Antioxidant, Anti-inflammatory, and Neuroprotective Effects of Novel Vinyl Sulfonate Compounds as Nrf2 Activator

Article abstract of DOI:10.1021/acsmedchemlett.9b00163

The main pathway responsible for cellular regulation against oxidative stress is nuclear factor E2-related factor-2 (Nrf2) signaling. We previously synthesized and reported a novel vinyl sulfone (1) as an Nrf2 activator with therapeutic potential for Parkinson's disease (PD). In this study, we changed the vinyl sulfone to vinyl sulfonamide or vinyl sulfonate to improve Nrf2 activating efficacy. We observed that the introduction of vinyl sulfonamide led to a reduction of the effects on Nrf2 activation, whereas vinyl sulfonate compounds exhibited superior activity compared to the vinyl sulfone compounds. Among the vinyl sulfonates, 3c exhibited 6.9- and 83.5-fold higher effects on Nrf2 activation than the corresponding vinyl sulfone (1) and vinyl sulfonamide (2c), respectively. Compound 3c was confirmed to induce expression of the Nrf2-dependent antioxidant enzymes at the protein level in cells. In addition, 3c mitigated PD-associated behavioral deficits by protecting DAergic neurons in the MPTP-induced mouse model of PD.

Full text of DOI:10.1021/acsmedchemlett.9b00163

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Letter
Anti-oxidant, Anti-inflammatory, and Neuroprotective Effects
of Novel Vinyl Sulfonate Compounds as Nrf2 Activator
Ji Won Choi, Su Jeong Shin, Hyeon Ji Kim, Jong-Hyun Park, Hyeon Jeong
Kim, Elijah Hwejin Lee, Ae Nim Pae, Yong-Sun Bahn, and Ki Duk Park
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00163 • Publication Date (Web): 03 Jun 2019
Downloaded from http://pubs.acs.org on June 9, 2019
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Anti-oxidant, Anti-inflammatory, and Neuroprotective Effects of Novel
Vinyl Sulfonate Compounds as Nrf2 Activator
a,†
a,b,†
a,b
a
a,b
Ji Won Choi , Su Jeong Shin , Hyeon Ji Kim , Jong-Hyun Park , Hyeon Jeong Kim , Elijah
a
a,c,d
b
a,c,d,
Hwejin Lee , Ae Nim Pae , Yong Sun Bahn , Ki Duk Park
*
a
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and
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Technology (KIST), Seoul 02792, Republic of Korea Department of Biotechnology, Yonsei University, Seoul 03722,
c
Republic of Korea Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and
d
Technology, Seoul 02792, Republic of Korea, KHU-KIST Department of Converging Science and Technology, Kyung Hee
University, Seoul 02447, Republic of Korea
Keywords: Parkinson’s disease, Anti-oxidant, Anti-inflammatory, Nrf2/Keap1 pathway, MPTP mouse model, Vinyl sulfone.
ABSTRACT: The main pathway responsible for cellular regulation against oxidative stress is nuclear factor E2-related factor-2 (Nrf2)
signaling. We previously synthesized and reported a novel vinyl sulfone (1) as an Nrf2 activator with therapeutic potential for
Parkinson’s disease (PD). In this study, we changed the vinyl sulfone to vinyl sulfonamide or vinyl sulfonate to improve Nrf2
activating efficacy. We observed that the introduction of vinyl sulfonamide led to a reduction of the effects on Nrf2 activation,
whereas vinyl sulfonate compounds exhibited superior activity than the vinyl sulfone compounds. Among the vinyl sulfonates, 3c
exhibited 6.9- and 83.5-fold higher effects on Nrf2 activation than the corresponding vinyl sulfone (1) and vinyl sulfonamide (2c),
respectively. 3c was confirmed to induce expression of the Nrf2 dependent antioxidant enzymes at the protein level in cells. In
addition, 3c mitigated PD-associated behavioral deficits by protecting DAergic neurons in the MPTP-induced mouse model of PD.
Parkinson’s disease (PD) is the most prevalent
neurodegenerative disorder featured clinically by motor
deficits. PD patients have clinical features such as tremor,
rigidity, slowness of movement and postural disability. Most
of the symptoms are caused by the death of dopaminergic
Although the precise mechanism associated with the Nrf2-
mediated anti-inflammation pathway has not been elucidated in
detail, recent studies reported that inflammatory mediators were
overexpressed in the Nrf2 knockout mice and deficiency in
transcription factor Nrf2 worsens inflammatory parameters in a
1
,2
1
8-20
(
DAergic) neurons that produce chemical messengers called
mouse model of neurodegenerative diseases.
