Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side chain and α,β-unsaturated ketone group

Article abstract of DOI:10.1080/14756366.2016.1243534

A new series of tertiary amine derivatives of chlorochalcone (4a～4l) were designed, synthesized and evaluated for the effect on acetylcholinesterase (AChE) and buthylcholinesterase (BuChE). The results indicated that all compounds revealed moderate or pot

Full text of DOI:10.1080/14756366.2016.1243534

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Tertiary amine derivatives of chlorochalcone
as acetylcholinesterase (AChE) and
buthylcholinesterase (BuChE) inhibitors: the
influence of chlorine, alkyl amine side chain and
α,β-unsaturated ketone group
Xiao-hui Gao, Chao Zhou, Hao-ran Liu, Lin-bo Liu, Jing-jing Tang & Xin-hua
Xia
To cite this article: Xiao-hui Gao, Chao Zhou, Hao-ran Liu, Lin-bo Liu, Jing-jing Tang & Xin-hua
Xia (2016): Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and
buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side chain and
α,β-unsaturated ketone group, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.1080/14756366.2016.1243534
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1243534
Published online: 01 Nov 2016.
Article views: 2
View supplementary material
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [FU Berlin]
Date: 06 November 2016, At: 18:50

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1243534
RESEARCH ARTICLE
Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and
buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side
chain and a,b-unsaturated ketone group
Xiao-hui Gaoa, Chao Zhoub, Hao-ran Liub, Lin-bo Liub, Jing-jing Tangb and Xin-hua Xiaa
b
aCollege of Pharmacy, Hu’nan University of Chinese Medicine, Changsha, PR China; College of Chemistry and Chemical Engineering, Hu’nan
University, Changsha, PR China
ABSTRACT
ARTICLE HISTORY
Received 29 April 2016
Revised 21 August 2016
Accepted 23 August 2016
Published online 21 October
2016
A new series of tertiary amine derivatives of chlorochalcone (4a~4l) were designed, synthesized and eval-
uated for the effect on acetylcholinesterase (AChE) and buthylcholinesterase (BuChE). The results indicated
that all compounds revealed moderate or potent inhibitory activity against AChE, and some possessed
high selectivity for AChE over BuChE. The structure–activity investigation showed that the substituted pos-
ition of chlorine significantly influenced the activity and selectivity. The alteration of tertiary amine group
also leads to obvious change in bioactivity. Among them, IC50 of compound 4l against AChE was
0.17 0.06 mmol/L, and the selectivity was 667.2 fold for AChE over BuChE. Molecular docking and enzyme
kinetic study on compound 4l suggested that it simultaneously binds to the catalytic active site (CAS) and
peripheral anionic site (PAS) of AChE. Further study showed that the pyrazoline derivatives synthesized
from chlorochalcones had weaker activity and lower selectivity in inhibiting AChE compared to that of
chlorochalcone derivatives.
KEYWORDS
Acetylcholinesterase inhibi-
tors; Alzheimer’s disease;
chlorochalcones; pyrazoline;
structure–activity
relationship
Introduction
Materials and methods
Alzheimer’s disease (AD), as one of most common diseases in the Chemistry
elderly population, is a chronic and progressive neurodegenerative
All chemicals and reagents were of analytical reagent grade and
used without further purification. The melting points were meas-
ured on a WRS-lA melting point detector1. H NMR spectra were
recorded on a Bruker 400 MHz instrument in CDCl3 with TMS as
the internal reference. Mass spectra were obtained on Finnigan
LCQ advantage MAX by electrospray ionization (ESI-MS). Infrared
spectrum (IR) was obtained on Shimadzu Infinity-1 infrared spec-
trometer. The purity of compounds was checked by Shimadzu LC-
20A high-performance liquid chromatography. Elemental analysis
was performed by elemental analyzer.
disorder1–3. Although the precise etiology of AD is not elucidated
enough, acetylcholinesterase (AChE) inhibitors were confirmed to
be as the primary drugs to slow down the progression of AD in
the present4,5
.
In recent, a lot of tertiary amine derivatives originated from nat-
ural products were synthesized and evaluated as AChE inhibitors6–9
.
The previous investigations in our laboratory suggested that chal-
cones with tertiary amine side chain, such as dimethylamine, dieth-
ylamine, dihydropyrrole or piperidine had more potent effects than
other nitrogen-containing chalcones on inhibiting AChE10–12
.
In order to explore the influence of substituent on inhibiting
AChE in chalcone derivatives, halogen atoms were considered to
introduce into the chalcone scaffold. Chlorine was selected to
modify the chalcone derivatives, considering chlorine exist in a lot
of drugs in clinic application, such as chlorphenamine, chlorpro-
mazine, chloroquine, etc. In the investigations searching for AChE
inhibitors, some chlorine-containing compounds revealed AChE
General method for synthesis of 4-hydroxy-chlorochalcones
(3a–3c)
Compounds 3a–3c were synthesized according to the relative refer-
ences15,16. 4-hydroxyacetophenone (1.36 g, 10 mmol) and ethanol
(20 mL) were mixed and stirred in the flask for 10 min until the solid
was dissolved. Then 30% NaOH (3 mL) was dropped into the mix-
ture, followed by stirring for 30 min in ice bath. Then chlorine ben-
zaldehyde (11 mmol) dissolving in ethanol (5 mL) was dropped into
the mixture, and the mixture continued to stir for about 36 h at
25 ꢀC monitoring by TLC until the reaction was completed. Then
the pH of the mixture was adjusted to 2–3 by HCl (1 mol/L).The mix-
ture was kept in ice bath for 2 h, followed by the appearance of pre-
cipitation. The precipitation was filtered and washed by distilled
water, and then dried at 50 ꢀC, refined by the recrystallization in
anhydrous ethanol. In result, compounds 3a–3c were gained.
inhibition properties13,14
.
