Chalcones and bis-chalcones: As potential α-amylase inhibitors; synthesis, in vitro screening, and molecular modelling studies

Article abstract of DOI:10.1016/j.bioorg.2018.05.003

Despite of a diverse range of biological activities associated with chalcones and bis-chalcones, they are still neglected by the medicinal chemist for their possible α-amylase inhibitory activity. So, the current study is based on the evaluation of this c

Full text of DOI:10.1016/j.bioorg.2018.05.003

Accepted Manuscript
Chalcones and Bis-Chalcones: As Potential α-Amylase Inhibitors; Synthesis, In
Vitro Screening, and Molecular Modelling Studies
Adebayo Tajudeen Bale, Khalid Mohammed Khan, Uzma Salar, Sridevi
Chigurupati, Tolulope Fasina, Farman Ali, Kanwal, Abdul Wadood,
Muhammad Taha, Sitanshu Sekhar Nanda, Mehreen Ghufran, Shahnaz Perveen
PII:
S0045-2068(18)30267-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.003
YBIOO 2356
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
17 March 2018
18 April 2018
4 May 2018
Please cite this article as: A. Tajudeen Bale, K. Mohammed Khan, U. Salar, S. Chigurupati, T. Fasina, F. Ali, Kanwal,
A. Wadood, M. Taha, S. Sekhar Nanda, M. Ghufran, S. Perveen, Chalcones and Bis-Chalcones: As Potential α-
Amylase Inhibitors; Synthesis, In Vitro Screening, and Molecular Modelling Studies, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.05.003
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Chalcones and Bis-Chalcones: As Potential α-Amylase Inhibitors; Synthesis, In Vitro
Screening, and Molecular Modelling Studies
Adebayo Tajudeen Bale,a Khalid Mohammed Khan,b,e Uzma Salar,b Sridevi Chigurupati,c
Tolulope Fasina,a Farman Ali,b Kanwal,a Abdul Wadood,d Muhammad Taha,e Sitanshu
Sekhar Nanda,f Mehreen Ghufran,d Shahnaz Perveeng
aDepartment of Chemistry, University of Lagos. Nigeria
bH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University,
Semeling, 08100 Bedong, Kedah, Malaysia
dDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
eDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi
Arabia
fDepartment of Chemistry, Myongji University, Yongin, South Korea
gPCSIR, Laboratories complex, Shahrah-e- Dr. Salimuzzaman, Karachi- 75280, Pakistan
Abstract: Despite of a diverse range of biological activities associated with chalcones and
bis-chalcones, they are still neglected by the medicinal chemist for their possible α-amylase
inhibitory activity. So, the current study is based on the evaluation of this class for the
identification of new leads as α-amylase inhibitors. For that purpose, a library of substituted
chalcones 1-13 and bis-chalcones 14-18 were synthesized and characterized by spectroscopic
1
techniques EI-MS and H-NMR. CHN analysis was carried out and found in agreement with
the calculated values. All compounds were evaluated for in vitro α-amylase inhibitory
activity and demonstrated good activities in the range of IC50 = 1.25 ± 1.05 - 2.40 ± 0.09 µM
as compared to the standard acarbose (IC50 = 1.04 ± 0.3 µM). Limited structure-activity
relationship (SAR) was established by considering the effect of different groups attached to
aryl rings on varying inhibitory activity. SMe group in chalcones and OMe group in bis-
chalcones were found more influential on the activity than other groups. However, in order to
predict the involvement of different groups in the binding interactions with the active site of
α-amylase enzyme, in silico studies were also conducted.
Keywords: Chalcones; bis-chalcones; in vitro α-amylase inhibitory activity; structure-
activity relationship; in silico studies
*Corresponding Author: drkhalidhej@gmail.com; khalid.khan@iccs.edu; Tel. 00922134824910; Fax.
00922134819018
1

Introduction
Diabetes mellitus is a chronic syndrome associated with the endocrine system. It affects the
metabolism of many molecules including carbohydrates, proteins, fat, electrolytes, and water.
Diabetes is mainly characterized by hyperglycemia, caused by either the deficiency of insulin
secretion or cells becomes irresponsive towards the production of insulin resulted in the
elevated blood glucose level. Amongst the different therapeutic approaches to treat diabetes,
one is to decrease postprandial hyperglycemia by the inhibition of hydrolyzing enzymes such
as α-amylase and α-glucosidase. α-Amylase is involved in the digestion of carbohydrates. It
is a metalloenzyme that breakdowns long chain carbohydrates into smaller molecules e.g.,
(conversion of starch into glucose and maltose) by hydrolyzing the α-1,4-glycosidic linkage.
It serves as the major digestive enzymes and facilitates intestinal absorption. The high blood
glucose level is associated with many pathological conditions such as diabetes, obesity, and
oral diseases. Diabetes in turn give rise to other pathological conditions including neuropathy,
retinopathy, microangiopathy, nephropathy, and cardiovascular diseases. Therefore, the
inhibitors for α-amylase is among the potential targets in the development of lead compounds
for the treatment of diabetes [1-10].
Chalcones, are abundantly found in edible plants and serves as a precursors for the synthesis
of flavonoids and isoflavonoids. The chalcones are α,β-unsaturated ketones having an enone
system between two aromatic rings. The keto-ethylenic group (-COCH=CH-) serves as the
reactive site and acts as a chromophore giving chalcones their characteristic color. The α,β-
unsaturation is responsible for the pharmacological properties of chalcones. These molecules
display interesting biological activities including antioxidant, antiinflammatory, cytotoxic,
anticancer, analgesic, antipyretic, antianginal, antihepatotoxic, antimicrobial, antimalarial,
and antiallergic activities. A common synthetic approach toward the synthesis of chalcones is
via the Knoevenagel condensation, many of which utilize microwave techniques [11-17].
Bis-chalcones have also got the considerable attention of biologists and synthetic chemists
due to their wide variety of biological activities. Their analogues are found to exhibit
potential antioxidant activities and are the potent NO production inhibitors. These compounds
are also reported for their anticancer potential against A549, DU145, KB, and KB-VIN
human cancer cell lines. Biphenyl based bis-chalcones have anticancer activity against MCF-
7 and MDA-MB-231 human breast cancer, HeLa cell lines, and human embryonic kidney
(HEK-293) cells. Some bis-chalcones have appreciable antibacterial and antifungal activities
2

