Synthesis and evaluation of coumarin/piperazine hybrids as acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-018-2185-x

A series of new coumarin/piperazine hybrids were synthesized and evaluated for anticholinesterase activity. Among them, compounds 4d and 4t exhibited potent human acetylcholinesterase (hAChE) inhibitory activity with IC₅₀ values of 2.42 and 9.89 μM, respectively, and 4t displayed highest selectivity toward hAChE over human butyrylcholinesterase (hBChE) by 9.8-fold. In addition, both compounds did not show observed cytotoxicity against SH-SY5Y neuroblastoma cell line at 100 μM. Kinetic analysis in tandem with molecular docking study revealed that these hybrids targeted both catalytic active site (CAS) and peripheral anionic site (PAS) of hAChE. The preliminary results highlighted 4t as an anti-Alzheimer’s disease lead compound worthy of further investigation.

Full text of DOI:10.1007/s00044-018-2185-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2185-x
ORIGINAL RESEARCH
Synthesis and evaluation of coumarin/piperazine hybrids as
acetylcholinesterase inhibitors
1
Juan Zhang1,2 Cheng-Shi Jiang
●
Received: 22 March 2018 / Accepted: 23 April 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of new coumarin/piperazine hybrids were synthesized and evaluated for anticholinesterase activity. Among them,
compounds 4d and 4t exhibited potent human acetylcholinesterase (hAChE) inhibitory activity with IC50 values of 2.42 and
9.89 μM, respectively, and 4t displayed highest selectivity toward hAChE over human butyrylcholinesterase (hBChE) by
9.8-fold. In addition, both compounds did not show observed cytotoxicity against SH-SY5Y neuroblastoma cell line at 100
μM. Kinetic analysis in tandem with molecular docking study revealed that these hybrids targeted both catalytic active site
(CAS) and peripheral anionic site (PAS) of hAChE. The preliminary results highlighted 4t as an anti-Alzheimer’s disease
lead compound worthy of further investigation.
●
●
●
●
Keywords Coumarin/piperazine hybrid Acetylcholinesterase inhibitor Molecular docking Cytotoxicity Alzheimer’s
disease
Introduction
be hydrolyzed by two types of cholinesterases, namely
acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE). AChE is 1013-fold more active than BChE, and
almost accounts for nearly 80% ACh hydrolysis in brain
(Feng et al. 2017). In this context, four out of five drugs
approved by the FDA for anti-AD therapy are AChE inhi-
bitors, i.e. donepezil, rivastigmine, galantamine, and tacrine
as shown in Fig. 1 (Herrmann et al. 2011). However, tacrine
was quickly withdrawn from the market due to its hepato-
toxicity (Barreiro et al. 2003). Although the others could
increase the concentration of ACh in brain to improve
cognitive impairment, none of them can cure or stop the
progress of AD (Weinstock 2012).
Compared with other AChE inhibitors on the market,
donepezil still gets a great of attention due to its noticeable
advantages, such as high bioavailability, few dose-limiting
hepatotoxicity, long half-life, and relative fewer side effects
(van Greunen et al. 2017). Therefore, lots of structural
optimizations and structure-activity relationship (SAR) stu-
dies have been conducted on donepezil in order to develop
more safe and effective AChE inhibitors. From the literature
most inhibitors maintained the N-benzyl-piperidine of
donepezil as this fragment could form important non-
covalent bonds with key amino acid residues in the active
site of AChE (Więckowska et al. 2015; Girisha et al. 2009).
To mimic the piperidine ring in donepezil, piperazine scaf-
fold as a bioisostere of piperidine have been extensively
Alzheimer’s disease (AD) is an age-related neurodegen-
erative disorder, which was characterized by progressive
memory loss, decline in language skills and cognitive
functions. Now, AD has already become the third leading
cause of death behind cancer and cardiovascular diseases.
Currently, about 46.8 million people worldwide are suf-
fering from AD, and this number will triple by 2050 with
the aging of the population (Yu et al. 2017).
Although AD pathogenesis is very complex and still not
fully understood, the low level of neurotransmitter acet-
ylcholine (ACh) in brain was mainly related to cognitive
and memory impairments of AD patients according to the
cholinergic hypothesis of AD (Bartus et al. 1982). ACh can
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2185-x) contains supplementary
material, which is available to authorized users.
* Cheng-Shi Jiang
jiangchengshi-20@163.com
1
School of Biological Science and Technology, University of Jinan,
Jinan 250022, China
2
Faculty of Ceilandia, University of Brasília, Brasília 72220275,
Brazil

Medicinal Chemistry Research
Fig. 1 Current and former AChE
inhibitors approved for AD
treatment
Fig. 2 Design of new coumarin/
piperazine hybrids
exploited and various piperazine-based AChE inhibitors
were developed (Demir Özkay et al. 2017). For example, a
piperazine derivative FK960 exerted beneficial effects on
memory deficits in AD rats and monkey (Matsuoka and
Aigner 1997; Inoue et al. 2001). In addition, ensaculin (Fig.
2), a piperazine/coumarin conjugate, had been used in clinic
as its HCl salt for treating AD for a long time (Hoerr and
Noeldner 2002). It was worth mentioning that coumarin
derivatives were usually found to have a broad-spectrum of
biological activities, such as anti-viral (Hassan et al. 2016),
anti-cancer (Dandriyal et al. 2016), anti-diabetic (Li et al.
2017), anti-inflammatory (Revankar et al. 2017), anti-
microbial (Bhat et al. 2013), and anti-neurodegenerative
activities (Jameel et al. 2016). In our project for seeking new
anti-AD drug candidates, hybridization of coumarin and
piperazine generated 21 derivatives as new candidates
against AD. Herein, in the present study we describe the
synthesis, biological evaluation and SAR study of this new
series of coumarin/piperazine hybrid AChE inhibitors. In
addition, the kinetic analysis and molecular docking were
performed, and the cytotoxicity against SH-SY5Y cell of
selected compounds were tested as well.
Commercially available reagents were used without further
purification. Organic solvents were evaporated with reduced
pressure using a Büchi R-100 evaporator. Reactions were
monitored by thin layer chromatography (TLC) using
GF254 silica gel plates (Yantai JingYou Silica Gel Inc.,
China). Silica gel column chromatography was performed
on Biotage Isolera One and silica gel (200–300 mesh) from
Qingdao Hailang Inc. (China). Purity data were recorded on
an Agilent 1260 HPLC using parameters as follows: H2O/
MeOH, 40/60 to 0/100 in 15 min, 0/100 in 5 min, +5 min
isocratic, flow rate of 1.0 mL/min, λ = 280 nm. NMR
spectra were measured on Bruker Avance III 600 MHz
spectrometers. Chemical shifts were expressed in δ (ppm)
and coupling constants (J) in Hz with solvent signals as
internal standards (CDCl3, δH 7.26 ppm and δC 77.2 ppm;
CD3OD, δH 3.31 ppm and δC 49.0 ppm). ESI-MS analyses
were performed on an Agilent 1260–6460 Triple Quard LC-
MS instrument, and the elemental analysis was recorded on
a Vario EL III (Elementar, Germany).
