Synthesis and anti-cholinesterase activity of new 7-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.05.056

A series of 7-hydroxycoumarin derivatives connected by an amidic linker to the different amines were designed and synthesized as cholinesterase inhibitors. Most compounds showed remarkable inhibitory activity against acetylcholinesterase (AChE) and butyry

Full text of DOI:10.1016/j.ejmech.2014.05.056

Accepted Manuscript
Synthesis and anti-cholinesterase activity of new 7-hydroxycoumarin derivatives
Masoumeh Alipour , Mehdi Khoobi , Alireza Moradi , Hamid Nadri , Farshad
Homayouni Moghadam , Saeed Emami , Zeinab Hasanpour , Alireza Foroumadi ,
Abbas Shafiee
PII:
S0223-5234(14)00480-2
10.1016/j.ejmech.2014.05.056
EJMECH 7014
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2014
Revised Date: 18 May 2014
Accepted Date: 23 May 2014
Please cite this article as: M. Alipour, M. Khoobi, A. Moradi, H. Nadri, F.H. Moghadam, S. Emami,
Z. Hasanpour, A. Foroumadi, A. Shafiee, Synthesis and anti-cholinesterase activity of new 7-
hydroxycoumarin derivatives, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.056.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and anti-cholinesterase activity of new 7-hydroxycoumarin derivatives
Masoumeh Alipour, Mehdi Khoobi, Alireza Moradi, Hamid Nadri, Farshad Homayouni
Moghadam, Saeed Emami, Zeinab Hasanpour, Alireza Foroumadi, Abbas Shafiee*
R1
H
N
R2
O
O
O
n
O
O
O
O
Ph
N
O
1
R = H, Me
R = amine
2
4r
n = 1 or 3
A series of 7-hydroxycoumarins were synthesized as cholinesterase inhibitors. Compound 4r
was the most potent compound against AChE (IC50 = 1.6 µM). Also, compound 4r significantly
protected neurons against H O -induced cell death.
2 2

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Highlights
•
•
New coumarin-derived cholinesterase inhibitors were synthesized from 7-OH-coumarins.
Among them, acetamide derivative 4r was the most potent compound against AChE.
•
•
Selectivity index of compound 4r against AChE vs. BuChE was about 26.
Compound 4r significantly protected neurons against H O -induced cell death.
2
2

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Synthesis and anti-cholinesterase activity of new 7-hydroxycoumarin
derivatives
a
a
b
b
Masoumeh Alipour , Mehdi Khoobi , Alireza Moradi , Hamid Nadri , Farshad Homayouni
c
d
a
a
a,
Moghadam , Saeed Emami , Zeinab Hasanpour , Alireza Foroumadi , Abbas Shafiee *
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b
Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
c
Neurobiomedical Research Center, School of Medicine, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A series of 7-hydroxycoumarin derivatives connected by an amidic linker to the
different amines were designed and synthesized as cholinesterase inhibitors. Most compounds
showed remarkable inhibitory activity against acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE). Among them, N-(1-benzylpiperidin-4-yl)acetamide derivative 4r
with IC value of 1.6 µM was the most potent compound against AChE. The selectivity index of
5
0
compound 4r for anti-AChE activity was about 26. Moreover, the compound 4r significantly
protected PC12 neurons against H O -induced cell death at low concentrations. The docking
2
2
study of compound 4r with AChE enzyme showed that both CAS and PAS are occupied by the
ligand.
Keywords: Alzheimer’s disease; Acetylcholinesterase; Coumarins; Docking study
*
Corresponding author. Tel: +98 21 66406757; Fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
1

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1
. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive
impairment, loss of memory and language deterioration [1]. It is the most common form of
dementia affecting more than 5% of the population over the age of 65 years [2].
At the molecular level, patients affected by AD show abnormal deposits of β-amyloid peptide
(Aβ) and abnormal spiral filaments in neurons, increased oxidative stress, and low levels of
acetylcholine (ACh) [3]. In general, the death of neurons during the progression of AD affects
the levels of brain neurotransmitters. Besides ACh, other neurotransmitter, e.g. glutamate and
serotonin are also affected later in the disease [4].
Currently, there is no cure for AD and treatment strategies being primarily symptomatic. The
main therapeutic strategy is based on the cholinergic hypothesis and specifically on
acetylcholinesterase (AChE) inhibition to improve cholinergic neurotransmission in the brain
[5]. The recent studies have shown that AChE involves in the extraneous non-cholinergic
function by binding to Aβ [6, 7]. The X-ray crystallography of AChE structure has demonstrated
that the active site of AChE is a deep and narrow gorge mainly composed of two distinct binding
sites: the Ser-His-Glu catalytic site located at the bottom of gorge, and the peripheral anionic
binding site (PAS) located at the gorge entrance [8]. The PAS has a crucial role in the
polymerization of Aβ plaques and fibrils formation [9].
In the recent decades, several AChE inhibitors such as donepezil, tacrine, rivastigmine, and
galantamine have been introduced for the treatment of mild to moderate AD [10]. Among these
agents, donepezil interacts with both active-site gorge and PAS, in which the dimethoxyindanone
part of donepezil binds to the PAS [11].
Structural development of AChE inhibitors resulted in introducing coumarin class of compounds
such as ensaculine and AP2238 as anti-Alzheimer’s agents (Fig. 1). Previous studies have
demonstrated that coumarin motif can bind primarily to the PAS of AChE [12]. Accordingly, we
have recently designed some novel coumarin-3-carboxamide derivatives linked to N-
benzylpiperidine as potent AChE inhibitors [13]. Furthermore, we described 4-hydroxycoumarin
derivatives in which an N-phenylpiperazine or N-benzylpiperidine moiety was connected with an
alkoxy amide spacer to the coumarin scaffold. Particularly, N-(1-benzylpiperidin-4-yl)acetamide
derivative displayed the highest AChE inhibitory activity [14]. In the search for finding new
coumarin-based AChE inhibitors, we decided to displace the side chain from 4- to 7-position of
2

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coumarin core structure. Thus, in continues of our previous works on the synthesis of novel
AChE inhibitors [13-15], we describe here synthesis and biological activity evaluation, as well as
docking study of 2-(2-oxo-2H-chromen-7-yloxy)amides 4a-s.
2
. Chemistry
As illustrated in Scheme 1, the target compounds 4a-s could be easily prepared starting from 7-
hydroxycoumarins 1a,b. 7-Hydroxycoumarin 1a was commercially available and 4-methyl-7-
hydroxycoumarin 1b was prepared by using Pechmann condensation [16]. Compound 1 was
converted to ethyl ester derivatives 2a-c by O-alkylation with ethyl 2-bromoacetate or ethyl 4-
bromobutanoate in the presence of K CO , in refluxing acetone. The esters 2 were hydrolyzed
2
3
with aqueous solution of sodium hydroxide to afford the corresponding acids 3a-c. Finally,
different procedure were screened for the condensation of the carboxylic acids 3 with appropriate
amines, such as application of dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N-
ethylcarbodiimide hydrochloride (EDC) with hydroxybenzotriazole (HBT) and N-
hydroxysuccinimide (NHS) in different solvents. Among these, the best result was obtained by
EDC/HBT in acetonitrile.
3
. Results and discussion
3
3
.1. Pharmacology
.1.1. Cholinesterase inhibitory activity
The cholinesterase inhibitory activity of synthesized compounds 4a-s in comparison with
standard drug donepezil was evaluated against AChE and BuChE enzymes and the results were
expressed as IC50 values in Table 1. The tested compounds showed IC50s ranging from 1.6 to 74
µM against AChE. Among them, benzylpiperidinylamino derivative 4r exhibited the most potent
inhibitory activity. However, compounds 4a-c, 4f, 4i, 4l, and 4q showed good activity against
AChE (IC50s <10 µM).
The obtained IC values of 4-phenylpiperazinyl derivative 4a-e against AChE revealed that the
5
0
2
substitution of phenyl ring by 2-OH, 2-F, 4-F and 3,4-Cl diminished the activity (4b-e vs 4a). In
contrast, in 4-methylcoumarin series 4h-l containing 4-phenylpiperazinyl moiety, the 2-OH, 2-F
and 3,4-Cl groups increased the inhibitory activity towards AChE.
2
3

