Synthesis, characterization, in silico studies and in vitro biological evaluation of isoniazid-hydrazone complexes

Article abstract of DOI:10.1016/j.molstruc.2020.128297

Isoniazid-hydrazone complexes (IHCs) were synthesized and thoroughly characterized by FT-IR, ¹H NMR and ¹³C NMR spectroscopic techniques. The anti-inflammatory and antidiabetic activities were tested for the IHCs using protein denatu

Full text of DOI:10.1016/j.molstruc.2020.128297

Journal Pre-proof
Synthesis, characterization, in silico studies and in vitro biological evaluation of
isoniazid-hydrazone complexes
M.P. Ramya Rajan, Rathikha Ramaswamy, Nithyabalaji Rajendran, Sribalan
Rajendran
PII:
S0022-2860(20)30622-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.128297
MOLSTR 128297
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 December 2019
Revised Date: 26 March 2020
Accepted Date: 17 April 2020
Please cite this article as: M.P.R. Rajan, R. Ramaswamy, N. Rajendran, S. Rajendran, Synthesis,
characterization, in silico studies and in vitro biological evaluation of isoniazid-hydrazone complexes,
Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128297.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

Table 1: DFT calculations of IHCs .
S.
Compoun
HOMO
(eV)
LUMO
(eV)
Band
Chemical
potential
Global
hardness
Global
softness
Electrophillicity
index
No d name
gap(∆E)
(eV)
(eV)
(eV-1)
0.3996
0.3647
0.4608
(eV)
(eV)
1.
2
3a
3b
3c
-5.7811
-6.0182
-5.5670
-3.2787
-3.2765
-3.3971
2.5024
2.7416
2.1698
-4.5299
-4.6474
-4.4820
1.2512
1.3708
1.0849
8.2003
7.8779
9.2581
3

Table 2: Molecular docking interaction of IHCs against α-amylase.
S.
No
Compound
Name
1HNY
Binding
energy
No. of hydrogen
bonding
Hydrogen bonded amino acid residue
(KJ/mol)
-294.42
1
3a
2
SER3, ARG421
SER3, TYR2
2
3
3b
3c
-315.83
-299.78
2
5
THR377, TRP388, ARG389,
GLN390,LYS322

•
•
•
•
•
The synthesis of IHCs is economically cheaper and simple method.
The IHCs proved as anti-inflammatory and antidiabetic agents at in vitro level.
Hydrazone-salicylate complex forms 5 hydrogen bonding interaction with 1HNY.
Electrophilicity index of hydrazone-salicylate complex is higher than others.
FMO studies showed the electron cloud localized in the benzoate and pyridinium.

Author contributions
Mrs. Ramya Rajan M.P.
: Synthesis and characterization of IHCs and Molecular docking.
: Research work design and scientific discussions.
: DFT studies.
Dr. Ramaswamy Rathikha
Mrs. Rajendran Nithyabalaji
Dr. Rajendran Sribalan
: Testing and analysis (in vitro biological studies).

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

Synthesis, characterization,insilicostudies and invitro biological evaluation of
Isoniazid-hydrazone complexes
RamyaRajanM.P,a*RamaswamyRathikha,bRajendranNithyabalaji,aRajendranSribalanc
a
Department of Physics, SRM Valliammai Engineering College, Kattankulathur, Chengalpet Dt., Tamil Nadu, India
bDepartment of Physics,Presidency College, Chennai, Tamil Nadu, India
c
Biochemie Innovations Lab, Tindivanam, Tamil Nadu, India.
*Corresponding author e-mail:ramyarajanmp@gmail.com
Abstract
Isoniazid-hydrazonecomplexes (IHCs)were synthesized and thoroughly characterized by
1
13
FT-IR, H NMR and C NMRspectroscopic techniques. The anti-inflammatory and antidiabetic
activities were tested for the IHCs using protein denaturation methods and the α-amylase
inhibitory method. The molecular modeling studies for IHCswere investigated and their
molecular parameters were calculated using Density Functional Theory methods. The IHCs
showed lower band gap and higher electrophilicity index values. Further, molecular docking
studies were investigated against the α-amylase enzyme to compare the experimental results.
The isoniazidhydrazone-salicylic acid complex (3c) showed five hydrogen bonding interactions
with aminoacid residues of the α-amylase enzyme (1HNY).
Keywords: Isoniazid, Anti-inflammatory, Antidiabetic, Molecular docking, DFT studies.
1. Introduction
Isoniazid is primarily used anti-tuberculosis drug [1] as well as antimicrobial drug, which
is an isomer of nicotinic hydrazide [2].The side effect of isoniazid is the elevation of liver
enzymes level in blood and hepatic toxicity appears as cellular necrosis, steatosis [3].
Metabolites of this drug also have toxic effects on liver cells [4].Hydrazine is one of the most

