Synthesis, biological evaluation and molecular modelling of 2′-hydroxychalcones as acetylcholinesterase inhibitors

Article abstract of DOI:10.3390/molecules21070955

A series of 2′-hydroxy- and 2′-hydroxy-4′,6′-dimethoxychalcones was synthesised and evaluated as inhibitors of human acetylcholinesterase (AChE). The majority of the compounds were found to show some activity, with the most active compounds having IC

Full text of DOI:10.3390/molecules21070955

molecules
Article
Synthesis, Biological Evaluation and Molecular
Modelling of 21-Hydroxychalcones as
Acetylcholinesterase Inhibitors
Sri Devi Sukumaran 1, Chin Fei Chee 1,2, Geetha Viswanathan 1, Michael J. C. Buckle 1,*,
Rozana Othman 1, Noorsaadah Abd. Rahman 2 and Lip Yong Chung 1,
*
1
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia;
shivanesri@yahoo.com (S.D.S.); cfchee@yahoo.com (C.F.C.); geethaviswa@gmail.com (G.V.);
rozanaothman@um.edu.my (R.O.)
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia;
noorsaadah@um.edu.my
2
*
Correspondence: buckle@um.edu.my (M.J.C.B.); chungly@um.edu.my (L.Y.C.);
Tel.: +60-3-7967-4959 (M.J.C.B. & L.Y.C.)
Academic Editor: Jean Jacques Vanden Eynde
Received: 16 March 2016; Accepted: 16 July 2016; Published: 22 July 2016
Abstract: A series of 21-hydroxy- and 21-hydroxy-41,61-dimethoxychalcones was synthesised and
evaluated as inhibitors of human acetylcholinesterase (AChE). The majority of the compounds were
found to show some activity, with the most active compounds having IC50 values of 40–85 µM.
Higher activities were generally observed for compounds with methoxy substituents in the A ring
and halogen substituents in the B ring. Kinetic studies on the most active compounds showed that
they act as mixed-type inhibitors, in agreement with the results of molecular modelling studies,
which suggested that they interact with residues in the peripheral anionic site and the gorge region
of AChE.
Keywords: Alzheimer’s disease; acetylcholinesterase; chalcones; molecular modelling
1. Introduction
Alzheimer’s disease (AD) is a degenerative brain condition characterised by severe memory loss
and cognitive impairment. It currently affects more than 30 million people worldwide, and these
numbers are set to quadruple by the year 2050 [1]. Following the cholinergic hypothesis, the current
first-line treatment for AD is the administration of acetylcholinesterase (AChE) inhibitors, which block
the enzymatic hydrolysis of acetylcholine (ACh), resulting in increased levels of the neurotransmitter
in the synapses between cholinergic neurons, thereby improving nerve transmission [
2]. The agents
that have been approved for clinical use to date have limited efficacy and may produce adverse effects,
such as nausea, vomiting, diarrhea, dizziness, and weight loss [
development of new AChE inhibitors.
3]. There is therefore a need for the
Crystallographic studies of AChE complexed with various inhibitors have shown that the enzyme
possesses two separate ligand binding sites—a catalytic site (CAS) and a peripheral anionic site
(PAS)—which are connected by a narrow gorge [4]. The two key binding residues in the CAS and PAS
are aromatic residues, Trp84 and Trp286. The classical hydrolytic role of AChE is performed at the CAS,
whereas the PAS has been associated with a number of non-classical roles, including the induction of
amyloid
these plaques are one of the main pathological hallmarks of AD, the use of inhibitors that bind to the
PAS and so inhibit both A aggregation and ACh hydrolysis by blocking the approach to the catalytic
β (Aβ) aggregation to form fibrils, which is a key step in the formation of Aβ plaques. Since
β
site has been proposed as a potential disease-modifying therapy [5]. Electron-rich aromatic rings are
Molecules 2016, 21, 955; doi:10.3390/molecules21070955
www.mdpi.com/journal/molecules

Molecules 2016, 21, 955
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a common feature of PAS-binding AChE inhibitors, which are believed to stack against the Trp286
residue of the PAS site, as exemplified in the recently-determined crystal structure of human AChE in
complex with donepezil [6]. However, in the case of compounds which contain aromatic rings that
interact with residues in the CAS or the gorge, electron-withdrawing substituents have been found to
increase AChE inhibition in comparison to unsubstituted compounds, especially when they are in the
para position [7,8].