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In the case of 4-unsubstituted coumarins with three carbons linker (n = 3), the (2-
hydroxyphenyl)piperazine, (2-fluorophenyl)piperazine, and 4-benzylpiperidine derivatives
(compounds 4b, 4c and 4f, respectively) showed similar activities. While, among the 4-
unsubstituted coumarins with n=1, (1-benzylpiperidin-4-yl)amino moiety was more favorable
than phenylpiperazine.
The comparison of 4-methylcoumarin derivatives 4h-m and 4-unsubstituted coumarin analogs
4
a-g in terms of anti-AChE activity, revealed that the effect of methyl group at 4-position
depended on the type of pendent group on the 7-position. For example, although the 4-methyl
derivative 4l was more potent than its unsubstituted analog 4e, but 4-methyl derivative 4h was
less potent than corresponding analog 4a.
As shown in Table 1, the length of side chain linker (n = 1 or 3) could affect the inhibitory
activity. In phenylpiperazine derivatives, the activity of compound 4a (n = 3) was higher than
compound 4n (n = 1). In addition, the anti-AChE activity of (2-fluorophenyl)piperazine
derivative 4c (n= 3) was superior than that of its analog 4o bearing shorter linker (n = 1).
Compounds 4e and 4f containing (3,4-dichlorophenyl)piperazine and benzylpiperidine were as
potent as 4p and 4q, respectively. Thus, the effect of amidic linker depended on the type of
substituted amine which attached to the linker. The survey of the IC50 values of
(fluorophenyl)piperazine derivatives against AChE demonstrated that the 2-fluoro substituent is
more favorable than the 4-fluoro one (4c vs. 4d and 4j vs. 4k).
It should be noted that the most of target compounds have distal tertiary amine that would be
protonated at the physiological pH. However, compounds 4f, 4g, 4m, 4q and 4s contain only
amidic nitrogen without distal amine. Interestingly amongst the latter compounds, 4-
benzylpiperidine derivatives 4f and 4q showed good activities (IC s <10 µM).
5
0
The IC50 values of compounds 4a-s against BuChE were in the range of 15 to 387 µM, which
were significantly higher than those of AChE. Among the tested compounds only (4-
fluorophenyl)piperazine derivative 4k showed more selective activity against BuChE. However,
the (2-fluorophenyl)piperazine derivative 4c was the most potent compound against BuChE.
Notably, the effect of structural modifications on BuChE activity was the same as AChE.
Nevertheless, the most potent compound against AChE (compound 4r) was not the most active
one against BuChE.
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The coumarin nucleus has been extensively considered in the design of AChE inhibitors,
resulted in introducing anti-Alzheimer’s agents such as ensaculine and AP2238. In order to
explore wide structure–activity relationships of coumarin derivatives, 3, 4, 6 or 7-substituted
coumarins have been studied more extensively. Attachment of functionalized amine via an
appropriate spacer to the mentioned positions of coumarin template is the typically explored
modifications. Recently, we have synthesized a series of coumarin-3-carboxamide derivatives
bearing N-benzylpiperidine side chain as potent AChE inhibitors [13]. Also, we have described
4
-hydroxycoumarin derivatives containing N-phenylpiperazine or N-benzylpiperidine residues.
Among them, N-(1-benzylpiperidin-4-yl)acetamide derivative displayed highest AChE inhibitory
activity (IC = 1.2 µM) with 37-fold selectivity for AChE respect to the BuChE [14]. In the
5
0
present work, we found that 7-hydroxycoumarin derivative 4r containing N-(1-benzylpiperidin-
-yl)acetamide derivative with IC value of 1.6 µM was the most potent compound against
4
5
0
AChE. These findings revealed that N-(1-benzylpiperidin-4-yl) substituent is a favorable scaffold
to attaching to the 4- or 7-hydroxycoumarin scaffold via an acetamido linker.
3
.1.2. Kinetic study of AChE inhibition
The most active compound 4r was subjected to kinetic studies. For this purpose, the rate of the
enzyme activity was measured at four different concentrations of compound 4r (0, 0.64, 1.27 and
2
.55 µM) using different concentration of the substrate acetylthiocholine (ATCh). In each case,
the initial velocity was measured at different concentrations of the substrate (S), and the
reciprocal of the initial velocity (1/v) was plotted against the reciprocal of [ATCh]. The double
reciprocal (Lineweavere-Burk) plot showed a pattern of increasing slopes and increasing
intercepts with higher inhibitor concentration (Fig. 2). This pattern indicates mixed-type
inhibition of the enzyme by compound 4r. The inhibitory constant (Ki) for the compound 4r was
calculated using secondary plot as depicted in Fig. 2 (Ki = 1.32 µM).
3
.1.3. Ferric reducing antioxidant power (FRAP)
Ferric reducing antioxidant power (FRAP) assay was used to measure total antioxidant activity
of the target compounds based on the method of Benzie and Strain [17]. In this method the
compounds act as reductants in a redox-linked colorimetric method. In principle ferric tripyridyl
triazine complex is reduced to ferrous form which has a deep blue color. The change of
5

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absorbance is measured at 585 nm. The antioxidant activity of the compounds is shown in Table
2
. Compounds 4s showed the highest antioxidant activity with FRAP value of 5.43. This
compound was less effective than the standard antioxidants ascorbic acid and quercetin (FRAP
2
+
value = 7.26 and 10.79 µmole Fe /g, respectively). Moreover, compounds 4b, 4i and 4p showed
significant antioxidant activity.
3
.1.4. Protective effect against H O -induced cell death in PC12 neurons
2 2
The neuroprotective activity of the compound 4r against oxidative stress-induced cell death in
differentiated PC12 cells was evaluated. The obtained data are shown in Fig. 3 in which the cell
viability was determined in comparison with H O -treated group. The neuroprotective activity of
2
2
the compound was evaluated at the concentrations of 1, 5, 10 µM. Moreover, quercetin was used
as reference compound at the concentration of 5 µM. Compound 4r exhibited no cytotoxic
activity towards PC12 cells at the tested concentrations. Based on the results, H O significantly
2
2
reduced the cell viability to ~49% compared to the control. Pretreatment of PC12 cells with the
compound 4r significantly protected neurons against cell death in all used concentrations (P
value <0.001).
As expected for standard agent quercetin, the observed results from both FRAP assay (Table 2)
and oxidative stress-induced cell death test in differentiated PC12 cells (Fig 3) revealed the high
potential of quercetin as neuroprotective and antioxidant agent. In contrast, the selected
compound 4r which showed good protection against oxidative stress-induced cell death in
differentiated PC12 cells was not an effective compound in FRAP assay. These results
demonstrated that the protection mechanism of compound 4r maybe different from that of
quercetin.
3
.2. Ligand-protein docking simulation
A molecular modeling study was performed to gain insight into the binding mode of target
compounds to the AChE enzyme. For this purpose, all tested compounds 4a-s were docked into
the active site of the enzyme using Autodock Vina program and the best docked poses in terms
of the free energy of binding were further analyzed to clarify interactions between ligands and
the target enzyme. The results showed that all compounds were similarly oriented in the active
site. In the case of the most active compound 4r (Fig. 4), the ligand was well accommodated in
6

