Structure–activity study of fluorine or chlorine-substituted cinnamic acid derivatives with tertiary amine side chain in acetylcholinesterase and butyrylcholinesterase inhibition

Article abstract of DOI:10.1002/ddr.21515

In this study, a series of new fluorine or chlorine-substituted cinnamic acid derivatives that contain tertiary amine side chain were designed, synthesized, and evaluated in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition. The resu

Full text of DOI:10.1002/ddr.21515

Received: 20 August 2018
DOI: 10.1002/ddr.21515
Revised: 28 December 2018
Accepted: 5 January 2019
R E S E A R C H A R T I C L E
Structure–activity study of fluorine or chlorine-substituted
cinnamic acid derivatives with tertiary amine side chain in
acetylcholinesterase and butyrylcholinesterase inhibition
Xiao-Hui Gao1 | Jing-Jing Tang2 | Hao-Ran Liu2 | Lin-Bo Liu2 | Ying-Zi Liu3
Enabling Technologies
Strategy, Management & Health Policy
Hit, Lead &
Candidate Discovery
Preclinical Research
& Development
Clinical
Research
Post-Market
Research
1Hu'nan Key Laboratory Cultivation Base of
Abstract
the Research and Development of Novel
Pharmaceutical Preparations, College of
Pharmacy, Changsha Medical University,
Changsha, China
In this study, a series of new fluorine or chlorine-substituted cinnamic acid derivatives that contain
tertiary amine side chain were designed, synthesized, and evaluated in acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) inhibition. The results show that almost all the derivatives contain-
ing tertiary amine side chain (compounds 4a–9d) exhibit moderate or potent activity in AChE inhibi-
tion. By contrast, their parent compounds (compounds 3a–3f) in the absence of tertiary amine
moitery exhibit poor inhibitory activity against AChE. For the compounds containing pyrroline or
piperidine side chain, the bioactivity in AChE inhibition is much intense than those containing
N,N-diethylamino side chain. The chlorine or fluorine substituted position produces a significant
effect on the bioactivity and selectivity in AChE inhibition. Most of the compounds that contain
para-substituted fluorine or chlorine exhibit potent activity against AChE and poor activity against
BChE, while ortho-substituted analogs show the opposite effect. It is worth noticing that the com-
pounds containing N,N-diethylamino side chain are exceptions to this pattern. Among the newly syn-
2College of Chemistry and Chemical
Engineering, Hu'nan University, Changsha,
China
3College of Medicine, Hu'nan Normal
University, Changsha, China
Correspondence
Hao-Ran Liu, College of Chemistry and
Chemical Engineering, Hu’nan University,
Changsha 410082, China.
Email: pharmaliu@126.com
and
Ying-Zi Liu, College of Medicine, Hu’nan
Normal University, Changsha 410013, China.
Email: liuyingzi111@sina.com
thesized compounds, compounds 6d are the most potent in AChE inhibition (IC50
=
1.11 0.08 μmol/L) with high selectivity for AChE over BChE (selectivity ratio: 46.58). An enzyme
kinetic study of compounds 6d suggests it produces a mixed-type inhibitory effect in AChE.
Funding information
Natural science foundation of Hu’nan province,
Grant/Award Numbers: No.2017JJ2050,
No.2018JJ3572; the grant of Key Laboratory
Breeding Base of Hu’nan Oriented
Fundamental and Applied Research of
Innovative Pharmaceutics, Grant/Award
Number: No.2016TP1029
KEYWORDS
cholinesterase inhibitors, cinnamic acid, halogen
treatment of Alzheimer's disease (AD) using cinnamic acid derivatives
(Estrada et al., 2016; Zhang et al., 2018). Acetylcholinesterase (AChE)
1
| INTRODUCTION
Cinnamic acid is a commonly occurring natural compound in Cinnamo-
mum cassia and other plants (Jayaprakasha & Rao, 2011). It has been
reported that cinnamic acid can be used as condiment and antioxidant
(Letizia, Cocchiara, Lapczynski, Lalko, & Api, 2005; Li et al., 2014).
Some recently synthesized cinnamic acid derivatives exhibit anti-
tyrosinase, hepatoprotective and α-glusidase inhibition effects, etc.
(Hu et al., 2016; Yan, Wang, Yen, Lee, & Yin, 2016; Song et al., 2016).
It has also drawn great attention of medicine researchers to the
inhibitors, as the major AD therapeutic drugs, exhibit adverse effects
such as vomiting, cardiotoxicity in clinical use yet (Hansen et al., 2008;
Birks, Grimley Evans, Iakovidou, Tsolaki, & Holt, 2009; Takaya et al.,
2009). Therefore, developing new acetylcholinesterase inhibitors has
become an area of intense interest in recent years. In our laboratory,
after screening more than 100 compounds, flavokawain B Mannich
base was found to have an inhibitory effect on AChE (Liu et al.,
2014a). As flavokawain B is a naturally occurring chalcone isolated
Drug Dev Res. 2019;1–8.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
GAO ET AL.
from the root of Piper methysticum (Jeong et al., 2015), a series of
chalcone with tertiary amine side chains were synthesized, which
were confirmed possessing inhibitory effect on AChE in subsequent
experiments. Some of them have better selectivity in inhibiting AChE
over butyrylcholinesterase (BChE) in our reports (Liu et al., 2014b; Liu
et al., 2016). Most recently, we have discovered cinnamic acid deriva-
tives similar to chalcone scaffold can inhibit AChE. The investigations
on the structure–activity relationship reveal that cinnamic acid deriva-
tives with para-substituted tertiary amine possess more potent bioac-
tivity in AChE inhibition than meta- or ortho-substituted ones (Gao
et al., 2018b).
2.2 | General method for synthesis of
halogen-substituted cinnamamides (3a–3f)
Compounds 3a–3f were synthesized according to previous reported
method (Gao et al., 2018a). Halogen-substituted cinnamic acid (2-,3-,
or 4-fluoro or chlorine cinnamic acid, 10 mmol), dichloromethane
(30 mL) and a small amount of dimethylformamide (DMF) were added
into the flask, followed by the addition of oxalyl chloride (2.55 mL,
30 mmol) drop by drop. The mixture was refluxed, and the reaction
process was monitored by thin layer chromatography (TLC) until the
reaction is completed. Then the solution was cooled to room tempera-
ture, dichloromethane (30 mL × 3) was added into the solution. After
the solvent and excess oxalyl chloride was removed by the evapora-
tion under reduced pressure, the halogen-substituted cinnamic acyl
chlorides were gained. These compounds were used as the intermedi-
ates to synthesize compounds 3a–3f without further purification.
