Design, synthesis and evaluation of novel cinnamic acid derivatives bearing N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer’s disease

Article abstract of DOI:10.1080/14756366.2016.1256883

A novel family of cinnamic acid derivatives has been developed to be multifunctional cholinesterase inhibitors against AD by fusing N-benzyl pyridinium moiety and different substituted cinnamic acids. In vitro studies showed that most compounds were endowed with a noteworthy ability to inhibit cholinesterase, self-induced Aβ (1–42) aggregation, and to chelate metal ions. Especially, compound 5l showed potent cholinesterase inhibitory activity (IC₅₀, 12.1 nM for eeAChE, 8.6 nM for hAChE, 2.6 μM for eqBuChE and 4.4 μM for hBuChE) and the highest selectivity toward AChE over BuChE. It also showed good inhibition of Aβ (1–42) aggregation (64.7% at 20 μM) and good neuroprotection on PC12 cells against amyloid-induced cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead compound for the treatment of Alzheimer’s diseases.

Full text of DOI:10.1080/14756366.2016.1256883

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and evaluation of novel cinnamic
acid derivatives bearing N-benzyl pyridinium
moiety as multifunctional cholinesterase
inhibitors for Alzheimer’s disease
Jin-Shuai Lan, Jian-Wei Hou, Yun Liu, Yue Ding, Yong Zhang, Ling Li & Tong
Zhang
To cite this article: Jin-Shuai Lan, Jian-Wei Hou, Yun Liu, Yue Ding, Yong Zhang, Ling Li & Tong
Zhang (2017) Design, synthesis and evaluation of novel cinnamic acid derivatives bearing N-benzyl
pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer’s disease, Journal of
Enzyme Inhibition and Medicinal Chemistry, 32:1, 776-788, DOI: 10.1080/14756366.2016.1256883
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1256883
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
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Date: 07 June 2017, At: 01:53

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 776–788
http://dx.doi.org/10.1080/14756366.2016.1256883
RESEARCH ARTICLE
Design, synthesis and evaluation of novel cinnamic acid derivatives bearing
N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for
Alzheimer’s disease
a
a
a
a
a
a
b
ꢀ
ꢀ
Jin-Shuai Lan , Jian-Wei Hou , Yun Liu , Yue Ding , Yong Zhang , Ling Li and Tong Zhang
aExperiment Center of Teaching & Learning, Shanghai University of Traditional Chinese Medicine, Shanghai, China; School of Pharmacy,
b
Shanghai University of Traditional Chinese Medicine, Shanghai, China
ABSTRACT
ARTICLE HISTORY
Received 26 July 2016
Revised 10 October 2016
Accepted 17 October 2016
A novel family of cinnamic acid derivatives has been developed to be multifunctional cholinesterase inhibi-
tors against AD by fusing N-benzyl pyridinium moiety and different substituted cinnamic acids. In vitro
studies showed that most compounds were endowed with a noteworthy ability to inhibit cholinesterase,
self-induced Ab (1–42) aggregation, and to chelate metal ions. Especially, compound 5l showed potent
cholinesterase inhibitory activity (IC50, 12.1 nM for eeAChE, 8.6 nM for hAChE, 2.6 lM for eqBuChE and
4.4lM for hBuChE) and the highest selectivity toward AChE over BuChE. It also showed good inhibition of
Ab (1–42) aggregation (64.7% at 20 lM) and good neuroprotection on PC12 cells against amyloid-induced
cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and
proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead com-
pound for the treatment of Alzheimer’s diseases.
KEYWORDS
Alzheimer’s disease;
b-amyloid aggregation;
benzylpyridinium; cinnamic
acid; metal chelator;
neuroprotection
Introduction
than Ab (1–40) does10,11. Preventing the formation and accumula-
tion of Ab is a probable therapeutic strategy for AD. The dysho-
meostasis of metal ions such as Cu, Fe and Zn is commonly
observed in many critical aspects of AD12. The Cu2þ present in
the brain at an abnormally high concentration interacts with
Ab, leading to the accelerated formation of neurofibrillary tan-
gles (NFTs) and generate reactive oxygen species (ROS), which
further induce oxidative impairment in the brain13. Moreover,
abnormally high levels of redox-active metal ions, such as Fe2þ
and Cu2þ, in the brain may lead to the formation of ROS14.
Alzheimer’s disease (AD) is a fatal neurodegenerative disorder that
is clinically associated with cognitive impairment, language skill
loss and dementia1. To date almost 48 million elderly people are
affected by AD, and this number is estimated to show unparal-
leled growth and increase to spread 131.5 million by 20502.
Although the etiopathogenesis of AD is unclear, multiple factors
such as amyloid-b (Ab) deposits, low levels of acetylcholine (ACh),
s-protein aggregation, dyshomeostasis of biometals and oxidative
stress, play a vital role in the pathogenesis of AD3. The current
therapeutic options against AD are composed by one N-methyl-D- Notably, compared with the normal levels of metal ions in
aspartate (NMDA) receptor antagonist and three acetylcholinester-
ase (AChE) inhibitors, namely memantine (NMDA), donepezil, riva-
stigmine and galantamine (AChE)4. Nevertheless, these marketed
drugs modestly alleviate the symptoms but cannot cure brain
damage or stop neuronal degeneration.
brain, the subtraction of physiologically essential metals may
lead to risk. Consequently, reducing the abnormally high con-
centration of metals in the brain by chelating the metals is an
additional logical approach for AD treatment. Oxidative stress
also plays a crucial role in the development of neurodegenera-
tive disorders such as AD. It has been hypothesised that the
antioxidant defence system cannot neutralize oxidative species
in elderly people15. The oxidative stress theory of ageing also
suggests that oxidative damage plays an important role in neur-
onal degeneration16. Therefore, drugs that can scavenge oxygen
radicals may be used to prevent AD.
Due to the pathological complication of AD, the multi-target-
directed ligand (MTDL) design strategy has been proposed, and a
range of compounds have been developed to act on various tar-
gets17–21. In AD, the effectiveness of multifunctional molecules
with two or more distinct pharmacological properties being prop-
AChE and BuChE play a role in cholinergic signaling. According
to the cholinergic hypothesis, the decrease in ACh levels results in
memory loss and cognitive impairment, and the clinical restoration
of cholinergic function is believed to alleviate AD symptoms5,6
.
Furthermore, studies have illustrated that AChE interacts with Ab
through the peripheral anionic site (PAS) promoting the formation
of steady AChE-Ab complexes, which are more toxic than single
Ab peptides7. Therefore, dual-site AChE inhibitors may be promis-
ing AD drug candidates8,9
.
Among multiple factors, neurotoxic Ab plaques in the brain are
a key contributing factor in the pathology of AD. Ab (1–40) and
Ab (1–42) are the key isoforms of Ab peptides. Ab (1–42) may
aggregate more rapidly and show stronger neuron cytotoxicity erly incorporated is higher than that of single-targeted drugs22.
CONTACT Tong Zhang
zhangtongshutcm@hotmail.com
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
Yue Ding
China.
dingyue-2001@hotmail.com Experiment Center of Teaching & Learning, Shanghai University of Traditional Chinese Medicine, Shanghai 201203,
ꢀ
These authors contributed equally to this work.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
777
Figure 1. Drug design strategy for multi-target-directed ligands.
Therefore, ChEIs with multiple effects, such as Ab disaggregation, instrument and uncorrected. IR (KBr-disc) spectra were recorded by
neuroprotection and oxidative load reduction may be a significant
approach for AD management.
Bruker Tensor 27 spectrometer (Billerica, MA).
1H NMR and 13C NMR spectra were measured on a BRUKER
AVANCE III spectrometer (Billerica, MA) at 25 ꢁC and referenced to
TMS. Chemical shifts are reported in ppm (d) using the residual
solvent line as internal standard. Splitting patterns are designed as
s, singlet; d, doublet; t, triplet; m, multiplet. The purity of all com-
pounds was confirmed to be higher than 95% through analytical
HPLC performed with Agilent 1200 HPLC System (Santa Clara, CA).