[18-19]. Chalcones and bis-chalcones have been studied in past years for α-glucosidase
inhibitory activity [20-24] (Figure-1).
Figure-1: Identified α-glucosidase inhibitors based on chalcones and bis-chalcones scaffold
Unfortunately, chalcones and bis-chalcones are rarely studied for α-amylase inhibitory
activity. Najafian et. al., reported one unsubstituted trans-chalcone as α-amylase competitive
inhibitor with in silico study [25]. By keeping in mind that there is utter need to explore this
class for α-amylase inhibitory potential, we decided to synthesize a library of substituted
chalcones and bis-chalcones to subject them for the afore-mentioned activity (Figure-2).
Figure-2: Overview of the structural features and α-amylase inhibitory activity of chalcones
1-13 and bis-chalcones 14-18
3

Results and Discussion
Chemistry
Substituted acetophenones were reacted with a variety of benzaldehydes to afford chalcone
(Scheme-1A) derivatives 1-13, however, bis-chalcones 14-18 were synthesized by treating
acetone with a variety of benzaldehydes (Scheme-1B). Both reactions were carried out at
room temperature under basic conditions (60% NaOH). The extent of reaction progress was
checked by TLC monitoring. Synthetic compound 1-18 were characterized by spectroscopic
1
techniques EI-MS and H-NMR. CHN analysis was also carried out. To the best of our
knowledge, structures of compounds 1, 2, 4, 5, and 7-16 [26-36] are known while remaining
derivatives are found to be new.
Scheme-1: Synthesis of (A) chalcones 1-13 and (B) bis-chalcones 14-18
1H- and 13C-NMR spectra of compound 18 were recorded in deuterated DMSO-d6 at 400
MHz machine. Being symmetrical in structure, 1H- and 13C-NMR signals are representing the
1
each half of the molecule. In H-NMR spectrum, two characteristic signals for H-1′ and -2′
were appeared at H 7.88 and H 7.12, respectively, showed trans coupling with each other
with a coupling value 15.6 Hz. H-4, H-5, and H-6 were resonated in a usual aromatic region
H 5.85-6.78. Two signals for methylene H (3.89) and methylH (1.28) groups of the ethoxy
moiety were appeared as quartet and triplet, respectively (Figure-3A).
Figure-3: 1H- (A) and 13C-NMR (B) chemical shifts of new compound 18
4

13C-NMR broad-band decoupled spectrum (DMSO-d6) showed total 11 carbon signals
including two methyl, two methylene, ten methine, and seven quarternary carbons (Figure-
3B).
EI-MS of compound 18 displayed the molecular ion peak [M]+ at m/z 354 representing the
formation of the desired molecule.
In vitro α-amylase inhibitory activity
Synthetic chalcones 1-13 and bis-chalcones 14-18 were screened for in vitro α-amylase
inhibitory activity. All compounds demonstrated a good α-amylase inhibitory activity in the
range of IC50 = 1.25 ± 1.05-2.40 ± 0.09 µM as compared to the standard acarbose (IC50 =
1.04 ± 0.3 µM) (Table-1).
Table-1: In vitro α-amylase inhibitory, DPPH and ABTS radical scavenging activities of
chalcones 1-13 and bis-chalcones 14-18
α-Amylase inhibitory
Compounds
R1
R2
activity
IC50 ± SEMa
2.06 ± 0.04
1.85 ± 0.09
1
2
2.07 ± 0.08
1.27 ± 0.7
3
4
1.99 ± 0.05
2.26 ± 0.07
5
6
5

1.92 ± 0.12
1.97 ± 0.08
7
8
1.98 ± 0.07
1.25 ± 1.05
9
10
2.15 ± 0.07
11
1.99 ± 0.09
2.00 ± 0.08
12
13
α-Amylase inhibitory
activity
Compounds
R2
IC50 ± SEMa
1.72 ± 0.1
1.80 ± 0.07
1.63 ± 0.18
2.40 ± 0.09
14
15
16
17
2.12 ± 0.1
18
6

Standards = Acarboseb
1.04 ± 0.3
SEMa (Standard error mean); Acarboseb (Standard inhibitor for α-amylase enzyme)
Structure-activity relationship (SAR)
Limited structure-activity relationship (SAR) was established by assuming that all structural
features such as enone moiety in chalcones 1-13, dienone moiety in bis-chalcones 14-18, and
aryl rings in all compounds (Figure-2), are responsible for exhibiting the α-amylase inhibitory
activity. However, the variation in the inhibitory activity is attributed by different groups
attached to aryl rings.
Chalcones 1-6, derived from 1-(p-tolyl)ethan-1-one showed inhibition potential in the range
of IC50 = 1.27 ± 0.7-2.26 ± 0.07 µM as compared to the standard acarbose (IC50 = 1.04 ± 0.3
µM). The difference in the inhibitory activity of these compounds is the result of different
substituents on aryl ring (R2). Amongst them, compound 4 (IC50 = 1.27 ± 0.7 µM) with 4′-
SCH3 group was found to be the most active and showed inhibitory activity comparable to
standard (IC50 = 1.04 ± 0.3 µM). Comparison of its activity with derivative 1 (IC50 = 2.06 ±
0.04 µM) having OCH3 instead of SCH3, showed less activity than compound 4, revealed that
the sulfur atom of SCH3 is taking part in the interaction with the active site of enzyme.
Similarly, replacing SCH3 to Cl and OBz in compounds 2 (IC50 = 1.85 ± 0.09 µM) and 5
(IC50 = 1.99 ± 0.05 µM), respectively, brought decline in the inhibitory activity. Furthermore,
compounds 3 (IC50 = 2.07 ± 0.08 µM) having 3′-OMe and 4′-OEt groups and compound 6
(IC50 = 2.26 ± 0.07 µM) having 2′-OMe and 3′-OEt groups on aryl ring (R2) showed
moderate inhibitory activity (Figure-4).
7

Figure-4: Comparison of α-amylase inhibitory activity of compounds 1-6
Chalcones 7-13, derived from 1-(4-methoxyphenyl)ethan-1-one demonstrated α-amylase
inhibitory activity ranging from IC50 = 1.25 ± 1.05-2.15 ± 0.07 µM. Different groups attached
to the aryl ring (R2) are responsible for varying inhibitory activity. Again in this group of
compounds, the 4′-SCH3 substituted derivative 10 (IC50 = 1.25 ± 1.05 µM) was found to be
the most active analog. Its activity may compared to distinctively similar compound 4 (IC50 =
1.27 ± 0.7 µM) having 4-CH3 instead of 4-OCH3 group on aryl ring (R1), showed almost
same activity which further confirmed the important role of SCH3 group in the inhibitory
potential. However, replacement of 4′-SCH3 with 4′-OMe, 4′-OEt, 4′-Ph, 4′-OBz, 4′-Br, and
2′,4′-di-Cl in compounds 7 (IC50 = 1.92 ± 0.12 µM), 13 (IC50 = 2.00 ± 0.08 µM), 12 (IC50 =
1.99 ± 0.09 µM), 11 (IC50 = 2.15 ± 0.07 µM), 9 (IC50 = 1.98 ± 0.07 µM), and 8 (IC50 = 1.97 ±
0.08 µM), respectively, showed decreased activity (Figure-5). It is important to note that
derivatives 7-9, and 11-13 showed much closed inhibitory potential which revealed that these
compounds have almost similar extent of interactions with the active site of α-amylase
enzyme.
8

