Synthesis and biological evaluation of 3-arylcoumarins as potential anti-Alzheimer's disease agents

Article abstract of DOI:10.1080/14756366.2019.1574297

Alzheimer's disease, a neurodegenerative illness, has the extremely complex pathogenesis. Accumulating evidence indicates there is a close relationship between several enzymes and Alzheimer's disease. Various substituted 3-arylcoumarin derivatives were synthesised, and their in vitro activity, including cholinesterase inhibitory activity, monoamine oxidase inhibitory activity, and antioxidant activity were investigated. Most of the compounds exhibited high activity; therefore 3-arylcoumarin compounds have the potential as drug candidates for the treatment of Alzheimer's disease.

Full text of DOI:10.1080/14756366.2019.1574297

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-
arylcoumarins as potential anti-Alzheimer's
disease agents
Jie Yang, Pingping Zhang, Yuheng Hu, Teng Liu, Jie Sun & Xiaojing Wang
To cite this article: Jie Yang, Pingping Zhang, Yuheng Hu, Teng Liu, Jie Sun & Xiaojing Wang
(2019) Synthesis and biological evaluation of 3-arylcoumarins as potential anti-Alzheimer's
disease agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 651-656, DOI:
10.1080/14756366.2019.1574297
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 651–656
https://doi.org/10.1080/14756366.2019.1574297
RESEARCH PAPER
Synthesis and biological evaluation of 3-arylcoumarins as potential
anti-Alzheimer’s disease agents
Jie Yanga,b,c,d, Pingping Zhanga,b,c,d, Yuheng Hua,b,c,d, Teng Liua,b,c,d, Jie Suna,b,c,d
Xiaojing Wanga,b,c,d
and
b
aSchool of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China; Institute of MateriaMedica,
c
d
Shandong Academy of Medical Sciences, Jinan, China; Key Laboratory for Biotech-Drugs Ministry of Health, Jinan, China; Key Laboratory for
Rare & Uncommon Diseases of Shandong Province, Jinan, China
ABSTRACT
ARTICLE HISTORY
Received 10 December 2018
Revised 18 January 2019
Accepted 22 January 2019
Alzheimer’s disease, a neurodegenerative illness, has the extremely complex pathogenesis. Accumulating
evidence indicates there is a close relationship between several enzymes and Alzheimer’s disease. Various
substituted 3-arylcoumarin derivatives were synthesised, and their in vitro activity, including cholinesterase
inhibitory activity, monoamine oxidase inhibitory activity, and antioxidant activity were investigated. Most
of the compounds exhibited high activity; therefore 3-arylcoumarin compounds have the potential as
drug candidates for the treatment of Alzheimer’s disease.
KEYWORDS
3-Arylcoumarin; Alzheimer’s
disease; cholinesterase
inhibitors; monoamine
oxidase inhibitors;
antioxidant activity
1. Introduction
antidepressants and anxiolytics, while MAO-B inhibitors can be
used alone or in combination to treat AD and Parkinson’s dis-
ease5. Accumulating evidence shows that cholinesterase (ChE) and
MAO are closely related to the disease symptoms and progression
of AD. Many studies have identified the role of various ChE/MAO
inhibitors in AD, while some showing positive results in clin-
ical trials6.
Alzheimer’s disease (AD) has become one of the major diseases
that threatening the health of elder people in modern society.
Alzheimer’s disease, a chronic progressive neurodegenerative dis-
ease, usually starts slowly and worsens over time. The most com-
mon early symptom is difficulty in remembering recent events. As
the disease advances, symptoms can include problems with lan-
guage, disorientation, loss of motivation, not managing and so
on1. Gradually, bodily functions are lost, ultimately leading to
death. AD is a refractory disease with complicated etiopathogene-
sis. Several hypotheses, such as cholinergic hypothesis, were pro-
posed to explain the cause of the disease. The most currently
available drug therapies were based on the cholinergic hypothesis,
which proposes that AD is caused by reduced synthesis of the
neurotransmitter acetylcholine (ACh)2.