Mass spectra were obtained on a MS Agilent 1100 Series LC/MSD
Trap mass spectrometer (ESI-MS) (Santa Clara, CA).
Cinnamic acid derivatives are naturally occurring compounds
that possess various pharmacological properties for diverse neuro-
logical disorders23. Ferulic acid (FA) and curcumin (Cur) are repre-
sentative bioactive compounds, and they can prevent Ab fibril
aggregation, inhibit Ab-mediated toxicity, scavenge ROS and
reduce inflammatory effects both in vitro and in vivo24–29
.
Consequently, cinnamic acid could serve as a beneficial fragment
in the designed MTDL, such as tacrine-FA hybrids, FA-memoquin
hybrids, FA-carbazole hybrids, donepezil-FA hybrids, cinnamic-
N-benzylpiperidine hybrids30–34
.
In the search for new cinnamic acid-based derivatives as anti-AD,
we have focused on the structure of benzylpyridinium salts (Figure
1), which may represent a privileged scaffold that can be used to
develop new AChE inhibitors35–37. Docking studies have shown that
the N-benzyl pyridinium moiety interacts with the CAS of AChE, and
the heterocyclic moiety interacts with the PAS of AChE forming
stacking interactions. Proceeding with our researches on natural
products with probable use as anti-AD38–40, we reasonably com-
bined the cinnamic acid with benzyl-pyridinium to achieve new
hybrids that are anticipated to be dual-acting AChE. In this study, all
designed compounds were synthesised and evaluated for their bio-
logical activities, including their anti-aggregation activity towards
Ab, ChE inhibition, antioxidant activity, metal chelating properties,
neuroprotective effects against Ab-induced PC12 cell injury and the
ability to cross the blood–brain barrier (BBB). Moreover, kinetic and
molecular modelling studies were performed to further explore their
mechanism of interaction with AChE and Ab.
General procedure for the preparation of compounds 3a–b
A mixture of compound 1 (10 mmol) and DMAP (10 mmol) in
20 mL anhydrous CH2Cl2 was stirred at 0 ꢁC for about 10 min and
EDCI (20 mmol) was added to the mixture and stirred at room
temperature for 1 h. The compound 2 (10 mmol) was added to the
solution, and stirring was continued overnight. The reaction mix-
ture was diluted with H2O and extracted with CH2Cl2. The organic
extracts were combined, washed with brine, and dried with
anhydrous Na2SO4, and the solvent was evaporated in vacuo to
give the crude product, which was purified by silica gel chroma-
tography with CH2Cl2:MeOH ¼15:1 as an eluent to afford corre-
sponding target compound as a yellow solid.
N-(pyridin-4-yl)cinnamamide (3a) Cinnamic acid was reacted with
4-aminopyridine following the general procedure to give the desired
product 3a with a yield of 85%. ESI/MS m/z: 225.1 [M þ H]þ; 1H NMR
(400 MHz, CDCl3) d 8.75 (s, 1H), 8.50 (dd, J ¼ 4.9, 1.4 Hz, 2H), 7.79 (d,
J ¼ 15.5 Hz, 1H), 7.64 (dd, J ¼ 4.8, 1.6 Hz, 2H), 7.49 (dd, J ¼ 6.6, 3.0 Hz,
2H), 7.40–7.32 (m, 3H), 6.63 (d, J ¼ 15.5 Hz, 1H).
Materials and methods
Materials
(E)-3-(3,4-dimethoxyphenyl)-N-(pyridin-4-yl) acrylamide (3b) (E)-3-
(3,4-dimethoxy -phenyl) acrylic acid was reacted with 4-aminopyri-
dine following the general procedure to give the desired product
3b with a yield of 88%. ESI/MS m/z: 285.2 [M þ H]þ; 1H NMR
(400 MHz, CDCl3) d 8.54 (d, J ¼ 5.3 Hz, 2H), 7.96 (s, 1H), 7.75 (d,
All chemicals (reagent grade) used were purchased from Sino Pharm
Chemical Reagent Co., Ltd. (Shanghai, China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on pre-
coated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-Dao,
China) plates and the spots were detected under UV light (254 nm).
Melting point was measured on an XT-4 micromelting point J ¼ 15.4 Hz, 1H), 7.62 (d, J ¼ 6.0 Hz, 2H), 7.15 (dd, J ¼ 8.3, 1.8 Hz,

778
J.-S. LAN ET AL
1H), 7.06 (d, J ¼ 1.8 Hz, 1H), 6.90 (d, J ¼ 8.3 Hz, 1H), 6.47 (d, 333.1 [M]þ; 1H NMR (400 MHz, DMSO-d6) d 11.84 (s, 1H), 8.95 (d,
J ¼ 15.4 Hz, 1H), 3.95 (s, 3H), 3.89 (s, 3H).
J ¼ 6.7 Hz, 2H), 8.23 (d, J ¼ 6.5 Hz, 2H), 7.79 (d, J ¼ 15.8 Hz, 1H), 7.70
(d, J ¼ 5.2 Hz, 2H), 7.63–7.57 (m, 2H), 7.49 (d, J ¼ 5.1 Hz, 3H), 7.31 (t,
J ¼ 8.4 Hz, 2H), 7.00 (d, J ¼ 15.8 Hz, 1H), 5.71 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) d 165.92, 163.15 (d, 1JCF¼246.44 Hz), 152.76,
145.61, 144.56, 137.03, 134.42, 131.62 (d, 3JCF¼7.98 Hz), 131.33,
129.65, 128.78, 120.66, 116.52 (d, 2JCF¼21.37 Hz), 115.77, 61.19.
General procedure for the preparation of compounds 5a–n
Compound 3 (10 mmol), appropriate benzyl chloride (12 mmol)
and a catalytic amount of KI in dry acetonitrile (20 mL) was
refluxed for 1–2 h. When the reaction was completed as indicated
by TLC, the mixture was then concentrated under reduced pres-
sure, and 20 mL of diethyl ether was added. On cooling, the pre-
cipitate was filtered and washed with diethyl ether to get the
target compounds 5a–n with high yields.
1-(3-Bromobenzyl)-4-cinnamamidopyridin-1-ium bromide (5f) Yield
92%; yellow solid; IR (KBr) m 3087, 3020, 2966, 1708, 1634, 1595,
1516, 1165, 1165, 1136, 966, 837 761, 703 cmꢂ1; m.p. >250 ꢁC; ESI/
1
MS m/z: 393.0, 395.0 [M]þ; H NMR (400 MHz, DMSO-d6) d 11.82 (s,
1H), 8.95 (d, J ¼ 6.8 Hz, 2H), 8.23 (d, J ¼ 6.5 Hz, 2H), 7.86–7.75 (m, 2H),
7.70 (d, J ¼ 4.8 Hz, 2H), 7.64 (d, J ¼ 8.3 Hz, 1H), 7.50 (dd, J ¼ 12.0,
6.4 Hz, 4H), 7.43 (t, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 15.8 Hz, 1H), 5.72 (s,
2H). 13C NMR (100 MHz, DMSO-d6) d 165.93, 152.85, 145.76, 145.76,
144.62, 137.60, 134.41, 132.52, 131.91, 131.81, 131.35, 129.66, 129.66,
128.79, 128.79, 128.20, 122.70, 120.64, 115.83, 115.83, 61.14.
1-Benzyl-4-cinnamamidopyridin-1-ium bromide (5a) Yield 91%; yel-
low solid; IR (KBr) m 3091, 3028, 2963, 1685, 1637, 1515, 1496, 120þ3,
1161, 966, 763, 752, 725 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 315.1 [M] ;
1H NMR (400 MHz, DMSO-d6) d 11.90 (s, 1H), 8.98 (d, J ¼ 6.7 Hz, 2H),
8.25 (d, J ¼ 6.5 Hz, 2H), 7.79 (d, J ¼ 15.7 Hz, 1H), 7.75–7.65 (m, 2H),
7.55–7.37 (m, 8H), 7.05 (d, J ¼ 15.8 Hz, 1H), 5.74 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) d 165.44, 152.24, 145.20, 145.20, 143.99, 134.68,
133.93, 130.81, 129.15, 129.15, 129.15, 129.15, 129.15, 129.15, 128.50,
128.50, 128.27, 128.27, 120.20, 115.21, 61.50.