Figure-5: Comparison of α-amylase inhibitory activity of compounds 7-13
Bis-chalcones 14-18 derived from acetone and demonstrated α-amylase inhibitory potential in
the range of IC50 = 1.63 ± 0.18 - 2.40 ± 0.09 µM as compared to the standard acarbose (IC50
= 1.04 ± 0.3 µM). Amongst these compounds, compound 16 (IC50 = 1.63 ± 0.18 µM) with
4/4′-OMe substitution on aryl rings R1/R2 was found to be the most active. Replacement of
4/4′-OMe with 4/4′-SMe as in compound 17 (IC50 = 2.4 ± 0.09 µM), resulted in slight
decreased in the inhibitory activity which might be due to slight low polarity of compounds
17 than 16. Activity of compound 16 may also compared with halogenated derivatives 14
(IC50 = 1.72 ± 0.1 µM) and 15 (IC50 = 1.8 ± 0.07 µM) having 4/4′-Cl and 4/4′-Br,
respectively, a comparable inhibitory activity was observed. Another compound 18 (IC50 =
2.12 ± 0.1 µM) having 2/2′-OH and 3/3′-OEt showed moderate inhibitory activity (Figure-6).
Figure-6: Comparison of α-amylase inhibitory activity of compounds 14-18
9

Precisely, it may extracted out from the limited SAR that in case of chalcones, SMe group is
playing a crucial role in the activity while other compounds with different substituents on aryl
ring R2 have almost same extent of inhibitory potential. However, in case of bis-chalcones,
OMe group has big influence on the activity than other groups as well as extended aromatic
part also showed the moderate inhibitory activity. Furthermore, by keeping in mind that
solely SAR is insufficient to have clear picture of the participation of different moieties of
compound in the inhibitory activity. So, in silico study was conducted to decipher the ligands
(synthetic compounds) interactions with active site of α-amylase enzyme which is as follows.
In silico studies
In order to predict the binding conformations of the synthetic chalcones 1-13 and bis-
chalcones 14-18 into the active site of α-amylase enzyme, MOE-Dock module implemented
in MOE program was used. The default parameters of MOE-Dock program were used in the
docking protocol. Best conformations of chalcones and bis-chalcones for hydrogen
bonding/arene-arene/arene-cation interactions were analyzed on the basis of docking score. It
was perceived from the docking calculation study that the top ranked conformations of
almost all chalcones and bis-chalcones were well fitted inside the active site of α-amylase
enzyme and were involved in several types of interactions with the active site residues of α-
amylase enzyme. i.e., Trp58, Trp59, Tyr62, Leu162, Arg195, Asp197, Glu233, Asp300,
His305, and Asp356. Table-2 represents the details of interactions including docking scores
for all chalcones and bis-chalcones. It is observed that the presence of electronegative groups
and electron rich species like -Cl, -Br, -S, and -O are the main structural features for the
active nature of compounds while bulky groups such as methyl and ethyl slightly lowered the
activity of some of the compounds. From the conformation of compound 10 (docking score=
-9.3467, Table-2), it was observed that this compound formed three hydrogen bonds and four
π-H linkages with the active site residues of α-amylase. Arg195 was observed in making H-
donor interaction with the oxygen atom of the methoxy moiety of the compound while
Glu233 and Asp356 established interactions with carbon and sulphur atoms of the compound,
respectively, as shown in Figure-7A. Trp59, Leu162, and Ala198 of active site residues
formed π-H interactions with the compound. The high potent inhibitory activity may be due
to the presence of the electron accepting group (carbonyl oxygen) and electronegative group
(MeO) which create an electron flow making the compound more active, polarizable, and
potent. Compound 4 (docking score = -9.0231) was observed to make three polar interactions
with the Glu233 and Asp300 residues of the binding pocket of the enzyme as shown in
10

Figure-7B. Trp59 formed arene-arene contact with benzene moiety of the same compound.
The inhibition of this compound might be due to the availability of the electron accepting
group (carbonyl oxygen) and electron donating groups (methyl group). Compounds 16 and
14 has docking scores of -8.4512 and -8.2893, respectively, against α-amylase enzyme.
Compound 16 is bound to the α-amylase enzyme in an adequate manner through four
hydrogen bonds and several hydrophobic interactions (Figure-7C). Whereas, compound 14
formed five polar and one arene-cation interactions with the active site residues of the α-
amylase enzyme (Figure-7D). The structural features observed in this group for the active
nature of compounds (16 and 14) are the presence of electronegative groups like halogen (-
Cl) and Methoxy (MeO) groups. The confirmations obtained after docking showed good
docking scores and demonstrated sound in silico inhibition of the α-amylase enzyme. Overall
a good correlation between the docking study and biological evaluation of active chalcones
and bis-chalcones was perceived. The correlation graph and the correlation coefficient values
are given in Figure-8.
Figure-7: Docking conformations of compounds on α-amylase enzyme. (A) 3D binding
mode of compound 10 as inhibitor of α-amylase enzyme. (B) 3D binding mode of compound
4. (C) 3D binding mode of compound 16. (D) 3D binding mode of compound 14 in binding
cavity of α-amylase enzyme. Ligands are shown in cyan color.
11

