Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as cholinesterase inhibitors against Alzheimer’s disease

Article abstract of DOI:10.1080/14756366.2017.1412314

Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat Alzheimer’s disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great importance. Herein, we report the synthesis, structure–activity relationship study and biological evaluation of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase, are evaluated for the amyloid β-protein self-aggregation inhibition and in vivo assays. The optimal compound 19, 27, and 30 (human AChE IC₅₀ = 10.2 ± 1.2, 16.5 ± 1.7, and 15.3 ± 1.8 nM, respectively) show good performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hepatotoxicity evaluation. These compounds deserve further evaluation for the development of new therapeutic agents against AD.

Full text of DOI:10.1080/14756366.2017.1412314

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and bioevaluation of new tacrine-
cinnamic acid hybrids as cholinesterase inhibitors
against Alzheimer’s disease
Yao Chen, Jie Zhu, Jun Mo, Hongyu Yang, Xueyang Jiang, Hongzhi Lin, Kai Gu,
Yuqiong Pei, Liang Wu, Renxiang Tan, Jing Hou, Jingyi Chen, Yang Lv, Yaoyao
Bian & Haopeng Sun
To cite this article: Yao Chen, Jie Zhu, Jun Mo, Hongyu Yang, Xueyang Jiang, Hongzhi Lin,
Kai Gu, Yuqiong Pei, Liang Wu, Renxiang Tan, Jing Hou, Jingyi Chen, Yang Lv, Yaoyao Bian
&
Haopeng Sun (2018) Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as
cholinesterase inhibitors against Alzheimer’s disease, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 290-302, DOI: 10.1080/14756366.2017.1412314
To link to this article: https://doi.org/10.1080/14756366.2017.1412314
©
2017 The Author(s). Published by Informa
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Date: 28 December 2017, At: 06:04

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 290–302
https://doi.org/10.1080/14756366.2017.1412314
RESEARCH PAPER
Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as
cholinesterase inhibitors against Alzheimer’s disease
a,b,c
d
d
d
e
d
d
a
a
Yao Chen , Jie Zhu , Jun Mo , Hongyu Yang , Xueyang Jiang , Hongzhi Lin , Kai Gu , Yuqiong Pei , Liang Wu ,
e
c
f
a
f
d
Renxiang Tan , Jing Hou , Jingyi Chen , Yang Lv , Yaoyao Bian and Haopeng Sun
a
b
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu Collaborative Innovation Center of Chinese Medicinal
c
Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, China; State Key Laboratory Cultivation Base for TCM Quality and
Efficacy, Nanjing University of Chinese Medicine, Nanjing, China; Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing,
China; Key Laboratory of Biomedical Functional Materials, School of Science, China Pharmaceutical University, Nanjing, China; School of
Nursing, Nanjing University of Chinese Medicine, Nanjing, China
d
e
f
ABSTRACT
ARTICLE HISTORY
Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat
Alzheimer’s disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great
importance. Herein, we report the synthesis, structure–activity relationship study and biological evaluation
of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in
vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase,
are evaluated for the amyloid b-protein self-aggregation inhibition and in vivo assays. The optimal com-
pound 19, 27, and 30 (human AChE IC50 ¼ 10.2 ± 1.2, 16.5 ± 1.7, and 15.3 ± 1.8 nM, respectively) show good
performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hep-
atotoxicity evaluation. These compounds deserve further evaluation for the development of new thera-
peutic agents against AD.
Received 18 July 2017
Revised 21 November 2017
Accepted 29 November 2017
KEYWORDS
Cholinesterase inhibitor;
multi-target ligand; tacrine
hybrid; cinnamic acid
Introduction
acetylcholinesterase inhibitors (AChEIs) can efficiently prevent the
2
0
hydrolysis of ACh and provide promising therapeutic effects . In
recent decades, the function of BuChE in the development of AD
is elucidated. In advanced AD, the level of AChE drops down to
90% compared to the healthy brain, leading to the loss of function
Alzheimer’s disease (AD) is one of the most prevalent forms of
late-life mental failure in humans. About 6% of the population
aged over 65 is affected by this disease , and it is estimated that
1
2
7
0 million people will suffer from AD by 2050 . Therefore, discov-
2
1
of AChEIs . While BuChE maintains the normal level or even up-
regulated as a compensatory feedback for the metabolism of ACh.
Inhibition of BuChE constitutes a promising target for clinical use,
ery of efficient anti-AD agents is of great importance for drug
3
,4
developers . So far, the mechanism for AD is still not clearly elu-
cidated, but it is well-accepted that AD is a multifactorial syn-
2
2
5
especially for progressed AD . Therefore, inhibitors of both AChE
and BuChE are expected to exert a potent therapeutic effect on
AD. Unfortunately, current ChEs inhibitors in clinical use, such as
drome deriving from a complex array of neurochemical factors .
Several hypotheses about AD pathogenesis are presented, such as
6
7
cholinergic dysfunction , amyloid cascade , hyperphosphorylation
2
3
8
9
donepezil and rivastigmine, only enable a palliative treatment .
Considering the multifactorial nature of AD, designing multi-tar-
get-directed ligands (MTDLs) that can simultaneously regulate
multiple targets in the development of AD, has emerged as a new
of s-protein , cell cycle hypothesis , and brain-derived neuro-
1
0
11
trophic factor hypothesis , oxidative stress , free radical forma-
1
2
13
14
tion , metal dyshomeostasis , and mitochondrial dysfunction .
These findings not only inspire the design of new anti-AD agents
with diverse mechanisms, but also depict a more complex AD
scenario.
2
4–26
strategy
, and many of MTDLs have been proved to show
2
7–31
promising pharmacological effects on AD
.
The enzymatic site of human AChE (hAChE) and BuChE
hBuChE) contains two binding sites: the catalytic active site (CAS)
So far, designing drugs targeting the cholinergic system is still
the most successful therapeutic strategy against AD. Many studies
(
have shown that the decline of acetylcholine (ACh) level results in at the bottom and the peripheral anionic site (PAS) near the
1
5–17
32,33
the cognitive and memory deficits
. Therefore, recovering cho- entrance of the gorge
. The two proteins share an almost 65%
linergic function by inhibiting cholinesterases (ChEs), which are in homologic amino acid sequences. Catalytic triads in CAS of hAChE
charge of the hydrolysis of ACh, is beneficial for the treatment of and hBuChE consist of conserved amino acids: Ser203, His447,
1
8,19
34
AD
AChE) and butyrylcholinesterase (BuChE). Under normal condition, hAChE is proved to be closely related to both hydrolysis of ACh
AChE is a dominant factor for ACh metabolism (80%), therefore, and neurotoxic cascade of AD through AChE-induced b-amyloid
. There are two types of ChEs, namely, acetylcholinesterase Glu334 in hAChE and Ser198, His438, Glu325 in hBuChE . PAS of
(
CONTACT Haopeng Sun
691@163.com
sunhaopeng@163.com
School of Nursing, Nanjing University of Chinese Medicine, Nanjing, China
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, China; Yaoyao Bian
1
Supplemental data for this article can be accessed here.
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,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
291
3
5
(
Ab) aggregation . As a result, designing MTDLs targeting both 1H), 7.35–7.31 (m, 4H), 6.36 (d, J ¼ 15.6 Hz, 1H), 6.02 (s, 1H), 3.97 (s,
CAS and PAS attracts the attentions of medicinal chemists 1H), 3.49 (d, J ¼ 5.4 Hz, 2H), 3.44–3.38 (m, 2H), 3.04 (s, 2H), 2.69 (s,
1
3
throughout the world.
Herein, we report our efforts in the design, synthesis and bioe- CDCl
valuation of a series of tacrine-cinnamic acid hybrids as acetyl- 128.81, 128.65, 128.37, 127.76, 123.80, 122.75, 120.80, 120.37,
2H), 2.03 (s, 2H), 1.93–1.85 (m, 4H), 1.68 (s, 2H). C NMR (500 MHz,
): d 166.20, 158.59, 150.56, 147.44, 140.90, 134.86, 129.65,
3
3
6,37
and BuChE inhibitors against AD
synthesised and evaluated for their ChEs inhibitory activities. The (ESI) m/z calcd for C26
structure-activity relationship (SAR) of these compounds is sum- HPLC (70% methanol in water with 0.5% H
marised and discussed. To avoid cytotoxicity, the antiproliferative 99.02%.
. The target compounds are 116.33, 48.94, 39.36, 34.01, 29.07, 27.24, 24.88, 23.04, 22.76. HRMS
þ
H
29
N
3
O [M þ H] 400.2380; found 400.2380.
3
PO
4
): t
R
¼ 3.92 min,
activity of the compounds are evaluated in PC-12 cells. Finally, the
N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)cinnamamide (12).
in vivo behavioural and hepatotoxic evaluations for the optimal Yellow powder, yield: 52%, mp 53–55 C. H NMR (300 MHz,
ꢁ
1
compounds are performed. Information gathering from these CDCl ): d 7.99 (d, J ¼ 9.3 Hz, 1H), 7.94 (d, J ¼ 8.1 Hz, 1H), 7.60–7.53
3
experiments will guide our further optimisation of new MTDLs (m, 2H), 7.49–7.41 (m, 2H), 7.36 (t, J ¼ 7.1 Hz, 1H), 7.04 (t, J ¼ 8.6 Hz,
against AD.
2H), 6.35 (d, J ¼ 15.6 Hz, 1H), 6.07 (s, 1H), 3.53 (t, J ¼ 7.2 Hz, 2H),
3
1
.40–3.34 (m, 2H), 3.07 (s, 2H), 2.70 (s, 2H), 1.96–1.84 (m, 4H),
1
3
.74–1.63 (m, 2H), 1.60–1.49 (m, 2H), 1.47–1.37 (m, 4H). C NMR
): d 166.06, 158.30, 150.93, 147.22, 140.78, 134.93,
Experimental sections
Chemistry
(
500 MHz, CDCl
3
1
1
2
4
0
29.60, 128.80, 128.49,128.42, 127.76, 123.71, 122.91, 120.90,
20.16, 115.83, 49.30, 39.55, 33.85, 31.63, 29.63, 26.65, 26.57, 24.82,
General experimental
þ
3.04, 22.74. HRMS (ESI) m/z calcd for C H N O [M þ H]
2
8 33 3
1
13
H-NMR and
C-NMR spectra were determined by a Bruker
28.2694; found 428.2694. HPLC (70% methanol in water with
Avance 300 MHz spectrometer at 300 K, with TMS as an internal
standard. MS spectra were determined on a Mariner Mass spec-
trum (ESI) or a LC/MSD TOF HR-MS spectrum. Melting points were
obtained using a Mel-TEMP II melting point apparatus. Thin-layer
chromatography was carried out on silica gel GF/UV 254 sup-
ported by glass plate, and the chromatograms were performed on
silica gel (200–300 mesh) visualised under UV light at 254 and
.5% H
3
PO
4
): t
R
¼ 4.97 min, 96.68%.
N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)cinnamamide (13).
