Synthesis, molecular docking and cholinesterase inhibitory activity of hydroxylated 2-phenylbenzofuran derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.11.043

We have designed, synthesized and evaluated a series of hydroxylated 2-phenylbenzofuran derivatives as potential cholinesterase inhibitors. Starting from a series of 2-phenylbenzofurans previously published, in this paper we present a complete synthesis and the influence on the activity of one or two hydroxyl groups located in meta or in meta and para positions respectively of the 2-phenyl ring and highlight the importance of position of hydroxyl groups. Moreover, simultaneous introduction of halogen at position 7 of the benzofuran scaffold resulted in an improved inhibitory activity against the enzyme. To further provide molecular insight and to identify the most probable ligand-binding site of the protein, docking studies were performed for the top-ranked compounds. Docking results revealed conserved ligand-binding residues and supported the role of catalytic site residues in enzyme inhibition.

Full text of DOI:10.1016/j.bioorg.2018.11.043

Accepted Manuscript
Synthesis, molecular docking and cholinesterase inhibitory activity of hydroxy-
lated 2-phenylbenzofuran derivatives
Antonella Fais, Amit Kumar, Rosaria Medda, Francesca Pintus, Francesco
Delogu, Maria J. Matos, Benedetta Era, Giovanna L. Delogu
PII:
S0045-2068(18)31193-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.043
YBIOO 2651
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2018
19 November 2018
24 November 2018
Please cite this article as: A. Fais, A. Kumar, R. Medda, F. Pintus, F. Delogu, M.J. Matos, B. Era, G.L. Delogu,
Synthesis, molecular docking and cholinesterase inhibitory activity of hydroxylated 2-phenylbenzofuran
derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.043
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, molecular docking and cholinesterase inhibitory activity of
hydroxylated 2-phenylbenzofuran derivatives
Antonella Faisa,#, Amit Kumarb,#, Rosaria Meddaa, Francesca Pintusa, Francesco Delogub,
Maria J. Matosc, Benedetta Eraa,§, Giovanna L. Delogua,§,*
a Department of Life and Environmental Sciences, University of Cagliari, 09042, Monserrato, Cagliari, Italy
b
Department of Mechanical, Chemical and Materials Engineering, University of Cagliari, via Marengo 2,
09123 Cagliari, Italy
c Department of Organic Chemistry, University of Santiago de Compostela, Santiago de Compostela, Spain
*Corresponding author Ph: +390706758566; e-mail: delogug@unica.it (G.L.Delogu)
#These authors should be considered joint first authors
§These authors should be considered joint last authors
ABSTRACT
We have designed, synthesized and evaluated a series of hydroxylated 2-
phenylbenzofuran derivatives as potential cholinesterase inhibitors. Starting from a series
of 2-phenylbenzofurans previously published, in this paper we present a complete
synthesis and the influence on the activity of one or two hydroxyl groups located in meta or
in meta and para positions respectively of the 2-phenyl ring and highlight the importance of
position of hydroxyl groups. Moreover, simultaneous introduction of halogen at position 7
of the benzofuran scaffold resulted in an improved inhibitory activity against the enzyme.
To further provide molecular insight and to identify the most probable ligand-binding site of
the protein, docking studies were performed for the top-ranked compounds. Docking
results revealed conserved ligand-binding residues and supported the role of catalytic site
residues in enzyme inhibition.
Keywords: 2-Phenylbenzofurans, Cholinesterase Inhibitors, Docking studies
1. Introduction
Benzofuran derivatives are an important class of organic compounds that occur in
natural products because of their pharmacological activities, including antitumoral
properties. They can be used as anti-inflammatory, antimicrobial, antagonists of the
angiotensin II receptor, analgesic, ligands of adenosine A1 receptor and so forth [1].

Phenylbenzofurans are a very important molecule skeleton due to their synthetic
versatility and their proved pharmacological properties [2]. They are synthetic compounds
in which an additional phenyl ring is present in any position of the benzofuran nucleus.
This could be easily obtained possible by two principal general methods: by a C-
phenylation of a benzofuran [3] or by the construction of the benzofuran nucleus with the
new ring already included on it [4]. In the present work we describe the second method, by
a classical Wittig reaction [5,6], perhaps the most direct and general method known for
preparing the desired 2-phenylbenzofurans.
Recently, benzofuran derivatives have showed enzymatic inhibition properties for
example on monoamine oxidase [7], acetylcholinesterase (AChE) [8-10] and
butyrylcholinesterase (BChE) [11, 12].
Acetylcholine (ACh) is a neurotransmitter that plays a role in the modulation of memory
function in normal and neurodegenerative conditions [13]. In the cholinergic system,
disruption in the levels of ACh is caused by hydrolytic action of cholinesterases (ChEs), a
family of enzymes that play a role in ACh regulation and in the cholinergic signaling [14].
AChE and BChE appear to be simultaneously active in the synaptic hydrolysis of ACh,
terminating its neurotransmitter action, and co-regulating levels of ACh. A well-
documented strategy to restore the neurotransmitter level involves the use of
cholinesterase inhibitors that suppress the ChEs enzymes and therefore increasing both
the level and duration of the neurotransmitter action [15-17].
In our efforts to contribute to the development of novel compounds that may be useful in
the treatment of neurodegenerative disorders such as Parkinson’s disease (PD) or
Alzheimer’s disease (AD), we are focusing on 2-phenylbenzofuran derivatives
[7,11,12,18,19].
In particular, with the aim of finding out structural features in the ChEs inhibitory activity,
in the present work, we describe the synthesis of hydroxylated 2-phenylbenzofuran
derivatives 7-bromine and 7-chlorine substituted and the importance of hydroxyl groups
substitution in the 2-phenyl ring. We recently developed a series of 2-phenylbenzofurans
which exhibited selective inhibitory property for BChE enzyme. Considering that the 7-
chlorine-2-(3,5-dihydroxyethoxyphenyl)benzofuran
and
7-bromine-2-(3,5-
dihydroxyethoxyphenyl)benzofuran displayed highest inhibitory activity towards BChE with
IC50 values of 6.23 μM and 3.57 μM respectively [11,12], in this paper we studied the
influence on the activity of one or two hydroxyl groups located in meta or in meta and para
positions respectively of the 2-phenyl ring in addition to the presence of a halogen at

