A new biological prospective for the 2-phenylbenzofurans as inhibitors of α-glucosidase and of the islet amyloid polypeptide formation

Article abstract of DOI:10.1016/j.ijbiomac.2020.12.117

In this study, we have investigated a series of hydroxylated 2-phenylbenzofurans compounds for their inhibitory activity against α-amylase and α-glucosidase activity. Inhibitors of carbohydrate degrading enzymes seem to have an important role as antidiabetic drugs. Diabetes mellitus is a wide-spread metabolic disease characterized by elevated levels of blood glucose. The most common is type 2 diabetes, which can lead to severe complications. Since the aggregates of islet amyloid polypeptide (IAPP) are common in diabetic patients, the effect of compounds to inhibit amyloid fibril formation was also determined. All the compounds assayed showed to be more active against α-glucosidase. Compound 16 showed the lowest IC₅₀ value of the series, and it is found to be 167 times more active than acarbose, the reference compound. The enzymatic activity assays showed that compound 16 acts as a mixed-type inhibitor of α-glucosidase. Furthermore, compound 16 displayed effective inhibition of IAPP aggregation and it manifested no significant cytotoxicity. To predict the binding of compound 16 to IAPP and α-glucosidase protein complexes, molecular docking studies were performed. Altogether, our results support that the 2-phenylbenzofuran derivatives could represent a promising candidate for developing molecules able to modulate multiple targets involved in diabetes mellitus disorder.

Full text of DOI:10.1016/j.ijbiomac.2020.12.117

International Journal of Biological Macromolecules 169 (2021) 428–435
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
A new biological prospective for the 2-phenylbenzofurans as inhibitors of
α-glucosidase and of the islet amyloid polypeptide formation
Giovanna Lucia Delogu a,1, Benedetta Era a,1, Sonia Floris a, Rosaria Medda a, Valeria Sogos b, Francesca Pintus a,
Gianluca Gatto c, Amit Kumar c, Gunilla Torstensdotter Westermark d, Antonella Fais a,
⁎
a
Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Cagliari 09042, Italy
Department of Biomedical Sciences, University of Cagliari, Monserrato, Cagliari 09042, Italy
Department of Electrical and Electronic Engineering, University of Cagliari, via Marengo 2, Cagliari 09123, Italy
b
c
d
Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Uppsala University, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2020
Received in revised form 1 December 2020
Accepted 15 December 2020
Available online 19 December 2020
In this study, we have investigated a series of hydroxylated 2-phenylbenzofurans compounds for their inhibitory
activity against α-amylase and α-glucosidase activity.
Inhibitors of carbohydrate degrading enzymes seem to have an important role as antidiabetic drugs.
Diabetes mellitus is a wide-spread metabolic disease characterized by elevated levels of blood glucose. The most
common is type 2 diabetes, which can lead to severe complications. Since the aggregates of islet amyloid poly-
peptide (IAPP) are common in diabetic patients, the effect of compounds to inhibit amyloid fibril formation
was also determined.
Keywords:
α-Glucosidase
All the compounds assayed showed to be more active against α-glucosidase. Compound 16 showed the lowest
IC50 value of the series, and it is found to be 167 times more active than acarbose, the reference compound.
The enzymatic activity assays showed that compound 16 acts as a mixed-type inhibitor of α-glucosidase. Further-
more, compound 16 displayed effective inhibition of IAPP aggregation and it manifested no significant
cytotoxicity.
Islet amyloid polypeptide
2-phenylbenzofuran, molecular docking
To predict the binding of compound 16 to IAPP and α-glucosidase protein complexes, molecular docking studies
were performed.
Altogether, our results support that the 2-phenylbenzofuran derivatives could represent a promising candidate
for developing molecules able to modulate multiple targets involved in diabetes mellitus disorder.
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
Type 1 (T1D) and Type 2 (T2D) diabetes are the two main types of
diabetes, and the latter type makes up about 90% of diabetes cases. In
In diabetic disease, controlling postprandial hyperglycaemia is
one of the main therapeutic targets. Inhibition of α-glucosidase
(EC 3.2.1.3) and α-amylase (EC 3.2.1.1), which are carbohydrate-
hydrolyzing enzymes, is enabled to reduce the rate of glucose absorp-
tion and control plasma glucose levels [1,2]. Therefore, in drug discovery
α-amylase and α-glucosidase is recognized as an important target [3–5].
