Design, synthesis, biological evaluation, and molecular modeling of coumarin-piperazine derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.201300242

Acetylcholinesterase (AChE) is an important drug target for the treatment of Alzheimer's disease. A novel series of coumarin-piperazine derivatives were synthesized and their potency to inhibit human AChE enzyme (hAChE) was studied. All the final compounds were characterized by infrared, ¹H NMR, ¹³C NMR, and elemental analysis. Docking experiments of the designed coumarin-piperazine derivatives were carried out in order to compare the theoretical and experimental binding affinities toward hAChE, to delineate the inhibitory mechanism. Subsequently, a structure-activity relationship (SAR) study using the molecular field method showed that the hydrophobic field and positive charge center conferred by the coumarin and piperazine moieties demonstrated an inhibitory mechanism. Among the compounds tested, 3f, 3j, and 3m were found to be the most potent inhibitors of hAChE. New coumarin-piperazine derivatives were synthesized and studied for their potency to inhibit the human acetylcholinesterase enzyme (hAChE). Docking experiments were carried out in order to compare the theoretical and experimental binding affinities toward hAChE. The hydrophobic field and positive charge center conferred by the coumarin and piperazine moieties demonstrated an inhibitory mechanism.

Full text of DOI:10.1002/ardp.201300242

Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
793
Full Paper
Design, Synthesis, Biological Evaluation, and Molecular
Modeling of Coumarin–Piperazine Derivatives as
Acetylcholinesterase Inhibitors
1
2
2
1
Rahul P. Modh , Sivakumar Prasanth Kumar , Yogesh T. Jasrai , and Kishor H. Chikhalia
1
Department of Chemistry, University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India
Department of Bioinformatics, Applied Botany Centre, University School of Sciences, Gujarat University,
Ahmedabad, Gujarat, India
2
Acetylcholinesterase (AChE) is an important drug target for the treatment of Alzheimer’s disease. A
novel series of coumarin–piperazine derivatives were synthesized and their potency to inhibit human
1
AChE enzyme (hAChE) was studied. All the final compounds were characterized by infrared, H NMR,
13
C NMR, and elemental analysis. Docking experiments of the designed coumarin–piperazine
derivatives were carried out in order to compare the theoretical and experimental binding affinities
toward hAChE, to delineate the inhibitory mechanism. Subsequently, a structure–activity relationship
(SAR) study using the molecular field method showed that the hydrophobic field and positive charge
center conferred by the coumarin and piperazine moieties demonstrated an inhibitory mechanism.
Among the compounds tested, 3f, 3j, and 3m were found to be the most potent inhibitors of hAChE.
Keywords: Alzheimer’s disease / Coumarin / Docking / Field-based activity model / hAChE / Piperazine
Received: June 28, 2013; Revised: August 13, 2013; Accepted: August 15, 2013
DOI 10.1002/ardp.201300242
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
activity. Drug development strategy has been efficiently
employed to inhibit this promising target by small-molecule
inhibitors that may enhance cholinergic transmission [3].
Regrettably, after many years of research in this area, several
of the approved drugs for treating these diseases only assuage
symptoms and have little effect on slowing disease progres-
sion. Numerous drug design experiments were carried out
using the crystal structures of AChE enzyme [4–6] reported in
recent years and promoted the development of potent AChE
inhibitors. Structure-based studies on hAChE protein revealed
a deep, narrow gorge containing a Ser-His-Glu catalytic triad
(Ser200-His440-Glu327) known as catalytic active site (CAS)
that localizes on the protein’s surface spanning 200 nm area.
This site is also sub-categorized into two functional sites called
anionic binding site (ABS) comprising five amino acids, viz.,
Trp86, Tyr130, Glu199, Phe330, and Tyr442 (at the middle of
the gorge) and peripheral anionic site (PAS) containing five
amino acids including Tyr70, Asp72, Tyr121, Glu278, Trp279,
Alzheimer’s disease (AD), the most common progressive and
devastating brain degenerative disease, is believed to be the
most general cause of dementia that primarily affects older
people [1]. According to the World Alzheimer Report 2012, 36
million people have been affected worldwide, and estimated
to reach 66 and 115 million by the years 2030 and 2050,
respectively. Despite the existence of several therapies on the
AD pathogenesis, existing drugs only delay the disease
progression, which poses
a major social and health
problem [2]. Biochemical studies hypothesized that the loss
of cortical cholinergic neurotransmission is associated with
the decline in cognitive mental functions of the affected
individuals wherein acetylcholinesterase (AChE), a metabolic
enzyme that breaks down ACh was found to lose its enzymatic
Correspondence: Prof. Kishor H. Chikhalia, Department of Chemistry,
School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380
0
09, Gujarat, India.
E-mail: chikhalia_kh@yahoo.com
Fax: þ91 79 26308545
Additional Correspondence: Rahul P. Modh,
E-mail: rahulmadh@gmail.com
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R. P. Modh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Figure 1. The outline of the various experimental and modeling techniques used in the study.
and Tyr334 (at the gorge’s entrance). Computational model-
ing and X-ray diffraction experiments on the AChE inhibitor
complexes showed molecules exhibit some specificity toward
interacting with any one (specific binders, e.g., rivastigmine
with CAS) or two (dual-binding inhibitors, e.g., doneprezil
with ABS and PAS) functional sites of hAChE. Different AChE
inhibitors including FDA-approved drugs that contain nitro-
gen atom acting as positive center (for example, doneprezil,
tacrine, propidium, rivastigmine, and ensaculin) have
exhibited good improvement in cognitive and memory
disorders [7]. Ensaculin is a coumarin molecule substituted
with a piperazine moiety, which showed memory-enhancing
effects in paradigms of passive and conditioned avoidance in
both normal and artificially amnesic rodents [8].
interlinked using acetamide linker with phenyl nucleus
where various chemical groups are introduced (Fig. 2).
Coumarin and its analogs have achieved huge therapeutic
significance in the field of medicinal chemistry for many years
since they display a fascinating array of pharmacological
properties [9]. Coumarin derivatives are commercialized as
We designed the coumarin–piperazine derivatives using
ensaculin as reference molecule, studied its hAChE inhibitory
potency, and validated using molecular modeling approaches
(
Fig. 1). Ensaculin has a basic coumarin and piperazine
pharmacophore that is connected adjacently and then
Figure 2. Design strategy of coumarin–piperazine derivatives.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Coumarin–Piperazine Derivatives as AChE Inhibitors
795
antibiotics (novobiocin and analogs) [10], anti-AIDS agents
depicted in Scheme 1. The main intermediate 4-(piperazin-1-
yl)-2H-chromen-2-one (1) was synthesized from 4-hydroxy
coumarin in excess piperazine at temperature 160°C [27]. The
synthesis of 2-chloro-N-(substituted aromatic)acetamides
(2a–2n) (intermediate-2) was achieved in good yield by the
reaction between appropriate aromatic amines and chlor-
oacetyl chloride in dichloromethane using triethylamine as a
base. The IR spectra of acetamides (2a–2n) revealed carbonyl
absorption and secondary N–H groups in the range of 1633–
(
calanolides) [11], anticoagulant (warfarin, phenprocou-
mon) [12], and antitumor drugs (geiparvarin) [13]. Some of
these drugs derived from 4-hydroxycoumarin scaffold have
been thoroughly explored [14]. In addition, piperazine
heterocycles are also important pharmacophore units in
drug research [15–18].
In recent times, electrochemical, membrane sensors, and
voltammetric methods have improved the drug delivery
system of neurological drugs by its efficient determina-
tion [19–22]. Amperometric biosensors with intercepted acetyl
cholinesterases are proved to be the promising tools for
evaluating many anticholinergic compounds and extended to
study available neurological drugs administered to human
subjects suffering from Alzheimer’s disease and myasthenia
gravis [23].
