Design, synthesis and biological evaluation of coumarin derivatives as novel acetylcholinesterase inhibitors that attenuate H2O2-induced apoptosis in SH-SY5Y cells

Article abstract of DOI:10.1016/j.bioorg.2016.07.013

A novel series of coumarin derivatives were designed, synthesized and investigated for inhibition of cholinesterase, including acetyl cholinesterase (AChE) and butyrylcholinesterase (BuChE). This biological study showed that these compounds containing piperazine ring had significant inhibition activities on AChE rather than BuChE. Further study suggested that 9x, as one of this kind of structure derivative, showed the strongest inhibition activity on AChE with an IC₅₀ value of 34?nM. Moreover, molecular docking, flow cytometry (FCM), and western blot assay suggested that 9x could induce cytoprotective autophagy to attenuate H₂O₂-induced cell death in human neuroblastoma SH-SY5Y cells. These findings highlight a new approach for the development of a novel potential neuroprotective compound targeting AChE with autophagy-inducing activity in future Alzheimer's disease (AD) therapy.

Full text of DOI:10.1016/j.bioorg.2016.07.013

Bioorganic Chemistry 68 (2016) 112–123
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of coumarin derivatives as
novel acetylcholinesterase inhibitors that attenuate H2O2-induced
apoptosis in SH-SY5Y cells
Dahong Yao a,1, Jing Wang b,1, Guan Wang a,1, Yingnan Jiang a, Lei Shang c, Yuqian Zhao a, Jian Huang a,
Shilin Yang a, , Jinhui Wang a, , Yamei Yu d,
⇑
⇑
⇑
a School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b Department of Neurology, Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University, Liaocheng 252000, China
c School of Pharmacy, China Medical University, Shenyang 110122, China
d Department of Emergency Radiology, Liaocheng People’s Hospital and Liaocheng Clinical School of Taishan Medical University, Liaocheng 252000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of coumarin derivatives were designed, synthesized and investigated for inhibition of
cholinesterase, including acetyl cholinesterase (AChE) and butyrylcholinesterase (BuChE). This biological
study showed that these compounds containing piperazine ring had significant inhibition activities on
AChE rather than BuChE. Further study suggested that 9x, as one of this kind of structure derivative,
showed the strongest inhibition activity on AChE with an IC50 value of 34 nM. Moreover, molecular
docking, flow cytometry (FCM), and western blot assay suggested that 9x could induce cytoprotective
autophagy to attenuate H2O2-induced cell death in human neuroblastoma SH-SY5Y cells. These findings
highlight a new approach for the development of a novel potential neuroprotective compound targeting
AChE with autophagy-inducing activity in future Alzheimer’s disease (AD) therapy.
Received 8 June 2016
Revised 20 July 2016
Accepted 25 July 2016
Available online 27 July 2016
Ó 2016 Published by Elsevier Inc.
1. Introduction
to prevent, halt, or reverse AD. However, in virtue of research
advances over the past decades, several therapeutic strategies have
Alzheimer’s disease (AD) is an overwhelming neurodegenera-
tive disorder that usually happens among over 60-year-old people,
which is characterized by progressive memory disorders, personal-
ity changes, language barrier and cognitive dysfunction [1–3]. Due
to the increasing aged population, AD has become one of the most
serious diseases that have aroused a wide social concern [4]. At the
molecular level, AD is a multifaceted neurodegenerative disorder
characterized by protein misfolding and aggregation, oxidative
stress, mitochondrial abnormality, and neuro-inflammatory pro-
cess [5–7]. According to the cholinergic hypothesis, AD is caused
by reduced level of the neurotransmitter acetylcholine in some
parts of the brain such as hippocampus, which is related to learn-
ing and cognition [8]. In addition, the accumulation of cellular
damage such as oxidized proteins is a feature common to all aging
cells. Effective elimination of such adverse factors is essential for
maintaining cellular homeostasis, which is primarily achieved by
autophagy [9–12]. By now, there are still no effective treatments
been developed to treat AD, including anti-cholinesterase, anti-
oxidant, anti-amyloid, anti-inflammatory and anti-apoptotic
approaches [13–16]. In particular, AD therapy has been much
improved along with the application of cholinesterase inhibitors.
At present, most prescribed drugs for treatment of AD are selective
AChE inhibitors, while new avenues for pharmacological interven-
tion against Ab in AD are still in development but not mature [17].
The majority of the therapeutic treatments of AD are aimed to
enhance acetylcholine level in brain by inhibiting AchE activity.
Drugs directly targeting AChE approved by the FDA are tacrine,
donepezil, Huperzine A and galanthamine, etc. (Fig. 1). Interest-
ingly, recent research has also shown that anti-diabetic drug gli-
mepiride could serve as AChE inhibitor for AD-diabetes dual
therapy [18]. Despite their AChE inhibitory activity, AChE inhibi-
tors also have a variety of side effects [19–21]. Therefore, the
development of efficient and low toxic AChE inhibitor bearing mul-
tiple biological functions is currently the main strategy for treat-
ment of AD.
AD is commonly accompanied by obvious oxidative damage in
neural cells [22]. At present, AD treatments are mostly focused
on the inhibition of cholinesterase, losing sight of the protection
of nerve cells. In this study, we did not only concern about the
⇑
Corresponding authors.
E-mail addresses: yangshilin@suda.edu.cn (S. Yang), profwjh@126.com
(J. Wang), yuyamei252000@163.com (Y. Yu).
1
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bioorg.2016.07.013
0045-2068/Ó 2016 Published by Elsevier Inc.

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
113
Fig. 1. Structures of several AChE inhibitors.
inhibition of cholinesterase, but also investigated whether the
potential compounds have protective effects against the oxidative
damage of nerve cells.
candidate compound targeting AChE with a potential to induce
protective autophagy for future AD therapy.
The crystal structure of AChE indicated that AChE possessed
two binding sites: a catalytic active site (CAS) located at the bot-
tom of the deep narrow gorge and a peripheral anionic site (PAS)
at the entrance [23]. The PAS was found to be related with the
non-catalytic function of AChE, accelerating b-amyloid (Ab) pep-
tide deposition and promoting the formation of Ab fibril. Therefore,
AChE might play a crucial role in amyloid deposition at an early
stage of AD [24–26]. Accordingly, the AChE inhibitors with dual
interaction on PAS and CAS could be more effective agents for
the management of AD. Recently, a large number of studies have
revealed a certain AChE inhibitory activity of coumarin, a natural
product which could form strong pi-pi bonds with AChE through
the interaction between its aromatic ring and the aromatic amino
acid residues of PAS [27].
2. Results and discussion
2.1. The chemical structure and synthesis of coumarin derivatives
The synthetic pathway of our designed compounds was shown
in Scheme 1. The starting material 1 was condensed with diethyl-
malonate in the presence of catalytic amount piperidine in ethanol
to afford 2, which was hydrolyzed to get acid 3. Halogenation of
acid 3 via thionyl chloride gave 4. The intermediate 5 was then
reacted with N-bromosuccinimide (NBS) in the presence of AIBN
to yield 6, which was then condensed with appropriate amines in
the presence of triethylamine in CH2Cl2 to afford 7. Hydrogenation
of compound 7 via Fe/NH4Cl obtained 8. Final products 9a-9aa
were obtained through addition of 8 to intermediate 4 in dry
CH2Cl2.
In our previous studies, we isolated a simple coumarin, ethyl 7-
methoxy-2-oxo-2H-chromene-3-carboxylate, bearing a weak inhi-
bitory activity for AChE (IC50 = 100 lM) from Ferula dissecta
(Ledeb.) Ledeb. Herein, based on the crystal structure of AChE, a
novel series of coumarin derivatives were designed and synthe-
sized, employing ethyl 7-methoxy-2-oxo-2H-chromene-3-carboxy
late as the leading compound. First, to explore the impact of R1 on
electric distribution, different electronegative groups are intro-
duced to the A ring of the scaffold. To extend the scaffold along
with the activity pocket, a substituted benzene was introduced to
the 3-position of coumarin by the amido bond to occupy the deep
narrow aromatic gorge and R2 was replaced by different isosteres.
In addition, in order to fully occupy the bottom of active pocket,
different secondary amines were grafted to cover the CAS area.
Finally, 27 coumarin derivatives were synthesized, most of which
possessed superior selective AChE inhibitory activity. Both docking
simulation and AChE assay suggested 9x as the most efficient one.
In order to further study the cell protective activity of 9x, flow
cytometry (FCM), MDC, Hoechst 33258 fluorescent staining and
western blot were performed. All the results suggested that 9x
could protect human neuroblastoma SH-SY5Y cells against H2O2-
induced cell death, which is an established in vitro cellular model
of AD. Furthermore, the protective effect of 9x was mainly due to
its ability to induce cytoprotective autophagy. Collectively, these
findings highlight a new approach for the development of a novel
2.2. AChE and BuChE assays
The inhibitory activities of 9a-9aa against AChE and BuChE
were evaluated according to the spectrophotometric method of Ell-
man [28]. For comparison, Huperzine A, an AChE inhibitor in clin-
ical use, was used as standard drug. The IC50 value for the
inhibition of AChE and BuChE were summarized in Table 1 and
Fig. 2. The results showed that all the synthetic compounds with
piperazine ring exhibited significant inhibitory activities on AChE,
with IC50 values in the nanomolar range. Furthermore, compounds
with N-ethylpiperazine showed higher efficiency than compounds
with N-methylpiperazine. The possible reason is that the size of
the ethyl group is larger than that of the methyl group, facilitating
occupation of the active site and thus preventing the substrate
from entering the catalytic center. Data showed that all the com-
pounds had almost no inhibition activities on BuChE, indicating
these compounds are selective inhibitors of AChE. Compounds
treated with morpholine or dibutylamine inhibited neither AChE
nor BuChE, suggesting that presentation of piperazine ring was
required for inhibitory activities against AChE. Also, by comparing
morpholine with piperazine, we can predict that the second N
atom plays a pivotal role in the maintenance of the inhibitory