The potential of acetylcholinesterase (AChE) inhibitors has
been well explored and developed as an anti-AD drug3. There are
currently five drugs used to treat AD cognitive problems, four of
which are AChE inhibitors (tacrine, donepezil, galantamine, riva-
stigmine). The AChE activity of AD patients decreased, but butyryl-
cholinesterase (BuChE) activity was stable or even increased; the
ratio of AChE to BuChE changed, indicating that BuChE may act
as a compensatory mechanism for ACh metabolism. Studies have
shown that selective BuChE inhibitors are beneficial for cognitive
dysfunction of AD, and dual AChE/BuChE inhibition can maximise
therapeutic efficacy4. Monoamine oxidase (MAO) is one of several
enzymes that cause oxidative stress and dementia behaviour and
3-Arylcoumarin refers to a class of compounds having a cou-
marin skeleton and having an aryl structure at the 3-position,
which is rich in biological activity such as antioxidant7, anti-inflam-
matory8, antitumour9, antiviral10, antifungal11, antidiabetic12, MAO
inhibitory activity13,14, ChE inhibitory activity15 and so on. The aeti-
ology of Alzheimer’s disease is complex, and for this multi-faceted
disease, the efficacy of multi-targeted drugs is better than that of
single-targeted drugs. Coumarin compounds, as dual inhibitors,
are promising compounds that control AD16–20. Patil et al.21
reviewed the synthesis and designed aspects of coumarin deriva-
tives as MAO inhibitors for AD, in which most 3-arylcoumarin
compounds selectively inhibit MAO-B and give some guidance in
modifying the structure. Wang et al.15 designed, synthesised, and
evaluated a series of 6-substituted 3-arylcoumarin derivatives as
dual AChE/MAO-B dual inhibitors for the treatment of AD, which
provided meaningful information for further development of
multifunctional drugs for AD treatment. Previous studies15,21–24
have designed coumarin derivatives as potential inhibitors of
MAO and ChE, and have achieved good results. These results
encourage us to further explore the potential of coumarins as can-
psychological symptoms in AD. MAO-A inhibitors can be used as didates for the treatment of AD.
CONTACT Jie Sun
of Medical Sciences, Jinan 250062, Shandong, China
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
sunjie310@126.com; Xiaojing Wang
xiaojing6@gmail.com
School of Medicine and Life Sciences, University of Jinan-Shandong Academy
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.

652
J. YANG ET AL.
modifications. The crude enzyme was extracted from the liver of
200–250 g of Wistar rats according to literature methods26–28. The
crude enzyme protein content was determined by the Bradford/
method using a Bradford Protein Assay Kit (Beyotime). Forty
microlitres of enzyme solution and 40 mL of sample solution were
added to a 96-well plate. The solution was then incubated at
37 ꢀC for 20 min. One-twenty microlitres of the 4-(trifluoromethyl)
benzylamine solution and 40 mL of the chromogenic agent
(1 mmol/L vanillic acid, 0.5 mmol/L 4-aminoantipyrine, 4U/mL
horseradish peroxidase, 0.2 M pH ¼ 7.6 PBS constant volume) were
added subsequently, and incubated at 37 ꢀC for 90 min. The
absorbance was measured at 490 nm using a microplate reader,
and the inhibition rate of MAO and the IC50 value of each sample
were calculated according to the formula. The control group
replaced the sample solution with PBS (0.2 M, pH ¼ 7.6), the posi-
tive control replaced the sample with the positive drug, and the
blank group replaced the substrate with PBS, and each group was
measured three times in parallel to average.
2. Experimental
2.1. Animals
The AB wild-type zebrafish was provided by Key Laboratory for
Drug Screening Technology of Shandong Academy of Sciences.
