Design, synthesis and pharmacological evaluation of chalcone derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.08.033

A novel series of chalcone derivatives (4a-8d) were designed, synthesized, and evaluated for the inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The log P values of the compounds were shown to range from 1.49 to 2.19, which suggested that they were possible to pass blood brain barriers in vivo. The most promising compound 4a (IC₅₀: 4.68 μmol/L) was 2-fold more potent than Rivastigmine against AChE (IC₅₀: 10.54 μmol/L) and showed a high selectivity for AChE over BuChE (ratio: 4.35). Enzyme kinetic study suggested that the inhibition mechanism of compound 4a was a mixed-type inhibition. Meanwhile, the result of molecular docking showed its potent inhibition of AChE and high selectivity for AChE over BuChE.

Full text of DOI:10.1016/j.bmc.2014.08.033

Bioorganic & Medicinal Chemistry 22 (2014) 6124–6133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacological evaluation of chalcone
derivatives as acetylcholinesterase inhibitors
a
a
a
b
a
Hao-ran Liu a, , Xian-jun Liu , Hao-qun Fan , Jing-jing Tang , Xiao-hui Gao , Wu-Kun Liu
⇑
a College of Chemistry and Chemical Engineering, Hu’nan University, Changsha 410082, China
b College of Pharmacy, Hu’nan University of Traditional Chinese Medicine (TCM), Changsha 410208, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2014
Revised 22 August 2014
Accepted 26 August 2014
Available online 4 September 2014
A novel series of chalcone derivatives (4a–8d) were designed, synthesized, and evaluated for the inhibi-
tion activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The logP values of
the compounds were shown to range from 1.49 to 2.19, which suggested that they were possible to pass
blood brain barriers in vivo. The most promising compound 4a (IC50: 4.68
lmol/L) was 2-fold more
potent than Rivastigmine against AChE (IC50: 10.54 mol/L) and showed a high selectivity for AChE over
l
BuChE (ratio: 4.35). Enzyme kinetic study suggested that the inhibition mechanism of compound 4a was
a mixed-type inhibition. Meanwhile, the result of molecular docking showed its potent inhibition of AChE
and high selectivity for AChE over BuChE.
Keywords:
Chalcone derivatives
AChE inhibitors
LogP
Ó 2014 Elsevier Ltd. All rights reserved.
Molecular docking
1. Introduction
of AD in the early to moderate stages.9 However, these AChE inhib-
itors are known to have side effects or demerits such as hepatotox-
Alzheimer’s disease (AD) is a progressive, chronic, neurodegen-
erative disorder, characterized by declining in memory and cogni-
tive abilities. It is estimated that about 6% of the population
worldwide aged over 65 currently suffer from AD.1,2 The etiology
of AD is still not full known, but many investigations have sug-
gested that reduced level of the neurotransmitter acetylcholine
(Ach), formation of b-amyloid peptide (Ab) plaques, increased
oxidative stress, inflammation and Tau-protein aggregation are
thought to play significant roles in the process of this disease.3–5
Current treatment of AD mainly focuses on the inhibition of
AChE activity aimed at rectifying the deficiency of cerebral acetyl-
choline.6 Based on the cholinergic hypothesis, the deterioration of
memory and cognition in AD patients is mainly caused by the
extensive decline of ACh, which is released into presynaptic neuron
to transport nervous signal and rapidly hydrolyzed by AChE.7
Moreover, recent studies have identified that AChE could also play
a key role in accelerating the assembly of b-amyloid into amyloid
fibrils.8 Up to now, several AChE inhibitors (such as donepezil,
Rivastigmine, galantamine and tacrine) have been approved by
European and US regulatory authorities for the clinical treatment
icity, short half life, and gastrointestinal tract excitement.10
Therefore, the investigation on searching for new and better AChE
inhibitors is still of great interest.
Recently, AChE inhibitors from natural products had attracted
significant attention because of their fewer side effects. Huperzine
A and galantamine are successful examples of AChE inhibitors from
natural products.11 However, it is not so easy to find the potent
compounds which could be potential drugs without any structure
modifications for many natural products had weak bioactivities
yet. In addition, the total chemical synthesis of some complicated
natural products is usual difficult and costly. So, it is highly desir-
able to synthesize some derivates with natural compounds back-
bone according to the idea of rational molecular design to gain
potential drugs for the therapy of AD. For the research and devel-
opment of AChE inhibitors, many investigations revealed that
nitrogen atom was important to the inhibition of AChE.12 Several
flavonoid and coumarin derivatives with terminal amine groups
(Fig. 1) have been successfully designed and synthesized and some
of them exhibited potent inhibitory activity against AChE.13–15
Among them, ensaculin, a coumarin derivative, is under clinical
investigation for potential AD management.16
Chalcones, natural compounds widely existed in fruits and veg-
etables, belong to the flavonoid family and consist of two aromatic
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterases; ACh,
acetylcholine; BuChE, butyrylcholinesterase; PAS, peripheral anionic sites; LogP,
octanol/water partition; CNS, central nervous system; BBB, blood brain barrier.
rings connected by an
a,b-unsaturated carbonyl group. Possibly
due to the flexible structure, chalcones can bind effectively to
many kinds of enzymes or receptors and exhibit diverse biological
⇑
Corresponding author. Tel.: +86 0731 83860540.
E-mail address: pharmaliu@126.com (H.-r. Liu).
http://dx.doi.org/10.1016/j.bmc.2014.08.033
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
6125
Figure 1. The chemical structure of flavonoid derivatives (A1, A2) and coumarin derivatives (B1, B2, Ensaculin) as the AChE inhibitors.
activities, such as anti-cancer, anti-infective, anti-inflammatory,
anti-oxidant and anti-angiogenic effects.17–20 But few investiga-
tions were conducted by pharmaceutical researchers about their
biological activities of inhibiting AChE. Thus, based on the design
experiences from existing AChE inhibitors, we designed and syn-
thesized a series of chalcones with different basic side chains
(n = 2–6) and evaluated their biological activity of inhibiting AchE.
Moreover, we explored the binding mode of the compounds to
AchE by kinetic experiments and measured the logarithm 1-octa-
nol/water partition coefficient (logP values). The logP value was
used to assess the ability of compounds to penetrate blood brain
barrier (BBB). Furthermore, molecular docking studies were carried
out to study the binding mode and selectivity of these compounds
against AChE.
to six carbons and K2CO3 in N,N-dimethylformamide (DMF) at
80 °C to generate compounds 3a–3e. Finally, the target compounds
4a–8d were obtained by refluxing 3a–3e with commercially
available secondary amines (dimethylamine, diethylamine,
dipropylamine and dibutylamine) in acetone in the presence of
K2CO3 and NaI. The structures of the designed compounds were
characterized by proton nuclear magnetic resonance spectroscopy
(1H NMR), infrared spectrum (IR) and mass spectrometry (MS).
Besides, the purities of all synthesized compounds were confirmed
to be higher than 95% by HPLC.
2.2. LogP values
For a drug to treat AD, the ability to penetrate the BBB is vital.
Although the factors that affect the penetration of a drug from
the systemic circulation into the central nervous system (CNS)
were complicated, logP was thought as an important physical
chemistry parameter to evaluate or predict the ability to cross
BBB, which widely used in medicinal chemistry investigation.22
Hansch23 presumed that the logP with optimum CNS penetration
was around 2 0.7. LogP values of the synthesized compounds
were measured by the classical shake-flask method24 using
RP-HPLC. As shown in Table 1, the logP values of the tested
2. Results and discussion
2.1. Chemistry
The synthetic routes to target compounds 4a-8d are outlined in
Scheme 1. Reaction of 4-hydroxyacetophenone 1 with benzalde-
hyde under the catalysis of NaOH/EtOH provided compound 2.21
Then, compound 2 was treated with dibromoalkanes bearing two
Scheme 1. Reagents and conditions: (a) benzaldehyde, NaOH, EtOH, rt; (b) Br(CH2)nBr, K2CO3, DMF, 80 °C; (c) second amine, K2CO3, NaI, acetone, reflux.