The aetiology could be different in diabetes mellitus as well as the
treatment, but the hyperglycaemia is the common feature. Chronic
hyperglycaemia causes high levels of oxygen free radicals in different
tissues of diabetic patients, and plays a significant part in the advance-
ments of various associated complications [6].
over 90% of patients afflicted with T2D, amyloid deposits are observed
in the extracellular space of pancreatic islets of Langerhans [7] and intra-
cellular in islet β-cells [8]. The islet amyloid polypeptide (IAPP) is a 37-
residue peptide hormone that aggregates to form amyloid fibrils [9],
which are seen mainly in association with T2D, but recently also
identified in the pancreas of individuals with T1D [10]. Aggregated
IAPP has cytotoxic properties and is believed to be of critical importance
for the loss of β-cells in T2D [11]. Therefore, the inhibition of its
formation has therapeutic potential. Indeed, inhibition approaches of
polypeptide-based and of small molecules have provided promising
results [12,13].
Benzofuran ring is a basic structure of various natural medicines
and synthetic chemicals. Numerous studies have shown that
2-arylbenzofurans have biological activities such as anti-cancer, anti-
bacterial, anti-viral, anti-oxidative, anti-fungal, and anti-microbial
⁎
Corresponding author.
E-mail address: fais@unica.it (A. Fais).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ijbiomac.2020.12.117
0141-8130/© 2020 Elsevier B.V. All rights reserved.

G.L. Delogu, B. Era, S. Floris et al.
International Journal of Biological Macromolecules 169 (2021) 428–435
2.2. Biological assays
anti-
inflammatory
anti-bacterial
anti-oxidative
2.2.1. Assay for α-amylase inhibitory activity
The inhibition of α-amylase activity by compounds 9–16 was deter-
mined by using 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) as
an artificial substrate. The solution containing 60 μL of 50 mM sodium
phosphate buffer at pH 7.0, 20 μL of NaCl (1 M) and 40 μL of α-
amylase from porcine pancreas (1 mg/mL), was mixed in microplate
multi-wells and incubated in the absence or presence of the sample at
37 °C for 10 min. Similarly, acarbose as a standard inhibitor was used
as a positive control. Following incubation, we added 80 μL of a
2.5 mM CNPG3 solution and the amount of 2-chloro-nitrophenol re-
leased by the enzymatic hydrolysis was monitored at 405 nm.
R1
anti-fungal
anti-tumoral
R
O
anti-microbial
anti-viral
anti-Alzheimer’s
disease
Fig. 1. Biological activities of 2-arylbenzofuran derivatives.
2.2.2. Assay for α-glucosidase inhibitory activity
Inhibitory activity of α-glucosidase was performed as described by
Fais et al. [23]. The solution was prepared in 0.1 M phosphate buffer
pH 6.8 and was mixed with 40 μL of enzyme from Saccharomyces
cerevisiae solution containing 0.125 U/mL.
activities (Fig. 1). Thus, there is a great interest in using benzofuran as a
building block of pharmacological agents.
Many benzofuran derivatives, of natural or synthetic origin, have
been approved clinically, and some of which are fused with other het-
erocyclic moieties [14].
Twenty microliters of test samples were mixed at various concentra-
tions with the enzyme solution and then incubated for 15 min at 37 °C.
After incubation, 20 μL of the substrate, p-nitrophenyl α-D-
glucopyranoside (pNPG), 5 mM solution in 0.1 M phosphate buffer
was added to the above-prepared mixture and incubated again
under the same experimental conditions. The subsequent addition
of 50 μL of 0.2 M sodium carbonate solution stops the reaction. The
amount of released p-nitrophenol was measured using a 96-well mi-
croplate reader at 405 nm. DMSO control was used whenever re-
quired, and the final concentration of DMSO was maintained below
8% v/v, concentration that does not affect the enzyme activity.
Acarbose was used as a positive control. The concentration of com-
pound needed to inhibit 50% of α-glucosidase activity under assay
conditions was defined as IC50 value.
Keeping in mind that molecules that hit more than one target may
possess in principle a safer profile compared to single-targeted ones
[15,16], a series of hydroxylated 2-phenylbenzofurans compounds,
with promising therapeutic targets [17–19] have been studied.
