Synthesis, in vitro α-amylase inhibitory, and radicals (DPPH & ABTS) scavenging potentials of new N-sulfonohydrazide substituted indazoles

Article abstract of DOI:10.1016/j.bioorg.2019.103410

Over-expression of α-amylase enzyme causes hyperglycemia which lead to many physiological complications including oxidative stress, one of the most commonly associated problem with diabetes mellitus. Marketed α-amylase inhibitors such as acarbose, voglibose, and miglitol used to treat type-II diabetes mellitus, but also linked to several harmful effects. Therefore, it is essential to explore new and nontoxic antidiabetic agents with additional antioxidant properties. In this connection, a series of new N-sulfonohydrazide substituted indazoles 1–19 were synthesized by multistep reaction scheme and assessed for in vitro α-amylase inhibitory and radical (DPPH and ABTS) scavenging properties. All compounds were fully characterized by different spectroscopic techniques including ¹H, ¹³C NMR, EI-MS, HREI-MS, ESI-MS, and HRESI-MS. Compounds showed promising α-amylase inhibitory activities (IC₅₀ = 1.23 ± 0.06–4.5 ± 0.03 μM) as compared to the standard acarbose (IC₅₀ 1.20 ± 0.09 μM). In addition to that all derivatives were found good to moderate scavengers of DPPH (IC₅₀ 2.01 ± 0.13–5.3 ± 0.11) and ABTS (IC₅₀ = 2.34 ± 0.07–5.5 ± 0.07 μM) radicals, in comparison with standard ascorbic acid having scavenging activities with IC₅₀ = 1.99 ± 0.09 μM, and IC₅₀ 2.03 ± 0.11 μM for DPPH and ABTS radicals. In silico molecular docking study was conducted to rationalize the binding interaction of α-amylase enzyme with ligands. Compounds were observed as mixed type inhibitors in enzyme kinetic characterization.

Full text of DOI:10.1016/j.bioorg.2019.103410

Journal Pre-proofs
Synthesis, In vitro α-Amylase Inhibitory, and Radicals (DPPH & ABTS)
Scavenging Potentials of New N-Sulfonohydrazide Substituted Indazoles
Rafaila Rafique, Khalid Mohammed Khan, Arshia, Sridevi Chigurupati,
Abdul Wadood, Ashfaq Ur Rehman, Uzma Salar, Vijayan Venugopal,
Shahbaz Shamim, Muhammad Taha, Shahnaz Perveen
PII:
S0045-2068(19)31595-0
DOI:
https://doi.org/10.1016/j.bioorg.2019.103410
YBIOO 103410
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 September 2019
28 October 2019
28 October 2019
Please cite this article as: R. Rafique, K. Mohammed Khan, Arshia, S. Chigurupati, A. Wadood, A. Ur Rehman,
U. Salar, V. Venugopal, S. Shamim, M. Taha, S. Perveen, Synthesis, In vitro α-Amylase Inhibitory, and Radicals
(DPPH & ABTS) Scavenging Potentials of New N-Sulfonohydrazide Substituted Indazoles, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103410
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Synthesis, In vitro α-Amylase Inhibitory, and Radicals (DPPH & ABTS) Scavenging
Potentials of New N-Sulfonohydrazide Substituted Indazoles
Rafaila Rafique,1 Khalid Mohammed Khan,1,2 Arshia,1 Sridevi Chigurupati,3 Abdul Wadood,4
Ashfaq Ur Rehman,4 Uzma Salar,5 Vijayan Venugopal,6 Shahbaz Shamim,1 Muhammad Taha,2
Shahnaz Perveen7
1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
2Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia.
3Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Qassim
University, Buraidah 52571, Kingdom of Saudi Arabia
4Department of Biochemistry, Shankar Campus, Abdul Wali Khan University, Mardan, Khyber Pukhtoonkhwa,
Pakistan
5Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
6Faculty of Pharmacy, AIMST University, Kedah-08100, Malaysia
7PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: Over-expression of α-amylase enzyme causes hyperglycemia which lead to many
physiological complications including oxidative stress, one of the most commonly associated
problem with diabetes mellitus. Marketed α-amylase inhibitors such as acarbose, voglibose, and
miglitol used to treat type-II diabetes mellitus, but also linked to several harmful effects. Therefore,
it is essential to explore new and nontoxic antidiabetic agents with additional antioxidant
properties. In this connection, a series of new N-sulfonohydrazide substituted indazoles 1-19 were
synthesized by multistep reaction scheme and assessed for in vitro α-amylase inhibitory and radical
(DPPH and ABTS) scavenging properties. All compounds were fully characterized by different
13
spectroscopic techniques including 1H, C-NMR, EI-MS, HREI-MS, ESI-MS, and HRESI-MS.
Compounds showed promising α-amylase inhibitory activities (IC50 = 1.23 ± 0.06 - 4.5 ± 0.03 µM)
as compared to the standard acarbose (IC50 1.20 ± 0.09 µM). In addition to that all derivatives were
found good to moderate scavengers of DPPH (IC50 2.01 ± 0.13 – 5.3 ± 0.11) and ABTS (IC50 =
2.34 ± 0.07 - 5.5 ± 0.07 µM) radicals, in comparison with standard ascorbic acid having scavenging
activities with IC50 = 1.99 ± 0.09 µM, and IC50 2.03 ± 0.11 µM for DPPH and ABTS radicals. In
silico molecular docking study was conducted to rationalize the binding interaction of α-amylase
Corresponding authors: khalid.khan@iccs.edu; drkhalidhej@gmail.com; Tel.: 00922134824910; Fax:
00922134819018
1

