Synthesis, structure–activity relationship and molecular docking of 3-oxoaurones and 3-thioaurones as acetylcholinesterase and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.10.016

The present study describes efficient and facile syntheses of varyingly substituted 3-thioaurones from the corresponding 3-oxoaurones using Lawesson's reagent and phosphorous pentasulfide. In comparison, the latter methodology was proved more convenient, giving higher yields and required short and simple methodology. The structures of synthetic compounds were unambiguously elucidated by IR, MS and NMR spectroscopy. All synthetic compounds were screened for their inhibitory potential against in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Molecular docking studies were also performed in order to examine their binding interactions with AChE and BChE human proteins. Both studies revealed that some of these compounds were found to be good inhibitors against AChE and BChE.

Full text of DOI:10.1016/j.bmc.2016.10.016

Bioorganic & Medicinal Chemistry 25 (2017) 100–106
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–activity relationship and molecular docking
of 3-oxoaurones and 3-thioaurones as acetylcholinesterase and
butyrylcholinesterase inhibitors
Ehsan Ullah Mughal a, , Amina Sadiq , Shahzad Murtaza , Hummera Rafique ,
⇑
b
a
a
c
a
d
e
Muhammad Naveed Zafar , Tauqeer Riaz , Bilal Ahmad Khan , Abdul Hameed ,
e
Khalid Mohammed Khan
a
Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
Department of Chemistry, Govt. College Women University, Sialkot 51300, Pakistan
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan
b
c
d
e
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes efficient and facile syntheses of varyingly substituted 3-thioaurones from the
corresponding 3-oxoaurones using Lawesson’s reagent and phosphorous pentasulfide. In comparison, the
latter methodology was proved more convenient, giving higher yields and required short and simple
methodology. The structures of synthetic compounds were unambiguously elucidated by IR, MS and
NMR spectroscopy. All synthetic compounds were screened for their inhibitory potential against
in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. Molecular docking stud-
ies were also performed in order to examine their binding interactions with AChE and BChE human pro-
teins. Both studies revealed that some of these compounds were found to be good inhibitors against AChE
and BChE.
Received 23 August 2016
Revised 8 October 2016
Accepted 12 October 2016
Available online 14 October 2016
Keywords:
Flavonoids
Aurones
3
-Thioaurones
Ó 2016 Elsevier Ltd. All rights reserved.
Lawesson’s reagent
AChE/BChE inhibitors
Molecular docking studies
1
. Introduction
-Benzylidenebenzofuran-3(2H)-one (aurone), is an interesting
classical methods for their synthesis were reviewed by Boumendjel
1
0
et al. Since then, a number of similar or refined methods have
1
1,12
2
been reported in literature
in order to synthesize bioactive aur-
subclass of flavonoids, found from natural or synthetic sources.
They have received less attention in comparison to the structurally
similar and widely investigated other flavonoids such as flavones
and flavonols. Nevertheless, they are equally important, and thus
display an important role in the pigmentation of some flowers,
fruits and particularly are responsible for giving bright yellow color
one derivatives. However, the corresponding 3-thio analogues of
aurones have been less studied due to limited number of methods
available for their preparation, although these sulfurated aurones
may offer new biological activities and may also be used as precur-
sors for the synthesis of other physiological active organic com-
pounds. Keeping in view the above-mentioned biological
importance of aurones and their derivatives, our continued interest
1
of flowers. They also display strong and wide variety of biological
2
13
activities. For instance, they have been demonstrated antifungal,
in the chemistry of thio-analogues of flavonoids and the less
insect antifeedant, inhibition of tyrosinase,4 antioxidants activi-
3
literature available on aurones encouraged us to introduce
expedient and general methods for the synthesis of 3-thioaurones
from 3-oxoaurones. To the best of our knowledge, there is no
example available in literature for the preparation of 3-thioau-
rones. We, therefore, design a methodology to transform variously
substituted aurones into the 3-thioaurones by using Lawesson’s
5
–8
ties etc.
number of reports on the preparation of 2-benzylidenebenzofuran-
-ones, despite having interesting biological properties. A very
Surprisingly, the literature search gives a rather narrow
3
accepted way to prepare aurones was developed by Varma et al.
which is based on the aldol-like condensation of benzofuran-3
9
(
2H)-ones with benzaldehydes. This, and some other more or less
reagent (LR) as well as phosphorous pentasulfide (P
2 5
S )/sodium
hydrogen carbonate (NaHCO ). Both of these reagents could
3
efficiently convert substituted as well as unsubstituted aurones
into their corresponding 3-thioanalogues. However, the latter
⇑
Corresponding author. Tel.: +92 333 8464959.