The sole FDA-
3
,4
dopamine in the substantia nigra (SN) pars compacta. The
etiology and pathogenesis of PD is not yet known, but several
studies have suggested that oxidative stress is partly responsible
for the loss of DAergic neurons and plays an important role in
approved Nrf2 activator, dimethyl fumarate (DMF, brand name
TM
Tecfidera ), shows effectiveness in treating multiple sclerosis
21,22
(MS) by regulating inflammation.
Taken together, targeting
the Nrf2 pathway is an attractive therapeutic strategy for
neurodegenerative diseases including PD through anti-
inflammatory and antioxidant effects.
5
-7
neurodegeneration of PD. Another factor contributing to the
pathogenesis of PD has been thought to be neuro-inflammation.
Microglial activation is linked to the loss of DAergic neurons,
suggesting that neuronal inflammation contribute to the
We previously synthesized and reported vinyl sulfone
23-25
scaffolds as an Nrf2 activator.
The lead compound (1) with
8
,9
progressive degeneration.
Several clinical studies have
the vinyl sulfone scaffold induced expression of Nrf2-
reported that the levels of inflammatory enzymes have been
dependent antioxidant enzyme and relieved the production of
elevated in mouse models of PD and brain of PD patients.10-12
23,24
inflammatory mediators through Nrf2 signaling.
Compound
Nuclear factor E2-related factor-2 (Nrf2), encoded by gene
NFE2L2, is a nuclear transcription factor that regulates cellular
defense system against oxidative stress and has been shown to
1 also showed promising efficacy in the 1-methyl-4-phenyl-
1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse model.
1
3,14
down-regulate inflammatory responses.
conditions, Nrf2 adheres to Kelch-like ECH-associated protein
(Keap1) as a complex and undergoes ubiquitination and
Under normal
OMe O
S
Cl
O
S
O
S
R1
A
R1
R2
A
N
O
A
O
B
H O
B
O
B
R2
1
proteasomal degradation in cytoplasm. Under oxidative stress
conditions, Nrf2 is liberated from Keap1 and translocated into
the nucleus, which binds to the antioxidant response element
(ARE) sequence in the DNA promoter region and begins
transcription of the ARE-regulated antioxidant enzyme such as
heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL),
and reduced nicotinamide adenine dinucleotide phosphate
1
2
3
In this study, we replaced the sulfone moiety of compound 1
with a sulfonamide or sulfonate. Vinyl sulfonamides (2) and
vinyl sulfonates (3) can be conveniently synthesized and also
have α,β-unsaturated sulfone group which is highly activated
Michael acceptor and may be responsible for activating Nrf2.
Here, we report the development, synthesis and biological
evaluation of a series of vinyl sulfonamides and vinyl
sulfonates. We observed that vinyl sulfonate scaffold enhanced
1
5-17
(
NAD(P)H) quinone oxidoreductase 1 (NQO1).
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Nrf2 activating efficacy. The most potent compound (3c) was
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3b
O
O
O
O
O
O
O
O
O
O
4-OMe
2-OMe
3-OMe
4-OMe
2-OMe
3-OMe
4-OMe
2-OMe
3-OMe
4-OMe
-H
1.480 ± 0.047
0.076 ± 0.009
0.165 ± 0.026
0.237 ± 0.015
0.698 ± 0.016
0.943 ± 0.050
0.948 ± 0.028
1.334 ± 0.024
1.778 ± 0.030
1.155 ± 0.036
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evaluated for the expression of anti-oxidant enzymes and
attenuated the production of inflammatory cytokines in BV-2
microglial cells and SH-SY5Y human neuroblastoma cells.
Finally, the compound was tested for in vivo therapeutic effects
on parkinsonism in the MPTP-induced mouse model.
3
c
2’-Cl
2’-Cl
2’-Cl
3’-Cl
3’-Cl
3’-Cl
4’-Cl
4’-Cl
4’-Cl
3
d
3e
3f
3
g
RESULTS AND DISCUSSION
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Scheme 1. Synthesis of compound 2 and 3
3j
NH2
3
k
O
S
R1
4
R1
3
O
S
2'
A
2'
a)
b)
1b
SFNc
R2
Cl
3'
R2
4'
N
3'
R2
4'
2-OMe
2’-Cl
0.530 ± 0.025
0.580 ± 0.024
O
R1 = H, OMe
2
H O
B
2
2
2
a: R1 = H, R2 = H
b: R1 = H, R2 = 2'-Cl
c: R1 = 2-OMe, R2 = 2'-Cl
4
a: R2 = H
5a: R2 = H
a
4b: R2 = 2'-Cl
5b: R2 = 2'-Cl
5c: R2 = 3'-Cl
5d: R2 = 4'-Cl
The Nrf2 functional assay was accomplished by a PathHunter®
4
4
c: R2 = 3'-Cl
d: R2 = 4'-Cl
2d: R1 = 3-OMe, R2 = 2'-Cl
U2OS Nrf2 nuclear translocation cell line (93-0821C3,
DiscoveRx). U2OS cells were plated at 13,000 cells/well in
triplicate with various compound concentrations for 6 h. Nrf2
translocation, which is activation-dependent, was determined using
a cell-based functional assay of the mean ± standard error half
maximal effective concentration (EC50) values. Compound 1
developed as an Nrf2 activators in previous study.
sulforaphane: a well-known potent Nrf2 activator.
