Journal of Saudi Chemical Society (2018) 22, 1003–1013

King Saud University

Journal of Saudi Chemical Society

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ORIGINAL ARTICLE

Structure elucidation, DNA binding, DFT,

molecular docking and cytotoxic activity studies on
novel single crystal (E)-1-(2-fluorobenzylidene)
thiosemicarbazide
Nasima Arshad a,*, Pervaiz Ali Channar b, Aamer Saeed b,**, Shahid Iqbal Farooqi a,
Aneela Javeed c, Fayaz Ali Larik b, Waqar Ahmad Abbasi b, Ulrich Flörke d

a
Department of Chemistry, Allama Iqbal Open University, 44000 Islamabad, Pakistan
b
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan
c
Healthcare Biotechnology, Atta-ur-Rehman School of Applied Biosciences, National University of Science and
Technology (NUST), Islamabad, Pakistan
d
Department Chemie, Fakultätfür Naturwissenschaften, Universität Paderborn, Warburgerstrasse 100, 33098 Paderborn, Germany

Received 6 February 2018; revised 23 April 2018; accepted 2 May 2018

Available online 21 May 2018

KEYWORDS Abstract Compound 3 {(E)-1-(2-fluorobenzylidene)thiosemicarbazide} – a new Schiff base of

Thiosemicarbazide Schiff
base;
Crystal structure;
DNA binding;
DFT calculations;
Molecular docking;
Huh-7 cell line activity
thiosemicarbazide has been synthesized, characterized and reported for crystal structure. Planer side
chain in the crystal structure was observed co-planer with aromatic ring plane and molecules were
connected into centrosymmetric dimmers via intermolecular hydrogen bonding. DFT geometry
optimization and the relevant quantum parameters indicated unstable and reactive nature of com-
pound 3. Experimental and theoretical findings for DNA binding by UV–visible, cyclic voltamme-
try and molecular docking studies showed consistency in kinetic (Kb) and thermodynamic (DG)
parameters and that compound 3 significantly interacted with DNA via intercalation. Viscometric
analysis further comprehended intercalation as possible binding mode of the compound with DNA
and non-denaturing of DNA in the presence of 10% aqueous DMSO. Docked parameters further

* Corresponding author at: Department of Chemistry, Faculty of Sciences, Allama Iqbal Open University, Islamabad 44000, Pakistan.
** Co-corresponding author at: Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan.
E-mail addresses: nasimaa2006@yahoo.com (N. Arshad), aamersaeed@yahoo.com (A. Saeed).
Peer review under responsibility of King Saud University.

