Design, synthesis, biological evaluation and docking study of 5-oxo-4,5-dihydropyrano[3,2-c]chromene derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.07.038

A series of fused coumarins namely 5-oxo-4,5-dihydropyrano[3,2-c]chromenes linked to N-benzylpyridinium scaffold were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The 1-(4-fluorobenzyl)pyridinium d

Full text of DOI:10.1016/j.ejmech.2013.07.038

Accepted Manuscript
Design, synthesis, biological evaluation and docking study of 5-oxo-4,5-
dihydropyrano[3,2-c]chromene derivatives as acetylcholinesterase and
butyrylcholinesterase inhibitors
Mehdi Khoobi, Masoumeh Alipour, Amirhossein Sakhteman, Hamid Nadri, Alireza
Moradi, Mehdi Ghandi, Saeed Emami, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(13)00486-8
10.1016/j.ejmech.2013.07.038
EJMECH 6325
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 May 2013
Revised Date: 12 July 2013
Accepted Date: 14 July 2013
Please cite this article as: M. Khoobi, M. Alipour, A. Sakhteman, H. Nadri, A. Moradi, M. Ghandi, S.
Emami, A. Foroumadi, A. Shafiee, Design, synthesis, biological evaluation and docking study of 5-
oxo-4,5-dihydropyrano[3,2-c]chromene derivatives as acetylcholinesterase and butyrylcholinesterase
inhibitors, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.07.038.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis, biological evaluation and docking study of 5-oxo-4,5-dihydropyrano[3,2-c]chromene
derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors
Mehdi Khoobi, Masoumeh Alipour, Amirhossein Sakhteman, Hamid Nadri, Alireza Moradi, Mehdi Ghandi,
Saeed Emami, Alireza Foroumadi , Abbas Shafiee *
A series of fused coumarins bearing N-benzylpyridinium scaffold were designed as AChE and BuChE
inhibitors. The 1-(4-fluorobenzyl)pyridinium derivative 6g showed the most potent anti-AChE activity and the
highest AChE/BuChE selectivity.

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and docking study of 5-oxo-4,5-
dihydropyrano[3,2-c]chromene derivatives as acetylcholinesterase and
butyrylcholinesterase inhibitors
a
b
c
c
Mehdi Khoobi , Masoumeh Alipour , Amirhossein Sakhteman , Hamid Nadri , Alireza Moradi
c
b
d
a,e
a,
,
Mehdi Ghandi , Saeed Emami , Alireza Foroumadi , Abbas Shafiee *
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b
School of Chemistry, University College of Science, University of Tehran, PO Box 14155-6455,
Tehran, Iran
c
Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
e
Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
Abstract: A series of fused coumarins namely 5-oxo-4,5-dihydropyrano[3,2-c]chromenes linked
to N-benzylpyridinium scaffold were synthesized and evaluated as acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) inhibitors. The 1-(4-fluorobenzyl)pyridinium derivative 6g
showed the most potent anti-AChE activity (IC50 value = 0.038 ꢀM) and the highest
AChE/BuChE selectivity (SI>48). The docking study permitted us to rationalize the observed
structure–affinity relationships and to detect possible binding modes.
Keywords: Alzheimer’s disease, Acetylcholinesterase, Chromenes, Coumarins, Docking study
*
Corresponding author. Tel: +98 21 66406757; Fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
1

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1
. Introduction
Alzheimer’s disease (AD) is the most common cause of dementia among the elderly patients,
which leads to progressive decline in cognition and memory [1]. This neurological disorder
characterized by reducing levels of acetylcholine (ACh) in the cortex and hippocampus regions
of brain [2]. However, the pathogenesis of AD is highly complicated, and the low levels of
acetylcholine also associated with the presence of numerous plaques of amyloid β-peptide (Aβ),
neurofibrillary tangles and oxidative stress [3,4].
Acetylcholine is primarily hydrolyzed by acetylcholinesterase (AChE) enzyme, and terminated
its action. Thus, one of the most promising approaches adopted for palliative treatment of AD is
the increasing of acetylcholine levels in the brain by targeting the AChE enzyme [5]. Recently, it
has been reported that in AD patients, AChE activity is greatly reduced in specific regions of the
brain while butyrylcholinesterase (BuChE) activity increases, likely as compensation for AChE
decrease. Due to the key role of BuChE in hydrolysis of Ach, preserving the AChE/BuChE
activity ratios in damaged brain is essential for successful treatment of AD. Thus, dual inhibition
of AChE and BuChE might improve the signs and symptoms in AD patients [6].
Several AChE inhibitors (Figure 1) such as rivastigmine, donepezil, galantamine, and tacrine are
the main agents for the improvement of cognitive function and the enhancement of memory in
AD patients [7,8]. Moreover, several studies have also suggested that AChE inhibitors interfere
with the synthesis, deposition and aggregation of Aβ [9,10]. Molecular study evidences have
shown that the interaction of some AChE inhibitors with the peripheral anionic binding site
(PAS) of AChE may be involved in Aβ deposition [11,12]. Indeed, the PAS and catalytic site
(CS) of AChE enzyme are characterized by two tryptophan residues located at the entrance and
2

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the bottom of the active-site gorge. Based on the dual binding model with the AChE, compounds
that interact with both CS and PAS could be effective agents for the management of AD [10,13].
A structural survey of AChE inhibitors revealed that AP2238 and ensaculin has a distinct
scaffold with a coumarin moiety (Figure 2). Moreover, a number of naturally occurring and
synthetic coumarin analogs exhibited potent AChE inhibitory activity [14]. It has been
demonstrated that AChE inhibitors with coumarin moiety primarily interact with the PAS of
AChE [15,16]. These findings prompt medicinal chemists to design dual inhibitors of AChE by
incorporating a catalytic site interacting moiety with coumarin through an appropriate linker
[
14].
In this regard, we have previously designed a series of coumarin and coumaranone AChE
inhibitors linked to benzyl pyridinium group (Figure 2) with the capability of interaction with
both sites of the enzyme [17]. In continuation of our study of developing new AChE inhibitors,
in this work we synthesized a series of fused coumarins 6a-o namely 5-oxo-4,5-
dihydropyrano[3,2-c]chromene derivatives linked to N-benzylpyridinium scaffold and evaluated
them as AChE and BuChE inhibitors.
2
. Chemistry
In this work, we describe synthesis of a novel series of pyranochromenes 6a–o bearing an N-
benzylpyridinium moiety in a rather convenient way, owing to the good accessibility of the
pyranochromenes nucleus. As outlined in Scheme 1, these compounds could be easily prepared
by the simple treatment of 2-amino-5-oxo-4-(pyridin-3 or 4-yl)-4,5-dihydropyrano[3,2-
c]chromene-3-carbonitriles 5a,b with different benzyl chlorides. Several methods have been
reported for the synthesis of dihydropyrano[c]chromenes. Recently, a green inorganic-organic
3

