An expedient, ionic liquid mediated multi-component synthesis of novel piperidone grafted cholinesterase enzymes inhibitors and their molecular modeling study

Article abstract of DOI:10.1016/j.ejmech.2013.06.054

Series of hitherto unreported piperidone grafted pyridopyrimidines synthesized through ionic liquid mediated multi-component reaction. These compounds were evaluated for their inhibitory activities against AChE and BChE enzymes. All the compounds displaye

Full text of DOI:10.1016/j.ejmech.2013.06.054

European Journal of Medicinal Chemistry 67 (2013) 221e229
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
An expedient, ionic liquid mediated multi-component synthesis of
novel piperidone grafted cholinesterase enzymes inhibitors and their
molecular modeling study
,
b
c,
Alireza Basiri a, Vikneswaran Murugaiyah a , Hasnah Osman , Raju Suresh Kumar
*
**
,
Yalda Kia b, Khalijah Binti Awang d, Mohamed Ashraf Ali e
a School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
b School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
c Department of Chemistry, College of Sciences, King Saud University, PO Box 2455, Riyadh, Saudi Arabia
d Department of Chemistry, Faculty of Science University of Malaya, 50603 Kuala Lampur, Malaysia
e Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of hitherto unreported piperidone grafted pyridopyrimidines synthesized through ionic liquid
mediated multi-component reaction. These compounds were evaluated for their inhibitory activities
against AChE and BChE enzymes. All the compounds displayed considerable potency against AChE with
Received 1 June 2013
Received in revised form
19 June 2013
Accepted 24 June 2013
Available online 4 July 2013
IC50 values ranging from 0.92 to 9.11
inhibitory activities compared to standard drug with IC50 values of 0.92, 1.29 and 2.07
all the compounds displayed higher BChE inhibitory activity compared to galantamine with IC50 values of
1.89e8.13 M. Molecular modeling, performed for the most active compounds using three dimensional
m
M, therein compounds 6a, 6h and 6i displayed superior enzyme
m
M. Remarkably,
m
Keywords:
AChE
BChE
Multi-component reaction
Ionic liquid
crystal structures of TcAChE and hBChE, disclosed binding template of these inhibitors into the active site
of their respective enzymes.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Molecular modeling
1. Introduction
and cognitive dysfunctions in AD patients, the so-called cholinergic
hypothesis, has gained interest [4].
According to World Alzheimer’s Report 2012, there are 36 million
people living with dementia worldwide, which is predicted to in-
crease up to 115 million by 2050 unless there is a cure or treatment to
delay the onset or progression of the disease [1]. Alzheimer’s disease
(AD) is the most common type of dementia. Biochemical deficits in
AD patients arise from degeneration of the cholinergic neurons
caused by the phosphorylation of tau proteins leading to develop-
Currently approved pharmacological treatments for AD are
limited to cholinesterase inhibitors (ChEI’s), working by inhibiting
cholinesterase enzymes from hydrolyzing acetylcholine to restore
the cholinergic function as well as the N-methyl
D-aspartate
receptor antagonist (e.g. memantine), which acts at the gluta-
minergic pathway [5e7]. Despite the tremendous efforts in search
of disease modifying agents working along with the b-amyloid or
ment of neurofibrillary tangles and formation of
b
-amyloid senile
tau pathways, none are clinically available due to their adverse
effects. Therefore, the search for new cholinesterase enzymes in-
hibitors is still ongoing worldwide.
plaques. This neurodegeneration leads to remarkable reduction of
neurotransmitter acetylcholine at the synaptic clefts [2,3]. Since
acetylcholine plays a major role in cognitive processes, increasing
acetylcholinelevelsto restore thesubstantial impairmentof memory
Both cholinesterase enzymes acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) are involved in the hydrolysis of
acetylcholine; however studies showed that as the disease pro-
gresses, the activity of AChE decreases while the activity of BChE
remains unaffected or even increases [8]. In the brain of advanced
staged AD patients, BChE can compensate for AChE when the activity
of AChE is inhibited by AChE inhibitors. Thus, BChE hydrolyses the
already depleted levels of ACh in these patients [9,10]. It has been
* Corresponding author. Tel./fax: þ6046534583.
** Corresponding author.
E-mail addresses: dr.Murugaiyah@gmail.com (V. Murugaiyah), sraju@ksu.edu.sa
(R.S. Kumar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.054

222
A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
Table 1
pyrimidine entities are reported to possess potent dual cholines-
terase and A -aggregation inhibitory properties [27e31]. Inspired
Residue composition of active sites in TcAChE and hBChE.
b
by the aforementioned inhibitory potential of pyrimidine de-
rivatives on cholinesterase enzymes activity and in search of new
potent AChE/BChE enzymes inhibitors, in the present study we
report an efficient method to synthesize biologically active piper-
idone grafted pyridopyrimidines via microwave assisted, multi-
component reaction methodology in ionic liquid and their cholin-
esterase enzyme inhibitory activities.
Entry Site name Residue composition in Residue composition in
TcAChE
hBChE [34]
1
2
Catalytic triad
Ser200, His440 and
Glu327
Trp84 and Phe330
His438, Ser198 and
Glu325
Trp82 and Phe329
Choline binding
site ( -anionic site)
Acyl-binding pocket Phe288 and Phe290
a
3
4
Leu286 and Val288
Gly116, Gly117 and
Ala199
Oxyanion hole
Gly118, Gly117 and
Ala201
5
Peripheral anionic
site (b-anionic site) Trp279 and Tyr334 and Phe398
Tyr 70, Asp72, Tyr 121, Trp231, Val288, Leu286
2. Result and discussion
2.1. Chemistry
also proposed that individuals with low-activity of BChE can sustain
cognitive functions better comparing to individuals with normal
BChE activity [11]. Furthermore, restoration of ACh levels by BChE
inhibition seems to occur without apparent adverse effects [9].
Molecular modeling plays an important role in the rational drug
design and is used to predict the bonding affinity, spatial orienta-
tion and total binding energy of the small molecule drug candidates
to the active site of their target enzymes [12]. Active site of AChE
and BChE enzymes is located deep in the center of the molecule
with a narrow gorge made up of five important regions to accom-
modate and hydrolyze the acetylcholine substrate, namely, cata-
lytic triad [13], oxyanion hole [14], choline binding site [15], acyl
binding pocket [16] and peripheral anionic site (Table 1) [17]. In
AChE, aromatic residues such as tryptophan (Trp) and phenylala-
nine (Phe) comprise the active site gorge, whilst in BChE the gorge
is lined with hydrophobic residues such as valine (Val), which al-
lows accommodation of bulkier substrates [17].