important metabolites of isoniazid [5].The derivatives of isoniazid may overcome these
problems.The hydrazones are simple derivatives of hydrazines. Moreover, hydrazones possess a
wide range of pharmacological activities such as antimicrobial [6], anticonvulsant [7],analgesics
[8], anti-inflammatory [9], antitubercular [10] and antitumor [11] activities. For instance,
isoniazid hydrazones are antitubercular agents [12] and alsothe incorporation of hydrazoneunit in
isoniazid maintaining antimycobacterial activity. Therefore hydrazones of isoniazid, retaining its
activity, avoid toxicity and it showed better effectiveness than isoniazid [13].
The combinations of drug molecules can help to improve therapeutic efficiency than
single agents[14]. Nowadays the drug combination therapy is a promising strategy for multiple
complex diseases [15]. Some of thesecombinations were found to be impressive because which
is reducing the toxicity and side effects. For example, both glyburide and metformin are used for
the treatment of type 2 diabetes. The glyburide reduces insulin resistance and metformin
increases insulin secretion respectively. In this combination of two drug molecules, therapeutic
efficacy improves due to their complementary mechanism [16-17]. The physical and chemical
properties of complexes are completely varied from the parent drugs. It is an important process
in drug discovery research because the complexation improves the solubility and stability of the
drug[18]. For example, the complex of theophylline with ethylenediamine (aminophylline) has
very good stability[19]. Similarly, the cyclodextrincomplexation process was used to improve
the stabilityin many drug developments[20].
Based on the above knowledge, the isoniazid hydrazone is focused onthe synthesis and
characterized. The isoniazid-hydrazones (IHCs) are planning for complexation with three
bioactive compounds like benzoic acid, aspirin and salicylic acid (Fig. 1). The complete

invitroexperimental and insilico studies were performed to investigate the biological importance
of IHCs.
2. Experimental section
2.1 Materials and methods
The analytical grade solvents were usedand purchasedfrom SpectrochemorSigma
Aldrich. Reactions were monitored by the TLC platewhich is on precoated silica gel 60 F254 in
TLC sheets (0.2mm thickness, Merck plate) and 60-120 mesh Merck silica gel used for column
1
chromatography. Petroleum ether and ethyl acetate wereused as the eluents. H and 13C NMR
spectra were recorded on Bruker 300 MHz and 75 MHz instruments, CDCl3and DMSO-d6were
used as an internal solvent. Chemical shift values were represented in δ (ppm) and coupling
constants are mentioned in terms of Hz with the internal reference TMS. ESI-MS spectra were
recorded in the LCQ fleet mass spectrometer. FT-IR spectra were recorded in Thermo Scientific
Nicolet iS50 FT-IR Spectrometer.
2.2 Synthetic procedure for (E)-N'-(4-chlorobenzylidene)isonicotinohydrazide (2)
The isonicotinic hydrazide (3.0 g, 21.89 mmol) and p-chlorobenzaldehye (3.06 g, 21.89)
were dissolved in ethanol (30 mL). To that solution glacial acetic acid (0.1 mL) was added. The
reaction mixture was refluxed for 3 hours at 80 °C. Then the reaction mixture was cooled to
room temperature. The obtained solid was filtered washed with cooled ethanol (5mL).
White solid.1H NMR (300 MHz, DMSO-d6+CDCl3) δ 8.73 (d, J = 5.4 Hz, 2H), 8.42 (s, 1H),
7.81 (d, J = 5.1 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 7.2 Hz, 2H).13C NMR (75 MHz,
CDCl3) δ 161.23, 148.96, 147.24, 139.40, 134.56, 131.51, 127.74, 127.63, 120.52.ESI-MS
calculated 259.05 found 260 [M+1] +.IR (KBr Disc) cm-1:3164.20, 1659.39, 1596.83, 1551.06,
816.46.