1-(4-Bromobenzyl)-4-cinnamamidopyridin-1-ium
bromide
(5g)
Yield 90%; yellow solid; IR (KBr) m 3020, 1707, 1624, 1514, 1460, 1336,
1141, 972, 837, 762 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 393.0, 395.0
[M]þ; 1H NMR (400 MHz, DMSO-d6) d 11.81 (s, 1H), 8.92 (d, J ¼ 7.4 Hz,
2H), 8.22 (d, J ¼ 7.2 Hz, 2H), 7.79 (d, J ¼ 15.8 Hz, 1H), 7.72–7.69 (m,
2H), 7.68–7.65 (m, 2H), 7.50–7.45 (m, 5H), 6.97 (d, J ¼ 15.8 Hz, 1H),
5.70 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d 165.94, 152.81, 145.73,
145.73, 144.54, 134.47, 134.43, 132.56, 132.56, 131.35, 131.35, 129.65,
129.65, 128.77, 128.77, 123.06, 120.69, 115.73, 115.73, 61.19.
4-Cinnamamido-1-(3-methylbenzyl) pyridin-1-ium bromide (5b)
Yield 90%; yellow solid; IR (KBr) m 3074, 3023, 2969, 1701, 1633,
1596, 1527, 1456, 1166, 965, 849, 743 cmꢂ1; m.p. >250 ꢁC; ESI/MS
1
m/z: 329.1 [M]þ; H NMR (400 MHz, DMSO-d6) d 11.80 (s, 1H), 8.94
(d, J ¼ 6.7 Hz, 2H), 8.22 (d, J ¼ 6.4 Hz, 2H), 7.80 (d, J ¼ 15.6 Hz, 1H),
7.70 (d, J ¼ 4.9 Hz, 2H), 7.49 (d, J ¼ 5.1 Hz, 3H), 7.33 (dd, J ¼ 15.5,
8.0 Hz, 2H), 7.28 (d, J ¼ 7.6 Hz, 1H), 7.24 (d, J ¼ 7.5 Hz, 1H), 6.99 (d,
J ¼ 15.7 Hz, 1H), 5.67 (s, 2H), 2.32 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 165.92, 152.72, 145.68, 145.68, 144.56, 139.03, 135.06,
134.42, 131.33, 131.03, 129.66, 129.66, 129.58, 129.51, 128.78,
128.78, 126.08, 120.66, 115.77, 115.77, 62.09, 21.39.
(E)-1-benzyl-4-(3-(3,4-dimethoxyphenyl) acrylamido) pyridin-1-ium
bromide (5h) Yield 91%; yellow solid; IR (KBr) m 3087, 3022, 2962,
1706, 1641, 1596, 1512, 1463, 1264, 1132, 1025, 965, 746,
703 cmꢂ1
;
m.p. >250 ꢁC; ESI/MS m/z: 375.1 [M]þ; 1H NMR
(400 MHz, DMSO-d6) d 11.74 (s, 1H), 8.95 (d, J ¼ 7.4 Hz, 2H), 8.22 (d,
J ¼ 7.3 Hz, 2H), 7.73 (d, J ¼ 15.6 Hz, 1H), 7.51–7.41 (m, 5H), 7.28 (dd,
J ¼ 6.4, 1.9 Hz, 2H), 7.06 (d, J ¼ 8.9 Hz, 1H), 6.89 (d, J ¼ 15.7 Hz, 1H),
5.72 (s, 2H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d 170.92, 157.60, 156.56, 154.20, 150.40, 150.40, 149.54, 139.97,
134.41, 134.41, 134.33, 133.72, 133.72, 131.95, 128.10, 122.89,
120.34, 117.04, 115.64, 115.64, 66.73, 60.90, 60.74.
4-Cinnamamido-1-(4-methylbenzyl) pyridin-1-ium bromide (5c)
Yield 88%; yellow solid; IR (KBr) m 3015, 1697, 1628, 1509, 1458,
1339, 1162, 975, 860, 761 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 329.1
[M]þ; 1H NMR (400 MHz, DMSO-d6) d 11.84 (s, 1H), 8.93 (d,
J ¼ 6.9 Hz, 2H), 8.23 (d, J ¼ 6.6 Hz, 2H), 7.79 (d, J ¼ 15.7 Hz, 1H), 7.70
(d, J ¼ 4.9 Hz, 2H), 7.49 (d, J ¼ 5.1 Hz, 3H), 7.40 (d, J ¼ 7.7 Hz, 2H),
7.26 (d, J ¼ 7.6 Hz, 2H), 7.01 (d, J ¼ 15.9 Hz, 1H), 5.67 (s, 2H), 2.31 (s,
3H). 13C NMR (100 MHz, DMSO-d6) d 165.91, 152.68, 145.58,
145.58, 144.52, 139.18, 134.43, 132.16, 131.32, 130.18, 130.18,
129.65, 129.65, 129.08, 129.08, 128.77, 128.77, 120.68, 115.72,
115.72, 61.90, 21.22.
(E)-4-(3-(3,4-dimethoxyphenyl) acrylamido)-1-(3-methylbenzyl) pyri-
din-1-ium bromide (5i) Yield 93%; yellow solid; IR (KBr) m 3016,
1697, 1626, 1509, 1458, 1257, 1134, 1015, 971, 840, 771, 709,
593 cmꢂ1
;
m.p. >250 ꢁC; ESI/MS m/z: 389.1 [M]þ; 1H NMR
(400 MHz, DMSO-d6) d 11.69 (s, 1H), 8.91 (d, J ¼ 7.4 Hz, 2H), 8.20 (d,
J ¼ 7.1 Hz, 2H), 7.73 (d, J ¼ 15.6 Hz, 1H), 7.36–7.22 (m, 6H), 7.06 (d,
J ¼ 8.9 Hz, 1H), 6.84 (d, J ¼ 15.7 Hz, 1H), 5.66 (s, 2H), 3.85 (s, 3H),
3.80 (s, 3H), 2.32 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 166.17,
152.81, 151.81, 149.44, 145.59, 145.59, 144.83, 139.03, 135.07,
130.22, 129.57, 129.49, 127.18, 126.05, 123.39, 118.09, 115.59,
115.59, 112.27, 110.86, 62.04, 56.14, 55.99, 21.38.
4-Cinnamamido-1-(3-fluorobenzyl) pyridin-1-ium bromide (5d)
Yield 89%; white solid; IR (KBr) m 3075, 3018, 2966, 1701, 1632,
1589, 1526, 1448, 1146, 965, 848, 750, 676 cmꢂ1; m.p. >250 ꢁC;
ESI/MS m/z: 333.2 [M]þ; 1H NMR (400 MHz, DMSO-d6) d 11.84 (s,
1H), 8.96 (d, J ¼ 6.7 Hz, 2H), 8.23 (d, J ¼ 6.5 Hz, 2H), 7.80 (d,
J ¼ 15.8 Hz, 1H), 7.70 (d, J ¼ 4.9 Hz, 2H), 7.54–7.45 (m, 4H), 7.42 (d,
J ¼ 9.7 Hz, 1H), 7.34 (d, J ¼ 7.6 Hz, 1H), 7.28 (t, J ¼ 8.6 Hz, 1H), 7.00
(d, J ¼ 15.8 Hz, 1H), 5.74 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d
165.93, 163.72 (d, 1JCF¼246.44 Hz), 152.85, 145.78, 145.78, 144.60,
137.61 (d, 3JCF¼7.58 Hz), 134.41, 131.80 (d, 3JCF¼8.29 Hz), 131.34,
129.66, 129.66, 128.79, 128.79, 120.64, 116.51 (d, 2JCF¼21.72 Hz),
116.07 (d, 2JCF¼22.15 Hz), 115.80, 61.27.
