Design, Synthesis, and Activity Study of Cinnamic Acid Derivatives as Potent Antineuroinflammatory Agents

Article abstract of DOI:10.1021/acschemneuro.0c00578

Neuroinflammatory cytokines are promising therapeutic targets for the treatment of Alzheimer's disease. Herein, we described our efforts toward the investigation of cinnamic acid derivatives as antineuroinflammatory agents. Intensive structural modificati

Full text of DOI:10.1021/acschemneuro.0c00578

pubs.acs.org/chemneuro
Research Article
Design, Synthesis, and Activity Study of Cinnamic Acid Derivatives
as Potent Antineuroinflammatory Agents
Shun Huang, Wenfeng Liu,* Yonglian Li, Kun Zhang, Xi Zheng, Hubing Wu, and Ganghua Tang
Cite This: ACS Chem. Neurosci. 2021, 12, 419−429
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ABSTRACT: Neuroinflammatory cytokines are promising therapeutic targets for
the treatment of Alzheimer’s disease. Herein, we described our efforts toward the
investigation of cinnamic acid derivatives as antineuroinflammatory agents. Intensive
structural modifications led to the identification of compound 4f as the most
effective antineuroinflammatory agent in vitro. The oral administration of compound
4f could reverse lipopolysaccharide (LPS)-induced memory disturbance and
normalize glucose uptake and metabolism in the brains of mice. Further biological
studies in vivo revealed that compound 4f was directly bound to the mitogen-
activated protein kinase (MAPK) signaling pathway, resulting in suppression of its
downstream signaling pathway by blocking neuroinflammatory progression.
Docking studies showed that compound 4f could be inserted into the active
pocket of interleukin-1β (IL-1β). Furthermore, it was confirmed that compound 4f
formed hydrogen bonds with SER84 to enhance the binding affinity. Taken
together, these results are of great importance in the development of cinnamic acid
derivatives for the treatment of Alzheimer’s disease.
KEYWORDS: Cinnamic acid derivatives, lipopolysaccharide, neuroinflammation, Alzheimer’s disease
Cinnamic acid, a major constituent of cinnamon (Cinnamo-
mum cassia), is responsible for the pharmacological properties
of cinnamon, which include anti-inflammatory, antimicrobial,
antigastric, and antitumor activities.14−17 Cinnamic acid is
widely used as a plant-derived spice and flavoring agent in
nutritional supplements. Cinnamic acid derivatives were
confirmed to exert significant neuroprotective and anti-
inflammatory effects by reducing Aβ-induced reactive oxygen
species (ROS) and proinflammatory cytokines.18,19 Accumu-
lating evidence has identified cinnamic acid and its derivatives
as potent antineuroinflammatory agents that could be
repositioned as therapeutic candidates to treat many neuro-
degenerative diseases, notably in AD.20,21 D-amino acids,
which have been used in the preparation of sweeteners and as
food and nutritional supplements,22,23 and their peptides have
received increasing attention in the treatment of inflammatory
diseases.24−26
INTRODUCTION
■
Alzheimer’s disease (AD), a devastating neurodegenerative
disease, is characterized by age-related memory loss and
progressive cognitive impairment.1 Accumulating evidence has
shown that neuroinflammation is one of the neuropathological
hallmarks of AD.2,3 Once stimulated, signal-dependent tran-
scription factors, including mitogen-activated protein kinase
(MAPK) and nuclear factor-κB (NF-κB), are activated in
neuroinflammatory processes to up-regulate the expression of
downstream proinflammatory cytokines, such as interleukin-1β
(IL-1β), IL-6, and tumor necrosis factor-α (TNF-α).4
Moreover, microglia-dominated neuroinflammation, in re-
sponse to various stimuli, has been shown to promote the
overexpression of various neuroinflammatory cytokines,
leading to astrocyte activation and neuronal damage.5−7
Under neuroinflammatory conditions, neuronal damage can
facilitate microglial and astrocyte activation, which, in turn,
produces a cycle of constant neuroinflammation.8−12 Impor-
tantly, sustained and chronic neuroinflammatory processes and
progressive neuronal damage expedite the injury of brain
tissue, which ultimately results in the development of
behavioral pathology.13 This suggests that the inhibition of
neuroinflammation is a potentially viable strategy for the
treatment of AD. However, few therapeutic agents modifying
neuroinflammation have been reported to relieve the
symptoms of AD. Therefore, new anti-AD agents are
demanded.
A recent study reported that modifying the structural
skeletons of active constituents from natural products was an
effective way to find lead compounds.27 To date, numerous
Received: September 3, 2020
Accepted: January 7, 2021
Published: January 13, 2021
https://dx.doi.org/10.1021/acschemneuro.0c00578
© 2021 American Chemical Society
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a
Scheme 1. Synthesis of the Target Compounds
a
Reagents and conditions: (a) methyl D-alaninate hydrochloride, EDC·HCl, HOAt, TEA, and DCM, at room temperature (rt) for 4 h; (b) LiOH
and MeOH-THF-H2O, at rt for 2 h, and then HCl; (c) methyl D-phenylalaninate hydrochloride, EDC·HCl, HOAt, TEA, and DCM, at rt for 4 h;
(d) LiOH and MeOH-THF-H2O, at rt for 2 h, and then HCl.
effective agents, natural compounds conjugated to other active
constituents, have been widely used to treat AD.28,29 Inspired
by those results, we proposed to design and synthesize 24
cinnamic acid derivatives containing D-amino acid or peptides,
with the aim of identifying unique antineuroinflammatory
agents against AD. Furthermore, the antineuroinflammatory
capacity of the cinnamic acid derivatives was assessed by their
ability to inhibit the production of nitric oxide (NO) and IL-1β
in an in vitro system and an in vivo model, and the
antineuroinflammatory mechanisms of compound 4f were
investigated to reveal new insight into its therapeutic potential
for AD.
Table 1. Effect of the Compounds on NO Production
inhibition at
20 μM (%)
NO IC50
a
ID
R1
R2
R3
R4
(μM)
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
H
H
H
CH3
10.69
25.06
21.73
14.52
17.51
29.46
14.39
24.83
24.94
31.26
9.90
29.35
31.09
11.08
20.62
42.57
20.09
62.72
16.19
12.41
13.75
26.61
14.06
11.83
77.01
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
CH3
OCH3
F
CF3
OCH3
H
CH3
OCH3
F
CF3
OCH3
H
CH3
OCH3
F
CF3
OCH3
H
CH3
OCH3
F
CF3
OCH3
RESULTS AND DISCUSSION
■
OCH3
H
OCH3
H
The synthetic route used for the preparation of cinnamic acid
derivatives is shown in Scheme 1. Briefly, the synthesis of
compounds 2a−f began with the amidation of substituted
cinnamic acids (1a−f) with methyl D-alaninate hydrochloride
and was followed by a hydrolytic reaction with lithium
hydroxide to yield intermediate 3a−f (Scheme 1). Then
compounds 3a−f were mixed with commercially available
methyl D-phenylalaninate hydrochloride to afford compounds
4a−f through an amidation reaction in the presence of
triethylamine. Similarly, compounds 4a−f were hydrolyzed
with lithium hydroxide to afford compounds 5a−f. Finally, 24
compounds were synthesized in the present study. The yields
ranged from 54.1 to 89.3%, and the purity of the target
compounds was >92%.
H
OCH3
H
OCH3
H
CH3
OCH3
H
OCH3
H
16.2
>20
H
>20
>20
>20
>20
>20
It has been demonstrated that cinnamic acid and its
derivatives can be effective therapeutical agents for the
treatment of AD by inhibiting neuroinflammatory activity.18−20
Herein, we investigated their inhibitory effect on NO
production in vitro. As shown in Table 1, resveratrol (Res)
at an initial concentration of 20 μM showed 77.01% inhibition
against NO production. Thus, we used Res as the positive
control and investigated the inhibitory effect of 24 compounds
at 20 μM on NO production. Compared to that of Res, most
of the synthesized compounds exhibited potent inhibitory
activity against NO production. Among them, compound 4f at
20 μM displayed potent inhibitory activity (62.72%) compared
to that of the other compounds, with an IC50 value of 16.2 μM.
Compound 4d showed moderate activity at 42.57% inhibition
(Table 1). Structure−activity relationship (SAR) studies
showed the activity of the target compounds against NO
production in lipopolysaccharide (LPS)-induced BV2 micro-
OCH3
OCH3
Res
11.9
a
Concentration (μM) for 50% inhibition of NO release in BV2 cells.
The IC50 values are the mean SD of at least three experimental
determinations.
glia cells in the descending order of 4f > 4d > 3d > 4a > 30%.
Compounds 4a, 4d, and 4f, with substituents at the R4 position
(R4 = CH3), exhibited significantly higher inhibition rates of
31.09, 42.57, and 62.72%, respectively, at 20 μM. Compound
4f with −OCH3 at the R1, R2, and R3 positions was the most
potent compound against LPS-induced NO production.
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Figure 1. Effects of compound 4f on LPS-induced memory impairment. (A) Movement tracks of mice in the place navigation (hidden-platform)
test. (B) The escape latency time of mice in the place navigation (hidden-platform) test. (C) The percentage of time the mice spent searching for
the target quadrant in the spatial probe test. (D) Movement tracks of the mice in the spatial probe test. (E) The number of times the mice crossed
the removed-platform area in the spatial probe test. The significance of differences from control group at *p < 0.05, from LPS group at #p < 0.05.