As part of our continuing pursuit focused to design new
leads with potent biological activities [24–26] and the
therapeutic importance of coumarin and piperazine chemical
groups/moieties, a series of coumarin–piperazine analogs
were designed based on the structural features of ensaculin.
The designed molecules were then synthesized and evaluated
for their potency to inhibit hAChE enzyme. Further,
molecular modeling studies were carried out using molecular
docking and molecular field-based QSAR modeling approaches
to understand the inhibition mechanism.
ꢀ
1
1
1693 and 3249–3324 cm , respectively. In H NMR, the NH
proton of amide type linkage appeared at d 8.24–8.74 ppm and
proton of (–CH –Cl) group appeared at d 4.18–4.26 ppm.
2
Subsequent coupling of this compounds (2a–2n) with the
piperazinyl-coumarin intermediate (1) under basic conditions
in dimethyl formamide at reflux temperature yielded the
corresponding 2-(4-(2-oxo-2H-chromen-4-yl)piperazin-1-yl)-N-
(substituted aromatic)acetamides (3a–3n), the desired target
compound. The appearance of (C–N) absorption in the range
ꢀ
1
1
1232–1272 cm was observed in IR spectra. In H NMR, there
is disappearance of (–CH –Cl) group peak whereas a signal for
Ar–H proton of coumarin ring appeared in the range of 7.70–
8.32 ppm and signal for (–CH ) group of piperazine ring in the
2
2
1
3
range of 2.5–3.5 ppm, while in C NMR, the appearance of
(C–N) peak of coumarin in the range of d 157.43–159.20 ppm
indicated the desired conversion.
Results and discussion
Chemistry
AChE inhibition evaluation
In view of the high pharmacological activity profile of
coumarin compounds, we have designed and synthesized
piperazine–coumarin derivatives via the synthetic strategy
The hAChE inhibitory potency of the synthesized coumarin–
piperazine analogs was measured using Ellman’s method to
determine its potential interest toward hAChE inhibitors [28].
Scheme 1. Synthetic pathway of 2-(4-(2-oxo-2H-chromen-4-yl)piperazin-1-yl)-N-(substituted aromatic)acetamide derivatives.
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R. P. Modh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Table 1. Anti-hAChE activities of the synthesized compounds.
electron withdrawing (–Cl) group at the meta position had an
IC50 of 21 mmol/L and the activity remarkably decreased if
shifted to ortho and para positions in 3a (310 mmol/L) and 3d
hAChE inhibition
a)
S. no.
Compound
R
(IC50 ꢁ SD) (mmol/L)
(287 mmol/L), respectively. The decrease in inhibitory activities
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
2-Cl
3-Cl
56 ꢁ 0.1
310 ꢁ 0.3
21 ꢁ 0.2
was observed when replacing phenyl ring by naphthalene
ring with –Cl at ortho position (3e ¼ 483 mmol/L).
4-Cl
287 ꢁ 0.3
483 ꢁ 0.1
8.3 ꢁ 0.04
41 ꢁ 0.3
2-Cl-C10
2-CH
3-CH
4-CH
2-NO
3-NO
4-NO
2-OCH
3-OCH
4-OCH
H
6
Computational methods
3
3
Molecular docking
3
69 ꢁ 0.03
59 ꢁ 0.2
Ensaculin is a compound of benzopyran with a piperazine
substituted moiety and being commercialized under the trade
name of KA-672 HCl [8]. We considered ensaculin as reference
molecule to design coumarin–piperazine analogs (3a–3n) and
the docked conformation of ensaculin within hAChE active
site cavity was used to understand the mode of interaction of
synthesized compounds 3a–3n. This reference-based docking
protocol made way to better understand the contribution of
various functional groups in the dataset contributing toward
catalytic activity. Docking simulations using Molegro Virtual
Docker (MVD) software revealed the binding specificity of
different functional groups of synthesized molecules (3a–3n),
viz., coumarin, piperazine, phenyl moieties/groups, and
acetamide linker interact with functional residues of PAS
site along with neighbor amino acids. We achieved a similar
docked pose of ensaculin as reported by Zhou et al. [29],
specifically interacting with functional residues of hAChE PAS
region, which indicates the reliability of the molecular
docking experiment presented in this study. Coumarin group
of ensaculin exhibits van der Waals interaction with PAS key
residues, Tyr124 and Tyr341, while the coumarin group of 3f
and 3j developed contacts with Tyr341 and Trp286. The
piperazine moieties of 3f and 3m interact with Trp286. The
electrostatic interaction was observed with coumarin group
with the following PAS important amino acids including
Trp286 (ensaculin) and Tyr341 (3j and 3m). H-bonding
specificity was noted with promising molecules (3f, 3j, and
3m) along with its reference, ensaculin making interactions
in the order: piperazine with Ser293, phenyl moiety with
Arg247, and acetamide linker with Gln369 (except ensaculin).
We expect that the contribution of aromatic features of
coumarin, piperazine, and phenyl moieties of the synthesized
molecules 3a–3n would develop P–P interaction with
aromatic residues of PAS. The coumarin group of ensaculin
and 3f showed P–P interactions with Trp286 while all the
molecule including ensaculin formed P contacts using
phenyl group with Arg296, a nearby residue localized in
PAS region. A list of hAChE PAS amino acids interacting with
different functional groups of the reported molecules are
given in Supplementary Table S1. The overlapping docked
conformations of ensaculin, along with molecules 3f, 3j, and
3m at the PAS region, is graphically shown in Fig. 3. Thus, it is
2
10
11
12
13
14
15
16
3j
3k
3l
2
5.5 ꢁ 0.02
58 ꢁ 0.1
2
3
19 ꢁ 0.05
4.9 ꢁ 0.03
90 ꢁ 0.1
3m
3n
Ensaculin
3
3
b)
0.36 ꢁ 0.01
0.11 ꢁ 0.01
b)
Doneprazil
a)
Values are expressed as the mean ꢁ standard error of the mean
of three experiments.
b)
Reference drug.
The compounds shown in bold face possess significant anti-
hAChE activity.
To allow comparison of the results, ensaculin was used as the
reference compound. The hAChE inhibitory activities of the
synthesized compounds are shown in Table 1. The data clearly
show that most of the designed compounds exhibited
moderate inhibitory activities toward hAChE enzyme. All
the synthesized compounds were found to be less potent than
the reference drugs, ensaculin and doneprazil, for hAChE
inhibition. The IC50 values differed due to the attachment of
different substituents to the phenyl ring of 3a–n compounds.
The substituted coumarin–piperazine compounds including
3
f
2-(4-(2-oxo-2H-chromen-4-yl)piperazin-1-yl)-N-o-tolyl-acet-
amide, 3j N-(3-nitrophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)piper-
azin-1-yl)acetamide, and 3m N-(3-methoxyphenyl)-2-(4-(2-oxo-
2
activities with IC50 values of 8.3, 5.5, and 4.9 mmol/L,
respectively. Among the newly synthesized analogs, the most
potent compound was 3m (4.9 mmol/L) having (–OCH ) group
3
at meta position. If (–OCH ) group was attached at ortho and
para positions, activity was further decreased as observed in 3l
and 3n with IC50 values of 19 and 90 mmol/L, respectively. The
second most potent compound against hAChE was 3f (IC50
H-chromen-4-yl)piperazin-1-yl)acetamide showed significant
3
value ¼ 8.3 mmol/L) possessing (–CH
3
) group at ortho position.