Synthesis of 2-oxo-2H-chromene-3-carboxylic acid (2)
In a round-bottomed flask 2-hydroxybenzaldehyde (2.44 g,
20 mmol) and Meldrum’s acid (3.46 g, 24 mmol) in water
(30 mL) were heated at reflux under stirring for 10 h. Then
the reaction mixture was cooled and filtered on Büchner
funnel. The products were purified by recrystallization from
methanol to yield compound 2 as a white solid (3.61 g, 95%
Materials and methods
Chemistry
1
Melting points were measured by a Melting Point YRY-3
apparatus (Tianjin Precision Apparatus Factory, China).
yield). m.p. 145–146 °C. H NMR (600 MHz, MeOD) δ
8.82 (s, 1H), 7.83 (dd, J = 8.1, 1.5 Hz, 1H), 7.76 (ddd, J =

Medicinal Chemistry Research
8.8, 7.5, 1.6 Hz, 1H), 7.45–7.41 (m, 2H). 13C NMR
(150 MHz, MeOD) δ 165.5, 160.8, 156.3, 151.1, 136.0,
131.4, 126.4, 119.7, 118.3, 117.6. ESI–MS m/z: 191.1
[M+H]+.
5.0 Hz, 2H), 2.55 (t, J = 5.0 Hz, 2H), 2.48 (brs, 2H). 13C
NMR (150 MHz, CDCl3) δ 163.5, 158.1, 154.2, 143.2,
137.7, 132.9, 129.3, 128.7, 128.5, 127.4, 125.4, 125.0,
118.4, 117.0, 62.9, 53.1, 52.6, 47.4, 42.3. ESI–MS m/z:
349.2 [M+H]+. Anal: Calcd. for C21H20N2O3: C 72.40, H
5.79, N 8.04; found: C 72.33, H 5.68, N 8.16.
4-(2-oxo-2H-chromene-3-carbonyl)piperazine-1-carboxylate
(3)
3-(4-(4-Methylbenzyl)piperazine-1-carbonyl)-2H-chromen-
To a solution of coumarin-3-carboxyl acid derivatives 2
(1.80 g, 9.5 mmol) in 30 mL dry dichloromethane (DCM)
was added EDCI (2.18 g, 11.4 mmol), HOBT (1.28 mg,
2-one (4b)
A light yellow solid, yield 77%, m.p. 125–126 °C, HPLC
purity: 98.54%, tR = 13.376 min. 1H NMR (600 MHz,
CDCl3) δ 7.90 (s, 1H), 7.58 (ddd, J = 8.3, 7.5, 1.4 Hz, 1H),
7.53 (dd, J = 7.7, 1.4 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H),
7.32 (dd, J = 7.7, 7.5 Hz, 1H), 7.20 (d, J = 7.9 Hz, 2H),
7.13 (d, J = 7.8 Hz, 2H), 3.78 (t, J = 5.0 Hz, 2H), 3.51 (s,
2H), 3.43–3.34 (t, J = 5.0 Hz, 2H), 2.53 (t, J = 5.0 Hz, 2H),
2.47 (brs, 2H), 2.33 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
163.5, 158.1, 154.2, 143.1, 137.1, 134.6, 132.9, 129.3,
129.2, 128.6, 125.4, 125.0, 118.4, 117.0, 62.6, 53.1, 52.5,
47.4, 42.3, 21.2. ESI–MS m/z: 363.2 [M+H]+. Anal: Calcd.
for C22H22N2O3: C 72.91, H 6.12, N 7.73; found: C 72.93,
H 6.10, N 7.71.
9.5 mmol)
and N,N-diisopropylethylamine
(2.45 g,
19 mmol) at 0 C. After 0.5 h, 1-Boc-piperazine (1.77 g,
9.5 mmol) was added, and the reaction mixture was stirred
at room temperature for 16 h. The mixtures were con-
centrated and the residue was extracted by ethyl acetate
(EtOAc) and water. The organic layers was combined, dried
over MgSO4, and evaporated under reduced pressure. The
residue was further purified by flash column chromato-
graphy with petroleum ether/EtOAc (1:1) to produced
compound 3 as a white solid (2.87 g, 85% yield). m.p.
1
153–154 °C. H NMR (600 MHz, CDCl3) δ 7.95 (s, 1H),
7.61 (ddd, J = 8.6, 7.4, 1.4 Hz, 1H), 7.55 (dd, J = 7.7,
1.2 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 (dd, J = 7.6,
7.4 Hz, 1H), 3.74 (s, 2H), 3.55 (t, J = 4.5 Hz, 2H), 3.50 (t, J
= 4.5 Hz, 2H), 3.36 (s, 2H), 1.47 (s, 9H). 13C NMR
(150 MHz, CDCl3) δ 163.8, 158.1, 154.6, 154.3, 143.8,
133.2, 128.8, 125.1, 125.0, 118.0, 117.0, 80.5, 47.2, 42.3,
28.5. ESI–MS m/z: 359.1 [M+H]+.
3-(4-(3,5-Dimethylbenzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4c)
A white solid, yield 89%, m.p. 219–220 °C, HPLC purity:
95.59%, tR = 14.569 min. H NMR (600 MHz, CDCl3) δ
1
7.90 (s, 1H), 7.58 (ddd, J = 8.5, 7.4, 1.1 Hz, 1H), 7.53 (dd,
J = 7.6, 1.1 Hz 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.32 (dd, J =
7.6, 7.4 Hz, 1H), 6.92 (s, 2H), 6.90 (s, 1H), 3.79 (brs, 2H),
3.47 (s, 2H), 3.40 (t, J = 5.0 Hz, 2H), 2.54 (t, J = 5.0 Hz,
2H), 2.47 (brs, 2H), 2.30 (s, 6H). 13C NMR (150 MHz,
CDCl3) δ 163.4, 158.1, 154.2, 143.1, 138.0, 137.5, 132.9,
129.0, 128.6, 127.2, 125.5, 125.0, 118.5, 117.0, 63.0, 53.2,
52.7, 47.4, 42.3, 21.4. ESI–MS m/z: 377.1 [M+H]+. Anal:
Calcd. for C23H24N2O3: C 73.38, H 6.43, N 7.44; found: C
73.36, H 6.46, N 7.41.
General procedure for synthesis of 4a–4u
To a stirred solution of 3 (100 mg, 0.28 mmol) in 5 mL
DCM was added excessive amount of CF3CO2H dropwise
under nitrogen at 0 °C, and the reaction was monitored by
TLC. The solvent was removed under reduced pressure to
yield de-protection product as light yellow oil, which was
used in next step without further purification.