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the gorge of AChE active site so that the benzylpiperidin moiety was leaning toward the catalytic
anionic subsite (CAS). Specifically, phenyl ring stacks against the Trp83 via T-shape (edge-to-
face) π-π interaction and the piperidine ring that is located at the vicinity of the Phe329 and
Phe330, formed a π-cation interaction. In this binding mode, the coumarin scaffold formed π-
stacking with the aromatic ring of Trp278 in the PAS and its carbonyl group was hydrogen-
bonded to Arg288. This binding mode is in agreement with mixed-mode inhibition pattern of 4r,
in which both CAS and PAS are occupied by the ligand.
4
. Conclusion
We designed and synthesized 7-hydroxycoumarin derivatives connected by an amidic linker to
the different amines such as (substituted)phenylpiperazine, 4-benzylpiperidine, 1-
benzylpiperidin-4-yl-amino-, 4-(substituted phenyl)-4-hydroxypiperidine. Most compounds
showed remarkable inhibitory activity against AChE and BuChE. Among them, N-(1-
benzylpiperidin-4-yl)acetamide derivative 4r with IC50 value of 1.6 µM was the most potent
compound against AChE. The FRAP assay of synthesized compounds 4a-s showed that some
compounds had good antioxidant activity. Although the selected compound 4r showed no
significant antioxidant activity as evaluated by FRAP assay, but could significantly protect
neurons against H O -induced cell death at low concentrations. The Kinetic study of AChE
2
2
inhibition indicated a mixed-type inhibition of the enzyme by compound 4r. The docking study
of compound 4r with AChE enzyme showed that both CAS and PAS are occupied by the ligand.
These results make the prototype compounds 4 as promising cholinesterase inhibitors for further
developments.
5
. Experimental
All starting materials, solvents and reagents were purchased from Sigma–Aldrich, Fluka, or
Merck, and used without further purification. Melting points were measured on a Kofler hot
stage apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR Magna 550
1
spectrometer (KBr disks). H NMR spectra were recorded on a Bruker 500 MHz NMR
instrument. The chemical shifts (δ) and coupling constants (J) are expressed in parts per million
(ppm) and Hertz (Hz), respectively. Mass spectra were obtained by using an HP (Agilent
technologies) 5937 Mass Selective Detector. Elemental analyses were carried out with a Perkin-
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Elmer model 240-C apparatus. The results of elemental analyses (C, H, N) were within ± 0.4%
of the calculated values.
5
.1. General procedure for the synthesis of compounds 3a-c
A mixture of 7-hydroxycoumarin (1a) or 4-methyl-7-hydroxycoumarin (1b) (5.0 mmol) and
potassium carbonate (5.5 mmol) in acetone (5 ml) was stirred at room temperature for several
minutes. Then, ethyl 2-bromoacetate or ethyl 4-bromobutanoate (5.2 mmol) was added dropwise
to the mixture under argon atmosphere and the mixture was refluxed for 7-9 h. After completion
of the reaction (monitored by TLC), the mixture was cooled to room temperature and diluted
with water. The precipitated product 2 was collected by filtration and washed with water, and
used without further purification. The crude product 2a-c was dissolved in aqueous 10% NaOH
(25 ml) and the solution was refluxed for 4 h. The reaction mixture was cooled and acidified with
aqueous 6% HCl. The precipitated white solid was filtered off and subsequently washed with
water to give compound 3.
5
.1.1. 2-((2-Oxo-2H-chromen-7-yl)oxy)acetic acid (3a)
-1
Yield 96%; white crystal; mp 180-182 ºC; IR (KBr, cm ) νmax: 1746 and 1623 (C=O), 3362
1
(
OH); H NMR (CDCl , 500 MHz,) δ 3.91 (s, 2H, CH O), 6.11 (d, 1H, H coumarin, J = 9.4
3
2
3
Hz), 6.82-6.84 (m, 2H, H6,8 coumarin), 7.27 (d, 1H, H
5 4
coumarin, J = 8.3 Hz,), 7.52 (d, 1H, H
coumarin, J = 9.4 Hz). Anal. Calcd for C H O : C, 60.00; H, 3.66. Found: C, 60.32; H, 3.40.
1
1
8
5
5
.1.2. 4-(2-Oxo-2H-chromen-7-yloxy)butanoic acid (3b)
-1
Yield 98%; white crystal; mp 182-184 ºC; IR (KBr, cm ) νmax: 1725 and 1679 (C=O), 3083
1
(
OH); H NMR (DMSO-d
6
, 500 MHz) δ 1.93-1.99 (m, 2H, CH
2
CH
2
CH
2
), 2.39 (t, 2H, CH
2
C=O,
J = 7.3 Hz), 4.09 (t, 2H, OCH , J = 6.4 Hz), 6.28 (d, 1H, H coumarin, J = 9.5 Hz), 6.94 (dd, 1H,
2
3
H
6
coumarin, J = 2.5 and 8.6 Hz), 6.97 (d, 1H, H
8
coumarin, J = 2.5 Hz), 7.61 (d, 1H, H
5
coumarin, J = 8.6 Hz), 7.98 (d, 1H, , H coumarin, J = 9.5 Hz). Anal. Calcd for C H O : C,
4
13 12
5
6
2.90; H, 4.87. Found: C, 63.24; H, 4.52.
5
.1.3. 4-(4-Methyl-2-oxo-2H-chromen-7-yloxy)butanoic acid (3c)
8

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-1
Yield 97%; white crystal; mp 168-170 ºC; IR (KBr, cm ) νmax: 1736 and 1684 (C=O), 3457
1
(
OH); H NMR (CDCl 500 MHz) δ 2.16-2.20 (m, 2H, CH CH CH ), 2.41 (s, 3H, CH ), 2.62 (t,
3
,
2
2
2
3
2
1
H, CH C=O, J = 6.9 Hz), 4.10 (t, 2H, OCH , J = 5.7 Hz), 6.15 (s, 1H, H coumarin), 6.81 (s,
2
2
3
H, H
8
coumarin), 6.86 (d, 1H, H
6
coumarin, J = 8.6 Hz), 7.50 (d, 1H, H
5
coumarin, J = 8.6 Hz).
Anal. Calcd for C H O : C, 64.12; H, 5.38. Found: C, 64.37; H, 5.64.
1
4
14
5
5
.2. General procedure for the synthesis of compounds 4a-s
Compound 3 (1.0 mmol), EDC (1.0 mmol) and HBT (1.0 mmol) were dispersed in dry
acetonitrile (5 ml). The mixture was stirred at room temperature for 1 h and then appropriate
amine (1.0 mmol) was added to the mixture. The mixture was stirred at room temperature for 24
h. After completion of the reaction (monitored by TLC), the solvent was evaporated and the
residue was washed with saturated sodium carbonate. The resulting crude product was purified
by crystallization from ethanol.
5
.2.1. 7-(4-Oxo-4-(4-phenylpiperazin-1-yl)butoxy)-2H-chromen-2-one (4a)
-1
1
Yield 87%, yellow crystal; mp 128-130 ºC. IR (KBr, cm ) νmax: 1721 and 1647 (C=O); H NMR
(
CDCl , 500 MHz) δ 2.19-2.23 (m, 2H, CH CH CH ), 2.59 (t, 2H, CH C=O, J = 6.7 Hz), 3.16-
3 2 2 2 2
3
.18 (m, 4H, 2CH N), 3.65-3.81 (m, 4H, 2CH N), 4.12 (t, 2H, OCH J = 5.4 Hz), 6.24 (d, 1H,
2
2
2,
H
3
coumarin, J = 9.3 Hz), 6.82-6.86 (m, 2H, H
8
coumarin and H phenyl), 6.90-6.93 (m, 3H, H
6
coumarin and 2H phenyl), 7.28 (t, 2H, 2H phenyl, J = 7.3 Hz), 7.37 (d, 1H, H coumarin, J = 8.4
5
1
3
Hz), 7.63 (d, 1H, H
4
coumarin, J = 9.3 Hz). C NMR (CDCl
4.8, 48.9, 49.2, 67.2, 101.1, 112.0, 112.6, 116.1, 120.1, 128.3, 128.7, 142.9, 150.3, 155.3, 160.7,
61.6, 169.9. Anal. Calcd for C H N O : C, 70.39; H, 6.16; N, 7.14. Found: C, 70.76; H, 6.49;
3
, 125 MHz) δ 24.0, 28.6, 41.0,
4
1
2
3
24
2
4
N, 7.48.
5
.2.2. 7-(4-(4-(2-Hydroxyphenyl)piperazin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4b)
-1
Yield 89%; yellow crystal; mp 151-153 ºC; IR (KBr, cm ) νmax: 1731 and 1624 (C=O), 3296
1
(
OH); H NMR (CDCl , 500 MHz) δ 2.20-2.24 (m, 2H, CH CH CH ), 2.61 (t, 2H, CH C=O, J =
3
2
2
2
2
6
6
.9 Hz), 2.85-2.87 (m, 4H, 2CH
2
2
N), 3.67-3.81 (m, 4H, 2CH N), 4.13 (t, 2H, OCH2, J = 5.6 Hz),
.26 (d, 1H, H coumarin, J = 9.4 Hz), 6.83-6.91 (m, 4H, OH and 3H phenyl), 6.97 (d, 1H, H
3
5
phenyl, J = 7.8 Hz), 7.07-7.13 (m, 2H, H6,8 coumarin), 7.38 (d, 1H, H coumarin, J = 8.4 Hz),
9

