2-Phenylbenzofuran derivatives as butyrylcholinesterase inhibitors: Synthesis, biological activity and molecular modeling

Article abstract of DOI:10.1016/j.bmcl.2016.03.039

A series of 2-phenylbenzofurans compounds was designed, synthesized and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect towards acetylcholinesterase (AChE) was detected. Among these benzofuran derivatives, compound 16 exhibited the highest BChE inhibition with an IC₅₀ value of 30.3 μM. This compound was found to be a mixed-type inhibitor as determined by kinetic analysis. Moreover, molecular dynamics simulations revealed that compound 16 binds to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE and it displayed the best interaction energy value, in agreement with our experimental data.

Full text of DOI:10.1016/j.bmcl.2016.03.039

Accepted Manuscript
2-Phenylbenzofuran derivatives as butyrylcholinesterase inhibitors: Synthesis,
biological activity and molecular modeling
Giovanna L. Delogu, Maria J. Matos, Maura Fanti, Benedetta Era, Rosaria
Medda, Enrico Pieroni, Antonella Fais, Amit Kumar, Francesca Pintus
PII:
S0960-894X(16)30259-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.039
BMCL 23682
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2016
9 March 2016
11 March 2016
Please cite this article as: Delogu, G.L., Matos, M.J., Fanti, M., Era, B., Medda, R., Pieroni, E., Fais, A., Kumar,
A., Pintus, F., 2-Phenylbenzofuran derivatives as butyrylcholinesterase inhibitors: Synthesis, biological activity and
molecular modeling, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.03.039
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2-Phenylbenzofuran derivatives as butyrylcholinesterase inhibitors:
Synthesis, biological activity and molecular modeling
Giovanna L. Delogu,a Maria J. Matos,b Maura Fanti,c Benedetta Era,a Rosaria Medda,a Enrico
Pieroni,d Antonella Fais,a* Amit Kumar,d,e* Francesca Pintusa
a Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
b
Department of Organic Chemistry, University of Santiago de Compostela, Santiago de
Compostela, Spain
c Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy
d Biomedicine Department, Center for Advanced Studies, Research and Development in Sardinia
(CRS4), Pula, Italy
e Department of Mechanical, Chemical and Materials Engineering, University of Cagliari, Cagliari,
Italy
Corresponding Authors *Ph: +390709250257; e-mail: amit369@gmail.com
*Ph: + 390706754506; e-mail: fais@unica.it

Abstract
A series of 2-phenylbenzofurans compounds was designed, synthesised and evaluated as
cholinesterase inhibitors. The biological assay experiments showed that most of the compounds
displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect
towards acetylcholinesterase (AChE) was detected. Among these benzofuran derivatives, compound
16 exhibited the highest BChE inhibition with an IC50 value of 30.3 µM. This compound was found
to be a mixed-type inhibitor as determined by kinetic analysis. Moreover, molecular dynamics
simulations revealed that compound 16 binds to both the catalytic anionic site (CAS) and peripheral
anionic site (PAS) of BChE and it displayed the best interaction energy value, in agreement with our
experimental data.
Keywords: Alzheimer's disease; Acetylcholinesterase; Butyrylcholinesterase; Benzofurans;
Cholinesterase inhibitors; Molecular dynamics; Docking

Alzheimer’s disease (AD) is an irreversible and progressive brain disorder, which is characterized
by progressive memory loss and a wide range of cognitive impairments.1 Although the precise cause
of AD is not completely known, there are some factors that have been described to play a significant
role in the pathogenesis of AD, such as: deficit of acetylcholine (ACh), presence of amyloid-β
deposits, τ-protein aggregation, oxidative stress and metal ions imbalance. Among these distinct
research approaches, the cholinergic hypothesis has been examined more extensively. In fact, low
levels of ACh appear to be a critical element in the development of cognitive and neurodegenerative
disorders in AD patients.2
Accordingly, one strategy in AD treatment is to restore the levels of ACh by inhibiting
acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8), which are
mainly responsible for ACh hydrolysis. These enzymes belong to the superfamily of α/β-hydrolase
fold proteins and they are able to hydrolyse ACh with different efficiencies.3 They are encoded by
two distinct human genes and display 65% homology in their amino acid sequences. These two
proteins also show a great similarity in both their tertiary structure and their overall architecture of
active sites.4,5 Both AChE and BChE have indeed a primarily hydrophobic active gorge into which
ACh diffuses and is cleaved.
Ligand binding specificity between the two enzymes has been related to differences in the residue’s
structural arrangement, which lead to the active site located near the bottom of a deep and narrow
gorge (Figure 1, Table S1 in Supplementary Material). The gorge is characterised by (a) a peripheral
site at the entrance, (b) an oxyanion hole, (c) a choline-binding site located within the entrance, and
(d) the active site constituted by an acyl pocket buried near the catalytic triad.
In fact, structural analysis had revealed that these enzymes have two major substrate-binding sites.
One is a peripheral anionic site (PAS) at the entrance of the gorge, acting as a regulatory site; the
other is the catalytic anionic site (CAS), which is located in the bottom of the gorge and it is
assigned to a Ser-His-Glu catalytic triad.6

