Synthesis and screening of (E)-3-(2-benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazine analogs as novel dual inhibitors of α-amylase and α-glucosidase

Article abstract of DOI:10.1016/j.bioorg.2020.103979

(E)-3-(2-Benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazines analogs 1–27 were synthesized by multi-step reaction scheme and subjected to in vitro inhibitory screening against α-amylase and α-glucosidase enzymes. Out of these twenty-seven synthetic analog

Full text of DOI:10.1016/j.bioorg.2020.103979

Journal Pre-proofs
Synthesis and Screening of (E)-3-(2-Benzylidenehydrazinyl)-5,6-diphen-
yl-1,2,4-triazine Analogs as Novel Dual Inhibitors of α-Amylase and α-Glu-
cosidase
Shahbaz Shamim, Khalid Mohammed Khan, Nisar Ullah, Sridevi
Chigurupati, Abdul Wadood, Ashfaq Ur Rehman, Muhammad Ali, Uzma
Salar, Ahmad Alhowail, Muhammad Taha, Shahnaz Perveen
PII:
S0045-2068(20)31276-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103979
YBIOO 103979
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
13 February 2020
16 May 2020
27 May 2020
Please cite this article as: S. Shamim, K. Mohammed Khan, N. Ullah, S. Chigurupati, A. Wadood, A. Ur
Rehman, M. Ali, U. Salar, A. Alhowail, M. Taha, S. Perveen, Synthesis and Screening of (E)-3-(2-
Benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazine Analogs as Novel Dual Inhibitors of α-Amylase and α-
Glucosidase, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103979
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Synthesis and Screening of (E)-3-(2-Benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazine
Analogs as Novel Dual Inhibitors of α-Amylase and α-Glucosidase
1
1,2
3
4
Shahbaz Shamim, Khalid Mohammed Khan, Nisar Ullah, Sridevi Chigurupati, Abdul
5
5
1
6
7
Wadood, Ashfaq Ur Rehman, Muhammad Ali, Uzma Salar, Ahmad Alhowail, Muhammad
2
8
Taha, Shahnaz Perveen
1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
2
3
Chemistry Department, King Fahd University of Petroleum & Minerals, Dhahran-31261, Saudi
Arabia
4
Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Qassim
University, Buraidah 52571, Kingdom of Saudi Arabia
Department of Biochemistry, Shankar Campus, Abdul Wali Khan University, Mardan, Khyber
5
Pukhtoonkhwa, Pakistan
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
6
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
Department of Pharmacology, Faculty of Pharmacy, Qassim University, Buraidah 51452, Saudi
7
8
Arabia
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: (E)-3-(2-Benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazines analogs 1-27 were
synthesized by multi-step reaction scheme and subjected to in vitro inhibitory screening against α-
amylase and α-glucosidase enzymes. Out of these twenty-seven synthetic analogs, ten compounds
1
4-17, 19, and 21-25 are structurally new. All compounds exhibited good to moderate inhibitory
potential in terms of IC values ranging (IC =13.02 ± 0.04 - 46.90 ± 0.05 µM) and (IC =13.09
5
0
50
50
±
±
0.08 - 46.44 ± 0.24 µM) in comparison to standard acarbose (IC = 12.94 ± 0.27 µM and 10.95
50
0.08 µM), for α-amylase and α-glucosidase, respectively. Structure-activity relationship
indicated that analogs with halogen substitution(s) were found more active as compared to
compounds bearing other substituents. Kinetic studies on most active α-amylase and α-glucosidase
inhibitors 5, 7, 9, 15, 24, and 27, suggested non-competitive and competitive types of inhibition
mechanism for α-amylase and α-glucosidase, respectively. Molecular docking studies predicted
Corresponding authors: khalid.khan@iccs.edu; drkhalidhej@gmail.com; Tel.: 00922134824910; Fax:
0
0922134819018
1

the good protein-ligand interaction (PLI) profile with key interactions such as arene-arene, H-ℼ ,
ℼ ℼℼ , and ℼ -H etc., against the corresponding targets.
Keywords: 1,2,4-Triazine, in vitro screening, α-amylase inhibition, α-glucosidase inhibition,
structure-activity relationship, kinetics, molecular modeling, acarbose
Introduction
The elevated blood glucose level (hyperglycemia) is the leading factor of diabetes mellitus (DM),
normally caused by the insulin resistance, relative insulin insufficiency, and increased endogenous
glucose production (EGP) [1]. Prolonged hyperglycemia leads to many diabetic complications,
including nephropathy, neuropathy, retinopathy, and cardiovascular diseases which in turn are the
major causal factors for mortality in diabetic patients [2-4]. Hyperglycemic condition leads to the
development of both micro and macro vascular dysfunctions [5]. Both type I and type II diabetes
have similar physio-pathological properties but differ in etiology. Type II diabetes is the most
expressive form of this disease as there are almost 430 million type II diabetic patients worldwide
and the number is likely to reach 630 million by 2045 [6,7].
+
2
α-Amylase (E.C.3.2.1.1) is a Ca ion containing metalloenzyme. Usually, this enzyme found in
microbes, plants, and higher organisms. In higher organism, it is secreted by the pancreas and
salivary glands. α-Amylase catalyzes the initial hydrolysis of polysaccharides into
oligosaccharides and monosaccharides by breaking α-1,4-glucosidic bonds. However, α-
glucosidase is another important enzyme which is present in the brush-border of small intestine. It
catalyzes the breakdown of oligosaccharides into monosaccharides and releases free α-D-glucose
which is mainly responsible to cause hyperglycemia. However, α-glucosidase activities are
associated with two small intestinal membrane-bound enzymes: maltase-glucoamylase (MGAM)
and sucrose-isomaltase (SI). As they are involved in the breakdown of dietary sugars and starches,
MGAM and SI are also the attractive targets for inhibition, as a means of controlling blood glucose
levels in individuals with type 2 diabetes [8-10].
DM can be treated by elevating the endogenous insulin secretion, enhancing the insulin’s action
at the target tissue, reducing the demand of insulin, and inhibiting the hydrolysis of oligo and
disaccharides to monosaccharides [9-14]. As α-amylase and α-glucosidase play a key role in the
carbohydrate digestion, inhibition of these enzymes offers possible therapeutic strategies for
2

postprandial hyperglycemia management [15]. Voglibose, acarbose, and migilitol are inhibitors of
α-amylase and α-glucosidase enzymes, and being clinically used to treat type II diabetes [13].
Regrettably, these clinical inhibitors cause serious side effects in human body such as diarrhea,
abdominal pain, flatulence, and liver disorder. Consequently, search for new α-amylase and α-
glucosidase inhibitors with no or less side effects is highly desirable [16].
Heterocyclic compounds are the key scaffolds for many biological evaluations, as those have been
implicated in the discovery of new biologically important molecules [17-19]. Heterocyclic
scaffolds bearing nitrogen and/or sulfur atom/s find widespread utility in the development of
antidiabetic therapeutics [20,21]. Triazine is one of the nitrogen-containing aromatic heterocycles
that possesses a wide range of biological applications such as analgesic, antituberculosis,
antifungal, anticancer, antiprotozoal, antimalarial, antiviral, antimicrobial, and antiinflammatory
activities [22,23]. In particular, 1,2,3-triazine and its derivatives are widely known for their
efficacy and fewer side effects [24]. It is pertinent to mention that 1,2,3-triazine derived scaffolds
are in the final phases of clinical investigations [25].
Our ongoing research is dedicated to the synthesis of bioactive compounds and we have reported
a number of α-amylase and α-glucosidase inhibitors based on different heterocyclic scaffolds [26-
3
2].
We
have
also
reported
2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-
hydroxyphenyl)ethan-1-one derivatives as potent α-glucosidase inhibitors [33]. Herein, we wish
to report the dual inhibitory potential of 1,2,4-triazine based compounds against α-amylase and α-
glucosidase enzymes in order to introduce the possible therapeutic leads to serve as antidiabetic
agents (Figure-1).
1
,2,4-Triazine
1
,2,4-Triazine
Aryl
Aryl
H
R
N
N
N
N
N
S
O
N
N
H
N
N
OH
H
Imine moiety
2
-((5H-[1,2,4]Triazino[5,6-b]indol-3-yl)thio)-1-(3-
Synthetic Analogues 4-27
hydroxyphenyl)ethan-1-one