In this study, a series of chlorochalcones with tertiary amine
side chain were synthesized and structure–activity relationship
investigation was performed to explore the influence of chlorine
in inhibiting AChE. In addition, four pyrazoline compounds were
synthesized from chalcones and evaluated for the bioactivity to
explore the influence of a,b-unsaturated ketone group. Compound
4l, which had the most potent inhibitory activity against AChE,
was selected to perform the kinetic and molecular docking studies
for exploring the binding mode or mechanism to AChE.
CONTACT Hao-ran Liu
pharmaliu@126.com
College of Chemistry and Chemical Engineering, Hu’nan University, Changsha, PR China; Xin-hua Xia
xiaxin-
hua001@163.com
College of Pharmacy, Hu'nan University of Chinese Medicine, Changsha, PR China
Supplemental data for this article can be accessed here
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
X. H. GAO ET AL.
4'-Hydroxy-2-chlorochalcone (3a)
C19H20ClNO2: C, 69.19; H, 6.11; N, 4.25; O, 9.70; found C, 69.32; H,
6.04; N, 4.19; O, 9.74.
Light yellow solid was gained with yield of 80.7%. It was a known
compound which was reported to have anticancer activity17.
(E)-3–(4-chlorophenyl)-1–(4-(2-
(dimethylamino)ethoxy)phenyl)prop-2-en-1-one (4c)
4'-Hydroxy-3-chlorochalcone (3b)
1
A white solid, yield: 75.6%; m.p: 2 3 3 ꢁ 234 ꢀC; H NMR (400 MHz,
Light yellow solid, yield: 73.5%; m.p: 1 2 0 ꢁ 122 ꢀC; 1H NMR
(400 MHz, CDCl3, ppm): 6.96–6.98 (2H, d, J ¼8.0 Hz, 30-H and 50-H),
7.26–7.34 (2H, m, 4-H and 5-H), 7.54–7.81 (3H, m, a-H, 2-H and 6-
H), 8.10–8.16 (3H, m, b-H, 20-H and 60-H), 11.10 (1H, s, OH). MS m/z
(ESI): [M þ H]þ 259. IR (KBr) m/cmꢂ1: 3426, 3016, 1667.2, 1617, 1583,
1446, 1340, 1288, 1215, 1172, 839 and 732. Anal. calcd for
C15H11ClO2: C, 69.64; H, 4.29; O, 12.37; found C, 69.52; H, 4.27; N,
12.42%.
CDCl3) d (ppm): 2.37 (6H, s, 2 ꢃ NCH3), 2.79 (2H, t, J ¼12.0 Hz,
OCH2CH2), 4.14 (2H, t, J ¼12.0 Hz, OCH2CH2), 7.00–7.02 (2H, m, 30-H
and 50-H), 7.38–7.40 (2H, m, 2-H and 6-H), 7.51 (1H, d, J ¼ 16.0 Hz,
a-H), 7.55–7.59 (2H, m, 3-H and 5-H), 7.73–7.77 (1H, d, J ¼ 16.0 Hz,
b-H), 8.02–8.04 (2H, m, 20-H and 60-H). MS m/z (ESI): [M þ H]þ 330.
IR (KBr) m/cmꢂ1: 2805, 2774, 2359, 1658, 1595, 1558, 1456, 1246,
1227, 1175, 1020 and 731. Anal. calcd for C19H20ClNO2: C, 69.19; H,
6.11; N, 4.25; O, 9.70; found C, 68.98; H, 6.07; N, 4.28; O, 9.72.
4'-Hydroxy -4-chlorochalcone (3c)
(E)-3–(2-chlorophenyl)-1–(4-(2-(diethylamino)ethoxy)phenyl)prop-
Light yellow solid was gained with yield of 85.6%. It is a known
compound which was reported to inhibiting monoamine
oxidases18.
2-en-1-one (4d)
Yellow viscous liquid was gained with yield of 78.1%. It is a known
compound with no reports about bioactivity20.
General method for synthesis of 4-amino alkyl-chlorochalcones
(4a–4l)
(E)-3–(3-chlorophenyl)-1–(4-(2-(diethylamino)ethoxy)phenyl)prop-
2-en-1-one (4e)
Compounds 4a–4l were synthesized according to the relative
references19. 4’-hydroxy chlorochalcones (0.2620 g, 1 mmol) and
potassium carbonate (0.4140 g, 3 mmol) were mixed in acetone
(25 mL) in oil bath with continuous stirring at 56 ꢀC for 30 min,
then chloroethyldimethylamine hydrochloride, chloroethyldiethyl-
amine hydrochloride, chloroethylpiperidine hydrochloride or chlor-
oethylpyrrolidine hydrochloride (3 mmol) and sodium iodide
(0.075 g, 0.5 mmol) were added into it. The mixture was refluxed
overnight, filtered and concentrated. The concentrate was
extracted with CH2Cl2 (3 ꢃ 30 mL), The CH2Cl2 phase was washed
by saturated NaHCO3 (2 ꢃ 30 mL), saturated salt water (3 ꢃ 30 mL),
dried with sodium sulfate anhydrous, followed by concentrating in
vacuum and then the residue was purified by silica gel to gain the
product with dichloromethane/methanol (80:1) as the eluent.
