Neuroprotective and Antineuroinflammatory Effects of Hydroxyl-Functionalized Stilbenes and 2-Arylbenzo[b]furans

Article abstract of DOI:10.1021/acs.jmedchem.7b00376

The drugs currently used to treat Alzheimer’s disease (AD) are limited in the benefits they confer, and no medication has been clearly proven to cure or delay the progression of AD. Most candidate AD drugs are meant to reduce the production, aggregation,

Full text of DOI:10.1021/acs.jmedchem.7b00376

Article
pubs.acs.org/jmc
Neuroprotective and Antineuroinflammatory Effects of
Hydroxyl-Functionalized Stilbenes and 2‑Arylbenzo[b]furans
Pei-Chun Chen,† Wei-Jern Tsai,‡ Yune-Fang Ueng,‡ Tsai-Teng Tzeng,† Hsiang-Ling Chen,† Pei-Ru Zhu,†
Chia-Hsiang Huang,‡ Young-Ji Shiao,*,‡,§ and Wen-Tai Li*,‡,§
†Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei 11221, Taiwan, R.O.C.
‡National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan, R.O.C.
S
* Supporting Information
ABSTRACT: The drugs currently used to treat Alzheimer’s disease (AD) are limited in the benefits they confer, and no
medication has been clearly proven to cure or delay the progression of AD. Most candidate AD drugs are meant to reduce the
production, aggregation, and toxicity of amyloid β (Aβ) or to promote Aβ clearance. Herein, we demonstrate the efficient
synthesis of hydroxyl-functionalized stilbene and 2-arylbenzo[b]furan derivatives and report on the neuroprotective and anti-
inflammatory effects of these phenolic compounds in vitro and in an animal model. Structure−activity relationships revealed that
the presence of an acrylate group on 2-arylbenzo[b]furan confers neuroprotective and anti-inflammatory effects. Furthermore,
compounds 11 and 37 in this study showed particular potential for development as disease-modifying anti-Alzheimer’s drugs,
based on their neuroprotective effects on neuron cells, their antineuroinflammatory effects on glial cells, and the ability to
ameliorate nesting behavior in APP/PS1 mice. These results indicate that 2-arylbenzo[b]furans could be candidate compounds
for the treatment of AD.
drugs are currently undergoing clinical evaluation. Most of these
drugs seek to reduce the production, aggregation, and toxicity of
Aβ or to promote Aβ clearance.7 However, oxidative stress is
among the earliest events in AD pathogenesis, and previous
research on free radicals and oxidative stress revealed that
oxidative damage is a crucial factor in neuronal degeneration.8
Furthermore, the interaction between Aβ and Cu2+ has been
shown to contribute to the production of reactive oxygen
species (ROS).9
Tournefolic acid B (TAB) and lithospermic acid (LSA) are
active components isolated from Tournefortia sarmentosa Lam.
(Boraginaceae), a medicinal plant commonly used in Taiwan
as a detoxicant, anti-inflammatory agent, and promotor of
circulation to prevent blood stasis.10 In previous studies, we
demonstrated that TAB and LSA derivatives effectively attenuate
neurotoxicity mediated by Aβ, glutamate, NMDA, and 1-methyl-
4-phenylpyridinium. Specifically, TAB and LSA achieve this by
abrogating calcium overload in mitochondria and retarding
the caspase 8-truncated bid−cytochrome c pathway.11−14 Other
polyphenols, such as resveratrol (a natural compound with
stilbene structure), have also been studied extensively as anti-AD
agents because of their antineuroinflammatory properties and
INTRODUCTION
■
Alzheimer’s disease (AD) is a common neurodegenerative
disorder that leads to cognitive decline, irreversible memory
loss, disorientation, and language impairment.1 The histopatho-
logical hallmarks of AD include extracellular amyloid deposition
of senile plaques and neurofibrillary tangles in various regions
of the brain. Senile plaques are massive extracellular deposits of
aggregated amyloid-β (Aβ) peptides, and neurofibrillary tangles
are listed among the neuropathological lesions characterizing
AD.2 The etiology of AD has yet to be fully elucidated; how-
ever, it is clear that a number of factors likely play important
roles in the development of the disease. These factors include the
presence of Aβ and hyperphosphorylated τ-proteins, oxidative
stress, and low levels of acetylcholine.3 The inflammation
response triggered by AD has also been shown to induce the
production of proinflammatory cytokines (such as IL-1β, TNFα,
and IL-6) by microglia and resident astrocytes.4
Two classes of drug are currently used to treat AD: acetyl-
cholinesterase inhibitors and N-methyl-D-aspartate (NMDA)
receptor antagonists.5 Nonetheless, these drugs confer only
limited benefits, and no medication has been clearly proven
to cure or delay the progression of AD. Much of the existing
research in this field has focused on the discovery of small
molecules capable of facilitating the development of novel
clinical treatments for AD.6 Indeed, a number of candidate
Received: March 11, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.7b00376
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Figure 1. Chemical structures of hydroxyl-functionalized tournefolic acid A and salvianolic acid F, as well as their structural relationships with
tournefolic acid B and lithospermic acid.
Scheme 1. Synthesis of Hydroxyl-Functionalized Stilbene 5
Scheme 2. Synthesis of Hydroxyl-Functionalized 2-Arylbenzo[b]furan 11
ability to inhibit Aβ aggregation by scavenging oxidants.15−17
Therefore, we hypothesize that polyphenolic compounds
presenting antioxidant activity could form the basis of novel
treatments for AD. However, the TAB molecule comprises a
seven-membered ring scaffold (belonging to the dibenzoxazepine
class), which makes it difficult to synthesize. Nonetheless,
the isosteres tournefolic acid A (TAA) and salvianolic acid
F (SAF) are characterized by planar phenolic stilbene and
2-arylbenzo[b]furan structures. Because of their antioxidant, anti-
inflammatory, and AD-modifying activities, we posit that TAA
and SAF could potentially be used as starting molecules in
the development of novel treatments for AD (Figure 1).18−22
In the current study, we demonstrate the efficient synthesis
of hydroxyl-functionalized stilbene and 2-arylbenzo[b]furan
derivatives. We also report on the neuroprotective and anti-
inflammatory effects of these planar phenolic compounds in vitro
and in an animal model. We hypothesize that hydroxyl-
functionalized stilbenes and 2-arylbenzo[b]furans could be a
useful starting point for the future development of treatments
for AD.
B
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C
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Table 2. Hydroxyl-Functionalized 2-Arylbenzo[b]furans for Neuroprotective and Antineuroinflammatory Effects
compound
R1
R2
R3
R4
R5
R6
R7
R8
R9
a
11
36
37
38
39
40
41
42
43
H
Br
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
OH
OH
H
H
CHCHCO2Et
H
-OCH2O-
H
a
CHCHCO2Et
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
Br
Br
Br
H
Br
OH
OH
OH
OH
OH
H
H
H
H
H
H
OH
H
a
H
OH
CHCHCO2Et
a
E isomer.
glutamate-mediated neurotoxicity, in accordance with standard
procedures (Tables 1 and 2). Specifically, the cell viability of
neurons was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) reduction analysis.
We determined that 5 of the 34 hydroxyl-functionalized
stilbenes and 2-arylbenzo[b]furans conferred neuroprotective
effects against fAβ25−35-mediated toxicity (Tables 3 and 4 and
Figure S1). Among those 5 neuroprotective compounds, 3 also
presented neuroprotective activities against glutamate-mediated
excitotoxicity.
Compounds 5, 11, and 37 (all of which are hydroxyl-
functionalized stilbene and benzo[b]furan derivatives) were
shown to confer potent protective benefits on cortical neurons
by reducing fAβ25−35- or glutamate-mediated neurotoxicity.
Specifically, compounds 5, 11, and 37 decreased fAβ25−35-
mediated cell death (Table 3 and Figure S1A). Cortical neurons
were treated with either vehicle (0.1% DMSO, v/v) or one
of the aforementioned compounds at various concentrations.
MTT reduction assays revealed that treatment with compounds
5, 11, and 37 at 50 μM decreased both fAβ25−35- and
glutamate-mediated neuronal death (Table 4 and Figure S1B).
Conversely, treatment with compounds 40 and 42 decreased
Aβ-mediated neuronal death and conferred a moderate
protective effect against glutamate-mediated neuronal death.
These experiments identified compound 37 as the most effective
at protecting neurons against fAβ25−35- and glutamate-mediated
toxicity (Table 4).
