Synthesis and biological evaluation of 3–(4-aminophenyl)-coumarin derivatives as potential anti-Alzheimer’s disease agents

Article abstract of DOI:10.1080/14756366.2019.1615484

The work is focused on the design of drugs that prevent and treat Alzheimer’s disease (AD) and its complications. A series of 3–(4-aminophenyl)-coumarin derivatives designed, synthesised, fully characterised and evaluated in vitro/vivo. The biological assay experiments showed that some compounds displayed a clearly selective inhibition for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among all compounds, compound 4m exhibited the highest AChE inhibition with an IC₅₀ value of 0.091 ± 0.011 μM and compound 4k exhibited the highest BuChE inhibition with an IC₅₀ value of 0.559 ± 0.017 μM. A zebrafish behaviour analyser (Zebrobox) was used to determine the behavioural effects of the active compound on the movement distance of the aluminium chloride-induced zebrafish. Compound 4m offered a potential drug design concept for the development of therapeutic or preventive agents for AD and its complications.

Full text of DOI:10.1080/14756366.2019.1615484

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of 3–(4-
aminophenyl)-coumarin derivatives as potential
anti-Alzheimer’s disease agents
Yu-Heng Hu, Jie Yang, Yun Zhang, Ke-Chun Liu, Teng Liu, Jie Sun & Xiao-Jing
Wang
To cite this article: Yu-Heng Hu, Jie Yang, Yun Zhang, Ke-Chun Liu, Teng Liu, Jie Sun &
Xiao-Jing Wang (2019) Synthesis and biological evaluation of 3–(4-aminophenyl)-coumarin
derivatives as potential anti-Alzheimer’s disease agents, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1083-1092, DOI: 10.1080/14756366.2019.1615484
To link to this article: https://doi.org/10.1080/14756366.2019.1615484
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Published online: 22 May 2019.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1083–1092
https://doi.org/10.1080/14756366.2019.1615484
RESEARCH PAPER
Synthesis and biological evaluation of 3–(4-aminophenyl)-coumarin derivatives as
potential anti-Alzheimer’s disease agents
a,b,c,d
e
e
Yu-Heng Hua,b,c,d , Jie Yang
, Yun Zhang , Ke-Chun Liu , Teng Liua,b,c,d, Jie Suna,b,c,d
and
ꢀ
ꢀ
Xiao-Jing Wanga,b,c,d
aSchool of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, PR China; Institute of Materia
b
c
d
Medica, Shandong Academy of Medical Sciences, Jinan, PR China; Key Laboratory for Biotech-Drugs Ministry of Health, Jinan, PR China; Key
e
Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan, PR China; Biology Institute, Qilu University of Technology (Shandong
Academy of Sciences), Jinan, PR China
ABSTRACT
ARTICLE HISTORY
Received 22 February 2019
Revised 26 April 2019
Accepted 29 April 2019
The work is focused on the design of drugs that prevent and treat Alzheimer’s disease (AD) and its
complications. A series of 3–(4-aminophenyl)-coumarin derivatives designed, synthesised, fully character-
ised and evaluated in vitro/vivo. The biological assay experiments showed that some compounds
displayed a clearly selective inhibition for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
Among all compounds, compound 4m exhibited the highest AChE inhibition with an IC50 value of
0.091 0.011 mM and compound 4k exhibited the highest BuChE inhibition with an IC50 value of
0.559 0.017 mM. A zebrafish behaviour analyser (Zebrobox) was used to determine the behavioural effects
of the active compound on the movement distance of the aluminium chloride-induced zebrafish.
Compound 4m offered a potential drug design concept for the development of therapeutic or preventive
agents for AD and its complications.
KEYWORDS
Alzheimer’s disease; 3–(4-
aminophenyl)-coumarin;
acetylcholinesterase; butyr-
ylcholinesterase; zebrafish
1. Introduction
terminate the excitatory effects of neurotransmitters on the post-
synaptic membrane, which may be related to the formation of
AD8. At present, there are no specific drugs for the treatment of
AD. AChE inhibitors are currently the only ones that are universally
recognised and have significant therapeutic effects in the world.
They have a good effect on the treatment of mild-to-moderate
AD. Among the mature AChE inhibitor drugs studied, there are
mainly tacrine, donepezil, rivastigmine, galantamine9 and so on.
Coumarin compounds are natural compounds with benzopyran
ring nucleus. Previous studies showed that Schiff bases derivatives
containing a triazole ring, uracil derivatives, aryl methanesulfonate
derivatives are potent AChE and BChE inhibitors10–12. In addition
to AChE, BuChE is important in the regulation of cholinergic sys-
tem and it is reported to efficiently catalyse the hydrolysis of
acetylcholine13. BuChE, an enzyme that breaks down artificial
butyrylcholine, is known to hydrolyse ACh and other ester deriva-
tives in the body14. BuChE, which is a tetrameric serine esterase
consisting of monomers of ꢁ90-kDa molecular mass, showed over
65% structural similarity to AChE15. BuChE is expressed in a dis-
tinct population of neurons, some of which contain AChE. The
importance of BuChE is further supported by the observation that
AChE-knockout mice survive to adulthood indicating that BuChE is
able to compensate for the lack of AChE, allowing the continued
regulation of cholinergic neurotransmission16.
Alzheimer’s disease is a degenerative disease of the central ner-
vous system that is mainly characterised by progressive memory
impairment1,2. AD has brought heavy burdens to society and fam-
ily because of the characteristics of progressive memory loss and
loss of acquired knowledge3. The main characteristic pathological
changes in the brain of patients with AD are the appearance of
senile plaques (SPs)4 with extracellular amyloid b-protein (Ab)
deposition, and intracellular Tau hyperphosphorylation, formation
of neurofibrillary tangles (NFTs)5, and loss of neurons6. Today
there are 47 million people living with dementia worldwide. By
2050, this number will have increased above 135 million.
Dementia affects 3.9 persons out of 1000 at the age ranging from
60 to 647.
Although some work has been done on the pathogenesis
of AD, there is no comprehensive and substantial cognition.
The pathological theory widely accepted in the world is the
“cholinergic hypothesis”. Acetylcholine is the neurotransmitter in
the brain. The cholinergic hypothesis proposes that AD is caused
by reduced synthesis of the neurotransmitter acetylcholine. ACh
deficiency may cause learning loss and memory decline in
patients. ACh levels and restoration of cholinergic nerve conduc-
tion can improve AD patients’ memory ability and cognitive level
to reduce their symptoms. Therefore, increasing the ACh content
in the brain of AD patients is a treatment for AD. AChE, a key
The drug’s efficacy depends on the interactions between the
enzyme in biological nerve conduction, can degrade ACh and ligand and the biological target. The type of interactions affects
CONTACT Jie Sun
sunjie310@126.com; Xiao-Jing Wang
xiaojing6@gmail.com
School of Medicine and Life Sciences, University of Jinan-Shandong
Academy of Medical Sciences, Jinan, Shandong, 250200, China
ꢀ
These authors contributed equally to this work.
Supplemental data for this article is available online at https://doi.org/10.1080/14756366.2019.1615484.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1084
Y.-H. HU ET AL.
the molecular mechanism of ligands inhibition of ChE activity. The 2.2. Biological evaluation
research of new ChE inhibitory compounds is nowadays urgently
2.2.1. In vitro cholinesterase inhibitory activity
18
needed17. Piazzi et al.
constructed a series of benzylpiperidine-
AChE belongs to serine hydrolase family of enzymes catalysing
the hydrolysis of neurotransmitter ACh into choline and acetic
acid, consequently causing cessation of cholinergic neurotransmis-
sion20. AChE is widely distributed in conducting tissues like nerves
and muscles, central and peripheral tissues, motor, and sensory
fibres in addition to cholinergic and non-cholinergic fibres21. It is
also present in plasma and blood cells22,23. As shown in Table 2,
only eight compounds (m, 4f, 4j, 4 l, 4o, 5a, 7 b, and 7d)
coumarin derivatives by linking a benzylpiperidine moiety and a
coumarin scaffold through a benzene ring. Here, we focus on the
synthesis of 3–(4-aminophenyl)-coumarin derivatives and prelimin-
ary study of their pharmacological activities. This work has
designed and synthesised a series of compounds and carried out
a series of active screening around the research of anti-AD.
Table 1. Compounds 4a–4s, 5a–5b, 6a–6b, and 7a–7d.
