Synthesis, structure-activity relationship and molecular docking studies of 3-O-flavonol glycosides as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.05.050

The prime objective of this research work is to prepare readily soluble synthetic analogues of naturally occurring 3-O-flavonol glycosides and then investigate the influence of various substituents on biological properties of synthetic compounds. In this context, a series of varyingly substituted 3-O-flavonol glycosides have been designed, synthesized and characterized efficiently. The structures of synthetic molecules were unambiguously corroborated by IR, ¹H, ¹³C NMR and ESI-MS spectroscopic techniques. The structure of compound 22 was also analyzed by X-ray diffraction analysis. All the synthetic compounds (21–30) were evaluated for in vitro inhibitory potential against cholinesterase enzymes. The results displayed that most of the derivatives were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with varying degree of IC₅₀ values. The experimental results were further encouraged by molecular docking studies in order to explore their binding behavior with the active pocket of AChE and BChE enzymes. The experimental and theoretical results are in parallel with one another.

Full text of DOI:10.1016/j.bmc.2018.05.050

Accepted Manuscript
Synthesis, Structure-Activity Relationship and Molecular Docking Studies of
3-O-Flavonol Glycosides as Cholinesterase Inhibitors
Ehsan Ullah Mughal, Asif Javid, Amina Sadiq, Shahzad Murtaza, Muhammad
Naveed Zafar, Bilal Ahmad Khan, Sajjad Hussain Sumra, Muhammad Nawaz
Tahir, Kanwal, Khalid Mohammed Khan
PII:
S0968-0896(18)30964-7
https://doi.org/10.1016/j.bmc.2018.05.050
BMC 14392
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 May 2018
30 May 2018
31 May 2018
Please cite this article as: Mughal, E.U., Javid, A., Sadiq, A., Murtaza, S., Zafar, M.N., Khan, B.A., Sumra, S.H.,
Nawaz Tahir, M., Kanwal, Khan, K.M., Synthesis, Structure-Activity Relationship and Molecular Docking Studies
of 3-O-Flavonol Glycosides as Cholinesterase Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.05.050
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Synthesis, Structure-Activity Relationship and Molecular Docking Studies of
3-O-Flavonol Glycosides as Cholinesterase Inhibitors
Ehsan Ullah Mughala, Asif Javida, Amina Sadiqb, Shahzad Murtazaa, Muhammad Naveed
Zafarc, Bilal Ahmad Khand, Sajjad Hussain Sumraa, Muhammad Nawaz Tahire, Kanwalf,
Khalid Mohammed Khanf,g
aDepartment of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
bDepartment of Chemistry, Govt. College Women University, Sialkot 51300, Pakistan
cDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan
dDepartment of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan
eDepartment of Physics, University of Sargodha, Sargodha, Pakistan
fH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi 75270, Pakistan
gDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
Abstract: The prime objective of this research work is to prepare readily soluble synthetic
analogues of naturally occurring 3-O-flavonol glycosides and then investigate the influence
of various substituents on biological properties of synthetic compounds. In this context, a
series of varyingly substituted 3-O-flavonol glycosides have been designed, synthesized and
characterized efficiently. The structures of synthetic molecules were unambiguously
1
corroborated by IR, H-, 13C-NMR and ESI-MS spectroscopic techniques. The structure of
compound 22 was also analyzed by X-ray diffraction analysis. All the synthetic compounds
(21-30) were evaluated for in vitro inhibitory potential against cholinesterase enzymes. The
results displayed that most of the derivatives were potent inhibitors of acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) with varying degree of IC50 values. The
experimental results were further encouraged by molecular docking studies in order to
explore their binding behavior with the active pocket of AChE and BChE enzymes. The
experimental and theoretical results are in parallel with one another.
Keywords: Flavonoids, flavonols, 3-hydroxyflavones, glycosides, cholinesterases,
AChE/BChE inhibitors, molecular docking studies
*Corresponding Author: ehsan.ullah@uog.edu.pk; Tel.: 0092-333-8464959
1

Introduction
Flavonoids constitute a large and significant group of naturally occurring secondary
metabolites. These ubiquitous compounds play important role in the growth and development
of plants, and are beneficial for human health [1,2]. They mostly occur in glycosylated form,
comprising an aglycone core and a sugar unit. The attachment of one or more sugar moieties
at different positions of the aglycone can give rise to structural variability in these
compounds, and thus accountable for increasing their solubility and rendering them to
demonstrate their inherent functions such as antioxidant capability and potential biological
properties [3] e.g., anticancer, antioxidant and antimicrobial bioactivities [4].
Among these metabolites, flavonol-O-glycosides which frequently occur in the form of 3-O-
glycosides (Figure-1) have attained increasing interest due to their immense medicinal and
pharmacological applications. These compounds are reported to possess several
pharmacological properties including anticarcinogenic, antiplatelet, antioxidant, antithrombic,
antimutagenic, and genoprotective effects, cytoprotective, cardioprotective and
vasoprotective activities [5-7]. They also play an important role as interspecies signaling
molecules in plants, and in humans they demonstrate a various activities such as
antimicrobial, anticancer and radical-scavenging activities [8], antidiarrheic, diuretic,
hemostatic, stomachic, uterine congestion, cardiovascular diseases, analgesic, and
antidiabetic etc [9,10]. Constructive and regulatory functions of these scaffolds are making
them intensively investigated class. The modifications are carried out on all the three rings,
particularly on ring C, due to which flavonoids represent one of the largest and the most
diverse class of plant secondary metabolites [11].
2