Considering that previous studies [20,21] indicated 2-arylbenzofuran
derivatives as antidiabetic agents, we have analysed a series of 2-
phenylbenzofuran derivatives, for which the anti-cholinesterase action
is already known [16], to improve the potential of this scaffold for
their inhibitory activities also towards α-glucosidase and α-amylase
enzymes.
Recently, the concept of metabolism-dependent neurodegeneration
mechanisms is gaining importance, as T2D patients have a higher risk of
developing Alzheimer's disease (AD) symptoms. The overlapping
mechanisms of AD and T2D offer a new perspective taking us towards
an entirely different approach, which involves targeting insulin signal-
ling, and glucose metabolism as a novel therapeutic strategy for AD [22].
To promote the usage of these inhibitors in drug discovery and de-
velopment process, their potential effect on the kinetic and structural
properties of IAPP have been analysed.
The Lineweaver-Burk double reciprocal plot was performed to deter-
mine the mode of inhibition of compound 16 on α-glucosidase. The
assay was performed by varying the concentration of inhibitor and pNPG.
2.2.3. Inhibition of IAPP aggregate formation
The formation of amyloid fibril was monitored by characteristic
changes in Thioflavin T (ThT) fluorescence intensity. IAPP stock solution
was obtained dissolving 2 mg of synthetic IAPP (Cambridge) in 250 μL
(2 mM) of hexafluoroisopropanol (HFIP). This stock solution was stored
at −20 °C. All solutions for these studies were prepared by adding a PBS
buffer thioflavin-T solution (1 mM) to IAPP peptide (in lyophilized dry
form) immediately before the measurement. The final concentration
of IAPP was 10 μM with 0.5% of DMSO. When compounds were present
at different concentration (100 μM, 50 μM, 10 μM, 1 μM, and 0.1 μM), the
IAPP to compound ratio was respectively 1:10, 1:5, 1:1, 1:0.1 and 1:0.01
by weight. ThT fluorescence was monitored at 480 nm with 440 nm ex-
citation at 37 °C on a FLUOstar Omega microplate reader. The experi-
ments performed in sextuplicates were repeated three times.
Finally, molecular docking study was performed to spot pivotal
structural aspects that influence the activity of compounds against α-
glucosidase protein and IAPP.
2. Materials and methods
2.1. Chemistry
All commercial reagents and materials were purchased from
commercial sources and used without further purification. Melting
points were measured on a Büchi 510 apparatus and were uncor-
rected. 1H NMR and 13C NMR spectra were recorded with a Varian
INOVA 500 spectrometer using deuterated chloroform (CDCl3) and
dimethyl sulfoxide (DMSO) as solvents. Chemical shifts (δ) are
given in parts per million (ppm) using TMS as an internal standard.
Coupling constants J are expressed in hertz (Hz). Spin multiplicities
were given as s (singlet), d (doublet), dd (doublet of doublets), m
(multiplet), and apparent triplet (app t). Using a Perkin Elmer 240B
microanalyser, elemental analyses were performed and were found
to be 0.4% of calculated values, in all the cases. Column chromatogra-
phy purifications were performed using Aldrich silica gel (60–120)
mesh size. For all compounds, the analytical results revealed 98% pu-
rity. We performed flash chromatography (FC) on silica gel (Merck
60, 230–400 mesh); analytical thin-layer chromatography (TLC)
was performed on pre-coated silica gel plates (Merck 60 F254), visu-
alized by exposure to UV light.
2.2.4. Cell viability assay
HeLa cells viability was detected by the colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
These cells were grown in Dulbecco's Modified Eagle Medium supple-
mented with 10% fetal bovine serum, 2 mM L-Glutamine, penicillin
(100 U/mL) and streptomycin (100 μg/mL) at 37 °C in 5% CO2.
The colorimetric MTT assay was utilized for measuring the activity of
mitochondrial enzymes in living cells that converted MTT into purple
formazan crystals. Briefly, 3 × 104/mL cells were seeded in a 96-well
plate and incubated with samples at concentrations ranging from 1 to
100 μM for 48 h. Since DMSO was used as a solvent for compounds,
cell viability was also performed in the presence of DMSO alone, as sol-
vent control. After incubation time, MTT solution (final concentration
of 0.5 mg/mL) was added to each well and incubated for 3 h at 37 °C.
429

G.L. Delogu, B. Era, S. Floris et al.
