Microwave assisted synthesis, cholinesterase enzymes inhibitory activities and molecular docking studies of new pyridopyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2013.03.058

A series of hitherto unreported pyrido-pyrimidine-2-ones/pyrimidine-2- thiones were synthesized under microwave assisted solvent free reaction conditions in excellent yields and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibitory activity. Among the pyridopyrimidine derivatives, 7e and 7l displayed 2.5- and 1.5-fold higher enzyme inhibitory activities against AChE as compared to standard drug, galanthamine, with IC₅₀ of 0.80 and 1.37 μM, respectively. Interestingly, all the compounds except 6k, 7j and 7k displayed higher inhibitory potential against BChE enzyme in comparison to standard with IC₅₀ ranging from 1.18 to 18.90 μM. Molecular modeling simulations of 7e and 7l was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) and human butyrylcholinesterase (hBChE) enzymes to disclose binding interaction and orientation of these molecule into the active site gorge of respective receptors.

Full text of DOI:10.1016/j.bmc.2013.03.058

Bioorganic & Medicinal Chemistry 21 (2013) 3022–3031
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microwave assisted synthesis, cholinesterase enzymes inhibitory
activities and molecular docking studies of new pyridopyrimidine
derivatives
b
c,
b
Alireza Basiri a, Vikneswaran Murugaiyah a, , Hasnah Osman , Raju Suresh Kumar , Yalda Kia ,
⇑
⇑
Mohamed Ashraf Ali 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 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:
A series of hitherto unreported pyrido-pyrimidine-2-ones/pyrimidine-2-thiones were synthesized under
microwave assisted solvent free reaction conditions in excellent yields and evaluated in vitro for their
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibitory activity. Among the
pyridopyrimidine derivatives, 7e and 7l displayed 2.5- and 1.5-fold higher enzyme inhibitory activities
Received 20 February 2013
Revised 19 March 2013
Accepted 21 March 2013
Available online 1 April 2013
against AChE as compared to standard drug, galanthamine, with IC50 of 0.80 and 1.37
Interestingly, all the compounds except 6k, 7j and 7k displayed higher inhibitory potential against BChE
enzyme in comparison to standard with IC50 ranging from 1.18 to 18.90 M. Molecular modeling simu-
lM, respectively.
Keywords:
l
Microwave assisted synthesis
Pyridopyrimidines
AChE and BChE activity
Molecular docking
lations of 7e and 7l was performed using three-dimensional structure of Torpedo californica AChE
(TcAChE) and human butyrylcholinesterase (hBChE) enzymes to disclose binding interaction and orienta-
tion of these molecule into the active site gorge of respective receptors.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
junction.6 Commercially available drugs such as galanthamine,
donepezil, rivastigmine and tacrine showed positive results in
According to the World Alzheimer report 2012, Alzheimer’s dis-
ease (AD) is among the most significant social, health and econom-
symptomatic improvements of mild to moderate AD patients.7
Donepezil and galanthamine being most selective for AChE while
rivastigmine inhibit AChE and BChE at the same extent and tacrine
shows lower selectivity for AChE than BChE.8
ical crisis of the 21st century.1 AD is
a complex disease
characterized by accumulation of b-amyloid (Ab) plaques and neu-
rofibrillary tangles composed of tau amyloid fibrils, associated
with synapses loss and neuro-degeneration leading to impairment
of memory and other cognitive dysfunctions.2 The cognitive deficit
is thought to be due to loss of cholinergic neurons in basal fore-
brain.3 One of the pharmacological approaches to restore choliner-
gic function is by blocking the breakdown of neurotransmitter in
the cholinergic neurons, acetylcholine (ACh), leading to the use
of cholinesterase inhibitors to treat AD.4
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
enzymes are involved in the breakdown of acetylcholine in the
brain and inhibition of these enzymes may increase the efficacy
of treatment and broaden the indications.5 Effects of cholinesterase
inhibitors are mainly due to enhancement of cholinergic transmis-
sion at cholinergic autonomic synapses and at the neuromuscular
The recent development of inhibitors includes drugs with high
selectivity for BChE, which also showed enhancement of ACh levels
in rats brain.9 BChE inhibitors were also found to reduce amyloid
precursor protein levels in animals with a cholinergic lesion in
the forebrain.10 These agents represent an additional advantage
for long-term stabilization of cognitive and behavioral symptoms
in patients with advanced AD.11 A selective BChE inhibitor, may
produce significant increase in brain ACh levels without triggering
severe peripheral or central cholinergic adverse effects.8
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 candi-
dates to the active site of their target enzymes.12 AChE’s active
site is located on the bottom of a long and narrow gorge, acetylcho-
line and substrate guidance down the gorge is facilitated by cat-
ion–
p interactions with aromatic side-chains residues such as
⇑
phenylalanine, tryptophan and tyrosine lining gorge wall.13 The
overall structure of human BChE is very similar to that of AChE
Corresponding authors. Tel./fax: +60 46534583.
E-mail addresses: vicky@usm.my (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.2013.03.058

A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
3023
1
2
+
4
+
Solid NaOEt
Heat or MWI
O
Ar
Ar
H2N
X
H
HCl.HN
X=O,S
O
N
X
4
NH2
(i) HCl, CH3COOH
1
+
HN
HN
NH
(ii) K2CO3
Solid NaOEt
Heat or MWI
Acetone/water
2 ArCHO
Ar
Ar
2
6: X=O
7: X=S
3
Scheme 1. Synthesis of 6a–l and 7a–l.
from Torpedo California. Most differences between BChE and
TcAChE are confined to the residues composing the gorge, whereby
in BChE it is replaced with hydrophobic Leu286 and Val288. These
changes make it possible for the binding of bulkier butyrate sub-
strate moiety and inhibitors in BChE.14
was filtered and dried in vacuo to afford the pyridopyrimidine-2-
thiones (7c) in 72% yield (Method A). The product obtained by this
method doesn’t require further purification as it is evident from
TLC and 1H NMR spectra. The same reaction was conducted under
microwave irradiation condition (Method B), as this method is
evolved as an alternative to conventional heating. Interestingly,
the reaction was completed in just 30 seconds affording the prod-
uct in excellent yield (95%) with high purity.
Natural and synthetic biologically active compounds with pyri-
midinone moiety, find applications in pharmaceutical and bio-
chemical fields15 as antihypertensive,16
a1a-adrenergic receptor
antagonists,17 antibacterial, anti-inflammatory and antitumor
agents.18 Compounds which constitute the core structural ele-
ments of pyrimidinone are commonly present in the polycyclic
marine alkaloids which show anti cancer, anti HIV and cytotoxic
properties.19–21 Inspired by the aforementioned significance of
pyrimidinone derivatives, in the present study two series of pyri-
midinone derivatives were synthesized and their cholinesterase
inhibitory activities were explored. Further molecular modeling
was performed on the most active compounds to understand the
possible reasons for their selectivity.
Alternatively, an attempt was made to synthesize the pyrido-
pyrimidine derivatives through one pot pseudo four-component
reaction of 4-piperidone hydrochloride monohydrate (1), 4-meth-
ylbenzaldehyde (2) and thiourea in a molar ratio 1:2:1, respec-
tively in presence of catalytic amount of solid sodium ethoxide
(Method C). The reaction mixture in a semi-micro boiling tube
was heated on a water bath with constant grinding. The reaction
took quite a longer time (10 min) than the earlier methods (Meth-
od A and B) for completion, resulting a yellow viscous liquid in 61%
yield. 1H NMR analysis of this liquid showed the presence of pyri-
dopyrimidine along with some non-characterizable impurities. In
order to reduce the impurities and to lessen the reaction time,
the same pseudo four-component reaction was conducted under
microwave irradiation condition (method D). As expected the reac-
tion was completed in 3–4 min affording the product along with
some impurities as method C. The above results disclose that a
maximum yield of pyridopyrimidine derivative was obtained with
high purity when the reaction was performed under microwave
irradiation method.
