Ionic liquid mediated synthesis of mono- and bis-spirooxindole- hexahydropyrrolidines as cholinesterase inhibitors and their molecular docking studies

Article abstract of DOI:10.1016/j.bmc.2014.01.002

One pot, three-component reaction of 1-acryloyl-3,5- bisarylmethylidenepiperidin-4-ones with isatin and sarcosine in molar ratios of 1:1:1 and 1:2:2 furnished to mono- and bis-spiropyrrolidine heterocyclic hybrids comprising functionalized piperidine, pyrrolidine and oxindole structural motifs. Both mono and bis-spiropyrrolidines displayed good inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 2.36-9.43 μM. For butyrylcholinesterase (BChE), mono-cycloadducts in series 8 with IC₅₀ values of lower than 10 μM displayed better inhibitory activities than their bis-cycloadduct analogs in series 9 with IC₅₀ values of 7.44-19.12 μM. The cycloadducts 9j and 8e were found to be the most potent AChE and BChE inhibitors with IC₅₀ values of 2.35 and 3.21 μM, respectively. Compound 9j was found to be competitive inhibitor of AChE while compound 8e was a mixed-mode inhibitor of BChE with calculated K _i values of 2.01 and 6.76 μM, respectively. Molecular docking on Torpedo californica AChE and human BChE showed good correlation between IC ₅₀ values and free binding energy values of the synthesized compounds docked into the active site of the enzymes.

Full text of DOI:10.1016/j.bmc.2014.01.002

Bioorganic & Medicinal Chemistry 22 (2014) 1318–1328
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ionic liquid mediated synthesis of mono- and
bis-spirooxindole-hexahydropyrrolidines as
cholinesterase inhibitors and their molecular
docking studies
b,
c
c
Yalda kia a, Hasnah Osman a, , Raju Suresh Kumar , Alireza Basiri , Vikneswaran Murugaiyah
⇑
⇑
a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
b Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh, Saudi Arabia
c School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
One pot, three-component reaction of 1-acryloyl-3,5-bisarylmethylidenepiperidin-4-ones with isatin and
sarcosine in molar ratios of 1:1:1 and 1:2:2 furnished to mono- and bis-spiropyrrolidine heterocyclic
hybrids comprising functionalized piperidine, pyrrolidine and oxindole structural motifs. Both mono
and bis-spiropyrrolidines displayed good inhibitory activity against acetylcholinesterase (AChE) with
Received 22 October 2013
Revised 23 December 2013
Accepted 1 January 2014
Available online 9 January 2014
IC50 values of 2.36–9.43
values of lower than 10
l
l
M. For butyrylcholinesterase (BChE), mono-cycloadducts in series 8 with IC50
M displayed better inhibitory activities than their bis-cycloadduct analogs in
Keywords:
Alzheimer’s disease
AChE
BChE
1,3-Dipolar cycloaddition
Spiropyrrolidines
Enzyme kinetic study and molecular
docking
series 9 with IC50 values of 7.44–19.12
AChE and BChE inhibitors with IC50 values of 2.35 and 3.21
be competitive inhibitor of AChE while compound 8e was a mixed-mode inhibitor of BChE with calcu-
lated Ki values of 2.01 and 6.76 M, respectively. Molecular docking on Torpedo californica AChE and
lM. The cycloadducts 9j and 8e were found to be the most potent
l
M, respectively. Compound 9j was found to
l
human BChE showed good correlation between IC50 values and free binding energy values of the synthe-
sized compounds docked into the active site of the enzymes.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
none is clinically available due to their side effects. Thus, the search
for new cholinesterase inhibitor is still ongoing worldwide.
Alzheimer’s disease (AD), the most common cause of dementia
among the elderly individuals is an irreversible neurodegenerative
disorder characterized by diverse cognitive impairments, neuro-
psychiatric symptoms and inability to perform routine activities.1
AD is associated with the degeneration of cholinergic neurons in
the basal forebrain, which leads to substantial decrease in genera-
tion of acetylcholine (ACh) neurotransmitter. ACh deficiency se-
verely affects the cognitive abilities, memory function and
emotional responses in AD patients. Pathogenesis of AD is largely
characterized by the presence of extracellular amyloid plaques
(Ab plaques) and intracellular neurofibrillary tangles (NFT) com-
posed of phosphorylated tau proteins.2–4 Based on so-called cho-
linergic hypothesis, one of the most promising therapeutic
approach to enhance cholinergic function is by the use of cholines-
terase inhibitors.5,6 In spite of tremendous efforts to develop novel
disease modifying agents working via b-amyloid or tau pathways,
Two major cholinesterases, acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE), involved in hydrolysis and regula-
tion of ACh in vertebrates.7 These two cholinesterases share similar
structure topology of possessing a 20 Å long, narrow active site
channel located in the center of the enzyme, ending to a catalytic
triad where the hydrolysis of acetylcholine takes place.8 This chan-
nel is composed of five major binding regions, namely (i) periphe-
ral anionic site,9 (ii) oxyanion hole,10 (iii) choline binding pocket,10
(iv) acyl binding site11 and (v) catalytic triad. In AChE, amino acid
residues having aromatic side chains such as tryptophan (Trp) and
tyrosine (Tyr) facilitate the insertion and transition of substrate or
inhibitor inside the active site cavity. In BChE these aromatic resi-
dues are mostly replaced with hydrophobic ones such as valine
(Val) and leucine (Leu) resulting in more spacious channel to
accommodate bulkier substrates.12
Ionic liquid mediated, multi-component reactions owing to
their unrivaled features such as high solvating abilities, interesting
catalytic behavior and recyclability have gained great importance
among the chemists for the synthesis of biologically active
heterocycles.13–15 Under this context and as a part of our ongoing
⇑
Corresponding authors. Tel.: +60 4 6533558; fax: +60 4 6574854.
E-mail addresses: osman.hasnah2012@yahoo.com (H. Osman), sraju@ksu.edu.sa
(R.S. Kumar).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.002

Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
1319
research in the construction and biological evaluation of novel
heterocyclic hybrids,16–19 we report ionic liquid mediated synthe-
sis of a library of novel mono- and bis-spiropyrrolidines and their
in vitro cholinesterases inhibitory activities. Mode of inhibition, en-
zyme kinetics and molecular docking studies were also conducted
for the most active derivatives to investigate their nature of inhibi-
tion and binding interaction template to the active site of the
enzymes.
to the steric hindrance exerted by the cycloadduct 9 for subsequent
cycloaddition. Performing aforementioned reactions in ionic sol-
vent had noticeable advantages over methanol in terms of reaction
time and yield. The high product yields were probably due to the
catalytic abilities of [bmim]Br that also improved the reaction
rates.
Structures of the mono and bis-spiropyrrolidines 8 and 9 are in
agreement with the combustion data, IR, 1D and 2D NMR spectro-
scopic data. The elemental analysis results are within 0.4% of the
theoretical values. In the 1H NMR spectrum of 8a, a doublet of dou-
blets at 5.00 ppm with J = 10.5 and 8.7 Hz is due to H-4 of the pyr-
rolidine ring. The HMQC correlation of H-4 assigns the carbon
signal at 44.0 ppm to C-4. Further, H-4 shows HMBC correlation
with the C@O of the piperidone ring at 196.5 ppm and the spiro
carbon C-300 at 63.2 ppm, besides showing correlation with the
adjacent carbon C-5 at 57.1 ppm. The HMQC correlation of C-5 as-
signs the triplets at 3.47 ppm (J = 8.7 Hz) and 4.10 ppm
(J = 10.5 Hz) to 5-CH2. The two doublets at 2.54 and 4.33 ppm with
J = 14.3 Hz are assigned to 200-CH2 while the doublet of doublets at
3.31 ppm with J = 18.8, 2.0 Hz and a doublet at 4.27 ppm with
J = 18.8 Hz are due to 600-CH2 of the piperidone ring. From HMQC
correlation, the carbon signals at 46.1 and 45.5 ppm are assigned
to C-200 and C-600, respectively (Fig. 1).
2. Results and discussion
2.1. Chemistry
The highly functionalized dipolarophiles viz. 1-acryloyl-3,5-dia-
rylmethylidenepiperidin-4-ones (5) required for the synthesis of
spiro-heterocycles were prepared by Claisen–Schmidt condensa-
tion of 4-piperidone hydrochloride (1) with a series of aromatic
aldehydes (2) to furnish N-unsubstituted 3,5-bis[(E)-arylmethyli-
dene]tetrahydro-4(1H)-pyridinones (3), which were subsequently
acylated with acryloyl chloride, following the previously reported
procedure.20 The dipolarophiles (5) are versatile synthons for the
construction of more complex spiro-heterocycles owing to pres-
ence of diverse dipolarophile functions such as three C@C and
two C@O groups.
