Design, synthesis and evaluation of novel 7-aminoalkyl-substituted flavonoid derivatives with improved cholinesterase inhibitory activities

Article abstract of DOI:10.1016/j.bmc.2015.12.031

A novel series of 7-aminoalkyl-substituted flavonoid derivatives 5a-5r were designed, synthesized and evaluated as potential cholinesterase inhibitors. The results showed that most of the synthesized compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities at the micromolar range. Compound 2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5q) showed the best inhibitory activity (IC₅₀, 0.64 μM for AChE and 0.42 μM for BChE) which were better than our previously reported compounds and the commercially available cholinergic agent Rivastigmine. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5q with AChE and BChE. Furthermore, molecular modeling study showed that 5q targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds (5a-5r) did not affect PC12 and HepG2 cell viability at the concentration of 10 μM. Consequently, these flavonoid derivatives should be further investigated as multipotent agents for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2015.12.031

Accepted Manuscript
Design, Synthesis and Evaluation of Novel 7-Aminoalkyl-Substituted Flavo-
noid Derivatives with Improved Cholinesterase Inhibitory Activities
Wen Luo, Ying Chen, Ting Wang, Chen Hong, Li-Ping Chang, Cong-Cong
Chang, Ya-Cheng Yang, Song-Qiang Xie, Chao-Jie Wang
PII:
S0968-0896(15)30197-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.031
BMC 12724
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 November 2015
16 December 2015
17 December 2015
Please cite this article as: Luo, W., Chen, Y., Wang, T., Hong, C., Chang, L-P., Chang, C-C., Yang, Y-C., Xie, S-
Q., Wang, C-J., Design, Synthesis and Evaluation of Novel 7-Aminoalkyl-Substituted Flavonoid Derivatives with
Improved Cholinesterase Inhibitory Activities, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
10.1016/j.bmc.2015.12.031
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Design, Synthesis and Evaluation of Novel 7-Aminoalkyl-Substituted
Flavonoid Derivatives with Improved Cholinesterase Inhibitory Activities
Wen Luo§,a,*, Ying Chen§,a, Ting Wanga, Chen Honga, Li-Ping Changa, Cong-Cong Changa, Ya-Cheng
Yanga, Song-Qiang Xieb, Chao-Jie Wang a,*
a
Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004,
People’s Republic of China
b Institute of Chemical biology, Henan University, Kaifeng 475004, People’s Republic of China
* Wen Luo: Email luowen83@henu.edu.cn, Tel 086 0371-22864665, Fax 086 0371-22864665
* Chao-Jie Wang: Email wcjsxq@163.com, Tel 086 0371-22864665, Fax 086 0371-22864665
§ The first two authors contributed equally to this work.
ABSTRACT
A novel series of 7-aminoalkyl-substituted flavonoid derivatives 5a-5r were designed, synthesized and
evaluated as potential cholinesterase inhibitors. The results showed that most of the synthesized
compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory
activities
at
the
micromolar
range.
Compound
2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl) octyloxy)-4H-chromen-4-one (5q) showed the best inhibitory
activity (IC50, 0.64 M for AChE and 0.42 M for BChE) which were better than our previously
reported compounds and the commercially available cholinergic agent Rivastigmine. The results from a
Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5q with AChE and BChE.

3
Furthermore, molecular modeling study showed that 5q targeted both the catalytic active site (CAS) and
the peripheral anionic site (PAS) of AChE. Besides, these compounds (5a-5r) did not affect PC12 and
HepG2 cell viability at the concentration of 10 M. Consequently, these flavonoid derivatives should be
further investigated as multipotent agents for the treatment of Alzheimer’s disease.
KEYWORDS
Flavonoid derivatives
Cholinesterase
Anti-Alzheimer agent
Molecular modeling
Cytotoxicity

4
1. Introduction
Alzheimer’s disease (AD) represents a progressive neurodegenerative brain disorder that is
characterized by dementia, memory loss and other cognitive impediments, particularly among elderly
individuals.1 A variety of factors are considered key pathological hallmarks of AD, such as low levels of
acetylcholine, -amyloid (A) deposits and oxidative damage.2 These factors provide a basis for
cholinergic, amyloid and oxidative stress hypotheses potentially explaining the origin of Alzheimer’s
disease.3, 4
Current treatment options for AD mainly focus on improving the cholinergic neurotransmission in
the brain, which is mostly based on the ‘cholinergic hypothesis’.5 Various approaches have explored this
hypothesis, however, cholinesterase inhibitors were the first and, to date, are the only agents that offer
promising results in the treatment of AD.6 Currently, four AChE inhibitors have been approved by the
European and US regulatory authorities: Tacrine, Donepezil, Rivastigmine and Galantamine. These
inhibitors exhibit beneficial effects on cognitive, functional and behavioural symptoms of AD. However,
accumulative side effects or demerits such as hepatotoxicity, periphery side effect, short half-life, or
gastrointestinal tract disorders diminish the overall clinical applicability.8 Therefore, the development of
efficient AChE inhibitors with low toxicity still continues to be a critical goal in AD therapy.
Two types of cholinesterase (ChE) species are found in the central nervous system,
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with both species able to hydrolyze
acetylcholine. In the healthy brain, AChE predominates over BChE activity, however, during the early
development of AD, AChE activity decreases in certain brain areas, whereas BChE levels remain
unchanged or may even rise with disease progression.9 Furthermore, BChE may also participate in the

5
aggregation of A. As a consequence, a good balance in the AChE and BChE inhibition profiles may
provide additional benefits in the treatment of AD.
Flavonoids represent a class of polyphenolic compounds found in plants, such as fruit, vegetables, tea
and soybeans.11 A wide range of physiological and biochemical functions are displayed by flavonoids,
including UV-protection, anti-tumor, antioxidant, neuroprotection and anti-inflammatory
characteristics.12,13 In general, flavonoids feature low toxicity and have received much attention by a
variety of research fields in medicinal chemistry.14 However, not many articles have been reported
focusing on new flavonoid derivatives as anti-AD agents. In recent years, only a few research groups
have selected flavonoids as lead compounds in an effort to optimize the structures for the development
of ChEs inhibitors as a treatment option for AD (Fig. 1, compounds A, B and C).15-17
Figure 1. Flavonoids as new drugs or imaging agents in the treatment of AD
For many years, our group has focussed on the synthesis of flavonoids and the further development of
ChE inhibitors.18,19 We have previously studied a series of N, N-dimethylated flavonoid derivatives as
multifunctional ChEs inhibitors and found that these derivatives feature a high inhibitory potency for
both AChE and BChE (Fig. 2).20 We now focus our interests on the development of novel flavonoid
derivatives with high potency to find use as ChE inhibitors. In the present study, a series of new
flavonoid derivatives with B ring modifications were designed, synthesized and evaluated for their
biological activity, including cytotoxicity studies and investigations on the inhibition characteristics of

