Discovery of novel isoflavone derivatives as AChE/BuChE dual-targeted inhibitors: synthesis, biological evaluation and molecular modelling

Article abstract of DOI:10.1080/14756366.2017.1347163

AChE and BuChE are druggable targets for the discovery of anti-Alzheimer’s disease drugs, while dual-inhibition of these two targets seems to be more effective. In this study, we synthesised a series of novel isoflavone derivatives based on our hit compound G from in silico high-throughput screening and then tested their activities by in vitro AChE and BuChE bioassays. Most of the isoflavone derivatives displayed moderate inhibition against both AChE and BuChE. Among them, compound 16 was identified as a potent AChE/BuChE dual-targeted inhibitor (IC₅₀: 4.60 μM for AChE; 5.92 μM for BuChE). Molecular modelling study indicated compound 16 may possess better pharmacokinetic properties, e.g. absorption, blood–brain barrier penetration and CYP2D6 binding. Taken together, our study has identified compound 16 as an excellent lead compound for the treatment of Alzheimer’s disease.

Full text of DOI:10.1080/14756366.2017.1347163

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of novel isoflavone derivatives as
AChE/BuChE dual-targeted inhibitors: synthesis,
biological evaluation and molecular modelling
Bo Feng, Xinpeng Li, Jie Xia & Song Wu
To cite this article: Bo Feng, Xinpeng Li, Jie Xia & Song Wu (2017) Discovery of novel isoflavone
derivatives as AChE/BuChE dual-targeted inhibitors: synthesis, biological evaluation and
molecular modelling, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 968-977, DOI:
10.1080/14756366.2017.1347163
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Date: 20 July 2017, At: 02:51

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 968–977
https://doi.org/10.1080/14756366.2017.1347163
RESEARCH PAPER
Discovery of novel isoflavone derivatives as AChE/BuChE dual-targeted inhibitors:
synthesis, biological evaluation and molecular modelling
a
b
a
a
ꢀ
ꢀ
Bo Feng , Xinpeng Li , Jie Xia and Song Wu
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Department of New Drug Research and Development, Institute
b
of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; Food and Drug
Administration of Beijing Yanqing District, Beijing 102100, China
ABSTRACT
ARTICLE HISTORY
Received 15 March 2017
Revised 13 June 2017
Accepted 20 June 2017
AChE and BuChE are druggable targets for the discovery of anti-Alzheimer’s disease drugs, while dual-
inhibition of these two targets seems to be more effective. In this study, we synthesised a series of novel
isoflavone derivatives based on our hit compound G from in silico high-throughput screening and then
tested their activities by in vitro AChE and BuChE bioassays. Most of the isoflavone derivatives displayed
moderate inhibition against both AChE and BuChE. Among them, compound 16 was identified as a potent
AChE/BuChE dual-targeted inhibitor (IC50: 4.60 lM for AChE; 5.92 lM for BuChE). Molecular modelling study
indicated compound 16 may possess better pharmacokinetic properties, e.g. absorption, blood–brain bar-
rier penetration and CYP2D6 binding. Taken together, our study has identified compound 16 as an excel-
lent lead compound for the treatment of Alzheimer’s disease.
KEYWORDS
Alzheimer’s disease;
isoflavone derivatives;
AChE/BuChE dual-targeted
inhibitor; molecular
modelling
Introduction
inhibition of both AChE and BuChE may be beneficial for treating
the cognitive deficits16,17
Such multifunctional cholinestarase
.
Alzheimer’s disease (AD) is one of the most common neurodege-
nerative diseases and accounts for more than 80% of dementia
cases worldwide in elderly people1,2. It is in particular character-
ised by a forebrain cholinergic neuronal loss, a progressive cogni-
tive decline and neuropsychiatric cholinergic disturbances, which
seems to be closely related to pathological formation of b-amyl-
oid3. Studies have demonstrated that memory impairment in
patients with dementia is due to the selective and irreversible defi-
ciency in the cholinergic functions4. Cholinesterase is the enzyme
which catalyses the hydrolysis of acetylcholine, and thus inhibition
of cholinesterase can be an effective way for the treatment of
inhibitors have been identified so far and were showing positive
clinical outcome for the treatment of AD18,19. For instance, the
AChE/BuChE dual-targeted inhibitor, i.e. a “blockbuster drug” riva-
stigmin (cf. Figure 1) displayed
a more potent effect than
donepezil20.
In order to identify new classes of AChE/BuChE dual-targeted
inhibitors, in silico high-throughput screening (HTS) was performed
to screen our in-house chemical library that contained more than
30,000 compounds. On the top-ranked compound list, an isofla-
vone derivative G (cf. Figure 2) attracted our attention as it dis-
played favourable binding modes with both AChE and BuChE (cf.
Supplementary Figure S1). To the best of our knowledge, the scaf-
folds of flavonoids21,22 and its closely related ones such as homoi-
solavonoids23,24, trihydroxyflavone25,26 were privileged structures
that showed beneficial effects on neurological disorders, e.g. anti-
inflammation, metal chelation, neuroprotection, Ab fibril formation
inhibition and free radical scavenging effect. In addition, a recent
study revealed a few compounds structurally similar to G were
AChE inhibitors21. These evidences prompted us to test the activ-
ity of G against both AChE and BuChE. Interestingly, the com-
pound G showed a dual-targeting effect, with an IC50 of 1.47 lM
for AChE and 3.37 lM for BuChE. In this study, we explored its
preliminary structure-activity relationship (SAR) by synthesising a
series of novel isoflavone derivatives and testing their inhibitory
effects on both AChE and BuChE. Then we carried out molecular
docking to predict binding modes of isoflavone derivatives to
AD5–7
.
Two types of the cholinesterase, namely, acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE) were discovered in the
nervous system8. However, AChE has a 1013 fold higher acetylcho-
line hydrolytic activity than BuChE does under the same condi-
tion9,10
. Many AChE inhibitors were discovered, of which
donepezil11 and galantamine12 (cf. Figure 1) have been approved
by Food and Drug Administration (FDA). Nevertheless, the clinical
use demonstrated they have modest memorial and cognitive
improving functions for patients with AD. Specifically, they only
provide
a symptomatic and palliative pharmacological effect
instead of curing AD, and their effects would “wear off” after a cer-
tain period of treatment time13,14. Evidences suggested that add-
itional inhibition of brain BuChE may become an important
therapeutic strategy for AD treatment. It was reported that the
BuChE had a key role that it could partly compensate for the
action of AChE15. In addition, studies indicated that a balanced AChE and BuChE. Moreover, we predicted the pharmacokinetic
CONTACT Jie Xia
jiexia@imm.ac.cn; Song Wu
ws@imm.ac.cn
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, No.2 Nanwei Road, Beijing 100050, China
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2017 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, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
969
Figure 1. Structures of currently marketed cholinesterase inhibitors.
175.9, 162.4, 159.8, 157.9, 152.2, 130.2, 128.2, 125.1, 124.2, 119.1,
114.7, 114.1, 101.2, 68.3, 55.5, 28.4. ESI-MS m/z 375.38 [M þ H]þ.
