Generation of potent Nrf2 activators via tuning the electrophilicity and steric hindrance of vinyl sulfones for neuroprotection

Article abstract of DOI:10.1016/j.bioorg.2020.104520

Oxidative stress is constantly involved in the etiopathogenesis of an ever-widening range of neurodegenerative diseases. As a consequence, effective repression of cellular oxidative stress to a redox homeostatic condition is a promising and feasible strategy to treat, or at least retard the progression of, such disorders. Nrf2, a primary orchestrator of cellular antioxidant response machine, is responsible for detoxifying and compensating for deleterious oxidative stress via transcriptional activation of a diverse array of antioxidant biomolecules. In the framework of our persistent interest in disclosing small molecules that interfere with cellular redox-regulating machinery, we report herein the synthesis, optimization, and biological assessment of 47 vinyl sulfone scaffold-bearing small molecules, most of which exhibit robust neuroprotective effect against H₂O₂-mediated lesions to PC12 cells. After initial screening, the most potent neuroprotective compounds 9b and 9c with marginal cytotoxicity were selected for the follow-up studies. Our results demonstrate that their neuroprotective effects are attributed to the up-regulation of a panel of antioxidant genes and corresponding gene products. Further mechanistic studies indicate that Nrf2 is indispensable for the cellular performances of 9b and 9c, arising from the fact that silence of Nrf2 gene drastically nullifies their protective action. Taken together, 9b and 9c discovered in this work merit further development as neuroprotective candidates for the treatment of oxidative stress-mediated pathological conditions.

Full text of DOI:10.1016/j.bioorg.2020.104520

Bioorganic Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Generation of potent Nrf2 activators via tuning the electrophilicity and
steric hindrance of vinyl sulfones for neuroprotection
Zi-Long Song, Yanan Hou, Feifei Bai, Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry & College of Chemistry and Chemical Engineering, Lanzhou University, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oxidative stress is constantly involved in the etiopathogenesis of an ever-widening range of neurodegenerative
diseases. As a consequence, effective repression of cellular oxidative stress to a redox homeostatic condition is a
promising and feasible strategy to treat, or at least retard the progression of, such disorders. Nrf2, a primary
orchestrator of cellular antioxidant response machine, is responsible for detoxifying and compensating for
deleterious oxidative stress via transcriptional activation of a diverse array of antioxidant biomolecules. In the
framework of our persistent interest in disclosing small molecules that interfere with cellular redox-regulating
machinery, we report herein the synthesis, optimization, and biological assessment of 47 vinyl sulfone
scaffold-bearing small molecules, most of which exhibit robust neuroprotective effect against H2O2-mediated
lesions to PC12 cells. After initial screening, the most potent neuroprotective compounds 9b and 9c with mar-
ginal cytotoxicity were selected for the follow-up studies. Our results demonstrate that their neuroprotective
effects are attributed to the up-regulation of a panel of antioxidant genes and corresponding gene products.
Further mechanistic studies indicate that Nrf2 is indispensable for the cellular performances of 9b and 9c, arising
from the fact that silence of Nrf2 gene drastically nullifies their protective action. Taken together, 9b and 9c
discovered in this work merit further development as neuroprotective candidates for the treatment of oxidative
stress-mediated pathological conditions.
Oxidative stress
Vinyl sulfone
Antioxidant
Nrf2
Neuroprotection
1. Introduction
high levels of transition metals, modest antioxidant response mecha-
nism, and considerable amounts of polyunsaturated fatty acids [5,9,10],
During various metabolic processes, aerobic organisms inevitably
produce reactive oxygen species (ROS), such as hydrogen peroxide,
superoxide anion and so on [1]. These vigorous species play both
beneficial and deleterious roles in cells [1–3]. Under physiological
conditions, ROS levels are under the control of antioxidant defense
system, and ROS are of paramount importance in various repertoires of
cellular signal transduction pathways [3,4]. However, once this equi-
librium is beyond of control, due to the insufficient antioxidant response
mechanism and/or excessive ROS generation, oxidative stress ensues
[1,4]. Oxidative stress elicits a wealth of detrimental effects on cellular
vital components, including proteins, lipids, and nucleic acids [1], and
contributes to numerous pathological conditions, such as neurodegen-
erative disorders [5], cancers [6], digestive system diseases [7], and
cardiovascular diseases [8].
all of which predispose brain neurons particularly vulnerable to oxida-
tive stress. Further, dopaminergic neurons might sustain an excessive
overload of oxidative stress, emanating from metabolism of dopamine
(DA) [11]. Enzymatic metabolism of DA by monoamine oxidase (MAO)
produces H2O2 as a byproduct. Alternatively, spontaneous oxidation of
DA leads to the generation of neuromelanin and dopamine quinones
(DAQs) [12], both of which result in the formation of ROS via redox-
cycling in the presence of reducing agents. In addition, DAQs could
either be engaged in electrophilic addition with cysteine-containing
proteins, nucleic acids, and thiol antioxidant glutathione (GSH) or un-
dergo cyclization process to form highly unstable and reactive dop-
aminochrome that is also prone to redox-cycling through consecutive
consumption of NADPH and concomitant release of superoxide anion
[13]. These convergences of evidences suggest that the brains are
extremely sensitive to oxidative stress that imparts cumulative damages
on brain neurons. Besides, owing to the limited regenerative capacity of
neurons resulting from their post-mitotic characteristic [5], progressive
Although the brains account for only 2 ~ 5% of whole body weight,
they voraciously consume about 25 ~ 30% of oxygen budget needed for
supporting the whole body [9]. Additionally, brains are equipped with
* Corresponding author.
E-mail address: fangjg@lzu.edu.cn (J. Fang).
https://doi.org/10.1016/j.bioorg.2020.104520
Received 21 March 2020; Received in revised form 30 October 2020; Accepted 26 November 2020
Available online 30 November 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Zi-Long Song, Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2020.104520

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
loss of brain neurons takes place, which leads to the development of
various neurodegenerative diseases, such as Parkinson’s disease (PD),
Alzheimer disease (AD), and Huntington’s disease (HD). As a conse-
quence, efficient elimination of excessive ROS and restoring the ho-
meostatic redox balance are intriguing and viable tactics to relieve the
symptoms or even retard the progression of these pathological condi-
tions [14–17]. Either exogenous or endogenous antioxidant molecules
are able to neutralize noxious ROS; however, exogenous antioxidant
molecules are usually characterized by short half-lives and narrow
therapeutical windows, precluding their practical application in clinical
studies [10]. Alternatively, activation of endogenous antioxidant
response machinery is more preferred as such a process is able to exert
sustainable beneficial effects through transcriptional up-regulation of a
plethora of antioxidant species, such as thioredoxin reductase (TrxR),
fumarate (DMF, Tecfidera, Chart 1) has been already approved by the US
Food and Drug Administration for the treatment of early multiple scle-
rosis (MS) via mitigating the oxidative stress in an Nrf2-dependent
manner [36]. The successful clinical application of DMF also encour-
ages our group to unveil more Nrf2-targeted molecules [37–47], and
several naturally occurring molecules and synthetic agents have been
found to exhibit potent Nrf2 activation capacity and thus show
impressive neuroprotective effect [48–51] (Chart 1). Vinyl sulfone
represents a chemical skeleton that is similar to α, β-unsaturated ketone
(also renowned as Michael acceptor) shared with many Nrf2 activators
[18,23,31] (Chart 1). Although several synthetic molecules bearing
vinyl sulfone motif had been documented by sporadic studies to show
neuroprotective activity [52–54], the molecular structural determinants
responsible for this activity and the underlying mechanism are not
entirely clarified. Herein, we report the synthesis, evaluation, and
mechanistic studies of 47 vinyl sulfone derivatives as Nrf2 activators in
protecting PC12 cells from H2O2-induced damages. Two compounds 9b
and 9c, discovered through elaborate structural optimization, were
identified to possess not only low cytotoxicity but also the highly neu-
roprotective activity, and therefore selected as hit compounds for
further evaluation. The follow-up studies indicate that 9b and 9c could
attenuate the H2O2-mediated insults via up-regulation of a wide range of
antioxidant/detoxifying enzymes and molecules, including TrxR, GSH,
NQO1, and HO-1. Further mechanistic studies reveal that activation of
Nrf2 is imperative for the cellular physiological action of 9b and 9c in
PC12 cells, since knock-down of Nrf2 expression almost abrogated the
neuroprotection.
GSH, NAD(P)H quinone oxidoreductase
1 (NQO1), and heme
oxygenase-1 (HO-1), all of which are referred as the “ultimate antioxi-
dants” [18–20].
Nuclear factor erythroid 2-related factor 2 (Nrf2), a member of basic
region leucine zipper transcription factors, belongs to Cap ‘n’ Collar
(CNC) family and is a primary executor of the cellular antioxidative
signaling network. An extensive range of intrinsic antioxidative species
(e.g. TrxR, GSH, NQO1, and HO-1) are all governed by Nrf2 at tran-
scriptional level [21]. Under physiological conditions, Nrf2 is main-
tained at a low basal level in quiescent cells, which is ascribed to its
natural repressor Kelch-like ECH-associated protein 1 (Keap1) that re-
tains Nrf2 in cytoplasm and sequentially shepherds it for degradation
through the ubiquitination-proteasome pathway [22,23]. Once in the
context of stressed conditions, a group of certain cysteine residues in
Keap1 are modified, and the Keap1-mediated ubiquitination of Nrf2 is
terminated. Sequentially, Nrf2 translocates and accumulates in nucleus,
where it heterodimerizes with members of small musculoaponeurotic
fibrosarcoma (sMAF) family of transcription factors, and binds to the
regulatory enhancer sequences termed “antioxidant response element”
(ARE) in the promoter region of many antioxidant genes, leading to the
transcriptional activation of ARE-directed expression of abundant
detoxifying/cytoprotective molecules [23–27]. In this regard, activation
of endogenous antioxidant mechanism of Nrf2-ARE emerges to provide
an inroad for therapeutical intervention, or at least delay of the pro-
gression, of neurodegenerative diseases [10,16–18,22,23,28–30].
During the last couple of decades, a broad range of researchers had
paid intensive attention to the identification and development of Nrf2-
ARE activators both from natural sources and synthetic community
[18,22–26,31–35]. Delightedly, the well-known Nrf2 activator dimethyl
2. Results and discussion
2.1. Chemistry
A concise chemical conversion to the synthesis of intermediates and
desired compounds was delineated in Scheme 1. First, intermediates 3
were obtained in high yields through a nucleophilic substitution reac-
tion between commercially available materials 1 and 2. Second,
oxidation of intermediates 3 with 3-chloroperoxybenzoic acid (m-CPBA)
gave the intermediates 4. Finally, the Horner–Wadsworth–Emmons
Reaction was employed to furnish the 47 target compounds in moderate
to excellent yields via exploiting intermediates 4 and various easily
accessible aromatic aldehydes as staring materials. All newly synthe-
sized compounds were characterized through 1H NMR (400 MHz), 13
C
NMR (100 MHz), and ESI-MS analysis. Purity determination of all target
Chart 1. Previously reported Nrf2 activators and our strategy for the discovery of potent Nrf2 activators based on the electrophilic double-bond bearing vinyl
sulfone scaffold.
2

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of intermediates and desired compounds.
compounds was analyzed by HPLC, and the purity (>95%) conform the
Journal requirement and were suitable for further medicinal chemistry
studies.
cytotoxicity (Fig. 2A and Table S3 in Supporting Information). Then,
these 8 compounds plus 6b were all evaluated for neuroprotection in the
same model as that employed before. As shown in Fig. 2B (also shown as
values in Table S4 in Supporting Information), 6b with R1 = H is more
neuroprotective than molecules (compounds 10a ~ c) with R1 = ortho-
OCH3 in neuroprotection. Further, compounds with R1 = ortho-Cl shows
more potent neuroprotective activity than their counterpart compounds
with R1 = H, respectively (9b > 6b, 9c > 6o, and 9a > 6n, Fig. 3 and
Table S4 in Supporting Information). What’s more, it is worth
mentioning that R2 = ortho-F might compromise the neuroprotective
activity of the compounds, and R2 = ortho-Cl is more favored than R2 =
ortho-Br (9b > 9c, 6b > 6o, and 10b > 10c, Fig. 3 and Table S4 in
Supporting Information). Obviously, 9b and 9c conferred the most
potent neuroprotection among all the tested compounds against the
H2O2-mediated assaults on PC12 cells.
2.2. Biological studies
2.2.1. Initial screening, structure–activity relationship (SAR), and
optimization studies
At the beginning of our research, 39 compounds were synthesized,
namely 6a ~ m, 7a ~ m, and 8a ~ m, and their cytotoxicity toward
PC12 cells were assessed by the MTT assay. As exhibited in Fig. 1A (also
shown as values in Table S1 in Supporting Information), all the 39
compounds showed weak cytotoxicity, even at high concentration of 50
μ
M. As a consequence, these 39 compounds were all selected for the
neuroprotective evaluation against H2O2-induced lesions to PC12 cells,
well-established cellular neurodegenerative diseases model.
a
2.2.2. Relief of PC12 cells from H2O2-induced lesions by 9b and 9c
PC12 cells have been demonstrated to possess neuronal character-
istics to a great extent and are generally employed as a model for PD
studies [39–41,53,55]. Consequently, an injury model based on PC12
cells was engendered to investigate whether 9b and 9c could protect the
cells from H2O2-induced insults. As illustrated in Fig. 4A (also shown as
values in Table S5 in Supporting Information), H2O2 caused evident
death of PC12 cells, and the cell viability treated with H2O2 only was
about half of the control group. However, when PC12 cells were pre-
treated with 9b or 9c, the H2O2-induced cell death was markedly
[24,39–41,53] The protection screening results (Fig. 1B and Table S2 in
Supporting Information) showed that 5 compounds (6a ~ b, 7b, and
8a ~ b) possessed apparent neuroprotective activity, with 6b being the
most potent one. As seen from these 5 most active compounds, the R2
group should be an assembly with an electron-withdrawing atom/
function group in close proximity to the vinyl group of the molecules,
and the Cl atom is more preferred than the CF3 group. On the other hand,
activity determinants of the R1 are complex, since the most active three
compounds (6b, 7b, and 8b) have substituents as H (electron-neutral),
CH3 (slightly electron-donating), and F (electron-withdrawing). Ac-
cording to the results attained above and enlightened by studies per-
formed previously by Park et al. [54], additional 8 compounds (6n ~ o,
9a ~ c, and 10a ~ c) were synthesized to further explore the SAR of R1
and R2 groups. The newly synthesized 8 compounds showed negligible
ameliorated at as low concentration as 0.5
μ
M. Both 9b and 9c could
elevate the cell viability up to ~ 75% at the concentration of 1
μ
M. When
cells were damaged, the lactate dehydrogenase (LDH) was released from
cytoplasm to culture medium on account of the impaired cell membrane
3