International Journal of Biological Macromolecules 169 (2021) 428–435
The cells were lysed with 100 μL of DMSO (≥99,7%), and the optical den-
sity was measured at 560 nm with an auto microplate reader (Multiskan
FC - Thermo Scientific). The mean values and standard deviations (SD)
were calculated from triplicates.
2.4. Statistical analyses
Statistical differences were evaluated using Graph Pad Version 8
software (San Diego, CA, USA). Comparison between groups was esti-
mated by one-way analysis of variance (one-way ANOVA) followed by
Tukey's multiple comparison test. The values with p < 0.001 were con-
sidered significant. Data displayed are as mean SD of three indepen-
dent experiments.
2.3. Molecular docking
The three-dimensional (3D) structure of IAPP [24] was obtained from
the protein data bank (PDB id: 2L86). However, due to the unavailability
of the experimental 3D structure of an α-glucosidase protein from Saccha-
romyces cerevisiae, a template-based homology modelling approach was
followed using Swiss-model web server [25] with 3D reference structure
of isomaltase from Saccharomyces cerevisiae (3AJ7) [26] having 72% se-
quence identity [27] with the target protein structure. The predicted pro-
tein tertiary structure model was evaluated by local quality estimates [28]
and Ramachandran plot of the dihedrals (see ESI† Fig. S1). The 3D struc-
ture of compound 16 was obtained using open-babel software [29]. Fur-
ther details of ligand preparation have been described in our previous
studies [30,31]. The docking experiment to generate a protein-ligand
complex was performed using a COACH-D web-server [32].
3. Results and discussion
3.1. Chemistry
Synthesis of 2-phenylbenzofurans was performed using an intramo-
lecular Wittig procedure, primarily due to its simplicity and accessibility
[33–35].
The synthesis of 2-substituted benzofurans 1–8 was carried out by
Wittig reaction, following the procedure shown in Fig. 2.
Starting with the conveniently substituted 2-hydroxybenzyl
alcohols a-b and PPh3·HBr in acetonitrile, at 82 °C, for 2 h
CHO
PPh3 HBr/CH3CN
NaBH4/EtOH
P+Ph3Br-
OH
a
OH
OH
0 °C to rt, 2 h
OH
82 °C, 2 h
R
R
R
a, c: R = Cl
b, d: R = Br
c-d
a-b
b
O
Cl
OMe
OH
HI/AcOH/Ac2O
OMe
O
O
toluene/NEt3
110 °C, 2 h
0°C to reflux, 4h
O
O
R
R
R
1-2
9-10
O
Cl
OMe
OMe
OH
OH
MeO
OMe
HI/AcOH/Ac2O
0°C to reflux, 4h
toluene/NEt3
110 °C, 2 h
P+Ph3Br-
R
11-12
3-4
O
Cl
OH
R
c-d
OMe
OMe
OMe
OH
toluene/NEt3
110 °C, 2 h
HI/AcOH/Ac2O
0°C to reflux, 4h
OMe
OH
O
O
O
O
R
R
R
R
13-14
15-16
5-6
7-8
OMe
OMe
OMe
O
Cl
OH
OH
OH
HI/AcOH/Ac2O
0°C to reflux, 4h
MeO
OMe
OMe
toluene/NEt3
110 °C, 2 h
1,3,5,7: R = Cl
2,4,6,8: R = Br
9,11,13,15: R = Cl
10,12,14,16: R = Br
Fig. 2. The synthetic route towards methoxylated 2-phenylbenzofurans 1–8 and hydroxylated 2-phenylbenzofurans 9–16.
430

G.L. Delogu, B. Era, S. Floris et al.
International Journal of Biological Macromolecules 169 (2021) 428–435
(Fig. 2a), the desired Wittig reagents were readily prepared. The
means for the development of the benzofuran moieties 1–8 were
accomplished through an intramolecular reaction between the appropri-
ate 2-hydroxybenzyltriphosphonium salt c-d and the commercially ob-
tainable benzoyl chlorides, in toluene, at 110 °C, for 2 h. To obtain the
corresponding hydroxy derivatives compounds 9–16 (Fig. 2b), hydrolysis
of methoxy groups of compounds 1–8 was carried out through the treat-
ment with hydrogen iodide in acetic acid/acetic anhydride at 0 °C and
then at reflux for 3 h [18,19] (see ESI† Spectrum S1 and S2).
of inhibition at most (data not shown). While, at the same concentra-
tion, compounds efficiently inhibited α-glucosidase activity, and the re-
sults are summarized in Table 1.
