Design, synthesis, and evaluation of isoflavone analogs as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.02.053

A series of novel isoflavone analogs were designed, synthesized, and evaluated as multitarget-directed ligands for the treatment of Alzheimer's disease. In vitro evaluations revealed that some ligands had multifunctional profiles, including potent blockage of histamine 3 receptor (H3R), excellent inhibition of acetylcholinesterase (AChE), neuroprotective effects and anti-neuroinflammatory properties. Among these derivatives, compound 9b exhibited the highest ability to block H3R (IC₅₀ = 0.27 μM) and good inhibitory activity against AChE (IC₅₀ = 0.08 μM). Additionally, compound 9b showed obvious neuroprotective effect on SH-SY5Y by preventing copper-induced neuronal damage and potent anti-neuroinflammatory activity by inhibiting the production of inflammatory factors on BV-2 cells. A molecular modeling study revealed that 9b acts as a mixed-type inhibitor that interacts simultaneously with H3R and AChE. Moreover, in vivo data revealed that compound 9b did not cause acute toxicity in mice at doses up to 1000 mg/kg, and had desirable pharmacokinetic properties, as well as a good blood-brain barrier (BBB) permeability (log BB = 1.24 ± 0.07). Further studies demonstrated that chronic oral treatment with 9b significantly improved cognitive dysfunction in scopolamine-induced AD mice in the step-down passive avoidance test. Taken together, the present study showed that compound 9b is a promising multifunctional drug candidate for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2019.02.053

Accepted Manuscript
Design, synthesis, and evaluation of isoflavone analogs as multifunctional agents for
the treatment of Alzheimer's disease
Dongmei Wang, Min Hu, Xinpeng Li, Dan Zhang, Chengjuan Chen, Junmin Fu, Shuai
Shao, Gaona Shi, Yu Zhou, Song Wu, Tiantai Zhang
PII:
S0223-5234(19)30167-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.053
EJMECH 11145
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 December 2018
Revised Date: 14 February 2019
Accepted Date: 17 February 2019
Please cite this article as: D. Wang, M. Hu, X. Li, D. Zhang, C. Chen, J. Fu, S. Shao, G. Shi, Y. Zhou,
S. Wu, T. Zhang, Design, synthesis, and evaluation of isoflavone analogs as multifunctional agents
for the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.02.053.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and evaluation of isoflavone analogs as
multifunctional agents for the treatment of Alzheimer's disease
a
a
,1
Dongmei Wang , Min Hua,1, Xinpeng Lib, Dan Zhangc, Chengjuan Chen , Junmin
Fua, Shuai Shaoa, Gaona Shia, Yu Zhoua, Song Wua,**, Tiantai Zhanga,*
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China;
b Food and Drug Administration of Beijing Yanqing District, Beijing 102100, China
c Department of Pharmacy, China-Japan Friendship Hospital, Beijing 100029, China
* Corresponding author. State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China.
**
Corresponding author. State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China.
E-mail address: ttzhang@imm.ac.cn (Tiantai Zhang); ws@imm.ac.cn (Song Wu).
1 These authors contributed equally to this work.
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Abstract
A series of novel isoflavone analogs were designed, synthesized, and evaluated as
multitarget-directed ligands for the treatment of Alzheimer’s disease. In vitro
evaluations revealed that some ligands had multifunctional profiles, including potent
blockage of histamine 3 receptor (H3R), excellent inhibition of acetylcholinesterase
(AChE), neuroprotective effects and anti-neuroinflammatory properties. Among these
derivatives, compound 9b exhibited the highest ability to block H3R (IC50 = 0.27 µM)
and good inhibitory activity against AChE (IC50 = 0.08 µM). Additionally, compound
9b showed obvious neuroprotective effect on SH-SY5Y by preventing
copper-induced neuronal damage and potent anti-neuroinflammatory activity by
inhibiting the production of inflammatory factors on BV-2 cells. A molecular
modeling study revealed that 9b acts as a mixed-type inhibitor that interacts
simultaneously with H3R and AChE. Moreover, in vivo data revealed that compound
9b did not cause acute toxicity in mice at doses up to 1000 mg/kg, and had desirable
pharmacokinetic properties, as well as a good blood-brain barrier (BBB) permeability
(log BB = 1.24 ± 0.07). Further studies demonstrated that chronic oral treatment with
9b significantly improved cognitive dysfunction in scopolamine-induced AD mice in
the step-down passive avoidance test. Taken together, the present study showed that
compound 9b is a promising multifunctional drug candidate for the treatment of
Alzheimer’s disease.
Key words: Alzheimer's disease; isoflavone analogs; histamine 3 receptor;
acetylcholinesterase; neuroprotection; multifunctional agents
1. Introduction
Alzheimer’s disease (AD) is the most common cause of dementia, and is
characterized by progressive memory loss and cognitive impairment. It affects more
than 24 million people worldwide, with an increasing trend in case numbers [1].
Although the exact etiology of AD is not fully known, there are diverse factors that
seem to play vital roles in the pathophysiology of the disease, including neuron loss,
β-amyloid (Aβ) deposits, tau protein hyperphosphorylation, metal ion dyshomeostasis,
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oxidative stress, neuroinflammation, and neurotransmitter system dysfunction [2-4].
Since the pathogenesis of AD is complicated, and multiple factors are involved in the
pathological process of AD, the underlying mechanism of AD progression remains
unclear and there is no current effective treatment for the disease. To date, there are
only four acetylcholinesterase (AChE) inhibitors and one N-methyl-D-aspartate
(NMDA) receptor antagonists approved to treat AD by the US Food and Drug
Administration (FDA). These drugs can improve the cognitive function symptoms,
but fail to reverse or delay the progression of AD.
Considering the complex pathogenesis of AD, multifunctional agents that
simultaneously interfere with two or more pathogenic AD factors might be good
candidates for treating AD. Thus, multitarget-directed ligands (MTDLs) that take into
account multiple biological factors of AD are a new strategy in the drug discovery for
AD [5, 6]. Based on this strategy, a number MTDLs were developed by modifying
commercial drugs or optimizing structure of natural product to simultaneously
modulate multi-targets and exert multiple beneficial activities against AD [7-9].
Among targets related to AD, AChE is considered as a primary target. It could be
combined with other targets to design and synthesis of MTDLs, such as AChE
combining with BuChE [10], AChE combing with BACE1 [11], AChE combing with
calcium channel [7], AChE combing with Monoamine oxidase [8].
Histamine and acetylcholine (ACh) are important neurotransmitters in the central
nervous system (CNS) that are associated with memory and cognitive function in AD
through the histamine 3 receptor (H3R) and AChE, respectively [12, 13]. H3Rs are
primarily found in the CNS, and mainly expressed in the globus pallidus, basal
ganglia, hippocampus and cortex [14]. It controls the synthesis and release of
histamine and other neurotransmitters including ACh, dopamine, glutamate, serotonin,
γ-aminobutyric acid and norepinephrine [15]. Constitutive H3R activation leads an
inhibition of neurotransmitter release and influences brain disorders like AD,
schizophrenia and epilepsy. Selective H3R antagonists such as ABT238, GSK-239512
and MK-3134 (Fig.1), have been linked to increases in cognition and spatial memory
in numerous animal experiments. Currently, four single-targeted AChE inhibitors
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(donepezil, tacrine, rivastigmine, and galantamine) are the main clinical drugs for the
treating AD.