ꢁ
1
Yellow powder, yield: 30%, mp 50–52 C. H NMR (300 MHz,
CDCl
): d 7.98 (d, J ¼ 6.0 Hz, 1H), 7.95 (d, J ¼ 5.7 Hz, 1H), 7.60 (d,
3
J ¼ 15.7 Hz, 1H), 7.57–7.50 (m, 1H), 7.47–7.44 (m, 2H), 7.40–7.25 (m,
5
3
H), 6.46 (d, J ¼ 15.6 Hz, 1H), 6.23 (s, 1H), 3.51 (t, J ¼ 7.0 Hz, 2H),
.38–3.31(m, 2H), 3.07 (s, 2H), 2.68 (s, 2H), 1.89 (s, 4H), 1.71–1.59
3
65 nm. Purity for final compounds was measured by HPLC with
13
(m, 2H), 1.56–1.47 (m, 2H), 1.29 (s, 8H). C NMR (500 MHz, CDCl3):
Agilent Technologies 1260 infinity C18 4.60 mm ꢀ150 mm column
d 166.07, 157.62, 151.46, 146.48, 140.55, 135.01, 129.51, 128.80,
using a mixture of solvent methanol/water at the flow rate of
1
4
2
4
0
28.77, 127.74, 127.71, 123.79, 123.10, 121.13, 119.73, 115.25,
0
.5 ml/min and peak detection at 254 nm under UV. All solvents
9.31, 39.72, 33.36, 31.65, 31.28, 29.61, 29.10, 26.79, 26.74, 24.69,
were reagent grade without special instruction. When necessary,
solvents were purified and dried. The vacuum distillation was per-
formed by using a rotary evaporator at a reduced pressure of ca.
þ
2.94, 22.56. HRMS (ESI) m/z calcd for C30
H
37
N
3
O [M þ H]
56.3006; found 456.3006. HPLC (70% methanol in water with
.5% H PO ): t ¼ 7.73 min, 98.24%.
3
4
R
2
0 Torr. Organic solutions were dried over anhydrous sodium
sulphate.
N-(2-((1,2,3,4-tetrahydroanthracen-9-yl)amino)ethyl)cinnamamide (9).
(
E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(o-tolyl)acry-
ꢁ
1
lamide (14). Yellow powder, yield: 33%, mp 55–57 C. H NMR
300 MHz, CDCl
ꢁ
ꢁ
44
1
(
3
): d 8.00 (t, J ¼ 7.6 Hz, 2H), 7.92 (d, J ¼ 15.5 Hz, 1H),
Yellow powder, yield: 39%, mp 55–57 C. 86–88 C . H NMR
300 MHz, CDCl ): d 7.96 (d, J ¼ 8.5 Hz, 1H), 7.88 (d, J ¼ 8.4 Hz, 1H),
7
1
.57 (t, J ¼ 7.5 Hz, 1H), 7.50 (d, J ¼ 7.6 Hz, 1H), 7.37 (t, J ¼ 7.6 Hz,
(
3
H), 7.24 (d, J ¼ 7.0 Hz, 1H), 7.19 (d, J ¼ 7.6 Hz, 2H), 6.33 (d,
7
.66 (d, J ¼ 15.6 Hz, 1H), 7.55–7.44 (m, 3H), 7.37–7.34 (m, 3H), 7.30
J ¼ 15.4 Hz, 1H), 5.94 (s, 1H), 3.57 (t, J ¼ 7.1 Hz, 2H), 3.42–3.36 (m,
(
d, J ¼ 7.1 Hz, 1H), 6.44 (s, 1H), 6.39 (s, 1H), 3.70 (s, 4H), 3.01 (s, 2H),
1
3
2H), 3.10 (s, 2H), 2.70 (s, 2H), 2.43 (s, 3H), 1.92 (s, 4H), 1.75–1.66
2
1
1
3
3
.70 (s, 2H), 1.86 (s, 4H). C NMR (500 MHz, CDCl ): d 167.61,
1
3
(
m, 2H), 1.63–1.56 (m, 2H), 1.45 (s, 4H). C NMR (500 MHz, CDCl
d 166.20, 157.33, 151.58, 146.06, 138.52, 137.43, 133.97, 130.70,
29.32, 128.96, 127.32, 126.15, 126.09, 123.88, 123.11, 122.10,
3
):
57.94, 151.06, 146.58, 141.52, 134.68, 129.85, 128.87, 128.76,
27.85, 127.67, 123.86, 122.85, 120.37, 119.73, 115.85, 49.85, 40.68,
3.43, 25.01, 22.95, 22.54. HRMS (ESI) m/z calcd for C H N O
1
2
4 25 3
þ
119.57, 115.14, 49.11, 39.51, 33.05, 31.50, 29.58, 26.59, 26.49, 24.68,
[
M þ H] 372.2065; found 372.2065. HPLC (70% methanol in water
with 0.5% H PO ): t
¼ 3.95 min, 97.85%.
N-(3-((1,2,3,4-tetrahydroanthracen-9-yl)amino)propyl)cinnama-
þ
2
4
2.88, 22.45, 19.82. HRMS (ESI) m/z calcd for C H N O [M þ H]
2
9 35 3
3
4
R
42.286; found 442.2862. HPLC (70% methanol in water with 0.5%
ꢁ
ꢁ
44
H PO ): t ¼ 6.62 min, 95.09%.
3
4
R
mide (10). Yellow powder, yield: 43%, mp 58–60 C. 130–132 C .
1
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(m-tolyl)a-
H NMR (300 MHz, CDCl
3
): d 8.12 (d, J ¼ 8.5 Hz, 1H), 8.01 (d,
ꢁ
1
crylamide (15). Yellow powder, yield: 27%, mp 53–55 C. H NMR
300 MHz, CDCl
J ¼ 8.4 Hz, 1H), 7.67 (d, J ¼ 15.7 Hz, 1H), 7.60–7.45 (m, 3H),
(
7
2
3
): d 7.95 (t, J ¼ 9.1 Hz, 2H), 7.59 (d, J ¼ 11.7 Hz, 1H),
.54 (d, J ¼ 10.1 Hz, 1H), 7.36 (d, J ¼ 7.8 Hz, 1H), 7.30 (d, J ¼ 8.6 Hz,
H), 7.23 (d, J ¼ 8.1 Hz, 1H), 7.15 (d, J ¼ 7.0 Hz, 1H), 6.37 (d,
7
3
6
1
1
2
3
0
.43–7.30 (m, 4H), 6.99 (s, 1H), 6.57 (d, J ¼ 15.7 Hz, 1H), 5.75 (s, 1H),
.66 (s, 2H), 3.59–3.46 (m, 2H), 3.08 (s, 2H), 2.77 (s, 2H), 1.89 (s,
1
3
3
H). C NMR (500 MHz, CDCl ): d 167.27, 156.36, 152.20, 144.83,
J ¼ 15.6 Hz, 1H), 5.77 (s, 1H), 3.50 (t, J ¼ 7.1 Hz, 2H), 3.34–3.33 (m,
41.03, 134.83, 129.69, 129.45, 128.80, 127.83, 126.03, 124.22,
23.19, 120.79, 119.13, 115.02, 44.93, 36.62, 32.37, 31.37, 24.95,
2.81, 22.18. HRMS (ESI) m/z calcd for C25
86.2223; found 386.2223. HPLC (70% methanol in water with
2
H), 3.07 (s, 2H), 2.70 (s, 2H), 2.35 (s, 3H), 1.91 (s, 4H), 1.73–1.62
1
3
þ
(
m, 2H), 1.60–1.52 (m, 2H), 1.40 (s, 4H). C NMR (500 MHz, CDCl
d 166.18, 157.95, 151.15, 140.87, 138.41, 134.88, 130.41, 128.67,
28.62, 128.49, 128.02, 124.88, 123.75, 122.98, 120.74, 119.94,
3
):
H N
27 3
O [M þ H]
1
.5% H PO ): t ¼ 4.04 min, 98.27%.
3
4
R
N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)cinnamamide (11). 115.55, 49.22, 39.52, 33.54, 31.59, 29.62, 26.62, 26.54, 24.77, 22.98,
ꢁ
ꢁ
29 1
þ
Yellow powder, yield: 36%, mp 73–75 C. 155–157 C . H NMR 22.63, 21.33. HRMS (ESI) m/z calcd for C29
300 MHz, CDCl
): d 7.91 (t, J ¼ 9.1 Hz, 2H), 7.62 (d, J ¼ 15.6 Hz, 1H), 442.285; found 442.2858. HPLC (70% methanol in water with 0.5%
.54 (t, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 3.6 Hz, 1H), 7.45 (d, J ¼ 2.1 Hz,
PO ): t
¼ 7.35 min, 95.17%.
H
35
N
3
O [M þ H]
(
7
3
H
3
4
R

2
92
Y. CHEN ET AL.
(
E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(p-tolyl) 49.23, 39.66, 33.76, 31.59, 29.48, 26.61, 26.51, 24.79, 23.01, 22.70.
ꢁ
1
þ
acrylamide (16). Yellow powder, yield: 33%, mp 52–53 C. H NMR HRMS (ESI) m/z calcd for C28
300 MHz, CDCl ): d 7.96–7.89 (m, 2H), 7.63–7.50 (m, 2H), 7.41–7.30 507.2165. HPLC (70% methanol in water with 0.5% H PO ):
H
31ClN
4
O
3
[M þ H] 507.216; found
(
3
3
4
(
m, 3H), 7.16 (d, J ¼ 8.0 Hz, 2H), 6.32 (d, J ¼ 15.6 Hz, 1H), 5.77 (s, t ¼ 6.78 min, 95.01%.
R
1
2
1
1
1
4
2
H), 3.47 (t, J ¼ 7.2 Hz, 2H), 3.39–3.33 (m, 2H), 3.06 (s, 2H), 2.71 (s,
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3–(2-(trifluoro-
H), 2.35 (s, 3H), 1.91 (t, J ¼ 3.2 Hz, 4H), 1.71–1.61 (m, 2H), methyl)phenyl)acrylamide (21). Yellow powder, yield: 43%, mp
1
3
ꢁ
1
.60–1.50 (m, 2H), 1.46–1.34 (m, 4H). C NMR (500 MHz, CDCl ): d 57–60 C. H NMR (300 MHz, CDCl ): d 8.00–7.91 (m, 3H), 7.69–7.61
3
3
66.26, 158.50, 150.79, 147.47, 140.73, 139.85, 132.16, 129.52, (m, 2H), 7.59–7.47 (m, 2H), 7.42 (t, J ¼ 7.4 Hz, 1H), 7.35 (t, J ¼ 7.5 Hz,
28.66, 128.33, 127.73, 123.65, 122.87, 120.28, 119.85, 115.97, 1H), 6.39 (d, J ¼ 15.4 Hz, 1H), 6.02 (s, 1H), 3.53 (t, J ¼ 7.0 Hz, 2H),
9.34, 39.53, 34.01, 31.66, 29.66, 26.68, 26.60, 24.85, 23.08, 22.80, 3.40–3.34 (m, 2H), 3.07 (s, 2H), 2.70 (s, 2H), 1.90 (s, 4H), 1.70–1.64
þ
13
1.40. HRMS (ESI) m/z calcd for C29
H
35
N
3
O [M þ H] 442.2853; (m, 2H), 1.61–1.51 (m, 2H), 1.41 (s, 4H). C NMR (500 MHz, CDCl
3
):
found 442.2860. HPLC (70% methanol in water with 0.5% H
3
PO
4
): d 165.35, 156.95, 151.78, 145.68, 136.09, 134.12, 131.95, 129.11,
t ¼ 6.42 min, 95.67%.
128.89, 128.47, 127.82, 126.94, 126.06, 126.02, 125.64, 123.93,
123.21, 119.36, 114.94, 48.96, 39.51, 32.86, 31.40, 29.46, 26.49,
R
(
E)-3-(2-nitrophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
1
hexyl)acrylamide (17). Yellow powder, yield: 23%, mp 62–63 C. H 26.37, 24.64, 22.82, 22.35. HRMS (ESI) m/z calcd for C29
H
32
F
3
N
3
O
þ
NMR (300 MHz, CDCl
3
): d 8.05–7.97 (m, 2H), 7.93 (t, J ¼ 9.4 Hz, 2H), [M þ H] 496.258; found 496.2580. HPLC (70% methanol in water
7
.66–7.56 (m, 2H), 7.55–7.48 (m, 2H), 7.35 (t, J ¼ 7.4 Hz, 1H), 6.33 with 0.5% H PO ): t ¼ 7.58 min, 95.34%.