Then para-aminophenol (1.1 g, 10 mmol) and acetonitrile (10 mL)
was added into the flask to dissolve by heating, followed by the addi-
tion of triethylamine (2.0 mL, 15 mmol) and halogen-substituted cin-
namic acyl-chloride solution. The mixture solution was refluxed and
the reaction process was monitored by TLC until the reaction was
completed. Then the reaction solution is cooled to room temperature,
followed by the addition of 10% NaOH solution. The solution was fil-
tered, adjusting the pH of filtrate to 4–5 by hydrochloric acid. Then a
large number of solid precipitates were produced. The precipitates
were collected by filtration, and were washed by water to neutral.
Finally solid products were gained with the yield more than 90%.
(E)-3-(2-fluoro phenyl)-N-(4-hydroxy phenyl) acrylamide (3a): white solid,
mp: 210.6–210.9 ꢀC (Lit.211–213 ꢀC). It is a known compound as com-
mercial block but no biological activity has been reported (Ramalakshmi,
Deepa, Sumanth Srinivas, Puratchikody, & Arunkumar, 2009).
Based on the above investigations, in consideration of important
effects of halogens in drug discovery and development (Lu et al.,
2012; Vulpetti & Dalvit, 2012), in this study a new series of fluorine or
chlorine-substituted cinnamic acid derivatives with para-substituted
tertiary amine side chain are designed, synthesized, and evaluated for
AChE and BChE inhibition (Figure 1). Compound 6d, which is con-
firmed having the most potent activity in AChE inhibition among new
synthesized compounds in subsequent bioactivity evaluation, is
selected for conducting an enzymatic kinetics study.
2
| MATERIALS AND METHODS
2.1 | Chemistry
All chemicals or reagents are of analytical reagent-grade and used
without further purification. The melting points of compounds were
measured on a WRS-lA melting point detector (temperature uncor-
rected). 1H NMR spectra were recorded on a Bruker 400 MHz instru-
ment in CDCl3 with tetramethylsilane (TMS) as the internal reference.
Mass spectra were obtained on Waters XEVO G2-XS QTOF
by electrospray ionization. Infrared spectrum was obtained by
Shimadzu Infinity-1 infrared spectrometer. The purity of compounds
was checked by Shimadzu LC-20A high performance liquid
chromatography.
(E)-3-(3-fluoro phenyl)-N-(4-hydroxy phenyl) acrylamide (3b): white
solid, mp: 227.7–228.5 ꢀC (Lit.228–230 ꢀC). It is a known compound
as commercial block but no biological activity has been reported
(Ramalakshmi et al., 2009).
FIGURE 1 Design pathway of halogen-substituted cinnamic acid derivatives. (a) Flavokawain B Mannich base derivatives; (b) chalcone
derivatives with tertiary amine moiety; (c) cinnamic acid derivatives with tertiary amine moiety; and (d) halogen-substituted cinnamic acid
derivatives with tertiary amine moiety

GAO ET AL.
3
(E)-3-(4-fluoro phenyl)-N-(4-hydroxy phenyl) acrylamide (3c): white
White powder, yield: 48.5%, mp: 108.5–108.9 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.38–1.39 (2H, m, piperidine-H),
1.47–1.51 (4H, m, piperidine-H), 2.44–2.51 (6H, m, 3× NCH2), 4.04
solid, mp: 215.5–216.3 ꢀC (Lit.217–218 ꢀC). It is a known compound
as commercial block but no biological activity has been reported
(Ramalakshmi et al., 2009).
(2H, t, J = 6.0 Hz, OCH2CH2), 6.90–6.93 (3H, m, Ar
H and
(E)-3-(2-chloro phenyl)-N-(4-hydroxy phenyl) acrylamide (3d): white
solid, mp: 225.3–226.2 ꢀC (Lit.226–228 ꢀC). It is a known compound
in a report possessing antibacterial activity (Ramalakshmi et al., 2009).
(E)-3-(3-chloro phenyl)-N-(4-hydroxy phenyl) acrylamide (3e): white
solid, mp: 237.5–238.3 ꢀC (Lit.239–240 ꢀC). It is a known compound
as commercial block but no biological activity has been reported
(Ramalakshmi et al., 2009).
Ar CH CH), 7.28–7.34 (2H, m, Ar H), 7.44–7.49 (1H, m, Ar H),
7.59–7.62 (3H, m, Ar H and Ar CH CH), 7.68–7.72 (1H, m, Ar H),
10.18 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for C22H25
FN2O2 + ([M]+): 368.2; found M + 1:369.2. IR (KBr) cm−1: 3317,
3,314, 1,666, 1,627, 1,558, 1,355, 1,232, 985, 750.
(E)-3-(2-fluoro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (4d)
Light green powder, yield:57.8%, mp: 180.9–182.3 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.84 (4H, br, pyrrolidine-H), 2.99 (4H,
br, pyrrolidine-H), 3.23 (2H, br, OCH2CH2), 4.17 (2H, t, J = 6.0 Hz,
OCH2CH2), 6.91–6.99 (3H, m, Ar H and Ar CH CH), 7.28–7.34
(E)-3-(4-chloro phenyl)-N-(4-hydroxy phenyl) acrylamide (3f): white
solid, mp: 226.6–227.2 ꢀC (Lit.228–230 ꢀC). It is a known compound
with antimicrobial activity (Ramalakshmi et al., 2009).
(2H, m, Ar H), 7.44–7.50 (1H, m, Ar H), 7.60–7.66 (3H, m, Ar
H
2.3 | General method for synthesis of compounds
4a–9d
and Ar CH CH), 7.69–7.72 (1H, m, Ar H), 10.21 (1H, s, NH). MS
(ESI-Q-TOFMS) m/z calculated for C21H23FN2O2 + ([M]+): 354.2;
found M + 1:355.2. IR (KBr) cm−1: 3259, 3,028, 1,668, 1,600, 1,558,
1,344, 1,294, 989, 754.
Compounds 4a–9d were synthesized according to previous reports
(Liu et al., 2017; Sang et al., 2017). One of cinnamic amides (3a–3f)
(1 mmol), anhydrous K2CO3 (0.68 g, 5 mmol) were added into a flask
with 25 mL acetone as the solvent, stirred and refluxed for 45 min.
Subsequently, one of aminoethyl chlorides hydrochloride (3 mmol)
and NaI (0.009 g, 0.05 mmol) were added to the flask and continued
to refluxed, monitoring the reaction process by TLC. After the reac-
tion was completed, the reaction mixture cooled to room temperature,
filtered, removed the solvent by rotary evaporation. Then residue was
dissolved by dichloromethane (30 mL), washed by saturated NaCl (aq,
30 mL × 2), followed by drying using anhydrous sodium sulfate. The
solution was filtered and the solvent was removed to afford the crude
product. The crude product was purified by column chromatography
with the eluent (V(MeOH):V(DCM) = 1:60~70).
