Synthesis and biological evaluation of 4-arylcoumarins as potential anti-Alzheimer's disease agents

Article abstract of DOI:10.1016/j.bmcl.2019.126900

Alzheimer's disease (AD) is a progressive neurological degenerative disease that has complex pathogenesis. A variety of studies in humans indicate that several enzymes inhibitors can be useful in the treatment of AD, including acetylcholinesterase (AchE), butyrylcholinesterase (BuChE) and monoamine oxidase (MAO). Various substituted 4-arylcoumarin derivatives were synthesised, and their activity in vitro were investigated, including AChE/BuChE inhibitory activity, MAO inhibitory activity, and antioxidant activity. Most of the compounds were found to exhibit high inhibitory activity, and individual compounds have extremely excellent activities. Therefore 4-arylcoumarins provides an idea for drugs design for the development of therapeutic or preventive agents for AD.

Full text of DOI:10.1016/j.bmcl.2019.126900

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Synthesis and biological evaluation of 4-arylcoumarins as potential anti-Alz-
heimer’s disease agents
Yinling Yun, Jie Yang, Yuhang Miao, Xiaojing Wang, Jie Sun
PII:
S0960-894X(19)30878-9
DOI:
https://doi.org/10.1016/j.bmcl.2019.126900
BMCL 126900
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Bioorganic & Medicinal Chemistry Letters
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25 November 2019
9 December 2019
Please cite this article as: Yun, Y., Yang, J., Miao, Y., Wang, X., Sun, J., Synthesis and biological evaluation of 4-
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https://doi.org/10.1016/j.bmcl.2019.126900
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Synthesis and biological evaluation of 4-arylcoumarins as
potential anti-Alzheimer’s disease agents
Yinling Yun a,b,c,d†, Jie Yang a,b,c,d, Yuhang Miao a,b,c,d, Xiaojing Wang a,b,c,d* and Jie Sun a,b,c,d*
a School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical
Sciences, Jinan, Shandong, China
b Institute of MateriaMedica, Shandong Academy of Medical Sciences, Jinan, Shandong, China
c Key Laboratory for Biotech-Drugs Ministry of Health, Jinan, Shandong, China
d Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan, Shandong, China
ABSTRACT：
Alzheimer's disease (AD) is a progressive neurological degenerative disease that
has complex pathogenesis. A variety of studies in humans indicate that several
enzymes inhibitors can be useful in the treatment of AD, including
acetylcholinesterase (AchE), butyrylcholinesterase (BuChE) and monoamine oxidase
(MAO). Various substituted 4-arylcoumarin derivatives were synthesised, and their
activity in vitro were investigated, including AChE/BuChE inhibitory activity, MAO
inhibitory activity, and antioxidant activity. Most of the compounds were found to
exhibit high inhibitory activity, and individual compounds have extremely excellent
activities. Therefore 4-arylcoumarins provides an idea for drugs design for the
development of therapeutic or preventive agents for AD.
Keywords: 4-Arylcoumarins; Alzheimer's disease; Cholinesterase inhibitors;
Monoamine oxidase; Antioxidant activity
One of the most feared and devastating aspects of aging is the deterioration of
[1]
memory and other psychological processes that occur frequently with age .
Alzheimer's disease (AD) has developed into one of the major diseases that jeopardize
the health of elder people in modern society. AD is a progressive neurological
degenerative disease that influences up to 5% of people over the age of 65, and this
[2]
proportion increases to 20% in people over 80 years of age . The most common early
symptom is that it is difficult to remember what happened recently. Typical symptoms
of AD patients are progressive memory impairment and damage of cognitive function,
and often accompanied by behavioral disorders such as aggression, depression and
[3]
paralysis . AD gradually weakens cognition, emotions, and physical ability, while at
the same time disrupting the lives of family caregivers who become their “second
1

