Design, synthesis and biological evaluation of novel donepezil-coumarin hybrids as multi-target agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2016.02.023

Combining N-benzylpiperidine moiety of donepezil and coumarin into in a single molecule, novel hybrids with ChE and MAO-B inhibitory activity were designed and synthesized. The biological screening results indicated that most of compounds displayed potent inhibitory activity for AChE and BuChE, and clearly selective inhibition to MAO-B. Of these compounds, 5m was the most potent inhibitor for eeAChE and eqBuChE (0.87 μM and 0.93 μM, respectively), and it was also a good and balanced inhibitor to hChEs and hMAO-B (1.37 μM for hAChE; 1.98 μM for hBuChE; 2.62 μM for hMAO-B). Molecular modeling and kinetic studies revealed that 5m was a mixed-type inhibitor, which bond simultaneously to CAS, PAS and mid-gorge site of AChE, and it was also a competitive inhibitor, which occupied the active site of MAO-B. In addition, 5m showed good ability to cross the BBB and had no toxicity on SH-SY5Y neuroblastoma cells. Collectively, all these results suggested that 5m might be a promising multi-target lead candidate worthy of further pursuit.

Full text of DOI:10.1016/j.bmc.2016.02.023

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Novel Donepezil-Coumarin
Hybrids as Multi-Target Agents for the Treatment of Alzheimer’s Disease
Sai-Sai Xie, Jin-Shuai Lan, Xiaobing Wang, Zhi-Min Wang, Neng Jiang, Fan
Li, Jia-Jia Wu, Jin Wang, Ling-Yi Kong
PII:
S0968-0896(16)30103-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.023
BMC 12825
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 November 2015
17 February 2016
18 February 2016
Please cite this article as: Xie, S-S., Lan, J-S., Wang, X., Wang, Z-M., Jiang, N., Li, F., Wu, J-J., Wang, J., Kong,
L-Y., Design, Synthesis and Biological Evaluation of Novel Donepezil-Coumarin Hybrids as Multi-Target Agents
for the Treatment of Alzheimer’s Disease, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.02.023
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Design, Synthesis and Biological Evaluation of Novel Donepezil-Coumarin
Hybrids as Multi-Target Agents for the Treatment of Alzheimer’s Disease
†
Sai-Sai Xie, † Jin-Shuai Lan, Xiaobing Wang, Zhi-Min Wang, Neng Jiang, Fan Li,
Jia-Jia Wu, Jin Wang, Ling-Yi Kong *
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China
* Corresponding Author. Tel/Fax: +86-25-83271405; E-mail: cpu_lykong@126.com;
†These authors contributed equally to this work.
Abstract
Combining N-benzylpiperidine moiety of donepezil and coumarin into in a single
molecule, novel hybrids with ChE and MAO-B inhibitory activity were designed and
synthesized. The biological screening results indicated that most of compounds
displayed potent inhibitory activity for AChE and BuChE, and clearly selective
inhibition to MAO-B. Of these compounds, 5m was the most potent inhibitor for
eeAChE and eqBuChE (0.87 μM and 0.93 μM, respectively), and it was also a good
and balanced inhibitor to hChEs and hMAO-B (1.37 μM for hAChE; 1.98 μM for
hBuChE; 2.62 μM for hMAO-B). Molecular modeling and kinetic studies revealed
that 5m was a mixed-type inhibitor, which bond simultaneously to CAS, PAS and
mid-gorge site of AChE, and it was also a competitive inhibitor, which occupied the
active site of MAO-B. In addition, 5m showed good ability to cross the BBB and had
no toxicity on SH-SY5Y neuroblastoma cells. Collectively, all these results suggested
that 5m might be a promising multi-target lead candidate worthy of further pursuit.
Keywords: Alzheimer’s disease, coumarin, donepezil, cholinesterase, monoamine
oxidase, docking.
1

1. Introduction
Alzheimer’s disease (AD) is the most common type of dementia clinically
characterized by progressive loss of memory and deficits in different cognitive
domains [1, 2]. It has been estimated that there were 36 million people with dementia
in 2010, and this number will rise to 42.3 million in 2020 and 81.1 million by 2040 [3,
4]. Although several factors including low levels of acetylcholine (ACh), the
formation of β-amyloid deposits, oxidative stress and dyshomeostasis of biometals
have been demonstrated to play significant roles in the pathogenesis of AD, the exact
etiology is still not fully understood [5, 6].
The current strategies for AD treatment are mainly based on cholinergic
hypothesis, which suggests that a decline of ACh levels in specific brain regions leads
to cognitive and memory deficits, and sustaining or recovering cholinergic function
can alleviate these symptoms [7, 8]. Supporting this notion that four cholinesterase
inhibitors (ChEIs), tacrine, rivastigmine, galanthamine and donepezil, have been
approved in clinical use [9]. However, due to the complex nature of AD, these drugs
can only reverse the symptoms for a short period of time instead of halting or curing
the neurodegeneration [10]. Thus, a more appropriate approach termed the
Multi-Target Directed Ligands (MTDLs) strategy has been proposed to face this
disease [11-13]. MTDLs mean a single compound that can simultaneously modulate
different targets involved in the neurodegenerative AD cascade [14, 15]. One of the
most widely adopted approaches for designing MTDLs is to modify a ChEI and
render it exert other biological properties useful for treating AD[16].
2

MAOs are flavin adenine dinucleotide (FAD)-containing enzymes responsible
for the oxidative deamination of endogenous monoamine neurotransmitters, trace
amines, and a number of amine xenobiotics [17, 18]. Two isoforms, namely MAO-A
and -B, have been identified based on substrate selectivity and inhibitor sensitivity
[19]. MAO-A preferentially deaminates serotonin, adrenaline and noradrenaline, and
is selectively and irreversibly inhibited by clorgyline, while MAO-B preferentially
deaminates β-phenylethylamine and benzylamine, and is irreversibly inhibited by
R-(-)-deprenyl [20, 21]. It was found that MAO-B activity increases with age.
Especially in AD, a significant rise in MAO-B activity was found in cerebral spinal
fluid (CSF), brain tissue as well as in platelets [22, 23]. The high levels of MAO-B in
neuronal tissue could lead to an increase in the level of H2O2 and oxidative free
radicals, which ultimately contribute to the etiology of AD [24]. Thus, selective
inhibition of MAO-B becomes another valuable approach for the treatment of AD.
In recent years, many MTDLs have been designed and synthesized by combining
the ChEI and MAO-B inhibitor into one molecule [25-33]. Because studies suggest
that simultaneous inhibition of MAO-B and ChE can not only improve the level of
ACh and reduce oxidative stress in brain but also can decrease β-amyloid deposition,
another hallmark in AD pathogenesis, which will be more effective against AD [34].
Among all these MTDLs, TV2236 (Ladostigil) has been approved for phase IIb
clinical trial, which prompt us to search new multi-target compounds with ChE and
MAO-B inhibitory activity.
Very recently, we have reported a series of tacrine-coumarin hybrids as
3