B
O
C
A
O
Sugar
O
Figure-1: Representative structure of 3-O-flavonolglycoside
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by dementia
and deficiency in daily life activities along with neuropsychiatric symptoms or behavioral
disturbance. The hallmarks of AD are dysfunction of cholinergic system, elevated
aggregation of β-amyloid peptides (Aβ) and loss of cognitive function [12]. The low level of
cholinergic neurotransmission in cortical and other regions of the human brain leads to the
loss of cognitive function in AD patients [13]. Studies have shown that by increasing
acetycholine (ACh) level through inhibiting acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) enzymes in the body offer a potential therapy for treating AD,
and thus improves both mental and memory symptoms in that disease. Standard AChE
inhibitors such as donepezil, galantamine, and rivastigmine are involved in improving
symptoms for most patients by increasing cholinergic neurotransmission level in the body.
However, these drugs are expensive as well as their long term use leads to various adverse
side effects and reoccurrence of symtoms [12]. Over the past decades, naturally occurring
AChE and BChE inhibitors have been isolated from the plants, and used as natural therapy
for AD treatment. In this context, flavonolO-glycosides have been reported as potent
cholinesterase inhibitors [12-14]. Possible high chelating capability, antioxidant activity and
low toxicity of these scaffolds render them promising compounds for developing multipotent
drugs against AD [15]. However, natural sources of such drugs are insufficient to cope with
requirements all over the globe. Therefore, there is urgent need to explore and design new
synthetic analogues of these natural drugs.
3

In view of immense biological significance of naturally occurring flavonol-3-O-glycosides,
several workers have reported their isolation from plant sources [16]. The process of isolation
and purification of these compounds are quite long, tedious, laborious, expensive, even then
small quantities are obtained for testing their biological activities. It is well known that these
glycosides are mostly hydroxylated, methoxylated and prenylated at rings A and B. In reality,
these substituents are responsible for their reported biological activities. It is also well known
that compounds having halogens, nitro, alkyl groups as substituents are also highly bioactive
[17,18], nevertheless, in nature no flavonoid glycosides have been reported with these
substituent. Therefore, a general synthetic method and combinatorial synthesis of a library of
these molecules would be very desirable.
In the light of above-mentioned reports and in order to explore new compounds which might
be used as drugs for the treatment of AD and also to find new structures for improving the
activity of these scaffolds, we initiated a project aiming to design and synthesize of varyingly
substituted acylated flavonol-3-O-glycosides in laboratory, since synthesis leads to the target
molecules in sufficiently large quantities. Moreover, it is also desire to study the effect of
various substituents on cholinesterase inhibitory potential of these compounds, and thus to
study the structure activity relationship (SAR) for each of the synthesized compound. The
experimental results have also been validated by molecular docking studies.
Material and Methods
All the chemicals were purchased from Merck (Germany) and Sigma-Aldrich (USA) and
used as delivered. Melting points were measured on an Electrothermal melting point
apparatus and are uncorrected. The IR spectra were recorded on a Bio-red spectrophotometer
using KBr discs. NMR spectra were measured on a Bruker DRX 300 instrument (1H, 300
MHz, 13C, 75 MHz). Accurate mass measurements were carried out with the Fisons VG
sector-field instrument (EI) and a FT-ICR mass spectrometer. The IR values are mentioned in
ῡ units and NMR chemical shift values were determined in ppm units. Absorption spectra
4

were recorded in dichloromethane on Jasco UV-VIS V-660 or Jasco UV-VIS V-670
instrument. Bruker Smart APEX-II CCD diffractometer was used for single crystal XRD
studies.
General Procedure for the synthesis of flavonol 3-O-glycosides
Methanolic solution of 2-hydroxyacetophenone (1.2 mL, 10 mmol) was mixed with 10 mL
sodium hydroxide (30%) and stirred for 30 min. Benzaldehyde (1.0 mL, 10 mmol) was then
added drop wise and the reaction mixture was further stirred for 24 h. Progression of reaction
was monitored by comparative TLC. Chalcone so formed was further cyclized by adding 1.5
mL H2O2 (35%) followed by stirring for 1 h. After completion, HCl (10%) was added to the
reaction mixture and precipitates of flavonol were formed. The precipitate were filtered
followed by thoroughly washing with water, the residue obtained was then dried and
crystallized from ethanol. Flavonol (238.23 mg, 1.0 mmol) was dissolved in 5.0 mL
chloroform containing anhydrous potassium carbonate (414.6 mg, 3.0 mmol), and tetrabutyl
ammonium bromide (TBAB 967.0 mg, 3.0 mmol). Then H2O (5.0 mL) was added drop by
drop to the above mixture and stirred for 30 min. Subsequently, peracetylglycosyl bromide
(592.0 mg, 1.5 mmol) of was added by further stirring at room temperature for 36 h. The
reaction progress was monitored with TLC. The reaction mixture was quenched with the
addition of water and neutralized by HCl (10%). The aqueous layer was then extracted with
dichloromethane (3×5 mL). The combined organic layer was washed with brine followed by
drying over anhydrous sodium sulfate, it was then filtered and concentrated under reduced
pressure. The residue was purified by column chromatography through silica gel (pet-
ether/ethylacetate (10:1 to 3:1) and furnished the product.
Enzyme Inhibition Essay
Enzyme inhibitory studies were carried out by using Ryan and Ellman method [18] with
slight modification. 100 µL of each sample (20, 40, 60, 80, 100 µM) was mixed with 50 µL
enzyme (AChE/BChE) and allowed it to stand for 10 minutes. 50 µL of substrate i.e.
5