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
1. Cytotoxicity and neuro-
Fig.
protection screening of vinyl sulfone
derivatives against PC12 cells. (A) PC12
cells were grown to adhere for 24 h
followed by incubation with target
compounds (10 μM, 20 μM, and 50 μM)
for further 24 h. MTT assay was per-
formed to determine the cell viability.
(B) PC12 cells were grown to adhere for
24 h, and then were treated with com-
pounds (1 μM) for another 24 h followed
by exposing to the fresh medium con-
taining 500 μM H2O2 for further 12 h.
Then MTT assay was performed to
determine the cell viability. Data are
presented as means ± SD from triplicate
experiments. **P
<
0.01 versus the
control group; ^P < 0.05 and ^^P < 0.01
versus H2O2-treated group.
Fig. 2. (A) Cytotoxicity screening of the succedently synthesized vinyl sulfone derivatives against PC12 cells. The experimental details are the same as those
described in Fig. 1A. (B) Neuroprotection screening of 6b and the succedently synthesized vinyl sulfone derivatives. The experimental details are the same as those
described in Fig. 1B. Data are presented as means ± SD from triplicate experiments. **P < 0.01 versus the control group; ^P < 0.05 and ^^P < 0.01 versus the H2O2-
treated group.
integrity. Thus a LDH leakage assay was conducted for the purpose of
validating the results attained above. As shown in Fig. 4B, the content of
LDH released in culture medium of solely H2O2-treated PC12 cells was
two folds more than that of the control group, while pretreatment of
PC12 cells with 9b or 9c remarkably alleviated the LDH leakage pro-
voked by H2O2-mediated damage. Consistently, the LDH leakage assay
results were in line with the MTT assay. Taken together, these results
pronounced the neuroprotective activity of 9b and 9c against H2O2-
mediated oxidative damages.
2.2.3. Clearance of ROS in PC12 cells by 9b and 9c treatment
Oxidative stress-mediated damages to vital cellular components are
attributed to the redox imbalance resulting from inadequate antioxidant
capacity against the excessive generation of ROS. On this account, we
therefore asked if 9b and 9c could diminish the ROS production to as-
suage the oxidative stress-mediated damages inflicted by H2O2.
Dichlorodihydrofluorescein diacetate (DCFH-DA), a non-fluorescent and
cell membrane-permeable probe, was employed to detect ROS. DCFH-
DA could easily diffuse into cells and then be hydrolyzed by esterases
4

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 3. Structures and neuroprotective activity of the most potent vinyl sulfone derivatives.
Fig. 4. Neuroprotection of 9b and 9c
against the H2O2-mediated injury to
PC12 cells. (A) Dose-dependent protec-
tion of 9b and 9c. The cells were seeded
to adhere for 24 h followed by incuba-
tion with 9b (0.5
μ
M and 1
M), and t-BHQ (0.5
M, and 40
μ
M), 9c (0.5
M, 1 M,
μM) for further
μ
M and 1
μ
μ
μ
10
μ
M, 20
μ
24 h, and subsequently the cells were
exposed to the fresh medium containing
400 μM H2O2 for 12 h. Then MTT assay
was performed to determine the cell
viability. (B) Validation of the neuro-
protection of 9b and 9c by measurement
of LDH activity in culture medium. The
cells were seeded to adhere for 24 h
followed by incubation of 9b (0.5
μM
and 1 μM) and 9c (0.5 μM and 1 μM) for
another 24 h, and then were further
exposed to fresh medium containing
500 μM H2O2 for 12 h. The LDH leakage
assay was performed to determine the
activity of LDH released in the culture
medium. Data are presented as means ±
SD from triplicate experiments. **P <
0.01 versus the control group; ^P < 0.05
and ^^P < 0.01 versus the H2O2-treated
group.
Fig. 5. Mitigation of ROS production by 9b and 9c in PC12 cells. The cells were bred to adhere for 24 h, and then were pretreated with 9b (0.5
(0.5 M and 1 M) for further 24 h before 500 M H2O2 was added. After the cells were cultured for 5 h, the ROS probe DCFH-DA (10 M) was loaded. The cells were
further cultured for 0.5 h, and the images were taken.
μM and 1 μM) and 9c
μ
μ
μ
μ
5

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
to give non-fluorescent dichlorodihydrofluorescin, which reacts with
ROS to afford fluorescent dichlorofluorescein. As depicted in Fig. 5,
strong fluorescence was observed in PC12 cells treated with H2O2 only,
whereas the control group showed marginal fluorescence signal.
Prominently, pretreatment of the cells with 9b or 9c could significantly
alleviate the fluorescence intensity, which indicates that clearance of
ROS contributes to the observed neuroprotective effect of 9b and 9c
(Fig. 4).
(Fig. 7C), and HO-1 (Fig. 7D) were all elevated to different extents in a
dose-dependent manner. In addition, TrxR activity was also confirmed
by the TRFS-Green (Fig. 7E), which is a fluorescence probe developed by
our group [56] to selectively in situ detect TrxR activity in live cells. The
fluorescence increased accompanied by the elevation of the compounds
concentration. Taken together, 9b and 9c were capable of efficient
activation of cellular antioxidant response system at both transcriptional
and translational levels.
2.2.4. Activation of antioxidant response machinery by 9b and 9c in PC12
cells
2.2.5. Involvement of Nrf2 for the biological performance of 9b and 9c
Owing to the activation of ARE-directed antioxidant/detoxifying
species by 9b and 9c, we thereby asked if this phenomenon was Nrf2-
dependent. After preparation of cytoplasmic and nuclear fractions of
PC12 cells treated with 9b or 9c, Western blots analysis was conducted
in order to interrogate whether 9b or 9c could instigate Nrf2 to migrate
from cytoplasm to nucleus, which is indispensable for the antioxidant
activity imparted by Nrf2. Although total Nrf2 content was not signifi-
cantly affected by compounds treatment, cytoplasmic and nuclear Nrf2
level varied gradually (Fig. 8). Consistently, cytoplasmic Nrf2 level
declined with concurrent accumulation of nuclear Nrf2, and the coin-
cidental result indicated that Nrf2 translocated from cytoplasm to nu-
cleus. With the intention to confirm the Nrf2-directed initiation of
transcription of ARE-dependent cytoprotective genes by 9b and 9c,
pARE-luciferase plasmid was applied to the transfection of PC12 cells to
generate a stable cell line. The classical Nrf2 activator tert-butylhy-
droquinone (t-BHQ) was used as a positive control. As shown in Fig. 9C,
both 9b and 9c activated the ARE-luciferase reporter in a dose-
dependent manner, and 9b induced a conspicuously stronger reporter
In consideration of the results that 9b and 9c could robustly protect
PC12 cells from H2O2-induced injuries (Fig. 4), we then speculated that
9b and 9c were capable of activation of the cellular antioxidative
response machinery via transcriptional activation of cytoprotective
genes and up-regulation of corresponding gene products. Afterwards,
qRT-PCR was employed to determine several antioxidant/detoxifying
genes including TrxR, GCLC [γ – glutamyl – cysteinyl – ligase (GCL)
catalytic subunit], GCLM (GCL modulatory subunit), NQO1, and HO-1
after 9b or 9c treatment. As displayed in Fig. 6, all these genes were
elevated to varying degrees, and elevation of HO-1 was the most distinct
among all the tested genes. 9b boosted both NQO1 and HO-1 at the
highest level after 6 h treatment, with 11-fold and 15-fold increase,
respectively, and the other three genes remained to be increased and
sustainable even at 12 h. Similarly, 9c promoted HO-1 at the peak level
after 6 h treatment, while the other four genes reached a relatively high
level at 12 h. Collectively, these results manifested that 9b and 9c effi-
ciently promoted the transcription of the indicated antioxidant/detoxi-
fying genes. Next, we were curious about whether these activated
antioxidant/detoxifying genes would be transformed to functional en-
zymes and molecules, so determination of the content or activity of the
corresponding gene products was conducted. GSH, a ubiquitous cellular
antioxidant molecule and cardinal thiol-containing tripeptide, is bio-
synthesized with the aid of GCL that is a heterodimer composed of
GCLM and GCLC. As shown in Fig. 7B, the content of GSH was promoted
to an impressively high level, and the activities of TrxR (Fig. 7A), NQO1
response at such a low concentration as 0.5
μM than t-BHQ did at 40 μM.
What’s more, even 9c at 1 M did the similar thing as t-BHQ did at 40
μ
μ
M. Collectively, 9b and 9c pronounced as exceedingly sensitive in-
ducers that activated Nrf2-ARE pathway.
To further consolidate the results above, two cell lines were estab-
lished exploiting short hairpin RNAs (shRNA) to accentuate the neces-
sity of Nrf2 in 9b- and 9c-mediated neuroprotection in response to
oxidative stress. In specific, shNT (non - targeting plasmid) and shNrf2
(Nrf2 - targeting plasmid) were transfected to PC12 cells to achieve two
stable cell lines, and the knockdown efficiency was determined as is
shown in Fig. 9A, B. Additionally, the knockdown efficiency was also
consistently confirmed by the fact that shNrf2 cells were more sensitive
to H2O2-only treatment than shNT cells (Fig. 9D). Therefore the neu-
roprotection of 9b and 9c in the newly generated two cell lines were
evaluated (Fig. 9D and Table S6 in Supporting Information), and the
neuroprotective effects imparted by 9b and 9c in PC12 shNT cells were
similar as those in the wild type (Fig. 4). However, this effect was
drastically abolished in PC12 shNrf2 cells, implying that Nrf2 was
definitely implicated in the cytoprotection of 9b and 9c against oxida-
tive stress in PC12 cells.
ROS are recognized as both physiologically functional and patho-
logically detrimental to cells that are endowed with exquisite antioxi-
dant systems to harness ROS for beneficial effects under normal
conditions. However, once ROS generation overwhelms the cellular
antioxidant response mechanism, oxidative stress ensues. At the same
time, the “dark side” of ROS emerges as damaging various cellular
components, which results in acceleration of cell senescence and death.
Neurodegenerative diseases affect increasing number of people world-
wide, while the etiology of the diseases remains controversial. However,
mounting evidences discovered in past years indicate that oxidative
stress is routinely involved in the etiology of the diseases. Oxidative
stress acts as a causal risk factor, or at least an essential auxiliary
component, in contributing to the progressive loss of neurons in pa-
tients, which is characteristic of neurodegenerative disorders. In this
sense, it is tempting to speculate that restoration of redox homeostasis
via chemotherapeutical intervention might be a promising and feasible
strategy to slow or even reverse the progression of the diseases. As a
central orchestrator in cellular antioxidant response machinery, Nrf2
Fig. 6. Transcriptional activation of the antioxidant genes by 9b and 9c in
PC12 cells. The cells were incubated with 9b (1 μM) and 9c (1 μM) for given
times, and then the cells were lysed to acquire total RNA. qRT-PCR was
employed to analyze the indicated genes expression with GAPDH as a reference.
Data are presented as means ± SD from triplicate experiments. *P < 0.05 and
**P < 0.01 versus the control group.
6