However, when (–CH ) was shifted to para and meta positions
3
the inhibitory activity was declined to 41 and 69 mmol/L in the
case of 3g and 3h. Compound 3j showed good inhibitory
potency when (–NO ) is attached at meta position and
2
decreased if shifted to ortho and para positions (3i ¼ 59
mmol/L and 3k ¼ 58 mmol/L). Compound 3c having strong
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Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Coumarin–Piperazine Derivatives as AChE Inhibitors
797
Figure 3. The binding poses of molecules 3f, 3j, 3m, and reference ensaculin in the hAChE active site (PDB 4EY7). The H bonds are shown
in dotted lines.
evident from the predicted dock poses of the designed
molecules that they exhibit similar mode of receptor binding
in comparison to the ensaculin pose.
molecules, exclusive of pivot molecule) were randomly
chosen as test set while remaining compounds participated
in training the SAR model (Fig. 4 and Table 2).
The equation of the developed SAR model is:
Molecular field-based QSAR model
Further, we performed molecular field-based QSAR model to
shed more lights over the structure–activity relationships
Predicted pIC50 ¼ 0:954 ðactual pIC50Þ þ 0:194
2
2
r ¼ 0:944; q ¼ 0:874
(
SAR) using Forge V10 (Cresset Biomolecular Discovery Ltd.). A
Graphical analysis over the field-based alignment of the
dataset (Fig. 5) yielded the following insights: The hydropho-
bicity conferred by aromatic groups in the synthesized
coumarin–piperazine molecules indicated the uniform distri-
bution of hydrophobic small fields over the entire surface
span; this property is very essential as the lining residues of
PAS established interaction with aromatic residues, which
may also additionally contribute P–P mode of interactions as
observed similarly in the docking experiments and it is
compatible with high anti-hAChE potency. The nitrogen
atoms in the piperazine group of the molecules act as positive
charge center, a molecular property observed in many potent
systematic conformational hunt was performed with the
dataset (3a–3n, ensaculin) and best conformers sorted by
conformational energies were retained. The experimental
activities (IC ) of the dataset were transformed into its
logarithmic scale, pIC50, and expressed as dependent variable.
The low-energy conformers of each molecule in the dataset
were selected by making an initial alignment with the pivot
molecule (ensaculin) and only those low-energy conformers
representing each molecule in the dataset were chosen for
final alignment whose molecular field base alignment score
was >0.5. Twenty-five percent of the molecules (four
5
0
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R. P. Modh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
interaction (supplementary Fig. S1). Further, the synthesized
compounds can be improved by introducing various chemo-
types in the phenyl ring having H bond ability so at interact
with Arg296. The topological arrangement of the coumarin
and piperazine moieties was found to be feasible while the
length of the aliphatic linker can be varied to develop potent
hAChE inhibitors.
Conclusion
A novel series of coumarin–piperazine derivatives were
designed and synthesized from 4-hydroxy coumarin in excess
piperazine, followed by substitution with various aromatic
acetamides; their structures were confirmed by spectral data
and elemental analysis. Further, we evaluated the inhibitory
potency of hAChE enzyme of synthesized compounds using
Ellman method. Varying IC50 values were observed due to the
attachment of different substituents to the phenyl ring of 3a–
n compounds. Among the compounds tested, the most potent
compounds are 3f 2-(4-(2-oxo-2H-chromen-4-yl)piperazin-1-yl)-
N-o-tolyl-acetamide, 3j N-(3-nitrophenyl)-2-(4-(2-oxo-2H-chro-
men-4-yl)piperazin-1-yl)acetamide, and 3m N-(3-methoxy-
phenyl)-2-(4-(2-oxo-2H-chromen-4-yl)piperazin-1-yl)acetamide,
which showed significant anti-hAChE activities with IC50
values of 8.3, 5.5, and 4.9 mmol/L, respectively. The objective of
this study was to understand the relationship between hAChE
inhibitory activity, docking results, and mapped molecular
field descriptors in a series of coumarin–piperazine deriva-
tives. We showed that the docked conformations of the
studied molecules were similar to that of published ensaculin
Figure 4. Scatter plot of actual versus predicted pIC50 values
obtained from the field-based QSAR equation [predicted
pIC50 ¼ 0.954 (actual pIC50) þ 0.194, r ¼ 0.944, q ¼ 0.874].
2
2
AChE inhibitors [30] which developed a cloud of positive
region at the middle of the aligned dataset conformers. In
addition, acetamide linker having H bond tendency owing to
its nitrogen and oxygen atoms can line the surface wall of
hAChE cavity. We anticipate that aromatic features of the
molecules in the required topological order, as observed in
the molecular alignment, discriminates the specificity of
various chemo types interacting with three hAChE biochemi-
cal sites, viz., CAS, ABS, and PAS, respectively. In addition, the
aromatic moieties along with the positive center specifically
interact with PAS key residues, which also signifies P–P
Table 2. Actual and predicted pIC50 values from molecular field-based QSAR model with docking energy.
Molecular field-based activity model
Molecular docking
Activity
a)
Compound
Actual pIC50
Predicted pIC50
Sim
MolDock score (kcal/mol)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
4.26
3.51
4.68
3.54
3.32
5.08
4.39
4.16
4.23
5.26
4.23
4.71
5.31
4.04
6.44
4.40
4.10
4.30
4.30
3.30
5.10
4.40
4.20
5.00
5.30
4.70
4.80
5.20
4.10
6.34
0.624
0.626
0.640
0.646
0.621
0.636
0.635
0.651
0.592
0.625
0.643
0.614
0.622
0.650
0.630
ꢀ42.9971
ꢀ32.3691
ꢀ13.5520
ꢀ63.8741
ꢀ22.0209
ꢀ9.9534
b)
b)
g
ꢀ17.9741
ꢀ59.5030
ꢀ18.5972
ꢀ63.6484
ꢀ74.4328
ꢀ7.1539
h
b)
i
j
k
l
b)
m
n
ꢀ57.5664
ꢀ45.6042
ꢀ28.7613
Ensaculin
a)
Sim, similarity score.
b)
Taken as test set to develop field-based activity model. Rest of the molecules were considered as training set.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Coumarin–Piperazine Derivatives as AChE Inhibitors
799
Figure 5. Molecular field-based alignment of
the designed molecules (3a–3n). The elec-
tronic fields are depicted in large spheres (light
gray – electropositive, dark – electronegative)
and van der Waals (light gray), and hydrophobic
fields (gray sphere enclosed in aromatic group)
are shown in small spheres.
pose. Docking experiments recognized the probable mode of
interaction developed by the designed compounds with
hAChE key residues. From molecular field-based activity
model, we conclude that aromatic features along with various
substituted groups of the molecules are essential features to
develop interaction with hAChE PAS site and are strongly
associated with the hAChE inhibitory activities.
reduced pressure, which afforded thick oil from which a
creamish solid was separated after frequent treatment with
diethyl ether and ethyl acetate. Pure compound was then
recrystallized from methanol [27]. Whitish solid, yield: 65%; m.p.
ꢀ1
2
(
90–292°C; IR (KBr, cm ): 3401 (Sec N–H), 1714 (C–O), 1487, 1557
–C–C–), 1243 (–C–N).
–
General synthetic procedure for 2-chloro-N-(substituted
aromatic)acetamides (2a–2n)
Chloroacetyl chloride (4.5 mL, 0.06 mol) was added dropwise to a
mixture of the appropriate aromatic amine (0.05 mol) and
triethyl amine (11.2 mL, 0.08 mol) in dichloromethane (25 mL)
at 0–5°C. The reaction mixture was stirred at 0–5°C for 30 min
and at room temperature (30°C) for a further 1 h. It was then
poured into crushed ice. The organic layer was separated, washed
Experimental section
Materials and methods
All solvents were dried over an appropriate drying agent and
purified by standard methods [31]. All the chemicals and reagents
used were of analytical grade from Sdfine, Sigma, and Merck
unless otherwise specified. Melting points were determined in
open capillaries on a Veego electronic apparatus VMP-D (Veego
Instrument Corporation, Mumbai, India) and are uncorrected. IR
3
successively with NaHCO solution and water, and dried over
anhydrous sodium sulfate. Evaporation under reduced pressure
gave solid compounds (2a–n). The progress of the reaction was
monitored by thin layer chromatography using toluene/acetone
ꢀ
1
spectra (KBr pellets) in cm were recorded on a “BOMMEN” IR
spectrophotometer. Analytical thin-layer chromatography was
performed on Merck precoated aluminum plates 60 F254 with a
(8:2) as solvent system [32].