As depicted in Scheme 1, three-component [3+2]-cycloaddition
reaction of dipolarophiles (5) with isatin (6) and sarcosine (7) in
[bmim]Br as ionic solvent afforded a library of spiro-cycloadducts
in good yields (Table 1). Initially, the reaction of an equimolar mix-
ture of 1-acryloyl-3,5-diphenylidenepiperidin-4-ones (5), isatin (6)
and sarcosine (7) was investigated under refluxing methanol for 5
h, furnishing to mono-spiropyrrolidine (8a) as the sole product in
62% yield. The reaction of 5, 6 and 7 in a molar ratio 1:2:2 for a
longer period of time (15 h) afforded more complex bis-spiropyrr-
olidine (9a) in moderate yield of 48%.
The above reactions were also performed in [bmim]Br, as a
green and eco-friendly reaction medium. By refluxing an equimo-
lar mixture of 5, 6, 7 and 1 molar equiv of [bmim]Br, the mono-
spiropyrrolidine (8a) was obtained in 30 min and 83% yield. The
bis-spiropyrrolidine (9a) was also afforded in a shorter reaction
time of 2 h and 71% yield by refluxing 1:2:2 mixture of the reac-
tants and 2 molar equiv of ionic solvent. In both reactions, the spi-
ropyrrolidines [(8a) and (9a)] were purified through flash column
chromatography. Further attempt to obtain the more complex
tri-spiropyrrolidine cycloadduct were unsuccessful, plausibly due
Further, the piperidone ring protons, 200-CH2 and 600-CH2 show
HMBC correlation with the carbonyl carbon of acryloyl moiety at
176.7 ppm. The doublet of doublets at 6.62 ppm is due to b-hydro-
gen of the acryloyl moiety whereas the doublets at 5.68 and
6.25 ppm are due to two
a-hydrogens. The singlets at 7.82 and
8.36 ppm are due to the arylmethylidene hydrogen and NH hydro-
gen of the oxindole, respectively. The aromatic hydrogens appear
as doublets and multiplets in region between 6.87 and 7.56 ppm.
The 1H and 13C NMR chemical shifts of the bis-spiropyrrolidines
(9) were also assigned by similar considerations. As the cycload-
duct 9 possess two oxindolo-pyrrolidine rings, to distinguish the
1H and 13C chemical shifts of these two rings, the ring attached
to C-300 of 4-piperidone moiety is considered as ring A and the
other ring attached to 100-NAC@O is considered as ring B. Further,
the structure and stereochemistry of mono and bis-spiropyrroli-
dines were confirmed by the single crystal X-ray crystallographic
analysis of 8a and 9a (Figs. 2 and 3).
The plausible mechanism for the formation of spiropyrrolidines
8 and 9 in ionic solvent is depicted in Scheme 2. The electron-
deficient hydrogen atom of [bmim]Br, forms hydrogen bonding
interaction with the carbonyl function of isatin, whereby facilitates
H
NH.HCl
O
N
2'
N
4
O
1
2
3
1"
O
5
6
, 1 eq.
2"
N
Ar
O
7, 1 eq.
2 ArCHO
H
O
6"
5"
HCl, CH3COOH
K2CO3, Acetone
4"
N
0.5 h
O
H
(+)-8
Ar
Ar
6
Ar
+
O
H
N
Cl
N
NH
Ar
6, 1 eq.
OH
O
O
4
7
7
, 1 eq.
O
O
K2CO3
[BMIM]Br
Reflux
Acetone
5
Ar
H
N
H4
5
1
O
N
1
2'
3
2
N 2
3
4
6
, 2 eq.
5
1"
O
2"
N
Ar
7, 2 eq.
2'
O
H
N
5"
6"
2 h
H
4"
O
Ar
(+)-9
Scheme 1. Synthesis of mono- and bis-spiropyrrolidines 8(a–k) and 9(a–k).

1320
Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
Table 1
AChE and BChE inhibitory activities of compounds 8(a–k) and 9(a–k)
Entry
Compound
Ar
Yield (%)
AChE inhibition IC50
g/mL
BChE inhibition IC50
g/mL
3.78 0.21
Selectivity
l
l
M
l
l
M
AChEa
BChEb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
9a
9b
9c
9d
9e
C6H5
83
71
75
77
71
82
80
77
81
77
85
71
60
64
52
54
57
69
65
61
64
72
—
4.39 0.14
1.28 0.11
1.41 0.15
2.93 0.12
3.71 0.14
3.16 0.09
3.92 0.15
3.98 0.14
4.79 0.19
4.23 0.11
4.58 0.21
1.78 0.21
6.65 0.19
3.49 0.22
3.88 0.17
4.41 0.25
1.81 0.18
3.60 0.15
5.41 0.25
4.59 0.28
1.76 0.14
3.84 0.28
0.60 0.09
8.74
3.97
4.10
5.12
6.88
5.34
6.14
7.49
8.40
7.80
7.59
2.63
9.43
4.73
5.19
6.18
2.47
4.42
7.67
6.14
2.35
4.93
2.09
7.52
0.86
0.97
1.03
0.82
0.47
1.45
0.87
1.24
0.81
0.43
1.17
3.55
1.33
2.20
1.81
1.20
5.00
4.05
1.47
1.54
3.33
3.88
3.47
1.16
1.03
0.97
1.22
2.14
0.69
1.15
0.81
1.24
2.30
0.86
0.28
0.75
0.45
0.55
0.83
0.20
0.25
0.68
0.65
0.30
0.26
0.28
2-CH3C6H4
2-(CH3O)C6H4
2-ClC6H4
2.05 0.14
2.37 0.19
2.41 0.15
1.72 0.12
4.58 0.21
3.40 0.19
4.92 0.22
3.87 0.24
1.82 0.14
5.32 0.25
6.31 0.21
8.80 0.24
7.69 0.18
6.97 0.17
5.29 0.22
9.45 0.26
14.53 0.17
7.94 0.27
6.94 0.18
5.56 0.21
14.83 0.29
5.55 0.15
3.86
4.22
4.20
3.21
7.74
5.32
9.28
6.77
3.39
8.85
9.34
12.51
10.42
9.38
2-FC6H4
3-(NO2)C6H4
2,4-Cl2C6H3
4-CH3C6H4
4-ClC6H4
4-FC6H4
1-Naphthyl
C6H5
2-CH3C6H4
2-(CH3O)C6H4
2-ClC6H4
2-FC6H4
7.44
9f
9g
9h
9i
9j
9k
3-(NO2)C6H4
2,4-Cl2C6H3
4-CH3C6H4
4-ClC6H4
4-FC6H4
1-Naphthyl
Galanthamine
12.34
17.90
11.29
9.44
7.82
19.12
19.34
Reference standard
a
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE).
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE).
b
2.19, s
2.54, d, J=14.35 Hz
4.33, d, J=14.35 Hz
46.1
34.5
3.47, t, J=8.79 Hz
4.10, t, J=10.59 Hz
57.1
167.2
H
O
O
O
5.68, d, J =11.89 Hz
6.25, d, J =16.88 Hz
129.6
NH
NH
β
H
74.9
α
N
H
N
2a
5"
1
2
5
1'
H
3
4
63.2
196.5
N
N
O
Ar
Ar
H
O
5.00, dd, J =10.59,
8.79 Hz
176.7
1"
6.62, dd,
3"
2"
6"
H
O
H
4"
J=16.88, 11.89 Hz
44.0
H
129.8
3.31, dd, J=18.81, 2.09 Hz
4.27, d, J=18.81 Hz
45.5
H
Ar
H
Ar
7.82, s
122.5
6.87-7.56, m,
H-aromatic
Figure 1. Selected HMBC correlations and 1H and 13C chemical shifts of 8a.
the formation of reactive azomethine ylide via the decarboxylative
condensation of isatin and sarcosine. The newly formed ylide,
attacks chemo-selectively to a C@C bond of piperidone ring of
dipolarophile furnishing the mono-spiropyrrolidine (8). To afford
bis-spiropyrrolidines (9), an additional azomethine ylide adds to
the C@C bond of acryloyl moiety of 8. These reactions proceeded
regio-selectively due to preferential addition of azomethine ylide
nucleophilic carbon to the b-carbon of enone moiety in piperidone
ring, and stereo-selectively owing to formation of only one
stereoisomer in both mono- and bis-cycloadducts.
10 lM (Table 1). In series 8, ortho-substituted derivatives viz. 8b
with o-CH3, 8c with o-methoxy, 8d with o-Cl and 8e bearing o-F
moiety on phenyl rings, with IC50 values in the range of
3.97–6.88
para-substituted derivatives (8h–j) with IC50 values in the range
of 7.49–8.40 M. Remaining compounds in this series also showed
good AChE inhibitory activity with IC50 values ranging from 7.49 to
8.74 M. Bis-spiropyrrolidine cycloadducts in series 9 also dis-
lM, displayed better AChE inhibitory activity than
l
l
played good AChE inhibitory activities. The un-substituted 9a, di-
chloro-substituted 9g and fluoro-substituted 9j showed potent
inhibitory activity with IC50 values of lower than 3
to the reference standard, galanthamine with IC50 value of
2.09 M. Among the synthesized derivatives, compound 9j dis-
played the most potent AChE inhibitory activity with IC50 value
of 2.36 M.