Accordingly, compound 4f was selected for further inves-
tigation of the antineuroinflammatory mechanism of action.
The systemic administration of LPS has been reported to
cause memory impairment and learning disabilities.30 Herein,
we performed the Morris water test (MWM) test to determine
the effects of compound 4f on LPS-induced memory
impairment and learning disability. As shown in Figure 1A,B,
LPS treatment caused a cognitive deficiency (p < 0.05) and an
increase in the escape latency time compared to that of the
control group, whereas the oral administration of compound 4f
dose-dependently decreased the escape latency time in the
second session (p < 0.05). In addition, the escape latency time
of the TTP488 group was lower than that of the LPS group
mice in the first, second, third, and fourth sessions. Moreover,
the results demonstrated that the movement tracks of the mice
were consistent with the escape latencies in 2 min. The
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Figure 2. Effects of compound 4f on LPS-induced cerebral metabolism. Data represent the mean SD (n = 6 mice per group). Significantly
different from the control group at *p < 0.05; from the LPS group at #p < 0.05.
compound 4f-treated groups found the platform by a shorter
route.
to increase 18F-FDG uptake in a dose-dependent manner.
Furthermore, mice, treated with TTP488 and compound 4f
(LPS + 100 mg/kg/d), markedly increased neuronal glucose
utilization and normalized glucose metabolism. Taken
together, these results demonstrated that compound 4f could
reverse memory disturbance and normalize glucose uptake and
metabolism in the LPS-induced Alzheimer’s mouse model.
To evaluate the effect of compound 4f on LPS-induced
neuron damage, Nissl staining was used to determine the
histopathologic changes. As shown in Figure 3, the results
demonstrated that LPS treatment resulted in the loss of Nissl
substance and dead neurons in the brains compared to the
brains of the control group. In contrast, compound 4f
administration markedly elevated Nissl substance in the
hippocampus of the mice, increased the number of neurons,
and protected normal neurons in the cortex and hippocampus
from damage by LPS.
Neuroinflammatory cytokines secreted by activated micro-
glia and astrocytes, which are crucial in neuroinflammatory
processes, lead to AD.13 Thus, the LPS-induced expression of
IL-1β in AD mice in the absence or presence of compound 4f
was tested to investigate the effect of compound 4f on cytokine
production. As shown in Figure 4, the marked overexpression
of IL-1β was observed in mice treated with LPS compared to
the controls. However, pretreatment with different doses of
In the spatial probe test, mice from all groups placed in the
water maze without the hidden platform were used to assess
memory performance. As illustrated in Figure 1C,D, the LPS-
treated mice showed significant memory impairment. How-
ever, the result was reversed in TTP488 and compound 4f-
treated mice. Furthermore, mice in the TTP488 and
compound 4f groups spent more time in the target quadrant
and had greater numbers of platform crossings compared to
those of the LPS group. These results also indicated that mice
treated with compound 4f showed dose-dependent increases in
the time spent in the target quadrant and crossing times.
Additionally, the recorded tracks of the mice from all groups
clearly confirmed these results (Figure 1E). LPS, a well-studied
immune stimulator, was reported to induce immune neuro-
inflammatory responses, cause cognitive impairment, and
dysregulate glucose metabolism in mice.31,32 Herein, to assess
the relevance of cerebral metabolism in cognitive impairment
in mice, we investigated the effects of compound 4f on brain
glucose uptake using an LPS-induced Alzheimer’s mouse
model. Micro-PET/CT imaging indicated that in mice treated
with LPS alone, the uptake of 18F-fluorodeoxyglucose (18F-
FDG) in the brain was significantly lower than that in the
control group mice (Figure 2). Compound 4f was also shown
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Figure 3. Effects of compound 4f on normal neurons in the cortex and hippocampus. Nissl staining and normal neuronal counts in the
hippocampus and cortex of each group. Scale bar = 50 μm. The data represent the mean SD (n = 6 mice per group). Significantly different from
the control group at *p < 0.05; from the LPS group at #p < 0.05.
compound 4f could prevent the cytotoxicity of LPS in the
brain. Moreover, IL-1β expression was dose-dependently
suppressed by compound 4f.
memory loss. In addition, it was shown that late-onset AD
decreased bilateral glucose metabolism.38 Therefore, we
designed and synthesized cinnamic acid derivatives containing
D-amino acids and determined the effects of these compounds
in vitro and in vivo. Moreover, the inhibitory effect of
compound 4f on cerebral glucose metabolism was investigated
by micropositron emission tomography (PET) imaging.
Additionally, the behavioral experiments clearly demonstrated
that compound 4f effectively attenuated LPS-induced cognitive
impairment, such as those on working memory and spatial
cognitive memory.
Accumulating evidence has indicated that changes in the
structure and function of the pyramidal cells in the
hippocampus and cortex were closely implicated in cognitive
and behavioral function.39,40 Herein, we determined the effect
of compound 4f on the LPS-induced pyramidal cells using
Nissl staining. The results demonstrated that the LPS
injections caused a considerable loss of Nissl-stained cells
compared with control group mice. However, cinnamic acid
derivative treatment reversed the pathological changes in the
hippocampus. Furthermore, compound 4f inhibited pyramidal
cells damage in a dose-dependent manner.
When stimulated by LPS, the MAPK and NF-κB signaling
pathways were activated and subsequently resulted in the
release of proinflammatory cytokines, which were confirmed to
be involved in the development of various inflammatory
disorders, such as AD.33 Thus, the antineuroinflammatory
mechanisms of action of compound 4f were investigated by
Western blotting. As illustrated in Figure 3, compound 4f dose-
dependently lowered the LPS-induced expression of MAPK
p38 and NF-κB p65, whereas treatment with compound 4f
showed no evident effect on the expression of TLR4 and
Myd88. Taken together, these results indicated that compound
4f suppressed LPS-induced IL-1β activation by inhibiting the
MAPK/NF-κB signaling pathway.
As described in Figure 5A,B, compound 4f was docked into
the binding pocket of IL-1β (PDB ID: 9ILB). Compound 4f
nested well into the active pocket of IL-1β. Furthermore, the
oxygen atom of the OCH3 group formed a hydrogen bond
with SER84 in the catalytic pocket to enhance the affinity for
IL-1β.
AD, a neurodegenerative disease, is closely related to
aging.34 A number of studies have indicated that a low-dosage
LPS, injection to mice, induced a series of behavioral changes,
such as cognitive disorders and memory loss, which well-mimic
the symptoms of AD patients.35 Furthermore, it was
demonstrated that α-, β- and γ-secretases, activated by LPS
triggered the overexpression of proinflammatory cytokines in
the brain and caused physiological symptoms and behavioral
symptoms.36,37 Interestingly, numerous studies confirmed that
changes in regional brain metabolism and energetics played a
vital role in the pathogenesis of cognitive disorders and
Previous studies indicated that neuroinflammation stimu-
lated by LPS correlated with the overexpression of
proinflammatory cytokines, which was confirmed to be one
of the causes of neuropathogenesis and cognitive impair-
ment.41,42 Moreover, LPS, well-known as a TLR4 ligand, led to
the initiation of a series of neuroinflammatory cascades, such as
the MAPK and NF-κB signaling pathway.43 Hence, it is
necessary to evaluate the inhibitory effect of compound 4f on
LPS-induced inflammatory processes. Our studies revealed that
inflammatory responses in the brain of the mice treated with
LPS were significantly exacerbated through the release of IL-
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inflammatory signaling pathway. Moreover, a molecular
docking study confirmed that compound 4f could be
effectively inserted into the active pocket of IL-1β protein
and formed a hydrogen bond with SER84. Collectively, these
results demonstrated that compound 4f could be a promising
lead for further optimization as a new antineuroinflammatory
agent against Alzheimer’s disease.
METHODS
■
Materials and Methods. LPS (Escherichia coli O55:B5) was
purchased from Sigma-Aldrich (St. Louis, MO, USA). TTP488,
referred to in a previous publication,44 was used as a standard
compound in the experiment. Antibodies against TLR4, Myd88,
MAPK p38, NF-κB p65, and IL-1β were obtained from Bioss
Biotechnology Co. (Beijing, China). Reagents were used without
1
further purification unless specified. H nuclear magnetic resonance
(NMR) and 13C NMR spectra were recorded using tetramethylsilane
(TMS) as an internal standard on a Bruker DPX-500 spectrometer.
Mass spectrometry analysis was performed on a liquid chromatog-
raphy−mass spectrometer (LC-MS, LCQ) with an ESI source.
Melting points were determined on a micro melting point apparatus
(Shanghai, China) and were not corrected. Optical rotations were
obtained using a WZZ-1S digital polarimeter (Shanghai, China) at
sodium D line (589 nm) and were reported in concentrations of 0.01
g/mL.
General Procedure for the Synthesis of Target Compounds.