E-mail address: ehsan.ullah@uog.edu.pk (E.U. Mughal).
http://dx.doi.org/10.1016/j.bmc.2016.10.016
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
0

E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
101
process was found to be more convenient, relatively cheaper and
offers higher yields.
reaction mixture and stirring was continued for 1–2 h. After the
completion of reaction (monitored by TLC), the reaction mixture
was poured into water and the resultant solid was filtered, washed
with water and crystallized from ethanol.
Disease-associated enzyme’s inhibition by a small molecular
drug has been emerged a promising strategy to cure human dis-
eases. Alzheimer’s disease (AD) is the most common type of
dementia which affects millions of people worldwide. AD has been
accounted in the loop of top ten death causing diseases, which
stimulate medicinal chemists to develop new lead molecule as
drug candidates. The various complications associated with AD
are slow memory deterioration, language skills impairment and
many other cognitive dysfunctions. Cholinesterases (ChEs) are con-
sidered to be a major focal point of pharmaceutical research for the
treatment of some of the symptoms of Alzheimer’s disease (AD).
Two ChEs found in humans, which are known as acetyl-
cholinesterase (AChE) and butyrylcholinesterase (BChE), are con-
sider as principal factors for AD complications. Both of these
enzymes are present in cholinergic synapses in the central nervous
system (CNS), in the parasympathic synapses in the periphery, and
in the neuromuscular junction. However, AChE is selective for ACh
hydrolysis and BChE hydrolyses acetylcholine and other choline
esters as a non-specific cholinesterase.14 These enzymes have
unique binding pockets which are well-suited for interactions with
small drug molecules. In consequence, the nature of the chemistry
of enzyme catalysis makes ChEs amenable to inhibition by small
molecular weight, drug-like molecules. Therefore, it is an urgent
need to investigate and develop such drug structures which could
efficiently inhibit ChEs in order to cure AD. Recently, aurone and
structurally resembled compound such as benzofurans, indanones
and coumarins were screened for their inhibition potential against
AChE and BChE enzymes.1
2.1.2. General method for synthesis of 3-thioaurones using
1
3
Lawesson’s reagent (LR):
A mixture of aurone (1.0 mmol) and LR (1.5 mmol) was refluxed
in anhydrous toluene (10 mL) under argon atmosphere. Initially,
the reaction mixture was stirred at room temperature for 15 min
and then 3 h at 90 °C. After the completion of reaction (TLC analy-
sis), the solvent was removed under reduced pressure. The residue
was then purified through silica gel column (n-hexane/dichloro-
methane; 7:3) to afford 3-thioaurones.
The spectroscopic data for all newly synthesized compounds is
2
given below. However, the spectral data of 3-oxoaurones 2, 4 and
2
0
5
have also been published in literature.
2.1.2.1.
(Z)-2-Benzylidene-6-bromobenzofuran-3(2H)-one
(1). This substance was obtained as yellow solid in 90% yield,
1
mp 127–129 °C. IR (KBr):
(500 MHz, DMSO-d ): d 7.95 (d, J = 8.0 Hz, 1H, ArH), 7.84 (d,
J = 2.0 Hz, 1H, ArH), 7.68 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.35–7.19
m
ꢀ ¼ 1718 (C@O), 1610 (C@C). H NMR
6
1
3
(m, 5H, ArH), 6.90 (s, 1H, benzylic). C NMR (125 MHz, DMSO-
): d 184.2, 166.1, 164.6, 162.4, 146.0, 137.0, 137.0, 133.7, 128.6,
d
6
125.0, 124.1, 122.0, 116.7, 113.1, 113.5; MS (ESI, +ve): m/z (%),
+
322 (100, [M+Na] ), 324 (97); accurate mass (ESI, +ve) of [M
+
+Na] : Calcd. for C15
H
9
2
BrO Na 322.9659; found 322.9640.
5–19
2.1.2.2.
(3). This compound was obtained as yellow sticky material in
83% yield. mp 121–123 °C IR (KBr):
ꢀ ¼ 1717 (C@O), 1608 (C@C).
): d 7.98 (dd, J = 8.1, 2.5 Hz, 1H,
(Z)-2-(4-Isobutylbenzylidene)benzofuran-3(2H)-one
The present research work is aimed at the syntheses of vary-
ingly substituted 3-oxoaurones, 3-thioaurones and determination
of their inhibitory potential against AChE and BChE enzymes.