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D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
Scheme 1. Reagents and conditions: (a) ethanol, piperidine, reflux; (b) NaOH, 90 °C, HCl; (c) SOCl2, DMF, reflux; (d) CHCl3, NBS, AIBN, r.t.; (e) CH2Cl2, triethylamine, r.t.; (f)
95% ethanol, Fe, NH4Cl, reflux; (g) CH2Cl2, triethylamine, r.t.
activity. In addition, different substituted coumarins had some
effect on the inhibition activity of AChE. In general, compounds
with stronger electron-withdrawing ability would exhibit more
potent inhibitory activity on AChE. The group R2 had little effect
on the activity of the compounds in the enzyme activity test, but
was related to the in vivo metabolic properties of the compounds.
In this study, we screened the compound 9x with nitro group,
N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-6-nitro-2-
oxo-2H-chromene-3-Carboxamide, which presented an IC50 value
of 34 nM, serving as the most potent compound for AChE inhibition
whose inhibitory activity was 19 folds than that of Huperzine A.
stable and whether they converged, low root-mean-square devia-
tion (RMSD) fluctuations of the systems observed indicated well-
behaved systems.
2.4. 9x protected SH-SY5Y cells from H2O2-induced apoptosis
Next, we tested whether 9x protected SH-SY5Y cells from
oxidative stress-induced cell death [29]. SH-SY5Y cells were
treated with 1 mM H2O2 and 9x solely at various concentrations
(0–3200 nM) or 1 mM H2O2 together with 9x at given concentra-
tions (Fig. 4A). 9x could obviously increase the cell viability
compared with cells treated with 1 mM H2O2 alone. Meanwhile,
9x did not affect the cell viability of SH-SY5Y at these concentra-
tions. To characterize whether the cell death induced by H2O2
was apoptosis, we observed the morphologic changes of
SH-SY5Y cells. When cells were treated with 1 mM H2O2, the
apoptotic alterations could be observed under inverted micro-
scopy and by fluorescence microscopy after Hoechst 33258
staining (Fig. 4B). When cells were pretreated with 100 nM
9x 1 h before 1 mM H2O2 treatment, the apoptotic DNA fragments
significantly decreased compared with cells treated with 1 mM
H2O2 solely. In addition, apoptosis was further evaluated by
Annexin-V/PI double staining. 9x markedly decreased the apop-
totic ratio in H2O2-treated SH-SY5Y cells (Fig. 4C and D). Previous
reports demonstrated that H2O2 could induce cell death through
oxidative stress-induced mitochondrial injury [30], therefore we
assessed whether mitochondria death pathway was involved in
the process. The level of Bcl-2 markedly decreased while the
expressions of cleaved caspase-3 and BAX remarkably increased
after 1 mM H2O2 administration. While cells were pretreated with
9x, the level of cleavage of caspase-3 decreased and the level of
Bcl-2 increased (Fig. 4E). Herein, conclusion could be drawn that
9x could protect SH-SY5Y cells from H2O2-induced apoptosis by
alleviating mitochondrial injury.
2.3. Docking and molecular dynamic (MD) stimulation of 9x with
AChE
To explore the interaction mode of the compound 9x (shown in
Fig. 3A) against AChE, a molecular modeling study was performed.
A detailed view of the interaction of compound 9x with AChE was
displayed in Fig. 3B and C. As presented, compound 9x could form
interaction with PAS of AChE with two amino acids Trp286 and
Arg296 involved, Trp286 as the key residue in ligand recognition.
In this case, Trp286 was responsible for binding the aromatic rings
of coumarin with four pi-pi stackings. Also, the lactone carbonyl
group of coumarin moiety could interact with Phe295 and
Arg296 to form two hydrogen bonds. Moreover, the central part
of the amide bond could also form hydrogen bond with Phe295
in the deep aromatic narrow gorge. In addition, the aniline moiety
formed two pi-pi stackings with Tyr341 and Trp286, respectively.
The ethylpiperazine moiety covered the CAS area through
hydrophobic interactions with Phe330 and Trp84. These results
showed that compound 9x could bind to AChE stably. To further
explore the stability of the complex compound 9x-AChE, 3 ns MD
simulations were successfully performed on compound 9x-AChE
complex (Fig. 3D). To gauge whether the MD simulations were

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
115
Table 1
Inhibition activities of compounds 9a-9aa on AChE and BuChE.
1
O
O
O
2
H
N
R1
3
R3
9a-9aa
R2
Compound
R1
R2
R3
IC50 (means SEM)a
AChE (nM)
BuChE (nM)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
9u
9v
9w
9x
1-OCH3
1-OCH3
1-OCH3
1-OCH3
1-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
1-H
1-H
1-H
1-H
1-H
3-Cl
3-Cl
3-Cl
3-Cl
3-Br
3-Br
3-Br
3-Br
3-NO2
3-NO2
3-NO2
3-NO2
Cl
Cl
Cl
CF3
H
Cl
Cl
Cl
H
CF3
Cl
Cl
Cl
H
CF3
Cl
Cl
Cl
H
Cl
Cl
Cl
H
Cl
Cl
Cl
H
Ethylpiperazine
Methylpiperazine
Morpholine
Methylpiperazine
Di-n-butylamine
Ethylpiperazine
Methylpiperazine
Morpholine
Di-n-butylamine
Methylpiperazine
Methylpiperazine
Ethylpiperazine
Morpholine
Di-n-butylamine
Methylpiperazine
Ethylpiperazine
Methylpiperazine
Morpholine
Di-n-butylamine
Ethylpiperazine
Methylpiperazine
Morpholine
Di-n-butylamine
Ethylpiperazine
Methylpiperazine
Morpholine
195.5 1.51
1224.3 0.72
ndb
ndb
1767.0 1.3
ndb
1452.6 3.13
ndb
ndb
ndb
122.0 0.46
1537.4 0.93
ndb
ndb
1677.0 0.98
ndb
ndb
ndb
590.7 1.26
1300.8 1.79
1301.0 1.25
ndb
ndb
1974.0 2.01
1587.0 1.66
ndb
ndb
ndb
618.2 0.68
221.6 1.01
840.9 0.78
ndb
ndb
ndb
1789.0 1.54
ndb
ndb
ndb
120.4 1.0
330.0 1.0
ndb
ndb
1889.5 1.62
ndb
ndb
ndb
34.0 1.00
806.9 1.16
ndb
ndb
9y
9z
9aa
1882.6 2.11
ndb
ndb
Di-n-butylamine
ndb
Huperzine A
Iso-OMPA
647.6 2.33
134.0 1.94
a
IC50: 50% inhibitory concentration (means SEM of three experiments).
nd: IC50 > 2000 nM (highest concentration tested).
b
2.5. 9x induced obvious autophagy to protect SH-SY5Y cells from
H2O2-induced apoptosis
3. Experimental section
3.1. Materials and measurements
Autophagy could protect neurons from cell death and maintain
their normal functions by effectively eliminating cellular
damage [9]. To determine how 9x protected SH-SY5Y cells from
H2O2-induced apoptosis, we further explored whether autophagy
were involved in this process. We found obvious autophagy in both
H2O2 and 9x + H2O2 treated SH-SY5Y cells (Fig. 5A). Additionally,
9x resulted in enhanced autophagy ratio compared with H2O2 trea-
ted group, which could also be testified by increased levels of LC3
and p62 (Fig. 5C–E). These results indicated that 9x could induce
autophagy in H2O2 treated SH-SY5Y cells. Meanwhile, we detected
the cell viability and level of cleaved-caspase3 of the control, H2O2,
9x + H2O2 and 9x + H2O2 + 3-MA treated groups (Fig. 5B and F).
These results together with the Annexin V-PI staining of different
groups in Fig. 4C revealed that 9x could induce protective autop-
hagy to protect SH-SY5Y cells from H2O2-induced apoptosis, thus
9x played as a protector in SH-SY5Y cells to resist H2O2-induced
oxidative damages.
1H NMR spectra were recorded at 400 MHz. The chemical
shifts were recorded in ppm relative totetramethylsilane and
with the solvent resonance as the internal standard. Data were
reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet), coupling
constants (Hz), integration. 13C NMR data were collected at
100 MHz with complete proton decoupling. Chemical shifts
were reported in ppm from the tetramethylsilane with the
solvent resonance as internal standard. ESI-HRMS spectra were
recorded on a commercial apparatus and methanol was used
to dissolve the sample. All chemicals were obtained from
commercial sources and used without further purification.
Column chromatography was carried out on silica gel
(300–400 mesh, Qingdao Marine Chemical Ltd, Qingdao, China).
Thin layer chromatography (TLC) was performed on TLC silica
gel 60 F254 plates.

