Synthesis, structure, carbohydrate enzyme inhibition, antioxidant activity, in silico drug-receptor interactions and drug-like profiling of the 5-styryl-2-aminochalcone hybrids

Article abstract of DOI:10.3390/molecules26092692

The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzalde-hyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry of the styryl and α,β-unsaturated carbonyl arms, and the presence of NH^… O intramolecular hydrogen bond were validated using¹ H-NMR and X-ray data. The 2-amino-5-styrylacetophenones and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit α-glucosidase and/or α-amylase activities. Their antioxidant properties were evaluated in vitro through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic studies of the most active derivatives from each series against α-glucosidase and/or α-amylase activities have been performed supported by molecular docking studies to determine plausible protein–ligand interactions on a molecular level. The key aspects of the pharmacokinetics of these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human epithelial (A549) cell line to establish their safety profile at least in vitro.

Full text of DOI:10.3390/molecules26092692

molecules
Article
Synthesis, Structure, Carbohydrate Enzyme Inhibition,
Antioxidant Activity, In Silico Drug-Receptor Interactions and
Drug-Like Profiling of the 5-Styryl-2-Aminochalcone Hybrids
Malose J. Mphahlele 1,
*
, Emmanuel Ndubuisi Agbo 1 and Yee Siew Choong 2,
*
1
Department of Chemistry, College of Science, Engineering and Technology, University of South Africa,
Private Bag X06, Florida 1710, South Africa; piruesbest@yahoo.com
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang 11800, Malaysia
Correspondence: mphahmj@unisa.ac.za (M.J.M.); yeesiew@usm.my (Y.S.C.)
2
*
Abstract: The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzalde-
hyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry
of the styryl and α,β
-unsaturated carbonyl arms, and the presence of NH . . . O intramolecular hy-
drogen bond were validated using 1H-NMR and X-ray data. The 2-amino-5-styrylacetophenones
and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit
α
-glucosidase and/or
through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging
assays. Kinetic studies of the most active derivatives from each series against -glucosidase and/or
-amylase activities have been performed supported by molecular docking studies to determine
α-amylase activities. Their antioxidant properties were evaluated in vitro
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
α
ꢀꢁꢂꢃꢄꢅꢆ
α
Citation: Mphahlele, M.J.; Agbo,
E.N.; Choong, Y.S. Synthesis,
plausible protein–ligand interactions on a molecular level. The key aspects of the pharmacokinetics of
these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated
at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated
for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human
epithelial (A549) cell line to establish their safety profile at least in vitro.
Structure, Carbohydrate Enzyme
Inhibition, Antioxidant Activity, In
Silico Drug-Receptor Interactions and
Drug-Like Profiling of the
5-Styryl-2-Aminochalcone Hybrids.
Molecules 2021, 26, 2692. https://
doi.org/10.3390/molecules26092692
Keywords: styryl-aminochalcones; intramolecular hydrogen bonding; α-glucosidase; α-amylase;
antioxidant; drug-receptor interaction; cytotoxicity
Academic Editors: Massimiliano Arca
and Lillian Barros
1. Introduction
Received: 29 March 2021
Accepted: 28 April 2021
Published: 4 May 2021
Non-insulin-dependent diabetes mellitus is a serious global health concern and ac-
cording to the World Health Organization’s (WHO’s) report of 2018, about 90% patients
suffering from diabetes have been diagnosed for type 2 diabetes mellitus (T2DM) [1]. This
metabolic disorder is characterized by an increased level of glucose in the blood known
as post-prandial hyperglycemia (PPHG), which causes vascular complications leading to
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published maps and institutional affil-
iations.
damage of vital organs [
fail to compensate for insulin resistance due to glucotoxicity and lipotoxicity, resulting in
T2DM [ ]. -Amylase is secreted from the pancreas, and this enzyme is responsible for
converting large starch and glycogen molecules into simpler absorbable sugars [
are, in turn, converted by -glucosidase into glucose for intestinal absorption [
inhibition of the activity of intestinal -glucosidase and pancreatic
carbohydrate digestion, in turn, delay glucose uptake leading to reduced blood sugar
levels [ ]. Glucosidase inhibitors continue to attract considerable interest in medicinal
2,3]. The pancreas becomes unable to produce enough insulin or
4
α
1,5] that
α
6,
7]. Dual
α
α-amylase suppresses
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1,8,9
chemistry due to their promising therapeutic potential in the treatment of disorders such as
diabetes, human immunodeficiency virus (HIV) infection, metastatic cancer, and lysosomal
storage diseases [10]. Oxidative stress is one of the most common factors underlying insulin
resistance and diabetes [11
–14]. Oxidative stress may result from the interaction of scav-
enger receptors, such as RAGE, with advanced glycoxidation end-products (AGEs) formed
Molecules 2021, 26, 2692. https://doi.org/10.3390/molecules26092692
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2692
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from the non-enzymatic glycation of proteins, lipids and nucleic acids with reducing sugars
and with the products of glucose metabolism and their oxidation products, a phenomenon
which is fostered by chronic hyperglycemia [15]. It is envisaged that compounds that
can delay, inhibit, or prevent the oxidative damage by scavenging free radicals will help
to ameliorate complications such as coronary heart disease or stroke, and non-vascular
pathologies such as cancer [16].
Nature-based small molecular weight ligands such as stilbenes (1,3-diphenylpropenes)
and chalcones (1,3-diphenylprop-2-ene-1-ones) are not only important for the inhibition of
cancer, but are also useful for the treatment of diabetes, cardiovascular and neurological dis-
eases as well as other chronic diseases [17,18]. Resveratrol (trans-3,4,5-trihydroxystilbene)
A
(Figure 1), for example, has been found to be capable of delaying the absorption of
carbohydrates and lower postprandial glucose concentrations in high-fat diet-induced
hyperglycemic mice [19]. Substituted chalcones either from natural or synthetic sources,
on the other hand, are well known insulin sensitizers, which modulate diverse antidiabetic
targets [20]. Several studies verified the effectiveness of chalcone-based compounds as
antihyperglycemic and/or hypoglycemic agents through in vitro and in vivo experimental
responses [21]. A series of hydroxychalcones (
toluenesulfonamide)chalcone ( ) derivatives have been evaluated for inhibitory effect
in vitro against -glucosidase, -amylase, and -amylase activities [22]. Among these
classes of -unsaturated carbonyl compounds, only the 4-aminochalcones and their
B and C), 4-aminochalcones and the 4-(p-
D
α
α
β
α,β
N-tosyl derivatives were found to exhibit significant and increased inhibitory activities
against these carbohydrate hydrolyzing enzymes, respectively. An in vivo study carried
out with alloxan induced diabetic Wister male albino rats (100 mg/kg) revealed the 4-
aminochalcones to exhibit significant antidiabetic efficacy with decreased blood glucose
levels in the diabetic rats compared with control rats [23]. Molecular docking (in silico)
study of the 4-aminochalcones into α-glucosidase binding sites revealed π–π stacked, π-
cationic, polar, electrostatic and hydrophobic bonding interactions with key residues in
the binding pockets of this enzyme [24]. The relatively planar conformation in the case of
flavonoid derivatives such as quercetin, for example, is considered to allow for increased
conjugative effect resulting in stronger α-glucosidase inhibitory and antioxidant activity
than the non-planar analogues [25]. Computational studies also showed that electron
donating groups on an aromatic ring increase the electron density at the ortho and para
positions of the π-system resulting in increased antioxidant activity while electron with-
drawing groups decrease the activity [24]. Electron donating groups in the case of stilbene
derivatives facilitate the addition of oxygen radicals to the ortho ring position [26].
Figure 1. Structures of resveratrol (A), hydroxychalcones (B,C) and 4-(p-toluenesulfonamide)chalcone (D) with antihyper-
glycemic properties.
Recently the concept of hybrid antihyperglycemic agents has attracted much attention
for treating complications associated with T2DM [27]. Molecular hybrids often act on
multiple therapeutic targets because of the presence of two or more different, covalently
fused pharmacophores. Despite the wide distribution of stilbenes and chalcones in plant
species, molecular hybrids merging these two pharmacophores seem not to have occurred

Molecules 2021, 26, 2692
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in nature. One of the goals of medicinal chemistry research and drug discovery is to
design and develop compounds that both show desired biological activities and are easily
accessible. The glucose lowering potentials of stilbene derivatives and aminochalcones
inspired us to link these two scaffolds based on 2-amino-5-iodoacetophenone as a template
for initial olefination at C-5 with phenylboronic acid derivatives followed by Claisen–
Schmidt aldol condensation of the intermediate 2-amino-5-styrylacetophenones with 3/4-
fluorobenzaldehyde. This rational design was inspired by the literature precedents that the
electron-withdrawing inductive effect of fluorine atom could help the drug molecules in
forming hydrogen and/or halogen bonding interactions with the protein targets, and thus
enhance biological activity [28]. Moreover, we envisaged that the presence of a fluorophenyl
ring on the conjugated scaffold would result in increased
π
-electron distribution to lead to
T-shaped, –alkyl, alkyl)
increased noncovalent or hydrophobic (e.g., stacking,
π–
π
π–
π
π
interactions of compounds with the receptors. In our view, increased conjugative effects
due to the presence styryl and potential hydrogen bonding 2-aminochalcone components
as well as the presence of various substituents on the phenyl groups could increase their
inhibitory effect against α-glucosidase and/or α-amylase activities. Since an ideal anti-
diabetic drug should exhibit hypoglycemic properties and inhibit oxidative stress, we
also evaluated the test compounds for antioxidant potential through the 2,2-diphenyl-
1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic
assays (in vitro) and molecular docking (in silico) were performed on the most active
compounds from each series against these carbohydrate hydrolyzing enzymes to determine
plausible protein-drug interactions on a molecular level. Moreover, the ADME (absorption,
distribution, metabolism, and excretion) properties of the most active derivatives were also
predicted using in silico methods.
2. Results and Discussion
2.1. Chemical Synthesis and Structural Analysis
The synthesis of the title compounds was achieved as outlined in Scheme 1 below via
initial Suzuki–Miyaura cross-coupling of 2-amino-5-iodoacetophenone (
boronic acid derivatives as coupling partners to afford the corresponding 2-amino-5-
styrylacetophenones 2a (Table 1 shows the designation of substituents). These com-
1) with phenyl-
–
d
pounds and their derivatives were characterized using a combination of NMR, FT-IR
and mass spectroscopic techniques complemented with single crystal X-ray diffraction
method. Copies of the NMR (1H- and 13C-) and FT-IR spectra have been included as
1
Figures S1 and S2 of the Supplementary Information (SI), respectively. The H NMR
spectra of 2a–d revealed a set of doublets in the aromatic region for the olefinic protons
with vicinal coupling constant (J) values in the range 15.0–16.5 ppm, which confirmed
the trans geometry of the styryl wing. The protons of the amino group resonate as an
intense singlet in the aromatic region around
δ 6.41 ppm. The trans geometry of the styryl
arm of compounds 2a was distinctly confirmed by XRD structure of compound 2a
–d
(CCDC 2053923) as a representative model (Figure 2a). XRD method also revealed the
presence of an intramolecular hydrogen bond between hydrogen atom of the amino group
and carbonyl oxygen with N(1)-H(1A)...O(1) bond distance of 1.98(3) Å and bond angle of
130(2)◦. Further transformation of compounds 2a
–
d
via Claisen-Schmidt aldol condensa-
tion with 3-fluorobenzaldehyde or 4-fluorobenzaldehyde under basic conditions afforded
the 5-styryl-2-aminochalcone derivatives 3a or 3e
, respectively. The 1H- and 13C NMR
spectra of compounds (refer to Figure S1 in SI) revealed the presence of increased number
of signals in the aromatic region compared to the corresponding substrates . A singlet
for NH2 of compounds still resonated in the aromatic region around = 7.61 ppm. The
presence of a singlet in the 13C-NMR spectra of these compounds around
= 191.0 ppm
for the carbonyl carbon further confirmed their -unsaturated carbonyl nature. The
–
d
–h
3
2
3
δ
δ
α,β
olefinic protons of the styryl and chalcone wings resonate as two sets of doublets among
the aromatic proton signals with vicinal coupling constant (J) values of 15.0–16.5 ppm
consistent with their trans geometry. The trans geometry of the styryl and chalcone arms