Yellow viscous liquid, yield: 73.1%; 1H NMR (400 MHz, CDCl3) d
(ppm): 1.12 (6H, t, J ¼12.0 Hz, 2 ꢃ NCH2CH3), 2.68–2.74 (4H, m,
2 ꢃ NCH2CH3), 2.96 (2H, t, J ¼12.0 Hz, OCH2CH2), 4.17 (2H, t,
J ¼12.0 Hz, OCH2CH2), 6.96–6.98 (2H, d, J ¼8.0 Hz, 30-H and 50-H),
7.26–7.35 (2H, m, 4-H and 5-H), 7.54–7.81 (3H, m, a-H, 2-H and 6-
H), 8.10–8.16 (3H, m, b-H, 20-H and 60-H). MS m/z (ESI):
[M þ H]þ 358. IR (KBr) m/cmꢂ1: 2970, 2810, 1661, 1508, 1337, 1246,
1173, 1024, 833 and 735. Anal. calcd for C21H24ClNO2: C, 70.48; H,
6.76; N, 3.91; O, 8.94; found: C, 70.63; H, 6.62; N, 3.85; O, 8.78.
(E)-3–(4-chlorophenyl)-1–(4-(2-(diethylamino)ethoxy)phenyl)prop-
2-en-1-one (4f)
Yellow viscous liquid was gained with yield of 72.4%. It is a known
compound with no reports about bioactivity21.
(E)-3–(2-chlorophenyl)-1–(4-(2-
(dimethylamino)ethoxy)phenyl)prop-2-en-1-one (4a)
(E)-3–(2-chlorophenyl)-1–(4-(2-(piperidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4g)
Light yellow solid, yield: 81.6%; m.p:
7
6 ꢁ 77 ꢀC; 1H NMR
(400 MHz, CDCl3) d (ppm): 1.09 (6H, s, 2 ꢃ NCH3), 2.92 (2H, t,
J ¼12.0 Hz, OCH2CH2), 4.14 (2H, t, J ¼12.0 Hz, OCH2CH2), 6.98–7.00
(2H, m, 30-H and 50-H), 7.27–7.34 (2H, m, 3-H and 6-H), 7.43–7.77
(3H, m, a-H and 4-H and 5-H), 8.02–8.05 (2H, m, 20-H and 60-H),
8.15–8.19 (1H, d, J ¼ 16.0 Hz, b-H). MS m/z (ESI): [M þ H]þ 330. IR
(KBr) m/cmꢂ1: 2806, 2769, 1661, 1611, 1576, 1458, 1261, 1227,
1176, 1020 and 730. Anal. calcd for C19H20ClNO2: C, 69.19; H, 6.11;
N, 4.25; O, 9.70; found C, 68.28; H, 6.05; N, 4.21; O, 9.63.
1
Light white solid, yield: 83.7%; m.p: 5 5 ꢁ 56 ꢀC; H NMR (400 MHz,
CDCl3) d (ppm): 1.24–1.28 (2H, m, piperidine-H), 1.22–1.36 (4H, m,
piperidine-H), 2.35–2.43 (4H, m, piperidine-H), 2.73(2H, t,
J ¼12.0 Hz, OCH2CH2), 4.12 (2H, t, J ¼12.0 Hz, OCH2CH2), 6.98–7.00
(2H, m, 30-H and 50-H), 7.27–7.34 (2H, m, 3-H and 6-H), 7.43–7.77
(3H, m, a-H and 4-H and 5-H), 8.02–8.05 (2H, m, 20-H and 60-H),
8.15–8.19 (1H, d, J ¼16.0 Hz, b-H). MS m/z (ESI): [M þ H]þ 370. IR
(KBr) m/cmꢂ1: 3904, 3853, 3838, 3821, 3802, 3726, 3725, 3649,
2960, 2930, 2360, 1608, 1558, 1508, 1458 and 730. Anal. calcd for
C22H24ClNO2: C, 71.44; H, 6.54; N, 3.79; O, 8.65 found C, 71.58; H,
6.41; N, 3.65; O, 8.72.
(E)-3–(3-chlorophenyl)-1–(4-(2-
(dimethylamino)ethoxy)phenyl)prop-2-en-1-one (4b)
1
Light white solid, yield: 82.6%; m.p: 6 2 ꢁ 63 ꢀC; H NMR (400 MHz,
CDCl3) d (ppm): 2.79 (6H, s, 2 ꢃ NCH3), 3.31 (2H, t, J ¼ 12.0 Hz,
OCH2CH2), 4.51 (2H, t, J ¼12.0 Hz, OCH2CH2), 6.96–6.98 (2H, d,
J ¼8.0 Hz, 30-H and 50-H), 7.26–7.35 (2H, m, 4-H and 5-H), 7.54–7.81
(E)-3–(3-chlorophenyl)-1–(4-(2-(piperidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4h)
(3H, m, a-H, 2-H and 6-H), 8.10–8.16 (3H, m, b-H, 20-H and 60-H), Light yellow solid, yield: 85.7%; m.p:
7
5 ꢁ 76 ꢀC; 1H NMR
MS m/z (ESI): [M þ H]þ 330. IR (KBr) m/cmꢂ1: 2805, 2774, 1678, (400 MHz, CDCl3)
d (ppm): 1.28–1.29 (2H, m, piperidine-H),
1626, 1576, 1458, 1246, 1227, 1175, 1020 and 722. Anal. calcd for 1.35–1.39 (4H, m, piperidine-H), 2.33–2.43 (4H, m, piperidine-H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
2.77 (2H, t, J ¼12.0 Hz, OCH2CH2), 4.15 (2H, t, J ¼12.0 Hz, OCH2CH2), (1H, m, NCH), 6.92–6.95 (2H, m, 30-H and 50-H), 7.03–7.28 (4H, m,
6.98–7.00 (2H, m, 30-H and 50-H), 7.27–7.34 (2H, m, 3-H and 6-H), 1-H and 2-H and 4-H and 5-H), 7.65–7.67 (2H, m, 20-H and 60-H).