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D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
Fig. 2. The inhibition effects on AchE activity of the potential compounds. Different concentration of the potential compounds (9a, 9b, 9d, 9f, 9g, 9j, 9k, 9l, 9o, 9p, 9q, 9t, 9u,
9x, 9y) on AchE activity compared with Huperzine A. The concentration ranged from 0 to 4 lM and the incubation environment was 30 min at 37 °C.
3.2. General procedure for the synthesis of compounds 2a-f
J = 2.4, 8.7 Hz), 6.82 (1H, d, J = 2.4 Hz), 4.40 (2H, q, J = 7.1 Hz),
3.91 (3H, s, OCH3), 1.40 (3H, t, J = 7.1 Hz).
To a solution of the appropriate salicylaldehyde derivatives
(20 mmol) in absolute ethyl alcohol (30 ml), diethylmalonate
(1.4 eq) and piperidine (0.1 eq) were added, and the resulting mix-
ture was allowed to warmed up to reflux, and then glacial acetic
acid (3 drops) was added. The mixture was refluxed for 4 h contin-
ually and ice water (13 ml) was added. The crude product was fil-
tered and purified by re-crystallization from ethyl alcohol and
water (70–90%). Yield 60–95%.
3.2.4. Ethyl 6-chloro-2-oxo-2H-chromene-3-carboxylate (2d)
White plate crystal, yield 76%, m.p. 173–174 °C, 1H NMR (CDCl3,
400 MHz), dH 8.44 (1H, s), 7.60 (1H, d, J = 2.4 Hz), 7.59 (1H, dd,
J = 2.4, 8.8 Hz), 7.32 (1H, d, J = 8.8 Hz), 4.43 (2H, q, J = 7.0 Hz),
1.41 (3H, t, J = 7.0 Hz).
3.2.5. Ethyl 6-bromo-2-oxo-2H-chromene-3-carboxylate (2e)
White needle crystal, yield 75%, m.p. 175–177 °C, 1H NMR
(CDCl3, 400 MHz), dH 8.44 (1H, s), 7.60 (1H, d, J = 2.4 Hz), 7.59
(1H, dd, J = 2.4, 8.7 Hz), 7.32 (1H, d, J = 8.7 Hz), 4.42 (2H, q,
J = 7.1 Hz), 1.42 (3H, t, J = 7.1 Hz).
3.2.1. Ethyl 8-methoxy-2-oxo-2H-chromene-3-carboxylate (2a)
White needle crystal, yield 88%, m.p. 87–90 °C, 1H NMR (CDCl3,
400 MHz), dH 8.50 (1H, s), 7.15–7.30 (3H, m), 4.41 (2H, q,
J = 7.1 Hz), 3.97 (3H, s, OCH3), 1.41 (3H, t, J = 7.1 Hz).
3.2.6. Ethyl 6-nitro-2-oxo-2H-chromene-3-carboxylate (2f)
white solid, yield 75%, m.p. 193–197 °C, 1H NMR (CDCl3,
400 MHz), dH 8.58 (1H, s), 8.56 (1H, d, J = 2.6 Hz), 8.50 (1H, dd,
J = 2.6, 9.0 Hz), 7.51 (1H, d, J = 9.0 Hz), 4.44 (2H, q, J = 7.2 Hz),
1.43 (3H, t, J = 7.2 Hz).
3.2.2. Ethyl 2-oxo-2H-chromene-3-carboxylate (2b)
White needle crystal, yield 90%, m.p. 87–90 °C, 1H NMR (CDCl3,
400 MHz), dH 8.53 (1H, s), 7.60–7.70 (2H, m), 7.30–7.37 (2H, m),
4.42 (2H, q, J = 7.0 Hz), 1.41 (3H, t, J = 7.0 Hz).
3.3. General procedure for the synthesis of compounds 4a-f
3.2.3. Ethyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (2c)
Light yellow solid, yield 82%, m.p. 132–134 °C, 1H NMR (CDCl3,
400 MHz), dH 8.51 (1H, s), 7.51 (1H, d, J = 8.7 Hz), 6.89 (1H, dd,
Compounds 3a-f (2 mmol) in aqueous sodium hydroxide
(50 ml, 20%) was heated to reflux for 2 h, then filtered. The filtrate

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
117
Fig. 3. Molecular modeling, docking and molecular dynamics (MD) simulations of AChE targeting compound 9x-AChE. (A) Molecular structures of compound 9x. (B)
Molecular docking of compound 9x. (C) The interactions of compound 9x and active pocket. (D) Molecular dynamics of compound 9x-AChE complex.
was allowed to cool 0 °C and acidified to pH = 2 with 1 N
hydrochloric acid, and filtered to give the crude product without
further purification. The crude product was dissolved in dichloro
sulfoxide (20 ml) and refluxed for 3 h, the solvent was removed
under reduced pressure to yield the desired product b1-b6 as fine
light yellow powder.
After removing the solvent under reduced pressure, the crude pro-
duct was purified by flash chromatography on silica gel, eluting
with petroleum ether and ethyl acetate (3–10%), yielding the title
compounds 7a-e (85–92%).
3.5.1. 1-(2-Chloro-4-nitrobenzyl)-4-methylpiperazine (7a)
Light yellow solid, yield 89%, 1H NMR (CDCl3, 400 MHz), dH 8.22
(1H, d, J = 2.3 Hz), 8.10 (1H, dd, J = 8.5, 2.3 Hz), 7.74 (1H, d,
J = 8.5 Hz), 3.68 (2H, s), 2.50–2.60 (8H, m), 2.31 (3H, s).
3.4. General procedure for the synthesis of compounds 6a-c
To a solution of appropriate p-nitrotoluene derivatives (2.5 g,
12.0 mmol) and 2, 2-azobisisobutyronitrile (AIBN, 34 mg,
0.203 mmol) in chloroform (30 ml), N-bromosuccinimide (NBS,
1.16 g, 6.5 mmol) was added in small batches, and the resulting
mixture was stirred about 36 h at 56 °C. The crude was filtered
and washed with ethyl acetate. The filtrate was collected and
washed with saturated aqueous sodium bicarbonate and brine,
and then dried over anhydrous sodium sulfate. After removing
the solvent under reduced pressure to give the crude product 6a-
c and used in the next step without further purification.
3.5.2. 1-(2-Chloro-4-nitrobenzyl)-4-ethylpiperazine (7b)
Light yellow solid, yield 93%, 1H NMR (CDCl3, 400 MHz), dH 8.22
(1H, d, J = 2.3 Hz), 8.10 (1H, dd, J = 8.5, 2.3 Hz), 7.76 (1H, d,
J = 8.5 Hz), 3.75 (4H, t, J = 4.6 Hz), 3.68 (2H, s), 2.54 (4H, t,
J = 4.6 Hz).
3.5.3. 4-(2-Chloro-4-nitrobenzyl)morpholine (7c)
Light yellow solid, yield 93%, 1H NMR (CDCl3, 400 MHz), dH 8.24
(1H, d, J = 2.2 Hz), 8.10 (1H, dd, J = 8.5, 2.3 Hz), 7.74 (1H, d,
J = 8.5 Hz), 3.68 (2H, s), 2.50–2.60 (8H, m), 2.44 (2H, q, J = 7.2 Hz),
1.09 (3H, t, J = 7.2 Hz).
3.5. General procedure for the synthesis of compounds 7a-e
The 4-nitrobenzyl bromide derivatives (6a-c, 18 mmol), triethy-
lamine (2.21 g, 22 mmol) and desired amine (22 mmol) were com-
bined in dichloromethane (30 ml) and stirred for 4–6 h at room
temperature. The solvent was then removed under reduced pres-
sure, and the resulting crude was dissolved in ethyl acetate,
washed with saturated aqueous sodium bicarbonate and brine.
3.5.4. 4-(4-Nitro-2-(trifluoromethyl)benzyl)morpholine (7d)
Light yellow solid, yield 89%, 1H NMR (CDCl3, 400 MHz), dH 8.30
(1H, s), 8.21 (1H, d, J = 8.5 Hz), 7.99 (1H, d, J = 8.5 Hz), 3.61 (2H, s),
2.34–2.40 (8H, m), 2.16 (3H, s).