Figure-8: A correlation graph for predicted docking score and IC50 values
Table-2: Docking scores and report of predicted interactions of docked conformations
Interaction Report
Docking
E
(kcal/mol)
-0.2
-0.3
-0.0
-0.3
-0.2
-0.9
-0.2
-0.1
-1.0
-0.5
-0.0
-0.0
-0.3
-0.2
-0.0
-0.0
-2.7
-0.3
-0.2
-0.3
-0.8
-0.4
-0.0
-0.3
-0.6
-0.0
-0.0
-0.5
-0.5
-0.5
Comp.
1
Ligand
Receptor
Interaction
Distance
Score
C1 1
C19 32
CL18 31
6-ring
OD2 ASP 300 (A)
6-ring TRP 59 (A)
OD2 ASP 356 (A)
CB TRP 59 (A)
CD1 LEU 162 (A)
CD2 LEU 162 (A)
CB TRP 59 (A)
OE1 GLU 233 (A)
OD2 ASP 300 (A)
OE1 GLU 233 (A)
6-ring TRP 59 (A)
5-ring TRP 59 (A)
OD2 ASP 356 (A)
OD1 ASP 197 (A)
5-ring TRP 59 (A)
6-ring TRP 59 (A)
OD1 ASP 300 (A)
OE1 GLU 233 (A)
CB TYR 62 (A)
OD1 ASP 300 (A)
6-ring TYR 62 (A)
5-ring TRP 59 (A)
6-ring TRP 59 (A)
OD1 ASP 300 (A)
OE1 GLU 233 (A)
5-ring TRP 59 (A)
6-ring TRP 59 (A)
OD2 ASP 356 (A)
OD1 ASP 197 (A)
OD2 ASP 197 (A)
H-donor
H-π
H-donor
π-H
3.36
3.61
3.24
3.56
4.57
3.79
3.72
3.82
3.13
2.20
3.76
3.86
3.52
3.50
3.56
3.95
2.99
3.59
4.10
3.45
3.88
4.66
3.45
3.55
3.21
3.67
3.70
3.45
3.47
3.88
-6.7845
-7.7641
-6.5211
2
3
6-ring
6-ring
6-ring
π-H
π-H
π-H
S18 31
S18 31
C19 32
6-ring
H-donor
H-donor
H-donor
π-π
-9.0231
4
6-ring
C4 6
π-π
H-donor
H-donor
π-π
C19 32
6-ring
6-ring
O18 30
C21 37
6-ring
C18 28
C18 28
C20 33
6-ring
-7.1093
-6.2198
-7.5432
5
6
7
π-π
H-donor
H-donor
π-H
H-donor
H-π
H-π
π-π
H-donor
H-donor
π-π
C13 19
CL17 26
6-ring
6-ring
C4 6
-7.4389
-7.3421
8
9
π-π
H-donor
H-donor
H-donor
BR17 27
BR17 27
12

C10 15
C19 29
6-ring
6-ring
C3 4
6-ring TRP 58 (A)
6-ring TRP 59 (A)
5-ring TRP 59 (A)
6-ring TRP 59 (A)
OE1 GLU 233 (A)
NH ARG 195 (A)
OD2 ASP 356 (A)
6-ring TRP 59 (A)
CB TRP 59 (A)
CD2 LEU 162 (A)
CB ALA 198 (A)
OD1 ASP 356 (A)
6-ring TRP 59 (A)
CB TYR 62 (A)
CB TRP 59 (A)
CD1 LEU 162 (A)
CD2 LEU 162 (A)
5-ring TRP 59 (A)
OD1 ASP 300 (A)
5-ring TRP 59 (A)
6-ring TRP 59 (A)
OD1 ASP 300 (A)
OD1 ASP 300 (A)
OD1 ASP 356 (A)
OD1 ASP 356 (A)
OD1 ASP 197 (A)
6-ring TRP 59 (A)
OD2 ASP 356 (A)
OD1 ASP 356 (A)
OD1 ASP 197 (A)
OD2 ASP 197 (A)
6-ring TRP 58 (A)
CG PRO 54 (A)
CB TRP 357 (A)
OD1 ASP 356 (A)
OD1 ASP 356 (A)
OD1 ASP 300 (A)
NH HIS 305 (A)
OD2 ASP 356 (A)
OD1 ASP 197 (A)
OD2 ASP 197 (A)
OD1 ASP 300 (A)
OD1 ASP 300 (A)
CB THR 163 (A)
6-ring TYR 62 (A)
OD1 ASP 300 (A)
OD1 ASP 300 (A)
OD2 ASP 300 (A)
OD1 ASP 197 (A)
OD1 ASP 356 (A)
OD1 ASP 197 (A)
OG1 ARG 195 (A)
6-ring TRP 59 (A)
H-π
H-π
π-π
4.78
4.70
3.46
3.88
2.39
3.84
3.60
3.67
3.85
3.83
4.71
3.53
3.61
3.58
3.73
4.11
3.78
3.54
3.57
3.65
3.67
2.62
3.62
2.30
3.00
3.12
3.78
3.61
3.57
3.46
3.83
4.78
4.54
4.58
2.21
2.42
3.33
3.15
3.50
3.27
3.64
3.37
3.66
3.29
3.90
3.42
3.09
2.96
2.67
2.83
2.60
2.81
3.64
-0.3
-0.2
-0.0
-0.0
-0.5
-0.0
-0.5
-0.8
-0.3
-0.7
-0.2
-0.2
-0.6
-0.2
-0.5
-0.2
-0.5
-0.0
-0.3
-0.0
-0.0
-0.3
-1.1
-0.3
-0.5
-0.4
-0.0
-0.3
-0.4
-0.4
-0.8
-0.2
-0.4
-0.2
-0.3
-0.3
-0.3
-0.3
-0.5
-0.3
-1.2
-0.3
-0.3
-0.2
-0.2
-0.7
-1.2
-0.8
-4.2
-3.3
-1.6
-1.8
-0.6
π-π
H-donor
H-donor
H-donor
H-π
O
S19 32
C20 33
6-ring
6-ring
6-ring
C23 38
C18 28
O8 12
6-ring
6-ring
6-ring
6-ring
C13 20
6-ring
6-ring
C4 6
C8 13
C12 19
C18 29
CL19 31
6-ring
C12 19
C18 29
BR19 31
BR19 31
C7 11
6-ring
-9.3467
10
π-H
π-H
π-H
H-donor
H-π
H-acceptor
π-H
π-H
π-H
π-π
H-donor
π-π
-7.1239
-7.1192
11
12
-7.0027
-8.2893
13
14
π-π
H-donor
H-donor
H-donor
H-donor
H-donor
π-π
H-donor
H-donor
H-donor
H-donor
H-π
-7.9887
15
π-H
π-H
6-ring
C4 6
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-acceptor
H-π
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-donor
H-π
C8 13
C12 19
O
C12 19
S21 36
S21 36
C16 22
C23 35
O11 15
C23 35
-8.4512
-5.5673
-6.1107
16
17
18
C
C
C
O
O
O
O
19
36
36
61
65
69
79
5
-9.8935
Standard
C
13

