A novel chalcone derivative as Nrf2 activator attenuates learning and memory impairment in a scopolamine-induced mouse model

Article abstract of DOI:10.1016/j.ejmech.2019.111777

Alzheimer's disease is a common neurodegenerative disease characterized by progressive degeneration and neuronal cell death, resulting in neural network dysfunction. As the underlying mechanisms, oxidative damage and neuroinflammation have been reported to contribute to the onset and deterioration of Alzheimer's disease. The nuclear factor E2-related factor 2-antioxidant responsive element signaling pathway is a pivotal cellular defense mechanism against oxidative stress. Nrf2, a transcription factor, regulates the cellular redox balance and is primarily involved in anti-inflammatory responses. In this study, we synthesized novel chalcone derivatives and found a highly potent Nrf2 activator, compound 20a. Compound 20a confirmed to activate Nrf2 and induce expression of the Nrf2-dependent enzymes HO-1 and GCLC at both mRNA and protein levels. It also suppressed the production of nitric oxide and downregulated inflammatory mediators in BV-2 microglial cells. We found that compound 20a effectively increased the expression level and the activity of superoxide dismutase in both BV-2 microglial cells and brain hippocampus region of the scopolamine-induced mouse model. In addition, compound 20a effectively recovered the learning and memory impairment in a scopolamine-induced mouse model.

Full text of DOI:10.1016/j.ejmech.2019.111777

Journal Pre-proof
A novel chalcone derivative as Nrf2 activator attenuates learning and memory
impairment in a scopolamine-induced mouse model
Hyeon Jeong Kim, Bo Ko Jang, Jong-Hyun Park, Ji Won Choi, Sun Jun Park, Seong
Rim Byeon, Ae Nim Pae, Yong Sup Lee, Eunji Cheong, Ki Duk Park
PII:
S0223-5234(19)30929-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111777
EJMECH 111777
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 July 2019
Revised Date: 1 October 2019
Accepted Date: 9 October 2019
Please cite this article as: H.J. Kim, B.K. Jang, J.-H. Park, J.W. Choi, S.J. Park, S.R. Byeon, A.N. Pae,
Y.S. Lee, E. Cheong, K.D. Park, A novel chalcone derivative as Nrf2 activator attenuates learning and
memory impairment in a scopolamine-induced mouse model, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.111777.
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A Novel Chalcone Derivative as Nrf2 Activator Attenuates
Learning and Memory Impairment in a Scopolamine-Induced
Mouse Model
a,b,1
a,1
a
a
a,c
Hyeon Jeong Kim , Bo Ko Jang , Jong-Hyun Park , Ji Won Choi , Sun Jun Park , Seong
d
a,c,e
e,f
b
a,c,e,
Rim Byeon , Ae Nim Pae , Yong Sup Lee , Eunji Cheong , and Ki Duk Park
*
a
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia,
Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
b
Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
c
Division of Bio-Medical Science & Technology, KIST School, Korea University of Science
and Technology, Seoul 02792, Republic of Korea
d
Kainos Medicine, Inc. Daewangpangyo-ro, Seongnam, Gyeonggi 13488, Republic of Korea
e
KHU-KIST Department of Converging Science and Technology, Kyung Hee University,
Seoul 02447, Republic of Korea
f
Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee
University, Seoul 02447, Republic of Korea
1
These authors contributed equally to this article.
*
Corresponding author
Ki Duk Park, PhD; Convergence Research Center for Diagnosis, Treatment and Care System
of Dementia, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu,
Seoul, Republic of Korea. TEL: 82-2-958-5132, Fax: +82-2-958-5189, E-mail:
kdpark@kist.re.kr
1

Abstract
Alzheimer’s disease is a common neurodegenerative disease characterized by progressive
degeneration and neuronal cell death, resulting in neural network dysfunction. As the
underlying mechanisms, oxidative damage and neuroinflammation have been reported to
contribute to the onset and deterioration of Alzheimer’s disease. The nuclear factor E2-related
factor 2-antioxidant responsive element (Nrf2-ARE) signaling pathway is a pivotal cellular
defense mechanism against oxidative stress. Nrf2, a transcription factor, regulates the cellular
redox balance and is primarily involved in anti-inflammatory responses. In this study, we
synthesized novel chalcone derivatives and found a highly potent Nrf2 activator, compound
2
0a. Compound 20a confirmed to activate Nrf2 and induce expression of the Nrf2-dependent
enzymes HO-1 and GCLC at both mRNA and protein levels. It also suppressed the
production of nitric oxide and downregulated inflammatory mediators in BV-2 microglial
cells. We found that compound 20a effectively increased the expression level and the activity
of superoxide dismutase (SOD) in both BV-2 microglial cells and brain hippocampus region
of the scopolamine-induced mouse model. In addition, compound 20a effectively recovered
the learning and memory impairment in a scopolamine-induced mouse model.
2

Keywords
Alzheimer’s disease; Nuclear factor erythroid 2-related factor 2; Chalcone; Oxidative stress;
Superoxide dismutase
Highlights
A novel series of chalcone derivatives were synthesized as Nrf2 activators.
Compound 20a increases antioxidant enzyme expression by nuclear translocation of Nrf2.
20a suppresses reactive oxygen species against oxidative stress in microglia.
20a increases expression level and activity of superoxide dismutase in vitro and in vivo.
20a restores scopolamine-induced memory impairment in mice.
3

1
. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disease with memory
deficits and cognitive impairment. It is characterized by the accumulation of amyloid plaques
and abnormally phosphorylated neurofibrillary tangles, mitochondrial abnormalities, and
oxidative stress, which cause neural network dysfunction and death of neurons, especially in
the cerebral cortex and hippocampus. [1, 2] In addition to these pathological hallmarks, many
studies have proposed that oxidative stress and neuroinflammation can cause and accelerate
AD. Chronic inflammatory processes are remarkably activated in the brains of AD patients.
[
3] Furthermore, the number of activated microglia that produce reactive oxygen species
ROS), nitric oxide (NO) and pro-inflammatory cytokines such as interleukin 6 (IL-6) and
(
tumor necrosis factor-α (TNF-α) is increased [4, 5], indicating that antioxidant and anti-
inflammatory therapeutic approaches can be beneficial in AD by suppressing neurotoxic
responses.
The nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE)
signaling pathway is a primary cellular defense mechanism against oxidative damage. Under
oxidative stressed conditions, the transcription factor Nrf2 regulates cellular redox balance
through the induction of antioxidant enzyme genes such as heme oxygenase-1 (HO-1),
NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase (GCL). [6, 7]
Nrf2 has also been shown to contribute to the anti-inflammatory process. It has been reported
that activation of Nrf2 suppresses the expression of cell adhesion molecules such as E-
selectin and vascular cell adhesion molecule 1 (VCAM-1) [8, 9], as well as the induction of
pro-inflammatory cytokines and enzymes, including cyclooxygenase-2 (COX-2) and
inducible nitric oxide synthase (iNOS). [10-13] The Nrf2 signaling inhibits ROS-associated
nuclear factor (NF)-κB activation by inducing the antioxidant responses and blocking the
4