positions 7 of the benzofuran scaffold, which has been revealed as adequate position for
substitution.
2. Experimental
2.1 Chemistry
2.1.1 General information
Starting materials and reagents were obtained from commercial suppliers (Sigma-
Aldrich) and were used without further purification. Melting points (mp) are uncorrected
and were determined with a Reichert Kofler thermopan or in capillary tubes in a Büchi 510
1
apparatus. H NMR and 13C NMR spectra were recorded with a Varian INOVA 500
spectrometer using CDCl3 as solvent. Chemical shifts (δ) are expressed in parts per
million (ppm) using TMS as an internal standard. Coupling constants J are expressed in
hertz (Hz). Spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets),
and m (multiplet). Elemental analyses were performed by using a Perkin Elmer 240B
microanalyzer and are within 0.4% of calculated values in all cases. The analytical results
indicate 98% purity for all compounds. Flash chromatography (FC) was performed on
silica gel (Merck 60, 230-400 mesh); analytical thin-layer chromatography (TLC) was
performed on pre-coated silica gel plates (Merck 60 F254). Organic solutions were dried
over anhydrous Na2SO4. Concentration and evaporation of the solvent after reaction or
extraction was carried out on a rotary evaporator (Büchi Rotavapor) operating under
reduced pressure.
2.1.2. General procedure for the preparation of 2-phenylbenzofuran (1-4).
A mixture of 2-hydroxybenzyltriphenylphosphonium bromide (0.50 g, 1.11 mmol) and
benzoyl chloride (0.12 mL, 1.11 mmol) in a mixed solvent (toluene 20 mL and Et3N 0.5
mL) was stirred under reflux for 2 h. The precipitate was removed by filtration. The filtrate
was concentrated, and the residue was purified by silica gel chromatography
(hexane/EtOAc 9:1) to give the desired compounds 1-4.
2.1.2.1. 7-chloro-2-(3-methoxyphenyl) benzofuran (1). It was obtained with a yield of 56%.
1
m.p. 43-45 °C. H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 3.91 (s, 3H, OCH3), 6.98-
6.93 (m, 1H, H-4'), 7.01 (d, J = 1.2, 1H, H-3), 7.17 (td, J = 7.5, 3.5, 1H, H-5), 7.34-7.30 (m,

13
1H, H-6'), 7.41-7.36 (m, 1H, H-6), 7.53-7.44 (m, 3H, H-4, H-2' and H-5'); C NMR (125
MHz, CDCl3), δ (ppm), J (Hz) = 55.46, 102.11, 110.65, 114.78, 116.73, 117.81, 119.50,
123.91, 124.53, 130.00, 130.88, 150.68, 131.20, 156.70, 160.04. Anal. calcd. for
C15H11ClO2: C, 69.64%; H, 4.29%. Found: C, 69.69%; H, 4.31%.
2.1.2.2. 7-bromine-2-(3-methoxyphenyl) benzofuran (2). It was obtained with a yield of
1
81%. m.p. 45-47 °C. H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 3.91 (s, 3H, OCH3),
6.97 (d, J = 8.1, 1H, H-4’), 7.23 (s, 1H, H-3), 7.30 (d, J = 7.8, 1H, H-6'), 7.52 (t, J = 7.9, 1H,
H-5’), 7.64 (s, 1H, H-2’), 7.45 (t, J = 7.7, 1H, H-5), 7.72 (d, J = 7.8, 1H, H-6), 7.83 (m, 1H,
H-4); 13C NMR (125 MHz, CDCl3), δ (ppm) = 56.10, 100.92, 103.94, 108.46, 111.73,
118.76, 119.94, 123.12, 124.32, 127.20, 130.93, 149.83, 151.22, 152.96, 157.83;. Anal.
calcd. for C15H11BrO2: C,59.43%; H, 3.66%. Found: C, 59.50%; H, 3.72%.
2.1.2.3. 7-chlorine-2-(3,4-dimetoxyphenyl) benzofuran (3). It was obtained with a yield of
51%. m.p. 117-120 °C; 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 4.30 (s, 3H, OCH3),
4.37 (s, 3H, OCH3), 7.26 (s, 1H, H-3), 7.28 (t, J = 6.2, 1H, H-5), 7.52 (d, J = 7.8, 1H, H-5'),
13
7.63 (d, J = 7.8, 1H, H-6'), 7.74 (d, J = 1.1, 1H, H-6), 7.85-7.78 (m, 2H, H-4 e H-2'); C
NMR (125 MHz, CDCl3), δ (ppm) = 55.97, 56.05, 100.41, 108.22, 111.37, 116.42, 118.32,
119.09, 122.85, 123.78, 123.97, 131.12, 149.25, 149.98, 150.42, 156.88; Anal. calcd. for
C16H13ClO3: C, 66.56%; H, 4.54%. Found: C, 66.61%; H, 4.57%
2.1.2.4. 7-bromine-2-(3,4-dimethoxyphenyl) benzofuran (4). It was obtained with a yield of
79%; m.p. 139-141 °C; 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 4.27 (s, 3H, OCH3),
4.32 (s, 3H, OCH3), 7.27 (d, J = 7.2, 2H, H-5' e H-6'), 7.41 (t, J = 7.7, 1H, H-5), 7.70 (s, 1H,
H-2'), 7.73 (d, J = 7.8, 1H, H-6), 7.83-7.78 (m, 2H, H-3 e H-4); 13C NMR (125 MHz, CDCl3),
δ (ppm) = 56.13, 56.19, 100.65, 103.88, 108.41, 111.52, 118.48, 119.81, 123.00, 124.28,
126.94, 130.87, 149.38, 150.12, 151.91, 156.88; Anal. calcd. for C16H13BrO3: C, 57.68%;
H, 3.93%. Found: C, 57.73%; H, 3.95%
2.1.3 General procedure for the preparation of hydroxylated 2-phenylbenzofurans (5-8)
A solution of the corresponding methoxy-2-phenylbenzofuran (0.11 g, 0.50 mmol) in
acetic acid (5.0 mL) and acetic anhydride (5.0 mL), at 0 °C, was prepared. Hydriodic acid
57% (10.0 mL) was added drop-wise. The mixture was stirred under reflux temperature for