In the context of green chemistry, ionic liquid (IL) mediated
multicomponent reactions gained much attentions as an efficient
synthetic tool from the viewpoint of evasion from intermediate
isolation and purification steps, effectively merged with unique
properties of green ionic solvents such as strong solvating ability,
catalytic behavior and recyclability [18,19]
Natural and synthetic biologically active compounds with
pyrimidine moiety, find wide applications in pharmaceutical field
[20] as antihypertensive [21], a1-adrenergic receptor antagonist
[22], antibacterial, anti-inflammatory, antitumor [23] and anti-HIV
agents [24e26]. Moreover, compounds comprising thiourea and
N-Substituted piperidine-4-ones 2/3 were prepared by refluxing
4-piperidone monohydrate hydrochloride (1) and ethyl bromide (2)/
4-(2-chloroethyl)morpholine (3) in ethanol (Scheme 1). With the
functionalized piperidones 2/3 in hand, the aim is to synthesize
pyridopyrimidine-2-thiones using ionic liquid as green solvent.
To optimize the reaction conditions, initially a model reaction of
1-ethylpiperidine-4-one, benzaldehyde and thiourea in 1:2:1 molar
ratio in 1 M equiv of [BMIM]Br was performed under conventional
heating, the reaction progress was monitored by TLC. After
completion of the reaction (60 min), the product was isolated (52%)
through flash column chromatography. Subsequently, the same
reaction was investigated also, under microwave irradiation which as
in the case of thermal reaction, the reactants in 1 M equiv of [BMIM]
Br was subjected to microwave irradiation. After completion of the
reaction (2 min), the pyridopyrimidine-2-thiones were isolated as
the single reaction product through column chromatography in 71%
yield. The above results clearly showed that microwave irradiation
led to an enhancement in the yield of the product and the reaction
time has also been reduced over the conventional thermal method.
Consequently, all other reactions were performed under microwave
irradiation.
The structure of the pyridopyrimidine-2-thiones 6/7 is in
accordance with the combustion data, 1D and 2D NMR spectro-
scopic data and IR spectroscopy (vide infra). In the 1H NMR spectrum
of 7c, the singlet at 5.58 ppm is readily assigned to H-4, which shows
HMBC correlations with the doublets at 2.90 and 3.18 ppm
(J ¼ 16.75 Hz) enabling their assignment to H-5a and H-5b. A
doublet and a multiplet at 3.40 (J ¼ 13.60 Hz) and 3.52e3.54 ppm
Scheme 1. Synthesis of 6(aej) and 7(aej).

A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
223
Fig. 1. Selected HMBC correlations and 1H and 13C chemical shifts of 7c.
can be assigned to H-7a and H-7b. From the HMBC correlations of
C-7, the multiplet around 2.25e2.33 ppm is assigned to CH2-10
while the other multiplet around 2.47e2.52 ppm is due to
CH2-20. The morpholine ring hydrogens appeared as multiplets at
2.25e2.33 ppm and 3.52e3.55 ppm. A hydrogen singlet at 6.68 ppm
is due to H-9 and two singlets at 6.87 and 7.79 ppm are due to
two NH while the aromatic hydrogens appear as multiplets in the
region 7.14e7.47 ppm. The assignment of carbon signals bearing
hydrogens has been done from the chemical shifts of hydrogens and
C,H-COSY correlations. These assignments are also supported by the
HMBC correlations (Fig. 1) of 7c. In addition, the structure and
stereochemistry of the pyridopyrimidine-2-thiones were confirmed
by the single crystal X-ray crystallographic analysis of 7b (Fig. 2)
[32]. The probable mechanism for the formation pyridopyrimidine-
2-thiones (6/7) in [BMIM]Br via Michael additionecondensatione
tautomerization domino sequence is shown in Scheme 2.
from electric eel and BChE enzyme from equine serum. As sum-
marized in Table 2, both series displayed remarkable AChE and
BChE inhibitory activities with IC50 values of lower than 10 mM.
Among the newly synthesized compounds, phenyl un-substituted
derivative 6a, displayed significant sub-micromolar AChE inhibi-
tory activity with IC50 value of 0.92
substituted derivative 7b, showed highest BChE inhibitory activity
with IC50 value of 1.89 M. Generally, all N-ethyl substituted in
mM while ortho-methyl
m
series 6 except for ortho-chloroderivative, displayed better AChE
inhibitory activity compared to their N-ethylmorpholino
substituted analogs in series 7.
In both series, para substituted derivatives displayed better AChE
inhibitory activities compared to their ortho substituted analogs. It is
worth to note that un-substituted derivatives 6a and 7a possessed
the highest AChE inhibitory potentials among their respective series
followed by para-chloro and para-fluoro derivatives. These results
clearly indicate that presence of electronegative atoms at para po-
sition of phenyl ring viz. chloro and fluoro have noticeable influence
on AChE inhibitory activities observed. However, di-chloro de-
rivatives, 6j and 7j displayed lower AChE enzyme inhibitory
compared to mono-chloro derivatives in both series. Besides, com-
pounds possessing electron donating moieties such as eCH3 and e
OCH3 displayed lower AChE inhibitory activities than the electron
withdrawing moieties. To compare the AChE inhibitory activities to
2.2. AChE and BChE inhibitory assay
All the newly synthesized compounds were evaluated for their
cholinesterase inhibitory activities against AChE enzyme derived
standard drug, un-substituted 6a (IC50 ¼ 0.92
substituted 6h (IC50 ¼ 1.29 M) and para-fluoro substituted deriva-
tive 6i (IC50 ¼ 2.07 M) displayed higher AChE inhibitory activities
than galantamine with IC50 value of 2.09 M.
mM), para-chloro
m
m
m
For BChE, compounds in series 7 displayed up to two fold higher
enzyme inhibitory activity compared to compounds in series 6,
except for 7f and 7j. In contrary to AChE, ortho-substituted de-
rivatives in series 6 and 7 displayed better BChE inhibitory activity
compared to their para-substituted analogs. Interestingly, all newly
synthesized compounds possessing either electron withdrawing or
electron donating substituents on phenyl ring displayed remark-
able BChE inhibitory activities with IC50 values of 1.89e8.13
mM that
is much higher than galantamine with IC50 value of 19.34
mM. It is
also worth to note that except for 6a, 6e, 6h and 6i, all the com-
pounds displayed 1.2e4.31 times more selectivity toward BChE
rather than AChE. As mentioned in the Introduction section, in-
hibitors with good balance between AChE and BChE inhibition such
as 6a, 6h and 6i or more selective toward BChE such as 7b, are
valuable therapeutic targets in AD therapy.