multi-target compounds for treatment of AD (Figure 1) [35]. The tacrine was used to
inhibit ChEs, and the coumarin moiety was chosen to inhibit MAOs. Among these
compounds, compound 1 displayed potent inhibitory activity toward AChE and
BuChE, and clearly selective inhibition for MAO-B. However, due to the presence of
tacrine moiety [36], this compound showed severe hepatotoxicity (unpublished work),
which hindered its further application.
Figure 1. Design strategy for donepezil-coumarin hybrids.
In order to continue our work to discovery new multi-target compounds with
both ChE and MAO-B inhibitory activity and to optimize the tacrine-coumarin
hybrids, in this study, we wanted to replace the tacrine with a N-benzylpiperidine
moiety based on donepezil to design a series of novel donepezil-coumarin hybrids as
multi-target compounds for the treatment of AD. Compared to tacrine, donepezil was
4

more potent and had no hepatotoxicity [37, 38]. The N-benzylpiperidine moiety, like
tacrine, could inhibit the ChEs through binding to the catalytic anionic site (CAS) of
ChEs. The design strategy of new compounds is shown in Figure 1. Similar to our
previous design, the flexible alkyl chain was retained to connect coumarin and
N-benzylpiperidine moiety, because such linker could be lodged by the AChE cavity,
allowing the hybrids simultaneously to interact with PAS and CAS of the enzyme [39,
40]. Meanwhile, the position of the linker tethered to coumarin was unchanged in
order to better evaluate the effect of this replacement design. In addition, to find the
optimal length for ChE inhibition, the linker length was varied in initial step. Once the
optimal length was obtained, different substituents were introduced to 3- and/or
4-position of coumarin ring to investigate the possible effects on both ChE and MAO
inhibition. All designed compounds were synthesized and evaluated for their ability to
inhibit ChEs and MAOs. The kinetic and molecular modeling studies were carried out
to investigate interaction mechanism of selected compounds with AChE and MAO-B.
The blood-brain barrier (BBB) permeation and the in vitro SH-SY5Y neuroblastoma
cell toxicity assays were also performed to test the preliminary drug-like properties of
selected compounds. Herein, we report the design, synthesis and evaluation of a series
of novel donepezil-couamrin hybrids as multi-target compounds for AD treatment.
2. Result and Discussion
2.1 Chemistry
The synthesis of the designed compounds 5a-m is illustrated in Scheme1. The
coumarin derivatives 2a-k were obtained according to the previous reported methods
5

[28, 29, 35, 41-43]. Reacting the compounds 2a-k with the corresponding α,
ω-dibromoalkanes afforded the key intermediates 3a-l, which was then treated with
commercially available compounds 4a-b in the presence of potassium carbonate in
acetonitrile to give the target compounds 5a-m.
Scheme 1. Synthesis of compounds 5a-m. Reagents and conditions: (a) Br(CH2)nBr, anhydrous
K2CO3, acetone, reflux, 4 h; (b) Anhydrous K2CO3, CH3CN, reflux, 8 h.
Scheme 2. Synthesis of compounds 9a-b. (a) Ethyl acetoacetate, conc. H2SO4 (cat.), 1,4-dioxane,
60oC, 4 h; (b) NBS, dry benzene, benzoyl peroxide, reflux., 16 h. (c) Anhydrous K2CO3, CH3CN,
reflux, 8 h.
6

For compounds 9a-b, they were easily synthesized by the route shown in
Scheme 2. Condensation of m-cresol with ethyl acetoacetate in the presence of a
catalytic amount of concentrated sulfuric acid in 1, 4-dioxane provided compound 7.
The obtained 7 was selectively brominated at the 7-methyl group by NBS in dry
benzene to give compound 8 [44]. Finally, using same condition for preparation of
compounds 5a-m, compound 8 was reacted with 4a-b to furnish the desired
compounds 9a-b.
2.2 In Vitro Inhibition of ChEs
The inhibitory activities of the test compounds 5a-m and 9a-b against eeAChE
(from electric eel) and eqBuChE (from equine serum) were determined according to
the spectrophotometric method described by Ellman et al [45]. For comparison
purpose, tacrine and donepezil were used as reference compounds. The IC50 values of
all test compounds and their selectivity index for eeAChE over eqBuChE are
summarized in Table 1. From the table, it is interesting to note that, unlike donepezil
which showed selectively inhibition for AChE (SI = 94.8), all compounds showed the
balanced inhibition for both AChE and BuChE. Considering increasing attention to
BuChE, these compounds might be more advantageous in AD treatment [46, 47].
The previous works demonstrated that the linker length connected the CAS and
PAS binding moieties is critical for ChE inhibition [24, 29, 41, 48, 49]. Therefore, to
get the optimal linker length, compounds 5a-c with varying linker length between
coumarin and N-benzyl piperidine moieties were synthesized in first step. The results
7

shown in Table 1 indicated compound 5a showed the most potent inhibitory activity
among these three compounds, which suggested that the short linker (n = 2 and m = 0)
was more beneficial for ChE inhibition and thus selected as the optimal linker length.
With the optimal length in hand, we next introduced different substituents to 3- and/or
4-position of coumarin moiety and investigated their possible effects on ChE
inhibition. Unlike the previous reports [29, 41, 42], introduction of mono-substituent
to 3- or 4-position of coumarin moiety did not remarkably influence the inhibitory
activity in present study. Compounds 5d and 5f-j exhibited the inhibitory activity
similar to their no substituted analogue 5a. Only compound 5e which have a chloro
group (IC50 = 1.50 μM for eeAChE; IC50 = 1.48 μM for eqBuChE) on the 4-position
of coumarin ring showed the activity higher than that of 5a (IC50 = 4.42 μM for
eeAChE; IC50 = 5.34 μM for eqBuChE). However, simultaneous substitution of 3- and
4-positon at coumarin moiety afforded compounds 5k-m, which could increase the
activity in both ChE inhibition. Especially, 3,4-cyclohexane-fused coumarin 5m
showed the most potent inhibitory activity in this series with IC50 value of 0.87 μM
and 0.93 μM against eeAChE and eqBuChE, respectively. In addition to the above
investigation, the effects on ChE inhibition of replacement of oxygen atom linked to
7-position of coumarin moiety with carbon atom were also explored. The results
indicated that compound 9b having a two-carbon linker (m = 2) between amino group
and N-benzyl piperidine moiety was more potent inhibitor for both ChEs than
compound 9a.
8