The zebrafish were cultured in an environment with a cycle of
14 h light and 10 h darkness, a pH of about 7.0, and a temperature
of about 28 ꢀC. The healthy zebrafish were mated in a tank one
day before the experiment, with a male to female ratio of 1:1. The
separator was taken the next day and the fertilised eggs were col-
lected 0.5 h later. The fertilised eggs were washed 3 times with
aquaculture water, then disinfected with methylene blue solution,
and transferred to clean culture water of about 28 ꢀC for light-con-
trol culture.
Wistar rat, weight 200–250 g, were obtained from Jinan Peng
Yue Experimental Animal Co. (License number: SCXK (Lu)
2014–0007), Ltd. The animals were housed under standard labora-
tory conditions and maintained on a standard pellet diet and
water ad libitum. All experiments involving living animals and their
care were performed in strict accordance with the National Care
and Use of Laboratory Animals by the National Animal Research
Authority (China) and guidelines of Animal Care and Use issued
by the University of Jinan Institutional Animal Care and Use
Committee. The experiments were approved by the Institutional
Animal Care and Use Committee of the School of Medicine and
Life Sciences, University of Jinan. All efforts were made to minim-
ise animal’s suffering and to reduce the number of animals used.
Monoamineoxidase inhibitory effect ð%Þ
¼ ½ðAC ꢁ ABÞ ꢁ ðAS ꢁ ASBÞꢂ=ðAC ꢁ ABÞ ꢃ 100%
where AC is the absorbance of control group; AB is the absorbance
of blank group; AS is the absorbance of sample group; ASB is the
absorbance of sample blank group.
2.4. In vitro antioxidant activity
2.2. In vitro cholinesterase inhibitory activity
The total antioxidant capacity of the 3-arylcoumarin compounds
was measured by the FRAP (the Ferric Reducing Ability of Plasma)
assay of Benzie et al.29 with slight modifications. This method is
based on the reduction of colourless Fe(III)-TPTZ(2,4,6-Tris(2-pyr-
idyl)-s-triazine) complex to coloured Fe(II)-TPTZ complex by the
compounds. FRAP working solution (300 mmol/L acetate buffer,
10 mmol/L TPTZ, 20 mmol/LFeCl3) was ready to use. A hundred
microlitres of the sample solution was added to 3 ml of FRAP
reagent and then incubated at 37 ꢀC for 15 min. The experiments
were repeated for three times. The absorbance was measured at
593 nm to clarify the changes. The standard curve was drawn with
FeSO4 as standard material, and the regression equation was
obtained. With 1.0 mmol/L FeSO4 as standard, the antioxidant
activity of the sample is expressed in millimoles of Fe2SO4
The anticholinesterase activity of the 3-arylcoumarin compounds
was determined by the method of Ellman et al.25 with slight mod-
ifications. AChE inhibitory activities were determined by AChE
from electric eel (Macklin). BuChE inhibitory activities were deter-
mined by BuChE from horse serum (Aladdin). To a 10 ml tube,
2.65 ml of phosphate buffer solution (0.1 M, pH ¼ 8.0), 100 mL of
5,5-dithiobis-2-nitrobenzoic acid (0.75 mM in 0.1 M phosphate buf-
fer solution, pH ¼ 8.0) were added sequentially, 50 mL AChE solu-
tion (0.2 U/mL in 0.1 M phosphate buffer solution, pH ¼ 8.0),
100 mL of different concentrations of the sample solution, shaken
well, and incubated at 37 ꢀC for 5 min. Then 100 mL of substrate
(1.5 mM in 0.1 M phosphate buffer solution, pH ¼ 8.0) were added,
shaken well, and incubated at 37 ꢀC for 20 min. After the reaction
was completed, 1 ml of sodium lauryl sulphate (4%, SDS in water)
was added. The absorbance at 412 nm of the samples was meas- required to achieve the same absorbance.
ured using a spectrophotometer, and the inhibition rate of cholin-
esterase and the IC50 value of each sample were calculated
according to the formula. Determination of the inhibitory activity
2.5. Zebrafish behavioural experiment
of BuChE is similar. The sample blank group replaced the sub-
strate with PBS, and the blank group replaced the sample solution
with a solvent. The sample solution was set to five concentration
gradients and the experiment was repeated three times.