acetylthiocholine iodide (0.71 mM) for AChE or butyrylthiocholine chloride (0.2 mM) for
BChE, 50 µL (0.5 mM) of DTNB [5,5´-dithio-bis(2-nitrobenzoic acid)] and 500 µL
phosphate buffer of pH 8 added to above mixture and incubated for 20 min at 37 ˚C. Solution
turned yellow due to formation of 5-thio-2-nitrobenzoate anion as a result of hydrolysis of
substrate. The hydrolysis of substrate is monitored spectrophotometrically by measuring
absorbance at wavelength of 400 nm and 412 nm for AChE and BChE, respectively.
Percentage inhibition was calculated by this formula:
(%) Inhibition =
Here A = absorbance of the enzyme with test sample; B = absorbance of enzyme without test
sample
Each experiment was repeated thrice and concordant value was used. IC50 value was
calculated through simple linear regression. Donepezil was used as reference compound.
Molecular Docking Assay [18]
Molecular docking studies were performed to predict the interaction of enzyme and ligand.
Crystal structure of AChE and BChE was obtained from (RCSB) protein data bank ACD
Chemsketch and 3D Pro 12.0 were used for the 3D optimization of given compounds and
were saved as SYBYL mol2 file format. AutoDock Tool v1.5.6 was used for docking, 100
different configurations were optimized. Discovery Studio Visualizerv 4.0 was used for
visualization of most potent and best poses of under study compounds.
Results and Discussion
Chemistry
Flavonols (11-20) were synthesized starting from 2-hydroxyacetophenone and varyingly
substituted aromatic aldehydes in two steps sequence through Algar-Flynn-Oyamada (AFO)
reaction [19]. 2'-Hydroxyacetophenone was condensed with various aldehydes in methanolic
solution of sodium hydroxide to produce 2'-hydroxychalcones. These intermediate were then
oxdatively cyclized with 30% hydrogen peroxide (H2O2) in the same solvent under basic
6

medium to afford 2-hydroxyflavones in excellent yields (Scheme 1). Flavonols so obtained
were directly used, without further purification, for the follow-up O-glycosylation reaction.
The final 3-O-glycosylation step was accomplished by treating flavonols with acetobromo-α-
D-glucose in the presence of potassium carbonate and dimethylformamide (DMF) under
phase transfer catalytic conditions.
Compound No.
21
R1
R2
H
S
H
H
22
23
F
H
H
24
25
H
26
7

H
27
H
H
28
29
30
CH3
Scheme-1: Synthesis of varyingly substituted 3-O-flavonolglycosides (21-30)
The target compounds (21-30) were synthesized in moderate to excellent yields and were
found readily soluble in common organic solvents such as methanol, ethanol, THF,
chloroform etc.
All the newly 3-O-acylated glycosides were purified by column chromatography and
subsequently characterized by FTIR, NMR spectroscopy and mass spectrometry. For
instance, in FTIR spectra, the disappearance of peaks around of 3420 cm-1 due to 3-OH and
appearance of new peaks around 1620 cm-1 due to (OAc) indicate successful attachment of
1
acylated sugar with aglycone part. Similarly, H-NMR spectra showed the peaks ranging
from 8.30 to 7.20 ppm for aromatic protons, the peaks ranging from 6.10 to 4.95 ppm for
aliphatic protons of acylated glucose moiety and two singlet peaks at 2.06 and 1.99 ppm for
two non-equivalent acetyl groups. Moreover, their 13C-NMR spectra exhibit two
characteristics signals around 173 and 170 pmm for flavonol ketonic group and ester acetyl
group, respectively. The molecular masses of the 3-O-flavonol acylated glycosides were
confirmed by electron spray ionization (ESI) mass spectrometry. Their ESI spectra showed
quasi molecular ion peaks corresponding to [M+H]. All the spectral data unequivocally
corroborate the structures of the newly synthesized molecules. The single crystal for the
8

compound 22 was grown via slow evaporation of dichloromethane solution, and its molecular
structure was resolved by X-ray single-crystal structure analysis and is depicted in Figure-2.
a)
b)
Figure-2: (a) Molecular structure 22, as determined by X-ray diffraction. Oxygen atoms: red;
sulfur: yellow, and hydrogen atoms have been omitted for clarity: (b) 3D view predicting
crystal packing pattern.
Crystal data
1846188
C27H26O12S
574.54
CCDC Number
Chemical Formula
Mr
9

Crystal system, space group
Temperature (K)
a, b, c (Å)
Orthorhombic, P212121
296
5.5797 (5), 21.600 (3), 22.668 (3)
V (Å3)
2732.1 (6)
Z
4
Mo Ka
Radiation type
m (mm-1)
0.18
Crystal size (mm)
0.40 × 0.18 × 0.16
Data collection
Diffractometer
Absorption correction
Tmin, Tmax
Bruker Kappa APEXII CCD
Multi-scan (SADABS; Bruker, 2005)
0.910, 0.960
No. of measured, independent and
observed [I > 2s(I)] reflections
Rint
9078, 5286, 2422
0.070
0.617
(sin q/l)max (Å-1)
Refinement
R[F2 > 2s(F2)], wR(F2), S
No. of reflections
No. of parameters
No. of restraints
0.067, 0.171, 0.96
5286
353
6
H-atom treatment
Dρmax, Dρmin (e Å-3)
Absolute structure
H-atom parameters constrained
0.20, -0.25
Flack x determined using 954 quotients [(I+)-(I-
10