2.3 Preparation of 3a-c
The intermediate 2 (10 mmol) and corresponding carboxylic acid (10 mmol) was
dissolved in ethanol (10 mL) and heated for 10 min and cooled to room temperature. The
evaporation of the solvent yielded target compounds.
2.3.1(E)-4-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)pyridin-1-ium benzoate (3a)
White solid.1H NMR (300 MHz, DMSO-d6) δ 12.17 (s, 1H), 8.78(d, J = 4.8 Hz, 2H), 8.49 (bs,
1H), 7.94 (d, J = 7.2 Hz, 2H), 7.83-7.76 (m, 4H), 7.63 – 7.49 (m, 5H).13C NMR (75 MHz,
DMSO-d6) δ 167.86, 162.19, 150.83, 150.07, 148.18, 140.85, 135.37, 133.45, 133.34, 131.31,
129.79, 129.44, 129.05, 122.06.IR (KBr Disc) cm-1: 3027.86, 1738.39, 1661.95, 1560.18,
847.46.706.71.
2.3.2 (E)-4-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)pyridin-1-ium 2-acetoxybenzoate
(3b)
White solid.1H NMR (300 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.79 (d, J = 4.8 Hz, 2H), 8.44 (s,
1H), 7.92 (br s, 2H), 7.81-7.77 (m, 3H), 7.66-7.62 (m, 2H), 7.54 (br s, 1H), 7.40 – 7.36 (m, 1H),
7.21 (brs, 1H), 2.24 (s, 3H).13C NMR (75 MHz, DMSO-d6) δ 172.55, 169.71, 166.12, 162.19,
150.85, 148.18, 140.87, 135.37, 134.32, 133.48, 131.90, 129.42, 129.01, 126.58, 124.54, 124.29,
122.06, 21.60.IR (KBr Disc) cm-1:3025.83, 1748.96, 1662.10, 1604.57, 1293.55, 851.62, 752.78.
2.3.3(E)-4-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)pyridin-1-ium 2-hydroxybenzoate
(3c)

White solid.1H NMR (300 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.85 (d, J = 4.8 Hz, 2H), 8.52 (s,
1H), 7.89 (d, J = 5.1 Hz, 2H), 7.88-7.85 (m, 3H), 7.61-7.54 (d, J = 8.1 Hz, 3H), 7.01-6.95 (m,
13
2H). C NMR (75 MHz, DMSO-d6) δ 172.50, 162.20, 161.70, 150.81, 148.21, 140.92, 136.11,
135.39, 133.47, 130.80, 129.47, 129.01, 127.46, 122.10, 119.67, 117.61, 113.51. IR (KBr Disc)
cm-1: 3027.36, 1738.45, 1659.56, 1591.85, 1216.91, 849.56, 751.88.
2.4 Anti-inflammatory activity
2.4.1 Protein denaturation technique
The IHCs were tested for anti-inflammatory activity using the inhibition of albumin
denaturation technique followed by the literature reports [21-23].
2.5 Antidiabetic Activity
2.5.1α- amylase inhibitory activity
The α-amylase inhibitory activity was carried out by following the reported literature
[24].
2.6 Molecular docking study
The molecular docking studies were performed followed by the reported literature
method [25]. Molecular docking of compounds was carried out with crystal structures of 1HNY
and performed using the Hex 8.0 software. The three dimensional structure of IHCswas
constructed usingChemBio 3D ultra 13.0 software and then they were energetically minimized
using MMFF94 with the maximum number of iteration of 5000 andminimum RMS gradient of
0.10 [26]. The crystal structure of the protein was taken from Protein Data bank (www.rcsb.org)
and chain A was selected for docking studies. All bound water and ligand were eliminated from

the protein and polar hydrogen was added to the protein as it is required for the electrostatics and
then nonpolar hydrogen atoms were merged. The ligand was docked in the active site of 1HNY
[27]. The docking parameters were used as default in Hex 8.0.
2.7 Computational calculations
All the computational calculations including representation of Highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) in the checkpoint
files were performed with the Gaussian 09W program using density functional theory [28]. The
chemical structure of the IHCs was optimized with B3LYP/6.311 ++ G(d,p) basis set. The Gauss
view software package was used to visualize the computed structures including HOMO, LUMO
and Molecular electrostatic potential (MEP) representations.
3. Results and Discussions
3.1 Chemistry
The reaction of isoniazid with p-chlorobenzaldehyde at reflux condition yielded the
required starting material 2.The complexation of 2 with corresponding carboxylic acids yielded
various IHCs. The overall synthetic route for IHCs was represented in Scheme 1.
3.1.1 Characterization
3.1.1.1 Compound 3a
1
The H NMR clearly showed that the compound 3a contains 14 numbers of protons (the
pyridinium proton is a labile proton, which may not appear in the spectrum). The appearance
singlet at 12.17 ppm for one proton indicates the presence of a hydrazide NH unit. The sharp
singlet for one proton at 8.49 ppm has appeared for the imine CH unit. The doublet at 8.79 ppm
for 2 protons appeared for 2a & 6a protons of pyridinium unit. Similarly, the doublet at 7.94 ppm
for 2 protons appeared for 3a & 5a protons of pyridinium unit. The signals around 7.83-7.76 ppm
appeared for 13a & 17a protons of the p-chlorophenylene unit. Similarly, the signals around

