Synthesis, structure analysis, biological activity and molecular docking studies of some hydrazones derived from 4-aminobenzohydrazide

Article abstract of DOI:10.1016/j.molstruc.2020.129354

(E)-4-Amino-N′-(substituted benzylidene) benzohydrazides (1-8) were synthesized by condensing 4-aminobenzohydrazide with an appropriate aldehyde in the molar ratio of 1:1 in methanol as solvent. Single crystal X-ray diffraction has been recorded for compo

Full text of DOI:10.1016/j.molstruc.2020.129354

Journal of Molecular Structure 1226 (2021) 129354
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, structure analysis, biological activity and molecular docking
studies of some hydrazones derived from 4-aminobenzohydrazide
S. Senthilkumara,∗, J. Seralathana, G. Muthukumaranb
a Department of Chemistry, Annamalai University, Annamalainagar, Chidambaram, Tamil Nadu 608 002, India
b Department of Chemistry, Krishnasamy College of Engineering and Technology, Cuddalore, Tamil Nadu 607 109, India
a r t i c l e i n f o
a b s t r a c t
ꢀ
Article history:
(E)-4-Amino-N -(substituted benzylidene) benzohydrazides (1-8) were synthesized by condensing 4-
Received 25 June 2020
aminobenzohydrazide with an appropriate aldehyde in the molar ratio of 1:1 in methanol as solvent.
Single crystal X-ray diffraction has been recorded for compound 1. For all the synthesized compounds,
FT-IR, 1H and 13 C NMR spectra have been recorded. Also antibacterial and docking studies have been done
for all compounds 1-8. Since for compound 1 only single crystal X-ray analysis has been carried out, its
structural characterization have been done in detail, characterisation of remaining compounds 2-8 have
been carried out by comparing their FT-IR, 1H and 13 C NMR spectral data with that of compound 1. Single
crystal X-ray analysis of 1 show that the compound exists in E-configuration about azomethine double
bond. The crystal state of the compound was stabilised by the hydrogen bond present in the molecule.
DFT calculations using the B3LYP/6-311++G method have been carried out for 1 to get the optimized
geometry, IR and NMR frequencies, Mulliken charge distribution, molecular electrostatic potential (MEP)
and energies of HOMO – LUMO. A good agreement between the DFT predicted and the experimentally
measured IR and NMR spectra of 1 was found. Analysis of MEP and Mulliken atomic charges bar diagram
of 1, confirms the chemical reactivity and existence of intermolecular hydrogen bonding of the molecule.
The result of antibacterial and antifungal activities shows that the compounds 2, 4, 7 and 8 showed good
antibacterial activity against the tested bacteria and the compounds 1, 3, 5 and 6 showed good antifun-
gal activities against all the three fungal strains. These results showed that electron donating and electron
withdrawing groups play important roles in antibacterial and antifungal activities of the compounds1-8,
respectively. Molecular docking studies of compounds 1-8 shows that the various ligand of compounds
1-8 shows glide score values lies between -5.923 to -7.298, among these the compounds 6 and 8 showed
excellent glide score values.
Revised 26 September 2020
Accepted 27 September 2020
Available online 28 September 2020
Keywords:
4-Aminobenzohydrazide
Crystal structure
Antimicrobial activity
Molecular docking
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
berculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobac-
terium microti and Mycobacterium canetti. Of these, Mycobacterium
Generally, Schiff bases were synthesized through a condensa-
tion reaction between a primary amine and an aldehyde or ketone
in an alcoholic solvent like methyl alcohol or ethyl alcohol [1]. Hy-
tuberculosis (M. tuberculosis) is the most contributing agent for TB,
caused by the slow-growing bacteria that are frequently found in
the lungs. Such kind of growing bacteria can be inhibited using
hydrazones. Moreover, many reports are evidence that the hydra-
zones acquire a range of biological activities [11–18] such as anti-
tuberculosis and antibacterial, at present a variety of research ac-
tivities regarding Schiff bases are in progress.
–
–
= –
drazones are characterized by the existence of HN N
C type of
linkage between two aromatic phenyl rings. These types of bases
have vast number of applications [2–7] such as catalysts, bioactive
molecules, organic dyes and liquid crystals. Also they have been
used as insecticides, sterilants and herbicides [8,9].
In an earlier study [19], we reported the synthesis and charac-
terization of (E)-4-amino-N’-(2,5-dimethoxybenzylidene) benzohy-
drazide using single crystal X-ray diffraction, FT-IR, FT-Raman, UV–
vis and NMR spectra. In the current study, we synthesised eight
M. tuberculosis has been the major health threat that remains
to be the leading cause of mortality [10]. Tuberculosis (TB) is
caused by five types of mycobacteria such as Mycobacterium tu-
ꢀ
(E)-4-amino-N -(substituted benzylidene) benzohydrazides (1-8).
Among these, only for compound 1 single crystal X-ray diffraction
have been recorded. For all the synthesized compounds 1-8, FT-IR,
1H and 13C NMR spectra have been recorded and also antibacte-
∗
Corresponding author.
