Design and synthesis of newer potential 4-(N-acetylamino)phenol derived piperazine derivatives as potential cognition enhancers

Article abstract of DOI:10.1016/j.bioorg.2015.04.004

A series of novel hybrids has been designed, synthesized and evaluated for cognition enhancing activities through the inhibition of acetylcholinesterase (AChE) and by passive avoidance mouse model. All the compounds showed excellent AChE inhibition activi

Full text of DOI:10.1016/j.bioorg.2015.04.004

Accepted Manuscript
Design and synthesis of newer potential 4-(N-acetylamino)phenol derived pi-
perazine derivatives as potential cognition enhancers
Poonam Piplani, Chhanda Charan Danta
PII:
S0045-2068(15)00032-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.04.004
YBIOO 1804
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
16 February 2015
Please cite this article as: P. Piplani, C.C. Danta, Design and synthesis of newer potential 4-(N-acetylamino)phenol
derived piperazine derivatives as potential cognition enhancers, Bioorganic Chemistry (2015), doi: http://dx.doi.org/
10.1016/j.bioorg.2015.04.004
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Design and synthesis of newer potential 4-(N-acetylamino)phenol derived piperazine
derivatives as potential cognition enhancers
Poonam Piplani a*, Chhanda Charan Danta a
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India
*Corresponding author
Dr Poonam Piplani
Email: ppvohra28in@yahoo.co.in
Mobile no. +91-9357036068
Professor of Pharmaceutical Chemistry
University Institute of Pharmaceutical Sciences
Chandigarh-160 014, India
6th April, 2015
1

ABSTRACT
A series of novel hybrids has been designed, synthesized and evaluated for cognition enhancing
activities through the inhibition of acetylcholinesterase (AChE) and by passive avoidance mouse
model. All the compounds showed excellent AChE inhibition activities and potentially reversed
the scopolamine induced memory deficit. Enzyme kinetic and molecular docking studies have
confirmed their dual binding affinity and mixed type inhibition. Among them, compounds 1b
and 2d displayed excellent IC50 values of 1.66 μM and 0.49 μM and competitive inhibitor
constant Ki 43.66 μM and 4.10 μM respectively. Ex vivo study confirmed their CNS penetration
and brain AChE inhibition abilities. Furthermore, 1b and 2d showed significant antiamnesic
activity at a dose of 1.0 mg/kg as compared to the reference compounds piracetam and
rivastigmine. The results indicate that these two compounds emerged to be developed as
cognition enhancers worthy of future pursuit.
Keywords: Acetylcholinesterase inhibitors, piperazine derivatives, cognition, passive avoidance
stepdown, antiradical activity, molecular docking
2

1. Introduction
Cognition is a combination of skills, including knowledge, acquisition, attention, memory,
learning, language, perception, skilled motor behaviours, decision making, goal setting, planning
and judgements [1]. Cognitive dysfunction is one of the most functionally infirm aspect of many
neuropsychiatric disorders and neurodegenerative disorders, such as Alzheimer’s disease (AD),
Parkinson disease (PD), schizophrenia, depression, seizure disorders and traumatic brain injury
[2]. It is a very common problem in the over 65 year age group and creates a symptomatic
clinical situation that a person has complaints about memory loss and shows the evidence for
cognitive impairment. It subsequently progresses towards the most devastating form of clinical
dementia, i.e. Alzheimer dementia, shown by around 5% of the population and can be considered
as the preclinical stage of AD and the risk factors for AD could also be risk factors for the
development and progression of cognitive dysfunction [3-8].
The most excited approach towards the discovery of new cognition enhancers has been based on
the functions of the central cholinergic system [9]. It has been found that central cholinergic
depletion is the hallmark of AD and experimentally induced cholinergic dysfunction produces
cognitive deficits both preclinically and clinically [10]. Although, in the past several decades
many cognition enhancers have been discovered, recently research intend has turned towards the
development of new acetylcholinesterase inhibitors (AChEIs) as these are capable of
suppressing the normal break down of ACh from the synaptic cleft, thereby increasing overall
level of ACh available to the relevant postsynaptic receptors [11-14].
The X-ray crystallographic structure of AChE has revealed that its active site gorge lined with
aromatic residues about 20 Å deep contains two ligand binding sites. i. e. the acylation site (AS)
and the peripheral anionic site (PAS). The acylation site (Phe288, Phe290, Phe299) lies at the
bottom of the gorge, consisting of a catalytic triad (His440-Glu327-Ser200), and the PAS
(Trp279, Tyr70, Tyr121, Asp72, Glu197, Phe290) is located at the entrance of the gorge. The AS
also has a quaternary ammonium binding locus consisting of Glu199, Phe330 and Trp84.
Ligands can bind selectively to either the acylation site or the peripheral site [15-17]. However
recent research has revealed that dual binding site inhibitors of AChE facilitate cholinergic
transmission as well as interfere with the synthesis, deposition and aggregation of toxic beta-
amyloid (Aβ). Rivastigmine, ensaculin and donepezil (Fig. 1) are well known clinically used
3

AChEIs for AD therapeutics [18]. Rivastigmine is a ‘pseudo-irreversible’ inhibitor of AChE and
approved for the treatment of mild to moderate cognition deficit [19,20]. Similarly, ensaculin, a
piperazine based molecule has shown improvement in memory and cognitive functions including
slow down of progressive neurodegeneration. It is reported a dual binding site inhibitor of AChE
and has been used to treat AD clinically for a long time [18]. Donepezil is a centrally acting
reversible AChEI and reported as a potent anti-amnesic and neuroprotective agent [21]. On this
basis, pharmacophoric hybrid (Fig. 2) approach has been made to design the novel molecules by
hybridizing the pharmacophoric fragments of rivastigmine and ensaculin. 4-(N-
acetylamino)phenol was selected for the aryloxy fragment with the hope of its possibility to form
favourable hydrogen bond, π-π stacking interactions and simultaneously may show free radical
scavenging activity. Therefore, p-(N-acetylamino)phenol derived piperazine derivatives have
been synthesized and screened for cognition enhancing activities.
H3C
H3C
H3C
CH3
CH3
N
N
O
N
OCH3
N
O
O
O
N
O
OCH3
Rivastigmine
Ensaculin
O
O
O
Donepezil
Fig. 1. Structure of clinically used marketed drugs for AD therapeutics.
4

N
O
O
O
N
O
H3C
H3C
CH3
CH3
O
O
N
O
N
O
O
CH3
R
O
N
N
N
O
N
N
H
O
R
N
H
Fig. 2. Pharmacophoric hybrid design strategy of the target compounds.
2. Results and discussion
2.1. Chemistry
Two series of 4-(N-acetylamino)phenol derived piperazine derivatives were designed and
synthesized as depicted in schemes 1 and 2. In both schemes, 4-(N-acetylamino)phenol was used
as the starting material, anhydrous K2CO3 as catalyst and ethyl methyl ketone as the solvent to
afford the key intermediates 1 and 2. The compounds have been synthesized by the fusion (an
excellent green chemistry approach; completely avoiding use of any organic solvent) of
intermediate 1 with different monosubstituted piperazines (Scheme 1) whereas the compounds of
scheme 2 were synthesized by refluxing the intermediate 2 with different monosubstituted
piperazines in the presence of anhydrous K2CO3 in ethyl methyl ketone. All the synthesized
1
13
compounds are in accordance with the IR, H NMR, C NMR, Mass and CHN analysis data.
The synthesized ligands are enlisted in Table 1 and Table 2.
5

R
N
N
OCH3
O
OH
O
O
O
b
a
HN
CH3
HN
CH3
HN
CH3
O
O
O
1a - 1g
1
Scheme 1. Reagents and reaction conditions: (a) methylchloroacetate, ethyl methyl ketone,
anhyd. K2CO3 (b) monosubstituted piperazines, heat
Table 1. Synthesized compounds of scheme 1.
Compounds
R
1a
1b
-CH2CH3
1c
F
1d
1e
F
N
N
1f
N
1g
O
O
6