3
4
R
(d, J ¼ 15.6 Hz, 1H), 6.03 (s, 1H), 3.50 (t, J ¼ 7.1 Hz, 2H), 3.41–3.34
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3–(3-(trifluoro-
(m, 2H), 3.06 (s, 2H), 2.72 (s, 2H), 1.93 (s, 4H), 1.75–1.64 (m, 2H), methyl)phenyl)acrylamide (22). Yellow powder, yield: 38%, mp
1
3
ꢁ
C NMR (500 MHz, CDCl ): d 48–54 C. H NMR (300 MHz, DMSO): d 8.13 (s, 1H), 8.10 (d,
3
1
1
1
1
1
2
4
0
.62–1.52 (m, 2H), 1.42 (s, 4H).
65.02, 158.37, 150.87, 148.32, 147.28, 135.74, 133.33, 131.12, J ¼ 5.1 Hz, 1H), 7.91–7.83 (m, 1H), 7.78–7.68 (m, 2H), 7.68–7.59 (m,
29.71, 129.10, 128.48, 128.40, 126.33, 124.81, 123.68, 122.89, 1H), 7.53–7.46 (m, 2H), 7.34 (t, J ¼ 7.4 Hz, 1H), 6.75 (d, J ¼ 15.7 Hz,
20.18, 115.90, 49.29, 39.65, 33.91, 31.61, 29.58, 26.62, 26.54, 24.82, 1H), 5.45 (s, 1H), 3.41 (s, 2H), 3.15 (d, J ¼ 5.9 Hz, 2H), 2.90 (s, 2H),
þ
13
3.05, 22.76. HRMS (ESI) m/z calcd for C28
H
32
N
4
O
3
[M þ H]
2.71 (s, 2H), 1.80 (s, 4H), 1.56 (s, 2H), 1.42 (s, 2H), 1.30 (s, 4H).
C
3
73.2547; found 473.2546. HPLC (70% methanol in water with NMR (500 MHz, CDCl ): d 165.43, 157.26, 151.60, 145.90, 139.03,
.5% H PO ): t ¼ 4.27 min, 95.56%.
135.77, 131.14, 131.07, 129.36, 128.99, 127.21, 125.96, 123.98,
123.95, 123.88, 123.07, 122.86, 119.49, 115.01, 49.04, 39.55, 32.90,
3
4
R
(
E)-3-(3-nitrophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
1
hexyl)acrylamide (18). Yellow powder, yield: 30%, mp 62–63 C. H 31.50, 29.54, 26.53, 26.43, 24.67, 22.86, 22.40. HRMS (ESI) m/z calcd
þ
NMR (300 MHz, CDCl
3
): d 8.34 (s, 1H), 8.19–8.16 (m, 1H), 7.96–7.88 for C29
H F
32 3
N
3
O [M þ H] 496.258; found 496.2580. HPLC (70%
(
(
m, 2H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.65 (d, J ¼ 15.6 Hz, 1H), 7.57–7.50 methanol in water with 0.5% H PO ): t ¼ 11.99 min, 96.20%.
3
4
R
m, 2H), 7.38–7.30 (m, 1H), 6.52 (d, J ¼ 15.6 Hz, 1H), 6.02 (s, 1H),
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3–(4-(trifluoro-
3
1
1
1
1
4
.48 (t, J ¼ 7.1 Hz, 2H), 3.42–3.36 (m, 2H), 3.05 (s, 2H), 2.70 (s, 2H), methyl)phenyl)acrylamide (23). Yellow powder, yield: 44%, mp
ꢁ
1
.91 (t, J ¼ 3.0 Hz, 4H), 1.72–1.62 (m, 2H), 1.62–1.54 (m, 2H), 58–62 C. H NMR (300 MHz, CDCl ): d 7.97 (d, J ¼ 8.7 Hz, 1H), 7.93
3
1
3
.42–1.37 (m, 4H). C NMR (500 MHz, CDCl
3
): d 165.17, 157.95, (d, J ¼ 8.6 Hz, 1H), 7.63 (d, J ¼ 19.4 Hz, 3H), 7.58–7.53 (m, 3H), 7.36
51.13, 148.57, 146.81, 137.88, 136.80, 133.88, 129.83, 128.61, (t, J ¼ 7.6 Hz, 1H), 6.47 (d, J ¼ 15.6 Hz, 1H), 6.01 (s, 1H), 3.51 (t,
27.94, 124.17, 123.76, 123.73, 123.00, 121.60, 119.92, 115.56, J ¼ 7.0 Hz, 2H), 3.42–3.36 (m, 2H), 3.08 (s, 2H), 2.72 (s, 2H), 1.93 (s,
1
3
9.20, 39.64, 33.56, 31.56, 29.48, 26.61, 26.51, 24.75, 22.97, 22.63. 4H), 1.68 (t, J ¼ 6.8 Hz, 2H), 1.64–1.57 (m, 2H), 1.43 (s, 4H).
C
þ
HRMS (ESI) m/z calcd for C28
H
32
N
4
O
3
[M þ H] 473.2547; found NMR (500 MHz, CDCl
3
): d 165.61, 158.16, 151.04, 147.06, 138.66,
): 138.48, 128.53, 128.07, 127.80, 125.65, 125.62, 123.77, 123.70,
123.01, 120.05, 115.65, 49.20, 39.60, 33.68, 31.56, 29.50, 26.65,
4
73.2554. HPLC (70% methanol in water with 0.5% H
3
PO
4
t ¼ 5.08 min, 95.08%.
R
(
32 3 3
E)-3-(4-nitrophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)- 26.52, 24.75, 22.96, 22.64. HRMS (ESI) m/z calcd for C29H F N O
ꢁ
1
þ
hexyl)acrylamide (19). Yellow powder, yield: 33%, mp 62–63 C. H [M þ H] 496.2571; found 496.2571. HPLC (70% methanol in water
NMR (300 MHz, CDCl ): d 8.22 (s, 1H), 8.19 (s, 1H), 7.97 (d, with 0.5% H PO ): t ¼ 8.34 min, 95.08%.
3
3
4
R
J ¼ 8.3 Hz, 1H), 7.91 (d, J ¼ 8.4 Hz, 1H), 7.66 (d, J ¼ 15.6 Hz, 1H), 7.61
(E)-3-(4-fluorophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
1
(
6
3
s, 1H), 7.58 (s, 1H), 7.54 (d, J ¼ 7.3 Hz, 1H), 7.35 (t, J ¼ 7.6 Hz, 1H), hexyl)acrylamide (24). Yellow powder, yield: 48%, mp 55–56 C. H
.51 (d, J ¼ 15.6 Hz, 1H), 6.11 (s, 1H), 3.50 (t, J ¼ 7.1 Hz, 2H), NMR (300 MHz, CDCl ): d 7.97 (d, J ¼ 8.4 Hz, 1H), 7.93 (d, J ¼ 8.5 Hz,
3
.42–3.36 (m, 2H), 3.06 (s, 2H), 2.71 (s, 2H), 1.93 (s, 4H), 1.72–1.63 1H), 7.63 (d, J ¼ 15.6 Hz, 1H), 7.56 (t, J ¼ 8.1 Hz, 1H), 7.50 (d,
1
3
(m, 2H), 1.63–1.55 (m, 2H), 1.41 (s, 4H). C NMR (500 MHz, CDCl
3
): J ¼ 3.9 Hz, 1H), 7.47 (d, J ¼ 1.9 Hz, 1H), 7.40–7.32 (m, 4H), 6.41 (d,
d 164.86, 158.50, 150.79, 148.10, 147.42, 141.21, 138.22, 128.61, J ¼ 15.6 Hz, 1H), 5.93 (s, 1H), 3.50 (t, J ¼ 7.1 Hz, 2H), 3.41–3.35 (m,
1
4
28.40, 128.30, 125.02, 124.11, 123.69, 122.84, 120.24, 115.96, 2H), 3.07 (s, 2H), 2.71 (s, 2H), 1.92 (s, 4H), 1.73–1.63 (m, 2H),
9.29, 39.70, 33.98, 31.63, 29.54, 26.61, 26.54, 24.85, 23.07, 22.79. 1.62–1.51 (m, 2H), 1.42 (s, 4H).
1
3
3
C NMR (500 MHz, CDCl ): d
þ
HRMS (ESI) m/z calcd for C28
H
32
N
4
O
3
[M þ H] 473.2547; found 165.91, 164.50, 162.51, 151.32, 146.54, 139.61, 131.13, 129.57,
4
73.2549. HPLC (70% methanol in water with 0.5% H PO ): 129.50, 128.80, 127.77, 123.84, 123.01, 120.60, 119.81, 115.98,
3 4
t
R
¼ 4.62 min, 95.83%.
115.81, 115.44, 49.18, 39.53, 33.37, 31.55, 29.60, 26.57, 26.50, 24.73,
þ
(
E)-3-(4-chloro-3-nitrophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9- 22.94, 22.56. HRMS (ESI) m/z calcd for C28
H
32FN
3
O
3
[M þ H]
yl)amino)hexyl)acrylamide (20). Yellow powder, yield: 46%, mp 446.2602; found 446.2601. HPLC (70% methanol in water with
5
ꢁ
1
5–60 C. H NMR (300 MHz, CDCl
3
): d 7.96 (s, 1H), 7.93 (d, 0.5% H
3
PO
4
): t
R
¼ 5.50 min, 94.98%.
J ¼ 4.4 Hz, 1H), 7.89 (s, 1H), 7.58 (d, J ¼ 5.6 Hz, 1H), 7.54 (s, 2H), 7.47
(E)-3-(2-chlorophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
1
(
5
d, J ¼ 10.8 Hz, 1H), 7.34 (t, J ¼ 7.8 Hz, 1H), 6.44 (d, J ¼ 15.6 Hz, 1H), hexyl)acrylamide (25). Yellow powder, yield: 51%, mp 53–55 C. H
.93 (s, 1H), 3.49 (t, J ¼ 7.0 Hz, 2H), 3.41–3.34 (m, 2H), 3.06 (s, 2H), NMR (300 MHz, CDCl
3
): d 7.98 (t, J ¼ 15.4 Hz, 3H), 7.56 (t, J ¼ 7.1 Hz,
2
.70 (s, 2H), 1.92 (s, 4H), 1.72–1.62 (m, 2H), 1.59–1.49 (m, 2H), 1.40 2H), 7.41–7.33 (m, 2H), 7.28–7.20 (m, 2H), 6.44 (d, J ¼ 15.6 Hz, 1H),
1
3
(
1
1
d, J ¼ 3.2 Hz, 4H). C NMR (500 MHz, CDCl ): d 164.83, 158.19, 6.08 (s, 1H), 3.53 (t, J ¼ 6.7 Hz, 2H), 3.39 (d, J ¼ 6.2 Hz, 2H), 3.08 (s,
3
50.97, 148.15, 147.07, 136.79, 135.21, 132.31, 132.11, 131.46, 2H), 2.71 (s, 2H), 1.92 (s, 4H), 1.69 (s, 2H), 1.59 (s, 2H), 1.43 (s, 4H).
28.52, 128.22, 127.36, 124.51, 123.70, 122.96, 120.05, 115.71,
1
3
3
C NMR (500 MHz, CDCl ): d 165.61, 158.24, 150.96, 147.14,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
293
1
3
1
1
3
36.63, 134.68, 133.24, 130.36, 130.13, 128.48, 128.32, 127.53, 4H), 1.70–1.61 (m, 2H), 1.58–1.49 (m, 2H), 1.44–1.34 (m, 4H).