Corresponding bromide (1 equiv) was added to a solu-
tion of above obtained oil and triethylamine (150 μL) in
acetonitrile (5 mL) and the reaction mixture was stirred
under reflux for 8 h. Subsequently, the solvent was evapo-
rated under reduced pressure to get residue, which was
purified by silica gel column chromatography to afford the
desired products 4a–4u, respectively.
3-(4-(3,5-Bis(trifluoromethyl)benzyl)piperazine-1-carbonyl)-
2H-chromen-2-one (4d)
A light yellow solid, yield 92%, m.p. 170–171 °C, HPLC
purity: 98.88%, tR = 14.883 min. 1H NMR (600 MHz,
CDCl3) δ 7.93 (s, 1H), 7.81 (s, 2H), 7.78 (s, 1H), 7.59 (ddd,
J = 8.5, 7.4, 1.4 Hz, 1H), 7.54 (dd, J = 7.6, 1.4 Hz, 1H),
7.36 (d, J = 8.5 Hz, 1H), 7.33 (dd, J = 7.6, 7.4 Hz, 1H),
3.82 (brs, 2H), 3.65 (s, 2H), 3.42 (t, J = 4.7 Hz, 2H), 2.58 (t,
J = 5.0 Hz, 2H), 2.51 (t, J = 4.7 Hz, 2H). 13C NMR
(150 MHz, CDCl3) δ 163.6, 158.1, 154.3, 143.5, 140.9,
133.1, 131.8 (q, J = 33.2 Hz), 128.9, 128.70, 125.2, 125.1,
123.4 (q, J = 129.1 Hz), 121.5 (m), 118.41, 116.97, 61.77,
3-(4-Benzylpiperazine-1-carbonyl)-2H-chromen-2-one (4a)
A white solid, yield 82%, m.p. 127–128 °C, HPLC purity:
1
97.20%, tR = 12.049 min. H NMR (600 MHz, CDCl3) δ
7.90 (s, 1H), 7.58 (ddd, J = 8.3, 7.4, 1.4 Hz, 1H), 7.53 (dd,
J = 7.7, 1.4 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.32(m, 5H),
7.27(m, 1H), 3.79 (brs, 2H), 3.55 (s, 2H), 3.40 (t, J =

Medicinal Chemistry Research
53.06, 52.70, 47.28, 42.20. ESI–MS m/z: 485.2 [M+H]+.
Anal: Calcd. for C23H18F6N2O3: C 57.03, H 3.75, N 5.78;
found: C 57.29, H 3.72, N 5.81.
117.0, 112.49 (t, J = 20.0, 20.0 Hz), 111.4 (dd, J = 21.7,
5.0 Hz), 52.3, 51.7, 48.7, 47.3, 42.2. ESI-MS m/z: 385.2 [M
+H]+. Anal: Calcd. for C21H18F2N2O3: C 65.62, H 4.72, N
7.29; found: C 65.60, H 4.74, N 7.33.
3-(4-(4-Fluoro-2-methylbenzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4e)
3-(4-(4-Bromo-2-fluorobenzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4h)
A light yellow solid, yield 85%, m.p. 120–121 °C, HPLC
purity: 97.58%, tR = 13.709 min. 1H NMR (600 MHz,
CDCl3) δ 7.90 (s, 1H), 7.58 (ddd, J = 8.6, 7.4, 1.5 Hz, 1H),
7.53 (dd, J = 7.6, 1.3 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H),
7.32 (dd, J = 7.6, 7.4 Hz, 1H), 7.17 (dd, J = 7.9, 6.4 Hz,
1H), 6.86 (dd, J = 9.7, 2.5 Hz, 1H), 6.81 (ddd, J = 8.4, 8.3,
2.6 Hz, 1H), 3.76 (brs, 2H), 3.46 (s, 2H), 3.37 (t, J = 4.2 Hz,
2H), 2.53 (brs, 2H), 2.46 (brs, 2H), 2.35 (s, 3H). 13C NMR
(150 MHz, CDCl3) δ 163.5, 162.9, 161.3, 158.1, 154.2,
143.2, 140.1 (d, J = 7.8 Hz), 133.0, 131.50 (d, J = 8.0 Hz),
128.7, 125.3, 125.0, 118.4, 117.2 (d, J = 21.0 Hz), 116.9,
112.2 (d, J = 20.7 Hz), 60.1, 53.0, 52.5, 47.4, 42.3, 19.45
(d, J = 1.1 Hz). ESI–MS m/z: 381.2 [M+H]+. Anal: Calcd.
for C22H21FN2O3: C 69.46, H 5.56, N 7.36; found: C 69.49,
H 5.57, N 7.34.
A light yellow solid, yield 79%, m.p. 181–182 °C, HPLC
purity: 98.15%, tR = 13.938 min. 1H NMR (600 MHz,
CDCl3) δ 7.91 (s, 1H), 7.59 (ddd, J = 8.3, 7.4, 1.5 Hz, 1H),
7.53 (dd, J = 7.7, 1.3 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H),
7.32 (dd, J = 7.7, 7.4 Hz, 1H), 7.26 (m, 2H), 7.22 (dd, J =
9.8, 8.7 Hz, 1H), 3.79 (brs, 2H), 3.57 (s, 2H), 3.39 (t, J =
4.7 Hz, 2H), 2.57 (brs, 2H), 2.50 (brs, 2H). 13C NMR
(150 MHz, CDCl3) δ 163.5, 162.1, 160.4, 158.1, 154.3,
143.4, 133.0, 132.46 (d, J = 5.0 Hz), 128.7, 127.5 (d, J =
3.6 Hz), 125.3, 125.1, 119.3, 119.1, 118.4, 117.0, 54.8,
52.9, 52.3, 47.3, 42.2. ESI–MS m/z: 445.0, 447.0 [M+H]+.
Anal: Calcd. for C21H18BrFN2O3: C 56.64, H 4.07, N 6.29;
found: C 56.62, H 4.10, N 6.25.
