Design, synthesis and biological evaluation of new coumarin-dithiocarbamate hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.01.055

A series of new coumarin-dithiocarbamate hybrids were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's Disease (AD). The biological assays indicated that most of them showed potent inhibition and excellent selectivity towards acetylcholinesterase (AChE), and could inhibit self-induced β-amyloid (Aβ) aggregation. Especially, compound 4n presented the highest ability to inhibit AChE (IC₅₀, 0.027 μM for hAChE) and good inhibition of Aβ aggregation (40.19% at 25 μM). Kinetic and molecular modeling studies revealed that 4n was a mixed-type inhibitor, which could interact simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. In addition, it also possessed specific metal-chelating ability, good BBB permeability and low toxicity on SH-SY5Y neuroblastoma cells. Moreover, compound 4n did not exhibit any acute toxicity in mice at doses up to 1000 mg/kg, and could reverse the cognitive dysfunction of scopolamine-induced AD mice. As far as we know, 4n was the first reported dithiocarbamate derivative with multifunctional activity. Its excellent profiles in vitro and effectivity in vivo highlight this structurally distinct compound as a potential lead compound in the research of innovative multifunctional drugs for AD.

Full text of DOI:10.1016/j.ejmech.2018.01.055

European Journal of Medicinal Chemistry 146 (2018) 287e298
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of new coumarin-
dithiocarbamate hybrids as multifunctional agents for the treatment
of Alzheimer's disease
,
b
,
,
,
Neng Jiang a 1, Qichun Huang b 1, Jing Liu c, Ningsheng Liang b *, Qing Li d, Qinghua Li e,
,
**
Sai-Sai Xie a
a National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine,
Nanchang 330006, PR China
b Department of Pharmacy, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi, PR China
c School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, PR China
d Pharmaceutical College, Guangxi Medical University, Shuangyong Road, Nanning 530021, Guangxi, PR China
e Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guilin Medical University, 109 North 2nd Huan Cheng Road, Guilin 541004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new coumarin-dithiocarbamate hybrids were designed, synthesized and evaluated as
multifunctional agents for the treatment of Alzheimer's Disease (AD). The biological assays indicated that
most of them showed potent inhibition and excellent selectivity towards acetylcholinesterase (AChE),
Received 24 November 2017
Received in revised form
29 December 2017
and could inhibit self-induced
highest ability to inhibit AChE (IC50, 0.027
at 25 M). Kinetic and molecular modeling studies revealed that 4n was a mixed-type inhibitor, which
b
-amyloid (A
b
) aggregation. Especially, compound 4n presented the
Accepted 17 January 2018
m
M for hAChE) and good inhibition of A
b
aggregation (40.19%
m
could interact simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) of
AChE. In addition, it also possessed specific metal-chelating ability, good BBB permeability and low
toxicity on SH-SY5Y neuroblastoma cells. Moreover, compound 4n did not exhibit any acute toxicity in
mice at doses up to 1000 mg/kg, and could reverse the cognitive dysfunction of scopolamine-induced AD
mice. As far as we know, 4n was the first reported dithiocarbamate derivative with multifunctional
activity. Its excellent profiles in vitro and effectivity in vivo highlight this structurally distinct compound
as a potential lead compound in the research of innovative multifunctional drugs for AD.
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Alzheimer's disease
Coumarin
Dithiocarbamate
Cholinesterase
Multifunctional agents
1. Introduction
peptide deposits, dyshomeostasis of biometals, oxidative stress and
hyperphosphorylated tau protein [4].
Alzheimer's disease (AD), characterized by memory loss, decline
in language skills and many other cognitive impairments, is an age-
related neurodegenerative disorder and a global public health issue
[1,2]. Today, the dementia number is estimated 46 million people
worldwide and that is expected to reach 131.5 million by 2050 [3].
Due to the complexity and unidentified etiopathogenesis, many
factors are thought to be related to the initiation and development
Among the various pathogenic factors of AD, current clinical
treatment of AD has mainly focused on deficits of acetylcholine
(ACh). The palliative drugs approved by the FDA consists of four
acetylcholinesterase inhibitors (AChEIs), tacrine (now withdrawn
the market due to hepatotoxicity), donepezil, rivastigmine and
galantamine (Fig. 1a) [5]. Base on cholinergic hypothesis, the
decline levels of ACh leads to memory deficits and cognitive im-
pairments, and reducing the ACh metabolism is beneficial to
improvement in memory and cognitive dysfunction [6]. There are
two types of cholinesterases that can hydrolyze ACh in the central
nervous system (CNS), namely acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) [7]. Although a few studies sug-
gested that BuChE played a significant role in AD pathophysiology,
the mechanism of BuChE was not yet completely identified. In
of AD, including deficits of acetylcholine (ACh), amyloid-
b (Ab)
* Corresponding author.
** Corresponding author.
E-mail addresses: Liangn01@163.com (N. Liang), xiesaisainanchang@hotmail.
com (S.-S. Xie).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2018.01.055
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

288
N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
Fig. 1. (a) AChE inhibitors used for the treatment of AD. (b) Design strategy for coumarin-dithiocarbamate hybrids.
addition, BuChE primarily distributes in peripheral system such as
plasma, liver and muscle tissues, and accordingly BuChE inhibitor
may potentially result in peripheral side effects. For example,
tacrine, a dual AChE and BuChE inhibitor, has worst hepatotoxicity
and other peripheral adverse reaction [8,9]. Therefore, discovery of
effective and selective AChE inhibitors with lower side effects may
be more valuable for the treatment of AD. The crystallographic
structure of AChE reveals 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 gorge [10,11]. Research suggest that AChE
inhibitors should contain a core ring system that binds to PAS, a
basic center that interact with CAS, and a linker, such as, CH2, eSe,
-O-, and CONH(CH2)n, etc., between the core ring system and the
basic center to fulfil the structural requirements [12].
biometal ions in the brain may be a potential therapeutic method
for the AD treatment.
So far, there are only four AChE inhibitors and one N-methyl-D-
aspartate receptor antagonist approved by FDA for clinical treat-
ment of AD. Although these drugs can temporarily improve the
cognitive and daily function, they cannot mitigate or halt the pro-
gression of AD. Due to the complex etiology disease networks, an
efficient therapy is more inclined to discover multifunctional drugs
that can simultaneously modulate the complex etiology networks.
Therefore, many medicinal chemists have made great efforts to
develop multifunctional molecules by incorporating several
different active structural fragments into one molecule for the
treatment of multi-factor neurodegenerative diseases [23,24].
Coumarins, an important group of naturally occurring com-
pounds with small molecular weight, exist in various plant species.
Coumarins have attracted increasingly attention in recent years due
to a wide of biological activities related to neurological disorders
Another significant hypothesis reveals that A
brain plays a crucial role in AD pathogenesis. The amyloid precursor
protein (APP) is hydrolyzed by -secretase enzymes to produce
peptides that can aggregate into monomers, oligomers and large
plaques [13,14]. These aggregates can initiate pathogenic
b plaques in the
a,
b
,
g
[24e28], such as AChE inhibition, anti-A
inhibition. Our previous studies demonstrated that coumarin was
able to bind to the PAS of AChE via aromatic stacking in-
b aggregation and MAO-B
A
A
b
b
p-p
cascade and ultimately lead to the neuronal loss and dementia [15].
The A peptides have two key isoforms: Ab1-40 and Ab1-42. Ab1-42
shows lower solubility and is more prone to aggregate into fibrils
than Ab1-40 [16]. The A plaques generated from Ab1-42 lead to
severe neuronal toxicity [17]. Hence, the prevention of Ab1-42 ag-
gregation could serves as a rational strategy for the treatment of
AD. Studies also reveal that AChE can facilitate amyloid fibril for-
mation through the interaction with the PAS of AChE, giving stable
teractions [24,29] and therefore could serve as one part of the dual-
binding mode of action. On the other hand, dithiocarbamate is a
versatile pharmacophore and has been widely used for drug design
[30]. However, to our knowledge, there are few reports about the
research on dithiocarbamate derivatives against AD. Recently, we
found that dithiocarbamate could serve as the other part that bind
to the CAS of AChE. Meanwhile, considering the broad activities of
dithiocarbamate, we reasoned that compounds containing this
group might exert multifunctional activity for the treatment of AD.
Therefore, to further find new dual-acting AChE inhibitors with
potential multifunctional activity, in this study, we attempted to
connect coumarins (binding to PAS) with the dithiocarbamate
moieties (binding to CAS) to design a series of new coumarin-
dithiocarbamate hybrids as multifunctional agents (Fig. 1b). All
designed compounds were synthesized and evaluated for ChE in-
b
b
AChE-Ab complexes, which are more toxic than single Ab peptides.
Dual-site inhibitors that bind simultaneously to the PAS and CAS of
AChE can not only stimulate the cholinergic system, but also inhibit
the A
b aggregation promoted by AChE [18]. Therefore, dual-site
inhibitors are thought to be more promising anti-AD drug
candidates.
