Synthesis, molecular structure, spectral analysis and cytotoxic activity of two new aroylhydrazones

Article abstract of DOI:10.1016/j.molstruc.2017.01.059

Two new aroylhydrazones viz 4-nitro-N′-(1-(pyridin-2-yl)ethylidene)benzohydrazide, NPHY (4) and 4-nitro-N′-(1-(thiophen-2-yl)ethylidene)benzohydrazide, NPHT (5) have been prepared and characterized by ¹H NMR, ¹³C NMR, FT-IR, UV–Visible spectroscopy and mass spectrometry. All quantum calculations were performed at DFT level of theory using B3LYP functional and 6-31G (d,p) as basis set. TD-DFT calculated electronic transitions are found to be in good agreement with experimental findings. The assignments for normal vibrational modes have been done by computing Potential Energy Distribution (PED) using Gar2ped. HOMO-LUMO analysis was performed and reactivity descriptors were also computed. Global electrophilicity index (ω) of 6.12–6.26 eV shows these aroylhydrazones to be strong electrophiles. Intramolecular interactions were analyzed by ‘Atoms in molecule’ (AIM) approach. Also, the computed first static hyperpolarizabilities (β₀) of these hydrazones indicate their future application as an attractive non-linear optical (NLO) material. Cytotoxicity evaluated by MTT assay, suggested that the synthesized aroylhydrazones significantly reduce the cell viability of breast cancer cell lines (MCF7) and human prostate adenocarcinoma (DU145) in a dose dependent manner. Cytotoxic potencies (IC₅₀) of these hydrazones against MCF7 and DU145 cell lines were found in range of 54.67–85.67 μM. The result of ROS activity provides supportive data for molecular mechanism of these hydrazones, which is related to apoptotic cellular death. Nuclear condensation assay performed by DAPI staining shows fragmented and condensed nuclei in MCF7 cells, suggesting cell death by apoptosis.

Full text of DOI:10.1016/j.molstruc.2017.01.059

Journal of Molecular Structure 1135 (2017) 82e97
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, molecular structure, spectral analysis and cytotoxic activity
of two new aroylhydrazones
,
Ravindra Kumar Singh b, Ashok Kumar Singh a *, Sahabjada Siddiqui c,
Mohammad Arshad c, Asif Jafri c
a Department of Chemistry, Faculty of Science, University of Lucknow, Lucknow, 226 007, UP, India
b Department of Chemistry, Jawaharlal Nehru Memorial P.G. College, Barabanki, 225 001, UP, India
c Molecular Endocrinology Lab, Department of Zoology, University of Lucknow, Lucknow, 226 007, UP, India
a r t i c l e i n f o
a b s t r a c t
Two new aroylhydrazones viz 4-nitro-N0-(1-(pyridin-2-yl)ethylidene)benzohydrazide, NPHY (4) and 4-
nitro-N0-(1-(thiophen-2-yl)ethylidene)benzohydrazide, NPHT (5) have been prepared and character-
ized by 1H NMR, 13C NMR, FT-IR, UVeVisible spectroscopy and mass spectrometry. All quantum calcu-
lations were performed at DFT level of theory using B3LYP functional and 6-31G (d,p) as basis set. TD-DFT
calculated electronic transitions are found to be in good agreement with experimental findings. The
assignments for normal vibrational modes have been done by computing Potential Energy Distribution
(PED) using Gar2ped. HOMO-LUMO analysis was performed and reactivity descriptors were also
Article history:
Received 9 September 2016
Received in revised form
29 December 2016
Accepted 18 January 2017
Available online 20 January 2017
Keywords:
computed. Global electrophilicity index (u) of 6.12e6.26 eV shows these aroylhydrazones to be strong
Aroylhydrazones
DFT-B3LYP
MTT assay
ROS activity
Apoptosis
electrophiles. Intramolecular interactions were analyzed by ‘Atoms in molecule’ (AIM) approach. Also,
the computed first static hyperpolarizabilities (b0) of these hydrazones indicate their future application
as an attractive non-linear optical (NLO) material. Cytotoxicity evaluated by MTT assay, suggested that
the synthesized aroylhydrazones significantly reduce the cell viability of breast cancer cell lines (MCF7)
and human prostate adenocarcinoma (DU145) in a dose dependent manner. Cytotoxic potencies (IC50) of
these hydrazones against MCF7 and DU145 cell lines were found in range of 54.67e85.67 mM. The result
of ROS activity provides supportive data for molecular mechanism of these hydrazones, which is related
to apoptotic cellular death. Nuclear condensation assay performed by DAPI staining shows fragmented
and condensed nuclei in MCF7 cells, suggesting cell death by apoptosis.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
antitubercular [6], antioxidant [7], antimicrobial [8] and anti-
malarial activity [9]. Several aroylhydrazones are known to find
The chemical and physiological properties of hydrazones have
been attracting researchers for a number of years. Acyl or aroyl-
hydrazones are Schiff bases having active pharmacophore (>C]
NeNHeCOe) group and prepared from the condensation of acyl-
hydrazine with an aldehyde or a ketone. They find wide range of
applications in pharmaceuticals and medicinal fields. They act as
intermediate for synthesis of various heterocyclic compounds such
as 1, 3, 4-oxadiazolines,1, 3, 4-oxadiazine,1, 2, 4etriazine, pyrazoles
[1] and 1, 3, 4eoxadiazolines, 2eazetidinones, 4ethiazolidinones
[2,3]. They are reported to show wide variety of biological prop-
erties such as anti-convulsant [4], anti-HIV, anticancer [5],
application in spectrophotometric identification of certain transi-
tion metals and used as metal extracting agents [10,11]. Many
hydrazones also finds application in optical storage [12] devices,
development of potential chemosensors [13], optoelectronic tech-
nology [14], photo-catalyst and new non-linear optical materials
[15e17]. Hydrazones have strong coordination ability towards
different metal ions [18,19]. Acylhydrazones are also known to
show dynamic response to various types of physical and chemical
stimulus [20,21]. They exhibit dynamic reversible E/Z photo-
isomerization in response to UV radiation [22] and represent sys-
tems with multiple dynamics suitable for information storage de-
vices and for the design of molecular photo switches.
In the recent years, density functional theory (DFT) has been
extensively used in theoretical modeling, drug and functional ma-
terial design. Many important chemical and physical properties of
* Corresponding author.
E-mail address: singhaks3@rediffmail.com (A.K. Singh).
http://dx.doi.org/10.1016/j.molstruc.2017.01.059
0022-2860/© 2017 Elsevier B.V. All rights reserved.

