Synthesis and exploration of in-silico and in-vitro α-glucosidase and α-amylase inhibitory activities of N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)arylamides

Article abstract of DOI:10.1007/s13738-018-01580-4

In a search for α-amylase and α-glucosidase inhibitors to treat type II diabetes, a new series of N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)arylamides were synthesized from 3-acetyl-2-methyl-4-phenylquinolines. Initially, nitro function of 1-(2-methyl-6-nitro-4-phenylquinolin-3-yl) ethanone was converted into the corresponding amine by grinding it with zinc dust and ammonium chloride (reducing agent) which in turn successfully converted into the N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl) arylamides by treating it with coupling reagents such as EDC, HATU, and DCC. All the synthesized compounds were found to afford excellent yields with HATU, moderate in EDC, and very less in DCC and hence, HATU was considered as a suitable coupling reagent. These analogs are structurally characterized by NMR, NMR-DEPT, and HRMS. All the synthesized compounds were evaluated for in-silico and in-vitro α-glucosidase and α-amylase inhibitory activity using acarbose as standard and all the compounds showed positive results by in-silico and in-vitro α-amylase inhibition assay. Among the tested compounds, compound 5c and 5d in α-glucosidase as well as in α-amylase are found to have least binding energy value. These compounds found to form more stable ligand–receptor complex amongst other compounds. In addition, in experimental part, also the compounds 5c and 5d exhibited 56.90 ± 0.77% and 59.46 ± 0.61% of the higher potent α-glucosidase inhibitory activity with IC₅₀ values 171.75 ± 3.95?μmol/mL and 171.67 ± 1.57?μmol/mL significantly (p < 0.05) compared to the remaining?seven test samples. And similarly, the compound 5c and 5d possessed α-amylase inhibitory activity at a concentration of 100?μg/mL (55.42 ± 0.42% and 55.42 ± 1.14%) with IC₅₀ values 138.92 ± 4.44?μmol/mL and 158.78 ± 2.34?μmol/mL.

Full text of DOI:10.1007/s13738-018-01580-4

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-01580-4
ORIGINAL PAPER
Synthesis and exploration of in-silico and in-vitro α-glucosidase
and α-amylase inhibitory activities of N-(3-acetyl-2-methyl-4-
phenylquinolin-6-yl)arylamides
L. Jyothish Kumar1 · Y. Suresh2 · R. Rajasekaran3 · S. Rajeswara Reddy2 · V. Vijayakumar1
Received: 24 May 2018 / Accepted: 24 December 2018
© Iranian Chemical Society 2019
Abstract
In a search for α-amylase and α-glucosidase inhibitors to treat type II diabetes, a new series of N-(3-acetyl-2-methyl-
4-phenylquinolin-6-yl)arylamides were synthesized from 3-acetyl-2-methyl-4-phenylquinolines. Initially, nitro function
of 1-(2-methyl-6-nitro-4-phenylquinolin-3-yl) ethanone was converted into the corresponding amine by grinding it with
zinc dust and ammonium chloride (reducing agent) which in turn successfully converted into the N-(3-acetyl-2-methyl-
4-phenylquinolin-6-yl) arylamides by treating it with coupling reagents such as EDC, HATU, and DCC. All the synthesized
compounds were found to afford excellent yields with HATU, moderate in EDC, and very less in DCC and hence, HATU was
considered as a suitable coupling reagent. These analogs are structurally characterized by NMR, NMR-DEPT, and HRMS.
All the synthesized compounds were evaluated for in-silico and in-vitro α-glucosidase and α-amylase inhibitory activity
using acarbose as standard and all the compounds showed positive results by in-silico and in-vitro α-amylase inhibition
assay. Among the tested compounds, compound 5c and 5d in α-glucosidase as well as in α-amylase are found to have least
binding energy value. These compounds found to form more stable ligand–receptor complex amongst other compounds.
In addition, in experimental part, also the compounds 5c and 5d exhibited 56.90 0.77% and 59.46 0.61% of the higher
potent α-glucosidase inhibitory activity with IC50 values 171.75 3.95 µmol/mL and 171.67 1.57 µmol/mL significantly
(p < 0.05) compared to the remaining seven test samples. And similarly, the compound 5c and 5d possessed α-amylase
inhibitory activity at a concentration of 100 µg/mL (55.42 0.42% and 55.42 1.14%) with IC50 values 138.92 4.44 µmol/
mL and 158.78 2.34 µmol/mL.
Keywords Arylamides · Coupling reagents (EDC, DCC and HATU) · Antidiabetic activity · α-Amylase inhibition activity ·
α-Glucosidase inhibition activity · Docking studies
Introduction
Preserving the living of people suffering from diabetes is
major impact on society, since the disease makes oxidative
stress consequential from glucose oxidation [1]. Diabetes
mellitus is a group of metabolic disorder and characterized
by pancreatic β-cells when does not make enough insulin or
is not proficient to utilize the insulin effectively, or both. The
hypersensitivity reactions of pancreas β-cells and the irregu-
lar digesting in carbohydrates, fat, and protein, and these
types of several factors are involved in the growth of diabe-
tes mellitus. The day-by-day diabetes mellitus, especially
in type 2 diabetes (NIDDM) cases, are increased and about
117 million are currently suffering worldwide, and the ratio
was estimated to the year of 2030 to reach 336 million [2].
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-018-01580-4) contains
supplementary material, which is available to authorized users.