Recently, many studies show that there are excessive bio-metal
ions (Cu, Zn, Fe) in the brains of AD patients, which is several-fold
higher than that of healthy person [19]. The excessive metal ion is
able to interact with Ab peptides and then accelerate the Ab ag-
gregates and neurofibrillary tangles, which lead to dysfunction and
neuron death [20]. On the other hand, excess of these redox-active
metals that interact with Ab is able to contribute to the production
hibition and anti-Ab aggregation. Besides, the outstanding com-
pound was selected for further evaluation including metal
chelation, the ability to cross the blood-brain barrier (BBB) in vitro,
acute toxicity and neuroprotective effects in scopolamine-induced
cognitive impairment in mice. Furthermore, kinetic and molecu-
lar modeling studies were also carried out to investigate the
binding mode of compounds with AChE.
of reactive oxygen species (ROS) and cause the oxidative damage of
the central nervous systems (CNS) [21,22]. Hence, modulating these

N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
289
2. Results and discussion
piperazine moiety only presented very weak inhibition for AChE.
However, it is interesting to note that the substitutes at 4-position
of piperazine moiety were very sensitive to inhibitory activity.
2.1. Chemistry
Compound 4i (IC50 ¼ 1.99 0.06
mM) bearing an isopropyl group
The synthetic route of the target compounds 4a-q is illustrated
in Scheme 1. Following our reported procedure [31], compound 2
was afforded by condensing resorcinol with ethyl acetoacetate in
the presence of a catalytic amount of concentrated sulfuric acid.
exerted much higher activity than compounds 4h and 4j possessing
methyl (36.84% inhibition) and cyclopropyl (18.69% inhibition)
substituents. Taken together, the above results indicated that
piperidinyl group was the optimal substitution pattern for AChE
inhibition, and compound 4d possessing relatively high activity
was selected for further optimization.
Then, 2 was reacted with the corresponding a, u-dibromoalkanes
to produce the intermediates 3a-f. Finally, using a one-pot pro-
cedure [32], the target compounds 4a-q was obtained in good
yields by the reaction of 3a-f with the appropriate secondary
amines, carbon disulfide and TEA in DMF.
After lengthening the linker length between coumarin and
dithiocarbamate moiety in compound 4d from two to eight carbon
atoms, the new obtained compound 4n with a four-carbon atom
linker exhibited the best inhibition for AChE in this series. It
showed the IC50 value of 0.027
than that of donepezil (IC50 ¼ 0.041 0.001
m
M, which was 1.5-fold more potent
2.2. In vitro inhibition of ChEs
mM).
Finally, all compounds displayed inactive or very weak activity
for BuChE. This result demonstrated that most of the present
compounds were selective inhibitors to AChE. In view of BuChE was
mainly localized in the peripheral tissues and very small amount
was present in the brain region [36]. Therefore, these compounds
would result in lesser degree of associated side effects and might be
more beneficial for AD treatment.
The inhibitory activities of the test compounds 4a-q against
hAChE and hBuChE were determined according to the spectro-
photometric method described by Ellman et al. [33]. For compari-
son purpose, the well-known cholinesterase inhibitors, donepezil
and tacrine, were used as reference compounds. The experimental
results were presented as IC50 (mM), or for poorly active com-
pounds, as the percentage of inhibition at 10
are summarized in Table 1.
mM. All these results
2.3. Kinetic studies for the inhibition of AChE
From the table, it can be seen that the terminal amine groups
connected with the dithiocarbamate moiety play an important role
in AChE inhibition. Compounds 4c and 4d with cyclic amine groups
(pyrrolidinyl and piperidinyl) in side chains showed more potent
activity than compounds 4a and 4b containing an alkyl amine
group (dimethylamino and diethylamino). And piperidinyl group
seemed more beneficial for AChE inhibition than pyrrolidinyl group
In order to investigate the inhibition mechanism of the present
compounds for AChE, the most potent inhibitor, compound 4n, was
selected for kinetic study. Analysis of the Lineweaver-Burk recip-
rocal plot (1/V vs 1/S) showed both increasing slopes and intercepts
at increasing concentration of the inhibitor (Fig. 2a). This pattern
indicated a mixed-type inhibition and therefore revealed that
compound 4n might be able to bind to the catalytic active site (CAS)
as well as peripheral anionic site (PAS) of AChE. Replots of the slope
versus concentration of 4n gave an estimate of competitive inhi-
bition constant, Ki, of 37.14 nM (Fig. 2b).
since compound 4d (IC50 ¼ 0.47 0.23
m
M) exhibited nearly 2-fold
more potent activity than compound 4c (IC50 ¼ 0.89 0.11
mM).
However, introducing another piperidinyl substituent to 4-position
of piperidinyl moiety afforded compound 4k, which gave rise to a
decrease in AChE inhibition (IC50 ¼ 1.16 0.12
mM). Morpholinyl, 4-
hydroxypiperidine and diethanolamino groups were not beneficial
for AChE inhibition, as compounds 4e-g showed the inhibitory
2.4. Molecular modeling study of AChE inhibition
activity, which was less than 50% at 10 mM. This result suggested
that the oxygen atom at the terminal moiety was not tolerated for
AChE inhibition. Unlike previous reports that indicated 4-
substitued piperazine could enhance the inhibitory activity for
AChE [34,35], the present compounds 4h-j and 4l did not show
improved activity compared to compound 4d. With exception of
compound 4i, most of the compounds (4h, 4j and 4l) containing
In order to explore more information about the binding in-
teractions between compound 4n and AChE, a molecular modeling
study was carried out using the Molecular Operating Environment
(MOE 2008.10) software package. The X-ray crystal structure of the
recombinant human acetylcholinesterase in complex with done-
pezil (hAChE, PDB code 4EY7) was applied to establish the starting
Scheme 1. Synthesis of compounds 4a-q. Reagents and conditions: (a) Ethyl acetoacetate, conc. H2SO4 (cat.), 1,4-dioxane, 60 ꢀC, 4 h. (b) Br(CH2)nBr, anhydrous K2CO3, acetone,
reflux, 4 h; (c) appropriate secondary amines, CS2, TEA, DMF, r.t., 12 h.

290
N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
Table 1
Inhibition of hAChE, hBuChE and self-induced Ab1-42 aggregation by compounds 4a-q.
Compd.
n
R
AChE
IC50
BuChE Inhibition (%)b
Ab
aggregation inhibition (%)d
(
m
M)/inhibition (%)a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
2
2
2
2
2
2
2
2
2
2
2
2
3
4
5
6
8
e
e
e
A
B
C
D
E
F
G
H
I
J
K
L
D
D
D
D
D
-
10.59 1.34
1.94 0.56
0.89 0.11
0.47 0.23
10.99 1.26%
n.a.c
34.49 3.08%
36.84 2.51%
1.99 0.06
18.69 2.00%
1.16 0.12
12.33 1.59%
0.29 0.07
0.027 0.002
0.21 0.03
0.61 0.08
n.a.c
n.a.c
24.55 1.25
29.08 0.98
33.22 1.67
37.88 2.49
20.02 0.99
17.83 1.02
16.12 0.07
34.02 2.15
35.44 1.33
34.07 2.55
43.53 3.11
27.39 0.95
37.03 1.65
40.19 2.39
40.03 1.05
33.86 2.01
10.02 0.07
n.a.e
10.89 1.58
n.a.c
37.24 5.13
n.a.c
n.a.c
n.a.c
n.a.c
28.10 2.82
n.a.c
24.27 2.06
n.a.c
31.24 3.12
15.29 1.95
10.10 1.02
6.21 0.99
n.a.c
4q
Donepezil
Tacrine
Curcumin
0.041 0.001
0.43 0.02
IC50 ¼ 4.22 0.20
mM
m
-
-
IC50 ¼ 0.021 0.001
M
e
39.62 3.35
a
The 50% inhibitory concentration of human AChE or percent inhibition with inhibitor at 10
m
M (means SD of three experiments).
M (means SD of three experiments).
n. a. ¼ no active. Compounds defined “no active” means that percent inhibition is less than 5.0% at a concentration of 10
b
c
The percent inhibition of human BuChE with inhibitor at 10
m
mM in the assay conditions.
d
e
Inhibition of self-induced Ab1-42 aggregation by tested inhibitors at 25
mM.
n.a. ¼ no active. Compounds defined “no active” means that percent inhibition was less than 5% at 25
m
M.
Fig. 2. (a) Kinetic study on the mechanism of AChE inhibition by compound 4n. Overlaid LineweaverꢁBurk reciprocal plots of AChE initial velocity at increasing substrate con-
centration (0.05e0.50 mM) in the absence of inhibitor and in the presences of different concentrations of 4n are shown. (b) The plot of the slopes of the Lineweaver-Burk plots
versus inhibitor concentration.
model of AChE. As shown in Fig. 3, the coumarin moiety was bound
to the PAS of the enzyme, establishing a stacking interaction
self-induced Ab1-42 aggregation [37]. Curcumin, a known active
natural product for inhibition of Ab1-42 self-aggregation, was used
as reference compound. From the results summarized in Table 1
and Fig. 4, it can be seen that most of the compounds show mod-
p-p
between its phenyl ring and the indole ring of Trp 286 (3.92 Å).