Molecules 2021, 26, 2692
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of compounds 3a
–h
were distinctly confirmed by the XRD structure of compound 3f
(CCDC 2035809) as a representative model within these series (Figure 2b). XRD method
revealed the existence of a six-membered intramolecular hydrogen bonded ring (graph set
descriptor, S(6)) involving hydrogen atom of the amino group and carbonyl oxygen with
N(1)-H(1A)...O(2) bond distance of 1.88(2) Å, which helps to stabilize the planarity of these
molecular constructs. Integrating intramolecular hydrogen bonding formation in drug
design has become an exciting challenge in medicinal or bioorganic chemistry especially
in the context of drug-receptor interactions. Intramolecularly hydrogen bonded drug-like
molecules have been found to exhibit favorable alignment with the protein pocket resulting
in increased ligand-receptor interactions [29]. Moreover, conformational restriction of small
drug molecules due to intramolecular hydrogen bonds results in increased lipophilicity,
membrane permeability and pharmacological activity.
O
O
O
I
Ar
Ar(OH)2, PdCl2(PPh3)2, PCy3,
K2CO3, DMF (aq), 90 oC
Ar
3/4-FC6H4CHO, KOH,
EtOH, RT, 24 h
CH3
F
CH3
NH2
NH2
NH2
1
2a d
3a-h
-
Scheme 1. Synthesis of the 5-styryl-2-aminochalcone hybrids 3a–h.
Table 1. Designation and substitution of aryl groups for 3a–h.
Ar
2a–d
3a–d
3e–h
C6H5-
4-FC6H4-
4-ClC6H4-
4-MeOC6H4-
2a
2b
2c
3-F (3a)
3-F (3b)
3-F (3c)
3-F (3d)
4-F (3e)
4-F (3f)
4-F (3g)
4-F (3h)
2d
(a)
(b)
Figure 2. Molecular structure of compound 2a
(a) and 3f (b) determined from single-crystal X-ray
data, showing the atom-labelling scheme, which differs from systematic numbering.

Molecules 2021, 26, 2692
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Compounds 3a
–
h
and their corresponding precursors 2a
–d
were, in turn, evaluated
for inhibitory properties against
α
-glucosidase and α-amylase, and also for DPPH and NO
free radical scavenging potential.
2.2. Biological Evaluation
The test compounds were evaluated for inhibitory activity in vitro against yeast
α
-glucosidase from Saccharomyces cervisiae using acarbose as a reference standard. Acar-
bose is a competitive
α-glucosidase inhibitor [30] which has been found to delay the
absorption of carbohydrate from the small intestine and to reduce postprandial hyper-
glycemia in patients with T2DM than metformin and sulfonylureas [31]. The half max-
imal inhibitory concentration (IC50) values were calculated from the dose-dependent
curves as the concentrations of inhibitors required to decrease 50% of the enzyme activ-
ity (Table 2). Only compound 2a within the series 2a
–
d
was found to exhibit significant
-glucosidase compared to acarbose (IC50 = 0.95 0.28 M)
0.10 M. The trend in activity of these compounds decreases
inhibitory effect against
with an IC50 value of 5.4
α
±
µ
±
µ
with the increasing size of the substituent at the para position of the styryl arm as fol-
lows: C6H5- (2a) > 4-FC6H4- (2b) > 4-ClC6H4- (2c) > 4-CH3OC6H4- (2d). A combination
of the 50-styryl group and 20-amino-3-fluorochalcone scaffold resulted in significantly
reduced inhibitory effect for 3a (IC50 = 17.8
ity against -glucosidase, on the other hand, was observed for 3b with a combination
of 50-(4-fluorostyryl) group and 20-amino-3-fluorochalcone scaffold, and its IC50 value
is 6.1 0.24
M. The analogous 50-(4-chlorostyryl) substituted derivative 3c exhibited
±
0.32 µM). An improved inhibitory activ-
α
±
µ
slightly improved activity (IC50 = 12.6
±
0.31 µM) compared to the corresponding substrate
2c. A combination of strongly lipophilic 50-(4-methoxystyryl) group and the 20-amino-
3-fluorochalcone moiety on the framework of 3d resulted in improved activity0against
0
α
-glucosidase (IC50 = 9.4 ± 0.50 µM). Compound 3e comprises the 5 -styryl and 2 -amino-
4-fluorochalcone moieties and this derivative exhibited the highest activity amongst the
5-styryl-2-aminochalcone hybrids with an IC50 value of 5.1
±
0.61 µM. Its activity is com-
parable to that of the corresponding substrate 2a. A combination of the 50-(4-fluorostyryl)
and 20-amino-4-fluorochalcone moieties resulted in significant inhibitory activity for 3f
0
(IC50 = 6.9
±
0.37
µ
M). The presence of the electron withdrawing 5 -(4-chlorostyryl) group
on the 20-amino-4-fluorochalcone scaffold, on the other hand, resulted in significantly
reduced activity for 3g (IC50 = 19.2
was also observed for hybrid 3h substituted with a strongly
methoxystyryl group with an IC50 value of 10.5
slightly less active than the isomeric 3d.
±
0.47
µ
M). An improved activity against
-electron delocalizing 4-
M. However, this compound is
α-glucosidase
π
±
0.18 µ
α
-Amylase assay of compounds
-amylase inhibitor screening kit using acarbose (IC50 = 1.03
-amylase inhibitor from Triticum aestivum (IC50 = 0.31 0.05 M) as reference standards
for the assay. 2-Amino-5-styrylacetophenone 2a was found to be the most active within the
category 2a and to exhibit significant inhibitory activity against -amylase compared
to the reference standards with an IC50 value of 2.3 0.20 M. Significantly reduced
inhibitory effect against -amylase activity was, however, observed for 2b substituted with
an electron withdrawing fluorine atom at the para position of the phenyl group with an
IC50 value of 19.3 0.42 M. The 4-chlorostyryl derivative 2c, on the other hand, exhibited
significant activity than 2b with an IC50 value of 5.8 0.51 M. The presence of a bulky
4-methoxystyryl arm on the scaffold of 2d resulted in moderate inhibitory effect against
amylase with an IC50 value of 9.0 0.31 M. The 5-styryl-2-aminochalcones 3a exhibited
2
and
3
was carried out following protocol enclosed
in
α
α
±
0.05 M) and specific
µ
±
µ
–
d
α
±
µ
α
±
µ
±
µ
α
-
±
µ
–h
different trend in activity depending on the position of fluorine on the B-ring of the
chalcone framework. A combination of the 50-styryl group and 20-amino-3-fluorochalcone
scaffold resulted in significantly reduced activity for the styryl-chalcone hybrid 3a with an
IC50 value of 10.7
other hand, exhibited significant inhibitory effect against
1.6 0.52 M. A similar trend in activity was observed for the 2 -amino-5 -(4-fluorostyryl)-
±
0.21 µ
M. The isomeric 20-amino-50-styryl-4-fluorochalcone 3e, on the
α
-amylase with an IC50 value of
0
0
±
µ