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hybrid magnetic nanocatalyst in aqueous medium was used successfully for the synthesis of 2-
amino-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitriles [18]. Accordingly, compounds
5
were synthesized using a commercial pyridine-3 or 4-carbaldehydes (1a or 1b) and
malononitrile 2 and subsequent reaction with 4-hydroxycoumarin (4) in the presence of catalytic
amount of magnetic nanocatalyst. The target compounds 6 were prepared through the reaction of
compounds 5 with benzyl chlorides having electron donating or withdrawing substitutions, in dry
acetonitrile under reflux condition. On cooling, the precipitate was filtered and washed with
diethyl ether. The notable features of this method are the easy product isolation with high yields
without application of column chromatography.
3
. Results and Discussion
3
.1. In vitro inhibition of AChE and BuChE
The target compounds 6a-o were evaluated for their in vitro inhibitory activities against AChE
and BuChE in comparison with marketed AChE inhibitor donepezil as standard drug. The anti-
cholinesterase activities (µM) of two series of compounds 4-pyridinium and 3-pyridinium
derivatives (6a-i and 6j-o, respectively) are shown in Table 1. The data are expressed as mean ±
S.E. of three independent experiments. The data were statistically analyzed by means of one-way
ANOVA (Analysis of Variance) followed by Newman-Kules post-test to compare each pair of
the compounds (P values < 0.05 were considered as significant).
The IC50 values of test compounds demonstrated that all compounds showed significant
inhibition against AChE at concentrations less than 7.5 µM. The most active compounds were 4-
fluoro- and 3,4-dichloro- analogs of 4-pyridinium series (compounds 6g and 6h) with IC values
5
0
≤
0.044 µM. Furthermore, compounds 6f, 6i and 6n exhibited good anti-AChE activity (IC50
4

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values = 0.48-0.831 µM. Although, compounds 6g-i were also the most active compounds
toward BuChE (IC values = 0.566-1.84 µM) but the selectivity of compounds 6g and 6h for
5
0
AChE was higher than 28. In contrast, compound 6i with high anti-cholinesterase activity
showed no significant selectivity for AChE/BuChE. In general, the anti-AChE activities of all
compounds with the exception of 6i and 6o were about 3 to 48 times more than their anti-BuChE
activities.
3
.2. Structure-activity relationships
The inhibitory activity of 4-pyridinium derivatives 6a-i against AChE demonstrated that para-
fluoro derivative 6g showed the most potent activity, while ortho- or meta-flouro derivatives (6a
and 6d) were less active halo-substituted compounds. Moreover, the introduction of 3-chloro,
3
,4-dichoro or 2,4-dichloro on N-benzyl group of 4-pyridinium series improved anti-AChE
potency as observed in compounds 6f, 6h and 6i. The comparison of IC values of 2- or 3-
50
chloro compounds 6b and 6f with their 2- or 3-fluoro analogues 6a and 6d revealed that the
chlorine atom is more effective than fluorine in the ortho and meta positions.
In the 3-pyridinium series (compounds 6j-o), the fluoro- substituted compounds 6j, 6l and 6o
showed approximately same inhibitory activity against AChE. This finding demonstrates that the
regioisomerism of fluorine substituent on the N-benzyl group of 3-pyridinum series has no
significant effect on inhibitory activity. In contrast, 2-chlorobenzyl derivative 6k was
significantly more active than 3-chlorobenzyl analogue 6m.
In the term of BuChE inhibitory activity in 4-pyridinium series, compounds 6g-i containing
halogen atom at para-position showed improved potency. By comparing the IC values of 2-
5
0
chloro derivative 6b with 2,4-dichloro analogue 6i or of 3-chloro derivative 6f with
5

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corresponding 3,4-dichloro compound 6h, it could be concluded that the introduction of second
chlorine atom onto the para-position of benzyl group increased the anti-BuChE activity. In
addition, the translocation of fluorine atom on the N-benzyl-4-pyridinium moiety had significant
effect on BuChE inhibitory activity, and para-fluoro was preferred. Replacement of fluorine
atom at ortho or meta positions with chlorine atom increased BuChE-inhibition potency
(compounds 6b and 6f vs. 6a and 6d, respectively).
The observed IC values of 3-pyridinium derivatives 6j-o against BuChE revealed that the better
5
0
result was obtained with 4-fluoro substituent (compound 6o). Similar to the 4-pyridinium series,
- or 3-chloro derivatives 6k and 6m were more active than 2- or 4-fluoro derivatives 6j and 6l
2
against BuChE.
The comparison of IC50 values of 3-pyridinium derivatives with their corresponding 4-
pyridinium regioisomers against AChE revealed that 3-pyridinium analogs 6j-l and 6n were
more active than their regioisomers. In contrast, the anti-AChE activity of 6m and 6o was less
than their 4-pyridinium regioisomers. In the 4-pyridinium series, 4-fluoro and 3,4-dichloro
derivatives were the most potent compounds, in the term of AChE inhibition. While amongst the
3
-pyridinium derivatives, 3-methyl analog showed highest inhibitory activity against AChE.
These results demonstrated that the effect of 3,4-regioisomerism of the pyridinium part depends
on the type and the position of substituent on benzyl group.
It is worthy to note that in the cases of dichlorobenzyl derivatives 6h and 6i, regioisomerism of
chlorine substitution could dramatically change the AChE/BuChE selectivity. While 2,4-
dichlorobenzyl derivative 6i has no significant selectivity, but 3,4-dichlorobenzyl counterpart 6h
shows relatively high selectivity for AChE.
6

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Interestingly, both 4-fluorobenzyl analogs 6g and 6o have similar activity against BuChE, but the
anti-AChE activity of 4-pyridinum derivative 6g is about 57-fold more than that of 3-pyridinum
congener 6o.
In overall, the representative compound 6g has a potent anti-AChE activity, which can inhibit
AChE at the nano-molar concentrations with proper selectivity.
3
.2. Docking studies
In order to explain the different activities of the target compounds and also to clarify their
interaction mode in the active site of AChE, docking studies were conducted using Autodock
Vina 1.1.1. Several Ligand-bounded crystallographic structures of AChE are available in the
RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). In this study, the PDB
structure of 3I6Z was retrieved for docking purposes. Since the AChE from electrophorus
electricus was used for the experimental section and there were not any high resolution (<2.5 Å)
crystallographic structures of this target in the PDB, therefore we used the high-resolution
homologue structure (3I6Z: 2.19 Å) with 64% overall identity towards the native protein and
1
00% identity in the active site. The best pose of each ligand in terms of the free energy of
binding was extracted from the 50 generated top scores. Since, compounds 6g and 6n were able
to represent the whole chemical space and were more potent than their counterparts were
subjected to further studies of binding mode. The best pose of the compounds in the gorge of
AChE is depicted in Figure 3.
As described, the chemical space is comprised of two fragments; a pyranochromen-2-one, and a
N-benzylpyridinium part. A common feature of all compounds is their ability to interact with the
two distinct regions in the active site, viz central anionic site (CAS) at the vicinity of the catalytic
7