E-mail address: ss08058@annamalaiuniversity.ac.in (S. Senthilkumar).
https://doi.org/10.1016/j.molstruc.2020.129354
0022-2860/© 2020 Elsevier B.V. All rights reserved.

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
rial, antifungal activities and docking studies have been done. For
compound 1 structural characterization has been done in detail,
characterisation of remaining compounds 2-8 have been carried
out by comparing their FT-IR, 1H and 13C NMR spectral data with
that of compound 1. Further, for all the synthesized compounds 1-
8 antibacterial, antifungal activities and docking studies have been
done.
2.1.7. Molecular docking studies
The following process was used for molecular docking stud-
ies. The ligand for docking studies was prepared using LigPrep,
Schrodinger. The ligand has been geometrically refined and as-
signed an appropriate protonation state at pH 7.0
2.0. The
strength minimization has been carried out using the aid of OPLS
2005 pressure subject. The target protein enoylacyl and carrier pro-
tein reductase (InhA) from Mycobacterium tuberculosis was down-
loaded from the Protein Data Bank (PDB id: 2NSD) and the active
site was chosen. The grid was assigned by picking the ligand as
the center of the grid and then the grid box was generated by ap-
plying default parameters. The docking change has been finished
using GLIDE, Schrodinger, GLIDE XP (extra precision) technique.
2. Experimental details
2.1. General
2.1.1. Melting point determination
Melting point was taken in open capillaries and it was uncor-
rected.
ꢀ
2.1.8. Synthesis of (E)-4-amino-N -(substituted benzylidene)
benzohydrazide (1-8)
2.1.2. IR spectrum
4-Aminobenzohydrazide (2 mmol) and appropriate aldehyde
(2 mmol) were dissolved in (25 ml) methanol along with few
drops of acetic acid to get a clear solution. The reaction mix-
ture was taken in a RB flask and refluxed on a water bath for
about 3 h [Scheme 1]. The progress of the reaction was moni-
tored by thin layer chromatography (TLC). After completion of the
reaction, methanol was removed by vacuum distillation. The sepa-
IR spectra were recorded on an AVATAR 330 FT-IR spectrometer
in KBr pellets.
2.1.3. NMR spectra
1H and 13C NMR spectra were recorded on a Bruker Avance
FT NMR spectrometer operating at 400.23 MHz for 1H and 100.62
MHz for 13C. Solutions for recording 1H and 13C NMR spectra were
prepared by dissolving about 10 mg and 50 mg of the compounds,
respectively, in 0.5 mL DMSO–d6. The number of data points for
1H and13C NMR was 16 K and 32 K, respectively. All NMR mea-
surements were made using 5 mm tubes.
ꢀ
rated (E)-4-amino-N -(substituted benzylidene) benzohydrazides 1-
8 were recrystallized from methanol. The melting points of the
crystallized products were determined in open capillaries.
3. Results and discussion
2.1.4. Single crystal X-ray crystallography
3.1. Synthesis and crystal structure
Determination of the unit cell and data collection were per-
formed on a Bruker AXS kappa APEX 3 CMOS diffractometer us-
ꢀ
(E)-4-Amino-N -(substituted benzylidene) benzohydrazides (1-
˚
ing graphite-monochromated Mo-Kα radiation (λ = 0.71073 A) at
8) have been synthesised using Scheme 1.
296 K. Multi scan absorption corrections were applied using the
SADABS (Bruker, 2016) program. The structure was solved and re-
fined using SHELXT-2014/5 (Sheldrick, 2014)) and SHELXL-2014/7
(Sheldrick, 2014), respectively. All non-hydrogen atoms were re-
fined anisotropically, whereas the hydrogen atoms were refined
isotropically. The positions of the hydrogen atoms are identified
from the difference electron density maps and their distances are
geometrically optimized.
3.2. Preparation of compound 1 crystal for X-ray crystallographic
study
ꢀ
The recystallized (E)-4-amino-N -(3-bromobenzylidene) benzo-
hydrazide (1) was dissolved in distilled methanol and left for evap-
oration. The crystal was formed after four days. The supernatant
liquid was decanted. The crystal suitable for X-ray analysis was
obtained after washing with methanol. Since single crystal X-ray
crystallography has been recorded for 1 only, its characterization
has been discussed in detail [23]. For compounds 2-8, assignment
of IR vibrational frequencies, 1H and 13C chemical shifts have been
carried out by comparing it with that for compound 1.
2.1.5. Computational methods
DFT calculations [20,21] were made using the B3LYP/6-311++G
method. The calculated frequencies of IR and NMR values confirm
the stability of compound. DFT calculations for optimized struc-
ture of 1 used to analysis the structural properties such as fron-
tier molecular orbitals, Mulliken charge and molecular electrostatic
map. Hirshfeld surfaces and 2D fingerprint plots of 1 were calcu-
lated using Crystal Explorer 3.0 [22].
