Design, synthesis, and biological evaluation of (E)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides as novel multifunctional neuroprotective agents

Article abstract of DOI:10.1021/jm500258v

Novel (E)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides were designed and synthesized as new analogues of 1, which showed interesting multifunctional neuroprotective effects, including antioxidative and antineuroinflammatory properties. Specifically, target compounds display excellent potency in scavenging reactive free radicals and demonstrate potent effects against various kinds of toxicities, including H₂O₂, 6-hydroxydopamine, and lipopolysaccharide in different types of neuronal cells. The antioxidative properties of the target compounds are more potent than that of 1, and the antineuroinflammatory properties are less strong than that of 1. According to the parallel artificial membrane permeation assay for the blood-brain barrier, target compounds possess greater blood-brain barrier (BBB) permeability than 1. In short, due to improvement of the antioxidative effect, stability, and BBB permeability, (E)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides can thus be considered as potential multifunctional neuroprotective agents and serve as new lead candidates in the treatment of neurodegenerative diseases.

Full text of DOI:10.1021/jm500258v

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of (E)‑3,4-
Dihydroxystyryl Aralkyl Sulfones and Sulfoxides as Novel
Multifunctional Neuroprotective Agents
Xianling Ning,† Ying Guo,† Xiaowei Wang,† Xiaoyan Ma,† Chao Tian,† Xueqi Shi,† Renzong Zhu,†
Can Cheng,∥ Yansheng Du,⊥ Zhizhong Ma,§ Zhili Zhang,*,† and Junyi Liu*,†,‡
†Department of Chemical Biology, School of Pharmaceutical Sciences, ‡State Key Laboratory of Natural and Biomimetic Drugs, and
§Department of the Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing
100191, China
∥Department of Pharmaceutical Chemistry, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
⊥Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States
S
* Supporting Information
ABSTRACT: Novel (E)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides were designed
and synthesized as new analogues of 1, which showed interesting multifunctional
neuroprotective effects, including antioxidative and antineuroinflammatory properties.
Specifically, target compounds display excellent potency in scavenging reactive free radicals
and demonstrate potent effects against various kinds of toxicities, including H2O2, 6-
hydroxydopamine, and lipopolysaccharide in different types of neuronal cells. The
antioxidative properties of the target compounds are more potent than that of 1, and the
antineuroinflammatory properties are less strong than that of 1. According to the parallel
artificial membrane permeation assay for the blood−brain barrier, target compounds
possess greater blood−brain barrier (BBB) permeability than 1. In short, due to
improvement of the antioxidative effect, stability, and BBB permeability, (E)-3,4-
dihydroxystyryl aralkyl sulfones and sulfoxides can thus be considered as potential
multifunctional neuroprotective agents and serve as new lead candidates in the treatment
of neurodegenerative diseases.
small molecule that possesses multifunctional activities acting
on more than one biological target has to be studied or
designed, which can reduce side effects and increase the
therapeutic index.
INTRODUCTION
■
Progressive neurodegenerative disorders, in which the nervous
system progressively and irreversibly deteriorates, such as
Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s (HD)
disease as well as amyotrophic lateral sclerosis (ALS), greatly
reduce the life quality of patients.1 Mechanisms of neuro-
degenerative disorders are complex, involving a lot of known
and unknown signaling cascades, oxidative and nitrosative
stress, inflammation, apoptosis, mitrochondrial injury, and
many more events.2 For example, oxidative stress induced by
overproduction of reactive oxygen species leads to injury of
essential components in neural cells.3 Inflammation is also an
important pathogenic factor to neurodegenerative disorders.
This process plays a vital role for normal function in the central
nervous system (CNS), but it may increase sharply with
overactivation of microglia and overproduction of proinflam-
matory cytokines and mediators such as cyclooxygenase-2
(COX-2), inducible nitric oxide synthase (iNOS), and tumor
necrosis factor α (TNF-α) that eventually give rise to cell
injury.4 Thus, the drug with a single-target mechanism of action
cannot always compensate for these complex pathways. The
strategy of a “cocktail” of two or more drugs which has been
applied in the clinical treatment has several defects, such as
multiple toxicities and side effects of the drugs.5 Therefore, a
1 (caffeic acid phenethyl ester, CAPE) (Figure 1) as the
active component of propolis of honeybee hives has been
shown to have antioxidative,6 anti-inflammatory,7 antiviral,8
antibacterial,9 antiatherosclerotic,10 immunostimulatory,11 and
antitumor12 properties. The antioxidative property of 1 is
related to repressing production of reactive oxygen species and
the xanthine/xanthine oxidase system,13 and the anti-inflam-
matory property is reflected through lessening leukotriene and
prostaglandin synthesis by suppressing cyclooxygenase enzyme
activity and its gene expression.14 It is speculated that both the
antioxidative and antiinflammatory properties of 1 are
conducive to its neuroprotective effects. Recent studies have
proved the effects of 1 against various toxicities in neural cells,
such as hydrogen peroxide (H2O2),15 6-hydroxydopamine (6-
OHDA),15 and glutamate.16 It is also demonstrated that 1 is
able to prevent hypoxia−ischemic brain damage17 and MPTP-
induced dopaminergic neurotoxicity18 in animal models. In
Received: February 17, 2014
Published: April 3, 2014
© 2014 American Chemical Society
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Figure 1. Structures of 1, (E)-3,4-dihydroxystyryl aralkyl ketones, and target compounds.
addition, these studies indicate that neuroprotective effects of 1
are relevant to suppression of oxidative damage, inflammation,
apoptosis, activation of caspase-3, phosphorylation of p38, and
release of mitochondrial cytochrome c.15−19 On the basis of the
above evidence, it is believed that 1 is a potent neuroprotective
agent with multiple target effects.
Table 1. In Silico log P Values of Compounds
compd
X
Y
log P
Many preclinical studies have proved that 1 possesses
primary safety properties and bioactivity in vitro and in animal
models, but pharmacokinetic studies indicate that 1 as a
carboxylic ester is remarkably decomposed to caffeic acid by
esterases in rats after the quick oral absorption.20 Therefore, in
this study we design and synthesize 1 analogues with better
stability and research their structure−activity relationships.
According to previous studies, 1 seems to reflect the
neuroprotective effect via its catechol ring functionality that
offers free radical scavenging and antioxidant effects and the
double bond of the side chain that increases the stabilization of
the phenolic radical.21 Therefore, (E)-3,4-dihydroxystyryl group
is reserved in the structure of designed target compounds. In
our laboratory, (E)-3,4-dihydroxystyryl aralkyl ketones (Figure
1) were designed and synthesized which reserved the (E)-3,4-
dihydroxystyryl group and introduced the ketone group instead
of the unstable ester group.22 The (E)-3,4-dihydroxystyryl
aralkyl ketones showed potential neuroprotective effects,
including greater anti-inflammatory effects than 1, which
indicated that enhancing stability was conducive to the increase
of the neuroprotective effect.22 To further investigate new
analogues of 1 and enhance the neuroprotective effect, sulfone
and sulfoxide analogues of 1 and (E)-3,4-dihydroxystyryl aralkyl
sulfone and sulfoxide (Figure 1) were designed, in which the
SO double bond in the sulfone and sulfoxide groups had
higher polarity than the CO double bond. Then we used the
Molinspiration property prediction software to predict partition
coefficient log P values of designed target compounds. The log
P value has a strong influence on the absorption, distribution,
metabolism, and excretion (ADME) properties of the drug, so
it is a major determinant of druggability. log P is also related to
crossing the blood−brain barrier, which is especially important
to neuroprotective agents. The predicted results of 1 and its
ketone, sulfone, and sulfoxide analogues are listed in Table 1,
which reveal that log P values of sulfone and sulfoxide
analogues of 1 are within the optimum range between 2 and 3.