* V. Vijayakumar
kvpsvijayakumar@gmail.com
1
Department of Chemistry, School of Advanced sciences,
Centre for Organic and Medicinal Chemistry, VIT
University, Vellore, Tamil Nadu 632 014, India
2
Division of Animal Biotechnology, Department
of Biotechnology, School of Herbal Studies and Naturo
Sciences, Dravidian University, Kuppam, AP 517 425, India
3
Department of Biotechnology, VIT University, Vellore,
Tamil Nadu 632 014, India
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
Hence, type 2 diabetes is a widespread metabolic disease
than the type 1 diabetes due to insulin resistance in the liv-
ing and marginal tissues [3]. Indeed, a number of in-vitro
and in-vivo studies have shown that the synthesized com-
pounds proceed to prevent diabetes mellitus is the inhibi-
tion activity of enzymes like α-amylase and α-glucosidase,
which are complex in the absorption of starch converted
in to maltose, absorption of glucose converted in to gly-
cogen, and in control of postprandial hyperglycemia [3].
An α-amylase enzyme is a well-known enzyme found in
the pancreatic juice and saliva, which divided into large
unsolvable starch molecules into edible molecules. There-
fore, inhibition of α-amylase enzymes plays a key role in
the management of degenerative diseases such as hypergly-
cemia. The α-glucosidase enzyme inhibition activity is one
of the significant therapeutic advances in the management
of hyperglycemia (type 2 diabetes) by slowing the digestion
system of glucose in the intestine [4]. Acarbose, miglitol,
and voglibose are the good inhibitors of α-glucosidase
enzyme and clinically used for the oral administration of
type 2 diabetes (INDDM), and these drugs are also used as
anticancer [5], anti-HIV [6], and anti-hepatitis agents [7].
Amide functionality is one of the basic structural motifs
in bio (organic) molecules such as peptides and proteins [8].
Many pharmaceutically active, naturally occurring [9] (e.g.,
β-lactams) and synthetic compounds [9] (e.g., anaesthetic
lidocaine) possess the amide group as the basis of their
biological efficacy. Comprehensively medicinal chemistry
applications exposed that the carboxamide or amide group
appears in more than 25% of known drugs [8]. Recently, aryl
amide-based organic compounds have gained considerable
scientific interest due to their wide range of applications
in heterocycles as well as in medicinal chemistry [8]. For
example, aminamide, pvacaine, and lidocaine exhibit inflam-
mation behaviours due to an amide linkage in their structural
unit. Amide-based drugs are well-known anticancer drugs;
for example, hydroxyl-pyrimidine showed anticancer activ-
ity and paracetamol used for pain and fever due to amide
linkage.
used for the design and synthesis of compounds with vari-
ous pharmacological properties such as antibacterial [13],
antifungal [14], antimalarial [15], anti-inflammatory [16],
anticancer [17], anti-tubercular [18], and anti-diabetic [19]
activities. So far, no reports are available on aryl amides
with 3-acetyl-2-methyl-4-phenylquinoline moiety in the
literature, and we also anticipated based on the diverse
biological activities of the quinolines and arylamides that
the introduction of aryl amides function into the 3-acetyl-
2-methyl-4-phenylquinoline moiety may lead to the new
derivatives with the enhanced activity. This prompted us
to attempt the present work in which we have synthesised
6-nitro-3-acetyl-2-methyl-4-phenylquinoline, subsequently
reduced to its corresponding amine which in turn (used as
a synthon) reacted with aryl acids to afford the correspond-
ing aryl amides using coupling reagents such as 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDC), hexafluoro
phosphate azabenzotriazole tetramethyl uronium (HATU),
and N,N′-dicyclohexylcarbodiimide (DCC).
Moreover, the inhibitory activity of 3-acetyl-4-phenlquin-
oline-based arylamides against α-amylase and α-glucosidase
were not yet investigated. Therefore, the present study was
deliberate to evaluate the inhibitory activity of new series
of arylamide based 3-acetyl-4-phenlquinolines along with
in-silico and in-vitro studies using the standard drug acar-
bose against α-amylase and α-glucosidase inhibition assay
followed by an examination of structure–activity relation-
ship (SAR).
Results and discussion
In the present work, a series of N-(3-acetyl-2-methyl-
4-phenylquinolin-6-yl)arylamides were synthesized by
coupling 6-amino-3-acetyl-2-methyl-4-phenylquinoline
with arylacids using coupling reagents such as HATU,
EDC, and DCC in the presence of DIEPA (Diisopropyleth-
H
H
N
O
H
N
O
N
H
N
H
N
N
N
N
HN
O
O
OH
O
O
HO
5-hydroxypyrimidine-
Aminamide
Lidocaine
Paracetamol
Pivacaine
2,4(1H,3H)-dione
Previously reported bioactive amide based drugs
There are reports available [10–12] on the formation of
aryl amide by condensing primary amines with a carbox-
ylic acid in the presence of coupling reagents [11, 12]. On
the other hand, 3-acetyl-4-phenylquinolines are frequently
ylamine) base. Initially, the ortho-nitro amino benzophe-
none was converted into 1-(2-methyl-6-nitro-4-phenylqui-
nolin-3-yl) ethanone 2 (Scheme 1) by reacting it with
acetylacetone in the presence of ortho-phosphoric acid
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Journal of the Iranian Chemical Society
O
O
Zinc dust
Ammonium chloride
O
O
H2N
O2N
O2N
O
Ethanol
Grinding for 20 mins
ortho-phosphoric acid
N
3
NH2
N
2
1
RT
O
Coupling Reagents
12 hrs
R
OH
Dry DMF
Reflux, 2 hrs
4a-i
Compounds
R
Compounds
R
4f, 5f
4g, 5g
4h, 5h
4i, 5i
3-Chloro
3-Fluoro
3-Iodo
-H
2-Chloro
4a, 5a
4b, 5b
4c, 5c
4d, 5d
4e, 5e
R
O
2-Iodo
2-Nitro
3-Bromo
H
N
4-Chloro
O
N
5a-i
Scheme 1 Synthesis of 1-(6-amino-2-methyl-4-phenylquinolin-3-yl) ethanone (3 and 5a–i)
(catalyst) in ethanol. In our earlier report [20], the nitro
function of compound 2 was reduced into the correspond-
ing amine 1-(2-methyl-6-amino-4-phenylquinolin-3-yl)