2.3. Molecular modeling
Molecular modeling simulation performed for the most potent
Fig. 2. ORTEP diagram of 7b.
AChE and BChE inhibitors, 6a and 7b, to disclose their binding

224
A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
Scheme 2. Plausible mechanism for the formation of novel pyridopyrimidine-2-thiones 6/7.
interactions and orientation template inside the active site gorge of
their respective enzymes, by using three dimensional crystal
structures of Torpedo california AChE and human BChE. As depicted
in Fig. 3, compound 6a, is completely accommodated inside the
active site gorge of TcAChE displaying mild polar interaction with
Ser200 and His440 at catalytic triad of this enzyme. At choline
binding site, it is stacked against Trp84 while simultaneously
showing hydrophobic interaction with Tyr334 residue. Compound
6a also showed mild polar interactions with Gly117 and Gly118 at
oxyanion hole, hydrophobic interactions with Phe288 and Phe290
at acyl binding pocket as well as hydrophobic interactions with
Trp279, Tyr121, Tyr334 and Phe331 residues comprising the
entrance of the gorge at peripheral anionic site. Based on this
binding interaction template, this inhibitor completely fills in the
gorge and prohibits substrate accommodation and hydrolysis in the
active site of the enzyme, which coincides with the significant sub
micromolar AChE inhibitory activity observed for this compound.
Moreover, the crystal structures of TcAChE enzyme in complex with
Table 2
Physical data, EeAChE and EqBChE activities of 6(aej) and 7(aej).
Entry
Compound
R0
Yielda (%)
Reaction time (min) MWI
AChE inhibition (IC50) (
m
M)
BChE inhibition (IC50) (
m
M)
Selectivity
AChEb
BChEc
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
2-CH3
2-Cl
71
63
65
62
73
63
70
68
64
61
69
67
63
68
74
58
72
71
69
62
e
2
0.92
6.17
8.22
5.54
4.92
7.88
5.02
1.29
2.07
8.75
4.49
8.15
6.75
9.32
8.39
9.11
7.16
5.49
6.72
8.67
2.09
4.20
3.93
3.57
4.48
8.13
5.11
4.73
5.88
5.98
3.61
3.23
1.89
2.44
2.97
5.93
5.16
3.58
4.28
3.41
5.87
19.34
4.56
0.64
0.43
0.81
1.65
0.65
0.94
4.56
2.76
0.41
0.72
0.23
0.36
0.32
0.71
0.57
0.50
0.78
0.51
0.68
9.25
0.22
1.57
2.30
1.24
0.61
1.54
1.06
0.22
0.36
2.42
1.39
4.31
2.77
3.14
1.42
1.77
2.00
1.28
1.97
1.47
0.11
2.5
2.5
2.5
<2
4
<2
<2
<2
5
3
3.5
4
4
2
5
3
3
5
5
e
2-F
2-OCH3
3-NO2
4-CH3
4-Cl
4-F
2,4-Cl2
H
2-CH3
2-Cl
2-F
2-OCH3
3-NO2
4-CH3
4-Cl
9
10
11
12
13
14
15
16
17
18
19
20
21
6j
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-F
2,4-Cl2
Galantamine
7j
e
a
Isolated yield after purification by column chromatography.
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE).
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE).
b
c

A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
225
moieties, amino acid residues involved in ligandereceptor complex
as well as their bonding types regarding compounds 6a and 7b are
summarized in Table 3.
3. Conclusion
Two series of novel piperidone grafted pyrimidine derivatives
as potential cholinesterase inhibitor agents have been synthesized
via an ionic liquid mediated multi-component reaction of N-
substituted 4-piperidones, aromatic aldehydes and thiourea under
microwave irradiation. The synthesized compounds were found to
be potent against both AChE and BChE enzymes with significant
IC50 values of less than 10
mM as compared to standard drug.
Further structural modification to improve their cholinesterase
enzyme inhibitory potency is being carried out in our laboratory.
4. Experimental
4.1. Chemistry
Fig. 3. Binding interactions of 6a with major active site residues of TcAChE receptor.
The chemicals used were obtained from Merck (Germany) and
Sigma Aldrich (USA). Melting points were determined by open tube
capillary method and are uncorrected. Purity of the compounds
was checked on thin layer chromatography (TLC) plates (silica gel
G) using the petroleum ether/ethyl acetate solvent system and the
spots were examined under UV light. IR spectra were obtained on a
PerkineElmer 1720 FT-IR spectrometer (KBr Pellets). 1H and 13C
NMR were performed on Bruker Avance 500 (1H: 500 MHz, 13C:
125 MHz) spectrometer in CDCl3, using TMS as internal standard.
Mass spectra were recorded on Varian 320-MS TQ LC/MS using ESI.
available Alzheimer’s drugs such as huperzine A, galantamine and
donepezil also show similar interactions of occupying the periph-
eral anionic site, stacking against Trp84 and interacting with resi-
dues at catalytic triad [33]
Molecular docking analysis of 7b into the active site of hBChE
also displayed the complete insertion and efficient accommodation
of this inhibitor into the active site gorge of the enzyme by dis-
playing p,p-stacking interaction with choline binding site residue,
Trp82 and mild polar interaction with catalytic triad residues,
His438 and Ser198, at the bottom of the gorge. This molecule also
showed mild polar interactions with Gly116 and Gly117 at oxyanion
hole in addition to hydrophobic interactions with Trp231, Leu286
comprising peripheral anionic site and Phe398 and Ala199
composing acyl binding pocket of the hBChE active site (Fig. 4). This
binding interaction template also, resembles the reported template
for hBChE receptor in complex with its substrate, butyrylcholine,
which obviously coincides with potent in vitro BChE inhibitory
activity observed for this compound [34]. The data representing
free binding energies, receptors interacting sites, ligand interacting
4.1.1. General procedure for synthesis of (6aej) and (7aej)
1 M equiv of [BMIM]Br was added to 1:2:1 ratio of 1-
ethylpiperidine-4-one (2)/1-(morpholinoethyl)piperidine-4-one
(3), aromatic aldehydes (4) and thiourea (5). The mixture mixed
well and irradiated for 2e5 min at maximum power level (300 W)
in a CEM microwave synthesizer during which period till comple-
tion of reaction as evident by TLC. Subsequently, mixture purified
using flash column chromatography to afford compounds 6aej and
7aej in good yields (Table 2).
