Design, and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one derivatives as the potential alpha-amylase inhibitors and antioxidants

Article abstract of DOI:10.1016/j.bioorg.2018.10.010

Diabetes is the most prevalent metabolic disorder causing a high rate of mortality and morbidity. Recently alpha-amylase is reported to be good drug design target for the treatment of diabetes mellitus. We have designed 116 molecules based on aza-Michael

Full text of DOI:10.1016/j.bioorg.2018.10.010

Accepted Manuscript
Design, and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one deriva-
tives as the potential alpha-amylase inhibitors and antioxidants
Roqia Bashary, Gopal L. Khatik
PII:
S0045-2068(18)30870-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.010
YBIOO 2544
Reference:
Bioorganic Chemistry
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4 October 2018
Please cite this article as: R. Bashary, G.L. Khatik, Design, and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-
one derivatives as the potential alpha-amylase inhibitors and antioxidants, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.10.010
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Design, and facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one derivatives as the
potential alpha-amylase inhibitors and antioxidants
Roqia Bashary1,2, Gopal L. Khatik2*
1Department of Pharmaceutical Chemistry, Kabul University, Kabul, Afghanistan.
2School of Pharmaceutical Sciences, Lovely Professional University, Jalandhar-Delhi G.T.
Road, Phagwara (Punjab), India-144411
*Corresponding author:
Dr. Gopal L. Khatik
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Lovely
Professional University, Phagwara (Punjab), India-144411
Email: gopal_niper@rediffmail.com; gopal.16803@lpu.co.in
1

Abstract
Diabetes is the most prevalent metabolic disorder causing a high rate of mortality and
morbidity. Recently alpha-amylase is reported to be good drug design target for the
treatment of diabetes mellitus. We have designed 116 molecules based on aza-Michael
adduct of trans-chalcone as 1,3 diaryl-3-(arylamino)propan-1-ones which were studied by
molecular docking and among them best six derivatives were synthesized easily via aza-
Michael addition on trans-chalcone using KOH as a catalyst and evaluated for alpha-
amylase inhibition along with antioxidant activity. It was observed that all compounds have
alpha-amylase inhibitory activity but at different extents. The molecule 3e is the most
potent alpha-amylase inhibitor of this series. 3a is the second most potent compound,
whereas only one molecule 3d has shown antioxidant activity.
Keywords: Diabetes; Antidiabetic; Alpha-amylase inhibitor; Aza-Michael addition;
Antioxidant
2

1. Introduction:
Diabetes mellitus is the most prevalent non-infective metabolic disorder among the
millions of people around the world[1]. Recent studies have shown that the prevalence of
the disease is growing, from a worldwide prevalence of 422 million persons in 2016 to 844
million persons in 2030[2]. Diabetes mellitus is classified into two types: type 1 and type
2[1]. Type 2 diabetes is responsible for 90 to 95 percent of diabetes cases around the world.
In type 2 diabetes, insulin is available but the body cannot use it which is referred to as
insulin resistance. On the progression of type 2 diabetes, it gets worse and leading to
produce less insulin. There are several factors cause diabetes, among them, the main risk
factor for type 2 diabetes is obesity and overweight[3]. Looking at the main drawback with
insulin and its preparations, various oral hypoglycemic agents were discovered as
antidiabetic agents including alpha-amylase inhibitors (Fig. 1)[4]. Pancreatic and salivary
glands in human are the main regions for production and secretion of alpha-amylase
enzyme[5]. The enzyme can be obtained from three main sources viz plants, animals, and
microorganisms have a number of industrial and biotechnological applications. Alpha-
amylases play a major role in the metabolism of starch and glycogen to control the blood
glucose level. In human salivary and pancreatic enzymes are alpha-amylase enzymes[6].
Both enzymes are studied well due to their importance in a number of disorders like
diabetes, pancreatitis, and parotitis. Inhibitors of alpha-amylase enzyme may reduce the
glucose levels that can occur after a meal by slowing the speed of conversion of starch to
monosaccharides[7]. Recently various newer alpha amylase inhibitors were identified as
small molecules like indole,[8] benzofuran hydrazine,[9] thiadiazole,[10] and chromone
[11].
We designed our target molecules employing aza-Michael addition reaction due to its
versatility in constructions of C–N bond via nucleophilic addition of aniline/amine to alpha,
beta-unsaturated
compounds
in
a
straightforward
manner.
There are several catalyst used for aza-Michael reaction like Lewis acidic catalysts
PtCl4·5H2O,[12] Cu(OTf)2,[13] InCl3,[14] Yb(OTf3),[15] LiClO4,[16] Bi(NO3),[17]
FeCl3.6H2O,[18] CeCl3.7H2O,[19] ZnO,[20] MgO,[21] silica supported perchloric
acid,[22,23] ionic liquid,[24] sulfated zirconia,[25] organocatalytic,[26] and bases like 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU)[27].
3