(E)-3-(3-fluoro phenyl)-N-(4-(2-[dimethylamino] ethoxy) phenyl)
acrylamide (5a)
White powder, yield: 48.6%, mp: 119.1–119.8 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.22 (6H, s, 2× NCH3), 2.61 (2H, t,
J = 6.0 Hz, OCH2CH2), 4.02 (2H, t, J = 4.0 Hz, OCH2CH2), 6.84 (H, d,
J = 16.0 Hz, Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.22–7.27 (1H, m,
Ar H), 7.44–7.50 (3H, m, Ar H and Ar CH CH), 7.52–7.51 (H, m,
Ar H), 7.58–7.62 (2H, m, Ar H), 10.27 (1H, s, NH). MS (ESI-Q-TOFMS)
m/z calculated for C19H21FN2O2 + ([M]+): 328.2; found M + 1:329.2.
IR (KBr) cm−1: 3288, 2,981, 1,664, 1,608, 1,581, 1,328, 1,276, 993, 775.
(E)-3-(3-fluoro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
amide (5b)
Light yellow powder, yield: 56.8%, mp: 139.2–140.5 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.00 (6H, t, J = 8.0 Hz, 2× NCH2CH3),
2.51 (2H, t, J = 4.0 Hz, OCH2CH2), 2.61–2.83 (4H, m, 2× NCH2CH3),
4.02 (2H, br, OCH2CH2), 6.84 (H, d, J = 16.0 Hz, Ar CH CH),
6.91–6.93 (2H, m, Ar H), 7.22–7.26 (1H, m, Ar H), 7.44–7.50 (3H,
m, Ar H and Ar CH CH), 7.52–7.54 (1H, m, Ar H), 7.58–7.62 (2H,
m, Ar H), 10.12 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for
(E)-3-(2-fluoro phenyl)-N-(4-(2-[dimethylamino] ethoxy) phenyl)
acrylamide (4a)
Grayish white powder, yield:49.3%, mp: 113.1–114.0 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 2.34 (6H, s, 2× NCH3), 2.76 (2H,
t, J = 6.0 Hz, OCH2CH2), 4.12 (2H, t, J = 4.0 Hz, OCH2CH2),
6.98–7.02 (3H, m, Ar H and Ar CH CH), 7.36–7.42 (2H, m, Ar H),
7.52–7.57 (1H, m, Ar H), 7.67–7.71 (3H, m, Ar H and Ar CH CH),
7.76–7.80 (1H, m, Ar H), 10.27 (1H, s, NH). MS (ESI-Q-TOFMS) m/z
calculated for C19H21FN2O2 + ([M]+): 328.2; found M + 1:329.2. IR
(KBr) cm−1: 3367, 3,007, 1,654, 1,600, 1,577, 1,350, 1,278, 972, 742.
(E)-3-(2-fluoro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
amide (4b)
Light yellow powder, yield:55.6%, mp: 112.6–113.4 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 0.98–1.04 (6H, m, 2× NCH2CH3), 2.50 (2H,
br, OCH2CH2), 2.71–2.92 (4H, m, 2× NCH2CH3), 4.06 (2H, br, OCH2CH2),
6.90–6.94 (3H, m, Ar H and Ar CH CH), 7.28–7.34 (2H, m, Ar H),
7.44–7.49 (1H, m, Ar H), 7.59–7.64 (3H, m, Ar H and Ar CH CH),
7.68–7.72 (1H, m, Ar H), 10.19 (1H, s, NH). MS (ESI-Q-TOFMS) m/z cal-
culated for C21H25FN2O2 + ([M]+): 356.2; found M + 1:357.2. IR (KBr)
cm−1: 3311, 2,970, 1,660, 1,625, 1,600, 1,336, 1,253, 968, 752.
(E)-3-(2-fluoro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (4c)
C21H25FN2O2 + ([M]+): 356.2; found M + 1:357.2. IR (KBr) cm−1
:
3288, 2,981, 1,662, 1,602, 1,583, 1,357, 1,294, 979, 783.
(E)-3-(3-fluoro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (5c)
Grayish white powder, yield: 47.8%, mp: 131.3–131.6 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1.75 (4H, br, piperidine-H),
2.49–2.51 (2H, m, piperidine-H), 2.72–2.97 (6H, m, 3× NCH2), 4.10
(2H, t, J = 6.0 Hz, OCH2CH2), 6.84 (1H, d, J = 16.0 Hz, Ar CH CH),
6.93–6.95 (2H, m, Ar H), 7.22–7.27 (1H, m, Ar H), 7.44–7.50 (3H,
m, Ar H and Ar CH CH), 7.52–7.58 (1H, m, Ar H), 7.61–7.63 (2H,
m, Ar H), 10.13 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for
C22H25FN2O2 + ([M]+): 368.2; found M + 1:369.2. IR (KBr) cm−1
:
3079, 2,933, 1,664, 1,602, 1,583, 1,357, 1,292, 987, 781.
(E)-3-(3-fluoro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (5d)

4
GAO ET AL.
Grayish white powder, yield: 59.1%, mp: 123.0–123.7 ꢀC; 1H
OCH2CH2), 4.05 (2H, t, J = 4.0 Hz, OCH2CH2), 6.87 (1H, d,
J = 16.0 Hz, Ar CH CH), 6.92–6.94 (2H, m, Ar H), 7.42–7.46 (2H,
m, Ar H), 7.55–7.57 (1H, m, Ar H), 7.60–7.63 (2H, m, Ar H),
7.75–7.77 (1H, m, Ar H), 7.85 (1H, d, J = 16.0 Hz, Ar CH CH),
10.21 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for C19H21
ClN2O2 + ([M]+): 344.1; found M + 1:355.1. IR (KBr) cm−1: 3296,
2,926, 1,670, 1,631, 1,606, 1,359, 1,298, 981, 823.
NMR (400 MHz, DMSO-d6) δ (ppm): 1.41–1.54 (4H, m, pyrrolidine-H),
2.50–2.51 (4H, m, pyrrolidine-H), 2.68 (2H, br, OCH2CH2), 4.08 (2H,
br, OCH2CH2), 6.84 (1H, d, J = 16.0 Hz, Ar CH CH), 6.93–6.96 (2H,
m, Ar H), 7.22–7.27 (1H, m, Ar H), 7.44–7.53 (3H, m, Ar H and
Ar CH CH), 7.53–7.58 (1H, m, Ar H), 7.60–7.63 (2H, m, Ar H),
10.12 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for
C
21H23FN2O2 + ([M]+): 354.2 found M + 1:355.2. IR (KBr) cm−1
3049, 2,931, 1,668, 1,604, 1,583, 1,357, 1,292, 987, 779.