Fig. 1. Structures of H3R antagonists in clinical trials.
Increasing evidences indicate that neuronal injury and neuroinflammation play
important roles in the progression of AD [16, 17]. Neuroprotection and
anti-neuroinflammation are also valuable tools to slow down the progression of AD.
Therefore, the successful protection of neuronal cells from damage and inhibiting
inflammatory response of microglial cells may potentially prevent the development of
AD. Consequently, strategies such as simultaneously targeting H3R and AChE,
protecting neuronal cells from damage, inhibiting the inflammatory response of the
nervous system have been envisaged for AD treatment.
Our previous studies have shown that isoflavone analogs exhibit cholinesterase
inhibitory activity [18, 19]. Inspired by these discoveries, we aimed to create a new
family of MTDL molecules that can inhibit AChE activity, block H3R, or possess
other biological activities. More than twenty compounds [18, 19] with inhibitory
activity towards AChE were selected to evaluate their antagonistic activity on H3R.
Encouragingly, four compounds (A, B, C, and D, Table 1) showed simultaneous
antagonistic activity on H3R. Preliminary structure-activity relationship (SAR)
analysis indicated that the introduction of piperidine or N-ethyl-N-methyl-alkylamine
at the 7-position generates good H3R antagonist activity (compound A, B, or D). The
four-carbon chain between the amino group and isoflavone core seems to be better
than three-carbon chain (compound B vs D). However, the introduction of
N-ethyl-N-methyl-alkylamine at the 7-position seems to reduce the inhibitory activity
of BuChE. In addition, the di-substitution at the 7, 4'-position of isoflavone appears to
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be helpful for exerting multitarget inhibitory activities (compound C). These findings
could aid us to develop new promising multifunctional agents that target H3R, and
AChE, and possess other activities, such as neuroprotection, anti-neuroinflammation,
and so on.
We did the rationale design following these four points. Firstly, different amine
groups were introduced to the 7-position of isoflavone core beyond piperidine or
N-ethyl-N-methyl-alkylamine, for example, primary amines, secondary amines, or
amide, to test the effect on the activities. Secondly, in order to find optimal linker
between the amino group and isoflavone core, we designed two- or four-carbon chain
or changed alkyl to alkoxy chain. Thirdly, tacrine, a good AChE and BuChE inhibitor,
was introduced into the 7-position of isoflavones to evaluate whether it contributed to
the multitarget inhibition activity. Besides, one of the important H3R antagonists
reported in the literature is the diamine-based ligand [20-22]. Those diamie-based
antagonists contain an aromatic ring and two basic functional groups flanking a
lipophilic core. Encouraged by the results and our discoveries, we reasoned that
incorporation of the isoflavone scaffold with the diamine groups could furnish a
viable platform for the development of H3R, AChE, and other functional effects.
In the current study, we have reported on the design, synthesis and biological
evaluation of 11 new isoflavone analogs (2a-9c) based on our previous developments.
After evaluating all compounds for the inhibition of ChE (including AChE and
BuChE) and antagonistic activity towards H3R, the outstanding compound, 9b, was
selected for further evaluation. We investigated its neuroprotective effects in vitro,
anti-neuroinflammatory effects, in vivo acute toxicity effects and pharmacokinetic
profile, including BBB permeability and cognitive function. The ability of 9b to cross
the BBB was also predicted in silico by the “ADMET Descriptors” module
implemented in DS2016. Furthermore, molecular modeling studies were performed to
investigate the binding mode and the structure-activity relationships of 9b with
targets.
Table 1. The results of first-round screening.
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IC50 (µM)a
AChEb
Comp. Chemical structures
H3R
BuChEc
A
B
C
D
0.73±0.01
3.98±0.02 8.83±0.02
0.43±0.002 9.47±0.04 n.a.d
2.07±0.01 0.71±0.02 4.55±0.02
0.52±0.008 22.60±0.05 n.a.d
a IC50 values represent the concentration of inhibitor required to decrease enzyme activity or H3R
activity by 50% and are the mean of three independent experiments, each performed in triplicate
(SD = standard deviation).
b AChE was extracted from mouse brains.
c BuChE was derived from mouse plasma.
d n.a.= no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a
concentration of 10 µM in the assay conditions.
2. Results and discussion
2.1. Chemistry
The synthetic route of the new isoflavone analogs is depicted in Schemes 1 and 2.
The bromo-substituted intermediates 1a and 1b were prepared using commercially
available formononetin and dibromodiethyl ether in acetone in the presence of
potassium carbonate. Thereafter, with various amine intermediates available, target
molecules of 2a-2e were synthesized in the presence of potassium carbonate [19]. In
addition, compound 3 was obtained by removal of tert-butoxycarbonyl groups from
compound 2e in the presence of hydrochloric acid. Compound 6 was obtained by
reaction of compound 3 with 9-chloro-1,2,3,4-tetrahydroacridine in the presence of
phenol and sodium iodide. The synthesis of starting materials A, B, and E shown in
Scheme 2 has been reported previously by our team [18]. The demethylation of A, B,
and E in the presence of 40% hydrobromic acid provided compounds 7a-7c, which
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were then reacted with 1,2-dibromoethane in acetone in the presence of potassium
carbonate to produce 8a-8c. Finally, reaction of 8a-8c with piperidine produced the
target compounds 9a-9c. The structures of all target compounds were confirmed by
1
high-resolution mass spectroscopy (HRMS) and H and 13C nuclear magnetic
resonance (NMR) spectroscopy.
Scheme 1. The synthesis of target compounds 2a-2e, 3, and 6. Reagents and
conditions: (I) K2CO3, acetone, Br(CH2)2Br or Br(CH2)2O(CH2)2Br, 60°C; (II) amines,
K2CO3, DMF/acetonitrile, 100°C, 3 h; (III) 30-40% HCl/EtOH, RT, 20 min (from 2e);
(IV) Pd/C, H2, CH3OH, RT, 8 h; (V) POCl3, cyclohexanone, refluxing, 1 h; (VI) NaI,
PhOH, Ar2, 180°C, 2 h.
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Scheme 2. The synthesis of target compounds 7a and 9a-9c. Reagents and conditions:
(I) 40% HBr, 120°C, 3 h; (II) K2CO3, acetone, Br(CH2)2Br, 60°C; (III) piperidine,
acetonitrile.