2. Results and discussion
Product
R1
R2
R3
R4
R5
R6
R7
Yield (%)
2.1. Chemistry
4a
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
OH
OH
H
OH
H
OH
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
F
F
H
H
H
CH3
Cl
H
H
CH3
F
H
H
H
Cl
CH3
H
H
Cl
H
–
–
–
–
–
–
–
–
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
66
70
58
77
85
81
72
80
72
75
78
68
70
81
87
82
77
62
58
70
75
78
82
65
72
65
72
The synthetic route of the 3–(4-aminophenyl)-coumarin derivatives 4b
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
–
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
CH3
CH2Cl
are summarised in (Scheme 1). Starting from substituted p-amino-
phenylacetic acids and substituted o-hydroxybenzaldehydes,
H
H
H
2a–2c was obtained by Perkin reaction, followed by acidification
with hydrochloric acid to give 3–(4-aminophenyl)-coumarin 3a–3c.
A series of substituted benzoyl chlorides were synthesised by
reference methods and used in the next reaction19. The amide
condensation of 3a–3c with an acid chloride in a mixed solution
of pyridine and acetone gives compounds 4a–4s (Table 1). Details
on the chemical and spectroscopic characterisations of com-
pounds 4a–4s were described in the Supporting Information.
Cl
H
H
F
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CF3
CH2Cl
4m
4n
4o
H
H
H
H
The synthetic route of 3–(4-aminophenyl)-coumarin deriva- 4p
4q
OCH3
tives bearing heterocycles is summarised in Scheme 2.
Hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide
4r
4s
Cl
H
–
–
–
–
–
–
–
–
H
CH2Cl
(DCC) was added into an anhydrous toluene solution containing
compounds 1–6. Then, compound 3a or 3b was added to the
reaction solution, respectively. The reaction mixture was stirred
at room temperature for 8–16 h to give compounds 5a–5b,
6a–6b, and 7a–7d (Table 1). Details on the chemical and spec-
troscopic characterisations of compounds 5a–5b, 6a–6b, and
7a–7d were described in the Supporting Information.
5a
5b
6a
6b
7a
7b
7c
7d
–
–
–
–
–
–
–
–
OCH3
H
OCH3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
H
OCH3
OCH3
OCH3
H
Scheme 1. General synthetic route to 3–(4-aminophenyl)-coumarin 3a–3c and compounds 4a–4s. Reagents and conditions: a) acetic anhydride, Et3N, 115 ꢂC; b) HCl
ethanol; c) (COCl)2, DCM, reflux; d) 3–(4-aminophenyl)-coumarin 3a–3c, acetone, pyridine, and RT.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1085
Scheme 2. General synthetic route to compounds 5a–5b, 6a–6b, and 7a–7d. Reagents and conditions: e) 3–(4-aminophenyl)-coumarin 3a–3b, DCC, HOBt, toluene,
and RT.
presented moderate to excellent AChE inhibitory activity. Notably, group at R1 position and a methyl group, a fluorine atom or a
4 m (IC50 ¼ 0.091 0.011 lM) had relatively strong activity, which chlorine atom (such as compounds 4g, 4k, 4m, 4o, 4p, and 4q),
was weaker than donepezil (IC50 ¼ 0.012 0.001 lM). The target have a certain AChE inhibitory activity.
In the human brain, BuChE is found in neurons and glial cells as
well as in neuritic plaques and tangles in AD patients24. It has been
recently reported that dual inhibition of AChE and BuChE might
reduce the symptoms of AD owing to the key role of BuChE in
hydrolysis of ACh25. As shown in Table 2, most of the compounds
exhibited moderate to excellent inhibitory activity against BuChE.
It is noteworthy that compounds 4b, 4f, 4i, 4j, 4k, 4n, and 7a
have relatively strong inhibitory activity, and their inhibitory activity
compounds with an aromatic heterocyclic ring (such as com-
pounds 5a, 7b, and 7d have a furan ring, a naphthalene ring, and
a thiophene ring, respectively) obtained by the amide condensa-
tion reaction and exhibited a good AChE inhibitory activity.
Compounds 4d, 4m, and 4s contain methyl chloride at the same
position. However, compound 4m with methoxy at R1 position
has much better AChE inhibitory activity than compound 4s with
methoxy at R1 position and hydroxyl groups at R2 position. At
the same time, the activity of compound 4m is better than that of is better than donepezil (IC50
¼
2.665 0.015lM), especially
the compound 4d contained hydroxyl groups at R2 position. compound 4k (IC50 ¼ 0.559 0.017 lM). Through comparing the
Compounds 4a, 4b, 4c, 4e, 4g, 4h, and 4i contained a hydroxyl compounds 4a, 4b, 4f, 4g, 4 h, 4j, 4k, 4p, and 4r, we find 3-aryl-
group at R2 position and contained methyl, fluorine or chlorine, coumarins with an amino group can enhance its BuChE inhibitory
and these compounds have no inhibitory activity against AChE. activity by introducing halogen-containing benzene ring by the
The results indicated that compounds containing a methoxy amide condensation reaction. We can find that the halogen-

1086
Y.-H. HU ET AL.
substituted compounds at the R4 and R5 positions are more active the fish in the 50.0 mg/L group were dead or malformed after
24 h administration; all fish died in the 50.0 mg/L group at 48 h,
and malformed and dead zebrafish began in the 10.0 mg/L group;
5.0 mg/L groups of zebrafish have individual deaths and deform-
ities after 72 h of administration.
than the R3-substituted compounds by comparing their IC50 values.
It can be seen that the halogen substitution position has a great
influence on the inhibitory activity of BuChE.
According to in vitro AChE/BuChE inhibition test results, we
decided to study the effects of compounds 4f, 4j, 4k, 4l, 4m,
4r, 5a, 7b, and 7d on the aluminium trichloride-induced behav-
ioural inhibition model of zebrafish larvae.
2.2.3. The effect of compounds on zebrafish model
Zebrafish motion retardation characteristics were used to simu-
late motion disorders in AD. The main purpose of this experiment
was to calculate the total distance of zebrafish movement
induced by aluminium trichloride in the corresponding time after
administration. As shown in Table 5, compounds 4f, 4j, 4m, and
7d have certain therapeutic effects on the behavioural inhibition
of zebrafish induced by aluminium trichloride. In particular, com-
pounds 4f, 4j, 4m, and 7d had significant effects compared with
the model group. In the whole experiment, compound 4m
showed significant therapeutic effect at 50.0 and 100.0 lg/mL
concentration, which could significantly increase the total dis-
tance of zebrafish juvenile movement induced by aluminium
trichloride. With the increase of the concentration of compound
4j, the total distance of zebrafish juvenile motion induced by alu-
minium trichloride increased first and then decreased. The total
distance of zebrafish juvenile motion at the maximum concentra-
tion (100.0 lg/mL) was decreased compared to the total distance
of zebrafish juvenile motion at 50.0 lg/mL. The results showed
that high doses of compounds might slow down the movement
distance of juvenile zebrafish. A more intuitive comparison can
be seen in Figure 1.
2.2.2. Aluminium trichloride-induced behavioural inhibition model
of zebrafish juveniles
AD causes the patient’s actions to become sluggish, and the
zebrafish behavioural model can mimic AD from a motor per-
spective. AD model of zebrafish juveniles is produced by alumin-
ium trichloride26,27. The zebrafish movement distance within
20 min was observed in a Zebrobox zebrafish behaviour analyser
(Viewpoint, Lyon, France) by setting different concentrations of
aluminium trichloride solution. The most suitable modelling con-
centrations were chosen, and then the effect of the compounds
on the behaviour of the aluminium trichloride-induced zebrafish
juveniles was explored. As shown in Table 3, we chose 5.0 mg/L
aluminium trichloride as the best modelling concentration.