The live/dead cell viability assay indicated that 10 μM
fAβ25−35 induced a 40% decrease in calcein staining (in terms
of both cell number and fluorescence intensity) (Figure 2).
However, when cells were treated with 50 μM of compounds 5,
11, 37, 40, or 42, the fAβ25−35-induced decrease in calcein
staining was significantly attenuated. Conversely, treatment with
30 μM glutamate induced a 50% decrease in calcein staining (in
terms of both cell number and fluorescence intensity); however,
treatment with 50 μM of compounds 5, 11, or 37 significantly
attenuated the decrease in calcein staining.
The fAβ25−35-induced cell death was shown to coincide
with a 44% reduction in MAP2 and a 36% reduction in tau
protein. Compounds 40 and 42 significantly attenuated
the decrease in MAP2 (Figure 3A,C); however, none of
these six compounds attenuated the decrease in tau protein
(Figure 3A,D). Glutamate provoked a decrease in the immuno-
reactivity of MAP2 (49%) and tau (81%). Aβ was also shown to
CHEMISTRY
■
Schemes 1 and 2 present the strategies used to synthesize novel
neuroprotective agents with a structural scaffold composed
of hydroxyl-functionalized stilbene and 2-arylbenzo[b]furan
derivatives. The series of hydroxyl-functionalized stilbene
derivatives was synthesized using the following method. First,
trimethoxybenzyl alcohol 1 was brominated with phosphorus
tribromide to produce trimethoxybenzyl bromide. Then,
trimethoxybenzyl bromide was reacted with triphenyl phos-
phine to produce trimethoxybenzyl phosphorus ylide 2.21,23
Subsequently, a Wittig reaction involving 4-bromobenzaldehyde
and phosphorus ylide 2 was performed, resulting in the forma-
tion of stilbene 3 with a selectivity of approximately E/Z = 3/1.
The E configuration of bromo-substituted stilbene 3 was then
coupled with ethyl acrylate via a palladium-catalyzed Heck
coupling reaction to produce (E)-ethyl acrylate substituted
stilbene 4. Finally, the methoxyl groups were deprotected from
compound 4 using boron tribromide to obtain the desired
hydroxyl-functionalized stilbene 5 (Scheme 1).
Similarly, 2-arylbenzo[b]furan 9 was prepared using the
Wittig reaction to convert substituted phosphonium ylide 7 and
salicylaldehyde into stilbene 8. Stilbene 8 was subsequently
cyclized in a basic iodine solution to obtain bromo-substituted
2-arylbenzo[b]furan 9, as shown in Scheme 2. The bromo-
substituted 2-arylbenzo[b]furan 9 was then coupled with ethyl
acrylate via a palladium-catalyzed Heck coupling reaction to
produce (E)-ethyl acrylate substituted 2-arylbenzo[b]furan 10.
Finally, the methoxyl groups were removed from compound 10
using the boron tribromide treatment described in Scheme 1
in order to obtain a satisfactory yield of hydroxyl-functionalized
2-arylbenzo[b]furan 11.21
RESULTS
■
Hydroxyl-Functionalized Stilbene and 2-Arylbenzo[b]-
furan Derivatives Confer Protective Effects on Cortical
Neurons by Reducing fAβ25−35- and Glutamate-
Mediated Neurotoxicity. The maximum nontoxic concen-
tration of each synthesized hydroxyl-functionalized stilbene and
2-arylbenzo[b]furan was determined before further analyses
were performed. (For details regarding these determinations,
refer to Table S1 in the Supporting Information.) Upon so
doing, the synthesized hydroxyl-functionalized stilbenes and
2-arylbenzo[b]furans were evaluated for neuroprotective
effects against fibril amyloid β protein 25−35 (fAβ25−35)- or
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we cultured mixed glial cells for 16 days, at which point
microglia exhibited a dendritic cell-like appearance.24 Glial
function was then characterized according to the production of
nitric oxide (NO) induced by 100 ng/mL lipopolysaccharides
(LPS). Interestingly, we found that the level of LPS-induced
NO production depended on the amount of time that elapsed
after the medium was refreshed. (For details pertaining to this
investigation, refer to Figure S2A.) Thus, the cultures that were
activated 1 and 2 days after the medium had been refreshed
were respectively used as cell models for screening the
promotion and inhibition of NO production. Treatment with
50 μM of compounds 18 and 36 was shown to increase NO
production by 39 and 90%, respectively, in cultures that were
activated 1 day after the medium was refreshed (Figure S2B).
Treating cells with 50 μM of any other compound led to
varying effects on NO production in culture that were activated
2 days after the medium was refreshed (Figure S2C). Table 4
lists (1) EC50 values that describe the degree to which selected
compounds inhibit NO production and (2) the antioxidant
capacity of selected compounds as determined by trolox
equivalent antioxidant capacity (TEAC).
In Vitro Pharmacokinetic Studies and Calculated
Physicochemical Properties. A preliminary assessment of
the in vitro ADMET for active compounds 5, 11, 37, and 40
was completed prior to performing animal assays. In so doing,
we found that CYP3A4 appears to be the most abundant
cytochrome P450 isoform in the human liver. To assess
potential interactions with CYP3A4 substrates, we sought to
determine the effects of hydroxyl-functionalized stilbene
(compound 5) and 2-arylbenzo[b]furans (compounds 11, 37,
and 40) on CYP3A4 activity. We observed that the interaction
between CYP3A4 and compound 5 was less pronounced than
the interaction between CYP3A4 and compounds 11, 37, and
40 (Table 4). Additionally, we calculated the physicochemical
properties of compounds 5, 11, and 37 in order to evaluate their
brain penetration capabilities and determine whether they meet
the overall pharmacokinetics and toxicological requirements to
qualify as a central nervous system drug candidate. Specifically,
the physicochemical properties were calculated using QikProp
(Table 5), which has proven to be highly effective in designing
molecules that maximize brain exposure. The physicochemical
properties we considered included lipophilicity, as expressed
by the calculated logarithm of the octanol/water partition
coefficient (QPlogPo/w), the brain/blood partition coefficient
(QPlogBB), Caco-2 cell permeability (QPPCaco), total solvent
accessible surface area (SASA), and human oral absorption
(HOA). The selected compounds presented an excellent drug-
like profile with no violations of either Lipinski’s rule of five or
Jorgensen’s rule of three.
Table 3. Neuroprotective and Anti-inflammatory
Activities of Hydroxyl-Functionalized Stilbenes and
2-Arylbenzo[b]furans on Cortical Neurons and Mixed Glial
a
Culture
Aβ-mediated
Glu-mediated
LPS-induced glial
neuronal death
neuronal death
NO production
b
b
b
compound
(%)
(%)
(% of vehicle)
vehicle
TAB
TABM
4
39.20 0.88
24.66 6.31***
40.12 7.23
44.43 4.65
19.23 4.66***
25.45 6.14***
39.67 0.84
38.18 0.03
41.76 3.36
38.78 0.74
38.07 1.97
38.17 1.53
39.08 2.78
41.24 0.94
36.56 1.28
42.53 2.89
41.59 6.59
41.57 0.98
41.19 2.36
35.98 3.64
41.50 0.26
41.62 4.44
36.93 0.60
45.74 0.41
37.67 0.49
46.98 2.17
43.51 1.77
45.46 1.90
48.16 3.08
43.98 2.86
21.18 3.17***
42.34 1.69
45.80 1.96
16.95 1.30***
30.10 7.00
4.11 0.13***
31.58 1.50**
41.22 3.40
42.43 5.02
20.38 9.20***
44.56 2.93
28.05 3.40**
21.00 0.89**
42.89 2.38
44.02 1.96
43.95 0.67
41.17 2.40
44.30 0.38
49.21 2.32
41.26 2.09
42.73 1.88
43.30 2.09
43.06 2.32
39.47 5.67
41.64 1.95
42.05 1.37
43.29 0.88
42.09 3.27
37.28 2.97
41.07 2.14
45.69 1.40
47.10 3.06
38.42 2.63
30.94 1.51
36.43 0.53
37.02 3.31
46.84 3.52
24.48 3.68***
41.89 1.48
43.67 1.13
31.45 2.42
33.02 3.01
40.83 0.98
43.50 1.45
100
98.77 9.32
99.63 7.55
91.44 16.18
72.93 12.67*
49.61 7.04*
102.71 3.44
95.92 2.51
5
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
36
37
38
39
40
41
42
43
127.76 22.65
94.57 29.33
106.26 2.19
70.98 16.18**
139.87 26.41***
89.35 22.23
73.27 1.36**
114.19 19.73
123.48 29.02
75.33 25.27
26.02 2.39***
75.60 11.13
88.34 14.03
74.88 19.64*
59.11 3.05***
48.79 6.21***
105.48 10.60
101.00 7.00
81.35 13.43
83.73 15.24
27.99 11.95*
202.71 26.3***
40.35 19.47**
24.92 7.68***
106.67 9.87
86.85 6.71
101.84 9.16
61.64 12.53**
38.13 11.25***
a
Cortical neurons were incubated with the vehicle (0.1% DMSO) or
with the maximum nontoxic concentration of hydroxyl-functionalized
stilbenes and 2-arylbenzo[b]furans for 2 h before being exposed to
Aβ (10 μM) for 48 h or glutamate (30 μM) for 24 h. After treating the
mixed glial cells with LPS for 48 h, culture medium was collected to
b
determine the nitrite content. Concentration of compounds used to
Compounds 5, 11, and 37 were selected for in vivo assays
due to their effectiveness in in vitro assays. For these assays, we
focused on nesting, as this behavior involves a broad network of
brain regions. Results of this investigation showed that APP/
PS1 mice had lower nesting scores and less nestlet shredding
than did WT mice. Furthermore, APP/PS1 mice treated with
compounds 11 and 37 (but not 5) registered higher nesting
scores than that treated with vehicle (Figure 4A) and presented
more pronounced nestlet shredding (Figure 4B).
