Synthesis, crystal structure, hirshfeld surface analysis, DFT calculations, anti-diabetic activity and molecular docking studies of (E)-N’-(5-bromo-2-hydroxybenzylidene) isonicotinohydrazide

Article abstract of DOI:10.1016/j.molstruc.2020.128800

In this present work, the newly synthesized compound E)-N’-(5-bromo-2-hydroxybenzylidene)isonicotinohydrazide (2) has been synthesized and characterized by IR, ¹H &¹³C NMR, ESI-MS and single crystal X-ray diffraction analysis using e

Full text of DOI:10.1016/j.molstruc.2020.128800

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Synthesis, crystal structure, hirshfeld surface analysis, DFT calculations, anti-diabetic
activity and molecular docking studies of (E)-N'-(5-bromo-2-hydroxybenzylidene)
isonicotinohydrazide
Khalid Karrouchi, Saad Fettach, Mukesh M. Jotani, Abir Sagaama, Smaail Radi,
Hazem A. Ghabbour, Yahia N. Mabkhot, Benacer Himmi, My El Abbes Faouzi,
Noureddine Issaoui
PII:
S0022-2860(20)31125-X
DOI:
https://doi.org/10.1016/j.molstruc.2020.128800
MOLSTR 128800
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 April 2020
Revised Date: 25 June 2020
Accepted Date: 29 June 2020
Please cite this article as: K. Karrouchi, S. Fettach, M.M. Jotani, A. Sagaama, S. Radi, H.A. Ghabbour,
Y.N. Mabkhot, B. Himmi, M. El Abbes Faouzi, N. Issaoui, Synthesis, crystal structure, hirshfeld
surface analysis, DFT calculations, anti-diabetic activity and molecular docking studies of (E)-N'-(5-
bromo-2-hydroxybenzylidene) isonicotinohydrazide, Journal of Molecular Structure (2020), doi: https://
doi.org/10.1016/j.molstruc.2020.128800.
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CRediT author Statement
Khalid Karrouchi: Conceptualization, Methodology, Original Draft, Writing - Review &
Editing.
Saad Fettach: Methodology, Investigation.
Mukesh M. Jotani: Theoretical, Calculations Writing - Original Draft.
Abir Sagaama: Methodology, Investigation.
Smaail Radi: Supervision, Project administration.
Hazem A. Ghabbour: Software, Formal analysis.
Yahia N. Mabkhot: Software, Formal analysis.
Benacer Himmi: Investigation.
My El Abbes Faouzi : Visualization, Investigation.
Noureddine Issaoui: Theoretical, Calculations Writing - Original Draft, Writing - Review &
Editing.

Synthesis, crystal structure, hirshfeld surface analysis, DFT calculations, anti-diabetic
activity and molecular docking studies of (E)-N'-(5-bromo-2-hydroxybenzylidene)
isonicotinohydrazide
a,
b
c
d
e
Khalid Karrouchi *, Saad Fettach , Mukesh M. Jotani , Abir Sagaama , Smaail Radi ,
f
g,h
i
b
Hazem A. Ghabbour , Yahia N. Mabkhot , Benacer Himmi , My El Abbes Faouzi ,
d,
Noureddine Issaoui *
a
Laboratory of Analytical Chemistry and Bromatology, Faculty of Medicine and Pharmacy,
Mohammed V University, Rabat, Morocco.
b
Biopharmaceutical and toxicological analysis research team, Laboratory of Pharmacology and
Toxicology, Pharmacokinetic Research Team, Faculty of Medicine and Pharmacy, University
Mohammed V of Rabat, Morocco.
c
Department of Physics, Bhavan’s Sheth R. A. College of Science, Ahmedabad, Gujarat 380001, India
d
University of Monastir, Laboratory of Quantum and Statistical Physics (LR18ES18), Faculty of
Sciences, Monastir 5079, Tunisia.
e
Laboratoire de Chimie Appliquée et Environnement (LCAE), Faculté des Sciences, Université
Mohamed I, 60000 Oujda, Morocco.
f
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P. O. Box
2457, Riyadh 11451, Saudi Arabia.
g
h
i
Departement of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha
1421, P. O. Box 960, 61421, Saudi Arabia.
6
Research Center for Advanced Materials Science (RCAMS), King Khalid University, Abha 61413, P.
O. Box 9004, Saudi Arabia.
Filière Techniques de Santé, Institut Supérieur des Professions Infirmières et Techniques de Santé de
Rabat, Ministère de la Santé, Morocco.
*
Corresponding authors:
Khalid.karrouchi@um5s.net.ma (K. Karrouchi)
issaoui_noureddine@yahoo.fr (N. Issaoui)
1

Abstract
In this present work, the newly synthesized compound E)-N'-(5-bromo-2-
hydroxybenzylidene)isonicotinohydrazide (2) has been synthesized and characterized by IR,
1
13
H & C NMR, ESI-MS and single crystal X-ray diffraction analysis using experimental and
theoretical methods. The molecular geometry, vibrational frequencies, frontier molecular
orbital (HOMO-LUMO) energies and thermodynamic properties of the title molecule were
explored using Density Functional Theory (DFT) calculation via B3LYP method with 6-
3
1++G (d,p) basis set. Moreover, Hirshfeld and Molecular electrostatic potential (MEP)
surfaces analyses were investigated. In addition, the title compound was evaluated for their in
vitro antidiabetic activity against α-glucosidase and α-amylase enzymes. Finally, Molecular
docking studies were performed between the title ligand and 1CLV/2ZE0 enzymes. Docking
calculations showed that the 2-2ZE0 complex is more stable than 2-1CLV complex, since it
has the best inhibitory impact whose their total energy score equal to -108.68 Kcal/mol.
Docking results reveal that the main interaction forces are H-bond and van der Waals
interactions.
Keywords: Isoniazid; X-ray analysis; DFT; Hirshfeld surface analysis; α-glucosidase; α-
amylase; Molecular docking.
1
. Introduction
Diabetes mellitus is a serious metabolic disorder of modern era and severe interminable health
complications are associated with it, and type-2 diabetes is widely spread kind of this disorder
[
1]. Hence, one of the therapeutic approaches in treating diabetes is to reduce postprandial
hyperglycemia by inhibiting major carbohydrate hydrolyzing enzymes. Two enzymes have
been implicated in processes leading to the development of diabetic symptoms, namely α-
glucosidase, which is also implicated in intestinal disease and α-amylase, which hydrolyzes α-
bonds of large α-linked polysaccharides. These enzymes are the key carbohydrate hydrolyzing
enzymes, located in the brush-border surface membrane of human intestinal cells, which plays
an important role in the carbohydrate digestion [2, 3]. Thus, discovery and development of
new α-glucosidase and α-amylase inhibitors have attracted great attention in recent years [4,
5
].
The N-acylhydrazone scaffold (-CO-NH-N=CH-) is represented by the fusion between imine
and amide subunits, which provides pharmacophoric interaction points including both
hydrogen-bond donor and acceptors sites and are capable of interacting with a large range of
active sites [6, 7]. Generally, the N-acylhydrazones are prepared by condensation of ketones,
2