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1004
assured the drug like characteristics of the investigated compound as fit in Lipinski’s criteria. Dose
dependant cytotoxic activity of compound 3 against human Huh-7 cell line indicated its anti-cancer
potential at 100 mg/ml concentration.
Ó 2018 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The thiosemicarbazide moiety serves as structural template for
the synthesis of a wide variety of N and S containing hetero-
cyclic compounds. Moreover, the presence of NH and C‚S
moieties plays essential role for the synthesis of various metal
complexes. Sulfur and nitrogen atoms of hydrazine in
thiosemicarbazides provide a platform by acting as chelating
ligands to form complexes of transition metal ions. Thiosemi-
carbazides have been evaluated over the last 50 years as antivi-
ral, antibacterial, and anticancer therapeutics and their
biological activities are a function of parent aldehyde or
ketone moiety [1–7].
Schiff bases are nitrogen analogs of the aldehydes or
ketones and their metal complexes are reported to have more
biological potentials than simple organic compounds [8]. Schiff
bases can be easily synthesized by reacting primary amines
with aldehyde or ketones in acidic or medium at neutral pH.
Azomethine group containing lone pair on its nitrogen atom
plays main role in biological processes via hydrogen bonding
interaction with active cell constituents [9–11]. Many biologi-
cally important Schiff bases have been reported for possessing
antibacterial [1–7], antifungal [12–14], antimicrobial [15–17]
and anti-HIV [18,19] activities. Schiff base have also been
investigated for antitumor and anti inflammatory activities
using animal tumors as well as by in-vitro biological and ana-
lytical methods [20–22]. Other applications of Schiff bases are
reported for their chemotherapeutic, catalytic, polymer stabi-
lizer and corrosion inhibition activities [10,11,23,24].
DNA damaging – the major cause of pathological changes
in living organisms like development of several types of can-
cers, may arise due to abnormal changes in the DNA structure
via metabolic disturbance under natural and environmental
influence. Chemotherapeutic drugs available in the market
though prohibit the process of cell division but also have sev-
ere side effects. This drawback is still a major challenge for
researcher to replace them with those having no or fewer side
effects.
Covalent binding of a compound with DNA is the main
cause to harm to the healthy cell; therefore research on the
compounds that interact with DNA non- covalently and rever-
sibly via electrostatic, groove binding and intercalation has
gained much attention. In this respect, several new compounds
have been explored to probe their potency as therapeutic
agents by reversible DNA binding interactions. Theoretical
and analytical studies are considered more appropriate for in
vitro investigations of anticancer/or antitumor activities of a
drug/ or drug like compound via interaction with DNA [25–
27]. In this paper, we are reporting newly synthesized Schiff
bases of thiosemicarbazide for crystal structure and DFT, its
DNA binding by UV–visible spectroscopy, cyclic voltamme-
try, viscometry and molecular docking and compound’s activ-
ity for cancer cell line.
N. Arshad et al.
2. Experimental
2.1. Materials and methods
Chemicals and reagents used in synthesis, analytical and bio-
logical studies were of analytical grade. All the necessary
purifications and drying of solvents were carried out according
to standard methods. The dried solvents were stored over
molecular sieves. For monitoring of the synthetic reactions;
thin layer chromatography (TLC) was conducted on 0.25
mm silica gel plates (60 F254, Merck). Visualization of chro-
matogram was made under UV-lamp at 365 and 254 nm. Rf
value was calculated using a solvent system of petroleum ether:
ethyl acetate in 4:1 ratio. The yield (%) given was on the basis
of 1.0 mM of each precursor used. ds.DNA extraction from
calf thymus gland was done by using Falcon protocol [28].
Autoclaved apparatus and water was used throughout the
extraction procedure. DNA threads were obtained and dis-
solved in water. Stock solution of DNA was further diluted
and monitored on UV–visible spectrophotometer for absor-
bance at kmax of 260 nm. DNA concentration was determined
from Lambert-Beer’s equation (A = eCL; where e260 (DNA)
= 6600 cm
1 M
1, C = DNA concentration, L = cuvette
path length) [28,29]. Further, absorbance ratio A260/A280 was
evaluated greater than 1.8 which assured the DNA purity
[30]. The stock solution of synthesized compound 3 was pre-
pared by dissolving it in 10% aqueous DMSO. For DNA
binding studies, compound’s concentration was optimized to
1 mM and kept constant while adding various diluted concen-
trations of DNA ranging from 40 mM to 320 mM under body
temperature of 37 °C. In viscometric experiments 10 lM fixed
concentration of DNA was used with varying concentrations
of compound 3. For cytotoxicity assay, Human Hepatocellular
Carcinoma cell line (Huh-7) was cultured in Dulbecco’s Mod-
ified Eagle Media DMEM – high glucose with 10% FBS
(Thermo Fischer Scientific, USA) and 1% pen-strep MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide
(Sigma Aldrich, USA).
2.2. Instrumentations
Melting point was recorded using a digital Gallenkamp
(SANYO) model MPD.BM 3.5 apparatus and is uncorrected.
1
H NMR and 13C NMR spectra were determined at 300 MHz
using a Bruker AM-300 spectrophotometer. FTIR and mass
spectra (EI, 70 eV) were recorded, respectively, on a Bio-
Rad-Excalibur Series Mode FTS 3000 MX spectrophotometer
(USA) in range of 4000–400 cm
1 and on a GC–MS Agilent
Technologies 6890N and an inert mass selective detector
5973 mass spectrometer technologies. Elemental analysis was
conducted using a LECO-183 CHNS analyzer. Shimadzu1800
spectrophotometer (TCC-240A, Japan) armed with tempera-
ture control device was used to record the electronic
Studies on single crystal (E)-1-(2-fluorobenzylidene)thiosemicarbazide
absorption spectra using 1.0 cm matched quartz cells. Hettich
EBA20 Portable Centrifuge C 2002 (Max. speed: 6000 min
1)
and a vortex machine were used during the extraction of DNA
from chicken blood. ‘‘AUTOLAB” PGSTAT-302 poten-
tiostat/galvanostat with the electrochemical software package
GPES version 4.9 (Eco Chemie, Utrecht, Netherlands) was
used for cyclic voltammetric experiments. Viscosity experi-
ments were carried out on an automated Schott Gerate vis-
cometer (Model; AVS 310). Theoretical quantum chemical
calculations were done by using Gaussian 09 software, while
for molecular docking simulation MOE-dock by Chemical
Computing Group Inc was used. Molecular modeling studies
were performed on Pentium 1.6 GHz workstation, 512 MB
memory with the Windows Operating System that applies a
two stage scoring process to sort out the best conformations
and orientations of the ligand based on its interaction pattern
with the DNA.
2.3. General procedure for synthesis
The thiosemicarbazide (0.138 g, 1.0 mmol) was introduced to a
stirred solution of suitably substituted benzaldehyde (1.0
mmol) in absolute ethanol (10 ml) along with 1–2 drops of
concentrated sulfuric acid. The reaction mixture was refluxed
for 3–6 h and completion of the reaction was checked by
TLC. The mixture was concentrated and resulted solid product
was separated and recrystallized from ethanol.
2.4. Characterization data
2.4.1. (E)-1-(2-Fluorobenzylidene)thiosemicarbazide –
compound 3
Yield: 75%: m.p 235 °C Rf: 0.52; Petroleum ether:ethyl acetate
(6:4) FTIR; (KBr, cm
1): 3375 (NH2), 3261 (NAH), 3117 (sp2
CH), 1624 (C‚N), 1558 (ArAC‚C), 1071 (C‚S), 1H NMR
(300 MHz, DMSO d6): d (ppm): 11.56 (s, 1H, NH), 8.27 (s, 1H,
CH‚N), 8.25–7.19 (m, 4H ArAH), 8.08 (s, 2H, ANH2); 13C
NMR (75 MHz, DMSO d6): d (ppm): 178.5, 162.9, 159.6,
135.2, 127.2, 125.1, 122.2, 116.0. GC–MS (EI, 70 eV): m/z
(%):197 (M+ 100).
2.5. Crystal structure determination of compound 3
C8H8FN3S, Mr = 197.2, colorless crystal, size 0.25  0.18 
0.08 mm3, orthorhombic space group Pbca with Z = 8, a =
11.845(2), b = 6.6894(14), c = 22.759(5) Å, V = 1803.3(6)
