Exploring 3-Benzyloxyflavones as new lead cholinesterase inhibitors: synthesis, structure–activity relationship and molecular modelling simulations

Article abstract of DOI:10.1080/07391102.2020.1803136

In this protocol, a series of 3-benzyloxyflavone derivatives have been designed, synthesized, characterized and investigated in?vitro as cholinesterase inhibitors. The findings showed that all the synthesized target compounds (1–10) are potent dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes with varying IC₅₀ values. In comparison, they are more active against AChE than BChE. Remarkably, amongst the series, the compound 2 was identified as the most active inhibitor of both AChE (IC₅₀ = 0.05 ± 0.01 μM) and BChE (IC₅₀ = 0.09 ± 0.02 μM) relative to the standard Donepezil (IC₅₀ = 0.09 ± 0.01 for AChE and 0.13 ± 0.04 μM for BChE). Moreover, the derivatives 5 (IC₅₀ = 0.07 ± 0.02 μM) and 10 (0.08 ± 0.02 μM) exhibited the highest selective inhibition against AChE as compared to the standard. Preliminary structure-activity relationship was established and thus found that cholinesterase inhibitory activities of these compounds are highly dependent on the nature and position of various substituents on Ring-B of the 3-Benzyloxyflavone scaffolds. In order to find out the nature of binding interactions of the compounds and active sites of the enzymes, molecular docking studies were carried out. (Figure presented.) HIGHLIGHTS 3-benzyloxyflavone analogues were designed, synthesized and characterized. The target molecules (1–10) were evaluated for their inhibitory potential against AChE and BChE inhibitory activities. Limited structure-activity relationship was developed based on the different substituent patterns on aryl part. Molecular docking studies were conducted to correlate the in?vitro results and to identify possible mode of interactions at the active pocket site of the enzyme. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1803136

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Exploring 3-Benzyloxyflavones as new
lead cholinesterase inhibitors: synthesis,
structure–activity relationship and molecular
modelling simulations
Ehsan Ullah Mughal , Amina Sadiq , Momna Ayub , Nafeesa Naeem , Asif
Javid , Sajjad Hussain Sumrra , Muhammad Naveed Zafar , Bilal Ahmad
Khan , Fouzia Perveen Malik & Ishtiaq Ahmed
To cite this article: Ehsan Ullah Mughal , Amina Sadiq , Momna Ayub , Nafeesa Naeem , Asif
Javid , Sajjad Hussain Sumrra , Muhammad Naveed Zafar , Bilal Ahmad Khan , Fouzia Perveen
Malik & Ishtiaq Ahmed (2020): Exploring 3-Benzyloxyflavones as new lead cholinesterase
inhibitors: synthesis, structure–activity relationship and molecular modelling simulations, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1803136
To link to this article: https://doi.org/10.1080/07391102.2020.1803136
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1803136
Exploring 3-Benzyloxyflavones as new lead cholinesterase inhibitors: synthesis,
structure–activity relationship and molecular modelling simulations
Ehsan Ullah Mughala, Amina Sadiqb, Momna Ayubb, Nafeesa Naeema, Asif Javida, Sajjad Hussain Sumrraa,
Muhammad Naveed Zafarc, Bilal Ahmad Khand, Fouzia Perveen Malike and Ishtiaq Ahmedf
b
aDepartment of Chemistry, University of Gujrat, Gujrat, Pakistan; Department of Chemistry, Govt. College Women University, Sialkot,
c
d
Pakistan; Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan; Department of Chemistry, University of Azad Jammu
e
f
and Kashmir, Muzaffarabad, Pakistan; RCMS, National University of Science and Technology, Islamabad, Pakistan; Department of
Engineering & Biotechnology, University of Cambridge, Cambridge, UK
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 7 May 2020
Accepted 13 July 2020
In this protocol, a series of 3-benzyloxyflavone derivatives have been designed, synthesized, characterized
and investigated in vitro as cholinesterase inhibitors. The findings showed that all the synthesized target
compounds (1–10) are potent dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) enzymes with varying IC50 values. In comparison, they are more active against AChE than BChE.
Remarkably, amongst the series, the compound 2 was identified as the most active inhibitor of both
KEYWORDS
3-Benzyloxyflavones;
Flavonoids; Alzheimer’s
disease; Cholinesterase
inhibitors; Benzyl chloride;
Molecular modelling studies
AChE (IC50 ¼ 0.05 0.01 lM) and BChE (IC50 ¼ 0.09 0.02 lM) relative to the standard Donepezil (IC50
¼
0.09 0.01 for AChE and 0.13 0.04 lM for BChE). Moreover, the derivatives 5 (IC50 ¼ 0.07 0.02 lM) and
10 (0.08 0.02 lM) exhibited the highest selective inhibition against AChE as compared to the standard.
Preliminary structure-activity relationship was established and thus found that cholinesterase inhibitory
activities of these compounds are highly dependent on the nature and position of various substituents
on Ring-B of the 3-Benzyloxyflavone scaffolds. In order to find out the nature of binding interactions of
the compounds and active sites of the enzymes, molecular docking studies were carried out.
HIGHLIGHTS
1. 3-benzyloxyflavone analogues were designed, synthesized and characterized.
2. The target molecules (1–10) were evaluated for their inhibitory potential against AChE and BChE
inhibitory activities.
3. Limited structure-activity relationship was developed based on the different substituent patterns
on aryl part.
4. Molecular docking studies were conducted to correlate the in vitro results and to identify possible
mode of interactions at the active pocket site of the enzyme.
Abbreviations: AChE: Acetylcholinesterase; Ala: Alanine; AD: Alzheimer disease; AFO:
Algar–Flynn–Oyamada; AI: Amyloid-including protein; BChE: Butyrylcholinesterase; DMF:
Dimethylformamide; DMSO: Dimethyl sulfoxide; ESI: Electrospray ionization; EtOH: Ethanol; FTIR:
Fourier transformed infrared radiations; His: Histamine; H2O2: Hydrogen peroxide; IR: Infrared; K2CO3:
Potassium carbonate; Leu: Leucine; MeOH: Methanol; Met: Methionine; NaOH: Sodium hydroxide; NMR:
CONTACT Ehsan Ullah Mughal
ehsan.ullah@uog.edu.pk
Department of Chemistry, Govt. College Women University, Sialkot-51300, Pakistan
Department of Chemistry, University of Gujrat, Gujrat, 50700, Pakistan; Amina Sadiq
amina.sadiq@gcwus.edu.pk
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1803136.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
E. U. MUGHAL ET AL.
Nuclear magnetic resonance; Pas: Peripheral anionic site; PBD: Protein drug bank; Phe: Phenylalanine;
Pro: Proline; Ser: Serine; SAR: Structure-activity relationship; TLC: Thin-layer chromatography; Trp:
Tryptophan; Tyr: Tyrosine; UV-VIS: Ultraviolet-Visible
Introduction
in the basal forebrain. The development of chemical agents
for the treatment of AD has been of great interest for several
years (Rampa et al., 1998). The enzymes such as AChE and
BChE were identified as essential targets for efficient AD
management by increasing the production of acetylcholine
in the brain and reducing the AI deposition as each of these
enzymes play an essential role at the beginning of geriatric
plaque development (Mughal et al., 2018; 2019). Deposition
of b-amyloid in the brain is hypothesized as the main cause
of neuronal cell death in AD patients, thus preventing the
accumulation of b-amyloid represents an alternative thera-
peutic strategy that specifically targets AD pathogenesis.
Several small molecules can interact with the mechanism of
b-amyloid aggregation and the functionalization of small aro-
matic molecules can be used as the logical nature of inhibi-
tors of aggregation. Although there is a significant amount
of literature reporting on AChE and BChE inhibitors (Agbo
et al., 2019; Asghar et al., 2020; Bajda et al., 2011; Barai et al.,
2019; Cavdar et al., 2019; Chen et al., 2017; Mughal, Sadiq,
Khan, et al., 2017; Mughal, Sadiq, Murtaza, et al., 2017; Faraji
et al., 2019; Mathew et al., 2019; Mishra et al., 2017;
Mphahlele et al., 2018; Mughal et al., 2018; Mughal et al.,
Flavonoids represent a group of polyphenolic compounds
which are widely distributed throughout the higher plants.