3 h. The solvent was evaporated under vacuum and the dry residue was purified by FC
(dichloromethane/methanol 9.8:0.2) to give the desired compound 5-8 [20].
2.1.3.1. 7-chlorine-2-(3-hydroxyphenyl) benzofuran (5). It was obtained with a yield of
94%. m.p. 110-112 °C. 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 6.88-6.81 (m, 1H, H-
4'), 7.27 (t, J = 7.8, 1H, H-5), 7.43-7.30 (m, 4H, H-6, H-2', H-5' e H-6'), 7.45 (s, 1H, H-3),
13
7.62 (d, J = 7.7, 1H, H-4), 9.75 (s, 1H, OH); C NMR (125 MHz CDCl3), δ (ppm): 102.52,
109.50, 111.32, 115.16, 115.82, 116.48, 120.16, 124.33, 124.45, 130.24, 130.68, 149.39,
156.22, 157.86; Anal. calcd. for C14H9ClO2: C, 68.72%; H, 3.71%. Found: C, 68.75%; H,
3.75%.
2.1.3.2. 7-bromine-2-(3-hydroxyphenyl) benzofuran (6). It was obtained with a yield of
93%. m.p. 92-95 °C. 1H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 5.40 (s, 1H, OH), 7.13
(d, J = 8.1, 1H, H-4'), 7.26 (s, 1H, H-3), 7.35 (t, J = 7.8, 1H, H-6'), 7.57 (t, J = 7.9, 1H, H-
5'), 7.65 (s, 1H, H-2'), 7.76-7.67 (m, 3H, H-4, H-5 and H-6); 13C NMR (125 MHz CDCl3), δ
(ppm): 102.39, 104.06, 112.06, 116.22, 117.98, 120.20, 124.37, 127.50, 130.32, 130.50,
131.46, 152.05, 156.01, 156.29; Anal. calcd. for C14H9BrO2: C, 61.78%; H, 3.58%. Found:
C, 61.80%; H, 3.62%.
2.1.3.3. 7-chlorine-2-(3,4-dihydroxyphenyl) benzofuran (7). It was obtained with a yield of
1
83%; m.p. 74-76 °C; H NMR (500 MHz, CDCl3), δ (ppm), J (Hz) = 4.57-5.58 (m, 2H, 2 x
OH). 7.26 (s, 1H, H-3), 7.38 (d, 1H, J = 8.3 Hz, H-5'), 7.53-7.58 (m, 1H, H-2'), 7.66 (d, 1H,
J = 8.3, H-6'), 7.77 (t, 1H, J = 11.4, 5.7 Hz, H-5), 7.83 (m, 2H, H-4 and H-6), 13C NMR (125
MHz, DMSO), δ (ppm ) = 100.54, 112.52, 115.97, 116.52, 118.81, 119.23, 123.40, 123.86,
124.08, 131.15, 143.82, 144.82, 150.47, 156.68; Anal. calcd. for C14H9ClO3: C, 64.51%; H,
3.48%. Found: C, 64.54%; H, 3.52%.
2.1.3.4. 7-bromine-2-(3,4-dihydroxyethoxyphenyl) benzofuran (8). It was obtained with a
yield of 87%; m.p. 74-76 °C; 1H NMR (500 MHz, DMSO), δ (ppm) = 3.12-4.06 (m, 2H, 2 x
OH), 7.33 (d, J = 8.3, 1H, H-5'), 7.45 (d, J = 7.8, 1H, H-6'), 7.64 (s, 1H, H-3), 7.71-7.82 (m,
3H, H-4, H-5 and H-2'), 7.83 (d, J = 7.7, 1H, H-6); 13C NMR (125 MHz, DMSO), δ (ppm ) =
103.1, 106.9(2C), 108.8, 117.4, 119.8, 124.5, 124.8, 125.3, 139.6, 155.1, 156.1,
159.3(2C); Anal. calcd. for C14H9BrO3: C, 64.51%; H, 3.48%. Found: C, 64.54; H, 3.52%.