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 7. Elevation of the antioxidant species by 9b and 9c in PC12 cells. The cells were seeded to adhere for 24 h followed by incubation of 9b (0.5
μM and 1 μM) and
9c (0.5 M and 1 M) for further 24 h, and then were lysed to prepare cell samples. Afterwards, the content of GSH (B), and activities of TrxR (A), NQO1 (C), and HO-
μ
μ
1 (D) were determined. (E) After incubation of PC12 cells with 9b (0.5 μM and 1 μM) and 9c (0.5 μM and 1 μM) for 24 h, TRFS-Green (10 μM) was added for
continuous 4 h culture. Then the images were taken. Data are presented as means ± SD from triplicate experiments. **P < 0.01 versus the control group. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
emerges as a pharmacological target for neurodegenerative diseases
therapeutics.
against H2O2-induced damages. Initial screening, SAR, and optimization
studies exhibited herein would be instrumental to further facilitate the
optimization and substantiation of this series of compounds. The
following studies showed that 9b and 9c protected PC12 cells from
H2O2-mediated insults (Fig. 4) via up-regulation of a diverse range of
antioxidant species (TrxR, NQO1, GSH, and HO-1) (Fig. 6 and Fig. 7).
Mechanistic studies demonstrated that 9b and 9c promoted the trans-
location of Nrf2 (Fig. 8), initiation of Nrf2-dependent activation of ARE
(Fig. 9C), and finally transcriptional activation of ample antioxidant
genes (Fig. 7). Further, silence of Nrf2 in PC12 cells strikingly blunted
the neuroprotection (Fig. 9A, B, and D), underpinning the indispensable
role of Nrf2 in cytoprotection of 9b and 9c.
Chalcones are a family of typical Nrf2 activators according to the
classification by Hu et al. [18], and the activation property is usually
ascribed to their electrophilic double bonds, which could covalently
modify the key cysteines in Keap1, eventually leading to the activation
of Nrf2. Scaffold of vinyl sulfone, structurally similar to chalcones, is
accordingly presumed to have the same properties as chalcones.
As part of our advancing work concerning the identification and
development of small redox-regulating molecules targeting Nrf2 acti-
vation, we described here the synthesis of vinyl sulfone derivatives and
discovered two hit compounds, 9b and 9c, with potent neuroprotection
7

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
Fig. 8. Variation of Nrf2 location stimulated by 9b
and 9c in PC12 cells. Incubation of the cells with 9b
(1 μM) and 9c (1 μM) for different given times, and
then the cells were lysed in preparation for different
part of protein samples. (A, C) Western blots analysis
(WB) of the total, nuclear, and cytosolic Nrf2 after
stimulation by 9b and 9c. (B, D) Quantification of WB
was also expressed. Data are presented as means ± SD
from triplicate experiments. *P < 0.05 and **P < 0.01
versus the control group.
Fig. 9. Essential involvement of Nrf2 in the
protection of PC12 cells by 9b and 9c. (A)
Western blots analysis of Nrf2 expression in
PC12-shNT cells and PC12-shNrf2 cells after
transfection of PC12 cells with corresponding
plasmids. (B) Corresponding quantification result
of Western blots analysis. (C) The luciferase re-
porter gene assay of Nrf2-ARE activation by 9b
and 9c with t-BHQ as a positive control. Trans-
fected PC12 cells were incubated with t-BHQ (20
μ
M and 40
μ
M), 9b (0.5
μM and 1 μM), and 9c
(0.5 M and 1
μ
μ
M) for 24 h, and then the cells
were lysed to prepare samples used for luciferase
activity assessment. (D) Neuroprotection of 9b
and 9c against H2O2-induced injuries in PC12-
shNT cells and PC12-shNrf2 cells. Data are pre-
sented as means ± SD from triplicate experi-
ments. *P < 0.05 and **P < 0.01 vs vehicle
group; ^^P < 0.01 vs H2O2-treated group.
3. Conclusion
species. The structural determinants responsible for neuroprotective
activity, mechanism of action, and cellular target of 9b and 9c were all
fully elucidated, which would provide invaluable information for
further development of them as potential chemotherapeutical candi-
dates. Further studies to optimize and improve the drug-like properties
of them are underway in our lab, and the corresponding in vivo experi-
mental results will be reported in due course.
As a whole, 47 vinyl sulfone derivatives were synthesized and eval-
uated, and two compounds, 9b and 9c, were identified to possess the
most potent neuroprotective effects. Mechanistic studies showed that 9b
and 9c activated, promoted the translocation of Nrf2, and thereafter
initiated the transcription and expression of a series of antioxidant
8

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
4. Experimental section
7.364 (d, 2H, J = 8 Hz), 4.200–4.127 (m, 4H), 3.756 (d, 2H, J = 16.8
Hz), 2.448 (s, 3H), 1.299 (t, 6H, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz)
δ: 145.163, 137.086, 129.703, 128.338, 63.366, 63.302, 54.510,
53.145, 21.654, 16.238, 16.175; MS-ESI m/z: 307.05 [M + H]+.
4.1. Chemistry
4.1.1. General information
All chemical reagents were purchased from Aladdin (Shanghai,
China) without purification, and the reaction solvents were from
Kemiou (Tianjin, China). Purification or drying of the reaction solvents
is in light of the standard methods. Silica-gel plates (GF254, TLC) and
Silica gel (200 ~ 300 mesh) were bought from Qingdao Haiyang
(Qingdao, China). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectras
of compounds in appropriate solvent were analyzed on a Bruker AMX
spectrometer with tetramethylsilane (TMS) as an internal reference.
Mass spectra (MS) were recorded on the Shimadzu LCMS-2020 system
(ESI-MS).
4.1.3.3. Diethyl (((4-fluorophenyl)sulfonyl)methyl)phosphonate (4c). 3c
(8.348 g) and m-CPBA (20.708 g) afforded 6.92 g of 4c as a white solid;
Yield: 74.3%; mp: 74–76 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.061–8.012
m, 2H), 7.283–7.226 (m, 2H), 4.209–4.136 (m, 4H), 3.790 (d, 2H, J =
16.8 Hz), 1.310 (t, 6H, J = 6.8 Hz); 13C NMR (CDCl3, 100 MHz) δ:
167.366, 164.812, 135.926, 135.893, 131.587, 131.490, 130.066,
128.242, 116.540, 116.314, 63.538, 63.474, 54.658, 53.287, 16.309,
16.246; MS-ESI m/z: 311.05 [M + H]+.
4.1.3.4. Diethyl (((2-chlorophenyl)sulfonyl)methyl)phosphonate (4d). 3d
(8.842 g) and m-CPBA (20.708 g) afforded 7.14 g of 4d as a colorless oil;
Yield: 72.8%; mp: colorless oil; 1H NMR (CDCl3, 400 MHz) δ: 8.128 (dd,
1H, J = 1.6 Hz, 8 Hz), 7.588–7.512 (m, 2H), 7.477–7.435 (m, 1H),
4.145–4.038 (m, 6H), 1.244 (t, 6H, J = 6.8 Hz); 13C NMR (CDCl3, 100
MHz) δ: 137.427, 135.148, 132.655, 131.825, 131.795, 127.413,
63.573, 63.508, 52.097, 50.737, 16.256, 16.193; MS-ESI m/z: 327.05
[M + H]+.
4.1.2. Purity determination of target compounds by HPLC
Purity analysis of the 47 target compounds were with the assistance
of HPLC through the Wondasil C-18 superb reversed-phase column (5
mm, 4.6 mm × 150 mm), which was conducted on the Shimadzu LC-20A
system. Water and methanol were both of HPLC grade and employed as
the mobile phase. Chromatographically pure methanol was used to
dissolve the target compounds that then were injected by 5 μL. The flow
rate was 0.6 mL/min, and the detective wavelength ranged from 190 nm
to 600 nm to search the maximum absorption wavelength of the tested
compounds. The purity of all target compounds is > 95%, which is
qualified for the medicinal research. The retention time (tR) and eluent
condition were given for each final compound.
4.1.3.5. Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (4e).
3e (8.709 g) and m-CPBA (20.708 g) afforded 6.21 g of 4e as a colorless
solid; Yield: 64.2%; mp: 58–60 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.952
(dd, 1H, J = 1.6 Hz, 7.6 Hz), 7.616–7.573 (m, 1H), 7.097 (t, 1H, J = 7.2
Hz), 7.037 (d, 1H, J = 8.4 Hz), 4.138–4.063 (m, 4H), 4.033 (d, 2H, J =
16.8 Hz), 3.981 (s, 3H), 1.244 (t, 6H, J = 7.2 Hz); 13C NMR (CDCl3, 100
MHz) δ: 157.261, 135.988, 130.397, 127.988, 120.770, 112.368,
63.375, 63.312, 56.525, 52.192, 50.829, 16.307, 16.244; MS-ESI m/z:
323.10 [M + H]+.
4.1.3. General synthesis of intermediates 3 (3a ~ e) and 4 (4a ~ e)
Intermediates 3 (3a ~ e) and 4 (4a ~ e) were synthesized according
to the methods described by Park et al. [54]. In brief, corresponding
thiophenol (1a 4.4 g, 1b 4.97 g, 1c 5.13 g, 1d 5.78 g, 1e 5.61 g, 40
mmol, 1 eq), commercially available intermediate 2 (15.47 g, 48 mmol,
1.2 eq), and Cs2CO3 (26.07 g, 80 mmol, 2 eq) were mixed in an
appropriate volume (~200 mL) of N, N-Dimethylformamide (DMF), the
mixture of which was stirred at 70 ◦C overnight. After concentration of
the reaction, the residue was washed with a large amount (~450 mL) of
H2O and extracted with ethyl acetate. The combined organic layer was
dried with MgSO4 and evaporated to afford oil materials as corre-
sponding intermediates 3 (3a ~ e), which were used for next without
further purification, and the yields ranged from 85.2% ~ 94.8%. The
corresponding oily 3 (3a 7.809 g, 3b 8.229 g, 3c 8.348 g, 3d 8.842 g, 3e
8.709 g, 30 mmol, 1 eq) was dissolved in an appropriate volume (~200
mL) of dichloromethane, and then the reaction was placed in ice. After it
was thoroughly cooled, 3-chloroperoxybenzoic acid (m-CPBA) (20.708
g, 120 mmol, 4 eq) was added portion-wise. After the addition was
completed, it was stirred at room temperature for ~ 5 h and quenched
with aqueous Na2S2O3 until 3 (3a ~ e) disappeared. Then it was washed
well with aqueous Na2S2O3 and water, and extracted with dichloro-
methane. The combined organic layer was concentrated to give a res-
idue, which was purified on column chromatography to afford pure
intermediates 4 (4a ~ e).
4.1.3.6. General synthesis of target compounds (6a ~ o, 7a ~ m, 8a ~ m,
9a ~ c, and 10a ~ c). The synthetic procedure for the target compounds
followed the method described by Park et at. [54]. Briefly, correspond-
ing intermediate 4 (4a 292 mg, 4b 306 mg, 4c 310 mg, 4d 327 mg, 4e
322 mg, 1 mmol, 1 eq) was dissolved in an appropriate volume (~20
mL) of anhydrous tetrahydrofuran (THF) in schlenk tube, and then it
was placed at ꢀ 78 ◦C. The tube was degassed three times with argon,
and then n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol, 1.2 eq) was injected slowly
to the reaction at ꢀ 78 ◦C. After the reaction was stirred at ꢀ 78 ◦C for 1 h,
corresponding aldehyde 5 (5a ~ o) (1.1 mmol, 1.1 eq) diluted in
anhydrous THF was injected to the tube at ꢀ 78 ◦C, at which temperature
the reaction was further stirred for 30 min. Later the reaction was stirred
at room temperature for ~ 1 h, which was quenched with saturated
aqueous NH4Cl and extracted with ethyl acetate. The combined organic
layer was then concentrated to obtain a residue, which was purified on
column chromatography to give the target compound.
4.1.3.7. (E)-1-(2-(phenylsulfonyl)vinyl)-2-(trifluoromethyl)benzene
(6a). 4a (292 mg, 1 mmol), 5a (0.15 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 286 mg of 6a as a white solid; Yield: 91.7%;
mp: 72–74 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.048 (dq, 1H, J = 15.2 Hz,
2 Hz), 7.973–7.943 (m, 2H), 7.719 (d, 1H, J = 7.6 Hz), 7.668–7.624 (m,
1H), 7.604–7.495 (m, 5H), 6.839 (d, 1H, J = 15.2 Hz); 13C NMR (CDCl3,
100 MHz) δ: 140.032, 138.545, 138.523, 138.501, 138.479, 133.825,
132.402, 131.943, 131.396, 131.378, 130.562, 129.556, 129.362,
129.056, 128.425, 127.977, 126.554, 126.499, 126.445, 126.391,
4.1.3.1. Diethyl ((phenylsulfonyl)methyl)phosphonate (4a). 3a (7.809 g)
and m-CPBA (20.708 g) afforded 6.10 g of 4a as a colorless solid; Yield:
69.6%; mp: 48–49 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.001 (d, 2H, J =
7.6 Hz), 7.681 (t, 1H, J = 7.2 Hz), 7.582 (t, 2H, J = 8 Hz), 4.202–4.126
(m, 4H), 3.826 (d, 2H, J = 16.8 Hz), 1.297 (t, 6H, J = 7.2 Hz); 13C NMR
(CDCl3, 100 MHz) δ: 140.018, 134.183, 129.200, 128.395, 63.539,
63.476, 54.501, 53.134, 16.326, 16.262; MS-ESI m/z: 293.05 [M + H]+.
125.152, 122.428; MS-ESI m/z: 313.05 [M + H]+; Purity: 99.39%, tR
=
5.87 min (20% water in methanol).
4.1.3.2. Diethyl (tosylmethyl)phosphonate (4b). 3b (8.229 g) and m-
CPBA (20.708 g) afforded 5.72 g of 4b as a white solid; Yield: 66.2%;
mp: 61–62 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.869 (d, 2H, J = 8.4 Hz),
4.1.3.8. (E)-1-chloro-2-(2-(phenylsulfonyl)vinyl)benzene (6b). 4a (292
mg, 1 mmol), 5b (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 237 mg of 6b as a white solid; Yield: 84.9%; mp:
9

