2-Benzyloxynaphthalene aminoalkylated chalcone designed as acetylcholinesterase inhibitor: Structural characterisation, in vitro biological activity and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2020.128898

The design of an acetylcholinesterase inhibitor with multifunctional properties became the perspective for the development of an effective drug against Alzheimer's disease. Towards this target, 1-{4-hydroxy-3-[(piperidin-1-yl)methyl]phenyl}ethan-1-one (chalcone 3) was prepared and studied as an acetylcholinesterase inhibitor. The novel chalcone 3 was synthesised via Claisen-Schmidt condensation reaction with 84percent yield and characterized using 1D and 2D NMR spectroscopy. The in vitro bioactivity studies of chalcone 3 demonstrated excellent inhibitory activity against AChE (IC₅₀ 1.0 nM) showing 33-fold better inhibition than donepezil, biometal chelating ability and moderate antioxidant activity. Chalcone 3 with these fascinating multifunctional proprieties can be a good candidate for the development of AD treatments. A molecular modelling investigation revealed that chalcone 3 showed dual binding inhibition of AChE enzyme. XRD shows short intra- and inter-molecular interactions with two chalcone 3 molecules per cell. Interesting Hirshfeld Surface Analysis (HSA) was conducted showing explicit agreement with the XRD analysis.

Full text of DOI:10.1016/j.molstruc.2020.128898

Journal Pre-proof
2-Benzyloxynaphthalene Aminoalkylated Chalcone Designed as
Acetylcholinesterase Inhibitor: Structural Characterisation, in vitro
Biological Activity and Molecular Docking Studies
Ghadah Aljohani , Adeeb Al-Sheikh Ali , Musa A. Said ,
David L. Hughes , Shaya Y. Alraqa , Syazwani Amran ,
Farediah Ahmad , Norazah Basar
PII:
S0022-2860(20)31223-0
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.128898
MOLSTR 128898
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
27 April 2020
11 July 2020
13 July 2020
Please cite this article as: Ghadah Aljohani , Adeeb Al-Sheikh Ali , Musa A. Said , David L. Hughes ,
Shaya Y. Alraqa , Syazwani Amran , Farediah Ahmad , Norazah Basar , 2-Benzyloxynaphthalene
Aminoalkylated Chalcone Designed as Acetylcholinesterase Inhibitor: Structural Characterisation, in
vitro Biological Activity and Molecular Docking Studies, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128898
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Highlights
 A novel chalcone was characterized using NMR, IR, UV-Vis, XRD
and HSA analysis.
 The chelating studies with some metal chloride were performed using
UV-vis.
 The chalcone shows biometal chelating ability and moderate
antioxidant activity.
 The bio-studies of chalcone revealed 33-fold better inhibition than
donepezil.
 A molecular modelling study of the chalcone shown dual binding
inhibition of AChE.

2-Benzyloxynaphthalene Aminoalkylated Chalcone Designed as
Acetylcholinesterase Inhibitor: Structural Characterisation, in vitro Biological
Activity and Molecular Docking Studies
Ghadah Aljohania¸b, Adeeb Al-Sheikh Alia, Musa A. Said a*, David L. Hughesc, Shaya Y. Alraqaa,
Syazwani Amrand, Farediah Ahmadb, Norazah Basarb*
a
Chemistry Department, Taibah University, P.O. Box 30002, Code 14177, Al-Madinah Al
Munawarah, Kingdom of Saudi Arabia
b
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor
Bahru, Johor, Malaysia
c School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom,
d
Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor
Bahru, Johor, Malaysia
Abstract:
The design of an acetylcholinesterase inhibitor with multifunctional properties became
the perspective for the development of an effective drug against Alzheimer’s disease.
Towards this target, 1-{4-hydroxy-3-[(piperidin-1-yl)methyl]phenyl}ethan-1-one
(chalcone 3) was prepared and studied as an acetylcholinesterase inhibitor. The novel
chalcone 3 was synthesised via Claisen-Schmidt condensation reaction with 84% yield
and characterized using 1D and 2D NMR spectroscopy. The in vitro bioactivity studies
of chalcone 3 demonstrated excellent inhibitory activity against AChE (IC50 1.0 nM)
showing 33-fold better inhibition than donepezil, biometal chelating ability and moderate
antioxidant activity. Chalcone 3 with these fascinating multifunctional proprieties can be
a good candidate for the development of AD treatments. A molecular modelling
investigation revealed that chalcone 3 showed dual binding inhibition of AChE enzyme.
XRD shows short intra- and inter-molecular interactions with two chalcone 3 molecules
per cell. Interesting Hirshfeld Surface Analysis (HSA) was conducted showing explicit
agreement with the XRD analysis.
Keywords:
Acetylcholinesterase (AChE); metal chelation; NMR titration, molecular docking, XRD,
Hirshfeld Surface Analysis
1. Introduction