Thus, sulfone and sulfoxide analogues of 1 display better
potential druggability than 1 and its ketone analogues. Some
currently clinical pharmaceuticals that have sulfoxide or sulfone
moieties, such as acedapsone and solasulfone, were first used as
antibacterials. Recently, styryl sulfones and sulfoxides were
found to have potential antitumor effects, and previous acute
toxicity studies in rats reveal that no detectable toxicity could be
caused by styryl aralkyl sulfone and sulfoxide analogues.23 In
1
CO
O
C
C
C
3.362
3.457
3.076
2.673
(E)-3,4-dihydroxystyryl aralkyl ketone
CO
5c
7c
OSO
SO
addition, the styryl sulfone and sulfoxide function is often
present in the skeletal structures of many promising neuro-
protective agents, including 5-HT6 receptor antagonists.24,25
Therefore, sulfone and sulfoxide analogues of 1 probably
become novel neuroprotective agents with high efficacy and
low toxicity. To elucidate the structure−activity relationships,
compounds with alkyl chains of different lengths and various
substituted groups on the aromatic ring were also designed.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic routes for designed compounds
(E)-3,4-dihydroxystyryl aralkyl sulfones 5a−h and sulfoxides
7a−h are respectively shown in Schemes 1 and 2.
Commercially available substituted aralkyl bromides 2a−h
served as starting materials reacted with mercaptoacetic acid in
methanol to provide key intermediates (aralkylsulfanyl)acetic
acids 3a−h under the catalysis of NaOH with excellent yields
(approximately 90%). The intermediates 3a−h were selectively
oxidized with 30% H2O2 aqueous solution at room temperature
to (aralkylsulfonyl)acetic acids 4a−h in acetic acid and to
(aralkylsulfinyl)acetic acids 6a−h in methanol, which is
probably because acetic acid can react with H2O2 to get
peroxyacetic acid with strong oxidizing capacities. Then
Knoevenagel condensation of the (aralkylsulfonyl)acetic acids
4a−h or (aralkylsulfinyl)acetic acids 6a−h with 3,4-dihydrox-
ybenzaldehyde using pyrrolidine and acetic acid as catalysts in
refluxed THF followed by chromatographic purification gave
(E)-3,4-dihydroxystyryl aralkyl sulfones 5a−h and sulfoxides
7a−h in yields of about 50% and 70%. Besides 5a−h, methyl
aralkyl sulfones as decarboxylated products of 4a−h were
detected in the reaction because of 4a−h having a higher
methylene activity than 6a−h. Therefore, β-aminopropionic
acid was used instead of pyrrolidine/acetic acid to increase the
condensation yields (about 80%) with 3,4-dihydroxybenzalde-
hyde by reducing the side reactions.26 The desired E geometry
of the target compounds was confirmed by the coupling
constants (J ≈ 16 Hz), and no Z geometry compounds were
separated in this reaction.
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a
Scheme 1. Synthesis of (E)-3,4-Dihydroxystyryl Aralkyl Sulfones
a
Reagents and conditions: (a) HSCH2COOH, NaOH, MeOH, rt, 84−93%; (b) 30% H2O2, acetic acid, rt, 70−87%; (c) 3,4-dihydroxybenzaldehyde,
β-aminopropionic acid, THF, reflux, 72−95%.
a
Scheme 2. Synthesis of (E)-3,4-Dihydroxystyryl Aralkyl Sulfoxides
a
Reagents and conditions: (d) 30% H2O2, MeOH, rt, 75−86%; (e) 3,4-dihydroxybenzaldehyde, pyrrolidine, acetic acid, THF, reflux, 60−75%.
Figure 2. Injuries of H2O2 and 6-OHDA to neuronal cells and LPS to microglia cells can contribute to neurodegeneration.
Biological Evaluation. The neuroprotective properties of
two series of target compounds were assessed through the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability
model and antioxidative and anti-inflammatory pharmacological
models on neural cells, including the neuronal protecting effect
against damage induced by H2O2 and 6-OHDA and nitric oxide
production inhibiting effect. The pathogenic mechanisms of
H2O2, 6-OHDA, and LPS as neurotoxins are shown in Figure 2.
H2O2 can generate exogenous free radicals, which are highly
reactive species that are capable of lipid, protein, and DNA
damage. 6-OHDA induces neuronal death via uncoupling
mitochondrial oxidative phosphorylation, resulting in energy
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deprivation, which is related to its ability to produce H2O2,
hydroxyl, and superoxide radicals. LPS activates microglia cells,
and the uncontrolled activation of microglia cells may result in
neuronal damage by the overproduction of NO, ROS, and
cytotoxic cytokines. These neurotoxins are able to induce
apoptosis and eventually lead to neurodegeneration.27
In addition, a parallel artificial membrane permeation assay
for the blood−brain barrier (PAMPA-BBB) was used to predict
BBB permeability of target compounds. The results showed
that two series of target compounds showed higher
antioxidative activities and stronger BBB permeability com-
pared with 1, and the primary structure−activity relationship
was also discussed.
Free Radical Scavenging Ability. Reactive species have
also been identified to be closely related to neurodegeneration.
Antioxidants can react with reactive species to invalidate them
and are of interest as potential therapeutics. DPPH as a stable
free radical existing in vitro can usually be used in preliminary
screening of compounds with the capability of scavenging
reactive free radicals. The free radical scavenging abilities of
target compounds were determined by the published method28
over the concentration range of 1−50 μM. The results are
shown in Table 2. Vitamin C is a common antioxidant, which is
Neuronal Protection Effect against Damage Induced
by H2O2. Cellular damage in the nervous system by free radical
species has been implicated in AD, PD, HD, and ALS.29 To
further confirm the antioxidant properties of target compounds
in neural cells, target compounds 5a−h and 7a−h were also
tested by the H2O2 model on PC12 cells. H2O2 can generate
exogenous free radicals, which are highly reactive species that
are capable of lipid, protein, and DNA damage (Figure 2).30
PC12 cells generally as a screening model for studying
neurodegenerative diseases were used in this test.31 The
protection effect against H2O2 can be determined by the cell
viability through an MTT assay. The cell viabilities attributable
to the protective efficacy of target compounds against H2O2 at
5 μM are shown in Figure 3. To further evaluate and compare
Table 2. Free Radical Scavenging Abilities of 1, 5a−h, and
a
7a−h by the DPPH Method
Figure 3. Protective effects of 1, 5a−h, and 7a−h against H2O2-
induced injury in PC12 cells at 5 μM. PC12 cells were pretreated by
the tested compounds for 3 h. Then the cells were treated with 500
μM H2O2 for 5 h. Cell viability was determined by the MTT assay.
The viability of untreated cells is defined as 100%. Data are expressed
as the mean SD, n = 3.
compd
IC50 (μM)
compd
7a
IC50 (μM)
5a
5b
5c
5d
5e
5f
8.1 0.2
10.5 0.3
9.9 0.2
17.2 0.4
8.8 0.1
9.5 0.4
5.8 0.3
9.1 0.2
12.1 0.3
14.7 0.4
11.6 0.2
14.4 0.3
16.2 0.5
8.1 0.1
9.9 0.2
7.8 0.2
11.5 0.3
25.7 0.5
7b
7c
7d
7e
7f
the activities, the protective efficacy of target compounds
possessing prominent activities in Figure 3 at 2.5, 5, and 10 μM
were also tested and are listed in Figure 4. Almost all of the
5g
5h
1
7g
7h
vitamin C
a
Data are expressed as the mean SD, n = 3.
usually used as the positive control in the DPPH model.
Compounds 1, 5a−h, and 7a−h exhibit significant free radical
quenching abilities, when compared with vitamin C. The
possible cause is that the conjugated system consisting of a
catechol group, a double bond, or an ester, a sulfone, or a
sulfoxide group in 1, 5a−h, and 7a−h can provide better
electron delocalization than vitamin C, and these compounds as
better hydrogen donors more easily scavenge free radicals. It is
observed that the majority of target compounds, except 5d, 7a,
7c, and 7d, show similar or stronger free radical scavenging
capacities compared to 1. Especially 5g (IC50 = 5.8 0.3 μM)
exhibits optimal radical scavenging activity among the
compounds that is 2-fold higher than that of 1 (IC50 = 12.1
0.3 μM). On the whole, activities of sulfones 5a−h are
greater than those of sulfoxides 7a−h. The reason may be that
sulfones can offer better electron delocalization for sulfoxides.
5d (IC50 = 17.2 0.4 μM) and 7d (IC50 = 16.2 0.5 μM) with
four-carbon alkyl chains show weaker free radical quenching
abilities compared with 5a−c and 7a−c with one- to three-
carbon alkyl chain. It is also notable that electron-withdrawing
trifluoromethyl (5g and 7g) and chloro (5e and 7e) substituted
compounds show more effective activities than electron-
donating tert-butyl (5f and 7f) and methoxyl (5h and 7h)
substituted compounds.