7.43–7.77 (3H, m, a-H and 4-H and 5-H), 8.02–8.05 (2H, m, 20-H MS m/z (ESI): [M þ H]þ 426. IR (KBr) m/cmꢂ1: 3442, 3419, 3062,
and 60-H), 8.15–8.19 (1H, d, J ¼16.0 Hz, b-H). MS m/z (ESI): 3030, 2933, 2853, 2787, 1653, 1608, 1510, 1422, 1251, 1223, 1175,
[M þ H]þ 370. IR (KBr) m/cmꢂ1: 3903, 3853, 3838, 3802, 3736, 3649, 1163, 1028, 988 and 814. Anal. calcd for C24H28ClN3O2: C, 67.67; H,
3566, 2960, 2934, 2359, 1659, 1611, 1578, 1236, 1175, 1026 6.63; N, 9.87; O, 7.51; found C, 67.78; H, 6.52; N, 9.81; O, 7.46.
and 730. Anal. calcd for C22H24ClNO2: C, 71.44; H, 6.54; N, 3.79; O,
8.65 found C, 71.52; H, 6.48; N, 3.71; O, 8.62.
1–(5-(2-Chlorophenyl)-3–(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4,5-
dihydro-1H-pyrazol-1-yl) ethan-1-one (5b)
(E)-3–(4-chlorophenyl)-1–(4-(2-(piperidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4i)
Yellow viscous liquid, yield:74.2%; 1H NMR (400 MHz, CDCl3) d
(ppm): 1.84–1.87 (6H, m, piperidine-H), 2.69–2.73 (3H, s, COCH3),
White solid was gained with the yield: 82.7%. It is a known com- 2.96–2.99 (4H, m, piperidine-H), 3.09 (2H, t, J ¼12.0 Hz, OCH2CH2),
pound with anticancer effect in a recent investigation22.
3.12 (1H, dd, J ¼4.0 Hz, 16.0 Hz, CH2-Ha), 3.76 (1H, dd, J ¼12.0 Hz,
16.0 Hz, CH2-Hb), 4.20 (2H, t, J ¼12.0 Hz, OCH2CH2), 5.76–5.80 (1H,
m, NCH), 6.90–6.93 (2H, m, 30-H and 50-H), 7.13–7.35 (4H, m, 3-H
and 4-H and 5-H and 6-H), 7.63–7.67 (2H, m, 20-H and 60-H).
MS m/z (ESI): [M þ H]þ 412. IR (KBr) m/cmꢂ1: 3446, 3421, 3063,
3032, 2935, 2858, 2779, 1642, 1590, 1512, 1339, 1243, 1219, 1173,
1020, 980 and 726. Anal. calcd for C23H26ClN3O2: C, 67.06; H, 6.36; N,
10.20; O, 7.77; found C, 66.91; H, 6.48; N, 10.09; O, 7.84.
(E)-3–(2-chlorophenyl)-1–(4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4j)
Yellow solid was gained with the yield of 80.7%. It is a known
compound with anticancer effect in a recent investigation22.
(E)-3–(3-chlorophenyl)-1–(4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4k)
1–(5-(3-Chlorophenyl)-3–(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethan-1-one (5c)
White solid, yield: 80.7%; m.p: 9 7 ꢁ 98 ꢀC; 1H NMR (400 MHz,
CDCl3) d (ppm): 1.72–1.75 (4H, m, pyrrolidine-H), 2.56–2.58 (4H, m,
pyrrolidine-H), 2.76 (2H, t, J ¼12.0 Hz, OCH2CH2), 4.01 (2H, t,
J ¼12.0 Hz, OCH2CH2), 6.98–7.00 (2H, m, 30-H and 50-H), 7.27–7.34
(2H, m, 3-H and 6-H), 7.43–7.77 (3H, m, a-H and 4-H and 5-H),
8.02–8.05 (2H, m, 20-H and 60-H), 8.15–8.19 (1H, d, J ¼16.0 Hz, b-H).
MS m/z (ESI): [M þ H]þ 356. IR (KBr) m/cmꢂ1: 2956, 2781, 1657,
1604, 1593, 1510, 1479, 1339, 1269, 1211, 1175, 1020, 980 and
735. Anal. calcd for C21H22ClNO2: C, 70.88; H, 6.23; N, 3.94; O, 8.99;
found C, 70.95; H, 6.27; N, 3.88; O, 8.91.
Yellow viscous liquid, yield:77.6%; 1H NMR (400 MHz, CDCl3) d
(ppm): 1.84–1.87 (6H, m, piperidine-H), 2.69–2.73 (3H, s, COCH3),
2.96–2.99 (4H, m, piperidine-H), 3.09 (2H, t, J ¼12.0 Hz, OCH2CH2),
3.13 (1H, dd, J ¼4.0 Hz, 16.0 Hz, CH2-Ha), 3.76 (1H, dd, J ¼12.0 Hz,
16.0 Hz, CH2-Hb), 4.20 (2H, t, J ¼12.0 Hz, OCH2CH2), 5.76–5.80
(1H, m, NCH), 6.83–6.85 (2H, m, 30-H and 50-H), 7.04–7.29 (4H, m,
2-H and 4-H and 5-H and 6-H), 7.66–7.70 (2H, m, 20-H and 60-H).
MS m/z (ESI): [M þ H]þ 412. IR (KBr) m/cmꢂ1: 3443, 3417, 3062,
3032, 2936, 2881, 1645, 1583, 1510, 1479, 1339, 1269, 1243, 1211,
1175, 1020, 980 and 735. Anal. calcd for C23H26ClN3O2: C, 67.06; H,
6.36; N, 10.20; O, 7.77; found C, 66.89; H, 6.45; N, 10.34; O, 7.61.