The death and deformity of zebrafish were observed. As
shown in Table 4, the blank group, 0.1, 0.5 mg/L group did not
cause malformation and death of zebrafish for 3 days. Most of
Table 2. Biological evaluation in vitro.
c
IC50 (lM)
Compound
AChEa
BuChEb
SId
3. Conclusions
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
>50
>50
>50
3.885 0.955
2.445 0.035
15.65 1.825
8.905 1.175
10.57 0.132
2.565 0.275
24.45 0.535
3.881 0.191
2.265 0.225
0.905 0.081
0.559 0.017
4.325 0.685
41.19 1.044
0.951 0.159
>50
<0.097
<0.050
<0.874
0.189
<0.211
1.319
<0.489
<0.078
<0.045
0.217
Twenty-seven 3–(4-aminophenyl)-coumarin derivatives were syn-
thesised by Perkin reaction, amide condensation reaction and
carboxylic acid condensation reaction. We then studied the
pharmacological activity of the synthesised compounds. The bio-
logical evaluation showed that most of the novel compounds
were good dual cholinesterase inhibitors in the micromolar
range. In particular, compound 4m showed the highest effective
47.21 3.566
>50
1.944 0.141
>50
>50
>50
4.165 0.161
27.97 2.560
5.604 0.132
0.091 0.011
>50
8.259 0.581
48.95 2.325
48.21 3.125
19.68 2.450
48.21 2.685
2.487 0.151
47.12 1.589
19.14 0.981
>50
0.020
0.772
AChE inhibitory activity (IC50
was slightly weaker to the positive drug donepezil (IC50
¼
0.091 0.011 lM), which
¼
452.637
<0.020
>6.054
0.326
>1.037
0.220
>1.037
3.842
0.098
>2.612
–
<0.052
341.87
0.176
0.700
222.08
0.012 0.001 lM). The inhibitory activities of compounds 4b, 4f,
4i, 4j, 4k, 4n, and 7a on BuChE were higher than the positive
drug donepezil (IC50 ¼ 2.665 0.015 lM). Especially, compound
4k showed the highest effective BuChE inhibitory activities (IC50
¼ 0.559 0.017 lM), which 5-times more active than donepezil
15.96 0.475
>50
4.335 0.125
>50
9.555 0.455
4.615 0.055
>50
4s
5a
5b
6a
6b
7a
7b
7c
Table 4. Effect of different concentrations of AlCl3 on mortality, deformity, and
mortality of zebrafish juveniles after 72 h of administration.
>50
Concentration (mg/L)
(Mortality rate (%)
Malformation rate (%)
>50
2.065 0.045
43.76 1.965
3.381 0.342
4.645 0.245
2.665 0.015
Control
0.1
0.5
1.0
5.0
10.0
50.0
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
1.11 0.17
0.00 0.00
0.00 0.00
0.00 0.00
1.11 0.17
2.22 0.17
16.67 0.29
100.00 0.00
0.128 0.011
19.19 1.640
6.631 0.120
0.012 0.001
7d
Donepezil
aAChE from Electrophorus electricus.
ꢀꢀ
ꢀꢀ
11.11 0.17
bBuChE from equine serum.
ꢀꢀ
ꢀꢀ
100.00 0.00
cEach value represents the mean SD (n¼ 3).
dSI: selectivity index (IC50 BuChE/IC50 AChE).
ꢀ
ꢀꢀ
Each value represents the mean SE, (n¼ 90), p<.05, p<.01 compared to control.
Table 3. Zebrafish movement distance (cm) at different concentrations of AlCl3 at 72 hpe (hours post-exposure).
Concentration (mg/L)
Control
0.1
0.5
1.0
5.0
10.0
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
Movement distance (cm)
290.2 7.3
292.4 6.7
236.8 9.4
224.3 10.8
181.3 9.6
204.6 12.8
ꢀ
ꢀꢀ
Mean SE, n ¼ 6, p<.05, and p<.01 compared to control.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1087
Table 5. Effects of different compounds on the average total distance of zebrafish juveniles.
Total movement distance (cm)
5 mg/L 10 mg/L
Control
Model
50 mg/L
100 mg/L
4f
4j
4k
4l
4m
4r
5a
7b
7d
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
316.4 14.8
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
190.5 9.3
205.7 4.5
208.8 9.6
194.9 5.5
179.9 10.1
198.3 10.1
223.2 16.4
236 11.5ꢀꢀ
244.8 13.6ꢀꢀ
ꢀꢀ
225.7 13.4
#
#
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
226 10.7ꢀꢀ
231.8 13.2ꢀꢀ
#
#
ꢀꢀ
ꢀꢀ
ꢀꢀ
205.7 9.5
186.7 7.7
194.6 13.6
210.2 6.0
182.4 4.6
188.8 14.7
278.4 11.4
208.4 9.7
181.8 6.9
211.5 20.ꢀ2ꢀ##
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
##
184.8 11.5ꢀꢀ
ꢀꢀ
230.8 6.2
247.1 12.3
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
178 11.2
196.4 11.7
194.4 9.4
187.2 6.2
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
194.2 6.5
272.7 8.9
202.9 6.2
215.5 17.1
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
227.4 8.1ꢀꢀ
272.1 10.4ꢀꢀ
#
#
ꢀꢀ
224.2 9.2
235.4 11.4
#
Each value represents the mean SE, (n ¼ 8), p<.05, p<.01 compared to control; p<.05, ##p<.01 compared to the model group.
ꢀ
ꢀꢀ
Figure 1. Effect of different compounds on the average total distance of zebrafish juveniles. p < 0.05, p<.01 compared to control; #p<.05, ##p<.01 compared to
the model group.
ꢀ
ꢀꢀ

1088
Y.-H. HU ET AL.
(IC50 ¼ 2.665 0.015 lM). Compounds 4 m, 4o, and 7b were reflux washing, allowed to stand at cold, then vacuum filtered and
highly selective for AChE and compounds 4a, 4 b, 4h, 4i, and 4k rinsed with cold ethanol to give 4a as a white solid (66% yield).
were highly selective for BuChE. We decided to study the effects Compounds 4 b–4s were obtained using the same procedure.
of compounds 4f, 4j, 4k, 4l, 4m, 4r, 5a, 7b, and 7d on the alu-
minium trichloride-induced behavioural inhibition model of
zebrafish larvae. The result showed that compounds 4f, 4j, 4m,
and 7d could alleviate the behavioural inhibition symptoms of
zebrafish juveniles induced by aluminium trichloride. In particular,
compound 4m at 50.0 and 100.0 lg/mL concentration has signifi-
cant effects compared with the model group. In conclusion, com-
pound 4m represents an interesting lead for drug design for the
development of therapeutic or preventive agents for AD.
4.1.2.1. 3-[4-(3-fluoro-benzoylamino)-phenyl]-7-hydroxy-coumarin
1
(4a). White solid, yield: 66%, mp308.2–309.3 ꢂC. H NMR (600 MHz,
DMSO-d6) d 10.59 (s, 1H), 10.43 (s, 1H), 8.18 (s, 1H), 7.89–7.82 (m,
3H), 7.83–7.77 (m, 1H), 7.76–7.70 (m, 2H), 7.61 (q, J ¼ 6.7 Hz, 2H),
7.51–7.41 (m, 1H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.77 (d, J ¼ 2.2 Hz,
1H). 13 C NMR (151 MHz, DMSO-d6) d 164.64, 163.22, 161.60,
160.59, 155.27, 140.83, 139.21, 137.61, 137.57, 131.12, 131.07,
130.94, 130.37, 129.00, 124.42, 124.40, 122.14, 120.39, 119.11,
118.97, 115.08, 114.93, 113.88, 112.56, 102.19.MS: m/z (%):397.8
[M þ 23] þ, 357.8.
4. Experimental
4.1. Synthesis
4.1.2.2. 3-[4-(4-fluoro-benzoylamino)-phenyl]-7-hydroxy-coumarin
1
(4b). White solid, yield: 70%, mp321.4–323.1 ꢂC. H NMR (600 MHz,
Melting points were determined using a Thiele tube and were
uncorrected. The 1H-NMR and 13 C-NMR spectra were recorded
with a Bruker AM-600 spectrometer (Billerica, MA) with TMS as the
internal standard. Chemical shifts were reported at room tempera-
ture on a scale (ppm) with DMSO-d6 as the solvents and J values
are given in Hertz. Mass spectra were obtained with an Agilent
Trap VL LC/MS spectrometer (Santa Clara, CA). The absorbance
was recorded by a Hitachi U-3000 UV spectrophotometer (Tokyo,
Japan). Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
DMSO-d6) d 10.59 (s, 1H), 10.38 (s, 1H), 8.17 (s, 1H), 8.12–8.03 (m,
2H), 7.88–7.82 (m, 2H), 7.76–7.70 (m, 2H), 7.61 (d, J ¼ 8.5 Hz, 1H),
7.44–7.34 (m, 2H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.77 (d, J ¼ 2.2 Hz,
1H).13C NMR (151 MHz, DMSO-d6) d 164.93, 163.77, 161.57, 160.60,
155.25, 140.77, 139.42, 131.74, 131.72, 130.96, 130.90, 130.76,
130.35, 128.98, 122.18, 120.33, 115.91, 115.77, 113.87, 112.56,
102.18. MS: m/z (%):375.9 [M þ 1] þ, 279.7, 357.8.
4.1.2.3. 3-[4-(4-methyl-benzoylamino)-phenyl]-7-hydroxy-coumarin
(4c). Light green solid, yield: 58%, mp307.5–308.9 ꢂC. 1H NMR
(600 MHz, DMSO-d6) d 10.59 (s, 1H), 10.28 (s, 1H), 8.17 (s, 1H), 7.90
(d, J ¼ 8.1 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 2H), 7.74–7.67 (m, 2H), 7.61
(d, J ¼ 8.5 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 2H), 6.83 (dd, J ¼ 8.5, 2.3 Hz,
1H), 6.76 (d, J ¼ 2.1 Hz, 1H), 2.40 (s, 3H).13C NMR (151 MHz, DMSO-
d6) d 167.82, 161.58, 160.62, 155.27, 140.75, 139.65, 135.11, 133.65,
130.76, 130.69, 130.36, 130.14, 129.11, 128.85, 127.54, 126.88,
126.03, 125.58, 125.53, 122.24, 119.83, 113.88, 112.57, 102.20. MS:
m/z (%):372.1 [M þ 1] þ, 251.9, 279.9.