Molecular docking was also performed in order to study the bind-
ing affinities of the synthesized compounds for previously men-
tioned human proteins.
m
1
H NMR (500 MHz, DMSO-d
6
ArH), 7.90–7.50 (m, 3H, ArH), 7.30 (d, J = 7.5 Hz, 2H, ArH), 7.08
(d, J = 7.5 Hz, 2H, ArH), 6.97 (s, 1H, C@C–H), 2.52 (d, J = 7.5 Hz,
1
3
2H, CH
(
1
3
(
2
), 1.88 (m, 1H, CH), 0.90 (d, J = 7.4 Hz, 6H, CH
125 MHz, DMSO-d ): d 185.2, 168.2, 162.5, 150.5,139.2, 135.0,
30.7, 126.2, 125.3, 125.0, 123.0, 119.0, 117.0, 115.0, 113.6, 45.0,
3
); C NMR
6
O
X
+Å
H
H
3
CO
CO
0.0, 23.0; MS (EI, 70 eV): m/z (%), 278 (35, [M] ), 235 (100), 221
22), 178 (7), 121 (8), 117 (23),; accurate mass (EI-MS) of [M] :
R
1
+Å
O
3
18 2
Calcd. for C19H O 278.1306; found 278.1309.
N
R
2
3
-Oxo/Thio-aurones
X = O or S (1-10)
2
.1.2.3.
(Z)-2-Benzylidene-6-bromobenzofuran-3(2H)-thione
ꢀ ¼ 1249
6
C@S), 1609 (C@C). H NMR (500 MHz, DMSO-d ): d 7.93 (d,
Donepezil
(
6). Pale yellow solid; mp 213–215 °C. IR (KBr):
m
1
(
J = 8.0 Hz, 1H, ArH), 7.82 (d, J = 2.0 Hz, 1H, ArH), 7.67 (dd, J = 8.0,
2
.5 Hz, 1H, ArH), 7.32–7.15 (m, 5H, ArH), 6.92 (s, 1H, benzylic).
1
3
2
2
. Materials and methods
.1. General
6
C NMR (125 MHz, DMSO-d ): d 208.1, 166.0, 164.4, 162.4,
1
45.5, 137.0, 136.2, 133.5, 128.5, 123.0, 123.5, 121.5, 116.21,
+Å
113.0, 113.2; MS (EI, 70 eV): m/z (%), 315 (100, [M] ), 317 (98,
+
[
M+2] ), 268 (58), 251 (30), 203 (15), 167 (35), 149 (90); accurate
+Å
Melting points were measured on an Electrothermal melting
mass (EI-MS) of [M] : Calcd. for C15
317.2008.
9
H BrOS 317.2004; found
point apparatus and are uncorrected. The IR spectra were recorded
on a Bio-red spectrophotometer using KBr discs. NMR spectra were
1
13
measured on a Bruker DRX 500 instrument ( H, 500 MHz, C,
25.7 MHz). Mass spectra were recorded on a Fisons VG Autospec
2.1.2.4. (Z)-2-(4-Methoxybenzylidene)benzofuran-3(2H)-thione
ꢀ ¼ 1249
1
(7). Dark yellow solid; mp 143–145 °C. IR (KBr): m
1
X double-focusing mass spectrometer. Accurate mass measure-
ments were carried out with the Fisons VG sector-field instrument
6
(C@S), 1608 (C@C). H NMR (500 MHz, DMSO-d ): d 8.00 (d,
J = 7.5 Hz, 2H, ArH), 7.81 (d, J = 7.5 Hz, 2H, ArH), 7.57 (m, 2H,
ArH), 7.33 (m, 1H, ArH), 7.10 (m, 1H, ArH), 6.99 (s, 1H, C@C–H),
(
EI) and a FT-ICR mass spectrometer. All chemicals were purchased
1
3
from Alfa Aesar or Sigma Aldrich and used as delivered.
3.85 (s, 3H, OCH
67.2, 160.5, 149.5, 138.2, 136.0, 129.0, 125.2, 125.0, 124.9,
124.0, 118.0, 116.5, 114.0, 113.1, 56.0; MS (EI, 70 eV): m/z (%),
3
);
6
C NMR (125 MHz, DMSO-d ): d 208.1,
1
2
.1.1. General method for synthesis of 3-thioaurones using P
A solution of P (1.5 mmol) in anhydrous THF (5 mL) was
added to a stirred solution of aurone (1.0 mmol) in THF (10 mL)
followed by the addition of solid NaHCO (6 equiv) to the same
2 5
S
+Å
S
5
268 (100, [M] ), 253 (21), 240 (7), 197 (15), 92 (6), 77 (10), 53
(4); accurate mass (EI-MS) of [M] : Calcd. for
268.3303; found 268.3306.