Chalcones are α,β unsaturated carbonyl compounds with a 1,3-diphenyl-2-propene-1-one
framework [1]. They are open-chain flavonoids with two aromatic rings linked by an
aliphatic three-carbon chain. Chalcones are an essential component in natural products
and play a key role in synthetic manipulations [2].
Although naphthalene has toxic effects [3], some of its derivatives are bestowed with
medicinal value [4], and several of them have been characterised as antioxidants [5].
Considering both the cytotoxic and antioxidant properties of naphthalene derivatives,
organic and medicinal chemists are trying to design drugs that have maximum therapeutic
index and minimum toxicity so that they can be used as therapeutics [6]. Some
investigations have been conducted to synthesise chalcones using naphthaldehyde [7,8].
Alzheimer's disease (AD) has received considerable attention because it is the most
common cause of dementia. According to the World Health Organization, it accounts for
60–70 per cent of dementia cases worldwide [9]. Cholinesterase inhibitors (ChEIs) are
the only class of agents that have so far consistently proved effective in treating the
cognitive and functional symptoms of AD [10]. Medications approved by the U.S. Food
and Drug Administration (FDA) for AD belong to a category of acetylcholinesterase
inhibitors, but most of them were found to have side effects [11].
A trend has developed recently to use naturally occurring and synthetic derivatives of
chalcones as cholinesterase inhibitors due to their slight side effects [12]. Recent studies
on AD have confirmed that a Mannich base and chalcone, when incorporated into a
single pharmacophore, displayed moderate inhibitory effect for AChE with excellent
multifunctional properties [13,14].
Metal chelators and reactive oxygen species (ROS) scavengers have demonstrated
potential therapeutic capacity in treating AD due to the increased level of metal ions in
the plaque [15]. Iron, copper and zinc are essential elements for properly maintaining
neurological functions in the brain. However, their abnormal levels have been observed
in several subcortical regions of the AD brain tissues [16]. N-terminal amino acid
residues contain metal binding sites that are capable of coordinating transition metal ions
with high affinity. These metal ions significantly affect the aggregation behaviour of
Amyloid β-protein (Aβ) [17,18]. Metal-binding at these sites can accelerate Aβ

aggregation and generate more amorphous aggregates, including neuro-toxic oligomers
[19]. A relationship between increased aluminium exposure and AD has been found in
some studies [20]. It has been also reported that aluminium can play a role in the
aetiology of AD because of its ability to increase oxidative stress caused by iron and
other metals [21].
This study is a follow-up of our comprehensive research on designing and synthesising
chalcone derivatives as acetylcholinesterase inhibitors using Mannich bases. Hence, the
spectroscopic data for 1-{4-hydroxy-3-[(piperidin-1-yl)methyl]phenyl}ethan-1-one
(chalcone 3) have been invesitgated to help understand the structural and geometrical
features of the novel chalcone in the crystal lattice. To investigate possible binding
interactions with the target enzyme, a molecular modelling study was performed using
the experimental crystal structure data of the title compound 3. Hirshfeld Surface
Analysis (HSA) clarifies details of intermolecular interactions derived from the X-ray
analysis.
2. Experimental
2.1 Synthesis of chalcone
2-Benzyloxy-1-naphthaldehyde (1) was obtained via an alkylation reaction of
commercially available 2-hydroxy-1-naphthaldehyde in a typical, published procedure
[22]. 1-{4-Hydroxy-3-[(piperidin-1-yl)methyl]phenyl}ethan-1-one (2) was synthesised
by means of the Mannich reaction of 4-hydroxyacetophenone with piperidine and
formaldehyde according to the method reported earlier [23]. The starting materials
underwent a Claisen-Schmidt condensation reaction to produce chalcone 3 as yellow
crystals in an excellent yield (84%) with melting point of 137-140 °C (Scheme 1) [24–
26].