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1
3
7
5
1
6
4 3
.64 (d, 1H, H coumarin, J = 9.4 Hz). C NMR (CDCl , 125 MHz) δ 24.0, 28.6, 41.7, 45.5,
1.8, 52.2, 67.1, 101.1, 112.1, 112.6, 113.9, 119.7, 120.8, 126.4, 128.3, 137.6, 142.8, 150.7,
55.3, 160.7, 161.6, 170.0. Anal. Calcd for C H N O : C, 67.63; H, 5.92; N, 6.86. Found: C,
2
3
24
2
5
7.30; H, 6.30; N, 7.18.
5
.2.3. 7-(4-(4-(2-Fluorophenyl)piperazin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4c)
-1
1
Yield 85%; white crystal; mp 111-113 ºC; IR (KBr, cm ) ν : 1723 and 1642 (C=O); H NMR
max
(
CDCl , 500 MHz) δ 2.19-2.23 (m, 2H, CH CH CH ), 2.59 (t, 2H, CH C=O, J = 6.6 Hz), 3.05-
3
2
2
2
2
3
.07 (m, 4H, 2CH
2
2
N), 3.66-3.82 (m, 4H, 2CH N), 4.12 (t, 2H, OCH2, J = 5.3 Hz), 6.25 (d, 1H,
H coumarin, J = 9.3 Hz), 6.83 (s, 1H, H phenyl), 6.85 (d, 1H, H coumarin, J =8.5 Hz), 6.90-
3
6
6
.93 (m, 1H, H phenyl), 6.94-6.99 (m, 1H, H phenyl), 7.03-7.07 (m, 2H, H
8
coumarin and H
1
3
phenyl), 7.37 (d, 1H, H coumarin, J = 8.5 Hz), 7.63 (d, 1H, H coumarin, J = 9.3 Hz). C NMR
5
4
(
CDCl , 125 MHz) δ 24.0, 28.6, 41.2, 45.0, 49.8, 50.4, 67.2, 101.1, 112.1, 112.6, 115.6, 115.8,
3
1
18.6, 122.6, 122.7, 124.0, 128.3, 142.9, 154.2, 155.3, 160.7, 161.6, 169.9. Anal. Calcd for
C H FN O : C, 67.31; H, 5.65; N, 6.83. Found: C, 67.68; H, 5.89; N, 6.37.
23
23
2
4
5
.2.4. 7-(4-(4-(4-Fluorophenyl)piperazin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4d)
-1
1
Yield 90%; yellow crystal; mp 138-140 ºC; IR (KBr, cm ) ν : 1721 and 1652 (C=O); H NMR
max
(
CDCl
3
, 500 MHz) δ 2.19-2.23 (m, 2H, CH
2
CH
2
CH
2
2
), 2.59 (t, 2H, CH C=O, J = 6.8 Hz), 3.06-
3
.08 (m, 4H, 2CH N), 3.65-3.79 (m, 4H, 2CH N), 4.12 (t, 2H, OCH J = 5.5 Hz), 6.25 (d, 1H,
2
2
2,
3
H coumarin, J = 9.4 Hz), 6.82-6.91 (m, 4H, H6,8 coumarin and 2H phenyl), 6.97 (m, 2H, 2H
1
3
phenyl), 7.37 (d, 1H, H coumarin, J = 8.4 Hz), 7.63 (d, 1H, H coumarin, J = 9.4 Hz). C NMR
5
4
(
CDCl , 125 MHz) δ 24.0, 28.6, 41.1, 44.9, 49.9, 50.2, 67.2, 101.1, 112.0, 112.6, 115.1, 115.3,
3
1
18.0, 118.1, 128.3, 142.8, 147.0, 155.3, 156.1, 158.0, 160.6, 161.6, 169.9. Anal. Calcd for
C H FN O : C, 67.31; H, 5.65; N, 6.83. Found: C, 67.70; H, 5.84; N, 6.40.
23
23
2
4
5
.2.5. 7-(4-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4e)
-1
1
Yield 95%; white crystal; mp 139-141 ºC; IR (KBr, cm ) ν : 1724 and 1650 (C=O); H NMR
max
(
CDCl
3
, 500 MHz) δ 2.19-2.23 (m, 2H, CH
2
CH
2
CH
2
2
), 2.59 (t, 2H, CH C=O, J = 6.6 Hz), 3.13-
3
.15 (m, 4H, 2CH N), 3.64-3.79 (m, 4H, 2CH N), 4.12 (t, 2H, OCH J = 5.3 Hz), 6.25 (d, 1H,
2
2
2,
3
H coumarin, J = 9.3 Hz), 6.74 (d, 1H, H phenyl, J = 8.0 Hz), 6.82 (s, 1H, H phenyl), 6.84 (d, 1H,
10

ACCEPTED MANUSCRIPT
H
6
coumarin, J = 8.6 Hz), 6.94 (s, 1H, H
8
coumarin), 7.29 (d, 1H, H phenyl, J = 8.0 Hz), 7.37 (d,
1
3
1
H, H coumarin, J = 8.6 Hz), 7.63 (d, 1H, H coumarin, J = 9.3 Hz). C NMR (CDCl , 125
5
4
3
MHz) δ 24.0, 28.6, 40.7, 44.5, 48.4, 48.5, 67.1, 101.1, 112.0, 112.6, 115.3, 117.3, 122.6, 128.3,
1
30.1, 132.4, 142.8, 149.7, 155.3, 160.6, 161.5, 169.9. Anal. Calcd for C23
H
22Cl
2
N
2
O
4
: C, 59.88;
H, 4.81; N, 6.07. Found: C, 60.21; H, 5.19; N, 6.35.
5
.2.6. 7-(4-(4-Benzylpiperidin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4f)
-1
1
Yield 85%; yellow crystal; mp 102-104 ºC; IR (KBr, cm ) ν : 1720 and 1630 (C=O); H NMR
max
(
CDCl
CHCH N), 2.15-2.19 (m, 2H, CH CH CH ), 2.51-2.57 (m, 5H, CH C=O, CHN and CH
2
3 2
, 500 MHz) δ 1.12-1.16 (m, 2H, 2CHCH N), 1.67-1.77 (m, 3H, CH piperidine and
2
2
2
2
2
2
benzyl), 2.91-2.98 (m, 2H, 2CHN), 3.85-3.87 (m, 1H, CHN), 4.10 (t, 2H, OCH2, J = 5.6 Hz),
.61-4.64 (m, 1H, CHN), 6.25 (d, 1H, H coumarin, J = 9.3 Hz), 6.82 (s, 1H, H coumarin), 6.85
4
3
8
(
d, 1H, H coumarin, J = 8.4 Hz), 7.13 (d, 1H, H phenyl, J = 7.0 Hz), 7.22 (t, 1H, H phenyl, J =
6
7
.0 Hz), 7.26-7.31 (m, 2H, H phenyl), 7.37 (d, 1H, H
5
coumarin, J = 8.4 Hz), 7.64 (d, 1H, H
4
1
3
coumarin, J = 9.3 Hz). C NMR (CDCl , 125 MHz) δ 24.1, 28.7, 31.3, 32.0, 37.7, 41.5, 42.4,
3
4
5.2, 67.3, 101.1, 112.0, 112.1, 112.5, 125.5, 127.8, 128.2, 128.5, 139.3, 142.9, 155.3, 160.7,
61.6, 169.6. Anal. Calcd for C H NO : C, 74.05; H, 6.71; N, 3.45. Found: C, 74.36; H, 6.42;
1
2
5
27
4
N, 3.78.
5
.2.7. 7-(4-(4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl)-4-oxobutoxy)-2H-chromen-2-one (4g)
-1
Yield 88%; white solid; mp 149-151 ºC; IR (KBr, cm ) νmax: 1725 and 1643 (C=O), 3450 (OH);
1
H NMR (CDCl , 500 MHz) δ 1.54-1.70 (m, 4H, 2CH ), 1.96-2.01 (m, 2H, CH CH CH ), 2.39
3
2
2
2
2
(
t, 2H, CH C=O, J = 6.5 Hz), 2.87-2.93 (m, 1H, CHN), 3.36-3.60 (m, 2H, CH N), 3.92 (t, 2H,
2
2
OCH2, J = 5.5 Hz), 4.32-4.36 (m, 1H, CHN), 4.48 (s, 1H, OH), 6.03 (d, 1H, H
3
coumarin, J = 9.3
Hz), 6.62 (s, 1H, H coumarin), 6.67 (d, 1H, H coumarin, J = 8.3 Hz), 7.15 (d, 2H, H phenyl, J =
8
6
8
.2 Hz), 7.21-7.27 (m, 3H, 2H phenyl and H
5
4
coumarin), 7.49 (d, 1H, H coumarin, J = 9.3 Hz).
1
3
C NMR (CDCl , 125 MHz) δ 24.0, 28.4, 36.9, 37.2, 37.9, 41.1, 67.1, 69.7, 100.8, 111.8, 112.0,
3
1
12.2, 119.9, 126.0, 128.3, 130.4, 143.0, 147.2, 155.1, 160.5, 161.5, 169.5. Anal. Calcd for
24 5
C H24BrNO : C, 59.27; H, 4.97; N, 2.88. Found: C, 59.58; H, 4.62; N, 3.17.
5
.2.8. 4-Methyl-7-(4-oxo-4-(4-phenylpiperazin-1-yl)butoxy)-2H-chromen-2-one (4h)
11