[4]
victims” .
A variety of studies in humans indicate that the dysfunction of acetylcholine
Ach) neurons in the brain has a major impact on cognitive decline in those with
advanced age and AD patients. The premise has become the basis for most of AD’s
(
[5]
treatment strategies and drug development methods to date . With regard to
cholinergic hypothesis”, one of the rational and effective methods for treating AD
“
symptoms is increasing the amount of Ach by inhibiting acetylcholinesterase (AchE),
which causes Ach to degrade in the presynaptic region. AChE inhibitors are currently
recognized drugs with significant therapeutic effects in the world. Based on these
studies, several AChE inhibitors containing different chemical backbones were
[6]
[7]
[8]
[9]
synthesized, there are mainly tacrine , donepezil , rivastigmine , galantamine
and so on. It is used clinically to prevent and treat the development of early AD.
While the AChE activity of AD patients decreases progressively,
[10]
butyrylcholinesterase (BuChE) activity shows a certain increase . It has been
reported that the differences in enzyme kinetics between AChE and BuChE result in a
change in the efficiency of ACh hydrolysis depending on substrate concentration, and
[11]
BuChE plays a key role in Ach hydrolysis . BuChE inhibition and AChE inhibition
[12]
are viable therapeutic strategies for cognitive dysfunction in AD . In fact, in the
damaged brain, it is critical to preserve the proportion of AChE/BuChE activity for
successful treatment of AD. Monoamine oxidase (MAO) inhibitors were first used as
antidepressants and have long been used. It now seems that some MAO inhibitors are
useful in the treatment of several neurodegenerative diseases, including Parkinson's
[
13]
disease and AD .
4
-Arylcoumarin refers to a class of compounds having a coumarin skeleton and
[14]
having an aryl structure at the 4-position . Studies have shown that 4-arylcoumarin
[15]
[16]
compounds are rich in biological activity, such as anti-diabetic , anti-oxidant ,
[17]
[18]
[19]
[20]
anti-tumour , anti-bacterial , inhibition of extracellular protein , anti-fungal ,
[21]
[22]
[23]
anti-inflammatory , anti-viral , anti-plasmodium and others . The etiology of
AD is complex, and there are currently five drugs used to treat AD cognitive
problems. Treatment with a single target is difficult for AD. Multi-target drugs may
be an important direction for future AD drug development. Coumarins are a large
class of natural and synthetic sources of compounds with a variety of pharmacological
[24]
[25]
activities, including MAO inhibition . Edmondson and co-workers
have
determined that coumarin derivatives competitively inhibit MAO-B. Bernard and
co-workers[26] have reported that 17 coumarins and 2 chromone derivatives having
2

known inhibitory activities against MAO-A and MAO-B can also be used as AChE
inhibitors. These findings encourage us to further explore the potential of
4
-arylcoumarins and its derivatives to be candidates for the treatment of AD. The
experiments we have performed provide an idea for the development of drug designs
for therapeutic or prophylactic agents for AD.
Accordingly, in this work we described the biological evaluation of
4
-arylcoumarins (synthesised by our research group) as selective ChE/MAO-B
inhibitors. The pharmacological profiles of these new compounds including AChE
and BuChE inhibition, MAO-B inhibition, the kinetics of enzyme inhibition, and
antioxidant.
The synthetic methods and chemicals previously reported by our research group
[27]
were used . Details on the synthetic routes (Scheme 1 and Scheme 2), chemical (Table
) and spectroscopic characterisations of compounds were described in the references.
1
R2
R3
R7
R1
COOH
R7
CHO
R6
R5
b
R6
a

R1
R3
OH
R1
R3
R2
R4
R5
O
O
R2
a-2e
R4
1
a-1e
3
3a-3d
2
1
2
4
6
7
5
1
a:R =R =H,R =OCH
3a:R =R =R =H,R =OH
1-12
3
1
2
3
4
6
5
7
1
1
1
1
b:R =OCH ,R =OH,R =H
3b:R =R =OH,R =R =H
3
1
2
3
4
6
5
7
c:R =R =OH,R =H
3c:R =R =H,R =R =OH
1
2
3
4
6
5
7
d:R =R =OCH ,R =H
3d:R =R =OH,R =R =H
3
1
3
2
e:R =R =H.R =OH
Scheme 1. General synthetic route to 4-aryl-3,4-dihydrocoumarin, and conditions: (a) malonic
acid, piperidine, pyridine, 90℃; (b) nitrobenzene or acetic acid, montmorillonite K-10, 100°C.
3