2e: R1 = 4-OMe, R2 = 2'-Cl
OH
R1
4
R1
3
O
A
2'
S
O
3'
R2
4'
R1 = H, OMe
2
O
B
b
24
c
SFN,
3a: R1 = H, R2 = H
3b: R1 = 4-OMe, R2 = H
3
3
c: R1 = 2-OMe, R2 = 2'-Cl
d: R1 = 3-OMe, R2 = 2'-Cl
The synthesized compounds were evaluated for Nrf2
activation in the optimized cell-based system. As we previously
reported, this assay system assesses the ability of synthesized
compounds to free Nrf2 from Keap1 and induce Nrf2
translocation to the nucleus using genetically engineered stable
3e: R1 = 4-OMe, R2 = 2'-Cl
3f: R1 = 2-OMe, R2 = 3'-Cl
3g: R1 = 3-OMe, R2 = 3'-Cl
3h: R1 = 4-OMe, R2 = 3'-Cl
3i: R1 = 2-OMe, R2 = 4'-Cl
3j: R1 = 3-OMe, R2 = 4'-Cl
k: R1 = 4-OMe, R2 = 4'-Cl
3
2
5,26
cell-line.
The efficacy of Nrf2 activation is described in
Reagents and conditions: a) SO
CH Cl , r.t, 5 h.
2 2 3
Cl , DMF, 0 °C to r.t, 3 h; b) Et N,
Table 1 as EC50 values. In this assay, the previously developed
compound 1 was confirmed to increase Nrf2 activation (1 EC50
= 0.530 μM).
2
2
We further optimized previously developed compound 1 by
modifying the α,β-unsaturated sulfone position according to
two categories: vinyl sulfonamides (2) or vinyl sulfonates (3).
The synthetic pathway of vinyl sulfonamides (2) and vinyl
sulfonates (3) are described in Scheme 1. Various styrenes (4a–
First, we replaced the sulfone moiety with sulfonamide, and
observed that Nrf2 activation was minimal. In our previous
study, we found that the addition of the methoxy (OMe) and
chloride (Cl) functional groups improved Nrf2 activation. We
systematically attached a methoxy group to the 2-, 3-, 4-
positions on the A ring and a chloride group to the 2’-, 3’-, 4’-
of the B ring. We found that addition of a methoxy group to the
4
2 2
d) were prepared by addition of sulfuryl chloride (SO Cl ) in
dimethylformamide (DMF) for 3 h at 90°C to produce (E)-2-
phenylethene-1-sulfonyl chlorides (5a–5d). Condensation of
5a–5d with various anilines or phenols in the presence of
triethylamine (TEA) in dichloromethane (CH Cl ) for 5 h at
2 2
room temperature provided the sulfonamides 2a–2e and the
sulfonates 3a–3k, respectively.
2
-position on the A ring and a chloride to the 2’-position on the
B ring enhanced the Nrf2 activation relative to compound 2a
(2c EC50 = 6.35 μM vs 2a EC50 > 10 μM). Next, we substituted
the sulfone moiety with sulfonate. Impressively, compound 3a
showed Nrf2 activation in comparison to that of compound 2a
(
3a EC50 = 1.04 μM). We again systematically introduced the
Table 1. EC50 values against Nrf2 activation of synthesized
compounds
methoxy and chloride functional groups and observed Nrf2
activation. Similar to the results of a previous study, the vinyl
sulfonates exhibited the most potent Nrf2 activation when 2-
OMe (2-OMe > 3-OMe > 4-OMe) was added to R
and 2′-Cl (2′-Cl > 3′-Cl > 4′-Cl) was inserted into R
ring. The compounds 3c–3e with 2’-Cl on B ring exhibited
potent Nrf2 activation than compound 1. In these eleven series
of compounds, 3c with 2-OMe on A ring showed about 7-fold
increase in Nrf2 activation than compound 1 (3c EC50 = 0.076
μM vs 1 EC50 = 0.530 μM). Thus, we selected 3c for further
biological studies because it exhibited the most potent effect on
Nrf2 activation (EC50 = 0.076 μM; Figure 1a) and favorable
metabolic stabilities (Table S1 in the Supporting Information)
4
O
S
R1
A
2'
1
of the A ring
of the B
3
X
3'
R2
4'
2
O
B
2
Compd.