Compound 1 was crystallized from methyl alcohol as single
crystals using the slow evaporation process and characterized by
the X-ray diffraction method. The parameters of unit cell for 1 are
˚
˚
˚
a = 9.1603(2) A, b = 10.0727(2) A, c = 14.3439(3) A, α = 90°,
˚
β = 90°, γ = 90°.and Z = 4, V = 1323.50(5) A3. An ORTEP view
2.1.6. Antibacterial activity
with atom labelling system of 1 is shown in Fig. 1. X-ray diffraction
data and its refinements are listed in Table 1. Compound 1 crystal-
lized in the Orthorhombic crystal system, with Pna21 space group
Antibacterial activity of the synthesized compounds 1-8 was
carried out using the standard disk diffusion method. For this
study Ciprofloxacin has been used as standard. The sample and
– =
–
bond.
and was found to adopt an E-configuration about its
C
N
the standard solutions were prepared by dissolving 100 μg
̸
mL in
In 1, the hydrazone linkage plays an important role. The bond
˚
=
=
DMSO. Nutrient agar medium was inoculated with different mi-
croorganisms and once the media was solidified, it was punched
with a 6 mm diameter well. To the agar plates containing bacteria
the sample and the standard solutions were added and incubated
at 37 °C for 24 h. After incubation, the diameter of the zone in-
hibition was measured. The inhibitory activity of DMSO was also
employed as a negative control. Then the antibacterial activity of
sample solutions was evaluated by comparing it with the standard
solution.
length of carbonyl (C8 O1) and C7 N1 is observed as 1.236 A and
˚
1.266 A, respectively, which is exactly matches with the literature
[24,25]. The bromo benzylidene phenyl and amino phenyl rings of
1 were found to joined together through an open chain carbonyl
– =
–
–
–
hydrazone ( C N NH CO ) system, in which all the atoms are in
the same plane. Besides, both amino benzoyl phenyl and bromo
benzylidene pheny rings of 1 are also lie in the same plane formed
– =
–
bond. The resulted bond lengths and bond angles are in
by
C
N
good agreement with the standard values.
2

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
ꢀ
Scheme 1. Synthesis of (E)-4-amino-N -(substituted benzylidene) benzohydrazides (1-8)
Fig. 1. ORTEP view with atom labelling system of 1.
Bond lengths and bond angles associated with various H-
Furthermore, the various bond lengths of both amino ben-
zoyl phenyl and bromobenzylidene phenyl rings of the optimized
geometry are listed in Table 3, which clearly indicates that all
the bond lengths of both the phenyl rings are in good agree-
ment between the calculated and experimental values. Also, it was
clear that all bond lengths of both phenyl rings are almost equal.
Moreover, analysis of various bond lengths apart from the phenyl
rings the following observations were obtained. The bond lengths
bonding [26–29] are presented in Table 2. Wherein, the atoms
C(7),N(2) and N(3) acted as proton donors and O(1) acted as pro-
ton acceptor, which indicates that, the molecule of 1 possess three
donor atoms and one acceptor atom for producing intermolecu-
lar H-bond. The observed C(7)−H(7)•••O(1); N(3)−H(3B)•••O(1);
and N(2)−H(2A)•••O(1) intermolecular hydrogen bonds stabilises
the crystal structure.
˚
˚
˚
–
–
–
–
of C12 N3,C8 N2,N1 N2 and C3 C7 are 1.370 A,1.363 A,1.373 A &
˚
1.479 A respectively, they are in accordance with the theoretical
3.3. DFT quantum chemical calculations for optimized structure
˚
values[23]. Since the values range between 1.363 and 1.479 A it in-
dicates that the above bonds are single bond in nature. Similarly,
For the optimized structure of 1 shown in Fig. 2, DFT quantum
chemical calculations have been carried out using the B3LYP/6-
311++G basis set. The values of bond lengths, bond angles and tor-
sion angles obtained by DFT calculation [23] were compared with
the experimental values. Since the calculated and theoretical val-
ues were in good agreement, it confirms that the Fig. 2 is most
stable optimized structures of 1.
˚
˚
–
–
the bond lengths of C8 O1 and C7 N1 are 1.236 A and 1.266 A re-
spectively, they are in accordance with the theoretical values [24].
These bond length values indicate the double bond nature.
Analysis of bond angle values gives an idea about the orienta-
tion of phenyl rings. Generally, if the phenyl rings are symmetry
in nature all the bond angles should be around the normal angle
3

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Table 1
Crystal data and structure refinement of compound 1
Parameters
Values
CCDC NO
1837652
C14 H12 Br N3
318.18
Empirical formula
Formula weight
O
Temperature
296(2) K
˚
Wavelength
0.71073 A
Crystal system
Orthorhombic
Pna21
Space group
˚
Unit
a = 9.1603(2) A
α= 90°.
β= 90°.
γ = 90°.