Scheme 1. Synthetic route to the title chalcone 3.
2.2 Crystal structure of chalcone 3 and Hirshfeld Surface Analysis
Recrystallisation of chalcone 3 using cold ethanol provided a suitable crystal for X-ray
analysis. Crystals are pale yellow plates and, from a sample under oil, one, ca 0.23 x 0.28
x 0.43 mm, was mounted on a glass fibre and fixed in the cold nitrogen stream on an
Oxford Diffraction Xcalibur-3/Sapphire3-CCD diffractometer, equipped with Mo-Kα
radiation and graphite monochromator. Intensity data were measured by thin-slice φ-
scans. Total no. of reflections recorded, to θmax = 27.5, was 23161 of which 6372 were
unique (Rint = 0.037); 4826 were observed with I > 2σI.
Data were processed using CrysAlis PRO 1.171.39.46 [27] for cell refinement and data
reduction. Program used to solve structure: SHELXT [28a]; program used to refine
structure: SHELXL2014/7 [28b]; molecular graphics: ORTEP [29]; software used to
prepare material for publication: SHELXL2014/7 [28b] and WinGX [29].
Crystal data: C32H31NO3, C2H6O, M = 523.64. Triclinic, space group P-1 (no. 2), a =
10.6946(5), b = 11.7088(5), c = 13.5975(5) Å, α = 96.356(4), β = 107.209(4), γ =
116.643(5) , V = 1393.45(12) Å3. Z = 2, Dc = 1.248 g cm-3, F(000) = 560, T = 140(1) K,
μ(Mo-Kα) = 0.81 cm-1, λ(Mo-Kα) = 0.71073 Å.
Hirshfeld surface analysis and the related 2D-fingerprint plots were calculated using
Crystal Explorer, which reads a structure input file in CIF format [30].

2.3 Metal-chelating studies
The metal chelating studies were performed on chalcone 3 using the Shimadzu
UV-1601 spectrophotometer and Bruker 400MHz NMR spectrometer. The UV−Vis
absorption spectra of chalcone 3 in methanol (50 μM) alone and in the presence of CuCl2,
ZnCl2, AlCl3, and FeCl2 (50 μM in methanol) were recorded with wavelength ranging
from 200 to 600 nm in situ at room temperature. The molar ratio method was used to
determine the stoichiometry of the complex 3-Fe2+ by titrating the methanol solution of
chalcone 3 in a fixed concentration (60 μM) with increasing concentration of FeCl2 (6-
100 μM). The UV−Vis spectra were recorded at 307 nm and treated by numerical
subtraction of FeCl2 and 3 at corresponding concentrations, plotted versus the mole
fraction of 3. For NMR titrations, chalcone 3 (9 mg, 0.019 mmol) was dissolved in
1
CD3OD (1 mL) for HNMR analysis using a micro NMR tube and the spectrum was
recorded before titration. Then FeCl2 (19 μM) solution (in D2O) was titrated
1
progressively using a micropipette within the NMR tube to monitor the HNMR in situ.
Spectra 2-6 were recorded upon the addition of the increasing equivalents of FeCl2.
2.4 In vitro bioactivity studies of chalcone 3
2.4.1 Antionxidant activity analysis
The antioxidant evaluation was performed against 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical based on the method described by Hamad et al. [31]. A series of dilutions
ranging from 10 μg/ml to 13 mg/ml was prepared in methanol. An aliquot of 40 μL of
chalcone 3 from each serial dilution was mixed with 160 μL of freshly prepared
methanolic solution of 100 μM DPPH radical and kept in the dark. After 30ꢀmin of
incubation, the decrease in absorbance at 517 nm was determined. The absorbance of the
DPPH radical without antioxidant (blank) and the reference compound ascorbic acid
were also measured. All the determinations were performed in three replicates and
averaged. The percentage inhibition of the DPPH radical was calculated according to the
formula:
Percentage Inhibition (%) =
100

