Insights into the discovery of novel neuroprotective agents: A comparative study between sulfanylcinnamic acid derivatives and related phenolic analogues

Article abstract of DOI:10.3390/molecules24234405

Exogenous antioxidants may be beneficial therapeutic tools to tackle the oxidative damage in neurodegenerative diseases by regulation of the redox state that is critical for cell viability and organ function. Inspired by natural plant polyphenols, a series of cinnamic acid-based thiophenolic and phenolic compounds were synthesized and their antioxidant and neuroprotective properties were studied. In general, our results showed that the replacement of the hydroxyl group (OH) by a sulfhydryl group (SH) increased the radical scavenging activity and enhanced the reaction rate with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^?) and galvinoxyl radical (GO^?). These results correlated well with the lower oxidation potential (E_p) values of thiophenols. However, a lower peroxyl radical (ROO^?) scavenging activity was observed for thiophenols in oxygen radical absorbance capacity (ORAC-FL) assay. Furthermore, the introduction of 5-methoxy and 5-phenyl groups in the aromatic ring of 4-thioferulic acid (TFA) 2 and ferulic acid (FA) 1 did not significantly improve their antioxidant activity, despite the slight decrease of E_p observed for compounds 5, 6, and 9. Concerning cinnamic acid amides, the antioxidant profile was similar to the parent compounds. None of the compounds under study presented significant cytotoxic effects in human differentiated neuroblastoma cells. Thiophenolic amide 3 stands out as the most promising thiophenol-based antioxidant, showing cellular neuroprotective effects against oxidative stress inducers (hydrogen peroxide and iron).

Full text of DOI:10.3390/molecules24234405

molecules
Article
Insights into the Discovery of Novel Neuroprotective
Agents: A Comparative Study between
Sulfanylcinnamic Acid Derivatives and Related
Phenolic Analogues
Daniel Chavarria 1,2,†, Carlos Fernandes 1,† , Brandon Aguiar 1, Tiago Silva 1,2, Jorge Garrido 3,
Fernando Remião 4 , Paulo J. Oliveira 2 , Eugenio Uriarte 5,6 and Fernanda Borges 1,
*
1
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto,
Portugal; danielf_chavarria@live.com.pt (D.C.); cevinhas@gmail.com (C.F.);
brandonlageaguiar@gmail.com (B.A.); ntb.silva@gmail.com (T.S.)
CNC–Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech, Biocant Park,
3060-197 Cantanhede, Portugal; pauloliv@cnc.uc.pt
Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic of Porto, 4200-072 Porto,
Portugal; jjg@isep.ipp.pt
UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy,
University of Porto, 4050-313 Porto, Portugal; remiao@ff.up.pt
Departamento de Química Orgánica, Facultad de Farmacia, 15782 Santiago de Compostela, Spain;
eugenio.uriarte@usc.es
Instituto de Ciencias Químicas Aplicadas, Universidad Autónoma de Chile, Santiago 8900000, Chile
Correspondence: fborges@fc.up.pt
2
3
4
5
6
*
†
Both authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Dimitra Hadjipavlou-Litina
Received: 23 October 2019; Accepted: 28 November 2019; Published: 2 December 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Exogenous antioxidants may be beneficial therapeutic tools to tackle the oxidative damage
in neurodegenerative diseases by regulation of the redox state that is critical for cell viability and
organ function. Inspired by natural plant polyphenols, a series of cinnamic acid-based thiophenolic
and phenolic compounds were synthesized and their antioxidant and neuroprotective properties
were studied. In general, our results showed that the replacement of the hydroxyl group (OH) by a
sulfhydryl group (SH) increased the radical scavenging activity and enhanced the reaction rate with
1,1-diphenyl-2-picrylhydrazyl radical (DPPH• ) and galvinoxyl radical (GO• ). These results correlated
well with the lower oxidation potential (Ep) values of thiophenols. However, a lower peroxyl radical
(ROO• ) scavenging activity was observed for thiophenols in oxygen radical absorbance capacity
(ORAC-FL) assay. Furthermore, the introduction of 5-methoxy and 5-phenyl groups in the aromatic
ring of 4-thioferulic acid (TFA)
activity, despite the slight decrease of Ep observed for compounds
2
and ferulic acid (FA)
1
did not significantly improve their antioxidant
6, and . Concerning cinnamic
5
,
9
acid amides, the antioxidant profile was similar to the parent compounds. None of the compounds
under study presented significant cytotoxic effects in human differentiated neuroblastoma cells.
Thiophenolic amide
3 stands out as the most promising thiophenol-based antioxidant, showing
cellular neuroprotective effects against oxidative stress inducers (hydrogen peroxide and iron).
Keywords: cinnamic acid; 4-thioferulic acid derivatives; ferulic acid derivatives; antioxidant activity;
cytotoxicity; neuroprotection
Molecules 2019, 24, 4405; doi:10.3390/molecules24234405
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4405
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1. Introduction
Oxidative stress is currently defined as the deregulation of the balance between the oxidants
and antioxidants in favor of the former, with consequential disruption of the redox signaling and/or
molecular damage [
inflicted by reactive species (RS) may ultimately lead to programmed cell death [
of evidence implicate oxidative stress on the neurodegeneration process observed in patients with
Alzheimer’s disease (AD) and Parkinson’s disease (PD) [ ]. Actually, markers of lipid peroxidation
and protein oxidation were detected in individuals with AD and PD [ ]. Both disorders were also
1]. The harmful effects on human cells associated with the oxidative damage
2
]. Several lines
3
4–8
associated with a decline of the pool of antioxidants, and a decrease of the activity of antioxidant
enzymes [9–12].
Given the role of oxidative stress on the pathogenesis of neurodegenerative diseases, the use
of exogenous antioxidants to assist the antioxidant defense system in upstream (prevention of RS
generation) or downstream (radical scavenging) antioxidant pathways may help to maintain neuronal
integrity and delay the neurodegenerative processes [13,14].
Ferulic acid (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid, FA, compound 1, Figure 1) is a
natural hydroxycinnamic acid (HCA) able to act directly by scavenging harmful oxidants, or indirectly by
downregulation of pathways involved in cell-death and upregulation of the expression of antioxidant
enzymes [15]. Neuroprotective properties in both in vitro and in vivo models, associated with
antioxidant and anti-inflammatory activities, were ascribed to FA [16–18]. However, the therapeutic
use of FA is hampered by its poor bioavailability, as a consequence of its low absorption and rapid
metabolism and excretion [19,20].
Figure 1. Rational design of new chemical entities based on FA (compound
2) scaffolds.
1) and TFA (compound
Ferulic acid has been used as a scaffold for the design and development of novel antioxidants
with potential therapeutic interest [21 23]. Within this framework, 4-thioferulic acid (TFA, compound
, Figure 1), a new antioxidant obtained by the isosteric replacement of the hydroxyl group (OH) by a
–
2
sulfhydryl group (SH) in FA, was reported by our group [24].
To gain insight on the advantages of thiophenol systems, herein we report the synthesis and
the biological screening of novel TFA derivatives (compounds
3–6, Figure 1). The corresponding
hydroxycinnamic acid derivatives (compounds 10, Figure 1) were synthesized to directly compare
7–
the effect of the OH to SH replacement. Following the studies of the antioxidant activity, we assessed
the cytotoxicity of the new derivatives in differentiated SH-SY5Y cells and their protective properties
against oxidative stress insults (H2O2, iron) in the same cellular model.