Molecules 2021, 26, 2692
6 of 19
0
0
3-fluorochalcone 3b and the isomeric 2 -amino-5 -(4-fluorostyryl)-4-fluorochalcone 3f with
0
0
IC50 values of 15.6
±
0.60
µ
M and 9.5
±
0.41 µ0M, respectively. 2 -Amino-5 -(4-chlorostyryl)-
0
3-fluorochalcone 3c and its isomer 2 -amino-5 -(4-chlorostyryl)-4-fluorochalcone 3g, on the
other hand, exhibited improved activity against this enzyme with IC50 values of 2.5
µM
and 1.7 0.25
±
µ
M, respectively. Both the 20-amino-50-(4-methoxystyryl)-3-fluorochalcone
3d and the isomer 3h were found to be moderately active against
values of 7.0 ± 0.45 µM and 7.6 ± 0.20 µM, respectively.
α-amylase with IC50
Table 2. α-Glucosidase and α-amylase inhibition, and antioxidant activity of 2a–d and 3a–h.
(IC50 (SD) µM)
Compound
α-Glucosidase
α-Amylase
DPPH
NO
2a
2b
2c
2d
3a
3b
3c
3d
3e
5.4 ± 0.10
12.7 ± 0.16
15.9 ± 0.34
30.7 ± 0.42
17.8 ± 0.32
6.1 ± 0.24
12.6 ± 0.31
9.4 ± 0.50
5.1 ± 0.61
6.9 ± 0.37
19.2 ± 0.47
10.5 ± 0.18
0.95 ± 0.28
2.3 ± 0.20
19.3 ± 0.42
5.8 ± 0.51
9.0 ± 0.31
10.7 ± 0.21
15.6 ± 0.60
2.4 ± 0.10
7.0 ± 0.45
1.6 ± 0.52
9.5 ± 0.41
1.7 ± 0.25
7.6 ± 0.20
1.03 ± 0.05
7.1 ± 0.32
15.8 ± 0.21
10.4 ± 0.15
9.3 ± 0.23
21.6 ± 0.32
6.6 ± 0.33
15.3 ± 0.10
3.9 ± 0.21
4.2 ± 0.43
18.5 ± 0.37
5.2 ± 0.53
25.8 ± 0.43
-
8.3 ± 0.22
7.2 ± 0.40
11.6 ± 0.43
25.9 ± 0.61
18.9 ± 0.54
5.3 ± 0.34
10.6 ± 0.30
7.9 ± 0.28
20.5 ± 0.46
8.8 ± 0.33
15.1 ± 0.19
20.2 ± 0.23
-
3f
3g
3h
Acarbose
α-Amylase
inhibitor
Ascorbic acid
-
0.31 ± 0.05
-
-
-
-
4.2 ± 0.27
6.14 ± 0.21
IC50 values (µM) were calculated from log dose inhibition curves and are expressed as means
±
standard deviation
(SD) of three independent experiments.
Hitherto, the 2-aminochalcones with different substitution pattern on the B-ring [32
]
and the 4-aminochalcones and the 4-(sulfonamido)-substituted chalcones [33] exhibited
no antioxidant activities by the DPPH radical scavenging method. Reduced or lack of
antioxidant activity of these compounds was attributed to the presence of intramolecu-
lar N-H···O hydrogen bond between amino and kenone group of the 2-aminochalcones
and their sulfanamido derivatives which is envisaged to deactivate the unsaturated moi-
ety [32,34]. The antioxidant properties of (E)-stilbenes, on the other hand, has been found
to be dependent on the electron-donating or electron-withdrawing nature of the functional
groups at the 4 and 40 positions, respectively [35]. A combination of the styryl and the
electrophilic α,β-unsaturated carbonyl arms on the same molecular framework encour-
aged us to evaluate the test compounds for antioxidant properties through the DPPH and
NO radical scavenging assays. Moderate free radical scavenging activity was observed
for the 2-amino-5-styrylacetophenones 2a
with the IC50 values in the range of 7.1
and IC50 values of 7.2 0.40 M to 25.9
potential to inhibit both -glucosidase and
stress. Significantly reduced antioxidant activity in the DPPH and NO radical scaveng-
ing assays was observed for 3a with IC50 values of 21.6 0.32 M and 18.9 0.54 M,
respectively. The presence of a moderately -electron delocalizing fluorine atom at the
para position of the styryl arm of 3b with significant -glucosidase inhibitory effect, on
the other hand, resulted in significantly increased free radical scavenging activity with
IC50 values of 6.6 0.33 M and 5.3 0.34 M in the DPPH and NO assays, respectively.
Reduced DPPH and NO free radical scavenging activities were observed for 3c with a
–
d
against ascorbic acid (IC50 = 4.2
0.32 M to 15.8 0.21 M in the DPPH assay
0.61 M in the NO assay. Compound 2a has
-amylase activities, and to reduce oxidative
±
0.27 µM)
±
µ
±
µ
±
µ
±
µ
α
α
±
µ
±
µ
π
α
±
µ
±
µ
0
0
combination of the 5 -(4-chlorostyryl) arm and 2 -amino-3-fluorochalcone framework with
IC50 values of 15.3 0.10 M and 10.6 0.30 M, respectively. A strongly -delocalization
4-methoxystyryl wing linked to position-5 of the 20-amino-3-fluorochalcone framework of
±
µ
±
µ
π

Molecules 2021, 26, 2692
7 of 19
3d resulted in significantly increased free radical scavenging activity for this compound
with an IC50 value of 3.9
3e against both carbohydrate hydrolyzing enzymes was found to exhibit comparable
±
0.21 µ
M. The most active 20-amino-50-styryl-4-fluorochalcone
free radical scavenging activity to ascorbic acid in the DPPH assay with an IC50 value
of 4.2
activity with an IC50 value of 20.5
for 20-amino-50-(4-fluorostyryl)-4-fluorochalcone 3f in the DPPH assay with an IC50 value
of 18.5 0.37 M. However, this compound exhibited significant NO radical scavenging
activity with an IC50 value of 8.8 0.33
M. A combination of 50-(4-chlorostyryl) group
and 20-amino-4-fluorochalcone scaffold resulted in significant DPPH scavenging activity
for 3g with an IC50 value of 5.2 0.53 M. This compound was, however, found to ex-
±
0.43
µ
M. However, 3e exhibited significantly reduced NO radical scavenging
±
0.46 M. Reduced antioxidant activity was observed
µ
±
µ
±
µ
±
µ
hibit reduced NO radical scavenging activity with an IC50 value of 15.1
±
0.19 µM. The
presence of a strongly electron donating 50-(4-methoxystyryl) arm on the scaffold of the
20-amino-4-fluorochalcone 3h resulted in significantly reduced activity for this compound
in both the DPPH (IC50 = 25.8
±
0.43
µ
M) and NO (IC50 = 20.2
±
0.23 µM) radical scaveng-
ing assays. The observed free radical scavenging activities of these molecular hybrids is
presumably due to the presence of styryl arm on the intramolecularly hydrogen bonded
2-aminochalcone scaffold.
Compounds 2a and 3e which exhibited relatively higher inhibitory effect against
α
-glucosidase and α-amylase activities and free radical scavenging properties, were in
turn, subjected to kinetic studies on both enzymes to elucidate the plausible molecular
mechanism of inhibition.
2.3. Kinetic Studies on 2a and 3e
The enzyme mode of inhibition of compounds 2a and 3e against
α-glucosidase and
α
-amylase were evaluated by constructing the Lineweaver-Burk and the Dixon plots
at increasing substrate and inhibitor concentrations. The Lineweaver-Burk plot of 1/V
versus 1/[S] in the presence of different concentrations of compound 2a in the case of
α
-glucosidase gave a series of straight lines that intersect on the x-axis (Figure 3a). The plot
shows an unchanged Michaelis constant (Km) value of 0.31 0.02 and a decrease in the
velocity of the reaction (Vmax) values (0.051–0.012 M/min). The Dixon plot (Figure 3b)
has straight lines that intersect on the x-axis with the calculated Ki value of 0.30 0.04 M,
±
µ
±
µ
and this observation is associated with non-competitive mode of inhibition confirming the
Lineweaver-Burk data. Compound 3e, on the other hand, showed increasing Km values
0.25–0.35) with relatively unchanged Vmax value of 0.01
Dixon plot of 3e (Figure 4b) showed set of straight lines intersecting above the x-axis with
Ki value of 1.48 0.05 M. Both the Lineweaver-Burk and the Dixon plots for compound
±
0.002 µM/min (Figure 4a). The
±
µ
3e suggested a mixed mode of inhibition for this compound. The compound probably
binds to the active site and other allosteric sites on the enzyme.
2.3.1. Molecular Docking Studies
Molecular docking (in silico) has been employed to obtain a theoretical/hypothetical
model for potential binding modes of the test compounds against
-amylase binding sites. The estimated binding free energy of these compounds (refer
to Table 3) show that 2b 3a 3b and 3h were the least favorable ligands against
glucosidase. On the other hand, derivatives 2b 2d and 3h represent the least favored
for binding into -amylase. Compounds 3e and 3g are predicted to have the strongest
α-glucosidase and
α
,
,
–
d
α-
,
α
and comparable binding affinities against
above-mentioned inhibition assay results.
α-amylase and these values correlate with the
The interactions between the test compounds and the enzymes were further analysed
to compare their conformation in the binding site of -glucosidase and -amylase. The
smaller size and volume of compounds 2a and 2b resulted in lower number of hydrophobic
interactions with -glucosidase compared to 3b 3e and 3g (Figure 5). A similar trend
is observed for 2c and 2d compared to derivatives 3a 3c 3d 3f and 3h Figure S3). It
α
α
α
,
,
,
,
(

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is also interesting to note that the most favorable compound in the first series, i.e., 2a
and series
-amylase (Figure 6). Such
compounds (2b and 3b). Relatively reduced hydrophobic interactions were observed for
the smaller 2c and 2d compared to hybrids 3a 3c 3d 3f and 3h in -amylase (Figure S4).
3
(namely, 3e and 3g) form
π–π stacking interaction with the side chain of
α
π–π
stacking interaction was not observed in the least favorable
,
,
,
α
The hydrogen bond was mainly contributed by the carbonyl group of the test compounds.
Figure 3. Lineweaver-Burk (a) and Dixon (b) plots for 2a against α-glucosidase.