ACCEPTED MANUSCRIPT
-1
1
Yield 89%; white crystal; mp 143-145 ºC; IR (KBr, cm ) νmax: 1715 and 1644 (C=O); H NMR
(
CDCl , 500 MHz) δ 2.19-2.23 (m, 2H, CH CH CH ), 2.40 (s, 3H, CH ), 2.60 (t, 2H, CH C=O, J
3 2 2 2 3 2
=
6.8 Hz), 3.15-3.17 (m, 4H, 2CH N), 3.66-380 (m, 4H, 2CH N), 4.12 (t, 2H, OCH J = 5.6 Hz),
2
2
2,
6
3
.14 (s, 1H, H
3
8
coumarin), 6.83 (s, 1H, H coumarin), 6.86-6.90 (m, 1H, H phenyl), 6.90-6.93 (m,
H, H coumarin and 2H phenyl), 7.28 (t, 2H, 2H phenyl, J = 7.3 Hz), 7.50 (d, 1H, H coumarin,
6
5
1
3
J = 8.7 Hz). C NMR (CDCl
3
, 125 MHz) δ 18.1, 24.0, 28.6, 41.0, 44.8, 48.9, 49.2, 67.1, 101.1,
1
11.5, 111.7, 113.1, 116.1, 120.1, 125.0, 128.7, 150.3, 152.0, 154.7, 160.8, 161.4, 169.9. Anal.
Calcd for C H N O : C, 70.92; H, 6.45; N, 6.89. Found: C, 70.56; H, 6.18; N, 7.27.
2
4
26
2
4
5
.2.9. 7-(4-(4-(2-Hydroxyphenyl)piperazin-1-yl)-4-oxobutoxy)-4-methyl-2H-chromen-2-one (4i)
-1
Yield 91%; white crystal; mp 158-160ºC; IR (KBr, cm ) νmax: 1725 and 1623 (C=O), 3326
1
(
OH); H NMR (CDCl , 500 MHz) δ 2.20-2.24 (m, 2H, CH CH CH ), 2.40 (s, 3H, CH ), 2.61 (t,
3
2
2
2
3
2
H, CH C=O, J = 6.9 Hz), 2.85-2.87 (m, 4H, 2CH N), 3.67-3.81 (m, 4H, 2CH N), 4.13 (t, 2H,
2 2 2
OCH2, J = 5.6 Hz), 6.14 (s, 1H, H
3
coumarin), 6.83-6.91 (m, 4H, OH and 3H phenyl), 6.97 (d,
1
H, H phenyl, J =7.8 Hz), 7.07-7.13 (m, 2H, H6,8 coumarin), 7.50 (d, 1H, H coumarin, J = 8.7
5
1
3
Hz). C NMR (CDCl
11.7, 113.1, 113.9, 119.7, 120.8, 125.0, 126.4, 137.6, 142.8, 150.7, 152.0, 160.7, 161.4, 170.0.
Anal. Calcd for C H N O : C, 68.23; H, 6.20; N, 6.63. Found: C, 67.95; H, 6.58; N, 6.29.
3
, 125 MHz) δ 18.1, 24.0, 28.6, 41.8, 45.5, 51.8, 52.2, 67.1, 101.1, 111.5,
1
2
4
26
2
5
5
.2.10. 7-(4-(4-(2-Fluorophenyl)piperazin-1-yl)-4-oxobutoxy)-4-methyl-2H-chromen-2-one (4j)
-1
1
Yield 90%; white crystal; mp 123-125ºC; IR (KBr, cm ) νmax: 1717 and 1654 (C=O); H NMR
(
DMSO-d , 500 MHz) δ 1.97-2.00 (m, 2H, CH CH CH ), 2.36 (d, 3H, CH J = 1.0 Hz), 2.52 (t,
6 2 2 2 3,
2
H, CH C=O, J = 7.2 Hz), 2.91-2.97 (m, 4H, 2CH N), 3.58-3.62 (m, 4H, 2CH N), 4.10 (t, 2H,
2 2 2
3
OCH2, J = 6.4 Hz), 6.17 (d, 1H, H coumarin, J = 1.0 Hz), 6.92-7.00 (m, 4H, H6,8 coumarin and
1
3
2
H phenyl), 7.06-7.15 (m, 2H, 2H phenyl), 7.64 (d, 1H, H coumarin, J = 9.0 Hz). C NMR
5
(
CDCl
3
, 125 MHz) δ 18.0, 24.1, 28.3, 41.0, 44.8, 50.0, 50.4, 67.6, 101.1, 111.0, 112.3, 113.0,
1
15.8, 116.0, 119.4, 122.7, 124.7, 126.3, 139.5, 153.3, 153.9, 154.7, 155.9, 160.1, 161.5, 170.0.
Anal. Calcd for C H FN O : C, 67.91; H, 5.94; N, 6.60. Found: C, 67.68; H, 5.83; N, 6.35.
2
4
25
2
4
5
.2.11. 7-(4-(4-(4-Fluorophenyl)piperazin-1-yl)-4-oxobutoxy)-4-methyl-2H-chromen-2-one (4k)
12