Å3; Dc = 1.453 Mg/m3, l = 0.328 mm
1, F(0 0 0) = 816.
The intensity data were recorded using a Bruker SMART
CCD area-detector diffractometer with graphite monochro-
mated MoKa radiation (k = 0.71073 Å) at T = 130(2) K.
15,834 reflections collected 1.8 > > 27.9°; 2151 independent
reflections I > 2r(I), Rint = 0.052. Structure solution by
direct methods, full-matrix least squares refinement based on
F2 and 118 parameters [31,32]. All but H-atoms were refined
anisotropically, hydrogen atoms were clearly located from dif-
ference Fourier maps and then refined at idealized positions
riding on the carbon atoms with isotropic displacement param-
eters Uiso(H) = 1.2Ueq(C/N) and CAH 0.95 Å, NAH 0.88 Å.
Refinement converged at R1 = 0.039[I > 2r(I)], wR2 =
0.098 [all data] and S = 1.04; min./max. DF
0.32/0.37 e/Å3.
Crystallographic data for the structure reported in this paper
1005
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication no. CCDC-
1567724. Copies of available material can be obtained free of
charge via www.ccdc.cam.ac.uk.
2.6. Experimental procedures for DNA binding investigations
2.6.1. Spectroscopic titrations
In order to investigate binding of the synthesized compound 3
with DNA; spectroscopic titrations were carried out on UV–
visible spectrophotometer by maintaining the temperature of
cell cuvette at 37 °C. Initially, spectra were run separately for
the compound and DNA. Then 40–320 mM DNA was titrated
in aliquot form with 1 mM fixed concentration of the com-
pound. Variations in the spectral responses were recorded
separately at 37 °C and absorbance data were obtained for
the compound before and after the addition of various concen-
trations of DNA.
2.6.2. Voltammetric titrations
Glassy carbon (d = 3 mm), saturated calomel (SCE; 3.5 M
KCl) placed in luggin capillary and a Pt wire as working,
reference and counter electrode, respectively, were used in
double wall Dr. Bob cell for electrochemical measure-
ments. Surface of glassy carbon electrode was polished
with powdered a-alumina, washed and dried before run-
ning each voltammetric experiment. Temperature of the
sample solution was maintained at 37 °C by circulating
water in double wall cell. In order to maintain oxygen free
environment in the cell; purging of argon gas (99.999%)
for 10–15 min and its blanketing was made before every
electrochemical assay.
Cyclic voltammetry of compound 3 alone (1 mM) and in
the presence of varying DNA concentrations (40–320 mM)
was carried out within potential range of
0.2 V to
1.4 V
at scan rate of 100 mV/s. Solvent purity was assured by run-
ning blank CV within same potential range.
2.6.3. Viscosity measurements
Before starting viscosity experiments; various samples with
fixed DNA concentration (10 lM) and varying compound
3 concentrations (10–80 mM; with the difference of 10
mM) were prepared separately. Viscosity was measured ini-
tially for DNA solution at its fixed concentration as go.
Then each sample was run separately and viscosity was
measured as g. During each measurement, temperature
was kept at 37 °C. The values of cube root of relative
specific viscosity i.e., (g/go)1/3 was determined and then
plotted against compound-DNA concentration ratio i.e.,
[compound]/[DNA].
2.7. Theoretical procedures
2.7.1. DFT method
Quantum semi imperial computational studies for com-
pound structure were carried out by density functional the-
ory (DFT) method. Molecular geometry of compound 3
was optimized by using Gaussian 09 at DFT/B3LYP/basis
set (6-31G) level and quantum computational parameters
were obtained.
1006
Scheme 1 Synthesis of compound 3; (E)-1-(2-fluorobenzylidene)thiosemicarbazide.
2.7.2. Molecular docking method
Structure of compound 3 was drawn and minimized on MOE
window using MOE builder and entered into MOE database.
The starting point of the docking simulation was the X-ray
structure of the DNA with PDB ID 1BNA obtained through
the protein data bank (PDB) and imported to MOE window.
All water molecules were removed from the complex with 12
base pairs running in 50 -30 direction. The base pair sequence
was (50 -D(CGCGAATTCGCG)-30 ): (50 -D(CGCGAATTCGC
G)-30 ) with molecular weight of 7326.84. All hydrogen atoms
were added to the structure with their standard geometry fol-
lowed by their energy optimization tool using MOPAC 7.0.
The resulting DNA model was subjected to systematic confor-
mational search at default parameters with RMS gradient of
0.01 kcal mol
1 using Site Finder. A number of runs were car-
ried out to get a final binding docking pose as accurate as pos-
sible. The best conformation was selected based on energetic
ground and the minimum final docking energy (DG) [25,26].
2.8. In vitro cytotoxic assay
Anticancer potential of the synthesized compound 3 was tested
by a colorimetric MTT assay as reported earlier [33]. Human
Hepatocellular Carcinoma cell line (Huh-7) was used to evalu-
ate the cytotoxic effect of compound 3. The cell lines were
grown and maintained in DMEM – high glucose supplemented
with 10% FBS (phosphate buffer saline) and 1% pen-strep
antibiotic. For MTT assay; 1  104 cells were plated into each
of 96 well plates and incubated for attachment overnight
before incubating them with varying concentration (25–
100ug/ml) of compound 3 in triplicates. PBS was added in con-
trol wells. Cells were incubated again for 24 h at 37 °C incuba-
tor with 5% CO2. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) reagent was added to each well,
including control. The plates were then incubated for 3 h until
purple precipitates of formazan were visible. After aspirating
all solutions from the wells, Formazan were dissolved in 100
ml of DMSO, by keeping the plates covered in the dark for
1 h at room temperature. The absorbance was recorded at
570 nm in a microplate reader. Percentage cytotoxicity was cal-
culated as;
% cell viability ¼ ðAs
Ab Þ=ðAc
Ab Þ  100
where As, Ab and Ac corresponds to absorbance value of sam-
ple, blank and control respectively. The IC50 value was extrap-
olated from the dose-response graph. The concentrations of
compound 3 that reduced the viability of the cells by 50% were
determined by plotting the triplicate data sets over various
concentration ranges. The values were calculated using regres-
sion analysis of PRISM program.
N. Arshad et al.
3. Results and discussion
3.1. Chemistry of synthesized compound 3
Synthesis of target compound was carried out according to
synthetic route outlined in Scheme 1. The thiosemicarbazide
in dry ethanol was allowed to react with suitably substituted
hetero aromatic aldehyde in the presence of sulfuric acid as
catalyst to obtain the desired compound 3 in good to high
yields. The compound obtained was purified by recrystalliza-
tion from aqueous ethanol.
In the typical case of compound 3, presence of the C‚S
functional group was marked by the appearance of stretching
bands at 1071 cm
1, for C‚N at 1624 cm
1, for ArAC‚C at
1558 cm
1 and a stretching around 3375 and 3261 cm
1 indi-
cating the presence of NH2 and NH linkage of the hydrazide
moiety. In 1H NMR spectrum the appearance of a singlet at
d 8.27 for azomethine proton (HC‚N) and singlets at d 11.5
and 8.08 were noticed for NH and NH2, respectively (See Sup-
plementary data). The characteristic azomethine protons were
observed in the range of 8.27 ppm in 1H NMR spectra. In 13C
NMR spectra, the signals at d 178.0 (C‚S) and 162 (C‚N)
confirmed the formation of the thiosemicarbazones.
3.2. Description of crystal structure
The molecular structure of synthesized compound 3 is repre-
sented in Fig. 1. The molecular structure was found closely
related to that of bis-fluoro substituted analogs [34]. It showed
that the side chain is almost co-planar with the aromatic ring
plane; the relevant torsion angles measured as
C3AC2AC1AN1 9.56(16)° and C2AC1AN1AN2 0.68(14)°.
The side chain itself was planar with largest deviation of 0.68
(1) Å for N2 from the best plane through C1 to S1. Pertinent
Fig. 1 Molecular structure of compound 3. Anisotropic dis-
placement ellipsoids are drawn at the 50% probability level.
Studies on single crystal (E)-1-(2-fluorobenzylidene)thiosemicarbazide 1007