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0 µM
0.1 µM
0.5 µM
1 µM
2.5 µM
5 µM
900
800
700
600
500
400
300
200
100
0
y = 54.815x + 154.11
y = 43.128x + 117.43
y = 33.73x + 103.94
y = 24.954x + 89.65
y = 18.458x + 65.088
y = 10.907x + 43.083
-6
-4
-2
0
2
4
6
8
10
12
-100
-200
(mM)
1/[SUBSTRATE]
(a)
5 mM
1 mM
0.5 mM
0.1 mM
800
700
600
500
400
300
200
100
0
y = 91.12x + 154.8
y = 54.477x + 105.71
y = 36.557x + 75.389
y = 18.886x + 46.022
-4
-3
-2
-1
0
1
2
3
4
5
6
7
-100
-200
(µM)
[INHIBITOR]
(b)
Figure 4. Lineweaver-Burk (a) and Dixon (b) plots for 3e against α-glucosidase.
2.3.2. Prediction of Pharmacokinetic Properties of Compounds 2a, 3e and 3g
In the last part of this investigation, we predicted the drug-likeness of the most active
compounds using an in silico method. Drug-likeness is a complex balance of various
molecular properties such as hydrophobicity, electronic distribution, hydrogen bonding
characteristics, molecule size and flexibility and the presence of various pharmacophoric
features [36]. In silico prediction of properties such as oral absorption, blood-brain barrier
penetration, toxicity, metabolism, aqueous solubility, logP, pKa, half-life, and plasma
protein binding contribute significantly to drug’s success in the drug discovery process,
and minimizes the pharmacokinetic failures at various clinical phase. Bioavailability of the
most active compound from each series, namely, 2a
, 3e and 3g is predicted at theoretical
level through the Lipinski rule of five (molecular weight (< 500), hydrogen-bond donor

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(< 5), hydrogen-bond acceptor (< 10) and cLogP (< 5), which describes the relationship
between physicochemical and pharmacokinetic properties of drugs (Table 4). Compound
2a did not violate any Lipinski rule of five, but compounds 3e and 3g violate one rule.
Metabolic diseases such as diabetes require a stable long-term therapy accompanied
by well tolerated and low toxicity, and this encouraged us to evaluate the safety pro-
file of 2a and 3e as potential antidiabetic agents at least in vitro. The compounds were
evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the
adenocarcinomic human epithelial (A549) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium (MTT) assay. Preliminary results of this study (Table 5) revealed no
effect of these compounds on the viability of the Vero and A549 cells compared doxorubicin
used as a reference standard at the same concentrations.
Table 3. The predicted binding free energy values of 2a–d and 3a–h obtained from docking simulation
of the test compounds against α-glucosidase and α-amylase.
Binding Free Energy (kcal/mol)
Compound
α-Glucosidase
α-Amylase
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
−7.78
−6.81
−7.63
−7.44
−6.70
−6.32
−6.75
−6.71
−7.30
−6.45
−7.87
−6.71
−7.06
−6.33
−7.03
−6.43
−8.40
−8.34
−8.50
−9.31
−9.37
−8.32
−9.66
−6.96
3g
3h
Figure 5. The interaction between the most (2a, 3e and 3g) and the least (2b and 3b) active compounds against α-glucosidase.

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Figure 6. The interaction between the most (2a, 3e and 3g) and the least (2b and 3b) active compounds against α-amylase.
Table 4. Pharmacokinetics properties predictions of compounds 2a, 3e and 3g.
Compound
Property
2a
3e
3g
miLogP
Topological polar surface area; TPSA (Å)
Absorption (%); 109-(0.345 × TPSA)
Number of atom
3.81
43.09
94.13
18
237.30
229.70
2
5.94
43.06
94.14
26
343.40
316.90
2
6.62
43.09
94.13
27
377.85
330.43
2
Molecular weight
Molecular volume
Hydrogen bond acceptor
Hydrogen bond donor
Rotatable bonds
2
3
2
5
2
5
Lipinski’s violation
0
1
1
Table 5. The cytotoxicity of compounds 2a and 3e on Vero and A549 cell lines.
Cytotoxicity, IC50 (µM)
Compound
Vero Cells
A549 Cells
2a
3e
164.56 ± 0.59
220.0 ± 0.57
0.66 ± 0.12
65.70 ± 0.14
204.00 ± 0.55
1.14 ± 0.23
Doxorubicin
3. Materials and Methods
3.1. Instrumentation
The melting point values of the test compounds were recorded on a Thermocouple
digital melting point apparatus (Mettler Toledo LLC, Columbus, OH, USA). The infrared
(IR) spectra were recorded using the thin-film method on a Bruker VERTEX 70 FT-IR
Spectrometer (Bruker Optics, Billerica, MA, USA) equipped with an ATR (diamond at-
tenuated total reflectance) accessory. Merck kieselgel 60 (0.063–0.200 mm) (Merck KGaA,
Frankfurt, Germany) was used as a stationary phase for column chromatography. The 1H
NMR and 13C NMR spectra were recorded as deuterated chloroform (CDCl3) or dimethyl
sulfoxide ((CD3)2SO) solutions using Agilent 500 MHz NMR spectrometer (Agilent Tech-

Molecules 2021, 26, 2692
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nologies, Oxford, UK) operating at 500 MHz and 125 MHz for 1H and 13C, respectively.
The chemical shifts are quoted relative to tetramethylsilane (TMS) used as an internal
reference standard (
follows: chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet
sept = septet m = multiplet, br = broad), coupling constant J (Hz), and integration. The
δ = 0.00 ppm) or to residual protonated solvent. Data are presented as
,
,
high-resolution mass spectra were recorded at an ionization potential of 70 eV using Micro-
mass Autospec-TOF (double focusing high resolution) instrument (Waters Corp., Milford,
MA, USA).
3.2. Synthesis of 2-Amino-5-Iodoacetophenone (1)
A stirred solution of 2-aminoacetophenone (5.00 g, 37.04 mmol, purity = 98%) in
acetonitrile (40 mL) was treated slowly with N-iodosuccinimide (8.32 g, 37.04 mmol,
purity = 95%) at room temperature. The mixture was stirred at room temperature for 1 h,
and then poured into an ice-cold saturated aqueous solution of sodium thiosulphate. The
resultant precipitate was filtered, washed with an ice cold water, and recrystallized from
ethanol to afford 2-amino-5-iodoacetophenone as a brown solid (8.70 g, 90%), mp 97−98 ◦C;
FTIR (ATR)
ν
= 517, 623, 671, 738, 821, 887, 956, 1292, 1310, 1419, 1460, 1530, 1572, 1602,
max
1627 (C=O), 3310, 3424 (NH2) cm−1; 1H NMR (CDCl3)
δ = 2.73 (3H, s, -CH3), 5.73 (1H, br s,
NH2), 6.64 (1H, d, J = 8.7 Hz, H-3), 7.65 (1H, d, J = 8.7 Hz, H-4), 8.14 (1H, s, H-6); 13C NMR
(CDCl3) δ = 28.1, 75.4, 119.6, 120.5, 140.5, 142.6, 149.7, 199.8 (C=O).
3.3. Typical Procedure for the Synthesis of 2-Amino-5-Styrylacetophenones 2a–d
A mixture of
1
(0.50 g, 1.92 mmol), PdCl2(PPh3)2 (0.07 g, 0.11 mmol, purity
≥
99%),
PCy3 (0.27 g, 0.20 mmol, purity = 95%) and K2CO3 (0.27 g, 2.30 mmol, purity
≥
99%) and
phenylboronic acid (0.40 g, 2.30 mmol, purity = 95%) in 3:1 DMF–water (v/v; 20 mL) in
a two-necked round bottom flask equipped with a condenser and rubber septum was
purged with nitrogen gas for 30 min. A balloon filled with nitrogen gas was connected to
◦
the top of the condenser, and the reaction mixture was stirred at 70 C for 3 h. The mixture
was quenched with an ice-cold water, and the product was extracted into chloroform.
The combined organic layers were washed with water and dried over anhydrous MgSO4.
The salt was filtered off and the solvent was evaporated under reduced pressure on a
rotary evaporator. The residue was purified by column chromatography on silica gel using
2:1 toluene-ethyl acetate (v/v) mixture as an eluent. Compounds 2a–d were prepared in
this fashion.
(E)-1-(2-Amino-5-styrylphenyl)ethan-1-one (2a)
Yellow solid (0.33 g, 73%), mp. 119–120 ◦C; FTIR (ATR)
ν
= 418, 537, 688, 818, 947,
max
1169, 1194, 1213, 1422, 1494, 1545, 1572, 1618, 1649, 3343, 3473 cm−1; 1H NMR (DMSO-d6)
δ
= 2.86 (3H, s, -CH3), 7.01 (1H, d, J = 8.5 Hz, H-40), 7.29 (1H, d, Jtrans = 16.3 Hz, H-
α
), 7.43
), 7.63 (3H, t, J = 7.5 Hz, H-3 ,5 ), 7.68 (2H, s, -NH2), 7.81 (2H, d,
= 28.3,
0
0
(1H, d, Jtrans = 17.0 Hz, H-
β
J = 7.0 Hz, H-2 ,6 ), 7.90 (1H, d, J = 8.0 Hz), 8.20 (1H, s, H-6); 13C NMR (DMSO-d6)
δ
0
0
117.9, 124.1, 124.5, 126.3, 127.2, 128.8, 129.1, 129.2, 131.0, 132.0, 138.2, 151.2, 200.6; HRMS
(ES): m/z [M + H]+ calc for C16H16NO: 238.1232; found 238.1233.
(E)-1-(2-Amino-5-(4-fluorostyryl)phenyl)ethan-1-one (2b)
◦
Yellow solid (0.34 g, 70%), mp. 149–150 C; FTIR (ATR)
ν
= 534, 598, 830, 926, 1209,
max
1226, 1496, 1506, 1544, 1570, 1614, 1650, 3347, 3482 cm−1; H NMR (DMSO-d6)
δ
= 2.63
(3H, s, -CH3), 6.40 (2H, s, -NH2), 6.66 (1H, d, J = 8.6 Hz, H-3), 6.85 (1H, d, Jtrans = 16.3 Hz,
H- ), 6.93 (1H, d, Jtrans = 16.3 Hz, H-
), 7.03 (2H, t, J = 8.7 Hz, H-30,50), 7.44 (2H, dd,
1
α
β
J = 5.4 Hz and 8.7 Hz, H-20,60), 7.52 (1H, dd, J = 2.0 Hz and 8.6 Hz, H-4), 7.76 (1H, d,
J = 2.0 Hz, H-6); 13C NMR (DMSO-d6)
δ
= 27.9, 115.6 (d, 2JCF = 21.3 Hz), 117.8, 118.0, 124.2,
3
4
125.2, 127.5 (d, JCF = 7.9 Hz), 127.7, 127.8, 130.9, 131.7, 133.8, (d, JCF = 2.2 Hz), 149.9,
162.0 (d, 1JCF = 245.0 Hz), 200.7; HRMS (ES): m/z [M + H]+ calc for C16H15FNO: 256.1138;
found 256.1141.