2.2. Determination of cholinesterase inhibition
The cholinesterase inhibition activity was performed using Ellman’s method and
analyzed as previously described [21] with slight modifications [11].
Acetylthiocholine iodide (AChI)/S-butyrylthiocholine iodide (BChI), and 5,5’-dithiobis-(2-
nitrobenzoic) acid (DTNB) (Sigma-Aldrich) were used for the determination of the
AChE/BChE activities. Acetylcholinesterase was from Electrophorus electricus (AChE),
while butyrylcholinesterase was from equine serum (BChE) (Sigma-Aldrich).
Total volume of the reaction mixture was 200 μL. It contained phosphate buffer (0.1 M,
pH 8.0), enzyme solution, AChE (0.3 U/ml) or BChE (0.15 U/ml, DTNB (1.5 mM final
concentration), and inhibitor, at different concentration, dissolved in 1% DMSO or DMSO
alone (control). Finally, ATCI or BTCI (1.5 mM final concentration) as the substrate was
added to the reaction mixture and the absorbance immediately monitored at 405 nm, with
a plate reader FLUOstar OPTIMA (BMG Labtech, Offenburg, Germany). The activity of the
enzymes was performed at 25 °C. Galantamine and tacrine were used as standard
inhibitors. IC50 values were obtained from activity (slope) versus compound concentration
plots. IC50 values displayed represent the mean ± standard deviation for three independent
assays.
2.3. Molecular modeling studies
Three-dimensional (3D) protein structure of human BChE (PDB id: 4TPK) was
accessed from protein data bank, and for the compounds the 3D-coordinates were
generated using Open Babel software. Molecular docking of the compounds into hBChE
protein was performed employing EADock DSS docking software incorporated in the
SwissDock web server [22]. The docking procedure considers the entire protein surface as
a potential target. Utilizing this procedure, a large number of ligand binding modes
(~15000) were generated, with the simultaneous rough interaction energy estimation. The
binding modes with favourable energies were then ranked and classified into different
clusters, based on the full fitness scoring function. The most probable and consistent
conformation was selected from 10 independent docking runs for each compound. The
accuracy and prediction of correct binding mode depends on the number of free dihedral
angles of the compound (only one in our case), with a success rate of 93% while
considering the top five binding modes.

3. Result and discussion
3.1. Chemistry
Benzofuran derivatives 1-8 were efficiently synthesized by a Wittig reaction, according
to the protocol outlined in Figure 1. The desired Wittig reagents were readily prepared from
the conveniently substituted ortho-hydroxybenzyl alcohols a-b [12,23,24] and PPh3·HBr.
(Figure 1, Scheme 1a). The key step for the formation of the benzofuran moiety was
achieved by an intramolecular reaction between the ortho-hydroxybenzyltriphosphonium
salt c-d [12] and the appropriate benzoyl chloride [5,6]. Hydrolysis of the methoxy groups
of compounds 1-4 was performed by treatment with hydrogen iodide in acetic acid/acetic
anhydride, to give the corresponding hydroxy derivatives 5-8 (Fig. 1, Scheme 2b) [20].
1
The benzofuran structures were confirmed by H NMR, 13C NMR, and elemental
analyses.
CHO
OH
PPh3 HBr/CH3CN
82 °C, 2 h.
NaBH4/EtOH
0 °C to rt, 2 h
P+Ph3Br-
OH
a
OH
OH
R
R
R
a, c: R = Cl
b, d: R = Br
c-d
a-b
O
Cl
OMe
OMe
OMe
OH
toluene/NEt3
HI/AcOH/Ac2O
0°C to reflux, 4h
b
110 °C, 2 h;
O
O
O
O
R
R
R
1-2
5-6
P+Ph3Br-
OH
R
OH
OH
toluene/NEt3
110 °C, 2 h;
c-d
HI/AcOH/Ac2O
OMe
0°C to reflux, 4h
O
Cl
R
7-8
3-4
5,7: R = Cl
6,8: R = Br
1,3: R = Cl
2,4: R = Br
OMe
OMe
Fig. 1. Protocol for synthesis of compounds 1-8. (a) scheme 1 (b) scheme 2.
3.2. Biological activity

The potential effects of the synthesized compounds 5-8 on AChE and BChE was
performed on animal enzymes AChE and BChE, due to their lower cost and high degree of
similarity with their respective human enzymes. Table 1 shows the inhibitory effect of the
newly synthesized compounds 5-8 compared with those previously published by us 9-14
[11,12].
AChE
BChE
Reference
Compounds
IC50 (µM)
OH
OH
OH
5
28.21±1.26
23.96±2.77
56.27±2.07
>100
13.42±2.57
7.96±0.28
15.54±2.62
10.86±0.35
O
Cl
6
O
Br
OH
7
O
Cl
OH
OH
8
O
Br
OH
OH
9
[12]
[12]
>100
>100
30.3±1.90
82.5±7.1
O
Cl
10
O
Br
OH
[11]
[11]
[11]
[11]
11
>100
6.23±0.43
3.57±0.25
25.7±1.60
18.41±0.93
O
OH
OH
Cl
12
100±6.10
O
OH
OH
Br
OH
13
50±3.30
37±2.6
O
OH
OH
Cl
OH
OH
14
O
Br