(E)-3-(4-fluoro phenyl)-N-(4-(2-[dimethylamino] ethoxy) phenyl)
acrylamide (6a)
:
(E)-3-(2-chloro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
amide (7b)
Light yellow powder, yield: 56.6%, mp: 113.7–114.6 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.91 (6H, br, 2× NCH2CH3), 3.04 (4H,
br, 2× NCH2CH3), 3.28 (2H, br, OCH2CH2), 4.25 (2H, t, J = 4.0 Hz,
OCH2CH2), 6.96 (1H, d, J = 16.0 Hz, Ar CH CH), 7.04–7.07 (2H, m,
Ar H), 7.50–7.55 (2H, m, Ar H), 7.62–7.65 (1H, m, Ar H),
7.72–7.74 (2H, m, Ar H), 7.83–7.86 (1H, m, Ar H), 7.93 (1H, d,
J = 16.0 Hz, Ar CH CH) 10.32 (1H, s, NH). MS (ESI) m/z calculated
White powder, yield: 49.6%, mp: 161.5–162.3 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.30 (6H, s, 2× NCH3), 2.70 (2H, t,
J = 4.0 Hz, OCH2CH2), 4.10 (2H, t, J = 4.0 Hz, OCH2CH2), 6.83 (1H, d,
J = 16.0 Hz, Ar CH CH), 6.99–7.01 (2H, m, Ar H), 7.34–7.39 (2H, m,
Ar H), 7.62 (1H, d, J = 16.0 Hz, Ar CH CH), 7.66–7.70 (2H, m, Ar H),
7.74–7.78 (2H, m, Ar H), 10.15 (1H, s, NH). MS (ESI-Q-TOFMS) m/z
calculated for C19H21FN2O2 + ([M]+): 328.2; found M + 1:329.2. IR
(KBr) cm−1: 3296, 2,935, 1,660, 1,624, 1,597, 1,334, 1,294, 985, 833.
(E)-3-(4-fluoro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
amide (6b)
Light yellow powder, yield: 58.2%, mp: 155.2–156.0 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.13–1.27 (6H, m, 2× NCH2CH3), 2.97
(2H, br, OCH2CH2), 2.75–2.97 (4H, m, 2× NCH2CH3), 4.71 (2H, br,
OCH2CH2), 6.76 (1H, d, J = 16.0 Hz, Ar CH CH), 6.95–6.97 (2H,m,
Ar H), 7.27–7.31 (2H, m, Ar H), 7.56 (1H, d, J = 16.0 Hz,
Ar CH CH), 7.63–7.65 (2H, m, Ar H), 7.66–7.70 (2H, m, Ar H),
10.11 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for
for C21H25ClN2O2 + ([M]+): 372.2 found M + 1:373.2. IR (KBr) cm−1
:
3298, 2,970, 1,658, 1,622, 1,601, 1,336, 1,294, 966, 819.
(E)-3-(2-chloro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (7c)
White powder, yield: 63.9%. mp:127.2–128.1 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.38–1.39 (2H, m, piperidine-H),
1.47–1.51 (4H, m, piperidine-H), 2.44–2.51 (6H, m, 3× NCH2), 4.03
(2H, t, J = 6.0 Hz, OCH2CH2), 6.90–6.93 (3H, m, Ar
H and
Ar CH CH), 7.28–7.34 (2H, m, Ar H), 7.44–7.49 (1H, m, Ar H),
7.59–7.62 (3H, m, Ar H and Ar CH CH), 7.68–7.72 (1H, m, Ar H),
10.18 (1H, s, NH). MS (ESI) m/z calculated for C22H25ClN2O2 + ([M]
+): 384.2 found M + 1:385.2. IR (KBr) cm−1: 3294, 2,954, 1,664,
1,627, 1,610, 1,356, 1,305, 981, 825.
C
21H25FN2O2 + ([M]+): 356.2; found M + 1:357.2. IR (KBr) cm−1
3294, 2,935, 1,683, 1,641, 1,600, 1,354, 1,294, 1,168, 829.
(E)-3-(4-fluoro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (6c)
Grayish white powder, yield: 46.4%, mp: 165.3–166.1 ꢀC; 1H NMR
(400 MHz, DMSO-d6) (ppm): 1.39–1.52 (4H, m, piperdine-H),
:
(E)-3-(2-chloro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (7d)
Light green powder, yield: 65.2%, mp: 157.7–158.4 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.84 (4H, br, pyrrolidine-H), 2.92 (4H, br,
pyrrolidine-H), 3.23 (2H, br, OCH2CH2), 4.18 (2H, t, J = 6.0 Hz,
OCH2CH2), 6.91–6.99 (3H, m, Ar H and Ar CH CH), 7.28–7.34 (2H,
m, Ar H), 7.44–7.50 (1H, m, Ar H), 7.60–7.66 (3H, m, Ar H and
Ar CH CH), 7.69–7.72 (1H, m, Ar H), 10.21 (1H, s, NH). MS (ESI) m/z
calculated for C21H23ClN2O2 + ([M]+): 370.1; found M + 1:371.1. IR
(KBr) cm−1: 3292, 2,968, 1,654, 1,622, 1,600, 1,348, 1,240, 970, 827.
(E)-3-(3-chloro phenyl)-N-(4-(2-[dimethylamino]ethoxy) phenyl) acryl-
amide (8a)
White powder, yield: 48.1%. mp: 145.6–146.3 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.22 (6H, s, 2× NCH3), 2.61 (2H, t,
J = 6.0 Hz, OCH2CH2), 4.02 (2H, t, J = 4.0 Hz, OCH2CH2), 6.84 (H, d,
J = 16.0 Hz, Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.22–7.27 (1H, m,
Ar H), 7.44–7.50 (3H, m, Ar H and Ar CH CH), 7.52–7.54 (1H, m,
Ar H), 7.58–7.62 (2H, m, Ar H), 10.27 (1H, s, NH). MS (ESI-Q-TOFMS)
m/z calculated for C19H21ClN2O2 + ([M]+): 344.1; found M + 1:345.1.
IR (KBr) cm−1: 3126, 2,926, 1,670, 1,631, 1,560, 1,359, 1,246, 981, 823.
(E)-3-(3-chloro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
amide (8b)
δ
2.50–2.51 (6H, m, 3× NCH2), 4.06 (2H, br, OCH2CH2), 6.74 (1H, d,
J = 16.0 Hz, Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.26–7.31 (2H, m,
Ar H), 7.55 (1H, d, J = 16.0 Hz, Ar CH CH), 7.57–7.61 (2H, m,
Ar H), 7.66–7.70 (2H, m, Ar H), 10.07 (1H, s, NH). MS (ESI-Q-TOFMS)
m/z calculated for C22H25FN2O2 + ([M]+): 368.2; found M + 1:369.2.
IR (KBr) cm−1: 3321, 2,954, 1,662, 1,625, 1,595, 1,332, 1,293, 983, 825.