COOH
COOCH CH
COOCH2CH3
Br
2
3
Br
CHO
a
c
d
R1
R3
R1
R3
R1
R3
R1
R3
R2
R2
R2
R2
1
f-1h
2f-2h
4f-4h
5f-5h
1
3
2
1
1
1
f:R =R =H,R =OCH
3
1
2
3
COOH
g:R =H,R =R =OCH
3
1
2
3
OCH3
h:R =R =R =OCH
OH
3
R1
R3
R1
R3
R7
R6
e
g
f
R7
R7

R1
R3
R6
R5
R6
R5
R5
OH
R2
f-6h
R4
6
O
O
O
O
3
a-3c
R4
R4
4
5
6
7
3
a:R =R =OH,R =R =H
4
6
5
7
3
b:R =R =H,R =R =OH
1
3-15
16-20
4
6
7
5
3
c:R =R =R =H,R =OH
Scheme 2. General synthetic route to 4-aryl coumarin, and conditions: (a) malonic acid,
piperidine, pyridine, 90℃; (c) EtOH, SOCl , reflux, 2h; (d) Br , CH Cl , 0℃, 20 min; (e) KOH,
2
2
2
2
EtOH, reflux, 6h; (f) nitrobenzene, montmorillonite K-10, 100℃; (g) I , Al, acetonitrile, reflux,
2
5
h.
Table 1. Compounds 1-20.
Product
R1
R2
R3
H
R4
H
R5
R6
H
R7
H
1
2
3
4
5
6
7
8
9
H
OCH3
OH
OH
OH
H
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OH
H
H
OH
H
H
H
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
H
H
OCH3
OH
H
H
H
H
OH
H
H
H
OCH3
OCH3
OH
H
H
OH
OH
OH
OH
H
OCH3
OCH3
H
H
H
H
H
H
H
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
OH
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
OH
OH
OH
OH
H
OH
OH
H
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OH
H
OH
OH
OH
OH
OH
H
OCH3
H
H
H
H
OH
H
H
H
H
H
OH
OH
H
H
4

1
1
2
8
9
0
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
OH
H
H
OH
In the human brain, AChE is mainly located in cholinergic axons and neuronal
cells, while BuChE is mainly distributed in glial cells. These two enzymes are also
present in neuritic plaques and tangles in AD patients. The proportion of ChEs present
[28]
in the human brain changes throughout the AD process . The inhibitory activities of
the all compounds on AChE (from electric eel) and BuChE (from equine serum) were
tested using the method of Ellman and co-workers[29] with donepezil as reference
standard. As shown in Table 2, less than half of the compounds presented inhibitory
activity for AChE/BuChE. Only nine compounds (6, 13, 14, 15, 16, 17, 18, 19 and 20)
exhibited moderate to excellent AChE inhibitory activity. Notably, compound 19
(
IC =0.025±0.01 µM) had relatively strong activity, which showed a stronger ability
50
than donepezil (IC =0.029±0.01 µM), and the compound exhibited selective AChE
5
0
inhibitory activity. Compound 13 (IC =0.052±0.02 µM) displayed weaker capacity
5
0
than donepezil but was of the same order of magnitude. The results showed that
1
2
3
4
-arylcoumarins with a R , R , R - trihydroxy group has a significantly stronger
2
2
3
inhibitory activity than the R - monohydroxy or R , R -dihydroxy 3-arylcoumarin.
4
5
And R , R -dihydroxyl is also crucial for inhibiting AChE. However, there is no
significant tendency for the inhibitory activity of BuChE.
Table 2. Biological evaluation in vitro.
.
-1
IC value (µM)
FRAP value(mmol g )
5
0
Product
AChE inhibitory activity
BuChE inhibitory activity
MAO-B inhibitory activity
antioxidant activity
1
2
3
4
5
6
7
8
9
>50
>300
>300
0.896±0.02
9.376±0.07
0.922±0.02
9.168±0.16
10.089±0.01
9.603±0.01
1.061±0.05
1.499±0.02
9.014±0.05
2.259±0.12
2.846±0.10
4.759±0.18
>50
>300
82.507±24.48
>300
>50
>300
21.629±0.71
10.938±0.47
0.283±0.04
>50
148.655±16.21
134.045±9.14
72.533±4.14
>300
2.183±0.03
0.257±0.15
0.430±0.11
83.700±3.42
140.510±22.16
55.943±10.75
19.383±1.90
75.427±3.84
26.573±7.18
>50
>300
>50
>300
1
1
1
0
1
2
>50
>300
>50
>300
>50
>300
5

