Discovery of vinyl sulfones as a novel class of neuroprotective agents toward Parkinson's disease therapy

Article abstract of DOI:10.1021/jm401788m

Although the etiology of Parkinson's disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.

Full text of DOI:10.1021/jm401788m

Article
pubs.acs.org/jmc
Discovery of Vinyl Sulfones as a Novel Class of Neuroprotective
Agents toward Parkinson’s Disease Therapy
Seo Yeon Woo,†,∥ Ji Hyun Kim,§,∥ Mi Kyeong Moon,§ Se-Hee Han,§ Seul Ki Yeon,† Ji Won Choi,†
Bo Ko Jang,† Hyo Jung Song,† Yong Gu Kang,† Jin Woo Kim,† Jaeick Lee,‡ Dong Jin Kim,†
Onyou Hwang,*,§ and Ki Duk Park*,†
†Center for Neuro-Medicine, Brain Science Institute, and ‡Doping Control Center, Korea Institute of Science and Technology, Seoul,
136-791, Republic of Korea
§Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, 138-736, Republic of Korea
S
* Supporting Information
ABSTRACT: Although the etiology of Parkinson’s disease (PD) remains elusive,
recent studies suggest that oxidative stress contributes to the cascade leading to
dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main
pathway responsible for cellular defense system against oxidative stress. Nrf2 is a
transcription factor that regulates environmental stress response by inducing
expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone
derivatives. They exhibited a broad range of activities in inducing HO-1, whose
gene expression is under the control of Nrf2. Among them, compound 12g was
confirmed to activate Nrf2 and induce expression of the Nrf2-dependent
antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and
protein levels in DAergic neuronal cells. This was accompanied by protection of
DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In
addition, compound 12g effectively resulted in attenuation of the PD-associated
behavioral deficits in the mouse model.
(GCLM) and catalytic (GCLC) subunits, and several members
of the glutathione S-transferase family.10−13
INTRODUCTION
■
Parkinson’s disease (PD) is a progressive neurodegenerative
movement disorder associated with a profound loss of
dopamine (DA) neurons in the substantia nigra (SN) pars
compacta. Although the cause and pathogenesis of PD remain
elusive, recent studies suggest that oxidative stress plays a
critical role in DAergic cell death in PD.1−8 Oxidative stress is
caused by an imbalance between the production of reactive
oxygen species (ROS) and cellular antioxidant defense
system.2−4 ROS are likely to be elevated in the brain and
neuronal cells because the excitatory amino acids and
neurotransmitters produce ROS through their metabolism.8,9
The DAergic neurons are thought to be under particularly high
oxidative stress due to the presence of DA.2 Furthermore,
superoxide and nitric oxide produced by microglia, which can
be elevated by molecules released from damaged DAergic
neurons, can also contribute to oxidative stress. Therefore,
attenuation of oxidative stress by induction of the antioxidant
defense system can be beneficial to survival of DAergic neurons.
The nuclear factor E2-related factor 2 (Nrf2) signaling is the
main pathway responsible for cellular defense system against
oxidative stress. Nrf2 is a transcription factor that regulates
environmental stress response by inducing expression of
antioxidant enzyme genes such as heme oxygenase 1 (HO-1),
NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate-
cysteine ligase (GCL), which consists of both the modifier
There is increasing evidence that the Nrf2 signaling is
associated with PD.14−18 Expression of the Nrf2-mediated
cytoprotective genes declines with age, which is the main risk
factor for PD.19,20 In the few surviving DAergic neurons of
post-mortem brains from PD patients, Nrf2 is localized in the
nucleus and is increased compared to control subjects,
suggesting an attempt to reduce oxidative stress through the
Nrf2-dependent transcription of the antioxidant enzyme
genes.21 In animal studies, Nrf2-deficient mice are hyper-
sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) induced neurodegeneration,14,18,19 whereas over-
expression of Nrf2 is protective.19,22 These findings indicate
that Nrf2 activation is a promising target for therapeutics aimed
at reducing or preventing DAergic neuronal death in PD.
Recently Kumar et al. reported that a chalcone derivative (1)
was a potent activator of the Nrf2 signaling pathway from a
study using a cell based assay.23 Chalcone contains a unique
structure, α,β-unsaturated ketone, that is responsible for its
various biological activities.24,25 In this study we introduced
either a vinyl sulfoxide or vinyl sulfone group to the α,β-
unsaturated carbonyl entity of chalcone (2, 3) and initially
Received: November 18, 2013
Published: January 27, 2014
© 2014 American Chemical Society
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screened the derivatives for their capacity to induce HO-1, one
of the antioxidant enzymes whose expressions are under the
control of Nrf2. We found that the introduction of a vinyl
sulfone group led to the most potent activities. Herein, we
report the design, synthesis, and biological evaluation of a series
of vinyl sulfone derivatives (3). One of the compounds with the
highest potency was further evaluated for its ability to induce
various antioxidant enzymes in DAergic neuronal cells and to
protect these cells from oxidative insults. In addition, the
compound was tested for its ability to attenuate the nigral
DAergc neurodegeneration and PD-associated motor deficits in
an animal model of PD.
synthesized by the base-catalyzed Claisen−Schmidt condensa-
tion of an aldehyde and ketone in polar solvents such as ethanol
or methanol. Synthesis of the compounds 9−16 followed a
standard protocol (Scheme 1). Substituted benzenethiols (5)
were coupled with (diethoxyphosphoryl)methyl 4-methylben-
zenesulfonate (4) in the presence of base to give the sulfides
(6). Oxidation of the sulfides with 1−2.2 equiv of m-
chloroperoxybenzoic acid (mCPBA) at −20 or 0 °C provided
the sulfoxides (7) or the sulfones (8), respectively. The
compounds 9−16 were obtained by utilizing Horner−Emmons
olefination reaction with commercially available substituted
benzaldehydes.
Initial Screening against Expression of HO-1 Gene. To
initially compare Nrf2 activating efficacy among various
compounds, we assessed their ability to induce expression of
HO-1, a major Nrf2-dependent gene.26 This was because we
had set up in our laboratory a sensitive cell based assay system
for detecting HO-1 protein using a sandwich enzyme-linked
immunosorbent assay (ELISA). This screening system had
been optimized for murine BV-2 cells, and our preliminary
studies had revealed that the pattern of Nrf2-dependent HO-1
induction was similar in BV-2 cells and DAergic neuronal cells.
For this reason, we used this system in the primary screening,
and the results were subsequently confirmed in DAergic
neuronal cells as shown below.
RESULTS AND DISCUSSION
First, to compare chalcone 1 with either the vinyl sulfoxide
compound 2 or the vinyl sulfone compound 3, we prepared
three compounds that included the same functional groups on
ring A and ring B, respectively. Comparison of HO-1 inducing
activities among the three compounds revealed that the vinyl
■
Chemistry. We used chalcone 1, which contained an α,β-
unsaturated ketone moiety, as our structural template and
modified the ketone position according to two categories:
sulfoxide or sulfone. Chalcone compounds (1) can be readily
Scheme 1. General Procedure for the Preparation of Vinyl Sulfoxide and Vinyl Sulfone Derivatives
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sulfone (10a) was the most potent, followed by the chalcone
(1) and the vinyl sulfoxide (9a) (Table 1). Accordingly, we
12g Activates Nrf2 and Induces NQO1, HO-1, GCL
Gene Expression in DAergic Cells. CATH.a cells, a mouse
DAergic neuronal cell line derived from the brain,27 were used
because these cells have previously been demonstrated by us to
readily induce NQO1 gene expression under a Nrf2-activating
condition.28 As Nrf2 activation leads to translocation into the
nucleus, where it acts as a transcription factor, we determined
whether such a phenomenon occurs upon exposure to 12g.
Western blot analysis on the nuclear fraction of cells treated
with 12g for 3 h revealed dose-dependent increases in the
amount of nuclear Nrf2 (Figure 1A). A concentration of 1 μM
was sufficient to cause a statistically significant change, and 10
Table 1. Effects of Compounds 1, 9a, and 10a on Expression
of the Nrf2-Dependent HO-1 Gene
μM resulted in a dramatic increase of (3.5
0.16)-fold.
Activation of Nrf2 requires the release of its cytosolic inhibitor
protein keap1, which renders Nrf2 no longer subject to
degradation by the ubiquitin proteasome system.10 Therefore,
the total amount of Nrf2, as well as the nuclear Nrf2, would be
elevated. Western blot on total cell lysate demonstrated that
Nrf2 was indeed accumulated following the 12g treatment
(Figure 1B). Again, a statistically significant increase was
observed at 1 μM, and further increases were observed in a
dose-dependent manner. Taken together, 12g evidently led to
cellular accumulation and nuclear translocation of Nrf2,
indicative of activation of the transcription factor.
a
BV-2 cells were plated in 96-well plates at 20 000 cells/well in
triplicate with test compounds (20 μM) for 24 h. Protein level of HO-
1 was determined using a sandwich ELISA assay and expressed as % of
vehicle treated control
sulforaphane (5 μM), a well-known potent activator of Nrf2.
b
c
SEM. Vehicle: 0.04% DMSO. SFN:
We further asked whether the target enzyme genes’
expressions are induced, as would be expected as a result of
Nrf2 activation. NQO1 activity is especially important in
protection of DAergic neurons, as it removes DA quinone, the
product of DA oxidation, which can damage the cell via a
number of mechanisms including protein modification and
ROS generation.2 RT-PCR against NQO1 on mRNA isolated
from cells treated with 12g showed a dramatic and dose-
dependent increase. Significant induction was observed at 1 μM
((1.95 0.20)-fold), and the degree of induction reached (5.67
0.13)-fold at 10 μM (Figure 2A). Western blot analysis
(Figure 2E) also revealed a similar pattern of elevation of the
NQO1 protein level, reaching a (4.43 0.04)-fold increase at
10 μM 12g.
12g also elevated HO-1 expression in DAergic neuronal cells
as well. HO-1, the enzyme responsible for the conversion of
heme to biliverdin and carbon monoxide, has been shown to
have antioxidant and neuroprotective properties.29 A slight but
statistically significant increase was observed at 1 μM 12g, and
at 10 μM, over 3-fold increases were observed at both mRNA
and protein levels (Figure 2B and Figure 2F).
subsequently synthesized 56 vinyl sulfone derivatives that
included various functional groups on both ring A and ring B
(Table 2, compounds 10−16). The first set of compounds
contained a methoxy group attached at the para position on
ring A. Although for 10a−m we did not observe a clear trend in
the effect of the electronic properties of the aryl substituent
(R2), substitutions on ring B with an electron-withdrawing
group (CF3, F, Cl, or OCF3) led to higher activities than an
electron-donating group (OMe). Next, we systematically placed
a methoxy group at the 2′-, 3′-, and 4′-positions on ring A. In
the three series of compounds listed in Table 2, the 2′-methoxy
derivatives (12) were more active (2.4−3.8 times higher than
vehicle) than the corresponding 3′- and 4′-methoxy derivatives
(10 and 11) (1.2−2.6 and 1.1−2.8 times higher than vehicle,
respectively). These results indicated that the position of OMe
substitution on ring A was crucial for the effect on HO-1
induction activity. Among compounds 12, 12g exerted the
highest activity (3.8-fold induction).
GCL is an important antioxidant enzyme because it plays a
critical role in the biosynthesis of glutathione, which is the
major antioxidant molecule in the cell. The enzyme consists of
two subunits, the modulatory (GCLM) and catalytic (GCLC)
subunits. RT-PCR against GCLM and GCLC showed that both
subunits were significantly and similarly induced starting at 2
μM ((1.43 0.16)- and (1.41 0.01)-fold, respectively). The
degrees of induction reached (2.56 0.07)- and (2.64 0.24)-
fold at 10 μM (Figure 2C and Figure 2D). Western blot
analysis (Figure 2G and Figure 2H) also revealed dose-
dependent elevations of the GCLM and GCLC protein levels
by 12g. Taken together, 12g was able to activate Nrf2 and
induce expression of the antioxidant enzymes NQO1, HO-1,
and GCL in DAergic neuronal cells.
When the methoxy group on ring A was replaced with
hydrogen (16), halogens (13, 14), or dimethoxy group (15),
the HO-1 inducing activity was similar or slightly decreased.
Interestingly, in this series, we found that 2″-trifluoromethyl
derivatives (13a, 14a, 15a, and 16a) showed better activities
than the corresponding 3″- and 4″-trifluoromethyl derivatives
(13b,c, 14b,c, 15b,c and 16b,c). These findings suggested that
the substitution of an electron-withdrawing group at 2″-
position on ring B in the vinyl sulfone derivatives led to
increased HO-1 inducing activity. A similar finding was
observed in compounds 10 and 11.
The above structure−activity relation study demonstrated an
excellent HO-1 inducing activity of 12g. Subsequent experi-
ments were carried out to examine whether this compound
could indeed lead to Nrf2 activation and production of various
antioxidant enzymes involved in cellular defense system in
DAergic neuronal cells and to protection of these cells from
oxidative damage.