enzyme with ligands. Compounds were observed as mixed type inhibitors in enzyme kinetic
characterization.
Keywords: Indazole; sulfonohydrazide; in vitro; α-amylase enzyme; ABTS and DPPH; in silico
Introduction
Diabetes mellitus (DM), a prolonged metabolic ailment associated with insufficient amount of
insulin secretion or its unsatisfactory activity [1-3]. These complications resulted the higher
glucose level or hyperglycemia in human body [4]. Non-insulin dependent diabetes mellitus
(NIDDM-type II DM) is also a threaten for all individuals due to its higher rate of occurrence.
According to WHO (2018) report, mostly 90% patients are suffering from type II DM around the
world. The hydrolysis of carbohydrate into monomer by catalytic hydrolase α-amylase enzyme (α-
1,4-glucan-4-glucanohydrolases, EC. 3.2.1.1) secreted from pancreas [5]. The hydrolysis of
carbohydrate resulted in the elevated blood glucose level after a meal known as post-prandial
hyperglycemia (PPHG) and it is a main warning sign [6]. The elevated blood glucose level
damaged many organs and prove to be one of the risk factors for several complications [7,8]. In
order to cure DM, one of the best therapies is to reduce the activity of α-amylase enzyme.
Acarbose, voglibose, and miglitol (Figure-1A) are marketed inhibitors of α-amylase enzyme and
used to treat type II diabetes mellitus. However, these agents have some adverse effects such as
flatulence, diarrhea, meteorism, and abdominal discomfort therefore always recommended with
other antidiabetic agents for more efficacy [9-11].
Hyperglycemia is one of the reasons to generate free radicals and other reactive oxygen species
which lead to oxidative damage to many tissues. These reactive oxygen species commonly linked
with diabetic complications [12]. Auto-oxidation and protein glycosylation of glucose is also
caused by the access production of free radicals [13, 14]. Free radicals are unstable chemical
species that contain unpaired electron. These free radicals start a chain reaction in a human body
by capturing an electron from the neighboring biomolecule thus they behave abnormally in living
system. Antioxidants are the molecules that convert the free radicals into non-harmful substances
by donating their electron to them. In short antioxidants are agents that protect the body from
oxidative stress by inhibiting the process of oxidation [15]. As the diabetic issues becoming more
complicated due to oxidative stress, there is still need to design a medicine to control type II DM
2

with more effectiveness and low or no side effects. Currently, it is demanding to discover entities
those possesses both hypoglycemic as well as antioxidants potentials to treat diabetic patients [16].
Figure-1: (A) Marketed inhibitors (drugs) for α-amylase enzyme, (B) Identified lead molecules
Heterocycles are important motifs of many biologically active compounds and marketed drugs.
Indazole nuclei, a nitrogen-containing heterocycle, is a center of attraction among the scientist in
the field of medicinal chemistry due to their broad spectrum of biological properties [17]. Indazole
is also known as benzopyrazole, a fused bicyclic aromatic compound based on pyrazole and
benzene ring. In pharmaceutical chemistry indazole is considered as an efficacious bioisosteres of
benzimidazole and indole due to two consecutive nitrogen atoms that enable the molecule to act
as an acceptor or donor with hydrophobic protein pockets through hydrogen bonded interaction
[18-20]. Indazole core have a wide spectrum of bioactivities such as antiinflammatory, analgesic,
antimicrobial, anticancer, and antidiabetic activities [21].
This study is focused on the evaluation of sulfonohydrazide substituted indazole derivatives for
their in vitro α-amylase inhibitory activity as well as antioxidant potentials against DPPH and
ABTS radicals. Herein, we report the synthesis, characterization, in silico, in vitro enzyme
inhibitory activities, and kinetics studies.
Our research group has been engaged in designing and evaluation of several classes of bioactive
molecule in order to search new leads of drug development for the cure of diabetes (Figure-1B)
[22-27]. We have also reported N-carbothioamide indazole as potential α-amylase and α-
3

glucosidase inhibitors [28]. The newly synthesized compounds have close structural features with
the N-carbothioamide indazole, therefore we decided to explore this new class for their possible
α-amylase inhibitory, and also radical scavenging activities (DPPH and ABTS) by keeping in mind
that free radical generation is also associated with the diabetic patients A rational of current study
is depicted in Figure-2.
Figure-2: Rational of the current study
Results and Discussion
Chemistry
A variety of sulfonohydrazide substituted indazoles 2-19 were synthesized by multi-step reaction.
In first step, 4-oxyindazole 1 was formed by reacting dimedone, dimethylformamide
dimethylacetal (DMF-DMA), phenylhydrazine in the presence of catalytic amount of CuCl2.2H2O
in ethanol for 3 h. In the next step, 4-oxyindazole 1 was treated with different sulfonylhydrazide
derivatives in ethanol in the presence of pyridine as catalyst and refluxed for 2 h to obtain the
desired sulfonohydrazide substituted indazoles 2-19 (Scheme-1). Reaction progress was checked
by TLC analysis (ethyl acetate/hexanes, 2:8). Formation of expected products (1-19) were
1
confirmed by spectroscopic data obtained from EI-MS, ESI-MS, HRESI-MS, and H-NMR
spectroscopic techniques. All synthetic compounds were new except derivative 1, which is
previously reported [28, 29].
4