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
102–103 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.096 (d, 1H, J = 15.6 Hz),
7.985–7.956 (m, 2H), 7.664–7.620 (m, 1H), 7.588–7.547 (m, 2H), 7.514
(dd, 1H, J = 1.6 Hz, 8 Hz), 7.435 (dd, 1H, J = 1.2 Hz, 8 Hz),7.341(td, 1H,
1.2 mmol) gave 260 mg of 6 h as a white solid; Yield: 94.7%; mp:
101–103 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.959–7.938 (m, 2H),
7.672–7.602 (m, 2H), 7.570–7.530 (m, 2H), 7.301 (t, 1H, J = 8 Hz),
7.075 (d, 1H, J = 7.6 Hz), 6.990–6.931 (m, 2H), 6.850 (d, 1H, J = 15.6
Hz), 3.809 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 160.065, 142.545,
140.760, 133.742, 133.511, 130.209, 129.450, 127.759, 127.625,
121.319, 117.238, 113.472, 55.464; MS-ESI m/z: 275.05 [M + H]+;
Purity: 98.55%, tR = 5.48 min (20% water in methanol).
J = 1.6 Hz, 8 Hz), 7.285–7.245 (m, 1H), 6.898 (d, 1H, J = 15.6 Hz); 13
C
NMR (CDCl3, 100 MHz) δ: 140.313, 138.443, 135.383, 133.671,
132.056, 130.688, 130.466, 130.031, 129.501, 128.312, 127.891,
127.322; MS-ESI m/z: 279.00 [M + H]+; Purity: 99.80%, tR = 6.23 min
(20% water in methanol).
4.1.3.9. (E)-1-methoxy-2-(2-(phenylsulfonyl)vinyl)benzene
(6c). 4a
4.1.3.15. (E)-1-(2-(phenylsulfonyl)vinyl)-4-(trifluoromethyl)benzene
(6i). 4a (292 mg, 1 mmol), 5i (0.16 mL, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 280 mg of 6i as a white solid; Yield: 89.8%; mp:
127–129 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.971–7.949 (m, 2H), 7.708
(d, 1H, J = 15.6 Hz), 7.664–7.634 (m, 3H), 7.605–7.555 (m, 4H), 6.955
(d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) δ: 140.551, 140.206,
135.847, 135.832, 133.845, 132.882, 132.554, 130.113, 129.595,
128.872, 127.929, 126.223, 126.185, 126.147, 126.109, 125.056,
122.349; MS-ESI m/z: 313.15 [M + H]+; Purity: 98.05%, tR = 5.78 min
(20% water in methanol).
(292 mg, 1 mmol), 5c (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 236 mg of 6c as a white solid; Yield: 91.8%; mp:
84–85 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.992–7.943 (m, 2H),
7.645–535 (m, 3H), 7.450–7.427 (dd, 1H, J = 1.2 Hz, 8 Hz), 7.301 (td,
1H, J = 1.2 Hz, 7.6 Hz), 7.232–7.167 (m, 2H), 6.784 (d, 1H, J = 15.2
Hz), 2.461 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 140.783, 140.219,
138.300, 133.467, 131.322, 131.152, 131.056, 129.446, 128.235,
127.745, 126.957, 126.587, 19.877; MS-ESI m/z: 259.05 [M + H]+;
Purity: 99.28%, tR = 6.03 min (20% water in methanol).
4.1.3.10. (E)-1-methyl-2-(2-(phenylsulfonyl)vinyl)benzene
(6d). 4a
4.1.3.16. (E)-1-chloro-4-(2-(phenylsulfonyl)vinyl)benzene (6j). 4a (292
mg, 1 mmol), 5j (155 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 238 mg of 6j as a white solid; Yield: 85.6%; mp: 125–126 ◦C;
1H NMR (CDCl3, 400 MHz) δ: 7.958–7.936 (m, 2H), 7.657–7.542 (m,
(292 mg, 1 mmol), 5d (150 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 259 mg of 6d as a white solid; Yield: 94.5%; mp:
84–86 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.969–7.940 (m, 2H), 7.894 (d,
1H, J = 15.6 Hz), 7.620–7.512 (m, 3H), 7.427–7.355 (m, 2H), 7.077 (d,
1H, J = 15.6 Hz), 6.978–6.907 (m, 2H), 3.879 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ: 158.893, 141.172, 138.661, 133.219, 132.624, 130.929,
129.319, 127.828, 127.651, 121.164, 120.841, 111.299, 55.581; MS-
ESI m/z: 275.05 [M + H]+; Purity: 95.73%, tR = 5.63 min (20% water
in methanol).
4H), 7.394 (dd, 4H, J = 8.4 Hz, 20.8 Hz), 6.838 (d, 1H, J = 15.6 Hz); 13
C
NMR (CDCl3, 100 MHz) δ: 141.020, 140.432, 137.236, 133.600,
130.839, 129.829, 129.457, 129.413, 127.871, 127.719; MS-ESI m/z:
279.00 [M + H]+; Purity: 99.49%, tR = 5.82 min (20% water in
methanol).
4.1.3.17. (E)-1-methoxy-4-(2-(phenylsulfonyl)vinyl)benzene (6 k). 4a
(292 mg, 1 mmol), 5 k (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6
mL, 1.2 mmol) gave 247 mg of 6 k as a white solid; Yield: 95.8%; mp:
130–132 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.956–7.934 (m, 2H),
7.678–7.523 (m, 4H), 7.379 (d, 2H, J = 8 Hz), 7.194 (d, 2H, J = 8 Hz),
6.804 (d, 1H, J = 15.2 Hz), 2.370 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ:
142.658, 141.973, 141.013, 133.377, 129.908, 129.695, 129.400,
128.693, 127.680, 126.142, 21.634; MS-ESI m/z: 259.05 [M + H]+;
Purity: 98.54%, tR = 6.00 min (20% water in methanol).
4.1.3.11. (E)-1-(2-(phenylsulfonyl)vinyl)-3-(trifluoromethyl)benzene
(6e). 4a (292 mg, 1 mmol), 5e (192 mg, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 279 mg of 6e as a white solid; Yield: 89.4%;
mp: 90–92 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.958–7.936 (m, 2H),
7.708–7.591 (m, 5H), 7.552–7.480 (m, 3H), 6.995 (d, 1H, J = 15.6 Hz);
13C NMR (CDCl3, 100 MHz) δ: 140.531, 140.131, 133.710, 133.148,
131.772, 131.630, 131.305, 130.975, 129.757, 129.474, 129.439,
128.831, 127.764, 127.567, 127.531, 127.493, 127.456, 125.093,
125.055, 125.017, 124.979, 124.951; MS-ESI m/z: 313.05 [M + H]+;
Purity: 96.90%, tR = 6.09 min (20% water in methanol).
4.1.3.18. (E)-1-methyl-4-(2-(phenylsulfonyl)vinyl)benzene (6 l). 4a
(292 mg, 1 mmol), 5 l (0.14 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 258 mg of 6 l as a white solid; Yield: 94.2%; mp:
113–115 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.942 (d, 2H, J = 7.6 Hz),
7.654–7.518 (m, 4H), 7.434 (d, 2H, J = 7.6 Hz), 6.897 (d, 2H, J = 8.8
Hz), 6.709 (d, 1H, J = 15.6 Hz), 3.831 (s, 3H); 13C NMR (CDCl3, 100
MHz) δ: 162.156, 142.377, 141.222, 133.261, 130.476, 129.357,
127.575, 125.026, 124.497, 114.600, 55.532; MS-ESI m/z: 275.05 [M +
H]+; Purity: 97.44%, tR = 5.23 min (20% water in methanol).
4.1.3.12. (E)-1-chloro-3-(2-(phenylsulfonyl)vinyl)benzene (6f). 4a (292
mg, 1 mmol), 5f (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 238 mg of 6f as a white solid; Yield: 85.3%; mp: 92–94 ◦C;
1H NMR (CDCl3, 400 MHz) δ: 7.958–7.935 (m, 2H), 7.660–7.546 (m,
4H), 7.469 (s, 1H), 7.401–7.260 (m, 3H), 6.872 (d, 1H, J = 15.2 Hz); 13
C
NMR (CDCl3, 100 MHz) δ: 140.831, 140.381, 135.209, 134.214,
133.709, 131.159, 130.454, 129.530, 128.959, 128.284, 127.844,
126.935; MS-ESI m/z: 279.00 [M + H]+; Purity: 97.98%, tR = 6.23 min
(20% water in methanol).
4.1.3.19. (E)-(2-(phenylsulfonyl)vinyl)benzene (6 m). 4a (292 mg, 1
mmol), 5 m (0.12 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 237 mg of 6 m as a colorless solid; Yield: 97.1%; mp:
72–74 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.971–7.942 (m, 2H), 7.694 (d,
1H, J = 15.2 Hz), 7.648–7.605 (m, 1H), 7.573–7.533 (m, 2H),
7.508–7.477 (m, 2H), 7.442–7.368 (m, 3H), 6.865 (d, 1H, J = 15.6 Hz),
13C NMR (CDCl3, 100 MHz) δ: 142.470, 140.633, 133.425, 132.262,
131.241, 129.357, 129.078, 128.585, 127.609, 127.223; MS-ESI m/z:
245.05 [M + H]+; Purity: 98.89%, tR = 5.08 min (20% water in
methanol).
4.1.3.13. (E)-1-methoxy-3-(2-(phenylsulfonyl)vinyl)benzene (6 g). 4a
(292 mg, 1 mmol), 5 g (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 237 mg of 6 g as a colorless solid; Yield: 91.9%; mp:
71–73 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.963–7.933 (m, 2H), 7.660 (d,
1H, J = 15.6 Hz), 7.634–7.594 (m, 1H), 7.564–7.524 (m, 2H), 7.379 (d,
2H, J = 8.4 Hz), 7.194 (d, 2H, J = 8.4 Hz), 6.805 (d, 1H, J = 15.6 Hz),
2.368 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 142.827, 140.910, 138.980,
133.465, 132.411, 132.193, 129.448, 129.273, 129.098, 127.762,
127.100, 125.963, 21.406; MS-ESI m/z: 259.05 [M + H]+; Purity:
98.38%, tR = 6.18 min (20% water in methanol).
4.1.3.20. (E)-1-bromo-2-(2-(phenylsulfonyl)vinyl)benzene (6n). 4a (292
mg, 1 mmol), 5n (0.12 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 246 mg of 6n as a colorless solid; Yield: 93.9%; mp:
72–73 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.960 (d, 2H, J = 7.6 Hz), 7.769
4.1.3.14. (E)-1-methyl-3-(2-(phenylsulfonyl)vinyl)benzene (6 h). 4a
(292 mg, 1 mmol), 5 h (150 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
10

