Design, synthesis and evaluation of quinolinone derivatives containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of Alzheimer’s disease

Article abstract of DOI:10.1080/14756366.2019.1687460

A series of novel quinolinone derivatives bearing dithiocarbamate moiety were designed and synthesised as multifunctional AChE inhibitors for the treatment of AD. Most of these compounds exhibited strong and clearly selective inhibition to eeAChE. Among them, compound 4c was identified as the most potent inhibitor to both eeAChE and hAChE (IC₅₀ = 0.22 μM for eeAChE; IC₅₀ = 0.16 μM for hAChE), and it was also the best inhibitor to AChE-induced Aβ aggregation (29.02% at 100 μM) and an efficient inhibitor to self-induced Aβ aggregation (30.67% at 25 μM). Kinetic and molecular modelling studies indicated that compound 4c was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4c had good ability to cross the BBB, showed no toxicity on SH-SY5Y neuroblastoma cells and was well tolerated in mice at doses up to 2500 mg/kg (po).

Full text of DOI:10.1080/14756366.2019.1687460

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and evaluation of quinolinone
derivatives containing dithiocarbamate moiety as
multifunctional AChE inhibitors for the treatment
of Alzheimer’s disease
Jie Fu, Fengqi Bao, Min Gu, Jing Liu, Zhipeng Zhang, Jiaoli Ding, Sai-Sai Xie &
Jinsong Ding
To cite this article: Jie Fu, Fengqi Bao, Min Gu, Jing Liu, Zhipeng Zhang, Jiaoli Ding, Sai-
Sai Xie & Jinsong Ding (2020) Design, synthesis and evaluation of quinolinone derivatives
containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of
Alzheimer’s disease, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 118-128, DOI:
10.1080/14756366.2019.1687460
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 118–128
https://doi.org/10.1080/14756366.2019.1687460
RESEARCH PAPER
Design, synthesis and evaluation of quinolinone derivatives containing
dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment
of Alzheimer’s disease
Jie Fua,b, Fengqi Baob, Min Gub, Jing Liud, Zhipeng Zhangc, Jiaoli Dingc, Sai-Sai Xiec
and Jinsong Dinga
b
aXiangya School of Pharmaceutical Sciences, Central South University, Changsha, China; Jiangsu Zeyun Pharmaceutical Co., Ltd, Xibei Town
c
Industrial Park, Wuxi, China; National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of
d
Traditional Chinese Medicine, Nanchang, China; School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, PR China
ABSTRACT
ARTICLE HISTORY
Received 24 July 2019
Revised 27 September 2019
Accepted 25 October 2019
A series of novel quinolinone derivatives bearing dithiocarbamate moiety were designed and synthesised
as multifunctional AChE inhibitors for the treatment of AD. Most of these compounds exhibited strong
and clearly selective inhibition to eeAChE. Among them, compound 4c was identified as the most potent
inhibitor to both eeAChE and hAChE (IC50 ¼ 0.22 lM for eeAChE; IC50 ¼ 0.16 lM for hAChE), and it was
also the best inhibitor to AChE-induced Ab aggregation (29.02% at 100lM) and an efficient inhibitor to
self-induced Ab aggregation (30.67% at 25 lM). Kinetic and molecular modelling studies indicated that
compound 4c was a mixed-type inhibitor, which could interact simultaneously with the catalytic anionic
site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4c had good ability to cross the BBB,
showed no toxicity on SH-SY5Y neuroblastoma cells and was well tolerated in mice at doses up to
2500 mg/kg (po).
KEYWORDS
Dithiocarbamate; quinoli-
none; cholinesterase;
Alzheimer’s disease;
multifunctional inhibitors
1. Introduction
butyrylcholinesterase (BuChE)6. However, in contrast to AChE,
BuChE is mainly distributed in the peripheral areas, and the inhib-
Alzheimer’s disease (AD), a progressive and irreversible neurode-
generative disorder, is the most common cause leading to mem-
ory impairment and dementia in elderly people1. According to
World Alzheimer Report, nearly 50 million people worldwide are
living with dementia in 2018, and the number is likely to reach
152 million by 20502. This disease has higher use and costs of
health care and long-term care services than other conditions3.
Therefore, such a large number of patients will bring heavy bur-
den to public health system in the future.
AD was first identified >100 years ago, but the exact aetiology
is still largely unknown. Many clinical hallmarks, including deficits
of acetylcholine (ACh), amyloid-b (Ab) peptide deposits, oxidative
stress, dyshomeostasis of biometals and hyperphosphorylated tau
protein, are thought to be associated with the initiation and
development of AD. Over the past years, although great efforts
have been devoted to develop novel drugs to treat AD by both
pharmaceutical companies and academic institutions, no new
drugs have entered the clinical market and >200 drug candidates
ended up with failure in advanced clinical trials. Currently, the
clinical treatment of AD is mainly based on the cholinergic
hypothesis, which states that improving the ACh level in brain
through inhibition of ACh hydrolysis can alleviate the memory
loss and cognitive decline related to AD4,5. At the neuronal level,
there are two types of cholinesterases (ChEs) that can catalyse
ition of BuChE may cause side effect in the peripheral system7,8
.
Thus, selective inhibition of AChE seems more beneficial for
AD treatment.
In addition to cholinergic hypothesis, another hypothesis, so-
called amyloid-b (Ab) pathological hypothesis, has attracted great
interest in recent years9. This hypothesis posits that the gradual
accumulation and aggregation of Ab peptide in the brain is a cen-
tral event that results in neurodegeneration and dementia10,11. Ab
peptides consist of 39–43 residues, which are generated via
sequential scission of the amyloid precursor protein (APP) by b-
and c-secretases. Ab peptides can aggregate into monomers,
oligomers and large Ab plaques, which initiate a neurodegenera-
tive cascade that involves inflammation, oxidative stress, and mito-
chondrial dysfunction, eventually leading to neuronal death and
cognitive dysfunction12. Therefore, prevention of Ab aggregation
provides another therapeutic approach to halt AD pathogenesis.
In fact, besides the vital role in nerve transmission, AChE also
plays an important role in processing of Ab peptides13. The crys-
tallographic structure of AChE shows that it has a nearly 20 Å
deep narrow gorge which consists of two binding sites: a catalytic
active site (CAS) at the bottom of the gorge and a peripheral
anionic site (PAS) near the entry of the gorge14. Generally, inhibi-
tors that bind to either one site can inhibit the AChE. However,
recent reports indicate that, apart from catalysing ACh hydrolysis,
ACh hydrolysis, namely acetylcholinesterase (AChE) and the PAS also has a close relationship with the Ab aggregation.
CONTACT Sai-Sai Xie
University of Traditional Chinese Medicine, Nanchang 330006, China; Jinsong Ding
South University, Changsha 410083, China
xiesaisainanchang@hotmail.com
National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi
dingjs0221@163.com
Xiangya School of Pharmaceutical Sciences, Central
Supplemental data for this article can be accessed here.
ß 2019 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
119
The PAS can accelerate the formation of Ab aggregation and
amyloid fibril. The interaction between Ab and the PAS produces
stable AChE-Ab complexes, which are more toxic than Ab peptide
aggregates15,16. Accordingly, dual binding site inhibitors able to
interact with both the CAS and PAS will be more promising for
treating AD, as they can not only improve cognition by inhibiting
AChE, but also provide additional benefit through reducing the
Ab aggregation induced by AChE. The basic precept of designing
dual binding site AChE inhibitors is to take three parts to fulfil the
structural requirements of AChE. The three parts are a PAS bind-
ing moiety, a CAS binding moiety and a linker connecting these
two moieties.
To date, there are four AChE inhibitors and one N-methyl-D-
aspartate receptor antagonist that have been approved for the
treatment of AD in clinic. However, due to the complex aetiology
of AD, these single-target drugs can only achieve some temporary
amelioration of the symptoms instead of slowing or halting the
disease progress. Thus, a more appropriate approach for treating
this disease is development of multifunctional molecules that can
simultaneously modulate multiple targets or pathways related to
AD. The most popular method for obtaining these molecules is to
design a dual binding site AChE inhibitor and make it exert add-
itional activities because of the fragments introduced.