Table1. Inhibition of eeAChE and eqBuChE by Compounds 5a-m and 9a-b.
IC50 (μM)
compd
R1
H
R2
H
n m
2 0
3 0
2 2
2 0
2 0
eeAChEa
4.42 0.12
11.71 0.92
8.91 0.63
5.62 0.22
1.50 0.27
6.29 0.31
6.34 0.25
9.18 0.32
9.65 0.45
6.03 0.59
1.48 0.31
1.58 0.21
0.87 0.09
7.28 0.61
0.91 0.17
0.04 0.003
0.11 0.01
eqBuChEb
5.34 0.45
9.86 0.91
9.79 0.16
4.38 0.42
1.48 0.17
5.64 0.55
6.95 0.22
10.22 1.07
8.93 0.79
7.53 0.62
1.67 0.18
1.42 0.29
0.93 0.44
10.83 1.01
0.96 0.03
3.79 0.01
0.02 0.001
SIc
5a
1.21
0.84
1.10
0.78
0.99
0.90
1.10
1.11
0.92
1.25
1.13
0.90
1.07
1.49
1.05
94.8
0.18
5b
H
H
5c
H
H
5d
H
Me
Cl
5e
H
5f
H
OMe 2 0
OEt 2 0
CF3 2 0
5g
H
5h
H
5i
5j
H
Ph
H
2 0
2 0
2 0
2 0
2 0
Me
Me
Cl
5k
Me
Me
5l
5m
-(CH2)4-
9a
H
Me
Me
-
-
-
-
-
0
2
-
9b
H
-
donepezil
tacrine
-
-
-
a The 50% inhibition at the indicated concentration (means SD of three experiments) of AChE
b
from electric eel. The 50% inhibition at the indicated concentration (means
SD of three
index
experiments)
of
BuChE
from
equine
serum.
cAChE
selectivity
=
IC50(eqBuChE)/IC50(eeAChE).
9

To further evaluate the inhibitory activity of the present compounds on ChEs,
compounds 5e, 5k-m and 9b with high inhibitory activity on ChEs of animal origin
were selected to test on human ChEs. It can be seen from the Table 2 that compounds
5e and 5k-l exhibit the inhibitory activity for hChEs in the same range as that for
animal ChEs. The inhibitory activity of compound 5m for hChEs was little decreased
in comparison to its inhibitory activity for animal ChEs. Interestingly, compound 9b
showed the most potent inhibition for hAChE in this series with IC50 value of 0.067
μM, which was 13.6-fold more efficient for inhibition of eeAChE. However, its
inhibitory activity for hBuChE was remarkably decreased (IC50 = 0.96 μM for
eqBuChE; IC50 = 3.45 μM for hBuChE), and thus making it a clearly selective
inhibitor for hAChE.
Table 2. Inhibition of hAChE and hBuChE by Compounds 5e, 5k-m and 9b.
IC50 (μM)
compd
hAChE (μM)a
2.04 0.11
1.69 0.09
1.82 0.14
1.37 0.05
0.067 0.001
0.01 0.002
hBuChE (μM)b
2.93 0.15
2.67 0.14
1.68 0.13
1.98 0.08
3.45 0.76
2.74 0.16
SIc
1.43
1.58
0.92
1.45
51.49
274
5e
5k
5l
5m
9b
donepezil
a
The 50% inhibition at the indicated concentration (means SD of three experiments) of human
b
AChE. The 50% inhibition at the indicated concentration (means SD of three experiments) of
human BuChE. c hAChE selectivity index = IC50(hBuChE)/IC50(hAChE).
10

2.3. In Vitro Inhibition of Human MAOs
To complete the multi-target biological profile of the target compounds, the
inhibitory activity against hMAO-A and –B was measured, and a non-selective MAO
inhibitor, iproniazide, was used as reference compound [50, 51]. As shown in Table 3,
all compounds can effectively inhibit MAO-B at micromolar range, while no
compound show significantly inhibitory activity for MAO-A at 100 μM, indicating all
compounds are clearly selective MAO-B inhibitors. Among these compounds,
compound 9a (IC50 = 1.93 μM) showed the most potent inhibitor activity for MAO-B
with IC50 value 4.1-fold more potent than that of iproniazide (IC50 = 7.97 μM).
However, different from our previous conclusion in tacrine-coumarin hybrids that
substituents at 3- or/and 4-position of couamrin moiety could largely influence the
inhibitory activity on MAOs [35], the present results did not give a clear
structure-activity relationship (SAR). In view of all above results, compound 5m
presenting the most potent inhibition for animal ChEs and efficient inhibition for
hChEs and hMAO-B was selected as promising compound for further study.
Table 3 Inhibition of hMAO-A and hMOA-B by Compounds 5a-m and 9a-b.
IC50 (μM)
compd
5a
hMAO-Aa
hMAO-Bb
8.39 0.91
12.6 1.2
30.4 2.5
2.75 0.22
33.9 2.1
SIc
N
N
N
N
N
> 11.91
> 7.94
> 3.29
> 36.36
> 2.95
5b
5c
5d
5e
11