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site (CS) and the peripheral anionic site (PAS) at the top the gorge of AChE. The most important
residues responsible for the interaction of the synthesized inhibitors included Phe330, Trp84 in
CAS and Trp279 in PAS, where Phe330 was the key residue in ligand recognition and
trafficking. The aromatic part in pyranochromenone fragment was oriented so that a π-π
interaction was possible with Phe330 and Trp84. In addition, the positively charged amino
substituent on the pyran ring was prone to negative area caused by a carboxylic side chain of
Asp72.
In the compounds with enhanced activities (i.e. 6g), an additional π-π interaction between the
benzyl group on pyridinium ring and Trp279 led to stabilization of the resulting complex.
In the 4-pyridinium series, 6g showed to be the most active compound and its binding mode in
the active site is displayed in Figure 4.
The presence of electron withdrawing groups such as fluoro or chloro on the proper position of
the benzylic pendent could lead to reinforcing π-π interaction as in compounds 6g, in comparison
with 6c. The order of activity is 4-F >2-F ≈ 3-F for fluoro- substitution and 3-Cl >2-Cl for
chloro- substitution. This can be explained by the more feasible π-π interactions in the more
potent compounds (Figure 5).
As described above, when two chlorine atoms were attached to the benzyl moiety, the activity
was largely dependent on their positions. The π-π interaction was destabilized in compound 6i
due to the disfavored orientation of lipophilic chlorine atom towards the hydrophilic pocket
composed of ser286, Ile287 and Phe287, as depicted in Figure 6.
In the case of compound 6e, the presence of methyl at the 3-position of benzyl moiety led to
unexpectedly increasing of the activity. This finding can be reasonably explained by π-H
interaction between methyl hydrogens at position 3 and the phenyl ring of Tyr70 (Figure 7).
8

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Among the 3-pyridinium derivatives, the most active compound was 6n with a 3-methylbenzyl
moiety. A π-H interaction similar to that observed for compound 6e was responsible for its
enhanced activity in comparison with compounds 6l and 6m containing 3-fluoro or 3-chloro
benzyl groups respectively (Figure 8).
The most active compound against BuChE (compound 6i) was used for docking with BuChE
(PDB ID: 4BDS), and the best-docked pose was extracted for further study. The binding mode
for the interaction of 6i with the target structure is depicted in Figure 9. The orientation of the
ligand in the active site facilitated formation of a π-π stacking between coumarin ring and Trp79.
Consequently, the partially charged amino and nitrile groups were exposed to oxyanionic hole
composed by carbonyl moieties of Gln64, Asn65, Asp67, Asn80, Thr117 and Gly118. Moreover,
the hydrophobic 2,4-dichlorophenyl moiety was well fitted in a hydrophobic pocket made by
Pro282, Leu283, Phe326, Tyr329 and Gly330.
3
.3. ADMET prediction
The ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties of the
target compounds were calculated using some web-based applications. BBB (Blood-Brain
Barrier) penetration, HIA (Human Intestinal Absorption), Caco-2 cell permeability and AMES
test were calculated using admetSAR [19] a web-base application and LC50 values (Lethal
concentration 50%) were predicted using http://lazar.in-silico.de/predict. The predicted ADMET
data were summarized in Table 2. Based on the predicted values for BBB penetration, all
compounds might be able to penetrate into the CNS and therefore, are considered as active
compound on the CNS. Moreover, all the compounds may not show either acute toxicity
according to the calculated LC50 values nor mutagenic effect with respect to the AMES test
9

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data. In addition, the Lipinski criteria for the target compounds were predicted and the data are
provided in the Supplementary data. According to the Lipinski criteria of drug likeness, all
compounds were within the range set by Lipinski’s rule-of-five and could be good candidate for
drug development.
3
.4. Kinetic study of AChE inhibition
The mechanism of the enzyme inhibition was investigated using the most active compound 6g.
Three fixed concentrations of the inhibitor (2.1 nM, 21 nM and 105 nM) were chosen and for
each concentration, the initial velocity (V) of the substrate hydrolysis was measured at different
substrate (S) concentrations in the range of 0.14-0.69 mM. A plot of the reciprocal of the initial
velocity (1/V) versus the reciprocal of the substrate concentration (1/S) was thereafter obtained.
The Lineweaver–Burk plot showed increasing slopes and intercepts with higher inhibitor
concentrations (Figure 10). It was therefore concluded that a mixed-type inhibition could be
attributed to the compound 6g. The kinetic study was in agreement with the docking studies in
which a dual binding site interaction with the receptor was observed.
4
. Conclusion
In conclusion, we described a series of fused coumarins namely 5-oxo-4,5-dihydropyrano[3,2-
c]chromenes linked to N-benzylpyridinium scaffold as new acetylcholinesterase inhibitors. Two
distinct 3-pyridinium and 4-pyridinium derivatives were synthesized from appropriate pyridine-
carbaldehyde, malononitrile and 4-hydroxycoumarin, and subsequent N-benzylation. The results
of anticholinesterase activity toward AChE and BuChE revealed that the type and position of
halogen on the benzyl pendent could regulate anti-AChE activity and AChE/BuChE selectivity.
1
0

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The 1-(4-fluorobenzyl) derivative 6g from 4-pyridinium series showed the most potent anti-
AChE activity (IC value = 0.038 ꢀM) and the highest AChE/BuChE selectivity (SI>48). Our
5
0
docking study demonstrated the capability of prototype compounds to interact with the two
distinct regions in the active site of AChE; the catalytic site and the peripheral anionic site.
Moreover, the docking simulations permitted us to rationalize the observed structure–affinity
relationships.
5
. Experimental
5
.1. General methods
All commercially available reagents were used without further purification. The inorganic–
organic hybrid magnetic nanocatalyst was synthesized following the protocol previously
described by our group [18]. TLC was conducted on silica gel 250 micron, F254 plates. Melting
points were measured with a Kofler hot stage apparatus and are uncorrected. The IR spectra were
1
taken using Nicolet FT-IR Magna 550 spectrographs (KBr disks). H NMR spectra were
recorded on an NMR instrument Bruker 500 MHz. The chemical shifts (δ) and coupling
constants (J) are expressed in parts per million and hertz, respectively. Mass spectra of the
products were obtained with an HP (Agilent Technologies) 5937 Mass Selective Detector.
Elemental analyses were carried out by a CHN-Rapid Heraeus elemental analyzer. The results of
elemental analyses (C, H, N) were within ± 0.4% of the calculated values.
5
.2. General procedure for the synthesis of 2-amino-5-oxo-4-(pyridin-3 or 4-yl)-4,5-
dihydropyrano[3,2-c]chromene-3-carbonitriles (5a or 5b)
1
1