investigate Aβ-mediated neuronal death and LPS-induced glial NO
production are listed in Table S1. Results are presented as the mean
SD from three independent experiments. Significant differences
between the cell group treated with the vehicle and other groups are
indicated as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
provoke such a decrease; however, it was less pronounced.
Nonetheless, treatment with compounds 5, 11, and 37 signifi-
cantly attenuated both of these decreases (Figure 3A,E,F).
Hydroxyl-Functionalized Stilbene and 2-Arylbenzo[b]-
furan Derivatives Show Antioxidant Activities and Confer
Anti-inflammatory Effects on a Mixed Glial Culture. To
examine the anti-inflammatory activities of the hydroxyl-
functionalized stilbene and 2-arylbenzo[b]furan derivatives,
DISCUSSION
■
In neuroprotection assays, five compounds (5, 11, 37, 40, and 42)
were found to protect neurons against Aβ-mediated neurotoxicity.
Among these five compounds, three (5, 11, and 37) were also
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Table 4. Neuroprotective and Anti-inflammatory Activities, Inhibition of CYP3A4, and Antioxidant Capacity of Hydroxyl-
a
Functionalized Stilbenes and 2-Arylbenzo[b]furans
Aβ-mediated neuronal death
(EC50, μM)
Glu-mediated neuronal death
LPS-induced glial NO
CYP3A4 inhibition
antioxidant capacity
(TEAC, mM)
compound
(EC50, μM)
(EC50, μM)
(IC50, μM)
TAB
TABM
5
35.67 8.39
n.d.
n.d.
n.d.
n.d.
1.27 0.20
1.48 0.31
0.14 0.01
0.01 0.01
n.d.
>MNTC
>MNTC
46.08 7.36
>MNTC
>MNTC
>MNTC
>MNTC
32.99 5.41
>MNTC
>MNTC
>MNTC
>MNTC
n.d.
n.d.
40.89 6.45
>MNTC
99.24 9.89
>MNTC
36.2 4.2
11.2 3.6
n.d.
11
17
>MNTC
66.96 17.51
48.94 14.02
84.81 4.68
42.95 5.47
56.60 11.34
39.24 11.94
>MNTC
24
>MNTC
n.d.
n.d.
29
>MNTC
n.d.
n.d.
34
>MNTC
n.d.
n.d.
37
52.95 6.33
>MNTC
21.7 2.2
n.d.
0.94 0.08
0.97 0.06
0.06 0.04
0.12 0.13
0.20 0.07
38
40
36.45 7.86
26.71 5.39
>MNTC
23.5 4.7
n.d.
42
>MNTC
43
82.55 19.07
n.d.
a
Results of this investigation are also expressed as EC50 values representing the mean SD of three independent experiments. n.d., not determined.
MNTC, maximum nontoxic concentration.
Figure 2. Hydroxyl-functionalized stilbene and 2-arylbenzo[b]furan protect cortical neurons against Aβ- and glutamate-induced neurite damage.
(A) Cortical neurons incubated with 50 μM of the indicated compounds for 2 h before being exposed to Aβ (10 μM) for 48 h or to glutamate
(30 μM) for 24 h. (B) Cell viability assessed using the live/dead cell viability assay. The number of live cells was calculated according to the number
of calcein AM positive cells. (C) Integrated density of live cells based on the average integrated density of calcein AM positive cells. Results are
expressed relative to cells treated with the vehicle alone and represent the mean SEM from three independent experiments. Significant differences
between cells treated with toxin and toxin plus compounds are indicated as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
found to confer protection against glutamate-mediated neuro-
toxicity. A comparison of compounds with the 3,4,5-trimethoxy
ring (4), pyrogallol ring (5), and pyrogallol ring but with
acrylate substituted at the R4 position (24) revealed that
compounds 4 and 24 do not confer neuroprotective effects.
This suggests that the pyrogallol ring and acrylate substituted
at the R3 position are responsible for the neuroprotective
effects of the stilbene scaffold. Moreover, as compared with
compound 11, compound 43 was shown to protect neurons
against fAβ25−35-mediated neurotoxicity, but not against
glutamate-mediated neurotoxicity. This suggests that the hydroxyl
substitution at the R4 position of compound 43 counteracts the
neuroprotective effects against glutamate-mediated neurotoxicity.
In addition, compound 39 differs from compound 37 in that it
has a bromide substituent in the R2 position. This substitution
eliminated the neuroprotective benefits shown by compound 37,
which suggests that the acrylate substitution in the R2 position is
responsible for the neuroprotective effects of the 2-arylbenzo[b]-
furan scaffold. Furthermore, unlike compound 40 with the
catechol ring on the 2-position of 2-arylbenzo[b]furan, pyrogallol
ring 39 and 4-hydroxyphenyl ring 41 did not appear to have
neuroprotective effects against Aβ-mediated neurotoxicity, which
suggests that the catechol ring of 40 is important for this activity.
Overall, compounds 5, 11, and 37 were more potent than the
reference compounds TAB and TABM against both Aβ- and
glutamate-mediated neurotoxicity (Table 3).
In the neurite protection assay, compounds 40 and 42 were
shown to protect neurites against Aβ-mediated neurotoxicity,
which suggests that the bromo substitution is also important
for the protection of neurites (Figure 3). Mixed glial cultures
F
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Figure 3. Hydroxyl-functionalized stilbene and 2-arylbenzo[b]furan protect neurites against Aβ- and glutamate-induced cell death. Cortical neurons
were incubated with 50 μM of the indicated compounds for 2 h before being exposed to either 10 μM Aβ for 48 h (A) or 30 μM glutamate for 24 h
(B). (A, B) Representative images showing the immunocytochemistry of MAP2 (red) and tau (green). The integrated density of MAP2 (C, D) and
tau protein (E, F) was also measured. Results are expressed relative to cells treated with vehicle alone and represent the mean SEM from three
independent experiments. Significant differences between cells treated with Aβ and control are indicated as follows: **, p < 0.01; ***, p < 0.001.
Significant differences between cells treated with Aβ plus vehicle and compounds are indicated as follows: #, p < 0.05; ##, p < 0.01; and
###, p < 0.001.
a
Table 5. Calculated Physicochemical Properties for Selected Compounds Using QikProp 2.2
b
c
d
e
f
g
h
compound
SASA
QPlogPo/w
QlogS
QPlogHERG
QPlogBB
QPPCaco
%HOA
5
655.9
583.8
652.2
2.95
3.19
1.88
−4.71
−4.38
−4.47
−6.29
−5.38
−6.01
−2.2
−1.2
−2.6
118.8
346.9
37.4
81.3
91.1
66.1
11
37
a
b
Calculated properties and their recommended range, according to the QikProp user manual. SASA: total solvent accessible surface area in Å2
c
d
(300−1000). QPlogPo/w: predicted octanol/water partition coefficient (−2.00 to 6.5). QPlogS: predicted aqueous solubility (S in mol dm−3;
e
f
log S −6.5 to 0.5). QPlogHERG: predicted IC50 value for blockage of HERG K+ channels (<−5). QPlogBB: predicted brain/blood partition
g
h
coefficient (−3.0 to −1.2). QPPCaco: predicted Caco-2 cell permeability in nm/s (>500, great; <25%, poor). %HOA: % human oral absorption
(>80%, high; <25%, poor).