3.3. DNA binding studies by UV–visible spectroscopy

UV–visible spectroscopy is the most significant technique to

Fig. 2 Unit cell with intermolecular H-bonding pattern as dotted
lines, viewed along b-axis. H atoms not involved are omitted.
bond geometries were N1AN2 1.3784(19), N1AC1 1.279(2),
N2-C8 1.347(2), N3AC8 1.325(2), C8AS1 1.6953(17) and
C3AF1 1.358(2) Å. The crystal packing is given in Fig. 2 which
showed intermolecular N3-H32A. . .S(-x + 1,
y,
z + 1)
hydrogen bonds with NAH 0.88 Å, H. . .S 2.53 Å and
NAH. . .S 170.4° that connected the molecules into centrosym-
metric dimers which are stacked along the b-axis. The crystal
data and structure refinement for compound 3 are provided
in Table 1 and selected bond lengths and angles are listed in
Table 2.
learn the binding mode of small molecules and has been
reported several times for DNA binding interactions, kinetics
and mechanism [20,25–27]. Compound–DNA interaction can
easily be assessed by monitoring spectral changes in the com-
pound’s spectra after DNA addition; hence is considered an
effective tool for recognition of their interplay, understanding
of binding mechanism and complex formation. The changes
observed in the UV-spectra of a compound upon DNA titra-
tions have provided evidences of the interactional modes. In
case of intercalative binding mode, p-p* stacking interactions
may appear and resultant spectral changes are observed as
decrease in the absorbance peak (hypochromism) [35]. Fur-
ther, bathochromic (red) shift in the spectral response after
DNA addition confirm stabilization of the DNA duplex [35].
The UV-spectra of investigated compound 3 were recorded
in 10% aqueous DMSO at physiological temperature 37 °C
before and after the addition of various concentrations of
CT-DNA, Fig. 3. Compound’s absorbance peak appeared at
kmax = 270.5 nm. Upon addition of various aliquots of
DNA, peak intensity drop up to 12.7% as calculated by
the following formula.
Afree
Abound
H% ¼  100:
Afree
A slight red shift of 1.5 nm was also observed which along
with hypochromic effect validated the formation of complex
via p-p* stacking interaction between the chromophoric part

Table 1 Crystal data and structure refinement for compound 3.

Empirical formula C8 H8 F N3 S
Formula weight 197.23
Temperature 130(2) K
Wavelength 0.71073 Å
Crystal system Orthorhombic
Space group Pbca
Unit cell dimensions a = 11.845(2) Å a = 90°
b = 6.6894(14) Å b = 90°
c = 22.759(5) Å c = 90°
Volume 1803.3(6) Å3
Z 8
Density (calculated) 1.453 Mg/m3
Absorption coefficient 0.328 mm
1
F(0 0 0) 816
Crystal size 0.25  0.18  0.08 mm3
Theta range for data collection 1.79 to 27.87°.
Index ranges
15h11,
8k8,
29l29
Reflections collected 15,834
Independent reflections 2151 [R(int) = 0.0523]
Completeness to theta = 27.87° 100.0%
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 0.9742 and 0.9225
Refinement method Full-matrix least-squares on F2
Data/restraints/parameters 2151/0/118
Goodness-of-fit on F2 1.038
Final R indices [I > 2sigma(I)] R1 = 0.0393, wR2 = 0.0909
R indices (all data) R1 = 0.0550, wR2 = 0.0987
Largest diff. peak and hole 0.368 and
0.323 e.Å
3
1008 N. Arshad et al.