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A mixture of pyridine-carbaldehyde 1 (1 mmol), malononitrile (2, 1.2 mmol), magnetic catalytic
system (1.5 mole %) and water (5 ml) was stirred at reflux for a few minutes. Then, 4-
hydroxycoumarin (4, 1 mmol) was added. The mixture was refluxed for 30 minutes. After
completion of the reaction which was monitored by TLC, it was allowed to cool at room
temperature. The reaction mixture was diluted with ethyl acetate and the catalyst was easily
separated with an external magnet and washed twice with ethyl acetate. The solvent was
removed in vacuo, and the resultant solid was purified by recrystallization from ethanol.
5
.2.1. 2-Amino-5-oxo-4-(pyridin-4-yl)-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (5a)
-1
Yield 70%; white solid; mp 270-272 ºC [18]; IR (KBr, cm ) ν : 3354 and 3273 (NH ), 2194
max
2
1
(
CN), 1715 (C=O); H NMR (500 MHz, DMSO-d ) δ: 8.51 (d, 2H, J = 4.5 Hz, pyridine), 7.91
6
(
d, 1H, J = 7.5 Hz, H ), 7.72 (t, 1H, J = 7.5 Hz, H ), 7.53 (s, 2H, NH ), 7.51-7.45 (m, 2H, H ),
1
0
8
2
7,9
1
3
7
1
.33 (d, 2H, J = 4.5 Hz, pyridine), 4.51 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 159.5,
4 6
58.1, 154.0, 152.2, 151.7, 149.6, 133.1, 124.6, 122.9, 122.5, 118.8, 116.5, 112.8, 102.4, 56.4.
5
.2.2. 2-Amino-5-oxo-4-(pyridin-3-yl)-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (5b)
-1
Yield 79%; white solid; mp 255-257 ºC [18]; IR (KBr, cm ) ν : 3374 and 3284 (NH ), 2193
max
2
1
(
CN), 1708 (C=O); H NMR (500 MHz, DMSO-d ) δ: 8.85 (br s, 1H, pyridine), 8.46 (br s, 1H,
6
pyridine), 7.89 (br s, 1H, pyridine), 7.70 (s, 2H, NH ), 7.50-7.35 (m, 5H, H
and pyridine),
2
7-10
1
3
4
1
.55 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 159.5, 158.0, 153.7, 152.1, 149.0, 148.2,
4 6
38.7, 135.4, 132.9, 124.6, 123.7, 122.5, 119.0, 116.5, 112.8, 102.9, 56.9.
1
2

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5
.3. General procedure for the synthesis of 3- or 4-(2-amino-3-cyano-5-oxo-4,5-
dihydropyrano[3,2-c]chromen-4-yl)-1-(benzyl)pyridinium chloride derivatives 6a-o
Compound 5 (1 mmol), appropriate benzyl chloride (1.2 mmol) and a catalytic amount of KI in
dry acetonitrile (5 ml) was refluxed for 10-16 h. When the reaction was completed as indicated
by TLC, the mixture was then concentrated under reduced pressure, and 5 ml of diethyl ether
was added. The products 6a-o were collected by filtration in good yields.
5
.3.1. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2-
fluorobenzyl)pyridinium chloride (6a)
Yield 90%; yellow solid; mp 191-194 ºC; IR (KBr, cm ) ν : 3423 and 3321 (NH ), 2189 (CN),
-1
max
2
1
1
718 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.12 (d, 2H, pyridine, J = 5.9 Hz), 8.23 (d, 2H,
6
pyridine, J = 5.9 Hz), 7.94 (d, 1H, H , J = 7.5 Hz), 7.85 (s, 2H, NH ), 7.76 (t, 1H, H , J = 7.5
1
0
2
8
Hz), 7.63 (t, 1H, phenyl, J = 7.1 Hz), 7.55-7.43 (m, 3H, H7,9 and phenyl), 7.33-7.29 (m, 2H,
1
3
phenyl), 5.95 (s, 2H, CH ), 4.93 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 162.0, 161.5,
2
4
6
1
1
59.8, 159.6, 158.4, 154.9, 152.4, 145.1, 133.5, 132.2, 131.6, 127.4, 125.3, 124.9, 122.8, 121.2,
+
18.5, 116.7, 116.2, 116.0, 112.9, 100.8, 57.4, 54.5. EI-MS (m/z, %) 426 (M , 2), 313 (61), 121
(
85), 109 (100), 83 (70). Anal. Calcd for C H ClFN O : C, 65.01; H, 3.71; N, 9.10. Found: C,
25 17 3 3
6
5.35; H, 3.54; N, 8.92.
5
.3.2. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2-
chlorobenzyl)pyridinium chloride (6b)
Yield 75%; orange solid; mp 146-149 ºC; IR (KBr, cm ) ν : 3451 and 3385 (NH ), 2193 (CN),
-1
max
2
1
1
717 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.06 (d, 2H, pyridine, J = 5.9 Hz), 8.23 (d, 2H,
6
1
3