We noted that the compounds with one hydroxyl substituent in
meta position of the phenyl ring, and chlorine or bromine atom in
position 7 of benzofuran scaffold (compounds 9 and 10) exerted a
weak α-glucosidase inhibitory activity when compared to other
compounds. Nevertheless, their IC50 values are better than the
value determined for acarbose (139 μM), the reference compound.
Interestingly, introduction of another hydroxyl group into the other
meta-position (compounds 11 and 12) of the phenyl ring increases
the inhibitory activity, which is higher for the compound 12 with
an IC50 value 4.3-fold better than the reference. It is worth
highlighting that the presence of two hydroxyl groups in the
meta and para-positions, compounds 13 and 14, significantly in-
creased the inhibitory activity of the 2-phenylbenzofuran scaffold
3.2. Enzyme inhibitory studies
The compounds 9–16 were evaluated for their inhibitory activity
against α-amylase and α-glucosidase activity. All the compounds, at a
concentration of 25 μM, were weak inhibitors of α-amylase giving 33%
with an IC50 value of 9.57
3.04 and 1.45
0.16 respectively.
Compounds 15 (IC50 = 5.19
0.36) and 16 (IC50 = 0.83
0.16), with three hydroxyl substituents in phenyl ring (3,4,5-posi-
tions), inhibited the enzyme with efficiency similar to compounds
13 and 14. Interestingly, α-glucosidase inhibitory activity displayed
by compound 16 was about 167 times higher than the reference
compound.
Thus, it establishes that number of hydroxyl groups and their posi-
tion in the 2-phenyl ring of the synthesized compounds could decrease
or increase the inhibitory activity of compounds against α-glucosidase.
Our results are in good agreement with other previous studies [19,36],
which have shown that the position and number of the hydroxyl groups
in the ligand can influence the magnitude of hydrogen bond interac-
tions with the protein, and consequently, their activity.
The mode of inhibition of compound 16 was determined by the
Lineweaver-Burk plot, as shown in Fig. 3. Increasing the concentration
of compound 16 resulted in a family of lines with different slope and in-
tercept, intersecting in the second quadrant. Thus, implying that com-
pound 16 was a mixed-type inhibitor, which can bind the free enzyme
and the enzyme-substrate (ES) with different equilibrium constants,
KI and KIS (0.83 and 3.55 μM, respectively). The equilibrium constants
values were obtained from the slope or the vertical intercept versus in-
hibitor concentration, (see ESI† Fig. S2).
Table 1
Inhibition of α-glucosidase enzyme by compounds 9–16.
Compounds
α-Glucosidase
IC50 (μM) SD
61.03
57.16
53.25
0.64a
9
3.56a
1.30a
10
11
32.37
2.21b
3.3. Inhibition of IAPP fibril formation
12
13
14
9.57
1.45
5.19
3.04c
To monitor the in vitro fibril formation, Thioflavin T (ThT) binding
assay has been used. When ThT binds to amyloid fibrils, it exhibits an
0.16c
0.36c
3.0
2.5
2.0
1.5
1.0
0.5
0.0
15
0.83
139
0.16c
16
-3
-2
-1
0
1
2
3
4
5
6
Acarbose$
11.3d
1/[pNPG] (mM)-1
IC50 values are expressed as the mean SD of three experiments. Different
letters indicate statistically significant differences between compounds
(p < 0.001).
Fig. 3. Lineweaver–Burk plots analysis of compound 16. Inhibitor concentrations were 0
(X), 0.4 μM (•), 0.6 μM (■), 0.8 μM (▲).
$
Positive control.
431

G.L. Delogu, B. Era, S. Floris et al.
International Journal of Biological Macromolecules 169 (2021) 428–435
30000
25000
20000
15000
10000
5000
0
IAPP 10
M
IAPP 10 M + compound 16 10
IAPP 10 M + compound 16 50
IAPP 10 M + compound 16 100
0
200
400
600
800
1000
Time (min)
Fig. 4. Thioflavin T fluorescence emission plot corresponding to the β-sheet formation of IAPP samples upon incubation with compound 16.
enhanced fluorescence and a characteristic blue shift in the emission
spectrum.