2. Results and discussion
2.1. Chemistry
In the present investigation, the reaction of a series of N-unsub-
stituted dibenzylidenepiperidine-4-ones with urea/thiourea in
presence of catalytic amount of solid sodium ethoxide afforded
the functionalized hitherto unreported pyridopyrimidine-2-ones
(6)/pyridopyrimidine-2-thiones (7) in excellent yields (Scheme 1).
The N-unsubstituted dibenzylidenepiperidine-4-ones (3) required
for the present study was synthesized following the literature re-
ported method by Dimmock et al.22
In order to find the optimal, efficient, eco-friendly and handy
protocol for the synthesis of pyridopyrimidine derivatives, the
reaction was examined under different conditions by choosing
the model reaction between an equimolar ratio of 3,5-bis(4-meth-
ylbenzylidene)piperidin-4-one and thiourea in the presence of cat-
alytic amount of solid sodium ethoxide. Initially, the neat reaction
mixture in a semi micro boiling tube was heated in a water bath,
for about 4–5 min during which period a transparent viscous liquid
formed showing completion of reaction and double examined by
TLC. Water (50 mL) was added to the mixture, precipitated solid
Structural elucidation of the pyrido-pyrimidine-2-ones (6)/
pyrimidine-2-thiones (7) was accomplished by FT-IR, 1D and 2D
Cl
Cl
6.60, s
119.41
3.51, d
3.66, d
J =15.09 Hz
H
H
9
8
45.79
8.08, s
H
H
N
H7
8a
N
S
S
H
1
2
174.35
HN6
H
3 NH
HN
NH
112.54
4a
2.12, brs
5
4
7.65, s
H
H
H
3.04, d
3.28, d
Cl
Cl
J=17.90 Hz
45.05
7.07-7.45
aromatic
5.47, s
54.78
Figure 1. Selected HMBCs and 1H and 13C chemical shifts of 7c.
Ar
Ar
Ar
Ar
H
N
H2N
X
H2N
O
N
X
X
O
X
NH2
-H2O
HN
HN
NH
HN
NH
HN
NH
H
Ar
Ar
Ar
Ar
6/7
Scheme 2. Mechanism for the formation of novel pyridopyrimidines 6/7.

3024
A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
NMR spectroscopy techniques and elemental analysis. The IR spec-
trum of 7c revealed the presence of NH and C@S around 3400 and
1650 cmꢀ1, respectively. In the 1H NMR spectrum of 7c, the singlet
at 5.47 ppm was readily assigned to H-4, which shows H,H-COSY
correlations with the doublets at 3.04 and 3.28 ppm
(J = 17.90 Hz) enabling their assignment to H-5a and H-5b. The
other doublets at 3.51 and 3.66 ppm (J = 15.09 Hz) can be assigned
to H-7a and H-7b. The one hydrogen singlet at 6.60 ppm is due to
H-9 and two singlets at 7.65 and 8.08 ppm is due to two NH hydro-
gens while the aromatic hydrogens appeared as multiplets in the
region 7.07–7.45 ppm. The assignment of signals of carbon bearing
hydrogens has been done from the chemical shifts of hydrogens
and C,H-COSY correlations. These assignments are also supported
by the HMBCs (Fig. 1) of 7c. The 1H and 13C chemical shifts of 7c
are depicted in Figure 1. The probable mechanism for the forma-
tion of pyridopyrimidine derivatives (6)/(7) via Michael addition-
condensation-tautormerization domino sequence is shown in
Scheme 2.
Comparing AChE inhibitory activities of 6a–l, comprising C@O
and 7a–l comprising C@S moieties disclosed that all derivatives
of 7, displayed better inhibitory activities than 6 except for 7c,
7d and 7f which showed 1.3-, 2.2- and 1.2-fold lower activities
contrasting to 6c, 6d and 6f. It is worth to note that, compounds
7e, 7i and 7l, significantly represented 50, 16 and 10 times more
AChE inhibitory activity than their analogous 6e, 6i and 6l, respec-
tively. Remaining compounds in series 7, also displayed 1.01–1.33
times higher inhibitory potential comparing to their resembling
derivatives in series 6 as depicted in Figure 2. Lower AChE inhibi-
tory activities observed in pyrimidinones (6) comparing to pyrim-
idinethiones (7) are probably attributable to hydrogen bonding
interactions of electronegative oxygen atom with amino acid resi-
dues located at the entrance of AChE active site gorge which pre-
vents the proper insertion and accommodation of inhibitors
inside the gorge which is essential to expose the enzyme inhibitory
activity. In series 7, owing to displacement of oxygen atom with
2.2. In vitro cholinesterase enzymes inhibitory activity
The cholinesterase enzymes inhibitory activities of the synthe-
sized compounds were summarized in Table 1. Among the twenty
four newly synthesized compounds, pyridopyrimidine-2-thiones
7e with ortho-methoxy and 7l with 1-naphthyl groups on the aryl
ring displayed 2.5- and 1.5-fold higher inhibitory potential against
AChE in contrast to galanthamine with IC50 = 0.8 and 1.37 lM,
respectively. In addition, compound 7i, possessing para-chloro
substituent on the aryl ring, also showed comparable AChE inhib-
itory potency to standard (IC50 = 2.25 lM).
Higher potency of 7e and 7l in contrast to standard presumably
is related to high aromatic content of active site gorge in AChE en-
zyme. Containing electron rich aromatic cores, viz. Compound 7e
(R = ortho-methoxy) or possessing more aromatic core units as
for 7l (R = 1-naphtyl) facilitate insertion/accommodation of com-
pound in the active site gorge of AChE by interactions with aro-
matic side chains residues that line the gorge wall.
Figure 2. AChE inhibitory activity comparison diagram (IC50 vs substitution on
aromatic ring) for 6a–l and 7a–l.
Table 1
Physical data, AChE and BChE activities of 6(a–l) and 7(a–l)
Entry
Compound
Ar
Yield (%)
AChE inhibition (IC50
)
BChE inhibition (IC50
)
Selectivity
Conv.