They are recognized by possessing 2-phenyl-c-benzopyrone
(C6-C3-C6) as a characteristic skeleton. They are further div-
ided into various sub-classes such as aurones, anthocyanins,
chalcones, flavones, flavonols, and isoflavones depending on
the saturation level and the absence or presence of the cen-
tral pyran ring (Patel & Shah, 2017; Sashidhara et al., 2012).
Studies performed on these compounds have confirmed that
they belong to the class of secondary plant metabolites
(Harborne & Williams, 2000; Hostetler et al., 2017) and are
well known for having a vast variety of bioactivities such as
anti-oxidant (Rice-Evans, 2001), anxiolytic and anti-cancer (Liu
et al., 2010), analgesic, anti-inflammatory and anti-microbial
(Mishra & Tiwari, 2011), anti-ulcer and thrombosis (Bhatt
et al., 2016).
Flavonols (3-hydroxy-2-phenyl-4H-chromen-4-ones) (Figure
1) constitute one of the most important sub-classes of flavo-
noids. Owing to the structural diversity of flavonols (3-
hydroxyflavones) and their ubiquitous occurrence in fruits
and vegetables, they have attracted the attention of medi-
cinal chemists because of their importance in a variety of
important pharmacological activities including health bene-
fits (Bozdag-Dundar et al., 2005; Graf et al., 2005; Tapas et al.,
2008). However, most of the flavonols have not been devel-
oped as clinical drugs because of poor bioavailability (less
than 5%), toxicity, limited occurrence in nature and induction
or inhibition of some metabolic enzymes (Jin et al., 2019; Li
et al., 2017).
€
2019; Ozil et al., 2019; Saxena & Dubey, 2019; Shaik et al.,
2016; Shaikh et al., 2020; Sun et al., 2019; Xie et al., 2016;
Yerdelen et al., 2015), but it has been observed that a great
deal of attention has been paid in recent years to the hunt
for new AChE inhibitors for the treatment of AD (Loizzo
et al., 2008). In this connection, during recent years, natural
flavonoids and their synthetic analogues have been identi-
fied as potential inhibitors of cholinesterase enzymes, as they
have the benefits of being more tolerable, inexpensive and
easier to occur in the natural environment (Agbo et al., 2019;
Anand & Singh, 2013; Daglia, 2012; Mughal, Sadiq, Khan,
et al., 2017; Mughal, Sadiq, Murtaza, et al., 2017; Uriarte-
Pueyo & I Calvo, 2011). Furthermore, flavonoids and related
compounds were reported in the literature as potent anti-
microbial agents (Genoux et al., 2011; Ibrahim, 2014;
Kamlesh et al., 2017; Mahmoud et al., 2017; Mughal et al.,
2006; Sarbu et al., 2019; Shakhatreh et al., 2016). Due to their
unique ability to modulate different enzyme systems, the
envisioned compounds exhibit a great diversity in their bio-
logical activities and therapeutic functions (Dymarska et al.,
2018). Additionally, the substitution pattern in flavone deriva-
tives plays a vital role in their biochemical and pharmaco-
logical properties.
To overcome those problems, the development of new
synthetic methods to produce structurally modified flavones
is now considered as an important goal in exploring their
diverse roles. This reason has increased the interest of medi-
cinal chemists to further study flavones as lead molecules to
treat various diseases. In this context, recently researchers
have focused on the synthesis and biological evaluation of
ꢀ
flavone-based ethers (Dıaz et al., 2017; Imran et al., 2016; Jin
et al., 2019; Li et al., 2017; Nhu et al., 2015; Wang et al.,
2018). However, the reported natural and synthetic flavone
derivatives containing ether linkage mostly present at ring A
or ring B, and such motifs have been reported to possess
potent enzyme inhibition activities (Imran et al., 2016).
Therefore, to the best of our knowledge, exploring the syn-
thesis and inhibitory potential of 3-O-benzylflavonol scaffolds
against cholinesterase enzymes remain an interesting goal.
Senile Alzheimer’s disease (AD) is a severe neurodegenera-
tive disorder with progressive cognitive impairments among
the older people in developed and some developing coun-
tries that eventually leads to death. AD is responsible for
Encouraged by above-mentioned biological potential of
flavone as a privileged scaffold and following our study on
exploring new structural motifs against cholinesterase
enzymes (Mughal, Sadiq, Khan, et al.2017; Mughal, Sadiq,
Murtaza, et al., 2017; Mughal et al., 2018; Mughal et al.,
2019), we have designed and synthesized a new set of 3-O-
protein aggregation, inflammation, amyloid-including protein benzylflavonol ethers with different functional groups for
(AI), oxidative stress, and acetylcholine signalling dysfunction determining their inhibitory capacity against the previously

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Figure 1. Representative structures of Flavonol (A) and 3-O-benzylflavonol or 3-benzyloxyflavone (B).
mentioned targets. The role of synthetic flavones as possible 30 min, benzyl chloride (2.0 mmol) was added and the reac-
inhibitors of cholinesterases remains an important target and tion mixture was stirred further at the same temperature for
a constant endeavour.
24 to 48 h. After reaction completion (analysed by TLC), the
reaction mixture was quenched by addition of water and
neutralized with HCl (10%). The phases were separated and
the aqueous phase was extracted with chloroform
(2 ꢁ 25 mL). The combined organic phase was washed with
brine, dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The crude product was
purified by recrystallization in ethanol to afford the desired
product in pure form.
Materials and methods
All the commercially available reagents were obtained from
Sigma-Aldrich and Merck and used as supplied. Melting
points were determined on an Electro-thermal melting point
apparatus and uncorrected. IR spectra were taken as KBr
discs using Bio-Rad spectrophotometer. NMR (1H, 300 MHz,
13C, 75 MHz) spectra were recorded on a Bruker spectrometer
with TMS as the internal standard. EI-MS spectrometric ana-
lysis was obtained using a Fisons VG sector-field instrument.
Enzyme inhibition assay
ꢁ
The IR values and chemical shifts are expressed in m units AChE and BChE inhibition activities were measured spectro-
and parts per million respectively. Reaction progress was photometrically by the Ellman method with minor adjust-
monitored using thin-layer chromatography (TLC) on silica ment. Five different concentrations of standard and test
gel pre-coated plates and spots were detected under UV
light. The absorption spectra have been recorded on the
Jasco UV-VIS V-670 instrument using QUARTZ cell in very
dilute solutions prepared in different solvents.
compounds were prepared by diluting their stock solutions
immediately before use. 100 mL of each sample was com-
bined with 50 mL AChE/BChE enzyme and allowed to stand
for 10 min. 50 mL of substratum i.e. acetylthiocholine iodide
(0.71 mM) for acetylcholinesterase or butyrylthiocholine chlor-
ide (0.2 mM) for butyrylcholinesterase, 50 mL (0.5 mM) of
DTNB and 500 mL phosphate buffer of pH 8 were added to
the above-mentioned mixture and the mixture was incu-
bated at retention time for 15 min at 37 ˚C. The solution
becomes yellow due to the hydrolysis of substrate causing
the formation of 5-thio-2-nitrobenzoate anion. The substrate
hydrolysis was determined using spectrophotometer by
measuring the increase in absorbance at 400 nm and 412 nm
for acetylcholinesterase and butyrylcholinesterase, respect-
ively. The percent enzyme inhibition (%) was calculated using
the following equation:
General procedures for the syntheses of 3-
hydroxyflavone and 3-benzyloxyflavone derivatives
Synthesis of 3-hydroxyflavone derivatives (F1-F10)
A mixture of 20-hydroxyacetophenone (1.2 mL, 10.0 mmol)
and 10 mL of an aqueous solution of NaOH (30%) was stirred
in MeOH (25 mL) for 30 min accompanied by dropwise add-
ition of substituted benzaldehyde (10.0 mmol). The reaction
mixture was stirred further at ambient temperature for 5-6 h.
The progress of reaction was checked by comparative TLC.
Chalcone thus formed, in situ, was cyclized further by the
addition of 1.5 mL of 35% H2O2 solution followed by stirring
for an additional 1 h at the same temperature. The reaction
mixture was neutralized by hydrochloric acid (HCl, 10%). The
precipitates formed were filtered, washed thoroughly with
water, dried and crystallized by ethanol to give pure
Flavonol (F1-F10).
