Cholinesterase inhibitory activity versus aromatic core multiplicity: A facile green synthesis and molecular docking study of novel piperidone embedded thiazolopyrimidines

Article abstract of DOI:10.1016/j.bmc.2013.11.020

Novel thiazolopyrimidine derivatives have been synthesized via microwave assisted, domino cascade methodology in ionic liquid and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Among the newly synthesized compounds 6d, 6a, 6e and 6b displayed higher AChE inhibitory activity than standard drug, galanthamine, with IC ₅₀ values of 0.53, 1.47, 1.62 and 2.05 μM, respectively. Interestingly, all the compounds except for 6m-r and 6x displayed higher BChE inhibitory potentials than galanthamine with IC₅₀ values ranging from 1.09 to 18.56 μM. Molecular docking simulations for 6d possessing the most potent AChE and BChE inhibitory activities, disclosed its binding interactions at the active site gorge of AChE and BChE enzymes.

Full text of DOI:10.1016/j.bmc.2013.11.020

Bioorganic & Medicinal Chemistry 22 (2014) 906–916
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cholinesterase inhibitory activity versus aromatic core multiplicity:
A facile green synthesis and molecular docking study of novel
piperidone embedded thiazolopyrimidines
b
c,
b
Alireza Basiri a, Vikneswaran Murugaiyah a, , Hasnah Osman , Raju Suresh Kumar , Yalda Kia ,
⇑
⇑
Alysha Hooda d, Richard B. Parsons d
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 Institute of Pharmaceutical Science, King’s College London, London SE1 9NH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel thiazolopyrimidine derivatives have been synthesized via microwave assisted, domino cascade
methodology in ionic liquid and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcho-
linesterase (BChE) inhibitory activities. Among the newly synthesized compounds 6d, 6a, 6e and 6b
displayed higher AChE inhibitory activity than standard drug, galanthamine, with IC50 values of 0.53,
Received 10 September 2013
Revised 8 November 2013
Accepted 9 November 2013
Available online 19 November 2013
1.47, 1.62 and 2.05
l
M, respectively. Interestingly, all the compounds except for 6m–r and 6x displayed
M.
higher BChE inhibitory potentials than galanthamine with IC50 values ranging from 1.09 to 18.56
l
Keywords:
Domino cascade synthesis
Ionic liquids
Thiazolopyrimidines
AChE and BChE inhibitory activity
Molecular modeling
Aromatic core multiplicity
Molecular docking simulations for 6d possessing the most potent AChE and BChE inhibitory activities,
disclosed its binding interactions at the active site gorge of AChE and BChE enzymes.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
affected more than 37 million people worldwide.17 The etiology
of AD is not completely known, however the deposition of
In the context of green chemistry, microwave assisted organic
synthesis (MAOS) has gained widespread acceptance in drug
discovery laboratories. Those reactions, which are assisted by
unrivaled features of ionic liquids such as remarkable catalytic
behavior, excellent chemical/thermal stability and good solvating
ability, displayed significant merits of reduced reaction time and
improved yields.1,2 Compounds comprising pyrimidine entity were
found to exhibit a wide range of biological activities suchlike
antitumor,3–5 anti-inflammatory,6 adenosine A2A receptor antago-
nists,7 antiviral8,9 as well as potent cholinesterase/Ab-aggregation
inhibitory properties.10–16 In our pervious study, we reported syn-
thesis and cholinesterase inhibitory activities of novel pyrimidine
and thiopyrimidine derivatives, comprising two aromatic rings.
These new dual core compounds displayed reasonable cholinester-
ase inhibitory activities with more selectivity toward BChE.16
Alzheimer’s disease (AD) is a neurodegenerative disorder that
based on the World Health Organization (WHO) report, has
extracellular b-amyloid plaques and formation of intracellular
neurofibrillary tangles are thought to play principal roles in the
pathophysiology of this disease, causing loss of cholinergic neurons
in the forebrain, cortex and hippocampus of AD patients.18–22 The
decline in acetylcholine (ACh) neurotransmitter level causes the
cognitive impairments seen in AD patients. Thus, increasing ACh
levels to restore the cholinergic neurotransmission and improve
the cognitive functions in AD patients are of prime importance.7,23
Current clinically approved treatments for AD are limited to
cholinesterase inhibitors (ChEI’s), working by inhibiting cholines-
terases from hydrolyzing ACh and N-methyl D-aspartate receptor
antagonists (e.g. memantine), which act at the glutaminergic
pathway.24–26 Despite the tremendous efforts in search of disease
modifying agents working via b-amyloid or tau pathways, none
are clinically available due to their adverse effects. Therefore, the
search for new cholinesterase enzymes inhibitors is still a promis-
ing approach and ongoing worldwide. Two cholinesterases, acetyl-
cholinesterase (AChE) and butyrylcholinesterase (BChE) are
responsible for degradation and regulation of ACh in human body,
however they differ in kinetics and substrate selectivity.27 Cholin-
ergic therapy for AD initially focused on AChE inhibition because
⇑
Corresponding authors. Tel./fax: +60 46534583.
E-mail addresses: vicky@usm.my, dr.Murugaiyah@gmail.com (V. Murugaiyah),
sraju@ksu.edu.sa (R.S. Kumar).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.020

A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
907
Table 1
Residue composition of active sites in hAChE and hBChE
Entry
Site name
Residue composition in hAChE
Residue composition in hBChE38
1
2
3
4
5
Catalytic triad
Ser203, His447 and Glu201
Trp86 and Phe338
Phe295 and Phe297
Gly121, Gly122 and Ala204
Tyr72, Asp74, Tyr124, Trp286, Tyr337 and Tyr341
His438, Ser198 and Glu325
Trp82 and Phe329
Leu286 and Val288
Gly116, Gly117 and Ala199
Trp231, Val288, Leu286 and Phe398
Choline binding site (
Acyl-binding pocket
Oxyanion hole
a-anionic site)
Peripheral anionic site (b-anionic site)
this is the main enzyme involved in the breakdown of ACh in the
normal brain.28 Studies have shown that as the disease progresses,
the activity of AChE decreases while the activity of BChE remains
unaffected or even increases.29 Moreover, in the brain of
advanced-staged AD patients, BChE can compensate for AChE
where the activity of AChE is inhibited and hydrolyze the already
depleted levels of ACh.30,31 Although overall AChE level is reduced
in the brain of AD patients, it is increased within and around the
amyloid plaques. Since both AChE and BChE hydrolyze ACh and in-
volved in amyloid plaques maturation, inhibition of both enzymes
using a dual inhibitor should results in the higher levels of ACh in
the brain that provides more significant clinical efficacy.28 Hence,
dual inhibitors are valuable therapeutic compounds in AD therapy.
The active site of human AChE enzyme is located at the bottom
of a 20 Å long, narrow gorge comprising five important regions to
accommodate and hydrolyze the acetylcholine, namely; catalytic
triad,32 oxyanion hole,33 choline binding site,34 acyl binding
pocket35 and peripheral anionic site,36 (Table 1). Acetylcholine or
inhibitors guidance inside the gorge is facilitated by hydrophobic
interactions with aromatic amino acid residues lining the gorge
wall viz. phenylalanine (Phe), tryptophan (Trp) and tyrosine
(Tyr).37 While the overall structure of human BChE is similar to
that of human AChE, the active site of hBChE has many of the chan-
nel-lining aromatic residues replaced by residues with aliphatic
side chains, such as leucine (Leu) and valine (Val), making BChE
better able to accommodate bulkier substrates and inhibitors.38
Inspired by aforementioned biological significance of dual AChE
and BChE inhibitors and in search for new potent Alzheimer’s dis-
ease modifying agents, in the present study we wish to report ionic
liquid mediated synthesis and cholinesterase inhibitory activity
study of novel thiazolopyrimidine derivatives comprised of three
aromatic rings. Subsequently, the effect of additional aromatic ring
on AChE and BChE inhibitory activities and selectivity is compared
to dual core inhibitors from our previous report. By the aid of
molecular docking studies, the plausible binding interactions
mechanisms of the most active derivatives at the active site of
AChE and BChE are also explored.