Unlike AChE, for BChE, compounds in series
better inhibitory activities than bis-cycloadducts in series 9. The
lM, comparable
2.2. Cholinesterase inhibitory activity
l
All the newly synthesized spiropyrrolidine derivatives were
evaluated in vitro for their inhibitory activities against AChE and
BChE. Both mono and bis-cycloadducts displayed good inhibitory
activity against AChE enzyme with IC50 values of lower than
l
8
displayed

Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
1321
hand, the bis-cycloadducts in series 9 showed weaker BChE inhib-
itory activities compared to their mono analogs in series 8 with
IC50 values ranging from 7.44 to 19.12 lM. Similar to that for series
8, fluoro embedded derivatives 9e and 9j, displayed highest BChE
inhibition followed by chloro- and un-substituted derivatives with
IC50 values of less than 10 lM.
In addition, except for compound 8e and 8j that showed more
selectivity for BChE, the rest of the compounds in series 8, dis-
played equal selectivity toward both AChE and BChE with selectiv-
ity indexes ranging from 0.81 to 1.24. Recent studies have showed
that as the AD progresses, the activity of AChE decreases while the
activity of BChE remains unaffected or even increases. Moreover, in
the advance staged AD patients, BChE can compensate for the AChE
to diminish the already depleted level of acetylcholine.21,22 Thus,
novel cholinesterase inhibitors with dual AChE and BChE inhibitory
activity or more selectivity toward BChE are valuable therapeutic
agents. Unlike series 8, the bis-spiropyrrolidines in series 9 were
more selective toward AChE with selectivity indexes ranging from
1.20 to 5.00 whereby compound 8f displayed highest selectivity for
AChE.
2.3. Enzyme inhibition kinetics
The types of inhibition and enzyme kinetics were investigated
for the most active AChE and BChE inhibitors, namely 9j and 8e.
The inhibition data and types of inhibitions of these compounds
are summarized as kinetic data (Table 2) and Lineweaver–Burk
plots (Figs. 4 and 5), respectively. Compound 9j was found to be
a competitive inhibitor of AChE while compound 8e was a
mixed-mode inhibitor of BChE. The calculated Ki values were
2.01 and 6.76 lM for 9j and 8e, respectively.
2.4. Molecular docking
Figure 2. ORTEP diagram of 8a.
To further validation of in vitro cholinesterase inhibitory results
and get better insight into the binding interactions at molecular le-
vel, all the newly synthesized compounds were docked into the ac-
tive site of AChE and BChE derived from crystal structure of
Torpedo californica AChE and human BChE enzymes. The free ener-
gies of binding interactions for the synthesized compounds
showed good correlations to their in vitro median inhibitory con-
centrations, with regression values of 0.667 and 0.712 for AChE
and BChE enzymes, respectively (Figs. 6 and 7).
Both mono- and bis-cycloadducts in series 8 and 9 displayed
good activity against AChE. Active site gorge of AChE is composed
of residues possessing aromatic side chains such as phenylalanine
(Phe) and tyrosine (Tyr), which due to its high aromatic content
establishes extensive hydrophobic interactions with the inhibitors
and substrates. Although the overall structure of BChE is similar to
that of AChE, but the active site of BChE has many of the aromatic
residues replaced by residues with aliphatic side chains, such as
leucine (Leu) and valine (Val). Due to this, the bis-cycloadducts
in series 9, comprising more aromatic cores, displayed better AChE
inhibitory activities than mono-cycloadducts in series 8, plausibly
owing to more
p–p and r–p interactions with aromatic channel-
lining residues. However, for BChE, both series displayed moderate
inhibition probably due to less favorable interactions with ali-
phatic residues lining the gorge wall in the BChE enzyme.
The molecular docking analysis of the most potent compounds,
namely 9j against AChE and 8e against BChE are depicted in Fig-
ures 6 and 7 and described in detail. Docking of compound 9j into
the active site of AChE disclosed that it preferentially binds to
peripheral anionic site (PAS) of the enzyme through hydrogen
bonding interactions with Tyr334 as well as hydrophobic interac-
tions with Trp279, Tyr70 and Tyr121. The effective binding to
PAS of the AChE efficiently blocked the entry of the channel and
Figure 3. ORTEP diagram of 9a.
mono-spiropyrrolidines bearing fluoro atom on either ortho or para
position of phenyl ring viz. 8e and 8j, showed the highest inhibi-
tions with IC50 values of 3.21 and 3.39 lM. Similar to AChE,
ortho-substituted derivatives in series 8 displayed higher inhibi-
tory activities compared to para-substituted analogs. On the other

1322
Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
Ar
Br
Bu
N
H
+
N
Br
N
N
N
O
H
O
N
O
O
O
+
-H2O
-CO2
N
N
H
O
O
H
H
OH
Ar
Bu
N
N
H
N
H
N
H
O
O
N
H
N
N
N
N
O
N
N
Ar
H
Ar
H
O
O
O
N
N
H
H
H
O
O
Ar
(+)-9
Scheme 2. Mechanism for the formation of mono- and bis-cycloadducts 8 and 9.
Ar
(+)-8
N
+
Bu
N
Br
Phe329 at choline binding site as well as hydrophobic interaction
with Leu286 and Val288 at the peripheral anionic site of the en-
zyme. Compound 8e also displayed mild polar interaction with
Gly116 and Gly117 composing oxyanion hole, in addition to
Ser198 and His438 at the catalytic triad of the enzyme (Fig. 9).
Based on the above findings, compound 8e completely filled in
the BChE channel and prevents substrate insertion and hydrolysis
at the active site of enzyme. The interaction data of the enzymes
with amino acid residues involved in ligand–receptor complex
and their bonding types are presented Table 3 for the most active
inhibitors 9j and 8e.
Table 2
Kinetic parameters for cholinesterase inhibition
Compound
Enzyme
AChE
l
M
Km (mM)
Vmax
(
D
OD412/min)
Ki
9j
0
0.331
0.462
0.560
0.150
0.221
0.497
0.021
0.021
0.021
0.011
0.012
0.015
2.01
0.70
1.40
0
0.90
1.81
8e
BChE
6.76
3. Conclusion
prohibits insertion and accommodation of substrate into the active
site of the enzyme (Fig. 8). This result completely coincided with
the kinetic findings, which indicates 9j is a competitive inhibitor
of AChE.
Docking analysis for compound 8e on BChE revealed that this
inhibitor is effectively anchored to the active site of BChE enzyme
Two novel series of piperidone-grafted, mono- and bis-spiropyrr-
olidine derivatives were prepared via one pot, three-component
reaction of azomethine ylide generated from isatin and sarcosine
with highly functionalized dipolarophiles in good yield and short
reaction time using [bmim]Br as ionic solvent. Both mono and bis-
cycloadducts in series 8 and 9 displayed good activity against AChE
through hydrophobic and p,p-stacking interactions with Trp82 and
Figure 4. Lineweaver–Burk plot of cholinesterase inhibition by compounds 9j on AChE.

Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
1323
Figure 5. Lineweaver–Burk plot of cholinesterase inhibition by compounds 8e on BChE.
-1.5
-1.3
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
-30
-32
-34
-36
-38
-40
-42
-44
-46
Free binding energy (kcal/mol)
Figure 6. Correlation diagram of pIC50 versus free binding energy of compounds
8(a–k) and 9(a–k) for AChE inhibitory activity.
-1.8
-1.6
-1.4
-1.2
-1
Figure 8. Binding orientations and interactions of compound 9j at the TcAChE
active site.
-0.8
-0.6
-0.4
and BChE inhibitors with IC50 values of 2.35 and 3.21 lM, respec-
tively. The docking analysis of the newly synthesized compounds
showed good correlations between IC50 values and free binding
energies of the docked compounds.
-0.2
0
-10
-15
-20
-25
-30
-35
-40
-45
-50
Free binding energy (kcal/mol)
4. Experimental
Figure 7. Correlation diagram of pIC50 versus free binding energy of compounds
8(a–k) and 9(a–k) for BChE inhibitory activity.
4.1. General methods
The melting points were measured in open capillary tubes and
are uncorrected. FT-IR spectra were recorded on a Perkin Elmer
2000 instrument. The 1H, 13C and the 2D NMR spectra were re-
corded on a Bruker (Avance) 500 MHz NMR instrument using
with IC50 values of lower than 10 lM. However mono-spiropyrroli-
dines better BChE inhibitory activity than the bis-spiropyrrolidines.