G
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EXPERIMENTAL SECTION
■
Compound Synthesis and Characterization. All reactions were
conducted using dried glassware in an oven at 120 °C overnight, after
which the samples were underwent cooling in a desiccator. Unless
otherwise specified, all reagents were used as received from
commercial suppliers. Dichloromethane (DCM) and N,N′-dimethyl-
formamide (DMF) were dried over calcium hydride for 48 h prior
to distillation. Tetrahydrofuran (THF) was distilled from sodium/
1
benzophenone ketyl under nitrogen. H NMR spectra were obtained
using Bruker Avance 400 (400 MHz), Varian Unity Inova 500 (500 MHz),
and Varian VNMRS600 (600 MHz) spectrometers. All NMR chemical
shifts are reported as δ values in parts per million (ppm), and coupling
constants (J) are given in hertz (Hz). The abbreviations of splitting
patterns are as follows: singlet (s); doublet (d); triplet (t); quartet (q);
broad (br); multiplet that remains unresolved due to the field strength
of the instrument (m); doublet of doublets (dd); doublet of triplets
(dt); and doublet of doublets of doublets (ddd). Melting points
were measured using a Yanaco MP-S3 micro melting point apparatus
and are not corrected. Fourier transform infrared spectra were col-
lected using an Avatar 320 spectrometer. Mass spectra and high-
resolution mass spectra were recorded on ThermoQuest Finnigan and
ThermoQExactive Focus mass spectrometers, respectively. Purification
was performed using preparative separations in flash column chromato-
graphy (Merck silica gel 60, particle size of 230−400 mesh). Analytical
TLC was conducted on precoated plates (Merck silica gel 60, F254).
Compounds analyzed on TLC plates were visualized using a UV light,
I2 vapor, or basic aqueous potassium permanganate (KMnO4) under
heating. All compounds submitted for biological testing were confirmed
to be of >95% purity, based on HPLC chromatograms obtained using a
Shimadzu VP series HPLC in conjunction with a Shiseido Capcell PAK
C18 column (250 mm × 4.6 mm, 5 μm, 1.0 mL/min flow rate).
General Procedure for the Synthesis of 5-(4-Bromostyryl)-
1,2,3-trimethoxybenzene (3) via Wittig Reaction. A solution of
phosphorus ylide 2 (354 mg, 0.68 mmol) in THF (30 mL) was cooled
to 0 °C under nitrogen, and lithium tert-butoxide (108 mg, 1.35 mmol)
was added portionwise. The mixture was stirred at 0 °C for 30 min.
A solution of 4-bromobenzaldehyde (126 mg, 0.68 mmol) in THF
(7 mL) was added dropwise at 0 °C, and the reaction mixture was
warmed to room temperature and stirred for 24 h. Saturated aqueous
NH4Cl solution was added to the reaction mixture, and the mixture was
extracted with EtOAc (20 mL × 3). The combined organic layers were
washed with brine, dried with MgSO4, filtered, and concentrated. The
residue was purified by silica gel column chromatography to yield 3
(164 mg, 69%, combined yield, E/Z = 3/1).
General Procedure for the Synthesis of Ethyl (E)-3-(4-((E)-
3,4,5-Trimethoxystyryl)phenyl)acrylate (4) via Palladium-Cata-
lyzed Coupling Reaction. A solution of (E)-5-(4-bromostyryl)-
1,2,3-trimethoxybenzene 3 (300 mg, 0.86 mmol), ethyl acrylate
(0.11 mL, 1.03 mmol), palladium(II) acetate (10 mg, 0.045 mmol),
copper(I) iodide (9 mg, 0.045 mmol), and triethylamine (0.24 mL) in
N,N′-dimethylformamide (10 mL) under a nitrogen atmosphere was
heated at 70 °C for 24 h until the reaction was complete, as indicated
by TLC. The reaction mixture was quenched with water and extracted
with ethyl acetate. The organic layers were combined, dried over
MgSO4, and concentrated. The residue was purified by column
chromatography to give ethyl (E)-3-(4-((E)-3,4,5-trimethoxystyryl)-
phenyl)acrylate 4 (203 mg, 64%) as a white solid. mp 178−180 °C;
1H NMR (500 MHz, CDCl3): δ 7.66 (d, J = 16.0 Hz, 1H), 7.49 (s,
4H), 7.07 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 16.0 Hz, 1H), 6.73 (s, 2H),
6.42 (d, J = 16.0 Hz, 1H), 4.24 (q, J = 7.5 Hz, 2H), 3.90 (s, 6H), 3.86
(s, 3H), 1.32 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3):
δ 167.0, 153.0, 144.0, 139.5, 133.2, 132.2, 131.3, 129.5, 129.1, 127.9,
118.0, 106.1, 60.9, 60.5, 55.9, 14.3; HRMS m/z (M + 1)+ calcd for
C22H25O5, 369.1697; found, 369.1693.
Figure 4. Compounds 11 and 37 were shown to restore the negative
nesting behavior of APP/PS1 mice. Five-month-old wild type mice
were orally administered vehicle (WT), whereas APP/PS1 mice were
orally administered vehicle (V) as well as compound 5 (5), 11 (11), or
37 (37) over a period of 60 days before conducting nest construction
tests. (A) Nesting scores and (B) the number of unshredded nestlets.
The results represent the mean SD of all WT or APP/PS1 mice.
Significant differences between WT and APP/PS1 groups are indicated
as follows: ***, p < 0.001. Significant differences between compound-
treated and control (V) groups are indicated as follows: #, p < 0.05;
##, p < 0.01; ###, p < 0.001.
activated by LPS to produce NO were used to evaluate the
antineuroinflammatory effects of the compounds.25,26 Results
of this assay revealed that 13 compounds (5, 11, 17, 20, 24,
27, 28, 29, 34, 37, 38, 42, and 43) possessed antineuroin-
flammatory activities and compounds 24, 34, 37, and 38 were
the most active (Tables 3 and 4).
APP/PS1 mice obtained lower nesting scores and presented
less nestlet shredding than did WT mice. Nonetheless,
compounds 11 or 37 (but not compound 5) were shown
to reverse this decline. These results may be due to the fact
that compound 5 is a stilbene, whereas 11 and 37 are
2-arylbenzo[b]furans. The molecular structure of hydroxyl-
functionalized stilbene 5 is close to the naturally occurring
polyphenol resveratrol. Resveratrol has a very limited half-life
in plasma and poor bioavailability.27 Therefore, despite the
fact that compound 5 was active in all in vitro assays, it may
not have been effective in the in vivo assay due to the same
issue. Further pharmacokinetic and metabolic studies on these
compounds are currently under active investigation and will be
reported in due course.
CONCLUSIONS
■
Much of the current research aimed at developing therapeutic
drugs for AD is based on the hypothesis that Aβ plays a
pivotal role in the onset and progression of AD and that the
secondary consequences of Aβ generation and deposition
(oxidation, inflammation, and excitotoxicity) exacerbate disease
progression. This leads to the conclusion that limiting
oxidation, excitotoxicity, and inflammation may be beneficial
as disease-modifying strategies.28 In the current study, we
demonstrate the efficient synthesis of hydroxyl-functionalized
stilbene and 2-arylbenzo[b]furan derivatives and report on
the neuroprotective and anti-inflammatory effects of these
planar phenolic compounds in vitro and in an animal model.
Hydroxyl-functionalized stilbenes and 2-arylbenzo[b]furans
appear to confer neuroprotective effects on neuron cells
and antineuroinflammatory effects on glial cells, whereas only
2-arylbenzo[b]furans were shown to ameliorate ADL in
APP/PS1 mice. Taken together, our findings suggest that
hydroxyl-functionalized 2-arylbenzo[b]furans could be a useful
starting point for future efforts to develop and improve treat-
ments for AD.
General Procedure for the Synthesis of 2-(2-Bromo-4,5-
dimethoxyphenyl)benzofuran (9). A solution of 2-(2-bromo-4,5-
dimethoxystyryl)phenol 8 (241 mg, 0.72 mmol) in 20 mL of THF
was mixed with potassium carbonate (594 mg, 4.3 mmol) and iodine
(1.09 g, 4.3 mmol). The mixture was stirred at room temperature
for 3 h until the reaction was complete, as indicated by TLC.