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
(d, 1H, J = 15.6 Hz), 7.650–7.541 (m, 3H), 7.486–7.369 (m, 2H),
7.194–7.074 (m, 2H), 7.024 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100
MHz) δ: 162.869, 160.332, 140.562, 135.580, 135.557, 133.575,
132.909, 132.821, 130.381, 130.355, 130.281, 130.197, 129.461,
127.817, 124.802, 124.765, 120.668, 120.555, 116.575, 116.359; MS-
ESI m/z: 263.05 [M + H]+; Purity: 99.65%, tR = 5.50 min (20% water
in methanol).
mmol) gave 288 mg of 7e as a white solid; Yield: 88.4%; mp: 96–98 ◦C;
1H NMR (CDCl3, 400 MHz) δ: 7.835 (d, 2H, J = 8.4 Hz), 7.714–7.643 (m,
4H), 7.539 (d, 1H, J = 7.6 Hz), 7.361 (d, 2H, J = 8 Hz), 6.931 (d, 1H, J =
15.6 Hz), 2.444 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 144.920,
141.943, 140.075, 137.296, 133.412, 131.928, 131.768, 131.600,
130.225, 129.922, 129.817, 129.048, 127.987, 127.583, 127.545,
125.099, 125.059, 21.768; MS-ESI m/z: 327.00 [M + H]+; Purity:
97.53%, tR = 7.22 min (20% water in methanol).
4.1.3.21. (E)-1-fluoro-2-(2-(phenylsulfonyl)vinyl)benzene (6o). 4a (292
mg, 1 mmol), 5o (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 305 mg of 6o as a colorless solid; Yield: 94.8%; mp:
4.1.3.27. (E)-1-chloro-3-(2-tosylvinyl)benzene (7f). 4b (306 mg,
1
mmol), 5f (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 239 mg of 7f as a white solid; Yield: 81.8%; mp: 91–93 ◦C; 1H NMR
(CDCl3, 400 MHz) δ: 7.821 (d, 2H, J = 8 Hz), 7.586 (d, 1H, J = 15.6 Hz),
7.454 (s, 1H), 7.390–7.303 (m, 5H), 6.856 (d, 1H, J = 15.2 Hz), 2.442 (s,
3H); 13C NMR (CDCl3, 100 MHz) δ: 144.766, 140.265, 137.424,
135.188, 134.342, 131.036, 130.426, 130.158, 129.334, 128.239,
127.907, 126.860, 21.753; MS-ESI m/z: 293.00 [M + H]+; Purity:
98.77%, tR = 7.40 min (20% water in methanol).
1
◦
89–90 C; H NMR (CDCl3, 400 MHz) δ: 8.061 (d, 1H, J = 15.6 Hz),
7.992–7.962 (m, 2H), 7.662–7.547 (m, 4H), 7.496 (dd, 1H, J = 2 Hz, 7.6
Hz), 7.311 (td, 1H, J = 1.2 Hz, 7.6 Hz), 7.272–7.230 (m, 1H), 6.846 (d,
1H, J = 15.2 Hz); 13C NMR (CDCl3, 100 MHz) δ: 140.968, 140.289,
133.639, 133.570, 132.537, 132.053, 130.248, 129.415, 128.271,
127.853, 125.574; MS-ESI m/z: 322.95 [M + H]+; Purity: 99.36%, tR
=
6.49 min (20% water in methanol).
4.1.3.22. (E)-1-(2-tosylvinyl)-2-(trifluoromethyl)benzene (7a). 4b (306
mg, 1 mmol), 5a (0.15 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 289 mg of 7a as a white solid; Yield: 88.7%; mp:
106–107 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.013 (dd, 1H, J = 2.4 Hz,
15.2 Hz), 7.830 (d, 2H, J = 8.4 Hz), 7.710 (d, 1H, J = 7.6 Hz),
7.591–7.484 (m, 3H), 7.358 (d, 2H, J = 8 Hz), 6.821 (d, 1H, J = 14.8
Hz), 2.441 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 144.891, 137.927,
137.905, 137.076, 132.376, 132.291, 131.489, 130.441, 130.167,
129.292, 128.987, 128.389, 128.006, 126.503, 126.449, 126.394,
126.340, 125.166, 122.442, 21.733; MS-ESI m/z: 327.00 [M + H]+;
Purity: 99.87%, tR = 6.90 min (20% water in methanol).
4.1.3.28. (E)-1-methoxy-3-(2-tosylvinyl)benzene (7 g). 4b (306 mg, 1
mmol), 5 g (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 246 mg of 7 g as a white solid; Yield: 90.4%; mp: 93–94 ◦C; 1H NMR
(CDCl3, 400 MHz) δ: 7.824 (d, 2H, J = 7.6 Hz), 7.627 (d, 1H, J = 15.6
Hz), 7.337 (d, 2H, J = 8 Hz), 7.280–7.261 (m, 3H), 7.247–7.205 (m,
1H), 6.831 (m, 1H, J = 15.2 Hz), 2.431 (s, 3H), 2.347 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ: 144.415, 142.206, 138.887, 137.917, 132.471,
132.025, 130.037, 129.180, 129.027, 127.772, 127.442, 125.856,
21.714, 21.360; MS-ESI m/z: 273.05 [M + H]+; Purity: 99.56%, tR
=
7.37 min (20% water in methanol).
4.1.3.29. (E)-1-methyl-3-(2-tosylvinyl)benzene (7 h). 4b (306 mg, 1
mmol), 5 h (150 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 270 mg of 7 h as a colorless solid; Yield: 93.8%; mp: 56–58 ◦C; 1H
NMR (CDCl3, 400 MHz) δ: 7.820 (d, 2H, J = 8.4 Hz), 7.626 (d, 1H, J =
15.6 Hz), 7.358–7.266 (m, 3H), 7.069 (d, 1H, J = 7.6 Hz), 6.977–6.940
(m, 2H), 6.840 (d, 1H, J = 15.2 Hz), 3.810 (s, 3H), 2.438 (s, 3H); 13C
NMR (CDCl3, 100 MHz) δ: 159.701, 144.237, 141.657, 137.488,
133.494, 129.885, 129.812, 127.710, 127.473, 121.025, 116.876,
113.058, 55.138, 21.415; MS-ESI m/z: 289.05 [M + H]+; Purity:
98.93%, tR = 6.42 min (20% water in methanol).
4.1.3.23. (E)-1-chloro-2-(2-tosylvinyl)benzene (7b). 4b (306 mg,
1
mmol), 5b (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 243 mg of 7b as a white solid; Yield: 83.1%; mp: 104–105 ◦C; 1H
NMR (CDCl3, 400 MHz) δ: 8.057 (d, 1H, J = 15.6 Hz), 7.842 (d, 2H, J =
8 Hz), 7.500 (dd, 1H, J = 2 Hz, 7.6 Hz), 7.427 (dd, 1H, J = 1.2 Hz, 8 Hz),
7.366–7.308 (m, 3H), 7.276–7.239 (m, 1H), 2.442 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ: 144.714, 137.930, 137.397, 135.351, 131.934,
130.846, 130.461, 130.441, 130.137, 128.295, 127.972, 127.303,
21.759; MS-ESI m/z: 293.00 [M + H]+; Purity: 99.86%, tR = 7.36 min
(20% water in methanol).
4.1.3.30. (E)-1-chloro-4-(2-tosylvinyl)benzene (7i). 4b (306 mg,
1
4.1.3.24. (E)-1-methoxy-2-(2-tosylvinyl)benzene (7c). 4b (306 mg, 1
mmol), 5c (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 253 mg of 7c as a white solid; Yield: 92.9%; mp: 81–83 ◦C; 1H NMR
(CDCl3, 400 MHz) δ: 7.940 (d, 1H, J = 15.2 Hz), 7.829 (d, 2H, J = 8.4
Hz), 7.426 (d, 1H, J = 7.6 Hz), 7.347 (d, 2H, J = 8 Hz), 7.293 (t, 1H, J =
6.8 Hz), 7.223–7.163 (m, 2H), 6.767 (d, 1H, J = 15.2 Hz), 2.453 (s, 3H),
2.440 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 144.448, 139.656, 138.213,
137.794, 131.403, 131.106, 130.925, 130.064, 128.570, 127.788,
126.907, 126.548, 21.724, 19.872; MS-ESI m/z: 273.05 [M + H]+; Pu-
rity: 98.98%, tR = 7.16 min (20% water in methanol).
mmol), 5i (0.16 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 301 mg of 7i as a white solid; Yield: 92.4%; mp: 127–128 ◦C; 1H
NMR (CDCl3, 400 MHz) δ: 7.835 (d, 2H, J = 8.4 Hz), 7.691–7.571 (m,
5H), 7.363 (d, 2H, J = 8 Hz), 7.940 (d, 1H, J = 15.6 Hz), 2.445 (s, 3H);
13C NMR (CDCl3, 100 MHz) δ: 144.955, 139.985, 137.137, 135.910,
130.362, 130.218, 128.806, 127.968, 126.174, 126.138, 126.100,
126.062, 21.772; MS-ESI m/z: 327.05 [M + H]+; Purity: 99.49%, tR
=
6.74 min (20% water in methanol).
4.1.3.31. (E)-1-methoxy-4-(2-tosylvinyl)benzene (7j). 4b (306 mg, 1
mmol), 5j (155 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 248 mg of 7j as a white solid; Yield: 84.8%; mp: 140–142 ◦C; 1H
NMR (CDCl3, 400 MHz) δ: 7.820 (d, 2H, J = 8.4 Hz), 7.603 (d, 1H, J =
15.6 Hz), 7.417–7.340 (m, 6H), 6.822 (d, 1H, J = 15.2 Hz), 2.439 (s,
3H); 13C NMR (CDCl3, 100 MHz) δ: 144.668, 140.490, 137.458,
137.149, 130.955, 130.112, 129.784, 129.420, 128.212, 127.811,
21.738; MS-ESI m/z: 293.00 [M + H]+; Purity: 95.03%, tR = 6.55 min
(20% water in methanol).
4.1.3.25. (E)-1-methyl-2-(2-tosylvinyl)benzene (7d). 4b (306 mg, 1
mmol), 5d (150 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2 mmol)
gave 261 mg of 7d as a white solid; Yield: 90.4%; mp: 70–73 ◦C; 1H NMR
(CDCl3, 400 MHz) δ: 7.882–7.815 (m, 3H), 7.416–7.316 (m, 4H), 7.065
(d, 1H, J = 15.6 Hz), 6.970–6.899 (m, 2H), 3.870 (s, 3H), 2.423 (s, 3H);
13C NMR (CDCl3, 100 MHz) δ: 158.770, 144.102, 138.128, 138.020,
132.462, 130.765, 129.894, 128.101, 127.624, 121.138, 120.750,
111.220, 55.500, 21.643; MS-ESI m/z: 289.05 [M + H]+; Purity:
96.75%, tR = 6.64 min (20% water in methanol).
4.1.3.32. (E)-1-methyl-4-((4-(trifluoromethyl)styryl)sulfonyl)benzene (7
k). 4b (306 mg, 1 mmol), 5 k (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 262 mg of 7 k as a white solid; Yield: 96.4%; mp:
148–150 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.821 (d, 2H, J = 8.4 Hz),
4.1.3.26. (E)-1-(2-tosylvinyl)-3-(trifluoromethyl)benzene (7e). 4b (306
mg, 1 mmol), 5e (192 mg, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
11