S
HN
SH
HO
N
H
O
Quinolinone
Dithiocarbamate
Flexible linker
CAS binding
R1
R2
S
HN
S
O
N
O
H
PAS binding
Versatile
pharmacophore
Figure 1. Rational design of quinolinone derivatives as multifunctional AChE
inhibitors for the treatment of AD.
structure-activity relationship (SAR) studies. All designed com-
pounds were synthesised and evaluated for their ability to inhibit
ChEs and cross the blood-brain barrier (BBB). The compounds with
potential activities were also chosen for studies including the
inhibitory evaluation of hAChE and Ab aggregation, kinetic study
of AChE inhibition, and molecular modelling studies of their bind-
ing modes with AChE and Ab1-42. Finally, the most promising
compound was selected to test its cytotoxicity in SH-SY5Y neuro-
blastoma cells and acute toxicity in mice. Herein, we report the
The quinolinone scaffold is prevalent in a variety of pharmaco-
logically active synthetic and natural compounds17. The reported
biological activities of quinolinone derivatives including antioxida-
tion, anti-osteoporosis, anti-influenza and anticancer activities,
etc.18. Recent studies indicated that several quinolinone deriva-
tives also presented potent AChE inhibitory activity, and molecular
docking studies showed that the quinolinone moiety could bind
to the PAS through a p-p stacking interaction19,20. Therefore, it
can serve as a PAS binding moiety for design of dual binding site design, synthesis and evaluation of a series of novel quinolinone
inhibitor. On the other hand, dithiocarbamate is a versatile
pharmacophore, which has been receiving intense attention in
recent years. Very recently, our group found that this moiety
could interact with CAS of AChE, and many compounds possess-
ing this moiety also displayed promising inhibitory activity for
self-induced Ab aggregation21,22. Meanwhile, unlike the benzyl
piperidine of donepezil or tacrine that have been widely used as
CAS binding group for design of multifunctional AChE inhibitors,
derivatives containing dithiocarbamate moiety as multifunctional
AChE inhibitors for AD treatment.
2. Experimental
2.1. Chemistry
All reagents and solvents were obtained from commercial suppli-
the dithiocarbamate is still rarely used. Therefore, it is interesting ers and used without further purification unless specified. The
to explore this scaffold as a CAS binding group to design multi-
functional AChE inhibitors.
reactions were monitored by analytical thin-layer chromatography
on Silica Gel aluminium sheets and visualised with a UV lamp.
Melting points were determined with an XT-4 micromelting point
apparatus and are uncorrected. The NMR spectra were recorded
on a Bruker ACF-600 spectrometer using the tetramethylsilane
(TMS) as internal standard. High resolution mass spectra (HRMS)
were taken on a Triple TOF 5600 spectrometer (AB Sciex).
Based on the facts and considerations mentioned above, in
present study, we attempted to connect quinolinone fragment
with dithiocarbamate moiety to design a series of new com-
pounds as multifunctional dual binding site AChE inhibitors for
the treatment of AD (Figure 1). The quinolinone was selected to
inhibit AChE-induced Ab aggregation by binding to PAS, while the
dithiocarbamate moiety was used to inhibit AChE through inter-
acting with the CAS. A flexible linker was exploited to connect
these two moieties, because such linker could allow designed
compounds simultaneously bind to both CAS and PAS. In add-
ition, since our previous studies indicated that compounds bear-
ing dithiocarbamate moiety could efficiently inhibit self-induced
Ab aggregation, these new compounds were also expected to
present similar activity, thereby exerting multifunctional effects.
Furthermore, in our initial step, a piperidine dithiocarbamate
moiety was first used as a starting fragment to explore the opti-
mal linker length, because the previous studies suggested that
this moiety could potently and selectively inhibit AChE. And once
the optimal length was determined, various secondary amine
2.2. General procedures for the preparation of compounds 3a-e
and 8a-b
The intermediates 2 and 7a-b were prepared according to the lit-
erature methods20,23,24, and the structures of these compounds
were characterised by mass spectra. Then, to a mixture of 2/7a-b
(5.0 mmol) and anhydrous K2CO3 (1.4 g, 10 mmol) in acetone
(15 mL) was added appropriate a, x-dibromoalkanes (50 mmol),
and the reaction was refluxed under stirring for 4 h. After complet-
ing, the mixture was filtered, and the filtrate was evaporated
under reduced pressure. The obtained residue was purified by sil-
ica gel chromatography with PE/EA (20:1) as eluent to give com-
groups were introduced to dithiocarbamate moiety for further pounds 3a-e and 8a-b as white solid.

120
J. FU ET AL.
2.2.1. 7–(2-bromoethoxy)quinolin-2(1H)-one (3a)
2.3.1. 2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethyl piperidine-1-car-
bodithioate (4a)
1
Yield 77%. H NMR (600 MHz, CDCl3) d 7.77 (d, J ¼ 9.3 Hz, 1H), 7.48
Yield 86%; mp 221–222 ꢀC; 1H NMR (600 MHz, DMSO-d6) d 11.56
(s, 1H), 7.81 (d, J ¼ 9.5 Hz, 1H), 7.57 (d, J ¼ 8.6 Hz, 1H), 6.83 (dd,
J ¼ 8.6, 2.4 Hz, 1H), 6.80 (d, J ¼ 2.5 Hz, 1H), 6.31 (dd, J ¼ 9.4, 1.1 Hz,
1H), 4.24 (t, J ¼ 6.2 Hz, 4H), 3.92 (s, 2H), 3.71 (t, J ¼ 6.3 Hz, 2H), 1.76
– 1.32 (m, 6H). 13 C NMR (150 MHz, DMSO-d6) d 193.33, 162.64,
160.30, 141.04, 140.44, 129.84, 119.24, 114.03, 111.07, 99.27, 66.68,
53.09, 51.49, 35.62, 26.29, 25.66, 23.99. HRMS: calcd for
C17H21N2O2S2 [M þ H]þ 349.1039, found 349.1028.
(d, J ¼ 8.6 Hz, 1H), 6.91 (d, J ¼ 2.4 Hz, 1H), 6.84 (dd, J ¼ 8.6, 2.4 Hz,
1H), 6.59 (d, J ¼ 9.3 Hz, 1H), 4.35 (t, J ¼ 6.3 Hz, 2H), 3.69 (t,
J ¼ 6.3 Hz, 2H).
2.2.2. 7–(3-bromopropoxy)quinolin-2(1H)-one (3 b)
1
Yield 79%. H NMR (600 MHz, CDCl3) d 7.79 (d, J ¼ 9.4 Hz, 1H), 7.50
(d, J ¼ 8.7 Hz, 1H), 6.93 (d, J ¼ 2.4 Hz, 1H), 6.86 (dd, J ¼ 8.7, 2.4 Hz,
1H), 6.61 (d, J ¼ 9.4 Hz, 1H), 4.24 (t, J ¼ 5.8 Hz, 2H), 3.65 (t,
J ¼ 6.4 Hz, 2H), 2.39 (p, J ¼ 6.1 Hz, 2H).
2.3.2. 3-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)propyl piperidine-1-
carbodithioate (4 b)
Yield 83%; mp 186–187 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.15 (s,
1H), 7.78 (d, J ¼ 9.3 Hz, 1H), 7.48 (d, J ¼ 8.6 Hz, 1H), 6.91 – 6.81 (m,
2H), 6.60 (d, J ¼ 9.4 Hz, 1H), 4.33 (br s, 2H), 4.20 (t, J ¼ 6.0 Hz, 2H),
3.93 (br s, 2H), 3.55 (t, J ¼ 7.1 Hz, 2H), 2.36 – 2.18 (m, 2H), 1.76 –
1.71 (m, 6H). 13 C NMR (150 MHz, CDCl3) d 195.24, 164.72, 161.27,
141.05, 140.18, 129.08, 117.75, 114.38, 112.85, 99.08, 66.84, 52.95,
51.38, 33.37, 28.56, 26.03, 25.44, 24.31. HRMS: calcd for
C18H23N2O2S2 [M þ H]þ 363.1195, found 363.1182.
2.2.3. 7–(4-bromobutoxy)quinolin-2(1H)-one (3c)
1
Yield 78%. H NMR (600 MHz, CDCl3) d 7.76 (d, J ¼ 9.3 Hz, 1H), 7.47
(d, J ¼ 8.6 Hz, 1H), 6.86 (d, J ¼ 2.3 Hz, 1H), 6.83 (dd, J ¼ 8.6, 2.3 Hz,
1H), 6.58 (d, J ¼ 9.4 Hz, 1H), 4.12 (t, J ¼ 6.0 Hz, 2H), 3.53 (t,
J ¼ 6.6 Hz, 2H), 2.12 (p, J ¼ 6.8 Hz, 2H), 2.04 – 1.99 (m, 2H).