)]/[(I+)+(I-)] (Parsons, Flack and Wagner, Acta Cryst.
B69 (2013) 249-259).
-0.09 (16)
Absolute structure parameter
The spectroscopic data of all the newly synthesized target compounds are given below:
2-Phenyl-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside (21)
Pale yellow solid; Yield: 70%; m.p. 175-177ºC; IR (νmax; KBr, cm-1): 3112, 2965, 1625,
1507, 1101, 1021; 1H NMR (300 MHz, DMSO-d6) δ: 9.61 (s, 1H, Ar-H), 8.11 (d, J = 8.1 Hz,
1H, Ar-H), 7.79 (m, J = 8.7, 1.2 Hz, 1H, Ar-H), 7.74 (d, J = 8.4 Hz, 1H, Ar-H), 7.56 (t, J =
7.5 Hz, 2H, Ar-H), 7.51 (t, J = 7.5 Hz, 1H, Ar-H), 7.46 (t, J = 8.7 Hz, 1H, Ar-H), 5.7 ( d, J =
6.3 Hz, 1H, Aliphatic) 5.31-5.24 (m, 1H, Aliphatic), 5.16 (dd, J = 9.6, 6.4 Hz, 1H, Aliphatic),
5.04 (t, J = 9.1 Hz, 1H, Aliphatic), 4.38-4.30 (m, 2H, Aliphatic), 4.18-4.11 (m, 1H,
Aliphatic), 2.08 (s, 3H, -COH3), 2.03 (s, 6H, -COCH3), 1.74 (s, 3H, -COCH3); 13C NMR (75
MHz, DMSO-d6) δ: 175.6, 171.1, 170.6, 170.4, 169.4, 161.1, 158.4, 154.5, 134.4, 134.2,
130.6, 127.0, 124.1, 123.0, 122.4, 118.3, 115.1, 97.5, 70.3, 69.9, 69.4, 67.3, 61.9, 20.7, 20.7,
20.7, 20.6; accurate mass (ESI, AcCN, +ve) of [M+H]+: Calcd for C29H29O12 569.1659;
found 569.1645.
Synthesis of 2-(thiophen-2-yl)-4H-chromen-4-one 3-O-2,3,4,6-O-tetraacetyl-β-D-
glucopyranoside (22)
º
Light yellow solid; Yield: 78%; m.p. 185-186 C; IR (νmax; KBr, cm-1): 3104, 2971, 1614,
1502, 1112, 1035; 1H NMR (300 MHz, DMSO-d6) δ: 8.17-7.97 (m, 3H, Ar-H), 7.92-7.69 (m,
2H, Ar-H), 7.52 (d, J = 1.01 Hz, 1H, Ar-H), 7.32 (dd, J = 4.95, J = 3.85 Hz, 1H, Ar-H), 6.06
(d, J = 7.70 Hz, 1H, Aliphatic), 5.44 (d, J = 9.54 Hz, 1H, Aliphatic), 5.35-5.21 (m, 1H,
Aliphatic), 5.05-4.90 (m, 1H, Aliphatic), 3.97-3.89 (m, 2H, Aliphatic), 2.06 (s, 3H, -COH3),
1.99 (s, 6H, -COCH3), 1.70 (s, 3H, -COCH3); 13C NMR (75 MHz, DMSO-d6) δ: 172.2,
169.8, 169.7, 169.6, 169.4, 154.5, 152.3, 134.6, 133.3, 133.2, 131.5, 131, 127.9, 125.5, 125.1,
11

123.4, 118.4, 97.8, 72.2, 71.8, 70.9, 68.1, 60.9, 20.7, 20.6, 20.5, 20.2; accurate mass (ESI,
AcCN, +ve) of [M+H]+: Calcd for C27H27O12S 575.1223; found 575.1205.
2-(4-Flourophenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(23)
Off-white solid; Yield: 66%; m.p. 171-173 ºC; IR (νmax; KBr, cm-1): 3110, 2970, 1615, 1512,
1115, 1015; 1H NMR (300 MHz, DMSO-d6) δ: 8.23-8.05 (m, 3H, Ar-H), 7.87 (ddd, J = 8.48,
6.92, 1.65 Hz, 1H, Ar-H), 7.77 (dd, J = 8.44, 0.73 Hz, 1H Ar-H), 7.54 (ddd, J = 7.98, 6.97,
1.19 Hz, 1H, Ar-H), 7.47-7.32 (m, 2H Ar-H), 5.73 (d, J = 7.89 Hz, 1H, Aliphatic), 5.48-5.31
(m, 1H, Aliphatic), 5.11-4.82 (m, 3H, Aliphatic), 4.02-3.77 (m, 3H, Aliphatic), 2.07 (s, 3H, -
COCH3), 1.97 (s, 6H, -COCH3), 1.82 (s, 3H, COCH3); 13C NMR (75 MHz, DMSO-d6) δ:
173.3, 170.1, 169.9, 169.7, 156.3, 155.2, 136.2, 132.1, 132, 127.2, 125.8, 123.8, 119, 115.9,
115.6, 98.9, 72.2, 71.8, 71, 68.3, 61.5, 20.9, 20.8, 20.6, 20.4; accurate mass (ESI, AcCN, +ve)
of [M+H]+: Calcd for C29H28O12F 587.1564; found 587.1551.
2-(4-Acetamidophenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-
glucopyranoside (24)
º
Yellow solid; Yield: 75%; m.p. 200-203 C; IR (νmax; KBr, cm-1): 3310, 3104, 2981, 1620,
1520, 1117, 1011; 1H NMR (300 MHz, DMSO-d6) δ: 8.37-8.25 (m, 1H, N -H), 8.22-8.12 (m,
1H, Ar-H), 7.75 (dd, J = 7.79, 1.56 Hz, 1H, Ar-H), 7.61 (d, J = 8.62 Hz, 1H, Ar-H), 7.45-7.30
(m, 1H, Ar-H), 6.88-6.73 (m, 2H, Ar-H), 6.54 (d, J = 8.8 Hz, 1H, Ar-H), 6.06 (d, J = 7.70 Hz,
1H, Aliphatic), 5.44 (d, J = 9.54 Hz, 1H, Aliphatic), 5.35-5.21 (m, 1H, Aliphatic), 5.05-4.90
(m, 1H, Aliphatic), 3.97-3.89 (m, 2H, Aliphatic), 2.02 (s, 3H, -COCH3), 1.98 (s, 6H, -
13
COCH3), 1.70 (s, 3H, -COCH3), 1.23 (s, 3H, Aliphatic); C NMR (75 MHz, DMSO-d6) δ:
171.8, 169.8, 169.4, 169.2, 167.4, 165.8, 161.6, 153.1, 150, 134.2, 131.2, 130.7, 130.1, 123.7,
118.1, 116.6, 112.5, 96.3, 72.2, 71.7, 71.2, 68, 62.4, 21.9, 20.6, 20, 20.5, 20.8; accurate mass
(ESI, AcCN, +ve) of [M+H]+: Calcd for C31H32O13N 626.1873; found 626.1850.
12

