Synthesis, evaluation and molecular dynamics study of some new 4-aminopyridine semicarbazones as an antiamnesic and cognition enhancing agents

Article abstract of DOI:10.1016/j.bmc.2013.06.003

Some new semicarbazones of 4-aminopyridine were synthesized and evaluated for antiamnesic, cognition enhancing and anticholinesterase activities. The results illustrated a significant cognition enhancing effect on elevated plus maze model with a significant reversal of scopolamine-induced amnesia. A significant inhibition in acetycholinesterase (AChE) activity by all the synthesized compounds in specific brain regions that is, prefrontal cortex, hippocampus and hypothalamus was observed. Compound 4APi exhibited significant antiamnesic and cognition enhancing activity which was comparable with standard drug donepezil. Its enzyme kinetic study revealed a non-competitive inhibition of AChE and a competitive inhibition of butyrylcholinesterase (BChE). Docking studies predicted the binding modes of these compounds in AChE active site, which were further processed for molecular dynamics simulation for calculating binding free energies using Molecular Mechanics-Generalized Born Surface Area (MM/GBSA). All the computational study confirmed their consensual interaction with AChE justifying the experimental outcome.

Full text of DOI:10.1016/j.bmc.2013.06.003

Bioorganic & Medicinal Chemistry 21 (2013) 5451–5460
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, evaluation and molecular dynamics study of some new
4-aminopyridine semicarbazones as an antiamnesic and cognition
enhancing agents
⇑
Saurabh K. Sinha, Sushant K. Shrivastava
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology, Banaras Hindu University, Varanasi, U.P. 221005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new semicarbazones of 4-aminopyridine were synthesized and evaluated for antiamnesic, cogni-
tion enhancing and anticholinesterase activities. The results illustrated a significant cognition enhancing
effect on elevated plus maze model with a significant reversal of scopolamine-induced amnesia. A signif-
icant inhibition in acetycholinesterase (AChE) activity by all the synthesized compounds in specific brain
regions that is, prefrontal cortex, hippocampus and hypothalamus was observed. Compound 4APi exhib-
ited significant antiamnesic and cognition enhancing activity which was comparable with standard drug
donepezil. Its enzyme kinetic study revealed a non-competitive inhibition of AChE and a competitive
inhibition of butyrylcholinesterase (BChE). Docking studies predicted the binding modes of these com-
pounds in AChE active site, which were further processed for molecular dynamics simulation for calcu-
lating binding free energies using Molecular Mechanics–Generalized Born Surface Area (MM/GBSA). All
the computational study confirmed their consensual interaction with AChE justifying the experimental
outcome.
Received 19 April 2013
Revised 2 June 2013
Accepted 4 June 2013
Available online 11 June 2013
Keywords:
4-Aminopyridine
Nootropic
Antiamnesic
Anticholinesterase
Enzyme kinetic study
Molecular dynamics
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
gands at the peripheral anionic site constituted by amino acid res-
idues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341.12,13
Alzheimer’s disease is a progressive neurodegenerative disor-
der, characterised by selective loss of cholinergic neurons and
accumulation of b-amyloid protein in the selective brain areas such
as cortex and hippocampus.1,2 Loss of memory, cognitive decline,
impaired performance of activities of daily life and behavioural
changes are hallmark of Alzheimer’s disease.3–5 Augmentation of
cholinergic neurotransmission in the brain plays a pivotal role in
the treatment strategy of Alzheimer’s disease. Various drugs, clin-
ically used to treat Alzheimer’s disease elevates the level of acetyl-
choline at the synapse by inhibiting the metabolising enzyme
AChE.6,7 Through various molecular docking and dynamics study
of different AChE inhibitors, it has been concluded that, the active
centre of human acetylcholinesterase (hAChE) consists of several
major domains such as, an esteratic site8 containing the catalytic
triad Ser-203, His-447 and Glu-334, an anionic site (Trp-86) that
Currently, derivatives of 4-aminopyridine (4AP) are under inten-
sive investigation owing to their antiacetylcholinesterase activity
which have shown promising effects in alleviating memory related
dysfunctions.14,15 Several carbamate derivatives of 4AP and Schiff
bases of styrylpyridine have been synthesized and evaluated for
their anticholinesterase activity.16,17 Some 4-aminobutyric acid
(GABA) and 2-indolinone derivatives of 4AP have been also re-
ported to possess antiamnesic activity.18 Also, the hydrazone
derivatives of dihydropyridine and indolinones have been reported
as potent anticholinesterase, antibutyrylcholinesterase and b-amy-
loid aggregation properties.19,20 3-Methylpyridinium and 2-thio-
naphthol derivatives of berberine have evaluated for AChE and
BChE inhibitory activity, respectively.21 Some quinazolinimines
have been shown selective butyrylcholinesterase inhibitory activ-
ity.22,23 A number of N-benzylpiperidine–purine derivatives have
been reported as dual inhibitors of AChE and BChE.24
binds through cation–p interactions with the quaternary ammo-
nium of choline and hydrophobic sites which binds aryl substrates
and other uncharged ligands.9–11 The allosteric modulation of
hAChE catalytic activity is possible through binding of some li-
In our earlier studies, we have reported some new schiff bases,
anilide and imide derivatives of 4AP for their cognition enhancing,
antiamnesic and anticholinesterase activities among which benzo-
phenone and imide derivatives were found to be highly significant.
In continuation of our previous research work25,26 we have de-
signed and synthesized some new semicarbazones of 4AP having
potential antiamnesic and cognition enhancing activities.
⇑
Corresponding author. Tel.: +91 945 2156527; fax: +91 542 2368428.
E-mail addresses: sksinha.rs.phe@itbhu.ac.in, skshrivastava.phe@itbhu.ac.in
(S.K. Shrivastava).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.003

5452
S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
tives to gain an insight on their nature of inhibition (Table 2).
2. Results and discussion
2.1. Chemistry
The most active compound 4APi demonstrated a non-competitive
inhibition for AChE (Ki = 0.082 0.60) and competitive inhibition
for BChE (Ki = 16.82 0.76) enzymes. The non-competitive inhibi-
tion29 is attributed to a possible interaction of compound with
the peripheral anionic site (PAS) of AChE and was also confirmed
by docking studies. The synthesized derivatives were then evalu-
ated for antiamnesic and cognition enhancing activities in rat ele-
vated plus maze (EPM) model30 which is a simple method for
assessment of learning and memory that depends upon measuring
the transfer latency of rats. Rats are known for their natural dislike
for open and high spaces. In these types of studies, animals usually
spend more time in enclosed arms rather than open arms in plus
maze test.31 This behaviour suggest that the transfer latency re-
duces day by day, if the animal have previous experience to move
from the open arms to the enclosed arms, which attributed to an
enhanced memory. Pre-treatment with tested compounds resulted
in reduced transfer latency as compared to control group on first
and second day of EPM exposure in significant and dose dependant
manner, indicating facilitated learning process (Fig. 1). Scopol-
amine (1.5 mg/kg), significantly increased the transfer latency as
compared to control group (p <0.001), resulting in amnesia which
was reversed significantly by tested compounds and donepezil32 (p
<0.001) (Table 3).