Cinnamic acid (1a−f) (2.0 mmol, 1 equiv), D-alanine methyl ester
hydrochloride (2.0 mmol, 1 equiv), EDC·HCl (2.4 mmol, 1.2 equiv),
and HOAt (2.4 mmol, 1.2 equiv) were dissolved in dichloromethane
(DCM, 20 mL), and then triethanolamine (TEA, 0.2 mmol, 0.1
equiv) was added slowly. The reaction mixture was stirred at room
temperature until the reaction was completed. Then, the reaction
mixture was quenched with saturated aqueous NaHCO3 solution and
extracted with ethyl acetate. The organic layer was subsequently
washed with water and brine, dried over anhydrous Na2SO4, and
concentrated. The obtained residues were purified by flash
chromatography (EtOAc/PE = 1/10) to yield compounds 2a−f.
The compounds 2a−f were reacted with LiOH using MeOH/THF/
H2O (3:1:1, 15 mL) as a solvent. After 2 h, 12 M HCl solution was
added to the reaction mixture. The solvent was removed under
reduced pressure to yield compounds 3a−f.
Figure 4. Anti-inflammatory mechanism of compound 4f against LPS-
induced neuroinflammation in mice. Significantly different from the
control group at *p < 0.05 and **p < 0.01; from the LPS group at #p
< 0.05 and ##p < 0.01.
Compounds 3a−f (1.5 mmol, 1 equiv), D-phenylalanine methyl
ester hydrochloride (1.5 mmol, 1 equiv), EDC·HCl (1.8 mmol, 1.2
equiv), and HOAt (1.8 mmol, 1.2 equiv) were dissolved in DCM (20
mL), and TEA (0.15 mmol, 0.1 equiv) was added slowly. The
reaction mixture was stirred at room temperature until the reaction
was completed. Then the reaction mixture was quenched with
saturated aqueous NaHCO3 solution and extracted with ethyl acetate.
The organic layer was subsequently washed with water and brine,
dried over anhydrous Na2SO4, and concentrated. The obtained
residues were purified by flash chromatography (EtOAc/PE = 1/5) to
provide compounds 4a−f. Compounds 4a−f were reacted with LiOH
using MeOH/THF/H2O (3:1:1, 15 mL) as a solvent. After 2 h, 12 M
HCl solution was added to the reaction mixture. The solvent was
removed under reduced pressure to provide compounds 5a−f.
Methyl-cinnamoyl-D-alaninate (2a). This compound is a white
solid; yield 89.1%; mp = 119−120 °C; [α]2D1 = −12.0 (c = 0.5,
CH2Cl2); 1H NMR (500 MHz, CDCl3): δ 7.57 (d, J = 15.6 Hz, 1H),
7.46−7.41 (m, 2H), 7.03 (t, J = 8.6 Hz, 2H), 6.48 (d, J = 7.2 Hz, 1H),
6.36 (d, J = 15.6 Hz, 1H), 4.78−4.68 (m, 1H), 3.77 (s, 3H), 1.87 (s,
1H), 1.46 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ
173.82, 165.24, 164.58, 162.59, 140.43, 130.89, 130.87, 129.72,
129.65, 119.78, 119.77, 116.01, 115.84, 52.63, 48.20, 18.54. HRMS
calcd for C13H15NO3, [M − H]− 232.0979, found 232.0972.
Methyl-(E)-(3-(p-tolyl)acryloyl)-D-alaninate (2b). This compound
is a white solid; yield 81.4%; mp = 171−173 °C; [α]2D1 = −13.7 (c =
Figure 5. Proposed binding model of the 4f-IL-1β complex. (A) The
yellow dashed lines represent hydrogen bonds. (B) The green dashed
lines represent hydrogen bonds.
1β. However, compound 4f suppressed the release of IL-1β in
a dose-dependent manner. In particular, our mechanistic
studies demonstrated that the systemic injection of LPS
activated MAPK and NF-κB signaling pathways, indicated by
the down-regulated expression of IL-1β in vivo.
In summary, we demonstrated that novel antineuroin-
flammatory agents could be discovered by assembling the
active fragments of cinnamic acid and D-amino acid. Among
the synthesized compounds, compound 4f significantly
inhibited neuroinflammation, and exerted good neuroprotec-
tive effects. The in vitro and in vivo experiments indicated that
compound 4f exerted suppressive effects in LPS-induced
neuroinflammation and cognitive impairment by inhibiting the
overexpression of IL-1β dependent upon the MAPK-mediated
1
0.5, CH2Cl2); H NMR (500 MHz, CDCl3): δ 7.59 (d, J = 15.6 Hz,
1H), 7.37 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 6.43 (dd, J =
19.7, 11.4 Hz, 2H), 4.77−4.71 (m, J = 7.2 Hz, 1H), 3.76 (s, 3H), 2.34
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(s, 3H), 1.46 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ
173.81, 165.62, 141.66, 140.13, 131.90, 129.55, 127.88, 119.00, 52.59,
48.19, 21.45, 18.60. HRMS calcd for C14H17NO3, [M − H]−
246.1136, found 246.1129.
1H), 7.65−7.60 (m, 2H), 7.43 (d, J = 15.8 Hz, 1H), 7.25 (t, J = 8.8
Hz, 2H), 6.66 (d, J = 15.8 Hz, 1H), 4.35−4.29 (m, J = 7.3 Hz, 1H),
1.32 (d, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 174.67,
165.05, 164.17, 162.20, 138.38, 131.92 (d, J = 2.8 Hz), 130.17 (d, J =
8.5 Hz), 122.08, 116.50, 116.32, 48.15, 17.80. HRMS calcd for
C12H12FNO3, [M − H]− 236.0728, found 236.0721.
Methyl-(E)-(3-(4-methoxyphenyl)acryloyl)-D-alaninate (2c). This
compound is a white solid; yield 83.8%; mp: = 144−146 °C; [α]D21
=
−9.1 (c = 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3): δ 7.57 (d, J =
15.6 Hz, 1H), 7.41 (t, J = 5.7 Hz, 2H), 6.89−6.84 (m, 2H), 6.40 (d, J
= 7.3 Hz, 1H), 6.32 (d, J = 15.6 Hz, 1H), 4.77−4.71 (m, J = 7.2 Hz,
1H), 3.81 (s, 3H), 3.76 (s, 3H), 1.46 (d, J = 7.2 Hz, 3H). 13C NMR
(126 MHz, CDCl3): δ 173.86, 165.75, 160.95, 141.30, 129.47, 127.38,
117.65, 114.23, 77.35, 77.09, 76.84, 55.36, 52.57, 48.16, 18.61. HRMS
calcd for C14H17NO4, [M − H]− 262.1085, found 262.1080.
Methyl-(E)-(3-(4-fluorophenyl)acryloyl)-D-alaninate (2d). This
compound is a white solid; yield 87.1%; mp = 189−190 °C; [α]D21
= −11.2 (c = 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3): δ 7.57 (d, J
= 15.6 Hz, 1H), 7.43−7.40 (m, 2H), 6.87−6.84 (m, 2H), 6.40 (d, J =
7.2 Hz, 1H), 6.33−6.30 (d, J = 15.6 Hz, 1H), 4.77−4.71 (m, 1H),
3.77 (s, 3H), 1.46 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3):
δ 173.82, 165.24, 164.58, 162.59, 140.43, 130.88 (d, J = 3.4 Hz),
129.68 (d, J = 8.6 Hz), 119.78 (d, J = 1.8 Hz), 116.01, 115.84, 52.63,
48.20, 18.54. HRMS calcd for C13H14FNO3, [M − H]− 250.0885,
found 250.0879.
(E)-(3-(4-(trifluoromethyl)phenyl)acryloyl)-D-alanine (3e). This
compound is a white solid; yield 71.9%; mp = 258−260 °C; [α]D21
1
= −10.9 (c = 0.5, MeOH); H NMR (500 MHz, -d6): δ 8.55 (d, J =
7.3 Hz, 1H), 7.85−7.73 (m, 1H), 7.52 (d, J = 15.9 Hz, 1H), 6.86 (d, J
= 15.9 Hz, 1H), 4.37−4.32 (m, J = 7.3 Hz, 1H), 1.34 (d, J = 7.3 Hz,
1H). 13C NMR (126 MHz, DMSO-d6): δ 174.55, 164.64, 139.39,
137.90, 129.84, 129.59, 128.64, 126.30 (d, J = 3.8 Hz), 125.65,
125.03, 123.49, 48.23, 17.78. HRMS calcd for C13H12F3NO3, [M −
H]− 286.0697, found 286.0693.
(E)-(3-(3,4,5-trimethoxyphenyl)acryloyl)-D-alanine (3f). Light yel-
low solid; yield 67.2%; mp = 196−198 °C; [α]2D1 = −13.5 (c = 0.5,
CH2Cl2); 1H NMR (500 MHz, DMSO): δ 8.32 (d, J = 7.3 Hz, 1H),
7.37 (d, J = 15.7 Hz, 1H), 6.90 (s, 2H), 6.67 (d, J = 15.8 Hz, 1H),
4.32 (t, J = 7.3 Hz, 1H), 3.81 (s, 6H), 3.68 (s, 3H), 1.32 (d, J = 7.3
Hz, 3H). 13C NMR (126 MHz, DMSO): δ 174.68, 165.19, 153.53,
139.65, 139.10, 130.95, 121.65, 105.34, 60.54, 56.29, 48.11, 17.82.