The cycloadducts 9j and 8e were found to be the most potent AChE

1324
Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
C, 76.47; H, 5.62; N, 8.36. Found: C, 75.25; H, 5.12; N, 7.96. 1H
NMR (500 MHz, CDCl3): d 2.19 (s, 3H, CH3), 2.54 (d, J = 14.35, H-
200), 3.31 (dd, J = 18.81, 2.09, 1H, H-600), 3.47 (t, J = 8.79, 1H, H-5),
4.10 (t, J = 10.59, 1H, H-5), 4.27 (d, J = 18.81, 1H, H-600), 4.33 (d,
J = 14.35, 1H, H-200), 5.00 (dd, J = 10.59, 8.79 1H, H-4), 5.68 (d,
J = 11.89 Hz, 1H, H-a), 6.25 (d, J = 16.88 Hz, 1H, H-a), 6.62 (dd,
J = 16.88, 11.89 Hz, 1H, H-b), 6.87–7.56 (m, 14H, H-aromatic),
7.82 (s, 1H, H-arylmethylidene), 8.36 (br. s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 34.53, 44.04, 45.52, 46.18, 57.19, 63.27,
74.91, 110.43, 122.55, 125.13, 126.74, 127.20, 127.45, 128.62,
128.88, 128.92, 129.60, 129.69, 129.78, 129.84, 130.40, 134.07,
137.47, 140.49, 142.80, 167.23, 176.75, 196.56.
4.1.1.2. 1-Methyl-4-(2-methylphenyl)pyrrolo-(spiro[2.30]oxin-
dole)-spiro[3.300]-500-(2-methylphenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8b).
White solid; (0.121 g, 71%); mp
150–153 °C; IR (KBr)
m
max: 3423, 1720, 1615 cmꢀ1; Anal. Calcd
for C34H32N3O3: C, 76.96; H, 6.08; N, 7.92. Found: C, 75.34; H,
6.17; N, 6.99. 1H NMR (500 MHz, CDCl3): d 2.11 (s, 3H, CH3), 2.23
(s, 3H, CH3), 2.25 (s, 3H, CH3), 2.46 (d, J = 14.30, H-200), 3.22 (dd,
J = 18.28, 2.39 Hz, 1H, H-600), 3.37 (t, J = 8.49 Hz, 1H, H-5), 3.98 (t,
J = 9.51 Hz, 1H, H-5), 4.18 (d, J = 18.28, 1H, H-600), 4.24 (d,
J = 14.30, 1H, H-200), 4.88 (dd, J = 9.51, 8.49 Hz, 1H, H-4), 5.63 (d,
Figure 9. Binding orientations and interactions of compound 8e at the hBChE active
site.
J = 10.54 Hz, 1H, H-a), 6.22 (d, J = 16.72 Hz, 1H, H-a), 6.51–6.57
TMS as internal standard, DMSO-d6 and CDCl3 as solvents. Chemi-
cal shifts are given in parts per million (d-scale) and the coupling
constants are given in Hertz. Silica gel-G plates (Merck) were used
for TLC analysis with a mixture of petroleum ether (60–80 °C) and
ethyl acetate as eluent. Elemental analyses were performed using a
Perkin Elmer 2400 Series II Elemental CHN analyzer.
(dd, J = 16.71, 10.54 Hz, 1H, H-b), 6.73–7.35 (m, 12H, H-aromatic)
7.70 (s, 1H, H-arylmethylidene), 9.01 (br. s, 1H, NH) 13C NMR
(125 MHz, CDCl3): d 20.02, 20.37, 33.49, 43.15, 44.15, 45.17,
56.27, 62.24, 73.91, 109.16, 121.54, 124.27, 125.73, 126.29,
127.77, 128.06, 128.27, 128.35, 128.55, 128.68, 129.59, 130.35,
133.35, 136.01, 139.06, 139.42, 141.47, 165.98, 175.64, 195.56.
4.1.1. General procedure for synthesis of 8(a–k) and 9(a–k)
1 molar equiv of [bmim]Br were added to equimolar mixture of
1-acryloyl-3,5-diarylidenepiperidin-4-ones (5, 0.364 mmol), isatin
(6, 0.364 mmol) and sarcosine (7, 0.364 mmol) and refluxed for
30 min. After completion of the reaction, as evident by TLC, the
mono-spiropyrrolidines (8) were afforded in pure form through
column chromatography. The same reaction was performed
employing 1:2:2 ratio of (5, 0.364 mmol), (6, 0.728 mmol) and (7,
0.728 mmol) in 2 molar equiv of refluxing [bmim]Br, which fur-
nished to bis-spiropyrrolidines (9) after 2 h that subsequently puri-
fied through column chromatography. The purity of both (8) and
(9) examined using TLC and 1H NMR techniques.
4.1.1.3. 1-Methyl-4-(2-methoxyphenyl)pyrrolo-(spiro[2.30]oxin-
dole)-spiro[3.300]-500-(2-methoxyphenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8c).
White solid; (0.134 g, 75%); mp
125–127 °C; IR (KBr)
m
max: 3419, 1711, 1601 cmꢀ1; Anal. Calcd
for C34H32N3O5: 72.58; H, 5.73; N, 7.47. Found: 72.07; H, 5.23; N,
7.07. 1H NMR (500 MHz, CDCl3): d 2.21 (s, 3H, CH3), 2.45 (d,
J = 14.05, H-200), 3.20 (dd, J = 17.95, 2.37 Hz, 1H, H-600), 3.39 (t,
J = 9.22 Hz, 1H, H-5), 3.70 (s, 3H, O–CH3), 3.72 (s, 3H, O–CH3),
3.95 (t, J = 8.27 Hz, 1H, H-5), 4.17 (d, J = 17.95, 1H, H-600), 4.22 (d,
J = 14.05, 1H, H-200), 4.85 (dd, J = 9.22, 8.27 Hz, 1H, H-4), 5.62 (d,
J = 10.51 Hz, 1H, H-a), 6.22 (d, J = 16.12 Hz, 1H, H-a), 6.57 (dd,
J = 16.12, 10.51 Hz, 1H, H-b), 6.72–7.43 (m, 12H, H-aromatic)
7.69 (s, 1H, H-arylmethylidene), 9.20 (br. s, 1H, NH) 13C NMR
(125 MHz, CDCl3): d 33.46, 43.20, 43.67, 45.16, 54.17, 54.27,
56.43, 62.00, 73.98, 109.13, 112.87, 113.12, 121.44, 124.18,
125.77, 126.16, 126.65, 127.74, 128.41, 128.46, 129.78, 131.61,
139.07, 141.40, 157.74, 159.68, 166.02, 175.71, 195.52.
4.1.1.1.
spiro[3.300]-500-(phenylmethylidene)-100-N-acrolylpiperidin-400-
one (8a). White solid; (0.137 g, 83%); mp 192–195 °C; IR
max: 3421, 1719, 1617 cmꢀ1; Anal. Calcd for C32H28N3O3:
1-Methyl-4-(phenyl)pyrrolo-(spiro[2.30]oxindole)-
(KBr)
m
Table 3
Binding interaction data for compounds 9j and 8e docked into active site of TcAChE and hBChE
Entry
1
Ligand
Enzyme
Interacting site
PASa
Amino acid residue
Bond type
9j
TcAChE
Trp279
Tyr70
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding (2.38 Å)
Hydrophobic
Hydrophobic
Mild polar
Tyr121
Tyr334
Leu286
Val288
Gly116 & 117
Phe329
Trp82
2
8e
hBChE
PAS
OHb
Choline binding site
p–p Stacking
Hydrophobic
Mild polar
Mild polar
CTc
His438
Ser198
a
b
c
Peripheral anionic site.
Oxyanion hole.
Catalytic triad.

Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
1325
4.1.1.4.
dole)-spiro[3.300]-500-(2-chlorophenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8d).
White solid; (0.144 g, 77%); mp
155–157 °C; IR (KBr)
max: 3420, 1717, 1621 cmꢀ1; Anal. Calcd
1-Methyl-4-(2-chlorophenyl)pyrrolo-(spiro[2.30]oxin-
43.87, 46.04, 56.95, 62.32, 76.03, 110.89, 123.01, 124.36, 126.71,
127.20, 127.35, 127.73, 128.96, 129.65, 129.98, 130.50, 131.52,
131.95, 132.61, 134.03, 134.41, 135.94, 136.19, 136.56, 136.88,
142.70, 167.05, 176.95, 197.63.
m
for C32H26Cl2N3O3: C, 67.25; H, 4.59; N, 7.35. Found: C, 66.51; H,
4.14; N, 7.08. 1H NMR (500 MHz, CDCl3): d 2.12 (s, 3H, CH3), 2.46
(d, J = 14.22, H-200), 3.20 (dd, J = 18.25, 2.52 Hz, 1H, H-600), 3.41 (t,
J = 8.32 Hz, 1H, H-5), 4.05 (t, J = 9.58 Hz, 1H, H-5), 4.13 (d,
J = 18.25, 1H, H-600), 4.42 (d, J = 14.22, 1H, H-200), 4.92 (dd, J = 9.58,
4.1.1.8. 1-Methyl-4-(4-methylphenyl)pyrrolo-(spiro[2.30]oxin-
dole)-spiro[3.300]-500-(4-methylphenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8h).