Fig. 1. Representative oral antidiabetic drugs.
2. Results & discussion:
Since trans-chalcone is the scaffold which can inhibit the alpha-amylase enzyme[28,29]
and reduces the absorption of smaller absorbable carbohydrates like glucose thus, we have
designed 116 molecules based on trans-chalcone via aza-Michael addition (Fig. 2) to
investigate potentiation in alpha-amylase inhibition. The designed molecules were screened
by molecular docking study.
Fig. 2. Design of alpha-amylase inhibitor based on trans-chalcone.
4

For the identification of potentially active ligands, 116 designed molecules as 1,3 diaryl-3-
(arylamino)propan-1-ones were analyzed by molecular docking using Auto dock Vina
1.5.6, a molecular docking software[30]. Alpha-amylase protein 4gqr was downloaded
from pdb data bank[31] and validated from the internal ligand (MYC504) using Autodock
Vina molecular docking software. For the preparation of protein, it was reloaded and
various problems were fixed such as missing bonds or atoms and removed extraneous
structures like water molecules. Polar hydrogens were added along with the Kollman
charges. After saving the macromolecule (as pdbqt file) the 4gqr.pdbqt was loaded and set
it as map type by choosing ligand and grid box was generated. The protein was validated
by the extraction of ligand and docking it in the same manner as an actual ligand. All the
designed molecules were drawn by prepared ChemDraw program and their geometry was
optimized using the MM2 method[32]. All the optimized ligands were saved in pdb format
and the validated protein was used for docking study to find out binding affinities for 116
designed molecules which revealed that these molecules were found good compared to
MYC504, trans-chalcone, and acarbose (Refer Table S1 in supplementary information).
The most active compounds which were having a high binding affinity towards protein
4gqr were identified and taking in account of binding affinity (Kcal/mol), availability, cost,
and feasibility of reaction, some of the trans-chalcone derivatives i.e. 3a-f were selected as
shown in Table 1.
Table 1
Selected trans-chalcone derivatives based on binding affinity to 4gqr.
S. No.
Code
Structure
Binding affinity on
4gqr (Kcal/mol)
1
2
-8.0
-7.4
3a
3b
5

3
-7.4
3c
4
5
6
-8.3
-8.2
-7.5
3d
3e
3f
7
8
9
Internal ligand MYC504
-7.8
-7.5
-6.9
Basic scaffold trans-Chalcone
Standard
Acarbose
For in-depth study, an analysis at the binding site of 4gqr protein was done with 3a
molecule to understand the binding interactions. As in the case of compound 3a which is
having aniline substituent at β-carbon was found to interact with indole ring of TRP59
through pi-pi interaction. Aryl ring of TYR62 was found to be linked to both aryl rings of
3a through the pi-pi bond, along with it amino acid, THR163 was also attached leading to
high binding affinity i.e, -8.0 Kcal/mol (Fig. 3).
6

Fig. 3 The interaction of 3a with human pancreatic alpha-amylase (4gqr).
Introductions of other substituents such as CH3, OH, Br, Cl, F, and OCH3 at different meta
and para positions of three aromatic rings will be leading to the formation of a number of
derivatives with higher affinities than acarbose (Refer Table S1 in supplementary
information). According to above assumptions, we can conclude that aniline and the other
aromatic rings at β carbon are the lipophilic ones which can make stronger interactions
with the active site, α carbon is the linker and aromatic keto is the phenyl part.
Synthesis of the synthetically feasible most potent molecule (3a-3f) was carried out using
the scheme depicted below (Scheme 1). In step 1 the trans-chalcones (1a-1b) were
prepared by the aldol condensation reaction using acetophenone with benzaldehyde (a) or
4-methoxy benzaldehyde (b) and sodium hydroxide as a base in ethanol with 98% and 57%
yield respectively. Further trans-chalcone were reacted with aniline (2a-2e) in presence of
potassium hydroxide as a catalyst in ethanol to afford 1,3 diaryl-3-(arylamino)propan-1-one
(3a-3f) in 68-97% yield via aza-Michael addition (Table 2). Optimization of reaction
condition for the aza-Michael addition was done with prototype reaction using trans-
chalcone and aniline (Table 3) which suggested that ethanol and potassium hydroxide to be
best to afford 1,3 diaryl-3-(arylamino)propan-1-one in 97% yield.
7

Scheme 1. Synthesis of 1,3 diaryl-3-(arylamino)propan-1-ones via aza-Michael addition.
Table 2
Synthesis of 1,3 diaryl-3-(arylamino)propan-1-one derivatives.*
S.
No.
1
trans-Chalcone
Aniline
Product
Yield
(%)
97
2
66
8