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D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
Fig. 4. 9x protected SH-SY5Y cells from H2O2 induced apoptosis. (A) Comparison of cell viability of 9x, H2O2, and 9x + H2O2 towards SH-SY5Y cells. (B) Effects of compound 9x,
H2O2, 9x together with H2O2 on cell morphology: bright-field microscopy images and fluorescence microscopic appearance of Hoechst 33258 staining nuclei of SH-SY5Y cells,
apoptotic cells were observed in fragmented fluorescent nuclei. (C) The results of Annexin-V/PI binding assay through flow cytometry for untreated control, H2O2, 9x + H2O2,
9x + H2O2 + 3-MA. (D) The percentage of apoptotic cells were statistics in the pie chart. (E) Western blot analysis demonstrating Bcl-2, BAX and cleaved-caspase3 levels in
different groups.
3.5.5. N-butyl-N-(4-nitrobenzyl)butan-1-amine (7e)
3.6.1. 3-Chloro-4-((4-methylpiperazin-1-yl)methyl)aniline (7a)
Prepared by method B, light yellow solid, yield 89%, 1H NMR
(CDCl3, 400 MHz), dH 7.26 (1H, s), 7.17 (1H, d, J = 8.2 Hz), 6.68
(1H, d, J = 2.4 Hz), 6.54 (1H, dd, J = 8.2, 2.4 Hz), 3.66 (2H, s), 3.51
(2H, s), 2.44–2.52 (8H, m), 2.23 (3H, s).
Light yellow oil, yield 93%, 1H NMR (CDCl3, 400 MHz), dH 8.16
(2H, d, J = 8.7 Hz), 7.52 (1H, d, J = 8.7 Hz), 3.63 (2H, s), 4.41 (4H, t,
J = 7.1 Hz), 1.44 (4H, m), 1.29 (4H, m), 0.88 (6H, t, J = 7.3 Hz).
3.6. General procedure for the synthesis of compounds 8a-e
3.6.2. 3-Chloro-4-((4-ethylpiperazin-1-yl)methyl)aniline (7b)
Prepared by method B, light yellow solid, yield 93%, 1H NMR
(CDCl3, 400 MHz), dH 7.17 (1H, d, J = 8.3 Hz), 6.68 (1H, d,
J = 2.3 Hz), 6.54 (1H, dd, J = 8.3, 2.3 Hz), 3.66 (2H, s), 3.53 (2H, s),
2.44–2.52 (8H, m), 2.40 (2H, q, J = 7.3 Hz), 1.07 (3H, t, J = 7.3 Hz).
Method A: To a solution of 7c or 7e (19 mmol) in 95% ethanol
(30 ml) respectively, iron powder (76 mmol), ammonium chloride
(NH4Cl, 66 mmol) and catalytic amount of glacial acetic acid were
added under strenuously stirring, and then the mixture was
allowed to warmed up to reflux for 4 h. The additional iron powder
was removed by filtration, and the filtrate was concentrated to dry-
ness to give crude product.
Method B: To a solution of 7a, 7b or 7d (2.5 mmol) in
methanol (30 ml) respectively was added pd-c (10%, 0.1 eq)
and concentrated hydrochloric acid (1 ml) under N2. The suspen-
sion was degassed under vacuum and purged with H2 three
times. The mixture was stirred under H2 balloon at room tem-
perature for about 1 h. The suspension was filtered through a
pad of Celite and the cake was washed with methanol. The
combined filtrates were concentrated to give the crude product.
The crude was purified by re-crystallization from methanol and
aether.
3.6.3. 3-Chloro-4-(morpholinomethyl)aniline (7c)
Prepared by method A, light yellow solid, yield 93%, 1H NMR
(CDCl3, 400 MHz), dH 7.17 (1H, d, J = 8.3 Hz), 6.68 (1H, d,
J = 2.3 Hz), 6.54 (1H, dd, J = 8.3, 2.3 Hz), 3.69 (2H, s), 3.66 (4H, t,
J = 4.5 Hz), 3.53 (2H, s), 2.48 (4H, t, J = 4.5 Hz).
3.6.4. 4-((4-Methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline
(7d)
Prepared by method B, light yellow solid, yield 89%, 1H NMR
(CDCl3, 400 MHz), dH 7.28 (1H, d, J = 8.3 Hz), 6.85 (1H, d,
J = 2.3 Hz), 6.75 (1H, dd, J = 8.3, 2.3 Hz), 5.43 (2H, s), 3.38 (2H, s),
2.31 (8H, m), 2.14 (3H, s).

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
119
Fig. 5. 9x induced obvious autophagy to protect SH-SY5Y cells from H2O2 induced-apoptosis. (A) The fluorescence microscopic appearance of MDC staining of H2O2, 9x
together with H2O2 treated SH-SY5Y cells. (B) Cell viability of control, H2O2, 9x + H2O2 and 9x + H2O2 + 3-MA groups. **P < 0.01 compared with H2O2-treated SH-SY5Y cells. (C)
Autophagy was determined by the analyses of MDC staining using flow cytometry. (D) Quantitative analysis of MDC positive ratio. Cells were collected after vehicle, H2O2 or
9x together with H2O2 treatment for 24 h, respectively. (D) SH-SY5Y cells were treated with vehicle, H2O2, or 9x together with H2O2 for 24 h, respectively, then the
expressions of p62 and LC3 were detected by western blot analysis. (E) At the same time, beside these groups, the level of cleaved-caspase3 were also detected in SH-SY5Y
cells which were co-incubated with 9x, H2O2 and 3-MA.
3.6.5. 4-((Dibutylamino)methyl)aniline (7e)
J = 2.1, 8.4 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.33 (1H, dd, J = 7.8,
7.7 Hz), 7.30 (1H, dd, J = 1.6, 7.8 Hz), 7.23 (1H, dd, J = 1.6, 7.7 Hz),
4.01 (3H, s, OCH3), 3.62 (2H, s), 2.59 (8H, br s), 2.45 (2H, q,
J = 7.2 Hz), 1.11 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz) dC,
161.3, 159.3, 149.3, 147.0, 144.1, 137.2, 134.6, 132.1, 131.0,
125.4, 121.2, 120.9, 119.2, 118.7, 118.4, 115.9, 58.7, 56.3,
52.8,52.8, 52.7, 52.7, 52.2, 11.8.
Prepared by method A, light yellow solid, yield 93%, 1H NMR
(CDCl3, 400 MHz), dH 7.09 (2H, d, J = 8.3 Hz), 6.64 (2H, d,
J = 8.3 Hz), 3.59 (2H, s), 3.45 (2H, s), 2.34 (4H, t, J = 7.4 Hz), 2.48
(4H, t, J = 4.5 Hz), 1.44 (4H, m), 1.29 (4H, m), 0.87 (6H, t, J = 7.3 Hz).
3.7. General procedure for the synthesis of compounds 9a-9aa
N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-8-met
hoxy-2-oxo-2H-chromene-3-carboxamide (9b) light yellow solid,
yield 88%, m.p. 205–207 °C; HR-ESI-MS m/z 442.1517 M+H+ (calcd.
for C23H25N3O4Cl 442.1534). 1H NMR (CDCl3, 400 MHz) dH, 10.86
(1H, s, NH), 8.98 (1H, s), 7.89 (1H, d, J = 2.1 Hz), 7.54 (1H, dd,
J = 2.1, 8.4 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.33 (1H, dd, J = 7.8,
7.7 Hz), 7.30 (1H, dd, J = 1.6, 7.8 Hz), 7.23 (1H, dd, J = 1.6, 7.7 Hz),
4.01 (3H, s, OCH3), 3.62 (2H, s), 2.56 (4H, br s), 2.47 (4H, br s),
2.30 (3H, s); 13C NMR (CDCl3, 100 MHz) dC, 161.3, 159.4, 149.3,
147.1, 144.1, 137.2, 134.6, 132.2, 130.9, 125.4, 121.2, 121.0,
119.2, 118.7, 118.4, 115.9, 58.7, 56.3, 55.1, 55.1, 52.9, 52.9, 45.9.
N-(3-chloro-4-(morpholinomethyl)phenyl)-8-methoxy-2-oxo-
2H-chromene-3-carboxamide (9c) light yellow solid, yield 81%,
m.p. 226–228 °C; HR-ESI-MS m/z 429.1232 M+H+ (calcd. for
To a solution of 8a-e (20 mmol) and triethylamine (40 mmol) in
dichloromethane (20 ml), a solution of 4a-f (1.5 eq) in dichloro-
methane (10 ml) was added dropwise at room temperature over
20 min and stirred overnight. The mixture was washed with satu-
rated aqueous sodium bicarbonate and brine. After removing the
solvent under reduced pressure, the crude product was purified
by flash chromatography on silica gel, eluting with dichloro-
methane and methanol (10–30%), yielding the title compounds.
N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-8-meth
oxy-2-oxo-2H-chromene-3-carboxamide (9a) light yellow solid,
yield 82%, m.p. 183–186 °C; HR-ESI-MS m/z 429.1223 M+H+ (calcd.
for C22H22N2O5Cl 429.1217). 1H NMR (CDCl3, 400 MHz) dH, 10.86
(1H, s, NH), 8.98 (1H, s), 7.89 (1H, d, J = 2.1 Hz), 7.54 (1H, dd,