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2. Results and Discussion
2.1. Chemistry
The synthesis of TFA (compound
2
) [24] and derivatives thereof (compounds
3
–6
) was performed
following the strategy depicted in Scheme 1. First, the reaction between 5-bromovanillin (compound
11) and phenylboronic acid in the presence of Pd(OAc)2 and K2CO3 under microwave (MW) irradiation
yielded 5-phenylvanillin (compound 12) (Scheme 1, step a). Then, the thiocarbamoylation of
4-hydroxybenzaldehyde derivatives 12
alkaline conditions afforded compounds 15
rearrangement under inert atmosphere of compounds 15
step c). Cinnamic acid derivatives 21 23 were obtained via Knoevenagel-Doebner condensation
between aldehydes 18 20 and malonic acid (Scheme 1, step d). The PyBOP-mediated amidation of
compound 21 with benzylamine or phenethylamine led to the obtainment of the amides 24 and 25
–
14 with dimethylthiocarbamoyl chloride (DMTCl) under
17 (Scheme 1, step b). A MW-assisted Newman-Kwart
17 yielded compounds 18 20 (Scheme 1,
–
–
–
–
–
,
respectively (Scheme 1, step e). Finally, the alkaline hydrolysis of compounds 21
derivatives 2–6 (Scheme 1, step f).
–25 afforded thiophenol
Scheme 1. Synthetic strategy followed for the synthesis of compounds
K2CO3, Pd(OAc)2, TBAB, DMF, 150 ◦C (microwave (MW) irradiation), 15 min; (
DMF, 70 ◦C, 3 h; ( ) diphenyl ether, argon, 240 ◦C (MW irradiation), 20 min; (
) malonic acid, anhydrous
pyridine, piperidine, 70 ◦C, 4–5 h; ( ) 1. DMF, DIPEA, PyBOP, CH2Cl2, 0 ◦C, 30 min; 2. benzylamine or
2-phenylethylamine, rt, overnight; (f) MeOH, aqueous solution of NaOH 2 M, reflux, 1.5–16 h.
2–
6. (a) phenylboronic acid,
b) DMTCl, DABCO,
c
d
e

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The corresponding hydroxycinnamic acid derivatives (compounds
7–
10, Figure 1) were
synthesized according to procedures previously reported [21,25].
2.2. Evaluation of Antioxidant Activity
Following the synthesis of TFA and FA derivatives (compounds 1–10), we evaluated their radical
scavenging activity against three synthetic radicals: 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•),
2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+) and galvinoxyl (GO•).
In these assays, after the generation of the synthetic colored radical, the radical scavenging activity
is evaluated by spectrophotometry [26]. Trolox was used as a reference antioxidant. The results are
presented as mean IC50 ± SD (n = 3) values (Table 1).
Table 1. Radical scavenging activities and redox potentials (Ep) of thiophenols 2–6 and phenols 1, 7–10.
The results obtained in DPPH•, ABTS•+, GO• assays are expressed as mean IC50 standard deviation
(n = 3).
IC50 (µM)
ORAC-FL
Index
Ep (mV)
Compound
•
+
DPPH•
47.8 ± 0.9a
49.8 ± 0.7
GO•
R1
R2
R3
ABTS
2 (TFA)
3
19.9 ± 1.0a
20.6 ± 0.7
4.25
±
0.10a 0.34
±
0.04a
271
198
4.50 ± 0.08
0.12 ± 0.02
4
47.3 ± 0.7
42.7 ± 2.1
50.7 ± 1.6
64.6 ± 1.9a
61.7 ± 0.8
19.9 ± 0.3
22.9 ± 0.6
27.0 ± 1.8
16.8 ± 0.4a
25.6 ± 1.8
4.41 ± 0.15
6.70 ± 0.07
6.66 ± 0.13
37.5 ± 2.5a
76.4 ± 3.6
0.15 ± 0.01
0.30 ± 0.03
0.34 ± 0.01
3.2 ± 0.1a
4.0 ± 0.4
186
218
245
5
6
1(FA)
7
344 b
327
8
9
61.2 ± 0.7
43.9 ± 1.6
69.7 ± 3.8
26.0 ± 1.7
16.6 ± 0.3
19.8 ± 0.8
70.7 ± 2.5
3.90 ± 0.1
31.2 ± 3.9
3.7 ± 0.3
3.2 ± 0.3
2.7 ± 0.1
338
291
351
10
Trolox
—-
—-
—-
24.6 ± 0.9
18.2 ± 0.5
2.98 ± 0.15
1.00 ± 0.02
110
a
b
Data from Chavarria et al. [24]. Data from Garrido et al. [23].
In general, thiophenols
10, while the scavenging of ABTS•+ radical was similar. The introduction of an additional 5-methoxy
group (compound ) was proven to be a structural modification with limited benefits, since it only
caused slight changes in the antioxidant activity of TFA (compound ) (a modest improvement in the
antioxidant activity was only observed in DPPH•assay). Conversely, the presence of an additional
aromatic ring (compound ) decreased the antioxidant activity of TFA. Although an additional
2–6
showed higher scavenging of DPPH• and GO• radicals than phenols 1,
7
–
5
2
6
aromatic ring can increase the electron delocalization and stabilize the generated thiophenoxyl radical
by resonance, it may reduce the accessibility of the compound to the center of the radicals and
thereby decrease the radical scavenging activity [27]. The same tendency was observed with the
phenolic analogues.
Considering that both 5-methoxy and 5-phenyl groups did not significantly increase the radical
scavenging activity of TFA, we then evaluated the effect of the introduction of substituents on the
carboxylic function of TFA. As shown in Table 1, benzyl and phenethyl amides of TFA (compounds
and 4, respectively) displayed similar IC50 values to the free carboxylic acid. The same tendency was
observed for the phenolic analogues (compounds and ). Therefore, the carboxylic acid amidation
with lipophilic substituents maintained the antioxidant activity of TFA.
3
7
8