3-(4-(4-Bromo-3-fluorobenzyl)piperazine-1-carbonyl)-2H-
3-(4-(2,4-Difluorobenzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4i)
chromen-2-one (4f)
A white solid, yield 85%, m.p. 208–209 °C, HPLC purity:
1
A light yellow solid, yield 81%, m.p. 101–102 °C, HPLC
purity: 98.80%, tR = 12.247 min. 1H NMR (600 MHz,
CDCl3) δ 7.92 (s, 1H), 7.60 (ddd, J = 8.3, 7.5, 1.5 Hz, 1H),
7.54 (dd, J = 7.7, 1.4 Hz, 1H), 7.37–7.31 (m, 3H), 6.86
(ddd, J = 8.3, 8.1, 1.7 Hz, 1H), 6.81 (ddd, J = 8.4, 8.3,
1.4 Hz, 1H), 3.82 (brs, 2H), 3.66 (brs, 2H), 3.43 (s, 2H),
2.64 (brs, 4H). 13C NMR (150 MHz, CDCl3) δ 163.5, 162.4
(d, J = 12.1 Hz), 160.7 (d, J = 12.2 Hz), 158.2, 154.3,
143.5, 133.1, 132.7, 128.7, 125.1, 118.4, 117.0 (d, J =
21.0 Hz), 111.5 (d, J = 22.5 Hz), 104.0 (dd, J = 25.7,
25.9 Hz), 54.5, 52.6, 52.1, 47.0, 41.9. ESI–MS m/z: 385.2
[M+H]+. Anal: Calcd. for C21H18F2N2O3: C 65.62, H 4.72,
N 7.29; found: C 65.65, H 4.70, N 7.26.
98.17%, tR = 13.741 min. H NMR (600 MHz, CDCl3) δ
7.91 (s, 1H), 7.59 (ddd, J = 8.3, 7.4, 1.4 Hz, 1H), 7.54 (dd,
J = 7.7, 1.2 Hz, 1H), 7.48 (dd, J = 7.8, 7.4 Hz, 1H), 7.36 (d,
J = 8.3 Hz, 1H), 7.32 (dd, J = 7.7, 7.4 Hz, 1H), 7.15 (dd, J
= 9.4, 1.5 Hz, 1H), 6.99 (dd, J = 8.1, 1.2 Hz, 1H), 3.79 (brs,
2H), 3.50 (s, 2H), 3.40 (t, J = 4.7 Hz, 2H), 2.54 (t, J =
5.0 Hz, 2H), 2.48 (t, J = 4.4 Hz, 2H). 13C NMR (150 MHz,
CDCl3) δ 163.5, 160.0, 158.5, 158.1, 154.2, 143.4, 140.0,
133.4, 133.0, 125.7 (d, J = 3.3 Hz), 125.3, 125.1, 119.3,
118.4, 117.0, 116.9 (d, J = 22.4 Hz), 61.8, 53.0, 52.6, 47.4,
42.2. ESI–MS m/z: 445.1, 447.0 [M+H]+. Anal: Calcd. for
C21H18BrFN2O3: C 56.64, H 4.07, N 6.29; found: C 56.65,
H 4.05, N 6.27.
3-(4-(2,6-Difluorobenzyl)piperazine-1-carbonyl)-2H-
3-(4-(2-(Trifluoromethyl)benzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4g)
chromen-2-one (4j)
A white solid, yield 88%, m.p. 145–146 °C, HPLC purity:
98.51%, tR = 11.876 min. H NMR (600 MHz, CDCl3) δ
A white solid, yield 91%, m.p. 80–81 °C, HPLC purity:
97.09%, tR = 14.035 min. H NMR (600 MHz, CDCl3) δ
1
1
7.88 (s, 1H), 7.58 (ddd, J = 8.3, 7.3, 1.4 Hz, 1H), 7.52 (dd,
J = 7.3, 1.4 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.31 (dd, J =
7.5, 7.3 Hz, 1H), 7.24 (dd, J = 8.0, 7.7 Hz, 1H), 6.89 (dd, J
= 7.7, 7.6 Hz, 2H), 3.78 (t, J = 4.5 Hz, 2H), 3.74 (s), 3.39
(t, J = 4.6 Hz, 2H), 2.61 (t, J = 4.6 Hz, 2H), 2.54 (brs, 2H).
13C NMR (150 MHz, CDCl3) δ 163.4, 162.2 (dd, J = 248.4,
8.2 Hz), 161.33 (d, J = 8.3 Hz), 158.1, 154.2, 143.2, 133.0,
129.7 (dd, J = 10.4, 10.3 Hz), 128.7, 125.3, 125.0, 118.4,
7.92 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz,
1H), 7.59 (ddd, J = 8.6, 7.0, 1.4 Hz, 1H), 7.54 (dd, J = 7.6,
1.3 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.6 Hz,
2H), 7.32 (dd, J = 7.6, 7.0 Hz, 1H), 3.81 (brs, 2H), 3.71 (s,
2H), 3.42 (t, J = 4.8 Hz, 2H), 2.58 (t, J = 4.8 Hz, 2H), 2.51
(brs, 2H). 13C NMR (150 MHz, CDCl3) δ 163.6, 158.1,
154.3, 143.3, 137.2, 133.0, 132.0, 130.4, 129.9, 128.90 (d,
J = 30.2 Hz), 128.7, 127.2, 126.0 (q, J = 5.7 Hz, 2H),

Medicinal Chemistry Research
125.4, 125.1, 118.4, 117.0, 58.2, 53.2, 52.7, 47.4, 42.4.
ESI–MS m/z: 417.2 [M+H]+. Anal: Calcd. for
C22H19F3N2O3: C 63.46, H 4.60, N 6.73; found: C 63.42, H
4.58, N 6.77.
ESI–MS m/z: 427.0 [M+H]+. Anal: Calcd. for
C21H19BrN2O3: C 59.03, H 4.48, N 6.56; found: C 59.09, H
4.50, N 6.60.
3-(4-(4-Bromobenzyl)piperazine-1-carbonyl)-2H-chromen-2-
3-(4-(4-(Trifluoromethyl)benzyl)piperazine-1-carbonyl)-2H-
one (4n)
chromen-2-one (4k)
A white solid, yield 65%, m.p. 213–214 °C, HPLC purity:
1
A white solid, yield 74%, m.p. 220–221 °C, HPLC purity:
95.77%, tR = 13.473 min. H NMR (600 MHz, CDCl3) δ
97.16%, tR = 13.710 min. H NMR (600 MHz, CDCl3) δ
1
7.91 (s, 1H), 7.59 (ddd, J = 8.3, 7.4, 1.5 Hz, 1H), 7.53 (dd,
J = 7.6, 1.3 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.36 (d, J =
8.3 Hz, 1H), 7.32 (t, J = 7.6, 7.4 Hz, 1H), 7.20 (d, J =
8.2 Hz, 2H), 3.76 (brs, 2H), 3.49 (s, 2H), 3.39 (t, J = 4.6 Hz,
2H), 2.53 (t, J = 4.8 Hz, 2H), 2.47 (brs, 2H). 13C NMR
(150 MHz, CDCl3) δ 163.50, 158.13, 154.25, 143.29,
136.87, 133.00, 131.62, 130.85, 128.68, 125.35, 125.07,
121.26, 118.44, 116.98, 62.16, 53.05, 52.55, 47.37, 42.29.
ESI–MS m/z: 427.1, 429.1 [M+H]+. Anal: Calcd. for
C21H19BrN2O3: C 59.03, H 4.48, N 6.56; found: C 58.98, H
4.44, N 6.51.