(E)-3–(4-chlorophenyl)-1–(4-(2-(pyrrolidin-1-
yl)ethoxy)phenyl)prop-2-en-1-one (4l)
1–(5-(4-Chlorophenyl)-3–(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4,5-
dihydro-1H-pyrazol-1-yl) ethan-1-one (5d)
Yellow solid was gained with the yield of 81.7%. It is a known
compound with anticancer effect in a recent investigation22.
Yellow viscous liquid, yield: 73.2%; 1H NMR (400 MHz, CDCl3) d
(ppm): 1.84–1.87 (6H, m, piperidine-H), 2.69–2.73 (3H, s, COCH3),
2.96–2.99 (4H, m, piperidine-H), 3.09 (2H, t, J ¼12.0 Hz, OCH2CH2),
General procedure of synthesis of compounds 5a–5d
Compounds 4i, 4j, 4k or 4l (1 mmol), 80% hydrazine hydrate 3.13 (1H, dd, J ¼4.0 Hz, 16.0 Hz, CH2-Ha), 3.76 (1H, dd, J ¼12.0 Hz,
(0.2 mL, 4 mmol) and acetic acid (5 mL) were mixed and refluxed23. 16.0 Hz, CH2-Hb), 4.20 (2H, t, J ¼12.0 Hz, OCH2CH2), 5.76–5.80 (1H,
When the reaction was completed, the mixture was cooled to m, NCH), 6.92–6.95 (2H, m, 30-H and 50-H), 7.03–7.28 (4H, m, 1-H
room temperature and 30 mL CH2Cl2 was added into it. The and 2-H and 4-H and 5-H), 7.65–7.67 (2H, m, 20-H and 60-H). MS m/
CH2Cl2 phase was washed by saturated K2CO3 solution, and dried z (ESI): [M þ H]þ 412. IR (KBr) m/cmꢂ1: 3442, 3419, 3060, 3031, 2930,
by anhydrous sodium sulfate. After the solution was filtered and 2883, 1640, 1585, 1510, 1307, 1263, 1230, 1221, 1175, 1032, 978
the solvent was evaporated under reduced pressure, the crude and 738. Anal. calcd for C23H26ClN3O2: C, 67.06; H, 6.36; N, 10.20;
product was gained. The refined product was achieved by the sil- O, 7.77; found C, 67.24; H, 6.18; N, 10.12; O, 7.65.
ica column chromatography with the eluent (methanol: methylene
chloride ¼1:20).
Log p measurement
Log p, defined as the logarithm of octanol-water partition coeffi-
1–(5-(4-Chlorophenyl)-3–(4-(2-(piperidin-1-yl)ethoxy)phenyl)-4,5-
cient is an important parameter to evaluate the lipophilicify of
compounds. It can be calculated by determining the concentration
dihydro-1H-pyrazol-1-yl) ethan-1-one (5a)
Yellow viscous liquid, yield: 70.6%; 1H NMR (400 MHz, CDCl3) d of compound in octanol phase and water phase until the partition
(ppm): 1.84–1.86 (6H, m, piperidine-H), 2.43 (3H, s, COCH3), equilibrium was completed. In present investigation, log p of com-
2.54–2.57 (4H, m, piperidine-H), 2.86 (2H, t, J ¼12.0 Hz, OCH2CH2), pounds 4a~4l was measured by the shake flask method with
3.07 (1H, dd, J ¼4.0 Hz, 16.0 Hz, CH2-Ha), 3.64 (1H, dd, J ¼12.0 Hz, slight modification, and PBS (pH ¼7.4) was used as the water
16.0 Hz, CH2-Hb), 4.20 (2H, t, J ¼12.0 Hz, OCH2CH2), 5.79–5.83 phase24. The mobile phase was methanol: 0.1% triethylamine

4
X. H. GAO ET AL.
(TEA)/80:20 (v/v), at a flow rate of 1.0 mL.minꢂ1 through a C18 col- scoring in MOE software was done by ASE scoring function.
umn (250 nm ꢃ4.6 mm, 5 mm) at 32 ꢀC with detect wavelength
318 nm. Experiments were conducted in triplicate and log p values
were calculated.
Results and discussion
Chemistry
AChE and BuChE inhibition assay
Three different chlorobenzaldehydes (compounds 2a–2c) reacted
with 4-hydroxyl chalcone in ethanol with sodium hydroxide as
catalyst to generate three 4'-hydroxy–chlorochalcones (compounds
3a–3c). Then compounds 3a–3c reacted with four different alkyl
amines to gain a series of amine alkyl – substituted chlorochal-
cone derivatives (compounds 4a–4l) in the presence of potassium
AChE/BuChE activity assays were conducted by Ellman method
with light modification25. The brain and serum of Sprague–Dawley
rat was used as the resource of AChE and BuChE, respectively.
Each compound was dissolved in Tween 80 and diluted with
water to various concentrations immediately before use. The reac-
tion mixture containing 40 mL AChE/BuChE, 100 mL acetylthiocho- carbonate, acetone and sodium iodide. The total synthetic route is
line iodide/S-Butyrylthiocholine iodide, 2.76 mL Na2HPO4/NaH2PO4
buffer (pH 8.0, 0.1 mol/L), and 100 mL different concentrations of
test compounds were incubated at 30 ꢀC for 25 min. Then the
reaction was terminated via adding 100 mL 20% sodium dodecyl
sulfate (SDS), then 100 mL 10 mmol/L 5, 5'-Dithiobis-(2-nitrobenzoic
acid) (DTNB) was added to generate the yellow anion 5-thio-2-
nitro-benzoic acid. The absorbance of each assay mixture was
measured at 412 nm by UV spectroscopy. The IC50 values were cal-
culated by Bliss method and expressed as mean SD of the repli-
cates. Rivastigmine was used as the control drug.
shown in Scheme 1.