Donepezil was used as a positive control.
The experimental sample group and the blank control group were
set in a 48-well plate, and 0.5 ml of aquaculture water and a
juvenile fish that developed to 72 hpf (hours post fertilisation)
were added to each well. Eight juvenile fish were set up for each
experimental group. Compounds 2, 20, 22 were set at four differ-
ent concentrations of 10 mg/ml, 50 mg/ml, 100 mg/ml, and 1000 mg/
ml. The 48-well plate was placed in the dark box of the zebrafish
behavioural analysis system. The fish were adapted to the environ-
ment for 10 min before the experiment. The zeblab software
(Viewpoint, Lyon, France) was used to collect the trajectories of
the juveniles in each group within 30 min, recorded every 5 min,
and the total parade distance and parade time were exported by
software. The average distance of each group of fish parades
ð
Þ
Cholinesterase inhibitory effect % ¼ ½A0 ꢁ ðA1 ꢁ A2Þꢂ=A0 ꢃ 100%
where A0 is the absorbance of blank group; A1 is the absorbance
of sample group; A2 is the absorbance of sample blank group.
2.3. In vitro monoamine oxidase inhibitory activity
The MAO inhibitory activity of the 3-arylcoumarin compounds was
determined by the method of Holt et al.26 with slight was calculated.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
653
Scheme 1. General synthetic route to 3-arylcoumarin, reagents, and conditions: (a) sulphur, p-toluenesulfonic acid, morpholine, 120ꢀC; (b) sodium hydroxide solution,
tetrabutylammonium bromide, 100 ꢀC; (c) acetic anhydride, triethylamine, 112ꢀC.
Table 1. Compounds 1–24.
2.6. Statistical analysis
Product
R1
R2
R3
R4
R5
R6
OH
H
OH
OH
H
R7
R8
Data were shown as mean SD Differences between individual
groups were analysed by using ANOVA followed by Dunnett’s
test. A difference with a p value of <.05 was considered to be
significant.
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
H
OH
H
OH
H
H
H
OH
H
OH
OH
H
OH
H
OH
H
OH
OH
OH
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
CH3
CH3
F
H
OH
OH
OCH3
H
3. Results and discussion
9
3.1. Chemistry
10
11
12
13
14
15
OCH3
OH
H
The synthetic methods and chemicals previously reported by our
research group were used30. The synthetic route was summarised
in Scheme 1. The substituted phenylacetic acid is synthesised by
substituting acetophenone, then the 3-arylcoumarin structure skel-
H
OCH3
H
OH
OH
H
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
eton is synthesised by Perkin reaction with substituted salicylalde- 16
17
hyde under the action of triethylamine and acetic anhydride, and
the target compound is obtained by acid hydrolysis (Table 1).
Details on the chemical and spectroscopic characterisations of
compounds were described in the references.
18
19
20
21
22
23
24
F
Br
Br
OH
H
OH
OH
CH3
H
3.2. Biological evaluation
3.2.1. In vitro cholinesterase inhibitory activity
3-arylcoumarin, and 3-arylcoumarin having a R5, R6-dihydroxy
group all showed high AChE inhibitory activity. However, there is
no significant tendency for the inhibitory activity of BuChE.
AChE is associated with neurons and axons, and is primarily
responsible for ACh hydrolysis and termination. Studies have
shown that long-term AChE inhibitor treatment can prevent the
progression of cognitive dysfunction in patients with AD31–33. In
the AD brain, BuChE may play a beneficial role by restoring cho-
linergic activity and/or by restoring AChE/BuChE activity ratio34.
The AChE/BuChE inhibitory activity of all compounds was deter-
mined by the method of Ellman25, and donepezil was used as a
reference compound in this assay. All the compounds were tested
AChE/BuChE inhibitory activity in vitro, as shown in Table 2, most
compounds presented inhibitory activity for AChE/BuChE. Most of
the compounds exhibited moderate AChE inhibitory activity.