2. Results and discussion
2.1. Chemistry
The highly functionalized a,b-unsaturated ketones (3) required
for the synthesis of (6) were prepared by the Claisen–Schmidt
condensation of 4-piperidone hydrochloride (1) with a series of
aromatic aldehydes in the presence of HCl in acetic acid.39 The
domino cascade reaction of 3,5-diarylidenepiperidin-4-ones (3),
thiourea (4) and a-Bromoacetophenones (5a–d) in the ionic liquid,
1-butyl-3-methylimidazolium bromide ([bmim]Br) furnished (E)-
9-arylidene-3,5-diaryl-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazol-
o[3,2-a]pyrimidines (6a–x) in good yield. (Scheme 1)
In order to find an optimal and efficient protocol for the synthe-
sis of 6(a–x), we initiated our study by performing the model
reaction of an equimolar ratio of para-methyl derivative of 3,
5-bis-(aryledene)piperidin-4-one (3), thiourea (4) and 2-bromo-
40-chloroacetophenone (5b), employing two different synthetic
approaches. In the first approach, the synthetic potential of a one
pot, three component reaction was investigated using one molar
equivalent of ([bmim]Br). Owing to the slow reaction rate of
3,5-bis-(aryledene)piperidin-4-one (3) with thiourea (4), in con-
trast to the relatively fast reaction of (4) and (5), this methodology
failed to furnish the proposed thiazolopyrimidines (6).
Thus, we carried out a domino cascade two-step reaction by
refluxing 3,5-bis-(aryledene)piperidin-4-one (3) and thiourea (4)
in 1 molar equivalent of [bmim]Br for 2 h, as the first step. In the
second step, 2-bromo-40-chloroacetophenone (5b) were added to
Ar
Ar
H
2 ArCHO
O
O
H2N
S
N
S
[bmim]Br
2
+
HCl.HN
HN
NH2
HN
NH
MW, 15 min
(i) HCl, CH3COOH
3
1
Ar
4
Ar
(ii) K2CO3
Acetone/water
intermediate
MW, 2 min
O
5a:R=C6H5
Br
R
5b:R=p-FC6H4
5c:R=p-ClC6H4
5d:R=p-OCH3C6H4
5(a-d)
Ar
12
10
11
9a
N
S1
8
9
6
2
HN7
N
5
3
5a
4
R
Ar
6(a-x)
Scheme 1. Synthesis of 6(a–x).

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A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
Structural elucidation of the thiazolopyrimidines (6a–x) was
7.37, s
122.9
accomplished using 1D and 2D NMR spectroscopy techniques as
well as elemental analysis. In the 1H NMR spectrum of 6q, the
one hydrogen singlet at 5.36 ppm is due to H-5 and from HMQC
the peak at 60.4 ppm was assigned to C-5. Further, H-5 shows
HMBCs with C-6 at 46.0 ppm besides showing correlation with
C-5a and C-11 at 114.4 and 162.0 ppm, respectively. From HMQC,
the doublets at 3.04 and 3.28 ppm (J = 17.00 Hz) was assigned to
H-6a and H-6b whilst the other doublets at 3.77 and 3.87 ppm
(J = 15.00 Hz) can be attributed to H-8a and H-8b. The singlets at
7.37 ppm and 5.96 ppm are due to H-12 and H-2, the later shows
HMBCs with C-3 at 137.4 ppm. The multiplets around 6.67–
7.34 ppm is due to aromatic hydrogens. These assignments are also
supported by the HMBC. The 1H and 13C chemical shifts as well as
HMBC correlations of 6q are depicted in Figure 1.
Cl
5.96, s
101.1
3.77, d, J=15.00 Hz
3.87, d, J=15.00 Hz
46.3
H
162.2
N
S
139.3
H
111.4
HN
N
1.91, br.s
137.4
H
3.04, d, J=17.00 Hz
3.28, d, J=17.00 Hz
46.0
6.67-7.34
Aromatic
5.36, s
Cl
Cl
60.4
Cl
A plausible mechanism for the formation of (6) in the presence
of ([bmim]Br) is described in Scheme 2. As ([bmim]Br) plays dual
role as both solvent and catalyst, it facilitates the reaction by form-
ing H-bond between the electron deficient hydrogen atom of ionic
liquid with the oxygen atom of carbonyl functionality in 3. Initial
H
12
10
N
1
9a
S
8
9
6
2
11
H
7
HN
N
4
5
5a
H
3
H
Michael addition of thiourea to b position of
a,b-unsaturated
ketone followed by sequential condensation/tautomerization
affords the intermediate A. In the next step, a five membered
thiazoline ring formed via tautomerization of NH–C@S bond to
negatively charged sulfur atom and its subsequent nucleophilic
addition to positively charged carbon atom of CH2–Br bond in
2-bromoacetophenones and eventually nucleophilic addition and
elimination of secondary amine to carbonyl moiety via enamine
pathway as depicted in scheme 2.
Cl
Cl
Figure 1. Selected HMBCs and 1H and 13C chemical shifts of 6q.
mixture without isolating the intermediates and reaction contin-
ued for additional 2 h. After completion of the reaction as evident
by TLC, the thiazolopyrimidine product was isolated in moderate
yield through column chromatography (57%). Consequently, the
same reaction was investigated under microwave irradiation as a
valuable alternative for conventional heating. As expected, the first
step of the reaction was completed in less than 15 min followed by
the next step, which interestingly was completed in less than
2 min.
The domino cascade methodology reinforced by the unique
properties of ionic liquid and features of microwave irradiation
method enabled us to furnish a library of hitherto unreported
thiazolopyrimidines rapidly and in good yield. The selection of
different aldehydes and 2-bromo-acetophenones is based on the
fact that these groups consist of a wide range of electron donating
and electron withdrawing moieties enabling us to investigate their
inhibitory effects on the AChE and BChE enzymes. On the other
hand, derivatives such as chloro, fluoro and methoxy showed
positive influence on the inhibitory activities of synthesized
compounds in our previous study.16
2.2. In vitro cholinesterase enzymes inhibitory activities
All the newly synthesized compounds were evaluated for their
cholinesterases inhibitory activities against AChE derived from
Electrophorus electricus and BChE from equine serum. The active
sites of cholinesterase enzymes establish extensive hydrophobic
interactions with their inhibitors/substrates owing to high aro-
matic content. In the present study, we aimed to evaluate a variety
of thiazolopyrimidines derivatives comprising three aromatic cores
to investigate how aromatic content and multiplicity affect AChE/
BChE inhibitors potencies.
The molecular structure of 6 is composed of three distinctive
regions, namely: R; comprising phenyl, p-fluoro phenyl, p-chloro
phenyl and p-methoxy phenyl moieties, Ar; ascribing versatile
phenyl derivatives, which both are engrafted to an aliphatic thiaz-
olopyrimidine core.
AChE inhibitory activities of thiazolopyrimidine derivatives are
summarized in Table 2. Compounds 6a–f, bearing un-substituted
N
N
Ar
Ar
+
N
+
Br
Br
O
H
N
N
Bu
H
H
S
N
S
Bu
Ar
Ar
7
H2N
HN
NH
HN
NH
-H2O
H2N
S
O
S
H
Ar
Ar
NH2
HN
HN
NH
Intermediate A
Ar
Ar
3
4
Ar
H
N
Ar
Ar
Br
H
N
S
5b
N
S
S
Br
Br
HN
NH
O
H
N
N
6
H
R
H
+
+
HN
N
HN
N
O
Ar
Bu
OH
R
N
N
H
H
H
R
Ar
Ar
Bu
Bu
Br
N
+
N
Scheme 2. Plausible mechanism for the formation of 6.