C
3
26.93, 123.82, 123.70, 122.92, 120.10, 115.78, 49.28, 39.60, 33.79, NMR (500 MHz, CDCl ): d 166.38, 160.81, 158.53, 150.74, 147.51,
1.61, 29.57, 26.63, 26.55, 24.80, 23.03, 22.72. HRMS (ESI) m/z calcd 140.35, 129.27, 128.70, 128.29, 127.62, 123.63, 122.86, 120.30,
þ
for C H ClN O [M þ H] 462.231; found 462.2314. HPLC (70% 118.52, 115.99, 114.22, 55.35, 49.34, 39.51, 34.07, 31.65, 29.67,
2
8
32
3
methanol in water with 0.5% H
3
PO
4
): t
R
¼ 6.43 min, 96.83%.
26.68, 26.60, 24.85, 23.08, 22.81. HRMS (ESI) m/z calcd for
þ
(
E)-3-(3-chlorophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
C
29
H
35
3
N O
2
[M þ H] 458.2802; found 462.2805. HPLC (70% metha-
ꢁ
1
hexyl)acrylamide (26). Yellow powder, yield: 32%, mp 55–59 C. H nol in water with 0.5% H PO ): t ¼ 4.42 min, 95.51%.
3
4
R
NMR (300 MHz, CDCl
1
3
): d 8.00 (d, J ¼ 4.0 Hz, 1H), 7.97 (d, J ¼ 4.0 Hz,
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3–(2,3,4-trime-
H), 7.58 (d, J ¼ 5.4 Hz, 1H), 7.51 (d, J ¼ 15.9 Hz, 2H), 7.38 (d, thoxyphenyl)acrylamide (31). Yellow powder, yield: 43%, mp
ꢁ
1
J ¼ 7.5 Hz, 1H), 7.33 (d, J ¼ 7.4 Hz, 3H), 6.41 (d, J ¼ 15.6 Hz, 1H), 5.89 56–60 C. H NMR (300 MHz, CDCl ): d 8.21 (d, J ¼ 8.5 Hz, 1H), 8.13
3
(
2
s, 1H), 3.55 (t, J ¼ 7.0 Hz, 2H), 3.37 (d, J ¼ 6.6 Hz, 2H), 3.10 (s, 2H), (d, J ¼ 8.2 Hz, 1H), 7.74 (d, J ¼ 15.8 Hz, 1H), 7.58 (t, J ¼ 7.7 Hz, 1H),
.71 (s, 2H), 1.93 (s, 4H), 1.75–1.65 (m, 2H), 1.63–1.53 (m, 2H), 1.44 7.37 (t, J ¼ 7.7 Hz, 1H), 7.20 (t, J ¼ 6.1 Hz, 1H), 6.54 (d, J ¼ 15.8 Hz,
1
3
(s, 4H).
C NMR (500 MHz, CDCl ): d 165.64, 157.32, 151.53, 1H), 6.32 (s, 1H), 3.89–3.85 (m, 9H), 3.74 (t, J ¼ 7.0 Hz, 2H),
3
1
1
3
46.03, 139.08, 136.84, 134.73, 130.03, 129.39, 128.94, 127.37, 3.40–3.34 (m, 2H), 3.17 (t, J ¼ 5.4 Hz, 2H), 2.65 (t, J ¼ 5.7 Hz, 2H),
27.30, 126.11, 123.86, 123.11, 122.49, 119.55, 115.09, 49.06, 39.54, 1.86 (d, J ¼ 4.3 Hz, 4H), 1.79–1.67 (m, 2H), 1.64–1.52 (m, 2H), 1.43
1
3
3.04, 31.50, 29.53, 26.56, 26.46, 24.69, 22.88, 22.44. HRMS (ESI) m/ (s, 4H).
3
C NMR (500 MHz, CDCl ): d 166.71, 156.74, 154.85,
þ
z calcd for C H ClN O [M þ H] 462.231; found 462.2316. HPLC 153.02, 151.92, 145.31, 142.41, 135.51, 129.16, 126.66, 123.92,
2
8
32
3
(70% methanol in water with 0.5% H
3
PO
4
): t
R
¼ 7.31 min, 95.25%.
123.19, 123.10, 122.01, 120.37, 119.21, 114.66, 107.59, 61.29, 60.88,
(
E)-3-(4-chlorophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
56.04, 48.93, 39.40, 32.52, 31.43, 29.60, 26.52, 26.42, 24.64, 22.80,
ꢁ
1
þ
hexyl)acrylamide (27). Yellow powder, yield: 39%, mp 56–57 C. H 22.27. HRMS (ESI) m/z calcd for C H N O [M þ H] 518.301;
3
1 39 3 4
NMR (300 MHz, CDCl
H), 7.60–7.52 (m, 2H), 7.42–7.34 (m, 3H), 7.34–7.29 (m, 2H), 6.41
d, J ¼ 15.6 Hz, 1H), 6.19 (s, 1H), 3.53 (t, J ¼ 7.2 Hz, 2H), 3.40–3.33
m, 2H), 3.07 (s, 2H), 2.69 (s, 2H), 1.91 (s, 4H), 1.72–1.62 (m, 2H), thoxyphenyl)acrylamide (32). Yellow powder, yield: 29%, mp
3
): d 7.99 (d, J ¼ 8.5 Hz, 1H), 7.94 (d, J ¼ 9.3 Hz, found 518.3019. HPLC (70% methanol in water with 0.5% H
¼ 4.44 min, 95.59%.
(E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3–(3,4,5-trime-
3 4
PO ):
1
(
(
t
R
1
3
ꢁ
1
1
1
1
1
2
4
0
.62–1.52 (m, 2H), 1.42–1.38 (m, 4H). C NMR (500 MHz, CDCl
3
): d 58–60 C. H NMR (300 MHz, CDCl
3
): d 7.96 (d, J ¼ 8.5 Hz, 1H), 7.92
65.69, 157.61, 151.37, 139.51, 139.45, 135.44, 133.43, 130.46, (d, J ¼ 8.5 Hz, 1H), 7.57 (d, J ¼ 4.8 Hz, 1H), 7.52 (d, J ¼ 5.1 Hz, 1H),
29.06, 128.92, 128.84, 128.54, 127.76, 123.85, 123.01, 121.38, 7.35 (t, J ¼ 7.5 Hz, 1H), 6.71 (s, 2H), 6.34 (d, J ¼ 15.5 Hz, 1H), 5.97 (d,
19.72, 115.34, 49.15, 39.53, 33.27, 31.53, 29.58, 26.55, 26.48, 24.71, J ¼ 5.4 Hz, 1H), 3.87 (d, J ¼ 4.7 Hz, 9H), 3.50 (t, J ¼ 7.1 Hz, 2H),
þ
2.93, 22.53. HRMS (ESI) m/z calcd for C28
H
32ClN
3
O [M þ H]
3.40–3.34 (m, 2H), 3.06 (s, 2H), 2.71 (s, 2H), 1.92 (s, 4H), 1.72–1.63
1
3
62.2307; found 462.2306. HPLC (70% methanol in water with (m, 2H), 1.60–1.52 (m, 2H), 1.41 (s, 4H). C NMR (500 MHz, CDCl3):
.5% H
3
PO
4
): t
R
¼ 8.09 min, 95.22%.
d 166.02, 158.19, 153.38, 150.95, 147.08, 140.62, 139.54, 130.54,
128.48, 128.26, 123.69, 122.91, 120.36, 120.07, 115.73, 104.98,
hexyl)acrylamide (28). Yellow powder, yield: 59%, mp 60–61 C. H 60.93, 56.12, 49.24, 39.52, 33.74, 31.60, 29.62, 26.63, 26.55, 24.79,
(E)-3-(4-bromophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
1
þ
NMR (300 MHz, CDCl
3
): d 7.97 (d, J ¼ 8.5 Hz, 1H), 7.93 (d, J ¼ 8.5 Hz, 23.01, 22.70. HRMS (ESI) m/z calcd for C31
H N
39 3
O
4
[M þ H]
1
3
3
H), 7.61–7.53 (m, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.36 (t, J ¼ 7.3 Hz, 518.3019; found 518.3019. HPLC (70% methanol in water with
H), 6.37 (d, J ¼ 15.6 Hz, 1H), 5.76 (s, 1H), 3.51 (t, J ¼ 7.1 Hz, 2H), 0.5% H PO ): t ¼ 4.00 min, 96.23%.
3
4
R
.41–3.35 (m, 2H), 3.08 (s, 2H), 2.72 (s, 2H), 1.93 (s, 4H), 1.74–1.63
(E)-3-(3-hydroxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)ami-
): no)hexyl)acrylamide (33). Yellow powder, yield: 13%, mp
1
3
(m, 2H), 1.62–1.50 (m, 2H), 1.42 (s, 4H). C NMR (500 MHz, CDCl
3
ꢁ
1
d 165.66, 158.36, 150.86, 147.27, 139.57, 133.83, 132.02, 129.16, 96–100 C. H NMR (300 MHz, CDCl
1
4
HRMS (ESI) m/z calcd for C28
5
3
): d 8.00 (d, J ¼ 7.8 Hz, 1H), 7.95
28.50, 128.43, 123.72, 123.70, 122.87, 121.47, 120.17, 115.88, (d, J ¼ 8.9 Hz, 1H), 7.60–7.52 (m, 1H), 7.45 (s, 1H), 7.41–7.35 (m, 1H),
9.30, 39.59, 33.89, 31.63, 29.60, 26.63, 26.56, 24.83, 23.05, 22.76. 7.19 (t, J ¼ 7.9 Hz, 1H), 6.90 (d, J ¼ 7.9 Hz, 2H), 6.80 (s, 1H), 5.88 (d,
þ
H32BrN
3
O [M þ H] 506.1802; found J ¼ 15.4 Hz, 1H), 5.58 (s, 1H), 3.60–3.53 (m, 2H), 3.34–3.31 (m, 2H),
06.1807. HPLC (70% methanol in water with 0.5% H PO ): 3.08 (s, 2H), 2.73 (s, 2H), 1.91 (s, 4H), 1.75–1.66 (m, 2H), 1.59–1.52
3 4
1
3
t
R
¼ 8.28 min, 97.37%.
(m, 2H), 1.49 –1.36 (m, 4H). C NMR (500 MHz, CDCl
3
): d 158.38,
(
E)-3-(4-cyanophenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
141.02, 129.94, 128.98, 127.90, 123.93, 122.74, 120.46, 119.88,
hexyl)acrylamide (29). Yellow powder, yield: 23%, mp 55–57 C. H 119.75, 117.64, 48.91, 39.19, 33.14, 31.05, 29.73, 26.09, 26.01, 24.94,
ꢁ
1
þ
NMR (300 MHz, CDCl
1
3
): d 7.97 (d, J ¼ 8.3 Hz, 1H), 7.92 (d, J ¼ 8.4 Hz, 22.92, 22.52. HRMS (ESI) m/z calcd for C28
H N
33 3
O
2
[M þ H]
H), 7.71 (s, 1H), 7.65 (d, J ¼ 7.3 Hz, 1H), 7.58 (d, J ¼ 5.1 Hz, 2H), 444.265; found 444.2657. HPLC (70% methanol in water with 0.5%
7
.53 (s, 2H), 7.49–7.39 (m, 1H), 7.39–7.29 (m, 1H), 6.49 (d, H PO ): t ¼ 2.45 min, 95.06%.