(E)-4-(3-(3,4-dimethoxyphenyl) acrylamido)-1-(4-methylbenzyl) pyri-
din-1-ium bromide (5j) Yield 94%; yellow solid; IR (KBr) m 3068,
3014, 2948, 1683, 1622, 1508, 1462, 1293, 1132, 975, 794,
594 cmꢂ1
;
m.p. >250 ꢁC; ESI/MS m/z: 389.1 [M]þ; 1H NMR
(400 MHz, DMSO-d6) d 11.40 (s, 1H), 8.65 (d, J ¼ 6.7 Hz, 2H), 8.52 (s,
2H), 7.74 (d, J ¼ 15.5 Hz, 1H), 7.48 (d, J ¼ 15.5 Hz, 1H), 7.31–7.27 (m,
4-Cinnamamido-1-(4-fluorobenzyl) pyridin-1-ium bromide (5e) 3H), 7.22–7.17 (m, 4H), 6.83 (d, J ¼ 8.3 Hz, 1H), 5.65 (s, 2H), 3.95 (s,
Yield 95%; yellow solid; IR (KBr) m 3083, 3022, 2969, 1626, 1509, 3H), 3.90 (s, 3H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d
1462, 1338, 1158, 964, 846, 767 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 166.15, 152.77, 151.81, 149.45, 145.51, 145.51, 144.82, 139.17,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
779
132.18, 130.18, 130.18, 129.07, 129.07, 127.02, 123.39, 118.10, acid) (Ellman’s reagent, DTNB), S-butyrylthiocholine iodide (BTCI),
115.57, 115.57, 112.29, 110.85, 61.84, 56.16, 55.99, 21.22.
acetylthiocholine iodide (ATCI) and donepezil hydrochloride were
purchased from Sigma-Aldrich (St. Louis, MO). The capacity of the
test compounds 5a–n to inhibit AChE and BuChE activities was
assessed by Ellman’s method41. Test compound was dissolved in a
minimum volume of DMSO (1%) and was diluted using the buffer
solution (50 mM Tris–HCl, pH ¼8.0, 0.1 M NaCl, 0.02 M
MgCl2ꢃ6H2O). In 96-well plates, 160 lL of 1.5 mM DTNB, 50 lL of
AChE (0.22 U/mL prepared in 50 mM Tris–HCl, pH ¼8.0, 0.1% w/v
bovine serum albumin, BSA) or 50 lL of BuChE (0.12 U/mL pre-
pared in 50 mM Tris–HCl, pH ¼8.0, 0.1% w/v BSA) were incubated
with 10 lL of various concentrations of test compounds
(0.001–100 lM) at 37 ꢁC for 6 min followed by the addition of the
substrate (30 lL) ATCI (15 mM) or BTCI (15 mM) and the absorb-
ance was measured at different time intervals (0, 60, 120 and
180 s) at a wavelength of 405 nm. The concentration of compound
producing 50% of enzyme activity inhibition (IC50) was calculated
by nonlinear regression analysis of the response-concentration
(log) curve, using the Graph-Pad Prism program package (Graph
Pad Software, San Diego, CA). Results are expressed as the
mean SEM of at least three different experiments performed in
triplicate.
(E)-4-(3-(3,4-dimethoxyphenyl) acrylamido)-1-(3-fluorobenzyl) pyri-
din-1-ium bromide (5k) Yield 88%; yellow solid; IR (KBr) m 3014,
2964, 1711, 1688, 1592, 1518, 1445, 1258, 1137, 1014, 969, 850,
752, 571 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 393.1 [M]þ; 1H NMR
(400 MHz, DMSO-d6) d 11.74 (s, 1H), 8.95 (d, J ¼ 7.4 Hz, 2H), 8.22 (d,
J ¼ 7.0 Hz, 2H), 7.74 (d, J ¼ 15.6 Hz, 1H), 7.55–7.47 (m, 1H), 7.42 (d,
J ¼ 9.8 Hz, 1H), 7.34 (d, J ¼ 7.7 Hz, 1H), 7.27 (dd, J ¼ 14.2, 4.5 Hz,
3H), 7.06 (d, J ¼ 8.9 Hz, 1H), 6.88 (d, J ¼ 15.7 Hz, 1H), 5.74 (s, 2H),
3.83 (d, J ¼ 4.1 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) d 173.89,
165.13 (d, 1JCF¼228.26 Hz), 161.48, 152.96, 151.82, 149.45, 145.71,
145.71, 144.85, 137.67 (d, 3JCF¼8.16 Hz), 131.79 (d, 3JCF¼8.27 Hz),
127.19, 125.19, 123.38, 118.10, 116.50 (d, 2JCF¼21.52 Hz), 116.06 (d,
2JCF¼22.38 Hz), 115.85, 115.62, 115.62, 112.28, 110.88, 61.19, 56.14,
55.98.
(E)-4-(3-(3,4-dimethoxyphenyl) acrylamido)-1-(4-fluorobenzyl) pyri-
din-1-ium bromide (5l) Yield 91%; yellow solid; IR (KBr) m 3080,
3015, 1703, 1621, 1597, 1506, 1461, 1263, 1131, 1021,þ964, 844,
802, 786, 596 cmꢂ1; m.p. >250 ꢁC; ESI/MS m/z: 393.1 [M] ; H NMR
1
(400 MHz, DMSO-d6) d 11.69 (s, 1H), 8.91 (d, J ¼ 7.3 Hz, 2H), 8.19 (d,
J ¼ 7.0 Hz, 2H), 7.72 (d, J ¼ 15.6 Hz, 1H), 7.58 (dd, J ¼ 8.7, 5.4 Hz,
2H), 7.31 (d, J ¼ 8.9 Hz, 1H), 7.28 (dd, J ¼ 4.2, 2.5 Hz, 2H), 7.05 (d,
J ¼ 8.9 Hz, 1H), 6.83 (d, J ¼ 15.6 Hz, 1H), 5.69 (s, 2H), 3.82 (d,
J ¼ 3.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) d 166.17, 162.94 (d,
1JCF¼251.49 Hz), 152.86, 151.82, 149.45, 145.55, 145.55, 144.82,
131.60 (d, 3JCF¼8.08 Hz), 131.41, 127.19, 123.37, 118.11, 116.51 (d,
2JCF¼21.47 Hz), 115.60, 112.28, 110.87, 61.13, 56.15, 55.98.
Kinetic analysis of AChE inhibition
To obtain the mechanism of action 5l, reciprocal plots of 1/vel-
ocity versus 1/substrate were constructed at different concentra-
tions of the substrate thiocholine iodide (0.05–0.5 mM) by using
Ellman’s method35. Three concentrations of 5l were selected for
the studies: 30.0, 15.0 and 7.5 nM for the kinetic analysis of AChE
inhibition. The plots were assessed by a weighted least-squares
analysis that assumed the variance of velocity (v) to be a constant
(E)-1-(3-bromobenzyl)-4-(3-(3, 4-dimethoxyphenyl) acrylamido) pyr- percentage of v for the entire data set. Slopes of these reciprocal
plots were then plotted against the concentration of 5l in a
weighted analysis and Ki was determined as the intercept on the
negative x-axis. Data analysis was performed with Graph Pad
Prism 4.03 software (Graph Pad Software Inc., San Diego, CA).
idin-1-ium bromide (5m) Yield 92%; yellow solid; IR (KBr) m 2928,
2831, 1592, 1511, 1460, 1313 1137, 9þ90, 859, 762, 710 cmꢂ1; m.p.