AChE and BChE appear to be simultaneously active in the synaptic hydrolysis of ACh, terminating
its neurotransmitter action and co-regulating levels of ACh.7 AChE has a very high catalytic
efficiency for ACh hydrolysis and it is mainly found in cholinergic synapses, while BChE has lower
efficiency and it is widely distributed in tissues and plasma. In a healthy brain, AChE predominates
and BChE is considered to play a minor role in regulating ACh levels. On the other hand, BChE
activity increases in the temporal cortex and hippocampus during the development of AD, while at
the same time AChE activity decreases.8
Since AD is characterized by a forebrain cholinergic neuron loss and a progressive decline in ACh,
a possible therapeutic strategy involves the use of cholinesterase (ChE) inhibitors to restore the
neurotransmitter levels and thus retard AD symptoms.9-11
These inhibitory molecules may act by binding the CAS site (competitive mechanism) or PAS (non-
competitive mechanism) or may exert a dual binding enzyme inhibition – acting as mixed-type
inhibitors.12 Moreover, since the oxidative stress may be a risk factor for the initiation and
progression of AD drugs, both antioxidant and inhibitory actions might be useful for either the
prevention or the treatment of AD.
Benzofuran scaffold (oxygen heterocycle) is a common moiety found in many biologically active
natural and synthetic products. Therefore, it represents a very important pharmacophore in drug
discovery.13 It is present in many medicinally important compounds which show biological activity,
including anticancer and anti-inflammatory properties.14,15 Benzofuran scaffold has drawn
considerable attention over the last few years due to its profound physiological and
chemotherapeutic properties.16 Some benzofuran derivatives are also known as monoamine oxidase
and 5-lipoxygenase inhibitors, antagonists of the angiotensin II receptor, blood coagulation factor
Xa inhibitors, ligands of adenosine A1 receptor, etc.13,17 Recent studies have also investigated their
inhibitory activity towards AChE.18-21
In this study, a series of 2-phenylbezonfurans was synthesized and their inhibitory activity towards
the ChE was investigated. To better understand the enzyme inhibition mechanisms, in relation to the

substituents and their positions in the presented compounds, molecular modeling studies were also
performed.
Figure 1. Cartoon representation for the enzymes under investigation. (A) Equine serum BChE
(B) Electrophorus electricus AChE. The residues lining the gorge of the two enzymes are shown.
The conserved residues between the two are shown in red (licorice representation) and non-
conserved in green. The catalytic triad residues are shown as ball-stick representation.
Compounds 1-16 were efficiently synthesized by an intramolecular Wittig reaction (Scheme 1 and
2). The desired Wittig reagent was readily prepared from the conveniently substituted ortho-
hydroxybenzyl alcohol a–g and triphenylphosphine hydrobromide (Scheme 1).22-31 The key step for
the formation of the benzofuran moiety was achieved by an intramolecular reaction between ortho-
hydroxybenzyltriphosphonium salts h–n and the appropriate benzoyl chlorides (Scheme 2)
(Supplementary Material).30,32-39