Conclusion
Synthetic chalcones 1-13 and bis-chalcones 14-18 showed significant in vitro α-amylase
inhibitory activity as compared to the standard acarbose. Limited structure-activity
relationship (SAR) suggested that SMe and OMe groups are playing crucial role in the
inhibitory activity in the case of chalcones and bis-chalcones, respectively. However, in silico
study predicted a number of interacting sites of the ligands (synthetic compounds) with the
active site of α-amylase enzyme. This study has identified a number of lead compounds
which can further explore in order to get a powerful inhibitor for α-amylase enzyme.
Experimental
Materials and methods
All chemicals were of analytical grade and purchased from Sigma-Aldrich, USA. Silica gel
coated aluminium plates (Kieselgel 60, 254, E. Merck, Germany) were used for thin layer
chromatography (TLC). Spots were visualized with a dual wavelength (254 and 365 nm) UV
light. Electron impact mass experiment were recorded on MAT 312 and MAT 113D mass
spectrometer. The 1H-NMR spectra were recorded on Bruker AM machines, operating at 300
and 400 MHz. The chemical shift are presented in ppm (δ) relative to tetramethylsilane
(TMS) as an internal standard and the coupling constant (J) are in Hz. Melting points of the
compounds were determined on Stuart® SMP10 melting point apparatus. CHN Analyses
were carried out on a Carlo Erba Strumentazione-Mod-1106, Italy.
General procedure for the synthesis of chalcones 1-13
To a stirred solution of 4-methyl/4-methoxy acetophenone (0.5 mmol) in EtOH (10 mL),
solution of NaOH (60%) (3 mL) was added drop-wise by maintaining the temperature at 0 °C.
Substituted benzaldehyde derivative (0.5 mmol) was added to the above-mentioned mixture
and further stirred for 2-3 h. Completion of reaction was monitored by TLC analysis. After
completion of reaction, it was kept in refrigerator for overnight and then diluted with ice cold
water, the resulting precipitates were filtered and washed with excess of distilled water. The
precipitates were crystallized from methanol [37]. Structures of compounds 1-13 were
1
deduced by EI-MS and H-NMR spectroscopic techniques. CHN analysis was also
performed.
14

(E)-3-(4-Methoxyphenyl)-1-(p-tolyl)prop-2-en-1-one (1)
Yellow solid; Yield: 72%; M.p.: 94-96 ºC; 1H-NMR (400 MHz, DMSO-d6):  8.05 (d, J2,3/6,5
= 8.4 Hz, 2H, H-2, H-6), 7.84 (d, J2′,3′/6′,5′ = 8.8 Hz, 2H, H-2′, H-6′), 7.80 (d, J2′′,1′′ = 14.4 Hz,
1H, H-2′′ Vinylic), 7.70 (d, J1′′,2′′ = 15.2 Hz, 1H, H-1′′ Vinylic), 7.37 (d, J3,2/5,6 = 8.0 Hz, 2H,
H-3, H-5), 7.01 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 3.81 (s, 3H, OCH3), 2.39 (s, 3H, CH3);
EI-MS: m/z (rel. abund. %), 252 (M+, 100), 237 (43), 161 (29), 108 (16), 90 (19), 64 (7);
Anal. Calcd for C17H16O2: C, 80.93; H, 6.39; Found: C, 80.95; H, 6.42.
(E)-3-(4-Chlorophenyl)-1-(p-tolyl)prop-2-en-1-one (2)
º
1
Yellow solid; Yield: 75%; M.p.: 122-124 C; H-NMR (300 MHz, DMSO-d6):  8.05 (d,
J2,3/6,5 = 7.8 Hz, 2H, H-2, H-6), 7.96 (d, J2′′,1′′ = 15.0 Hz, 1H, H-2′′ Vinylic), 7.91 (d, J2′,3′/6′,5′
=
7.2 Hz, 2H, H-2′, H-6′), 7.70 (d, J1′′,2′′ = 15.6 Hz, 1H, H-1′′ Vinylic), 7.50 (d, J3′,2′/5′,6′ = 8.1
Hz, 2H, H-3′, H-5′), 7.36 (d, J3,2/5,6 = 8.1 Hz, 2H, H-3, H-5), 2.37 (s, 3H, CH3); EI-MS: m/z
(rel. abund. %), 256 (M+, 100), 258 (M+2, 30), 241 (20), 221 (23), 165 (18), 119 (40), 91
(24); Anal. Calcd for C16H13ClO: C, 74.86; H, 5.10; Found: C, 74.88; H, 5.13.
(E)-3-(4-Ethoxy-3-methoxyphenyl)-1-(p-tolyl)prop-2-en-1-one (3)
º
1
Yellow solid; Yield: 73%; M.p.: 113-115 C; H-NMR (400 MHz, DMSO-d6):  8.06 (d,
J2,3/6,5 = 8.4 Hz, 2H, H-2, H-6), 7.81 (d, J2′′,1′′ = 15.6 Hz, 1H, H-2′′ Vinylic), 7.68 (d, J1′′,2′′
=
15.6 Hz, 1H, H-1′′ Vinylic), 7.52 (d, J2′,6′ = 1.6 Hz, 1H, H-2′), 7.37 (ovp, 3H, H-3, H-5, H-6′),
7.00 (d, J5′,6′ = 8.4 Hz, 1H, H-5′), 4.09 (q, 2H, O-CH2CH3), 3.85 (s, 3H, OCH3), 2.39 (s, 3H, -
CH3), 1.35 (t, 3H, O-CH2CH3); 13C-NMR (100 MHz, DMSO-d6): δC 188.9, 149.5, 149.3,
145.3, 144.0, 134.7, 129.6, 129.6, 129.3, 129.3, 126.4, 122.0, 121.2, 118.0, 111.2, 64.7, 56.2,
21.5, 14.6; EI-MS: m/z (rel. abund. %), 296 (M+, 100), 281 (6), 267 (76), 119 (20), 91 (18);
Anal. Calcd for C19H20O3: C, 77.00; H, 6.80; Found: C, 77.02; H, 6.83.
(E)-3-(4-(Methylthio)phenyl)-1-(p-tolyl)prop-2-en-1-one (4)
Yellow solid; Yield: 69%; M.p.: 96-98 ºC; 1H-NMR (400 MHz, DMSO-d6):  8.06 (d, J2,3/6,5
= 8.0 Hz, 2H, H-2, H-6), 7.89 (d, J2′′,1′′ = 15.6 Hz, 1H, H-2′′ Vinylic), 7.82 (d, J2′,3′/6′,5′ = 8.4
Hz, 2H, H-2′, H-6′), 7.70 (d, J1′′,2′′ = 15.6 Hz, 1H, H-1′′ Vinylic), 7.37 (d, J3,2/5,6 = 8.0 Hz, 2H,
H-3, H-5), 7.31 (d, J3′,2′/5′,6′ = 8.4 Hz, 2H, H-3′, H-5′), 2.51 (s, 3H, -SCH3) 2.39 (s, 3H, -CH3);
EI-MS: m/z (rel. abund. %), 268 (M+, 100), 253 (77), 221 (76), 177 (31), 119 (38), 91 (40);
Anal. Calcd for C17H16OS: C, 76.08; H, 6.01; Found: C, 76.10; H, 6.04.
15