>250 ꢁC; ESI/MS m/z: 453.0, 455.0 [M] ; 1H NMR (400 MHz, DMSO-
d6) d 11.72 (s, 1H), 8.93 (d, J ¼ 6.9 Hz, 2H), 8.21 (d, J ¼ 6.5 Hz, 2H),
7.80 (s, 1H), 7.74 (d, J ¼ 15.6 Hz, 1H), 7.64 (d, J ¼ 8.0 Hz, 1H), 7.51
(d, J ¼ 7.8 Hz, 1H), 7.42 (t, J ¼ 7.9 Hz, 1H), 7.29 (s, 2H), 7.06 (d,
J ¼ 8.5 Hz, 1H), 6.85 (d, J ¼ 15.6 Hz, 1H), 5.70 (s, 2H), 3.83 (d,
J ¼ 3.8 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) d 166.17, 152.96,
151.83, 149.45, 145.68, 144.90, 137.63, 132.51, 131.90, 131.81,
128.18, 127.17, 123.41, 122.70, 118.06, 115.68, 112.28, 110.87,
61.09, 56.15, 55.98.
Molecular modeling studies
Molecular modeling calculations and docking studies were per-
formed using Molecular Operating Environment (MOE) software
version 2008.10 (Chemical Computing Group, Montreal, Canada)43.
The X-ray crystallographic structures of AChE (PDB code 1EVE) and
Ab (1–42) (PDB code PDB 1IYT) were obtained from the Protein
Data Bank. All water molecules in PDB files were removed and
hydrogen atoms were subsequently added to the protein. The
compound 5l was built using the builder interface of the MOE
program and energy minimized using MMFF94x force field. Then
the 5l was docked into the active site of the protein by the
“Triangle Matcher” method, which generated poses by aligning
the ligand triplet of atoms with the triplet of alpha spheres in cav-
ities of tight atomic packing. The Dock scoring in MOE software
was done using ASE scoring function and force field was selected
as the refinement method. The best 10 poses of molecules were
retained and scored. After docking, the geometry of resulting
complex was studied using the MOE’s pose viewer utility.
(E)-1-(4-bromobenzyl)-4-(3-(3,4-dimethoxyphenyl) acrylamido) pyri-
din-1-ium bromide (5n) Yield 88%; yellow solid; IR (KBr) m 3012,
2948, 1681, 1618, 1508, 1460, 1268, 1130, 975, 841, 797 cmꢂ1; m.p.
>250 ꢁC; ESI/MS m/z: 453.0, 455.0 [M]þ; 1H NMR (400 MHz, DMSO-
d6) d 11.71 (s, 1H), 8.91 (d, J ¼ 7.3 Hz, 2H), 8.20 (d, J ¼ 6.9 Hz, 2H),
7.73 (d, J ¼ 15.6 Hz, 1H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.46 (d, J ¼ 8.4 Hz,
2H), 7.32–7.25 (m, 2H), 7.06 (d, J ¼ 8.9 Hz, 1H), 6.85 (d, J ¼ 15.7 Hz,
1H), 5.69 (s, 2H), 3.82 (d, J ¼ 3.7 Hz, 6H). 13C NMR (100 MHz,
DMSO-d6) d 166.17, 152.91, 151.83, 149.45, 145.66, 145.66, 144.87,
134.51, 132.57, 132.57, 131.32, 131.32, 127.18, 123.40, 123.05,
118.08, 115.62, 115.62, 112.28, 110.86, 61.18, 56.14, 55.98.
Biological activity
ABTS radical cation scavenging activity assay49
2,20-Azino-bis-2-ethybenz-thiazoline-6-sulfonic acid (ABTS) was dis-
In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (E.C. 3.1.1.7) from electric eel and human solved in purified water to a 7 mM concentration. ABTS radical cat-
erythrocytes, butyrylcholinesterase (BuChE, E.C. 3.1.1.8) from ion (ABTS.þ) was produced by reacting ABTS stock solution with
equine serum and human serum, 5, 50-dithiobis-(2-nitrobenzoic 2.45 mM potassium persulfate (final concentration) and allowing

780
J.-S. LAN ET AL
the mixture to stand in the dark at room temperature for at least Cell culture and measurement of cell viability50
18 h before use. The stock solution of ABTS was serially diluted
with sodium phosphate buffer (50 mM, pH 7.4) to 100 lM. Trolox
and 5a–n at different concentrations (total volume of 50 lL) were
added to 150 lL of 100 lM ABTS solution, respectively. After the
addition of either trolox or another antioxidant to the ABTS solu-
tion, complete mixing of reactants was achieved by bubbling
three to four times using plastic pipettes. The optical absorbance
of ABTS at 415 nm was measured at 6 min after addition and equi-
librated at 30 ꢁC. Each individual treatment was repeated for three
times and the results of the experiments were compared.
The toxicity effect of the tested compounds on the rat pheochro-
mocytoma (PC12) cells was examined according to previously
reported methods. PC 12 cells was obtained from the Cell Bank of
the Chinese Academy of Sciences (Shanghai, China) and routinely
grown at 37 ꢁC in a humidified incubator with 5% CO2 in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% bovine calf serum, 100 units per mL penicillin, and 100 units
per mL of streptomycin. Cells were sub-cultured in 96-well plates
at a seeding density of 5000 cells per well and allowed to adhere
and grow. When cells reached the required confluence, they were
placed into serum-free medium and treated with compound 5l.
Twenty-four hours later the survival of cells was determined by
MTT assay. Briefly, after incubation with 20 lL of MTT at 37 ꢁC for
4 h, living cells containing MTT formazan crystals were solubilized
in 200 lL DMSO. The absorbance of each well was measured using
a microculture plate reader with a test wavelength of 570 nm and
a reference wavelength of 630 nm. Results are expressed as the
mean SD of three independent experiments.
PC 12 cells were grown in RPMI-1640 medium containing 10%
(v/v) foetal bovine serum, 100 U penicillin/mL and 100 mg strepto-
mycin/mL under 5% CO2 at 37 ꢁC. The culture media were
changed every other day. After pretreatment with different con-
centrations of test compound (0, 6, 12.5, 25, 50 lM) for 2 h, PC 12
cells were incubated with 25 lM of Ab (1–42) for 24 h. The cell via-
bility was evaluated using MTT assay. Briefly, the cells were treated
with 10 lL MTT (5 mg/mL in PBS) for 4 h at 37 ꢁC. Then, 200 lL of
DMSO was added to dissolve the dark blue formazan crystals
formed in intact cells, and the absorbance at 570 nm was detected
by a microplate reader. PC12 cells were cultured without test com-
pound or Ab (1–42) as control group and the results were
expressed by percentage of control.
Inhibition of Ab (1–42) self-induced aggregation
Inhibition of self-induced Ab (1–42) aggregation was measured
using a Thioflavin T (ThT)-binding assay47. HFIP pretreated Ab
(1–42) samples (Anaspec Inc., Fremont, CA) were resolubilized with
a 50 mM phosphate buffer (pH 7.4) to give a 25 lM solution. Each
tested compound was firstly prepared in dimethyl sulfoxide
(DMSO) at a concentration of 10 mM and 1 lL of each was added
to the well of black, opaque Corning 96-well plates such that the
final solvent concentration was 10%. The final concentration of
each compound was 20 lM and was prepared in independent trip-
licates. The solvent control was also included. Then, 9 lL of 25 mM
Ab (1–42) sample was added to each well and the samples mixed
by gentle trapping. Plates were covered to minimize evaporation
and incubated in dark at room temperature for 46–48 h with no
agitation.
After the incubation period, 200 lL of 5 lM ThT in 50 mM
glycine–NaOH buffer (pH 8.0) was added to each well.
Fluorescence was measured on a SpectraMax M5 (Molecular
Devices, Sunnyvale, CA) multi-mode plate reader with excitation
and emission wavelengths at 446 nm and 490 nm, respectively.
The fluorescence intensities were compared and the percent inhib-
ition due to the presence of the inhibitor was calculated by the
following formula: 100 ꢂ (IFi/IFoꢄ100) where IFi and IFo are the
fluorescence intensities obtained for Ab (1–42) in the presence
and in the absence of inhibitor, respectively.
