Synthesis and biological evaluation of 3-arylbenzofuranone derivatives as potential anti-Alzheimer’s disease agents

Article abstract of DOI:10.1080/14756366.2020.1740694

Multi-target drugs can better address the cascade of events involved in oxidative stress and the reduction in cholinergic transmission that occur in Alzheimer’s disease than cholinesterase inhibitors alone. We synthesised a series of 3-arylbenzofuranone derivatives and evaluated their antioxidant activity, cholinesterase inhibitory activity, and monoamine oxidase inhibitory activity. 3-Arylbenzofuranone compounds exhibit good antioxidant activity as well as selective acetylcholinesterase inhibitory activity. The IC₅₀ value of anti-acetylcholinesterase inhibition of Compound 20 (0.089 ± 0.01 μM) is similar to the positive drug donepezil (0.059 ± 0.003 μM). According to the experimental results, Compounds 7, 13 show a certain effect in the in?vitro evaluation performed and have the potential as drug candidates for the treatment of Alzheimer’s disease.

Full text of DOI:10.1080/14756366.2020.1740694

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-
arylbenzofuranone derivatives as potential anti-
Alzheimer’s disease agents
Jie Yang, Yinling Yun, Yuhang Miao, Jie Sun & Xiaojing Wang
To cite this article: Jie Yang, Yinling Yun, Yuhang Miao, Jie Sun & Xiaojing Wang (2020)
Synthesis and biological evaluation of 3-arylbenzofuranone derivatives as potential anti-Alzheimer’s
disease agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 805-814, DOI:
10.1080/14756366.2020.1740694
To link to this article: https://doi.org/10.1080/14756366.2020.1740694
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Published online: 18 Mar 2020.
Submit your article to this journal
View related articles
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
2020, VOL. 35, NO. 1, 805–814
https://doi.org/10.1080/14756366.2020.1740694
RESEARCH PAPER
Synthesis and biological evaluation of 3-arylbenzofuranone derivatives as
potential anti-Alzheimer’s disease agents
a,b,c,d
Jie Yanga,b,c,d , Yinling Yun
, Yuhang Miaoa,b,c,d, Jie Sunb,c,d
and
ꢀ
ꢀ
Xiaojing Wangb,c,d
b
aSchool of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Shandong, Jinan, China; Institute of
c
Materia Medica, Shandong First Medical University and Shandong Academy of Medical Sciences, Shandong, Jinan, China; Key Laboratory for
d
Biotech-Drugs Ministry of Health, Shandong, Jinan, China; Key Laboratory for Rare and Uncommon Diseases of Shandong Province, Shandong,
Jinan, China
ABSTRACT
ARTICLE HISTORY
Received 24 December 2019
Revised 14 February 2020
Accepted 2 March 2020
Multi-target drugs can better address the cascade of events involved in oxidative stress and the reduction in
cholinergic transmission that occur in Alzheimer’s disease than cholinesterase inhibitors alone. We synthesised
a series of 3-arylbenzofuranone derivatives and evaluated their antioxidant activity, cholinesterase inhibitory
activity, and monoamine oxidase inhibitory activity. 3-Arylbenzofuranone compounds exhibit good antioxidant
activity as well as selective acetylcholinesterase inhibitory activity. The IC50 value of anti-acetylcholinesterase
inhibition of Compound 20 (0.089 0.01 lM) is similar to the positive drug donepezil (0.059 0.003 lM).
According to the experimental results, Compounds 7, 13 show a certain effect in the in vitro evaluation per-
formed and have the potential as drug candidates for the treatment of Alzheimer’s disease.
KEYWORDS
3-Arylbenzofuranone deriva-
tives; Alzheimer’s disease;
cholinesterase inhibitors;
monoamine oxidase
inhibitors; antioxi-
dant activity
Introduction
AD drug. MAO has a major role in brain development and function,
and its inhibitors have found clinically as antidepressant and anti-
Parkinsonian drugs6. MAO regulates the concentration of exogen-
ous and endogenous amines (including neurotransmitters) in the
central nervous system and peripheral tissues, which exacerbates
subsequent oxidative stress and neurodegeneration.
Benzofuranone and its derivatives are important heterocyclic com-
pounds and are widely used in pesticides, dyes, foods, polymer proc-
essing, and other fields. Benzofuranone and its derivatives have rich
biological activities, including anticholinesterase8, antibacterial9, anti-
HIV10, anti-allergic and anti-inflammatory activity11, antioxidant, anti-
nociceptive activity12, and other activity, it can be used as a substrate
for b-lactamase13 and insulin amyloid fibrosis inhibitor14 and is also
potential antipsychotics15. The current study describes the prepar-
ation and in vitro activity of 3-arylbenzofuranone derivatives as ChE
inhibitors, MAO B inhibitors, and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavengers. The purpose of the research is to screen com-
pounds that inhibit ChE and MAO, which have the potential for the
treatment of AD and other neurodegenerative diseases.
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder
characterised by the destruction of nerve cells, the rapid deterior-
ation of memory and other important cognitive functions1. The
two major hallmarks of AD are intracellular neurofibrillary tangles
composed of hyperphosphorylated T protein2 and senile plaques
containing aggregated amyloid b-peptide (Ab)3. There are other
factors that cause synaptic dysfunction and neurodegeneration,
which leads to a decrease in neurotransmitter acetylcholine (ACh)
levels4, leading to memory and cognitive deficits5. Monoamine
oxidase (MAO) is one of the several enzymes that contribute to
the behavioural and psychological symptoms of dementia in AD6.
Accumulating evidence indicates a close contact between several
enzymes and AD. Cholinesterases (ChEs) and MAOs are closely
related to the disease symptomatology and progression.
ChEs, enzymes that terminate cholinergic neurotransmission in
the brain, act by catalysing the hydrolysis of ACh, so their inhibition
can be used to alleviate memory and cognitive deficits in AD. Two
types of ChEs, acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE), are known. The difference between the two types is
related to their respective preference for the substrate: the former
hydrolyses ACh more rapidly; the latter hydrolyses butyrylcholine
more rapidly. Experimental studies have found that the mainten-
ance of AChE/BuChE activity ratio in the healthy brain can improve
the symptoms of AD7. Inhibition of AChE can result in an increase
in BuChE activity, which causes hydrolysis of AChE in a novel man-
Experimental
Synthesis
Materials and methods
Melting points were determined using a Thiele tube and were
1
ner. Therefore, a double ChE inhibitor may be a more effective anti- uncorrected. The HNMR and 13CNMR spectra were recorded with
CONTACT Jie Sun
sunjie310@126.com; Xiaojing Wang
xiaojing6@gmail.com
Institute of Materia Medica, Shandong First Medical University and Shandong
Academy of Medical Sciences, Shandong, Jinan 250062, China.
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2020 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.

806
J. YANG ET AL.
a Bruker AM-600 spectrometer (Billercia, MA, USA) with TMS as J ¼ 11.3 Hz), 39.98 ꢃ 39.62 (m), 39.55 (s), 39.52 (s), 39.38 (s), 39.29
the internal standard. Chemical shifts were reported at room tem- (s), 39.17 (d, J ¼ 21.0 Hz). MS: m/z (%) [M þ Na]þ 279.0.
6-Methoxy-3–(40-methoxyphenyl)-benzofuranone (3). White
perature on a scale (ppm) with DMSO-d6 as the solvents and J val-
solid, yield 85.32%, m.p. 156–157 ꢁC. 1H NMR (600 MHz, DMSO-d6)
ues are given in Hertz. Mass spectra were obtained with an
d 7.10 (dd, J ¼ 11.4, 4.6 Hz, 3H), 6.96 ꢃ 6.92 (m, 3H), 6.75 (dd,
Agilent Trap VL LC/MS spectrometer (Santa Clara, CA, USA). The
absorbance was recorded by RZ-9618 Microplate Reader. Unless
otherwise noted, all solvents and reagents were commercially
available and used without further purification.