or aldehydes with hydrazides [8-10]. Changes of the subunits bonded to both acyl and imine
functions resulted in several N-acylhydrazone derivatives that are capable to modulate
different molecular targets, providing a surprisingly wide range of pharmacological activities
such as antiviral, analgesic, antimicrobial, anti-inflammatory, anti-cancer, antioxidant,
vasodilator, trypanocidal, antiplatelet, antiprotozoal, cathecolase and antinociceptive agents
[
11-23].
Given the importance of therapeutic properties of N-acylhydrazone derivatives, the study of
the molecular structure, electronic and thermodynamic properties are essential to know the
effect of different substituents on molecular structures in order to find the relationship of these
groups with their biological activities. In this context, DFT calculations have become a very
important and widely used tool to develop a close relationship between theoretical and
experimental data by giving clues related to molecular geometry structure and to electronic
and thermodynamic properties. Consequently, these techniques have become very reliable in
the prediction of many molecular properties with great precision [24-30].
In this study, we report the synthesis (E)-N'-(5-bromo-2-hydroxybenzylidene) isonicotino-
hydrazide to be evaluated as a new anti-diabetic agent. The newly synthesized compound was
1
13
characterized by IR, H- and C-NMR, ESI-MS and the (E)-configuration of azomethine
N=CH) group was confirmed by single-crystal X-ray diffraction analysis. In addition, the
(
molecular geometry, HOMO-LUMO energies, molecular electrostatic potential (MEP) and
thermodynamic properties of the title molecule were explored using DFT/B3LYP method
with 6-31++G (d,p) basis set. The in vitro antidiabetic activity of the target compound was
evaluated against two enzymes α-glucosidase and α-amylase. Finally, Molecular docking
studies were performed between the title ligand and 1CLV/2ZE0 enzymes.
2
. Experimental
2
.1. General methods
Chemical reagents were purchased from Fluka, Sigma and Aldrich chemicals. Reactions were
checked with TLC using aluminum sheets with silica gel 60 F254 from Merck. Melting points
were measured using a Buchi B-545 digital capillary melting point apparatus and used
without correction. The FT-IR spectrum was recorded with Perkin-Elmer VERTEX 70 FT-IR
-1
spectrometer covering field 400–4.000 cm . NMR spectroscopies were recorded in dry
3

1
deuterated DMSO on a Bruker AC spectrometer at 200 MHz for H NMR and 50 MHz for
1
3
C NMR; δ is expressed in ppm related to TMS (0 ppm) as internal standard. Splitting
patterns are designated as follow: s (singlet), d (doublet), t (triplet), m (multiplet). Coupling
constants (J values) are listed in Hertz (Hz). Mass spectra were obtained using an API 3200
LC/MS/MS system equipped with an ESI source and the samples were diluted in methanol.
2
.2. Synthesis
General procedure for the synthesis of (E)-N'-(5-bromo-2-hydroxybenzylidene)isonicotino-
hydrazide (2)
The title compound was prepared according to the previously published protocol [31-33].
(Scheme 1).To a solution of Isoniazid (137 mg, 1 mmol) and 5-bromo-2-
hydroxybenzaldehyde (201 mg, 1 mmol) in 10 ml of ethanol was added two drops acetic acid.
Then, the reaction mixture was poured in cold water, and the precipitate formed was filtered
out and recrystallized from ethanol to afford the product (2) as yellow crystals. Yield (75%);
-1
1
m.p. 252-254 °C; IR (ν(cm )) : 3173 (NH), 3138 (OH), 1675 (C=O), 1613 (C=N); H NMR
200 MHz, DMSO-d ) δ : 12.37 (s, 1H, -CONH-), 11.15 (s, 1H, OH), 8.81 (d, J = 6.1 Hz, 1H,
(
6
H2 & H6 pyridyl), 8.66 (s, 1H, N=CH), 7.86 (d, J = 6.1 Hz, 1H, H3 & H5 pyridyl), 7.83 (d, J
2.5 Hz, 1H, H6-Ar), 7.44 (dd, J = 8.8, 2.6 Hz, 1H, H4-Ar), 6.92 (d, J = 8.7 Hz, 1H, H3-
=
1
3
Ar). C NMR (50 MHz, DMSO-d ) δ : 160.96, 155.94, 149.84, 145.87, 139.36, 133.36,
6
+
+
1
29.64, 121.00, 120.75, 118.17, 110.03; ESI-MS: m/z = 320.3[M+H] , m/z = 342.3 [M+Na] .
Scheme 1. Synthesis of the title compound 2.
2
.3. X-ray analysis
X-ray single crystal data were collected by using graphite monochromatic MoKα radiation (λ
0.71075 Å) radiation on a Bruker APEX-II D8 Venture area diffractometer. Bruker SAINT
=
was harnessed for data reduction and cell refinement and SHELXT [34, 35] was utilized for
structure solving. Full-matrix least-square technique was utilized for final refinement with the
4