12g Protects DAergic Neuronal Cells. Whether the
ability of 12g to induce the antioxidant enzymes might afford
neuroprotection was then assessed. For this, CATH.a cells
exposed to tetrahydrobiopterin (BH4) was used, as this system
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Table 2. Effects of Vinyl Sulfone Derivatives on Expression of the Nrf2-Dependent HO-1 Gene
a
a
compd
R1
R2
HO-1 (%)
compd
R1
R2
HO-1 (%)
10a
10b
10c
10d
10e
10f
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
4′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
3′-OMe
2′-OMe
2′-OMe
2′-OMe
2′-OMe
2′-OMe
2′-OMe
2″-CF3
3″-CF3
4″-CF3
2″-F
3″-F
4″-F
2″-Cl
3″-Cl
4″-Cl
2″-OMe
3″-OMe
4″-OMe
2″-OCF3
2″-NH3Cl
2″-CF3
3″-CF3
4″-CF3
2″-F
3″-F
4″-F
2″-Cl
3″-Cl
257.5 6.7
213.8 6.2
153.7 25.5
174.1 20.2
203.4 19.8
177.0 1.2
157.5 22.1
124.8 14.6
168.9 8.1
125.5 13.9
145.9 21.9
132.7 17.1
212.5 35.6
140.6 7.6
251.1 56.0
204.1 25.3
147.9 16.4
148.4 10.6
136.2 7.1
159.7 14.2
284.2 12.2
111.1 1.8
227.8 5.7
328.2 31.8
256.1 39.8
357.1 11.7
372.7 11.1
353.3 23.4
259.1 19.6
12g
12h
12i
2′-OMe
2′-OMe
2′-OMe
4′-F
4′-F
4′-F
4′-F
4′-F
4′-F
4′-F
2″-Cl
3″-Cl
4″-Cl
2″-CF3
3″-CF3
4″-CF3
2″-F
382.2 12.6
244.7 17.6
330.4 19.8
248.1 6.0
158.2 17.5
124.7 3.5
240.7 26.1
177.4 4.3
141.3 30.8
225.3 9.8
136.8 4.1
111.8 11.4
242.5 30.7
110.8 8.0
139.8 12.6
107.4 14.9
104.9 14.8
111.2 4.6
168.6 7.4
126.6 17.0
116.7 8.7
204.1 25.7
153.0 10.4
119.1 10.6
193.2 24.5
187.8 11.1
154.7 6.2
100.0
13a
13b
13c
13d
13e
13f
10g
10h
10i
3″-F
4″-F
10j
13g
13h
13i
2″-Cl
3″-Cl
4″-Cl
2″-CF3
3″-CF3
4″-CF3
2″-F
10k
10l
4′-F
4′-F
10m
10n
11a
11b
11c
11d
11e
11f
14a
14b
14c
14d
14e
14f
4′-Cl
4′-Cl
4′-Cl
4′-Cl
4′-Cl
4′-Cl
4′-Cl
4′-Cl
4′-Cl
3′,4′-OMe
3′,4′-OMe
3′, 4′-OMe
H
3″-F
4″-F
14g
14h
14i
2″-Cl
3″-Cl
4″-Cl
2″-CF3
3″-CF3
4″-CF3
2″-CF3
3″-CF3
4″-CF3
11g
11h
11i
15a
15b
15c
16a
16b
16c
4″-Cl
12a
12b
12c
12d
12e
12f
2″-CF3
3″-CF3
4″-CF3
2″-F
3″-F
4″-F
H
H
b
vehicle
c
SFN
229.3 5.8
a
BV-2 cells were plated in 96-well plates at 20 000 cells/well in triplicate with test compounds (20 μM) for 24 h. Protein level of HO-1 was
b
c
determined using a sandwich ELISA assay and expressed as % of vehicle treated control SEM. Vehicle: 0.04% DMSO. SFN: sulforaphane (5
μM), a well-known potent activator of Nrf2.
has been extensively used in previous studies to examine
DAergic cell death by oxidative damage.28,30−35 Co-treatment
with various concentrations of 12g provided protection from
the BH4-induced cell death, as demonstrated in two different
cell death assays. In both ATP assay (Figure 3A) and LDH
activity assay (Figure 3B), a concentration as low as 0.1 μM
provided statistically significant protection, and the degree of
protection was further increased at higher concentrations of
12g. Interestingly, this concentration did not result in a
statistically significant increase in the antioxidant enzymes at
the mRNA and protein levels (Figure 2). It is possible that the
combination of small, seemingly insignificant increases of the
enzymes may have been sufficient in providing cytoprotection
of these cells. The cells exposed to 12g alone in the same
concentration range for 72 h did not cause changes in cell
viability (Figure 3C and Figure 3D), demonstrating that 12g
itself has no cytotoxicity.
12g Protects the Nigral DAergic Neurons and Striatal
DAergic Terminals in the PD Animal Model. Whether 12g
could serve as a potential therapeutic agent for PD through
neuroprotection was also tested in an animal model of PD
generated by administration of the DAergic toxin MPTP. The
MPTP treatment led to dramatic disappearance of the tyrosine
Figure 1. 12g induces Nrf2 nuclear translocation and elevates Nrf2
protein level. CATH.a cells were exposed to various concentrations of
12g. (A) After 3 h, the cells were harvested and the nuclear fraction
was subjected to Nrf2 Western blot with B-lamin as a nuclear marker.
(B) After 24 h, the cells were harvested and the cell lysate was
subjected to Nrf2 Western blot with β-actin used as an internal
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 < 0.05; (∗∗) P < 0.01 vs untreated
control.
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Figure 2. 12g induces gene expression of antioxidant enzymes in DAergic neuronal cells. CATH.a cells were exposed to various concentrations of
12g and harvested after 6 h for RT-PCR (A−D) and 24 h for Western blot (E−H) analyses. GAPDH and β-actin were used as respective internal
controls. All experiments were performed at least in duplicate cultures, and the data were averaged and expressed as fold induction of untreated
control SEM: (∗) P < 0.05; (∗∗) P < 0.01 vs untreated control.
to the vehicle-treated control (Figure 4A, left panel).
Quantitative analysis revealed that the number of TH-
immunopositive neurons was decreased by MPTP to
Figure 3. 12g protects DAergic neuronal cells from oxidative demise.
CATH.a cells were exposed to various concentrations of 12g, and cell
viability was estimated by ATP assay (A, C) and LDH activity assay
(B, D). (A, B) After exposure to 12g for 20 h, the cells were further
treated with 200 μM BH4 for an additional 24 h. (C, D) The cells
Figure 4. 12g protects the nigrostriatal system against MPTP-elicited
were exposed to 12g alone for 72 h (C, D). All experiments were
neurodegeneration in PD mouse model. (A) SN and striatal sections
performed at least in duplicate cultures, and the data were averaged
of mice treated with MPTP alone or with 12g (10 mg/kg) were
and expressed as percent of untreated control SEM: (A, B) (∗∗) P <
examined for immunoreactive TH to detect DAergic neurons and
0.01 vs BH4-treated; (C, D) the 12g alone-treated were not
fibers, respectively. Typical immunomicrographs are shown. Scale bars
significantly different from the untreated controls.
= 200 μm. (B) The TH-immunopositive neurons in the SN sections
were counted. (C) The TH-immunopositive fibers in the striatal
sections were quantitated by densitometry. The data are expressed as
hydroxylase (TH) immunopositive DAergic neurons in the SN.
On the other hand, in the MPTP animals co-treated with 12g
(10 mg/kg), the DAergic neurons remained at the level similar
% vehicle-treated control. The results are shown as the mean SEM
(n = 10 per group): (∗) P < 0.05; (∗∗) P < 0.01 vs vehicle-treated
control; (#) P < 0.05; (##) P < 0.01 vs MPTP-treated.
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approximately half of vehicle-administered control group
(Figure 4B). On the other hand, the animals coadministered
with 12g had the DArgic neurons whose number was not
significantly different from the control. 12g alone had no
apparent effect on TH-immunoreactive cells (data not shown).
DAergic terminals in the striatum, the brain region to which
the nigral DAergic neurons project their fibers and where
DAergic neurodegeneration first occurs in PD, were also
examined (Figure 4A, right panel). A dramatic decrease in the
density of TH-immunoreactive fibers was noted in the striatum
of the MPTP-treated animals (43% compared to vehicle-treated
control by densitometric analysis), but this was prevented in
the animals administered with 12g (Figure 4C). Therefore, 12g
protected the nigral DAergic neurons and their terminals in the
striatum in the PD animal model. On the basis of the present
findings that 12g is able to induce the Nrf2 activation and
expression of a number of cytoprotective enzyme genes, it is
probable that this pathway played a major role in the DAergic
neuroprotection. However, one cannot eliminate the possibility
that pathway(s) other than the Nrf2 signaling also contributed
to the protection.
In a vertical grid test, another behavioral test we previously
demonstrated to provide a reliable measure of PD-related
motor activity,36 the MPTP mice took much longer to turn
around and to climb down on the vertical grid apparatus (12.4
1.8 s and 22.4 3.9 s, respectively) compared to the vehicle-
treated control (6.3
0.9 s and 8.3
0.7 s, respectively)
(Figure 5B and Figure 5C). The animals treated with 12g
significantly improved their performance on the vertical grid, as
they took a shorter time to turn (4.0 0.7 s) and climb down
(6.3 0.7 s). In addition, whereas the total time taken on the
grid apparatus was much longer for the MPTP mice (34.8 5.4
s) compared to the control (14.7 1.4 s), the co-treatment
with 12g prevented this deficit (10.3 1.3 s) (Figure 5D). The
number of successful steps taken on the apparatus was lower in
the MPTP animals (75.4 6.2%), but this also returned to the
vehicle-treated control upon co-treatment with 12g (96.9
5.5%) (Figure 5E). In all test analyses, the 12g-coadministered
group was not significantly different from the vehicle control.
Taken together, 12g was found to effectively prevent the motor
deficits that are associated with PD.
CONCLUSION
■
12g Alleviates Motor Deficits of PD Animal Model.
Whether 12g’s neuroprotective effects might be accompanied
by symptomatic alleviation was tested in the same animal
model. The animals were subjected to behavioral tests for
evaluation of their motor activity. In hindlimb test (Figure 5A),
the MPTP-treated animals in general showed weakness of
postural balance and received an average score of 2.21 0.21,
We prepared and evaluated 56 vinyl sulfone compounds for
their activity to induce expression of HO-1, which is an Nrf2-
dependent gene. Significant activity was observed for many of
these compounds. Comparison of methoxy substituted aryl
regioisomers (2′, 3′, 4′) on ring A demonstrated that 2′-
methoxy derivatives exhibited the most potent activity. The
SAR indicated that electron-withdrawing group substitution on
ring B led to increased activity. The compound with highest
activity (12g) was demonstrated to activate Nrf2 and induce
the expression of Nrf2-dependent antioxidant enzymes NQO-1,
GCLC, and GLCM as well as HO-1 in DAergic neuronal cells
in a dose-dependent manner. 12g significantly protected
DAergic neurons from cytotoxic damage both in vitro and in
vivo and attenuated the PD-associated motor deficits in MPTP-
induced mouse model of PD. In conclusion, we present a novel
vinyl sulfone compound with a therapeutic potential for PD.
compared to 3.58
0.15 of the vehicle-treated control. In
comparison, the animals treated with 12g exhibited consid-
erable improvement, with an average score of 3.00 0.22.
EXPERIMENTAL SECTION
■
General Methods. Melting points were determined in open
capillary tubes using an OptiMelt melting point apparatus (Stanford
Research System, Inc.) and are uncorrected. NMR spectra were
obtained at 400 MHz (1H) and 100 MHz (13C) using TMS as an
internal standard. Chemical shifts (δ) are reported in parts per million
(ppm) from tetramethylsilane (TMS). High-resolution mass spec-
trometry was performed on a LTQ Orbitrap (Thermo Electron
Corporation) instrument. Analytical HPLC was performed using a
Waters E2695 system equipped with the following column: SunFire
C18 column (4.6 mm × 150 mm; 5 μm). HPLC data were recorded
using following parameters: H2O/MeCN, 90/10 → 0/100 in 17 min,
+3 min isocratic, flow rate of 1.0 mL/min, λ = 254 and 280 nm.
Reactions were monitored by analytical thin-layer chromatography
(TLC) plates (Merck, catalog no. 1.05715) and analyzed with 254 nm
light. The samples were purified by column chromatography using
silica gel (Merck, catalog nos. 1.07734 and 1.09385). All chemicals and
solvents were reagent grade and used as obtained from commercial
sources without further purification. Yields reported are for purified
products and were not optimized. Compounds were checked by TLC,
1H and 13C NMR, HRMS. The TLC, NMR, and the analytical data
confirmed that the purity of the products was ≥95%.
Figure 5. 12g alleviates motor deficits in MPTP-induced PD mice.
Motor deficits of animals treated with MPTP alone or co-treated with
12g (10 mg/kg) were assessed by the hindlimb test (A) and by various
indices on their behavior on vertical grid apparatus (B−E). The results
are shown as the mean SEM (n = 10 per group): (∗∗) P < 0.01 vs
vehicle-treated control, (#) P < 0.05; (##) P < 0.01 vs MPTP-treated.