J ¼ 8.3, 2.3 Hz, 1H), 5.20 (s, 1H), 3.79 (s, 3H), 3.74 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) d 176.03 (s), 160.14 (s), 158.87 (s), 154.16 (s),
129.33 (s), 128.13 (s), 125.62 (s), 119.61 (s), 114.42 (s), 110.19 (s),
97.22 (s), 55.66 (s), 55.15 (s), 47.86 (s), 39.87 (d, J ¼ 21.0 Hz), 39.66
(s), 39.52 (s), 39.38 (s), 39.24 (s), 39.10 (s). MS: m/z (%) [M þ Na]þ
293.0, [M þ H]þ 271.0.
General method for synthesis of compounds 3a-3d
5-Methoxy-3–(40-methoxyphenyl)-benzofuranone (4). White
solid, yield 76.32%, m.p. 126–127 ꢁC. 1H NMR (600 MHz, DMSO-d6)
d 7.23 (d, J ¼ 8.8 Hz, 1H), 7.12 (d, J ¼ 8.7 Hz, 2H), 6.97 ꢃ 6.92 (m,
3H), 6.79 ꢃ 6.74 (m, 1H), 5.28 (s, 1H), 3.75 (s, 3H), 3.70 (s, 3H). 13C
NMR (151 MHz, DMSO-d6) d 175.92 (s), 158.90 (s), 156.37 (s),
146.97 (s), 129.46 (s), 129.13 (s), 127.74 (s), 114.47 (s), 114.22 (s),
111.11 (s), 110.70 (s), 55.68 (s), 55.15 (s), 48.95 (s), 39.94 (s), 39.73
(d, J ¼ 21.0 Hz), 39.52 (s), 39.39 (s), 39.38 (s), 39.24 (s), 39.10 (s). MS:
m/z (%) [M þ Na]þ 293.0, [M þ H]þ 271.0.
Taking the synthesis of 3, 4, 5-trimethoxy mandelic acid as an
example. Other mandelic acids were obtained using the same pro-
cedures. 3,4,5-Trimethoxybenzaldehyde 39.2 g (0.2 mol), TBAB 3.2 g
(10 mmol), and chloroform 240 ml were added to a 500 ml three-
necked flask equipped with a dropping funnel and a reflux con-
denser. The mixture was thoroughly stirred to completely dissolve,
and the temperature was raised to 40 ꢁC. A 50% NaOH solution
(40 g of NaOH dissolved in 40 g of water) was slowly added drop-
wise through a dropping funnel to maintain a temperature of
45–50 ꢁC. After the TLC detection reaction was completed, it was
allowed to stand for cooling and suction filtration. The filter cake
was washed with chloroform 40 ml ꢂ 3. The resulting solid mix-
ture was acidified with hydrochloric acid, extracted with ethyl
acetate, dried over anhydrous sodium sulphate, concentrated, and
recrystallisation from ethyl acetate/petroleum ether gave a white
solid (yield: 70.5%).
6,7-Dihydroxy-3–(40-methoxyphenyl)-benzofuranone (5). White
solid, yield 91.91%, m.p. 138–140 ꢁC. 1H NMR (600 MHz, DMSO-d6)
d 7.12 ꢃ 7.09 (m, 2H), 6.94 ꢃ 6.91 (m, 2H), 6.59 (d, J ¼ 8.0 Hz, 1H),
6.43 (dd, J ¼ 8.0, 1.1 Hz, 1H), 5.15 (s, 1H), 3.74 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) d 176.16 (s), 158.81 (s), 146.82 (s), 141.77 (s),
129.93 (s), 129.35 (s), 128.56 (s), 119.11 (s), 114.67 (s), 114.39 (s),
111.45 (s), 55.18 (s), 48.53 (s), 39.94 (s), 39.81 (s), 39.80 ꢃ 39.52 (m),
39.39 (s), 39.31 (d, J ¼ 21.0 Hz), 39.10 (s). MS: m/z (%) [M þ Na]þ
295.0, [M þ H]þ 273.0.
6-Hydroxy-3–(30,40-dimethoxyphenyl)-benzofuranone (6). White
solid, yield 60.47%, m.p. 171–172 ꢁC. 1H NMR (600 MHz, DMSO-d6)
d 9.88 (s, 1H), 7.00 (dd, J ¼ 8.2, 0.9 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H),
6.81 (d, J ¼ 2.0 Hz, 1H), 6.67 (d, J ¼ 2.2 Hz, 1H), 6.64 (dd, J ¼ 8.3,
2.0 Hz, 1H), 6.59 (dd, J ¼ 8.2, 2.2 Hz, 1H), 5.12 (s, 1H), 3.73 (s, 3H),
3.71 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 158.29 (s), 154.03 (s),
148.95 (s), 148.44 (s), 128.68 (s), 125.64 (s), 120.14 (s), 117.76 (s),
112.08 (d, J ¼ 15.6 Hz), 111.31 (s), 98.25 (s), 55.54 (d, J ¼ 3.3 Hz),
48.19 (s), 39.87 (d, J ¼ 20.9 Hz), 39.67 (s), 39.59 (d, J ¼ 21.0 Hz),
39.41 (s), 39.38 (s), 39.17 (d, J ¼ 21.0 Hz). MS: m/z (%)
[M þ Na]þ 308.9.
General method for the synthesis of 3-arylbenzofuranone 1–23
Taking the synthesis of 6-hydroxy-3–(30,40,50-trimethoxyphenyl)-
benzofuranone as an example. Other 3-arylbenzofuranone com-
pounds were obtained using the same procedures. 3,4,5-
Trimethoxymandelic acid 4.84 g (20 mmol), resorcin 2.64 g
(24 mmol), and boron trifluoride-diethyl ether 20 ml were added
to a 100 ml three-necked flask equipped with a reflux condenser
and a drying tube. The raw material was stirred well to completely
dissolve, and maintain the temperature at 30–35 ꢁC continuous
stirring. After the TLC detection reaction was completed, the reac-
tion was allowed to stand for cooling. The reaction solution was
poured into a beaker containing 100 ml of ice water and thor-
oughly stirred. After a large amount of white solid was precipi-
tated, it was allowed to stand, and suction filtered. The filter cake
was washed with saturated sodium bicarbonate solution, then
washed with distilled water until near neutral, dried to give a pale
pink solid, and recrystallised from methanol to yield white solid
(yield: 92.72%).
5-Hydroxy-6-methoxy-3–(40-methoxyphenyl)-benzofuranone (7).
White solid, yield 83.92%, m.p. 151–153 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 8.92 (s, 1H), 7.11 ꢃ 7.08 (m, 2H), 7.00 (s, 1H),
6.95 ꢃ 6.92 (m, 2H), 6.57 (d, J ¼ 0.7 Hz, 1H), 5.14 (s, 1H), 3.81 (s,
3H), 3.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 176.46 (s),
158.82 (s), 148.21 (s), 145.91 (s), 143.58 (s), 129.30 (s), 128.29 (s),
118.50 (s), 114.39 (s), 111.34 (s), 96.47 (s), 56.13 (s), 55.15 (s), 48.54
(s), 40.00 (d, J ¼ 16.4 Hz), 39.87 (d, J ¼ 21.0 Hz), 39.66 (s), 39.52 (s),
39.38 (s), 39.24 (s), 39.10 (s). MS: m/z (%) [M þ Na]þ 309.0.
