
activates the FOXO homolog DAF-16 stress response, up-regulating
the levels of SOD-3 and CTL-1,2,3, partially neutralizing the ROS
formation induced by paraquat. This hormetic response could
protect against the subsequent oxidative damage induced by the
powerful prooxidant paraquat, an agent that affects survival,
reproduction and longevity parameters in C. elegans.
Focusing at the safe use of the thioquinolines, we have primarily
assessed their toxicological profile. Organochalcogens toxicity has
been linked to the stability of the carbon-chalcogen bond [7], which
could explain the behavior of the thioquinolines studied in this
4. Discussion
work. Compound 3g had a higher toxicity (18 mM) probably because
this compound has a fluor atom, a weak deactivating with high
electronegativity, which facilitates reaction with another bio-
molecules. On the other hand, compound 3h, which has the neutral
In this study we investigated the toxicity and pharmacological
potential of a series of novel compounds based on the sulfur con-
taining quinoline compounds in C. elegans. We found that these

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W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
with the lower ROS levels found 48 h after thioquinolies treatment
(compounds 3beh) (Fig. 5B). It has been reported that primaquine,
an anti-malarial drug based on the quinoline ring, has pro-oxidant
activity against Plasmodium falciparum in the red blood cells. Pri-
maquine can generate hydroxyl radicals from oxidation of pyridine
nucleotides, such as NADH and NADPH [32]. This can explain the
elevated amounts of ROS in the worms following thioquinolines
treatment, per se. Interestingly, we verified the absence of toxic
effects following thioquinolines treatment on longevity (Fig. 3A and
B)and reproduction endpoints (Fig. 4B). In fact, we have found that
compounds 3f and 3h prolonged lifespan of treated worms, per se.
This effect can be attributed to the theory that small ROS formation
could have beneficial effects in some ROS-associated situations,
such as aging or even physical exercise, where there is an imbalance
of antioxidant/prooxidant homeostasis [33e36], a mechanism
known by hormesis. More studies are required to clarify these
point, but we suggest a possibly involvement of DAF-16 trans-
location to the nucleus, since that the superexpression of this one
can extend the longevity of C. elegans in non-stress conditions [40].
Paraquat toxicity mechanisms involve the generation of the
superoxide anion, which can lead to the formation of more toxic
reactive oxygen species, such as hydrogen peroxide and hydroxyl
radical; and the oxidation of the cellular NADPH, the major source
of reducing equivalents for the intracellular reduction of paraquat,
which results in the disruption of important NADPH, required in
biochemical processes [37]. In our study we could observe that
paraquat reduced significantly the survival (Fig. 2A and B) the
longevity (Fig. 3C and D) and reproduction (Fig. 4B) in the acute
treatment. Besides, the ROS levels were significantly higher than in
the non-treated worms (Fig. 5C and D). Thus, our findings suggest
that ROS formation induced by paraquat can negatively affect sur-
vival, longevity and reproduction in C. elegans, which is in agree-
ment to previous studies [38,39]. Notably, ROS formation induced
by the thioquinolines did not affect any of these parameters.
In agreement to the hormetic response theory, five of the
compounds (3bed, 3f and 3h) protected the worms against the
injury caused by paraquat on survival, and only three of these (3c,
3f and 3h) were able to offer protection against the reduced life-
span induced by paraquat. We have tested whether the thio-
quinolines could reverse the oxidative damage, however only 3h
was able to increase survival after paraquat exposure (3h in Fig. 2B).
This is an additional finding that supports the hormetic mechanism
provided by the compounds, as they could not decrease mortality
once the damage-induced by paraquat is already present.
Fig. 6. Antioxidant enzymes levels following thioquinolines treatment A) SOD-3:GFP,
B) CTL-1,2,3 :GFP. Data are expressed as mean ꢄ SEM. *, ** and *** indicate, respec-
tively, p < 0.05, p < 0.01 or p < 0.0001 compared to the control group.
and stable naphthalene heterocycle bond to the chalcogen, pre-
sented the lowest toxicity (2500 mM). The pairs 3b/3d and 3e/3f are
In C. elegans, brood size represents the reproductive capacity
and can be used as a parameter of toxicity [38]. 3c and 3h were able
to completely recover from paraquat-induced reproductive toxicity
(Fig. 4B). We also observed that paraquat toxicity in C. elegans could
be associated to high and persistent ROS formation (Fig. 5C and D),
whereas 3c and 3h pre-treatment prevented the increased ROS
levels in those two treated groups (Fig. 5D).
Antioxidant compounds that enhance stress resistance without
prolonging lifespan of C. elegans, as 3b and 3d, have been described
in the literature [41,42], demonstrating again the controversial role
of the ROS in the aging process. On the other hand 3c, 3f and 3h
were able to protect from the oxidative damage-induced by para-
quat in survival and lifespan endpoints, in accordance to the hy-
pothesis that the oxidative state in C. elegans could modulate these
two parameters at the same time [43].
constitutional isomers. As both 3d and 3e showed lower toxicity of
each pair and have activating substituents that guide the reaction to
the orto and para position, we believe that the para position is
blocked by the calchogen atom and consequently the formation of a
possible toxic product is diminished. 3b and 3f have activating
substituents that guide the reaction to the same positions and, in
that case, they are free to react. Compound 3c has a methoxy
substituent that could guide the reaction to the position blocked by
the chalcogen, but in this case the high electron donating-ability of
this group, in comparison with the methyl group, seems to
contribute to this effect, taking into account its elevated toxicity
when compared to the others (230
comparison to the others thioquinolines (120
because of the absence of substituents that could stabilize the
structure. As the lowest lethal concentration was 18 M (3g), we
decided to test a nonlethal concentration of 10 M for all the
mM). 3a has high toxicity in
mM), probably
In addition, the putative hormetic thioquinolines response may
act by modulating some intracellular process that modulate anti-
oxidant defenses, so we further investigated whether thioquino-
lines caused the translocation of the transcription factor DAF-16
into the nucleus. Four thioquinolines that protected against
paraquat-induced mortality and reduced ROS levels induced by this
m
m
compounds. We verified that, at this concentration, all the thio-
quinolines induced ROS formation in recently L1-treated worms
(Fig. 5A) without causing any mortality (Fig. 1B). This is consistent