For the synthesis of 4’-hydroxy–chlorochalcones, the concentra-
tion of sodium hydroxide dramatically influences the yield and
purity of the products. As a result, 30% sodium hydroxide was
chosen as the catalyst. For the synthesis of compounds 4a–4l, the
solvent for the reaction is important. In this reaction, acetone was
selected as the solvent for its easy operation and low toxicity. In
addition, sodium iodide was used as the catalyst to enhance the
reaction activity and accelerate the reaction process. For the syn-
thesis of different tertiary amine substituted chlorochalcones, the
reaction time ranged from 5 to 8 h. Pyrazoline compounds were
synthesized from chlorochalcones, hydrazine and acetic acid
(Scheme 2). High-yield desired compounds were gained via this
reaction.
Kinetic studies
Kinetic experiment was conducted by a reported method with
some modifications26. Compound 4l was added into the assay
solution and pre-incubated with the enzyme at 30 ꢀC, followed by
the addition of 100-mL acetylthiocholine iodide including five con-
centrations. The assay solution contained 100 mL compound 4l,
100 mL DTNB, 2.76 mL 0.1 mol/L Na2HPO4/NaH2PO4 buffer (pH 8.0).
Kinetic profile of AChE was determined by UV spectrophotometer
at 412 nm. Additionally, the blank control experiment was con-
ducted with the vehicle to replace compound 4l solution.
New synthetic compounds were characterized by proton
nuclear magnetic resonance spectroscopy (1H NMR), IR and mass
spectrometry (MS) and elemental analysis. The purities of all syn-
thesized compounds were confirmed to be higher than 98% by
HPLC.
Bioactivity evaluation
Tertiary amine group, which is thought as a versatile pharmaco-
phore appeared in the structures of many drugs in clinic practice,
such as local anesthetics lidocaine, estrogen receptor modulator,
tamoxifen, antipsychotic drug chlorpromazine and antimalarial
chloroquine. Interestingly, in this investigation, some of synthe-
sized compounds are known compounds. Compounds 3a and 3c
were reported to be as anticancer agent and monoamine oxidase
Inhibitor, respectively17,18. Compounds 4d and 4f were patent pro-
tected compounds three decades ago (US Patent 4342782, US
Patent 2668813), but there are no reports on the bioactivity of
Molecular docking
Molecular docking was performed by molecular operating environ-
ment (MOE) software package (Chemical Computing Group Inc.,
ꢀ
Montreal, Canada). The X-ray crystallographic structures of AChE
(PDB code: 1EVE) and BuChE (PDB code: 1P0I) were gained from
protein data bank. 3D structure of compound 4l as the strongest
AChE inhibitor in the present investigation was established by vir-
tue of the builder interface of MOE program, and docked into the
active site of the protein after energy being minimized. The dock
Scheme 1. The synthesis of compounds 4a–4l (I) 30% NaOH, EtOH, 25 ꢀC, stirring 36 h; (II) K2CO3, acetone, NaI, 56 ꢀC, reflux.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
Scheme 2. The synthesis of compounds 5a–5d.
showed 11–62 folds inhibitory activity as that of Rivastigmine
(IC50 ¼10.54 mmol/L). In addition, compound 4l possessed high
selectivity for AChE (Ratio: 667.2).
Table 1. AChE and BuChE inhibitory activity and log p of chlorochalcone
derivatives.
ꢄ
IC50 (lmol/L)
Based on the data in Table 1, it seemed that the variation of
amino-alkyl side chain markedly influence the inhibitory potency
of compounds against AChE. Generally, the order of the inhibitory
potency of these derivatives was as followed: pyrrolidine group-
> piperidine group > dimethylamine group > diethylamine group.
Compounds 4j, 4k and 4l, which were substituted by pyrrolidine
group, exhibited potent inhibitory activity with IC50 values less
than 1.0 mmol/L. On other hand, the substituted position of chlor-
ine atom in chalcone scaffold was very important for the inhibitory
and selectivity for AChE. For dimethylamine or diethylamine sub-
stituted chlorochalcone derivatives the order of inhibitory potency
against AChE was: Para > Meta > Ortho, while for piperidine or pyr-
rolidine substituted chlorochalcone derivatives the order was:
Para > Orto > Meta. In addition, all Para-substituted chlorochalcone
derivatives had the highest selectivity in inhibiting AChE over
BuChE. Among them, compound 4l had the highest selectivity as
667 fold. Compared with the tertiary amine derivatives of fluoro-
chalcone in our previous report, compound 4l showed higher
selectivity against AChE than that of fluorochalcone derivatives27.
Compound 4l was selected for enzyme kinetic studies. The lin-
ear Lineweaver–Burk equation was applied to evaluate the inhib-
ition profile. The graphical analysis of the steady-state inhibition
data of compound 4l was shown in Supplement data: Figure 1.
According to the analysis, compound 4l presented a mixed-type
inhibition for that Km increased and Vmax decreased with the
increasing of the concentration of compound 4l. The competitive
inhibition constant (Ki) and the noncompetitive constant (Ki’) are
0.38 and 2.95 mmol/L, respectively (Supplement data: Table 2).