HRMS calcd for C15H19NO6, [M − H]− 308.1140, found 308.1136.
Methyl-cinnamoyl-D-alanyl-D-phenylalaninate (4a). This com-
pound is a white solid; yield 80.4%; mp = 160−161 °C; [α]2D1 = +19.9
(c = 0.5, CH2Cl2); 1H NMR (400 MHz, CDCl3): δ 7.63 (d, J = 15.7
Hz, 1H), 7.48 (dd, J = 6.5, 2.9 Hz, 2H), 7.36−7.28 (m, 4H), 7.23−
7.18 (m, 2H), 7.13 (dd, J = 8.8, 5.8 Hz, 1H), 7.09 (s, 1H), 6.97 (d, J =
7.7 Hz, 1H), 6.50 (d, J = 15.7 Hz, 1H), 4.86 (dd, J = 13.8, 6.8 Hz,
1H), 4.78 (d, J = 7.3 Hz, 3H), 3.14 (dd, J = 13.8, 5.8 Hz, 1H), 3.05
(dd, J = 13.8, 6.9 Hz, 1H), 1.42 (d, J = 7.0 Hz, 3H). 13C NMR (126
MHz, CDCl3): δ 172.35, 171.75, 165.43, 140.21, 135.69, 133.21,
129.36, 128.61, 127.12, 120.78, 53.52, 52.43, 48.81, 37.89, 18.44.
HRMS calcd for C22H24N2O4, [M − H]− 379.1663, found 379.1659.
Methyl-((E)-3-(p-tolyl)acryloyl)-D-alanyl-D-phenylalaninate (4b).
This compound is a white solid; yield 88.6%; mp = 125−126 °C;
Methyl-(E)-(3-(4-(trifluoromethyl)phenyl)acryloyl)-D-alaninate
(2e). This compound is a white solid; yield 79.3%; mp = 212−213 °C;
[α]2D1 = −14.5 (c = 0.5, CH2Cl2); H NMR (500 MHz, CDCl3): δ
1
7.63−7.51 (m, 5H), 6.63 (d, J = 7.3 Hz, 1H), 6.52 (d, J = 15.6 Hz,
1H), 4.78−4.72 (m, J = 7.2 Hz, 1H), 3.77 (s, 3H), 1.47 (d, J = 7.2 Hz,
3H). 13C NMR (126 MHz, CDCl3): δ 173.78, 164.77, 139.94, 138.04,
131.41, 131.15, 127.98, 125.77 (d, J = 3.7 Hz), 124.95, 122.79,
122.51, 77.33, 77.07, 76.82, 52.67, 48.28, 18.45. HRMS calcd for
C14H14F3NO3, [M − H]− 300.0853, found 300.0848.
Methyl-(E)-(3-(3,4,5-trimethoxyphenyl)acryloyl)-D-alaninate (2f).
This compound is a white solid; yield 83.8%; mp = 137−138 °C;
[α]2D1 = −13.1 (c = 0.5, CH2Cl2); H NMR (500 MHz, CDCl3): δ
1
7.50 (d, J = 15.5 Hz, 1H), 6.68 (s, 2H), 6.47 (d, J = 7.4 Hz, 1H), 6.36
(d, J = 15.5 Hz, 1H), 4.76−4.70 (m, J = 7.2 Hz, 1H), 3.85 (d, J = 1.5
Hz, 9H), 3.76 (s, 3H), 1.45 (d, J = 7.2 Hz, 3H). 13C NMR (126 MHz,
CDCl3): δ 173.85, 165.33, 153.33, 141.57, 139.50, 130.26, 119.45,
104.88, 60.96, 56.08, 52.61, 48.17, 18.57. HRMS calcd for
C16H21NO6, [M − H]− 322.1296, found 322.1292.
[α]2D1 = +16.2 (c = 0.5, CH2Cl2); H NMR (500 MHz, CDCl3): δ
1
7.60 (d, J = 15.6 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 7.2 Hz,
2H), 7.17 (d, J = 7.6 Hz, 3H), 7.11−7.07 (m, 2H), 6.90 (s, 1H), 6.37
(dd, J = 15.5, 8.0 Hz, 2H), 4.90−4.81 (m, 1H), 4.65 (dd, J = 12.3, 5.1
Hz, 1H), 3.72 (d, J = 11.0 Hz, 3H), 3.15 (dd, J = 14.0, 5.6 Hz, 1H),
3.05 (dd, J = 13.9, 6.8 Hz, 1H), 2.37 (s, 2H), 1.39 (d, J = 7.0 Hz, 3H).
13C NMR (126 MHz, CDCl3): δ 172.02, 171.72, 165.87, 141.78,
Cinnamoyl-D-alanine (3a). This compound is a white solid; yield
61.5%; mp = 169−171 °C; [α]2D1 = −15.1 (c = 0.5, MeOH); 1H NMR
(500 MHz, DMSO-d6): δ 7.68−7.64 (d, J = 7.3 Hz, 1H), 7.48 (t, J =
7.1 Hz, 2H), 7.34 (dd, J = 10.8, 10.3 Hz, 3H), 6.48−6.44 (d, J = 15.9
Hz, 1H), 4.71−4.67 (t, J = 7.3 Hz, 1H), 1.52 (d, J = 7.3 Hz, 3H), 1.25
(s, 1H). 13C NMR (126 MHz, DMSO-d6): δ 174.63, 165.09, 139.54,
135.34, 129.98, 129.43, 128.00, 122.30, 48.17, 17.86. HRMS calcd for
C12H13NO3, [M − H]− 218.0823, found 218.0815.
140.26, 135.73, 131.87, 129.64, 129.27, 128.62, 127.89, 127.10,
118.94, 53.39, 52.45, 48.71, 37.88, 21.49, 18.12. HRMS calcd for
C23H26N2O4, [M − H]− 393.1820, found 393.1817.
Methyl-((E)-3-(4-methoxyphenyl)acryloyl)-D-alanyl-D-phenylala-
ninate (4c). This compound is a white solid; yield 85.1%; mp = 147−
1
149 °C; [α]2D1 = +21.7 (c = 0.5, CH2Cl2); H NMR (500 MHz,
(E)-(3-(p-tolyl)acryloyl)-D-alanine (3b). Thisc compound is a white
CDCl3): δ 7.58 (d, J = 15.6 Hz, 1H), 7.44 (dd, J = 8.6, 3.9 Hz, 2H),
7.20 (d, J = 7.5 Hz, 2H), 7.17−7.13 (m, 1H), 7.09 (d, J = 7.0 Hz,
2H), 7.04 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.49 (d, J =
7.6 Hz, 1H), 6.30 (dd, J = 15.6, 5.5 Hz, 1H), 4.84 (dd, J = 13.6, 6.7
Hz, 1H), 4.67 (dd, J = 14.7, 7.4 Hz, 1H), 3.82 (s, 3H), 3.71 (d, J =
10.4 Hz, 3H), 3.14 (dd, J = 13.8, 5.6 Hz, 1H), 3.06−3.00 (m, 1H),
1.39 (d, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ 172.21,
171.75, 166.07, 161.02, 141.38, 135.79, 129.50, 129.28, 128.60,
127.37, 127.07, 117.65, 114.30, 55.38, 53.44, 52.43, 48.70, 37.89,
18.15. HRMS calcd for C23H26N2O5, [M − H]− 409.1769, found
409.1765.
solid; yield 54.8%; mp = 240−241 °C; [α]2D1 = −12.5 (c = 0.5,
1
MeOH); H NMR (500 MHz, DMSO-d6): δ 8.43 (d, J = 7.3 Hz,
1H), 7.45 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 15.8 Hz, 1H), 7.22 (d, J =
8.0 Hz, 2H), 6.66 (d, J = 15.8 Hz, 1H), 4.35−4.29 (m, J = 7.3 Hz,
1H), 2.31 (s, 3H), 1.31 (d, J = 7.3 Hz, 3H). 13C NMR (126 MHz,
DMSO-d6): δ 174.33, 164.93, 139.41, 139.17, 132.15, 129.67, 127.63,
120.77, 47.74, 21.05, 17.40. HRMS calcd for C13H15NO3, [M − H]−
232.0979, found 232.0972.
(E)-(3-(4-methoxyphenyl)acryloyl)-D-alanine (3c). This com-
pound is a white solid; yield 66.2%; mp = 219−220 °C; [α]D21
=
−13.8 (c = 0.5, MeOH); 1H NMR (500 MHz, DMSO-d6): δ 8.35 (d,
J = 7.3 Hz, 1H), 7.56−7.47 (m, 2H), 7.38 (d, J = 15.8 Hz, 1H), 7.01−
6.92 (m, 2H), 6.57 (d, J = 15.8 Hz, 1H), 4.32 (t, J = 7.3 Hz, 1H), 3.78
(s, 3H), 1.31 (d, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ
179.53, 170.18, 165.58, 144.01, 134.34, 132.60, 124.47, 119.62, 60.46,
52.87, 22.60. HRMS calcd for C13H15NO4, [M − H]− 248.0928,
found 248.0923.