4-Aminopyridine urea (4APa) was synthesized by treating 4-
aminopyridine with sodium cyanate in presence of glacial acetic
acid. The urea derivative on condensation with hydrazine hydrate
with ethanol in the presence of sodium hydroxide yielded the 4-
aminopyridine semicarbazide (4APb). The semicarbazones
(4APc–j) were prepared by reaction of the appropriate aldehyde
or ketone with 4-aminopyridine semicarbazide as represented in
Scheme 1. The purity of compounds was checked by TLC and all
synthesized derivatives were characterised by FT-IR, 1H, 13C NMR
and elemental analysis. In IR, peak of C@N and NH streching vibra-
tions was observed at 1590 cmꢀ1 and 3433–3326 cmꢀ1, respec-
tively. In 1H NMR spectra, compounds 4APa and 4APb showed
peak at d 6.51 ppm due to the presence of ꢀNH2 proton. In subse-
quent steps of synthesis, both the protons of ꢀNH2 were substi-
tuted by different groups which resulted into the formation of
N@CH (4APc–h) and N@C (4APi–j) bond that caused the disap-
pearance of peak at d 6.51 ppm, confirming the substitution. The
13C NMR value of d 155–158 ppm also confirmed the formation
of N@CH and N@C bond.
Prefrontal cortex, hippocampus and hypothalamus are three
specific brain regions involved in processing of memory with high
innervations of cholinergic neurons. It is reported that 50–90%
reduction in ChAT, an enzyme which synthesizes acetylcholine is
observed in hippocampus, cortex and hypothalamus in patients
with dementia of Alzheimer’s Type (DAT).33,34 Hippocampus is per-
manently involved in memories that have to be acquired and re-
trieved. It is also temporarily involved in the process of memory
2.2. Biological activity
The IC50 values of all the derivatives on AChE and BChE was
determined as per Ellman method.27 For AChE inhibitory activity,
4APi was found to elicit comparable activity (0.052 0.01
and selectivity (355) with respect to standard donepezil
(0.04 0.012 M), (381) among all the derivatives (Table 1). Fur-
ther, enzyme kinetics study28 was also performed for all deriva-
lM)
l
Scheme 1. The synthetic pathway of the compounds here presented.

S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
5453
Table 1
Cholinesterase activity and binding free energy of synthesized derivatives and donepezil
b
Compound
AChE
IC50
BChE
IC50
Selectivity for AChEa
pIC50 (M) AChE
DGGBSA (kcal/mol)
(
lM) SEM
(
lM) SEM
4Apa
4Apb
4Apc
4Apd
4Ape
4Apf
4Apg
4Aph
4Api
274.3 3.978
211.3 2.812
82.3 1.333
32.5 1.359
4.26 1.756
48.3 1.485
12.7 1.480
18.7 1.756
0.052 0.01
0.145 0.842
0.04 0.012
>1000
>1000
—
—
3.56
3.67
4.08
4.48
5.37
4.31
4.89
4.72
7.28
6.83
7.39
ꢀ33.8719
ꢀ36.1949
ꢀ45.7731
ꢀ51.0306
ꢀ60.4941
ꢀ48.9383
ꢀ55.4814
ꢀ53.6457
ꢀ77.0588
ꢀ69.8529
ꢀ78.1427
845.2 4.672
245.8 1.854
58.25 1.269
786.5 2.272
426.4 2.642
528.4 4.472
18.46 0.86
0.358 0.64
15.24 0.88
10.26
7.56
13.67
16.28
33.57
28.25
355
4Apj
Donepezil
2.46
381
a
Selectivity for AChE is defined as IC50 (BChE)/IC50 (AChE).
Binding free energy calculated by molecular mechanics-generalized born surface area (MM/GBSA) present in PRIME 3.1 (Desmond software, Schrödinger, LLC, USA),
b
pIC50 = log(1/IC50).
Table 2
Enzyme kinetics study of synthesized derivatives and donepezil
Compound
Inhibition
AChE
Inhibition
BChE
Ki (l
M) SEM
Ki (lM) SEM
4Apa
4Apb
4Apc
4Apd
4Ape
4Apf
4Apg
4Aph
4Api
nt
nt
c
c
c
c
c
c
nt
nt
nt
nt
c
nc
nc
c
c
nc
c
nt
nt
88.6 2.27
25.6 1.480
3.56 0.88
685.2 11.7
212.4 4.85
72.68 1.75
624.425 6.8
282.62 6.8
446.48 6.47
16.82 0.76
0.628 0.84
12.54 0.76
35.46 2.4
10.254 1.285
20.52 0.90
0.082 0.60
0.246 0.66
0.056 0.016
nc
nc
nc
4Apj
Donepezil
c
nc
c = competitive, nc = non-competitive, nt = not tested.
consolidation and as an area for the temporary storage of the to-
be-consolidated information.35 This information is further relayed
to prefrontal cortex, where short-term memory is converted into
long term memory by a process called consolidation.36 Several
investigations have also reported that the stimulation of hypothal-
amus facilitates hippocampus dependent learning and memory
processes in both young and aged rats.37 Therefore, ex vivo AChE
activity was determined, which showed that all the synthesized
compounds inhibited AChE activity in distinct brain regions that
is, prefrontal cortex, hippocampus and hypothalamus compared
to control (p <0.01, p <0.05 and p <0.001), respectively (Table 4).
These results also confirmed the ability of semicarbazones of 4AP
to enhance cholinergic neurotransmission in these distinct brain
regions and attributed to enhanced learning and memory functions
due to their anti-cholinesterase activity. Acute toxicity studies did
not show any signs of toxicity and mortality up to a dose of
100 mg/kg of body weight as evident through a normal behav-
ioural pattern of rats for the desired period of observation.
Figure 1. (A) Effect of synthesized derivatives and donepezil on learning impair-
ment on elevated plus maze in wistar rats at dose (5 mg/kg), (B) at dose (10 mg/kg).