7-(3-Bromopropoxy)-3–(4-methoxyphenyl)-4H-chromen-4-one (a2)
The compound was obtained as white solid in 91.5% yield. Purity
99.2% (by HPLC); mp 121.5–123.6 ꢁC; 1H NMR (400 MHz, CDCl3) d
8.22 (d, J ¼ 8.8 Hz, 1H), 7.92 (s, 1H), 7.50 (d, J ¼ 8.8 Hz, 2H),
7.02–6.91 (m, 3H), 6.87 (d, J ¼ 2.4 Hz, 1H), 4.22 (t, J ¼ 6.0 Hz, 2H),
3.84 (s, 3H), 3.63 (t, J ¼ 6.0 Hz, 2H), 2.38 (m, J ¼ 6.0 Hz, 2H); 13C
NMR (100 MHz, CDCl3) d 176.0, 163.1, 159.7, 158.0, 152.2, 130.3,
128.0, 125.1, 124.3, 118.7, 114.8, 114.1, 100.9, 66.1, 55.5, 32.1, 29.7.
ESI-MS m/z 389.34 [M þ H]þ.
Figure 2. Chemical Structure of the hit compound G.
profile of those dual-targeting isoflavone derivatives, i.e. absorp-
tion, distribution, metabolism and excretion (ADME) properties.
Materials and methods
7-(4-Bromobutoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (a3)
The compound was obtained as white solid in 88.6% yield. Purity
98.7% (by HPLC); mp 152.8–154.0 ꢁC; 1H NMR (400 MHz, CDCl3) d
8.21 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.50 (d, J ¼ 8.8 Hz, 2H),
6.99–6.96 (m, 3H), 6.84 (d, J ¼ 2.4 Hz, 1H), 4.10 (t, J ¼ 6.0 Hz, 2H),
3.84 (s, 3H), 3.51 (t, J ¼ 6.0 Hz, 2H), 2.14–1.98 (m, 4H); 13C NMR
(100 MHz, CDCl3) d 175.8, 163.2, 159.6, 157.9, 152.0, 130.1, 127.8,
124.9, 124.2, 118.5, 114.7, 114.0, 100.6, 67.6, 55.3, 33.2, 29.3, 27.6.
ESI-MS m/z 403.31 [M þ H]þ.
Chemistry
All melting points (mps) were measured by a Melting Point YRT-3
apparatus (Tianjin precision apparatus factory, China) and were
uncorrected. NMR spectra were performed using 300 or 400 MHz
spectrometers (Varian Mercury, USA) with TMS as an internal
standard. High resolution mass spectra were determined by
Thermo Scientific Exactive Plus mass spectrometry with ESI
method (Thermo, USA). Some compounds were purified by
medium-pressure preparative column chromatography (Shimadzu,
Japan). The purity of all these synthesised compounds was deter-
mined by HPLC analysis (Waters, USA). All the materials were
obtained from commercial suppliers and used without purification,
unless otherwise specified. Yields were not optimised. TLC analysis
was carried out on silica gel plates GF254 (Yantai chemical
research institute, China). Column chromatography was performed
on silica gel (200–300 mush; Qingdao Marine Chemical Inc.).
7-(2-Bromoethoxy)-5-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-
one (a4)
The procedure is the same as above method, however, the for-
mononetin was replaced by 5-hydroxyl formononetin. The com-
pound was obtained as white solid in 94.4% yield. Purity
99.6% (by HPLC); mp 157.1–159.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d 12.87 (s, 1H), 7.88 (s, 1H), 7.46 (d, J ¼ 8.8 Hz, 2H), 6.98 (d,
J ¼ 8.8 Hz, 2H), 6.40 (dd, J ¼ 2.0, 15.2 Hz, 2H), 4.36 (t, J ¼ 6.0 Hz,
2H), 3.85 (s, 3H), 3.66 (t, J ¼ 6.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3) d 181.0, 164.0, 163.1, 160.0, 158.0, 152.9, 130.2, 123.9,
123.0, 114.3, 106.8, 98.7, 93.2, 68.3, 55.5, 28.4. ESI-MS m/z
391.38[M þ H]þ.
General procedure for the preparation of intermediates (a1–a4)
Formononetin (5 g, 18.6 mmol), finely grounded anhydrous K2CO3
(30 g, 270 mmol) and 1,2-dibromoethane (17.2 ml, 199 mmol) or
1,3-dibromopropane (20.1 ml, 199 mmol) or 1,4-dibromobutane
(24 ml, 198 mmol) were added into 500 ml acetone solution. The
mixture was refluxed for 10 h. The residues were then added into
100 ml water and stirred for 1 h and filtrated by suction. After that,
the pastry was washed by 5 ml water for three times. The solid
was finally dried in vacuum at 50 ꢁC to give the desired product
without further purification.
7-(2-Bromoethoxy)-3-(4-(2-bromoethoxy)phenyl)-4H-chromen-4-one
(a5)
The procedure for the preparation of a5 was the same with that
for a1–a4, except that the formononetin was replaced with daid-
zein and the final product was purified by silico column and
eluted by CH2Cl2. The compound obtained as white solid in 26.3%
yield. mp 170.2–172.4 ꢁC; 1H NMR (400 MHz, CDCl3) d 8.23 (d,
J ¼ 9.2 Hz, 1H), 7.93 (s, 1H), 7.50 (d, J ¼8.4 Hz, 2H), 7.03–6.97 (m,
3H), 6.87 (d, J ¼ 1.6 Hz, 1H), 4.40 (t, J ¼ 6.4 Hz, 2H), 4.33 (d,
J ¼6.4 Hz, 2H), 3.69 (t, J ¼ 6.4 Hz, 2H), 3.66 (t, J ¼ 6.4 Hz, 2H); 13C
NMR (100 MHz, CDCl3) d: 174.6, 162.2, 157.7, 157.3, 153.6, 130.1,
127.1, 124.6, 123.3, 117.9, 115.0, 114.4, 101.4, 68.5, 67.8, 31.4, 31.0.
ESI-MS (m/z): 467.44 [M þ H]þ.
7-(2-Bromoethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-one (a1)
The compound was obtained as white solid in 93.4% yield. Purity
98.5% (by HPLC); mp 177.7–179.6 ꢁC; 1H NMR (400 MHz, CDCl3) d
8.23 (d, J ¼ 8.8 Hz, 1H), 7.92 (s, 1H), 7.50 (d, J ¼ 8.8 Hz, 2H),
7.02–6.96 (m, 3H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.39 (t, J ¼ 6.4 Hz, 2H),
3.84 (s, 3H), 3.69 (t, J ¼ 6.4 Hz, 2H); 13C NMR (100 MHz, CDCl3) d

970
B. FENG ET AL.
General procedure for the synthesis of compounds 1–15
7-(3-(Ethyl(methyl)amino)propoxy)-3-(4-methoxyphenyl)-4H-chro-
men-4-one (5)
Intermediate a1 (1.87 g, 5 mmol) or a2 (1.88 g, 5 mmol) or a3
(2.02 g, 5 mmol) or a4 (100 mg, 0.26 mmol) were dissolved in
150 ml acetonitrile or 70 ml DMF. Then K2CO3 (13.8 g, 100 mmol)
and various substituted amino-group (16.4 mmol) were added. The
mixture was stirred and refluxed for 3 h and monitored by TLC.