4.1.1. General method for synthesis of compounds 3a–3c
Compound 1a (1.38 g, 10 mmol) and P-aminophenylacetic acid
(1.51 g, 10 mmol) were added to triethylamine (5.56 g, 55 mmol)
and acetic anhydride (6.12 g, 60 mmol) and then the mixture was
heated at 115 ꢂC for 6 h. The resulting mixture was quenched by
the addition of water, and a large amount of solid precipitated.
The filtrate was vacuum filtered and the resulting solid was
washed three times with water. The obtained solid 2a was dis-
solved in 10 mL of absolute ethanol, and then reacted by adding
hydrochloric acid (40 mL, 10%) at 80 ꢂC for 6 h (TLC monitoring).
The reaction solution was poured into 50 mL of ice water and
stirred to precipitate a large amount of light green solid. After
standing and filtration, the solid was washed with water until
the pH of the wash solution was nearly neutral. The solid
was recrystallised from ethanol/water to obtain compound 3a
(1.78 g, 70.4% yield). Compounds 3b–3c were obtained using the
same procedures.
4.1.2.4.
3-[4-(4-chloromethyl-benzoylamino)-phenyl]-7-hydroxy-
coumarin (4d). Light yellow, yield: 77%, mp264.9–267.0 ꢂC. 1H
NMR (600 MHz, DMSO-d6) d 10.60 (s, 1H), 10.40 (s, 1H), 8.17 (s, 1H),
7.99 (d, J ¼ 8.1 Hz, 2H), 7.86 (d, J ¼ 8.6 Hz, 2H), 7.73 (d, J ¼ 8.5 Hz,
2H), 7.61 (d, J ¼ 8.3 Hz, 3H), 6.83 (dd, J ¼ 8.4, 1.7 Hz, 1H), 6.77 (d,
J ¼ 1.3 Hz, 1H), 4.86 (s, 2H).13C NMR (151 MHz, DMSO-d6) d 165.60,
161.58, 160.60, 155.25, 141.62, 140.77, 139.44, 135.15, 130.75,
130.35, 129.28, 128.98, 128.57, 122.18, 120.30, 113.87, 112.56,
102.18. MS: m/z (%):406.0 [M þ 1] þ, 351.8, 369.9, 387.9.
4.1.2.5. 3-[4-(3-methyl-benzoylamino)-phenyl]-7-hydroxy-coumarin
(4e). Light yellow solid, yield: 85%, mp265.2–266.7 ꢂC. 1H NMR
(600 MHz, DMSO-d6) d 10.59 (s, 1H), 10.33 (s, 1H), 8.17 (s, 1H), 7.86
(d, J ¼ 8.7 Hz, 2H), 7.80 (s, 1H), 7.77 (d, J¼ 6.5 Hz, 1H), 7.72 (d,
J¼ 8.7 Hz, 2H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.43 (d, J ¼ 6.6 Hz, 2H), 6.83
(dd, J ¼ 8.5, 2.2 Hz, 1H), 6.76 (d, J ¼ 2.2 Hz, 1H), 2.42 (s, 3H).13CNMR
(151 MHz, DMSO-d6) d 166.16, 161.56, 160.61, 155.24, 140.72,
139.57, 138.20, 135.32, 132.70,130.62, 130.34, 128.96, 128.79,
128.63, 125.34, 122.21, 120.26, 113.86, 112.57, 102.18, 56.50. MS:
m/z (%):371.9 [M þ 1] þ, 251.8, 279.8.
4.1.2. General method for synthesis of compounds 4a–4s
The 3-fluorobenzoyl chloride was synthesised from 3-fluorobenzoic
acid (0.28 g, 2 mmol) with excess oxalyl chloride (1.28 mL, 15 mmol)
in CH2Cl2 (20 mL) containing N, N-dimethylformamide (DMF) as a
catalyst. The mixture was refluxed for about 2 h until the dis-
appearance of the 3-fluorobenzoic acid monitoring by TLC, and
then, the mixture was cooled to room temperature. The redundant
oxalyl chloride was evaporated under vacuum. The crude benzoyl
chloride was used in the following reaction without further purifi-
cation. The 3-fluorobenzoyl chloride was dissolved in acetone
(5 mL). Compound 3a (0.51 g, 2 mmol) was added to 20 mL of acet-
one, and then 15 mL of pyridine was added, and 3-fluorobenzoyl
4.1.2.6. 3-[4-(3-chloro-benzoylamino)-phenyl]-7-hydroxy-coumarin
chloride was added dropwise at 0–5 ꢂC. The reaction was carried (4f). Yellow solid, yield: 81%, mp304.9–306.0 ꢂC.1H NMR (600 MHz,
out for 7 h. The reaction solution was poured into ice water to pre- DMSO-d6) d 10.60 (s, 1H), 10.47 (s, 1H), 8.18 (s, 1H), 8.04 (t,
cipitate a solid which was vacuum filtered, washed with water 3 J ¼ 1.8 Hz, 1H), 7.98–7.90 (m, 1H), 7.88–7.79 (m, 2H), 7.77–7.71 (m,
times, and the solid was dried. The solid was added to ethanol for 2H), 7.68 (ddd, J ¼ 8.0, 2.1, 1.0 Hz, 1H), 7.60 (dd, J ¼ 15.8, 8.1 Hz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1089
2H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.77 (d, J ¼ 2.2 Hz, 1H).13C NMR 2H), 7.40–7.27 (m, 3H), 3.94 (s, 3H).13C NMR (151 MHz, DMSO-d6) d
(150 MHz, DMSO-d6) d 164.55, 161.60, 160.59, 155.27, 140.83,
139.21, 137.26, 133.71, 131.96, 130.96, 130.92, 130.37, 129.01,
127.92, 127.02, 122.14, 120.39, 113.88, 112.56, 102.19. MS: m/z
(%):413.9 [M þ 23]þ, 373.8.
164.95, 159.95, 146.72, 142.62, 140.44, 139.71, 130.47, 129.36,
129.15, 126.86, 125.92, 125.89, 125.02, 120.61, 120.38, 120.24,
114.26, 56.57. MS: m/z (%):461.9 [M þ 23]þ, 419.9.
4.1.2.13. 3-[4-(4-chloromethyl-benzoylamino)-phenyl]-8-methoxy-
coumarin (4m). Yellow solid, yield: 70%, mp274.2–275.6 ꢂC.1H
NMR (600 MHz, DMSO-d6) d 10.43 (s, 1H), 8.26 (s, 1H), 8.03–7.96
(m, 2H), 7.91–7.86 (m, 2H), 7.81–7.76 (m, 2H), 7.61 (d, J ¼ 8.3 Hz,
2H), 7.37–7.29 (m, 3H), 4.86 (s, 2H), 3.94 (s, 3H).13C NMR (151 MHz,
DMSO-d6) d 165.66, 159.96, 146.72, 142.60, 141.66, 140.33, 140.01,
135.10, 130.15, 129.30, 128.59, 126.91, 125.02, 120.62, 120.27,
120.23, 114.23, 56.57. MS: m/z (%):419.9 [M þ 1]þ, 383.3.
4.1.2.7. 3-[4-(2-fluoro-benzoylamino)-phenyl]-7-hydroxy-coumarin
(4g). Light red, solid yield: 72%, mp268.9–372.4 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.59 (s, 1H), 10.54 (s, 1H), 8.16 (s, 1H), 7.80
(d, J ¼ 8.7 Hz, 2H), 7.71 (dd, J ¼ 17.1, 5.1 Hz, 3H), 7.61 (d, J ¼ 8.5 Hz,
2H), 7.36 (dd, J ¼ 10.0, 2.5 Hz, 2H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H),
6.77 (d, J ¼ 2.2 Hz, 1H).13C NMR (151 MHz, DMSO-d6) d 163.29,
161.59, 160.59, 155.27, 140.80, 139.17, 130.90, 130.42, 130.40,
130.36, 129.13, 125.07, 125.05, 122.14, 119.73, 116.73, 116.59,
113.87, 112.55, 102.18. MS: m/z (%):376.0 [M þ 1]þ, 279.8, 357.9.