7.91 (s, 1H), 7.60–7.56 (m, 3H), 7.54 (d, J = 7.7, 1.4 Hz,
1H), 7.45 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.3 Hz, 1H), 7.32
(dd, J = 7.7, 7.5 Hz, 1H), 3.80 (brs, 2H), 3.60 (s, 2H), 3.40
(t, J = 4.7 Hz, 2H), 2.54 (t, J = 5.0 Hz, 2H), 2.49 (t, J =
4.7 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 163.5, 158.1,
154.2, 143.3, 142.1, 133.0, 129.7 (q, J = 32.3 Hz), 129.3,
128.7, 125.4 (q, J = 3.6 Hz), 125.3, 125.1, 124.2 (q, J =
271.9 Hz), 118.4, 117.0, 62.3, 53.1, 52.6, 47.4, 42.3.
ESI–MS m/z: 417.2 [M+H]+. Anal: Calcd. for
C22H19F3N2O3: C 63.46, H 4.60, N 6.73; found: C 63.44, H
4.63, N 6.70.
Methyl 4-((4-(2-oxo-2H-chromene-3-carbonyl)piperazin-1-
3-(4-(2-Bromo-4-methoxybenzyl)piperazine-1-carbonyl)-2H-
yl)methyl)benzoate (4o)
chromen-2-one (4l)
A light yellow solid, yield 78%, m.p. 180–181 °C, HPLC
purity: 95.38%, tR = 11.742 min. 1H NMR (600 MHz,
CDCl3) δ 8.00 (d, J = 8.2 Hz, 2H), 7.91 (s, 1H), 7.59 (ddd,
J = 8.2, 7.4, 1.4 Hz, 1H), 7.53 (dd, J = 7.6, 1.4 Hz, 1H),
7.42 (d, J = 6.9 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.32 (dd,
J = 7.6, 7.4 Hz, 1H), 3.91 (s, 2H), 3.81 (brs, 2H), 3.61 (brs,
2H), 3.42 (brs, 2H), 2.57 (brs, 2H), 2.51 (brs, 2H). 13C
NMR (150 MHz, CDCl3) δ 167.1, 165.6, 163.5, 158.2,
154.2, 143.4, 133.0, 129.9, 129.1, 128.7, 125.3, 125.1,
118.4, 117.0, 62.4, 53.1, 52.6, 52.3, 47.3, 42.2. ESI–MS m/
z: 407.2 [M+H]+. Anal: Calcd. for C23H22N2O5: C 67.97, H
5.46, N 6.89; found: C 67.94, H 5.50, N 6.94.
A light yellow solid, yield 92%, m.p. 164–165 °C, HPLC
purity: 99.54%, tR = 14.096 min. 1H NMR (600 MHz,
CDCl3) δ 7.91 (s, 1H), 7.58 (ddd, J = 8.7, 7.5, 1.1 Hz, 1H),
7.53 (dd, J = 7.7, 1.1 Hz, 1H), 7.41 (d, J = 8.7 Hz, 1H),
7.34 (t, J = 7.5 Hz, 1H), 7.32 (dd, J = 7.7, 7.5 Hz, 1H), 7.04
(d, J = 3.0 Hz, 1H), 6.68 (dd, J = 8.7, 3.1 Hz, 1H), 3.80
(brs, 2H), 3.78 (s, 3H), 3.59 (s, 2H), 3.41 (t, J = 4.6 Hz,
2H), 2.62 (t, J = 5.0 Hz,2H), 2.55 (brs, 2H). 13C NMR
(150 MHz, CDCl3) δ 163.52, 159.05, 158.08, 154.20,
143.22, 138.09, 133.43, 132.95, 128.66, 125.29, 125.03,
118.40, 116.92, 116.30, 115.02, 114.36, 61.64, 55.58,
53.05, 52.53, 47.39, 42.32. ESI–MS m/z: 374.2 [M+H]+.
Anal: Calcd. for C22H21BrN2O4: C 57.78, H 4.63, N 6.13;
found: C 57.83, H 4.60, N 6.17.
3-(4-(4-(Methylsulfonyl)benzyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4p)
3-(4-(3-Bromobenzyl)piperazine-1-carbonyl)-2H-chromen-2-
one (4m)
A light yellow solid, yield 78%, m.p. 174–175 °C, HPLC
purity: 95.62%, tR = 7.873 min. 1H NMR (600 MHz,
CDCl3) δ 7.91–7.88 (m, 3H), 7.91 (s, 1H), 7.59 (ddd, J =
8.5, 7.4, 1.4 Hz, 1H), 7.56–7.53 (m, 13H), 7.35 (d, J =
8.5 Hz, 1H), 7.32 (dd, J = 7.6, 7.4 Hz, 1H), 3.79 (brs, 2H),
3.63 (s, 2H), 3.41 (brs, 2H), 3.05 (s, 3H), 2.56 (brs, 2H),
2.50 (brs, 2H). 13C NMR (150 MHz, CDCl3) δ 163.5, 158.1,
154.2, 147.5, 143.4, 139.6, 133.1, 129.8, 128.7, 127.6,
127.3, 125.1, 118.4, 116.9, 62.1, 53.1, 52.6, 52.3, 47.3,
44.6, 42.2. ESI–MS m/z: 427.1 [M+H]+. Anal: Calcd. for
C22H22N2O5S: C 61.96, H 5.20, N 5.20, S 7.52; found: C
67.94, H 5.50, N 6.94.
A yellow solid, yield 77%, m.p. 118–119 °C, HPLC purity:
1
95.85%, tR = 13.697 min. H NMR (600 MHz, CDCl3) δ
7.91 (s, 1H), 7.59 (ddd, J = 8.6, 7.4, 1.5 Hz, 1H), 7.54 (dd,
J = 7.6, 1.5 Hz, 1H), 7.50 (s, 1H), 7.40 (d, J = 8.3 Hz, 1H),
7.36 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 7.6, 7.4 Hz, 1H),
7.24–7.21 (m, 1H), 7.20–7.18 (m, 1H), 3.80 (brs, 2H), 3.52
(s, 2H), 3.41 (brs, 2H), 2.56 (brs, 2H), 2.49 (brs, 2H). 13C
NMR (150 MHz, CDCl3) δ 163.5, 158.2, 154.2, 143.3,
133.0, 132.1, 130.8, 130.2, 130.1, 128.7, 127.8, 125.4,
125.1, 122.7, 118.4, 117.0, 62.2, 53.0, 52.6, 47.3, 42.2.