˚
cell
b = 10.0727(2) A
˚
dimensions
c = 14.3439(3) A
˚
Volume
1323.50(5) A3
Z
4
Density (calculated)
Absorption coefficient
F(000)
1.597 Mg/m3
3.101 mm-1
640
Crystal size
0.150 × 0.100 × 0.100 mm3
3.006–28.747°
Theta range for data collection
Index ranges
−12<=h<=12, −13<=k<=13, −18<=l<=19
Reflections collected
Independent reflections
Completeness to theta = 25.242°
Absorption correction
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2sigma(I)]
R indices (all data)
Absolute structure parameter
Extinction coefficient
Largest diff. peak and hole
30724
3335 [R(int) = 0.0375]
99.3%
Semi-empirical from equivalents
0.7453 and 0.5805
Full-matrix least-squares on F2
3335/5/182
1.048
R1 = 0.0376, wR2 = 0.0875
R1 = 0.0494, wR2 = 0.0935
−0.001(4)
n/a
˚
0.584 and −1.316 e.A-3
Fig. 2. Optimized structure of 1 crystal.
Table 2
Hydrogen bonds of compound 1 [A and °].
–
–
metry in nature. Whereas the bond angles of C10 C9 C8 (123.1°)
˚
–
–
is greater than that of C14 C9 C8 (118.3°), which indicates clearly
that the amino benzoyl phenyl ring is not in symmetry that it is
slightly tilting towards the carbonyl group due to some force of
attraction.
–
H...A
–
D
d(D H)
d(H...A)
d(D...A)
<(DHA)
–
C(7) H(7)...O(1)#1
0.93
2.66
3.438(5)
3.011(6)
3.000(4)
142.0
–
N(3) H(3B)…O(1)#2
0.87(2)
0.82(2)
2.17(3)
2.18(3)
164(5)
170(4)
–
N(2) H(2A)...O(1)#1
3.4. Vibrational frequencies analysis
Symmetry transformations used to generate equivalent atoms:
#1 x+1/2, -y+1/2, z #2 -x+1, -y, z-1/2 #3 x, y, z-1
The solid phase FT-IR spectrum [30–36] of compound
1
(Plate 1) was recorded and the vibrational frequencies were com-
pared with that obtained through a calculation using B3LYP/6-
311++G basis set (Plate 2). The experimental and calculated vi-
–
–
–
–
of 120°. Here, the bond angles of C4 C3 C7 (120.8°) and C2 C3 C7
(119.7°) indicates that the bromo benzylidene phenyl ring is sym-
4

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Table 3
The theoretical and experimental bond lengths for both phenyl rings of 1.
Aminobenzoyl phenyl ring
Bromobenzylidene phenyl ring
˚
˚
˚
˚
Bond
Bond length A (calculated)
Bond length A (experimental)
Bond
Bond length A (calculated)
Bond length A (experimental)
–
–
C9 C10
1.402
1.383
1.406
1.404
1.387
1.401
1.391(5)
1.378(6)
1.399(6)
1.383(6)
1.385(6)
1.392(6)
C1 C2
1.384
1.405
1.4
1.387(5)
1.399(6)
1.395(5)
1.376(6)
1.389(6)
1.364(6)
–
–
C2 C3
C10 C11
–
–
C3 C4
C11 C12
–
–
C12 C13
C4 C5
1.392
1.392
1.395
–
–
C5 C6
C13 C14
–
–
C1 C6
C9 C14
Plate 1. Experimentally recorded FT-IR spectrum of 1.
Plate 2. FT-IR spectrum of 1 crystal predicted at the B3LYP/6-311++G basis set.
3.5. Analysis using 1H NMR and 13 C NMR spectra
brational wave numbers of 1 were tabulated in Table 4. Since the
calculated and experimental vibrational wave numbers of 1 are al-
most matches with each other, it confirms the optimized structure
(Fig. 2) of compound 1.
1H and 13C NMR spectra have been recorded for compounds 1-
8. Assignments of 1H NMR signals have been made using the posi-
tion, integral values and coupling constants of the signals [37]. The
1H NMR spectrum of compounds 1 is displayed in Plate 3, there
is a signal at 5.81 ppm corresponding to two protons is due to
the amino protons. Another signal at 8.35 ppm corresponds to one
Furthermore, the solid phase FT-IR spectra of compound 2-8
were recorded and the assignments of vibrational frequencies were
made by comparing it with that of compound 1 and were tabu-
lated along with its physical data in Table 5.
5

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Plate 3. 1H NMR spectrum of 1.
proton is due to the azomethine proton. The signal at 11.58 ppm
corresponding to one proton is due to the NH proton. There are
five signals in the range 6.59 ppm to 7.89 ppm corresponding to
eight protons are due to aromatic protons.
Table 4
Comparison of some selected experimental and calculated vibrational
wave numbers (cm−1) of 1 at the B3LYP/6-311++G level of theory
Assignments
Experimental values
Calculated values
From the observed frequencies of lines due to aromatic protons,
the various proton-proton coupling constants were determined.
These are as follows:
–
–
C
C
C
C
N
N
H str.