When the reaction was finished, the reaction solution was poured
into 600 ml water and stirred for 5 min. After that, the mixture was
filtrated by suction and the pastry was washed by water, then,
dried by vacuum at 50 ꢁC to yield the desired products with chro-
matography, recrystallisation, or without any further purification.
The compound was obtained as white solid in 26.4% yield. Purity
98.7% (by HPLC); mp 100.7–101.4 ꢁC; 1H NMR (400 MHz, CDCl3) d
8.19 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.50 (d, J ¼ 8.8 Hz, 2H),
6.99–6.96 (m, 3H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.12 (t, J ¼ 6.4 Hz, 2H),
3.84 (s, 3H), 2.56 (t, J ¼ 6.8 Hz, 2H), 2.47 (q, J ¼ 7.2 Hz, 2H), 2.27 (s,
3H), 2.05–1.99 (m, 2H), 1.08(t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 176.0, 163.6, 159.7, 158.1, 152.2, 130.3, 127.9, 125.0, 124.4,
118.5, 115.0, 114.1, 100.8, 67.1, 55.5, 53.7, 51.6, 41.8, 27.1, 12.3.
HRMS: calcd for C22H26NO4[M þ H]þ, 368.1862, found: 368.1859.
5-Hydroxy-3-(4-mehtoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-
chromen-4-one (1)
7-(4-(Ethyl(methyl)amino)butoxy)-3-(4-methoxyphenyl)-4H-chro-
men-4-one (6)
The compound was obtained as white solid in 85.7% yield. Purity
99.3% (by HPLC); mp 81.3–83.1 ꢁC. 1H NMR (400 MHz, CDCl3) d:
12.83 (s, 1H), 7.86 (s, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 6.98 (d, J ¼ 8.4 Hz,
2H), 6.40 (d, J ¼ 13.2 Hz, 2H), 4.20 (brs, 2H), 3.84 (s, 3H), 2.83 (brs,
2H), 2.56 (brs, 4H), 1.65 (brs, 4H), 1.47 (brs, 2H); 13C NMR (100 MHz,
CDCl3) d 181.0, 164.9, 162.8, 159.9, 158.1, 152.8, 130.2, 123.8, 123.1,
114.2, 106.4, 983.9, 93.1, 66.8, 57.7, 55.5, 55.2, 26.0, 24.2. HRMS:
calcd for C23H26NO5[M þ H]þ, 396.1811, found: 396.1807.
The final compound was obtained as white solid in 11.7% yield
purified by medium-pressure preparative column chromatography
method (CH2Cl2: CH3OH ¼97: 3). Purity 99.1% (by HPLC); mp
128.3–139.9 ꢁC; 1H-NMR (400 MHz, CDCl3) d 8.17 (d, J ¼ 8.8 Hz, 1H),
7.88 (s, 1H), 7.48 (d, J ¼ 8.8 Hz, 2H), 6.96–6.94 (m, 3H), 6.81 (d,
J ¼ 2.0 Hz, 1H), 4.05 (t, J ¼ 6.0 Hz, 2H), 3.81 (s, 3H), 2.48–2.41 (m,
4H), 2.24 (s, 3H), 1.88–1.81 (m, 2H), 1.71–1.64 (m, 2H), 1.07 (t,
J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 175.9, 163.5, 159.6,
158.0, 152.1, 130.2, 127.7, 124.9, 124.3, 118.3, 114.9, 114.0, 100.6,
68.5, 56.8, 55.4, 51.5, 41.5, 27.0, 23.8, 12.2. HRMS: calcd for
C23H28NO4 [M þ H]þ, 382.2018, found: 382.2018.
3-(4-Methoxyphenyl)-7-(3-(piperidin-1-yl)propoxy)-4H-chromen-4-
one (2)
The compound was obtained as white solid in 7% yield.
Purity 98.8% (by HPLC); mp 152.8–154.0 ꢁC; 1H NMR (400 MHz, 7-(2-((2-(5-Methoxy-1H-indol-3-yl)ethyl)amino)ethoxy)-3-(4-methox-
CDCl3) d 8.19 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.50 (d, J ¼ 8.8 Hz,
yphenyl)-4H-chr-omen-4-one (7)
The compound was obtained as pale yellow solid in 10.5% yield.
2H), 6.99–6.96 (m, 3H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.12 (t, J ¼ 6.4 Hz,
Purity 99.6% (by HPLC); mp 236.0 –238.3 ꢁC; 1H NMR (400 MHz,
2H), 3.84 (s, 3H), 2.50 (t, J ¼ 7.2 Hz, 2H), 2.42 (brs, 4H), 2.07–2.00
(m, 2H), 1.64–1.58 (m, 4H), 1.48–1.44 (m, 2H); 13C NMR (100 MHz,
CD3OD) d 8.14 (d, J ¼ 0.8 Hz, 1H), 8.03 (d, J ¼ 8.8 Hz, 1H), 7.46 (d,
CDCl3) d 175.9, 163.5, 159.6, 158.0, 152.0, 130.1, 127.7, 124.8,
J ¼ 8.8 Hz, 2H), 7.20 (d, J ¼ 8.8 Hz, 1H), 7.05 (s, 1H), 7.01 (d,
124.3, 118.3, 114.9, 114.0, 100.6, 67.2, 55.7, 55.3, 54.7, 26.6, 26.0,
J ¼ 2.4 Hz, 1H), 6.96 (d, J ¼ 8.8 Hz, 2H), 6.89–6.85 (m, 2H), 6.72 (dd,
24.4. HRMS: calcd for C24H28NO4[M þ H]þ, 394.2018, found:
J ¼ 8.8, 2.4 Hz, 1H), 4.13 (t, J ¼ 4.2 Hz, 2H), 3.81 (s, 3H), 3.75 (s, 3H),
3.03 (t, J ¼ 4.2 Hz, 2H), 2.97 (t, J ¼ 3.6 Hz, 4H); 13C NMR (100 MHz,
394.2017.
CD3OD) d 177.9, 164.8, 161.1, 159.5, 154.9, 154.9, 133.5, 131.3,
128.9, 128.2, 125.9, 125.4, 124.4, 119.2, 116.2, 114.8, 113.0, 112.8,
3-(4-Methoxyphenyl)-7-(4-(piperidin-1-yl)butoxy)-4-H-chromen-4-
one (3)
112.6, 101.9, 101.2, 68.3, 56.2, 55.7, 50.3, 48.7, 26.0. HRMS: calcd
for C29H29N2O5 [M þ H]þ, 485.2076, found: 485.2077.
The compound was obtained as white solid in 93.6% yield.