General Procedure for the Vinyl Sulfone Derivatives. To a
cooled anhydrous tetrahydrofuran (THF) solution (−78 °C) of 8 was
added 2 M n-BuLi solution in cyclohexane (1.05 equiv). The reaction
mixture was stirred at −78 °C (1 h), and then the desired substituted
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benzaldehyde (1.1 equiv) was added at −78 °C. The reaction mixture
was stirred at room temperature (1 h) and then quenched with H2O
(same volume of THF). The reaction mixture was diluted with EtOAc
(∼200 mL) and washed with H2O (2 × ∼ 200 mL) and brine (2 ×
∼200 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column
chromatography on SiO2.
(E)-1-(2-((4-Methoxyphenyl)sulfinyl)vinyl)-2-trifluoromethyl-
benzene (9a). Diethyl (4-methoxyphenylsulfinyl)methylphosphonate
(0.27 g, 0.88 mmol), 2 M n-BuLi solution in cyclohexane (0.49 mL,
0.92 mmol), and 2-(trifluoromethyl)benzaldehyde (0.13 mL, 0.97
mmol) gave 0.07 g (22%) of 9a as a colorless oil. Rf = 0.25 (n-hexane/
EtOAc 2/1); 1H NMR (400 MHz, CDCl3) δ 7.68−7.75 (m, ArH, (E)-
isomeric CH), 7.62 (d, J = 8.8 Hz, 2ArH), 7.50−7.57 (m, 2ArH), 7.44
(t like due to dd, J = 7.5 Hz, ArH), 7.02 (d, J = 8.8 Hz, 2ArH), 6.79 (d,
J = 15.2 Hz, (E)-isomeric CH), 3.85 (s, OCH3); 13C NMR (100 MHz,
CDCl3) δ 162.4, 138.3, 134.6, 133.2, 132.2, 131.6, 129.2, 128.5 (q, JC−F
111.0−113.0 °C; HPLC purity, 12.9 min, 99.3%; 1H NMR (400 MHz,
CDCl3) δ 7.88 (d, J = 9.0 Hz, 2ArH), 7.71 (d, J = 15.6 Hz, (E)-
isomeric CH), 7.45 (m, ArH), 7.36−7.41 (m, ArH), 7.14−7.19 (m,
ArH), 7.08−7.13 (m, ArH), 6.98−7.03 (m, 2ArH, (E)-isomeric CH),
3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 161.5 (d, JC−F
= 253.5 Hz, ArC-F), 134.3, 132.6 (d, J = 8.8 Hz), 131.9, 130.8 (d, JC−F
= 8.1 Hz), 130.1, 130.0, 124.7 (d, JC−F = 3.2 Hz), 120.6 (d, JC−F = 11.3
Hz), 116.3 (d, JC−F = 21.5 Hz), 114.6 (12ArC, 2CH), 55.7 (OCH3);
HRMS (M + H)+ (ESI+) 293.0645 [M + H]+ (calcd for
C15H13FO3SH+ 293.0648).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene
(10e). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g
(85%) of 10e as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp
1
91.0−93.0 °C; HPLC purity, 12.9 min, 99.6%; H NMR (400 MHz,
CDCl3) δ 7.87 (d, J = 8.9 Hz, 2ArH), 7.58 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.36 (td like due to ddd, J = 5.8, 7.9 Hz, ArH), 7.24−
7.26 (m, ArH), 7.14−7.18 (m, ArH), 7.08−7.12 (m, ArH), 7.01 (d, J =
8.9 Hz, 2ArH), 6.84 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 162.9 (d, JC−F = 246.2
Hz, ArC-F), 139.8, 134.7 (d, JC−F = 7.6 Hz), 131.8, 130.7 (d, JC−F = 8.2
Hz), 130.0, 129.5, 124.5 (d, JC−F = 2.0 Hz), 117.9 (d, JC−F = 21.2 Hz),
114.7 (d, JC−F = 21.7 Hz), 114.6 (12ArC, 2CH), 55.7 (OCH3); HRMS
(M + H)+ (ESI+) 293.0645 [M + H]+ (calcd for C15H13FO3SH+
293.0648).
= 30.4 Hz), 128.4, 127.2, 126.3 (q, JC−F = 5.3 Hz), 124.1 (q, JC−F
=
272.2 Hz, CF3), 115.2 (12ArC, 2CH), 55.7 (OCH3); HRMS (M +
H)+ (ESI+) 327.0670 [M + H]+ (calcd for C16H13F3O2SH+ 327.0667).
( E) - 1 - ( 2 - ( ( 4 - M e t h o x y p h e n y l ) s u l f o n y l ) v i n y l ) - 2 -
trifluoromethylbenzene (10a). Diethyl (4-methoxyphenyl-
sulfonyl)methylphosphonate (0.49 g, 1.52 mmol), 2 M n-BuLi
solution in cyclohexane (0.80 mL, 1.60 mmol), and 2-
(trifluoromethyl)benzaldehyde (0.22 mL, 1.67 mmol) gave 0.34 g
(66%) of 10a as a white solid. Rf = 0.40 (n-hexane/EtOAc 2/1); mp
1
94.0−95.0 °C; HPLC purity, 13.8 min, 99.8%; H NMR (400 MHz,
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-fluorolbenzene
(10f).37 Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50
g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.38 g
(83%) of 10f as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
112.0−114.0 °C; HPLC purity, 12.7 min, 97.0%;1H NMR (400 MHz,
CDCl3) δ 7.86 (d, J = 8.8 Hz, 2ArH), 7.59 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.46 (dd, J = 5.3, 8.8 Hz, 2ArH), 7.07 (t, J = 8.8 Hz,
2ArH), 7.01 (d, J = 8.8 Hz, 2ArH), 6.77 (d, J = 15.4 Hz, (E)-isomeric
CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.2 (d, JC−F
= 251.0 Hz, ArC-F), 163.6, 140.0, 132.1, 130.5 (d, JC−F = 8.6 Hz),
129.9, 128.8 (d, JC−F = 2.8 Hz), 127.8, 116.3 (d, JC−F = 21.8 Hz), 114.6
(12ArC, 2CH), 55.7 (OCH3); HRMS (M + H)+ (ESI+) 293.0647 [M
+ H]+ (calcd for C15H13FO3SH+ 293.0648).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-chloromethyl-
benzene (10g).38 Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
(0.82 mL, 1.63 mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71
mmol) gave 0.36 g (76%) of 10g as a white solid. Rf = 0.30 (n-hexane/
EtOAc 3/1); mp 148.0−150.0 °C; HPLC purity, 13.6 min, 99.3%; 1H
NMR (400 MHz, CDCl3) δ 8.03 (d, J = 15.4 Hz, (E)-isomeric CH),
7.89 (d, J = 9.0 Hz, 2ArH), 7.50 (dd, J = 1.7, 7.9 Hz, ArH), 7.43 (dd, J
= 1.3, 7.9 Hz, ArH), 7.30−7.35 (m, ArH), 7.24−7.28 (m, ArH), 7.02
(d, J = 9.0 Hz, 2ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 137.4, 135.3, 131.9,
131.8, 131.0, 130.9, 130.5, 130.2, 128.3, 127.3, 114.8 (12ArC, 2CH),
55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0349 [M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
CDCl3) δ 7.98 (d, J = 15.2 Hz, (E)-isomeric CH), 7.88 (d, J = 8.9 Hz,
2ArH), 7.71 (d, J = 7.6 Hz, ArH), 7.48−7.59 (m, 3ArH), 7.02 (d, J =
8.9 Hz, 2ArH), 6.82 (d, J = 15.2 Hz, (E)-isomeric CH), 3.88 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 137.2, 132.5, 132.3,
131.5, 131.4, 130.3, 130.1, 129.0 (d, JC−F = 30.5 Hz), 128.3, 126.3 (q,
JC−F = 5.4 Hz), 121.0 (q, JC−F = 272.4 Hz, CF3), 114.7 (12ArC, 2CH),
55.7 (OCH3); HRMS (M + H)+ (ESI+) 343.0612 [M + H]+ (calcd for
C16H13F3O3SH+ 343.0616).
( E) - 1 - ( 2 - ( ( 4 - M e t h o x y p h e n y l ) s u l f o n y l ) v i n y l ) - 3 -
trifluoromethylbenzene (10b). Diethyl (4-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-
(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.46 g
(87%) of 10b as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
104.0−106.0 °C; HPLC purity, 13.8 min, 99.4%; 1H NMR (400 MHz,
CDCl3) δ 7.88 (d, J = 9.0 Hz, 2ArH), 7.71 (s, ArH), 7.63−7.66 (m,
2ArH, (E)-isomeric CH), 7.51−7.55 (m, ArH), 7.02 (d, J = 9.0 Hz,
2ArH), 6.92 (d, J = 15.5 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C
NMR (100 MHz, CDCl3) δ 163.8, 139.4, 133.4, 131.6, 131.5 (q, JC−F
= 32.6 Hz), 130.2, 130.1, 129.7, 127.3 (d, JC−F = 3.2 Hz), 124.9 (d,
JC−F = 3.7 Hz), 123.6 (q, JC−F = 271.0 Hz, CF3), 114.7 (12ArC, 2CH),
55.7 (OCH3); one signal was not detected and is believed to overlap
with nearby peaks; HRMS (M + H)+ (ESI+) 343.0613 [M + H]+
(calcd for C16H13F3O3SH+ 343.0616).
( E) - 1 - ( 2 - ( ( 4 - M e t h o x y p h e n y l ) s u l f o n y l ) v i n y l ) - 4 -
trifluoromethylbenzene (10c). Diethyl (4-methoxyphenylsulfonyl-
methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in
cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)-
benzaldehyde (0.23 mL, 1.71 mmol) gave 0.47 g (88%) of 10c as a
white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp 135.0−137.0 °C;
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-chloromethyl-
benzene (10h). Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
(0.82 mL, 1.63 mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71
mmol) gave 0.34 g (71%) of 10h as a white solid. Rf = 0.35 (n-hexane/
1
HPLC purity, 14.0 min, 99.8%; H NMR (400 MHz, CDCl3) δ 7.88
(d, J = 8.9 Hz, 2ArH), 7.57−7.66 (m, 4ArH, (E)-isomeric CH), 7.02
(d, J = 8.9 Hz, 2ArH), 6.93 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 139.3, 136.0, 132.3 (q,
JC−F = 32.5 Hz), 131.5, 130.7, 130.1, 128.7, 126.0, 123.7 (ArC, trans
CH) (q, JC−F = 270.9 Hz, CF3), 114.7 (12ArC, 2CH), 55.7 (OCH3);
HRMS (M + H)+ (ESI+) 343.0613 [M + H]+ (calcd for
C16H13F3O3SH+ 343.0616).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene
(10d). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.36 g
(74%) of 10d as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
1
EtOAc 3/1); mp 71.0−72.0 °C; HPLC purity, 13.6 min, 99.1%; H
NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz, 2ArH), 7.56 (d, J =
15.4 Hz, (E)-isomeric CH), 7.45 (s, ArH), 7.32−7.39 (m, 3ArH), 7.02
(d, J = 8.8 Hz, 2ArH), 6.85 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.8, 139.7, 135.2, 134.4,
131.9, 130.9, 130.4, 130.1, 129.7, 128.2, 126.8, 114.8 (12ArC, 2CH),
55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0349 [M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-chloromethyl-
benzene (10i).39 Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
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(0.82 mL, 1.63 mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol)
gave 0.43 g (89%) of 10i as a white solid. Rf = 0.35 (n-hexane/EtOAc
3/1); mp 129.0−133.0 °C (lit.39 121.0−123.0 °C); HPLC purity, 13.9
(0.49 mL, 0.98 mmol), and 2-aminobenzaldehyde (0.12 g, 1.02 mmol)
gave 0.18 g (70%) of 10n as a yellow solid. Rf = 0.38 (n-hexane/EtOAc
1/3); mp 177.0−180.0 °C; HPLC purity, 11.4 min, >99.0%; 1H NMR
(400 MHz, MeOD) δ 7.92 (d, J = 9.0 Hz, 2ArH), 7.82−7.87 (m, ArH,
(E)-isomeric CH), 7.58 (td like due to ddd, J = 1.4, 7.6 Hz, ArH),
7.43−7.51 (m, 2ArH, (E)-isomeric CH), 7.15 (d, J = 9.0 Hz, 2ArH),
3.90 (s, OCH3); 13C NMR (100 MHz, MeOD) δ 164.2, 133.2, 132.2,
132.1, 131.2, 130.6, 129.9, 129.0, 128.4, 127.0, 123.8, 114.5 (12ArC,
2CH), 55.0 (OCH3); HRMS (M + H)+ (ESI+) 290.0854 [M + H]+
(calcd for C15H15NO3SH+ 290.0851).