MWI
(l
g/ml) M)
(
l
(l
g/ml) M)
(
l
AChEa
BChEb
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
C6H5
78
75
72
69
72
75
71
69
72
77
69
74
73
72
72
72
79
69
75
74
72
76
68
67
—
94
91
90
89
93
91
88
90
92
94
91
92
94
85
95
89
94
88
93
95
92
90
91
92
—
21.79 0.22
13.94 0.14
9.28 0.16
6.56 0.17
15.17 0.11
11.51 0.12
7.49 0.15
16.05 0.15
14.24 0.21
15.61 0.20
13.59 0.22
5.49 0.21
6.42 0.14
14.36 0.21
13.02 0.17
14.91 0.23
0.32 0.05
14.52 0.24
5.80 0.18
13.51 0.21
1.14 0.19
13.76 0.25
12.27 0.16
0.59 0.024
0.60 0.012
68.73
40.42
24.14
18.59
40.23
28.27
15.86
46.52
36.84
44.23
29.86
13.16
19.27
39.72
32.39
40.43
0.80
34.31
11.88
37.47
2.25
37.22
26.25
1.37
0.91 0.11
1.80 0.17
0.97 0.21
1.17 0.12
4.04 0.11
4.21 0.24
2.94 0.17
2.45 0.14
6.46 0.19
6.67 0.21
10.77 0.18
2.25 0.11
1.26 0.14
1.11 0.08
1.17 0.09
2.40 0.10
0.46 0.05
3.42 0.21
0.81 0.06
2.27 0.15
2.52 0.10
10.64 0.16
18.38 0.22
2.42 0.17
5.55 0.01
2.86
5.22
2.51
3.31
10.70
10.34
6.22
7.09
16.73
18.90
23.51
5.39
3.78
3.07
2.91
6.50
1.18
8.09
1.65
6.27
6.26
28.82
49.8
5.58
19.34
0.04
0.13
0.10
0.18
0.27
0.37
0.39
0.15
0.45
0.43
0.79
0.41
0.20
0.08
0.09
0.16
1.47
0.24
0.14
0.17
2.78
0.77
1.50
4.07
3.47
24.01
7.74
9.57
5.61
3.75
2.73
2.54
6.56
2.20
2.34
1.26
2.44
5.09
12.94
11.13
6.21
0.67
4.25
7.20
5.95
0.35
1.29
0.67
0.25
0.28
2-CH3C6H4
2-ClC6H4
2-FC6H4
2-(OCH3)C6H4
3-(NO2)C6H4
4-BrC6H4
4-CH3C6H4
4-ClC6H4
4-FC6H4
2,4-Cl2C6H3
Naphthyl
C6H5
2-CH3C6H4
2-ClC6H4
2-FC6H4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-(OCH3)C6H4
3-(NO2)C6H4
4-BrC6H4
4-CH3C6H4
4-Cl2C6H4
4-FC6H4
2,4-Cl2C6H3
Naphthyl
Galanthamine
2.09
a
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE).
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE).
b

A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
3025
more polarizable sulfur atom, these unfavorable hydrogen bonding
interactions are avoided and inhibitors are relatively better en-
grafted into the active site gorge of the enzyme resulting in higher
inhibitory potencies.
ortho-methoxy phenyl derivatives, 7e and 6e, with IC50 = 0.8 versus
40.23 M, for AChE and IC50 = 1.18 versus 10.70 M, for BChE,
respectively.
l
l
In conclusion, newly synthesized pyridopyrimidines 6 and 7
owing to their planar spatial configuration which is necessary for
proper insertion into the enzymes active site and high aromatic
content to facilitate accommodation of inhibitor inside the gorge
through hydrophobic interaction with aromatic residues lining
the active site, represented their inhibitory activity and therefore
are good candidates to be used as cholinesterase inhibitor agents.
Among 24 synthesized compounds, 7e and 7l displayed 2.5 and
1.5 times more AChE inhibitory potential compared to standard
drug. It can also be noted that most of the pyridopyrimidines
tested in our study were found to be 2–16 times more active and
selective toward BChE in contrast to galanthamine which among
them compounds 7e and 7g showed 16 and 12 time more activity
than standard. Selective BChE may be useful in ameliorating a cho-
linergic deficit in AD due to increased BChE activity and its involve-
ment in formation and maturation of b-amyloid plaques.9,23
As for BChE, interestingly, all the synthesized pyridopyrimi-
dines represented selectivity toward BChE and also displayed bet-
ter BChE inhibitory potential in comparison to standard drug
except for 6k, 7j and 7k. Analyzing IC50 data represented in Table 1
disclosed that un-substituted and ortho-substituted derivatives in
these two series demonstrated better BChE inhibitory activities
in contrast to their meta para and di-substituted analogous. Addi-
tionally, compound 6a, showed remarkable selectivity for BChE
with 24 times more tendency toward this enzyme compared to
AChE.
The replacement of C@O in series 6 with C@S moiety in series 7,
resulted in relatively better BChE inhibitory activity with ten com-
pounds displaying IC50 lower than 10 lM in 7 contrasting to seven
compounds in 6. Compound 6a, 6c and 6d, relatively displayed bet-
ter inhibitory activity compared to their analogous 7a, 7c and 7d.
On the other hand, meta- and para-phenyl substituted 7e–i,
showed better inhibitory activity in contrast to their analogous in
series 6. Compounds 7e and 7g also displayed 9 and 4 times better
activities than 6e and 6g, respectively. Compound 6j with para-flu-
oro phenyl and 6k with ortho-, para-dichloro phenyl rings, rela-
tively displayed higher potency compared to 7j and 7k (Fig. 3).
As mentioned earlier, unlike AChE, BChE active site wall is lined
by hydrophobic residues such as leucine and valine. These changes
provide more room for insertion/accommodation of substrates and
inhibitors inside the BChE active site. Based on this principle,
replacement of oxygen with sulfur atom in C@X moiety, does not
ensue remarkable difference in inhibitory activities among similar
derivatives in contrary to AChE enzyme. In both assays the maxi-
mum difference in activities among two series were observed for
2.3. Docking simulation
In order to gain functional and structural insight into the mech-
anism of most active compounds, molecular docking simulation
was performed by the aid of Maestro™ docking software. Active
sites in TcAChE and human BChE due to different roles can be clas-
sified into five regions as mentioned in the introduction part. Ami-
no acid residue compositions of these five sites for both enzymes
are summarized in Table 2.
Molecular docking simulations were performed on TcAChE
receptor for compound 7e and 7l which displayed superior
AChE inhibitory activity in comparison to galanthamine and on
hBChE receptor for compound 7e which possessed highest BChE
inhibitory activity among the newly synthesized pyridopyrimi-
dines. This simulation may assist to reveal binding orientation
and interaction of these molecules with amino acid residues com-
posing active site gorge in these two enzymes. The interaction
sites, residue involved and bonding types as well as the ligand
interacting moiety are summarized in Table 3.
Molecular modeling analysis for 7e indicated that hydrogen
bonding, hydrophobic and mild polar interactions are three major
interactions incorporating the attachment of this ligand to TcAChE
receptor. In brief, 7e at peripheral anionic site, represented hydro-
phobic interaction with Trp279, Tyr70, Tyr121 and Tyr334, at acyl
binding pocket displayed hydrophobic interaction with Phe288
and Phe290 and at oxyanion hole it bound to Gly118 and Gly119
by mild polar interactions. Two
action (2.334 Å) with Phe330 together with
p
–
p
stacking and H-bonding inter-
stacking interac-
p–p
tion with Trp84 at choline binding site, anchored this compound
to the bottom of TcAChE active site gorge. In addition to interaction
with residues composing peripheral anionic site, oxanion hole, acyl
binding pocket and choline binding site, this compound also dis-
played mild polar interactions with Glu199, Ser200 and His440
amino acid residues composing catalytic triad. Interaction of 7e
with all 5 major sites of active site gorge may be a good explana-
tion for its remarkable AChE inhibitory activity against this enzyme
Figure 3. BChE inhibitory activity comparison diagram (IC50 vs substitution on
aromatic ring) for 6a–l and 7a–l.