2.2. Inhibition of the histamine H3R
Compounds 2a-9c were evaluated for H3R antagonistic activity by analyzing
U2OS cells stably expressing human recombinant H3R with a LiveBLAzer
FRET-B/G loading Kit (Invitrogen, USA). The H3R antagonist thioperamide was
used as a positive control agent. The antagonistic potencies of tested compounds are
presented in Table 2. It was clear that compounds 9a-9c showed antagonistic activity,
with IC50 values of 0.41, 0.27 and 1.94 µM, respectively. The IC50 values were
compared with the thioperamide as a positive control agent. The SAR study indicated
that N-ethyl-N-methyl-alkylamine at the 7-position of isoflavone exhibited apparent
higher H3R antagonistic activity than piperidine substitution (compound B vs A;
compound 9b vs 9a). Regarding the linker length between the amino group and
isoflavone core, the optimal carbon number seems to be four (compound B vs D). In
addition, the di-substitution at the 7,4'-position led to a clearly increase in activity;
when compared to corresponding mono-substituted compounds (compounds 9a vs A;
9a vs 7a; 9b vs B).
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Table 2 Biological evaluation of the inhibitory activity of 2a-2e, 3, 6, 7a, and 9a-9c
against H3R, AChE, and BuChE activity.
Inhibition (%) at 10 µMa
IC50 (µM)b
AChEc
Comp.
H3R
AChEc
35.03
78.49
11.19
73.71
-0.17
16.95
85.80
63.38
85.07
92.75
82.79
NTf
BuChEd
12.41
80.31
-9.87
66.00
0.12
H3R
>50
BuChEd
>50
30.32
47.12
-15.63
41.02
-197.9
-6.69
60.49
6.88
>50
2a
>50
4.50±0.02
>50
5.98±0.01
n.a.e
2b
n.a.e
>50
2c
5.00±0.03
n.a.e
21.59±0.12
n.a.e
2d
n.a.e
n.a.e
>10
2e
4.82
>50
n.a.e
3
97.12
50.25
86.37
81.30
91.55
NTf
0.96±0.01
26.79±0.14
0.20±0.003
0.38±0.01
24.77±0.11
2.49±0.01
6
>50
7a
72.36
75.24
71.45
71.12
NTf
0.41±0.01
9a
0.27±0.004 0.081±0.0002 2.89±0.02
9b
1.94±0.01
1.03±0.01
NTf
0.35±0.01
NTf
2.92±0.01
NTf
9c
Thioperamide
Donepezil
Rivastigmine
88.99
NTf
NTf
0.084±0.0003 NTf
NTf
0.058±0.001
NTf
93.15
NTf
a Data are presented as the mean of three independent experiments
b IC50 values represent the concentration of inhibitor required to decrease enzyme activity or H3R
activity by 50% and are the mean of three independent experiments, each performed in triplicate
(SD = standard deviation).
c AChE was extracted from mouse brains.
d BuChE was derived from mouse plasma.
e n.a.= no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a
concentration of 10 µM in the assay conditions.
f NT = not tested.
2.3. Cholinesterase (ChE) inhibitory activity
The cholinesterase inhibitory activities of compounds 2a-9c were determined using
Ellman’s assay on AChE and BuChE [23]. The well-known ChE inhibitors Donepezil
and rivastigmine were selected as reference compounds of AChE and BuChE,
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respectively. The results are summarized as IC50 value and percentage of inhibition at
10 µM in Table 2.
The data in Table 2 indicates that the activity decreased to almost zero after the
alkyl chain between the amino group and isoflavone core of compound A was
substituted with the same length of alkoxy chain (2a). This indicates that the alkyl
chain is essential for the retention of inhibitory activity. The optimal carbon number
between the amino group and isoflavone core seems to be four (compound B vs D; 9a
vs 9c).
In addition, the terminal groups NR1R2 of the side chain exerted significant effects
on ChE inhibitory activities. Several amine-containing compounds, including
secondary amines and amide were introduced to formononetin (2a, 2b, 2c, 2d, 2e, 3
and 6) to optimize ChE inhibitory activities. The results show most compounds
decrease in activity in comparison with compound A which contains the piperidine
group. As expected, compound 6, bearing 9-amino-1,2,3,4-tetrahydroacridine,
exhibited more potent inhibition of AChE (IC50 = 0.96 µM) and BuChE (IC50 = 0.38
µM) than compound A. 9-amino-1,2,3,4-tetrahydroacridine, known as Tacrine, is an
FDA-approved acylcholinesterase inhibitor that is clinically used to treat AD (now
discontinued). N-ethyl-N-methyl-alkylamine at the 7-position of isoflavone exhibited
apparent higher AChE antagonistic activity than piperidine substitution (compound B
vs A; 9b vs 9a).
Three bi-substituted compounds (9a-9c) were synthesized. Encouragingly, these
compounds exhibited pronounced dual-inhibitory effects on AChE and BuChE
activity, and their inhibitory activities were very similar at the micromolar level.
Compound 9b exhibited the highest inhibitory activity on AChE (IC50 = 0.081 µM),
with moderate inhibitory activity on BuChE (IC50 = 2.89 µM). The inhibitory activity
of 9b on AChE was equivalent to the positive control donepezil (0.084 µM). Those
results also confirmed that the di-substitution at the 7, 4'-position contributed to the
increase in ChE inhibitory activity compared to corresponding mono-substituted
compounds.
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2.4. Antioxidant activity
Oxidative stress can induce cellular damage and promote neurodegenerative
diseases [24]. Antioxidant activity is therefore beneficial for AD treatment. The
antioxidant activities of compounds were evaluated by the oxygen radical absorbance
capacity assay method using fluorescein (ORAC-FL) [25, 26]; the vitamin E analogue
trolox was used as a standard. All the isoflavone analogs exhibited potent peroxyl
radical absorbance capacities, ranging from 0.01 to 0.88 trolox equivalents, as shown
in Table 3. Most compounds presented moderate antioxidant activity, with an activity
of 0.11 trolox equivalents.
Table 3 Antioxidant activity of isoflavone analogs A-D, 2a-2e, 3, 6, 7a, and 9a-9c.
Comp.
A
Trolox equivalenta
Comp.
2e
Trolox equivalenta
0.05±0.001
0.08±0.001
0.01±0.0003
0.88±0.003
0.11±0.002
0.01±0.0001
0.14±0.004
0.07±0.001
B
3
0.11±0.002
C
6
0.14±0.002
D
7a
0.12±0.003
2a
2b
2c
9a
0.01±0.0001
0.01±0.0002
0.01±0.0004
9b
9c
2d
Trolox
0.01±0.0004
1±0.0001
a Results are expressed as
µM of Trolox equivalent/µM of tested compound and data are presented
as the mean ± SD of three independent experiments.
2.5. Molecular modeling studies
To further elucidate the binding interactions between compound 9b and AChE or
H3R, a molecular modeling study was performed using the Gold 3.0.1 software
package [27]. AChE (PDB: 1EVE) or homology model of H3R in complex with
compound 9b were pretreated by the Discovery Studio program (BIOVIA Inc, San
Diego, CA). The Discovery Studio 2016 and PyMol programs were used to display
2D and 3D models from the molecular docking and show the different interaction
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types between compound 9b and AChE or H3R.