-Glucosidase inhibitors
-Amylase and -Glucosidase Inhibitors
Figure-1: Rationale for the design of target compounds
3

Results and Discussion
Chemistry
Synthesis of target compounds (4-27) was achieved as outlined in scheme-1. In brief, condensation
of benzil with thiosemicarbazide in acetic acid under heating afforded 5,6-diphenyl-1,2,4-triazine-
3
-thiol 1 in 80% yield. The S-alkylation of 1 with methyl iodide under basic condition yielded 3-
(methylthio)-5, 6-diphenyl-1,2,4-triazine 2, which in turn was refluxed with hydrazine in ethanol
to produce the desired key intermediate 3-hydrazineyl-5, 6-diphenyl-1,2,4-triazine 3 in 75% yield.
Finally, condensation of 3 with a variety of substituted aryl benzaldehydes rendered the target
compounds (4-27) in good to excellent yields (Scheme-1). The chemical structures of the
1
synthesized target compounds were fully characterized by spectroscopic methods including H-,
13C-NMR and EI-MS. Specifically, geometric isomerism of iminic double bond was deduced with
the help of NOESY analysis. Among all the synthetic analogues, ten compounds 14-17, 19, and
2
1-25 are structurally new derivatives.
O
N
N
SH
N
S
MeI, NaOH
EtOH, 6 hrs
CH3
AcOH
0 C, 6 hrs
N
O
N
°
8
N
+
H N NH
1
2
2
N H
EtOH,
NH2
2
N H
S
6
2 .
hrs
H
2
O
R
N
O
N
N
N
EtOH
H N
H
2
N
+
N
N
°
N
N
4 hrs, 80 C
H
H
H
R
3
(
4-27)
Scheme-1: Synthetic scheme for (E)-3-(2-benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazine
analogs 4-27
4

S
O
HN
N
NH2
O
S
AcOH
AcOH
-H2O
OH
NH
OH

HN
H N
80 C, 6 hrs
2
N
NH2
H
HO
S
NH2
N
SH
N
N
S
N
S
-
H O
2
HO
N
NH
NH
N
N
H
NaOH -H O
2
1
H N-NH .H O
2
2
2
H
N
N
SNa
N
N
S
N
N
N
EtOH
NaI
CH3
NH2
H C
I
EtOH
3
O
N
N
N
-
-CH SH
3
H
2
3
R
CH CN
3
H
N
OH
N
N
H
N
N
N
-
H O
N
2
N
H
N
R
N
R
4-27
Figure-2: Proposed mechanism for the synthesis of compounds 1-27
1
The H-NMR of most active compound 15 exhibited two down field singlets resonated at δ 12.41
H
ppm (NH) and δ 8.68 ppm (CH) of the imine moiety. Likewise, the spectrum showed two doublets
H
due to H-2', H-5' at δ 8.77 (J = 2.7 Hz) and 7.84 ppm (J = 9.0 Hz), respectively. H-4' resonated
H
at δ 8.21 ppm as doublet of doublet (dd). The resonances for the remaining aromatic protons
H
appeared as an overlapping multiplet in the region between δ 7.50-7.36 ppm (Figure-3A).
H
1
3
Similarly, C-NMR spectrum of compound 15 displayed characteristic signals owing to triazine
ring and imine carbons at 158.2 (C-3), 156.6 (C-5), 151.7 (C-6), and 137.4 (imine C), respectively.
Rest of aromatic carbon resonances appeared in the usual aromatic region from δ 146.7 to 120.7
C
ppm (Figure-3B). The E stereochemistry of the iminic double bond was deduced with the help of
5

NOESY wherein the NH and proton on the iminic carbon showed the NOESY interactions (Figure-
C).
3
7
.44-7.36 (m)
H
1
29.0
5

8.68 (s)
H
H
H
6

128.2
135.8
1
2.41 (brs)
128.6
129.0
Cl
H
Cl
4

4
N
H
7.84 (d)
H
H
N
158.2
1

6
133.5 137.4
N
N
5
3
N
1
3

124.3
H
2

H
N
156.6
128.2
128.2 146.7
N 137.4
5

H

6

2

120.7
H
N2
N
5
131.5
4

129.4
6
N
H
H
1

3
138.2
1
35.6
1
8.21 (dd)
8
.77 (d)
4

NO2
NO2
130.4
1
28.2
2

H
H 7.50-7.46 (m)
3

1
29.4
H
A
B
tion
Interac
NOESY
H
H
N
N
NO2
N
N
N
E-Isomer
C
1
13
Figure-3: (A) H-NMR chemical shifts; (B) C-NMR chemical shifts of compound 15; (C)
Characteristic NOESY interaction between NH and proton on the iminic carbon in E-isomer
+
The EI-MS spectrum of compound 15 showed the molecular ion peak [M ] at m/z 430 for
molecular formula C H ClN O . The spectrum further displayed a fragment ion at m/z 295 due
2
2
15
6
2
to the loss of chlorine. In addition, the significant base peak of 1,2-diphenylethyne fragments
appeared at m/z 178 (Figure-4).
6

H
Cl
H
H
H
H
N
N
N
H
N
N
N
N
N
N
N
N
N
N
N
N
NO2
•+
NO2
C H ClN O
2
•
22 15
+
6
C H N
•
1
6
12
5
C H N O
2
[
M ] m/z = 430
22 16
6
m/z = 274
m/z = 395
•
C H
C H
14 10
6
5
m/z = 77
m/z = 178
Figure-4: Key EI-MS fragmentation pattern of compound 15
In vitro α-amylase and α-glucosidase inhibitory screening
All the synthesized 1,2,4-triazine analogs 1-27 were assessed for their α-amylase and α-
glucosidase inhibitory activities in vitro as shown in Table-1. These compounds exhibited
significant activities against both enzymes. In comparison to the standard acarbose (IC s = 12.94
5
0
±
0.27 µM and 10.95 ± 0.08 µM for α-amylase and α-glucosidase, respectively), compounds 5, 7,
9
, 15, 24, and 27 showed significant α-amylase and α-glucosidase inhibitory activities with IC50
values ranging from 13.02 ± 0.04 to 14.77 ± 0.02 µM, and 13.09 ± 0.08 to 14.87 ± 0.04 µM,
respectively.
Table-1: In vitro α-amylase and α-glucosidase inhibitory activities of 1,2,4-triazine derivatives
1
-27.
α-Amylase
α-Glucosidase
Intermediates
Structures
Inhibitory Activity
Inhibitory Activity
a
a
(µM ± SEM )
(µM ± SEM )
N
SH
1
36.88 ± 0.24
36.47 ± 0.04
N
N
7

N
N
S
CH3
2
3
36.95 ± 0.08
26.52 ± 0.09
36.4 ± 0.25
N
N
H
N
N
N
NH2
26.42 ± 0.07
H
R
H
N
N
N
N
N
α-Amylase
α-Glucosidase
Compounds
R
Inhibitory Activity
Inhibitory Activity
a
a
(µM ± SEM )
(µM ± SEM )
4
5
26.96 ± 0.07
26.44 ± 0.22
13.09 ± 0.08
13.02 ± 0.04
26.28 ± 0.24
14.48 ± 0.08
36.79 ± 0.20
Cl
Cl
6
7
8
9
26.59 ± 0.06
14.55 ± 0.04
36.55 ± 0.22
Cl
Cl
Cl
F
14.10 ± 0.28
36.97 ± 0.08
36.24 ± 0.08
14.29 ± 0.25
36.47 ± 0.08
36.57 ± 0.20
CF3
OH
1
1
0
1
HO
OH
8

1
2
45.02 ± 0.04
45.09 ± 0.08
OH
OH
NO2
1
1
3
4
25.44 ± 0.20
26.44 ± 0.07
25.65 ± 0.20
26.57 ± 0.22
OH
O N
2
NO2
Cl
1
1
5
6
14.42 ± 0.08
36.47 ± 0.27
14.47 ± 0.02
36.52 ± 0.04
Cl
H N
2
1
1
7
8
36.46 ± 2.06
46.90 ± 0.05
36.64 ± 0.09
46.44 ± 0.24
OCH3
OH
OCH3
1
2
2
2
9
0
1
2
26.97 ± 0.07
27.44 ± 0.09
26.05 ± 0.40
34.40 ± 0.05
26.55 ± 0.07
27.45 ± 0.07
26.55 ± 0.49
34.57 ± 0.09
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
Br
OCH3
OCH3
H CO
3
Br
Br
2
3
27.49 ± 0.20
27.55 ± 0.27
HO
OCH3
9