Table 2
Residue composition of active sites in TcAChE and hBChE
Entry
Site name
Residue composition in TcAChE28,29
Residue composition in human BChE14
1
2
3
4
5
Catalytic triad
Ser 200, His 440 & Glu 327
Trp 84 & Phe 330
Phe 288 & Phe 290
Gly118, Gly119 & Ala201
Tyr 70, Asp 72, Tyr 121, Trp 279 & Tyr 334
His438, Ser198 & Glu325
Trp82 & Phe329
Trp230, Leu286 & Val288
Gly116, Gly117 & Ala199
Trp 231, Leu 286 & Phe 398
Choline binding site (
Acyl-binding pocket
Oxyanion hole
a-anionic site)
Peripheral anionic site (b-anionic site)

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A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
Table 3
Binding interaction data for 7e and 7l docked into active site gorge of AChE/7e docked into BChE receptors
Entry Ligand Enzyme Binding energy (kcal) Interacting site
Residue
Bond type
Residue interacting moiety Ligand interacting moiety
1
2
3
7e
TcAChE
TcAChE
hBChE
ꢀ10.1
ꢀ8.3
ꢀ8.7
PAS*
Trp 279
Tyr 70
Tyr 334
Tyr 121
Hydrophobic Indole
Hydrophobic Aryl ring
Hydrophobic Aryl ring
Hydrophobic Methoxy
Nitrogen
Hydrophobic Aryl ring
Ring 4
Methoxy ring 4
Methoxy ring 4
Arylidene, methoxy ring 1
Ring 1 & 2
Ring 4, Methoxy ring 1
Nitrogen ring 3
Ring 1, 2 & 3
Ring 1
OH**
Gly 117 & 118 Mild polar
Acyl binding pocket Phe 290
Choline binding site Phe 330
H-bonding,
C@O
p
p
–
–
p
p
stacking Aryl ring
stacking Indole
Trp 84
His 440
CT***
PAS
Mild polar
Nitrogen
Ring 1
7l
Tyr 70
Tyr 121
Tyr 334
Gly 117 & 118 Mild polar
Ala 201 Mild polar
Hydrophobic Aryl ring
Hydrophobic Aryl ring
Hydrophobic Aryl ring
Ring 4
Naphthyl
Ring 4
Ring 1 & naphthyl
Naphthyl
Naphthyl
Nitrogen ring 3
Ring 1
OH
Nitrogen
Nitrogen
Acyl binding pocket Phe 290
Choline binding site Phe 330
Trp 84
Hydrophobic Aryl ring
H-bonding C@O
stacking Indole
p–p
CT
His 440
Ser 200
Mild polar
Mild polar
Nitrogen
Methoxy
Ring 1
Naphthyl
7e
PAS
Trp 231
Phe 398
Val 288
Leu 286
Hydrophobic Indole
Hydrophobic Aryl ring
Hydrophobic Methyl
Hydrophobic CH2 & CH3
Ring 4
Methoxy
Ring 4
Ring 4
OH
Gly 116 & 117 Mild polar
Ala 199 Mild polar
Nitrogen
Nitrogen
Ring 2 & 3
Methoxy
Ring 2
Choline binding site Phe 329
Trp 82
Hydrophobic Aryl ring
Hydrophobic Indole
Ring 1 & Methoxy
*
Peripheral anionic site.
Oxyanion hole.
Catalytic triad.
**
***
R
4
H
H
N
X
2
3
HN
NH
1
R
in contrast to standard drug. AChE structure complexed with
available Alzheimer’s drugs such as huperzine A, galanthamine
and tacrine also shows similar interactions of occupying of the
peripheral anionic site, stacking against Trp 84 and interacting
with residues at catalytic triad (Fig. 4).24
Docking profile of compound 7l closely resembles to 7e, in
which at peripheral anionic site it bound to Tyr70, Tyr121,
Tyr334 and Trp279 residues through hydrophobic interactions
and strongly anchored to choline binding site through hydropho-
bic and hydrogen bonding (2.30 Å) interactions with Phe330 be-
In conclusion, information gathered from docking simulation
analysis for 7e and 7l were in accordance with the IC50 values ob-
tained from the cholinesterase inhibition assay. In general, better
accommodation of inhibitor inside the active site gorge due to more
appropriate interactions with its amino acid residues, ensue better
inhibitory activities and lower IC50 values during In vitro assay.
3. Conclusion
In conclusion a series of pyridopyrimidine derivatives were syn-
thesized employing a facile microwave irradiation protocol and
were assayed for their AChE and BChE activities. Compound 7e dis-
played the highest inhibition for both AChE and BChE enzymes
side
p–p stacking interaction with Trp84. This compound also
showed mild polar interaction with Gly188, Gly119 and Ala201
at oxyanion hole and Ser200 and His440 at catalytic triad. As
mentioned earlier these key interactions are also observable in
AChE crystal structure complexed with available Alzheimer’s
drugs (Fig. 5).
Docking of compound 7e on hBChE receptor showed hydropho-
bic interaction with Trp231, Phe398, Val288 and Leu286 at periph-
eral anionic site beside Phe329 and Trp82 at choline binding site
together with mild polar interactions with Gly 116, Gly 117 and
Ala 199 at oxyanion hole. This compound also attached to side
chain residues such as Tyr 332 and Trp 430 at the bottom of the
gorge (Fig. 6).
with IC50 of 0.8 and 1.18
played higher inhibitory potential for AChE in comparison to galan-
thamine with IC50 = 1.37 M. 21 out of 24 compounds in this
series, represented higher inhibitory activities toward BChE in con-
trast to galanthamine ranging from 1.18 to 18.90 M. Molecular
lM, respectively. Compound 7l also dis-
l
l
docking of 7e and 7l to TcAChE and hBChE receptors completely
coincided with the activities observed. The presence of NH, C@S/
O and C@C functionalities in these pyridopyrimidine derivatives
makes it a valuable synthon for the construction of more complex
heterocycles of biological importance.

A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
3027
Figure 4. Binding interaction of 7e with active site residues of TcAChE receptor.
examined by TLC. Water was added to the mixture, precipitated so-
lid was filtered and dried in vacuo to afford compounds 6a–l and
7a–l in excellent yields (Table 1).
4. Experimental
4.1. General methods
4.2.1. (E)-8-Benzylidene-4-phenyl-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6a)
The chemicals used were obtained from Merck (Germany) and
Sigma Aldrich (USA). Melting points were determined by open tube
capillary method and are uncorrected. A CEM microwave synthe-
sizer (Model: Discover-S 908860) operating at 180/240 V and 50/
60 Hz with consumption of 1100 W with microwave power maxi-
mum level of 300 W and microwave frequency of 2455 MHz was
employed for the irradiation done in this work. Purity of the com-
pounds was checked on thin layer chromatography (TLC) plates
(silica gel G) using the petroleum ether/ethyl acetate solvent sys-
tem and the spots were examined under UV light. 1H and 13C
NMR were performed on Bruker Avance 500 (1H: 500 MHz, 13C:
125 MHz) spectrometer in CDCl3, using TMS as internal standard.
Yellow solid; mp 130–132 °C; Anal. Calcd for C20H19N3O: C,
75.69; H, 6.03; N, 13.24. Found: 75.24; H, 5.62; N, 13.02. 1H NMR
(500 MHz, CDCl3): dH 2.96 (d, 1H, J = 17.25 Hz, H-5a), 3.13 (d, 1H,
J = 17.25 Hz, H-5b), 3.60 (d, 1H, J = 15.10 Hz, H-7a), 3.76 (d, 1H,
J = 15.10 Hz, H-7b), 4.80 (s, 1H, H-4), 6.34 (s, 1H, NH), 6.71 (s, 1H,
H-9), 7.13–7.35 (m, 10H, H-aromatic, NH), 7.95 (s, 1H, NH); 13C
NMR (125 MHz, CDCl3): dC 45.29, 45.38, 50.09, 110.77, 121.87,
126.99, 128.27, 128.99, 129.35, 136.24, 142.34, 154.28.