where Ablank = absorbance of the blank solution (containing DPPH solution without
sample) and Asample = absorbance of a sample solution. The concentration affording 50%
inhibition (IC50) values were calculated using GraphPad Prism for Windows (version
8.3.0) software.
2.4.2 Inhibition of AChE enzyme activity
The acetylcholinesterase inhibitory activity of chalcone 3 was determined by Ellman's
microplate assay described by Koay et al.(2014) [32]. 140 µl of 0.1 M sodium phosphate
buffer (pH 8) was added first followed by 20 µl of the test sample (in 10% methanol) and
20 µl of 0.09 unit/ml AChE from Electrophorus electricus (Electric eel). After pre-
incubation at room temperature, 10 µl of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid)
DTNB was added into each well followed by 10 µl of 14 mM acetylthiocholine iodide as
substrate. The absorbance of the coloured product was measured using a microplate
reader at 412 nm following 30 minutes incubation. Donepezil hydrochloride (Sigma-
Aldrich) was used as a positive control. Percentage inhibition was calculated using the
following formula for eight different concentrations:
Percentage Inhibition (%) =
100
Three replicates of the sample were used for statistical analysis with values reported as
mean ± S.D. Standard curves were generated and the 50% inhibitory concentration (IC50)
values were calculated using GraphPad Prism.
2.5 Molecular docking
A docking study was carried out using AUTODOCK 4.2 [33]. The X-ray crystal structure
of the acetylcholinesterase complexed with donepezil (PDB code: 1EVE) was obtained
from the Protein Data Bank (https://www.rcsb.org/structure/1eve). All ligands and water
molecules were removed from the retrieved protein using Discovery Studio Visualizer
v17.2.0.16349 [34]. The experimental crystal structure of chalcone 3 was converted to
PDB file and used for docking after the removal of the co-solvent (ethanol). The

chalcone-protein interactions were analysed and visualised in two-dimensional (2D)
diagrams using Discovery Studio Visualizer.
3. Results and discussion
3.1 Spectral analysis
The 1H NMR spectrum of chalcone 3 showed an AB spin system at ẟH 7.97 and 8.36 (d, J
= 15.4 Hz) attributed to H-β and H-α, respectively as presented in Fig. 1. These two
doublets are characteristic of the trans-olefinic protons of chalcone. The remaining
aromatic protons were assigned based on the COSY spectrum (Fig. 2). The 13C NMR and
DEPT spectra displayed twenty-eight carbon signals, including five methylenes, fourteen
methines, eight quaternaries and one carbonyl carbon (Fig. S1). The HMQC spectrum
displayed direct correlations between protons and carbons in the compound, thus
confirmed the assignment of the protonated carbon peaks at δC 22.78 (C-9ʹ/11), 71.20 (C-
1ʺ), 128.67 (C-5ʺ), 128.74 (C-4ʺ/6ʺ), 129.10 (C-3ʺ/7ʺ) (Fig. S2). HMBC spectrum was
reported to confirm the assignment of protons and carbons. The spectrum Fig. S3
exhibited the presence of correlation between A2B2 aromatic protons at δH 7.57-7.63 (H-
3", H-7") with C-4",6 " (δC 128.74), while protons at δH 7.38-7.47 (H-4ʺ, H-6ʺ) were
correlated with C-3",7 " (δC 129.10) and C-5" (δC 128.67). The complete 1H, 13C, COSY,
HMQC and HMBC spectral data of chalcone 3 are listed in Table 1.

Fig. 1 1H NMR spectrum of chalcone 3
H-6, H-6ʹ
H-3ʺ, H-7ʺ
H-α
H-8
H-7, H-4ʺ,
H-6ʺ, H-5ʺ
H-5ʹ
H-2ʹ
H-5
H-β
H-3
H-4
ppm
7.0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
8.6
8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9
ppm
Fig. 2 1H-1H COSY spectrum of chalcone 3
Table 1 NMR Spectroscopic data of chalcone 3
Position δH
δC
COSY
HMBC