1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
0
0.052±0.02
0.299±0.09
7.915±0.72
8.857±0.61
9.454±0.64
7.048±0.29
0.025±0.01
7.002±0.30
0.029±0.01
>300
>300
9.895±0.07
9.648±0.03
1.374±0.02
9.710±0.08
9.887±0.02
10.503±0.01
10.101±0.04
10.458±0.01
>300
>300
>300
104.053±4.12
>300
>300
>300
293.377±23.29
189.080±7.92
1.987±0.10
22.173±7.45
188.414±17.68
106.183±2.05
207.984±12.59
2.600±1.49
Donepezil
Rasagiline
0.230±0.05
Ascorbic Acid
9.868±0.01
The compound 19 was selected for kinetic measurements because it showed a
good inhibitory activity against AChE. The graphical analysis of the steady-state
inhibition data of 19 against AChE is shown in Figure 1. The results indicate that both
slopes and intercepts increased with increasing inhibitor concentration. This pattern
indicates that compound 19 was a mixed-type inhibitor.
1
1
1
4000
2000
0000
8
6
4
2
000
000
000
000
0
5
5
0
0
0 nM
nM
.5 nM
nM
-30
-20
-10
0
10
20
30
-
-
-
-
2000
4000
6000
8000
-
1
-1
[
S] nM
Figure 1. Kinetic study of the mechanism of AChE inhibition by compound 19.
Three-dimensional (3D) protein structure of AChE (PDB id: 6O4W) was
accessed from protein data bank, and for the compounds the 3D-coordinates were
generated using ChemDraw software. Molecular docking of the compounds into
hAChE protein was performed employing CDOCKER semi-flexible docking
algorithm incorporated in the Discovery Studio 2019. Based on the experimental
results, we performed docking calculations for the compound 19 (Figure 2). In the
6

binding pocket, amino acid residues Phe295 and Arg296 (amino acid backbone,
non-residue) and a water molecule (797) form hydrogen bonds with the parent
carbonyl. In addition, the water molecule 875 forms a hydrogen bond with the ligand
B ring phenolic hydroxyl group, and the water molecule 788 forms a hydrogen bond
with the ligand A ring phenolic hydroxyl group. Trp286 and Tyr124 and Ligand A
ring form a Pi-Pi stack, while Phe338 and Tyr337 have a Pi-donor hydrogen bond
with the ligand.
Figure 2. Docking of compound 19 to hAChE protein
It has been reported that MAO is also an important target for the treatment of
AD. MAO-B accelerates the oxidative deamination of neurotransmitters, resulting in
[30]
enhanced production of free radicals thus causing oxidative stress . As a result,
selective MAO-B inhibitors are useful in the treatment of AD. The inhibitory
activities were evaluated of all compounds against the MAO-B in the way of Holt and
co-workers[31] by references. The selective MAO-B inhibitor rasagiline was used as a
reference compound in this assay. The MAO used in the experiment was selfmade.
The protein content standard curve was drawn according to the absorbance of the
protein standards at different concentrations at 595nm, 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 3.78mg per mL of protein. It can
be seen from Table 2 that most of the compounds showed MAO-B inhibitory activity.
Notably, compound 5 (IC =0.257±0.15 µM) had strongest activity, which displayed
5
0
weaker capacity than rasagiline (IC =0.230±0.05 µM). The compound 6
5
0
(
IC =0.430±0.11 µM) was slightly weaker than the positive control drug rasagiline.
50
The experimental results show that the 4-aryl-3,4-dihydrocoumarin compounds with
4
5
4
R , R -dihydroxy group have better MAO inhibitory activity, indicating that R ,
7