BꢂA
ð Þ
% Inhibition ¼
ꢁ 100
B
Here A ¼ enzyme absorbance with test sample;
B ¼ enzyme absorbance without test sample. Every experi-
ment was performed in triplicate and the average value was
taken. IC50 values were estimated by linear regression ana-
lysis. Donepezil was used as a standard (Agbo et al., 2019;
Asghar et al., 2020; Barai et al., 2019; Mughal, Sadiq, Khan,
Synthesis of 3-benzyloxyflavone derivatives (1–10)
The pure synthesized flavonol product (1.0 mmol) was dis- et al., 2017; Mughal, Sadiq, Murtaza, et al., 2017; Faraji et al.,
solved in dimethylformamide (5.0 mL) containing anhydrous 2019; Mishra et al., 2017; Mphahlele et al., 2018; Mughal
€
K2CO3 (5.0 mmol), and tetra-butyl ammonium bromide et al., 2018; Mughal et al., 2019; Ozil et al., 2019; Shaikh
(3.0 mmol). After stirring the reaction mixture at 50-60 ꢀC for et al., 2020; Sun et al., 2019).

4
E. U. MUGHAL ET AL.
two characteristics signals around d 173.0 ppm and 75.0 ppm
for ketonic and oxymethylene (-OCH2-) structural features,
respectively. Likewise, the structures of other derivatives
were also characterized by a similar strategy. In spite of hav-
ing all NMR data, the complete assignment of each signal
was not achieved. Nevertheless, all spectroscopic data are
in good agreement with assumed structures of the
desired compounds.
The spectral data of all the synthesized flavonols except
F2 are given in the literature as well as in supporting infor-
mation (SI) (Gunduz et al., 2012; Gupta et al., 2014; Khanna
et al., 2015; Singh et al., 2017; You et al., 2020). However, the
complete spectroscopic data of their ether derivatives (1–10)
and the flavonol (F2) are as under:
Molecular docking studies
Docking assay was conducted to determine the enzyme-lig-
and interactions. AChE (PDB ID: 4BDT) and BChE (PDB ID:
4BDS) crystal structures were obtained from ACDz
Chemsketch protein database (RCSB) and 3 D Pro 12.0 was
used to optimize the compounds in 3 D orientation and
processed as SYBYL mol 2 file format. The AutoDock software
v1.5.6 has been used for docking purposes. Discovery Studio
Visualizer v 4.0 was used to represent the most active and
best position of compounds under analysis (Mughal, Sadiq,
Khan, et al.2017; Mughal, Sadiq, Murtaza, et al., 2017; Mughal
et al., 2018; Mughal et al., 2019).
Results and discussion
Chemistry
2-(4-(Diphenylamino) phenyl)-3-hydroxy-4H-chromen-4-
one (F2)
Given the extensive literature on the Algar–Flynn–Oyamada
(AFO) reaction and the reported synthesis of close analogues
to those we required (Mughal et al., 2018; Nhu et al., 2015),
our initial attempts to prepare 3-O-Benzylated flavonol deriv-
atives (1–10) employed the conventional two-step one-pot
approach: generation of the requisite 20-hydroxychalcone as
key intermediates via the Claisen–Schmidt condensation, and
subsequent oxidative cyclization with basic hydrogen perox-
ide (35%) solution (Scheme 1). In the event, 20-hydroxychal-
cones were obtained through base-catalysed condensation
of 20-hydroxyacetophenone with different aryl aldehydes in
methanol-sodium hydroxide solution. The resultant 20-
hydroxychalcone derivatives were then subjected to conven-
tional AFO conditions (hydrogen peroxide and sodium
hydroxide) in the same solvent to produce the flavonols (F1-
F10) as outlined in Scheme 1. These intermediate compounds
were purified through recrystallization by ethanol and there-
after analysed by FTIR, UV-Vis and NMR spectroscopic techni-
ques only. The desired compounds (1–10) were synthesized
through a single-step reaction of 3-hydroxyflavone (flavonol)
with benzyl chloride in the presence of K2CO3 dissolved in
DMF. All 3-benzyloxyflavone derivatives (1–10) were
obtained in moderate to good yields and purified by recrys-
tallization in EtOH.
Mustard-yellow crystalline solid; Yield: 90%; m.p. 188-190 ꢀC;
Rf (Ethyl acetate: n-hexane 1:3) ¼ 0.7; UV kmax 5 293,
393 nm (CH3OH); FTIR (cmꢂ1): 3221, 1686, 1481, 1410, 1329,
1
1154, 1134, 824; H NMR (300 MHz, CDCl3): d 9.74 (s, 1H, OH),
7.63–7.58 (m, 2H, Ar-H), 7.30–7.24 (m, 6H, Ar-H), 7.12-7.07 (m,
8H, Ar-H), 6.96 (d, J ¼ 9.0 Hz, 2H, Ar-H), 13C NMR (75 MHz,
CDCl3): d 181.5, 153.4, 146.1, 131.4, 129.7, 129.3, 126.3, 126.2,
125.0, 118.3, the other carbons are isochronous; accurate
mass (EI-MS) of [M]þꢃ: Calcd. for C27H19NO3 405.13649;
found 405.13640.
3-(Benzyloxy)-2-phenyl-4H-chromen-4-one (1) (Peres
et al., 2017)
Dark-brown crystalline solid; Yield: 86%; m.p. 116–118 ꢀC; Rf
(Ethyl acetate: n-hexane 1:3) ¼ 0.8; UV kmax ¼ 296 nm
(EtOAc); FTIR (cmꢂ1): 3029, 2895, 1719, 1596, 1282; 1H NMR
(300 MHz, CDCl3): d 7.70–7.65 (m, 4H, Ar-H), 7.42-7.25 (m, 5H,
Ar-H), 7.25–7.15 (m, 5H, Ar-H), 5.05 (s, 2H, CH2-Ph); 13C NMR
(75 MHz, CDCl3): d 175.5, 157.3, 156.0, 142.1, 136.4, 135.0,
132.4, 131.3, 130.4, 129.2, 129.0, 128.7, 128.4, 127.6, 127.3,
127.0, 125.4, 124.7, 123.8, 117.7, 73.5; accurate mass (ESI,
AcCN, þve) of [M þ H]þ: Calcd. for C22H17O3 329.11776;
found 329.11770.
The structures of all newly synthesized compounds were
deduced from various spectroscopic techniques (UV-Vis, FTIR,
NMR spectroscopic techniques). Their molecular masses were
confirmed by Electrospray Ionization (ESI) method. For
instance, in the IR spectrum, there are signs of a successful
benzyl group attachment with chromone part by the dis-
appearance of peak around 3300 cmꢂ1 due to the 3-OH and
the appearance of new peak around 1300 cmꢂ1 due to ben-
zylic moiety. Similarly, the count of a number of protons and
3-(Benzyloxy)-2-(4-diphenylamino) phenyl)-4H-chromen-
4-one (2)
Yellow crystalline solid; Yield: 80%; m.p. 147–150 ꢀC; Rf (Ethyl
acetate: n-hexane) ¼ 0.9; UV kmax ¼ 298 nm (EtOAc); FTIR
(cmꢂ1): 3110, 2850, 1685, 1501, 1402, 1328, 1284;1H NMR
(300 MHz, CDCl3): d 7.67–7.60 (m, 4H, Ar-H), 7.40–7.26 (m, 6H,
Ar-H), 7.22–7.15 (m, 8H, Ar-H), 7.26-7.15 (m, 5H, Ar-H), 5.06 (s,
2H, CH2-Ph); 13C NMR (75 MHz, CDCl3): d 179.7, 155.2, 148.3,
135.7, 133.7, 132.6, 132.1, 131.0, 130.6, 130.2, 129.8, 129.1,
128.7, 128.4, 128.1, 128.0, 126.7, 125.8, 124.7,124.2, 123.7, 123
118.4, 74.2, the other carbons are isochronous; accurate mass
(ESI, AcCN, þve) of [M þ H]þ: Calcd. for C34H26NO3 496.19126;
1
the number of carbon resonances in their H-NMR and 13C-
NMR spectra, respectively, were also in agreement with the
suggested molecular formulas. There is one characteristic
singlet peak because of oxymethylene (-OCH2-) group
1
appearing around d 5.00 ppm in H-NMR spectra of the envi-
1
sioned compounds. Additionally, the H NMR spectra showed
downfield peaks for aromatic protons ranging from 8.23 to
7.15 ppm. Furthermore, their 13C NMR spectra manifested found 496.19116.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Scheme 1. Synthesis of flavonols (F1-F10) and 3-O-benzylflavonol derivatives (1–10).