degradation of inhibitor of κB α (IκBα). [14-16] In addition, recent studies suggested that
activation of Nrf2 inhibits NLRP3 inflammasome assembly and activation. [17, 18] These
results demonstrate that Nrf2 plays a crucial role in anti-inflammatory responses as well as
antioxidant defenses.
Based on this cytoprotective role of Nrf2, its signaling pathway is believed to be
involved in protection against various neurodegenerative diseases, including AD, Parkinson’s
disease (PD), and multiple sclerosis (MS). [19, 20] Nrf2-deficient mice exhibit enhanced
sensitivity to a variety of chemical and biological stresses and neurotoxicants and they are
highly susceptible to inflammatory diseases. [7, 14] Nuclear Nrf2 and its target genes are also
downregulated in the brains of AD patients [21] and mouse models of AD. [22, 23]
Conversely, overexpression of Nrf2 suppressed the pathology of AD with memory
improvement in a mouse model of AD. [24] Thus, Nrf2 activation is an attractive therapeutic
target for AD through anti-inflammatory and antioxidant effects.
Small molecules that activate Nrf2 signaling have been investigated for the
development of potential anti-inflammatory agents, but dimethyl fumarate (DMF, brand name
TM
Tecfidera ), developed to treat patients with relapsing-remitting multiple sclerosis (RRMS),
is the only drug approved by the FDA. Chalcone, which contains a unique α,β-unsaturated
ketone, is a representative compound that activates Nrf2 and has been reported to expand to
many disease drugs including anti-inflammatory, anti-cancer, neuroprotective and beta-
amyloid disaggregate agents (Figure 1). [25-32] In particular, novel chalcone derivatives have
recently been reported for AD-related targets. [33, 34] In the present study, we synthesized a
novel series of chalcone derivatives as Nrf2 activators and evaluated their Nrf2-activating
efficacies, discovering an excellent Nrf2 activating compound (20a) (Figure 1). Compound
2
0a was further evaluated for its antioxidant and anti-inflammatory effects in activated
5

microglia. We also investigated whether Nrf2 activation by compound 20a could restore the
memory deficits using a scopolamine-induced amnesia mouse model, an acute model with
oxidative stress, neuroinflammation and cognitive impairment. [35-39]
6

2
2
. Results
.1. Compound 20a is a new and potent Nrf2 activator
Kumar et al reported that novel chalcone derivatives effectively activated Nrf2
through Keap1 modification and the most potent derivative (21, Table S1 in the Supporting
Information) was further evaluated for its ability to induce Nrf2-dependent antioxidant genes.
[
26] In our previous study, [40] we optimized vinyl sulfones by introducing nitrogen
heterocycles to improve the Nrf2 activating efficacy and druglike properties. In the present
study, in order to optimize chalcone scaffold as Nrf2 activators, we synthesized chalcone
derivatives containing heterocyclic amines such as morpholine, pyrrolidine and N-
methylpiperazine (Scheme 1 and 2). To evaluate the Nrf2-activating ability of the synthesized
compounds, we assessed their ability to release Nrf2 from Kelch-like ECH-associated protein
1
(Keap1) and translocate Nrf2 into the nucleus using our previously reported cell-based
assay system. [40, 41] The potency of Nrf2 activation is shown in Table 1 as half maximal
effective concentration (EC ) values. Most derivatives (5, 6a-e, 6g, 9, 11, 14, 17, 20)
5
0
significantly activated Nrf2 nuclear translocation compared to that of DMF (EC : 5, 6a-e, 6g,
5
0
9
, 11, 14, 17, 20 = 0.63–3.33 µM vs DMF = 5.11 µM). First, we introduced a
propylmorphline group into the 2-, 3-, and 4-OH of ring A (4-6). In these three series of
compounds, substitution the 4-position substituted derivatives (6) showed more potent Nrf2
translocation effects than the corresponding 2- and 3-position derivatives (4, 5) (6a = 1.36
µM > 5a = 2.30 µM & 6d = 1.63 µM > 5b = 1.72 µM > 4 = 5.52 µM). We also found that 2’-
position substituted derivatives (6a: 2’-CF , EC = 1.36 µM; 6d: 2’-OMe, EC = 1.63 µM)
3
50
50
exhibited better activities than the corresponding 3’- and 4’-position substituted derivatives
(
6b: 3’-CF , EC = 2.06 µM; 6e: 3’-OMe, EC = 2.58 µM; 6c: 4’-CF , EC = 2.43 µM; 6f:
3 50 50 3 50
4
’-OMe, EC = 6.97 µM). In addition, the replacement of propylmorpholine at 4-hydroxyl
50
7

group of ring A with propylpyrrolidine slightly enhanced the Nrf2 activation effect (9b: EC50
0.99 µM vs 6d: EC50 = 1.63 µM). Next, we synthesized derivatives (14, 17, 20) that
=
weakened the basicity of heterocyclic amines, including ketone group, based on previously
the most active compound 6a. All synthesized derivatives exerted significant Nrf2 activation
effects (EC50 = 0.63 µM–1.91 µM). Among them, compound 20a has the highest Nrf2
activating potency and is superior to the well-known strong Nrf2 activator, sulforaphane
(
SFN) (EC : 20a = 0.63 µM vs SFN = 0.88 µM). In our previous study, [42] we analyzed the
50
expression level of HO-1, a major Nrf2-dependent gene, using a sandwich enzyme-linked
immunosorbent assay (ELISA) to compare Nrf2 activating efficacy among the synthesized
compounds. To confirm the Nrf2 activating efficacy of the present class, we performed an
ELISA assay for positive controls and the selected nine compounds. We observed that the
Nrf2 activation effect confirmed by ELISA analysis tended to be the same as in the Keap1-
Nrf2 functional assay (Table S1 in the Supporting Information).
In addition, we examined in vitro ADME/Tox properties such as CYP inhibition,
metabolic stabilities and blood-brain barrier (BBB) permeability. The selected compound 20a
showed low inhibitory effect on 5 major CYP isotypes and favorable metabolic stabilities
(Table S2 in the Supporting Information). We also performed a parallel artificial membrane
permeability assay-BBB test using a commercial kit and found that the permeability of 20a
might be favorable for CNS drugs (Table S2 in the Supporting Information). Subsequent
experiments were performed to investigate whether the most potent compound could exhibit
both antioxidant and anti-inflammatory properties through Nrf2 activation in BV-2 microglial
cells.
8

2
.2. Compound 20a induces Nrf2 nuclear translocation
The most potent compound 20a showed concentration-dependent activation of Nrf2
nuclear translocation with an EC50 of 0.634 µM in the Keap1-Nrf2 nuclear translocation
assay (Figure 2A). To further investigate the in vitro efficacies of compound 20a, we first
evaluated its cytotoxic effect on BV-2 microglial cells. It was confirmed that compound 20a
did not affect cell viability up to 10 µM (Figure 2B). Next, to identify whether compound 20a
induces nuclear translocation of Nrf2 in BV-2 microglial cells, we measured the expression
level of Nrf2 in the nucleus after treatment with compound 20a in a time-dependent manner.
The nuclear Nrf2 level increased to maximum at ~2 h when compound 20a was treated at 10
µM in BV-2 microglial cells (Figure 2C). We also measured Nrf2 levels in nuclear fraction
and whole cell lysate after 6 h exposure to various concentrations of compound 20a. Western
blot data indicated that compound 20a concentration-dependently increased the amount of
nuclear Nrf2, with a significant increase at 0.1 µM (Figure 2C). Because activated Nrf2 is
released from Keap1 and is no longer subject to proteosomal degradation, the total Nrf2 level,
as well as nuclear Nrf2, are expected to increase. Indeed, total cellular Nrf2 accumulated
following treatment with compound 20a (Figure 2D). Taken together, we confirmed that
compound 20a effcectively induced the Nrf2 release from Keap1 and Nrf2 nuclear-
translocation in BV-2 microglial cells.
2
.3. Compound 20a induces gene expression of antioxidant enzymes and attenuates
inflammatory responses in activated microglia
In previous studies Nrf2 activators have been reported to induce expression of
antioxidant genes and suppress inflammatory responses as well as antioxidant defenses. [40-
9