Figure 4. Protective effects of 1 and target compounds with prominent
activities at 5 μM, 5b, 5c, 5e, 5f, 7c, 7e, 7f, and 7g, against H2O2-
induced injury in PC12 cells at 2.5, 5, and 10 μM. PC12 cells were
pretreated by the tested compound for 3 h. Then the cells were treated
with 500 μM H2O2 for 5 h. Cell viability was determined using the
MTT assay. The viability of untreated cells is defined as 100%. Data
are expressed as the mean SD, n = 3.
target compounds significantly show protection effects against
damage induced by H2O2 at 5 μM as shown in Figure 2.
Especially 5c and 5f display approximately 2-fold higher
activities than 1 at 2.5 and 5 μM as shown in Figure 3. On
the whole, activities of sulfones 5a−h are higher than those of
sulfoxides 7a−h, which is consistent with the result of the
DPPH model. It seems that chloro (5e and 7e), tert-butyl (5f
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and 7f), and trifluoromethyl (5g and 7g) substituted
compounds display more effective activities compared with
unsubstituted (5a and 7a) and methoxyl substituted (5h and
7h) compounds.
compared to 1 as shown in Figure 6. Sulfones do not reveal
higher protection effects against 6-OHDA than sulfoxides. It is
also seen that electron-withdrawing chloro (5e and 7e) and
trifluoromethyl (5g and 7g) substituted compounds exhibit
more protection effects than unsubstituted (5a and 7a) and
electron-donating tert-butyl (5f and 7f) and methoxyl (5h and
7h) substituted compounds, which is completely consistent
with the result of the DPPH model and is basically consistent
with the result of the H2O2 model. The tert-butyl substituted
compounds (5f and 7f) show weak activity in this model, but
significant activity in the H2O2 model. The different results of
the two models are likely caused by the different mechanisms,
and additional studies will be carried out to clarify this
phenomenon.
Inhibiting Nitric Oxide Production of LPS-Stimulated
BV2 Microglial Cells. Activated microglial cells can release an
inflammatory mediator, including nitric oxide, the over-
production of which can result in neural cell death in
neurodegenerative disorders (Figure 2).32 Therefore, detecting
the suppression effect of NO production is an effective method
to evaluate the antineuroinflammatory properties of target
compounds 5a−h and 7a−h. The suppression effect of NO
production was measured by the Griess assay.33 The results are
listed in Table 3. Some target compounds show significant
Neuronal Protection Effect against Damage Induced
by 6-OHDA. PD is a neurodegenerative disease characterized
by progressive loss of dopaminergic (DA) neurons of the
substantia nigra pars compacta. 6-OHDA has been widely used
to generate PD-like models. It induces neuronal death via
uncoupling mitochondrial oxidative phosphorylation, resulting
in energy deprivation, which is related to its ability to produce
H2O2, hydroxyl, and superoxide radicals (Figure 2).19 Recently,
it has been shown that 6-OHDA is also able to induce
apoptosis in various cell types. Therefore, the 6-OHDA model
was used to evaluate the effects of target compounds on PD.
The protection effects against 6-OHDA can be assessed by the
cell viability using the MTT assay. The cell viabilities
attributable to the protective efficacy of target compounds
against 6-OHDA at 40 μM are listed in Figure 5, and those of
Table 3. Nitric Oxide Production Inhibiting Effect of 1, 5a−
a
h, and 7a−h in LPS-Stimulated BV2 Microglial Cells
compd
IC50 (μM)
compd
IC50 (μM)
5a
5b
5c
5d
5e
5f
11.6 0.4
30.6 0.3
10.5 0.1
19.8 0.3
11.7 0.2
12.6 0.5
15.9 0.2
11.9 0.2
6.4 0.4
7a
7b
7c
7d
7e
7f
50.3 0.4
42.6 0.6
28.6 0.2
36.8 0.4
14.0 0.1
9.3 0.2
Figure 5. Protective effects of 1, 5a−h, and 7a−h against 6-OHDA-
induced injury in PC12 cells at 40 μM. PC12 cells were pretreated by
the tested compound for 3 h. Then the cells were treated with 400 μM
6-OHDA for 48 h. Cell viability was determined by the MTT assay.
The viability of untreated cells is defined as 100%. Data are expressed
as the mean SD, n = 3.
5g
5h
1
7g
7h
15.5 0.3
33.0 0.5
a
Data are expressed as the mean SD, n = 3.
target compounds showing pointed activities at 5, 10, and 40
μM are listed in Figure 6. Except 5f and 7f, target compounds
significantly display protection effects against damage induced
by 6-OHDA at 40 μM as shown in Figure 5. It is observed that
5g and 7b show more remarkable activities at 5 and 10 μM
inhibition effects of NO production in LPS-stimulated BV2
cells, although they do not display higher activities than 1. By
and large, sulfones 5a−h possess stronger inhibition effects of
NO production than sulfoxides 7a−h, which is consistent with
the result of the H2O2 model. It seems that substituted
compounds show similar effective activities compared with
unsubstituted compounds for sulfones, but higher effective
activities than unsubstituted compounds for sulfoxides. Besides,
in all cases cell viabilities are more than 90% by the MTT assay
in this experiment, so the selected concentrations of target
compounds do not result in any prominent cytotoxicity, and to
a certain extent, target compounds exhibit relatively low toxicity
to neural cells. It is also confirmed that the inhibition effect of
NO production in LPS-stimulated BV2 microglial cells is not a
result of the cytotoxicity of target compounds.
In Vitro Evaluation of the BBB Permeability. BBB
penetration capacity is important for the effectiveness of CNS
drugs. The parallel artificial membrane permeability assay
(PAMPA) as a high-throughput technique was used to study
the penetration of target compounds into BBB. The in vitro
permeability (Pe) of target compounds 5a−h and 7a−h and
control drugs verapamil, hydrocortisone, and clonidine through
the porcine polar brain lipid were detected using PBS as the
Figure 6. Protective effects of 1 and target compounds showing
pointed activities, 5b, 5c, 5e, 5g, 7b, 7c, 7e, and 7g, against 6-OHDA-
induced injury in PC12 cells at 5, 10, and 40 μM. PC12 cells were
pretreated by the tested compound for 3 h. Then the cells were treated
with 400 μM 6-OHDA for 48 h. Cell viability was determined by the
MTT assay. The viability of untreated cells is defined as 100%. Data
are expressed as the mean SD, n = 3.
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a
Table 4. Permeability Results of 1, Target Compounds, and Control Drugs from the PAMPA-BBB Assay
compd
5a
Pe (10−6 cm s−1
)
prediction
compd
Pe (10−6 cm s−1
)
prediction
10.1 0.5
5.7 0.2
4.1 0.2
4.1 0.3
3.3 0.2
4.7 0.5
4.3 0.2
6.4 0.4
1.8 0.3
16.9 0.4
CNS+
CNS+
CNS+
CNS+
CNS
7a
7b
7c
7d
7e
7f
4.9 0.5
5.0 0.2
9.1 0.6
4.5 0.4
4.1 0.2
5.8 0.8
4.2 0.3
4.3 0.2
1.8 0.2
5.2 0.5
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS-
5b
5c
5d
5e
5f
CNS+
CNS+
CNS+
CNS-
CNS+
5g
7g
7h
5h
1
hydrocortisone
clonidine
verapamil
CNS+
a
Data are expressed as the mean SD, n = 3. PBS was used as the solvent.
solvent in accordance with the method described by Di et al.34
According to the following ranges for BBB permeation
prediction, the compounds can be classified: Pe (10−6 cm
s−1) > 4.0, “CNS+” (high BBB permeation); Pe (10−6 cm s−1) <
2.0, “CNS−” (low BBB permeation); Pe (10−6 cm s−1) from 4.0
to 2.0, “CNS ” (BBB permeation uncertain). From the results
in Table 4, Pe values of control drugs are in accord with the
results of Di et al., which indicates that the assay used by us is
reliable. On the bsais of the results in Table 4 and the ranges
above, we can consider that the majority of target compounds
are able to cross the BBB [Pe (10−6 cm s−1) > 4] and only 5e [2
< Pe (10−6 cm s−1) < 4] exhibits weaker permeability.
group chloro and trifluoromethyl substituted compounds show
more effective neuroprotective effects compared with unsub-
stituted and electron-donating group methoxyl substituted
compounds in almost all tested models. It is also notable that
tert-butyl substituted compounds display similar NO inhibition
effects and protection effects against damage induced by H2O2,
but weaker DPPH free radical scavenging capacities and
protection effects against damage induced by 6-OHDA
compared to electron-withdrawing group chloro and trifluor-
omethyl substituted compounds. The above results indicate
that neuropretective activities of target compounds are closely
related to substituted groups on the aromatic ring; thus, the
electronic or steric hindrance effect on the aromatic ring has an
impact on biological activity.