4.1.1.1. (E)-8-Benzylidene-6-ethyl-4-phenyloctahydropyrido[4,3-d]
pyrimidine-2(1H)-thione (6a). Yellow solid; mp 164e167 ꢀC; IR
(KBr) nmax: 2973, 1542, 1487 cmꢁ1. Anal. calcd. for C22H25N3S: C,
72.69; H, 6.93; N, 11.56. Found: C, 72.12; H, 7.21; N, 10.94. 1H NMR
(500 MHz, CDCl3): dH 0.92e0.96 (m, 3H, CH3), 2.38e2.42 (m, 2H,
CH2), 2.67 (d, 1H, J ¼ 16.07 Hz, H-5a), 3.07 (d, 1H, J ¼ 16.07 Hz, H-
5b), 3.30 (d, 1H, J ¼ 13.55 Hz, H-7a), 3.63 (d, 1H, J ¼ 13.55 Hz, H-7b),
5.01 (s, 1H, H-4), 6.64 (s, 1H, H-9), 7.20e7.36 (m, 12H, H-aromatic,
NH). 13C NMR (125 MHz, CDCl3): dC 11.84, 50.73, 51.64, 51.72, 59.12,
110.79, 123.11, 125.66, 127.20, 127.53, 128.40, 128.85, 129.14, 129.52,
130.37, 135.64, 140.83, 173.52.
4.1.1.2. (E)-6-Ethyl-8-(2-methylbenzylidene)-4-(o-tolyl)octahy-
dropyrido[4,3-d]pyrimidine-2(1H)-thione (6b). Yellow solid; mp
160e162 ꢀC; IR (KBr) nmax: 2974, 1546, 1489 cmꢁ1. Anal. calcd. for
C24H29N3S: C, 73.62; H, 7.46; N, 10.73. Found: C, 72.92; H, 7.05; N,
10.19. 1H NMR (500 MHz, CDCl3): dH 0.99e1.04 (m, 3H, CH3), 2.26e
2.40 (m, 5H, CH2 and CH3), 2.76 (d, 1H, J ¼ 16.15 Hz, H-5a), 3.04 (d,
1H, J ¼ 16.15 Hz, H-5b), 3.26 (d, 1H, J ¼ 13.47 Hz, H-7a), 3.65 (d, 1H,
J ¼ 13.47 Hz, H-7b), 4.97 (s, 1H, H-4), 6.61 (s, 1H, H-9), 7.08e7.21 (m,
10H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.18, 21.18,
21.21, 21.27, 48.77, 50.99, 51.27, 51.46, 59.20, 106.20, 122.38, 125.84,
127.17, 127.23, 129.14, 129.21, 129.68, 129.80, 132.82, 137.45, 138.20,
138.56, 138.76, 173.92.
Fig. 4. Binding interactions of 7b with major active site residues of hBChE receptor.

226
A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
Table 3
Binding Interaction data for 6a and 7b docked into active site gorge of TcAChE and hBChE receptors.
Entry
1
Ligand
Enzyme
Binding energy (kcal)
Interacting site
PASa
Amino acid residue
Bond type
Ligand interacting moiety
Ring 1
6a
TcAChE
ꢁ8.0
Trp279
Tyr334
Asp72
Hydrophobic
Hydrophobic
Mild polar
Hydrophobic
Mild polar
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Mild polar
Ring 2
N-ethyl
Ring 4
Ring 3
Tyr121
OHb
Gly117 and 118
Phe288
Phe290
Phe330
Trp84
Acyl binding pocket
Choline binding site
CTc
Ring 2
Ring 4
Ring 4
Ser200
His 440
Mild polar
2
7b
hBChE
ꢁ7.7
Side chain
PAS
Pro285
Ser287
Thr120
Tyr332
Leu286
Val288
Gly116 and 117
Phe329
Trp82
Hydrophobic
Mild polar
Mild polar
Hydrophobic
Hydrophobic
Hydrophobic
Mild polar
Ring 1
Ring 4
Ring 1
Morpholine moiety
OH
Ring 4
Morpholine moiety
Ring 4
Choline binding site
Hydrophobic
pep stacking
CT
His438
Ser198
Mild polar
Mild polar
Morpholine moiety
Morpholine moiety
a
Peripheral anionic site.
Oxyanion hole.
Catalytic triad.
b
c
4.1.1.3. (E)-8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-
6-ethyloctahydropyrido[4,3-d]pyrimidine-2(1H)-thione (6c).
Yellow solid; mp 137e139 ꢀC; IR (KBr) nmax: 2971, 1547, 1488 cmꢁ1
121.64, 126.27, 126.95, 128.37, 130.61, 133.79, 134.35, 159.01,
159.98, 173.88.
.
Anal. calcd. for C22H23Cl2N3S: C, 61.11; H, 5.36, N, 9.72. Found: C,
62.12; H, 5.07, N, 8.94. 1H NMR (500 MHz, CDCl3): dH 0.87e0.95 (m,
3H, CH3), 2.37e2.44 (m, 2H, CH2), 2.80 (d, 1H, J ¼ 16.27 Hz, H-5a),
3.11 (d, 1H, J ¼ 16.27 Hz, H-5b), 3.29 (d, 1H, J ¼ 13.39 Hz, H-7a), 3.44
(d, 1H, J ¼ 13.39 Hz, H-7b), 5.59 (s, 1H, H-4), 6.67 (s, 1H, H-9), 7.14e
7.48 (m, 10H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.09,
31.11, 50.90, 51.27, 51.71, 52.53 55.20, 110.79, 119.18, 126.47, 127.15,
127.32, 128.24, 128.67, 128.82, 129.25, 129.58, 129.76, 130.18, 131.74,
133.77, 137.26,174.60.
4.1.1.6. (E)-6-Ethyl-8-(3-nitrobenzylidene)-4-(3-nitrophenyl)octahy-
dropyrido[4,3-d]pyrimidine-2(1H)-thione (6f). Yellow solid; mp
158e161 ꢀC; IR (KBr) nmax: 2969, 1542, 1457, 779 cmꢁ1. Anal. calcd.
for C22H23N5O4S: C, 58.26; H, 5.11; N,15.44. Found: C, 57.45; H, 4.67;
N, 15.09. 1H NMR (500 MHz, CDCl3): dH 0.93 (t, 3H, J ¼ 7.25 Hz, CH3),
2.45e2.51 (m, 2H, CH2), 2.60 (d, 1H, J ¼ 15.92 Hz, H-5a), 3.04 (d, 1H,
J ¼ 15.92 Hz, H-5b), 3.17 (d, 1H, J ¼ 13.55 Hz, H-7a), 3.49 (d, 1H,
J ¼ 13.39 Hz, H-7b), 5.39 (s, 1H, H-4), 5.85 (s, 1H, H-9), 7.16e8.21 (m,
10H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.07, 50.97,
52.34, 52.53, 54.45, 109.45, 119.91, 121.64, 126.49, 126.89, 128.40,
130.81, 134.93, 158.71, 159.12, 174.42.