6
cholinesterase.
Figure 2. Our previous work and present work
2. Results and discussion
2.1 Chemistry
The synthetic route to the target compounds (5a–5r) starting from commercially available
1,3-benzenediol (1) was shown in Scheme 1. Condensation of 1,3-benzenediol (1) with
chloroacetonitrile in the presence of ZnCl2 in ether was followed by hydrolysis in water and produced
ketone in 87% yield. The ketone 2 was treated with aryl aldehydes in the presence of excess NaOH in
EtOH, was followed by acidification with aqueous HCl and resulted in the flavonoids 3 in satisfactory
yield.20 The alkylation of 3 with 1,8-dibromooctane in acetone provided 4 in good yields. Finally, the
reaction of 4 with pyrrolidine produced the targets compounds 5a-5r (except 5h, 5j and 5l) in 42-65%
yields. 5h, 5j and 5l were obtained by deprotection from compounds 5g, 5i and 5k. The structures of the
target compounds were confirmed by 1H NMR, 13C NMR and MS.

7
Scheme 1. Synthesis of flavonoid derivatives. Reagents and conditions: (a) ClCH2CN, ZnCl2, EtOEt, HCl, 0 C; (b)
HCl, H2O, reflux; (c) aryl aldehyde, 10% NaOH, rt, EtOH; (d) 1,8-dibromooctane, K2CO3, acetone, reflux; (e)
pyrrolidine, K2CO3, CH3CN, 60 C, (f) HOAc/HCl, 80 C.
2.2 In vitro inhibition studies on AChE and BChE
The synthesized compounds 5a-5r were evaluated for their in vitro inhibitory activities towards AChE
and BChE using the Ellman method.21 The results were subsequently compared to the commercially
available standard cholinergic agent Rivastigmine. The anti-cholinesterase activities and their selectivity
indexes are summarized in Table 1.
The IC50 values obtained reveal that all of the tested compounds 5a-5r exhibit very potent inhibitory
activities (IC50 values = 0.64-4.80 µM), far superior to the standard drug Rivastigmine (IC50 values =
6.35 µM). Particularly the 2-methoxy derivative 5b appears to be the most potent compound against

8
AChE, with an IC50 value of 0.64 µM. Concurrently, this compound proves to be about 10-fold more
potent than Rivastigmine and 3-fold more potent than our previously reported compound (5s). Moreover,
the 3-methoxy, 2,3-dimethoxy and the 1-naphthyl derivatives 5c, 5e and 5q with IC50 values of 0.67,
0.83 and 0.72 µM, respectively, also feature high activities against AChE. Comparing the unsubstituted
compound 5a to the ortho-, meta- or para-substituted analogs 5b, 5c and 5d shows that the introduction
of a methoxy group in 2- or 3- but not in 4-position on the B ring in the flavonoid scaffold significantly
improved the inhibitory activity against AChE. Furthermore, a bigger or smaller group such as a
benzyloxy group (5g and 5i) or a hydroxy group (5h and 5j) in 2- or 3-position leads to a decrease in the
AChE inhibitory activity. Comparing the the para-substituted analogs 5d, 5k-5p, 5r and 5s, revealed
that the introduction of a larger group in 4-position (5k, 5o and 5r) does in fact not increase the AChE
inhibitory activity.
The observed IC50 values of the target compounds against BChE indicate that all compounds with the
exception of 5b, 5h and 5q were determined to be less potent than the standard drug Rivastigmine. Most
impressively, the anti-BChE activity of the most potent compound 5q was determined to be 4 times
higher than that of Rivastigmine and 6 times higher than our previously reported compound (5s). The
2-methoxy and 2-hydroxy derivatives 5b and 5h with IC50 values of 0.76 and 1.26 µM, respectively, also
showed significant anti-BChE activities. However, most of the substituted benzyl derivatives were not
less potent than the substituted analog 5a against BChE. Taken in concert, these results demonstrate that
an unsuitable substituent and/or functional group on the B ring results in a detrimental effect on the
anti-BChE activity.
As calculated in Table 1, most of compounds (except 5h and 5q) showed a high selectivity for AChE
(SI > 1) over BChE. The most active species (5q) against BChE features a similar selectivity index (SI =
0.58) as Rivastigmine (SI = 0.27).

9
Table 1. Inhibition of ChE activity and selectivity index.
IC50a for AChE IC50b for BChE
Ar
Compds
SIc
(M)
(M)
2.05
5a
1.09±0.03
2.24±0.01
1.19
3.42
3.19
5b
5c
5d
0.64±0.04
0.67±0.05
1.70±0.05
0.76±0.02
2.29±0.04
5.42±0.13
3.43±0.22
4.13
2.04
2.62
5e
5f
0.83±0.01
1.08±0.02
2.01±0.03
2.20±0.06
5.26±0.21
5g
0.41
>15.8
4.39
5h
5i
3.07±0.21
1.58±0.12
1.12±0.05
1.26±0.01
>25
5j
4.92±0.67

10
13.16
5k
1.90±0.13
>25
3.79
1.54
3.09
4.77
5l
5m
5n
5o
1.21±0.08
1.25±0.01
1.12±0.02
4.58±0.08
1.92±0.003
3.46±0.23
10.25±0.27
2.150.12
2.97
0.58
5.21
5p
5q
5r
1.38±0.11
4.10±0.43
0.42±0.01
>25
0.720.03
4.80±0.21
5s d
1.31
0.27
1.99±0.08
6.35±1.50
2.60±0.03
Rivastigmine
-
1.71±0.09
a AChE from electric eel; IC50, 50% inhibitor concentration (means ± SEM of three experiments).
b BChE from equine serum; IC50, 50% inhibitor concentration (means ± SEM of three experiments).
c SI: Selectivity Index =IC50 (BChE)/ IC50 (AChE)
d Data of ChEs inhibition were taken from ref. [20].
2.3 Kinetic characterization of ChE inhibition
The compound that appeared to be the most successful at inhibiting ChEs was 5q. We further