R
CHO
OH
R
R
PPh3 HBr/CH3CN
82 °C, 2 h.
NaBH4/EtOH
0 °C to rt, 2 h
OH
OH
P+Ph3Br-
OH
h-n
R1
R1
R1
a, h: R = H, R1 = H
a-g
b, i: R = Me, R1 = H
c, j: R = H, R1 = Me
d, k: R = Br, R1 = H
e, l: R = H, R1 = Br
f, m: R = Cl, R1 = H
g, n: R = H, R1 = Cl
Scheme 1
O
Cl
R
R
R
P+Ph3Br-
toluene/NEt3
110 °C, 2 h;
HI/AcOH/Ac2O
0°C to reflux, 4h
R2
OH
+
O
O
OH
R2
R2 = H, OMe
R1
R1
R1
h-n
1 R, R1, R2 = H
2 R2 = OMe; R, R1 = H
10 R, R1 = H
11 R = Me; R1 = H
12 R1 = Me; R = H
13 R = Br; R1 = H
14 R1 = Br; R = H
15 R = Cl; R1 = H
16 R1 = Cl; R = H
3 R = Me; R2 = OMe; R1 = H
4 R1 = Me; R2 = OMe; R = H
5 R = Br; R2 = OMe; R1 = H
6 R1 = Br; R2 = OMe; R = H
7 R = Cl; R2 = OMe; R1 = H
8 R1 = Cl; R2 = OMe; R = H
9 R1 = Br; R, R2 = H
Scheme 2
Hydrolysis of the methoxy groups of compound 2–8 was performed by treatment with hydrogen
iodide in acetic acid/acetic anhydride, to gave the corresponding hydroxy derivatives 10–16.30,33,40-
1
13
42 We report the H NMR, C NMR and mass spectrometry analysis (Supplementary Material Fig.
S1-S3) of our more active compound 16.
The AChE and BChE inhibitory activity43 of all synthetized compounds was firstly evaluated at
compound concentration of 100 µM (Supplementary Material). As observed, only compounds 9 and
11 exerted a very weak inhibitory activity towards AChE, while all the compounds, except
compounds 2 and 10, inhibited BChE enzymatic activity with a varying efficiency (Table 1). In

particular, compounds 12, 14 and 16 showed the highest inhibition percentages and the lowest IC50
values.
Table 1. Cholinesterase inhibitory activity of compound 1, 2, 8-16.
% Inhibition at 100 μM IC50 ± SDa (μM)
Compound
AChE
BChE
6
BChE
> 100
-
-
1
-
> 100
2
-
5
> 100
8
4
-
12
-
> 100
9
> 100
10
4.8
-
19
58
16.6
54
15
77
> 100
11
77 ± 6.7
> 100
12
-
13
-
82.5 ± 7.1
> 100
14
-
15
-
30.3 ± 1.9
28.3 ± 2.1
16
Galantamine
a Data represent the mean (± standard deviation, SD) of three independent experiments
Compound 16 was found to be the best BChE inhibitor with an IC50 value of 30.3 µM. Thus, it has
been chosen for the kinetic studies. We investigated the kinetic behaviour of BChE at different
concentrations of inhibitor and substrate (BTCI). In the presence of compound 16, the Lineweaver–
Burk plots showed that this compound was a mixed-type inhibitor, since increasing the
concentration of compound 16 resulted in a family of straight lines which intersected in the second
quadrant (Figure 2). In this case, the inhibitor can bind not only with the free enzyme but also with
the enzyme-substrate complex. The equilibrium constants for binding with the free enzyme (KI) and

with the enzyme–substrate complex (KIS) were obtained either from the slope or the Vmax values (y-
intercepts) plotted against inhibitor concentration, respectively. The values of KI and KIS of
compound 16 were determined to be 5.0 and 87.4 μM, respectively.
Figure 2. Kinetic study of the mechanism of BChE inhibition displayed by compound 16. In
(A) Overlaid Lineweaver-Burk reciprocal plots of BChE initial velocity at increasing substrate
concentration (0.05-0.50 mM) in the absence and in the presence of compound 16. The
concentrations of inhibitor were 0 (○), 10 (■), 20 (□) and 30 (●) μM; (B) Secondary plot of slopes
versus compound 16 concentrations and (C) Secondary plot of 1/Vmax values versus compound 16
concentrations.
Our in depth analysis showed that the simplest 2-phenylbenzofuran (compound 1), without any
substitution, displayed no inhibitory activity towards AChE and BChE. The introduction of a
methoxy or a hydroxy substituent in the para position of the 2-phenyl ring led to compounds 2 and
10, respectively. Both compounds proved to be inactive against both ChE enzymes.
We then studied the effect of the introduction of an electron-donating group (methyl) or an electron-
withdrawing group (bromine and chlorine) at positions 5 or 7 of the benzofuran nucleus. The
introduction of a methyl substituent at these positions of the 2-(4-hydroxyphenyl)benzofuran (10)