2-(2-Carboxyphenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(25)
º
Light yellow solid; Yield: 45%; m.p. 230-233 C; IR (νmax; KBr, cm-1): 3450, 3108, 2988,
1619, 1526, 1109, 1030; 1H NMR (300 MHz, DMSO-d6) δ: 8.04 (dd, J = 8.1, 1.5 Hz, 1H, Ar-
H), 7.84 (ddd, J = 20, 7.8, 1.6 Hz, 2H, Ar-H), 7.75-7.61 (m, 3H, Ar-H), 7.54 (ddd, J = 8.3,
7.2, 1.4 Hz, 1H, Ar-H), 7.46 (ddd J = 7.6, 1.7 Hz, Ar-H), 5.79 (d, J = 6.4 Hz, 1H, Aliphatic),
5.82 (dd, J = 9.5, 9 Hz, 1H, Aliphatic), 5.1-5 (m, 2H, Aliphatic), 4.39-4.31 (m, 2H), 4.18-4.11
(m, 1H, Aliphatic), 2.10 (s, 3H, -COCH3), 2.05 (s, 6H, -COCH3), 1.75 (s, 3H, -COCH3); 13C
NMR (75 MHz, DMSO-d6) δ: 192.4, 170.6, 168.7, 168.5, 168, 166.5, 141, 140.8, 136.4,
132.7, 125.9, 124.4, 123.6, 117.8, 113.7, 98.9, 71.6, 71.1, 71, 69.1, 62.4, 22.3, 20.6, 20.3,
20.2, 20.1; accurate mass (ESI, AcCN, -ve) of [M-H]-: Calcd for C30H27O14 611.1400; found
611.1390.
2-(Naphthalen-1-yl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(26)
º
Off-white solid; Yield: 73%; m.p. decompose above 185 C; IR (νmax; KBr, cm-1): 3141,
1
3011, 1627, 1536, 1107, 1038; H NMR (300 MHz, DMSO-d6) δ: 8.26-8.17 (m, 1H, Ar-H),
8.04 (dd, J = 8.1, 1.5 Hz, 1H, Ar-H), 7.95-7.86 (m, 3H, Ar-H), 7.71 (dd, J = 8.3, 1.3 Hz, 1H,
Ar-H), 7.65 (ddd, J = 8.3, 7.1, 1.5 Hz, 1H, Ar-H), 7.61-7.51 (m, 2H, Ar-H), 7.53-7.43 (m,
2H, Ar-H), 5.5 (d, J = 6.0 Hz, 1H, Aliphatic), 5.44 (m, J = 9.54 Hz, 1H, Aliphatic), 5.21-5.35
(m, 1H, Aliphatic), 4.9-5.05 (m, 1H, Aliphatic), 3.99-3.98 (m, 2H, Aliphatic), 2.08 (s, 3H, -
COH3), 1.95 (s, 6H, -COCH3), 1.74 (s, 3H, -COCH3); 13C NMR (75 MHz, DMSO-d6) δ:
173.5, 170.6, 170.2, 169.6, 159.1, 155.8, 138.1, 135.0, 133.5, 131, 128, 126.8, 124.4, 119.1,
99.2, 89.5, 72.8, 72.2, 71.6, 71.0, 69.0, 66.8, 61.6, 60.8, 29.5, 21, 21, 20.8, 20.7, 14; accurate
mass (ESI, AcCN, +ve) of [M+H]+: Calcd for C33H30O12 618.1737; found 618.1708.
13

