Identification of new chromenone derivatives as cholinesterase inhibitors and molecular docking studies

Article abstract of DOI:10.2174/1573406414666180222091833

Background: Alzheimer's Disease (AD) is the leading cause of dementia among the aging population. This devastating disorder is generally associated with the gradual memory loss, specified by a decrease of acetylcholine level in the cortex hippocampus of t

Full text of DOI:10.2174/1573406414666180222091833

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Medicinal Chemistry, 2018, 14, 809-817
RESEARCH ARTICLE
ISSN: 1573-4064
eISSN: 1875-6638
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Identification of New Chromenone Derivatives as Cholinesterase Inhibi-
tors and Molecular Docking Studies
Jamshed Iqbala,*, Muhammad S.A. Abbasib, Sumera Zaiba, Saifullah Afridia, Norbert Furtmannc,
Jürgen Bajorathc and Peter Langerb
aCentre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad, Abbottabad-22060,
b
c
Pakistan; Institut für Chemie, Universität Rostock, Albert Einstein Str. 3A, 18059 Rostock, Germany; Department of
Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-
Wilhelms-Universität, Dahlmannstr. 2, D-53113 Bonn, Germany
Abstract: Background: Alzheimer's Disease (AD) is the leading cause of dementia among the
aging population. This devastating disorder is generally associated with the gradual memory loss,
specified by a decrease of acetylcholine level in the cortex hippocampus of the brain due to
hyperactivation of cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE)).
Objective: Therefore, inactivation of AChE and BChE by inhibitors can increase the acetylcholine
level and hence may be an encouraging strategy for the treatment of AD and related neurological
problems.
Method: In this contribution, two series of chromenone-based derivatives were tested by Ell-
A R T I C L E H I S T O R Y
mann’s calorimetric method for AChE and BChE inhibition.
Received: September 16, 2017
Revised: January 27, 2018
Accepted: January 29, 2018
Results: All the compounds showed inhibitory activity against cholinesterases and some of them
exhibited dual inhibition of AChE as well as BChE. The most potent inhibitor of AChE was 2l
having an IC50 value of 0.08 0.03 ꢀM, while 3q inhibited the BChE with an IC50 value of 0.04
0.01 ꢀM. In case of dual inhibition, 3h showed an inhibitory concentration of 0.15 0.01 ꢀM for
AChE, and 0.09 0.01 ꢀM for BChE. Molecular docking studies were performed to explore the
probable binding modes of the most potent dual inhibitors.
DOI:
10.2174/1573406414666180222091833
Conclusion: It can be hypothesized that the inhibitors are able to target cholinesterase pathways
and may emerge as a suitable outset for the further development process.
Keywords: Alzheimer’s disease, amino acid, chromosome, chromenonederivatives, cholinesterase inhibitor, molecular docking.
1. INTRODUCTION
volved in neurotransmission located at all autonomic gan-
glia, cholinergic brain synapses and neuromuscular junc-
tions, and plays a crucial role in the termination of signal
transmission by hydrolyzing ACh [3, 5, 6]. AChE also acts
as an adhesion protein in bone matrix, as well as supporting
neurite growth and amyloid fibrils production, mostly found
in the brain cells of Alzheimer’s patients [3, 7, 8]. Currently,
the inhibition of ChEs represents a promising strategy for the
symptomatic treatment and management of AD [9]. Cho-
linergic neurons are mainly located in regions of the fore-
brain that are associated with learning and memory [10] and
hippocampus [11]. By inhibiting AChE the degradation of
ACh is reduced, and in this way, ACh levels can be elevated,
which results in improved learning and memory functions.
The irreversible deficiency of cholinergic transmitters is the
main cause of memory impairment in patients suffering from
senile dementia, therefore, AChE inhibitors can be employed
at the early stage of AD treatment [12].