3
4
R
J ¼ 15.6 Hz, 1H), 6.36 (s, 1H), 3.51 (d, J ¼ 6.2 Hz, 2H), 3.37 (d,
(E)-3-(4-hydroxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
ꢁ
J ¼ 5.8 Hz, 2H), 3.05 (s, 2H), 2.68 (s, 2H), 1.89 (s, 4H), 1.67 (s, 2H), hexyl)acrylamide (34). Yellow powder, yield: 15%, mp 108–110 C.
1
3
1
1
1
1
1
2
4
0
.57 (s, 2H), 1.40 (s, 4H). C NMR (500 MHz, CDCl
3
): d 165.22,
3
H NMR (300 MHz, CDCl ): d 8.15 (s, 1H), 7.97–7.89 (m, 2H),
57.51, 151.44, 146.28, 138.01, 136.32, 132.45, 132.05, 130.67, 7.57–7.49 (m, 1H), 7.37–7.26 (m, 2H), 6.84 (d, J ¼ 8.5 Hz, 1H), 6.14
29.71, 128.89, 127.46, 123.85, 123.71, 123.12, 119.65, 118.36, (d, J ¼ 11.6 Hz, 1H), 5.76 (s, 1H), 3.50 (t, J ¼ 6.9 Hz, 2H), 3.40–3.14
15.26, 113.07, 49.11, 39.60, 33.22, 31.50, 29.47, 26.56, 26.46, 24.70, (m, 2H), 3.05 (s, 2H), 2.70 (s, 2H), 2.05–1.74 (m, 4H), 1.72–1.60 (m,
þ
13
2.90, 22.50. HRMS (ESI) m/z calcd for C H N O [M þ H]
2H), 1.57–1.48 (m, 2H), 1.45–1.29 (m, 4H).
C NMR (500 MHz,
2
9 32 4
53.2648; found 453.2648. HPLC (70% methanol in water with CDCl ): d 161.23, 158.42, 158.31, 150.95, 150.84, 147.26, 147.15,
3
.5% H
3
PO
4
): t
R
¼ 3.57 min, 96.58%.
129.50, 128.50, 128.35, 123.72, 122.88, 120.12, 116.25, 115.80,
49.28, 37.98, 33.74, 31.65, 29.50, 26.52, 26.50, 24.81, 23.03, 22.72.
(
E)-3-(4-methoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)ami-
ꢁ
þ
no)hexyl)acrylamide (30). Yellow powder, yield: 26%, mp 53–54 C. HRMS (ESI) m/z calcd for C28
H
33
N
3
O
2
[M þ H] 444.2649; found
1
3 3 4
H NMR (300 MHz, CDCl ): d 7.96–7.88(m, 2H), 7.59–7.51 (m, 2H), 444.2649. HPLC (70% methanol in water with 0.5% H PO ):
7
6
2
.42 (d, J ¼ 8.7 Hz, 2H), 7.38–7.29 (m, 1H), 6.87 (d, J ¼ 8.7 Hz, 2H), t ¼ 3.00 min, 95.43%.
R
.24 (d, J ¼ 15.5 Hz, 1H), 5.79 (s, 1H), 3.82 (s, 3H), 3.47 (t, J ¼ 7.1 Hz,
(E)-3-(4-phenoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)ami-
ꢁ
H), 3.39–3.32 (m, 2H), 3.05 (s, 2H), 2.70 (s, 2H), 1.91 (t, J ¼ 3.0 Hz, no)hexyl)acrylamide (35). Yellow powder, yield: 28%, mp 47–49 C.

2
1
94
Y. CHEN ET AL.
H NMR (300 MHz, CDCl
3
): d 7.99 (t, J ¼ 7.8 Hz, 2H), 7.60–7.54 (m, human, B4186), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB,
2
H), 7.44 (d, J ¼ 5.4 Hz, 3H), 7.42 (d, J ¼ 1.9 Hz, 2H), 7.41 (d, D218200), acetylthiocholine iodide (ATC, A5751), and butyrylthio-
J ¼ 0.8 Hz, 1H), 7.39–7.38 (m, 1H), 7.36 (d, J ¼ 1.6 Hz, 1H), 6.95 (d, choline iodide (BTC, B3253) were obtained from Sigma-Aldrich
J ¼ 8.8 Hz, 2H), 6.29 (d, J ¼ 15.5 Hz, 1H), 5.92 (d, J ¼ 6.0 Hz, 1H), 5.09 (St. Louis, MO).
(
(
s, 2H), 3.54 (t, J ¼ 7.1 Hz, 2H), 3.41–3.34 (m, 2H), 3.09 (s, 2H), 2.70
The inhibitory effects of the synthesised compounds in this
s, 2H), 1.92 (t, J ¼ 2.9 Hz, 4H), 1.69 (m, 2H), 1.60–1.54 (m, 2H), paper were evaluated according to our previously reported
13
28
1
1
1
1
2
5
0
.43–1.39 (m, 4H). C NMR (500 MHz, CDCl
3
): d 166.35, 160.06, method . Briefly, AChE/BuChE stock solution was diluted to give
58.41, 150.90, 147.32, 140.47, 136.59, 129.33, 128.68, 128.51, 2.5 units/mL (for eeAChE, eqBuChE, and huAChE) or 0.5 units/mL
28.44, 128.15, 127.85, 127.48, 123.70, 122.90, 120.21, 118.53, for huBuChE. ATC/BTC iodide solution (0.075 M) was prepared in
15.89, 115.17, 70.10, 49.33, 39.53, 33.89, 31.65, 29.68, 26.65, 26.59, deionised water. DTNB solution (0.01 M) was prepared using water
þ
4.84, 23.06, 22.77. HRMS (ESI) m/z calcd for C H N O [M þ H]
containing 0.15% (w/v) sodium bicarbonate. The assay buffer was
3
5 39 3 2
34.3119; found 534.3119. HPLC (70% methanol in water with prepared as follows: potassium dihydrogen phosphate (1.36 g,
.5% H PO ): t
¼ 11.98 min, 97.02%.
10 mmol) was dissolved in 100 ml of water. The pH of the solution
E)-3-(4-benzyl-3-methoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9- was then adjusted to 8.0 ± 0.1 with KOH. Stock solutions of the
3
4
R
(
yl)amino)hexyl)acrylamide (36). Yellow powder, yield: 33%, mp test samples were dissolved in ethanol to give a final concentra-
1
ꢂ4
ꢁ
5
(
(
5
3
7–64 C. H NMR (300 MHz, CDCl
3
): d 8.11 (d, J ¼ 8.3 Hz, 1H), 8.04 tion of 10 M when diluted to the final volume of 3.32 ml. For
d, J ¼ 8.6 Hz, 1H), 7.62–7.48 (m, 3H), 7.46–7.31 (m, 6H), 7.12–6.99 each compound, a dilution series of at least five different concen-
ꢂ5 ꢂ9
m, 2H), 6.86 (t, J ¼ 5.7 Hz, 1H), 6.30 (d, J ¼ 15.5 Hz, 1H), 5.86 (s, 1H), trations (normally 10 –10 M) were prepared.
.17 (s, 2H), 3.89 (s, 3H), 3.64 (t, J ¼ 6.9 Hz, 2H), 3.41–3.29 (m, 2H),
For measurement, a cuvette containing 3.0 ml of phosphate buf-
.14 (s, 2H), 2.66 (s, 2H), 1.89 (s, 4H), 1.77–1.63 (m, 2H), 1.62–1.49 fer, 100 mL of AChE or BuChE, 100 mL of DTNB, and 100 mL of the
m, 2H), 1.42 (s, 4H). C NMR (500 MHz, CDCl
3
): d 166.29, 157.31, test compound solution were added in sequence. The reaction was
1
3
(
initiated after adding 20 mL of ATC or BTC, and the solution was
mixed immediately. Two minutes (eeAChE and eqBuChE) or 15 min
huAChE and huBuChE) after ATC or BTC addition, the absorption
1
1
1
2
51.55, 149.72, 149.67, 140.49, 136.71, 128.96, 128.62, 128.36,
28.00, 127.35, 127.26, 123.88, 123.09, 121.64, 119.55, 119.03,
15.14, 113.64, 110.47, 77.33, 56.03, 49.08, 45.99, 39.43, 31.49,
(
ꢁ
ꢁ
was measured at 25 C (eeAChE and eqBuChE) or 37 C (huAChE
and huBuChE) at 412 nm, using a Shimadzu 160 spectrophotom-
eter. For the reference value, 100 mL of water replaced the test com-
pound solution. For the blank control, additionally 100 mL of water
replaced the enzyme solution. The measurement for each concen-
tration was assayed in triplicate. GraphPad Prism 5 was used for
data processing. The inhibition curve was fitted by plotting percent-
age enzyme activity (100% for the reference) versus logarithm of
test compound concentration. The IC50 values were calculated
according to the curve, and the data were shown in mean ± SEM.
9.63, 26.54, 26.46, 24.68, 22.89, 22.45. HRMS (ESI) m/z calcd for
þ
C
36
H
41
N
3
O
3
[M þ H] 564.3221; found 564.3216. HPLC (70% metha-
nol in water with 0.5% H PO ): t ¼ 9.66 min, 96.84%.
3
4
R
(
E)-3-(4-(4-chlorophenoxy)-3-methoxyphenyl)-N-(6-((1,2,3,4-tetrahy-
droacridin-9-yl)amino)hexyl)acrylamide (37). Yellow powder, yield:
7%, mp 68–70 C. H NMR (300 MHz, CDCl
ꢁ
1
1
2
6
2
2
3
): d 7.96 (t, J ¼ 9.2 Hz,
H), 7.59–7.46 (m, 2H), 7.33 (d, J ¼ 8.6 Hz, 6H), 7.07–6.95 (m, 2H),
.81 (d, J ¼ 8.0 Hz, 1H), 6.28 (d, J ¼ 15.3 Hz, 1H), 5.85 (s, 1H), 5.12 (s,
H), 3.88 (s, 3H), 3.53 (t, J ¼ 6.3 Hz, 2H), 3.38–3.32 (m, 2H), 3.06 (s,
H), 2.68 (s, 2H), 1.90 (s, 4H), 1.66 (d, J ¼ 5.5 Hz, 2H), 1.56 (d,
1
3
J ¼ 4.3 Hz, 2H), 1.41 (d, J ¼ 22.8 Hz, 4H). C NMR (500 MHz, CDCl
3
):
d 166.17, 149.79, 149.43, 140.58, 135.25, 133.85, 128.98, 128.83,
Kinetic studies of AChE inhibition
1
1
2
28.77, 128.64, 127.40, 124.00, 123.91, 123.05, 121.59, 119.12,
19.05, 113.76, 110.49, 70.25, 56.02, 49.10, 39.45, 31.46, 30.23,
9.72, 26.52, 26.46, 24.68, 22.89, 22.45. HRMS (ESI) m/z calcd for
Kinetic studies were performed according to methods reported
4
5
previously . The concentrations of the substrate ATC or BTC were
prepared as 25, 50, 90, 150, 226, and 452 mM. Different concentra-
tions of compound 19 with 0, 1, 2, 4, 10 nM, were also prepared
by stock solution. For measurement, the enzymatic reaction was
extended to 20 min (huAChE and huBuChE) before the determin-
ation of the absorption. Graph Pad Prism 5 was applied for data
processing. Vmax and Km values of the Michaelis–Menten kinetics
were determined by nonlinear regression from substrate-velocity
curves, while Lineweaver–Burk plots were fitted by using linear
regression method.
þ
C
36
H
40ClN
3
O
3
[M þ H] 598.2834; found 598.2834. HPLC (70%
methanol in water with 0.5% H PO ): t ¼ 16.25 min, 95.53%.