2.2.4. 7-((5-bromopentyl)oxy)quinolin-2(1H)-one (3d)
1
Yield 76%. H NMR (600 MHz, CDCl3) d 7.76 (d, J ¼ 9.4 Hz, 1H), 7.46
(d, J ¼ 9.3 Hz, 1H), 6.87 (dd, J ¼ 9.3, 2.4 Hz, 1H), 6.83 (d, J ¼ 2.4 Hz,
1H), 6.56 (d, J ¼ 9.4 Hz, 1H), 4.10 (t, J ¼ 6.1 Hz, 2H), 3.45 (t,
J ¼ 6.1 Hz, 2H), 1.98 (br s, 2H), 1.87 (br s, 2H), 1.67 (br s, 2H).
2.3.3. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl piperidine-1-car-
bodithioate (4c)
Yield 84%; mp 151–152 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.24 (s,
1H), 7.76 (d, J ¼ 9.4 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz, 1H), 6.86 (d,
J ¼ 2.3 Hz, 1H), 6.83 (dd, J ¼ 8.6, 2.3 Hz, 1H), 6.58 (d, J ¼ 9.4 Hz, 1H),
4.32 (br s, 2H), 4.12 (t, J ¼ 5.8 Hz, 2H), 3.92 (br s, 2H), 3.42 (t,
J ¼ 6.9 Hz, 2H), 2.07 – 1.87 (m, 4H), 1.75 – 1.70 (m, 6H). 13 C NMR
(150 MHz, CDCl3) d 195.66, 164.89, 161.36, 140.94, 140.31, 129.04,
117.85, 114.26, 112.74, 99.05, 67.84, 52.88, 51.38, 36.76, 28.37,
25.54, 24.35. HRMS: calcd for C19H25N2O2S2 [M þ H]þ 377.1352,
found 377.1341.
2.2.5. 7-((6-bromohexyl)oxy)quinolin-2(1H)-one (3e)
1
Yield 74%. H NMR (600 MHz, CDCl3) d 7.76 (d, J ¼ 9.4 Hz, 1H), 7.46
(d, J ¼ 9.3 Hz, 1H), 6.89 – 6.72 (m, 2H), 6.56 (d, J ¼ 9.4 Hz, 1H), 4.07
(t, J ¼ 6.3 Hz, 2H), 3.44 (t, J ¼ 6.8 Hz, 2H), 1.97 – 1.73 (m, 4H), 1.53
(p, J ¼ 3.6 Hz, 4H).
2.2.6. 7–(4-bromobutoxy)-4-methylquinolin-2(1H)-one (8a)
1
Yield 77%. H NMR (600 MHz, CDCl3) d 7.59 (d, J ¼ 8.7 Hz, 1H), 6.85
(br s, 1H), 6.83 (br s, 1H), 6.46 (s, 1H), 4.11 (t, J ¼ 6.0 Hz, 2H), 3.51
(t, J ¼ 6.5 Hz, 2H), 2.48 (s, 3H), 2.10 (p, J ¼ 6.8 Hz, 2H), 1.99 (p,
J ¼ 6.8 Hz, 2H).
2.3.4. 5-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)pentyl piperidine-1-
carbodithioate (4d)
Yield 80%; mp 148–149 ꢀC; 1H NMR (600 MHz, CDCl3) d 11.61 (s,
1H), 7.78 (d, J ¼ 9.4 Hz, 1H), 7.48 (d, J ¼ 8.7 Hz, 1H), 6.85 (dd,
J ¼ 8.6, 2.3 Hz, 1H), 6.82 (d, J ¼ 2.3 Hz, 1H), 6.58 (d, J ¼ 9.4 Hz, 1H),
4.33 (br s, 2H), 4.09 (t, J ¼ 6.4 Hz, 2H), 3.92 (br s, 2H), 3.54 – 3.24
(m, 2H), 1.92 – 1.80 (m, 5H), 1.76 – 1.64 (m, 7H). 13 C NMR
(150 MHz, CDCl3) d 195.86, 164.48, 161.52, 141.04, 140.14, 129.12,
117.72, 114.26, 112.81, 98.97, 68.20, 52.88, 51.30, 36.98, 28.69,
28.56, 25.44, 24.35. HRMS: calcd for C20H27N2O2S2 [M þ H]þ
391.1508, found 391.1527.
2.2.7. 7–(4-bromobutoxy)-3,4-dimethylquinolin-2(1H)-one (8 b)
1
Yield 75%. H NMR (600 MHz, CDCl3) d 7.63 (d, J ¼ 9.0 Hz, 1H), 6.85
(dd, J ¼ 9.0, 2.5 Hz, 1H), 6.70 (d, J ¼ 2.5 Hz, 1H), 4.12 (t, J ¼ 6.0 Hz,
2H), 3.53 (t, J ¼ 6.5 Hz, 2H), 2.45 (s, 3H), 2.27 (s, 3H), 2.09 (p,
J ¼ 6.8 Hz, 2H), 1.98 (p, J ¼ 6.8 Hz, 2H).
2.3. General procedure for the preparation of compounds 4a-m
and 9a-b
2.3.5. 6-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)hexyl piperidine-1-car-
bodithioate (4e)
To a solution of appropriate secondary amine (1.2 mmol) and TEA
(131 mg, 1.3 mmol) in DMF (2 mL) was added CS2 (99 mg, Yield 85%; mp 118–120 ꢀC; 1H NMR (600 MHz, DMSO-d6) d 11.57
1.3 mmol) drop wise. After stirring for 5 min, another solution con- (s, 1H), 7.80 (d, J ¼ 9.4 Hz, 1H), 7.55 (d, J ¼ 9.4 Hz, 1H), 6.78 (d,
taining corresponding 2-quinolinone derivatives 3a-e or 8a-b J ¼ 7.1 Hz, 2H), 6.29 (d, J ¼ 9.4 Hz, 1H), 4.22 (br s, 2H), 4.00 (t,
(1.2 mmol) in DMF (3 mL) was added, and the mixture was stirred J ¼ 6.5 Hz, 2H), 3.88 (br s, 2H), 3.24 (t, J ¼ 7.2 Hz, 2H), 1.74 (q,
at room temperature for 12 h. After completing, the mixture was J ¼ 6.7 Hz, 2H), 1.71 – 1.60 (m, 4H), 1.56 (tq, J ¼ 8.2, 5.3, 4.3 Hz, 4H),
diluted with water, and the standard extraction procedure was 1.48 – 1.38 (m, 4H). 13 C NMR (150 MHz, DMSO-d6) d 194.42,
carried out to afford the crude product, which was purified by sil- 162.70, 160.91, 141.13, 140.48, 129.71, 118.94, 113.72, 111.31,
ica gel chromatography with PE/EA (10:1) as eluent to obtain the 98.99, 68.07, 52.67, 51.22, 36.60, 28.85, 28.52, 25.52, 24.06. HRMS:
target compounds.
calcd for C21H29N2O2S2 [M þ H]þ 405.1665, found 405.1685.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
121
2.3.6. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl 4-methylpiperi- 29.17, 28.56, 28.27, 25.70. HRMS: calcd for C20H27N2O3S2 [M þ H]þ
407.1457, found 407.1494.
dine-1-carbodithio-ate (4f)
Yield 84%; mp 143–144 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.54 (s,
1H), 7.76 (d, J ¼ 9.4 Hz, 1H), 7.45 (d, J ¼ 8.6 Hz, 1H), 6.86 (d,
J ¼ 2.2 Hz, 1H), 6.82 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.57 (d, J ¼ 9.4 Hz, 1H),
5.56 (br s, 1H), 4.63 (br s, 1H), 4.11 (t, J ¼ 5.8 Hz, 2H), 3.42 – 3.40
(m, 2H), 3.18 (br s, 1H), 3.09 (br s, 1H), 1.98 – 1.92 (m, 4H), 1.79 –
1.74 (m, 3H), 1.29 (br s, 2H), 0.99 (d, J ¼ 6.0 Hz, 3H). 13 C NMR
(150 MHz, CDCl3) d 195.73, 165.06, 161.32, 140.87, 140.36, 129.00,
117.89, 114.22, 112.66, 99.03, 67.80, 52.11, 50.40, 36.81, 34.01,
33.52, 30.99, 28.38, 25.52, 21.30. HRMS: calcd for C20H27N2O2S2
[M þ H]þ 391.1508, found 391.1534.