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H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
compounds ranged from 1.49 to 2.19, which indicated that all of
the compounds were possible sufficiently lipophilic to pass BBB
in vivo.
n = 5, 7.65; 8a, n = 6, 7.02; 4b, n = 2, 13.11; 5b, n = 3, 4.44; 6b,
n = 4, 4.31; 7b, n = 5, 2.93; 8b, n = 6, 1.98). However, a different
trend was observed for the compounds linked with dipropylamine
(4c, 5c, 6c, 7c, 8c) and dibutylamine (4d, 5d, 6d, 7d, 8d). Clearly,
compounds 6c and 6d, which combined dipropylamine or dibutyl-
amine group with chalcone backbone by a four methylene spacer,
showed better inhibition activity against AChE (6c, 16.92; 6d,
4.56). Moreover, for compounds containing the same terminal
group, the one with higher inhibition against AChE showed higher
selectivity for AChE in the meantime (4a, ratio: 4.35; 4b, ratio:
4.14; 6c, ratio: 1.50; 6d, ratio: 1.91).
2.3. In vitro inhibition of AChE and BuChE
It has been reported that butyrylcholinesterase (BuChE) can
non-specifically hydrolyzes Ach and its level is unchanged or even
rises in advanced AD.25,26 Thus, the inhibition of BuChE may also be
beneficial to improve AD symptoms.27
The inhibitory effects of the synthesized compounds on AChE
and BuChE were evaluated using Ellman method, with Rivastig-
mine as the positive control. The half maximal inhibitory concen-
tration (IC50 values) for AChE and BuChE inhibition and the
selectivity for AChE were summarized in Table 1. According to
the data, all of the synthesized compounds exhibited higher
inhibitory activities against AChE than the precursor compound 2
2.4. Kinetic studies
Compound 4a was selected for further kinetic measurement
since it is the most active one against AChE. The linear Linewe-
aver–Burk equation which is a double reciprocal form of the
Michaelis–Menten equation was converted to evaluate the inhibi-
tion mechanism. The graphical analysis of the steady-state inhibi-
tion data of compound 4a was shown in Figure 3. These results
showed that increasing the concentration of compound 4a resulted
in different slopes and intercepts. According to Table 2, higher
concentrations of compound 4a resulted in increases of Km, while
Vmax decreased, which was conformed to the characteristics of
mixed-type inhibition. It was therefore concluded that a mixed-
type inhibition could be attributed to the compound 4a. This
behavior showed that this compound can bind both with the cata-
lytic site and the non-catalytic site of the enzyme, with different
equilibrium constants. The inhibition constants for the inhibitor
binding with the catalytic site, Ki, and with the non-catalytic site,
(IC50 >500
of 4.68, 7.63, 8.95, 5.91
activities compared to the reference compound Rivastigmine
(IC50 = 10.54 mol/L). Specifically, the most promising compound
l
mol/L). Compounds 4a, 4b, 6a and 6c with IC50 values
lmol/L, respectively, showed potent
l
4a was 2-fold more active than Rivastigmine and also showed a
high selectivity for AChE over BuChE (ratio: 4.35).
Based on the screening data (Fig. 2), it appeared that variation of
the length of the chain linking chalcone backbone with terminal
amine groups influenced their AChE and BuChE inhibitory activity
significantly. In general, for compounds containing diethylamine or
dimethylamine group, the inhibition activity against AChE was
decreased as the chain length increased (4a, n = 2, inhibitory
potency (=1/IC50*100): 21.37; 5a, n = 3, 9.55; 6a, n = 4, 11.17; 7a,
Table 1
In vitro inhibition of AChE/BuChE and logP values
a
Compound
R
n
IC50
(
lM)
Selectivity for AChEb
LogPc
AChE
BuChE
2
.>500
>500
4a
5a
2
3
4.68 0.13
10.47 1.22
20.36 1.84
32.29 2.41
4.35
3.08
1.49
1.59
6a
4
8.95 0.59
11.75 1.18
1.32
1.64
N
N
7a
8a
4b
5b
5
6
2
3
13.07 1.23
14.24 1.35
7.63 0.82
22.51 2.15
17.42 1.38
10.42 0.82
31.59 3.33
20.12 1.81
1.33
0.73
4.14
0.89
1.73
1.80
1.61
1.71
6b
4
23.21 2.47
10.24 0.76
0.44
1.74
7b
8b
4c
5c
5
6
2
3
34.14 2.81
50.44 5.86
64.55 5.39
24.63 3.02
26.33 3.06
18.47 1.53
52.58 4.37
10.13 0.53
0.77
0.37
0.81
0.41
1.84
1.91
1.86
1.91
6c
4
5.91 0.28
8.84 0.55
1.50
1.95
N
N
7c
8c
4d
5d
5
6
2
3
29.48 2.31
44.05 4.19
252.96 35.76
151.83 14.53
39.65 6.57
40.74 4.47
279.11 32.52
68.25 6.25
2.09
0.92
1.10
0.45
2.01
2.07
2.00
2.06
6d
4
21.94 2.64
41.91 3.66
1.91
2.09
7d
8d
5
6
76.87 6.53
162.59 40.87
10.54 0.86
96.92 21.49
75.09 7.85
0.26 0.081
3.86
0.46
0.03
2.14
2.19
Rivastigmine
a
b
c
IC50: 50% inhibitory concentration (means SD of three experiments).
Selectivity for AChE is defined as IC50 (BuChE)/IC50 (AChE).
The partition coefficients of in the octanol/buffer solution at pH 7.4 were determined by the classical shake-flask method.

H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
6127
Figure 2. (A) Effects of alkyl chain lengths on anti-AChE activities; (B) Effects of alkyl chain lengths on anti-BuChE activities; (C) Effects of alkyl chain lengths on selectivity for
AChE.
In order to study the binding mode of our compounds with two
cholinesterases, molecular modeling was carried out using the
docking program MOE2008 and the results were shown in Figure 4.
Compound 4a, with strong inhibitory activity and high selectivity
against AChE, exhibited multiple points binding modes with AChE.
The aromatic moiety adopted an appropriate orientation for its
Table 2
Kinetic parameters of AChE inhibited by compound 4a
4a
M)
Michaelis–Menten
equation
Km
(mM)
vmaꢀx1
/
Ki
Ki0
(l
D
Amin
(l
M)
(l
M)
0
1.5
3
1/v = 125.89/[S] + 14.10
1/v = 187.08/[S] + 34.42
1/v = 208.00/[S] + 40.78
1/v = 241.88/[S] + 51.25
8.92
5.44
5.10
4.72
0.0709
0.029
0.024
0.020
7.99
3.52
binding to PAS, via the
p–p stacking interaction with Trp279 and
Phe330, and the ring-to-ring distance was 4.02 Å and 4.17 Å. More-
over, the conformation of the side chain could fit well with the
shape of the gorge and the positively-charged nitrogen atom of
6
dimethylamine made a cation–
in the CAS. In contrast, Compound 4a could only have a cation–
p interaction (3.85 Å) with Trp84
Ki0, are obtained from the replots of the slope and intercept versus
concentration of compound 4a, respectively, which are both linear
p
interaction between Trp82 (4.26 Å) and the nitrogen atom of
dimethylamine with BuChE. Meanwhile, the calculated binding free
energy of compound 4a with AChE is lower than that with BuChE
(ꢀ30.7299 J/(K*mol/l and ꢀ25.8991 J/(K*mol/l, respectively). These
results may rationalize the potent inhibition of compound 4a for
AChE and the high selectivity for AChE over BuChE.
in Figure 3. The Ki and Ki0 value are 7.99
lmol/L and 3.52 lmol/L,
respectively, which indicated that the inhibition mechanism of
compound 4a against AChE includes competitive and uncompeti-
tive inhibition.