Methyl-((E)-3-(4-fluorophenyl)acryloyl)-D-alanyl-D-phenylalani-
nate (4d). This compound is a white solid; yield 75.6%; mp = 173−
175 °C; [α]2D1 = +15.0 (c = 0.5, CH2Cl2); H NMR (500 MHz,
1
DMSO-d6): δ 8.45 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 7.8 Hz, 1H),
7.66−7.59 (m, 2H), 7.44 (d, J = 15.8 Hz, 1H), 7.31−7.25 (m, 4H),
7.25−7.18 (m, 3H), 6.71 (d, J = 15.8 Hz, 1H), 4.55−4.43 (m, 2H),
3.61 (d, J = 20.3 Hz, 3H), 3.05 (dd, J = 13.9, 5.7 Hz, 1H), 2.97−2.90
(m, 1H), 1.23 (d, J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO-d6):
δ 172.88, 172.29, 164.87, 164.14, 162.18, 138.21, 137.55, 132.02,
130.13 (d, J = 8.4 Hz), 129.63 (d, J = 14.1 Hz), 128.68 (d, J = 10.4
(E)-(3-(4-fluorophenyl)acryloyl)-D-alanine (3d). This compound is
a white solid; yield 70.1%; mp = 233−234 °C; [α]2D1 = −12.1 (c = 0.5,
1
MeOH); H NMR (500 MHz, DMSO-d6): δ 8.44 (d, J = 7.3 Hz,
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Research Article
Hz), 127.02, 122.36, 116.49, 116.32, 54.08, 52.33, 48.32, 36.97, 18.86.
HRMS calcd for C22H23FN2O4, [M − H]− 397.1569, found 397.1565.
Methyl-(E)-(2-(3-(4-(trifluoromethyl)phenyl)acrylamido)-
acryloyl)-D-phenylalaninate (4e). This compound is a white solid;
yield 80.2%; mp = 191−192 °C; [α]2D1 = +19.7 (c = 0.5, CH2Cl2); 1H
NMR (500 MHz, CDCl3): δ 7.64 (s, 1H), 7.62−7.55 (m, 5H), 7.24−
7.20 (m, 2H), 7.17 (ddd, J = 7.4, 3.6, 1.3 Hz, 1H), 7.11−7.07 (m,
2H), 6.92 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H), 6.51 (d, J =
15.6 Hz, 1H), 4.87 (dt, J = 7.8, 6.2 Hz, 1H), 4.70 (dd, J = 14.5, 7.2
Hz, 1H), 3.72 (d, J = 7.2 Hz, 3H), 3.15 (dd, J = 13.9, 5.7 Hz, 1H),
3.07 (dd, J = 13.9, 6.5 Hz, 1H), 1.42 (d, J = 7.0 Hz, 3H). 13C NMR
(126 MHz, CDCl3): δ 172.05, 171.68, 164.97, 139.93, 138.09, 135.63,
129.26, 128.64, 127.99, 127.17, 125.85 (d, J = 3.7 Hz), 122.60, 53.44,
52.47, 48.88, 37.88, 18.44. HRMS calcd for C23H23F3N2O4, [M −
H]− 447.1537, found 447.1534.
3.11−2.99 (m, 1H), 2.95−2.78 (m, 1H), 1.21 (d, J = 7.1 Hz, 3H),
1.03 (d, J = 7.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δ 173.17,
172.69, 164.93, 138.18, 137.94, 130.15 (d, J = 8.4 Hz), 129.67,
128.62, 126.87, 122.38, 116.50, 116.32, 53.98, 48.49, 36.97, 18.79.
HRMS calcd for C21H21FN2O4, [M − H]− 383.1413, found 383.1405.
((E)-3-(4-(trifluoromethyl)phenyl)acryloyl)-D-alanyl-D-phenylala-
nine (5e). This compound is a white solid; yield 69.1%; mp = 226−
227 °C; [α]2D1 = +17.3 (c = 0.5, MeOH); 1H NMR (500 MHz,
DMSO-d6): δ 8.44−8.35 (m, 1H), 8.28 (d, J = 7.9 Hz, 1H), 7.82−
7.74 (m, 3H), 7.49 (d, J = 15.9 Hz, 1H), 7.27−7.20 (m, 4H), 7.18
(td, J = 5.9, 2.6 Hz, 1H), 6.91 (d, J = 15.9 Hz, 1H), 4.50−4.39 (m,
2H), 3.07 (dd, J = 13.8, 4.9 Hz, 1H), 2.92 (dd, J = 13.8, 9.0 Hz, 1H),
1.23 (d, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ 13C
NMR (126 MHz, DMSO) δ 173.21, 172.56, 164.44, 137.97, 137.69,
129.65, 128.62 (d, J = 2.8 Hz), 126.88, 126.33, 125.31, 53.97, 48.46,
36.99, 18.87. HRMS calcd for C22H21F3N2O4, [M − H]− 433.1381,
found 433.1373.
((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)-D-alanyl-D-phenylala-
nine (5f). This compound is a light yellow solid; yield 77.2%; mp =
185−187 °C; [α]2D1 = +13.3 (c = 0.5, CH2Cl2); 1H NMR (500 MHz,
DMSO-d6): δ 12.77 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.11 (d, J = 7.8
Hz, 1H), 7.35 (d, J = 15.7 Hz, 1H), 7.26 (d, J = 7.4 Hz, 1H), 7.23
(dd, J = 5.3, 3.0 Hz, 3H), 7.19 (d, J = 6.9 Hz, 1H), 6.90 (d, J = 3.7 Hz,
2H), 6.72 (d, J = 15.7 Hz, 1H), 4.48−4.37 (m, 2H), 3.80 (d, J = 2.0
Hz, 6H), 3.68 (d, J = 1.5 Hz, 3H), 3.06 (dd, J = 13.9, 5.0 Hz, 1H),
2.91 (dd, J = 13.8, 8.9 Hz, 1H), 1.21 (d, J = 7.0 Hz, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 164.97, 153.52, 137.91, 131.03, 129.65,
128.65, 126.91, 121.92, 105.29, 60.55, 56.27, 53.87, 48.37, 37.03,
18.95. HRMS calcd for C24H28N2O7, [M + H]+ 457.1956, found
457.1959.
Cell Culture. Murine microglial (BV-2) cells (Cell Resource
Center of Chinese Academy of Medical Science, Beijing, China) were
grown in Dulbecco Modified Eagle Medium (DMEM; Gibco, CA,
USA) containing 10% fetal bovine serum (FBS, Hyclone, CA, USA)
and 1% penicillin/streptomycin (Hyclone, CA, USA) in a 37 °C
humidified atmosphere containing 5% CO2. These cells were
subcultured every two days.
NO Production in LPS-Induced BV2 Microglial Cells. NO
production was determined by a colorimetric method using the Griess
reagent as previously described.45 Briefly, the BV2 cells were
preincubated for 30 min in the absence or presence of all compounds
at different concentrations before adding LPS (1 μg/mL, 10 μL) for
24 h at 37 °C. The cell medium was centrifuged, and the supernatant
(50 μL) was dispensed into 96-well plates and then mixed with an
equal volume of Griess reagent (Beyotime Biotechnology, Beijing,
China). The plates were incubated at room temperature for 10 min,
and the absorbance at 540 nm was measured using a multifunctional
microplate reader. The inhibition rate of NO production was
calculated by the following equation:
Methyl-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)-D-alanyl-D-phe-
nylalanin-eate (4f). This compound is a white solid; yield 84.5%; mp
= 183−184 °C; [α]2D1 = +17.6 (c = 0.5, CH2Cl2); H NMR (500
1
MHz, CDCl3): δ 7.56 (d, J = 15.5 Hz, 1H), 7.31−7.23 (m, 3H),
7.22−7.17 (m, 1H), 7.12 (d, J = 6.8 Hz, 2H), 6.96 (d, J = 7.9 Hz,
1H), 6.74 (d, J = 2.6 Hz, 2H), 6.52 (d, J = 7.6 Hz, 1H), 6.36 (d, J =
15.5 Hz, 1H), 4.89−4.82 (m, 1H), 4.70 (dd, J = 14.1, 6.8 Hz, 1H),
3.89 (t, J = 2.3 Hz, 9H), 3.74 (d, J = 6.9 Hz, 3H), 3.17 (dd, J = 13.9,
5.7 Hz, 1H), 3.08 (dd, J = 13.8, 6.6 Hz, 1H), 1.42 (d, J = 7.0 Hz, 3H).
13C NMR (126 MHz, CDCl3): δ 172.10, 171.72, 165.57, 153.40,
141.68, 139.61, 135.72, 130.23, 129.27, 128.62, 127.12, 119.42,
104.92, 61.00, 56.11, 53.44, 52.44, 48.77, 37.90, 18.39. HRMS calcd
for C25H30N2O7, [M − H]− 469.1980, found 469.1974.