Cholinesterases (ChEs) are a family of esterase/lipase en-
zymes that share extensive sequence homology and catalyze
the hydrolysis of acetylcholine (ACh). To terminate the de-
sired cholinergic-transmission, ChEs catalyze the acetylcho-
line breakdown into choline and acetic acid [1-3]. Based on
specificities of substrates and inhibitors, different vertebrate
ChEs include acetylcholinesterase (AChE) and butyrylcho-
linesterase (BChE). Amino acid sequence homology in
AChE and BChE is 65% although they are encoded by dif-
ferent genes on the human chromosome (AChE: 7q22,
BChE: 3q26) [4]. AChE (EC 3.1.1.7) is a key enzyme in-
*Address correspondence to this author at the Centre for Advanced Drug
Research, COMSATS Institute of Information Technology, Abbottabad,
Postal Code-22060, Pakistan; Tel: +92-992-383591-96; Fax: +92-992-
383441; E-mails: drjamshed@ciit.net.pk; jamshediqb@googlemail.com
1875-6638/18 $58.00+.00
© 2018 Bentham Science Publishers

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Medicinal Chemistry, 2018, Vol. 14, No. 8
Iqbal et al.
2.4. Cholinesterase Inhibition Studies
Pseudocholinesterase or BChE (E.C. 3.1.1.8), also known
as plasma cholinesterase, is mostly found in the liver and
participates in the control of neurotransmission [7]. It is re-
ported that BChE contributes about 20% and AChE accounts
for 80% of total cholinesterase activity in healthy brains. In
patients suffering from AD, the activity of BChE is found to
increase to about 40% [4, 7].
The capacity to inhibit cholinesterase activity was deter-
mined by Ellmann’s calorimetric method [19] with slight
modifications [5]. The reaction mixture contained 60 ꢂL
phosphate buffer (KH2PO4/ KOH), pH 7.7, 10 ꢂL of enzyme
(0.015 units per well for AChE & BChE) and 10 ꢂL of test
compound (prepared in 1% DMSO and diluted in the buffer).
All the contents were mixed thoroughly and incubated for 10
min at 37 °C. The enzymatic reaction was initiated by the
addition of 10 ꢂL of respective substrates (1 mM acetylthio-
choline chloride or butyrylthiocholine chloride) to their en-
zyme solutions. After that 10 ꢀL of coloring reagent 5,5’-
dithiobis(2-nitrobenzoic acid) (DTNB; 0.5 mM) was added.
The mixture was incubated at 37 °C for 20 min and the ab-
sorbance was measured at 405 nm using a 96-well mi-
croplate reader (BioTek ELx800, Instruments, Inc. USA).
Blank controls without enzymes were also prepared in order
to check the non-enzymatic hydrolysis of substrates. The
experiments were carried out in triplicate. Galanthamine (0.1
mM) was used as a standard inhibitor. Similarly, to deter-
mine the enzyme activity, the reaction without inhibitor was
performed. The percentage inhibition was calculated by the
following formula:
A characteristic feature of AD patients is the presence of
neurofibrillary tangles (NFT) and ꢁ-amyloid plaques, in
which large quantities of ChEs are found, more specifically
BChE. Therefore, the possible involvement of ChEs in the
formation of tangles and plaques activates the microglia and
thus hydrolyzes acetylcholine [13]. Hence, ChE inhibitors
not only play a role to enhance the function of the damaged
brain, but also help in the prevention of further degeneration
[12, 14]. AChE and BChE inhibitors are considered for the
treatment of AD patients. While existing medications (done-
pezil, tacrine and rivastigmine) target AChE, there is cur-
rently no medication available targeting BChE [4]. There-
fore, there is a growing interest in the development of cho-
linesterase inhibitors for the therapy of Alzheimer’s and re-
lated neurological problems.
Very recently, hybrid compounds such as acridine-
chromenones, quinoline-chromenones, and triazole-chrome-
nones have been developed as new anti-Alzheimer’s agents
[15, 16]. However, most of these hybrid compounds exhibit
only moderate potency against AChE. In view of these find-
ings, and to expand the scope of the chromenone skeleton in
medicinal chemistry as anti-Alzheimer’s agents, we intro-
duce herein structurally diverse chromenone derivatives with
modular substitution pattern. These compounds were more
easy to access compared to larger hybrid molecules reported
in the literature [15, 16]. Cholinesterase inhibitory activity
was evaluated and molecular docking studies were carried
out to explore the probable binding modes of potent dual
inhibitors identified in this study.