1
min, >99.0%; H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz,
2ArH), 7.5662−7.66 (d, J = 15.4 Hz,(E)-isomeric CH), 7.39 (d, J =
8.6 Hz, 2ArH), 7.34 (d, J = 8.6 Hz, 2ArH), 7.00 (d, J = 8.8 Hz, 2ArH),
6.82 (d, J = 15.4 Hz, (E)-isomeric CH), 3.86 (s, OCH3); 13C NMR
(100 MHz, CDCl3) δ 163.8, 140.0, 137.1, 132.1, 131.2, 130.1, 129.8,
129.5, 128.7, 114.8 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+
(ESI+) 309.0349 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-methoxy-
benzene (10j). Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
(0.82 mL, 1.63 mmol), and 2-methoxybenzaldehyde (0.21 mL, 1.71
mmol) gave 0.44 g (91%) of 10j as a white solid. Rf = 0.25 (n-hexane/
(E ) - 1 - ( 2 - ( ( 3 - M e t h o x y p h e n y l) sulf onyl )viny l)- 2-
trifluoromethylbenzene (11a). Diethyl (3-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-
(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.41 g
(77%) of 11a as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
1
EtOAc 3/1); mp 83.0−85.0 °C; HPLC purity, 12.8 min, 96.8%; H
1
NMR (400 MHz, CDCl3) δ 7.82−7.89 (m, 2ArH, (E)-isomeric CH),
7.34−7.42 (m, 2ArH), 7.05 (d, J = 15.5 Hz, (E)-isomeric CH), 7.00
(d, J = 8.9 Hz, 2ArH), 6.90−6.97 (m, 2ArH), 3.88 (s, OCH3), 3.87 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.5, 158.9, 137.6, 132.9,
132.4, 130.7, 129.9, 128.7, 121.5, 120.9, 114.6, 111.4 (12ArC, 2CH),
55.8 (OCH3), 55.6 (OCH3); HRMS (M + H)+ (ESI+) 305.0845 [M +
H]+ (calcd for C16H16O4SH+ 305.0848).
74.0−75.0 °C; HPLC purity, 14.0 min, 99.4%; H NMR (400 MHz,
CDCl3) δ 8.05 (dd, J = 2.0, 15.2 Hz, (E)-isomeric CH), 7.72 (d, J =
7.3 Hz, ArH), 7.43−7.60 (m, 6ArH), 7.15 (ddd, J = 1.0, 2.6, 8.2 Hz,
ArH), 6.83 (d, J = 15.2 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C
NMR (100 MHz, CDCl3) δ 160.3, 141.3, 138.5, 132.4, 132.0, 131.4,
130.6, 130.5, 129.2 (q, JC−F = 30.6 Hz), 128.4, 126.4 (d, JC−F = 5.2
Hz), 123.8 (q, JC−F = 272.2 Hz, CF3), 120.5, 120.1, 112.1 (12ArC,
2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 343.0615 [M + H]+
(calcd for C16H13F3O3SH+ 343.0616).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-methoxy-
benzene (10k). Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
(0.82 mL, 1.63 mmol), and 3-methoxybenzaldehyde (0.21 mL, 1.71
mmol) gave 0.46 g (98%) of 10k as a yellow oil. Rf = 0.30 (n-hexane/
(E ) - 1 - ( 2 - ( ( 3 - M e t h o x y p h e n y l) sulf onyl )viny l)- 3-
trifluoromethylbenzene (11b). Diethyl (3-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-
(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.43 g
(80%) of 11b as a white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp
1
EtOAc 3/1); HPLC purity, 12.7 min, 97.3%; H NMR (400 MHz,
CDCl3) δ 7.87 (d, J = 9.0 Hz, 2ArH), 7.59 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.29 (t like due to dd, J = 7.9 Hz, ArH), 7.06 (d, J = 7.6
Hz, ArH), 7.01 (d, J = 9.0 Hz, 2ArH), 6.93−6.98 (m, 2ArH), 6.83 (d, J
= 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3), 3.81 (s, OCH3); 13C
NMR (100 MHz, CDCl3) δ 163.7, 160.1, 141.4, 133.9, 132.3, 130.1,
130.0, 128.3, 121.2, 117.0, 114.7, 113.4 (12ArC, 2CH), 55.8 (OCH3),
55.4 (OCH3); HRMS (M + H)+ (ESI+) 305.0845 [M + H]+ (calcd for
C16H16O4SH+ 305.0848).
1
73.0−75.0 °C; HPLC purity, 14.1 min, 99.7%; H NMR (400 MHz,
CDCl3) δ 7.66−7.73 (m, 3ArH, (E)-isomeric CH), 7.52−7.56 (m,
2ArH), 7.45−7.49 (m, 2ArH), 7.16 (ddd, J = 0.8, 2.4, 8.2 Hz, ArH),
6.94 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR
(100 MHz, CDCl3) δ 160.3, 141.5, 140.6, 133.3, 131.8, 131.7 (q, JC−F
= 32.6 Hz), 130.7, 129.8, 129.6, 127.6, 125.1, 123.7 (q, JC−F = 270.6
Hz, CF3), 120.2, 120.1, 112.4 (12ArC, 2CH), 55.8 (OCH3); HRMS
(M + H)+ (ESI+) 343.0619 [M + H]+ (calcd for C16H13F3O3SH+
343.0616).
(E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-methoxy-
benzene (10l).39 Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane
(0.82 mL, 1.63 mmol), and 4-methoxybenzaldehyde (0.21 mL, 1.71
mmol) gave 0.43 g (91%) of 10l as a white solid. Rf = 0.20 (n-hexane/
EtOAc 3/1); mp 127.0−129.0 °C (lit.39 108.0−109.0 °C); HPLC
(E ) - 1 - ( 2 - ( ( 3 - M e t h o x y p h e n y l) sulf onyl )viny l)- 4-
trifluoromethylbenzene (11c). Diethyl (3-methoxyphenylsulfonyl)-
methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in
cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)-
benzaldehyde (0.23 mL, 1.71 mmol) gave 0.46 g (87%) of 11c as a
white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp 101.0−102.0 °C;
HPLC purity, 14.1 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.69
(d, J = 15.4 Hz, (E)-isomeric CH), 7.66 (d, J = 8.5 Hz, 2ArH), 7.60 (d,
J = 8.4 Hz, 2ArH), 7.53 (td like due to ddd, J = 1.4, 7.7 Hz, ArH), 7.48
(d, J = 8.0 Hz, ArH), 7.45 (t like due to dd, J = 2.1 Hz, ArH), 7.16
(ddd, J = 1.1, 2.6, 8.5 Hz, ArH), 6.95 (d, J = 15.4 Hz, (E)-isomeric
CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.3, 141.4,
140.8, 135.9, 132.7 (q, JC−F = 32.6 Hz), 130.7, 130.1, 128.9, 126.1 (d,
JC−F = 3.4 Hz), 123.7 (q, JC−F = 270.8 Hz, CF3), 120.2, 120.1, 112.4
(12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 343.0616 [M
+ H]+ (calcd for C16H13F3O3SH+ 343.0616).
1
purity, 12.6 min, 97.8%; H NMR (400 MHz, CDCl3) δ 7.86 (d, J =
9.0 Hz, 2ArH), 7.58 (d, J = 15.4 Hz, (E)-isomeric CH), 7.42 (d, J = 8.7
Hz, 2ArH), 7.00 (d, J = 9.0 Hz, 2ArH), 6.89 (d, J = 8.7 Hz, 2ArH),
6.69 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3), 3.83 (s,
OCH3); 13C NMR (100 MHz, CDCl3) δ 163.5, 162.0, 141.3, 132.8,
130.4, 129.8, 125.3, 125.2, 114.6 (12ArC, 2CH), 55.8 (OCH3), 55.5
(OCH3); one signal was not detected and is believed to overlap with
nearby peaks; HRMS (M + H)+ (ESI+) 305.0847 [M + H]+ (calcd for
C16H16O4SH+ 305.0848).
( E) - 1 - ( 2 - ( ( 4 - M e t h o x y p h e n y l ) s u l f o n y l ) v i n y l ) - 2 -
trifluoromethoxybenzene (10m). Diethyl (4-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-
(trifluoromethoxy)benzaldehyde (0.24 mL, 1.71 mmol) gave 0.48 g
(87%) of 10m as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene
(11d). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g
(85%) of 11d as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1);
1
75.0−77.0 °C; HPLC purity, 14.0 min, 99.1%; H NMR (400 MHz,
CDCl3) δ 7.87 (d, J = 8.9 Hz, 2ArH), 7.82 (d, J = 15.5 Hz, (E)-
isomeric CH), 7.54 (d, J = 7.0 Hz, ArH), 7.44 (t like due to dd, J = 7.8
Hz, ArH), 7.30 (t like due to dd, J = 7.5 Hz, 2ArH), 7.02 (d, J = 8.9
Hz, 2ArH), 6.92 (d, J = 15.5 Hz, (E)-isomeric CH), 3.88 (s, OCH3);
13C NMR (100 MHz, CDCl3) δ 163.9, 147.7, 134.4, 132.2, 131.7,
1
HPLC purity, 13.1 min, 98.7%; H NMR (400 MHz, CDCl3) δ 7.75
(d, J = 15.6 Hz, (E)-isomeric CH), 7.53 (td like due to ddd, J = 1.3, 7.8
Hz, ArH), 7.43−7.49 (m, 3ArH), 7.37−7.43 (m, ArH), 7.09−7.20 (m,
3ArH), 7.02 (d, J = 15.6 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C
NMR (100 MHz, CDCl3) δ 161.6 (d, JC−F = 253.7 Hz, ArC-F), 160.2,
141.7, 135.5, 132.9 (d, JC−F = 8.8 Hz), 130.6, 130.3, 130.1 (d, J = 8.3
Hz), 124.8, 120.6 (d, JC−F = 11.2 Hz), 120.0, 116.5 (d, JC−F = 21.5
Hz), 112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+)
293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648).
131.3, 130.1, 128.8, 127.3, 124.3, 121.5, 120.5 (q, J = 257.5 Hz, OCF3),
114.7 (12ArC. 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+)
359.0565 [M + H]+ (calcd for C16H13F3O4SH+ 359.0565).
(E)-2-(2-((4-Methoxyphenyl)sulfonyl)vinyl)aniline Hydro-
chloride (10n). Diethyl (4-methoxyphenylsulfonyl)methyl-
phosphonate (0.30 g, 0.93 mmol), 2 M n-BuLi solution in cyclohexane
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(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene
(11e). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.42 g
(93%) of 11e as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1);
solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-
(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.49 g
(92%) of 12a as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
1
116.0−119.0 °C; HPLC purity, 13.4 min, >99.0%; H NMR (400
MHz, CDCl3) δ 8.08 (dd, J = 1.8, 15.2 Hz, (E)-isomeric CH), 8.04
(dd, J = 1.8, 7.8 Hz, ArH), 7.72 (d, J = 7.6 Hz, ArH), 7.65 (d, J = 7.6
Hz, ArH), 7.56−7.61 (m, 2ArH), 7.51 (t like due to dd, J = 7.6 Hz,
ArH), 7.13 (t like due to dd, J = 7.4 Hz, ArH), 7.01−7.06 (m, ArH,
(E)-isomeric CH), 3.95 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ
1
HPLC purity, 12.9 min, 96.9%; H NMR (400 MHz, CDCl3) δ 7.63
(d, J = 15.4 Hz, (E)-isomeric CH), 7.43−7.53 (m, 5ArH), 7.12−7.15
(m, ArH), 7.08 (t like due to dd, J = 8.6 Hz, 2ArH), 6.79 (d, J = 15.4
Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3)
δ 163.0 (d, JC−F = 246.3 Hz, ArC-F), 160.2, 141.6, 141.0, 134.6 (d,
JC−F = 7.5 Hz), 130.8, 130.7, 130.6, 128.9, 124.7, 120.1 (d, JC−F = 21.2
Hz), 118.2 (d, JC−F = 21.2 Hz), 114.9 (d, JC−F = 22.1 Hz), 112.3
(12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 293.0650 [M
+ H]+ (calcd for C15H13FO3SH+ 293.0648).
157.6, 138.9, 135.8, 132.6, 131.6, 131.3, 130.3, 129.9, 129.3 (q, JC−F
=
30.6 Hz), 128.4, 127.6, 126.4 (q, JC−F = 5.4 Hz), 123.8 (q, JC−F = 272.4
Hz, CF3), 120.7, 112.4 (12ArC, 2CH), 56.2 (OCH3); HRMS (M +
H)+ (ESI+) 343.0616 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616).
