Experimental and theoretical studies of a pyrazole-thiazolidin-2,4-di-one hybrid

Article abstract of DOI:10.1016/j.molstruc.2017.03.100

The present work describes synthesis, characterization and biological evaluations of a hybrid compound 10 composed of two intriguing scaffolds pyrazole and thiazolidin-2,4-di-one. The title compound was obtained via multi-step reaction and characterized by a number of techniques (viz. IR, UV–Visible, ¹H-NMR, ¹³C-NMR and MS) including X-ray crystallography. The structural and photophysical data of compound 10 were well supported by theoretical calculations performed at density functional (DFT) level. In-vitro anticancer studies on different human cancer cell lines indicated moderate to low activity of the compounds. The molecular target of the compound was predicted through in-silico studies. Finding of the studies are presented herein.

Full text of DOI:10.1016/j.molstruc.2017.03.100

Accepted Manuscript
Experimental and theoretical studies of a pyrazole-thiazolidin-2,4-di-one hybrid
Md. Mushtaque, Fernando Avecilla, Ashanul Haque, Ahmed Perwez, Md. Shahzad
Khan, M. Moshahid Alam Rizvi
PII:
S0022-2860(17)30389-7
10.1016/j.molstruc.2017.03.100
MOLSTR 23596
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 October 2016
Revised Date: 25 March 2017
Accepted Date: 26 March 2017
Please cite this article as: M. Mushtaque, F. Avecilla, A. Haque, A. Perwez, M.S. Khan, M.M.A. Rizvi,
Experimental and theoretical studies of a pyrazole-thiazolidin-2,4-di-one hybrid, Journal of Molecular
Structure (2017), doi: 10.1016/j.molstruc.2017.03.100.
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Experimental and Theoretical Studies of a Pyrazole-Thiazolidin-2,4-di-one
Hybrid
Md. Mushtaquea*,#, Fernando Avecillab, Ashanul Haquec, Ahmed Perwezd, Md. Shahzad Khand,
M. Moshahid Alam Rizvie*
aSchool of Physical and Molecular Sciences, Department of Chemistry, Al-Falah University,
Dhauj, Faridabad, Haryana, 121004, India.
bDepartamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira s/n,
15008, A Coruña, Spain.
cDepartment of Chemistry, Sultan Qaboos University, P.O. Box 36, Al Khod 123, Sultanate of
Oman.
dDepartmentof Biosciences, Jamia Millia Islamia, New Delhi, 110025, India.
eAdvancedMaterial Research Group, ABV-Indian Institute of information technology and
Management Gwalior, 474015, India.
Abstract:
The present work describes synthesis, characterization and biological evaluations of a hybrid
compound 10 composed of two intriguing scaffolds pyrazole and thiazolidin-2,4-di-one. The title
compound was obtained via multi-step reaction and characterized by a number of techniques
1
(viz. IR, UV-Visible, H-NMR, 13C-NMR and MS) including X-ray crystallography. The
structural and photophysical data of compound 10 were well supported by theoretical
calculations performed at density functional (DFT) level. In-vitro anticancer studies on different
human cancer cell lines indicated moderate activity of the compounds. The molecular target of
the compound was predicted through in-silico studies. Finding of the studies are presented
herein.
Keywords: Synthesis, Molecular docking, MTT-assay, Quantum chemical studies
*Corresponding authors: mush.chem@gmail.com; GSM: +91-9891717992
Email: rizvi_ma@yahoo.com
#Author current affiliation: Department of Pharmacy and pharmacology, University of Bath,
Claverton Down, BA2 7YA, UK. mm2494@bath.ac.uk; GSM: +44-07424873711

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1. Introduction
Heterocyclic compounds, commonly defined as aliphatic or aromatic cyclic moieties composed
of two or more different type of elements, are indeed the most versatile class of compounds
human race have ever seen. This fact is well supported by the ubiquitous occurrence of
functional heterocyclic molecules in biomolecules (viz. DNA, proteins and amino acids)and
synthetic agents (viz. drugs, paints, etc.). Due to the intriguing chemical, physical and biological
features endowed by heteroatom (like N, O, S) bearing compounds, an extensive amount of work
have been performed by the researchers across the globe [1-4]. Compounds based on
heterocyclic scaffolds are being exploited by pharmaceutical and materials industries to produce
goods for daily life. Among large number of classes and sub-classes,one, two or three N-
containing compounds are the most versatile members of heterocyclic compounds. They are
considered as one of the backbone of organic synthesis, natural products, medicinal agents, and
polymers.
Pyrazole, a 5-membered two nitrogen containing heterocyclic ring, is a well-known scaffold
among medicinal chemists and is the fragment which is found in many drugs (for ex. Celecoxib,
stanolol, AZ-037, Crizotinib, Cyantraniliprole, F-2545920, Mepiprazole, Lonazolac etc.),
pesticides (Isopropylmethylpyrazolyldimethylcarbamate), and dyes (Pyrazole-72) etc. Due to the
diverse medicinal properties of pyrazole ring, number of pyrazole-based compounds have been
reported time to time with hypoglycemic[5], anti-fungal[6],anti-cancer, anti-microbial[7]and
anti-inflammatory[8, 9]. In the quest of new and effective pharmacologically active compounds,
researchers now exploring pyrazole hybrids along with their biological activities[10, 11]. In
addition to the pyrazole, last few decades also witnessed a significant amount of interest in
thiazolidinone-based compounds. Thiazolidinone derivatives have been found to display various
biological activities viz, anti-amoebic activity[12], anti-microbial[13] anti-proliferative
[14]antiviral [15], anti-convulsant[16]and anti-cancer properties[12, 17, 18]. Due to the unique
features these two class of molecules, hybrid compounds composed these two promising
scaffolds have also been reported withanti-microbial, anti-cancer and antiviral activity (Fig.1a-
c)[19-22]Considering the intriguing features of hybrids and uniqueness of these two fragments,
we report herein the synthesis, characterization and pharmacology of pyrazole-thiazolidinone
conjugates (10). In addition to this, theoretical calculation (quantum chemical and docking) has
also been presented.