4
2] Therefore, we evaluated whether Nrf2 activation with compound 20a could have these
beneficial effects. After nuclear translocation, the transcription factor Nrf2 binds to its
specific promoter sequences, AREs, and induces the expression of several cytoprotective
genes, which are involved in glutathione synthesis, ROS scavenging, and detoxification of
xenobiotics against oxidative stress. [6] To confirm whether these Nrf2-dependent enzyme
genes are induced by compound 20a because of Nrf2 activation in microglial cells, we
measured the gene expression level of HO-1, which is the enzyme that converts heme to
biliverdin, ferrous iron, and carbon monooxide, and glutamate-cysteine ligase regulatory
subunit (GCLM), the regulatory subunit of GCL responsible for glutathione synthesis.
Pretreatment with compound 20a for 8 h significantly upregulated the relative mRNA
expression levels of Ho-1 and Gclm in BV-2 microglial cells (Figure 3A, B). We also
measured the protein levels of HO-1 and GCLM in a time-dependent manner after treatment
with compound 20a. At a concentration of 3 µM, compound 20a significantly increased the
expression levels of HO-1 and GCLM with a maximum increase at ~6 h (Figure 3C–E). Next,
western blot analysis revealed that both HO-1 and GCLM were concentration-dependently
increased after 12 h exposure to various concentrations of compound 20a, with >7-fold and
>15-fold upregulation observed at 10 µM, respectively. (Figure 3F–H). These results indicate
that compound 20a significantly induces the Nrf2-dependent gene expression by activating
Nrf2 in BV-2 microglial cells.
The Nrf2 signaling pathway have been reported to contributes to the anti-
inflammatory process by suppressing inflammatory mediators including NO, pro-
inflammatory cytokines, and inflammatory enzymes such as COX-2 and iNOS. [13] It also
has been suggested that the Nrf2 down-regulates inflammatory responses through increasing
expression of HO-1, the major Nrf2-dependent antioxidant enzyme, and neutralizing ROS in
1
0

several studies using cell cultures and Nrf2 knockout mouse model. [43- 45] We further
investigated whether compound 20a can reduce lipopolysaccharide (LPS)-induced
inflammatory responses in BV-2 microglial cells. LPS stimulation significantly increased NO
production compared to that of the untreated control and this aberrant NO production was
concentration-dependently reduced by compound 20a (Figure 4A). To determine whether
compound 20a regulates the expression of iNOS resulting in inhibition of NO production, we
measured protein expression levels of iNOS, an NO-synthesizing enzyme, after pretreatment
of the LPS-stimulated BV-2 microglial cells with compound 20a for 3 h. Western blot
analysis revealed that up-regulated gene expression of iNOS due to LPS stimulation was also
concentration-dependently inhibited by compound 20a, with almost complete inhibition at 10
µM compared to the levels in the LPS-untreated control (Figure 4B). Next, we assessed the
anti-inflammatory effects of compound 20a on the production of proinflammatory cytokines.
LPS stimulation markedly increased the secretion of TNF-α and IL-6 in BV-2 microglial cells,
whereas preincubation of the cells with compound 20a for 3 h significantly suppressed the
levels of both proinflammatory cytokines in a concentration-dependent manner (Figure 4C,
D). Taken together, these results indicate that compound 20a effectively down-regulates LPS-
induced inflammation in microglia by suppressing inflammatory mediators, such as iNOS,
TNF-α, and IL-6.
2
.4. Compound 20a reduces ROS production in microglia
ROS, one of the main indicators of oxidative stress, are known to contribute to
neurodegeneration by causing inflammation and neurotoxicity when overproduced. [46] To
examine wheter coumpound 20a can effectively reduce ROS in oxidative condition, we
1
1

measured the amount of ROS produced after hydrogen peroxide (H O ) treatment in cells
2
2
pretreated with or without compound 20a, using 2’, 7’-Dichlorodihydrofluorescin diacetate
DCFH-DA), a fluorescent probe for intracellular ROS. Treatment with H O significantly
(
2
2
increased intracellular ROS production compared to that of the untreated control (Figure 5A).
In contrast, pretreatment with compound 20a for 24 h concentration-dependently inhibited
this ROS production in microglial cells (Figure 5A). Furthermore, pretreatment with 10 µM
compound 20a also suppressed the increase in ROS production induced by H O treatment at
2
2
all concentrations (Figure 5B). In addition, we detected intracellular ROS accumulation in
BV-2 microglial cells through fluorescence imaging, which confirmed that excessive ROS
accumulation due to H O treatment was dramatically reduced by pretreatment with
2
2
compound 20a in a similar concentration-dependent manner (Figure 5C). These results show
that compound 20a suppresses ROS accumulation in microglial cells through its potent
antioxidant activity.
2
.5. Compound 20a increases expression and activity of SOD in vitro and in vivo
Superoxide dismutase (SOD) is an important Nrf2-dependent antioxidant enzyme
that catalyzes the dismutation of superoxide radicals into H O . The expression level and
2
2
activity of SOD have been reported to be reduced in the brain and plasma of mild cognitive
impairment and AD patients. [47-51] It indicates that oxidative stress occurs from the early
stage of disease and compromised antioxidant defense system contribute to the progression
and pathology of AD. To investigate whether compound 20a upregulate the gene expression
of SOD1, one of three human SODs located in the cytoplasm, we measured the protein levels
of SOD1 after treatment with compound 20a for 12 h. Similar to that of other Nrf2-regulated
1
2

enzymes (Figure 3F), the expression of SOD1 increased in a concentration-dependent manner
Figure 6A). We next examined the effect of compound 20a on SOD activity. The activity of
(
SOD in BV-2 microglial cells after treating with compound 20a for 24 h increased in a
concentration-dependent manner, which indicates that compound 20a effectively upregulates
the gene expression as well as the activity of this antioxidant enzyme (Figure 6B). We also
examined the antioxidant effect of compound 20a in H O -treated BV-2 microglial cells. We
2
2
found that SOD activity was reduced to approximately 50% after 18 h exposure to 400 µM
H O in the microglia (Figure 6C). Pretreatment with compound 20a remarkably restored the
2
2
reduced SOD activity to a higher level than that of the H O -untreated control (Figure 6C). In
2
2
addition, we determined whether compound 20a could increase in vivo SOD activity in the
brain hippocampus using the scopolamine-induced amnesia model (Figure 6D). Scopolamine,
a nonselective muscarinic acetylcholine receptor antagonist, has been known to cause
memory impairment by blocking central cholinergic signaling. [37-39] Furthermore, recent
studies have reported that the scopolamine-induced amnesia model is accompanied by
oxidative stress [38, 52], neuroinflammation [35, 36], upregulation of several AD-associated
genes [53], and accumulation of amyloid β and phosphorylated tau proteins [52, 54], all of
which are major pathological features of AD. The activity of SOD was reduced in the
scopolamine-treated mice (2 mg/kg, i.p. injection) but was significantly restored to normal
levels by pretreatment with compound 20a (30 mg/kg/day, p.o. for 5 days) (Figure 6E). These
results means that compound 20a actually afforded antioxidant effects against oxidative stress
by enhancing expression and activity of SOD in vitro and in vivo.
2
.6. Compound 20a restores learning and memory in vivo
1
3

To examine if Nrf2 activation with compound 20a could lead to in vivo therapeutic
efficacy in cognitive disorders, we performed behavioral tests using the scopolamine-induced
amnesia model. We assessed spatial working memory using the Y-maze spontaneous
alternation test. We treated mice with compound 20a (30 mg/kg/day, p.o.) for 5 days and
injected scopolamine (2 mg/kg, i.p. injection) before the test on the last day (Figure 7A). The
scopolamine-treated mice showed a reduced percentage of spontaneous alternations
compared to that of the vehicle-treated control mice (Figure 7B). However, treatment with
compound 20a remarkably recovered the reduced alternation percentage (Figure 7B). There
was no difference in the total number of arm entries among the experimental groups,
confirming that the changes in percent alternation were not caused by hyperactivity of the
mice (Figure 7B). Next, we treated mice with compound 20a (30 mg/kg/day, p.o.) for 5 days
and learning and memory performance was examined using the passive avoidance test in the
scopolamine-induced mouse model (1 mg/kg, i.p. injection on day 4) (Figure 7C). As shown
in Figure 7D, the scopolamine-treated mice showed markedly shorter step-through latency
than the vehicle-treated control mice did, indicating memory deficits induced by scopolamine.
In contrast, the mice treated with compound 20a showed improved latency to more than 70%
of the vehicle-treated control level (Figure 7D). These results indicate that compound 20a
effectively restores learning and memory in the scopolamine-induced amnesia model,
suggesting a potential therapeutic effect in AD.
1
4