aid of anisotropic thermal data for non-hydrogen atoms on F. The details of the X-ray crystal
data and the structure solution as well as the refinement are given in Table 1.
2
.4. Computational details
.4.1. DFT
2
All geometry optimizations and frequency calculations of the desired product 2 were carried
out using the Gaussian 09 software [36] and visualizations of modeling were performed with
GaussView5 software [37]. The GaussView5 program is designed for the 3-D design of the
molecule and to visually identify the properties of the molecule. Density functional theory
with the Becke three parameters hybrid functional (DFT-B3LYP/6-31++G (d,p)) [38, 39].
calculations were performed for all atoms of the title compound in the gas phase and DMSO,
separately. Vibrational frequencies were calculated at the same level to ensure that each
stationary point was a true minimum. The optimized molecular structure is used to perform
harmonic vibrational frequencies, NMR shifts and UV-Vis data, HOMO and LUMO
distributions and MEP analysis of the studied compound.
2
.4.2. Molecular docking studies
Analysis for the docking simulation two programs was used: iGEMDOCK [40] and
Discovery Studio Visualizer [41]. The first software gives the total energy value on the basic
of the proteins-ligand interactions. Molecular docking parameters selected for iGEMDOCK
program were as follows: population size 800, generations 80 and number of solutions 10.
The second software determines the representation of the best docked poses of ligand inside
protein and their different interactions (VDW, H-bond…). The protein structure of α-
glucosidase GSJ enzyme (pdb: 2ZE0) [42] and ꢀ-amylase (pdb: 1CLV) [43] were download
from the Research Collaboratory for Structural Bioinformatics (RSCB) Protein Data Bank
(http://www.pdb.org/pdb/home/home.do).
2
.5. Antidiabetic activity
The α-glucosidase and α-amylase inhibition assays were carried out according to the
previously published methods [23, 44].
3
3
. Result and discussion
.1. X-ray structure description and optimized geometry
The details of the X-ray crystal data and the structure solution as well as the refinement of the
title compound are given in Table 1. Supplementary data are deposited at CCDC under
5

deposition number 1836618. The title compound crystalized in the monoclinic system, space
group P2 /c. The molecular structure of 2 sees (5-bromo-2-hydroxy benzylidene) linked with
1
isonicotine through hydrazine, Fig. 1. The benzylidene-C9 and carbonyl-C8 atoms have the
trans configuration about N1-N2 bond characterized from the value of torsional angle C7-N1-
N2-C8 to be 176.3(2)°. The twist of (C1-C6) ring about C3-C7 (N1-C7-C3-C4 = -5.2(3) ° and
that of N3-containing pyridine ring about C8-C9 bond as the torsion angle of 24.0(3)° had
resulted 11.16(11)° dihedral angle between these two aromatic residues. The hydroxyl oxygen
atom O1 and bromine Br1 areout of least square plane of (C1-C6) benzene ring and have the
deviation of 0.035 Å and -0.034 Å respectively.
The molecular packing of the title molecule features intermolecular hydrazinyl-N—H···N
(pyridyl) hydrogen bond which give rise to C(8) chain, whereas the intramolecular hydroxyl-
O—H···N (hydrazine) forms S(6) graph-set motif (Fig. S1, Table 2). Further, the stability to
the layers is provided by aromatic cycle stacking between inversion related (C1-C6) benzene
and N3-containing pyridin rings [inter-centroid distance = 3.6712(13) Å at (-x, -y,-z)]. The
intermolecular benzene-C—H···π(benzene) contacts between the layers, Table 1, had resulted
the three-dimensional architecture in the crystal of 2.
The optimized geometry of compound 2 is illustrated in Fig. 2. A summary of selected bond
lengths [Å] and angles [°] compared to the experimental data on the crystal structure are given
in Table 3. The molecular structure of the title compound composed of two rings. These rings
are pyridine and 5-bromo-2-hydroxybenzyl which are connected through an acylhydrazone (-
CO-NH-N = CH-) group. In the following discussion, the molecular structure and atomic
numbering scheme adopted for the present study was taken from the optimized structure (Fig.
2
1
and Table 3). The C-C bond lengths in the pyridine ring are reported [45] at the range
.390-1.402 Å and in the present study, The C-C bond lengths (XRD/DFT) are found at the
range 1.377-1.503 Å/1.392-1504Å. The C-N bond length is found at 1.344 Å for C22-N6 and
.333 Å for C24-N6 by XRD, these values are very closer to the calculated values 1.338 Å
1
and 1.335 Å, respectively. In this molecule, the calculated C─C bond length of the benzyl
ring varies from 1.382-1.418 Å, which is slightly greater than that of experimental values
(1.373-1.403 Å). In carbohydrazide group, the bond length of C18-O3, C18-N5, N4-N5, C16-
N4 and C18-C19 are calculated 1.212, 1.358, 1.385, 1.286 and 1.504 Å. The experimental
bond lengths of above are observed about 1.205, 1.370, 1.375, 1.275 and 1.503 Å.The
calculated N─H bond length in carbohydrazide group is N5─H28 = 1.015 Å by DFT method,
which is 0.206 Å deviate from the experimental value (0.809 Å).The calculated C─H bond
6