Cinnamoyl-D-alanyl-D-phenylalanine (5a). This compound is a
white solid; yield 72.0%; mp = 203−204 °C; [α]2D1 = +18.6 (c = 0.5,
1
MeOH); H NMR (500 MHz, DMSO): δ 8.31 (d, J = 7.8 Hz, 1H),
8.21 (s, 1H), 7.56 (d, J = 7.1 Hz, 2H), 7.45−7.39 (m, 3H), 7.39−7.35
(m, 1H), 7.26−7.21 (m, 5H), 7.18−7.13 (m, 1H), 6.77 (d, J = 15.8
Hz, 1H), 4.42 (ddd, J = 13.4, 11.7, 6.2 Hz, 2H), 3.06 (dd, J = 13.8, 5.0
Hz, 1H), 2.94−2.89 (m, 1H), 1.21 (d, J = 7.1 Hz, 3H). 13C NMR
(126 MHz, DMSO): δ 173.20, 172.64, 164.95, 139.37, 137.99,
135.38, 129.95, 129.67, 129.43, 128.61, 127.99, 126.85, 122.52, 53.98,
48.45, 37.03, 18.83. HRMS calcd for C21H22N2O4, [M − H]−
365.1507, found 365.1501.
((E)-3-(p-tolyl)acryloyl)-D-alanyl-D-phenylalanine (5b). This com-
pound is a white solid; yield 74.1%; mp = 220−221 °C; [α]2D1 = +15.0
(c = 0.5, MeOH); 1H NMR (500 MHz, DMSO-d6): δ 8.34 (d, J = 7.8
Hz, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.37 (d, J
= 15.8 Hz, 1H), 7.24 (s, 1H), 7.23 (d, J = 3.0 Hz, 4H), 7.21 (s, 1H),
7.20−7.15 (m, 1H), 6.71 (d, J = 15.8 Hz, 1H), 4.53−4.38 (m, 2H),
3.05 (dd, J = 13.8, 5.0 Hz, 1H), 2.92 (dd, J = 13.8, 9.0 Hz, 1H), 2.31
(s, 3H), 1.20 (d, J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO-d6): δ
173.17, 172.74, 165.17, 139.74, 139.37, 137.91, 132.57, 130.03,
129.67, 128.63, 127.98, 126.88, 121.40, 53.94, 48.45, 36.97, 21.42,
18.75. HRMS calcd for C22H24N2O4, [M − H]− 379.1663, found
379.1658.
inhibition rate (%) = (ALPS − ACompound)/(ALPS − AControl
× 100%
)
((E)-3-(4-methoxyphenyl)acryloyl)-D-alanyl-D-phenylalanine
(5c). This compound is a white solid; yield 65.6%; [α]2D1 = +16.3 (c =
0.5, MeOH); mp = 198−199 °C; 1H NMR (500 MHz, DMSO-d6): δ
12.81 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H),
7.54−7.48 (m, 2H), 7.38 (d, J = 15.7 Hz, 1H), 7.28−7.22 (m, 4H),
7.19 (dd, J = 6.2, 2.5 Hz, 1H), 6.98 (dd, J = 9.0, 2.5 Hz, 2H), 6.61 (d,
J = 15.8 Hz, 1H), 4.57−4.30 (m, 2H), 3.78 (d, J = 1.8 Hz, 3H), 3.10−
2.99 (m, 1H), 2.91 (d, J = 8.8 Hz, 1H), 1.22 (d, J = 7.1 Hz, 3H). 13C
NMR (126 MHz, DMSO-d6): δ 173.23, 172.75, 165.28, 160.80,
139.13, 137.92, 129.65, 129.57, 128.64, 127.93, 126.88, 119.97,
114.87, 55.71, 53.91, 48.34, 37.03, 18.83. HRMS calcd for
C22H24N2O5, [M − H]− 395.1612, found 395.1607.
where AConpound was the absorbance of the BV2 cells treated with
target compounds and LPS, ALPS was the absorbance of the BV2 cells
treated with LPS alone, and AControl was the absorbance of normal
BV2 cells.
Animals and Comound 4f Treatment. Female C57 mice (5−6
weeks) were obtained from the Center of Animal Testing of Southern
Medical University (SCXK/20160041, Guangzhou, China). All
animals were randomly divided into five groups with six mice in
each group and housed (25 1 °C at 50% relative humidity) under
12 h/12 h light−dark cycle conditions. Food and water were provided
ad libitum. All animal care and experimental procedures were carried
out in strict accordance with the National Institute of Health’s Guide
for the Care and Use of Laboratory Animals.
((E)-3-(4-fluorophenyl)acryloyl)-D-alanyl-D-phenylalanine (5d).
This compound is a white solid; yield 70.9%; mp = 217−219 °C;
[α]2D1 = +18.2 (c = 0.5, MeOH); H NMR (500 MHz, DMSO-d6): δ
1
As previously published,46 the LPS-induced Alzheimer’s disease
model was established as follows:
8.37 (dd, J = 11.8, 8.2 Hz, 1H), 8.29 (dd, J = 25.5, 7.9 Hz, 1H), 7.62
(ddd, J = 8.6, 5.4, 2.9 Hz, 2H), 7.41 (d, J = 15.8 Hz, 1H), 7.28 (t, J =
6.3 Hz, 1H), 7.26−7.24 (m, 2H), 7.23 (d, J = 3.6 Hz, 3H), 7.17 (dd, J
= 8.5, 4.1 Hz, 1H), 6.73 (d, J = 15.8 Hz, 1H), 4.51−4.33 (m, 2H),
(1) Control group: Mice in control group were injected with
phosphate-buffered saline (PBS) once daily for 14 days.
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(2) LPS group: Mice in the LPS group were injected with PBS
once daily for 7 days. After 7 days, the mice were
intraperitoneally (i.p.) administered LPS (250 μg/kg) once
daily for 7 days after PBS treatment.
(3) TTP488 group: Mice in the TTP488 group were injected with
TTP488 (5 mg/kg) once daily for 7 days. After 7 days, the
mice were administered LPS (250 μg/kg) i.p. once daily for 7
days after TTP488 (5 mg/kg) treatment.
(4) 4f-50 group: Mice in the 4f-50 group were orally administered
compound 4f (50 mg/kg) once daily for 7 days. After 7 days,
the mice were administered LPS (250 μg/kg) i.p. once daily
for 7 days after compound 4f (50 mg/kg) treatment.
(5) 4f-100 group: Mice in the 4f-100 group were orally
administered compound 4f (100 mg/kg) once daily for 7
days. After 7 days, the mice were administered LPS (250 μg/
kg) i.p. once daily for 7 days after compound 4f (100 mg/kg)
treatment.
Western Blotting. A total of 20 mg of brain proteins from each
group was subjected to sodium dodecyl sulfate−polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose
membranes. Immunoblotting was performed using anti-IL-1β
(1:5000; WB Preferred), NF-κB p65 (1:5000), and MAPK p38
(1:5000). Immunodetection was done using an enhanced chem-
iluminescence detection kit (MultiSciences Biotech, Hangzhou,
China). The band density was quantified using Quantity One analysis
software (Bio-Rad, Hercules, CA, USA) by calculating the average
optical density in each field.
Molecular Modeling. AutoDock4.2 was used to simulate the
interaction between compound 4f and IL-1β. After hydrogen atoms
were added to compound 4f using Babel and partial charges were
generated using MOPAC, the hydrogen bonding energy of compound
4f was minimized using the Lenard-Jones 6/12 potential mode and
optimized with iterative minimization. In addition, a maximum of 300
iterations was used to optimize the energy gradient, and a total of 200
docking runs were performed in each cycle of the iterative
minimization. The crystal structure of IL-1β was retrieved from the
RCSB Protein Data Bank (PDB: 9ILB). All water molecules were
removed, and hydrogen atoms were added to achieve IL-1β protein
protonation. The active pocket of IL-1β protein was simulated by the
automatic mode. Other settings were used in the default settings.
Statistical Analysis. The quantitative data are expressed as the
mean standard deviation (SD). Statistical analysis of the data was
performed using Graph Pad Prism 5 software (La Jolla, CA, USA) for
comparisons using the Student’s t-test or one- or two-way ANOVA to
determine statistical significance. The p values of <0.05 and <0.01
were considered to indicate statistically significant differences.
Micropositron Emission Tomography/Computed Tomog-
raphy Imaging. The change in brain glucose uptake was determined
as previously described.46 Briefly, after 2 weeks of drug administration,
PET scans and image analysis were used to determine brain glucose
uptake by compound 4f in mice in each group. First, after fasting for
12 h, the mice were injected i.p. with 18F-FDG under general
anesthesia with 7% chloral hydrate. Ten-minute static PET scans of
the mice were obtained 1 h after 18F-FDG injections, followed by a
low-dose CT scan (for attenuation correction and localization) using
an Inveon Micro-PET/CT system (Siemens Healthiness, Munich,
Germany). Second, the images were reconstructed using the Micro-
PET/CT manager (Siemens Medical Solutions Inc., Pittsfield, USA)
with an attenuation correction. A region-of-interest (ROI), placed on
the inflammatory brains, was drawn and measured using Inveon
Research Workplace software. Quantification of the 18F-FDG uptake
in the brains of each group was calculated to obtain the image-ROI-
derived percent injected dose per gram (% ID/g).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00578.