O
N
O
Cl
OH
N
b
a
R
HN
CH3
HN
CH3
HN
CH3
O
O
2
O
2a - 2g
Scheme 2.
Reagents and reaction conditions: (a) bromochloropropane, ethyl methyl ketone, anhyd.
K2CO3 (b) monosubstituted piperazines, ethyl methyl ketone, anhyd. K2CO3
Table 2. Synthesized compounds of scheme 2.
Compounds
R
1a
1b
-CH2CH3
1c
F
1d
1e
F
Cl
1f
N
N
1g
N
2.3. Docking
Docking studies of all the hybrids (1a-1g and 2a-2g) on TcAChE demonstrated that they
emerged as good dual binding site inhibitors. The ligands were docked with TcAChE through H-
bond interaction and π-π stacking interaction at both AS and PAS. The carbonyl oxygen and
nitrogen atoms of 4-(N-acetylaminophenoxy) moiety was involved in H-bond interaction with
the amino acids His440, Arg289, Phe228, Tyr70, Tyr130 and Trp84, Phe228, Ser122, Ser286
respectively. Similarly, the aromatic fragments of the ligands showed π-π stacking interaction
7

with Trp84, Trp279, Phe330, Phe331 and Tyr334. The N4 atom of piperazine ring showed H-
bond interaction with Glu199 (1a, 2a), Phe288 (2a) and His440 respectively. The F atom of 1c
also involved in H-bonding with Glu199. The phenoxy oxygen atom of compound 2b showed H-
bond interaction with Tyr121. Molecular docking images of most active compounds of both
series 1b and 2d are given in (Fig. 3 and Fig. 4) respectively.
Fig. 3. Molecular docking of compound 1b into the active sites of TcAChE. Ligand is in green
colour, dotted green show H-bond interaction and dotted yellow show π-π stacking interaction.
Fig. 4. Molecular docking of compound 2d into the active sites of TcAChE. Ligand is in green
colour, dotted green show H-bond interaction and dotted yellow show π-π stacking interaction.
8

2.4. Inhibition of AChE
2.4.1. In vitro AChE Inhibition
To determine the AChE inhibitory activity of the synthesized compounds (1a-1g and 2a-2g), in
vitro assay was conducted using Ellman spectrophotometric method [22]. Enzyme kinetics study
was also performed to elucidate the mechanism of AChE inhibition. The IC50 values and the
kinetic parameters were calculated using GraphPad Prism 5 (Table 3). All the compounds
exhibited excellent IC50 values. Compounds 1b and 2d showed IC50 values of 1.66 µM and 0.49
µM respectively. It affirmed that the relationship between substrate concentration and reaction
velocity was in good agreement with Michaelis-Menten kinetics. Overlaid reciprocal
Lineweaver-Burk plots were drawn for compounds 1b and 2d (Fig. 5 and Fig. 6) and their
graphical analysis showed increasing slopes (decreased Vmax) and increasing intercepts (higher
Km) with the increase in inhibitor concentrations which indicated of a mixed type inhibition.
However, graphical analysis of rivastigmine showed unaffected Vmax and increased Km which
confirmed competitive type inhibition. The kinetic results of the synthesized ligands strongly
supported that these are dual binding site inhibitors. The competitive inhibition constant Ki of
compounds 1b and 2d are calculated as 43.66 µM and 4.10 µM respectively.
Table 3. Enzyme Kinetics of the synthesized compounds (1a-2g) on AChE activity
Compd/Inhibitor
IC50 SEM
(µM)
Km SEM
Vmax SEM
(µmol/L/min)
Ki SEM
Type of
inhibition
(µM)
(µM)
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
57.28 0.04
1.66 0.03
30.24 0.02
43.63 0.05
51.12 0.03
15.58 0.03
10.66 0.03
79.80 0.03
1.39 0.02
1.36 0.13
0.49 0.84
10.29 0.07
1.16 2.26
13.32 3.37
1.26 1.93
2.16 3.25
3.78 3.36
1.28 2.54
2.09 1.97
1.56 1.92
1.52 1.27
1.81 2.34
29.95 2.43
19.30 1.13
55 1.33
51 1.65
90 2.39
48 2.78
73 1.77
46 1.67
65 2.35
106 3.67
88 3.45
140 3.95
206 2.33
451 1.55
15.77 0.03
43.66 1.25
46.81 0.39
5.45 0.91
8.95 2.23
34.83 3.42
19.47 1.52
117.0 3.33
2.28 3.43
2.54 2.51
4.10 2.31
9.90 1.33
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
2d
2e
9

2f
2g
16.68 0.16
27.48 0.31
5.34 0.37
6.18 1.11
1.78 3.35
1.82 0.9
216 2.45
88 1.75
54 2.7
2.26 1.09
9.04 1.03
130.9 0.6
Mixed
Mixed
Competitive
Rivastigmine
8
6
4
2
25 μM
ꢀ
ꢀ
∇
15 μM
10 μM
0 μM
ꢀ
∇
∇
ꢁ
ꢀ
ꢀ
ꢁ
∇
ꢁ
ꢀ
ꢁ
ꢀ
ꢀ
-0.02
0.02
0.04
0.06
1/[S] mM-1
-2
Fig. 5. Kinetic study on the mechanism of AChE inhibition by 1b. Overlaid Lineweaver-Burk
reciprocal plots of AChE initial velocity at increasing substrate concentration (ATChI, 0.2 – 0.8
mM)) in the absence of inhibitor and in the presence of 1b (0 - 25 µM) are shown.
4
3
2
25 μM
ꢀ
15 μM
10 μM
0 μM
ꢀ
ꢁ
ꢁ
ꢀ
1
ꢀ
ꢀ
ꢁ
ꢀ
°
ꢀ
ꢁ
ꢀ
°
°
°
-0.02
0.02
0.04
0.06
1/[S] mM-1
-1
Fig. 6. Kinetic study on the mechanism of AChE inhibition by 2d. Overlaid Lineweaver-Burk
reciprocal plots of AChE initial velocity at increasing substrate concentration (ATChI, 0.2 – 0.8
mM) in the absence of inhibitor and in the presence of 2d (0 - 25 µM) are shown.
10

2.4.1. Ex vivo AChE Inhibition
Primarily to investigate the CNS penetration ability of the synthesized compounds, 1a and 2d are
selected for their central AChE inhibition studies by ex vivo assay using Ellman method. The
percentage of brain AChE inhibition versus untreated controls was measured as depicted in (Fig.
7 and Fig. 8) which showed both 1a and 2d exhibited dose-dependent brain AChE inhibition.
60
40
20
ꢀꢀꢀ
ꢀꢀꢀ
0
1b
CONTROL
Fig. 7. Ex vivo determination of AChE inhibitory activity of 1b. Data are expressed as mean
SEM. Significance is determined by Student’s t test. ٭٭٭p < 0.001 as compared to control.
60
40
20
ꢀꢀꢀ
ꢀꢀꢀ
0
2d
CONTROL
11