Besides, the carbonyl oxygen of coumarin moiety also formed a
hydrogen bond with Ser 293 in PAS (2.97 Å). The piper-
idinyldithiocarbamate moiety of compound 4n was located into the
CAS and showed a hydrophobic interaction with residues Gly 448,
Trp 86, Tyr337 and Gly 121. In addition, the side chain between
coumarin and dithiocarbamate moiety folded in a conformation in
the gorge that allowed it to interact with Phe 338, Tyr 341, Tyr 124
and Phe 295 via hydrophobic interactions. Taken together, all these
results indicated that compound 4n occupied the entire enzymatic
CAS, the mid-gorge site and the PAS, and could bind simultaneously
to both the PAS and CAS.
erate to good potencies (10.02e43.53% at 25
m
M) compared with
M). Compounds 4k, 4n and 4o (43.53%,
M, respectively) were the best inhibitors
curcumin (39.62% at 25
40.19%, and 40.03% at 25
m
m
in this series, which showed the percent inhibition better than that
of curcumin. Unlike the AChE inhibition, compounds with different
terminal secondary amines and varied linker length did not give a
clear trend in Ab1-42 aggregation inhibition. However, it is inter-
esting to note that 4-piperidinopiperidine group seemed more
beneficial for inhibitory activity, as compound 4k gave the highest
inhibitory activity for Ab1-42 aggregation. Given all above results,
compound 4n with the most potent inhibitory activity for hAChE
and the second highest inhibition potency for Ab1-42 aggregation
was selected as promising compound for further study.
2.5. Inhibition of self-induced Ab1-42 aggregation
All compounds tested for ChEs inhibition were also evaluated by
a thioflavin T-based fluorometric assay for their ability to inhibit

N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
291
Fig. 3. (a) 3D docking model of compound 4n with hAChE. Atom colours: greenecarbon atoms of 4n, greyecarbon atoms of residues of hAChE, dark blueenitrogen atoms,
redeoxygen atoms, yellowesulfur atom. The dashed lines represent the interactions between the protein and the ligand. (b) 2D schematic diagram of docking model of compound
4n with hAChE. The figure was prepared using the ligand interactions application in MOE. (For interpretation of the references to colour in this figure legend, the reader is referred
to the Web version of this article.)
2.6. Metal-chelating studies
strength enhancement, while Cu2þ and Fe2þ had relativity weak
effects on the absorbance at the same concentration and condi-
tions. However, when Zn2þ were added, no significant change was
observed for UVevis spectra. These results indicated that com-
pound 4n could selectively chelate the metals and was more
The ability of compound 4n to chelate biometals, such as Cu2þ
,
Zn2þ, Fe2þ and Fe3þ was evaluated by a UVevis spectroscopy assay
[38]. As shown in Fig. 5a, Fe3þ caused an obvious absorption

292
N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
Table 2
Permeability Pe ( ꢂ 106 cm/s) in the PAMPA-BBB assay for 10 commercial drugs in the
experiment validation.
Commercial drugs
Bibliographya
Experimentb
Testosterone
Verapamil
Imipramine
Progesterone
Clonidine
Corticosterone
Piroxicam
Caffeine
17.0
16.0
13.0
9.3
5.3
5.1
2.5
1.3
1.1
0.8
24.0 0.9
19.8 0.8
15.1 1.5
16.2 0.6
5.1 0.2
6.3 0.5
4.5 0.3
3.3 0.1
2.9 0.2
1.1 0.1
Lomefloxacin
Ofloxacin
a
Taken from Ref. [42].
b
Experimental data are the mean SD of 3 independent experiments, using PBS:
EtOH (70:30) as solvent.
Fig. 4. Inhibition of self-induced Ab1-42 aggregation by compounds 4a-q comparing
with that of curcumin. The thioflavin-T fluorescence method was used and the mea-
surements were carried out in the presence of 25
mM test compound. The mean SD
values from three independent experiments were shown.
Therefore, these results proved that compound 4n was low cyto-
toxicity to SH-SY5Y cells.
sensitive to chelate Fe3þ
.
To further understand the interactions between compound 4n
and Fe3þ, Job's method was performed to the determine the stoi-
chiometry of the 4n-Fe3þ complex [39]. After keeping total con-
2.8. In vitro bloodebrain barrier permeation assay
certation of compound and Fe3þ at 50
mM and changing the molar
Blood-brain barrier (BBB) permeability is a first requirement for
successful CNS drugs. Therefore, to determine whether the selected
compound 4n could be able to penetrate into the brain, the parallel
artificial membrane permeation assay for blood-brain barrier
(PAMPA-BBB) was performed [42]. After comparing experimental
permeabilities of 10 commercial drugs with reported values
(Table 2), a plot of experiment data versus the bibliographic values
gave a good linear correlation: Pe (exp.) ¼ 1.2653 Pe (bibl.) þ 0.7955
(R2 ¼ 0.9483) (Fig. 6). From this equation and taking into account
the limits established by Di et al. for BBB permeation, we classified
compounds as follows:
ratio of Fe3þ from 0.1 to 0.9, the absorbance of Job's plot curve got to
maximum when the molar fraction of Fe3þ was 0.5, which revealed
a 1:1 stoichiometry for the complex (Fig. 5b). Moreover, the asso-
ciation constant (Ka) of the complex was also calculated using the
Benesi-Hildebrand equation [39,40], and the value of Ka was
1.69 ꢂ 103 Mꢁ1, suggesting that compound 4n could moderately
chelate Fe3þ
.
2.7. SH-SY5Y neuroblastoma cell toxicity
To investigate the cytotoxicity of compound 4n, the human
neuroblastoma cell line SH-SY5Y were exposed to 4n at different
(a) ‘CNSþ’ (high BBB permeation predicted): Pe (10ꢁ6 cm/
s) > 5.86
concentrations (12.5, 25, 50 and 100
mM) for 24 h, and the cell
(b) ‘CNS-’ (low BBB permeation predicted): Pe (10ꢁ6 cm/s) < 3.33
(c) ‘CNSþ/-’ (uncertain BBB permeation): 3.33 < Pe (10ꢁ6 cm/
s) < 5.86
viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium (MTT) assay [41]. The results indicated that
compound 4n showed negligible cell death up to the concentration
of 50
m
M
(12.5
m
M: 99.2 1.4%; 25
m
M: 101.1 10.3%; 50
M, com-
M: 80.3 1.5%).
m
M:
According to the measured permeability, compound 4n showed
a Pe value of 9.21 ꢂ 10 ꢁ6 cm/s, which indicated that it could
penetrate the BBB and reach the biological targets in the CNS.
90.1 2.4%). After increasing the concentration to 100
pound 4n showed a decrease of cell viability (100
m
m
Fig. 5. (a)The UVevis absorption spectra of compound 4n (25
m
M) alone or in the presence of 50
mM of CuCl2, ZnCl2, FeSO4 and Fe2(SO4)3 in MeOH solution. (b) Job's plot of
compound 4n with Fe3þ in MeOH solution. Total concentration was kept constant at 50
m
M and the absorbance at 373 nm was used.

N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
293
control group (##p < .01). Treatment with compound 4n (20.6, 10.3
and 5.2 mg/kg) could increase the latency and reduce the number
of errors in a dose-dependent manner. The high dose group of
compound 4n (20.6 mg/kg) presented a longer latency (192 s) and
less number of errors (1.75) than donepezil group (181 s; 2.00).
Meanwhile, the medium dose group (10.3 mg/kg) also showed the
comparable effects (182 s; 1.89) to donepezil group. However, for
the low dose group (5.2 mg/kg), it didn't exhibit significant differ-
ence compared with model group, but the latency (151 s) and
number of errors (2.5) were largely improved. All in all, these re-
sults demonstrated that compound 4n could cross the blood-brain
barrier and reverse cognitive deficit by increasing brain cholinergic
activity due to the inhibition of AChE.
3. Conclusion
Fig. 6. Lineal correlation between experimental and reported permeability of com-
mercial drugs using the PAMPA-BBB assay. Pe (exp.) ¼ 1.2653 Pe (bibl.)
þ
0.7955
(R2 ¼ 0.9483).
To develop effective drugs for the treatment of AD, a series of
new multifunctional hybrids 4a-q by connecting coumarin with
dithiocarbamate moiety were designed, synthesized and evaluated.
The results indicated that most of these compounds showed potent
AChE inhibitory activity and high selectivity for AChE over BuChE.
2.9. Acute toxicity test
The acute toxicity profile is an important criterion in the
development of new drugs. Therefore, the most potent compound
4n was selected for evaluation in KM mice according to the re-
ported method [43,44]. After administration of the compound
(1000 mg/kg), mice were monitored continuously for the first 4 h
for any abnormal behavior and mortality changes, intermittently
for the next 24 h, and occasionally thereafter for 14 days to monitor
the onset of any delayed effects. During the experimental period, no
acute toxicity, such as significant abnormal changes in water or
food consumption, or body weight reduction or mortality, was
observed. Furthermore, all mice were sacrificed on the 14th day
after drug administration and macroscopically examined for
possible damage to the heart, liver and kidneys. The results indi-
cated that compound 4n did not develop any acute toxicity and well
tolerated at doses up to 1000 mg/kg.
Compound 4n (IC50 ¼ 0.027
mM) showed the best inhibitory activity
to hAChE, which was 1.5-fold more potent than that of donepezil.
Kinetic and molecular modeling study suggested that compound
4n was a mix-typed inhibitor, which could bind simultaneously to
PAS and CAS of AChE. In addition, compound 4n could effectively
inhibit Ab aggregation (40.19% at 25 mM) and selectively chelate
Fe3þ ion. It also showed good ability to penetrate the BBB in vitro
and exhibited low toxicity to SH-SY5Y cells. More importantly, our
in vivo study proved that 4n did not display acute toxicity in mice at
doses up to 1000 mg/kg, and mice treated with 4n (20.6 and
10.3 mg/kg, p.o.) could significantly prolong the latency and reduce
number of errors in the step-down passive avoidance test. To the
best of our knowledge, compound 4n was the first reported com-
pound containing a dithiocarbamate moiety, which have multi-
functional activity. Such excellent properties rendered compound
4n a potential lead compound deserved for further study, and
dithiocarbamate moiety could be used as a new scaffold for
designing innovative multifunctional drugs for the treatment of AD.
