Syntheses, in vitro α-amylase and α-glucosidase dual inhibitory activities of 4-amino-1,2,4-triazole derivatives their molecular docking and kinetic studies

Article abstract of DOI:10.1016/j.bmc.2020.115467

Thirty-three 4-amino-1,2,4-triazole derivatives 1–33 were synthesized by reacting 4-amino-1,2,4-triazole with a variety of benzaldehydes. The synthetic molecules were characterized via ¹H NMR and EI-MS spectroscopic techniques and evaluated for

Full text of DOI:10.1016/j.bmc.2020.115467

Journal Pre-proofs
Syntheses, in vitro α-amylase and α-glucosidase dual inhibitory activities of
4-amino-1,2,4-triazole derivatives their molecular docking and kinetic studies
Emmanuel Oloruntoba Yeye, Kanwal, Khalid. Mohammed Khan, Sridevi
Chigurupati, Abdul Wadood, Ashfaq Ur Rehman, Shahnaz Perveen, Mari
Kannan Maharajan, Shahbaz Shamim, Shehryar Hameed, Sherifat A. Aboaba,
Muhammad Taha
PII:
S0968-0896(20)30281-9
https://doi.org/10.1016/j.bmc.2020.115467
BMC 115467
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 December 2019
20 March 2020
25 March 2020
Please cite this article as: E. Oloruntoba Yeye, Kanwal, Khalid. Mohammed Khan, S. Chigurupati, A. Wadood,
A. Ur Rehman, S. Perveen, M. Kannan Maharajan, S. Shamim, S. Hameed, S.A. Aboaba, M. Taha, Syntheses, in
vitro α-amylase and α-glucosidase dual inhibitory activities of 4-amino-1,2,4-triazole derivatives their molecular
docking and kinetic studies, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115467
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Syntheses, in vitro α-amylase and α-glucosidase dual inhibitory activities of 4-amino-
1,2,4-triazole derivatives their molecular docking and kinetic studies
Emmanuel Oloruntoba Yeye,a,b Kanwal,a Khalid. Mohammed Khan,a,c Sridevi Chigurupati,d
Abdul Wadood,e Ashfaq Ur Rehman,e Shahnaz Perveen,f Mari Kannan Maharajan,g Shahbaz
Shamim,a Shehryar Hameed,a Sherifat A. Aboaba,b Muhammad Tahac
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bOrganic Unit, Chemistry Department, University of Ibadan, Nigeria
cDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
dDepartment of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Qassim
University, Buraidah 52571, Kingdom of Saudi Arabia
eDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
fPCSIR Laboratories Complex, Karachi, Shahra-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
gSchool of Pharmacy, International Medical University, Kuala Lumpur, 57000, Malaysia
Abstract: Thirty-three 4-amino-1,2,4-triazole derivatives 1-33 were synthesized by reacting 4-
amino-1,2,4-triazole with a variety of benzaldehydes. The synthetic molecules were characterized
1
via H-NMR and EI-MS spectroscopic techniques and evaluated for their anti-hyperglycemic
potential. Compounds 1-33 exhibited good to moderate in vitro α-amylase and α-glucosidase
inhibitory activities in the range of IC50 values 2.01 ± 0.03 - 6.44 ± 0.16 and 2.09 ± 0.08 - 6.54 ±
0.10 µM as compared to the standard acarbose (IC50 = 1.92 ± 0.17 µM) and (IC50 = 1.99 ± 0.07
µM), respectively. The limited structure-activity relationship suggested that different substitutions
on aryl part of the synthetic compounds are responsible for variable activity. Kinetic study
predicted that compounds 1-33 followed mixed and non-competitive type of inhibitions against α-
amylase and α-glucosidase enzymes, respectively. In silico studies revealed that both triazole and
aryl ring along with different substitutions were playing an important role in the binding
interactions of inhibitors within the enzyme pocket. The synthetic molecules were found to have
dual inhibitory potential against both enzymes thus they may serve as lead candidates for the drug
development and research in the future studies.
Corresponding Authors: khalid.khan@iccs.edu; drkhalidhej@gmail.com, Tel.: +922134824910, Fax: +922134819018
1

Introduction:
Triazoles belongs to the nitrogen containing heterocyclic compounds and are comprised of a five-
membered ring system with two carbon and three nitrogen atoms in the ring having alternate π-
bonds [1]. It exists in two isomeric forms, 1,2,3-triazole and 1,2,4-triazole. Triazoles are
pharmacologically active molecules due to their structural properties, like moderate dipole
character, hydrogen bonding capability, ion-dipole, π-π stacking, cation-π, hydrophobic effect, van
der Waal forces, rigidity, and stability. These characteristic structural features make them more
stable under in vivo conditions which lead to their superior pharmacological activities [2-5].
Enzyme inhibition studies remain an important area of pharmacology. The activity of natural
substrates towards their respective enzymes have been blocked or altered by the action of enzyme
inhibitors [6]. These enzyme inhibitors suppress the over-expression of an enzyme thus preventing
different disorders. Therefore, the search of new and potential enzyme inhibitors has attracted the
considerable attention of medicinal chemists. 1,2,4-Triazoles and their derivatives has been widely
known for their synthetic and biological importance [7] and this ring system is also a part of wide
variety of therapeutically important drug candidates [8] (Figure-1).
H3C
CH3
N
Cl
N
OH
O
O
N
F
Cl
N
N
C8H17
N
N
H2N
H2N
O
C2H5
N
N
N
N
N
N
N
N
S
N
Cl
F
F
Cl
Cl
Fluconazole derivative
(antifungal agent)
Carboxyamidotriazole, CDI
(anticancer)
Anti-diabetic
Anti-diabetic
Figure-1: 1,2,4-triazole containing drugs and their bioactivities
Diabetes mellitus is amongst the most prevailing chronic metabolic disorders and is usually
characterized by hyperglycemia which is caused due to less insulin action or secretion or both. The
chronic hyperglycemia is associated with microvascular complications thus causing the eyes,
kidneys, and nerves, as well as it leads to an increased risk for cardiovascular disease (CVD) [9].
α-Glucosidase belongs to the family of hydrolase enzyme and is found in small intestine. It
hydrolyses carbohydrates to glucose during digestion which is then absorbed into blood stream.
The excess amount of glucose in the blood increases postprandial blood glucose level thus leads
to diabetes. Therefore, to control the postprandial hyperglycemia in type-II- diabetes mellitus, the
2