2
-Chloro-N-phenyl acetamide (2a)
0
.2 mm layer of silica gel-G, and spots were visualized under UV
ꢀ1
White solid, yield: 78%; m.p. 135–138°C; IR (KBr, cm ): 3281
irradiation. NMR spectra were recorded on a 400 and 500 MHz
spectrometer (Bruker AMX 400, Bruker AV 400, or a Bruker DRX
N–H), 1680 (C–O); 1H NMR (400 MHz, DMSO-d
(
6
): d 8.56 (s, 1H,
NH), 7.66 (m, 2H), 7.40 (m, 2H), 7.14 (m, 1H), 4.22 (s, 2H, –CH –Cl).
–
2
5
00) using DMSO as a solvent and TMS (Me
H resonant frequency of 500 MHz and
C resonant frequency of 125 MHz. All H and C NMR chemical
4
Si) as an internal
1
standard, with
2
-Chloro-N-(2-chlorophenyl)acetamide (2b)
1
3
1
13
ꢀ1
Pale yellow solid, yield: 83%; m.p. 161–164°C; IR (KBr, cm ): 3275
shifts are quoted in ppm and were calibrated on solvent signals.
Multiplicities are given as s (singlet), d (doublet), dd (doublet–
doublet), q (quadruplet), t (triplet), and m (multiplet). Elemental
analyses (C, H, and N) were performed using a GmbH Vario Micro
cube Elementar Analyzer (Germany).
N–H), 1676 (C–O); 1H NMR (400 MHz, DMSO-d
(
6
): d 8.35 (s, 1H,
NH), 8.04 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.63 (dd, J ¼ 7.5, 1.5 Hz, 1H),
.31 (m, 2H), 7.13 (m, 1H), 4.21 (s, 2H, –CH –Cl).
–
7
2
2
-Chloro-N-(3-chlorophenyl)acetamide (2c)
ꢀ1
Pale yellow solid, yield: 87%; m.p. 170–174°C; IR (KBr, cm ): 3281
1
Synthetic procedure for 4-(piperazin-1-yl)-2H-chromen-2-
one (1)
A mixture of 4-hydroxy-2H-chromen-2-one (5.0 g, 0.03 mol) and
excess piperazine (17.5 g, 0.20 mol) was stirred at 160°C for 1 h,
and then the final hot solution was poured into ice water. The
resulting solution was exhaustively extracted with chloroform,
–
(
N–H), 1693 (C–O); H NMR (400 MHz, DMSO-d
–NH), 7.99 (m, 1H), 7.49 (m, 1H), 7.37 (m, 1H), 7.23 (m, 1H), 4.26
(s, 2H, –CH –Cl).
6
): d 8.46 (s, 1H,
2
2-Chloro-N-(4-chlorophenyl)acetamide (2d)
ꢀ1
Pale yellow solid, yield: 78%; m.p. 168–170°C; IR (KBr, cm ):
3292 (N–H), 1672 (C–O); 1H NMR (400 MHz, DMSO-d ): d 8.35
dried over anhydrous Na
2 4
SO , and evaporated to dryness at
6
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
00
R. P. Modh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
(
–
s, 1H, –NH), 7.63 (m, 2H), 7.45 (m, 2H), 7.19 (m, 1H), 4.24 (s, 2H,
2-Chloro-N-(4-methoxyphenyl)acetamide (2n)
ꢀ1
CH –Cl).
2
White solid, yield: 76%; m.p. 119–120°C; IR (KBr, cm ): 3311
N–H), 1668 (C–O). 1H NMR (400 MHz, DMSO-d
–
(
6
): d 8.27 (s, 1H,
NH), 7.60 (d, J ¼ 7.5 Hz, 2H), 7.44 (d, J ¼ 7.1 Hz, 2H), 4.24 (s, 2H,
2
-Chloro-N-(2-chloronaphthalen-1-yl)acetamide (2e)
ꢀ1
2 3
–CH –Cl), 3.77 (s, 3H, –OCH ).
Pale yellow solid, yield: 83%; m.p. 182–184°C; IR (KBr, cm ): 3256
1
(
N–H), 1633 (C–O). H NMR (400 MHz, DMSO-d
): d 10.01 (s, 1H,
NH), 8.07 (dd, J ¼ 7.5, 1.6 Hz, 1H), 8.02 (m, 1H), 7.54–7.53 (m, 2H),
6
Synthetic procedure for 2-(4-(2-oxo-2H-chromen-4-yl)-
piperazin-1-yl)-N-(substituted aromatic)-
acetamides (3a–3n)
To 4-(piperazin-1-yl)-2H-chromen-2-one (1) (1.0 g, 4.3 mmol) in
–
7
–
.51 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.16 (d, J ¼ 7.5 Hz, 1H), 4.26 (s, 2H,
–Cl).
CH
2
1
0 mL of dimethyl formamide (DMF), appropriate 2-chloro-N-
2
-Chloro-N-o-tolylacetamide (2f)
ꢀ
1
(substituted aromatic)acetamides (2a–n) (4.7 mmol) and potassi-
um carbonate (0.65 g, 4.7 mmol) were added and the reaction
mixture was refluxed for 10–12 h; then, after cooling to room
temperature, it was slowly poured into 100 mL of ice water and
the resulting solid was separated by filtration and washed
successively with water. The product was dried to get compounds
Off-white solid, yield: 85%; m.p. 164–166°C; IR (KBr, cm ): 3288
N–H), 1664 (C–O). 1H NMR (400 MHz, DMSO-d
(
6
): d 8.37 (s, 1H,
NH), 7.38 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.30 (dd, J ¼ 7.5, 1.6 Hz, 1H),
.24 (m, 1H), 7.07 (m, 1H), 4.21 (s, 2H, –CH –Cl), 2.12 (s, 3H,
CH ).
–
7
–
2
3
(
3a–3n). The progress of the reaction was monitored by TLC using
2
-Chloro-N-m-tolylacetamide (2g)
n-hexane/ethyl acetate (8:2) as solvent system.
ꢀ
1
Off-white solid, yield: 83%; m.p. 168–170°C; IR (KBr, cm ): 3269
N–H), 1683 (C–O). 1H NMR (400 MHz, DMSO-d
(
6
): d 8.32 (s, 1H,
NH), 7.54 (dd, J ¼ 7.5, 1.6 Hz, 1H), 7.42 (m, 1H), 7.31 (dd, J ¼ 7.5,
.6 Hz, 1H), 6.97 (m, 1H), 4.24 (s, 2H, –CH –Cl), 2.34 (s, 3H, –CH ).
2
-(4-(2-Oxo-2H-chromen-4-yl)piperazin-1-yl)-N-phenyl
–
acetamide (3a)
1
2
3
ꢀ1
Yield: 78%; m.p. 210–214°C; IR (KBr): cm 3311 (NH), 1735 (CO,
1
coumarin), 1668 (C–O), 1232 (–C–N); H NMR (500 MHz, DMSO-d
):
6
2
-Chloro-N-p-tolylacetamide (2h)
d (ppm) 10.90 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5, 1.6 Hz, 1H, ArH,
coumarin), 7.70–7.55 (m, 3H, ArH), 7.45–7.26 (m, 4H, ArH), 7.07 (t,
ꢀ1
White solid, yield: 80%; m.p. 161–163°C; IR (KBr, cm ): 3295
N–H), 1671 (C–O). 1H NMR (400 MHz, DMSO-d
(
6
): d 8.30 (s, 1H,
NH), 7.52–7.49 (m, 2H), 7.34–7.36 (m, 2H), 4.21 (s, 2H, –CH –Cl),
.19 (s, 3H, –CH ).