Scheme-1: Synthesis of sulfonohydrazide substituted indazole derivatives 1-19
Structure elucidation of the most active compound (6)
1H- and 13C-NMR
1H-NMR of compound 6 was taken in deuterated CDCl3 at 300 MHz. The most downfield and
characteristic proton H-3 of indazole ring was appeared as a singlet at δ 8.30. The molecule also
possesses a five aromatic protons, in which protons H-2′, H-3′, H-5′, H-6′ were resonated at δ 7.49-
7.46 as an overlapped multiplet, respectively. While the proton H-4′ was also appeared as a
multiplate at δ 7.39-7.35. Protons related to the indazole ring H-5 and H-7 were appeared at δ 2.72,
and 2.67, as a singlet, respectively. Two methyl protons were appeared as a singlet at δ 1.11
(Figure-3A).
13C-NMR broad-band decoupled spectrum of compound 6 showed total fourteen carbon signals.
Six signals observed for quaternary carbons including C-4, C-6, C-8, C-9, C-1ʹ, and CF3, four
signals corresponds to six methine carbons CH-3, CH-2′/6′, CH-3′/5′, and CH-4ʹ, two signals for
methylene carbons CH2-5 and CH2-7, and one signal for two methyl carbon 2CH3-6 appeared, as
designated in figure-3B
5

Figure-3: 1H- & 13C-NMR chemical shifts of compound 6
To know about the stereochemical arrangement of imine bond. NOESY (nuclear Overhauser
enhancement spectroscopy) was recorded for compound 6. On the basis of NOESY interactions
compounds were identified as (E)-stereoisomer (Figure-4).
Figure-4: NOESY interactions
In vitro biological screening
All synthetic hybrid sulfonohydrazide substituted indazoles 1-19 were assessed for their α-amylase
inhibitory, DPPH and ABTS radical scavenging activities. Table-1 illustrated that all molecules
showed good to moderate α-amylase inhibitory activities with IC50 values ranging from 1.23 ±
0.06 µM to 4.5 ± 0.031 µM, when compared to standard acarbose (IC50 = 1.20 ± 0.09 µM). All
6

compounds displayed good DPPH and ABTS radical scavenging abilities with IC50 values 2.01 ±
0.13 - 5.3 ± 0.11 µM and 2.34 ± 0.07 - 5.5 ± 0.07 µM, respectively, when compared to the standard
ascorbic acid (IC50 = 1.99 ± 0.09 µM and IC50 = 2.03 ± 0.11 µM).
Table-1: In vitro α-amylase inhibitory and radical (DPPH and ABTS) scavenging activities of
derivatives 1-19
α-Amylase
inhibitory
activity
IC50 ± SEMa
DPPH radical
scavenging
activity
IC50 ± SEMa
ABTS radical
scavenging
activity
IC50 ± SEMa
Compounds
Structures
4.5 ± 0.03
5.3 ± 0.11
5.5 ± 0.07
1
Category “A”
3.91 ± 0.05
4.78 ± 0.09
4.99 ± 0.19
2
3.52 ± 0.03
4.23 ± 0.12
4.54 ± 0.09
3
7

2.92 ± 0.08
3.99 ± 0.89
4.04 ± 0.01
4
1.99 ± 0.12
2.77 ± 0.12
2.87 ± 0.02
5
1.23 ± 0.06
2.01 ± 0.13
2.34 ± 0.07
6
Category “B”
3.97 ± 0.08
4.82 ± 0.12
4.93 ± 0.04
7
8

3.72 ± 0.11
4.67 ± 0.11
4.79 ± 0.21
8
3.63 ± 0.12
4.63 ± 0.08
4.82 ± 0.21
9
3.83 ± 0.11
4.96 ± 0.07
4.99 ± 0.21
10
9

3.41 ± 0.16
4.47 ± 0.07
4.62 ± 0.12
11
1.66 ± 0.08
2.65 ± 0.09
2.87 ± 0.32
12
1.37 ± 0.03
2.56 ± 0.08
2.78 ± 0.08
13
10

1.93 ± 0.09
2.78 ± 0.07
2.96 ± 0.13
14
1.38 ± 0.11
2.43 ± 0.15
2.56 ± 0.11
15
1.91 ± 0.11
2.65 ± 0.09
2.76 ± 0.06
16
1.52 ± 0.09
2.56 ± 0.11
2.72 ± 0.12
17
11

2.51 ± 0.09
3.89 ± 0.05
3.93 ± 0.14
18
1.97 ± 0.03
2.91 ± 0.13
2.98 ± 0.07
19
Acarboseb
1.20 ± 0.09
-
-
-
Standards
Ascorbic acidc
1.99 ± 0.09
2.03 ± 0.11
SEMa (Standard error mean), Acarboseb (standard inhibitor for α-amylase inhibitory activity), Ascorbic acidc
(Standard for DPPH and ABTS radical scavenging activity).
Structure-activity relationship (SAR) for α-amylase enzyme inhibition
Mostly electronic and steric effects are responsible for variation in enzyme inhibitory activities of
the compounds 1-19. Structure-activity relationship of this class was rationalized by considering
that obtained inhibitory activity is resultant of common structure features and different substituent
present at the sulfonylhydrazide. For the sake of convenience, this series of compounds were
divided in two categories i.e. category “A” (where R is an alkyl group) and category “B” (where
R is an aryl group). However, trivial disparities in the α-amylase inhibitory activities within a
specific category could be attributed by the diverse substitution pattern on sulfonylhydrazide
moiety (Table-1).
12