2.10. In vivo assay
As compound 4n showed excellent results on the above studies,
it was necessary to determine whether it could improve memory
impairment in vivo. After establishing the classical scopolamine-
induced cognitive deficit mouse model, a step-down passive
avoidance test was performed to evaluate the effect of compounds
4n on cognitive improvement [45,46], and donepezil was used as
positive control. It can be seen from Fig. 7 that the model group
exhibit much shorter latency and more number of errors than the
4. Experimental section
4.1. Chemistry
All chemical reagents used in synthesis were obtained from
Sinopharm Chemical Reagent Co., Ltd. (China). The reactions were
monitored by TLC on glass-packed precoated silica gel GF254
Fig. 7. Effects of compound 4n on the (a) latency (s) and (b) number of errors in the step-down test by the scopolamine-induced cognitive impairment. The data shown are
mean SEM (n ¼ 8). ##P < .01 vs. control group, *P < .05, **P < .01 vs. model group.

294
N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
(Qingdao Haiyang Chemical Plant, Qingdao, China) plates. Column
chromatography was performed on silica gel (90e150 m; Qingdao
Marine Chemical Inc.). Melting point was measured on an XT-4
micromelting point instrument and uncorrected. 1H NMR spectra
(600 MHz) and 13C NMR spectra (151 MHz) were recorded on a
Bruker ACF-600 spectrometer at 25 ꢀC. Chemical shifts are reported
J ¼ 8.8 Hz, 1H), 6.86 (d, J ¼ 8.8, 2.0 Hz, 1H), 6.81 (d, J ¼ 2.0 Hz, 1H),
6.14 (s, 1H), 4.03 (t, J ¼ 6.3 Hz, 2H), 3.45 (t, J ¼ 7.2 Hz, 2H), 2.41 (s,
3H), 1.92 (br s, 2H), 1.85 (br s, 2H), 1.54 (br s, 4H); ESI-MS m/z: 339.1
[MþH]þ.
m
4.3.6. 7-((8-bromooctyl)oxy)-4-methyl-2H-chromen-2-one (3f)
in ppm (
d) using the TMS as internal standard, and the coupling
Yield 85%; m.p. 49e50 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.49 (d,
constants are reported in hertz (Hz). The purity of all compounds
for biological evaluation was confirmed to >95% by analytical HPLC
conducted on an Agilent 1200 HPLC System. Mass spectra were
carried out on a Thermo Scientific LCQ Fleet mass spectrometer
(ESI-MS) and an AB Sciex Triple TOF 5600 spectrometer (HR-ESI-
MS), respectively.
J ¼ 8.8 Hz,1H), 6.86 (d, J ¼ 9.8 Hz,1H), 6.80 (d, J ¼ 2.5 Hz,1H), 6.13 (s,
1H), 4.02 (t, J ¼ 5.8 Hz, 2H), 3.42 (t, J ¼ 6.3 Hz, 2H), 2.41 (s, 3H), 1.87
(br s, 2H), 1.82 (br s, 2H), 1.48 (br s, 4H), 1.38 (br s, 4H); ESI-MS m/z:
367.2 [MþH]þ.
4.4. General procedure for the preparation of compounds 4a-q
4.2. 7-Hydroxy-4-methyl-2H-chromen-2-one (2)
CS2 (99 mg, 1.3 mmol) was added dropwise to the mixture of the
secondary amine (1.2 mmol) and TEA (131 mg, 1.3 mmol) in DMF
(2 mL). After stirring for 5 min, a solution of the corresponding
coumarin derivatives 3a-f (1.2 mmol) in DMF (3 mL) was added.
Then, the reaction mixture was stirred at room temperature for
12 h. When the reaction was completed, water (20 mL) was added
and the mixture was extracted with ethyl acetate (10 mL ꢂ 3), the
combined organic phase was washed with water (15 mL ꢂ 3), dried
over anhydrous Na2SO4 and concentrated under vacuum to afford
the crude product. The crude product was purified by silica gel
chromatography with PE/EA (10:1) as eluent to obtain the target
compounds 4a-q.
To an ice-cold solution of resorcinol (5.5 g, 50 mmol) in dioxane,
conc. H2SO4 (2 mL) was added dorpwise under 25 ꢀC. After the
addition of concentrated sulfuric acid, ethylacetoacetate (7 mL) was
added and the mixture was heated to 60 ꢀC for 4 h. Then, the
mixture was poured into cold water and the precipitate was filtered
and dried under reduced pressure. The resulting mixture was
recrystallized from methanol to give 2 as white needle crystals,
yield 91%; m.p. 186e187 ꢀC; 1H NMR (600 MHz, acetone-d6)
d 7.61
(1H, d, J ¼ 9.0 Hz), 6.85 (1H, dd, J ¼ 9.0, 2.5 Hz), 6.75 (1H, d,
J ¼ 2.5 Hz), 6.09 (1H, s), 2.40 (3H, s). ESI-MS m/z: 175.0 [MꢁH]ꢁ.
4.3. General procedure for the preparation of compounds 3a-f
4.4.1. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl
dimethylcarbamodithioate (4a)
A mixture of 2 (5.0 mmol) with suitable
a
,
u
-dibromoalkanes
Yield 85%; yellow solid; m.p. 111e112 ꢀC; 1H NMR (600 MHz,
(50 mmol) and anhydrous K2CO3 (1.4 g, 10 mmol) in acetone
(15 mL) was refluxed under stirring for 4 h. After cooling, the re-
action mixture was filtered, and the filtrate was evaporated under
reduced pressure. The obtained residue was purified by silica gel
chromatography with PE/EA (15:1) as eluent to give compounds
3a-f as white solid.
CDCl3)
d
7.52 (d, J ¼ 8.8 Hz, 1H), 6.93 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.88 (d,
J ¼ 2.4 Hz, 1H), 6.16 (s, 1H), 4.33 (t, J ¼ 6.3 Hz, 2H), 3.78 (t, J ¼ 6.3 Hz,
2H), 3.60 (s, 3H), 3.43 (s, 3H), 2.42 (s, 3H). 13C NMR (151 MHz, CDCl3)
d
196.06, 161.59, 161.37, 155.28, 152.46, 125.30, 113.54,112.33,112.17,
102.06, 66.92, 45.68, 41.48, 35.92, 18.64. HRMS: calcd for
C
15H18NO3S2 [M þ H]þ 324.0723, found 324.0760.
4.3.1. 7-(2-bromoethoxy)-4-methyl-2H-chromen-2-one (3a)
4.4.2. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl
diethylcarbamodithioate (4b)
Yield 86%; m.p.104e105 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.53 (d,
J ¼ 8.8 Hz, 1H), 6.91 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.83 (d, J ¼ 2.5 Hz, 1H),
6.17 (s, 1H), 4.37 (t, J ¼ 6.1 Hz, 2H), 3.70 (t, J ¼ 6.1 Hz, 2H), 2.40 (s,
3H); ESI-MS m/z: 283.1 [MþH]þ.
Yield 82%; white solid; m.p. 126e128 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.54 (d, J ¼ 8.8 Hz, 1H), 6.91 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.84 (d,
J ¼ 2.4 Hz, 1H), 6.18 (s, 1H), 4.07 (t, J ¼ 6.0 Hz, 2H), 3.84e3.74 (m,
4H), 3.70 (t, J ¼ 6.1 Hz, 2H), 2.43 (s, 3H), 1.38e1.21 (m, 6H). 13C NMR
4.3.2. 7-(3-bromopropoxy)-4-methyl-2H-chromen-2-one (3b)
(151 MHz, CDCl3) d 194.37, 161.59, 161.02, 155.21, 152.46, 125.76,
Yield 92%; m.p.82e83 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.51 (d,
113.85,112.34,112.14,102.08, 67.05, 49.87, 46.89, 35.92, 18.70,12.53,
12.56. HRMS: calcd for C17H22NO3S2 [M þ H]þ 352.1036, found
352.1053.
J ¼ 8.8 Hz, 1H), 6.88 (d, J ¼ 8.8 Hz,1H), 6.84 (d, J ¼ 1.0 Hz,1H), 6.15 (s,
1H), 4.19 (t, J ¼ 5.8 Hz, 2H), 3.63 (t, J ¼ 6.3 Hz, 2H), 2.40 (s, 3H),
2.39e2.35 (m, 2H); ESI-MS m/z: 297.1 [MþH]þ.
4.4.3. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl pyrrolidine-
1-carbodithioate (4c)
4.3.3. 7-(4-bromobutoxy)-4-methyl-2H-chromen-2-one (3c)
Yield 87%; m.p.58e60 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.52 (d,
Yield 87%; yellow solid; m.p. 149e151 ꢀC; 1H NMR (600 MHz,
J ¼ 8.8 Hz, 1H), 6.86 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.80 (d, J ¼ 2.5 Hz, 1H),
6.14 (s, 1H), 4.07 (t, J ¼ 6.1 Hz, 2H), 3.51 (t, J ¼ 6.6 Hz, 2H), 2.40 (s,
3H), 2.16e2.05 (m, 2H), 2.03e1.95 (m, 2H); ESI-MS m/z: 311.1
[MþH]þ.