(E)-3-(4-fluoro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (6d)
Grayish white powder, yield: 61.2%. mp: 189.7–190.6 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 2.24 (4H, br, pyrrolidine-H),
2.50–2.51 (4H, m, pyrrolidine-H), 2.64 (2H, t, J = 4.0 Hz, OCH2CH2),
4.02 (2H, t, J = 4.0 Hz, OCH2CH2), 6.80 (1H, d, J = 16.0 Hz,
Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.50–7.53 (2H, m, Ar H),
7.57 (1H, d, J = 16.0 Hz, Ar CH CH), 7.59–7.66 (4H, m, Ar H),
10.09 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated for
C
21H23FN2O2 + ([M]+): 354.2; found M + 1:355.2. IR (KBr) cm−1
3284, 2,945, 1,675, 1,635, 1,600, 1,355, 1,244, 970, 825.
(E)-3-(2-chloro phenyl)-N-(4-(2-[dimethylamino]ethoxy) phenyl) acryl-
amide (7a)
:
Light yellow powder, yield: 57.7%, mp: 155.6–156.9 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 1.00 (6H, t, J = 8.0 Hz, 2× NCH2CH3), 2.61–2.83 (6H, m, 3×
NCH2CH3), 4.02 (2H, br, OCH2CH2), 6.84 (H, d, J = 16.0 Hz,
White powder, yield: 47.9%. mp: 106.4–106.9 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.46 (6H, s, 2× NCH3), 2.67 (2H, br,

GAO ET AL.
5
Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.22–7.26 (H, m, Ar H),
7.44–7.50 (3H, m, Ar H and Ar CH CH), 7.52–7.54 (1H, m, Ar H),
7.58–7.62 (2H, m, Ar H), 10.12 (1H, s, NH). MS (ESI) m/z calculated
+): 344.1; found M + 1:345.1. IR (KBr) cm−1: 3271, 2,980, 1,658,
1,622, 1,597, 1,294, 1,231, 981, 821.
(E)-3-(4-chloro phenyl)-N-(4-(2-[diethylamino] ethoxy) phenyl) acryl-
for C21H25ClN2O2 + ([M]+): 372.2 found M + 1:373.2. IR (KBr) cm−1
:
amide (9b)
Light yellow powder, yield: 58.4%, mp: 183.8–185.6 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.32–1.68 (6H, m, J = 4.0 Hz, 2×
NCH2CH3), 2.58–2.59 (6H, m, 2× NCH2CH3 and OCH2CH2), 4.23
(2H, br, OCH2CH2), 6.89 (1H, d, J = 16.0 Hz, Ar CH CH), 7.03–7.04
(2H, m, Ar H), 7.58–7.65 (3H, Ar CH CH and Ar H), 7.69–7.74
(3H, m, Ar H), 10.20 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated
3261, 2,929, 1,678, 1,654, 1,598, 1,296, 1,236, 970, 823.
(E)-3-(3-chloro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (8c)
Grayish white powder, yield: 52.8%, mp: 173.2–174.0 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1.75 (4H, br, piperidine-H),
2.49–2.51 (2H, m, piperidine-H), 2.72–2.97 (6H, m, 3× NCH2), 4.10
(2H, t, J = 6.0 Hz, OCH2CH2), 6.84 (1H, d, J = 16.0 Hz, Ar CH CH),
6.93–6.95 (2H, m, Ar H), 7.22–7.27 (1H, m, Ar H), 7.44–7.49 (3H,
m, Ar H and Ar CH CH), 7.52–7.58 (1H, m, Ar H), 7.61–7.63 (2H,
m, Ar H), 10.13 (1H, s, NH). MS (ESI) m/z calculated for
for C21H25ClN2O2 + ([M]+): 372.2 found M + 1:373.2. IR (KBr) cm−1
:
3271, 2,970, 1,658, 1,624, 1,597, 1,294, 1,232, 977, 823.
(E)-3-(4-chloro phenyl)-N-(4-(2-[piperidin-1-yl] ethoxy) phenyl) acryl-
amide (9c)
Grayish white powder, yield: 57.6%, mp: 179.7–181.3 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1.74 (4H, br, piperdine-H),
2.50–2.51 (2H, m, piperdine-H), 2.69 (4H, br, piperdine-H), 2.94 (2H,
br, OCH2CH2), 4.08 (2H, t, J = 4.0 Hz, OCH2CH2), 6.80 (1H, d,
J = 16.0 Hz, Ar CH CH), 6.92–6.95 (2H, m, Ar H), 7.50–7.57 (3H,
m, Ar CH CH and Ar H), 7.60–7.65 (3H, m, Ar H), 10.10 (1H, s,
NH). MS (ESI-Q-TOFMS) m/z calculated for C22H25ClN2O2 + ([M]+):
384.2 found M + 1:385.2. IR (KBr) cm−1: 3288, 2,933, 1,660, 1,624,
1,597, 1,292, 1,232, 985, 823.
C
22H25ClN2O2 + ([M]+): 384.2 found M + 1:385.2. IR (KBr) cm−1
3284, 2,933, 1,668, 1,622, 1,533, 1,294, 1,236, 970, 823.
(E)-3-(3-chloro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (8d)
:
Grayish white powder, yield: 54.3%. mp: 166.3–167.1 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1.41–1.54 (4H, m, pyrrolidine-H),
2.50–2.51 (4H, m, pyrrolidine-H), 2.68 (2H, br, OCH2CH2), 4.08 (2H,
br, OCH2CH2), 6.84 (1H, d, J = 16.0 Hz, Ar CH CH), 6.93–6.95 (2H,
m, Ar H), 7.22–7.27 (1H, m, Ar H), 7.44–7.51 (3H, m, Ar H and
Ar CH CH), 7.53–7.58 (1H, m, Ar H), 7.60–7.63 (2H, m, Ar H),
10.12 (1H, s, NH). MS (ESI) m/z calculated for C21H23ClN2O2 + ([M]
+): 370.1; found M + 1:371.1. IR (KBr) cm−1: 3280, 2,824, 1,653,
1,620, 1,598, 1,296, 1,236, 970, 819.
(E)-3-(4-chloro phenyl)-N-(4-(2-[pyrrolidin-1-yl] ethoxy) phenyl) acryl-
amide (9d)
Grayish white powder, yield: 55.1%. mp: 184.3–185.2 ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 2.27 (6H, br, pyrrolidine-H), 2.50–2.51 (2H, m, pyrrolidine-H),
2.68 (2H, t, J = 4.0 Hz, OCH2CH2), 4.04 (2H, t, J = 4.0 Hz, OCH2CH2),
6.87 (1H, d, J = 16.0 Hz, Ar CH CH), 6.92–6.94 (2H, m, Ar H),
7.43–7.47 (2H, m, Ar H), 7.55–7.57 (1H, m, Ar H), 7.61–7.63 (2H,
m, Ar H), 7.75–7.77 (1H, m, Ar H), 7.85 (1H, d, J = 16.0 Hz,
Ar CH CH), 10.20 (1H, s, NH). MS (ESI-Q-TOFMS) m/z calculated
(E)-3-(4-chloro phenyl)-N-(4-(2-[dimethylamino]ethoxy) phenyl) acryl-
amide (9a)
White powder, yield: 54.5%, mp: 180.3–181.2 ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.24 (6H, s, 2× NCH3), 2.64 (2H, t,
J = 6.0 Hz, OCH2CH2), 4.02 (2H, t, J = 6.0 Hz, OCH2CH2), 6.80 (1H,
d, J = 16.0 Hz, Ar CH CH), 6.91–6.93 (2H, m, Ar H), 7.50–7.57
(3H, m, Ar CH CH and Ar H), 7.59–7.65 (4H, m, Ar H), 10.09 (1H,
s, NH). MS (ESI-Q-TOFMS) m/z calculated for C19H21ClN2O2 + ([M]
for C21H23ClN2O2 + ([M]+): 370.1; found M + 1:371.1. IR (KBr) cm−1
:
3292, 2,924, 1,660, 1,622, 1,595, 1,294, 1,232, 987, 825.