5f
N
33.6 0.9
31.9 1.9
28.5 1.7
45.6 3.6
2.38 0.11
23.4 1.1
14.7 1.5
2.62 0.81
1.93 0.33
40.5 4.1
7.97 1.5
0.009c
> 2.98
> 3.13
> 3.51
> 2.19
> 42.01
> 4.27
> 6.80
> 38.17
> 51.81
> 2.47
0.82
5g
N
5h
N
5i
N
5j
N
5k
N
5l
5m
N
N
N
9a
9b
N
iproniazide
safinamide
6.57 2.1
45.00c
5000
a The 50% inhibition at the indicated concentration (means SD of three experiments) of human
b
MAO-A. The 50% inhibition at the indicated concentration (means SD of three experiments)
c
of human MAO-B. hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B). N = no
inhibitory activity was observed on MAO-A at 100 µM.c Values obtained from Ref. 52.
2.4. Kinetic Study of ChE Inhibition
In order to investigate the inhibition mechanism of compound 5m, an enzyme
kinetic study was carried out. The type of inhibition was elucidated from the analysis
of Lineweaver-Burk plots, which were reciprocal rates versus reciprocal substrate
concentrations for the different inhibitor concentrations resulting from the
substrate-velocity curves. For AChE, the plots (Figure 2) showed that both increasing
slopes and intercepts at increasing inhibitor concentration. This pattern indicated a
mixed-type inhibition and therefore revealed that compound 5m might be able to bind
to the catalytic active site (CAS) as well as peripheral anionic site (PAS) of AChE.
12

Figure 2 Kinetic study on the mechanism of eeAChE inhibition by compound 5m. Overlaid
Lineweaver−Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.05−0.50 mM) in the absence of inhibitor and in the presences of different concentrations of 5m
are shown.
In contrast, a different plot for BuChE was obtained (Figure 3), showing
increasing slopes and constant intercepts in different inhibitor concentrations. This
suggested a competitive inhibition, which revealed that compound 5m might compete
for the same binding site as the substrate acetylcholine.
Figure 3 Kinetic study on the mechanism of eqBuChE inhibition by compound 5m. Overlaid
13

Lineweaver−Burk reciprocal plots of BuChE initial velocity at increasing substrate concentration
(0.05−0.50 mM) in the absence of inhibitor and in the presences of different concentrations of 5m
are shown.
2.5. Reversibility and Kinetic Study of MAO-B Inhibition
From a therapeutic point of view, reversible inhibition of MAO-B may have
significant advantages over the irreversible inactivation of the enzyme. Therefore, to
investigate whether compound 5m is a/an reversible or irreversible inhibitor of
MAO-B, the recovery of enzymatic activity after dilution of the enzyme–inhibitor
complexes was evaluated, and an irreversible inhibitor, pargyline, was used as
reference compound [53]. MAO-B was pre-incubated with compounds 5m at
concentrations of 0, 10 and 100 × IC50 for 30 min and then diluted 100-fold to yield
concentrations of 0, 0.1 and 1 × IC50. For reversible inhibition, enzymatic activity is
expected to recover to approximately 90% after dilution to 0.1 × IC50, and 50% after
dilution to 1 × IC50. For an irreversible inhibitor, enzyme activity is expected not to
recover after diluting the enzyme–inhibitor complex. From the Figure 4, it can be seen
that, after the dilution of 5m to 0.1 × IC50, the MAO-B catalytic activities are
recovered to levels of 86% of the control value (recorded in absence of inhibitor).
After dilution to 1× IC50, the MAO-B catalytic activities are recovered to levels of
47%. This behavior is consistent with a reversible interaction of the compound with
MAO-B. After similar incubation of MAO-B with the irreversible inhibitor pargyline
at 10 × IC50, and dilution of the enzyme–inhibitor complex to 0.1× IC50, the MAO-B
14

activities were not fully recovered (less than 10% of control).
Figure 4 Recovery of enzyme activity after dilution. Human MAO-B were preincubated with
compound 5m at concentrations equal to 10 × IC50 and 100 × IC50 for 30 min and then diluted to
0.1 × IC50 and 1 × IC50, respectively. The residual enzyme activities were subsequently measured.
To further characterize the interaction of compound 5m with MAO-B, the type
of enzyme inhibition was also determined by enzyme kinetic study. The initial rates of
the MAO-B-catalyzed oxidation of the substrate p-tyramine, applied at six different
concentrations, were measured in the presence of three different concentrations of
compound 5m. The results are depicted as double reciprocal Lineweaver−Burk plots
in Figure 5. The plots for compound 5m were linear and intersected at the y-axis with
the plot for the uninhibited enzyme. These results led us to conclude that the
compound 5m was competitive inhibitor, which occupies the substrate binding site of
MAO-B, in agreement with its reversible mode of interaction.
15

Figure 5 Kinetic study on the mechanism of human MAO-B inhibition by compound 5m.
Overlaid Lineweaver−Burk reciprocal plots of the inhibition of human MAO-B in the presence of
different concentrations of 5m with p-tyramine (0.05-3.0 mM) as substrate are shown.
2.6. Docking Study
To study the binding modes of compound 5m with AChE and MAO-B, docking
study was carried out using the Molecular Operating Environment (MOE 2008.10)
software package, and the Marvin software package [http://www.chemaxon.com] was
performed to calculate the protonation level of the ligand in physiological pH.
2.6.1 Docking Study of Compounds 5m with AChE
The binding mode of compound 5m with respect to AChE was investigated
based on the X-ray crystal structure of the recombinant human acetylcholinesterase in
complex with donepezil (PDB code 4EY7)[54]. As shown in Figure 6, the coumarin
moiety binds to the PAS of the enzyme, establishing a π-π stacking interaction
between its phenyl ring and the indole ring of Trp 286 (3.59 Å distance). Besides, the
16

carbonyl oxygen of coumarin moiety also formed a hydrogen bond with Ser 293 in
PAS (2.18 Å distance). Similarly to donepezil, the N-benzylpiperidine moiety located
at CAS of AChE, which showed a π-π stacking interaction (3.70 Å distance) with Trp
86. In the middle of the gorge, the protonated nitrogen linked to piperidine also
contributed to AChE binding. It formed a cation-π interaction with Tyr 341 (4.87 Å
distance).
Figure 6 (a) 3D docking model of compound 5m with hAChE. Atom colours: yellow–carbon
atoms of 5m, cyan–carbon atoms of residues of hAChE, dark blue–nitrogen atoms, red–oxygen
atoms. The dashed lines represent the interactions between the protein and the ligand. (b) 2D
schematic diagram of docking model of compound 5m with hAChE. The figure was prepared
17

using the ligand interactions application in MOE.
2.6.2 Docking Study of Compounds 5m with MAO-B
The binding mode of compound 5m with respect to MAO-B was investigated
based on the X-ray crystal structure of the human monoamine oxidase B in complex
with 7-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB code 2V61)[55].
It can be seen from the Figure 7 that coumarin moiety occupy the substrate cavity,
which left the lactone function close to the FAD cofactor, and it was stabilized by
hydrophobic interactions with Tyr 398, Phe 343 and Tyr 435. The N-benzylpiperidine
moiety was located into entrance cavity and interacted with Thr 201, Thr 202, Phe
168, Leu 167, Leu 164, Pro 102, Pro 103, Ile 199 through van der waals and
hydrophobic interactions.
18