2.5. Molecular modeling
We also performed the docking study of compound 2 (IC50
>500
stacking interactions between the chalcone backbone and
Trp279, Phe330 were observed, and no cation– interaction
between chalcone backbone and the CAS was found. This result
supported that a tertiary amino group on the alkyl side chain is
the key requirement for the potent AChE inhibition, because it is
an important functional group which can made interactions with
lmol/L) with AChE. From the docking result, only the p–p
The X-ray crystallographic structure of AChE showed that it
contains two separate ligand binding sites—a catalytic active site
(CAS) and a peripheral cationic site (PAS), located at the entrance
and the bottom of the active-site gorge, respectively.28 The simul-
taneous binding to both the CAS and PAS has been suggested to be
important in designing powerful and selective AChE inhibitors.
p

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H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
3. Conclusion
In summary, a series of chalcone derivatives were designed,
synthesized and tested for their biological activity. Some of them
exhibited AChE inhibition activity compared with chalcone in the
preliminary bioassay. Among them, compound 4a, which con-
tained a dimethylamine group linked to chalcone backbone by a
two-carbon spacer, exhibited the most potent AChE inhibitory
activity and high selectivity for AChE over BuChE. The kinetic study
on compound 4a suggested that it conducted its effect as a mixed-
type inhibition including competitive and uncompetitive inhibi-
tion against AChE. Molecular docking study on compound 4a
indicated that it could bind to both the CAS and PAS of AChE simul-
taneously. Overall, Compound 4a might serve as a potential agent
for the treatment of AD.
4. Experimental
4.1. Chemistry
Figure 3. Lineaweaver–Burk plot for the inhibition of AChE by compound 4a.
All chemicals and reagents were of analytical reagent grade and
were used without further purification. 1H NMR spectra were
recorded on an INORA400 MHz instrument in CDCl3 with TMS as
the internal reference. Infrared spectrum was obtained by
Shimadzu Infinity-1 infrared spectrometer. Mass spectra were
obtained on Finnigan LCQ Advantage MAX by electrospray ioniza-
tion (ESI-MS). The purity of the compounds was checked on Shima-
dzu LC-20A high performance liquid chromatography.
the amino acids in the CAS. Additionally, molecular docking study
of compound 4d (IC50 = 252.96 mol/L) with AChE indicated that
compound 4d could bind to PAS via the stacking interaction
with Trp279. However, the positively-charged nitrogen atom of
dibutylamine did not bind to CAS via the cation– interaction with
Trp84, but revealing a cation– interaction with Phe330. Due to
l
p–p
p
p
the lack of interaction with CAS, this binding mode caused its
lower inhibitory activities against AChE.
Figure 4. Molecular modeling of compound 4a with AChE (A) and BuChE; (B) compound 2 with AChE; (C) and compound 4d with AChE; (D) generated with MOE2008.

H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
6129
4.2. Synthesis of 40-hydroxychalcone (2)
2.07–2.14 (2H, m, CH2CH2Br), 3.51 (2H, t, J = 6.0 Hz, CH2CH2Br),
4.09 (2H, t, J = 6.0, OCH2CH2), 6.98 (2H, d, J = 8.0 Hz, 30-H and 50-
H), 7.40–7.43 (3H, m, 3-H and 4-H and 5-H), 7.55 (1H, d,
A mixture of 4-hydroxyacetophenone (1.36 g, 10 mmol), benz-
aldehyde (1.3 mL, 11 mmol) and sodium hydroxide (1 g, 25 mmol)
were dissolved in ethanol/water (4:1 v/v, 20 mL) and stirred at
room temperature for a period of 24 h. 10% HCl was added to
adjust the solution to pH = 3 and the product precipitated. Filtra-
tion of the reaction mixture gave a light yellow solid product with
a yield of 85.3%. Mp 173–175 °C. 1H NMR (400 MHz, CDCl3) d
(ppm): 6.95 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.41–7.44 (3H, m,
J = 16.0 Hz,
J = 16.0 Hz, b-H), 8.05 (2H, d, J = 8.0 Hz, 20-H and 60-H). MS m/z
(ESI): 359 [M+H]+. IR (KBr) /cmꢀ1: 3029, 2951, 2848, 2786,
a-H), 7.64–7.66 (2H, m, 2-H and 6-H), 7.81 (1H, d,
m
1653, 1601, 1586, 1505, 1340, 1219, 1175, 835, 768.
4.3.4. (E)-1-(4-(5-Bromopentyloxy)phenyl)-3-phenylprop-2-en-
1-one (3d)
3-H and 4-H and 5-H), 7.55 (1H, d, J = 16.0 Hz,
a
-H), 7.64–7.66
It was synthesized from compound 2 (1.12 g, 5 mmol) with
K2CO3 (2.07 g, 15 mmol) in DMF (15 mL) and 1,5-dibromopentane
(4.1 mL, 30 mmol), and purified using silica-gel column chroma-
tography with ethyl acetate/petroleum ether (1:16, v/v) as elution
to give a white solid product with a yield of 71.5%. Mp 73–75 °C. 1H
NMR (400 MHz, CDCl3) d (ppm): 1.63–1.70 (2H, m, CH2CH2CH2),
1.82–1.88 (2H, m, CH2CH2Br), 1.92–1.99 (2H, m, OCH2CH2), 3.45
(2H, t, J = 6.0 Hz, CH2CH2Br), 4.06 (2H, t, J = 6.0, OCH2CH2), 6.98
(2H, d, J = 8.0 Hz, 30-H and 50-H), 7.39–7.44 (3H, m, 3-H and 4-H
(2H, m, 2-H and 4-H), 7.82 (1H, d, J = 16.0 Hz, b-H), 8.01 (2H, d,
J = 8.0 Hz, 20-H and 60-H). MS m/z (ESI): 225 [M+H]+. IR (KBr)
m/
cmꢀ1: 3319, 3038, 2968, 1658, 1604, 1576, 1449, 1337, 1222,
1171, 835, 766.