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
83
biological and chemical systems can be predicted by various
computational methods [23,24]. In the view of above significance,
we here report the synthesis of two new aroylhydrazones having
pyridyl and thineyl moiety. The synthesized hydrazones (4) and (5)
are well characterized using various experimental and theoretical
spectroscopic methods. Quantum chemical calculation using DFT
approach has been performed on molecular structure, conforma-
tional analysis, MEP, HOMO-LUMO analysis, spectroscopic and NLO
properties. AIM theory has been used to classify and understand
intramolecular hydrogen bonding interactions in the synthesized
hydrazones. Furthermore, the cytotoxic activity of these hydra-
zones have been studied by MTT assay, Reactive oxygen species
(ROS) activity and Nuclear condensation assay by DAPI staining.
These heterocyclic acylhydrazones will provide future opportunity
for synthesis of new derivatives and their metal complexes with
diverse biological and material properties.
The reaction mixture was cooled at room temperature and then
filtered. The precipitate was washed with methanol followed by
ether and then dried in air, giving NHPY (4) as white amorphous
powder. Yield: 72%, M.p. 212e214 ꢁC, MS (m/z): calcd. 284.09. obs.
285.17 (100%), for [Mþþ1], calcd. 569.18. obs. 569.32 (50%) for
[2Mþþ1]. Anal. calcd. for C14H12N4O3 (284.27): C 59.15, H 4.25 and
N 19.71; Found: C 59.29%, H 4.33% and N 19.62%. 1H NMR (400 MHz,
DMSO-d6)
dexp/calcd 11.16/9.25 (1H, H-17, s, -NH), 8.36e8.34/
8.83e8.74 (2H, d, H-9, H-10, J ¼ 8.0 Hz), 8.15e8.12/8.52e8.74 (2H, d,
H-7, H-8, J ¼ 8.0 Hz), 7.98e7.45/8.95e7.60 (4H, H-27, H28, H-29, H-
30, m, pyridyl), 2.48/2.61 (3H, H-31, H-32, H-33, s, -CH3). 13C NMR
(100 MHz, DMSO-d6) dexp/calcd C14e162.75/145.64, C25e156.19/
140.55, C19e154.87/139.50, C5-149.18/136.80, C21e139.28/134.38,
C2-136.68/127.52, C22e124.37/110.52, C23e130.44/122.12, C1,C3-
129.30/115.24, C24e120.52/107.10, C4,C6-123.40/111.17, C20e
12.93/-0.87. Mass spectrum of NHPY (4) is given as Supplementary
Fig. S1. The 1H and 13C NMR spectrum is shown as Supplementary
Figs. S2 and S3.
2. Experimental
2.1. Materials and methods
2.2.3. Synthesis of 4-nitro-N0-(1-(thiophen-2-yl)ethylidene)
benzohydrazide, NHTP (5)
All the reagents and chemicals used were of analytical and high
purity grade. The solvent was purified and dried according to
standard procedures [25]. MEM, RPMI 1640, fetal bovine serum
(FBS), MTT (3 -(4,5-dimethylthiazol- 2-yl) ꢀ2,5-diphenyltetrazo-
lium bromide) dye, and antibiotic solution were purchased from
Himedia, India. DAPI (40, 60-diamidino-2 phenylindole) and DCFH-
DA (2,7-dichlorodihydrofluorescein diacetate) dye were pur-
chased from Sigma-Aldrich, USA. Thin layer chromatography (TLC)
was performed on Silica Gel ‘G’ (Merck, India) coated plates for
monitoring the progress of reaction. Melting points (ꢁC) were
determined in an open capillary by electro-thermal melting point
apparatus and were uncorrected. The DART-mass spectrum of
compounds was recorded on JEOL-Acc TOF JMS-T100LC mass
spectrometer in ESIþ mode. Elemental analysis (C, H, N or S) was
performed on Varian Elementar e III analyzer. Infrared (FTIR)
spectra were recorded in KBr pellets on Bruker FTIR-spectrometer
in range of 4000e500 cm-1 1H NMR spectra (at 400 MHz) and 13C
NMR spectra (at 100 MHz) was recorded in DMSO-d6 on Bruker
model NMR spectrometer using (TMS) as internal reference.
UVeVisible spectra were taken on LABTRONICS LT-2900 spectro-
photometer equipped with a 1.0 cm quartz cell.
To the equimolar solution of 2-acetylthiophene (1 mmol,
0.11 ml) and 4-nitrobenzohydrazide (1 mmol, 181 mg) dissolved in
15 ml methanol, added 1e2 drops of conc. HCl acid as catalyst and
reaction mixture was then refluxed for about 4 h resulting in yellow
color precipitate. The progress of reaction was monitored by TLC.
The reaction mixture was cooled at room temperature and then
filtered. The precipitate was washed with methanol followed by
ether and then dried in air, giving NHTP (5) as yellow amorphous
fibrous powder. Yield: 69%, M.p. 201e203 ꢁC, MS (m/z): calcd.
289.06. obs. 290.13 (100%) for [Mþþ1], calcd. 579.10. obs. 579.25
(100%) for [2Mþþ1]. Anal. calcd. for C13H11N3O3S (289.310): C 53.97
H 3.83 N 14.52 and S 11.08; Found: C 53.60%, H 3.97% and N 14.39%
and S 11.02 .1H NMR (400 MHz, DMSO-d6)
dexp/calcd 11.07/8.93
(1H, H-17 s, -NH), 8.35e8.33/8.71e8.72 (2H, d, H-9, H-10, J ¼ 7.2
Hz), 8.11e8.09/8.46e8.13 (2H, d, H-7, H-8, J ¼ 7.2 Hz), 7.95e7.05/
7.64e7.33 (3H, H-28, H-30, H-31, m, thineyl), 2.40/2.36 (3H, H-21,
H-22, H-23, s, -CH3); 13C NMR (100 MHz, DMSO-d6)
dexp/calcd
C14e162.02/145.01, C5-153.81/136.66, C24e149.08/136.09, C19e
142.84/133.13, C2-139.71/127.79, C29e130.79/120.76, C25e128.77/
114.07, C1,C3-129.35/115.03, C27e127.6/112.2, C4, C6-123.43/111.17,
C20e15.14/2.61. Mass spectrum of NHTP (5) is given as Supple-
mentary Fig. S4. The 1H and 13C NMR spectrum is shown as Sup-
plementary Figs. S5 and S6.
2.2. Synthesis
2.2.1. Preparation of 4-nitrobenzohydrazide (1)
3. Quantum chemical calculations
Ethyl 4-nitrobenzoate was synthesized from 4-nitrobenzoic acid
by following reported esterification method [25]. Ethyl 4-
nitrobenzoate (1.30 g, 6.66 mmol) was dissolved in 25 ml meth-
anol and then added hydrazine hydrate (100%) (0.48 mL, 10 mmol)
and stirred for 10 min. The reaction mixture was then refluxed for
about 3 h for completion of reaction. The progress of reaction was
monitored by TLC. The solution is cooled at room temperature and
kept overnight in refrigerator, resulting in whitish solid. The pre-
cipitate was then filtered off, washed and recrystallized from
ethanol to give pure 4-nitrobenzohydrazide (1) product as creamy
white solid, Yield 79%; M.p. 208e210 ꢁC.
All quantum calculations including geometry optimization have
been carried out with Gaussian 09 software package [26] using
Density Functional Theory (DFT) and B3LYP functional with 6e31G
(d, p) as basis set in gas phase [27,28]. Molecular geometry of lower
energy conformers were fully optimized by Berny's algorithm using
redundant coordinates. The energies and intensities of 25 spin
allowed electronic transitions were calculated using TD-DFT theory
with B3LYP method in dichloromethane (DCM) solvent using IEF-
PCM model [29]. The normal mode analysis was performed and
the potential energy distribution (PED) was done using Gar2ped
program [30] to assign various vibrational modes. The first static
hyperpolarizability (b0) was calculated by the finite field pertur-
bation method in vacuum [29,31]. Using the x, y and z components
2.2.2. Synthesis of 4-nitro-N0-(1-(pyridin-2-yl)ethylidene)
benzohydrazide, NHPY (4)
To the equimolar solution of 2-acetylpyridine (1 mmol, 0.11 ml)
and 4-nitrobenzohydrazide (1 mmol, 181 mg) dissolved in 15 ml
methanol, added 1e2 drops of conc. HCl acid as catalyst and reac-
tion mixture was then refluxed for about 5 h, resulting in white
color precipitate. The progress of reaction was monitored by TLC.
of b obtained from Gaussian 09 output, the magnitude of the mean
first hyperpolarizability tensor can be calculated. Since the value of
the polarizability (ja0j), first hyperpolarizability (b0) of Gaussian 09
output are reported in atomic unit (a.u.) and these values are
converted into electrostatic unit (esu) using converting factors as