(E ) - 1 - ( 2 - ( ( 2 - M e t h o x y p h e n y l) sulf onyl )viny l)- 3-
trifluoromethylbenzene (12b). Diethyl (2-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-
(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.51 g
(96%) of 12b as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp
(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-4-fluorobenzene
(11f). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.42 g
(93%) of 11f as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1);
1
1
HPLC purity, 13.0 min, 99.6%; H NMR (400 MHz, CDCl3) δ 7.63
138.0−139.0 °C; HPLC purity, 13.6 min, >99.0%; H NMR (400
(d, J = 15.4 Hz, (E)-isomeric CH), 7.52 (dt like due to ddd, J = 1.4, 7.8
Hz, ArH), 7.44−7.48 (m, 2ArH), 7.37 (td like due to ddd, J = 5.8, 7.8
Hz, ArH), 7.26−7.28 (m, ArH), 7.09−7.17 (m, 3ArH), 6.86 (d, J =
15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz,
CDCl3) δ 164.4 (d, JC−F = 251.4 Hz, ArC-F), 160.2, 141.9, 141.2,
MHz, CDCl3) δ 8.01 (dd, J = 1.8, 7.8 Hz, ArH), 7.63−7.72 (m, 3ArH,
(E)-isomeric CH), 7.57 (ddd, J = 1.0, 1.8, 7.5 Hz, ArH), 7.52 (t like
due to dd, J = 7.8 Hz, ArH), 7.19 (d, J = 15.5 Hz, (E)-isomeric CH),
7.10 (td like due to ddd, J = 1.0, 8.4 Hz, ArH), 7.01 (d, J = 8.0 Hz,
ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.5, 141.4,
135.8, 133.7, 131.6, 131.5 (q, JC−F = 32.6 Hz), 129.8, 129.6, 129.2,
128.1, 127.4, 125.0, 123.7 (q, JC−F = 270.8 Hz, CF3), 120.9, 112.6
(12ArC,2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 343.0618 [M
+ H]+ (calcd for C16H13F3O3SH+ 343.0616).
(E ) - 1 - ( 2 - ( ( 2 - M e t h o x y p h e n y l) sulf onyl )viny l)- 4-
trifluoromethylbenzene (12c). Diethyl (2-methoxyphenylsulfonyl)-
methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in
cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)-
benzaldehyde (0.23 mL, 1.71 mmol) gave 0.52 g (94%) of 12c as a
white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 113.0−114.0 °C;
HPLC purity, 13.7 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.03
(dd, J = 1.8, 7.7 Hz, ArH), 7.71 (d, J = 15.5 Hz, (E)-isomeric CH),
7.66 (d, J = 8.3 Hz, 2ArH), 7.58−7.62 (m, 3ArH), 7.22 (d, J = 15.5 Hz,
(E)-isomeric CH), 7.13 (td like due to ddd, J = 0.8, 8.3 Hz, ArH), 7.03
(d, J = 8.3 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ
157.5, 141.4, 136.4, 135.8, 132.4 (q, JC−F = 32.5 Hz), 129.8, 129.7,
128.8, 128.2, 126.1, 123.8 (q, JC−F = 270.6 Hz, CF3), 120.9, 112.6
(12ArC, 2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 343.0617 [M
+ H]+ (calcd for C16H13F3O3SH+ 343.0616).
130.7 (d, JC−F = 8.7 Hz), 130.6, 128.7, 127.1, 119.9, 116.4 (d, JC−F
=
21.9 Hz), 112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+
(ESI+) 293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648).
(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-2-chlorobenzene
(11g). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.39 g
(81%) of 11g as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp
1
88.0−89.0 °C; HPLC purity, 13.9 min, >99.0%; H NMR (400 MHz,
CDCl3) δ 8.08 (d, J = 15.4 Hz, (E)-isomeric CH), 7.50−7.56 (m,
2ArH), 7.43−7.48 (m, 3ArH), 7.34 (td like due to ddd, J = 1.6, 7.6 Hz,
ArH), 7.25−7.29 (m, ArH), 7.15 (ddd, J = 0.8, 2.6, 8.2 Hz, ArH), 6.90
(d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100
MHz, CDCl3) δ 160.2, 141.5, 138.4, 135.3, 132.0, 130.6, 130.4, 130.0,
128.3, 127.3, 120.0, 112.3 (12ArC, 2CH), 55.8 (OCH3); HRMS (M +
H)+ (ESI+) 309.0350 [M + H]+ (calcd for C15H13ClO3SH+309.0352).
(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-3-chlorobenzene
(11h). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.36 g
(74%) of 11h as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1);
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene
(12d). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.38 g
(85%) of 12d as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
110.0−113.0 °C; HPLC purity, 12.5 min, 99.0%; 1H NMR (400 MHz,
CDCl3) δ 8.03 (dd, J = 1.7, 7.8 Hz, ArH), 7.78 (d, J = 15.6 Hz, (E)-
isomeric CH), 7.58 (ddd, J = 1.7, 7.5, 8.7 Hz, ArH), 7.48 (td like due
to ddd, J = 1.7, 7.5 Hz, ArH), 7.38−7.42 (m, 1H), 7.27 (d, J = 15.6 Hz,
(E)-isomeric CH), 7.18 (td like due to ddd, J = 0.9, 7.5 Hz, ArH),
1
HPLC purity, 14.0 min, 98.9%; mp 71.0−72.0 °C; H NMR (400
MHz, CDCl3) δ 7.59 (d, J = 15.4 Hz, (E)-isomeric CH), 7.50−7.53
(m, ArH), 7.43−7.47 (m, 3ArH), 7.30−7.38 (m, 3ArH), 7.14 (ddd, J =
1.0, 2.6, 8.1 Hz, ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH); 13C
NMR (100 MHz, CDCl3) δ 160.3, 141.6, 140.8, 135.3, 134.3, 131.2,
130.7, 130.5, 129.0, 128.3, 126.9, 120.2, 120.1, 112.3 (12ArC, 2CH),
55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0353 [M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
7.09−7.14 (m, 2ArH), 7.02 (d, J = 8.2 Hz, ArH), 3.98 (s, OCH3); 13
C
(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-4-chlorobenzene
(11i). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol) gave 0.33 g
(70%) of 11i as a white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp
NMR (100 MHz, CDCl3) δ 161.4 (d, JC−F = 253.3 Hz, ArC-F), 157.4,
136.4, 135.6, 132.6 (d, JC−F = 8.8 Hz), 130.0, 129.8 (d, JC−F = 7.9 Hz),
129.5, 128.4, 124.7 (d, JC−F = 3.1 Hz), 121.0 (d, JC−F = 11.3 Hz),
120.7, 116.3 (d, JC−F = 21.6 Hz), 112.5 (12ArC,2CH), 56.2 (OCH3);
HRMS (M + H)+ (ESI+) 293.0647 [M + H]+ (calcd for
C15H13FO3SH+ 293.0648).
1
85.0−87.0 °C; HPLC purity, 13.9 min, >99.0%; H NMR (400 MHz,
CDCl3) δ 7.61 (d, J = 15.4 Hz, (E)-isomeric CH), 7.52 (dt like due to
ddd, J = 1.3, 7.7 Hz, ArH), 7.43−7.48 (m, 3ArH), 7.31−7.40 (m,
3ArH), 7.15 (ddd, J = 1.0, 2.6, 8.2 Hz, ArH), 6.87 (d, J = 15.4 Hz, (E)-
isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.2,
141.7, 141.0, 137.3, 130.9, 130.6, 129.8, 129.5, 127.9, 120.0, 119.9,
112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+)
309.0352 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352).
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene
(12e). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.37 g
(82%) of 12e as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp
117.0−119.0 °C; HPLC purity, 12.5 min, 99.0%; 1H NMR (400 MHz,
CDCl3) δ 8.02 (dd, J = 1.7, 7.9 Hz, ArH), 7.65 (d, J = 15.5 Hz, (E)-
isomeric CH), 7.58 (ddd, J = 1.7, 7.4, 8.4 Hz, ArH), 7.37 (td like due
to ddd, J = 5.8, 7.9 Hz, ArH), 7.28 (d, J = 7.9 Hz, ArH), 7.19 (dt like
( E) - 1 - ( 2 - ( ( 2 - M e t h o x y p h e n y l ) s u l f o n y l ) v i n y l ) - 2 -
trifluoromethylbenzene (12a). Diethyl (2-methoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi
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due to ddd, J = 2.0, 9.5 Hz, ArH), 7.08−7.16 (m, 2ArH, (E)-isomeric
CH), 7.02 (d, J = 8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100
MHz, CDCl3) δ 162.9 (d, JC−F = 246.0 Hz, ArC-F), 157.4, 141.8,
135.6, 135.0 (d, JC−F = 7.5 Hz), 130.7 (d, JC−F = 8.1 Hz), 129.5, 128.5,
128.3, 124.5, 120.8, 117.9 (d, JC−F = 21.3 Hz), 114.7 (d, JC−F = 22.0
Hz), 112.5 (12ArC, 2CH), 56.3 (OCH3); HRMS (M + H)+ (ESI+)
293.0648 [M + H]+ (calcd for C15H13FO3SH+ 293.0648).
7.50−7.60 (m, 3ArH), 7.22−7.26 (m, 2ArH), 6.82 (d, J = 15.2 Hz,
(E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.9 (d, JC−F
=
255.0 Hz, ArC-F), 138.7, 136.2 (d, JC−F = 2.3 Hz), 132.4, 131.9, 131.3,
130.8 (d, JC−F = 9.6 Hz), 130.6, 130.2, 129.2 (q, JC−F = 30.6 Hz),
128.4, 126.5 (q, JC−F = 272.4 Hz, CF3), 116.8 (d, JC−F = 22.6 Hz)
(12ArC, 2CH); HRMS (M + H)+ (ESI+) 331.0411 [M + H]+ (calcd
for C15H10F4O2SH+ 331.0416).
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-4-fluorobenzene
(12f). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g
(87%) of 12f as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp
122.0−124.0 °C; HPLC purity, 12.5 min, 98.9%;1H NMR (400 MHz,
CDCl3) δ 8.02 (dd, J = 1.7, 7.8 Hz, ArH), 7.65 (d, J = 15.5 Hz, (E)-
isomeric CH), 7.57 (ddd, J = 1.7, 7.4, 9.1 Hz, ArH), 7.50 (dd, J = 5.3,
8.8 Hz, 2ArH), 7.01−7.13 (m, 3ArH, (E)-isomeric CH), 7.02 (d, J =
8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.3
(d, JC−F = 251.0 Hz, ArC-F), 157.4, 142.0, 135.5, 130.6 (d, JC−F = 8.5
Hz), 129.5, 129.1, 128.5, 126.7, 120.8, 116.3 (d, JC−F = 21.9 Hz), 112.5
(12ArC, 2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 293.0648 [M
+ H]+ (calcd for C15H13FO3SH+ 293.0648).
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-2-chlorobenzene
(12g). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.44 g
(91%) of 12g as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
119.0−121.0 °C; HPLC purity, 13.3 min, >99.0%; 1H NMR (400
MHz, CDCl3) δ 8.11 (d, J = 15.4 Hz, (E)-isomeric CH), 8.04 (dd, J =
1.7, 7.7 Hz, ArH), 7.55−7.61 (m, 2ArH), 7.43 (dd, J = 1.4, 7.7 Hz,
ArH), 7.33 (dd, J = 1.7, 7.7 Hz, ArH), 7.28 (dd, J = 1.4, 7.7 Hz, ArH),
7.10−7.13 (m, ArH, (E)-isomeric CH), 7.01 (d, J = 8.4 Hz, ArH), 3.98
(s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.3, 141.7, 136.9, 135.5,
131.3, 129.7, 129.4, 129.3, 128.3, 127.6, 120.7, 112.5 (12ArC, 12CH),
56.3 (OCH3); HRMS (M + H)+ (ESI+) 309.0352 [M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-trifluoromethyl-
benzene (13b). Diethyl (4-fluorophenylsulfonyl)methylphosphonate
(0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL,
1.69 mmol), and 3-(trifluoromethyl)benzaldehyde (0.24 mL, 1.77
mmol) gave 0.50 g (93%) of 13b as a white solid. Rf = 0.40 (n-hexane/
EtOAc 5/1); mp 110.0−112.0 °C; HPLC purity, 14.0 min, 99.5%; 1H
NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 5.0, 8.7 Hz, 2ArH), 7.73 (s,
ArH), 7.70 (d, J = 15.5 Hz, (E)-isomeric CH), 7.65−7.68 (m, 2ArH),
7.55 (t like due to dd, J = 7.8 Hz, ArH), 7.24 (t like due to dd, J = 8.7
Hz, 2ArH), 6.93 (d, J = 15.5 Hz, (E)-isomeric CH); 13C NMR (100
MHz, CDCl3) δ 165.9 (d, JC−F = 255.0 Hz, ArC-F), 136.4, 133.2,
131.9, 131.8 (q, JC−F = 32.6 Hz), 130.8 (d, JC−F = 9.0 Hz), 129.9,
129.5, 127.7 (d, JC−F = 3.1 Hz), 125.1 (d, JC−F = 3.5 Hz), 123.7 (q,
JC−F = 270.9 Hz, CF3), 117.0, 116.8 (12ArC, 2CH); HRMS (M + H)+
(ESI+) 331.0415 [M + H]+ (calcd for C15H10F4O2SH+ 331.0416).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-trifluoromethyl-
benzene (13c). Diethyl (4-fluorophenylsulfonyl)methylphosphonate
(0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL,
1.69 mmol), and 4-(trifluoromethyl)benzaldehyde (0.24 mL, 1.77
mmol) gave 0.49 g (92%) of 13c as a white solid. Rf = 0.50 (n-hexane/
EtOAc 5/1); mp 140.0−141.0 °C; HPLC purity, 14.1 min, 99.4%; 1H
NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 5.0, 8.9 Hz, 2ArH), 7.70 (d, J
= 15.4 Hz, (E)-isomeric CH), 7.66 (d, J = 8.4 Hz, 2ArH), 7.60 (d, J =
8.4 Hz, 2ArH), 7.25 (t like due to dd, J = 8.6 Hz, 2ArH), 6.93 (d, J =
15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 166.0 (d,
JC−F = 255.2 Hz, ArC-F), 140.7, 136.4, 135.8, 132.8 (q, JC−F = 32.7
Hz), 130.8 (d, JC−F = 9.6 Hz), 130.0, 128.9, 126.2 (d, JC−F = 3.3 Hz),
123.7 (q, JC−F = 271.0 Hz, CF3), 116.9 (d, JC−F = 22.6 Hz) (12ArC,
2CH); HRMS (M + H)+ (ESI+) 331.0422 [M + H]+ (calcd for
C15H10F4O2SH+ 331.0416).