Parent compound 1 (IC50 = 4.5 ± 0.03 µM) having ketone functionality directly attached to
indazole nucleus was identified as least active compound among the series as compared to the
standard acarbose (IC50 = 1.20 ± 0.09 µM). Category “A” contains compounds 2-6 (IC50 = 1.23 ±
0.06 - 3.91 ± 0.05 µM) having alkyl substitutions at sulfonohydrazide group. Amongst this
category, compound 6 (IC50 = 1.23 ± 0.06 µM) with trifluoromethyl group attached to sulfone
moiety of indazole was found to be significantly potent. Comparing the inhibitory activity of
compound 6 with parent compound 1 (IC50 = 4.5 ± 0.06 µM) showed increased activity which
suggests that addition of a substituent on sulfonohydrazide moiety play significant role in the
inhibitory potential. Whereas, comparing compound 6 with 2 (IC50 = 3.91 ± 0.05 µM) lacking
fluoro groups showed decreased activity, suggested that fluoro groups are itself taking significant
part in the enzyme inhibition. Compound 5 (IC50 = 1.99 ± 0.12 µM) with isopropyl group showed
good inhibition against α-amylase enzyme as compared to compound 4 (IC50 = 2.92 ± 0.08 µM)
having n-butyl chain. Compounds 3 (IC50 = 3.52 ± 0.03 µM) having ethyl substitution exhibited
nearly same inhibitory activity as and 2 (IC50 = 3.91 ± 0.05 µM) (Figure-5).
Figure-5: α-Amylase inhibitory activity of compounds 2-6
13

Analogs 7-19 (IC50 = 1.37 ± 0.03 - 3.97 ± 0.08 µM) include in category “B”, having phenyl and
naphthyl rings with or without substitution. Compounds 7 (IC50 = 3.97 ± 0.08 µM), having phenyl
ring without substitution found to be least active among the whole series. While different alkyl
substitution on phenyl as in compounds 8 (IC50 = 3.72 ± 0.11 µM), and 9 (IC50 = 3.63 ± 0.12 µM),
displayed moderate activities which are comparable to each other. Compounds 10 (IC50 = 3.83 ±
0.11 µM) and 11 (IC50 = 3.41 ± 0.16 µM) have naphthyl ring instead of phenyl ring. Relatively
increased inhibitory activity of compound 11 might be due to the presence of the tertiary amine on
naphthyl ring (Figure-6).
Figure-6: α-Amylase inhibitory activity of compounds 7-11
Compounds 12-14 having nitro and combination of nitro with methyl substituents. Among them,
compound 13 (IC50 = 1.37 ± 0.03 µM) with p-nitro group was found to be the second most active
compound of the series. As the nitro group switched to ortho position, a slight decrease in
inhibition potential was observed in compound 12 (IC50 = 1.66 ± 0.08 µM) which suggests that
nitro at para position better interacts with active site of enzyme. In case of compound 14 (IC50 =
1.93 ± 0.09 µM) with methyl and nitro groups para to each other, a slight decreased inhibition was
observed. Concisely, groups at ortho and meta positions are not taking part as actively as para
position (Figure-7).
14

Figure-7: α-Amylase inhibitory activity of compounds 12-13
Dichlorinated derivatives 15 (IC50 = 1.38 ± 0.11 µM) and 16 (IC50 = 1.91 ± 0.11 µM) have almost
similar structures. Compound 15 having one of the chloro groups at para was found to be better
active than compound 16 with one of the chloro at meta position. Which clearly suggests that para
position is also playing some important role in the inhibitory potential. Compound 17 (IC50 = 1.52
± 0.09 µM) having both chlorine moieties at meta positions in combination of hydroxy at ortho
position showed better activity than compound 16. When single chlorine atom was present at meta
in combination with methoxy at ortho position as in molecule 18 (IC50 = 2.51 ± 0.09 µM) showed
decreased activity as compared to their dichloro counter parts which revealed that combination of
methoxy with single chlorine is not really effective for inhibitory activity. Compound 19 (IC50 =
1.97 ± 0.03 µM) having solely a methoxy group at para positon showed inhibitory activity
comparable to other analogs (Figure-8).
15

Figure-8: α-Amylase inhibitory activity of compounds 15-19
Precisely, SAR revealed that in comparison to indazole, sulfonohydrazide substituted indazole
derivatives were far better active. Whereas amongst sulfonohydrazide substituted indazole
derivatives, the varying groups do not any substantial effect on the inhibition and the slight
disparity in the IC50 values might be due to the nature and positions of the substitutions. In fact,
merely SAR investigation is just given an insight related to the influence of diverse groups. To
find out the inhibition mechanism as well as type of interactions between compounds and active
site of enzyme, kinetic studies as well as in silico study were also performed.
Structure-activity relationship (SAR) for DPPH and ABTS radical scavenging activities
Limited SAR point out the influence of various pharmacophore present in a molecule. However,
substitution pattern on phenyl ring deduced the variable antioxidant activities (Figure-9).
16