N str.
3053
1142
1606
1630
950
3046
1138
1597
1624
1068
3334
1471
=
=
N str.
O functional group
N str.
J5,4 & J5,6 [Bromobenzylidene phenyl] = 8.0 Hz [from the signals
at 6.59 and 7.41 ppm]
–
–
H str.
3337
1473
J2,3[Aminobenzoyl phenyl]
7.60 ppm]
=
8.8Hz [from the signal at
=
C
C str.
Str.= stretching.
Table 5
IR Vibrational frequency (cm−1) and physical data of compounds 1-8.
Compound
Assignments
1
2
3
4
5
6
7
8
–
–
C
C
C
C
N
N
H (aromatic)
N str.
3053
1142
1606
1630
950
3035
1133
1598
1630
1030
3313
1456
-
3026
1130
1592
1621
1080
3339
1488
618
3035
1142
1503
1627
1080
3334
1364
-
3035
1145
1595
1627
1098
3343
1473
615
3029
1136
1598
1624
1062
3340
1482
704
3065
1136
1598
1648
1027
3378
1491
-
2991
1130
1605
1640
1022
3343
1462
-
=
=
N str.
O
–
–
N str.
H str.
3337
1473
831
=
C
C str.
Halogens
mp.a (°C)
Yield %
210-212
80
210-212
80
210-212
84
210-212
90
208-210
78
216-218
80
208-210
74
220-222
80
a
The reported melting points are uncorrected Str.= stretching
6

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Plate 4. 13 C NMR spectrum of 1.
J5,6[Aminobenzoyl phenyl] =8.8 Hz [from the signal at
7.68 ppm]
The signal at 142.45 ppm is due to the amino bearing carbon of
aminobenzoyl phenyl ring. The high intense signals at 127.38 and
117.80 ppm are due to the ortho and meta carbons of aminoben-
zoyl phenyl ring, respectively. The signals at 136.19 and 131.23 ppm
are assigned to the ortho carbons of the bromobenzylidene phenyl
ring. Among these, one of the ortho carbon is deshielded due to
magnetic anisotropic effect of loan pair present in the bromine,
hence the deshielded signal at 136.19 ppm is assigned to the or-
tho carbon C-2 of bromobenzylidene phenyl ring. The low intense
signal at 134.71 ppm is due to the ipso carbon of bromobenzyli-
dene phenyl ring. The signal at 137.35 ppm is due to the bromo
bearing carbon. The signal at 134.01 ppm is due to the para car-
bon of bromobenzylidene phenyl ring. The signal at 124.42 ppm is
due to the meta carbon of bromobenzylidene phenyl ring.
The assigned 1H and 13C NMR signals of compound 1 are in
accordance with the chemical shift values obtained through theo-
retical using the B3LYP/6-311++G basis set [38–41]. The assigned
NMR values of 1 confirm the proposed structure shown in Fig. 2.
The 1H and 13C NMR chemical shift values of 1 along with its the-
oretical values were tabulated in Table 6.
Thus, the individual assignments of aromatic signals were
made using the coupling constants. Since the signals at 6.59
and 7.41 ppm are having the same value of coupling constant,
which indicates that, these signals belong to the bromobenzyli-
dene phenyl protons. Hence, the doublet at 6.59 ppm correspond-
ing to two protons is assigned to the H-4 & H-6 protons and the
triplet at 7.41 ppm corresponding to one proton is assigned to the
H-5 proton of bromobenzylidene phenyl ring. The singlet at 7.89
ppm corresponding to one proton is assigned to the H-2 proton
of bromobenzylidene phenyl ring. Similarly, the signals at 7.60 and
7.67 ppm are having the same coupling constant, which indicates
that, these signals belong to the aminobenzoyl phenyl ring pro-
tons. The doublet at 7.60 ppm corresponding to one proton is as-
signed to the H-3 proton of aminobenzoyl phenyl ring. The signal
at 7.67 ppm corresponding to three protons seems to be a merged
signal, is due to H-2, H-5 and H-6 protons of aminobenzoyl phenyl
ring.
Assignments of 13C NMR signals have been made using the po-
sition and intensity of the signals. The 13C NMR spectrum of 1 is
displayed in Plate 4, there is a signal at 168.31 ppm which is due to
carbonyl carbon and the signal at 152.65 ppm is assigned to azoni-
Assignments of 1H NMR signals for compounds 2-8 have been
made using the position, integral values and also has been com-
pared with the compound 1. In the 1H NMR spectra of compounds
2-8, there is a signal in the range 5.70–5.81 ppm corresponding
to two protons. This signal is assigned to the amino protons. An-
other signal in the range 8.26–8.38 ppm corresponds to one pro-
=
trile carbon (C N). The signals obtained in the range of 117.80–
149.12 ppm are due to aromatic carbons. Among these the signal at
149.12 ppm is due to the ipso carbon of aminobenzoyl phenyl ring.
7

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Table 6
The experimental and calculated 1H and 13 C chemical shifts of 1.