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Figure 1:
Synergistic effects of pyrazole and thiazolidinone compounds (a) anti-microbial,
(b)anti-cancer (c) antiviral.
2.
Experimental
2.1. Materials and Methods
All the required chemicals were purchased from Merck and Aldrich Chemical Company (USA).
Precoated aluminium sheets (Silica gel 60 F254, Merck Germany) were used for thin-layer
chromatography (TLC) and spots were visualized under UV light. UV–Visible spectrum was
obtained with a Perkin-Elmer Lambda 40 UV-Vis. spectrometer (CT 06859 USA) in DMSO
solvent. FT-IR spectra were recorded on Perkin Elmer model 1600 FT-IR RX1
1
spectrophotometer as KBr discs. H-NMR spectra were recorded on Bruker Spectrospin DPX
300 MHz spectrometer respectively using CDCl3 as a solvent and trimethylsilane (TMS) as an
internal standard. Splitting patterns are designated as follows; s, singlet; d, doublet; m, multiplet.
Chemical shift values are given in ppm. X-ray data were collected on Bruker SMART Apex
CCD diffractometer (SAI, Universidade da Coruña).
2.2. Synthesis
2.2.1. Synthesis of 3-(3-nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde(4)
1.0 mole of hydrazone (3) was dissolved in minimum amount of DMF, and 2.0 mole of DMF
were taken in a round bottom flask and 2.0 mole of POCl3 was poured in DMF dropwise at ice
temperature and mixture was kept for 20 min. for preparing Veilsmeyer reagent. After 20
minutes, hydrazone (3) was poured in Vilsmeier reagent slowly and stirred at room temperature
then refluxed for 10-12 h.The progress of the reaction was monitored by TLC in 5 % (MeOH:

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CHCl3). After the completion of the reaction, the reaction mixture was cooled and poured in ice
cold water and treated with 2N NaOH and stirred, creamy solid was appeared. The solid was
filtered and dried and recrystallized in CHCl3.IR(νmax, cm-1): 3142(Ar-H); 3082 (Ar-H); 1685(-
1
CHO), 1596(C-N); H-NMR(CD-Cl3)(ppm): 10.006(s, 1H, aldehydic proton), 9.396(s, 1H,
Pyrazole proton), 8.852(s, 1H, Ar-H), 8.456(d, 1H, J = 7.5 Hz, Ar-H), 8.316(d, 1H, J = 7.8 Hz,
Ar-H), 7.996(d, 2 H, J = 7.5 Hz, Ar-H), 7.813(t, 1H, J = 7.8Hz, Ar-H), 7.611(t, 2H, J = 7.2 Hz,
Ar-H), 7.473(t, 1H, J = 7.2 Hz, Ar-H); C16H11N3O3: Calc mass: 293; m/z; [M+H]+= 294.
2.2.2. Synthesis of 2, 4-thiazolidi-one (7)
1 mole of thiourea was dissolved in water and 1 mole of chloroacetic acid was added followed
by the addition of concentrated hydrochloric acid and refluxed. The progress of reaction was
monitored by TLC. After the completion of reaction, the reaction mixture was kept at room
temperature for overnight. Crystalline solid was obtained and filtered and washed with water.
The structure was confirmed by X-ray single crystal structure.
2.2.3. Synthesis of 3-(2, 4-dioxo-1, 3-thiazolidin-3-yl)propanenitrile (9)
1 mol of compound (7) was dissolved in dry DMF followed by the addition of sodium hydride
and added 1.5 mol of acrlyonitrile. The reaction mixture was refluxed for an hour and after the
completion of the reaction, the reaction mixture became light brown. The mixture was poured in
water and back extracted with dichloromethane and dry over sodium sulphate and evaporated on
rotatory vacuum. The brown colored liquid obtained was left for crystallization for a day. Light
0
brown crystalline solid was obtained.M.P:70 C; FT-IR; 2250 (CN), 1749(C=O), 3006 (CH2);
1HNMR (CDCl3)(PPm): 4.025(s, 2H, -S-CH2), 3.964(t, 2H, J = 6.6 Hz, CH2), 2.768(t, 2H, J
=6.6 Hz, -CH2-); Calc.mass of C3H3NO2S:170; exp. Mass observed at [M+NH4]+= 188.2
2.2.4. Synthesis of 3-[(5Z)-5-{[3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]methylidene
2,4-dioxo-1,3-thiazolidin-3-yl]propanenitrile(10)
1 mol of compound (9) was dissolved in absolute ethanol in round bottom followed by the
addition of 1mol pipiridine base. The reaction mixture was stirred at for few minutes and then 1
mol of compound (4) was added. The reaction mixture was stirred at reflux and the progress of
reaction mixture monitored by TLC 5% (Methanol:Chloroform). After the completion of
reaction, yellow solid product was appeared and filtered on Buckner funnel. The compound was
0
recrystallized in chloroform and methanol. M.P: 139 C; IR(λmax)cm-1: 3026(Ar-H); 2941 (Ar-
H); 2250(CN), 1741 (-C=O), 1656(C-N), 1562 (H-C=C-); 1H-NMR(CD-Cl3)(ppm): 8.766(s, 1H,