2
+Å
16 12 2
C H O S
3

1
02
E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
2
(
1
.1.2.5. (Z)-2-(4-Isobutylbenzylidene)benzofuran-3(2H)-thione
8). Red solid; mp 192–194 °C. IR (KBr):
ꢀ ¼ 1254 (C@S),
598 (C@C). 1H NMR (500 MHz, DMSO-d
): d 7.92 (d, J = 8.0, 1H,
(i.e. AChE/BChE). Then reaction mixture was allowed to stand for
10 min so that enzyme and sample could mix well. After incuba-
tion, the buffer solution (500 L) with concentration of 50 mM
and pH 7.7 was added followed by the addition of 50 L substrate
m
6
l
ArH), 7.80 (m, 1H, ArH), 7.57 (m, 2H, ArH), 7.32 (d, J = 7.5, 2H,
ArH), 7.20 (d, J = 7.5, 2H, ArH), 6.96 (s, 1H, C@C–H), 2.49 (d,
l
(i.e. acetylthiocholine iodide (AChI) and butyrylthiocholine chlo-
0
J = 7.5, 2H, CH
2
), 1.82 (m, 1H, CH), 0.86 (d, J = 7.5 Hz, 6H, CH
3
);
ride (BChCl)) and DTNB [5,5 -dithiobis(2-nitrobenzoic acid)]
1
3
C NMR (125 MHz, DMSO-d ): d 208.12, 167.2, 160.5, 149.5,
6
(50 lL) in it. The resulting solution was incubated for 20 min at
1
1
38.2, 136.0, 129.0, 125.2, 125.0, 124.9, 124.0, 118.0, 116.5,
37 °C. Finally, spectrophotometric absorbances were measured at
400 nm (for AChE) and 412 nm (for BChE) by using UV–Vis spec-
trophotometer and percentage inhibition was calculated by follow-
ing formula:
14.0, 113.1, 44.5, 29.0, 23.0; MS (EI, 70 eV): m/z (%), 294 (65,
+Å
[
M] ), 251 (100), 223 (14), 195 (2), 165 (6), 152 (4), 125 (3); accu-
+Å
rate mass (EI-MS) of [M] : Calcd. for C19H18OS 294.4106; found
2
94.4101.
Inhibition ð%Þ ¼ ðB ꢀ AÞ=B ꢁ 100
2
.1.2.6. (Z)-2-(Benzylidene)benzofuran-3(2H)-thione (9). Orange
Here A = absorbance of the enzyme with test sample and
B = absorbance of the enzyme without test sample. Each experi-
ment was repeated in triplicate and mean values were calculated.
Inhibition was also estimated by calculating the IC50 values. IC50 is
the concentration at which a compound shows 50% inhibition of
the enzyme. To calculate IC50 values, each selected sample (1 mM
1
solid; mp 94–96 °C. IR (KBr):
m
ꢀ ¼ 1226 (C@S), 1608 (C@C). H NMR
): d 7.92 (m, 2H, ArH), 7.80 (m, 2H, ArH), 7.57
m, 2H, ArH), 7.32 (m, 1H, ArH), 7.20 (m, 2H, ArH), 6.96 (s, 1H,
(
500 MHz, DMSO-d
6
(
1
3
C@C–H); C NMR (125 MHz, DMSO-d
6
): d 208.1, 167.2, 160.5,
1
1
2
49.5, 138.2, 135.8, 128.7, 125.2, 125.0, 124.9, 124.5, 118.0, 116.5,
14.0, 113.1; MS (EI, 70 eV): m/z (%), 238 (45, [M] ), 237 (100),
05 (9), 207 (8), 176 (9), 165 (11), 77 (8); accurate mass (EI-MS) of
+Å
in DMSO) was assayed at five concentrations (20
lL, 40 lL, 60 lL,
80 L, 100 L). IC50 values were obtained from dose–effect curves
l
l
+Å
[
M] : Calcd. for C15
H
10OS 238.3043; found 238.3054.
by linear regression.
2
(
(
.1.2.7.
10).