To explore the possible interacting mode between the chloro-
chalcone derivatives and AChE, molecular docking was performed
for compound 4l with software MOE2008. As shown in
Supplement data: Figure 2, this compound exhibited a multiple
points-binding mode with AChE (Supplement data: Figure 2A). In
the top of the gorge, the aromatic moiety adopted an appropriate
orientation for its binding to peripheral anionic site (PAS), via the
p–p stacking interaction with Trp279. The side chain interacted
with Tyr334 in the mid-gorge zone. In the bottom of the gorge,
the nitrogen of pyrrolidine ring binds to Trp84 via a cation–p in
catalytic active site (CAS). Compared to the interaction between
compound 4l and AChE mentioned above, compound 4l can only
bind with BuChE via Tyr257 and Gly311 that were not the import-
ant amino acids in the catalytic process of BuChE (Supplement
data: Figure 2B). These results may partially explain its potent
inhibition and high selectivity for AChE. As a potential compound
for treatment of AD, log p was thought as an important physical
chemistry parameter to valuate or predict the ability to cross
Compound
R1
R2
AChE
BuChE
Selectivity† Log p
3a
3b
3c
4a
2-Cl
3-Cl
4-Cl
2-Cl
–
–
–
>500
>500
>500
>500
>500
>500
–
–
–
–
–
–
2.11 0.38 41.12 2.31
3.76 0.26 32.14 7.16
1.55 0.16 43.62 6.94
5.42 0.20 63.83 9.21
5.83 0.42 80.21 7.18
3.78 0.39 101.03 17.23
1.80 0.48 35.62 7.31
19.5
1.75
4b
4c
4d
4e
4f
3-Cl
4-Cl
2-Cl
3-Cl
4-Cl
2-Cl
8.5
28.1
11.8
13.8
26.7
19.8
1.83
1.61
1.63
1.62
1.69
1.75
4g
4h
4i
3-Cl
4-Cl
2-Cl
3-Cl
4-Cl
4-Cl
2-Cl
3-Cl
4-Cl
–
1.31 0.17 24.51 6.14
18.7
1.72
1.79
1.85
1.81
1.83
–
0.91 0.09 114.21 26.15 125.5
4j
0.81 0.03 32.08 5.71
0.28 0.05 31.06 3.41
39.6
4k
4l
110.9
0.17 0.06 113.43 18.22 667.2
5a
5b
5c
5d
5.94 0.27 97.64 3.78
6.31 0.16 29.91 1.25
2.41 0.34 27.62 2.58
2.04 0.26 108.03 5.56
16.4
4.74
–
11.5
–
52.96
–
Rivastigmine‡
–
10.54 0.86
0.26 0.08
0.025
–
ꢄ
IC50: 50% inhibitory concentration (means SD of three experiments).
†Selectivity for AChE is defined as IC50 (BuChE)/IC50 (AChE).
‡Used for positive control.
them up to now20,21. Compounds 4i, 4j and 4l were reported as
anticancer agents in pharmacology experiments22.
Although some known compounds contained in the present
investigation, the bioactivity of them in inhibiting AChE was not
be reported. As shown in Table 1, the data indicated that all
amino alkyl substituted chlorochalcones exhibited better inhibitory
activities against AChE than the precursor compounds 3a–3c
(IC50 >500 mmol/L). Among them, compounds 4i, 4j, 4k and 4l
with IC50 values of 0.91, 0.81, 0.28 and 0.17 mmol/L respectively, blood brain barrier (BBB). It was reported that the log p with

6
X. H. GAO ET AL.
optimum central nervous system (CNS) penetration was around
0.728. As shown in Table 1, log p values of new synthesized
5. Molinuevo JL, Gauthier S. Benefits of combined cholinester-
ase inhibitor and memantine treatment in moderate-severe
Alzheimer’s disease. Alzheimers Dement 2013;9:326–31.
6. Mukherjee PK, Kumar V, Mal M, et al. Acetylcholinesterase
inhibitors from plants. Phytomedicine 2007;14:289–300.
7. Li RS, Wang XB, Hu XJ, et al. Design, synthesis and evalu-
ation of flavonoid derivatives as potential multifunctional
acetylcholinesterase inhibitors against Alzheimer’s disease.
Bioorg Med Chem Lett 2013;23:2636–41.
2
compounds ranged from 1.61 to 1.83, which indicated that all the
compounds had sufficiently lipophilicify to pass the BBB in vivo.
In further study to explore the influence of chalcone scaffold
on bioactivity, pyrazoline derivatives were synthesized from chlor-
ochalcones. The bioactivity evaluation showed that the inhibition
potency of pyrazoline derivatives against AChE dramatically
decreased compared to that of chlorochalcone derivatives
(Table 1). It indicated that a,b-unsaturated ketone group in chal-
cone possibly pay an important role for the inhibitory activity
against AChE. In addition, the selectivity of pyrazoline derivatives
to inhibiting AChE over BuChE also decreased significantly.
8. Asadipour A, Alipour M, Jafari M, et al. Novel coumarin-3-car-
boxamides bearing N-benzylpiperidine moiety as potent
acetylcholinesterase inhibitors. Eur
J
Med Chem
2013;70:623–30.
9. Silva T, Reis J, Teixeira J, et al. Alzheimer's disease,
enzyme targets and drug discovery struggles: from nat-
ural products to drug prototypes. Ageing Res Rev
2014;15:116–45.
Conclusion
In the present investigation, a series of chlorochalcones and pyra-
zoline derivatives were synthesized and evaluated in inhibiting
AChE and BuChE. The result showed that the substituted position
of chlorine significantly influenced the activity and selectivity of
compounds in inhibiting AChE. For those compounds with the
same amine alkyl side chain, Para-substituted chlorochalcone had
the highest activity and selectivity. The pyrazoline derivatives syn-
10. Liu HR, Huang XQ, Lou DH, et al. Synthesis and acetylcholin-
esterase inhibitory activity of Mannich base derivatives flavo-
kawain B. Bioorg Med Chem Lett 2014;24:4749–53.
11. Liu HR, Liu XJ, Fan HQ, et al. Design, synthesis and
pharmacological evaluation of chalcone derivatives as
acetylcholinesterase
inhibitors.