ACCEPTED MANUSCRIPT
-1
1
Yield 92%; yellow crystal; mp 162-164 ºC; IR (KBr, cm ) νmax: 1716 and 1653 (C=O); H NMR
(
CDCl , 500 MHz) δ 2.19-2.23 (m, 2H, CH CH CH ), 2.39 (s, 3H, CH ), 2.59 (t, 2H, CH C=O, J
3 2 2 2 3 2
=
6.6 Hz), 3.06-3.08 (m, 4H, CH N), 3.65-3.79 (m, 4H, 2CH N), 4.12 (t, 2H, OCH J = 5.3 Hz),
2
2
2,
6
.13 (s, 1H, H
3
coumarin), 6.81 (s, 1H, H
8
coumarin), 6.86-6.88 (m, 3H, H
6
coumarin and 2H
1
3
phenyl), 6.95-6.99 (m, 2H, 2H phenyl), 7.49 (d, 1H, H coumarin, J = 8.6 Hz). C NMR (CDCl ,
5
3
1
1
25 MHz) δ 18.1, 24.0, 28.6, 41.1, 44.9, 49.9, 50.2, 67.1, 101.1, 111.4, 111.7, 113.1, 115.2,
15.3, 118.0, 118.1, 125.0, 147.0, 152.0, 154.7, 156.1, 158.0, 160.7, 161.4, 169.9. Anal. Calcd
for C H FN O : C, 67.91; H, 5.94; N, 6.60. Found: C, 67.54; H, 6.29; N, 6.28.
2
4
25
2
4
5
.2.12. 7-(4-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-4-oxobutoxy)-4-methyl-2H-chromen-2-one
4l)
Yield 94%; white crystal; mp 143-145 ºC; IR (KBr, cm ) ν : 1711 and 1649 (C=O); H NMR
(
-1
1
max
(
CDCl , 500 MHz) δ 2.19-2.23 (m, 2H, CH CH CH ), 2.40 (s, 3H, CH ), 2.59 (t, 2H, CH C=O, J
3
2
2
2
3
2
=
2 2
6.8 Hz), 3.13-3.15 (m, 4H, 2CH N), 3.65-3.79 (m, 4H, 2CH N), 4.12 (t, 2H, OCH2, J = 5.5
Hz), 6.14 (s, 1H, H coumarin), 6.74 (d, 1H, H phenyl, J = 8.0 Hz), 6.82 (s, 1H, H phenyl), 6.86
3
(
d, 1H, H
6
coumarin, J = 8.6 Hz), 6.94 (s, 1H, H
8
coumarin), 7.30 (d, 1H, H phenyl, J = 8.0 Hz),
1
3
7
4
1
.50 (d, 1H, H coumarin, J = 8.6 Hz). C NMR (CDCl , 125 MHz) δ 18.1, 24.0, 28.6, 40.7,
5
3
4.5, 48.4, 48.5, 67.0, 101.1, 111.5, 111.7, 113.1, 115.3, 117.3, 122.7, 125.0, 130.1, 132.4, 149.7,
51.9, 154.7, 160.7, 161.3, 170.0. Anal. Calcd for C24 : C, 60.64; H, 5.09; N, 5.89.
2 2 4
H24Cl N O
Found: C, 60.31; H, 5.35; N, 6.29.
5
2
.2.13. 7-(4-(4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl)-4-oxobutoxy)-4-methyl-2H-chromen-
-one (4m)
-1
Yield 88%; white solid; mp 149-151 ºC; IR (KBr, cm ) νmax: 1704 and 1638 (C=O), 3457 (OH);
1
H NMR (DMSO-d , 500 MHz) δ 1.56-1.84 (m, 4H, 2CH ), 1.96-2.01 (m, 2H, CH CH CH ),
6
2
2
2
2
2
.39 (s, 3H, CH
3
2
), 2.53-2.57 (m, 2H, CH CO), 2.89-2.95 (m, 1H, CHN), 3.38-3.41 (m, 1H,
CHN), 3.77-3.80 (m, 1H, CHN), 4.11 (t, 2H, OCH J = 5.5 Hz), 4.34-4.38 (m, 1H, CHN), 5.25
2
,
(
s, 1H, OH), 6.21 (s, 1H, H coumarin), 6.95-6.99 (m, 2H, H coumarin), 7.39 (d, 2H, H phenyl,
3 6,8
1
3
J = 7.9 Hz), 7.47 (d, 2H, H phenyl, J = 7.8 Hz), 7.67 (d, 1H, H
5
coumarin, J = 8.5 Hz). C NMR
(DMSO, 125 MHz) δ 18.1, 24.2, 28.2, 37.3, 37.9, 41.2, 67.9, 69.9, 101.1, 111.0, 112.3, 113.0,
13

ACCEPTED MANUSCRIPT
1
19.4, 126.4, 127.1, 130.6, 148.7, 153.3, 154.7, 160.1, 161.6, 169.6. Anal. Calcd for
C H BrNO : C, 60.01; H, 5.24; N, 2.80. Found: C, 59.78; H, 5.61; N, 2.44.
25
26
5
5
.2.14. 7-(2-Oxo-2-(4-phenylpiperazin-1-yl)ethoxy)-2H-chromen-2-one (4n)
-1
1
Yield 90%; white solid; mp 178-179 ºC; IR (KBr, cm ) ν : 1728 and 1651 (C=O); H NMR
max
3 2 2
(CDCl , 500 MHz,) δ 3.16-3.20 (m, 4H, 2CH N), 3.70-3.80 (m, 4H, 2CH N), 4.82 (s, 2H,
CH O), 6.29 (d, 1H, H coumarin, J= 9.3 Hz), 6.85-6.87 (m, 1H, H phenyl), 6.91-6.94 (m, 4H,
2
3
H6,8 coumarin and 2H phenyl), 7.27-7.30 (t, 2H, 2H phenyl, J= 7.4 Hz), 7.39 (d, 1H, H5
1
3
coumarin, J = 8.3 Hz,), 7.63 (d, 1H, H
4
coumarin, J= 9.3 Hz). C NMR (CDCl
3
, 125 MHz) δ
4
1
6
1.5, 44.6, 48.8, 49.3, 66.8, 95.3, 101.5, 112.0, 112.8, 113.2, 116.2, 120.2, 128.5, 128.7, 142.7,
50.2, 155.1, 160.4, 164.8. Anal. Calcd for C21
8.94; H, 5.81; N, 7.83.
20 2 4
H N O : C, 69.22; H, 5.53; N, 7.69. Found: C,
5
.2.15. 7-(2-(4-(2-Fluorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (4o)
-1
1
Yield 85%; white crystal; mp 198-199 ºC; IR (KBr, cm ) ν : 1725 and 1651(C=O); H NMR
max
(
CDCl
3
, 80 MHz) δ 2.81-3.23 (m, 4H, 2CH
2
N), 3.42-3.81 (m, 4H, 2CH
2
2
N), 5.02 (s, 2H, OCH ),
6
.33 (d, 1H, H coumarin, J = 9.4 Hz), 6.89-7.14 (m, 6H, 4H phenyl and H coumarin), 7.65 (d,
3 6,8
1
H, H coumarin, J = 9.0 Hz), 7.93 (d, 1H, H coumarin, J = 9.4 Hz). Anal. Calcd for
5
4
C
21
H
19FN
2
O
4
: C, 65.96; H, 5.01; N, 7.33. Found: C, 66.19; H, 5.29; N, 7.17.
5
.2.16. 7-(2-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (4p)
-1
1
Yield 85%; white solid; mp 174-176 ºC; IR (KBr, cm ) ν : 1731 and 1648 (C=O); H NMR
max
(
DMSO-d , 500 MHz,) δ 3.13-3.22 (m, 4H, 2CH N), 3.60-3.61 (m, 4H, 2CH N), 5.04 (s, 2H,
6
2
2
CH
2
3
O), 6.29 (d, 1H, H coumarin, J= 9.5 Hz), 6.80-6.83 (m, 1H, H phenyl), 6.96-6.98 (m, 2H,
H coumarin and H phenyl), 7.01 (d, 1H, H coumarin, J = 2.3 Hz), 7.22-7.26 (m, 1H, H phenyl),
6
8
1
3
7
.62 (d, 1H, H
5
4
coumarin, J = 8.7 Hz,), 7.99 (d, 1H, H coumarin, J= 9.5 Hz). C NMR (DMSO-
d6, 125 MHz,) δ 41.0, 43.8, 48.2, 48.05, 65.9, 95.3, 101.4, 112.5, 112.8, 115.8, 119.3, 128.9,
1
29.2, 140.3, 144.2, 150.7, 155.1, 160.2, 161.2, 165.1. Anal. Calcd for C H C N O : C, 58.21;
21 18 l2 2 4
H, 4.19; N, 6.47. Found: C, 58.42; H, 4.09; N, 6.30.
5
.2.17. 7-(2-(4-Benzylpiperidin-1-yl)-2-oxoethoxy)-2H-chromen-2-one (4q)
14