84
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
(for a0
:
1
a.u.
¼
0.1482
ꢂ
10ꢀ24 esu; for b0
:
1
was measured with a multiwell microplate reader (Synergy H1
Hybrid Multi-Mode Microplate Reader, BioTek) at an excitation
wavelength of 485 nm and at an emission wavelength of 528 nm.
Values were expressed as the percentage of fluorescence intensity
relative to the control wells.
a.u ¼ 0.008639 ꢂ 10ꢀ30 esu). Presentation graphics and visualiza-
tions were done with the help of Gauss view 5.0 [32]. AIMALL
software [33] was used to prepare molecular graph and compute
topological parameters at bond critical point [34,35].
4.4. Nuclear condensation assay by DAPI staining
4. Anticancer activity studies
The apoptotic effect of synthesized aroylhydrazones at different
4.1. Cell line and culture
concentrations (25 and 75 mM) was analyzed by nuclear fluorescent
DAPI following previous protocol [37]. The cells were seeded and
treated for 24 h in a 96 well plate. After the treatment period, cells
were washed with PBS and fixed in 4% paraformaldehyde for
10 min. Subsequently the cells were permeabilized with per-
meabilization buffer (3% paraformaldehyde and 0.5% Triton X-100)
and stained with DAPI. After staining, the images were taken with a
fluorescent microscope. Quantification of cells was performed in
triplicate for each treatment and 100 cells per sample were counted
in different fields (at least 10 random fields per sample) to score for
percent apoptotic cells as reported earlier [38] using a fluorescent
microscope (Nikon ECLIPSE Ti-S, Japan).
MCF-7 (Human, breast carcinoma) and human prostate adeno-
carcinoma (DU145) cell lines were obtained from cell repository-
National Centre for Cell Sciences, Pune, India. MCF7 cells were
cultured in RPMI-1640 medium and DU145 cells were maintained
in MEM media. Media were supplemented with 2.0 mM L-gluta-
mine, 1.5 g/l NaHCO3, 0.1 mM non-essential amino acids and
1.0 mM sodium pyruvate and supplemented to contain 10% (v/v)
fetal calf serum (HiMedia). Cells were grown at 37 ꢁC, 5% CO2 in a
humidified air.
4.2. MTT assay for in vitro cytotoxicity
% of apoptotic cell ¼ (apoptotic cells þ late apoptotic cells) / (total
no of cells) ꢂ 100
This assay is based on the enzymatic reduction phenomenon
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) dye. To evaluate the cytotoxic effect of aroylhydrazones (4)
and (5), MTT assay was performed with slight modification [36].
Cancer cells MCF-7 and DU145 (1 ꢂ 104) were seeded in 100
ml
4.5. Statistical analysis
complete medium in each well of 96-well culture plate for over-
night at 37 ꢁC and 5% CO2. Stock solutions of test hydrazones was
prepared and diluted to obtain desired concentrations (10, 25, 50,
Data of the cell viability, ROS activity and nuclear apoptosis were
expressed as the mean
SEM from three independent experi-
75 and 100
mM) in culture medium. This solution was then added to
ments. One-way ANOVA and Dunnett's Multiple Comparison Test
was performed using Graph Pad prism (Version 5.01) software for
significance test, using a p value ꢃ 0.05.
the wells as per experimental design. After 24 h of treatment, 10
m
l
of MTT (5 mg/ml of media without phenol red and serum) solution
was added in each well and the plate was further incubated for
3 h at 37 ꢁC until formazan blue crystal developed. Then, the su-
5. Results and discussion
pernatant was discarded from each well and 100 ml of DMSO was
added to solubilise formazan crystals for 10 min at 37 ꢁC. The
absorbance was recorded at 540 nm by a microplate reader (BIO-
RAD-680). The percentage viability was calculated by using the
formula:
5.1. Conformational analysis, molecular geometries and topological
study
The synthetic route for preparation of aroylhydrazones NHPY (4)
and NHTP (5) is shown in Scheme 1. The structure of synthesized
hydrazones was confirmed by various spectroscopic techniques
and was then fully optimized. In order to trace conformational
flexibility and various possible conformers, a relaxed PES scan along
CeC single bond was performed. Two separate PES scan was carried
out for hydrazone NHPY (4) and NHTP (5), along dihedral angle
N18eC19eC25eC24 and N18eC19eC24eC25 respectively. The
energy profile as the function of torsion angle and global minima
obtained on PES curve is shown as Supplementary Figs. S7 and S8. As
seen from figures, two conformers are obtained for each PES profile,
which corresponds to esyn and eanti form (as two minima). In syn-
conformer, ring hetero-atom (N or S) is on same side of methyl
group whereas in anti-conformer, the hetero atom is opposite to
methyl group. The aroylhydrazone NHPY (4) exists as syn-
conformer with N18eC19eC25eC24 dihedral angle of 0.14ꢁ while
hydrazone NHTP (5) exists as anti-conformer with N18eC19e
C24eC25 dihedral angle of ꢀ179.51ꢁ in its lowest energy stable
ground state. The minimum energy conformers were further
optimized at same level of theory, without imposing any symmetry
constraints on the molecule. The optimized structure of syn and
anti conformer of both aroylhydrazones is shown as Fig.1. The eigen
values obtained from scan output reveals that for hydrazone NHPY
(4), that -syn conformer is 8.16 kcal/mol more stable than its anti-
conformer. The esyn conformer has geometrical stability due to
% Cell viability ¼ [(OD of treated)̸ (OD of control)] ꢂ 100
The plot of % cell viability versus concentrations was used to
calculate the concentration lethal to 50% of the cells (IC50). The
cellular morphological changes were observed under inverted
phase contrast microscopy (Nikon ECLIPSE Ti-S, Japan).
4.3. Reactive oxygen species (ROS) activity
Microscopic fluorescence imaging was used to study ROS gen-
eration in MCF7 cells after exposure to different concentrations of
hydrazone compounds [37]. Cells (1 ꢂ 104 per well) were seeded in
96-well culture plate and allowed to adhere for 24 h in a CO2
incubator at 37 ꢁC. Cells were then exposed to one lower concen-
tration (25 mM) and other higher concentration (75 mM) for 24 h.
After exposure, cells were incubated with DCFH-DA (10 mM) as the
fluorescence agent for 30 min at 37 ꢁC. The reaction mixture was
aspirated and replaced by 200 ml of phosphate-buffered saline (PBS)
in each well. The plates were kept on a shaker for 10 min at room
temperature in the dark. An inverted fluorescence microscope with
a camera (Nikon ECLIPSE Ti-S, Japan) was used to analyze intra-
cellular fluorescence of cells. For quantitative fluorometric analysis,
cells (1 ꢂ 104 per well) were seeded and treated with test com-
pound in 96-well black bottom culture plate. Fluorescence intensity