3
4
71
70
5
6
82
68
*trans-chalcone (1eq, 4.8mmol), dissolved in 10 mL ethanol and added aniline (1eq,
4.8mmol) and potassium hydroxide (0.5eq, 2.4 mmol) were added and stirred at rt (room
temperature) for 16 hrs
Table 3 Effects of reagents and solvents on the aza-Michael addition of trans-chalcone and
aniline*
S. No.
Reagent (0.5 equiv)
Triethylamine
Triethylamine
Triethylamine
Triethylamine
Triethylamine
Solvent
Ethanol
Yield (%)
Time (hours)
1
2
3
4
5
69
17
NA
4
24
20
16
12
12
Methanol
Water
Acetonitrile
Benzyl
23
9

alcohol
Hexane
Toluene
DMF
6
Triethylamine
Triethylamine
Triethylamine
Triethylamine
NaOH
NA
NA
NA
48
12
12
12
12
16
20
12
16
24
12
12
24
7
8
9
Chloroform
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
10
11
12
13
14
15
16
17
83
LiOH.H2O
69
Pyridine
NA
97
KOH
Triethylamine
AlCl3
69
NA
17
Silica gel 100-200
Lithium perchlorate
NA
*trans-chalcone (1eq, 4.8mmol), dissolved in 5 mL ethanol and added aniline (1eq,
4.8mmol) and reagent (0.5eq, 2.4 mmol) were added and stirred at rt (room temperature);
NA = No reaction
All six synthesized 1,3 diaryl-3-(arylamino)propan-1-ones (3a-3f) were tested for their
alpha-amylase inhibitory activity with little modification described by Sudha et al.[33] and
others [34-36]. It was observed that all of them found active inhibtors of alpha-amylase
enzyme at different extents but better than acarbose (Table 4, Fig. 4). Log concentrations
versus % inhibition curves were plotted and IC50 values were calculated for all molecules
(Fig. 5). Among these 3e has shown highest activity against alpha-amylase enzyme with
IC50 23.17 μg/mL whereas 3a and 3f were the second and third most potent alpha-amylase
inhibitors with IC50 25.70 μg/mL and 48.17 μg/mL respectively. All these were found
better tha acarbose which was having IC50 891.25 μg/mL.
10

Fig. 4 % inhibition of alpha-amylase enzyme by 1,3 diaryl-3-(arylamino)propan-1-one and
acarbose at different concentrations.
Fig. 5 Comparison of alpha-amylase inhibitory activity of 1,3 diaryl-3-(arylamino)propan-
1-ones with acarbose
11

Table 4 IC50 values obtained by alpha-amylase inhibition and antioxidant activity
S. No.
1,3 diaryl-3-
IC50 (μg/mL)
Alpha-amylase
inhibition
IC50 (μg/mL)
(arylamino)propan-1-one
Antioxidant (DPPH
assay)
N
1
25.70
3a
2
3
4
5
6
7
8
225.89
177.82
251.18
23.17
48.17
891.25
-
N
3b
N
3c
79.43
3d
N
3e
N
3f
Acarbose
Ascorbic acid
-
831.76
*N = Low activity
Further, all 1,3 diaryl-3-(arylamino)propan-1-ones (3a-3f) were evaluated for their
antioxidant activity which was compared with a standard reducing agent i.e. ascorbic
acid[37]. Among all molecules, the only 3d has reduced DPPH with higher potency (IC50 =
79.43 μg/mL) whereas other found less potent compared to ascorbic acid. These results
suggested that substitution on all three phenyl rings have variant effect particularly un-
substituted or electron withdrawing groups tend to increase the potency for alpha amylase
whereas this is reverse in case of antioxidant activity.
3. Materials and methods:
3.1. Molecular docking
For the identification of potentially active ligands, 116 designed molecules as 1,3 diaryl-3-
(arylamino)propan-1-ones were analyzed by molecular docking using Auto dock Vina
1.5.6 software, a molecular docking software[30]. For the extraction and preparation of
ligands, 4gqr (alpha-amylase) protein was downloaded from the protein data bank (PDB
2017)[31]. For the preparation of protein, it was reloaded and various problems were fixed
12

such as missing bonds or atoms and removed extraneous structures like water molecules.
Polar hydrogens were added along with the Kollman charges. After saving the
macromolecule (as pdbqt file) the 4gqr.pdbqt was loaded and set it as map type by
choosing ligand and grid box was generated. The protein was validated by the extraction of
ligand and docking it in the same manner as an actual ligand. The designed molecules were
drawn by ChemDraw Ultra and converted to 3D structures. The geometry of all compounds
was optimized by semi-empirical MM2 method (ChemBioDraw 2012)[32]. Molecular
docking was performed on the optimized structure of the protein.
3.2. Chemistry
1
The H-NMR spectra were recorded at 400 MHz on a Bruker Avance 400 (400 MHz)
1
spectrometer in DMSO using TMS as an internal standard. The chemical shifts (δ) for H
are given in ppm. The following abbreviations are used to indicate the multiplicity: s,
singlet; d, doublet; m, multiplet. The reactions were monitored by TLC (Merck).
Evaporation of solvents was performed under reduced pressure using rotator evaporator
commercial grade reagents and solvents were used without further purification. The open
capillary method was employed for the determination of melting points and was
uncorrected. Fourier Transformed Infrared (FT-IR) spectroscopic data was recorded on a
spectrophotometer (Perkin Elmer 783, Pyrogen 1000 Spectrophotometer, USA) employing
the KBr disc method. Mass spectra of all the synthesized compounds were recorded on a
JEOL GC Mass spectrometer (Japan).
3.2.1. The general synthetic procedure of trans-chalcone:
trans-Chalcones (1a / 1b) were prepared by dissolving acetophenone (1eq, 8.3 mmol) in 10
ml of ethanol in a single-necked RBF fitted on a magnetic stirrer. Then aryl aldehyde (1eq,
8.3 mmol) and sodium hydroxide (0.1 eq, 0.83 mmol) were added separately while stirring.
The mixture was stirred for 5 hrs at room temperature and reaction progress was monitored
by TLC. The white precipitate was formed which was collected by vacuum filtration.
Thereafter the crude trans-chalcones were recrystallized from ethanol and characterized by
melting point and FTIR.
(E)-1,3-diphenyl-prop-2-en-1-one (1a)
Yield: 98%; white solid; m.p. 55˚C; FTIR (KBr, υ cm-1): 1659(C=O); 1603(C=C); Rf
(EtOAc : hexane; 05:95) = 0.69.
(E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (1b)
Yield: 57%; yellow solid; m.p. 62˚C; FTIR (KBr, υ cm-1): 1700(C=O); 1655(C=C);
1262(C-O). Rf (EtOAc : hexane; 10:90) = 0.73.
13