activity of enzyme should be suppressed by using different inhibitors [10-16]. Several α-
glucosidase inhibitors such as miglitol, voglibose, and acarbose have been reported in the literature
and are used to decrease the postprandial blood glucose levels [17-18]. α-Amylase (EC 3.2.1.1) is
another enzyme of hydrolase family bearing calcium atom as a metal co-factor. It catalyzes the
hydrolysis of polysaccharides to monosaccharides [19]. The absoprtion of starch in human body
is catalyzed by the hydrolytic action of α-amylase and α-glucosidase enzymes [20]. Hence, the
development of potent α-amylase inhibitors that can be used as chemotherapeutic agents might be
a better approach for the treatment of diabetes [19,21].
Since our research group had already reported the diverse biological activities of compounds
derived from 1,2,4-triazoles [22-28], therefore, in continuation of our previous work we had
selected and modified another compound from this class in order to explore its potential. It is
worth-noting that our research group has reported various classes of compounds including
benzotriazoles, pyrazolones, and other heterocyclic molecules as dual and potential inhibitors of
α-amylase and α-glucosidase enzymes. In the current study 1,2,4-triazole derivatives 1-33 were
synthesized and subjected for their α-amylase and α-glucosidase inhibitory activities and the
results showed that these compounds were found to exhibit potential inhibitory activities against
both enzymes and may act as lead molecules (Figure-2). To the best of our knowledge compounds
1, 5-7, 10-12, 13, 17, 19-23, 25-28, and 32 are new while others are already reported.
3

Representative lead molecules
H3C
Me
H
H
N
N
N
N
F
N
O
N
N
R
R
N
N
H
H3C
N
H
Br
O
Cl
-Glucosidase and
-Amylase inhibitior


-Glucosidase inihibitors

-Glucosidase inihibitors

-Glucosidase and
-Amylase inhibitior


Dual inhibitors of -amylase and -glucosidase


Imine Bond
R
N
N
N
1,2,4-Triazole moeity
N
Aryl part
Currently synthetic scaffold 1-33
Figure-2: Rational of the current study.
Results and Discussion
Chemistry
Schiff base derivatives of 4-amino-1,2,4-triazole 1-33 were synthesized by refluxing 4-amino-
1,2,4-triazole with different substituted benzaldehydes in equimolar proportion in ethanol. H2SO4
was added in catalytic amount and reaction was refluxed for 2-4 h. The reaction progress was
periodically observed by thin layer chromatography (TLC). After completion of reaction, the
solvent was evaporated on a rotary evaporator. Further purifications of synthetic molecules was
1
done by the crystallization from ethanol (Scheme-1). Spectroscopic techniques EI-MS and H-
NMR were used to elucidate the structures of synthetic compounds.
CHO
N
N
N
N
Ethanol, H2SO4
Reflux, 4 h
N
N
+
R
N
NH2
R
(1-33)
Scheme-1: Synthesis of Schiff base derivatives of 4-amino-1,2,4-triazole 1-33.
In vitro α-amylase and α-glucosidase activities:
The synthetic derivatives 1-33 displayed good α-amylase and α-glucosidase inhibitory activities,
having IC50 values in the range of 2.01 ± 0.03 – 6.44 ± 0.16 µM and 2.09 ± 0.08 – 6.54 ± 0.10 µM
4

as compared to the standard acarbose (IC50 = 1.92 ± 0.17) and (IC50 = 1.99 ± 0.07 µM),
respectively. Generally, the synthetic molecules were comprised of 1,2,4-triazole ring, aryl ring,
and imine bond. However, the aryl ring had different substitutions ‘‘R” which apparently played
their role in the inhibitory potential of compounds and variations in the inhibitory activities are
attributed to the type of substitutions and their respective positions on the aryl part (Table-1)
(Figure-3).
1,2,4-Triazole moeity
N
N
Aryl ring
N
N
R
Imine bond
1-33
Cl,Br,F,Me,OH,OCH3,NO2,NH2 etc
Figure-3: General structure of synthetic compounds
Table-1: In vitro α-amylase and α-glucosidase inhibition of Schiff base derivatives of 4-amino-1,2,4-
triazole (1-33)
α-Amylase inhibition α-Glucosidase Inhibition
Compound No.
Structures
IC50 ± SEMa (μM)
IC50 ± SEMa (μM)
N
N
OH
N
1
2
2.99 ± 0.13
2.62 ± 0.08
N
N
OH
HO
N
N
5.69 ± 0.1
5.44 ± 0.11
N
HO
N
3
4
5.95 ± 0.07
5.27 ± 0.11
5.44 ± 0.07
5.37 ± 0.11
N
OH
N
N
N
OH
N
N
N
HO
5

OH
HO
N
5
6
5.92 ± 0.15
4.37 ± 0.02
5.74 ± 0.05
4.28 ± 0.08
N
OH
N
N
H3CO
N
N
OCH3
N
N
H3CO
N
N
N
7
4.37 ± 0.11
4.29 ± 0.1
N
H3CO
OCH3
OCH3
OCH3
N
N
8
9
N
N
5.12 ± 0.08
5.24 ± 0.05
5.46 ± 0.1
5.36 ± 0.1
N
N
N
OC2H5
OCH3
N
N
N
OCH3
OC2H5
N
10
11
N
5.22 ± 0.07
4.34 ± 0.06
5.47 ± 0.11
4.27 ± 0.16
N
N
CH3
N
N
H3C
N
N
N
12
13
6.44 ± 0.16
4.36 ± 0.1
6.37 ± 0.02
4.28 ± 0.03
N
N
N
Cl
Cl
N
N
Cl
N
14
15
2.18 ± 0.12
5.97 ± 0.08
2.49 ± 0.05
5.37 ± 0.08
N
N
N
N
N
N
N
NO2
6

N
N
N
N
NO2
OH
N
16
17
2.36 ± 0.1
2.47 ± 0.11
6.34 ± 0.06
N
6.23 ± 0.09
N
N
OCH3
OH
N
N
18
19
5.94 ± 0.07
3.31 ± 0.08
5.3 ± 0.14
N
N
N
N
N
N
OCH3
OH
OCH3
3.27 ± 0.01
OCH3
Br
N
N
20
N
2.25 ± 1.05
2.52 ± 0.09
N
OCH3
Br
N
21
22
2.27 ± 0.01
5.25 ± 1.05
2.37 ± 0.02
5.52 ± 0.09
N
OH
N
N
OCH3
HO
N
N
N
N
Br
H3CO
N
N
23
24
3.3 ± 0.04
2.9 ± 0.04
3.46 ± 0.09
2.33 ± 0.13
N
N
Br
F
N
N
OCH3
N
N
7

OCH3
F
N
25
26
2.33 ± 0.13
4.46 ± 0.09
2.27 ± 0.11
4.37 ± 0.11
N
N
N
Cl
N
N
N
N
N
NO2
NO2
N
N
27
2.41 ± 0.09
2.31 ± 0.07
N
HO
H2N
N
N
28
29
2.28 ± 0.08
5.04 ± 0.3
2.44 ± 0.03
5.44 ± 0.29
N
N
N
N
N
O
N
N
N
N
OCH3
N
N
O
30
6.23 ± 0.1
6.54 ± 0.10
N
N
31
32
5.27 ± 0.16
6.41 ± 0.03
5.41 ± 0.03
6.3 ± 0.04
N
N
N
N
N
N
8