% age inhibition = 100-(Ac/Af) ꢃ 100
Where “Ac” and “Af” are the absorbance of inhibitor and
control (without inhibitor) minus their respective pre-read
absorbance. The compounds that revealed more than 50%
inhibition for AChE/BChE were further diluted to eight dif-
ferent concentrations for the assessment of IC50 values
(GraphPad PRISM 5.0, San Diego, California, USA).
2.5. Molecular Docking Studies
The X-ray structures of human AChE (PDB ID: 4BDT)
[20] in complex with huprine W and BChE (PDB ID: 4BDS)
[20] in complex with tacrine were obtained from the RCSB
Protein Bank [21]. These structures were used as templates
for flexible ligand docking with AutoDock 4.2 [22]. In the
case of AChE, residue Tyr337 was flexible during docking
while all other residues were held fixed in their crystallo-
graphic conformation. In the case of BChE, the entire active
site was kept rigid including residue Ala328, which corre-
sponds to Tyr337 in AChE. Prior to docking, crystallo-
graphic ligand and water molecules were removed from the
X-ray structures and explicit hydrogen atoms were added
using the Molecular Operating Environment (MOE 2012.10)
[23]. Atomic partial charges were calculated using Auto-
Dock Tools [22]. Selected chromone derivatives were
docked into the active sites of AChE and BChE using Auto-
Dock 4.2 with standard parameter settings. Based on visual
inspection, high-scoring docking poses were selected as pu-
tative binding modes.
2. MATERIALS AND METHODS
2.1. Synthesis of 6-oxo-6H-benzo[c]chromene-8,10-dicar-
boxylate Derivatives
A general scheme and procedure for the synthesis of a
novel series of 6-oxo-6H-benzo[c]chromene-8,10-dicarbo-
xylate derivatives (2a-n) have been reported in a previous
publication [17].
2.2. Synthesis of 6H-benzo[c]chromen-6-ones Derivatives
A general scheme and procedure for the synthesis of a
novel series of 6H-benzo[c]chromen-6-ones (3a-w) have
also been reported previously [18].
3. RESULTS AND DISCUSSION
2.3. Materials and Chemicals
3.1. Synthesis of 6-oxo-6H-benzo[c]chromene-8,10-
dicarboxylate Derivatives
AChE (from electric eel) BChE (from horse serum), ace-
tylthiocholine chloride and butyrylthiocholine chloride and
DTNB were purchased from Sigma Aldrich (St. Louis, MO,
USA) and other chemicals were of analytical grade.
The synthetic route adopted for the preparation of 6-oxo-
6H-benzo[c]chromene-8,10-dicarboxylate derivatives (2a-n)
is illustrated in Scheme 1. The physicochemical properties

Chromenone Derivatives as Cholinesterase Inhibitors
Medicinal Chemistry, 2018, Vol. 14, No. 8 811
Cl
O
O
H
O
(1)
O/R1 O
O
O
O
R1
(2 equiv.)
OR2
R2O
OR2
(1 equiv.)
For 2c-2n
For 2a & 2b
i
i
O
R1
O
R2O
OR2
O
O
2(a-n)
Scheme 1. Synthesis of 6H-benzo[c]chromen-6-ones 2(a-n); Reagents and conditions: (i) K2CO3, THF, 50 °C, 2h (2a &2b) and 4-8 h (2c-
2n).
R1
Cl
O
i
H
O
O
R1
(a)
O
O
H
O
(1)
(2)
O
R2
(b)
R1
R2
O
O
3(a-w)
Scheme 2. Synthesis of compounds 3(a-w); Reagents and Conditions; (i) PdCl2(PPh3)2, CuI, THF, K2CO3, 20-50 °C, 5-10 h.
and their spectroscopic data were reported in our previous
publication [17]. All variations of substituents R1 & R2 are
shown in Table 1.