2
2
4
5
14.46 ± 2.06
26.45 ± 0.06
14.64 ± 0.09
26.47 ± 0.20
F
OCH3
O
2
2
6
7
36.47 ± 0.22
36.50 ± 0.23
O
14.77 ± 0.02
12.94 ± 0.27
14.87 ± 0.04
10.95 ± 0.08
b
Standard
Acarbose
a
b
SEM (standard error mean); Acarbose (Standard for α-amylase and α-glucosidase inhibitory activities).
Structure-activity relationship (SAR)
Ostensibly, all structural units of the active compounds 1-27 have a pivotal role in demonstrating
the inhibitory potential. However, different substitution/s on the phenyl ring is presumably
responsible for the variation in the activities. Thus, a limited SAR is explained on the basis of
varying features on the phenyl ring.
Among all the 1,2,4-triazines, the parent compound 1 (α-amylase IC50 = 36.88 ± 0.24, α-
glucosidase IC = 36.47 ± 0.04 µM) showed less inhibitory strength. The methylated analogue 2
5
0
(
α-amylase IC = 36.95 ± 0.08, α-glucosidase IC = 36.40 ± 0.25 µM) demonstrated almost same
50 50
inhibitory potential like its parent analog against both enzymes. However, hydrazine incorporated
compound 3 (α-amylase IC = 26.52 ± 0.09, α-glucosidase IC = 26.42 ± 0.07 µM) results in
5
0
50
much enhanced inhibitory activities as compared to compounds 1 and 2. Previously, we have
identified the amino (-NH2) bearing 2-((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)-1-(3-
hydroxyphenyl)ethan-1-one derivative B as moderately active inhibitor of α-glucosidase [33]. In
comparison to compounds B, analog 3 was found to be much better dual inhibitor. Good activity
of compound 3 might be accredited due to less carbon load which helps to fit well into the binding
pocket of the enzymes.
1
0

Eventually, incorporation of phenyl part as in compound 4 (α-amylase IC = 26.96 ± 0.07, α-
5
0
glucosidase IC = 26.44 ± 0.22 µM) also showed good inhibitory potential than compounds 1 and
5
0
2
, but almost same as analog 3 which confirms the vital role of hydrazine moiety in inhibition of
both enzymes. Similarly, in our previous study, phenyl bearing compound A also recognized as
good α-glucosidase inhibitor (Figure-5).
O
O
H
H
N
N
S
N
N
S
N
N
N
(
Me
N
NH2
A)
(B)
Moderate -Glucosidase Inhibitor [33]
Good -Glucosidase Inhibitor [33]
N
SH
H
N
S
N
N
CH3
NH2
N
N
N
N
N
N
1
2
3

-Amylase  IC50 = 36.88 ± 0.24 
IC50 = 36.95 ± 0.08 
IC50 = 36.40 ± 0.25 
IC50 = 26.52 ± 0.09 
IC50 = 26.42 ± 0.07 
-Glucosidase  IC50
= 36.47 ± 0.04 
H
H
N
N
N
N
N
4
IC50 = 26.96 ± 0.07 
IC50 = 26.44 ± 0.22 
Figure-5: SAR of compounds 1-4 for α-amylase and α-glucosidase inhibitory activities
On the hands, compounds bearing Cl and F substituents displayed good to moderate α-amylase
and β-glucosidase inhibitions. For example, p-chloro substituted compound 5 (α-amylase IC =
5
0
1
3.02 ± 0.04, α-glucosidase IC = 13.09 ± 0.08 µM) showed α-amylase as well as α-glucosidase
50
inhibitory activities, comparable to standard acarbose (α-amylase IC50 = 12.94 ± 0.27, α-
glucosidase IC = 10.95 ± 0.08 µM). Comparison of activities of compound 5 with its precursor
5
0
4
which does not possess any substitution, revealed that the incorporation of chloro group at para
position leads to increased inhibitory potential. Similarly, o,p-dichloro substituted compound 7
showed slightly decreased activity (α-amylase IC = 14.48 ± 0.08, α-glucosidase IC = 14.55 ±
5
0
50
1
1

0
.04 µM), which suggested that extra chloro at the ortho position has not much influenced on the
inhibitory activity. Likewise, the activity of compound 6 (α-amylase IC = 26.28 ± 0.24, α-
5
0
glucosidase IC = 26.59 ± 0.06 µM) with o,o-dichloro substitution showed further decreased
5
0
inhibitory activities which confirmed that the chloro at ortho position showed less potential.
Furthermore, o-fluoro substituted compound 8 (α-amylase IC = 36.79 ± 0.20, α-glucosidase IC
50
5
0
=
36.89 ± 0.22 µM) also showed less inhibitory potential. In contrast, compound C with m-fluoro
substitution was identified as the good α-glucosidase inhibitor in our previous study. Ortho
position for fluoro in compound 8 might be less favorable to interact with the binding pocket of
enzymes [33]. Compound 9 (α-amylase IC = 14.10 ± 0.28 α-glucosidase IC = 14.29 ± 0.25 µM)
5
0
50
with the inclusion of tri-fluoro methyl group at para position showed an inhibitory potential
comparable to the standard acarbose (α-amylase IC = 12.94 ± 0.27 µM, α-glucosidase IC =
5
0
50
1
0.95 ± 0.08 µM). Limited inhibitory activity pattern of different halogen substituted compounds
revealed that substitution at para position has an indispensable role in the inhibition of both
enzymes (Figure-6).
O
H
N
N
S
F
N
H
H
Cl
N
H
N
H
N
N
N
N
N
N
N
(
C)
N
Good -Glucosidase Inhibitor [33]
N
Cl
Cl
Cl
7
6
H
H
IC50 = 14.48 ± 0.08 
IC50 = 14.55 ± 0.04 
IC50 = 26.28 ± 0.24 
IC50 = 27.28 ± 0.25 
N
N
N
N
N
Cl
5
H
H
H
H
IC50 = 13.02 ± 0.04 
IC50 = 13.02 ± 0.04 
N
N
N
N
N
N
N
N
N
N
CF3
F
9
8
IC50 = 14.10 ± 0.28 
IC50 = 14.29 ± 0.25 
IC50 = 36.79 ± 0.20 
IC50 = 36.55 ± 0.22 
Figure-6: SAR of compounds 5-9 for α-amylase and α-glucosidase inhibitory activities
Among hydroxy containing compounds 10-12, p-hydroxy bearing molecules 10 (α-amylase IC50
=
36.97 ± 0.08, α-glucosidase IC = 36.47 ± 0.08µM) showed moderate activity. Incorporation of
50
1
2