X
R
1
R
2
Nrf2 EC50 (μM)a
2
a
N
N
N
N
N
O
-H
-H
-H
> 10
> 10
2
b
2’-Cl
2’-Cl
2’-Cl
2’-Cl
-H
2
c
2-OMe
3-OMe
4-OMe
-H
6.350 ± 0.039
> 10
2d
2e
> 10
The cytotoxic potential of 3c was assessed and nearly 100%
cell survival was observed up to a concentration of 10 μM (98.0
3
a
1.040 ± 0.015
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1.2% at 1 μM, 97.6 ± 2.4% at 3 μM, 95.9 ± 1.6% at 10 μM;
±
1
2
3
4
5
6
7
8
9
Figure 1b). We then examined the effects on Nrf2 activation
and expression of anti-oxidant enzymes in vitro. The results
indicated that 3c increased Nrf2 activation in a concentration-
dependent manner. Figure 1c shows that treatment with 3c
significantly increased nuclear Nrf2 protein levels in a
concentration-dependent manner (4.0 ± 0.01-fold at 0.3 μM,
1
7.6 ± 0.04-fold at 10 μM). We also confirmed that total Nrf2
protein levels increased in a concentration-dependent manner
3.3 ± 0.02-fold at 0.3 μM, 6.7 ± 0.03-fold at 10 μM; Figure 1d).
(
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Figure 2. 3c induces gene expression of Nrf2-dependent
antioxidant enzymes in BV-2 cells. (a) BV-2 cells were exposed to
various concentrations of 3c for 3 h followed by RT-qPCR analysis
of HO-1, GCLM, and GCLC expression. (b) BV-2 cells were
treated with 3c for 12 h, and protein levels of HO-1, GCLM, and
GCLC were measured via Western blot analysis. The expression
levels of antioxidant enzymes were normalized to β-actin. All
experiments were performed at least in duplicate cultures, and the
data were averaged and expressed as fold induction of untreated
control ± SEM. **P < 0.01, *** P < 0.005 (paired t-test).
NQO1 is a crucial factor in the metabolism and toxicity of
quinone, and acts as a detoxification agent in mitochondrial
phase 2 metabolism. Western blot analysis revealed that the
protein level of NQO1 increased 4.7-fold by treating with 3c
(Figure 3). As observed in BV-2 cells, GCL protein levels were
also concentration-dependently increased by treatment with 3c
in SH-SY5Y cells. Both GCLM and GCLC protein levels were
significantly upregulated by treatment with 3c, reaching 2.9-
and 1.5-fold induction, respectively. These results indicate that
Figure 1. Nrf2 translocation to the nucleus is activated by 3c. (a)
The Nrf2 translocation assay of engineered U2OS cells were
treated with various concentrations of 3c. (b) Viability assay of BV-
3
c quantitatively upregulates the Nrf2/ARE pathway in BV-2
cells and SH-SY5Y.
2
cells treated with 3c for 6 h. (c) After treatment with 3c (0.3‒10
μM), Nrf2 levels were analyzed by Western blotting using whole
BV-2 microglial cells (d) and nuclear extracts. All experiments
were performed at least in duplicate cultures, and the data were
averaged and expressed as fold induction of untreated control ±
SEM. ****P < 0.001 vs untreated control (paired t-test).
We investigated whether the expression of ARE-targeted anti-
oxidant enzymes were induced by Nrf2 activation. The enzyme
HO-1 degrades heme to carbon monoxide, iron ions and
biliverdin. HO-1 activity ultimately has important physiological
2
7,28
functions associated with cell protection.
GCL, an
antioxidant enzyme in the biosynthetic pathway for major cells,
consists of modulatory (GCLM) and catalytic (GCLC)
2
9
subunits. Real-time quantitative polymerase chain reaction
RT-qPCR) analysis revealed that the mRNA levels of HO-1,
GCLM and GCLC were upregulated by treatment with 3c
10.7-, 5.3- and 2.3-fold, respectively, Figure 2a). Western blot
(
Figure 3. 3c effects gene expression of Nrf2-dependent antioxidant
enzymes in SH-SY5Y cells. SH-SY5Y cells were treated with 3c
for 6 h or 24 h and harvested for Western blot analysis. Nqo1,
GCLM, and GCLC protein levels were presented as relative
amounts using β-actin as a loading control. All experiments were
performed at least in duplicate cultures, and the data were averaged
and expressed as fold induction of untreated control ± SEM. **P <
0.01, *** P < 0.005, **** P < 0.001 (paired t-test).
(
analysis indicated that HO-1 and GCLM protein levels was
significantly increased by 10.3-fold and 5.6-fold following
treatment with 3c, respectively (Figure 2b).