Compared to 1 (Pe = 1.8
0.3 × 10−6 cm s−1), all target
compounds possess greater BBB permeability. Therefore, the
BBB permeability of target compounds with sulfone and
sulfoxide groups instead of a carboxylic ester group is improved.
Above all, due to improvement of the activity, stability, and
BBB permeability, (E)-3,4-dihydroxystyryl aralkyl sulfones and
sulfoxides can thus be considered as potential multifunctional
neuroprotective agents and serve as new lead candidates in the
treatment of neurodegenerative diseases.
CONCLUSION
■
There has been growing interest in the neuroprotective activity
of 1 and its derivatives in recent years. In our previous studies,
(E)-3,4-dihydroxystyryl aralkyl ketones as ketone derivatives of
1 showed greater antineuroinflammatory.
EXPERIMENTAL SECTION
■
Chemistry. The structural characterization of compounds was
performed by NMR and high-resolution mass spectrometry (HRMS).
NMR spectra were recorded on a Bruker AVANCE III-400
spectrometer with tetramethylsilane (Me4Si) as an internal standard,
and chemical shifts are reported in δ (ppm). HRMS spectra were
recorded on a Bruker Apex IV FTMS spectrometer. The purity of
target compounds was determined by HPLC (Agilent 1260 infinity
HPLC system). All assayed compounds displayed a purity ≥95%
(Table S1 in the Supporting Information). All reactions were
monitored by TLC, which was performed on precoated silica gel
F254 plates. Detection was done by iodine vapor staining and UV light
irradiation (UV lamp, model UV-IIB). Column chromatography was
performed with silica gel H (200−300 or 500 mesh). Melting points
were determined on an X4-type apparatus and were uncorrected.
Unless otherwise stated, all reagents were purchased from commercial
sources. If necessary, they were purified and dried by standard
methods. Organic solutions were dried over anhydrous sodium sulfate.
General Procedure for the Synthesis of (Aralkylsulfanyl)-
acetic Acids 3a−h. The aralkyl bromide (5 mmol) was added to a
solution of mercaptoacetic acid (5 mmol) in methanol (15 mL) and
the resulting solution stirred. Then a solution of NaOH (10 mmol) in
methanol (5 mL) was added slowly, and the final mixture was stirred
at rt until absence of the aralkyl bromide (checked by TLC). The
reaction mixture was concentrated in vacuo, diluted with H2O, and
neutralized with HCl (1 mol/L). Then the obtained reaction mixture
was extracted with ethyl acetate. The combined organic fractions were
washed with brine, dried (Na2SO4), and concentrated under reduced
pressure. Purification of the crude residue by column chromatography
(petroleum ether/ethyl acetate) afforded the title compound.
In this work, we continued our study on the CO position
of 1. According to the property prediction, log P values of
sulfone and sulfoxide analogues of 1 are within the optimum
range between 2 and 3. Therefore, we synthesized novel (E)-
3,4-dihydroxystyryl aralkyl sulfones and sulfoxides and tested
their multifunctional neuroprotective effects, including anti-
oxidative and antineuroinflammatory properties. Specifically,
target compounds display excellent potency in scavenging
reactive free radicals and demonstrate potent effects against
various kinds of toxicities which are important elements taking
part in pathogenesis of neurodegenerative disorders. The
antioxidative properties of the target compounds are more
potent than that of (E)-3,4-dihydroxystyryl aralkyl ketones and
1. In addition, sulfone derivatives possess stronger neuro-
protective effects than sulfoxide derivatives. According to the
PAMPA-BBB assay, the target compounds possess greater BBB
permeability than 1.
On the basis of the results of our study, the compounds to be
highlighted are 5c and 5g showing a high degree of
multifunctional activities related to neuroprotective effects.
The activities of compounds possessing various lengths of alky
chains (n = 1−4) have no obvious regularity in the H2O2, 6-
OHDA, and NO models; however, compounds possessing
longer alkyl chains, 5d and 7d, show weaker free radical
scavenging capacities compared with shorter alkyl chain
compounds in the DPPH model. The electron-withdrawing
4307
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Data for (benzylsulfanyl)acetic acid (3a): white solid (93%); mp
62−63 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H, COOH),
7.24−7.35 (m, 5H, ArH), 3.81 (s, 2H, SCH2COOH), 3.12 (s, 2H,
ArCH2S).
Data for (phenethylsulfanyl)acetic acid (3b): white solid (85%);
mp 52−53 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.53 (s, 1H,
COOH), 7.21−7.30 (m, 5H, ArH), 3.27 (s, 2H, SCH2COOH), 2.84
(s, 4H, SCH2CH2Ar).
Data for ((4-tert-butylbenzyl)sulfonyl)acetic acid (4f): white solid
(82%); mp 179−180 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.48 (s,
1H, COOH), 7.31−7.45 (m, 4H, ArH), 4.58 (s, 2H, SO2CH2COOH),
4.17 (s, 2H, ArCH2SO2), 1.29 (s, 9H, C(CH3)3).
Data for ((4-(trifluoromethyl)benzyl)sulfonyl)acetic acid (4g):
white solid (87%); mp 158−159 °C; 1H NMR (400 MHz, DMSO-d6)
δ 13.57 (s, 1H, COOH), 7.62−7.81 (m, 4H, ArH), 4.78 (s, 2H,
SO2CH2COOH), 4.24 (s, 2H, ArCH2SO2).
Data for ((4-methoxybenzyl)sulfonyl)acetic acid (4h): white solid
(81%); mp 155−156 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.47 (s,
1H, COOH), 6.97−7.33 (m, 4H, ArH), 4.55 (s, 2H, SO2CH2COOH),
4.12 (s, 2H, ArCH2SO2), 3.77 (s, 3H, CH3OAr).
Data for ((3-phenylpropyl)sulfanyl)acetic acid (3c): colorless
liquid (90%); 1H NMR (400 MHz, DMSO-d6) δ 12.51 (s, 1H,
COOH), 7.16−7.28 (m, 5H, ArH), 3.22 (s, 2H, SCH2COOH), 2.63
(t, 2H, SCH2CH2CH2Ar), 2.57 (t, 2H, SCH2CH2CH2Ar), 1.81 (m,
2H, SCH2CH2CH2Ar).
General Pprocedure for the Synthesis of (E)-3,4-Dihydrox-
ystyryl Aralkyl Sulfones 5a−h. 3,4-Dihydroxybenzaldehyde (2
mmol) and β-aminopropionic acid (2 mmol) were added to a solution
of the (aralkylsulfonyl)acetic acid (2 mmol) solution in THF (15 mL)
and the resulting solution heated to reflux until absence of the
(aralkylsulfonyl)acetic acid (checked by TLC). The reaction mixture
was concentrated in vacuo, diluted with H2O, and extracted with ethyl
acetate. The combined organic fractions were washed with brine, dried
(Na2SO4), and concentrated under reduced pressure. Purification of
the crude residue by column chromatography (petroleum ether/ethyl
acetate) afforded the title compound.
Data for (E)-4-[2-((phenylmethyl)sulfonyl)vinyl]benzene-1,2-diol
(5a): white solid (72%); mp 133−134 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.74 (s, 1H, p-ArOH), 9.24 (s, 1H, m-ArOH), 6.76−7.41
(m, 10H, ArH, CHCH), 4.50 (s, 2H, ArCH2SO2); 13C NMR (100
MHz, DMSO-d6) δ 149.34, 146.11, 144.27, 131.56, 129.57, 128.76,
128.68, 124.21, 122.43, 122.19, 116.20, 115.48, 60.52; HR-MS (ESI+)
m/z 291.06856 [M + H]+, found 291.06797 [M + H]+, 308.09453 [M
+ NH4]+, 313.04980 [M + Na]+.