1
2
3
4
5
6
7
8
-
-
132.93
156.84
132.54
115.15
123.19
128.36
124.53
129.30
129.18
116.77
8.06 (d, J = 9.2 Hz)
7.67 (d, J = 9.2 Hz)
8.20 (d, J = 8.8 Hz)
7.57-7.63 (m)
7.38-7.47 (m)
7.94 (d, J = 8.8 Hz)
-
H-4
C-2, C-1
H-3
H-6
H-5, H-7
H-6, H-8
H-7
C-2, C-9, C-10
C-7, C-9, C-10
C-8
C-5
C-1, C-6, C-10
9
10
α
-
7.97 (d, J = 15.8 Hz) 126.76
8.36 (d, J = 15.8 Hz) 136.06
H- β
H- α
C=O, C-1
C=O, C-2, C-1, C-α
3.2
β
Crystal
structure
analysis
of
1ʹ
2ʹ
3ʹ
4ʹ
5ʹ
6ʹ
7ʹ
8ʹ, 12
9ʹ, 11
10ʹ
-
129.74
134.91
117.17
161.76
116.06
132.16
54.02
52.32
22.78
21.66
8.16 (d, J = 2.0 Hz)
-
-
7.02 (d, J = 8.8 Hz)
7.57-7.63 (m)
4.21 (s)
2.97 (br s)
1.75 (br s)
1.50 (br s)
C-4ʹ, C-6ʹ, C-7ʹ
chalcone
3 and
Hirshfeld
Surface
Analysis
H-6ʹ
H-5ʹ
C-1ʹ, C-3ʹ, C4ʹ
C-4ʹ, C-2ʹ
C-2ʹ, C-3ʹ, C4ʹ, C-8ʹ
H-3ʺ, H-7ʺ
C-2, C-2ʺ, C-3ʺ, C-
7ʺ
Details
of data
collectio
n,
1ʺ
5.43 (s)
71.20
2ʺ
-
137.17
129.10
128.74
128.67
187.88
3ʺ, 7ʺ
4ʺ, 6ʺ
5ʺ
7.57-7.63 (m)
7.38-7.47 (m)
7.38-7.47 (m)
-
C-4ʺ, C-6ʺ
C-3ʺ, C- 7ʺ, C-5ʺ
paramete
rs,
C=O
Experimental data measured at 400 MHz in DMSO
crystallographic data and final agreement parameters are collected in Table 2. Selected
bond angles and distances are collected in Table 3. The molecular structure of 3 with
atomic numbering is shown in Fig. 3(a) and Table 4. With one co-crystallized solvent
molecule, X-ray single-crystal structural analysis revealed that compound 3 is triclinic,
space group P-1, with two chalcone 3 molecule per cell. The bonds around the piperidine
N atom, C30–N31, N31–C36, N31–C32 are 1.4736 (15) 1.4705 (16) and 1.4740 (16) Å
respectively which are slightly shorter than the normal C–N bond, 1.50
Table 2 Crystal data and structure refinement details for 2-Benzyloxynaphthylchalcone 3

C32H31NO3C2H6O
F(000)= 560
Mr = 523.64
Triclinic, P-1 (no. 2)
a = 10.6946 (5) Å
Dx = 1.248 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 7929 reflections,
b = 11.7088 (5) Å
θ = 3.3–32.4
c = 13.5975 (5) Å
α = 96.356 (3)°
β = 107.209 (4)°
γ = 116.643 (5)°
V = 1393.45 (12) Å3
Z = 2
θ = 3.3–32.4°
µ = 0.08 mm−1
T = 140 K
Wedge, pale yellow
0.43 × 0.28 × 0.23 mm
Oxford Diffraction Xcalibur 3/Sapphire3 CCD
diffractometer
6372 independent reflections
Radiation source: Enhance (Mo) X-ray Source 4826 reflections with I > 2σ(I) ‘observed’
Graphite monochromator
Rint = 0.037
Detector resolution: 16.0050 pixels mm-1
θmax = 27.5°, θmin = 3.7°
H atoms treated by a mixture of independent
and constrained refinement
R1(‘observed’ data) = 0.045
Weighting: w = 1/[σ2(Fo2) + (0.059P)2 +
0.130P]
where P = (Fo2 + 2Fc2)/3
wR2(all data) = 0.125
S = 1.09
(Δ/σ)max = 0.001
Δρmax = 0.41 e Å−3
Δρmin = −0.29 e Å−3
6372 reflections
360 parameters
Table 3 Selected geometric parameters (Å, °) of compound 3
C2—O2
1.3627 (15)
1.4366 (17)
1.2293 (16)
1.3574 (14)
C30—N31
N31—C36
N31—C32
O41—C42
1.4736 (15)
1.4705 (16)
1.4740 (16)
1.429 (2)
O2—C20
C13—O13
C17—O17