Proton chemical shift, ppm
Carbon chemical shift, ppm
Theoretical values
B3LYP/
Theoretical values
B3LYP/
Experimental
values
Experimental
values
Proton
6-311++G(d,p)
Carbon
6-311++G(d,p)
NH2
H-4 & H-6b
H-5b
5.818
6.59
7.41
4.22
4.24
7.28
metaa
metab
orthoa
ortho C-6b
parab
117.80
124.42
127.38
131.23
134.01
134.71
136.19
137.35
142.45
149.12
157.65
117.6
128.8
130.0
131.1
133.1
137.1
138.9
141.5
143.0
149.2
155.4
H-3a
7.60
7.67
7.81
8.18
ipsob
orthoC-2b
Br-bearing
NH2 -bearing
ipsoa
H-2,H-5 &
H-6a
b
=
H-2
7.896
8.353
11.589
8.94
8.97
9.42
C
N
=
CH
NH
N
=
C
O
168.31
163.5
a
Aminobenzoyl phenyl.
b
Bromobenzylidene phenyl.
Table 7
1H NMR Chemical shifts (ppm) of 1-8.
Compound
Proton
1
2a
3a
4a
5a
6a
7a
8a
Aromatic
Methoxyl
Methyl
NH2
6.58–7.89
6.57–7.66
3.80
6.58–7.72
6.58–7.68
-
6.59–7.74
6.59–7.69
6.58–7.99
3.83
6.51–7.61
3.74 & 3.73
-
-
-
-
-
-
-
-
2.34
-
-
-
5.81
8.35
11.58
5.76
5.80
8.38
11.51
5.77
5.81
8.37
11.59
5.81
8.37
11.54
5.79
5.70
=
CH
NH
N
8.34
8.36
8.36
8.26
11.32
11.38
11.48
11.30
a
Assignment were made compared with compound 1
Table 8
13 C NMR Chemical shifts (ppm) of 1-8.
Compound
Carbon
1
2a
3a
4a
5a
6a
7a
8a
Methyl
Methoxy
Aromatic
=
-
-
-
21.47
-
-
-
-
-
-
-
-
55.74
-
55.73
56.01& 55.88
108.53–129.74
152.67
117.80–149.12
157.65
113.03–129.70
152.63
160.98
113.80–137.49
152.53
113.07–139.87
152.71
113.67–137.48
152.89
113.07–137.49
113.22–138.48
152.79
C
C
N
0
152.85
163.51
=
168.31
163.53
163.44
163.63
163.20
163.39
a
Assignment were made compared with compound 1.
1.48 × 10−30esu., respectively. Since, the first order hyperpolariz-
ability of 1 is greater than that of urea (β = 0.3728 × 10−30esu)
[19,40], it confirms the nonlinearity characteristics and electronic
properties of 1.
ton and it is due to the azomethine proton. There is a signal in the
range 11.30–11.59 ppm corresponding to one proton. This is due to
the NH protons. The signals in the range 6.51–7.99 ppm are due to
aromatic protons. The 1H NMR chemical shifts of 1-8 are given in
Table 7.
Similarly, the assignments of 13C NMR signals have been made
for compounds 2-8 using the position and intensity of the signals.
In the 13C NMR spectra of compounds 2-8, there is a signal in the
range 160.98–168.31 ppm is due to carbonyl carbon and the signal
in the range 152.53–157.65 ppm is assigned to azonitrile carbon
3.7. Frontier molecular orbitals
Frontier molecular orbitals (FMOs) play a vital role [23] in de-
termining the stability, optical properties and chemical reactivity
of molecules. Hence, we calculated the energies of highest occu-
pied molecular orbital (HOMO), lowest unoccupied molecular or-
bital (LUMO) using DFT-B3LYP/6-311++G.
=
(C N). The signals obtained in the range 108.53–149.12 ppm are
due to aromatic carbons. The 13C NMR chemical shifts of 1-8 are
given in Table 8.
The highest occupied molecular orbital (HOMO) acts as an elec-
tron donor and the lowest unoccupied molecular orbitals (LUMO)
acts as an electron acceptor. If a molecule possesses lower energy
gap between the HOMO–LUMO, this indicates that the molecule
is lower stability, high polarizability and greater chemical reac-
tivity in nature. Whereas, if a molecule possesses higher energy
gap between the HOMO–LUMO), this indicates that the molecule
is higher stability, lower polarizability and chemical reactivity in
nature.