White solid; (0.117 g, 77%); mp
139–141 °C; IR (KBr)
m
max: 3421, 1707, 1618 cmꢀ1; Anal. Calcd
8.32 Hz, 1H, H-4), 5.92 (d, J = 10.19 Hz, 1H, H-
a
), 6.31 (d,
for C34H32N3O3: C, 76.96; H, 6.08; N, 7.92. Found: C, 75.12; H,
5.99; N, 6.81. 1H NMR (500 MHz, CDCl3): d 2.12 (s, 3H, CH3), 2.28
(s, 3H, CH3), 2.30 (s, 3H, CH3), 2.45 (d, J = 14.39, H-200), 3.25 (dd,
J = 18.32, 2.77 Hz, 1H, H-600), 3.40 (t, J = 7.98 Hz, 1H, H-5), 4.01 (t,
J = 8.87 Hz, 1H, H-5), 4.23 (d, J = 18.32, 1H, H-600), 4.27 (d,
J = 14.39, 1H, H-200), 4.90 (dd, J = 8.87, 7.98 Hz, 1H, H-4), 5.69 (d,
J = 16.51 Hz, 1H, H- ), 6.68 (dd, J = 16.51, 10.19 Hz, 1H, H-b),
a
6.70–7.57 (m, 12H, H-aromatic) 7.75 (s, 1H, H-arylmethylidene),
9.25 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 34.28, 43.68,
44.48, 48.02, 56.40, 62.08, 75.46, 110.48, 122.65, 126.01, 126.70,
127.78, 129.28, 129.40, 129.55, 130.37, 130.84, 131.47, 132.21,
132.47, 134.74, 136.29, 136.32, 140.23, 141.14, 177.03, 179.89,
198.60.
J = 11.15 Hz, 1H, H-a), 6.22 (d, J = 17.19 Hz, 1H, H-a), 6.57 (dd,
J = 17.19, 11.15 Hz, 1H, H-b), 6.84–7.51 (m, 12H, H-aromatic)
7.72 (s, 1H, H-arylmethylidene), 8.97 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 20.05, 20.40, 33.52, 43.17, 44.19, 45.17,
56.26, 62.28, 73.92, 110.11, 121.52, 124.31, 125.82, 126.32,
127.71, 128.11, 128.34, 128.42, 128.62, 128.74, 129.68, 130.44,
133.41, 136.12, 139.15, 139.47, 141.51, 165.74, 175.64, 195.72.
4.1.1.5.
dole)-spiro[3.300]-500-(2-fluorophenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8e).
White solid; (0.107 g, 71%); mp
145–148 °C; IR (KBr)
max: 3423, 1717, 1618 cmꢀ1; Anal. Calcd
1-Methyl-4-(2-fluorophenyl)pyrrolo-(spiro[2.30]oxin-
m
for C32H26F2N3O3: C, 71.36; H, 4.87; N, 7.80. Found: C, 70.52; H,
4.38; N, 7.92. 1H NMR (500 MHz, CDCl3): d 2.20 (s, 3H, CH3), 2.74
(d, J = 14.22, H-200), 3.13 (d, J = 18.09, 1H, H-600), 3.47 (t,
J = 8.15 Hz, 1H, H-5), 4.08 (t, J = 9.71 Hz, 1H, H-5), 4.12–4.18 (m,
2H, H-200, H-600), 5.20 (dd, J = 9.71, 8.15 Hz, 1H, H-4), 5.65 (d,
4.1.1.9.
dole)-spiro[3.300]-500-(4-chlorophenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8i).
White solid; (0.127 g, 81%); mp
154–157 °C; IR (KBr)
max: 3422, 1729, 1615 cmꢀ1; Anal. Calcd
1-Methyl-4-(4-chlorophenyl)pyrrolo-(spiro[2.30]oxin-
m
J = 16.64 Hz, 1H, H-
a
), 6.23 (d, J = 10.43 Hz, 1H, H-
a
), 6.55 (dd,
for C32H26Cl2N3O3: C, 67.25; H, 4.59; N, 7.35. Found: C, 66.71; H,
4.21; N, 7.19. 1H NMR (500 MHz, CDCl3): d 2.19 (s, 3H, CH3), 2.54
(d, J = 14.17, H-200), 3.24 (dd, J = 18.12, 2.41 Hz, 1H, H-600), 3.49 (t,
J = 8.11 Hz, 1H, H-5), 4.02 (t, J = 9.24 Hz, 1H, H-5), 4.20 (d,
J = 18.12, 1H, H-600), 4.27 (d, J = 14.17, 1H, H-200), 4.95 (dd, J = 9.24,
J = 16.64, 10.43 Hz, 1H, H-b), 6.81–7.65 (m, 12H, H-aromatic),
7.88 (s, 1H, H-arylmethylidene), 8.78 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 34.90, 37.69, 44.49, 46.13, 56.36, 62.85,
75.53, 110.83, 115.52, 115.82, 116.05, 116.34, 116.47, 123.22,
124.41, 124.45, 124.79, 125.01, 126.96, 127.69, 129.19, 130.24,
131.07, 131.69, 131.81, 132.27, 133.53, 142.53, 167.01, 176.81,
196.31.
8.11 Hz, 1H, H-4), 5.72 (d, J = 10.25 Hz, 1H, H-
a), 6.26 (d,
J = 17.32 Hz, 1H, H- ), 6.59 (dd, J = 17.32, 10.25 Hz, 1H, H-b),
a
6.82–7.52 (m, 12H, H-aromatic) 7.75 (s, 1H, H-arylmethylidene),
9.17 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 34.83, 44.37,
45.26, 46.49, 57.71, 63.56, 75.29, 110.73, 123.04, 125.32, 126.96,
127.58, 129.19, 129.35, 129.52, 130.18, 130.49, 131.58, 132.03,
132.80, 133.73, 136.22, 136.35, 139.52, 142.80, 167.45, 176.97,
196.55.
4.1.1.6.
dole)-spiro[3.300]-500-(3-nitrophenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8f).
White solid; (0.135 g, 82%); mp
157–159 °C; IR (KBr)
max: 3294, 1712, 1615 cmꢀ1; Anal. Calcd
1-Methyl-4-(3-nitrophenyl)pyrrolo-(spiro[2.30]oxin-
m
for C32H26N5O7: C, 64.86; H, 4.42; N, 11.82. Found: C, 64.17; H,
4.79; N, 12.25. 1H NMR (500 MHz, CDCl3): d 2.20 (s, 3H, CH3),
2.54 (d, J = 14.07, H-200), 3.25 (dd, J = 18.25, 2.07 Hz, 1H, H-600),
3.57 (t, J = 8.38 Hz, 1H, H-5), 4.07 (t, J = 9.19 Hz, 1H, H-5), 4.25 (d,
J = 18.25, 1H, H-600), 4.29 (d, J = 14.07, 1H, H-200), 5.05 (dd, J = 9.19,
4.1.1.10. 1-Methyl-4-(4-fluorophenyl)pyrrolo-(spiro[2.30]oxin-
dole)-spiro[3.300]-500-(4-fluorophenylmethylidene)-100-N-acro-
lylpiperidin-400-one (8j).
White solid; (0.142 g, 77%); mp
181–184 °C; IR (KBr)
m
max: 3423, 1725, 1615 cmꢀ1; Anal. Calcd
8.38 Hz, 1H, H-4), 5.74 (d, J = 10.52 Hz, 1H, H-
a
), 6.27 (d,
for C32H26F2N3O3: C, 71.36; H, 4.87; N, 7.80. Found: C, 70.87; H,
4.21; N, 7.89. 1H NMR (500 MHz, CDCl3): d 2.09 (s, 3H, CH3), 2.45
(d, J = 14.50, H-200), 3.20 (dd, J = 18.05, 2.45 Hz, 1H, H-600), 3.40 (t,
J = 8.35 Hz, 1H, H-5), 3.94 (t, J = 9.45 Hz, 1H, H-5), 4.16 (d,
J = 18.05, 1H, H-600), 4.20 (d, J = 14.35, 1H, H-200), 4.86 (dd, J = 9.45,
J = 16.24 Hz, 1H, H- ), 6.55 (dd, J = 16.24, 10.52 Hz, 1H, H-b),
a
6.83–8.41 (m, 13H, H-aromatic, H-arylmethylidene), 8.79 (s, 1H,
NH). 13C NMR (125 MHz, CDCl3): d 34.88, 44.42, 45.30, 46.53,
57.76, 63.62, 75.34, 110.74, 123.08, 125.32, 126.96, 127.77,
129.14, 129.42, 129.57, 130.25, 130.54, 131.71, 132.07, 132.80,
133.73, 136.22, 136.35, 139.52, 142.80, 167.44, 176.92, 196.53.
8.35 Hz, 1H, H-4), 5.63 (d, J = 10.71 Hz, 1H, H-
a), 6.19 (d,
J = 16.86 Hz, 1H, H- ), 6.52 (dd, J = 16.86, 10.71 Hz, 1H, H-b),
a
6.74–7.44 (m, 12H, H-aromatic) 7.69 (s, 1H, H-arylmethylidene),
9.52 (s, 1H, NH) 13C NMR (125 MHz, CDCl3):d 33.43, 44.00, 44.70,
46.18, 56.45, 62.07, 73.90, 109.42, 114.36, 114.53, 114.77, 115.04,
121.50, 124.10, 125.70, 126.17, 128.07, 128.46, 128.60, 129.23,
130.31, 131.44, 131.51, 132.17, 138.25, 141.81, 160.11, 161.12,
162.07, 163.13, 166.31, 175.76, 195.19.