Data are expressed as mean SEM (n = 6). Data were statistically analyzed by one-
way ANOVA. ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 as compared to control (vehicle treated)
group.
against the starting structures. The plot suggests that, RMSD of
the backbone has a significant stability over the course of MD sim-
ulation and reaches equilibrium within 2 ns with average fluctua-
tion of two RMSD. In most of the snapshots of the production
2.3. Molecular docking and dynamics simulations
To elucidate the importance of protein flexibility in ligand bind-
ing, the active compounds were docked into pocket of AChE and
the highest scoring pose along with protein was subjected to
molecular dynamics simulations. Docking was performed using
Glide extra precision (XP) and further molecular dynamics simula-
tion was processed in explicit aqueous solution for 5 ns. The stabil-
ity of the system under simulation was evaluated by the root-
mean-square deviations (RMSD) of the backbone atoms. Figure 2
shows the RMSD values of the backbone of system computed
phase, pyridine ring of 4APi displayed
p–p stacking with the six-
member ring of the Trp-286 indole and in some snaps of the sim-
ulation amide NH of the ligand was showing hydrogen bond with
backbone carbonyl oxygen of Tyr-341. While analysing the root
mean square fluctuation (RMSF) for finding high fluctuating
areas in molecule, the amino acids of two major loops one near
the C-termina region viz. 70–80 amino acids and other extending
from amino acids 285 to 303, were displaying fluctuations ranging

5454
S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
Table 3
Reverse effect of synthesized derivative on scopolamine-induced amnesia on elevated plus maze in rats
Treatment [dose(mg/kg)]
Transfer latency (s)
Average transfer latency
(Day 1 and Day 2)
Day 1
Day 2
Control
SCP (1.5)
67.83 0.94
87.00 0.89
80.83 0.79
77.83 0.60
78.00 0.57
75.50 0.56
74.17 0.47
70.33 0.66
70.17 0.60
67.33 0.88
65.57 0.47
65.17 0.88
70.17 0.60
67.83 0.83
67.17 0.60
64.83 0.60
68.17 0.47
65.00 0.57
64.57 0.84
61.83 0.60
65.33 0.49
64.17 0.60
64.20 0.76
61.25 0.47
62.17 0.47
81.17 0.60a
75.00 0.73b
73.00 0.57b
71.83 0.60b
69.83 0.47b
69.33 0.49b
66.17 0.60b
65.00 0.60b
63.67 0.49b
61.83 0.60b
57.67 0.66b
65.83 0.57b
63.33 0.66b
62.17 0.60b
57.17 0.60b
64.50 0.76b
57.87 0.76b
56.13 0.88b
54.17 0.60b
60.50 0.76b
56.83 0.70b
55.42 0.49b
53.86 0.60b
65
84.085
77.915
75.41
74.91
72.66
71.75
68.25
67.58
65.5
63.7
61.42
68
65.58
64.67
61
66.33
61.43
60.35
58
4APa (5.0) + SCP(1.5)
4APa (10.0) + SCP
4APb (5.0) + SCP
4APb (10.0) + SCP
4APc (5.0) + SCP
4APc (10.0) + SCP
4APd (5.0) + SCP
4APd (10.0) + SCP
4Ape (5.0) + SCP
4Ape (10.0) + SCP
4APf (5.0) + SCP
4APf (10.0) + SCP
4APg (5.0) + SCP
4APg (10.0) + SCP
4APh (5.0) + SCP
4APh (10.0) + SCP
4APi (5.0) + SCP
4APi (10.0) + SCP
4APj (5.0) + SCP
4APj (10.0) + SCP
Donepezil (5.0) + SCP
Donepezil (10.0) + SCP
62.91
60.5
59.81
57.55
Data are expressed as mean SEM (n = 6). Data were statistically analyzed by one way ANOVA.
SCP = Scopolamine.
a
Significantly different from control (vehicle treated) group p <0.001.
Significantly different from scopolamine treated group p <0.001.
b
Table 4
Effect of synthesized derivatives on acetylcholinesterase (AchE) activity on different region of rat brain
Treatment [dose(mg/kg)]
‘n’ moles of substrate hydrolyzed/min/mg protein
Prefrontal cortex
Hippocampus
Hypothalamus
Control
45.83 0.94
47.50 0.84
40.50 0.76
4APa (5.0)
4APa (10.0)
4APb (5.0)
4APb (10.0)
4APc (5.0)
4APc (10.0)
4APd (5.0)
4APd (10.0)
4Ape (5.0)
4Ape (10.0)
4APf (5.0)
4APf (10.0)
4APg (5.0)
4APg (10.0)
4APh (5.0)
4APh (10.0)
4APi (5.0)
43.00 0.73*
44.50 0.76*
37.50 0.56*
37.33 0.49*
37.47 0.60*
37.17 0.60*
36.50 0.99**
35.83 0.90***
36.33 0.49***
34.67 0.80***
34.83 0.49***
32.50 0.84***
35.17 0.47***
36.83 0.49***
35.33 0.84***
33.17 0.57***
35.83 0.60***
33.67 0.70***
31.00 0.57***
29.17 0.60***
34.13 0.60***
31.50 0.92***
28.54 0.49***
26.82 0.57***
42.50 0.76**
42.33 0.60**
42.17 0.66**
42.03 0.66**
40.50 0.42***
39.33 0.49***
37.50 0.42***
36.17 0.79***
34.83 0.60***
39.50 0.42***
37.83 0.60***
36.87 0.76***
35.17 0.94***
36.62 0.60***
35.50 0.42***
32.67 0.49***
30.00 0.57***
35.50 0.42***
33.83 0.01***
31.25 0.42***
28.24 0.76***
44.17 0.60**
44.50 0.76**
43.67 0.66**
42.33 0.66***
41.00 0.57***
39.17 0.60***
36.83 0.47***
37.50 0.76***
35.03 0.60***
40.33 0.49***
37.50 0.42***
38.17 0.60***
35.83 0.60***
39.50 0.42***
36.83 0.49***
33.67 0.49***
32.00 0.57***
36.33 0.49***
34.17 0.60***
32.38 0.60***
30.86 0.60***
4APi (10.0)
4APj (5.0)
4APj (10.0)
Donepezil (5.0)
Donepezil (10.0)
Data are expressed as mean SEM (n = 6). Data were statistically analyzed by one way ANOVA.
*
p <0.05 as compared to control (vehicle treated) group.
p <0.01 as compared to control (vehicle treated) group.
p <0.001 as compared to control (vehicle treated) group.
**
***
2.5 Å. Due to these fluctuations the ligand was adopting numerous
orientations. To explore the best possible conformation of the
receptor and the ligand, while they are binding, all the snapshots
of the simulation were clustered according to RMSD.
2.3.1. Binding free energy calculation and docking analysis
All the compounds were docked into the pocket of the cluster
representatives and their binding affinities were calculated using
the MM/GBSA method. The correlation between experimental

S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
5455
Figure 2. RMSD values of the backbone of system computed against the starting
structures.
Figure 3. Correlation between MM/GBSA predicted binding affinities and experi-
mentally determined pIC50 values against AChE.
Figure 4. The binding mode of (A) 4APi and (B) donepezil in the AChE binding
pocket. The hydrogen bonds are displayed as dashed yellow.
activities and calculated binding free energies of all the models are
shown in Figure 3. The cluster-3 has generated highest correlation
of 0.91252, where binding free energies of this model displayed a
good correlation with respect to the experimental activity and
hence this confirmation was further considered for analysing the
structure–activity relationship of the inhibitors with respect to
the receptor.