4.3. General procedure for the synthesis of 3a–3e
To a solution of compound 2 (1.12 g, 5 mmol) and K2CO3 (2.07 g,
15 mmol) in DMF (15 mL), an appropriate amount of
a
,
x
-dibro-
and 5-H), 7.55 (1H, d, J = 16.0 Hz,
and 6-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.05 (2H, d, J = 8.0 Hz, 20-
H and 60-H). MS m/z (ESI): 373 [M+H]+. IR (KBr) /cmꢀ1: 3030,
2953, 2850, 2784, 1656, 1602, 1588, 1507, 1343, 1221, 1177,
834, 767.
a-H), 7.63–7.67 (2H, m, 2-H
moalkane was added. After stirring at 80 °C for 2–10 h until the
material 2 disappeared, the solvent was poured into ice water
(100 mL), extracted with EtOAc (2 ꢁ 70 mL) and washed with sat-
urated aq NaCl solution (2 ꢁ 80 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated in vacuum. Then the res-
idue was purified by a silica-gel column chromatography to afford
product.
m
4.3.5. (E)-1-(4-(6-Bromohexyloxy)phenyl)-3-phenylprop-2-en-
1-one (3e)
It was synthesized from compound 2 (1.12 g, 5 mmol) with
K2CO3 (2.07 g, 15 mmol) in DMF (15 mL) and 1,6-dibromohexane
(4.6 mL, 30 mmol), and purified using silica-gel column chroma-
tography with ethyl acetate/petroleum ether (1:18, v/v) as elution
to give a white solid product with a yield of 75.8%. Mp 68–70 °C. 1H
NMR (400 MHz, CDCl3) d (ppm): 1.50–1.58 (4H, m, CH2CH2CH2CH2
and CH2CH2CH2CH2), 1.81–1.95 (4H, m, CH2CH2Br and OCH2CH2),
3.43 (2H, t, J = 6.0 Hz, CH2CH2Br), 4.05 (2H, t, J = 6.0, OCH2CH2),
6.97 (2H, d, J = 8.0 Hz, 30-H and 50-H), 7.40–7.44 (3H, m, 3-H and
4.3.1. (E)-1-(4-(2-Bromoethyl)phenyl)-3-phenylprop-2-en-1-
one (3a)
It was synthesized from compound 2 (1.12 g, 5 mmol) with
K2CO3 (2.07 g, 15 mmol) in DMF (15 mL) and 1,2-dibromoethane
(2.6 mL, 30 mmol), and purified using silica-gel column chroma-
tography with ethyl acetate/petroleum ether (1:10, v/v) as elution
to give a white solid product with a yield with a yield of 45.1%. Mp
89–91 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 3.72 (2H, t, J = 6.0 Hz,
BrCH2), 4.39 (2H, t, J = 6.0 Hz, OCH2), 7.01 (2H, d, J = 8.8 Hz, 30-H
and 50-H), 7.41–7.44 (3H, m, 3-H and 4-H and 5-H), 7.55 (1H, d,
4-H and 5-H), 7.55 (1H, d, J = 16.0 Hz,
H and 6-H), 7.80 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d, J = 8.8 Hz,
20-H and 60-H). MS m/z (ESI): 387 [M+H]+. IR (KBr) /cmꢀ1: 3031,
a-H), 7.62–7.66 (2H, m, 2-
J = 16.0 Hz,
J = 16.0 Hz, b-H), 8.05 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z
(ESI): 331 [M+H]+. IR (KBr) /cmꢀ1: 3028, 2952, 2846, 1659,
a-H), 7.64–7.67 (2H, m, 2-H and 4-H), 7.82 (1H, d,
m
2958, 2851, 2782, 1655, 1603, 1588, 1506, 1342, 1220, 1176,
836, 765.
m
1601, 1576, 1341, 1221, 1182, 833, 764.
4.4. General procedure for the synthesis of 4a–8d
4.3.2. (E)-1-(4-(3-Bromopropyl)phenyl)-3-phenylprop-2-en-1-
one (3b)
A mixture of compounds 3a–3c (1 mmol), secondary amines
(dimethylamine, diethylamine, dipropylamine and dibutylamine),
K2CO3 (0.414 g, 3 mmol), NaI (0.008 g, 0.05 mmol), acetone
(20 mL) was heated under reflux for 8 h. The solvent was removed
and the residue was dissolved with EtOAc (30 mL). Then the
organic extracts was washed with saturated aq NaCl solution
(2 ꢁ 20 mL), dried over anhydrous Na2SO4 and evaporated in vac-
uum, giving a crude product which was chromatographed on silica
gel with different ratio of methanol/dichloromethane as elution to
afford the compounds 4a–8d.
It was synthesized from compound 2 (1.12 g, 5 mmol) with
K2CO3 (2.07 g, 15 mmol) in DMF (15 mL) and 1,3-dibromopropane
(3.1 mL, 30 mmol), and purified using silica-gel column chroma-
tography with ethyl acetate/petroleum ether (1:12, v/v) as elution
to give a white solid product with a yield of 56.3%. Mp 84–86 °C. 1H
NMR (400 MHz, CDCl3) d (ppm): 2.33–2.56 (2H, m, CH2CH2Br), 3.63
(2H, t, J = 6.0 Hz, CH2CH2Br), 4.20 (2H, t, J = 6.0, OCH2CH2), 6.99 (2H,
d, J = 8.8 Hz, 30-H and 50-H), 7.41–7.43 (3H, m, 3-H and 4-H and 5-
H), 7.56 (1H, d, J = 16.0 Hz,
7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and 60-
H). MS m/z (ESI): 345 [M+H]+. IR (KBr) /cmꢀ1: 3031, 2956, 2843,
a-H), 7.64–7.67 (2H, m, 2-H and 6-H),
m
4.4.1. (E)-1-(4-(2-(Dimethylamino)ethoxy)phenyl)-3-
2781, 1655, 1603, 1589, 1508, 1344, 1221, 1173, 833, 767.
phenylprop-2-en-1-one (4a)
According to the general method, the reaction of compound 3a
(0.33 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:35, v/v) as
elution to give a light yellow solid product with a yield of 77.3%.
Mp 73–75 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 2.37 (6H, s,
2 ꢁ NCH3), 2.79 (2H, t, J = 6.0 Hz, NCH2CH2), 4.14 (2H, t, J = 6.0 Hz,
OCH2CH2), 7.01 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.41–7.44 (3H,
4.3.3. (E)-1-(4-(4-Bromobutyl)phenyl)-3-phenylprop-2-en-1-
one (3c)
It was synthesized from compound 2 (1.12 g, 5 mmol) with
K2CO3 (2.07 g, 15 mmol) in DMF (15 mL) and 1,4-dibromobutane
(3.6 mL, 30 mmol), and purified using silica-gel column chroma-
tography with ethyl acetate/petroleum ether (1:14, v/v) as elution
to give a white solid product with a yield of 67.7%. Mp 78–80 °C. 1H
NMR (400 MHz, CDCl3) d (ppm): 1.96–2.04 (2H, m, OCH2CH2),
m, 3-H and 4-H and 5-H), 7.56 (1H, d, J = 16.0 Hz, a-H), 7.63–7.66

6130
H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
(2H, m, 2-H and 4-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d,
J = 8.0 Hz, 20-H and 60-H). MS m/z (ESI): 296 [M+H]+. IR (KBr)
m
1219, 1173, 836, 764. Purity: 98.8% by HPLC (MeOH/0.1% triethyl-
amine (TEA) 85:15 (v/v); tR 3.28 min).
as elution to give a light yellow solid product with a yield of
63.3%. Mp 68–70 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 2.01–
2.05 (2H, m, NCH2CH2CH2), 2.32 (6H, s, 2 ꢁ NCH3), 2.53 (2H, t,
J = 6.0 Hz, NCH2CH2), 4.12 (2H, t, J = 6.0 Hz, OCH2CH2), 6.99 (2H,
d, J = 8.0 Hz, 30-H and 50-H), 7.42–7.44 (3H, m, 3-H and 4-H and
/cmꢀ1: 3031, 2951, 2801, 2769, 1660, 1607, 1588, 1512, 1339,
5-H), 7.56 (1H, d, J = 16.0 Hz,
H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and
60-H). MS m/z (ESI): 310 [M+H]+. IR (KBr) /cmꢀ1: 3032, 2951,
a-H), 7.64–7.67 (2H, m, 2-H and 4-
4.4.2. (E)-1-(4-(2-(Diethylamino)ethoxy)phenyl)-3-phenylprop-
2-en-1-one (4b)
m
According to the general method, the reaction of compound 3a
(0.33 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:40, v/v) as
elution to give a light yellow oil product with a yield of 67.8%. 1H
NMR (400 MHz, CDCl3) d (ppm): 1.12 (6H, t, J = 6.8 Hz, 2 ꢁ NCH2CH3),
2.68–2.74 (4H, m, 2 ꢁ NCH2CH3), 2.96 (2H, t, J = 6.0 Hz, NCH2CH2),
4.17 (2H, t, J = 6.0 Hz, OCH2CH2), 6.99 (2H, d, J = 8.0 Hz, 30-H and
50-H), 7.41–7.44 (3H, m, 3-H and 4-H and 5-H), 7.56 (1H, d,
2870, 2772, 1655, 1609, 1589, 1503, 1346, 1223, 1175, 831, 773.