Medicinal Chemistry Research
4-((4-(2-Oxo-2H-chromene-3-carbonyl)piperazin-1-yl)
methyl)benzonitrile (4q)
8.32–8.26 (m, 1H), 7.90 (s, 1H), 7.85 (dd, J = 7.8, 1.5 Hz,
1H), 7.79 (dd, J = 6.8, 2.5 Hz, 1H), 7.59 (ddd, J = 8.4, 7.4,
1.5 Hz, 1H), 7.55–7.48 (m, 3H), 7.42–7.38 (m, 2H), 7.35 (d,
J = 8.4 Hz, 1H), 7.32 (dd, J = 7.6, 7.4 Hz, 1H), 3.95 (s,
2H), 3.78 (brs, 2H), 3.38 (t, J = 4.7 Hz, 2H), 2.63 (t, J =
5.0 Hz, 2H), 2.55 (brs, 2H). 13C NMR (150 MHz, CDCl3) δ
163.5, 158.1, 154.2, 143.1, 134.0, 133.5, 132.9, 132.6,
128.6, 128.7, 128.4, 127.8, 126.0, 125.9, 125.4, 125.2,
125.0, 124.8, 118.4, 117.0, 61.2, 53.2, 52.8, 47.4, 42.4.
ESI–MS m/z: 427.0 [M+H]+. Anal: Calcd. for C25H22N2O3:
C 75.36, H 5.57, N 7.03; found: C 75.30, H 5.60, N 7.05.
A light yellow solid, yield 87%. m.p. 210–211 °C, HPLC
purity: 97.77%, tR = 10.200 min. 1H NMR (600 MHz,
CDCl3) δ 7.92 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.59 (ddd,
J = 8.3, 7.4, 1.4 Hz, 1H), 7.54 (dd, J = 7.6, 1.4 Hz, 1H),
7.46 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.3 Hz, 1H), 7.33 (dd,
J = 7.6, 7.4 Hz, 1H), 3.80 (brs, 2H), 3.59 (s, 2H), 3.40 (t, J
= 4.6 Hz, 2H), 2.55 (t, J = 5.0 Hz, 2H), 2.49 (t, J = 4.7 Hz,
2H). 13C NMR (150 MHz, CDCl3) δ 163.5, 158.1, 154.3,
143.7, 143.4, 133.1, 132.4, 129.6, 128.7, 125.3, 125.1,
119.0, 118.4, 117.0, 111.3, 62.3, 53.1, 52.7, 47.3, 42.3.
ESI–MS m/z: 374.2 [M+H]+. Anal: Calcd. for C22H19N3O3:
C 70.76, H 5.13, N 11.25; found: C 70.70, H 5.16, N 11.28.
3-(4-([1,1′-Biphenyl]-4-ylmethyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4u)
A white solid, yield 89%, m.p. 178–179 °C, HPLC purity:
1
Methyl (E)-3-(4-((4ethyl (E)-3-(4-((-(2-oxo-2H-chromene-3-
98.54%, tR = 13.376 min. H NMR (600 MHz, CDCl3) δ
carbonyl)piperazin-1-yl)methyl)phenyl)acrylate (4r)
7.91 (s, 1H), 7.59 (m, 3H), 7.56 (d, J = 8.2 Hz, 2H), 7.54
(dd, J = 7.8, 1.5 Hz, 1H), 7.44 (dd, J = 8.0, 7.4 Hz, 2H),
7.39 (d, J = 8.2 Hz, 2H), 7.35 (m, 3H), 3.81 (brs, 2H), 3.59
(s, 2H), 3.42 (t, J = 4.8, 2H), 2.59 (t, J = 4.8, 2H), 2.53 (brs,
2H). 13C NMR (150 MHz, CDCl3) δ 163.5, 158.1, 154.2,
143.2, 141.0, 140.4, 136.8, 133.0, 129.7, 128.9, 128.7,
127.4, 127.2, 127.2, 125.4, 125.0, 118.4, 117.0, 62.6, 53.2,
52.6, 47.4, 42.4. ESI–MS m/z: 425.1 [M+H]+. Anal: Calcd.
for C27H24N2O3: C 76.40, H 5.70, N 6.60; found: C 76.35,
H 5.74, N 6.58.
A white solid, yield 78%, m.p. 212–213 °C, HPLC purity:
1
99.09%, tR = 12.760 min. H NMR (600 MHz, CDCl3) δ
7.91 (s, 1H), 7.68 (d, J = 16.0, 1H), 7.59 (ddd, J = 8.4, 7.6,
1.4 Hz, 1H), 7.53 (dd, J = 7.7, 1.1 Hz 1H), 7.48 (d, J =
8.0 Hz, 2H), 7.36–7.3 (m, 3H), 7.32 (dd, J = 7.6, 7.4 Hz,
1H), 6.43 (d, J = 16.0, 1H), 3.80 (s, 3H), 3.79 (brs, 2H),
3.56 (s, 2H), 3.40 (t, J = 4.5 Hz, 2H), 2.55 (t, J = 4.8 Hz,
2H), 2.49 (brs, 2H). 13C NMR (150 MHz, CDCl3) δ 167.6,
163.5, 158.1, 154.2, 144.7, 143.3, 140.5, 133.6, 133.0,
129.6, 128.7, 128.3, 125.4, 125.1, 118.4, 117.7, 117.0,
62.5, 53.1, 52.6, 51.9, 47.4, 42.3. ESI–MS m/z: 433.2 [M
+H]+. Anal: Calcd. for C25H24N2O5: C 69.43, H 5.59, N
4.68; found: C 69.49, H 5.56, N 4.66.
Evaluation of hAChE and hBChE inhibitory activity
AChE and BChE inhibitory activities of all the target
compounds were determined by using modified Ellman’s
method (Ellman et al. 1961; Talesa 2001. Human recom-
binant AChE, human plasmatic BChE, 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB), phosphate buffer solution
(PBS, pH 8.0), acetylthiocholine (ATC) iodide, and butyr-
ylthiocholine (BUC) iodide were purchased from Sigma-
Aldrich (Steinheim, Germany). Donepezil was used as
positive control. Enzyme solutions were prepared at 2.0 U/
mL in 2 mL aliquots. The assay medium consisted of 10 μL
of enzyme, 40 μL of PBS, 20 μL of 0.01 M DTNB and 10
μL of tested compound. Assayed solutions of tested com-
pounds were pre-incubated with corresponding ChE for
5 min. The reaction was initiated by addition of 20 μL of
0.01 M substrate (ATC or BUC). The activity was deter-
mined by measuring the increase in absorbance at 410 nm at
37 °C in 2 min intervals using Tecan Spark multimode
microplate reader (Switzerland). The percentage of inhibi-
tion (I) was calculated from the measured data as follows: I
= (Ac−Ai)/Ac × 100%, where Ai and Ac represent the
change in the absorbance in the presence of inhibitor and
without inhibitor, respectively.