2.3.11. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl 4-methylpiper-
azine-1-car-bodithioate (4k)
Yield 86%; mp 182–184 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.29 (s,
1H), 7.74 (d, J ¼ 9.4 Hz, 1H), 7.45 (d, J ¼ 8.6 Hz, 1H), 6.84 (d,
J ¼ 2.3 Hz, 1H), 6.82 (dd, J ¼ 8.6, 2.4 Hz, 1H), 6.57 (d, J ¼ 9.4 Hz, 1H),
4.39 (br s, 2H), 4.11 (t, J ¼ 5.7 Hz, 2H), 3.99 (br s, 2H), 3.42 (t,
J ¼ 6.8 Hz, 2H), 2.52 – 2.46 (m, 4H), 2.35 (br s, 3H), 2.00 – 1.90 (m,
4H). 13 C NMR (150 MHz, CDCl3) d 197.05, 164.95, 161.28, 140.83,
140.35, 129.03, 117.97, 114.20, 112.58, 99.01, 67.76, 54.41, 50.30,
49.61, 45.64, 36.73, 28.36, 25.48. HRMS: calcd for C19H26N3O2S2
[M þ H]þ 392.1461, found 392.1498.
2.3.7. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl 4-isopropylpi-
peridine-1-carbodithi-oate (4 g)
Yield 80%; mp 126–127 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.29 (s,
1H), 7.74 (d, J ¼ 9.4 Hz, 1H), 7.45 (d, J ¼ 8.6 Hz, 1H), 6.84 (d, 2.3.12. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl morpholine-4-
J ¼ 2.3 Hz, 1H), 6.82 (dd, J ¼ 8.6, 2.4 Hz, 1H), 6.57 (d, J ¼ 9.4 Hz, 1H),
carbodithioate (4 l)
Yield 88%; mp 156–158 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.46 (s,
4.38 (s, 2H), 4.11 (t, J ¼ 5.8 Hz, 2H), 3.98 (s, 2H), 3.43 (t, J ¼ 6.8 Hz,
2H), 2.77 (p, J ¼ 6.4 Hz, 1H), 2.62 (br s, 4H), 2.05 – 1.87 (m, 4H),
1.84 (s, 1H), 1.08 (s, 3H), 1.07 (s, 3H). 13 C NMR (150 MHz, CDCl3) d
196.57, 164.95, 161.29, 140.83, 140.35, 129.02, 117.97, 114.19,
112.59, 99.01, 67.77, 54.39, 51.44, 50.11, 48.16, 36.66, 28.36, 25.50,
18.42. HRMS: calcd for C22H31N2O2S2 [M þ H]þ 419.1821,
found 420.1809.
1H), 7.77 (d, J ¼ 9.4 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz, 1H), 6.87 (d,
J ¼ 2.4 Hz, 1H), 6.82 (dd, J ¼ 8.6, 2.4 Hz, 1H), 6.58 (d, J ¼ 9.3 Hz, 1H),
4.37 (br s, 2H), 4.12 (t, J ¼ 5.6 Hz, 2H), 3.98 (br s, 2H), 3.78 (br s,
4H), 3.44 (t, J ¼ 6.7 Hz, 2H), 2.00 – 1.94 (m, 4H). 13 C NMR (150 MHz,
CDCl3) d 197.65, 165.17, 161.30, 140.97, 140.33, 129.02, 114.29,
112.72, 99.07, 67.75, 66.41, 51.28, 50.43, 36.59, 28.35, 25.47. HRMS:
calcd for C18H23N2O3S2 [M þ H]þ 379.1144, found 379.1186.
2.3.8. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl [1,40-bipiperi-
dine]-10-carbodithio -ate (4 h)
2.3.13. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl pyrrolidine-1-
carbodithioate (4 m)
Yield 79%; mp 129–130 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.39 (s,
1H), 7.75 (d, J ¼ 9.4 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz, 1H), 6.85 – 6.81 (dd,
J ¼ 8.1, 2.5 Hz, 2H), 6.57 (d, J ¼ 9.5 Hz, 1H), 5.60 (s, 1H), 4.69 (s, 1H),
4.11 (t, J ¼ 5.9 Hz, 2H), 3.41 (s, 2H), 3.14 (br s, 2H), 2.64 (s, 1H), 2.52
(s, 4H), 2.02 – 1.83 (m, 6H), 1.60 (br s, 6H), 1.45 (br s, 2H). 13 C
NMR (150 MHz, CDCl3) d 196.08, 165.00, 161.29, 140.82, 140.37,
129.02, 118.00, 114.19, 112.57, 99.01, 67.78, 62.07, 51.12, 50.26,
49.29, 36.94, 28.36, 27.97, 27.33, 26.30, 25.50, 24.65. HRMS: calcd
for C24H34N3O2S2 [M þ H]þ 460.2087, found 460.2129.
Yield 86%; mp 170–172 ꢀC; 1H NMR (600 MHz, CDCl3) d 12.26 (s,
1H), 7.75 (d, J ¼ 9.3 Hz, 1H), 7.46 (d, J ¼ 8.6 Hz, 1H), 6.85 (d,
J ¼ 2.3 Hz, 1H), 6.83 (dd, J ¼ 8.6, 2.3 Hz, 1H), 6.59 (d, J ¼ 9.4 Hz, 1H),
4.11 (t, J ¼ 5.8 Hz, 2H), 3.96 (t, J ¼ 7.2 Hz, 2H), 3.67 (t, J ¼ 6.6 Hz,
2H), 3.42 (t, J ¼ 6.9 Hz, 2H), 2.12 – 2.05 (m, 2H), 2.02 – 1.87 (m, 6H).
13 C NMR (150 MHz, CDCl3) d 192.80, 164.86, 161.34, 140.91,
140.30, 129.03, 117.84, 114.24, 112.69, 99.03, 67.82, 54.97, 50.63,
36.02, 28.28, 26.04, 25.69, 24.31. HRMS: calcd for C18H23N2O2S2
[M þ H]þ 363.1195, found 363.1234.
2.3.9. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl 4-hydroxypiperi-
dine-1-carbodithio -ate (4i)
2.3.14. 4-((4-methyl-2-oxo-1,2-dihydroquinolin-7-yl)oxy)butylpiperi-
dine-1-carbodithioate (9a)
Yield 89%; mp 197–198 ꢀC; 1H NMR (600 MHz, DMSO-d6) d 11.58
(s, 1H), 7.81 (d, J ¼ 9.4 Hz, 1H), 7.55 (d, J ¼ 9.0 Hz, 1H), 6.80 – 6.77
(m, 2H), 6.29 (d, J ¼ 9.4 Hz, 1H), 4.89 (d, J ¼ 4.2 Hz, 1H), 4.56 (s,
1H),4.11 (br s, 1H), 4.03 (t, J ¼ 6.0 Hz, 2H), 3.94 (br s, 1H), 3.84 –
3.81(m, 1H), 3.69 (br s, 1H), 3.31 (t, J ¼ 7.1 Hz, 2H), 1.87 – 1.77 (m,
6H), 1.45 – 1.39 (m, 2H). 13 C NMR (150 MHz, DMSO-d6) d 194.56,
162.68, 160.80, 141.09, 140.47, 129.71, 118.98, 113.76, 111.26,
99.06, 67.69, 64.94, 49.03, 47.46, 36.50, 34.46, 33.94, 28.25, 25.68.
HRMS: calcd for C19H25N2O3S2 [M þ H]þ 393.1301, found 393.1328.
Yield 84%; mp 168–169 ꢀC; 1H NMR (600 MHz, CDCl3) d 7.60 (d,
J ¼ 8.8 Hz, 1H), 6.86 (d, J ¼ 8.8 Hz, 1H), 6.80 (s, 1H), 6.46 (s, 1H),
4.33 (br s, 2H), 4.13 (t, J ¼ 5.9 Hz, 2H), 3.92 (br s, 2H), 3.43 (t,
J ¼ 6.9 Hz, 2H), 2.50 (s, 3H), 2.00 – 1.94 (m, 4H), 1.75 – 1.71 (m,
6H). 13 C NMR (150 MHz, CDCl3) d 195.63, 161.17, 149.59, 139.71,
125.89, 114.87, 112.44, 99.29, 67.85, 52.93, 51.33, 36.73, 28.35,
25.55, 24.35, 19.24. HRMS: calcd for C20H27N2O2S2 [M þ H]þ
391.1508, found 391.1529.