led to compounds 11 and 12, respectively. Compound 12, which only differs from compound 11 in
its respective position of methyl group (position 7 compared to position 5, Scheme 2), showed an
increase in its BChE inhibitory activity. Interestingly, substitution of bromine at position 7
(compound 14), increases the enzymatic inhibition respecting to substitution at position 5
(compound 13), which is consistent with the trend observed on methyl substitutions. Furthermore,
substitution with chlorine at position 7 (compound 16) significantly improved the inhibitory activity
towards BChE, if compared with compound 15 (chlorine at position 5).
In summary, our data for 2-phenylbenzofuran derivatives with hydroxyl at para position and
substitution with methyl, bromine and chlorine at position 7, resulted in a better inhibition value
regarding to the same substitution at position 5.
Therefore, the most relevant structural feature of the synthesized 2-phenylbenzofurans is the
contemporary presence of a hydroxy group at para position of the 2-phenyl ring and a substitution
at position 7; all these compounds (12, 14 and 16) being active against BChE.
Since oxidative stress is closely associated with the development of AD,44 antioxidant activity of the
described compounds was evaluated by measuring radical scavenging capacity by ABTS assay
(Supplementary Material).45 As expected, compounds without hydroxylic groups in their structure
(compounds 1, 2, 8 and 9) did not present radical scavenging activity (data not shown). Compounds
12, 14 and 16, which are the best BChE inhibitors, exhibited the highest antioxidant activity (Table
2). Taking into account these results, and the structural characteristics of the compounds, we
observed that the concurrently presence of a hydroxyl group in the para position of the phenyl ring
and the substitution at position 7 of the benzofuran scaffold, appeared to play an important role in
determining the desired antioxidant activity.
In addition to the above biological evaluation, the potential cytotoxicity effect of compound 16 on
the cell line NSC-34 (motor-neuron) was evaluated to determine the safety of this molecule
(Supplementary Material). After treating the cells with this compound at different concentrations
(5-100 μM) for 48 h, the cell viability was determined (Figure 3). The results indicate that this

inhibitor exhibited no considerable cytotoxic effect on NSC-34 neuron like cell at the concentration
at which it inhibits the ChE activity.
Table 2. Radical scavenging activity of compounds 10-16.
ABTS scavenging
Compound
% Inhibition at 100 μM
IC50 ± SDa (μM)
24
31
-
10
-
11
72
53.5 ± 4.7
-
12
15
13
53
93.6 ± 6.4
-
14
15
15
62.4
67.6 ± 4.5
13 ± 1.1
16
Troloxb
a Data represent the mean (± standard deviation, SD) of three independent experiments
b Trolox was used as the positive control.
Figure 3. The effect of compound 16 on NSC-34 cell viability. Cells were treated with different
concentrations of compound (5-100 μM) and studied by MTS reagent. Data is expressed as a
percentage of the control.

To better understand the impact of substitutions (benzofuran moiety and 2-phenyl ring) on enzyme
inhibitory ability, compounds 8, 15 and 16 were selected for computational modeling (Fig. S5
Supplementary Material). Molecular docking results showed that the three compounds were able to
interact with CAS residues in BChE and with PAS in AChE with very similar docking energies (~6-
7 kcal/mol). To investigate the structural and dynamical aspects upon ligand binding we simulated
the two enzymes for 50 ns in the free and ligand bound configurations. In the case of AChE, all the
compounds (8, 15 and 16) did not interact with any CAS residues. Therefore no enzymatic
inhibition was observed. On the other hand, in the case of BChE, the three compounds interacted
with at least one of the three CAS residues, displaying enzymatic inhibition. Therefore, the
simulations of our models suggested the interaction of 2-phenylbenzofurans with CAS residues to
be crucial for enzymatic inhibition. However, the levels of BChE inhibition varied in the three
compounds, displaying compound 16 the highest value (77%). Based on these results, we decided
to focus our attention on the interaction of these three compounds with BChE.
Enzyme dynamics was investigated in the presence of these compounds using Prody software.46 The
results obtained (Fig. S6, Supplementary Material) highlighted the importance of the substitution at
position 7 in enzyme dynamics. We further estimated the binding strength between the enzyme
residues and the three compounds. Interestingly, compound 16 displayed the best interaction energy
value and also possessed the highest percentage of its total surface area buried in the gorge (Table
S2, Supplementary Material). To better understand the origin of these differences, we carefully
inspected the binding mode of the ligands in complex with BChE (Figure 4), and with AChE
(Figure 5, shown for comparison) using Ligplot.47