11
investigated 5q using graphical analysis of steady state inhibition data (Fig. 2, A). The Lineweaver–Burk
plots showed both increasing slope (decreased Vmax) and increasing intercept (higher Km) for higher
inhibitor concentrations, indicating a mixed-type inhibition. Replots of the slope versus concentration of
compound 5q gave an extimate of the inhibition constant, Ki of 0.206 M. This suggested that
compound 5q was able to bind both CAS and PAS of AChE and supported the results of the molecular
modeling study. The same inhibition type between 5q and BChE was found in graphical analysis (Fig. 2,
B).
Non inhibitor
[5q]= 0.5 M
[5q]= 1.0 M
[5q]= 2.0 M
Non inhibitor
[5q]= 0.5 M
[5q]= 1.0 M
[5q]= 2.0 M
B
A
20
15
10
5
15
10
5
0
0
-10
-5
0
5
10
-1
15
20
-10
-5
0
5
10
-1
15
20
-1
-1
[S] mM
[S] mM
Figure 2. Lineweaver-Burk plot for the inhibition of AChE (A) and BChE (B) by compound 5q.
2.4 Molecular modeling study
In order to obtain functional and structural insight into the binding interactions between compound 5q
22
and TcAChE (PDB code: 1ACJ), molecular docking simulation was performed using Autodock 4.0
and PyMOL software as shown in Figure 3. Regarding the docking studies, compound 5q occupied the
enzymatic CAS, mid-gorge and PAS, the charged nitrogen of pyrrolidine showed a cation- interaction
with Trp84 and Phe330, and a hydrogen bond (3.6 Å) was found with His440 at the CAS. At the PAS,
the naphthalene ring of flavonoid stacked against the Trp279 through - stacking with a distance of 3.8

12
Å. The docking studies showed that compound 5q exhibited a mixed type of inhibition, consistent with
our kinetic analysis result. Since the crystal structure of BChE from equine serum has not been reported,
so we did not perform the docking study for compound 5q with BChE.
Figure 3. Docking model of 5q-TcAChE complex.
2.5. MTT assay of PC12 and HepG2 cells viability
To investigate the effect of the new compounds on cell viability, a MTT assay was conducted using
PC12 and HepG2 cell lines. The cells were incubated for 48 hours with varying target compound
concentrations (1 and 10 µM). At a concentration of 1 µM, all of the compounds were shown to be
nontoxic and had no effect on the cell viability of PC12 and HepG2 cells. At a concentration of 10 µM,
all of the compounds except for 5r were shown to be nontoxic to PC12 cells, and most of the
compounds showed no obvious effect on the cell viability of HepG2 cells.
Table 2. Inhibition rate (%)a of PC12 and HepG2 cells
PC12
10 M
HepG2
10 M
Compd.
1 M
-2.83
3.08
1 M
11.31
5.9
1.37
3.4
8.97
5a
5b
24.32

13
3.53
-0.15
7.29
-4.47
0.47
-7.71
0.79
-4.43
-2.83
0.70
-0.55
6.27
2.50
0.29
3.00
0.46
4.62
-18.19
-6.72
-2.94
-13.85
22.86
6.61
16.22
6.69
23.66
21.48
76.04
41.39
34.08
60.58
30.60
43.87
54.34
21.39
77.08
25.66
10.83
19.03
18.9
5c
5d
13.48
16.26
19.73
5.92
5e
5f
5g
5h
-2.53
-6.56
1.37
6.63
5i
19.02
10.41
18.52
9.17
5j
5k
7.42
5l
-0.03
-1.7
5m
5n
6.39
-12.49
-9.56
9.01
13.32
5.74
5o
5p
4.42
5q
68.90
13.91
71.92
5r
10.24
5.78
15.05
Tacrine
a The values are the mean  SD of three independent experiments.
2.6. ADMET prediction
The in silico prediction of physicochemical, ADME and toxicity properties of compounds 5b and 5q
23
were calculated using the ACD/Labs Percepta Platform (License#58830) and results were compared
to those obtained for donepezil and tacrine.
As demonstrated in Table 3, the druglikeness of compound 5b and 5q was similar to donepezil and
different from tacrine, with no violations of Lipinsky's rule of 5 (except rotatable bonds). Regardless of
the poor solubility, compounds 5b and 5q were showed to be highly or moderately permeable based on
predicted Log P, permeability across Caco-2 monolayers (Pe) and human intestinal absorption (HIA) test.
The drug safety profile of 5b and 5q was also projected using Program ACD/Percepta, based on
probabilistic predictors. The metabolic stability, the inhibition of hERG (the human
Ether-a-go-go-Related Gene) and the mutagenic profile were calculated and the results converted in the

14
so called classification scores (Table 4). As depicted in Table 3 compounds 5b and 5q were predicted as
undefined (score > 0.33 and < 0.67) in HLM (human liver microsomes) and hERG, which were similar
with donepezil and tacrine. However, tacrine was predictable to be mutagenic (score > 0.67), and
compounds 5b and 5q were predicted as undefined (score > 0.33 and < 0.67).
Table 3. Physico-chemistry properties and ADMET profile of compounds 5b, 5q, donepezil and tacrine calculated
using the Program ACD/Percepta.
Compounds
Predicted properties
Donepezil
Tacrine
5b
5q
MW (g/mol)
H-Donors
H-Acceptor
Rot. Bonds
Rings
449.58
469.61
379.49
198.26
0
0
0
2
5
4
11
4
2
12
6
0
4
1
5
4
0
3
0
Lipinski
Solubility
Log P
1 violation
0.02 mg/ml
7.05
0.05 mg/ml
6.43 mg/ml
6 mg/ml
5.77
4.23
2.86
Pe=3010-6 cm/s Pe=210-6 cm/s Pe=19410-6 cm/s Pe=6310-6 cm/s
Caco-2
PPB
99%
-3.48
100%
0.62
99%
-4.57
100%
0.57
95%
-2.65
100%
0.54
78%
-2.59
100%
0.52
CNS
HIA
HLM
AMES
0.43
0.44
0.26
0.85
hERG
0.58
0.54
0.53
0.45
Table 4. The meaning of classification score range values for ADMET properties using Program
ACD/Percepta.
Predicted ADMET properties
hERG
Classification
scores
HLM
Stable
AMES
≤ 0.33
>0.33 and ≤ 0.67
> 0.67
Non-inhibitor
Undefined
Inhibitor
Non-mutagenic
Undefined
Undefined
Undefined
Mutagenic