ACCEPTED MANUSCRIPT
-1
1
Yield 87%; yellow crystal; mp 101-103 ºC. IR (KBr, cm ) νmax: 1728 and 1620(C=O); H NMR
(
CDCl , 80 MHz) δ 1.10-1.41 (m, 2H, 2CHCH N), 1.46-3.21 (m, 2H, 2CHCH N), 3.21-3.40 (m,
3
2
2
5
6
1
C
H, CH piperidine, 2CHN and CH benzyl), 3.41-3.82 (m, 2H ,2CHN) ,4.93 (m, 2H, OCH ),
2
2
3
.27 (d, 1H, H coumarin, J = 9.4 Hz), 6.86-7.46 (m, 7H, 5H phenyl and H6,8 coumarin), 7.55 (d,
H, H coumarin, J = 9.2 Hz), 7.92 (d, 1H, H coumarin, J = 9.4 Hz). Anal. Calcd for
5
4
23
H
23NO
4
: C, 73.19; H, 6.14; N, 3.71;. Found: C, 72.89; H, 6.41; N, 3.82.
5
.2.18. N-(1-Benzylpiperidin-4-yl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (4r)
-1
Yield 82%; yellow crystal; mp 142-144 ºC. IR (KBr, cm ) νmax: 1735 and 1620 (C=O), 3258
1
(
NH); H NMR (DMSO-d , 80 MHz) δ 1.06-1.83 (m, 4H, 2CH CH N), 2.75-3.19 (m, 4H,
6
2
2
2
CH
2
2 2
N), 3.31-3.49 (m, 1H, CH piperidine), 3.44 (s, 2H, CH benzyl) ,4.57 (s, 2H, OCH ), 6.32
(
d, 1H, H coumarin, J = 9.5 Hz), 6.82-7.48 (m, 7H, 5H phenyl and H coumarin), 7.55 (d, 1H,
3
6
,
8
H coumarin, J = 9.3 Hz), 8.01 (d, 1H, H coumarin, J = 9.5 Hz). Anal. Calcd for C H N : C,
5
4
23 24
2
7
0.39; H, 6.16; N, 7.14. Found: C, 70.68; H, 5.83; N, 7.39.
5
.2.19. N-(3,4-Dimethoxyphenethyl)-2-((2-oxo-2H-chromen-7-yl)oxy)acetamide (4s)
-1
Yield 84%; white crystal; mp 148-150 ºC; IR (KBr, cm ) νmax: 1734 and 1670 (C=O), 3364
1
(
NH); H NMR (DMSO-d , 500 MHz) δ 2.41-2.67 (m, 2H, CH ), 3.12-3.46 (m, 2H ,CH N) ,
6
2
2
3
1
.64-3.65 (m, 6H, OCH
3
2 3
), 4.58 (s, 2H, OCH ), 6.31 (d, 1H, H coumarin, J = 9.5 Hz), 6.69 (d,
H, H phenyl, J = 8.5 Hz), 6.79 (s, 1H, H phenyl), 6.83 (d, 1H, H phenyl, J = 8.5 Hz), 6.95-6.97
coumarin, J = 8.3 Hz), 8.00 (d, 1H, H coumarin, J = 9.5
Hz), 8.18-8.20 (m, 1H, NH). C NMR (DMSO-d , 125 MHz) δ 30.6, 34.5, 55.2, 55.4, 67.1,
(m, 2H, H6,8 coumarin), 7.64 (d, 1H, H
5
4
1
3
6
1
1
01.6, 111.7, 112.3, 112.6, 112.8, 120.3, 129.4, 131.5, 144.2, 147.1, 148.5, 155.1, 160.1, 160.7,
66.7. Anal. Calcd for C21 : C, 65.79; H, 5.52; N, 3.65. Found: C, 65.95; H, 5.22; N, 3.86.
H21NO
6
5
.3. AChE and BuChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000
unit), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum) and butyrylthiocholine
iodide (BTCh) were provided from Sigma-Aldrich. 5,5’-Dithiobis-(2-nitrobenzoic acid)
(DTNB), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium
hydroxide, sodium hydrogen carbonate, and acetylthiocholine iodide (ATCh) were purchased
15

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from Fluka. The stock solutions of the target compounds were prepared in a mixture of DMSO
(
1 ml) and ethanol (9 ml) and diluted with 0.1 M KH PO / K HPO buffer (pH = 8.0) to obtain
2 4 2 4
final concentrations. 20 µl of substrate (acetylthiocholine iodide 0.075 M) was added to the test
solution to obtain final concentration of 466 µM. All experiments were performed based on the
previously described method [18]. Spectrophotometric measurements were performed on a UV
Unico Double Beam Spectrophotometer. The same method was also taken for BuChE inhibition
assay.
5
.4. Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed based on the method reported by Benzie and Strain [16] with
slightly modification. 300 mM acetate buffer (3.1 g C NaO .3H O and 16 ml C ), pH 3.6
2
H
3
2
2
2 4 2
H O
and 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM hydrochloric acid and 20 mM
ferric chloride hexahydrate solution were mixed (FRAP solution). The temperature of the
solution was raised to 37 ºC before the assay. After that 300 µl of the compound stock solution
was added the test solution was incubated for 15 min. Then, the change of absorbance was
monitored at the 585 nm. Results are expressed in µmole ferrous/g dry mass of compounds
according to the plotted standard curve of ferrous sulfate. Ascorbic acid was used as reference
compound.
5
.5. Cell culture, differentiation, and treatment conditions for cell viability assay
Rat undifferentiated PC12 cells were cultured in RPMI 1640 media with 10% FCS containing
1
00 units/ml penicillin and 100 µg/ml streptomycin (All from GIBCO, Grand Island, NY, USA).
4
Cells were treated with trypsin and 10 cells were placed on each well of 96-well culture plates
in RPMI 1640 media. For induction of neuronal differentiation, PC12 cells were cultured in
serum-free media (RPMI 1640 media containing 100 units/ml penicillin and 100 µg/ml
streptomycin) for 2 days, thereafter the medium changed to aforementioned serum free medium
containing NGF (50 ng/ml, Sigma) and continued for 5 days until neurite outgrowth was
observed by inverted microscope [19]. Differentiated PC12 cells were incubated with different
2 2
concentrations (1, 5 and 10 µM) of the compounds for 3 h before treatment with H O (300 µM).
The occurrence of apoptosis was established after staining with DAPI, and cell viability was
measured after 24 h by using the MTT assay as reported previously [20].
16

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5
.6. Docking studies
The crystal structure of Torpedo californica acetylcholinesterase (PDB entery code 1eve) in
complex with E2020 was obtained from Protein Data Bank [21]. Then, the co-crystallized ligand
and waters were removed from the protein. For docking experiments, ligand molecule was drawn
using MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and energy
minimized to a minimum root mean standard deviation gradient of 0.100 using Chem3D Ultra.
The structures were converted to 3D by means of Openbabel 2.3.1 [22]. Afterwards, the protein
and optimized structures of ligands were converted to required pdbqt format using Autodock
Tools 1.5.4 [23]. Docking simulations were performed with Auto Dock vina 1.1.1 [24]. A box of
1
5×15×15 Å in x, y and z directions was used to cover all binding sites of the active site. The
center of box was set to x = 2.023, y = 63.295, z = 67.062 (geometrical center of co-crystallized
ligand). The exhaustiveness was set to 100 and the default settings were used for all other
parameters. At the end of docking, the best dock poses with lowest binding free energies were
further analyzed to find out possible interactions between the AChE and inhibitors. Molecular
visualizations were carried out in DS Viewer Pro (Accelrys, Inc., San Diego, CA).
Acknowledgments
This work was supported by a grant from the Research Council of Tehran University of Medical
Sciences.
References
[
[
[
[
[
[
1] Alzheimer’s Society: http://www.alzheimers.org.uk/.
2] L. Fratiglioni, B. Winblad, E. von Strauss, Physiol. Behav. 92 (2007) 98–104.
3] E. Scarpini, P. Scheltens, H. Feldman, Lancet Neurol. 2 (2003) 539–547.
4] C. Holmes, D. Wilkinson, Adv. Psychiatr. Treat. 6 (2000) 193–200.
5] P.N. Tariot, H. Federoff, J. Alzheimer Dis. Assoc. Disord. 17 (2003) 105–113.
6] M.C. Dinamarca, D. Weinstein, O. Monasterio, N.C. Inestrosa, Biochem. 50 (2011) 8127–
8
137.
[7] X.-C. He, S. Feng, Z.-F. Wang, Y. Shi, S. Zheng, Y. Xia, H. Jiang, X.-C. Tang, D. Bai,
Bioorg. Med. Chem. 15 (2007) 1394–1408.
17