SCHEME 1 Reagents and conditions: (I). (COCl)2, DMF, DCM; (II). Aminophenol, TEA, acetonitrile, 60 ꢀC; (III) Cl(CH2)2NR, K2CO3, NaI, acetone
reflux

6
GAO ET AL.
TABLE 1 Effect of compound 3a–3f on inhibitory activity against
AChE and BChE
IC50 a(μmol/L)
Selectivity
for AChEb
Compound
X
AChE
>500
>500
>500
>500
>500
>500
10.54
BChE
>500
>500
>500
>500
>500
>500
0.26
3a
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
–
–
3b
–
3c
–
3d
–
3e
–
3f
–
Rivastigminec
0.02
0.86
0.08
a
b
c
IC50 values of compounds represent the concentration that caused 50%
enzyme activity loss.
The selectivity is defined as the ratio of IC50 of compounds inhibiting
BChE/AChE.
FIGURE 2 Lineweaver–Burk plots for AChE kinetic profile in the
Used for positive control for IC50
.
presence of compound 6d
2.4 | AChE and BChE inhibition assay
assay mixture consists of 50 μL AChE(BChE), 100 μL S-acetylthiocholine
iodide (S-butyrylthiocholine iodide), 2.76 mL Na2HPO4/NaH2PO4 buffer
(pH 8.0, 0.1 mol/L), and 100 μL various concentrations of test com-
pounds, it was incubated at 30 ꢀC for 25 min. Then 100 μL 20% sodium
dodecylsulfate (SDS) and 100 μL 10 mmol/L 5,50-dithiobis-(2-nitrobenzoic
acid) (DTNB) was added into the assay mixture to terminate the reaction
AChE or BChE activity assays were performed with Ellman method
(Ellman, Courtney, Andres, & Featherstone, 1961). The brain and serum
of Sprague–Dawley rat was used as the resource of AChE and BChE,
respectively. Every compound was dissolved in a little Tween 80 and
diluted by water to various concentrations immediately before use. The
TABLE 2 Effect of compound 4a–9d on inhibitory activity against AChE and BChE
IC50a (μmol/L)
Compound
X
R
AChE
BChE
Selective for AChEb
14.28
0.60
4a
2-F
2-F
2-F
2-F
3-F
3-F
3-F
3-F
4-F
4-F
4-F
4-F
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
–
N(CH3)2
2.80 0.25
71.35 4.16
3.72 0.20
1.46 0.13
2.14 0.12
26.96 2.89
3.12 0.14
1.30 0.05
1.72 0.04
131.02 13.33
1.75 0.23
1.11 0.08
2.65 0.17
22.26 1.26
4.25 0.18
1.91 0.09
1.98 0.13
20.91 1.46
3.44 0.19
1.73 0.13
1.45 0.14
18.54 2.46
2.52 0.05
1.40 0.11
10.54 0.86
39.97 1.82
42.58 3.77
224.65 9.78
27.69 0.78
27.85 0.93
>500
4b
N(CH2CH3)2
Piperidine
Pyrrolidine
N(CH3)2
4c
60.39
18.97
13.01
>18.55
6.20
4d
5a
5b
N(CH2CH3)2
Piperidine
Pyrrolidine
N(CH3)2
5c
19.33 0.96
27.21 1.92
243.03 8.64
47.15 1.42
62.99 2.66
51.7 3.26
35.37 2.48
34.71 2.81
98.64 7.45
13.34 1.02
27.56 2.45
30.12 2.31
76.90 4.37
27.16 2.44
19.22 0.98
29.41 1.77
44.13 3.12
45.6 3.10
0.26 0.081
5d
20.93
141.30
0.36
6a
6b
N(CH2CH3)2
Piperidine
Pyrrolidine
N(CH3)2
6c
35.99
46.58
13.35
1.56
6d
7a
7b
N(CH2CH3)2
Piperidine
Pyrrolidine
N(CH3)2
7c
22.17
6.98
7d
8a
13.92
1.44
8b
N(CH2CH3)2
Piperidine
Pyrrolidine
N(CH3)2
8c
22.35
15.70
13.26
1.59
8d
9a
9b
N(CH2CH3)2
Piperidine
Pyrrolidine
–
9c
17.51
32.57
0.02
9d
Rivastigminec
a
b
c
IC50 values of compounds represent the concentration that caused 50% enzyme activity loss.
The selectivity is defined as the ratio of IC50 of compounds inhibiting BChE/AChE.
Used for positive control for IC50
.

GAO ET AL.
7
TABLE 3 Kinetics parameters assay of compound 6d inhibiting AChE
C (μM)
0
Michaelis–Menten equation
1/v = 121.36/[S] + 35.49
1/v = 210.81/[S] + 43.40
1/v = 314.84/[S] + 54.71
1/v = 495.98/[S] + 69.96
Km (mM)
3.42
Vmax (ΔAmin−1
0.028
)
Ki (μM)
Ki0 (μM)
0.35
0.88
1.76
4.86
0.023
0.61
1.86
5.75
0.018
7.09
0.014
and generate yellow product. The absorbance of each assay mixture was
measured at 412 nm by UV spectroscopy. Then the IC50 values were cal-
culated by Bliss method and expressed as mean SD.
So cinnamic acids derivatives in the present study can be confirmed as
trans-conformation (3JHH = 16 Hz). In addition, there are some broad
peaks in 1H NMR spectra of some compounds, such as compounds 4b,
5c, 6c. The hydrogen atoms that are adjacent to oxygen or nitrogen
atoms with larger electronegativity exhibit broad peaks. It is speculated
that proton exchange among these hydrogen atoms leads to the decou-
pling phenomenon, showing a broad peak in the spectra.
2.5 | Kinetic studies
Kinetic study was conducted according to a reported method with some
modifications (Skrzypek, Matysiak, Niewiadomy, Bajda, & Szymanski,
2013). The assay solution consists of 100 μL compound 6d, 100 μL
DTNB, 2.76 mL 0.1 mol/L Na2HPO4/NaH2PO4 buffer (pH 8.0). It was
preincubated with AChE at 30 ꢀC, followed by the addition of 100 μL
acetylthiocholine iodide with five various concentrations in parallel.