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The kinetic data obtained in DPPH•, ABTS•+, and GO• assays can provide information concerning
the reactivity of the compounds under study (Figures S1–S3). As previously reported for TFA [24],
thiophenols 3–6 reacted almost instantaneously with the radicals, leading to fast absorbance decays
and reaching the steady state within the first minutes of reaction (Figures S1A–E, S2A–E and S3A–E).
These results pointed out that the reaction rate was increased by the OH to SH replacement. Compared
to TFA, the reactions of compounds
5 and 6 with the radicals occurred slower, requiring more time to
reach the steady state (Figures S1D,E, Figures S2D,E and Figures S3D,E). Therefore, the presence of
5-methoxy and 5-phenyl groups in thiophenols delayed the reaction progress, possibly due to increased
steric hindrance. On the other hand, compounds
3 and 4 displayed the same kinetic profile of TFA
(Figures S1B,C, Figures S2B,C and Figures S3B,C), indicating that the reactivity of TFA was preserved
with the functionalization of the carboxylic acid with lipophilic groups. Like ferulic acid
1
[
24], phenols
•
+
7
–
10 displayed hyperbolic kinetic curves indicating moderate reactivity towards DPPH•, ABTS
,
and GO• (Figures S1F–J, S2F–J and S3F–J).
We also studied the antioxidant activity toward peroxyl radicals (ROO•) using the oxygen radical
absorbance capacity (ORAC-FL) assay. In this assay, ROO• radicals are scavenged by hydrogen atom
transfer (HAT) or by radical addition processes [27]. Phenols (compounds
1, 7–10) showed ORAC-FL
indexes ranging between 2.7–4.0 (Table 1) and displayed higher ORAC-FL indexes than thiophenols
(compounds 2–6). Thiophenols presented ORAC-FL indexes below 1 (Table 1). Therefore, contrary
to what was observed in DPPH•, ABTS•+ and GO•assays, these results show that phenols are better
ROO• radical scavengers than thiophenols. The dissimilarities between the two sets of compounds
may be related to the differences on the acidity of the phenol and the thiophenol moieties. Since the
thiophenol group of TFA
1 is significantly more acidic than the phenol group of FA (pKa thiophenol = 5.6;
pKa phenol = 9.1) [24], there is a high amount of thiophenolate anions at pH 7.3 while the phenol group
remains undissociated. Therefore, in opposition to phenols
2–6 were poor scavengers of ROO• radicals.
1 and 7–10, the deprotonated thiophenols
2.3. Electrochemical Studies
Voltammetric measurements are particularly useful to estimate the redox properties of bioactive
systems, thus enabling a prompt screening of their antioxidant capacity [28]. In this context, to obtain
complementary information concerning the antioxidant activity of the compounds under study,
differential pulse voltammetry (DPV) experiments were performed to study the electrochemical
properties of compounds 2–6 and 7–10. Experiments were carried out at a physiological pH of 7.4
using a glassy carbon-working electrode. The oxidation potential (Ep) values obtained are depicted in
Table 1.
All compounds showed well-defined anodic waves at physiological pH, which may be attributed
to the oxidation of the thiophenol or the phenol groups. Thiols and phenols can undergo one-electron
oxidation processes with the formation of thiyl and phenoxyl radicals, respectively [29
values of thiol-containing compounds ranged between +186 mV and +271 mV, while phenols
and 10 displayed higher Ep values, ranging between +291 mV and +351 mV (Table 1 . The results
,30]. The Ep
2–
6
1
7–
)
obtained showed that the replacement of the OH by an SH group shifted the peak potentials toward
less positive values, suggesting that thiophenols are more prone to oxidation than phenols.
The introduction of 5-methoxy (compounds
did not significantly change the Ep values when compared to FA and TFA, although a slight decrease
was observed for compounds 6, and . The lower potential values may result from the increased
stabilization of the thiophenoxyl/phenoxyl radicals, which is associated with the electron-donating
properties of the methoxy group [31] (compounds and ) or the increased electron delocalization
provided by the additional aromatic ring (compound 6).
The voltammetric behavior of the cinnamic acid amides
for the parent compounds (TFA and FA). The introduction of lipophilic groups at the side chain did
5 and 9) or 5-phenyl (compounds 6 and 10) groups
5
,
9
5
9
3, 4, 7, and 8 was similar to that observed

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not significantly affect the redox properties of phenol and thiophenol groups and, therefore, no major
changes in the redox potentials were observed.
2.4. Cellular Studies
2.4.1. Evaluation of Cytotoxicity Profile
The cytotoxicity profile of thiophenols
SH-SY5Y cells differentiated into a dopaminergic phenotype [32]. After 24 h of exposure with the
compounds at three different concentrations (1, 10 and 50 M), the cell viability was evaluated using two
3–6 and phenols 7–10 was assessed in human neuroblastoma
µ
methods: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay, a method
that relies on the reduction of MTT into the respective formazan by cells with active metabolism [33],
and the neutral red (NR) uptake assay, which is based on the lysosomal uptake of the dye NR [34].
The results, expressed as mean MTT reduction (% of control) SEM and NR uptake (% of control)
SEM (n = 3), are depicted in Figure 2.
±
Figure 2. Cellular viability of differentiated SH-SY5Y cells after a 24 h treatment with thiophenols 3–6
and phenols
using two methods: MTT reduction (
as the mean % of untreated controls
the parametric method of one-way ANOVA, followed by the Dunnett’s multiple comparisons test.
In all cases, p values lower than 0.05 were considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001,
**** p < 0.0001 vs untreated cells).
7
–
10 at three different concentrations (1, 10 and 50
µ
M). Cellular viability was evaluated
A
,
C) and NR uptake (
B
,
D) assays. Results are expressed
±
SEM (n = 3). Statistical comparisons were performed using
Thiophenols
MTT reduction and in NR uptake at concentrations up to 50
>85%), which is in accordance with the previous data obtained for FA
3
–
6
(Figure 2A,B) and phenols
7
–
10 (Figure 2C,D) did not cause marked decreases in
M (MTT reduction and NR uptake were
and TFA 24]. Based on these
µ
1
2 [
results, we can conclude that the cytotoxicity profile within the tested concentration range was upheld
with the OH to SH replacement, the introduction of 5-methoxy and 5-phenyl substituents, and the
functionalization of the carboxylic acid group (benzyl or phenethyl amides).