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(E)-1-(2-Amino-5-(4-chlorostyryl)phenyl)ethan-1-one (2c)
Yellow solid (0.41 g, 78%), mp. 137–138 ◦C; FTIR (ATR)
ν
= 567, 625, 817, 851, 964,
max
1191, 1215, 1426, 1507, 1546, 1616, 1642, 3348, 3482 cm−1; 1H NMR (DMSO-d6)
δ
= 2.62 (3H,
s, -CH3), 6.41 (2H, s, -NH2), 6.65 (1H, d, J = 8.6 Hz, H-3), 6.83 (1H, d, Jtrans = 16.3 Hz, H- ),
7.00 (1H, d, Jtrans = 16.3 Hz, H-
), 7.30 (2H, d, J = 8.5 Hz, H-30,50), 7.39 (2H, d, J = 8.5 Hz,
H-20,60), 7.51 (1H, dd, J = 2.0 Hz and 8.6 Hz, H-4), 7.75 (1H, d, J = 2.0 Hz, H-6); 13C NMR
α
β
(DMSO-d6) δ = 27.9, 117.8, 118.0, 124.0, 125.0, 127.6, 128.6, 128.9, 131.1, 131.8, 132.6, 136.1,
150.1, 200.7; HRMS (ES): m/z [M + H]+ calc for C16H15ClNO: 272.0848; found 272.0842.
(E)-1-(2-Amino-5-(4-methoxystyryl)phenyl)ethan-1-one (2d)
◦
Yellow solid (0.35 g, 69%), mp. 139–140 C; FTIR (ATR)
ν
= 520, 806, 851, 956, 1029,
δ = 2.55 (3H, s, -CH3), 3.75
max
1243, 1511, 1619, 1651, 3357, 3490 cm−1; 1H NMR (DMSO-d6)
(3H, s, -OCH3), 6.77 (1H, d, J = 8.5 Hz, H-3), 6.90 (2H, d, J = 8.5 Hz, H-30,50), 6.93 (1H,
d, Jtrans = 16.5 Hz, H-
α), 7.00 (1H, d, Jtrans = 16.5 Hz, H-β), 7.33 (2H, s, -NH2), 7.45 (2H,
d, J = 8.5 Hz, H-20,60), 7.57 (1H, dd, J = 2.0 Hz and 9.0 Hz, H-4), 7.84 (1H, d, J = 1.5 Hz,
H-6); 13C NMR (DMSO-d6)
δ = 28.4, 55.4, 114.6, 117.1, 117.9, 124.2, 124.5, 126.5, 127.5, 130.8,
131.3, 131.8, 150.9, 158.8, 200.6; HRMS (ES): m/z [M + H]+ calc for C17H18NO2: 268.1338;
found 268.1337.
3.4. Typical Procedure for the Claisen-Schmidt Aldol Condensation of 2a–d to Afford 3a–h
A mixture of
(3 pellets, purity
2
≥
(1 equiv.), 4-fluorobenzaldehyde (1.2 equiv., purity = 98%) and KOH
85%) in absolute ethanol (2.5 mL/mmol of ) was stirred for 24 h at
2
room temperature, and then quenched with an ice-cold water. The precipitate was filtered
and purified by column chromatography on silica gel using toluene as an eluent. The
following products were prepared in this fashion.
(E)-1-(2-Amino-5-((E)-styryl)phenyl)-3-(3-fluorophenyl)prop-2-en-1-one (3a)
◦
Orange solid (0.09 g, 66%), mp. 129–130 C; FTIR (ATR)
ν
= 534, 622, 701, 826, 954,
max
1189, 1210, 1545, 1567, 1614, 1649, 2922, 3347, 3465 cm−1; 1H NMR (DMSO-d6) δ0= 6.91 (1H,
d, J = 9.0 Hz, H-3), 7.06 (1H, d, Jtrans = 16.0 Hz, H-
α
), 7.28 (1H, d, J = 8.5 Hz, H-6 ), 7.29 (1H,
d, Jtrans = 16.50Hz, H-
β
), 7.34 (1H, t, J = 9.0, H-4”), 7.42 (2H, t, J = 7.5, H-4”), 7.56 (1H, d,
0
J = 8.0 Hz, H-2 ), 7.60 (2H, d, J = 8.0 Hz, H-2 ), 7.71 (2H, s, NH2), 7.73 (1H, d, Jtrans = 15.5 Hz
,
H-
α
0), 7.75 (2H, t, J = 7.5, H-4”), 7.91 (1H, dd, J = 6.0 Hz and 9.0 Hz, H-4), 8.15 (1H, d,
Jtrans = 15.0 Hz, H-β0), 8.30 (1H, s, H-6); 13C NMR (DMSO-d6)
δ
= 115.3 (d, 2JCF = 21.75 Hz),
117.5 (d, 2JCF = 20.75 Hz), 117.7, 118.3, 124.6, 124.8, 125.4, 126.1 (d, 4JCF = 2.0 Hz), 126.5,
127.5, 129.1, 129.4, 131.5, 131.6 (d, 4JCF = 1.9 Hz), 132.3, 138.3 (d, 3JCF = 7.6 Hz), 138.5, 141.4
(d, 4JCF = 2.75 Hz), 152.7, 136.2 (d, 1JCF = 242.5 Hz), 190.9; HRMS (ES): m/z [M + H]+ calc
for C23H19FNO: 344.1451; found 344.1451.
(E)-1-(2-Amino-5-((E)-4-fluorostyryl)phenyl)-3-(3-fluorophenyl)prop-2-en-1-one (3b)
◦
Orange solid (0.10 g, 73%), mp. 175–177 C; FTIR (ATR)
ν
= 468, 544, 790, 827, 999,
max
1168, 1186, 1241, 1484, 1507, 1567, 1616, 1646, 3391, 3480 cm−1; 1HNMR (DMSO-d6
)
δ
= 6.84
(1H, d, J = 8.6 Hz, H-3), 7.03 (1H, d, Jtrans = 16.3 Hz, H-
α
), 7.14 (1H, d,
J
trans = 17.0 Hz, H- ),
β
7.18 (2H, t, J = 8.5 Hz, H-30,50), 7.26 (1H, t, J = 9.5 Hz, H-6”), 7.48 (1H, dd, J = 2.0 Hz
and 7.5 Hz, H-2”), 7.44 (2H, dd, J = 5.5 Hz and 8.5 Hz, H-20,60), 7.63 (2H, s, -NH2),
7.66 (1H, d,
J
trans = 17.0 Hz, H-
trans = 15.5 Hz, H-
α
0), 7.65–7.68 (2H, m, Ph), 7.83 (1H, dd, J = 2.0 Hz and
9.0 Hz, H-4), 8.04 (1H, d,
J
β
0), 8.20 (1H, s, H-6); 13C NMR (DMSO-d6)
δ
= 114.9 (d, 2JCF = 21.8 Hz), 115.9 (d, 2JCF = 21.8 Hz), 117.2 (d, 2JCF = 21.8 Hz), 117.5, 118.1,
3
3
123.5, 124.3, 125.2, 125.8, 128.0 (d, JCF = 7.6 Hz), 128.8, 131.2 (d, JCF = 9.5 Hz), 132.0,
4
3
4
134.8 (d, JCF = 2.9 Hz), 138.1 (d, JCF = 7.5 Hz), 141.2 (d, JCF = 2.9 Hz), 152.4, 161.3 (d,
1JCF = 242.8 Hz), 163.0 (d, JCF = 241.75 Hz), 190.6; HRMS (ES): m/z [M + H]+ calc for
1
C23H18F2NO: 362.1356; found 362.1348.