A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
909
Table 2
Physical data, AChE and BChE inhibitory activities of 6(a–x)
Entry
Compound
R
Ar
Yield (%)
AChE inhibition (IC50
)
BChE inhibition (IC50
)
Selectivity
AChEa BChEb
(
l
g/ml) M)
(
l
(
l
g/ml) M)
(l
1
2
3
4
5
6
6a
6b
6c
6d
6e
6f
C6H5
C6H5
86
79
82
84
81
85
0.6 0.04
1.47
2.05
4.23
0.53
1.62
4.04
0.70 0.03
1.34 0.07
2.02 0.11
0.79 0.04
1.09 0.08
1.85 0.12
1.74
2.68
4.10
1.71
2.18
3.95
1.19
1.30
0.97
3.21
1.35
0.98
0.84
0.77
1.03
0.31
0.74
1.02
o-ClC6H4
o-(OCH3)C6H4
p-CH3C6H4
p-ClC6H4
p-FC6H4
1.03 0.11
2.08 0.15
0.24 0.02
0.81 0.05
1.89 0.09
7
8
9
10
11
12
6g
6h
6i
6j
6k
6l
p-FC6H4
C6H5
81
73
75
79
71
68
9.17 0.15
3.14 0.11
2.81 0.09
3.10 0.18
2.98 0.12
6.41 0.22
20.32
6.03
5.50
6.47
5.73
13.16
3.57 0.14
2.41 0.08
4.47 0.19
2.35 0.17
8.21 0.22
7.68 0.25
7.91
4.63
8.75
4.91
15.80
15.77
0.39
1.30
1.59
0.76
2.75
1.20
2.57
0.76
0.63
1.32
0.36
0.83
o-ClC6H4
o-(OCH3)C6H4
p-CH3C6H4
p-ClC6H4
p-FC6H4
13
14
15
16
17
18
6m
6n
6o
6p
6q
6r
p-ClC6H4
C6H5
79
82
81
83
85
78
11.16 0.11
2.78 0.05
2.68 0.07
10.78 0.15
2.87 0.10
5.16 0.11
23.85
5.20
5.08
21.74
5.37
10.27
20.75 0.22
4.10 0.16
15.45 0.27
16.14 0.17
14.70 0.18
11.18 0.20
44.34
7.65
29.28
32.56
27.43
22.24
1.86
1.47
5.76
1.50
5.11
2.16
0.54
0.68
0.17
0.67
0.20
0.46
o-ClC6H4
o-(OCH3)C6H4
p-CH3C6H4
p-ClC6H4
p-FC6H4
19
20
21
22
23
24
6s
6t
6u
6v
6w
6x
p-(OCH3)C6H4
Galanthamine
C6H5
80
76
79
84
87
83
7.36 0.14
4.24 0.12
6.26 0.21
4.90 0.10
4.49 0.17
11.18 0.19
15.92
7.97
11.97
10.44
8.44
4.82 0.17
6.68 0.25
9.7 0.22
5.14 0.18
6.81 0.19
13.25 0.24
10.41
12.56
18.56
10.48
12.79
26.55
0.65
1.58
1.55
1.01
1.52
1.18
1.52
0.63
0.64
0.99
0.66
0.84
o-ClC6H4
o-(OCH3)C6H4
p-CH3C6H4
p-ClC6H4
p-FC6H4
22.41
25
0.60 0.01
2.09
5.55 0.01
19.34
3.47
0.28
a
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE).
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE).
b
phenyl moiety in region ‘R’, displayed highest inhibitory activities
with IC50 values of lower than 4.5 M. Therein, compound 6d with
(Ar = p-methylphenyl), showed highest AChE inhibitory activity
with sub-micromolar IC50 value of 0.53 M, which is four times
higher than standard drug, galanthamine with IC50 value of
2.09 M.
Structure activity relationship (SAR) study for compounds 6a–x
clearly showed that substitution on region ‘R’ is a major determi-
nant of AChE inhibitory activity. In addition, the planar configura-
tion of thiazolopyrimidine compounds provides less hindered
insertion and better accommodation inside the narrow AChE active
site channel. Moreover, the polarizable sulfur atom of thiazoline
ring and free nitrogen atom of piperidone ring effectively interact
with electron rich aromatic gorge of AChE through mild polar
(dipole–induced dipole) interactions. ‘Dipole–induced dipole’
interactions, mostly occurs between a permanent dipole such as
l
Ar
H
N
S
l
HN
NH
l
Ar
Dual core inhibitors
7a: Ar=
, AChE IC50=19.27 M; BChE IC = 3.78 M
µ µ
50
Cl
7c: Ar=
, AChE IC50=32.29 µM; BChE IC50= 2.91µM
, AChE IC50=0.8 µM; BChE IC50= 1.18 µM
OCH3
C@O, C–S or C–N and molecules with
p electrons, like benzene
rings of phenylalanine and tyrosine.
7e: Ar=
Moving from 6a–f with (R = phenyl) to 6g–l with (R = p-fluoro-
phenyl) then 6m–r with (R = p-chlorophenyl) and finally 6s–x with
(R = p-methoxyphenyl), a decline in AChE inhibitory activities is
observed. This is presumably due to the enlargement of ‘R’ that
led to more hindered insertion and improper accommodation of
inhibitors inside the active site channel of the AChE enzyme.
On the other hand, substitution on region ‘Ar’ showed only a
minor effect on the AChE inhibition of these derivatives. However,
the presence of a chloro atom on either ortho or para position of the
phenyl ring of ‘Ar’ improved the inhibitory potencies, with IC50 val-
ues ranging from 1.62 to 8.44 lM. In addition, compounds 6i and
6o with Ar = o-methoxyphenyl and 6j with Ar = p-methylphenyl
also displayed good inhibitory activities with IC50 values of lower
than 10 lM.
7h
: Ar=
, AChE IC50=37.47 µM; BChE IC50= 6.27 µM
CH3
7i: Ar=
7j: Ar=
, AChE IC50=2.25 µM; BChE IC50= 6.26 µM
Cl
, AChE IC50=37.22 M; BChE IC = 28.82
M
µ
µ
50
F
Regarding BChE inhibitory activities, compounds 6a–j and 6n
Figure 2. Schematic representation of dual core inhibitors, their derivatives, AChE
displayed remarkable potencies with IC50 values of lower than
and BChE inhibitory activities.

910
A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
Figure 3. Binding interaction of 6d with major active site residues of hAChE.
9
l
M, therein compound 6d displayed highest activity with IC50
value of 1.71 M, almost 10 times stronger than the standard drug.
Rest of the compounds displayed moderate activities with IC50 val-
ues ranging from 10.41 to 44.34 M. The effect of ‘R-substitution’
‘Ar’, viz. 7a, (Ar = phenyl, IC50 = 19.27); 7c, (Ar = o-chlorophenyl,
IC50 = 32.39); 7e, (Ar = o-methoxyohenyl, IC50 = 0.8); 7h, (Ar =
p-methylphenyl, IC50 = 37.47); 7i, (Ar = p-chlorophenyl, IC50
2.25) and 7j, (Ar = p-fluorophenyl, IC50 = 37.22 M) (Fig. 2). The
l
=
l
l
on BChE inhibitory activities was similar to AChE except for com-
pounds 6m–r with R = p-methoxyphenyl which displayed better
activities than 6g–l with R = p-chlorophenyl. This effect is probably
due to the larger active site of BChE enzyme, which preferably
accommodates more energetically favorable inhibitors irrespective
to their size.
results indicated that, except for compounds 6c and 6i, the rest of tri-
ple core thiazolopyrimidines in this study displayed 1.5 to 70 fold
higher AChE inhibitory activities than their dual core analogs. Com-
pounds 6a–f ascribing phenyl as the additional aromatic moiety in
‘R’, represented highest variation in the activities observed, therein
compound 6d displayed almost 70 times higher AChE inhibitory
In our previous study, we reported synthesis and cholinesterase
inhibitory activities of novel pyridopyrimidothione derivatives
comprising two aromatic rings.16 To get better insight into the
effect of aromaticity on cholinesterase inhibitory activities, com-
pounds 6a–x composed of three aromatic cores compared to their
dual core analogs possessing similar substituent on phenyl ring of
activity than dual core 7h (IC50 = 0.53 vs 37.47 lM).
This phenomenon is presumably due to the high aromatic con-
tent of AChE enzyme active site enabling it to establish strong
hydrophobic and
p,p-stacking interactions with the substrate or
inhibitors, employing its aromatic amino acid residues. So, the
inhibitors with three aromatic cores were found to interact more
Figure 4. Orientation of 6d superimposed with galanthamine in active site of hAChE enzyme.

A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
911
Figure 5. Binding interaction of 6d with major active site residues of hBChE.
considerably decreased activities for the remaining compounds
comparing to their dual core analogs.