Figure 4. Molecular interaction picture of equine serum BChE bound to the three compounds.
In (A) compound 16 (B) compound 15 and (C) compound 8.
Figure 5. Molecular interaction picture of Electrophorus electricus AChE bound to the three
compounds. In (A) compound 16 (B) compound 15 and (C) compound 8.

Previous biochemical and molecular studies48 found the difference in the inhibitory property of
E2020 towards AChE and BChE to be related to simultaneous participation of CAS and PAS
binding residues (present only for AChE). Our modeling results confirm a similar effect for 2-
phenylbenzofurans (compound 16, Figure 4). Interestingly, in all three complexes, the compounds
globally interacted with CAS residues (S198, H438). However, only in compound 16 we found
benzofuran moiety to interact with PAS residues (Q119, Y332) and 2-phenyl ring moiety to interact
with CAS residues and with residues W82, E197 and G439. Therefore, explaining the high
interaction energy (Table S2, Supplementary Material) and the related high inhibition (Table 1). In a
recent study, residue Q119 was described to be important towards selective inhibition of mouse
BChE by two biscarbamates compounds49 and residue W82 (W86 in AChE) as a crucial component
of the anionic site6 and as a controller for opening and closing of CAS.50 Other experimental studies
further analyzed the relevance of residue E197 (E202 in AChE) in substrate inhibition51 and of
residue Y332 (Y341 in AChE) in substrate binding.52 Overall, jointly with all these previous
proposals and findings, our current experimental and modeling results confirm and explain the
highest BChE inhibitory activity noticed for compound 16.
In conclusion, in this study a series of 2-phenylbenzofurans has been designed, synthesized and
evaluated for its ChE inhibitory activity. These compounds showed no inhibition against AChE,
while inhibiting BChE with various levels of efficiency. Compounds 12, 14 and 16 proved to be the
most potent inhibitors. These compounds also displayed the highest antioxidant activity as well.
According to our results, the contemporary presence of a hydroxy group in the para position of the
phenyl ring and a substitution at position 7 of the benzofuran scaffold, improved the inhibitory
activity, regarding to the other synthesized compounds. In particular, compound 16 exhibited the
highest BChE inhibition. In addition, kinetic analysis indicated that compound 16 is a mixed-type
inhibitor with no considerable cytotoxic effect on NSC-34 cells.
Molecular modeling demonstrated that the interaction of 2-phenylbenzofurans with CAS residues is
crucial for the enzymatic inhibition. Our simulation also revealed that compound 16 binds both

CAS and PAS sites in BChE, in accordance to the experimental data, which showed that this
compound acts as a mixed-type inhibitor.
We can therefore conclude that the combination of biological assays and molecular dynamics
simulations allowed highlighting the molecular basis of the selective BChE inhibitory activity by
the benzofuran scaffold.
Since the BChE activity progressively increases in patients with AD, while AChE activity remains
unchanged or declines during the disease course, the use of molecules that selectively interact with
BChE might have a relevant role in treatment of patients with advanced AD. In this scenario, our
findings could be extended to design and develop new potentially useful selective therapeutic
molecules.
Acknowledgements
This work was supported by University of Cagliari, CRS4 and its HPC staffs for help and access to
computational facility. G. L. Delogu is grateful to R. Mascia (University of Cagliari) for his
technical assistance. Authors also thank Dr. Gavin Brelstaff for language polishing.
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