6-Methoxy-7-hydroxy-3–(40-methoxyphenyl)-benzofuranone (8).
White solid, yield 86.36%, m.p. 138–139 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 9.44 (s, 1H), 7.11 (d, J ¼ 8.5 Hz, 2H), 6.93 (d, J ¼ 8.6 Hz,
2H), 6.77 (d, J ¼ 8.2 Hz, 1H), 6.57 (d, J ¼ 8.2 Hz, 1H), 5.20 (s, 1H),
3.80 (s, 3H), 3.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 175.87
(s), 158.82 (s), 148.87 (s), 141.31 (s), 130.99 (s), 129.37 (s), 128.31
(s), 121.27 (s), 114.43 (d, J ¼ 13.2 Hz), 108.07 (s), 56.27 (s), 55.16 (s),
48.44 (s), 39.94 (s), 39.80 (s), 39.66 (s), 39.52 (s), 39.38 (s), 39.24 (s),
39.10 (s). MS: m/z (%) [M þ Na]þ 309.0, [M þ H]þ 287.0.
6-Hydroxyl-3–(40-methoxyphenyl)-benzofuranone (1). White
solid, yield 94.14%, m.p. 188–190 ꢁC. 1H NMR (600 MHz, DMSO-d6)
d 9.88 (s, 1H), 7.11 (s, 2H), 7.02 ꢃ 6.86 (m, 3H), 6.63 (d, J ¼ 46.1 Hz,
2H), 5.13 (s, 1H), 3.74 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d
176.19 (s), 158.82 (s), 158.30 (s), 154.05 (s), 129.30 (s), 128.36 (s),
125.61 (s), 117.82 (s), 114.38 (s), 111.32 (s), 98.24 (s), 55.14 (s),
47.84 (s), 39.94 (s), 39.80 (s), 39.66 (s), 39.60 ꢃ 39.11 (m), 39.10 (s),
39.09 ꢃ 38.90 (m). MS: m/z (%) [M þ Na]þ 278.9.
5-Hydroxyl-3–(40-methoxyphenyl)-benzofuranone (2). White
solid, yield 95.12%, m.p. 164–165 ꢁC. 1H NMR (600 MHz, DMSO-d6)
d 9.38 (s, 1H), 7.13 ꢃ 7.08 (m, 3H), 6.96 ꢃ 6.93 (m, 2H), 6.74 (ddd,
J ¼ 8.7, 2.6, 0.7 Hz, 1H), 6.55 (dd, J ¼ 2.5, 0.9 Hz, 1H), 5.23 (s, 1H),
6-Methoxy-3–(30,40-dimethoxyphenyl)-benzofuranone (9). White
3.75 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 176.09 (s), 158.85 (s), solid, yield 62.81%, m.p. 125–126 ꢁC. 1H NMR (600 MHz, DMSO-d6)
154.32 (s), 145.74 (s), 129.40 (s), 128.99 (s), 127.94 (s), 115.10 (s), d 7.12 (d, J ¼ 7.7 Hz, 1H), 6.99 ꢃ 6.89 (m, 2H), 6.83 (s, 1H), 6.75 (d,
114.44 (s), 111.74 (s), 111.02 (s), 55.15 (s), 48.89 (s), 40.02 (d, J ¼ 7.4 Hz, 1H), 6.65 (d, J ¼ 7.0 Hz, 1H), 5.18 (s, 1H), 3.80 (s, 3H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
807
3.76 ꢃ 3.68 (m, 6H). 13C NMR (151 MHz, DMSO-d6) d 175.87 (s), 175.47 (s), 156.38 (s), 153.23 (s), 146.95 (s), 137.16 (s), 131.26 (s),
160.11 (s), 154.13 (s), 148.73 (d, J ¼ 72.9 Hz), 128.42 (s), 125.62 (s),
128.71 (s), 114.43 (s), 111.18 (s), 110.71 (s), 105.72 (s), 60.01 (s),
120.16 (s), 119.53 (s), 112.10 (d, J ¼ 11.7 Hz), 110.16 (s), 97.20 (s),
55.97 (s), 55.76 (s), 49.89 (s), 39.94 (s), 39.80 (s), 39.66 (s), 39.52 (s),
39.38 (s), 39.24 (s), 39.10 (s). MS: m/z (%) [M þ H]þ 330.9.
55.59 (d, J ¼ 16.4 Hz), 48.19 (s), 39.97 ꢃ 39.30 (m), 39.17 (d,
J ¼ 21.0 Hz). MS: m/z (%) [M þ Na]þ 323.0, [M þ H]þ 301.1,
[2M þ Na]þ 623.1.
6-Methoxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
(16).
White solid, yield 90.16%, m.p. 153–154 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.17 (d, J ¼ 8.4 Hz, 1H), 6.96 (d, J ¼ 2.2 Hz, 1H), 6.76
(dd, J ¼ 8.3, 2.2 Hz, 1H), 6.49 (s, 2H), 5.20 (s, 1H), 3.80 (s, 3H), 3.72
(s, 6H), 3.65 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 175.54 (s),
160.18 (s), 154.15 (s), 153.20 (s), 137.12 (s), 131.66 (s), 125.67 (s),
119.18 (s), 110.19 (s), 105.59 (s), 97.24 (s), 59.99 (s), 55.93 (s), 55.65
(s), 48.78 (s), 39.94 (s), 39.73 (d, J ¼ 21.0 Hz), 39.57 (s), 39.52 (s),
39.38 (s), 39.24 (s), 39.10 (s). MS: m/z (%) [M þ H]þ 331.1,
[M þ Na]þ 353.1, [2M þ Na]þ 683.2.
5,6-Dimethoxy-3–(40-methoxyphenyl)-benzofuranone
(10).
White solid, yield 91.11%, m.p. 128–130 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.12 (d, J ¼ 8.7 Hz, 2H), 7.07 (s, 1H), 6.94 (d, J ¼ 8.8 Hz,
2H), 6.79 (d, J ¼ 0.5 Hz, 1H), 5.18 (s, 1H), 3.81 (s, 3H), 3.75 (s, 3H),
3.67 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 176.34 (s), 158.87 (s),
149.74 (s), 147.12 (s), 146.14 (s), 129.38 (s), 128.13 (s), 117.96 (s),
114.43 (s), 108.67 (s), 96.44 (s), 56.28 (s), 56.09 (s), 55.15 (s), 48.66
(d, J ¼ 15.2 Hz), 40.00 (d, J ¼ 18.1 Hz), 39.80 (s), 39.59 (d,
J ¼ 21.0 Hz), 39.43 (s), 39.38 (s), 39.24 (s), 39.10 (s). MS: m/z (%)
[M þ Na]þ 323.0, [M þ H]þ 301.1, [2M þ Na]þ 623.1.
5-Methoxy-3–(20,30,40-trimethoxyphenyl)-benzofuranone
(17).
White solid, yield 84.85%, m.p. 132–135 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.17 (d, J ¼ 8.7 Hz, 1H), 7.13 (d, J ¼ 8.4 Hz, 1H),
6.89 ꢃ 6.86 (m, 1H), 6.84 (d, J ¼ 8.6 Hz, 1H), 6.59 ꢃ 6.57 (m, 1H),
5.14 (s, 1H), 3.81 (s, 3H), 3.69 (s, 3H), 3.67 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) d 176.56 (s), 156.57 (s), 154.30 (s), 147.54 (s),
142.16 (s), 130.53 (s), 125.91 (s), 122.95 (s), 114.01 (s), 111.07 (s),
110.34 (s), 108.04 (s), 60.69 (s), 60.42 (s), 56.36 (s), 56.11 (s), 43.89
(d, J ¼ 1014.6 Hz), 40.52 (s), 40.57 ꢃ 40.18 (m), 40.18 ꢃ 39.79 (m),
39.81 ꢃ 39.79 (m), 39.69 (s), 39.55 (s). MS: m/z (%) [M þ Na]þ 353.1,
[2M þ Na]þ 683.2.