C@S), 1606 (C@C). H NMR (500 MHz, DMSO-d
(Z)-2-(4-Fluorobenzylidene)benzofuran-3(2H)-thione
Orange red solid; mp 76–78 °C. IR (KBr):
ꢀ ¼ 1222
): d 7.97 (d,
2.3. Molecular docking
m
1
Molecular docking analysis was performed in order to investi-
gate the interaction of synthetic compounds with AChE and BChE.
The protein was exported from Protein Data Bank. Molecular struc-
tures of the envisioned compounds were drawn by ACD/ChemS-
ketch and were 3D optimized by using Chem 3D Pro 12.0 and
was saved as SYBYL mol2 file format. Docking of Protein was done
by using AutoDock Tool v1.5.6. In this docking process, hundred
different possess were produced and selected one of the best pose
after visualizing each pose by using Discovery Studio Visualizer
6
J = 7.5 Hz, 2H, ArH), 7.85 (d, J = 7.5 Hz, 2H, ArH), 7.60 (m, 2H,
1
3
ArH), 7.36 (m, 1H, ArH), 7.22 (m, 1H, ArH), 6.97 (s, 1H, CH);
NMR (125 MHz, DMSO-d ): d 208.4, 167.2, 160.5, 150.0, 138.2,
36.0, 128.1, 125.0, 125.2, 125.1, 124.0, 118.3, 117.0, 114.0,
C
6
1
1
+Å
13.1; MS (EI, 70 eV): m/z (%), 256 (100, [M] ), 228 (17), 199
(
32), 170 (11), 123 (14), 95 (17), 76 (10); accurate mass (EI-MS)
+Å
of [M] : Calcd. for C15
.2. Enzyme inhibition assay
Enzyme inhibition studies were based on the method adopted
9
H FOS 256.2948; found 256.2955.
2
3
2
v4.0.
3. Results and discussion
2
1
22
by Ryan and Ellman with slight modifications. 100
sample solution (0.001 M/1 mM in DMSO) was placed into a small
test tube followed by the addition of 50 L corresponding enzyme
lL of each
It is reported that P2S5 could be efficiently used as thiation
agent particularly for the conversion of flavones, chromones and
l
O
O
O
aq. KOH, EtOH
H
R
1
OH
R
2
R
1
OH
R
2
Hg(OAc)
2
/
Pyridine, reflux
S
O
i) Lawesson's reagent
toluene, reflux, 3 h
O
O
R
1
R
or
1
ii) P2S5, NaHCO , THF
3
1
-2 h
3
-Thioaurones (6-10)
3-Oxoaurones (1-5)
R
2
R
2
3
-Oxoaurones 3-Thioaurones
Substituents
Substituents
R
1
R
2
1
6
Br
H
2
3
4
5
7
H
H
H
H
OMe
iBu
H
8
9
10
F
Scheme 1. Synthesis of 3-oxoaurones and 3-thioaurones.

E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
103
Table 1
2 5
A comparison of obtained yields by using P S and LR
Aurone
3-Thioaurone
R
1
R
2
Yield (%) by P
S
2 5
Yield (%) by LR
1
2
3
4
5
6
7
8
9
Br
H
H
H
H
H
73
88
92
90
78
70
79
80
79
72
OCH
3
i
Bu
H
F
10
related structures into their thio analogues.24 Interestingly, when
we applied this reagent in the presence of sodium bicarbonate in
anhydrous tetrahydrofuran (THF) to the varyingly substituted
and unsubstituted aurones, an exciting reaction took place to pro-
duce 3-thioaruones 6–10 from their oxo-analogues. Furthermore,
sulfuration of aurones 1–5 was also achieved by treating with
indicates that the sulfur analogs may not be ignored they may be
useful, if there is an appropriate substituent installed on it. There-
fore, we cannot rule out the sulfur containing analogs as AChE and
BChE inhibitors. In case of sulfur containing compounds the sulfur
atom has also influence to control the activity; however position
and nature of the substituent appeared to be more influential in
the inhibition activities. The difference in activities could be
rationalized on the basis of enzymes nature and substitution and
their position on the 3-oxoaurones or 3-thioaurones molecules.
This could be attributed to enhanced binding interaction between
non-polar molecular parts and hydrophobic pocket of the enzymes.
These findings could further be supported by molecular docking
analysis. In the light of our current study, we are planning to con-
tinue the synthesis of more 3-oxoaurone and-thioaurone mole-
cules along with some chemical modifications on compounds 2,
3, 9, and 10 to explore this class of compound as a potent class
of AChE and BChE inhibitors.