(AChE & BChE) level occurred in specific regions of the
brain [7, 24]. For AChE as well as BChE enzymes, initially,
all compounds were screened at 0.1 mM concentration to
assess the % inhibition. Compounds with more than 50%
inhibition were further analyzed at multiple concentrations
and their IC50 values were calculated. Galanthamine was
used as a standard inhibitor for both enzymes.
3.2. Synthesis of 6H-benzo[c]chromen-6-ones Derivatives
The synthetic route adopted for the preparation of 6H-
benzo[c]chromen-6-ones derivatives (3a-w) is illustrated in
Scheme 2. The physicochemical properties and their spectro-
scopic data were reported in our previous publication [18].
All variations of substituents R1 & R2 are shown in Table 2.
As described earlier, the topology of these compounds
can be easily modulated by the use of various readily acces-
sible starting materials generating chromenone derivatives
with different substitution sites. These newly developed
chromenones might be advantageous in the development of
cholinesterase inhibitors compared to the literature examples
that largely lacked structural diversity and included bulky
molecules to inhibit cholinesterase. Screening of chrome-
none derivatives (2a-n & 3a-w) against AChE and BChE
showed that these derivatives exhibited potent inhibitory
3.2. In vitro Cholinesterase Inhibition Studies
Ellman’s colorimetric method was employed to deter-
mine the cholinesterase inhibition potential of the syn-
thesized compounds (2a-n & 3a-w) against AChE and BChE
enzymes. Recent studies suggested that in Alzheimer’s pa-
tients having severe pathology, fluctuation in cholinesterases

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Medicinal Chemistry, 2018, Vol. 14, No. 8
Iqbal et al.
Table 1. Cholinesterase inhibition efficacy of compounds 2(a-n).
O
R1
O
R2O
OR2
O
O
2(a-n)
AChE
BChE
Sr. No.
R1
R2
IC50 SEM (ꢀM)
2a
Me
Et
Me
2.03 0.06
0.29 0.01
0.26 0.01
1.78 0.03
0.98 0.02
0.92 0.01
1.51 0.03
1.45 0.02
0.76 0.01
0.25 0.01
0.53 0.01
0.08 0.03
0.26 0.01
1.54 0.03
0.62 0.01
2.94 0.08
3.82 0.09
0.16 0.01
0.49 0.01
0.24 0.01
0.51 0.02
0.43 0.02
1.39 0.03
3.63 0.01
0.13 0.02
3.92 0.02
3.11 0.03
0.22 0.02
0.93 0.03
0.87 0.03
2b
Me
2c
OH
Me
2d
OH
Et
2e
CH2Cl
Me
Me
2f
Et
2g
Me
i-Pr
2h
Me
(CH2)2OMe
2i
Me
Allyl
Et
2j
Pr
2k
Me
CH2Ph
Et
2l
Ph
2m
2n
CH2Cl
CH2OMe
-
Et
Me
-
Galanthamine
activity (Tables 1 & 2). Among them, compounds 2l, 3h, 3l,
3o, 3q, 3u and 3w were potent inhibitors of AChE, as re-
vealed by experimental data. Diethyl 6-oxo-9-phenyl-6H-
benzo[c]chromene-8,10-dicarboxylate (2l) having an IC50
value of 0.08 0.03 ꢀM was found to be the most active
AChE inhibitor, whereas, 3h, 3l, 3o, 3q, 3u and 3w showed
significant and comparable IC50 values of 0.15 0.01, 0.15
0.01, 0.12 0.01, 0.13 0.01, 0.11 0.01 and 0.11 0.01
ꢀM, respectively. All these inhibitors were more active than
mene-8-carboxylate) and 3q (tert-butyl 9-(4-(tert-butyl)
phenyl)-6-oxo-6H-benzo[c]chromene-8-carboxylate) were
most potent. The IC50 values of 3h and 3q were 0.15 0.01
and 0.13 0.01 ꢀM, respectively for AChE and 0.09 0.01
and 0.04 0.01 ꢀM, respectively for BChE (Tables 1 & 2).