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2.4.2. Protection against H2O2- and Ferric Iron-Induced Oxidative Damage
The protective properties of compounds
were also examined. Hydrogen peroxide (H2O2) and ferric nitrilotriacetate (FeNTA) were used as
inducers of oxidative stress [32]. After the pre-treatment with the test compounds at 10 and 50
1–10 against oxidative stress in the same cellular model
µM
for 24 h, differentiated SH-SY5Y cells were exposed to H2O2 (1.5 mM) or FeNTA (4 mM) for 30 min
and 24 h, respectively. The cellular viability was assessed using the MTT reduction assay. The results
obtained are illustrated in Figures 3 and 4.
Figure 3. Protection against H2O2-induced damage in differentiated SH-SY5Y cells by thiophenols
) and phenols 10 ). After a pre-treatment with the compounds under study (10 and 50
for 24 h, cells were exposed H2O2 1.5 mM for additional 30 min. Cellular viability was evaluated by
the MTT reduction assay. Results are expressed as the mean % of untreated controls SEM (n = 3).
2–6
(
A
1,
7–
(
B
µM)
±
Statistical comparisons were estimated using the nonparametric method of Kruskal–Wallis (one-way
ANOVA on ranks), followed by Dunn’s post hoc test (* p < 0.05, ** p < 0.01, compared with cells treated
only with H2O2; $$ p < 0.01, $$$ p < 0.001; $$$$ p < 0.0001 compared with CTRL). Positive control with
caffeic acid (50 µM) was performed under the same conditions described above. The result is depicted
in Supplementary Information (Figure S4A).
Figure 4. Protection against FeNTA-induced damage in differentiated SH-SY5Y cells by thiophenols
2
–
6
(
A
) and phenols
for 24 h, cells were exposed FeNTA 4 mM for additional 24 h. Cellular viability was evaluated by
the MTT reduction assay. Results are expressed as the mean % of untreated controls SEM (n = 3).
1, 7–10 (B). After a pre-treatment with the compounds under study (10 and 50 µM)
±
Statistical comparisons were estimated using the nonparametric method of Kruskal–Wallis (one-way
ANOVA on ranks), followed by Dunn’s post hoc test (* p < 0.05 compared with cells treated only with
FeNTA; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001; $$$$ p < 0.0001 compared with CTRL). Positive control with
caffeic acid (50 µM) was performed under the same conditions described above. The result is depicted
in Supplementary Information (Figure S4B).
The incubation of differentiated SH-SY5Y cells with H2O2 (1.5 mM) decreased the MTT reduction
to 50.7
±
1.9% (p < 0.0001) in comparison to control cells (Figure 3A,B). The damage inflicted by H2O2

Molecules 2019, 24, 4405
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was attenuated by thiophenols and phenols containing benzyl amide (compounds
3
and
7
, p < 0.05) and
5-methoxy groups (compound
protection against H2O2-induced cell death was also observed with pre-treatment of neuroblastoma
cells with TFA (10 M, p < 0.01; 50 M, p < 0.05, Figure 3A) and compound 10 (p < 0.05, Figure 3A) at
5, p < 0.05, and compound 9, p < 0.01) at 10 µM (Figure 3A,B). A mild
µ
µ
both tested concentrations. The data obtained for TFA
by Chavarria et al. [24].
2 and FA 1 at 50 µM were previously reported
Transition metals like iron accumulate with age in specific brain areas, such as the substantia
nigra and the hippocampus [35]. FeNTA was used to mimic iron overload conditions in SH-SY5Y
cells. Treatment with FeNTA 4 mM decreased the MTT reduction to 47.2
comparison to control cells (Figure 4A,B). Remarkably, TFA derivative
±
1.2% (p < 0.0001) in
significantly protected
3
neuroblastoma cells from the deleterious effects of FeNTA at both tested concentrations (p < 0.05,
Figure 4A). The FeNTA-induced decrease in MTT reduction was also attenuated in neuroblastoma
cells pre-treated with TFA 2 (p < 0.05, Figure 4A) and compound 10 (p < 0.05, Figure 4B) at 50 µM.
Taking these data together, we concluded that TFA
significantly protected neuroblastoma cells against oxidative stress, both by the direct action of H2O2
and by high concentrations of iron. In addition, 5-methoxy derivatives (compounds and ) and FA
benzyl amide (compound ) were able to prevent the damage induced by H2O2, but were ineffective
2, TFA benzyl amide 3, and FA derivative 10
5
9
7
against the harmful effects caused by high concentrations of iron.
2.5. Estimation of Drug-Like Properties
In early drug discovery, the balancing of key physicochemical properties within optimal ranges may
improve the compounds’ quality and thereby increase the likelihood of therapeutic success [36]. In this
context, we calculated several physicochemical parameters of TFA (compound 2) and compounds 3 and
10 to predict their “drug-likeness”. These compounds were considered the most promising cinnamic
acid derivatives because they displayed significant neuroprotective activity against two oxidative
stressors in neuroblastoma cells. The predicted physicochemical properties included topological polar
surface area (TPSA in Å), number of hydrogen acceptors (HBA), number of hydrogen donors (HBD),
number of rotatable bonds (RB), and logarithm of the ratio of the concentration of a drug in the brain
and in the blood (logBB). The results obtained are presented in Table 2.
Table 2. Predicted physicochemical properties of TFA (compound 2) and compounds 3 and 10.
cLogPb
Compound
MW a
TPSA a
HBA a
HBD a
RB
LogBB
2
3
10
210.25
299.39
270.28
1.97
3.25
2.76
46.53
38.33
66.76
4
4
4
2
2
2
3
6
4
−0.9373
0.02948
-1.511
CNS+ drugs
< 500 c
2–5 c
< 90 c
<−1
< 7 d
< 3 d
< 8 d
a
Properties predicted using the Stardrop software. MW: molecular weight; TPSA: topological polar surface area;
HBA: number of H-bond acceptor atoms; HBD: number of H-bond donor atoms; RB: number of rotatable bonds;
b
logBB: logarithm of the ratio of the concentration of a drug in the brain and in the blood. Properties predicted
using the SwissADME (http://swissadme.ch/index.php); clogP: logarithm of the octanol/water partition coefficient.
c
d
Data from Hitchcock et al. [37]. Data from Pajouhesh et al. [38].
The values obtained for compound
central nervous system (CNS). On the other hand, TFA and compound 10 presented cLogP and LogBB
values below or close to the recommended limits. Therefore, TFA and compound 10 are less likely to
3 are within the optimal ranges of drugs able to act in the
2
cross the blood-brain barrier by passive diffusion and thereby attain the CNS. The presence of a free
carboxylic acid may restrict the access to the brain, as previously observed for similar systems [21].
Overall, these results suggest that TFA benzyl amide (compound
drug-like properties among the selected candidates.
3) presents the most favorable