Purity 99.4% (by HPLC); mp 152.8–154.0 ꢁC; 1H NMR (400 MHz,
CDCl3) d 8.19 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.51 (d, J ¼ 8.8 Hz,
7-(3-((2-(5-Methoxy-1H-indol-3-yl)ethyl)amino)propoxy)-3-(4-
methoxyphenyl)-4H-chromen-4-one (8)
2H), 6.98–6.96 (m, 3H), 6.83 (d, J ¼ 2.0 Hz, 1H), 4.08 (t, J ¼ 6.4 Hz,
2H), 3.84 (s, 3H), 2.44 (brs, 6H), 1.90–1.83 (m, 2H), 1.78–1.73 (m,
2H), 1.65–1.63 (m, 4H), 1.46 (brs, 2H); 13C NMR (100 MHz, CDCl3)
d 175.9, 163.4, 159.6, 158.0, 152.0, 130.1, 127.8, 124.9, 124.3,
118.3, 114.8, 114.0, 100.6, 68.4, 58.8, 55.4, 54.5, 27.1, 25.7, 24.2,
23.1. HRMS: calcd for C25H30NO4[M þ H]þ, 408.2175, found:
408.21738.
The compound was obtained as yellow solid in 51.8% yield. Purity
98.7% (by HPLC); mp 108.8–109.5 ꢁC; 1H NMR (400 MHz, CD3OD) d
8.17 (d, J ¼ 0.8 Hz, 1H), 8.04 (d, J ¼ 8.8 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz,
2H), 7.21 (d, J ¼ 8.4 Hz, 1H), 7.06–7.05 (m, 2H), 6.98 (d, J ¼ 8.8 Hz,
2H), 6.90 (d, J ¼ 2.0 Hz, 1H), 6.82 (dd, J ¼ 8.8, 2.0 Hz, 1H), 6.75 (dd,
J ¼ 8.8, 2.0 Hz, 1H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.82 (s, 3H), 3.80 (s, 3H),
2.98 (s, 4H), 2.87 (t, J ¼ 6.8 Hz, 2H), 2.05–1.99 (m, 2H); 13C NMR
(100 MHz, CD3OD) d 178.0, 165.0, 161.2, 159.6, 155.0, 154.9, 133.5,
131.4, 128.9, 128.2, 125.9, 125.4, 124.4, 119.1, 116.4, 114.9, 113.1,
112.7, 112.7, 101.7, 101.3, 68.4, 56.3, 55.7, 50.7, 47.5, 29.3, 25.8.
HRMS: calcd for C30H31N2O5 [M þ H]þ, 499.2233, found: 499.2231.
7-(2-(Ethyl(methyl)amino)ethoxy)-3-(4-methoxyphenyl)-4H-chro-
men-4-one (4)
The compound was obtained as pale yellow solid in 40% yield.
Purity 99.2% (by HPLC); mp 110.3–112.4 ꢁC; 1H NMR (400 MHz,
CDCl3) d 8.20 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J ¼ 8.8 Hz, 2H),
7.01–6.95 (m, 3H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.18 (t, J ¼ 6.0 Hz, 2H),
3.84 (s, 3H), 2.87 (t, J ¼ 6.0 Hz, 2H), 2.58 (q, J ¼ 7.2 Hz, 2H), 2.38 (s,
3H), 1.12 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 176.0,
163.3, 159.7, 158.0, 152.2, 130.3, 127.9, 125.0, 124.4, 118.6, 115.0,
114.1, 100.9, 67.0, 55.6, 55.5, 52.1, 42.4, 12.3. HRMS: calcd for
C21H24NO4 [M þ H]þ, 354.1705, found: 354.1701.
7-(4((2-(5-Methoxy-1H-indol-3-yl)ethyl)amino)butoxy)-3-(4-methox-
yphenyl)-4H-chromen-4-one (9)
The compound was purified by silica column (CH2Cl2:
CH3OH¼ 400: 9) and obtained as yellow solid in 26.1% yield. Purity
99.2% (by HPLC); mp 76.8–79.5 ꢁC; 1H NMR (400 MHz, CD3OD) d
8.14 (s, 1H), 8.06 (d, J ¼ 6.8 Hz, 1H), 7.45 (d, J ¼ 8.4 Hz, 2H), 7.21

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
971
(d, J ¼ 8.8 Hz, 1H), 7.04 (s, 2H), 6.97–6.95 (m, 3H), 6.75 (dd, J ¼ 8.8, 3-(4-Hydroxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-
one.HBr (14)
2.4 Hz, 1H), 4.03 (t, J ¼ 6.0 Hz, 2H), 3.80 (s, 3H), 3.80 (s, 3H), 2.98
(brs, 4H), 2.70 (t, J ¼ 7.2 Hz, 2H), 1.82–1.75 (m, 2H), 1.71–1.64 (m,
The product from the prior step (569 mg, 1.5 mmol) was dissolved
into 10 ml 40% methanol solution of HBr and refluxed at 80 ꢁC for
3 h. After cooling down to 0 ꢁC, the reactant was filtrated by suc-
tion and washed by methanol (3 ml) for three times. Then, the
liquid was removed by vacuum and the residues were recrystal-
lised by methanol. The compound was obtained as white solid in
15.8% yield. Purity 99.3% (by HPLC); mp 241.2–246.5 ꢁC; 1H NMR
(400 MHz, DMSO-d6) d 8.39 (s, 1H), 8.04 (d, J ¼ 8.8 Hz, 1H), 7.40 (d,
J ¼ 8.0 Hz, 2H), 7.21 (s, 1H), 7.10 (d, J ¼ 8.8 Hz, 1H), 6.82 (d,
J ¼ 8.0 Hz, 2H), 4.34 (t, J ¼ 4.2 Hz, 2H), 3.03 (brs, 2H), 2.77 (brs, 4H),
1.64–1.58 (m, 4H), 1.44 (brs, 2H); 13C NMR (100 MHz, D2O/CD3OD)
d 178.7, 163.4, 159.2, 157.4, 155.5, 131.5, 128.2, 125.6, 124.2, 119.1,
116.4, 116.4, 102.4, 63.3, 56.4, 54.7, 23.7, 22.1. HRMS: calcd for
C22H24NO4 [M ꢂ Br]þ, 366.1705, found: 366.1694.
2H); 13C NMR (100 MHz, CD3OD) d 178.0, 165.3, 161.2, 159.7, 155.0,
154.9, 133.5, 131.4, 128.9, 128.2, 125.9, 125.4, 124.3, 119.0, 116.4,
114.9, 113.0, 113.0, 112.7, 101.8, 101.3, 69.6, 56.3, 55.7, 50.7, 49.9,
27.7, 26.8, 25.9. HRMS calcd for C31H33N2O5 [M þ H]þ, 513.2389,
found: 513.2380.