J ¼ 7.5 Hz, 1H, ArH), 5.02 (s, 1H, –CH, coumarin), 3.33 (s, 2H, –CH ),
2
–
2
3.24 (t, J ¼ 5.0 Hz, 4H, –CH , piperazine), 2.55 (d, J ¼ 10.2 Hz, 4H,
2
1
3
2
3
2
–CH , piperazine); C NMR (125 MHz, DMSO): d (ppm) 169.64 (1C,
–
CONH–), 169.16 (1C, –C–O, coumarin), 157.43 (1C, –C–N,
coumarin), 151.77, 130.49, 125.60, 125.28, 117.60, 115.79,
84.42 (7C, coumarin), 139.39, 129.13, 120.17, 119.08 (6C, Ar–C),
2
-Chloro-N-(2-nitrophenyl)acetamide (2i)
ꢀ
1
Yellow solid, yield: 69%; m.p. 176–180°C; IR (KBr, cm ): 3249
N–H), 1647 (C–O). 1H NMR (400 MHz, DMSO-d
59.99 (1C, –CH –CO–NH–), 52.77 (2C, piperazine), 50.24 (2C,
2
(
6
): d 8.37 (s, 1H,
NH), 8.24 (dd, J ¼ 7.5, 1.6 Hz, 1H), 8.08 (dd, J ¼ 7.5, 1.6 Hz, 1H),
.82 (m, 2H), 7.78 (m, 2H), 4.18 (s, 2H, –CH –Cl).
21 3 3
piperazine). Anal. calcd. for C21H N O (363.41) C, 69.41; H, 5.82;
N, 11.56; O, 13.21. Found: C, 69.21; H, 5.83; N, 11.53; O, 13.20.
–
7
2
N-(2-Chlorophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
2
-Chloro-N-(3-nitrophenyl)acetamide (2j)
piperazin-1-yl)acetamide (3b)
ꢀ
1
Yellow solid, yield: 73%; m.p. 181–183°C; IR (KBr, cm ): 3295
ꢀ1
Yield: 72%; m.p. 222–225°C; IR (KBr): cm 3401 (NH), 1714 (CO,
1
(
N–H), 1674 (C–O). H NMR (400 MHz, DMSO-d
1
1
6
): d 8.74 (dd, J ¼ 1.5,
.5 Hz, 1H), 8.41 (s, 1H, –NH), 8.00 (m, 2H), 7.69 (dd, J ¼ 7.5, 7.5 Hz,
H), 4.26 (s, 2H, –CH –Cl).
1
coumarin), 1681 (C–O), 1243 (–C–N), 613 (C–CI); H NMR
(
1
500 MHz, DMSO-d
.6 Hz, 1H, ArH), 7.68 (d, 2H, ArH), 7.42 (dd, J ¼ 7.5, 1.6 Hz, 1H,
6
): d (ppm) 9.90 (s, 1H, –NH), 8.29 (dd, J ¼ 7.5,
2
ArH), 7.36 (ddd, 1H, ArH, coumarin), 7.28 (dd, J ¼ 7.5, 1.6 Hz, 1H,
ArH, coumarin), 7.16 (dd, J ¼ 7.5, 1.6 Hz, 1H, ArH, coumarin), 7.12
(s, 1H, ArH, coumarin), 5.95 (s, 1H, ArH, coumarin), 5.10 (s, 2H,
–CH ), 3.34 (d, J ¼ 10.2 Hz, 4H, –CH , piperazine), 3.24 (t, J ¼ 5.0 Hz,
2
-Chloro-N-(4-nitrophenyl)acetamide (2k)
ꢀ
1
Yellow solid, yield: 75%; m.p. 185–186°C; IR (KBr, cm ): 3283
N–H), 1669 (C–O). 1H NMR (400 MHz, DMSO-d
(
6
): d 8.41 (s, 1H,
2
2
1
3
–
NH), 7.53–7.41 (m, 4H), 4.18 (s, 2H, –CH
2
–Cl).
2
4H, –CH , piperazine); C NMR (125 MHz, DMSO): d (ppm) 169.43
(
1C, –CONH–), 169.16 (1C, –C–O, coumarin), 157.43 (1C, –C–N,
2
-Chloro-N-(2-methoxyphenyl)acetamide (2l)
coumarin), 151.77, 130.49, 125.60, 125.28, 117.60, 115.79, 84.42
(7C, coumarin), 134.79, 129.97, 128.39, 127.60, 122.49 (5C, Ar–C),
ꢀ
1
White solid, yield: 76%; m.p. 124–128°C; IR (KBr, cm ):
1
3
306 (N–H), 1655 (C–O). H NMR (400 MHz, DMSO-d
s, 1H, –NH), 7.85 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.08 (dd, J ¼ 7.5, 1.5 Hz,
H), 7.05–6.99 (m, 2H), 4.22 (s, 2H, –CH –Cl), 3.83 (s, 3H,
OCH ).
6
): d 8.29
127.45 (1C, Ar–C, C–Cl), 59.99 (1C, –CH
piperazine), 50.24 (2C, piperazine). Anal. calcd. for C21
2
–CO–NH–), 52.77 (2C,
3 3
20ClN O
(
H
1
–
2
(397.85) C, 63.40; H, 5.07; N, 10.56; O, 12.06. Found: C, 63.50; H,
5.06; N, 10.53; O, 12.03.
3
2
-Chloro-N-(3-methoxyphenyl)acetamide (2m)
N-(3-Chlorophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
ꢀ
1
Off-white solid, yield: 71%; m.p. 121–122°C; IR (KBr, cm ): 3324
piperazin-1-yl)acetamide (3c)
N–H), 1676 (C–O). 1H NMR (400 MHz, DMSO-d
ꢀ1
(
6
): d 8.24 (s, 1H,
NH), 7.74 (dd, J ¼ 1.5, 1.5 Hz, 1H), 7.32 (dd, J ¼ 7.5, 7.5 Hz, 1H),
.17 (m, 2H), 6.73 (m, 2H), 4.18 (s, 2H, –CH –Cl), 3.80 (s, 3H,
OCH ).
Yield: 76%; m.p. 230–232°C; IR (KBr): cm 3393 (NH), 1742 (CO,
1
–
coumarin), 1657 (C–O), 1262 (–C–N), 633 (C–CI); H NMR
7
–
2
(500 MHz, DMSO-d ): d (ppm) 10.17 (s, 1H, –NH), 8.32 (dd,
6
3
J ¼ 7.5, 1.6 Hz, 1H, coumarin), 7.75 (m, 1H, ArH), 7.72–7.66 (m,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Coumarin–Piperazine Derivatives as AChE Inhibitors
801
1
–
H, ArH), 7.45–7.30 (m, 4H, ArH), 7.16 (m, 1H, ArH), 5.02 (s, 1H,
CH, coumarin), 3.33 (s, 2H, –CH
), 3.24 (t, J ¼ 5.0 Hz, 4H, –CH
piperazine), 2.55 (d, J ¼ 10.2 Hz, 4H, –CH
125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C, –C–O,
coumarin), 157.43 (1C, –C–N, coumarin), 151.77, 130.49, 125.60,
–CH
17.27 (1C, –CH
2
–CO–NH–), 52.77 (2C, piperazine), 50.24 (2C, piperazine),
). Anal. calcd. for C22 (377.44) C, 70.01; H,
2
2
,
3
23 3 3
H N O
1
3
2
, piperazine); C NMR
6.14; N, 11.13; O, 12.72. Found: C, 70.16; H, 6.12; N, 11.11; O, 12.8.
