Synthesis, crystal structure, DNA interaction and anticancer evaluation of pyruvic acid derived hydrazone and its transition metal complexes

Article abstract of DOI:10.1002/aoc.3851

A novel tridentate chelating ligand, Ethyl 2-(2-(2-chlorobenzoyl)hydrazono)propanoate and its late transition metal complexes were synthesized, characterized and evaluated for anticancer behavior. The structures were elucidated with the help of elemental analyses, spectral (vibrational, electronic, NMR and mass) and thermo-gravimetric techniques. Single crystal X-ray crystallographic studies of the ligand suggest an orthorhombic lattice structure with Pna21 space group. The interaction of ligand and complexes with DNA (CT-DNA) has been extensively studied using absorption, emission, viscosity and thermal denaturation studies with E. coli DNA. The DNA cleavage ability of ligand and metal complexes was tested using plasmid pBR322 DNA by gel electrophoresis method. The ligand and its copper complex have been evaluated for their in vitro anticancer activity against human cancer cells of different origin such as KB (Oral), A431 (Skin), Mia-Pa-Ca (Pancreases), K-549 (Lung), K-562 (Leukemia), MCF-7 (Breast) and VERO by MTT assay and the apoptosis assay was carried out with acridine orange/ethidium bromide (AO/EB) staining method. The studies suggest that ligand and copper complex exhibit significant cytotoxic activity on KB, MCF-7, A-431, Mia-Pa-Ca-2 an d A-549 cell lines compared to K-562 and VERO cell lines.

Full text of DOI:10.1002/aoc.3851

Received: 3 January 2017
DOI: 10.1002/aoc.3851
Revised: 21 March 2017
Accepted: 26 March 2017
F U L L PA P E R
Synthesis, crystal structure, DNA interaction and anticancer
evaluation of pyruvic acid derived hydrazone and its transition
metal complexes
1
2
2
2,3
1
Divya Hegde | Suneel Dodamani | Vijay Kumbar | Sunil Jalalpure | Kalagouda B. Gudasi
1
Department of Chemistry, Karnatak
A novel tridentate chelating ligand, Ethyl 2‐(2‐(2‐chlorobenzoyl)hydrazono)
propanoate and its late transition metal complexes were synthesized, characterized
and evaluated for anticancer behavior. The structures were elucidated with the
help of elemental analyses, spectral (vibrational, electronic, NMR and mass)
and thermo‐gravimetric techniques. Single crystal X‐ray crystallographic studies
of the ligand suggest an orthorhombic lattice structure with Pna21 space group.
The interaction of ligand and complexes with DNA (CT‐DNA) has been
extensively studied using absorption, emission, viscosity and thermal denaturation
studies with E. coli DNA. The DNA cleavage ability of ligand and metal
complexes was tested using plasmid pBR322 DNA by gel electrophoresis
method. The ligand and its copper complex have been evaluated for their
in vitro anticancer activity against human cancer cells of different origin such
as KB (Oral), A431 (Skin), Mia‐Pa‐Ca (Pancreases), K‐549 (Lung), K‐562
University, Pavate Nagar, Dharwad‐580003,
Karnataka, India
2
Dr. Prabhakar Kore Basic Science Research
Center, KLE University, Nehru Nagar,
Belgaum‐590010, Karnataka, India
3
KLE University's College of Pharmacy,
Nehru Nagar, Belgaum‐590010, Karnataka,
India
Correspondence
Kalagouda B. Gudasi, Department of
Chemistry, Karnatak University, Pavate
Nagar, Dharwad‐580003, Karnataka, India.
Email: drkbgudasi@kud.ac.in
(Leukemia), MCF‐7 (Breast) and VERO by MTT assay and the apoptosis assay
was carried out with acridine orange/ethidium bromide (AO/EB) staining method.
The studies suggest that ligand and copper complex exhibit significant cytotoxic
activity on KB, MCF‐7, A‐431, Mia‐Pa‐Ca‐2 an d A‐549 cell lines compared to
K‐562 and VERO cell lines.
KEYWORDS
anticancer studies, DNA binding and cleavage studies, hydrazone, transition metal complexes, pyruvic acid
1
| INTRODUCTION
Schiff bases, and many transition metal complexes have
attracted much attention due to their promising cancer
Cancer is a malignant disease responsible for high levels
of mortality and morbidity throughout the world.
inhibiting properties.
Now a days researcher mainly focuses on simple drugs
without or less side effects, cost effective and easy
preparative methods. Para‐amino benzoic acid (PABA) is
a water soluble naturally occurring compound and is essential
for microorganisms and some animals. Few reports suggest
that, PABA inhibit enzymes such as tyrosinase involved in
melanogenesis, or bind to PXR (Pregnane X Receptor)/
SXR (Xenobiotic Sensing Nuclear Receptor) receptors to
[
1,2]
Chemotherapy is one of the most notable healing
approaches. Numerous chemotherapeutic agents developed
to date often failed, as drug‐resistant tumour cells became
predominant. But drug‐based approaches for the cancer
therapy are still among the most widely used methods due
[
4]
[3]
to the invention of new and better drugs. Among them,
simple benzoic acid derivatives, nitrogen and sulphur
heterocycles, aliphatic carboxylic/pyruvic acid derivatives,
[
5]
regulate genes involved in cell cycle progression.
Appl Organometal Chem. 2017;e3851.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 19
https://doi.org/10.1002/aoc.3851