4.2.2. (E)-8-(2-Methylbenzylidene)-4-p-tolyl-3,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6b)
Yellow solid; mp 154–156 °C; Anal. Calcd for C22H23N3O: C,
76.49; H, 6.71; N, 12.16. Found: 76.94; H, 6.12; N, 12.21. 1H NMR
(500 MHz, CDCl3): dH 2.31 (s, 3H, CH3), 2.45 (s, 3H, CH3), 2.98 (d,
1H, J = 17.14 Hz, H-5a), 3.18 (d, 1H, J = 17.14 Hz, H-5b), 3.57 (d,
1H, J = 15.04 Hz, H-7a), 3.69 (d, 1H, J = 15.04 Hz, H-7b), 5.26 (s,
1H, H-4), 5.63 (s, 1H, NH), 6.69 (s, 1H, H-9), 7.03–7.35 (m, 9H, aro-
matic, NH); 13C NMR (125 MHz, CDCl3): dC 19.51, 20.50, 45.75,
45.92 56.65, 110.55, 120.87, 125.80, 127.31, 127.93, 128.40,
128.68, 128.84, 129.77, 130.45, 131.56, 135.51, 135.87, 137.36,
139.75, 154.13.
4.2. General procedure for synthesis of 6 and 7
Conventional method: Catalytic amount of sodium ethoxide was
added to an equimolar dry mixture of unsaturated ketones (3) and
urea/thiourea (4 and 5), the mixture was ground uniformly in a
semi-micro boiling tube on the water bath for about 4–5 min dur-
ing which period a transparent viscous liquid formed showing
completion of reaction and double examined by TLC. Water was
added to the mixture, precipitated solid was filtered and dried in
vacuo to afford compounds 6a–l and 7a–l in moderate to good
yields (Table 1).
4.2.3. (E)-8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-
Microwave irradiation method: Catalytic amount of sodium eth-
oxide was added to an equimolar dry mixture of unsaturated ke-
tones (3) and urea/thiourea (4 and 5), the mixture was ground
well in a semi-micro boiling tube and irradiated for 0.5 min in a
CEM microwave synthesizer during which period a transparent
viscous liquid formed showing completion of reaction and double
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6c)
Yellow solid; mp 155–157 °C; Anal. Calcd for C20H17Cl2N3O C,
62.19; H, 4.44; N, 10.88. Found: 62.37; H, 4.12; N, N, 10.77. 1H
NMR (500 MHz, CDCl3): dH 3.11 (d, 1H, J = 17.46 Hz, H-5a), 3.36
(d, 1H, J = 17.46 Hz, H-5b), 3.60 (d, 1H, J = 14.94 Hz, H-7a), 3.72

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A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
Figure 5. Binding Interaction of 7l with active site residues of TcAChE receptor.
(d, 1H, J = 14.94 Hz, H-7b), 5.50 (s, 1H, H-4), 5.73 (s, 1H, NH), 6.73
(s, 1H, H-9), 7.12–7.47 (m, 9H, aromatic, NH); 13C NMR (125 MHz,
CDCl3): dC 45.68, 45.82, 55.20, 110.86, 119.33, 126.76, 128.37,
128.59, 129.18, 129.59, 129.77, 130.01, 130.20, 131.25, 133.04,
134.44, 139.12, 154.07.
4.2.6. (E)-8-(3-Nitrobenzylidene)-4-(3-nitrophenyl)-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6f)
Yellow solid; mp 176–178 °C; Anal. Calcd for C20H17N5O5 C,
58.97; H, 4.21; N, 17.19. Found: C, 58.17; H, 4.29; N, 17.11. 1H
NMR (500 MHz, CDCl3): dH 2.73 (d, 1H, J = 18.06 Hz, H-5a), 3.01
(d, 1H, J = 18.06 Hz, H-5b), 3.34 (d, 1H, J = 15.24 Hz, H-7a), 3.54
(d, 1H, J = 15.24 Hz, H-7b), 4.99 (s, 1H, H-4), 5.48 (s, 1H, NH),
6.02 (s, 1H, H-9), 7.10–8.08 (m, 8H, aromatic), 8.31 (s, 1H, NH);
13C NMR (125 MHz, CDCl3): dC 45.73, 45.97, 111.03, 119.99,
122.96, 125.36, 125.45, 125.94, 126.24, 126.33, 126.49, 127.09,
127.38, 128.22, 128.74, 129.46, 129.70, 131.10, 132.29, 133.62,
133.87, 134.49, 154.47.
4.2.4. (E)-8-(2-Fluorobenzylidene)-4-(2-fluorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6d)
Yellow solid; mp 146–149 °C; Anal. Calcd for C20H17F2N3O C,
67.98; H, 4.85; N, 11.89. Found: C, 67.74; H, 4.75; N, 12.10. 1H
NMR (500 MHz, CDCl3): dH 3.13 (d, 1H, J = 17.46 Hz, H-5a), 3.32
(d, 1H, J = 17.46 Hz, H-5b), 3.57 (d, 1H, J = 15.09 Hz, H-7a), 3.71
(d, 1H, J = 15.09 Hz, H-7b), 5.30 (s, 1H, H-4), 6.00 (s, 1H, NH),
6.65 (s, 1H, H-9), 6.97–7.40 (m, 8H, H-aromatic), 7.73 (s, 1H,
NH); 13C NMR (125 MHz, CDCl3): dC 45.57, 45.97, 110.54, 114.91,
115.82, 115.90, 116.12, 116.18, 124.14, 124.19, 124.31, 125.38,
125.43, 128.28, 129.07, 129.11, 129.28, 129.57, 129.67, 130.26,
130.37, 131.25, 131.28, 154.54, 158.65, 158.82, 161.92, 162.11.
4.2.7. (E)-8-(4-Bromobenzylidene)-4-(4-bromophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-one (6g)
Yellow solid; mp 174–176 °C; Anal. Calcd for C20H17Br2N3O C,
48.90; H, 3.49; N, 8.55. Found: C, 49.25; H, 4.05; N, 8.21. 1H NMR
(500 MHz, CDCl3): dH 2.91 (d, 1H, J = 11.05 Hz, H-5a), 3.09 (d, 1H,
J = 11.05 Hz, H-5b), 3.54 (d, 1H, J = 9.15 Hz, H-7a), 3.64 (d, 1H,
J = 9.15 Hz, H-7b), 4.87 (s, 1H, H-4), 6.39 (s, 1H, NH), 6.61 (s, 1H,
H-9), 7.05–7.31 (m, 8H, H-aromatic), 8.01 (s, 1H, NH); 13C NMR
(125 MHz, CDCl3): dC 45.22, 45.29, 58.46, 110.69, 120.72, 127.25,
128.31, 128.39, 128.52, 129.17, 130.44, 133.05, 134.21, 134.52,
140.54, 154.07.