another hydroxy at ortho position as in compound 11 (α-amylase IC50 = 36.24 ± 0.08, α-
glucosidase IC = 36.57 ± 0.20 µM) has no significant effect on the activity which indicates
5
0
presence of any group at ortho position has no or least role in the inhibition. Similarly, compound
2 (α-amylase IC = 45.02 ± 0.04, α-glucosidase IC = 45.09 ± 0.08 µM) with second hydroxyl
1
5
0
50
group at meta position results in further decrease in inhibitory activities which suggested that
substituting the meta position would result in the loss of inhibition strength. Interestingly, the
hydroxy bearing analog D was identified as the potent α-glucosidase inhibitor in our previous
study [33] (Figure-7).
O
H
N
N
S
OH
N
N
(D)
Potent -Glucosidase Inhibitor [33]
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
HO
OH
OH
OH
10
11
12
IC50 = 36.97 ± 0.08 
IC50 = 36.47 ± 0.08 
IC50 = 36.24 ± 0.08 
IC50 = 36.57 ± 0.20 
IC50 = 45.02 ± 0.04 
IC50 = 45.09 ± 0.08 
Figure-7: SAR of compounds 10-12 for α-amylase and α-glucosidase inhibitory activities
Nitro containing compounds 13-16 proved to be good to moderate inhibitors too. Amongst 13-16,
compound 15 (α-amylase IC = 14.42 ± 0.08 µM, α-glucosidase IC = 14.47 ± 0.02 µM) with
5
0
50
nitro along with chloro, has shown inhibitory potential comparable to standard acarbose (α-
amylase IC = 12.94 ± 0.27 µM, α-glucosidase IC = 10.95 ± 0.08 µM). Only the absence of
5
0
50
chloro in compound 13 (α-amylase IC = 25.44 ± 0.20 µM, α-glucosidase IC = 25.65 ± 0.20
5
0
50
µM) leads to decreased inhibitory potential. In addition, compound 14 (α-amylase IC = 26.44 ±
5
0
0
.07 µM, α-glucosidase IC = 26.57 ± 0.22 µM) with combination of nitro and hydroxyl groups
50
showed almost same activities as compound 13. Similarly, compound 16 (α-amylase IC = 36.47
5
0
±
0.27 µM, α-glucosidase IC = 36.52 ± 0.04 µM) having chloro and amino para to each other
50
showed moderate activity. It demonstrates that nitro in combination with halogen showed better
role in inhibition as compared to only nitro and nitro with hydroxyl group (Figure-8).
1
3

H
H
H
H
H
N
H
N
N
N
NO2
N
N
Cl
N
N
N
NO2
N
N
N
N
N
N
H N
Cl
2
1
3
15
16
IC50 = 25.44 ± 0.20 
IC50 = 25.65 ± 0.20 
IC50 = 14.42 ± 0.08 
IC50 = 14.47 ± 0.02 
IC50 = 36.47 ± 0.27
IC50 = 36.52 ± 0.04 
H
H
N
N
N
OH
N
N
O N
2
14
IC50 = 26.44 ± 0.07 
IC50 = 26.57 ± 0.22 
Figure-8: SAR of compounds 13-16 for α-amylase and α-glucosidase inhibitory activities
Furthermore, compounds 17-23 having combination of methoxy with fluoro, chloro, and bromo
groups were also found to be good to moderate inhibitors. Compound 24 (α-amylase IC = 14.46
5
0
±
2.06 µM, α-glucosidase IC = 14.64 ± 0.09 µM) with methoxy and fluoro substitutions ortho to
50
each other, demonstrated inhibition comparable to standard and its good activity might be
ascribing due to electronegative fluorine atom at para position. Similarly, compound 19 (α-
amylase IC = 26.97 ± 0.07 µM, α-glucosidase IC = 26.55 ± 0.07 µM) having fluoro and chloro
5
0
50
ortho to each other, revealed less inhibitory potential. Furthermore, presence of bromo, hydroxyl,
and methoxy groups meta to each other as in compound 23 (α-amylase IC = 27.49 ± 0.20 µM,
5
0
α-glucosidase IC = 27.55 ± 0.27 µM) tends to decrease the activity as compared to compound
5
0
2
4. In compounds 21 (α-amylase IC = 26.05 ± 0.40 µM, α-glucosidase IC = 26.55 ± 0.49 µM)
50 50
and 22 (α-amylase IC = 34.40 ± 0.05µM, 34.57 ± 0.09 µM) presence of bromine with two
5
0
methoxy groups, ortho and meta to each other, respectively, not showed any pronounce effect in
exhibiting the inhibitory potential. While in previous study, p-bromo substituted derivative G
revealed good inhibitory potential against α-glucosidase enzyme [33]. Compounds 17 (α-amylase
IC = 36.46 ± 2.06 µM, α-glucosidase IC = 36.64 ± 0.09 µM) and 20 (α-amylase IC = 27.44
5
0
50
50
±
0.09µM, α-glucosidase IC = 27.45 ± 0.07 µM) were found to be moderately active. Compound
50
1
8 (α-amylase IC = 46.9 ± 0.05 µM, α-glucosidase IC = 46.44 ± 0.24 µM) with o-methoxy
50 50
substitution resulted in sharp drop in the activity compare to the rest of the methoxy substituted
analogs. Previously, o-methoxy substituted analog E was found to be moderately active while p-
1
4

methoxy substituted analog F was recognized as the potent α-glucosidase inhibitor [33] (Figure-
).
9
O
H
N
N
S
N
N
O
OMe
O
OMe
H
H
N
N
S
N
N
N
S
N
(
F)
Potent -Glucosidase Inhibitor [33]
N
N
Br
(
E)
H
H
(G)
Moderate -Glucosidase Inhibitor [33]
Good -Glucosidase Inhibitor [33]
N
N
N
OCH3
OCH3
N
N
H
H
H
OCH3
H
N
N
N
N
OCH3
N
N
N
20
N
IC50 = 27.44 ± 0.09 
IC50 = 27.45 ± 0.07 
N
N
OCH3
OH
1
7
18
IC50 = 36.46 ± 2.06 
IC50 = 36.66 ± 2.02 
IC50 = 46.9 ± 0.05 
IC50 = 46.44 ± 0.24 
H
H
N
N
N
H
H
N
H
H
OCH3
N
N
N
N
N
N
N
N
F
N
N
OCH3
N
2
4
Cl
H CO
Br
3
IC50 = 14.46 ± 2.06 
IC50 = 14.64 ± 0.09 
19
OCH3
22
IC50 = 26.97 ± 0.07 
IC50 = 26.55 ± 0.07 
IC50 = 34.40 ± 0.05
IC50 = 34.57 ± 0.09


H
H
Br
H
H
N
N
N
N
OCH3
Br
N
N
N
N
N
N
HO
OCH3
OCH3
21
23
IC50 = 26.05 ± 0.40 
IC50 = 26.55 ± 0.49 
IC50 = 27.49 ± 0.20 
IC50 = 27.55 ± 0.27 
Figure-9: SAR of compounds 17-24 for α-amylase and α-glucosidase inhibitory activities
Compounds 25-27 have heterocycles like furan and extended ring systems, exhibited good to
moderate inhibitory activities. Compound 27 (α-amylase IC = 14.77 ± 0.02 µM, α-glucosidase
5
0
IC = 14.87 ± 0.04 µM) was found one of the most active compounds of the series having a furan
5
0
ring which means that insertion of a heterocyclic ring in the core motif could be responsible for
enhancing the inhibitory potential. Similarly, coumarin bearing analog I in our previous work was
also recognized as the good α-glucosidase inhibitor [33]. Compounds 25 (α-amylase IC = 26.45
5
0
1
5

±
0.06 µM, α-glucosidase IC = 26.47 ± 0.20 µM) and 26 (α-amylase IC = 36.47 ± 0.22 µM, α-
50 50
glucosidase IC = 36.47 ± 0.23 µM) showed moderate activity might be due to extended ring
5
0
system. These extended ring systems might create steric hindrance to well fit into the active site
of both enzymes. Despite that, analog H with biphenyl ring was found to be good α-glucosidase
inhibitor. Its good activity might be due to attainment of favorable conformation to fit well into
the active site of α-glucosidase enzyme (Figure-10).
O
O
H
H
N
N
S
N
N
S
N
N
N
N
O
O
(H)
(I)
Good -Glucosidase Inhibitor [33]
Good -Glucosidase Inhibitor [33]
H
H
H
H
N
N
H
H
N
N
N
N
N
N
N
N
O
N
N
N
N
N
O
2
7
2
6
2
5
IC50 = 14.77 ± 0.02 
IC50 = 14.87 ± 0.04 
IC50 = 36.47 ± 0.22 
IC50 = 36.60 ± 0.23 
IC50 = 26.45 ± 0.06
IC50 = 26.47 ± 0.20 
Figure-10: SAR of compounds 25-27 for α-amylase and α-glucosidase inhibitory activities
Kinetic studies of α-amylase enzyme inhibition
Kinetic studies on the most active α-amylase and α-glucosidase inhibitors including 5, 7, 9, 15, 24,
and 27 were conducted to interpret the inhibition mechanism. The mechanism of enzyme inhibition
was analyzed by graph fitting analysis, using Sigma-Plot enzyme kinetic software version 14.0.
By using various kinetics plots such as Lineweaver-Burk, Hill, Hanes-Woolf, Eadie-Hofstee,
2
Dixon and Scatchard models, the kinetic parameters Vmax, Km, AICc and R values were
calculated (Table-2 & -3). Based on curve fitting model and low AICc value, the kinetic studies
results showed that all selected compounds following noncompetitive type inhibitory mechanism
for α-amylase and competitive type inhibitory mechanism in case of α-glucosidase enzyme.
1
6