7.63-7.49 ppm appeared for 14a & 16a protons of the p-chlorophenylene unit. Another protons
signals around 7.63-7.49 & 7.83-7.76 ppm appeared for the benzoate unit.
The 13C NMR showed that the compound 3a contains 14 sets of carbon signals. The
appearance of the carbon signal at 167.86 ppm indicates the presence of carboxylate carbon and
the peak appeared at 162.19 ppm indicates the presence of hydrazide carbon. The appearance of
a peak at 148.18 ppm has appeared for imine carbon. The carbon signal at 150.83 ppm has
appeared for 2a and 6a carbons for the pyridinium unit. Similarly, 3a and 5a carbons of
pyridinium unit appeared at 122.06 ppm respectively. The p-chlorophenylene carbons 13a & 17a
appeared at 129.05 and 14a & 16a carbons appeared at 129.44 ppm respectively. The benzoate
phenyl carbons like 20a-24a appeared in the region of 130.11 to 131.63 ppm correspondingly.
The FT-IR spectrum gave some additional evidence for compound formations. The
appearance of the medium absorption band at 3460 cm-1 indicates the presence of a hydrazide
NH unit. Similarly, the appearance of the medium absorption band at 3400 cm-1 indicates the
presence of the pyridinium NH unit. The stretching frequencies at 3029 and 2970 cm-1 indicate
the presence of aromatic rings which appeared for the stretching frequency of aromatic CH units.
The strong absorbance at 1738 cm-1indicates the presence of carboxylate and hydrazide carbonyl
units. The imine stretching frequency has appeared at 1661 cm-1.The stretching frequency for the
C=N unit of pyridiniumhas appeared at 1591 cm-1 and the stretching frequency of C=C for
aromatic rings appeared at 1560 cm-1. The stretching frequency around 1215 cm-1 indicates the
presence of the C-O unit which is present in the carboxylate of benzoate moiety. The C-Cl
stretching frequency has appeared at 707 cm-1.

3.1.1.2 Compound 3b
1
The compound 3b has 16 numbers of protons, the H NMR spectrum of 3b clearly
showed proton signals for 15 protons (the pyridiniumNH is a labile proton, which may not
appear in the spectrum). The appearance of a doublet at 8.79 ppm for 2 protons and the
appearance of a signal at 7.94 ppm for 2 protons indicate the presence of a pyridinium unit.
These signals have appeared for 2b, 6b, 3b and 5b protons of pyridinium unit correspondingly.
The singlets at 8.44 ppm and 12.15 ppm appeared for hydrazide and imine unit respectively. The
proton signals for 13a & 17a in the region of 7.81-7.77 ppm and 14a & 16a protons appeared at
7.66-7.62 ppm correspondingly which belongs to the p-chlorophenylene unit. The sharp singlet
for 3 protons at 2.24 ppm appeared for acetate CH3 unit. The signals around 7.49-7.31& 7.21
ppm appeared for aspirin phenylene protons.
Similarly, the compound 3b contains 18 sets of carbon signals, the 13C NMR spectrum
also showed 18 sets of carbon signals. The peak at 172.55 ppm appeared for the carboxylate unit
of aspirin. The carbon signal at 166.12 ppm appeared for acetate carbon of the aspirin unit. The
appearance of the carbon signal at 162.19 ppm indicates the presence of hydrazide which is
present in the pyridinium hydrazide unit. The carbon signal at 148.18 ppm appeared for the
imine unit. The appearance of carbon signals at 150.85 & 122.06 ppm indicates the presence of
pyridinium unit which belongs to 2b, 6b, 3b and 5b carbons. The appearance of carbon signals at
129.01 & 129.42 ppm indicates the presence of the p-chlorophenylene unit.
In the FT-IR spectrum, the medium absorption band at 3490 cm-1 indicates the presence
of hydrazide stretching frequency. Similarly, the appearance of stretching frequency at 3400 cm-1
indicates the presence of the pyridinium NH unit. The absorption band at 3075, 3026 and 2846
cm-1 is appeared for aromatic CH stretching vibrations. The absorption band at 1746 cm-1has