7-(2-(Benzyl(methyl)amino)ethoxy)-3-(4-methoxyphenyl)-4H-chro-
men-4-one (10)
The compound was obtained as white solid in 76.1% yield. Purity
98.8% (by HPLC); mp 135.0–136.6 ꢁC; 1H NMR (400 MHz, DMSO-
d6) d: 8.42 (s, 1H), 8.02 (d, J ¼ 8.8 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 2H),
7.32–7.24 (m, 5H), 7.18 (s, 1H), 7.08 (d, J ¼ 8.8 Hz, 1H), 7.00 (d,
J ¼ 8.4 Hz, 2H), 4.26 (t, J ¼ 5.6 Hz, 2H), 3.79 (s, 3H), 3.59 (s, 2H),
2.79 (t, J ¼ 5.6 Hz, 2H), 2.25(s, 3H); 13C NMR (100 MHz, DMSO-d6)
d 174.6, 162.9, 159.0, 157.4, 153.5, 138.9, 130.1, 128.1, 126.9,
126.9, 124.1, 123.4, 117.6, 115.1, 113.6, 101.1, 66.8, 61.6, 55.1,
54.9, 42.3. HRMS: calcd for C26H26NO4[M þ H]þ, 416.1862, found:
416.1855.
3-(4-Hyroxyphenyl)-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one
.HBr (15)
The procedure for the synthesis of compound 15 was the same as
that of 14. The compound was obtained as white solid in 15.8%
yield. Purity 98.9% (by HPLC); mp 234.4–236.8 ꢁC; 1H NMR
(400 MHz, D2O/CD3OD) d 8.15(s, 1H), 8.01 (d, J ¼ 8.8 Hz, 1H), 7.36
(d, J ¼ 8.4 Hz, 2H), 7.12 (d, J ¼ 8.8 Hz, 1H), 7.07 (s, 1H), 6.93 (d,
J ¼ 8.4 Hz, 2H), 4.38 (t, J ¼ 4.4 Hz, 2H), 3.62 (t, J ¼ 4.4 Hz, 2H), 3.45
(brs, 4H), 2.14 (brs, 4H); 13C NMR (100 MHz, D2O/CD3OD) d 178.4,
163.4, 159.0, 157.4, 155.3, 131.4, 128.2, 125.4, 124.1, 119.0, 116.3,
116.3, 102.3, 64.6, 55.6, 54.5, 23.7. HRMS: calcd for C21H22NO4
[M ꢂ Br]þ, 352.1543, found: 352.1541.
7-(2-(Dibenzylamino)ethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-
one (11)
The crude product was recrystallised by ethyl acetate. The final
product was obtained as white solid in 49.9% yield. Purity 98.4%
(by HPLC); mp 118.9–120.0 ꢁC; 1H NMR (400 MHz, DMSO-d6) d:
8.42 (s, 1H), 8.02 (d, J ¼ 8.8 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 2H), 7.39
(d, J ¼ 7.6 Hz, 4H), 7.32 (d, J ¼ 7.6 Hz, 4H), 7.23 (t, J ¼ 7.2 Hz, 2H),
7.09 (s, 1H), 7.05–6.99 (m, 3H), 4.25 (t, J ¼ 5.6 Hz, 2H), 3.79 (s, 3H),
3.68 (s, 4H), 2.83 (t, J ¼ 5.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
d 174.6, 162.8, 159.0, 157.4, 153.4, 139.2, 130.0, 128.5, 128.2,
126.9, 126.9, 124.1, 123.3, 117.5, 115.1, 113.6, 101.0, 66.7, 57.9,
55.1, 51.2. HRMS: calcd for C32H30NO4 [M þ H]þ, 492.2175, found:
492.2169.
7-(2-(Piperidin-1-yl)ethoxy)-3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-
4H-chrom en-4-one (16)
Intermediate a5 (932 mg, 2 mmol) was dissolved in 50 ml aceto-
nitrile. Then K2CO3 (4.60 g, 33.2 mmol) and piperidine (0.31 ml,
3.13 mmol) were added. The mixture was refluxed and stirred
overnight. When the reaction ended, the reaction mixture was
poured into 200 ml water and stirred for 30 min. After that, the
mixture was filtrated by suction and the pastry was washed by
water (6 ml) for three times. The crude product was purified by
3-(4-Methoxyphenyl)-7-(2-(prop-2-yn-1-ylamino)ethoxy)-4H-chro-
men-4-one (12)
The compound was obtained as yellow solid in 54.1% yield. Purity 200–300 mush silica column (CH2Cl2:CH3OH ¼20:3). The purified
99.4% (by HPLC); mp 127.8–130.7 ꢁC; 1H NMR (400 MHz, compound was obtained as white solid in 13.6% yield. Purity
CD3COCD3) d 8.22 (s, 1H), 8.11 (d, J ¼ 8.4 Hz, 1H), 7.57 (d, 99.6% (by HPLC); mp 148.7–152.2 ꢁC; 1H NMR (400 MHz, CDCl3) d
J ¼ 7.6 Hz, 2H), 7.09–6.97 (m, 4H), 4.28 (t, J ¼ 4.8 Hz, 2H), 3.31 (s, 8.19(d, J ¼ 9.2 Hz, 1H), 7.91 (s, 1H), 7.48 (d, J ¼ 8.8 Hz, 2H),
3H), 3.49 (s, 2H), 3.12 (t, J ¼ 4.8 Hz, 2H), 2.66 (s, 1H); 13C NMR 7.00–6.96 (m, 3H), 6.86 (d, J ¼ 2.4 Hz, 1H), 4.20 (t, J ¼ 6.0 Hz, 2H),
(100 MHz, CD3COCD3) d 175.6, 164.4, 160.5, 158.8, 153.6, 131.0, 4.15 (t, J ¼ 6.0 Hz, 2H), 2.84–2.79 (m, 4H), 2.53 (brs, 8H),
128.1, 125.4, 125.1, 119.2, 115.7, 114.4, 101.7, 83.2, 72.7, 69.4, 55.6, 1.65–1.59 (m, 8H), 1.46 (brs, 4H); 13C NMR (100 MHz, CDCl3) d
47.7, 38.5. HRMS: calcd for C21H30NO4[M þ H]þ, 350.1392, found: 176.0, 163.3, 158.9, 158.0, 152.2, 130.2, 127.9, 125.0, 124.4, 118.6,
350.1383.
115.1, 114.8, 100.9, 66.9, 66.1, 58.0, 57.8, 55.3, 55.2, 26.1, 26.0,
24.3, 24.3. HR MS: calcd for C29H37N2O4 [M þ H]þ, 477.2753,
found: 477.2743.
7-(2-(((3 R,5 S,7r)-3,5-dimethyladamantan-1-yl)amino)ethoxy)-3-(4-
methoxyphenyl)-4H-chromen-4-one (13)
3-(4-Methoxyphenyl)-7-(2-oxo-2-(piperidin-1-yl)ethoxy)-4H-chro-
men4-one (17)
The compound was obtained as pale yellow solid in 41% yield.