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-3-chlorobenzene
(12h). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.44 g
(91%) of 12h as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp
116.0−117.0 °C; HPLC purity, 13.4 min, 99.8%; 1H NMR (400 MHz,
CDCl3) δ 8.02 (dd, J = 1.7, 7.9 Hz, ArH), 7.56−7.65 (m, ArH,(E)-
isomeric CH), 7.48 (s, ArH), 7.32−7.40 (m, 3ArH), 7.09−7.16 (m,
ArH, (E)-isomeric CH), 7.02 (d, J = 8.3 Hz, ArH), 3.98 (s, OCH3);
13C NMR (100 MHz, CDCl3) δ 157.5, 141.9, 137.1, 135.6, 131.5,
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-fluorobenzene
(13d). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), and 2-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.37 g
(82%) of 13d as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
1
72.0−74.0 °C; HPLC purity, 13.1 min, 98.8%; H NMR (400 MHz,
CDCl3) δ 7.97 (dd, J = 5.0, 8.9 Hz, 2ArH), 7.76 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.47 (td like due to ddd, J = 1.7, 7.6 Hz, ArH), 7.38−
7.44 (m, ArH), 7.16−7.25 (m, 3ArH), 7.12 (ddd, J = 0.9, 8.3, 10.8 Hz,
ArH), 7.00 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz,
CDCl3) δ 165.8 (d, JC−F = 254.6 Hz, ArC-F), 161.7 (d, JC−F = 253.9
Hz, ArC-F), 136.7, 135.8, 133.0 (d, JC−F = 8.9 Hz), 130.7 (d, JC−F = 9.5
Hz), 130.2 (d, JC−F = 8.5 Hz), 124.8 (d, JC−F = 3.1 Hz), 120.6 (d, JC−F
= 11.3 Hz), 116.9, 116.7, 116.4 (12ArC, 2CH); HRMS (M + H)+
(ESI+) 281.0445 [M + H]+ (calcd for C14H10F2O2SH+ 281.0448).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-fluorobenzene
(13e). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), and 3-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.42 g
(93%) of 13e as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
129.8, 129.7, 129.5, 128.6, 127.7, 120.9, 112.6(12ArC, 2CH), 56.5
(OCH3); HRMS (M + H)+ (ESI+) 309.0352[M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
(E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-4-chlorobenzene
(12i). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g,
1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63
mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol) gave 0.44 g
(91%) of 12i as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp
125.0−126.0 °C; HPLC purity, 13.5 min, >99.0%; 1H NMR (400
MHz, CDCl3) δ 8.02 (dd, J = 1.7, 7.7 Hz, ArH), 7.64 (d, J = 15.5 Hz,
(E)-isomeric CH), 7.58 (td like due to ddd, J = 1.7, 7.7 Hz, ArH), 7.43
(d, J = 8.6 Hz, 2ArH), 7.37 (d, J = 8.6 Hz, 2ArH), 7.09−7.13 (m, ArH,
(E)-isomeric CH), 7.02 (d, J = 8.2 Hz, ArH), 3.97 (s, OCH3); 13C
NMR (100 MHz, CDCl3) δ 157.4, 141.6, 135.7, 135.1, 134.7, 130.9,
130.4, 129.6, 128.6, 128.1, 126.8, 120.8, 112.6 (12ArC, 2CH), 56.4
(OCH3); HRMS (M + H)+ (ESI+) 309.0353 [M + H]+ (calcd for
C15H13ClO3SH+ 309.0352).
1
52.0−54.0 °C; HPLC purity, 13.0 min, 98.4%; H NMR (400 MHz,
CDCl3) δ 7.96 (dd, J = 5.0, 9.2 Hz, 2ArH), 7.64 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.34 (td like due to ddd, J = 5.7, 8.0 Hz, ArH), 7.21−
7.28 (m, 3ArH), 7.18 (td like due to ddd, J = 2.0, 9.2 Hz, ArH), 7.13
(ddd, J = 0.8, 2.5, 8.3 Hz, ArH), 6.84 (d, J = 15.4 Hz, (E)-isomeric
CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.8 Hz, ArC-
F), 163.0 (d, JC−F = 246.5 Hz, ArC-F), 141.2, 136.6, 134.6, 130.9,
130.7 (d, JC−F = 9.5 Hz), 128.8, 124.8, 118.3 (d, JC−F = 21.2 Hz), 116.8
(d, JC−F = 22.5 Hz), 114.9 (d, JC−F = 22.0 Hz) (12ArC, 2CH); HRMS
(M + H)+ (ESI+) 281.0446 [M + H]+ (calcd for C14H10F2O2SH+
281.0448).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-trifluoromethyl-
benzene (13a). Diethyl (4-fluorophenylsulfonyl)methylphosphonate
(0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL,
1.69 mmol), and 2-(trifluoromethyl)benzaldehyde (0.23 mL, 1.77
mmol) gave 0.50 g (93%) of 13a as a white solid. Rf = 0.30 (n-hexane/
1
EtOAc 5/1); mp 78.0−79.0 °C; HPLC purity, 13.9 min, 99.1%; H
NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 1.8, 15.2 Hz, (E)-isomeric
CH), 7.97 (dd, J = 5.0, 8.8 Hz, 2ArH), 7.72 (d, J = 7.6 Hz, ArH),
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-fluorobenzene
(13f).37 Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
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1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), and 4-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.44 g
(97%) of 13f as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
(0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL,
1.61 mmol), and 3-(trifluoromethyl)benzaldehyde (0.22 mL, 1.68
mmol) gave 0.50 g (95%) of 14b as a white solid. Rf = 0.50 (n-hexane/
EtOAc 5/1); mp 159.0−161.0 °C; HPLC purity, 14.9 min, >99.0%;
1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.7 Hz, 2ArH), 7.65−7.73
(m, 3ArH, (E)-isomeric CH), 7.53−7.57 (m, 3ArH), 6.92 (d, J = 15.4
Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.1, 140.5,
138.9, 133.2, 131.9, 131.8 (q, JC−F = 32.7 Hz), 129.9, 129.4, 129.2,
127.8, 125.2, 123.6 (q, JC−F = 270.5 Hz, CF3) (12ArC, 2CH); one
signal was not detected and is believed to overlap with nearby peaks;
HRMS (M + H)+ (ESI+) 347.0121 [M + H]+ (calcd for
C15H10ClF3O2SH+ 347.0120).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-trifluoromethyl-
benzene (14c). Diethyl (4-chlorophenylsulfonyl)methylphosphonate
(0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL,
1.61 mmol), and 4-(trifluoromethyl)benzaldehyde (0.23 mL, 1.68
mmol) gave 0.49 g (91%) of 14c as a white solid. Rf = 0.55 (n-hexane/
EtOAc 5/1); mp 147.0−149.0 °C; HPLC purity, 14.9 min, 99.0%; 1H
NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.6 Hz, 2ArH), 7.65−7.73 (m,
2ArH, (E)-isomeric CH), 7.59 (d, J = 8.3 Hz, 2ArH), 7.55 (d, J = 8.6
Hz, 2ArH), 6.92 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100
MHz, CDCl3) δ 141.1, 140.6, 138.8, 135.7, 132.8 (q, JC−F = 32.6 Hz),
1
83.0−84.0 °C; HPLC purity, 12.9 min, 99.5%; H NMR (400 MHz,
CDCl3) δ 7.96 (dd, J = 5.2, 8.8 Hz, 2ArH), 7.65 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.49 (dd, J = 5.2, 8.8 Hz, 2ArH), 7.23 (t like due to dd,
J = 8.6 Hz, 2ArH), 7.09 (t like due to dd, J = 8.6 Hz, 2ArH), 6.77 (d, J
= 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.7
(d, JC−F = 254.5 Hz, ArC-F), 164.5 (d, JC−F = 251.6 Hz, ArC-F), 141.4,
136.8, 130.7 (d, JC−F = 8.7 Hz), 130.6 (d, JC−F = 9.5 Hz), 128.6 (d,
JC−F = 2.7 Hz), 127.0, 116.7 (d, JC−F = 22.6 Hz), 116.4 (d, JC−F = 22.0
Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 281.0445 [M + H]+
(calcd for C14H10F2O2SH+ 281.0448).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-chlorobenzene
(13g). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), 2-chlorobenzaldehyde (0.20 mL, 1.77 mmol) gave 0.40 g
(84%) of 13g as a white solid. Rf = 0.40 (n-hexane/EtOAc 5/1); mp
107.0−108.0 °C; HPLC purity, 13.7 min, 99.3%; 1H NMR (400 MHz,
CDCl3) δ 8.08 (d, J = 15.5 Hz, (E)-isomeric CH), 7.98 (dd, J = 5.1,
8.8 Hz, 2ArH), 7.51 (dd, J = 1.6, 7.7 Hz, ArH), 7.44 (dd, J = 1.1, 8.0
Hz, ArH), 7.35 (td like due to ddd, J = 1.6, 7.7 Hz, ArH), 7.22−7.29
(m, 3ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100
MHz, CDCl3) δ 165.8 (d, JC−F = 254.8 Hz, ArC-F), 138.7, 136.5,
135.4, 132.1, 130.9, 130.8 (d, JC−F = 9.5 Hz), 130.5, 130.0, 128.4,
127.4, 116.8 (d, JC−F = 22.5 Hz) (12ArC, 2CH); HRMS (M + H)+
(ESI+) 297.0149 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-chlorobenzene
(13h). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), and 3-chlorobenzaldehyde (0.20 mL, 1.77 mmol) gave 0.37 g
(76%) of 13h as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
107.0−109.0 °C; HPLC purity, 13.8 min, 99.4%; 1H NMR (400 MHz,
CDCl3) δ 7.96 (dd, J = 5.0, 9.0 Hz, 2ArH), 7.61 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.47 (s, ArH), 7.32−7.41 (m, 3ArH), 7.24 (t like due to
dd, J = 9.0 Hz, 2ArH), 6.85 (d, J = 15.4 Hz, (E)-isomeric CH); 13C
NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.9 Hz, ArC-F), 141.0,
136.6, 135.3, 134.1, 131.2, 130.8 (d, JC−F = 9.5 Hz), 130.5, 128.9,
128.3, 127.0, 116.8 (d, JC−F = 22.6 Hz) (12ArC, 2CH); HRMS (M +
H)+ (ESI+) 297.0152 [M + H]+ (calcd for C14H10ClFO2SH+
297.0152).
129.9, 129.8, 129.4, 128.9, 126.2 (d, JC−F = 3.4 Hz), 123.7 (q, JC−F
=
270.8 Hz, CF3) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 347.0113
[M + H]+ (calcd for C15H10ClF3O2SH+ 347.0120).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-fluorobenzene
(14d).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.42 g
(91%) of 14d as a white solid. Rf = 0.50 (n-hexane/EtOAc 5/1); mp
1
89.0−91.0 °C; HPLC purity, 14.0 min, 99.4%; H NMR (400 MHz,
CDCl3) δ 7.89 (d, J = 8.6 Hz, 2ArH), 7.76 (d, J = 15.6 Hz, (E)-
isomeric CH), 7.53 (d, J = 8.6 Hz, 2ArH), 7.46 (td like due to ddd, J =
1.6, 7.6 Hz ArH), 7.38−7.44 (m, ArH), 7.18 (td like due to ddd, J =
0.9, 7.6 Hz, ArH), 7.12 (td like due to ddd, J = 0.9, 8.3 Hz, ArH), 7.00
(d, J = 15.6 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ
161.7 (d, JC−F = 254.0 Hz, ArC-F), 140.4, 139.2, 136.2, 133.1 (d, JC−F
= 8.9 Hz), 130.6, 130.0, 129.8, 129.4, 124.9 (d, JC−F = 3.3 Hz), 120.5
(d, JC−F = 11.2 Hz) (12ArC, 2CH); one signal was not detected and is
believed to overlap with nearby peaks; HRMS (M + H)+ (ESI+)
297.0150 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152).