In vitro BBB permeation assay
Brain penetration of compounds was evaluated using a parallel
artificial membrane permeation assay (PAMPA) in a similar manner
as described by Di et al.51 Commercial drugs were purchased from
Sigma (St. Louis, MO) and Alfa Aesar (Haverhill, MA). The porcine
brain lipid (PBL) was obtained from Avanti Polar Lipids (Alabaster,
AL). The donor microplate (PVDF membrane, pore size 0.45 mm)
Metal-chelating study
The study of metal chelation was performed in methanol at 298 K
using UV–vis spectrophotometer (SHIMADZU UV-2450PC, Kyoto, and the acceptor microplate were both from Millipore (Darmstadt,
Germany). The 96-well UV plate (COSTAR@) was from Corning
Japan) with wavelength ranging from 200 to 500 nm. Due to the
low solubility in PBS, the compounds were tested in methanol.
The absorption spectra of compound 5l (50 lM, final concentra-
tion) alone or in the presence of CuSO4 and FeSO4 (100 lM, final
concentration) for 30 min in methanol were recorded 1 cm-quartz
cells. A fixed amount of compound 5l (50 lM) was mixed with
growing amounts of Cu2þ (10–80 lM) and UV spectra were
recorded. Variation of absorbance at 363 nm was used to monitor
Incorporated (Harrodsburg, KY). The acceptor 96-well microplate
was filled with 300 lL of PBS/EtOH (7:3), and the filter membrane
was impregnated with 4 lL of PBL in dodecane (20 mg/mL).
Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-
fold in PBS/EtOH (7:3) to achieve a concentration of 100 mg/mL,
200 lL of which was added to the donor wells. The acceptor filter
plate was carefully placed on the donor plate to form a sandwich,
which was left undisturbed for 16 h at 25 ꢁC. After incubation, the
donor plate was carefully removed and the concentration of com-
pound in the acceptor wells was determined using an UV plate
reader (Flexstation@ 3). Every sample was analyzed at five wave-
lengths, in four wells, in at least three independent runs, and the
results are given as the mean SD. In each experiment, nine qual-
ity control standards of known BBB permeability were included to
validate the analysis set.
the formation of 5l/Cu2þ complex44,45
.
Inhibition of Cu2þ-induced Ab (1–42) aggregation
For the inhibition of Cu2þ-induced Ab (1–42) aggregation experi-
ment, the Ab was diluted in 20 lM HEPES (pH 6.6) with 150 lM
NaCl. The mixture of the peptide (10 lL, 25 lM, final concentra-
tion) with or without copper (10 lL, 25 lM, final concentration)
and the test compound (10 lL, 50 lM, final concentration) were
incubated at 37 ꢁC for 24 h. The 20 lL of the sample was diluted
to a final volume of 200 lL with 50 lM glycine–NaOH buffer (pH Ex vivo brain penetration52
8.0) containing ThT (5 lM). The detection method was the same as
Fifteen male OF1 mice, weighing 25 g were used. Animals were
housed under controlled light (with a 12-h light/12-h dark cycle,
that of self-induced Ab aggregation experiment47.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
781
lights on at 7:00 a.m.) at 25 ꢁC and proper humidity. Rats were activity in brain of the drug-treated mice with activity from
given food and tap water ad libitum.
untreated controls.
Donepezil hydrochloride and compound 5l were dissolved 10%
DMSO. Animals were divided into three experimental handling
groups: mice administered with (i) control (10% DMSO, n ¼ 5); (ii)
donepezil (10 lmol/kg, n ¼ 5); (iii) compound 5l (10 lmol/kg,
n ¼ 5). Groups of 15 mice were treated i.p. with each compound.
The animals were sacrificed 10 min later and brains were quickly
removed and frozen on dry ice. Residual AChE activity was deter-
mined as previously described by the method of Ellman et al.41
using brain homogenate preparations as a source of the enzyme:
A homogenate of brain samples (10%, w/v) in 0.03 M sodium
phosphate buffer (pH 7.4) was prepared. The brain homogenate in
volume of 200 lL was mixed with 1% Triton X-100 and centrifuged
at 3000 rpm at 4 ꢁC for 10 min. Just before analysis of the enzym-
atic activity, an amount of 100 lL of homogenate was diluted 2.5
times in 0.1 M phosphate-buffered solution (pH 8.0). The reaction
took place in a final volume of 300 lL of 0.1 M phosphate-buffered
solution (pH 8.0) containing 100 lL of diluted homogenate and
333 lM DTNB solution. To avoid interferences between AChE and
BChE activities, 100 lM ISOOMPA (specific BChE inhibitor) was pre-
sent in the incubation medium. Prior to the addition of the sub-
strate acetylthiocholine, a preincubation period of 5 min was used
to eliminate the endogenous ACh present in the homogenates.
Result and discussion
Chemistry
As shown in Scheme 1, the target compounds 5a–n were synthe-
sized. First of all, the commercially available cinnamic acid deriva-
tives 1 were activated with 4-dimethylaminopyridine (DMAP)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI) at 0 ꢁC in dichloromethane solutions, and subsequently con-
densation with compound 2 at room temperature overnight
afforded the intermediates 3. Finally, the benzyl pyridinium brom-
ide salts 5 were efficiently obtained by refluxing proper benzyl
bromides 4 with the intermediates 3 in dry acetonitrile.
In vitro cholinesterase inhibitory activity and preliminary SAR
studies
The activities of compounds 5a–n and the relative compound cin-
namic acid against AChE (from electric eel) and BuChE (from
equine serum) were examined using the spectrophotometric
method of Ellman et al.41 Donepezil was used as a standard com-
The reaction was started by the addition of ATCI (450 lM) and the pound for comparison. From Table 1, the novel cinnamic acid
absorbance at 414 nm was evaluated 2 min after the substrate derivatives showed high activity towards AChE with IC50 values in
addition. Percent of inhibition was calculated by comparing AChE the nanomolar range, and high selectivity for AChE over BuChE,
O
O
N
NH2
i
OH
+
N
H
R1
N
R1
1
3
2
R1
H
N
ii
Br
Br
R2
+
R2
O
N
4
5
Scheme 1. Synthesis of cinnamic acid derivatives 5a–n. Reagents and conditions: (i) DMAP/EDCI, CH2Cl2, rt., 12 h; (ii) CH3CN, reflux, 1–3 h.
Table 1. Inhibition of ChEs activity and selectivity index of compounds 5a–n.
a
IC50
Compound
R1
R2
eeAChE (nM)
eqBuChE (lM)
Selectivity index (SI)b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
–
–
–
–
–
–
–
–
3-CH3
4-CH3
3-F
54.1 8.2
102.6 9.6
1263.8 1.2
93.5 7.7
90.8 9.2
153.6 2.6
1450.5 4.3
135.2 4.8
51.4 18
90.7 3.2
20.6 1.0
12.1 1.8
92.6 9.0
50.3 8.3
>100 000
40.2 3.6
6.3 0.6
8.1 1.3
25.0 0.7
7.4 0.5
10.0 0.4
2.5 0.3
2.1 0.8
4.5 0.7
2.7 0.8
3.8 0.9
3.1 0.5
2.6 1.6
2.5 0.6
1.9 0.5
>100
116.5
78.9
19.8
79.1
110.1
16.3
1.5
33.3
52.3
41.9
150.5
214.9
27.0
37.8
–
4-F
3-Br
4-Br
–
3-CH3
4-CH3
3-F
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
–
4-F
3-Br
4-Br
–
Cinnamic acid
Donepezil
–
–
4.5 0.2
112.5
aIC50: 50% inhibitory concentration (means SD of three experiments).
bSelectivity Index ¼ IC50 (eqBuChE)/IC50 (eeAChE).

782
J.-S. LAN ET AL
indicating that these derivatives are selective AChE inhibitors. Kinetic study of AChE inhibition
Among the cinnamic acid derivatives, compound 5l (IC50¼12.1 nM)
To explore the AChE inhibitory mechanism of action of the cin-
showed the most potent inhibitory activity against AChE, which
was 3.3-fold higher than that of donepezil (IC50¼40.2 nM).