1
3
Lawesson’s reagent in refluxing anhydrous toluene.
reagents worked efficiently in order to furnish 3-thioaurones 6–
0 in moderate to good yields and have no influence on substitu-
Both
1
tion pattern of aurones 1–5 (Scheme 1). However, the former
methodology was found to be relatively cheap, high yielding and
has simple workup. A comparison of yields by both the reagent is
given in Table 1.
Aurones 1–5 were synthesized according to the literature pro-
1
,12b
cedure
2 5 3
and then allowed to react with P S /NaHCO /THF and
LR/toluene to give 3-thioaurones 6–10. The structures of synthetic
compounds were elucidated by IR, mass spectrometry and NMR
spectroscopy. For example, in IR spectrum, the disappearance of
the C@O signal and appearance of C@S new signal around 1200–
3.2. Molecular docking
ꢀ
1
1
250 cm unequivocally confirm the replacement of oxygen by
a sulfur atom. This result was further supported by the C@S signal
Molecular docking analyses of the series of compounds 1–10
were accomplished into the receptor site of the crystal structures
of AChE and BChE in order to elucidate the probable mechanism
by which the title compounds could induce enzyme inhibition
activities and to figure out the ligand–protein interaction at molec-
ular level for establishing structure–activity relationships. The
lowest possible binding energies of the synthetic compounds 1–
10 obtained after docking studies are shown in Table 3.
1
3
observed in their C NMR spectra between 200–208 ppm. The
molecular masses of the compounds were confirmed by Electron
Ionization mass spectrometry (EIMS). Their mass spectra showed
the presence of molecular ion peaks as base peaks and give charac-
1
teristics fragmentation pattern of aurones. All spectral data ( H and
1
3
C and EIMS spectra) are in good agreement with the skeleton of
3
-oxoaurones 1 & 3 and 3-thioaurones 6–10.
Inhibitors were analyzed on the bases of their affinity energy
(Table 3) and type of interactions they made with protein residues.
As more amino acids of active site are hydrophobic in nature so
they show more non-ionic interactions. The visualization of the
most potent compound 2 inside AChE enzyme revealed several
important interactions. In the active pocket of AChE this compound
forms a hydrogen bonding interaction with amino acid residues
3
.1. Enzyme inhibition assays
In vitro, the compounds 1–10 were screened for their enzyme
inhibition potential against acetylcholinesterase and butyryl-
cholinesterase by using spectrophotometric method. The results
of enzyme inhibition activities are presented in Table 2. Their
IC50 values were also calculated and donepezil was used as a pos-
THR83, TYR72, GLN71 and ASN87. This also forms a
action with amino acid ASP74. The catalytic amino acid TYR337
and TRP86 were also observed to form stacking interactions
p-anion inter-
2
1,22
itive control.
It is evident from the results that compounds 2, 3 and 9 showed
inhibition activities against AChE whereas, compounds whereas 3
and 10 against BChE. Their IC50 values are given in Table 2. It is
noteworthy that the (Z)-2-(4-isobutylbenzylidene)benzofuran-3
p–p
with aryl ring of the envisioned compound. The putative binding
mode of this can be found in Figure 1.
Similarly, the visualization of the other most potent compound
3 inside AChE enzyme exhibited enormous important interactions.
In the active pocket of AChE the compound 3 forms hydrogen
bonding interaction with amino acid residues like SER125 and
TYR337. It also forms pi-anion interaction with amino acid
ASP74. The catalytic amino acid GLY121 and GLY122 were also
observed to form amide-pi staking interactions with phenyl ring
of this compound. It has also been seen that the compound 2
makes pi-alkyl interactions with amino acids ALA204, TRP236,
PHE295, PHE297 and HIS447. The putative binding mode of the
compound 3 can be found in Figure 2.