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D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
C22H22N2O5Cl 429.1217). 1H NMR (CDCl3, 400 MHz) dH, 10.86 (1H,
s, NH), 8.98 (1H, s), 7.89 (1H, d, J = 2.0 Hz), 7.54 (1H, dd, J = 2.0,
8.4 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.33 (1H, dd, J = 7.8, 7.7 Hz), 7.30
(1H, dd, J = 1.6, 7.8 Hz), 7.23 (1H, dd, J = 1.6, 7.7 Hz), 4.01 (3H, s,
OCH3), 3.75 (4H, br t, J = 4.3 Hz), 3.62 (2H, s), 2.54 (4H, br s); 13C
NMR (CDCl3, 100 MHz) dC, 161.3, 159.4, 149.4, 147.1, 144.1,
137.4, 134.7, 131.7, 131.1, 125.4, 121.3, 121.0, 119.3, 118.8,
118.4, 115.9, 67.0, 67.0, 59.2, 56.4, 53.5, 53.5.
8-Methoxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoro
methyl)phenyl)-2-oxo-2H-chromene-3-carboxamide (9d) light
yellow solid, yield 82%, m.p. 176–178 °C; HR-ESI-MS m/z
476.1796 M+H+ (calcd. for C24H25N3O4F3 476.1797). 1H NMR
(CDCl3, 400 MHz) dH, 10.95 (1H, s, NH), 9.00 (1H, s), 8.03 (1H, d,
J = 2.0 Hz), 7.88 (1H, dd, J = 2.0, 8.4 Hz), 7.77 (1H, d, J = 8.4 Hz),
7.33 (1H, dd, J = 7.8, 7.7 Hz), 7.30 (1H, dd, J = 1.6, 7.8 Hz), 7.24
(1H, dd, J = 1.6, 7.7 Hz), 4.01 (3H, s, OCH3), 3.65 (2H, s), 2.52 (8H,
br s), 2.31 (3H, s); 13C NMR (CDCl3, 100 MHz) dC, 161.3, 159.6,
149.4, 147.1, 144.1, 136.3, 133.9, 131.2, 125.4, 123.5, 121.0,
119.2, 118.3, 118.0, 117.9, 115.9, 57.8, 56.3, 55.1, 55.1, 52.9, 52.9,
45.9.
N-(4-((dibutylamino)methyl)phenyl)-8-methoxy-2-oxo-2H-chr
omene-3-carboxamide (9e) light yellow solid, yield 85%, m.p. 118–
119 °C, HR-ESI-MS m/z 437.2423 M+H+ (calcd. for C26H33N2O4
437.2420). 1H NMR (CDCl3, 400 MHz) dH, 10.82 (1H, s, NH), 8.99
(1H, s), 7.68 (2H, d, J = 8.2 Hz), 7.35–7.28 (4H, m), 7.77 (1H, d,
J = 8.4 Hz), 7.22 (1H, dd, J = 1.6, 7.5 Hz), 4.01 (3H, s, OCH3), 3.55
(2H, s), 2.42 (4H, br t, J = 6.6 Hz), 1.46 (4H, m), 1.30 (4H, m), 0.88
(6H, t, J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) dC, 161.3, 159.2,
148.9, 147.1, 144.1, 136.3, 129.5, 129.5, 125.3, 125.3, 120.9,
120.3, 120.3, 119.3, 118.8, 115.7, 58.0, 56.3, 53.4, 53.4, 29.0, 29.0,
20.6, 20.6, 14.0, 14.0.
N-(4-((dibutylamino)methyl)phenyl)-7-methoxy-2-oxo-2H-chr
omene-3-carboxamide (9i) light yellow solid, yield 82%, m.p. 161–
162 °C; HR-ESI-MS m/z 437.2423 M+H+ (calcd. for C26H33N2O4
437.2420). dH, 10.81 (1H, s, NH), 8.93 (1H, s), 7.88 (1H, d,
J = 2.0 Hz), 7.63 (1H, d, J = 8.6 Hz), 7.52 (1H, dd, J = 2.0, 8.4 Hz),
7.44 (1H, d, J = 8.4 Hz), 6.97 (1H, dd, J = 2.3, 8.6 Hz), 6.90 (1H, d,
J = 2.3 Hz), 3.94 (3H, s, OCH3), 3.53 (2H, s), 2.40 (4H, br t,
J = 7.2 Hz), 1.46 (4H, m), 1.30 (4H, m), 0.88 (6H, t, J = 7.3 Hz); 13C
NMR (CDCl3, 100 MHz) dC, 165.1, 162.2, 159.7, 156.7, 148.7,
141.9, 136.4, 131.1, 129.4, 129.3, 120.2, 114.9, 114.2, 113.2,
112.5, 100.4, 58.1, 56.1, 53.5, 53.5, 29.2, 29.2, 20.6, 20.6, 14.1, 14.1.
7-Methoxy-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoro
methyl)phenyl)-2-oxo-2H-chromene-3-carboxamide (9j) light yel-
low solid, yield 85%, m.p. 194–196 °C; HR-ESI-MS m/z 476.1790 M
+H+ (calcd. for C24H25N3O4F3 476.1797). dH, 10.81 (1H, s, NH), 8.93
(1H, s), 8.01 (1H, d, J = 2.0 Hz), 7.87 (1H, dd, J = 2.0, 8.4 Hz), 7.77
(1H, d, J = 8.4 Hz), 7.63 (1H, d, J = 8.7 Hz), 6.98 (1H, dd, J = 2.4,
8.7 Hz), 6.91 (1H, d, J = 2.4 Hz), 3.94 (3H, s, OCH3), 3.64 (2H, s),
2.52 (8H, br s), 2.31 (3H, s); 13C NMR (CDCl3, 100 MHz) dC, 165.3,
162.2, 160.2, 156.8, 149.1, 136.6, 133.6, 131.3, 131.2, 123.4,
117.9, 117.8, 114.4, 114.4, 112.5, 100.4, 57.8, 56.1, 55.2, 55.2,
53.0, 53.0, 45.9.
N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-2-oxo-
2H-chromene-3-carboxamide (9k) light yellow solid, yield 84%,
m.p. 190–192 °C; HR-ESI-MS m/z 412.1411 M+H+ (calcd. for
C22H23N3O3Cl 412.1428). dH, 10.85 (1H, s, NH), 9.01 (1H, s), 7.89
(1H, d, J = 2.1 Hz), 7.73 (2H, m), 7.54 (1H, dd, J = 2.1, 8.4 Hz), 7.45
(2H, m), 7.44 (1H, d, J = 8.4 Hz), 3.61 (2H, s), 2.59 (4H, br s), 2.53
(4H, br s), 2.33 (3H, s); 13C NMR (CDCl3, 100 MHz) dC, 161.8,
159.4, 154.5, 149.2, 137.3, 134.6, 134.5, 132.2, 131.0, 131.0,
125.0, 121.3, 118.7, 118.6, 118.3, 116.7, 58.7, 55.1, 55.1, 52.9,
52.9, 45.9.
N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-2-oxo-
2H-chromene-3-carboxamide (9l) light yellow solid, yield 82%,
m.p. 176–178 °C; HR-ESI-MS m/z 426.1583 M+H+ (calcd. for
N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-7-meth
oxy-2-oxo-2H-chromene-3-carboxamide (9f) yellow solid, yield
83%, m.p. 177–179 °C, HR-ESI-MS m/z 442.1517 M+H+ (calcd. for
C
23H25N3O4Cl 442.1534). 1H NMR (CDCl3, 400 MHz) dH, 10.81
(1H, s, NH), 8.93 (1H, s), 7.88 (1H, d, J = 2.0 Hz), 7.63 (1H, d,
J = 8.6 Hz), 7.52 (1H, dd, J = 2.0, 8.4 Hz), 7.44 (1H, d, J = 8.4 Hz),
6.97 (1H, dd, J = 2.3, 8.6 Hz), 6.90 (1H, d, J = 2.3 Hz), 3.94 (3H, s,
OCH3), 3.62 (2H, s), 2.59 (8H, br s), 2.45 (2H, q, J = 7.2 Hz), 1.11
(3H, t, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz) dC, 165.3, 162.2,
160.0, 156.8, 149.0, 137.5, 134.6, 131.9, 131.2, 131.0, 121.2,
118.6, 114.6, 114.4, 112.5, 100.4, 58.8, 56.1, 53.0, 53.0, 52.8, 52.8,
52.3, 11.9.
N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-7-met
hoxy-2-oxo-2H-chromene-3-carboxamide (9g) light yellow solid,
yield 92%, m.p. 198–200 °C; HR-ESI-MS m/z 442.1537 M+H+ (calcd.
for C23H25N3O4Cl 442.1534). 1H NMR (CDCl3, 400 MHz) dH, 10.81
(1H, s, NH), 8.93 (1H, s), 7.88 (1H, d, J = 2.0 Hz), 7.63 (1H, d,
J = 8.6 Hz), 7.52 (1H, dd, J = 2.0, 8.4 Hz), 7.44 (1H, d, J = 8.4 Hz),
6.97 (1H, dd, J = 2.3, 8.6 Hz), 6.90 (1H, d, J = 2.3 Hz), 3.94 (3H, s,
OCH3), 3.62 (2H, s), 2.59 (8H, br s), 2.56 (4H, br s), 2.47 (4H, br
s), 2.30 (3H, s); 13C NMR (CDCl3, 100 MHz) dC, 165.3, 162.2,
160.0, 156.8, 149.0, 137.4, 134.6, 131.9, 131.2, 131.0, 121.2,
118.6, 114.5, 114.4, 112.5, 100.4, 58.8, 56.1, 55.1, 55.1, 52.9, 52.9,
45.9.
C23H25N3O3Cl 426.1584). dH, 10.85 (1H, s, NH), 9.01 (1H, s), 7.89
(1H, d, J = 2.1 Hz), 7.73 (2H, m), 7.54 (1H, dd, J = 2.1, 8.4 Hz), 7.45
(2H, m), 7.44 (1H, d, J = 8.4 Hz), 3.61 (2H, s), 2.80 (8H, br s), 2.76
(2H, q, J = 7.2 Hz), 1.27 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3,
100 MHz) dC, 161.8, 159.4, 154.5, 149.2, 137.3, 134.6, 134.5,
132.2, 131.0, 131.0, 125.0, 121.3, 118.7, 118.6, 118.3, 116.7, 58.7,
56.3, 52.8, 52.8, 52.7, 52.7, 52.2, 11.8.
N-(3-chloro-4-(morpholinomethyl)phenyl)-2-oxo-2H-chromene-
3-carboxamide (9m) light yellow solid, yield 82%, m.p. 197–198 °C;
HR-ESI-MS m/z 399.1120 M+H+ (calcd. for C21H20N2O4Cl 399.1112).
dH, 10.85 (1H, s, NH), 9.01 (1H, s), 7.89 (1H, d, J = 2.1 Hz), 7.73 (2H,
m), 7.54 (1H, dd, J = 2.1, 8.4 Hz), 7.45 (2H, m), 7.44 (1H, d,
J = 8.4 Hz), 3.72 (4H, br t, J = 4.6 Hz), 3.61 (2H, s), 2.52 (4H, br t,
J = 4.6 Hz); 13C NMR (CDCl3, 100 MHz) dC, 161.8, 159.4, 154.5,
149.2, 137.3, 134.6, 134.5, 132.2, 131.0, 131.0, 125.0, 121.3,
118.7, 118.6, 118.3, 116.7, 67.0, 67.0, 59.2, 56.4, 53.5, 53.5.
N-(4-((dibutylamino)methyl)phenyl)-2-oxo-2H-chromene-3-ca
rboxamide (9n) light yellow solid, yield 82%, m.p. 145–146 °C, HR-
ESI-MS m/z 407.2320 M+H+ (calcd. for C25H30N2O3 407.2318). dH,
10.85 (1H, s, NH), 9.01 (1H, s), 7.73 (2H, m), 7.66 (2H, d,
J = 8.4 Hz), 7.45 (2H, m), 7.34 (2H, d, J = 8.4 Hz), 3.61 (2H, s), 2.40
(4H, br t, J = 7.2 Hz), 1.46 (4H, m), 1.30 (4H, m), 0.88 (6H, t,
J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) dC, 161.8, 159.4, 154.5,
149.2, 137.5, 134.8, 134.6, 134.6, 131.3, 131.0, 125.6, 121.3,
118.8, 118.6, 118.3, 116.8, 58.1, 53.5, 53.5, 29.2, 29.2, 20.6, 20.6,
14.1, 14.1.
N-(3-chloro-4-(morpholinomethyl)phenyl)-7-methoxy-2-oxo-
2H-chromene-3-carboxamide (9h) light yellow solid, yield 82%,
m.p. 224–226 °C; HR-ESI-MS m/z 429.1223 M+H+ (calcd. for
C22H22N2O5Cl 429.1217). dH, 10.81 (1H, s, NH), 8.93 (1H, s), 7.88
(1H, d, J = 2.0 Hz), 7.63 (1H, d, J = 8.6 Hz), 7.52 (1H, dd, J = 2.0,
8.4 Hz), 7.44 (1H, d, J = 8.4 Hz), 6.97 (1H, dd, J = 2.3, 8.6 Hz), 6.90
(1H, d, J = 2.3 Hz), 3.94 (3H, s, OCH3), 3.72 (4H, br t, J = 4.6 Hz),
3.60 (2H, s), 2.52 (4H, br t, J = 4.6 Hz); 13C NMR (CDCl3, 100 MHz)
dC, 165.3, 162.2, 160.0, 156.8, 149.0, 137.7, 134.7, 131.2, 131.2,
131.2, 121.2, 118.6, 114.5, 114.4, 112.5, 100.4, 67.0, 67.0, 59.2,
56.1, 53.5, 53.5.
N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phe-
nyl)-2-oxo-2H-chromene-3-carboxamide (9o) light yellow solid,
yield 80%, m.p. 160–162 °C; HR-ESI-MS m/z 446.1695 M+H+ (calcd.
For C23H23N3O3F3 446.1692). dH, 10.94 (1H, s, NH), 9.02 (1H, s), 8.02
(1H, br s), 7.88 (1H, d, J = 8.4 Hz), 7.78 (2H, d, J = 8.4 Hz), 7.73 (2H,