Notably, compound 22 (IC50¼3.04 0.32 mM) had relatively strong
activity, which displayed weaker capacity than donepezil, and
compound 20 exhibited selective AChE inhibitory activity. Only
compound 7 (IC50¼2.76 0.57 mM) had stronger BuChE activity
than donepezil, and compounds 2, 5, 10 exhibited selective
BuChE inhibitory activity. The results showed that 3-arylcoumarins
with a R5, R6-dihydroxy group has a significantly stronger inhibi-
3.2.2. In vitro monoamine oxidase inhibitory activity
MAO plays an important role in neurotransmitter inactivation, and
MAO dysfunction is considered to be the cause of many mental
and neurological diseases. MAO plays an important role in AD path-
ology, and the progress of AD has a close correlation with MAO
activity35,36. All the synthesised compounds were evaluated for their
MAO inhibitory activity in the way of Holt26 by references. The
selective MAO-B inhibitor rasagiline was used as a reference com-
pound in this assay. The MAO used in the experiment was self-
made. The protein content standard curve was drawn according to
the absorbance of the protein standards at different concentrations
at 595 nm, and then the crude enzyme protein content was
obtained by a regression equation according to the absorbance of
the crude enzyme. The crude enzyme obtained was 0.157mg per
mg of protein. Nearly half of the compounds showed MAO-B inhibi-
tory activity than the R6- or R5-position mono-substituted tory activity. Among them, Compound 22 had the strongest

654
J. YANG ET AL.
Table 2. Biological evaluation in vitro.
IC50 value (lM)
FRAP value(mmol/g)
Antioxidant activity
Product
AChE inhibitory activity
BuChE inhibitory activity
MAO-B inhibitory activity
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
78.49 2.84
>100
17.97 1.13
105.42 2.92
>100
44.82 2.35
13.06 0.03
16.15 2.18
34.71 1.87
14.39 1.94
21.17 0.65
2.76 0.57
18.86 0.43
30.14 3.06
10.57 0.85
75.60 4.01
24.81 3.81
70.70 5.75
46.35 6.78
26.53 0.98
21.82 2.54
13.15 0.35
29.27 4.41
25.76 3.61
>100
>700
>700
73.45 11.97
>700
>700
>700
204.61 6.92
65.75 4.61
>700
>700
>700
63.36 4.04
>700
503.05 12.56
562.06 7.13
>700
>700
>700
231.62 17.19
>700
0.51 0.09
0.16 0.05
15.43 0.19
7.73 0.12
0.42 0.01
0.66 0.08
2.29 0.02
10.51 0.23
0.67 0.04
1.47 0.04
0.41 0.08
11.75 0.12
0.61 0.11
2.98 0.22
10.40 0.48
0.35 0.05
13.16 0.68
0.38 0.07
12.75 0.65
0.42 0.06
14.64 0.53
41.42 0.35
12.84 0.13
8.41 0.11
68.25 0.72
16.39 0.19
13.13 1.35
100.59 2.02
>100
40.37 2.09
8.45 0.16
42.44 3.16
84.57 1.67
13.31 2.26
82.81 1.43
12.58 1.28
74.29 3.72
27.12 0.66
19.21 0.68
8.75 0.63
20.25 1.60
8.37 0.68
9.92 0.54
70.92 4.22
>700
27.03 0.50
>700
3.04
0.32
13.74 0.32
30.90 1.27
330.04 6.49
Donepezil
Rasagiline
Ascorbic Acid
0.021
0.001
4.10
0.18
0.125 0.005
3.79 0.08
Each value represents the mean SD (n ¼ 3).
ꢄ
Figure 1. Effect of compounds 2, 20, 22 on the average total distance of zebrafish juveniles. (Compared with the control group, p < .05 had a significant difference,
#
p < .01 had a very significant difference; compared with the model group, p < .05 had a significant difference, ##p < .01 had a very significant difference).