4.1.1.4. (E)-6-Ethyl-8-(2-fluorobenzylidene)-4-(2-fluorophenyl)octa-
hydropyrido[4,3-d]pyrimidine-2(1H)-thione (6d). Yellow solid; mp
131e134 ꢀC; IR (KBr) nmax: 3696, 2981, 1506, 1055 cmꢁ1. Anal. calcd.
for C22H23F2N3S: C, 66.14; H, 5.80; N, 10.52. Found: C, 67.12; H, 5.21;
N, 10.07. 1H NMR (500 MHz, CDCl3): dH 0.78e0.98 (m, 3H, CH3),
2.38e2.45 (m, 2H, CH2), 2.78 (d, 1H, J ¼ 16.23 Hz, H-5a), 3.11 (d, 1H,
J ¼ 16.23 Hz, H-5b), 3.26 (d, 1H, J ¼ 13.55 Hz, H-7a), 3.47 (d, 1H,
J ¼ 13.55 Hz, H-7b), 5.60 (s, 1H, H-4), 6.62 (s, 1H, H-9), 6.91e7.47 (m,
10H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.16, 50.97,
51.87, 51.90, 52.30, 108.65, 115.04, 115.06, 115.54, 115.55, 115.71,
115.73,123.75, 123.78, 125.07, 125.09, 127.84, 128.64,128.75, 128.85,
128.88, 129.24, 129.30,129.49,129.95, 130.01,130.76, 130.78, 153.74,
159.16, 174.21.
4.1.1.7. (E)-6-ethyl-8-(4-methylbenzylidene)-4-(p-tolyl)octahy-
dropyrido[4,3-d]pyrimidine-2(1H)-thione (6g). Yellow solid; mp
164e166 ꢀC; IR (KBr) nmax: 2967, 1552, 1467 cmꢁ1. Anal. calcd. for
C24H29N3S: C, 73.62; H, 7.46; N, 10.73. Found: C, 72.77; H, 7.12; N,
10.27. 1H NMR (500 MHz, CDCl3): dH 0.92 (t, 3H, J ¼ 7.25 Hz, CH3),
2.32 (s, 3H, CH3), 2.33 (s, 1H, CH3), 2.36e2.39 (m, 2H, CH2), 2.61 (d,
1H, J ¼ 15.76 Hz, H-5a), 2.99 (d, 1H, J ¼ 15.76 Hz, H-5b), 3.25 (d, 1H,
J ¼ 13.55 Hz, H-7a), 3.63 (d,1H, J ¼ 13.55 Hz, H-7b), 4.93 (s,1H, H-4),
5.76 (s, 1H, H-9), 6.63 (s, 1H, NH), 6.84e7.34 (m, 9H, H-aromatic,
NH). 13C NMR (125 MHz, CDCl3): dC 12.24, 21.16, 21.22, 51.03, 52.11,
52.15, 59.33, 109.05, 122.10, 126.68, 126.73, 127.01, 127.07, 129.01,
129.17, 129.23, 129.65, 133.37, 137.01, 138.16, 139.22, 174.15.
4.1.1.5. (E)-6-Ethyl-8-(2-methoxybenzylidene)-4-(2-methoxyphenyl)
octahydropyrido[4,3-d]pyrimidine-2(1H)-thione (6e). Yellow solid;
mp 138e140 ꢀC; IR (KBr) nmax: 3691, 2973, 1509, 1042 cmꢁ1. Anal.
calcd. for C24H27N3O2S: C, 68.38; H, 6.46; N, 9.97. Found: C, 67.89;
H, 6.15; N, 9.52. 1H NMR (500 MHz, CDCl3): dH 0.93 (t, 3H,
J ¼ 7.25 Hz, CH3), 2.35e2.43 (m, 2H, CH2), 2.61 (d, 1H, J ¼ 15.92 Hz,
H-5a), 2.97 (d, 1H, J ¼ 15.92 Hz, H-5b), 3.27 (d, 1H, J ¼ 13.39 Hz, H-
7a), 3.62 (d, 1H, J ¼ 13.39 Hz, H-7b), 3.79 (s, 3H, OeCH3), 3.80 (s,
3H, OeCH3), 4.93 (s, 1H, H-4), 5.60 (s, 1H, H-9), 6.58 (s, 1H, NH),
6.86e7.25 (m, 9H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC
12.25, 51.09, 52.07, 52.18, 55.26, 55.31, 59.05, 108.80, 114.33,
4.1.1.8. (E)-8-(4-chlorobenzylidene)-4-(4-chlorophenyl)-
6-ethyloctahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(6h).
Yellow solid; mp 162e165 ꢀC; IR (KBr) nmax: 2971, 1547, 1488,
1090 cmꢁ1. Anal. calcd. for C22H23Cl2N3S: C, 61.11; H, 5.36, N,
9.72. Found: C, 62.22; H, 5.16, N, 8.71. 1H NMR (500 MHz,
CDCl3): dH 0.93 (t, 3H, J ¼ 7.09 Hz, CH3), 2.32e2.41 (m, 2H, CH2),
2.57 (d, 1H, J ¼ 16.07 Hz, H-5a), 2.93 (d, 1H, J ¼ 16.07 Hz, H-5b),
3.23 (d, 1H, J ¼ 13.39 Hz, H-7a), 3.50 (d, 1H, J ¼ 13.39 Hz, H-7b),
4.89 (s, 1H, H-4), 6.11 (s, 1H, H-9), 6.67 (s, 1H, NH), 7.09e7.37
(m, 9H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.17,

A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
227
51.10, 51.97, 58.79, 109.15, 127.32, 128.05, 128.33, 128.48,
129.22, 130.54, 133.07, 134.28, 134.65, 140.54, 174.79.
J ¼ 16.75 Hz, H-5a), 3.18 (d, J ¼ 16.75 Hz, H-5b), 3.40 (d, J ¼ 13.60 Hz,
H-7a), 3.52e3.55 (m, 5H, H-7b, CH2-50), 5.58 (s, 1H, H-4), 6.68 (s, 1H,
H-9), 6.87 (s, 1H, NH), 7.11-7.21 (m, 8H, H-aromatic), 7.79 (s, 1H, NH).
13C NMR (125 MHz, CDCl3): dC 52.49, 52.62, 53.24, 53.90, 55.13, 56.82,
66.76, 110.50, 120.07, 126.51, 127.58, 128.25, 129.23, 129.58, 129.79,
130.04, 130.23, 130.69, 132.71, 133.78, 134.18, 137.30, 174.78.