Fig. 8. Ex vivo determination of AChE inhibitory activity of 2d. Data are expressed as mean
SEM. Significance is determined by Student’s t test. ٭٭٭p < 0.001 as compared to control.
2.5. Passive avoidance test (The reversal of scopolamine induced memory deficit)
Over the past few decades, many efforts have been made to develop new piperazine derivatives
as cognition enhancers. Some of them like DM232 and DM235 have shown outstanding potency
and able to prevent amnesia at doses as low as 0.001 mg kg-1 subcutaneous, being several
thousand times more potent than the reference compound piracetam [23,24]. Even though their
pharmacological mechanism is not known, they are able to ameliorate the scopolamine induced
memory deficit in animal models and can be developed as new cognition enhancer as piracetam.
Likewise Ensaculin, a piperazine containing molecule acts as a cognition enhancer through the
inhibition of AChE [18]. Thus, the synthesized compounds were screened using the passive
avoidance step-down mice model [25] against piracetam and rivastigmine as standard drugs. The
basal latency results of 1b and 2d are given in (Fig. 9 and Fig. 10). Both the compounds 1b and
2d showed significant improvement in cognition as compare to control and scopolamine treated
groups. The results of memory parameters (latency and number of mistakes) of 1b and 2d are
included in (Table 4).
12

100
80
60
40
20
0
ꢀꢀꢀ
≠≠≠
ꢀꢀꢀ
≠≠≠
ꢀ
≠≠≠
Treatment
CONTROL
1b
SCO
Fig. 9. Cognition enhancing activity of compound 1b on mouse passive avoidance step down test
against scopolamine induced memory loss. Data are expressed by mean SEM. Significance
was determined by Two-way ANOVA and followed by Bonferroni posttest. It showed
significant improvement in cognition: (≠≠≠) p < 0.0001 as compare to scopolamine; (٭٭٭) p <
0.0001 and (٭) p < 0.05 as compare to control.
100
80
60
40
20
ꢀꢀꢀ
≠≠≠
ꢀꢀꢀ
≠≠≠
ꢀꢀꢀ
≠≠≠
0
Treatment
SCO
CONTROL
2d
Fig. 10. Cognition enhancing activity of compounds 2d on mouse passive avoidance step down
test against scopolamine induced memory loss. Data are expressed by mean SEM. Significance
13

was determined by Two-way ANOVA and followed by Bonferroni posttest. It showed
significant improvement in cognition: (≠≠≠) p < 0.0001 as compare to scopolamine and (٭٭٭) p <
0.0001 as compare to control.
Table 4. Cognition enhancing effect of piperazine derivatives on mouse passive avoidance test
against scopolamine induced memory loss.
Experimental
Group*
Treatment Groups
(Dose = mg/kg, i.p.)
1st day
2nd day
Memory parameters
(Mean S.E.M)
Memory parameters
(Mean S.E.M)
Latency
(sec)
No. of
mistakes
Latency
(sec)
No. of
mistakes
1b
2d
1.0
1.0
---
5.6 3.14
2
0.95
3.4 1.69 1.2 0.49
2.2 0.58
9.0 0.63
7.8 2.82 1.4 0.24 5.0 2.84
6.6 0.98 6.0 1.30 6.2 1.11
Control
Scopolamine
Piracetam
0.5
2.5
25.8 1.77 15.4 0.87 21.8 0.66 9.4 4.20
2.6 0.51
1.2 0.97 2.2 0.74 1.6 2.73
Rivastigmine
1.0
1.8 0.37
0.4 0.40 2.6 0.93 1.0 2.61
*Each group contains 5 animals (n = 5)
2.6. Antiradical activity
Free radicals are high energetic reactive oxygen/nitrogen species (ROS/RNS). They are
responsible for causing onset of life threatening disease like AD and it progresses towards the
cognitive dysfunction. They also cause oxidative stress (OS) in human body which shows
detrimental effects on neurons and is the basic reason of damage of nerve cells and nerve cell
functions. Therefore, till date many natural and synthetic antioxidants have been developed as
neuroprotective agents and they have shown beneficial results in the treatment of cognitive
dysfunction [26]. Thus, to determine the antiradical capability of the synthesized compounds
DPPH method was followed as reported by Shimada et al. [27]. The result is summarized in
(Table 5).
14

Table 5. Antiradical activity of the synthesized compounds (1a-2g).
Compds
Antiradical activity (% Inhibition)
(Mean S.E.M)
IC50
(µg/ml)
1 (µg/ml)
5 (µg/ml)
25 (µg/ml)
50 (µg/ml)
100 (µg/ml)
1a
1b
1c
1d
1e
51.07 0.34 51.84 0.35 53.23 0.14 55.62 0.04 57.05 0.03 81.26 0.36
75.56 0.21 75.84 0.30 75.87 0.06 77.81 0.10 81.46 0.04 1.76 0.61
50.23 0.08 51.31 0.10 52.11 0.01 52.82 0.02 54.51 0.01 123.8 0.37
73.61 0.01 74.92 0.02 75.08 0.03 75.44 0.03 76.58 0.00 96.64 0.21
49.61 0.01 51.98 0.02 52.31 0.01 53.31 0.01 56.02 0.02 281.4 0.86
48.32 0.02 51.02 0.02 51.51 0.01 56.13 0.02 57.22 0.02 63.46 0.56
1f
1g
2a
2b
2c
64.47 0.05 77.75 0.01 81.01 0.01
49.68 0.15 50.30 0.03 51.58 0.02
47.56 0.24 49.08 0.02 49.82 0.08
47.32 0.12 50.84 0.11 52.88 0.34
81.74 0.02 82.95 0.02
51.66 0.02 51.90 0.04
51.56 0.01 54.24 0.04
51.80 0.06 52.55 0.02
0.51 0.44
8.46 0.11
274.4 0.48
0.80 0.44
2d
2e
2f
2g
51.82 0.08 52.08 0.02 52.56 0.02 52.94 0.01 53.86 0.02 3.09 0.08
45.03 0.02 51.32 0.01 52.91 0.01 53.04 0.02 54.96 0.02 0.95 0.28
48.63 0.18 48.98 0.01 49.28 0.14 49.37 0.14 49.84 0.02 57.78 0.14
50.32 0.32 51.60 0.01 53.03 0.04 53.99 0.02 55.55 0.02 4.18 0.13
75.51 0.04 90.41 0.01 95.47 0.03 96.39 0.01 97.51 0.01 0.93 0.32
Trolox
3. Conclusion
Two series of novel hybrids have been designed and all the hybrids were synthesized without
any difficulty. All the compounds were afforded with higher percentage yields ranging from 65.0
to 90.0%. They emerged as potential dual binding site inhibitors that have been proved by both
enzyme kinetics and molecular docking studies. Based on the design approach and molecular
structures, it can be strongly conclude that the presence of 4-(N-acetylaminophenoxy) moiety
and N4-substituted piperazine fragment seemed to enhance interactions as well as inhibitory
activity, confirming the designed rationale. Ex vivo studies have suggested the good brain
penetration and central AChE inhibition abilities of the compounds. The capability of
ameliorating the scopolamine induced memory deficit has been identified from in vivo studies
using rodent memory evaluator in mice. DPPH assay has shown good antiradical properties
suggesting that they may develope as neuroprotective agents in near future. Compounds 1b and
15