5
R -dihydroxyl is very important for inhibiting MAO.
Compound 5 was used to investigate the mode of MAO-B inhibition because it
showed strongest inhibitory activity against MAO-B. The catalytic rates were
measured at eight different 4-(trifluoromethyl) benzylamine solution concentrations,
and each plot was constructed at four different concentrations of 5. The graphical
analysis of the steady-state inhibition data of 5 against MAO-B is shown in Figure 3.
The results show that the plots for different concentrations of 5 were linear and
intersected at the x-axis. This pattern indicated that 5 is a noncompetitive MAO-B
inhibitor.
1
1
200
000
8
6
4
2
00
00
00
00
0
5
0
5
5
00
0
-30
-20
-10
0
10
20
30
-
-
-
200
400
600
-
1
-1
[
S] µM
Figure 3. Kinetic study of the mechanism of MAO inhibition by compound 5.
3
D protein structure of MAO-B (PDB id: 4A79) was accessed from protein data
bank. Based on the experimental results, we performed docking calculations for the
compound 5 (Figure 4). The Tyr435 in the binding pocket and a water molecule
(2183) form a hydrogen bond with the carbonyl group of the ligand core. Another
water molecule in the pocket (2348) also forms a hydrogen bond with the hydroxyl
group at the 8-position of the ligand. In addition, Tyr398 and the ligand A ring,
Tyr326 and ligand C ring became Pi-Pi stacking. Leu171 and Ile199 form a
hydrophobic interaction with the ligand, Cys172 and the ligand C ring form a
Pi-sulfur interaction.
8

Figure 4. Docking of compound 5 to MAO-B protein
The antioxidant activity of the all compounds were tested using the method of
Benzie et al.[32] with vitamin C as reference standard. The standard curve was drawn
with FeSO as standard material, and the antioxidant capacity of the sample is
4
expressed as FRAP value. It can be seen from Table 2 that more than half of the
compounds presented good antioxidant activity. Notably, six compounds (5, 13, 17,
1
8, 19 and 20) showed a stronger ability than vitamin C (FRAP value=9.868±0.01
.
-1
. -1
mmol g ). Compound 18 (FRAP value=10.503±0.01 mmol g ) has the strongest
antioxidant capacity. The antioxidant capacity of compound is related to the position
and number of hydroxyl substitution, and the experimental results show that the
4
5
4
-arylcoumarins compounds with R , R -dihydroxy group have better antioxidant
activity.
In conclusion, we study the pharmacological activities against AD of
-arylcoumarins synthesised by our research group, including cholinesterase
4
inhibitory activity, monoamine oxidase inhibitory activity and antioxidant activity.
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, and the compound 18 with several hydroxyl groups have
excellent antioxidant activity. According to ChE inhibitory and MAO inhibitory test
results, most of the compounds showed moderate to excellent inhibitory activity. The
selective AChE inhibitory compound 19 (IC =0.025±0.01 µM) showed a stronger
5
0
ability than positive drug. The same is true for the MAO inhibitory compound 5
IC =0.257±0.15 µM). From the perspective of structure, the maximum inhibitory
(
5
0
4
5
activity was displayed by compounds with R , R -dihydroxy group, indicating that
4
5
4
-arylcoumarins with R , R -dihydroxyl is very important for inhibiting AChE and
9