Chalcones, or 1,3-diaryl-2-propen-1-ones, are a class of polyphenolic compounds belonging to
the flavonoid family which possess a wide range of pharmacological activities, including anti-cancer,
anti-inflammatory, and anti-oxidant activities [
9]. However, there have been relatively few previous
reports of chalcones and their derivatives with AChE inhibitory activity [10
–
16], and the effects of
different substituents on the phenyl rings have not yet been extensively investigated. In the present
1
work, we chose to study 2 -hydroxychalcones, as compounds with this scaffold have been previously
reported to show good activity against AChE with strong selectivity compared to butyrylcholinesterase
(BChE) [10]. Furthermore, it has been recently reported that good AChE inhibitory activity has
been exhibited by some derivatives of the naturally-occurring 21-hydroxy-41,61-dimethoxychalcone,
flavokawain B [15], and that compounds with this substitution pattern in the A ring are non-cytotoxic
at 100 µM concentration towards human embryonic kidney (HEK-293) cells [17], which resemble
developing neuronal cells [18]. Hence, we decided to synthesise and compare the AChE inhibitory
activities of a series of 21-hydroxy- and 21-hydroxy-41,61-dimethoxychalcones with a range of
substituents attached to the B ring. Furthermore, kinetic and molecular modelling studies were carried
out on the most active compounds to investigate their mode of inhibition and binding interactions
with AChE.
2. Results and Discussion
1
1
1–14 were prepared from the corresponding 2 -hydroxyacetophenones and
2 -hydroxychalcones
benzaldehydes via the Claisen–Schmidt reaction (Scheme 1). The physicochemical and spectroscopic
data obtained from the new compounds and 14 were in agreement with their structures, and those
8
from the known compounds were in agreement with published data. Examination of the predicted
Lipinski rule of five parameters of the compounds (Table 1) shows that they all obey both the rule of
five and the more restrictive blood-brain permeability considerations (molecular weight < 450, number
of hydrogen bond donors (HBD) < 3, log P 2–5, polar surface area (PSA) < 90 Å2) [19], suggesting that
they should be orally active and able to reach the central nervous system.
Table 1. Lipinski rule of five parameters for 21-hydroxychalcones 1–14.
PSA/Å2
Compound
MW
HBD
HBA
A log P
1
2
3
4
5
6
7
8
9
10
11
12
13
224.26
238.28
303.15
258.70
267.32
254.28
303.15
309.14
298.33
363.20
327.37
314.33
363.20
369.20
379.49
1
1
1
1
1
1
1
2
1
1
1
1
1
2
0
2
2
2
2
3
3
2
3
4
4
5
5
4
5
4
3.46
3.95
4.21
4.12
3.62
3.44
4.21
4.55
3.91
4.18
3.59
3.41
4.18
4.51
4.57
38.12
38.12
38.12
38.12
41.49
47.05
38.12
58.93
55.98
55.98
59.33
64.91
55.98
76.79
38.51
14
Donepezil
MW: molecular weight; A log P: logarithm of octanol–water partition coefficient; HBD: number of hydrogen
bond donors; HBA: number of hydrogen bond acceptors; PSA: polar surface area.