ACCEPTED MANUSCRIPT
pyridine, J =5.9 Hz), 7.92 (d, 1H, H , J = 7.5 Hz), 7.82 (s, 2H, NH ), 7.77 (t, 1H, H , J = 7.5
1
0
2
8
1
3
Hz), 7.61-7.48 (m, 6H, H7,9 and phenyl), 5.97 (s, 2H, CH ), 4.96 (s, 1H, H ). C NMR (125
2
4
MHz, DMSO-d ) δ: 162.4, 161.1, 155.0, 153.1, 144.9, 144.0, 133.2, 131.9, 131.7, 131.4, 131.2,
6
1
30.1, 128.1, 127.8, 125.9, 125.3, 122.6, 121.7, 119.4, 115.8, 65.6, 60.4. Anal. Calcd for
C H Cl N O : C, 62.77; H, 3.58; N, 8.78. Found: C, 62.54; H, 3.44; N, 9.06.
25
17
2
3
3
5
.3.3. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2-
methylbenzyl)pyridinium chloride (6c)
Yield 91%; yellow solid; mp 233-236 ºC; IR (KBr, cm ) ν : 3427 and 3239 (NH ), 2189 (CN),
-1
max
2
1
1
727 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.05 (d, 2H, pyridine, J = 6.5 Hz), 8.23 (d, 2H,
6
pyridine, J = 6.5 Hz), 7.97 (d, 1H, H , J = 7.5 Hz), 7.88 (s, 2H, NH ), 7.77 (t, 1H, H , J = 7.5
10
2
8
Hz), 7.54-7.48 (m, 2H, H ), 7.36-7.27 (m, 3H, phenyl), 7.17 (d, 1H, phenyl, J = 7.4 Hz), 5.94
7
,9
1
3
(
s, 2H, CH ), 4.94 (s, 1H, H ), 2.26 (s, 3H, CH ). C NMR (125 MHz, DMSO-d ) δ: 161.8,
2 4 3 6
1
1
4
59.7, 158.4, 154.8, 152.4, 145.0, 136.9, 133.4, 132.2, 130.9, 129.4, 129.2, 127.2, 126.6, 124.8,
22.8, 118.4, 116.6, 112.8, 100.7, 60.9, 54.4, 18.7. Anal. Calcd for C H ClN O : C, 68.20; H,
2
6
20
3
3
.40; N, 9.18. Found: C, 67.92; H, 4.11; N, 9.32.
5
.3.4. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
fluorobenzyl)pyridinium chloride (6d)
Yield 93%; yellow solid; mp 247-250 ºC; IR (KBr, cm ) ν : 3469 and 3395 (NH ), 2193 (CN),
-1
max
2
1
1
719 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.25 (d, 2H, pyridine, J = 5.8 Hz), 8.23 (d, 2H,
6
pyridine, J = 5.8 Hz), 7.95 (d, 1H, H , J = 7.6 Hz), 7.84 (s, 2H, NH ), 7.76 (t, 1H, H , J = 7.6
10
2
8
Hz), 7.55-7.37 (m, 5H, H7,9 and phenyl), 7.32-7.25 (m, 1H, phenyl), 5.87 (s, 2H, CH ), 4.92 (s,
2
1
4

ACCEPTED MANUSCRIPT
1
3
1
1
5
9
H, H ). C NMR (125 MHz, DMSO-d ) δ: 161.8, 159.7, 158.4, 154.9, 152.4, 144.9, 133.4,
4 6
31.4, 127.3, 125.1, 124.8, 122.8, 118.4, 116.6, 116.4, 116.2, 116.1, 115.9, 112.9, 100.7, 61.8,
4.4. Anal. Calcd for C H ClFN O : C, 65.01; H, 3.71; N, 9.10. Found: C, 65.28; H, 3.64; N,
2
5
17
3
3
.29.
5
.3.5. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
methylbenzyl)pyridinium chloride (6e)
Yield 92%; yellow solid; mp 244-247 ºC; IR (KBr, cm ) ν : 3472 and 3243 (NH ), 2194 (CN),
-1
max
2
1
1
722 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.22 (d, 2H, pyridine, J = 6.5 Hz), 8.22 (d, 2H,
6
pyridine, J = 6.5 Hz), 7.95 (d, 1H, H , J = 7.8 Hz), 7.85 (s, 2H, NH ), 7.76 (t, 1H, H , J = 7.8
10
2
8
Hz), 7.52 (t, 1H, H , J = 7.8 Hz), 7.48 (d, 1H, H , J = 7.8 Hz), 7.40 (s, 1H, phenyl), 7.37 (d, 1H,
9
7
phenyl, J = 7.5 Hz), 7.32 (t, 1H, phenyl, J = 7.5 Hz), 7.23 (d, 1H, phenyl, J = 7.5 Hz), 5.84 (s,
1
3
2
1
1
H, CH ), 4.91 (s, 1H, H ), 2.30 (s, 3H, CH ). C NMR (125 MHz, DMSO-d ) δ: 161.6, 159.7,
2 4 3 6
58.3, 155.8, 154.9, 152.3, 144.8, 138.6, 134.1, 133.4, 130.0, 129.4, 129.1, 127.3, 125.9, 124.8,
+
22.8, 118.4, 116.6, 112.8, 100.7, 62.6, 54.4, 20.8. EI-MS (m/z, %) 422 (M , 3), 239 (36), 162
(
38), 121 (63), 105 (100), 91 (73), 77 (67). Anal. Calcd for C H ClN O : C, 68.20; H, 4.40; N,
26 20 3 3
9
.18. Found: C, 68.42; H, 4.14; N, 9.02.
5
.3.6. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
chlorobenzyl)pyridinium chloride (6f)
Yield 90%; yellow solid; mp 240-242 ºC; IR (KBr, cm ) ν : 3465 and 3395 (NH ), 2193 (CN),
-1
max
2
1
1
718 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.22 (d, 2H, pyridine, J = 5.9 Hz), 8.22 (d, 2H,
6
pyridine, J = 5.9 Hz), 7.94 (d, 1H, H , J = 7.5 Hz), 7.83 (s, 2H, NH ), 7.76-7.74 (m, 2H, H and
1
0
2
8
1
5

ACCEPTED MANUSCRIPT
1
3
phenyl), 7.55-7.48 (m, 5H, H7,9 and phenyl), 5.83 (s, 2H, CH ), 4.92 (s, 1H, H ). C NMR (125
2
4
MHz, DMSO-d ) δ: 161.8, 159.7, 158.3, 154.9, 152.3, 144.9, 136.3, 133.6, 133.4, 131.1, 129.4,
6
1
4
29.0, 127.7, 127.4, 124.8, 122.7, 118.4, 116.6, 112.8, 100.7, 61.8, 54.4, 36.5. EI-MS (m/z, %)
+
42 (M , 2), 223 (100), 150 (95), 127 (24), 125 (63), 91 (91), 57 (92), 43 (74). Anal. Calcd for
C H Cl N O : C, 62.77; H, 3.58; N, 8.78. Found: C, 63.05; H, 3.74; N, 8.92.
25
17
2
3
3
5
.3.7. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(4-
fluorobenzyl)pyridinium chloride (6g)
Yield 89%; yellow solid; mp > 250 ºC; IR (KBr, cm ) ν : 3478 and 3395 (NH ), 2194 (CN),
-1
max
2
1
1
713 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.23 (d, 2H, pyridine, J = 5.9 Hz), 8.23 (d, 2H,
6
pyridine, J =5.9 Hz), 7.95 (d, 1H, H , J = 7.5 Hz), 7.85 (s, 2H, NH ), 7.78-7.70 (m, 3H, H , 2H
10
2
8
phenyl), 7.54-7.47 (m, 2H, H ), 7.37-7.27 (m, 2H, phenyl), 5.84 (s, 2H, CH ), 4.92 (s, 1H, H ).
7
,9
2
4
Anal. Calcd for C H ClFN O : C, 65.01; H, 3.71; N, 9.10. Found: C, 65.20; H, 3.63; N, 9.38.
2
5
17
3
3
5
.3.8. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3,4-
dichlorobenzyl)pyridinium chloride (6h)
Yield 70%; orange solid; mp > 250 ºC; IR (KBr, cm ) ν : 3431 and 3375 (NH ), 2193 (CN),
-1
max
2
1
1
717 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.26 (d, 2H, pyridine, J = 5.9 Hz), 8.22 (d, 2H,
6
pyridine, J = 5.9 Hz), 8.02 (s, 1H, phenyl), 7.95 (d, 1H, H , J = 7.5 Hz), 7.86 (s, 2H, NH ), 7.74
1
0
2
(t, 1H, H , J = 7.5 Hz), 7.64 (d, 1H, phenyl, J = 7.5 Hz), 7.53-7.49 (m, 4H, H and phenyl), 5.87
8 7,9
(
s, 2H, CH ), 4.92 (s, 1H, H ). Anal. Calcd for C H Cl N O : C, 58.56; H, 3.15; N, 8.19.
2
4
25 16
3
3
3
Found: C, 58.35; H, 3.27; N, 8.34.
1
6