Moreover, compound 5l exhibited the highest selectivity level
towards AChE over BuChE (SI ¼214.9). By contrast, compound 5n
exhibited the highest inhibitory activity against BuChE
(IC50¼1.9 lM), resulting 2.3-times more potent than that of done-
pezil (IC50¼4.5 lM). However, the AChE inhibitory activity of cin-
namic acid was remarkably low (IC50>100 lM), suggesting that the
N-benzyl pyridinium moiety is unavoidably required for higher
activity. The AChE inhibition by benzyl pyridinium bromide was
assayed to be in the micromolar range40, which was lower than
that of novel cinnamic acid derivatives. This finding suggests that
the cinnamic acid skeleton was also essential for AChE activity.
To improve the inhibitory activity of the compounds against
ChEs, we introduced substituents with different sizes and elec-
tronic properties on the benzene ring of cinnamic acid and on the
benzyl group of the N-benzyl pyridinium moiety were varied. The
IC50 value of compounds 5a–n indicated that the presence of
methoxy groups at the positions 3 and 4 of the N-benzyl pyridi-
nium moiety increased the ChE activity. For example, the ChE
inhibitory activity of compounds 5i–n was higher than that of
compounds 5b–g. Moreover, AChE inhibition was also affected by
the substituents on the benzyl group. Compared with
compound 5a, the introduction of methyl, fluorine and bromine
on the meta-, para-position reduced AChE inhibitory activity (com-
pounds 5b–g). For example, compound 5a (IC50¼54.1 nM for
AChE) was more potent than compound 5g (IC50¼1450.5 nM for
AChE) possessing 4-Br on the benzyl group. By contrast, compared
with compound 5h, compounds 5i–n with different substituents
on the benzyl group showed an increased AChE inhibitory activity.
For example, compound 5l bearing 4-F on the benzyl group
exhibited more potent AChE inhibition (11-fold) than did com-
pound 5h. Similar to compound 5a–n for BuChE inhibition, com-
pounds 5f (IC50¼2.5 lM), 5g (IC50¼2.1 lM), 5l (IC50¼2.5 lM) and
5m (IC50¼1.9 lM) that were characterized by a Br substituent
showed more potent inhibition. Therefore, we can rationally
deduce that the size of a substituent more crucially affects BuChE
inhibition than its electronic properties.
namic acid derivatives, the most potent inhibitor, compound 5l,
was selected for a kinetic study using Lineweaver–Burk plots39.
Graphical analysis (Figure 2) revealed both increasing slopes and
increasing intercepts with increasing inhibitor concentration.
According to this pattern, compound 5l is a mixed-type inhibitor
for AChE and might be able to bind to the CAS and the PAS
of AChE.
Molecular modeling study of AChE inhibition
To further study the dual-site mode of compound 5l for AChE, a
molecular docking study was performed using the software pack-
age MOE 2008.1042,43. The X-ray crystal structure of the TcAChE
complex with donepezil (hAChE, PDB code 1EVE) was applied to
establish the starting model of AChE. As shown in Figure 3, the
N-benzyl pyridinium moiety of compound 5l was bound to the
CAS of AChE, via aromatic p–p stacking interactions with the phe-
nyl ring from Trp 84 with the ring-to-ring distance of 3.88 Å and
the pyridine ring from Phe 330 with the ring-to-ring distance of
349 Å. Moreover, the charged nitrogen of the pyridine ring bound
to the CAS was via a cation–p interaction with Trp 84 and Phe
330. The cinnamic acid moiety occupied the PAS formed by Trp
279 and Gln 74. All these results obviously indicated that com-
pound 5l could simultaneously bind to the PAS and CAS of AChE.
Metal-chelating properties of compound 5l
The chelating ability of compound 5l with biometals such as Cu2þ
and Fe2þ was studied using UV–vis spectroscopy (Figure 4)44,45. As
shown in the UV–vis spectrum (Figure 4(A)), compound 5l without
metal ions showed the absorption maximum at 216 nm and a
shoulder at 353 nm. Upon the addition of CuSO4, a bathochromic
shift in the maximum absorption from 216 nm to 224 nm and in
the shoulder from 353 nm to 363 nm occurred, suggesting the for-
mation of the 5l–Cu2þ complex. When FeSO4 was added, a red
shift in the maximum absorption from 216 nm to 226 nm occurred,
suggesting that compound 5l coordinates with Fe2þ. The com-
plexation ability of compound 5l might be ascribed to the dime-
thoxy group on the cinnamic acid moiety and to the amide
Finally compounds 5i–n were selected for evaluation on human
AChE. As listed in Table 2, all tested compounds presented IC50
values in the nanomolar range and were slightly more potent
inhibitors for hAChE than for eeAChE. The SARs for hAChE were
similar to those drawn for eeAChE inhibition (Table 1). Compound
5l (IC50¼8.6 nM for hAChE) displayed the highest inhibition, which
was fourfold higher than that of standard donepezil
(IC50¼33.5 nM).
moiety of the compound46,33
.
To determine the stoichiometry of the 5l–Cu2þ complex, the
molar ratio method was used, for which solutions of compound 5l
with accumulative amounts of CuSO4 were prepared. The UV spec-
tra (Figure 4(B)) showed that the absorbance at 363 nm, related to
Table 2. Inhibition of ChEs activity and selectivity index of com-
pounds 5i–n.
a
IC50
Compounds
hAChE (nM)
hBuChE (lM)
Selectivity indexb
5i
5j
5k
5l
43.7 0.5
96.5 2.8
19.2 1.6
8.6 0.9
79.4 1.5
51.1 4.8
33.5 3.8
4.9 0.7
5.6 1.9
4.8 0.5
4.4 1.2
2.1 0.6
1.9 0.5
7.6 1.9
112.1
58.0
250.0
511.6
26.5
37.2
226.9
5m
5n
Donepezil
Figure 2. Kinetic study on the mechanism of EeAChE inhibition by compound 5l.
Overlaid Lineweaver–Burk reciprocal plots of AChE initial velocity at increasing
substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the
presence of compound 5l are shown. Lines were derived from a weighted least-
squares analysis of the data points.
aIC50
: 50% inhibitory concentration (means SD of three
experiments).
bSelectivity Index ¼ IC50 (hBuChE)/IC50 (hAChE).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
783
Figure 3. (A) 3D docking model of compound 5l with TcAChE. Atom colors: yellow – carbon atoms of 5l, gray – carbon atoms of residues of TcAChE, dark blue –
nitrogen atoms, red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) 2D schematic diagram of docking model of
compound 5l with TcAChE. The figure was prepared using the ligand interactions application in MOE.
the formation of the Cu–5l complex, initially increased at increas- (65.6%, 64.7% and 66.3%, respectively, at 20 lM) showed the high-
ing concentrations of CuCl2 and then plateaued, and the two
straight lines intersected at a mole fraction of 1.02. Thus a 1:1 stoi-
chiometry was hypothesised for the 5l–Cu2þ complex.
est potency. Interestingly, compounds 5h–n with the dimethoxy
group on the cinnamic acid moiety exhibited high inhibition
against self-induced Ab (1–42) aggregation with inhibition ranging
from 53.9 to 66.3% at 20 lM. For example, compound 5l (64.7% at
20 lM) was more potent than that of compound 5e (52.8% at
20 lM). This finding led to the hypothesis that the dimethoxy
group might favour Ab aggregation inhibition. However, substitu-
ents on the benzyl group (see compounds 5i–n) did not seem to
play a role in the inhibition of Ab (1–42) self-aggregation.