respectively. From the plot of Ao/(A
Ao) to 1/[DNA],

Table 2 Bond lengths (Å) and angles (°) for Compound 3.
the ratio of the intercept to the slope gave the value of
Bond distance (Å) binding constant, Kb.
S(1)AC(8) 1.6953(17) C(2)AC(3) 1.389(2) The evaluated binding constant (Kb = 6.02  103 M
1)
F(1)AC(3) 1.358(2) C(2)AC(7) 1.398(3) further depicted intercalative binding of compound 3 with
N(1)AC(1) 1.279(2) C(3)AC(4) 1.378(3) DNA as this value was found comparable with reported inter-
N(1)AN(2) 1.3784(19) C(4)AC(5) 1.377(3) calator, isoxazocucumine (6.3  103 M
1) [37]. Using the value
N(2)AC(8) 1.347(2) C(5)AC(6) 1.391(3) of binding constant in equation (DG =
RT lnKb); free
N(3)AC(8) 1.325(2) C(6)AC(7) 1.384(3) energy change, DG, was evaluated as negative value which
C(1)AC(2) 1.460(2)
indicated spontaneous binding of the compound with DNA.
Bond angles (°)
C(1)AN(1)AN(2) 114.75(14) C(4)AC(3)AC(2) 123.40(18) 3.4. DNA binding studies by cyclic voltammetry
C(8)AN(2)AN(1) 119.91(14) C(5)AC(4)AC(3) 118.25(18)
N(1)AC(1)AC(2) 120.14(16) C(4)AC(5)AC(6) 120.62(17)
C(3)AC(2)AC(7) 116.93(16) C(7)AC(6)AC(5) 119.92(19) Cyclic voltammetry is the most versatile, flexible and conve-
C(3)AC(2)AC(1) 120.26(16) C(6)AC(7)AC(2) 120.85(17) nient way for kinetic, analytical, thermodynamic and mech-
C(7)AC(2)AC(1) 122.80(15) N(3)AC(8)AN(2) 117.80(15) anistic studies. Interactional studies in particular to
F(1)AC(3)AC(4) 118.15(16) N(3)AC(8)AS(1) 123.30(13) compound binding with DNA could easily be assessed by
F(1)AC(3)AC(2) 118.43(15) N(2)AC(8)AS(1) 118.90(13) cyclic voltammetry in term of current - potential variations
in the voltammetric response of the redox active compound
[20,25,27].
DNA binding of compound 3 was studied in 10% aqueous
DMSO at 37 °C. Cyclic voltammogram of compound showed
a single irreversible reduction peak at
0.727 V, while scan-
ning the voltammograms in the presence of DNA concentra-
tions showed a significant decrease in the reduction peak
current, Fig. 4. The current drop was evaluated 10.3% using
following equation;
Io
I
%Current ¼  100
Io