ACCEPTED MANUSCRIPT
5
.3.9. 4-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2,4-
dichlorobenzyl)pyridinium chloride (6i)
Yield 77%; orange solid; mp > 250 ºC; IR (KBr, cm ) ν : 3451 and 3385 (NH ), 2193 (CN),
-1
max
2
1
1
717 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.06 (d, 2H, pyridine, J = 5.9 Hz), 8.23 (d, 2H,
6
pyridine, J = 5.9 Hz), 7.94 (d, 1H, H , J = 7.5 Hz), 7.87 (s, 2H, NH ), 7.85-7.77 (m, 2H, H and
1
0
2
8
phenyl), 7.66-7.49 (m, 4H, H and phenyl), 5.98 (s, 2H, CH ), 4.96 (s, 1H, H ). Anal. Calcd for
7
,9
2
4
C H Cl N O : C, 58.56; H, 3.15; N, 8.19. Found: C, 58.35; H, 3.27; N, 8.34.
25
16
3
3
3
5
.3.10. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2-
fluorobenzyl)pyridinium chloride (6j)
Yield 92%; green solid; mp 222-225 ºC; IR (KBr, cm ) ν : 3433 and 3388 (NH ), 2190 (CN),
-1
max
2
1
1
5
708 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.37 (s, 1H, pyridine), 9.03 (d, 1H, pyridine, J =
6
.8 Hz), 8.73 (d, 1H, pyridine, J = 6.8 Hz), 8.17-8.15 (m, 1H, pyridine), 7.96 (d, 1H, H , J = 7.6
10
Hz), 7.81-7.76 (m, 3H, NH and H ), 7.55-7.40 (m, 4H, H and phenyl), 7.45-7.21 (m, 2H,
2
8
7,9
1
3
phenyl), 5.98 (s, 2H, CH ), 4.92 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 159.7, 158.3,
2
4
6
1
54.5, 152.3, 145.6, 144.7, 144.0, 143.8, 133.3, 131.9, 130.8, 128.2, 125.1, 124.7, 122.8, 118.5,
16.6, 116.0, 115.8, 112.8, 100.9, 57.9, 54.9. Anal. Calcd for C H ClFN O : C, 65.01; H, 3.71;
1
25
17
3
3
N, 9.10. Found: C, 65.28; H, 3.56; N, 8.89.
5
.3.11. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(2-
chlorobenzyl)pyridinium chloride (6k)
Yield 87%; green solid; mp 222-225 ºC; IR (KBr, cm ) ν : 3458 and 3385 (NH ), 2189 (CN),
-1
max
2
1
1
709 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.41 (s, 1H, pyridine), 8.94 (d, 1H, pyridine, J =
6
1
7

ACCEPTED MANUSCRIPT
6
.1 Hz), 8.76 (d, 1H, pyridine, J = 7.7 Hz), 8.17-8.15 (m, 1H, pyridine), 7.97 (d, 1H, H , J = 7.4
10
Hz), 7.82 (s, 2H, NH ), 7.76 (t, 1H, H , J = 7.4 Hz), 7.60-7.42 (m, 5H, H and phenyl), 7.32 (d,
2
8
7,9
1
3
1
1
1
H, phenyl, J = 7.4 Hz), 6.01 (s, 2H, CH ), 4.93 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ:
2 4 6
59.7, 158.3, 154.5, 152.3, 145.6, 145.3, 143.9, 143.8, 133.4, 133.0, 131.6, 131.2, 130.8, 130.0,
28.2, 128.0, 124.8, 122.8, 118.6, 116.6, 112.8, 100.9, 61.3, 55.0. Anal. Calcd for
C H Cl N O : C, 62.77; H, 3.58; N, 8.78. Found: C, 62.41; H, 3.75; N, 8.73.
25
17
2
3
3
5
.3.12. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
fluorobenzyl)pyridinium chloride (6l)
Yield 89%; yellow solid; mp 188-190 ºC; IR (KBr, cm ) ν : 3442 and 3395 (NH ), 2194 (CN),
-1
max
2
1
1
6
7
713 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.46 (s, 1H, pyridine), 9.13 (d, 1H, pyridine, J =
6
.1 Hz), 8.73 (d, 1H, pyridine, J = 8.0 Hz), 8.17-8.15 (m, 1H, pyridine), 7.94 (dd, 1H, H , J =
1
0
.9 and 1.5 Hz), 7.79 (s, 2H, NH ), 7.76 (dt, 1H, H , J = 7.9 and 1.5 Hz), 7.55-7.45 (m, 3H, H
7,9
2
8
and phenyl), 7.39-7.35 (m, 1H, phenyl), 7.31 (d, 1H, phenyl, J = 7.7 Hz), 7.28-7.24 (m, 1H,
1
3
phenyl), 5.87 (s, 2H, CH ), 4.90 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 163.1, 161.1,
2
4
6
1
1
59.8, 158.4, 154.6, 152.4, 145.5, 144.4, 144.1, 143.8, 136.7, 133.4, 131.2, 128.4, 124.8, 124.5,
22.8, 118.7, 116.6, 116.3, 115.5, 115.4, 112.9, 100.9, 62.4, 55.0. Anal. Calcd for
C H ClFN O : C, 65.01; H, 3.71; N, 9.10. Found: C, 65.24; H, 3.58; N, 9.33.
25
17
3
3
5
.3.13. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
chlorobenzyl)pyridinium chloride (6m)
Yield 90%; yellow solid; mp 217-221 ºC; IR (KBr, cm ) ν : 3467 and 3398 (NH ), 2193 (CN),
-1
max
2
1
1
715 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.48 (s, 1H, pyridine), 9.17 (d, 1H, pyridine, J =
6
1
8