(
l
2H)-one 3 was found to be the dual inhibitor of AChE (IC50 = 0.98 -
M) and BChE (IC50 = 1.02 M) which indicated that this com-
l
pound may be used as inhibitor for both enzymes at the same
time and may cure over expression of two enzymes. It is isobutyl-
benzylidene derivative of aurone and fine tuning by chemical mod-
ification may results in single inhibitor of both enzymes. However,
compound (Z)-2-(4-methoxybenzylidene)benzofuran-3(2H)-one 2
(
IC50 = 1.26 lM) found to have considerable selective activity
against AChE and may serve as lead compound for the develop-
ment of powerful inhibitor for AChE. In general, 3-oxoaurones 1–
5
6
are appeared to be more potent as compared to 3-thioaurones
–10. The replacement of oxygen with sulfur caused decreased in
In analogy to previous discussion, it is manifested from the Fig-
ure 3 that the highly potent compound 3 inside BChE enzyme
exhibited several valuable interactions. For example, in the active
pocket of BChE the compound 3 forms hydrogen bonding interac-
tion with amino acid residues GLY116, GLY117, ALA199 and
the activities such compounds 2, and 3 when transformed 6 and
they completely lost the AChE activity. However, weak activities
of compounds 9 and 10 against AChE and BChE, respectively clearly
7

1
04
E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
Table 2
Structures and IC50 of the synthetic compounds 1–10 against AChE and BChE
Compound No.
Structure
AChE IC50 ± SEMa
(
lM)
BChE IC50 ± SEMa
(lM)
O
1
NAb
NAb
O
Br
O
O
NAb
2
3
1.26
0.98
OMe
O
O
1.02
O
4
5
6
7
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
O
O
O
F
S
O
Br
S
O
OMe
S
O
8
9
NAb
NAb
S
6.39
NAb
NAb
5.27
O
S
O
1
0
F
O
H
3
CO
DonepezilSt
H CO
0.09c
0.13c
3
N
St
Standard inhibitor for AChE and BChE.
a
IC50 values (mean ± standard error of mean).
NA: Not active.
b
c
Standard IC50 values for AChE and BChE.

E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
105
Table 3
amino acid ALA328 and MET437 form interaction with alkyl group.
With all these interactions this inhibitor shows best orientation
with minimum energy of ꢀ8.97 kJ/mol.
Binding energies of the selective poses against human AChE and BChE
Compound hAChE lowest binding energy
hBChE lowest binding energy
DG in KJ mol
ꢀ
1
ꢀ1
D
G in KJ mol
1
2
3
4
5
6
7
8
9
1
ꢀ8.72
ꢀ8.75
ꢀ8.40
ꢀ8.31
ꢀ8.24
ꢀ9.14
ꢀ8.73
ꢀ10.20
ꢀ8.79
ꢀ8.53
ꢀ8.42
ꢀ7.79
ꢀ8.97
ꢀ7.91
ꢀ7.81
ꢀ8.85
ꢀ7.81
ꢀ8.39
ꢀ8.15
ꢀ8.03
4. Conclusion
In summary, we have developed a simple, versatile and rapid
methodology to synthesize 3-thioaurones from 3-oxoaurones
using P S /NaHCO and Lawesson’s reagent. Both reagents offer
2 5 3
high functional groups tolerance. However, the former methodol-
ogy avoids tedious workup, and thus provides a convenient and
an efficient access to 3-thio derivatives of substituted and unsub-
stituted 3-oxoaurones. The synthetic compounds bearing hetero-
cyclic moiety were screened for their inhibitory potential against
AChE and BChE. The results revealed that the compounds 2, 3, 9,
and 10 show weak to good inhibitory potential, in particular, the
aurone 3 was identified as dual inhibitor for both enzymes. Herein,
the molecular docking was used to get insights into the interaction
of synthesized inhibitory compounds with AChE and BChE
0
Standard
ꢀ10.82 (HUW)
ꢀ6.90 (THA)
SER198. This inhibitor also forms
TRP82. –Alkyl interaction has also been observed with amino acid
TRP82, TRP430, TRP440 and LEU286. T-shaped interaction was
observed with TRP82, TRP231, PHE398 and HIS438. The catalytic
p–p interactions with amino acid
p
p–p
Figure 1. Putative binding of compound 2 inside AChE active pocket, hydrogen bonding interactions are shown as green dashed lines and
as solid yellow lines while -anion interaction are shown as orange dashed lines.
p–p staking interaction are shown
p
Figure 2. Most probable binding mode of compound 3 inside AChE enzyme.

1
06
E. U. Mughal et al. / Bioorg. Med. Chem. 25 (2017) 100–106
Figure 3. Most probable binding mode of compound 3 inside BChE enzyme.
enzymes. The studies performed have important implications for
the design of AChE and BChE inhibitors. It could be worth exploring
to make further structural modifications of existing scaffolds in
order to enhance the inhibitory potential of the presented series
of 3-oxoaurone and-thioaurone derivatives.
8. Harkat, H.; Blanc, A.; Weibel, J. M.; Pale, P. J. Org. Chem. 2008, 73, 1620.
Varma, R. S.; Varma, M. Tetrahedron Lett. 1992, 33, 5937.