DPPH & ABTS Radical Scavenging Activities
6
5
4
3
2
1
0
DPPH
ABTS
Compounds
Figure-9: Comparison of radical scavenging activities (DPPH & ABTS) of compounds.
Parent compound 1 showed DPPH (IC50 = 5.3 ± 0.11 µM) and ABTS (IC50 = 5.5 ± 0.07 µM)
radical scavenging activities, respectively, and found to be least radical scavenger in the series as
compared to the standard ascorbic acid (DPPH; IC50 = 1.99 ± 0.09 µM and ABTS; IC50 = 2.03 ±
0.11 µM).
Category “A” contains various alkyl chain substituted derivatives, showed significant to moderate
DPPH (IC50 = 2.01 ± 0.13 - 4.32 ± 0.17 µM) and ABTS scavenging activities (IC50 = 2.34 ± 0.07
- 4.99 ± 0.19 µM), respectively. Compound 6 bearing trifluoromethyl group displayed highest
degree of DPPH (IC50 = 2.01 ± 0.13 µM) and ABTS scavenging activities (IC50 = 2.34 ± 0.07 µM),
respectively, among the series. Compounds 5 (DPPH IC50 = 2.77 ± 0.12 µM; ABTS IC50 = 2.87 ±
0.02 µM), 12 (DPPH IC50 = 2.65 ± 0.09 µM; ABTS IC50 = 2.87 ± 0.32 µM), 13 (DPPH IC50 =
2.56 ± 0.08 µM; ABTS IC50 = 2.78 ± 0.08 µM), 14 (DPPH IC50 = 2.78 ± 0.07 µM; ABTS IC50 =
2.96 ± 0.13 µM), 15 (DPPH IC50 = 2.43 ± 0.15 µM; ABTS IC50 = 2.56 ± 0.11 µM), 16 (DPPH
IC50 = 2.65 ± 0.09 µM; ABTS IC50 = 2.76 ± 0.06 µM), 17 (DPPH IC50 = 2.56 ± 0.11 µM; ABTS
IC50 = 2.72 ± 0.12 µM), and 19 (DPPH IC50 = 2.91 ± 0.13 µM; ABTS IC50 = 2.98 ± 0.07 µM) also
showed better ability to scavenged radicals with respect to their α-amylase inhibitory activities.
Kinetic studies for α-amylase inhibition
Kinetic studies on the most active compounds 6, and 12-17 were performed to investigate the mode
of inhibition. The enzyme inhibitory mechanism was scrutinized through graph fitting study via
Sigma-Plot enzyme kinetic software. With the help of several kinetics plots i.e. Lineweaver-Burk
17

(Figure-10A), Hill (Figure-10B), Hanes-Woolf (Figure-10C), Eadie-Hofstee (Figure-10D), Dixon
(Figure-10E), and Scatchard models (Figure-10F), the kinetic parameters like Vmax, Km, AICc
and R2 values were also calculated (Table-2). On the basis of curve fitting model and low AICc
value, the kinetic data of the selected compounds revealed that they are mixed type inhibitors for
α-amylase enzyme.
Table-2: Kinetic characterization of active compounds 6, and 12-17
Vmax
(µM/min)1
182.5
Km
(mM)
3.4
3.8
3.6
3.7
3.5
3.4
3.5
Compounds
AICc
R2
Type of inhibition
102.8
105.1
100.3
109.3
115.2
105.2
103.1
0.984
0.908
0.902
0.906
0.912
0.912
0.923
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
6
169.7
165.8
173.3
162.4
168.8
157.2
12
13
14
15
16
17
Figure-10A: Lineweaver-Burk plot of 1/[S] Vs 1/Rate with different concentration
18

Figure-10B: Hill plot, different concentration of [S] Vs Rate (Vmax-Rate) with different
concentration of inhibitor.
19

Figure-10C: Hanes-Woolf plot of compound 6 at various concentration of [S] Vs [S]/Rate in the
different concentration of inhibitor.
20

Figure-10D: Eadie-Hofstee plot of compound 6 by rate/[S] Vs Rate in the different
concentration of mixed-type inhibitor.
21

Figure-10E: Dixon plot of compound 6 at different concentration of mixed inhibitor Vs 1/Rate.
22

Figure-10F: Scatchard plot of compound 6 by Rate Vs Rate/[S] in the different concentration of
mixed-type inhibitor.
Docking studies
The blind docking (BL) approach was performed in order to discover the more possibilities for
binding sites due to mixed inhibition nature of compounds against α-amylase enzyme. The mixed
inhibition is a type in which the inhibitor may bind to the enzyme whether or not the enzyme has
already bound the substrate but has a greater affinity for one state or the other. The results of BL
revealed that all compounds bind to a distinct site with various affinity other than the substrate
binding site as shown in Figure-11A. Moreover, on the basis of visualization, the binding of mixed
inhibitors eventually get vital conformation changes within the substrate binding site, and further
rescue the enzyme to produce the product. Commonly, the active site of α-amylase enzyme
includes Trp58-59, Tyr151, Arg195, Leu162, Thr163, Asp197-198, 300, Lys200-201, and Glu233
(Figure-11B). Afterward, the blind docking showed that the most active derivatives usually
adopted side chain and backbone donor/acceptor hydrogen interaction with residue Arg267,
Arg319, Asp433, and Asn431, respectively. In case of most active derivative 6 in the series adopt
23