length of the 5-bromo-2-hydroxybenzyl and pyridine ring varies from 1.083-1.086 Å which is
slightlygreater than that of experimental values (0.930 Å).In the pyridine moiety of the title
compound, the bond angles (XRD/DFT) C19-C20-C22, C20-C19-C26and C26-C24-N6 are
1
18.7°/118.7°, 118.1°/118.0° and 124.1°/123.6°, respectively. For benzyl ring, the bond
angles lie between 118.9-120.7° experimentally, these bond angles were predicted at the
range 119.1-121.0° with DFT-B3LYP/6-31++G (d,p). From the results we can say that, the
DFT/B3LYP functional/method estimated the bond lengths and bond angles in good
agreement with experimental results.
3
.2. FT-IR study
The experimental infrared spectrum of the title compound was recorded in a solid state using
reflectance (ATR) mode (Fig. 3). The OH and NH stretching vibrations are generally
-1
observed around 3500–3300 cm [46]. This absorption is strongly influenced by the chemical
environment, in particular when OH or NH groups are involved in the intramolecular or
intermolecular hydrogen bond [46]. González-Baró et al. [47] reported the NH stretching
-
mode (ν ) of the isonicotinoylhydrazone of 2-hydroxy-3-methoxybenzaldehyde at 3157 cm
NH
1
.
On the other hand, the intermolecular bond H leads in many cases to a broadening of the
band, as shown by the IR spectrum in the case of NH absorption of the title molecule, which
-
1
has been attributed to the low IR band at 3173 cm .
Benković et al. [48] reported the stretching of the OH groups, involved in the intramolecular
hydrogen bond with the nitrogen atom of the group C=N, of hydrazones with hydroxyl
-1
groupin position 2 of phenyl ring at 3142 cm . In the present study, the IR band appears at
−
1
3
138 cm has been assigned to stretching modes of OH involved in the intramolecular
hydrogen bond. In general, the OH in-plane deformation vibration for phenols lies in the
-1
region 1440-1260 cm [49], Arunagiri et al. [50] reported the in-plane deformation vibrations
-1
of two OH at 1242 and 1220 cm . In this work, the observed in-plane deformation vibrations
-1
of OH appear at 1285 cm . The in-plane and out-of-plane deformation vibrations of NH
-1
modes are observed at 1210 and 875 cm , respectively [51]. In our present study, the bands
-1
observed at 1210 and 888 cm is assigned as these modes.
The C-H stretching vibrations of aromatic rings give rise to bands in the region 3200–3000
-1
cm in aromatic compounds [52]. For the title molecule, a series of infrared absorptions
-1
between 3115 and 2819 cm were assigned as CH stretching modes of the pyridyl and benzyl
-1
rings. The C-H in-plane deformation vibrations are observed in the region 1300–1000 cm
and are usually of medium to weak intensity [52]. Galić et al. [53] reported the in-plane CH
7

-1
deformations of the pyridyl and phenyl moieties in the range of 1200–1000 cm , and the out
of plane CH deformations and the skeletal torsions of the aromatic rings between 1000 and
-1
7
00 cm . In the present work, the bands due to C-H in-plane bending vibration interact
somewhat with C–C stretching vibrations, are observed as a number of bands in the region
−
1
1
9
330 and 1067 cm in IR. The out-of-plane CH deformations are observed between 600 and
-1
00 cm [52]. Generally, the CH out-of-plane deformations with the highest wavenumbers are
weaker than those absorbing at lower wavenumber. In our case, the observed wavenumbers
-1
with weak intensity at 956, 931, 869 and 704 cm are identified as CH out-of-plane
deformation vibration.
The C=O stretching vibrations generally appear in a wave number region between 1800–1600
-1
cm , they are one of the most representative in an infrared spectrum [52]. In our study, the
-1
strongest band in the infrared spectrum, at 1675 cm , is attributed to C=O stretching
-1
vibrations. This mode was assigned at 1655 cm by Sheeja et al. [51] for quinoline-2-
-1
carbaldehyde benzoyl hydrazine and at 1656 cm by de Freitas et al. [46] for 8-
hydroxyquinoline-2-carboxaldehyde isonicotinoylhydrazone. The in-plane and out-of-plane
-1
deformation vibrations of C=O are expected in the regions 625 ± 70 and 540 ± 80 cm ,
-1
respectively [51]. Here, these modes are observed at 779 and 611 cm respectively.
-1
The stretching vibrations νC=N reported at 1604 and 1603 cm by Galić et al. [53] for
-1
aroylhydrazones and the moderate bands at 1364 and 1367 cm are assigned as the C-N
-1
stretching. For the title molecule, the C=N stretching mode is observed at 1619 cm and the
-1
medium band observed at 1379 cm in the IR spectrum is assigned as C-N stretching mode.
-1
-1
The N─N stretching has been reported at 1156 cm by de Freitas et al. [46], at 1118 cm by
-1
Sheejaet al. [51] and at 1112 cm by Arunagiri et al. [50]. For our title compound ν
N─N
-1
vibrations observed at medium intensity band 1052 cm in IR spectrum.The aromatic C=C-C
stretching vibrations of phenyl and pyridyl are very much important and occur in the region
-1
-1
1
200-1650 cm [46]. In this case, the infrared bands at 1411, 1434, 1476, and 1554 cm are
assigned as C=C stretching mode in pyridyl and phenyl rings (Fig. S1).
1
13
3
.3. H- and C-NMR studies
1
The H NMR spectra of the title molecule displayed a two doublets at δ 6.92 and 7.83 ppm,
anddoublet of doublets at δ 7.44 ppm due to 5-bromo-2-hydroxybenzyl protons. The chemical
shifts of the pyridyl protons appeared as two doublets at δ 7.86 and 8.81 ppm. The chemical
shifts of the azomethine (N=CH) proton appeared as singlet at δ 8.66 ppm. The chemical
8