Figure 7 (a) 3D docking model of compound 5m with MAO-B. Atom colours: yellow–carbon
atoms of 5m, cyan–carbon atoms of residues of AChE, green-carbon atoms of FAD, dark
blue–nitrogen atoms, red–oxygen atoms. The dashed lines represent the interactions between the
protein and the ligand. (b) 2D schematic diagram of docking model of compound 5m with
MAO-B. The figure was prepared using the ligand interactions application in MOE.
2.7. In Vitro Blood-Brain Barrier Permeation Assay
Brain penetration is a first requirement for successful CNS drugs. Therefore, to
evaluate whether selected compound 5m could be able to penetrate into the brain, we
used a parallel artificial membrane permeation assay for blood-brain barrier
19

(PAMPA-BBB). This model was established by Di et al. [56], which is a simple and
rapid method to predict passive BBB permeation. The in vitro permeabilities (Pe) of
compound 5m and 9 commercial drugs through a lipid extract of porcine brain were
determined using PBS/EtOH (70:30). Assay validation was made by comparing the
experimental permeability with the reported values of these commercial drugs (Table
4), which gave a good lineal correlation, Pe (exp.) = 1.2086 Pe (bibl.) - 0.2915 (R2 =
0.9260). From this equation and taking into account the limit established by Di et al.
for BBB permeation, we determined that compounds with permeabilities above 4.54 ×
10 −6 cm s −1 could cross the BBB. Compound 5m showed a Pe value of 16.21 × 10 −6
cm s −1, which indicated that it could cross the BBB and reach the therapeutic targets
in the CNS.
Table 4. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB assay for compound 5m and 9
commercial drugs used in the experiment validation.
compd
Testosterone
Verapamil
β-Estradiol
Clonidine
Bibliographya
Experimentb
16.81 1.23
21.01 0.75
17.03 0.44
8.14 0.26
3.11 0.32
1.32 0.07
1.41 0.14
1.81 0.22
1.31 0.01
16.21 0.21
17
16
12
5.3
5.1
2.5
1.9
1.1
0.8
Corticosterone
Piroxicam
Hydrocortisone
Lomefloxacin
Ofloxacin
5m
a Taken from Ref.56.b Data are the mean SD of three independent experiments.
20

2.8. SH-SY5Y Neuroblastoma Cell Toxicity
In addition to the above biological evaluation, the potential cytotoxicity effect of
compound 5m on the human neuroblastoma cell line SH-SY5Y was also evaluated.
After exposing the cells to this compound for 24 h, the cell viability was determined
by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay [57, 58].
The result indicated that 5m show negligible cell death at 1-50 μM (1 μM: 99.6
1.7 %; 5 μM: 101.7 10.3 %; 10 μM: 97.5 10.1 %; 25 μM: 94.6 9.6 %; 50 μM:
92.6 2.6 %), which suggested that 5m was nontoxic to SH-SY5Y cells and might be
a suitable multi-target agent for the treatment of AD.
2.9. Hepatotoxicity of Compound 5m on HepG2 Cells
Finally, to verify whether the replacement of tacrine with N-benzylpiperidine
moiety could eliminate the hepatotoxicity, an in vitro hepatotoxicity assay for
compound 5m was performed on HepG2 cells [59]. After 24 h incubation at 6.25-50
μM, no obvious variation in cell viability was found for compound 5m (6.25 μM:
100.7 2.3 %; 12.5 μM: 101.4 2.1 %; 25 μM: 105.1 6.1 %; 50 μM: 104.2
5.3 %), which indicated 5m did not show hepatotoxicity on HepG2 cells and the
replacement design was rational.
3. Conclusion
A series of donepezil-coumarin hybrids with ChEs and MAO-B inhibitory
activity as multi-target agents have been designed and synthesized. Most of these
compounds showed sub-micro to micromolar inhibitory activity toward AChE and
BuChE, and clearly selective inhibition for MAO-B. Among these compounds,
21

compound 5m presenting the most potent inhibition for animal ChEs and efficient
inhibition for hChEs and hMAO-B was selected as the most promising compound for
kinetic and molecular modeling studies. The results revealed that 5m was a
mixed-type inhibitor, binding simultaneously to CAS, PAS and mid-gorge site of
AChE, and it was also a competitive inhibitor, which occupied the active site of
eqBuChE and MAO-B. In addition, 5m showed no toxicity to SH-SY5Y cells at 1-50
μM, good ability to penetrate the CNS and no hepatotoxicity on HepG2 cells. Overall,
all these biological properties highlighted the hybrid 5m as a potential multi-target
agent for the treatment of AD.
4. Experimental section
4.1. Chemistry
All chemical reagents used in synthesis were obtained from Sinopharm Chemical
Reagent Co., Ltd. (China). The reactions were monitored by TLC on glass-packed
precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates.
Column chromatography was performed on silica gel (90-150 μm; Qingdao Marine
1
Chemical Inc.). H NMR spectra (500 MHz) and 13C NMR spectra (125 MHz) were
recorded on a Bruker ACF-500 spectrometer at 25°C. Chemical shifts are reported in
ppm (δ) using the TMS as internal standard, and the coupling constants are reported in
hertz (Hz). The purity of all compounds for biological evaluation was confirmed to >
95% by analytical HPLC conducted on an Agilent 1200 HPLC System. Mass spectra
were carried out on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer
(ESI-MS) and an Agilent 6520B Q-TOF spectrometer (HR-ESI- MS), respectively.
22