(E)-3-(4-(Benzyloxy)phenyl)-1-(p-tolyl)prop-2-en-1-one (5)
º
1
Yellow solid; Yield: 72%; M.p.: 110-112 C; H-NMR (400 MHz, DMSO-d6):  8.05 (d,
J2,3/6,5 = 8.0 Hz, 2H, H-2, H-6), 7.84 (d, J2′,3′/6′,5′ = 8.8 Hz, 2H, H-2′, H-6′), 7.80 (d, J2′′,1′′
=
15.6 Hz, 1H, H-2′′ Vinylic), 7.70 (d, J1′′,2′′ = 15.6 Hz, 1H, H-1′′, Vinylic), 7.47 (d, J3,2/5,6 = 7.2
Hz, 2H, H-3, H-5), 7.41 (ovp, 5H, H-2′′′, H-3′′′, H-4′′′, H-5′′′, H-6′′′), 7.09 (d, J3′,2′/5′,6′ = 8.8
Hz, 2H, H-3′, H-5′), 5.17 (s, 2H, -OCH2C6H5), 2.39 (s, 3H, -CH3); EI-MS: m/z (rel. abund.
%), 328 (M+, 75), 237 (15), 119 (22), 91 (100), 65 (16); Anal. Calcd for C23H20O2: C, 84.12;
H, 6.14; Found: C, 84.15; H, 6.16.
(E)-3-(3-Ethoxy-2-hydroxyphenyl)-1-(p-tolyl)prop-2-en-1-one (6)
Red solid; Yield: 70%; M.p.; 278-280 ºC; 1H-NMR (400 MHz, DMSO-d6):  8.02 (d, J2′′,1′′
=
14.8 Hz, 1H, H-2′′ Vinylic), 7.86 (ovp, 3H, H-2, H-6, H-1′′ Vinylic), 7.29 (d, J3,2/5,6 = 7.6 Hz,
2H, H-3, H-5), 6.92 (d, J6′,5′ = 7.6 Hz, 2H, H-6′), 6.49 (d, J4′,5′ = 6.8 Hz, 2H, H-4′), 5.95 (bd.t,
1H, H-5′), 3.91 (q, 2H, -O-CH2CH3), 2.36 (s, 3H, -CH3), 1.28 (s, 3H, -O-CH2CH3); 13C-NMR
(100 MHz, DMSO-d6): δC 188.8, 151.3, 147.7, 144.0, 141.2, 134.7, 129.7, 129.7, 129.3,
129.3, 121.5, 121.1, 120.4, 117.0, 115.2, 64.7, 21.5, 14.6; EI-MS: m/z (rel. abund. %), 282
(M+, 73), 265 (55), 119 (100), 91 (38); Anal. Calcd for C18H18O3: C, 76.57; H, 6.43; Found:
C, 76.59; H, 6.45.
(E)-1,3-Bis(4-methoxyphenyl)prop-2-en-1-one (7)
º
1
Yellow solid; Yield: 76%; M.p.:101-103 C; H-NMR (400 MHz, DMSO-d6):  8.15 (d,
J2,3/6,5 = 8.8 Hz, 2H, H-2, H-6), 7.84 (d, J2′,3′/6′,5′ = 8.8 Hz, 2H, H-2′, H-6′), 7.81 (d, J2′′,1′′
=
15.2 Hz, 1H, H-2′′ Vinylic), 7.68 (d, J1′′,2′′ = 15.2 Hz, 1H, H-1′′ Vinylic), 7.08 (d, J3,2/5,6 = 8.8
Hz, 2H, H-3, H-5), 7.01 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 3.85 (s, 3H, -OCH3), 3.81 (s,
3H, -OCH3); EI-MS: m/z (rel. abund. %), 268 (M+, 100), 253 (48), 237 (28), 225 (33), 161
(33), 135 (50), 77 (17); Anal. Calcd for C17H16O3: C, 76.10; H, 6.01; Found: C, 76.12; H,
6.04.
(E)-3-(2,4-Dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (8)
º
1
Brownish yellow solid; Yield: 73%; M.p.:134-136 C; H-NMR (400 MHz, DMSO-d6): 
8.26 (d, J6′,5′ = 8.4 Hz, 1H, H-6′), 8.18 (d, J2,3/6,5 = 5.2 Hz, 2H, H-2, H-6), 8.04 (d, J2′′,1′′ = 15.2
Hz, 1H, H-2′′ Vinylic), 7.94 (d, J1′′,2′′ = 15.2 Hz, 1H, H-1′′ Vinylic), 7.74 (d, J3′,5′ = 2.0 Hz,
1H, H-3′), 7.55 (dd, J5′,3′ = 2.0 Hz, J5′,6′ = 10.0 Hz, 1H, H-5′), 7.09 (d, J3,2/5,6 = 8.8 Hz, 2H, H-
16