6-Hydroxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
(11).
White solid, yield 92.72%, m.p. 191–193 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 9.90 (s, 1H), 7.04 (dd, J ¼ 8.2, 1.0 Hz, 1H), 6.67 (d,
J ¼ 2.2 Hz, 1H), 6.60 (dd, J ¼ 8.2, 2.2 Hz, 1H), 6.47 (s, 2H), 5.14 (s,
1H), 3.72 (s, 6H), 3.65 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d
175.71 (s), 158.35 (s), 154.04 (s), 153.17 (s), 137.05 (s), 131.93 (s),
125.68 (s), 117.42 (s), 111.35 (s), 105.53 (s), 98.31 (s), 60.00 (s),
55.92 (s), 48.76 (s), 39.94 (s), 39.80 (s), 39.66 (s), 39.52 (s), 39.31 (d,
J ¼ 21.0 Hz),
39.24 ꢃ 39.23
(m),
39.10
(s).
MS:
m/z
(%)[M þ Na]þ 339.3.
6-Methoxy-3–(20,30,40-trimethoxyphenyl)-benzofuranone
(18).
6-Methoxy-7-hydroxy-3–(30,40-dimethoxyphenyl)-benzofuranone
(12). White solid, yield 78.13%, m.p. 135–137 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 9.43 (s, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 6.82 (d,
J ¼ 1.9 Hz, 1H), 6.77 (d, J ¼ 8.3 Hz, 1H), 6.66 (dd, J ¼ 8.3, 1.9 Hz, 1H),
6.60 (d, J ¼ 8.2 Hz, 1H), 5.18 (s, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 3.71
(s, 3H). 13C NMR (151 MHz, DMSO-d6) d 175.72 (s), 148.91 (d,
J ¼ 13.3 Hz), 148.45 (s), 141.30 (s), 130.95 (s), 128.60 (s), 121.17 (s),
120.25 (s), 114.49 (s), 112.10 (d, J ¼ 8.6 Hz), 108.00 (s), 56.25 (s),
55.55 (s), 48.78 (s), 48.62 (s), 39.87 (d, J ¼ 21.0 Hz), 39.66 (s),
39.64 ꢃ 39.62 (m), 39.52 (s), 39.38 (s), 39.24 (s), 39.10 (s). MS: m/z
(%) [M þ Na]þ 339.0, [M þ H]þ 317.1, [2M þ Na]þ 655.1.
White solid, yield 75.76%, m.p. 100–101 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.12 (d, J ¼ 8.5 Hz, 1H), 6.92 (dd, J ¼ 9.6, 1.6 Hz, 2H),
6.83 (d, J ¼ 8.6 Hz, 1H), 6.66 (dd, J ¼ 8.3, 2.4 Hz, 1H), 5.06 (s, 1H),
3.81 (s, 3H), 3.77 (s, 3H), 3.69 (s, 3H), 3.33 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) d 176.68 (s), 160.31 (s), 154.61 (s), 154.22 (s),
151.07 (s), 142.21 (s), 125.71 (s), 124.98 (s), 123.39 (s), 121.12 (s),
109.91 (s), 108.05 (s), 97.47 (s), 60.68 (s), 60.44 (s), 56.36 (s), 56.04
(s), 46.15 (s), 40.33 (d, J ¼ 21.0 Hz), 40.12 (s), 39.98 (s), 39.84 (s),
39.70 (s), 39.56 (s). MS: m/z (%) [M þ Na]þ 353.1, [2M þ Na]þ 683.2.
5,6-Dimethoxy-3–(30,40-dimethoxyphenyl)-benzofuranone (19).
White solid, yield 84.85%, m.p. 149–151 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.06 (s, 1H), 6.94 (d, J ¼ 8.3 Hz, 1H), 6.83 ꢃ 6.80 (m,
2H), 6.66 (d, J ¼ 8.2 Hz, 1H), 5.17 (s, 1H), 3.81 (s, 3H), 3.74 (s, 3H),
3.72 (s, 3H), 3.68 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 176.18
(s), 149.71 (s), 148.95 (s), 148.49 (s), 147.10 (s), 146.09 (s), 128.39
(s), 120.24 (s), 117.85 (s), 112.11 (d, J ¼ 11.0 Hz), 108.68 (s), 96.40
(s), 56.29 (s), 56.06 (s), 55.53 (s), 54.91 (s), 49.03 (s), 39.87 (d,
J ¼ 21.0 Hz), 39.66 (s), 39.52 (s), 39.52 (s), 39.38 (s), 39.24 (s), 39.10
(s). MS: m/z (%) [M þ Na]þ 353.1, [2M þ Na]þ 683.2.
5-Hydroxy-6-methoxy-3–(30,40-dimethoxyphenyl)-benzofuranone
(13). White solid, yield 79.11%, m.p. 154–155 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 8.91 (d, J ¼ 1.5 Hz, 1H), 7.00 (d, J ¼ 1.8 Hz,
1H), 6.93 (dd, J ¼ 8.3, 1.8 Hz, 1H), 6.81 (s, 1H), 6.63 (d, J ¼ 8.2 Hz,
1H), 6.59 (s, 1H), 5.12 (s, 1H), 3.81 (d, J ¼ 1.8 Hz, 3H), 3.73 (dd,
J ¼ 10.9, 1.8 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) d 176.31 (s),
148.96 (s), 148.45 (s), 148.19 (s), 145.89 (s), 143.53 (s), 128.61 (s),
120.13 (s), 118.43 (s), 112.10 (d, J ¼ 13.5 Hz), 111.35 (s), 96.43 (s),
56.11 (s), 55.55 (s), 48.88 (s), 39.94 (s), 39.90 ꢃ 39.44 (m), 39.38 (s),
39.24 (s), 39.10 (s). MS: m/z (%) [M þ Na]þ 339.0, [2M þ Na]þ 655.1.
6,7-Dihydroxyꢃ3–(30,40,50-trimethoxyphenyl)-benzofuranone (20).
White solid, yield 87.65%, m.p. 170–172 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 9.49 (s, 1H), 9.26 (s, 1H), 6.60 (d, J ¼ 8.1 Hz, 1H), 6.50
(dd, J ¼ 8.1, 1.1 Hz, 1H), 6.47 (s, 2H), 5.15 (s, 1H), 3.72 (s, 6H), 3.65 (s,
3H). 13C NMR (151MHz, DMSO-d6) d 175.61 (s), 153.15 (s), 146.86
(s), 141.74 (s), 132.06 (s), 129.89 (s), 118.62 (s), 114.72 (s), 111.45 (s),
105.53 (s), 60.01 (s), 55.91 (s), 49.41 (s), 39.87 (d, J ¼ 21.0Hz),
39.76 ꢃ 39.72 (m), 39.66 (s), 39.52 (s), 39.38 (s), 39.24 (s), 39.10 (s).
MS: m/z (%) [M þ H]þ 332.9.
5-Hydroxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
(14).