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
121
m), 7.45 (2H, m), 3.64 (2H, s), 2.52 (4H, br s), 2.47 (4H, br s), 2.30
(3H, s); 13C NMR (CDCl3, 100 MHz) dC, 161.8, 159.6, 154.5, 149.3,
136.3, 134.6, 134.0, 131.3, 131.0, 125.6, 123.4, 118.6, 118.2,
118.0, 117.9, 116.8, 57.8, 55.2, 55.2, 53.1, 53.1, 46.0.
s), 2.58 (4H, br s), 2.51 (4H, br s), 2.32 (3H, s); 13C NMR (CDCl3,
100 MHz) dC, 161.2, 158.8, 153.3, 147.7, 137.2, 137.1, 134.7,
132.4, 132.0, 131.1, 121.2, 120.0, 119.4, 118.7, 118.5, 118.2, 58.8,
55.1, 55.1, 52.9, 52.9, 45.9.
6-Chloro-N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-
2-oxo-2H-chromene-3-carboxamide (9p) light yellow solid, yield
82%, m.p. 193–195 °C, HR-ESI-MS m/z 460.1123 M+H+ (calcd. For
6-Bromo-N-(3-chloro-4-(morpholinomethyl)phenyl)-2-oxo-2H-
chromene-3-carboxamide (9v) light yellow solid, yield 81%, m.p.
219–220 °C; HR-ESI-MS m/z 477.0215 M+H+ (calcd. For C21H19N2
O4ClBr 477.0217). 1H NMR (CDCl3, 400 MHz) dH, 10.75 (1H, s,
NH), 8.92 (1H, s), 7.88 (1H, d, J = 2.1 Hz), 7.87 (1H, d, J = 2.3 Hz),
7.78 (1H, dd, J = 2.3, 8.8 Hz), 7.52 (1H, dd, J = 2.1, 8.4 Hz), 7.46
(1H, d, J = 8.4 Hz), 7.35 (1H, d, J = 8.8 Hz), 3.72 (4H, br t,
J = 4.6 Hz), 3.62 (2H, s), 2.52 (4H, br t, J = 4.6 Hz); 13C NMR (CDCl3,
100 MHz) dC, 161.2, 158.8, 153.3, 147.8, 137.3, 137.2, 134.8, 132.0,
132.0, 131.1, 121.3, 120.1, 119.4, 118.7, 118.4, 118.3, 67.0, 67.0,
59.2, 56.4, 53.4, 53.4.
C
23H23Cl2N3O3 460.1119). 1H NMR (CDCl3, 400 MHz) dH, 10.76
(1H, s, NH), 8.93 (1H, s), 7.88 (1H, d, J = 2.0 Hz), 7.72 (1H, d,
J = 2.4 Hz), 7.65 (1H, dd, J = 2.4, 8.8 Hz), 7.52 (1H, dd, J = 2.0,
8.4 Hz), 7.44 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 8.8 Hz), 3.62 (2H,
s), 2.59 (8H, br s), 2.45 (2H, q, J = 7.2 Hz), 1.11 (3H, t, J = 7.2 Hz);
13C NMR (CDCl3, 100 MHz) dC, 161.2, 158.8, 152.8, 147.8, 137.1,
134.7, 134.4, 132.4, 131.1, 131.0, 128.9, 121.2, 119.6, 119.4,
118.6, 118.2, 58.8, 52.9, 52.9, 52.7, 52.7, 52.3, 11.9.
6-Chloro-N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-
2-oxo-2H-chromene-3-carboxamide (9q) light yellow solid, yield
88%, m.p. 213–216 °C, HR-ESI-MS m/z 446.1015 M+H+ (calcd. For
C22H21Cl2N3O3 446.1012). 1H NMR (CDCl3, 400 MHz) dH, 10.76
(1H, s, NH), 8.93 (1H, s), 7.88 (1H, d, J = 2.0 Hz), 7.72 (1H, d,
J = 2.4 Hz), 7.65 (1H, dd, J = 2.4, 8.8 Hz), 7.52 (1H, dd, J = 2.0,
8.4 Hz), 7.44 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 8.8 Hz), 3.62 (2H,
s), 2.58 (4H, br s), 2.51 (4H, br s), 2.32 (3H, s); 13C NMR (CDCl3,
100 MHz) dC, 161.2, 158.8, 152.8, 147.9, 137.1, 134.7, 134.4,
132.5, 131.1, 130.9, 128.9, 121.2, 119.6, 119.4, 118.6, 118.2, 58.8,
55.1, 55.1, 52.9, 52.9, 45.9.
6-Bromo-N-(4-((dibutylamino)methyl)phenyl)-2-oxo-2H-chro-
mene-3-carboxamide (9w) light yellow solid, yield 87%, m.p. 156–
157 °C; HR-ESI-MS m/z 485.1443 M+H+ (calcd. For C25H30N2O3Br
485.1440). dH, 10.72 (1H, s, NH), 8.93 (1H, s), 7.86 (1H, d,
J = 2.3 Hz), 7.77 (1H, dd, J = 2.3, 8.8 Hz), 7.66 (2H, d, J = 8.4 Hz),
7.34 (1H, d, J = 8.8 Hz), 7.33 (2H, d, J = 8.4 Hz), 3.53 (2H, s), 2.40
(4H, br t, J = 7.2 Hz), 1.45 (4H, m), 1.29 (4H, m), 0.88 (6H, t,
J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) dC, 161.2, 158.6, 153.2,
147.4, 136.9, 136.1, 131.9, 129.5, 129.5, 120.3, 120.3, 120.2,
119.8, 118.4, 118.1, 58.1, 56.1, 53.5, 53.5, 29.2, 29.2, 20.6, 20.6,
14.1, 14.1.
6-Chloro-N-(3-chloro-4-(morpholinomethyl)phenyl)-2-oxo-2H
-chromene-3-carboxamide (9r) light yellow solid, yield 81%, m.p.
211–212 °C; HR-ESI-MS m/z 433.0729 M+H+ (calcd. For C21H19N2
O4Cl2 433.0722). 1H NMR (CDCl3, 400 MHz) dH, 10.76 (1H, s, NH),
8.93 (1H, s), 7.88 (1H, d, J = 2.0 Hz), 7.72 (1H, d, J = 2.4 Hz), 7.65
(1H, dd, J = 2.4, 8.8 Hz), 7.52 (1H, dd, J = 2.0, 8.4 Hz), 7.44 (1H, d,
J = 8.4 Hz), 7.41 (1H, d, J = 8.8 Hz), 3.72 (4H, br t, J = 4.6 Hz), 3.60
(2H, s), 2.52 (4H, br t, J = 4.6 Hz); 13C NMR (CDCl3, 100 MHz) dC,
161.2, 158.8, 152.8, 147.8, 137.1, 134.7, 134.4, 132.4, 131.1,
131.0, 128.9, 121.2, 119.6, 119.4, 118.6, 118.2, 67.0, 67.0, 59.2,
56.4, 53.5, 53.5.
N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-6-nitro-
2-oxo-2H-chromene-3-carboxamide (9x) yellow solid, yield 83%,
m.p. 220–222 °C; HR-ESI-MS m/z 471.1438 M+H+ (calcd. For C23
-
H
24N4O5Cl 471.1435). 1H NMR (CDCl3, 400 MHz) dH, 10.59 (1H, s,
NH), 9.08 (1H, s), 8.67 (1H, d, J = 2.5 Hz), 8.55 (1H, dd, J = 2.5,
9.1 Hz), 7.86 (1H, d, J = 2.0 Hz), 7.61 (1H, d, J = 9.1 Hz), 7.52 (1H,
dd, J = 2.0, 8.4 Hz), 7.48 (1H, d, J = 8.4 Hz), 3.62 (2H, s), 2.59 (8H,
br s), 2.45 (2H, q, J = 7.2 Hz), 1.11 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3, 100 MHz) dC, 160.4, 158.1, 157.4, 147.8, 144.8, 136.9,
134.8, 132.4, 131.2, 128.7, 125.6, 121.3, 120.5, 118.7, 118.6,
118.1, 57.8, 51.9, 51.9, 51.7, 51.7, 51.3, 10.8.
6-Chloro-N-(4-((dibutylamino)methyl)phenyl)-2-oxo-2H-chro-
mene-3-carboxamide (9s) yellow solid, yield 82%, m.p. 147–
149 °C; HR-ESI-MS m/z 441.1949 M+H+ (calcd. For C25H30N2O3Cl
441.1945). 1H NMR (CDCl3, 400 MHz) dH, 10.76 (1H, s, NH), 8.93
(1H, s), 7.72 (1H, d, J = 2.4 Hz), 7.66 (2H, d, J = 8.3 Hz), 7.65 (1H,
dd, J = 2.4, 8.8 Hz), 7.41 (1H, d, J = 8.8 Hz), 7.34 (2H, d, J = 8.8 Hz),
3.53 (2H, s), 2.40 (4H, br t, J = 7.2 Hz), 1.45 (4H, m), 1.29 (4H, m),
0.88 (6H, t, J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) dC, 161.2,
158.6, 152.8, 147.5, 136.0, 134.1, 132.4, 130.9, 129.5, 129.5,
128.9, 120.3, 120.3, 119.8, 119.7, 118.2, 58.1, 56.1, 53.5, 53.5,
29.2, 29.2, 20.6, 20.6, 14.1, 14.1.
N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-6-nitro-
2-oxo-2H-chromene-3-carboxamide (9y) yellow solid, yield 88%,
m.p. 210–212 °C; HR-ESI-MS m/z 457.1297 M+H+ (calcd. For
C22H22N4O5Cl 457.1279). 1H NMR (CDCl3, 400 MHz) dH, 10.59
(1H, s, NH), 9.08 (1H, s), 8.67 (1H, d, J = 2.5 Hz), 8.55 (1H, dd,
J = 2.5, 9.1 Hz), 7.86 (1H, d, J = 2.0 Hz), 7.61 (1H, d, J = 9.1 Hz),
7.52 (1H, dd, J = 2.0, 8.4 Hz), 7.48 (1H, d, J = 8.4 Hz), 3.62 (2H, s),
2.58 (4H, br s), 2.51 (4H, br s), 2.32 (3H, s); 13C NMR (CDCl3,
100 MHz) dC, 160.4, 158.1, 157.4, 147.8, 144.8, 136.9, 134.8,
132.4, 131.2, 128.7, 125.6, 121.3, 120.5, 118.7, 118.6, 118.1, 58.8,
55.1, 55.1, 53.0, 53.0, 46.0.
6-Bromo-N-(3-chloro-4-((4-ethylpiperazin-1-yl)methyl)phenyl)-
2-oxo-2H-chromene-3-carboxamide (9t) light yellow solid, yield
86%, m.p. 182–184 °C; HR-ESI-MS m/z 504.0625 M+H+ (calcd. For
N-(3-chloro-4-(morpholinomethyl)phenyl)-6-nitro-2-oxo-2H-
chromene-3-carboxamide (9z) yellow solid, yield 84%, m.p. 266–
268 °C; HR-ESI-MS m/z 444.0945 M+H+ (calcd. For C21H19N3O6Cl
444.0962). 1H NMR (CDCl3, 400 MHz) dH, 10.59 (1H, s, NH), 9.08
(1H, s), 8.67 (1H, d, J = 2.5 Hz), 8.55 (1H, dd, J = 2.5, 9.1 Hz), 7.86
(1H, d, J = 2.0 Hz), 7.61 (1H, d, J = 9.1 Hz), 7.52 (1H, dd, J = 2.0,
8.4 Hz), 7.48 (1H, d, J = 8.4 Hz), 3.72 (4H, br t, J = 4.6 Hz), 3.62
(2H, s), 2.52 (4H, br t, J = 4.6 Hz); 13C NMR (CDCl3, 100 MHz) dC,
160.4, 158.1, 157.4, 147.8, 144.8, 136.9, 134.8, 132.4, 131.2,
128.7, 125.6, 121.3, 120.5, 118.7, 118.6, 118.1, 67.0, 67.0, 59.2,
56.4, 53.6, 53.6.
C
23H23BrClN3O3 504.0622). 1H NMR (CDCl3, 400 MHz) dH, 10.75
(1H, s, NH), 8.92 (1H, s), 7.88 (1H, d, J = 2.1 Hz), 7.87 (1H, d,
J = 2.3 Hz), 7.78 (1H, dd, J = 2.3, 8.8 Hz), 7.52 (1H, dd, J = 2.1,
8.4 Hz), 7.46 (1H, d, J = 8.4 Hz), 7.35 (1H, d, J = 8.8 Hz), 3.62 (2H,
s), 2.59 (8H, br s), 2.45 (2H, q, J = 7.2 Hz), 1.11 (3H, t, J = 7.2 Hz);
13C NMR (CDCl3, 100 MHz) dC, 161.2, 158.8, 153.3, 147.7, 137.2,
137.1, 134.7, 132.4, 132.0, 131.1, 121.2, 120.0, 119.4, 118.7,
118.5, 118.2, 58.8, 52.9, 52.9, 52.7, 52.7, 52.3, 11.9.
6-Bromo-N-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-
2-oxo-2H-chromene-3-carboxamide (9u) light yellow solid, yield
80%, m.p. 203–205 °C; HR-ESI-MS m/z 490.0526 M+H+ (calcd. For
N-(4-((dibutylamino)methyl)phenyl)-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide (9aa) yellow solid, yield 82%, m.p. 212–
213 °C; HR-ESI-MS m/z 452.2135 M+H+ (calcd. For C25H29N3O5
452.2131). 1H NMR (CDCl3, 400 MHz) dH, 10.59 (1H, s, NH), 9.08
(1H, s), 8.67 (1H, d, J = 2.5 Hz), 8.55 (1H, dd, J = 2.5, 9.1 Hz), 7.66
(2H, d, J = 8.4 Hz), 7.60 (1H, d, J = 9.1 Hz), 7.35 (2H, d, J = 8.4 Hz),
3.53 (2H, s), 2.40 (4H, br t, J = 7.2 Hz), 1.45 (4H, m), 1.29 (4H, m),
C
22H22N3O3ClBr 490.0510). 1H NMR (CDCl3, 400 MHz) dH, 10.75
(1H, s, NH), 8.92 (1H, s), 7.88 (1H, d, J = 2.1 Hz), 7.87 (1H, d,
J = 2.3 Hz), 7.78 (1H, dd, J = 2.3, 8.8 Hz), 7.52 (1H, dd, J = 2.1,
8.4 Hz), 7.46 (1H, d, J = 8.4 Hz), 7.35 (1H, d, J = 8.8 Hz), 3.62 (2H,