4.1.1.9. (E)-6-Ethyl-8-(4-fluorobenzylidene)-4-(4-fluorophenyl)octa-
hydropyrido[4,3-d]pyrimidine-2(1H)-thione (6i). Yellow solid; mp
150e152 ꢀC; IR (KBr) nmax: 3696, 2973, 1505, 1055 cmꢁ1 Anal. calcd.
for C22H23F2N3S: C, 66.14; H, 5.80; N, 10.52. Found: C, 67.29; H, 5.34;
N, 10.15. 1H NMR (500 MHz, CDCl3): dH 0.81e0.99 (m, 3H, CH3),
2.35e2.44 (m, 2H, CH2), 2.75 (d, 1H, J ¼ 16.19 Hz, H-5a), 3.09 (d, 1H,
J ¼ 16.19 Hz, H-5b), 3.31 (d, 1H, J ¼ 13.47 Hz, H-7a), 3.52 (d, 1H,
J ¼ 13.47 Hz, H-7b), 5.52 (s,1H, H-4), 6.53 (s,1H, H-9), 6.87e7.39 (m,
10H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.12, 50.87,
51.91, 51.93, 52.13, 109.05, 115.02, 115.07, 115.49, 115.51, 115.68,
115.71, 123.89, 123.97, 125.17, 125.21, 127.77, 128.69, 128.78, 128.94,
129.12, 129.38, 129.45, 129.69, 130.07, 130.15, 130.88, 130.94, 153.72,
159.24, 175.16.
4.1.1.14. (E)-8-(2-Fluorobenzylidene)-4-(2-fluorophenyl)-6-(2-
morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7d). Yellow solid; mp 112e115 ꢀC; IR (KBr) nmax: 3695, 2981, 1517,
1055 cmꢁ1. Anal. calcd. for C26H30F2N4OS: C, 64.44; H, 6.24; N,11.56.
Found: C, 64.12; H, 5.91; N,11.19.1H NMR (500 MHz, CDCl3): dH 2.15e
2.37 (m, 6H, CH2-10, CH2-40), 2.36e2.43 (m, 2H, CH2-20), 2.67 (d,
J ¼ 16.07 Hz, H-5a), 3.01 (d, J ¼ 16.07 Hz, H-5b), 3.34 (d, J ¼ 13.55 Hz,
H-7a), 3.43e3.51 (m, 4H, CH2-50), 3.63 (d, 1H, J ¼ 13.55 Hz, H-7b),
5.49 (s, 1H, H-4), 6.60 (s, 1H, H-9), 6.75 (s, 1H, NH), 7.12 (s, 1H, NH),
7.15-7.32 (m, 8H, H-aromatic). 13C NMR (125 MHz, CDCl3): dC 51.68,
51.78, 52.41, 52.86, 55.88, 58.56, 65.74,107.86,121.44,125.89,126.13,
126.27, 127.30, 127.50, 128.03, 128.25, 135.09, 141.03, 173.99.
4.1.1.10. (E)-8-(2,4-Dichlorobenzylidene)-4-(2,4-dichlorophenyl)-
6-ethyloctahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(6j).
Yellow solid; mp 142e145 ꢀC; IR (KBr) nmax: 2977, 1548,
1452 cmꢁ1. Anal. calcd. for C22H21Cl4N3S: C, 52.71; H, 4.22; N,
8.38. Found: C, 52.34; H, 3.99; N, 8.12. 1H NMR (500 MHz,
CDCl3): dH 0.95 (t, 3H, J ¼ 7.25 Hz, CH3), 2.38e2.44 (m, 2H, CH2),
2.78 (d, 1H, J ¼ 16.07 Hz, H-5a), 3.11 (d, 1H, J ¼ 16.07 Hz, H-5b),
3.30 (d, 1H, J ¼ 13.71 Hz, H-7a), 3.44 (d, 1H, J ¼ 13.71 Hz, H-7b),
4.78 (s, 1H, H-4), 5.55 (s, 1H, H-9), 6.65 (s, 1H, NH), 7.17e7.50 (m,
7H, H-aromatic, NH). 13C NMR (125 MHz, CDCl3): dC 12.14, 51.00,
51.93, 52.14, 55.14, 109.05, 126.33, 128.04, 128.11, 128.84, 129.32,
129.62, 129.88, 130.65, 132.72, 134.26, 138.53, 174.61.
4.1.1.15. (E)-8-(2-Methoxybenzylidene)-4-(2-methoxyphenyl)-6-(2-
morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7e). Yellow solid; mp 108e110 ꢀC; IR (KBr) nmax: 3699, 2923, 1544,
1035 cmꢁ1. Anal. calcd. for C28H36N4O3S: C, 66.11; H, 7.13; N, 11.01.
Found: C, 65.43; H, 6.82; N, 10.75. 1H NMR (500 MHz, CDCl3): dH
2.23e2.36(m, 6H, CH2-10, CH2-40), 2.44e2.53(m, 2H, CH2-20), 2.75 (d,
J ¼ 16.25 Hz, H-5a), 3.07 (d, J ¼ 16.25 Hz, H-5b), 3.43 (d, J ¼ 13.47 Hz,
H-7a), 3.53e3.61 (m, 4H, CH2-50), 3.72 (d, J ¼ 13.47 Hz,1H, H-7b), 3.81
(s, 3H, OeCH3), 3.83 (s, 3H, OeCH3), 4.96(s,1H, H-4), 6.56 (s,1H, H-9),
6.89 (s, 1H, NH), 6.91 (s, 1H, NH), 7.11-7.28 (m, 8H, H-aromatic). 13
C
4.1.1.11. ( E ) - 8 - B e n z y l i d e n e - 6 - ( 2 - m o r p h o l i n o e t h y l ) -
NMR (125 MHz, CDCl3): dC 52.61, 52.68, 53.91, 55.34, 55.39, 56.87,
4-phenyloctahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7a).
59.00, 66.79, 113.95, 114.55, 128.58, 130.66, 132.79, 160.07.
Yellow solid; mp 110e112 ꢀC; IR (KBr) nmax: 3185, 2967, 1543,
1454 cmꢁ1. Anal. calcd. for C26H32N4OS: C, 69.61; H, 7.19; N,
12.49. Found: C, 69.17; H, 6.78; N, 12.18. 1H NMR (500 MHz,
CDCl3): dH 2.21e2.31 (m, 6H, CH2-10, CH2-40), 2.44e2.49 (m, 2H,
CH2-20), 2.91 (d, J ¼ 16.55 Hz, H-5a), 3.21 (d, J ¼ 16.55 Hz, H-5b),
3.38 (d, J ¼ 13.72 Hz, H-7a), 3.49e3.54 (m, 5H, H-7b, CH2-50), 5.41
(s, 1H, H-4), 6.65 (s, 1H, H-9), 6.85 (s, 1H, NH), 7.81 (s, 1H, NH),
7.24e7.39 (m, 10H, H-aromatic). 13C NMR (125 MHz, CDCl3): dC
52.37, 52.54, 53.17, 53.82, 55.09, 56.78, 66.71, 109.98, 119.17,
125.44, 126.91, 127.45, 128.32, 128.92, 129.34, 130.12, 130.34,
130.72, 132.84, 134.12, 134.29, 137.48, 174.69.