Data for ((4-phenylbutyl)sulfanyl)acetic acid (3d): colorless liquid
1
(91%); H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H, COOH),
7.15−7.30 (m, 5H, ArH), 3.20 (s, 2H, SCH2COOH), 2.55−2.62 (m,
4H, SCH2CH2CH2CH2Ar), 1.64 (m, 2H, SCH2CH2CH2CH2Ar), 1.54
(m, 2H, SCH2CH2CH2CH2Ar).
Data for ((4-Chlorobenzyl)sulfanyl)acetic acid (3e): white solid
1
(93%); mp 51−52 °C; H NMR (400 MHz, DMSO-d6) δ 12.62 (s,
1H, COOH), 7.32−7.50 (m, 4H, ArH), 3.82 (s, 2H, SCH2COOH),
3.14 (s, 2H, ArCH2S).
Data for ((4-tert-butylbenzyl)sulfanyl)acetic acid (3f): white solid
1
(84%); mp 75−76 °C; H NMR (400 MHz, DMSO-d6) δ 12.58 (s,
1H, COOH), 7.22−7.36 (m, 4H, ArH), 3.77 (s, 2H, SCH2COOH),
3.12 (s, 2H, ArCH2S), 1.27 (s, 9H, C(CH3)3).
Data for ((4-(trifluoromethyl)benzyl)sulfanyl)acetic acid (3g):
white solid (88%); mp 64−65 °C; 1H NMR (400 MHz, DMSO-d6) δ
12.68 (s, 1H, COOH), 7.55−7.71 (m, 4H, ArH), 3.92 (s, 2H,
SCH2COOH), 3.16 (s, 2H, ArCH2S).
Data for ((4-Methoxybenzyl)sulfanyl)acetic acid (3h): white solid
Data for (E)-4-[2-((2-phenylethyl)sulfonyl)vinyl]benzene-1,2-diol
(5b): white solid (78%); mp 142−143 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.75 (s, 1H, p-ArOH), 9.23 (s, 1H, m-ArOH), 6.79−7.34
(m, 10H, ArH, CHCH), 3.43−3.47 (m, 2H, ArCH2CH2SO2),
2.95−3.00 (m, 2H, ArCH2CH2SO2); 13C NMR (100 MHz, DMSO-
d6) δ 149.35, 146.11, 143.97, 138.68, 128.95, 126.97, 124.32, 122.50,
122.45, 116.17, 115.71, 55.37, 28.71; HR-MS (ESI+) m/z 305.08421
[M + H]+, found 305.08341 [M + H]+, 322.10988 [M + NH4]+.
Data for (E)-4-[2-((3-phenylprop-1-yl)sulfonyl)vinyl]benzene-1,2-
1
(86%); mp 47−48 °C; H NMR (400 MHz, DMSO-d6) δ 12.57 (s,
1H, COOH), 6.88−7.23 (m, 4H, ArH), 3.75 (s, 2H, SCH2COOH),
3.74 (s, 3H, CH3O), 3.09 (s, 2H, ArCH2S).
General Procedure for the Synthesis of (Aralkylsulfonyl)-
acetic Acids 4a−h. The (aralkylsulfanyl)acetic acid (3 mmol) was
added to a solution of 30% H2O2 aqueous solution (2 mL) in acetic
acid (5 mL) and the resulting solution stirred at rt until absence of the
(aralkylsulfanyl)acetic acid (checked by TLC). The reaction mixture
was diluted with H2O and extracted with ethyl acetate. The combined
organic fractions were washed with brine, dried (Na2SO4), and
concentrated under reduced pressure. Purification of the crude residue
by column chromatography (petroleum ether/ethyl acetate) afforded
the title compound.
Data for (benzylsulfonyl)acetic acid (4a): white solid (82%); mp
138−139 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.51 (s, 1H,
COOH), 7.40 (s, 5H, ArH), 4.62 (s, 2H, SO2CH2COOH), 4.15 (s,
2H, ArCH2SO2).
1
diol (5c): white solid (71%); mp 117−118 °C; H NMR (400 MHz,
DMSO-d6) δ 9.75 (s, 1H, p-ArOH), 9.21 (s, 1H, m-ArOH), 6.78−7.30
(m, 10H, ArH, ArCHCHSO2), 3.10 (t, 2H, SO2CH2CH2CH2Ar),
2.70 (t, 2H, SO2CH2CH2CH2Ar), 1.94 (m, 2H, SO2CH2CH2CH2Ar);
13C NMR (100 MHz, DMSO-d6) δ 149.33, 146.11, 143.87, 141.22,
128.91, 128.82, 126.56, 124.30, 122.56, 122.49, 116.16, 115.78, 54.03,
33.86, 24.72; HR-MS (ESI+) m/z 319.09986 [M + H]+, found
319.09927 [M + H]+, 341.08147 [M + Na]+.
Data for (E)-4-[2-((4-phenylbut-1-yl)sulfonyl)vinyl]benzene-1,2-
Data for (phenethylsulfonyl)acetic acid (4b): white solid (72%);
mp 80−81 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.45 (s, 1H,
COOH), 7.25−7.33 (m, 5H, ArH), 4.30 (s, 2H, SO2CH2COOH),
3.58 (m, 2H, SO2CH2CH2Ar), 3.05 (m, 2H, SO2CH2CH2Ar).
Data for ((3-phenylpropyl)sulfonyl)acetic acid (4c): white solid
1
diol (5d): white solid (78%); mp 99−100 °C; H NMR (400 MHz,
DMSO-d6) δ 9.75 (s, 1H, p-ArOH), 9.23 (s, 1H, m-ArOH), 6.79−7.28
( m , 1 0 H , A r H , A r C H  C H S O 2 ) , 3 . 1 5 ( m , 2 H ,
SO2CH2CH2CH2CH2Ar), 2.58 (m, 2H, SO2CH2CH2CH2CH2Ar),
1.65−1.67 (m, 4H, SO2CH2CH2CH2CH2Ar); 13C NMR (100 MHz,
DMSO-d6) δ 149.29, 146.11, 143.72, 142.11, 128.78, 128.71, 126.21,
124.32, 122.66, 122.42, 116.17, 115.70, 60.23, 54.20, 34.93, 29.94; HR-
MS (ESI+) m/z 333.11551 [M + H]+, found 333.11499 [M + H]+.
Data for (E)-4-[2-(((4-chlorophenyl)methyl)sulfonyl)vinyl]-
benzene-1,2-diol (5e): white solid (78%); mp 166−167 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H, p-ArOH), 9.23 (s, 1H, m-
ArOH), 6.76−7.45 (m, 9H, ArH, CHCH), 4.53 (s, 2H,
ArCH2SO2); 13C NMR (100 MHz, DMSO-d6) δ 149.47, 146.12,
144.53, 133.60, 133.31, 128.82, 128.74, 124.14, 122.52, 121.94, 116.20,
115.53, 59.63; HR-MS (ESI+) m/z 325.02958 [M + H]+, found
325.02885 [M + H]+, 342.05537 [M + NH4]+.
1
(70%); mp 96−97 °C; H NMR (400 MHz, DMSO-d6) δ 13.40 (s,
1H, COOH), 7.19−7.31 (m, 5H, ArH), 4.26 (s, 2H, SO2CH2COOH),
3.25 (t, 2H, SO2CH2CH2CH2Ar), 2.70 (t, 2H, SO2CH2CH2CH2Ar),
2.01 (m, 2H, SO2CH2CH2CH2Ar).
Data for ((4-phenylbutyl)sulfonyl)acetic acid (4d): white solid
1
(78%); mp 76−77 °C; H NMR (400 MHz, DMSO-d6) δ 13.43 (s,
1H, COOH), 7.17−7.31 (m, 5H, ArH), 4.24 (s, 2H, SO2CH2COOH),
3.32 (m, 2H, SO2CH2CH2CH2CH2Ar), 2.61 (m, 2H,
S O 2 C H 2 C H 2 C H 2 C H 2 A r ) , 1 . 6 8 − 1 . 7 3 ( m , 4 H ,
SO2CH2CH2CH2CH2Ar); 13C NMR (100 MHz, DMSO-d6) δ
165.20, 142.04, 128.77, 126.27, 57.80, 52.95, 35.00, 30.03, 21.09;
MS (ESI) m/z 257.0970 [M + H]+, 279.1076 [M + Na]+.