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
85
Scheme 1. Synthetic route for formation of acid hydrazide precursors and products (4) and (5).
intramolecular hydrogen bonding interaction between pyridyl (N)
and hydrogen of methyl group, which lies on same side. In case of
hydrazone NHTP (5), the anti-conformer is more stable than syn-
conformer by energy 1.13 kcal/mol. This low energy barrier in-
dicates the presence of both conformers at room temperature in gas
phase. The stability of optimized geometry was confirmed by the
absence of any negative frequencies. Both esyn and eanti con-
formers of the hydrazones exists in E-configuration about C]N
bond. Both the studied hydrazones possesses C1 symmetry. The
lower energy syn-conformer (E ¼ ꢀ985.30008 a.u.) for hydrazone
NHPY (4) and anti-conformer (E ¼ ꢀ1290.01252 a.u.) of hydrazone
NHTP (5) is considered for all quantum calculations, including
molecular properties and vibrational wavenumbers calculations.
These aroylhydrazones, predominantly exists in keto-form and are
known to show keto-enol tautomerism due to intramolecular
proton transfer. The proton NMR spectra of these compounds taken
in DMSO-d6, shows small shoulders next to almost all signals,
suggesting the existence of keto-enol tautomerism in synthesized
hydrazones. Calculated small energy difference between two forms,
indicates their rapid inter-conversions at room temperature. The
enolic transformation is also confirmed by the presence of small
thiophene ring vary in range 1.37e1.42 Å. The C]N (1.29 Å) and C]
O (1.22 Å) bond lengths are similar for both the compounds and are
in consistent with earlier reported values for such bonds [22].
The theory of AIM has been efficiently used to describe strength
and nature of various inter and intra-molecular interactions. Mo-
lecular graph of the compound NHPY (4) and NHTP (5), using AIM
program at B3LYP/6-31G (d, p) level is shown in Supplementary
Fig. S9. Calculated geometrical as well as topological parameters for
bonds of interacting atoms are given in Table 1. The various type
interactions visualized in molecular graph are classified on the
basis of geometrical, topological and energetic parameters. In this
article, the Bader's theory application is used to estimate hydrogen
bond energy (E). The aroylhydrazone NHPY (4) shows one weak
intra-molecular hydrogen bond interaction between N26/H32
and aroylhydrazone NHTP (5) shows H/H interaction between
H22/H28. Using Espinosa approach [39], the energy of N26/H32
intramolecular hydrogen bond in NHPY (4) was calculated to
be ꢀ3.73 kcal/mol, while H22/H28 interaction energy in NHTP (5)
was calculated to be ꢀ1.76 kcal/mol.
5.2. Assignments of vibrational bands
signal at d
16.04 ppm for OeH proton in 1H NMR spectrum of
aroylhydrazone NHPY (4). The optimized geometrical parameters
of synthesized aroylhydrazones, are comparable with experimental
parameters of skeletally similar molecules [22]. Selected optimized
geometrical parameters of these aroylhydrazones are tabulated in
Supplementary Tables S1 and S2. As seen from the optimized
structure of NHPY (4), the nitro substituted phenyl ring is out of
molecular plane with C1eC2eC14eO15 torsion angle of 25.19ꢁ. The
B3LYP calculated CeC bond lengths in the phenyl ring of NHPY (4)
vary in the range 1.39e1.40 Å whereas CeC bond lengths in het-
erocyclic pyridine ring vary in range 1.39e1.41 Å. Similarly for
hydrazone NHTP (5), the nitro substituted phenyl ring is out of
plane with C1eC2eC14eO15 torsion angle of 25.44ꢁ. The calcu-
lated CeC bond lengths in the phenyl ring of NHTP (5) also vary in
the range 1.39e1.40 Å; whereas CeC bond lengths in heterocyclic
In order to obtain spectroscopic features of synthesized aroyl-
hydrazones, vibrational frequency calculations for normal modes
have been performed at DFT-B3LYP level of theory. The number of
atoms in hydrazone NHPY (4) and NHTP (5) are 33 and 31,
respectively; which gives 93 and 87 fundamental vibrational bands
(3n-6) respectively. The detailed assignments of experimental
wavenumbers and their comparisons with theoretically calculated
(scaled and selected) wave numbers for the normal modes along
with their %PED are given in Supplementary Tables S3 and S4. Since
the calculated wavenumbers for majority of vibrational bands are
larger than the observed wavenumbers, they were scaled down by
single scaling factor of 0.9608 to discard the anharmonicity present
in real system [40,41]. The comparison of experimental FT-IR
spectra of these aroylhydrazones with their simulated IR spectra

86
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
Fig. 1. DFT-B3LYP optimized structure of aroylhydrazones NHPY (4) and NHTP (5) as esyn and eanti conformers.
is shown as Fig. 2. The value of correlation coefficient obtained
between DFT computed theoretical and experimental wave-
numbers for aroylhydrazone NHPY (4) (R2 ¼ 0.999) and NHTP (5)
(R2 ¼ 0.999) shows good harmony with experimental results. The
correlation plot between of experimental and theoretical IR
wavenumbers is shown in Supplementary Figs. S10 and S11.

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
87
Table 1
Geometrical parameters (bond length) and topological parameters for bonds of interacting atoms: ellipticity (ε), electron density r(BCP), Laplacian of electron density V2r(BCP)
,
electron kinetic energy density G(BCP), electron potential energy density V(BCP), total electron energy density H(BCP) at bond critical point (BCP) and estimated interaction energy
(Eint.) (in Kcal/mol) of aroylhydrazones.
Interaction
Bond length(Å)
Ellipticity(ε)
r(BCP)
V
2r(BCP)
G(BCP)
V(BCP)
H(BCP)
E int.
NHPY (4)
N26/H32
NHTP (5)
H22/H28
2.3110
2.1062
0.4398
0.9903
0.0176
0.0102
0.0644
0.0428
0.0139
0.0082
ꢀ0.0119
ꢀ0.0056
ꢀ0.0021
ꢀ0.0025
ꢀ3.7337
ꢀ1.7570
Fig. 2. Comparison of experimental FT-IR spectra of aroylhydrazones (4) and (5) with the calculated spectra.