N
N
N
33
2.01 ± 0.03
1.92 ± 0.17
2.09 ± 0.08
1.99 ± 0.07
N
N
H
Standardb = Acarbose
IC50 SEMa = Mean ± Standard error of mean; Standardb = Inhibitor for α-amylase, α-glucosidase inhibitory activity
Structure-activity relationship (SAR) for α-amylase and α-glucosidase inhibitory activities
All structural features such as, triazole moiety, imine bond, and different substituted aryl parts are
equally playing their role in the α-amylase and α-glucosidase inhibition. Nonetheless, different
chemical entities on aryl ring resulted in variable activity of the synthetic molecules. Based on
variable substitutions and their different positions and numbers on aryl ring a limited structure-
activity relationship was rationalized. However, all synthetic compounds were found to be dual
inhibitors of α-amylase and α-glucosidase.
Compounds 1-5 having variable numbers and positions of hydroxy substituents have displayed
good inhibitory activities against α-amylase and α-glucosidase enzymes, respectively. Among
them, compound 1 (IC50 = 2.99 ± 0.13 μM) (IC50 = 2.62 ± 0.08 μM) with para hydroxy substitution
was more active as compared to other derivatives. Compound 2 (IC50 = 5.69 ± 0.1 μM) (IC50 =
5.44 ± 0.11 μM) having two hydroxy groups at meta and para positions of aryl ring was found to
be two folds less active in comparison with compound 1. Compound 3 (IC50 = 5.95 ± 0.07 μM)
(IC50 = 5.44 ± 0.07 μM) with ortho, para hydroxy substitution displayed decreased α-amylase
activity, however, α-glucosidase inhibition was similar to compound 2. Another dihydroxy
substituted compound 4 (IC50 = 5.27 ± 0.11 μM), (IC50 = 5.37 ± 0.11 μM) with hydroxy substituents
para to each other showed good activity as compared to its structurally similar compounds 2 and
3. The trihydroxy substituted compound 5 (IC50 = 5.92 ± 0.15 μM), (IC50 = 5.74 ± 0.05 μM) with
three hydroxy groups adjacent to each other exhibited comparable α-amylase activity to compound
3, however, it was less active against α-glucosidase enzyme. Thus it can be observed that
substitution of hydroxy group at different positions of aryl ring was responsible for variable
activity of synthetic molecules, might be the change in positions alter the binding interaction of
molecules within the active site of enzyme. The different numbers of hydroxy groups are also
found to be responsible for the variable activity, with increasing the number of hydroxy groups on
aryl ring a sharp change in activity was observed (Figure-4).
9

OH
HO
N
N
N
OH
N
N
OH
N
N
N
2
3
IC50 = 5.69 ± 0.1
IC50 = 5.44 ± 0.11

glucosidase inhibition


IC50 = 5.95 ± 0.07
IC50 = 5.44 ± 0.07

glucosidase inhibition


amylase inhibition

amylase inhibition

N
N
N
OH
N
1
IC50 = 2.99 ± 0.13
IC50 = 2.62 ± 0.08


amylase inhibition
glucosidase inhibition


OH
OH
HO
N
N
N
N
N
OH
N
N
N
HO
4
5
IC50 = 5.27 ± 0.11
IC50 = 5.37 ± 0.11

glucosidase inhibition


IC50 = 5.92 ± 0.15
IC50 = 5.74 ± 0.05

glucosidase inhibition


amylase inhibition

amylase inhibition

Figure-4: Structure-activity relationship of compounds 1-5
The methoxy substituted compounds 6-10 also displayed variable activity and were found to be
less active in comparison with hydroxy substituted compounds. Compound 6 (IC50 = 4.37 ± 0.02
μM), (IC50 = 4.28 ± 0.08 μM) with ortho and para methoxy substituents displayed similar activity
with its positional isomer 7 (IC50 = 4.37 ± 0.11 μM), (IC50 = 4.29 ± 0.1 μM) having both methoxy
groups at ortho positions. The activity was decreased with an addition of methoxy group on the
aryl ring in compound 8 (IC50 = 5.12 ± 0.08 μM), (IC50 = 5.46 ± 0.1 μM). Compounds 9 (IC50 =
5.24 ± 0.05 μM), (IC50 = 5.36 ± 0.1 μM) and 10 (IC50 = 5.22 ± 0.07 μM), (IC50 = 5.47 ± 0.11 μM)
were positional isomers of each other and bear methoxy and ethoxy groups. These compounds
displayed comparable α-amylase activity with each other, nevertheless, slight variation in the α-
glucosidase activity might be due to the changed positions of methoxy and ethoxy groups. Thus,
it showed that increasing the number of substituents or replacing those with bulkier groups resulted
in decreased inhibition due to which the molecules might not able to interact effectively within the
active pocket of enzyme (Figure-5).
10

OCH3
H3CO
N
N
N
N
N
N
N
N
OCH3
H3CO
6
7
IC50 = 4.37 ± 0.02
IC50 = 4.28 ± 0.08


IC50 = 4.37 ± 0.11
IC50 = 4.29 ± 0.1


amylase inhibition
glucosidase inhibition


amylase inhibition
glucosidase inhibition


OCH3
OCH3
N
N
N
N
N
N
OC2H5
OCH3
N
N
OCH3
9
8
IC50 = 5.12 ± 0.08
IC50 = 5.46 ± 0.1

glucosidase inhibition


IC50 = 5.24 ± 0.05
IC50 = 5.36 ± 0.1

glucosidase inhibition


amylase inhibition

amylase inhibition

N
N
N
OCH3
N
OC2H5
10
IC50 = 5.22 ± 0.07
IC50 = 5.47 ± 0.11

glucosidase inhibition


amylase inhibition

Figure-5: Structure-activity relationship of compounds 6-10
Compounds 11 and 12 with different alkyl substituents were also found to have moderate activity
but they were less active in comparison with hydroxy and methoxy substituted compounds.
Amongst them, compound 11 (IC50 = 4.34 ± 0.06 μM), (IC50 = 4.27 ± 0.06 μM) with two methyl
substituents meta to each other displayed good inhibition in comparison with compound 12 (IC50
= 6.44 ± 0.16 μM), (IC50 = 6.37 ± 0.02 μM) having para substituted t-butyl group. This pattern
again showed that the compounds with bulkier substituents are less active. Amongst halogen
substituted analogs 13-14, compound 13 (IC50 = 4.36 ± 0.10 μM) (IC50 = 4.28 ± 0.03 μM) with
para chloro substitution displayed moderate inhibition against both enzymes. Nevertheless,
compound 14 (IC50 = 2.18 ± 0.11 μM), (IC50 = 2.49 ± 0.05 μM) with dichloro substituents at ortho
and para positions displayed potential inhibition as compared to compound 13 and was found to
be second most active molecule of the series (Figure-6).
11