9.
1
1
0. Boumendjel, A. Curr. Med. Chem. 2003, 10, 2621.
1. Rambabu, D.; Srinivas, S.; Manjulatha, K.; Basavoju, S.; Rao, M. V. B.; Pal, M.
Mol. Cryst. Liq. Cryst. 2013, 577, 83.
1
2. (a) Ameta, K. L.; Rathore, N. S.; Kumar, B.; Malaga, E. S.; Verastegui, M.; Gilman,
R. H.; Verma, B. L. Int. J. Org. Chem. 2012, 2, 295; (b) Sekizaki, H. Bull. Chem. Soc.
Jap. 1988, 61, 1407.
Acknowledgement
13. Mughal, E. U.; Ayaz, M.; Hussain, Z.; Hasan, A.; Sadiq, A.; Riaz, M.; Malik, A.;
Hussain, S.; Choudhary, M. I. Bioorg. Med. Chem. 2006, 14, 4704.
1
4. Altıntop, M. D.; Gurkan-Alp, A. S.; Özkay, Y.; Kaplancıklı, Z. A. Arch. Pharm.
The authors are thankful to Prof. Dr. Norbert Sewald, Depart-
ment of Chemistry, Bielefeld University, Germany for his help in
spectroscopic measurements and carrying out synthetic work in
his laboratory.
Chem. Life Sci. 2013, 346, 571. and references quoted therein.
15. Lee, Y. H.; Shin, M. C.; Yun, Y. D.; Shin, S. Y.; Kim, J. M.; Seo, J. M.; Kim, N. J.; Ryu,
J. H.; Lee, Y. S. Bioorg. Med. Chem. 2015, 23, 231.
6. Nam, S. O.; Park, D. H.; Lee, Y. H.; Ryu, J. H.; Lee, Y. S. Bioorg. Med. Chem. 2014,
1
2
2, 1262.
1
1
7. Zhou, X.; Li, M.; Wang, X. B.; Wang, T.; Kong, L. Y. Molecules 2010, 15, 8593.
8. Baharloo, F.; Moslemin, M. H.; Nadri, H.; Asadipour, A.; Mahdavi, M.; Emami, S.;
Firoozpour, L.; Mohebat, R.; Shafiee, A.; Foroumadi, A. Eur. J. Med. Chem. 2015,
References and notes
9
3, 196.
1
2
.
.
Haudecoeur, R.; Boumendjel, A. Curr. Med. Chem. 2012, 19, 2861.
Morimoto, M.; Fukumoto, H.; Nozoe, T.; Hagiwara, A.; Komai, K. J. Agric. Food.
Chem. 2007, 55, 700.
Venkateswarlu, S.; Panchagnula, G. K.; Subbaraju, G. V. Biosci. Biotechnol.
Biochem. 2004, 68, 2183.
Hadjeri, M.; Barbier, M.; Ronot, X.; Mariotte, A. M.; Boumendjel, A.; Boutonnat,
J. J. Med. Chem. 2003, 46, 2125.
Schoepfer, J.; Fretz, H.; Chaudhuri, B.; Müller, L.; Seeber, E.; Meijer, L.; Lozach,
O.; Vangrevelinghe, E.; Furet, P. J. Med. Chem. 2002, 45, 741.
Cheng, H.; Zhang, L.; Liu, Y.; Chen, S.; Cheng, H.; Lu, X.; Zheng, Z.; Zhou, G. C.
Eur. J. Med. Chem. 2010, 45, 5950.
1
9. Sheng, R.; Xu, Y.; Hu, C.; Zhang, J.; Lin, X.; Li, J.; Yang, B.; He, Q.; Hu, Y. Eur. J.
Med. Chem. 2009, 44, 7.
0. Chen, H.; Dong Qi, X.; Qiu, P. Bangladesh J. Pharmacol. 2014, 9, 501.
1. Ryan, W.; Kenneth, R.; Earl, M.; Sultan, D. Biochim. Biophys. Acta 2011, 1810,
2
2
3
4
5
6
7
.
.
.
.
.
1
230.
2
2
2
2. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
3. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
4. Mughal, E. U.; Rasheed, L.; Hasan, A. Heterocycl. Commun. 2005, 11, 445. and
references quoted therein.
Kim, D.; Li, Y.; Horenstein, B. A.; Nskanishi, K. Tetrahedron Lett. 1990, 31, 7119.