Purity 98.9% (by HPLC); mp 127.5–129.5 ꢁC; 1H NMR (400 MHz,
CDCl3) d 8.20 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J ¼ 8.8 Hz, 2H), The piperidine (4 ml, 80 mmol) which was diluted in THF (5 ml)
7.00–6.96 (m, 3H), 6.84 (d, J ¼ 1.6 Hz, 1H), 4.15 (t, J ¼ 5.6 Hz, 2H), was added dropwise into the chloroacetyl chloride (3.1 ml,
3.84 (s, 3H), 3.04 (t, J ¼ 5.6 Hz, 2H), 2.18–2.15 (m, 1H), 1.52 (d, 40 mmol) dissolved in THF (10 ml) and stirred for 10 h at room
J ¼ 2.0 Hz, 2H), 1.35–1.27 (m, 9H), 2.21 (dd, J ¼ 12.4, 7.2 Hz, 2H), temperature, the mixture was condensed by reduced pressure to
0.86 (s, 6H); 13C NMR (100 MHz, CDCl3) d 176.0, 163.4, 159.7, 158.0, dryness and the residue was dissolved in 50 ml acetone. Then, for-
152.2, 130.3, 127.9, 125.0, 124.4, 118.6, 115.0, 114.1, 100.8, 69.5, mononetin (1 g, 3.73 mmol) and K2CO3 (4.60 g, 33.2 mmol) were
55.5, 52.4, 51.1, 49.2, 43.1, 41.4, 39.8, 32.6, 30.5, 30.4. HRMS calcd added into the solution. The reactant was refluxed overnight.
for C30H36NO4 [M þ H]þ, 474.2644, found: 474.2638.
When the reaction finished, the reaction solution was poured into

972
B. FENG ET AL.
200 ml water, stirred and filtrated by suction. The pastry was 67.0, 63.1, 55.3, 44.9, 25.6, 24.3. HRMS: calcd for C24H26NO6
[M þ H]þ, 424.1760, found: 424.1751.
washed by water and purified by 200–300 mush silica column
(CH2Cl2:CH3OH ¼100:1) .The compound was obtained as pale yel-
low solid in 45.3% yield. Purity 99.0% (by HPLC); mp
1
144.8–145.8 ꢁC; H NMR (400 MHz, CDCl3) d 8.16 (d, J ¼ 8.8 Hz, 1H),
2-((3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)ethyl prop-2-
yn-1-ylc-arbamate (20)
7.87 (s, 1H), 7.45 (d, J ¼ 8.8 Hz, 2H), 6.99 (d, J ¼ 8.8 Hz, 1H),
The procedure was the same as above. However, the piperidine
was replaced with prop-2-yn-1-amine (0.226 ml, 3.30 mmol). The
final compound was obtained as pale yellow solid in 18.7%
yield. Purity 99.4% (by HPLC); mp 155.1–157.4 ꢁC; 1H NMR
(400 MHz, DMSO-d6) d 8.42 (s 1H), 8.03 (d, J ¼ 8.8 Hz, 1H), 7.78
(t, J ¼ 5.6 Hz, 1H), 7.53 (d, J ¼ 8.8, 2.0 Hz, 2H), 7.20 (d, J ¼ 2.0 Hz,
1H), 7.10 (dd, J ¼ 8.8, 2.0 Hz, 1H), 7.01–6.97 (m, 2H), 4.37–4.32
(m, 4H), 3.79 (s, 5H), 3.10 (t, J ¼ 2.4 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) d 174.6, 162.6, 159.0, 157.4, 155.8, 153.5, 130.1, 127.0,
124.0, 123.4, 117.7, 115.0, 113.6, 101.2, 81.3, 73.0, 67.1, 62.5,
55.1, 29.8. HRMS: calcd for C22H20NO6 [M þ H]þ, 394.1291, found:
394.1286.
6.93–6.89 (m, 3H), 4.75 (s, 2H), 3.79 (s, 3H), 3.53 (brs, 2H), 3.42 (brs,
2H), 1.62–1.51 (m, 6H); 13C NMR (100 MHz, CDCl3) d 175.7, 165.0,
162.3, 159.5, 157.7, 152.2, 130.1, 127.9, 124.8, 124.1, 118.9, 114.6,
113.9, 101.4, 67.5, 55.3, 46.3, 43.2, 26.5, 25.5, 24.3. HRMS calcd for
C25H30NO5 [M þ H]þ, 424.2124, found: 424.2124.
3-(4-Methoxyphenyl)-7-(3-oxo-3-(piperidin-1-yl)propoxy)-4H-chro-
men-4-one (18)
K2CO3 (7.5 g, 54.3 mmol), 3-bromopropionic acid (6 g, 39.2 mmol)
and formononetin (2 g, 7.4 mmol) were dissolved in 100 ml acet-
one and refluxed for 2 days. After that, the reactant was con-
densed by reduced pressure to dryness and then the residue was
poured into 400 ml water. In order to precipitate the product, the
solution should be acidified with HCl (1 N) to pH 5. Then, the pre-
cipitated white solid was filtrated by suction and washed with
water (5 ml) for three times. 3-((3-(4-Methoxyphenyl)-4H-chromo-
gen 4-one-7-yl)oxy)propanoic acid crude product (2.465 g) was
obtained.
The obtained crude product was dissolved in 50 ml DMF,
then, N,N-diisopropylethylamine (1.02 ml, 5.85 mmol), piperidine
(0.5 ml, 5.1 mmol) and hydrochloro-1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide salt were added and stirred overnight at
room temperature. When the reaction finished, the reactant was
poured into 200 ml water and extracted with CH2Cl2, washed
with saturated K2CO3 and brine respectively. The organic phase
was taken, dried with Na2SO4, filtrated by suction and the fil-
trate was condensed by reduced pressure. The residues were
purified by silica column (petroleum ether:ethyl acetate ¼2:1).
The final compound was obtained as pale yellow solid in 26.9%
yield. Purity 99.5% (by HPLC); mp 161.0–163.0 ꢁC; 1H NMR
(400 MHz, CDCl3) d 8.19 (d, J ¼ 8.8 Hz, 1H), 7.91 (s, 1H), 7.50 (d,
J ¼ 8.4 Hz, 2H), 6.96 (brd, J ¼ 8.4 Hz, 3H), 6.90 (s, 1H), 4.42 (t,
J ¼ 6.0 Hz, 2H), 3.84 (s, 3H), 3.54 (brd, 4H), 2.89 (t, J ¼ 6.0 Hz,
2H), 1.70–1.60 (m, 6H); 13C NMR (100 MHz, DMSO-d6) d 174.6,
167.6, 162.9, 159.0, 157.4, 153.5, 130.1, 126.9, 124.1, 123.3,
117.5, 115.0, 113.6, 101.0, 65.1, 55.1, 45.9, 42.0, 31.8, 26.0, 25.3,
24.0. HRMS: calcd for C24H26NO5 [M þ H]þ, 408.1811, found:
408.1805.
AChE/BuChE bioassay
To measure in vitro AChE/BuChE activity, modified Ellman's
method27–30 was performed using a 96-well plate reader (BioTek
ELx808). AChE (0.5 U/mg) was extracted from rat cortex while
BuChE (3.4 U/mg) was obtained from human plasma. Each well
contained 50 ll potassium phosphate buffer (KH2PO4/K2HPO4,
0.1 M, pH 8.0), 25 ll test compounds and 25 ll enzyme. Notably,
the test compounds were firstly dissolved in the mixture of 50%
methanol and 50% DMSO, and eventually diluted so that the
final concentration of both solvents was less than 1% in the
assay. They were pre-incubated for 60 min at 37 ꢁC, and then
125 ll 5,50-dithiobis-2-nitrobenzoic acid (DNTB, 3 mM in buffer)
was added. Characterisation of the hydrolysis of acetylthiocholine
iodide or butyrylthiocholine chloride catalysed by AChE/BuChE
was performed spectrometrically at 412 nm, followed by the add-
ition of substrate (acetylthiocholine iodide or butyrylthiocholine
chloride 3 mM in water, respectively). The activity was deter-
mined by measuring the increase in absorbance at 412 nm after
15 min. For those compounds with inhibition rate >50% at the
concentration of 50 lM, the IC50 values were further determined.