ACCEPTED MANUSCRIPT
6
.1 Hz), 8.72 (d, 1H, pyridine, J = 7.7 Hz), 8.17-8.15 (m, 1H, pyridine), 7.97 (d, 1H, H , J = 7.7
10
Hz), 7.81 (s, 2H, NH ), 7.76 (t, 1H, H , J = 7.7 Hz ), 7.63 (s, 1H, phenyl), 7.54-7.44 (m, 5H,
2
8
1
3
phenyl and H ), 5.88 (s, 2H,), 4.90 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 159.7,
7
,9
4
6
1
1
3
58.3, 154.5, 152.3, 145.4, 144.4, 144.1, 143.7, 136.5, 133.6, 133.4, 131.0, 129.2, 128.4, 127.1,
24.7, 122.8, 118.6, 116.6, 112.8, 100.9, 62.3, 54.9. Anal. Calcd for C H Cl N O : C, 62.77; H,
2
5
17
2
3
3
.58; N, 8.78. Found: C, 62.93; H, 3.44; N, 8.86.
5
.3.14. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(3-
methylbenzyl)pyridinium chloride (6n)
Yield 90%; yellow solid; mp 195-198 ºC; IR (KBr, cm ) ν : 3426 and 3343 (NH ), 2202 (CN),
-1
max
2
1
1
6
719 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.47 (s, 1H, pyridine), 9.10 (d, 1H, pyridine, J =
6
.1 Hz), 8.71 (d, 1H, pyridine, J = 7.5 Hz), 8.18-8.16 (m, 1H, pyridine), 7.96 (d, 1H, H , J = 7.6
10
Hz), 7.81-7.76 (m, 3H, NH and H ), 7.55-7.48 (m, 2H, H ), 7.35-7.21 (m, 5H, phenyl), 5.83 (s,
2
8
7,9
1
3
2
1
1
6
H, CH ), 4.91 (s, 1H, H ), 2.27 (s, 3H, Me). C NMR (125 MHz, DMSO-d ) δ: 159.7, 158.3,
2 4 6
54.6, 152.4, 145.2, 144.3, 144.1, 143.6, 138.5, 134.3, 133.4, 129.7, 129.0, 128.6, 128.3, 125.2,
24.8, 122.7, 118.6, 116.6, 112.9, 100.9, 63.2, 55.0, 20.8. Anal. Calcd for C H ClN O : C,
2
6
20
3
3
8.20; H, 4.40; N, 9.18. Found: C, 68.33; H, 4.24; N, 9.03.
5
.3.15. 3-(2-Amino-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-4-yl)-1-(4-
fluorobenzyl)pyridinium chloride (6o)
Yield 74%; yellow solid; mp > 250 ºC; IR (KBr, cm ) ν : 3428 and 3379 (NH ), 2194 (CN),
-1
max
2
1
1
5
713 (C=O); H NMR (500 MHz, DMSO-d ) δ: 9.45 (s, 1H, pyridine), 9.13 (d, 1H, pyridine, J =
6
.3 Hz), 8.71 (d, 1H, pyridine, J = 7.5 Hz), 8.16-8.14 (m, 1H, pyridine), 7.96 (d, 1H, H , J = 7.4
10
1
9

ACCEPTED MANUSCRIPT
Hz), 7.80-7.75 (m, 3H, H and NH ), 7.59-7.47 (m, 4H, H and phenyl), 7.30-7.26 (m, 2H,
8
2
7,9
1
3
phenyl), 5.86 (s, 2H, CH ), 4.90 (s, 1H, H ). C NMR (125 MHz, DMSO-d ) δ: 163.4, 161.5,
2
4
6
1
59.7, 158.3, 154.5, 152.4, 145.3, 144.2, 144.0, 143.5, 133.4, 131.0, 130.5, 128.3, 124.8, 122.7,
18.6, 116.6, 116.1, 115.9, 112.9, 100.9, 62.4, 55.0. Anal. Calcd for C H ClFN O : C, 65.01;
1
25
17
3
3
H, 3.71; N, 9.10. Found: C, 65.20; H, 3.63; N, 9.38.
5
.4. Pharmacology
The inhibitory potency of target compounds on AChE and BuChE was determined using
Ellman’s method [20]. Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder,
from electric eel, 1000 units), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum)
and butyrylthiocholine iodide were obtained from Sigma–Aldrich. 5,5′-Dithiobis-(2-nitrobenzoic
acid) (DTNB), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium
hydroxide, sodium hydrogen carbonate, and acetylthiocholine iodide were purchased from Fluka.
The stock solutions of testing compounds were prepared by dissolving them in DMSO (1 ml)
-3
and phosphate buffer (9 ml). Five concentrations of the testing compounds in the range from 10
-7
to 10 M were tested. The reaction mixture included 100 µl of DTNB 0.1 M, 50 µl of enzyme (2
U/ml), 50 µl of inhibitor solution and 10 µl of substrate (acetylthiocholine iodide or
butyrylthiocholine iodide) 0.15 M. The changing of the absorbance was measured at 412 nm for
2
min. The IC50 values were determined graphically from inhibition curves (log inhibitor
concentration vs. percent of inhibition). A UV-2100 Rayleigh Double Beam Spectrophotometer
was used for spectrophotometric measurements.
5
.5. Molecular docking study
2
0

ACCEPTED MANUSCRIPT
The pdb structure of 3I6Z was retrieved for docking purposes as a complex co-crystallized with
inhibitor N-sacharinohexyl-galantamine. Then, the amino acid chain was kept and the water
molecules and co-crystallized ligand were removed and subsequently the missing atom types
were repaired using Modeller 9.11. Afterward, the polar hydrogen was added to the receptor and
the resulting protein was subjected to minimization using OPLS2005 force field. The prepared
protein was saved in pdbqt format using Autodock Tools 1.5.4 [21]. The ligand coordinates were
generated using MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com), which
was converted to 3D structure using Openbabel version (2.3.1) [22]. Finally the pdbqt formats
(The input format of docking software) of the ligands were prepared with Autodock Tools 1.5.4
using default parameters. Autodock Vina (ver. 1.1.1) [23] was used for docking calculations with
default parameters except for exhaustiveness that was set to 80. For all the docking calculations,
a grid box size of 40 × 40 × 40, centered at the geometrical center of co-crystallized ligand was
used. The coordinates x, y, z for the center of grid box were 3.755, 63.699, 65.983, respectively.
To validate our docking procedure, the co-crystallized ligand was re-docked into the active site
of the enzyme and the reasonable RMSD value of 1.947 Å was obtained. Finally, the
conformations with the most favorable free energy of binding were selected for analyzing the
interactions between the AChE and inhibitor. All the 3D models are generated using the Chimera
1
.6 software [24]. The same procedure was applied in the case of BuChE.
Acknowledgments
This work was supported by grants from the Research Council of Tehran University of Medical
Sciences, Iran National Elite Foundation (INEF) and the Iran National Science Foundation
(
INSF). Molecular graphics and analyses were performed with the UCSF Chimera package.
2
1