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
7.625 (d, 1H, J = 15.2 Hz), 7.376–7.325 (m, 4H), 7.186 (d, 2H, J = 8
Hz), 6.788 (d, 1H, J = 15.2 Hz), 2.431 (s, 3H), 2.366 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ: 144.343, 142.083, 141.805, 138.033, 130.017,
129.867, 129.774, 128.619, 127.727, 126.488, 21.701, 21.608; MS-ESI
m/z: 273.05 [M + H]+; Purity: 98.95%, tR = 7.12 min (20% water in
methanol).
4.1.3.38. (E)-1-chloro-2-(2-((4-fluorophenyl)sulfonyl)vinyl)benzene
(8d). 4c (310 mg, 1 mmol), 5d (150 mg, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 254 mg of 8d as a white solid; Yield: 86.8%;
mp: 98–100 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.984–7.935 (m, 2H),
7.876 (d, 1H, J = 15.6 Hz), 7.427–7.368 (m, 2H), 7.236–7.179 (m, 2H),
7.065 (d, 1H, J = 15.2 Hz), 6.985–6.913 (m. 2H), 3.883 (s, 3H); 13C
NMR (CDCl3, 100 MHz) δ: 166.573, 164.036, 158.736, 138.607,
137.170, 137.139, 132.635, 130.780, 130.366, 130.271, 127.522,
120.799, 120.706, 116.538, 116.313, 111.202, 55.432; MS-ESI m/z:
293.00 [M + H]+; Purity: 95.49%, tR = 5.76 min (20% water in
methanol).
4.1.3.33. (E)-1-methyl-4-((4-methylstyryl)sulfonyl)benzene (7 l). 4b
(306 mg, 1 mmol), 5 l (0.14 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 275 mg of 7 l as a white solid; Yield: 95.6%; mp:
92–93 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.816 (d, 2H, J = 8 Hz), 7.602
(d, 1H, J = 15.2 Hz), 7.421 (d, 2H, J = 8.8 Hz), 7.330 (d, 2H, J = 7.6 Hz),
6.891 (d, 2H, J = 8.8 Hz), 6.694 (d, 1H, J = 15.6 Hz), 3.829 (s, 3H),
2.427 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 162.060, 144.227, 141.826,
138.259, 130.406, 129.995, 127.652, 125.131, 124.877, 114.575,
4.1.3.39. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-3-(trifluoromethyl)
benzene (8e). 4c (310 mg, 1 mmol), 5e (192 mg, 1.1 mmol), and n-BuLi
(c = 2 M, 0.6 mL, 1.2 mmol) gave 285 mg of 8e as a white solid; Yield:
86.5%; mp: 106–107 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.004–7.955 (m,
2H), 7.735–7.660 (m, 4H), 7.550 (t, 1H, J = 8 Hz), 7.277–7.220 (m, 2H),
6.933 (d, 1H, J = 15.2 Hz); 13C NMR (CDCl3, 100 MHz) δ: 167.196,
164.646, 140.815, 136.388, 136.357, 133.174, 132.310, 131.984,
131.858, 131.658, 131.331, 130.873, 130.777, 129.888, 129.413,
127.838, 127.803, 127.766, 127.728, 125.177, 125.139, 125.100,
125.015, 122.306, 119.596, 117.033, 116.807; MS-ESI m/z: 331.20 [M
+ H]+; Purity: 99.62%, tR = 6.10 min (20% water in methanol).
55.535, 21.708; MS-ESI m/z: 289.05 [M + H]+; Purity: 99.08%, tR
=
6.12 min (20% water in methanol).
4.1.3.34. (E)-1-fluoro-4-(styrylsulfonyl)benzene (7 m). 4b (306 mg, 1
mmol), 5 m (0.12 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 247 mg of 7 m as a colorless solid; Yield: 95.6%; mp:
118–119 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.830 (d, 2H, J = 8.4 Hz),
7.659 (d, 1H, J = 15.6 Hz), 7.488–7.464 (m, 2H), 7.414–7.333 (m, 5H),
7.848 (d, 1H, J = 15.6 Hz), 2.435 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ:
144.493, 142.025, 137.830, 132.536, 131.204, 130.070, 129.158,
128.619, 127.805, 127.717, 21.724; MS-ESI m/z: 259.10 [M + H]+;
Purity: 99.12%, tR = 5.88 min (20% water in methanol).
4.1.3.40. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-3-methoxybenzene
(8f). 4c (310 mg, 1 mmol), 5f (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 241 mg of 8f as a white solid; Yield: 81.5%; mp:
105–107 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.988–7.938 (m, 2H), 7.618
(d, 1H, J = 15.6 Hz), 7.473 (s, 1H), 7.412–7.322 (m, 3H), 7.260–7.210
(m, 2H), 6.858 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) δ:
167.122, 164.573, 141.035, 136.522, 136.490, 135.283, 134.107,
131.287, 130.799, 130.704, 130.499, 128.819, 128.317, 126.975,
4.1.3.35. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-2-(trifluoromethyl)
benzene (8a). 4c (310 mg, 1 mmol), 5a (0.15 mL, 1.1 mmol), and n-BuLi
(c = 2 M, 0.6 mL, 1.2 mmol) gave 294 mg of 8a as a white solid; Yield:
89.1%; mp: 82–83 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.060 (dq, 1H, J = 2
Hz, 15.2 Hz), 8.010–7.960 (m, 2H), 7.743 (d, 1H, J = 7.6 Hz),
7.626–7.524 (m, 3H), 7.289–7.232 (m, 2H), 6.836 (d, 1H, J = 15.2 Hz);
13C NMR (CDCl3, 100 MHz) δ: 167.202, 164.652, 138.759, 138.738,
138.716, 136.214, 136.182, 132.445, 131.871, 131.310, 131.294,
130.930, 130.834, 130.657, 129.705, 129.399, 129.094, 128.788,
128.441, 127.889, 126.607, 126.552, 126.497, 126.442, 125.165,
122.441, 117.009, 116.784; MS-ESI m/z: 331.00 [M + H]+; Purity:
99.60%, tR = 6.27 min (20% water in methanol).
116.978, 116.753; MS-ESI m/z: 297.00 [M + H]+; Purity: 98.38%, tR
=
6.22 min (20% water in methanol).
4.1.3.41. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-3-methylbenzene (8
g). 4c (310 mg, 1 mmol), 5 g (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 247 mg of 8 g as a colorless solid; Yield: 89.3%;
mp: 55–57 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.987–7.949 (m, 3H), 7.437
(d, 1H, J = 8 Hz), 7.331–7.291 (m, 1H), 7.252–7.177 (m, 4H), 6.764 (d,
1H, J = 15.2 Hz), 2.462 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 166.891,
164.347, 142.913, 138.926, 137.052, 137.022, 132.246, 132.210,
130.580, 130.484, 129.213, 129.045, 126.979, 125.918, 116.756,
4.1.3.36. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-2-methoxybenzene
(8b). 4c (310 mg, 1 mmol), 5b (0.13 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 247 mg of 8b as a white solid; Yield: 83.4%;
mp: 104–105 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.082 (d, 1H, J = 15.6
Hz), 8.006–7.957 (m, 2H), 7.512 (dd, 1H, J = 1.6 Hz, 7.6 Hz), 7.441 (dd,
1H, J = 1.2 Hz, 8 Hz), 7.348 (td, 1H, J = 1.6 Hz, 7.6 Hz), 7.292–7.210
(m, 3H), 6.884 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) δ:
167.068, 164.521, 138.630, 136.555, 136.523, 135.402, 132.123,
130.830, 130.734, 130.640, 130.496, 130.030, 128.375, 127.357,
116.531, 21.276; MS-ESI m/z: 277.00 [M + H]+; Purity: 98.98%, tR
=
6.21 min (20% water in methanol).
4.1.3.42. (E)-1-chloro-3-(2-((4-fluorophenyl)sulfonyl)vinyl)benzene (8
h). 4c (310 mg, 1 mmol), 5 h (150 mg, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 257 mg of 8 h as a colorless solid; Yield: 88.1%;
mp: 66–68 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.988–7.939 (m, 2H), 7.646
(d, 1H, J = 15.6 Hz), 7.310 (t, 1H, J = 8 Hz), 7.253–7.195 (m, 2H), 7.077
(d, 1H, J = 8 Hz), 6.986–6.955 (m, 2H), 6.833 (d, 1H, J = 15.2 Hz),
3.0.815 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 166.876, 164.333,
160.007, 142.636, 136.842, 136.811, 133.543, 130.582, 130.487,
130.148, 127.433, 121.270, 117.261, 116.760, 116.535, 113.440,
55.375; MS-ESI m/z: 293.05 [M + H]+; Purity: 98.85%, tR = 5.57 min
(20% water in methanol).
116.905, 116.680; MS-ESI m/z: 297.00 [M + H]+; Purity: 96.57%, tR
=
6.15 min (20% water in methanol)%.
4.1.3.37. (E)-1-(2-((4-fluorophenyl)sulfonyl)vinyl)-2-methylbenzene
(8c). 4c (310 mg, 1 mmol), 5c (0.13 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 237 mg of 8c as a colorless solid; Yield:
1
◦
85.8%; mp: 76–77 C; H NMR (CDCl3, 400 MHz) δ: 7.987–7.940 (m,
3H), 7.438 (d, 1H, J = 8 Hz), 7.311 (td, 1H, J = 1.2 Hz, 7.6 Hz),
7.252–7.178 (m, 4H), 6.766 (d, 1H, J = 15.2 Hz), 2.462 (s, 3H); 13C
NMR (CDCl3, 100 MHz) δ: 166.772, 164.231, 140.170, 138.206,
136.754, 136.723, 131.049, 130.997, 130.531, 130.436, 127.877,
126.839, 126.493, 116.727, 116.502, 19.709; MS-ESI m/z: 277.05 [M +
H]+; Purity: 99.70%, tR = 6.07 min (20% water in methanol).
4.1.3.43. (E)-1-chloro-4-(2-((4-fluorophenyl)sulfonyl)vinyl)benzene
(8i). 4c (310 mg, 1 mmol), 5i (0.16 mL, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 273 mg of 8i as a white solid; Yield: 82.7%; mp:
136–137 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.995–7.962 (m, 2H),
7.724–7.590 (m, 5H), 7.272–7.230 (m, 2H), 6.935 (d, 1H, J = 15.6 Hz);
13C NMR (CDCl3, 100 MHz) δ: 167.207, 164.655, 140.735, 136.297,
136.265, 135.705, 132.988, 132.660, 130.881, 130.785, 129.939,
12

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
128.898, 126.257, 126.219, 126.182, 126.143, 125.034, 122.327,
135.145, 134.735, 132.551, 132.124, 131.820, 130.643, 130.393,
130.214, 128.302, 127.890, 127.463, 127.261; MS-ESI m/z: 313.05 [M
+ H]+; Purity: 99.50%, tR = 7.54 min (20% water in methanol).
117.048, 116.823; MS-ESI m/z: 331.25 [M + H]+; Purity: 99.52%, tR
=
5.80 min (20% water in methanol).
4.1.3.44. (E)-1-fluoro-4-((4-(trifluoromethyl)styryl)sulfonyl)benzene
4.1.3.50. (E)-1-chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)benzene
(9c). 4d (327 mg, 1 mmol), 5o (0.13 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 330 mg of 9c as a colorless solid; Yield:
92.6%; mp: 77–78 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.235 (dd, 1H, J =
1.6 Hz, 8 Hz), 8.166 (d, 1H, J = 15.6 Hz), 7.635 (dd, 1H, J = 1.2 Hz, 8
Hz), 7.583–7.461 (m, 4H), 7.334 (td, 1H, J = 0.8 Hz, 8 Hz), 7.290–7.247
(m, 1H), 7.039 (d, 1H, J = 15.2 Hz); 13C NMR (CDCl3, 100 MHz) δ:
143.806, 137.885, 134.806, 133.677, 132.835, 132.523, 132.308,
131.971, 130.866, 128.490, 128.232, 127.964, 127.561, 125.688; MS-
ESI m/z: 356.90 [M + H]+; Purity: 99.46%, tR = 7.82 min (20% water
in methanol).
(8j). 4c (310 mg, 1 mmol), 5j (155 mg, 1.1 mmol), and n-BuLi (c = 2 M,
0.6 mL, 1.2 mmol) gave 258 mg of 8j as a white solid; Yield: 87.2%; mp:
132–133 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.983–7.934 (m, 2H), 7.629
(d, 1H, J = 15.2 Hz), 7.431–7.363 (m, 4H), 7.253–7.203 (m, 2H), 6.817
(d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) δ: 167.089, 164.542,
141.285, 137.523, 136.676, 136.644, 130.816, 130.750, 130.654,
129.894, 129.568, 127.821, 116.961, 116.736; MS-ESI m/z: 297.00 [M
+ H]+; Purity: 99.86%, tR = 5.87 min (20% water in methanol).
4.1.3.45. (E)-1-fluoro-4-((4-methoxystyryl)sulfonyl)benzene (8 k). 4c
(310 mg, 1 mmol), 5 k (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6
mL, 1.2 mmol) gave 267 mg of 8 k as a white solid; Yield: 96.2%; mp:
112–113 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.980–7.931 (m, 2H), 7.649
(d, 1H, J = 15.2 Hz), 7.379 (d, 2H, J = 8.4 Hz), 7.237–7.193 (m, 4H),
6.780 (d, 1H, J = 15.2 Hz), 2.375 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ:
166.905, 164.364, 142.839, 142.121, 137.132, 137.100, 130.583,
130.488, 129.944, 129.562, 128.721, 125.968, 116.796, 116.571,
21.638; MS-ESI m/z: 277.00 [M + H]+; Purity: 99.69%, tR = 6.05 min
(20% water in methanol).
4.1.3.51. (E)-1-bromo-2-(2-((2-methoxyphenyl)sulfonyl)vinyl)benzene
(10a). 4e (327 mg, 1 mmol), 5n (0.12 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 266 mg of 10a as a white solid; Yield: 91.2%;
mp: 108–109 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.028 (dd, 1H, J = 1.6
Hz, 8 Hz), 7.786 (d, 1H, J = 15.6 Hz), 7.596–7.552 (m, 1H), 7.483 (td,
1H, J = 2 Hz, 7.6 Hz), 7.417–7.360 (m, 1H), 7.270 (d, 1H, J = 15.6 Hz),
7.176 (td, 1H, J = 1.2 Hz, 7.6 Hz), 7.135–7.085 (m, 2H), 7.017 (d, 1H, J
= 8.4 Hz), 3.969 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 162.786,
160.252, 157.458, 136.455, 136.430, 135.574, 132.624, 132.536,
130.151, 130.124, 129.914, 129.833, 129.601, 128.478, 124.754,
124.717, 121.126, 121.010, 120.806, 116.503, 116.288, 112.520,
56.303; MS-ESI m/z: 293.00 [M + H]+; Purity: 99.70%, tR = 5.38 min
(20% water in methanol).
4.1.3.46. (E)-1-fluoro-4-((4-methylstyryl)sulfonyl)benzene (8 l). 4c
(310 mg, 1 mmol), 5 l (0.14 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL,
1.2 mmol) gave 270 mg of 8 l as a white solid; Yield: 92.4%; mp:
118–119 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.920–7.871 (m, 2H), 7.571
(d, 1H, J = 15.6 Hz), 7.380 (d, 2H, J = 8.4 Hz), 7.208–7.125 (m, 2H),
6.848 (d, 2H, J = 8.8 Hz), 6.635 (d, 1H, J = 15.2 Hz), 3.780 (s, 3H); 13
C
4.1.3.52. (E)-1-chloro-2-(2-((2-methoxyphenyl)sulfonyl)vinyl)benzene
(10b). 4e (327 mg, 1 mmol), 5b (0.13 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 299 mg of 10b as a white solid; Yield: 97.4%;
mp: 115–116 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.112 (d, 1H, J = 15.6
Hz), 8.041 (dd, 1H, J = 2 Hz, 8 Hz), 7.603–7.543 (m, 2H), 7.432 (dd, 1H,
J = 1.6 Hz, 8 Hz), 7.336 (td, 1H, J = 1.6 Hz, 7.6 Hz), 7.291 (dd, 1H, J =
1.6 Hz, 7.6 Hz), 7.134–7.090 (m, 2H), 7.018 (d, 1H, J = 8.4 Hz), 3.978
(s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 157.494, 139.469, 135.671,
135.204, 131.802, 131.225, 130.391, 129.742, 129.698, 128.341,
128.204, 127.314, 120.799, 112.493, 56.340; MS-ESI m/z: 309.00 [M +
H]+; Purity: 99.72%, tR = 6.02 min (20% water in methanol).
NMR (CDCl3, 100 MHz) δ: 166.734, 164.196, 162.199, 142.499,
137.345, 137.313, 130.467, 130.397, 130.302, 124.843, 124.302,
116.664, 116.439, 114.584, 55.467; MS-ESI m/z: 293.05 [M + H]+;
Purity: 99.02%, tR = 5.32 min (20% water in methanol).
4.1.3.47. (E)-1-methyl-4-(styrylsulfonyl)benzene (8 m). 4c (310 mg, 1
mmol), 5 m (0.12 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 267 mg of 8 m as a colorless solid; Yield: 91.4%; mp:
77–79 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 7.988–7.939 (m, 2H), 7.685 (d,
1H, J = 15.6 Hz), 7.500–7.477 (m, 2H), 7.449–7.374 (m, 3H),
7.253–7.196 (m, 2H), 6.845 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100
MHz) δ: 166.944, 164.400, 142.739, 136.958, 136.925, 132.313,
131.395, 130.628, 130.533, 129.186, 128.675, 127.258, 116.809,
116.584; MS-ESI m/z: 293.05 [M + H]+; Purity: 98.96%, tR = 5.11 min
(20% water in methanol).
4.1.3.53. (E)-1-fluoro-2-(2-((2-methoxyphenyl)sulfonyl)vinyl)benzene
(10c). 4e (327 mg, 1 mmol), 5o (0.13 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 341 mg of 10c as a white solid; Yield: 96.6%;
mp: 114–115 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.081 (d, 1H, J = 15.2
Hz), 8.043 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.626 (dd, 1H, J = 1.2 Hz, 8 Hz),
7.605–7.534 (m, 2H), 7.328 (td, 1H, J = 1.2 Hz, 7.6 Hz), 7.271–7.229
(m, 1H), 7.132–7.091 (m, 1H), 7.065 (d, 1H, J = 15.6 Hz), 7.019 (d, 1H,
J = 8.4 Hz), 3.986 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ: 157.197,
141.455, 135.572, 133.319, 132.470, 131.824, 129.463, 129.285,
128.121, 127.780, 127.678, 125.175, 120.480, 112.358, 56.175; MS-
ESI m/z: 352.95 [M + H]+; Purity: 99.80%, tR = 6.19 min (20% water
in methanol).
4.1.3.48. (E)-1-bromo-2-(2-((2-chlorophenyl)sulfonyl)vinyl)benzene
(9a). 4d (327 mg, 1 mmol), 5n (0.12 mL, 1.1 mmol), and n-BuLi (c = 2
M, 0.6 mL, 1.2 mmol) gave 266 mg of 9a as a colorless solid; Yield:
89.7%; mp: 101–102 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.225 (dd, 1H, J
= 1.6 Hz, 8 Hz), 7.851 (d, 1H, J = 15.6 Hz), 7.576–7.387 (m, 5H), 7.234
(d, 1H, J = 15.6 Hz), 7.209–7.098 (m, 2H); 13C NMR (CDCl3, 100 MHz)
δ: 162.751, 160.211, 138.174, 138.150, 137.935, 134.691, 133.095,
133.007, 132.722, 131.892, 130.718, 130.313, 130.288, 128.144,
128.060, 127.503, 124.776, 124.740, 120.487, 120.374, 116.463,
116.248; MS-ESI m/z: 297.00 [M + H]+; Purity: 98.75%, tR = 6.61 min
(20% water in methanol).
4.2. Biological studies
4.2.1. Cell culture
The PC12 cell line was purchased from the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. It was
cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 2
mM glutamine, and 100 units/mL penicillin/streptomycin, and was
maintained at 37 ◦C in a humidified incubator with 5% CO2.
4.1.3.49. (E)-1-chloro-2-((2-fluorostyryl)sulfonyl)benzene (9b). 4d (327
mg, 1 mmol), 5b (0.13 mL, 1.1 mmol), and n-BuLi (c = 2 M, 0.6 mL, 1.2
mmol) gave 299 mg of 9b as a colorless solid; Yield: 95.8%; mp:
71–72 ◦C; 1H NMR (CDCl3, 400 MHz) δ: 8.232 (d, 1H, J = 7.6 Hz), 8.186
(d, 1H, J = 15.2 Hz), 7.581–7.430 (m, 5H), 7.374–7.269 (m, 2H), 7.052
(d, 1H, J = 15.2 Hz); 13C NMR (CDCl3, 100 MHz) δ: 141.008, 137.657,
13