Then the absorbance of assay solution was measured spectrometrically
at 412 nm, which is proportional to the activity of AChE to catalyze
acetylthiocholine iodide. The linear Lineweaver–Burk plot based on
Michaelis–Menten equation was used to evaluate the inhibition profile
and kinetic parameters. Additionally, the parallel control experiment was
conducted in the absence of compound 6d in the assay solution.
3.2 | Bioactivity evaluation
In this study newly synthesized compounds were evaluated in AChE
and BChE inhibition with the results shown in Tables 1 and 2. Cinnamic
amides without tertiary amine side chain (compounds 3a–3f) exhibit
poor activity in AChE and BChE inhibition (IC50 > 500 μmol/L). By con-
trast, compounds containing tertiary amine side chain (compounds 4a–
9c) exhibit moderate or potent activity against AChE. All compounds
containing dimethylamine, piperidine, diethylamine or pyrrolidine side
chain exhibit potent activity in AChE inhibition (IC50 < 5.0 μmol/L)
except the compounds with diethylamine side chain showing weak bio-
activity (IC50 > 18.0 μmol/L). In addition, almost all the cinnamic deriva-
tives with para-fluoro or chloro substituent are more potent in AChE
inhibition than meta- or ortho-substituted compounds. For those com-
pounds with piperidine or pyrrolidine side chain, fluoro-substituted
compounds are more potent in AChE inhibition than chloro-substituted
compounds. The results above indicate tertiary side chain may be
important in AChE or BChE inhibition. Also, the substitution of fluorine
or chlorine in cinnamic acid poses significant influence on the inhibitory
effect against AChE and BChE.
3
| RESULTS AND DISCUSSION
3.1 | Chemistry
The route of synthesis of compounds 4a–9d is shown in Scheme 1.
Firstly, the chloride compounds were prepared using condensation
reaction of halogen-substituted cinnamic acids and oxalyl chloride
with DMF as the catalyst. Then, cinnamic acid chlorides reacted with
p-aminophenol to synthesize amide derivatives respectively, which
subsequently reacted with four commercially available aminoethyl
chlorides (chloroethyldimethylamine hydrochloride, chloroethyldiethy-
lamine hydrochloride, chloroethylpiperidine hydrochloride, and chlor-
oethylpyrrolidine hydrochloride) in the presence of K2CO3 and NaI for
gaining desired compounds.
Among those newly synthesized compounds, compounds 6d pos-
sess inhibitory potency 9.5 times greater than Rivastigmine
(IC50 = 10.54 μmol/L) with IC50 values of 1.11 μmol/L. Due to the
high selectivity for AChE inhibition (Ratio: 46.58), it was selected for
further kinetic studies. The analysis result of compounds 6d in linear
Lineweaver–Burk plot is shown in Figure 2. It exhibits a mixed-type
inhibitory manner according to the plot. The competitive inhibition
constant (Ki) and the noncompetitive constant (Ki0) are 0.61 μmol/L
and 1.86 μmol/L, respectively (Table 3).
Compounds 4a–9d were gained by Williamson reaction between
aminoethyl chlorides and hydroxy cinnamamide. For various tertiary
amine substituents, the overall reaction times were arrayed in descend-
ing order as: diethylamine > dimethylamine > pyrrolidine > piperidine.
However, there is no significant difference in the yield of final products.
New synthetic compounds were characterized by proton nuclear
magnetic resonance spectroscopy (H1NMR), infrared spectrum (IR) and
mass spectrometry (MS). The compounds were measured by HPLC,
and the purity is over 98.0%. In this study, according to H1NMR spec-
trum, the cis- or trans-conformation of cinnamic acid derivatives can be
distinguished. Hydrogen spectrum shows that the 3JHH couplings of
trans-cinnamic amide are larger than that of cis-cinnamic amide. The
former is about 16–17 Hz, while the latter is about 12–13 Hz accord-
ing to previous report (Hanai, Kuwae, Takai, Senda, & Kunimoto, 2001).
4
| CONCLUSION
In the present study, a series of chlorine or fluorine cinnamic amide
derivatives were synthesized with their bioactivity in AChE and BChE
inhibition evaluated. The results show the tertiary amine side chain
exerts a significant effect on the bioactivity. Moreover, for the com-
pounds containing tertiary amine side chain, the chlorine or fluorine
substituted position also produces marked influences on the bioactivity
and selectivity in AChE inhibition. Most of the compounds that contain

8
GAO ET AL.
Li, Y., Dai, F., Jin, X. L., Ma, M. M., Wang, Y. H., Ren, X. R., & Zhou, B.
(2014). An effective strategy to develop active cinnamic acid-directed
antioxidants based on elongating the conjugated chains. Food Chemis-
try, 158(18), 41–47.
para-substituted fluorine or chlorine are shown to have potent activity
against AChE and poor activity against BChE, while the opposite effect
exhibit in the ortho-substituted analogs. This result may provide valu-
able information for future research on new cholinesterase inhibitors.
Liu, H. R., Fan, H. Q., Gao, X. H., Huang, X. Q., Liu, X. J., Liu, L. B., …
Liu, W. K. (2016). Design, synthesis and preliminary structure–activity
relationship investigation of nitrogen-containing chalcone derivatives
as acetylcholinesterase and butyrylcholinesterase inhibitors: A further
study based on Flavokawain B Mannich base derivatives. Journal of
Enzyme Inhibition and Medicinal Chemistry, 31(4), 580–589.
Liu, H. R., Huang, X. Q., Lou, D. H., Liu, X. J., Liu, W. K., & Wang, Q. A.
(2014a). Synthesis and acetylcholinesterase inhibitory activity of Man-
nich base derivatives flavokawain B. Bioorganic & Medicinal Chemistry
Letters, 24(19), 4749–4753.
ACKNOWLEDGMENTS
This research study was supported by “Natural Science Foundation of
Hu'nan Province” (No. 2017JJ2050 and No. 2018JJ3572) and Grant
(2016TP1029) awarded by Hu'nan Key Laboratory Cultivation Base of
the Research and Development of Novel Pharmaceutical Preparations.
Liu, H. R., Liu, L. B., Gao, X. H., Liu, Y., Xu, W. J., He, W., … Xia, X. H.
(2017). Novel ferulic amide derivatives with tertiary amine side chain
as acetylcholinesterase and butyrylcholinesterase inhibitors: The influ-
ence of carbon spacer length, alkylamine and aromatic group. European
Journal of Medicinal Chemistry, 126(1), 810–822.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
Liu, H. R., Liu, X. J., Fan, H. Q., Tang, J. J., Gao, X. H., & Liu, W. K. (2014b).