A control experiment was performed under the same condition
without any inhibitor and the blank contained buffer, water,
DTNB and substrate. The described method was also performed
for BuChE bioassay. In the bioassay, donepezil (an AChE inhibitor)
and tetraisopropyl pyrophosphoramide (Iso-OMPA,
inhibitor) were used as positive controls of AChE and BuChE,
respectively.
a BuChE
2-((3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)ethyl
dine-1-carboxylate (19)
piperi-
Molecular docking
Intermediate a1 (0.748 mg, 2 mmol), K2CO3 (4.60 g, 33.3 mmol) was
dissolved in DMF (70 ml), then, piperidine (0.302 ml, 3.30 mmol)
was poured into the reactant liquid, stirred and refluxed for 3 h.
When the reaction finished, the mixture was poured into water
(300 ml), stirred for 30 min and filtrated by suction. The pastry was
washed by water (5 ml) for three times and purified by 200–300
mush silica column eluted by CH2Cl2. The compound was obtained
as pale yellow solid in 12.6% yield. Purity 99.1% (by HPLC); mp
In order to understand the protein-ligand interactions between
the synthesised isoflavone derivatives and AChE/BuChE, the most
potent inhibitor as an example was docked against AChE and
BuChE. The ligand structure was built and prepared by DS 2016
(Discovery Studio version 2016, San Diego, CA, USA). Hydrogen
atoms were added to the structure and its ionisation states at pH
7.3 to 7.5 were generated. Besides, its lowest-energy conformation
was generated prior to docking. The X-ray structures of AChE (PDB
ID: 5EIH)31 and BuChE (PDB ID: 4BDS)32 were retrieved from the
Protein Data Bank. Both of them were high-resolution structures
of protein-ligand complexes and their organisms were consistent
with those of the enzymes used for bioassay. After stripping the
1
104.5–109.4 ꢁC. H NMR (400 MHz, CDCl3) d 8.19 (d, J ¼ 8.8 Hz, 1H),
7.90 (s, 1H), 7.48 (d, J ¼ 8.8 Hz, 2H), 7.00 (dd, J ¼ 8.8, 2.0 Hz, 1H),
6.95 (d, J ¼ 8.8 Hz, 2H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.46 (t, J ¼ 6.0 Hz,
2H), 4.27 (t, J ¼ 6.0 Hz, 2H), 3.82 (s, 3H), 3.41 (brs, 4H), 1.56–1.51
(m, 6H); 13C NMR (100 MHz, CDCl3) d 175.1, 163.0, 159.5, 157.8,
155.0, 152.1, 130.1, 127.8, 124.8, 124.2, 118.6, 114.8, 113.9, 100.9, cognate ligand from each complex, molecular docking was carried

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
973
Table 1. Chemical structures of synthesised isoflavone derivatives.
out by GOLD 3.0.1 in which the target protein was kept rigid,
while the ligands were left flexible to explore the conformational
space inside the binding site. After that, the docking poses of the
ligand were visually inspected.
In silico prediction of pharmacokinetic properties
Compound
n
R1
R2
R
Pharmacokinetic properties, in particular blood–brain barrier
penetration (BBB), are quite important for the drugs for the treat-
ment of AD. Therefore, the pharmacokinetic properties of AChE/
BuChE inhibitors were predicted by the “ADMET Descriptors”
module implemented in DS2016. The pharmacokinetic properties
available in this module were aqueous solubility, BBB, CYP2D6
binding, hepatotoxicity, intestinal absorption and plasma protein
binding.
1
2
OH
CH3
CH3
CH3
2
3
3
4
H
H
4
5
6
7
2
3
4
2
H
H
H
H
CH3
CH3
CH3
CH3
Results and discussion
Chemistry
In total, 20 isoflavone derivatives (cf. Table 1) were synthesised
and their chemical structures were confirmed by melting point, HR
1
MS, H NMR and 13C NMR.
The synthetic route of new isoflavone derivatives was designed
and depicted in Scheme 1 according to earlier reports33,34. Briefly,
the bromo-substituted intermediates a1–a5 were prepared
through commercially available isoflavones and dibromoalkane in
acetone in the presence of potassium carbonate. Reaction
between a1–a5 and corresponding amines in acetonitrile afforded
compounds 1–16. At first, we synthesised compounds 1–9 and
evaluated their biological activity. Structure-activity relationship
(SAR) of these compounds demonstrated the optimal length of
the linker between the amino group and isoflavone core structure
seems to be 2. Previous reports indicated that various amino
groups can form a cation–pi interaction with the aromatic amino
acid residues (i.e. Trp84 in AChE and Try82 in BuChE) located in
the catalysis active site of the cholinesterases35. This observation
prompted us to design and synthesise compounds 10–20 by sub-
stitution of different amino groups.
8
9
3
4
H
H
CH3
CH3
10
11
2
3
H
H
CH3
CH3
12
13
4
2
H
H
CH3
CH3
It is worth noting that carbamate substituted compounds 19
and 20 can be produced by the reaction of amines and bromo-
substituted isoflavone in the presence of DMF and K2CO3 rather
than the reported reaction of acyl chloride with hydroxyl-substi-
tuted isoflavone36 in acetonitrile. Interestingly, compound 17 can
be synthesised by step 1 and step 2 as shown in Scheme 1,
however this synthetic route was not suitable for compound 18
because of the poor reactive activity of 3-chloro-1-(piperidin-1-
yl)propan-1-one which has a longer distance between reactive
site and amide compared to 2-chloro-1-(piperidin-1-yl) ethan-1-
one. Therefore, the intermediate 3-((3-(4-methoxyphenyl)-4H-
chromogen-4-one-7-yl)oxy)propanoic acid was obtained by the
reaction of formononetin with 3-bromopropionic acid. Piperidine
was then added to the intermediate to produce the final com-
pound 18.
14
2
H
H
H
15
16
17
2
2
1
H
H
H
CH3
CH3
CH3
CH3
18
19
20
2
2
2
H
H
H
In vitro inhibition of AChE and BuChE
Both in vitro AChE and BuChE inhibitory activity were evaluated
for all the synthesised isoflavone derivatives. The results are shown
in Table 2. To be noted donepezil and Iso-OMPA were used as the
positive drugs.