3
. Discussion and Conclusion
Oxidative stress and neuroinflammation have been proposed as major contributors or
driving forces to the pathogenesis and progression of AD. [1, 3] There is no currently
available disease-modifying treatment that consistently maintains cognitive function by
suppressing the progression of neurodegeneration in AD. Thus, inducing antioxidant and anti-
inflammatory effects via Nrf2 activation can be an attractive therapeutic strategy in AD. In
this study, we presented a novel synthetic compound 20a with highly potent efficacy to
activate Nrf2. Compound 20a showed antioxidant and anti-inflammatory effects in vitro and
effectively ameliorated learning and memory impairment in the scopolamine-induced mouse
model.
Keap1, a negative regulator of Nrf2, normally sequesters Nrf2 in the cytoplasm and
causes its continuous degradation through the ubiquitin-proteasome pathway. Under oxidative
stressed conditions, oxidants and electrophiles chemically modify several critical cysteine
residues within Keap1, causing a conformational change and inactivation of the protein. As a
result, Nrf2 is released from Keap1, translocates into the nucleus, and induces the expression
of several cytoprotective genes. [6, 7] Through initial screening against nuclear translocation
activity of Nrf2 (Table 1), we found that compound 20a has potent Nrf2-activating efficacy.
Compound 20a also contains a unique chalcone structure, an α,β-unsaturated ketone, that is
highly reactive with cellular nucleophiles such as the thiol group of cysteine. Several studies
have shown that chalcone derivatives are potent activators of the Nrf2-ARE signaling
pathway. [26, 55] We also previously reported that the α,β-unsaturated sulfone group activate
Nrf2 via the Nrf2-Keap1 signaling pathway. [42] Accordingly, compound 20a appears to
induce nuclear translocation of Nrf2 due to the reactivity of the cysteine thiol group in Keap1.
1
5

Although the mechanism underlying Nrf2-dependent anti-inflammation is still not
clear, previous studies have reported that Nrf2 essentially contributes to anti-inflammatory
responses through crosstalk between Nrf2 and NF-κB pathways or elimination of ROS by
Nrf2-dependent antioxidant enzymes. [13] A recent study also revealed a novel mechanism
by which Nrf2 directly suppresses the induction of proinflammatory cytokine transcription by
binding to the promoter regions of proinflammatory cytokine genes. [12] The anti-
inflammatory properties of compound 20a are believed to be due to Nrf2 activation.
Compound 20a does not directly scavenge free radicals (data not shown). Therefore, Nrf2
activation in microglia by compound 20a induces the expression of Nrf2-dependent
antioxidant enzymes and the elimination of ROS, resulting in inhibition of the LPS-induced
inflammatory responses.
We have also demonstrated in vivo therapeutic effects of compound 20a in terms of
cognitive deficit-related behaviors. Compound 20a effectively restored SOD activity and
memory performance in the scopolamine-induced amnesia model. Previous in vivo studies
have shown that the Nrf2 signaling pathway has protective effects by modulating oxidative
stress in various neurodegenerative diseases, including AD. [19, 21] The scopolamine-
induced amnesia model is accompanied by oxidative stress, resulting in memory impairment,
which is ameliorated by suppression of free radicals and lipid peroxidation. [38, 52] Recent
studies have reported the neuroprotective effects of natural compounds or their metabolites
extracted from leaves and food on scopolamine-induced memory deficits. [35, 36, 39, 56, 57]
However, the molecular modes of action in which an Nrf2 activator directly affords
protective effects against the scopolamine-induced model have rarely been evaluated. Our
study suggests that Nrf2 activators represent potential therapeutic strategies for cognitive
impairment recovery in the scopolamine-induced mouse model.
1
6

1
7

4
4
. Materials and Methods
.1. General method
The synthetic scheme for final compounds is described in Scheme 2 and Scheme 3. The
reaction was monitored using analytical thin-layer chromatography (TLC) plates (Merck,
Cat# 1.05715) at 254 nm UV light. The reactants were purified by silica gel column
chromatography (Merck, Cat# 1.09385). Melting points were determined using an OptiMelt
melting point equipment (Stanford Research System, Inc.). NMR (Nuclear magnetic
resonance) spectra were obtained using Bruker spectrometers at 400 MHz / 300 MHz (1H) or
1
00 MHz / 75 MHz (13C). High-resolution mass spectrometry (HRMS) data was obtained
using electron scatter ionization (ESI) on a LTQ Orbitrap (Thermo Electron Corporation)
instrument. All chemicals, solvents and reagents were purchased from commercial reagent
sources as reagent grade without further purification.
4
.1.1. General procedure for the compounds (Method A)
To an ethanol (EtOH) solution of the 3, 8, 10, 13, 16 and 18 (1.0 equiv) was added either
lithium hydroxide (LiOH) (1.0 ~ 1.5 equiv) or lithium hydroxide monohydrate (1.0 ~ 1.5
equiv). The reaction mixture was stirred at room temperature (15 min) and then treated with
the desired benzaldehyde (1.5 ~ 3.0 equiv). The reaction mixture was stirred at room
temperature (2 h) and diluted with EtOAc (~150 mL) and washed with H O (3 × ~150 mL).
2
The organic layer was dried with anhydrous Na SO and concentrated in vacuo. The resulting
2
4
residue was purified using column chromatography on SiO to afford free form products. The
2
salt form compounds (4, 5, 6, 9, 11, 20) were prepared by addition of 4.0 M HCl to EtOAc
solutions.
1
8

4
.1.2.
(E)-3-(2-Methoxyphenyl)-1-(2-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydrochloride (4)
Using Method A, 3a (0.85 g, 3.23 mmol), 2-methoxybenzaldehyde (0.44 g, 3.29 mmol),
lithium hydroxide monohydrate (0.13 g, 3.23 mmol) and ethanol (15 mL, 0.25 M) gave 4b as
1
a pale gray solid (1.13 g, 84%). mp: 163.0–165.0 °C; H NMR (400 MHz, DMSO-d ) δ 12.16
6
+
(
br s, N H), 8.14−7.85 (m, 3 Ar H, 2 trans H), 7.45−7.41 (m, Ar H), 7.10−6.99 (m, 4 Ar H),
4
.20, (t, J = 5.92 Hz, CH ), 3.88−3.30 (m, OCH , 8 piperazine H), 2.82 (s, NCH ), 2.25 (t, J
2 3 3
13
=
7.48 Hz, CH ); C NMR (100 MHz, DMSO-d ) δ 187.4, 162.0, 158.1, 137.6, 132.0, 130.7,
2 6
1
30.6, 128.3, 123.0, 121.7, 120.6, 114.4, 111.7, 65.2, 55.6, 52.9, 49.5, 48.0, 41.9, 23.1.
4
.1.3 (E)-1-(3-(3-Morpholinopropoxy)phenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-
one hydrochloride (5a)
Using Method A, 3b, (0.60 g, 2.28 mmol), 2-trifluoromethylbenzaldehyde (0.36 mL, 2.73
mmol), lithium hydroxide (0.12 g, 2.73 mmol) and ethanol (10 mL, 0.25 M) gave 5a as a
1
white powder (0.08 g, 48%). mp: 130.5–132.5 °C; H NMR (400 MHz, DMSO-d ) δ 11.11
6
+
(
br s, N H), 8.37 (d, J = 7.8 Hz, Ar H), 8.06−7.96 (m, 2 trans H), 7.87−7.79 (m, 3 Ar H),
7
.71−7.67 (m, 2 Ar H), 7.54 (t, J = 7.96 Hz, Ar H), 7.31−7.28 (m, Ar H), 4.20 (t, J = 5.84 Hz,
CH ), 3.99−3.80 (m, 4 piperazine H), 3.47 (d, J = 12.3 Hz, CH ), 3.31−3.26 (m, 2 piperazine
2
2
1
3
H), 3.13−3.06 (m, CH ); C NMR (100 MHz, DMSO-d ) δ 189.1, 159.1, 139.0, 138.4, 133.4,
2
6
1
33.2, 131.0, 130.6, 129.4, 128.0 (q, JC-F = 29.5 Hz), 126.7, 126.3 (q, JC-F = 5.4 Hz), 124.6 (q,
JC-F = 272.4 Hz), 122.0, 120.5, 114.4, 65.9, 63.6, 53.9, 51.5, 23.4.
1
9