Galantamine
Tacrine
3.12±0.15
26.06±6.19
0.024±0.024
0.42±0.032
Table 1. Inhibition of AChE and BChE enzymes by compounds 5-14. ChE inhibition is expressed as the
mean ± SD (n = 3 experiments).
The experimental results showed that most of the compounds tested are selective
BChE inhibitors with a varying efficiency. The IC50 values of BChE inhibition are similar to
those showed by the most potent compounds previously described by us. As a rule, 2-
phenylbenzofurans substituted at the 7-position were found to be more active molecules
as BChE inhibitors than the corresponding 5-substituted derivatives or those derivatives
unsubstituted in the benzene ring [12].
The compounds 6 and 8 bromine-substituted resulted somewhat better active respect to
compounds chlorine-substituted 5 and 7 (Table 1).
Considering that 2-phenylbenzofurans with a bromine atom at position 5 in the
benzofuran ring were inactive, the obtained results point out that the location of this
substituent is at least as important as its nature for the activity [11,12].
In addition, in comparison to the previously obtained data, while the hydroxyl substituent
in the para position on the 2-aryl (compounds 9 and 10, IC50 value 30.3 and 82.5 and µM
respectively) leads to much worse results, when that substituent moved in meta position
(compounds 5 and 6, IC50 value 13.42 and 7.96 µM respectively), the activity improve
considerably [12].
Among all the benzofuran derivatives analyzed, maximum inhibitory activity against
eqBChE enzyme were displayed by compounds 11 (IC50 = 6.23 μM) and 12 (IC50 = 3.57
μM), with two hydroxyl substituents in meta position on the phenyl-ring and with presence
of chlorine and bromine atoms respectively at position 7 of benzofuran scaffold. EqBChE
inhibitory activity displayed by these compounds was about 4- and 8- times higher than the
reference compound, galantamine (IC50 = 26.06 μM).
The 2-phenylbenzofuran derivatives 7 and 8 with two hydroxyl substituents in meta and
para positions of the 2-phenyl ring displayed lower inhibitory activity toward eqBChE (IC50
= 15.54 μM and IC50 = 10.86 μM), compared to isomers 11 and 12 with two hydroxyl
groups in meta positions.
In the compounds with three hydroxyl substituents in the 2-phenyl ring (compounds 13
and 14), we found lower inhibitory activity against eqBChE (IC50 = 25.70 and 18.41 μM)

[11]. This fact suggests that little differences in the positions and number of hydroxyl
groups in the 2-phenyl ring of the synthesized compounds could decrease or increase the
inhibitory activity of the compounds against eqBChE. It has been shown previously that the
position and number of hydroxyl group in the ligand can influence the magnitude of
hydrogen bond interactions with the protein [25].
3.3. Molecular and physicochemical properties
Based on the experimental results, we performed docking calculations for the molecules
5-8 against protein hBChE.
In Table 2, we summarize the molecular and physicochemical properties of compounds
that were calculated using SwissADME web tool [26] and compare them with reference
drug molecules such as galantamine and tacrine.
Properties such as molecular weight (MW), volume, number of rotatable bonds [27],
number of hydrogen bond (H-bond) acceptor and donor atoms, and polar surface [28] area
(PSA), lipophilicity [29], water solubility [30] and drug likeliness [31,32] of the compounds
are essential parameters that should be considered in drug development.
5/9
6/10
7/11
8/12
13
14
Galantamine Tacrine
MW (g/mol)
244.67
289.12
260.67
305.12
276.67
321.12
287.35
198.26
Volume (Å3)
202.56
1
206.91
1
210.58
1
214.93
1
218.60
1
222.95
1
268.19
1
191.53
0
Rotatable bonds
H-bond acceptor
atoms
2
1
2
1
3
2
3
2
4
3
4
3
4
1
1
1
H-bond donor
atoms
Polar surface
area (Å2)
33.37
3.65
33.37
3.73
53.60
3.24
53.60
3.34
73.83
2.93
73.83
2.91
41.93
38.91
Lipophilicity
(logP)
1.91
Soluble
Yes
2.59
Soluble
Yes
Water Solubility
Moderate Moderate Moderate Moderate Moderate Moderate
Yes Yes Yes Yes Yes Yes
Drug likeliness
(Lipsinky)
Table 2. Molecular properties of compounds under investigation

The molecules investigated differ not only in the number of hydroxyl groups but also in
their position in the phenyl ring and include a contemporary presence of either chlorine (5,
7, 9, 11, 13) or bromine (6, 8, 10, 12, 14) at position 7 of the benzofuran scaffold.
Molecules with identical halogen substitution of benzofuran moiety, number of hydroxyl
group in the phenyl ring, but different in their position displayed same physicochemical
properties. For example, molecules 5 and 9 differ only in the substitution position of
hydroxyl group. All the molecules have only one rotatable bond each, thus displaying
similar flexibility. With increasing the number of hydroxyl groups in the phenyl ring, the
molecular weight, volume, number of hydrogen bond (H-bond) acceptor/donor atoms and
polar surface area values also increased. While, an opposite trend was found for
lipophilicity that provides measure the hydrophobic nature of the compound. Furthermore,
all 2-phenylbenzofuran derivatives investigated here displayed moderate water solubility
characteristics, and better lipophilicity with respect to the reference drug molecules.
Overall, based on the physiochemical and structure features investigated, all the
molecules displayed druglikeness characteristics.
Fig. 2. A BOILED-Egg graphical output using polar surface area (PSA) vs lipophilicity (LogP) for the
molecules investigated in this study. The region in yellow (yolk) represents high probability of brain
penetration, while white represents the physicochemical space of gastrointestinal absorption. Blue dots
represent molecule as substrate and red dots as non-substrates of the permeability glycoprotein.
Novel Brain Or IntestinaLEstimateD permeation (BOILED-Egg) method was employed
to predict two crucial pharmacokinetic parameters namely human gastrointestinal
absorption (HIA) and blood brain barrier (BBB) simultaneously, by utilizing two