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
4.2.2. MTT assay
electron acceptor in the reduction assay was employed by measuring the
absorbance at 600 nm and the total protein content was quantified by
Bradford method. Briefly, the reduction assay was conducted by mixing
The PC12 cells were seeded to adhere for 24 h, and addition of target
compounds (10 μM, 20 μM and 50 μM) was followed. After 24 h the cell
survival was determined by MTT assay. For the H2O2 injury cellular
model, PC12 cells were grown to adhere for 24 h and addition of target
compounds of indicated concentrations was followed. After 24 h the
the cell samples (5
μg of total proteins), NADH (200
μM), Tris–HCl (20
mM, pH 7.4), and DCPIP (40
μ
M), with/without addition of 20 μM
dicoumarol (a selective inhibitor of NQO1). Then absorbance decrease
at 600 nm was determined every 8 s for 2 min at room temperature. The
NQO1 activity was calculated by using the dicoumarol-inhibitable part
of DCPIP reduction. The same amount of DMSO was added to the control
group and the NQO1 activity was expressed as the percentage of the
control.
cells were damaged by H2O2 (500 μM) for 12 h followed by MTT assay.
The more detailed information on the procedure was based on our
previous work [39–41].
4.2.3. LDH leakage determination
PC12 cells were seeded to adhere for 24 h, and then the cells were
For the evaluation of cellular HO-1 activity, a spectrophotometric
method was employed and the total protein content was quantified by
Bradford method. Briefly, the cellular samples was added to buffer A
consisting of sucrose (0.25 M), Tris-HCl (20 mM, pH 7.4), phenyl-
methylsulfonyl fluoride (1 mM), and Na3VO4 (1 mM). Then the cellular
incubated with 9b (0.5 μM and 1 μM) and 9c (0.5 μM and 1 μM) for 24 h.
Afterwards, the old culture mediums were replaced with newly prepared
medium having 500 μM H2O2, and the cells were cultured for additional
12 h. Then measurement of LDH activity was conducted. The more
detailed information on the procedure was based on our previous work
[39–41].
samples containing 50
μg of total proteins was incubated with NADPH
(1 mM), hemin (25 M), and excessive bovine liver extracts (lysed in
μ
buffer A containing biliverdin reductase) in a final volume of 100 μL at
4.2.4. Determination of the cellular ROS level
37 ◦C for 30 min in dark. The blank group was treated in the same
manner without addition of NADPH, and the same amount of DMSO was
added to the control group. Then the absorbance of 464 nm was deter-
mined, and the HO-1 activity was expressed as the percentage of the
control.
PC12 cells were bred to adhere for 24 h and then the cells were
incubated with 9b (0.5 μM and 1 μM) and 9c (0.5 μM and 1 μM) for 24 h.
Afterwards 500 μM H2O2 was added and the cells were incubated for 5 h.
After the culture medium was discarded, 10 μM DCFH-DA in fresh FBS-
free medium was added, the culture mixture of which was maintained at
◦
37 C for 30 min in dark. Afterwards, photos were obtained by using
4.2.7. Cellular TrxR activity determination by TRFS-Green
fluorescence microscope (Life Technology).
PC12 cells were bred to adhere for 24 h, and then were treated with
9b (0.5 μM and 1 μM) and 9c (0.5 μM and 1 μM) for further 24 h fol-
4.2.5. Real-time quantitative reverse transcription-polymerase chain
reaction (qRT-PCR)
lowed by addition of 10 μM TRFS-Green. 4 h later, the activity of TrxR
was determined and the photos were taken from a fluorescence micro-
scope (Life Technology).
PC12 cells were bred to adhere for 24 h followed by incubation of 9b
(1 μM) and 9c (1 μM) for indicated time. The treated cells at the indi-
cated time were used to extract the total RNA by using RNA extraction
kit (TaKaRa, China). Reverse transcription kit (TaKaRa, China) was
exploited to reversely transcribe the extracted RNA to cDNA. PCR
Master Mix (TaKaRa, China) was utilized for qRT-PCR analysis on the
Mx3005P RT-PCR system (Agilent Technologies). The sequences of
genes primers were the same as we used before [39–41].
4.2.8. Western blots analysis
The PC12 cells were treated with 9b (1 μM) and 9c (1 μM) for 24 h,
and then the cellular fractions from nuclei, cytoplasm and whole cells
were prepared following our published procedures [39–41]. The protein
expression levels were determined by standard Western blots analysis.
Briefly, the whole cell fraction was prepared by lysing PC12 cells with
RIPA buffer. The cellular fractions from nuclei and cytoplasm were
obtained by lysing PC12 cells with buffer A containing Hepes (10 mM,
4.2.6. Assessment of content of GSH, and activity of TrxR, NQO1, and HO-
1
pH 7.9), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 μM),
PC12 cells were bred to adhere for 24 h, and then they were incu-
followed by addition of 1 μM of protease inhibitor cocktail. 15 min later,
bated with 9b (0.5
μM and 1
μ
M) or 9c (0.5
μ
M and 1
μM) for 24 h. Then
Nonidet-P40 (10
μ
M) was added, and the mixture was vortexed for 15 s,
◦
the cells were collected and lysed to prepare cellular extracts for each
antioxidant species determination. The detailed information on the
procedure was based on our previous work [39–41].
followed by centrifugation for 10 min (1000 g, 4 C). Thereafter, the
cellular nuclear fractions were separated from the cytosolic part. The
pellet was resuspended in 100 μM of buffer B containing Hepes (20 mM,
For the determination of total GSH, an enzymatic method was
pH 7.9), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 μM), fol-
employed. Briefly, a solution (120
μL) of DTNB (0.33 mg/mL) and
lowed by addition of 1 μM of protease inhibitor cocktail. After incuba-
glutathione reductase (1.66 units/mL) was added to each cellular sam-
tion on ice for 15 min, the mixture was vortexed for 15 s every 2 min,
◦
ple (20
μL) followed by addition of NADPH (60
μL of 0.66 mg/mL). And
followed by centrifugation for 10 min (20000 g, 4 C), and the super-
the same amount of DMSO was added to the control. Then the absor-
bance of 412 nm was determined every 10 s for 2 min.
natant was collected as the cellular nuclear fractions. The total protein
content was quantified by Bradford method, and the protein samples
were loaded on SDS-PAGE for electrophoresis. Then the separated pro-
teins were transferred on PVDF membranes, followed by blocking with
5% nonfat milk for 1 h at room temperature. Afterwards the PVDF
membranes were incubated with primary antibodies overnight at 4 ◦C,
and then they were incubated with corresponding secondary antibodies
for 1 h at room temperature. Subsequently, they were incubated with the
enhanced chemiluminescence kit, and the bands were recorded and the
intensities were analyzed.
For the evaluation of cellular TrxR activity, the endpoint insulin
reduction assay was conducted and the total protein content was
quantified by Bradford method. Briefly, the cellular samples containing
20
insulin (0.3 mM), NADPH (660
M) in a final volume of 50 μ
L at 37 ◦C for 30 min. Then 200
μg of total proteins was incubated with Tris–HCl (100 mM, pH 7.6),
μ
M), EDTA (3 mM), and E. coli Trx (15
μ
μ
L of DTNB
(1 mM in 6 M guanidine hydrochloride, pH 8.0) was used to terminate
the reaction. The blank group was treated in the same manner without
addition of Trx, and the same amount of DMSO was added to the control
group. Then the absorbance of 412 nm was determined, and the blank
value was subtracted from the experimental group. The TrxR activity
was expressed as the percentage of the control.
4.2.9. Establishment of Nrf2 knockdown PC12 cell line
The detailed information for the generation of stable Nrf2 knock-
down PC12 cells were in the light of the methods published by our group
[39–41]. In short, mice Nrf2 gene was specifically interfered with
shNrf2-842, and shNT was used as a control. G418 (0.5 mg/mL) was
For the determination of cellular NQO1 activity, a spectrophoto-
metric method using 2, 6-dichlorophenol-indophenol (DCPIP) as an
14