Table-2: Kinetic study of active compounds for α-amylase inhibition.
Vmax
Km
AICc
meanꢀ±ꢀSEM)
Comp.
(meanꢀ±ꢀSEM, (meanꢀ±ꢀSEM
R2
Type of inhibition
(
1
µM/min)
mM)
5
7
9
666.5 ± 0.07
560.5 ± 0.11
499.2 ± 0.09
608.3 ± 0.12
510.4 ± 0.16
598.1 ± 0.17
5.4 ± 0.09
5.3 ± 0.21
5.4 ± 0.2
5.0 ± 0.16
5.2 ± 0.07
5.4 ± 0.17
5.6 ± 0.09
207.5 ± 0.21
502.6 ± 0.30
641.1 ± 0.27
510.5 ± 0.09
625.1 ± 0.12
452.3 ± 0.20
198.5 ± 0.21
0.990 Non-competitive type
0.905 Non-competitive type
0.911 Non-competitive type
0.942 Non-competitive type
0.937 Non-competitive type
0.924 Non-competitive type
0.991 Non-competitive type
1
2
2
5
4
7
Acarbose 680.5 ± 0.12
Table-3: Kinetic study of active compounds for α-glucosidase inhibition.
Vmax
Km
AICc
meanꢀ±ꢀSEM)
Comp.
(meanꢀ±ꢀSEM, (meanꢀ±ꢀSEM
R2
Type of inhibition
(
1
µM/min)
mM)
5
7
9
597.45 ± 0.12
385.31 ± 0.2
471.24 ± 0.17
511.28 ± 0.2
356.10 ± 0.09
405.42 ± 0.11
5.2 ± 0.07
4.8 ± 0.11
5.2 ± 0.12
5.0 ± 0.09
5.0 ± 0.14
5.2 ± 0.08
5.4 ± 0.19
225.5 ± 0.07
292.2 ± 0.2
250.4 ± 0.19
249.0 ± 0.06
282.7 ± 0.1
277.3 ± 0.31
196.5 ± 0.09
0.989
0.924
0.917
0.908
0.977
0.904
0.992
Competitive type
Competitive type
Competitive type
Competitive type
Competitive type
Competitive type
Competitive type
1
2
2
5
4
7
Acarbose 689.5 ± 0.02
1
7

Lineweaver-Burk
Hill
A
B
0
.0026
.0024
.0022
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
1
00
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
-200
-150
-100
-50
0
50
100
0.01
0.1
1
10
1
/[Substrate] (µmol/mL)
[Substrate] (µmol/mL)
I = 0
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
I = 0.4
Hanes-Woolf
Eadie-Hofstee
C
D
0
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
800
00
00
00
400
00
00
00
0
7
0
6
0
5
0
0
0
3
0
2
0
1
0
0
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
10000
20000
30000
40000
50000
60000
Rate (µmol/min/mg)/[Substrate] (µmol/mL)
[Substrate] (µmol/mL)
I = 0
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
I = 0.4
Dixon
E
F
Scatchard
0
.0026
.0024
.0022
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
0
60000
50000
0
0
0
0
40000
0
0
30000
0
0
20000
0
0
10000
0
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0
0
100
200
300
400
500
600
700
800
[Inhibitor] (µmol/mL)
Rate (µmol/min/mg)
S = 0.02
S = 0.2
S = 0.4
S = 0.8
S = 1
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
Figure-11: (A) Lineweaver-Burk plot of 1/[S] Vs 1/Rate in the different concentration of non-
competitive type inhibitor. (B) Hill plot of different concentration of [S] Vs Rate (Vmax-Rate) in
the different concentration of non-competitive inhibitor. (C) Hanes-Woolf plot of compound 5 at
different concentration of [S] Vs [S]/Rate in the different concentration of non-competitive type
inhibitor. (D) Eadie-Hofstee plot of compound 5 by rate/[S] Vs Rate in the different concentration
of non-competitive type inhibitor. (E) Dixon plot of compound 5 at different concentration of non-
1
8

competitive type inhibitor Vs 1/Rate. (F) Scatard plot of compound 5 by Rate Vs Rate/[S] in the
different concentration of non-competitive type inhibitor. [Compound 5: 2-(4-chlorobenzylidene)-
1
-(5,6-diphenyl-1,2,4-triazin-3-yl)hydrazine]
Lineweaver-Burk
Hill
A
B100
0
.0026
.0024
.0022
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
0
0
0
0
0
0
10
0
0
0
0
0
0
1
0.01
0.1
[
1
10
-100
-80
-60
-40
-20
0
20
40
60
Substrate] (µmol/mL)
1
/[Substrate] (µmol/mL)
I = 0
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
I = 0.4
Hanes-Woolf
Eadie-Hofstee
C
D
0
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
800
00
00
00
400
00
00
00
0
7
0
6
0
5
0
0
0
3
0
2
0
1
0
0
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
10000
20000
30000
40000
50000
60000
[Substrate] (µmol/mL)
Rate (µmol/min/mg)/[Substrate] (µmol/mL)
I = 0
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
I = 0.4
Dixon
Scatchard
E
F
60000
0
.0026
.0024
.0022
.0020
.0018
.0016
.0014
.0012
.0010
.0008
.0006
.0004
.0002
0
0
50000
0
0
40000
0
0
30000
0
0
0
20000
10000
0
0
0
0
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0
100
200
300
400
500
600
700
800
[Inhibitor] (µmol/mL)
Rate (µmol/min/mg)
S = 0.02
S = 0.2
S = 0.4
S = 0.8
S = 1
I = 0
I = 0.0165
I = 0.125
I = 0.2
I = 0.4
Figure-12: (A) Lineweaver-Burk plot of 1/[S] Vs 1/Rate in the different concentration of
competitive type inhibitor. (B) Hill plot of different concentration of [S] Vs Rate (Vmax-Rate) in
1
9

the different concentration of competitive type inhibitor. (C) Hanes-Woolf plot of compound 5 at
different concentration of [S] Vs [S]/Rate in the different concentration of competitive type
inhibitor. (D) Eadie-Hofstee plot of compound 5 by rate/[S] Vs Rate in the different concentration
of competitive type inhibitor. (E) Dixon plot of compound 5 at different concentration of
competitive inhibitor Vs 1/Rate. (F) Scatard plot of compound 5 by Rate Vs Rate/[S] in the
different concentration of competitive type inhibitor. [Compound 5: 2-(4-chlorobenzylidene)-1-
(5,6-diphenyl-1,2,4-triazin-3-yl)hydrazine]
Docking study on α-amylase enzyme
Blind docking methodology was applied to explore the possible binding mode of the synthetic
inhibitors in the binding site other than the substrate binding site due to the non-competitive type
of inhibition against the α-amylase enzyme. Subsequently, it was observed that the top-ranked
conformations of all the compounds were well accommodated in the cryptic sites and were
involved in various types of interactions with the active site residues of α-amylase enzyme. i.e.,
F157, F158, T215, R312, and D408 etc. The docking conformation of the most active compounds
5
, 24, and 27 showed different modes of binding pattern (Figure-13A). The protein-ligand
interactions (PLI) profile and correlation between IC50 and docking scores for all the target
compounds illustrated in Table-4 and Figure-13B. Generally, the following active compounds
possess electron-donating and withdrawing groups, i.e., chloro (para)-benzene, fluoro (para)-
benzene plus alkoxy group and 2-methyl furan which ultimately trigger the potential of inhibition
of the corresponding compound at certain level. The most active compound 5 in the series bearing
p-chlorophenyl exhibited the best inhibitory potential as well as well-fit binding potential as
compared to other compounds (Figure-13C). The high potency of compound 5 may be attributed
to the presence of electron withdrawing chloro function, which induces partial positive charge over
benzene and hence promotes its πℼπ or π-H interaction. Likewise, compounds 24 and 27 also
showed better inhibitory potential both in in silico and in vitro. The protein-ligand interaction
profile for both 24 and 27 revealed similar binding potential. A total of three interactions were
observed for 24 adopted by the 1,2,4-triazine moiety with K35 and I253 and two interactions for
2
7 with A438 and S480 as shown in Figures-13D and -13E. The low PLI profile of both 24
compared to 5 might be due to the fluorine at para position which has the very weak amplitude of
activation. Similarly, for compound 27 which bears the aromatic furan group.
2
0