shifts of the hydroxy (-OH) and amide (NHCO) protons appear as a singlet at 11.15 and 12.37
1
3
ppm, respectively (Fig. S2). The C NMR spectra of the title compound showed the chemical
shifts of C=O and C-OH are at 160.96 and 155.94 ppm, respectively. The signals at 145.87
ppm are clearly assigned for azomethine group chemical shifts C=N. The signals at 121.00,
1
39.36 and 149.84 ppm are assigned for pyridyl carbons, while that the aromatic carbon
chemical shifts of the compound occurred in the range of 110.03-133.36 ppm (Fig. S3).
3
.4. ESI-MS study
The ESI-MS spectra show molecular ion peaks with m/z values 320.3 and 322.3 correspond
+
to the molecular weight [M+H] . The m/z value 342.2 and 344.2 correspond to the sodiated
+
molecular ion peak [M+Na] . These values are in good agreement with the proposed
composition for the title molecule (Fig. S4).
3
.5. Hirshfeld surfaces Analysis
The Hirshfeld surfaces calculated for the crystal structure of 2 in accord with the established
procedures [54] provide an additional insight on the influence of weak intermolecular
interactions on its molecular packing. In addition to broad and bright-red spots near amino-H
atom H2N and pyridine-N atom N3 recognizing donor and acceptor of potential N-H…N
hydrogen bond respectively on the Hirshfeld surfaces mapped over dnorm in - 0.151 to +
1
.197 arbitrary unit range (Fig. S5), the presences of diminutive and faint-red spots on the
surface characterize the weak intermolecular interactions in the crystal. In the crystal packing
of (2) the effect of short interatomic Br…O/O…Br and C…H/H…C contacts (Table S1) are
viewed as the diminutive-red spots whereas the presence of weak intermolecular C-H…O and
short interatomic C-C contacts with the faint-red spots (Fig. S5). The donors and acceptors of
intermolecular interactions are also viewed as the blue and red regions on Hirshfeld surfaces
mapped over electrostatic potential in -0.113 to +0. 153 a.u ranges around the respective
atoms corresponding to positive and negative potentials (Fig. S6). The donors and acceptors
of intermolecular C-H...π and its reciprocal i.e. π…H-C contact for 2, influential on the
packing are also illustrated through black-dotted lines joining blue-bump and light red-
concave regions around them on the shape-index mapped Hirshfeld surfaces in Fig. 4a. The
presence of π-π stacking interactions is illustrated through red-dotted lines in Fig. 4b.
The overall two dimensional fingerprint plot for 2, Fig. 5a, and those delineated into H...H,
O…H/H…O, N…H/H…N, C…C, C...H/H...C, Br...H/H...Br and Br...O/O…Br contacts [55]
is illustrated in Fig. 5b-e, respectively. The quantitative summary of percentage contribution
9

from different interatomic contacts to the hirshfeld surfaces of 2 are summarized in Table S2.
The pair of jaws-like peaks at de + di ~ 2.2 Å in fingerprint plot delineated into H...H contacts
(Fig. 5b) indicate the presence of such short interatomic contact (Table S1) between
methylene-H7and pyridyl-H11 atoms. The pair of long and thin spikes at de + di ~ 2.7 Å in
the fingerprint plot delineated into N...H/H...N contacts in Fig. 5d had resulted are due to the
formation of significant intermolecular N-H…N hydrogen bond. The effect of weak
intermolecular C-H…O contact in the crystal structure of 2 is also evident as the pair of
spikes having tips at de + di ~ 2.5 Å in the finger print delineated into O…H/H…O contacts,
Fig. 5c, through such short interatomic contacts (Table S1). The presence of intermolecular
of π-π stacking in the crystals of 2 is justified from the arrow-like distribution of points with
the greater density around de = di ~ 1.8 Å in the fingerprint delineated into C…C contacts
(
Fig. 4e). The fingerprint plot delineated into C...H/H...C contacts (Fig. 5f) characterize short
interatomic C…H/H…C contact and intermolecular C-H...π contact with the pairs of tips at de
di ~ 2.7 Å and ~ 2.8 Å respectively. The bromine substituent at phenol ring forms short
+
interatomic contacts with pyridyl hydrogen-H13 and carbonyl oxygen-O2 showing forceps-
like and needle shaped tips at de + di ~ 2.9 Å and at de + di ~ 3.1 Å with the asymmetric
distribution of points in the respective delineated fingerprint plots (Fig. 5g, 5h). The small
contributions from other interatomic contacts to the Hirshfeld surfaces summarized in Table
S2 have negligible effects on the crystal packing.
The pairwise interaction energies between the molecules within the crystal are calculated by
summing up four energy components comprising electrostatic (E ), polarization (E ),
ele
pol
dispersion (E ) and exchange - repulsion (E ) [56]. These energies were obtained by using
dis
rep
the wave function calculated at HF/STO-3G level theory. The quantitative summary of the
strength and nature of intermolecular interactions in the crystals of 2 is illustrated in Table 4.
It is observed from the interaction energies calculated between the reference molecule and
the inversion related molecule at –x, -y, -z that maximum energy from dispersive components
had resulted greatest total energy of interaction in the formation of π-π stacking. From
interaction energies calculated between the reference molecule and the molecule at -1/2+x,-
1
/2-y, 1/2+z, the electrostatic energy component is significant due to intermolecular N-H...N
hydrogen bond whereas dispersive components are dominant due to short interatomic contacts
related with the same molecules. The intermolecular C-H…O interaction between phenyl-C12
and hydroxyl-O1 is stronger compared to intermolecular C-H…π contacts. The other short
1
0

interatomic contacts involving bromine Br1 as summarized in Table 4 have small interaction
energies due to greater contribution from repulsive components.
The magnitudes of intermolecular energies are represented graphically in the energy
frameworks for both the analogues down-c axis in Fig. 6. Here the supramolecular
architecture of crystals is viewed through the cylinders joining centroids of molecular pairs
by using red, green and blue colour codes for the components E , E and E respectively.
ele
disp
tot
The radius of the cylinder is proportional to the magnitude of interaction energy which is
-1
adjusted to same scale factor 5 kJ mol within 4 × 4 × 4 unit cells. From the energy
frameworks illustrated in the supramolecular associations viewed down-c axis in Fig. 6 for the
crystal structure of 2, it is clearly seen that the electrostatic and dispersive energy components
are dominant between different groups of atoms.
3
.6. Molecular electrostatic potential (MEP)
Electrostatic potential is a very important property because it provides us much information
on the chemical reactivity of the studied compound especial to describe intermolecular
interactions. Based on a color code to detect and display regions and molecular properties of
the electrostatic potential. The blue color indicates positive values, red for negative values and
green indicate the neutral zone. The blue color indicates the strongest attraction and the red
indicates the strongest repulsion. It is very useful for the qualitative interpretation of
electrophilic and nucleophilic reactions and hydrogen bond interactions. The total electron
density surface mapped with electrostatic potential of compound 2 constructed using
B3LYP/6-311++G(d,p) level is shown in Fig. 7. The regions of negative potentials (excess in
electrons) are associated with the hydrogen atoms especial that which is linked the nitrogen
atoms whereas the regions of positive potentials (deficient in electrons) are associated with
the oxygen and the bromine atoms. Indeed the electrophilic and nucleophilic sites confirm
…
…
well the existence of N-H N and C-H O hydrogen bonds interactions between the entities of
our compound and explain their important role in the stabilization of the crystal.
3
.7. Frontier molecular orbital analysis
The electronic properties and the chemical reactivity behaviors of the chosen molecule have
been performed via TD-DFT approach [57, 58]. The Highest Occupied Molecular Orbital
(HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO), called frontier molecular
orbitals (FMOs), are the most important orbital in molecules. These two orbitals specify the
manner in which the molecule interacts with species. The energy of the HOMO is linked to
the ionization potential, while LUMO energy is linked to the electron affinity. The 3D plots of
1
1