88
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
5.2.1. NeH vibrations
t
-NO2 for aromatic nitro compounds is reported to be at 70 20
According to internal coordinate system, the imino group
(XꢀNHꢀY) associates with four types of vibrational frequencies
[42]. The NeH stretching vibrations are reported in literature to
and 65 10 cmꢀ1 [48,49]. In the present case, this deformation
mode is calculated at wavenumbers in range 62e65 cmꢀ1
.
give rise to band in the region 3390
60 cmꢀ1 [43]. In FT-IR
5.2.6. CH3 group vibrations
spectrum of aroylhydrazone NHPY (4) and NHTP (5), the
N16eH17 stretching of C]NeH are observed as weak band at 3455
and 3459 cmꢀ1 respectively, whereas these bands are theoretically
According to the Internal coordinate system recommended by
Pulay et al. [42], methyl (-CH3) group associates with five types of
vibrational frequencies, namely; asymmetric and symmetric
stretches, bending, rocking and torsion modes. In aromatic com-
pounds, the CH3 asymmetric stretching vibrations are expected
around 2980 cmꢀ1 and the symmetric CH3 stretching vibrations is
expected around 2810 cmꢀ1 [48,50]. The observed symmetric
stretching vibration (ns) of methyl group at 2878 cmꢀ1, is compa-
calculated at 3417 and 3420 cmꢀ1 respectively. The N-H rocking (
r)
and wagging (u
) bands assigned at 1492-1495 cmꢀ1 and 451-
492 cmꢀ1, which correlates well with experimentally observed
bands at 1507-1518 cmꢀ1 and 504-511 cmꢀ1 respectively.
5.2.2. >C]O group vibrations
rable with the experimental values observed at 2908-2915 cmꢀ1
.
In the simulated FTIR spectra of NHPY (4) and NHTP (5), the
calculated wavenumbers assigned for C (14) ¼ O (15) carbonyl
group stretching at 1722-1723 cmꢀ1, matches well with the
experimentally observed wavenumbers at 1668-1669 cmꢀ1. The
red shifting observed in ArꢀCOeNH stretching indicates its
involvement in intermolecular hydrogen bonding.
The calculated nasCH3 at wavenumber 2958-2966 cmꢀ1, corre-
sponds to experimentally observed wavenumbers at 2965-
2967 cmꢀ1. These assignments are also supported by literature [51].
The symmetric deformation mode of methyl group calculated at
1349-1363 cmꢀ1 is observed at 1344-1351 cmꢀ1 in FT-IR spectra of
these hydrazone compounds. The asymmetric deformations are
generally expected in the range 1400e1485 cmꢀ1 [52]. In the pre-
sent study, the asymmetric deformation is calculated at 1426-
1438 cmꢀ1 and these bands are experimentally observed at
1431 cmꢀ1. The observed methyl rocking at 1015-1018 cmꢀ1, agrees
5.2.3. C]N and CeN vibrations
The Schiff base linkage
assigned at wavenumbers 1592-1613 cmꢀ1, corresponds to the
observed wavenumber at 1602 cmꢀ1. The presence of
(C]N) band
n(C]N) for NHPY (4) and NHTP (5),
n
well with the calculated wavenumbers at 1015-1016 cmꢀ1
.
in both the synthesized aroylhydrazone confirms the presence
hydrazone linkage. The calculated bands for C]N also matches
with the reported wavenumbers at 1602 cmꢀ1 in literature [44].
The CeN stretch observed at 1181-1239 cmꢀ1 agrees well with
5.2.7. Aromatic ring vibrations
The synthesized aroylhydrazones have substituted phenyl ring
and heterocyclic pyridyl or thineyl ring. Their spectral region pre-
dominantly involves CeH, CeC and C]C stretching and CeCeC as
well as HeCeC bending vibrations. The calculated weak to medium
theoretically calculated wavenumbers at 1214-1217 cmꢀ1
.
5.2.4. NeN and CeC vibrations
bands for n(CeH) for aromatic phenyl and heterocyclic rings at
The NeN stretching vibrations calculated at wave numbers
1134-1139 cmꢀ1, agree well with experimental bands at wave
number 1113-1138 cmꢀ1.This also agrees with reported values in
literature [45]. The calculated/observed CeC stretching wave-
numbers for C25eC19 (at 1295/1293 cmꢀ1), C19eC20 (at 1349/
1351 cmꢀ1), C14eC2 (at 1214/1181 cmꢀ1) for NHPY (4) and
C24eC19 (at 1284/1298 cmꢀ1), C19eC20 (at 1284/1298 cmꢀ1),
C14eC2 (at 1217/1239 cmꢀ1) for NHTP (5) shows good agreement
with experimental wave numbers.
3069-3173 cmꢀ1 matches well with observed wavenumbers at
3065-3196 cmꢀ1. Vibrations involving in-plane CeH bending are
found throughout the region 1500-1000 cmꢀ1. These bending
modes are not pure but are mixed with other vibrations. The
dominant (PED > 20%) in-plane bending for NHPY (4) calculated at
wavenumbers 1451, 1412, 1275, 1271, 1157, 1133 and 1086 cmꢀ1, are
in agreement with experimental wavenumbers at 1459, 1411, 1268,
1150, 1138 and 1092 cmꢀ1. For aroylhydrazone NHTP (5), observed
major CeH in-plane bending at wavenumbers 1298, 1239, 1151,
1086 and 1070 cmꢀ1 matches well with calculated wavenumbers at
1271, 1217, 1156, 1087 and 1074 cmꢀ1. The CeH out-of plane
bending vibrations in aromatic rings appears in range of
1000e675 cmꢀ1 [53]. The dominant CeH out-of plane bending
vibrations for NHPY (4) are calculated at 980, 965, 945, 889, 856,
826, 775 and 732 cmꢀ1 whereas it is observed at 987, 864, 796, 783
and 731 cmꢀ1. Similarly, for NHTP (5), these out-plane bending
vibrations are observed at 1001, 937, 864, 852, 814, 780 and
689 cmꢀ1 and are theoretically computed at wavenumbers 972,
948, 884, 856, 826, 824, 799 and 690 cmꢀ1. The ring skeletal vi-
brations mainly involving carbon to carbon stretching are generally
expected in the range from 1650 to 1200 cmꢀ1 [43]. In the present
case, these vibrations are computed as mixed bands in range
1602e1275 cmꢀ1 and are comparable with experimentally
observed wavenumbers. The ring torsional modes generally appear
in the low wavenumber regions. The aromatic rings torsional
modes with more than 30% contribution are found at 658, 408 and
402 cmꢀ1 for NHPY (4) and for hydrazone NHTP (5), these modes is
5.2.5. NO2 group vibrations
The molecules under investigation possess one nitro group each
at para position of phenyl ring. The nitro group shows two types of
stretching vibrations as asymmetric (nas) and symmetric (ns). The
nas stretches are always observed at higher wavenumber than ns
stretches. In nitro compounds N]O stretching vibrations are
located in the regions 1550e1300 cmꢀ1 [43]. The nas and ns of nitro
group calculated at 1605-1604 cmꢀ1 and 1344 cmꢀ1, matches well
with observed wavenumbers at 1602 cmꢀ1 and 1337-1344 cmꢀ1
,
respectively. The nas and ns stretching vibrations of nitro group are
also reported in literature at 1588, 1331 cmꢀ1, respectively [46]. The
scissoring band (dsc-NO2) for nitrobenzene is reported at wave-
numbers 852 cmꢀ1 and for 1, 3-dinitrobenzene at wavenumbers
904 and 834 cmꢀ1 [47]. For the title hydrazones, these bands are
observed at 852-864 cmꢀ1
,
whereas this was calculated at
829 cmꢀ1. In aromatic nitro compounds the wagging (
u
-NO2) mode
is expected in the region 740
50 cmꢀ1 [47] with moderate to
strong intensity. This wagging mode observed at 696-701 cmꢀ1 is
found to be in good agreement with theoretically calculated
found at wavenumbers 752, 709, 657, 616, 539 and 409 cmꢀ1
.
wavenumbers at 698-701 cmꢀ1. The rocking mode (
r
NO2) is active
in the region 540 70 cmꢀ1 in aromatic nitro compounds [47]. This
NO2) mode calculated at 496-499 cmꢀ1 was observed in experi-
mental spectra at wavenumbers 504-511 cmꢀ1. The torsion mode of
5.3. NMR and UVeVis spectroscopy
(r
Quantum chemical calculations using density functional (DFT)
approach is found accurate enough in prediction of NMR spectra