4.1.2.14. 3-[4-(3-chloro-benzoylamino)-phenyl]-8-methoxy-couma-
rin (4n). White solid, yield: 81%, mp265.3–266.7 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.50 (s, 1H), 8.27 (s, 1H), 8.04 (s, 1H), 7.95
(d, J ¼ 7.5 Hz, 1H), 7.88 (d, J ¼ 8.2 Hz, 2H), 7.79 (d, J ¼ 8.2 Hz, 2H),
7.69 (d, J ¼ 7.8 Hz, 1H), 7.59 (t, J ¼ 7.8 Hz, 1H), 7.41–7.24 (m, 3H),
3.94 (s, 3H).13C NMR (151 MHz, DMSO-d6) d 164.63, 159.96, 146.73,
142.62, 140.41, 139.78, 137.22, 133.72, 132.01, 130.94, 130.36,
129.33, 129.31, 127.94, 127.05, 126.88, 125.03, 120.62, 120.36,
120.24, 114.27, 56.58. MS: m/z (%):428.0 [M þ 23]þ, 265.9, 293.9.
4.1.2.8. 3-[4-(4-chloro-benzoylamino)-phenyl]-7-hydroxy-coumarin
(4h). Light green solid, yield: 80%, mp313.2–315.0 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.59 (s, 1H), 10.43 (s, 1H), 8.17 (s, 1H),
8.08–7.98 (m, 2H), 7.89–7.82 (m, 2H), 7.78–7.69 (m, 2H), 7.67–7.57
(m, 3H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.76 (d, J ¼ 2.2 Hz, 1H).13C
NMR (151 MHz, DMSO-d6) d 164.93, 161.58, 160.59, 155.26, 140.80,
139.31, 136.96, 133.99, 130.86, 130.36, 130.15, 128.99, 128.97,
122.16, 120.36, 113.87, 112.56, 102.18. MS: m/z (%):391.9 [M þ 1]þ,
279.8, 373.8.
4.1.2.15. 3-[4-(3-methyl-benzoylamino)-phenyl]-8-methoxy-couma-
rin (4o). Light yellow solid, yield: 87%, mp244.4–247.0 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.36 (s, 1H), 8.26 (s, 1H), 7.93–7.85 (m, 2H),
7.78 (dd, J ¼ 14.6, 7.8 Hz, 4H), 7.48–7.39 (m, 2H), 7.36–7.27 (m, 3H),
3.94 (s, 3H), 2.42 (s, 3H).13C NMR (151 MHz, DMSO-d6) d 166.23,
159.97, 146.72, 142.59, 140.28, 140.14, 138.21, 135.28, 132.74,
130.02, 129.27, 128.80, 128.65, 126.93, 125.36, 125.01, 120.63,
120.23, 114.21, 56.57. MS: m/z (%):386.2 [M þ 1]þ, 265.9, 293.9.
4.1.2.9. 3-[4-(3, 5-dimethyl-benzoylamino)-phenyl]-7-hydroxy-cou-
marin (4i). White solid, yield: 72%, mp250.2–252.3 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.58 (s, 1H), 10.28 (s, 1H), 8.17 (s, 1H), 7.85
(d, J ¼ 8.7 Hz, 2H), 7.75–7.66 (m, 2H), 7.64–7.53 (m, 3H), 7.23 (s,
1H), 6.83 (dd, J ¼ 8.5, 2.2 Hz, 1H), 6.76 (d, J ¼ 2.2 Hz, 1H), 4.35 (t,
J ¼ 5.1 Hz, 1H), 2.37 (s, 6H).13C NMR (151 MHz, DMSO-d6) d 166.27,
161.55, 160.61, 155.24, 140.70, 139.61, 138.05, 135.35, 133.39,
130.56, 130.34, 128.94, 125.86, 122.23, 120.23, 113.86, 112.57,
102.18, 56.50. MS: m/z (%):386.1 [M þ 1]þ, 251.9, 279.9.
4.1.2.16. 3-[4-(2-fluoro-benzoylamino)-phenyl]-8-methoxy-couma-
rin (4p). White solid, yield: 82%, mp243.1–245.2 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.58 (s, 1H), 8.25 (s, 1H), 7.83 (d, J ¼ 8.7 Hz,
2H), 7.78 (d, J ¼ 8.7 Hz, 2H), 7.70 (d, J ¼ 1.5 Hz, 1H), 7.60 (d,
J ¼ 8.1 Hz, 1H), 7.44–7.24 (m, 5H), 3.94 (s, 3H).13C NMR (151 MHz,
DMSO-d6) d 163.37, 159.95, 146.72, 142.61, 140.38, 139.72, 133.14,
130.43, 130.41, 130.32, 129.45, 126.87, 125.41, 125.31, 125.09,
125.07, 125.02, 120.60, 120.24, 119.71, 116.75, 116.60, 114.26,
56.57. MS: m/z (%):411.9 [M þ 23]þ, 293.9, 371.9.
4.1.2.10. 3-[4-(3-fluoro-benzoylamino)-phenyl]-8-methoxy-couma-
rin (4j). White solid, yield: 75%, mp259.8–262.4 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.47 (s, 1H), 8.27 (s, 1H), 7.91–7.86 (m, 2H),
7.85 (d, J ¼ 7.9 Hz, 1H), 7.80 (ddd, J ¼ 11.3, 6.3, 2.1 Hz, 3H), 7.61 (td,
J ¼ 8.0, 5.9 Hz, 1H), 7.48 (dd, J ¼ 8.5, 2.3 Hz, 1H), 7.38–7.27 (m, 3H),
3.94 (s, 3H).13C NMR (151 MHz, DMSO-d6) d 164.70, 161.60, 159.96,
146.72, 142.61, 140.40, 139.78, 137.56, 131.13, 131.08, 130.34,
129.32, 126.88, 125.02, 124.44, 124.42, 120.61, 120.36, 120.23,
115.10, 114.95, 114.25, 56.57. MS: m/z (%):390.1 [M þ 1]þ,
293.9, 372.0.
4.1.2.17. 3-[4-(2-methoxy-benzoylamino)-phenyl]-8-methoxy-cou-
marin (4q). Light yellow solid, yield: 77%, mp205.9–206.3 ꢂC.1H
NMR (600 MHz, DMSO-d6) d 10.27 (s, 1H), 8.24 (s, 1H), 7.84 (d,
J ¼ 8.7 Hz, 2H), 7.76 (d, J ¼ 8.7 Hz, 2H), 7.66 (dd, J ¼ 7.5, 1.6 Hz, 1H),
7.58–7.48 (m, 1H), 7.34 (dd, J ¼ 6.6, 4.1 Hz, 1H), 7.31 (dd, J ¼ 4.6,
3.0 Hz, 2H), 7.20 (d, J ¼ 8.3 Hz, 1H), 7.08 (td, J ¼ 7.5, 0.8 Hz, 1H),
3.94 (s, 3H), 3.92 (s, 3H).13C NMR (151 MHz, DMSO-d6) d 165.13,
159.97, 156.98, 146.71, 142.59, 140.23, 139.94, 132.61, 130.16,
129.94, 129.38, 126.92, 125.34, 125.00, 120.99, 120.62, 120.22,
119.64, 114.21, 112.50, 56.57. MS: m/z (%):462.0 [M þ 1]þ, 134.9.
4.1.2.11. 3-[4-(4-fluoro-benzoylamino)-phenyl]-8-methoxy-couma-
rin (4k). White solid, yield: 78%, mp296.5–297.8 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.42 (s, 1H), 8.26 (s, 1H), 8.07 (s, 2H), 7.88
(d, J ¼ 8.1 Hz, 2H), 7.78 (d, J ¼ 8.2 Hz, 2H), 7.39 (t, J ¼ 7.6 Hz, 2H),
7.36–7.26 (m, 3H), 3.94 (s, 3H).13C NMR (151 MHz, DMSO-d6) d
165.00, 163.79, 159.97, 146.72, 142.60, 140.34, 139.99, 131.70,
130.99, 130.93, 130.16, 129.30, 126.91, 125.02, 120.62, 120.30,
120.23, 115.93, 115.79, 114.24, 56.58. MS: m/z (%):390.1 [M þ 1]þ,
293.9, 371.9.