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3. Materials and Methods
3.1. Chemistry
3.1.1. Synthesis
I. Synthesis of 6-Hydroxy-5-methoxy-[1,10-biphenyl]-3-carbaldehyde (12)
Synthesis and structural characterization are described in literature [21].
II. Thiocarbamoylation of Phenols
In a bottom flask with the phenol (1 mmol) dissolved in DMF, DABCO (4 mmol), and DMTCl
(2 mmol) were added. The mixture was heated at 70 ◦C for 3 h. Upon completion, the mixture was
poured onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was collected
by filtration under reduced pressure and washed with water. The general procedure was adapted
from Nowakowska et al. [39].
O-(4-Formyl-2-methoxyphenyl) dimethylcarbamothioate (15). Synthesis and structural analysis
are described in literature [24,39].
O-(4-Formyl-2,6-dimethoxyphenyl) dimethylcarbamothioate (16). The reaction mixture was poured
onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was collected by
1
filtration under reduced pressure and washed with water.
3.40 (s, 3H, NCH3), 3.49 (s, 3H, NCH3), 3.93 (s, 6H, 2
η
= 93%. H NMR (CDCl3-d1):
δ
(ppm) =
O).
H3), 106.3 (C2, C6), 134.2 (C1),
×
OCH3), 7.19 (s, 2H, H2, H6), 9.94 (s, 1H, C
H3), 56.5 (2
H
13C NMR (CDCl3-d1):
136.9 (C4), 153.4 (C3, C5), 186.7 (
δ
(ppm) = 38.9 (N
C
H3), 43.5 (N
C
×
O
C
CSO), 191.1
(C
HO). EI/MS m/z (%): 269.0 (M•+, 69), 88.0 (99), 72.0 (100).
O-(5-Formyl-3-methoxy-[1,10-biphenyl]-2-yl) dimethylcarbamothioate (17). The reaction mixture
was poured onto water (15 mL) cooled on an ice bath and acidified with HCl 6 M. The product was
1
collected by filtration under reduced pressure and washed with water.
δ
η
= 75%. H NMR (CDCl3-d1):
(ppm) = 3.17 (s, 3H, NCH3), 3.34 (s, 3H, NCH3), 3.94 (s, 3H, OC-H3), 7.39 (m, 3H, H30, H40, H50),
7.52 (m, 4H, H2, H6, H20, H60), 9.98 (s, 1H, C O). 13C NMR (CDCl3-d1):
H δ (ppm) = 38.7 (NCH3), 43.3
(NCH3), 56.4 (OC
H3), 109.7 (C2), 126.6 (C6), 128.0 (C40), 128.2 (C20, C60), 129.1 (C30, C50), 134.5 (C5),
136.4 (C1), 137.2 (C10), 145.0 (C4), 153.1 (C3), 186.4 (
243.0 (29), 88.0 (100), 72.0 (89).
CSO), 191.2 (C
HO). EI/MS m/z (%): 315.1 (M•+, 38),
III. Microwave-Assisted Newman-Kwart Rearrangement
O-phenyl-N,N-dimethylthiocarbamates 15 17 were placed onto a 5 mL microwave glass vial.
–
The vial was sealed with a septum and the content was maintained under an argon atmosphere
for 30 min at room temperature. After the addition of diphenyl ether, the vial was placed into the
microwave cavity. The reaction took place under microwave irradiation at 240 ◦C for 20 min.
S-(4-Formyl-2-methoxyphenyl) dimethylcarbamothioate (18). Synthesis and structural analysis
are described in the literature [24].
S-(4-Formyl-2,6-dimethoxyphenyl) dimethylcarbamothioate (19). Compound 19 was isolated
by flash column chromatography (ethyl acetate/dichloromethane (3:7)) and recrystallized in
1
dichloromethane/petroleum ether.
(bs, 3H, NC 3), 3.94 (s, 6H, 2 OC
(ppm) = 37.1 (2 H3), 56.7 (2
η
= 91%. H NMR (CDCl3-d1):
δ
(ppm) = 3.00 (bs, 3H, NCH3), 3.18
O). 13C NMR (CDCl3-d1):
H3), 105.0 (C2, C6), 112.7 (C4), 138.7 (C1), 161.8 (C3, C5), 164.5
H
×
H
3), 7.16 (s, 2H, H2, H6), 9.97 (s, 1H, C
H
δ
×
N
C
×
O
C
(COS), 191.5 (CHO). EI/MS m/z (%): 269.0 (M•+, 48), 72.1 (100).
S-(5-Formyl-3-methoxy-[1,10-biphenyl]-2-yl) dimethylcarbamothioate (20). Compound 20 was
isolated by flash column chromatography (ethyl acetate/dichloromethane (3:7)) and recrystallized in
1
dichloromethane/petroleum ether.
3.98 (s, 3H, OC
1H, H6), 10.01 (s, 1H, C
η
= 83%. H NMR (DMSO-d6):
δ
(ppm) = 2.96 (bs, 6H, 2
×
NCH3),
H
3), 7.38 (m, 5H, H20, H30, H40, H50, H60), 7.46 (d, J = 1.6 Hz, 1H, H2), 7.48 (d, J = 1.6 Hz,
O). 13C NMR (DMSO-d6):
(ppm) = 37.2 (2 H3), 56.8 (O H3), 108.2
H
δ
×
NC
C