H
DOI: 10.1021/acs.jmedchem.7b00376
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Journal of Medicinal Chemistry
Article
Saturated NaHSO3 aqueous solution was added to the solution, and
the mixture was extracted with ethyl acetate. The organic layers were
combined and dried over MgSO4. The residue was purified by flash
column chromatography to afford the title compound (194 mg, 81%)
7.07 (d, J = 1.5 Hz, 1H), 6.93 (dd, J = 8.0, 1.5 Hz, 1H), 6.86 (d, J =
16.0 Hz, 1H), 6.77 (dd, J = 8.0, 1.5 Hz, 1H), 6.25 (d, J = 16.0 Hz, 1H),
6.00 (s, 1H), 5.95 (s, 2H), 4.23 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 1.30
(t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 167.1, 148.1,
147.5, 147.4, 143.8, 143.5, 136.1, 132.1, 127.0, 124.5, 121.7, 119.5,
119.4, 118.0, 109.2, 108.3, 105.7, 101.1, 60.3, 56.1, 14.3; HRMS m/z
(M + 1)+ calcd for C21H21O6, 369.1333; found, 369.1326.
Ethyl (E)-3-(3-((E)-2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-4-hydroxy-5-
methoxyphenyl)acrylate (18). Yellow solid; mp 155−157 °C;
1H NMR (500 MHz, CDCl3): δ 7.63 (d, J = 15.5 Hz, 1H), 7.32 (d,
J = 1.5 Hz, 1H), 7.19 (d, J = 16.5 Hz, 1H), 7.10 (d, J = 16.5 Hz, 1H),
7.09 (d, J = 1.5 Hz, 1H), 6.95 (dd, J = 8.0, 1.5 Hz, 1H), 6.92 (d, J =
1.5 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.32 (d, J = 16.0 Hz, 1H), 6.14
(s, 1H), 5.96 (s, 1H), 4.25 (d, J = 7.5 Hz, 2H), 3.93 (s, 3H), 1.32 (t,
J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 167.2, 148.1, 147.4,
146.9, 145.3, 144.8, 135.2, 135.1, 132.1, 130.4, 129.9, 127.9, 126.4,
124.0, 121.7, 120.4, 120.2, 115.9, 108.4, 107.5, 105.7, 101.1, 60.4, 56.2,
14.4; HRMS m/z (M + 1)+ calcd for C21H21O6, 369.1333; found,
369.1327.
(E)-Ethyl 3-(2-((E)-2-Hydroxy-3-methoxystyryl)-4,5-
dimethoxyphenyl)acrylate (19). Amorphous powder; 1H NMR
(500 MHz, CDCl3): δ 8.10 (d, J = 16.0 Hz, 1H), 7.53 (d, J =
16.0 Hz, 1H), 7.19 (d, J = 16.0 Hz, 1H), 7.16 (dd, J = 8.0, 1.0 Hz, 1H),
7.10 (s, 1H), 7.02 (s, 1H), 6.84 (t, J = 8.0 Hz, 1H), 6.77 (dd, J = 8.5,
1.5 Hz, 1H), 6.27 (d, J = 16.0 Hz, 1H), 6.01 (s, 1H), 4.25 (q, J =
7.5 Hz, 2H), 3.95 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 1.32 (t, J =
7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 167.2, 150.9, 148.8,
146.7, 143.5, 141.8, 132.1, 125.9, 125.3, 125.3, 123.6, 119.6, 119.1,
117.6, 109.7, 108.8, 108.8, 60.4, 56.1, 56.0, 55.9; HRMS m/z (M + 1)+
calcd for C22H25O6, 385.1646; found, 385.1638.
1
as a yellowish solid. H NMR (600 MHz, CDCl3): δ 7.60 (d, J =
7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.45 (s, 1H), 7.44 (s, 1H), 7.29
(dt, J = 7.8, 1.8 Hz, 1H), 7.23 (dt, J = 7.8, 1.8 Hz, 1H), 7.13 (s, 1H),
3.96 (s, 3H), 3.91 (s, 3H); 13C NMR (150 MHz, CDCl3): δ 154.0,
153.2, 149.4, 148.4, 129.0, 124.5, 123.5, 122.9, 121.2, 116.7, 111.9,
111.5, 111.0, 105.9, 56.2, 56.2; HRMS m/z (M + 1)+ calcd for
C16H14BrO3, 333.0121; found, 333.0119.
General Procedure for the Synthesis of (E)-Ethyl 3-(2-
(benzofuran-2-yl)-4,5-dihydroxyphenyl)acrylate (11). To a
solution of 10 (254 mg, 0.72 mmol) in dry dichloromethane (20 mL)
at −60 °C under N2 was added BBr3 (0.40 mL, 4.32 mmol) dropwise.
The reaction mixture was then allowed to warm to −40 °C and stirred
for another 2 h until complete, as indicated by TLC. The reaction was
carefully mixed with saturated aqueous NaHCO3 (30 mL) at 0 °C and
stirred for 30 min. This mixture was extracted with ethyl acetate twice
(20 mL × 2), and the organic portion was combined, washed further with
brine, and dried with MgSO4. The residue was filtered, concentrated, and
purified by silica gel column chromatography to yield 11 as a white solid
(168 mg, 72%). mp 87−89 °C; 1H NMR (600 MHz, CDCl3): δ 8.11 (d,
J = 16.0 Hz, 1H), 7.58 (dd, J = 8.0, 1.2 Hz, 1H), 7.48 (dd, J = 8.0, 0.4 Hz,
1H), 7.34 (s, 1H), 7.27 (dt, J = 8.0, 0.4 Hz, 1H), 7.22−7.17 (m, 1H),
7.17 (s, 1H), 6.70 (s, 1H), 6.28 (d, J = 16.0 Hz, 1H), 5.95 (br, 1H), 5.87
(br 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3): δ 167.3, 154.7, 153.4, 145.4, 144.4, 143.0, 129.1,
126.4, 124.6, 124.5, 123.0, 121.1, 118.6, 115.1, 113.9, 111.1, 106.7, 60.6,
14.3; HRMS m/z (M − 1)− calcd for C19H15O5, 323.0914; found,
323.0918.
Ethyl (E)-3-(2-((E)-2-Hydroxystyryl)-4,5-dimethoxyphenyl)acrylate
1
(20). Yellow solid; mp 158−161 °C; H NMR (500 MHz, CDCl3): δ
(E)-2-(2-Bromo-4,5-dimethoxystyryl)-6-methoxyphenol (12).
1
8.10 (d, J = 16.2 Hz, 1H), 7.52 (dd, J = 7.8, 1.8 Hz, 1H), 7.45 (d, J =
16.2 Hz, 1H), 7.14 (dt, J = 7.8, 1.8 Hz, 1H), 7.13 (d, J = 16.2 Hz, 1H),
7.07 (s, 1H), 7.03 (s, 1H), 6.94 (dt, J = 7.8, 1.2 Hz, 1H), 6.79 (dd, J =
8.4, 1.2 Hz, 1H), 6.27 (d, J = 16.2 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H),
3.95 (s, 3H), 3.91 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H); 13C NMR
(125 MHz, CDCl3): δ 167.3, 153.1, 150.1, 148.9, 141.9, 131.9, 128.9,
127.5, 126.5, 125.7, 125.4, 124.6, 121.2, 117.7, 116.0, 109.0, 108.8,
60.5, 56.0, 56.0, 14.4; HRMS m/z (M + 1)+ calcd for C21H23O5,
355.1540; found, 355.1533.
White solid; mp 156−158 °C; H NMR (500 MHz, CDCl3): δ 7.00
(s, 1H), 6.75 (d, J = 12.0, 1H), 6.72−6.65 (m, 3H), 6.60 (t, J = 8.0 Hz,
1H), 5.77 (s, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.43 (s, 3H); 13C NMR
(125 MHz, CDCl3): δ 148.8, 147.6, 146.6, 143.5, 129.6, 129.5, 124.8,
123.0, 122.1, 119.1, 115.0, 114.4, 113.2, 109.5, 56.0, 56.0, 55.6; HRMS
m/z (M + 1)+ calcd for C17H18BrO4, 365.0383; found, 365.0378.