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
89
and exploring the relationship between molecular structure and
their chemical shifts [54]. The chemical shifts for fully optimized
structure have been calculated using DFT-B3LYP method with
6e31G (d, p) as basis set, employing GIAO approach. The IEFPCM
model with DMSO solvent (relative permittivity (ε) 46.7) was used
to consider solvent induced effects. The experimental and calcu-
lated chemical shifts of title aroylhydrazones are given in Table 2,
along with their assignments. In the experimental 1H NMR spectra
of NHPY (4) and NHTP (5), a singlet for eNH proton is observed at
agreement with experimental wavelength at 281 nm and is mainly
composed of (H-1 / L) electronic transition. The observed band
with
l max 232 nm is also correlates well with calculated vertical
excitation at 223 nm, with major contribution of (H-4 / Lþ1)
transition. Similarly, for compound NHTP (5), the calculated broad
and weak band with excitation energy of 458 nm, is found to
originate mainly from (H / L) transition. This weak band is not
observed in experimental spectrum of NHTP (5). The high intensity
absorption band observed at
l max 308 nm, is theoretically calcu-
d
11.16 and
methyl (C-20) proton are found at
Aromatic protons multiplet for NHPY and NHTP is observed in re-
gion 8.63e7.45 ppm and 8.35e7.05 ppm, respectively. Addi-
d
11.07 ppm respectively. Three proton singlets for
lated to arise from vertical excitation at energy of 321 nm
(f ¼ 0.2085) and 315 nm (f ¼ 0.4830). This band originates from (H-
2 / L) and (H / Lþ1) electronic transitions. Experimentally
d
2.48 and 2.40 ppm respectively.
d
d
observed absorption band at l max 267 nm is in agreement with
tional support for the structure of synthesized compounds was
provided by its 13C NMR spectra. The chemical shift value of the
carbon attached to nitrogen C19 ¼ N18 for these hydrazones, found
calculated (H-5 / L) electronic transition at 274 nm. The weak
band found at 234 nm is theoretically calculated to mainly arise
from (H-1 / Lþ1) transition with excitation energy of 254 nm. The
calculated molecular orbital coefficients and molecular orbital plots
show that the FMOs are mainly composed of p-atomic orbital's and
at
d 154.87 ppm (for NHPY) and d 142.84 ppm (for NHTP), corrob-
orated well with reported similar hydrazone compounds [22]. The
values of correlation coefficient for 1H and 13C NMR (r2 ¼ 0.909,
0.992) for NHPY (4) and (r2 ¼ 0.902, 0.996) for NHTP (5) show that
there are good agreement between experimental and calculated
results. In addition to 1H, 13C NMR chemical shifts, Mass spectrum,
also confirms the presence of molecular ion peak at 285.17 and
290.13 for Mþ1, corresponds to the molecular formula C14H12N4O3
and C13H11N3O3S of NHPY(4) and NHTP (5), respectively.
so the nature of major electronic excitations are assigned to be
p /
p* and are mainly composed of py and pz orbitals. Overall, TD-DFT
calculated electronic excitations give rise to the same pattern of
bands and are in good agreements with the experimental
spectrum.
5.4. HOMO-LUMO analysis and electronic descriptors
To understand the nature of various electronic excitations, the
UVevisible spectra of synthesized aroylhydrazones NHPY (4) and
NHTP (5) have been studied and TD-DFT excitations were calcu-
lated in the solvent phase. The comparison of experimental and
calculated UVeVis spectrum in dichloromethane is shown in Fig. 3.
The observed and calculated electronic transitions of high oscilla-
tory strength, along with experimental wavelength in solvent
(DCM) are presented in Table 3. For hydrazone NHPY (4), the
The highest occupied molecular orbital HOMO and lowest un-
occupied molecular orbital (LUMO) and their band gap are very
important quantum parameters which reflects the chemical sta-
bility of the molecule [55,56]. These molecular orbital plays an
important role in the optical and electric properties, as well as in
quantum chemistry and UVeVis spectra. The energy gap is a
measure of electron conductivity and explains the charge transfer
interaction within the molecule, which influences the biological
activity of the molecule. The calculated bad gap, frontier molecular
orbitals (FMOs), HOMO and LUMO plots along with and other
molecular orbitals plot, active in the electronic transition in solvent
DCM and their transition energy (nm) are presented in Fig. 4. For
observed weak and broad absorption band at
l max 403 matches
well with TD-DFT calculated vertical excitation at energy 402 nm,
with oscillator strength (f) of 0.1786. This excitation mainly origi-
nates from (H / L) electronic transition. The high oscillator
strength (f ¼ 0.8653) excitation at 292 nm was found to be in good
Table 2
Calculated and experimental 1H and 13C NMR chemical shifts (
d/ppm)) in DMSO for aroylhydrazones NHPY (4) and NHTP (5).
Atom no.
Exp. 1H NMR
Calcd 1H NMR
Atom no.
Exp. 13C NMR
Calcd 13C NMR
NHPY(4)
H-17(s)
H-9, 10 (d)
H-7, 8 (d)
H-27, 28, 29, 30 (m)
H-31, 32, 33 (s)
11.16, -NꢀH
9.25
8.79
8.63
8.95e7.60
2.61
C-14
C-25
C-19
C-5
C-21
C-2
C-23
C-1, C-3
C-22
C-4, C-6
C-24
C-20
162.75
156.19
154.87
149.18
139.28
136.68
130.44
129.30
124.37
123.40
120.52
12.93
145.64
140.55
139.50
136.80
134.38
127.52
122.12
115.24
110.52
111.17
107.10
ꢀ0.87
8.36e8.34, phenyl, (J ¼ 8.0 Hz)
8.15e8.12, phenyl, (J ¼ 8.0 Hz)
7.98e7.45 (pyridyl)
2.48 (methyl)
NHTP(5)
H-17(s)
H-9, 10 (d)
H-7, 8 (d)
H-28, 30, 31 (m)
H-21, 22, 23 (s)
11.07, -NꢀH
8.93
8.72
8.30
7.64e7.33
2.36
C-14
C-5
C-24
C-19
C-2
C-29
C-25
C-1, C-3
C-27
C-4, C-6
C-20
162.02
153.81
149.08
142.84
139.71
130.79
128.77
129.35
127.6
145.01
136.66
136.09
133.13
127.79
120.76
114.07
115.03
112.2
8.35e8.33, phenyl, (J ¼ 7.2 Hz)
8.11e8.09, phenyl, (J ¼ 7.2 Hz)
7.95e7.05 (thineyl)
2.40 (methyl)
123.43
15.14
111.17
2.61
s ¼ singlet; d ¼ doublet; m ¼ multiplet.

90
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
Fig. 3. Comparison of experimental UVeVis spectra of aroylhydrazones (4) and (5) with TD-DFT calculated simulated spectra.
Table 3
TD-DFT calculated excitations and approximate assignments for aroylhydrazones NHPY (4) and NHTP (5) in solvent dichloromethane using PCM model.
State
Excitation energy (eV/nm)
O.S (f)
CI Composition & major Transitions
l
max (exp.)
Assignments
NHPY (4)
S0 / S1
S0 / S6
S0 / S20
NHTP (5)
S0 / S1
S0 / S3
S0 / S5
S0 / S9
S0 / S13
3.09/402
4.25/292
5.86/223
0.1786
0.8653
0.1030
(0.71), H(74) / L(75)
(0.64), H-1(73) / L(75)
(0.59), H-4(70) / Lþ1(76)
403
281
232
p
p
p
/
/
/
p
p
p
*
*
*
2.71/458
3.86/321
3.94/315
4.53/274
4.89/254
0.1702
0.2085
0.4830
0.1616
0.0756
(0.71), H(75) / L(76)
Not obs.
308
p
p
p
p
p
/
/
/
/
/
p
p
p
p
p
*
*
*
*
*
(0.44), H-2(73) / L(76)
(0.60), H(75) / Lþ1(77)
(0.64), H-5 (70)/ L(76)
(0.62), H-1(74) / Lþ1(77)
e
267
234
hydrazone NHPY(4), HOMO lies at ꢀ6.344 eV and is delocalized
over the pyridine ring, carbonyl group, imine and methyl group
except p-nitro phenyl ring and whereas LUMO is located at ꢀ2.873
eV and is delocalized over the p-nitro phenyl ring and the carbonyl
group. This shows that charge transfer occurs within the molecule
and the frontier orbital energy gap is 3.471 eV in solvent phase. For
hydrazone NHTP (5), HOMO lies at ꢀ5.917 eV and is delocalized
over the thineyl ring, carbonyl group, and imine except p-nitro
phenyl ring and whereas LUMO is located at ꢀ2.848 eV and is
delocalized mainly over the p-nitro phenyl ring and the carbonyl
group. The HOMO-LUMO energy band gap for NHTP (5) in DCM
solvent is found to be ꢀ3.068 eV, indicating substantial charge
transfer.
index (u) for NHPY (4) and NHTP (5) of 6.12 eV and 6.26 eV,
respectively indicates strong electrophilic power of synthesized
hydrazones.
5.5. Molecular electrostatic potential surface (MEPS) and molar
refractivity (MR)
The electrostatic potential V(r) depicts the size, shape, charge
density and reactive sites of the molecule and is important for
studying drug-receptor and enzyme-substrate interactions [59,60].
It provides a visual method to understand the relative polarity of a
molecule. The MEP is related to the electron density and is very
useful descriptor in determining the sites for electrophilic and
nucleophilic reactions as well as hydrogen bonding interactions
[61]. The different values of electrostatic potential at the surface are
represented by different colors as red represents the region of most
electronegative electrostatic potential, blue represents region of
most positive electrostatic potential and green represents region of
zero potential. To predict reactive sites for electrophilic and
nucleophilic attack for the investigated molecule, the MEP at the
B3LYP/6-31G (d, p) optimized geometry was calculated. The elec-
tron density isosurface on to which the electrostatic potential
surface has been mapped is shown in Fig. 5. As can be seen from
figure, the negative electrostatic potential regions are mainly
localized over oxygen atoms of nitro and carbonyl groups. The
positive regions are localized near theeNH group as possible sites
for nucleophilic attack. These sites give information about region
where the compound can have intermolecular interactions.
The chemical reactivity and site selectivity of the molecular
systems have been determined on the basis of Koopman's theorem
[57]. The global reactivity descriptors are highly useful in predicting
global reactivity trends of a molecule. On the basis of Koopman's
theorem, by using the HOMO and LUMO energy values, the global
chemical reactivity descriptors such as electronegativity (
2(εLUMO þ εHOMO), chemical potential ( ) ¼ 1/2 (εLUMO þ εHOMO),
global hardness (
) ¼ 1/2 (εLUMO e εHOMO), global softness (S) ¼ 1/
and electrophilicity index ( can be defined. According
) ¼
2/2
to Parr et al., electrophilicity index ( ) [58] is a new reactivity index
c
) ¼ ꢀ1/
m
h
2
h
u
m
h
u
which quantifies the global electrophilic power of the molecule and
measures the stabilization in energy when the system acquires an
additional electronic charge (DN) from the environment. Calculated
various global reactivity descriptors for aroylhydrazones NHPY (4)
and NHTP (5) are listed in Table 4. Calculated global electrophilicity