O2—C2—C1
116.23 (11)
121.49 (12)
121.20 (11)
120.66 (11)
117.94 (12)
105.48 (11)
C30—N31—C32
C2—O2—C20
111.00 (10)
120.63 (10)
110.18 (11)
112.99 (17)
121.79 (11)
O2—C2—C3
O13—C13—C12
O13—C13—C14
O17—C17—C18
O2—C20—C21
N31—C36—C35
O41—C42—C43
O17—C17—C16
Table 4 Hydrogen-bond geometry (Å, °) of compound 3
D—H···A
D—H
0.93
H···A
2.13
D···A
D—H···A
C12—H12···O2
C20—H20B···O41i
O17—H17O···N31
2.7297 (16) 121
3.4797 (18) 153
0.97
2.59
0.93 (2)
0.92 (3)
1.73 (2)
1.90 (3)
2.6165 (16) 157.5 (16)
2.7980 (17) 168 (2)
O41—H41O···O13
Symmetry code: (i) x−1, y−1, z.
Å [35]. The molecule exhibits short intra- and inter-molecular interactions. An
intramolecular hydrogen bond links the hydroxyl group of O(17) with the piperidine
N(31) atom, and the solvent ethanol molecule forms a good hydrogen bond with the
carbonyl O(13) atom, Table 4 and Fig. 3(a). The molecule comprises three planar
aromatic ring groups and a chair-shaped piperidine ring. The hinge atoms between the
planar groups are C(20) and C(12), with the piperidine group hinged at C(30). The
naphthyl group lies parallel (at a distance of 3.476 Å), and related about a centre of
symmetry, to a neighbouring group with extensive π…π interactions. The chain of C(11,
12, 13, 14, 19, 18, 17) and O(17) overlies a symmetry-related chain, also with close π…π
interactions and at a distance of 3.375 Å. Fig. 4(b). Fig. 3(b) illustrates the molecular
interactions in compound 3 by showing red, white and blue colors; the presence of red
areas around some atoms indicates strong internal and external interactions.

‘Fingerprint plots’ from the Hirshfeld surface analysis, Fig, 5, show that H...H contacts
have the highest number of interactions in compound 3, (62.4%). The C…H/H…C
contacts act as secondary interactions with sharp peaks (20.8%). H…N/N…H contacts
show no role (0.0%), and O…H/H…O contacts account for 10.4% of interactions (di + de
= 2.4 Å), (Fig. 5). Interestingly, C…C interactions for 3 account for 5.2% of contacts,
indicating good π…π stacking.

(c)
(b)
(a)
N
N
OH
O
OH
O
O
O
Fig. 3 (a) An ORTEP diagram of 3 showing the atom-labelling scheme. Thermal ellipsoids are shown at 50% probability, (b) Hirshfeld surfaces
views of the molecule 3 mapped over dnorm with enabled surface transparency. The red and blue regions represent negative and positive electrostatic
potentials, respectively, (c) Mesh diagram of
3 showing the molecule with the extent of the electron density around it.

(a)
Fig. 4 (a) A projected view of the molecular packing of 3, (b) A pair of overlaying molecules of 3
showing the distance between calculated centroids. The distance between the overlapping rings is
ca 3.42 Å. This view was drawn using Mercury [36]. Hydrogen atoms are omitted for clarity and
were not included in the distance calculation.

Fig. 5 Two-dimensional fingerprint plots for compound 3: for Total, H...All, H...H, H...C, H...O and
H…N interactions. The outline of the full fingerprint is shown in gray. di is the closest internal
distance from a given point on the Hirshfeld surface and de is the closest external distance.
3.3 Metal-chelating properties
Considering the implications of metals in AD pathogenesis, the chelating ability of
chalcone 3 toward biometals such as Cu2+, Fe2+, Al3+, and Zn2+, was evaluated using
ultraviolet-visible (UV–Vis) spectrometry. The metal complexation capacity of chalcone
3 was investigated in situ using UV−Vis absorption. Interestingly, the maximum
absorption at 338 nm was shifted to 307 nm after the addition of FeCl2, which indicated
the possible formation of a 3-Fe2+ complex. The difference in the UV−Vis absorption
peaks (31 nm) between the free ligand and the Fe(II) complex is probably due to changes
in the molecular/electronic conformation effect caused by complexation, Figs. 6 and 8