3.2.2. General synthetic procedure of aza-Michael addition to prepare 1,3 diaryl-3-
(arylamino)propan-1-ones
1,3-diphenyl-3-(arylamino)propan-1-ones (3a-3e) were prepared by dissolving trans-
chalcone (1a or 1b) [1eq, 4.80 mmol] in ethanol in a single-necked RBF fitted on a
magnetic stirrer. Then aniline (2a-2c) [1eq, 4.80 mmol] and potassium hydroxide [0.5 eq,
2.4 mmol] were added separately and stirred well. The mixture was stirred for 16 hours at
room temperature and reaction progress was monitored by TLC. The white colored
precipitate which was formed collected by vacuum filtration. Thereafter the crude product
was recrystallized from ethanol and was obtained in good yield.
1,3 diphenyl-3-(phenylamino)propan-1-one (3a)
Yield: 97%; white crystalline solid; m.p. 165˚C; IR (KBr, υ cm-1): 3384 (NH), 1668 (C=O);
1H NMR (400 MHz), DMSO-d6, δ(ppm): 3.2 (dd, J1,2= 4.6 Hz, J1,3= 16.9 Hz; 1H), 3.6 (dd,
J1,2= 8.8Hz, J1,3= 16.9Hz, 1H), 4.98-5.03 (m, 1H), 6.1 (d, J1,2= 7.4Hz, 1H, NH), 6.5 (t, J1,2=
7.3Hz, 1H), 6.5 (d, J1,2= 7.7Hz, 2H), 6.96 (t, J1,2= 0.9 Hz, 2H), 7.1 (t, J1,2= 1Hz, 1H), 7.4 (t,
J1,2= 12.5Hz, 2H), 7.42-7.60 (m, 5H), 7.9 (d, J1,2= 1.3Hz, J1,3= 5.9 Hz, 2H); 13C NMR (400
MHz), DMSO-d6, δ(ppm): 46.42, 52.81, 112.79, 115.77, 126.61, 126.68, 128.62, 128.28,
128.67, 133.15, 136.76, 144.01, 147.79, 197.30 (C=O); MS-TOF (m/z) = 302 (M+H); anal
C21H19NO calcd. C, 83.69; H, 6.35; N, 4.65. Found C, 83.71; H, 6.42; N, 4.68.
1,3-diphenyl-3-(p-tolylamino)propan-1-one (3b)
1
Yield: 66%; white solid m.p. 170˚C; IR (KBr, υ Cm-1): 3401(NH), 1660(C=O); H NMR
(400 MHz), DMSO-d6, δ(ppm): 2.07 (s, 3H, CH3), 3.3 (dd, J1,2= 4.6Hz, J1,3= 4.8Hz, 1H),
3.6 (dd, J1,2= 8.6Hz, J1,3= 16.7Hz, 1H), 4.94-4.97 (m, 1H), 5.98 (d, J1,2= 7.9Hz, 1H, NH),
6.4 (d, J1,2= 8.3Hz, 2H), 6.8 (d, J1,2= 8.2Hz, 2H), 7.2 (t, J1,2= 7.3Hz, 1H), 7.3 (t, J1,2= 7.7Hz,
2H), 7.4 (d, J1,2= 7.3Hz, 2H), 7.5 (t, J1,2= 7.5Hz, 2H), 7.63 (t, J1,2= 7.4Hz, 1H, Ar H), 7.9 (d,
13
J1,2= 7.3Hz, 2H, Ar H); C NMR (400 MHz), DMSO-d6, δ(ppm): 19.98, 46.44, 53.16,
113.03, 124.18, 126.63, 128.01, 128.22, 128.66, 129.11, 133.12, 136.79, 144.11, 145.49,
197.44(C=O); MS-TOF (m/z) = 316 (M+H); anal C22H21NO calcd. C, 83.78; H, 6.71; N,
4.44. Found C, 83.84; H, 6.79; N, 4.61.
3-(4-methoxyphenyl)-1-phenyl-3-(phenylamino)propan-1-one (3c)
Yield: 71%; light yellow crystalline solid; m.p.155 ˚C; IR (KBr, υ Cm-1): 3376(NH),
1
1666(C=O), 1286(C-O); H NMR (400 MHz), DMSO-d6, δ(ppm): 3.3 (dd, J1,2= 4.8Hz,
J1,3= 11.9Hz; 1H), 3.6 (dd, J1,2= 8.6Hz, J1,3= 8.2Hz, 1H), 3.7 (s, 3H, OCH3), 4.92-4.97 (m,
1H), 6.2 (d, J1,2= 7.76, Hz, 1H, NH), 6.46 (t, J1,2= 7.3Hz, 1H), 6.5 (d, J1,2= 7.8Hz, 2H), 6.85
14