The compound 4APi formed strong hydrophobic and hydrogen
bond interactions with the amino acids binding site of AChE. The
protonated nitrogen of pyridine ring showed a hydrogen bond with
backbone oxygen of Thr-75, while the amine groups of urea moiety
formed a bifurcated hydrogen bond with backbone oxygen of Tyr-
341. Donepezil was observed to establish H-bond with Phe-295
backbone and showed interaction with the internal amino acid res-
idue Trp-286 by means of a p–p interaction (Fig. 4). From the IC50
values and selectivity data presented in Table 1, it is inferred that
substitution of the second hydrogen from imine carbon of semicar-
bazone increases the activity. In 4APi, where hydrogen atom was
replaced by a phenyl moiety, a greater AChE inhibitory activity
was observed. Similarly in derivative 4APj, where methyl substitu-
tion replaces the hydrogen, an increase in both AChE as well as
BChE inhibitory activity was seen. In case of 4APi, two phenyl rings
of biphenyl group were well placed in the hydrophobic pockets
formed by Tyr-341, Phe-338, Val-294, Phe-297, Trp-286, Leu-289,
Pro-290 and exhibited strong hydrophobic interactions (Figs. 5
and 6). Due to these interactions, 4APi showed a lower binding free
energy (ꢀ73.0588 kcal/mol) and therefore, had more favourable
Figure 5. Hydrophobic interactions of 4APi in the AChE binding pocket.
binding affinity than other compound (Table 1). In case of other
compounds 4APc, 4APd, 4APe, 4APf, 4APg and 4APh a reduced
biological activity was observed which may be attributed to the
lack of biphenyl ring and thus missed the strong hydrophobic

5456
S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
Figure 6. Surface representation of the binding pocket of AChE and colored by
binding property; White = neutral surface, Green = hydrophobic surface,
Red = hydrogen bonding acceptor potential, and Blue = hydrogen bond donor
potential.
interactions with receptor (Fig. in Supplementary data). Since 4APj
showed good activity against BChE and 4APi against AChE so to
understand the differences in interactions the compounds 4APi
and 4APj were also docked to the active site of BChE. In AChE,
the acyl-binding pocket is formed by two phenylalanine molecules
(Phe-295 and Phe-297), while in BChE, these two aromatic amino
acid residues are replaced by two smaller amino acid residues,
Leu-286 and Val-288. The key peripheral anionic site (PAS) resi-
dues i.e. Trp-286, Tyr-124 and Tyr-341 of AChE are replaced by
Tyr-332 and Ala-277. This structural difference causes a conforma-
tional change, thus providing a larger space in the deepest area of
the BChE cleft. It was observed that, both the compounds were
properly positioned into the enzyme gorge Ser-287, Ser-72, Ala-
277, Tyr-341, Val-288, Pro-285, Lue-286, Gln-71 and Glu-276.
4APj showed interaction with the internal amino acid residue
Tyr-332 (analogous to Tyr-341 in hAChE) and Ala-277 (analogous
to Trp-286 in hAChE) by means of a
p–p interaction. In addition,
the carbonyl oxygen of 4APj was also involved in forming a hydro-
gen bond with Ser-72 (Fig. 7). However, from the overall results
4APJ was found to be predominant for BChE and 4APi for AChE
inhibitory activity.
3. Conclusions and future directions
Thus, from the above study we have successfully identified a
new class of potent cognition enhancing and antiamnesic drugs.
Among the identified compounds, compound 4APi deserves fur-
ther studies which can lead to a discovery of a new lead having a
potent cognition enhancing and antiamnesic properties.
Figure 7. The binding mode of (A) 4APi and (B) 4APj in the BChE binding pocket.
The hydrogen bonds are displayed as dashed yellow.
4. Experimental
4.1. General
DMSO-d6. Elemental analysis was performed using Exeter CE-440
elemental analyzer. For statistical analysis one-way analysis of var-
iance (ANOVA) followed by Dunnet’s post hoc test was performed
using the Graph Pad Prism version 5.0 for Windows (Graph Pad
Software, Inc., 2009).
All reagents and solvents used in the study were of analytical
grade purity and were procured from Sigma–Aldrich (India). Melt-
ing point of the compounds was determined in open capillary
tubes using a BI 9300 Bumstead/electrothermal Stuart (SMPIO)
melting point apparatus and is uncorrected. The reaction progress
was monitored by thin layer chromatography with chloroform:
methanol (6:4) as the mobile phase on TLC silica gel 60 F254 alu-
minium sheets (Merck, India). UV spectra analysis was performed
on JASCO (Model 7800) UV–vis spectrophotometer. FTIR spectra
were recorded on a Shimadzu FTIR 8400S spectrophotometer at
4.2. Chemistry
4.2.1. General procedure for the synthesis of compounds
4APa–b
4-Aminopyridine (0.01 mol) was dissolved in 5 ml of glacial
acetic acid and was diluted to 25 ml with distilled water. To this
equimolar (0.01 mol) quantity of NaCNO in 25 ml of warm water
was added with continuous stirring. The reaction mixture was al-
lowed to stand for 4 h and the resultant product 1-(pyridin-4-
yl)urea (4APa) was collected by filtration, then washed with water
the scanning range of 400–4000 cmꢀ1
were recorded on a JEOL AL 300 FT-NMR spectrophotometer in
.
1H and 13C NMR spectra

S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
5457
and recrystallized from 95% ethanol.38 Equimolar quantities
(0.01 mol) of 4APa and hydrazine hydrate were dissolved in 5 ml
of ethanol and refluxed for 3 h in presence of 0.01 mol sodium
hydroxide. The resultant precipitate was filtered, dried and recrys-
tallised in 95% ethanol to yield 4-(pyridin-4-yl)semicarbazide
(4APb).39
4.2.2.4. (E)-1-(4-Methoxybenzylidene)-4-(pyridin-4-yl)semicarb-
azide (4APf). Yield: 68.2%, mp: 242–244 °C, Rf 0.40, IR (KBr,
m
cmꢀ1): 3433, 3326 (NH), 3061 (CH, CH3), 1680 (C@O), 1590, 1580
(C@N), 1475 (C@C, aromatic); 1H NMR (DMSO-d6) (d ppm): 10.58,
9.53 (br s, 2H, NH), 8.35 (d, J = 4.5, 2H, pyridine), 7.94 (s, 1H, N@CH),
7.65–7.49 (m, 4H, aromatic), 6.75 (d, J = 4.5, 2H, pyridine), 4.35 (s,
3H, CH3); 13C NMR (d ppm): 169.23 (C@O), 155.0 (N@CH), 154.35,
150.5, 109.12 (pyridine), 139.66, 130.82, 129.78, 128.28 (aromatic),
55.80 (CH3); Anal. Calcd (%) for C14H14N4O2: C, 62.21; H, 5.22; N,
20.73; found (%) C, 62.12; H, 5.19; N, 20.65.
4.2.1.1. 1-(Pyridin-4-yl)urea (4APa). Yield: 86.0%, mp: 212–
214 °C, Rf 0.65, IR (KBr,
m
cmꢀ1): 3433, (NH), 3326, 3121 (doublet
NH2), 1680 (C@O), 1590, 1580 (C@N), 1475 (C@C, aromatic); 1H
NMR (DMSO-d6) (d ppm): 8.93 (br s, 1H, NH), 8.37 (d, J = 4.8, 2H,
pyridine), 6.77 (d, J = 4.8, 2H, pyridine), 6.51 (s, 2H, NH2); 13C
NMR (d ppm): 169.46 (C@O), 154.25, 150.0, 109.53 (pyridine);
Anal. calcd (%) for C6H7N3O: C, 52.55; H, 5.14; N, 30.64; found
(%) C, 52.32; H, 5.20; N, 30.55.