2.3.10. 4-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)butyl 4-(hydroxyme-
thyl)piperidine-1-ca -rbodithioate (4j)
2.3.15. 4-((3,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)oxy)butylpi-
peridine-1-carbodithioate (9b)
Yield 87%; mp 214–215 ꢀC; 1H NMR (600 MHz, DMSO-d6) d 11.58
(s, 1H), 7.81 (d, J ¼ 9.5 Hz, 1H), 7.56 (d, J ¼ 9.5 Hz, 1H), 6.81 – 6.77
(m, 2H), 6.30 (d, J ¼ 9.4 Hz, 1H), 5.32 – 5.30 (m, 1H), 4.56 – 4.48 (m,
2H), 4.03 (t, J ¼ 6.1 Hz, 2H), 3.32 – 3.26 (m, 4H), 3.17 (t, J ¼ 13.4 Hz,
1H), 1.90 – 1.73 (m, 7H), 1.19 – 1.13 (m, 2H). 13 C NMR (150 MHz,
DMSO-d6) d 194.34, 162.68, 160.81, 141.09, 140.47, 129.71, 118.98,
Yield 85%; mp 136–138 ꢀC; 1H NMR (600 MHz, CDCl3) d 7.64 (d,
J ¼ 9.0 Hz, 1H), 6.86 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.70 (d, J ¼ 2.5 Hz, 1H),
4.33 (br s, 2H), 4.11 (t, J ¼ 6.0 Hz, 2H), 3.92 (br s, 2H), 3.43 (t,
J ¼ 7.1 Hz, 2H), 2.47 (s, 3H), 2.28 (s, 3H), 2.08 – 1.91 (m, 4H), 1.77 –
1.71 (m, 6H). 13 C NMR (150 MHz, CDCl3) d 195.61, 163.42, 160.13,
113.76, 111.26, 99.06, 67.70, 65.40, 51.72, 50.20, 38.36, 36.36, 144.22, 137.59, 125.76, 115.33, 112.11, 98.89, 67.76, 52.89, 51.26,

122
J. FU ET AL.
36.72, 29.72, 28.36, 25.56, 24.34, 15.49, 12.55. HRMS: calcd for reciprocal plots were constructed by plotting 1/velocity against 1/
C21H29N2O2S2 [M þ H]þ 405.1665, found 405.1724.
[substrate] at varying concentrations of the substrate acetylthio-
choline (0.05 – 0.5 mM). All measurements were performed in trip-
licate and data analysis was performed with Graph Pad Prism 4.03
software (San Diego, CA, USA).
2.4. Biological evaluation
2.4.1. In vitro inhibition experiments of ChEs
The in vitro inhibitory activities of test compounds against AChE
and BuChE were determined by the spectrophotometric method
of Ellman25. Acetylcholinesterase (AChE, from electric eel and
human erythrocytes), butyrylcholinesterase (BuChE, from equine
serum), S-butyrylthiocholine iodide (BTCI), acetylthiocholine iodide
(ATCI), 5, 50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB) and the reference compounds (tarcine, donepezil and gal-
anthamine) were obtained from Sigma-Aldrich (St. Louis, MO,
USA). The compounds were first prepared in DMSO and then
diluted with Tris-HCl buffer (50 mM, pH ¼ 8.0, 0.1 M NaCl, 0.02 M
MgCl2ꢁ6H2O) to yield corresponding test concentration (DMSO
<0.01%). For each assay, 160 lL of 1.5 mM DTNB, 50 lL of AChE
(0.22 U/mL for eeAChE; 0.05 U/mL for hAChE) or eqBuChE (0.12 U/
mL) were incubated with 10 lL of different concentrations of test
compounds at 37 ꢀC for 6 min. After this, 30 lL of substrate
(15 mM, ATCI or BTCI) was added, and the absorbance was meas-
ured with a wavelength of 405 nm at different time intervals (0,
60, 120, and 180 s). The concentration of each compound was
tested in triplicate, and IC50 values were calculated graphically
from percent inhibition curves, using the Graph Pad Prism 4.03
software (San Diego, CA, USA).
2.4.4. Molecular docking studies
The molecular docking studies were performed using the
Molecular Operating Environment (Montreal, Canada, version
2015.10) software package. The crystal structures of hAChE (PDB
code: 4EY7), TcAChE (PDB code: 2CKM) and Ab1-42 (PDB code:
1IYT) were downloaded from PBD. All water molecules from the
protein were removed, and hydrogen atoms were added. The
ligands were constructed using the builder module in MOE and
were further minimised via MMFF94X force field. After that, the
optimised geometry of ligand was saved as a (.mdb) file and
docked into the binding pocket of the protein using the MOE-
Dock programme. The London dG and Rigid Receptor were
selected as initial and final scoring methods, respectively. The best
5 poses of each ligand were retained and scored. The MOE’s pose
viewer utility was used to analyse the geometry of result-
ing complex.
2.4.5. Inhibition of AChE-induced Ab1-42 aggregation
The inhibitory activities of compounds 4c-f and 9a-b on AChE-
induced Ab1-42 aggregation were evaluated by a Thioflavin T
(ThT)-binding assay27,28. 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol
(HFIP) and Thioflavin T (ThT) were obtained from TCI (Shanghai)
Development. b-Amyloid1-42 (Ab1-42), supplied as trifluoroacetate
salt, and eeAChE were purchased from Royobiotech Co., Ltd
(Shanghai, China) and Sigma-Aldrich (St. Louis, MO, USA), respect-
ively. For co-incubation experiments, aliquots of Ab1-42 peptide
and AChE in presence or absence of the test compounds were
incubated for 48 h at 37 ꢀC. The final concentrations of Ab (dis-
solved in DMSO and diluted 0.215 M sodium phosphate buffer, pH
8.0), AChE (dissolved in 0.215 M sodium phosphate buffer, pH 8.0)
and test compounds were 200 lM, 2 lM and 100 lM, respectively.
After co-incubation, 200 lL of 5 lM ThT in 50 mM glycine-NaOH
buffer (pH 8.0) was added, and the fluorescence was determined
on a multi-mode plate reader (SpectraMax M5, Molecular Devices,
Sunnyvale, CA, USA) at kex ¼ 446 nm and kem ¼ 490 nm, respect-
ively. The percent inhibition of the AChE-induced Ab aggregation
due to the presence of inhibitor was calculated by the following
2.4.2. In vitro blood-brain barrier permeation assay
The parallel artificial membrane permeation assay (PAMPA) for
blood-brain-barrier was performed to predict the BBB penetration
of test compounds26. Before the experiments, all compounds were
prepared in DMSO, and the stock solutions were diluted in PBS/
EtOH (70:30) to make secondary stock solutions (25 lg/mL). After
the pre-treatment, the filter membrane on the 96-well filtration
plate (PVDF membrane, pore size 0.45 mm, Millipore) was coated
with 4 lL of PBL (Avanti Polar Lipids) in dodecane (20 mg/mL,
Sigma-Aldrich). Then, 300 lL of PBS/EtOH (70:30) and 200 lL of
diluted solution containing the corresponding drugs or test com-
pounds were added to corresponding acceptor well and donor
well, respectively. Afterwards, the acceptor filter plate was care-
fully placed on the donor plate to make the coated membrane
touch both donor solution and acceptor buffer. After incubation
for 18 h at 25 ꢀC, the concentrations of test compounds in refer-
ence, acceptor and donor wells were determined by a UV plate
reader (SpectraMax Plus 384, Molecular Devices). Pe was calculated
by the following expression: Pe ¼ fꢂVdVa/[(Vd þ Va)At]gln (1-dru-
gacceptor/drugequilibrium), where Vd is the volume of donor well,
Va is volume in acceptor well, A is the filter area, t is the perme-
ation time, drugacceptor is the absorbance obtained in the acceptor
well and drugequilibrium is the theoretical equilibrium absorbance.
Each compound was analysed at five wavelengths in four wells at
least three independent runs, and the results were expressed
as mean SD.
ꢃ
formula: 100 – (IFi/IFo 100), where IFi and IFo are the fluorescence
intensities obtained for Ab1-42 aggregation in the presence and in
the absence of inhibitor, respectively. Each assay was run in tripli-
cate and each reaction was repeated at least three independ-
ent times.
2.4.6. Inhibition of self-induced Ab1-42 aggregation
The inhibitory activities of compounds 4c-f and 9a-b on self-
induced Ab1-42 aggregation were also measured by the ThT
assay29. HFIP and ThT were purchased from TCI (Shanghai)
Development. Ab1-42 was obtained from Royobiotech Co., Ltd
(Shanghai, China). The monomeric Ab1-42 samples were prepared
by HFIP and diluted with a 50 mM phosphate buffer (pH 7.4) to
give a 25 lM solution. The test compound was dissolved in DMSO
(250 lM) for storage and needed not to be diluted prior to use. In
each well, 1 lL of test compound at final concentration of 25 lM
2.4.3. Kinetic study of AChE inhibition
To determine the inhibition mechanism of present compounds,
compound 4c was taken for kinetic analysis by Ellman’s method25.