It is also worth to mention that expect for compounds 6g, 6j and
6s, which showed more selectivity toward BChE, all the newly
synthesized thiazolopyrimidines had more selectivity for AChE.
In addition, compounds 6a–f except 6d displayed potent dual
inhibitory properties. AChE specific inhibitors might be useful at
the earlier stages of AD whereby AChE enzyme has dominant role
in breaking down the ACh. However, as the disease progresses,
BChE role become more prominent and therefore a dual inhibitor
is expected to be beneficial at both stage.
2.3. Molecular docking studies
Molecular docking simulations were performed on compounds
6d that displayed the highest AChE and BChE inhibitory activities
among the compounds 6a–x to gain functional and structural
insight into its binding interaction at the active sites of human
AChE and BChE enzymes.
These molecular docking analysis for 6d disclosed that the
majority of its binding interactions with hAChE receptor were mild
polar and hydrophobic in nature. As mentioned before, mild polar
interaction can be defined as the ‘dipole–induced dipole interac-
tions’ which mostly occurs between a permanent dipole and
Figure 6. Orientation of 6d superimposed with BCh molecule in active site of hBChE
enzyme.
efficiently with the AChE channel wall, facilitating their insertion
and accommodation into the gorge. However, the size of the addi-
tional aromatic core has to be chosen wisely due to constriction of
AChE gorge. As in our case, compounds 6a–f bearing relatively
small phenyl moiety as third aromatic core, displayed highest
inhibitory activity in contrast to larger ‘R’ substituents viz. fluoro,
chloro and methoxy phenyl.
Unlike AChE, for BChE most of the triple core thiazolopyrimi-
dines displayed lower activity than their dual core analogs namely;
7a, 7c, 7e, 7h, 7i, and 7j having IC50 values of 3.78, 2.91, 1.18, 6.27,
molecules with
The ‘Ar’ moieties of this compound viz. p-methylphenyl, displayed
relatively strong -stacking interaction with aromatic rings of
Tyr72 and Tyr341 at peripheral anionic as well as Phe338 at cho-
line binding site of the enzyme located at the entrance of the gorge.
p electrons, such as phenylalanine and tyrosine.
p,p
Besides, the phenyl group of ‘R’, also established a
p,p-stacking
interaction with another choline binding site residue, Trp84, at
the bottom of the gorge. Hydrophobic interactions with Phe295
and Phe297 at acyl binding pocket as well as mild polar interac-
tions with Gly121 and Gly122 at oxyanion hole and His447,
Ser203 at catalytic triad are the other dominant interactions of
6d with the active site residues of hAChE receptor (Fig. 3). This
binding template including all the five main sub-sites of AChE
receptor obviously coincided with significant inhibitory activity
of 6d contrasting to standard drug. Crystal structure of AChE in
complex with available Alzheimer’s drug galanthamine, also
showed similar template of occupying the peripheral anionic site,
6.26 and 28.82 lM, respectively. As mentioned earlier, in the active
site of BChE, aromatic residues such as tyrosine and tryptophan are
mostly replaced with hydrophobic valine and leucine residues.
These changes reduces BChE active site ability to form hyrdophobic
and
p,p-stacking interactions with aromatic cores comparing to
AChE. Although, engraftment of new aromatic core slightly
increased the activities for 6a, 6b, 6d, 6e, 6f, 6i and 6l, but it

912
A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
Table 3
Binding interaction data for 6d docked into active site gorge of hAChE and hBChE receptors
1
H
N
3
S
4
2
HN
N
H
6
5
Entry
1
Ligand
Enzyme
Docking score
Interacting site
PASa
Amino acid residue
Bond type
Ligand interacting moiety
Ring 1
6d
hAChE
ꢀ9.2
Trp286
Tyr72
Hydrophobic
p–p stacking
Tyr341
Tyr124
Gly121 and 122
Ala204
Phe295
Phe297
Phe338
Trp86
Hydrophobic
Hydrophobic
Mild polar
Hydrophobic
Hydrophobic
Hydrophobic
Ring 2
Ring 4
OHb
Acyl binding pocket
Choline binding site
CTc
p
p
–
–
p
p
Stacking
Stacking
Ring 6
Ring 4
Ser203
Mild polar
His447
Mild polar
2
6d
hBChE
ꢀ7.7
Side chain
Asn68
Ser287
Thr120
Leu286
Gly116 and 117
Phe329
Trp82
His438
Ser198
Mild polar
Mild polar
H-bonding
Hydrophobic
Mild polar
Hydrophobic
Ring 1
Ring 2, NH
Ring 5
PAS
OH
Choline binding site
Ring 6
Ring 4
Ring 6
Ring 5
p
p
–
–
p
p
Stacking
Stacking
CT
Mild polar
a
b
c
Peripheral anionic site.
Oxyanion hole.
Catalytic triad.
stacking against Trp86 and interaction with residues at catalytic
triad (Fig. 4).40
aromatic entity, due to constriction of AChE gorge and its sensivity
to the size of inhibitors. In general, triple core inhibitors in the
present study displayed better inhibition and selectivity toward
AChE in comparison to dual core ones, which mostly displayed bet-
ter selectivity for BChE. However, addition of un-substituted phe-
nyl moiety as the third aromatic core significantly increased both
AChE and BChE inhibitory activities in contrast to their previously
reported dual core analogs.
Regarding molecular docking simulation on BChE receptor, aro-
matic cores of ‘R’ and ‘Ar’ in 6d displayed effective
p,p-stacking
interaction with His438 in the catalytic triad and Trp82 at the
choline binding site of BChE enzyme, along with H-bonding inter-
action with the side chain hydroxyl of Thr120. This binding orien-
tation, in addition to the mild polar interaction with oxyanion hole
residues Gly116 and Gly117 as well as catalytic triad residue
Ser198 confirmed the complete insertion and efficient accommo-
dation of this inhibitor inside the active site gorge of the BChE
receptor (Fig. 5). Moreover, the crystal structure of BChE enzyme
in complex with its substrate, butrylylcholine, also showed the
similar interaction pattern of occupying choline binding site and
stacking against Trp82 as well as interaction with the residues
composing catalytic triad of the enzyme (Fig. 6).
4. Experimental
4.1. Chemistry
Melting points were recorded using Stuart Scientific (SMP1)
apparatus and are uncorrected. A CEM microwave synthesizer
(Model: Discover-S908860) operating at 180/240 V and 50/60 Hz
with consumption of 1100 W with microwave power maximum
level of 300 W and microwave frequency of 2455 MHz was em-
ployed for the irradiation done in this work. The 1H, 13C and the
2D NMR spectra were recorded on a Bruker (Avance) 500 MHz
NMR instrument using TMS as internal standard and CDCl3 as sol-
vent. Bruker Topspin 3.0 software was used to process the NMR data.
Chemical shifts are given in parts per million (d-scale) and the cou-
pling 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 accomplished
on a Perkin Elmer 2400 Series II Elemental CHN analyzer.
The data representing free binding energies, receptor interac-
tion sites, ligand interacting moieties, amino acid residues involved
in ligand–receptor complex as well as bonding types of compound
6d are summarized in Table 3.
3. Conclusion
A library of hitherto unreported pyrimidine grafted heterocy-
cles comprising three aromatic cores were synthesized via an effi-
cient methodology in ionic liquid [bmim]Br, with good yields and
investigated to disclose the effect of aromatic core multiplicity
on cholinesterases inhibition. In vitro cholinesterase inhibitory
results revealed that engraftment of the third aromatic core signif-
icantly improved AChE inhibitory activities up to 70 fold. However,
this phenomenon was inversely correlated to the size of embedded
4.1.1. General procedure for the synthesis of compounds (6a–x)
An equimolar mixture of (3E,5E)-3,5-bis-arylidene-piperidin-
4-one (3) and thiourea (4) were allowed to react under microwave

A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
913
irradiation (100 °C) by using 1 molar equivalent of [bmim]Br as io-
nic liquid. After 15 min 2-bromoacetophenones (5a–d) were added
to the mixture without isolating the intermediate and reaction
continued for another 2 min. After completion of the reaction as
evident by TLC, the products (6a–x) were isolated in good yields
via purification by column chromatography using 7.5:2.5 petro-
leum ether/ethyl acetate solvent system.