6
E. U. MUGHAL ET AL.
Scheme 1. Continued.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Scheme 1. Continued.
3084, 2925, 1724, 1527, 1497, 1288; 1H NMR (300 MHz,
CDCl3): d 8.80 (d, J ¼ 3.0 Hz, 1H, Ar-H), 8.48 (m, 1H, Ar-H), 8.39
(t, J ¼ 9.0 Hz, 1H, Ar-H), 8.00 (d, J ¼ 9.0 Hz, 1H, H-5), 7.82-7.76
(m, 4H, Ar-H), 7.30-7.21 (m, 5H, Ar-H), 5.03 (s, 2H, CH2-Ph);
13C NMR (75 MHz, CDCl3): d 175.5, 158.4, 155.1, 146.2, 134.7,
132.7, 131.4, 130.5, 130.0, 129.6, 128.8, 128.5, 128.3, 127.8,
127.1, 126.0 (CH, 125.5, 125.0, 124.0, 122.8, 118.1, 74.2; accur-
ate mass (ESI, AcCN, þve) of [M þ H]þ: Calcd. for C22H16NO5
374.10284; found 374.10275.
3-(Benzyloxy)-2-(4-methylphenyl)-4H-chromen-4-one (3)
Off-white solid; Yield: 75%; m.p. 110–112 ꢀC; Rf (Ethyl acetate:
n-hexane 1:3) ¼ 0.85; UV kmax ¼ 316 nm (EtOAc); FTIR (cmꢂ1):
1
3031, 2995, 2887, 1718, 1597, 1293; H NMR (300 MHz, CDCl3):
d 8.23 (dd, J ¼ 3.0, 9.0 Hz, 1H, Ar-H), 7.91–7.86 (m, 2H, Ar-H),
7.64–7.57 (m, 1H, Ar-H), 7.46–7.43 (m, 1H, Ar-H), 7.36–7.27 (m,
3H, Ar-H), 7.23-7.15 (m, 5H, Ar-H), 5.05 (s, 2H, CH2-Ph), 2.36 (s,
3H, Me); 13C NMR (75 MHz, CDCl3): d 175.0, 156.5, 155.3, 141.0,
139.7, 136.7, 133.4, 129.0, 128.93, 128.90, 128.8, 128.3, 128.2,
128.1, 125.7, 124.4, 124.2, 118.0, 74.2, 24.3,the other carbons
are isochronous; accurate mass (ESI, AcCN, þve) of [M þ H]þ:
Calcd. for C23H19O3 343.13342; found 343.13328.
3-(Benzyloxy)-2-(4-nitrophenyl)-4H-chromen-4-one (7)
Brown solid; Yield 58%; m.p. 104 ꢀC; Rf (Ethyl acetate: n-hex-
ane 1:3) ¼ 0.65; UV kmax ¼ 274 nm (EtOAc); FTIR (cmꢂ1):
3124, 2800, 1722, 1567, 1522, 1280; 1H NMR (300 MHz,
CDCl3): d 8.38 (d, J ¼ 9.0 Hz, 2H, Ar-H), 8.29 (J ¼ 9.0 Hz, 2H, Ar-
H), 8.12–8.01 (m, 2H, Ar-H), 7.90–7.80 (m, 5H, Ar-H), 5.06 (s,
2H, CH2-Ph); 13C NMR (75 MHz, CDCl3): d 175.4, 159.8,
156.7,148.8, 137.7, 135.1, 130.2, 129.8, 129.6, 129.0, 128.8,
128.7, 128.5, 128.1, 127.9, 127.2, 125.8, 125.5, 124.7,124.0,
118.1, 74.2; accurate mass (ESI, AcCN, þve) of [M þ H]þ:
Calcd. for C22H16NO5 374.10284; found 374.10271.
3-(Benzyloxy)-2-(thiophen-1-yl)-4H-chromen-4-one (4)
(Kamboj et al., 2013)
Orange solid; Yield: 80%; m.p. 154–156 ꢀC; Rf (Ethyl acetate: n-
hexane 1:3) ¼ 0.7; UV kmax¼ 340 nm (EtOAc); FTIR (cmꢂ1): 3029,
2960, 1964, 1720, 1598, 1288, 1024; 1H NMR (300 MHz, CDCl3): d
8.20–7.98 (m, 3H, Ar-H), 7.87–7.66 (m, 2H, Ar-H), 7.47–7.30 (m,
2H, Ar-H), 7.24–7.15 (m, 5H, Ar-H), 5.03 (s, 2H, CH2-Ph); 13C NMR
(75 MHz, CDCl3): d 175.2, 155.1, 144.5, 135.7, 131.7, 130.5, 129.3,
129.0, 128.9, 128.7, 128.4, 128.1, 127.9, 127.4, 127.0, 125.8,
125.3, 123.5, 118.6, 74.18; accurate mass (ESI, AcCN, þve) of
[M þ H]þ: Calcd. for C20H17O3S 337.08984; found 337.08971.
3-(Benzyloxy)-2-(4-methoxyphenyl)-4H-chromen-4-one
(8) (Kavitha, 2012; Rao & Kumar, 2014)
Orange solid; Yield: 71%; m.p.135–137 ꢀC; Rf (Ethyl acetate: n-
hexane 1:3) ¼ 0.7; UV kmax ¼ 280 nm (EtOAc); FTIR (cmꢂ1):
3028, 2880, 1720, 1501, 1423, 1200; 1H NMR (300 MHz,
CDCl3): d 8.02 (d, J ¼ 9.0 Hz, 2H, Ar-H), 7.98 (d, J ¼ 9.0 Hz, 2H,
Ar-H), 7.80 (m, 1H, Ar-H), 7.67–7.47 (m, 3H, Ar-H), 5.04 (s, 2H,
CH2-Ph);13C NMR (75 MHz, CDCl3): d 175.2, 160.3,159.7,155.2,
135.4, 134.3, 130.4, 129.5, 129.1, 128.8, 128.5, 128.2, 128.0,
127.5,127.3, 127.0, 126.0 125.6,124.8, 123.1,118.1, 74.3, 55.6;
accurate mass (ESI, AcCN, þve) of [M þ H]þ: Calcd. for
C23H19O4 359.12833; found 359.12820.
3-(Benzyloxy)-2-(4-chlorophenyl) -4H-chromen-4-one (5)
Off-white solid; Yield: 82%; m.p. 143–145 ꢀC; Rf (Ethyl acetate:
n-hexane 1:3) ¼ 0.81; UV kmax ¼ 350 nm (EtOAc); FTIR
(cmꢂ1): 3025, 2920, 1720, 1571, 1289, 752;1H NMR (300 MHz,
CDCl3): d 8.24 (d, J ¼ 3.0, 9.0 Hz, 1H, Ar-H), 7.88 (m, 2H, Ar-H),
7.65-7.59 (m, 1H, Ar-H), 7.46-7.43 (m, 1H, Ar-H), 7.38–7.33 (m,
3H, Ar-H), 7.25-7.15 (m, 5H, Ar-H), 5.08 (s, 2H, CH2-Ph); 13C
NMR (75 MHz, CDCl3): d 175.1, 155.2, 138.1, 136.6, 135.6,
133.4, 129.7, 127.3, 125.2, 124.5, 122.0, 118.2,72.1, other car-
bons are isochronous; accurate mass (ESI, AcCN, þve) of
[M þ H]þ: Calcd. for C22H16ClO3 363.07879; found 363.07865.
3-(Benzyloxy)-2-(furan-2-yl)-4H-chromen-4-one (9)
Dark-brown solid; Yield: 66%; m.p. 95–97 ꢀC; Rf (Ethyl acetate:
n-hexane 1:3) ¼ 0.6; UV kmax ¼ 329 nm (EtOAc); FTIR (cmꢂ1):
3021, 2789, 1709, 1592, 1235, 1170; 1H NMR (300 MHz,
CDCl3): d 8.35–8.25 (m, 1H, Ar-H), 8.05–7.98 (m, 2H, Ar-H),
3-(Benzyloxy)-2-(3-nitrophenyl)-4H-chromen-4-one (6)
Light-brown solid; Yield: 74%; m.p. 123–125 ꢀC; Rf (Ethyl acet-
ate: n-hexane) ¼ 0.6; UV kmax ¼ 298 nm (EtOAc); FTIR (cmꢂ1): 7.77–7.50 (m, 4H, Ar-H), 7.27–7.18 (m, 5H, Ar-H), 5.05 (s, 2H,

8
E. U. MUGHAL ET AL.
Table 1. AChE and BchE the enzyme inhibition efficiency of the target com-
pounds (1–10).