Decrease in the peak current showed less availability of

Fig. 3 Absorption spectrum of compound 3 in the absence (a)
and presence of 40(b), 80(c), 120(d), 160(e), 200(f), 240(g), 280(h),
320(i) mM DNA. The arrow direction indicates the increasing
conc. of DNA. The inset graph represents the plot of A0/A
A0
vs. 1/[DNA](mM)
1 for the calculation of binding constant (Kb)
and Gibb’s free energy(DG).
of compound 3 and DNA base pairs [35]. Spectral findings
obtained from UV-experiments advised that the compound
may bind to CT-DNA via intercalation [20,25,27]. Further-
more, binding constant (Kb) for the compound–DNA complex
was obtained by plotting Ao/(A
Ao) vs. 1/[DNA] and by fit-
ting the data in Benesi-Hildebrand equation [36].
Ao eG eG 1 through which it could bind with DNA. The results obtained
¼ þ
A
Ao eH
G
eG eH
G
eG Kb ½DNA
where, Ao and A are the absorbance of free and DNA
bound complex respectively, eG and eH
G the molar
extinction coefficients of free and DNA bound complex
free compound in the bulk solution due to formation of
slowly diffusing compound–DNA complex. A small shift
toward more negative potential (
0.20 V) has indicated the
difficulty in reduction process at the surface due to com-
pound–DNA interaction [38]. Binding constant (Kb) for
the compound–DNA complex by cyclic voltammetry was
evaluated through peak current variations using following
equation [39];
1
I2p ¼ ðI2
I2p Þ þ I2po
½DNA
Kb ½DNA po
where Kb is the binding constant, Ipo and Ip are peak currents
before and after the addition of DNA. A plot of I2p vs. I2po
I2p/
[DNA] gave a straight line with a slope equal to the reciprocal
of binding constant, Kb. The binding constant and free energy
change were evaluated 6.86  103 M
1 and
18.74 kJ mol
1,
respectively. These values were found comparable with that
obtained from spectroscopic studies.
3.5. Viscometric analysis for DNA binding confirmation
Viscosity determination of DNA in the presence of com-
pound’s concentration is the most appropriate and simple
way to verify the possible mode of interaction of a compound
from viscometric analysis provide best compliments to other
experimental techniques used in DNA binding studies. Fur-
ther, this technique could justify the state of DNA either dena-
tured or not if the medium mostly consists of non aqueous
solvent like DMSO [40].
Studies on single crystal (E)-1-(2-fluorobenzylidene)thiosemicarbazide
Fig. 4 Voltammetric responses of compound 3 in the absence (a) and presence of 40(b), 80(c), 120(d), 160(e), 200(f), 240(g), 280(h), 320
(i) mM DNA. Arrow direction indicates the increasing conc. of DNA. The inset graph represents the plot of I2p vs. I2po- I2p/[DNA] for the
calculation of binding constant (Kb) and Gibb’s free energy (DG).
Fig. 5 Plot of relative specific viscosity vs. [Compound]/[DNA]
by adding increasing concentrations of compound 3 i.e., 10–80 mM
upon fixed 10 mM DNA concentration at 37 °C.
Due to non-dissolving nature of investigated compound
3 in aqueous medium; present studies were carried out in
mixed solvent of 10% aqueous DMSO (DMSO-H2O in
9:1v/v ratio). As denaturation of DNA (i.e., ds.DNA con-
verted into ss.DNA) could arise in the presence of DMSO;
viscosity measurement was considered mandatory in present
work.
Upon increasing compound 3 concentration, the rela-
tive viscosity of DNA was increased gradually, Fig. 5.
This increase in viscosity represented widening of DNA
duplex as compound 3 molecules accommodated into
the base pairs pockets [41]. Further, linear rise in the rel-
ative viscosity was suggestive of intercalative binding of
compound 3 with DNA [40]. Since DNA base pairs are
separated during its denaturing; there should be lowering
in the viscosity values. In present work enhancement in
the viscosity was observed with the investigated com-
pound 3; hence the possibility of DNA denaturing could
be excluded.
1009
3.6. Computational structure analysis by DFT studies
Density functional theory (DFT) is a quantum mechanical
semi – imperial approach for theoretical investigations on elec-
tronic structure of a molecule. Maximum charge distribution
and various computational parameters including HOMO-
LUMO energies, HOMO-LUMO energy gap, binding energy,
dipole moment, ionization potential, electron affinity, elec-
tronegativity, global hardness etc can be calculated from the
optimized geometries. Interpretation of these theoretical
results could help to understand the molecular structure and
possibilities of interactions with biological macromolecules
like DNA.
The molecular geometry of compound 3 was optimized by
DFT/ B3LYP/6-31G level using Gaussian 09 software. The
charge distributions on the optimized geometry and HOMO-
LUMO orbital’s are provided in Fig. 6, while computed
parameters are given in Table 3. Optimized structure indicated
that compound 3 has planar geometry which is property of an
intercalator, Fig. 5. The greater negative charge (
0.317) on
fluorine atom substituted on ring indicated the possibility of
its interaction with hydrogen of DNA base pairs while planar
part of the molecule intercalated between the DNA base pairs.
HOMO and LUMO energies are directly related to ioniza-
tion potential and electron affinity, respectively, as electron is
donated by HOMO to the LUMO orbital. Stability,
polarizability and reactivity of a molecule could be decided
on the basis of HOMO-LUMO energy gap and binding
energy. If energy gap is small and binding energy is negative
in value, the molecule is considered unstable/or less stable,
more polarizable and more reactive [42]. The greater value of
HOMO (
2.07 eV) than LUMO energy and very small value
for HOMO-LUMO energy gap showed electron donation abil-
ity from HOMO to LUMO and that the investigated molecule
is less stable and more reactive [42]. The negative binding
energy further assured compound’s instability. More reactive
nature of the tested compound 3 could further be rationalized
on the basis of small value of hardness and greater value of
dipole moment [43].
1010 N. Arshad et al.

Fig. 6 The frontier molecule orbital density distribution of compound 3 after optimization at DFT/B3LYP/6-31G level. Top; optimized
structure, Bottom left; Bonding molecular orbital (HOMO), Bottom right; Anti-bonding molecular orbital (LUMO).

evaluated as 2.89 Å and 2.99 Å respectively, which may render

Table 3 Quantum-chemical parameters of compound 3 com-
its greater binding strength and complex stability [26].
puted with DFT at B3LYP/6-31G level.
Theoretical values of binding constant (Kb) and free energy
Parameters Computed values change (DG) were evaluated 2.51  103 M
1 and
20.18 kJ
E Homo(ev)
0.207 mol
1, respectively and showed consistency with that calcu-
E Lumo(ev)
0.072 lated experimentally form UV–visible spectroscopy and cyclic
DE (eV) 0.135 voltammetry. The other electronic and steric descriptors were
Ionization potential (eV) 0.207 calculated from molecular docking data and given in Table 4.
Electron affinity (eV) 0.072 The values evaluated for these descriptors revealed significant
Binding energy (a.u)
971.76401827 potential of compound 3 for binding with one or the more
Dipole moment (D) 9.969
DNA base pairs.
Electronegativity (X) 0.1395
Global hardness (H) 0.0675
Point group C1 3.8. Drug like characters of compound 3