2d are emerged as most potent of these series which can be considered for the future drug
development in search of new cognition enhancers.
4. Experimental section
4.1. Instrumentation and chemicals
All the chemicals used in the synthesis were purchased from Sigma Aldrich. The reaction
progress was monitored using thin layer chromatography (TLC). TLC plates were prepared with
silica gel G obtained from Merck company using ethyl acetate as solvent and activated at 110 0C
for 10 min. Iodine was used for the colour visualization of the spots. Melting points were
recorded on a Veego melting point apparatus and were uncorrected. Elemental analyses were
done on a Perkin-Elmer-2400 model CHN analyser using acetanilide as standard. The purity of
compounds was ascertained by TLC, melting point and elemental analysis methods. The
1
molecular structure of newly synthesized compounds was analyzed by FTIR, H NMR, 13C
NMR and Mass instrumental techniques. The FTIR spectra were obtained from a PerkinElmer
Spectrum Version 10.03.08. The NMR spectra were obtained using a Bruker Avance II 400 MHz
spectrometer in deuterated chloroform and deuterated dimethylsulfoxide as solvent and are
recorded in parts per million (ppm) downfield from tetramethylsilane (Me4Si) as internal
reference. The spin multiplicities were described as singlet (s), broad singlet (br s), doublet (d),
double doublet (dd), triplet (t), doublet of triplets (dt), triplet of double doublet (tdd) etc. The
MASS spectrum was obtained from a TOF MS ESI mass spectrometer, and was recorded on an
electrospray ionization mass spectrometer as the value m/z.
4.2. Syntheses
The syntheses of 4-(N-acetylamino)phenol derived piperazine derivatives 1a-1g and 2a-2g were
carried out using the procedures as given in Schemes 1 and 2.
4.2.1. Methyl 2-(4-acetamidophenoxy)acetate (1)
A mixture of N-(4-hydroxyphenyl)acetamide (0.5 g, 3.31 mmol) and methyl chloroacetate (0.8
ml, 9.13 mmol) in the presence of anhydrous potassium carbonate (1.0 g) was refluxed in ethyl
methyl ketone (15 ml) with continuous stirring at 90-100 0C for 18 h. The slurry was filtered and
16

the excess solvent was removed under reduced pressure and the solid obtained was crystallized
from ethanol to afford 1, 0.6 g, yield 81.1%, m.p. 133-135 0C. FTIR (KBr, cm-1): 3370.2, 3100.5,
1
2950.8, 1750.0, 1680.6, 1605.8, 1430.2, 1540.9, 1225.6, 1080.2, 830.9, 690.8. H NMR (400
MHz, DMSO-d6): δ 9.60 (br s, 1H, NH). 7.49 (d, 2H, Jo = 10.52 Hz, ArH), 6.80 (d, 2H, Jo =
10.54 Hz, ArH), 4.63 (s, 2H, OCH2), 3.76 (s, 3H, COOCH3), 2.07 (s, 3H, COCH3).
Calcd for C11H13NO4 : C, 59.19; H, 5.83; N, 6.28. Found: C, 59.09; H, 5.73; N, 6.16%.
4.1.2. N-[4-{2-(4-ethylpiperazin-1-yl)-2-oxoethoxy}phenyl]acetamide (1a)
A mixture of N-ethylpiperazine (0.9 ml, 7.05 mmol) and compound 1 (1.0 g, 4.48 mmol) was
0
thermally fused at 125-130 C for 3 h with continuous stirring. Crushed ice was added to the
reaction mixture and the solid obtained was crystallized from acetone to afford 1a, 1.33 g, yield
0
65.0%, m.p. 141 C. FTIR (KBr, cm-1): 3256.58, 3198.61, 3076.47, 2967.27, 2804.58, 1644.25,
1
1549.26, 1508.06, 1373.81, 1222.05, 1083.90, 1019.16, 829.45, 772.35. H NMR (400 MHz,
CDCl3): δ 8.65 (br s, 1H, NH), 7.38 (m, 2H, ArH), 6.80 (d, 2H, Jo = 9.0 Hz, ArH), 4.65 (s, 2H,
OCH2), 3.64 (t, 2H, J = 4.80 Hz, N1(CH2)2), 3.57 (t, 2H, J = 4.96 Hz, N1(CH2)2), 2.41 (m, 6H, -
13
(CH2)2N4CH2), 2.12 (s, 3H, COCH3), 1.08 (t, 3H, J = 7.18 Hz, CH3); C NMR (100 MHz,
CDCl3): δ 168.91, 166.45, 154.24, 132.53, 121.82, 114.74, 67.51, 52.42, 45.20, 42.08, 24.08,
11.90. MS (ESI+), m/z: Calcd. 305.2 [M] +; Obsvd. 306.3 [M + H] +. Calcd for C16H23N3O3 : C,
62.93; H, 7.59; N, 13.76. Found: C, 62.32; H, 7.43; N, 13.66%.
4.1.3. N-[4-{2-oxo-2-(4-phenylpiperazin-1-yl)ethoxy}phenyl]acetamide (1b)
A mixture of N-phenylpiperazine (0.6 ml, 3.80 mmol) and compound 1 (1.0 g, 4.48 mmol) was
0
thermally fused at 125-130 C for 1 h with continuous stirring. Crushed ice was added to the
reaction mixture and the solid obtained was crystallized from ethanol to afford 1b, 1.35 g, yield
0
85.44%, m.p. 197-198 C. FTIR (KBr, cm-1): 3259.44, 3056.23, 2841.73, 1663.22, 1599.72,
1
1445.27, 1512.68, 1333.70, 1155.09, 1110.52, 1027.51, 825.15, 750.50. H NMR (400 MHz,
DMSO-d6): δ 9.58 (br s, 1H, NH), 7.44 (d, 2H, Jo = 8.96 Hz, ArH), 7.18 (m, 2H, ArH), 6.82 (m,
5H, ArH), 4.69 (s, 2H, OCH2), 3.65 (s, 4H, N1(CH2)2), 3.11 (d, 4H, J = 18.88 Hz, N4(CH2)2),
2.00 (s, 3H, COCH3). 13C NMR (100 MHz, DMSO-d6): δ 167.92, 165.85, 153.49, 150.46,
132.84, 128.73, 120.63, 119.66, 115.9, 114.25, 66.76, 49.00, 44.38, 23.63. MS (ESI+), m/z:
Calcd. 353.2 [M] +; Obsvd. 354.3 [M + H] +, 376.2 [M + Na] +. Calcd for C20H23N3O3 : C, 67.97;
H, 6.56; N, 11.89. Found: C, 67.67; H, 6.35; N, 11.78%.
17

4.1.4. N-(4-(2-(4-(2-fluorophenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)acetamide (1c)
A mixture of N-(2-Fluorophenyl)piperazine (0.3 g, 1.66 mmol) and compound 1 (1.0 g, 4.48
0
mmol) was thermally fused at 140-150 C for 7 h with continuous stirring. Crushed ice was
added to the reaction mixture and the solid obtained was crystallized from acetone to afford 1c,
0
1.11 g, yield 66.86%, m.p. 128-129 C. FTIR (KBr, cm-1): 3206.24, 3140.28, 3111.51, 3009.46,
2909.58, 2856.24, 1671.13, 1626.79, 1553.24, 1513.56, 1485.01, 1428.79, 1377.56, 1328.47,
1182.92, 1111.08, 1016.60, 830.02 and743.80 cm-1. 1H NMR (100 MHz, CDCl3): δ 7.52 (br s,
1H, NH), 7.32 (m, 2H, ArH), 6.94 (m, 2H, ArH), 6.84 (m, 2H, ArH), 6.78 (m, 2H, ArH), 4.54 (s,
2H, OCH2), 3.68 (m, 4H, N1(CH2)2), 2.99 (m, 4H, N4(CH2)2), 2.05 (s, 3H, CH3). 13C NMR (100
MHz, CDCl3,): δ 168.52, 166.58, 156.96, 154.43, 139.41, 132.14, 124.60, 123.30, 121.88,
119.25, 116.28, 116.28, 114.96, 65.61, 50.98, 45.54, 24.31. MS (ESI+), m/z: Calcd. 371.2 [M] +;
Obsvd. 372.3 [M + H] +. Calcd for C20H22FN3O3 : C, 64.68; H, 5.97; N, 11.31. Found: C, 64.49;
H, 5.91; N, 11.11%.
4.1.5. N-(4-(2-(4-(4-fluorophenyl)piperazin-1-yl)-2-oxoethoxy)phenyl)acetamide (1d)
A mixture of N-(4-fluorophenyl) piperazine (0.6 g, 3.32 mmol) and compound 1 1.0 g, 4.48
0
mmol) was thermally fused at 125-130 C for 1 h with continuous stirring. Crushed ice was
added to the reaction mixture and the solid obtained was crystallized from ethanol to afford 1d
(1.41 g, 84.94%), m.p. 210 0C. FTIR (KBr, cm-1): 3257.15, 3124.62, 3048.04, 2841.98, 1661.32,
1
1515.66, 1445.63, 1324.98, 1156.74, 1110.26, 1029.16, 822.69, 740.91. H NMR (400 MHz,
DMSO-d6): δ 9.78 (br s, 1H, NH), 7.48 (dd, 2H, Jo = 7.14 Hz, Jm = 1.90 Hz, ArH), 7.07 (m, 2H,
ArH), 6.98 (m, 2H, ArH), 6.88 (m, 2H, ArH), 4.81 (s, 2H, OCH2), 3.61 (t, 4H, J = 5.04 Hz,
N1(CH2)2), 3.08 (m, 4H, J = 18.88 Hz, N4(CH2)2), 2.01 (s, 3H, COCH3). 13C NMR (100 MHz,
DMSO-d6): δ 167.74, 165.95, 157.45, 155.10, 147.64, 132.88, 120.40, 117.72, 115.32, 114.58,
66.22, 49.42, 49.04, 44.09, 41.09, 23.75. MS (ESI+), m/z: Calcd. 371.2 [M] +; Obsvd. 372.3
[M + H] +. Calcd for C20H22FN3O3 : C, 64.68; H, 5.97; N, 11.31. Found: C, 64.47; H, 5.93; N,
11.12%.
4.1.6. N-(4-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethoxy)phenyl)acetamide (1e)
A mixture of 1-(2-Pyridyl)piperazine (0.3 ml, 1.96 mmol) and compound 1 (1.0 g, 4.48 mmol)
was thermally fused at 110-120 0C for 1 h with continuous stirring. Crushed ice was added to the
reaction mixture and the solid obtained was crystallized from ethanol to afford 1e, 1.15 g,
18