Noteworthy, the compound 2 was recognised as the most
active inhibitor of both AChE (IC50 ¼ 0.05 0.01 lM) and
BChE (IC50 ¼ 0.09 0.02 lM), and thus exhibited highest
inhibition against both enzymes among the series, even
more than the standard Donepezil (IC50¼ 0.09 0.01 AChE
and 0.13 0.04 lM BChE). This lead compound of the series
was roughly two times more active than standard regarding
in vitro AChE-inhibitory activity and one and half times more
potent against in vitro BChE-inhibitory activity. The presence
of diphenylamino group at 40 position of ring B enables the
compound 2 to show the highest inhibition activity. Owing
to the bulky size and hydrophobic nature of the diphenyla-
mino substituent, this derivative interacts strongly with active
pockets of both enzymes.
Furthermore, the compound 5 (IC50 ¼ 0.07 0.02 for
AChE and IC50 ¼ 2.13 0.08 lM for BChE) was the second
potent selective inhibitor of AChE. The presence of only one
chloro group at para-position (Ring B) in that derivative
markedly enhance the inhibitory activity against AChE than
BChE, because this nicely fits in the active pocket of former
enzyme than latter.
In addition, the next most potent selective inhibitor of
AChE (IC50 ¼ 0.08 0.02 lM) was found 3-benzyloxyflavone
10, analogous to the derivative 2, relative to the standard
Donepezil (IC50 ¼ 0.09 0.01 lM). The presence of a dimethy-
lamino substitution at 40 position of the phenyl ring (ring B)
and non-polar and electron-rich structural core of this com-
pound are accountable to develop strong intermolecular
interactions with the electrophilic active site of the
AChE enzyme.
Compound No.
AChE IC50 SEMa (mM)
BChE IC50 SEMa (mM)
1
2
3
4
5
6
7
8
0.12 0.02
0.05 0.01
1.95 0.15
10.02 0.78
0.07 0.02
2.85 0.35
1.52 0.15
1.01 0.01
11.60 0.03
0.08 0.02
0.09 0.01
3.10 0.30
0.09 0.02
3.25 0.21
27.11 0.35
2.13 0.08
9.25 0.90
14.37 0.65
3.14 0.70
12.23 0.74
1.50 0.10
0.13 0.04
9
10
DonepezilSt
aIC50 values (mean standard error of mean); StStandard inhibitor for cholin-
esterase enzymes.
CH2-Ph); 13C NMR (75 MHz, CDCl3): d 175.3, 155.2, 145.0,
136.1, 131.8, 131.7, 130.3, 129.1, 129.0, 128.7, 128.5, 128.2,
128.0, 127.5, 127.1, 125.7, 125.5, 123.6, 118.6, 74.2; accurate
mass (ESI, AcCN, þve) of [M þ H]þ: Calcd. for C20H15O4
319.09703; found 319.09698.
3-(Benzyloxy)-2-(4-(dimethylamino)phenyl)-4H-chromen-
4-one (10)
Dark-red solid; Yield: 85%; m.p. 75–78 ꢀC; Rf (Ethyl acetate: n-
hexane) ¼ 0.8; UV kmax ¼ 318 nm (EtOAc); FTIR (cmꢂ1): 3021,
2750, 1710, 1650, 1603, 1203, 1047; 1H NMR (300 MHz,
CDCl3): d 8.21 (d, J ¼ 3.0, 9.0 Hz, 1H, Ar-H), 7.87 (m, 2H, Ar-H),
7.67–7.57 (m, 1H, Ar-H), 7.47–7.42 (m, 1H, Ar-H), 7.40–7.30
(m, 3H, Ar-H), 7.24–7.14 (m, 5H, Ar-H), 5.07 (s, 2H, CH2-Ph),
2.85 (N(CH3)2; 13C NMR (75 MHz, CDCl3): d 174.8, 156.2, 139.0,
137.2, 136.8, 133.4, 129.6, 128.7, 128.5, 128.6, 128.4, 128.0,
127.7, 125.5, 124.6, 123.6, 122.8, 118.3, 74.0, 40.3, the other
carbons are isochronous; accurate mass (ESI, AcCN, þve) of
[M þ H]þ: Calcd. for C24H22NO3 372.15996; found 372.15985.
The compounds bearing electron-donating groups (-OCH3,
-CH3 etc.) such as 3 (IC50 ¼ 1.95 0.15 for AChE and IC50
¼
3.25 0.21 lM for BChE) and 8 also displayed better inhibi-
tory activity against both enzymes. This might be attributed
to positions of those groups and effective interactions with
the pockets of enzymes. However, introducing strongly elec-
tron-withdrawing groups (-NO2) at the same position of ring
In vitro cholinesterase inhibition activity
Continuing our efforts to study the enzyme inhibition B distinctly resulted in decreased inhibitory activity, for
(Mughal, Sadiq, Khan, et al.2017; Mughal, Sadiq, Murtaza,
et al., 2017; Mughal et al., 2018; Mughal et al., 2019), all the
3-benzyloxyflavone derivatives (1–10) have been examined,
in vitro, for their inhibitory activity against commercially
accessible electric eel AChE and horse serum BChE enzymes.
Donepezil was used as a standard for comparing results,
and estimates of the half-maximum inhibitory concentration
(IC50) were calculated and are given in Table 1. From the
data in Table 1, several conclusions can be drawn concerning
the structure-activity relationship and the impact of the sub-
stituent in the phenyl ring (Ring B) of flavone moiety.
example, in case of the flavones 6 and 7. Interestingly, the
meta nitro substituted compound 6 (IC50 ¼ 2.85 0.35 for
AChE and IC50
¼
9.25 0.90 lM for BChE) disclosed
decreased activity relative to its para analogue 7 (IC50
¼
1.52 0.15 for AChE and IC50 ¼ 14.37 0.65 lM for BChE).
Perhaps, the former compound offers steric hindrance to fit
in the active site of cholinesterase enzymes. Moreover,
replacing aryl ring B with other heterocyclic rings such as thi-
ophene and furan in compound 4 (IC50 ¼ 10.02 0.78 for
AChE and IC50 ¼ 27.11 0.35 lM for BuChE) and 9 (IC50
¼
11.60 0.03 for AChE and IC50 ¼ 12.23 0.74 lM for BChE)
respectively resulted in significantly decreased inhibitory
activity as compared to the standard.
Structure-activity relationship
Overall, it was noted that all the compounds (1–10) are
potent inhibitors against AChE in comparison to BChE. These
findings showed that the nature and substitution pattern at
rings B & C improves the inhibitory activity of these com-
pounds against both suggested enzymes relative to unsubsti-
The synthesized compounds (1–10) were screened to con-
firm their importance as potent inhibitors of the cholinester-
ase enzymes. It is interesting to note that all synthesized
compounds (1–10) showed inhibition against both enzymes
(AChE and BChE). However, they are relatively more active tuted 3-benzyloxyflavone 1 (IC50 ¼ 0.12 0.02 for AChE and
against acetylcholinesterase than butyrylcholinesterase. IC50 ¼ 3.10 0.30 lM for BChE). Although all the structural

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 2. Structure-activityrelationship for cholinesterase enzymes activity of 3-O-benzylflavonol derivatives. Downward red arrows indicate a decrease in activity;
upward green arrows indicate an increase in activity.
Table 2. Lowest binding energies of the compounds (1–10) against various
selective modes.
Based on above-mentioned findings, these multi-func-
tional 3-benzyloxyflavone derivatives (1–10) could use as
lead compounds for the designing and development of new
inhibitors against cholinesterase enzymes. The conclusions of
the SAR studies are summarized in Figure 2.
h AChE Lowest
binding energy
DG (kcal/mol)
h BChE Lowest
binding energy
DG (kcal/mol)
Compound
No.