Figures 2 and 3 shows compound 9b localizes in the long tunnel of the active site,
which occurs through intermolecular hydrophobic and hydrophilic interactions. Our
studies showed compound 9b bonds to key amino acid residues Tyr70 and Gly117 via
hydrogen bonding. The aromatic ring structure of compound 9b also displays π-π
overlap interactions with Tyr34 and Phe331 in the catalytic cleft. Significantly,
Trp279 was found to bind to the nitrogen atoms on the piperazine group of compound
9b by π-cation interactions. These results show compelling evidence that compound
9b can stably combine with AChE (Fig. 2).
Fig. 2. (a) The predicted binding mode of compound 9b with AChE; (b) the binding
pocket of AChE by the surface representation; (c) 2D schematic diagram of potential
interactions between compound 9b and AChE.
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The homology model of H3R was built based on the crystal structure of the human
histamine 1 receptor (PDB:3RZE) in complex with the doxepin antagonist [19]. As
shown in Fig. 3, the isoflavone core structure of small molecule ligands bind to
Trp371, Tyr115, and Tyr374 by π-π interaction. Additionally, a salt bridge with
Glu206 also contributes to the stabilization of protein–ligand interaction. Again, these
interactions contribute to small molecule ligands adopting the appropriate orientation
to bind effectively to the active site.
Fig. 3. (a) The best homology model of H3R generated by DS MODELER; (b) the
binding pocket of H3R by surface representation; (c) 2D schematic diagram of
potential interactions between compound 9b and H3R.
2.6. ADMET in silico prediction
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As shown in Table 4, all compounds appeared to have very poor solubility in
aqueous media and have high hydrophobicity, whereas they possessed adequate
absorption except compound 6. Fortunately, three compounds with greater inhibitory
activity (9a-9c) were predicted to penetrate the BBB, which is a desirable property for
the treatment of AD. These compounds also may not bind to CYP2D6, which is
beneficial for ensuring therapeutic efficacy and avoiding side effects. In this prediction,
however, most compounds exhibit hepatotoxicity, therefore further biological
experiments are required to obtain additional data for testing this.
Table 4 Predicted pharmacokinetic properties of isoflavone analogs A~D, 2a-2e, 3, 6,
7a, and 9a-9c.
Solubility
Absorption BBB
Comp. AlogP98. PSA-2D
PPB
CYP2D6 Hepatotoxic
Level
Level
Level
A
4.532
3.963
3.433
3.889
3.758
3.838
4.295
4.039
3.6
47.443
47.443
47.443
47.443
56.373
56.901
56.901
82.045
83.132
70.631
68.162
59.328
50.796
50.796
50.796
2
2
2
2
2
2
2
2
2
3
1
2
2
2
2
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
2
2
3
4
1
0
1
1
true
true
true
true
true
true
true
true
false
true
true
true
false
true
true
true
true
true
true
false
true
true
true
B
C
D
2a
2b
2d
2c
2e
3
false true
true
true
true
true
true
true
true
true
true
true
true
true
false
false
false
false
true
2.004
5.997
4.306
5.588
5.019
4.945
6
7a
9a
9b
9c
false
false
false
AlogP98: Lipophilicity descriptor.
PSA-2D: Polar surface area.
Solubility Level: (0, good; 1, moderate; 2, poor; 3, very poor).
Absorption Level: (0, good; 1, moderate; 2, poor; 3, very poor).
BBB Level: (0, very high blood–brain barrier penetration; 1, high; 2, medium; 3, low).
PPB Prediction: The classification describing whether a compound is highly bound (>= 90%
bound) to plasma proteins using the cutoff Bayesian score of -2.209 (obtained by minimizing the
total number of false positives and false negatives).
CYP2D6 Prediction: The classification describing whether a compound is a CYP2D6 inhibitor
using the cutoff Bayesian score of 0.161 (obtained by minimizing the total number of false
positives and false negatives).
Hepatotoxic Prediction: The classification describing whether a compound is hepatotoxic using
the cutoff Bayesian score of -4.154 (obtained by minimizing the total number of false positives
and false negatives).
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2.7. Cytotoxicity and neuroprotective effects of 9b
Cytotoxic activity of the 9b was tested using human neuroblastoma cells,
SH-SY5Y. The results showed that 9b has a low cytotoxicity (CC50 > 100 µM) and
high antagonism effect for H3R (IC50 = 0.27µM), inhibition activity for AChE (IC50 =
0.081µM) and BuChE (IC50 =2.89 µM), resulting in a high selectivity index (SI,
CC50/IC50) of > 370.37, > 1234.56 and > 34.6, respectively (Table 5).
Table 5 In vitro cytotoxicity, H3R antagonism activity, AChE and BuChE inhibitory
activity and selectivity index of compound 9b.
Comp.
SH-SY5Y
H3R
AChE
IC50 (µM)
0.27±0.004 >370.37 0.081±0.0002 >1234.56
BuChE
IC50 (µM)
CC50 (µM)a IC50 (µM)b
SIc
SI
SI
>100
2.89±0.02
NT
>34.6
9b
85.79±0.9
25.06±0.4
67.82±0.7
1.03±0.01
NT
83.29
NTd
0.084±0.0003
NT
-
298.33
-
-
-
Thioperamide
Donepezil
-
-
NT
NT
0.058±0.001 1169.3
Rivastigmine
a
CC50 50% cytotoxic concentrations, values represented as average of at least triplicate measurements (mean ±
SD).
b
IC50 50% inhibitory concentration, values represent the concentration of inhibitor required to decrease enzyme
activity or H3R activity by 50% and are the mean of three independent experiments, each performed in triplicate
(mean ± SD).
c SI (selectivity index) = CC50/IC50.
d NT = not tested.
To further assess the therapeutic potential of 9b for AD, the neuroprotective
activity of 9b was assessed by measuring copper induced cell viability against
SH-SY5Y-APPsw cell line, which is a useful AD model by overexpresses the Swedish
mutant form of human APP [17]. As shown in Fig. 4a, 9b did not affect cell viability
up to the concentration of 100 µM. The results suggest that compound of 9b exhibits a
wide therapeutic safety range when it was administered to SH-SY5Y-APPsw cells.
Moreover, these preliminary results could encourage further studies to explain the
neuroprotective mechanisms of 9b.
The human neuroblastoma cell line SH-SY5Y-APPsw can highly express the
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APP gene after exposure by copper or other metal ions, and this model can imitate the
cytotoxicity of Aβ aggregation [17]. As shown in Fig. 4b, incubation of
SH-SY5Y-APPsw cells with copper caused significant toxicity, and cell viability was
approximately 46% lower than that of the control group (p < 0.005). Compound 9b
(10 µM) treatment significantly prevented copper-induced Aβ aggregate toxicity,
9b-treated cells displayed significantly greater viability than the cells treated only by
copper (p < 0.01). The positive control donepezil did not prevent the cytotoxicity of
Aβ aggregates induced by copper. Overall these experiments clarified the ability of
compound 9b to protect SH-SY5Y-APPsw cells from toxic Aβ species induced by
copper.