Molecules 2016, 21, 955
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The inhibitory activity of the prepared 21-hydroxychalcones at human recombinant AChE was
evaluated at 10 M using Ellman’s colorimetric assay method [20], and the IC50 values of the
µ
compounds that showed more than 20% inhibition were determined. The results are summarised
in Table 2. The majority of the compounds showed some AChE inhibitory activity, with the most
active compounds having IC50 values in the range of 40–85
to that observed for the standard PAS-binding AChE inhibitor propidium, but somewhat lower than
that obtained for the standard CAS-binding inhibitor tacrine. Compounds 14, which bear methoxy
groups at positions C-4 and C-6 in the A ring, were generally more active than the corresponding
compounds , which lack these substituents. This observation is consistent with previous work
which has shown the beneficial effect of electron-donating moieties in PAS-binding AChE inhibitors
with different scaffolds [21 22]. In contrast, when examining the effect of substitution in the B ring,
compounds 10 13, and 14 with halogen substituents (Br/Cl) showed the greatest activity, although
not all the compounds with halogen substituents in the B ring had measurable activity at 10 M.
µM. This level of activity is comparable
9
–
´
´
1–8
,
7
,
,
µ
Such an effect due to halogen substituents has also been previously observed in different series of
AChE inhibitors and has been explained in terms of additional hydrogen bonding or dipole-dipole
interactions, or increased van der Waals interactions [7,8].
Scheme 1. Synthesis of 21-hydroxychalcones.
Table 2. Substituent patterns and human acetylcholinesterase (AChE) inhibitory activity of
21-hydroxychalcones 1–14.
% Inhibition
IC50/µM b
Compound
R4´
R6´
R2
R3
R4
R5
at 10 µM a
1
2
3
4
5
6
7
8
9
10
11
12
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
Cl
H
Me
Br
H
H
H
H
H
H
H
Cl
H
H
H
H
H
Cl
11
na d
na d
na d
28
nd c
nd c
nd c
Cl
nd c
NMe2
OMe
H
H
Me
Br
NMe2
OMe
H
266 ˘ 48
H
6
nd c
Br
OH
H
H
H
H
Br
OH
38
55 ˘ 12
nd c
na d
23
354 ˘ 33
41 ˘ 13
nd c
30
5
7
23
51
50
91
nd c
13
85 ˘ 16
73 ˘ 22
11 ˘ 3
0.19 ˘ 0.04
14
Propidium e
Tacrine f
a
H
Values are expressed as mean of triplicate; b Values are expressed as mean
˘
SEM; c Not determined; d No
activity observed at 10 µM; e Standard PAS-binding AChE inhibitor; f Standard CAS-binding AChE inhibitor.
To investigate the mode of inhibi1tion of the most active compounds and to determine their
apparent inhibition constant (Ki and Ki ) values, kinetic studies were carried out on their steady state
inhibition of AChE. For each of the compounds, Lineweaver–Burk plots showed increasing slopes and
increasing intercepts at higher inhibitor concentrations and intersections in the second quadrant, above

Molecules 2016, 21, 955
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the x-axis and to the left of the y-axis, suggesting that they are all mixed-type inhibitors (see Figure 1).
1
The apparent Ki and Ki values obtained from nonlinear regression analysis are shown in Table 3.
Figure 1. Lineweaver-Burk plot for the inhibition of AChE hydrolysis of acetylthiocholine at different
concentrations of compound 10. Each point is the mean of triplicate determinations.
Table 3. Apparent human AChE inhibition constant (Ki and Ki1) values, equine butyrylcholinesterase
(BChE) inhibitory activity and selectivity for AChE for the most active compounds.
Human AChE
Equine BChE
IC50 /µM a
Selectivity
for AchE b
Compound
1
Ki /µM
Ki /µM
7
40
31
113
56
59
47
165
92
108 ˘ 18
59 ˘ 12
71 ˘ 14
81 ˘ 18
2.0
1.4
0.8
1.1
10
13
14
a
Values are expressed as mean
(AChE) are shown in Table 2.
˘
SEM; b Selectivity is defined as IC50 (BChE)/IC50 (AChE); values for IC50
The IC50 values of the four compounds that gave the greatest inhibition of AChE were also
determined for equine BChE, which is readily available and is considered a good model for human
BChE [23], as the equine form has 90% sequence identity compared to the human form. The compounds
all showed appreciable BChE inhibitory activity, but generally at lower levels than for AChE, giving
selectivities for AChE ranging from 1.1–2.0 (see Table 3). The exception was compound 13, which
showed some selectivity for BChE. Interestingly, BChE has been proposed as a therapeutic target for
advanced AD, when BChE takes over the role of ACh hydrolysis from AChE, as neuronal AChE is
depleted [24]. Hence, compounds that act as dual inhibitors of both AChE and BChE may also be
clinically useful.