1
2
3
4
5
6
7
8
ꢂ10.76
ꢂ12.80
ꢂ9.32
ꢂ12.37
ꢂ9.60
ꢂ11.01
ꢂ9.86
ꢂ9.42
Molecular docking simulations
ꢂ11.02
ꢂ9.85
ꢂ10.23
ꢂ9.65
In an attempt to understand the enzyme inhibition activity
of the synthesized derivatives, molecular docking study was
carried out to determine the binding modes of synthesized
ligands with cholinesterase enzymes. The human X-ray struc-
tures of AChE (PDB ID: 4BDT) and BChE (PDB ID: 4BDS) were
chosen as models for this analysis. In due course, for each
conformer with best (minimum) docking score was saved in
preferences. The binding energies are listed in Table 2. The
ligand developing most stable drug-receptor complex is the
ꢂ10.42
ꢂ9.41
ꢂ10.75
ꢂ9.35
9
10
Standard
ꢂ9.85
ꢂ9.01
ꢂ10.65
ꢂ9.45
ꢂ10.00 (HUW)
ꢂ6.83 (THA)
features are actively involved in inhibitory behaviour.
However, the differentiation of various groups on the key
structural motif was in fact responsible for altering the inhibi- one which is having minimum docking score. The drug-
tory activity. Since, all synthesized compounds have a com- receptor complexes of some potent ligands were analysed
mon skeleton of 3-O-benzylflavonol in their structures, the for various types of interactions such as hydrogen
activity was mainly due to different functional groups bonding, hydrophobic type interactions and van der Waals
attached to the main framework of 3-benzyloxyflavone.
interactions etc.

10
E. U. MUGHAL ET AL.
Figure 3. Putative binding interactions between ligand 2 and AChE.
For example, one of the under-study the most potent com- synthesized using precedent methodologies starting from 20-
pound 2 with binding energy (-12.80 kcal molꢂ1) expresses the hydroxyacetophenone and different substituted aromatic
ability to block the active sites of AChE. It develops the hydro- aldehydes over three steps. All the synthesized 3-benzyloxy-
phobic p ꢂ p stacked type associations with Trp86 and Tyr337 flavones (1–10) were evaluated against cholinesterase inhibi-
of catalytic triad amino acid residues. Trp439, Met443, Tyr341, tory potential. All analogues were found the active dual
Ser125, Pro88 and His447 amino acid residues of active pock- inhibitors of acetylcholinesterase and butyrylcholinesterase.
ets of acetylcholinesterase develop hydrophobic p ꢂ p stacked, However, the enzyme inhibition study disclosed that most of
p-sulphur, hydrogen bond and hydrophobic p-alkyl type inter- the compounds are comparatively more active against AChE
actions, as shown in Figures 3 and 4.
relative to BChE. Noteworthy, among the series, the com-
pound 2 displayed potent dual inhibitory activity (IC50
0.05 0.01 for AChE and IC50 ¼ 0.09 0.02 lM for BChE),
demonstrating that 2 was about threefold more effective
AChE inhibitor and fourfold more effective BChE inhibitor as
Additionally, the derivative 2 reveals its inhibitory poten-
tial against BChE by forming fruitful types of electrostatic
interactions. This compound builds up hydrophobic p-alkyl
type interaction Leu286 of acyl binding pocket inside the
BChE. Trp82 of peripheral anionic site (PAS) develops the
hydrophobic p ꢂ p stacked type associations inside the active
pocket of butyrylcholinesterase. This ligand also exhibits
p-lone pair, hydrophobic p ꢂ p T-Shaped and hydrophobic
p-alkyl type associations with Pro285, Phe329 and Ala328
amino acid residues inside the pockets butyrylcholinesterase
as shown in Figures 5 and 6.
¼
compared to the reference compound, Donepezil (IC50
¼
0.09 0.01 for AChE and IC50 ¼0.13 0.04 lM for BChE). In sil-
ico computational studies of all compounds also augmented
the in vitro analysis, whereby these molecules exhibited
strong interactions with the target protein and formed stable
complexes with AChE and BChE. The structure-activity rela-
tionship analysis discovered that cholinesterase inhibition of
these derivatives is strongly depending on the nature and
positions of the functional group on the aromatic ring B. In
this way, a number of exciting lead compounds have been
Conclusions
In conclusion, we have demonstrated the synthesis, charac- explored as cholinesterase inhibitors and thus enabling fur-
terization and biological assessment of 3-benzyloxyflavone ther development of novel drugs for the treatment of
derivatives. The desired 3-benzyloxyflavones (1–10) were Alzheimer’s disease.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 4. Compound 2 interactions with acetylcholinesterase in 3 D orientation. Interactions with different amino acid residues are depicted in the box. The 3 D rib-
bon along with the interaction of AChE amino acids represents an enzyme-stick configuration with the lowest energy content of the inhibitor 2.
Figure 5. Putative binding interactions between ligand 2 and BchE.

12
E. U. MUGHAL ET AL.
Figure 6. Compound 2 interactions with butyrylcholinesterase in 3 D orientation. Interactions with different amino acid residues are depicted in the box. The 3 D
ribbon along with the interaction of BChE amino acids represents an enzyme-stick configuration with the lowest energy content of the inhibitor 2.
Bajda, M., Guzior, N., Ignasik, M., & Malawska, B. (2011). Multi-target-
Acknowledgements
directed ligands in Alzheimer’s disease treatment. Current Medicinal
Chemistry, 18(32), 4949–4975. https://doi.org/10.2174/0929867117975
35245
We are highly thankful to Dr. Renhao Dong, Department of Chemistry,
Dresden University, Germany for his kind help in spectroscopic
measurements.
Barai, P., Raval, N., Acharya, S., Borisa, A., Bhatt, H., & Acharya, N. (2019).
Neuroprotective effects of bergenin in Alzheimer’s disease:
Investigation through molecular docking, in vitro and in vivo studies.
Behavioural Brain Research, 356, 18–40. https://doi.org/10.1016/j.bbr.
Disclosure statement
2018.08.010
No potential conflict of interest was reported by the author(s).
Bhatt, D., Soni, R., Sharma, G. K., & Dashora, A. (2016). Synthesis and
pharmacological activities of flavones: A review. American Journal of
Pharmaceutical Research, 6(02), 4345.
Bozdag-Dundar, O., Ceylan-Unlusoy, M., Altanlar, N., & Ertan, R. (2005).
Funding
This research work was financially supported by Higher Education
Commission of Pakistan under the NRPU Project No. 6484.
Synthesis and biological activity of some new flavone derivatives.
Arzneimittel-Forschung, 55(2), 102–106. https://doi.org/10.1055/s-0031-
1296830
Cavdar, H., Senturk, M., Guney, M., Durdagi, S., Kayik, G., Supuran, C. T., &
Ekinci, D. (2019). Inhibition of acetylcholinesterase and butyrylcholi-
nesterase with uracil derivatives: Kinetic and computational studies.
Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 429–437.
https://doi.org/10.1080/14756366.2018.1543288
Chen, Y., Lin, H., Yang, H., Tan, R., Bian, Y., Fu, T., Li, W., Wu, L., Pei, Y., &
Sun, H. (2017). Discovery of new acetylcholinesterase and butyrylcho-
linesterase inhibitors through structure-based virtual screening. RSC
Advances, 7(6), 3429–3438. https://doi.org/10.1039/C6RA25887E
Daglia, M. (2012). Polyphenols as antimicrobial agents. Current Opinion in
Biotechnology, 23(2), 174–181. https://doi.org/10.1016/j.copbio.2011.
08.007
References
Agbo, E. N., Gildenhuys, S., & Mphahlele, M. J. (2019). Inhibitory effects
of novel 7-substituted 6-iodo-3-o-flavonol glycosides against cholines-
terases and b-secretase activities, and evaluation for potential antioxi-
dant properties. Molecules, 24(19), 3500–3553. https://doi.org/10.3390/
molecules24193500
Anand, P., & Singh, B. (2013). Flavonoids as lead compounds modulating
the enzyme targets in Alzheimer’s disease. Medicinal Chemistry
Research, 22(7), 3061–3075. https://doi.org/10.1007/s00044-012-0353-y
Asghar, A., Yousuf, M., Fareed, G., Nazir, R., Hassan, A., Maalik, A., Noor,
T., Iqbal, N., & Rasheed, L. (2020). Synthesis, acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE) activities, and molecular
docking studies of a novel compound based on combination of flur-
biprofen and isoniazide. RSC Advances, 10(33), 19346–19352. https://
doi.org/10.1039/D0RA02339F
ꢀ
ꢀ
Dıaz, J. E., Martinez, D. C., Lopez, L. V., Mendez, G. M., Vera, R., & Loaiza,
A. E. (2017). Synthesis and in vitro antiproliferative activity of flavone
and 6-hydroxyflavone oxime ethers derivatives. Journal of the
Brazilian Chemical Society, 29(1), 177–184. https://doi.org/10.21577/
0103-5053.20170128

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Dymarska, M., Janeczko, T., & Kostrzewa-Susłow, E. (2018). Glycosylation
of 3-hydroxyflavone, 3-methoxyflavone, quercetin and baicalein in
fungal cultures of the genus Isaria. Molecules, 23(10), 2477. https://doi.
org/10.3390/molecules23102477
dithiolane, thiophene, coumarin, and 2-pyridone derivatives. Synthetic
Communications, 47(17), 1591–1600. https://doi.org/10.1080/00397911.