We also confirmed the antioxidant effects of 3c in SH-SY5Y
cells. SH-SY5Y cells have been extensively used to investigate
DAergic cell death induced by oxidative damage in PD
3
0
studies.
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Figure 4. The expression levels of inflammation-related enzymes are decreased by 3c in the concentration dependent manner. (a‒b) BV-2
cells were treated with 3c for 6 h and subsequently exposed to 1 μg/mL LPS for 12 h. Protein and mRNA levels were quantitated and
presented as relative levels. Data are presented as mean ± SD (n = 3). (c–d) BV-2 cells were treated with 3c and exposed to 1 μg/mL LPS
for 24 h. All experiments were performed at least in duplicate cultures, and the data were averaged and expressed as relative values compared
with the LPS-stimulated control ± SEM. **P < 0.01, *** P < 0.005, **** P < 0.001 vs LPS-stimulated control (paired t-test).
We investigated whether 3c relieves lipopolysaccharide (LPS)-
induced inflammation to induced inflammation to the
downregulation of nitric oxide (NO) synthase, iNOS, COX-2,
IL-1β, and TNF-α in microglial cells. To induce an
IL-1β induced by LPS were also significantly reduced by 3c at
mRNA levels (Figure 4b). NO, which plans an important role
in the pathogenesis of inflammation, was used as an
inflammatory marker. We found the increase in NO
production induced by LPS was significantly diminished by
treatment with 3c by ELISA (Figure 4c). In addition, LPS-
induced elevation of IL-6 and TNF-α were attenuated by 3c in
a concentration-dependent manner (Figure 4d).
3
1
30
inflammatory response, BV-2 cells were stimulated with LPS.
As shown in Figure 4, 3c significantly attenuated LPS-induced
inflammatory enzymes and cytokines in BV-2 cells. Dramatic
upregulation of iNOS and COX-2 by LPS were suppressed by
3c at protein levels (Figure 4a). The upregulation of iNOS and
Figure 5. 3c attenuates motor dysfunction in MPTP-treated mice. (a) Experimental schedule and behavior test diagram. Mice were
administered 3c (20 mg/kg) or vehicle and treated with MPTP (20 mg/kg) four times after 24 h. (b‒c) The behavioral deficit recovery of the
mice was evaluated by the vertical grid and coat-hanger tests. Results are shown as mean ± SEM (n = 10‒12 per group): *P < 0.05, ** P <
0
.01, *** P < 0.001, **** P < 0.0001, vs. the MPTP-treated group (one-way ANOVA with Tukey’s multiple comparisons test).
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We investigated whether 3c improves motor deficits using
and the number of TH-positive neurons in the SN were analyzed.
Both results are shown as mean  SEM. ****P < 0.0001, vs. the
MPTP-treated group (one-way ANOVA with Tukey’s multiple
comparisons test).
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the MPTP-induced mouse model for PD. We evaluated the
ability of mice to restore their motor ability by treatment with
3c (20ꢀmg/kg, p.o.) for 3 consecutive days prior and subsequent
to MPTP administration (4 injections of MPTP, 2-h intervals;
We used the MPTP-induced PD model to determine whether
3c can suppress neuroinflammation in vivo. An ionized
calcium-binding adapter molecule 1 (Iba-1) as a microglial
marker, was observed in the DAergic degeneration area of the
SN. Immunofluorescence intensity of Iba-1 in MPTP-injected
mice was 4-fold higher than that of saline-injected mice.
Nonetheless, MPTP-injected mice treated with 3c significantly
decreased Iba-1 immunoreactivity. In addition, TH-positive
DAergic neurons were abundantly detected only in the absence
of activated microglia in the merged image (Figure 7a and 7b).
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0ꢀmg/kg, i.p. injection, Figure 5a).
PD-related motor dysfunction was tested using a coat-hanger
3
2
test that included a vertical grid test established by our group.
In behavioral tests, motor deficits in the MPTP-induced PD
model were prevented by treatment with 3c. Specifically, the
turning time, descent time, and total time in MPTP-treated mice
were restored to control levels by 3c (Figure 5b).
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Similarly, coat-hanger tests also exhibited that 3c-treated
mice improved abnormal movement than MPTP-treated mice,
and showed similar scores to the vehicle control (Figure 5c).
MPTP-treated mice remained in their bases or moved
horizontally (score: 1.9 ± 0.1), whereas mice treated with 3c
(
score: 3.0 ± 0.2) showed a similar score to the vehicle control
(score: 3.2 ± 0.2). Therefore, all data demonstrate that 3c is
highly effective in preventing PD-related movement disorders
in MPTP-treated mouse models of PD (detailed information for
statistical analysis is shown in the Supporting Information,
Table S2).