(d, J1,2= 8.7 Hz, 2H), 6.97 (t, J1,2= 7.5Hz, 2H), 7.4 (d, J1,2= 8.7, Hz, 2H), 7.5 (t, J1,2= 7.4Hz,
2H), 7.62 (t, J 1,2= 6.3, 1H) 7.95 (d, J1,2= 1.3Hz, J1,3= 8.5Hz, 2H); 13C NMR (400 MHz),
DMSO-d6, δ(ppm): 46.51, 52.28, 54.92, 112.87, 113.64, 115.72, 127.68, 128.00, 128.66,
133.12, 135.76, 136.80, 147.81, 158.00, 197.46(C=O); MS-TOF (m/z) = 332 (M+H); anal
C22H21NO2 calcd. C, 79.73; H, 6.39; N, 4.23. Found C, 79.82; H, 6.45; N, 4.29.
3-((4-methoxyphenyl)amino)-1,3-diphenylpropan-1-one (3d)
Yield: 70% light green solid; m.p. 145˚C; IR (KBr, υ Cm-1): 3371(NH), 1666 (C=O),
1
1276(C-O); H NMR (400 MHz), DMSO-d6, δ(ppm): 3.3(dd, J1,2= 4.8Hz, J1,3=
16.6Hz;1H), 3.5 (s, 3H, OCH3), 3.6(dd, J1,2= 7.2Hz, J1,3= 16.6Hz, 1H), 4.89-4.94 (m, 1H),
5.8 (d, J= 8.1Hz, 1H, NH), 6.5(d, J1,2= 10.3Hz, 2H), 6.6 (d, J1,2= 10.3Hz, 2H), 7.15 (t, J1,2=
0.9Hz, 1H), 7.3 (t, J1,2= 7.7Hz, 2H), 7.4 (d, J1,2= 7.1Hz, 2H), 7.51(t, J1,2= 5.9Hz, 2H), 7.6
13
(t, J1,2= 7.4, 1H), 7.9 (d, J1,2= 4.2Hz, J1,3= 12.7Hz, 2H); C NMR (400 MHz), DMSO-d6,
δ(ppm): 46.52, 53.78, 55.17, 113.99, 114.38, 126.62, 126.66, 128.02, 128.22, 128.66,
133.11, 136.82, 141.93, 144.16, 150.69, 197.53(C=O); MS-TOF (m/z) = 332 (M+H); anal
C22H21NO2 calcd. C, 79.73; H, 6.39; N, 4.23. Found C, 79.81; H, 6.42; N, 4.27.
3-((4-chlorophenyl)amino)-1,3-diphenylpropan-1-one (3e)
Yield: 82%; white crystalline solid; m.p. 160˚C; IR (KBr, υ Cm-1): 3370(NH), 1664(C=O),
1
681(C-Cl); H NMR (400 MHz), DMSO-d6, δ(ppm): 3.31 (dd, J1,2= 4.5Hz, J1,3= 17Hz;
1H), 3.6 (dd, J1,2= 8.9Hz, J1,3= 17Hz, 1H), 4.94-4.99 (m, 1H), 6.4 (d, J1,2= 7.6Hz, 1H, NH),
6.5 (d, J1,2= 2Hz, 2H), 7.0 (d, J1,2= 3.3Hz, 2H), 7.2 (t, J1,2= 1.1 Hz, 1H), 7.3(t, J1,2= 7.8Hz,
2H), 7.4 (d, J1,2= 7.1Hz, 2H), 7.5 (t, J1,2= 17.1Hz, 2H), 7.6 (t, J1,2= 1Hz, 1H), 7.95(d, J1,2=
13
1.4Hz, J1,3= 8.5 Hz, 2H); C NMR (400 MHz), DMSO-d6, δ(ppm): 46.33, 52.84, 114.11,
119.04, 126.58, 126.80, 128.01, 128.34, 128.39, 128.67, 133.18, 136.69, 143.55, 146.73,
197.10(C=O); MS-TOF (m/z) = 336 (M+H); anal C21H18ClNO calcd. C, 75.11; H, 5.40; Cl,
10.56; N, 4.17. Found C, 75.21; H, 5.51; Cl, 10.61; N, 4.21.
3-((4-bromophenyl)amino)-1,3-diphenylpropan-1-one (3f)
Yield: 68%; greenish-white crystalline solid; m.p. 175˚CIR (KBr, υ Cm-1): 3369(NH),
1665(C=O), 565(C-Br); 1H NMR (400 MHz), DMSO-d6, δ(ppm): 3.3 (dd, J1,2= 4.6Hz, J1,3=
17.08Hz; 1H), 3.6 (dd, J1,2= 7.8Hz, J1,3= 17.0Hz, 1H), 4.93-4.98 (m, 1H), 6.4(d, J1,2=
1.96Hz, 1H, NH), 6.5 (d, J1,2= 2.4Hz, 2H), 7.1 (d, J1,2= 1.8Hz, 2H), 7.2 (t, J1,2= 7.3 Hz,
1H), 7.3 (t, J1,2= 7.8Hz, 2H), 7.4 (d, J1,2= 7.2Hz, 2H), 7.5 (t, J1,2= 1.6Hz, 2H), 7.6 (t, J1,2=
1.1Hz, 1H), 7.95 (d, J1,2= 1.3Hz, J1,3= 8.5 Hz, 2H); 13C NMR (400 MHz), DMSO-d6,
δ(ppm): 46.31, 52.74, 106.43, 114.69, 126.57, 126.80, 128.01, 128.35, 128.67, 131.20,
133.18, 136.68, 143.49, 147.08, 197.08(C=O); MS-TOF (m/z) = 382 (M+2); anal
15