4
.1.4.
(E)-3-(2-Methoxyphenyl)-1-(3-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydrochloride (5b)
Using Method A, 3b (0.88 g, 3.34 mmol), 2-methoxybenzaldehyde (0.46 g, 3.41 mmol),
lithium hydroxide monohydrate (0.14 g, 3.34 mmol) and ethanol (15 mL, 0.25 M) gave 5 as a
1
pale yellow solid (1.25 g, 89%). mp: 141.0–143.0 °C; H NMR (400 MHz, DMSO-d ) δ
6
+
1
1.53 (br s, N H), 8.06 (d, J = 15.8 Hz, trans H), 8.00 (dd, J = 1.4 Hz, 7.7 Hz, ArH), 7.86 (d,
J = 15.8 Hz, trans H), 7.75 (d, J = 7.8 Hz, ArH), 7.59 (s, ArH), 7.52−7.43 (m, 2 ArH), 7.25
dd, J = 2.2 Hz, 8.1 Hz, Ar H), 7.12 (d, J = 8.2 Hz, Ar H), 7.03 (t, J = 7.5 Hz, Ar H), 4.18 (t,
J = 6.0 Hz, 2 H), 3.95 (d, J = 9.7 Hz, 2 H), 3.89 (s, 3 H), 3.86 (d, J = 11.4 Hz, 2 H), 3.45 (d,
(
13
J = 12.0 Hz, 2 H), 3.33−3.24 (m, 2 H), 3.18−3.04 (m, 2 H), 2.29−2.22 (m, 2 H); C NMR
100 MHz, DMSO-d ) δ 188.9, 158.3 (d, J = 30.4 Hz), 139.1, 138.5, 132.3, 129.9, 128.4,
(
6
1
22.8, 121.7, 121.1, 120.6, 119.4, 113.6, 111.7.
4
.1.5
(E)-3-(2-Chlorophenyl)-1-(3-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydrochloride (5c)
Using Method A, 3b (0.60 g, 2.28 mmol), 2-chlorobenzaldehyde (0.31 mL, 2.73 mmol),
lithium hydroxide (0.12 g, 2.73 mmol) and ethanol (10 mL, 0.25 M) gave 5c as a white
1
powder (0.12 g, 72%). mp: 165.0–167.0 °C; H NMR (400 MHz, DMSO-d ) δ 11.19 (br s,
6
+
N H), 8.28−8.25 (m, Ar H), 8.08−7.98 (m, 2 trans H), 7.82 (d, J = 7.72 Hz, Ar H), 7.66−7.65
(
(
(
m, Ar H), 7.60−7.45 (m, 4 Ar H), 7.30−7.27 (m, Ar H), 4.20 (t, J = 6.0 Hz, CH ), 3.99−3.95
2
m, 2 piperazine H), 3.87−3.81 (m, 2 piperazine H), 3.47 (d, J = 12.3 Hz, CH ), 3.31−3.26
2
1
3
m, 2 piperazine H), 3.14−3.05 (m, 2 piperazine H), 2.29−2.22 (m, CH ); C NMR (100
2
2
0

MHz, DMSO-d ) δ 189.2, 159.1, 139.2, 139.1, 134.9, 132.7, 132.5, 130.5, 130.4, 129.2,
6
1
28.2, 125.3, 121.9, 120.4, 114.4, 65.8, 63.6, 53.9, 51.5, 23.4.
4
1
.1.6. (E)-1-(4-(3-Morpholinopropoxy)phenyl)-3-(2-(trifluoromethyl)phenyl)pro-2-pen-
-one hydrochloride (6a)
Using Method A, 3c (17.4 g, 65.9 mmol), 2-trifluorobenzaldehyde (8.86 g, 67.2 mmol),
lithium hydroxide monohydrate (2.76 g, 65.9 mmol) and ethanol (525 mL, 0.125 M) gave 6a
o
1
as a white solid (24 g, 87%). mp: 209.0–211.0 C; H NMR (400 MHz, DMSO-d ) δ 11.54
6
+
(
br s, N H), 8.39 (d, J = 7.8 Hz, ArH), 8.25 (d, J = 8.8 Hz, 2 ArH), 8.09 (d, J = 15.3 Hz, trans
H), 7.98 (d, J = 15.3 Hz, trans H), 7.88 (d, J = 7.8 Hz, ArH), 7.83 (t, J = 7.8 Hz, ArH), 7.70
t, J = 7.4 Hz, ArH), 7.16 (d, J = 8.8 Hz, 2 ArH), 4.26 (t, J = 5.9 Hz, 2 H), 4.00 (d, J = 12.2
Hz, 2 H), 3.91 (t, J = 12.1 Hz, 2 H), 3.50 (d, J = 12.1 Hz, 2 H), 3.33−3.28 (m, 2 H),
(
1
3
3
1
2
.16−3.09 (m, 2 H), 2.30−2.29 (m, 2 H); C NMR (100 MHz, DMSO-d ); δ 187.0, 162.6,
6
37.1, 133.0, 132.9, 131.2, 130.3, 130.2, 128.8, 127.5 (q, J = 29.4 Hz), 126.1, 124.2 (q, J =
72.4 Hz), 114.6, 65.6, 63.2, 53.3, 51.1, 22.9.
4
1
.1.7. (E)-1-(4-(3-Morpholinopropoxy)phenyl)-3-(3-(trifluoromethyl)phenyl)pro-2-pen-
-one hydrochloride (6b)
Using Method A, 3c (0.42 g, 1.59 mmol), 3-trifluorobenzaldehyde (0.22 mL, 1.63 mmol),
lithium hydroxide monohydrate (0.07 g, 1.59 mmol) and ethanol (5 mL, 0.25 M) gave 6b as a
1
white solid (0.22 g, 30%). mp: 193.0–194.0 °C; H NMR (400 MHz, MeOD) δ 8.16 (d, J =
8
.9 Hz, 2 ArH), 8.03 (t, J = 7.8 Hz, 2 ArH), 7.90 (d, J = 15.7 Hz, trans H), 7.80 (d, J = 15.7
2
1

Hz, 2 trans H), 7.73 (d, J = 7.6 Hz, ArH), 7.65 (t, J = 7.6 Hz, ArH), 7.11 (d, J = 8.8 Hz, 2
ArH), 4.25 (t, J = 5.7 Hz, 2 H), 4.18−3.97 (br m, 2 H), 3.91−3.71 (m, 2 H), 3.65−3.45 (br m,
1
3
2
H), 3.29 (t, J = 8.0 Hz, 2 H), 3.28−3.14 (br m, 2 H), 2.34−2.29 (m, 2 H); C NMR (100
MHz, DMSO-d ) δ 187.3, 162.5, 141.4, 136.0, 132.8, 131.2, 130.5, 129.9, 129.8 (q, J = 31.9
6
Hz), 126.5, 125.1, 124.1 (q, J = 270.9 Hz), 124.0, 114.6, 65.6, 63.2, 53.4, 51.1, 22.9.
4
1
.1.8. (E)-1-(4-(3-Morpholinopropoxy)phenyl)-3-(4-(trifluoromethyl)phenyl)pro-2-pen-
-one hydrochloride (6c)
Using Method A, 3c (0.44 g, 1.67 mmol), 4-trifluorobenzaldehyde (0.23 mL, 1.70 mmol),
lithium hydroxide monohydrate (0.07 g, 1.67 mmol) and ethanol (7 mL, 0.25 M) gave 6c as a
1
pale green solid (0.20 g, 29%). mp: 203.0–205.0 °C; H NMR (400 MHz, MeOD) δ 8.14 (m,
2
ArH), 7.89−7.95 (m, trans H, 2 ArH), 7.72−7.81 (m, trans H, 2 ArH), 7.09−7.11 (m, 2
ArH), 4.24 (t, J = 5.7 Hz, 2 H), 4.09 (d, J = 12.3 Hz, 2 H), 3.81 (t, J = 12.5 Hz, 2 H), 3.58 (d,
J = 12.2 Hz, 2 H), 3.41 (t, J = 8.0 Hz, 2 H), 3.21 (t, J = 12.2 Hz, 2 H), 2.35−2.28 (m, 2 H);
1
3
C NMR (100 MHz, DMSO-d ) δ 187.2, 162.4, 141.1, 138.8, 131.1, 130.3, 130.0, 129.9 (d,
6
J = 31.7 Hz), 129.3, 125.6, 124.7, 124.0 (q, J = 270.4 Hz), 114.5, 65.5, 63.1, 53.3, 51.0, 22.8.
4
.1.9.
(E)-3-(2-Methoxyphenyl)-1-(4-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydro chloride (6d)
Using Method A, 3c (0.30 g, 1.14 mmol), 4-trifluorobenzaldehyde (0.12 mL, 1.17 mmol),
lithium hydroxide monohydrate (0.05 g, 1.14 mmol) and ethanol (5 mL, 0.25 M) gave 6d as a
1
white solid (0.12 g, 28%). mp: 211.0–213.0 °C; H NMR (400 MHz, DMSO-d ) δ 11.75 (s,
6
+
N H), 8.15 (d, J = 8.8 Hz, 2 ArH), 7.97−8.05 (m, ArH, trans H), 7.89 (d, J = 15.7 Hz, trans
2
2