4.2.2.5. (E)-1-(3,4-Dimethoxybenzylidene)-4-(pyridin-4-yl)semi-
carbazide (4APg). Yield: 58.6%, mp: 220–222 °C, Rf 0.37, IR (KBr,
m
cmꢀ1): 3433, 3326 (NH), 3061 (CH, CH3), 1680 (C@O), 1590,
1580 (C@N), 1475 (C@C, aromatic); 1H NMR (DMSO-d6) (d ppm):
10.53, 9.52 (br s, 2H, NH), 8.46 (d, J = 4.8, 2H, pyridine), 7.92 (s,
1H, N@CH), 7.61–7.50 (m, 3H, aromatic), 6.81 (d, J = 4.8, 2H, pyri-
dine), 4.34 (s, 6H, CH3); 13C NMR (d ppm): 169.85 (C@O), 155.76
(N@CH), 154.28, 150.16, 109.58 (pyridine), 139.12, 130.62, 129.38,
128.26 (aromatic), 55.56 (CH3); Anal. Calcd (%) for C14H14N4O2: C,
62.21; H, 5.22; N, 20.73; found (%) C, 62.14; H, 5.18; N, 20.65.
4.2.1.2. 4-(Pyridin-4-yl)semicarbazide (4APb). Yield: 82.3%,
mp: 230–232 °C, Rf 0.50, IR (KBr,
m
cmꢀ1): 3433, (NH), 3326,
3121 (doublet NH2), 1680 (C@O), 1590, 1580 (C@N), 1475 (C@C,
aromatic); 1H NMR (DMSO-d6) (d ppm): 10.51, 9.35 (br s, 2H,
NH), 8.37 (d, J = 4.8, 2H, pyridine), 6.77 (d, J = 4.8, 2H, pyridine),
6.51 (s, 2H, NH2); 13C NMR (d ppm): 159.10 (C@O), 154.35,
150.61, 109.43 (pyridine); Anal. calcd (%) for C6H8N4O: C, 47.36;
H, 5.30; N, 36.82; found (%) C, 47.18; H, 5.26; N, 36.88.
4.2.2.6. (E)-1-(4-Hydroxy-3-methoxybenzylidene)-4-(pyridin-4-
yl)semicarbazide (4APh). Yield: 62.4%, mp: 247–249 °C, Rf 0.52,
IR (KBr,
m
cmꢀ1): 3517 (OH), 3433, 3326 (NH), 1680 (C@O), 1590,
4.2.2. General procedure for the synthesis of compounds 4APc–j
To the solution of 4APb (0.456 g, 0.003 mol) in 5 ml of ethanol,
an equimolar quantity of the appropriate aldehyde or ketone was
added. The pH of the reaction mixture was adjusted between 5
and 6 by adding glacial acetic acid. The reaction mixture was re-
fluxed for 2 h. The product obtained after cooling was filtered,
dried and crystallized in 95% ethanol.40
1580 (C@N), 1475 (C@C, aromatic); 1H NMR (DMSO-d6) (d ppm):
11.25 (s, 1H, OH), 10.59, 9.54 (br s, 2H, NH), 8.42 (d, J = 4.5, 2H, pyr-
idine), 7.92 (s, 1H, N@CH), 7.62–7.52 (m, 3H, aromatic), 6.80 (d,
J = 4.5, 2H, pyridine), 4.35 (s, 3H, CH3); 13C NMR (d ppm): 169.27
(C@O), 155.12 (N@CH), 154.62, 150.30, 109.56 (pyridine), 139.63,
130.41, 129.12, 128.0 (aromatic), 55.63 (CH3); Anal. Calcd (%) for
C14H14N4O3: C, 58.73; H, 4.93; N, 19.57; found (%) C, 58.64; H,
4.96; N, 19.48.
4.2.2.1. (E)-1-Benzylidene-4-(pyridin-4-yl)semicarbazide
(4APc). Yield: 76.6%, mp: 182–184 °C, Rf 0.58, IR (KBr,
m
4.2.2.7. 1-(Diphenylmethylene)-4-(pyridin-4-yl)semicarbazide
cmꢀ1): 3433, 3326 (NH), 1680 (C@O), 1590, 1580 (C@N), 1475
(C@C, aromatic); 1H NMR (DMSO-d6) (d ppm): 10.52, 9.52 (br s,
2H, NH), 8.45 (d, J = 5.1, 2H, pyridine), 7.93 (s, 1H, N@CH), 7.62–
7.41 (m, 5H, aromatic), 6.76 (d, J = 5.1, 2H, pyridine); 13C NMR (d
ppm): 169.85 (C@O), 155.22 (N@CH), 154.18, 150.35, 109.55 (pyr-
idine), 130.75, 129.12, 128.35 (aromatic); Anal. Calcd (%) for
(4APi). Yield: 66.8%, mp: 230–232 °C, Rf 0.60, IR (KBr, m
cmꢀ1):
3433, 3326 (NH), 1680 (C@O), 1590, 1580 (C@N), 1475(C@C, aro-
matic); 1H NMR (DMSO-d6) (d ppm): 10.51, 9.51 (br s, 2H, NH),
8.41 (d, J = 4.2, 2H, pyridine), 7.57–7.35 (m, 10H, aromatic), 6.78
(d, J = J = 4.2, 2H, pyridine); 13C NMR (d ppm): 169.75 (C@O),
156.54 (N@C), 154.35, 150.14, 109.62 (pyridine), 130.26, 129.63,
128.15 (aromatic); Anal. Calcd (%) for C19H16N4O: C, 72.13; H,
5.10; N, 17.71; found (%) C, 72.04; H, 5.06; N, 17.65.
C13H12N4O: C, 64.99; H, 5.03; N, 23.32; found (%) C, 64.74; H,
5.10; N, 23.25.
4.2.2.8.
yl)semicarbazide (4APj). Yield: 70.2%, mp: 212–214 °C, Rf
0.62, IR (KBr,
cmꢀ1): 3517 (OH), 3433, 3326 (NH), 3061 (CH,
(E)-1-(1-(4-Hydroxyphenyl)ethylidene)-4-(pyridin-4-
4.2.2.2. (E)-1-(2-Hydroxybenzylidene)-4-(pyridin-4-yl)semicarb-
azide (4APd). Yield: 72.8%, mp: 196–198 °C, Rf 0.48, IR (KBr,
m
m
cmꢀ1): 3517 (OH), 3433, 3326 (NH), 1680 (C@O), 1590, 1580
(C@N), 1475 (C@C, aromatic); 1H NMR (DMSO-d6) (d ppm): 11.16
(s, 1H, OH), 10.51, 9.55 (br s, 2H, NH), 8.39 (d, J = 4.5, 2H, pyridine),
7.93 (s, 1H, N@CH), 7.63–7.53 (m, 4H, aromatic), 6.73 (d, J = 4.5, 2H,
pyridine); 13C NMR (d ppm): 169.0 (C@O), 155.24 (N@CH), 154.15,
150.63, 109.0 (pyridine), 139.23, 130.61, 129.52, 128.86 (aromatic);
Anal. Calcd (%) for C13H12N4O2: C, 60.93; H, 4.72; N, 21.86; found (%)
C, 60.72; H, 4.69; N, 21.75.