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
[14] P.C. Trippier, K. Jansen Labby, D.D. Hawker, J.J. Mataka, R.B. Silverman, Target-
and mechanism-based therapeutics for neurodegenerative diseases: strength in
numbers, J. Med. Chem. 56 (2013) 3121–3147.
used to screened to generate stable PC12-shNrf2 cells and PC12-shNT
cells.
[15] C.J. Schmidlin, M.B. Dodson, L. Madhavan, D.D. Zhang, Redox regulation by NRF2
in aging and disease, Free Radical Biol. Med. 134 (2019) 702–707.
[16] M.J. Calkins, D.A. Johnson, J.A. Townsend, M.R. Vargas, J.A. Dowell, T.
P. Williamson, A.D. Kraft, J.M. Lee, J. Li, J.A. Johnson, The Nrf2/ARE pathway as a
potential therapeutic target in neurodegenerative disease, Antioxid. Redox
Signaling 11 (2009) 497–508.
4.2.10. ARE-luciferase reporter gene assay
This experiment was in accordance with the method established in
our recent published paper [38]. In a word, PC12 cells were transfected
with the pARE-luciferase plasmid (Beyotime, China), and then they were
screened with G418 (0.5 mg/mL) to generate a stable transfected cell
[17] A. Cuadrado, P. Moreno-Murciano, J. Pedraza-Chaverri, The transcription factor
Nrf2 as a new therapeutic target in Parkinson’s disease, Expert Opin. Ther. Targets
13 (2009) 319–329.
line. t-BHQ (20
μ
M and 40
μ
M, utilized as a positive control), 9b (0.5
μM
and 1 M), and 9c (0.5
μ
μ
M and 1 μ
M) were added to incubate with the
[18] S. Magesh, Y. Chen, L. Hu, Small molecule modulators of Keap1-Nrf2-ARE pathway
as potential preventive and therapeutic agents, Med. Res. Rev. 32 (2012) 687–726.
[19] M. Dodson, M.R. de la Vega, A.B. Cholanians, C.J. Schmidlin, E. Chapman, D.
D. Zhang, Modulating NRF2 in Disease: Timing Is Everything, Annu. Rev.
Pharmacol. Toxicol. 59 (2019) 555–575.
transfected cells for 24 h followed by preparation of cell samples. The
luciferase activity was determined with the aid of Firefly Luciferase
Reporter Gene Assay Kit (Beyotime, China).
[20] C.J. Schmidlin, M.B. Dodson, LalithaMadhavan, D.D. Zhang, Redox regulation by
NRF2 in aging and disease, Free Radical Biol. Med. 134 (2019) 702–707.
[21] T. Suzuki, H. Motohashi, M. Yamamoto, Toward clinical application of the Keap1-
Nrf2 pathway, Trends Pharmacol. Sci. 34 (2013) 340–346.
4.2.11. Statistics
Student’s t-test was used to check the difference between two groups,
and differences among multiple groups were checked by one-way
analysis of variance. Data are expressed as means ± standard devia-
tion (SD). It was considered to be statistically significant when P value is
< 0.05.
[22] A.J. Wilson, J.K. Kerns, J.F. Callahan, C.J. Moody, Keap calm, and carry on
covalently, J. Med. Chem. 56 (2013) 7463–7476.
[23] M.C. Lu, J.A. Ji, Z.Y. Jiang, Q.D. You, The Keap1-Nrf2-ARE Pathway As a Potential
Preventive and Therapeutic Target: An Update, Med. Res. Rev. 36 (2016) 924–963.
[24] Z.Y. Jiang, M.C. Lu, Q.D. You, Discovery and Development of Kelch-like ECH-
Associated Protein 1. Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2)
Protein-Protein Interaction Inhibitors: Achievements, Challenges, and Future
Directions, J. Med. Chem. 59 (2016) 10837–10858.
Declaration of Competing Interest
[25] Z.Y. Jiang, M.C. Lu, Q.D. You, Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)
Inhibition: An Emerging Strategy in Cancer Therapy, J. Med. Chem. 62 (2019)
3840–3856.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[26] J.S. Pallesen, K.T. Tran, A. Bach, Non-covalent Small-Molecule Kelch-like ECH-
Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2)
Inhibitors and Their Potential for Targeting Central Nervous System Diseases,
J. Med. Chem. 61 (2018) 8088–8103.
[27] D.D. Zhang, M. Hannink, Distinct cysteine residues in Keap1 are required for
Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by
chemopreventive agents and oxidative stress, Mol. Cell Biol. 23 (2003) 8137–8151.
[28] B. Halliwell, Oxidative stress and neurodegeneration: where are we now?
J. Neurochem. 97 (2006) 1634–1658.
Acknowledgements
Financial supports from the National Natural Science Foundation of
China (21778028, 22077055), the Natural Science Foundation of Gansu
Province (20JR5RA311, 18JR4RA003), and the 111 project are
acknowledged.
[29] B.J.J.o.n. Halliwell, Reactive oxygen species and the central nervous system,
J. Neurochem. 59 (1992) 1609–1623.
[30] M. Dodson, R. Castro-Portuguez, D.D. Zhang, NRF2 plays a critical role in
mitigating lipid peroxidation and ferroptosis, Redox Biol. 23 (2019).
[31] W. Hur, N.S. Gray, Small molecule modulators of antioxidant response pathway,
Curr. Opin. Chem. Biol. 15 (2011) 162–173.
Appendix A. Supplementary data
[32] P. Liu, W. Tian, S. Tao, J. Tillotson, E.M.K. Wijeratne, A.A.L. Gunatilaka, D.
D. Zhang, E. Chapman, Non-covalent NRF2 Activation Confers Greater Cellular
Protection than Covalent Activation, Cell Chem. Biol. 26 (2019) 1427–1435 e1425.
[33] K.M. Lee, T.Y. Kwon, U. Kang, E.K. Seo, J.H. Yun, C.W. Nho, Y.S. Kim,
Tussilagonone-induced Nrf2 pathway activation protects HepG2 cells from
oxidative injury, Food Chem. Toxicol. 108 (2017) 120–127.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104520.
[34] R. Joo Choi, M.S. Cheng, Y. Shik Kim, Desoxyrhapontigenin up-regulates Nrf2-
mediated heme oxygenase-1 expression in macrophages and inflammatory lung
injury, Redox Biol. 2 (2014) 504–512.
References
[1] B.C. Dickinson, C.J. Chang, Chemistry and biology of reactive oxygen species in
signaling or stress responses, Nat. Chem. Biol. 7 (2011) 504–511.
[2] M.P. Murphy, A. Holmgren, N.G. Larsson, B. Halliwell, C.J. Chang,
B. Kalyanaraman, S.G. Rhee, P.J. Thornalley, L. Partridge, D. Gems, T. Nystrom,
V. Belousov, P.T. Schumacker, C.C. Winterbourn, Unraveling the biological roles of
reactive oxygen species, Cell Metab. 13 (2011) 361–366.
[35] J. Lee, K. Song, E. Huh, M.S. Oh, Y.S. Kim, Neuroprotection against 6-OHDA
toxicity in PC12 cells and mice through the Nrf2 pathway by a sesquiterpenoid
from Tussilago farfara, Redox Biol. 18 (2018) 6–15.
[36] R.H. Scannevin, S. Chollate, M.Y. Jung, M. Shackett, H. Patel, P. Bista, W. Zeng,
S. Ryan, M. Yamamoto, M. Lukashev, K.J. Rhodes, Fumarates promote
cytoprotection of central nervous system cells against oxidative stress via the
nuclear factor (erythroid-derived 2)-like 2 pathway, J. Pharmacol. Exp. Ther. 341
(2012) 274–284.
[3] K.M. Holmstrom, T. Finkel, Cellular mechanisms and physiological consequences
of redox-dependent signalling, Nat. Rev. Mol. Cell Biol. 15 (2014) 411–421.
[4] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing,
Nature 408 (2000) 239–247.
[37] Z.L. Song, F. Bai, B. Zhang, J. Fang, Synthesis of Dithiolethiones and Identification
of Potential Neuroprotective Agents via Activation of Nrf2-Driven Antioxidant
Enzymes, J. Agric. Food Chem. 68 (2020) 2214–2231.
[5] K.J. Barnham, C.L. Masters, A.I. Bush, Neurodegenerative diseases and oxidative
stress, Nat. Rev. Drug Discov. 3 (2004) 205–214.
[38] Y. Hou, X. Li, S. Peng, J. Yao, F. Bai, J. Fang, Lipoamide Ameliorates Oxidative
Stress via Induction of Nrf2/ARE Signaling Pathway in PC12 Cells, J. Agric. Food
Chem. 67 (2019) 8227–8234.
[6] M. Rojo de la Vega, E. Chapman, D.D. Zhang, NRF2 and the Hallmarks of Cancer,
Cancer Cell 34 (2018) 21–43.
´
[7] J. Gracia-Sancho, M.J. Salvado, Gastrointestinal Tissue: Oxidative Stress and
[39] J. Yao, B. Zhang, C. Ge, S. Peng, J. Fang, Xanthohumol, a polyphenol chalcone
present in hops, activating Nrf2 enzymes to confer protection against oxidative
damage in PC12 cells, J. Agric. Food Chem. 63 (2015) 1521–1531.
[40] J. Yao, C. Ge, D. Duan, B. Zhang, X. Cui, S. Peng, Y. Liu, J. Fang, Activation of the
phase II enzymes for neuroprotection by ginger active constituent 6-dehydrogin-
gerdione in PC12 cells, J. Agric. Food Chem. 62 (2014) 5507–5518.
[41] S. Peng, B. Zhang, X. Meng, J. Yao, J. Fang, Synthesis of piperlongumine analogues
and discovery of nuclear factor erythroid 2-related factor 2 (Nrf2) activators as
potential neuroprotective agents, J. Med. Chem. 58 (2015) 5242–5255.
[42] S. Peng, Y. Hou, J. Yao, J. Fang, Activation of Nrf2 by costunolide provides
neuroprotective effect in PC12 cells, Food Funct. 10 (2019) 4143–4152.
[43] S. Peng, Y. Hou, J. Yao, J. Fang, Activation of Nrf2-driven antioxidant enzymes by
cardamonin confers neuroprotection of PC12 cells against oxidative damage, Food
Funct. 8 (2017) 997–1007.
Dietary Antioxidants, Academic Press, 2017.
[8] T.R. Dugas, Unraveling mechanisms of toxicant-induced oxidative stress in
cardiovascular disease, Curr. Opin. Toxicol. 7 (2018) 1–8.
[9] J.N. Cobley, M.L. Fiorello, D.M. Bailey, 13 reasons why the brain is susceptible to
oxidative stress, Redox Biol. 15 (2018) 490–503.
[10] H.E. de Vries, M. Witte, D. Hondius, A.J. Rozemuller, B. Drukarch, J. Hoozemans,
J. van Horssen, Nrf2-induced antioxidant protection: a promising target to
counteract ROS-mediated damage in neurodegenerative disease? Free Radical Biol.
Med. 45 (2008) 1375–1383.
[11] R. Franco, J.A. Doorn, J.-C. Rochet, Oxidative stress and redox signalling in
Parkinson’s disease, Royal Society of Chemistry, 2017.
[12] S. Fahn, G. Cohen, The oxidant stress hypothesis in Parkinson’s disease: evidence
supporting it, Ann. Neurol. 32 (1992) 804–812.
[13] F.L. van Muiswinkel, H.B. Kuiperij, The Nrf2-ARE Signalling Pathway: Promising
Drug Target to Combat Oxidative Stress in Neurodegenerative Disorders, Curr.
Drug Targets: CNS Neurol. Disord. 4 (2005) 267–281.
15

Z.-L. Song et al.
Bioorganic Chemistry xxx (xxxx) xxx
[44] S. Peng, J. Yao, Y. Liu, D. Duan, X. Zhang, J. Fang, Activation of Nrf2 target
enzymes conferring protection against oxidative stress in PC12 cells by ginger
principal constituent 6-shogaol, Food Funct. 6 (2015) 2813–2823.
[45] S. Peng, B. Zhang, J. Yao, D. Duan, J. Fang, Dual protection of hydroxytyrosol, an
olive oil polyphenol, against oxidative damage in PC12 cells, Food Funct. 6 (2015)
2091–2100.
[51] V. Kumar, S. Kumar, M. Hassan, H. Wu, R.K. Thimmulappa, A. Kumar, S.K. Sharma,
V.S. Parmar, S. Biswal, S.V. Malhotra, Novel chalcone derivatives as potent Nrf2
activators in mice and human lung epithelial cells, J. Med. Chem. 54 (2011)
4147–4159.
[52] J.W. Choi, S. Kim, J.H. Park, H.J. Kim, S.J. Shin, J.W. Kim, S.Y. Woo, C. Lee, S.
M. Han, J. Lee, A.N. Pae, G. Han, K.D. Park, Optimization of Vinyl Sulfone
Derivatives as Potent Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators
for Parkinson’s Disease Therapy, J. Med. Chem. 62 (2019) 811–830.
[53] X. Ning, Y. Guo, X. Wang, X. Ma, C. Tian, X. Shi, R. Zhu, C. Cheng, Y. Du, Z. Ma,
Z. Zhang, J. Liu, Design, synthesis, and biological evaluation of (e)-3,4-
dihydroxystyryl aralkyl sulfones and sulfoxides as novel multifunctional
neuroprotective agents, J. Med. Chem. 57 (2014) 4302–4312.
[46] F. Bai, B. Zhang, Y. Hou, J. Yao, Q. Xu, J. Xu, J. Fang, Xanthohumol Analogues as
Potent Nrf2 Activators against Oxidative Stress Mediated Damages of PC12 Cells,
ACS Chem. Neurosci. 10 (2019) 2956–2966.
[47] Y. Hou, S. Peng, X. Li, J. Yao, J. Xu, J. Fang, Honokiol Alleviates Oxidative Stress-
Induced Neurotoxicity via Activation of Nrf2, ACS Chem. Neurosci. 9 (2018)
3108–3116.
[48] M. Dumont, E. Wille, N.Y. Calingasan, D. Tampellini, C. Williams, G.K. Gouras,
K. Liby, M. Sporn, C. Nathan, M. Flint Beal, M.T. Lin, Triterpenoid CDDO-
methylamide improves memory and decreases amyloid plaques in a transgenic
mouse model of Alzheimer’s disease, J. Neurochem. 109 (2009) 502–512.
[49] B. Wei, J. Zhou, J.J. Xu, J. Cui, F.F. Ping, J.J. Ling, Y.J. Chen, Discovery of
coumarin-derived imino sulfonates as a novel class of potential cardioprotective
agents, Eur. J. Med. Chem. 184 (2019).
[54] S.Y. Woo, J.H. Kim, M.K. Moon, S.H. Han, S.K. Yeon, J.W. Choi, B.K. Jang, H.
J. Song, Y.G. Kang, J.W. Kim, J. Lee, D.J. Kim, O. Hwang, K.D. Park, Discovery of
vinyl sulfones as a novel class of neuroprotective agents toward Parkinson’s disease
therapy, J. Med. Chem. 57 (2014) 1473–1487.
[55] C. Malagelada, L.A. Greene, PC12 cells as a model for Parkinson’s disease research
(Chapter 29 in Parkinson’s Disease), Elsevier, 2008, 375-387.
[56] L. Zhang, D. Duan, Y. Liu, C. Ge, X. Cui, J. Sun, J. Fang, Highly selective off-on
fluorescent probe for imaging thioredoxin reductase in living cells, J. Am. Chem.
Soc. 136 (2014) 226–233.
[50] R. Kachadourian, B.J. Day, S. Pugazhenti, C.C. Franklin, E. Genoux-Bastide,
G. Mahaffey, C. Gauthier, A. Di Pietro, A. Boumendjel, A synthetic chalcone as a
potent inducer of glutathione biosynthesis, J. Med. Chem. 55 (2012) 1382–1388.
16