2-(4-Isobutylphenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(27)
º
Light yellow solid; Yield: 83%; m.p. 172-175 C; IR (νmax; KBr, cm-1): 3110, 2990, 1643,
1
1516, 1112, 1023; H NMR (300 MHz, DMSO-d6) δ: 8.15 (m, 1 H, Ar-H), 8.12 (d, J = 3.0
Hz, 1H, Ar-H), 7.97 (m, 1H, Ar-H), 7.49 (d, J = 6.0 Hz, 1H, Ar-H), 7.47 (d, J = 3.0 Hz, 1H,
Ar-H), 5.5 (d, J = 6 Hz, 1H, Aliphatic), 5.44 (m, J = 9.5 Hz, 1H, Aliphatic), 5.35-5.21 (m,
1H, Aliphatic), 5.05-4.90 (m, 1H, Aliphatic), 3.99-3.90 (m, 2H, Aliphatic), 2.40 (d, J = 9.0
Hz, 2 H, Aliphatic), 2.08 (s, 3H, -COH3), 1.95 (s, 6H, -COCH3), 1.92 (s, 3H, -COCH3), 1.90
(m, 1 H, -CH-), 1.74 (d, J = 9.0 Hz, 2H, -CH2- ) 1.05 (d, J = 9.0 Hz, 6 H, -(CH3)2); 13C NMR
(75 MHz, DMSO-d6) δ: 192.2, 168.8, 169.5, 169.3, 161.7, 142.5, 136.2, 136, 129.6, 127.0,
126.0, 121.6, 121.0, 119.0, 118.0, 98.4, 79.6, 71.5, 71.2, 70.6, 68.6, 67.3, 62.3, 45.1, 44.6,
30.2, 29.8, 28.3, 23.2, 22.4, 22.3, 20.4, 20.2, 20.2, 13.8, 10.7; accurate mass (ESI, AcCN,
+ve) of [M+H]+: Calcd for C33H37O12 625.2285; found 625.2274.
2-(4-Bromophenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(28)
º
Yellow solid; Yield: 63%; m.p. 190-192 C; IR (νmax; KBr, cm-1): 3112, 2967, 1620, 1524,
1
1117, 1018; H NMR (300 MHz, DMSO-d6) δ: 8.3 (s, 1H, Ar-H), 8.1 (d, J = 15.0 Hz, 2H,
Ar-H), 7.8-7.6 (m, 3H, Ar-H), 7.55-7.40 (m, 2H, Ar-H), 5.8 (dd, J = 9.0, 3.0 Hz, 1H,
Aliphatic), 5.45-5.35 (m, 1H, Aliphatic), 5.05-4.85 (m, 4H, Aliphatic), 2.11 (s, 3H, -COH3),
1.97 (s, 6H, -COCH3), 1.72 (s, 3H, -COCH3); 13C NMR (75 MHz, DMSO-d6) δ: 173.0, 169.6,
169.2, 167.0, 155.0, 154.7, 136.1, 133.5, 132.3, 131.3, 130.3, 128.0, 125.3, 123.3, 121.3,
118.6, 98.4, 71.6, 71.3, 70.6, 67.8, 67.3, 61.2, 29.8, 28.3, 23.2, 22.3, 20.4,20.2, 20.2; accurate
mass (ESI, AcCN, +ve) of [M+H]+: Calcd for C29H27O12Br 646.0685 (monoisotopic); found
646.0660.
14

2-(4-Methoxyphenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-glucopyranoside
(29)
Yellowish green solid; Yield: 70%; m.p. 169-171 ºC; IR (νmax; KBr, cm-1): 3123, 2991, 1601,
1
1530, 1124, 1037; H NMR (300 MHz, DMSO-d6) δ: 8.03 (dd, J = 8.1, 1.5 Hz, 1H, Ar-H),
8.01-7.95 (m, 2H, Ar-H), 7.7 (dd, J = 8.2, 1.4 Hz, 1H), 7.65 (ddd, J=8.3, 7.1, 1.5 Hz, 1H, Ar-
H), 7.54 (ddd, J = 8.3, 7.1, 1.4 Hz, 1H, Ar-H), 7.25-7.19 (m, 2H, Ar-H), 5.6 (dd, J = 8.0, 3.0
Hz, 1H, Aliphatic), 5.55-5.45 (m, 1H, Aliphatic) 5.20-4.90 (m, 4H, Aliphatic), 3.84 (s, 3H,
OMe), 2.11 (s, 3H, -COH3), 1.97 (s, 6H, -COCH3), 1.76 (s, 3H, -COCH3); 13C NMR (75
MHz, DMSO-d6) δ: 175.6, 171.1, 170.6, 170.4, 169.4, 161.1, 158.4, 154.5, 134.4, 134.2,
131.2, 126.9, 124.1, 122.9, 122.4, 118.3, 115.1, 97.5, 70.3, 69.9, 69.4, 67.3, 61.9, 55.8, 55.3,
20.7, 20.7, 20.7, 20.6; accurate mass (ESI, AcCN, +ve) of [M+H]+: Calcd for C30H31O13
599.17646; found 599.1740.
7-Methyl-2-(4-bromophenyl)-4H-chromen-4-one-3-O-2,3,4,6-O-tetraacetyl-β-D-
glucopyranoside (30)
º
Yellow solid; Yield: 75%; m.p. 194-196 C; IR (νmax; KBr, cm-1): 3105, 2960, 1601, 1503,
1098, 1026; 1H NMR (300 MHz, DMSO-d6) δ: 8.05-7.97 (m, 2H, Ar-H), 7.86 (d, J = 8.4 Hz,
1H), 7.70 (d, J = 2.2 Hz, 1H), 7.61-7.52 (m, 3H, Ar-H), 7.42 (dd, J = 8.4, 2.2 Hz, 1H, Ar-H),
5.81 (d, J = 6.4 Hz, 1H, Aliphatic), 5.28 (dd, J = 9.5, 3.0 Hz, 1H), 5.13 (dd, J = 9.5, 6.4 Hz,
1H), 5.04 (t, J = 9.1 Hz, 1H, Aliphatic), 4.38-4.30 (m, 2H, Aliphatic), 4.18-4.11 (m, 1H,
Aliphatic), 2.50 (s, 3H, Me), 2.11 (s, 3H, -COH3), 1.97 (s, 6H, -COCH3), 1.72 (s, 3H, -
13
COCH3); C NMR (75 MHz, DMSO-d6) δ: 175.6, 171.1, 170.6, 170.4, 169.4, 158, 154.8,
134.2, 130.6, 130.4, 129.2, 128.6, 128.2, 127.5, 124, 123.8, 120.1, 97.5, 71.3, 70.6, 67.8,
67.3, 61.2, 20.8, 20.4, 20.2, 20.2, 20.0; accurate mass (ESI, AcCN, +ve) of [M+H]+: Calcd.
for C30H31O12 662.0998 (monoisotopic); found 662.0970.
15