0
72.78%, m.p. 172-173 C. FTIR (KBr, cm-1): 3298.16, 3196.31, 3134.55, 3071.81, 2918.05,
2828.77, 1655.45, 1591.80, 1539.23, 1507.71, 1474.10, 1432.31, 1373.98, 1312.69, 1215.27,
1
1110.14, 1016.22, 823.74, 774.30. H NMR (400 MHz, CDCl3): δ 8.12 (dd, 1H, Jo = 4.96 Hz,
Jm = 1.28 Hz, PyrH), 7.44 (m, 1H, PyrH), 7.38 (br s, 1H, NH), 7.32 (m, 2H, ArH), 6.82 (m, 2H,
ArH), 6.60 (m, 2H, PyrH), 4.64 (s, 2H, OCH2), 3.64 (m, 4H, N1(CH2)2), 3.52 (m, 2H, N4(CH2)2),
3.44 (t, 2H, J = 5.20 Hz, N4(CH2)2), 2.06 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3,): δ 168.35,
166.70, 158.97, 154.44, 148.00, 137.76, 132.07, 121.89, 114.94, 114.07, 107.32, 67.99, 45.16,
+
+
41.83, 24.36. MS (ESI+), m/z: Calcd. 354.2 [M] ; Obsvd. 355.3 [M + H] . Calcd for
C19H22N4O3 : C, 64.39; H, 6.26; N, 15.81. Found: C, 64.93; H, 6.21; N, 15.75%.
4.1.7. N-(4-(2-oxo-2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethoxy)phenyl)acetamide (1f)
A mixture of 1-(2-Pyrimidyl)piperazine (0.25 ml, 1.71 mmol) and compound 1 (1.0 g, 4.48
0
mmol) was thermally fused at 130-135 C for 1.30 h with continuous stirring. Crushed ice was
added to the reaction mixture and the solid obtained was crystallized from ethanol to afford 1f,
0
1.33 g, 83.64%, m.p. 132 C. FTIR (KBr, cm-1): 3247.55, 3134.55, 3079.20, 2915.76, 1654.74,
1
1585.10, 1553.16, 1509.61, 1438.85, 1363.56, 1234.70, 1076.12, 1030.33, 980.83, 835.00. H
NMR (400 MHz, CDCl3,): δ 8.25 (d, 2H, J = 4.76 Hz, Pyr), 7.52 (br s, 1H, NH), 7.32 (m, 2H,
ArH), 6.82 (m, 2H, ArH), 6.47 (t, 1H, J = 4.76 Hz, Pyr), 4.64 (s, 2H, OCH2), 3.76 (m, 4H,
N1(CH2)2), 3.60 (m, 4H, N4(CH2)2), 2.06 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 168.43,
166.81, 161.44, 157.81, 154.39, 132.15, 121.91, 114.92, 110.60, 68.00, 45.24, 43.90, 43.45,
+
+
42.05, 24.31. MS (ESI+), m/z: Calcd. 355.2 [M] ; Obsvd. 356.3 [M + H] . Calcd for
C18H21N5O3 : C, 60.83; H, 5.96; N, 19.71. Found: C, 60.71; H, 5.88; N, 19.51%.
4.1.8. N-(4-(2-(4-(furan-2-carbonyl)piperazin-1-yl)-2-oxoethoxy)phenyl)acetamide (1g)
A mixture of 1-(2-Furoyl)piperazine (0.3 g, 1.66 mmol) and compound 1 (1.0 g, 4.48 mmol) was
0
thermally fused at 100-110 C for 2 h with continuous stirring. Crushed ice was added to the
reaction mixture and the solid obtained was crystallized from acetone to afford 1g, 1.21 g,
0
72.89%, m.p. 199-200 C. FTIR (KBr, cm-1): 3306.92, 3206.24, 3111.51, 2909.58, 2856.24,
1671.13, 1626.79, 1553.24, 1513.56, 1485.01, 1428.79, 1377.56, 1328.47, 1236.09, 1182.92,
1
1111.08, 1081.92, 1016.60, 830.02, 743.80. H NMR (400 MHz, CDCl3,): δ 7.49 (d, 1H, J =
4.76 Hz, furanoyl), 7.41 (d, 2H, J = 9.00 Hz, ArH), 7.16 (br s, 1H, NH), 7.06 (d, 1H, J = 3.6 Hz,
furanoyl), 6.91 (d, 2H, J = 9.00 Hz, ArH), 6.50 (q, 1H, J = 1.74 Hz, furanoyl), 4.71 (s, 2H,
19