ꢄꢄ
inhibitory activity (IC50¼27.03 0.50mM), which was weaker than from 10.40 0.48 to 41.42 0.35 mmol/g, which were 3–11 folds
the positive control drug rasagiline (IC50¼0.125 0.005mM). The of the value of vitamin
C
(FRAP value ¼ 3.79 0.08mmol/g).
experimental results show that the 3-arylcoumarin compounds with Compound 22 (FRAP value ¼ 41.42 0.35 mmol/g) has the strongest
R5, R6-dihydroxy group have better MAO inhibitory activity, indicat- antioxidant capacity. The antioxidant capacity of the compound is
ing that R5, R6-dihydroxyl is very important for inhibiting MAO.
related to the position and number of hydroxyl substitution, and
the R5, R6-dihydroxy substituted compound has a strong antioxi-
dant capacity.
3.2.3. In vitro antioxidant activity
Oxidative damage is involved in the pathogenesis of neuronal
degeneration in AD, which may represent a potential therapeutic 3.2.4. Zebrafish behavioural experiment
target for slowing the progression of AD or possibly preventing the To investigate the toxicity of compounds to zebrafish, the death
onset of AD37. Oxidative stress is a key feature in determining AD. and deformity of zebrafish were observed. Treatment in the blank
All the synthesised compounds were evaluated for their antioxidant group, 10, 50, 100, 1000 mg/ml group for three days did not cause
activities by means of the Ferric Reducing Ability of Plasma. Vitamin malformation and death of zebrafish. Healthy zebrafish were AlCl3
C was used as a reference in this assay. The standard curve was inducted after 72 h of fertilisation to construct a model of zebra-
drawn with FeSO4 as standard material, and the antioxidant fish Alzheimer’s disease. Compound 2 is a selective BuChE inhibi-
capacity of the sample is expressed as FRAP value. As shown in tor and Compound 20 is a selective AChE inhibitor that did not
Table 2, most of the compounds exhibited moderate to excellent show significant effects in zebrafish behavioural experiments.
antioxidant activities, even better than vitamin C. Especially, the Compound 22 is not only a dual ChE inhibitor but also a MAO-B
FRAP values of compounds 3, 8, 12, 15, 17, 19, 21, 22, 23 were inhibitor, which exhibits certain effects in zebrafish behavioural

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
655
Figure 2. Statistical graph of the total trajectory of zebrafish juveniles exposed to different concentrations of compound 22 for 72 hpf.
experiments. In the range of 10–100 mg/ml, the total distance of
zebrafish movement increases with the concentration of the com-
pound increases. However, the high doses of compounds might
cut down the movement distance of zebrafish (Figures 1 and 2).
Funding
The authors are grateful to support from the Science and
Technology Research Programme of Shandong Academy of
Medical Sciences (2017–16), Shandong Provincial Natural Science
Foundation [ZR2018LH021] and the Innovation Project of
Shandong Academy of Medical Sciences.
4. Conclusions
We continue to study the pharmacological activities of 3-arylcou-
marins synthesised by our research group. All the synthesised
compounds were evaluated for their antioxidant activities in the
way of the Ferric Reducing Ability of Plasma. Most compounds
demonstrated moderate to high activity, among which compound
22 with several hydroxyl groups showed an excellent activity in
this aspect. According to in vitro ChE inhibitory and MAO inhibi-
tory test results, we selected selective AChE inhibitory compound
20, selective BuChE inhibitory compound 2, and dual ChE and
MAO inhibitory compound 22 to study their anti-AD activity in
vivo. Notably, compound 22 showed a certain effect in vivo
experiments. Multi-target anti-AD compounds can modulate mul-
tiple signalling pathways or targets associated with AD, potentially
producing significant clinical effects.
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
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Ethical statement
All experiments involving living animals and their care were per-
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Disclosure statement
No potential conflict of interest was reported by the authors.

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