15
3. Conclusion
In conclusion, we have developed a series of 7-aminoalkyl-substituted flavonoid derivatives 5a-5r as
AChE and BChE inhibitors. These synthetic derivatives feature a higher inhibitory potency for both
AChE and BChE compared to other compounds we published previously as well as Rivastigmine.
Compounds 5b, 5c, 5e and 5q (IC50 < 1.0 µM) were found to exhibit the best AChE inhibitory activity.
Compounds 5b and 5q (IC50 < 1.0 µM) exhibited the highest inhibitory effect for BChE. Kinetic
analysis studies revealed that compound 5q features a mixed-type inhibition activity on both AChE and
BChE. Furthermore, molecular modeling studies indicate that compound 5q is able to bind to both CAS
and PAS in AChE. Moreover, at a concentration of 10 µM, most of these compounds demonstrate no
obvious toxicity effects on the cell viability of PC12 and HepG2. Our results indicated this new type of
flavonoid derivatives may provide a useful template for further optimization in the field of AD
pharmacotherapy.
4. Experimental section
4.1 Chemistry
1
Melting points (mp) were determined using an X-6 hot stage microscope and were not corrected. H
and 13C NMR spectra were recorded using TMS as the internal standard in CDCl3 with a Bruker
AV-300 spectrometer. MS spectra were recorded on a Shimadzu LCMS-2010A instrument with an ESI
mass selective detector. Elemental analyses were performed on a Gmbe VarioEL Elemental Instrument.
Flash column chromatography was performed with silica gel (200 - 300 mesh) purchased from Qingdao
Haiyang Chemical Co. Ltd.
4.2 General procedures for the synthesis of intermediate 3
To a mixture of acetophenone 2 (3.0 mmol) and different benzaldehyde (3.0 mmol) in ethanol (25 mL)

16
was added 10% sodium hydrate aqueous (5 mL), the mixture was stirred for 24 h at room temperature.
The solution was acidified with 1N HCl and filtered, and the filter cake was recrystallized from ethanol
got 3 which was used without further purification.
4.3 General procedures for the preparation of intermediate 4
To a solution of intermediate 3 (2.0 mmol) and anhydrous K2CO3 (1.38 g, 10.0 mmol) in acetone (10
mL), 1, 8-dibromooctane (4.0 mmol) was added. After reflux for 10 h until the starting material 3
disappeared, the solvent was removed under vacuum, the residue was poured into water and extracted
with EtOAc, the solution was dried over Na2SO4 and then concentrated, the compounds 4 were
crystallized from ethanol which were used without further purification.
4.4 General procedures for the preparation of compounds 5a-5r (except 5h, 5j and 5l)
To a solution of 4 (0.5 mmol) in CH3CN (10 mL) was added pyrrolidine (1.0 mmol) and anhydrous
K2CO3 (2.5 mmol). After stirring at 60 °C for 8 h, the solvent was removed under vacuum, the mixture
was diluted with CHCl3 and then washed with water and brine. The organic layer was dried over Na2SO4,
filtered, concentrated in vacuo, and purified by column chromatography with CHCl2/MeOH/NH4OH
(30:1:0.5%) elution, and then crystallized from ethanol.
4.5 General procedures for the preparation of compounds 5h, 5j and 5i
Compounds 5g, 5i or 5k (1 mmol) and a mixture of acetic acid (7 mL) and concentrated hydrochloric
(3.7 mL) were heated on a steam bath for 1.5 h. The solution was brought to neutrality with NaOH and
then diluted with CHCl3. The organic layer was dried over Na2SO4, filtered, concentrated in vacuo, and
purified by column chromatography with CHCl2/MeOH (15:1) elution.
4.5.1 2-phenyl-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5a)
Light yellow solid (56% yield), mp 96-98 C; 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.2 Hz, 2H),
7.71 (d, J = 8.3 Hz, 1H), 7.46 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 7.3 Hz, 1H), 6.83 (s, 1H), 6.80 – 6.71 (m,

17
2H), 4.08 (t, J = 6.5 Hz, 2H), 2.66 (d, J = 49.4 Hz, 6H), 1.95 – 1.79 (m, 6H), 1.64 (s, 2H), 1.48 (d, J =
7.4 Hz, 2H), 1.38 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 183.06, 168.64, 167.07, 147.89, 132.46,
131.31, 129.60, 128.86, 125.79, 114.56, 112.68, 111.75, 96.99, 68.92, 56.30, 54.00, 29.28, 29.14, 28.88,
27.31, 25.86, 23.38. MS (ESI+): m/z: 420.1[M+H]+. Elemental Anal. Calcd for C27H33NO3: C, 77.29; H,
7.93; N, 3.34. Found: C, 77.45; H, 7.90; N, 3.46.
4.5.2 2-(2-methoxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5b)
1
Yellow solid (58% yield), mp 101-103 C; H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 7.7 Hz, 1H),
7.64 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.01 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.68
(s, 2H), 3.99 (t, J = 6.2 Hz, 2H), 3.86 (s, 3H), 2.47 (s, 4H), 2.44 – 2.37 (m, 2H), 1.76 (s, 6H), 1.47 (dd, J
= 15.7, 7.4 Hz, 4H), 1.33 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 182.83, 168.29, 166.75, 158.52, 147.83,
131.74, 131.07, 125.54, 121.31, 120.69, 114.69, 112.42, 110.59, 105.73, 96.84, 68.86, 56.65, 55.47,
54.23, 29.48, 29.23, 29.07, 28.90, 27.63, 25.86, 23.37. MS (ESI+): m/z: 450.3[M+H]+. Elemental Anal.
Calcd for C28H35NO4: C, 74.80; H, 7.85; N, 3.12. Found: C, 75.06; H, 7.93; N, 3.04.
4.5.3 2-(3-methoxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5c)
Yellow solid (62% yield), mp 72-74 C; 1H NMR (300 MHz, CDCl3) 7.63 (d, J = 9.1 Hz, 1H), 7.41 (d,
J = 8.2 Hz, 2H), 7.31 (t, J = 8.2 Hz, 1H), 6.89 (d, J = 9.1 Hz, 1H), 6.73 – 6.66 (m, 3H), 4.00 (t, J = 6.5
Hz, 2H), 3.83 (s, 3H), 2.65 (s, 4H), 2.57 – 2.50 (m, 2H), 1.87 – 1.75 (m, 6H), 1.57 (d, J = 6.7 Hz, 2H),
13
1.42 – 1.31 (m, 8H). C NMR (75 MHz, CDCl3) δ 182.95, 168.59, 167.08, 159.69, 147.96, 133.63,
129.77, 125.72, 124.03, 116.34, 115.26, 114.46, 112.69, 111.55, 96.98, 68.94, 56.39, 55.32, 54.08, 29.33,
29.16, 28.88, 28.17, 27.40, 25.85, 23.37. MS (ESI+): m/z: 450.2[M+H]+. Elemental Anal. Calcd for
C28H35NO4: C, 74.80; H, 7.85; N, 3.12. Found: C, 74.80; H, 7.93; N, 3.26.