4.1.1.7.
oxindole)-spiro[3.300]-500-(2,4-dichlorophenylmethylidene)-100-
N-acrolylpiperidin-400-one (8g).
White solid; (0.144 g, 80%);
mp 171–174 °C; IR (KBr)
max: 3419, 1701, 1640 cmꢀ1; Anal. Calcd
for C32H24Cl4N3O3: C, 60.02; H, 3.78; N, 6.56. Found: C, 60.45; H,
4.28; N, 7.81. 1H NMR (500 MHz, CDCl3): d 2.17 (s, 3H, CH3), 2.70
(d, J = 14.40, H-200), 2.81 (d, J = 17.40, 1H, H-600), 3.59 (t,
J = 8.27 Hz, 1H, H-5), 4.05 (t, J = 9.49 Hz, 1H, H-5), 4.06 (d,
J = 17.40, 1H, H-600), 4.09 (d, J = 14.40, 1H, H-200), 5.07 (dd, J = 9.49,
1-Methyl-4-(2,4-dichlorophenyl)pyrrolo-(spiro[2.30]
m
4.1.1.11. 1-Methyl-4-(1-naphthyl)pyrrolo-(spiro[2.30]oxindole)-
spiro[3.300]-500-(1-naphthylmethylidene)-100-N-acrolylpiperidin-
400-one (8k).
(KBr) m
max: 3429, 1716, 1617 cmꢀ1; Anal. Calcd for C40H32N3O3:
White solid; (0.177 g, 85%); mp 171–173 °C; IR
8.27 Hz, 1H, H-4), 5.67 (d, J = 11.07 Hz, 1H, H-
a
), 6.12 (d,
J = 17.15 Hz, 1H, H- ), 6.79 (dd, J = 17.15, 11.07 Hz, 1H, H-b),
a
79.71; H, 5.35; N, 6.97. Found: 80.15; H, 6.12; N, 7.49. 1H NMR
(500 MHz, CDCl3): d 2.15 (s, 3H, CH3), 2.57 (d, J = 14.21, H-200),
3.39 (d, J = 18.07, 1H, H-600), 3.55 (t, J = 7.87 Hz, 1H, H-5), 4.27 (t,
6.89–7.41 (m, 10H, H-aromatic) 7.76 (s, 1H, H-arylmethylidene),
8.02 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 34.65, 42.06,

1326
Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
J = 9.12 Hz, 1H, H-5), 4.41 (d, J = 18.07, 1H, H-600), 4.48 (d, J = 14.21,
1H, H-200), 5.15 (dd, J = 9.12, 7.87 Hz, 1H, H-4), 5.71 (d, J = 11.02 Hz,
1H, H-a), 6.13 (d, J = 17.21 Hz, 1H, H-a), 6.32 (dd, J = 17.21,
11.02 Hz, 1H, H-b), 6.41–7.69 (m, 16H, H-aromatic) 7.79 (s, 1H,
H-arylmethylidene), 8.24 (s, 1H, NH). 13C NMR (125 MHz, CDCl3):
d 29.07, 47.86, 48.02, 51.81, 56.83, 61.17, 76.34, 112.14, 125.53,
126.57, 127.44, 128.33, 128.80, 129.86, 130.26, 130.42, 131.62,
131.74, 132.68, 133.44, 134.41, 136.67, 137.41, 137.81, 138.06,
139.15, 139.47, 143.65, 175.08, 180.96, 198.42.
8.02 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 24.51, 29.32, 38.29,
38.44, 41.17, 47.96, 48.67, 51.71, 56.92, 59.43, 64.31, 66.52, 76.17,
78.90, 113.22, 114.51, 118.45, 119.17, 119.36, 119.42, 125.59,
126.54, 127.91, 127.97, 128.19, 128.37, 129.11, 129.89, 130.31,
132.44, 132.59, 133.14, 134.12, 134.68, 135.54, 135.76, 135.97,
136.55, 144.71, 146.39, 175.11, 180.59, 182.15, 199.27.
4.1.1.15.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(2-chlorophenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(2-chlo-
4.1.1.12.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
rophenyl) pyrrolidine (9d).
147–149 °C; IR (KBr)
max: 3412, 1719, 1617 cmꢀ1, Anal. Calcd for
42H36Cl2N5O4: C, 67.65; H, 4.87; N, 9.39. Found: C, 68.19; H,
White solid; (0.105 g, 52%); mp
[2.30]oxindole-N-methyl pyrrolo)-500-(phenylmethylidene)tetra-
hydro-400-(1H)-pyridinone-1-methyl-4-(phenyl) pyrrolidine
m
C
(9a).
White solid; (0.125 g, 71%); mp 160–162 °C; IR (KBr)
5.29; N, 9.71. 1H NMR (500 MHz, CDCl3): d 1.95 (s, 3H, CH3), 2.15–
2.18 (s, 4H, CH3, H-4 of ring A), 2.52 (d, J = 16.80, 1H, H-600), 2.71–
2.81 (m, 1H, H-4 of ring A), 3.05–3.25 (m, 3H, CH2-5 of ring A, H-5
of ring B), 3.35 (d, J = 13.10 Hz, 1H, H-200), 3.40 (d, J = 16.80 Hz, 1H,
H-600), 3.47 (d, J = 13.10, 1H, H-200), 3.67–3.73 (m, 1H, H-3 of ring
A), 4.04–4.11 (m, 1H, H-5 of ring B), 4.86–4.92 (m, 1H, H-4 of ring
B), 6.06–7.54 (m, 16H, H-aromatic) 7.61 (s, 1H, H-arylmethylidene),
8.13 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 26.24, 34.82, 35.15,
42.52, 44.11, 44.93, 48.89, 53.53, 56.99, 62.96, 72.51, 75.77, 76.97,
77.39, 77.59, 77.81, 109.10, 110.67, 122.02, 123.40, 125.10,
126.83, 127.53, 127.94, 128.77, 129.64, 129.81, 129.87, 130.41,
130.70, 131.31, 132.20, 132.71, 132.95, 135.21, 136.89, 137.21,
140.46, 170.89, 179.17, 181.19, 198.73.
m
max: 3321, 1712, 1615 cmꢀ1; Anal. Calcd for C42H38N5O4: C,
74.54; H, 5.66; N, 10.35. Found: C, 75.12; H, 6.11; N, 11.21. 1H
NMR (500 MHz, CDCl3): d 0.48 (d, 1H, J = 14.45 Hz, H-600), 1.63 (s,
3H, CH3), 2.02 (s, 3H, CH3), 2.08–2.14 (m, 1H, H-4 of ring A),
2.38–2.44 (m, 2H, H-200, H-4 of ring A), 2.91-3.01 (m, 2H, CH2-5
of ring A), 3.21 (d, 1H, J = 14.45 Hz, H-600), 3.25 (d, 1H,
J = 14.50 Hz, 200), 3.36–3.40 (m, 1H, H-3 of ring A), 4.12 (t, 1H,
J = 9.00 Hz, H-5 of ring B), 5.21 (t, 1H, J = 9.00 Hz, H-4 of ring A),
5.50–8.08 (m, 19H, H-aromatic, H-arylmethylidene), 8.09 (s, 1H,
NH), 10.61 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 24.63,
34.01, 34.12, 43.22, 43.40, 45.58, 51.83, 56.61, 62.68, 72.18,
74.77, 108.55, 110.21, 119.19, 121.83, 122.52, 124.54, 124.65,
124.99, 125.27, 125.43, 125.77, 126.03, 126.24, 126.51, 126.79,
127.27, 128.09, 128.49, 129.15, 129.49, 129.76, 130.75, 130.78,
132.59, 132.74, 132.76, 133.44, 133.59, 137.43, 141.12, 142.97,
170.21, 176.38, 178.01, 199.51.
4.1.1.16.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(2-fluorophenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(2-fluo-
rophenyl) pyrrolidine (9e).
White solid; (0.115 g, 54%); mp
4.1.1.13.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
171–174 °C; IR (KBr)
m
max: 3425, 1702, 1618 cmꢀ1; Anal. Calcd
[2.30]oxindole-N-methyl pyrrolo)-500-(2-methylphenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(2-methyl-
for C42H36F2N5O4: C, 70.77; H, 5.09; N, 9.83. Found: C, 71.29; H,
5.44; N, 10.29. 1H NMR (500 MHz, CDCl3): d 2.05 (s, 3H, CH3),
2.10 (s, 3H, CH3), 2.22–2.29 (m, 1H, H-4 of ring A), 2.64–2.70 (m,
2H, H-600, H-4 of ring A), 3.12–3.20 (m, 2H, CH2-5 of ring A),
3.30–3.32 (m, 1H, H-5 of ring B), 3.35 (d, J = 13.70 Hz, 1H, H-200),
3.44 (d, J = 17.15 Hz, 1H, H-600), 3.55 (d, J = 13.70, 1H, H-200), 3.72–
3.75 (m, 1H, H-3 of ring A), 4.00–4.04 (m, 1H, H-5 of ring B),
4.88–4.90 (m, 1H, H-4 of ring B), 6.02–7.48 (m, 16H, H-aromatic)
7.56 (s, 1H, H-arylmethylidene), 7.86 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 24.77, 29.14, 38.07, 38.56, 41.05, 47.93,
48.60, 51.54, 56.84, 59.64, 64.54, 66.03, 76.67, 78.89, 113.19,
114.25, 118.90, 119.08, 119.36, 119.53, 125.54, 126.51, 127.81,
127.84, 128.29, 128.37, 128.97, 129.89, 130.62, 132.37, 132.44,
133.08, 133.94, 134.68, 135.33, 135.39, 135.96, 136.77, 144.71,
146.29, 175.46, 180.93, 183.05, 199.17.
phenyl) pyrrolidine (9b).