Data for ((4-chlorobenzyl)sulfonyl)acetic acid (4e): white solid
(82%); mp 144−145 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.53 (s,
1H, COOH), 7.41−7.51 (m, 4H, ArH), 4.65 (s, 2H, SO2CH2COOH),
4.19 (s, 2H, ArCH2SO2).
Data for (E)-4-[2-(((4-tert-butylphenyl)methyl)sulfonyl)vinyl]-
benzene-1,2-diol (5f): white solid (72%); mp 206−207 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H, p-ArOH), 9.24 (s, 1H, m-
ArOH), 6.77−7.39 (m, 9H, ArH, CHCH), 4.44 (s, 2H, ArCH2SO2),
1.27 (s, 9H, (CH3)3C); 13C NMR (100 MHz, DMSO-d6) δ 151.12,
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149.36, 146.12, 144.13, 131.28, 126.32, 125.60, 124.28, 122.51, 122.41,
116.20, 115.53, 60.14, 34.78, 31.53; HR-MS (ESI+) m/z 347.13116 [M
+ H]+, found 347.13086 [M + H]+, 364.15711 [M + NH4]+.
Data for (E)-4-[2-(((4-(trifluoromethyl)phenyl)methyl)sulfonyl)-
vinyl]benzene-1,2-diol (5g): white solid (95%); mp 202−203 °C;
1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H, p-ArOH), 9.24 (s, 1H,
m-ArOH), 6.77−7.76 (m, 9H, ArH, CHCH), 4.66 (s, 2H,
ArCH2SO2); 13C NMR (100 MHz, DMSO-d6) δ 149.53, 146.13,
144.73, 134.45, 132.36, 125.65, 125.61, 124.11, 122.59, 121.92, 116.20,
115.57, 59.93; HR-MS (ESI+) m/z 359.05594 [M + H]+, found
359.05518 [M + H]+.
Data for (E)-4-[2-(((4-methoxyphenyl)methyl)sulfonyl)vinyl]-
benzene-1,2-diol (5h): white solid (75%); mp 205−206 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H, p-ArOH), 9.23 (s, 1H, m-
ArOH), 6.76−7.28 (m, 9H, ArH, CHCH), 4.41 (s, 2H, ArCH2SO2),
3.74 (s, 3H, CH3O); 13C NMR (100 MHz, DMSO-d6) δ 159.70,
149.34, 146.11, 144.09, 132.73, 124.27, 122.39, 122.26, 121.29, 116.20,
115.47, 114.24, 59.85, 55.54; HR-MS (ESI+) m/z 338.10567 [M +
NH4]+, found 338.10517 [M + NH4]+.
General Procedure for the Synthesis of (Aralkylsulfinyl)-
acetic Acids 6a−h. The (aralkylsulfanyl)acetic acid (3 mmol) was
added to a solution of 30% H2O2 aqueous solution (2 mL) in MeOH
(6 mL) and the resulting solution stirred at rt until absence of the
(aralkylsulfanyl)acetic acid (checked by TLC). The reaction mixture
was concentrated in vacuo and extracted with ethyl acetate. The
combined organic fractions were washed with brine, dried (Na2SO4),
and concentrated under reduced pressure. Purification of the crude
residue by column chromatography (petroleum ether/ethyl acetate)
afforded the title compound and a small amount (10−20%) of
(aralkylsulfonyl)acetic acids 4a−h.
SOCH2COOH), 3.89 (d, J = 14.4 Hz, 1H, ArCH2SO), 3.54 (d, J =
14.4 Hz, 1H, ArCH2SO).
Data for ((4-methoxybenzyl)sulfinyl)acetic acid (6h): white solid
(75%); mp 134−135 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.12 (s,
1H, COOH), 6.94−7.26 (m, 4H, ArH), 4.18 (d, J = 13.2 Hz, 1H,
SOCH2COOH), 4.01 (d, J = 13.2 Hz, 1H, SOCH2COOH), 3.81 (d, J
= 15.4 Hz, 1H, ArCH2SO), 3.76 (s, 3H, CH3OAr), 3.51 (d, J = 15.4
Hz, 1H, ArCH2SO).
General Procedure for the Synthesis of (E)-3,4-Dihydrox-
ystyryl Aralkyl Sulfoxides 7a−h. 3,4-Dihydroxybenzaldehyde (2
mmol), pyrrolidine (catalytic amount), and acetic acid (catalytic
amount) were added to a solution of the (aralkylsulfinyl)acetic acid (2
mmol) solution in THF (15 mL) and the resulting solution heated to
reflux until absence of the (aralkylsulfinyl)acetic acid (checked by
TLC). The reaction mixture was concentrated in vacuo, diluted with
H2O, and extracted with ethyl acetate. The combined organic fractions
were washed with brine, dried (Na2SO4), and concentrated under
reduced pressure. Purification of the crude residue by column
chromatography (petroleum ether/ethyl acetate) afforded the title
compound.
Data for (E)-4-[2-((phenylmethyl)sulfinyl)vinyl]benzene-1,2-diol
(7a): white solid (61%); mp 175−176 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.42 (s, 1H, p-ArOH), 9.12(s, 1H, m-ArOH), 6.73−7.36
(m, 10H, ArH, CHCH), 4.25 (d, J = 16.4 Hz, 1H, ArCH2SO), 4.00
(d, J = 16.4 Hz, 1H, ArCH2SO); 13C NMR (100 MHz, DMSO-d6) δ
147.68, 145.96, 136.70, 131.46, 130.91, 128.72, 128.37, 128.16, 126.00,
120.61, 116.12, 114.68, 59.51; HR-MS (ESI+) m/z 275.07364 [M +
H]+, found 275.07355 [M + H]+.
Data for (E)-4-[2-((2-phenylethyl)sulfinyl)vinyl]benzene-1,2-diol
(7b): white solid (60%); mp 152−153 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.43 (s, 1H, p-ArOH), 9.10 (s, 1H, m-ArOH), 6.75−7.33
(m, 10H, ArH, CHCH), 2.87−3.21 (m, 4H, ArCH2CH2SO); 13C
NMR (100 MHz, DMSO-d6) δ 147.62, 145.98, 139.88, 136.31, 129.05,
128.97, 128.45, 126.81, 126.08, 120.63, 116.11, 114.86, 53.98, 27.69;
HR-MS (ESI+) m/z 289.08929 [M + H]+, found 289.08885 [M + H]+.
Data for (E)-4-[2-((3-phenylprop-1-yl)sulfinyl)vinyl]benzene-1,2-
Data for (benzylsulfinyl)acetic Acid (6a): white solid (75%); mp
126−127 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H,
COOH), 7.32−7.40 (m, 5H, ArH), 4.24 (d, J = 12.8 Hz, 1H,
SOCH2COOH), 4.07 (d, J = 12.8 Hz, 1H, SOCH2COOH), 3.85 (d, J
= 14.4 Hz, 1H, ArCH2SO), 3.55 (d, J = 14.4 Hz, 1H, ArCH2SO).
Data for (phenethylsulfinyl)acetic acid (6b): white solid (76%);
1
diol (7c): white solid (69%); mp 114−115 °C; H NMR (400 MHz,
1
mp 110−111 °C; H NMR (400 MHz, DMSO-d6) δ 13.11 (s, 1H,
DMSO-d6) δ 9.42 (s, 1H, p-ArOH), 9.08 (s, 1H, m-ArOH), 6.74−7.30
(m, 10H, ArH, CHCH), 2.69−2.90 (m, 4H, SOCH2CH2CH2Ar),
1.91 (m, 2H, SOCH2CH2CH2Ar); 13C NMR (100 MHz, DMSO-d6) δ
147.57, 145.97, 141.53, 135.95, 128.86, 128.82, 128.59, 126.46, 126.05,
120.57, 116.11, 114.84, 55.39, 52.40, 34.46, 23.78; HR-MS (ESI+) m/z
303.10494 [M + H]+, found 303.10420 [M + H]+.
COOH), 7.24−7.35 (m, 5H, ArH), 3.95 (d, 1H, J = 14.4 Hz,
SOCH2COOH), 3.70 (d, 1H, J = 14.4 Hz, SOCH2COOH), 3.10 (m,
2H, SOCH2CH2Ar), 2.99 (m, 2H, SOCH2CH2Ar).