White solid, yield 50.68%, m.p. 188–189 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 9.38 (s, 1H), 7.10 (d, J ¼ 8.7 Hz, 1H), 6.75 (ddd, J ¼ 8.7,
2.6, 0.7 Hz, 1H), 6.61 (dd, J ¼ 2.5, 0.9 Hz, 1H), 6.49 (s, 2H), 5.23 (s,
1H), 3.73 (s, 6H), 3.66 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d
175.61 (s), 154.29 (s), 153.21 (s), 145.70 (s), 137.14 (s), 131.52 (s),
128.65 (s), 115.23 (s), 111.74 (s), 111.08 (s), 105.67 (s), 60.02 (s),
55.97 (s), 49.83 (s), 39.87 (d, J ¼ 21.0 Hz), 39.68 (s), 39.66 (s), 39.52
(s), 39.38 (s), 39.17 (d, J ¼ 21.0 Hz). MS: m/z (%) [M þ H]þ 317.1,
[M þ Na]þ 339.1, [2M þ Na]þ 655.2.
5-Hydroxy-6-methoxy-3–(30,40,50-trimethoxyphenyl)-benzofura-
none (21). White solid, yield 83.24%, m.p. 178–180 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 8.92 (s, 1H), 7.00 (s, 1H), 6.63 (d, J ¼ 0.9 Hz,
1H), 6.47 (s, 2H), 5.14 (s, 1H), 3.81 (s, 3H), 3.72 (s, 6H), 3.66 (s, 3H).
13C NMR (151 MHz, DMSO-d6) d 175.97 (s), 153.19 (s), 148.27 (s),
145.90 (s), 143.52 (s), 137.09 (s), 131.88 (s), 118.11 (s), 111.33 (s),
5-Methoxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
(15).
White solid, yield 78.79%, m.p. 141–142 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.23 (d, J ¼ 8.8 Hz, 1H), 6.95 (ddd, J ¼ 8.8, 2.7, 0.7 Hz,
1H), 6.84 (dd, J ¼ 2.7, 1.0 Hz, 1H), 6.49 (s, 2H), 5.28 (s, 1H), 3.73 (s,
6H), 3.72 (s, 3H), 3.66 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d 105.55 (s), 96.44 (s), 60.01 (s), 56.09 (s), 55.95 (s), 49.49 (s), 39.94

808
J. YANG ET AL.
Cholinesterase inhibitory effect ð%Þ ¼ ½A0 ꢃ ðA1 ꢃ A2Þꢄ=A0 ꢂ 100%
(s), 39.73 (d, J ¼ 21.0 Hz), 39.52 (s), 39.38 (s), 39.24 (s), 39.10 (s). MS:
m/z (%) [M þ H]þ 347.1, [M þ Na]þ 369.1, [2M þ Na]þ 715.2.
6-Methoxy-7-hydroxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
where A0 is the absorbance of blank group; A1 is the absorbance
of sample group; A2 is the absorbance of sample blank group.
1
(22). White solid, yield 76.59%, m.p. 200–202 ꢁC. H NMR (600 MHz,
DMSO-d6) d 9.44 (s, 1H), 6.78 (d, J ¼ 8.3 Hz, 1H), 6.64 (dd, J ¼ 8.2,
1.1 Hz, 1H), 6.49 (s, 2H), 5.21 (d, J ¼ 0.7 Hz, 1H), 3.81 (s, 3H), 3.72 (s,
In vitro MAO inhibitory activity
6H), 3.65 (s, 3H). 13C NMR (151 MHz, DMSO-d6)
d 175.83
The MAO inhibitory activity of the 3-arylbenzofuranone com-
pounds was determined by the method of Holt et al.17 with slight
modifications. The crude enzyme was extracted from the liver of
200–250 g of Wistar rats according to literature methods18,19. The
crude enzyme protein content was determined by the Bradford
method using a Bradford Protein Assay Kit (Beyotime). The assay
was performed in 0.2 M potassium phosphate buffer pH 7.6 on
96-well plates in 240 ll total volume. The chromogenic solution
containing 1 mM vanillic acid, 0.5 mM 4-aminoantipyrine, and 4 U/
ml horseradish peroxidase in 0.2 M potassium phosphate buffer
pH 7.6 was mixed anew for each measurement. 40 lL of enzyme
solution and 40 lL of sample solution were added to a 96-well
plate. The solution was then incubated at 37 ꢁC for 20 min. 120 lL
of the 4-(trifluoromethyl) benzylamine solution and 40 lL of the
chromogenic solution 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 positive control replaced the sample with the posi-
tive drug, and the blank group replaced the substrate with PBS,
and each group was measured three times in parallel to average.
(s), 153.62 (s), 149.38 (s), 141.75 (s), 137.53 (s), 132.27 (s), 131.39
(s), 121.24 (s), 115.00 (s), 108.41 (s), 106.08 (s), 60.45 (s), 56.67 (s),
56.39 (s), 49.82 (s), 40.39 (s), 40.25 (s), 40.11 (s), 39.90 (d,
J ¼ 21.0 Hz), 39.69 (s), 39.55 (s). MS: m/z (%) [M þ H]þ 347.1044,
[M þ Na]þ 369.0866, [2M þ Na]þ 715.1836.
5-Hydroxy-6-methoxy-3–(30,40,50-trimethoxyphenyl)-benzofuranone
(23). White solid, yield 84.26%, m.p. 183–184 ꢁC. 1H NMR (600 MHz,
DMSO-d6) d 7.07 (s, 1H), 6.86 (d, J ¼ 0.6 Hz, 1H), 6.49 (s, 2H), 5.19 (s,
1H), 3.81 (s, 3H), 3.73 (s, 6H), 3.69 (d, J ¼ 4.3 Hz, 3H), 3.66 (s, 3H). 13C
NMR (151MHz, DMSO-d6) d 175.83 (s), 153.19 (s), 149.79 (s), 147.15
(s), 146.12 (s), 137.10 (s), 131.57 (s), 117.40 (s), 108.74 (s), 105.61 (s),
96.40 (s), 59.97 (s), 56.34 (s), 55.98 (d, J ¼ 18.2 Hz), 49.58 (s), 39.87 (d,
J ¼ 21.0 Hz), 39.67 (s), 39.59 (d, J ¼ 21.0 Hz), 39.38 (s), 39.25 (s), 39.17
(d, J ¼ 21.0 Hz). MS: m/z (%) [M þ H]þ 361.1, [M þ Na]þ 383.1,
[2M þ Na]þ 743.2.
Biological activity
Animals
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 condi-
tions and maintained on a standard pellet diet and water ad libi-
tum. All experiments involving living animals and their care were
performed in strict accordance with the National Care and Use of
Monoamine oxidase inhibitory effect ð%Þ
¼ ½ðAC – ABÞ – ðAS – ASB Þꢄ=ðAC – ABÞ ꢂ 100%
Laboratory Animals by the National Animal Research Authority where AC is the absorbance of control group; AB is the absorbance
(China) and guidelines of Animal Care and Use issued by
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 suf-
fering and to reduce the number of animals used.
of blank group; AS is the absorbance of sample group; ASB is the
absorbance of sample blank group.
In vitro antioxidant activity
The assay provides an assessment of the antioxidant activity due
to free radical scavenging by measuring hydrogen atom (or one
electron) donating activity. DPPH is a purple stable free radical
that is reduced to a yellow-coloured diphenylpicryl hydrazine by
an antioxidant. The spectrophotometric assay was carried out as
described by the literature method with a slight modification20.
100 lL of a sample solution was added to 100 lL of DPPH solu-
tion. Following 30 min of incubation at 37 ꢁC and protection from
light, the absorbance at 517 nm was determined using a micro-
plate reader. Each group was measured three times in parallel to
average. IC50 values were calculated. The percentage of DPPH rad-
ical scavenging rate of the target compound is calculated as fol-
lows:
In vitro ChE inhibitory activity
The anticholinesterase activity of the 3-arylbenzofuranone com-
pounds was determined by the method of Ellman et al.16 with
slight modifications. The in vitro inhibition assays of AChE
from electric eel and BChE from equine serum were run in phos-
phate buffer 0.1 M, at pH 8.0. Acetylthiocholine iodide and butyr-
ylthiocholine iodide were used as substrates respectively.