2017.1336776
Mathew, B., Parambi, D. G., Mathew, G. E., Uddin, M. S., Inasu, S. T., Kim,
Faraji, L., Nadri, H., Moradi, A., Bukhari, S. N. A., Pakseresht, B.,
H., Marathakam, A., Unnikrishnan, M. K., & Carradori, S. (2019).
Moghadam, F. H., Moghimi, S., Abdollahi, M., Khoobi, M.,
Foroumadi, A. (2019). Aminoalkyl-substituted flavonoids: Synthesis,
cholinesterase inhibition, b-amyloid aggregation, and neuroprotective
&
Emerging therapeutic potentials of dual-acting MAO and AChE inhibi-
tors in Alzheimer’s and Parkinson’s diseases. Archiv Der Pharmazie,
352(11), 1900177. https://doi.org/10.1002/ardp.201900177
study. Medicinal Chemistry Research, 28(7), 974–983. https://doi.org/10. Mishra, C. B., Kumari, S., Manral, A., Prakash, A., Saini, V., Lynn, A. M., &
1007/s00044-019-02350-4
Tiwari, M. (2017). Design, synthesis, in-silico and biological evaluation
of novel donepezil derivatives as multi-target-directed ligands for the
treatment of Alzheimer’s disease. European Journal of Medicinal
Chemistry, 125, 736–750. https://doi.org/10.1016/j.ejmech.2016.09.057
Mishra, B. B., & Tiwari, V. K. (2011). Natural products: An evolving role in
future drug discovery. European Journal of Medicinal Chemistry, 46(10),
4769–4807. https://doi.org/10.1016/j.ejmech.2011.07.057
Genoux, E., Nicolle, E., & Boumendjel, A. (2011). Flavonoids as anticancer
agents: Recent progress and state of the art?. Current Organic
Chemistry, 15(15), 2608–2615. https://doi.org/10.2174/13852721179
6367363
Graf, B. A., Milbury, P. E., & Blumberg, J. B. (2005). Flavonols, flavones, fla-
vanones, and human health: Epidemiological evidence. Journal of
Medicinal Food, 8(3), 281–290. https://doi.org/10.1089/jmf.2005.8.281
Gunduz, S., Goren, A. C., & Ozturk, T. (2012). Facile syntheses of 3-
hydroxyflavones. Organic Letters, 14(6), 1576–1579. https://doi.org/10.
1021/ol300310e
Mphahlele, M. J., Agbo, E. N., & Gildenhuys, S. (2018). Synthesis and
evaluation of the 4-substituted 2-hydroxy-5-iodochalcones and their
7-substituted 6-iodoflavonol derivatives for inhibitory effect on choli-
nesterases and b-secretase. International Journal of Molecular Sciences,
19(12), 4112. https://doi.org/10.3390/ijms19124112
Gupta, V. K., Mergu, N., & Singh, A. K. (2014). Fluorescent chemosensors
for Zn2þ ions based on flavonol derivatives. Sensors and Actuators B: Mughal, E. U., Ayaz, M., Hussain, Z., Hasan, A., Sadiq, A., Riaz, M., Malik,
Chemical, 202, 674–682. https://doi.org/10.1016/j.snb.2014.05.133
Harborne, J. B., & Williams, C. A. (2000). Advances in flavonoid research
since 1992. Phytochemistry, 55(6), 481–504. https://doi.org/10.1016/
S0031-9422(00)00235-1
A., Hussain, S., & Choudhary, M. I. (2006). Synthesis and antibacterial
activity of substituted flavones, 4-thioflavones and 4-iminoflavones.
Bioorganic & Medicinal Chemistry, 14(14), 4704–4711. https://doi.org/
10.1016/j.bmc.2006.03.031
Hostetler, G. L., Ralston, R. A., & Schwartz, S. J. (2017). Flavones: Food Mughal, E. U., Javid, A., Sadiq, A., Murtaza, S., Zafar, M. N., Khan, B. A.,
sources, bioavailability, metabolism, and bioactivity. Advances in
Nutrition (Bethesda, MD), 8(3), 423–435. https://doi.org/10.3945/an.116.
012948
Sumrra, S. H., Tahir, M. N., & Khan, K. M. (2018). Synthesis, structure-
activity relationship and molecular docking studies of 3-O-flavonol
glycosides as cholinesterase inhibitors. Bioorganic
Chemistry, 26(12), 3696–3706.
& Medicinal
Ibrahim, N. S. (2014). Antimicrobial activities of some synthetic flavo-
noids. IOSR Journal of Applied Chemistry, 7(5), 01–06., & Ahmed, F. Mughal, E. U., Sadiq, A., Ashraf, J., Zafar, M. N., Sumrra, S. H., Tariq, R.,
https://doi.org/10.9790/5736-07520106
Mumtaz, A., Javid, A., Khan, B. A., Ali, A., & Javed, C. O. (2019).
Flavonols and 4-thioflavonols as potential acetylcholinesterase and
butyrylcholinesterase inhibitors: Synthesis, structure-activity relation-
ship and molecular docking studies. Bioorganic Chemistry, 91,
103124https://doi.org/10.1016/j.bioorg.2019.103124
Imran, S., Taha, M., Ismail, N. H., Kashif, S. M., Rahim, F., Jamil, W., Wahab,
H., & Khan, K. M. (2016). Synthesis, in vitro and docking studies of
new flavone ethers as a-glucosidase inhibitors. Chemical Biology &
Drug Design, 87(3), 361–373. https://doi.org/10.1111/cbdd.12666
Jin, L., Wang, M. L., Lv, Y., Zeng, X.-Y., Chen, C., Ren, H., Luo, H., & Pan, Mughal, E. U., Sadiq, A., Khan, B. A., Zafar, M. N., Ahmed, I., & Zubair, M.
W. D. (2019). Design and synthesis of flavonoidal ethers and their
anti-cancer activity in vitro. Molecules, 24(9), 1749. https://doi.org/10.
3390/molecules24091749
(2017). Synthesis, molecular docking studies and biological evaluation
of 3-iminoaurones as acetylcholinesterase and butyrylcholinesterase
inhibitors. Letters in Drug Design
& Discovery, 14(9), 1035–1041.
Kamboj, R. C., Pratibha, P., Kumar, D., Sharma, G., Arora, R., Kumar, P.,
https://doi.org/10.2174/1570180814666170106123959
Kumar, S., Kumar, R., & Kamal, R. (2013). Intramolecular cyclization of Mughal, E. U., Sadiq, A., Murtaza, S., Rafique, H., Zafar, M. N., Riaz, T.,
photolabile thienylchromenones (I): Synthesis of angular tetracyclic
compounds (II). ChemInform, 44(46), no–249. https://doi.org/10.1002/
chin.201346136
Kamlesh, K., Sivakumar, T., & Afroze, A. (2017). Antimicrobial activity of
flavone analogues. Journal of Applied Pharmacy, 09 (01), 2. https://doi.
org/10.21065/1920-4159.1000232
Kavitha, P. (2012). Ultrasonic studies on molecular interactions in binary
mixtures of sesame oil with organic solvents. Indian Journal of
Advances in Chemical Science, 5(12), 148–154.
Khanna, R., Kumar, R., Dalal, A., & Kamboj, R. C. (2015). Absorption and
fluorescent studies of 3-hydroxychromones. Journal of Fluorescence,
25(5), 1159–1163. https://doi.org/10.1007/s10895-015-1623-0
Li, X., Lee, M., Chen, G., Zhang, Q., Zheng, S., Wang, G., & Chen, Q.-H.