ACCEPTED MANUSCRIPT
Chimera is developed by the 25 Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco.
References
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1] A.V. Terry, Jr., J.J. Buccafusco, The cholinergic hypothesis of age and Alzheimer's disease-
related cognitive deficits: recent challenges and their implications for novel drug
development, J. Pharmacol. Exp. Ther. 306 (2003) 821-7.
[
2] M.G. Cardozo, T. Kawai, Y. Iimura, H. Sugimoto, Y. Yamanishi, A.J. Hopfinger,
Conformational analyses and molecular-shape comparisons of a series of indanone-
benzylpiperidine inhibitors of acetylcholinesterase, J. Med. Chem. 35 (1992) 590-601.
[
[
3] M.R. Roberson, L.E. Harrell, Cholinergic activity and amyloid precursor protein metabolism,
Brain Res. Brain Res. Rev. 25 (1997) 50-69.
4] R.E. Fine, The biochemistry of Alzheimer disease, Alzheimer Dis. Assoc. Disord. 13 Suppl 1
(1999) S82-S87.
[
[
5] J.L. Cummings, Alzheimer's disease, N. Engl. J. Med. 351 (2004) 56-67.
6] a) E. Giacobini, Cholinesterase inhibitors: new roles and therapeutic alternatives, Pharmacol.
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Figure Captions:
Figure 1. Structures of well known AChE inhibitors.
Figure 2. Structures of coumarin containing AChE inhibitors, and designed fused coumarins 6a-
o as new inhibitors.
Figure 3. The best pose of the compounds 6g and 6n obtained from docking study in the active
site of AChE. The catalytic site (CS), central anionic site (CAS) and peripheral anionic site
(PAS) were shown in the receptor. The blue and yellow colors were attributed to the 6g and 6n
respectively.
Figure 4. A representative model for interaction of compound 6g and the AChE. The π-π
interaction is illustrated as a cyan dashed line. The calculated electrostatic potential map for
Asp72, Val71, Tyr70 and Tyr121 is shown and partial charge for an amino group as well. (Red
color: negatively charged region; Blue color: positively charged region).
Figure 5. The effect of halogen position on the orientation of phenyl ring toward Trp279. Left:
2
2
-fluoro and 4-fluoro derivatives (6a and 6g) are shown in brown and green respectively. Right:
-chloro and 3-chloro analogs (6b and 6f) are illustrated in red and yellow, respectively.
Figure 6. Illustration of binding mode of 6h (blue) and 6i (red) in the active site of the enzyme.
The calculated molecular surface for three amino acids in terms of their lipophilicity is shown.
(
The more hydrophilic surface is represented as pink)
Figure 7. Compound 6e in the active site of the AChE enzyme. The probable Pi-H interaction of
-methyl group with Tyr70 is shown by dashed line.
3
Figure 8. An additional Pi-H interaction of compound 6n with the Tyr70.
Figure 9. The predicted binding mode of compound 6i in the active site of BuChE.
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5

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Figure 10. The steady state inhibition of compound 6g, against AChE. Reciprocal plot was
based on the initial velocity and substrate concentration on increasing inhibitor concentrations.
Lines were obtained from weighted least square analysis of the data points.
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Table 1
Inhibition of AChE from Electrophorus electricus (eelAChE) and horse serum butyrylcholinesterase
(
eqBuChE) by compounds 6a-o in comparison with donepezil.
a
b
Compound
R
IC50 (µM)
eelAChE
2.9 ± 0.17
IC (µM)
Selectivity
5
0
eqBuChE
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
2-F
36 ± 2.28
12.4
3.2
2-Cl
2-CH
3-F
1.5 ± 0.09
4.8 ± 0.3
3.7 ± 0.21
17.8 ± 1.13
>50
4.8
3
3
2.8 ± 0.16
>17.8
2.6
3-CH
3-Cl
4-F
1.9 ± 0.11
5.5 ± 0.35
21.8 ± 1.38
1.84 ± 0.12
1.27 ± 0.08
0.566 ± 0.004
41 ± 2.6
0.48 ± 0.03
0.038 ± 0.002
0.044 ± 0.002
0.831 ± 0.048
1.8 ± 0.1
45.4
48.4
28.9
0.68
22.8
11.2
>25
5.2
g
h
i
3,4-Cl
2,4-Cl
2-F
j
k
l
2-Cl
3-F
1.0 ± 0.06
11.2 ± 0.71
>50
2.0 ± 0.12
m
n
o
3-Cl
7.4 ± 0.43
38.4 ± 2.4
14.3 ± 0.9
2.17 ± 0.14
5.38 ± 0.34
3-CH
4-F
0.79 ± 0.04
2.17 ± 0.12
0.014± 0.0008
18.1
1
3
Donepezil
384.3
a
Data are expressed as Mean ± S.E. of at least three different experiments.
Selectivity for AChE = IC (BuChE)/IC (AChE).
b
5
0
50
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Table 2
Predicted ADMET properties of the target compounds 6a-o.
a
Compound
R
BBB penetration
HIA
%
Caco-2
AMES
Acute Toxicity
b
c
d
Permeability
Toxicity
LC50 (µmol)
%
CNS activity
6
6
a
2-F
2-Cl
2-CH
3-F
0.84
0.80
0.80
0.84
0.80
0.80
0.84
0.80
0.80
0.84
0.80
0.84
0.80
0.80
0.84
0.99
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.98
0.98
0.99
0.98
0.99
0.98
0.98
0.98
0.98
0.98
0.98
0.98
0.98
0.99
0.98
0.99
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.85
1.90
2.65
2.85
2.65
1.90
2.85
1.73
1.73
2.72
1.81
2.72
1.81
2.54
2.72
3
b
6
c
3
3
+
-
6
d
6
e
3-CH
3-Cl
4-F
+
-
6
f
6
6
g
-
h
3,4-Cl
2,4-Cl
2-F
-
6
i
-
6
j
-
6
k
2-Cl
3-F
-
6
l
-
6
m
3-Cl
-
6
n
o
3-CH
4-F
-
3
+
-
6
Donepezil
+
a
Human Intestinal Absorption
b
The values more than 50 was considered as +
c
Mutagenic potential of chemicals
d
EPA v4b Fathead Minnow Acute Toxicity
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