Purity: 98.3% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 3.96 min).
4.4.6. (E)-1-(4-(3-(Diethylamino)propoxy)phenyl)-3-
phenylprop-2-en-1-one (5b)
According to the general method, the reaction of compound 3b
(0.344 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:40, v/v) as
elution to give a light yellow solid product with a yield of 70.7%.
Mp 55–57 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.11 (6H, t,
J = 6.8 Hz, 2 ꢁ NCH2CH3), 2.04–2.06 (2H, m, NCH2CH2CH2), 2.63–
2.74 (6H, m, 3 ꢁ NCH2CH2), 4.12 (2H, t, J = 6.0 Hz, OCH2CH2), 6.98
(2H, d, J = 8.0 Hz, 30-H and 50-H), 7.41–7.44 (3H, m, 3-H and 4-H
J = 16.0 Hz,
J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z
(ESI): 324 [M+H]+. IR (KBr) /cmꢀ1: 3028, 2956, 2816, 2789,
a-H), 7.64–7.66 (2H, m, 2-H and 4-H), 7.81 (1H, d,
m
1656, 1605, 1586, 1509, 1337, 1221, 1175, 833, 768. Purity:
98.6% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 4.63 min).
and 5-H), 7.56 (1H, d, J = 16.0 Hz,
and 4-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-
H and 60-H). MS m/z (ESI): 338 [M+H]+. IR (KBr) /cmꢀ1: 3033,
2952, 2873, 2769, 1656, 1607, 1587, 1505, 1337, 1221, 1171,
829, 769. Purity: 97.8% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
5.52 min).
a-H), 7.64–7.67 (2H, m, 2-H
4.4.3. (E)-1-(4-(2-(Dipropylamino)ethoxy)phenyl)-3-
phenylprop-2-en-1-one (4c)
m
According to the general method, the reaction of compound 3a
(0.33 g, 1 mmol) with dipropylamine (0.41 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:45, v/v) as
elution to give a light yellow oil product with a yield of 71.3%. 1H
NMR (400 MHz, CDCl3) d (ppm): 0.91 (6H, t, J = 6.8 Hz, 2 ꢁ NCH2-
CH2CH3), 1.48–1.54 (4H, m, 2 ꢁ NCH2CH2CH3), 2.51 (4H, t,
J = 6.0 Hz, 2 ꢁ NCH2CH2CH3), 2.91 (2H, t, J = 6.0 Hz, NCH2CH2),
4.12 (2H, t, J = 6.0 Hz, OCH2CH2), 6.99 (2H, d, J = 8.0 Hz, 30-H and
50-H), 7.41–7.45 (3H, m, 3-H and 4-H and 5-H), 7.56 (1H, d,
4.4.7. (E)-1-(4-(3-(Dipropylamino)propoxy)phenyl)-3-
phenylprop-2-en-1-one (5c)
According to the general method, the reaction of compound 3b
(0.344 g, 1 mmol) with dipropylamine (0.41 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:45, v/v) as
elution to give a light yellow solid product with a yield of 61.9%.
Mp 43–45 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 0.88 (6H, t,
J = 6.8 Hz, 2 ꢁ NCH2CH2CH3), 1.45–1.51 (4H, m, 2 ꢁ NCH2CH2CH3),
1.95–1.98 (2H, m, NCH2CH2CH2), 2.42 (4H, t, J = 7.2 Hz, 2 ꢁ NCH2
CH2CH3), 2.63 (2H, t, J = 6.0 Hz, NCH2CH2), 4.11 (2H, t, J = 6.0 Hz,
OCH2CH2), 6.98 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.41–7.44 (3H,
J = 16.0 Hz,
J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z
(ESI): 352 [M+H]+. IR (KBr) /cmꢀ1: 3031, 2959, 2872, 2776,
a-H), 7.64–7.67 (2H, m, 2-H and 4-H), 7.81 (1H, d,
m
1657, 1607, 1589, 1508, 1339, 1220, 1175, 832, 770. Purity:
97.8% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 8.81 min).
4.4.4. (E)-1-(4-(2-(Dibutylamino)ethoxy)phenyl)-3-phenylprop-
2-en-1-one (4d)
m, 3-H and 4-H and 5-H), 7.57 (1H, d, J = 16.0 Hz,
a-H), 7.64–7.67
(2H, m, 2-H and 4-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d,
According to the general method, the reaction of compound 3a
(0.33 g, 1 mmol) with dibutylamine (0.5 mL, 3 mmol) produced the
crude product. Then it was purified using silica-gel column chro-
matography with methanol/dichloromethane (1:45, v/v) as elution
to give a light yellow oil product with a yield of 59.2%. 1H NMR
(400 MHz, CDCl3) d (ppm): 0.93 (6H, t, J = 6.8 Hz, 2 ꢁ NCH2CH2CH2CH3),
1.26–1.33 (4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.35–1.49 (4H, m,
2 ꢁ NCH2CH2CH2CH3), 2.54 (4H, t, J = 6.0 Hz, 2 ꢁ NCH2CH2CH2CH3),
2.89 (2H, t, J = 6.0 Hz, NCH2CH2), 4.11 (2H, t, J = 6.0 Hz, OCH2CH2),
6.98 (2H, d, J = 8.0 Hz, 30-H and 50-H), 7.41–7.44 (3H, m, 3-H and
J = 8.8 Hz, 20-H and 60-H). MS m/z (ESI): 366 [M+H]+. IR (KBr)
m/
cmꢀ1: 3034, 2956, 2868, 2776, 1655, 1605, 1591, 1507, 1342,
1225, 1173, 831, 770. Purity: 98.1% by HPLC (MeOH/0.1% TEA
85:15 (v/v); tR 10.64 min).
4.4.8. (E)-1-(4-(3-(Dibutylamino)propoxy)phenyl)-3-
phenylprop-2-en-1-one (5d)
According to the general method, the reaction of compound 3b
(0.344 g, 1 mmol) with dibutylamine (0.5 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:45, v/v) as
elution to give a light yellow oil product with a yield of 72.5%. 1H
NMR (400 MHz, CDCl3) d (ppm): 0.89 (6H, t, J = 6.8 Hz, 2 ꢁ NCH2
CH2CH2CH3), 1.25–1.34 (4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.39–1.46
(4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.94–1.98 (2H, m, NCH2CH2CH2),
2.44 (4H, t, J = 6.0 Hz, 2 ꢁ NCH2CH2CH2CH3), 2.62 (2H, t,
J = 6.0 Hz, NCH2CH2), 4.11 (2H, t, J = 6.0 Hz, OCH2CH2), 6.98 (2H,
d, J = 8.8 Hz, 30-H and 50-H), 7.41–7.43 (3H, m, 3-H and 4-H and
4-H and 5-H), 7.56 (1H, d, J = 16.0 Hz,
H and 4-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz,
20-H and 60-H). MS m/z (ESI): 380 [M+H]+. IR (KBr) /cmꢀ1: 3035,
a-H), 7.64–7.67 (2H, m, 2-
m
2955, 2870, 2781, 1655, 1607, 1589, 1508, 1340, 1219, 1176,
831, 769. Purity: 96.9% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
14.63 min).