(
2-(4-(2-Oxo-2H-chromen-4-yl)piperazin-1-yl)-
N-m-tolylacetamide (3g)
Yield: 77%; m.p. 240–243°C; IR (KBr): cm 3394 (NH), 1759 (CO,
1
1
25.28, 117.60, 115.79, 84.42 (7C, coumarin), 138.69, 128.44,
26.61, 120.51, 119.77 (5C, Ar–C), 131.74 (1C, Ar–C, C–Cl), 59.99
ꢀ1
(1C, –CH
2
–CO–NH–), 52.77 (2C, piperazine), 50.24 (2C, piperazine).
20ClN (397.85) C, 63.40; H, 5.07; N, 10.56;
1
coumarin), 1685 (C–O), 1243 (–CH
500 MHz, DMSO-d
.6 Hz, 1H, coumarin), 7.74–7.62 (m, 2H, ArH), 7.46–7.33 (m, 3H,
ArH), 7.17 (t, J ¼ 7.5 Hz, 1H, ArH), 6.97 (m, 1H, ArH), 5.02 (s, 1H,
CH, coumarin), 3.33 (s, 2H, –CH
), 1211 (–C–N); H NMR
3
Anal. calcd. for C21
O, 12.06. Found: C, 63.25; H, 5.08; N, 10.55; O, 12.08.
H
3 3
O
(
1
6
): d (ppm) 10.17 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5,
N-(4-Chlorophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
–
2
), 3.24 (t, J ¼ 5.0 Hz, 4H, –CH
2
,
piperazin-1-yl)acetamide (3d)
piperazine), 2.55 (d, J ¼ 10.2 Hz, 4H, –CH , piperazine), 2.29 (s, 3H,
2
ꢀ1
13
Yield: 80%; m.p. 226–228°C; IR (KBr): cm 3387 (NH), 1706 (CO,
3
–CH ); C NMR (125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–),
1
coumarin), 1674 (C–O), 1222 (–C–N), 645 (C–CI); H NMR
169.16 (1C, –C–O, coumarin), 159.20 (1C, –C–N, coumarin),
151.77, 130.49, 125.60, 125.28, 117.60, 115.79, 84.42 (7C,
coumarin), 138.68, 137.60, 127.91, 127.09, 122.22, 118.59 (6C,
6
(500 MHz, DMSO-d ): d (ppm) 10.90 (s, 1H, –NH), 8.32 (dd,
J ¼ 7.5, 1.6 Hz, 1H, coumarin), 7.74–7.62 (m, 3H, ArH), 7.45–7.32
(m, 4H, ArH), 5.02 (s, 1H, –CH, coumarin), 3.33 (s, 2H, –CH
2
), 3.24
Ar–C), 59.99 (1C, –CH
(2C, piperazine), 20.78 (1C, –CH
(377.44) C, 70.01; H, 6.14; N, 11.13; O, 12.72. Found: C, 69.81; H,
6.13; N, 11.10; O, 12.69.
2
–CO–NH–), 52.77 (2C, piperazine), 50.24
23 3 3
). Anal. calcd. for C22H N O
(
t, J ¼ 5.0 Hz, 4H, –CH , piperazine), 2.55 (d, J ¼ 10.2 Hz, 4H, –CH
2
2
,
3
1
3
piperazine); C NMR (125 MHz, DMSO): d (ppm) 169.64 (1C,
CONH–), 169.16 (1C, –C–O, coumarin), 159.20 (1C, –C–N,
coumarin), 151.77, 130.49, 125.60, 125.28, 117.60, 115.79,
4.42 (7C, coumarin), 139.73, 129.12, 120.47 (5C, Ar–C), 131.97
1C, Ar–C, C–Cl), 59.99 (1C, –CH –CO–NH–), 52.77 (2C, piperazine),
0.24 (2C, piperazine). Anal. calcd. for C21 20ClN (397.85) C,
–
8
(
5
6
1
2-(4-(2-Oxo-2H-chromen-4-yl)piperazin-1-yl)-
N-p-tolylacetamide (3h)
Yield: 71%; m.p. 229–231°C; IR (KBr): cm 3424 (NH), 1733 (CO,
2
H
3
O
3
ꢀ1
3.40; H, 5.07; N, 10.56; O, 12.06. Found: C, 63.57; H, 5.06; N,
0.54; O, 12.05.
1
coumarin), 1662 (C–O), 1243 (–C–N), 1235 (–CH
500 MHz, DMSO-d
); H NMR
3
(
1
4
6
): d (ppm) 10.90 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5,
.6 Hz, 1H, coumarin), 7.70 (m, 1H, ArH, coumarin), 7.47–7.33 (m,
H, ArH), 7.13–7.07 (m, 2H, ArH), 5.02 (s, 1H, –CH, coumarin), 3.33
N-(2-Chloronaphthalen-1-yl)-2-(4-(2-oxo-2H-chromen-
4
-yl)piperazin-1-yl)acetamide (3e)
(s, 2H, –CH ), 3.24 (t, J ¼ 5.0 Hz, 4H, –CH , piperazine), 2.55 (d,
2
2
ꢀ1
13
Yield: 70%; m.p. 261–264°C; IR (KBr): cm 3401 (NH), 1714 (CO,
J ¼ 10.2 Hz, 4H, –CH
2
3
, piperazine), 2.32 (s, 3H, –CH ); C NMR
1
coumarin), 1681 (C–O), 1243 (–C–N), 652 (C–CI); H NMR
(125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C, –C–O,
coumarin), 157.43 (1C, –C–N, coumarin), 151.77, 130.49, 125.28,
119.62, 117.60, 115.79, 84.42 (7C, coumarin), 138.88, 136.34,
6
(500 MHz, DMSO-d ): d (ppm) 10.01 (s, 1H, –NH), 8.36 (dd,
J ¼ 7.3, 1.4 Hz, 1H, ArH), 8.01 (dd, J ¼ 7.5, 1.3 Hz, 1H, ArH,
coumarin), 7.85 (dd, J ¼ 7.5, 1.6 Hz, 1H. ArH), 7.70 (m, 1H, ArH,
coumarin), 7.52 (m, 3H, ArH), 7.45–7.33 (m, 3H, ArH), 5.02 (s, 1H,
128.54, 125.60 (6C, Ar–C), 59.99 (1C, –CH
piperazine), 50.24 (2C, piperazine), 21.28 (1C, –CH
for C22 (377.44) C, 70.01; H, 6.14; N, 11.13; O, 12.72.
2
–CO–NH–), 52.77 (2C,
3
). Anal. calcd.
–
CH, coumarin), 3.33 (s, 2H, –CH
2
), 3.24 (m, 4H, –CH
2
, piperazine),
23 3 3
H N O
1
3
2
.55 (m, 4H, –CH , piperazine); C NMR (125 MHz, DMSO): d
2
Found: C, 70.12; H, 6.13; N, 11.10; O, 12.71.
(
(
ppm) 169.16 (1C, –C–O, coumarin), 168.78 (1C, –CONH–), 157.43
1C, –C–N, coumarin), 151.77, 130.49, 125.60, 125.28, 117.60,
N-(2-Nitrophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
1
1
15.79, 84.42 (7C, coumarin), 133.27, 132.15, 130.71, 128.32,
27.16, 126.81, 126.78, 125.84, 125.66, (9C, Ar–C), 127.58 (1C,
piperazin-1-yl)acetamide (3i)
ꢀ1
Yield: 88%; m.p. 246–248°C; IR (KBr): cm 3401 (NH), 1714 (CO,
Ar–C, C–Cl), 59.99 (1C, –CH
5
6
2
–CO–NH–), 52.77 (2C, piperazine),
0.24 (2C, piperazine). Anal. calcd. for C25 22ClN (447.91) C,
7.04; H, 4.95; N, 9.38; O, 10.72. Found: C, 66.95; H, 4.96; N, 9.41;
1
coumarin), 1681 (C–O), 1350 (N–O), 1243 (–C–N); H NMR
H
3 3
O
6
(500 MHz, DMSO-d ): d (ppm) 10.01 (s, 1H, –NH), 8.53 (dd,
J ¼ 7.3, 1.4 Hz, 1H, ArH), 8.19 (dd, J ¼ 7.5, 1.3 Hz, 1H, ArH,
coumarin), 8.04 (dd, J ¼ 7.5, 1.6 Hz, 1H, coumarin), 7.71 (m, 2H,
ArH), 7.57 (m, 1H, ArH), 7.45–7.33 (m, 2H, ArH), 5.48 (s, 1H, –CH,
coumarin), 3.33 (s, 2H, –CH ), 2.96 (m, 4H, –CH , piperazine), 2.30
O, 10.73.