(2017). 3-O-Substituted-30, 40, 50-trimethoxyflavonols: Synthesis and
Khan, B. A., Hameed, A., & Khan, K. M. (2017). Synthesis, structure-
activity relationship and molecular docking of 3-oxoaurones and 3-
thioaurones as acetylcholinesterase and butyrylcholinesterase inhibi-
tors. Bioorganic & Medicinal Chemistry, 25(1), 100–106. https://doi.org/
10.1016/j.bmc.2016.10.016
Nhu, D., Hawkins, B. C., & Burns, C. J. (2015). Phase transfer catalysis
extends the scope of the Algar–Flynn–Oyamada synthesis of 3-
hydroxyflavones. Australian Journal of Chemistry, 68(7), 1102–1107.
https://doi.org/10.1071/CH14620
€
€
Ozil, M., Balaydın, H. T., & S¸enturk, M. (2019). Synthesis of 5-methyl-2, 4-
dihydro-3H-1, 2, 4-triazole-3-one’s aryl Schiff base derivatives and
investigation of carbonic anhydrase and cholinesterase (AChE, BuChE)
inhibitory properties. Bioorganic Chemistry, 86, 705–713. https://doi.
org/10.1016/j.bioorg.2019.02.045
cell-based evaluation as anti-prostate cancer agents. Bioorganic & Patel, S., & Shah, U. (2017). Synthesis of flavones from 2-hydroxy aceto-
Medicinal Chemistry, 25(17), 4768–4777. https://doi.org/10.1016/j.bmc.
2017.07.022
phenone and aromatic aldehyde derivatives by conventional methods
and green chemistry approach. Asian Journal of Pharmaceutical and
Clinical Research, 10(2), 403–576. https://doi.org/10.22159/ajpcr.2017.
v10i2.15928
Liu, H. L., Jiang, W. B., & Xie, M. X. (2010). Flavonoids: Recent advances
as anticancer drugs. Recent Patents on anti-Cancer Drug Discovery,
5(2), 152–164. https://doi.org/10.2174/157489210790936261
Loizzo, M. R., Tundis, R., Menichini, F., & Menichini, F. (2008). Natural
products and their derivatives as cholinesterase inhibitors in the treat-
ment of neurodegenerative disorders: An update. Current Medicinal
Chemistry, 15(12), 1209–1228. https://doi.org/10.2174/09298670878
4310422
Peres, B., Nasr, R., Zarioh, M., Lecerf, F., Di Pietro, A., Baubichon, H., &
Boumendjel, A. (2017). Ferrocene-embedded flavonoids targeting the
Achilles heel of multidrug-resistant cancer cells through collateral sen-
sitivity. European Journal of Medicinal Chemistry, 130, 346–353. https://
doi.org/10.1016/j.ejmech.2017.02.064
Rampa, A., Bisi, A., Valenti, P., Recanatini, M., Cavalli, A., Andrisano, V.,
Cavrini, V., Fin, L., Buriani, A., & Giusti, P. (1998). Acetylcholinesterase
inhibitors: Synthesis and structure-activity relationships of x-[N-
Mahmoud, M. R., Abu El-Azm, F. S., Ali, A. T.,
& Ali, Y. M.
(2017). Synthesis and antimicrobial evaluation of some novel

14
E. U. MUGHAL ET AL.
Methyl-N-(3-alkylcarbamoyloxyphenyl)-methyl] aminoalkoxyheteroaryl Singh, A. K., Saxena, G., & Arshad, M. (2017). Synthesis, characterization
derivatives. Journal of Medicinal Chemistry, 41(21), 3976–3986. https://
doi.org/10.1021/jm9810046
and biological evaluation of ruthenium flavanol complexes against
breast cancer. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, 180, 97–104. https://doi.org/10.1016/j.saa.2017.02.056
Sun, K., Bai, Y., Zhao, R., Guo, Z., Su, X., Li, P., & Yang, P. (2019).
Neuroprotective effects of matrine on scopolamine-induced amnesia
via inhibition of AChE/BuChE and oxidative stress. Metabolic Brain
Disease, 34(1), 173–181. https://doi.org/10.1007/s11011-018-0335-y
Tapas, A. R., Sakarkar, D., & Kakde, R. (2008). Flavonoids as nutraceuticals:
A review. Tropical Journal of Pharmaceutical Research, 7(3), 1089–1099.
https://doi.org/10.4314/tjpr.v7i3.14693
Uriarte-Pueyo, I., & I Calvo, M. (2011). Flavonoids as acetylcholinesterase
inhibitors. Current Medicinal Chemistry, 18(34), 5289–5302. https://doi.
org/10.2174/092986711798184325
Wang, T. Y., Li, Q., & Bi, K. S. (2018). Bioactive flavonoids in medicinal
plants: Structure, activity and biological fate. Asian Journal of
Pharmaceutical Sciences, 13(1), 12–23. https://doi.org/10.1016/j.ajps.
2017.08.004
Rao, M. L., & Kumar, A. (2014). Pd-catalyzed atom-economic couplings of
triarylbismuth reagents with 2-bromo-and 2, 6-dibromochromones
and synthesis of medicinally important fisetin. Tetrahedron Letters,
55(42), 5764–5770. https://doi.org/10.1016/j.tetlet.2014.08.081
Rice-Evans, C. (2001). Flavonoid antioxidants. Curr. Med. Chem, 8(7),
797–807. https://doi.org/10.2174/0929867013373011
Sarbu, L., Bahrin, L., Babii, C., Stefan, M., & Birsa, M. (2019). Synthetic fla-
vonoids with antimicrobial activity:
A review. Journal of Applied
Microbiology, 127(5), 1282–1290. https://doi.org/10.1111/jam.14271
Sashidhara, K. V., Kumar, M., & Kumar, A. (2012). A novel route to synthe-
sis of flavones from salicylaldehyde and acetophenone derivatives.
Tetrahedron Letters, 53(18), 2355–2359. https://doi.org/10.1016/j.tetlet.
2012.02.108
Saxena, M., & Dubey, R. (2019). Target enzyme in Alzheimer’s disease:
Acetylcholinesterase inhibitors. Current Topics in Medicinal Chemistry,
19(4), 264–275. https://doi.org/10.2174/1568026619666190128125912
Shaik, J. B., Palaka, B. K., Penumala, M., Kotapati, K. V., Devineni, S. R.,
Eadlapalli, S., Darla, M. M., Ampasala, D. R., Vadde, R., & Amooru, G. D.
(2016). Synthesis, pharmacological assessment, molecular modeling
and in silico studies of fused tricyclic coumarin derivatives as a new
family of multifunctional anti-Alzheimer agents. European Journal of
Medicinal Chemistry, 107, 219–232. https://doi.org/10.1016/j.ejmech.
2015.10.046
Shaikh, S., Dhavan, P., Ramana, M., & Jadhav, B. (2020). Design, synthesis
and evaluation of new chromone-derived aminophosphonates as
potential acetylcholinesterase inhibitor. Molecular Diversity, 1–15.
Shakhatreh, M. A. K., Al-Smadi, M. L., Khabour, O. F., Shuaibu, F. A.,
Hussein, E. I., & Alzoubi, K. H. (2016). Study of the antibacterial and
antifungal activities of synthetic benzyl bromides, ketones, and corre-
sponding chalcone derivatives. Drug Design, Development and
Therapy, 10, 3653–3660. https://doi.org/10.2147/DDDT.S116312
Xie, S.-S., Lan, J.-S., Wang, X., Wang, Z.-M., Jiang, N., Li, F., Wu, J.-J.,
Wang, J., & Kong, L.-Y. (2016). Design, synthesis and biological evalu-
ation of novel donepezil–coumarin hybrids as multi-target agents for
the treatment of Alzheimer’s disease. Bioorganic
& Medicinal
Chemistry, 24(7), 1528–1539. https://doi.org/10.1016/j.bmc.2016.02.023
Yerdelen, K. O., Koca, M., Anil, B., Sevindik, H., Kasap, Z., Halici, Z.,
Turkaydin, K., & Gunesacar, G. (2015). Synthesis of donepezil-based
multifunctional agents for the treatment of Alzheimer’s disease.
Bioorganic & Medicinal Chemistry Letters, 25(23), 5576–5582. https://
doi.org/10.1016/j.bmcl.2015.10.051
You, J., Fu, H., Zhao, D., Hu, T., Nie, J., & Wang, T. (2020). Flavonol dyes
with different substituents in photopolymerization. Journal of
Photochemistry and Photobiology A: Chemistry, 386, 112097. https://
doi.org/10.1016/j.jphotochem.2019.112097