4.4.5. (E)-1-(4-(3-(Dimethylamino)propoxy)phenyl)-3-
phenylprop-2-en-1-one (5a)
5-H), 7.57 (1H, d, J = 16.0 Hz,
4-H), 7.81 (1H, d, J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H
and 60-H). MS m/z (ESI): 394 [M+H]+. IR (KBr) /cmꢀ1: 3033,
2955, 2870, 2772, 1661, 1607, 1586, 1508, 1337, 1219, 1169,
a-H), 7.64–7.67 (2H, m, 2-H and
According to the general method, the reaction of compound 3b
(0.344 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol) pro-
duced the crude product. Then it was purified using silica-gel col-
umn chromatography with methanol/dichloromethane (1:40, v/v)
m

H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
6131
831, 766. Purity: 97.9% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
17.89 min).
2 ꢁ NCH2CH2CH2CH3), 1.25–1.31 (4H, m, 2 ꢁ NCH2CH2CH2CH3),
1.33–1.45 (4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.62–1.65 (2H, m, NCH2CH2),
1.81–1.84 (2H, m, OCH2CH2), 2.43–2.46 (6H, m, 3 ꢁ NCH2CH2), 4.07
(2H, t, J = 6.0 Hz, OCH2CH2), 6.98 (2H, d, J = 8.8 Hz, 30-H and 50-H),
7.41–7.43 (3H, m, 3-H and 4-H and 5-H), 7.57 (1H, d, J = 16.0 Hz,
4.4.9. (E)-1-(4-(4-(Dimethylamino)butoxy)phenyl)-3-
phenylprop-2-en-1-one (6a)
According to the general method, the reaction of compound 3c
(0.358 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol)
produced the crude product. Then it was purified using silica-gel
column chromatography with methanol/dichloromethane (1:40,
v/v) as elution to give a light yellow solid product with a yield of
a
-H), 7.64–7.67 (2H, m, 2-H and 4-H), 7.81 (1H, d, J = 16.0 Hz,
b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z (ESI): 408
[M+H]+. IR (KBr) /cmꢀ1: 3032, 2955, 2870, 2782, 1658, 1607,
m
1587, 1511, 1339, 1228, 1173, 835, 771. Purity: 97.7% by HPLC
(MeOH/0.1% TEA 85:15 (v/v); tR 19.81 min).
73.5%. Mp 81–83 °C. 1H NMR (400 MHz, CDCl3)
d (ppm):
1.70–1.75 (2H, m, NCH2CH2CH2), 1.84–1.88 (2H, m, OCH2CH2CH2),
2.29 (6H, s, 2 ꢁ NCH3), 2.40 (2H, t, J = 6.0 Hz, NCH2CH2), 4.07 (2H, t,
J = 6.0 Hz, OCH2CH2), 6.98 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.41–
4.4.13. (E)-1-(4-(5-(Dimethylamino)pentyloxy)phenyl)-3-
phenylprop-2-en-1-one (7a)
According to the general method, the reaction of compound 3d
(0.372 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol) pro-
duced the crude product. Then it was purified using silica-gel
column chromatography with methanol/dichloromethane (1:45,
v/v) as elution to give a light yellow solid product with a yield of
74.2%. Mp 186–188 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.60–
1.66 (2H, m, CH2CH2CH2), 1.87–1.92 (2H, m, NCH2CH2), 1.95–2.01
(2H, m, OCH2CH2), 2.86 (6H, s, 2 ꢁ NCH3), 3.11 (2H, t, J = 6.0 Hz,
NCH2CH2), 4.08 (2H, t, J = 6.0 Hz, OCH2CH2), 6.97 (2H, d,
J = 8.0 Hz, 30-H and 50-H), 7.40–7.44 (3H, m, 3-H and 4-H and
7.44 (3H, m, 3-H and 4-H and 5-H), 7.56 (1H, d, J = 16.0 Hz, a-H),
7.64–7.67 (2H, m, 2-H and 4-H), 7.81 (1H, d, J = 16.0 Hz, b-H),
8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z (ESI): 324 [M+H]+.
IR (KBr) m
/cmꢀ1: 3031, 2951, 2808, 2756, 1655, 1603, 1588, 1339,
1260, 1175, 835, 768. Purity: 98.5% by HPLC (MeOH/0.1% TEA
85:15 (v/v); tR 4.52 min).
4.4.10. (E)-1-(4-(4-(diethylamino)butoxy)phenyl)-3-
phenylprop-2-en-1-one (6b)
According to the general method, the reaction of compound 3c
(0.358 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:45, v/v) as
elution to give a light yellow solid product with a yield of 75.8%.
Mp 57–59 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.22 (6H, t,
J = 6.8 Hz, 2 ꢁ NCH2CH3), 2.85–2.88 (4H, m, NCH2CH2 and
OCH2CH2), 2.79–2.86 (6H, m, 3 ꢁ NCH2CH2), 4.09 (2H, t,
J = 6.0 Hz, OCH2CH2), 6.97 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.41–
5-H), 7.55 (1H, d, J = 16.0 Hz,
H), 7.80 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d, J = 8.0 Hz, 20-H and
60-H). MS m/z (ESI): 338 [M+H]+. IR (KBr) /cmꢀ1: 3030, 2954,
2867, 2779, 1657, 1605, 1589, 1505, 1340, 1225, 1175, 833, 768.
Purity: 98.4% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 5.74 min).
a-H), 7.62–7.67 (2H, m, 2-H and 4-
m
4.4.14. (E)-1-(4-(5-(Diethylamino)pentyloxy)phenyl)-3-
phenylprop-2-en-1-one (7b)
According to the general method, the reaction of compound 3d
(0.372 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:50, v/v) as
elution to give a white solid product with a yield of 78.7%. Mp
143–145 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.44 (6H, t,
J = 6.8 Hz, 2 ꢁ NCH2CH3), 1.58–1.66 (2H, m, CH2CH2CH2), 1.86–
1.99 (4H, m, NCH2CH2 and OCH2CH2), 3.07 (2H, t, J = 6.0 Hz,
NCH2CH2), 3.20 (4H, m, 2 ꢁ NCH2CH3), 4.08 (2H, t, J = 6.0 Hz,
OCH2CH2), 6.97 (2H, d, J = 8.8 Hz, 30-H and 50-H), 7.39–7.43 (3H,
7.44 (3H, m, 3-H and 4-H and 5-H), 7.56 (1H, d, J = 16.0 Hz, a-H),
7.64–7.67 (2H, m, 2-H and 4-H), 7.82 (1H, d, J = 16.0 Hz, b-H),
8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z (ESI): 352 [M+H]+.
IR (KBr) m
/cmꢀ1: 3029, 2957, 2872, 2785, 1658, 1605, 1589, 1511,
1341, 1227, 1169, 831, 766. Purity: 98.2% by HPLC (MeOH/0.1%
TEA 85:15 (v/v); tR 6.43 min).