[37]. The addition of AlCl3 and CuCl2 exhibited marginal blue shift of the maximum absorption
from 338 to 329 nm. However, no significant change was observed with the addition of ZnCl2.
To consider the stoichiometry of the resulting complex 3-Fe2+ using the molar ratio
method, chalcone 3 in a fixed concentration (60 μM) was mixed with increasing
concentrations of FeCl2 (6-100 μM). The absorbance of the resulting complex of FeCl2
and 3 was recorded using a UV-Vis spectrometer at 307 nm. As displayed in Fig 7, the
absorbance increased linearly at first, before plateauing when the mole fraction of Fe2+ to
3 was about 0.9, indicating a 1:1 stoichiometry for complex 3-Fe2+.
3
2.5
2
1.5
1
0.5
0
200
250
300
350
400
450
500
550
600
Wavelength (nm)
3-Cu 3-Zn
3-Al
3
3-Fe
Fig. 6 UV−Vis spectra of chalcone 3 in methanol (60 μM) alone and titrated with CuCl2, FeCl2,
ZnCl2 or AlCl3 (60 μM in methanol)

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Mole fraction of Fe (II) to compound 3
Fig. 7 The stoichiometry of complex 3-Fe2+ via the molar ratio method
To highlight the metal chelating sites a deuterium methanolic solution of chalcone 3 (19
μM) was titrated in situ with increasing equivalents of FeCl2 solution (19 μM). The
progress of the complexation was monitored using1H NMR spectroscopy as presented in
Fig. 8. The spectra indicated that the signals of the piperidine protons have been split to
five different signals due to the coordination of Fe+2 with the nitrogen atom; compared to
1
the crystal structure H NMR spectrum, which showed three broad singlet signals
attributed to three sets of equivalent CH2 groups of the piperidine moiety, this suggests
that the chalcone conformation was changed upon complexation. Moreover, the
methylene protons (Hb) have been shifted upfield upon complexation. Similarly, the
benzyloxy protons have been shifted upfield which indicated coordination via its oxygen.
The structure involved the ferrous(II) ion being coordinated through the lone pair
electrons of both nitrogen and oxygen atoms, correlated to Mannich base and benzyloxy
moieties respectively, and forming, probably, an octahedral structure, the common
geometric structure for Fe(II) in the presence of water/methanol molecules in solution
[38]. Because it was not possible to obtain a suitable single crystal for X-ray study, the
geometric structure for Fe(II) may not be fully confirmed. The IR spectra have proven to

be a suitable tool to provide enough information for us to describe the way of bonding of
the ligands. Therefore, a detailed elucidation of IR spectra of the chalcone ligand and the
effect of binding of iron(II) on the vibration frequencies of the chalcone ligand are
highlighted to conclude that the coordination mode involves chelation. The IR spectrum
of the precipitated complex (3-Fe2+) confirmed the coordination sites due to the presence
of a broad absorption band at 3432 cm-1 for the hydroxyl group and a very weak
stretching vibration at 2946 cm-1 for the aliphatic C-H. Additionally, it showed an
absorption frequency at 1604 cm-1 implying the presence of a conjugated carbonyl group
adjacent to the C=C group, with characteristic olefinic peak at 1641 cm-1 (Fig S4).
FeCl2
CD3OD
1:1 Iron (II) complex
Chalcone 3
Fig. 8 1H-NMR titration of compound 3 with FeCl2 in CD3OD. Spectra 2-6 recorded upon the
addition of increasing eq. of FeCl2
3.4 In vitro bioactivity evaluations of chalcone 3
The novel chalcone 3 exhibited potent inhibitory activity against Electric eel AChE with
IC50 1.0 nM showing better inhibition than donepezil (IC50. 33.4 nM). Furthermore, it
showed comparable scavenging inhibitory activity towards DPPH comparable to that of
ascorbic acid with a 3-fold difference, as displayed in Table 5.