MAO. Thus, we selected compound 19 and 5 to study kinetic experiment of enzyme
inhibition. Our study illustrates the importance of the number and position of
hydroxyl groups on the aromatic ring, and among the compounds tested, two hydroxyl
4
5
substituents in R , R to be the most functional for the AChE and MAO inhibition. In
summary, the experiment we did about 4-arylcoumarins provides an idea for drugs
design for the development of therapeutic or preventive agents for AD.
Ethical statement
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 minimise animal’s suffering and to reduce the number of animals used.
Acknowledgments
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.
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-aryl-3,4-dihydrocoumarins. Biochimie, 2014. 107 Pt B: p. 203-10.
Chen, H., et al., Design, synthesis, and anti-tumor activities of novel
triphenylethylene-coumarin hybrids, and their interactions with Ct-DNA. Bioorg Med Chem
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The AChE/BuChE inhibitory activity of the 4-arylcoumarin compounds was
determined by the method of Ellman and co-workers with slight modifications. To a
9
5
6-well plate, 120 µL of phosphate buffer solution (0.1 M, pH=8.0), 20 µL of
,5-dithiobis-2-nitrobenzoic acid (3.3 mM in 0.1 M phosphate buffer solution,
.
-1
pH=8.0) were added sequentially, 20 µL AChE solution (0.2 U mL in 0.1 M
phosphate buffer solution, pH=8.0), 20 µL of different concentrations of the sample
.
-1
solution (10.0, 100.0, 500.0, and 1000.0 µg mL ), shaken well, and incubated at 37℃
for 5min. Then 20 µL of substrate (5 mM in distilled water) were added, shaken well,
and incubated at 37 ℃ for 20 min. The absorbance at 412 nm of the samples was
measured using a microplate reader, and the inhibition rate of cholinesterase and the
IC value of each sample were calculated according to the formula. Determination of
5
0
the inhibitory activity of BuChE is similar. The sample blank group replaced the
substrate with distilled water, and the blank group replaced the sample solution with a
solvent. The sample solution was set to four concentration gradients and the
experiment was repeated three times. Donepezil was used as a positive control.
Cholinesterase inhibitory effect (%) = [A －( A －A )]/A ×100%
0
1
2
0
where A is the absorbance of blank group; A is the absorbance of sample group; A
0
1
2
is the absorbance of sample blank group.
The compound that showed better inhibitory activity against AChE/BuChE was
selected for kinetic measurements. To obtain the mechanism of action of compound,
reciprocal plots of [velocity]-1 versus [substrate]-1 were constructed at different
substrate solution concentrations by using Ellman and co-workers method. The
experiment were measured at eight different substrate solution concentrations (0.039,
0
.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 mM) and four different concentrations of
target compound (0, 0.5, 5 and 50 nM). To a 96-well plate, phosphate buffer solution,
,5-dithiobis-2-nitrobenzoic acid, AChE solution and target compound were added
5
sequentially, shaken well, and incubated at 37 ℃ for 5 min. Then substrate were
added, shaken well, and incubated at 37 ℃ for 20 min. The absorbance at 412 nm of
1
2