18
4.5.4 2-(4-methoxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5d)
Yellow solid (65% yield), mp 77-79 C; 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.8 Hz, 2H), 7.67
(d, J = 9.1 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.78 (s, 1H), 6.75 – 6.69 (m, 2H), 4.05 (t, J = 6.5 Hz, 2H),
3.86 (s, 3H), 2.57 (s, 4H), 2.51 – 2.44 (m, 2H), 1.87 – 1.77 (m, 6H), 1.61 – 1.52 (m, 2H), 1.52 – 1.43 (m,
2H), 1.36 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 182.84, 168.27, 166.80, 160.77, 146.85, 133.09,
125.59, 125.21, 114.88, 114.41, 112.44, 111.95, 96.94, 68.90, 56.58, 55.35, 54.19, 29.42, 29.19, 28.92,
28.72, 27.54, 25.88, 23.40. MS (ESI+): m/z: 450.2[M+H]+. Elemental Anal. Calcd for C28H35NO4: C,
74.80; H, 7.85; N, 3.12. Found: C, 74.71; H, 7.91; N, 3.32.
4.5.5 2-(3,4-dimethoxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5e)
Yellow solid (63% yield), mp 69-71 C; 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.27 (m, 2H), 6.78 (d, J
= 8.4 Hz, 1H), 6.58 (dd, J = 10.7, 2.1 Hz, 3H), 3.91 (dd, J = 8.1, 4.8 Hz, 2H), 3.84 (s, 3H), 3.80 (s, 3H),
2.55 (s, 4H), 2.46 – 2.40 (m, 2H), 1.75 – 1.66 (m, 6H), 1.52 – 1.44 (m, 2H), 1.38 – 1.31 (m, 2H), 1.23 (s,
13
6H). C NMR (100 MHz, CDCl3) δ 182.51, 168.02, 166.69, 150.43, 148.84, 146.71, 125.51, 125.37,
125.27, 114.64, 113.42, 112.34, 112.03, 111.03, 96.84, 68.81, 56.33, 55.81, 54.02, 29.28, 29.10, 28.85,
28.26, 27.34, 25.79, 23.30. MS (ESI+): m/z: 480.3[M+H]+. Elemental Anal. Calcd for C29H37NO5: C,
72.62; H, 7.78; N, 2.92. Found: C, 72.43; H, 7.67; N, 3.09.
4.5.6 7-(8-(pyrrolidin-1-yl)octyloxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (5f)
Yellow solid (51% yield), mp 73-75 C; 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.5 Hz, 1H), 6.98
(s, 2H), 6.65 – 6.52 (m, 3H), 3.92 (t, J = 6.3 Hz, 2H), 3.81 (d, J = 3.4 Hz, 9H), 2.45 (s, 4H), 2.40 – 2.33
13
(m, 2H), 1.70 (s, 6H), 1.41 (d, J = 34.2 Hz, 4H), 1.25 (s, 6H). C NMR (100 MHz, CDCl3) δ 182.51,
168.16, 166.86, 153.09, 147.22, 139.48, 127.75, 125.46, 114.42, 112.45, 111.76, 108.43, 96.88, 68.86,
60.85, 56.47, 56.02, 54.09, 29.36, 29.14, 28.87, 28.68, 27.46, 25.81, 23.30. MS (ESI+): m/z:

19
510.4[M+H]+. Elemental Anal. Calcd for C30H39NO6: C, 70.70; H, 7.71; N, 2.75. Found: C, 70.53; H,
7.55; N, 2.41.
4.5.7 2-(2-(benzyloxy)phenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5g)
Yellow oil (53% yield); 1H NMR (300 MHz, CDCl3) δ 8.20 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 9.2 Hz,
1H), 7.36 –7.7.32 (m, 4H), 7.26 (d, J = 7.1 Hz, 1H), 7.24 – 7.17 (m, 2H), 6.96 (t, J = 7.5 Hz, 1H), 6.85
(d, J = 8.3 Hz, 1H), 6.64 –6.61 (m, 2.1 Hz, 2H), 5.08 (s, 2H), 3.94 (s, 2H), 2.54 (s, 4H), 2.47 – 2.40 (m,
2H), 1.77 –1.70 (m, 6H), 1.49 (d, J = 6.6 Hz, 2H), 1.35 (d, J = 6.3 Hz, 2H), 1.26 (d, J = 3.2 Hz, 6H). 13C
NMR (75 MHz, CDCl3) δ 182.91, 168.41, 166.87, 157.68, 148.03, 136.62, 131.97, 131.04, 128.69,
128.00, 127.16, 125.68, 121.85, 121.08, 114.81, 112.51, 112.37, 105.92, 96.95, 70.36, 68.91, 56.47,
54.12, 29.38, 29.19, 28.91, 28.40, 27.47, 25.88, 23.39. MS (ESI+): m/z: 526.3[M+H]+. Elemental Anal.
Calcd for C34H39NO4: C, 77.68; H, 7.48; N, 2.66. Found: C, 77.55; H, 7.70; N, 2.55.
4.5.8 2-(2-hydroxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5h)
1
Light yellow solid (42% yield), mp 120-122 C; 1H NMR (400 MHz, CDCl3) δ H NMR (300 MHz,
MeOD) δ 8.10 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.28 (s, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.93 –
6.60 (m, 4H), 4.05 (t, J = 6.3 Hz, 2H), 3.09 – 2.99 (m, 2H), 1.97 (t, J = 7.0 Hz, 4H), 1.81 – 1.69 (m, 2H),
13
1.62 (s, 2H), 1.43 (s, 2H), 1.33 (s, 4H), 1.19 (s, 6H). C NMR (75 MHz, CDCl3) δ 184.70, 170.04,
169.01, 158.96, 148.72, 132.80, 126.54, 120.86, 120.57, 116.52, 115.61, 114.12, 108.34, 98.10, 70.18,
56.35, 55.14, 33.08, 30.11, 29.98, 27.54, 27.17, 26.93, 23.95. MS (ESI+): m/z: 436.2[M+H]+. Elemental
Anal. Calcd for C27H33NO4: C, 74.45 H, 7.64; N, 3.22. Found: C, 74.59; H, 7.39; N, 3.37.
4.5.9 2-(3-(benzyloxy)phenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5i)
Light yellow solid (53% yield), mp 84-86 C; 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J = 9.2 Hz, 1H),