(E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-chlorobenzene
(13i). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,
1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69
mmol), and 4-chlorobenzaldehyde (0.25 g, 1.77 mmol) gave 0.38 g
(79%) of 13i as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
134.0−136.0 °C; HPLC purity, 13.9 min, 98.9%; 1H NMR (400 MHz,
CDCl3) δ 7.96 (dd, J = 5.0, 8.8 Hz, 2ArH), 7.62 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.42 (d, J = 8.6 Hz, 2ArH), 7.38 (d, J = 8.8 Hz, 2ArH),
7.23 (t like due to dd, J = 8.6 Hz, 2ArH), 6.82 (d, J = 15.4 Hz, (E)-
isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.7
Hz, ArC-F), 141.2, 137.5, 136.7, 130.8 (d, JC−F = 12.0 Hz), 130.6,
129.9, 129.5, 127.9, 116.8 (d, JC−F = 22.5 Hz) (12ArC, 2CH); HRMS
(M + H)+ (ESI+) 297.0150 [M + H]+ (calcd for C14H10ClFO2SH+
297.0152).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-trifluoromethyl-
benzene (14a). Diethyl (4-chlorophenylsulfonyl)methylphosphonate
(0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL,
1.61 mmol), and 2-(trifluoromethyl)benzaldehyde (0.22 mL, 1.68
mmol) gave 0.45 g (85%) of 14a as a white solid. Rf = 0.45 (n-hexane/
EtOAc 5/1); mp 155.0−157.0 °C; HPLC purity, 14.8 min, 99.6%; 1H
NMR (400 MHz, CDCl3) δ 8.06 (dd, J = 2.0, 15.2 Hz, (E)-isomeric
CH), 7.89 (d, J = 8.6 Hz, 2ArH), 7.73 (d, J = 7.3 Hz, ArH), 7.51−7.59
(m, 5ArH), 6.81 (d, J = 15.2 Hz, (E)-isomeric CH); 13C NMR (100
MHz, CDCl3) δ 140.5, 139.1, 138.7, 132.4, 131.6, 131.2, 130.7, 129.9,
129.4, 129.2 (q, JC−F = 30.5 Hz), 128.5, 126.5 (q, JC−F = 5.4 Hz), 123.8
(q, JC−F = 272.3 Hz, CF3) (12ArC, 2CH); HRMS (M + H)+ (ESI+)
347.0114 [M + H]+ (calcd for C15H10ClF3O2SH+ 347.0120).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-trifluoromethyl-
benzene (14b). Diethyl (4-chlorophenylsulfonyl)methylphosphonate
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-fluorobenzene
(14e). Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.45 g
(99%) of 14e as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp
1
101.0−102.0 °C; HPLC purity, 13.9 min, >99.0%; H NMR (400
MHz, CDCl3) δ 7.88 (d, J = 8.7 Hz, 2ArH), 7.64 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.54 (d, J = 8.7 Hz, 2ArH), 7.38 (td like due to ddd, J =
5.7, 8.0 Hz, ArH), 7.28 (s, ArH), 7.11−7.19 (m, 2ArH), 6.83 (d, J =
15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 163.0 (d,
JC−F = 246.4 Hz, ArC-F), 141.6, 140.3, 139.0, 134.4 (d, JC−F = 7.6 Hz),
130.8 (d, JC−F = 8.1 Hz), 129.8, 129.3, 128.5, 124.8, 118.3 (d, JC−F
=
21.1 Hz), 114.9 (d, JC−F = 22.2 Hz) (12ArC, 2CH); HRMS (M + H)+
(ESI+) 297.0156 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-fluorobenzene
(14f).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.45 g
(99%) of 14f as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
117.0−120.0 °C (lit.39 100.0−102.0 °C); HPLC purity, 13.8 min,
1
99.7%; H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.6 Hz, 2ArH),
7.65 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.6 Hz, 2ArH),
7.49 (dd, J = 5.3, 8.6 Hz, ArH), 7.09 (t like due to dd, J = 8.6 Hz,
2ArH), 6.76 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz,
CDCl3) δ 164.6 (d, JC−F = 251.9 Hz, ArC-F), 140.8, 140.3, 139.3,
130.8 (d, JC−F = 8.7 Hz), 129.8, 129.3, 128.6 (d, JC−F = 2.9 Hz), 126.8,
116.5 (d, JC−F = 22.0 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+)
297.0155 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152).
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(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-chlorobenzene
(14g).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.68 mmol) gave 0.44 g
(91%) of 14g as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
139.0−141.0 °C (lit.39 139.5−140.5 °C); HPLC purity, 14.8 min,
+ H)+ (ESI+) 373.0723 [M + H]+ (calcd for C17H15F3O4SH+
373.0721).
(E)-1-(2-((3,4-Dimethoxyphenyl)sulfonyl)vinyl)-4-
trifluoromethylbenzene (15c). Diethyl (3,4-dimethoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi
solution in cyclohexane (0.75 mL, 1.49 mmol), and 4-
(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.50 g
(94%) of 15c as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
190.0−192.0 °C; HPLC purity, 13.4 min, 99.5%; 1H NMR (400 MHz,
CDCl3) δ 7.56−7.66 (m, 5ArH, (E)-isomeric CH), 7.38 (d, J = 2.0 Hz,
ArH), 6.99 (d, J = 8.5 Hz, ArH), 6.94 (d, J = 15.4 Hz, (E)-isomeric
CH), 3.95 (s, 2OCH3); 13C NMR (100 MHz, CDCl3) δ 152.7, 148.6,
138.4, 135.0, 131.6 (q, JC−F = 32.6 Hz), 130.7, 129.7, 127.8, 125.2,
125.1, 122.7 (q, JC−F = 270.6 Hz, CF3), 121.3, 110.2, 109.1 (12ArC,
2CH), 55.4 (2OCH3); HRMS (M + H)+ (ESI+) 373.0724 [M + H]+
(calcd for C17H15F3O4SH+ 373.0721).
1
>99.0%; H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 15.4 Hz, (E)-
isomeric CH), 7.90 (d, J = 8.6 Hz, 2ArH), 7.54 (d, J = 8.6 Hz, 2ArH),
7.51 (dd, J = 1.6, 7.8 Hz, ArH), 7.45 (dd, J = 1.2, 8.0 Hz, ArH), 7.35
(td like due to ddd, J = 1.6, 7.8 Hz, ArH). 7.26−7.30 (m, ArH), 6.88
(d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ
140.4, 139.1, 139.0, 135.5, 132.2, 130.6, 130.5, 129.8, 129.7, 129.4,
128.4, 127.4 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 312.9854 [M +
H]+ (calcd for C14H10Cl2O2SH+ 312.9857).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-chlorobenzene
(14h).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.68 mmol) gave 0.46 g
(97%) of 14h as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp
128.0−130.0 °C (lit.39 130.0−131.0 °C); HPLC purity, 14.8 min,
(E)-1-(2-((Phenyl)sulfonyl)vinyl)-2-trifluoromethylbenzene
(16a). Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71
mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol),
and 2-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.41
g (76%) of 16a as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
1
98.7%; H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.5 Hz, 2ArH),
1
72.0−74.0 °C; HPLC purity, 13.7 min, 99.7%; H NMR (400 MHz,
7.62 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.5 Hz, 2ArH),
7.47 (s, ArH), 7.32−7.41 (m, 3ArH), 6.84 (d, J = 15.4 Hz, (E)-
isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.4, 140.4, 139.0,
135.3, 134.1, 131.3, 130.5, 129.8, 129.3, 128.6, 128.3, 127.0 (12ArC,
2CH); HRMS (M + H)+ (ESI+) 312.9851 [M + H]+ (calcd for
C14H10Cl2O2SH+ 312.9857).
CDCl3) δ 8.05 (dd, J = 1.9, 15.2 Hz, (E)-isomeric CH), 7.96 (d, J =
7.4 Hz, 2ArH), 7.72 (d, J = 7.4 Hz, ArH), 7.49−7.66 (m, 6ArH), 6.84
(d, J = 15.2 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ
140.1, 138.5, 133.8, 132.4, 132.0, 131.4, 130.5, 129.5, 129.2 (q, JC−F
=
30.5 Hz), 128.4, 127.9, 126.5 (12ArC, 2CH), 123.8 (q, JC−F = 272.3
Hz, CF3); HRMS (M + H)+ (ESI+) 313.0510 [M + H]+ (calcd for
C15H11F3O2SH+ 312.0432).
(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-chlorobenzene
(14i).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g,
1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61
mmol), and 4-chlorobenzaldehyde (0.24 g, 1.68 mmol) gave 0.47 g
(98%) of 14i as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp
163.0−165.0 °C (lit.39 167.0−167.5 °C); HPLC purity, 14.8 min,
(E)-1-(2-((Phenyl)sulfonyl)vinyl)-3-trifluoromethylbenzene
(16b). Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71
mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol),
and 3-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.48
g (89%) of 16b as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp
1
98.4%; H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.7 Hz, 2ArH),
1
93.0−95.0 °C; HPLC purity, 13.8 min, 98.8%; H NMR (400 MHz,
7.63 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.7 Hz, 2ArH),
7.37−7.43 (m, 4ArH), 6.81 (d, J = 15.4 Hz, (E)-isomeric CH); 13C
NMR (100 MHz, CDCl3) δ 141.6, 140.3, 139.1, 137.6, 130.8, 129.9,
129.8, 129.5, 129.3, 127.6 (12ArC, 2CH); HRMS (M + H)+ (ESI+)
312.9853 [M + H]+ (calcd for C14H10Cl2O2SH+ 312.9857).
CDCl3) δ 7.96 (d, J = 7.3 Hz, 2ArH), 7.52−7.73 (m, 7ArH, (E)-
isomeric CH), 6.95 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100
MHz, CDCl3) δ 140.6, 140.3, 133.8, 133.3, 131.8, 131.7 (q, JC−F = 32.8
Hz), 129.8, 129.7, 129.6, 127.9, 127.6, 125.2 (12ArC, 2CH), 123.7 (q,
JC−F = 271.0 Hz, CF3); HRMS (M + H)+ (ESI+) 313.0510 [M + H]+
(calcd for C15H11F3O2SH+ 312.0432).
(E)-1-(2-((3,4-Dimethoxyphenyl)sulfonyl)vinyl)-2-
trifluoromethylbenzene (15a). Diethyl (3,4-dimethoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi
solution in cyclohexane (0.75 mL, 1.49 mmol), and 2-
(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.37 g
(69%) of 15a as a white solid. Rf = 0.25 (n-hexane/EtOAc 3/1); mp
174.0−175.0 °C; HPLC purity, 13.1 min, >99.0%; 1H NMR (400
MHz, CDCl3) δ 7.99 (dd, J = 2.1, 15.2 Hz, (E)-isomeric CH), 7.71 (d,
J = 7.6 Hz, ArH), 7.49−7.60 (m, 4ArH), 7.36 (d, J = 2.1 Hz, ArH),
6.98 (d, J = 8.8 Hz, ArH), 6.81 (d, J = 15.2 Hz, (E)-isomeric CH), 3.95
(s, OCH3), 3.94 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 152.6,
148.6, 136.6, 131.6, 131.4, 130.6, 130.5, 129.4, 128.1 (q, JC−F = 30.5
Hz), 127.4, 125.4 (q, JC−F = 5.4 Hz), 122.9 (q, JC−F = 272.2 Hz, CF3),
121.2, 110.0, 119.0 (12ArC, 2CH), 55.4 (OCH3), 55.3 (OCH3);
HRMS (M + H)+ (ESI+) 373.0720 [M + H]+ (calcd for
C17H15F3O4SH+ 373.0721).
(E)-1-(2-((3,4-Dimethoxyphenyl)sulfonyl)vinyl)-3-
trifluoromethylbenzene (15b). Diethyl (3,4-dimethoxyphenyl-
sulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi
solution in cyclohexane (0.75 mL, 1.49 mmol), and 3-
(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.46 g
(87%) of 15b as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp
154.0−156.0 °C; HPLC purity, 13.3 min, 99.1%; 1H NMR (400 MHz,
CDCl3) δ 7.72 (s, ArH) 7.63−7.67 (m, 2ArH, (E)-isomeric CH), 7.57
(dd, J = 2.2, 8.5 Hz, ArH), 7.53 (t like due to dd, J = 7.7 Hz, ArH),
7.38 (d, J = 2.2 Hz, ArH), 6.99 (d, J = 8.5 Hz, ArH), 6.94 (d, J = 15.4
Hz, (E)-isomeric CH), 3.95 (s, 2OCH3); 13C NMR (100 MHz,
CDCl3) δ 152.6, 148.5, 138.4, 135.0, 132.4, 130.7, 130.6, 130.5 (q, JC−F
= 32.5 Hz), 129.1, 128.7, 126.4, 124.0, 122.6 (q, JC−F = 271.0 Hz,
CF3), 121.2, 110.1, 109.0 (12ArC, 2CH), 55.3 (2OCH3); HRMS (M
(E)-1-(2-((Phenyl)sulfonyl)vinyl)-4-trifluoromethylbenzene
(16c).40 Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71
mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol),
and 4-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.50
g (94%) of 16c as a white solid. Rf = 0.40 (n-hexane/EtOAc 5/1); mp
129.0−131.0 °C (lit.40 130.0−131.0 °C); HPLC purity, 13.9 min,
1
99.4%; H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.1 Hz, 2ArH),
7.56−7.72 (m, 7ArH, (E)-isomeric CH), 6.95 (d, J = 15.4 Hz, (E)-
isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.6, 140.3, 135.9,
133.8, 132.8 (q, JC−F = 32.6 Hz), 130.2, 129.6, 128.9, 127.9, 126.2
(12ArC, 2CH), 123.7 (q, JC−F = 270.6 Hz, CF3); HRMS (M + H)+
(ESI+) 313.0510 [M + H]+ (calcd for C15H11F3O2SH+ 312.0432).