Figure-13: The PLI profile for non-competitive compounds against the α-amylase enzyme. (A)
The surface representation of the enzyme with different binding potential of the compound
(indicated by arrow each site) due to the non-competitive behavior of the synthesized derivatives.
(
B) A correlation graph for IC values vs predicted docking score. The 3D binding mode of
5
0
compound 5 (C) for compound 24 (D) and compound 27 (E).
Table-4: PLI-profile for α-amylase
Interactions detail
S. No.
Docking
score
Ligands Receptor Interaction Distance E cal/mol
Residue
GLU78 (A)
GLU78 (A)
ARG20 (A)
ARG20 (A)
S 19
S 19
N 16
N 16
N 16
N 20
N 20
N 16
N 16
C 6
OE1
OE2
NE
NH2
NE
O
OE2
NE
NH2
NE2
CG2
NE2
CD1
OG1
NE2
NE2
CG2
H-donor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-donor
H-acceptor
H-acceptor
H-acceptor
ℼ -H
4.07
3.92
3.04
3.48
3.69
3.19
3.38
3.01
3.56
3.14
3.71
3.61
3.81
3.35
3.33
3.50
3.98
-1.0
-0.8
-4.3
-0.9
-1.6
-1.0
-0.6
-5.2
-0.6
-1.0
-1.3
-0.8
-0.7
-0.6
-2.0
-1.9
-0.7
1
2
3
-5.06812477
ARG20 (A) -5.43538284
SER73 (A)
GLU78 (A)
-5.45487261
ARG20 (A)
ARG20 (A)
4
5
6
7
GLN63 (A) -6.54943132
6
-ring
6-ring
-ring
THR163 (A)
ℼ -H
ℼ -H
HIS201 (A) -7.10441732
ILE235 (A)
THR163 (A) -6.23625708
6
N 15
N 16
N 20
H-acceptor
H-acceptor
H-acceptor
ℼ -H
GLN63 (A)
GLN63 (A) -6.33572083
VAL107 (A)
6
-ring
2
1

N 19
N 16
N 19
OE2
CA
NH2
OG
O
CD1
O
O
CD1
OG
NH2
CA
N
H-donor
H-acceptor
H-acceptor
ℼ -H
H-donor
ℼ -H
H-donor
H-donor
ℼ -H
H-acceptor
H-acceptor
ℼ -H
H-acceptor
H-acceptor
ℼ -H
H-acceptor
H-acceptor
ℼ - ℼ
H-donor
ℼ -H
H-donor
H-donor
ℼ -H
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-donor
ℼ -H
3.41
3.41
3.01
3.60
3.25
3.56
3.00
3.11
3.54
3.59
3.08
3.70
3.60
3.04
3.60
3.28
3.03
3.43
3.28
3.51
3.28
3.07
3.48
3.51
3.19
3.07
3.20
3.36
3.82
3.17
3.16
3.05
3.60
3.77
3.74
3.49
3.53
3.55
3.28
3.80
-1.3
-0.9
-3.0
-0.6
-2.0
-0.8
-1.1
-1.0
-0.9
-0.6
-4.3
-0.7
-0.7
-2.7
-3.60
-1.0
-6.8
-0.0
-2.6
-0.7
-0.6
-0.5
-0.8
-1.3
-1.5
-2.6
-0.9
-2.8
-0.8
-1.8
-2.3
-2.1
-0.7
-1.0
-0.5
-0.5
-0.5
-1.0
-1.3
-0.6
GLU76 (A)
LYC68 (A)
ARG421 (A)
SER289 (A)
PRO34 (A)
ILE396 (A)
LEU211 (A)
GLY249 (A)
ILE396 (A) -6.32366037
SER3 (A)
ARG92 (A) -6.16662121
ASN5 (A)
LYS200 (A)
ILE235 (A) -6.88277388
HIS201 (A)
ARG343 (A)
LYS322 (A) -6.39212179
TRP388 (A)
PRO34 (A)
-6.36274624
ILE396 (A)
HIS476 (A) -6.25136709
PRO34 (A)
-6.61354113
ILE396 (A)
TRP221 (A)
-6.12138653
ARG92 (A)
ARG424 (A)
-5.64057207
ARG424 (A)
ARG424 (A)
-6.29311371
GLY9 (A)
ARG424 (A)
-6.29573059
ARG424 (A)
PRO34 (A)
-6.06567335
ILE396 (A)
LYS35 (A)
LYS35 (A)
ILE396 (A)
ILE396 (A) -5.74217653
ILE396 (A) -6.47250462
SER480 (A)
-6.65311193
ALA483 (A)
8
9
-6.0324111
-6.1724782
-6.40529776
-5.89396429
6
-ring
N 19
-ring
1
0
6
N 29
O 29
6-ring
N 16
O 31
1
1
1
2
1
1
1
3
4
5
6
-ring
O 31
O 32
N
6
-ring
NE2
NH2
NZ
5-ring
O
CD1
O
O
CD1
O
NH2
NH2
NH2
NH2
CA
HN2
HN2
O
N 18
O 30
6
-ring
N 19
-ring
1
1
1
6
7
8
6
O 29
N 19
6
-ring
CL 28 N
1
2
2
2
2
9
0
1
2
3
1
6
N 15
N 16
N 15
N 18
N 15
N 16
N 19
6
-ring
CD1
CE
N 15
N 16
H-acceptor
H-acceptor
ℼ -H
ℼ -H
ℼ -H
H-acceptor
ℼ -H
2
4
CA
CD1
CD1
CD1
N
-6.81212091
6
-ring
2
2
5
6
6-ring
6-ring
N 16
2
7
6
-ring
CB
Docking study on α-glucosidase enzyme
Based on the curve fitting model and low AICc value, the kinetic studies results revealed that all
selected compounds following the competitive type inhibitors mechanism against α-glucosidase
enzyme. Molecular docking study was performed on all compounds in the active site of the
homology model (Table-5), as previously described by Taha M et al. [34] using well-developed
2
2

modeling software MOE. Interestingly, we observed that the compounds showed best inhibitory
potential against α-glucosidase enzyme (Figure-14A and -14B) and shared similar mechanism of
inhibition i.e., the most active compound 12 showed total five interactions with catalytic residues
which might have role in enzymatic activity enhancement, i.e., R312, T215, F157, F158, D408
(Figure-14C). In case of other active compounds 15 and 21 shared similar binding modes of
interaction with catalytic residues, i.e., D408, F158, and T215 (Figure-14D).
A
C
B
Figure-14: The PLI profile for non-competitive compounds against the α-glucosidase enzyme.
A) The surface representation of the enzyme with zoomed binding site. The 3D binding mode of
(
compound 5 (B), for compound 24 (C).
These results delineate that the compounds bearing the electron-withdrawing group especially at
para position, potentially deactivate the π-system which subsequently make the aromatic ring very
+
+
electron-poor (δ ) relative to benzene, hence that δ benzene ring make interactions with specific
residues to gain the stability and this way enhances the inhibitory potential. While in case of
another compound which possesses an electron-donating group and showed less inhibitory
potential might be due the attached substituted group having weak amplitude of activation, hence
showed less enzymatic activity against both the enzymes. Our experimental activity best correlate
with molecular docking as shown in Figure-15.
2
3