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(E)-1-(2-Amino-5-((E)-4-Chlorostyryl)phenyl)-3-(3-fluorophenyl)prop-2-en-1-one (3c)
◦
Orange solid (0.10 g, 74%), mp. 187–188 C; FTIR (ATR)
954, 1170, 1210, 1245, 1260, 1544, 1570, 1615, 1645, 2852, 2930. 3325, 3478 cm−1; 1H NMR
(DMSO-d6) = 6.85 (1H, d, J = 7.5 Hz, H-3), 7.03 (1H, d, Jtrans = 16.5 Hz, H- ), 7.22 (1H, d,
Jtrans = 17.0 Hz, H-
), 7.26 (2H, t, J = 2.0 Hz, H-2”,6”), 7.39 (2H, d, J = 8.5 Hz, H-30,50), 7.50
(1H, dd, J = 2.0 Hz and 7.5 Hz, H-4”), 7.54 (2H, d, J = 8.5 Hz, H-20,60), 7.63 (2H, s, -NH2),
7.64 (1H, d, Jtrans = 15.5 Hz, H-
0), 7.66 (1H, t, J = 8.5 Hz, H-5”), 7.83 (1H, dd, J = 2.0 Hz
and 9.0 Hz, H-4), 8.07 (1H, d, Jtrans = 15.5 Hz, H-β0), 8.30 (1H, s, H-6); 13C NMR (DMSO-d6)
ν
= 433, 542, 775, 847,
max
δ
α
β
α
δ
= 114.9 (d, 2JCF = 20.9 Hz), 117.2 (d, 2JCF = 20.9 Hz), 117.5, 118.1, 123.2, 124.1, 125.2, 125.8,
125.8, 127.9, 129.1, 129.8, 131.3 (d, 3JCF = 7.6 Hz), 131.5, 132.1, 137.2, 138.1 (d, 3JCF = 7.6 Hz),
141.2 (d, 4JCF = 1.9 Hz), 152.5, 163.1 (d, 1JCF = 242.8 Hz), 190.6; HRMS (ES): m/z [M + H]+
calc for C23H18ClFNO: 378.1061; found 378.1063.
(E)-1-(2-Amino-5-((E)-4-methoxystyryl)phenyl)-3-(3-fluorophenyl)prop-2-en-1-one (3d)
◦
Orange solid (0.10 g, 73%), mp. 154–155 C; FTIR (ATR)
ν
= 534, 685, 782, 824,
max
955, 1165, 1211, 1245, 1508, 1614, 1646, 3328, 3480 cm−1; H NMR (DMSO-d6)
δ = 3.75
1
(3H, s, -OCH3), 6.84 (1H, d, J = 8.5 Hz, H-3), 6.91 (2H, d, J = 8.5 Hz, H-30,50), 7.00 (1H, d,
J
trans = 16.0 Hz, H-α), 7.03 (1H, d, Jtrans = 16.5 Hz, H-β), 7.25 (1H, t, J = 8.0 Hz, H-5”), 7.46
(2H, d, J = 9.0 Hz, H-20,60), 7.50 (1H, d, J = 7.5 Hz, H-6”), 7.58 (2H, s, -NH2), 7.62–7.65 (2H,
m, Ar), 7.66 (1H, d, Jtrans = 15.0 Hz, H-α0), 7.84 (1H, dd, J = 2.0 Hz and 9.0 Hz, H-4), 8.07 (1H,
d, Jtrans = 15.0 Hz, H-β δ = 55.6, 114.6,
0), 8.16 (1H, d, J = 1.5 Hz, H-6); 13C NMR (DMSO-d6)
114.9 (d, 2JCF = 20.7 Hz), 117.3 (d, 2JCF = 20.87 Hz), 117.5, 118.1, 124.4, 124.7, 125.2, 125.8 (d,
4JCF = 1.9 Hz), 126.9, 127.5, 130.7, 130.8, 131.6 (d, 3JCF = 7.6 Hz), 138.1 (d, 3JCF = 7.6 Hz),
141.1, 141.2, 152.1, 158.9, 163.2 (d, 1JCF = 241.9 Hz), 190.6; HRMS (ES): m/z [M + H]+ calc
for C24H21FNO2: 374.1556; found 374.1557.
(E)-1-(2-Amino-5-((E)-styryl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3e)
◦
Orange solid (0.09 g, 69%), mp. 161–162 C; FTIR (ATR)
ν
= 422, 524, 714, 822, 953,
max
1169, 1210, 1469, 1545, 1595, 1616, 1649, 3347, 3479 cm−1; H NMR (DMSO-d6)
δ
= 6.85
), 7.07 (1H, d, J = 9.0 Hz, H-4),
7.20 (1H, t, J = 4.5 Hz, H-30), 7.22 (1H, d, Jtrans = 16.5 Hz, H-
), 7.28 (2H, t, J = 9.0 Hz,
H-300,500), 7.34 (1H, t, J = 8.0 Hz, H-40), 7.53 (2H, d, J = 8.5 Hz, H-30,50), 7.61 (2H, s, NH2),
7.63 (1H, d, Jtrans = 15.5 Hz, H-
0), 7.83 (1H, d, J = 9.0 Hz, H-50), 7.96 (2H, dd, J = 6.0 Hz
and 9.0 Hz, H-200,600), 8.00 (1H, d, Jtrans = 15.5 Hz, H- 0), 8.21 (1H, s, H-6); 13C NMR
(DMSO-d6)
= 116.2 (d, 2JCF = 20.8 Hz), 117.6, 118.1, 124.3, 124.5, 126.3, 127.2, 128.9, 129.1,
1
(1H, d, J = 8.5 Hz, H-3), 7.04 (1H, d, Jtrans = 16.0 Hz, H-
α
β
α
β
δ
3
4
131.2, 131.4 (d, JCF = 8.5 Hz), 131.9, 132.2 (d, JCF = 2.9 Hz), 132.3, 138.2, 141.4, 152.3,
163.4 (d, 1JCF = 246.5 Hz), 190.7; HRMS (ES): m/z [M + H]+ calc for C23H19FNO: 344.1451;
found 344.1457.
(E)-1-(2-Amino-5-((E)-4-fluorostyryl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3f)
◦
Orange solid (0.10 g, 70%), mp. 181–182 C; FTIR (ATR)
ν
= 471, 534, 827, 954,
max
1158, 1179, 1217, 1506, 1566, 1615, 1648, 3309, 3493 cm−1; H NMR (DMSO-d6)
δ
= 6.46
), 7.00 (1H, d,
), 7.04 (2H, t, J = 8.7 Hz, H-30,50), 7.13 (2H, t, J = 8.7 Hz, H-300,500), 7.45
(2H, dd, J = 5.4 Hz and 8.7 Hz, H-20,60), 7.57 (1H, dd, J = 2.0 Hz and 8.7 Hz, H-4), 7.62
(1H, d, Jtrans = 16.3 Hz, H-
0), 7.66 (2H, dd, J = 5.4 Hz and 8.7 Hz, H-200,600), 7.73 (1H, d,
Jtrans = 16.3 Hz, H-
= 115.6 (d, 2JCF
1
(2H, s, NH2), 6.72 (1H, d, J = 8.6 Hz, H-3), 6.90 (1H, d, Jtrans = 16.3 Hz, H-
α
J
trans = 16.3 Hz, H-
β
α
β
0), 7.86 (1H, d, J = 2.0 Hz, H-6); 13C NMR (DMSO-d6)
δ
= 21.0 Hz), 116.1 (d, 2JCF = 21.9 Hz), 117.9, 118.7, 122.5 (d, 4JCF = 2.8), 124.3, 125.4, 127.6
(d, 3JCF = 8.0 Hz), 127.8 (d, 4JCF = 2.1), 129.8, 130.1 (d, 3JCF = 8.3 Hz), 131.4, 131.6, 133.8,
142.0, 150.7, 162.1 (d, 1JCF = 245.1 Hz), 163.9 (d, 1JCF = 249.8 Hz), 191.3; HRMS (ES): m/z
[M + H]+ calc for C23H18F2NO: 362.1356; found 362.1357.

Molecules 2021, 26, 2692
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(E)-1-(2-Amino-5-((E)-4-chlorostyryl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3g)
Orange solid (0.09 g, 74%), mp. 197–198 ◦C; FTIR (ATR)
ν
δ
= 471, 549, 829, 1180,
max
1223, 1279, 1530, 1650, 3369, 3494 cm−1; 1H NMR (DMSO-d6)
= 6.48 (2H, s, NH2), 6.72
),
7.12 (2H, t, J = 8.6 Hz, H-300,500), 7.30 (2H, d, J = 8.7 Hz, H-30,50), 7.41 (2H, d, J = 8.5 Hz,
H-20,60), 7.56 (1H, dd, J = 1.9 Hz and 8.0 Hz, H-4), 7.57 (1H, d, Jtrans = 16.3 Hz, H- 0), 7.66
(2H, dd, J = 5.4 Hz and 8.7 Hz, H-200,600), 7.73 (1H, d, Jtrans = 15.5 Hz, H- 0), 7.87 (1H, d,
(1H, d, J = 8.6 Hz, H-3), 6.87 (1H, d, Jtrans = 16.3 Hz, H-α), 7.00 (1H, d, Jtrans = 16.3 Hz, H-β
α
β
J = 1.8 Hz, H-6); 13C NMR (DMSO-d6) = 116.1 (d, 2JCF = 22.6 Hz), 118.0, 118.7, 122.5 (d,
δ
4JCF = 3.0), 124.2, 125.2, 127.3, 128.6, 128.8, 130.1, 130.3 (d, 3JCF = 8.5 Hz), 131.4, 131.6, 132.6,
1
136.1, 142.1, 150.8, 163.9 (d, JCF = 249.7 Hz), 191.3; HRMS (ES): m/z [M + H]+ calc for
C23H18ClFNO: 378.1061; found 378.1062.
(E)-1-(2-Amino-5-((E)-4-methoxystyryl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3h)
◦
Orange solid (0.11 g, 73%), mp. 153–154 C; FTIR (ATR)
ν
= 446, 472, 548, 827, 959,
max
1008, 1179, 1209, 1505, 1543, 1615, 1647, 3309, 3510 cm−1; H NMR (DMSO-d6)
δ
= 3.75
(3H, s, -OCH3), 6.84 (1H, d, J = 8.5 Hz, H-3), 6.91 (2H, d, J = 8.5 Hz, H-30,50), 7.00 (1H,
d, Jtrans = 16.5 Hz, H- ), 7.01 (1H, d, Jtrans = 15.5 Hz, H- ), 7.28 (2H, t, J = 9.0 Hz, H-
300,500), 7.46 (2H, d, J = 8.5 Hz, H-20,60), 7.57 (2H, s, NH2), 7.62 (1H, d, J = 9.0 Hz, H-4),
7.67 (1H, d, Jtrans = 15.5 Hz, H-
0), 7.96 (2H, dd, J = 6.0 Hz and 9.0 Hz, H-200,600), 8.00
(1H, d, Jtrans = 15.5 Hz, H- = 55.5, 114.6,
1
α
β
α
β
0), 8.17 (1H, s, H-6); 13CNMR (DMSO-d6)
δ
116.2 (d, 2JCF = 21.9 Hz), 117.6, 118.0, 123.6, 124.3, 124.7, 126.7, 127.5, 130.6, 130.9, 131.4 (d,
3JCF = 8.5 Hz), 131.8, 132.2 (d, 4JCF = 2.87), 141.3, 152.0, 158.8, 163.4 (d, 1JCF = 246.5 Hz),
190.8; HRMS (ES): m/z [M + H]+ calc for C24H21FNO2: 374.1556; found 374.1556.
3.5. α-Glucosidase Inhibition Assays of 2a–d and 3a–h
All the tests and analyses were performed in triplicates following a modified published
protocol described in our previous study [37]. The stock solution (200 µM) of the test
compounds and the reference standard, acarbose, were prepared in DMSO and further
diluted with phosphate buffer (100 mM, pH 6.8) to obtain final concentrations of 1. 2.5, 5,
10, 25 and 50
pH 6.8; 30 L) and the test sample (20
the designated wells, and the reaction mixtures were incubated at 37 ◦C for 10 min. 2 mM
p-nitrophenyl- -D-glucopyranoside (20 L) was added to each of the wells containing
µM.
α
-Glucosidase solution (0.48 u/mL, 20
µL), phosphate buffer (100 mM,
µ
µL) as well as the reference standard were added in
α
µ
reaction mixture to initiate the reaction. The reaction was pre-incubated for 30 min at
◦
37 C. The reaction was stopped by the addition of 20
µL of a solution of sodium carbonate
(200 mM) to each well. Five absorbance readings were recorded for each triplicate run
at a wavelength of 400 nm using Varioskan flash microplate spectrophotometer (Thermo
Scientific, Waltham, MA, USA). The average values obtained from the readings were used
to determine the IC50 and standard deviation values. The values were calculated by the
nonlinear regression analysis and expressed as the mean SD of three distinct experiments
using Graph Pad Prism software.
3.6. α-Amylase Inhibition Assays of 2a–d and 3a–h
The α-amylase assay was performed in triplicate using a 96-well plate following
the procedure by the manufacturer as outlined in the
(Catalog No. K482; Bio Vision). The stock solution (100
α
-Amylase Inhibitor Screening Kit
M) of the test compounds, specific
µ
α
-amylase inhibitor from Triticum aestivum and acarbose were prepared in DMSO, and
further diluted with
α
-amylase assay buffer to obtain final concentrations of 1, 2.5, 5, 10, 25
L of -amylase inhibitor and 40 L of assay buffer
were added to 3 wells), enzyme control (50 L of assay buffer was added to three wells) and
50 L of the test compounds were added into the remaining wells. A solution of -amylase
enzyme (50 L) prepared by adding 490 L of assay buffer to 10 L of -amylase enzyme
and 50
µM. For inhibitor control (10
µ
α
µ
µ
µ
α
µ
µ
µ
α
was added to each of the wells containing the reaction mixture to initiate the reaction. The
plate was incubated for 10 min at room temperature in the dark. Five different absorbance