Theoretical descriptors evaluated from docking data, Table 4,

3.7. Molecular docking studies
Molecular docking is the most suitable way for theoretical
understanding of molecular mechanism and for the elucidation
of binding mode/modes of a compound with DNA through
non-covalent interactions. This theoretical approach is consid-
ered most appropriate for structure based- drug design. Molec-
ular docking studies along with experimental studies could
help to explore a compound as a potential drug candidate.
Molecular docking of compound 3 was performed to pre-
dict its binding with DNA. The docked conformation of the
compound with lowest free energy and pose view image are
shown in Fig. 7. In Fig. 7 (right) dotted lines in curve shapes
showed solvent contact and blue blurred regions explained
direct exposure of the ligand to hydrophobic core of 1BNA.
Docked complex indicated intercalation of the compound with
1BNA as shown in Fig. 7 (left). However, in 2D lig plot (Fig. 7,
right) two additional hydrogen bonds were observed between
two hydrogen atoms at NH2 of thiosemicarbazide moiety of
compound 3 and cytosine (DC 13) and adenine (DA 14) bases
of DNA. The bond lengths H  DC13 and H  DA14 were
could further help to investigate the pharmacological behavior
of a compound if obeying the criteria of Lipinski’s rule of five
[44]. Compound 3 was found to have hydrogen bond (HB)
donor atoms (the total number of nitrogen–hydrogen and oxy-
gen–hydrogen bonds) < 5, HB acceptor atoms (all nitrogen or
oxygen atoms) < 10, molecular mass < 500 g/mol and octa-
nol–water partition coefficient S logP < 5. These values were
in accordance with Lipinski’ criteria for a compound to behave
like a drug. However, molar refractivity (MR) value was eval-
uated 5.3916 which were lesser than Lipinski’s limits i.e., 40–
130. Since Lipinski’s criteria allow only one violation of the
five rules for an orally active drug; compound 3 may exhibit
drug like characteristics. However, further studies on its phar-
macological and pharmaceutical activities may lead it to be
used as drug.
3.9. Cytotoxic activity
The synthesized compound 3 was tested for in vitro anti-cancer
activity against Human Hepatocellular Carcinoma (Huh-7)
cell line. Incorporation of cell culture studies offer a gold stan-
dard to evaluate the anticancer potential of the compounds
Studies on single crystal (E)-1-(2-fluorobenzylidene)thiosemicarbazide 1011

Fig. 7 Molecular docked complex of compound 3 with IBNA 3D complex (left); 2D lig plot showing interactions of compounds with
base pairs of 1BNA (right).

Table 4 Electronic and steric descriptors calculated from molecular docking data.
Electronic descriptors
Complex EHOMO ELUMO Eele Evander EIP ETotal
kcal/mol kcal/mol kcal/mol kcal/mol kcal/mol kcal/mol
Comp. 3 – 1BNA
8.8178
1.1053
254066.29 19.5233 8.8178
50316.14
Steric descriptors
Complex Hf Mr S logP Vsurf Dipole HB donor atoms HB acceptor atoms
kcal/mol
Comp. 3 – 1BNA 48.0713 5.3916 0.9928 210.8906 6.3717 2 2

Fig. 8 In vitro cell viability (left) and cytotoxicity (right) of compound 3 on Huh-7 Cell line at various concentrations.