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Pyrazole proton), 8.402(s, 1H, Ar-H), 8.343(d, 1H, J = 6.9 Hz, Ar-H), 8.076-8.001(m, 3H, Ar-
H), 7.861(t- 1H, J = 7.5 Hz, Ar-H), 7.681(s, 1H, H-C=C, alkenic proton), 7.580(d, 1 H, J = 6.0
Hz, ar-H), 7.449(d, 1H, J = 6.6 Hz, Ar-H), 4.530(t, 2H, J = 6.3 Hz, CH2), 2.940(t, 2H, J= 5.7 Hz,
CH2); C22H15N5O4S:Calc mass: 445; Observed: m/z; [M+H]+ = 446;
3. Cytotoxicity studies (MTT assay)
3.1. Cell culture
Human colon cancer cell line (SW-480), Breastcancer cell (MCF-7) and Human Embryonic
Kidney cell line (HEK-293) were procured from National Curator of Cell Sciences (NCCS)
Pune, India. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 %
fetal bovine serum with 100 units/mL penicillin,100 ꢀg/mL streptomycin, and 2.5 ꢀg/mL
amphotericin B, at 37 0C in a relative humidity 80 %, 5 % CO2.[19].
3.2. MTT assay
Cytotoxicity of the compounds was evaluated through MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyl tetrazolium bromide, M2128 Sigma Aldrich) assay on SW-480, MCF-7, and HEK-293
cells. MTT is a validated assay for the in vitro cytotoxicity of any natural, synthetic compounds
and extracts [20-21]. The cell count 1.2x104 cells/well was seeded in 96 well plate (150 ꢀL/well).
After the overnight incubation, cells were treated with different concentration of compounds for
48 hrs. After the 48 hrs of treatment, the medium was remove and incubated with 20 ꢀL of MTT
solution (5 mg/mL in Phosphate saline buffer) for 4 hours. The formazan crytals were form by
mitochondrial enzyme redution, finally solublized in DMSO (150 ꢀL/ well) and absorbance was
recorded at 570 nm through the Microplate reader (iMark, BIORAD, S/N 10321). Percent
viability was defined as the relativeabsorbance of treated versus untreated control cells.
4. Computational details
4.1. DFT studies
The ground state geometry optimizations have been performed using DFT with Becke-3-Lee-
Yang-Parr (B3LYP) exchange-correlation functional [22-23] in combination with 6-
311++G(d,p) basis sets using Gaussian03 package [24] without any symmetry constraint. The
calculated IR spectrum has been analyzed using GAUSSVIEW, and different modes of
vibrational frequencies have been assigned by VEDA 4 program with high degree of accuracy.
For electronic excited state calculations TD-DFT/B3LYP method was executed with same basis