N
N
N
CH3
N
N
N
N
N
H3C
11
12
IC50 = 6.37 ± 0.02
IC50 = 4.34 ± 0.06
IC50 = 4.27 ± 0.06


IC50 = 6.44 ± 0.16


amylase inhibition
glucosidase inhibition


amylase inhibition
glucosidase inhibition


Cl
N
N
N
Cl
N
N
N
Cl
N
N
14
13
IC50 = 4.36 ± 0.1
IC50 = 4.28 ± 0.03

glucosidase inhibition


IC50 = 2.18 ± 0.12
IC50 = 2.49 ± 0.05

glucosidase inhibition


amylase inhibition

amylase inhibition

Figure-6: Structure-activity relationship of compounds 11-15
The nitro substituted compounds 15 (IC50 = 5.97 ± 0.08 μM), (IC50 = 5.37 ± 0.08 μM) and 16 (IC50
= 2.36 ± 0.10 μM), (IC50 = 2.47 ± 0.11 μM) bearing nitro groups at meta and para positions,
respectively, displayed variable inhibition. A sharp change in activity of compound 16 was
observed and it might be attributed to the para position of nitro group. The addition of chloro at
ortho position along with nitro group resulted in increased inhibition as in compound 26 (IC50 =
4.46 ± 0.09 μM) (IC50 = 4.37 ± 0.09 μM) as compared to compound 15 bearing meta substituted
nitro group. Compound 27 (IC50 = 2.41 ± 0.09 μM) (IC50 = 2.31 ± 0.09 μM) with combination of
hydroxy and nitro groups meta to each other also displayed increased potential in comparison with
compounds 15 and 26. However, all these compounds were less active in comparison with
compound 16 which demonstrated that substitution of nitro group at para position favors the
binding interactions of enzyme with the molecule (Figure-7).
12

N
N
N
N
N
NO2
N
N
N
NO2
15
16
IC50 = 2.47 ± 0.11
IC50 = 5.97 ± 0.08
IC50 = 5.37 ± 0.08



glucosidase inhibition
IC50 = 2.36 ± 0.1


amylase inhibition

amylase inhibition
glucosidase inhibition


NO2
Cl
N
N
N
N
N
N
N
N
OH
NO2
27
26
IC50 = 4.46 ± 0.09
IC50 = 4.37 ± 0.09

glucosidase inhibition


IC50 = 2.41 ± 0.09
IC50 = 2.31 ± 0.09

glucosidase inhibition


amylase inhibition

amylase inhibition

Figure-7: Structure-activity relationship of compounds 15-16 and 26-27
Compounds 17-25 having mixed substitution of hydroxy, methoxy, and halogens, displayed
variable activities according to the nature and position of substituents. Amongst them, compound
17 (IC50 = 6.32 ± 0.09 μM) (IC50 = 6.34 ± 0.06 μM) with para hydroxy, and two t-butyl groups
para to each other was less active might be the presence of two bulky groups resulted in the
increased steric hindrance. However, combination of hydroxy and methoxy substituents in
compound 18 (IC50 = 5.94 ± 0.07 μM) (IC50 = 5.3 ± 0.14 μM) at para and meta positions,
respectively, displayed good activity. Interestingly, addition of another methoxy group ortho to
hydroxy in compound 19 (IC50 = 3.31 ± 0.08 μM) (IC50 = 3.27 ± 0.01 μM) resulted in increased
inhibition as compared to compound 18. Replacement of hydroxy group with bromo in compound
20 (IC50 = 2.25 ± 1.05 μM) (IC50 = 2.52 ± 1.05 μM) displayed further increased activity. However,
compound 21 (IC50 = 2.27 ± 0.01 μM) (IC50 = 2.37 ± 0.01 μM) with bromo and hydroxy groups
ortho to each other displayed superior activity in comparison with compounds 17-21, it showed
that bromo and hydroxy groups are actively participating in the activity. Nevertheless, changing
the position of bromo and hydroxy substituents and addition of methoxy group on aryl ring resulted
in decreased activity in compound 22 (IC50 = 5.25 ± 1.05 μM) (IC50 = 5.52 ± 0.09 μM). Compound
23 (IC50 = 3.30 ± 0.04 μM) (IC50 = 3.46 ± 0.09 μM) with bromo and methoxy groups para with
respect to each other was less active as compared to compounds 24 (IC50 = 2.90 ± 0.04 μM) (IC50
= 2.33 ± 0.13 μM) and 25 (IC50 = 2.33 ± 0.13 μM) (IC50 = 2.27 ± 0.11 μM) with combination of
methoxy and fluorine. The above results revealed that combination of fluoro with methoxy and
13

bromo with hydroxy have displayed potential inhibition, however, slight variations in activity of
these compounds was due to different positions of methoxy, bromo, and fluoro groups (Figure-8).
OCH3
OH
N
N
OH
N
N
N
N
N
N
18
IC50 = 5.3 ± 0.14
glucosidase inhibition