Data for ((3-phenylpropyl)sulfinyl)acetic acid (6c): white solid
(84%); mp 111−112 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.06 (s,
1H, COOH), 7.17−7.29 (m, 5H, ArH), 3.88 (d, 1H, J = 14.4 Hz,
SOCH2COOH), 3.61 (d, 1H, J = 14.4 Hz, SOCH2COOH), 2.79 (m,
2H, SOCH2CH2CH2Ar), 2.69 (m, 2H, SOCH2CH2CH2Ar), 1.93 (m,
2H, SOCH2CH2CH2Ar).
Data for ((4-phenylbutyl)sulfinyl)acetic acid (6d): white solid
(86%); mp 40−41 °C; 1H NMR (400 MHz, DMSO-d6) δ = 13.14 (s,
1H, COOH), 7.17−7.29 (m, 5H, ArH), 3.89 (d, 1H, J = 14.4 Hz,
SOCH2COOH), 3.63 (d, 1H, J = 14.4 Hz, SOCH2COOH), 2.84 (m,
2H, SOCH2CH2CH2CH2Ar), 2.61 (m, 2H, SOCH2CH2CH2CH2Ar),
1.66−1.73 (m, 4H, SOCH2CH2CH2CH2Ar); 13C NMR (100 MHz,
DMSO-d6) δ 168.04, 142.19, 128.76, 126.24, 56.60, 51.41, 35.13,
30.48, 22.11; MS (ESI) m/z 239.2291 [M − H]−.
Data for ((4-chlorobenzyl)sulfinyl)acetic acid (6e): white solid
(82%); mp 146−147 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.14 (s,
1H, COOH), 7.31−7.44 (m, 4H, ArH), 4.22 (d, J = 12.4 Hz, 1H,
SOCH2COOH), 4.05 (d, J = 12.4 Hz, 1H, SOCH2COOH), 3.83 (d, J
= 14.8 Hz, 1H, ArCH2SO), 3.50 (d, J = 14.8 Hz, 1H, ArCH2SO).
Data for ((4-tert-butylbenzyl)sulfinyl)acetic acid (6f): white solid
(79%); mp 145−146 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.14 (s,
1H, COOH), 7.24−7.42 (m, 4H, ArH), 4.21 (d, J = 12.8 Hz, 1H,
SOCH2COOH), 4.02 (d, J = 12.8 Hz, 1H, SOCH2COOH), 3.86 (d, J
= 14.4 Hz, 1H, ArCH2SO), 3.56 (d, J = 14.4 Hz, 1H, ArCH2SO), 1.29
(s, 9H, C(CH3)3).
Data for (E)-4-[2-((4-phenylbut-1-yl)sulfinyl)vinyl]benzene-1,2-
1
diol (7d): white solid (70%); mp 84−85 °C; H NMR (400 MHz,
DMSO-d6) δ 9.41 (s, 1H, p-ArOH), 9.09 (s, 1H, m-ArOH), 6.74−7.28
(m, 10H, ArH, CHCH), 2.90 (m, 1H, SOCH2CH2CH2CH2Ar),
2.71 (m, 1H, SOCH2 CH2 CH2 CH2 Ar), 2.60 (m, 2H,
S O C H 2 C H 2 C H 2 C H 2 A r ) , 1 . 5 9 − 1 . 7 0 ( m , 4 H ,
SOCH2CH2CH2CH2Ar); 13C NMR (100 MHz, DMSO-d6) δ
147.55, 145.97, 142.24, 135.89, 128.77, 128.72, 126.19, 126.08,
120.54, 116.11, 114.81, 52.77, 35.14, 30.51, 21.49; HR-MS (ESI+)
m/z 317.12059 [M + H]+, found 317.12073 [M + H]+.
Data for (E)-4-[2-(((4-Chlorophenyl)methyl)sulfinyl)vinyl]-
benzene-1,2-diol (7e): white solid (68%); mp 190−191 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H, p-ArOH), 9.11 (s, 1H, m-
ArOH), 6.69−7.42 (m, 9H, ArH, CHCH), 4.26 (d, J = 12.4 Hz, 1H,
ArCH2SO), 4.02 (d, J = 12.4 Hz, 1H, ArCH2SO); 13C NMR (100
MHz, DMSO-d6) δ 147.72, 145.97, 136.40, 132.96, 132.76, 130.29,
128.62, 128.00, 125.96, 120.64, 116.12, 114.69, 58.14; HR-MS (ESI+)
m/z 309.03467 [M + H]+, found 309.03483 [M + H]+.
Data for (E)-4-[2-(((4-tert-butylphenyl)methyl)sulfinyl)vinyl]-
benzene-1,2-diol (7f): white solid (67%); mp 206−207 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H, p-ArOH), 9.12 (s, 1H, m-
ArOH), 6.74−7.37 (m, 9H, ArH, CHCH), 4.22 (d, J = 12.4 Hz, 1H,
ArCH2SO), 3.92 (d, J = 12.4 Hz, 1H, ArCH2SO), 1.26 (s, 9H,
(CH3)3C); 13C NMR (100 MHz, DMSO-d6) δ 150.59, 147.67, 145.98,
136.55, 130.57, 128.66, 128.61, 126.04, 125.85, 120.62, 116.13, 114.73,
59.48, 34.74, 31.56; HR-MS (ESI+) m/z 331.13624 [M + H]+, found
331.13626 [M + H]+.
Data for ((4-(trifluoromethyl)benzyl)sulfinyl)acetic acid (6g):
1
white solid (81%); mp 112−113 °C; H NMR (400 MHz, DMSO-
d6) δ 13.18 (s, 1H, COOH), 7.51−7.75 (m, 4H, ArH), 4.34 (d, J =
12.8 Hz, 1H, SOCH2COOH), 4.16 (d, J = 12.8 Hz, 1H,
4309
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Data for (E)-4-[2-(((4-(trifluoromethyl)phenyl)methyl)sulfinyl)-
vinyl]benzene-1,2-diol (7g): white solid (75%); mp 216−217 °C;
1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H, p-ArOH), 9.12 (s, 1H,
m-ArOH), 6.73−7.71 (m, 8H, ArH, ArCHCHSO), 6.71 (d, J = 15.2
Hz, 1H, ArCHCHSO), 4.39 (d, J = 12.4 Hz, 1H, ArCH2SO), 4.12
(d, J = 12.4 Hz, 1H, ArCH2SO); 13C NMR (100 MHz, DMSO-d6) δ
147.76, 145.98, 136.96, 136.14, 131.77, 127.88, 125.92, 125.42, 125.39,
120.67, 116.12, 114.71, 58.35; HR-MS (ESI+) m/z 343.06103 [M +
H]+, found 343.06116 [M + H]+.
Data for (E)-4-[2-(((4-methoxyphenyl)methyl)sulfinyl)vinyl]-
benzene-1,2-diol (7h): white solid (62%); mp 190−191 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H, p-ArOH), 9.12 (s, 1H, m-
ArOH), 6.73−7.23 (m, 9H, ArH, CHCH), 4.18 (d, J = 12.8 Hz, 1H,
ArCH2SO), 3.94 (d, J = 12.8 Hz, 1H, ArCH2SO), 3.73 (s, 3H, CH3O);
13C NMR (100 MHz, DMSO-d6) δ 159.36, 147.66, 145.97, 136.70,
132.09, 128.42, 126.07, 123.13, 120.60, 116.13, 114.68, 114.19, 58.85,
55.51; HR-MS (ESI+) m/z 305.08421 [M + H]+, found 305.08429 [M
+ H]+, 327.06628 [M + Na]+.
Biological Assays. DPPH Free Radical Scavenging Ability. The
free radical scavenging ability was studied by DPPH.28 DPPH was
purchased from Sigma. The effect of test compounds on free radical
scavenging was reflected by decolorization monitoring of DPPH
radical. The reaction was performed in 96-well microplates. DPPH and
test compounds were dissolved in ethanol. A 100 μL sample of the test
solutions and 100 μL of DPPH solution (0.2 mM) were mixed in each
well. The mixture of neat ethanol and DPPH was used as the control.