We found that 3c demonstrated anti-oxidant and anti-
inflammatory effects in vitro, so we then tested the
neuroprotective effects on DAergic neurons in vivo using the
MPTP-induced PD mouse model. The number of tyrosine
hydroxylase (TH)-negative neurons was reduced in MPTP-
treated mice (up to 39 ± 8.3% of the treated vehicle), but not in
Figure 7. 3c inhibited microglial activation in the MPTP-induced
mouse model. Double immunofluorescence staining for TH (green)
and Iba-1 (Red) in the brain 10 days after MPTP injection (i.p.). (a)
TH and Iba-1 immunostaining was evident on the coronal section
of SN. (b) Quantitative comparison of relative Iba-1 staining
intensity between wild type (n = 9), 3c-treated (n = 9), and MPTP-
treated (n = 9) mice at high magnification (×4). Data are shown as
mean ± SEM. ***P < 0.001, **** P < 0.0001, vs. the MPTP-
treated group (one-way ANOVA with Tukey’s multiple
comparisons test).
3
c-co-treated mice (P > 0.001 vs. vehicle treatment). While the
TH-positive cell density in striatum and SN was reduced after
MPTP injection compared to controls, the cell density was
significantly restored in 3c-treated mice (Figure 6a and b). 3c
protected DAergic neurons in the striatum of the PD mouse
model.
In conclusion, vinyl sulfonamide and vinyl sulfonate
derivatives were developed and synthesized, and their effects
on Nrf2 translocation and Nrf2 activation were biologically
evaluated. Among them, the new vinyl sulfonate (3c) exhibited
excellent Nrf2 activation (3c EC50 = 76 nM). We demonstrated
that selected 3c induces the expression of Nrf2-dependent anti-
oxidant enzymes, and prevents the production of inflammatory
mediators. In addition, 3c displayed potent in vivo therapeutic
efficacy against motor dysfunction in MPTP-induced PD mice.
The recovery of TH expression and anti-inflammatory
responses in the SN and striatum were observed in this mouse
model. In summary, the novel Nrf2 activator 3c has potential as
an effective therapeutic agent for the treatment of PD.
EXPERIMENTAL SECTION
Detailed synthetic procedures and analytical data for all
compounds and experimental methods for biological studies are
described in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Figure 6. 3c protects DAnergic neurons in the MPTP-induced
mouse model. (a) TH protein staining was performed using striatal
and SN tissue sections of mice treated with vehicle or 3c (collected
7 days later). (b) Density of TH-positive neurons in the striatum
Experimental methods for synthesis and biological studies;
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Instrumental data for the synthesized compounds; 1H- & 13C-
NMR spectra of the final compounds (PDF).
9. Amor, S.; Puentes, F.; Baker, D.; van der Valk, P. Inflammation in
neurodegenerative diseases. Immunology, 2010, 129 (2), 154-69.
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0. Bartels, A.L.; Leenders, K.L. Cyclooxygenase and
neuroinflammation in Parkinson's disease neurodegeneration. Curr
Neuropharmacol, 2010, 8 (1), 62-8.
AUTHOR INFORMATION
Corresponding Author
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1. Lambert, T.W.; Soskolne, C.L.; Bergum, V.; Howell, J.; Dossetor,
J.B. Ethical perspectives for public and environmental health: fostering
autonomy and the right to know. Environ Health Perspect, 2003, 111
(2), 133-7.
12. Meredith, G.E.; Rademacher, D.J. MPTP mouse models of
Parkinson's disease: an update. J Parkinsons Dis, 2011, 1 (1), 19-33.
13. Tebay, L.E.; Robertson, H.; Durant, S.T.; Vitale, S.R.; Penning,
T.M.; Dinkova-Kostova, A.T.; Hayes, J.D. Mechanisms of activation
of the transcription factor Nrf2 by redox stressors, nutrient cues, and
energy status and the pathways through which it attenuates
degenerative disease. Free Radic Biol Med, 2015, 88 (Pt B), 108-146.
*
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E-mail: kdpark@kist.re.kr. Office: +82-2-9585132. Fax: +82-2-
585189.
ORCID
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Ki Duk Park: 000-0002-7753-214X
Author Contributions
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4. Kobayashi, E.H.; Suzuki, T.; Funayama, R.; Nagashima, T.;
J.W.C., and S.J.S. contributed equally to this work.
Hayashi, M.; Sekine, H.; Tanaka, N.; Moriguchi, T.; Motohashi, H.;
Nakayama, K.; Yamamoto, M. Nrf2 suppresses macrophage
inflammatory response by blocking proinflammatory cytokine
transcription. Nat Commun, 2016, 7, 11624.
Notes
The authors declare no competing financial interest.
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5. Baird, L.; Dinkova-Kostova, A.T. The cytoprotective role of the
Keap1-Nrf2 pathway. Arch Toxicol, 2011, 85 (4), 241-72.