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set as in optimization. The TD-DFT calculations for titled compound are made in the background
of dimethyl sulphoxide (DMSO) using PCM model [25-26].
4.2. DNA docking studies
Docking studies were performed at Intel(R) Core(TM) i3 CPU (2.3 GHz) with XP-based
operating system (Windows 2007). 3D Structure of compound (10) was drawn by Marvin
software and saved in pdb file format. The preparation of compound was done by assigning
Gastegier charges, merging non-polar hydrogens, and saving it in PDBQT file format using
AutoDock Tools (ADT)4.2[27-28]. The X-ray crystal structure of DNA (PDB ID: 1BNA) was
obtained from the Protein Data Bank [Protein Data Bank. Available online at:
http://www.rcsb.org/pdb]. Using ADT 4.2, DNA was saved in PDB file format leaving
heteroatoms (water). Gastegier charges were assigned to DNA and saved in PDBQT file format
using ADT. Preparation of parameter files for grid and docking was done using ADT. Docking
was performed with Auto-Dock4.2 (Scripps Research Institute, USA), considering all the
rotatable bonds of ligand as rotatable and receptor as rigid[29] Grid box size of 80 × 80 × 60 Å
with 0.375 Å spacing was used that included the whole DNA. In the present work we describe a
plug in for PyMOL which allows carrying out molecular docking, virtual screening and binding
site analysis with PyMOL. The plug in represents an interface between PyMOL and two popular
docking programs, Autodock[27-28], AutodockVina[29] and also the extensive use of a Python
script collection (Autodock Tools) [30].
5. Results and discussion
5.1. Chemistry
The thiazolidin-2, 4-di-one derivative was synthesized as described in Scheme (1) by reported
1
method [10, 12]. The synthesized compounds were confirmed by FT-IR, H-NMR, mass
spectrometer and X-ray single crystal structure. The melting point of final compound (10) was
recorded as 139 0C and is uncorrected.The appearance and disappearance of significant bands in
spectra confirm the formation of a particular compound. Here, the intermediate compounds were
confirmed by IR and 1H-NMR and mass spectroscopy. In case of compound (4) intermediate, the
presence of characteristic peak at 1685 cm-1due to aldehydic functional group (H-C=O)
confirmed the formation of compound. Additionally, the presence of other supporting functional
groups present in the molecule at 1596 cm-1 due to -C=N-, and 3142 cm-1 and 3082 cm-1 due to
1
Ar-H indicated the formation of compound (4). In H-NMR, the presence of chemical shift due

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to aldehydic proton (-CHO) at 10.006 ppm, and pyrazole proton at chemical shift 9.396 ppm
displayed that compound (4) was formed. Furthermore, in mass spectrum,the presence of m/z;
[M+H]+= 294 confirmed the formation of compound (4).Second intermediate compound (7) was
characterized x-ray single crystal structure.The third intermediate compound (9) was confirmed
by IR, as the presence of characteristic peak (CN) was at 2250 cm-1. It was also confirmed by 1H-
NMR and mass spectroscopy and the data are given in experimental section.
Scheme (1): (a) Absolute ethanol, Reflux(b) DMF, POCl3, reflux(c) H2O Aq. HCl (3:2),
reflux(d) NaH, DMF, reflux for an hour(e) piperidine, Ethanol, reflux.
The structure of final compound (10) was determined by IR,1H-NMR and mass spectrometer. In
IR, the appearance significant band in the range of 1500-1600 cm-1 due to -C=C- (exocyclic
alkenic bond) showed the presence of compound (10). The characteristic peaks due to carbonyl
group(-C=O),nitrile (-CN) and alkenic carbon(-C=C-) were present at 1749 cm-1 , 2250 cm-1 and
1
1562 cm-1 respectively in IR, which clearly indicated the formation of compound (10). In H-
NMR, significant chemical shift appeared at 8.766 ppm and 8.402 ppm due to pyrazole proton
and alkenic proton pointed out that compound (10) was formed. Additionally, the presence of
m/z at [M+H]+ = 446 also confirmed of compound(10).
5.2. Discussion of X-ray single crystal structure
The compound 2,4-thiazolidi-one (7)crystallized from water as colourless blocks and Fig.(FS1)
depicts an ORTEP representation of the asymmetric unit. Table (TS1) provides a selection of
distances and angles in the structure. The structure of the compound contains planar molecules
[mean deviation from plane, C(1), C(2), C(3), N(1), S(1), 0.0014(15) Å] which interact by

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intermolecular hydrogen bonds between protonated nitrogen atoms, N(1), and oxygen atoms of
the oxido group, O(2), (see Table 1). In the crystal packing the structure presents intermolecular
associations in dimers by hydrogen bonds (see Fig. (4). π-π interactions are not present in the
structure. The Crystal structure of the compound (7) has already been reported [31].The
compound is identical and the cell parameters too. The only difference is the order in the crystal
packing. In our crystal packing the dimmers appear in layers. In the crystal packing published in
Acta appear in zigzag packing. For comparative studies, the crystal packing structures have been
given in Fig. (4). It can be seen from the Figures that in the early reported crystal, the molecules
packed as hydrogen-bonded dimmers in ab plane and were stacked in a column along c; N-
°
H…O(2) bonds of 2.847 A and held the molecules together. It was also reported by the author
that methylene H atoms were not located with sufficient accuracy to be include in the structural
°
°
details, but the two O(4)-C(5) distances of 3.198 A and 3.226 A suggested C-H..O Bond.
However, in our studied compound (7), the hydrogen bonds are present between oxygen and
nitrogen, resulted in dimeric form due to hydrogen bond. Method has been given in supporting
information.
Figure 4: View of a section of crystal packing of the compound 2,4-thiazolidi-one (7). Dimers
associated by intermolecular hydrogen bonds appear in the structure.
Table (1):- Hydrogen bonds [Å] in the compound 2,4-thiazolidi-one (7).#
D
-H...A
___________________________________________________________________
N(1)-H(1N)...O(2)#1 0.84(5) 2.00(5) 2.836(3) 175(4)
d(D-H)
d(H...A)
d(D...A)
<(DHA)