D
Figure-15: A correlation graph for IC values vs predicted docking score
5
0
Table-5: PLI-profile for α-glucosidase enzyme
Interaction details
Ligands Receptor Interaction Distance E cal/mol Residue
Docking
score
S. No.
S 19
S 19
OE1
O
H-donor
H-donor
ℼ -ℼ
H-doner
H-donor
H-donor
H-donor
H-ℼ
H-ℼ
ℼ -H
H-donor
ℼ -H
ℼ -H
3.33
3.24
3.92
3.40
2.92
3.13
2.88
3.78
3.96
4.41
3.32
4.24
4.64
4.22
3.63
3.36
3.44
3.91
3.65
4.19
4.07
3.57
4.49
3.31
4.22
4.20
3.78
3.77
4.32
-2.7
-2.1
-0.0
-0.8
-1.9
-1.2
-1.7
-0.6
-0.8
-0.5
-3.4
-0.6
-0.9
-0.8
-0.8
-2.4
-1.2
-0.5
-1.0
-0.6
-0.6
-0.5
-1.0
-3.3
-0.6
-0.8
-0.6
-0.7
-0.5
GLU304
SER308
HIS239
SER156
GLU
1
2
3
-5.69577312
-5.96104622
-6.02973223
6
-ring
5-ring
O
OE1
OG1
O
6-ring
5-ring
CB
OD2
CE1
CE1
CG2
CB
OE2
OD1
5-ring
OD2
6-ring 2
CG2
CB
S 19
N 19
N 20
N 20
C 21
C 25
THR
SER
PHE157
HIS245
PHE157
ASP408
PHE157
PHE158
THR215
ARG312
GLU304
ASN241
HIS245
ASP408
PHE157
THR215
ARG312
PHE157
ASP408
PHE158
THR215
ARG312
HIS245
HIS245
4
5
6
-6.97443962
-8.00046825
-7.0944953
6
-ring
N 19
-ring
6-ring
6
6
6
-ring
-ring
ℼ -H
ℼ -H
N 19
CL 29
C 27
N 19
C 3
H-donor
H-donor
H-ℼ
H-donor
H-ℼ
ℼ -H
ℼ -H
H-ℼ
H-donor
ℼ -H
7
8
9
-7.31348944
-6.91528463
-7.36655712
6
6
-ring
-ring
6-ring
N 19
CA
OD2
CE1
CG2
CB
5-ring
5-ring
6
6
6
-ring
-ring
-ring
ℼ -H
ℼ -H
H-ℼ
H-ℼ
C 25
O 29
1
0
-6.8123126
2
4

6
-ring
N
ℼ -H
H-acceptor
H-acceptor
ℼ -H
ℼ -H
H-donor
3.83
3.40
3.18
4.34
4.12
2.79
-0.5
-0.6
-1.8
-0.7
-0.6
-9.4
ARG312
PHE157
HIS245
PHE158
THR215
GLU304
C 21
C 25
6
6
N 19
O 29
N 16
C 28
6-ring
5-ring
CE1
CG2
OE2
1
1
-6.96574306
-ring
-ring
1
1
2
3
-7.15614939
-7.14630222
O
ND2
-ring
H-donor
H-acceptor
H-ℼ
3.36
3.13
3.98
-0.7
-2.4
-1.5
GLU276
ASN241
HIS245
1
4
-7.2024107
5
H-donor
ℼ -H
ℼ -H
C 25
6-ring
OE1
CA
N
3.34
3.90
4.67
-0.8
-0.5
-0.8
GLU276
PHE157
ARG312
1
1
5
6
-7.5251317
6
-ring
ℼ -H
N 16
CL 29
C 27
ND2
N
5-ring CD
H-acceptor
H-acceptor
H-ℼ
3.14
3.40
3.97
4.33
3.27
2.91
3.99
3.89
3.31
4.18
3.58
3.91
4.33
4.43
4.03
3.60
4.34
4.11
3.58
4.22
4.26
4.43
2.97
3.98
4.71
-2.5
-0.5
-0.5
-0.5
-1.9
-0.9
-0.6
-1.0
-3.4
-.06
-1.1
-0.7
-0.6
-0.6
-0.5
-1.0
-0.7
-0.6
-1.0
-1.5
-0.9
-0.9
-1.5
-0.9
-0.9
ASN241
GLY217
HIS245
ARG312
ASN241
ASN241
HIS245
ASN241
ASP408
PHE157
PRO309
PHE157
THR215
PHE157
THR215
ASP408
PHE158
THR215
HIS279
PHE157
PHE157
PHE158
ASN241
ASN241
ARG312
-7.09421015
1
1
7
8
-6.77096987
-7.20406246
6
-ring
ℼ -H
N 16
N 19
C 28
ND2
OD1
5-ring
ND2
OD2
CA
H-acceptor
H-donor
H-ℼ
ℼ -H
H-donor
ℼ -H
H-donor
H-ℼ
ℼ -H
H ℼ
ℼ -H
H-donor
ℼ -H
ℼ -H
ℼ -H
ℼ -H
ℼ -H
ℼ -H
H-donor
ℼ -H
1
9
-7.21798372
6
-ring
N 19
-ring
2
2
2
0
1
2
-6.9724226
-7.78707218
-7.49799204
6
BR 29
C 2
O
6-ring
CG2
6-ring
CG2
OD2
CE1
CG2
CD2
CD1
CD1
CE1
OD1
ND2
N
6
-ring
C 21
-ring
2
3
-7.49100208
6
N 19
6-ring
2
2
2
4
5
6
-7.94695091
-7.21506643
-7.44134808
6
-ring
6-ring
-ring
6-ring
-ring
6
6
N 19
6-ring
2
7
-7.55965996
6
-ring
ℼ -H
Conclusion
A library of 27 derivatives of 1,2,4-triazine were synthesized and subjected to α-amylase and α-
glucosidase inhibitory activities. In vitro screening results and SAR revealed that the positions of
the substituted analogs played a remarkable role. The para substituted derivative founds to be more
active in both α-amylase and α-glucosidase inhibition as compared to the standard. The kinetic
studies revealed that all selected compounds followed non-competitive for α-amylase and
2
5

competitive type inhibitory mechanism for α-glucosidase enzyme. Docking results of the most
active compound 4 showed good protein-ligand-interaction (PLI) profile against the corresponding
target. In this ongoing study, we have identified a series of lead molecules as a dual inhibitors of
α-amylase as well as α-glucosidase enzymes which would helpful in future for the designing and
development of drugs for the treatment of diabetic complications.
Experimental
Materials and Methods
All reagents were purchased from Merck, (Germany) and Sigma Aldrich, (USA). Enzymes, α-
amylase from Aspergillus oryzae and α-glucosidase from Saccharomyces cerevisiae were obtained
from Sigma-Aldrich Co. LLC (St. Louis, MO). The NMR (300, 400 MHz) spectra were recorded
on Bruker AM spectrometers. Mass spectra (EI) and HR-MS (EI) were recorded on MAT 312 and
MAT 113D mass spectrometer (Germany) mass spectrometers. Melting points were determined
by using Büchi Melting Point-560 apparatus. Thin layer chromatography was carried out using
pre-coated silica gel-254 Merck, Germany. Spots were visualized under UV light at 366 and 254
nm. Chemical shift values are recorded in ppm with respective to TMS as internal standard and
coupling constant are presented in Hz.
Kinetic studies of α-amylase and α-glucosidase Inhibition
To determine the mechanism of enzyme inhibition, the kinetic studies were performed by using
varying concentration of α-amylase and α-glucosidase inhibitors (0.0625, 0.125, 0.2, and 0.4 mM)
with varying concentrations such as 0.02, 0.2, 0.4, 0.8 and 1.0 mM, of inhibitor substrate (p-
nitrophenyl R-D-maltoside (NPM)). The enzyme was dissolved in de-ionized water to produce the
concentration 0.2 mg/mL for each well. PIPES buffer was used as adjusted the pH of 7.0. Reaction
º
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
2
kinetic parameters Vmax, Km, AICc, and R values of all the α-amylase inhibitors were determined
by graph fitting analysis using Sigma-Plot enzyme kinetic software 14.0 version [35, 36].
2
6