CDCl3)
d
7.51 (d, J ¼ 9.0 Hz, 1H), 6.93 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.86 (d,
J ¼ 2.4 Hz, 1H), 6.14 (s, 1H), 4.31 (t, J ¼ 6.6 Hz, 2H), 3.95 (t, J ¼ 6.6 Hz,
2H), 3.77 (t, J ¼ 6.6 Hz, 2H), 3.69 (t, J ¼ 7.2 Hz, 2H), 2.40 (s, 3H),
2.10e2.09 (m, 2H), 2.01e1.99 (m, 2H). 13C NMR (151 MHz, CDCl3)
d
191.56, 161.57, 161.29, 155.20, 152.47, 125.58, 113.86, 112.32, 112.14,
4.3.4. 7-((5-bromopentyl)oxy)-4-methyl-2H-chromen-2-one (3d)
102.07, 67.07, 55.31, 50.73, 34.88, 26.08, 24.30, 18.69. HRMS: calcd
Yield 90%; m.p.59e60 ꢀC; 1H NMR (600 MHz, CDCl3)
d
7.48 (d,
for C17H20NO3S2 [M þ H]þ 350.0879, found 350.0904.
J ¼ 8.8 Hz, 1H), 6.84 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.79 (d, J ¼ 2.5 Hz, 1H),
6.12 (s, 1H), 4.04 (t, J ¼ 6.1 Hz, 2H), 3.45 (t, J ¼ 6.1 Hz, 2H), 2.41 (s,
3H), 1.94 (br s, 2H), 1.85 (br s, 2H), 1.66 (br s, 2H); ESI-MS m/z: 325.2
[MþH]þ.
4.4.4. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl piperidine-
1-carbodithioate (4d)
Yield 84%; white solid; m.p. 78e79 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.53 (d, J ¼ 8.8 Hz, 1H), 6.93 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.88 (d,
4.3.5. 7-((6-bromohexyl)oxy)-4-methyl-2H-chromen-2-one (3e)
J ¼ 2.5 Hz, 1H), 6.16 (s, 1H), 4.33 (t, J ¼ 6.3 Hz, 2H), 3.93 (br s, 2H),
Yield 82%; m.p. 58e59 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.50 (d,
3.81e3.79 (m, 2H), 3.71e3.68 (m, 2H), 2.42 (s, 3H), 1.74 (br s, 6H).

N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
295
13C NMR (151 MHz, CDCl3)
125.75, 113.85, 112.56, 112.13, 102.06, 67.11, 53.36, 51.44, 35.47,
28.47, 24.22, 18.69. HRMS: calcd for C18H22NO3S2 [M þ H]þ
364.1036, found 364.1062.
d
194.36, 161.61, 161.02, 155.21, 152.46,
4H), 3.81 (t, J ¼ 6.3 Hz, 2H), 2.74 (br s, 4H), 2.41 (s, 3H), 2.06 (m, 1H),
0.50 (br s, 4H). 13C NMR (151 MHz, CDCl3)
195.32, 161.58, 160.97,
155.03, 152.35, 125.60, 113.89, 112.33, 112.18, 102.04, 66.90, 52.56,
39.93, 35.54, 18.65, 6.00. HRMS: calcd for C20H25N2O4S2 [M þ H]þ
405.1301, found 405.1302.
d
4.4.5. 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl
morpholine-4-carbodithioate (4e)
4.4.11. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl [1,40-
Yield 81%, white solid; m.p. 138e139 ꢀC; 1H NMR (600 MHz,
bipiperidine]-10-carbo dithioate (4k)
CDCl3)
d
7.51 (d, J ¼ 8.4 Hz, 1H), 6.91 (dd, J ¼ 9.0, 1.8, 1H), 6.88 (d,
Yield 89%; yellow oil; 1H NMR (600 MHz, CDCl3)
d 7.54 (d,
J ¼ 1.8 Hz 1H), 6.14 (s, 1H), 4.37 (br s, 2H), 4.32 (t, J ¼ 6.2 Hz, 2H),
J ¼ 9.0 Hz,1H), 6.95 (dd, J ¼ 9.0 Hz, J ¼ 2.4 Hz,1H), 6.89 (d, J ¼ 2.4 Hz,
1H), 6.17 (s, 1H), 4.34 (t, J ¼ 6.6 Hz, 2H), 3.82 (br s, 2H), 3.25 (br s,
3H), 2.70 (br s, 1H), 2.57 (br s, 4H), 2.43 (s, 3H), 2.00 (br s, 3H), 1.65
3.98 (br s, 2H), 3.81 (t, J ¼ 6.6 Hz, 2H), 3.78 (br s, 4H) 2.40 (s, 3H). 13
C
NMR (151 MHz, CDCl3)
d 196.37, 161.47, 161.23, 155.20, 152.43,
125.61, 113.92, 112.32, 112.20, 101.99, 66.83, 66.35, 66.10, 51.61,
50.49, 35.38, 18.69. HRMS: calcd for C17H20NO4S2 [M þ H]þ
366.0828, found 366.0865.
(br s, 6H),1.49 (br s, 2H). 13C NMR (151 MHz, CDCl3)
d 194.86, 161.56,
161.30, 155.20, 152.49, 125.60, 113.87, 112.35, 112.15, 102.04, 67.01,
61.97, 60.63, 50.28, 35.69, 29.71, 26.11, 25.66, 24.53, 22.09, 18.69.
HRMS: calcd for C23H31N2O3S2 [M þ H]þ 447.1771, found 447.1766.
4.4.6. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl 4-
hydroxypiperidine-1-carbo dithioate (4f)
4.4.12. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl 4-
(pyrimidin-2-yl)piperazine- 1-carbodithioate (4l)
Yield 83%; yellow solid; m.p. 160e162 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.52 (d, J ¼ 8.8 Hz, 1H), 6.93 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.88 (d,
Yield 86%; white solid; m.p. 195e196 ꢀC; 1H NMR (600 MHz,
J ¼ 2.4 Hz, 1H), 6.16 (s, 1H), 4.62 (s, 1H), 4.35 (t, J ¼ 6.3 Hz, 2H),
4.25e4.21 (m, 2H), 4.14e4.08 (m, 1H), 3.80 (m, 2H), 3.79 (t,
J ¼ 5.4 Hz, 2H), 2.42 (s, 3H), 1.79e1.62 (m, 4H). 13C NMR (151 MHz,
CDCl3)
d
8.47 (d, J ¼ 4.8 Hz, 2H), 7.53 (d, J ¼ 8.4 Hz, 1H), 6.94 (dd,
J ¼ 8.4 2.4 Hz, 1H), 6.88 (d, J ¼ 2.4 Hz, 1H), 6.73 (t, J ¼ 4.8 Hz, 1H),
6.16 (s, 1H), 4.48 (br s, 4H), 4.35 (t, J ¼ 6.6 Hz, 2H), 4.11 (br s, 4H),
3.84 (t, J ¼ 6.6 Hz, 2H), 2.42 (s, 3H). 13C NMR (151 MHz, CDCl3)
CDCl3) d 194.95, 161.61, 161.32, 155.19, 152.51, 125.59, 113.88, 112.43,
112.14, 102.03, 67.02, 66.06, 48.74, 46.98, 35.77, 18.69. HRMS: calcd
d 196.26,161.50,161.24,157.83,155.21,152.43,125.60,113.91,112.33,
for C18H22NO4S2 [M þ H]þ 380.0985, found 380.1024.
112.19, 110.76, 102.01, 68.86, 42.97, 38.73, 35.50, 29.80, 28.93, 18.68.
HRMS: calcd for C21H23N4O3S2 [M þ H]þ 443.1206, found 443.1153.
4.4.7. 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl bis(2-
hydroxyethyl)carbamodithioate (4g)
4.4.13. 3-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)propyl
piperidine-1-carbodithioate (4m)
Yield 82%; white solid; m.p. 100e101 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.51 (d, J ¼ 8.4 Hz, 1H), 6.90 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.87 (d,
Yield 89%; white solid; m.p. 87e88 ꢀC; 1H NMR (600 MHz,
J ¼ 2.4 Hz, 1H), 6.14 (s, 1H), 4.35 (t, J ¼ 6.6 Hz, 2H), 4.32 (br s, 2H),
4.16 (br s, 2H), 4.06 (br s, 2H), 3.96 (br s, 2H), 3.76 (t, J ¼ 6.6 Hz, 2H),
CDCl3)
d
7.54 (d, J ¼ 9.0 Hz, 1H), 6.90 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.86 (d,
J ¼ 2.4 Hz, 1H), 6.17 (s, 1H), 4.32 (br s, 2H), 4.16 (t, J ¼ 6.0 Hz, 2H),
2.40 (s, 3H). 13C NMR (151 MHz, CDCl3)
d 198.18, 161.97, 161.77,
3.92 (br s, 2H), 3.54 (t, J ¼ 6.6 Hz, 2H), 2.42 (s, 3H), 2.30e2.26 (m,
155.43, 152.97, 125.90, 114.16, 112.94, 112.36, 102.16, 66.85, 61.00,
59.47, 58.04, 36.24, 19.00. HRMS: calcd for C17H22NO5S2 [M þ H]þ
384.0934, found 384.0940.
2H),1.76e1.69 (br s, 6H). 13C NMR (151 MHz, CDCl3)
d 195.12, 161.93,
155.29, 152.48, 125.61, 113.82, 112.44, 111.97, 101.60, 67.95, 53.10,
51.27, 38.68, 28.90, 24.26, 23.00, 18.59. HRMS: calcd for
C
19H24NO3S2 [M þ H]þ 378.1187, found 378.1209.