appeared for hydrazide carbonyl and carboxylate carbonyl units. The strong absorption band at
1660 cm-1has appeared for the C=N stretching vibration of imine moiety. The band at 1603 cm-1
and 1560 cm-1appeared for the C=NH unit of pyridinium and C=C unit of aromatic rings. The
band at 1293 and 1216 cm-1 appeared for C-O stretching vibrations of carboxylate units. The C-
Cl stretching vibration appeared at 751 cm-1 as a strong absorption band.
3.1.1.3 Compound 3c
1
The compound 3c contains 16 numbers of protons, the H NMR spectrum showed the
signals for 14 protons (the phenolic proton and pyridinium protons are labile protons which may
not appear in the spectrum). The singlets at 12.23 and 8.52 ppm indicate the presence of
hydrazide NH and imine CH units. The proton signal at 8.85 ppm for 2 protons appeared for 2c
& 6c protons of pyridinium unit. Similarly, the appearance of the proton signal at 7.89 ppm for 2
protons appeared for 3c & 5c protons of pyridinium unit. The appearance of protons signals at
7.84 & 7.61-7.54 ppm indicates the presence of p-chlorophenylene unit which belongs to
13c,17c,14c and 16c protons of the p-chlorophenylene moiety. The protons signals 7-01-6.95 &
7.61-7.54 ppm appeared for 23c & 22c protons of the salicylic acid unit.
13
Similarly, the compound 3c contains 16 sets of carbons, the C NMR spectrum showed
the 16 sets of carbon signals. The carbon signal at 172.50 ppm appeared for the carboxylate
carbon of thesalicylylunit. The carbon signal at 162.20 ppm appeared for the carbon of hydrazide
unit and the peak at 148.21 ppm appeared for imine carbon. The appearance of carbon signals at
150.81 & 122.10 ppm indicates the presence of a pyridinium unit. Similarly, the appearance of
carbon signals at 129.01 & 129.47 ppm appeared for the p-chlorophenylene unit
correspondingly. The carbon signals at 133.46 & 136.11 ppm appeared for 20c & 22c’s carbon

1
13
which is present in the salicylyl unit. The selected H and CNMR chemical shiftsof IHCs are
represented in Fig. 2.
In the FT-IR spectrum, the stretching frequency for hydrazide NH appeared at 3491 cm-1.
Similarly, the stretching frequency for the pyridiniumNH unit has appeared at 3396 cm-1. The
aromatic ring CH vibrations appeared at 3074 cm-1and 3027 cm-1 correspondingly. The
absorption band at 1738 cm-1 appeared for the carbonyl stretching frequency which is present in
the hydrazide moiety. The strong absorbance band at 1660 cm-1appeared for C=N stretching
vibrations of imine moiety. The band at 1590 cm-1 and 1557 cm-1appeared for the C=N unit of
pyridinium and C=C unit of aromatic rings. The band at 1216 cm-1 appeared for C-O stretching
vibrations of carboxylate units. The C-Cl stretching vibration has appeared at 752 cm-1 as a
strong absorption band.
3.2 Computational study
3.2.1 Frontier molecular orbitals
Highest occupied molecular orbital andlowest unoccupied molecular orbital are used to
predict the electrical properties, chemical properties, biological activity, stability and reactivity
of thecompounds[29-30].Themolecular orbitals playan important role in calculating the HOMO,
LUMO, bandgap and other parameters. In all IHCs, the electron cloud localized as same as each
other. In HOMO the electron density is localized in benzoate units and in LUMO the electron
density is localized in isoniazid moiety. Comparatively, all the IHCs have more electron density
in the region of carboxylate and isoniazid moiety. So the carboxylate and the isoniazid region is
a more negative region that could be ready to form the hydrogen bonding interaction with a
biomolecule. The same results were obtained in the molecular docking studies since the
isoniazid and carboxylate moieties form several hydrogen bonding interactions with the α-