122
D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
0.88 (6H, t, J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz) dC, 160.4, 157.9,
157.4, 147.4, 136.2, 135.8, 129.5, 129.5, 128.5, 125.5, 120.9, 120.3,
120.3, 118.7, 118.0, 58.1, 53.5, 53.5, 29.2, 29.2, 20.6, 20.6, 14.1,
14.1.
(MD) simulation. For this system, energy minimization and MD
simulation were performed by using the Sander module of the
AMBER 12.0. Prior to MD simulations, the entire system was sub-
jected to energy minimization in two stages to remove bad con-
tacts between the complex and the solvent molecules. Firstly, the
water molecules and counter ions were minimized by freezing
3.8. Determination of AChE and BuChE inhibitory activities and SAR
analysis
the solute using
a harmonic constraint of the strength of
100 kcal molÀ1 ÅÀ2. Secondly, the entire system was minimized
without restriction. Each stage consisted of a 5000-step steepest
descent and a 2500-step conjugate gradient minimization. Finally,
3 ns MD simulations were carried out for each system in an
isothermal isobaric ensemble (NPT) under periodic boundary
conditions.
The AChE and BuChE inhibition activity was accomplished by
the spectrophotometric Ellman’s method with own modifications.
The reaction mixture was consisted of 100
To the solution of PBS (pH 8.0, 30 L) consisted of KH2PO4 and
K2HPO4 in 96-well plate, the solution of enzyme AChE (15 L,
0.132 U/mL or BuChE (15 L, 0.04 U/mL) in the PBS was added
and followed by the addition of the synthesized compounds
(20 mol/L, 25 L DMSO). The resulting mixture were thoroughly
mixed, pre-incubated for 10 min at 37 °C and pre-read at
412 nm. The reaction started by the addition of 15 L of 4 mM
well-1substrate (acetylthiocholine iodide-ATCI or butyrylthio-
choline chloride-BuSCH), followed by the incorporation of 15
lL of the total volume.
l
l
l
3.11. MTT assay
l
l
Briefly, SH-SY5Y (5 Â 103 per well) were seeded in 96-well
plates and cultured for 24 h, followed by compounds treatment
for 24 h with different concentration. MTT was added per well
and incubated for another 4 h at 37 °C, then the supernatant fluid
was removed and DMSO was added 150 mL per well for 5 min.
The absorptions (OD) were measured at 570 nm with Spectra
MAX M5 microplate spectrophotometer (Molecular Devices).
l
lL
of 6 mM well-1 Ellman solution (DTNB). After 30 min of incubation
at 37 °C, absorbance was recorded at 412 nm by 96-well plate
reader Bio-Rad Laboratories Inc. All experiments were performed
with their respective standards (controls) in triplicate. As a positive
control Huperzine A (0.8 lM well-1) or Iso-OMPA (16.0 lM well-1)
was practiced. The percent inhibition was measured by following
3.12. Hoechst and MDC staining
Briefly, cells (1 Â 105 per well) were seeded in 6-well plates and
cultured for 24 h, followed by H2O2, 9x or H2O2 together with 9x
treatment for another 24 h. After rinsing with PBS 3 times, the cells
were treated with Hoechst 33258 or MDC with the appropriate
concentration for 20 min at 37 °C. Then the morphological change
of cell was examined by fluorescence microscope.
equation:
Inhibition
(control-blank) Â 100
Control: 55 L PBS, 15
Blank: 70 L PBS, 15
Test: 30 L PBS, 15
ATCI and 15 L DTNB
Sample blank: 75 L PBS, 25 lL tested compound
(%) = [(control-blank) À (test-sample blank)]/
l
l
L AChE, 15 L ATCI and 15 lL DTNB
l
l
l
l
L ATCI and 15 L DTNB
l
l
L AChE, 25 L tested compound, 15 lL
l
l
3.13. Flow cytometric analysis
l
For Annexin V/PI or MDC assays, 1 Â 105 cells were seeded in 6-
well plates for 24 h and then were treated with H2O2, 9x or H2O2
together with 9x. Then cells were collected and washed twice with
PBS and stained with 5 mL of Annexin V-FITC and 2.5 mL of PI
(5 mg/mL) in 1X binding buffer (10 mM HEPES, pH 7.4, 140 Mm
NaOH, 2.5 mM CaCl2) or appropriate concentration of MDC in PBS
for 30 min at room temperature in the dark. Then the cells were
checked with Flow cytometry (BD).
3.9. Molecular docking
The molecular docking method was used for the analysis of 9x
binding with AChE, the Discovery Studio 3.5 docking program was
adopted here. In the current study, the CHARMm force field was
used for energy minimization of the ligands. The binding sphere
was primarily defined as all residues of the target within 10 Å from
the ligand binding site [31]. The X-ray crystal structure of AChE
(PDB ID: 1EVE) was taken from PDB database. The preparation of
protein structure, including adding hydrogen atoms, removing
water molecules, and assigning CHARMm force field. A sphere con-
taining the residues in CAS and PAS binding pocket of protein
structure were defined as the binding site. Gold score was selected
as the score function, and the other parameters were set as default.
For each docking study, a total of 20 docking poses were retained.
The root-mean square deviation (RMSD) between docking poses
were calculated.
3.14. Western blot analysis
The SH-SY5Y cells were harvested, washed twice with cold PBS
and then lysed in whole cell lysis buffer, supplemented with the
proteinase inhibitors 100 lg/mL at 4 °C for 1 h. After 12,000g cen-
trifugation at 4 °C for 10 min, the protein concentration was deter-
mined by a BCA Protein Assay Kit (CWBIO, Beijing, China). Equal
amounts of total proteins were separated by 12% SDS-PAGE, and
transferred onto Immobilon-P Transfer Membrane (Millipore Cor-
poration, Billerica, MA, USA). The membranes were blocked with
5% skimmed milk at room temperature for 1 h, incubated with
indicated primary antibodies at 4 °C overnight and horseradish
peroxidase (HRP)-conjugated secondary antibody at room temper-
ature for 2 h, then visualized by using ECL reagents [32].
3.10. Molecular dynamics (MD) simulations
In the following molecular mechanics (MM) minimizations and
MD simulations, the AMBER99 force field and the general AMBER
force field (gaff) were used to establish the potential of receptors
and ligands, respectively. An appropriate number of sodium coun-
ter ions were placed around AChE-ligand complex to neutralize the
charges of the systems. Finally, the whole system was solvated in a
cubic periodic box of TIP3P water molecules, and the distance
between the edges of the water box and the closest atom of the
solutes was at least 10 Å. To avoid edge effects, periodic boundary
conditions were applied during the whole molecular dynamics
4. Conclusions
Alzheimer’s disease is a progressive, multifaceted neurodegen-
erative disease, which is characterized by progressive loss of neu-
rons and synapses in the cerebral cortex, resulting in loss of
memory and disability of learning and thinking. It is regrettable