13
OCH2), 3.80 (br s, 4H, N4(CH2)2), 3.69 (br s, 4H, N1(CH2)2), 2.16 (s, 3H, CH3). C NMR (100
MHz, CDCl3,): δ 168.91, 166.53, 155.12, 153.71, 147.11, 144.12, 132.17, 122.21, 118.97,
114.45, 111.93, 68.31, 48.91, 47.53, 24.31. MS (ESI+), m/z: Calcd. 371.1 [M] +; Obsvd. 372.2
[M + H] +. Calcd for C19H21N3O5 : C, 61.45; H, 5.70; N, 11.31. Found: C, 61.32; H, 5.51; N,
11.11%.
4.1.9. N-{4-(3-chloropropoxy)phenyl}acetamide (2)
A mixture of N-(4-hydroxyphenyl)acetamide (1.0 g, 4.39 mmol) and 1-Bromo-3-chloropropane
(1.2 ml, 8.39 mmol) in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium
0
carbonate (2.0 g) at 90-100 C with continuous stirring for 11 h. The slurry was filtered and the
excess solvent was removed under reduced pressure and the solid obtained was crystallized from
0
ethanol to afford 2, 1.3 g, yield 85.5%, m.p. 120-123 C. FTIR (KBr, cm-1): 3288.23, 3134.14,
2934.49, 1660.01, 1609.61, 1407.86, 1553.69, 1509.76, 1237.70. 1H NMR (400 MHz, CDCl3):
δ 7.48 (br s, 1H, NH), 7.38 (dd, 2H, Jo = 6.84 Hz, Jm = 2.16 Hz, ArH), 6.84 (dd, 2H, Jo = 6.84 Hz,
Jm = 2.16 Hz, ArH), 4.08 (t, 2H, J = 5.84 Hz, OCH2), 3.74 (t, 2H, J = 6.32 Hz, CH2Cl), 2.21
13
(pent, 2H, OCH2CH2CH2Cl), 2.14 (s, 3H, COCH3). C NMR (100 MHz, CDCl3): δ 168.48,
155.54, 131.22, 121.99, 114.78, 64.52, 41.56, 32.24, 24.33. Calcd for C11H14ClNO2 : C, 58.03;
H, 6.20; N, 6.15. Found: C, 57.99; H, 6.17; N, 6.13%.
4.1.10. N-(4-(3-(4-ethylpiperazin-1-yl)propoxy)phenyl)acetamide (2a)
A mixture of compound 2 (1.0 g, 4.39 mmol) and N-Ethyl piperazine (0.7 ml, 5.48 mmol) was
refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium carbonate (2.0 g)
at 90-100 0C with continuous stirring for 38 h. The slurry was filtered and the excess solvent was
removed under reduced pressure and the solid obtained was crystallized from acetone to afford
0
2a, 1.1 g, yield 82.08%, m.p. 190-191 C. FTIR (KBr, cm-1): 3236.37, 3182.62, 3118.65,
3055.97, 2952.66, 2832.48, 1672.71, 1600.74, 1540.92, 1511.27, 1407.55, 1308.06, 1239.76,
1
1173.90, 1139.37, 1039.77, 990.24, 829.33. H NMR (400 MHz, DMSO-d6,): δ 9.76 (br s, 1H,
NH), 7.48 (d, 2H, J = 8.96 Hz, ArH), 6.79 (d, 2H, Jo = 8.96 Hz, ArH), 3.97 (t, 2H, J = 6.04 Hz,
OCH2), 3.01 (br s, 8H, N1(CH2)4N4), 2.6 (s, 2H, CH2CH3), 2.54 (t, 2H, J = 1.72 Hz,
O(CH2)2CH2N1), 2.11 (s, 3H, COCH3), 1.97 (q, 2H, J = 14.92 Hz, OCH2CH2), 1.27 (s, 3H, CH3).
13C NMR (100 MHz, DMSO-d6): δ 167.67, 154.21, 132.47, 120.58, 114.09, 65.33, 53.49, 50.80,
50.28, 49.40, 30.52, 23.67, 9.10. MS (ESI+), m/z: Calcd. 305.2 [M] +; Obsvd. 306.3 [M + H] +.
Calcd for C17H27N3O2 : C, 58.03; H, 6.20; N, 6.15. Found: C, 57.99; H, 6.17; N, 6.13%.
20

4.1.11. N-[4-{3-(4-phenylpiperazin-1-yl)propoxy}phenyl]acetamide (2b)
A mixture of compound 2 (1.0 g, 4.39 mmol) and N-phenyl piperazine (0.7 ml, 4.44 mmol) was
refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium carbonate (2.0 g)
at 90-100 0C with continuous stirring for 30 h. The slurry was filtered and the excess solvent was
removed under reduced pressure and the solid obtained was crystallized from acetone to afford
2b, 1.2 g, yield 77.4%, m.p. 167-169 0C. FTIR (KBr, cm-1): 3296.68, 3129.44, 2828.32, 1655.00,
1
1602.06, 1402.24, 1508.78, 1239.40. H NMR (400 MHz,CDCl3): δ 7.34 (br s, 1H, NH), 7.30
(dd, 2H, Jo = 6.88 Hz, Jm = 6.92 Hz, ArH), 7.19 (m, 2H, ArH), 6.86 (d, 2H, Jo = 7.92 Hz, ArH),
6.78 (m, 3H, ArH), 3.93 (t, 2H, J = 6.32 Hz, OCH2), 3.14 (t, 4H, J = 4.98 Hz, N4(CH2)2), 2.56 (t,
4H, J = 4.98 Hz, N1(CH2)2), 2.50 (t, 2H, J = 7.38 Hz, O(CH2)2CH2N1), 2.06 (s, 3H, COCH3),
13
1.92 (pent, 2H, -OCH2CH2CH2N1). C NMR (100 MHz, CDCl3): δ 168.41, 155.86, 151.31,
130.95, 129.13, 121.98, 119.72, 116.06, 114.77, 66.48, 55.20, 53.30, 49.14, 26.77, 24.35. MS
(ESI+), m/z: Calcd. 353.2 [M] +; Obsvd. 354.3 [M + H] +. Calcd for C21H27N3O2 : C, 71.36; H,
7.70; N, 11.89. Found: C, 71.23; H, 7.56; N, 11.77%.
4.1.12. N-(4-(3-(4-(2-fluorophenyl)piperazin-1-yl)propoxy)phenyl)acetamide (2c)
A mixture of compound 2 (1.0 g, 4.39 mmol) and N-(2-Fluorophenyl)piperazine (0.5 g, 2.76
mmol) was refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium
0
carbonate (2.0 g) at 90-100 C with continuous stirring for 24 h. The slurry was filtered and the
excess solvent was removed under reduced pressure and the solid obtained was crystallized from
methanol to afford 2c, 1.3 g, yield 80.1%, m.p. 109-110 0C. FTIR (KBr, cm-1): 3285.21, 3133.48,
3072.37, 2935.60, 2817.16, 1656.81, 1609.96, 1510.12, 1407.93, 1372.81, 1241.26, 1172.05,
1039.06, 941.44, 831.82, 751.32. 1H NMR (400 MHz, CDCl3): δ 7.52 (br s, 1H, NH), 7.32 (m,
2H, ArH), 6.94 (m, 2H, ArH), 6.83 (m, 2H, ArH), 6.77 (m, 2H, ArH), 4.54 (s, 2H, OCH2), 3.71
(s, 4H, N4(CH2)2), 3.67 (t, 4H, J = 4.90 Hz, N1(CH2)2), 2.99 (m, 2H, CH2N1), 2.06 (s, 3H,
COCH3), 1.78 (pent, 2H, OCH2CH2CH2N1). 13C NMR (100 MHz, CDCl3): δ 168.51, 156.96,
154.43, 139.41, 132.13, 124.60, 123.30, 121.88, 119.25, 116.29, 114.91, 65.61, 52.32, 50.98,
50.32, 45.54, 42.22, 27.77, 24.31. MS (ESI+), m/z: Calcd. 371.2 [M] +; Obsvd. 372.3 [M + H] +.
Calcd for C21H26FN3O2 : C, 67.90; H, 7.06; N, 11.31. Found: C, 67.86; H, 6.98; N, 11.11%.
4.1.13. N-(4-(3-(4-(4-fluorophenyl)piperazin-1-yl)propoxy)phenyl)acetamide (2d)
A mixture of compound 2 (1.0 g, 4.39 mmol) and N-(4-Fluorophenyl)piperazine (0.6 g, 3.32
mmol) was refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium
21