20
7.47 (t, J = 2.0 Hz, 1H), 7.42 – 7.28 (m, 6H), 7.22 (d, J = 10.9 Hz, 1H), 6.92 (dd, J = 8.0, 2.6 Hz, 1H),
6.69 – 6.62 (m, 3H), 5.04 (s, 2H), 3.96 (t, J = 6.5 Hz, 2H), 2.61 – 2.53 (m, 4H), 2.50 – 2.43 (m, 2H),
1.81 – 1.71 (m, 6H), 1.52 (t, J = 7.7 Hz, 2H), 1.40 – 1.34 (m, 2H), 1.27 (d, J = 4.1 Hz, 4H), 1.18 (s, 2H).
13C NMR (400 MHz, CDCl3) δ182.96, 168.60, 167.09, 158.93, 147.97, 136.76, 133.69, 129.80, 128.67,
128.12, 127.61, 125.74, 124.37, 117.20, 116.22, 114.46, 112.75, 111.55, 96.97, 70.11, 68.97, 56.43,
54.09, 29.37, 29.20, 28.91, 28.29, 27.45, 25.88, 23.38. MS (ESI+): m/z: 526.4[M+H]+. Elemental Anal.
Calcd for C34H39NO4: C, 77.68; H, 7.48; N, 2.66. Found: C, 78.00; H, 7.57; N, 2.49.
4.5.10 2-(3-hydroxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5j)
Light yellow solid (41% yield), mp 179-181 C; 1H NMR (300 MHz, CDCl3) δ 7.85 (s, 1H), 7.60 (d,
J = 8.6 Hz, 1H), 7.37 (t, J = 2.0 Hz, 1H), 7.30 (dt, J = 7.7, 1.3 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.86 (d,
J = 2.0 Hz, 1H), 6.81 (dd, J = 8.0, 1.5 Hz, 1H), 6.75 (dd, J = 8.6, 2.1 Hz, 1H), 6.66 (s, 1H), 4.06 (t, J =
6.3 Hz, 2H), 3.25 (p, J = 1.7 Hz, 4H), 3.21 (s, 2H), 3.07 – 3.01 (m, 2H), 2.02 – 1.96 (m, 4H), 1.77 (t, J =
13
7.2 Hz, 2H), 1.36 (s, 6H), 1.22 (d, J = 1.8 Hz, 2H). C NMR (75 MHz, CDCl3) δ 181.67, 168.05,
166.83, 157.70, 147.18, 133.06, 129.89, 125.53, 117.59, 117.24, 113.71, 111.26, 97.38, 68.87, 53.83,
52.74, 28.48, 26.08, 25.15, 22.71. MS (ESI+): m/z: 436.1[M+H]+. Elemental Anal. Calcd for C27H33NO4:
C, 74.45; H, 7.64; N, 3.22. Found: C, 74.82; H, 6.68; N, 3.08.
4.5.11 2-(4-(benzyloxy)phenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5k)
Light yellow solid (62% yield), mp 74-76 C; 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.8 Hz, 2H),
7.67 (d, J = 9.2 Hz, 1H), 7.39 (ddd, J = 10.9, 9.3, 7.4 Hz, 5H), 7.02 (d, J = 8.8 Hz, 2H), 6.77 (s, 1H),
6.75 – 6.63 (m, 2H), 5.10 (s, 2H), 4.03 (t, J = 6.5 Hz, 2H), 2.59 (d, J = 5.5 Hz, 4H), 2.53 – 2.46 (m, 2H),
13
1.88 – 1.77 (m, 6H), 1.59 – 1.55 (m, 2H), 1.51 – 1.31 (m, 8H). C NMR (100 MHz, CDCl3) δ 182.87,
168.28, 166.82, 159.93, 146.89, 136.46, 133.13, 128.68, 128.17, 127.49, 125.61, 125.42, 115.28, 114.85,