4.4.11. (E)-1-(4-(4-(Dipropylamino)butoxy)phenyl)-3-
phenylprop-2-en-1-one (6c)
According to the general method, the reaction of compound 3c
(0.358 g, 1 mmol) with dipropylamine (0.41 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:45, v/v) as
elution to give a light yellow oil product with a yield of 67.2%. 1H
NMR (400 MHz, CDCl3) d (ppm): 0.89 (6H, t, J = 6.8 Hz, 2 ꢁ NCH2
CH2CH3), 1.46–1.52 (4H, m, 2 ꢁ NCH2CH2CH3), 1.66–1.68 (2H, m,
NCH2CH2), 1.82–1.86 (2H, m, OCH2CH2), 2.41–2.43 (4H, t,
J = 6.0 Hz, 2 ꢁ NCH2CH2CH3), 2.53 (2H, t, J = 6.0 Hz, NCH2CH2),
4.07 (2H, t, J = 6.0 Hz, OCH2CH2), 6.98 (2H, d, J = 8.8 Hz, 30-H and
50-H), 7.41–7.43 (3H, m, 3-H and 4-H and 5-H), 7.56 (1H, d,
m, 3-H and 4-H and 5-H), 7.54 (1H, d, J = 16.0 Hz, a-H), 7.63–7.67
(2H, m, 2-H and 4-H), 7.79 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d,
J = 8.0 Hz, 20-H and 60-H). MS m/z (ESI): 366 [M+H]+. IR (KBr)
m
/cmꢀ1: 3032, 2958, 2871, 2776, 1656, 1605, 1587, 1508, 1342,
1223, 1175, 835, 767. Purity: 98.7% by HPLC (MeOH/0.1% TEA
85:15 (v/v); tR 8.09 min).
4.4.15. (E)-1-(4-(5-(Dipropylamino)pentyloxy)phenyl)-3-
phenylprop-2-en-1-one (7c)
According to the general method, the reaction of compound 3d
(0.372 g, 1 mmol) with dipropylamine (0.41 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:50, v/v) as
elution to give a white solid product with a yield of 65.9%. Mp
129–131 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.02 (6H, t,
J = 6.0 Hz, 2 ꢁ NCH2CH2CH3), 1.56–1.63 (2H, m, CH2CH2CH2),
1.83–2.01 (8H, m, NCH2CH2 and OCH2CH2 and 2 ꢁ NCH2CH2CH3),
2.99–3.12 (6H, m, 3 ꢁ NCH2CH2), 4.07 (2H, t, J = 6.0 Hz, OCH2CH2),
6.96 (2H, d, J = 8.0 Hz, 30-H and 50-H), 7.40–7.45 (3H, m, 3-H and
J = 16.0 Hz,
J = 16.0 Hz, b-H), 8.04 (2H, d, J = 8.8 Hz, 20-H and 60-H). MS m/z
(ESI): 380 [M+H]+. IR (KBr) /cmꢀ1: 3030, 2953, 2872, 2789,
a-H), 7.64–7.67 (2H, m, 2-H and 4-H), 7.81 (1H, d,
m
1656, 1607, 1590, 1508, 1341, 1228, 1169, 829, 768. Purity:
97.6% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 11.91 min).
4.4.12. (E)-1-(4-(4-(Dibutylamino)butoxy)phenyl)-3-
phenylprop-2-en-1-one (6d)
According to the general method, the reaction of compound 3c
(0.358 g, 1 mmol) with dibutylamine (0.5 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:50, v/v) as
elution to give a light yellow oil product with a yield of 61.9%. 1H
4-H and 5-H), 7.54 (1H, d, J = 16.0 Hz,
H and 4-H), 7.79 (1H, d, J = 16.0 Hz, b-H), 8.02 (2H, d, J = 8.0 Hz,
20-H and 60-H). MS m/z (ESI): 394 [M+H]+. IR (KBr) /cmꢀ1: 3033,
2961, 2875, 2778, 1656, 1603, 1586, 1507, 1340, 1225, 1173,
836, 768. Purity: 97.2% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
14.42 min).
a-H), 7.63–7.68 (2H, m, 2-
m
NMR (400 MHz, CDCl3)
d (ppm): 0.92 (6H, t, J = 6.8 Hz,

6132
H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
4.4.16. (E)-1-(4-(5-(Dibutylamino)pentyloxy)phenyl)-3-
phenylprop-2-en-1-one (7d)
J = 6.0 Hz, 2 ꢁ NCH2CH2CH3), 1.46–1.58 (4H, m, CH2CH2CH2CH2
and CH2CH2CH2CH2), 1.68–1.79 (6H, m, 2 ꢁ NCH2CH2CH3 and
NCH2CH2), 1.80–1.87 (2H, m, OCH2CH2), 2.76–2.84 (6H, m,
3ꢁNCH2CH2), 4.05 (2H, t, J = 6.0 Hz, OCH2CH2), 6.96 (2H, d,
J = 8.0 Hz, 30-H and 50-H), 7.40–7.45 (3H, m, 3-H and 4-H and
According to the general method, the reaction of compound 3d
(0.372 g, 1 mmol) with dibutylamine (0.5 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:55, v/v) as
elution to give a light yellow oil product with a yield of 60.8%. 1H
NMR (400 MHz, CDCl3) d (ppm): 1H NMR (400 MHz, CDCl3) d
(ppm): 0.93 (6H, t, J = 6.0 Hz, 2 ꢁ NCH2CH2CH2CH3), 1.22–1.32
(4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.45–1.52 (2H, m, CH2CH2CH2),
1.72–1.89 (8H, m, NCH2CH2 and OCH2CH2 and 2 ꢁ NCH2CH2CH2CH3),
2.66–2.74 (6H, m, 3 ꢁ NCH2CH2), 4.05 (2H, t, J = 6.0 Hz, OCH2CH2),
6.96 (2H, d, J = 8.0 Hz, 30-H and 50-H), 7.41–7.45 (3H, m, 3-H and
5-H), 7.54 (1H, d, J = 16.0 Hz,
4-H), 7.79 (1H, d, J = 16.0 Hz, b-H), 8.02 (2H, d, J = 8.0 Hz, 20-H
and 60-H). MS m/z (ESI): 408 [M+H]+. IR (KBr) /cmꢀ1: 3031,
a-H), 7.63–7.68 (2H, m, 2-H and
m
2957, 2877, 2776, 1656, 1606, 1588, 1509, 1341, 1223, 1171,
838, 769. Purity: 97.7% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
18.51 min).
4.4.20. (E)-1-(4-(6-(Dibutylamino)hexyloxy)phenyl)-3-
4-H and 5-H), 7.55 (1H, d, J = 16.0 Hz,
2-H and 4-H), 7.79 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d, J = 8.0 Hz,
20-H and 60-H). MS m/z (ESI): 422 [M+H]+. IR (KBr) /cmꢀ1: 3032,
2958, 2873, 2779, 1656, 1608, 1589, 1508, 1341, 1226, 1172,
836, 770. Purity: 97.8 % by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
23.98 min).