2-(4-(2-Oxo-2H-chromen-4-yl)piperazin-1-yl)-
2
2
1
3
N-o-tolylacetamide (3f)
(m, 4H, –CH
2
, piperazine); C NMR (125 MHz, DMSO): d (ppm)
O, coumarin), 157.43 (1C,
ꢀ
1
169.64 (1C, –CONH–), 169.16 (1C, –C––
Yield: 74%; m.p. 233–236°C; IR (KBr): cm 3423 (NH), 1772 (CO,
1
coumarin), 1642 (C–O), 1260 (–CH
500 MHz, DMSO-d ): d (ppm) 9.60 (s, 1H, –NH), 8.05 (dd, 3H, ArH),
.67 (d, 3H, ArH), 7.40 (dd, 3H, ArH), 7.34 (dd, 1H, ArH, coumarin),
), 1253 (–C–N); H NMR
–C–N, coumarin), 151.77, 130.49, 125.60, 125.28, 117.60, 115.79,
84.42 (7C, coumarin), 140.21, 139.21, 130.26, 126.64, 122.74,
3
(
6
7
7
1
117.41 (6C, Ar–C), 59.99 (1C, –CH
zine), 50.24 (2C, piperazine). Anal. calcd. for C21H N O
2
–CO–NH–), 52.77 (2C, pipera-
(408.41)
.22 (d, 1H, ArH, coumarin), 7.20 (dd, 1H, ArH, coumarin), 7.12 (d,
H, ArH, coumarin), 5.93 (s, 1H, –CH, coumarin), 5.05 (s, 2H,
20 4 5
C, 61.76; H, 4.94; N, 13.72; O, 19.59. Found: C, 61.61; H, 4.93; N,
13.68; O, 19.54.
–
CH
2
), 3.35 (s, 3H, –CH
3
), 2.32 (t, J ¼ 5.0 Hz, 4H, –CH
2
, piperazine),
, piperazine); C NMR (125 MHz,
1
3
2
.21 (t, J ¼ 5.0 Hz, 4H, –CH
2
DMSO): d (ppm) 169.43 (1C, –CONH–), 169.16 (1C, –C–O,
coumarin), 157.43 (1C, –C–N<, coumarin), 151.77, 130.45,
N-(3-Nitrophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
piperazin-1-yl)acetamide (3j)
ꢀ1
1
1
25.60, 125.28, 117.60, 115.79, 84.42 (7C, coumarin), 137.76,
34.32, 130.48, 127.60, 126.92, 122.04 (6C, Ar–C), 59.99 (1C,
Yield: 85%; m.p. 253–256°C; IR (KBr): cm 3396 (NH), 1725 (CO,
1
coumarin), 1646 (C–O), 1378 (N–O), 1261 (–C–N); H NMR
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
02
R. P. Modh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
(
1
500 MHz, DMSO-d
H, ArH), 8.15 (dd, J ¼ 7.5, 1.6 Hz, 1H, ArH, coumarin), 7.91 (m, 2H,
ArH), 7.70 (m, 1H, ArH, coumarin), 7.61 (t, J ¼ 7.5 Hz, 1H, ArH),
.45–7.33 (m, 2H, ArH), 5.47 (s, 1H, –CH, coumarin), 3.33 (s, 2H,
CH , piperazine), 3.06 (t, J ¼ 4.9 Hz,
), 3.28 (t, J ¼ 4.9 Hz, 2H, –CH
H, –CH , piperazine),
, piperazine), 2.93 (t, J ¼ 4.8 Hz, 2H, –CH
, piperazine); C NMR (125 MHz,
6
): d (ppm) 11.06 (s, 1H, –NH), 8.59 (t, J ¼ 1.4 Hz,
coumarin), 158.58 (1C, –C–N, coumarin), 151.77, 130.49, 125.60,
125.28, 117.60, 115.79, 84.42 (7C, coumarin), 159.18, 139.00,
129.94, 117.48, 112.35, 108.04 (6C, Ar–C), 59.99 (1C,
7
–CH
2
–CO–NH–), 55.52 (1C, –OCH
3
), 52.77 (2C, piperazine),
(393.44) C,
–
2
2
2
2
50.24 (2C, piperazine). Anal. calcd. for C22H N O
23 3 4
2
2
67.16; H, 5.89; N, 10.68; O, 16.27. Found: C, 67.34; H, 5.90; N,
10.65; O, 16.28.
1
3
.49 (t, J ¼ 4.9 Hz, 2H, –CH
2
DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C, –C–O,
coumarin), 159.14 (1C, –C–N<, coumarin), 151.77, 130.49,
N-(4-Methoxyphenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
1
1
25.60, 125.28, 117.60, 115.79, 84.42 (7C, coumarin), 146.15,
39.36, 128.37, 122.53, 121.35, 117.98 (6C, Ar–C), 59.99 (1C,
piperazin-1-yl)acetamide (3n)
ꢀ1
Yield: 73%; m.p. 206–208°C; IR (KBr): cm 3401 (NH), 1726 (CO,
–
CH
Anal. calcd. for C21
9.59. Found: C, 61.60; H, 4.95; N, 13.74; O, 19.64.
2
–CO–NH), 52.77 (2C, piperazine), 50.24 (2C, piperazine).
coumarin), 1681 (C–O), 1243 (–C–N), 1075 (C–O–C); 1H NMR
20 4 5
H N O
(408.41) C, 61.76; H, 4.94; N, 13.72; O,
(
500 MHz, DMSO-d
.6 Hz, 1H, ArH, coumarin), 7.73 (m, 1H, ArH, coumarin), 7.48–
.33 (m, 4H), 6.89–6.83 (m, 2H), 5.02 (s, 1H), 3.79 (s, 3H, –OCH
.33 (s, 2H), 3.24 (t, J ¼ 5.0 Hz, 4H), 2.55 (d, J ¼ 10.2 Hz, 4H);
6
): d (ppm) 10.90 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5,
1
1
7
3
3
13
),
N-(4-Nitrophenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
C
piperazin-1-yl)acetamide (3k)
NMR (125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C,
–C–O, coumarin), 159.20 (1C, –C–N, coumarin), 151.77, 130.49,
ꢀ1
Yield: 79%; m.p. 247–250°C; IR (KBr): cm 3423 (NH), 1737 (CO,
1
coumarin), 1681 (C–O), 1339 (N–O), 1243 (–C–N); H NMR
125.60, 125.28, 117.60, 115.79, 84.42 (7C, coumarin), 159.02,
(
3
7
500 MHz, DMSO-d
6
): d (ppm) 10.90 (s, 1H, –NH), 8.20–8.13 (m,
H, ArH), 7.82 (d, J ¼ 6.9 Hz, 2H, ArH), 7.70 (m, 1H, ArH, coumarin),
.45–7.33 (m, 2H, ArH), 5.66 (s, 1H, –CH, coumarin), 3.33 (s, 2H,
CH ), 2.99 (m, 4H, –CH
, piperazine), 2.67 (t, J ¼ 5.1 Hz, 2H, –CH
piperazine), 2.31 (t, J ¼ 5.1 Hz, 2H, –CH
125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C, –C–O,
coumarin), 158.85 (1C, –C–N, coumarin), 151.77, 130.49, 125.60,
136.82, 120.30, 114.85, 114.85 (6C, Ar–C), 59.99 (1C,
–CH
2
–CO–NH–), 55.15 (1C, –OCH
3
), 52.77 (2C, piperazine),
(393.44) C,
50.24 (2C, piperazine). Anal. calcd. for C22
23 3 4
H N O
–
2
2
2
,
67.16; H, 5.89; N, 10.68; O, 16.27. Found: C, 67.04; H, 5.88; N,
10.67; O, 16.24.