4.2.5. (E)-8-(2-Methoxybenzylidene)-4-(2-methoxyphenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6e)
Yellow solid; mp 182–184 °C; Anal. Calcd for C22H23N3O3 C,
70.01; H, 6.14; N, 11.13. Found: C, 70.42; H, 6.01; N, 11.64. 1H
NMR (500 MHz, CDCl3): dH 3.23 (d, 1H, J = 17.58 Hz, H-5a), 3.34
(d, 1H, J = 17.58 Hz, H-5b), 3.70 (d, 1H, J = 15.06 Hz, H-7a), 3.82
(d, 1H, J = 15.06 Hz, H-7b), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
5.35 (s, 1H, H-4), 5.61 (s, 1H, NH), 6.65 (s, 1H, H-9), 6.87–7.30
(m, 9H, aromatic, NH); 13C NMR (125 MHz, CDCl3): dC 46.05,
52.19, 55.74, 55.77, 110.30, 110.85, 110.99, 116.84, 120.48,
121.48, 125.17, 127.99, 128.45, 128.86, 129.33, 129.66, 129.71,
130.79, 154.60, 157.00, 157.63.
4.2.8. (E)-8-(4-Methylbenzylidene)-4-p-tolyl-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6h)
Yellow solid; mp 170–173 °C; Anal. Calcd for C22H23N3O C,
76.49; H, 6.71; N, 12.16. Found: C, 76.91; H, 6.77; N, 11.85. 1H
NMR (500 MHz, CDCl3): dH 2.35 (br.s, 6H, CH3), 3.04 (d, 1H,
J = 17.28 Hz, H-5a), 3.21 (d, 1H, J = 17.28 Hz, H-5b), 3.69 (d, 1H,

A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
3029
Figure 6. Binding interaction of 7e with active site of hBChE receptor.
J = 15.09 Hz, H-7a), 3.85 (d, 1H, J = 15.09 Hz, H-7b), 4.88 (s, 1H, H-
4), 5.80 (s, 1H, NH), 6.60 (s, 1H, H-9), 7.07–7.36 (m, 8H, H-aro-
matic); 13C NMR (125 MHz, CDCl3): dC 21.53, 21.60, 45.73, 45.86,
59.38, 111.10, 121.70, 127.29, 127.98, 129.41, 129.66, 130.07,
133.60, 137.51, 138.59, 139.70, 154.20.
4.2.11. (E)-8-(2,4-Dichlorobenzylidene)-4-(2,4-dichlorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6k)
Yellow solid; mp 160–162 °C; Anal. Calcd for C20H15Cl4N3O C,
52.78; H, 3.32; N, 9.23. Found: C, 52.31; H, 3.82; N, 8.99. 1H NMR
(500 MHz, CDCl3): dH 3.08 (d, 1H, J = 18.00 Hz, H-5a), 3.34 (d, 1H,
J = 18.00 Hz, H-5b), 3.56 (d, 1H, J = 14.07 Hz, H-7a), 3.67 (d, 1H,
J = 14.07 Hz, H-7b), 4.70 (s, 1H, H-4), 5.87 (s, 1H, NH), 6.66 (s, 1H,
H-9), 7.03–7.41 (m, 6H, H-aromatic), 7.72 (s, 1H, NH); 13C NMR
(125 MHz, CDCl3): dC 45.56, 45.76, 54.81, 110.78, 118.62, 127.14,
127.60, 128.63, 128.75, 129.90, 130.05, 130.48, 131.82, 133.13,
133.62, 134.38, 135.10, 135.28, 137.70, 153.99.
4.2.9. (E)-8-(4-Chlorobenzylidene)-4-(4-chlorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6i)
Yellow solid; mp 170–172 °C; Anal. Calcd for C20H17Cl2N3O C,
62.19; H, 4.44; N, 10.88. Found: C, 62.72; H, 4.25; N, 10.57. 1H
NMR (500 MHz, CDCl3): dH 2.96 (d, 1H, J = 10.29 Hz, H-5a), 3.15
(d, 1H, J = 10.29 Hz, H-5b), 3.62 (d, 1H, J = 8.97 Hz, H-7a), 3.75 (d,
1H, J = 8.97 Hz, H-7b), 4.83 (s, 1H, H-4), 6.37 (s, 1H, NH), 6.64 (s,
1H, H-9), 7.07–7.27 (m, 8H, H-aromatic), 8.02 (s, 1H, NH); 13C
NMR (125 MHz, CDCl3): dC 45.27, 45.34, 58.46, 110.76, 120.81,
127.31, 128.33, 128.45, 128.60, 129.24, 130.58, 133.08, 134.29,
134.56, 140.63, 154.10.
4.2.12. (E)-4-(Naphthalen-1-yl)-8-(naphthalen-1-ylmethylene)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6l)
Yellow solid; mp 175–177 °C; Anal. Calcd for C28H23N3O C,
80.55; H, 5.55; N, 10.06. Found: C, 80.27; H, 5.19; N, 10.21. 1H
NMR (500 MHz, CDCl3): dH 3.14 (d, 1H, J = 17.05 Hz, H-5a), 3.27
(d, 1H, J = 17.05 Hz, H-5b), 3.82 (d, 1H, J = 15.14 Hz, H-7a), 3.97
(d, 1H, J = 15.14 Hz, H-7b), 4.92 (s, 1H, H-4), 5.85 (s, 1H, NH),
6.63 (s, 1H, H-9), 6.87–7.92 (m, 15H, H-aromatic, NH); 13C NMR
(125 MHz, CDCl3): dC 45.67, 45.74, 59.41, 111.17, 121.74, 127.31,
125.6, 125.8, 126.44, 127.98, 129.48, 129.76, 130.07, 133.35,
133.69, 137.51, 138.71, 139.77, 153.12.
4.2.10. (E)-8-(4-Fluorobenzylidene)-4-(4-fluorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-one (6j)
Yellow solid; mp 156–158 °C; Anal. Calcd for C20H17F2N3O C,
67.98; H, 4.85; N, 11.89. Found: C, 67.78; H, 4.81; N, 11.88. 1H
NMR (500 MHz, CDCl3): dH 2.98 (d, 1H, J = 10.32 Hz, H-5a), 3.16
(d, 1H, J = 10.32 Hz, H-5b), 3.64 (d, 1H, J = 9.03 Hz, H-7a), 3.75 (d,
1H, J = 9.03 Hz, H-7b), 4.85 (s, 1H, H-4), 6.33 (s, 1H, NH), 6.648 (s,
1H, H-9), 6.92–7.24 (m, 8H, H-aromatic), 8.00 (s, 1H, NH); 13C
NMR (125 MHz, CDCl3): dC 45.27, 45.41, 58.36, 110.61, 115.15,
115.32, 115.84, 116.01, 120.90, 127.26, 127.82, 128.65, 128.72,
130.95, 131.02, 132.22, 132.24, 138.08, 138.10, 154.15, 160.85,
161.64, 162.82, 163.61.
4.2.13. (E)-8-Benzylidene-4-phenyl-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7a)
Yellow solid; mp 130–132 °C; Anal. Calcd for C20H19N3S C,
72.04; H, 5.74; N, 12.60. Found: C, 71.68; H, 5.57; N, 12.15. 1H
NMR (500 MHz, CDCl3): dH 3.01 (d, 1H, J = 17.07 Hz, H-5a), 3.21
(d, 1H, J = 17.07 Hz, H-5b), 3.60 (d, 1H, J = 15.18 Hz, H-7a), 3.82

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A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
(d, 1H, J = 15.18 Hz, H-7b), 4.90 (s, 1H, H-4), 6.59 (s, 1H, H- 9), 7.10–
7.38 (m, 11H, H-aromatic, NH), 7.94 (s, 1H, NH); 13C NMR
(125 MHz, CDCl3): dC 45.40, 45.49, 59.40, 112.90, 122.32, 126.08,
127.38, 127.52, 127.99, 128.84, 129.22, 129.52, 129.67, 135.88,
141.23, 174.45.