ethanone 3 using zinc/ammonium chloride in dioxane sol-
vent [20]. In the present work, the above procedure was
slightly modified; the compound 2 was converted into 3
Table 1 Optimization for
different coupling reagents
used in the formation of
arylamide-based 3-acetyl-4-
phenylquinolines
S. no.
Coupling reagents
R1
R2
Time (h)
Compounds
Yield (%)
1
2
3
4
5
6
7
8
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
EDCI
HATU
DCC
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4a
4a
4a
4b
4b
4b
4c
4c
4c
4d
4d
4d
4e
4e
4e
4f
1
5a
5a
5a
5b
5b
5b
5c
5c
5c
5d
5d
5d
5e
5e
5e
5f
92
22
55
78
17
27
88
29
58
92
30
67
85
28
65
87
21
49
95
14
69
89
10
45
98
15
55
24
12
1
24
12
1
24
12
1
24
12
1
24
12
1
24
12
1
24
12
1
24
12
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4f
4f
5f
5f
4g
4g
4g
4h
4h
4h
4i
5g
5g
5g
5h
5h
5h
5i
EDCI
HATU
DCC
4i
4i
24
12
5i
5i
EDCI
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Journal of the Iranian Chemical Society
through mechano-chemical procedure by grinding the
compound 2 with zinc/ammonium chloride using pestle
and mortar for 20 min without solvent (Scheme 1) which
in turn converted N-(3-acetyl-2-methyl-4-phenylquino-
lin-6-yl)arylamides 5a–i (Scheme 1) by coupled it with
various aryl acids 4a–i in the presence of DIEPA base
using coupling reagents such as HATU, DCC, and EDC.
The conversion of 3 into 5a–i reaction failed to afford the
desired products, while we employed other bases such as
K2CO3, NaOH, KOH, CS2CO3, and NaOMe by keeping
EDC, HATU, and DCC as coupling reagents. The com-
pound 5i showed high yield (98%) and the compound
5b showed less yield (78%), when HATU was used as a
coupling reagent (Scheme 1). In case of DCC, compound
5d (30%) showed high yield and compound 5h (10%)
showed less yield. In case of EDC, compound 5g (69%)
showed high yield and compound 5b (27%) showed fewer
yields. Overall, it was observed that the yields were more
with HATU, moderate in EDC and very less in DCC, and
hence, HATU was considered as a suitable coupling rea-
gent (Table 1).
protons] coupled with ortho proton of phenyl ring at C-4
with a coupling constant of 7.53 d [J=7.60 Hz, one proton].
The 13C-NMR spectrum exhibited the following chemical
shift values δ 205.0, 165.9, 152.6, 144.9, 143.4, 136.3, 135.3,
134.5, 132.0, 129.7, 129.0, 128.7, 127.0, 128.8, 125.5, 124.4,
115.1, 31.9, and 23.7 ppm. The up-field signal at δ 23.7 has
been assigned as methyl carbon at C-2 and the other signal
at δ 31.9 ppm has been assigned to the methyl of the acetyl
group at C-3. The extreme down field signal at δ 205.0 ppm
has been assigned to the carbonyl carbon of acetyl group at
C-15. And one more down field signal at d 165.9 ppm has
been assigned to carbonyl carbon of acetyl group at C-23.
The aliphatic CH3 carbons at (C-14 and C-16) carbons were
distinguished using DEPT-135 spectrum of the compound at
(δ 23.7 and 31.9 ppm). Remaining aromatic carbon-attached
hydrogen appears from the aromatic region from δ 132.0 to
115.1 ppm. The signal at 132.0 ppm has been assigned to the
C-8 and C-7 respectively. The C-5 carbon was identified as
d 127.0 ppm. Similarly, the ortho, meta, and para carbons
of phenyl group at C-4 were identified as δ 124.4, 129.7
and 129.0 ppm, respectively. In addition, one more phenyl
ring at C-6 was identified as δ 128.7 and 128.8 ppm, respec-
tively. The signals at δ 152.6, 135.3, 125.5, 144.9, 165.9, and
205.0 ppm were due to the non-proton bearing carbons at C-2,
C-23, C-3, C-17, C-24, C-15, and ipso positions. The proton
and carbon chemical shift assignments of compound 5a has
been summarized and are given as Figs. 1, 2. The formation
of compound 5a also been supported by the observation of
m/z value at 381.1603 in the mass spectrum.
All the newly synthesized arylamides 5a–i were charac-
terized using 1H NMR, 13C NMR, DEPT-135, H, H-COSY,
HSQC, and HR-MS (ESI), and the detailed data were
included in the experimental part. Consequently, all the
newly derived target molecules 5a–i subjected to in-silico
and in-vitro studies by α-glucosidase and α-amylase inhibi-
tory activity. The compound 5a has been considered as a
representative example and its spectral characterization is
described as follows.