To investigate possible binding interactions, molecular modelling studies were performed by
randomly docking the most active compounds
v4.0. The recently-solved X-ray crystal structure of the mixed-type inhibitor, donepezil, with human
AChE (PDB ID: 4EY7) [ ] was used as the starting model. All the compounds were found to exhibit
7, 10, 13, and 14 into human AChE using AutoDock
6
binding poses similar to those of donepezil occupying the PAS and, to varying extents, the gorge
connecting it to the CAS (see Figure 2 and Supplementary Figures S1–S3). Specifically, in the case of
compound 10, aromatic rings A and B formed
Tyr 341 (at the entrance to the gorge leading to that CAS), respectively. The other three compounds
showed stacking or non-polar interactions with one or other of these two residues and/or Tyr337
and Phe338 in the gorge. These interactions with residues in the gorge region may account for the Ki
π–π stacking interactions with Trp286 (in the PAS) and
π–π

Molecules 2016, 21, 955
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1
values obtained from the kinetic studies being less than the Ki values—i.e., the competitive part of the
mixed-type inhibition was more pronounced than the uncompetitive part. Furthermore, in the case of
compounds
7 and 10, the carbonyl oxygen atom and the ortho hydroxyl group on the A ring were also
observed to form hydrogen bonds with backbone atoms from acyl pocket residues, Phe295 (2.8–3.0 Å)
and Arg296 (2.7–2.9 Å), respectively. This may be one of the reasons to account for the greater activity
of these two compounds, as compounds 13 and 14 only interacted with Arg296. Hydrogen bonds to
Phe295 have previously been observed in crystal structures of AChE in complex with donepezil and
other inhibitors [8,25,26]. The modelling studies also suggested that the beneficial effect of the halogen
substituents in the active compounds might be due to interactions with aromatic residues in the CAS
and gorge. Such interactions have previously been observed in a number of other protein–ligand
complexes [27].
Figure 2. (A–C) 3D representations of the binding poses of standard and test compounds in complex
with human AChE (PDB ID: 4EY7). The hydrophobic surfaces of the interacting residues are shown
in blue relief. Ligand–protein interactions are depicted with dotted lines: hydrogen bonds (green),
π
-cation interactions (orange),
tacrine; ( ) compound 10; (
10. Hydrogen bonds and
π-halogen interactions (purple). (A) donepezil; (B) propidium and
) Schematic representation of the binding interactions of compound
π–π stacking interactions are depicted with black and green dotted lines,
C
D
respectively. The green curve represents other non-polar interactions.
Molecular modelling studies were also performed by randomly docking the most active
compounds
7, 10, 13, and 14 into a model derived from the recently-solved X-ray crystal structure
of tacrine in complex with human BChE (PDB ID: 4BDS) [28]. Although all four compounds were

Molecules 2016, 21, 955
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found to bind, the calculated binding energy was 1.1–1.8 kcal/mol lower for AChE compared to BChE.
These results are consistent with the slight selectivity for AChE compared to BChE that was observed
for three out of the four most active compounds in the inhibition studies.
3. Materials and Methods
3.1. General Information
All melting points were taken on a Stuart melting point apparatus SMP30 (Staffordshire, UK).
NMR spectra were obtained using Jeol ECA 400 (400 MHz) and EX 270 (270 MHz) NMR spectrometers
(Jeol Ltd., Akishima, Tokyo, Japan) with tetramethylsilane as the internal standard. All chemical
shifts are reported in ppm. MS analysis was performed on an Agilent 6500 series accurate mass
Q-TOF (Agilent Technologies, Santa Clara, CA, USA) Analytical thin-layer chromatography (TLC)
was carried out on pre-coated aluminum silica gel sheets (Kieselgel 60 F254) from Merck (Darmstadt,
Germany). Column chromatography was performed with silica gel 60 (230–400 mesh) from Merck.