D. Yao et al. / Bioorganic Chemistry 68 (2016) 112–123
123
that there is no efficient means to prevent, cure or slow the pro-
Appendix A. Supplementary material
gression of AD. Although several research strategies have been
envisaged in recent decades, increasing cholinergic neurotransmis-
sion by inhibiting AChE still represents one of the mainstream
treatment options for AD. In this study, we have designed and syn-
thesized a new series of coumarin derivatives and evaluated their
cholinesterase inhibitory activities. The results suggested that
most of these compounds exhibited pronounced AChE inhibitory
activity with IC50 values in the nanomolar range and had no obvi-
ous inhibitory activity against BuChE. The most potent compound
9x displayed remarkable inhibitory activity against AChE with an
IC50 value of 34 nM, which is much stronger than Huperzine A.
Molecular docking and molecular dynamics simulation con-
tributed to speculating the interaction mode of compound 9x with
AChE and further supported 9x as a potent selective AChE inhibi-
tor. Furthermore, we found 9x could protect SH-SY5Y cells from
oxidative stress-induced cell death by decreasing mitochondrial
injury. 9x could induce protective autophagy in hydrogen peroxide
damaged SH-SY5Y cells, contributing to the resistance of hydrogen
peroxide induced apoptosis. These results suggested that 9x had
dual functions to increase acetylcholine level by inhibiting AChE
activity and to maintain cell vitality by decreasing oxidative dam-
age in SH-SY5Y cells. Notably, protective autophagy induced by 9x
has a potential to enhance neuron activity to resist other aging
related adverse factors, which would lead to a better prognosis
for AD patients. Collectively, 9x could serve as a promising precur-
sor agent for future AD therapy.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.07.
013.
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Conflicts of interest
The authors declare no conflict of interest.
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Acknowledgements
This work was supported by grants from National Natural
Science Fund (Nos. 81303270, U1303124, 81202403, 81573290),
Liaoning Science and Technology Project (No. 2013226027-4), Jilin
Science and Technology Progress Project (No. 20130204058YY),
Applied Basic Research Project of Sichuan Science and Technology
Department (No. 2013JY0154) and Shenyang Pharmaceutical
University Scientific Research Fund (No. ZCJJ2013407).
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