2
of 19
HEGDE ET AL.
Accordingly, identification of lead compounds with these
activities will likely lead to improved tumour specific
2 | EXPERIMENTAL
[
6]
treatment for numerous cancers including melanoma.
Structure–activity relationship study of a novel aspirin
2.1 | Materials and methods
[7,8]
Ethyl pyruvate, tris‐base, tris–HCl, calf thymus DNA and
ethidium bromide (EB) were obtained from Sigma
Aldrich and were of analytical grade. Hydrazine hydrate,
derivative shows strong anticancer activity.
A series of
benzyl esters (ABEs) and their inhibitory activity against
human colon (HT‐29 and SW480) and pancreatic (BxPC‐3
and MIA PaCa‐2) cancer cell lines were evaluated. The
benzyl esters bearing an aspirin or acyloxy group at the
meta or para position of the benzyl ring have shown better
2‐chlorobenzoic acid and metal chlorides are of AR grade
and were obtained from Spectrochem, India. Plasmid
DNA‐pBR322 was purchased from Chromous Biotech,
India. Agarose was obtained from Lonza (India). DMEM
[
9]
activity. It is proposed that, nature of the salicyloyl/
acyloxy function, the leaving group, and the additional
substituent, affecting the electron density of the benzyl ring,
are influential determinants of the inhibitory activity on
cancer cell growth.
(Dulbecco's Modified Eagle's Medium) medium, FBS, fetal
calf serum are purchased from Gibco, Thermofisher
Scientific, Antibiotic‐Antimycotic 100X solution and dual
fluorescent staining solution AO/EB were purchased from
Thermofisher Scientific. Oral cell line was purchased from
NCCS Pune. All the solvents were of either HPLC or AR
grade and obtained from S.D. Fine chemicals.
Ethyl pyruvate/pyruvic acid and its derivatives have
[
10,11]
attracted much attention in anticancer research.
Preliminary evidence for the anti‐inflammatory effects of
ethyl pyruvate came from studies carried out by Yang et al.
Metal content of Co(II), Ni(II) and Cu(II) complexes was
determined volumetrically and while that of Zn(II) complex
gravimetrically. Conductance measurements were recorded
in DMF (10− M) using ELICO‐CM‐82 conductivity bridge
with cell type CC‐01 and cell constant 0.53. The IR spectra
were recorded on a Nicolet 170SX FT‐IR spectrometer with
the fixed range of 4000–400 cm− using KBr disks. C, H,
N were determined using Thermo quest elemental analyzer.
[6–11]
and Venkataraman et al.
They have demonstrated that
resuscitation with a solution containing ethyl pyruvate
down‐regulated activation of the pro‐inflammatory
transcription factor, NF‐kB, as well as the expression of
several pro‐inflammatory proteins, such as TNF, IL‐6,
cyclooxygenase (COX)‐2, and inducible nitric oxide
3
1
[
6]
synthase (iNOS), in liver and intestinal mucosa. The
treatment with ethyl pyruvate/pyruvic acid has shown to
improve survival and/or ameliorate organ dysfunction in
a wide variety of preclinical models of critical illnesses,
such as severe sepsis, acute respiratory distress syndrome,
acute pancreatitis and stroke. Increasingly, however, it is
becoming apparent that certain pyruvate esters have
pharmacological effects, such as suppression of inflammation,
that are quite distinct from those exerted by pyruvate
1
13
H and C NMR spectra of ligand and zinc complex were
recorded in DMSO‐d on a Bruker 500 MHz spectrometer.
6
Electronic spectra were recorded on a Hitachi‐U‐3310
UV–visible spectrophotometer in the 200–1100 nm range
using DMF (10− M) as the solvent. The fluorescence
experiment was carried out on F‐2500 spectrofluorometer
3
(Hitachi, Japan) using 1.0 cm quartz cell. Thermogravimetric
study was carried out over a temperature range of 25–800 °C
using TGAQ500 analyzer with a heating rate of 10 °C min .
−
1
[
11]
anion.
Ethyl pyruvate (EP) also interfered with the
angiogenic cascade, including growth, invasion, migration,
and tube formation. Activation of NF‐kB by vascular
endothelial cell growth factor was reduced by ethyl
2.2 | Synthesis
[
10]
pyruvate.
The data demonstrated that EP has a strong
2.2.1 | Preparation of ligand [IV]
anti‐angiogenic activity and may be useful as a multi‐
functional drug for treatment of variety of diseases, including
inflammation, sepsis, hemorrhagic shock, coagulation, and
ischemia/reperfusion, as well as diseases related to
angiogenesis.
The ligand was synthesized in four simple steps as shown in
Scheme 1. All the intermediates were characterised by NMR
and GC mass analyses. Synthesis of the ligand involves
following steps.
The synthesis of structurally related compounds may
increase the anticancer activity drastically. Hence we
designed new substituted benzoic acid and ethyl pyruvate
based ligand and tested it for anticancer activity. The use
of transition metal complexes for the treatment of cancer
Synthesis of methyl‐2‐chlorobenzoate (II)
The desired ester (II) was prepared using literature procedure
[
17]
with slight modification.
To the methanolic solution of
2‐chlorobenzoic acid (I) (15.60 g, 100 mM), a catalytic
[12–16]
is known in medicinal field.
Considering this,
amount of conc.H SO was added and refluxed for 10 hours.
2
4
transition metal complexes of the new ligand were
synthesised, ligand and its copper complex were studied
for their anticancer activity.
After completion of the reaction, methanol was evaporated
and the resulting reaction mixture was extracted with ethyl
acetate and organic layer was washed with sodium