3.6. First order hyperpolarizibility analysis
The first order hyperpolarizability (β0), dipole moment (μ)
and polarizability (α0) of 1 was calculated using B3LYP/6-311++G
(d,p) and they are reproduced in Table 9. The mean polarizabil-
ity α0 and total polarizabilityꢀαof 1 were −1.7 × 10−30esu and
6.12 × 10−30esu., respectively. The total molecular dipole mo-
ment and first order hyperpolarizability were 7.0929 Debye and
8

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Table 9
The carbonyl carbon atom C(14) of 1 bear the highest neg-
ative charge of −0.814 a.u., and the C(6),C(8),C(18) and C(15)
atoms also bear high negative charges of −0.430,−0.328, −0.321
and −0.311 a.u., respectively. Similarly, the carbon atom C(5) of
the amino benzoyl phenyl ring and C(13) of bromo benzylidene
phenyl ring of 1 bear maximum positive charges of 0.744 a.u.
and 0.516 a.u., respectively, and the N(11), H(31), H(25), H(24) and
H(22) atoms also bear high positive charges of 0.279, 0.280, 0.246,
0.246 and 0.210 a.u., respectively. Thus, the presence of atoms with
high negative charges (electron donor) and the atoms with high
positive charges (electron acceptor) confirms the existence of in-
termolecular hydrogen bonding in 1.
The molecular electric dipole moment μ (Debye), po-
larizability (α0), hyperpolarizability (β0) energies of
compound 1.
Parameters
B3LYP/6-311++G(d,p)
Dipole moment (μ)
μx
μy
μz
Debye
1.6485
4.7013
−2.8789
μ
7.0929
Polarizability (α0)
αxx
αxy
αyy
αxz
αyz
×10−30 esu
−87.9303
0.6341
−105.3837
−10.3227
2.3984
3.9. Molecular electrostatic potentials
αzz
α0
−116.8643
−1.7
Molecular electrostatic potential (MEP) map shows the distri-
butions of electron density on the surface of a molecule. MEP
maps are used to analyse the electrophilic and nucleophilic re-
actions, interactions due to hydrogen bonding and chemical reac-
tivities [23,42]. Molecular electrostatic potential for the optimized
crystal structure of 1 has been calculated using B3LYP/6-311 ++G
(d,p) basis set [43]. The molecular electrostatic potential map of 1
contains positive and negative potentials which were represented
by blue and red colour, respectively. Since, the electrophiles are at-
tracted towards the negative regions and the nucleophiles are at-
tracted towards the positive regions, in 1 the carbonyl oxygen and
bromo at bromobenzylidene phenyl ring acts as electrophilic sites
and undergo electrophilic interaction.
ꢀα
Hyperpolarizability (β0)
6.12
×10−30 esu
21.7774
−3.4919
8.4570
βxxx
βxxy
βxyy
βyyy
βxxz
βxyz
βyyz
βxzz
βyzz
βzzz
β0
17.0647
−110.4726
−2.9977
−7.7185
26.3591
2.4365
0.6537
1.48
esu – electrostatic unit;
Standard value for urea
β0 = 0.3728× 10−30 esu.
=
1.3732 Debye;
4. Antimicrobial activity
4.1. Antibacterial activity
Antibacterial activity [44–47] of the synthesized compounds 1-
8 was carried out using standard disk diffusion method. In this
study the compounds 1-8 were screened against two gram positive
bacterial strains viz., Staphylococcus aureus, Streptococcus pyogens
and two gram negative bacterial strains viz.,Escherichia coli (ATCC
25922) [48] and Pseudomonas aeruginosa along with Ciprofloxacin
as standard. The antibacterial activity of the sample solution was
evaluated by comparing it with the standard solution.
The analysis shows that the compounds 1-8 have shown mod-
erate to excellent inhibitory activity against the tested bacteria.
Among these, the compound 8 showed excellent antibacterial ac-
tivity and compounds 2, 4 and 7 showed good antibacterial activ-
ity against all four bacterial pathogens. Further, the compounds 1,
3, 5and 6 showed less activity against all four bacterial pathogens
comparatively. These results provided clear evidence that the elec-
tronic property of the compounds has a close relationship to their
biological activity [49,50]. That is, the compounds (2, 4, 7 and 8)
having electron donating [51,52] groups (OH and OCH3) on the
benzene ring showed good antibacterial activity against the tested
bacteria, whereas, the compounds (1,3, 5 and 6) having electron
withdrawing groups (Cl and Br) on the benzene ring showed less
antibacterial activity against the tested bacteria. This evidence con-
firmed that suitable functional groups on phenyl ring were neces-
sary for better antibacterial activities in drug design [53]. Hence,
these results implied that electron donating groups play important
roles in the antibacterial activities of the compounds 1-8.
Fig. 3. Bar diagram of Mulliken atomic charges for 1 crystal.
For 1, the calculated energy of HOMO and LUMO is
−2.94086 eV and −8.0743 eV, respectively. From these, the en-
ergy band gap between HOMO–LUMO, is calculated as 3.64179 eV.
Since, the obtained energy band gap (Eg) value is high, which con-
firm the compound is more stable.
3.8. Mulliken charge distributions
To understand the dipole moments, polarizabilities, electronic
structures and chemical reactivities of molecules, partial atomic
charges [23] play a vital role. Hence, we calculated the atomic
4.2. Molecular docking studies
To find a suitable inhibitor for Mycobacterium tuberculosis dock-
ing studies [44,54] was carried out for InhA protein. InhA catalyzes
the reduction of lengthy chain trans-2-enoyl-ACP inside the kind II
fatty acid biosynthesis pathway of M.Tuberculosis [55]. Inhibition of
charges and their distributions of
1 using the DFTB3LYP/6-
311++G(d,p) method. The bar diagram of Mulliken atomic charges
is shown in Fig. 3.