(d, 1H, J = 15.00 Hz, H-7b), 5.59 (s, 1H, H-4), 7.12 (s, 1H, H-9),
7.15–8.06 (m, 9H, aromatic and NH), 8.21 (s, 1H, NH); 13C NMR
(125 MHz, CDCl3): dC 45.49, 45.80, 112.84, 120.22, 122.58,
125.12, 125.57, 126.02, 126.53, 126.59, 126.67, 126.84, 127.51,
127.59, 128.75, 128.84, 129.00, 129.68, 130.07, 131.00, 132.13,
132.85, 133.96, 134.51, 135.60, 174.76.
4.2.14. (E)-8-(2-Methylbenzylidene)-4-o-tolyl-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7b)
Yellow solid; mp 128–130 °C; Anal. Calcd for C22H23N3S C,
73.09; H, 6.41; N, 11.62. Found: C, 72.88; H, 6.21; N, 11.77. 1H
NMR (500 MHz, CDCl3): dH 2.26 (s, 3H, CH3), 2.34 (s, 3H, CH3),
2.85 (d, 1H, J = 17.25 Hz, H-5a), 3.11 (d, 1H, J = 17.25 Hz, H-5b),
3.36 (d, 1H, J = 15.09 Hz, H-7a), 3.57 (d, 1H, J = 15.09 Hz, H-7b),
5.18 (s, 1H, H-4), 6.59 (s, 1H, H- 9), 6.59–7.28 (m, 9H, H-aromatic,
NH), 7.73 (s, 1H, NH); 13C NMR (125 MHz, CDCl3): dC 19.68, 20.54,
45.19, 45.45, 56.48, 112.26, 121.32, 125.96, 126.25, 127.40, 128.26,
129.04, 129.13, 129.79, 130.54, 131.64, 134.93, 136.02, 137.15,
138.60, 174.24.
4.2.19. 4.2.19.(E)-8-(4-Bromobenzylidene)-4-(4-bromophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione(7g)
Yellow solid; mp 139–142 °C; Anal. Calcd for C20H17Br2N3S C,
50.55; H, 3.61; N, 8.84. Found: C, 50.15; H, 3.21; N, 8.49;. 1H
NMR (500 MHz, CDCl3): dH 2.93 (d, 1H, J = 11.09 Hz, H-5a), 3.12
(d, 1H, J = 11.09 Hz, H-5b), 3.57 (d, 1H, J = 9.05 Hz, H-7a), 3.69 (d,
1H, J = 9.05 Hz, H-7b), 5.39 (s, 1H, H-4), 7.08 (s, 1H, H-9), 7.07–
7.49 (m, 8H, H-aromatic), 8.17 (s, 1H, NH); 13C NMR (125 MHz,
CDCl3): dC 45.41, 45.82, 59.11, 111.69, 121.82, 127.94, 128.82,
129.12, 129.46, 129.89, 131.43, 133.75, 134.79, 135.21, 140.54,
175.15.
4.2.15. (E)-8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7c)
Yellow solid; mp 130–133 °C; Anal. Calcd for C20H17Cl2N3S C,
59.70; H, 4.26; N, 10.44. Found: C, 59.19; H, 3.74; N, 10.21. 1H
NMR (500 MHz, CDCl3): dH 2.12 (br.s 1H, NH), 3.04 (d, 1H,
J = 17.90 Hz, H-5a), 3.28 (d, 1H, J = 17.90 Hz, H-5b), 3.51 (d, 1H,
J = 15.09 Hz, H-7a), 3.66 (d, 1H, J = 15.09 Hz, H-7b), 5.47 (s, 1H,
H-4), 6.60 (s, 1H, H-9), 7.07–7.45 (m, 9H, aromatic, H-arylmethyl-
idene), 7.65 (s, 1H, NH), 8.08 (s, 1H, NH); 13C NMR (125 MHz,
CDCl3): dC 45.05, 45.79, 54.78, 112.54, 119.41, 126.54, 128.13,
129.12, 129.56, 129.67, 129.95, 130.03, 130.87, 132.59, 133.73,
133.91, 137.61, 174.35.
4.2.20. (E)-8-(4-Methylbenzylidene)-4-p-tolyl-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7h)
Yellow solid; mp 134–137 °C; Anal. Calcd for C22H23N3S C,
73.09; H, 6.41; N, 11.62. Found: C, 72.51; H, 6.18; N, 11.45. 1H
NMR (500 MHz, CDCl3): dH 2.34 (s, 3H, CH3), 2.36 (s, 3H, CH3),
3.04 (d, 1H, J = 17.68 Hz, H-5a), 3.21 (d, 1H, J = 17.68 Hz, H-5b),
3.63 (d, 1H, J = 14.37 Hz, H-7a), 3.83 (d, 1H, J = 14.37 Hz, H-7b),
4.88 (s, 1H, H-4), 6.55 (s, 1H, H-9), 6.75–7.49 (m, 9H, aromatic
and NH), 7.87 (s, 1H, NH); 13C NMR (125 MHz, CDCl3): dC 21.57,
21.64, 45.40, 45.59, 59.17, 112.70, 122.13, 125.98, 126.71, 127.35,
127.44, 129.55, 129.63, 130.17, 130.23, 133.00, 137.93, 138.34,
139.07, 174.42.
4.2.16. (E)-8-(2-Fluorobenzylidene)-4-(2-fluorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7d)
4.2.21. (E)-8-(4-Chlorobenzylidene)-4-(4-chlorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7i)
Yellow solid; mp 142–147 °C; Anal. Calcd for C20H17Cl2N3S, C,
59.70; H, 4.26; N, 10.44. Found: C, 59.92; H, 4.42; 10.31. 1H NMR
(500 MHz, CDCl3): dH 3.00 (d, 1H, J = 17.58 Hz, H-5a), 3.23 (d, 1H,
J = 17.58 Hz, H-5b), 3.58 (d, 1H, J = 15.06 Hz, H-7a), 3.79 (d, 1H,
J = 15.06 Hz, H-7b), 4.89 (s, 1H, H-4), 6.58 (s, 1H, H-9), 7.05–7.34
(m, 8H, aromatic), 7.93 (s, 1H, NH), 8.07 (s, 1H, NH); 13C NMR
(125 MHz, CDCl3): dC 45.39, 45.51, 58.63, 112.93, 126.22, 127.72,
128.87, 129.08, 129.80, 130.94, 133.95, 134.26, 135.12, 139.69,
174.31.
Yellow solid; mp 128–130 °C; Anal. Calcd for C20H17F2N3S C,
65.02; H, 4.64; N, 11.37. Found: C, 65.71; H, 4.39; N, 11.28. 1H
NMR (500 MHz, CDCl3): dH 3.15 (d, 1H, J = 17.76 Hz, H-5a), 3.33
(d, 1H, J = 17.76 Hz, H-5b), 3.57 (d, 1H, J = 15.09 Hz, H-7a), 3.72
(d, 1H, J = 15.09 Hz, H-7b), 5.33 (s, 1H, H-4), 6.57 (s, 1H, H-9)
7.00–7.38 (m, 8H, aromatic), 7.42 (s, 1H, NH), 7.98 (s, 1H, NH); dC
45.32, 45.72, 52.01, 52.07, 112.26, 115.14, 115.17, 115.96, 116.10,
116.25, 116.39, 123.46, 123.65, 124.30, 124.35, 125.60, 125.65,
126.81, 127.80, 127.98, 129.24, 129.29, 130.00, 130.11, 130.84,
130.95, 131.15, 131.19, 158.55, 158.81, 161.83, 162.10, 175.22.