Molecules 2021, 26, 2692
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readings were recorded for each triplicate run at a wavelength of 405 nm using Varioskan
flash microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA). The IC50 and
SD values were calculated using graph pad prism.
3.7. Free Radical Scavenging Assays
3.7.1. Determination of Reducing Activity of the Stable DPPH Radical by 2a–d and 3a–h
The antioxidant activities of the test compounds against ascorbic acid (Sigma Aldrich,
Saint Louis, Missouri, USA) as a positive control were evaluated using 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay developed by Zhu et al. as described in our
previous study [37]. Triplicate solutions (20
DMSO (final concentrations: 5, 10, 25, 50, and 100
well of a 96-well plate. A solution of 0.20 mM DPPH (20
µ
L) of the test compounds and ascorbic acid in
M) were added into each designated
L) in methanol was added to
µ
µ
each well, and the 96-well plate was wrapped with aluminum foil and incubated in the
dark for 45 min. Five absorbance readings were recorded at 512 nm using Varioskan flash
microplate spectrophotometer reader. The average values obtained from the absorbance
readings were used to determine the IC50 and standard deviation values.
3.7.2. NO Free Radical Scavenging Assay
Nitric oxide was generated from sodium nitroprusside and measured by Griess’
reaction following the literature method [38]. The experiment was done in triplicate with
ascorbic acid (vitamin C) used as a positive control for the assay. In a 96 well plate, 5
the test compounds (0.1, 0.5, 1.0, 5.0 and 10.0 M in methanol) or the positive control were
mixed with 5 L of 10 mM sodium nitroprusside prepared in phosphate buffered saline
(pH = 7.4) and allowed to incubate at room temperature for 2.5 h. Afterwards, 30 L of the
µL of
µ
µ
µ
Griess reagent (a mixture of 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride and 2%
sulfanilamide in 5%) was added to each well and the mixtures were and allowed to stand
at room temperature for 30 min. Five absorbance readings were recorded at 546 nm using
Varioskan flash microplate spectrophotometer reader.
3.8. Kinetic Studies
Enzyme kinetics studies were performed on compounds 2a and 3e with increasing
inhibitor and substrate concentrations. In the experimental procedure, inhibitors concen-
trations of 0.1, 0.5, 1, 2.5
were used. 5 L of the inhibitor and 2
each well of the 96-well plate. The reaction was incubated for 20 min at room temperature.
18 L of the substrate, PNPG (4-nitrophenyl -D-glucopyranoside) was then added and
µM, and final substrate concentration were 0.1, 0.5, 1 and 5 mM
µ
µ
L of the enzyme ( -glucosidase) were added to
α
µ
α
absorbance readings recorded after every 2 min at wavelength of 400 nm using a Varioskan
flash microplate spectrophotometer reader. Lineweaver-Burk plot (the inverse of velocity
(1/v) against the inverse of the substrate concentration (1/[S]) were used to determining
the type of inhibition, while the inhibitor constant was obtained from the Dixon plot (the
inverse of velocity (1/v) against concentration of inhibitor at each substrate concentration)
3.9. Molecular Docking of Test Compounds against α-Glucosidase and α-Amylase
We performed docking simulation of the test compounds against both
α-glucosidase
and
α-amylase. The initial coordinates for the test compounds were generated using
Avogadro program [39]. The polar hydrogen atoms of the test compounds were retained
and Gasteiger charges and torsional angles were assigned by AutoDockTools [40]. The
crystal structures of α-glucosidase (PDB code: 5NN8) and α-amylase (PDB code: 5E0F)
were used as the acceptors for the test compounds. The heteroatoms and water molecules
were first removed prior to the addition of polar hydrogen atoms. Kollman–Amber united
atom partial charges and solvation parameters were then assigned using AutoDockTools.
Docking simulation by AutoDock4.2.6 [40] was performed within 50
centered at ligand active site. AutoDock4.2.6 was used to apply 100 Lamarckian genetic
algorithm docking runs with the following parameters: energy evaluation of 2,500,000,
×
50
×
50 grid points

Molecules 2021, 26, 2692
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maximum of 27,000 generation, population of 150, mutation rate of 0.02 and crossover
rate of 0.8. Further interaction analysis using a Protein–Ligand Interaction Profiler was
performed on the ligand in the most populated cluster with most favourable binding
free energy.
3.10. Physicochemical Parameters of 2a, 3e and 3g
The bioactivity score and the molecular properties of the test compounds were calcu-
lated by Molinspiration (www.molinspiration.com, accessed on 4 April 2021). The Lipin-
ski’s rule of five was used to evaluate the drug-likeliness of the test compounds.
3.11. Evaluation of Cytotoxicity (MTT assay) of 2a and 3e on Vero and A549 Cells
The cytotoxicity activity of compounds 2a and 3e was evaluated against doxorubicin
(positive control) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay following a modification of a literature method by Mosmann [41]. The Vero
and A549 cells were maintained in Dulbecco0s modified Eagle0s medium (DMEM; Gibco)
supplemented with 10% Fetal bovine serum (FBS) and 1% penicillin/streptomycin in cul-
◦
ture flasks and incubated at 37 C in 5% CO2. When the cells reached 85% confluency, they
were detached using 2% trypsin. Cell count was performed using a handheld automated
cell counter (Scapter 3.0
bated overnight to allow cell attachment. After 24 h, the cells were treated with different
concentrations (0, 2.5, 5, 10, 25, 50, 100 M) of the test compounds and the reference
stand. The cells were incubated for 24 h followed by treatment with 20 L of MTT solution
(5 mg/mL). After 4 h incubation period, DMSO (100 L) was added to each well to dissolve
™
, Merck). The cells were seeded at 1
×
105 cells/well and incu-
µ
µ
µ
the formazan crystals. Five absorbance readings were recorded at 517 nm using Varioskan
flash microplate spectrophotometer reader.
4. Conclusions
A new set of 5-styryl-2-aminochalcone hybrids has been synthesized and their molec-
ular constructs confirmed using a combination of spectroscopic techniques complemented
with single crystal X-ray diffraction (XRD) method. The co-planarity of the molecular frame-
works of intermediates 2a–d and their styryl-chalcone derivatives 3a–h has been found to
be further stabilized by the presence of a thermodynamically favorable six-membered in-
tramolecular hydrogen bonding interactions between the carbonyl oxygen and the aryl-NH.
Two compounds, namely, 1-(2-amino-5-styrylphenyl)ethan-1-one (2a) and 1-(2-amino-5-
styryl)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3e) exhibited significant inhibitory effect
against both carbohydrate hydrolyzing enzymes (α-glucosidase and α-amylase) and an-
tioxidant activity. These compounds have potential to suppress carbohydrate digestion, in
turn, delay glucose uptake to lead to reduced blood sugar levels. Their antioxidant activity,
on the other hand, make them suitable candidates to delay, inhibit, or prevent the oxidative
damage by scavenging free radicals. Their safety profile was evaluated in vitro against the
Vero and A549 cells, and the preliminary results showed no effect on the viability of both
normal and cancerous cells after 24 h. These compounds will probably inhibit the activi-
ties of α-glucosidase and α-amylase, and also suppress some of the inflammatory effects
mediated by free radicals with minimal or no cytotoxic effects on normal cells. Further
cellular-based in vitro and in vivo studies including bioavailability and cell permeability
would help to clarify the mechanism of action of these compounds in the body. Structural
modifications of these stilbene-chalcone hybrids involving varying substitution pattern
on the styryl and/or chalcone scaffolds can further enhance effectiveness against enzyme
activity resulting in increased antihyperglycemic activity.
Supplementary Materials: Copies of 1H- and 13C NMR spectra (Figure S1) and FT-IR spectra
(Figure S2) of the text compounds, and interactions compounds 2c, 2d
α-glucosidase (Figure S3) and α-amylase (Figure S4).
, 3a, 3c, 3d, 3f and 3h with