9

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Fig. 4. Compound 1 docked in active site of 2NSD and interaction details.
Table 10
Molecular docking results of compound 1 with 2NSD.
Compound Glide gscore Glide evdw
Glide ecoul
Glide energy
Interacting residues
1
−6.556
−40.338
−7.301
−1.198
−47.64
H- Bond interaction with SER 94
H- Bond interaction with HOH
H- Bond interaction withASP 64
Pi-Pi stacking with PHE 41
-
2
−6.184
−39.598
−40.796
3
4
5
6
−6.695
−5.923
−6.688
−7.298
−38.266
−38.17
−4.741
−5.042
−5.553
−6.197
−43.007
−43.212
−45.501
−43.121
-
−39.947
−36.923
Halogen bond with ILE194
H- Bond interaction with HOH
H- Bond interaction withSER 94
H- Bond interaction with HOH
H- Bond interaction with HOH
H- Bond interaction withSER 94
Pi-Pi stacking with PHE 149, LYS 165, Water hydration.
7
8
−6.331
−6.951
−38.432
−40.609
−4.48
−42.913
−46.454
−5.845
Isoniazid(standard)
−13.042
−50,841
−6.158
−57.000
glide evdw = van der Waals interaction energies, glide ecoul = Coulomb interaction energies.
InhA disrupts the biosynthesis of the mycolic acids that had been
valuable components of the mycobacterial cellular wall.
Molecular docking studies have been carried out for compounds
1-8. The docking interaction of compound 1 is shown in Fig. 4.
The results of the docking studies and their interaction energy are
shown in Table 10. Analysis of the results shows that the various
ligand of compounds 1-8 shows a high score of glide score values
lies between −5.923 and −7.298. Among these the compounds 6
and 8 showed excellent glide score values compare to others due
to the H-bond interaction with water and SER 94. Therefore based
on the docking studies, the compounds 1-8 may be appropriate to
overcome the drug resistance of Mycobacterium tuberculosis InhA
protein.
ing electron withdrawing group showed good antifungal activities.
Molecular docking studies indicated that all the compounds 1-8
show good glide score values, among these the compounds 6 and 8
showed excellent glide score values due to the existence of H-bond
interaction.
Declaration of Competing Interest
We wish to confirm that there are no known conflicts of inter-
est associated with this publication and there has been no signifi-
cant financial support for this work that could have influenced its
outcome. We confirm that the manuscript has been read and ap-
proved by all named authors and that there are no other persons
who satisfied the criteria for authorship. We further confirm that
the order of authors listed in the manuscript has been approved by
all of us. We understand that the Corresponding Author is the sole
contact for the Editorial process (including Editorial Manager and
direct communications with the office). He is responsible for com-
municating with the other authors about progress, submissions of
revisions and final approval of proofs. We confirm that we have
provided a current, correct email address which is accessible by
the Corresponding Author.
5. Conclusions
ꢀ
We report eight new synthesized (E)-4-amino-N -(substituted
benzylidene) benzohydrazides (1-8). Single crystal X-ray crystal-
lographic analysis of
1 shows that the molecule exists in E-
– =
–
bond. The crystal state of the
configuration about the
C
N
compound was stabilised by the hydrogen bond present in the
molecule. For 1 the optimized geometry, IR and NMR frequen-
cies, Mulliken charge distribution, molecular electrostatic po-
tential (MEP) and energies of HOMO
– LUMO were calcu-
lated through density functional theory (DFT) using the B3LYP/6-
311++G method. A good agreement between the DFT predicted
and the experimentally measured IR and NMR spectra of 1 was
found. The result of antibacterial and antifungal activities of com-
pounds 1-8 showed that the compounds having electron donat-
ing group had good antibacterial activity and the compounds hav-
CRediT authorship contribution statement
S. Senthilkumar: Supervision. J. Seralathan: Conceptualization,
Methodology. G. Muthukumaran: Writing - original draft.
10

S. Senthilkumar, J. Seralathan and G. Muthukumaran
Journal of Molecular Structure 1226 (2021) 129354
Acknowledgements
[26] J. Li, B. Li, M. Pan, B. Liu, J. Cheng, R. Li, X. Gao, S. Wang, H. Hou, Z. Liu, Cryst.
Growth Des. 17 (2017) 2975.
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The authors are thankful to SAIF Laboratory, IIT (M), Chennai,
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[29] J. Li, X. Wang, R. Li, W. Zhang, H. Bai, Y. Liu, Z. Liu, T. Yu, Z. Liu, Y. Yang, Y. Zhu,
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Supplementary materials
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found, in the online version, at doi:10.1016/j.molstruc.2020.129354.
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11