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
91
Fig. 4. Selected M.O's of aroylhydrazones involved in vertical electronic transitions and their TD-DFT calculated excitation energy of states (nm) along with experimental values, in
solvent dichloromethane using PCM model.
Molar Refractivity (MR) is directly related to the refractive index,
molecular weight and density of steric bulk and responsible for the
lipophilicity and binding property of investigated system. It is
important property used for the study of quantitative structure
activity (QSAR) relationships and molecular modeling. The molar
refractivity is a constitutive-additive property and can be calculated
by the LorentzeLorentz equation [62,63] which holds for both
liquid and solid state of system, and defined as:
MR ¼ [(n2 e 1) (n2 þ 2)]. (MW /
r) ¼ 1.333
pNa0
where, n e refractive index, MW e molecular weight,
(MW/ ) e molar volume, N e Avogadro Number, 0 e polarizability
of molecular system. Molar refractivity is found to be related to the
r e density,
r
a
Table 4
Calculated EHOMO, ELUMO, energy band gap (EL- EH), chemical potential (
m
), electronegativity (
c
), global hardness (
h
), global softness (S) and global electrophilicity index (
u
) (in
eV) for aroylhydrazones NHPY (4) and NHTP (5) at B3LYP/6-31G (d, p) level.
Compounds
EHOMO
ELUMO
EH e EL
c
m
h
S
u
NHPY (4)
NHTP (5)
ꢀ6.3445
ꢀ5.9168
ꢀ2.8734
ꢀ3.0685
ꢀ3.4711
ꢀ3.0685
4.6089
4.3825
ꢀ4.6089
ꢀ4.3825
1.7355
1.5343
0.2881
0.3259
6.1197
6.2592

92
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
Fig. 5. MESP of aroylhydrazones mapped with isodensity shown as 3-D surface.
London dispersive forces that act in the drugereceptor interaction
5.7. Cytotoxic activity evaluation and morphological study
ꢀ
(Padron et al., 2002). Using the above equation and DFT calculated
polarizability, the molar refractivity (MR) for NHPY (4) and NHTP
(5) has been calculated as 76.96 and 78.28 esu, respectively.
Cellular toxicity of synthesized aroylhydrazones against MCF7
and DU145 cells were evaluated by MTT assay at 24 h period as
depicted in Fig. 6. The cell viability data of MCF7 cells showed that
10, 25, 50, 75 and 100 mM doses of hydrazone NHPY (4) results in
5.6. Non-linear optical (NLO) response
significant reduction in cell viability approximately 89.11, 72.15,
47.69, 28.29 and 19.26% (p < 0.001) respectively, as compared to
control. Similarly, hydrazone NHTP (5) at different dose concen-
Direct measurement of hyperpolarizability is a difficult task and
computational calculations with DFT approach is an alternative
choice to measure extensive properties of materials [64]. Theoret-
ical calculations on molecular hyperpolarizability are useful in
describing relationship between the electronic structure and non-
linear response of a molecule. The response of a system in
applied external field is described in terms of polarizabilities and
hyperpolarizabilities [64]. The components of (b) are defined as the
coefficients in the Taylor series expansion of the energy in the weak
external electric field [65,66]. The first static hyperpolarizability
tration (10, 25, 50, 75 and 100 mM) resulted in decrease in % of
viable cells approximately 94.19, 80.58, 66.20, 53.38 and 33.47%
(p < 0.001) respectively, as compared to control. Additionally,
cytotoxicity evaluated against DU145 cell lines showed that
hydrazone NHPY (4), results in significant decrease in cell viability
approximately 92.27, 85.96, 70.15, 57.51 and 33.39% (p < 0.001) as
compared to control, with increase in concentration of compound
(10, 25, 50, 75 and 100
mM), respectively. Similarly, NHTP (5) at
different doses (10, 25, 50, 75 and 100
mM) also resulted in signif-
(
b0) of this molecular system and related properties (ja0j and Da
)
icant reduction of % viable cell approximately 93.49, 88.37, 76.11,
63.99 and 40.89% (p < 0.001), as compared to control. These results
suggested that treatment of MCF7 and DU145 cells with synthe-
sized aroylhydrazones significantly reduce the cell viability of
breast cancer cell lines in a dose dependent manner. The IC50 value
of the standard drug Tamoxifen against MCF7 cell line is reported in
are calculated using DFT-B3LYP/6-31G (d,p) based on the finite field
perturbation approach in vacuum and their calculated values are
given in Table 5. The larger value of particular component of the
polarizability and hyperpolarizability indicates substantial delo-
calization of charge in these directions. The calculated first
static hyperpolarizability (b0) of aroylhydrazone NHPY (4) and
NPHT (5) in vacuum is found to be 23.804 ꢂ 10ꢀ30 esu and
53.028 ꢂ 10ꢀ30 esu respectively. Calculated b0 value for NHPY (4)
was found to be 2.1 times and b0 value for NPHT (5) is found to be
4.6 times greater than the b0 value of reference p-Nitroaniline (p-
NA) (b0 ¼ 11.54 ꢂ 10ꢀ30 esu). Therefore, investigated molecules will
show non-linear optical response and might be used as non-linear
optical (NLO) material in future.
literature as 5
and NHTP (5) was estimated to be 54.67
respectively, in MCF7 cells respectively. For DU145 cell lines, the
IC50 value for NHPY (4) and NHTP (5) was calculated to be 80.32
and 85.67 M respectively. Cellular morphological changes were
m
M [67]. The IC50 value of test compounds NHPY (4)
mM and 76.16
mM
m
M
m
observed in MCF7 and DU145 cells at 24 h after the treatment of
aroylhydrazones at various concentrations (10, 25, 50, 75 and
100 mM) by inverted phase contrast microscopy. As shown in figure,
the unexposed control cells remained smooth and flat and also
exhibited even cell surface, indicating normal healthy cells. How-
ever, there was drastic change in the morphology of the cells
treated with hydrazones (NHPY and NHTP) as shown by the
photomicrograph as Fig. 7. Cells treated with hydrazone com-
pounds displayed typical apoptotic feature including nuclear
condensation, extensive cytoplasmic vacuolization, acquired round
shape showing cellular shrinkage as compared to the unexposed
cells.
Table 5
Calculated Dipole moment (m0), Polarizability (ja0j), anisotropy of Polarizability (Da),
First hyperpolarizability (b0) and their components for NHPY (4) and NHTP (5) at
B3LYP/6-31G (d,p) level.
NHPY(4)
NHTP(5)
NHPY(4)
NHTP(5)
mx
my
mz
m0
axx
ayy
azz
ꢀ1.828
ꢀ0.666
0.146
1.783
1.635
0.327
2.441
366.100
183.159
81.480
31.122
37.022
bxxx
bxxy
bxyy
byyy
bxxz
bxyz
byyz
bxzz
byzz
bzzz
2675.069
128.904
67.331
10.759
ꢀ5.462
ꢀ11.516
1.760
10.382
ꢀ19.950
11.193
23.804
ꢀ6061.659
ꢀ268.656
ꢀ63.671
12.861
ꢀ22.562
ꢀ8.425
10.033
1.951
359.597
181.439
78.359
30.529
36.522
5.8. Intracellular ROS generation
j
a0
Da
j
ꢀ7.981
ROS function as signaling molecules in various pathways regu-
lating both cell survival and cell death [68]. However, excessive ROS
production can lead to oxidation of macromolecules and play an
important role in DNA mutations, ageing, and cell death
12.067
5.738
53.028
(b0)
mo in Debye (D); ja0j and Da in10ꢀ24 esu; b0 in10ꢀ30 esu.