4.2.22. (E)-8-(4-Fluorobenzylidene)-4-(4-fluorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7j)
Yellow solid; mp 140–142 °C; Anal. Calcd for C20H17F2N3S C,
65.02; H, 4.64; N, 11.37. Found: C, 65.29; H, 4.91; N, 11.15. 1H
NMR (500 MHz, CDCl3): dH 2.98 (d, 1H, J = 17.55 Hz, H-5a), 3.20
(d, 1H, J = 17.55 Hz, H-5b), 3.57 (d, 1H, J = 15.05 Hz, H-7a), 3.79
(d, 1H, J = 15.05 Hz, H-7b), 4.89 (s, 1H, H-4), 6.60 (s, 1H, H-9),
6.97–7.27 (m, 8H, aromatic), 8.00 (s, 1H, NH), 8.09 (s, 1H, NH);
13C NMR (125 MHz, CDCl3): dC 45.33, 45.44, 58.53, 112.74,
115.74, 116.03, 116.36, 116.65, 121.61, 126.19, 127.04, 129.24,
127.04, 129.24, 129.35, 131.31, 131.42, 131.88, 131.92, 137.12,
137.17, 160.78, 161.53, 164.07, 164.81, 174.18.
4.2.17. (E)-8-(2-Methoxybenzylidene)-4-(2-methoxyphenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7e)
Yellow solid; mp 190–192 °C; Anal. Calcd for C22H23N3O2S C,
67.15; H, 5.89; N, 10.68. Found: C, 67.22; H, 5.93; N, 10.67. 1H
NMR (500 MHz, CDCl3): dH 3.22 (d, 1H, J = 17.85 Hz, H-5a), 3.30
(d, 1H, J = 17.85 Hz, H-5b), 3.67 (d, 1H, J = 15.99 Hz, H-7a), 3.79
(d, 1H, J = 15.99 Hz, H-7b), 3.84 (s, 3H, OCH3), 3.85 (s, 3H, OCH3),
5.38 (s, 1H, H-4), 6.67 (s, 1H, H-9), 6.88–7.32 (m, 9H, aromatic
and NH), 7.82 (s, 1H, NH); 13C NMR (125 MHz, CDCl3): dC 45.78,
45.92, 52.35, 55.74, 55.86, 110.86, 111.15, 111.73, 117.56, 120.51,
121.62, 124.67, 127.25, 127.59, 128.27, 128.35, 129.40, 129.63,
130.22, 130.59, 130.79, 156.91, 157.65, 175.03.
4.2.23. (E)-8-(2,4-Dichlorobenzylidene)-4-(2,4-dichlorophenyl)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione
(7k)
4.2.18. (E)-8-(3-Nitrobenzylidene)-4-(3-nitrophenyl)-3,4,5,6,7,8-
hexahydropyrido[4,3-d]pyrimidine-2(1H)-thione (7f)
Yellow solid; mp 180–183 °C; Anal. Calcd for C20H17N5O4S C,
56.73; H, 4.05; N, 16.54. Found: C, 56.17; H, 3.72; N, 16.02. 1H
NMR (500 MHz, CDCl3): dH 2.83 (d, 1H, J = 17.79 Hz, H-5a), 3.14
(d, 1H, J = 17.79 Hz, H-5b), 3.39 (d, 1H, J = 15.00 Hz, H-7a), 3.61
Yellow solid; mp 136–139 °C; Anal. Calcd for C20H15Cl4N3S C,
50.98; H, 3.21; N, 8.92. Found: C, 51.55; H, 3.08; N, 8.69. 1H NMR
(500 MHz, CDCl3): dH 3.05 (d, 1H, J = 17.91 Hz, H-5a), 3.32 (d, 1H,
J = 17.91 Hz, H-5b), 3.49 (d, 1H, J = 15.00 Hz, H-7a), 3.65 (d, 1H,
J = 15.07 Hz, H-7b), 5.44 (s, 1H, H-4), 6.61 (s, 1H, H-9) 7.02–7.49
(m, 6H, aromatic), 7.74 (s, 1H, NH), 8.12 (s, 1H, NH); dC 45.21,

A. Basiri et al. / Bioorg. Med. Chem. 21 (2013) 3022–3031
3031
45.47, 54.75, 112.56, 119.07, 126.99, 127.34, 128.94, 129.14,
129.99, 130.16, 130.81, 131.87, 132.59, 133.59, 134.74, 135.02,
135.73, 136.64, 174.76.
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 respective
ligands. Docking stimulations were carried out on bioactive com-
pounds, handed in five poses per ligand, in which the best pose
with highest score was displayed for each ligand.
4.2.24. (E)-4-(Naphthalen-1-yl)-8-(naphthalen-1-ylmethylene)-
3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidin-2(1H)-thione (7l)
Yellow solid; mp 141–143 °C; Anal. Calcd for C28H23N3S C,
77.57; H, 5.35; N, 9.69. Found: C, 78.29; H, 5.94; N, 8.45; 1H
NMR (500 MHz, CDCl3): dH 3.09 (d, 1H, J = 17.15 Hz, H-5a), 3.19
(d, 1H, J = 17.15 Hz, H-5b), 3.71 (d, 1H, J = 15.07 Hz, H-7a), 3.94
(d, 1H, J = 15.07 Hz, H-7b), 5.49 (s, 1H, H-4), 6.63 (s, 1H, H-9),
6.81–8.17 (m, 16H, H-aromatic, NH); 13C NMR (125 MHz, CDCl3):
dC 45.71, 45.78, 55.17, 112.34, 122.43, 128.11, 126.75, 127.12,
127.44, 127.98, 129.48, 129.76, 129.96, 133.12, 133.54, 137.17
138.32, 139.62, 174.49.
Acknowledgments
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.
4.3. In vitro cholinesterase enzymes inhibitory activity
Supplementary data
The test samples for cholinesterase enzymes inhibitory poten-
tial was evaluated using modified Ellman’s method as described
by Ahmed and Gilani.25 Galanthamine was used as positive control.
Solutions of test samples and galanthamine were prepared in
DMSO at an initial concentration of 1 mg/mL (1000 ppm). The con-
centration of DMSO in final reaction mixture was 1%. At this con-
centration, DMSO has no inhibitory effect on both
acetylcholinesterase and butyrylcholinesterase enzymes.26
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.058.
References and notes
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bation at 25 °C, 10
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l
ylthiocholine iodide. Thirty min after the initiation of enzymatic
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from equine serum and S-butyrylthiocholine chloride were used.
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Absorbance of control ꢀ Absorbance of Sample
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Percent of inhibition ¼
Absorbance of control
ꢁ 100
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To date, there are a total of 145 AChE and 29 BChE co-crystal
structures and NMR structures available in Protein Data Bank
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Using Glide, (version 5.7, Schrödinger, LLC, New York, NY, 2011),
most active compounds were docked onto the active site of TcAChE
derived from three-dimensional structure of the enzyme complex
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to BChE derived from complex of the enzyme with Tabun analogue
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M.; Bonacorso, H. G.; Zanatta, N. Neurochem. Res. 2005, 30, 379.
27. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
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Water molecules and hetero groups were deleted from receptor
beyond the radius of 5 Å of reference ligand (E2020 or Tabun),
resulting protein structure refined and minimized by Protein
29. Silman, I.; Sussman, J. L. Chem. Biol. Interact. 2008, 175, 3.