amylase enzyme.The negative energies of HOMO (-5.5670 to -6.0182) and LUMO (-3.2765 to -
3.3971) indicate the IHCsare stable molecules.
The bandgap has been used to predict the stability and chemical reactivity of IHCs.The
bandgap values of IHCs are in the range of 2.1698 to 4.6474 eV correspondingly. Amid IHCs,
the calculated bandgap of compound 3b is higher than others which ismore stable than 3a and 3c.
The electrophilicity index (ω) is the ability of the molecule to accept the electrons from the
environment. Especially, the higher value of electrophilicity index has a higher ability to accept
electrons
from
biomolecules.
The
results
suggested
that
the
compound
3cexhibitedhigherelectrophilicityindex than others. The calculated electrophilicity index is found
to be 9.2581eV which is represented the highest capacity to accept the electrons. Thus the
compound 3c has the number of hydrogen bonding interactions in molecular docking studies.
Also the other DFT parameters like chemical potential, hardness and softness calculated and
presented in Table 1.The representation of HOMO and LUMO forIHCs was represented in Fig.
3, 4and 5.
Bandgap
∆E = ELUMO – EHOMO
Chemical potential µ = (EHOMO + ELUMO)/2
Global hardness
Global softness
ƞ = (ELUMO-EHOMO)/2
ζ = 1/2ƞ
Electrophilicity index ω = µ2/2ƞ
3.3 Molecular electrostatic potential
Molecular electrostatic potential was used to identify the bindingregion of a ligand with
the biomolecules.In molecular electrostatic potential, the blue color indicates the most positive
potential and the red color indicates the most negative potential which willbe very useful for
understanding the region responsible for the biological activity of the molecule [31]. Particularly,

the negative potential region of themolecule is more important because it is ready to make
hydrogenbonding interaction with protein.In the present research, the molecular electrostatic
potential is performed for the synthesized IHCs. The results indicate that the carbonyl present in
the molecule showed negative potential. Especially, the carboxylate units displayed the highest
negative potential. From these studies, the research is concluded that the carbonyl oxygen and
benzoate oxygens are responsible for hydrogen bonding interactions with proteins. The same
results obtained in molecular docking studies. Most of the hydrogen bonding interactions formed
between the oxygen of IHCs with the α-amylase enzyme. In compound 3c, the negative potential
is occupied in many regions which were identified at carbonyl oxygen of hydrazide, carboxylate
oxygen and phenolic OH unit. Also, the whole ring of the salicylate unit covered by the negative
potential which clearly displays the red colour. Thus the compound 3c showed 5 numbers of
hydrogen bonding interactions with theprotein. The hydrogen bonding interactions were
identified at carbonyl hydrazide, carboxylate oxygen and phenolic hydroxyl regions
respectively.In the case of compounds 3a and 3b, only two hydrogen bonding interactions were
identified in the docked complex. The molecular electrostatic potential of IHCs is represented in
Fig. 6.
3.4 Molecular docking studies
Molecular docking is the most frequently used method which is used to design the
structure-based drug, predict the binding conformation of small molecules to the applicable
target binding site and to develop the new drug candidate[31].α-amylase is a key enzyme in the
digestive system and catalyzes the initial step in starch hydrolysis.Its inhibitors possess an
important role in controlling diabetes. Hence the research is decided to choose the
enzymes for the molecular docking studies[32].
α-amylase

The obtained docking results indicated that compound3cexhibited strong interaction with
the α-amylase enzyme.The compound 3a showed two hydrogen bonding interactions with
ARG421 and SER3. The binding energy of 3a is found to be -294.42 KJ/mol. The compound
3bformedtwo hydrogen bondinginteractions withTYR2 and SER3 amino acid with good binding
energy. Similarly, the compound 3c showed five hydrogen bonding interactions with THR377,
TRP388, ARG389, GLN390 andLYS322amino acids with good binding energy. The calculated
binding energies of 3b and 3c are found to be -315.24 and -299.78 KJ/mol respectively. In the
docked complex of 3a, the chlorine forms a hydrogen bonding interaction with quinidine NH of
ARG421. Similarly, the carboxylate oxygen forms a hydrogen bonding interaction with OH of
SER3.
In the docked complex of 3b, the carbonyl of hydrazide forms the hydrogen bonding
interaction with CH of SER3 and NH of TYR2. In the case of 3c, the imine nitrogen forms a
hydrogen bonding interaction with guanidine NH of ARG389 and NH of GLN390. The
hydrazide carbonyl forms a hydrogen bonding interaction with NH of GLN390 and NH of
LYS322. Similarly, the carboxylate oxygen forms a hydrogen bonding interaction with indole
NH of TRP388. The OH of 3c forms hydrogen bonding interaction with NH of THR377.
Based on the docking results, the research is concluded that the IHCsare good anti-
diabetic active compounds.The various binding interactions of IHCs with α-amylaseare
represented in Fig.7 and binding energy, thenumber of hydrogen bonding interactions are
represented in Table 2.
3.5 Biological Evaluation
3.5.1 BSA denaturation technique