W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
457
Fig. 7. Thioquinolines modulate DAF-16 pathway per se: A) Subcellular localization of DAF-16 was considered in three levels: cytosolic, intermediate and nuclear, 30 worms per
group were visualized and scored. B) Thioquinolines effects on survival of knockdown mutants lacking daf-16. C) Thioquinolines/paraquat treatment effect above survival of mutants
lacking daf-16. D) ROS levels of L1 daf-16 mutants soon after the thioquinolines treatment. E) ROS levels of L4 daf-16 mutants 48 h after the thioquinolines treatment. F) ROS levels of
L4 daf-16 mutants 48 h after the thioquinolines/paraquat treatment. Arbitrary units of fluorescence/
when compared to paraquat group. # indicates difference when compared to control group. *, ** and *** indicate, respectively, p < 0.05, p < 0.01 or p < 0.0001 compared to the
m
g of protein. Data are expressed as mean ꢄ SEM. *, ** or *** indicates difference
control group.
pesticide (3c, 3d, 3f, and 3h) were able to modulate the subcellular
localization of DAF-16, increasing its translocation to the nucleus
(Fig. 7A). Furthermore, 3c and 3h up-regulated the expression of
both SOD-3::GFP and CTL-1,2,3::GFP (Fig. 6A and B), classical tar-
gets of DAF-16-mediated stress response [44,45], possibly as a
consequence of its migration to the nucleus. To confirm whether
thioquinolines were indeed modulating the IGF-1 pathway at the
DAF-16 level, we used a knockdown strain for daf-16 (mu86) and
verified that the compounds induced mortality, per se (Fig. 7B).
Besides that, the protection against paraquat in the survival
parameter was completely lost (Fig. 7C). We also verified that these
mutants had significantly more ROS levels than wild-type animals
soon after the thioquinolines treatment and 48 h after (Fig. 7D and
E and Table 3 for comparison between strains) Furthermore, none
of the compounds were able to prevent the paraquat-induced ROS
formation in the absence of daf-16 (Fig. 7F). Considering such ob-
servations we concluded that DAF-16 plays a vital role in the thi-
oquinolines response, probably by detoxifying the ROS generated
by them and, furthermore, protecting worms against paraquat
toxicity, which could explain the per se toxicity and the absence of
protection in daf-16 mutant. Many studies have demonstrated that
DAF-16 acts in the hormetic response against many toxicants by
activating mechanisms that repair damage and protect the organ-
ism from further injury. This oxidative-stress response has been
associated with the up-regulation of the mitochondrial superoxide
dismutase SOD-3 (leading into account that mitochondria is the

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W.G. Salgueiro et al. / European Journal of Medicinal Chemistry 75 (2014) 448e459
Table 3
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