In‑silico studies by α‑glucosidase and α‑amylase
inhibition assay
Spectral characterization of compound (5a)
1
The compound 5a also further characterized by H-NMR,
Methodology
13C-NMR, DEPT-135, H, H-COSY, C, H-COSY, and HR-MS
(ESI) spectral data. The 1H-NMR spectrum of compound 5a
exhibited the following chemical shifts δ 8.14 s [one proton],
8.04 d [J=4.00 Hz, two protons], 7.88 s [one proton], 7.82 d
[J=7.60 Hz, two protons], 7.53 d [J=7.60 Hz, one proton],
7.49 d [J=7.20 Hz, three protons], 7.43 t [J=8.00 Hz, two
protons], 7.36 d [J=7.60 Hz, two protons], 2.67 s [three pro-
tons], and 1.98 s [three protons]. Also this compound shows
the absence of –NH proton in deuterium exchanging at C-6
(containing amide bond) when D2O used as a solvent along
with CDCl3. The examination of H, H-COSY spectrum of the
molecule shows that the singlets at δ 2.67 ppm and 1.98 ppm
which are interesting for three protons of each are due to the
methyl groups of the molecule. The relatively up-field signal
δ 1.98 ppm is assigned to C-14 and the relatively down-field
signal δ 2.67 ppm is due to the methyl of the acetyl group
at C-16. The signal at δ 8.04 d [J=4.00 Hz, two protons]
coupled with the signal at δ 8.14 s [one proton] and hence
assigned as C-8. The signal at δ 7.82 d [J=7.60 Hz, two
Modelled structures of α-glucosidase and α-amylase were
retrieved from Swiss model repository [21] and (inhibitors)
.
7 49
.
7 49
.
7 49
11
12
.
7 43
10
.
7 36
.
7 53
13
.
.
7 82 7 36
21
19
17
9
O
22
8
8
.
H
N
.
2 67
23
18
4
7
3
a
5
24
6
8
.
7 43
.
15
20
5
1
16
4
7
O
.
1 98
14
2
a
8
N
.
7 82
8
1
.
8 04
.
8 04
Fig. 1 Summary of proton chemical shifts of 5a
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Journal of the Iranian Chemical Society
124
Table 2 Docking studies of compounds (5a–i) for α-glucosidase and
.
4
α-amylase
.0
9
2
1
.0
9
.7
12
9
2
Test compounds
Binding energy (BE) kcal/mol
1
12
8
.
7
12
.
8
8
.7
9
9
.
12
α-Glucosidase inhibition α-Amylase
12
.
8
7
44
1
0
O
assay
inhibition
assay
0
.
3
.
5
.
.
4
7
1
.
5
H
N
5
0
2
5
1
1
3
3
2
1
1
1
1
.
31 9
1
1
3
2
6
6
5a
−6.86
−6.21
−7.98
−6.88
−6.67
−5.15
−5.39
−5.33
−6.61
−3.4
−6.36
−6.04
−7.59
−6.63
−6.47
−6.41
−5.46
−5.85
−6.48
−3.4
5
.
4
12
.
.
8
7
.
5
3
2
O
5b
1
1
4
3
12
.
8
8
N
5
2
.
23 7
.
5c
4
.
6
.
129 9
5d
.
132 0
5e
5f
Fig. 2 Summary of carbon chemical shifts of 5a
5g
5h
small molecules were constructed using Marvin sketch. Fur-
thermore, the modelled enzymes and the small molecules
were subjected to energy minimization using Gromacs [22];
followed by the docking analysis which was carried out using
Auto dock 4.2 [23] program, where the XYZ grid points were
set to 50 each with a grid spacing of 0.558 Å. Population size
was set to 150. The number of grid points for X, Y, and Z was
126 each. Grid box centre for X, Y, and Z coordinates were
6.427, 29.225, and 50.704, respectively. Docking was carried
out for 10 GA (genetic algorithm) runs which produced ten
different conformations of each inhibitor (small molecules).
The conformations were then ranked based on their binding
energies and complex structures with least energies were
depicted in figures. The enzyme structure for Glucosidase
(PDB ID:5NN3) and amylase (PDB ID: 5U3A) were obtained
from Protein Data Bank [24]. The number of grid points for
Glucosidase is X=96, Y=96, and Z=126 with the grid spac-
ing of 0.978 Å, and the number of grid points for amylase is
X=98, Y=126, and Z=100 with the grid spacing of 0.647 Å.
5i
Acarbose
binding energies for docked derivatives were found to be in
range of −7.98 and −5.15 kcal/mol. All the compounds 5a–i
showed good binding energies with both α-glucosidase and
α-amylase as well as in in-vitro α-glucosidase and α-amylase
inhibition assay. Among the tested compounds, compound
5c and 5d in α-amylase as well as in α-glucosidase are found
to least binding energy value (Figs. 3, 4, 5 and 6). These
compounds found to form more stable ligand–receptor com-
plex amongst other compounds, and hence, these two com-
pounds were identified as lead molecules.
Interaction between receptor and ligand visualized
through discovery studio visualizer
In‑silico α‑glucosidase and α‑amylase inhibition
assay
In the recent years, the in-silico method for molecular dock-
ing plays an important role in the drug designing as well as
in the systematic study of docking a drug molecule into the
binding site or active site of the target protein [25]. Auto
dock is a flexible docking program which enables us to pre-
dict the favourable protein–ligand complex structures with
reasonable speed and accuracy. The docking technique will
undoubtedly continue to play an important role in drug dis-
covery. In this study, we docked the synthesized arylamides
5a–i into the active site of α-glucosidase and α-amylase
enzymes to predict their binding modes, binding affinities,
and the orientation of these compounds.