Fig. 4. MTT measurement of cell viability of compound 9b in SH-SY5Y-APPsw
cells. (a) Cell viability was assessed by MTT assay, SH-SY5Y-APPsw cells were
treated with compound 9b at a different concentration of 0-100 µΜ for 72h, the
results are expressed as the percentage of viable cells. (b) Protective effect of
compound 9b and donepezil from copper-induced Aβ aggregation neurotoxicity. The
data are shown as the mean ± SD from three different experiments, ###p < 0.005 vs.
control group, **p < 0.01 vs. the copper model group.
2.8. In vitro anti-inflammatory activity evaluation
Neuroinflammation is one of the hallmarks of AD pathogenesis. Therefore,
suppression of neuroinflammatory processes may have good potential as a therapeutic
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approach against AD [28-30]. Thus, compound 9b was evaluated for its in vitro
anti-inflammatory effects. Cell viability assay was performed to determine the
cytotoxicity of 9b on BV-2. The result indicated that 9b (0 - 100 µM) did not show
any significant effect on the proliferation of normal cultures of BV-2 in 72 h
incubation time period (Fig 5a). After pretreating BV-2 cells for 1 h with 9b and
lipopolysaccharide (LPS) for 24 h, the production of interleukin-6 (IL-6) and TNF-α
were determined by ELISA. As shown in Fig. 5b-c, LPS induced a significant
increase in pro-inflammatory IL-6 and TNF-α (p < 0.005), whereas 9b clearly
suppressed the production of IL-6 and TNF-α in LPS-stimulated BV-2 microglia (p <
0.005). Donepezil did not display the anti-inflammatory effect.
Fig. 5. Effects of 9b on the expression of pro-inflammatory factors in LPS-stimulated
BV-2 microglia. (a) Cell viability was assessed by MTT assay, BV-2 cells were
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treated with compound 9b at a different concentration of 1-100 µΜ for 72 h, the
results are expressed as the percentage of viable cells. For the anti-inflammatory
effects of 9b, cells were pre-treated with 9b at 10 µM for 1 h, and then subjected to
treatment with LPS (100 ng/mL) for 24 h. The amounts of IL-6 (b) and TNF-α (c)
###
were measured by ELISA,
The data are shown as the mean ± SEM from three
different experiments, p < 0.005, vs. control group. ***p < 0.005, vs. model group.
2.9. Acute toxicity of 9b
Safety is a primary concern in the development of new drugs. The acute toxicity
of compound 9b, the most promising multifunctional anti-AD agent, was determined
in ICR mice by orally administering doses of 0, 100, 200, 500, or 1000 mg/kg (n = 5
per group) [31]. After 14 days monitoring, all mice remained alive and no acute
toxicity phenomena were observed, including significant abnormal behavior, altered
water or food consumption, or marked body weight changes. All animals were
euthanized on the 14th day and no macroscopic abnormities of the heart, liver, and
kidneys were detected. These results indicate that the treated animals did not develop
acute toxicity symptoms, and tolerated compound 9b at doses up to 1000 mg/kg.
2.10. Pharmacokinetic profile and log BB value
To further understand the pharmacokinetics of 9b, a preliminary pharmacokinetic
analysis was performed in mice. Results in Table 6 display the plasma concentration
of 9b vs the time following a 30 mg/kg dose. The maximum plasma concentration
(Cmax) was 51.95 µg/L at 0.65 h (Tmax) after of oral administration. After Cmax, there
was a slow decline and elimination beyond 48 h (Fig. 6). The approximate T1/2 was
13.89 h. The area under curve until the last measurable concentration (AUC(0-t)), and
area under curve to infinite time (AUC(0-∞)) were 324.69 µg/L*h and 352.15 µg/L*h,
respectively. Interestingly, the level of 9b was 47.24 µg/L in the plasma and 762.10
µg/L in the brain after 40 min, which displayed a blood/brain partitioning (log BB)
value of 1.24 ± 0.07. The log BB value indicates compound 9b penetrated the BBB
and could therefore be liable to having anti-AD effects. Overall, these in vivo
pharmacokinetic results facilitate the evaluation of drug-like properties of 9b as a
potential AD treatment.
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Table 6 Pharmacokinetic parameters of 9b.
p.o.a
T1/2(h)
Tmax (h)
Cmax (µg/L)
AUC(0-t) (µg/L*h)
AUC(0-∞) (µg/L*h)
352.15±32.16
log BB
13.89±2.51 0.65±0.08 51.95±12.20
324.69±31.57
9b
p.o.b
9b
plasma(µg/L)
brain(µg/L)
762.10±31.56
47.24±7.80
1.24±0.07
The data are expressed as mean ± SEM.
a p.o., oral administration, dose is 30 mg/kg, n = 6.
b40 min after oral administration, dose is 30 mg/kg, n = 6.
Fig. 6. Mean plasma concentration-time profiles of 9b (30 mg/kg, p.o.) in ICR mice
(n = 6). The data are expressed as mean ± SEM.
2.11. In vivo assay
Cognitive improvement is the utmost criterion for evaluating anti-AD drugs. The
ability of 9b to improve scopolamine-induced cognitive impairment in ICR mice was
determined using a step-down passive avoidance test [32]. As shown in Fig. 7, in the
training test there was no difference regarding the latency among the different groups.
During the test stage, the latency of step-down of mice treated with scopolamine was
significantly less (5 s) than that of control mice (116 s) (p < 0.005). Compound 9b
significantly prolonged the scopolamine-induced latency in a dose-dependent manner
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(Fig. 7a). Especially, the latency of mice treated with 30 mg/kg compound 9b was
markedly greater than control mice, with latency scores of 53 seconds and 5 seconds,
respectively. This indicates compound 9b was clearly linked to improved memory in
mice experiencing scopolamine-induced cognitive impairment.
Our data reveals that compound 9b, as a multifunctional agent, might be a good
potential candidate for treating AD. Our results also showed that the positive control
agent donepezil demonstrated a strong therapeutic effect for scopolamine-induced
mice.
The index of error time is widely used to evaluate the ability of learning and
memory in animal models. Our results indicate that there was no difference regarding
the error times between the different groups during the training stage. However, in the
subsequent retention trial, the error times of mice treated with scopolamine alone (1
mg/kg, model group) were significantly greater than those of the vehicle-treated mice
(control group) (p < 0.01). Compared to the model mice, the mice receiving 9b
treatment showed the less error times in a dose-dependent manner (10 mg/kg, p <
0.05; 30 mg/kg, p < 0.01; Fig 7b). These results indicate that compound 9b might
have decreased the cognitive deficit induced by scopolamine, which could be linked
to an increase in brain-cholinergic activity.
Fig. 7. Effects of compound 9b on scopolamine-induced memory deficit by
step-down passive avoidance test in mice. During the training period, no significant
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differences were observed in latency and error times of mice among groups. In the test
stage, the mice were pretreated with compounds 9b (10 and 30 mg/kg, p.o.) or
donepezil (5.0 mg/kg, p.o.) for 1 h, then scopolamine (3 mg/kg, i.p.) were orally given
for another 30 min. Latency (a) and error times (b) were measured by step-down
passive avoidance was tested. The data are shown as the mean ± SEM (n = 15). ###p <
0.005 vs. control group. *p < 0.05, **p < 0.01 and ***p < 0.005 vs. the model group.