The fluorescence intensities of the dye were measured as a function
of time, and the corresponding spectrum was recorded. Any deviation
from the positive control sample along the time scale indicates inhibi-
tion or activation of the aggregation processes.
Compounds 9–16 were tested for their ability to inhibit amyloid fi-
bril formation. We added the compounds at the different ratios to
IAPP solutions and monitored the kinetics of IAPP amyloid formation
by ThT fluorescence emission.
Co-incubation of IAPP at different concentrations with each com-
pound 9–12 did not show any deviation from IAPP alone in ThT fluores-
cence intensity, and results are comparable (data not shown). The rate
120
100
80
60
40
20
0
0
1
10
25
50
100
of fibril growth does not appear to be significantly affected by these
compounds.
Compound 16 [ M]
Interestingly, the influence of compounds 13–16 on IAPP aggrega-
tion is almost opposite to that shown by compounds 9–12. Indeed, the
compounds prevented any increase in ThT signal, reflecting complete
prevention of aggregation, and the inhibiting effect lasted for a
prolonged time (>150 h). Compound 16, which displayed the best in-
hibitory activity against the α-glucosidase enzyme, also resulted as an
Fig. 5. The effect of compound 16 on HeLa cell viability. Cells were treated with different
concentrations of compound (1–100 μM), and their viability was evaluated by MTT
assay. Data depict the mean ( standard deviation) of three independent experiments.
Fig. 6. IAPP-Compound 16 complex. A. The top docked conformation of compound 16 to IAPP. The IAPP is shown as molecular surface and cartoon representation, ligand (magenta) in ball-
stick representation and the participating residues (blue) in licorice. B. Compound 16 interactions with the residues of IAPP mediated by hydrogen bonds (H-bonds), and by hydrophobic
(H-phobic) contacts. H-bonds are indicated by dashed lines between the atoms involved, while H-phobic contacts by an arc with spokes radiating towards the participating ligand atoms,
obtained using Ligplot [37].
432

G.L. Delogu, B. Era, S. Floris et al.
International Journal of Biological Macromolecules 169 (2021) 428–435
effective inhibitor of IAPP aggregation at a concentration of 50 μM
(Fig. 4).
The docking results predicted binding energy of −6.5 kcal/mol for
compound 16 against IAPP. A detailed examination of the binding site
allowed identifying key interacting residues, as shown in Fig. 6.
To comprehend and provide information concerning mixed-type in-
hibition property of compound 16 against the α-glucosidase protein,
docking for the complex was also performed (Fig. 7).
In a mixed-type inhibition, the ligand binds to an allosteric protein
site, which is different from the active site [39] (Fig. 7A). Indeed, the pre-
dicted binding site for compound 16 (with a binding energy value of
−7.5 kcal/mol) was noted in the region different from the active site
(Fig. 7B), in agreement with the kinetic binding data. Moreover, we ob-
served a rich protein-ligand interaction network (Fig. 7C) mediated by
H-bonds interactions (between hydroxyl groups of the ligand and resi-
dues Glu304, Thr307), and H-phobic interactions (Lys155, Phe157) and
pi-stacking interactions (Phe157, His239) (Fig. 7D).
3.4. Cell viability
After obtaining encouraging results from the assay experiments, the
potential cytotoxicity effect of the promising compound 16 was further
evaluated using the MTT test. Hela cells were treated with compound 16
at concentrations ranging from 1 to 100 μM for 48 h, and the potential
cytotoxic effect was determined.
Results indicate that compound 16 exhibited no significant cytotox-
icity at the concentration in which α-glucosidase activity is inhibited
(Fig. 5).
3.5. Molecular and physicochemical properties of compound 16
An important aim during early drug development is to identify
promising compounds as well as to investigate their pharmacokinetics
parameters [40,41]. Therefore, the presence of pan-assay interference
compounds (PAINS) and the conformity to Lipinski's rule was investi-
gated by utilizing free web tool Swiss ADME [42] (see ESI† Table S1).
Furthermore, we employed a novel Brain Or IntestinaLEstimateD per-
meation (BOILED-Egg) method to predict simultaneously two crucial
The tertiary structure of human IAPP was obtained from protein data
bank (PDB id: 2L86), while for the α-glucosidase protein from Saccharo-
myces cerevisiae was generated using homology modelling approach,
using the tertiary template structure (PDB id: 3AJ7) of isomaltase. Com-
pound 16 was docked alternatively to the two proteins by utilizing the
COACH-D web-server [32].