The arylamides 5a–i was successfully docked onto the
active site of α-glucosidase and α-amylase enzymes, and
their binding energies are represented in Table 2. The
Fig. 3 Test compound 5c (α-glucosidase)
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Journal of the Iranian Chemical Society
Fig. 4 Test compound 5d (α-glucosidase)
Fig. 6 Test compound 5d (α-amylase)
Statistical analysis
Statistical analysis was performed using SPSS 16.0 statisti-
cal package. The data were analysed by one-way analysis
of variance (ANOVA) followed by Duncan. All the results
were expressed as mean SD for triplicate determinations.
IC50 values were calculated using Graph Pad Prism 7 version
(Graph pad software, Inc., La Jolla, CA, USA) statistical
software.
Study of α‑glucosidase inhibition assay
The α-glucosidase inhibitory activity was determined by
published modified method [26]. 1 mg of α-glucosidase
(Saccharomyces cerevisiae isolated) was dissolved in
100 mL of pH 6.8 neutral phosphate buffer saline which
contains BSA (bovine serum albumin) 200 mg. The various
concentrations (20–100 µg/mL) of test samples were consist-
ing of reaction mixture: 10 µL was premixed with pH 6.8
phosphate buffer; 490 µL and 5 mM concentrated 250 µL of
p-NPG (p-nitro phenyl α-d-glucopyranoside). After comple-
tion of 5 min preincubation at 37 °C, add 250 µL (0.15 unit/
mL) of α-glucosidase and incubated at 37 °C for 15 min.
The reaction was stopped by the adding 2 mL of 200 mM
sodium carbonate. The enzyme activity was read at 405 nm
on UV–Vis spectrophotometer by calculating the quantity
of p-NPG released p-nitrophenol. Acarbose was used as
a standard drug for in this assay. Calculate the percentage
inhibition of α-glucosidase by using the following formula:
Fig. 5 Test compound 5c (α-amylase)
In‑vitro antidiabetic activity by α‑glucosidase
and α‑amylase inhibition assay
Chemicals and reagents used
α-Amylase and α-glucosidase enzymes, acarbose, 3,5-dini-
tro salicylic acid, and p-nitro phenyl α-d-glucopyranoside
(p-NPG) were purchased from Sigma-Aldrich (Bangalore,
India). Sodium phosphate buffer and potassium phosphate
buffers were prepared freshly in required PH. All other chem-
ical reagents were used as analytical grade in this study.
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Journal of the Iranian Chemical Society
(
) (
)
arylamides showed low potent 28.35 0.93% at the concen-
tration of 100 µg/mL α-amylase inhibitory activity with an
IC50 value of 460.79 12.53 µmol/mL.
Inhibition percentage = ControlA − SampleA / ControlA × 100,
where A=absorbance.
Study of α‑amylase inhibition assay
In‑vitro α‑amylase inhibition assay
The inhibition of α-amylase activity was carry out by the
procedure elucidate by [27]. Add 1 mL of 1% phosphate
buffer; prepared starch solution was incubated with 500 µL
of enzyme (α-amylase) solution for 10 min at 37 °C. In the
test sample, 1 mL (20–100 µg/mL) was added to enzyme
solution. 2 M of NaOH solution is apply to prevent the reac-
tion. 1 mL of DNS (Dinitro salicylic acid) solution is mixed
and solution is retained in hot water bath for 5 min. The test
tubes was cooled with running tap stream, the final volume
was prepared up to 10 mL using distilled water, and OD was
read at 540 nm against control/blank (without test sample)
using UV–Vis spectrophotometer. Acarbose was used as
reference chemical. Calculate the percentage inhibition of
α-amylase using the following formula:
Acarbose showed 76.57 0.57% inhibitory effects at a con-
centrations 100 µg/mL on the α-amylase activity with an
IC50 value 30.32 2.76 µmol/mL. The arylamide-based
3-acetyl-4-phenyl quinoline derivates of 5c and 5d exhibited
55.42 0.42% and 55.42 1.14% of higher potent α-amylase
inhibitory activity with an IC50 values 138.92 4.44 µmol/
mL and 158.78 2.34 µmol/mL significantly (p < 0.05)
compared to remaining seven test samples (Table 3). The
test sample 5g of arylamide-based 3-acetyl-4-phenyl qui-
noline derivate showed low potent 39.61 0.87% at the
concentration of 100 µg/mL α-amylase inhibitory activity
with an IC50 value 434.32 31.50 µmol/mL. However, 5e
(172.16 4.02 µmol/mL) and 5i (193.73 8.21 µmol/mL)
test samples of arylamides IC50 values were significantly
(p < 0.05) appreciable; α-amylase inhibitory effect was
showed when compared with remaining samples. The 100%
α-amylase inhibition of arylamide-based 3-acetyl-4-phenyl
quinoline graphs is shown in Table 3.
(
) (
)
Inhibition percentage = ControlA − SampleA / ControlA × 100,
where A=absorbance.
In‑vitro α‑glucosidase inhibition assay
The in-vitro α-glucosidase inhibitory activity of the aryla-
mides 5a–i is summarized in Table 3. The test sample 5d
possessed α-glucosidase inhibitory activity at a concentra-
tion of 100 µg/mL (59.46 0.61%). The test samples 5d
and 5c are most potent, with IC50 of 171.67 1.57 µmol/
mL and 171.75 3.95 µmol/mL significantly (p<0.05) com-
pared to the IC50 values of remaining seven test samples.