4.4.8. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl 4-
methylpiperazine-1-carbodi thioate (4h)
4.4.14. 4-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)butyl piperidine-
1-carbodithioate (4n)
Yield 80%; white solid; m.p. 98e99 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.52 (d, J ¼ 9.0 Hz, 1H), 6.93 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.88 (d,
Yield 83%; white solid; m.p. 91e92 ꢀC;1H NMR (600 MHz, CDCl3)
J ¼ 2.4 Hz, 1H), 6.13 (s, 1H), 4.35 (t, J ¼ 6.3 Hz, 2H), 4.23 (br s, 4H),
d
7.49 (d, J ¼ 9.0 Hz, 1H), 6.87 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.81 (d,
3.93 (br, 4H), 3.74 (t, J ¼ 6.5 Hz, 2H), 2.40 (s, 3H), 1.99 (s, 3H). 13C
J ¼ 2.4 Hz, 1H), 6.13 (s, 1H), 4.30 (br s, 2H), 4.06 (t, J ¼ 6.6 Hz, 2H),
3.90 (br s, 2H), 3.40 (t, J ¼ 7.2 Hz, 2H), 2.40 (s, 3H), 1.97e1.92 (m,
NMR (151 MHz, CDCl3)
d 194.55, 161.46, 161.20, 155.17, 152.48,
125.64, 113.96, 112.45, 112.21, 101.88, 66.83, 53.95, 50.89, 49.58,
45.45, 35.52, 18.78. HRMS: calcd for C18H23N2O3S2 [M þ H]þ
379.1145, found 379.1170.
4H), 1.72e1.69 (m, 6H). 13C NMR (151 MHz, CDCl3)
d 195.55, 162.06,
161.37, 155.29, 152.57, 125.50, 113.52, 112.64, 111.90, 101.43, 67.99,
52.97, 51.27, 36.63, 28.24, 25.56, 24.33, 18.69. HRMS: calcd for
C
20H26NO3S2 [M þ H]þ 392.1349, found 392.1346.
4.4.9. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl 4-
isopropylpiperazine-1-carbod ithioate (4i)
4.4.15. 5-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)pentyl
piperidine-1-carbodithioate (4o)
Yield 88%; yellow solid; m.p. 93e94 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.52 (d, J ¼ 8.8 Hz, 1H), 6.93 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.88 (d,
Yield 80%; white solid; m.p. 88e89 ꢀC;1H NMR (600 MHz, CDCl3)
J ¼ 2.5 Hz, 1H), 6.16 (s, 1H), 4.40 (br s, 2H), 4.33 (t, J ¼ 6.3 Hz, 2H),
4.02 (br s, 2H), 3.81 (t, J ¼ 6.3 Hz, 2H), 2.81 (s, 1H), 2.66 (br s, 4H),
2.42 (s, 3H), 1.10 (s, 3H), 1.09 (s, 3H). 13C NMR (151 MHz, CDCl3)
d
7.51 (d, J ¼ 8.8 Hz, 1H), 6.87 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.82 (d,
J ¼ 2.5 Hz, 1H), 6.15 (s, 1H), 4.32 (s, 2H), 4.05 (t, J ¼ 6.4 Hz, 2H), 3.92
(br s, 2H), 3.36 (t, J ¼ 6.4 Hz, 2H), 2.42 (s, 3H), 1.92e1.86 (m, 2H),
1.84e1.79 (m, 2H), 1.77e1.58 (m, 8H). 13C NMR (151 MHz, CDCl3)
d
195.37, 161.53, 161.26, 155.21, 152.45, 125.60, 113.89, 112.33, 112.17,
102.04, 66.97, 54.60, 51.62, 50.14, 42.97, 35.45, 18.69, 18.34. HRMS:
d 195.80, 162.16, 161.39, 155.31, 152.59, 125.50, 113.47, 112.64, 111.87,
calcd for C20H27N2O3S2 [M þ H]þ 407.1458, found 407.1456.
101.41, 68.31, 52.79, 51.32, 36.91, 28.57, 28.56, 25.40, 24.34, 18.69.
HRMS: calcd for C21H28NO3S2 [M þ H]þ 406.1505, found 406.1491.
4.4.10. 2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl 4-
cyclopropylpiperazine-1-car bodithioate (4j)
4.4.16. 6-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)hexyl
piperidine-1-carbodithioate (4p)
Yield 76%; yellow solid; m.p. 139e140 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.52 (d, J ¼ 8.8 Hz, 1H), 6.93 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.88 (d,
Yield 87%; white solid; m.p. 64e65 ꢀC; 1H NMR (600 MHz,
J ¼ 2.5 Hz, 1H), 6.16 (s, 1H), 4.33 (t, J ¼ 6.7 Hz, 2H), 4.03e3.87 (m,
CDCl3)
d
7.50 (d, J ¼ 9.0 Hz, 1H), 6.86 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.80 (d,

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N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
J ¼ 2.4 Hz, 1H), 6.13 (s, 1H), 4.30 (br s, 2H), 4.03 (t, J ¼ 6.6 Hz, 2H),
3.90 (br s, 2H), 3.32 (t, J ¼ 7.2 Hz, 2H), 2.40 (s, 3H), 1.85e1.81 (m,
2H), 1.77e1.68 (m, 8H), 1.53e1.51 (m, 4H). 13C NMR (151 MHz,
molecules in PDB files were removed and hydrogen atoms were
subsequently added to the protein. The compound 4n was con-
structed using the MOE builder module and energy minimized
using Merck Molecular force field (MMFF94x, RMSD gradient:
0.05 kcal molꢁ1Åꢁ1). The protonation level of the compound in
physiological pH was calculated by the Marvin 14.12.8 2014 soft-
ware package, ChemAxon [http://www.chemaxon.com]. Then the
4n was docked into the active site of the protein by the “Triangle
Matcher” method, which generated poses by aligning the ligand
triplet of atoms with the triplet of alpha spheres in cavities of tight
atomic packing. The Dock scoring in MOE software was done using
ASE scoring function and Force field was selected as the refinement
method. The best 10 poses of molecules were retained and scored.
After docking, the geometry of resulting complex was studied using
the MOE's pose viewer utility.
CDCl3)
d 196.31, 162.56, 161.75, 155.68, 152.95, 125.84, 113.80,
113.05, 112.20, 101.73, 68.81, 53.18, 51.59, 37.42, 29.21, 29.04, 29.03,
25.95, 24.71, 19.05. HRMS: calcd for C22H30NO3S2 [M þ H]þ
420.1662, found 420.1638.
4.4.17. 8-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)octyl piperidine-
1-carbodithioate (4q)
Yield 86%; white solid; m.p. 60e61 ꢀC; 1H NMR (600 MHz,
CDCl3)
d
7.51 (d, J ¼ 8.8 Hz, 1H), 6.87 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.83 (d,
J ¼ 2.5 Hz, 1H), 6.15 (s, 1H), 4.32 (br s, 2H), 4.03 (t, J ¼ 6.5 Hz, 2H),
3.91 (br s, 2H), 3.35e3.29 (m, 2H), 2.42 (s, 3H), 1.87e1.80 (m, 2H),
1.75e1.72 (m, 2H), 1.54e1.25 (m, 14H). 13C NMR (151 MHz, CDCl3)
d
196.09,162.25,161.42,155.33,152.60,125.47,113.42,112.71,111.85,
101.37, 68.55, 52.74, 51.22, 37.24, 30.37, 29.16, 29.07, 28.96, 28.93,
28.68, 25.89, 24.35, 23.75,18.69. HRMS: calcd for C24H34NO3S2 [M þ
H]þ 448.1975, found 448.1901.
5.4. Inhibition of Ab1-42 self-induced aggregation
Inhibition of self-induced Ab1-42 aggregation was measured
using a Thioflavin T (ThT)-binding assay [37]. HFIP pretreated Ab1-
5. Biological evaluation
samples (Anaspec Inc) were resolubilized with a 50 mM phos-
42
5.1. Inhibition experiments of ChEs
phate buffer (pH 7.4) to give a 25
mM solution. Each tested com-
pound was firstly prepared in dimethyl sulfoxide (DMSO) and 1
m
L
Acetylcholinesterase (AChE, E.C. 3.1.1.7) from human erythro-
cytes, butylcholinesterase (BuChE, E.C. 3.1.1.8) from human serum,
S-butylthiocholine iodide (BTCI), acetylthiocholine iodide (ATCI), 5,
50-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent, DTNB), done-
pezil hydrochloride was purchased from Sigma-Aldrich (St. Louis,
MO, USA). The inhibitory activities of test compounds 4a-q were
evaluated by Ellman's method [33]. The compounds were dissolved
in DMSO and diluted with the buffer solution (50 mM Tris-HCl,
pH ¼ 8.0, 0.1 M NaCl, 0.02 M MgCl2$6H2O) to yield corresponding
test concentrations (DMSO less than 0.01%). In each well of the
of each was added to the well of black, opaque Corning 96-well
plates such that the final solvent concentration was 10%. The final
concentration of each compound was 25
independent triplicates. The solvent control was also included.
Then, 9 L of 25 mM Ab1-42 sample was added to each well and the
mM and was prepared in
m
samples mixed by gentle trapping. Plates were covered to minimize
evaporation and incubated in dark at room temperature for
46e48 h with no agitation.