3, H-5), 3.86 (s, 3H, -OCH3); EI-MS: m/z (rel. abund. %), 306 (M+, 54), 308 (M+2, 30), 310
(M+4, 7), 291 (6), 271 (100), 135 (14); Anal. Calcd for C16H12Cl2O2: C, 62.56; H, 3.94;
Found: C, 62.58; H, 3.96.
(E)-3-(4-Bromophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (9)
º
1
Intense yellow solid; Yield: 71%; M.p.:152-154 C; H-NMR (400 MHz, CDCl3):  8.02 (d,
J2,3/6,5 = 8.8 Hz, 2H, H-2, H-6), 7.73 (d, J2′′,1′′ = 15.6 Hz, 1H, H-2′′ Vinylic), 7.54 (ovp, 5H, H-
3, H-5, H-2′, H-6′, H-1′′ Vinylic), 6.98 (d, J3′,2′/5′,6′ = 9.2 Hz, 2H, H-3′, H-5′), 3.87 (s, 3H, -
OCH3); EI-MS: m/z (rel. abund. %), 316 (M+, 97), 318 (M+2, 100), 301 (19), 288 (17), 237
(29), 135 (82); Anal. Calcd for C16H13BrO2: C, 60.59; H, 4.13; Found: C, 60.61; H, 4.15.
(E)-1-(4-Methoxyphenyl)-3-(4-(methylthio)phenyl)prop-2-en-1-one (10)
º
1
Intense yellow solid; Yield: 67%; M.p.: 97-101 C; H-NMR (400 MHz, CDCl3):  8.02 (d,
J2,3/6,5 = 8.8 Hz, 2H, H-2, H-6), 7.76 (d, J2′′,1′′ = 15.6 Hz, 1H, H-2′′ Vinylic), 7.54 (d, J2′,3′/6′,5′
=
= 8.4 Hz, 2H, H-2′, H-6′), 7.50 (d, J1′′,2′′ = 15.6 Hz, 1H, H-1′′ Vinylic), 7.24 (d, J3,2/5,6 = 8.4
Hz, 2H, H-3, H-5), 6.97 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 3.87 (s, 3H, -OCH3), 2.50 (s,
3H, -SCH3); EI-MS: m/z (rel. abund. %), 284 (M+, 100), 269 (43), 237 (40), 135 (53), 77
(18); Anal. Calcd for C17H16O2S: C, 71.80; H, 5.67; Found: C, 71.83; H, 5.65.
(E)-3-(4-(Benzyloxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (11)
º
1
Yellow solid; Yield: 74%; M.p.:135-137 C; H-NMR (400 MHz, DMSO-d6):  7.74 (d,
J2,3/6,5 = 8.4 Hz, 2H, H-2, H-6), 7.72 (d, J2′′,1′′ = 15.6 Hz, 1H, H-2′′ Vinylic), 7.46-7.33 (ovp,
9H, H-3, H-5, H-2′, H-6′, H-2′′′, H-3′′′, H-4′′′, H-5′′′, H-6′′′), 7.19 (d, J1′′,2′′ = 15.6 Hz, 1H, H-
1′′ Vinylic), 7.10 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 5.17 (s, 2H, -CH2C6H5), EI-MS: m/z
(rel. abund. %), 344 (M+ 7), 327 (3), 300 (10), 91 (100); Anal. Calcd for C23H20O3: C, 80.21;
,
H, 5.85; Found: C, 80.23; H, 5.88.
(E)-3-([1,1′-Biphenyl]-4-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (12)
º
1
Yellow solid; Yield: 76%; M.p.: 203-206 C; H-NMR (400 MHz, DMSO-d6):  7.90 (d,
J2,3/6,5 = 8.4 Hz, 2H, H-2, H-6), 7.97 (d, J2′′,1′′ = 16.0 Hz, 1H, H-2′′ Vinylic), 7.79 (d, J2′,3′/6′,5′
=
= 8.0 Hz, 2H, H-2′, H-6′), 7.75 (d, J2′′′,3′′′/6′′′,5′′′ = = 7.2 Hz, 2H, H-2′′′, H-6′′′), 7.51 (ovp, 8H, H-
3, H-5, H-3′, H-5′, H-1′′ Vinylic, H-3′′′, H-5′′′), 3.30 (s, 3H, -OCH3); EI-MS: m/z (rel. abund.
17

%), 314 (M+ 43), 178 (81), 386 (100); Anal. Calcd for C22H18O2: C, 84.05; H, 5.77; Found:
,
C, 84.03; H, 5.75.
(E)-3-(4-Ethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (13)
º
1
Yellow solid; Yield: 78%; M.p.:105-107 C; H-NMR (400 MHz, DMSO-d6):  8.15 (d,
J2,3/6,5 = 8.8 Hz, 2H, H-2, H-6), 7.84 (d, J2′,3′/6′,5′ = 8.8 Hz, 2H, H-2′, H-6′), 7.81 (d, J2′′,1′′
=
15.2 Hz, 1H, H-2′′ Vinylic), 7.68 (d, J1′′,2′′ = 15.2 Hz, 1H, H-1′′ Vinylic), 7.08 (d, J3,2/5,6 = 8.8
Hz, 2H, H-3, H-5), 7.01 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 3.91 (q, 2H, -OCH2-CH3), 3.85
(s, 3H, -OCH3), 1.28 (s, 3H, -O-CH2CH3); EI-MS: m/z (rel. abund. %), 282 (M+ 23), 265
,
(24), 253 (19), 237 (17), 200 (45); Anal. Calcd for C18H18O3: C, 76.57; H, 6.43; Found: C,
76.57; H, 6.43.
General procedure for the synthesis of bis-chalcones 14-18
To a stirred solution of aldehyde (1.0 mmol) and acetone (0.5 mmoL) in EtOH (10 mL), 3
°
mL solution of NaOH (60%) was added by maintaining the temperature at 0 C. Reaction
mixture was stirred for 2-3 h. Completion of reaction was monitored by TLC analysis. After
completion of reaction, it was kept in refrigerator for overnight and then diluted with ice cold
water, the resulting precipitates were filtered and washed with excess of distilled water. The
precipitates were crystallized from methanol. Structures of compounds 14-18 were deduced
by EI-MS and 1H-NMR spectroscopic techniques. CHN analysis was also carried out.
(1E,4E)-1,5-Bis(4-chlorophenyl)penta-1,4-dien-3-one (14)
Brown solid; Yield: 69%; M.p.:192-194 ºC; 1H-NMR (400 MHz, DMSO-d6):  7.82 (d, J3,2/5,6
= 6.4 Hz, 2H, H-3, H-5), 7.80 (d, J2′,1′ = 16.0 Hz, 1H, H-2′ Vinylic), 7.54 (d, J2,3/6,5 = 8.4 Hz,
2H, H-2, H-6), 7.37 (d, J1′,2′ = 16.0 Hz, 1H, H-1′ Vinylic); EI-MS: m/z (rel. abund. %), 302
(M+, 100), 304 (M+2, 70), 306 (M+4, 15), 267 (62), 239 (23), 204 (55), 165 (61), 137 (38),
101 (7), 75 (18); Anal. Calcd for C17H12Cl2O: C, 67.35; H, 3.99; Found: C, 67.33; H, 3.97.
(1E,4E)-1,5-Bis(4-bromophenyl)penta-1,4-dien-3-one (15)
º
1
Brown solid; Yield: 66%; M.p.:148-150 C; H-NMR (400 MHz, DMSO-d6):  7.78 (ovp,
3H, H-2, H-6, H-2′ Vinylic), 7.67 (d, J3,2/5,6 = 8.4 Hz, 2H, H-3, H-5), 7.38 (d, J1′,2′ = 16.0 Hz,
1H, H-1′ Vinylic); EI-MS: m/z (rel. abund. %), 390 (M+, 70), 392 (M+2, 100), 394 (M+4,
67), 311 (72), 209 (27), 204 (75), 102 (35); Anal. Calcd for C17H12Br2O: C, 52.08; H, 3.09;
Found: C, 52.06; H, 3.12.
18