The standard acarbose was exhibited 81.06 0.97% inhibi-
tion with IC50 of 48.24 0.97 µmol/mL. The 5 g derivate of
Experimental
Methods and materials
Melting points (mp) reported in this work was recorded in
Elchem microprocessor-based DT apparatus in open cap-
illary tubes and is incorrect. H NMR and 13C NMR are
1
recorded in Bruker 400 MHz NMR spectrometer with
TMS as an internal reference. The chemical shift values are
Table 3 α-Amylase and
Compound code
100% Inhibition
α-Amylase IC50
100% Inhibition of α-Glucosidase IC50
α-glucosidase inhibition activity
of synthesis of new N-(3-acetyl-
2-methyl-4-phenylquinolin-6-yl)
arylamides (5a–i)
of α-amylase
values (µmol/mL)
α-glucosidase
values (µmol/mL)
Acarbose
76.57 0.57%
45.14 1.14%
45.14 1.51%
55.42 0.42%
55.42 1.14%
53.14 0.57%
51.23 1.18%
39.61 0.87%
39.61 1.31%
53.71 0.98%
30.32 2.76a
318.59 20.90f
282.70 3.95f
138.92 4.44b
158.78 2.34b
172.16 4.02c
239.63 22.25e
434.32 31.50g
281.64 2.14f
193.73 8.21d
81.06 0.63%
49.33 0.5%
36.54 0.70%
56.90 0.77%
59.46 0.61%
48.61 0.77%
31.32 1.07%
28.35 0.93%
34.08 0.63%
45.34 0.61%
48.24 0.97a
267.68 14.88c
307.17 4.10d
171.75 3.95b
171.67 1.57b
253.06 12.03c
398.74 19.04e
460.79 12.53f
305.07 16.74d
289.29 7.17d
5a
5b
5c
5d
5e
5f
5g
5h
5i
Results are expressed as mean SD (n=3). Values in the column followed by the same superscript letter
(a–g) are significantly the same (p<0.05). α-Amylase and α-glucosidase inhibitory activity determined by
in-vitro antidiabetic assay was measured using Spectrophotometer. Values of % inhibition are mean of three
independent determinations at 100 µg/mL concentration of the test samples
1 3

Journal of the Iranian Chemical Society
reported in parts per million (δ, ppm) from internal stand-
ard TMS. Mass spectra are obtained from HRMS (Maxis
10138) analyzer. All reagents were purchased from Aldrich
and used as received. Solvents were removed under vacuum.
Organic extracts were dried with anhydrous Na2SO4. Silica
gel 60F254 aluminium sheets were used in analytical thin-
layer chromatography. Visualization of spots on TLC plates
was effected by UV illumination, exposure to iodine vapour,
and heating the plates dipped in the KMnO4 stain. In column
chromatography, the silica gel with 230–400 mesh size was
used for the purification.
product 5a. The solvent was removed in vacuum and the
crude was taken up in ethyl acetate, and the mixture was
diluted with ethyl acetate (25 mL) and washed with 2N HCl,
Sat. NaCl, dried with sodium sulphate and concentrated. The
resulting crude was purified by silica gel chromatography
with hexane: ethyl acetate (7:1). The similar procedure was
repeated for 3 with different substituted benzoic acids 4b–i
in the presence of DCC to get the product 5b–i.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)benzamide
(5a)
General procedure for the synthesis of compounds
(5a–i) in the presence of EDC (coupling reagent)
1
Yellow solid, yield 92%, m.p.: 148–150 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.14 (s, 1H), 8.04 (d,
J = 4.00 Hz, 2H), 7.88 (s, 1H), 7.82 (d, J = 7.60 Hz, 2H),
7.53 (d, J = 7.60 Hz, 1H), 7.49 (d, J = 7.20 Hz, 3H), 7.43
(t, J = 8.00 Hz, 2H), 7.36 (d, J = 7.60 Hz, 2H), 2.67 (s,
3H), 1.98 (s, 3H); 13C NMR (100 MHz, CDCl3): δ ppm
204.0, 165.9, 152.6, 144.9, 143.4, 136.3, 135.3, 134.9,
132.0, 129.7, 129.0, 128.7, 127.0, 128.6, 125.5 124.4,
115.1, 31.9, 23.7. HRMS-ESI (m/z) calcd for C25H20N2O2
[M+H]+ =381.1603, found=381.1603.
A mixture of EDC (0.12 g, 0.0006 M) N, N-diisopropyleth-
ylamine (0.15 mL, 0.1224 M), 1-(6-amino-2-methyl-4-phe-
nylquinolin-3-yl) ethanone 3 (0.1 g, 0.0003 M), benzoic acid
4a (0.08 g, 0.0003 M), and HOBt (0.1 g, 0.0006 M) in DMF
solvent (10 mL) stirred for 12 h at room temperature under
nitrogen atmosphere which afford the product 5a. The com-
pletion of the reaction was checked by TLC. The solvent was
removed in vacuum and the crude was purified by silica gel
column chromatography with hexane: ethyl acetate (7:1).