4.1.2.18. 3-[4-(2, 3-dichloro-benzoylamino)-phenyl]-8-methoxy-cou-
marin (4r). White solid, yield: 62%, mp258.6–261.7 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.76 (s, 1H), 8.25 (s, 1H), 7.83–7.74 (m, 5H),
7.61 (dd, J ¼ 7.6, 1.5 Hz, 1H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.37–7.28 (m,
3H), 3.94 (s, 3H).13C NMR (151 MHz, DMSO-d6) d 164.72, 159.95,
4.1.2.12. 3-[4-(4-trifluoromethyl-benzoylamino)-phenyl]-8-methoxy-
coumarin (4l). White solid, yield: 68%, mp298.1–301.5 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.63 (s, 1H), 8.27 (s, 1H), 8.18 (d, J ¼ 8.1 Hz,
2H), 7.94 (d, J ¼ 8.3 Hz, 2H), 7.90 (d, J ¼ 8.8 Hz, 2H), 7.84–7.76 (m, 146.73, 142.63, 140.44, 139.54, 132.58, 131.91, 130.51, 129.54,

1090
Y.-H. HU ET AL.
129.22, 128.62, 127.90, 126.86, 125.03, 120.59, 120.25, 119.59, 4.1.3.4. 3-[4-(1-naphthoylamino)-phenyl]-8-methoxy-coumarin (6b).
114.29, 56.58. MS: m/z (%):391.9 [M þ 1]þ, 293.9, 421.9.
White solid, yield: 82%, mp275.4–277.3 ꢂC.1H NMR (600 MHz,
DMSO-d6) d 10.75 (s, 1H), 8.26 (s, 1H), 8.25–8.18 (m, 1H), 8.10 (d,
J ¼ 8.3 Hz, 1H), 8.07–7.99 (m, 1H), 7.92 (d, J ¼ 8.6 Hz, 2H), 7.84–7.74
4.1.2.19. 3-[4-(4-Chloromethyl-benzoylamino)-phenyl]-7, 8-dihy-
(m, 3H), 7.68–7.55 (m, 3H), 7.40–7.25 (m, 3H), 3.94 (s, 3H).13C NMR
droxy-coumarin (4s). Yellow solid, yield: 58%, mp285.3–286.7 ꢂC.1H
(151 MHz, DMSO-d6) d 167.89, 159.98, 146.73, 142.62, 140.32,
NMR (600 MHz, DMSO-d6) d 10.40 (s, 1H), 10.13 (s, 1H), 9.41 (s, 1H),
140.20, 135.05, 133.65, 130.73, 130.17, 130.13, 129.43, 128.85,
8.13 (s, 1H), 7.99 (d, J ¼ 8.2 Hz, 2H), 7.86 (d, J ¼ 8.7 Hz, 2H), 7.74 (d,
127.56, 126.97, 126.89, 126.07, 125.57, 125.53, 125.03, 120.63,
J ¼ 8.7 Hz, 2H), 7.61 (d, J ¼ 8.3 Hz, 2H), 7.11 (d, J ¼ 8.5 Hz, 1H), 6.84
120.24, 119.81, 114.25, 56.58. MS: m/z (%):422.2 [M þ 1]þ, 155.0,
(d, J ¼ 8.4 Hz, 1H), 4.86 (s, 2H).13C NMR (151 MHz, DMSO-d6) d
266.0, 294.0.
165.59, 160.57, 149.94, 143.54, 141.61, 141.34, 139.39, 135.15,
132.28, 130.85, 129.28, 129.01, 128.57, 121.92, 120.29, 119.55,
113.35, 113.28. MS: m/z (%):422.0 [M þ 1]þ, 368.0, 385.9, 403.9.
4.1.3.5. 3-[4-(2-naphthoylamino)-phenyl]-7-hydroxy-coumarin (7a).
Light yellow solid, yield: 65%, mp305.6–307.2 ꢂC.1H NMR (600 MHz,
DMSO-d6) d 10.60 (s, 1H), 10.56 (s, 1H), 8.62 (s, 1H), 8.19 (s, 1H),
8.13–8.10 (m, 1H), 8.09–8.01 (m, 3H), 7.92 (d, J ¼ 8.7 Hz, 2H),
4.1.3. General method for synthesis of compounds 5a–5b, 6a–6b,
7.78–7.72 (m, 2H), 7.64 (ddd, J ¼ 17.6, 11.8, 5.0 Hz, 3H), 6.84 (dd,
and 7a–7d
J ¼ 8.4, 2.3 Hz, 1H), 6.77 (d, J ¼ 2.2 Hz, 1H).13C NMR (151 MHz,
HOBt (2 mmol) and dicyclohexyl carbodiimide (DCC, 2 mmol) were
added into an anhydrous toluene solution (10 mL) containing
compounds 1–6 (2 mmol) at 0 ꢂC, and the mixture was stirred for
45 min. Then, compounds 3a–3b (2.2 mmol) were added to the
reaction solution, respectively. There action mixture was stirred at
DMSO-d6) d 166.10, 161.57, 160.62, 155.26, 140.76, 139.60, 134.79,
132.62, 132.56, 130.71, 130.36, 129.46, 129.01, 128.53, 128.36,
128.17, 127.37, 124.94, 122.21, 120.32, 113.88, 112.58, 102.19,
56.50. MS: m/z (%):408.1 [M þ 1]þ, 251.9, 279.9.
room temperature for 8–16 h until compounds 1–6 disappeared.
4.1.3.6. 3-[4-(2-naphthoylamino)-phenyl]-8-methoxy-coumarin (7b).
Yellow solid, yield: 72%, mp242.1–243.5 ꢂC.1H NMR (600 MHz,
DMSO-d6) d 10.60 (s, 1H), 8.63 (s, 1H), 8.28 (s, 1H), 8.07 (ddd,
J ¼ 24.6, 15.8, 7.5 Hz, 4H), 7.98–7.93 (m, 2H), 7.85–7.78 (m, 2H),
7.65 (td, J ¼ 7.4, 1.4 Hz, 2H), 7.33 (ddd, J ¼ 7.8, 4.7, 2.6 Hz, 3H), 3.94
(s, 3H).13C NMR (151 MHz, DMSO-d6) d 166.17, 159.98, 146.73,
142.60, 140.32, 140.17, 134.81, 132.58, 132.56, 130.11, 129.47,
129.33, 128.57, 128.54, 128.38, 128.18, 127.38, 126.93, 125.02,
124.94, 120.64, 120.29, 120.23, 114.23, 56.57. MS: m/z (%):422.1
[M þ 1]þ, 265.9, 293.9.
The reaction mixture was filtered and the toluene was evaporated
under reduced pressure. The residue was dissolved in 10% NaOH
and filtered, and then 15% HCl was added to adjust pH of the
solution to 3–4, followed by the production of precipitation. The
precipitation washed with water 3 times, and the solid was dried.
The solid was added to ethanol for reflux washing, allowed to
stand at cold, then vacuum filtered and rinsed with cold ethanol
to give compounds 5a–5b, 6a–6b, and 7a–7d.
4.1.3.1.
3-[4-(furan-2-formylamino)-phenyl]-7-hydroxy-coumarin
(5a). Light yellow solid, yield: 70%, mp242.6–244.3 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.59 (s, 1H), 10.30 (s, 1H), 8.18–8.15 (m,
1H), 7.96 (dd, J ¼ 5.6, 0.9 Hz, 1H), 7.83 (d, J ¼ 8.7 Hz, 2H), 7.71 (d,
J ¼ 8.7 Hz, 2H), 7.60 (d, J ¼ 8.5 Hz, 1H), 7.38–7.36 (m, 1H), 6.83 (dd,
J ¼ 8.5, 2.2 Hz, 1H), 6.76 (d, J ¼ 2.1 Hz, 1H), 6.74–6.70 (m, 1H).13C
NMR (150 MHz, DMSO-d6) d 161.57, 160.59, 156.69, 155.25, 147.92,
146.30, 140.78, 138.89, 130.76, 130.35, 129.00, 122.18, 120.31,
115.37, 113.87, 112.69, 112.56, 102.18. MS: m/z (%):347.9 [M þ 1]þ,
279.8, 347.8.