21
112.51, 111.92, 96.92, 70.05, 68.91, 56.55, 54.17, 29.41, 29.20, 28.92, 28.60, 27.52, 25.89, 23.40. MS
(ESI+): m/z: 526.4[M+H]+. Elemental Anal. Calcd for C34H39NO4: C, 77.68; H, 7.48; N, 2.66. Found: C,
77.98; H, 7.28; N, 3.83.
4.5.12 2-(4-hydroxyphenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5l)
Light yellow solid (55% yield), mp 70-72 C; 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 8.7 Hz, 2H),
7.68 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 1.9 Hz, 1H), 6.91 (s, 1H), 6.90 – 6.78 (m, 3H), 4.17 (t, J = 6.3 Hz,
2H), 3.08 (s, 4H), 2.99 – 2.88 (m, 2H), 2.07 – 1.96 (m, 4H), 1.86 (dd, J = 14.3, 6.7 Hz, 2H), 1.67 (d, J =
13
7.1 Hz, 2H), 1.55 (s, 2H), 1.44 (s, 6H). C NMR (75 MHz, CDCl3) δ 182.99, 168.28, 167.37, 160.13,
146.14, 133.47, 124.95, 123.33, 115.80, 114.25, 113.29, 112.64, 96.56, 68.75, 55.24, 53.55, 28.85, 28.63,
26.50, 26.40, 25.54, 22.63. MS (ESI+): m/z: 436.2[M+H]+. Elemental Anal. Calcd for C27H33NO4: C,
74.45; H, 7.64; N, 3.22. Found: C, 74.79; H, 7.47; N, 3.16.
4.5.13 7-(8-(pyrrolidin-1-yl)octyloxy)-2-p-tolyl-4H-chromen-4-one (5m)
Light yellow solid (64% yield), mp 75-77 C; 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H),
7.69 (d, J = 8.8 Hz, 1H), 7.24 (s, 1H), 6.80 (s, 1H), 6.74 (d, J = 8.2 Hz, 2H), 4.06 (t, J = 6.5 Hz, 2H),
2.62 (s, 4H), 2.58 – 2.49 (m, 2H), 2.40 (s, 3H), 1.84 (t, J = 5.1 Hz, 6H), 1.58 (d, J = 6.5 Hz, 2H), 1.50 –
13
1.32 (m, 8H). C NMR (75 MHz, CDCl3) δ 183.05, 168.51, 166.97, 147.53, 140.09, 131.34, 129.69,
129.65, 125.72, 114.74, 112.57, 112.03, 96.98, 68.93, 56.54, 54.16, 29.40, 29.19, 28.92, 28.51, 27.50,
25.89, 23.40, 21.63. MS (ESI+): m/z: 434.3[M+H]+. Elemental Anal. Calcd for C28H35NO3: C, 77.56; H,
8.14; N, 3.23. Found: C, 77.46; H, 7.95; N, 3.27.
4.5.14 2-(4-chlorophenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5n)
Orange solid (43% yield), mp 89-91 C; 1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8.5 Hz, 2H), 7.69

22
(d, J = 9.1 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 6.80 – 6.70 (m, 3H), 4.06 (t, J = 6.5 Hz, 2H), 2.53 (s, 4H),
2.48 – 2.42 (m, 2H), 1.85 – 1.77 (m, 6H), 1.58 – 1.45 (m, 4H), 1.36 (s, 6H). 13C NMR (75 MHz, CDCl3)
δ 182.87, 168.59, 167.20, 148.04, 135.45, 132.40, 131.00, 129.13, 125.87, 114.42, 112.81, 110.27, 97.00,
69.02, 56.67, 54.26, 29.48, 29.23, 28.94, 28.91, 27.61, 25.90, 23.39. MS (ESI+): m/z: 454.2[M+H]+.
Elemental Anal. Calcd for C27H32ClNO3: C, 71.43; H, 7.10; N, 3.09. Found: C, 71.25; H, 7.03; N, 3.34.
4.5.15 2-(4-bromophenyl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5o)
1
Light yellow solid (47% yield), mp 108-111 C; H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 8.5 Hz,
2H), 7.69 (d, J = 9.1 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 6.76 – 6.72 (m, 3H), 4.07 (t, J = 6.5 Hz, 2H),
3.05 (s, 2H), 2.88 – 2.79 (m, 2H), 2.04 (s, 4H), 1.86 – 1.77 (m, 4H), 1.48 – 1.33 (m, 8H). 13C NMR (75
MHz, CDCl3) δ 182.85, 168.57, 167.17, 148.11, 132.58, 132.09, 131.38, 125.86, 123.90, 114.42, 112.81,
110.31, 97.04, 68.93, 55.95, 53.78, 29.08, 29.06, 28.85, 27.00, 26.65, 25.83, 23.39. MS (ESI+): m/z:
498.1[M+H]+. Elemental Anal. Calcd for C27H32BrNO3: C, 65.06; H, 6.47; N, 2.81. Found: C, 64.88; H,
6.57; N, 2.71.
4.5.16 7-(8-(pyrrolidin-1-yl)octyloxy)-2-(4-(pyrrolidin-1-yl)phenyl)-4H-chromen-4-one (5p)
1
Dark red solid (42% yield), mp 96-98 C; H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 8.6 Hz, 2H),
7.66 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.70 (d, J = 8.2 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H), 4.02 (t, J = 6.4
Hz, 2H), 3.34 (s, 4H), 2.67 (s, 4H), 2.59 – 2.51 (m, 2H), 2.02 (s, 4H), 1.89 – 1.77 (m, 6H), 1.61 (s, 2H),
13
1.47 – 1.30 (m, 8H). C NMR (75 MHz, CDCl3) δ 182.46, 167.54, 166.17, 148.68, 145.61, 133.47,
125.29, 119.51, 115.48, 114.30, 112.06, 111.85, 96.78, 68.74, 56.57, 54.17, 47.52, 29.41, 29.20, 28.94,
28.66, 27.53, 25.89, 25.45, 23.36. MS (ESI+): m/z: 489.4[M+H]+. Elemental Anal. Calcd for
C31H40N2O3: C, 76.19; H, 8.25; N, 5.73. Found: C, 76.29; H, 8.13; N, 5.41.