As compound 5l showed good inhibitory activity against Ab
(1–42) self-aggregation and favourable chelating properties, its
ability to inhibit Cu2þ-induced Ab (1–42) aggregation was investi-
gated by a ThT-binding assay48. Clioquinol was employed as a ref-
Inhibition of self-induced and Cu2þ-induced Ab (1–42)
self-induced aggregation
All compounds tested for ChEs inhibition were also evaluated by a
ThT-based fluorometric assay for their ability to inhibit self-induced
Ab (1–42) aggregation47. Curcumin was used as a reference,
because of its known inhibitory activity against Ab (1–42) self-
aggregation. The results are gathered in Table 3. Compounds
5a–n exhibited good potencies (46.3–65.6% at 20 lM) compared
with Cur (54.6% at 20 lM). Notably, compounds 5k, 5l and 5n erence compound. As shown in Figure 5, the fluorescence of Ab

784
J.-S. LAN ET AL
treated with Cu2þ is 160.3% that of Ab alone, which points out CQ, 60.2% inhibition). These results indicated that our compound
that Cu2þ hastens Ab aggregation. In comparison, the fluorescence could inhibit Cu2þ-induced Ab aggregation by effectively
of Ab treated with Cu2þ and the tested compound decreased dra- chelating Cu2þ
matically (5l, 68.6% inhibition of Cu2þ-induced Ab aggregation;
.
Docking study of compound 5l with Ab (1–42) peptide
To further study the interaction mode with compound 5l for Ab
(1–42), a molecular docking study was performed using the soft-
ware package MOE 2008.1043. The X-ray crystal structure of the
protein Ab structure (PDB 1IYT) from the Protein Data Bank was
used. As revealed in Figure 6, the benzene ring of cinnamic acid
interacted with the His 6 via a p–p stacking interaction. A hydro-
gen bond interaction was also observed between the acid amides
group of compound 5l and Glu 3. These results indicated that the
p–p stacking and the hydrogen bond interactions played crucial
roles in the stability of the 5l/Ab (1–42) complex.
Figure 5. Inhibition of Cu2þ-induced Ab (1–42) aggregation by compound 5l
comparing with that of clioquinol (CQ) ([Ab ¼ 25 lM, [5l] ¼ 50 lM, [CQ] ¼ 50 lM,
[Cu2þ] ¼ 25 lM, 37 ꢁC, 24 h). Values are reported as the mean SD of three inde-
Figure 4. (A) UV absorbance spectrum of 5l (50lM) alone (red) or in the pres-
ence of 100 lM CuSO4 (green) and 100lM FeSO4 (yellow) in MeOH. (B)
Determination of the stoichiometry of complex 5lꢂCu2þ by molar ratio method.
ꢀꢀ
pendent experiments. #p < .05, p < .01.
Table 3. Inhibition of Ab (1–42) self-induced aggregation and ABTS radical by target compounds.
Ab (1–42) aggregation
ABTS assay
Compound
R1
R2
inhibition (%)a
(trolox equiv)b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
Curcumin
–
–
–
–
–
–
–
–
3-CH3
4-CH3
3-F
46.3 1.6
48.5 1.8
55.1 1.5
53.6 2.3
52.8 1.8
48.8 2.1
57.2 1.1
55.7 3.2
53.9 0.9
63.4 2.8
65.6 2.3
64.7 1.6
62.8 1.4
66.3 2.9
54.6 2.6
Nc
N
N
N
N
N
N
N
N
N
N
N
N
N
1.65
4-F
3-Br
4-Br
–
3-CH3
4-CH3
3-F
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
3,4-di-OCH3
–
4-F
3-Br
4-Br
–
aInhibition of Ab (1–42) self-induced aggregation, the thioflavin-T fluorescence method was used, the mean SD
of at least three independent experiments and the measurements were carried out in the presence of 20 lM
compounds.
bData are expressed as (mmol trolox)/(mmol tested compound).
cN means <0.05.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
785
Figure 6. Docking study of compound 5l with Ab (1–42) generated with MOE. Atom colors: yellow – carbon atoms of 5l, gray – carbon atoms of residues of Ab
(1–42), dark blue – nitrogen atoms, red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand.
Figure 7. (A) Effects of compound 5l on cell viability in PC12 cells. The cell viability was determined by the MTT assay after 24 h of incubation with various concentra-
tions. The results were expressed as a percentage of control cells. Values are reported as the mean SD of three independent experiments. (B) Neuroprotection against
Ab (1–42) toxicity. Compound 5l was tested for neuroprotective activity against Ab (1–42) toxicity in PC12 cells. Data represent the mean SD of three observations.
ꢀ
ꢀꢀ
p < .05 and p < .01 compared to the Ab (1–42)-treated control group.
Evaluation of compounds for antioxidant activity
compound 5l against Ab-induced neuronal death of PC12 cells,
the data were recorded after the cells were exposed to increasing
concentrations of compound 5l (6, 12.5, 25 and 50 lM) for 24 h. As
can be seen in Figure 7(B), treatment of cells with Ab (1–42)
(25 lM) to the growth medium markedly reduced cell viability to
44.2% compared with the untreated cells (control). Compound 5l
exhibited neuroprotective effects at concentrations ranging from 6
to 50 lM (6 lM: 47.6 4.2%; 12.5 lM: 57.5 1.4%; 25 lM:
63.2 3.5%; 50 lM: 66.6 2.5%). These observations further
showed that novel cinnamic acid derivatives bearing the N-benzyl
pyridinium moiety can inhibit Ab (1–42) self-aggregation for the
treatment of AD.
The target compounds were evaluated for their antioxidant effi-
cacy by using ABTS (ferric reducing antioxidant power) assays49
.
Compared with Cur, results showed that tested compounds exhib-
ited no antioxidant ability (Table 3). This finding might be attrib-
uted to the absence of the hydroxy group of cinnamic acid
moiety.
Cytotoxicity of compound 5l in PC12 cells and neuroprotection
against Ab (1–42)-induced toxicity
On the basis of the aforementioned screening results, the poten-
tial toxicity effect of compound 5l in PC12 cells was studied50.
After incubating the cells with compound 5l for 24 h, the cell via-
bility was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium (MTT) assay. As shown in Figure 7(A), the result
revealed that compound 5l at 3–50 lŒ did not significantly affect
cell viability, indicating that compound 5l was nontoxic to neuro-
blastoma PC12 cells.
In vitro BBB permeation assay
For successful central nervous system (CNS) drugs, the first
requirement is crossing the BBB to reach brain. The potential abil-
ity of these compounds to penetrate into the brain was evaluated
using a PAMPA as described by Di et al.51 Assay validation was
completed by comparing the experimental permeabilities of nine
commercial drugs with previously reported values (Table 4)51. A
In the pathology of AD, Ab-induced neuronal cell death is a
serious event. To evaluate the neuroprotective effects of plot of the experimental data versus bibliographic values gave a

786
J.-S. LAN ET AL
Table 4. Permeability (Peꢄ10ꢂ6 cm sꢂ1) in the
deemed as a multifunctional cholinesterase inhibitor and serve as
a novel lead compound for treating AD.
PAMPA-BBB assay for nine commercial drugs, used
in the experiment validation.
PBS:EtOH
Commercial drugs
Bibla
(70:30)b
Disclosure statement
Testosterone
Verapamil
beta-Estradiol
Progesterone
Corticosterone
Piroxicam
Hydrocortisone
Ofloxacin
Dopamine
17
16
12
9.3
5.1
2.5
1.9
0.8
0.2
16.44
16.88
11.93
5.34
4.06
1.42
1.86
0.47
0.17
The authors declare no conflicts of interest.
Funding
National Natural Science Foundation of China, 10.13039/
501100001809 [No.81173561, 81274200, 81403175] Youth Talent
Sail Plan from Shanghai Committee of Science and Technology
[No. 14YF1411300] Project from Shanghai Committee of Science
and Technology [No. 13401900301]
aTaken from Ref.49
bData are the mean SD of three independent
experiments.
good linear correlation, Pe (exp.) ¼ 1.06 Pe (bibl.) ꢂ 1.69 (R2¼.94).
From this equation and taking into account the limit established
by Di et al. for blood–brain barrier permeation, we classified the
compounds as follows:
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