4.1.3.7. 3-[4-(6-chloro-N-nicotinamide)-phenyl]-8-methoxy-couma-
rin (7c). Light yellow solid, yield:65%, mp310.9–313.2 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.64 (s, 1H), 8.98 (d, J¼ 2.3 Hz, 1H), 8.39
(dd, J ¼ 8.3, 2.5 Hz, 1H), 8.27 (s, 1H), 7.87 (d, J ¼ 8.7 Hz, 2H),
7.82–7.78 (m, 2H), 7.73 (d, J ¼ 8.3 Hz, 1H), 7.36–7.29 (m, 3H), 3.94
(d, J ¼ 4.7 Hz, 3H).13C NMR (151 MHz, DMSO-d6) d 163.50, 149.88,
149.86, 146.73, 140.50, 139.61, 129.41, 129.38, 125.05, 124.65,
120.61, 120.36, 120.33, 120.26, 114.30, 56.58. MS: m/z (%):429.0
[M þ 23]þ, 265.9, 293.9.
4.1.3.2.
3-[4-(furan-2-formylamino)-phenyl]-8-methoxy-coumarin
4.1.3.8. 3-[4-(thiophene-2-formylamino)-phenyl]-8-methoxy-couma-
rin (7d). Yellow solid, yield: 72%, mp263.5–265.4 ꢂC.1H NMR
(600 MHz, DMSO-d6) d 10.38 (s, 1H), 8.26 (s, 1H), 8.07 (dd, J ¼ 3.8,
0.9 Hz, 1H), 7.89 (dd, J ¼ 5.0, 0.9 Hz, 1H), 7.85 (d, J ¼ 1.7 Hz, 1H),
7.84 (d, J ¼ 1.8 Hz, 1H), 7.79 (d, J ¼ 1.8 Hz, 1H), 7.77 (d, J ¼ 1.8 Hz,
1H), 7.37–7.28 (m, 3H), 7.25 (dd, J ¼ 4.9, 3.8 Hz, 1H), 3.94 (s, 3H).13C
NMR (151 MHz, DMSO-d6) d 160.44, 159.95, 146.72, 142.60, 140.37,
140.35, 139.65, 132.61, 130.15, 129.80, 129.34, 128.61, 126.86,
125.02, 120.62, 120.25, 120.23, 114.23, 56.57. MS: m/z (%):399.9
[M þ 23]þ, 265.9, 293.9.
(5b). Yellow solid, yield: 75%, mp268.1–269.5 ꢂC.1H NMR (600MHz,
DMSO-d6) d 10.34 (s, 1H), 8.25 (s, 1H), 8.00–7.94 (m, 1H), 7.91–7.83
(m, 2H), 7.80–7.72 (m, 2H), 7.39–7.37 (m, 1H), 7.35–7.29 (m, 3H), 6.73
(dd, J ¼ 3.5, 1.7 Hz, 1H), 3.94 (d, J ¼ 5.1 Hz, 3H).13C NMR (151 MHz,
DMSO-d6) d 159.96, 156.73, 147.88, 146.72, 146.36, 142.60, 140.34,
139.47, 130.16, 129.31, 126.90, 125.02, 120.61, 120.27, 120.22, 115.46,
114.24, 112.70. MS: m/z (%):362.0 [M þ 1]þ, 298.0.
4.1.3.3. 3-[4-(1-naphthoylamino)-phenyl]-7-hydroxy-coumarin (6a).
Light green solid, yield: 78%, mp287.5–289.3 ꢂC.1H NMR (600MHz,
DMSO-d6) d 10.70 (s, 1H), 10.60 (s, 1H), 8.21 (dd, J ¼ 7.0, 2.5 Hz, 1H),
8.18 (s, 1H), 8.10 (d, J ¼ 8.3 Hz, 1H), 8.06–8.00 (m, 1H), 7.89 (d,
J ¼ 8.6 Hz, 2H), 7.79 (d, J ¼ 6.3 Hz, 1H), 7.75 (d, J ¼ 8.7 Hz, 2H),
7.65–7.58 (m, 4H), 6.84 (dd, J ¼ 8.4, 2.3Hz, 1H), 6.77 (d, J ¼ 2.2 Hz,
1H).13C NMR (151 MHz, DMSO-d6) d 167.82, 161.58, 160.62, 155.27,
140.75, 139.65, 135.11, 133.65, 130.76, 130.69, 130.36, 130.14, 129.11,
128.85, 127.54, 126.88, 126.03, 125.58, 125.53, 122.24, 119.83, 113.88,
112.57, 102.20. MS: m/z (%):408.1 [M þ 1]þ, 251.9, 279.9.
4.2. Biological activity
4.2.1. Preparation of main reagents
In the experiment, 0.05 mol/L phosphate buffered saline (PBS)
of pH 8.0 was prepared first, and then the following solutions
were prepared: 1.5 mmol/L Acetylthiocholine iodide (ATChI);
0.75 mmol/L 5, 5’-Dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma
Aldrich, St. Louis, MO); 0.2 U/mL Acetylcholinesterase (AChE,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1091
from electric squid, Macklin); 0.5 U/mL Butyrylcholinesterase 3 times, disinfect the fertilised eggs with methylene blue solution,
and then move into clean zebrafish culture water at about 28 ꢂC
culture in a light incubator (HPG-280BX).
(BuChE, from equine serum, Aladdin, Shanghai, China);
1.5 mmol/L S-Butyrylthiocholine iodide (BTChI, Sigma Aldrich,
St. Louis, MO). 4% sodium dodecyl sulphate (SDS) was prepared
with secondary water. The test compounds and the positive con-
trol Donepezil (Sigma Aldrich, St. Louis, MO) were formulated
with DMSO to a gradient of 0.1, 1.0, 10.0, 50.0, 100.0, 500.0, and
1000.0 lg/mL. The above reagents were all analytically pure,
and the test water was all distilled water. All solutions were
stored in a refrigerator at 4 ꢂC.
4.2.3.2. Model of exercise retardation in Zebrafish juveniles made
of aluminium trichloride. The 72 hpf zebrafish were placed in a
petri dish, and the normal developing juveniles were selected
under a microscope in a 12-well plate. The concentration of AlCl3
in the experiment was set to 0, 0.1, 0.5, 1.0, 5.0, 10.0, and
50.0 mg/L (prepared for aquaculture water), three replicate wells
were set in each concentration group and then placed in a con-
stant temperature incubator at 28 ꢂC to continue the culture. The
continuous drug solution was exposed for 3 days. The different
concentrations of AlCl3 and the blank control group were placed
in 48-well plates. Each well was juvenile fish developed to 72 h
post-exposure (hpe) and added 0.5 mL of culture water and 8
juveniles were set in each experimental group. The well plate was
placed in the dark box of the zebrafish behaviour analysis system.
The fish was first adapted to 10 min at the beginning of the
4.2.2. In vitro AChE/BuChE inhibitory activity
The AChE/BuChE inhibitory activity of the 3–(4-aminophenyl)-
coumarin derivatives was determined by the method of Ellaman28
with slight modifications. The experiment was divided into control
group, sample blank group and sample group. Take a series of
test tubes, the reagents were loaded in test tubes at the dose of
Table 6. After incubation at 37 ꢂC for 20 min, quickly add 1 mL of
4% SDS to stop the reaction. Measuring ultraviolet absorbance
immediately at 412 nm. All experiments were run in triplicate. The experiment. The trajectories of the juveniles in each group were
absorbance was determined by UV spectrophotometer, and the
inhibition rate of AChE and the IC50 value of each sample were
calculated according to the formula.
collected using zeblab software for 10 min, recorded every 5 min,
and exported by software. Then calculate the total distance of
each group of zebrafish juveniles. The effects of different concen-
trations of aluminium trichloride on the death and teratogenicity
of zebrafish juveniles were observed by microscopy.
Inhibition rate % ¼ f½ðAC ꢃ ASÞ ꢃ ASBꢄ=ACg ꢅ 100%;
where AC (no sample added) is the absorbance of control group;
AS is the absorbance of sample group; ASB (add sample solution
but no substrate ATChI added) is the absorbance of sample
blank group.
The method for determining BuChE is the same as the method
for measuring AChE. The sample can be added with reference to
Table 6, AChE is changed to BuChE, and the substrate ATChI is
changed to substrate BTChI. The order of loading and the amount
of loading are the same as Table 6. The absorbance was deter-
mined by UV spectrophotometer, and the inhibition rate of BuChE
and the IC50 value of each sample were calculated according to
the formula. The formula used is the same as AChE.