5,50-Dithiobis(2-nitrobenzoic acid) (DTNB) was used as the chromo-
phoric reagent. To a 96-well plate, 120 mL of phosphate buffer
solution (0.1 M, pH ¼ 8.0, PBS), 20 mL of DTNB (3.3 mM in 0.1 M
PBS, pH ¼ 8.0) were added sequentially, 20 mL AChE solution
(0.2 U/mL in 0.1 M PBS, pH ¼ 8.0), 20 mL of different concentrations
of the sample solution, shaken well, and incubated at 37 ꢁC for
5 min. Then, 20 mL of substrate (5 mM in 0.1 M PBS, pH ¼ 8.0) were
added, shaken well, and incubated at 37 ꢁC for 20 min. The
absorbance at 412 nm of the samples was measured using a spec-
trophotometer, and the inhibition rate of ChE and the IC50 value
of each sample were calculated according to the formula. BChE
inhibitory activity was assessed similarly using butyrylthiocholine
iodide as the substrate. The sample solution was set to five con-
centration gradients and the experiment was repeated three
times. Donepezil was used as a positive control.
DPPH free radical scavenging effect ð%Þ
¼ ½A0 – ðA1 ꢃ A2Þꢄ=A0 ꢂ 100%
where A0 is the absorbance of blank group; A1 is the absorbance
of sample group; A2 is the absorbance of sample blank group.
Kinetic characterisation of ChEs inhibition
To obtain the mechanism of action of 13, reciprocal plots of 1/vel-
ocity versus 1/[substrate] were constructed at different concentra-
tions of the substrate thiocholine iodide by using Ellman’s
method. Compound 13 was added to the assay solution and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
809
Scheme 1. General synthetic route to 3-arylbenzofuranone, reagents and conditions: (a) CHCl3, TBAB, NaOH, 40–50 ꢁC; (b) H3Oþ; (c) BF3ꢅEt2O, 30 ꢁC.
preincubated with the enzyme at 37 ꢁC for 5 min, followed by the
Table 1. Compounds 1–23.
addition of the substrate. Kinetic characterisation catalysed by
enzyme was achieved spectrometrically at 412 nm. A parallel con-
trol experiment was carried out without compound 13 in the mix-
ture. Lineweaver–Burk plot was made based on the reaction rate
and substrate concentration to determine the type of inhibition of
the ChE by the compound.
Product
R1
R2
R3
R4
R5
R6
OH
H
OCH3
H
R7
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
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OCH3
H
OH
H
OH
OH
OCH3
H
H
H
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
H
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
OH
H
H
H
9
OCH3
H
Molecular modelling
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H
H
OCH3
H
The docking of the compounds was performed using the Accelrys
Discovery Studio 2019 (DS, Accelrys Software Inc., San Diego, CA,
USA) CDOCKER docking protocol. A simulation system was con-
structed based on the structure obtained from the protein data-
base (PDB: 6O4W for AChE; 6EP4 for BuChE; 4A79 for MAO-B).
Preparing the protein using prepare protein method building in
DS retains water in the protein-binding pocket. The active site is
defined by the ligand of the protein crystal structure, and then
the original crystal ligand is docked again, and the RMSD value
obtained is less than 1. Prior to the docking calculation, the ori-
ginal ligand was removed. The 3D structure of the compound was
generated and optimised using DS.
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
OH
H
H
OH
OH
OCH3
H
OCH3
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OH
H
H
OH
H
OCH3
on the chemical and spectroscopic characterisations of compounds
1–23 were described in the Supporting Information.
TOPKAT prediction
Compared with the original method, the ratio of mandelic acid
compounds to phenolic compounds was adjusted from 1:1 to
1:1.2 to ensure that the mandelic acid compounds fully reacted.
The completion of the reaction is monitored by thin layer chroma-
tography (TLC), not just the reaction time. Compounds with differ-
ent substituents differ greatly in reaction time. Excessive reactions
can affect compound yield and purity. Several polyhydroxy com-
pounds were synthesised by microwave reactions. The previous
reaction by heating in an oil bath required a reaction for 8 h, and
now the reaction time is shortened to 15 min by 200 W microwave
heating. The yield of the compound obtained by microwave heat-
ing is also slightly improved. Most 3-arylbenzofuranone com-
pounds can be purified by methanol recrystallisation, which is
efficient and fast.
TOPKAT protocol in DS was also used to predict the rat oral LD50
and potential toxicity (i.e. carcinogenicity and mutagenicity) of the
compounds (Table 3).
Statistical analysis
Data were shown as mean SD Differences between individual
groups were analysed by using ANOVA followed by Dunett’s test.
A difference with a p values of <0.05 was considered to be
significant.
Results and discussion
Chemistry
The routes for the synthesis of 3-arylbenzofuranone derivatives are
shown in Scheme 1 and the final products are listed in Table 1.
The synthesis method of the 3-arylbenzofuranone compound was
based on Lv et al. and optimised slightly21. Substituted mandelic
acid 3a–3d were synthesised with substituted benzaldehyde
1a–1d. The substituted mandelic acid and substituted phenol were
used as starting materials to obtain the corresponding compounds
Biology
In vitro ChE inhibitory activity
AChE is responsible for the hydrolysis of ACh in the synaptic cleft,
and AChE inhibitors may help increase ACh levels in damaged
cholinergic neurons22. Some of the most common untoward
1–23 by esterification and intramolecular alkylation reaction. Details effects of ChEs inhibition therapy are gastrointestinal complaints

810
J. YANG ET AL.
Table 2. Biological evaluation in vitro.
IC50 value (lM)
BuChE inhibitory activity
Product
AChE inhibitory activity
MAO-B inhibitory activity
Antioxidant activity
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
50.58 3.12
4.15 1.02
80.48 3.41
0.74 0.06
0.48 0.05
24.38 1.21
0.52 0.07
9.24 0.62
0.35 0.03
28.65 1.44
48.24 0.34
5.25 0.44
0.28 0.01
3.37 0.17
2.54 0.19
4.07 0.19
42.26 4.01
123.88 2.17
36.50 0.51
0.089 0.01
2.38 0.20
2.03 0.09
25.96 1.26
0.059 0.003
>200
24.92 0.96
>200
>200
>200
>200
32.92 0.76
>200
>200
>200
61.26 0.79
14.67 0.78
298.43 5.4
383.45 5.08
22.48 0.22
48.59 2.02
91.13 0.70
40.62 1.92
354.19 5.05
493.39 7.10
38.61 0.42
22.85 0.73
11.24 0.72
69.65 1.26
215.22 2.27
>500
82.68 1.31
180.34 1.32
145.56 1.83
149.21 3.39
>500
30.27 0.65
43.89 1.26
13.28 0.27
>100
11.78 0.18
33.07 1.45
5.51 0.20
/
2.62 0.21
5.96 0.14
17.17 0.58
13.89 0.11
11.22 0.24
1.60 0.10
5.33 0.08
5.11 0.13
32.58 0.42
18.01 0.48
29.17 0.31
64.48 1.29
12.56 0.14
42.56 2.58
5.35 0.33
6.44 0.12
11.94 0.16
>200
180.91 1.48
9.91 0.48
128.33 4.65
>200
>200
>200
>200
>200
>200
145.89 3.05
>200
>200
4.67 0.16
Donepezil
Rasagiline
Ascorbic acid
0.104 0.002
7.69 0.10
Each value represents the mean SD (n ¼ 3).