Human recombinant AChE and equine BChE were purchased from Sigma-Aldrich (St. Louis, MO,
USA). All other chemicals were purchased from Sigma-Aldrich or Merck and were of analytical grade.
The Lipinski rule of five parameters of the synthesised compounds were determined using Discovery
Studio v3.1 (Accelrys Inc., San Diego, CA, USA).
3.2. Synthesis of Chalcones 1–14
A mixture of the corresponding acetophenone (1 equiv) and benzaldehyde (1 equiv) in EtOH
(5 mL/1 mmol of acetophenone) was stirred at room temperature for 30 min. Then, a solution of
50% w/w aqueous KOH (1 mL/1 mmol) was added. The reaction mixture was stirred at rt until all
benzaldehyde was consumed when monitored on TLC. Afterwards, the mixture was poured into
ice-water acidified with 3 N HCl. In cases in which the chalcones precipitated, they were filtered,
purified using column chromatography, and crystallised from ethanol (yields: 65%–90%).
1
˝
˝
2 -Hydroxychalcone (1). Yellow crystals, mp 89–90 C, lit. 89–90 C [29]; for NMR data see ref. [30].
1
˝
˝
2 -Hydroxy-4-methylchalcone (
2
) Yellow needles, mp 117–118 C, lit. 116–117 C [31]; for NMR data see
ref. [31].
1
δ
˝ ˝
3
). Yellow needles, mp 138–139 C, lit. 138–139 C [32]; 1H-NMR (400 MHz,
4-Bromo-2 -hydroxychalcone (
CDCl3):
12.67 (s, 1H, OH), 7.80 (dd, J = 8.0, 1.5 Hz, 1H, H-61), 7.73 (d, J = 16.0 Hz, 1H, Hβ), 7.53 (d,
J = 16.0 Hz, 1H, Hα), 7.47 (d, J = 8.1 Hz, 2H, H-2, H-6), 7.41 (d, J = 7.8 Hz, 2H, H-3, H-5), 7.40 (m, 1H,
H-41), 6.94 (dd, J = 8.0, 1.5 Hz, 1H, H-31), 6.85 (m, 1H, H-51); 13C-NMR (100 MHz, CDCl3):
δ 193.44,
163.65, 143.96, 136.56, 133.53, 132.31, 129.97, 129.62, 125.27, 120.71, 119.94, 118.92, 118.70.
1
˝
˝
4-Chloro-2 -hydroxychalcone (
4
). Yellow needles, mp 145–146 C, lit. 148–151 C [29]; for NMR data see
ref. [31].
21-Hydroxy-4-(dimethylamino)chalcone ( ). Orange powder, mp 170–171 ˝C, lit. 172 ˝C [33]; for NMR
5
data see ref. [33].
21-Hydroxy-4-methoxychalcone ( ). Orange needles, mp 92–93 ˝C, lit. 92–94 ˝C [29]; for NMR data see
6
ref. [31].
1
˝ ˝
2-Bromo-2 -hydroxychalcone (7 ,
). Yellow needles, mp 104–105 C, lit. 103–104 C [32]; 1H-NMR (400 MHz
1
CDCl3): δ 12.63 (s, 1H, OH), 8.12 (d, J = 15.3 Hz, 1H, Hβ), 7.77 (dd, J = 8.1, 1.8 Hz, 1H, H-6 ), 7.61 (dd,
J = 8.1, 1.8 Hz, 1H, H-3), 7.51 (dd, J = 8.2, 1.9 Hz, 1H, H-41), 7.44 (d, J = 15.4 Hz, 1H, Hα), 7.38 (m,
1H, H-5), 7.25 (t, J = 7.3 Hz, 1H, H-6), 7.13 (m, 1H, H-4), 6.90 (dd, J = 8.0, 1.8 Hz, 1H, H-31), 6.82 (m,
1H, H-51); 13C-NMR (100 MHz, CDCl3):
128.03, 127.81, 126.16, 122.93, 119.90, 118.97, 118.68.