23
4.5.17 2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5q)
1
Light yellow solid (50% yield), mp 128-131 C; H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 7.0 Hz,
1H), 8.29 (d, J = 8.4 Hz, 1H), 7.87 (dd, J = 8.1, 1.4 Hz, 2H), 7.70 (d, J = 9.1 Hz, 1H), 7.60 (dd, J = 5.0,
1.7 Hz, 2H), 7.58 – 7.50 (m, 2H), 6.76 – 6.69 (m, 2H), 4.02 (t, J = 6.5 Hz, 2H), 2.76 (s, 4H), 2.66 – 2.58
(m, 2H), 1.94 – 1.85 (m, 4H), 1.85 – 1.76 (m, 2H), 1.68 – 1.60 (m, 2H), 1.49 – 1.40 (m, 2H), 1.34 (d, J =
13
3.6 Hz, 6H). C NMR (100 MHz, CDCl3) δ 182.83, 168.74, 167.09, 148.75, 133.72, 132.23, 130.23,
129.96, 128.94, 128.39, 126.99, 126.16, 125.85, 125.58, 123.43, 114.68, 112.73, 107.26, 97.08, 68.95,
56.29, 54.02, 29.28, 29.15, 28.88, 27.83, 27.31, 25.86, 23.38. MS (ESI+): m/z: 470.3[M+H]+. Elemental
Anal. Calcd for C31H35NO3: C, 79.28; H, 7.51; N, 2.98. Found: C, 79.58; H, 7.19; N, 3.13.
4.5.18 2-(biphenyl-4-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5r)
1
Light yellow solid (45% yield), mp 144-146 C; H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.3 Hz,
2H), 7.69 – 7.62 (m, 6H), 7.46 (t, J = 7.5 Hz, 2H), 7.38 (d, J = 7.3 Hz, 1H), 6.82 (s, 1H), 6.76 – 6.70 (m,
2H), 4.03 (t, J = 6.5 Hz, 2H), 2.77 (s, 4H), 2.65 – 2.60 (m, 2H), 1.93 – 1.87 (m, 4H), 1.82 (dd, J = 14.2,
7.2 Hz, 2H), 1.68 – 1.61 (m, 2H), 1.45 (d, J = 7.2 Hz, 2H), 1.35 (d, J = 3.2 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 182.88, 168.54, 167.06, 147.98, 142.13, 140.17, 131.78, 131.49, 128.95, 127.88, 127.44,
127.06, 125.74, 114.62, 112.68, 111.36, 97.06, 68.96, 56.29, 54.01, 29.30, 29.16, 28.91, 27.84, 27.32,
25.87, 23.40. MS (ESI+): m/z: 496.3[M+H]+. Elemental Anal. Calcd for C33H37NO3: C, 79.97; H, 7.52;
N, 2.83. Found: C, 80.09; H, 7.52; N, 2.85.
4.6 Biological activity
4.6.1 Inhibition assays on AChE and BChE in vitro
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyrylcholinesterase (BChE, E.C.

24
3.1.1.8, from equine serum), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride
(ATC), butylthiocholine chloride (BTC) were purchased from Sigma Aldrich. Test compounds were
dissolved in a minimum volume of DMSO (1%) and then diluted in 0.1 M KH2PO4/K2HPO4 buffer (pH
8.0) to provide a final concentration range.
All the assays were under 0.1 M KH2PO4/K2HPO4 buffer, pH 8.0, using a Shimadzu UV-2450
Spectrophotometer. Enzyme solutions were prepared to give 2.0 units/mL in 2 mL aliquots. The assay
medium contained phosphate buffer, pH 8.0 (1 mL), 50 L of 0.01 M DTNB, 10 L of enzyme, and 50
L of 0.01 M substrate (ATC). The substrate was added to the assay medium containing enzyme, buffer,
and DTNB with inhibitor after 15 min of incubation time. The activity was determined by measuring the
increase in absorbance at 412 nm at 1 min intervals at 37 C. Calculations were performed according to
the method of the equation in Ellman et al.21 In vitro BChE assay use the similar method described
above. Each concentration was assayed in triplicate.
4.6.2 Kinetic characterization of AChE and BChE inhibition
Kinetic characterization of AChE was performed using a reported method.24,25 Six different
concentrations of substrate were mixed in the 1 mL 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0), containing
50 L of DTNB, 10 L AChE, and 50 L substrate. Test compound was added into the assay solution
and pre-incubated with the enzyme at 37C for 15 min, followed by the addition of substrate. Kinetic
characterization of the hydrolysis of ATC catalyzed by AChE was done spectrometrically at 412 nm. A
parallel control with no inhibitor in the mixture, allowed adjusting activities to be measured at various
times. BChE assay use the similar method described above.
4.6.3 Molecular modeling
The crystal structure of the torpedo AChE (code ID: 1ACJ) were obtained in the Protein Data Bank

25
after eliminating the inhibitor and water molecules. The 3D structure of compounds 5q was built and
performed geometry optimization by molecular mechanics. Further preparation of substrates was
according to the previous report. 20
Docking studies were carried out using the AUTODOCK 4.0 program using ADT, polar hydrogen
atoms were added and Gasteiger charges were assigned to the enzyme. The resulting enzyme structure
was used as an input for the AUTOGRID program. AUTOGRID performed a precalculated atomic
affinity grid maps for each atom type in the ligand plus an electrostatics map and a separate desolvation
map present in the substrate molecule. All maps were calculated with 0.375 Å spacing between grid
points. The centre of the grid box was placed at the bottom of the active site gorge (AChE [2.781 64.383
67.971]. The dimensions of the active site box were set at 50  46  46 Å. Flexible ligand docking was
performed for the compounds. Docking calculations were carried out using the Lamarckian genetic
algorithm (LGA) and all parameters were the same for each docking.
4.6.4 MTT assay of HepG2 and PC12 cell viability
MTT assay of HepG2 was conducted according to the previous report. 19
PC12 cells were grown in DMEM, supplemented with 10% heat-inactivated fetal calf serum at 37 ºC
under 5% CO2 in a humidified chamber. The antiproliferative ability of compounds was evaluated in
PC12 cells by the conversion of MTT to a purple formazan precipitate. The cells were seeded into
96-well plates at 5 × 103 cells/well, after 12 h, 1.0, 10 M of compounds were subsequently added and
incubated for 48 h. The cell viability was determined by using MTT colorimetry, measuring the
absorption at 590 nm. Controls were taken as having 100% viability. Each concentration was tested in
triplicate.

26
Acknowledgements
We thank the Natural Science Foundation of China (No. 21302041), the Postdoctoral Science
Foundation of China (No. 2012M521391), the Educational Commission of Henan Province (No.
14A350008), and the Postdoctoral Science Foundation of Henan Province (No. 2011015) for financial
support of this study.
Conflict of Interest
We declare that we have no conflict of interest.
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Design, Synthesis and Evaluation of 7-Aminoalkyl-Substituted Flavonoid Derivatives with
Improved Cholinesterase Inhibitory Activities
Wen Luo*, Ying Chen, Ting Wang, Chen Hong, Li-Ping Chang,
Cong-Cong Chang, Ya-Cheng Yang, Song-Qiang Xie, Chao-Jie
Wang*
Key Laboratory of Natural Medicine and Immuno-
Engineering, Henan University, Kaifeng 475004, People’s
Republic of China
Institute of Chemical biology, Henan University,
Kaifeng 475004, People’s Republic of China
Eighteen 7-aminoalkyl-substituted flavonoid derivatives were synthesized and evaluated as potential cholinesterase
inhibitors, these compounds (5a-5r) exhibited better inhibitory activity than our previously reported compounds. They
showed a mixed-type inhibition for AChE and did not affect PC12 and HepG2 cell viability at the concentration of 10 M.