After 30 min of incubation at room temperature, the reduction in the
number of free radicals was detected by reading the absorbance at 517
nm. Vitamin C was used as a reference. Every sample was analyzed in
three wells and in three independent runs. The percentage of
inhibition was obtained from the following formula: [(A0 − Ac)/A0] ×
100, where A0 is the absorbance of the control and Ac is the
absorbance of the test sample.
Protection of PC12 Cells against H2O2- and 6-OHDA-Induced Cell
Injury. The PC12 cells were purchased from the Shanghai Institute of
Cell Biology, Chinese Academy of Sciences. Fetal bovine serum,
penicillin streptomycin, and trypsin−EDTA were purchased from
Invitrogen. Donor equine serum and high-glucose DMEM were
purchased from Thermo Scientific. MTT and DMSO were obtained
from AMRESCO. H2O2 and 6-OHDA were purchased from Sigma.
PC12 cells were cultured in high-glucose DMEM supplemented with
10% fetal bovine serum, 5% horse serum, 100 U mL−1 penicillin, and
100 U mL−1 streptomycin in a humidified atmosphere of 5% CO2 at
37 °C. PC12 cells were plated in 96-well microplates at a density of 4
× 105 cells mL−1 (100 μL per well). After cell attachment, PC12 cells
were preincubated with compounds dissolved in DMSO and diluted
with medium to the final concentrations for 3 h. Afterward, 20 μL of
H2O2 (diluted with medium to a final concentration of 500 μM) or 6-
OHDA (diluted with medium to a final concentration of 400 μM)
solution was added. After 5 and 48 h, respectively, the cell viability was
measured by MTT assay. The absorbance was detected at 570 nm.
The viability of cells treated with drugs is obtained by the following
formula: ODdrug‑treated/ODnormal cells × 100.
percentage of inhibition of NO was obtained from the following
formula: [(RL − R0 − Rc)/(RL − R0)] × 100, where RL is the release
amount of only the LPS-treated group, R0 is the release amount of the
normal control, and Rc is the release amount of the test compound and
LPS-treated group. The IC50 values of test compounds were calculated
by linear regression plots.
In Vitro Blood−Brain Barrier Permeability Assay. The ability to
cross the BBB was predicted and evaluated using PAMPA.34 The 96-
well filter plate (catalog no. MAIPN4550) and the donor plate (catalog
no. MATRNPS50) were both purchased from Millipore. Filter PDVF
membrane units were obtained from Symta. The porcine polar brain
lipid was purchased from Avanti Polar Lipids. Dodecane was obtained
from Alfa Aesar. Verapamil, clonidine, and hydrocortisone were
purchased from Sigma. Tested compounds were dissolved in DMSO
at 5 mg mL−1 as stock solutions. A 10 μL sample of stock solution was
diluted in PBS to make a secondary stock solution (final concentration
25 μg mL−1). These solutions were filtered. A 300 μL sample of
secondary stock solution was added to the donor well. The porcine
polar brain lipid was dissolved in dodecane at 20 mg mL−1. The filter
membrane was coated with 4 μL of porcine polar brain lipid solution,
and the acceptor well was filled with 150 μL of PBS. The acceptor
filter plate was carefully put on the donor plate to form a “sandwich”
which was composed of the donor with tested compounds on the
bottom, artificial lipid membrane in the middle, and the acceptor on
the top. The sandwich was incubated undisturbed at room
temperature for 18 h. The donor plate was removed after incubation.
The concentrations of tested compounds in the acceptor and reference
solutions were determined by a UV plate reader. Reference solutions
were prepared by diluting the sample secondary stock solution to the
same concentration as that with no membrane barrier. Every sample
was analyzed at three wavelengths, in three wells, and in three
independent runs. The Pe was obtained by the following equation:35
⎛
⎞
⎟
⎠
V V
[drug]ac
[drug]ref
dn ac
P = −
ln⎜1 −
⎝
e
st(V + V )
dn
ac
where Vdn (mL) = volume of the donor compartment, Vac (mL) =
volume of the acceptor compartment, [drug]ac = optical density of the
solution of the acceptor compartment, [drug]ref = optical density of
the reference solution, s (cm2) = membrane area, and t (s) =
incubation time.
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC data for target compounds (5a−h and 7a−h). This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86-10-82805203. E-mail: lilybmu@bjmu.edu.cn.
*Phone/fax: +86-10-82805203. E-mail: jyliu@bjmu.edu.cn.
■
Inhibiting NO Production of LPS-Stimulated BV2 Microglial Cells.
BV2 microglial cells were obtained from the Institute of Basic
Medicine, Chinese Academy of Medical Sciences. LPS was purchased
from Sigma. BV2 microglial cells were maintained in high-glucose
DMEM supplemented with 10% fetal bovine serum, 100 U mL−1
penicillin, and 100 U mL−1 streptomycin in a humidified atmosphere
of 5% CO2 at 37 °C. BV2 microglial cells were plated in 96-well
microplates at a density of 4 × 105 cells mL−1 (100 μL per well). After
cell attachment, the BV2 microglial cells were preincubated with
compounds dissolved in DMSO and diluted with medium to the final
concentrations for 3 h. Afterward, 20 μL of LPS solution (diluted with
medium to a final concentration of 100 nM) was added. The NO assay
kit was used to carry out nitrite assays after 24 h. The NO assay kit was
purchased from Applygen. The culture medium was mixed with the
same volume of reagent of the NO assay kit in 96-well plates. The
absorbance was detected at 540 nm. The release amount of NO was
calculated by the offered linear equation of the NO assay kit. The
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the National Natural Science
Foundation of China (Grants 20972011, 21042009, and
21172014) and Ministry of Science and Technology of China
(Grant 2009ZX09301-010).
ABBREVIATIONS USED
■
CAPE, caffeic acid phenethyl ester; AD, Alzheimer’s disease;
PD, Parkinson’s disease; HD, Huntington’s disease; ALS,
amyotrophic lateral sclerosis; iNOS, inducible nitric oxide
synthase; COX-2, cyclooxygenase-2; TNF, tumor necrosis
4310
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(16) Wei, X.; Ma, Z.; Fontanilla, C. V.; Zhao, L.; Xu, Z. C.;
Taggliabraci, V.; Johnstone, B. H.; Dodel, R. C.; Farlow, M. R.; Du, Y.
Caffeic acid phenethyl ester prevents cerebellar granule neurons
(CGNs) against glutamate-induced neurotoxicity. Neuroscience 2008,
155, 1098−1105.
(17) (a) Wei, X.; Zhao, L.; Ma, Z.; Holtzman, D. M.; Yan, C.; Dodel,
R. C.; Hampel, H.; Oertel, W.; Farlow, M. R.; Du, Y. Caffeic acid
phenethyl ester prevents neonatal hypoxic-ischaemic brain injury.
Brain 2004, 127, 2629−2635. (b) Altug, M. E.; Serarslan, Y.; Bal, R.;
Kontas, T.; Ekici, F.; Melek, I. M.; Aslan, H.; Duman, T. Caffeic acid
phenethyl ester protects rabbit brains against permanent focal ischemia
by antioxidant action: a biochemical and planimetric study. Brain Res.
2008, 1201, 135−142.
factor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;
IC50, half-maximal inhibitory concentration; BBB, blood−
brain barrier; PAMPA, parallel artificial membrane permeability
assay; PAMPA-BBB, parallel artificial membrane permeation
assay for the blood−brain barrier; DPPH, 1,1-diphenyl-2-
picrylhydrazyl; NO, nitric oxide; LPS, lipopolysaccharide;
PC12, rat pheochromocytoma cells; H2O2, hydrogen peroxide;
6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species;
DA, dopaminergic; CNS, central nervous system; TLC, thin-
layer chromatography; HRMS, high-resolution mass spectrom-
etry; HPLC, high-performance liquid chromatography
(18) Fontanilla, C. V.; Ma, Z.; Wei, X.; Klotsche, J.; Zhao, L.;
Wisniowski, P.; Dodel, R. C.; Farlow, M. R.; Oertel, W. H.; Du, Y.
Caffeic acid phenethyl ester prevents 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-induced neurodegeneration. Neuroscience 2011,
188, 135−141.
(19) Noelker, C.; Bacher, M.; Gocke, P.; Wei, X.; Klockgether, T.;
Du, Y.; Dodel, R. The flavanoide caffeic acid phenethyl ester blocks 6-
hydroxydopamine-induced neurotoxicity. Neurosci. Lett. 2005, 383,
39−43.
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