6. Magesh, S.; Chen, Y.; Hu, L. Small molecule modulators of Keap1-
ACKNOWLEDGMENT
1
This study was supported by a National Research Council of
Science & Technology grant by the South Korean government
(MSIP, No. CRC-15-04-KIST) and the National Research
Foundation of Korea (NRF-2018M3A9C8016849).
Nrf2-ARE pathway as potential preventive and therapeutic agents. Med
Res Rev, 2012, 32 (4), 687-726.
1
7. Done, A.J.; Traustadottir, T. Nrf2 mediates redox adaptations to
exercise. Redox Biol, 2016, 10, 191-199.
18. Suzuki, T.; Yamamoto, M. Stress-sensing mechanisms and the
physiological roles of the Keap1-Nrf2 system during cellular stress. J
Biol Chem, 2017, 292 (41), 16817-16824.
ABBREVIATIONS
ARE, anti-oxidant response element; DAergic, dopaminergic;
GCLC, glutamate-cysteine ligase catalytic subunit; GCLM,
glutamate-cysteine ligase regulatory subunit; HO-1, heme
oxygenase; Iba-1, ionized calcium-binding adapter molecule 1;
1
9. Rojo, A.I.; Pajares, M.; Rada, P.; Nunez, A.; Nevado-Holgado,
A.J.; Killik, R.; Van Leuven, F.; Ribe, E.; Lovestone, S.; Yamamoto,
M.; Cuadrado, A. NRF2 deficiency replicates transcriptomic changes
in Alzheimer's patients and worsens APP and TAU pathology. Redox
Biol, 2017, 13, 444-451.
Keap1, Kelch-like
lipopolysaccharide;
ECH-related
protein
1;
LPS,
MPTP, 1-methyl-4-phenyl-1,2,3,6-
2
0. Rojo, A.I.; Pajares, M.; Garcia-Yague, A.J.; Buendia, I.; Van
tetrahydropyridine; MS, multiple sclerosis; NO, nitric oxide;
NQO-1, NAD(P)H dehydrogenase; Nrf2, Nuclear factor E2-
related factor-2; PD, Parkinson disease; SFN, sulforaphane; SN,
substantia nigra; TH, tyrosine hydroxylase.
Leuven, F.; Yamamoto, M.; Lopez, M.G.; Cuadrado, A. Deficiency in
the transcription factor NRF2 worsens inflammatory parameters in a
mouse model with combined tauopathy and amyloidopathy. Redox
Biol, 2018, 18, 173-180.
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1. Burness, C.B.; Deeks, E.D. Dimethyl fumarate: a review of its use
in patients with relapsing-remitting multiple sclerosis. CNS Drugs,
2014, 28 (4), 373-87.
22. Gold, R.; Kappos, L.; Arnold, D.L.; Bar-Or, A.; Giovannoni, G.;
Selmaj, K.; Tornatore, C.; Sweetser, M.T.; Yang, M.; Sheikh, S.I.;
Dawson, K.T.; Investigators, D.S. Placebo-controlled phase 3 study of
oral BG-12 for relapsing multiple sclerosis. N Engl J Med, 2012, 367
REFERENCES
1
. Aarsland, D.; Creese, B.; Politis, M.; Chaudhuri, K.R.; Ffytche, D.-
H.; Weintraub, D.; Ballard, C. Cognitive decline in Parkinson disease.
Nat Rev Neurol, 2017, 13 (4), 217-231.
2
. Gratwicke, J.; Jahanshahi, M.; Foltynie, T. Parkinson's disease
dementia: a neural networks perspective. Brain, 2015, 138 (Pt 6), 1454-
(12), 1098-107.
7
3
6.
23. Lee, J.A.; Kim, J.H.; Woo, S.Y.; Son, H.J.; Han, S.H.; Jang, B.K.;
Choi, J.W.; Kim, D.J.; Park, K.D.; Hwang, O. A novel compound
VSC2 has anti-inflammatory and antioxidant properties in microglia
and in Parkinson's disease animal model. Br J Pharmacol, 2015, 172
. Branch, S.Y.; Chen, C.; Sharma, R.; Lechleiter, J.D.; Li, S.;
Beckstead, M. J. Dopaminergic neurons exhibit an age-dependent
decline in electrophysiological parameters in the mitopark mouse
model of Parkinson's disease. J Neurosci, 2016, 36 (14), 4026-37.
(4), 1087-100.29.