14H, H-aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 46.32,
46.60, 60.71, 100.83, 111.60, 123.37, 128.20, 128.71, 128.93,
129.09, 129.16, 129.65, 130.70, 130.96, 132.69, 132.76, 136.68,
136.20, 139.07, 139.83, 162.76.
4.1.1.6. (E)-9-(4-Fluorobenzylidene)-5-(4-fluorophenyl)-3-phe-
nyl-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimi-
dine (6f).
Yellow solid; yield: 85%, mp 161–164 °C; Anal.
4.1.1.1. (E)-9-Benzylidene-3,5-diphenyl-6,7,8,9-tetrahydro-5H-
Calcd for C28H21F2N3S: C, 71.62; H, 4.51; N, 8.95; Found: C,
71.72; H, 4.57; N, 8.97; 1H NMR (500 MHz, CDCl3): dH 2.06 (br
s, 1H, NH), 3.47 (d, J = 16.45 Hz, 1H, H-6a), 3.58 (d, J = 16.45 Hz,
1H, H-6b), 3.85 (d, J = 14.16 Hz, 1H, H-8a), 3.96 (d, J = 14.16 Hz,
1H, H-8b), 5.57 (s, 1H, H-5), 6.04 (s, 1H, H-2), 6.63–7.76 (m,
14H, H-aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 52.91,
53.49, 60.64, 101.45, 108.98, 125.07, 128.24, 128.53, 128.65,
128.90, 129.25, 129.71, 131.21, 131.32, 130.96, 133.78, 135.92,
139.35, 162.78.
pyrido[4,3-d]thiazolo[3,2-a]pyrimidine (6a).
Yellow solid;
yield: 86%, mp 216–219 °C; Anal. Calcd for C28H23N3S: C, 77.57;
H, 5.35; N, 9.69; Found: C, 76.17; H, 5.25; N, 9.55; 1H NMR
(500 MHz, CDCl3): dH 2.04 (br s, 1H, NH), 3.01 (d, J = 17.20 Hz,
1H, H-6a), 3.22 (d, J = 17.20 Hz, 1H, H-6b), 3.75 (d, J = 15.00 Hz,
1H, H-8a), 3.92 (d, J = 15.00 Hz, 1H, H-6b), 5.39 (s, 1H, H-5), 5.89
(s, 1H, H-2), 6.55–7.39 (m, 15H, H-aromatic), 7.41 (s, 1H, H-12).
13C NMR (125 MHz, CDCl3): dC 45.61, 45.79, 60.69, 100.11,
110.38, 124.51, 126.34, 128.39, 128.74, 128.95, 129.03, 129.18,
129.28, 130.32, 130.54, 134.09, 134.31, 136.41, 137.97, 138.21,
139.08, 162.57.
4.1.1.7.
6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine
(6g). Yellow solid; yield: 81%, mp 192–194 °C; Anal. Calcd
(E)-9-(Benzylidene)-5-phenyl-3-(4-fluorophenyl)-
4.1.1.2. (E)-9-(2-Chlorobenzylidene)-5-(2-chlorophenyl)-3-phe-
nyl-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine
for C28H22FN3S: C, 74.48; H, 4.91; N, 9.31; Found: C, 73.22; H,
4.31; N, 8.94; 1H NMR (500 MHz, CDCl3): dH 2.14 (br s, 1H,
NH), 3.08 (d, J = 17.37 Hz, 1H, H-6a), 3.30 (d, J = 17.37 Hz, 1H,
H-6b), 3.81 (d, J = 16.61 Hz, 1H, H-8a), 3.93 (d, J = 16.61 Hz, 1H,
H-8b), 5.38 (s, 1H, H-5), 5.93 (s, 1H, H-2), 6.74–7.56 (m, 15H,
H-aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 46.47, 46.73,
52.98, 53.79, 61.68, 111.84, 115.70, 115.99, 124.31, 126.68,
127.03, 128.47, 129.02, 131.04, 131.19, 132.43, 134.47, 137.87,
138.21, 141.45, 161.79, 165.10, 195.44.
(6b).
Yellow solid; yield: 79%, mp 162–165 °C; Anal. Calcd for
C28H21Cl2N3S: C, 66.93; H, 4.21; N, 8.36; Found: C, 66.88; H, 4.19;
N, 8.27; 1H NMR (500 MHz, CDCl3): dH 2.04 (br s, 1H, NH), 3.04
(d, J = 17.27 Hz, 1H, H-6a), 3.30 (d, J = 17.27 Hz, 1H, H-6b), 3.77
(d, J = 15.48 Hz, 1H, H-8a), 3.89 (d, J = 15.48 Hz, 1H, H-8b), 5.44
(s, 1H, H-5), 5.96 (s, 1H, H-2), 6.55–7.41 (m, 13H, H-aromatic),
7.43 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC 46.29, 46.58,
60.65, 100.71, 111.39, 123.17, 128.10, 128.55, 128.65, 128.93,
129.02, 129.44, 130.50, 130.72, 132.51, 132.72, 134.68, 136.10,
139.07, 139.75, 162.71.
4.1.1.8. (E)-9-(2-Chlorobenzylidene)-5-(2-chlorophenyl)-3-(4-
fluorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-
a]pyrimidine (6h).
Yellow solid; yield: 73%, mp 177–179 °C;
4.1.1.3. (E)-9-(2-Methoxybenzylidene)-5-(2-methoxyphenyl)-3-
phenyl-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimi-
Anal. Calcd for C28H20Cl2FN3S: C, 64.62; H, 3.87; N, 8.07; Found: C,
64.15; H, 3.59; N, 7.87; 1H NMR (500 MHz, CDCl3): dH 2.11 (br s,
1H, NH), 2.94 (d, J = 16.85 Hz, 1H, H-6a), 3.43 (d, J = 16.85 Hz,
1H, H-6b), 3.69 (d, J = 16.85 Hz, 1H, H-8a), 3.77 (d, J = 16.85 Hz,
1H, H-8b), 5.91 (s, 1H, H-5), 5.99 (s, 1H, H-2), 6.90–7.85 (m,
13H, H-aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 52.40,
52.81, 61.81, 100.40, 115.40, 115.56, 123.09, 126.03, 126.21,
127.95, 128.06, 128.91, 129.30, 129.37, 130.64, 130.68, 130.76,
130.87, 130.94, 131.82, 132.25, 133.68, 134.03, 135.85, 137.61,
138.90, 162.25, 162.47, 164.24, 164.67, 166.69, 195.01.
dine (6c).
Yellow solid; yield: 82%, mp 172–174 °C; Anal. Calcd
for C30H27N3O2S: C, 73.00; H, 5.51; N, 8.51; Found: C, 73.07; H, 5.59;
N, 8.54; 1H NMR (500 MHz, CDCl3): dH 2.86 (d, J = 16.55 Hz, 1H, H-
6a), 3.42 (d, J = 16.55 Hz, 1H, H-6b), 3.62 (s, 3H, methoxy), 3.80 (s,
1H, methoxy), 3.82-3.89 (m, 2H, H-8), 5.41 (s, 1H, H-5), 5.96 (s, 1H,
H-2), 6.48-7.75 (m, 14H, H-aromatic, H-12). 13C NMR (125 MHz,
CDCl3): dC 52.41, 53.38, 55.07, 55.24, 60.57, 100.57, 108.18, 125.11,
127.72, 127.89, 128.41, 128.62, 128.82, 129.11, 130.66, 133.15,
135.75, 139.11, 162.37.
4.1.1.9. (E)-9-(2-Methoxybenzylidene)-5-(2-methoxyphenyl)-3-
(4-fluorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,
4.1.1.4. (E)-9-(4-Methylbenzylidene)-3-phenyl-5-p-tolyl-6,7,8,9-
tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine (6d).
Yel-
2-a]pyrimidine (6i).