Molecules 2019, 24, 4405
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(C2), 124.1 (C4), 125.8 (C6), 127.7 (C40), 127.8 (C20, C60), 129.4 (C30,), 137.8 (C5), 140.3 (C1), 149.6 (C10),
161.0 (C3), 165.0 (
(13), 72.1 (100).
COS), 191.7 (C
HO). EI/MS m/z (%): 315.3 (M•+, 21), 243.2 (6), 200.1 (7), 171.1 (9), 84.0
IV. Knoevenagel-Doebner Condensation
In a round bottom flask with the aldehyde (1 mmol) dissolved in anhydrous pyridine (11.0 mmol),
malonic acid (1.5 mmol) and piperidine (0.35 mmol) were added. The mixture was stirred at 70 ◦C for
4–5 h. The general procedure was adapted from Teixeira et al. [40] with some modifications.
(E)-3-(4-((Dimethylcarbamoyl)thio)-3-methoxyphenyl)acrylic acid (21). Synthesis and structural
analysis are described in literature [24].
(E)-3-(4-((Dimethylcarbamoyl)thio)-3,5-dimethoxyphenyl)acrylic acid (22). Once the reaction was
complete, the reaction mixture was poured onto water (15 mL), cooled on an ice bath and acidified
with HCl 6 M. The product was collected by filtration under reduced pressure and washed with water.
After suspension in methanol, the solid was centrifuged at 3000 g for 20 min. The supernatant was
1
discarded, yielding the purified product.
3.06 (bs, 3H, NC 3), 3.80 (s, 6H, 2 OC
= 16.0 Hz, 1H, H ), 12.46 (bs, 1H, COO
H3), 104.7 (C2, C6), 107.0 (C4), 121.3 (C
(COOH). EI/MS m/z (%): 311.1 (M•+, 40), 84.0 (9), 72.1 (100).
η
= 88%. H NMR (DMSO-d6):
δ
(ppm) = 2.88 (bs, 3H, NC
), 7.05 (s, 2H, H2, H6), 7.59 (d, J
(ppm) = 36.9 (2 H3), 56.8 (2
), 161.6 (C3, C5), 164.6 ( OS), 168.0
H3),
H
β
×
H3), 6.71 (d, J = 16.0 Hz, 1H, Hα
H
). 13C NMR (DMSO-d6):
), 138.0 (C1), 144.2 (C
δ
×
N
C
×
O
C
α
β
C
(E)-3-(6-((Dimethylcarbamoyl)thio)-5-methoxy-[1,10-biphenyl]-3-yl)acrylic acid (23). Once the
reaction was complete, the reaction mixture was poured onto water (15 mL), cooled on an ice bath,
and acidified with HCl 6 M. The product was collected by filtration under reduced pressure and
washed with water. After suspension in methanol, the solid was centrifuged at 3000 g for 20 min.
1
The supernatant was discarded, yielding the purified product.
= 2.88 (bs, 6H, 2 NCH3), 3.87 (s, 3H, OCH3), 6.72 (d, J = 16.0 Hz, 1H, H
7.28 (m, 2H, H20, H60), 7.39 (m, 3H, H30, H40, H50), 7.45 (d, J = 1.6 Hz, 1H, H6), 7.63 (d, J = 16.0 Hz, 1H,
), 12.46 (bs, 1H, COO (ppm) = 36.9 (2 H3), 57.0 (O H3), 110.0 (C2),
). 13C NMR (DMSO-d6):
118.3 (C4), 121.5 (C
), 123.1 (C6), 127.9 (C40), 128.2 (C20, C60), 129.0 (C30, C50), 137.2 (C5), 140.9 (C1),
143.6 (C OS), 167.9 (
), 149.0 (C10), 161.1 (C3), 165.0 (
OOH). EI/MS m/z (%): 357.3 (M•+, 46), 268.2
(28), 72.1 (100).
η
= 59%. H NMR (DMSO-d6):
δ
(ppm)
×
α
), 7.24 (d, J = 1.5 Hz, 1H, H2),
Hβ
H
δ
×
N
C
C
α
β
C
C
V. PyBOP-Mediated Cinnamic Acid Amidation
In a round bottom flask, compound 21 (1 mmol) was dissolved in DMF and DIPEA (1.01 mmol).
The mixture was cooled at 0 ◦C on an ice bath, and a solution of PyBOP (1.01 mmol) in dichloromethane
(2.25 mL) was added slowly. The mixture was stirred for 30 min, and the amine (1.01 mmol) was added.
The general procedure was adapted from Gaspar et al. [41] with some modifications.
(E)-S-(4-(3-(Benzylamino)-3-oxoprop-1-en-1-yl)-2-methoxyphenyl) dimethylcarbamothioate (24).
Flash column chromatography was performed in ethyl acetate/dichloromethane (3:7). Recrystallization
1
in dichloromethane/petroleum ether yielded a white solid.
2.90 (bs, 3H, NC 3), 3.10 bs, 3H, NC 3), 3.86 (s, 3H, OC
J = 15.6 Hz, 1H, H ), 6.44 (t, J = 5.5 Hz, 1H, CON ), 6.96 (d, J = 1.5 Hz, 1H, H2), 7.04 (dd, J = 1.5 Hz, 7.9
Hz, 1H, H6), 7.30 (m, 5H, H20, H30, H40, H50, H60), 7.39 (d, J = 7.9 Hz, 1H, H5), 7.53 (d, J = 15.6 Hz,
1H, H (ppm) = 37.0 (2 H3), 43.9 (NH H2Ph), 56.1 (O H3), 110.9 (C2),
). 13C NMR (CDCl3-d1):
118.1 (C4), 120.2 (C
), 122.0 (C6), 127.4 (C40), 128.0 (C20, C60), 128.6 (C30, C50), 138.0 (C5) 138.3 (C1),
138.4 (C10), 140.03 (C
), 160.1 (C3), 165.5 ( OS), 166.3 (
ONH). EI/MS m/z (%): 370.3 (M•+, 59), 281.2
(22), 91.1 (32), 72.1 (100).
η
= 95%. H NMR (CDCl3 -d1):
δ
(ppm) =
H
H
H
3), 4.53 (d, J = 5.7 Hz, 2H, NHC
H
2Ph), 6.24 (d,
α
H
β
δ
×
NC
C
C
α
β
C
C
(E)-S-(2-Methoxy-4-(3-oxo-3-(phenethylamino)prop-1-en-1-yl)phenyl) dimethylcarbamothioate
25). Flash column chromatography was performed in ethyl acetate/dichloromethane (3:7).
(
Recrystallization in dichloromethane/petroleum ether yielded a white solid.
η
= 80%. 1H NMR
3),
(CDCl3-d1): (ppm) = 2.87 (t, J = 7.0 Hz, 2H, NHCH2C 2Ph), 3.00 (bs, 3H, NC 3), 3.13 (bs, 3H, NCH
δ
H
H