3
4
R
(
E)-3-(3-methoxy-4-(p-tolyloxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacri-
din-9-yl)amino)hexyl)acrylamide (38). Yellow powder, yield: 52%,
ꢁ
1
mp 63–65 C. H NMR (300 MHz, CDCl
3
): d 8.00 (d, J ¼ 3.8 Hz, 1H),
7
(
7
6
2
.97 (d, J ¼ 3.9 Hz, 1H), 7.59–7.53 (m, 1H), 7.53–7.48 (m, 1H), 7.34
d, J ¼ 9.6 Hz, 1H), 7.30 (d, J ¼ 7.9 Hz, 2H), 7.19–7.09 (m, 2H),
.03–6.94 (m, 2H), 6.82 (d, J ¼ 8.2 Hz, 1H), 6.30 (d, J ¼ 15.5 Hz, 1H),
.04 (t, J ¼ 5.6 Hz, 1H), 5.12 (s, 2H), 3.86 (s, 3H), 3.56 (t, J ¼ 7.1 Hz,
H), 3.37–3.30 (m, 2H), 3.08 (s, 2H), 2.65 (s, 2H), 2.34 (s, 3H), 1.87
(
s, 4H), 1.71–1.62 (m, 2H), 1.58–1.48 (m, 2H), 1.39 (d, J ¼ 7.0 Hz, 4H).
Inhibition of self-induced Ab1–42 aggregation
1
3
3
C NMR (500 MHz, CDCl ): d 166.35, 156.69, 151.97, 149.73,
Inhibitory effects of the compounds on self-induced Ab1–42 aggre-
gation were evaluated using a Thioflavin T (ThT)-(T3516, Sigma-
Aldrich, St. Louis, MO) binding assay according to the previously
1
1
1
2
49.71, 140.44, 137.76, 133.68, 129.30, 129.27, 129.25, 128.29,
27.37, 126.58, 123.97, 123.20, 121.67, 119.17, 119.04, 114.59,
13.61, 110.44, 70.86, 56.01, 48.88, 39.40, 32.44, 31.44, 29.60, 26.49,
43
reported method . Briefly, aliquots of 2.0 mL of Ab1–42 (AS-
6.41, 24.65, 22.79, 22.24, 21.21. HRMS (ESI) m/z calcd for
64129–05 Anaspec Inc.) containing 2 mg/mL HFIP (1,1,1,3,3,3-hexa-
þ
C
H
37 43
N
3
O
3
[M þ H] 578.3385; found 578.3385. HPLC (70% metha-
fluoro-2-propanol, 52517, Sigma-Aldrich, St. Louis, MO) were
stocked in DMSO. They were then diluted to the final concentra-
tion of 500 mM with 0.215 M sodium phosphate buffer (pH 8.0).
Test compounds were dissolved in DMSO and then diluted by buf-
fer to give a final concentration of 25 mM. The Ab1–42 and the test
sample solutions were incubated for 24 h at the room tempera-
nol in water with 0.5% H PO ): t ¼ 14.16 min, 96.28%.
3
4
R
Biological determinations
In vitro inhibitory evaluations on AChE and BuChE
AChE (EC 3.1.1.7, Type VI-S, from electric eel, C3389; from human, ture. After the incubation, the test samples were diluted to a final
C1682) and BuChE (EC 3.1.1.8, from equine serum, C0663; from volume of 150 mL with 50 mM glycine-NaOH buffer (pH 8.5)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
295
containing 5 mM Thioflavin T. Fluorescence intensity was read Hepatotoxicity studies
(
excitation wavelength 450 nm, emission wavelength 485 nm) on a Hepatotoxicity was evaluated according to the method previously
4
7
SpectraMax Paradigm Multimode Reader (Molecular Device).
described by using adult male ICR mice (8–10 weeks old, weigh-
The inhibitory rate of Ab1–42 self-induced aggregation was cal- ing 20–25 g) obtained from the Yangzhou University Medicine
culated according to the following equation: (1-IFi/IFc) ꢀ 100%. IFi Centre (Yangzhou, China). Tacrine and the test compounds
and IFc were the fluorescence intensities in the presence and were dissolved in a sodium carboxymethyl cellulose (CMC-Na)
absence of inhibitors, respectively, after subtracting the back- solution (0.5 g CMC-Na in 100 ml distilled water). Concentration of
ground fluorescence of the 5 mM Thioflavin T solution. Each meas-
urement was repeated in triplicate. The inhibitory rate of the test
compound was shown in mean ± SD.
3
mg/100 g body wt. of tacrine, corresponding to 151.5 mmol/kg
body wt., was administered intragastrically (ig). Equimolar dose of
test compounds to that of tacrine was administered ig. 8, 22, and
36 h after the administration, heparinised serum was collected
from the retrobulbar plexus and subjected to hepatotoxicity evalu-
ation. The activity of AST and ALT, two indicators of liver damage,
was determined using corresponding assay kit (EF551 and EF550
for ALT, EH027, and EF548 for AST, Wako, Japan). The data were
processed by Biochemical Analyzer (HITACHI 7020, Japan).
One hour after the collection of retrobulbar blood, mice were
sacrificed and livers were harvested for morphological studies by
using immunohistochemical method. Two 3 mm sections of each
liver extending from the hilus to the margin of the left lateral lobe
were isolated by Ultra-Thin Semiautomatic Microtome (Leica
RM2245, Germany) and immediately placed in 10% buffered for-
maldehyde, fixed for two days, and embedded together in one
paraffin block by using Paraffin Embedding Station (Leica
EG1150H, Germany). Subsequently, 5 mm sections were prepared
from these paraffin blocks. They were deparaffinated and stained
with hematoxylin and eosin or by means of the periodic acid-
Schiff procedure for glycogen.
Behavioural studies
Behavioural studies were carried out according to the method
4
6
reported previously . Briefly, the adult male ICR mice (8–10 weeks
old, weight 20–25 g) were obtained from the Yangzhou University
Medical Center (Yangzhou, China). Scopolamine hydrobromide
was purchased from Aladdin Reagents (H1507073, Shanghai,
China). Tacrine that was synthesised in our lab with >95% purity
was used as the positive control.
The mice were separated into six groups as follows: (i) vehicle
as a blank control group, (ii) scopolamine as a model group, (iii)
tacrine plus scopolamine as a positive control, (iv) compound 19
plus scopolamine as a test group, (v) compound 27 plus scopol-
amine as a test group; and (vi) compound 30 plus scopolamine as
a test group. Each group contains six mice. Tacrine and the syn-
thesised compounds were orally administered (20 mmol/kg body
weight) to mice in groups (iii), (iv), (v), and (vi), 30 min before the
ip administration of scopolamine (1 mg/kg) or saline for 10 con-
secutive days.
The cognitive function of the mice was evaluated by the Morris
water maze test, determined by analysis-management system
Molecular docking studies
The molecular docking was completed by CDOCKER module
implemented in Discovery Studio . The co-crystal structures of
huAChE and huBuChE with small molecular ligands were down-
(
Panlab SMART 3.0, America), according to the method previously
4
8
4
3
described . The maze was placed in a lit room with visual cues at
ꢁ
2
5 C. An escape platform (10 cm diameter) was at the centre of
one quadrant of the circular pool (120 cm diameter, 60 cm height) loaded from Protein Data Bank (PDB, ID: 4EY7, 4TPK). The struc-
with a depth of 40 cm water. For measurement of the cognitive tures were initially processed by “Prepare Protein” module in DS
function, each mouse included 5 days of learning and memory to give the structures suitable for docking. Missed sidechains of
training, followed by a probe trial on day 6. The starting positions the proteins were added and the water molecules were removed,
faced to the pool wall and were pseudorandomised for each trial. then the structures were protonated at pH 7.4. “Prepare Ligands”
For the cognitive evaluation, each mouse was individually eval- module in DS was applied for the structural preparation of
uated on both visible-platform (days 1–2) and hidden-platform the test compounds, which were then protonated at pH 7.4. The
(days 3–5) versions of the water maze. All mice received nonspa- resulted molecules were subsequently minimised by “Minimise
tial pretraining during the first two training days, which prepared Ligands” module. The “Smart Minimiser” algorithm was used to
them for the subsequent spatial learning test. During the two carry out the minimisation, with max steps set to 2000, RMS
days, mice were trained to find the platform that was labelled by Gradient set to 0.01. Other parameters were set as default.
a small flag (5 cm tall). The hidden-platform version was used to
For molecular docking, the binding site was defined as a site
determine the retention of memory to find the platform. During sphere (in 10 Å radius) around the original ligands in the co-crystal
the hidden-platform training trials, the escape platform was placed structures. The simulated annealing parameters were set as fol-
1
cm below the surface of the water. On each day, the animal was lows: heating steps and cooling steps were set to 2000 and 5000,
subjected to two trials, each of which lasted for 90 s. The time for
the mouse to find the platform (a successful escape) was recorded.
If a mouse failed to reach the platform within 90 s, the test was
terminated and the animal was gently navigated to the platform
by hand. Whether a mouse was successful or failed to reach the
platform within 90 s, it was kept on the platform for 30 s. On the
last day (day 6), the platform was removed from its location and
respectively, while heating and cooling temperature were set to
00, and 310, respectively. Other parameters were kept as default.
Ten top-ranked conformations for each docked compound were
retained for binding pattern analysis, which were visualised and
depicted in DS.
7
the animals were given a probe trial in which they had 90 s to Results and discussions
search for the platform. The time taken to reach the missing plat-
form and the number of times the animals crossed the platform
Compound design and chemistry
location were recorded.
The cinnamic acid is a naturally originated compound with diverse
Data for the time of escape latency, the trajectory travelled, biological activity. We notice that several derivatives of cinnamic
and the number of platform location crossings were recorded by acid, such as ferulic acid, caffeic acid, are reported to benefit the
Panlab SMART 3.0 and processed by Graphpad Prism 5.
treatment of AD for many reasons. More importantly, we observe

2
96
Y. CHEN ET AL.
O
O
Cl
HN
NH2
HN
N
N
n
n H
COOH
a
b
c
+
NH2
N
N
4
~8
9~13
n= 2, 3, 4, 6, 8
1
2
3
n= 2, 3, 4, 6, 8
1
1
2
4, R = 2-CH ; 15, R = 3-CH ; 16, R = 4-CH ;
3
3
3
O
N
7, R = 2-NO ; 18, R = 3-NO ; 19, R = 4-NO ;
2
2
2
0, R = 4-Cl-3-NO ; 21, R = 2-CF ; 22, R = 3-CF ;
HN
NH2
HN
N
2
3
3
6
6
H
R
23, R = 4-CF ; 24, R = 4-F; 25, R = 2-Cl;
3
R
c
HO
2
2
3
3
6, R = 3-Cl; 27, R = 4-Cl; 28, R = 4-Br;
9, R = 3-CN; 30, R = 4-OCH ; 31, R = 2,3,4-triOCH 3;
2, R = 3,4,5-triOCH ; 33, R = 3-OH; 34, R = 4-OH;
5, R = OBn; 36, R = 3-OCH -4-OBn;
+
3
N
O
3
7
cinnamic acid derivatives
1
4~36
3
R
O
HN
NH2
O
HN
N
N
H
6
6
c
37, R = Cl; 38, R = CH3;
HO
O
+
O
O
N
O
R
7
cinnamic acid derivatives
37~38
3 2 2 n 2
Scheme 1. Synthetic route of tacrine-cinnamic acid hybrids. Reagents and conditions: (a) POCl , reflux, 3 h; (b) pentanol, NH (CH ) NH , NaI, reflux, 18 h; (c) PyBop,
DIPEA, anhydrous CH Cl , room temp, 24 h.