a
-H), 7.62–7.66 (2H, m,
phenylprop-2-en-1-one (8d)
According to the general method, the reaction of compound 3e
(0.386 g, 1 mmol) with dibutylamine (0.5 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:55, v/v) as
elution to give a light yellow oil product with a yield of 63.7%. 1H
NMR (400 MHz, CDCl3) d (ppm): 0.93 (6H, t, J = 6.0 Hz, 2 ꢁ NCH2
CH2CH2CH3), 1.25–1.31 (4H, m, 2 ꢁ NCH2CH2CH2CH3), 1.45–1.57
(4H, m, CH2CH2CH2CH2 and CH2CH2CH2CH2), 1.62–1.78 (6H, m,
2 ꢁ NCH2CH2CH2CH3 and NCH2CH2), 1.83–1.90 (2H, m, OCH2CH2),
2.70–2.77 (6H, m, 3 ꢁ NCH2CH2), 4.05 (2H, t, J = 6.0 Hz, OCH2CH2),
6.96 (2H, d, J = 8.0 Hz, 30-H and 50-H), 7.41–7.45 (3H, m, 3-H and 4-
H and 5-H), 7.55 (1H, d, J = 16.0 Hz,
and 4-H), 7.79 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d, J = 8.0 Hz, 20-
H and 60-H). MS m/z (ESI): 436 [M+H]+. IR (KBr) /cmꢀ1: 3032,
2955, 2876, 2778, 1654, 1607, 1586, 1507, 1343, 1221, 1169,
836, 770. Purity: 97.3% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR
28.37 min).
m
4.4.17. (E)-1-(4-(6-(Dimethylamino)hexyloxy)phenyl)-3-
phenylprop-2-en-1-one (8a)
According to the general method, the reaction of compound 3e
(0.386 g, 1 mmol) with diethylamine (0.31 mL, 3 mmol) produced
the crude product. Then it was purified using silica-gel column
chromatography with methanol/dichloromethane (1:50, v/v) as
elution to give a white solid product with a yield of 75.6%.
Mp 139–141 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.48–1.61
(4H, m, CH2CH2CH2CH2 and CH2CH2CH2CH2), 1.80–1.96 (4H, m,
NCH2CH2 and OCH2CH2), 2.86 (6H, s, 3 ꢁ NCH3), 3.09 (2H, t,
J = 6.0 Hz, NCH2CH2), 4.05 (2H, t, J = 6.0, OCH2CH2), 6.97 (2H, d,
J = 8.0 Hz, 30-H and 50-H), 7.40–7.44 (3H, m, 3-H and 4-H and 5-H),
a-H), 7.62–7.66 (2H, m, 2-H
m
7.55 (1H, d, J = 16.0 Hz,
7.79 (1H, d, J = 16.0 Hz, b-H), 8.03 (2H, d, J = 8.0 Hz, 20-H and
60-H). MS m/z (ESI): 352 [M+H]+. IR (KBr) /cmꢀ1: 3033, 2962,
2877, 2781, 1655, 1606, 1587, 1510, 1340, 1224, 1173, 836, 771.
Purity: 98.3% by HPLC (MeOH/0.1% TEA 85:15 (v/v); tR 7.13 min).
a
-H), 7.61–7.66 (2H, m, 2-H and 4-H),
4.5. Enzyme inhibition assays
m
AChE/BuChE inhibitory activities of the synthesized compounds
were assayed by the spectrophotometric method developed by Ell-
man et al. with slight modification.29 The tested compounds were
dissolved in tween 80 (final concentration was 0.06% in each reac-
tion) and followed by dilution in 2% tween 80-water solution to
obtain final assay concentrations. Five different concentrations
were tested for each compound in triplicate to obtain the range
of 20–80% inhibition for AChE and BuChE. The reaction mixture
4.4.18. (E)-1-(4-(6-(Diethylamino)hexyloxy)phenyl)-3-
phenylprop-2-en-1-one (8b)
According to the general method, the reaction of compound 3e
(0.386 g, 1 mmol) with dimethylamine (0.34 mL, 3 mmol) pro-
duced the crude product. Then it was purified using silica-gel col-
umn chromatography with methanol/dichloromethane (1:50, v/v)
as elution to give a white solid product with a yield of 68.3%. Mp
165–167 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 1.44 (6H, t,
J = 6.8 Hz, 2 ꢁ NCH2CH3), 1.49–1.60 (4H, m, CH2CH2CH2CH2 and
CH2CH2CH2CH2), 1.81–1.95 (4H, m, NCH2CH2 and OCH2CH2), 3.05
(2H, t, J = 6.0 Hz, NCH2CH2), 3.19 (4H, m, 2 ꢁ NCH2CH3), 4.04 (2H,
t, J = 6.0, OCH2CH2), 6.96 (2H, d, J = 8.80 Hz, 30-H and 50-H), 7.40–
containing 40
or butyrylthiocholine iodide, 2.76 mL Na2HPO4/NaH2PO4 buffer
(pH 8.0), 100 L different concentrations of tested compounds
was incubated at 30 °C for 25 min. Then the reaction was termi-
nated by adding 100 L 20% Sodium dodecyl sulfate (SDS) and
100
10 mmol/L 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB)
lL AChE or BuChE, 100 lL acetylthiocholine iodide
l
l
lL
was added to generate the yellow anion 5-thio-2-nitro-benzoic
acid. The absorbance of each tested mixture was measured at
412 nm by UV spectroscopy. The IC50 values were calculated by
Bliss method and expressed as mean SD of the replicates.
7.44 (3H, m, 3-H and 4-H and 5-H), 7.54 (1H, d, J = 16.0 Hz, a-H),
7.61–7.66 (2H, m, 2-H and 4-H), 7.79 (1H, d, J = 16.0 Hz, b-H),
8.03 (2H, d, J = 8.0 Hz, 20-H and 60-H). MS m/z (ESI): 380 [M+H]+.
IR (KBr)
m
/cmꢀ1: 3031, 2960, 2875, 2778, 1657, 1605, 1589,
4.6. Kinetic assay
1508/, 1342, 1225, 1172, 837, 769. Purity: 98.1% by HPLC
(MeOH/0.1% TEA 85:15 (v/v); tR 10.17 min).
Kinetic characterization of AChE was performed using a
reported method.30 Compound 4a was added into the assay solu-
tion and preincubated with the enzyme at 30 °C for 25 min, fol-
lowed by the addition of Acetylthiocholine iodide. The assay
4.4.19. (E)-1-(4-(6-(Dipropylamino)hexyloxy)phenyl)-3-
phenylprop-2-en-1-one (8c)
According to the general method, the reaction of compound 3e
(0.386 g, 1 mmol) with dipropylamine (0.41 mL, 3 mmol) pro-
duced the crude product. Then it was purified using silica-gel col-
umn chromatography with methanol/dichloromethane (1:55, v/v)
as elution to give a white solid product with a yield of 71.5%. Mp
120–122 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 0.97 (6H, t,
solution contained 100
Na2HPO4/NaH2PO4 buffer (pH 8.0) and 100
iodide. Kinetic characterization of the hydrolysis of Acetylthiocho-
line iodide catalyzed by AChE was done spectrometrically at
412 nm. The parallel control experiment was carried out without
compound 4a in the mixture.
l
L compound 4a, 100
lL DTNB, 2.76 mL
lL Acetylthiocholine

H.-r. Liu et al. / Bioorg. Med. Chem. 22 (2014) 6124–6133
6133
4.7. Molecular docking
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Octanol–water partition coefficients of compounds 4a–8d were
measured by the shake flask method with slight modification31 and
PBS (pH = 7.4) was used as the aqueous phase. The mobile phase
was methanol: 0.1% TEA /85:15(v/v), at a flow rate of 1.0 mL minꢀ1
through a C18 column (250 nm ꢁ 4.6 mm, 5
lm) at 32 °C with
detect wavelength 319 nm . Experiments were conducted in tripli-
cate and log P values were calculated.
Acknowledgment
The present investigation was supported by the Grand of ‘‘The
project of science and technology of Hu’nan Province’’ (No.
2012SK3183) and the Grand of ‘‘the Fundamental Research Funds
for the Central Universities’’ of Hu’nan University.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.08.033.