As shown in Table 2, five isoflavone derivatives, i.e. 1, 3, 14, 15
and 16, showed both AChE and BuChE inhibitory effect. Notably,

974
B. FENG ET AL.
Scheme 1. The general procedure for the synthesis of compounds 1–20. (i): K2CO3, acetone, Br(CH2)nBr, 60 ꢁC; (ii): RH (amines), K2CO3, DMF/Acetonitrile, 100ꢁC, 3 h;
(iii): 40% HBr, 120ꢁC, 3 h; (iv): piperidine, THF r.t. 10 h; (v): formononetin, K2CO3, acetone; (vi) K2CO3, 3-bromopropionic acid, acetone, 60 ꢁC, 2 days; (vii) DMF, N, N-diiso-
propylethylamine, piperidine, 12 h, r.t.
compound 16 displayed equivalent inhibitory activity to two sin-
gle-targeting drugs (Donepezil and Iso-OMPA) as well as our hit
compound G.
Table 2. In vitro inhibition of AChE and BuChE for compounds 1–20.
Inhibition % at 50 lM IC50 (lM)
Name
AChE
BuChE
AChE
BuChE
Based on the chemical structures and their biological activity, it
is clear that AChE and BuChE inhibitory activities changed mark-
edly because of the change of amino substitutes. Compounds
would manifest inhibitory effect when C7 was substituted by
piperidine or N-methylethanamide. In the meanwhile, the length
of the side chain also played a significant role in maintaining the
inhibitory activity. Generally, compounds with side chains of two
or four carbon atoms can display a dual-targeting inhibition
whereas those with three carbon atoms selectively inhibited
BuChE. Besides, compounds with a side chain of four carbon
atoms showed stronger inhibition for BuChE than those with two
carbon atoms. However, ester-substituted compounds displayed
no AChE inhibitory effect. The alternative isoflavone core changed
the inhibition level for both AChE and BuChE. C5 hydroxyl substi-
G
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Donepezil
Iso-OMPA
n.d.a
71.10
28.34
85.65
58.66
25.38
63.76
3.68
n.d.
95.74
97.00
98.11
84.13
91.32
94.77
45.74
60.63
37.92
2.72
ꢂ18.48
ꢂ16.87
ꢂ6.62
99.86
99.88
99.74
ꢂ2.83
5.59
1.47
10.3
n.d.
46.39
100.4
n.d.
106.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.37
4.12
23.2
11.1
141.3
103.6
68.08
n.d.
15.6
n.d.
n.d.
n.d.
n.d.
2.42
0.72
3.34
ꢂ6.27
5.47
17.61
79.38
75.22
100.34
0.54
ꢂ2.36
ꢂ11.56
ꢂ4.84
64.82
n.d.
n.d.
n.d.
9.75
57.74
4.60
n.d.
n.d.
n.d.
n.d.
1.05
n.d.
7.66
7.19
5.92
n.d.
n.d.
9.59
n.d.
0
tution improved the inhibitory effect. When C4 was substituted
for 2-piperidineethoxyl group, the most potent AChE inhibitor 16
was obtained. All the information provided us with clues for fur-
ther lead optimisation.
53.03
ꢂ5.49
n.d.
n.d.
5.78
94.28
an.d.: not determined.
Binding modes of isoflavone derivatives
As compound 16 was the most potent AChE/BuChE dual-targeted
inhibitor, it was selected as an example and used in the docking
simulation. As shown in Figure 3, compound 16 was able to bind
both AChE and BuChE. Regarding AChE, the piperidine moiety in
C4’ position occupied the “entrance cavity” by forming p–cation
interaction with the key amino acid residues TRP286 and TYR341,
while the isoflavone core structure was well accommodated in the
catalytic cleft through p–p T-shaped interactions with PHE297/
TYR341 and hydrogen bonds with TYR124/TYR337. Furthermore,
the p–cation interaction between the piperidine ring in C7 position
with HIS447 also contributed to the stabilisation of protein–ligand
interaction.
Protein–ligand interactions between compound 16 with
BuChE were similar to that for AChE (cf. Figure 4). It deserved to
mention that the piperidine moiety in C7 position formed a
hydrogen bond with the key amino acid residue TYR332 which
can also interact with the isoflavone structure through p–p stack-
ing and p–p T-shape. Protonated amino group in C4 position can
stabilise the complex through forming a salt bridge with both
with TRP86, the salt bridge with GLU202 and the hydrogen bond TRP82.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
975
Figure 3. (a) 2D schematic diagram of potential interactions between compound 16 and AChE. (b) The predicted binding mode of compound 16 with AChE.
Predicted pharmacokinetic properties
in silico HTS. The in vitro AChE/BuChE bioassay has shown 5
out of 20 isoflavone derivatives were AChE/BuChE dual-targeted
inhibitors. SAR analysis has demonstrated the length of the
side chain and the type of amino-substituted group played an
essential role in AChE inhibition, whereas both factors had lit-
tle effect on the BuChE inhibition. Among these derivatives,
compound 16 possessed the greatest AChE inhibition as well
as strong BuChE inhibitory activity. The molecular docking
study demonstrated that p–cation and p–p interactions were
essential for compound 16 to display its dual-targeting effect.
In addition, the in silico prediction of pharmacokinetic proper-
ties has indicated 1) compounds 3, 14 and 16 were able to
penetrate the BBB and 2) compound 16 may be more effect-
ive and less toxic due to no binding to CYP2D6. Taken
together, compound 16 may warrant further development as a
potential drug-like AChE/BuChE dual-targeted inhibitor for the
treatment of AD.
As shown in Table 3, all the AChE/BuChE dual-targeted inhibitors
reported in this study appeared to have poor solubility in aqueous
media whereas possess good absorption. Like compound G, com-
pounds 3, 14 and 16 were predicted to penetrate the BBB, which
was a favoured property for drugs to treat neurodegenerative dis-
eases, e.g. AD. Encouragingly, compound 16 may not bind to
CYP2D6, which was different from other compounds, including the
hit compound G. This unique feature would be beneficial for
ensuring the efficacy of compound 16 and avoiding the side-
effect. These data prove our hit-to-lead optimisation strategy
seems to be also effective in optimising pharmacokinetic property.
Conclusion
In this study, we report the synthesis of a series of novel iso-
flavone derivatives based on our hit compound G identified by

976
B. FENG ET AL.
Figure 4. (a) 2D schematic diagram of potential interactions between compound 16 and BuChE. (b) The predicted binding mode of compound 16 with BuChE.
Table 3. Predicted pharmacokinetic properties of compounds 1, 3, 14, 15 and 16.
Compound
AlogP98
PSA-2D
Solubility level
Absorption level
BBB level
PPB
CYP2D6
1
3
14
15
16
G
3.647
4.532
3.664
3.208
4.945
4.267
68.259
47.443
59.328
59.328
50.796
47.443
2
2
2
2
2
2
0
0
0
0
0
0
2
1
1
2
1
1
True
True
True
True
True
True
True
True
True
True
False
True
AlogP98: Lipophilicity descriptor; PSA-2D: Polar surface area; AlogP98: Lipophilicity descriptor; PSA-2D: Polar surface area; Solubility Level: (0, Good; 1, Moderate; 2, Poor;
3, Very poor); Absorption Level: (0, Good; 1, Moderate; 2, Poor; 3, Very poor); BBB Level: (0, very high blood–brain barrier penetration; 1, high; 2, medium; 3, low).
Disclosure statement
References
The authors declare no conflict of interest in this work.
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