2
2
that the cinnamic acid moiety can serve as a good scaffold to hexamethylendiamine was considered to be the optimal linker for
insert into the narrow groove of the AChE active site, forming further structural modifications.
intermolecular interactions with residues in the PAS. Therefore, we
Next, we examined the optimal substituents on cinnamic acid
consider that cinnamic acid moiety is a good fragment for design- moiety. Methyl substitution at meta- or ortho- position of cinnamic
ing CAS-PAS dual site ChEs inhibitor. However, as a PAS binder, acid (14 and 15, respectively) led to the reduced activities com-
the structural modification of this moiety is not fully analysed pre- pared to compound 12, but the para-substituted compound 16
viously. Herein, we designed a series of tacrine-cinnamic acid showed comparable activity to 12. We further examined the
hybrids, and discussed the SAR for these compounds as dual site inductive effects of the substituents on the ChEs inhibitory activity.
ChEs inhibitors.
The nitro group was introduced to act as an electron-withdrawing
The synthesis of the tacrine-cinnamic acid hybrids is described group. Data showed that the nitro group could remarkably
in Scheme 1. Anthranilic acid 1 was condensed with cyclohexa- improve the activity of the compounds. The para-substituted 19
3
8,39
none 2 to yield chloro acridine 3
ent diamine led to 4–8, which were condensed with cinnamic acid IC50 ¼ 6.5 ± 0.6 nM). When the nitro group was replaced by -CF
to result in the target compounds 9–13. Compound 7 was con- compounds were less potent than 12, except for the para-substi-
. Treatment of 3 with differ- was the most potent compound (AChE IC50 ¼ 2.7 ± 0.4 nM, BuChE
,
3
tuted compound 23. Interestingly, the effects of methyl, nitro and
densed with different cinnamic acid analogues to obtain target
-
3
CF substitution on ChEs activity showed a consistent manner: for
molecule 14–38.
AChE, the activity was para- > meta- > ortho-, while it showed an
opposite manner on BuChE. The results indicated that the para-
position was optimal for the design of potent AChE inhibitor.
We subsequently designed compounds with halogen substitu-
tion. When substituted by Cl (25–27), the impact was very
similar to the above-mentioned results. When substituted by
different halogens, the activities on AChE and BuChE were
ChEs inhibitory activity and SAR analysis
The inhibitory effects on ChEs of all the target compounds were
4
0
determined according to Ellman’s method . AChE from electroph-
orus electricus (eeAChE) and BuChE from equine serum (eqBuChE)
were used in the assays. The activity was quantified by IC50 values
2
7
28
24
4-Cl > 4-Br > 4-F .
(
Table 1). All the compounds exhibited potent inhibitory activities
Next, we synthesised several methoxyl-substituted analogues to
on ChEs, with IC50 values in the nanomolar range. We firstly eval-
evaluate the impact of electro-donating effect on activity. For
4
1,42
uated the optimal linker between tacrine
and cinnamic acid mono-substituted compound 30, the activities on both AChE and
moiety, by introducing different diamines (compound 9–13). BuChE were obviously improved (AChE IC50 ¼ 3.7 ± 1.5 nM, BuChE
When n ¼ 2–4, compounds showed low activities on AChE, but IC50 ¼ 22.5 ± 5.9 nM) compared 12. For multi-substituted com-
when the diamine linker was extended to six or eight carbon pounds (31 and 32), the 3,4,5-triOCH was much more active than
3
atoms, compounds showed much improved activities (AChE the 2,3,4-triOCH3.
IC50 ¼ 26.5 ± 10.7 and 22.8 ± 9.2 nM for 12 and 13, respectively).
We subsequently introduced the hydroxy group (33–34) to the
We also observed that compound 10 and 11 exhibited high cinnamic acid moiety. Such substitution was favourable for AChE
selectivity on BuChE (selective index ¼28.7 and 71.17, respect- inhibition, the two compounds exhibited a good selectivity on
ively), indicating that they may serve as lead compounds for the AChE. We next introduced benzyloxy group at the para-position of
3
5
discovery of selective BuChE inhibitors. As 12 showed comparable the cinnamic acid moiety . Compared to 12, the activities of the
activity to 13, while its molecular weight was lower than 13, the three compounds on AChE were remarkably reduced, while the

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297
Table 1. AChE and BuChE inhibitory effects (shown as IC50 values), and selectivity index (SI) of the synthesised compounds.
O
O
O
HN
N
H
HN
N
N
H
HN
N
H
n
6
6
R
O
O
N
N
R
9
, n = 2; 10, n = 3; 11, n = 4;
2, n = 6; 13, n = 8;
14~36
37~38
1
a
IC50 (nM) ± SEM
b
c
d
Compound
R
AChE
BuChE
SI
9
–
–
–
–
–
2-CH
3-CH
4-CH
2-NO
3-NO
4-NO
328.2 ± 121.7
100.3 ± 5.2
209.3 ± 76.0
26.5 ± 10.7
22.8 ± 9.2
36.0 ± 12.3
53.1 ± 6.9
22.0 ± 3.7
17.5 ± 3.7
3.6 ± 0.5
69.1 ± 27.3
3.5 ± 0.4
2.9 ± 0.4
34.4 ± 16.6
7.8 ± 4.7
0.2
0.03
0.01
1.3
0.3
0.2
0.7
2.5
0.2
1.9
10
11
12
13
14
15
16
17
18
19
3
3
3
6.0 ± 1.5
37.3 ± 7.2
54.4 ± 11.7
3.6 ± 0.3
2
2
2
6.8 ± 0.6
2.7 ± 0.4
0.2 ± 1.2
6.5 ± 0.6e
2.4
0.6
d
1
6.3 ± 0.3
20
21
22
23
24
25
26
27
4-Cl-3-NO
2
6.2 ± 2.0
41.7 ± 8.5
61.3 ± 16.7
8.1 ± 0.3
15.4 ± 3.3
14.2 ± 3.2
16.2 ± 6.5
11.1 ± 2.2
2.4 ± 0.6
81.1 ± 15.7
16.4 ± 1.8
65.9 ± 21.4
8.7 ± 2.7
1.8
0.04
1.3
2.0
4.3
0.6
0.6
1.9
0.3
2-CF
3-CF
4-CF
4-F
3
3
3
2-Cl
3-Cl
4-Cl
9.2 ± 2.5
6.9 ± 1.2
12.9 ± 1.7
d
d
1
6.5 ± 1.7
5.7 ± 0.3
28
29
30
4-Br
3-CN
4-OCH
19.1 ± 2.0
5.1 ± 0.8
3.7 ± 1.5
29.9 ± 1.8
68.5 ± 13.6
22.5 ± 5.9
1.6
13.4
6.1
0.5
3
d
e
1
5.3 ± 1.8
8.0 ± 1.1
3
3
3
3
3
3
3
3
1
2
3
4
5
6
7
8
2,3,4-tri-OCH
3,4,5-tri-OCH
3-OH
4-OH
4-OBn
3-OMe-4-OBn
Cl
3
24.1 ± 8.1
6.4 ± 2.2
6.4 ± 2.1
2.2 ± 0.3
128.0 ± 32.3
86.3 ± 30.0
104.3 ± 26.4
127.8 ± 13.3
69.8 ± 11.1
8.1 ± 2.0
3.4 ± 0.4
0.3
0.5
3.9
19.8
0.2
0.3
4.610.2
0.5
3
25.1 ± 6.5
43.5 ± 9.3
29.0 ± 11.7
29.8 ± 7.3
22.6 ± 7.1
58.4 ± 7.5
10.6 ± 1.1
-CH
3
Tacrine
–
0.2
a
Concentration of the compound required for 50% inactivation of ChEs, data were shown in mean ± SEM of three experiments.
b
AChE (EC 3.1.1.7) from electric eel.
BuChE (EC 3.1.1.8) from horse serum.
c
d
e
AChE (EC 3.1.1.7) from human.
BuChE (EC 3.1.1.8) from human.
f
Selectivity index (SI) ¼ BuChE IC50/AChE IC50
.
BuChE activities were maintained. In addition, we synthesised Kinetic study of huAChE and huBuChE inhibition
three derivatives with 3-OMe-4-OBn substitution (36–38) on the
Next, we selected compound 19, which exhibited the very potent
ChEs inhibitory activity, to analyse its binding manner with
huAChE and huBuChE by using kinetic studies as described previ-
ously . Lineweaver–Burk reciprocal plots were applied to describe
the type of inhibition by 19. For AChE inhibition as shown in
Figure 1(A), when increase the concentration of 19 (1, 2, 4, and
cinnamic acid moiety, they also exhibited much reduced activities
against ChEs. As the binding site of BuChE was larger than AChE,
we inferred that the large benzyloxy group can be tolerated by
BuChE, while it was repelled by AChE.
To further validated the inhibitory activities of the target com-
pounds on human ChEs, the active representatives, 19, 27, and
4
3
10 nM), both the slopes and the intercepts were increased, indicat-
30, were selected for validation (Table 1). 19 exhibited huAChE
ing a decreased Vmax and a higher K . The intersection point
m
IC50 ¼ 10.2 ± 1.2 nM, huBuChE IC50 ¼ 6.3 ± 0.3 nM; 27 exhibited
huAChE IC50 ¼ 16.5 ± 1.7 nM, huBuChE IC50 ¼ 5.7 ± 0.3 nM. 30 exhib-
ited huAChE IC50 ¼ 15.3 ± 1.8 nM, huBuChE IC50 ¼ 8.0 ± 1.1 nM. The
located at the Y-axis, suggesting a noncompetitive binding
manner of 19 on huAChE. For BuChE inhibition (Figure 1(B)), the
activities were very similar to that in the eeAChE or eqBuChE intersection point located at the second quadrant, indicating a
assays. Therefore, we concluded that the synthesised compounds mixed-type inhibition of 19 on huBuChE. The detailed values of
can efficiently inhibit the activities of human ChEs, further con-
firmed their activities as ChEs inhibitors.
V
max and K
m
at different concentrations in the kinetic studies are
summarised in Table 2.

2
98
Y. CHEN ET AL.
Figure 1. Lineweaver–Burk plots resulting from subvelocity curves of huAChE and huBuChE activity with different substrate concentrations (25–450lM) in the absence
and presence of 1, 2, 4, 10 nM of 19.
Table 2. The Vmax and K
m
values for compound 19 in kinetic studies.
1,2,3,4-tetrahydroacridin ring formed a salt bridge with the side-
chain of Asp70. The tricyclic ring also interacted with the sidechain
of Tyr332 through p–p stacking contact. The oxygen atom of
amide moiety formed two hydrogen-bonds with Gly116 and
Gly117. For the cinnamic acid moiety, the phenyl ring interacted
with Trp82 and Ala328 through p–p stacking and p-alkyl contact,
respectively. The chlorine atom formed additional p-alkyl contacts
with Trp430, which further improved the binding affinity of 27.
Multiple van der Waals contacts were observed between com-
pound 27 and different residues such as Ser120, Leu286, Trp231,
Phe398, and His438, providing strong binding affinity.
Concentration (nM)
V
max (lM/min)
K
m
(lM)
R square
a
19 on AChE
0
1
2
4
2.0 ± 0.2
1.4 ± 0.1
1.2 ± 0.2
0.9 ± 0.1
0.5 ± 0.1
131.2 ± 34.0
115.9 ± 28.6
141.4 ± 60.8
169.2 ± 45.8
136.1 ± 40.6
0.98
0.98
0.94
0.98
0.97
1
0
b
19 on BuChE
0
1
2
4
0.47 ± 0.05
0.40 ± 0.03
0.25 ± 0.01
0.44 ± 0.08
0.19 ± 0.01
420.6 ± 84.6
406.1 ± 55.9
299.6 ± 40.8
741.4 ± 208.2
355.1 ± 47.8
0.99
0.99
0.99
0.99
0.99
1
0
Data are shown in mean ± SD of three experiments.