After the incubation period, 200 mL of 5 mM ThT in 50 mM
glycine-NaOH buffer (pH 8.0) was added to each well. Fluorescence
was measured on a SpectraMax M5 (Molecular Devices, Sunnyvale,
CA, USA) multi-mode plate reader with excitation and emission
wavelengths at 446 nm and 490 nm, respectively. The fluorescence
intensities were compared and the percent inhibition due to the
presence of the inhibitor was calculated by the following formula:
100 - (IFi/IFo ꢂ 100) where IFi and IFo are the fluorescence in-
tensities obtained for Ab1-42 in the presence and in the absence of
inhibitor, respectively.
plate, 160
mL of 1.5 mM DTNB, 50
mL of AChE (0.05 U/mL hAChE) or
50
m
L of BuChE (0.024 U/mL hBuChE) were incubated with 10 mL of
different concentrations of test compounds at 37 ꢀC for 6 min. After
this period, acetylthiocholine iodide (15 mM) or S-butyrylthiocho-
line iodide (15 mM) as the substrate (30 mL) was added and the
absorbance was measured with a wavelength of 405 nm at different
time intervals (0, 60, 120, and 180 s). IC50 values were calculated as
concentration of compound that produces 50% enzyme activity
inhibition, using the Graph Pad Prism 4.03 software (San Diego, CA,
USA). Results are expressed as the mean SD of at least three
different experiments performed in triplicate.
5.5. Metal-chelating studies
5.2. Kinetic study of AChE inhibition
The metal-chelating ability of compound 4n were studied using
a UVevis spectrophotometer at wavelength ranging from 200 to
500 nm [38]. The UV absorption of compound, alone or in the
presence of CuCl2, ZnCl2, FeSO4 and Fe2(SO4)3, were recorded after
incubating for 30 min in menthol at room temperature. The final
volume of reaction mixture was 3 mL. The final concentrations of
The kinetic study of AChE was performed by Ellman's method
with three different concentrations (12.5, 25 and 50 nM) of com-
pound 4n. LineweavereBurk reciprocal plots were constructed by
plotting 1/velocity against 1/[substrate] at varying concentrations
of the substrate acetylthiocholine (0.05e0.50 mM). The plots were
assessed by a weighted least-squares analysis that assumed the
variance of velocity (v) to be a constant percentage of v for the
entire data set. Data analysis was performed with Graph Pad Prism
4.03 software (San Diego, CA, USA).
compound 4n and metals were 25 mM and 50 mM, respectively.
The stoichiometry of the 4n-Fe3þcomplex was determined by
Job's method [39]. The method is that keeping the total concen-
tration of compound and Fe3þ at 50
mM, and varying the molar ratio
of Fe3þ from 0.1 to 0.9. The absorbance change at 373 nm were
plotted versus the mole fraction, showing a maximum at 0.5, which
revealed a stoichiometry of 1:1 for 4n-Fe3þ complex. The associa-
tion constant (Ka) was calculated by the Benesi-Hildebrand equa-
tion as follows [39,40]: 1/A-A0 ¼ 1/Ka (Amax-A0) [Fe3þ] þ 1/Amax-A0,
where A and A0 represent the absorbance of compound in the
presence and absence of Fe3þ, respectively. Amax is the saturated
absorbance of the compound in the presence of excess amount of
[Fe3þ]; [Fe3þ] is the concentration of Fe3þ ion added.
5.3. Molecular modeling studies
Molecular modeling calculations and docking studies were
performed using Molecular Operating Environment (MOE) soft-
ware version 2008.10 (Chemical Computing Group, Montreal,
Canada). The X-ray crystallographic structure of AChE (PDB code
4EY7) was obtained from the Protein Data Bank. All water

N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
297
5.6. SH-SY5Y neuroblastoma cell toxicity
suspended in 0.5% carboxymethyl cellulose sodium (CMC-Na) salt
solution and orally administered according to the divided groups.
Food and water were provided later. After administration of 4n, the
mice were observed continuously for the first 4 h for any abnormal
behavior and mortality changes, intermittently for the next 24 h,
and occasionally thereafter for 14 days for the onset of any delayed
effects. All animals were sacrificed after being anaesthetized by
ether on the 14th day after drug administration.
The cytotoxicity effect of test compound on the human neuro-
blastoma SH-SY5Y cells was evaluated by MTT assay according to a
described method [41]. 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% fetal bovine serum (FBS), 100
U/mL penicillin and 100 m
g/mL streptomycin in 5% CO2 at 37 ꢀC. For
cell viability assay, cells were placed into 96-well plates at a seeding
density of 10000 cells/well and incubated with compound 4n for
5.9. Step-down passive avoidance test
24 h. After this incubation, 20
Then, the medium was removed, and 200
m
L of MTT at 37 ꢀC was added for 4 h.
mL of DMSO was added to
Donepezil hydrochloride was purchased from the Energy
Chemical Co., Ltd (Shanghai, China). Scopolamine was obtained
from Suicheng Pharmaceutical Co. Ltd. (Zhengzhou, China). Male
kunming mice weighting 18e22 g were supplied by Hunan SJA
Laboratory Animal Co., Ltd (eligibility certification no. SCXK [xiang]
2017e0002). Animals were maintained in our animal house
controlled at constant temperature of 23 2 ꢀC with a relative
humidity of 55 5% and a 12 h light/dark cycle. They were allowed
free access to tap water and standard laboratory chow.
The step-down test was carried in the passive avoidance
chamber with a steel grid floor [45]. A plastic platform was placed
in bottom right corner of the chamber. Illumination was available in
the light box through LED lights at 250 lx. The mice underwent two
separate trials: a training trial and a recall trial. For the training trial,
each mouse was allowed to get familiar with the chamber for 5 min.
Then the power was on and the mouse was placed on the platform.
Once the mouse stepped down, it would receive an electric shock
(24 V, 0.5 mA), which caused it to return to the platform. We used a
total of 48 mice in the passive avoidance test with 8 mice were used
per treatment. Compounds 4n (20.6, 10.3 and 5.2 mg/kg, p.o.) or
donepezil (5.0 mg/kg, p.o.) as a positive control were orally given
1 h before each training trial. After 30 min, memory impairment
was induced by administering scopolamine (3 mg/kg, i.p.). After a
24 h interval, the recall trial was performed, and the mice were
placed on the platform again. The latency to step down on the grid
for the first time and the errors that resulted in a shock within
5 min were measured as the learning and memory performance.
dissolve the formazan crystal formed. The absorbance was
measured in a microculture plate reader with a test wavelength of
570 nm and
a reference wavelength of 630 nm. Results are
expressed as the mean SD of three independent experiments.
5.7. In vitro blood-brain barrier permeation assay
The ability of test compounds that penetrate into brain was
evaluated using a parallel artificial membrane permeation assay
(PAMPA) for blood-brain-barrier according to the method estab-
lished by Di et al. [42]. Commercial drugs, PBS (pH ¼ 7.4), DMSO and
dodecane were obtained from Sigma and Aladdin. Porcine brain
lipid (PBL) was purchased from Avanti Polar Lipids. The donor
microplate (96-well filter plate, PVDF membrane, pore size is
0.45 mm) and the acceptor microplate (indented 96-well plate)
were both from Millipore. The 96-well UV plate (COSTAR) was ac-
quired from Corning Inc. The compound was firstly dissolved in
DMSO at a concentration of 5 mg/mL. Then, it was diluted 200-fold
with a mixture of PBS/EtOH (70:30) to give a final concentration of
25
with PBL dissolved in dodecane (4
of diluted solution and 300 L of PBS/EtOH (70:30) were added to
mg/mL. The filter membrane in donor microplate was coated
mL, 20 mg/mL). After that, 200
mL
m
the donor wells and the acceptor wells, respectively. The donor
filter plate was carefully placed on the acceptor plate to make the
underside of filter membrane can contact with buffer solution.
After leaving this sandwich assembly undisturbedly for 16 h at
25 ꢀC, the donor plate was carefully removed, and the concentra-
tions of test compound in the acceptor, donor and reference wells
were measured with a UV plate reader (SpectraMax Plus 384,
Molecular Devices, Sunnyvale, CA, USA). Each sample was analyzed
at least three independent runs in four wells, and the results are
given as the means SD. Pe was calculated using the following
expression: Pe ¼ {- VdVa/[(Vd þ Va)At]}ln (1 - drugacceptor/drugequi-
librium), where Vd is the volume of donor well, Va is the volume in
the acceptor well, A is the filter area, t is the permeation time, drug
acceptor is the absorbance obtained in the acceptor well, and drug
equilibrium is the theoretical equilibrium absorbance. A plot of the
experimental Pe values of 10 standard drugs versus their biblio-
graphic values provided a good linear correlation, Pe (exp.) ¼ 1.2653
Pe (bibl.) þ 0.7955 (R2 ¼ 0.9483).
Acknowledgement
This research work was financially supported by the Program of
the National Natural Science Foundation of China [Grant No.
81760622, 31600265]; the Program of Natural Science Foundation
of Guangxi Province of China [grant No. 2017GXNSFAA198077]; the
Program of Natural Science Foundation of Jiangxi Province of China
[grant No. 20171BAB215064]; Research Fund for the Doctoral Pro-
gram of Jiangxi University of Traditional Chinese Medicine [Grant
No. 2015BS008]; the Program of Basic ability enhancement of
young teachers in guangxi colleges and universities [Grant
No.KY2016YB091]; Guangxi Key Laboratory of Brain and Cognitive
Neuroscience, Guilin Medical University [07010150001]; Project
from Health and Family planning Commission of Jiangxi province
[Grant No. 2016A048, 20173013].