H), 7.42−7.47 (m, ArH), 7.01−7.12 (m, 4 ArH), 4.21 (t, J = 6.0 Hz, 2 H), 3.87−3.98 (m, 7 H),
13
3
.46 (d, J = 12.2 Hz, 2 H), 3.24−3.29 (m, 2 H), 3.05−3.13 (m, 2 H), 2.24−2.31 (m, 2 H); C
NMR (100 MHz, DMSO-d ) δ 188.0, 162.6, 158.6, 138.2, 132.6, 131.3, 131.2, 128.9, 123.5,
6
1
22.2, 121.1, 115.0, 112.3, 65.9, 63.6, 56.2, 53.8, 51.4, 23.3.
4
.1.10.
(E)-3-(3-Methoxyphenyl)-1-(4-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydrochloride (6e)
Using Method A, 3c (0.35 g, 1.33 mmol), 3-methoxybenzaldehyde (0.16 mL, 1.36 mmol),
lithium hydroxide monohydrate (0.06 g, 1.33 mmol) and ethanol (5 mL, 0.25 M) gave 6e as a
1
white solid (0.43 g, 77%). mp: 212.0–214.0 °C; H NMR (400 MHz, DMSO-d ) δ 11.52 (br s,
6
+
N H), 8.19 (d, J = 8.9 Hz, 2 ArH), 7.95 (d, J = 15.6 Hz, trans H), 7.67 (d, J = 15.6 Hz, trans
H), 7.47 (s, ArH), 7.43 (d, J = 7.7 Hz, ArH), 7.36 (t, J = 7.9 Hz, ArH), 7.09 (d, J = 8.9 Hz, 2
ArH), 7.01 (dd, J = 2.1 Hz, 7.9 Hz, ArH), 4.20 (t, J = 6.1 Hz, 2 H), 3.95 (d, J = 11.5 Hz, 2 H),
3
3
1
5
.88 (d, J = 12.2 Hz, 2 H), 3.82 (s, 3 H), 3.45 (d, J = 12.2 Hz, 2 H), 3.30−3.20 (m, 2 ArH),
1
3
.14−3.02 (m, 2 H), 2.29−2.22 (m, 2 H); C NMR (100 MHz, DMSO-d ) δ 187.4, 162.3,
6
59.7, 143.2, 136.2, 131.0, 130.6, 130.0, 122.3, 121.6, 116.5, 114.5, 113.4, 65.4, 63.1, 55.3,
3.3, 51.0, 22.9.
4
.1.11.
(E)-3-(4-Methoxyphenyl)-1-(4-(3-morpholinopropoxy)phenyl)prop-2-en-1-one
hydrochloride (6f)
Using Method A, 3c (0.37 g, 1.41 mmol), 4-methoxybenzaldehyde (0.17 mL, 1.43 mmol),
lithium hydroxide monohydrate (0.06 g, 1.41 mmol) and ethanol (6 mL, 0.25 M) gave 6f as a
1
white solid (0.30 g, 52%). mp: 192.0–194.0 °C; H NMR (400 MHz, DMSO-d ) δ 11.53 (br s,
6
+
N H), 8.15 (d, J = 8.7 Hz, 2 ArH), 7.79−7.85 (m, trans H, 2 ArH), 7.67 (d, J = 15.5 Hz, trans
2
3

H), 7.08 (d, J = 8.8 Hz, 2 ArH), 7.01 (d, J = 8.7 Hz, 2 ArH), 4.19 (t, J = 6.1 Hz, 2 H), 3.95 (d,
J = 11.5 Hz, 2 H), 3.88 (d, J = 12.1 Hz, 2 H), 3.81 (s, 3 H), 3.44 (d, J = 12.1 Hz, 2 H),
13
3
.32−3.20 (m, 2 H), 3.11−3.04 (m, 2 H), 2.29−2.22 (m, 2 H); C NMR (100 MHz, DMSO-
d6) δ 187.2, 162.1, 161.2, 143.2, 130.9, 130.8, 130.7, 127.4, 119.5, 114.4, 114.3, 65.4, 63.1,
5
5.4, 53.3, 51.0, 22.9.
4
.1.12. (E)-3-(2-Chlorophenyl)-1-(4-(3-morpholinopropoxy)phenyl)prop-2-en-1-one (6g)
Using method A, 3c (0.60 g, 2.28 mmol) and 2-chlorobenzaldehyde (0.31 mL, 2.73 mmol),
lithium hydroxide (0.12 g, 2.73 mmol) and ethanol (10 mL, 0.25 M) gave 5c as a white
1
powder (0.63 g, 72%); H NMR (400 MHz, DMSO-d ) δ 8.24−8.17 (m, 3 Ar H), 8.01 (s, 2
6
Ar H), 7.59−7.44 (m, Ar H, 2 trans H), 7.09 (d, J = 8.64 Hz, 2 Ar H), 4.14 (t, J = 6.24 Hz,
CH ), 3.59−3.57 (m, 4 piperazine H), 2.45−2.37 (m, CH , 4 piperazine H), 1.95−1.88 (m,
2
2
13
CH ); C NMR (100 MHz, DMSO-d ) δ 187.6, 163.4, 138.2, 134.7, 132.9, 132.3, 131.6,
2
6
1
30.5, 129.0, 128.1, 125.3, 115.0, 66.8, 66.7, 55.2, 53.8, 26.2.
4
.1.13.
(E)-1-(4-(3-Morpholinopropoxy)phenyl)-3-(pyridin-2-yl)prop-2-en-1-one
hydrochloride (6h)
Using Method A, 3c (0.30 g, 1.14 mmol) and 2-pyridinecarboxaldehyde (0.11 mL, 1.16
mmol) and lithium hydroxide (0.05 g, 1.14 mmol) gave 6g as a yellow powder (0.10 g, 25%);
1
mp: 243.0–245.0 °C; H NMR (400 MHz, CDCl ) δ 11.45 (br s, 1H), 8.80 (d, J = 5.1 Hz,
3
ArH), 8.46 (d, J = 15.6 Hz, trans H), 8.19−8.28 (m, 4 ArH), 7.72−7.80 (m, trans H, ArH),
7
.13 (d, J = 9.0 Hz, 2 ArH), 4.22 (t, J = 6.04 Hz, 2 H), 3.83−3.98 (m, 4 H), 3.45 (d, J = 12.1
1
3
Hz, 2 H), 3.23−3.28 (m, 2 H), 3.03−3.12 (m, 2 H), 2.23−2.30 (m, 2 H); C NMR (100 MHz,
2
4