Yellow solid; yield: 75%, mp 188–
low solid; yield: 84%, mp 140–142 °C; Anal. Calcd for C30H27N3S: C,
78.06; H, 5.90; N, 9.10; Found: C, 78.16; H, 5.95; N, 9.17; 1H NMR
(500 MHz, CDCl3): dH 2.22 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.56 (br s,
1H, NH), 3.07 (d, J = 17.25 Hz, 1H, H-6a), 3.28 (d, J = 17.25 Hz, 1H, H-
6b), 3.80 (d, J = 15.10 Hz, 1H, H-8a), 3.97 (d, J = 15.10 Hz, H-8b), 5.41
(s, 1H, H-5), 5.92 (s, 1H, H-2), 6.55–7.39 (m, 13H, H-aromatic), 7.45 (s,
1H, H-12). 13C NMR (125 MHz, CDCl3): dC 45.62, 45.82, 60.72, 100.13,
110.40, 124.50, 126.34, 128.37, 128.75, 128.92, 129.04, 129.20, 129.27,
130.33, 130.60, 134.13, 134.34, 136.45, 137.99, 138.17, 139.02, 162.54.
190 °C; Anal. Calcd for C30H26FN3O2S: C, 70.43; H, 5.12; N, 8.21;
Found: C, 70.19; H, 4.88; N, 7.95; 1H NMR (500 MHz, CDCl3): dH
1.83 (br s, 1H, NH), 3.06 (d, J = 17.33 Hz, 1H, H-6a), 3.26 (d,
J = 17.33 Hz, 1H, H-6b), 3.72 (s, 3H, O–CH3), 3.80 (s, 3H, O–CH3),
3.82 (d, J = 15.29 Hz, 1H, H-8a), 3.91 (d, J = 15.29 Hz, 1H, H-8b),
5.30 (s, 1H, H-5), 5.89 (s, 1H, H-2), 6.63–7.36 (m, 13H, H-aromatic,
H-12). 13C NMR (125 MHz, CDCl3): dC 46.29, 46.42, 55.20, 55.22,
60.55, 100.22, 111.32, 113.59, 113.88, 115.37, 115.56, 123.22,
127.69, 130.62, 133.46, 134.12, 137.86, 158.27, 159.59, 161.93,
162.05, 164.03.
4.1.1.5. (E)-9-(4-Chlorobenzylidene)-5-(4-chlorophenyl)-3-phe-
nyl-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimi-
4.1.1.10. (E)-9-(4-Methylbenzylidene)-5-(4-methylphenyl)-3-(4-
fuorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]
dine (6e).
Yellow solid; yield: 81%, mp 165–167 °C; Anal.
Calcd for C28H21Cl2N3S: C, 66.93; H, 4.21; N, 8.36; Found: C,
66.82; H, 4.27; N, 8.44; 1H NMR (500 MHz, CDCl3): dH 2.06 (br
s, 1H, NH), 3.06 (d, J = 17.37 Hz, 1H, H-6a), 3.31 (d, J = 17.37 Hz,
1H, H-6b), 3.79 (d, J = 15.48 Hz, 1H, H-8a), 3.92 (d, J = 15.48 Hz,
1H, H-8b), 5.46 (s, 1H, H-5), 5.99 (s, 1H, H-2), 6.62–7.45 (m,
pyrimidine (6j).
Yellow solid; yield: 79%, mp 119–121 °C;
Anal. Calcd for C30H26FN3S: C, 75.13; H, 5.46; N, 8.76; Found: C,
74.46; H, 5.19; N, 8.39; 1H NMR (500 MHz, CDCl3): dH 2.24 (s, 3H,
CH3), 2.35 (s, 3H, CH3), 3.09 (d, J = 16.99 Hz, 1H, H-6a), 3.31 (d,
J = 16.99 Hz, 1H, H-6b), 3.75 (d, J = 14.91 Hz, 1H, H-8a), 4.06 (d,

914
A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
J = 14.91 Hz, 1H, H-8b), 5.42 (s, 1H, H-5), 5.93 (s, 1H, H-2), 6.57–7.52
(m, 13H, H-aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 21.47,
21.61, 45.23, 45.40, 61.36, 100.92, 109.05, 115.72, 116.00, 126.51,
129.47, 129.59, 129.75, 131.17, 131.28, 134.15, 134.22, 137.23,
134.22, 137.23, 138.22, 138.42, 138.76, 161.84, 162.92, 165.15.
7.37 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC 46.16, 46.29,
55.24, 60.52, 100.65, 111.11,123.46, 127.72, 128.66, 129.92,
130.08, 130.44, 132.62, 133.25, 134.22, 135.22, 137.77, 158.33,
159.65, 161.99.
4.1.1.16. (E)-9-(4-Methylbenzylidene)-5-(4-methylphenyl)-3-(4-
chlorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-
4.1.1.11. (E)-9-(4-Chlorobenzylidene)-5-(4-chlorophenyl)-3-(4-
fluorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]
a]pyrimidine (6p).
Yellow solid; yield: 83%, mp 124–127 °C;
pyrimidine (6k).
Yellow solid; yield: 71%, mp 139-141 °C;
Anal. Calcd for C30H26ClN3O2S: C, 72.64; H, 5.28; N, 8.47; Found:
C, 72.61; H, 5.12; N, 8.32; 1H NMR (500 MHz, CDCl3): dH 2.21 (s,
3H, CH3), 2.31 (s, 3H, CH3), 3.24 (d, J = 17.05 Hz, 1H, H-6a), 3.60
(d, J = 17.05 Hz, 1H, H-6b), 3.84 (d, J = 15.10 Hz, 1H, H-8a), 4.01
(d, J = 15.10 Hz, 1H, H-8b), 5.47 (s, 1H, H-5), 5.96 (s, 1H, H-2),
6.56–7.36 (m, 12H, H-aromatic), 7.56 (s, 1H, H-12). 13C NMR
(125 MHz, CDCl3): dC 21.04, 21.08, 43.94, 44.07, 60.71, 101.80,
117.88, 125.23, 126.15, 128.67, 129.01, 129.17, 129.21, 129.70,
130.14, 130.18, 133.27, 133.75, 135.37, 137.25, 138.61, 162.97.
Anal. Calcd for C28H20Cl2FN3S: C, 64.62; H, 3.87; N, 8.07; Found: C,
64.17; H, 3.44; N, 7.82; 1H NMR (500 MHz, CDCl3): dH 1.90 (s, 1H,
NH), 3.05 (d, J = 17.00 Hz, 1H, H-6a), 3.29 (d, J = 17.00 Hz, 1H, H-
6b), 3.78 (d, J = 15.00 Hz, 1H, H-8a), 3.89 (d, J = 15.00 Hz, 1H, H-
8b), 5.37 (s, 1H, H-5), 5.96 (s, 1H, H-2), 6.61–7.83 (m, 13H, H-aro-
matic, H-12). 13C NMR (125 MHz, CDCl3): dC 45.92, 46.18, 60.53,
100.73, 115.58, 115.75, 123.13, 127.58, 127.66, 128.33, 128.78,
128.89, 130.55, 130.72, 134.22, 134.43, 135.78, 137.56, 139.46,
162.16.
4.1.1.17. (E)-9-(4-Chlorobenzylidene)-3,5-bis(4-chlorophenyl)-
6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine
4.1.1.12. (E)-9-(4-Fluorobenzylidene)-5-(4-fluorophenyl)-3-(4-
fluorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]
(6q).
Yellow solid; yield: 85%, mp 148–150 °C; Anal. Calcd
pyrimidine (6l).
Yellow solid; yield: 68%, mp 157–159 °C;
for C28H20Cl3N3S: C, 62.64; H, 3.75; N, 7.83; Found: C, 62.55; H,
3.62; N, 7.81; 1H NMR (500 MHz, CDCl3): dH 1.94 (br s, 1H,
NH), 3.05 (d, J = 17.40 Hz, 1H, H-6a), 3.28 (d, J = 17.40 Hz, 1H,
H-6b), 3.77 (d, J = 15.29 Hz, 1H, H-8a), 3.87 (d, J = 15.29 Hz, 1H,
H-8b), 5.36 (s, 1H, H-5), 5.96 (s, 1H, H-2), 6.66–7.34 (m, 12H,
H-aromatic), 7.37 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC
46.04, 46.31, 60.46, 101.09, 111.43, 122.92, 127.71, 128.32,
128.76, 128.83, 128.93, 129.92, 130.57, 132.37, 132.47, 134.36,
134.47, 135.51, 135.84, 137.44, 139.33, 162.05.