C21H18BrNO calcd. C, 66.33; H, 4.77; Br, 21.01; N, 3.68. Found C, 66.41; H, 4.81; Br,
21.09; N, 3.74.
3.3. In-vitro alpha-amylase inhibitory activity assay
Alpha-amylase inhibitory activity assay was done by iodine-starch method[33-36].
Acarbose was used as a standard. The test is based on the formation of a complex between
iodine and starch which is blue in color and has a maximum absorbance at 580 nm. In
positive control solution, we added both alpha-amylase enzyme and starch but not any
inhibitor so that there should be 100% enzymatic activity and minimum absorbance (since
maximum starch is degraded by an alpha-amylase enzyme). On the other hand, negative
control solution did not contain alpha-amylase enzyme and inhibitor but an only starch
solution which upon addition of I2 solution a dark green colored complex was formed
which has shown maximum absorbance and no enzymatic activity. Overall, the absorbance
of test solutions should be in between the absorbance of positive and negative control
solutions and color intensity of them as well[33].
3.3.1. Preparation of phosphate buffer (0.02M, pH=6.9)
Solutions of 0.02M of Na2HPO4.12H2O and NaH2PO4.H2O were prepared separately and
their pH was detected by pH meter, thereafter the solution with lower pH (NaH2PO4.H2O)
was poured into solution with higher pH (Na2HPO4.12H2O) and rechecked pH of the
solution again, and adjusted by drop-wise addition of 0.006M NaCl or 1% NaOH solution
to 6.9.
3.3.2. Preparation of (0.5mg/mL) alpha-amylase solution
12.5 mg of aspergillus alpha-amylase was dissolved in minimum quantity of prepared
phosphate buffer (pH 6.9) in a 25 mL volumetric flask, thereafter complete solubility;
volume was made up to 25 mL by the same solution.
3.3.3. Preparation of 1% starch solution
1g of potato starch was dissolved in 100 mL of distilled water and boiled for 30 seconds on
a hot plate. The solution was cooled at room and filtered through simple filter paper.
Filtrates were used for the alpha-amylase inhibitory assay.
3.3.4. Preparation of 1% Iodine solution
1 g of iodine and 2 g of KI were dissolved in 50 ml of distilled water in a baker and stirred
on a magnetic stirrer overnight for complete solubility of I2, thereafter solution was
transferred to a 100 mL volumetric flask and volume was made up to 100 mL by the same
solvent and used for the assay.
16