HO-1 Induction Assay. BV-2 cells were cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 100 IU/L
penicillin, 10 μg/mL streptomycin, and 10% fetal bovine serum (FBS).
The cells (2.0 × 104 cells/mL) in a 96-well plate were treated with test
compounds (20 μM) for 24 h and washed. The cell pellet was
combined with 50 μL of lysis solution (150 mM NaCl, 50 mM Tris-
HCl, pH 8.0, and 1% Nonidet-P40), incubated on ice for 20 min, and
then centrifuged (3000g, 15 min). The supernatant was diluted 10
times with 50 mM Tris buffer (90 μL, pH 8.0).
U-shaped 96-well plates were coated with 100 μL of mouse
polyclonal HO-1 capture antibody (Enzo Life Sciences, Farmindale,
NY) that had been diluted (1:250) with coating buffer (10 mM
sodium phosphate, pH 7.4, and 15 mM NaCl). After incubation at
room temperature overnight, the solution was replaced by 200 μL
blocking buffer [10 mM sodium phosphate, pH 7.4, 15 mM NaCl, and
1% bovine serum albumin (BSA)], and the sample was incubated for 1
h. The blocking buffer was then removed, and the diluted supernatant
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(10 μL) combined with 90 μL of assay buffer (100 mM sodium
phosphate, pH 7.4, 150 mM NaCl, 1% BSA, and 0.1% Tween-20) was
added to each well. After incubation for 1 h and four washes in
washing buffer (10 mM sodium phosphate, pH 7.4, 15 mM NaCl, and
0.1% Tween-20), 100 μL of mouse polyclonal HO-1 detection
antibody (Enzo Life Sciences), diluted (1:250) with assay buffer, was
added. The resulting solution was incubated for 1 h and washed as
before. Then 100 μL of horseradish peroxidase labeled streptavidin,
diluted (1:600) with assay buffer, was added to each well, and the
samples were incubated for 1 h and washed. After addition of 100 μL
of Amplex-Red reagent [0.05 mM Amplex-Red (Invitrogen, Seoul,
Korea), 0.0068% H2O2 in phosphate buffered saline (PBS)] and
incubation for 30 min, fluorescence was measured using a fluorescence
spectrophotometer (SPECTRA MAX 340 pc; Molecular Devices,
Menlo Park, CA) at excitation at 530 nm and emission at 590 nm.
RT-PCR. CATH.a cells were grown in RPMI 1640 containing 8%
horse serum, 4% FBS, 100 IU/L penicillin, and 10 μg/mL
streptomycin at 37 °C in 95% air and 5% CO2 in humidified
atmosphere. The cells were treated with various concentrations of 12g
and incubated for 6 h. Total RNA was isolated, and reverse
transcription reactions (RT) were performed following the manu-
facturer’s directions (MBI Fermentas, Ontario, Canada). Polymerase
chain reaction (PCR) was performed using the following primers:
NQO1 (forward, 5′-CCATTCTGAAAGGCTGGTTTG-3′; reverse,
5′-CTAGCTTTGATCTGGTTGTC-3′); HO-1 (forward, 5′-
AGCAGGACATGGCCTTCT -3′; reverse, 5′-TCTGTCAG-
CATCACCTGCAG-3′); GCLC (forward, 5′-ATGACTGT-
TGCCAGGTGGATGAGA-3′; reverse, 5′-ACACGCCATCCTAAA-
CAGCGATCA-3′); GCLM (forward, 5′-AGCTGGACTC-
To obtain cell lysate, CATH.acells were treated with 12g and
incubated for 24 h. The cells were washed with ice-cold PBS and lysed
on ice in RIPA buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2
mM EDTA, and 0.5% NP-40) containing protease inhibitor cocktail.
After centrifugation (14000g, 10 min), the supernatant was obtained.
Western Blot Analyses. Protein concentrations were determined,
and equal amounts of protein were separated on a 10% SDS
polyacrylamide gel and transferred onto polyvinylidene difluoride−
nitrocellulose filters. After treatment in blocking solution [6% skim
milk in TTBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and
0.4% Tween-20)] for 1 h, the membrane was incubated overnight with
primary antibodies [NQO1 (Ab Frontier, Seoul, South Korea),
1:1000; HO-1 (Enzo Life Sciences, Farmingdale, NY), 1:1000; GCLC
(Novus Biologicals, Littleton, CO), 1:3000; GCLM (Santa Cruz
Biotechnology, Santa Cruz, CA), 1:200; β-actin (Sigma-Aldrich, St.
Louis, MO), 1:20000; and lamin B (Santa Cruz Biotechnology),
1:200] at 4 °C followed by horseradish peroxidase conjugated
secondary antibodies for 1 h at room temperature. Protein bands
were detected by chemiluminescence. Densitometric analysis was
performed using Image Gauge 4.0 program (Fujifilm, Tokyo, Japan),
and the data were normalized against β-actin, used as loading control.
Production of PD Animal Model and Treatment. All
procedures were preapproved by the Animal Experiment Review
Committee of the Asan Institute for Life Sciences and performed in
compliance with the U.S. National Institutes of Health Guide for the
Care and Use of Laboratory Animals (2011). All efforts were made to
minimize animal suffering. Male C57Bl/6 mice weighing 23−25 g
were maintained in a temperature- and humidity-controlled room with
a 12 h light−dark cycle and food and water available ad libitum. The
animals (n = 10 per group) were orally administered with 12g
suspended in N-methyl-2-pyrrolidone and 20% Tween 80 in saline at
10 mg/kg body weight per day every 24 h for 3 consecutive days.
MPTP (administered in four 20 mg/kg doses 2 h apart, dissolved in
saline) was injected intraperitoneally in a single day. The first MPTP
injection was made 24 h after the first 12g administration.
Immunohistochemistry. The animals were sacrificed 7 days after
the first MPTP injection. They were deeply anesthetized (with 15 mg/
kg tiletamine hydrochloride, 15 mg/kg zolazepam hydrochloride, and
1 mg/kg xylazine, injected intraperitoneally) and transcardially fixed in
4% paraformaldehyde as described previously.41 Brains were promptly
removed and postfixed in 4% paraformaldehyde. After cryoprotection,
the brain tissues were cut into 20 μm (nigral sections) or 40 μm
(striatal sections), and the sections were stored in 0.08% sodium azide
in PBS at 4 °C until analysis.
Immunostaining of the nigral and striatal brain tissue sections was
performed as described previously,42 using polyclonal antisera against
TH (1:5,000; Protos, New York, NY), Vectastain ABC kit, and
biotinylated secondary antibodies. The samples were visualized by
incubation in 0.05% 3,3′-diaminobenzidine and 0.003% H2O2. The
TH-immunopositive cells were manually counted on five sections, 80
μm apart, from all animals with the aid of a computer program
(Mousotron 3.8.3, Blacksun Software). Quantitative analysis of striatal
sections was performed using Image Gauge 4.0 (Fuji Photo Film Co.,
Tokyo, Japan) on five sections, 80 μm apart, from all animals.
Hindlimb Test. At 6 days after the last injection of MPTP, the
animals were subjected to the hindlimb test as previously
reported.41The animals were suspended by the tail and scored on a
scale of 0−4 based on the position of their hindlimbs. Each mouse
received a score of 4, from which the score of 1 was deducted for each
abnormal hindlimb movement of the joint or limb.
T G T G A T C A T G G C T T - 3 ′ ;
r e v e r s e ,
5 ′ -
CAAAGGCAGTCAAATCTGGTGGCA-3′); glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH, forward, 5′-CACCACCATGGAGA-
AGGCTGG-3′; reverse, 5′-TTGTCATGGATGACCTTGGCCAGG-
3′). GAPDH was used as an internal control. Analysis of each PCR
product on 1% agarose gel showed a single band with the expected
size. Densitometric analysis was performed using Image Gauge 4.0
program, and the data were normalized against GAPDH.
ATP Assay. CATH.a cells were seeded on a 96-well plate at a
density of 2.4 × 104 cells/well and treated with various concentrations
of 12g. To estimate cell viability, ATP level in live cells was measured
using CellTiter-Glo luminescent cell viability assay kit (Promega
Corp., Madison, WI). The cultured cells were mixed with the reagent
and lysed for 2 min, and the samples were incubated for 10 min at
room temperature. Then 100 μL of mixture was transferred onto a
black plate, and luminescence was measured for 1 min using a
microplate luminometer (MicroLumat Plus LB96 V, Berthold
Technologies GmbH & Co. KG, Bad Wildbad, Germany).
Lactate Dehydrogenase (LDH) Activity Assay. CATH.a cells
were seeded on a 96-well plate at a density of 2.4 × 104 cells/well and
treated with various concentrations of 12g. Aliquots (50 μL) of cell
culture medium were incubated at room temperature in the presence
of 0.26 mM NADH, 2.87 mM sodium pyruvate, and 100 mM
potassium phosphate buffer (pH 7.4) in a total volume of 200 μL. The
rate of NAD+ formation was monitored for 5 min at 2 s intervals at
340 nm using a microplate spectrophotometer (SPECTRA MAX 340
pc; Molecular Devices).
Prerapartion of Proteins for Western Blot. For nuclear
fractionation, CATH.a cells were treated with 12g for 3 h, harvested
in PBS, and resuspended in 100 μL of cold buffer containing 10 mM
HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, and 0.5 mM PMSF by gentle pipetting. The cells were
incubated on ice for 15 min, after which 7 μL of 10% NP-40 was added
and mixed for 10 s. The homogenate was then centrifuged (17800g, 2
min), and the supernatant was removed. This step was repeated once
again. The nuclear pellet was resuspended in 50 μL of ice-cold buffer
containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF. The
sample was mixed thoroughly and placed on a rotary shaker for 10
min. After centrifugation (17800g, 10 min), the soluble fraction
containing nuclear proteins was obtained.
Vertical Grid Test. Vertical grid test was performed as described
previously.36 Prior to the drug administration, mice were allowed to
acclimate to the vertical grid apparatus once a day for 3 consecutive
days: they were placed at the bottom of the apparatus facing upward so
that they would climb up the apparatus and then descend. On day 6,
the same vertical grid trials were made and videotaped. The videos
were replayed to analyze the time taken to climb down and also to
make a complete turn.
Data Analyses. Data are expressed as the mean
SEM of
independent experiments. Comparisons of three or more groups were
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analyzed by one-way ANOVA (analysis of variance) and post
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Synthetic procedures for the preparation of 1, 4, and 6−8; H
and 13C NMR spectra of compounds 1, 4, and 6−16 reported
in this study. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*O.H.: phone, 82-2-3010-4279; fax, 82-2-3010-4248; e-mail,
oyhwang@amc.seoul.kr.
*K.D.P.: phone, 82-2-958-5132; fax, 82-2-958-5189; e-mail,
kdpark@kist.re.kr.
Author Contributions
∥S.Y.W. and J.H.K. contributed equally to the work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by funds from the Korea Institute of
Science and Technology (KIST) (Grants 2E23870 and
2E22520, K.D.P) and by grants from the Korea Health
Technology R&D Project, the Ministry of Health and Welfare
(Grant HI12C1022, K.D.P), and the National Agenda Project
from Korea Research Council of Fundamental Science and
Technology (O.H.), and in part by the National Research
Foundation of Korea (NRF-2009-0081674, D.J.K, NRF-2009-
0081675, O.H.).
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ABBREVIATIONS USED
■
BH4, tetrahydrobiopterin; BSA, bovine serum albumin; DA,
dopamine; DAergic, dopaminergic; DMEM, Dulbecco’s
modified Eagle medium; ELISA, enzyme-linked immunosorb-
ent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-
phosphate dehydrogenase; GCL, glutamate-cysteine ligase;
GCLC, glutamyl-cysteine ligase catalytic subunit; GCLM,
glutamyl-cysteine ligase modifier subunit; HO-1, heme oxygen-
ase 1; LDH, lactate dehydrogenase; mCPBA, m-chloroperox-
ybenzoic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr-
idine; NQO1, NAD(P)H quinone oxidoreductase; Nrf2, Nf-
E2-related factor 2; PBS, phosphate buffered saline; PD,
Parkinson’s disease; ROS, reactive oxygen species; RT-PCR,
reverse transcription polymerase chain reaction; SFN, sulfor-
aphane; SN, substantia nigra; TH, tyrosine hydroxylase
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