Table 5 Cholinesterase and DPPH activities of chalcone 3
Compound
3
AChE IC50 (nM) ± SD
DPPH Radical IC50 µg/ml
1.0 0.003
33.4  0.002
-
55.52  1.1
-
Donepezil
AA
17.96  0.4
IC ±SD: Inhibitor concentration (mean ± S.D. of three experiments) needed for 50% inhibition
50
of the enzyme; AA (Ascorbic acid)
3.5 Binding prediction of chalcone 3 using molecular docking
Docking analysis was carried out using the elucidated crystal structure data of chalcone 3
to screen its binding affinity on the Torpedo california acetylcholinesterase (TcAChE)
(PDB 1EVE). Chalcone 3 displayed tight binding against the TcAChE enzyme with low
binding energy -13.29 kcal/mol in comparison to donepezil (-10.52 kcal/mol).
Superimposition of 3 (yellow stick) and donepezil (grey stick) as shown in Fig. 9
demonstrates the benzylic moiety in ring B is directed towards the peripheral anionic site
(PAS), while the three planar aromatic rings of chalcone 3 turned the piperidine ring
towards the catalytic anionic site (CAS) similar to donepezil. We note that the molecular
structure of 3 has changed drastically after binding with the protein when compared to the
free molecule. Comparison of the interactions of chalcone 3 and donepezil within the
active site of AChE revealed that there is considerable flexibility of the chalcone 3
molecule that facilitates the interaction between piperidine and the binding site for the
quaternary nitrogen of AChE, TRP84 via π-alkyl interactions with distances of 5.30 and
5.01 Å in line with donepezil (Fig. 9). TRP84 was considered the principal component of
the anionic site [39]. However, two hydrogen bonds were found between the hydroxyl
group at ring A of 3 and residues GLY118, and GLU199 at the oxyanion site with
distances 2.87 and 2.18 Å respectively Fig. 10(a). Moreover, a hydrogen bond was found
between the hydroxyl group of TYR121 at the PAS site and benzyloxy group at ring B
with distance 2.65 Å. Furthermore, hydrophobic interaction with the PAS was observed
between the benzyl moiety of chalcone 3 and the residue ASP70. This residue is a vital
component of the PAS site as several studies have reported its role as a trap for charged

substrate, thus contributing to catalytic efficiency. Similar transient binding also occurs
with cationic organophosphates [40,41]. It is also worth noting that at the bottom of the
gorge, a CH···π T-shaped hydrophobic interaction was observed between ring A of
chalcone 3 and the phenyl moiety of HIS440, a residue of the catalytic triad in the active
site of AChE [42]. These interactions were not observed in donepezil and might
contribute to the affinity to the enzyme hence improving the inhibition effect of chalcone
3. Overlay of the crystallographic structure (magenta) and the docked (green)
conformations of chalcone 3 suggests that the presence of methylene groups might be
responsible for the chalcone structural flexibility as presented in Fig. 11.
Therefore, we propose that the novel chalcone 3 shows potent in vitro inhibitory activity
towards AChE when compared with donepezil.
(a)
(b)
Fig. 9 Docking model of TcAChE-3 complex showing superimposition of the ligand 3 (yellow
stick) with donepezil (grey stick) in the active site of 1EVE (a). The interacting mode of chalcone
3 in the active gorge of TcAChE, (b)

(b)
(a)
Fig. 10 2D representations of interactions in the TcAChE- 3 complex (a) and in the TcAChE-donepezil complex(b)

Fig. 11 Overlay of crystallographic (magenta) and docked (green) conformations of chalcone 3
4. Conclusion
Interesting HS analysis was accomplished for the 2-benzyloxynaphthalene
aminoalkylated chalcone 3. The in vitro analysis of chalcone 3 showed potent inhibitory
activity against AChE with moderate scavenging ability toward DPPH. Moreover, the
chalcone 3 was able to coordinate with the biometal FeCl2 in a stoichiometry ratio 1:1 to
form the complex 3-Fe2+ based on the UV−Vis and the NMR analysis. Molecular
docking analysis was used to further explore the binding mode of 3 with AChE and to
investigate the efficacy of introducing Mannich base and benzyl moieties into a
naphthalene-based chalcone. The molecular modelling proves the potential inhibition
activity of 3 against AChE due to the presence of three hydrogen bonds with the PAS and
oxyanion site of 1EVE. Also, different critical hydrophobic interactions were observed
with the CAS amino acids and HIS440, a crucial residue of the catalytic triad. Taken
together, these preliminary findings present a starting point for development of
multifunctional agents for the treatment of AD in future.
5. Supplementary data:
CCDC 1973469 contains the crystallographic data for compound 3. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_ request/cif or from the Cambridge

Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (þ44)
1223 336 033; or e-mail: deposit@ccdc.cam.ac.u.k.
6. Acknowledgment: The authors are grateful for the financial support from Taibah
University and the technical assistance from UTM (4F830). M. A. Said is thankful to
Alexander von Humboldt Stiftung, Germany, for the opportunities provided.
Credit Author Statement
Author Contributions:
Conceptualization, N.B, A.A.A. S.Y.A. and G. A.
Methodology and Docking, G.A.
All authors, Writing-Review & Editing
D.L. and M.A.S; X-ray work
M.A.; HSA work.
CONFLICT OF INTEREST
NO CONFLICT OF INTEREST
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GRAPHICAL ABSTRACT