3.3.5. Preparation of 1M HCl solution
8.6 mL of concentrated HCl solution was diluted to 100 mL with distilled water.
3.3.6. Preparation of stock sample solution
10 mg of each compound was dissolved in 10 mL methanol in a volumetric flask and was
labeled as a stock sample solution 1000 ppm.
3.3.7 Preparation of working sample solution
From a solution of stock (1000 ppm), 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL were
transferred to separate 10 mL volumetric flasks and volumes were made up by methanol,
thereafter labeled as working sample solutions 50 ppm, 100 ppm, 150 ppm, 200 ppm, and
250 ppm.
Enzyme activity and %inhibition were calculated using the formula below:
Enzyme activity = (Abs.of negative control - Abs.of positive control)-Abs. of test sample×100
(Abs. of negative control-Abs. of positive control)
Inhibition (%) = 100-enzyme activity
Where, the absorbance of the sample (test sample + alpha-amylase), the absorbance of
negative control (no alpha-amylase, 0% enzymatic activity, and 100% absorbance), and the
absorbance of positive control (no inhibitor, 100 enzymatic activity, and 0% absorbance).
Inhibition concentration (IC50): It is the concentration of the drugs at which 50% inhibition
takes place.
3.4. In-vitro antioxidant activity assay
The assay has been carried out according to Murti et al[37]. Ascorbic acid was used as a
standard. The test is based on the reduction of DPPH which is a free radical with dark
purple color and the maximum absorbance at 516 nm. As the DPPH is getting reduced with
an antioxidant it will turn its color to yellow, means with an increase in the concentration of
antioxidant the absorbance should be decreased.
3.4.1. Preparation of DPPH solution
A solution of DPPH (1.3 mg/mL) in methanol was prepared and was kept in dark for 2
hours, and used freshly in the same day.
3.4.2. Preparation of stock sample solution
10 mg of each compound was dissolved in 10 mL methanol in a volumetric flask and was
labeled as a stock sample solution 1000 ppm.
3.4.3. Preparation of working sample solution
17

From a solution of stock (1000 ppm), 0.5 mL, 1mL, 1.5 mL, 2 mL, and 2.5 mL were
transferred to separate 10 mL volumetric flasks and volumes were made up of methanol,
thereafter labeled as working sample solutions 50 ppm, 100 ppm, 150 ppm, 200 ppm, and
250 ppm.
The DPPH free radical scavenging activity was calculated using the following formula:
% scavenging = Abs. of positive control -Abs. of test sample×100
Abs. of positive control
Where; positive control is absorbance of a DPPH solution without compound; the sample is
the absorbance of the test molecules with DPPH.
4. Conclusion:
In conclusion we have explored the aza-Michael adducts of trans- chalcone by taking help
of Autodock Vina a molecular docking software to identify the best molecules. The
identified best molecules 1,3 diaryl-3-(arylamino)propan-1-one derivatives (3a-f) were
synthesized in a simpler manner using KOH as a catalyst via aza-Michael addition. Further
biological evaluation revealed that these were found active as alpha-amylase inhibitors and
antioxidants. The molecule 3e was found to be the most potent alpha-amylase inhibitor
whereas 3a was to be second most potent. Wheras DPPH antioxidant assay showed that
only 3d has potent antioxidant. Thus these 1,3 diaryl-3-(arylamino)propan-1-ones
derivative would be considered as potential antidiabetic agents and further study could lead
to the development of the better antidiabetic drug in future.
Conflict of interest
The authors confirm that this article content has no conflicts of interest.
Acknowledgment
Gopal L. Khatik is grateful to SERB- Department of Science & Technology (DST) Govt.
of India for research funding. Roqia Bashary is thankful of World Bank’s Higher Education
Development Program (HEDP), Afghanistan for the scholarship.
Supplementary Information
Available free online, it includes experimental and spectral data.
References:
[1] E. Adeghate, Open Med. Chem. J. 5 (2011) 68-69.
18

[2] G.L. Khatik, A.K. Datusalia, W. Ahsan, P. Kaur, M. Vyas, A. Mittal, S.K. Nayak, Curr.
Drug Discov. Technol.15 (2018) 163-177.
[3] H.W.Baynest, J. Diabetes Metab. 6 (2015) 1-9.
[4] M. Taha, N.H. Ismail, W. Jamil, S. Imran, F. Rahim, S.M. Kashif, M. Zulkefeli, Med.
Chem. Res.25 (2016) 225-234.
[5] L.Kandra, J. Mol. Struct. (Theochem) 666-667 (2003) 487-498.
[6] S. Das, S. Singh, V. Sharma, M.L.Soni, Int. J. Pharma. Bio. Sci. 2 (2011) 486-496.
[7] M B.Narkhede, Asian J. Pharm. Clin. Res. 5 (2012) 75-76.
[8] M. Taha, M.S. Baharudin, N.H. Ismail, S. Imran, M.N. Khan, F. Rahim, M. Selvaraj, S.
Chigurupati, M. Nawaz, F. Qureshi, S. Vijayabalan, Bioorg. Chem. 80 (2018) 36-42.
[9] M. Taha, S.A.A. Shah, S. Imran, M. Afifi, S. Chigurupati, M. Selvaraj, F. Rahim, H.
Ullah, K. Zaman, S. Vijayabalan, Bioorg. Chem. 75 (2017) 78-85.
[10] M. Taha, M.T. Javid, S. Imran, M. Selvaraj, S. Chigurupati, H. Ullah, F. Rahim, F.
Khan, J.I. Mohammad, K.M. Khan, Bioorg. Chem. 74 (2017)179-186.
[11] P. Valentina, K. Ilango, S. Chander, S. Murugesan, Bioorg Chem. 74 (2017)158-165.
[12] S. Kobayashi, K. Kakumoto, M. Sugiura, Org. Lett. 4 (2002) 1319-1322.
[13] L.-W. Xu, J.- W. Li, C.-G. Xia, S.-L. Zhou, X.-X. Hu, Synlett (2003) 2425-2427.
[14] T.P. Loh, L.-L. Wei, Synlett 1998, 975-976.
[15] G. Jenner, Tetrahedron Lett. 36 (1995) 233-236.
[16] N. Azizi, M. R. Said, Tetrahedron 60 (2004) 383-387.
[17] N. Srivastava, B. K. Banik, J. Org. Chem. 68 (2003) 2109-2114.
[18] L.-W. Xu, L. Li, C.-G. Xia, Helv. Chim. Acta. 87 (2004) 1522-1526.
[19] G. Bartoli, M. Bosco, E. Marcantoni, M. Petrini, L. Sanbri, E. Torregiani, J. Org.
Chem. 66 (2001) 9052-9055.
[20] A. Zare, A. Hasaninejad, A. Khalafi-Nezhad, A. R. M. Zare, A. Parhami, G.R.
Nejabat, Arkivoc i (2007) 58-69.
[21] A. Zare, A. Hasaninejad, A. Khalafi-Nezhad, A. R. M. Zare, A. Parhami, Arkivoc xiii
(2007) 105-115.
[22] G.L. Khatik, G. Sharma, R. Kumar, A.K. Chakraborti, Tetrahedron 63 (2007) 1200-
1210.
19