(E)-5-(2,3-Dimethoxystyryl)benzo[d][1,3]dioxole (13). White solid;
mp 143−145 °C; 1H NMR (500 MHz, CDCl3): δ 6.76 (d, J = 8.0 Hz,
1H), 6.75−6.72 (m, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.62−6.57 (m,
4H), 6.52 (dd, J = 8.0, 2.0 Hz, 1H), 5.88 (s, 2H), 3.84 (s, 3H), 3.82 (s,
3H); 13C NMR (125 MHz, CDCl3): δ 147.3, 145.8, 131.1, 129.9,
129.3, 125.3, 123.5, 121.9, 121.6, 111.2, 109.9, 109.5, 109.2, 108.1,
100.8, 60.8, 55.7; HRMS m/z (M + 1)+ calcd for C17H17O4, 285.1121;
found, 285.1117.
(E)-Ethyl 3-(3-Hydroxy-4-methoxy-2-((E)-3,4,5-trimethoxystyryl)-
phenyl)acrylate. (21). White solid; mp 174−176 °C; 1H NMR
(500 MHz, CDCl3): δ 8.00 (d, J = 16.0 Hz, 1H), 7.15 (d, J = 16.5 Hz,
1H), 7.11 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 16.5 Hz, 1H), 6.77 (d, J =
8.5 Hz, 1H), 6.72 (s, 1H), 6.27 (d, J = 16.0 Hz, 1H), 6.08 (s, 1H), 4.21
(q, J = 7.5 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 6H), 3.84 (s, 3H), 1.27 (t,
J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 167.0, 153.2, 147.4,
143.7, 143.6, 138.1, 136.3, 133.3, 126.9, 124.3, 120.8, 119.4, 118.0,
109.3, 103.7, 60.8, 60.2, 56.0, 56.0, 14.5; HRMS m/z (M + 1)+ calcd
for C23H27O7, 415.1751; found, 415.1747.
Ethyl (E)-3-(6-((E)-3,4,5-Trimethoxystyryl)benzo[d][1,3]dioxol-5-
1
yl)acrylate (14). Yellow solid; mp 123−125 °C; H NMR (600 MHz,
CDCl3): δ 8.04 (d, J = 15.6 Hz, 1H), 7.27 (d, J = 15.6 Hz, 1H), 7.03 (d,
J = 6.6 Hz, 1H), 6.77 (d, J = 16.2 Hz, 1H), 6.71 (s, 2H), 6.23 (d, J =
15.6 Hz, 1H), 6.00 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.90 (s, 6H), 3.85
(s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 167.1,
153.4, 149.7, 147.8, 141.5, 138.2, 133.1, 132.9, 131.5, 126.8, 124.6, 118.1,
106.3, 106.0, 103.8, 101.6, 61.0, 60.5, 56.2, 14.3; HRMS m/z (M + 1)+
calcd for C23H25O7, 413.1595; found, 413.1586.
(E)-Ethyl 3-(4,5-Dimethoxy-2-((E)-3,4,5-trimethoxystyryl)phenyl)-
1
acrylate (22). White solid; mp 116−118 °C; H NMR (500 MHz,
CDCl3): δ 7.83 (d, J = 16.0 Hz, 1H), 7.04 (s, 1H), 6.70 (s, 1H), 6.67
(d, J = 12.0 Hz, 1H), 6.62 (d, J = 12.0 Hz, 1H), 6.31 (s, 1H), 6.18 (d,
J = 16.0 Hz, 1H), 4.18 (q, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H),
3.70 (s, 3H), 3.58 (s, 6H), 1.28 (t, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3): δ 167.0, 152.7, 150.6, 148.4, 142.3, 137.3, 132.2,
132.1, 131.8, 127.1, 125.4, 116.9, 112.2, 108.5, 106.3, 60.8, 60.3, 55.9,
55.8, 55.8, 14.3; HRMS m/z (M + 1)+ calcd for C24H29O7, 429.1908;
found, 429.1902.
(E)-Ethyl 3-(2-((E)-3,4-Dimethoxystyryl)-3-hydroxy-4-
methoxyphenyl)acrylate (16). White solid; mp 108−112 °C;
1H NMR (500 MHz, CDCl3): δ 8.01 (d, J = 15.5 Hz, 1H), 7.14 (d,
J = 8.5 Hz, 1H), 7.13 (d, J = 16.5 Hz, 1H), 7.07−7.04 (m, 2H), 6.88
(d, J = 16.5 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.5 Hz,
1H), 6.27 (d, J = 16.0 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H),
3.92 (s, 3H), 3.89 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3): δ 167.1, 149.1, 149.0, 147.4, 143.8, 143.5, 136.4,
130.7, 127.0, 124.7, 120.1, 119.4, 119.3, 118.0, 11.1, 109.2, 109.0, 60.3,
56.2, 56.0, 55.9, 14.3; HRMS m/z (M + 1)+ calcd for C22H25O6,
385.1646; found, 385.1642.
Ethyl (E)-3-(6-((E)-2-Hydroxy-3-methoxystyryl)benzo[d][1,3]-
1
dioxol-5-yl)acrylate (23). White solid; mp 168−170 °C; H NMR
(500 MHz, CDCl3): δ 8.08 (d, J = 16.0 Hz, 1H), 7.50 (d, J = 16.0 Hz,
1H), 7.17 (d, J = 16.0 Hz, 1H), 7.15 (dd, J = 8.0, 1.0 Hz, 1H), 7.13 (s,
1H), 7.00 (s, 1H), 6.83 (t, J = 8.0 Hz, 1H), 6.77 (dd, J = 8.0, 1.5 Hz,
1H), 6.22 (d, J = 16.0 Hz, 1H), 6.00 (s, 1H), 5.98 (s, 2H), 4.23 (q, J =
7.0 Hz, 2H), 3.90 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3): δ 167.2, 149.7, 147.6, 146.7, 143.6, 141.7, 133.8,
126.8, 125.7, 125.7, 123.5, 119.6, 119.2, 117.9, 109.7, 106.2, 105.9,
Ethyl (E)-3-(2-((E)-2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-3-hydroxy-4-
methoxyphenyl)acrylate. (17). Yellow solid; mp 168−170 °C;
1H NMR (500 MHz, CDCl3): δ 8.00 (d, J = 16.0 Hz, 1H), 7.12
(d, J = 8.0 Hz, 1H), 7.09 (d, J = 16.0 Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H),
I
DOI: 10.1021/acs.jmedchem.7b00376
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Journal of Medicinal Chemistry
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101.5, 60.4, 56.1, 14.3; HRMS m/z (M + 1)+ calcd for C21H21O6,
369.1333; found, 369.1326.
1H), 6.76 (d, J = 8.0 Hz, 1H), 6.74 (dd, J = 8.0, 1.5 Hz, 1H),
5.95 (s, 2H), 3.89 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 148.1,
147.1, 146.7, 143.3, 132.5, 129.1, 123.8, 121.4, 121.3, 119.5, 118.6,
109.2, 108.3, 105.7, 101.0, 56.1; HRMS m/z (M + Na)+ calcd for
C16H14O4Na, 293.0784; found, 293.0736.
(E)-Ethyl 3-(6-(Benzofuran-2-yl)-2,3,4-trihydroxyphenyl)acrylate
(24). Yellow solid; mp 168−170 °C; 1H NMR (500 MHz,
CD3OD): δ 7.68 (d, J = 16.2, Hz, 1H), 7.68 (s, 1H), 7.52 (d, J =
7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.01 (d,
J = 16.2 Hz, 1H), 6.89 (d, J = 16.2 Hz, 1H), 6.59 (s, 2H), 6.55 (d, J =
16.2 Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H); 13C
NMR (125 MHz, CD3OD): δ 168.6, 147.1, 146.1, 140.1, 136.1, 134.8,
131.3, 130.3, 129.9, 128.9, 127.5, 127.1, 125.9, 119.2, 107.1, 61.6, 14.6;
HRMS m/z (M − 1)− calcd for C19H17O5, 325.1071; found, 325.1066.
(E)-Ethyl 3-(3-((E)-3,4,5-Trimethoxystyryl)phenyl)acrylate (25).
(E)-5-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-2-methoxyphenol (32).
1
White solid; mp 139−141 °C; H NMR (500 MHz, CDCl3): δ 7.01
(d, J = 19.0 Hz, 2H), 6.97 (d, J = 8.0 Hz, 1H), 6.91−6.84 (m, 4H),
6.77 (d, J = 8.0 Hz, 1H), 5.95 (s, 2H), 5.61 (s, 1H), 3.93 (s, 3H);
13C NMR (125 MHz, CDCl3): δ 148.1, 147.0, 146.7, 145.4, 132.1,
130.1, 127.0, 126.2, 121.0, 120.2, 114.5, 108.4, 108.1, 105.4, 101.1,
55.9; HRMS m/z (M + 1)+ calcd for C16H15O4, 271.0965; found,
271.0916.