backbone donor and acceptor hydrogen interaction with residue Arg267 and Asn431, while side
chain donor with Asn481and Arg319 (Figure-11C). Based on visualization, we observed that the
binding of this compound, produce minor changes in the conformation of substrate binding site
and hence might unable the enzyme to produce the product. More fascinatingly, the most active
compound in the series 13 and 15, exposed same binding mode with residues Arg319 and Asn481
(Figure-11D-E). The only difference found not only the sidechain donor and acceptors interaction
adopted by residue Arg319 (common residue), but also the compound 13 showed one additional
interaction with the negatively charged sidechain residue Asp433. In case of other potent
derivatives 17 and 12 also showed same interaction with the residues found common in
comparison was sidechain negatively/positively charged Arg319 and Asp433 (Figure-11F-G).
Overall, the BL and enzyme kinetics study results demonstrates the nature of compounds were
mixed type inhibition. The binding of these active derivatives to the distinct site other than the
substrate binding site of α-amylase enzyme fetch an energetic conformational sidechain 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.
Figure-11: The ligand-protein interaction profile for mixed type inhibitors against α-amylase
enzyme. (11A) The surface representation of α-amylase enzyme (PDB ID 3BAJ), the magenta
24

color line shows the distinict binding site (allosteric site). (11B) while the blue color expresses the
substrate binding site. (11C) The binding mode of the most potent compound 6, (11D) compound
13, (11E) compound 15, (11F) compound 17, and (11G) compound 12.
Table 3: Interaction Detail of all compounds 1-19 against α₋ amylase enzyme.
Interactions details
Docking
Score (S)
E
(kcal/mol)
-1.0
-0.7
-3.6
-2.8
-4.7
-2.2
-3.6
-0.5
-4.5
-3.3
-0.6
-0.7
-4.5
-0.5
-7.6
-1.3
-3.7
-0.7
-1.4
-0.9
-0.2
-3.8
-9.7
-3.8
-4.1
-4.9
-0.8
-2.5
-1.1
-1
Compounds
Ligand
Receptor
Interactions Distance
O
12 OE1 GLU 484
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-donor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-acceptor
H-donor
3.15
3.33
3.15
2.93
3.14
3.07
3.17
3.3
3.03
3.09
3.34
3.58
2.91
3.75
2.95
3.16
3.04
3.37
3.46
3.1
3.30
3.57
3.03
3.28
3.18
2.99
3.39
3.15
3.06
3.68
3.19
3.58
3.59
3.2
-4.245
-5.036
-4.897
-5.210
-5.060
1
2
3
4
5
N
9
NH2 ARG 392
O
O
O
O
O
O
O
O
16 NH2 ARG 319
17 NH1 ARG 319
16 NH1 ARG 267
17 NH1 ARG 267
16 NH1 ARG 267
16 NE1 TRP 269
16 NH1 ARG 267
17 NE1 TRP 269
NH1 14
O
CA
ASN 431
ARG 267
N
8
-7.5954
6
O
16 NH1 ARG 319
17 ND2 ASN 481
O
N
O
O
O
O
O
O
O
O
N
N
O
O
O
O
13 NZ
16 CE
16 NZ
16 CA
16 CE
17 CE
17 OE1 GLU 484
16 NZ
17 NZ
14 NH1 ARG 267
14 OD1 ASP 433
16 NH1 ARG 319
30 OG
17 NE1 TRP 269
32 NH1 ARG 267
LYS 268
LYS 268
LYS 268
ARG 267
LYS 268
LYS 268
-4.236
-4.325
7
8
-5.321
9
-4.921
-5.214
-5.854
10
11
12
LYS 268
LYS 268
-7.267
13
SER 481
-4.581
-6.542
-5.254
14
15
16
N
O
N
8
OG
17 NH1 ARG 319
NZ LYS 268
SER 481
-1.1
0
8
O
N
O
O
N
C
O
17 NH1 ARG 267
14 OD1 ASP 433
16 NH1 ARG 319
17 NH1 ARG 267
14 OD1 ASP 433
24 OD1 ASP 433
16 NH1 ARG 319
-0.5
-4.4
-4.8
-1.7
-2.1
-0.5
-5
-6.158
-5.109
17
18
H-acceptor
H-acceptor
H-donor
2.97
2.98
3
-5.556
H-donor
H-acceptor
3.43
2.86
19
25

Conclusion
Synthetic indazoles 1-19 were tested for their in vitro α-amylase enzyme inhibition and radical
scavenging (DPPH and ABTS) activities. Limited structure-activity relationship showed that
synthetic molecules with halogens substituted have good potential for inhibition than other
substituents i.e. OH, NO2, and MeO. Almost all compounds exhibited good inhibition potential as
compared to the standard drug (acarbose). It is worth-mentioning that these compounds are also
found to be good radical scavenger in DPPH and ABTS assays. Compounds 6 found to be leading
potent α-amylase enzyme inhibitor and radical scavenger (DPPH and ABTS). Synthetic analogs
5, and 12-19 showed promising activities as well in α-amylase enzyme inhibition and radical
scavenging (DPPH and ABTS) assays. In silico molecular docking was performed to identify the
enzymatic binding interactions with the analogs. Mode of enzyme inhibition revealed by kinetic
study that demonstrated that all compounds were mixed type α-amylase inhibitors.
Current study has identified a series of lead molecules in order to conduct future research for
development of an efficient inhibitor for α-amylase for the development of insulin-independent
antidiabetic agents. These would also be used as a potential antioxidant agent to cure many
complications associated with oxidative stress in diabetic patients
Experimental
Materials and Methods
High purity chemicals were purchased from Sigma/Aldrich, USA. Bruker 300, and 400 MHz 1H-
and 13C-NMR spectrometers were used to conduct NMR experiments. Mass spectra were recorded
on JEOL JMS-600H and Finnigan MAT-311A mass spectrometers. ESI experiment were
conducted on Bruker Daltonics maxis II, and amazon speed Bruker daltonics (Germany) mass
spectrometers. Thin layer chromatography was performed on per-coated silica gel aluminium
plates (GF-25, Merck, Germany). Ultraviolet light of 254 and 365 nm wavelengths were used to
visualized the chromatograms. Melting points were noted on Stuart® SMP10 apparatus.
General procedure for the synthesis of 4-oxyindazole (1)
Dimedone (1 mmol), dimethylformaidedimethylacetal (DMF-DMA) (1 mmol), phenyl hydrazine
(1 mmol), and CuCl2.2H2O (0.5 mmol) were added in ethanol (10 mL) in the 50 mL round-
26