17
IC50 = 6.34 ± 0.06
glucosidase inhibition

IC50 = 5.94 ± 0.07


IC50 = 6.23 ± 0.09


amylase inhibition

amylase inhibition

OCH3
OH
OCH3
Br
N
N
N
N
N
N
N
N
OCH3
OCH3
19
20
IC50 = 3.31 ± 0.08
IC50 = 3.27 ± 0.01


IC50 = 2.25 ± 1.05
IC50 = 2.52 ± 1.05


amylase inhibition
glucosidase inhibition


amylase inhibition
glucosidase inhibition


Br
OCH3
HO
Br
N
N
N
N
N
N
N
OH
N
21
22
IC50 = 2.27 ± 0.01
IC50 = 2.37 ± 0.01

glucosidase inhibition


IC50 = 5.25 ± 1.05
IC50 = 5.52 ± 0.09

glucosidase inhibition


amylase inhibition

amylase inhibition

14

H3CO
F
N
N
N
N
N
N
OCH3
N
N
Br
IC50 = 3.46 ± 0.09
glucosidase inhibition

23
24
IC50 = 3.3 ± 0.04


IC50 = 2.9 ± 0.04
IC50 = 2.33 ± 0.13
glucosidase inhibition



amylase inhibition

amylase inhibition

OCH3
F
N
N
N
N
25
IC50 = 2.33 ± 0.13
IC50 = 2.27 ± 0.11

glucosidase inhibition


amylase inhibition

Figure-8: Structure-activity relationship of compounds 17-25
Compounds 28-33 bear different aryl rings including heterocyclic rings and bulky conjugated ring
systems therefore, these molecules displayed quite variable results. Compound 28 (IC50 = 2.28 ±
0.08 μM) (IC50 = 2.44 ± 0.03 μM) bearing 2-amino pyridine ring exhibited good inhibition against
both enzymes. However, compound 29 (IC50 = 5.04 ± 0.3 μM) (IC50 = 5.44 ± 0.29 μM) with
benzyloxy group at the aryl part was less active might be the incorporation of bulky group make
it more difficult to interact within the active site of enzyme. Addition of methoxy group along with
benzyloxy group resulted in further declined activity in compound 30 (IC50 = 6.23 ± 0.1 μM) (IC50
= 6.54 ± 0.10 μM). Compounds 31 (IC50 = 5.27 ± 0.16 μM) (IC50 = 5.41 ± 0.03 μM) and 32 (IC50
= 6.41 ± 0.03 μM) (IC50 = 6.3 ± 0.04 μM) with anthracene and pyrene rings, respectively, also
showed weak inhibition which showed that bulky groups are creating a steric hindrance thus the
molecules are unable to interact within the active site of enzyme. Compound 33 (IC50 = 2.01 ±
0.03 μM) (IC50 = 2.09 ± 0.08 μM) having indole ring as aryl part was most active molecule of the
series and displayed good inhibition as compared to the standard acarbose, might be the pyrrole
ring of indole is capable of forming the binding interactions with the active site of enzyme (Figure-
9).
15

H2N
N
N
N
N
O
N
N
N
N
N
29
28
IC50 = 2.44 ± 0.03
IC50 = 2.28 ± 0.08


IC50 = 5.04 ± 0.3
IC50 = 5.44 ± 0.29


amylase inhibition
glucosidase inhibition


amylase inhibition
glucosidase inhibition


N
O
O
N
N
N
N
N
N
N
31
30
IC50 = 6.54 ± 0.10
IC50 = 6.23 ± 0.1


glucosidase inhibition

IC50 = 5.27 ± 0.16

IC50 = 5.41 ± 0.03

glucosidase inhibition


amylase inhibition

amylase inhibition
N
N
N
N
N
N
N
N
N
H
33
32
IC50 = 6.41 ± 0.03
IC50 = 6.3 ± 0.04

glucosidase inhibition


IC50 = 2.01 ± 0.03
IC50 = 2.09 ± 0.08

glucosidase inhibition


amylase inhibition

amylase inhibition

Figure-9: Structure-activity relationship of compounds 29-33
Compound 1-21
10
5
0
Compound numbers
α-Amylase inhibition
α-Glucosidase Inhibition
Figure-11: Comparison of α-amylase and α-glucosidase activities of compound 1-21
16

Compound 22-33
10
5
0
Compound numbers
α-Amylase inhibition
α-Glucosidase Inhibition
Figure-12: Comparison of α-amylase and α-glucosidase activities of compound 22-33
Kinetic characterization of α-amylase inhibition
Kinetic studies on the most active α-amylase inhibitors 1, 14, 16, 20, 21, 24, 25, 27, 28, and 33
were carried out to interpret the inhibition mechanism of these compounds. The enzyme inhibition
mechanism analyzed by using Sigma-Plot enzyme kinetic software through various parameters as
shown in Figure-13 and Table-2. The kinetic studies revealed that the synthetic compounds
showed mixed type of inhibition.
Table-2: Kinetic studies of active compounds for α-amylase inhibition
S. No
Compound. No Vmax (µM/min)1 Km (mM) AICc
R2
Type of inhibition
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
1
2
3
4
5
6
7
8
9
1
237.4
220.8
206.8
241.5
220.8
237.7
240.8
231.9
212.7
256.1
1.3
1.2
0.9
1.3
1.2
1.3
1.3
1.3
1.0
1.5
110.6 0.912
108.3 0.915
106.4 0.902
110.2 0.935
112.3 0.905
108.3 0.922
108.4 0.923
110.3 0.905
118.2 0.975
121.7 0.992
14
16
20
21
24
25
27
28
33
10
17

Figure-13:A) Lineweaver-Burk plot of 1/[S] Vs 1/Rate in the different concentration of mixed-type
inhibitor. B) Hill plot of different concentration of [S] Vs Rate (Vmax-Rate) in the different
concentration of mixed-type inhibitor.
Figure-13:C)
different concentration of mixed-type inhibitor.
33
D)
Hanes-Woolf plot of compound
at different concentration of [S] Vs [S]/Rate in the
33
Eadie-Hofstee plot of compound
by rate/[S] Vs
Rate in the different concentration of mixed-type inhibitor.
Figure-13:E)
33
F)
at different concentration of mixed inhibitor Vs 1/Rate.
Dixon plot of compound
33
Scatard plot of compound
by Rate Vs Rate/[S] in the different concentration of mixed-type inhibitor.
18

Kinetic characterization of α-glucosidase inhibition
Kinetic studies on the most active α-glucosidase inhibitors1, 14, 16, 20, 21, 24, 25, 27, 28, and 33
were carried out to interpret the inhibition mechanism of these compounds. The enzyme inhibition
mechanism analyzed by using Sigma-Plot enzyme kinetic software through various parameters as
shown in Figure-14 and Table-3. The kinetic studies revealed that the synthetic compounds
showed uncompetitive type of inhibition.
Table-3: Kinetic studies of active compounds for α-glucosidase inhibition
S. No Compound. No Vmax (µM/min)1 Km (mM) AICc
R2
Type of inhibition
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
Uncompetitive type
1
2
1
701.4
684.7
680.2
690.5
698.3
701.2
697.1
695.3
684.8
702.7
0.4
0.5
0.5
0.7
0.4
0.5
0.5
0.5
0.5
0.3
201.4 0.985
200.4 0.994
212.1 0.914
213.5 0.984
214.2 0.989
205.2 0.988
200.8 0.901
211.7 0.991
205.1 0.988
199.4 0.994
14
16
20
21
24
25
27
28
33
3
4
5
6
7
8
9
10
Figure-14:A)
Lineweaver-Burk plot of 1/[S] Vs 1/Rate in the different concentration of mixed-type
Hill plot of different concentration of [S] Vs Rate (Vmax-Rate) in the different
B)
inhibitor.
concentration of mixed-type inhibitor.
19