CDCl ) δ 187.4, 163.2, 150.7, 146.9, 142.0, 138.1, 131.7, 130.5, 128.8, 126.4, 126.3, 115.2,
3
6
6.0, 63.6, 53.7, 51.5, 23.3.
4
.1.14.
(E)-1-(4-(3-Morpholinophoxy)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one
hydrochloride (6i)
Using Method A, 3c (0.30 g, 1.14 mmol) and 2-pyridinecarboxaldehyde (0.11 mL, 1.16
mmol) and lithium hydroxide (0.05 g, 1.14 mmol) gave 6h as an ivory powder (0.15 g, 37%);
1
mp: 115.0–116.0 °C; H NMR (400 MHz, CDCl ) δ 11.59 (br s, 1 H), 9.40 (s, ArH), 8.96 (d,
3
J = 8.3 Hz, ArH), 8.87 (d, J = 4.9 Hz, ArH), 8.32 (d, J = 15.8 Hz, trans H), 8.23 (d, J = 8.8
Hz, 2 ArH), 8.01−8.05 (m, Ar H), 7.81 (d, J = 15.7 HZ, trans H), 7.13 (d, J = 8.9 Hz, 2 ArH),
7
3
1
.23 (t, J = 6.0 Hz, 2 H), 3.85−3.97 (m, 5 H), 3.45 (d, J = 12.2 Hz, 2 H), 3.23−3.25 (m, 2 H),
13
.04−3.11 (m, 2 H), 2.23−2.30 (m, 2 H); C NMR (100 MHz, CDCl ) δ 187.3, 163.2, 144.0,
3
43.7, 143.0, 137.3, 134.2, 131.8, 130.5, 127.4, 127.1, 115.1, 66.0, 63.6, 53.7, 51.5, 23.3.
4
.1.15. (E)-1-(4-(3-(Pyrrolidin-1-yl)propoxy)phenyl)-3-(2-(trifluoromethyl)phenyl)prop-
2
-en-1-one hydrochloride (9a)
Using Method A, 8 (1.00 g, 4.04 mmol), 2-(trifluoromethyl)benzaldehyde (1.00 mL, 8.08
mmol) and lithium hydroxide (0.15 g, 6.06 mmol) gave 9a as an ivory solid (0.75 g, 42%).
o
1
+
mp: 164.0–166.0 C; H NMR (400 MHz, DMSO-d ) δ 10.76 (br s, N H), 8.35 (d, J = 7.8 Hz,
6
1
7
5
ArH), 8.22 (d, J = 8.7 Hz, 2 ArH), 8.06 (d, J =15.3 H, trans H), 7.96 (d, J =15.3 H, trans H),
.86−7.79 (m, 2 ArH), 7.68 (t, J = 7.6 Hz, 1 Ar H), 7.13 (d, J = 8.7 Hz, 2Ar H), 4.23 (t, J =
.9 Hz, OCH ), 3.56 (br s, 2 pyrrolidine H), 3.30 (t, J =8.04 Hz, NCH ), 3.01 (br s, 2
2
2
pyrrolidine H), 2.24−2.18 (m, CH CH CH ), 2.01 (br s, 2 pyrrolidine H), 1.91 (br s, 2
2
2
2
13
pyrrolidine H); C NMR (100 MHz, CDCl ) δ 187.4, 163.0, 137.5, 133.4, 131.7, 131.3,
3
2
5

1
1
31.1, 130.8, 130.5, 129.2, 127.9 (q, JC-F = 29.3 Hz), 126.7, 126.6, 124.7 (q, JC-F = 272.0 Hz),
55.1, 65.9, 53.3, 51.5, 25.5, 23.2.
4
.1.16. (E)-3-(2-Methoxyphenyl)-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)prop-2-en-1-
one hydrochloride (9b)
Using Method A, 8 (0.42 g, 1.70 mmol), 2-methoxybenzaldehyde (0.24 g, 1.73 mmol),
lithium hydroxide monohydrate (0.07 g, 1.70 mmol) and ethanol (15 mL, 0.25 M) gave 9 as a
1
pale brown solid (0.33 g, 48%). mp: 158.0–160.0 °C; H NMR (400 MHz, DMSO-d ) δ 11.09
6
+
(
br s, N H), 8.14 (d, J = 8.6 Hz, 2 ArH), 8.02 (d, J = 15.8 Hz, trans H), 7.97 (d, J = 7.5 Hz,
ArH), 7.88 (d, J = 15.7 Hz, trans H), 7.44 (t, J = 7.4 Hz, 1H), 7.10 (t, J = 8.1 Hz, 3 ArH),
.03 (t, J = 7.5 Hz, ArH), 4.20 (t, J = 5.9 Hz, 2H), 3.89 (s, OCH ), 3.60−3.50 (m, 2 H), 3.27
7
3
(q, J = 6.8 Hz, 2 H), 3.09−2.98 (m, 2 H), 2.22−2.18 (m, 2 H), 1.99−1.93 (m, 2 H), 1.90−1.87
1
3
(
m, 2 H); C NMR (100 MHz, DMSO-d ) δ 187.3, 161.9, 157.9, 137.5, 131.9, 130.6, 130.5,
6
1
28.2, 122.8, 121.5, 120.5, 114.3, 111.6, 65.1, 55.5, 52.6, 50.8, 24.9, 22.5.
4
.1.17. (E)-3-(2-Chlorophenyl)-1-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)prop-2-en-1-one
hydrochloride (9c)
Using Method A, 8 (1.00 g, 4.04 mmol), 2-(trifluoromethyl)benzaldehyde (1.00 mL, 8.08
mmol) and lithium hydroxide (0.15 g, 6.06 mmol) gave 9c an ivory solid (0.73 g, 41%) as a
o
1
+
Ivory solid. mp: 184.0–186.0 C; H NMR (400 MHz, DMSO-d ) δ 10.59 (br s, N H),
6
8
.25−8.20 (m, 3 Ar H), 8.03 (s, 2 Ar H), 7.60−7.58 (m, 1 Ar H), 7.52−7.45 (m, 2 Ar H), 7.12
(
d, J = 8.9 Hz, 2 Ar H), 4.23 (t, J = 6.0 Hz, OCH ), 3.56 (br s, 2 pyrrolidine H), 3.30 (t, J =
2
7
.4 Hz, 2 NCH ), 3.02 (br s, 2 pyrrolidine H), 2.24−2.17 (m, CH CH CH ), 2.02 (br s, 2
2 2 2 2
2
6

1
3
pyrrolidine H), 1.90 (br s, 2 pyrrolidine H); C NMR (100 MHz, CDCl ) δ 187.6, 162.9,
3
1
2
38.2, 134.7, 132.8, 132.3, 131.6, 130.7, 130.4, 129.0, 128.1, 125.2, 115.0, 65.8, 53.3, 51.5,
5.5, 23.2.
4
.1.18. (E)-3-(2-Methoxyphenyl)-1-(4-(3-(4-methylpiperazin-1-yl)propoxy)phenyl)prop-
2
-en-1-one dihydrochloride (11)
Using Method E, 10 (0.36 g, 1.30 mmol), 2-methoxybenzaldehyde (0.18 g, 1.33 mmol),
lithium hydroxide monohydrate (0.05 g, 1.30 mmol) and ethanol (10 mL, 0.25 M) gave 11 as
1
a pale yellow solid (0.55 g, 89%). mp: 232.0–234.0 °C; H NMR (400 MHz, DMSO-d ) δ
6
+
1
2.16 (br s, 2 N H), 8.13 (d, J = 8.8 Hz, 2 ArH), 8.01 (d, J = 15.7 Hz, trans H), 7.96 (d, J =
7
.7 Hz, ArH), 7.87 (d, J = 15.7 Hz, trans H), 7.44−7.40 (m, ArH), 7.09 (dd, J = 8.3 Hz, 8.8
Hz, 3 ArH), 7.01 (t, J = 7.5 Hz, ArH), 4.20 (t, J = 6.0 Hz, 2H), 3.88 (s, OCH ), 3.81 (br s,
3
2
H), 3.77 (br s, 2H), 3.69−3.49 (m, 4H), 3.30 (br s, 2H), 2.82 (s, 3H), 2.26−2.23 (m, 2H);
1
3
C NMR (100 MHz, DMSO-d ) δ 187.4, 162.0, 158.0, 137.6, 132.0, 130.7, 130.6, 128.3,
6
1
23.0, 121.7, 120.6, 114.4, 111.7, 65.2, 55.6, 52.9, 49.5, 48.0, 23.1.
4
.1.19.
(E)-N,N-Dimethyl-2-(4-(3-(2-(trifluoromethyl)phenyl)acryloyl)phenoxy)
acetamide (14a)
Using Method A, 13 (0.20 g, 0.90 mmol) and 2-(trifluoromethyl)benzaldehyde (0.10 mL,
1
.08 mmol) and LiOH (0.02 g, 1.08 mmol) gave 14a as a light yellow solid (0.15 g, 45%). Rf
o
1
=
0.45 (Only EtOAc); mp: 122.0–123.0 C; H NMR (400 MHz, CDCl ) δ 8.01–8.06 (m,
3
trans H), 7.92–7.96 (m, 2H), 7.74 (d, J = 7.8 Hz, ArH), 7.64 (d, J = 7.7 Hz, ArH), 7.53 (t, J =
2
7