5.8. Acute toxicity test [43,44]
Twenty-four Kunming mice (20 2 g) were supplied by Hunan
SJA Laboratory Animal Co., Ltd (eligibility certification no. SCXK
[xiang] 2016e0004). Animals were housed in stainless steel cages
by sex in a ventilated animal room and maintained under standard
conditions with a 12 h:12 h light/dark cycle. Distilled water and
sterilized food for mice were available ad libitum. They were
acclimated to this environment for 5 days prior to dosing.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at https://doi.org/10.1016/j.ejmech.2018.01.055.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
Animals were randomly divided into two groups: control group
and experimental group (1000 mg/kg, n ¼ 12 per group). Before
treatment, animals were fasted overnight. Compound 4n was
References
[1] M. Goedert, M.G. Spillantini, Sci. (New York, N.Y.) 314 (2006) 777e781.

298
N. Jiang et al. / European Journal of Medicinal Chemistry 146 (2018) 287e298
[2] S. Salloway, J. Mintzer, M.F. Weiner, J.L. Cummings, Alzheimers Dement 4
Curr. Med. Chem. 18 (2011) 3929e3951.
(2008) 65e79.
[26] P. Anand, B. Singh, N. Singh, Bioorg. Med. Chem. 20 (2012) 1175e1180.
[27] J.B. Shaik, B.K. Palaka, M. Penumala, K.V. Kotapati, S.R. Devineni, S. Eadlapalli,
M.M. Darla, D.R. Ampasala, R. Vadde, G.D. Amooru, Eur. J. Med. Chem. 107
(2016) 219e232.
[28] J.B. Shaik, B.K. Palaka, M. Penumala, S. Eadlapalli, M.D. Mark, D.R. Ampasala,
R. Vadde, D.A. Gangaiah, Chem. Biol. Drug Des. 88 (2016) 43e53.
[29] J.S. Lan, Y. Ding, Y. Liu, P. Kang, J.W. Hou, X.Y. Zhang, S.S. Xie, T. Zhang, Eur. J.
Med. Chem. 139 (2017) 48e59.
[3] H. Tang, H.T. Zhao, S.M. Zhong, Z.Y. Wang, Z.F. Chen, H. Liang, Bioorg. Med.
Chem. Lett 22 (2012) 2257e2261.
[4] E. Scarpini, P. Schelterns, H. Feldman, Lancet Neurol. 2 (2003) 539e547.
^
[5] N. Herrmann, S.A. Chau, I. Kircanski, K.L. Lanctot, Drugs 71 (2011) 2031e2065.
ꢀ
ꢀ
[6] J. Godyn, J. Jonczyk, D. Panek, B. Malawska, Pharmacol. Rep. 68 (2016)
127e138.
[7] R.T. Bartus, R.L. Dean, B. Beer, A.S. Lippa, Science 217 (1982) 408e417.
[8] B.Z. Kurt, I. Gazioglu, L. Basile, F. Sonmez, T. Ginex, M. Kucukislamoglu,
S. Guccione, Eur. J. Med. Chem. 102 (2015) 80e92.
[30] Y.C. Duan, Y.C. Ma, E. Zhang, X.J. Shi, M.M. Wang, X.W. Ye, H.M. Liu, Eur. J. Med.
Chem. 62 (2013) 11e19.
[9] A. Romero, R. Cacabelos, M.J. Oset-Gasque, A. Samadi, J. Marco-Contelles,
Bioorg. Med. Chem. Lett 23 (2013) 1916e1922.
[31] S.S. Xie, X.B. Wang, J.Y. Li, L. Yang, L.Y. Kong, Eur. J. Med. Chem. 64 (2013),
540e533.
[10] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman,
Science 253 (1991) 872e879.
[32] R.D. Li, H.L. Wang, Y.B. Li, Z.Q. Wang, X. Wang, Y.T. Wang, Z.M. Ge, R.T. Li, Eur.
J. Med. Chem. 93 (2015) 381e391.
[11] Y. Chen, J. Sun, L. Fang, M. Liu, S. Peng, H. Liao, J. Lehmann, Y. Zhang, J. Med.
Chem. 55 (2012) 4309e4321.
[33] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, Biochem. Phar-
macol. 7 (1961) 88e95.
[12] R. Leurs, R.A. Bakker, H. Timmerman, I. de Esch, J. Nat. Rev. Drug Discov. 4
(2005) 107e120.
[34] M.D. Altintop, A.S. Gurkan-Alp, Y. Ozkay, Z.A. Kaplancikli, Arch. Pharm. 346
(2013) 571e578.
[13] I. Peretto, S. Radaelli, C. Parini, M. Zandi, L.F. Raveglia, G. Dondio, L. Fontanella,
P. Misiano, C. Bigogno, A. Rizzi, B. Riccardi, M. Biscaioli, S. Marchetti, P. Puccini,
S. Catinella, I. Rondelli, V. Cenacchi, P.T. Bolzoni, P. Caruso, G. Villetti,
F. Facchinetti, E. Del Giudice, N. Moretto, B.P. Imbimbo, J. Med. Chem. 48
(2005) 5705e5720.
[35] U.A. Mohsen, Z.A. Kaplancikli, Y. Ozkay, L. Yurttas, Drug Res. 65 (2015)
176e183.
[36] M. Rosini, E. Simoni, M. Bartolini, A. Cavalli, L. Ceccarini, N. Pascu,
D.W. McClymont, A. Tarozzi, M.L. Bolognesi, A. Minarini, V. Tumiatti,
V. Andrisano, I.R. Mellor, C. Melchiorre, J. Med. Chem. 51 (2008) 4381e4384.
[37] M. Bartolini, C. Bertucci, M.L. Bolognesi, A. Cavalli, C. Melchiorre, V. Andrisano,
Chembiochem 8 (2007) 2152e2161.
[14] Y. Li, X. Qiang, L. Luo, X. Yang, G. Xiao, Q. Liu, J. Ai, Z. Tan, Y. Deng, Eur. J. Med.
Chem. 126 (2017) 762e775.
[15] J. Hardy, D.J. Selkoe, Science 297 (2002) 353e356.
[16] A.K. Sharma, S.T. Pavlova, J. Kim, D. Finkelstein, N.J. Hawco, N.P. Rath, J. Kim,
L.M. Mirica, J. Am. Chem. Soc. 134 (2012) 6625e6636.
[17] N. Jiang, X.B. Wang, Z.R. Li, S.Y. Li, S.S. Xie, M. Huang, L.Y. Kong, RSC Adv. 5
(2015) 14242e14255.
[38] M. I. Fernandez-Bachiller, C. Perez, G. C. Gonzalez-Munoz, S. Conde, M. G.
Lopez, M. Villarroya, A. G. Garcia, M. I. Rodriguez-Franco, J. Med. Chem. 53
4927e4937.
[39] L.Y. Wang, D.C. Ye, D.R. Cao, Spectrochim. Acta, Part A 90 (2012) 40e41.
[40] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703e2707.
[41] X. Li, H. Wang, Z. Lu, X. Zheng, W. Ni, J. Zhu, Y. Fu, F. Lian, N. Zhang, J. Li,
H. Zhang, F. Mao, J. Med. Chem. 59 (2016) 8326e8344.
[42] L. Di, E.H. Kerns, K. Fan, O.J. McConnell, G.T. Carter, Eur. J. Med. Chem. 38
(2003) 223e232.
ꢀ
[18] R. Leon, A.G. Garcia, J. Marco-Contelles, Med. Res. Rev. 33 (2013) 139e189.
[19] G. Pepeu, M.G. Giovannini, Curr. Alzheimer Res. 6 (2009) 86e96.
[20] J. Dong, C.S. Atwood, V.E. Anderson, S.L. Siedlak, M.A. Smith, G. Perry,
P.R. Carey, Biochemistry 42 (2003) 2768e2773.
[21] F. Gu, M. Zhu, J. Shi, Y. Hu, Z. Zhao, Neurosci. Lett. 440 (2008) 44e48.
[22] D.A. Butterfield, A.M. Swomley, R. Sultana, Antioxidants Redox Signal. 19
(2013) 823e835.
[23] N. Jiang, S.Y. Li, S.S. Xie, Z.R. Li, K.D. Wang, X.B. Wang, L.Y. Kong, Eur. J. Med.
Chem. 87 (2014) 540e551.
[43] C. Lu, Y. Guo, J. Yan, Z. Luo, H.B. Luo, M. Yan, L. Huang, X. Li, J. Med. Chem. 56
(2013) 5843e5859.
[44] J.S. Lan, T. Zhang, Y. Liu, J. Yang, S.S. Xie, J. Liu, Z.Y. Miao, Y. Ding, Eur. J. Med.
Chem. 133 (2017) 184e196.
[45] I. Klinkenberg, A. Blokland, Neurosci. Biobehav. Rev. 34 (2010) 1307e1350.
[46] Z. Sang, W. Pan, K. Wang, Q. Ma, L. Yu, Y. Yang, P. Bai, C. Leng, Q. Xu, X. Li,
Z. Tan, W. Liu, Eur. J. Med. Chem. 130 (2017) 379e392.
[24] S.S. Xie, X. Wang, N. Jiang, W. Yu, K.D. Wang, J.S. Lan, Z.R. Li, L.Y. Kong, Eur. J.
Med. Chem. 95 (2015) 153e165.
[25] I. Kostova, S. Bhatia, P. Grigorov, S. Balkansky, V.S. Parmar, A.K. Prasad, L. Saso,