Three concentrations of 4c were selected for the study, and
hAChE was chosen as enzyme source. Following a similar method and 9 lL of Ab1-42 sample were added, and the obtained mixture
mentioned above in enzyme inhibition assay, Lineweaver–Burk was incubated in dark at room temperature for 46–48 h with no

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
123
a
c
b
O
N
OH
O
N
O
Br
n
H2N
O
H
H
1
3a-e n = 2-6
2
S
4a-e n = 2-6 R = A
4f-m n = 4 R = B-I
O
N
O
S
R
n
H
R1
R1
R1
NH2
R2
O
R2
O
R2
d
e
b
Br
N
H
OH
N
H
NH2
O
N
H
O
NH2
8a R1 = Me R2 = H
8b R1 = R2 = Me
6a R1 = Me R2 = H
6b R1 = R2 = Me
7a R1 = Me R2 = H
7b R1 = R2 = Me
5
R1
R2
O
c
9a R1 = Me R2 = H
9b R1 = R2 = Me
S
N
N
H
O
S
R=
A=
N
N
N
N
N
N
N
F=
B=
C=
E=
D=
OH
OH
N
N
N
I=
G=
H=
O
N
Scheme 1 Synthesis of compounds 4a-m and 9a-b. Reagents and conditions: (a) (i) cinnamoyl chloride, dry dichloromethane, 4 h, reflux; (ii) AlCl3, chlorobenzene, 8 h,
reflux; (b) a, x-dibromoalkanes, K2CO3, acetone, reflux, 4 h; (c) appropriate secondary amines, CS2, TEA, DMF, r.t., 12 h. (d) ethyl acetoacetate for 6a, ethyl 2-methylace-
toacetate for 6 b, 150ꢀC, 48 h; (e) (i) NaNO2, H2SO4, 0 ꢀC; (ii) 10 M, H2SO4, reflux, 10 min.
agitation. Then, 200 lL of 5 lM ThT in 50 mM glycine-NaOH buffer Animal Co., Ltd. and randomly divided into corresponding groups.
(pH 8.0) was added, and the percent inhibition was calculated by They were maintained under a 12 h light/dark cycle and allowed
free access to tap water and standard laboratory chow. The room
was maintained at temperature of 23 2 ꢀC with a relative humid-
ity of 55 5%. Prior to dosing, all animals were acclimated to this
environment for 5 days. Compound 4c was suspended in 0.5% car-
boxymethyl cellulose sodium (CMC-Na) salt solution and given via
oral administration to the divided experimental groups (0, 625,
1250 and 2500 mg/kg, n ¼ 10 per group). After administration, ani-
mals were observed continuously for the first 4 h for any abnormal
behavioural changes or deaths, then intermittently for the next
24 h, and occasionally thereafter for 14 days for the onset of any
delayed effects. On the 14th day, all mice were sacrificed after
being anaesthetized by ether, and the possible toxic damage to
liver, heart and kidneys was examined macroscopically.
the method same as that of AChE-induced Ab1–42 experiment.
2.4.7. Cytotoxicity study of human neuroblastoma SH-SY5Y cells
The cytotoxicity of test compound on the human neuroblastoma
SH-SY5Y cells was evaluated by MTT assay30. The SH-SY5Y cells
were grown in a 1:1 mixture of Eagle’s minimum essential
medium (EMEM) and ham’s F-12 medium supplemented with 10%
foetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL
streptomycin in 5% CO2 at 37 ꢀC. For assay, cells were seeded into
96-well plates at a density of 10000 cells/well and treated with
compound 4c for 24 h. After this treatment, 20 lL of MTT at 37 ꢀC
was added. Four hours later, the medium was removed, and
200 lL of DMSO was added to dissolve the formazan crystal
formed. The absorbance was measured using a microculture plate
reader with a test wavelength of 570 nm and a reference wave-
length of 630 nm. Results are expressed as the mean SD of three
independent experiments.
3. Results and discussion
3.1. Chemistry
The synthetic pathways of target derivatives are illustrated in
Scheme 1. According to the previously reported methods, the 2-
quinolinone cores were obtained through different procedures
and starting materials20,23,24. The compound 2 was prepared from
2.4.8. Acute oral toxicity studies
A total of 40 Kunming mice (half male and half female) were used
to determine the acute toxicity of compound 4c31,32. All mice the condensation of trans-cinnamic acid chloride with m-anisidine,
weighting 18–25 g were obtained from Hunan SJA Laboratory followed by an intramolecular Friedel-Crafts acylation/dearylation

124
J. FU ET AL.
Table 1. Inhibition of eeAChE and eqBuChE by compounds 4a-m and 9a-b.
eeAChEa eqBuChEb
R1 R2 IC50 (lM)/Inhibition (%) IC50 (lM)/Inhibition (%)
Compd.
n
R
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
9a
9b
2
3
4
5
6
4
4
4
4
4
4
4
4
4
4
–
–
–
A
A
A
A
A
B
C
D
E
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
45.3 0.35%
3.96 0.71 lM
0.22 0.03 lM
0.24 0.02 lM
0.35 0.02 lM
0.69 0.22 lM
9.61 1.12 lM
3.80 0.35 lM
20.17 3.27%
14.52 2.44%
23.14 5.54%
12.31 1.76%
31.43 1.65%
0.30 0.12 lM
0.34 0.01 lM
0.041 0.002 lM
1.65 0.14 lM
0.21 0.02 lM
16.69 2.36%
16.60 4.01%
45.37 8.34%
20.95 2.61%
17.49 2.17%
37.22 1.22%
31.43 3.15%
2.00 0.23 lM
15.81 2.45%
23.91 2.03%
28.65 3.44%
30.47 4.11%
7.63 0.92%
F
G
H
I
A
A
–
–
–
18.31 3.15%
21.20 1.60%
4.22 0.20 lM
11.68 3.49 lM
0.057 0.013 lM
Me Me
Figure 2. Lineal correlation between experimental and reported permeability of
commercial drugs using the PAMPA-BBB assay. Pe (exp.) ¼ 0.9082Pe(bibl.) –
0.3034 (R2 ¼ 0.9410).
Donepezil
Galanthamine
Tacrine
–
–
–
–
–
–
aThe 50% inhibitory concentration of eeAChE or percent inhibition with inhibitor
at 10 lM (means SD of three experiments).
length of the alkyl chain was varied. From the IC50 values, com-
pound 4c containing a four-carbon atom linker showed better
inhibitory activity than the other compounds, which suggested
that the suitable linker length between the two anchoring groups
for AChE inhibition was four carbon atoms.
With the optimal length in hand, different terminal amine
groups were introduced to investigate their possible influences on
AChE inhibition. The no substituted piperidine moiety seemed to
bThe 50% inhibitory concentration of eqBuChE or percent inhibition with inhibi-
tor at 10 lM (means SD of three experiments).
reaction in refluxing chlorobenzene with aluminium chloride as
Lewis acid, while the compounds 7a-b were prepared from m-
phenylenediamine and ethyl acetoacetate/ethyl 2-methylacetoace-
tate by three steps including Pechmann condensation reaction,
diazotisation, and hydrolysis. When these key intermediates were
finished, they were treated with corresponding A, x-dibromoal-
kanes to provide compounds 3a-e and 8a-b. Finally, reacting com-
pounds 3a-e and 8a-b with the appropriate secondary amines,
carbon disulphide and TEA in DMF afforded the target com-
pounds 4a-m and 9a-b.
be essential for AChE inhibitory activity. Compound 4f (IC50
¼
0.69 lM) bearing a 4-methyl group on the piperidine ring showed
3-fold less inhibitory activity than the no substituted compound
4c (IC50 ¼ 0.22 lM). Replacement of the methyl group with an iso-
propyl group led to a largely drop in AChE inhibition. Compound
4 g (IC50 ¼ 9.61 lM) only gave a micromolar inhibition to AChE.