1
White solid; mp 177−179 °C; H NMR (500 MHz, CDCl3): δ 7.68
(d, J = 16.2 Hz, 1H), 7.62 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.39 (d,
J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.04 (d, J = 16.2 Hz, 1H),
6.97 (d, J = 16.2 Hz, 1H), 6.72 (s, 2H), 6.46 (d, J = 15.6 Hz, 1H), 4.25
(q, J = 7.2 Hz, 2H), 3.89 (s, 6H), 3.85 (s, 3H), 1.32 (t, J = 7.2 Hz,
3H); 13C NMR (125 MHz, CDCl3): δ 166.8, 153.4, 144.3, 138.1,
137.8, 134.8, 132.6, 129.5, 129.1, 128.0, 127.2, 127.0, 125.9, 118.5,
103.6, 60.9, 60.5, 56.0, 14.2; HRMS m/z (M + 1)+ calcd for C22H25O5,
369.1697; found, 369.1693.
(E)-5-(3,4,5-Trimethoxystyryl)benzo[d][1,3]dioxole (33). White
solid; mp 129−131 °C; H NMR (500 MHz, CDCl3): δ 6.77 (m,
1
2H), 6.69 (dd, J = 7.5, 0.5 Hz, 1H), 6.49 (s, 2H), 6.44 (d, J = 12.0 Hz,
1H), 6.39 (d, J = 12.0 Hz, 1H), 5.89 (s, 2H), 3.82 (s, 3H), 3.69 (s,
6H); 13C NMR (125 MHz, CDCl3): δ 152.9, 147.3, 146.6, 137.2,
132.5, 131.1, 129.4, 129.1, 122.9, 109.0, 108.1, 106.0, 100.9, 60.9, 55.9;
HRMS m/z (M + 1)+ calcd for C18H19O5, 315.1227; found, 315.1223.
3-(3,4-Dimethoxystyryl)-4-hydroxy-5-methoxybenzaldehyde
1
(E)-4-Bromo-2-(3,4-dimethoxystyryl)-6-methoxyphenol (26). Yel-
(34). White solid; mp 157−159 °C; H NMR (500 MHz, CDCl3): δ
1
low solid; mp 105−107 °C; H NMR (600 MHz, CDCl3): δ 7.29 (d,
9.85 (s, 1H), 7.69 (d, J = 1.5 Hz, 1H), 7.28 (d, J = 1.5 Hz, 1H), 7.26−
7.18 (m, 2H), 7.10−7.07 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.50 (s,
1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H); 13C NMR (125 MHz,
CDCl3): δ 191.1, 149.2, 149.1, 148.6, 147.3, 130.6, 130.4, 129.2, 124.4,
124.1, 120.2, 119.5, 111.2, 108.9, 106.9, 56.3, 55.9, 55.9; HRMS m/z
(M + 23)+ calcd for C18H18O5Na, 337.1046; found, 337.1042.
Ethyl (E)-3-(3-((E)-3,4-Dimethoxystyryl)-4-hydroxy-5-
methoxyphenyl)acrylate (35). White solid; mp 171−173 °C;
1H NMR (500 MHz, CDCl3): δ 7.64 (d, J = 16.0 Hz, 1H), 7.34 (d,
J = 1.5 Hz, 1H), 7.22 (d, J = 16.5 Hz, 1H), 7.13 (d, J = 16.5 Hz, 1H),
7.09−7.06 (m, 2H), 6.91 (d, J = 1.5 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H),
6.32 (d, J = 16.0 Hz, 1H), 6.18 (br, 1H), 3.93 (s, 3H), 3.92 (s, 3H),
3.89 (s, 3H), 3.79 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 167.6,
149.1, 149.0, 146.9, 145.3, 145.1, 130.6, 130.1, 126.3, 124.1, 120.3,
120.1, 120.0, 115.4, 111.2, 108.9, 107.4, 56.2, 55.9, 55.9, 51.6; HRMS
m/z (M + 1)+ calcd for C21H23O6, 371.1489; found, 374.1488.
Biological Procedures. Cell Culture. Primary cultures of neonatal
cortical neurons were prepared as described previously.27 The Institu-
tional Animal Care and Use Committee at the National Research
Institute of Chinese Medicine approved the animal protocol. In brief,
the cortex was isolated from Sprague−Dawley rat pups by decapitation
and digested in 0.5 mg/mL papain at 37 °C for 15 min. The tissue was
dissociated in Hibernate A medium (containing B27 supplement) by
aspirating trituration. Cells were plated and maintained in Neuro-
basal medium containing B27 supplement, 10 units/mL penicillin,
10 μg/mL streptomycin, and 0.5 μg/mL glutamine for 3 days. Cells
were then exposed to cytosine-β-D-arabinofuranoside (5 μM) for 1 day
to eliminate the proliferation of non-neuronal cells. The cells were
used for Aβ and glutamate experiments at days in vitro (DIV) 5 and
DIV10, respectively. Neurons are vulnerable to Aβ-mediated toxicity at
DIV5 and expression of glutamate receptor at DIV10. Primary mixed
glial cells were prepared from the cerebral cortex and maintained in
DMEM/F12 medium containing 10% FBS for 3 days. The medium
was changed with fresh culture medium, and cells were incubated for
4 days. Thereafter, the cells were cultured in Neurobasal medium/B27
supplement for 9 more days.
J = 2.4 Hz, 1H), 7.18 (d, J = 16.2 Hz, 1H), 7.08−7.04 (m, 3H), 6.85−
6.83 (m, 2H), 5.84 (s, 1H), 3.93 (s, 3H), 3.89 (s, 6H); 13C NMR
(150 MHz, CDCl3): δ 149.1, 149.0, 147.2, 142.6, 131.6, 130.5, 130.3,
125.5, 121.1, 120.2, 119.6, 112.3, 111.7, 111.2, 108.9, 56.4, 56.0;
HRMS m/z (M + 1)+ calcd for C17H18BrO4, 365.0384; found,
365.0379.
Ethyl (E)-3-(6-((E)-4-Methoxystyryl)benzo[d][1,3]dioxol-5-yl)-
acrylate (27). White solid; mp 84−86 °C; 1H NMR (600 MHz,
CDCl3): δ 8.07 (d, J = 15.6 Hz, 1H), 7.43 (dd, J = 7.2, 2.4 Hz, 2H),
7.25 (d, J = 15.6 Hz, 1H), 7.04 (s, 1H), 7.00 (s, 1H), 6.88 (dd, J = 7.2,
1.8 Hz, 2H), 6.81 (d, J = 15.6 Hz, 1H), 6.22 (d, J = 15.6 Hz, 1H), 5.98
(s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 3.82 (s, 3H), 1.31 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3): δ 167.2, 159.5, 149.7, 147.5,
141.6, 133.5, 131.0, 130.4, 129.9, 127.9, 126.6, 122.8, 117.9, 114.2,
113.6, 106.0, 105.9, 101.5, 60.4, 55.3, 14.3; HRMS m/z (M + 1)+ calcd
for C21H21O5, 353.1384; found, 353.1379.
Ethyl (E)-3-(2,3,4-Trimethoxy-6-((E)-4-methoxystyryl)phenyl)-
acrylate (28). Yellow solid; mp 83−85 °C; 1H NMR (600 MHz,
CDCl3): δ 7.92 (d, J = 16.2 Hz, 1H), 7.43 (d, J = 6.6 Hz, 2H), 7.16 (d,
J = 16.2 Hz, 1H), 6.89 (d, J = 6.6 Hz, 2H), 6.85 (s, 1H), 6.84 (d, J =
16.2 Hz, 1H), 6.40 (d, J = 16.2 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 3.93
(s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 1.31 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3): δ 167.7, 159.5, 154.3, 153.2,
141.7, 138.5, 134.7, 131.0, 130.0, 127.9, 124.9, 122.3, 120.3, 114.2,
105.8, 61.0, 60.8, 60.3, 56.0, 55.3, 14.3; HRMS m/z (M + 1)+ calcd for
C23H27O6, 399.1802; found, 399.1798.
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-4-bromo-6-methoxyphe-
1
nol (29). White solid; mp > 250 °C; H NMR (600 MHz, CDCl3): δ
7.26 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 16.5 Hz, 1H), 7.07 (d, J = 1.5 Hz,
1H), 7.03 (d, J = 16.5 Hz, 1H), 6.93 (dd, J = 8.0, 1.5 Hz, 1H), 6.83
(d, J = 2.5 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 5.95 (s, 2H), 3.88
(s, 3H); 13C NMR (150 MHz, CDCl3): δ 148.1, 147.3, 142.3, 131.9,
130.1, 125.3, 121.8, 121.0, 119.8, 112.4, 111.6, 108.4, 105.7, 101.1,
56.4; HRMS m/z (M − 1)− calcd for C16H12BrO4, 346.9913; found,
346.9920.