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______________________________________________________________________
#Symmetry transformations used to generate equivalent atoms: 1 -x,-y+1,-z+1.
5.3. Computational study
5.3.1. NBO analysis
NBO analysis centralizes to describe the role of inter and intramolecular bonding. In a molecular
system, it provides a key platform to find charge transfer or hyper conjugative interactions. There
is an energy difference in between interacting orbitals which is proportional to stabilization of
orbital interaction. Thus, the strongest interaction of stabilization is foundbetween main donors
(i) and acceptors (j). Actually, NBO describes interaction between bonding and anti-bonding
molecular orbitals quantitatively, which is expressed by the second order perturbation interaction
energyE(2)[32]. This energy depicts an estimate of the off diagonal NBO Fock matrix element.
The stabilization energy is expressed by the following mathematical expression.
ꢀ(ꢁ,ꢂ)ꢃ
E2 = ∆Ei, j= qi
(1)
ꢄꢁꢅꢄꢂ
Where qi= donor orbital occupancy εi and εj= orbital energy; F(i, j) = off diagonal Fock matrix.
In our reported compound (10), second lone pair (lp) of electron of sulphur delocalized to an
anti-bonding orbital of π*(C17-O21) through hyper conjugation, resulting the stabilization energy
26 kcal/mol. The lone pair of electron of N9 delocalized to the anti-bonding orbitals of π*(C7-
N8) and π*(C10-C11) through hyper conjugation and resonance, releasing the stabilization
energy of 26 kcal/mol and 14 kcal/mol. Additionally, Lone pair (lp) of electron of N18
delocalized to the anti-bonding orbitals of π*(C17-O21) and π*(C19-O20) through hyper
conjugation, furnished 57 Kcal/mol and 49 Kcal/mol stabilization energy. Furthermore, O20 lone
pair delocalized to an anti-bonding orbital of σ*(N18-C19) through resonance and gave
stabilization energy 20 Kcal/mol. The lone pair electrons present on O21 and O32 delocalized to
anti-bonding orbitals σ*(S16-C17) and σ*(C17-N18), and π*(N12-O31) through hyper
conjugation, and resonance, as consequence hyper conjugative and resonance stabilization
energy were found 35 Kcal/mol and 25 Kcal/mol, and 166 Kcal/mol respectively. The bonding
orbitals π(C3-C4), π(C3-C4) and π(C1-C2) interacted with the anti-bonding orbitals of π*(C1-
C2), π*(C5-C6) and π*(N12-O31) through resonance and caused stabilization energy 23
Kcal/mol, 19 Kcal/mol and 23 Kcal/mol. Thus, molecule is stabilized, and stabilization energy
has been given in Table (2).

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Table (2): Second order perturbation theory analysis of fock matrix in NBO basis for larger E2
value
Donor(i)
LP(2)S16
LP(1)N9
LP(1)N9
LP(1)N18
LP(1)N18
LP(2)O20
LP(2)O21
LP(2)O21
LP(3)O32
π(C3-C4)
π (C3-C4)
π (C1-C2)
Acceptor(j)
E2(in Kcal/mol)
26
26
41
57
49
π* (C17-O21)
π* (C7-N8)
π* (C10-C11)
π*(C17-O21)
π* (C19-O20)
σ* (N18-C19) 20
σ* (S16-C17)
σ*(C17-N18)
π* (N12-O31)
π*(C1-C2)
35
25
166
23
19
23
π*(C5-C6)
π*(N12-O31)
5.3.2. IR analysis
The3-[(2Z,5Z)-5-{[3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]methylidene}-4-oxo-2-
(phenylimino)-1,3-thiazolidin-3-yl]propanenitrile(10) consists of 47 atoms which gives to 135
normal modes of fundamental vibrations, out of which 46 modes are stretching, 45 bending
vibrations, 35 torsion and 9 out of plane. This molecule belongs to C1 symmetry. The
experimental and quantum chemically simulated IR-spectrum have been shown in Fig. (6).the
fundamental modes of vibration with B3LYP have been provided in Table (3). In our study, we
have scaled the B3LYP calculation with 0.98 to correct the theoretical error.