4.1.1.16. (E)-6-(2-Morpholinoethyl)-8-(3-nitrobenzylidene)-4-
(3-nitrophenyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7f).
Yellow solid; mp 182e184 ꢀC; IR (KBr) nmax: 2967, 1542, 1454,
1035 cmꢁ1. Anal. calcd. for C26H30N6O5S: C, 57.98; H, 5.61; N, 15.60.
Found: C, 57.25; H, 5.39; N, 15.12. 1H NMR (500 MHz, CDCl3): dH
2.21e2.31 (m, 6H, CH2-10, CH2-40), 2.53e2.61 (m, 2H, CH2-20), 2.97 (d,
J ¼ 16.49 Hz, H-5a), 3.21 (d, J ¼ 16.49 Hz, H-5b), 3.38 (d, J ¼ 13.72 Hz,
H-7a), 3.44e3.59 (m, 5H, H-7b, CH2-50), 5.61 (s, 1H, H-4), 6.59 (s, 1H,
H-9), 6.91 (s, 1H, NH), 7.19 (s, 1H, NH), 7.25-7.39 (m, 8H, H-aromatic).
13C NMR (125 MHz, CDCl3): dC 52.11, 52.29, 52.91, 53.28, 54.49, 56.36,
66.72, 111.09, 121.18, 127.03, 127.43, 128.11, 129.09, 129.44, 129.79,
130.12, 130.27, 130.71, 133.12, 133.69, 134.07, 140.29, 176.12.
4.1.1.12. (E)-8-(2-Methylbenzylidene)-6-(2-morpholinoethyl)-4-
(o-tolyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7b).
Yellow solid; mp 120e122 ꢀC; IR (KBr) nmax: 3178, 2958, 1551,
1033 cmꢁ1. Anal. calcd. for C28H36N4OS: C, 70.55; H, 7.61; N,
11.75. Found: C, 70.12; H, 7.21; N, 11.09. 1H NMR (500 MHz,
CDCl3): dH 2.24e2.34 (m, 6H, CH2-10, CH2-40), 2.35 (s, 3H, CH3),
2.37 (s, 3H, CH3), 2.44e2.52 (m, 2H, CH2-20), 2.78 (d,
J ¼ 16.21 Hz, H-5a), 3.09 (d, J ¼ 16.21 Hz, H-5b), 3.42 (d,
J ¼ 13.54 Hz, H-7a), 3.55e3.63 (m, 4H, CH2-50), 3.73 (d, 1H,
J ¼ 13.54, H-7b), 4.94 (s, 1H, H-4), 6.74 (s, 1H, H-9), 7.11 (s, 1H,
NH), 7.16-7.24 (m, 8H, H-aromatic), 7.79 (s, 1H, NH). 13C NMR
(125 MHz, CDCl3): dC 21.02, 21.10, 52.42, 52.93, 53.67, 56.37,
58.74, 66.54, 110.20, 123.16, 125.03, 125.50, 127.06, 129.10,
129.72, 132.71, 137.53, 137.95, 138.69, 173.45.
4.1.1.17. (E)-8-(4-Methylbenzylidene)-6-(2-morpholinoethyl)-
4-(p-tolyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7g).
Yellow solid; mp 158e160 ꢀC; IR (KBr) nmax: 3185, 2965, 1551,
1464 cmꢁ1. Anal. calcd. for C28H36N4OS: C, 70.55; H, 7.61; N, 11.75.
Found: C, 70.21; H, 7.29; N, 11.17. 1H NMR (500 MHz, CDCl3): dH
2.21e2.33 (m, 6H, CH2-10, CH2-40), 2.34 (s, 3H, CH3), 2.38 (s, 3H,
CH3), 2.41e2.49 (m, 2H, CH2-20), 2.69 (d, J ¼ 16.45 Hz, H-5a), 3.02
(d, J ¼ 16.45 Hz, H-5b), 3.37 (d, J ¼ 13.35 Hz, H-7a), 3.49e3.58 (m,
4H, CH2-50), 3.69 (d, 1H, J ¼ 13.35, H-7b), 4.87 (s, 1H, H-4), 6.62 (s,
1H, H-9), 7.07 (s,1H, NH), 7.12e7.21 (m, 8H, H-aromatic), 7.54 (s,1H,
NH). 13C NMR (125 MHz, CDCl3): dC 21.02, 21.10, 52.42, 52.93, 53.67,
56.37, 58.74, 66.54, 110.20, 123.16, 125.03, 125.50, 127.06, 129.10,
129.72, 132.71, 137.53, 137.95, 138.69, 173.45.
4.1.1.13. (E)-8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-6-(2-
morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7c). Yellow solid; mp 130e132 ꢀC; IR (KBr) nmax: 2974, 1548, 1478,
1092cmꢁ1. Anal. calcd. for C26H30Cl2N4OS: C, 60.34; H,13.70;N,10.83.
Found: C, 60.11; H, 13.24; N, 10.52. 1H NMR (500 MHz, CDCl3): dH
2.25e2.33 (m, 6H, CH2-10, CH2-40), 2.47e2.52 (m, 2H, CH2-20), 2.90 (d,
4.1.1.18. (E)-8-(4-Chlorobenzylidene)-4-(4-chlorophenyl)-6-(2-
morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7h). Yellow solid; mp 128e130 ꢀC; IR (KBr) nmax: 3175, 2962, 1544,
1485 cmꢁ1. Anal. calcd. for C26H30Cl2N4OS: C, 60.34; H,13.70;N,10.83.
Found: C, 60.21; H, 13.12; N, 10.49. 1H NMR (500 MHz, CDCl3): dH

228
A. Basiri et al. / European Journal of Medicinal Chemistry 67 (2013) 221e229
2.19e2.27 (m, 6H, CH2-10, CH2-40), 2.44e2.49 (m, 2H, CH2-20), 2.85 (d,
J ¼ 16.73 Hz, H-5a), 3.05 (d, J ¼ 16.73 Hz, H-5b), 3.44 (d, J ¼ 13.19 Hz,
H-7a), 3.58e3.69 (m, 5H, H-7b, CH2-50), 5.67 (s, 1H, H-4), 6.71 (s, 1H,
H-9), 6.97 (s, 1H, NH), 7.11-7.21 (m, 8H, H-aromatic), 7.34 (s, 1H, NH).