3. Conclusion
In the present work, a series of novel isoflavone analogs 2a-9c were designed,
synthesized and evaluated as multifunctional agents against AD by their targeting of
H3R and AChE, and protecting neuronal cells from damage, suppressing
neuroninflammatory response. The most interesting agent within our 11 compounds
was 9b, which showed an obvious multifunctional profile in blocking H3R with an
IC50 value of 0.27 µM and significantly inhibiting activities against AChE with an
IC50 value of 0.08 µM, but weakly inhibiting effect for BuChE (IC50 value of 2.89
µM). Molecular docking results provided an explanation for mix-type inhibition, as
9b could bind simultaneously to H3R and AChE. Moreover, Compound 9b displayed
a modest antioxidant activity and obvious anti-neuroinflammatory property, exhibited
a low toxicity and strong neuroprotective effects on SH-SY5Y-APPsw cells. More
importantly, our in vivo study revealed that compound 9b does not exhibit any acute
toxicity in mice at doses up to 1000 mg/kg; also that 9b oral treatment (10 and 30
mg/kg) significantly prevented scopolamine-induced memory deficits in mice by
prolonging the latency and reducing the error times in the step-down passive
avoidance test. It also possessed reasonable pharmacokinetic properties, with
substantial metabolic stability (T1/2 = 13.89 h in vivo) and salient log BB values (log
BB = 1.24) for penetration of the CNS in mouse. Therefore, the current study suggests
that compound 9b is a reliable and promising multifunctional drug candidate for
treating AD and deserves further investigation into its mechanisms of action.
4. Experimental
4.1. Chemistry
Unless otherwise indicated, all solvents and organic reagents were obtained from
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commercially available sources and were used without further purification. The
reaction process was monitored using ultra-performance liquid chromatography-mass
spectrometry (UPLC-MS). Yields were not optimized. 1H NMR spectra was recorded
on a Varian Mercury-400 or 500 MHz instrument, while 13C NMR was recorded on a
Varian Mercury-400 or 600 MHz instrument using DMSO-d6, CDCl3, CD3OD, or
CD3COCD3 as a solvent and tetramethylsilane (TMS) as an internal standard (1H
13
NMR and C NMR were recorded at different times). High-resolution mass spectra
(HRMS) were recorded on an Agilent Technologies LC/MSD TOF spectrometer.
Thin-layer chromatography (TLC) analysis was carried out on silica gel plates GF254
(Yantai Chemical Research Institute, China). Column chromatography was performed
on silica gel (90-150 mm; Qingdao Marine Chemical Inc.).
4.1.1. General procedure for the preparation of intermediates (1a, 1b)
Formononetin (5.0 g, 18.6 mmol), finely grounded anhydrous K2CO3 (30 g, 217
mmol) and 1,2-dibromoethane (16.0 mL, 186 mmol) or 2,2'-dibromodiethyl ether
(23.4 mL, 186 mmol) were added to 500 mL acetone. The mixture was refluxed for 10
h. The residues were then added into 100 mL water and stirred for 1 h then filtered by
suction. The filter was then washed three times with 5 mL water. Finally, the solid was
dried in a vacuum at 50 °C to give 1a and 1b without further purification.
4.1.1.1. 7-(2-(2-Bromoethoxy)-ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (1a)
White solid, 93% yield. mp 112.1-113.7 °C. 1H NMR (400 MHz, CDCl3): δ 8.21
(d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.03-6.96 (m, 3H), 6.87 (d,
J = 2.0 Hz, 1H), 4.24 (t, J = 4.8 Hz, 2H), 3.95-3.89 (m, 4H), 3.84 (s, 3H), 3.51 (t, J =
6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 175.9, 163.1, 159.7, 157.9, 152.2, 130.3,
128.0, 125.0, 124.3, 118.8, 114.9, 114.1, 101.1, 71.6, 69.4, 68.1, 55.5, 30.3. ESI-MS
(m/z): 419.13 [M+H]+.
4.1.1.2. 7-(2-Bromoethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (1b)
White solid, 93% yield, mp 177.7-179.6 °C. Data for this compound were in
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agreement with those reported in the literature [18].
4.1.2. General procedure for the synthesis of compounds 2a-2e
To a mixture of intermediate 1a (1.25 g, 3.0 mmol) or 1b (1.13 g, 3.0 mmol) and
K2CO3 (13.8 g, 100 mmol) in 100 mL CH3CN, the corresponding amines (6.0 mmol)
and DMF (30 mL) were added, and the resulting solution was refluxed and monitored
by UPLC-MS. When the reaction was finished, the reaction solution was poured into
ice water and extracted with CH2Cl2. The combined organic layers were washed with
water and dried over Na2SO4. After concentrated in vacuo, the residue was purified by
column chromatography using CH2Cl2/MeOH (97:3) as eluents to yield the pure
compounds 2a-2e and 3.
4.1.2.1.
3-(4-methoxyphenyl)-7-(2-(2-(piperidin-1-yl)ethoxy)ethoxy)-4H-chromen
-4-one (2a)
Light yellow solid, 16％ yield. mp 138.0-139.9 °C. 1H NMR (400 MHz, CDCl3):
δ 8.20 (d, J = 8.8 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.02-6.95 (m, 3H),
6.86 (d, J = 2.4 Hz, 1H), 4.22 (t, J = 4.8 Hz, 2H), 3.86 (t, J = 4.8 Hz, 2H), 3.83 (s, 3H),
3.71 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 2.45 (m, 4H), 1.62-1.56 (m, 4H),
1.45-1.39 (m, 2H) ; 13C NMR (100 MHz, CDCl3): δ 175.9, 163.3, 159.7, 157.9, 152.2,
130.2, 127.9, 125.0, 124.4, 118.6, 115.0, 114.1, 101.0, 69.5, 69.3, 68.2, 58.6, 55.5,
55.2, 26.0, 24.3. HRMS: C25H30NO5 [M+H]+: m/z Calcd: 424.2124; Found: 424.2124.