˚
bottomed flask at 70 C for 3 h. Reaction progress was examined by TLC. Precipitate formed,
which were collected and washed with hexane to get products and crystallized from ethanol [28].
6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-one (1)
Yield: 65%, M.P.: 119-121 ºC; Rf: 0.3 (ethyl acetate/hexanes, 3:7); 1H-NMR (400 MHz, DMSO-
d6): δH 8.04 (s, 1H, CH), 7.61-7.55 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.49 (m, 1H, H-4′), 2.91 (s, 2H,
H-5), 2.35 (s, 2H, H-7), 1.01 (s, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): C 144 (C=N), 142
(C=O), 139(C‒N), 137 (N-C=C), 129 (C-3′, C-5′), 127 (C-4′), 123 (C-2′, C-6′), 117 (N-C=C), 38
(C-7), 36 (C-5), 33 (C-6), 28 (CH3, CH3); EI-MS m/z (% rel abund.): 240 (M+, 98), 225 (11), 197
(3), 148 (100), 156 (12), 77 (51).
General procedure for the synthesis of sulfonylhydrazide derivatives
In a 50 mL round-bottomed flask, different substituted sulfonyl chloride (1 mmol) were treated
with hydrazine hydrate (5 mL) in ethanol (10 mL) in the presence of few drops of pyridine at 70
˚C for 2 h. Reaction progress was checked by TLC periodically. The pure products was obtained
via collection of the precipitate by filtration, washing with hexane and water to remove impurities.
Extra pure products were obtained by crystallization from ethanol.
General procedure for the synthesis of sulfonohydrazide substituted indazoles (2-19)
4-Oxyindazole 1 (1 mmol) and substituted sulfonyl hydrazide (1 mmol), ethanol (1mL) with 4-5
drops of glacial acetic acid were added into a 100 mL reaction flask and refluxed for 2 h. Reaction
was monitored occasionally by TLC. Precipitates were appeared, washed with hexane and
crystallized from ethanol to obtained pure products.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-
ylidene)methanesulfonohydrazide (2)
Yield: 76%, M.P.: 209-212 ºC; Rf: 0.5 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, DMSO-
d6): δH 9.76 (s, 1H, NH), 7.94 (s, 1H, CH), 7.55-7.53 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.44-7.41
(m, 1H, H-4′), 3.02 (s, 3H, S-CH3), 2.76 (s, 2H, H-6), 2.41 (s, 2H, H-7), 0.98 (s, 6H, 2-CH3); 13C-
NMR (100 MHz, DMSO-d6): C 146.5( C=N Iminic), 144.0 (C=N), 139.7 (C‒N), 134.9 (N-C=C),
129.3 (C-3′, C-5′), 125.1 (C-4′), 124.9 (C-2′, C-6′), 114.6 (N-C=C), 44.0 (C-7), 42.9 (C-S), 31.6
27

(C-5), 30.5 (C-6), 28.5 (CH3, CH3); FAB (Pos.) MS m/z = 333 [M-H]+1; HRFAB-MS Calcd for
C16H21O2N4S: m/z = 333.1385, Found 333.1382.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-
ylidene)ethanesulfonohydrazide (3)
Yield: 71%, M.P.: 265-268 ºC; Rf: 0.21 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, CDCl3):
δH 8.31 (s, 1H, CH), 7.51-7.46 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.40-7.37 (m, 1H, H-4′), 3.38-3.31
(m, 2H, CH2-S), 2.73 (s, 2H, H-6), 2.66 (s, 2H, H-7), 1.41 (m, 3H, CH2-CH3), 1.12 (s, 6H, 2-CH3);
13C-NMR (100 MHz, CDCl3): C 146.5 (C=N Iminic), 144.0 (C=N), 139.7 (C‒N), 134.9 (N-C=C),
129.3 (C-3′, C-5′), 125.1 (C-4′), 124.9 (C-2′, C-6′), 114.6 (N-C=C), 50.9 (C-S), 44.0 (C-7), 31.6
(C-5), 30.5 (C-6), 28.5 (CH3, CH3), 20.6 (CH3); FAB (Pos.) MS m/z = 347 [M-H]+1; HRFAB-MS
Calcd for C17H23O2N4S: m/z = 347.1542, Found 347.1559.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-ylidene)butane-1-
sulfonohydrazide (4)
Yield: 67%, M.P.: 275-277 ºC; Rf: 0.44 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, CDCl3):
δH 8.30 (s, 1H, CH), 7.51-7.46 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.40-7.35 (m, 1H, H-4′), 3.46 (m,
2H, CH2-S), 2.82 (s, 2H, H-6), 2.75-2.72 (m, 4H, CH2-CH2), 2.66 (s, 2H, H-7), 1.12 (s, 6H, 2-
CH3), 1.08 (s, 3H, CH2-CH3);13C-NMR (100 MHz, CDCl3): C 146.5 (C=N Iminic), 144.0 (C=N),
139.7 (C‒N), 134.9 (C‒C=C), 129.3 (C-3′, C-5′), 125.1 (C-4′), 124.9 (C-2′, C-6′), 114.6 (C‒C=C),
57.7 (C-S), 44.0 (C-7), 31.6 (C-5), 30.5 (C-6), 28.5 (CH3, CH3), 21.5 (CH2-CH2-CH3), 21.0 (CH2-
CH2-CH3), 16.8 (CH2-CH3); ESI (Neg.) MS m/z = 373 [M-H]-1; HRESI-MS Calcd for
C19H25O2N4S: m/z = 373.1704, Found 373.1702.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-ylidene)propane-2-
sulfonohydrazide (5)
Yield: 82%, M.P.: 285-287 ºC; Rf: 0.32 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, CDCl3):
δH 8.31 (s, 1H, CH), 7.50-7.46 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.40-7.35 (m, 1H, H-4′), 2.77 (m,
1H, CH-S), 2.73 (s, 2H, H-6), 2.67 (s, 2H, H-7), 1.12 (s, 6H, 2-CH3), 1.08 (s, 6H, 2CH-CH3); 13C-
NMR (100 MHz, CDCl3): C 146.5 (C=N Iminic), 144.0 (C=N), 139.7 (C-N), 134.9 (N-C=C),
129.3 (C-3′, C-5′), 125.1 (C-4′), 124.9 (C-2′, C-6′), 114.6 (N-C=C), 60.0 (C-S), 44.0 (C-7), 31.6
28