1
3
2
, piperazine); C NMR
(
Biological assay
In vitro AChE inhibition assay
The AChE activity of the newly synthesized molecules (3a–3n) was
determined quantitatively in human blood by Ellman’s
method [28] with some modifications proposed by Skrinjaric-
Spoljar et al. [33]. A series of newly synthesized coumarin
derivatives were tested as AChE inhibitors. Initially, venous blood
1
1
25.28, 117.60, 115.79, 84.42 (7C, coumarin), 145.38, 143.73,
24.91, 119.16 (6C, Ar–C), 59.99 (1C, –CH –CO–NH–), 52.77 (2C,
2
piperazine), 50.24 (2C, piperazine). Anal. calcd. for C21
408.41) C, 61.76; H, 4.94; N, 13.72; O, 19.59. Found: C, 61.70; H,
.95; N, 13.69; O, 19.58.
20 4 5
H N O
(
4
N-(2-Methoxyphenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
(100 mL) was collected into dried heparinized tubes and subjected
piperazin-1-yl)acetamide (3l)
to centrifugation at 2000 rpm. It was ensured that all the plasma
was withdrawn and then the remnant erythrocytes were
suspended in water (100 mL). The suspended erythrocytes were
subjected to 60-fold dilution using buffer (0.1 M phosphate
buffer pH 7.4) and subsequently frozen so as to get hemolyzed.
The suspension was again diluted with buffer after thawing and
ꢀ
1
Yield: 74%; m.p. 217–220°C; IR (KBr): cm 3416 (NH), 1713 (CO,
1
coumarin), 1657 (C–O), 1232 (–C–N), 1063 (C–O–C); H NMR
(
1
(
500 MHz, DMSO-d
.6 Hz, 1H, ArH, coumarin), 7.94 (dd, J ¼ 7.4, 1.7 Hz, 1H, ArH), 7.63
m, 1H, ArH, coumarin), 7.45–7.33 (m, 2H, ArH), 7.21–7.05 (m, 3H,
ArH), 5.02 (s, 1H, –CH, coumarin), 3.85 (s, 3H, –OCH ), 3.33 (s, 2H,
CH , piperazine), 2.55 (d, J ¼ 10.2 Hz,
), 3.24 (t, J ¼ 5.0 Hz, 4H, –CH
H, –CH , piperazine); C NMR (125 MHz, DMSO): d (ppm) 169.64
1C, –CONH–), 169.16 (1C, –C–O, coumarin), 157.43 (1C, –C–N,
coumarin), 151.77, 130.49, 125.60, 125.28, 117.60, 115.79, 84.42
7C, coumarin), 151.03, 127.90, 124.59, 124.33, 121.50, 114.03 (6C,
Ar–C), 59.99 (1C, –CH –CO–NH–), 56.43 (1C, –OCH ), 52.77 (2C,
piperazine), 50.24 (2C, piperazine). Anal. calcd. for C22
393.44) C, 67.16; H, 5.89; N, 10.68; O, 16.27. Found: C, 67.01; H,
.87; N, 10.70; O, 16.30.
6
): d (ppm) 10.01 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5,
0
3
then Ellman’s reagent DTNB (5,5 -dithiobis-(2-nitrobenzoic acid)
–
2
2
was added (final DTNB conc. during enzyme assay 0.33 mM).
Acetylthiocholine (ATCh) was added (final ATCh conc. during
enzyme assay 1.0 mM) after 10 min of DTNB treatment. The
absorbance was measured at 412 nm using Jasco V-570 spectro-
photometer with a modified Zijlstra reagent consisting of
potassium ferricyanide, sodium bicarbonate and Triton X-100
against a blank containing suspended hemolyzed erythrocytes
and the enzymatic activities were recorded at 37°C. The overall
procedure made the final assay volume to 3 mL. It should be noted
that the final dilution of erythrocytes was 600-fold relative to
whole blood and of plasma 150-fold during enzyme assay. The
final reaction volume was 1.5 mL and the final erythrocyte
dilution 120-fold. The absorbance was recorded against buffer at
546 nm at 37°C. The inhibitory activities of AChE were expressed
in micromoles hydrolyzed ATCh per minute and per milliliter
whole blood.
1
3
4
(
2
(
2
3
23 3 4
H N O
(
5
N-(3-Methoxyphenyl)-2-(4-(2-oxo-2H-chromen-4-yl)-
piperazin-1-yl)acetamide (3m)
ꢀ1
Yield: 61%; m.p. 213–215°C; IR (KBr): cm 3389 (NH), 1764 (CO,
1
coumarin), 1666 (C–O), 1272 (–C–N), 1048 (C–O–C); H NMR
(
1
500 MHz, DMSO-d
.6 Hz, 1H, ArH, coumarin), 7.82 (m, 1H, ArH, coumarin), 7.52 (t,
6
): d (ppm) 10.17 (s, 1H, –NH), 8.32 (dd, J ¼ 7.5,
J ¼ 1.5 Hz, 1H, ArH), 7.45–7.33 (m, 2H, ArH), 7.31–7.22 (m, 2H,
Computational method
ArH), 6.65 (m, 1H, ArH), 5.02 (s, 1H, –CH, coumarin), 3.77 (s, 3H,
–
OCH
piperazine), 2.55 (d, J ¼ 10.2 Hz, 4H, –CH
125 MHz, DMSO): d (ppm) 169.64 (1C, –CONH–), 169.16 (1C, –C–O,
3
), 3.33 (s, 2H, –CH
2
), 3.24 (t, J ¼ 5.0 Hz, 4H, –CH
2
,
Molecular docking
Molecular docking was carried out using Molegro Virtual Docker
(MVD) version 4.3.0 (CLC Bio, Denmark) [34] to study coumarin–
1
3
2
, piperazine); C NMR
(
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 793–804
Coumarin–Piperazine Derivatives as AChE Inhibitors
803
piperazine derivatives interaction with hAChE enzyme. The
crystal structure of recombinant hAChE in complex with
donepezil (PDB id: 4EY7) [35] was retrieved from PDB. The
compounds were drawn in MarvinSketch 5.9.3 (ChemAxon LLC)
chemical drawing software and geometrically optimized in 3D.
The protein was preprocessed by removing crystallographic
waters and energy minimization was performed prior to
docking. Cavity prediction was initially performed using “Detect
Cavities” module of Molegro with expanded van der Waals probe
The authors are very thankful to Prof. Nisha K. Shah, Head, Department
of Chemistry, School of Sciences, Gujarat University, Ahmedabad, India
for her kind cooperation for providing support and research facility.
The authors wish to offer their deep gratitude to Dr. Bipin Pandey,
Zydus Research Centre, Ahmedabad, India for carrying out spectral
analysis. Mr. Rahul Modh would like to acknowledge UGC New Delhi,
for Junior Research Fellowship. Mr. Sivakumar Prasanth Kumar
acknowledges support from Department of Science and Technology,
New Delhi, India as INSPIRE Fellowship.
to find an accessible region. The maximum number of cavities
ꢂ
was set to 10 with probe size of 1.20 A , and minimum and
ꢂ
3
maximum cavity volume of 10 and 10,000 A . This module
utilizes a simple grid-based cavity prediction dependent on
molecular surface and/or van der Waals radii to detect regions of
accessibility. Protein dataset was then imported using the
The authors have declared no conflict of interest.
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