physicochemical descriptors (lipophilicity, polarity) of the molecule. It is encouraging to
note that all the molecules investigated are classified in the same physiochemical space of
tacrine and galantamine drugs, which indicate high probability of the molecules being
absorbed in the brain (Fig. 2).
The molecules considered here displayed better lipophilicity and molecular flexibility
with respect to galantamine and tacrine. Moreover, molecules with two or more hydroxyl
groups displayed higher polar surface area and number of H-bond donor and acceptor
atoms. Indeed, the relevance of molecular flexibility and polar surface area values of the
molecules with oral bioavailability in rats has been tested for 434 pharmacia compounds
[33]. All the molecules displayed druglikeness characteristic, a parameter that qualitatively
assesses the possibility for a compound to become an oral drug. Interestingly, we found
that all the molecules possess likelihood to become an oral drug.
Based on the experimental results and to further identify the most probable ligand-
binding site of the protein hBChE, we performed docking calculations for the molecules 5-
8. Docking results marked all the molecules to bind at the same protein site (Fig. 3), and
displaying similar binding energy value ranging between 7.4 to 7.8 kcal/mol.
Fig. 3. Docking of molecules to hBChE protein. The most favourable docked position of molecules 5-8 in
binding site of hBChE protein. The active site residues and molecules are shown in licorice and the protein in
cartoon representation. On the right panel, zoomed view of the interaction region is shown.

To explain the origin of the similarity in the binding energy values, we carefully probed
the interaction site of the molecules in complex with hBChE protein, using Ligplot [34]
software.
Fig. 4. Molecular interaction picture of hBChE protein complexes with molecule: (A) 5, (B) 6, (C) 7 and (D) 8.
The binding site of hBChE protein is characterized by an acyl pocket, located at the
bottom of a deep catalytic gorge; the oxyanion hole; the peripheral site, located at the lip of
the gorge; and the choline-binding site also located within the gorge. In all the four ligand-
protein complexes, we found involvement of catalytic triad residues (Ser198, His438), H-
bond interactions with residue Glu197, acyl pocket residues (Leu286, Val288) and
oxyanion hole residue (Gly117). Choline binding-site residue Trp82 was involved in
interactions with molecules 5, 7 and 8 (Fig. 4).

The docking binding energy values for these four complexes are quite similar,
consistent with the not so different IC50 values calculated in our experiments. In
accordance with our previously published results [11,12], the 2-pheynlbenzofuran
derivatives with hydroxyl group/s in the phenyl ring exhibited rich interaction network
involving the catalytic triad residues, which we propose to be an essential component for a
molecule to display inhibitory activity against BChE enzyme. Different substitution position
of hydroxyl group in the 2-phenyl group resulted in different interaction patterns, but
remained consistent with participation of key residues to binding.
4. Conclusion
In this study, we have used the Wittig reaction as a key step for the efficient and general
synthesis of a series of hydroxylated 2-phenylbenzofuran derivatives. All the compounds
have been evaluated for their cholinesterase inhibitory activity. The experimental results
showed that most of the benzofurans tested are selective BChE inhibitors with a varying
efficiency. The IC50 values of BChE inhibition indicated compounds 6 and 8 with the
presence of bromine at position 7 of the benzofuran scaffold as the most potent inhibitors
with an IC50 value of 7.96 and 10.86 µM respectively. However, a thorough analysis of the
results obtained, revealed that the maximum inhibitory activity was displayed by
benzofurans derivatives with two hydroxyl substituents in meta position on the phenyl-ring
and chlorine and bromine atoms respectively at position 7 of benzofuran scaffold
(compounds 11 and 12). Thus, our study provides a complete picture of the importance of
number and position of the hydroxyl groups in the 2-phenyl ring, and among the
compounds tested, two hydroxyl substituents in meta position to be the most functional for
the
cholinesterase
inhibition.
Moreover,
comparative
physicochemical
and
pharmacokinetic properties of the compounds with the commonly prescribed drugs
revealed high probability of all the compounds to be absorbed in the brain and exhibiting
promising druglikeness characteristics. Finally, molecular docking simulations assisted in
explaining the structure-activity relationships of this type of compounds.
Conflict of interest
The authors declare no conflict of interest.