0
carbonate (2.0 g) at 90-100 C with continuous stirring for 32 h. The slurry was filtered and the
excess solvent was removed under reduced pressure and the solid obtained was crystallized from
0
ethanol to afford 2d, 1.41 g, yield 86.5%, m.p. 180 C. FTIR (KBr, cm-1): 3292.11, 3056.27,
2957.16, 2829.69, 1657.50, 1607.53, 1509.74, 1374.69, 1237.56, 1152.46, 819.12, 914.33,
711.01.1H NMR (400 MHz, CDCl3): δ 7.13 (br s, 1H, NH), 7.30 (dd, 2H, Jo = 6.86 Hz, Jm = 2.10
Hz, ArH), 6.89 (m, 2H, ArH), 6.80 (m, 4H, ArH), 3.94 (t, 2H, J = 6.30 Hz, OCH2), 3.06 (t, 4H,
J = 4.94 Hz, N4(CH2)2), 2.56 (t, 4H, J = 4.94 Hz, CH2N1(CH2)2), 2.51 (t, 2H, J = 7.36 Hz,
CH2N1), 2.08 (s, 3H, COCH3), 1.92 (pent, 2H, OCH2CH2CH2N1). 13C NMR (100 MHz, CDCl3):
δ 168.26, 158.34, 155.87, 147.98, 130.89, 121.93, 117.80, 115.51, 114.80, 66.45, 55.13, 53.28,
50.16, 26.77, 24.39. MS (ESI+), m/z: Calcd. 371.2 [M] +; Obsvd. 372.3 [M + H] + . Calcd for
C21H26FN3O2 : C, 67.90; H, 7.06; N, 11.31. Found: C, 67.85; H, 6.99; N, 11.11%.
4.1.14. N-(4-(3-(4-(3-chlorophenyl)piperazin-1-yl)propoxy)phenyl)acetamide (2e)
A mixture of compound 2 (1.0 g, 4.39 mmol) and N-(3-Chlorophenyl)piperazine hydrochloride
(1.3 g, 4.53 mmol) was refluxed in ethyl methyl ketone (30 ml) in the presence of anhydrous
0
potassium carbonate (3.0 g) at 90-100 C with continuous stirring for 13 h. The slurry was
filtered and the excess solvent was removed under reduced pressure and the solid obtained was
0
crystallized from ethanol to afford 2e, 1.5 g, yield 90.0%, m.p. 139-140 C. FTIR (KBr, cm-1):
3290.20, 3133.45, 3073.38, 2947.57, 2835.59, 1648.92, 1595.23, 1546.93, 1506.58, 1406.61,
1251.66, 1126.94, 1035.24, 993.47, 948.18, 829.26, 758.38. 1H NMR (400 MHz, CDCl3): δ 7.64
(br s, 1H, NH), 7.40 (m, 2H, ArH), 7.17 (t, 1H, Jo = 8.12 Hz, ArH), 6.84 (m, 5H, ArH), 4.02 (t,
2H, J = 6.30 Hz, OCH2), 3.21(t, 4H, J = 5.0 Hz, N4(CH2)2), 2.60 (m, 6H, CH2N1(CH2)2), 2.14 (s,
3H, COCH3), 1.98 (pent, 2H, OCH2CH2CH2N1). 13C NMR (100 MHz, CDCl3): δ 168.56, 155.81,
152.33, 134.91, 131.03, 130.06, 122.01, 119.21, 115.67, 114.74, 113.84, 66.40, 55.12, 53.07,
48.62, 26.76, 24.31. MS (ESI+), m/z: Calcd. 387.2 [M] +; Obsvd. 388.3 [M + H] +. Calcd for
C21H26ClN3O2 : C, 65.02; H, 6.76; N, 10.83. Found: C, 64.97; H, 6.53; N, 10.71%.
4.1.15. N-(4-(3-(4-(pyridin-2-yl)piperazin-1-yl)propoxy)phenyl)acetamide (2f)
A mixture of compound 2 (1.0 g, 4.39 mmol) and 1-(2-Pyridyl)piperazine (0.3 ml, 1.96 mmol)
was refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium carbonate
0
(2.0 g) at 90-100 C with continuous stirring for 24 h. The slurry was filtered and the excess
solvent was removed under reduced pressure and the solid obtained was crystallized from
ethanol to afford 2f, 1.39 g, yield 89.1%, m.p. 120-121 0C. FTIR (KBr, cm-1): 3278.26, 3071.30,
22

2937.12, 2836.02, 1657.87, 1602.57, 1557.52, 1510.29, 1476.63, 1439.69, 1374.14, 1311.30,
1
1243.11, 1169.55, 1143.27, 1055.82, 936.87, 826.68, 777.36. H NMR (400 MHz, CDCl3): δ
8.18 (dd, 1H, Jo = 4.88 Hz, Jm = 1.32 Hz, PyrH), 7.47 (m, 1H, PyrH), 7.44 (br s, 1H, NH), 7.36
(m, 2H, ArH), 6.84 (dd, 2H, Jo = 12.06 Hz, Jm = 3.14 Hz, ArH), 6.62 (m, 2H, ArH), 4.0 (t, 2H, J
= 6.30 Hz, OCH2), 3.56 (t, 4H, J = 5.06 Hz, N4(CH2)2), 2.58 (m, 6H, CH2N1(CH2)2), 2.13(s, 3H,
COCH3), 2.01 (pent, 2H, OCH2CH2CH2N1). 13C NMR (100 MHz, CDCl3): δ 168.34, 159.53,
155.84, 147.92, 137.50, 131.02, 121.93, 114.80, 113.35, 107.14, 66.45, 55.22, 53.06, 45.19,
+
+
26.69, 24.29. MS (ESI+), m/z: Calcd. 354.2 [M] ; Obsvd. 355.3 [M + H] . Calcd for
C20H26N4O2 : C, 67.77; H, 7.39; N, 15.81. Found: C, 67.53; H, 7.27; N, 15.79%.
4.1.16. N-(4-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)phenyl)acetamide (2g)
A mixture of compound 2 (1.0 g, 4.39 mmol) and 1-(2-Pyrimidyl)piperazine (0.5 ml, 3.42
mmol) was refluxed in ethyl methyl ketone (20 ml) in the presence of anhydrous potassium
0
carbonate (2.0 g) at 90-100 C with continuous stirring for 23.30 h. The slurry was filtered and
the excess solvent was removed under reduced pressure and the solid obtained was crystallized
0
from ethanol to afford 2g, 1.39 g, yield 89.1%, m.p. 180-181 C. FTIR (KBr, cm-1): 3287.64,
3150.30, 2906.96, 2808.76, 1655.22, 1592.74, 1514.35, 1440.23, 1391.32, 1360.55, 1312.94,
1
1253.70, 1169.51, 1140.27, 1051.26, 1012.94, 981.72, 832.63, 790.21. H NMR (400 MHz,
CDCl3): δ 8.23 (d, 2H, Jo = 4.76 Hz, PyrimH), 7.31(dd, 2H, Jo = 6.94 Hz, Jm = 2.02 Hz, ArH),
7.20 (br s, 1H, NH), 6.78 (dd, 2H, Jo = 6.90 Hz, Jm = 2.06 Hz, ArH), 6.41 (t, 1H, Jo = 4.74 Hz,
PyrimH), 3.94 (t, 2H, J = 6.28 Hz, OCH2), 3.78 (t, 4H, Jo = 10.12 Hz, N4(CH2)2), 2.48 (m, 6H,
13
CH2N1(CH2)2), 2.07 (s, 3H, COCH3), 1.94 (m, 2H, OCH2CH2CH2N1). C NMR (100 MHz,
CDCl3): δ 168.25, 161.63, 157.73, 155.84, 130.95, 121.91, 114.80, 109.89, 66.40, 55.25, 53.11,
43.59, 26.65, 24.36. MS (ESI+), m/z: Calcd. 355.2 [M] +; Obsvd. 356.3 [M + H] +. Calcd for
C19H25N5O2 : C, 64.20; H, 7.09; N, 19.70. Found: C, 63.98; H, 7.03; N, 19.55%.
4.3. Computational study
4.3.2. Docking
23