ACCEPTED MANUSCRIPT
[
8] N. Mishra, D. Sasmal, Bioorg. Med. Chem. Lett. 23 (2013) 702–705.
9] A. Alvarez, R. Alarcón, C. Opazo, E.O. Campos, F.J. Muñoz, F.H. Calderón, F. Dajas, M.K.
Gentry, B.P. Doctor, F.G. De Mello, J. Neurosci. 18 (1998) 3213–3223.
10] R.A. Hansen, G. Gartlehner, A.P. Webb, L.C. Morgan, C.G. Moore, D.E. Jonas, Clin.
Interv. Aging 3 (2008) 211–225.
[
[
[
[
11] S. Rizzo, M. Bartolini, L. Ceccarini, L. Piazzi, S. Gobbi, A. Cavalli, M. Recanatini, V.
Andrisano, A. Rampa, Bioorg. Med. Chem. 18 (2010) 1749–1760.
12] a) P. Anand, B. Singh, N. Singh, Bioorg. Med. Chem. 20 (2012) 1175–1180. b) Z. Radic, P.
Taylor, J. Appl. Toxicol. 21 (2001) S13–14. c) V. Simeon-Rudolf, Z. Kovarik, Z. Radic, E.
Reiner, Chem. Biol. Interact. 119–120 (1999) 119–128.
[
13] A. Asadipour, M. Alipour, M. Jafari, M. Khoobi, S. Emami, H. Nadri, A. Sakhteman, A.
Moradi, V. Sheibani, F. Homayouni Moghadam, A. Shafiee, A. Foroumadi, Eur. J. Med.
Chem. 70 (2013) 623–630.
[
14] S.F. Razavi, M. Khoobi, H. Nadri, A. Sakhteman, A. Moradi, S. Emami, A. Foroumadi, A.
Shafiee, Eur. J. Med. Chem. 64 (2013) 252–259.
[
15] a) H. Akrami, B.F. Mirjalili, M. Khoobi, H. Nadri, A. Moradi, A. Sakhteman, S. Emami, A.
Foroumadi, A. Shafiee, Eur. J. Med. Chem. 2014, in press. DOI:
1
0.1016/j.ejmech.2014.01.017.
b) M. Khoobi, M. Alipour, A. Moradi, A. Sakhteman, H. Nadri, S.F. Razavi, M. Ghandi, A.
Foroumadi, A. Shafiee, Eur. J. Med. Chem. 68 (2013) 291–300.
c) M. Khoobi, M. Alipour, A. Sakhteman, H. Nadri, A. Moradi, M. Ghandi, S. Emami, A.
Foroumadi, A. Shafiee, Eur. J. Med. Chem. 68 (2013) 260–269.
d) M. Alipour, M. Khoobi, H. Nadri, A. Sakhteman, A. Moradi, M. Ghandi, A. Foroumadi,
A. Shafiee, Arch. Pharm. Chem. Life Sci. 346 (2013) 577–587.
e) M. Alipour, M. Khoobi, A. Foroumadi, H. Nadri, A. Moradi, A. Sakhteman, M. Ghandi,
A. Shafiee, Bioorg. Med. Chem. 20 (2012) 7214–7222.
[
[
[
16] B. Kiskhan, Y. Yagci, J. Polym. Sci. 45 (2007) 1670–1676.
17] F.F. Benzie, J.J. Strain, Methods Enzymol. 299 (1999) 15–23.
18] H. Nadri, M. Pirali-Hamedani, A. Moradi, A. Sakhteman, A. Vahidi, V. Sheibani, A.
Asadipour, N. Hosseinzadeh, M. Abdollahi, A. Shafiee, A. Foroumadi, Daru J. Pharm. Sci.
2
1 (2013) 15.
18

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[
19] S.H. Koh, S.H. Kim, H. Kwon, Y. Park, K.S. Kim, C.W. Song, J. Kim, M.H. Kim, H.J. Yu,
J.S. Henkel, H.K. Jung, Brain Res. Mol. Brain Res. 118 (2003) 72–81.
[20] D. Zsolt, A. Juhász, M. Gálfi, K. Soós, R. Papp, D. Zádori, B. Penke, Brain Res. Bull. 62
(2003) 223–229.
[
21] RCSB Protein Data Bank. http://www.rcsb.org/pdb/home/home.do, 2012.
22] N.M. O’Boyle, M. Banck, C.A. James, C. Morley, T. Vandermeersch, G.R. Hutchison, J.
Cheminform. 3 (2011) 33.
[
[23] M.F. Sanner, J. Mol. Graph. Model. 17 (1999) 57–61.
[24] O. Trott, A.J. Olson, J. Comput. Chem. 31 (2010) 455–461.
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Captions:
Figure 1. Examples of known AChE inhibitors: donepezil as indanone derivative, and
ensaculine and AP2238 as coumarin derivatives.
Figure 2. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 4r at different
concentrations of acetylthiocholine (ATCh), Right: Secondary plot of the enzyme for calculation
of steady-state inhibition constant (Ki=1.32 µM) of 4r.
2 2
Figure 3. Neuroprotective activity of compound 4r against H O -induced cell death in
differentiated PC12 cells (*** P < 0.001).
Figure 4. The schematic 2D and 3D representations (top and bottom, respectively) of compound
r, docked in the active site of AChE.
4
Scheme 1. Synthesis of compounds 4a-s. Reagents and conditions: (a) Ethyl 2-bromoacetate or ethyl
-bromobutanoate, K CO , acetone, reflux; (b) 10% NaOH (aq), reflux and then 6% HCl (aq); (c)
4
2
3
appropriate amine, EDC, HBT, CH CN.
3
20

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Table 1
The enzyme inhibitory activity (IC , µM) of the target compounds 4a-s against AChE and BuChE.
50
R1
O
R2
n O
O
O
1
2
Compound
R
R
n
AChE
BuChE
4
4
a
H
3
3
3.43 ± 0.19
7 ± 0.38
120 ± 7
b
H
H
280 ± 16
15 ± 0.85
4
c
3
6.9 ± 0.37
4
4
d
e
H
H
3
3
50 ± 2.8
75 ± 4.4
80 ± 5.1
26 ± 1.77
4
4
f
H
3
6.9 ± 0.4
387 ± 23
g
H
3
3
3
73 ± 4
180 ± 10
68 ± 3.3
184±8.3
4
4
h
i
CH
CH
3
3
20.8 ± 1.8
6.2 ± 0.18
4
j
CH
3
3
16.4 ± 0.98
29 ± 2.61
4
4
k
l
CH
CH
3
3
3
3
41.6 ± 2.3
24 ± 1.4
250 ± 15
4.38 ± 0.24
4
m
CH
3
3
34.6 ± 2.26
141 ± 5.23
21

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4
4
n
o
H
H
1
1
33 ± 1.8
50 ± 2.9
86 ± 5
34.5 ± 1.9
4
p
H
1
26.8 ± 1.5
120 ± 7
4
4
q
r
H
H
1
1
9.8 ± 0.54
46 ± 2.7
42 ± 2.4
1.6 ± 0.088
4
s
H
-
1
-
74 ± 4
163 ± 9.5
7.3 ± 0.42
Donepezil
-
0.022 ± 0.001
a
Data are expressed as Mean ± SE (three independent experiments)
22

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Table 2
The total antioxidant activity of the compounds 4a-s determined by FRAP assay
Compounds
FRAP Values
2+
(
µmole Fe /g)
4
a
1.13
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
2.82
0.15
1.37
0.78
1.61
0.03
0.07
2.01
0.10
0.08
0.05
0.06
1.94
0.07
2.01
0.05
0.06
5.43
Ascorbic acid
Quercetin
7.26
10.79
23

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O
N
H CO
3
H CO
3
Donepezil
N
CH3
N
H CO
3
N
O
CH3
O
H CO
3
CH3
H CO
O
3
H CO
O
O
3
Ensaculine
AP2238
Figure 1. Examples of known AChE inhibitors: donepezil as indanone derivative, and
ensaculine and AP2238 as coumarin derivatives.
24

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Figure 2. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 4r at different
concentrations of acetylthiocholine (ATCh), Right: Secondary plot of the enzyme for calculation
of steady-state inhibition constant (Ki=1.32 µM) of 4r.
25

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Figure 3. Neuroprotective activity of compound 4r against H O -induced cell death in
2
2
differentiated PC12 cells (*** P < 0.001).
26

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Figure 4. The schematic 2D and 3D representations (top and bottom, respectively) of compound
4
r, docked in the active site of AChE.
27