δ 193.42, 163.66, 143.65, 136.61, 134.70, 133.67, 131.69, 129.78,

Molecules 2016, 21, 955
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1
˝
8
). Yellow powder, mp 125–126 C. 1H-NMR (400 MHz, DMSO-d6):
3,5-Dichloro-2,2 -dihydroxychalcone (
12.48 (s, 1H, OH), 10.43 (s, 1H, OH), 8.26 (d, J = 7.8 Hz, 1H, H-61), 8.12 (d, J = 15.5 Hz, 1H, Hβ), 8.06
(d, J = 15.5 Hz, 1H, Hα), 7.57 (m, 1H, H-41), 7.55 (d, J = 2.3 Hz, 1H, H-4), 7.53 (d, J = 2.3 Hz, 1H, H-6),
δ
7.00 (m, 1H, H-3 ), 6.98 (m, 1H, H-5 ); 13C-NMR (100 MHz, DMSO-d6):
δ 193.99, 162.45, 151.89, 138.05,
1
1
136.93, 131.43, 131.38, 126.80, 126.19, 124.59, 123.82, 123.59, 121.11, 119.60, 118.20; HREIMS m/z 309.0085
[M + H]+ (calculated for C15H11O3Cl2, 309.0080).
21-Hydroxy-41,61-dimethoxy-4-methylchalcone ( ). Orange needles, mp 130–131 ˝C, lit. 132–133 ˝C [31];
9
for NMR data see ref. [31].
4-Bromo-21-hydroxy-41,61-dimethoxychalcone (10). Orange powder, mp 150–151 ˝C, lit. 150–151 ˝C [34];
for NMR data see ref. [34].
21-Hydroxy-41,61-dimethoxy-4-(dimethylamino)chalcone (11). Brown crystals, mp 163–165 ˝C; for NMR
data see ref. [17].
21-Hydroxy-4,41,61-trimethoxychalcone (12). Orange powder, mp 112–113 ˝C, lit: 112 ˝C [35]; for NMR
data see ref. ref. [35].
2-Bromo-21-hydroxy-41,61-dimethoxychalcone (13). Orange powder, mp 147–148 ˝C, lit. 146–147 ˝C [36];
for NMR data see ref. [36].
˝
1
3,5-Dichloro-2,21-dihydroxy-41,61-dimethoxychalcone (14). Yellow needles, mp 192–193 C. H-NMR
(
400 MHz, DMSO-d6):
δ
13.22 (s, 1H, OH), 7.84 (d, J = 15.6 Hz, 1H, Hβ),17.80 (d, J = 15.6 Hz, 1H, Hα),
1
7.68 (d, J = 2.4 Hz, 1H, H-4), 7.58 (d, J = 2.4 Hz, 1H, H-6), 6.15 (s, 1H, H-5 ), 6.13 (s, 1H, H-3 ), 3.88, 3.83
(s, 3H each, OCH3); 13C-NMR (100 MHz, CDCl3):
δ 192.87, 166.07, 165.66, 162.35, 151.55, 136.15, 130.69,
130.46, 127.21, 126.68, 124.42, 123.46, 107.01, 94.36, 91.56, 56.70, 56.16; HREIMS m/z 369.0303 [M + H]+
(calculated for C17H15O5Cl2, 369.0297).
3.3. Enzyme Inhibition Studies
AChE and BChE inhibition studies were performed in 96 well plates by the method of
Ellman, et al. [20]. 110
by 20 L of test compound, 50
of AChE or BChE (0.15 units/mL). The mixture was preincubated for 20 min at 37 C before addition
of 50 L (0.120 mM) of the substrate, acetylthiocholine (ATC) iodide or butyrylthiocholine (BTC) iodide
µL of sodium phosphate buffer (pH 8.0) was added to each well followed
1
µL of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) (0.126 mM) and 20 µL
µ
˝
µ
(depending on the enzyme). The hydrolysis of ATC or BTC was monitored using an Infinite® M200
PRO multimode reader (Tecan Group Ltd., Männedorf, Switzerland) by measuring the absorbance due
to yellow 5-thio-2-nitrobenzoate anion at 412 nm every 30 s for 25 min. Each assay was performed in
triplicate. Propidium iodide and tacrine were used as standard inhibitors. The percentage of inhibition
was calculated using the expression:
pE-Sq{E ˆ 100
where E is the activity of the enzyme without test compound and S is the activity of the enzyme with
test compound.