■
sı
Morris Water Maze Test. The behavioral assessment after 2
weeks of drug administration was carried out using MWM as reported
earlier.46 The water maze consisted of a circular pool with a diameter
of 120 cm and a hidden platform (1 cm under the water) with a
diameter of 10 cm. The pool was filled with opaque water (titanium
white powder was added to the water, to enable distinguishing of the
1
Experimental Section with H and 13C NMR spectra of
2a−5f and HRMS spectra of 2a−5f (PDF)
AUTHOR INFORMATION
Corresponding Author
■
black C57 mice) at a temperature of 24
1 °C. A video camera,
connected to the SMART video-tracking and analysis system (TSE,
Bad Homburg, Germany), was located above the pool and used to
track the animal’s movement. The MWM test consisted of 4 days of
training, 1 day of navigation testing, and 1 day of spatial probe testing.
During the 4 days, the hidden platform was kept constant at the
midpoint of target quadrant and the mice were trained in the MWM
twice daily. The training consisted of four sessions of trials (one
session/day, one trial/session). Each trial was performed for 60 s until
the mouse climbed onto the hidden platform target. In the navigation
test, the mice were free to swim in the pool for 120 s to locate the
platform. The escape latency time (s) to find the hidden platform and
the time spent in the target quadrant (s) were recorded. During the
probe trials, the platform in the quadrant was removed and the mice
were put into the pool and swam for 120 s. The mice were monitored
with the camera and the percentage (%) of time spent in each
quadrant and the number of times the mice crossed the platform area
were recorded using the video-tracking and analysis system.
Nissl Staining. The brains of mice from all groups were removed
in toto, washed twice with 0.01 M PBS, and postfixed in 4%
paraformaldehyde (PFA). Paraffin-embedded brain specimens were
sectioned coronally (4 μm). For the detection of normal neurons,
tissue slices were washed with xylene for 5 min, progressively
dehydrated in 100% alcohol and 75% alcohol, consequently stained
with a warmed 0.1% toluidine blue solution for 3−5 min, and finally
rinsed with distilled water. The slides were imaged and captured using
an Olympus confocal microscope (IX-73, Tokyo, Japan). Normal
neurons had prominent Nissl granules and a light nucleus and were
examined with a 400× objective. The analysis of normal neuronal cells
was conducted on three nonoverlapping fields per slice.
Wenfeng Liu − School of Biotechnology and Health Sciences,
Wuyi University, Jiangmen 529020, China; International
Healthcare Innovation Institute (Jiangmen), Jiangmen
529020, China; orcid.org/0000-0002-7169-843X;
Email: wyuchemlwf@126.com
Authors
Shun Huang − Nanfang PET Center, Nanfang Hospital,
Southern Medical University, Guangzhou, Guangdong
510515, China
Yonglian Li − School of Eco-environment Technology,
Guangdong Industry Polytechnic, Guangzhou 510300, China
Kun Zhang − School of Biotechnology and Health Sciences,
Wuyi University, Jiangmen 529020, China
Xi Zheng − School of Biotechnology and Health Sciences, Wuyi
University, Jiangmen 529020, China; Susan Lehman
Cullman Laboratory for Cancer Research, Department of
Chemical Biology, Ernest Mario School of Pharmacy, Rutgers,
The State University of New Jersey, Piscataway, New Jersey
08854, United States
Hubing Wu − Nanfang PET Center, Nanfang Hospital,
Southern Medical University, Guangzhou, Guangdong
510515, China
Ganghua Tang − Nanfang PET Center, Nanfang Hospital,
Southern Medical University, Guangzhou, Guangdong
510515, China
427
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ACS Chem. Neurosci. 2021, 12, 419−429

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Walker, D. G., Webster, S. P., Wegrzyniak, B., Wenk, G. L., and Coray,
T. W. (2000) Inflammation and Alzheimer’s disease. Neurobiol. Aging
21 (3), 383−421.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00578
(11) Wyss-Coray, T. (2006) Inflammation in Alzheimer disease:
driving force, bystander or beneficial response? Nat. Med. 12 (9),
1005−15.
Author Contributions
S.H. and W.F.L. conceived the study. Y.L.L. and W.F.L.
designed and performed the target compounds. S.H. and
W.F.L. designed and performed experiments. X.Z., K.Z.,
H.B.W., G.H.T., S.H., and W.F.L. analyzed and interpreted
data. S.H. and W.F.L. wrote the manuscript.
(12) Ballatore, C., Lee, V. M. Y., and Trojanowski, J. Q. (2007) Tau-
mediated neurodegeneration in Alzheimer’s disease and related
disorders. Nat. Rev. Neurosci. 8 (9), 663−72.
(13) Morales, I., Guzman-Martinez, L., Cerda-Troncoso, C., Farias,
G., and Maccioni, R. B. (2014) Neuroinflammation in the
pathogenesis of Alzheimer’s disease: A rational framework for the
search of novel therapeutic approaches. Front. Cell. Neurosci. 8, 112.
(14) Liao, J. C., Deng, J. S., Chiu, C. S., Hou, W. C., Huang, S., Shie,
P. H., and Huang, G. J. (2012) Anti-inflammatory activities of
cinnamomum cassia constituents in vitro and in vivo. Evid. Based
Complement. Alterna. Med. 2012 (2012), 1−12.
Funding
Financial support was provided by Natural Science Foundation
of Guangdong-Guangzhou Joint Fund (No. :
2019A1515110941), National Natural Science Foundation of
China (No.: 81901781), Foundation for Young Talents (No.:
2016KQNCX174, 2017GkQNCX006, and 2017KTSCX184),
Foundation for Department of Education of Guangdong
Province (No.: 2017KSYS010), President Foundation Project
of Nanfang Hospital, Southern Medical University (No.:
2018Z003), the Basic and Theoretical Research Programs of
Science and Technology Foundation of Jiangmen (No.:
2020JC01013), and the Joint Fund of Wuyi University-
Macau (No.: 2019WGALH01).
(15) Guzman, J. D. (2014) Natural cinnamic acids, synthetic
derivatives and hybrids with antimicrobial activity. Molecules 19 (12),
19292−349.
(16) Nanjundaiah, S. M., Annaiah, H. N., and Dharmesh, S. M.
(2011) Gastroprotective effect of ginger rhizome (Zingiber officinale)
extract: Role of gallic acid and cinnamic acid in H(+), K(+)-ATPase/
H. pylori inhibition and anti-oxidative mechanism. Evid. Based
Complement. Alternat. Med. 2011 (2011), 1−12.
Notes
(17) De, P., Baltas, M., and Bedosbelval, F. (2011) Cinnamic acid
derivatives as anticancer agents-a review. Curr. Med. Chem. 18 (11),
1672−703.
The authors declare no competing financial interest.
REFERENCES
(18) Perluigi, M., Joshi, G., Sultana, R., Calabrese, V., De Marco, C.,
Coccia, R., Cini, C., and Butterfield, D. A. (2006) In vivo protective
effects of ferulic acid ethyl ester against amyloid-beta peptide 1−42-
induced oxidative stress. J. Neurosci. Res. 84 (2), 418−26.
(19) Wang, Y., Wang, Y., Li, J., Hua, L. L., Han, B., Zhang, Y. Z.,
Yang, X. P., Zeng, Z. L., Bai, H. Y., Yin, H. L., and Lou, J. Y. (2016)
Effects of caffeic acid on learning deficits in a model of Alzheimer’s
disease. Int. J. Mol. Med. 38 (3), 869−75.
■
̈
̈
(1) Alzheimer, A. (1911) Uber eigenartige krankheitsfalle des
spateren alters. Z. Gesamte Neurol. Psychiatr. 4 (1), 356.
̈
(2) Heneka, M. T., Carson, M. J., El Khoury, J., Landreth, G. E.,
Brosseron, F., Feinstein, D. L., Jacobs, A. H., Wysscoray, T., Vitorica,
J., Ransohoff, R. M., Herrup, K., Frautschy, S. A., Finsen, B., Brown,
G. C., Verkhratsky, A., Yamanaka, K., Koistinaho, J., Latz, E., Halle, A.,
Petzold, G. C., Town, T., Morgan, D., Shinohara, L., Perry, V. H.,
Holmes, C., Bazan, N. G., Brooks, D. J., Hunot, S., Joseph, B.,
Deigendesch, N., Garaschuk, O., Boddeke, E., Dinarello, C. A.,
Breitner, J. C. S., Cole, G. M., Golenbock, D. T., and Kummer, M. P.
(2015) Neuroinflammation in Alzheimer’s disease. Lancet Neurol. 14
(4), 388−405.
(20) Zhang, X., He, X., Chen, Q., Lu, J. F., Rapposelli, S., and Pi, R.
(2018) A review on the hybrids of hydroxycinnamic acid as multi-
targetdirected ligands against Alzheimer’s disease. Bioorg. Med. Chem.
26 (3), 543−50.
(21) Chakrabarti, S., Jana, M., Roy, A., and Pahan, K. (2018)
Upregulation of suppressor of cytokine signaling 3 in microglia by
cinnamic acid. Curr. Alzheimer Res. 15 (10), 894−904.
(22) Mazur, R. H., Reuter, J. A., Swiatek, K. A., and Schlatter, J. M.
(1973) Synthetic sweeteners. 3. Aspartyl dipeptide esters from L-and
D-alkylglycines. J. Med. Chem. 16 (11), 1284−7.
(23) Glowaky, R. C., Hendrick, M. E., Smiles, R. E., and Torres, A.