Bioorg
Med
Chem
2014;22:6124–33.
thesized from chlorochalcones with the cyclization of 12. Liu HR, Fan HQ, Gao XH, et al. Design, synthesis and prelim-
inary structure–activity relationship investigation of nitro-
gen-containing chalcone derivatives as acetylcholinesterase
and butyrylcholinesterase inhibitors: a further study based
on Flavokawain B Mannich base derivatives. J Enzyme Inhib
Med Chem 2016;3:580–9.
a,b-unsaturated ketone group had weaker activity and lower
selectivity in inhibiting AChE compared to that of chlorochalcone
derivatives, suggesting that a,b-unsaturated ketone group was
important for inhibiting AChE. Among them, compound 4l
revealed the strongest AChE inhibitory activity (IC50: 0.17 mmol/L)
and highest selectivity (Ratio: 667.2). Enzyme kinetic study sug-
gested that compound 4l was a mixed-type inhibitor. Molecular
docking supported the mixed-type inhibition mechanism.
Compound 4l might serve as a potential agent for the treatment
of AD.
13. Oztas¸kın N, Cetinkaya Y, Taslimi P, et al. Antioxidant and
acetylcholinesterase inhibition properties of novel bromo-
phenol derivatives. Bioorg Chem 2015;60:49–57.
_
14. Aksu K, Topal F, Gulcin I, et al. Acetylcholinesterase inhibi-
tory and antioxidant activities of novel symmetric sulfamides
derived
from
phenethylamines.
Arch
Pharm
2015;348:446–55.
15. Burmaoglu S, Algul O, Anil DA, et al. Synthesis and anti-pro-
liferative activity of fluoro-substituted chalcones. Bioorg Med
Chem Lett 2016;26:3172–6.
16. Cole AL, Hossain S, Cole AM, et al. Synthesis and bioevalua-
tion of substituted chalcones, coumaranones and other fla-
vonoids as anti-HIV agents. Bioorg Med Chem
2016;24:2768–76.
Disclosure statement
The authors confirm that this article content has no conflict of
interest.
Funding
This study was financial supported by "The innovative investiga- 17. Syam S, Abdelwahab SI, Al-Mamary MA, et al. Synthesis of
chalcones
with
anticancer
activities.
Molecules
tion funds for post-graduated students of Hu’nan province" (No.
2012;17:6179–95.
CX2015424).
18. Chimenti F, Fioravanti R, Bolasco A, et al. Chalcones: a valid
scaffold for monoamine oxidases inhibitors. J Med Chem
2009;52:2818–24.
References
19. Lee YH, Shin MC, Yun YD, et al. Synthesis of aminoalkyl-sub-
stituted aurone derivatives as acetylcholinesterase inhibitors.
Bioorg Med Chem 2015;23:231–40.
20. Edward J, Cragoe J, Rahway NJ. 1-(Substituted-aminoalkoxy-
phenyl)-2-methylene-1-alkanones, compositions and their
use. US 4342782[P] Aug 3, 1982.
21. Goldbeg WM, Montclair U, Rachlin AI, et al. Basic ketones
and salts thereof. US 2668813[P] Feb 9, 1954.
22. Mokale SN, Dube PN, Bhavale SA, et al. Synthesis, in-vitro
screening, and docking analysis of novel pyrrolidine and
piperidine-substituted ethoxy chalcone as anticancer agents.
Med Chem Res 2014;24:1842–56.
1. Pivtoraiko VN, Abrahamson EE, Leurgans SE, et al. Cortical
pyroglutamate amyloid-b levels and cognitive decline in
Alzheimer's disease. Neurobiol Aging 2015;36:12–19.
2. Anand R, Gill KD, Mahdi AA. Therapeutics of Alzheimer’s dis-
ease: past, present and future. Neuropharmacology
2014;76:27–50.
ꢀ
3. Garcıa-Alberca JM. Cognitive intervention therapy as treat-
ment for behavior disorders in Alzheimer disease: evidence
ꢀ
on efficacy and neurobiological correlations. Neurologıa
(English Edition) 2015;30:8–15.
4. Small G, Bullock R. Defining optimal treatment with cholin-
esterase inhibitors in Alzheimer's disease. Alzheimers
Dement 2011;7:177–84.
23. Insuasty B, Montoya A, Becerra D, et al. Synthesis of novel
analogs of 2-pyrazoline obtained from [(7-chloroquinolin-4-yl)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
amino]chalcones and hydrazine as potential antitumor and
AChE and BuChE inhibitors. Eur
J
Med Chem
antimalarial agents. Eur J Med Chem 2013;67:252–62.
2013;62:311–19.
24. Yu H, Li WM, Kan KKW, et al. The physicochemical properties 27. Liu HR, Zhou C, Fan HQ, et al. Novel potent and selective
and the in vivo AChE inhibition of two potential anti-
Alzheimer agents, bis(12)-hupyridone and bis(7)-tacrine. J
Pharmaceut Biomed Anal 2008;46:75–81.
acetylcholinesterase inhibitors as potential drugs for the
treatment of Alzheimer's disease: synthesis, pharmacological
evaluation, and molecular modeling of amino-alkyl-substi-
tuted fluoro-chalcones derivatives. Chem Biol Drug Des
2015;86:517–22.
25. Ellman GL, Courtney KD, Andres VJ, et al. A new and rapid
colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol 1961;7:88–95.
28. Glave WR, Hansch CJ. Relationship between lipophilic char-
26. Skrzypek A, Matysiak J, Niewiadomy A, et al. Synthesis and
biological evaluation of 1,3,4-thiadiazole analogues as novel
acter
1972;61:589–91.
and
anesthetic
activity.
J
Pharm
SCI-US