[23] C. Mukherjee, A. K. Misra, Lett. Org. Chem. 4 (2007) 54-59.
[24] S.R. Roy, A.K. Chakraborti, Org, lett. 12 (2010) 3866-3869.
[25] B. M. Reddy, M. K. Patil, B. T. Reddy, Catal. Lett. 126 (2008) 413-418.
[26] J. Wang, P. Li, P.Y. Choy, A.S.C. Chan, F.Y. Kwong, Chem. Cat. Chem. 4 (2012)
917-925.
[27] M. Baidya, H. Mayr, Chem. Commun. (2008) 1792-1794.
[28] A.T. Bale, K.M. Khan, U. Salar, S. Chigurupati, T. Fasina, F. Ali, A. Wadood, M.
Taha, S.S. Nanda, M. Ghufran, S. Perveen, Bioorg. Chem 79 (2018)179-189.
[29] M. Najafian, A. Ebrahim-Habibi, N. Hezareh, P. Yaghmaei, K. Parivar, B. Larijani,
Mol. Biol. Reports, 38(2011)1617-1620.
[30] O. Trott, A.J. Olson, J. Comput. Chem. (2010) 455-461.
[31] PDB is accessed from: https://www.rcsb.org/pdb/home/home.do Accessed on 5 Oct
2017, 17:22.
[32] ChemBioDraw. Energy minimizations were performed MM2 Interface program on
ChemBio3D Ultra 12.0, and structures were drawn by ChemBioDraw Ultra 12.0
(CambridgeSoft).
[33] P. Sudha, S.S. Zinjarde, S.Y. Bhargava, A.R. Kumar, BMC Complement Altern Med.
11 (2011) 1.
[34] J. Al-Asri, E. Fazekas, G. Lehoczki, A. Perdih, C. Görick, M.F. Melzig, G. Gyémánt,
G. Wolber, J. Mortier, Bioorg. Med. Chem. 23 (2015) 6725-6732.
[35] B. Prabha, S. Neethu, S.L. Krishnan, D.R. Sherin, M. Madhukrishnan, R.
Ananthakrishnan, K.B. Rameshkumar, T.K. Manojkumar, P. Jayamurthy, K.V.
Radhakrishnan. Bioorg. Med. Chem. 26 (2018) 3461-3467.
[36] Z.S. Toh, H. Wang, Y.M. Yip, Y. Lu, B.J. Lim, D. Zhang, D. Huang, Bioorg Med.
Chem. 23 (2015) 7641-7649.
[37] Y. Murti, A. Goswami, P. Mishra, Int. J. Pharm. Tech. Res. 5 (2013) 811-818.
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Graphical Abstract
Leave this area blank for abstract info.
Design, and facile synthesis of 1,3 diaryl-
3-(arylamino)propan-1-one derivatives as the
potential alpha-amylase inhibitors and
antioxidants
Roqia Bashary1,2, Gopal L. Khatik2*
1Department of Pharmaceutical Chemistry, Kabul University, Kabul, Afghanistan; 2School of Pharmaceutical
Sciences, Lovely Professional University, Jalandhar-Delhi G.T. Road, Phagwara (Punjab), India-144411
21

Highlights
 Autodock Vina a moleccular docking a tool to facilitate molecule design
 Facile synthesis of 1,3 diaryl-3-(arylamino)propan-1-one
 Identification of novel alpha-amylase inhibitors and antioxidants
 3e found to be most potent alpha-amylase inhibitors with IC50 23.17 μg/mL as
antidiabetic
 3d has reduced DPPH with higher potency (IC50 = 79.43 μg/mL) as antioxidant
22