In general, all the derivatives showed strong inhibitory po-
tential for AChE and most of them were also potent inhibi-
tors of BChE. Several AChE inhibitors showed improved
inhibitory activity compared to standard inhibitor gal-
anthamine and some of the BChE inhibitors displayed the
same trend.
the standard galanthamine (0.62
0.01 ꢀM). Substitution
patterns of these compounds represented a balance of elec-
tronic and steric effects. Similarly, compounds 2c, 2j, 2m,
3h, 3p, 3q, 3r and 3w displayed potent BChE inhibitory ac-
tivity. Among these, 3q (tert-butyl 9-(4-(tert-butyl)phenyl)-
6-oxo-6H-benzo[c]chromene-8-carboxylate) having an IC50
value of 0.04 0.01 ꢀM was found to be the most potent
BChE inhibitor, while the remaining compounds 2c, 2j, 2m,
3h, 3p, 3r and 3w exhibited IC50 values of 0.16 0.01, 0.13
0.02, 0.22 0.02, 0.09 0.01, 0.15 0.02, 0.07 0.01 and
0.33 0.02 ꢀM, respectively.
The cholinesterase inhibitory profile of the tested com-
pounds was dependent on several factors including the na-
ture of the substituents and their presence at different posi-
tions of the basic skeleton. Among the series 2a-n, com-
pound 2l was the most potent inhibitor with a phenyl ring at
R1 position while carrying an ethyl group at R2 position. The
presence of a phenyl group increased the inhibition of 2l to
~8 fold compared to galanthamine. A similar trend was ob-
served for compounds 3a-w, when a phenyl ring was substi-
tuted at R1, irrespective of the substituent attachment at R2,
an enhanced inhibitory activity was observed (3d, 3e, 3k). At
the same time, all compounds of both series carrying a
Most of the compounds in this series were found to ex-
hibit dual inhibitory activity against AChE and BChE,
among which 3h (ethyl 6-oxo-9-propyl-6H-benzo[c]chro-

Chromenone Derivatives as Cholinesterase Inhibitors
Medicinal Chemistry, 2018, Vol. 14, No. 8 813
Table 2. Cholinesterase inhibition efficacy of compounds 3(a-w).
R1
R2
O
O
3(a-w)
AChE
BChE
Sr. No.
R1
R2
IC50 SEM (ꢀM)
3a
4-MeC6H4
Ph
CO2Me
CO2Me
CO2Me
CO2Et
CO2-i-Pr
CO2Et
CO2Me
CO2Et
CO2-i-Pr
COMe
COMe
COMe
CO2Et
COMe
CO2Me
CO2Et
CO2-t-Bu
CO2Me
CO2Me
CO2Me
CO2Et
CO2Me
CO2Et
-
0.76 0.01
1.67 0.03
0.45 0.01
0.36 0.01
0.29 0.02
0.50 0.01
0.42 0.01
0.15 0.01
0.65 0.01
0.40 0.01
0.25 0.01
0.15 0.01
0.42 0.01
0.41 0.01
0.12 0.01
0.22 0.01
0.13 0.01
0.21 0.01
0.43 0.01
1.85 0.02
0.11 0.01
0.21 0.01
0.11 0.01
0.62 0.01
0.71 0.02
2.83 0.09
3.41 0.02
4.37 0.03
2.79 0.02
3.61 0.01
2.98 0.03
0.09 0.01
0.77 0.01
0.74 0.02
1.49 0.01
3.53 0.04
4.87 0.04
3.45 0.02
2.73 0.03
0.15 0.02
0.04 0.01
0.07 0.01
7.19 0.05
5.67 0.04
4.46 0.03
3.24 0.09
0.33 0.02
0.87 0.03
3b
3c
n-Pent
3d
Ph
3e
Ph
3f
n-Pent
3g
n-Pr
3h
n-Pr
3i
n-Pr
3j
n-Pr
3k
Ph
3l
n-Pent
3m
4-MeC6H4
4-MeC6H4
t-BuC6H4
t-BuC6H4
t-BuC6H4
2-MeC6H4
4-n-PrC6H4
4-FC6H4
4-FC6H4
4-(MeO)C6H4
4-(MeO)C6H4
-
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
Galanthamine
phenyl moiety at R1 showed significant inhibition of AChE
but lower activity against BChE. Moreover, in the series 2a-
n, an ethyl substituent had a greater influence on inhibition
against both ChEs and the inhibitory activity of the com-
pounds was found to be higher than that of the standard in-
hibitor galanthamine. Overall, the compounds in series 2a-n
were found to be more potent against BChE, whereas, com-
pounds of series 3a-w exhibited significant inhibition of
AChE. Although both series shared the same basic skeleton,
they contained different substituents, which was a likely rea-
son for variation in the inhibitory activities of both sets of
compounds. In the case of 3a-w, the larger substituents were
attached at both positions (R1 & R2) while smaller substitu-
ents were present in 2a-n. Substitution patterns are reported
in Tables 1 and 2.