the samples was measured using a microplate reader, and the experiment was repeated
three times. Slopes of these reciprocal plots were then plotted against the
concentration of compound in a weighted analysis and Ki was determined as the
intercept on the negative x-axis.
The MAO inhibitory activity of the 4-arylcoumarin compounds was determined
by the method of Holt and co-workers with slight modifications. The crude enzyme
was extracted from the liver of Wistar rats. 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 laboratory conditions
and maintained on a standard pellet diet and water ad libitum. After extracting the
crude enzyme, the crude enzyme protein content was determined by the
Bradford/method using a Bradford Protein Assay Kit (Beyotime). 40 µL of enzyme
.
-1
solution and 40 µL of sample solution (125.0, 250.0, 500.0, and 1000.0 µg mL ) were
added to a 96-well plate. The solution was then incubated at 37 ℃ for 20 min. 120 µL
of the 4-(trifluoromethyl) benzylamine solution and 40 µL of the chromogenic agent
.
-1
(
1 mM vanillic acid, 0.5 mM 4-aminoantipyrine, 4 U mL horseradish peroxidase, 0.2
M pH=7.6 phosphate buffer solution constant volume) were added subsequently, and
incubated at 37 ℃ for 90 min. The absorbance was measured at 490 nm using a
microplate reader, and the inhibition rate of MAO and the IC value of each sample
5
0
were calculated according to the formula. The control group replaced the sample
solution with a solvent, the positive 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.
Monoamineoxidase inhibitory effect (%)= [(A －A )－(A －A )]/(A －A )×100%
1
2
3
4
1
2
where A is the absorbance of blank group; A is the absorbance of blank background
1
2
group; A is the absorbance of sample group; A is the absorbance of sample
3
4
background group.
Data processing method of the kinetic characterization of MAO inhibition is
similar to the kinetic measurements of AChE. The experiment were measured at eight
different substrate solution concentrations (0.039, 0.078, 0.156, 0.312, 0.625, 1.25,
2
.5, 5 mM) and four different concentrations of target compound (0, 5, 50 and 500
µM). To a 96-well plate, enzyme solution and target compound were added
sequentially. The solution was then incubated at 37 ℃ for 20 min. The
4
-(trifluoromethyl) benzylamine solution and the chromogenic agent were added, and
incubated at 37 ℃ for 90 min. The absorbance was measured at 490 nm using a
1
3

microplate reader, and the experiment was repeated three times.
The total antioxidant capacity of the 4-arylcoumarin compounds was measured
by the FRAP (the Ferric Reducing Ability of Plasma) assay of Benzie and
co-workers[32] with slight modifications. This method is based on the reduction of
colourless Fe(III)-TPTZ(2,4,6-Tris(2-pyridyl)-s-triazine) complex to coloured
Fe(II)-TPTZ complex by the compounds. FRAP working solution (300 mM acetate
buffer, 10 mM TPTZ, 20 mM FeCl ) was ready to use. 180 µL of FRAP reagent and 5
3
µL of sample solution were added to a 96-well plate, and then incubated at 37 ℃ for 5
min. The experiments set up 3 groups of parallel groups. The absorbance was
measured at 593 nm using a microplate reader. The standard curve was drawn with
FeSO as standard material, and the regression equation was obtained. With 1.0 mM
4
FeSO as standard, the antioxidant activity of the sample is expressed in millimoles of
4
FeSO required to achieve the same absorbance.
4
Computer simulation molecular docking steps are as follows. The ligand is first
treated, and the method is Discovery Studio 2019 built-in prepare ligand method. The
molecule does not form tautomerism and isomers, the protonation pH is set to 7, and
the remaining parameters are default parameters. The energy optimization of the
formed ligand is carried out by Discovery Studio 2019 built-in minimization, and the
force field used is the MMF force field. After the above steps are completed, the
resulting ligand is treated for molecular docking. The protein required for docking
was treated by Discovery Studio 2019 built-in prepare protein method, and the main
purpose was to hydrogenate the protein. Considering the structure of the docking
ligand, we retain the water molecules in the protein binding pocket as well as the
endogenous ligand, and other parameters are set to default. Based on the active sites
included in the PDB database and the location of the ligands in the original eutectic
complex, the coordinates and radius of the active sites are determined. The specific
parameters are as follows: hAChE: X=89.214403, Y=85.422867, Z=-5.545143, r=11;
MAO-B: X=52.076244, Y=157.208276, Z=30.314829, r=11. Molecular docking of
the compounds into protein was performed employing CDOCKER semi-flexible
docking algorithm incorporated in the Discovery Studio 2019.
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4

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5