Design, synthesis and pharmacological evaluation of chalcone deriva-
tives as acetylcholinesterase inhibitors. Bioorganic & Medicinal Chemis-
try, 22(21), 6124–6133.
Lu, Y. X., Liu, Y. T., Xu, Z. J., Li, H. Y., Liu, H. L., & Zhu, W. L. (2012). Halo-
gen bonding for rational drug design and new drug discovery. Expert
Opinion on Drug Discovery, 7(5), 375–383.
Ramalakshmi, N., Deepa, S., Sumanth Srinivas, K., Puratchikody, A., &
Arunkumar, S. (2009). Synthesis, characterization and biological
screening of some novel 1,3,5-trisubstituted 2-pyrazolines. Rasayan
Journal of Chemistry, 2(2), 393–396.
ORCID
Hao-Ran Liu
https://orcid.org/0000-0002-0622-5310
REFERENCES
Birks, J., Grimley Evans, J., Iakovidou, V., Tsolaki, M., & Holt, F. E. (2009).
Rivastigmine for Alzheimer's disease. Cochrane Database of Systematic
Reviews, 15(2), CD001191.
Ellman, G. L., Courtney, K. D., Andres, V. J., & Featherstone, R. M. (1961).
A new and rapid colorimetric determination of acetylcholinesterase
activity. Biochemical Pharmacology, 7(2), 88–95.
Sang, Z. P., Pan, W. L., Wang, K. R., Ma, Q. G., Yu, L. T., Yang, Y., …
Liu, W. M. (2017). Design, synthesis and evaluation of novel ferulic
acid-O-alkylamine derivatives as potential multifunctional agents for
the treatment of Alzheimer's disease. European Journal of Medicinal
Chemistry, 130(4), 379–392.
Estrada, M., Herrera-Arozamena, C., Pérez, C., Viña, D., Romero, A.,
Morales-García, J. A.,
… Rodríguez-Franco, M. I. (2016). New
cinnamic–N-benzylpiperidine and cinnamic–N,N-dibenzyl(N-methyl)
amine hybrids as Alzheimer-directed multitarget drugs with antioxi-
dant, cholinergic, neuroprotective and neurogenic properties. European
Journal of Medicinal Chemistry, 121(10), 376–386.
Skrzypek, A., Matysiak, J., Niewiadomy, A., Bajda, M., & Szymanski, P.
(2013). Synthesis and biological evaluation of 1,3,4-thiadiazole ana-
logues as novel AChE and BuChE inhibitors. European Journal of Medic-
inal Chemistry, 62(7), 311–319.
Gao, X. H., Liu, L. B., Liu, H. R., Tan, J. J., Kang, L., Wu, H. N., … Yan, J. Y.
(2018a). Structure–activity relationship investigation of benzamide and
picolinamide derivatives containing dimethylamine side chain as acetyl-
cholinesterase inhibitors. Journal of Enzyme Inhibition and Medicinal
Chemistry, 33(1), 110–114.
Gao, X. H., Tang, J. J., Liu, H. R., Liu, L. B., Kang, L., & Chen, W. (2018b).
Structure–activity relationship investigation of tertiary amine deriva-
tives of cinnamic acid as acetylcholinesterase and butyrylcholinester-
ase inhibitors: Compared with that of phenylpropionic acid, sorbic acid
and hexanoic acid. Journal of Enzyme Inhibition and Medicinal Chemistry,
33(1), 519–524.
Hanai, K., Kuwae, A., Takai, T., Senda, H., & Kunimoto, K.-K. (2001). A com-
parative vibrational and NMR study of cis-cinnamic acid polymorphs
and trans-cinnamic acid. Spectrochimica Acta Part A, 57(3), 513–519.
Hansen, R. A., Gartlehner, G., Webb, A. P., Morgan, L. C., Moore, C. G., &
Jonas, D. E. (2008). Efficacy and safety of donepezil, galantamine and
rivastigmine for the treatment of Alzheimer‘s disease: A system review
and meta-analysis. Clinical Interventions in Aging, 3(2), 211–225.
Hu, Y. H., Chen, Q. X., Cui, Y., Gao, H. J., Xu, L., Yu, X. Y., … Wang, Q.
(2016). 4-Hydroxy cinnamic acid as mushroom preservation: Anti-
tyrosinase activity kinetics and application. International Journal of Bio-
logical Macromolecules, 86(5), 489–495.
Song, Y. H., Kim, D. W., Curtis-Long, M. J., Park, C., Son, M., Kim, J. Y., …
Park, K. H. (2016). Cinnamic acid amides from Tribulus terrestris display-
ing uncompetitive α-glucosidase inhibition. European Journal of Medici-
nal Chemistry, 114(5), 201–208.
Takaya, T., Okamoto, M., Yodoi, K., Hata, K., Kijima, Y., Nakajima, H., …
Kawashima, S. (2009). Torsades de pointes with QT prolongation
related to donepezil use. Journal of Cardiology, 54(3), 507–511.
Vulpetti, A., & Dalvit, C. (2012). Fluorine local environment: From screen-
ing to drug design. Drug Discovery Today, 17(15–16), 890–897.
Yan, S. L., Wang, Z. H., Yen, H. F., Lee, Y. J., & Yin, M. C. (2016). Reversal
of ethanol-induced hepatotoxicity by cinnamic and syringic acids in
mice. Food and Chemistry Toxicology, 98(12), 119–126.
Zhang, X., He, X. X., Chen, Q. H., Lu, J. F., Rapposelli, S., & Pi, R. B. (2018).
A review on the hybrids of hydroxycinnamic acid as multi-target-
directed ligands against Alzheimer's disease. Bioorganic & Medicinal
Chemistry, 26(3), 543–550.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Sup-
porting Information section at the end of the article.
Jayaprakasha, G. K., & Rao, L. J. (2011). Chemistry, biogenesis, and biologi-
cal activities of Cinnamomum zeylanicum. Critical Reviews in Food Sci-
ence and Nutrition, 51(6), 547–562.
Jeong, H. J., Lee, C. S., Choi, J., Hong, Y. D., Shin, S. S., Park, J. S., … Ko, J.
(2015). Flavokawains B and C, melanogenesis inhibitors, isolated from
the root of Piper methysticum and synthesis of analogs. Bioorganic &
Medicinal Chemistry Letters, 25(4), 799–802.
Letizia, C. S., Cocchiara, J., Lapczynski, A., Lalko, J., & Api, A. M. (2005).
Fragrance material review on cinnamic acid. Food and Chemical Toxicol-
ogy, 43(6), 925–943.
How to cite this article: Gao X-H, Tang J-J, Liu H-R, Liu L-B,
Liu Y-Z. Structure–activity study of fluorine or chlorine-
substituted cinnamic acid derivatives with tertiary amine side
chain in acetylcholinesterase and butyrylcholinesterase inhibi-
tion. Drug Dev Res. 2019;1–8. https://doi.org/10.1002/ddr.
21515