3.4. Molecular Docking
For docking analysis, X-ray structures of human AChE
(PDB ID: 4BDT) and BChE (PDB ID: 4BDS) were selected
as templates because structures of electric eel AChE were
only available at very low crystallographic resolutions (> 4

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Medicinal Chemistry, 2018, Vol. 14, No. 8
Iqbal et al.
Gln119
Tyr124
Ala328
Met437
Pro446
Fig. (1). Comparison of active sites. A superposition of active site residues of X-ray structures of AChE (cyan) in complex with huprine W
(yellow) and BChE (orange) in complex with tacrine (magenta) is shown. Circled in red are residue differences in the active sites, as dis-
cussed in the text. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).
Tyr124
Asp74
Gln119
Tyr341
Asp70
297
Val288
332
Ser125
Thr120
Phe29
Gly
2
Phe
Leu286
Gly117
Gly116
Gly121
Trp236
38
Phe329
Gly
Tyr337
Ala328
Trp82
Gly120
Gly115
Trp86
Ser203
His447
Trp430
Met434
Ser198
His438
Trp439
Met44
Leu1
Tyr
Met437
Pro446
Glu202
Glu19
Tyr440
Tyr449
Fig. (2). Putative binding modes. Docking poses of compounds 3h (light green) and 3q (dark green) in the active sites of AChE (left) and
BChE (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).
Å) and a structure of equine BChE was not available. A
comparison of the active sites of AChE and BChE including
bound ligands is shown in Fig. (1). The residue Tyr337 in
AChE is replaced by Ala328 in BChE. For docking, the side
chain of Tyr337 in AChE was treated flexibly (as the only
residue in both structures) because different X-ray structures
revealed multiple side chain conformations of Tyr337 de-
pending on co-crystallized compounds. The corresponding
BChE residue Ala328 has no side chain flexibility.
(BChE) show similar binding modes within the active sites
of these enzymes. Huprine W is sandwiched between the
aromatic side chains of residues Tyr337 and Trp86 and
formed ꢀ-ꢀ interactions. Due to the replacement of Tyr337
by Ala328 in BChE, formation of the aromatic sandwich is
not possible. Nevertheless, the aromatic ring system of
tacrine is oriented similarly to the corresponding moiety in
huprine W [20].
The best dual AChE/BChE inhibitors identified herein,
compounds 3h and 3q, were docked into the active sites of
the two enzymes. Fig. (2) shows an overlay of the putative
binding modes of compounds 3h and 3q in AChE and
BChE. The docking poses are similar, yielding favorable
positions of the 6H-benzo[c]chromen-6-one ring systems for
interactions with side chains of several aromatic residues.
Another notable difference between the active sites is the
replacement of residue Pro446 in AChE at the bottom of the
hydrophobic pocket by the bulkier residue Met437 in BChE.