Figure-14:C)
33
Hanes-Woolf plot of compound
different concentration of Competitive -type inhibitor.
rate/[S] Vs Rate in the different concentration of Competitive-type inhibitor.
at different concentration of [S] Vs [S]/Rate in the
D) 33
Eadie-Hofstee plot of compound
by
Figure-14:E)
33
Dixon plot of compound
33
Scatard plot of compound
at different concentration of competitive inhibitor Vs
F)
1/Rate.
by Rate Vs Rate/[S] in the different concentration of
Competitive-type inhibitor.
Molecular Docking (MD) Studies
α-Amylase molecular study
The blind docking (BD) approach was performed to examine the possible binding site of these
compounds other than the substrate binding site due to their mixed inhibition nature against the α-
amylase enzyme. In the mixed type of inhibition, the inhibitor might bind to the protein either the
substrate has already linked to the protein or not. The BD study revealed that the derivatives bind
to a distinct site other than the substrate binding site with various affinity, as shown in (Figure-
15: A) (left side). Based on visual inspection, it was perceived that slight conformational changes
in the backbone and side-chain of substrate binding residues had been bought, thus gladly
supporting the nature of synthetic molecules. In general, the active site of the α-amylase enzyme
20

includes Trp58-59, Tyr151, Leu162, Thr163, Arg195, Asp197-198, 300, Lys200-201, and Glu233
as shown in (Figure-15: A) (right side). Afterwards, the BD results revealed that the top active
derivatives in the series commonly adopted side-chain donor/acceptor and arene-H interactions
including; residue Lys265, Arg319, Asp433, Asn431, and Pro486, respectively. In case of most
active derivative 33 in the series adopts side-chain donor and acceptor hydrogen interaction with
residue Asn481 and Asp433, while residue Arg319 formed an arene-H bond with the indole
pyrrhole ring of the compound (Figure-15: C). Based on the visual inspection, we observed that
the binding of this compound, bring slight conformational changes in the substrate-binding site
and hence might unable the enzyme to produce the product. Derivative 33, the most active in the
series which showed a considerable inhibition as compared to the standard acarbose was
presumably an indole ring that exhibits not only favourable interaction with the active site of the
enzyme but also showed good docking score comparatively. More interestingly, the 2nd active
compound 14 showed a similar binding pattern as was observed for compound 25, the only
difference found was the additional arene-H interaction of D433 adopted with compound 33. In
case of 3rd most active derivatives, compounds 14, 20, 21, and 28 showed similar binding mode
with residues Pro485, Asn481, Asp433, and Lys268 (Figure-15: D-F), these compounds possess
only differences in activity, that might be due to the strength of the bonding interaction as well the
distance among the residue and compound. In case of other active derivatives in the series (1 and
30) also showed similar interaction with the residues found common Lys268, and with Arg319
compound 16, and with Pro485 compound 25 as shown in (Figure-15: G-H). Overall, the BD and
enzyme kinetics study results demonstrate and support the mixed inhibition nature of these
derivatives. The binding of these active derivatives to the distinct site other than the substrate-
binding site of α-amylase enzyme fetch an active conformational side-chain and backbone changes
in the key residues and hence the consequences of changes in the side and backbone chain further
reduce the affinity of the substrate against the enzyme. The detail protein-ligand interaction profile
listed in (Table-4).
21

Figure-15: The ligand-protein interaction profile for mixed inhibitors against the α-amylase
enzyme. (A) The surface representation of the alpha-amylase enzyme (PDB ID 3BAJ), the left
side surface representation indicates the distinct binding site (cryptic site) while the right substrate
binding site. (B) The binding mode of the most potent compound 33, (C) for compound 14, (D)
for compound 20, (E) for compound 21, (F) for compound 28, (G) for compound 16 and (H) for
compound 25.
Table-4: Interaction Detail for all derivatives against the α-amylase enzyme.
S. No.
Interaction details
Docking
score
ligands receptor
Distance E cal/mol
residue
ARG 319
ASP 433
ASN 431
ARG 267
ASP 433
LYS 268
LEU 320
ILE 266
PRO 486
ASP 433
ASN 481
ASN 481
LEU 320
LYS 268
ARG 319
ASN 481
ASN 481
PRO 486
ARG 319
ASN 481
LYS 268
ARG 319
LEU 320
PRO 486
ARG 319
1
2
5-ring
O
NH1
OD1
O
π-cation
H-donor
H-donor
H-acceptor
H-donor
H-acceptor
π-H
H-donor
H-acceptor
H-donor
H-acceptor
H-acceptor
π-H
3.89
2.94
3.34
3.58
2.76
3.56
3.85
2.86
3.48
2.83
3.54
3.75
3.83
3.35
3.62
3.54
3.55
3.45
3.89
3.66
3.42
3.91
3.70
3.58
3.89
-2.7
-4.4
-0.6
-0.7
-5.1
-1.4
-0.5
-1.0
-0.5
-5.4
-0.8
-0.5
-1.2
-0.8
-0.9
-0.8
-0.5
-0.5
-3.1
-0.6
-7.9
-2.2
-1.0
-0.5
-1.9
-4.71
-4.64
N
N
CA
3
4
O
OD1
NZ
-4.99
-4.82
N
5-ring
O
CD1
O
N
CG
5
6
O
OD1
CB
-4.00
-4.78
O
O
ND2
CD1
NZ
7
8
5-ring
O 18
5-ring
O
-4.67
-4.01
H-acceptor
π-H
CB
9
10
CB
H-acceptor
H-acceptor
H-acceptor
π-cation
H-acceptor
H-acceptor
π-cation
π-H
-4.24
-4.080
N 1
N 2
5-ring
N
CB
CG
NH1
CB
11
12
-4.83
-5.03
N
NZ
5-ring
6-ring
N
NH1
CD1
CG
H-acceptor
π-cation
5-ring
NH1
22

13
14
5-ring
NH1
N 1
5-ring
5-ring
N
NH1
OD1
CB
NH1
NH1
O
CA
NH1
CB
NH1
O
NH2
OD1
NZ
OD1
CB
CB
CG
OD1
CG
CB
π-cation
H-donor
H-acceptor
π-cation
π-cation
H-donor
3.88
3.30
3.78
3.77
3.87
3.34
3.58
2.91
3.72
3.87
3.37
3.59
2.90
3.91
2.89
3.55
3.67
3.53
3.50
3.48
3.56
2.82
3.59
3.26
3.34
3.58
2.91
3.55
3.64
3.88
3.59
3.67
3.41
3.32
2.99
2.95
3.87
3.49
3.60
3.86
3.57
3.42
3.91
3.72
3.95
3.73
3.70
3.55
3.92
3.86
3.42
-2.4
-1.6
-0.6
-1.3
-1.0
-0.6
-0.7
-4.5
-0.6
-1.2
-0.6
-1.3
-3.3
-0.6
-3.0
-0.6
-0.5
-0.5
-2.6
-0.5
-0.5
-5.5
-0.6
-8.4
-0.6
-0.7
-4.5
-0.5
-0.6
-2.0
-3.5
-2.4
-0.5
-0.7
-4.0
-3.0
-1.7
-0.5
-0.5
-2.9
-0.7
-7.3
-2.4
-1.0
-4.7
-0.5
-0.6
-0.6
-2.2
-0.5
-2.3
ARG 319
ASP 433
ASN 481
ARG 319
ARG 319
ASN 431
ARG 267
ARG 319
ASN 481
ARG 319
ARG 267
ARG 319
ASP 433
LYS 268
ASP 433
ARG 319
ASN 481
PRO 486
ASP 433
PRO 486
ASN 481
ASP 433
PRO 486
LYS 268
ASN 431
ARG 267
ARG 319
PRO 486
ASN 481
ARG 319
LYS 268
ARG 319
ASN 481
ASN 431
ASN 481
ASP 433
LYS 268
PRO 486
ASN 481
ARG 319
ASN 481
LYS 268
ARG 319
LEU 320
ARG 319
ARG 319
ARG 319
ASN 481
ARG 319
ASP 433
ASP 433
-4.73
-5.92
15
-4.82
N
H-acceptor
H-acceptor
H-acceptor
π-cation
O
16
17
18
19
20
N 1
5-ring
O 14
N 3
O 7
6-ring
O
-5.32
-4.97
-4.82
-4.81
-5.77
H-donor
H-acceptor
H-donor
π-cation
H-donor
5-ring
N 1
N 2
NH1
N
N
O 14
O 2
π-H
H-acceptor
H-acceptor
H-donor
21
22
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
π-cation
H-acceptor
π-cation
H-accepter
H-donor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
π-cation
-5.59
OD1
CG
-4.030
O
6
NZ
23
24
25
N
N
O
O
-4.77
-4.71
-5.49
CA
NH1
CG
N 2
N 3
5-ring
N
5-ring
N
CB
NH1
NZ
NH1
CB
OD1
ND2
OD1
NZ
CG
CB
26
27
-4.64
-4.76
O 7
O 10
N 14
N 6
N 1
N 2
5-ring
N 1
N 6
5-ring
6-ring
5-ring
5-ring
6-ring
N 3
5-ring
5-ring
NH1
28
29
-5.51
-4.44
NH1
CB
NZ
30
H-acceptor
H-acceptor
π-cation
-4.49
NH1
CD1
NH1
NH2
CD
π-H
31
π-cation
-4.83
π-cation
32
33
π-H
-4.97
-6.53
CB
H-acceptor
π-cation
NH1
NH2
OE1
H-acceptor
π-cation
23