(C-5), 30.5 (C-6), 28.5 (CH3, H3), 15.8 (CH-2CH3); ESI (Neg.) MS m/z = 359 [M-H]-1; HRESI-
MS Calcd for C18H23O2N4S: m/z = 359.1547, Found 359.1555.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-ylidene)-1,1,1-
trifluoromethanesulfonohydrazide (6)
Yield: 79%, M.P.: 263-265 ºC; Rf: 0.38 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, CDCl3):
δH 8.30 (s, 1H, CH), 7.49-7.46 (m, 4H, H-2′, H-3′, H-5′, H-6′), 7.39-7.35 (m, 1H, H-4′), 2.72 (s,
13
2H, H-6), 2.67 (s, 2H, H-7), 1.11 (s, 6H, 2-CH3); C-NMR (100 MHz, CDCl3): C 159.5(CF3),
146.5 (C=N iminic), 144.0 (C=N), 139.7 (C-N), 134.9 (N-C=C), 129.3 (C-3′, C-5′), 125.1 (C-4′),
124.9 (C-2′, C-6′), 114.6 (N-C=C), 44.0 (C-7), 31.6 (C-5), 30.5 (C-6), 28.5 (CH3, CH3); ESI (Neg.)
MS m/z = 385 [M-H]-1; HRESI-MS Calcd for C16H16F3O2N4S: m/z = 385.0952, Found 385.0952.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-
ylidene)benzenesulfonohydrazide (7)
Yield: 85%, M.P.: 217-219 ºC; Rf: 0.29 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, CDCl3):
δH 8.01 (d, J2′′,6′′ = 6.9 Hz 2H, H-2′′, H-6′′), 7.93 (s, 1H, CH), 7.58 (t, J3′′,5′′ = 6.9 Hz 2H, H-3′′, H-
5′′), 7.54 (m, 1H, H-4′′), 7.49 (d, J2′,6′ = 9.3 Hz 2H, H-2′, H-6′), 7.43-7.35 (m, 3H, H-3′, H-4′, H-
13
5′), 2.64 (s, 2H, H-6), 2.30 (s, 2H, H-7), 1.01 (s, 6H, 2-CH3); C-NMR (100 MHz, CDCl3): C
146.5 (C=N iminic), 144.0 (C=N), 139.7 (C-N), 138.9 (C-S) 134.9 (N-C=C), 131.9 (C-4′′) 129.3
(C-3′, C-5′), 128.9 (C-2′′, C-6′′), 127.3 (C-3′′, C-5′′), 125.1 (C-4′), 124.9 (C-2′, C-6′), 114.6 (N-
C=C), 44.0 (C-7), 31.6 (C-5), 30.5 (C-6), 28.5 (CH3, CH3); EI-MS m/z = 394 (M+, 16), 253 (100),
225 (68), 209 (67), 182 (21), 77 (37); HREI-MS Calcd for C21H22O2N4S: m/z = 394.1463, Found
394.1469.
(E)-N'-(6,6-Dimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-ylidene)-4-
methylbenzenesulfonohydrazide (8)
Yield: 68%, M.P.: 288-291 ºC; Rf: 0.47 (ethyl acetate/hexanes, 2:8); 1H-NMR (300 MHz, DMSO-
d6): δH 10.19 (s, 1H, NH), 8.26 (s, 1H, CH), 7.81 (d, J2′′/3′′,6′′/5′′ = 9 Hz 2H, H-2′′, H-6′′), 7.56-7.50
(m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′), 7.40 (d, J3′′/2′′,5′′/6′′ = 9 Hz 2H, H-3′′, H-5′′), 2.70 (s, 2H, H-5),
2.36 (s, 3H, Ph-CH3), 2.23 (s, 2H, H-7), 0.93 (s, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): C
146.5 (C=N iminic), 144.0 (C=N), 143.0 (C-S), 139.7 (C-N), 138.6 (C-4′′), 134.9 (N-C=C), 129.3
29