Figure 1. Kinetic study of the mechanism of ChEs inhibition by compound 13. Overlaid Lineweaver–Burk reciprocal plots of ChEs initial velocity at increasing substrate
concentration in the absence of inhibitor and in the presence of 13 are shown. A is a double reciprocal plot of compound 13 inhibition of AChE. B is a double recipro-
cal plot of compound 13 inhibition of BuChE.
resulting from stimulation of peripheral autonomic cholinergic sys- respectively lacking one hydroxyl group at R5 than compound 5
tems. The pre-clinical data indicate that more selective AChE (IC50 ¼ 0.48 0.05 lM) and 20 (IC50 ¼ 0.089 0.005 lM), so we specu-
inhibitors produce fewer peripheral cholinergic signs than non- lated that the R5, R6-dihydroxy substituted compound is better than
selective ChE inhibitors23. Dual ChE inhibitors may increase the the R6 monosubstituted compound at AChE inhibitory activity. The
incidence of adverse reactions.
activities of the compounds
7
(IC50 ¼ 0.52 0.07 lM), 13
The ChE inhibitory activity of all compounds was evaluated by the (IC50 ¼ 0.28 0.01 lM) are respectively superior to those of the com-
method of Ellman16. Donepezil was used as a reference compound in pounds 10 (IC50 ¼ 28.65 1.44 lM), 19 (IC50 ¼ 36.50 0.51 lM), so we
this assay. As shown in Table 2, all compounds presented AChE presumed that the R6 methoxy-substituted and the R7 hydroxy-substi-
inhibitory activity. Notably, compound 20 (IC50 ¼ 0.089 0.005 lM) tuted compound is superior to that of the R6, R7-dimethoxy-substi-
had relatively strong activity, which displayed little weaker capacity tuted compound. Nearly half of the compounds have BuChE
than donepezil (IC50 ¼ 0.059 0.003 lM). Compounds 4, 5, 7, 9, 13, inhibitory activity. The IC50 value of compound 13
20 have IC50 values less than 1 lM. Compound
1
(IC50 ¼ 9.91 0.48 lM) is similar to positive drug donepezil
(IC50 ¼ 50.58 3.12 lM) and 11 (IC50 ¼ 48.24 0.34 lM) were (IC50 ¼ 4.67 0.16 lM). Compounds 2, 7, and 13 have IC50 values less

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
811
Figure 2. Schematic presentations of the putative AChE binding modes with compound 20 and compound 11.
Figure 3. Schematic presentations of the putative BuChE binding modes with compound 13 and compound 19.

812
J. YANG ET AL.
Figure 4. Schematic presentations of the putative MAO-B binding modes with compound 9 and compound 12.
Table 3. TOPKAT prediction results
TD50 value (mg/kg)
LD50 value(g/kg)
Compound
Mutagenicity
Carcinogenicity (Mouse)
Carcinogenicity (Rat)
Acute oral toxicity (Rat)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Donepezil
Rasagiline
0.495
0.535
0.631
0.586
0.569
0.406
0.583
0.571
0.522
0.528
0.211
0.522
0.533
0.274
0.323
0.33
0.443
0.474
0.516
0.293
0.336
0.292
0.275
0.22
184
184
89
7.9
7.9
0.719
0.998
0.784
1.95
0.842
2.62
0.488
0.732
2.89
1.62
1.76
1.83
1.75
1.37
1.76
1.73
3.63
1.73
2.75
2.59
1.07
1.95
1.67
0.392
0.427
1.49
2.08
7.11
11.8
5.81
5.81
2.09
2.09
6.87
8.55
8.55
9.24
1.64
2.08
2.08
1.49
2.19
6.11
3.89
8.46
0.994
1.56
20.2
89
83.6
252
209
127
100
100
268
268
268
268
106
106
106
106
128
198
158
282
75.4
99.8
94.9
0.598
than 50 lM. As shown in Table 2, most of the compounds have evaluated for their MAO inhibitory activity in the way of Holt by
selective AChE inhibitory activity, which may have a lower incidence references17. Among them, compound 13 (IC50 ¼ 11.24 0.72 lM)
had the best inhibitory activity of MAO-B but was still weaker
than the positive drug rasagiline (IC50 ¼ 0.104 0.002 lM).
Compounds 8, 12, 22 all have a hydroxyl group at R5 and a
methoxy group at the R6 position, all exhibiting good activity. All
of the tested compounds showed good activity in the substitution
of R5 with a hydroxyl group, so we speculated that the hydroxyl
of adverse reactions than the double ChE inhibitor.
In vitro MAO inhibitory activity
MAO plays a major role in brain development and function, and
its inhibitors are used clinically as antidepressants and anti-
Parkinson’s drugs. MAO-B activity is increased in the brains of substitution at the R5 position may contribute to an increase in
Alzheimer’s patients24. All the synthesised compounds were the MAO-B inhibitory activity of the compound.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
813
In vitro antioxidant activity
agents, which have inhibitory activity against ChEs and MAO-B
and antioxidant activity. All the synthesised compounds were eval-
uated for their antioxidant activities in the way of DPPH free rad-
ical scavenging experiment. Most compounds demonstrated
moderate to high activity. According to the results of in vitro ChEs
inhibition assay, we selected compound 13 with better ChE inhib-
ition to study the kinetics of ChE inhibition. According to the kin-
etic experiments, the type of action of compound 13 on ChE
inhibition is reversible inhibition, indicating that our design strat-
egy is reasonable. Previously, our research group also carried out
anti-AD research on 3-arylcoumarin compounds, and most of
them have double ChE inhibitory activity26. Compared with 3-aryl-
coumarin compounds, 3-arylbenzofuranone compounds have bet-
ter AChE and MAO-B inhibitory activity than 3-arylcoumarin
compounds and have selective AChE inhibitory activity. Selective
AChE compounds have fewer adverse reactions than double ChE
inhibition, so screening for compounds with better selective AChE
inhibitory activity also has a certain significance. Multi-target anti-
AD compounds can modulate multiple signalling pathways or tar-
gets associated with AD, potentially producing significant clinical
effects. All in all, the multifunctional effects of these 3-arylbenzo-
furanone derivatives qualify them as potential anti-AD agents, and
they are more promising than 3-arylcoumarin compounds in
anti-AD.
Alzheimer’s patients exhibit extensive oxidative stress throughout
the body. Oxidative stress is an early and prominent symptom of
AD, which plays an important role in AD20. All the synthesised
compounds were evaluated for their antioxidant activities in the
way of scavenging DPPH25. The 3-arylbenzofuranone compounds
exhibit good antioxidant capacity. The antioxidant activity of the
compounds 5, 7, 8, 12, 13, 14, 21, 22 was superior to the positive
drug vitamin C (IC50 ¼ 7.69 0.10 lM). Compounds 8, 12, 22 are
substituted at the R5 position with a hydroxy group and at the R6
position with a methoxy group. Compounds 7, 13, and 21 are
substituted at the R6 with a methoxy group and at the R7 position
with a hydroxy group. Therefore, we speculated that the hydroxy-
methoxy ortho-substitution facilitates the improvement of the
antioxidant capacity of the compound.
Kinetic study of ChEs inhibition
In order to gain insight into the mechanism of action of these
derivatives on ChEs, compound 13, showing good inhibitory activ-
ity against ChEs, was selected for kinetic measurements. The
graphical analysis of the steady-state inhibition data of 13 against
ChEs is shown in Figure 1. According to the figure, it can be
judged that the inhibition mode of compound 13 against ChEs is
reversible inhibition.