α-Glucosidase molecular study
The blind docking (BD) approach was performed to explore the possible binding mode of the
synthetic inhibitors in the binding site other than the substrate binding site due to the uncompetitive
inhibition type of the synthesis derivatives against the α-glucosidase enzyme. The BD results
revealed that all the derivatives bind to a site; cryptic site, other than substrate-binding site with
various affinity due to the uncompetitive nature as shown in (Figure-16A) and (Table-5). The
deep insight protein-ligand interaction (PLI) analysis revealed that mostly the active inhibitors
which showed high potency against the corresponding enzyme adopt various interaction with
acidic, basic and polar residues, amino acid includes Asp68, Asp214, Glu276, Asp408, and
Glu304, basic include Arg439 and His111 which is polar and with the benzyl side chain of the
non-polar, essential amino acid Phe157, respectively. More interestingly, the indole pyrrhole ring
of all the inhibitors showed similar interaction with the acidic residue Asp214 this might play a
crucial role in enhancing the catalytic activity of the enzyme. In case of the most potent inhibitor
compound, 33 in the series showed favourable binding mode in the cryptic site of against α-
glucosidase enzyme and adopted side-chain donor interaction only with acidic residues including
Asp214, Asp68, Glu276 and Glu304 (Figure-16B). Moreover, the other active derivative in the
series compound 25 showed similar binding mode with residues acidic side chain residues Asp68,
Asp214, and hydrophobic side chain residue Phe157 (Figure-16C). The only differences found
was the additional interaction adopted by compound 25 instead of Asp68 with the aliphatic amino
acid Lys155. In case of other active compounds 27 and 24 found in adopting side-chain acceptor
and donor interaction with the acidic residue Asp214 and with polar side chain His111 (Figure-
16D-F). Overall, the high potency of these compounds might be based on the favourable
interaction adopted by the indole pyrrhole ring of the compounds with the acidic residue Asp214
and with another residue. Additionally, the BD study and enzyme kinetics study results
demonstrate and support the uncompetitive nature of these compounds.
24

Figure-16:
The ligand-protein interaction (LPI) profile for uncompetative inhibitors against
-

(A)
glucosidase enzyme.
The surface representation of the cryptic site aprt from substrate binding site
due to the uncompetative type of inhibiton of the compounds
Figure-16: (B)
33 (C)
25
for compound
The binding mode of the most potent compound
25

Figure-16:
(D)
27 (E)
24
for compound
The binding mode of the most potent compound
for compound
Table-5: Interaction Detail for all compounds against α-glucosidase enzyme.
Interaction Report
Docking
score
S. No
Ligand
Receptor
Interaction
Distance E (kcal/mol)
Residue
1
2
O 14
OD1
H-donor
3.10
-1.3
ASN 412
-5.33
O 14
O 14
O 15
O 15
O 16
C 10
C 1
C 7
C 13
N 3
N 1
N 3
N 1
N 3
N 1
O 15
N 1
C 1
O 7
N 3
OD1
OD2
OE1
N
H-donor
H-donor
H-donor
H-acceptor
H-donor
H-π
2.84
3.18
2.84
3.10
2.97
3.92
3.48
4.31
4.08
3.01
3.00
3.05
2.92
3,12
3.02
3.05
3.09
3.14
2.77
3.07
3.79
3.03
4.02
3.44
3.61
-5.0
-0.5
-3.3
-1.1
-1.4
-0.5
-0.6
-0.6
-1.7
-1.2
-1.0
-1.2
-1.2
-1.0
-1.2
-1.3
-1.1
-0.8
-4.4
-1.1
-0.5
-1.1
-0.8
-4.6
-0.6
ASP 214
ASP 214
GLU 276
TRP 136
THR 307
HIS 245
GLU 276
HIS 245
HIS 245
HIS 111
HIS 111
HIS 111
HIS 111
HIS 111
HIS 111
HIS 111
HIS111
ASP 214
GLU 276
HIS 111
LEU 218
HIS 111
PHE 157
ASP 214
GLU 214
-5.31
3
4
5
6
7
-5.04
-4.93
-5.10
-5.44
-5.24
OG1
5-RING
OE1
5-RING
5-RING
NE2
NE2
NE2
NE2
NE2
NE2
NE2
NE2
OD1
OE1
NE2
CD2
NE2
CA
H-donor
H-π
8
-5.93
H-π
9
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-donor
H-acceptor
π-H
-6.32
-6.34
-5.58
-5.81
-4.93
-5.54
-5.72
-5.52
-5.61
-5.41
-5.79
-5.65
-5.48
-5.64
-5.48
-5.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5-RING
N 3
6-RING
BR 14
C 4
H-acceptor
π-H
H-donor
H-donor
OD1
OE2
26