Molecular docking protocol
MOE-dock module of Molecular Operating Environment (MOE) software was used to dock all
the non-competitive (blind docking) and competitive (substrate-based docking) against α-amylase
and α-glucosidase enzyme. Due to the unavailability of crystallographic structure of the
corresponding enzyme, we used the homology modeling structural coordinates described by Taha
M et al. [34] for α-glucosidase enzyme while for α-amylase enzyme, the crystallographic structure
was retrieved from protein data bank using the PDB code: 3BAJ respectively. Next, for blind
docking (non-competitive) against α-amylase, the whole protein was considered as a binding site,
while for competitive inhibitor docking, the substrate-binding site of α-glucosidase enzyme was
selected including the residues F157, F158, T215, R312, and D408, respectively. Briefly, both the
structural coordinates were subjected for 3D protonation and energy minimization up to 0.05
Gradient using MMFF94s forcefield implemented in MOE software. The 3D structures were built
by using Molecular Builder Module in MOE. Lastly, a total of 10 different conformations for each
inhibitor allowed to generate. The ligands are flexible during docking, to obtain the minimal
energy protein-ligand complex. Later, the docked complexes were ranked based on the docking
scores. Finally, the predicted ligand-protein complexes were analyzed for molecular interactions
using PyMol v 1.7.
Synthesis of 5,6-diphenyl-1,2,4-triazine-3-thiol (1)
Benzil (1 mmol) in acetic acid (10 mL) was taken into a 100 mL round-bottomed flask. Mixture
°
was heated at 80 C with continuous stirring. After 5 minutes, thiosemicarbazide (2 mmol) was
added in the reaction mixture and refluxed for 6 h. Reaction progress was monitored by TLC. After
completion, reaction was cooled. On cooling, precipitates were formed which were filtered and
washed with cold acetic acid, hexane, and distilled water. Crude product was recrystallized from
ethanol to afford pure product [37, 38].
º
1
Yield: 80%; M.P.: 234-236 C; H-NMR (300 MHz, DMSO-d ): δ 15.15 (s, 1H, SH), 7.47-7.40
6
H,
(m, 4H, H-2, H-6, H-2', H-6'), 7.38-7.32 (m, 6H, H-3, H-4, H-5, H-2', H-3', H-4' H-5'); EI-MS m/z
+
(
% rel abund.): 265 (M , 98), 178 (100), 152 (36), 89 (65), 76 (57).
2
7

Synthesis of 3-(methylthio)-5, 6-diphenyl-1,2,4-triazine (2)
°
Sodium hydroxide (1 mmol) was dissolved in ethanol (10 mL) and mixture was warm at 100 C.
After dissolving NaOH, basic solution was cooled to room temperature and 5, 6-diphenyl-1,2,4-
triazine-3-thiol (1 mmol) was added into the cooled basic solution and stirred for 15 minutes. After
that, iodomethane (2.2 mmol) was added drop wise to the reaction mixture and stirred at room
temperature for 4-6 h. Precipitates were appeared in the reaction mixture which were filtered and
washed with distilled water. Crude product was recrystallized from ethanol to give pure product
[33, 34].
º
1
Yield: 85%; M.P.: 154-157 C; H-NMR (300 MHz, DMSO-d ): δ 7.49-7.44 (m, 5H, H-2, H-3,
6
H,
H-6, H-2', H-6'), 7.43-7.34 (m, 5H, H-4, H-5, H-3', H-4' H-5'), 2.70 (s, 3H, SCH ); EI-MS m/z (%
3
+
rel abund.): 265 (M , 98), 178 (100), 152 (36), 89 (65), 76 (57).
Synthesis of 3-hydrazineyl-5, 6-diphenyl-1,2,4-triazine (3)
A mixture of compound 2 (3-(methylthio)-5,6-diphenyl-1,2,4-triazine) (1 mmol) and hydrazine
monohydride (NH NH .H O) (2 mL, 10 mmol) was taken in ethanol (10 mL) into a 100 mL
2
2
2
round-bottomed flask and refluxed for 12 h. After completion, reaction mixture kept in fridge to
cool. Upon cooling, precipitates were appeared in the reaction mixture which were filtered and
washed with little volume of chilled ethanol and recrystallized from ethanol to give 3-hydrazineyl-
5
,6-diphenyl-1,2,4-triazine [37, 38].
º
1
Yield: 75%; M.P.: 182-185 C: H-NMR (300 MHz, DMSO-d ): δ 8.90 (brs, 1H, NH), 7.44-7.39
6
H,
+
(
m, 3H, H-2, H-6, H-2'); EI-MS m/z (% rel abund.): 265 (M , 98), 178 (100), 152 (36), 89 (65), 76
(57).
General procedure for the synthesis of compounds 4-27
Compound 3 (3-hydrazineyl-5, 6-diphenyl-1,2,4-triazine) (1 mmol) was taken in ethanol (10 mL)
into a 100 mL round-bottommed flask. Then, substituted aryl aldehydes (1 mmol) was added in
the reaction mixture and heated at 70 to 80 ˚C for 3-4 h with continuous stirring. The resultant
mixture was monitored through TLC in order to check the reaction progress. As the reaction
proceeded, precipitates were appeared in the reaction mixture. After completion, resultant
precipitates were filtered and washed thoroughly with hexane to afford pure products. Compounds
2
8

1
13
4
-27 (Table-1) were characterized by different spectroscopic techniques including H-, C-NMR,
EI-, and HREI-MS.
(E)-3-(2-Benzylidenehydrazinyl)-5,6-diphenyl-1,2,4-triazine (4) [39]
º
1
Yield: 72%; M.P.: 240-241 C; H-NMR (300 MHz, DMSO-d ): δ 11.95 (brs, 1H, NH), 8.28 (s,
6
H
1
H, CH), 7.73 (d, J2',3' = J6',5' = 6.9 Hz, 2H, H-2', H-6'), 7.49 (m, 3H, H-4', H-2'', H-6''), 7.42-7.35
(m, 10H, H-3', H-5', H-3", H-4". H-5", H-2"', H-3"', H-4"', H-5"', H-6"'); EI-MS m/z (% rel abund.):
+
3
51 (M , 45), 274 (43), 248 (75), 178 (100), 165 (19).
(E)-3-(2-(4-Chlorobenzylidene)hydrazinyl)-5,6-diphenyl-1,2,4-triazine (5) [39]
º
1
Yield: 75%; M.P.:255-256 C; H-NMR (300 MHz, DMSO-d ): δ 12.02 (brs, 1H, NH), 8.26 (s,
6
H
1
5
H, CH), 7.76 (d, J2',3' = J 6',5' = 7.8 Hz, 2H, H-2', H-6'), 7.51 (d, J3',4' = J5',6' = 8.4 Hz, 2H, H-3', H-
'), 7.46-7.45 (m, 2H, H-2", H-6''), 7.40-7.35 (m, 8H, H-3", H-5", H-4". H-2"', H-3"', H-4"', H-5"',
+
H-6"'); EI-MS m/z (% rel abund.): 385 (M , 18), 387 (M+2, 6), 274 (16) 248 (36), 178 (100), 165
(13).
(E)-3-(2-(2,6-Dichlorobenzylidene)hydrazinyl)-5,6-diphenyl-1,2,4-triazine (6) [39]
º
1
Yield: 78%; M.P.: 233-235 C; H-NMR (300 MHz, DMSO-d ): δ 12.22 (brs, 1H, NH), 8.46 (s,
6
H
1
7
H, CH), 7.58 (d, J3',4' = J 5',4' = 8.4 Hz, 2H, H-3', H-4'), 7.47-7.44 (m, 3H, H-4', H-2", H-6''), 7.47-
.35 (m, 8H, H-3", H-4", H-5", H-2"', H-3"', H-4"', H-5"', H-6"'); EI-MS m/z (% rel abund.): 419
+
(
M , 11), 421 (M+2, 8), 423 (M+4, 2), 384 (12), 274 (25) 248 (42), 178 (100), 165 (18).
(E)-3-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-5,6-diphenyl-1,2,4-triazine (7) [39]
º
1
Yield: 72%; M.P.: 242-243 C; H-NMR (300 MHz, DMSO-d ): δ 12.24 (brs, 1H, NH), 8.62 (s,
6
H
1
H, CH), 8.06 (d, J6',5' = 8.7 Hz, 1H, H-6'), 7.70 (d, J3',5' = 2.1 Hz, 1H, H-3'), 7.53 (dd, J5',3' = 1.8
Hz, J5',6' = 6.9 Hz, 1H, H-5'), 7.49-7.35 (m, 8H, H-3", H-4", H-5", H-2"', H-3"', H-4"', H-5"', H-
+
6
"'); EI-MS m/z (% rel abund.): 419 (M , 11), 421 (M+2, 7), 423 (M+4, 1), 384 (5), 274 (13), 248
(35), 178 (100), 165 (12).
2
9