Molecules 2019, 24, 4405
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3.62 (m, 2H, NHCH2CH2Ph), 3.86 (s, 3H, OCH3), 5.93 (t, J = 5.7 Hz, 1H, N
H
), 6.22 (d, J = 15.6 Hz, 1H,
Hα
), 6.98 (d, J = 1.5 Hz, 1H, H2), 7.06 (dd, J = 1.5 Hz, 7.9 Hz, 1H, H6), 7.28 (m, 5H, H20, H30, H40, H50,
H60), 7.42 (d, J = 7.9 Hz, 1H, H5), 7.52 (d, J = 15.6 Hz, 1H, H
(NHCH2 H2Ph), 37.0 (2 H3), 40.8 (NH H2CH2Ph), 56.1 (O
), 122.1 (C6), 126.5 (C40), 128.7 (C20, C60), 128.8 (C30, C50), 138.1 (C5) 138.25 (C1), 139.0 (C10), 140.0
), 160.1 (C3), 165.6 ( OS), 166.1 (
ONH). EI/MS m/z (%): 384.3 (M•+, 47), 295.2 (13), 105.1 (16),
β
). 13C NMR (CDCl3-d1):
δ (ppm) = 35.0
C
×
NC
C
C
H3), 110.8 (C2), 118.3 (C4), 120.1
(C
(C
α
β
C
C
72.1 (100).
VI. Alkaline Hydrolysis
To a solution of the S-phenyl-N,N-dimethylthiocarbamate (1 mmol) in methanol (1.5 mL), aqueous
solution of sodium hydroxide 2 M (5–17.5 mmol) was added dropwise. The reaction mixture was
stirred at 90 ◦C until the TLC analysis showed maximal substrate consumption.
(E)-3-(4-Sulfanyl-3-methoxyphenyl)acrylic acid (TFA,
2). Synthesis and structural analysis is
described in literature [24].
(E)-N-Benzyl-3-(4-sulfanyl-3-methoxyphenyl)acrylamide (
3
). Compound was obtained by an
3
alkaline hydrolysis of compound 24 with the following conditions: compound 24 (0.82 g, 2.20 mmol),
methanol (4.5 mL), and aqueous solution of NaOH 2 M (5.50 mL, 11.0 mmol). The mixture was
protected from the light and stirred at 90 ◦C for 1.5 h. Upon completion, the mixture was allowed to
cool at room temperature, then poured onto cooled aqueous saturated solution of NH4Cl (15 mL) and
acidified with aqueous solution of acetic acid 5 M added dropwise. The solid was filtered under reduced
pressure and washed with water. TLC analysis was performed in ethyl acetate/dichloromethane/formic
1
acid (3:7:0.01).
η
= 53%. H NMR (MeOD-d4):
δ
(ppm) = 3.91 (s, 3H, OC
), 7.05 (dd, J = 1.5 Hz, 8.0 Hz, 1H, H6), 7.10 (d, J = 1.3 Hz, 1H, H2), 7.24 (m,
2H, H5 H40), 7.32 (m, 4H, H20, H30, H50, H60), 7.51 (d, J = 15.7 Hz, 1H, H ). 13C NMR (MeOD-d4):
), 120.5 (C6), 124.3 (C4), 126.9 (C40), 127.2
H3), 4.49 (s, 2H, NHCH2Ph),
6.60 (d, J = 15.7 Hz, 1H, H
α
β
δ
(ppm) = 42.9 (NHCH2Ph), 55.0 (OCH3), 109.2 (C2), 119.4 (Cα
(C20, C60), 128.2 (C30, C50), 128.7 (C5) 133.0 (C1), 138.5 (C10), 140.4 (C
β), 155.1 (C3), 167.2 (
CONH).
ESI/MS m/z (%): 300 (M+ + 1, 100), 284 (55).
(E)-3-(4-Sulfanyl-3-methoxyphenyl)-N-phenethylacrylamide (
4
). Compound was obtained by
4
an alkaline hydrolysis of compound 25 with the following conditions: compound 25 (0.42 g, 1.1 mmol),
methanol (1.6 mL), and an aqueous solution of NaOH 2 M (2.7 mL, 5.5 mmol). The mixture was
protected from the light and stirred at 90 ◦C for 1.5 h. Upon completion, the mixture was allowed to
cool at room temperature, then poured onto a cooled aqueous saturated solution of NH4Cl (15 mL) and
acidified with aqueous solution of acetic acid 5 M added dropwise. The solid was filtered under reduced
pressure and washed with water. TLC analysis was performed in ethyl acetate/dichloromethane/formic
1
acid (3:7:0.01).
η
= 45%. H NMR (CDCl3-d1):
δ
(ppm) = 2.89 (t, J = 7.0 Hz, 2H, NHCH2C
), 6.29 (d, J = 15.5 Hz, 1H, H
J = 1.5 Hz, 1H, H2), 6.99 (dd, J = 1.5 Hz, 8.0 Hz, 1H, H6), 7.23 (m, 4H, H5, H30, H40, H50), 7.32 (m,
H20, H60), 7.53 (d, J = 15.5 Hz, 1H, H ). 13C NMR (CDCl3-d1):
(ppm) = 35.7 (NHCH2 H2Ph), 40.8
), 120.8 (C6), 123.4 (C4), 126.6 (C40), 128.7 (C20,
H
2Ph), 3.66
(m, 2H, NHCH2CH2Ph), 3.90 (s, 3H, OCH3), 5.66 (m, 1H, N
H
α), 6.92 (d,
β
δ
C
(NHCH2CH2Ph), 55.9 (OCH3), 109.5 (C2), 119.9 (Cα
C60), 128.8 (C30, C50), 129.1 (C5), 133.2 (C1), 138.9 (C10), 140.7 (C
β), 154.8 (C3), 165.9 (CONH). ESI/MS
m/z (%): 314 (M+ + 1, 100).
(E)-3-(4-Sulfanyl-3,5-dimethoxyphenyl)acrylic acid (5). Compound 5 was obtained by an alkaline
hydrolysis of compound 22 with the following conditions: compound 22 (0.64 g, 2.05 mmol), methanol
(2 mL) and aqueous solution of NaOH 2 M (12.5 mL, 2.5 mmol). The mixture was protected from
◦
the light and stirred at 90 C for 9 h. Upon completion, the mixture was allowed to cool at room
temperature, then poured onto a cooled aqueous saturated solution of NH4Cl (15 mL) and acidified
with aqueous solution of acetic acid 5 M added dropwise. The solid was filtered under reduced
pressure and washed with water. TLC analysis was performed in ethyl acetate/dichloromethane/formic
1
acid (3:7:0.01).
(d, J = 15.9 Hz, 1H, H
η
= 79.6%. H NMR (CDCl3-d1):
δ
(ppm) = 3.94 (s, 6H, 2
×
OC
β
H3), 4.19 (s, 1H, SH), 6.40
α
), 6.74 (s, 2H, H2, H6), 7.72 (d, J = 16.0 Hz, 1H, H
). 13C NMR (CDCl3-d1):
δ

Molecules 2019, 24, 4405
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(ppm) = 56.3 (2
172.2 (COOH). ESI/MS m/z (%): 241 (M+ + 1, 100).
(E)-3-(6-Sulfanyl-5-methoxy-[1,10-biphenyl]-3-yl)acrylic acid (
×
O
C
H3), 103.8 (C2, C6), 114.2 (C4), 116.3 (C
α
), 131.0 (C1), 147.1 (C
β
), 155.4 (C3, C5),
6). Compound 6 was obtained by
an alkaline hydrolysis of compound 23 the following conditions: compound 23 (0.36 g, 1.01 mmol),
methanol (2 mL) and aqueous solution of NaOH 2 M (8.75 mL, 17.5 mmol). The mixture was protected
from the light and stirred at 90 ◦C for 16 h. Upon completion, the mixture was allowed to cool at room
temperature, then poured onto cooled aqueous saturated solution of NH4Cl (15 mL) and acidified with
aqueous solution of acetic acid 5 M added dropwise. The solid was filtered under reduced pressure
and washed with water. TLC analysis was performed in ethyl acetate/dichloromethane/formic acid
1
(3:7:0.01).
η
= 73%. H NMR (MeOD-d4):
δ
(ppm) = 3.99 (s, 3H, OCH3), 6.47 (d, J = 15.9 Hz, 1H, H
α
),
7.03 (d, J = 1.4 Hz, 1H, H2), 7.20 (d, J = 1.7 Hz, 1H, H6), 7.40 (m, 5H, H20 H30, H40, H50, H60), 7.63 (d, J
= 15.9 Hz, 1H, Hβ δ (ppm) = 55.6 (OCH3), 107.7 (C2), 117.5 (Cα), 122.6 (C6),
). 13C NMR (MeOD-d4):
124.3 (C4), 127.6 (C40), 128.1 (C20, C60), 128.7 (C30, C50), 131.5 (C5), 140.2 (C1), 141.2 (C10), 144.4 (C
β),
154.5 (C3), 169.2 (COOH). ESI/MS m/z (%): 287 (M+ + 1, 100).
3.2. Radical Scavenging Activity
3.2.1. Spectrophotometric Methods
I. DPPH• Radical Assay
DPPH• radical scavenging activity was performed as described by Teixeira et al. [40] (see
Supplementary Information).
II. ABTS• Radical Cation Assay
+
ABTS• scavenging activity was evaluated as described by Teixeira et al. [40] (see Supplementary
+
Information).
III. GO• Radical Assay
GO• radical scavenging protocol was adapted from the literature [42
Supplementary Information.
–44] and described in
3.2.2. Fluorometric Methods (ORAC-FL Assay)
The ORAC-FL analyses were performed using an experimental protocol adapted from the
literature [45,46] and described in Supplementary Information.
3.3. Electrochemical Measurements
DPV experiments were performed as described elsewhere [23] (see Supplementary Information).
3.4. In Vitro Toxicology
3.4.1. SH-SY5Y Cell Culture
Human SH-SY5Y neuroblastoma cells were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA). SH-SY5Y cell culture and differentiation were performed as previously
described by Fernandes et al. [32] and in Supplementary Information.
3.4.2. Cytotoxicity
Stock solutions of the test compounds (1, 10 and 50 mM) were freshly prepared in ethanol absolute.
Final test concentrations of the compounds under study were obtained by diluting into culture medium
immediately before use, giving a final maximum concentration of 0.1% ethanol.