the FMOs of the compound 2 are displayed in Fig. 8. The DFT calculated reactivity
descriptors: energy band gap, ionization potential, electron affinity, chemical hardness,
chemical softness, electronegativity, chemical potential, electrophilicity index and the
maximum charge transfer index are determined in Table 5. Results reveal the energy level of
the HOMO orbital is equal to -6.395 eV and the energy level of the LUMO orbital is equal to
-2.575 eV, which gives an energy gap about -3.820 eV. This gap energy describes well the
molecular chemical reactivity [59, 60]. All the parameters given in the table 7 are related to
the energies of HOMO and LUMO orbitals by the following relations: the ionization potential
I = −EHOMO, the electron affinity A= −ELUMO and the electronic chemical potential μ=
1
/2(I+A). These parameters are equal to -3.820, 6.395 and -4.485 eV, respectively. The
chemical hardness (η) can be estimated as ELUMO–EHOMO= (I−A) and found about 1.910 eV.
-1
Additionally, our molecule has a chemical softness (ζ = 1/2η) equal to 0.262 eV . The
Electronegativity (χ = (I+A)/2) is computed based on the HOMO and LUMO energies and
2
found to be 4.485 eV. Also, the compound 2 has an electrophilicity index ω = μ /2η about
1
0.534 eV. Finally, the maximum charge transfer index (ΔNmax= –μ/η) has been calculated
and found to be 2.349.
3
.8. Thermodynamic properties
The thermodynamic functions such as enthalpy change (H), entropies (S) and heat capacity at
a constant pressure (Cp) were determined for the title compound using B3LYP/6-
3
11++G(d,p) at different temperatures 100-1000K and as observed in Fig. 9 and Table 6. It
can be shown that these thermodynamic functions increase with temperature as predicted that
the molecular vibrational intensities change with rise of temperature. The correlation
equations between heat capacities, entropies, enthalpy changes and temperatures were fitted
by quadratic formulas, and the corresponding fitting factors (R2) for these thermodynamic
properties are 0.99961, 0.99997 and 0.99951, respectively. The corresponding fitting
equations are as follow:
-4
2
2
C = 19.69087+0.96165T -4.27323 × 10 T (R = 0.99961)
p
-4
2
2
S = 275.35652+ 1.05962T -2.63098 × 10 T (R = 0.99997)
-4
2
2
H = -12.72168 +0.1309T + 2.45152 × 10 T (R = 0.99951)
These equations above will be helpful for deepened studies of the title compound. We note
that all thermodynamic calculations were done in the gas phase and could not be used in
solution.
1
2

3
.9. Anti-diabetic activity
The antidiabetic activity of the title compound has been systematically evaluated against two
enzymes at different concentration. The α-glucosidase and α-amylase inhibition capacity of
the compound under investigation was fixed. Table 7 illustrates the IC values of the in vitro
5
0
antidiabetic profile of the title molecule, compared to acarbose as reference drug. In vitro
study on the inhibition of α-glucosidase shows that compound 2 has significant activity with
an IC value of 280.45 ± 1.55 µM. This value is 2.85 and 22.08-fold lower than those of
5
0
acarbose (IC = 98.12 µM) and resveratrol (IC = 12.70 µM) [61, 62], well-known as α-
5
0
50
glucosidase inhibitors, respectively. The results of the α-amylase test reveal that the
compound 2 has a good inhibitory activity with an IC value of 135.02 ± 1.43 µM, compared
5
0
with that of the reference acarbose (IC = 2.29 ± 0.21 µM) (Table 7).
5
0
In summarize, building on the results obtained from the comparison performed between 2 and
acarbose on front of α-glucosidase and α-amylase enzymes, the title compound might be used
as potent α‐ glucosidase inhibitor.
3
.10. Molecular docking
The prediction of the ligand-protein conformation was performed using iGEMDOCK
program, in order to confirm the potency of inhibition activities of (E)-N'-(5-bromo-2-
hydroxybenzylidene)isonicotinohydrazide (2) compound against Alpha-glucosidase GSJ and
ꢀ
-amylase enzymes. Also to give vision into the mechanism of action of potential antidiabetic
drugs."α-glucosidase GSJ" receptor represents an extremely common enzyme, whose role
appears in anti-bacterial defenses and the breakdown of complex carbohydrates. Several
diseases are linked to deficiencies of this protein, which can lead to problems during
development and sometimes can lead to death. The second receptor "ꢀ-amylase"is a digestive
enzyme; it is a constituent of saliva and pancreatic juice. This enzyme is linked to bacteria
that can ferment the glucose that it produces into organic acid. In this work, docking analysis
was performed to explore the binding mechanism of the molecule 2 to the ꢀ-glucosidase and
ꢀ
-amylase enzymes to allow the design of new 2-based 1CLV/2ZE0 inhibitors.
In Table 8, we present the docking energies and their distributions (VDW, H-bond, electronic
interactions and Averconpair). The best docked conformation of the ligand 2 in the binding
sites of 2ZE0 and 1CLV proteins and their 2D interactions are illustrated in Fig. 10,
respectively. Docking calculations predicted good total energy score of the title compound
with the selected target. 2-2ZE0 is more stable than 2-1CLV complexes since it has the best
1
3