due to controlled conditions and automated procedures. The
% cytotoxicity of test compound was evaluated by using
MTT assays using various concentrations of compound 3,
Fig. 8 and Table 5. The % viability of the cells decreased with
increasing concentrations of the compound and at 100 mg/ml
concentration cell viability reduced to less than 50%, while
all other concentrations showed greater percentage viability
(above 50%). The results clearly indicated that the compound
has shown anticancer (cytotoxic) potential in dose dependent
manner. The maximum anticancer potential was observed with
100 mg/ml concentration and the extent of cytotoxicity was
58.556%. The dose dependent inhibition in cell growth was
1012
Table 5 % cell viability and % cytotoxicity data.
Conc. (mg/ml) % Cell Viability % Cytotoxicity
0 100 0
25 98.17949 1.82051
50 71.78632 28.21368
75 62.23077 37.76923
100 41.444 58.556
found more significant at 100 mg/ml of test compound and
revealed its potency against liver cancer cell line at this
concentration.
IC50 value of test compound was determined to be 72.94
mg/ml (14.37 mM) which is greater than that reported for refer-
ence standard anticancer drug Tamoxifen (IC50 mM; 5.8 and
10.5 for the cell growth inhibition of MCF-7, SaOS-2 cell lines,
respectively) [45]. The result thus clearly indicated that
compound 3 has a potential anticancer activity against the
hepatocellular carcinoma and could be further considered for
its anticancer potential evaluation for other type of cancers
using other relevant cell lines. Furthermore; compound 3 can
also be tested in vivo for its anticancer potential using animal
models.
4. Conclusions
Compound 3 {(E)-1-(2-fluorobenzylidene)thiosemicarbazide}
was synthesized, characterized and its structure was deter-
mined by X-ray single crystal analysis. DFT charge distribu-
tion and chemical reactivity descriptors indicated that the
synthesized compound is reactive. Spectroscopic, voltammet-
ric, viscometric and docking studies revealed intercalation as
a prominent mode for compound 3-DNA interaction. Experi-
mental and theoretical findings were in agreement. Computed
parameters showed drug likeness of compound 3. IC50 value
evaluated for 100 mg/ml of compound 3 against Human Hepa-
tocellular Carcinoma cell line (Huh-7) showed its potency
against liver cancer.
Acknowledgments
The authors would like to thanks Chemistry Departments of
Allama Iqbal Open University and Quaid-i-Azam University
and Healthcare Biotechnology Department of NUST for
experimental and theoretical facilities in Pakistan and Depart-
ment Chemie Paderborn Germany for crystal structure.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.jscs.2018.05.
002.
References
[1] H. Beraldo, D. Gambino, Mini-Rev. Med. Chem. 4 (2004) 31–
39.
[2] J.S. Casas, M.S. Garcı́a-Tasende, J. Sordo. Coord. Chem. Rev.
209 (2000) 197–261.
N. Arshad et al.
[3] H.A. Tang, L.F. Wang, R.D. Yang, Transit. Met. Chem. 28
(2003) 395–398.
[4] M. Aggarwal, B. Kondeti, R. McKenna, Expert Opin Ther Pa.
23 (2013) 717–724.
[5] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De
Simone, Chem. Rev. 112 (2012) 4421–4468.
[6] A.R. Brash, J. Biol Chem. 274 (1999) 23679–23682;
A.B. Camargo, E. Marchevsky, J.M. Luco, J. Agric. Food
Chem. 55 (2007) 3096–3103.
[7] L. Dailey, P. Imming, Curr. Med. Chem. 6 (1999) 389–398.
[8] Z.H. Chohan, M. Praveen, A. Ghaffar, Met. Based Drugs 4
(1997) 267–272.
[9] S. Patai, The Chemistry of Carbon–Nitrogen Double Bond,
Interscience, New York, 1970, pp. 149–180.
[10] K.N. Venugopala, B.S. Jayashree, Indian J. Heterocycl. Chem.
12 (2003) 307–310.
[11] K. Vashi, H.B. Naik, Eur. J. Chem. 1 (2004) 272–276.
[12] A. Nunez-Montenegro, R. Carballo, E.M. Vazquez-Lopez,
Polyhedron 27 (2008) 2867.
[13] A. Nunez-Montenegro, R. Carballo, E.M. Vazquez-Lopez,
Polyhedron 27 (2008) 3915.
[14] K. Alomar, M.A. Khan, M. Allain, G. Bouet, Polyhedron 28
(2009) 1273.
[15] P.M. Krishna, K.H. Reddy, Inorg. Chim. Acta 362 (2009) 4185.
[16] M. Belicchi-Ferrari, F. Bisceglie, E. Buluggiu, G. Pelosi, P.
Tarasconi, Polyhedron 28 (2009) 1160.
[17] T.S. Lobana, P. Kumari, R.J. Butcher, Inorg. Chem. Commun.
11 (2008) 11.
[18] T.S. Lobana, R. Sharma, R.J. Butcher, Polyhedron 28 (2009)
1103.
[19] T.S. Lobana, S. Khanna, G. Hundal, R.J. Butcher, A.
Castineiras, Polyhedron 28 (2009) 3899.
[20] N. Arshad, M. Ahmad, M.Z. Ashraf, H. Nadeem, J.
Photochem. Photobiol B: Biol. 138 (2014) 331–346.
[21] E.M. Eudnett, W.J. Dunn, J. Med. Chem. 15 (1972) 339.
[22] E.M. Eudnett, P.D. Mooney, J. Med. Chem. 13 (1970) 786.
[23] D.N. Dhar, C.L. Taploo, J. Sci.i Ind. Res. 41 (1982) 501–506.
[24] S. Li, S. Chen, S. Lei, H. Ma, R. Yu, Corrosion Sci. 41 (1999)
1273–1287.
[25] N. Arshad, M. Zafran, Z. Ashraf, F. Perveen, J. Photochem.
Photobiol. B: Biol. 169 (2017) 134–147.
[26] N. Arshad, F. Perveen, P.A. Channar, S.I. Farooqi, A. Saeed, F.
A. Larik, H. Ismail, B. Mirza, J. Mol. Struct. 1139 (2017) 371–
380.
[27] N. Arshad, M.H. Bhatti, S.I. Farooqi, S. Saleem, B. Mirza,
Arab. J. Chem. 9 (2016) 451–462.
[28] T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, New York, 1989.
[29] M.E. Reichmann, S.A. Rice, A. Thomas, P. Doty, J. Am. Chem.
Soc. 76 (1954) 3047–3053.
[30] S.S. Babkina, N.A. Ulakhovich, Anal. Chem. 77 (2005) 5678–
5685.
[31] Bruker, SMART (Version 5.63), SAINT (Version 6.02), Bruker
AXS Inc., Madison, Wisconsin, USA, 2002.
[32] G.M. Sheldrick, Acta Cryst. 112 (2008) A64.
[33] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B.
Mitchell, Cancer Res. 47 (1987) 936–942.
[34] J.L. Bautista, M. Flores-Alamo, J. Tiburcio, R. Vieto, H.
Torrens, Molecules 18 (2013) 13111.
[35] M. Sirajuddin, S. Ali, N.A. Shah, M.R. Khan, N.M. Tahir,
Spectrochim. Acta Part A: Mol. Biomol. Spectros. 94 (2012)
134–142.
[36] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71 (1949)
2703–2707.
[37] N. Shahabadi, A. Fatahi, J. Mol. Struct. 970 (2010) 90–95.
[38] I. Kocak, U. Yildiz, B. Coban, A. Sengul, J. Solid State
Electrochem. 19 (2015) 2189–2197.
[39] J. Niu, G. Cheng, S. Dong, Electrochim. Acta 39 (1994) 2455.
Studies on single crystal (E)-1-(2-fluorobenzylidene)thiosemicarbazide
[40] N.K. Janjuaa, A. Shaheen, A. Yaqub, F. Perveen, et al,
Spectrochim. Acta Part A 79 (2011) 1600–1604.
[41] G. Zhang, J. Guo, J. Pan, X. Chen, J. Wang, J. Mol. Struct. 923
(2009) 114–119.
[42] C.S. Abraham, J.C. Prasana, S. Muthu, Spectrochim. Acta Part
A: Mol. Biomol. Spectros. 181 (2017) 153–163.
1013
[43] S. Špirtović-Halilović, M. Salihović, E. Veljović, A. Osmanović,
S. Trifunović, D. Završnik, Bull. Chem. Techool. Bosnia
Herzegovina 43 (2014) 57–60.
[44] A. Leo, C. Hansch, D. Elkins, Chem. Rev. 71 (1971) 525–616.
[45] D. Chowrasia, C. Karthikeyan, L. Choure, M.G. Sahabjada, M.
d. Arshad, P. Trivedi, Arab. J. Chem. 10 (2017) S2424–S2428.