Ethical statement
Molecular modelling
To clarify the mechanism of the compound’s inhibitory activity on
the enzyme, the binding model of the active and inactive com-
pounds to the active site was compared. The active site binding pat-
terns of compounds 20, 11, and 4A79 are shown in Figure 2,
respectively. It can be seen from Figure 2 that the main difference
between the two compounds and the 4A79 binding model is the
interaction between compound 20 and His447. This supports the R5,
R6-dihydroxy substituted compound is better than the R6 monosub-
stituted compound at AChE inhibitory activity. The active site binding
patterns of compounds 13, 19, and 6EP4 are shown in Figure 3,
respectively. The effect of substituents on BuChE inhibition cannot
be summarised by IC50 value and molecular docking results. The
active site binding patterns of compounds 9, 12, and 4A79 are
shown in Figure 4, respectively. It can be seen from Figure 4 that
the main difference between the two compounds and the 6O4W
binding model is the interaction between the hydroxyl group at the
R5 position of compound 12 and two molecules of water. This sup-
ports the hydroxyl substitution at the R5 position may contribute to
an increase in the MAO-B inhibitory activity of the compound.
All experiments involving living animals and their care were per-
formed 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
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 suf-
fering and to reduce the number of animals used.
Disclosure statement
The authors declare that they have no competing interests.
Funding
The authors are grateful to support from the Science and
Shandong Provincial Natural Science Foundation [ZR2018LH021],
the Innovation Project of Shandong Academy of Medical Sciences
and Academic promotion programme of Shandong First Medical
University [No. 2019LJ003].
TOPKAT prediction
Toxicity Prediction by Komputer assisted technology (TOPKAT)
protocol in DS was used to predict the potential toxicity of the
compound and the acute oral toxicity effects in rats. According to
the analysis of the results in Table 3, all compounds were non-
mutagenic. Table 3 summarises the acute oral toxicity effects of
rats, indicating that a low level of toxicity with values ranging
from 0.488 to 3.63 g/kg, which are all greater than the posi-
tive drug.
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
References
1. Burns A, Iliffe S. Alzheimer’s disease. BMJ 2009;338:b158.
2. Morris M, Knudsen GM, Maeda S, et al. Tau post-translational
modifications in wild-type and human amyloid precursor
protein transgenic mice. Nat Neurosci 2015;18:1183–9.
Conclusion
In conclusion, a series of 3-arylbenzofuranone derivatives were
designed, synthesised, and evaluated as multi-targeting anti-AD

814
J. YANG ET AL.
3. Glabe CC. Amyloid accumulation and pathogensis of
Alzheimer’s disease: significance of monomeric, oligomeric
and fibrillar Ab. Alzheimer’s Dis 2005;38:167–77.
related to insulin amyloid fibrillation: a structure-function
study. Chem Biol Drug Des 2011;78:659–66.
~
15. Aranda R, Villalba K, Ravina E, et al. Synthesis, binding affin-
4. Scarpini E, Schelterns P, Feldman H. Treatment of
Alzheimer’s disease; current status and new perspectives.
Lancet Neurol 2003;2:539–47.
ity, and molecular docking analysis of new benzofuranone
derivatives as potential antipsychotics. J Med Chem 2008;51:
6085–94.
5. Perry EK, Tomlinson BE, Blessed G, et al. Correlation of cho- 16. Ellman GL, Courtney KD, Andres V, et al. A new and rapid
linergic abnormalities with senile plaques and mental test
colorimetric determination of acetylcholinesterase activity.
scores in senile dementia. Br Med J 1978;2:1457–9.
Biochem Pharmacol 1961;7:88–95.
6. Youdim MB, Edmondson D, Tipton KF. The therapeutic 17. Holt A, Sharman DF, Baker GB, et al. A continuous spectro-
potential of monoamine oxidase inhibitors. Nat Rev
Neurosci 2006;7:295–309.
7. Karlsson D, Fallarero A, Brunhofer G, et al. The exploration
photometric assay for monoamine oxidase and related
enzymes in tissue homogenates. Anal Biochem 1997;244:
384–92.
of thienothiazines as selective butyrylcholinesterase inhibi- 18. Youdim MBH, Gross A, Finberg JPM. Rasagiline [N-propargyl-
tors. Eur J Pharm Sci 2012;47:190–205.
8. Nadri H, Pirali-Hamedani M, Moradi A, et al. 5,6-
Dimethoxybenzofuran-3-one derivatives: a novel series of
1R(þ)-aminoindan], a selective and potent inhibitor of mito-
chondrial monoamine oxidase B. Br J Pharmacol 2001;132:
500–6.
€
dual acetylcholinesterase/butyrylcholinesterase inhibitors 19. Stafford GI, Pedersen PD, Jager AK, et al. Monoamine oxi-
bearing benzyl pyridinium moiety. Daru J Pharm Sci 2013;
21:15.
dase inhibition by southern African traditional medicinal
plants. S Afr J Bot 2007;73:384–90.
9. Khaleghi M, Navidpour L, Sepehrizadeh Z, et al. The sensi- 20. Wang XL, Wang WZ, Li L, et al. Oxidative stress and mito-
tization of legionella pneumophila to some antibiotics by
reserpine and anti-legionella effects of different benzofura-
none derivatives. J Med Bacteriol 2013;2:35–40.
chondrial dysfunction in Alzheimer’s disease. Biochim
Biophys Acta 2014;1842:1240–7.
21. Lv ZL, Gao Y, Li J, et al. Synthesis and antitumor activities of
3-arylbenzofunanone analogues. Chem J Chin Univ 2013;34:
2531–9.
10. Gu Q, Wang RR, Zhang XM, et al. A new benzofuranone and
anti-HIV constituents from the stems of Rhus chinensis.
Planta Med 2007;73:279–82.
22. Richman DP, Agius MA. Treatment of autoimmune myasthe-
nia gravis. Neurology 2003;61:1652–61.
11. de Souza Nunes JP, Da Silva KA, Da Silva GF, et al. The anti-
hypersensitive and antiinflammatory activities of a benzofur- 23. Liston DR, Nielsen JA, Villalobos A, et al. Pharmacology of
anone derivative in different experimental models in mice:
the importance of the protein kinase C pathway. Anesth
Analg 2014;119:836–46.
selective acetylcholinesterase inhibitors: implications for use
in Alzheimer’s disease. Eur J Pharmacol 2004;486:9–17.
24. Kennedy BP, Ziegler MG, Alford M, et al. Early and persistent
alterations in prefrontal cortex MAO A and B in Alzheimer’s
disease. J Neural Transm 2003;110:789–801.
12. Padaratz P, Fracasso M, De Campos-Buzzi F, et al.
Antinociceptive activity of a new benzofuranone derived
from a chalcone. Basic Clin Pharmacol Toxicol 2009;105: 25. Liang XL, Wang XL, Li Z, et al. Improved in vitro assays of
257–61.
superoxide anion and 1,1-diphenyl- 2-picrylhydrazyl (DPPH)
radical-scavenging activity of isoflavones and isoflavone
metabolites. J Agric Food Chem 2010;58:11548–52.
13. Adediran SA, Cabaret D, Drouillat B, et al. The synthesis and
evaluation of benzofuranones as b-lactamase substrates.
Bioorg Med Chem 2001;9:1175–83.
14. Rabiee A, Ebrahim-Habibi A, Navidpour L, et al.
Benzofuranone derivatives as effective small molecules
26. Yang J, Zhang PP, Hu YH, et al. Synthesis and biological
evaluation of 3-arylcoumarins as potential anti-Alzheimer’s
disease agents. J Enzyme Inhib Med Chem 2019;34:651–6.