inhibitory impact whose their total energy score equal to -108.68 Kcal/mol, while it is a little
weak for the 2-1CLV complex (E=-94.82 Kcal/mol). Docking results reveal that the main
interaction forces are H-bond and VDW interaction (as shown in Table 8). The two
complexes have very nearby VDW interactions, but 2-2ZE0 take the strongest VDW
interaction which is equal to -96.447 kcal/mol. Likewise, this complex presents the strongest
Hydrogen bonding interaction (-12.233 kcal/mol). While 2-1CLV complexe have the highest
AverConPair (25.52 kcal/mol). In addition, the 2 dimensional representations demonstrate the
interactions existing between our molecule and the amino acid of the selected proteins. For
2
ZE0-2, the VDW interactions were observed between 2 and A:LYS:2, A:ARG:315,
A:GLY:270, A:ASN:273 and A:TRP:317 amino residues. The A:GLU:271 and A:GLY:316
form two H-bonds with oxygen atoms (O2 and O3) of 5-bromo-2-hydroxybenzyl ring and
acylhydrazone group, respectively. Then the pyridine and 5-bromo-2-hydroxybenzyl ring
form pi-donor hydrogen bonds with A:GLU:272 and A:ASN:314 residues. Concerning 1CLV
protein, our molecule interact with the following amino acids: A:ASN:468, A:GLN:35,
A:TYR:368, A:GLY:369, A:ARG:365, A:LEU:432, A:HIS:466, A:GLY:36 and A:GLY:9
creating nine Van der Waals interactions. The carbon atom C16 of acylhydrazone group
participate with A:GLY:372 in carbon hydrogen bond. In the other hand, there are also other
types of contacts existing among the both complexes like; pi-anion, pi-cation, Amide-pi
stacked.... In order to better understand the binding of these complexes, the hydrogen bonding
interactions were also investigated and depicted in Fig. S7. This latter show that, the 5-bromo-
2
-hydroxybenzyl ring participate as an electron donor-acceptor in the same time for the both
complexes. Whereas, the pyridine ring and acylhydrazone (-CO-NH-N=CH-) group are
involved like electron acceptor in 2-2ZE0 and 2-1CLV. The main conclusion can be drawn
from these results is that the title molecule promotes inhibition of enzyme activity, i.e. it
contributes greatly and effectively to the treatment of diabetic diseases.
4
. Conclusions
In summary, new crystal, (E)-N'-(5-bromo-2-hydroxybenzylidene)isonicotinohydrazide (2)
1
13
has been synthesized and characterized by FT-IR, H- and C-NMR, ESI-MS and the (E)-
configuration of azomethine (N=CH) group was comfirmed using single-crystal X-ray
diffraction analysis. Based on the optimized structure analysis, the theoretical results
(DFT/B3LYP) are in agreement with the experimental ones. Intra-molecular interaction of the
compound was investigated via Hirshfeld surface analysis. The compound is a good non-
linear optical material candidate and the most reactive site of the compound is around the
1
4

nitrogen atoms (electrophilic attack region), based on the MEP surface. The in vitro
antidiabetic activity of the title compound was evaluated against α-glucosidase and α-amylase
enzymes, and compared to acarbose building on IC50 value demonstrating an important
inhibitory effect on front of the both enzymes especially for α-glucosidase. A molecular
docking studies showed that the 2-ꢀ-glucosidase GSJ complex is more stable than 2-ꢀ-
amylase complex, since it has the best inhibitory impact whose their total energy score equal
to -108.68 Kcal/mol. Docking results reveal that the main interaction forces are H-bond and
van der Waals interactions.
Conflicts of Interest
The authors declare no conflict of interest.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University for its funding this prolific research group no. (R.G.P. 2/115/41). This work was
also supported by PPR2-MESRSFC-CNRST-P10 project (Morocco) as well as the UM5R.
Supplementary crystallographic data: CCDC 1836620 for 2 contain the supplementary
crystallographic data for these compounds, and can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Supporting Information Available: Figures S1-S7, Tables S1 and S2.
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2
1

Caption Figures
Figure 1. The molecular structure of 2 showing the atomic labeling scheme and displacement
ellipsoids at 50 % probability level.
Figure 2. The optimized geometry of compound 2 using B3LYP/6-311++G(d,p) theory.
Figure 3. The FT-IR experimental spectrum of compound 2.
Figure 4. Views of Hirshfeld surface about reference molecule of 2, (a) mapped with the
shape-index property highlighting C-H…π/π…H-C contacts, (b) mapped over electrostatic
potential highlighting of π-π stacking interactions.
Figure 5. The full two-dimensional fingerprint plot for 2 and those delineated into H...H,
O…H/H…O, N…H/H…N, C…C, C...H/H...C, Br...H/H...Br and Br...O/O…Br contacts.
Figure 6. The energy frameworks composed of (a) electrostatic potential force, (b) dispersion
force and (c) totalenergy for cluster about a reference molecule of 2. The energy frameworks
were adjusted to the same scale factor of 80 with a cut-off value of 5 kJ mol−1 within 4 × 4 ×
4
unit cells.
Figure 7. Calculated molecular electrostatic potential contour map of 2.
Figure 8. Frontier’s molecular orbital HOMOs-LUMOs plots of the title compound with
B3LYP/6-311++G(d,p).
Figure 9. Variation of entropy (S), heat capacity (C) and enthalpy (H) with temperature for
the title molecule.
Figure 10. The best docked poses of the ligand 2 in the proteins 2ZE0 (a) and 1CLV (b) and
their 2D interactions.
2
2