7
4
1
1
.5 Hz, ArH), 7.42 (t, J = 7.6 Hz, ArH), 7.34 (d, J = 15.5, trans H), 6.94–6.98 (m, 2ArH),
1
3
.71 (s, OCH ), 3.02 (s, NCH ), 2.92 (s, NCH ); C NMR (75 MHz, CDCl ) δ 188.5, 167.1,
3
3
3
3
61.6, 139.5 (d, J = 1.92 Hz), 134.2, 132.1, 131.4, 131.0, 129.8, 129.2 (q, J = 30.2 Hz), 128.0,
26.4, 126.3 (q, J = 5.58 Hz), 124.0 (q, JC-F = 272.4 Hz), 114.7, 67.2, 36.5, 35.7; HRMS (M +
+
+
+
+
H) (ESI ) 378.1310 [M + H] (calcd for C H F NO H 378.1312).
2
0
18
3
3
4
.1.20. (E)-2-(4-(3-(2-Methoxyphenyl)acryloyl)phenoxy)-N,N-dimethyl acetamide (14b)
Using Method A, 13 (0.50 g, 2.26 mmol) and 2-methoxybenzaldehyde (0.46 g, 3.39 mmol)
and LiOH (0.09 g, 2.26 mmol) gave 14b as a light yellow solid (0.57 g, 100%). R = 0.34
f
o
1
(
EtOAc/n-hexane 5/1); mp: 118.0–119.0 C; H NMR (400 MHz, DMSO-d ) δ 8.10 (d, J =
6
8
.8 Hz, 2 ArH), 7.95–8.03 (m, ArH, trans H), 7.87 (d, J = 15.7 Hz, trans H), 7.42–7.47 (m,
ArH), 7.12 (d, J = 8.3 Hz, ArH), 7.02–7.06 (m, 3 ArH), 4.94 (OCH ), 3.90 (OCH ), 2.91 (s,
2
3
13
NCH ), 2.86 (s, NCH ); C NMR (100 MHz, CDCl ) δ 189.3, 167.1, 161.6, 158.8, 139.7,
3
3
3
1
32.1, 131.6, 130.8, 129.2, 124.0, 122.7, 120.7, 114.5, 111.3, 67.2, 55.5, 36.5, 35.7; HRMS
+
+
+
+
(
M + H) (ESI ) 340.1542 [M + H] (calcd for C H N O H 340.1543).
20 21 2 4
4
.1.21. (E)-N, N-Dimethyl-2-(4-(3-(pyridin-2-yl)acryloyl)phenoxy)acetamide (14c)
Using Method A, 13 (0.20 g, 0.90 mmol), 2-pyridinecarboxaldehyde (0.26 mL, 2.70 mmol)
and LiOH·H O (0.05 g, 1.10 mmol) gave 14c as a yellow solid (0.11 g, 39%). R = 0.10 (Only
2
f
1
EtOAc); H NMR (400 MHz, DMSO-d ) δ 8.69 (s, ArH), 8.07–8.16 (m, trans H, 2 ArH),
6
7
.91 (s, 2 ArH), 7.69 (d, J = 15.4 Hz, trans H), 7.44 (s, ArH), 7.07 (d, J = 8.2 Hz, 2 ArH),
1
3
6
.94 (d, 3.2 Hz, ArH), 4.98 (s, OCH ), 3.01 (s, NCH ), 2.86 (s, NCH ); C NMR (100 MHz,
2
3
3
2
8

DMSO-d ) δ 188.1, 167.1, 162.9, 153.4, 150.5, 142.8, 137.6, 131.2, 130.9, 130.7, 125.7,
6
+
+
+
1
25.2, 125.1, 115.3, 66.2, 36.0, 35.4; HRMS (M + H) (ESI ) 311.1383 [M + H] (calcd for
+
C H N O H 311.1390).
18
18
2
3
4
.1.22. (E)-N, N-Dimethyl-2-(4-(3-(pyridin-3-yl)acryloyl)phenoxy)acetamide (14d)
Using Method A, 13 (0.20 g, 0.90 mmol), 3-pyridinecarboxaldehyde (0.25 mL, 2.70 mmol)
and LiOH·H O (0.05 g, 1.10 mmol) gave 14d as a white solid (0.13 g, 47%). R = 0.10 (Only
2
f
o
1
EtOAc); mp: 143.5–145.0 C; H NMR (400 MHz, CDCl ) δ 8.87–8.88 (m, ArH), 8.64–8.66
3
(m, ArH), 8.04–8.08 (m, 2 ArH), 7.95–7.98 (m, ArH), 7.84 (d, J = 15.8 Hz, trans H), 7.66 (d,
J = 15.7 Hz, trans H), 7.37–7.40 (m, ArH), 7.06–7.09 (m, 2 ArH), 4.82 (s, OCH ), 3.13 (s,
2
13
NCH ), 3.02 (s, NCH ); C NMR (100 MHz, CDCl ) δ 188.0, 167.0, 162.0, 151.0, 149.9,
3
3
3
+
1
40.3, 134.5, 131.4, 130.8, 123.8, 123.7, 114.7, 100.9, 67.2, 36.5, 35.7; HRMS (M + H)
+
+
+
(
ESI ) 311.1383 [M + H] (calcd for C H N O H 311.1390).
18 18 2 3
4
.1.23. (E)-N,N-Dimethyl-2-(4-(3-(pyridin-4-yl)acryloyl)phenoxy)acetamide (14e)
Using Method A, 13 (0.20 g, 0.90 mmol), 4-pyridinecarboxaldehyde (0.26 mL, 2.70 mmol)
and LiOH·H O (0.05 g, 1.10 mmol) gave 14e as a light yellow solid (0.10 g, 35%). R = 0.05
2
f
o
1
(
Only EtOAc); mp: 146.0–147.5 C; H NMR (400 MHz, DMSO-d ) δ 8.67 (d, J = 5.8 Hz, 2
6
ArH), 8.14–8.18 (m, 2 ArH, trans H), 7.84 (d, J = 5.8 Hz, 2 ArH), 7.65 (d, J = 15.6 Hz, trans
1
3
H), 7.08 (d, J = 8.8 Hz, 2 ArH), 4.99 (s, OCH ), 3.02 (s, NCH ), 2.86 (s, NCH ); C NMR
2
3
3
(
100 MHz, DMSO-d ) δ 187.7, 167.0, 163.0, 150.8, 142.4, 140.7, 131.4, 130.6, 126.9, 122.9,
6
+
+
+
+
1
15.3, 66.2, 36.0, 35.4; HRMS (M + H) (ESI ) 311.1382 [M + H] (calcd for C H N O H
18 18 2 3
2
9