CH3), 1680 (C@O), 1590, 1580 (C@N), 1475 (C@C, aromatic); 1H
NMR (DMSO-d6) (d ppm): 10.55, 9.62 (br s, 2H, NH), 8.44 (d,
J = 4.5, 2H, pyridine), 7.73–7.62 (m, 3H, aromatic), 6.76 (d, J = 4.5,
2H, pyridine), 6.16 (s, 1H, OH), 1.25 (s, 3H, CH3); 13C NMR (d
ppm): 169.25 (C@O C@O), 157.63 (N@C), 154.13, 150.39, 109.61
(pyridine), 139.29, 130.52, 129.12, 128.21 (aromatic), 27.85
(CH3); Anal. Calcd (%) for C14H14N4O2: C, 62.21; H, 5.22; N, 20.73;
found (%) C, 62.32; H, 5.20; N, 20.69.
4.2.2.3. (E)-1-(2,4-Dihydroxybenzylidene)-4-(pyridin-4-yl)semi-
carbazide (4APe). Yield: 64.6%, mp: 206–208 °C, Rf 0.42, IR (KBr,
m
4.3. Biological studies
cmꢀ1): 3517 (OH), 3433, 3326 (NH), 1680 (C@O), 1590, 1580
4.3.1. Estimation of cholinesterase activity (in vitro)
(C@N), 1475 (C@C, aromatic); 1H NMR (DMSO-d6) (d ppm): 11.45
(s, 2H, OH), 10.52, 9.56 (br s, 2H, NH), 8.48 (d, J = 4.5, 2H, pyridine),
7.99 (s, 1H, N@CH), 7.66–7.57 (m, 3H, aromatic), 6.78 (d, J = 4.5, 2H,
pyridine); 13C NMR (d ppm): 169.54 (C@O), 155.14 (N@CH),
154.45, 150.0, 109.92 (pyridine), 139.26, 135.51, 130.12, 129.0
(aromatic); Anal. Calcd (%) for C13H12N4O3: C, 57.35; H, 4.44; N,
20.58; found (%) C, 57.14; H, 4.46; N, 20.55.
The ability of all tested compounds to inhibit acetylcholinester-
ase from electric eel (E.C. 3.1.1.7) and butyrylcholinesterase from
human serum (E.C. 3.1.1.8) was tested and their effectiveness in
inhibition could be conclusive through their IC50 values. The IC50
values were determined by the Ellman’s spectrophotometric meth-
od27 which is performed by recording the rate of increase in the
absorbance at 412 nm for 5 min. The stock solution of AChE was

5458
S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
prepared by dissolving AChE in 0.1 M phosphate buffer (pH 8.0). In
case of BChE stock solution was prepared by dissolving the lyoph-
ilized powder in an aqueous solution of gelatine 0.1%. The compo-
sition of final solution for assay consisted of 0.1 M phosphate
buffer pH 8.0, with the addition of 340 mM 5,50-dithio-bis(2-nitro-
benzoic acid), 0.02 unit/mL of AChE, or BChE and 550 mM of sub-
strate (acetylthiocholine iodide, ATCh or butyrylthiocholine
iodide, BTCh, respectively). Five different concentrations of inhibi-
tors between 20% and 80% (test compounds) were selected in order
to obtain inhibition of the enzymatic activity. From the aliquots
with water ad libitum prior to test. On the day of experiment,
derivatives were administered at graded doses upto 100 mg/kg
p.o. in 0.3% carboxymethyl cellulose as vehicle. All the animals
were monitored continuously for 30 min, 2 h and up to 48 h to de-
tect any changes in the autonomic or behavioral responses and also
for tremors, convulsions, salivation, diarrhoea, sleep, lacrimation,
heart rate, pulse rate, blood pressure and feeding behaviour as a
sign of acute toxicity.
4.3.5. Drug treatment
(50
l
L), increasing concentration of the inhibitors were added to
The synthesized compounds and donepezil were suspended in
0.3% carboxymethyl cellulose and administered orally at a dose
of 5 mg/kg and 10 mg/kg for 8 days. Scopolamine (1.5 mg/kg, i.p.)
was given 1 h after test procedure on day 7 only to induce the
amnesia. All animals of control groups were treated 0.3% carboxy-
methyl cellulose equal to volume of experimental drugs. Refer to
Table 5 for graphical time line of treatment schedule.
the assay solution and were preincubated for 20 min at 37 °C with
the enzyme followed by the addition of substrate. Blank used in the
assays consisted of all the components except AChE or BChE in or-
der to account for the non-enzymatic reaction. Reaction rates of
the assay were then compared and percent inhibition due to the
presence of increasing concentrations of inhibitor was calculated.
The concentration of each test compound was analyzed in tripli-
cate, and IC50 values were determined graphically from log concen-
tration percent inhibition curves.41,42
4.3.6. Elevated plus maze task
The elevated plus maze (EPM) test is regarded to be a simple
method for evaluation of learning and memory processes by mea-
suring transfer latency (TL).30,31 The plus maze used in the study,
consisted of two opposite open arms, 50 ꢁ 10 cm, crossed with
two enclosed arms of same dimensions having walls with a height
4.3.2. Enzyme kinetics study
Ellman’s method of spectrophotometric analysis was used to
determine the type of inhibition. Acetylcholine iodide and butyryl-
thiocholine iodide was used as a substrate at various concentra-
tions both below and above but near Km in a phosphate buffer at
pH 8 keeping a fixed amount of cholinesterase in the absence or
presence of different inhibitors. The concentration of the inhibitor
was kept close to one which corresponds to 50% inhibition of the
enzyme activity (IC50) and their inhibitory kinetics were evaluated
by the Lineweaver and Burk method.28
of 40 cm. The arms were connected with
a central square
(10 ꢁ 10 cm) to give the apparatus a plus sign appearance. The
maze was kept in a dimly lit room at an elevated height of 50 cm
above the floor. On day 7, individual rats from each group were
placed on the end of one of the open arms, facing away from the
centre. Time taken by the animal to enter one of the closed arms
(transfer latency) was recorded with the help of a stop watch
and considered as day 1. The rat was left in the enclosed arm for
10–15 s and returned to its home cage. On day 8 that is, second
day of EPM exposure the same procedure was repeated and TL
was recorded.44
4.3.3. Animals
Charles Foster rats of either sex (weighing 120–150 g) were pro-
cured from Central Animal House, Institute of Medical Sciences,
Banaras Hindu University, Varanasi (Registration No. 542/02/ab/
CPCSEA). They had free access to water ad libitum and were fed
with semi synthetic balanced diet, with occasional supply of green
vegetables (salad leaves). Six rats were housed per cage at temper-
ature (25 2 °C) and 45–55% relative humidity. Twelve hours of
light and dark cycles was strictly followed in a fully ventilated
room. Prior permission of Institutional Animal Ethical Committee
was obtained (No. Dean/11-12/CAEC/329).