(1991) Development and uses of Alitame: A novel dipeptide amide
sweetener. Sweeteners 450, 57−67.
(24) Fujihara, S. M., Cleaveland, J. S., Grosmaire, L. S., Berry, K. K.,
Kennedy, K. A., Blake, J. J., Loy, J., Rankin, B. M., Ledbetter, J. A., and
Nadler, S. G. (2000) A D-amino acid peptide inhibitor of NF-κB
nuclear localization is efficacious in models of inflammatory disease. J.
Immunol. 165 (2), 1004−112.
(25) Kim, E. Y., Rajasekaran, G., and Shin, S. Y. (2017) LL-37-
derived short antimicrobial peptide KR-12-a5 and its d-amino acid
substituted analogs with cell selectivity, anti-biofilm activity,
synergistic effect with conventional antibiotics, and anti-inflammatory
activity. Eur. J. Med. Chem. 136, 428−41.
(26) Li, J., Kuang, Y., Gao, Y., Du, X. W., Shi, J. F., and Xu, B.
(2013) D-amino acids boost the selectivity and confer supramolecular
hydrogels of a nonsteroidal anti-inflammatory drug (NSAID). J. Am.
Chem. Soc. 135 (2), 542−5.
(27) Guo, Z. (2017) The modification of natural products for
medical use. Acta Pharm. Sin. B 7 (2), 119−36.
(3) Wang, Q., Liu, Y., and Zhou, J. (2015) Neuroinflammation in
Parkinson’s disease and its potential as therapeutic target. Transl.
Neurodegener. 4 (1), 19.
(4) Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C., and Gage,
F. H. (2010) Mechanisms underlying inflammation in neuro-
degeneration. Cell 140 (6), 918−34.
(5) Cacquevel, M., Lebeurrier, N., Cheenne, S., and Vivien, D.
(2004) Cytokines in neuroinflammation and Alzheimer’s disease.
Curr. Drug Targets 5 (6), 529−34.
(6) Hensley, K. (2010) Neuroinflammation in Alzheimer’s disease:
mechanisms, pathologic consequences, and potential for therapeutic
manipulation. J. Alzheimer's Dis. 21 (6), 1−14.
(7) Mcgeer, E. G., and Mcgeer, P. L. (2010) Neuroinflammation in
Alzheimer’s disease and mild cognitive impairment: a field in its
infancy. J. Alzheimer's Dis. 19 (1), 355−61.
(8) Jucker, M., and Walker, L. C. (2013) Self-propagation of
pathogenic protein aggregates in neurodegenerative diseases. Nature
501 (7465), 45−51.
(9) Gonzalezscarano, F., and Baltuch, G. (1999) Microglia as
mediators of inflammatory and degenerative diseases. Annu. Rev.
Neurosci. 22 (1), 219−40.
(10) Akiyama, H., Barger, S., Barnum, S., Bradt, B. M., Bauer, J.,
Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M. R.,
Fiebich, B. L., Finch, C. E., Frautschy, S. A., Griffin, W. S. T., Hampel,
H., Hull, M., Landreth, G. E., Lue, L., Mrak, R. E., Mackenzie, I. R. A.,
Mcgeer, P. L., Obanion, M. K., Pachter, J. S., Pasinetti, G. M.,
Salaman, C. P., Rogers, J., Rydel, R. E., Shen, Y., Streit, W. J.,
Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F. L., Veerhuis, R.,
(28) Dias, K. S., de Paula, C. T., Dos Santos, T., Souza, I. N. O.,
Boni, M. S., Guimaraes, M. J. R., Da Silva, F. M. R., Castro, N. G.,
Neves, G., De Carvalho Veloso, C., Coelho, M. M., Melo, I. S. F.,
Giusti, F. C. V., Giustipaiva, A., Da Silva, M. L., Dardenne, L. E.,
428
https://dx.doi.org/10.1021/acschemneuro.0c00578
ACS Chem. Neurosci. 2021, 12, 419−429

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Guedes, I. A., Pruccoli, L., Morroni, F., Tarozzi, A., and Viegas, C.
(2017) Design, synthesis and evaluation of novel feruloyl-donepezil
hybrids as potential multitarget drugs for the treatment of Alzheimer’s
disease. Eur. J. Med. Chem. 130, 440−57.
(29) Cheng, B., Lin, Y. S., Kuang, M., Fang, S., Gu, Q., Xu, J., and
Wang, L. Y. (2015) Synthesis and anti-neuroinflammatory activity of
lactone benzoyl hydrazine and 2-nitro-1-phenyl-1H-Indole derivatives
as p38α MAPK inhibitors. Chem. Biol. Drug Des. 86 (5), 1121−30.
(30) Chen, M., Chang, Y., Huang, S., Xiao, L. H., Zhou, W., Zhang,
L. Y., Li, C., Zhou, R. P., Tang, J., Lin, L., Du, Z. Y., and Zhang, K.
(2018) Aromatic-turmerone attenuates LPS-induced neuroinflamma-
tion and consequent memory impairment by targeting TLR4-
dependent signaling pathway. Mol. Nutr. Food Res. 62 (2), 1−9.
(31) Kapogiannis, D., and Mattson, M. P. (2011) Disrupted energy
metabolism and neuronal circuit 32 dysfunction in cognitive
impairment and Alzheimer’s disease. Lancet Neurol. 10 (2), 187−98.
(32) Block, M. L., Zecca, L., and Hong, J. S. (2007) Microglia-
mediated neurotoxicity: uncovering the molecular mechanisms. Nat.
Rev. Neurosci. 8 (1), 57−69.
(33) Lu, Y. C., Yeh, W. C., and Ohashi, P. S. (2008) LPS/TLR4
signal transduction pathway. Cytokine 42 (2), 145−51.
(34) Raz, N., Ghisletta, P., Rodrigue, K. M., Kennedy, K. M., and
Lindenberger, U. (2010) Trajectories of brain aging in middle-aged
and older adults: regional and individual differences. Neuroimage 51
(2), 501−11.
(35) Sparkman, N. L., Martin, L. A., Calvert, W. S., and Boehm, G.
W. (2005) Effects of intraperitoneal lipopolysaccharide on Morris
maze performance in year-old and 2-month-old female C57BL/6J
mice. Behav. Brain Res. 159 (1), 145−51.
(36) Semmler, A., Hermann, S., Mormann, F., Weberpals, M.,
Paxian, S. A., Okulla, T., Schafers, M., Kummer, M. P., Klockgether,
T., and Heneka, M. T. (2008) Sepsis causes neuroinflammation and
concomitant decrease of cerebral metabolism. J. Neuroinflammation 5
(1), 38.
(37) Lee, J. W., Lee, Y. K., Yuk, D. Y., Choi, D. Y., Ban, S. B., Oh, K.
W., and Hong, J. T. (2008) Neuro-inflammation induced by
lipopolysaccharide causes cognitive impairment through enhancement
of beta-amyloid generation. J. Neuroinflammation 5 (1), 37.
(38) Reitz, C., Brayne, C., and Mayeux, R. (2011) Epidemiology of
Alzheimer disease. Nat. Rev. Neurol. 7 (3), 137−52.
(39) Eichenbaum, H. (2000) A cortical-hippocampal system for
declarative memory. Nat. Rev. Neurosci. 1 (1), 41−50.
(40) Eichenbaum, H. (2004) Hippocampus: cognitive processes and
neural representations that underlie declarative memory. Neuron 44
(1), 109−20.
(41) Palop, J. J., and Mucke, L. (2010) Amyloid-beta-induced
neuronal dysfunction in Alzheimer’s disease: From synapses toward
neural networks. Nat. Neurosci. 13 (7), 812−8.
(42) Wang, Q., Liu, Y., and Zhou, J. (2015) Neuroinflammation in
Parkinson’s disease and its potential as therapeutic target. Transl.
Neurodegener. 4 (1), 19.
(43) Qin, L., Wu, X., Block, M. L., Liu, Y., Breese, G. R., Hong, J. S.,
Knapp, D. J., and Crews, F. T. (2007) Systemic LPS causes chronic
neuroinflammation and progressive neurodegeneration. Glia 55 (5),
453−62.
(44) Davidowitz, E. J., Moe, J. G., Reitz, A. B., Bian, H., Gluchowski,
C., Hendrix, J., Yehaskel, A. S., Mcdonnell, M. E., and Loughran, M.
H. (2019) Novel quinazolinones that inhibit the formation of tau
oligomers and their method of use. U.S. Patent Application No. 16/
471 363.
(45) Fang, Y., Zhou, H., Gu, Q., and Xu, J. (2019) Synthesis and
evaluation of tetrahydroisoquinoline-benzimidazole hybrids as multi-
functional agents for the treatment of Alzheimer’s disease. Eur. J. Med.
Chem. 167 (2019), 133−45.
(46) Liu, W. F., Huang, S., Li, Y. L., Zheng, X., and Zhang, K. (2019)
Suppressive effect of glycyrrhizic acid against lipopolysaccharide-
induced neuroinflammation and cognitive impairment in C57 mice
via toll-like receptor 4 signaling pathway. Food Nutr. Res. 63 (63),
1516−28.
429
https://dx.doi.org/10.1021/acschemneuro.0c00578
ACS Chem. Neurosci. 2021, 12, 419−429