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Table (3): Comparative study of theoretical and experimental IR of 3-[(2Z,5Z)-5-{[3-(3-
nitrophenyl)-1-phenyl-1H-pyrazol-4-yl]methylidene-4-oxo-2-(phenylimino)-1,3-thiazolidin-3-
yl]propanenitrile (10).
Experimental IR
Theoretical
IR
Assignments
3281
ν(C10–C37)99
3228
3227
3222
3205
ν(C6 –H36)99
ν(C2–H33)96
ν(C25–H43)96
ν(C3–H34)85,ν(C4–H35)85,ν(C4–H35)12, ν(C3–H34)13
ν(C26–H44)92, ν(C27–H45)92, ν(C28–H46)92,
ν(C29–H47)92
3200
3192
3182
3173
3166
3149
3157
3108
ν(C26–H44)90, ν(C27–H45)90, ν(C29–H47)90
ν(C28–H46)86, ν(C29–H47)86
ν(C26–H44)89, ν(C27–H45)89, ν(C28–H46)89
ν(C14–H38)99
ν(C22–H39)96, ν(C22–H40)96
3065
3094
3092
ν(C23–H41)91, ν(C23–H42)91
ν(C22–H39)91, ν(C22–H40)91
3015
2257
1749
1702
1674
3052
2364
1820
1763
1665
ν(C23–H41)97
ν(C23–H42)90, ν (N30–C24) 90
ν(O20–C19)79, ν (O21–C17)79
ν(O20–C19)78, ν (O21–C17)78
ν(C1–C2) 64, ν(C1–C6)64, ν(C3–C4)64 + δ(H33–C2–
C3)10
1655
1652
1642
ν(C11–C14)65, ν(C14–C15)65+ δ(H38–C14–C15)10
ν(C26–C27)63, ν(C28–C29)63, ν(C25–C26)63,
ν(C27–C28)63
1642
δ(H43–C25–C26)11, δ(H44–C26–C27)11, δ(H46–C28–
C29)11, δ(H47–C29–C28)11
1637
1624
1605
1567
ν(C13–C25) 58, ν(C26–C27)58
ν(C2–C3)69, ν(C5–C6)69
ν(C1–C2)76, ν(C2–C3)76, ν (C3–C4)76, ν (C5– C6)76
δ (H37–C10–C11)12
1562

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1520
1510
1533
1529
δ (H43–C25–C26)30, δ (H47–C29–C28)30
ν (C5–C6)14+ δ (H33–C2–C3)18+ δ (H34–C3–C4)18+
δ (H36–C6–C5)18
1499
1480
δ(H43–C25–C26)43
1488
1454
ρ(H39–C22–N18–C17)13, ρ(H40–C22–N18–C17)13,
ρ(H41– C23–C24–N30)13, ρ(H42–C23–C24–N30)13
δ(H40–C22–H39)77, δ (H42–C23–H41)77
ρ(H39–C22–N18–C17)18,ρ(H40–C22–N18–C17)18,
ρ(H41–C23–C24–N30)18, ρ(H42–C23–C24–N30)18
ν(N9–C13)39, ν(N9–C10)39, ν(N12–C1)39
δ(C7–N8–N9)12
1473
1425
1444
1398
1381
1379
1364
1358
δ(O32–N12–O31)12
δ(H39–C22–C23)56
ν(N18–C22)18+ ν(N18–C19)15
δ(H47–C29–C28)68, δ(H43–C25–C26)68, δ(H46–C28–
C29) 68
1348
ν(C25–C26)10, ν(C26–C27)10, ν(C27–C28)10,
ν (C28–C29)10
1345
1290
1267
1238
ν(C1–C6)54, ν(C2–C3)54, ν(C3–C4)54, ν(C5–C6)54
ρ(H39–C22–N18–C17)13, ρ(H41–C23–C24–N30)13
ν(N9–C13)22, ν(N9–C10)22 ,ν(N12–C1)22
δ(C11–C10–N9)14
1128
1043
1190
1185
δ(H33–C2–C3)69, δ(H34–C3–C4)69
δ(H44–C2–C27)71, δ(H45–C27–C28)71
1065
1023
1017
1011
1008
991
δ(C1–C2–C3)16
ρ(H30–C24 –C23–N22)10
δ(C2–C3–C4)50
ρ(C2–C1–C6–N5)15
ρ(H44 –C26–C27–C28)75
δ(C15–C19–N18)11
ρ(H43 –C25–C26–C27)68
δ(C10–N9–N8)25
984
972

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964
953
935
ρ(H33–C2–C3–C4)71
ρ(H33– C2–C3–C4)84
ρ(H36–C6–C5–C7)80
922
864
929
846
828
ρ(H43–C2 –C26–C27)84
ρ(H43–C25–C26–C27)90
ρ(H33–C2–C3–C4)67
818
776
764
752
711
705
668
657
648
607
597
ρ(H37–C10–C1–C7)62
ρ(H43–C25–C26–C27)48+ σ(N9–C25–C29–C13)18
σ(O20–C15–N18–C19)20
σ(O20–C15–N18–C19)64
δ(C5–C7–N8)12
750
ρ(C27–C26–C25–C13)47
δ(C11–C14–N15)26
689
672
σ(O21–N18–S16–C17)81
σ(C10–C13–N8–N9)47
δ(C22–C23–N24)39
δ(C19–C15–S16) 13
υ-stretching; δ-bending; τ-torsion, σ Out of plane.
Figure 6: Theoretical FT-IR of compound(10).