13C NMR (125 MHz, CDCl3): dC 52.44, 52.51, 53.23, 53.79, 55.07, 56.75,
67.01, 110.39, 121.19, 126.44, 127.67, 128.21, 129.24, 129.78, 129.45,
131.14, 131.23, 131.67, 132.11, 132.78, 133.28, 137.49, 175.17.
was 1%. At this concentration, DMSO has no inhibitory effect on both
acetylcholinesterase and butyrylcholinesterase enzymes.
The initial screening was carried out at 10 mg/mL of test samples
in 1% DMSO and each test was conducted in triplicates. Absor-
bencies of the test samples were corrected by subtracting the
absorbance of their respective blank. Percentage enzyme inhibition
is calculated using the following formula:
Absorbance of control ꢁ Absorbance of sample
Percentage of inhibition ¼
ꢂ 100
Absorbance of control
4.1.1.19. (E)-8-(4-Fluorobenzylidene)-4-(4-fluorophenyl)-6-(2-
morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7i). Yellow solid; mp 148e150 ꢀC; IR (KBr) nmax: 3681, 2923, 1507,
1037 cmꢁ1. Anal. calcd. for C26H30F2N4OS: C, 64.44; H, 6.24; N,11.56.
Found: C, 64.32; H, 5.78; N, 11.25. 1H NMR (500 MHz, CDCl3): dH
2.12e2.35 (m, 6H, CH2-10, CH2-40), 2.29e2.37 (m, 2H, CH2-20), 2.64
(d, J ¼ 16.27 Hz, H-5a), 3.09 (d, J ¼ 16.27 Hz, H-5b), 3.31 (d,
J ¼ 13.49 Hz, H-7a), 3.41e3.49 (m, 4H, CH2-50), 3.58 (d, 1H,
J ¼ 13.49 Hz, H-7b), 5.77 (s, 1H, H-4), 6.65 (s, 1H, H-9), 6.89 (s, 1H,
NH), 7.19 (s, 1H, NH), 7.23-7.41 (m, 8H, H-aromatic). 13C NMR
(125 MHz, CDCl3): dC 51.63, 51.72, 52.321, 53.11, 55.65, 58.44, 65.77,
108.19, 121.49, 125.73, 126.29, 126.48, 127.15, 127.53, 128.07, 128.35,
135.12, 144.52, 174.16.
Subsequently, the determination of IC50 was carried out using a
set of five concentrations.
4.3. Molecular modeling
In order to investigate possible interactions between the syn-
thesized compounds and the active site of acetylcholinesterase and
butyrylcholinesterase, molecular docking study was performed.
Using Glide, (version 5.7, Schrödinger, LLC, New York, NY, 2011),
compounds 6a and 7b were docked onto the active site of TcAChE
derived from three-dimensional structure of the enzyme complex
with anti-Alzheimer’s drug, donepezil (PDB ID: 1EVE) and 7b to
BChE derived from complex of the enzyme with its substrate (PDB
code: 1P0P).
4.1.1.20. (E)-8-(2,4-Dichlorobenzylidene)-4-(2,4-dichlorophenyl)-6-
(2-morpholinoethyl)octahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7j). Yellow solid; mp 118e120 ꢀC; IR (KBr) nmax: 2923, 1517,
1055 cmꢁ1. Anal. calcd. for C26H28Cl4N4OS: C, 53.25; H, 4.81; N, 9.55.
Found: C, 52.55; H, 4.27; N, 9.02. 1H NMR (500 MHz, CDCl3): dH
2.21e2.32 (m, 6H, CH2-10, CH2-40), 2.53e2.58 (m, 2H, CH2-20), 2.97
(d, J ¼ 16.79 Hz, H-5a), 3.15 (d, J ¼ 16.79 Hz, H-5b), 3.58 (d,
J ¼ 13.10 Hz, H-7a), 3.71e3.82 (m, 5H, H-7b, CH2-50), 5.73 (s, 1H, H-
4), 6.88 (s, 1H, H-9), 7.11 (s, 1H, NH), 7.22-7.47 (m, 6H, H-aromatic),
7.83 (s, 1H, NH). 13C NMR (125 MHz, CDCl3): dC 52.58, 52.69, 53.46,
54.19, 55.27, 57.11, 66.89, 110.25, 122.17, 127.49, 127.87, 128.19,
129.43, 129.85, 130.32, 132.24, 132.45, 132.69, 133.19, 133.88, 134.07,
137.55, 174.48.
Water molecules and hetero groups were deleted from receptor
beyond the radius of 5 A of reference ligand (donepezil or BCh),
ꢀ
resulting protein structure refined and minimized by Protein
Preparation Wizard using OPLS-2005 force field. Receptor 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 ligands. Docking
stimulations were carried out on bioactive compounds resulting in
5 poses per ligand, in which the best pose with highest score was
displayed for each ligand.
Acknowledgments
We would like to thank the Malaysian Government and Uni-
versiti Sains Malaysia (USM) for the Research University Grant
(1001/PFARMASI/813031). AB is supported by the USM Graduate
Assistant Scheme from the Institute for Postgraduate Studies (IPS)
of Universiti Sains Malaysia.
4.2. AChE and BChE inhibitory assays
Cholinesterase inhibitory activity of the synthesized compounds
was evaluated using the Ellman’s microplate assay following method
described by Ahmed and Gilani [35]. For acetylcholinesterase (AChE)
inhibitory assay, 140
first added to a 96-well microplate followed by 20
and 20 L of 0.09 units/mL acetylcholinesterase enzyme from Elec-
trophorus electricus (Sigma). After 15 min of incubation at 25 ꢀC,10
of 10 mM 5,50-dithiobis-2-nitrobenzoic acid (DTNB) was added into
each well followed by 10 L of acetylthiocholine iodide (14 mM). At
m
L of 0.1 M sodium phosphate buffer (pH 8) was
Appendix A. Supplementary data
mL of test samples
m
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.
06.054.
mL
m
30 min after the initiation of enzymatic reaction, absorbance of the
colored end-product was measured using BioTek PowerWave X 340
Microplate Spectrophotometer at 412 nm. For butyrylcholinesterase
(BChE) inhibitory assay, the same procedures were followed except
for the use of enzyme and substrate, whereby butyrylcholinesterase
from equine serum and S-butyrylthiocholine chloride (14 mM) were
used, respectively.
Galantamine was used as positive control. Test samples and gal-
antamine were prepared in DMSO at an initial concentration of 1 mg/
mL (1000 ppm). The concentration of DMSO in final reaction mixture
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