Inhibition of self-induced Ab1–42 aggregation by selected
compounds
a
AChE (EC 3.1.1.7) from human.
b
BuChE (EC 3.1.1.8) from human.
Compound 19, 27, and 30 that showed potent activity on ChEs
inhibition were further evaluated for their inhibitory capacity on
self-induced Ab1–42 aggregation based on a thioflavin T-based
fluorometric assay. Under the concentration of 25 mM, the three
compounds inhibited the aggregation of Ab1–42 with the inhibitory
rate 31.82, 42.22, and 34.57%, respectively (Table 3). 25 mM of
Resveratrol was used a reference compound, which showed
Binding mode analysis by molecular modelling
To further investigate the binding pattern of the synthesised com-
pounds with ChEs, we next performed molecular docking studies
by using Discovery Studio (DS) (version 3.0, BIOVIA) (Figure 2).
Bind mode of huAChE-19 and huBuChE-27 were analysed. The
two compounds were selected as representatives because they
30.36% inhibitory rate. The results suggested that these ChEs
exhibited very potent activity on AChE or BuChE. As shown in inhibitors had the multi-target potential in the treatment of AD.
Figure 3(A), 19 simultaneously occupied both the CAS and PAS of Therefore, the in vivo activities of the three compounds were fur-
AChE. The 1,2,3,4-tetrahydroacridin core located at the CAS, and ther evaluated.
formed p–p stacking interactions with Trp86 and His447. The -NH-
group on 1,2,3,4-tetrahydroacridin ring interacted with the back-
bone of Tyr337 through a hydrogen bond. The carbonyl group of
Behavioural studies
the cinnamic acid moiety formed two hydrogen bonds with the
sidechain of Phe295 and Arg296. Such polar interactions stabilised
the binding pattern, resulting in the location of cinnamic acid moi-
ety in the PAS site. The phenyl ring interacted with multiple resi-
dues through van der Waals interactions, such as Trp286, Leu289,
Ser293, and Val294.
To further investigate the ability of the synthesised compound to
ameliorate the cognitive ability, three representatives, 19, 27, and
30, were selected for in vivo behavioural analysis by using a
Morris water maze test in scopolamine-induced cognition-impaired
adult ICR mice (male mice, 8–10 weeks old, weight 20–25 g).
Tacrine (30 mg/kg body weight) was used as positive control. After
the treatment of the test compounds, the mean escape latency
Compared to the linear conformation of 19 when it bound to
huAChE, compound 27 exhibited a U-shaped conformation (Figure values of all the groups were shown in Figure 3 (the latency val-
(B)), which was obviously different from 19. Different from the ues of the mice in the training process are shown in Table S1). It
3
narrow and long binding site of AChE, BuChE contains a much was clear that administration of scopolamine led to a remarkable
larger and broader binding site, therefore, the U-shaped conform- delay of the latency to target (12.6 ± 1.1 s vs. 44.3 ± 3.1 s) as com-
ation of 27 can better occupy the active site of BuChE. Such phe- pared to the control group. Tacrine reduced this time to 34.5 ± 1.
nomenon also indicated the importance of the flexible linker in 3 s. All of the three synthesised compounds exhibited much
this series of compounds. The protonated nitrogen atom on improved cognitive function in the ICR mice, as the time of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
299
Figure 2. Binding mode prediction of the representative compounds with ChEs. Visualisation of 19 on huAChE (A, PDB id: 4EY7), and visualisation of 27 on huBuChE
B, PDB id: 4TPK) were shown in the figure. Compounds were shown in stick mode coloured in blue. Key residues were labelled as thin stick mode in yellow.
(
Intermolecular interactions were described as dot lines in different colours according to the type of the interaction: green, hydrogen bond; light green, hydrophobic
contact; purple, p–p stacking; pink, p-alkyl contact.
Figure 3. Effects of oral administration of tacrine (15 mg/kg), 19, 27, and 30 (15mg/kg) on scopolamine-induced cognitive impairment in ICR mice determined by
ꢃ
ꢃꢃ
ꢃꢃꢃꢃ
p < .0001 vs.
tt ha ce ri nM eo rgr ri so uwp a) .t er maze test. Tacrine (15 mg/kg) was used as positive control. Data are presented as the mean ± SEM (n ¼ 6; p < .05, p < .01,
Table 3. Inhibition of self-induced Ab1–42 aggregation and anti-proliferative that the electron-withdrawing groups might play significant role
activities of the synthesised compounds.
for the activity, as the nitro as R group showed the best perform-
ance. Halogen substitution at this position was also acceptable,
while electron-donating groups reduced the activity of the
compounds.
Inhibitory rate of compounds (25 lM) on self-induced Ab1-42 aggregation (%)
Compound
19
27
30
Resveratrol
30.36
Inhibitory rate
31.82
42.22
34.57
Hepatotoxicity studies
latency to target obviously reduced compared to tacrine. Among
For preliminary safety evaluation, we next investigated the pos-
sible drug-induced hepatotoxicity of 19, 27, and 30 by determin-
ing the levels of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST), two known markers of liver damage
them, compound 19 and 27 showed comparable performance (25.
ꢃꢃ ꢃ
± 1.5 s, p < .01, 25.6 ± 1.8 s, p < .05, respectively), while 30 was
0
slightly less positive (29.4 ± 1.9 s). The data of latency to target
were also supported by the analysis of the trajectories of the mice
in each group. For the mice in scopolamine model group (Figure
(
Table 4 and Figure 5). Heparinised serum was collected after the
treatment of the compounds at 8, 22, and 36 h. Generally, all the
three synthesised compounds did not show remarkable hepato-
4
(B)), the trajectory was very long and disordered as compared to
the control group (Figure 4(A)). The performance of tacrine group toxicity, as the levels of ALT and AST were comparable to those
was improved, but still much longer than the control group from the control group at the three time points. Therefore, these
(
Figure 4(C)). For mice treated with 19, 27, and 30 (Figure 4(D–F)), compounds showed preliminary safety as expected. In addition,
they showed much shortened distances as compared to tacrine the morphologic results of the three compounds by immunohisto-
group, with a similar orientation and distance to that of the nor- chemical staining were in accordance with the ALT and AST data.
mal mice. The results indicated that the cognitive function of mice Treatment of tacrine (Figure 6(C)), 19 (Figure 6(D)), 27 (Figure 6(E))
in these three groups were much recovered.
or 30 (Figure 6(F)) did not cause remarkable morphologic changes
The results confirmed that the synthesised compounds were in liver compared to the control group (Figure 6(A)). Taken
in vivo active and had the potential as anti-AD agents through together, our synthesised compounds were safe for further
ameliorating the cognitive function. Besides, it also indicated development.

3
00
Y. CHEN ET AL.
Figure 4. The trajectories of mice in control (A), model (B), tacrine (C), 19 (D), 27 (E), and 30 (F) group in the Morris water maze test.
Table 4. The determination of ALT and AST level (U/L) after the administration of test compounds.
Group
8 h
22 h
36 h
ALT (U/L)
Control
Model
Tacrine
31.2 ± 3.2
38.3 ± 2.5
38.0 ± 3.5
36.0 ± 2.9
35.4 ± 4.2
35.4 ± 4.7
39.7 ± 2.6
39.9 ± 4.4
38.7 ± 5.2
44.4 ± 3.9
39.4 ± 3.7
43.4 ± 2.5
41.1 ± 3.4
40.1 ± 4.1
40.9 ± 3.5
40.2 ± 2.5
37.1 ± 3.5
39.4 ± 2.7
1
2
3
9
7
0
AST (U/L)
Control
Model
Tacrine
118.9 ± 14.4
132.2 ± 7.6
134.5 ± 9.9
119.6 ± 10.8
123.3 ± 15.6
116.7 ± 16.9
128.9 ± 13.1
134.1 ± 9.6
125.8 ± 16.2
130.6 ± 8.2
133.9 ± 8.4
128.3 ± 5.9
132.9 ± 9.9
139.7 ± 5.9
138.7 ± 8.5
133.9 ± 13.5
130.6 ± 8.3
129.2 ± 12.1
1
2
3
9
7
0
Tacrine (30 mg/kg) was used as the reference compound. Values were expressed as the Mean ± SD (n ¼ 6).
Conclusions
further provide structural information in designing potent ChEIs.
In the present study, with the aim to identify multi-target directed The binding manner of compound 19 to huAChE and huBuChE
compounds as new anti-AD agents, a series of tacrine-cinnamic was also analysed by kinetic and molecular docking studies. The
acid hybrids were synthesised. Most of the compounds potently representatives, compound 19, 27, and 30, effectively inhibited
inhibited both AChE and BuChE, with the IC50 values in the nano- the self-induced Ab1–42 aggregation. Subsequent in vivo evaluation
molar range. The SAR study indicated that the optimal linker of the three compounds showed that they remarkably reduced
between the tacrine and cinnamic acid moiety was the six-carbon the scopolamine-induced cognitive impairment in the Morris water
alkyl chain. Extension the length of the linker, or introduction of maze test. In addition, the compounds exhibited preliminary safety
bulky group such as benzyl group to the cinnamic acid moiety, in hepatotoxicity studies, without improving the level of ALT and
resulted in much reduced ChEs inhibitory effects. Our findings AST. Our findings enlarge the SAR of tacrine-based hybrids, and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
301
Figure 5. The determination of ALT (A) and AST (B) level after the treatment of test compounds. Values are expressed as mean ± SEM (n ¼ 6).
Figure 6. The histomorphological appearance of livers of male mice after treatment with the solvent only (control, A), or 36 h after administration of scopolamine (B),
tacrine (C), 19 (D), 27 (E) and 30 (F). HE staining, original magnificationꢀ200.
provide promising lead compounds for further optimisation of
new therapeutic agents on AD.
acetylcholinesterase-induced
Bioorg Med Chem Lett 2012;22:2257–61.
beta-amyloid
aggregation.
3
.
.
Spilovska K, Zemek F, Korabecny J, et al. Adamantane – a
lead structure for drugs in clinical practice. Curr Med Chem
2016;23:3245–66.
Disclosure statement
4
Kuca K, Soukup O, Maresova P, et al. Current approaches
The authors declare no competing financial interests.
against Alzheimer’s disease in clinical trials. J Braz Chem Soc
2016;27:641–9.
Funding
5. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy.
Physiol Rev 2001;81:741–66.
We gratefully thank the support from the grants 81402851,
6.
7.
8.
9.
Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic
hypothesis of Alzheimer’s disease: a review of progress.
J Neurol Neurosurg Psychiatry 1999;66:137–47.
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Ballatore C, Lee VM, Trojanowski JQ. Tau-mediated neurode-
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Nat Rev Neurosci 2007;8:663–72.
8
1573281, and 81603529 of National Natural Science Foundation
of China, BK20140957 of Natural Science Foundation of Jiangsu
Province, Natural Science Foundation of the Jiangsu Higher
Education Institutions (16KJB360002). We also thank the support
from Fundamental Research Funds for the Central Universities
(2015ZD009), Jiangsu Qing Lan Project, Top-notch Academic
Programs Project of Jiangsu Higher Education Institutions (TAPP-
PPZY2015A070) and Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
Webber KM, Raina AK, Marlatt MW, et al. The cell cycle in
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