142–144 °C; IR (KBr)
White solid; (0.118 g, 60%); mp
m
max: 3432, 1714, 1619 cmꢀ1; Anal. Calcd
for C44H42N5O4: C, 74.98; H, 6.01; N, 9.94. Found: C, 75.51; H,
6.44; N, 10.32. 1H NMR (500 MHz, CDCl3): d 2.07 (s, 3H, CH3),
2.15 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.34–2.38
(m, 1H, H-4 of ring A), 2.71–2.79 (m, 2H, H-600, H-4 of ring A),
3.17–3.29 (m, 2H, CH2-5 of ring A), 3.48–3.53 (m, 1H, H-5 of ring
B), 3.45 (d, J = 14.07 Hz, 1H, H-200), 3.59 (d, J = 17.32 Hz, 1H, H-600),
3.90 (d, J = 14.07, 1H, H-200), 3.95–4.02 (m, 1H, H-3 of ring A),
4.15–4.21 (m, 1H, H-5 of ring B), 5.21–5.27 (m, 1H, H-4 of ring
B), 6.17–7.52 (m, 16H, H-aromatic), 7.72 (s, 1H, H-arylmethyli-
dene), 7.91 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 21.45,
21.74, 26.15, 34.61, 35.42, 44.32, 45.25, 45.52, 49.02, 54.32,
57.21, 63.29, 73.52, 75.19, 109.16, 110.12, 122.21, 122.75, 124.45,
125.32, 126.58, 127.27, 129.36, 129.60, 130.09, 130.28, 130.43,
130.89, 131.78, 135.47, 136.68, 140.18, 140.37, 141.32, 142.69,
171.88, 177.65, 179.50, 198.19.
4.1.1.17.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(3-nitrophenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(3-nitrophenyl)
pyrrolidine (9f).
White solid; (0.105 g, 57%); mp 160–162 °C;
4.1.1.14.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
IR (KBr)
m
max: 3427, 1707, 1619 cmꢀ1; Anal. Calcd for C42H36N7O8:
[2.30]oxindole-N-methyl pyrrolo)-500-(2-methoxyphenylmethy-
lidene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(2-methoxy-
C, 65.79; H, 4.73; N, 12.79. Found: C, 65.95; H, 4.92; N, 12.58. 1H
NMR (500 MHz, CDCl3): d 1.78 (s, 3H, CH3), 2.19 (s, 3H, CH3),
2.20–2.24 (m, 1H, H-4 of ring A), 2.49–2.62 (m, 2H, H-600, H-4 of
ring A), 2.98–3.02 (m, 1H, CH2-5 of ring A), 3.13–3.18 (m, 1H, H-
5 of ring B), 3.28 (d, J = 13.95 Hz, 1H, H-200), 3.47–3.53 (m, 2H, H-
200, H-600), 3.64–3.68 (m, 1H, H-3 of ring A), 4.30–4.34 (m, 1H, H-5
of ring B), 5.01–5.05 (m, 1H, H-4 of ring B), 6.12–8.03 (m, 17H,
H-aromatic, H-arylmethylidene), 8.14 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 21.54, 22.15, 26.39, 31.25, 37.19, 37.61,
45.19, 46.51, 46.97, 53.29, 64.79, 68.03, 76.19, 78.42, 111.19,
122.47, 125.79, 127.21, 127.72, 128.14, 128.56, 129.79, 130.44,
130.96, 131.12, 131.67, 132.17, 132.23,132.44, 136.21, 138.71,
143.20, 145.32, 177.15, 179.21, 181.29, 199.05.
phenyl) pyrrolidine (9c).
164–166 °C; IR (KBr)
max: 3430, 1718, 1617 cmꢀ1; Anal. Calcd for
44H42N5O6: C, 71.72; H, 5.75; N, 9.50. Found: C, 72.41; H, 6.22;
White solid; (0.138 g, 64%); mp
m
C
N, 10.17. 1H NMR (500 MHz, CDCl3): d 1.98 (s, 3H, CH3), 2.15 (s,
3H, CH3), 2.19–2.25 (m, 1H, H-4 of ring A), 2.62–2.74 (m, 2H,
H-600, H-4 of ring A), 3.13–3.22 (m, 2H, CH2-5 of ring A), 3.34–3.38
(m, 1H, H-5 of ring B), 3.39 (d, J = 13.43 Hz, 1H, H-200), 3.49 (d,
J = 17.05 Hz, 1H, H-600), 3.69 (d, J = 13.43, 1H, H-200), 3.72 (s, 3H,
O–CH3), 3.76 (s, 3H, O–CH3), 3.80–3.85 (m, 1H, H-3 of ring A),
4.12–4.17 (m, 1H, H-5 of ring B), 5.09–5.16 (m, 1H, H-4 of ring B),
6.25–7.71 (m, 16H, H-aromatic), 7.65 (s, 1H, H-arylmethylidene),

Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
1327
4.1.1.18.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
2H, H-600, H-4 of ring A), 3.04–3.15 (m, 2H, 5-CH2 of ring A),
3.43–3.48 (m, 1H, H-5 of ring B), 3.62 (d, J = 13.55 Hz, 1H, H-200),
3.75 (d, J = 17.49 Hz, 1H, H-600), 4.04 (d, J = 13.55, 1H, H-200), 4.12–
4.19 (m, 1H, H-3 of ring A), 4.25–4.28 (m, 1H, H-5 of ring B),
4.79–4.84 (m, 1H, H-4 of ring B), 6.11–7.56 (m, 16H, H-aromatic),
7.54 (s, 1H, H-arylmethylidene), 8.19 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): d 24.25, 29.32, 38.19, 38.76, 41.29, 48.24,
48.92, 51.77, 57.12, 60.21, 64.92, 66.25, 76.77, 79.31, 112.20,
113.25, 117.90, 119.25, 119.41, 119.72, 125.81, 126.41, 127.83,
127.87, 128.42, 128.64, 129.12, 129.89, 130.52, 132.76, 132.56,
133.19, 134.12, 134.79, 135.56, 135.91, 135.99, 136.79, 144.82,
146.58, 174.26, 181.31, 182.94, 198.15.
[2.30]oxindole-N-methyl pyrrolo)-500-(2,4-dichlorophenylmethy-
lidene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(2,4-
dichlorophenyl) pyrrolidine (9g).
69%); mp 151–153 °C; IR (KBr)
White solid; (0.138 g,
m
max: 3429, 1711, 1621 cmꢀ1, Anal.
Calcd for C42H34Cl4N5O4: C, 61.93; H, 4.21; N, 8.60. Found: C, 62.44;
H, 4.79; N, 9.07. 1H NMR (500 MHz, CDCl3): d 1.99 (s, 3H, CH3), 2.12
(s, 3H, CH3), 2.30–2.34 (m, 1H, H-4 of ring A), 2.72–2.79 (m, 2H, H-
600, H-4 of ring A), 2.94–3.03 (m, 2H, CH2-5 of ring A), 3.06–3.14 (m,
1H, H-5 of ring B), 3.29–3.35 (m, 1H, H-200), 3.45–3.53 (m, 1H, H-
600), 3.73 (d, J = 14.10, 1H, H-200), 3.79 (d, J = 17.42, 1H, H-600),
3.89–3.94 (m, 1H, H-3 of ring A), 3.96–4.02 (m, 1H, H-5 of ring
B), 4.74–4.80 (m, 1H, H-4 of ring B), 6.08–7.32 (m, 14H, H-aro-
matic), 7.53 (s, 1H, H-arylmethylidene), 8.03 (s, 1H, NH). 13C
NMR (125 MHz, CDCl3): d 22.44, 22.91, 26.43, 35.34, 36.02, 45.05,
45.72, 46.19, 54.18, 57.69, 64.49, 64.71, 72.64, 74.91, 109.79,
110.44, 123.78, 124.91, 128.18, 128.85, 129.51, 131.29, 131.60,
141.45, 141.61, 173.77, 178.80, 181.05, 199.45.
4.1.1.22.