4.3.7. Estimation of acetylcholinesterase activity in brain tissue
One hour after the last doses of test compounds and donepezil,
all rats were quickly sacrificed and the specific brain regions (pre-
frontal cortex, hippocampus and hypothalamus) were isolated
from the brain. The above brain regions were homogenized in ice
cold 0.1 M phosphate buffer (pH 8.0) in 1:10 ratio and centrifuged
at 1000ꢁg for 10 min at 4 °C, and supernatant was used as a source
of enzyme. Further, the activity of BChE was blocked by using
0.25 mM tetraisopropyl pyrophosphoramide (iso-OMPA) and AChE
activity was measured by adopting Ellman’s spectrophotometric
method.26 The enzyme activity was determined and was expressed
as the ‘n’ moles of substrate hydrolyzed/min/mg of protein.45
4.3.4. Acute toxicity evaluation
Acute toxicity studies for the derivatives were performed as per
OECD guidelines.43 Nulliparous, non-pregnant, healthy female albi-
no rats weighing between 120 and 150 g were fasted overnight
Table 5
Time line and treatment schedule of scopolamine induced amnesic experiments
Treatment days
Treatment schedule for amnesic model on EPM
Tested compounds (5 and 10 mg/kg) groups Donepezil (5 and 10 mg/kg) treated groups
Control (0.3%
carboxymethyl
cellulose)
Tested
Tested compounds + SCP
Donepezil
Donepezil + SCP
compounds
Pre-treatment period
1–6
Vehicle treated
Compounds
treatment
Compounds treatment
Donepezil
treatment
Donepezil treatment
Experimental manipulation
7th day was considered
day 1 in EPM Test
8th day was considered
day 2 in EPM Test
Vehicle treated
Vehicle treated
Compounds
treatment
Compounds
treatment
SCP (1.5 mg/kg i.p.) given 1 h after
compounds treatment
Compounds treatment
Donepezil
treatment
Donepezil
treatment
SCP (1.5 mg/kg i.p.) given 1 h after
Donepezil treatment
Donepezil treatment
SCP = Scopolamine.

S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
5459
4.3.8. Molecular docking and dynamics simulation
using the local optimization strategy to calculate ligand binding
energies. The best scoring pose was selected for each molecule.
4.3.8.1. Preparation of the small molecules. All the molecules
with their inhibition activities were taken and 3D structures were
sketched using Maestro 9.3 and geometrically minimized with
Macromodel 9.9 based on OPLS-2005 force field.
The binding free energy
as:
DGbind of ligand and protein was estimated
D
Gbind
¼
D
EMM
þ
D
Gsol ꢀ T
D
S
ð1Þ
where,
D
EMM is the difference in energy using the OPLS-2005 force
4.3.8.2. Preparation of the protein. The crystal structure of
AChE and BChE with high resolution was retrieved from the pro-
tein data bank (pdb code: 1B41 and 1POI, respectively).46 The
structure was prepared in the following procedures by protein
preparation wizard in Maestro 9.3, including adding hydrogens,
assigning partial charges using the OPLS-2005 force field and
assigning protonation states, restrained, partial energy minimiza-
tion and the resulting structure was used as the receptor model
in the following studies.
field,
DGsol is the difference in the GBSA and non polar (estimated
by the solvent accessible surface area) solvation energy, and T
represents the entropy term. Corrections for entropic changes were
not considered.
DS
Acknowledgements
The authors gratefully acknowledge the University Grants Com-
mission (UGC), New Delhi, India for the financial support to Mr.
Saurabh K. Sinha. Grant no. R/Dev./IX-Sch./(SRF-JRF) Pharm./
15402.
4.3.8.3. Molecular docking. For the receptor structure, crystal-
lographic and trajectory water molecules, ions and ligand com-
pounds were removed. Proteins were prepared using Schrodinger
software, Maestro 9.3 and Glide 5.8. The Glide XP algorithm was
employed using a grid box volume of 10_10_10 Å. All the struc-
tures were fitted in binding pocket and the lowest energy pose
for each docking run was retained.47
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.003.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.3.8.4. MD simulations. The docked complex of AChE and 4APi
were further optimized using molecular dynamics simulations of
Desmond software48 with OPLS-AA force field in explicit solvent
with the TIP3P water model. The docked complex was soaked in
to the TIP3P water molecules adequately, the dimensions of each
orthorhombic water box were 63 Å_69 Å_79 Å approximately and
the systems were neutralized by adding Cl counter ions to balance
the net charges of the systems. The generated solvent model for the
docked complex was containing about 54000 atoms. Before MD
simulations, the systems were minimized and pre-equilibrated
using the default relaxation routine implemented in Desmond.
The minimization was done for 7000 steps, in which the first
2000 steps were steepest descents and the last 5000 were L-BFGS.
MD simulations were performed using the minimized structures
and the Particle-mesh Ewald (PME) method was used to handle
long-range electrostatic interactions. Time step of simulation was
2.0 fs and a 10 Å cut off was used for non-bonded interactions.
Shake algorithm was employed to keep all bonds involving hydro-
gen atoms rigid. Constant-Volume (NVT) MD simulation was per-
formed for the first 100 ps during which temperature of the
system was raised from 0 to 300 K and for further simulation the
temperature was maintained at 300 K. Subsequently, the system
was equilibrated in NPT which is composed of minimization and
short MD simulation (12 and 24 ps) to relax the model system.
After that, long equilibration MD simulation was performed for
first 2 ns and long production MD simulation for 5 ns. The produc-
tion run was carried out using Langevin dynamics to maintain the
temperature at 300 K and a pressure of 1.0132 bar. Data were col-
lected every 1 ps during the MD runs.49
References and notes
1. Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, B. E. Neuropathol. Appl.
Neurobiol. 1978, 4, 273.
2. Gil-Bea, F. J.; Garcia-Alloza, M.; Dominguez, J.; Marcos, B.; Ramirez, M. J.
Neurosci. Lett. 2005, 375, 37.
3. Cummings, J. L. N. Engl. J. Med. 2004, 351, 56.
4. Weinstock, M. Neurodegeneration 1995, 4, 349.
5. Mohamed, T.; Rao, P. P. N. Curr. Med. Chem. 2011, 18, 4299.
6. Hakansson, L. Acta. Neurol. Scand. Suppl. 1993, 149, 7.
7. Lahiri, D. K.; Farlow, M. R.; Greig, N. H.; Sambamurti, K. Drug Dev. Res. 2002, 56,
267.