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5.3.3. C-H vibrations
The different theoretical modes of C-H stretching vibrations of compound (10) lie in the region
of 3000-3280cm-1. Theoretical mode of vibration of C-H of pyrazole was calculated at 3280 cm-
1, however, its experimental counterpart was observed at 3157cm-1. The asymmetric and
symmetric vibrations, due to aliphatic groups C22-H39 and C22-H40, were observed at 3149 cm-
1 and 3092 cm-1 respectively. C23-H41 and C23-H42 showed asymmetric vibration at 3094 cm-1
and symmetric vibration at 3052 cm-1, theoretically, however, their experimental values for same
were found at 3065 cm-1 and 3015 cm-1 respectively. The stretching mode of vibrations of C-H
of aromatic ring were calculated at 3228 cm-1, 3227 cm-1, 3222 cm-1, 3205 cm-1, 3200 cm-1, 3192
cm-1, 3186 cm-1, 3182 cm-1 and 3173 cm-1 respectively, however, its experimental value was
observed at 3108 cm-1. Moreover, the exocyclic methylene group C14-H38 showed wave
number at 3165 cm-1 and experimental value was at 2889 cm-1. Other modes of vibration have
been given in Table (3).
5.3.4. CN Vibration
The theoretical mode of vibration of CN was calculatedat 2364 cm-1 and its experimental
counterpart value was observed at 2257 cm-1.
5.3.5. O=C vibrations
The carbonyl C=O groups contributedsimulated wave numbers at 1820 cm-1 and 1736 cm-1 and
its experimental values were observed at 1749 cm-1 and 1702 cm-1 respectively.
5.3.6. C=C vibrations
The theoretical and experimental mode of vibration of exocyclic alkenic carbon (C=C) were
found at 1652 cm-1 and 1655 cm-1. Besides of it, C=C of aromatic ring contributed its values at
1552 cm-1 and 1520 cm-1experimentally, and its calculated values were found at 1642 cm-1, 1637
cm-1, 1625 cm-1 and 1605 cm-1.
5.3.7. C-N vibrations
The simulatedstretching vibration of C-N of thiazolidinone ring was found at 1444 cm-1. But for
same functional group experimental value was observed at 1665 cm-1. Other modes of vibrations
associated with C-N have been demonstrated in given in Table (3).
5.4. Frontier Molecular Orbitals
The biological potency of a molecule and some important features viz. ionization energy and
electron affinity, of the under studied compound (10) can be obtained from frontier molecular

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orbital HOMO (highest occupied molecular orbital) and LUMO (least unoccupied molecular
orbital). Molecules having smaller interfrontier orbitals are more active chemically and have
lower stability [33-35]. The Ionization enthalpy is obtained from to highest occupied molecular
orbital (HOMO), however, electron gain enthalpy is from the lowest unoccupied molecular
orbital. The ionization energy and electron affinity are expressed as I= -EHOMO(6.58eV)& A= -
E
LUMO(2.78eV).
5.5. UV-Visible Spectra
The theoretical and experimental comparative studies of electronic transition have been given in
Table (4), and spectra have been shown in Fig. 7(a-b). In order to calculate theoretical electronic
transition, TDDFT calculation has been performed with basic setsat TD-DFT/B3LYP/6-
311G++(d, p) same level of theory as used in optimization for twelve lowest singlet state with
DMSO background. Fig.(8) Contained the interfrontier molecular orbitals obtained from TDFFT
calculation at the contour value of 0.02 have been used to analyze the electronic transitions
behavior for reported peaks in Table (4).The calculated electronic transition at 371 nm
corresponds to the experimental transition at 373 nm. This transition is due to nitro group
attached to the aromatic ring of pyrazole. The nature of transition isn→π* and the molecular
orbitals involved HOMO to LUMO (90%). The observed electronic transition at 296 nm
corresponds to the theoretical value at 294 nm and carbonyl group(-C=O) attached to 2,4-
thiazolididione ring is responsible this transition. The nature of a transition is n→π*, and major
contribution of orbital is from HOMO-4 to LUMO (23%).Additionally, experimental electronic
transition was observed at 284 nm. However, its theoretical counterpart was observed at 275 nm.
The electronic transition involves π→π* transition and this transition is mainly contributed from
HOMO-2 to LUMO (49%). Hence, It can be inferred that the theoretical and experimental results
are almost similar.