4.2. General procedure for the preparation of compounds 5a-m and 9a-b.
A mixture of 4a-b (1.5 mmol) and the corresponding coumarin derivatives 3a-l/8
(1.6 mmol) in dry acetonitrile (15 mL) was refluxed in the presence of anhydrous
K2CO3 (1.7 mmol) for 8 h. After cooling to the room temperature, the mixture was
filtered, and the filtrate was evaporated under reduced pressure. The residue was
purified by silica gel chromatography using CHCl3/MeOH (50:1) as eluent to obtain
target compounds 5a-m as yellow oil.
4.2.1. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-2H-chromen-2-one (5a).
Yield 69.1 %. 1H NMR (500 MHz, DMSO) δ 7.98 (d, J = 9.5 Hz, 1H), 7.61 (d, J
= 8.5 Hz, 1H), 7.33 – 7.25 (m, 4H), 7.23 (d, J = 7.0 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H),
6.94 (dd, J = 8.5, 2.0 Hz, 1H), 6.27 (d, J = 9.5 Hz, 1H), 4.10 (t, J = 5.5 Hz, 2H), 3.42
(s, 2H), 2.92 (t, J = 5.5 Hz, 2H), 2.74 (d, J = 11.5 Hz, 2H), 2.48 – 2.41 (m, 1H), 1.94
(t, J = 11.0 Hz, 2H), 1.78 (d, J = 11.5 Hz, 2H), 1.26 (td, J = 13.5, 3.5 Hz, 2H).13C
NMR (125 MHz, DMSO) δ 162.28, 160.78, 155.87, 144.79, 139.12, 129.96, 129.21,
129.21, 128.58, 128.58, 127.26, 113.22, 112.94, 112.85, 101.74, 68.96, 62.68, 54.80,
52.28, 52.28, 45.28, 32.48, 32.48. ESI/MS m/z: 379.2 [M + H]+; HRMS: calcd for
C23H27N2O3 [M + H]+ 379.2016, found 379.2013.
4.2.2. 7-(3-((1-benzylpiperidin-4-yl)amino)propoxy)-2H-chromen-2-one (5b).
Yield 68.2 %.1H NMR (500 MHz, DMSO) δ 8.01 (d, J = 9.5 Hz, 1H), 7.64 (d, J
= 8.5 Hz, 1H), 7.38 – 7.27 (m, 5H), 7.25 (t, J = 7.0 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H),
6.96 (dd, J = 8.5, 2.0 Hz, 1H), 6.30 (d, J = 9.5 Hz, 1H), 4.15 (t, J = 6.0 Hz, 2H), 3.44
(s, 3H), 2.75 (dd, J = 14.0, 8.0 Hz, 4H), 2.47 (d, J = 10.0 Hz, 1H), 1.95 (t, J = 11.5 Hz,
23

2H), 1.92 – 1.85 (m, 2H), 1.80 (d, J = 10.0 Hz, 2H), 1.35-1.21 (m, 2H). 13C NMR
(125 MHz, DMSO) δ 161.92, 160.46, 155.57, 144.49, 138.84, 129.67, 128.91, 128.91,
128.25, 128.25, 126.93, 112.92, 112.67, 112.58, 101.48, 67.96, 62.71, 53.46, 47.89,
46.71, 36.07, 33.33, 32.20, 32.20. ESI/MS m/z: 393.2 [M + H]+; HRMS: calcd for
C24H29N2O3 [M + H]+ 393.2173, found 393.217.
4.2.3.
(5c).
7-(2-((2-(1-benzylpiperidin-4-yl)ethyl)amino)ethoxy)-2H-chromen-2-one
Yield 67.3 %.1H NMR (500 MHz, DMSO) δ 8.01 (d, J = 9.5 Hz, 1H), 7.64 (d, J
= 8.5 Hz, 1H), 7.36–7.26 (m, 4H), 7.25 (d, J = 7.0 Hz, 2H), 7.01 (s, 1H), 6.97 (d, J =
10.5 Hz, 1H), 6.30 (t, J = 9.5 Hz, 1H), 4.14 (t, J = 5.5 Hz, 3H), 3.42 (s, 2H), 2.92 (t, J
= 5.0 Hz, 3H), 2.76 (d, J = 11.0 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.87 (t, J = 11.0 Hz,
2H), 1.60 (d, J = 12.0 Hz, 2H), 1.37 (dd, J = 13.5, 7.0 Hz, 2H), 1.19-1.04 (m, 2H). 13C
NMR (125MHz, DMSO) δ 162.22, 160.76, 155.87, 144.79, 139.14, 129.97, 129.21,
129.21, 128.55, 128.55, 27.23, 113.22, 112.97, 112.88, 101.78, 68.26, 63.01, 53.76,
53.76, 48.19, 47.01, 36.37, 33.63, 32.50, 32.50. ESI/MS m/z: 407.2 [M + H]+; HRMS:
calcd for C25H31N2O3 [M + H]+ 407.2329, found 407.2328.
4.2.4. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-methyl-2H-chromen-2-one
(5d).
Yield 66.4 %.1H NMR (500 MHz, DMSO) δ 7.66 (d, J = 9.5 Hz, 1H), 7.36 –
7.25 (m, 4H), 7.22 (t, J = 7.0 Hz, 1H), 6.99 – 6.91 (m, 2H), 6.19 (s, 1H), 4.10 (t, J =
5.5 Hz, 2H), 3.42 (s, 2H), 2.92 (t, J = 5.5 Hz, 2H), 2.74 (d, J = 11.5 Hz, 2H), 2.48 –
2.41 (m, 1H), 2.38 (s, 3H), 1.94 (t, J = 10.5 Hz, 2H), 1.78 (d, J = 11.5 Hz, 2H), 1.32 –
24