Fig. 7. Modelling α-glucosidase - compound 16 complex. A. 3D model of protein (as a cartoon), catalytic residues (as licorice), and active site highlighted in red. B. Compound 16 binding
region identified from docking. C. The zoomed view of predicted ligand binding site. D. Compound 16 interactions with the protein residues mediated by H-bonds (blue), H-phobic con-
tacts (dashed grey lines), parallel pi-stacking (green dashed line), and perpendicular pi-stacking (dark green dashed line), obtained using PLIP program [38].
433

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International Journal of Biological Macromolecules 169 (2021) 428–435
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pharmacokinetic parameters, namely gastrointestinal absorption (GA)
and brain penetration (BA), employing two important physicochemical
descriptors (lipophilicity and polarity) of compound 16 (see ESI†
Fig. S3).
4. Conclusions
A series of benzofuran derivatives were efficiently synthesized, and
they showed excellent inhibitory activities against α-glucosidase in
comparison to the standard drug. Among these derivatives, compound
16 was the most potent one with an IC50 value of 0.83 0.16 μM. The
kinetic analysis showed that it inhibited α-glucosidase activity with a
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compound 16 in preventing amyloid fibril formation. Our results
highlighted a relationship between the number of hydroxyl groups
and their position in the 2-phenylbenzofuran scaffold and α-
glucosidase inhibitory activity.
Furthermore, docking studies revealed that the ligand-binding re-
gion is located far from the active site, thus supporting the mixed-type
inhibitory characteristic of compound 16 against the α-glucosidase
protein.
Overall, the findings obtained from the present study will encourage
us to continue our efforts towards optimising the pharmacological pro-
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molecule can modulate multiple targets simultaneously is potentially
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CRediT authorship contribution statement
Giovanna L. Delogu: Methodology, Investigation, Writing - review &
editing, Visualization Benedetta Era: Conceptualization, Methodology,
Investigation, Writing - review & editing, Visualization Sonia Floris:
Investigation, review & editing Rosaria Medda: Methodology, Review
& editing Valeria Sogos: Methodology, Investigation Francesca Pintus:
Methodology, Review & editing Gianluca Gatto: Funding acquisition,
Resources Amit Kumar: Methodology, Investigation, Writing - review
& editing, Data Curation Gunilla Torstensdotter Westermark: Method-
ology, Investigation, Funding acquisition Antonella Fais: Conceptuali-
zation, Methodology, Investigation, Writing
Visualization, Supervision.
- Original Draft,
Declaration of competing interest
[21] I. Ali, R. Rafique, K.M. Khan, S. Chigurupati, X. Ji, A. Wadood, A.U. Rehman, U. Salar,
M.S. Iqbal, M. Taha, S. Perveen, B. Ali, Potent alpha-amylase inhibitors and radical
(DPPH and ABTS) scavengers based on benzofuran-2-yl(phenyl)methanone deriva-
tives: syntheses, in vitro, kinetics, and in silico studies, Bioorg. Chem. 104 (2020),
104238, .
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[22] J. Madhusudhanan, G. Suresh, V. Devanathan, Neurodegeneration in type 2 diabetes:
Alzheimer's as a case study, Brain and Behavior 10 (5) (2020).
Acknowledgment
[23] A. Fais, B. Era, A. Di Petrillo, S. Floris, D. Piano, P. Montoro, C.I.G. Tuberoso, R. Medda,
F. Pintus, Selected enzyme inhibitory effects of Euphorbia characias extracts,
Biomed. Res. Int. 2018 (2018) 1–9.
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brane orientation of IAPP in its natively amidated form at physiological pH in a
membrane environment, Biochim. Biophys. Acta Biomembr. 1808 (10) (2011)
2337–2342.
The present work was partially supported by FIR (Fondo Integrativo
per la Ricerca), University of Cagliari, and by the Novo Nordisk (grant
number: NNF 180c0034256) and Swedish Diabetes Foundation (grant
number: DIA 2017-296) to GTW.
[25] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.T. Heer,
T.A P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS-MODEL: homol-
ogy modelling of protein structures and complexes, Nucleic Acids Res.. 46(W1)
(2018) W296-W303.
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from Saccharomyces cerevisiae and in complex with its competitive inhibitor malt-
ose, FEBS J. 277 (20) (2010) 4205–4214.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2020.12.117.
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