2-((4-(2-Oxo-2H-chromene-3-carbonyl)piperazin-1-yl)
methyl)benzonitrile (4s)
A yellow solid, yield 91%, m.p. 164–165 °C, HPLC purity:
1
99.56%, tR = 10.582 min. H NMR (600 MHz, CDCl3) δ
7.97 (s, 1H), 7.70 (dd, J = 7.8, 0.8 Hz, 1H), 7.67–7.58 (m,
3H), 7.56 (dd, J = 7.7, 1.4 Hz, 1H), 7.46 (td, J = 7.6,
1.1 Hz, 1H), 7.38–7.31 (m, 2H), 4.04 (s, 2H), 3.92 (brs,
2H), 3.54 (t, J = 4.6, 2H), 2.91 (brs, 4H). 13C NMR
(150 MHz, CDCl3) δ 163.5, 158.3, 154.3, 144.4, 133.4,
133.4, 133.3, 131.3, 129.3, 128.9, 125.2, 124.5, 118.3,
117.6, 117.0, 113.9, 59.4, 52.5, 51.9, 46.0, 41.0. ESI–MS
m/z: 374.2 [M+H]+. Anal: Calcd. for C22H19N3O3: C 70.76,
H 5.13, N 11.25; found: C 70.80, H 5.09, N 11.30.
3-(4-(Naphthalen-1-ylmethyl)piperazine-1-carbonyl)-2H-
chromen-2-one (4t)
A yellow solid, yield 77%, m.p. 118–119 °C, HPLC purity:
1
95.28%, tR = 14.574 min. H NMR (600 MHz, CDCl3) δ

Medicinal Chemistry Research
Scheme 1 Synthesis of 4a–4u.
Reaction conditions and
reagents: a Meldrum’s acid,
H2O, reflux, 10 h; b 1-Boc-
piperazine, EDCI, HOBT,
DCM, DIEPA, 16 h; c (1)
CF3CO2H, DCM, 0 °C; (2)
RCH2Br, Et3N, acetone, 80 °C
for 8 h
Kinetic assay
The formazan dye product was measured by the absorbance
at 490 nm on a Tecan Spark multimode microplate reader
(Switzerland).
Kinetic studies of inhibition on AChE were performed by
using Ellman’s method as described above. The con-
centrations of used substrates were 0.07813, 0.1563,
0.3125, and 0.625 μM. Linear regression was used for cal-
culation of Lineweaver-Burk plots, and all the calculations
were performed using GraphPad Prism 5.0 software.
Results and discussion
Chemical synthesis
Molecular docking
The synthesis of coumarin/piperazine hybrids 4a–4u was
shown in Scheme 1. Coumarin-3-carboxylic acid 2 was
prepared from the reaction of Meldrum’s acid with 2-
hydroxybenzaldehyde. Then compound 3 was obtained by
EDCI condensation in the presence of 2 and Boc-
piperazine. Finally, the reaction between deprotection pro-
duct of 3 and different bromides yielded the corresponding
target compounds 4a–4u. All the prepared compounds were
confirmed by using 1H NMR, 13C NMR, MS and elemental
analysis data. All the spectra of compounds have been
provided in supporting information.
Molecular docking were performed by using the Autodock
4.2 program (Morris et al. 1998; Huey et al. 2007). The
crystal structure of the hAChE complexed with donepezil
(PDB 4EY7) was obtained from the Protein Data Bank after
removing the inhibitor and water molecules. The 3D
Structure of 4t was built by performimng MM2 minimize
energy in ChemBio3D Ultra 11.0. Using Autodock Tools
1.5.6, preparation of receptor was made by addition of
hydrogen atoms and Gasteiger charges, and finally assign-
ment of atomic types as AD4 type, and then autotorsions
was used to define the rotatable bonds in the ligand pre-
paration. The resulting enzyme structure was used as an
input for the Autogrid program, which performed a pre-
calculated atomic affinity grid maps for each atom type in
the ligand plus an electrostatics map and a separate deso-
lvation map presented in the substrate molecule. The
dimensions of the box were set to 60 × 60 × 60 with grid
spacing of 0.375 Å. Rigid ligand docking was performed for
the compounds. Docking calculations were carried out
using the Lamarckian genetic algorithm.
AChE and BChE inhibition assay
The inhibitory activities of hybrids 4a–4u against human
recombinant AChE (hAChE) and human plasmatic BChE
(hBChE) were evaluated with donepezil as reference com-
pound. The results were summarized in Table 1. Nearly all
the tested compounds showed inhibition on hAChE with
IC50 values ranging 2.42 to 80.78 μM, except 4r and 4u (IC50
values > 100 μM). Compared with compound 4a, the methyl
substituent(s) in 4b and 4c did not show significant effect on
the activity. Since the fluorinated functionalities were
reported as key pharmacophore found in about 20% phar-
maceutical on the market (Furuya et al. 2011), the F-
substituted analogs 4d–4k were then prepared. Among them,
compound 4d with 3,5-bistrifluoromethyl displayed the most
potent activity (IC50 = 2.42 μM), while compound 4k with 4-
trifluoromethyl exhibited decreased activity (IC50 = 62.87
μM) and compounds 4o–4r with electron-withdrawing
groups (cyano, methyl carboxylate, methylsulfonyl, and
methyl acrylate) at para-position showed week activity. This
result indicated that strong electron-withdrawing group at
In vitro cytotoxicity evaluation
Cytotoxicity of selected compounds were evaluated against
SH-SY5Y neuroblastoma cell line according to a reported
protocol (Mosmann 1983), with denopezil as positive con-
trol. SH-SY5Ycell were inoculated into 96-well plates.
After incubation for 24 h, the cells were treated with dif-
ferent concentrations of tested compounds for 24 h and then
were incubated with 10 μL of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) at 37 °C for 2 h.