1.18 (m, 2H). 13C NMR (125 MHz, DMSO) δ 162.17, 160.61, 155.21, 153.83, 139.14,
129.18, 128.56, 127.24, 126.88, 113.60, 112.90, 111.60, 101.74, 68.99, 62.70, 54.82,
52.30, 52.30, 45.33, 32.57, 32.57, 18.57. ESI/MS m/z: 392.2 [M + H]+; HRMS: calcd
for C24H29N2O3 [M + H]+ 392.2173, found 392.2175.
4.2.5. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-chloro-2H-chromen-2-one
(5e).
Yield 65.2 %.1H NMR (500 MHz, DMSO) δ 7.77 (d, J = 9.0 Hz, 1H), 7.35 –
7.25 (m, 4H), 7.23 (t, J = 7.0 Hz, 1H), 7.14 – 6.97 (m, 2H), 6.70 (s, 1H), 4.15 (t, J =
5.5 Hz, 2H), 3.43 (s, 2H), 2.94 (t, J = 5.5 Hz, 2H), 2.74 (d, J = 11.5 Hz, 2H), 2.49 –
2.40 (m, 1H), 1.95 (t, J = 10.5 Hz, 2H), 1.79 (d, J = 11.5 Hz, 2H), 1.27 (td, J = 13.5,
3.5 Hz, 2H).13C NMR (125 MHz, DMSO) δ 163.44, 159.16, 154.81, 149.06, 139.15,
129.18, 129.18, 129.18, 128.57, 128.57, 128.57, 127.25, 126.89, 113.91, 112.41,
111.35, 101.97, 69.29, 62.68, 54.80, 52.29, 52.29, 45.24, 32.53, 32.53. ESI/MS m/z:
413.1 [M + H]+; HRMS: calcd for C23H26ClN2O3 [M + H]+ 413.1626, found
413.1624.
4.2.6. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-methoxy-2H-chromen-2-one
(5f).
Yield 63.2 %.1H NMR (500 MHz, DMSO) δ 7.69 (d, J = 9.0 Hz, 1H), 7.37 –
7.27 (m, 4H), 7.25 (t, J = 7.0 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.95 (dd, J = 9.0, 2.0
Hz, 1H), 5.76 (s, 1H), 4.13 (t, J = 5.5 Hz, 2H), 4.00 (s, 3H), 3.45 (s, 2H), 2.95 (t, J =
5.5 Hz, 2H), 2.76 (d, J = 11.5 Hz, 2H), 2.48 (dd, J = 9.0, 5.0 Hz, 1H), 1.97 (t, J = 10.8
Hz, 2H), 1.81 (d, J = 11.0 Hz, 2H), 1.29 (td, J = 13.5, 3.5 Hz, 2H). 13C NMR (125
25

MHz, DMSO) δ 166.71, 162.67, 162.51, 155.04, 139.11, 129.20, 129.20, 128.58,
128.58, 127.27, 124.41, 112.96, 108.81, 101.63, 88.12, 68.82, 62.65, 57.35, 54.82,
52.25, 45.22, 32.35. ESI/MS m/z: 409.2 [M + H]+; HRMS: calcd for C24H29N2O4 [M
+ H]+ 409.2122, found 409.2121.
4.2.7. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-ethoxy-2H-chromen-2-one
(5g).
Yield 60.6 %.1H NMR (500 MHz, DMSO) δ 7.68 (d, J = 9.0 Hz, 1H), 7.36 –
7.26 (m, 4H), 7.23 (t, J = 7.0 Hz, 1H), 6.99 – 6.91 (m, 2H), 5.71 (s, 1H), 4.24 (q, J =
7.0 Hz, 2H), 4.12 (t, J = 5.5 Hz, 2H), 3.44 (s, 2H), 2.96 (t, J = 5.5 Hz, 2H), 2.76 (d, J
= 11.5 Hz, 2H), 2.48 (s, 1H), 1.96 (t, J = 11.0 Hz, 2H), 1.81 (d, J = 11.5 Hz, 2H), 1.42
(t, J = 7.0 Hz, 3H), 1.29 (dd, J = 20.5, 10.5 Hz, 2H). 13C NMR (125 MHz, DMSO) δ
165.76, 162.59, 162.54, 155.07, 139.06, 129.19, 129.19, 128.57, 128.57, 127.27,
124.44, 112.92, 108.90, 101.57, 88.35, 68.66, 65.71, 62.62, 54.83, 53.30, 52.21, 45.17,
32.21, 14.40, 14.40. ESI/MS m/z: 423.2 [M + H]+; HRMS: calcd for C25H31N2O4 [M
+ H]+ 423.2278, found 409.228.
4.2.8.
7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-(trifluoromethyl)-2H-chromen-2
-one (5h).
Yield 68.5 %.1H NMR (500 MHz, DMSO) δ 7.64 (d, J = 8.5 Hz, 1H), 7.31 (dd, J
= 13.5, 7.0 Hz, 5H), 7.26 (d, J = 6.5 Hz, 1H), 7.18 (s, 1H), 7.09 (d, J = 9.0 Hz, 1H),
6.88 (s, 1H), 4.18 (s, 2H), 3.46 (s, 2H), 2.99 (s, 2H), 2.78 (d, J = 11.0 Hz, 2H), 1.99
(dd, J = 25.0, 10.5 Hz, 3H), 1.83 (d, J = 12.0 Hz, 2H), 1.34 - 1.25 (m, 2H).13C NMR
26

(125 MHz, DMSO) δ 162.70, 159.17, 159.12, 156.33, 138.50, 129.28, 129.28, 129.19,
128.62, 128.62, 127.39, 126.37, 114.21, 113.76, 107.03, 102.76, 68.08, 68.08, 62.42,
52.00, 52.00, 44.76, 31.39, 29.47. ESI/MS m/z: 447.2 [M + H]+; HRMS: calcd for
C24H26F3N2O3 [M + H]+ 447.189, found 413.1888.
4.2.9. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-4-phenyl-2H-chromen-2-one
(5i).
Yield 62.5 %.1H NMR (500 MHz, DMSO) δ 7.63 – 7.57 (m, 3H), 7.54 (dd, J =
7.0, 2.5 Hz, 2H), 7.36 (d, J = 9.0 Hz, 1H), 7.35 – 7.27 (m, 4H), 7.25 (t, J = 7.0 Hz,
1H), 7.11 (d, J = 2.5 Hz, 1H), 6.96 (dd, J = 9.0, 2.5 Hz, 1H), 6.26 (s, 1H), 4.15 (t, J =
5.5 Hz, 2H), 3.45 (s, 2H), 2.97 (t, J = 5.5 Hz, 2H), 2.77 (d, J = 11.5 Hz, 2H), 2.48 (d,
J = 10.0 Hz, 1H), 1.97 (t, J = 10.5 Hz, 2H), 1.82 (d, J = 11.0 Hz, 2H), 1.28 (dt, J = 9.0,
8.0 Hz, 2H). 13C NMR (125 MHz, DMSO) δ 162.28, 160.45, 155.95, 155.64, 139.08,
135.50, 130.13, 129.34, 129.34, 129.20, 129.20, 128.89, 128.89, 128.58, 128.58,
128.32, 127.27, 113.28, 112.33, 111.82, 102.26, 68.83, 62.63, 54.82, 52.23, 52.23,
45.19, 32.27, 32.27. ESI/MS m/z: 455.2 [M + H]+; HRMS: calcd for C29H31N2O3 [M
+ H]+ 455.2329, found 455.2327.
4.2.10. 7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-3-methyl-2H-chromen-2-one
(5j).
Yield 64.1 %.1H NMR (500 MHz, DMSO) δ 7.80 (s, 1H), 7.52 (d, J = 8.5 Hz,
1H), 7.35 – 7.26 (m, 4H), 7.23 (t, J = 7.0 Hz, 1H), 6.96 (d, J = 2.0 Hz, 1H), 6.92 (dd,
J = 8.5, 2.5 Hz, 1H), 4.09 (t, J = 5.5 Hz, 2H), 3.43 (s, 2H), 2.91 (t, J = 5.5 Hz, 2H),
2.74 (d, J = 11.5 Hz, 2H), 2.48 – 2.40 (m, 1H), 2.06 (s, 3H), 1.95 (t, J = 11.0 Hz, 2H),
27