[2.30]oxindole-N-methyl
400-(1H)-pyridinone-1-methyl-4-(1-naphthyl) pyrrolidine
(9k). White solid; (0.172 g, 72%); mp 195–197 °C; IR (KBr)
max: 3406, 1711, 1618 cmꢀ1; Anal. Calcd for C50H42N5O4: C,
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
pyrrolo)-500-(1-naphthyl)tetrahydro-
m
77.30; H, 5.45; N, 9.01. Found: C, 77.81; H, 5.92; N, 9.28. 1H NMR
(500 MHz, CDCl3): 1.89 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.20–2.25
(m, 1H, H-4 of ring A), 2.50–2.57 (m, 2H, H-600, H-4 of ring A),
3.15–3.22 (m, 2H, CH2-5 of ring A), 3.38–3.44 (m, 1H, H-5 of ring
A), 3.59 (d, J = 13.84 Hz, 1H, H-200), 3.92 (d, J = 17.35 Hz, 1H, H-
600), 4.13 (d, J = 13.84, 1H, H-200), 4.24–4.27 (m, 1H, H-3 of ring A),
4.80–4.86 (m, 1H, H-4 of ring B), 5.60–5.64 (m, 1H, H-5 of ring
B), 6.13–7.47 (m, 24H, H-aromatic) 7.66 (s, 1H, H-arylmethyli-
dene), 8.02 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 25.12,
30.22, 38.19, 38.79, 44.09, 46.51, 46.92, 53.24, 64.12, 67.03,
75.91, 112.19, 125.72, 127.52, 128.39, 129.64, 129.88, 129.96,
130.12, 130.74, 130.99,131.44, 136.17, 138.54, 142.44, 145.32,
176.46, 179.83, 182.25, 198.35.
4.1.1.19.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(4-methylphenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(4-methyl-
phenyl) pyrrolidine (9h).
121–124 °C; IR (KBr)
White solid; (0.119 g, 65%); mp
m
max: 3429, 1712, 1613 cmꢀ1; Anal. Calcd
for C44H42N5O4: C, 74.98; H, 6.01; N, 9.94. Found: C, 75.71; H,
6.52; N, 10.44. 1H NMR (500 MHz, CDCl3): d 2.03 (s, 3H, CH3),
2.11 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.27–2.34
(m, 1H, H-4 of ring A), 2.69–2.75 (m, 2H, H-600, H-4 of ring A),
3.14–3.22 (m, 2H, CH2-5 of ring A), 3.36–3.43 (m, 1H, H-5 of ring
B), 3.48 (d, J = 13.82 Hz, 1H, H-200), 3.69 (d, J = 17.24 Hz, 1H, H-600),
3.83 (d, J = 13.82, 1H, H-200), 4.02–4.07 (m, 1H, H-3 of ring A),
4.10–4.14 (m, 1H, H-5 of ring B), 4.67–4.72 (m, 1H, H-4 of ring
B), 6.15–7.53 (m, 14H, H-aromatic) 7.52 (s, 1H, H-arylmethyli-
dene), 7.80 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 21.52,
21.83, 26.28, 34.75, 35.33, 44.54, 45.19, 45.43, 48.87, 53.48,
57.03, 63.82, 73.22, 74.78, 109.46, 110.58, 122.18, 122.96, 124.98,
125.74, 126.96, 127.51, 129.36, 129.63, 130.01, 130.20, 130.33,
130.86, 131.68, 135.45, 136.48, 140.28, 140.48, 141.19, 142.64,
172.75, 177.71, 179.85, 197.18.
4.2. AChE and BChE inhibitory assay
The cholinesterase enzymes inhibitory potential was evaluated
using modified Ellman’s method as described by Ahmed and Gilan-
i.23 Galanthamine was used as reference standard. Solutions of test
samples and galanthamine were prepared in DMSO at an initial
concentration of 1 mg/mL (100% DMSO). The concentration of
DMSO in final reaction mixture was 1%. At this concentration,
DMSO has no inhibitory effect on both acetylcholinesterase and
butyrylcholinesterase enzymes.24
4.1.1.20.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(4-chlorophenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(4-chlo-
rophenyl) pyrrolidine (9i).
White solid; (0.132 g, 61%); mp
For acetylcholinesterase (AChE) inhibitory assay, 140
0.1 M sodium phosphate buffer of pH 8 was first added to a 96-well
microplate followed by 20 L of test samples and 20 of
0.09 units/mL acetylcholinesterase. After 15 min of incubation at
25 °C, 10
L of 10 mM 5,50-dithiobis-2-nitrobenzoic acid (DTNB)
was added into each well followed by 10 L of 14 mM acetylthi-
ocholine iodide. Absorbance of the colored end-product was mea-
sured using BioTek PowerWave 340 microplate
spectrophotometer at 412 nm for thirty min after the initiation of
enzymatic reaction. For butyrylcholinesterase (BChE) inhibitory as-
say, the same procedure described above was followed, except for
the use of enzyme and substrate, instead of which, butyrylcholin-
esterase from equine serum and S-butyrylthiocholine chloride
were used.
lL of
170–172 °C; IR (KBr)
m
max: 3431, 1718, 1611 cmꢀ1; Anal. Calcd
for C42H36Cl2N5O4: C, 67.65; H, 4.87; N, 9.39. Found: C, 68.22; H,
5.37; N, 9.78. 1H NMR (500 MHz, CDCl3): d 1.99 (s, 3H, CH3), 2.12
(s, 3H, CH3), 2.30–2.35 (m, 1H, H-4 of ring A), 3.15–3.35 (m, 4H,
H-600, H-4 of ring A, 5-CH2 of ring A), 3.71–3.80 (m, 2H, H-5 of ring
B, H-200), 3.38–4.05 (m, 2H, H-200, H-600), 4.26–4.32 (m, 1H, H-3 of
ring A), 4.54–4.60 (m, 1H, H-5 of ring B), 4.74–4.82 (m, 1H, H-4
of ring B), 6.09–7.32 (m, 16H, H-aromatic) 7.54 (s, 1H, H-arylmeth-
ylidene), 9.22 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): d 21.11,
21.46, 25.93, 34.34, 34.90, 44.05, 44.72, 44.93, 53.18, 56.56,
63.49, 72.54, 74.35, 108.79, 109.79, 122.71, 124.74, 127.18,
128.85, 129.22, 130.08, 130.54, 140.45, 140.61, 171.77, 179.10,
182.07, 197.42.
l
lL
l
l
X
Each test was conducted in triplicate. Absorbance of the test
samples were corrected by subtracting the absorbance of their
respective blank. Percentage inhibition was calculated using the
following formula:
4.1.1.21.
Spiro-[2.30]-oxindole-spiro[3.300]-100-carbonyl(spiro
[2.30]oxindole-N-methyl pyrrolo)-500-(4-fluorophenylmethylid-
ene)tetrahydro-400-(1H)-pyridinone-1-methyl-4-(4-fluo-
rophenyl) pyrrolidine (9j).
169–171 °C; IR (KBr)
for C42H36F2N5O4: C, 70.77; H, 5.09; N, 9.83. Found: C, 71.86; H,
5.52; N, 10.38. 1H NMR (500 MHz, CDCl3): d 2.04 (s, 3H, CH3),
2.12 (s, 3H, CH3), 2.19–2.25 (m, 1H, H-4 of ring A), 2.59–2.65 (m,
White solid; (0.129 g, 64%); mp
m
max: 3422, 1707, 1618 cmꢀ1; Anal. Calcd
Percentage of inhibition
Absorbance of control ꢀ Absorbance of sample
¼
ꢁ 100
Absorbance of control

1328
Y. kia et al. / Bioorg. Med. Chem. 22 (2014) 1318–1328
4.2.1. Enzyme kinetics and mode of inhibition
References and notes
Lineweaver–Burk plot analysis was performed to determine the
mode of enzyme inhibition and enzyme kinetics of compounds 9j
and 8e on AChE and BChE, respectively. The enzyme inhibition
kinetics was carried out in the absence and presence of 9j (0,
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including Michaelis–Menten (Km) constant and maximum velocity
(Vmax) were derived from Lineweaver–Burk plot while inhibition
constant (Ki) was derived from Dixon plot.
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4.3. Molecular docking study
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Molecular docking was performed using Glide software (version
5.7, Schrödinger, LLC, New York, NY, 2011). All the synthesized
compounds were docked into the active site of TcAChE derived
from three-dimensional structure of the enzyme complex with
anti-Alzheimer’s drug, E2020 (Aricept™) (PDB ID: 1EVE) and BChE
derived from complex of the enzyme with its substrate BCh (PDB
code: 1P0P). Water molecules and hetero groups were deleted
from active site beyond the radius of 5 Å of reference ligand
(E2020 or BCh), resulting protein structure refined and minimized
by Protein Preparation Wizard using OPLS-2005 force field. Recep-
tor Grid Generation program were used to prepare AChE and BChE
grid and all the ligands were optimized by LigPrep program by
using OPLS-2005 force field to generate lowest energy state of
respective ligands. Docking stimulations were carried out on all
compounds, handed in 5 poses per ligand, in which the best pose
with highest score was displayed for each ligand.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.002.