Molecular docking study of the synthesized compounds (1a-1g and 2a-2g) was performed
against Torpedo californica acetylcholinesterase (TcAChE, PDB ID: 1EVE) to study the binding
interactions using VLife MDS 4.3 package.
4.4. Biochemical study
4.4.1. Inhibition of AChE
4.4.1.1. In vitro AChE Inhibition
The inhibition reactions of AChE were determined by the Ellman method. This method is based
on the use of acetylthiocholine iodide (ATChI) as synthetic substrate and 5, 5'-dithiobis-(2-
nitrobenzoic acid) (DTNB) to generate a yellow chromophore (5-mercapto-2-nitrobenzoic acid)
which is colorimetrically detected. Whole mouse brains of LAKA strain (25-30 g) were
decapitated and rinsed with ice cold normal saline solution and homogenized in a glass Teflon
homogenizer (REMI MOTORS, India) containing 50 volumes of sodium phosphate buffer ( pH
8.0, 0.1 M) and centrifuged (Research centrifuge, REMI, R-24) at 10,000 rpm for 20 min at 4 0C
to prepare supernatant. The obtained supernatant was used as a source of enzyme for the assay.
Initially stock solutions of each test drug were prepared and diluted to various concentrations
immediately before use. An aliquot of diluted solution at five different concentration (1, 5, 10, 15
and 25 µl) levels was mixed with phosphate buffer (pH 8.0, 3.0 ml), 100 µl of buffered Ellman’s
reagent, 100 µl of acetylthiocholine iodide solution and 50 µl of supernatant and the change in
optical density was measured immediately at 412 nm for 3 minutes in a PERKIN ELMER
UV/VIS spectrophotometer. The AChE activity was calculated using the formula: AChE degree
= (0.05 x change in optical density) / mg protein. Additionally, the concentration of each
compound required to inhibit acetylcholinesterase activity by 50% (IC50) was calculated by non-
linear regression of the log(concentration)-response curve using GraphPad Prism 5. Rivastigmine
was used as standard drug. The protein estimation was carried out using Biuret method using
Biuret reagent prepared by mixing sodium potassium tartrate (4.5 g ) in 40 ml of 0.2 N NaOH,
cupric sulphate pentahydrate and potassium iodide (0.5 g) and made up the volume 100 ml with
0.2 N NaOH solution. The working solution was prepared by mixing 50 µl of supernatant, 2.9 ml
of normal saline and 3 ml of Biuret reagent and incubated at room temperature for 10 minutes
24

and measured the optical density at 540 nm in the above mentioned UV-VIS spectrophotometer.
The amount of protein content (in mg) was calculated using the standard table based on optical
density. Kinetic studies were performed using six concentrations of substrate ATChI (0.25 – 2.0
mM) in the presence and absence of above mentioned same concentrations of inhibitors. Kinetic
parameters were calculated from the ‘Enzyme kinetic’ module of GraphPad Prism 5.
4.4.1.1. Ex vivo AChE Inhibition
To evaluate the brain permeability of the selected compounds, ex vivo AChE Inhibition assay
was performed. Each compound was intraperitoneally (i.p.) injected to mice at doses of 1.0
mg/kg and 3 mg/kg. After 1 h animals were killed by decapitation, brain was immediately
dissected, washed with ice cold saline and kept in phosphate buffer (pH 8.0). Whole brain was
then homogenate and centrifuged to get the supernatant. The obtained supernatant was used for
assay following Ellman method to measure the protein content and percentage of brain AChE
Inhibition.
4.5. Pharmacological Evaluation (In vivo assay)
4.5.1. Animals
Albino mice (Laka strain, 25-30 g) of both sexes were purchased from Central Animal House,
Panjab University, Chandigarh, India. All the research protocols were approved by the
Institutional Animal Ethical Committee of University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh, India. The treatment and maintenance of animals were conducted
in accordance with the Principle of Laboratory Animal Care. Animals were housed 5 per cage
with food and water available ad libitum and maintained at constant temperature (25 1 0C) and
humidity (60 10%) under a 12 h light / dark cycle. All behavioural task paradigms were
conducted between 10.00 h and 16.00 h.
25

4.5.2. Passive avoidance step-down task paradigm
Passive avoidance test were carried out in a rodent memory evaluator (IMCORP, Ambala Cantt.
India). All the synthesized compounds excluding the intermediates (1 and 2) were evaluated for
antiamnesic activity using mice. Piracetam (2.5 mg/kg, obtained from Brown and Burk
Pharmaceuticals) and rivastigmine (1.0 mg/kg, obtained from Sun Pharmaceutical Industries Ltd.
Silvassa, India) were used as standard drugs. Scopolamine (0. 5 mg/kg i.p.) was used for memory
disruption as amnesic agent. The test drugs were suspended in 0.25% carboxymethylcellulose
(CMC) and administered intraperitoneally (i.p.) to animals. All of the mice were treated with
scopolamine before experiments. Control group was not treated by any drug.
The rodent memory evaluator consists of a fabricated box with electric grid and a centrally
located shock free zone (SFZ). It has an electric display system where the voltage current is
controlled and the timer is displayed. By keeping the equipment switch off, each mouse was
placed individually on the electric grid and allowed to explore the apparatus for about one
minute. The equipment was turned on and switch on the latency mode. A proper voltage current
(20 V) for foot shock was selected and put the animal on the electric grid and the start button was
pressed. Noted down the latency (in seconds) i.e. the time taken by the mouse to reach the SFZ
as displayed in the timer. The animals were rejected which took more than 2 minutes to reach
SFZ. Put the animal back on the electric grid and repeated it thrice to get three basal readings to
acquainted the animals with the task (trained). After one hour of the training, placed the animal
on the electric grid one more time and noted the latency. Then turned on the mistake mode and
count down the number of mistakes made by animal in 15 minutes as soon as touched the
electric grid and felt shock. Compared the basal latency (recorded three readings I, II and III for
each animal from each group) in reaching SFZ of each compound by control and scopolamine
treated animals. Recorded basal latency data were analyzed and significance was determined by
applying two-way ANOVA statistical technique followed by Bonferroni posttest for each
compound with respect to control group and scopolamine treated group in a GraphPad Prism 5.
Data are expressed by mean SEM. Memory parameters (latency and no. of mistakes) are
analyzed by non-parametric method using GraphPad Prism.
26

4.6. Antiradical activity
Antiradical activities of the synthesized compounds were examined using DPPH (1, 1- diphenyl-
2-picrylhydrazyl) method. Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid)
was used as standard. 1 ml solution of each compound at different concentration (1, 5, 25, 50 and
100 µg/ml) in methanol was mixed with 2 ml of DPPH solution (0.5 mM). The mixture was
shaken vigorously and allowed to stand at room temperature for 30 min. The absorbance was
measured at 517 nm against methanol as the blank in a spectrophotometer. Higher antiradical
activity observed by the lower absorbance of the reaction mixture. Experiment was accomplished
in triplicate. The antiradical activity was calculated in percentage as per the following formula:
antiradical activity (%) = (absorbance of control - absorbance sample / absorbance of control) x
100.
Statistical study
Statistical analysis was performed using GraphPad Prism 5. Statistical significance was accepted
for P values of < 0.05.
Acknowledgements
This work was supported by University Grant Commission (UGC), New Delhi, India in a
Research Fellowship for Meritorious Student (RFMS) Scheme. We are also thankful to
Sophisticated Advanced Instrumentation Facilities (SAIF), Panjab University, Chandigarh, India
for providing Mass and NMR data.
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28

Graphical abstract
O
O
O
N
N
H3C
N
H
Compound 1b: Mouse AChE, IC50 = 1.66 µM, Ki = 43.66 µM
F
N
O
N
O
H3C
N
H
Compound 2d: Mouse AChE, IC50 = 0.49 µM, Ki = 4.10 µM
29