Anal. Calcd for C28H20F3N3S: C, 68.98; H, 4.13; N, 8.62; Found: C,
68.54; H, 3.97; N, 8.25; 1H NMR (500 MHz, CDCl3): dH 3.20 (d,
J = 16.70 Hz, 1H, H-6a), 3.39 (d, J = 16.70 Hz, 1H, H-6b), 3.80 (d,
J = 14.97 Hz, 1H, H-8a), 3.90 (d, J = 14.97 Hz, 1H, H-8b), 5.48 (s, 1H,
H-5), 5.97 (s, 1H, H-2), 6.67–7.26 (m, 13H, H-aromatic, H-12). 13C
NMR (125 MHz, CDCl3): dC 52.64, 53.20, 60.39, 61.96, 101.97,
115.43, 115.60, 115.91, 128.08, 130.60, 130.68, 130.86, 137.73,
164.84, 194.75.
4.1.1.13.
6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine
(6m). Yellow solid; yield: 79%, mp 152–155 °C; Anal. Calcd
(E)-9-(Benzylidene)-5-phenyl-3-(4-chlorophenyl)-
4.1.1.18. (E)-9-(4-Fluorobenzylidene)-5-(4-fluorophenyl)-3-(4-
chlorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-
a]pyrimidine (6r).
Yellow solid; yield: 78%, mp 160–163 °C;
for C28H22ClN3S: C, 71.86; H, 4.74; N, 8.98; Found: C, 71.81; H,
4.749; N, 8.90; 1H NMR (500 MHz, CDCl3): dH 2.03 (br s, 1H,
NH), 3.09 (d, J = 17.37 Hz, 1H, H-6a), 3.31 (d, J = 17.37 Hz, 1H,
H-6b), 3.82 (d, J = 15.48 Hz, 1H, H-8a), 3.93 (d, J = 15.48 Hz, 1H,
H-8b), 5.39 (s, 1H, H-5), 5.95 (s, 1H, H-2), 6.75–7.36 (m, 13H,
H-aromatic), 7.45 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC
46.46, 46.72, 60.62, 101.12, 111.92, 124.34, 126.71, 126.93,
128.48, 128.84, 129.02, 129.06, 129.38, 129.75, 132.37, 134.51,
135.65, 137.83, 138.08, 141.26, 162.55.
Anal. Calcd for C28H20ClF2N3S: C, 66.73; H, 4.00; N, 8.34; Found:
C, 66.65; H, 4.01; N, 8.12; 1H NMR (500 MHz, CDCl3): dH 2.14 (br
s, 1H, NH), 3.06 (d, J = 17.20 Hz, 1H, H-6a), 3.32 (d, J = 17.20 Hz,
1H, H-6b), 3.62 (d, J = 15.65 Hz, 1H, H-8a), 3.86 (d, J = 15.65 Hz,
1H, H-8b), 5.39 (s, 1H, H-5), 5.97 (s, 1H, H-2), 6.64-7.47 (m, 12H,
H-aromatic), 7.70 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC
52.52, 53.11, 60.50, 101.03, 108.46, 124.34, 128.03, 128.10,
128.72, 128.79, 129.49, 129.91, 129.94, 130.79, 130.85, 130.91,
133.92, 135.38, 136.67, 137.53, 139.64, 162.18.
4.1.1.14. (E)-9-(2-Chlorobenzylidene)-5-(2-chlorophenyl)-3-(4-
chlorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-
4.1.1.19.
6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,2-a]pyrimidine
(6s). Yellow solid; yield: 80%, mp 145–147 °C; Anal. Calcd
(E)-9-(Benzylidene)-5-phenyl-3-(4-methoxyphenyl)-
a]pyrimidine (6n).
Yellow solid; yield: 82%, mp 182–185 °C;
Anal. Calcd for C28H20Cl3N3S: C, 62.64; H, 19.81; N, 7.83; Found: C,
62.51; H, 19.72; N, 7.69; 1H NMR (500 MHz, CDCl3): dH 2.10 (br s,
1H, NH), 2.94 (d, J = 16.55 Hz, 1H, H-6a), 3.43 (d, J = 16.55 Hz, 1H,
H-6b), 3.69 (d, J = 15.25 Hz, 1H, H-8a), 3.76 (d, J = 15.25 Hz, 1H,
H-8b), 5.92 (s, 1H, H-5), 5.99 (s, 1H, H-2), 6.84–7.76 (m, 12H, H-
aromatic, H-12). 13C NMR (125 MHz, CDCl3): dC 52.38, 52.81,
61.86, 100.58, 108.74, 123.15, 126.17, 128.09, 128.65, 128.71,
128.96, 129.27, 129.39, 129.49, 130.17, 130.63, 131.77, 133.68,
134.03, 134.10, 135.53, 135.82, 137.48, 139.53, 162.47.
for C29H25N3OS: C, 75.13; H, 5.44; N, 9.06; Found: C, 74.22; H,
5.09; N, 9.21; 1H NMR (500 MHz, CDCl3): dH 2.54 (br s, 1H,
NH), 3.10 (d, J = 17.28 Hz, 1H, H-6a), 3.32 (d, J = 17.28 Hz, 1H,
H-6b), 3.81 (d, J = 15.12 Hz, 1H, H-8a), 3.84 (s, 3H, methoxy),
3.95 (d, J = 15.12 Hz, 1H, H-8b), 5.42 (s, 1H, H-5), 5.89 (s, 1H,
H-2), 6.74-7.47 (m, 13H, H-aromatic), 7.54 (s, 1H, H-12). 13C
NMR (125 MHz, CDCl3): dC 46.26, 46.55, 55.77, 61.42, 99.89,
111.47, 114.14, 123.25, 124.50, 126.77, 126.93, 128.49, 128.67,
128.93, 129.74, 130.55, 132.06, 134.55, 137.81, 139.07, 141.52,
160.58, 162.93.
4.1.1.15. (E)-9-(2-Methoxybenzylidene)-5-(2-methoxyphenyl)-
3-(4-chlorophenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazol-
4.1.1.20. (E)-9-(2-Chlorobenzylidene)-5-(2-chlorophenyl)-3-(4-
methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,
o[3,2-a]pyrimidine (6o).
Yellow solid; yield: 81%, mp 198–
201 °C; Anal. Calcd for C30H26ClN3S: C, 68.23; H, 4.96; N, 7.96;
Found: C, 68.15; H, 4.75; N, 7.82; 1H NMR (500 MHz, CDCl3): dH
1.91 (br s, 1H, NH), 3.07 (d, J = 17.15 Hz, 1H, H-6a), 3.28 (d,
J = 17.15 Hz, 1H, H-6b), 3.72 (s, 3H, O–CH3), 3.81 (s, 3H, O–CH3),
3.84 (d, J = 15.48 Hz, 1H, H-8a), 3.92 (d, J = 15.48 Hz, 1H, H-8b),
5.32 (s, 1H, H-5), 5.92 (s, 1H, H-2), 6.63–7.33 (m, 12H, H-aromatic),
2-a]pyrimidine (6t).
Yellow solid; yield: 76%, mp 172–
174 °C; Anal. Calcd for C29H23Cl2N3OS: C, 65.41; H, 4.35; N, 7.89;
Found: C, 65.12; H, 4.12; N, 8.17 1H NMR (500 MHz, CDCl3): dH
2.59 (br s, 1H, NH), 3.28 (d, J = 16.45 Hz, 1H, H-6a), 3.64 (d,
J = 16.45 Hz, 1H, H-6b), 3.69 (s, 3H, methoxy), 3.78 (s, 3H, meth-
oxy), 3.83 (s, 3H, methoxy) 3.86 (d, J = 15.92 Hz, 1H, H-8a), 4.30

A. Basiri et al. / Bioorg. Med. Chem. 22 (2014) 906–916
915
(d, J = 15.92 Hz, 1H, H-8b), 5.51 (s, 1H, H-5), 5.94 (s, 1H, H-2), 6.58-
7.27 (m, 11H, H-aromatic), 7.61 (s, 1H, H-12). 13C NMR (125 MHz,
CDCl3): dC 43.59, 55.23, 55.26, 55.41, 60.29, 100.38, 113.89, 114.05,
114.16, 114.33, 122.39, 126.78, 127.68, 128.16, 128.46, 130.37,
130.70, 132.57, 138.93, 159.03, 159.77, 160.35, 163.17.