(E)-2-(2-Bromo-3,4,5-trimethoxystyryl)phenol (30). White solid;
1
Preparation and Biochemical Characterization of fAβ25−35.
fAβ25−35 was prepared by dissolving Aβ25−35 in H2O at 1 mM
and aging for 1 week at 37 °C. A diluted solution of Aβ was spotted
onto a mica slide and scanned using an Agilent 5400 atomic
microscope (Molecular Imaging Coorporation, Temp, A2) as describe
previously.24
Treatment with fAβ25−35 or Glutamate and Synthetic
Compounds. Cortical neurons were pretreated with vehicle [0.1%
dimethyl sulfoxide (DMSO)] or the synthetic compounds for 30 min.
Then, fAβ25−35 or glutamate was added directly into the medium and
the cells were incubated for 48 or 24 h, respectively.
mp 147−149 °C; H NMR (600 MHz, CDCl3): δ 7.54 (dd, J = 7.5,
1.5 Hz, 1H), 7.44 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 16.0 Hz, 1H),
7.17−7.13 (m, 1H), 7.01 (s, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.79 (d, J =
8.0 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 6H); 13C NMR (150 MHz,
CDCl3): δ 153.1, 152.8, 150.9, 142.8, 133.2, 128.8, 128.6, 127.5, 125.2,
124.4, 121.2, 121.1, 116.0, 111.1, 105.4, 103.6, 61.2, 60.9, 56.2; HRMS
m/z (M + 1)+ calcd for C17H18BrO4, 365.0383; found, 365.0378.
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-6-methoxyphenol (31).
1
White solid; mp 141−143 °C; H NMR (500 MHz, CDCl3): δ 7.23
(d, J = 15.0 Hz, 1H), 7.12 (dd, J = 8.0, 1.5 Hz, 1H), 7.09 (s, 1H), 7.08
(d, J = 15.0 Hz, 1H), 6.94 (dd, J = 8.0, 1.5 Hz, 1H), 6.82 (t, J = 8.0 Hz,
J
DOI: 10.1021/acs.jmedchem.7b00376
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Measurement of Cell Viability. The live/dead cell viability assay
and the reduction of MTT were used to evaluate cell viability. Cells
were loaded with 1 μM of both calcein AM and ethidium homo-
dimer-1 at room temperature for 30 min. The cells were observed by
confocal laser scanning microscopy (Jena, Germany). The reduction
of MTT was used to evaluate cell viability. Cells were incubated with
0.5 mg/mL MTT for 1 h. The formazan particles were dissolved in
DMSO. OD600 nm was measured using an enzyme-linked immuno-
sorbent assay reader.
nestlets were determined after an overnight period. The nest quality
was assessed on a 1−5 rating scale, with 1 indicating that the nestlet
was not noticeably touched, 2 indicating that the nestlet was partially
shredded and scattered on the floor, 3 indicating that the nestlet was
mostly shredded but only a flat nest was built, 4 indicating that the
nest was identifiable but flat, and 5 indicating that the nest was built in
a burrow.
Statistical Analysis. The results are expressed as the mean
standard error of the mean (SEM) and were analyzed by analysis of
variance (ANOVA) with post hoc Bonferroni multiple comparison tests.
Immunocytochemistry. Treated cells were fixed with 4% para-
formaldehyde at room temperature for 15 min and permeabilized with
0.5% Triton X-100 for 10 min. Cells were blocked with 10% control
donkey serum at room temperature for 2 h. Thereafter, cells were
exposed to anti-MAP2 monoclonal antibody (1:100, Invitrogen) or
goat antit-au antibody (1:100, Santa Cruz) at 4 °C overnight. After
washing, the cells were incubated in staining solution containing
fluorescein isothiocyanate-conjugated donkey anti-mouse IgG and RRX-
conjugated donkey anti-goat IgG (1:200; Jackson ImmunoResearch)
in the dark overnight at 4 °C. Cells were then washed with PBS
and mounted with Aqua Poly/Mount (Polyscience Inc., Warrington,
PA, USA). The excitation/emission wavelengths used for fluorescein
isothiocyanate and RRX were 488/520 and 570/590, respectively.
Fluorescence intensity was quantified using MetaMorph software.
Measurement of Nitrite. Nitrite content (NO release) was measured
by incubating culture medium with an equal volume of Griess reagent
(0.05% N-(1-naphthyl)-ethylene-diamine dihydrochloride, 0.5% sulfani-
lamide, and 1.25% phosphoric acid). After incubation, the optical density
was detected at a wavelength of 540 nm using a microplate reader with
NaNO2 as the standard.
Cytochrome P450 3A4 (CYP3A4) Activity Assay. The wild-type
CYP3A4 constructs with N-terminal modifications were generously
provided by Dr. F. Peter Guengerich (Nashville, TN, USA).29 Enzyme
expression and membrane preparation were performed as described
before.30 Cytochrome P450 content was determined using a spectral
method reported by Omura and Sato.31 Nifedipine oxidation
activity of CYP3A4 was determined following the method reported
by Guengerich et al.32 The mean of 3−4 determinations of CYP3A4
activity in the presence of increasing concentrations of hydroxyl-
functionalized stilbenes or 2-arylbenzo[b]furans was used for
calculating the IC50 (the concentration required for 50% inhibition
of catalytic activity). IC50 values were calculated by curve fitting using
a Grafit software (start at 0, defined end; Erithacus Software Ltd.,
Staines, UK).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.7b00376.
■
S
Cell toxicity assay results for compounds 4, 5, and
1
11−43 on cortical neurons and mixed glial culture; H
and 13C NMR spectra of compounds 4, 9, and 11−35
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: yshiao@nricm.edu.tw. Phone: 886-2-2820-1999 ext.
4163 (Y.-J.S.).
*E-mail: wtli@nricm.edu.tw. Phone: 886-2-2820-1999 ext.
8261 (W.-T.L.).
■
ORCID
Wen-Tai Li: 0000-0002-7610-1778
Author Contributions
§Y.-J.S. and W.-T.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was supported by the Ministry of Science and
Technology (MOST 103-2320-B-077-004-MY3; MM10501-0274).
■
Evaluation of Antioxidant Activity. The antioxidant activity of the
synthetic compounds was determined by trolox equilibrium antioxidant
capacity (TEAC) as described previously.33
Predicted Physicochemical Properties. QikProp application was
used for predicting the physicochemical properties of compounds
presented in this study.34
ABBREVIATIONS USED
■
Aβ, β-amyloid peptide; AD, Alzheimer’s disease; APP/PS1,
APPswe/PS1ΔE9; ADL, activities of daily living; DCM,
dichloromethane; DMSO, dimethyl sulfoxide; EthD-1, ethi-
dium homodimer-1; MAP2, microtubule associated protein 2;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NFT, neurofibrillary tangle; ROS, reactive oxygen
species; NMDA, N-methyl-D-asparate; NO, nitric oxide; TAB,
tournefolic acid B; TABM, tournefolic acid B methyl ester; WT,
wild type
Animal Management and Administration. The Institutional
Animal Care and Use Committee at the National Research Institution
of Chinese Medicine approved the animal protocol (IACUC nos.
100-A-04 and 102-417-3). The APPswe/PS1ΔE9 double transgenic
mouse model (APP/PS1) of AD was purchased from Jackson
Laboratory (no. 005864); this mouse expresses a chimeric mouse/
human APP695 harboring the Swedish K670M/N671L mutations
(APPswe) and human PS1 with the exon-9 deletion mutation
(PS1ΔE9). The gender ratio for breeding was one male with two
females in one cage. Experiments were conducted using WT siblings
and AD transgenic female C57BL/6J mice. The animals were housed
under controlled temperature (24 1 °C) and humidity (55−65%)
with a 12:12 h (07:00−19:00) light−dark cycle. All mice were provided
with commercially available rodent normal chow diet and water ad
libitum. To explore the effects of the synthetic compounds in vivo,
5 month old APP/PS1 mice were fed with the synthetic compounds
(30 mg/kg/day) and examined using behavioral tests.
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DOI: 10.1021/acs.jmedchem.7b00376
J. Med. Chem. XXXX, XXX, XXX−XXX