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
93
Fig. 6. The column graph representation for cytotoxicity (as % cell viability) at different concentrations of aroylhydrazones NHPY (4) and NHTP (5), against MCF7 (A) and DU145 (B)
cell line evaluated by MTT assay at 24 h incubation.
[69].Therefore, increased intracellular ROS production might be
implicated in cellular damage of MCF7 cells. Fig. 8, depicted the
effect of aroylhydrazones-induced intracellular ROS generation in
cultured MCF7 cells. The treatment of MCF7 cells with these
hydrazones for 24 h resulted in a dose-dependent enhancement in
ROS generation compared with untreated cells, which was evident
by the increase in intensity of DCF fluorescence. The results from
quantitative measurement of ROS intensity showed that 25
conc. of aroylhydrazone NHPY (4) and NHTP (5) induced 169.11%
(***p < 0.001) and 131.82% (***p < 0.001) enhancement in ROS
production, respectively as compared to control. Moreover, ROS
production for aroylhydrazone NHPY (4) and NHTP (5) increased by
mM

94
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
Fig. 7. Photomicrograph showing morphological changes in MCF7 (A) and DU145 (B) cells at 24 h after the treatment with aroylhydrazones NHPY (4) and NHTP (5) at various
concentrations (10, 25, 50, 75 and 100 M) by inverted phase contrast microscopy.
m
approximately to 216.52% (***p < 0.001) and 171.69% (***p < 0.001)
respectively, at the concentrations of 75 M (Fig. 9) when compared
to untreated cells. This result provides supportive data for
molecular mechanism of these hydrazones, which is related to
apoptotic cellular death. Further, inhibition of the antioxidant
system in cancer cells could cause a substantial increase of ROS due
m
Fig. 8. Photomicrograph showing the effect of aroylhydrazones NHPY (4) and NHTP (5) induced intracellular ROS generation in cultured MCF7 cells at two different concentrations.

R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
95
Fig. 9. Column graph for ROS generation in cultured MCF7 compared with untreated (control) cells, for aroylhydrazones NHPY (4) and NHTP (5) showing increase in intensity of DCF
fluorescence (as % DCFDA).
to the high basal ROS output in these cells and lead cell death [70].
Hence, it might be speculated that these hydrazones may inhibit
antioxidant system in cancer cells and consequently lead to ROS
accumulation in these cells and trigger cell death.
Column graph showing % apoptotic cells at 25 and 75 mM of test
compounds is shown as Fig. 11. Furthermore, approximately 22.3
and 38.67% of apoptotic cells were observed when MCF7 cells were
treated with 25 and 75
Similarly, hydrazone NHTP (5) showed, approximately 11.67 and
21.0% of apoptotic cells with 25 and 75 M of test compound
mM of hydrazone NHPY (4), respectively.
m
5.9. Nuclear condensation and apoptosis
respectively. Fragmented and condensed nuclei in MCF7 cells
suggest that these aroylhydrazones caused cell death by an
apoptotic process. Our study also supported that these hydrazones
induces nuclear condensation and leads to apoptosis.
Apoptosis is characterized by a series of typical morphological
features, such as shrinkage of the cell and fragmentation into
membrane-bound apoptotic bodies [71]. As depicted from photo-
micrograph of MCF7 cells (Fig. 10), synthesized aroylhydrazones
demonstrated apoptosis in MCF7 cells in a dose dependent manner
as evident by their increased permeability to DAPI with the pres-
ence of nuclear apoptotic bodies and chromatin condensation.
6. Conclusions
Two new aroylhydrazones have been synthesized and well
Fig. 10. Photomicrograph of MCF7 cells, treated with aroylhydrazones NHPY (4) and NHTP (5) at two different concentrations, showing morphological changes, nuclear
condensation and cellular apoptosis.

96
R.K. Singh et al. / Journal of Molecular Structure 1135 (2017) 82e97
Fig. 11. Column graph showing % apoptotic cells in cultured MCF7 at 25 and 75
mM of test compounds NHPY (4) and NHTP (5).
characterized by 1H NMR, 13C NMR, FT-IR, UVeVisible spectros-
copy and mass spectrometry. Conformational analysis through
relaxed PES scan shows that aroylhydrazone NHPY (4) exists in
esyn conformer and NHTP (5) as eanti conformer in its lowest
ground state. A combined spectroscopic and quantum chemical
studies performed on these hydrazones suggested that experi-
mentally observed chemical shifts, electronic transitions and
vibrational wavenumbers are in good agreement with theoreti-
cally calculated values. The molecular orbital coefficients and
molecular orbital plots analysis suggests that the nature of
Acknowledgements
Author A.K Singh; thankfully acknowledge University Grant
Commission (UGC) (F. No. 39-701/2010-SR), New Delhi for financial
support. Author R.K. Singh; acknowledge SAIF-CDRI Lucknow and
IIT Kanpur, for providing spectral measurement facilities. Author
Sahabjada Siddiqui is thankful to ICMR, New Delhi, India for the
award of Senior Research Fellowship (No. 45/26/2013/BMS/TRM).
We are also grateful to chemistry department, University of Luck-
now for providing Central facility for computational research.
electronic excitations involved in these hydrazones are
p / p*
type. AIM calculations and topological parameters at bond crit-
ical points (BCP) confirm the presence of a weak intramolecular
hydrogen bond between N (26) and H (32) in aroylhydrazone
NHPY (4) of energy ꢀ3.73 kcal/mol. These compounds show
strong intramolecular charge transfer (ICT) due to movement of
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2017.01.059.
p
eelectron cloud from donor to acceptor and is responsible for
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