4.2.3.3. Effects of compounds on the inhibition of movement abil-
ity of zebrafish caused by aluminium trichloride. The zebrafish
with 72 hpf after fertilisation is placed in a petri dish. Under nor-
mal conditions, the normal developing juvenile fish are selected in
a 12-well plate, and 15 juveniles per hole are set. Control group
(0.5% DMSO), model group (5.0 mg/L AlCl3), 5.0 mg/L AlCl3 þ test
compound 5 lg/mL group, 5.0 mg/L AlCl3 þ test compound 10 lg/
mL group, 5.0 mg/L AlCl3 þ test compound 50lg/mL group,
5.0 mg/L AlCl3 þ test compound 100 lg/mL group, three duplicate
wells were set at each concentration. After the administration,
the zebrafish was placed in a constant temperature incubator at
28 ꢂC to continue the culture for 3 days. Changing the liquid (aqua-
culture water) every 24 h and removing dead young fish.
Behavioural observations are consistent with AlCl3 modelling.
4.2.3. Behavioural experiment of zebrafish juveniles
4.2.3.1. Zebrafish juveniles. AB wild-type zebrafish is provided by
the Key Laboratory of Drug Screening Technology of Shandong
Academy of Sciences. The zebrafish culture was carried out in a
light cycle of 14 h light and 10 h dark at a temperature of 28 ꢂC.
At 16:00 the day before the experiment, the healthy zebrafish
were mated in a tank with a male to female ratio of 1:1 or 2:1.
The next day at 8:30 to extract the separator, after 1–2 h, collect
4.2.4. Statistical analysis
The experimental data were processed by the statistical software
SPSS version 16.0 (SPSS Inc., Chicago, IL). All the experimental
data were expressed by mean SD or mean SE. The statistical
the fertilised eggs, wash the fertilised eggs with culture water for differences were analysed by ANOVA. The comparison between
groups was performed by Tukey test. p < .05 was considered as
significant difference. p < .01 was a very significant difference.
Table 6. The amount and order of each reactant of acetylcholinesterase inhib-
ition test.
Volume (lL)
Disclosure statement
Reagents
Control group
Sample blank group
Sample group
The authors declare that they have no competing interests.
PBS
AChE
2650
50
2650
50
2650
50
DNTB
100
0
100
100
100
0
100
100
0
Compounds
DMSO
Funding
Mix well and incubate at 37 ꢂC for 5 min
The authors are grateful to support from the Science and
Technology Research Program of Shandong Academy of Medical
Sciences (2017–16), Shandong Provincial Natural Science Foundation
[ZR2018LH021], and the Innovation Project of Shandong Academy
of Medical Sciences.
ATChI
PBS
100
0
0
100
100
0
Mix well and react at 37 ꢂC for 20 min
SDS
1000
1000
1000

1092
Y.-H. HU ET AL.
14. Nguyen VT, Zhao BT, Seong SH, et al. Inhibitory effects of
serratene-type triterpenoids from Lycopodium complanatum
on cholinesterases and b-secretase 1. Chem Biol Interact
2017;274:150–7.
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
15. Huang YJ, Huang Y, Baldassarre H, et al. Recombinant
human butyrylcholinesterase from milk of transgenic ani-
mals to protect against organophosphate poisoning. Proc
Natl Acad Sci USA 2007;104:13603–8.
16. Li B, Stribley JA, Ticu A, et al. Abundant tissue butyrylcholi-
nesterase and its possible function in the acetylcholinester-
ase knockout mouse. J Neurochem 2002;75:1320–31.
References
1. Camps P, El Achab R, Morral J, et al. New tacrine-huperzine
a hybrids (huprines): highly potent tight-binding acetyl-
cholinesterase inhibitors of interest for the treatment of
Alzheimer’s disease. J Med Chem 2000;43:4657–66.
2. Sippl W, Contreras JM, Parrot I, et al. Structure-based 3D
QSAR and design of novel acetylcholinesterase inhibitors. J
Comput-Aided Mol Des 2001;15:395–410.
3. Lendon CL, Ashall F, Goate AM. Exploring the etiology of
Alzheimer disease using molecular genetics. JAMA 1997;277:
825–31.
4. Karran E, Mercken M, Strooper BD. The amyloid cascade
hypothesis for Alzheimer’s disease: an appraisal for the
development of therapeutics. Nat Rev Drug Discov 2011;10:
698–712.
5. DE Calignon A, Polydoro M, Suarez-Calvet MS, et al.
Propagation of Tau pathology in model of early
Alzheimer’s disease. Neuron 2012;73:685–97.
6. Lemmens MAM, Sierksma ASR, Rutten BPF, et al. Age-related
changes of neuron numbers in the frontal cortex of a trans-
genic mouse model of Alzheimer’s disease. Brain Struct
Funct 2011;216:227–37.
7. Czarnecka K, Girek M, Maciejewska K, et al. New cyclopenta-
quinoline hybrids with multifunctional capacities for the
treatment of Alzheimer’s disease. J Enzyme Inhib Med Chem
2018;33:158–70.
8. Terry RD, Masliah E, Salmon DP, et al. Physical basis of cog-
nitive alterations in Alzheimer’s disease: synapse loss is the
major correlate of cognitive impairment. Ann Neurol 1991;
30:572–80.
ꢀ
17. Zaton AML, Ferrer JM, Gordoa J, et al. Binding of coumarins
to site I of human serum albumin. Effect of the fatty acids.
Chem Biol Interact 1995;97:169–74.
18. Piazzi L, Rampa A, Bisi A, et al. 3-(4-f[benzyl(methyl)amino]-
methylgphenyl)-6,7-dimethoxy-2H-2-chromenone (AP2238)
inhibits both Acetylcholinesterase and Acetylcholinesterase-
induced b-amyloid aggregation: a dual function lead for
Alzheimer’s disease therapy. J Med Chem 2003;46:2279–82.
(https://pubs.acs.org/doi/abs/10.1021/jm0340602)
19. Gao XH, Liu LB, Liu HR, et al. Structure-activity relationship
investigation of benzamide and picolinamide derivatives
containing dimethylamine side chain as acetylcholinesterase
inhibitors. J Enzyme Inhib Med Chem 2018;33:110–4.
20. Massoulie J, Pezzementi L, Bon S, et al. Molecular and cellu-
lar biology of cholinesterases. Prog Neurobiol 1993;41:31–91.
21. Brimijoin S. Molecular forms of acetylcholinesterase in
brain, nerve and muscle: nature, localization and dynamics.
Prog Neurobiol 1983;21:291–322.
22. Heller M, Hanahan DJ. Human erythrocyte membrane bound
enzyme acetylcholinesterase. BBA Biomembranes 1972;17:
251–72.
23. Szelenyi JG, Bartha E, Hollan SR. Acetylcholinesterase activity
of lymphocytes: an enzyme characteristic of T-cells. Br J
Haematol 1982;50:241–5.
24. Asadipour A, Alipour M, Jafari M, et al. Novel coumarin-3-
carboxamides bearing N-benzylpiperidine moiety as potent
acetylcholinesterase inhibitors. Eur J Med Chem 2013;14:
623–30.
25. Giacobini E. Cholinesterase inhibitors: new roles and thera-
peutic alternatives. Pharmacol Res 2004;50:433–40.
26. Senger MR, Seibt KJ, Ghisleni GC, et al. Aluminum exposure
alters behavioral parameters and increases acetylcholinester-
ase activity in zebrafish (Danio rerio) brain. Cell Biol Toxicol
2011;27:199–205.
ꢀ
a
9. Pearson VE. Galantamine: a new Alzheimer drug with a past
life. Ann Pharmacother 2001;35:1406–13.
€
€
10. Ozil M, Balaydın HT, S¸enturk M. Synthesis of 5-methyl-2,4-
dihydro-3H-1,2,4-triazole-3-one’s aryl Schiff base deriva-
tives and investigation of carbonic anhydrase and cholin-
esterase (AChE, BuChE) inhibitory properties. Bioorg Chem
2019;86:705.
11. Cavdar H, Senturk M, Guney M, et al. Inhibition of acetyl-
cholinesterase and butyrylcholinesterase with uracil deriva-
tives: kinetic and computational studies. J Enzyme Inhib
Med Chem 2019;34:429–37.
27. Newman M, Verdile G, Martins RN, et al. Zebrafish as a tool
in Alzheimer’s disease research. Biochim Biophys Acta 2011;
1812:346–52.
12. Kani Z, Nashia S, Murat G, et al. Effects of aryl methanesul-
fonate derivatives on acetylcholinesterase and butyrylcholi-
nesterase. J Biochem Mol Tox 2018;32:e22210.
28. Ellaman G. A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem Pharmacol 1961;7:
88–95.
13. Ali R, Sheikh IA, Jabir NR, et al. Comparative review of deca-
de’s research on cholinesterase inhibition. Am J Neuroprot
Neuroregen 2012;4:136–44.