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Table 4:Theoretical and experimental UV-Visible of compound (10).
Experimental
TDDFT-6311++g(d,p)
Assignments
Abs.
λ (nm)
λ (nm)
Oscillator
strength
--
399
373
1.33
1.33
--
--
371
0.04
0.01
0.05
HOMO->LUMO (90%)
n→π*
296
284
2.49
2.37
294
275
H-4->LUMO (23%)
π→π*
H-2->LUMO (49%)
π→π*
7(a)
7(b)
Figure 7(a-b): Experimental (b) theoretical UV-Visible of compound (10)

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Figure 8: Frontier molecular orbitals of compound(10).
5.6. MTT Assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is an important dye
reduced by succinate dehydrogenase enzyme of mitochondrial living cells to produce water
insoluble purple formazan crystals[36-37]. MTT assay has widely been used to assess the

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cytotoxic effects of synthetic compounds/Natural compounds/Extract [20, 38]. The cytotoxicity
of compounds (4 & 10) was screened against MCF-7, SW-480 and HEK-293 cell lines for 48
hours treatment at concentration range 0-400 ꢀg/ml as demonstrated in Fig.9(a-c). In our study,
it was found that compounds (4 & 10) were found non-cytotoxic against the cancerous cell lines
MCF-7 (IC50 = 254.89 ꢀg/ml & 282.32 ꢀg/ml), and SW-480 cells (IC50= 232.15 ꢀg/ml & 200
ꢀg/ml). The compounds (4 & 10) were also found non-toxic against normal human HEK-293
(IC50 = 634.58 ꢀg/ml & 404.52 ꢀg/ml) cells. Hence, our results depicted that compounds (4 &
10) were found non-cytotoxic against MCF-7 and SW-480 cells upto the concentration
282.32ꢀg/ml and 200ꢀg/ml. At higher concentrations, these compounds may behave anti-cancer
agent.
Figure 9(a-c): Cells viability of the compounds (4 &10) against MCF-7, SW-480 and HEK-
293 Cells at the concentration range 10-500 µg/ml.
5.7. Molecular docking
Molecular docking or computational studies have become common to find molecular target for a
particular disease. From this, one can easily predict the biological potential and active sites of the

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target. Organic and inorganic molecules are being exploited on large scale to know the behavior
with target before synthesis and further biological activity. The computational studies have been
a very important tool for drug development [39] owning to its quick results, cheap price, free and
easy availability of software’s [30]. DNA docking studies have performed with the help of Auto
Dock-Vina tool in order to get binding energy, residues with studied compounds [27-28]. The
molecular docking study of compound (10) was performed with DNA dodecamers
d(CGCGAATTCGCG)2 (PDB ID: 1BNA). The binding energy of the docked compound (10)
was found -7.2 Kcal/mol. It was found that molecule preferred to interact with base pair of DNA
as exhibited in Fig.10(a-d) and made polar and several no-polar interactions Fig. 10(a-d).This
molecular orientation of compoundled to the formation of one H-bonds hydrogen bonds with
chain B (B: DG’16/O4::O of 2,4-thiozolidi-di-one (3.2°A). The interaction, bond distance, chain
of DNA and interacting residue of DNA has been presented in Fig.6(a-d).Ali et. al reported in
his paper that 3-nitro substituted pyrazole derivative associated with curcumine displayed
docking energy -7.44 kcal/mol as minor grove binder and acted as anticancer agent[10]. But our
reported compound (10) showed binding energy -7.20 kcal/mol, acting as intercalating agent and
may act as anti-cancer agent.

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Figure10(a-d): Molecular docking of compound (10) with known receptor (PDBID: 1BNA).
6. Conclusion
From our studies, it can be concluded that the ionization energy and electron affinity of the
compound (10) were found 6.58 eV and 2.78 eV. Other quantum chemical studies UV-Visible
and FT-IR of reported compound (10) suggested that experimental and theoretical data were
almost in good agreement with each other. Molecular docking revealed that binding energy of
the compound (10) was -7.2 Kcal/mol and behaved intercalating agent which is a good indication
for anti-cancer agent. But MTT-assay data showed that compounds (4 &10) were non-toxic up to
the concentrations 254.89 µg/ml and 282.32 µg/ml against cancerous MCF-7 cell line and 232.15
µg/ml and 200 µg/ml against Siha cells. Thus, it may be concluded that at higher concentration
compounds may behave as anti-cancer agent.

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Acknowledgement
One of the authors. Dr. Md. Mushtaque is thankful to Science and Engineering research Board
(SERB), Govt. of India for financial assistance (SB/OS/-092/2015-16) and Prof. Stephen
Husbands for his help in the manuscript preparation. Also thankful to the chancellor(Mr. Jawad
Ahmed Siddiqui) of Al-Falah University for his continuous support and also thankful to Dr.
Mohd.Shahid Khan to permit me to use his license of Gaussian 03 software, Department of
Physics, Jamia Millia Islamia, New Delhi, 110025, India.
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•
•
•
•
DNA-binding affinity was found -7.2 Kcal/mol
Compound(10) occupied Z-configuration about exocyclic alkenic bond.
Compounds (4 and 10) both were found non-toxic against cancerous and normal cell lines.
Crystal structure of compound(7) was found in dimeric form.