Molecules 2019, 24, 4405
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In cytotoxicity experiments, differentiated SH-SY5Y cells were exposed to the test compounds (1,
10 and 50 M) for 24 h. Controls were treated with culture media containing 0.1% ethanol. Cell viability
µ
was estimated using the MTT reduction assay [32] and the NR uptake assay [47] (see Supplementary
Information).
3.4.3. Protection against Oxidative Stress Inducers
To evaluate the ability of phenols and thiophenols to protect neuronal cells from oxidative stress,
hydrogen peroxide (H2O2) and ferric nitrilotriacetate (FeNTA) were used as inducers of oxidative
damage. Differentiated SH-SY5Y cells were pre-treated with the compounds under study for 24 h and
exposed to the oxidative stressors.
I. Cytoprotective Properties against H2O2-Induced Damage
Differentiated SH-SY5Y cells were pretreated with the test compounds (10 and 50
µM) for
24 h. Then, the culture medium was removed and replaced with medium containing H2O2 1.5 mM,
and cells were incubated for additional 30 min at 37 ◦C in a humidified, 5% CO2
Cell viability was estimated by the MTT reduction assay [32].
−
95% air atmosphere.
II. Cytoprotective Properties against FeNTA-Induced Damage
Nitrilotriacetic acid disodium salt solution (500 mM) and nitrilotriacetic acid trisodium salt
solution (500 mM) were mixed until pH 7 was achieved and diluted to obtain a concentration of
250 mM. This solution was added to the required amount of iron(III) chloride to obtain the NTA:iron(III)
molar ratio of 2.5:1 ([Fe(III) = 100 mM). Then, the ferric nitrilotriacetic acid (FeNTA) solution was
diluted in culture medium to obtain a final iron(III) solution with concentration of 4 mM.
Differentiated SH-SY5Y cells were pretreated with the test compounds (10 and 50 µM) for 24 h.
Then, the culture medium was removed and replaced with medium containing FeNTA 4 mM. Cells
were incubated for additional 24 h at 37 ◦C in a humidified, 5% CO2
was estimated by the MTT reduction assay [32].
−
95% air atmosphere. Cell viability
3.4.4. Statistical Analysis
Data analysis for all the studies are specified in Supplementary Information.
3.5. Estimation of Drug-Like Properties
The calculation of molecular weight (MW), topological polar surface area (TPSA), number of
hydrogen bond donors (HBD) and acceptors (HBA), number of rotatable bonds (RB) and logarithm of
the ratio of the concentration of a drug in the brain and in the blood (logBB) was performed using the
Stardrop software. The calculation of octanol/water partition coefficient (clogP) was performed using
the SwissADME (http://swissadme.ch/index.php).
4. Conclusions
In this work, we successfully obtained a series of TFA and FA derivatives, and evaluated their
antioxidant activity, as well as their cytotoxicity and cytoprotective profiles. Structural diversity was
attained by incorporating 5-methoxy or 5-phenyl groups at the aromatic ring, or benzyl and phenethyl
amide groups at the carboxylic acid group.
Thiophenols displayed remarkable radical scavenging properties. Compared to phenols,
thiophenols reacted faster and more efficiently with DPPH• and GO• radicals, and presented lower
Ep values at physiological pH. However, unlike phenols, thiophenols were unable to scavenge ROO•
radicals in ORAC-FL assay. The presence of different substituents directly attached to the aromatic
ring did not improve the antioxidant profile of the parent compounds (TFA and FA). Although a
slight decrease of Ep values was observed, 5-methoxy and 5-phenyl derivatives showed similar or

Molecules 2019, 24, 4405
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lower radical scavenging activity, and slower reaction rates than the unsubstituted analogues. On the
other hand, the carboxylic acid amidation was proven to be a beneficial structural modification of the
cinnamic acid scaffold, since it preserved the antioxidant profile of the parent acids while improving the
drug-like properties of the antioxidants. The cytotoxicity of FA and TFA derivatives in differentiated
SH-SY5Y cells was similar to that of the parent compounds at concentrations up to 50
µM. Among
the tested compounds, only TFA , TFA benzyl amide , and 5-phenylferulic acid 10 attenuated
2
3
the oxidative damage inflicted by H2O2 and iron overload. Compound
antioxidant of the series with favorable physicochemical properties.
3 is highlighted as the best
Overall, the results obtained in this work emphasized the dissimilar biological outline of
thiophenols and phenols, as well as the effect of the structural modification of FA and TFA on
their antioxidant properties. The data may be useful for the development of new drugs able to tackle
the events that lead to the progression of neurodegenerative disorders.
Supplementary Materials: The following are available online, Experimental details, Statistics, Figure S1: DPPH•
•
+
kinetic curves of thiophenols
2
–
6
and phenols
1
,
7–
10; Figure S2
:
ABTS kinetic curves of thiophenols
2
–
6
and
phenols
against (
1,
7
–10; Figure S3
:
GO• kinetic curves of thiophenols
2
–
6
and phenols
1,
7–
10; Figure S4: Protection
A
) H2O2- and (
B
) FeNTA-induced damage in differentiated SH-SY5Y cells by caffeic acid (CA); Figure S5:
1H and 13C NMR spectra of final compound
3
(NMR spectrum obtained in MeOD-d4); Figure S6: 1H and 13
(NMR spectrum obtained in MeOD-d4); Figure S7: 1H and 13C NMR spectra of
5
(NMR spectrum obtained in MeOD-d4); Figure S6: 1H and 13C NMR spectra of final compound
C
NMR spectra of final compound
final compound
6 (NMR spectrum obtained in MeOD-d4).
4
Author Contributions: Performing of experiments, data acquisition and analysis D.C., C.F., B.A., T.S. and J.G.;
D.C., C.F., B.A., T.S., J.G., F.R., P.J.O., E.U. and F.B. contributed for writing and reviewing of manuscript.
Funding: This project was supported by Foundation for Science and Technology (FCT) and FEDER/COMPETE
research grants (UID/QUI/00081, NORTE-01–0145-FEDER-000028, POCI-01–0145-FEDER-029164).
D.
Chavarria, C. Fernandes and T. Silva grants are supported by FCT, POPH and FEDER/COMPETE and
NORTE-01-0145-FEDER-000028.This article is based upon work from COST Action CA15135.
Conflicts of Interest: The authors declare no conflict of interest.
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