However, it should be noted that introduction of another piperi-
dine to 4-postion of piperidine ring resulted in a significant
improvement in BuChE inhibition. Compound 4 h (IC50 ¼ 3.80 lM
for AChE; IC50 ¼ 2.00 lM for BuChE) was the only compound with
BuChE inhibitory activity in this series. Installation of a hydroxy
group on piperidine ring was not tolerated, as compounds 4i
(20.17% inhibition) and 4j (14.52% inhibition) did not show any
inhibitory activity on AChE. Besides, replacement of the piperidine
group with other cyclic amines were also not beneficial for the
AChE inhibition. Compounds 4k (23.14% inhibition) and 4 l
(12.31% inhibition), possessing a 4-methylpiperazine moiety and a
morpholinyl moiety, respectively, displayed neglected inhibition
on AChE. Unlike our previous study that contraction of the piperi-
dine ring to pyrrole ring had little impact on the inhibitory activ-
ity21, in present work compound 4 m showed the inhibitory
3.2. Biological evaluation
3.2.1. In vitro inhibition studies of AChE and BuChE
The inhibitory activities of target compounds 4a-m and 9a-b
against eeAChE (from electric eel) and eqBuChE (from equine
serum) were determined according to the spectrophotometric
method described by Ellman et al.25. For comparison purposes,
three well-known cholinesterase inhibitors, donepezil, galanth-
amine and tacrine, were used as reference compounds. The
experimental results were presented as IC50 (lM), or for poorly
active compounds, as the percentage of inhibition at 10 lM. All
these results are summarised in Table 1. It can be seen from the
table that most of compounds show moderate to good inhibitory
activity on AChE with IC50 values ranging from micromolar to sub-
micromolar. Among these compounds, compound 4c displayed
the highest inhibitory activity to AChE with IC50 value of 0.22 lM,
which was similar to that of tacrine (IC50 ¼ 0.21 lM) and 7.5 times
more potent than that of galanthamine (IC50 ¼ 1.65 lM). With
exception of compound 4 h, no significant BuChE inhibitory activ-
ity was observed for the present compounds. Considering inhib-
ition of BuChE may lead to unexpected side effects in the
peripheral tissues, these compounds will be more beneficial for
AD treatment.
activity less than 50% at 10 lM. Moreover, compound 9a (IC50
0.30 lM) with mono-methyl group and compound 9b (IC50
0.34 lM) with dimethyl groups on coumarin ring were also syn-
thesised to further extend the SARs. The results indicated that
introduction of methyl group to coumarin slightly reduced the
inhibitory activity on AChE in comparison to their analogue 4c.
¼
¼
3.2.2. In vitro blood-brain barrier permeation assay
The ability to penetrate the blood-brain barrier (BBB) is the first
requirement for successful central nervous system (CNS) drugs
36,37. Therefore, to determine the BBB penetration of our present
compounds, a parallel artificial membrane permeability (PAMPA)
The previous studies indicated that the linker length between
PAS and CAS binding moieties played a crucial role in AChE inhib-
ition33–35. Therefore, to determine the optimal length for the pre-
sent compounds, compounds 4a-e with different linker lengths
(n ¼ 2–6) were prepared in the initial step. As shown in the table, assay was performed. This assay was established by Di et al. and
the inhibitory activities for AChE changed significantly as the has been successfully applied to predict the passive BBB

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
125
Table 2. Permeability Pe(ꢄ10ꢂ6) in the PAMPA-BBB assay for 9 commercial Table 3. Permeability Pe (ꢄ106 cm/s) in the PAMPA-BBB assay for target com-
drugs in the experiment validation.
Commercial drugs
pounds and their predicted penetration into CNS.
Bibliographya
Experimentb
Compound
Pe (ꢄ106 cm/s)a
Predictionb
Testosterone
Estradiol
Progesterone
Chlorpromazine
Corticosterone
Hydrocortisone
Caffeine
17.0
12.0
9.3
6.5
5.1
1.9
1.3
1.02
0.1
14.2 3.6
10.2 2.5
10.4 1.9
7.3 2.3
2.4 0.8
0.46 0.12
1.2 0.2
0.19 0.23
0.16 0.07
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
9a
9b
8.57 1.23
8.78 1.11
8.91 2.03
8.82 1.34
9.85 0.98
6.30 0.86
7.14 1.97
6.42 1.02
2.83 0.65
3.25 0.82
4.54 0.84
7.35 1.05
10.00 2.63
8.79 2.10
8.67 1.76
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNS
Atenolol
Theophylline
aAdapted from Di et al.26
.
CNS
bExperimental data are expressed as mean SD from three independent experi-
ments, using PBS: EtOH (70:30) as solvent.
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
permeation of a multitude of diverse compounds26. After compar-
ing experimental permeabilities with the reported values of 9
commercial drugs, a plot of experiment data versus the biblio-
graphic values gave a good linear correlation: Pe (exp) ¼ 0.9082 Pe
(bibl.) – 0.3034 (R2 ¼ 0.9410) (Figure 2 and Table 2). From this
equation and considering the limit established by Di et al. for BBB
permeation, we determined that compounds with permeabilities
above 3.33 ꢄ 10ꢂ6 cm sꢂ1 could cross the BBB. As shown in the
Table 3, with exception of compounds 4i and 4j, all compounds
showed the Pe value higher than 3.33, which suggested that they
were able to cross the BBB and might reach the biological targets
located in the CNS.
aPermeability Pe (ꢄ106 cm/s) values are expressed as mean SD from three
independent experiments, using PBS: EtOH (70:30) as solvent.
bCNS þ is predicted as high BBB permeation with Pe (ꢄ106 cm/s) > 3.33, CNS
is uncertain for BBB permeation with 1.51 < Pe (ꢄ106 cm/s) < 3.33.
Table 4. Inhibition of hAChE, and AChE- and self-induced Ab1-42 aggregation by
selected compounds.
hAChE
Inhibition of Ab1–42 aggregation (%)
Compd.
IC50 (lM)a
AChE-inducedb
Self-inducedc
4c
4d
4e
4f
9a
9b
Donepezil
Tacrine
Curcumin
0.16 0.02
0.57 0.01
0.38 0.02
1.21 0.33
0.76 0.10
0.39 0.01
0.021 0.001
0.47 0.03
–
29.02 2.06
24.11 3.04
20.28 2.21
12.06 3.18
14.19 2.85
24.37 3.03
26.16 2.55
–
30.67 6.54
26.01 3.01
25.82 1.43
20.35 3.22
33.36 4.85
35.29 2.17
–
3.2.3. In vitro inhibition studies of hAChE
Since almost all compounds could cross the BBB, we then selected
compounds 4c-f and 9a-b with sub-micromolar inhibitory activity for
further evaluation on hAChE. As shown in Table 4, all selected com-
pounds having potent inhibition on eeAChE also exhibit high inhib-
ition to hAChE. It is worth noting that compounds 4c, 4e and 9b did
not display better activity on eeAChE than tacrine but showed stron-
–
–
37.27 2.76
aThe 50% inhibitory concentration of hAChE (means SD of three experiments).
bInhibition of AChE-induced Ab1–42 aggregation. The thioflavin-T fluorescence
method was used, and the concentration of the tested inhibitor was 100 lM
(means SD of three experiments).
cInhibition of self-induced Ab1-42 aggregation. The thioflavin-T fluorescence
ger inhibition on hAChE than tacrine, especially for compound 4c method was used, and the measurements were carried out in the presence of
25 lM inhibitor (means SD of three experiments).
(IC50 ¼ 0.16 lM); it presented nearly 3-fold potent inhibitory activity
on hAChE than tacrine (IC50 ¼ 0.47 lM), which was largely improved
docking results (see Supporting Information, Supplementary Table
S1–S2) indicated that compounds 4c-f and 9a-b with potent inhibi-
tory activities on both hAChE and eeAChE showed lower S value
than the other compounds in corresponding crystal structure of
AChE. Inspection of the docking orientations found that most of
these compounds exhibited the binding modes with hAChE and
TcAChE similar to those of donepezil. The most potent compound 4c
showed the lowest S value on both hAChE and TcAChE. As shown in
Figure 4, the interaction mode of compound 4c with hAChE indicate
that compound 4c fits well into the active site of hAChE, which can
occupy the entire enzymatic CAS, mid-gorge site and PAS. The qui-
nolinone moiety bound to the PAS, forming a p-p stacking inter-
action with Trp 286, while the terminal piperidine group located into
the CAS and established a ‘arene-H’ interaction with Trp 86. In add-
ition, the polymethylene chain was folded in a conformation to
make it interact with the mid-gorge site through a ‘arene-H’ inter-
action with Tyr 341. All these results demonstrated that the present
compounds were dual binding site inhibitors of AChE, which was in
agreement with our results obtained from the kinetic analysis.
in comparison to its inhibitory activity on eeAChE.