19

(1E,4E)-1,5-Bis(4-methoxyphenyl)penta-1,4-dien-3-one (16)
º
1
Yellow solid; Yield: 74%; M.p.: 130-132 C; H-NMR (400 MHz, DMSO-d6):  7.74 (d,
J2,3/6,5 = 8.8 Hz, 2H, H-2, H-6), 7.73 (d, J2′,1′ = 15.6 Hz, 1H, H-2′ Vinylic), 7.19 (d, J1′,2′
=
15.6 Hz, 1H, H-1′ Vinylic), 7.02 (d, J3,2/5,6 = 8.8 Hz, 2H, H-3, H-5), 3.80 (s, 3H, OCH3); EI-
MS: m/z (rel. abund. %), 294 (M+, 100), 279 (28), 186 (47), 161 (39), 133 (25), 121 (20);
Anal. Calcd for C19H18O3: C, 77.53; H, 6.16; Found: C, 77.55; H, 6.18.
(1E,4E)-1,5-Bis(4-(methylthio)phenyl)penta-1,4-dien-3-one (17)
º
1
Yellow solid; Yield: 76%; M.p.: 143-145 C; H-NMR (400 MHz, DMSO-d6):  7.74 (ovp,
3H, H-2, H-6, H-2′ Vinylic), 7.32 (ovp, 3H, H-3, H-5, H-1′ Vinylic), 2.51 (s, 3H, -SCH3);
13C-NMR (100 MHz, DMSO-d6): δC 187.3, 141.8, 141.8, 138.4, 138.4, 131.8, 131.8, 128.7,
128.7, 128.7, 128.7, 127.0, 127.0, 127.0, 127.0, 123.0, 123.0, 14.5, 14.5; EI-MS: m/z (rel.
abund. %), 326 (M+, 100), 311 (6), 279 (34), 204 (38), 177 (10), 134 (19), 102 (11); Anal.
Calcd for C19H18OS2: C, 69.90; H, 5.56; Found: C, 69.93; H, 5.58.
(1E,4E)-1,5-Bis(3-ethoxy-2-hydroxyphenyl)penta-1,4-dien-3-one (18)
Reddish brown solid; Yield: 79%; M.p.: >300 ºC; 1H-NMR (400 MHz, DMSO-d6):  7.88 (d,
J2′,1′ = 15.6 Hz, 1H, H-2′ Vinylic), 7.12 (d, J1′,2′ = 15.6 Hz, 1H, H-1′ Vinylic), 6.78 (d, J6,5
=
7.6 Hz, 1H, H-6), 6.41 (d, J4,5 = 7.2 Hz, 1H, H-4), 5.85 (t, J5(4,6) = 7.2 Hz, 1H, H-5), 3.89 (q,
4H, -O-CH2CH3), 1.28 (t, 3H, -O-CH2CH3); 13C-NMR (100 MHz, DMSO-d6): δC 187.5,
151.9, 151.9, 150.9, 150.9, 148.0, 148.0, 123.1, 123.1, 121.4, 121.4, 121.0, 121.0, 117.3,
117.3, 115.2, 115.2, 64.7, 64.7, 14.6, 14.6; EI-MS: m/z (rel. abund. %), 354 (M+, 16), 336
(73), 321 (100), 279 (56), 163 (52), 134 (23), 43 (52); Anal. Calcd for C21H22O5: C, 71.17; H,
6.26; Found: C, 71.19; H, 6.28.
In vitro α-amylase inhibitory assay
The α-amylase inhibitory activity was determined as method reported by Uzma et al. [38]. A
volume of 500 µL of test sample (100, 200, 400, 800, 1000 µg/mL) and 500 µL of α-amylase
°
solution (0.5 mg/mL) in 0.2 mM phosphate buffer (pH 6.9) were incubated at 25 C for 10
min. After pre-incubation, 500 μL of 1% starch solution in 0.02 M sodium phosphate buffer
°
(pH 6.9) was added to each tube and incubated at 25 C for 10 minutes. The reaction was
arrested with 1 mL of dinitrosalicylic acid colour reagent. The tubes were then incubated in
20

boiling water for 5 min and cooled to room temperature. The solutions were diluted after
adding 10 mL distilled water and the absorbance was measured at 540 nm [39-40].
The percentage of inhibition was calculated as illustrated:
% Inhibition = (Absorbance Control – Absorbance Sample) / Absorbance Control x 100
The IC50 values, concentration required to inhibit the α-amylase activity by 50% were
calculated by a non-linear regression graph plotted between percentage inhibition (x axis)
versus concentrations (y axis), using a Graph Pad Prism Software (Version 5).
In Silico Methodology
To understand the binding modes of the chalcones and bis-chalcones derivatives in the active
site of α-amylase enzyme, all compound were docked into the binding site of α-amylase
enzyme. The 3D structure of α-amylase (PDB ID: 1HNY) was obtained from Protein Data
Bank. Water molecules were removed and the 3D protonation of the protein molecule was
carried out. Energy of the protein molecule was minimized with the help of energy
minimization algorithm implemented in MOE (Molecular Operating Environment) software
and the minimized structure was used for docking. The 3D structures of ligands were built
using builder tool in MOE (www.chemcomp.com). All the built structures were 3D
protonated and were energy minimized. The 3D structure were saved in mdb file format as
input file for docking.
Acknowledgment: Authors are thankful to the Higher Education Commission (HEC),
Pakistan under National Research Program for Universities (NRPU) for the financial support
to Research Project No. 20-1910 and NAM S&T for awarding fellowship 2015-2016 to
Adebayo Tajudeen Bale to visit H. E. J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan.
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Highlights:

Substituted chalcones 1-13 and bis-chalcones 14-18 were synthesized and structurally
characterized.



Compounds 1-18 were screened for in vitro α-amylase inhibitory activity.
Limited structure-activity relationship was established.
In silico study was conducted to predict the binding interactions of compounds with
the active site of α-amylase enzyme.
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