Cholinesterase Enzymes Inhibition Assay
In continuation to our efforts on enzyme inhibition studies [18], all the target compounds (21-
30) were screened in vitro, for their inhibitory potential against commercially available
electric eel acetylcholinesterase (AChE) and horse serum butrylcholinesterase (BChE)
enzymes by using spectrophotometric method.
The experimental results are reported in Table 1. Their IC50 values were also calculated, and
Donepezil was used as standard.
Table-1: Cholinesterase inhibition efficacy of compounds 21-30
Compound
No.
Compound Structure
AChE IC50 ± SEMa BChE IC50 ± SEMa
(µM)
(µM)
102.91±0.96
45.86±0.39
21
22
23
18.52±0.81
29.23±0.56
25.51±0.08
49.83±0.72
38.70±0.00
34.05±0.01
39.70±0.87
48.43±0.03
24
25
16

36.35±1.30
18.69±0.01
26
02.05±0.11
10.20±0.10
05.42±0.33
03.12±0.35
27
28
20.70±0.15
22.36±0.10
0.09±0.19
26.00±0.16
30.58±0.18
0.13±0.22
29
30
DonepezilSt
St
aIC50 values (mean ± standard error of mean); Standard inhibitor for AChE and BChE
enzymes
All the tested derivatives (21-30) displayed potent variable degree of inhibition (IC50 = 02.05
± SEM to 102.91 ± SEM µM) as compared to standard drug donepezil (IC50 = 0.09 ± SEM
(AChE) and 0.13 ± SEM µM (BChE). Most of the derivatives are potent selective inhibitor of
either AChE or BChE enzyme. The experimental results showed that these compounds are
comparatively more inhibitor for AChE as compared to BChE. This is attributed to relatively
smaller active site of AChE enzyme that can assimilate the smaller groups unlike the larger
17

active site of BChE enzyme. Even though all the structural features were actively taking part
in inhibitory activity, however, the variation of different groups on main structural motif was
actually accountable for alteration in inhibitory potential. The compound 27 was found to be
the most active dual inhibitor (IC50 = 02.05 µM for AChE and 05.42 µM for BChE) among
the series having isobutyl at 4' position of the phenyl ring. The high inhibitory potential of
this compound is mainly due to hydrophobic interactions of that alkyl group with non-polar
pockets of the envisioned enzymes. The next most potent dual inhibitor is compound 28
((IC50 = 10.20 µM for AChE and 03.12 µM for BChE) with bromo group at 4' position of the
phenyl ring. This derivative is relatively selective for BChE. Its inhibitory potential is due to
the formation of strong non-bonding interactions with active pockets of the envisioned
enzymes. The un-substituted derivative 21 was appeared to be about least inhibitor against
both enzymes among the series (Table-1). This reflects the fact that mostly inhibitor potential
of these compounds is due to the presence of different substituents on the main scaffold.
Overall, it has been observed that all the compounds (21-30) are comparatively better potent
inhibitor against AChE as compare to BChE (Table-1). These results showed that substitution
at the ring B increase the inhibitory potential of these compounds in comparison to un-
substituted compound 21. Moreover, sulfur containing ring, for example compound 22,
makes strong interaction with active pockets of AChE (IC50 = 18.52 µM) rather than BChE
(IC50 = 25.51 µM). It is noted that proper substitution on the ring A, B and glycosylation
control the activity of these compounds against cholinesterase enzymes. Since, all envisioned
compounds have common 3-O-glycosylated flavone skeleton in their structures; the activity
was mainly due to different functional groups attached on the main framework of flavonol.
These findings have been further studied by in silico studies (molecular docking). These
results manifest that such multifunctionalized structural motifs are promising candidates for
designing and the development of new scaffolds for the cure of Alzheimer disease.
18

Molecular Docking Studies
All the compounds (21-30) were analyzed by molecular docking studies in order to find out
plausible binding interactions of inhibitors with the active pocket sizes of the enzymes, and
also to figure out protein-ligand interactions at molecular level for establishing SAR studies.
For this studies, X-ray structures of human AChE (PDB ID: 4BDT) and BChE (PDB ID:
4BDS) were selected as templates. The lowest bonding energies of the compounds (21-30)
obtained after docking analyzing are given in Table-2.
Table-2: Binding energies of selective posses against human AChE and BChE
Compound No.
h AChE Lowest Binding Energy h BChE Lowest Binding Energy ΔG
ΔG in KJ mol-1
-6.04
in KJ mol-1
-8.81
21
-5.06
-8.40
22
-5.32
-9.08
23
+2.38
-8.38
24
-0.37
-7.97
25
+0.96
-8.97
26
-8.24
-9.48
27
28
-3.93
-8.98
+1.05
-8.71
29
+0.55
-9.81
30
-10 (HUW)
-6.83 (THA)
Standard
The docking analysis demostrated the top ranked conformations of all compounds were well
accommodated inside the active site of AChE and BChE enzymes and comprised of different
types of interactions with the active site residues of the envisioned enzymes. For instance, the
visualization of compound 22 inside the active pockets of human AChE enzyme revealed
19

several important interactions as shown in Figure-3. This compound forms hydrogen bonding
interaction with amino acid residues Tyr124, Gly121, Ser125, Tyr337, hydrophobic π-alkyl
interactions with Trp86 and hydrophobic π-π stacked interactions with Trp439, Tyr337, and
Trp86. Moreover, π-lone pair interactions with Trp86 and π-sulfur interactions with MET443,
Tyr341 are involved with AChE enzyme.
Similarly, the visualization of the same potent compound 22 showed several important
interactions inside the active pocket of BChE enzyme as displayed in Figure-4. This
derivative establishes H-bonding with amino acid residues Pro285, Ser198, hydrophobic π-π
T shaped interactions with Trp82, Trp231, hydrophobic amide-π stacked interactions with
Gly116 and π-sulfur interactions with Trp231 inside the active pocket of human BChE.
Figure-3: Putative binding interactions of compound 22 against AChE
20