4.1.2.2. 7-(2-(cyclopentylamino)ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (2b)
Light yellow solid, 17% yield. mp 110.5-112.7 °C. 1H NMR (400 MHz, CD3OD):
δ 8.23 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.13-7.11 (m, 2H),
6.99 (d, J = 8.4 Hz, 2H), 4.23 (t, J = 4.2 Hz, 2H), 3.83 (s, 3H), 3.20-3.13 (m, 1H),
3.04 (t, J = 4.2 Hz, 2H), 2.00-1.92 (m, 2H), 1.78-1.70 (m, 2H), 1.64-1.55 (m, 2H),
1.45-1.36 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 176.0, 163.4, 159.7, 158.0, 152.2,
130.3, 127.9, 125.0, 124.4, 118.6, 114.9, 114.1, 100.8, 68.6, 59.9, 55.5, 47.4, 33.4,
24.2. HRMS: C23H26NO4 [M+H]+: m/z Calcd: 380.1862; Found: 380.1861.
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4.1.2.3. 2-(2-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)ethyl)isoindoline-1,
3-dione (2c)
1
White solid, 82% yield. mp 170.8-172.5 °C. H NMR (400 MHz, DMSO-d6): δ
8.40 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.90-7.83 (m, 4H), 7.51 (d, J = 8.8 Hz, 2H),
7.17 (d, J = 2.4 Hz, 1H), 7.02-6.98 (m, 3H), 4.39 (t, J = 5.6 Hz, 2H), 4.02 (t, J = 5.6
Hz, 2H), 3.78 (s, 3H) ; 13C NMR (100 MHz, DMSO-d6): δ 174.6, 167.7, 162.3, 159.0,
157.3, 153.5, 134.5, 131.5, 130.1, 127.0, 124.0, 123.4, 123.2, 117.8, 115.1, 113.6,
101.2, 65.5, 55.1, 36.7. HRMS: C26H20NO6 [M+H]+: m/z Calcd: 442.1291; Found:
442.1282.
4.1.2.4. 7-(2-(cyclohexylamino)ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (2d)
Light yellow solid, 74％ yield. mp 119.2-121.7 °C. 1H NMR (400 MHz, CDCl3):
δ 8.20 (d, J = 8.8 Hz, 1H), 7.91 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.01-6.96 (m, 3H),
6.85 (d, J = 2.4 Hz, 1H), 4.17 (t, J = 4.2 Hz, 2H), 3.84 (s, 3H), 3.09 (t, J = 4.2 Hz, 2H),
2.55-2.48 (m, 1H), 1.95-1.92 (m, 2H), 1.79-1.75 (m, 2H), 1.66-1.63 (m, 2H),
1.34-1.07 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 176.0, 163.4, 159.7, 158.0, 152.2,
130.3, 127.9, 125.1, 124.4, 118.7, 114.9, 114.1, 100.9, 68.8, 56.9, 55.5, 45.7, 33.8,
26.3, 25.2. HRMS: C24H28NO4 [M+H]+: m/z Calcd: 394.2018; Found: 394.2012.
4.1.2.5. Tert-butyl (2-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)ethyl)
carbamate (2e)
1
White solid, 96% yield. mp 98.6-101.9 °C. H NMR (400 MHz, CD3COCD3): δ
8.23 (s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.57 (d, J =7.6 Hz, 2H), 7.08 (d, J = 7.6 Hz,
2H), 6.99 (d, J = 8.0 Hz, 2H), 4.24 (t, J = 5.6 Hz, 2H), 3.84 (s, 3H), 3.56-3.52 (m, 2H),
13
1.42 (s, 9H); C NMR (100 MHz, CD3COCD3): δ 175.6, 164.2, 160.5, 158.7, 156.8,
153.6, 131.0, 128.1, 125.4, 125.1, 119.2, 115.7, 114.4, 101.7, 79.0, 68.5, 55.6, 40.5,
28.6. HRMS: C23H26NO6 [M+H]+: m/z Calcd: 412.1760; Found: 412.1747.
4.1.3. 7-(2-aminoethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one hydrochloride (3)
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Compound 2e (0.41 g, 1.00 mmol) was stirred in hydrochloride ethanol
solution (30ml, 30-40% ) at room temperature for 20 min. The mixture was
evaporated to dryness under reduced pressure. The residue was washed with acetone
1
twice to give the title compound as a white solid, 82% yield. mp>250 °C. H NMR
(400 MHz, CD3OD): δ 8.24 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H),
7.19-7.16 (m, 2H), 7.00 (d, J = 9.2 Hz, 2H), 4.39 (t, J = 5.2 Hz, 2H), 3.83 (s, 3H),
3.44 (t, J = 5.2 Hz, 2H); 13C NMR (100 MHz, CD3OD): δ 177.9, 164.1, 161.3, 159.5,
155.1, 131.4, 128.6, 126.1, 125.2, 119.8, 116.2, 114.9, 102.5, 66.1, 55.8, 40.1. HRMS:
C18H18NO4 [M+H]+: m/z Calcd: 312.1236; Found: 312.1235.
4.1.4. 2-aminobenzoic acid (4)
A solution of 2-nitrobenzoic acid (8.00 g, 47.9 mmol) in methanol (120 mL) was
added to Pd/C, and the resulting mixture was stirred under hydrogen atmosphere at
room temperature for 8 h. The resulting mixture was filtered and the filtrate was
concentrated in vacuo to give the desired product 4 as a light yellow solid in yield of
1
43%. mp 140.8-142.8 °C. H NMR (400 MHz, CD3OD): δ 7.80 (dd, J = 6.4, 1.2 Hz,
13
1H), 7.24-7.20 (m, 1H), 6.72 (d, J = 6.4 Hz, 1H), 6.58-6.55 (m, 1H); C NMR (100
MHz, CD3OD): δ171.7, 152.8, 134.9, 132.7, 117.7, 116.6, 111.9. ESI-MS (m/z):
138.04 [M+H]+.
4.1.5. 9-chloro-1,2,3,4-tetrahydroacridine (5)
A mixture of 4 (2.06 g, 15 mmol) and cyclohexanone (1.55 mL, 15 mmol) was
added to phosphorus oxychloride (15 mL, 164 mmol) in an ice bath for 2 min, then
the mixture was refluxed for 1 h. After evaporation of the solvent, the residue was
dissolved in dichloromethane (10 mL). The combined organic phases were washed
with potassium carbonate solution and saturated aqueous sodium chloride, dried over
sodium sulfate, and filtered. The filtrate was evaporated to dryness under reduced
pressure. The residue was recrystallized from acetone to give the 5 as a yellow solid
in yield of 57%. mp 67.0-68.0 °C. 1H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 8.4 Hz,
1H), 7.98 (d, J = 8.8 Hz, 1H), 7.68-7.64 (m, 1H), 7.55-7.51 (m, 1H), 3.14-3.11 (m,
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13
2H), 3.04-3.01 (m, 2H), 1.98-1.92 (m, 4H); C NMR (100 MHz, CDCl3): δ 159.6,
146.8, 141.6, 129.4, 129.0, 128.7, 126.6, 125.5, 123.8, 34.3, 27.6, 22.8, 22.8. ESI-MS
(m/z): 218.16 [M+H]+.
4.1.6. 3-(4-methoxyphenyl)-7-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethoxy)-4H-
chromen-4-one (6)
The compound 3 (1.40 g,4.04 mmol) was dissolved in water and an aqueous
solution of sodium hydroxide (10%) was added dropwise to the mixture until the
aqueous solution became basic. The mixture was extracted with dichloromethane (20
mL). The combined organic phases were washed with saturated aqueous sodium
chloride (30 mL), dried over sodium sulfate, and filtered. The solvent was evaporated
to dryness under reduced pressure. The residue was added to compound 5 (0.88 mg,
4.04 mmol), phenol (2.30 g, 24.4 mmol) and NaI (0.10 mg, 0.67 mmol). The reaction
mixture was warmed to 180-185 °C and stirred for 2 h under an argon atmosphere.