4
. Vogt Weisenhorn, D.M.; Giesert, F.; Wurst, W. Diversity matters -
24. Woo, S.Y.; Kim, J.H.; Moon, M.K.; Han, S.H.; Yeon, S.K.; Choi,
J.W.; Jang, B.K.; Song, H.J.; Kang, Y.G.; Kim, J.W.; Lee, J.; Kim,
D.J.; Hwang, O.; Park, K.D. Discovery of vinyl sulfones as a novel
class of neuroprotective agents toward Parkinson's disease therapy. J
Med Chem, 2014, 57 (4), 1473-87.
heterogeneity of dopaminergic neurons in the ventral mesencephalon
and its relation to Parkinson's disease. J Neurochem, 2016, 139 Suppl
1
, 8-26.
5. Asaithambi, A.; Ay, M., Jin, H.; Gosh, A., Anantharam, V.;
Kanthasamy, A.; Kanthasamy, A.G. Protein kinase D1 (PKD1)
phosphorylation promotes dopaminergic neuronal survival during 6-
OHDA-induced oxidative stress. PLoS One, 2014, 9 (5), e96947.
2
5. Choi, J.W.; Kim, S.; Park, J.H.; Kim, H.J.; Shin, S.J.; Kim, J.W.;
Woo, S.Y.; Lee, C.; Han, S.M.; Lee, J.; Pae, A.N.; Han, G.; Park, K.D.
Optimization of vinyl sulfone derivatives as potent nuclear factor
erythroid 2-related factor 2 (Nrf2) activators for Parkinson's disease
therapy. J Med Chem, 2019, 62, 811-830.
6
. Dias, V.; Junn, E.; Mouradian, M.M. The role of oxidative stress in
Parkinson's disease. J Parkinsons Dis, 2013, 3 (4), 461-91.
7. Hou, Y.; Peng, S.; Li, X.; Yao, J.; Xu, J.; Fang, J. Honokiol alleviates
oxidative stress-induced neurotoxicity via activation of Nrf2. ACS
Chem Neurosci, 2018, 9, 3108-16.
2
6. Park, J.H.; Choi, J.W.; Ju, E.J.; Pae, A.N.; Park, K.D. Antioxidant
and anti-Inflammatory activities of natural compound,
shizukahenriol, through Nrf2 activation. Molecules, 2015, 20 (9),
a
8
. Sarkar, S.; Raymick, J.; Imam, S. Neuroprotective and therapeutic
1
2
5989-6003.
strategies against Parkinson's disease: recent perspectives. Int J Mol
Sci, 2016, 17 (6), 904.
7. Loboda, A.; Damulewicz, M.; Pyza, E.; Jozkowicz, A.; Dulak, J.
Role of Nrf2/HO-1 system in development, oxidative stress response
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
and diseases: an evolutionarily conserved mechanism. Cell Mol Life
Sci, 2016, 73 (17), 3221-47.
8. Li, H.; Wood, J.T.; Whitten, K.M.; Vadivel, S.K.; Seng, S.;
Makriyannis, A.; Avraham, H.K. Inhibition of fatty acid amide
hydrolase activates Nrf2 signalling and induces heme oxygenase 1
transcription in breast cancer cells. Br J Pharmacol, 2013, 170 (3), 489-
505.
Nrf2/antioxidant response element signaling pathway in both neuronal
and microglial cells: therapeutic relevance to neurodegenerative
disease. J Nutr Biochem, 2012, 23 (10), 1314-23.
31. Sharma, J.N.; Al-Omran, A.; Parvathy, S.S. Role of nitric oxide in
inflammatory diseases. Inflammopharmacology, 2007, 15 (6), 252-
9.40.
32. Kim, S.T.; Son, H.J.; Choi, J.H.; Ji, I.J.; Hwang, O. Vertical grid
test and modified horizontal grid test are sensitive methods for
evaluating motor dysfunctions in the MPTP mouse model of
Parkinson's disease. Brain Res, 2010, 1306, 176-83.
1
2
3
4
5
6
7
8
9
2
2
9. Takahashi, S.; Hisatsune, A.; Kurauchi, Y.; Seki, T.; Katsuki, H.
Insulin-like growth factor 1 specifically up-regulates expression of
modifier subunit of glutamate-cysteine ligase and enhances glutathione
synthesis in SH-SY5Y cells. Eur J Pharmacol, 2016, 771, 99-106.
30. Kim, S.S.; Lim, J.; Bang, Y.; Gal, J.; Lee, S.U.; Cho, Y.C.; Yoon,
G.; Kang, B.Y.; Cheon, S.H.; Choi, H.J. Licochalcone E activates
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Table of Content
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Anti-oxidant, Anti-inflammatory, and Neuroprotective Effects
of Novel Vinyl Sulfonate Compounds as Nrf2 Activator
Ji Won Choia,†, Su Jeong Shina,b,†, Hyeon Ji Kima,b, Jong-Hyun Park , Hyeon Jeong Kima,b,
a
Elijah Hwejin Lee , Ae Nim Pae , Yong Sun Bahn , Ki Duk Parka,c,d,*
a
a,c,d
b
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