IC50 values were obtained from concentration-inhibition experiments by nonlinear regression
analysis using PRISM® v5.0 (GraphPad Inc., San Diego, CA, USA).
Kinetic characterisation of AChE inhibition was performed under similar conditions to the
AChE assay using different concentrations of test compounds (0–100 µM) at substrate concentrations
ranging from 0.1 mM to 1.0 mM. Each assay was performed in triplicate. The mode of inhibition
was investigated using Lineweaver–Burk plots of 1/initial reaction rate (1/V) against 1/substrate
concentration (1/[S]). Apparent inhibition constant (Ki and Ki1) values were determined using the
nonlinear regression mixed-model inhibition method in PRISM® v5.0 (GraphPad Inc.).

Molecules 2016, 21, 955
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3.4. Molecular Modelling Studies
The X-ray crystal structures of human AChE and BChE in complex with donepezil and tacrine,
respectively, were retrieved from the Protein Data Bank (PDB IDs: 4EY7 and 4BDS). Ligands and
water molecules were removed using Discovery Studio v3.1. Hydrogen atoms were added and
double coordinates were corrected using Hyperchem Pro v6.0 (Hypercube Inc., Gainesville, FL, USA).
Hydrogen atoms were again added, non-polar hydrogen atoms were merged, and missing atoms
were repaired using AutoDock Tools v4 [37]. Gasteiger charges were added and AutoDock v4.0 type
atoms were assigned to the protein. Three dimensional structural models of the test compounds were
built using Chem Bio-3D v13 (CambridgeSoft, Cambridge, MA, USA) and saved in MOL2 format.
The compounds were then prepared in protonated form, where appropriate for physiological pH, and
minimised using Hyperchem Pro v6.0 (geometry optimisation using MM+) to give the lowest energy
conformation. Test compounds were randomly docked into the protein structures with AutoDock
v4.0 using a hybrid Lamarckian Genetic Algorithm [38], with an initial population size of 150 and
a maximum number of 2,500,000 energy evaluations. The grid box was set to cover the entire protein
with a spacing of 0.375 Å. The root mean square deviation (RMSD) tolerance was set to 2.0 Å for the
clustering of docked results and the docked pose of each ligand was selected on the basis of free energy
of binding and cluster analysis. Ligand-protein interactions were analysed using Discovery Studio
v3.1 and PoseView [39].
4. Conclusions
In conclusion, a series of 21-hydroxychalcones was synthesised and evaluated as inhibitors of
human AChE. Compounds with methoxy substituents on the A ring and halogen substituents on the
B ring were generally found to be more potent, with activities that are comparable to the standard
PAS-binding AChE inhibitor, propidium. Kinetic studies on the most active compounds showed
that they act as mixed-type inhibitors. This finding was consistent with molecular modelling studies,
which suggested that the compounds interact with residues in the PAS and gorge region of AChE.
Further investigations into the use of 21-hydroxychalcone derivatives as potential disease-modifying
drug candidates for the treatment of AD are in progress.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/7/955/s1.
Acknowledgments: The research was supported by grants from the Ministry of Education, Malaysia
(ER002-2012A and TR001C-2014A) and the University of Malaya (RP002-2012E and PG034-2014A).
Author Contributions: S.D.S.: biological evaluation, molecular modelling and manuscript preparation. C.F.C.:
chemical synthesis. G.V.: biological evaluation, M.J.C.B.: project design and co-ordination and manuscript
preparation. R.O. and N.A.R.: project co-ordination. L.Y.C.: project design and co-ordination.
Conflicts of Interest: The authors declare no conflict of interest.
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