The similar procedure was repeated for 3 with different sub-
stituted benzoic acids 4b–i in the presence of EDC to get the
product 5b–i.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-2-chloroben-
zamide (5b)
General procedure for the synthesis of compounds
(5a–i) in presence of HATU (coupling reagent)
Pale yellow solid, yield 78%, m.p.: 145–147 °C; 1H NMR
(400 MHz, CDCl3): δ ppm 8.08 (d, J=8.00 Hz, 2H), 7.99
(d, J=7.60 Hz, 1H), 7.92 (s, 1H), 7.69 (d, J=6.00 Hz, 1H),
7.50 (s, 3H), 7.37 (t, J = 9.20 Hz, 5H), 2.68 (s, 3H), 1.99
(s, 3H); 13C NMR (100 MHz, CDCl3): δ ppm 205.6, 152.8,
145.1, 143.5, 135.8, 135.3, 135.0, 135.9, 131.8, 130.6,
130.3, 130.0, 129.0, 128.8, 127.2, 125.5, 124.1, 115.2,
31.8, 23.7. HRMS-ESI (m/z) calcd for C25H19ClN2O2
[M+H]+ =415.1213, found=415.1215.
To a solution of the benzoic acid (0.08 g, 0.0003 M) 4a in
DMF (10 mL) was added DIEPA (0.15 mL, 0.0006 M) fol-
lowed by HATU (0.17 g, 0.0006 M). The mixture was stirred
at RT for 5 min; after some time, the dark clear mixture
was appeared, which is further treated with the 1-(6-amino-
2-methyl-4-phenylquinolin-3-yl) 3 (0.1 g, 0.0003 M). The
reaction mixture was stirred at RT for 1 h to get product
5a. The mixture was diluted with ethyl acetate (25 mL) and
washed with 2N HCl, Sat. NaCl, dried with sodium sulphate
and concentrated. The resulting crude was purified silica gel
column chromatography with hexane: ethyl acetate (7:1).The
similar procedure was repeated for 3 with different substi-
tuted benzoic acids 4b–i in the presence of HATU to get the
product 5b–i.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-2-iodobenza-
mide (5c)
1
Colourless solid yield; 88%, m.p.: 147–149 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.14 (d, J=8.80 Hz, 1H), 8.07 (d,
J=8.80 Hz, 1H), 7.87 (t, J=7.20 Hz, 2H), 7.68 (s, 1H), 7.50
(s, 4H), 7.39 (s, 3H), 7.12 (t, J=7.20 Hz, 1H), 2.69 (s, 3H),
1.99 (s, 3H); 13C NMR (100 MHz, CDCl3): δ ppm 205.4,
167.2, 144.9, 143.7, 140.0, 135.4, 134.9, 131.6, 130.0,
129.7, 129.1, 128.8, 128.6, 128.3, 125.5, 124.2, 115.1,
92.2, 31.8, 23.5. HRMS-ESI (m/z) calcd for C25H19IN2O2
[M+H]+ =507.0569, found=507.0569.
General procedure for the synthesis of compounds
(5a–i) in the presence of DCC (coupling reagent)
To a solution of the 1-(6-amino-2-methyl-4-phenylquinolin-
3-yl) 3 (0.1 g, 0.0003 M), benzoic acid 4a (0.08 g, 0.0003 M)
in THF (3 mL) at 0 °C was added DCC (0.18 g, 0.0006 M).
The resulting mixture was stirred at RT for 24 h to get the
1 3

Journal of the Iranian Chemical Society
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-2-nitrobenza-
mide (5d)
122.6, 119.1, 118.8, 115.5, 114.6, 114.4, 31.8, 23.6. HRMS-
ESI (m/z) calcd for C25H19FN2O2 [M + H]+ = 399.1509,
found=399.1508.
1
Brown solid, yield 92%, m.p.: 161–162 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.57 (s, 1H), 8.04 (s, 3H), 7.85
(s, 1H), 7.64 (d, J=6.80 Hz, 1H), 7.56 (d, J=4.80 Hz, 2H),
7.46 (s, 3H), 7.36 (s, 2H), 2.65 (s, 3H), 1.98 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ ppm 205.7, 164.6, 152.8, 146.1,
145.0, 143.5, 136.1, 135.3, 134.9, 133.8, 130.7, 129.8,
129.0, 128.8, 125.5, 124.4, 124.2, 115.4, 31.8, 23.6. HRMS-
ESI (m/z) calcd for C25H19N3O4 [M + H]+ = 426.1454,
found=426.1453.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-3-iodobenza-
mide (5h)
1
Colourless solid, yield 89%, m.p: 156–158 °C; H NMR
(400 MHz, CDCl3): δ ppm 7.53 (d, J=1.60 Hz, 1H), 7.51
(dd, J = 1.20, 3.80 Hz, 1H), 7.48 (d, J = 13.60 Hz, 1H),
7.48 (d, J=12.00 Hz, 1H), 7.43 (d, J=5.60 Hz, 2H), 7.41
(d, J = 5.60 Hz, 1H), 7.37 (dd, J = 2.40, 4.20 Hz, 2H),
7.36–7.23 (m, 1H), 7.23 (d, J = 0.80 Hz, 2H), 7.21 (dd,
J=0.80, 2.40 Hz, 1H), 2.68 (s, 3H), 2.00 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ ppm 204.7, 164.8, 152.8, 145.0,
143.4, 138.3, 136.0, 135.3, 136.0, 135.3, 135.0, 132.9,
129.9, 129.8, 129.1, 129.0, 128.8, 128.5, 125.5, 124.4,
115.3, 31.9, 23.7. HRMS-ESI (m/z) calcd for C25H19IN2O2
[M+H]+ =507.0569, found=507.0569.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-3-bromoben-
zamide (5e)
1
Brown solid, yield 85%, m.p.: 157–159 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.24 (d, J=8.80 Hz, 1H), 8.12
(s, 1H), 8.05 (s, 1H), 7.98 (d, J = 8.40 Hz, 1H), 7.92 (d,
J = 7.20 Hz, 2H), 7.67 (d, J = 7.20 Hz, 1H), 7.55 (s, 3H),
7.38 (d, J = 4.80 Hz, 3H), 2.32 (s, 3H), 1.69 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ ppm 210.1, 169.4, 156.7, 149.4,
142.4, 141.9, 140.1, 139.9, 135.5, 135.0, 134.8, 133.8,
133.8, 131.7, 129.9, 129.8, 126.8, 126.0, 36.7, 28.4. HRMS-
ESI (m/z) calcd for C25H19BrN2O2 [M + H]+ = 459.0708,
found=459.0708.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-4-chloroben-
zamide (5i)
1
Yellow solid, yield 98%, m.p.: 146–147 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.08 (d, J=8.40 Hz, 2H), 8.02
(d, J=8.80 Hz, 1H), 7.86 (s, 1H), 7.77 (d, J=8.40 Hz, 2H),
7.51 (d, J = 6.40 Hz, 3H), 7.41 (d, J = 8.40 Hz, 2H), 7.36
(t, J = 1.60 Hz, 2H), 2.68 (s, 3H), 2.00 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ ppm 205.7, 164.8, 152.8, 145.0, 143.4,
138.3, 136.0, 135.3, 135.0, 132.9, 129.9, 129.8, 129.1,
129.0, 128.8, 128.5, 125.5, 124.4, 115.3, 31.9, 23.7. HRMS-
ESI (m/z) calcd for C25H19ClN2O2 [M + H]+ = 415.1213,
found=415.1215.