Galanthamine was used as reference standard. Solutions of test
samples and galanthamine were prepared in DMSO at an initial
concentration of 1 mg/mL (1000 ppm). The concentration of DMSO
in final reaction mixture was 1%. At this concentration, DMSO has
no inhibitory effect on both AChE and BChE enzymes.42
For acetylcholinesterase (AChE) inhibitory assay, 140
0.1 M sodium phosphate buffer of pH 8 was first added to a
96-wells 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 of 14 mM
acetylthiocholine iodide. Absorbance of the colored end-product
was measured using BioTek PowerWave 340 Microplate
Spectrophotometer at 412 nm for 30 min after the initiation of
enzymatic reaction. For butyrylcholinesterase (BChE) inhibitory
assay, the same procedure described above was followed, except
for the use of enzyme and substrate, instead of which, butyrylcho-
line esterase from equine serum and S-butyrylthiocholine chloride
were used.
lL of
4.1.1.21. (E)-9-(2-Methoxyphenylbenzylidene)-5-(2-methoxyl-
phenyl)-3-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,
l
lL
3-d]thiazolo[3,2-a]pyrimidine (6u).
Yellow solid; yield:
79%, mp 132–135 °C; Anal. Calcd for C31H29N3O3S: C, 71.10; H,
5.58; N, 8.02; Found: C, 71.54; H, 6.17; N, 8.72; 1H NMR
(500 MHz, CDCl3): dH 2.49 (br s, 1H, NH), 3.46 (d, J = 17.49 Hz,
1H, H-6a), 3.54 (d, J = 17.49 Hz, 1H, H-6b), 3.72 (d, J = 16.07 Hz,
1H, H-8a), 3.80 (s, 3H, methoxy), 3.86 (d, J = 16.07 Hz, 1H, H-
8b), 5.61 (s, 1H, H-5), 5.97 (s, 1H, H-2), 6.75-7.36 (m, 11H, H-aro-
matic), 7.69 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC 46.72,
46.93, 55.79, 61.56, 101.22, 111.52, 114.23, 123.39, 124.57,
126.82, 127.12, 128.54, 128.75, 129.11, 129.78, 130.69, 132.17,
134.63, 137.92, 139.18, 141.62, 160.71, 162.97.
l
lL
X
Each test was conducted in triplicate. Absorbance of the test
samples was corrected by subtracting the absorbance of their
respective blank. Percentage inhibition was calculated using the
following formula:
4.1.1.22. (E)-9-(4-Methylbenzylidene)-5-(4-methylphenyl)-3-(4-
methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,
2-a]pyrimidine(6v).
Yellow solid; yield: 84%, mp 158–
161 °C; Anal. Calcd for C31H29N3OS: C, 75.73; H, 5.95; N, 8.55;
Found: C, 76.49; H, 6.64; N, 9.05; 1H NMR (500 MHz, CDCl3): dH
2.24 (s, 3H, CH3), 2.35 (s, 3H, CH3), 3.11 (d, J = 16.99 Hz, 1H, H-
6a), 3.32 (d, J = 16.99 Hz, 1H, H-6b), 3.79 (d, J = 14.73 Hz, 1H, H-
8a), 3.86 (s, 3H, methoxy), 4.02 (d, J = 14.73 Hz, 1H, H-8b), 5.41
(s, 1H, H-5), 5.89 (s, 1H, H-2), 6.58–7.14 (m, 11H, H-aromatic),
7.51 (s, 1H, H-12). 13C NMR (125 MHz, CDCl3): dC 21.55, 21.62,
45.50, 45.68, 55.79, 61.07, 100.01, 109.67, 114.17, 123.22, 125.53,
126.65, 129.40, 129.68, 130.62, 134.45, 137.05, 138.39, 138.58,
139.20, 160.60, 163.15.
Absorbance of controlꢀAbsorbance of Sample
Percentage of inhibition ¼
Absorbance of control
ꢁ100:
4.3. Molecular docking studies
Using Glide™, (version 5.7, Schrödinger, LLC, New York, NY,
2011), the most active compound, 6d, were docked onto the
active site of hAChE derived from the crystal structures of the
enzyme complexed with the anti-Alzheimer’s drug, galanthamine
(PDB ID: 4EY6) and to hBChE derived from the crystal structure of
the complex of the enzyme with its substrate BCh (PDB code:
1P0P).
4.1.1.23. (E)-9-(4-Chlorobenzylidene)-5-(4-chlorophenyl)-3-(4-
methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,
2-a]pyrimidine(6w).
Yellow solid; yield: 87%, mp 169–
171 °C; Anal. Calcd for C29H23Cl2N3OS: C, 65.41; H, 4.35; N, 7.89;
Found: C, 65.12; H, 4.71; N, 8.21; 1H NMR (500 MHz, CDCl3): dH
2.41 (br s, 1H, NH), 3.39 (d, J = 16.65 Hz, 1H, H-6a), 3.67 (d,
J = 16.65 Hz, 1H, H-6b), 3.77 (d, J = 14.65 Hz, 1H, H-8a), 3.85 (s,
3H, methoxy), 4.29 (d, J = 14.65 Hz, 1H, H-8b), 5.60 (s, 1H, H-5),
5.99 (s, 1H, H-2), 6.60–7.29 (m, 11H, H-aromatic), 7.58 (s, 1H, H-
12). 13C NMR (125 MHz, CDCl3): dC 43.57, 55.12, 55.32, 55.67,
60.31, 100.07, 113.87, 114.17, 114.25, 114.33, 122.51, 126.79,
127.82, 128.22, 128.54, 130.49, 130.71, 132.62, 140.11, 159.27,
159.85, 160.19, 163.24.
Water molecules and hetero groups were deleted from the en-
zyme beyond the radius of 5 Å of the reference ligand (galantha-
mine or BCh), and the resulting protein structure refined and
minimized by Protein Preparation Wizard™ using the OPLS-
2005 force field. The Receptor Grid Generation program was used
to prepare hAChE and hBChE grid and all the ligands were opti-
mized by LigPrep™ using the OPLS-2005 force field to generate
the lowest energy state of the respective ligands. Docking stimu-
lations were carried out on bioactive compounds to generate 5
poses per ligand, and the best pose (with the highest score) dis-
played for each.
4.1.1.24. (E)-9-(4-Fluorobenzylidene)-5-(4-fluorophenyl)-3-(4-
methoxyphenyl)-6,7,8,9-tetrahydro-5H-pyrido[4,3-d]thiazolo[3,
Acknowledgments
2-a]pyrimidine (6x).
Yellow solid; yield: 83%, mp 212–
We would like to thank the Malaysian Government and Univer-
siti Sains Malaysia (USM) for the Research University Grant (1001/
PFARMASI/813031). A.B. is supported by the USM Graduate Assis-
tant Scheme from the Institute for Postgraduate Studies (IPS) of
Universiti Sains Malaysia.
215 °C; Anal. Calcd for C29H23F2N3OS: C, 69.72; H, 4.64; N, 8.41;
Found: C, 70.72; H, 4.19; N, 8.21; 1H NMR (500 MHz, CDCl3): dH
2.05 (br s, 1H, NH), 3.05 (d, J = 17.35 Hz, 1H, H-6a), 3.28 (d,
J = 17.35 Hz, 1H, H-6b), 3.35 (d, J = 16.20 Hz, 1H, H-8a), 3.71 (d,
J = 14.20 Hz, 1H, H-6b), 3.85 (s, 3H, methoxy), 5.40 (s, 1H, H-5),
5.89 (s, 1H, H-2), 6.67–7.47 (m, 11H, H-aromatic), 7.80 (s, 1H, H-
12). 13C NMR (125 MHz, CDCl3): dC 46.10, 52.65, 55.40, 60.24,
99.63, 108.59, 113.61, 113.84, 114.93, 115.10, 115.43, 122.74,
124.05, 128.12, 130.09, 130.30, 130.84, 132.04, 135.55, 134.33,
137.05, 138.52, 160.29, 163.58.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.020.
4.2. In vitro cholinesterase enzymes inhibitory assay
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