N1
6-RING
N 4
O 16
C 17
N 13
6-RING
5-RING
N 1
N 1
C 4
6-RING
C 1
OD1
CA
NZ
NE2
OD1
NE2
CA
H-donor
π-H
3.2
3.08
3.5
-4.3
-1.3
-2.9
-0.7
-0.5
-1.3
-1.0
-0.8
-1.4
-1.0
-0.9
-0.7
-0.6
-4.8
-0.5
-4.5
ASP 214
PHE 157
LYS155
HIS 111
ASP 214
HIS 111
PHE 157
ARG 312
GLY 217
HIS 111
ASP 408
HIS 279
ASP 214
GLU 276
ASP 214
GLU 276
25
-6.15
H-acceptor
H-acceptor
H-donor
H-acceptor
π-H
26
27
3.20
3.38
3.09
4.02
4.24
3.56
3.08
3.50
4.07
3.20
3.04
3.10
3.14
-5.92
-5.54
28
-4.73
CB
N
π-H
29
30
31
32
H-acceptor
H-acceptor
H-donor
π-H
H-donor
H-donor
H-donor
H-donor
-6.16
-6.83
-5.77
-6.20
NE2
OD2
CD2
OD1
OE1
OD1
OE1
N 9
C2
N3
33
-7.29
Conclusion:
Novel derivatives of 4-amino-1,2,4-triazole (1-33) have been synthesized by reacting 4-amino-
1,2,4-triazole with different substituted benzaldehydes in ethanol under reflux. The synthetic
molecules were characterized via 1H-NMR and EI-MS spectroscopic techniques and evaluated for
their α-amylase and α-glucosidase inhibition. These compounds demonstrated good inhibitory
activity against both enzymes and found to be dual and potential inhibitors of α-amylase and α-
glucosidase enzymes. Among thirty three derivatives, ten compounds 1, 14, 16, 20, 21, 24, 25, 27,
28, and 33 showed superior activity in the range of IC50 value of 2.01-2.09 against α-amylase and
2.09-2.62 μM against α-glucosidase enzymes, respectively, however, other compounds were
moderately active. Compound 33 (IC50 = 2.01 ± 0.03 μM) (IC50 = 2.09 ± 0.08 μM) with an indole
ring as aryl part was found to be the most potential inhibitor of both enzymes might be due to the
better interactions of indole ring with the active site of enzyme. Kinetic studies revealed that the
synthetic analogs have exhibited mixed type of inhibition against α-amylase and uncompetitive
type of inhibition against α-glucosidase enzymes. The in silico studies have supported and
identified the binding interactions of these synthetic molecules with the enzyme. In conclusion,
these compounds may serve as lead molecules for further drug development studies that may help
to control hyperglycemia thus in future they may serve as drug candidates for the treatment of
diabetes.
27

Materials and methods
Thin layer chromatography was carried out on pre-coated silica gel, GF-254 (Merck, Germany).
Spots were visualized under ultraviolet light at 254, 366 nm or iodine vapors. EI-MS spectra were
1
recorded on MAT 312 and MAT 113D mass spectrometers. The H-NMR were recorded on a
Bruker AM spectrometers, operating at 300 and 400 MHz. The chemical shift values 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 a Stuart® SMP10 melting point
apparatus and are uncorrected.
Kinetic Study Assay
α-Amylase inhibition assay
To determine the mechanism of enzyme inhibition, the kinetic studies were performed by using
varying concentration of α-amylase inhibitors (0.0625, 0.125, 0.3 and 0.4 mM) with varying
concentrations of inhibitor substrate (p-nitrophenyl-R-D-maltoside (NPM)) such as 0.1, 0.2, 0.4,
0.8, and 1.0 mM. The enzyme was dissolved in de-ionized water to produce the concentration 0.2
mg/mL for each well. PIPES buffer was adjusted at the pH of 7.0, the reaction mixture was
incubated for the period of 30 minutes at 25 ˚C. The absorbance was measured using ELISA reader
by keeping the 96-well plate (BioTek XL-800, USA). The kinetic parameters Vmax, Km, AICc
and R2 values of all the α-amylase inhibitor activities were determined by graph fitting analysis
using Sigma-Plot enzyme kinetic software 14.0 version [29-30]
α-Glucosidase inhibition assay
To determine the mechanism of α-glucosidase enzyme inhibition, the kinetic studies were
performed by using varying concentration of α-glucosidase inhibitors (0.0625, 0.125, 0.3 and 0.4
mM) with varying concentrations of inhibitor substrate (p-nitrophenyl R-D-maltoside (NPM))
such as 0.1, 0.2, 0.4, 0.8 and 1.0 mM. The enzyme was dissolved in de-ionized water to produce
the concentration 0.2 mg/mL for each well. PIPES buffer was used at the pH of 7.0. The reaction
mixture was incubated for the period of 30 minutes at 25 ˚C for 1-minute interval. The absorbance
was measured using ELISA reader by keeping the 96-well plate (BioTek XL-800, USA). The
kinetic parameters Vmax, Km, AICc and R2 values of all the α-glucosidase inhibitors were
determined by graph fitting analysis using Sigma-Plot enzyme kinetic software 14.0 version [27].
28

Enzyme Inhibition Assays
α-Amylase Inhibition assay
The α-amylase inhibitory activity was determined by an assay modified from Kwon, Apostolidis
and Shetty. A volume of 500 µL of α-amylase solution (0.5 mg/mL) in 0.2 mM phosphate buffer
(pH 6.9) and 500 µL of test sample (100, 200, 400, 800, 1000 µg/mL) were incubated for 10 min
˚
at 25 C. After pre-incubation, 1% starch solution (500 μL) in 0.02 M sodium phosphate buffer
(pH 6.9) was added to each tube and incubated at 25 ˚C for 10 min. 1 mL of dinitrosalicylic acid
color reagent was then added to the reaction mixture and the tubes were incubated in boiling water
for 5 min, and finally cooled to room temperature. The solutions were diluted by adding 10 mL
distilled water and the absorbance was measured at 540 nm [24]. The percentage inhibition was
calculated as illustrated,
% Inhibition = (Absorbance Control – Absorbance Sample)/Absorbance Control × 100
α-Glucosidase Inhibition assay
Rat intestinal acetone powder in typical saline (100:1; w/v) was sonicated appropriately and the
supernatant was used as a source of basic intestinal α-glucosidase after centrifugation. In short,10
mL of test samples of 5 mg/mL in DMSO solution were reconstituted in 100 mL of 100 mM-
phosphate buffer at pH 6.8 in 96-well micro-plate and incubated with 50 mL of basic intestinal α-
glucosidase for five min before 50 mL substrate (5 mM, p-nitrophenyl-α-D-glucopyranoside
prepared in same buffer) was added p-nitro-phenol released was measured at 405 nm
spectrophotometrically (Spectra Max plus384, Molecular Devices Corporation, Sunnyvale, CA,
USA) 5 min after incubation with the substrate. Individual blanks for test samples were prepared
to accurate background absorbance where the substrate was changed with 50 mL of buffer. Control
sample contained 10 mL DMSO beside test samples. Percentage of enzyme inhibition was
measured as (1B/A) 100 where [A] represents absorbance of control exclusive of test samples, and
[B] corresponding to absorbance in presence of test samples [15].
29