Figure-4: Putative binding interactions of compound 22 against BChE
Furthermore, visualization of most potent compound 27 shows hydrogen bonding with amino
acid residues of catalytic triad of AChE, as ‘O’ atom of Ser125 form hydrogen bond with
acetyl group of glycone portion of under observed compound. This compound also shows
hydrogen bonding with Tyr341. The overall this derivative shows hydrophobic π-π stacked
interaction with Met443, Trp86, Tyr337 with AChE. Trp86 shows hydrophobic π-π T-
shaped, hydrophobic π-alkyl type of interaction with AChE. Tyr133 shows hydrophobic π-
alkyl type interaction with manifested compound as depicted in Figure-5.
21

Figure-5. Interactions of the compound 27 with AChE at 3D space. Interactions with specific
amino acid residues are shown in the box. The 3D ribbon represents the enzyme, stick model
is the lowest energy conforms of the inhibitor 27 along with amino acids of AChE interacting
with it.
In continuation, the carbonyl group of other potent compound 27 present in the ring C shows
hydrogen bonding with catalytic triad amino acid residue Ser198 inside BChE. Glycone
portion of given compound reveals its inhibitory potential by developing hydrogen bonding
with catalytic triad (His438) of BChE. Gly116, Gly117 of oxyanion hole also shows
hydrophobic amide π-stacked interaction with this ligand in BChE. Leu286 of acyl binding
site of BChE exhibit hydrophobic π-alkyl association with this compound. Trp82 of
peripheral anionic site (PAS) shows hydrogen bonding with the glycone portion of
envisioned compound. Overall Phe329 and Trp82 of BChE shows hydrophobic π-π T- shape
and π-sigma type forces of attractions, respectively, with this ligand as shown in Figure-6.
22

Figure-6. Interactions of the compound 27 with BChE at 3D space. Interactions with specific
amino acid residues are shown in the box. The 3D ribbon represents the enzyme, stick model
is the lowest energy conforms of the inhibitor 27 along with amino acids of BChE interacting
with it.
Moreover, docking studies of compound 28 against AChE reveals several types of
associations. Thr83, Ser125, Gly121, Gly122 shows hydrogen bonding with that ligand. Trp
86 of catalytic triad of AChE shows hydrophobic π-π T-shaped association with ligand 28.
This compound also exhibits hydrophobic π-π stacked type of associations with amino acid
residue Trp439 and Tyr337. Additionally, this shows hydrophobic π-alkyl interaction with
Pro446 and Trp86. Met443 shows π-sulfur type of associations with ligand 28 inside the
active pocket of AChE enzyme. The interaction mode has been shown in Figure-7.
Similarly, the ligand 28 expresses its inhibitory potential against BChE by showing π-lone
pair interaction with Trp82 of peripheral anionic site (PAS) with ‘O’ atom of acetyl group of
the ligand. Gly117 of oxyanion hole shows hydrogen bonding with the ligand 28. Leu286 of
acyl binding site shows hydrophobic π-alkyl interaction with aromatic ring A of under study
compound. His438 of catalytic triad shows hydrophobic π-alkyl interaction with glycone
portion of this ligand. Overall Trp231 and Phe329 exhibit hydrophobic π-π T-shaped type of
interactions with the 28 as depicted in Figure-8.
23

Figure-7. Interactions of 28 with AChE at 3D space. Interactions with specific amino acid
residues are shown in the box. The 3D ribbon represents the enzyme, stick model is the
lowest energy conforms of the inhibitor 28 along with amino acids of AChE interacting with
it.
Figure-8. Interactions of 28 with BChE at 3D space. Interactions with specific amino acid
residues are shown in the box. The 3D ribbon represents the enzyme, stick model is the
lowest energy conforms of the inhibitor 28 along with amino acids of BChE interacting with
it.
24

Conclusion
In conclusion, an efficient synthesis, characterization and biological evaluation of 3-O-
flavonol glycosides as potent AChE and BChE inhibitors have been reported. The
compounds 21-30 were synthesized starting from 2-hydroxyacetophenone and varyingly
substituted aromatic aldehydes by employing literature known procedures after little
modifications. These derivatives were screened for their potential against cholinesterase
enzymes. Enzymes inhibition assay revealed that most of the compounds are relatively more
activity against AChE in comparison to BChE. The SAR studies were established on the
basis of substitution pattern on flavonol scaffold. The experimental results were further
supported by molecular docking analysis. The theoretical results are in accordance with
experimental results. These findings have paved the way to design and develop new potent
cholinesterase enzymes inhibitor on the basis of 3-O-flavonol glycosides for the treatment of
Alzheimer disease, and thus may also be of interest for applications in medicinal chemistry.
Acknowledgements
The financial support for this work is gratefully acknowledged by Higher Education
Commission of Pakistan under the SRGP Project. 282 and NRPU Project. 6484. Also, we are
thankful to Prof. Dr. Lukas J. Gooßen, Department of Chemistry, Bochum University,
Germany for his kind help in getting spectroscopic measurements.
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28

Graphical Abstract
Synthesis, Structure-Activity Relationship and Molecular Docking
Studies of 3-O-Flavonol Glycosides as Cholinesterase Inhibitors
Ehsan Ullah Mughal, Asif Javid, Amina Sadiq, Shahzad Murtaza, Muhammad Naveed Zafar,
Bilal Ahmad Khan, Sajjad Hussain Sumra, Muhammad Nawaz Tahir, Kanwal, Khalid Mohammed
Khan
29