Table 1
Crystal data and structure refinement for (2).
CCDC Deposition Number
1836620
Crystal data
Chemical formula
C H BrN O
2
1
3
10
3
Mr
320.15
Crystal system, space group
Monoclinic, P2 /n
1
Temperature (K)
a, b, c (Å)
β (°)3
293
8.5812 (5), 15.9004 (10), 9.3872 (6)
100.472 (2)
1259.50 (13)
4
V (Å )
Z
Radiation type
µ (mm )
Mo Kα
3.27
-
1
Crystal size (mm)
Data collection
Diffractometer
Absorption correction
0.51 × 0.28 × 0.18
Bruker APEX-II CCD
Multi-scan
SADABS Bruker 2014
0.943, 0.987
Tmin, Tmax
No. of measured, independent and observed [I> 2σ(I)] 29233, 5059, 2488
reflections
Rint
sin θ/λ)max (Å )
Refinement
0.087
0.783
−1
(
2
2
2
R[F > 2σ(F )], wR(F ), S
0.052, 0.110, 1.00
No. of reflections
5059
No. of parameters
180
No. of restrains
2
No. H-atom treatments
H atoms treated by a mixture of
independent and constrained
refinement
−3
Δρmax, Δρmin (e Å )
0.44, −0.63

Table 2
Hydrogen-bond geometry (Å, °) for (2).
D—H···A
D—H
0.829 (17)
0.830 (16)
0.93
H···A
1.83 (2)
2.225 (17)
2.85
D···A
D—H···A
149(4)
167 (2)
126
O1—H1O···N1i
2.581 (3)
3.039 (3)
3.477 (3)
N2—H2N···N3
ii
C5—H5···Cg1
Symmetry code:(i)x+1/2, -y-1/2, z+1/2; (ii) –x+1/2, y+1/2, -z-1/2

Table 3
Optimized bond lengths and bond angles (Å, °) parameters of compound (2).
Theo.
Exp.
∆(Τheor-Exp.)
Bond lengths
C1-Br1
O2-C8
O2-H
C8=O2
N1-N2
N1=C7
N1-H1O
N2-C8
N2-H2N
N3-C12
N3-C11
C4-C5
C4-C3
C5-H5
C5-C6
C6-H6
C6-C1
C1-C2
C2-H
C2-C3
C3-C7
C7-H7
C8-C9
C9-C13
C9-C10
C13-H13
C13-C12
C12-H12
C11-H11
C11-C10
C10-H10
RMSD
1.918
1.342
0.982
1.212
1.358
1.286
1.820
1.385
1.015
1.338
1.335
1.400
1.419
1.083
1.387
1.083
1.397
1.382
1.084
1.408
1.451
1.096
1.504
1.396
1.397
1.083
1.392
1.086
1.086
1.394
1.084
0.090
1.892
1.205
0.867
1.205
1.370
1.275
1.830
1.357
0.830
1.335
1.332
1.387
1.402
0.930
1.378
0.930
1.380
1.374
0.930
1.391
1.460
0.930
1.503
1.392
1.377
0.930
1.377
0.930
0.930
1.379
0.930
0.026
0.109
0.116
0.008
-0.013
0.011
0.010
0.028
0.185
0.003
0.003
0.014
0.017
0.153
0.009
0.152
0.017
0.008
0.154
0.016
-0.009
0.167
0.001
0.004
0.020
0.153
0.014
0.156
0.156
0.015
0.155
Bond angles
C4-O1-H1O
N2-N1-C7
N1-N2-C8
N1-N2-H2N
C8-N2-H2N
C12-N3-C11
O1-C4-C5
O1-C4-C3
C5-C4-C3
C4-C5-H5
C4-C5-C6
C6-C5-H
C5-C6-H
C5-C6-C1
C1-C6-H6
Br1-C1-C6
Br1-C1-C2
C6-C1-C2
C1-C2-H2
C1-C2-C3
C3-C2-H2
109.8
118.8
120.1
119.3
120.1
117.3
117.7
123.1
119.1
118.2
121.0
120.8
120.1
119.7
120.2
119.7
119.8
120.5
120.2
120.5
119.3
107.0
119.5
115.3
120.0
124.0
116.4
117.9
122.5
119.6
119.6
120.7
119.7
120.2
119.6
120.2
119.8
119.4
120.6
119.8
120.5
119.8
2.800
-0.700
4.761
-0.700
-3.900
0.900
-0.200
0.600
-0.500
-1.400
0.343
1.112
-0.127
0.136
-0.009
-0.118
0.391
-0.100
0.439
0.000
-0.510

C2-C3-C4
119.1
122.1
118.8
121.4
121.4
117.3
123.0
122.7
114.3
118.0
124.1
118.0
119.9
118.7
121.3
123.7
116.1
120.2
116.2
123.6
120.1
118.7
122.0
119.3
0.197
119.0
121.9
119.1
118.7
120.6
120.7
122.2
121.5
116.3
116.9
124.9
118.0
120.6
118.7
120.7
123.9
118.1
118.0
117.9
124.2
117.9
118.7
120.6
120.6
0.100
0.200
-0.301
2.672
0.800
-3.400
0.800
1.200
-1.966
1.020
-0.875
0.000
-0.721
0.074
0.646
-0.232
-1.964
2.196
-1.693
-0.600
2.200
0.000
1.359
-1.321
C4-C3-C7
C2-C3-C7
N1-C7-C3
N1-C7-H7
C3-C7-H7
O2-C8-N2
O2-C8-C9
N2-C8-C9
C8-C9-C13
C8-C9-C10
C10-C9-C13
C9-C13-H13
C9-C13-C12
C12-C13-H13
N3-C12-C13
N3-C12-H12
C13-C12-H12
N3-C11-H11
N3-C11-C10
C10-C11-H11
C9-C10-C11
C9-C10-H10
C11-C10-H10
RMSD

Table 4
Summary of energies calculated for intermolecular interactions in (2) (kJ mol ).
-1
Contact
R(Å) Eele Epol Edis Erep Etot
N2-H2N...N3
H7...H11
9.89
4.40
-43.1 -15.7 -22.8 37.9 -43.9
H7...C11
Cg(C1-C6)…...Cg (C9-C11/N3/C12-C13)
C6…C12
-34.6
-5.8
-7.2
-1.2
-6.3
-2.4
-89.0 52.1 -77.9
-19.7 12.5 -14.3
9
.31
C5-H5…Cg (C9-C11/N3/C12-C13)
O1…H12
9.78
-20.3
-4.1
-18.7
7.8
-35.3
-5.1
Br1…H13
1
1.51
-11.8 13.9
Br1…O2