Acknowledgements
This work was partially supported by University of Cagliari
References
[1] Y. Jiang, B. Gao, W. Huang, Y. Liang, G. Huang, Y. Ma, Simple, Convenient, and
Efficient Synthesis of 2-Arylsubstituted Benzo[b]furans, Synthetic Commun., 2009, 39,
197-204. doi:10.1080/00397910701860323.
[2] G. Khodarahmi, P. Asadi, F. Hassanzadeh, E. Khodarahmim, Benzofuran as a
promising scaffold for the synthesis of antimicrobial and antibreast cancer agents: A
review, Journal of Research in Medical Sciences, 2015, 20, 1094-1104.
doi:10.4103/1735-1995.172835.
[3] J. Cao, Z-L Chen, S-M Li, G-F Zhu, Y-Y Yang, C. Wang, W-Z Chen, J-T Wang, J-Q
Zhang and L. Tang, Palladium-Catalyzed Regioselective C-2 Arylation of Benzofurans
with N′-Acyl Arylhydrazines, Eur. J. Org. Chem., 2018, 2774–2779.
doi:10.1002/ejoc.201800374.
[4] A.A. Abu-Hashem, H.A.R. Hussein, A.S. Aly, and M.A. Gouda, Synthesis of benzofuran
derivatives via different methods, Synthetic Communications, 2014, 44, 2285-2312.
doi:10.1080/00397911.2014.894528.
[5] A. Hercouet and M. Le Corre, Une nouvelle voie d'accds aux benzofuranne,
Tetrahedron Lett., 1979, 23, 2145-2148.
[6] L.J. Twyman, D. Allsop, A short synthesis of the β-amyloid (Aβ) aggregation inhibitor 3-
p-toluoyl-2-[4′-(3-diethylaminopropoxy)-phenyl]-benzofuran., Tetrahedron Lett., 1999,
40, 52, 9383-9384.
[7] G.L. Delogu, F. Pintus, L. Mayán, M.J. Matos, S. Vilar, J. Munín, A. Fontenla, G.
Hripcsak, F. Borges and D. Viña, MAO inhibitory activity of bromo-2-
phenylbenzofurans: synthesis, in vitro study, and docking calculations, Med. Chem.
Commun., 2017, 8, 1788-1796. doi:10.1039/C7MD00311K.
[8] X. Zha, D. Lamba, L. Zhang, Y. Lou, C. Xu, D. Kang, L. Chen, Y. Xu, L. Zhang, A. De
Simone, S. Samez, A. Pesaresi, J. Stojan, M.G. Lopez, J. Egea, V. Andrisano, and M.
Bartolini, Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for
the Treatment of Alzheimer’s Disease: Design, Synthesis, Biological Evaluation, and
X-ray
Crystallography.
J.
Med.
Chem.,
2016,
59,
114-131.
doi:10.1021/acs.jmedchem.5b01119.
[9] W-Y Wu, Y-C Dai, N-G Li, Z-X Dong, T. Gu, Z-H Shi, X. Xue, Y-P Tanga and J-A Duan,
Novel multitarget-directed tacrine derivatives as potential candidates for the treatment
of Alzheimer’s disease.J. Enzyme Inhib. Med. Chem., 2017, 32, 572-587.
doi:10.1080/14756366.2016.1210139.
[10] F. Baharloo, M.H. Moslemin, H. Nadri, A. Asadipour, M. Mahdavi, S. Emami, L.
Firoozpour, R. Mohebat, A. Shafiee, A. Foroumadi, Benzofuran-derived
benzylpyridinium bromide as potent acetylcholinesterase inhibitors. Euro. J. Med.
Chem., 2015, 93, 196-201. doi:10.1016/j.ejmech.2015.02.009.
[11] A. Kumar, F. Pintus, A. Di Petrillo, R. Medda, P. Caria, M.J. Matos, D. Viña, E.
Pieroni, F. Delogu, B. Era, G.L. Delogu, A. Fais, Novel 2-pheynlbenzofuran derivatives

as selective butyrylcholinesterase inhibitors for Alzheimer’s disease, Sci. Rep., 2018,
8, 4424. doi:10.1038/s41598-018-22747-2.
[12] G.L. Delogu, M.J. Matos, M. Fanti, B. Era, R. Medda, E. Pieroni, A. Fais, A. Kumar, F.
Pintus, 2-Phenylbenzofuran derivatives as butyrylcholinesterase inhibitors: Synthesis,
biological activity and molecular modeling, Bioorg. Med. Chem. Lett., 2016, 26, 2308-
2313. doi:10.1016/j.bmcl.2016.03.039.
[13] J. Micheau, & A. Marighetto, Acetylcholine and memory: A long, complex and chaotic
but still living relationship, Behav. Brain Res., 2011, 221, 424-429.
doi:10.1016/j.bbr.2010.11.052.
[14] M. Mesulam, A. Guillozet, P. Shaw, & B. Quinn, Widely spread butyrylcholinesterase
can hydrolyze acetylcholine in the normal and Alzheimer brain. Neurobiol. Dis., 2002,
9, 88-93. doi:10.1006/nbdi.2001.0462.
[15] N. Oztaskin, Y. Cetinkaya, P. Taslimi, S. Goksu, I. Gulcin, Antioxidant and
acetylcholinesterase inhibition properties of novel bromophenol derivatives, Bioorg.
Chem., 2015, 60. doi:10.1016/j.bioorg.2015.04.006.
[16] C. Bayrak, P. Taslimi, I. Gulcin, A. Menzek, The first synthesis of 4-phenylbutenone
derivative bromophenols including natural products and their inhibition profiles for
carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase enzymes, Bioorg.
Chem., 2017, 72, 359-366. doi: 10.1016/j.bioorg.2017.03.001.
[17] P. Anand, & B. Singh, A review on cholinesterase inhibitors for Alzheimer’s disease.
Arch. Pharmacal Res., 2013, 36, 375-399. doi:10.1007/s12272-013-0036-3.
[18] G. Ferino, E. Cadoni, M.J. Matos, E. Quezada, E. Uriarte, L. Santana, S. Vilar, N.P.
Tatonetti, M. Yáñez, D. Viña, C. Picciau, S. Serra and G. Delogu, MAO inhibitory
activity of 2-arylbenzofurans versus 3-arylcoumarins: synthesis, in vitro study, and
docking calculations, ChemMedChem, 2013, 8, 956. doi:10.1002/cmdc.201300048.
[19] G.L. Delogu, S. Serra, E. Quezada, E. Uriarte, S. Vilar, N.P. Tatonetti and D. Viña,
Monoamine Oxidase (MAO) Inhibitory Activity: 3-Phenylcoumarins versus 4-Hydroxy-
3-phenylcoumarins,
ChemMedChem,
2014,
9,
8,
1672-1676.
doi:10.1002/cmdc.201402010.
[20] E. Quezada, G. Delogu, C. Picciau, L. Santana, G. Podda, F. Borges, V. García
Morales, D. Viña, F. Orallo, Synthesis and Vasorelaxant and Platelet Antiaggregatory
Activities of a New Series of 6-Halo-3-phenylcoumarins, Molecules, 2010, 15, 270-
279. doi:10.3390/molecules15010270.
[21] G.I. Ellman, K.D. Courtney, V. Andres jr, R.M. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 1961,
7, 88-95. doi:10.1016/0006-2952(61)90145-9.
[22] A. Grosdidier, V. Zoete, & O. Michielin, SwissDock, a protein-small molecule docking
web service based on EADock DSS. Nucleic Acids Res., 2011, 39, W270–W277.
doi:10.1093/nar/gkr366.
[23] C. Meier, C. Ducho, H. Jessen, D. Vukadinović-Tenter, & J. Balzarini, Second-
GenerationcycloSal-d4TMP Pronucleotides Bearing Esterase-Cleavable Sites - Te
“Trapping” Concept, Eur. J. Org. Chem., 2006, 197-206. doi:10.1002/ejoc.200500490.
[24] B. Assoah, J.R. Vale, E. Kalenius, L.F. Veiros, and N.R. Candeias, Lewis Base
Catalyzed Intramolecular Reduction of Salicylaldehydes by Pinacol-Derived