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-3-chloroben-
zamide (5f)
1
Yellow solid, yield 87%, m.p.: 167–169 °C; H NMR
(400 MHz, CDCl3): δ ppm 10.45 (s, 1H), 8.24 (d,
J = 8.00 Hz, 1H), 8.08 (d, J = 8.00 Hz, 1H), 7.99 (d,
J=7.60 Hz, 1H), 7.92 (s, 1H), 7.69 (d, J=6.00 Hz, 1H), 7.67
(d, J=1.20 Hz, 1H), 7.50 (s, 3H), 7.38 (d, J=4.80 Hz, 3H),
2.62 (s, 3H), 1.99 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
ppm 210.1, 169.4, 156.7, 149.4, 147.8, 142.4, 141.9, 140.1,
139.2, 135.5, 135.0, 134.8, 133.8, 133.8, 133.6, 131.7,
129.9, 129.8, 126.8, 120.0, 28.4. HRMS-ESI (m/z) calcd
for C25H19ClN2O2 [M+H]+ =415.1213, found=415.1215.
Conclusion
In summary, we have synthesized 3-acetyl-4-phenylquin-
oline based aryl amides from 1-(2-methyl-6-amino-4-phe-
nylquinolin-3-yl)ethanone using coupling reagents such as
HATU, EDCI, and DCC, and it was observed that the yields
were more with HATU, moderate in EDC, and very less
in DCC, and hence, HATU was considered as a suitable
coupling reagent. In addition, all the newly synthesized
arylamides were subjected to in-silico and in-vitro anti-
diabetic activity by α-glucosidase and α-amylase inhibition
assay using acarbose as standard. Among the tested com-
pounds, compound 5c and 5d in α-glucosidase as well as
in α-amylase are found to least binding energy value in in-
silico studies. These compounds found to form more stable
ligand–receptor complex amongst the other compounds.
And in experimental part, also the compounds 5c and 5d
exhibited 56.90 0.77% and 59.46 0.61% of the higher
N-(3-acetyl-2-methyl-4-phenylquinolin-6-yl)-3-fluoroben-
zamide (5g)
1
Yellow solid, yield 95%, m.p: 151–153 °C; H NMR
(400 MHz, CDCl3): δ ppm 8.00 (t, J =9.60 Hz, 2H), 7.89
(s, 1H), 7.57 (d, J=7.20 Hz, 2H), 7.51 (d, J=8.80 Hz, 3H),
7.45 (d, J = 3.60 Hz, 4H), 7.18 (s, 1H), 2.62 (s, 3H), 1.99
(s, 3H); 13C NMR (100 MHz, CDCl3): δ ppm 205.7, 164.7,
163.9, 161.5, 152.8, 145.0, 143.5, 136.8, 136.7, 136.0,
135.3, 134.9, 130.4, 130.3, 129.9, 128.8, 125.4, 124.4,
1 3

Journal of the Iranian Chemical Society
9. A.A. Bekhit, O.A. El-Sayed, E. Aboulmagd, J.Y. Park, Eur. J.
Med. Chem. 39, 249–255 (2004)
potent α-glucosidase inhibitory activity with IC50 values
171.75 3.95 µmol/mL and 171.67 1.57 µmol/mL sig-
nificantly (p<0.05) compared to remaining seven test sam-
ples. And, similarly, the compound 5c and 5d possessed
α-amylase inhibitory activity at a concentration of 100 µg/
mL (55.42 0.42% and 55.42 1.14%) with IC50 values
138.92 4.44 µmol/mL and 158.78 2.34 µmol/mL to find
their efficacy, and the potent lead molecules were found.
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Acknowledgements Authors are thankful to the administration, VIT
University, Vellore, India, for providing facilities to carry research
work and also thankful to SIF-Chemistry for providing NMR facility.
Authors are thankful to University of Hyderabad [Network resource
centre (UGC-NRC)] for HRMS facility and also NMR facilities. The
authors also thankful to the Division of Animal Biotechnology, Depart-
ment of Biotechnology, School of Herbal Studies and Nature Sciences,
Dravidian University. Author L. Jyothish Kumar is thankful to the VIT
University for providing research associate ship.
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