Furthermore, Tyr124 in AChE is replaced by Gln119 in
BChE, which adopts different side chain orientations. The
crystallographic ligands huprine W (AChE) and tacrine

Chromenone Derivatives as Cholinesterase Inhibitors
Medicinal Chemistry, 2018, Vol. 14, No. 8 815
Asp74
Asp70
Gln119
Tyr124
Tyr341
Thr120
Phe297
Ser125
Val288
Tyr332
Phe123
Gly117
Gly122
Gly121
Gly116
Leu286
e338
Trp236
Phe329
Trp82
231
Trp86
Le
Gly
Tyr337
Ala328
Gly12
Trp439
Leu130
Trp430
Ser203
Ser198
His447
Met443
His438
Met434
Met437
Pro446
Tyr449
Glu197
Tyr133
Glu202
Tyr440
Fig. (3). Putative versus crystallographic binding modes. Overlays of docking poses of compound 3q (dark green) and the X-ray structure
of huprine W (yellow) within the active site of AChE (left) and the X-ray structure of tacrine (magenta) within the active site of BChE (right)
are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).
Fig. (4). Comparison of predicted binding modes. Shown is a superposition of active site residues of AChE (cyan) and of BChE (orange)
with the respective docking poses of compound 3q (magenta: 3q docked into AChE, yellow: 3q docked into BChE). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this paper).
In addition, the ring carbonyl groups of both compounds
are in potential hydrogen bonding distance to the side chain
hydroxyl group of Tyr341 (AChE). In BChE, the ring sys-
tems of 3h and 3q adopt slightly different positions than in
AChE due to the replacement of Tyr337 (AChE) with
Ala328 (BChE). The smaller alanine residue leads to a minor
ring re-orientation bringing 3h in possible hydrogen bonding
distance to residues Ser198 and His438 of the catalytic triad
of BChE. Furthermore, the ethoxy substituent is directed
towards a lipophilic pocket formed by residues Val288,
Leu286, Phe329 and Trp231. Comparisons of the predicted
binding modes of 3q and the crystallographic huprine W
within the active site of AChE and in the active site of
hBChE are shown in Fig. (3).

816
Medicinal Chemistry, 2018, Vol. 14, No. 8
Iqbal et al.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
In AChE, both 3q and huprine W adopt a parallel orienta-
tion between Tyr337, Trp86 and Trp439, while the ring sys-
tems of tacrine and 3q are not in a parallel arrangement with
the active site of BChE. Fig. (4) shows a superposition of the
putative binding modes of compound 3q within the active
sites of AChE and BChE. Compound 3q fits more deeply
into the hydrophobic pocket formed by the residues
Trp86/Trp82 (AChE/BChE), Tyr337/Ala328, Tryp439/
Trp430, Pro446/Met437, and Tyr449/Tyr440 in case of
AChE. The reason for the slightly different position is the
exchange of the residue Pro446 in AChE at the bottom of the
hydrophobic pocket with the bulkier residue Met437 in
BChE. Nevertheless, the 6H-benzo[c]chromen-6-one ring
systems can interact with the aromatic residues of both en-
zymes. As a consequence of this rearrangement, different
interactions are likely for the inhibitor in the active sites of
AChE and BChE, i.e., alternative hydrogen bonds with
Tyr341 (AChE) and Thr120 (BChE) might be formed. In
addition, the tertiary butyl phenyl substituent of the inhibitor
is placed into a hydrophobic pocket formed by residues
Val288, Trp231, and Leu286 in the active site of BChE.
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base
of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Jamshed Iqbal is thankful to the Organization for the
Prohibition of Chemical Weapons (OPCW), The Hague, The
Netherlands and Higher Education Commission of Pakistan
for the financial support through Project No. 20-3733/
NRPU/R&D/14/520. N.F. was supported by a fellowship
from the Jürgen Manchot Foundation, Düsseldorf, Germany.
This orientation would not be possible within the active
site of AChE because of short contacts between the inhibitor
and Tyr337, which restricts the available space in AChE
compared to BChE and might thus be responsible for partly
different compound binding modes.
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