Chlorohydrosilane,
Eur.
J.
Org.
Chem.,
2018,
2910-2917.
doi:10.1002/ejoc.201800544.
[25] P. Attri, K.Y. Baik, P. Venkatesu, I.T. Kim, E. H. Choi, Infuence of Hydroxyl Group
Position and Temperature on Termophysical Properties of Tetraalkylammonium
Hydroxide Ionic Liquids with Alcohols. PLoS One, 2014, 9, 86530.
doi:10.1371/journal.pone.0086530.
[26] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci. Rep., 2017, 7, 42717. doi:10.1038/srep42717.
[27] A. Kumar, R. Cardia, G. Cappellini, Electronic and optical properties of chromophores
from bacterial cellulose, Cellulose, 2018, 25(4), 2191-2203. doi:org/10.1007/s10570-
018-1728-0
[28] P. Ertl, B. Rohde, P. Selzer, Fast calculation of molecular polar surface area as a sum
of fragment-based contributions and its application to the prediction of drug transport
properties, J. Med. Chem., 2000, 43(20), 3714-3717. doi:10.1021/jm000942e.
[29] A. Daina, O. Michielin, V. Zoete, iLOGP: a simple, robust, and efficient description of
n-octanol/water partition coefficient for drug design using the GB/SA approach, J.
Chem. Inf. Model., 2014, 54(12), 3284-3301. doi:10.1021/ci500467k.
[30] J. Ali, P. Camilleri, M.B. Brown, A.J. Hutt, S.B. Kirton, Revisiting the general solubility
equation: in silico prediction of aqueous solubility incorporating the effect of
topographical polar surface area, J. Chem. Inf. Model., 2012, 52(2), 420-428.
doi:10.1021/ci200387c.
[31] U. Ammarah, A. Kumar, R. Pal, N.C. Bal, G. Misra, Identification of new inhibitors
against human Great wall kinase using in silico approaches, Sci. Rep. 2018, 8(1),
4894 doi:org/10.1038/s41598-018-23246-0.
[32] A. Fais, B. Era, S. Asthana, V. Sogos, R. Medda, L. Santana, E. Uriarte, M.J. Matos,
F. Delogu, A. Kumar, Coumarin derivatives as promising xanthine oxidase inhibitors,
Int.
J.
Biol.
Macromol.
2018,
120(Pt
A),
1286-1293.
doi:org/10.1016/j.ijbiomac.2018.09.001.
[33] J. Lu, K. Crimin, J.T. Goodwin, P. Crivori, C. Orrenius, L. Xing, P.J. Tandler, T.J.
Vidmar, B.M. Amore, A.G. Wilson, P.F. Stouten, P.S. Burton, Influence of molecular
flexibility and polar surface area metrics on oral bioavailability in the rat, J. Med.
Chem., 2004, 47(24), 6104-6107. doi:10.1021/jm0306529.
[34] R.A. Laskowski, M.B. Swindells, LigPlot+: multiple ligand-protein interaction diagrams
for drug discovery, J. Chem. Inf. Model., 2011, 51, 10, 2778-86.
doi:10.1021/ci200227u.

Graphical abstract

Highlights
 Series of hydroxylated 2-phenylbenzofuran were synthesized.
 Compounds displayed activity against butyrylcholinesterase enzyme.
 Importance of number and position of hydroxyl subsitution in enzyme activity.
 Physicochemical properties of derivatives were characterised.
 Relationship between physicochemical properties and activity was found.
 Docking studies assisted in explaining the structure-activity.