Medicinal Chemistry Research
Table 1 In vitro inhibition on
hAChE and hBChE
Compounds
x,y
IC50 (μM) SDa
Selectivity for hAChEb
hAChE
hBChE
4a
H
32.28 0.34
32.79 0.06
30.75 0.03
2.42 0.15
27.04 0.12
15.54 0.06
20.78 0.56
18.96 0.31
42.04 0.34
15.53 0.24
62.87 0.44
18.66 0.81
12.03 0.01
42.04 0.34
55.28 0.17
57.78 0.32
80.78 0.17
>100
41.96 0.22
20.51 0.01
15.67 0.12
3.87 0.23
32.45 0.78
34.19 0.01
29.09 0.33
9.48 0.22
46.24 0.85
27.22 0.08
69.16 0.19
14.93 0.04
21.04 0.33
17.63 0.67
82.92 1.33
75.11 0.89
>100
1.3
0.6
0.5
1.6
1.2
2.2
1.4
0.5
1.1
1.8
1.1
0.8
1.7
2.4
1.5
1.3
–
4b
4c
4-CH3
3-CH3, 5-CH3
3-CF3, 5-CF3
2-CH3, 4-F
2-F, 4-F
2-F, 6-F
2-F, 4-Br
3-F, 4-Br
2-CF3
4d
4e
4f
4g
4h
4i
4j
4k
4l
4-CF3
2-Br, 4-OCH3
3-Br
4m
4n
4o
4-Br
4-CO2Me
4-SO2Me
4-CN
4p
4q
4r
4,
>100
–
4s
2-CN
33.28 0.66
8.78 0.22
>100
16.64 0.27
86.04 0.76
>100
0.5
9.8
–
4t
–
4u
donepezil
–
0.03 0.01
4.31 0.76
144
aIC50 (hBChE)/IC50 (hAChE)
bResults are expressed as the mean of 3 independent experiments
meta-position of benzene ring might be favorable for
increased AChE inhibitory activity, and the electron-
withdrawing group at para-position of benzene resulted in
decreased inhibition on AChE. Next, the effect of aromatic
property of substituent on bioactivity was investigated, the
naphthalene group was first introduced to the piprazine based
on the observation that naphthalene was capable of increas-
ing the inhibitory activity and selectivity toward AChE in our
previous study (Li et al. 2016). Interestingly, although
compound 4t with naphthalene group showed slightly
weaker inhibition on AChE than 4d, it had the highest
selectivity toward AChE over BChE by 9.8-fold. However,
the introduction of another aromatic system, biphenyl, made
the activity loss (4u, IC50 > 100 μM), probably due to its
steric hindrance. Compared with the activity of donepezil,
further structural modifications on this series of hybrids to
improve their potency and selectivity are still need.
obtain information about the inhibition mode and binding
site. The mechanism of AChE inhibition was analyzed by
recording substrate concentration-enzyme velocity curves in
the presence of different concentration of 4t.
As shown in Fig. 3a, it revealed both decreasing Vmax and
increasing Kmax at rising concentrations of 4t (Fig. 3). The
equilibrium constants for binding with the free enzyme (KI)
and with the enzyme-substrate complex (KIS) were obtained
from the slope and the 1/Vmax values plotted against inhibitor
concentration, respectively (Delogu et al. 2016). The values
of KI (Fig. 3b) and KIS (Fig. 3c) of 4t were determined to be
3.29 and 11.47 μM for hAChE, respectively. These patterns
indicated that compound 4t was a linear-mixed type AChE
inhibitor, suggesting that 4t may not only compete with the
substrate for the catalytic active site (CAS) but also interact
with the second binding site, peripheral anionic site (PAS).
This was in agreement with the subsequent result from
molecular modeling study of 4t (Fig. 4).
Kinetic assay
Molecular modeling study
Since compound 4t showed promising inhibitory activity
and highest selectivity toward hAChE, it was selected as a
representative compound of this series for kinetic assay to
The probable binding mode of compound 4t with the cat-
alytic domain of AChE (PDB: 4EY7) (Cheung et al. 2012)

Medicinal Chemistry Research
Fig. 3 Kinetic assay on AChE inhibition displayed by 4t. a Lineweaver–Burk reciprocal plots of initial velocity and increasing substrate
concentrations (0.078–0.625 mM); b Secondary plot of slopes versus 4t concentrations; c Secondary plot of 1/Vmax versus 4t concentrations
Fig. 4 The docking of 4t (carbon in green) in the catalytic site of AChE with only the primary interactions shown
was investigated by molecular docking, which was per-
formed by using Autodock 4.2 (Morris et al. 1998; Huey
et al. 2007). The structure images was created by Pymol 1.5.
As shown in Fig. 4, compound 4t was bound very well to
the active-gorge of AChE, with coumarin fragment oriented
in the PAS and naphthalene moiety located in the CAS.
There are three kinds of primary interactions between 4t and
the key amino acid residues in the active-site of AChE,
including H-bond, π- π and salt bridge interactions. For
example, the coumarin and naphthalene moieties could
form π- π interactions with Trp286 and Trp86, respectively,
and the carbonyl in coumarin interacts with Phe295 via
forming H-bond. The protonated nitrogen atom in the
piperazine ring could form H-bond interaction with Try341
and salt bridge with Asp74. In addition, the hydrophobic
interactions between 4t and the surrounding key amino acid
residues (Try337, Trp286, Phe338, Try341, and Trp 86)
also contribute to the affinity of ligand and AChE.
residues in active site of BChE compared with the binding
mode of 4t/AChE. This was speculated to be a reason that 4t
exhibited good selectivity toward AChE over BChE.
In vitro cytotoxicity
The toxicity profile of active compound is usually con-
sidered throughout the process of drug research and
development. Herein, the in vitro cytotoxicity of com-
pounds 4d and 4t, which showed IC50 values less than 10
μM in AChE assay, were selected to be evaluated against
SH-SY5Y neuroblastoma cell line, using MTT assay with
donepezil as reference compound. The results in Table 2
demonstrated that both hybrids did not exhibit observed
cytotoxic effect on SH-SY5Y cell, with very low inhibition
rate at 100 μM, indicating that they are much less cytotoxic
than donepezil (IC50 = 20.8 μM).
In addition, molecular docking was also performed for 4t/
BChE by using Autodock 4.2 to explain its selectivity toward
AChE and BChE. The result of docking result of 4t/BChE
(Fig. S1, ESI+) indicated that 4t could also be protonated in
the active pocket of BChE (PDB: 4BDS) (Nachon et al.
2013) but it showed fewer interactions with the amino acid
Conclusion
In summary, 21 novel derivatives were prepared based on
the hybridization of coumarin and piperazine pharmaco-
phores, and were subjected to the in vitro hAChE and

Medicinal Chemistry Research
Table 2 The cytotoxicity of compounds
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Compound
SH-SY5Y
100 μMa
IC50
(μM)
>100b
>100
20.8
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4d
5.4%
4.4%
89.1%
4t
donepezil
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trifluoromethylation. Nature 473:470–477
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bCompound with inhibition ratio less than 50% at 100 μM was not
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tested further
hBChE inhibition assay. Among the synthesized analogs,
compounds 4d and 4t afforded IC50 values ranging from
2.42 to 9.89 μM against hAChE with no observed cyto-
toxicity against SH-SY5Y cell at 100 μM. Especially, 4t
showed high selectivity toward hAChE over hBChE by 9.8-
fold. The kinetic assay and molecular modeling study
revealed these compounds as dual binding site AChE
inhibitors. The present study demonstrated that these cou-
marin/piperazine hybrids serve as a new template for
developing novel potent and safe AChE inhibitors.
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Acknowledgements This research work was financially supported by
the Natural Science Foundation of China (No. 21672082) and Shan-
dong Key Development Project (No. 2016GSF201209).
Jameel E, Umar T, Kumar J, Hoda N (2016) Coumarin: a privileged
scaffold for the design and development of antineurodegenerative
agents. Chem Biol Drug Des 87:21–38
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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