1.79 (d, J = 11.5 Hz, 2H), 1.31 – 1.20 (m, 2H). 13C NMR (125 MHz, DMSO) δ
162.02, 161.24, 154.80, 140.25, 139.15, 129.19, 129.19, 128.93, 128.93, 128.56,
127.24, 121.52, 113.35, 113.05, 101.44, 68.94, 62.70, 54.80, 52.30, 45.36, 32.58,
32.58, 16.91. ESI/MS m/z: 393.2 [M + H]+; HRMS: calcd for C24H26F3N2O3 [M + H]+
393.2173, found 393.2174.
4.2.11.
7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-3,4-dimethyl-2H-chromen-2-one
(5k).
Yield 68.2 %.1H NMR (500 MHz, DMSO) δ 7.74 – 7.64 (m, 1H), 7.38 – 7.27 (m,
4H), 7.25 (t, J = 7.0 Hz, 1H), 6.96 (dd, J = 5.5, 2.5 Hz, 2H), 4.10 (t, J = 5.5 Hz, 2H),
3.44 (s, 2H), 2.93 (t, J = 5.5 Hz, 2H), 2.76 (d, J = 11.5 Hz, 2H), 2.45 (t, J = 10.0 Hz,
1H), 2.37 (s, 3H), 2.09 (s, 3H), 1.97 (t, J = 11.0 Hz, 2H), 1.80 (d, J = 11.5 Hz, 2H),
1.27 (d, J = 11.5 Hz, 2H). 13C NMR (125 MHz, DMSO) δ 161.71, 161.14, 153.50,
147.28, 139.21, 129.17, 129.17, 128.56, 128.56, 127.23, 126.60, 118.32, 114.08,
112.78, 101.46, 100.09, 69.02, 62.72, 54.81, 52.34, 45.43, 32.73, 15.36, 13.36.
ESI/MS m/z: 407.2 [M + H]+; HRMS: calcd for C25H31N2O3 [M + H]+ 407.2329,
found 407.2327.
4.1.12.
7-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-3-chloro-4-methyl-2H-chromen-2
-one (5l).
Yield 68.3 %.1H NMR (500 MHz, DMSO) δ 7.75 (t, J = 15.0 Hz, 1H), 7.31 –
7.22 (m, 4H), 7.20 (t, J = 7.0 Hz, 1H), 7.04 – 6.95 (m, 2H), 4.10 (t, J = 5.0 Hz, 2H),
28

3.40 (s, 2H), 2.92 (t, J = 5.0 Hz, 2H), 2.72 (d, J = 11.0 Hz, 2H), 2.47 (s, 3H),
2.47-2.39 (m, 1H), 1.92 (t, J = 11.0 Hz, 2H), 1.77 (d, J = 11.5 Hz, 2H), 1.24 – 1.16 (m,
2H). 13C NMR (125 MHz, DMSO) δ 160.73, 156.83, 153.09, 152.99, 149.29, 129.61,
129.61, 128.53, 128.53, 127.58, 117.22, 113.95, 113.75, 102.25, 101.74, 79.39, 79.01,
63.16, 62.74, 52.34, 45.32, 29.34, 29.34, 16.60. ESI/MS m/z: 427.2 [M + H]+; HRMS:
calcd for C24H28ClN2O3 [M + H]+ 427.1783, found 427.1781.
4.1.13.
3-(2-((1-benzylpiperidin-4-yl)amino)ethoxy)-7,8,9,10-tetrahydro-6H-benzo[c]
chromen-6-one (5m).
Yield 65.1 %.1H NMR (500 MHz, DMSO) δ 7.60 (d, J = 9.5 Hz, 1H), 7.34 –
7.25 (m, 4H), 7.23 (t, J = 7.0 Hz, 1H), 6.97 – 6.87 (m, 2H), 4.10 (t, J = 5.5 Hz, 2H),
3.43 (s, 2H), 2.94 (t, J = 5.5 Hz, 2H), 2.82 – 2.68 (m, 4H), 2.47 (t, J = 10.0 Hz, 1H),
2.40 (t, J = 6.0 Hz, 2H), 1.95 (t, J = 11.0 Hz, 2H), 1.88 – 1.58 (m, 6H), 1.33 – 1.20 (m,
2H). 13C NMR (125 MHz, DMSO) δ 161.35, 160.99, 153.44, 147.99, 139.14, 129.18,
129.18, 128.57, 128.57, 127.25, 125.40, 119.93, 113.63, 112.71, 101.56, 68.76, 62.67,
54.84, 52.27, 45.32, 32.46, 32.46, 25.12, 24.02, 21.70, 21.35. ESI/MS m/z: 427.2 [M
+ H]+; HRMS: calcd for C24H28ClN2O3 [M + H]+ 427.1783, found 427.1781.
4.1.14. 7-(((1-benzylpiperidin-4-yl)amino)methyl)-4-methyl-2H-chromen-2-one
(9a).
Yield 64.6 %.1H NMR (500 MHz, DMSO) δ 7.72 (d, J = 8.0 Hz, 1H), 7.38 (d, J
= 9.0 Hz, 2H), 7.31 (dt, J = 14.5, 7.0 Hz, 4H), 7.25 (t, J = 7.0 Hz, 1H), 6.36 (s, 1H),
3.86 (s, 2H), 3.44 (s, 2H), 2.75 (d, J = 12.0 Hz, 2H), 2.44 (s, 3H), 2.48 – 2.35 (m, 1H),
29