When the reaction was over (TLC analysis), ethyl acetate was added and the organic
layer was washed with aqueous sodium hydroxide,water, then saturated aqueous
sodium chloride, then dried over sodium sulfate and filtered. The solvent was
evaporated to dryness under reduced pressure. The residue was purified on a silica gel
chromatography using dichloromethane/ethyl acetate (20:1) as eluent to produce the
1
light yellow solid in the yield of 52%. mp 149.7-152.3 °C. H NMR (400 MHz,
CD3OD): δ 8.18-8.15 (m, 2H), 8.06 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.58
(t, J = 7.6 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.40 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.4
Hz, 2H), 6.93-6.93 (m, 2H), 4.25 (t, J = 4.8 Hz, 2H), 3.98 (t, J = 4.8 Hz, 2H), 3.83 (s,
13
3H), 2.99 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H), 1.90-1.85 (m, 4H); C NMR
(100 MHz, CD3OD): δ 175.9, 162.7, 159.8, 159.0, 157.9, 152.2, 149.7, 147.5, 130.2
(2C), 129.1, 128.6, 128.2, 125.1, 124.5, 124.2, 122.5, 121.1, 119.0, 118.6, 114.7,
114.1 (2C), 101.0, 68.2, 55.5, 48.1, 34.2, 24.8, 23.1, 22.9. HRMS: C31H29N2O4
[M+H]+: m/z Calcd: 493.2127; Found: 493.2115.
4.1.7. General procedure for the synthesis of A–E
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Compounds A–E were synthesized according to our previously reported method
and the spectral data were matched with our previous publication [18].
4.1.8. General procedure for the synthesis of 7a–7c
Compound A (4.08 g, 10 mmol), B (3.81 g, 10 mmol) or E (3.79 g, 10 mmol)
was added into 60 mL 40% hydrobromic acid. After stirring at 120 °C for 3 h, the
reaction mixture was cooled to 0 °C and filtered. The filtered residue was purified by
recrystallization with methanol to give pure intermediate 7a–7c.
4.1.8.1. 3-(4-hydroxyphenyl)-7-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one
hydrobromide (7a)
Off-white solid, 81% yield. mp 225.3-228.5 °C. 1H NMR (400 MHz,
D2O/CD3OD): δ 8.15 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H),
7.06-6.94 (m, 4H), 4.10 (t, J = 4.8 Hz, 2H), 3.54 (m, 2H), 3.16 (m, 2H), 2.94 (t, J
=10.8 Hz, 2H), 2.00-1.72 (m, 10H); 13C NMR (100 MHz, D2O/CD3OD): δ 178.6,
164.7, 159.3, 157.4, 155.3, 131.4, 127.9, 125.4, 124.3, 118.4, 116.6, 116.3, 101.9,
69.0, 57.6, 54.2, 26.7, 23.9, 22.3, 21.6. HRMS: C24H28NO4 [M+H]+: m/z Calcd:
394.2018; Found: 394.2019.
4.1.8.2. 7-(4-(ethyl(methyl)amino)butoxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one
hydrobromide (7b)
1
White solid, 76% yield. mp 234.4-236.8 °C. H NMR (500 MHz, DMSO-d6): δ
8.38 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 2.0 Hz,
1H), 7.10 (dd, J = 9.0 Hz, J =2.0 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 4.14 (t, J = 6.5 Hz,
2H), 2.31 (m, 4H), 2.12 (s, 1H), 1.79-1.73 (m, 2H), 1.59-1.53, 0.97 (t, J = 7.5 Hz,
3H) ; 13C NMR (125 MHz, DMSO-d6): δ 175.4, 163.7, 158.1(2C), 153.8, 130.7 (2C),
130.3, 127.6, 124.3, 122.8, 118.1, 115.7 (2C), 114.0, 101.6, 69.1, 56.8, 51.5, 41.8,
27.0, 23.8, 12.8. HRMS: C22H26NO4 [M+H]+: m/z Calcd: 368.4530; Found: 368.4525.
4.1.8.3. 3-(4-hydroxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one
27

ACCEPTED MANUSCRIPT
hydrobromide (7c)
1
White solid, 60 mg, 21% yield. mp 241.2-246.5°C. H NMR (400 MHz,
DMSO-d6):δ 8.39 (s, 1H), 8.04 (d, J = 8.8Hz, 1H), 7.40 (d, J = 8.0Hz, 2H), 7.21 (s,
1H), 7.10 (d, J = 8.8Hz, 1H), 6.82 (d, J = 8.0Hz, 2H), 4.34 (t, J = 4.2Hz, 2H), 3.03 (t,
J = 4.2Hz, 2H), 2.77 (m, 4H), 1.64-1.58 (m, 4H) , 1.44 (m, 2H) ; 13C NMR (100 MHz,
DMSO-d6): δ 178.7, 163.4, 159.2, 157.4, 155.5, 131.5, 128.2, 125.6, 124.2, 119.1,
116.4, 116.4, 102.4, 63.3, 56.4, 54.7, 23.7, 22.1. HRMS: C22H24NO4 [M+H]+: m/z
Calcd: 366.1705; Found: 366.1694.
4.1.9. General procedure for the synthesis of intermediates 8a–8c
The suspension of intermediate 7a (2.37 g, 5.0 mmol) or 7b (2.24 g, 5.0 mmol) or
7c (2.23 g, 5.0 mmol) was stirred at 70 °C for 2 d in potassium carbonate (4.15 g, 30
mmol) and 1,2-dibromoethane (2.16 mL, 25 mmol). Then the solvent was evaporated
under vacuum. Water (50 mL) was added to the residue. The precipitate was filtered
and washed twice with water. The crude product was dried in an oven and used in the
next step without further purification.
4.1.10. General procedure for the synthesis of target compounds 9a-9c
The suspension of compound 8a (1.00 g, 2.0 mmol) or 8b (0.95 g, 2.0 mmol) or
8c (0.94 g, 2.0 mmol) was stirred at 100 °C for 2h with potassium carbonate (2.76 g,
20 mmol) and piperidine (1.82 mL, 20 mmol) in acetonitrile (50 mL). Then the
reaction mixture was poured into ice water and then filtered. The filtered residues
were dried in an oven and purified by column chromatography using DCM/MeOH
(20:3) as eluent to yield target compounds 9a-9c.
4.1.10.1 7-(4-(piperidin-1-yl)butoxy)-3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-4H-
chromen-4-one (9a)
Light yellow solid, 75% yield. mp 127.6-130.6 °C. 1H NMR (400MHz, CDCl3): δ
8.19 (d, J = 8.4 Hz, 1H ), 7.91 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 6.97 (m, 3H), 6.83 (d,
J = 2.0 Hz, 1H), 4.14 (t, J = 8.4 Hz, 2H), 4.08 (t, J = 8.4 Hz, 2H), 2.79 (t, J = 8.4 Hz,
28