8. Gibney, G.; Camp, S.; Dionne, M.; Mac Phee-Quigley, K.; Taylor, P. Proc. Natl.
Acad. Sci. USA 1990, 87, 7546.
9. Kreienkamp, H. J.; Weise, C.; Raba, R.; Aaviksar, A.; Hucko, F. Proc. Natl. Acad. Sci.
USA 1991, 88, 6117.
10. Weise, C.; Kreienkamp, H. J.; Raba, R.; Pedak, A.; Aaviksar, A.; Hucko, F. EMBO J.
1990, 9, 3385.
11. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I.
Science 1991, 253, 872.
12. Barak, D.; Kronman, C.; Ordentlich, A.; Naomi, A.; Bromberg, A.; Marcus, D.;
Lazar, A.; Velan, B.; Shafferman, A. J. Biol. Chem. 1994, 269, 6296.
13. Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P. EMBO J. 2003, 22, 1.
14. Polman, C. H.; Bertelsmann, F. W.; van Loenen, A. C.; Koeteier, J. C. Arch. Neurol.
1994, 51, 292.
15. Murray, N. M.; Newsome-Davis, J. Neurology 1981, 31, 265.
16. Scipione, L.; De Vita, D.; Musella, A.; Flammini, L.; Bertoni, S.; Barocelli, E.
Bioorg. Med. Chem. Lett. 2008, 18, 309.
17. Cavallito, C. J.; Yun, H. S.; Edwards, M. L.; Foldes, F. F. J. Med. Chem. 1971, 14,
130.
18. Andreani, A.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.; Pietra, C.; Villetti,
G. Eur. J. Med. Chem. 2000, 35, 77.
19. Alptuzun, V.; Prinz, M.; Horr, V.; Scheiber, J.; Radacki, K.; Fallarero, A.; Vuorela,
P.; Engels, B.; Braunschweig, H.; Erciyas, E.; Holzgrabe, U. Bioorg. Med. Chem.
2010, 18, 2049.
4.3.8.5. Dynamics trajectory clustering, docking and molecular
mechanics–generalized born surface area (MM/GBSA) calcula-
tion. The complexes present in trajectory file after production
phase of MD simulations, were clustered according to the RMSD
of backbone. The hierarchical cluster average linkage method was
used to generate the clusters. The representative structures from
the entire cluster were further considered for ensemble docking
of all the inhibitors. The docked poses were re-ranked using MM/
GBSA present in PRIME 3.1.50 The protein structure was relaxed
and the energies were calculated using the OPLS-2005 force field
and the GBSA continue model. On the basis of the flexible region
within 10 Å around the ligand, the docked poses were minimized
20. Campagna, F.; Catto, M.; Purgatorio, R.; Altomare, C. D.; Carotti, A.; De Stradis,
A.; Palazzo, G. Eur. J. Med. Chem. 2011, 46, 275.
21. Huang, L.; Luo, Z.; He, F.; Lu, J.; Li, X. Bioorg. Med. Chem. 2010, 18, 4475.
22. Chen, X.; Tikhonova, G.; Decke, M. Bioorg. Med. Chem. 2011, 19, 1222.
23. Decker, M.; Kraus, B.; Heilmann, J. Bioorg. Med. Chem. 2008, 16, 4252.
24. Rodríguez-Franco, M. I.; Fernández-Bachiller, M. I.; Pérez, C.; Castro, A.;
Martínez, A. Bioorg. Med. Chem. 2005, 13, 6795.
25. Sinha, S. K.; Shrivastava, S. K. Med. Chem. Res. 2012, 21, 4395.
26. Sinha, S. K.; Shrivastava, S. K. Bioorg. Med. Chem. Lett. 2013. http://dx.doi.org/
10.1016/j.bmcl.2013.03.026.
27. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
28. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658.
29. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem. Pharmacol. 2003,
65, 407.

5460
S. K. Sinha, S. K. Shrivastava / Bioorg. Med. Chem. 21 (2013) 5451–5460
30. Sharma, A. C.; Kulkarni, S. K. Prog. Neuropsychopharmacol. Biol. Psychiatry. 1992,
16, 117.
down procedure). Available from: http://www.oecd.org [last accessed on 2006
Mar 23].
31. Itoh, J.; Nabeshima, T.; Kameyama, T. Psychopharmacology (Berl). 1990, 101, 27.
32. Snyder, P. J.; Bednar, M. M.; Cromer, J. R.; Maruff, P. Alzheimers Dement. 2005, 1, 126.
33. Davies, P.; Maloney, A. J. Luncet 1976, 25, 1403.
34. Harley, C. W.; Lacaille, J. C.; Galway, M. Behav. Brain Res. 1983, 270, 335.
35. Knowlton, B. J.; Fanselow, M. S. Curr. Opin. Neurobiol. 1998, 8, 293.
36. Tronel, S.; Matthijs, G. P. F.; Susan, J. S. Learn. Mem. 2004, 11, 453.
37. Soriano-Mas, C.; Redolar-Ripoll, D.; Aldavert-Vera, L.; Morgado-Bernal, I.;
Segura-Torres, P. Behav. Brain Res. 2005, 160, 141.
38. Dains, F. B.; Wertheim, E. Allophanic. Ester. 1920, 42, 2303.
39. Pandeya, S. N.; Aggarwal, N.; Jain, J. S. Pharmazie 1999, 54, 300.
40. Pandeya, S. N.; Yogeeswari, P.; Stables, J. P. Eur. J. Med. Chem. 2000, 35, 879.
41. Belluti, F.; Piazzi, L.; Bisi, A.; Gobbi, S.; Bartolini, M.; Cavalli, A.; Valenti, P.;
Rampa, A. Eur. J. Med. Chem. 2009, 44, 1341.
44. Ojha, R.; Sahu, A. N.; Muruganandam, A. V.; Singh, G. K.; Krishnamurthy, S.
Brain Cogn. 2010, 74, 1.
45. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
46. Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar, A.; Kronman, C.;
Barak, D.; Ariel, N.; Shafferman, A.; Silman, I.; Sussman, J. L. Acta. Crystallogr.,
Sect. D 2000, 56, 1385.
47. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49,
6177.
48. Desmond Molecular Dynamics System, version 3.1, D. E. Shaw Research, New
York, NY, 2012. Maestro-Desmond Interoperability Tools, version 3.1,
Schrödinger, New York, NY, 2012.
42. Zdrazilova, P.; Stepankova, S.; Komers, K.; Ventura, K.; Cegan, A. Z. Naturforsch
2004, 59, 293.
49. Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.; Sherman, W. J.
Chem. Theory. Comput. 2010, 6, 1509.
43. Organization for Economic Co-operation and Development (OECD), 2006.
OECD guidelines for the testing of chemicals-425 (Acute oral toxicity up and
50. Rapp, C.; Kalyanaraman, C.; Schiffmiller, A.; Schoenbrun, E. L.; Jacobson, M. P. J.
Chem. Inf. Model. 2011, 51, 1634.