3.2.4. Kinetic study of hAChE inhibition
To investigate the inhibition mechanism of present compounds
with AChE, compound 4c, the most potent inhibitor in this series,
was taken as a representative for enzyme kinetic study. It can be
seen from the Lineweaver-Burk plots (Figure 3(a)) that both slopes
and intercepts are increased as the concentrations of the inhibitor
are increased. This pattern indicated a mixed-type inhibition,
which suggested that the present compounds might be dual
binding site inhibitors, being able to bind simultaneously to the
PAS and CAS of AChE. Replots of the slope versus concentration
of 4c gave an estimate of competitive inhibition constant, Ki, of
0.225 lM (Figure 3(b)).
3.2.5. Molecular modelling study with AChE
To further determine the interaction mode of present compounds
with AChE, molecular modelling studies were performed with a
Molecular Operating Environment (MOE) software. The crystal struc-
tures of hAChE complexed with donepezil (PDB code 4EY7) and
3.2.6. Inhibition of AChE-induced Ab1-42 aggregation
TcAChE complexed with bis(7)-tacrine were used to establish the It has been established that the PAS mediates AChE-triggered Ab
starting model. Donepezil was used as a reference compound. The aggregation. Thus, compounds capable of interacting with the

126
J. FU ET AL.
Figure 3. Kinetic study on the inhibition mechanism of hAChE by compound 4c. (a) Overlaid Lineweaver-Burk reciprocal plots of hAChE initial velocity at increasing
substrate concentrations (0.05–0.50mM) in the absence of inhibitor and in the presence of different concentrations (0.4, 0.2 and 0.1lM) of 4c are shown. (b) The plot
of the slopes of the Lineweaver-Burk plots versus inhibitor concentration.
compounds can efficiently inhibit the Ab aggregation with inhib-
ition rates ranging from 12.06% to 29.02% at 100 lM. Generally,
compounds with potent inhibition on AChE also exhibited high
inhibitory activity for Ab aggregation. Compound 4c that dis-
played the most potent inhibitory activity on both eeAChE and
hAChE exerted the strongest effect on inhibition of Ab aggrega-
tion. It showed the inhibition rate of 29.02%, which was more
potent than that of donepezil (26.16% inhibition).
3.2.7. Inhibition of self-induced Ab1-42 aggregation
The capacities of these selected compounds to inhibit self-induced
Ab1–42 aggregation were also investigated by employing the same
ThT-based fluorometric assay29. Curcumin, a natural inhibitor of self-
induced Ab aggregation, was used as positive control. From the
results summarised in Table 4, all compounds exhibited moderate
inhibitory activity for Ab aggregation at a concentration of 25 lM.
The relatively long linkers were not beneficial for the inhibitory activ-
ity, as compounds 4d-f showed lower activity than their short linker
analogues 4c and 9a-b. Among them, compound 9b with dimethyl
groups on quinolinone ring presented the most potent inhibitory
activity for Ab aggregation with inhibition rate of 35.29% at 25 lM.
3.2.8. Molecular modelling study with Ab1-42
Since most of the selected compounds could efficiently inhibit Ab1-42
aggregation, their possible binding modes with Ab1-42 were also
investigated by molecular docking studies. The studies were per-
formed on the Molecular Operating Environment (MOE) software,
and compound 9b with most potent inhibitory activity for self-
induced Ab1-42 aggregation was chosen for docking into the struc-
ture of Ab1-42 (PDB code 1IYT). As shown in Figure 5, compound 9b
Figure 4. (a) 3 D docking model of compound 4c with hAChE. Atom colours:
green-carbon atoms of 4c, gray-carbon atoms of residues of hAChE, dark blue-
nitrogen atoms, red-oxygen atoms, yellow-sulfur atoms. (b) 2 D schematic dia-
gram of docking model of compound 4c with hAChE. The figure was prepared
using the ligand interactions application in MOE.
is transversely fitted along the major helix, and forms a p-cation
interaction between its quinolinone ring and Lys 16 of Ab1-42
Hydrophobic interactions also play an important role in the stability
of 9b/Ab1-42 complex. From the Figure 5(a), it can be seen that com-
pound 9b establishes hydrophobic interactions with residues Leu 17,
Phe 20, Ala 21, Val 24, Ile 31, Leu 34 and Met 35.
.
PAS may have the ability to reduce Ab aggregation. Because our
compounds have been demonstrated to be dual binding site
inhibitors of AChE, compounds 4c-f and 9a-b with potent inhibi-
tory activity on AChE were also selected for testing their inhibitory
activities on AChE-induced Ab aggregation by a thioflavin T (ThT)
fluorescence method 27,28. It can be seen from the Table 4 that, in
3.2.9. Cytotoxicity on human neuroblastoma SH-SY5Y cells
After all above evaluation, compound 4c with potent inhibitory
activity for both eeAChE and hAChE as well as good BBB perme-
comparison to the reference compound donepezil, most ability and Ab aggregation inhibitory effect was considered as the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
127
select for acute toxicity study in vivo to further investigate its
safety property. After delivering compound 4c to Kunming (KM)
mice with different dosages (625, 1250 and 2500 mg/kg) by oral
administration, no evident toxicity signs such as death, significant
weight loss, and drastically altered consumption of water or food,
were observed during the two-week experimental period. Besides,
at the end of the experiment, all used animals were sacrificed,
and the possible toxic damage on their organs was also examined.
The results indicated that compound 4c did not cause any toxicity
effect at a dose up to 2500 mg/kg.
4. Conclusion
A series of novel quinolinone derivatives containing dithiocarba-
mate moiety were designed and synthesised as multifunctional
AChE inhibitors for the treatment of AD. Most of these com-
pounds exhibited potent eeAChE inhibitory activity and excellent
selectivity to eeAChE over eqBuChE. Among them, compound 4c
having a four-carbon atom linker and a piperidine moiety at ter-
minal position displayed the highest activity to inhibit eeAChE,
and it was also the strongest inhibitor of hAChE with IC50 value of
0.16 lM, which was nearly 3-fold more potent than that of tacrine
(IC50 ¼ 0.47 lM). Kinetic and molecular docking analyses revealed
that compound 4c was a mix-typed inhibitor able to bind simul-
taneously to PAS and CAS of hAChE. Besides, compound 4c pre-
sented the best ability to inhibit AChE-induced Ab aggregation
(29.02% at 100 lM) and could efficiently inhibit self-induced Ab
aggregation (30.67% at 25 lM). More importantly, it showed negli-
gible toxicity to SH-SY5Y cells, good ability to penetrate the BBB
and no significant acute toxicity in mice at doses up to 2500 mg/
kg. Overall, we propose that compound 4c is a promising candi-
date deserved for further study.
Figure 5. (a) 3 D docking model of compound 9b with Ab1-42. Atom colours:
green-carbon atoms of 9b, gray-carbon atoms of residues of Ab, dark blue-nitro-
gen atoms, red-oxygen atoms, yellow-sulfur atoms. (b) 2 D schematic diagram of
docking model of compound 9b with Ab. The figure was prepared using the lig-
and interactions application in MOE.
Disclosure statement
The authors declare no conflicts of interest.
Funding
This work was supported by the National Natural Science
Foundation of China [Grant No. 81573374, 21807052], Hunan
Engineering Research Centre for Optimisation of Drug Formulation
and Early Clinical Evaluation [Grant No. 2015TP2005], Jiangxi
Outstanding Youth Talent Support Programme [Grant No.
20192BCB23018] and Natural Science Foundation of Jiangxi, China
[Grant No. 20192ACBL21034].
Figure 6. Cytotoxicity of compound 4c on human neuroblastoma cells SH-SY5Y.
SH-SY5Y cells were incubated with different concentrations of compound 4c
(12.5–100 mM) for 24 h. The results are shown as the percentage of viable cells
after treatment with compound 4c vs untreated control cells. Date are expressed
as mean SD from three independent experiments.
ORCID
most promising compound for further study. To determine
whether compound 4c have potential toxicity on nerve cells, its
cytotoxicity on human neuroblastoma SH-SY5Y cells was meas-
ured by the 3–(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
(MTT) assay30. As shown in Figure 6, compound 4c did not show
a significant effect on cell viability at doses up to 100 lM, which
indicated that it might be a safe agent for treating AD.
Sai-Sai Xie
http://orcid.org/0000-0002-8274-0066
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