Molecules 2021, 26, 2692
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Author Contributions: Conceptualization, literature review, analyses and writing of the original
draft, M.J.M.; synthesis and bioassays, E.N.A.; docking studies Y.S.C.; revision and editing M.J.M.
and Y.S.C. All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded by the University of South Africa and the National Research
Foundation (NRF) in South Africa (NRF GUN: 118554) as well as Universiti Sains Malaysia (RUi
grant: 1001/CIPPM/8011051). The views and opinions expressed herein are those of the authors and
not of the host institutions or the funding bodies.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The CIF files containing complete information on 2a (CCDC 2053923)
and 3f (CCDC 2035809), and are freely available upon request from the following website: www.
ccdc.cam.ac.uk/datarequest/cif, accessed on 4 April 2021 or by contacting the Cambridge Crys-
tallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033; email:
deposit@ccdc.cam.ac.uk.
Acknowledgments: We are grateful to the University of Stellenbosch Central Analytical Facility
(CAF) for mass spectrometric data. Single crystal X-ray analysis was performed at the University of
the Witwatersrand with equipment funded by the NRF Equipment Programme (UID: 78572).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
References
1.
2.
3.
Rafique, R.; Khan, K.M.; Arshia; Kanwal; Chigurupati, S.; Wadood, A.; Rehman, A.U.; Karunanidhi, A.; Hameed, S.; Taha, M.;
et al. Synthesis of new indazole based dual inhibitors of -glucosidase and -amylase enzymes, their in vitro, in silico and
kinetics studies. Bioorg. Chem. 2020, 94, 103195. [CrossRef] [PubMed]
Kawde, A.-N.; Taha, M.; Alansari, R.S.; Almandil, N.B.; Anouar, E.H.; Uddin, N.; Rahim, F. Exploring efficacy of indole-based
dual inhibitors for α-glucosidase and α-amylase enzymes: In silico, biochemical and kinetic studies. Int. J. Biol. Macromol. 2020,
154, 217–232. [CrossRef] [PubMed]
α
α
Rao, R.R.; Tiwari, A.K.; Reddy, P.P.; Babu, K.S.; Suresh, G.; Ali, A.Z.; Madhusudana, K.; Agawane, S.B.; Badrinarayan, P.; Sastry,
G.N.; et al. Synthesis of antihyperglycemic, α-glucosidase inhibitory, and DPPH free radical scavenging furanochalcones. Med.
Chem. Res. 2020, 21, 760–774.
4.
5.
6.
7.
Poitout, P.; Robertson, R.P. Minireview: Secondary β-cell failure in type 2 diabetes– a convergence of glucotoxicity and lipotoxicity.
Endocrinology 2002, 143, 339–342. [CrossRef]
Imran, S.; Taha, M.; Selvaraj, M.; Ismail, N.H.; Chigurupati, S.; Mohammad, J.I. Synthesis and biological evaluation of indole
derivatives as α-amylase inhibitor. Bioorg. Chem. 2017, 73, 121–127. [CrossRef]
Zhao, X.; Tao, J.; Zhang, T.; Jiang, S.; Wei, W.; Han, H.; Shao, Y.; Zhou, G.; Yue, H. Resveratroloside alleviates postprandial
hyperglycemia in diabetic mice by competitively inhibiting α-glucosidase. J. Agric. Food Chem. 2019, 67, 2886–2893. [CrossRef]
Alqahtani, A.S.; Hidayathulla, S.; Rehman, M.T.; ElGamal, A.A.; Al-Massarani, S.; Razmovski-Naumovski, V.; Alqahtani, M.S.; El
Dib, R.A.; Al Ajmi, M.F. Alpha-amylase and alpha-glucosidase enzyme inhibition and antioxidant potential of 3-oxolupenal and
katononic acid isolated from Nuxia oppositifolia. Biomolecules 2020, 10, 61. [CrossRef]
8.
9.
Aispuro-Pérez, A.; López-Ávalos, J.; García-Páez, F.; Montes-Avila, J.; Corrales, L.A.P.; Ochoa-Terán, A.; Bastidas, P.; Montaño,
S.; Calderón-Zamora, L.; Osuna-Martínez, U.O.; et al. Synthesis and molecular docking studies of imines as
α-amylase inhibitors. Bioorg. Chem. 2020, 94, 103491. [CrossRef]
α-glucosidase and
Ghabi, A.; Brahmi, J.; Alminderej, F.; Messaoudi, S.; Vidald, S.; Kadrie, A.; Aouadi, K. Multifunctional isoxazolidine derivatives as
α-amylase and α-glucosidase inhibitors. Bioorg. Chem. 2020, 98, 103713. [CrossRef]
10. Balbaa, M.; El-Hady, N.A.; Taha, N.; Rezki, N.; El Ashry, E.S.H. Inhibition of
α-glucosidase and α-amylase by diaryl derivatives
of imidazole-thione and 1,2,4-triazole-thiol. Eur. J. Med. Chem. 2017, 46, 2596–2601. [CrossRef]
11. Furukawa, S.; Fujita, T.; Shimabukuro, M.; Iwaki, M.; Yamada, Y.; Nakajima, Y.; Nakayama, O.; Makishima, M.; Matsuda, M.;
Shimomura, I. Increased oxidative stress in obesity and its impact on metabolic syndrome. J. Clin. Investig. 2004, 114, 1752–1761.
[CrossRef]
12. McGarry, T.; Biniecka, T.; Veala, G.J.; Rearon, U. Hypoxia, oxidative stress and inflammation. Free Rad. Biol. Med. 2018, 125, 15–24.
13. Tangvarasittichai, S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J. Diabetes 2015, 6,
456–480. [CrossRef]
14. Pisoschi, A.M.; Pop, A.; Iordache, F.; Stanca, L.; Predoi, G.; Serban, A.I. Oxidative stress mitigation by antioxidants—An overview
on their chemistry and influences on health status. Eur. J. Med. Chem. 2020, 209, 112891.

Molecules 2021, 26, 2692
19 of 19
15. Chetan, S.; Amarjeet, K.; Thind, S.S.; Baljit, S.; Shiveta, R. Advanced glycation End-products (AGEs): An emerging concern for
processed food industries. J. Food Sci. Technol. 2015, 52, 7561–7576.
16. Vieira, R.; Souto, S.B.; Sánchez-López, E.; Machado, A.l.; Severino, P.; Jose, S.; Santini, A.; Fortuna, A.; Garcia, M.L.; Silva, A.M.;
et al. Sugar-lowering drugs for type 2 diabetes mellitus and metabolic syndrome– review of classical and new compounds: Part-I.
Pharmaceuticals 2019, 12, 152. [CrossRef]
17. Singh, P.; Anand, A.; Humar, V. Recent developments in biological activities of chalcones: A mini review. Eur. J. Med. Chem. 2015
85, 758–777.
,
18. Zhang, A.J.; Rimando, A.M.; Mizuno, C.S.; Mathews, S.T. α-Glucosidase inhibitory effect of resveratrol and piceatannol. J. Nutr.
Biochem. 2017, 47, 86–93. [CrossRef]
19. Rimando, A.M.; Suh, N. Biological/chemopreventive activity of stilbenes and their effect on colon cancer. Planta Med. 2008, 74,
1635–1643.
20. Hgani, U. Re-exploring promising α-glucosidase inhibitors for potential development into oral anti-diabetic drugs: Finding
needle in the haystack. Eur. J. Med. Chem. 2015, 103, 133–162.
21. Zhou, B.; Xing, C. Diverse molecular targets for chalcones with varied bioactivities. Med. Chem. 2015, 5, 388–404. [CrossRef]
22. Seo, W.D.; Kim, J.H.; Kang, J.E.; Ryu, H.W.; Curtis-Long, M.J.; Lee, H.S.; Yang, M.S.; Park, K.H. Sulfonamide chalcone as a new
class of α-glucosidase inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 5514–5516.
23. Rammohan, A.; Bhaskar, B.V.; Venkateswarlu, N.; Gu, W.; Zyryanov, G.V. Design, synthesis, docking and biological evaluation of
chalcones as promising antidiabetic agents. Bioorg. Chem. 2020, 95, 103527. [CrossRef]
24. Najafi, M.; Najafi, M.; Najafi, H. Theoretical study of the substituent and solvent effects on the reaction enthalpies of the
antioxidant mechanisms of tyrosol derivatives. Bull. Chem. Soc. Jpn. 2013, 86, 497–509. [CrossRef]
25. Moalin, M.; Van Strijdonck, G.P.; Beckers, M.; Hagemen, G.J.; Borm, P.J.; Bast, A.; Haenen, G.R. A planar conformation and
the hydroxyl groups in the B and C rings play a pivotal role in the antioxidant capacity of quercetin and quercetin derivatives.
Molecules 2011, 16, 9636–9650.
26. Stojanovic´, S.; Sprinz, H.; Brede, O. Efficiency and mechanism of the antioxidant action of trans-resveratrol and its analogues in
the radical liposome oxidation. Arch. Biochem. Biophys. 2001, 391, 79–89. [CrossRef] [PubMed]
27. Bhutani, R.; Pathak, D.P.; Kapoor, G.; Husain, A.; Iqbal, M.A. Novel hybrids of benzothiazole-1,3,4-oxadiazole-4-thiazolidinone:
Synthesis, in silico ADME study, molecular docking and in vivo anti-diabetic assessment. Bioorg. Chem. 2018, 83, 6–9. [CrossRef]
28. Lu, Y.; Liu, Y.; Xu, Z.; Li, H.; Liu, H.; Zhu, W. Halogen bonding for rational drug design and new drug discovery. Expert Opin.
Drug Discov. 2012, 7, 375–383. [CrossRef]
29. Kuhn, B.; Mohr, P.; Stahl, M. Intramolecular hydrogen bonding in medicinal chemistry. J. Med. Chem. 2018, 53, 2601–2611.
[CrossRef]
30. Ag, H.B.B. Pharmacology of α-glucosidase inhibition. Eur. J. Clin. Investig. 1994, 24, 3–10.
31. Rosak, C.; Mertes, G. Critical evaluation of the role of acarbose in the treatment of diabetes: Patient considerations. Diabetes Metab.
Syndr. Obes. 2012, 5, 357–367. [CrossRef] [PubMed]
32. Ruanwas, P.; Chantrapromma, S.; Fun, H.-K. Synthesis, characterization, antioxidant, and antibacterial activities of 2-
aminochalcones and crystal structure of (2E)-1-(2-aminophenyl)-3-(4-ethoxyphenyl)-2-propen-1-one. Mol. Cryst. Liq. Cryst. 2015
609, 126–139. [CrossRef]
,
33. Iqbal, H.; Prabhakar, V.; Sangith, A.; Chandrika, B.; Balasubramanian, R. Synthesis, anti-inflammatory and antioxidant activity of
ring-A-monosubstituted chalcone derivatives. Med. Chem. Res. 2014, 23, 4383–4394. [CrossRef]
34. Sulpizio, C.; Roller, A.; Giester, G.; Rompel, A. Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted
0
2 -aminochalcones. Monatsh. Chem. 2016, 147, 1747–1757. [CrossRef]
0
35. Garcia, G.X.; Larsen, S.W.; Pye, C.; Glabreath, M.; Isovitsch, R.; Fradenger, E.A. The functional group on (E)-4,4 –disubstituted
stilbenes influences toxicity and antioxidant activity in differentiated PC-12 cells. Bioorg. Med. Chem. Lett. 2013, 23, 6355–6359.
[CrossRef]
36. Walters, W.P.; Murcko, A.A.; Murcko, M.A. Recognizing molecules with drug-like properties. Curr. Opin. Chem. Biol. 1999, 3,
384–387. [CrossRef]
37. Olomola, T.O.; Mphahlele, M.J.; Gildenhuys, S. Benzofuran-selenadiazole hybrids as novel α-glucosidase and cyclooxygenase-2
inhibitors with antioxidant and cytotoxic properties. Bioorg. Chem. 2020, 100, 103945. [CrossRef]
38. Leone, A.M.; Francis, P.L.; Rhodes, P.; Moncada, S. A rapid and simple method for the measurement of nitrite and nitrate in
plasma by high performance capillary electrophoresis. Biochem. Biophys. Res. Commun. 1994, 200, 951–957. [CrossRef]
39. Hanwell, M.D.; Curtis, D.E.; Lonie, D.C.; Vandermeersch, T.; Zurek, E.; Hutchison, G.R. Avogadro: An advanced semantic
chemical editor, visualization, and analysis platform. J. Cheminform. 2012, 4, 17. [CrossRef]
40. Morris, V.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and AutoDockTools4:
Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 40, 2785–2791. [CrossRef]
41. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63. [CrossRef]