HEGDE ET AL.
3 of 19
washed with methanol and air dried. All the complexes were
obtained in good yields.
CoL. Yield 65%; Anal. Calcd for [Co(C H Cl N O )].
20
16
2 4 6
H O (%): C, 43.19; H, 3.26; N, 10.07; Co, 10.60. Found
2
−
1
(
%): C, 43.17; H, 3.24; N, 10.04; Co, 10.59. IR (cm ):
amide carbonyl ν(C5 = O3) 1649, azomethine ν(C = N)
1
570, νasym(C3O2O1) 1593, νsym(C3O2O1) 1376.
NiL. Yield 62%; Anal. Calcd for [Ni(C H Cl N O )].
20
16
2 4 6
H O (%): C, 43.21; H, 3.26; N, 10.08; Ni, 10.56. Found
2
−
1
(
%): C, 43.18; H, 3.24; N, 10.02; Ni, 10.55. IR (cm ): amide
carbonyl ν(C5 = O3) 1620, azomethine ν(C = N) 1570,
νasym(C3O2O1) 1592, νsym(C3O2O1) 1384.
SCHEME 1 Synthetic route for ligand and its numbering scheme
CuL. Yield 70%; Anal. Calcd for [Cu(C H Cl N O )].
20
16
2 4 6
bicarbonate, then with brine solution and dried over
anhydrous magnesium sulphate. The solvent was removed
invacuo to get the desired product. (Yield: 60%).
2H O (%):C, 41.50; H, 3.48; N, 9.68; Cu, 10.98. Found
2
−1
(%): C, 41.51; H, 3.45; N, 9.64; Cu, 11.06. IR (cm ): amide
carbonyl ν(C5 = O3) 1647, azomethine ν(C = N) 1583,
νasym(C3O2O1) 1593, νsym(C3O2O1) 1383.
Synthesis of 2‐Chlorobenzohydrazide (III)
To the methanolic solution of methyl‐2‐chlorobenzoate
ZnL. Yield 61%; Anal. Calcd for [Zn(C H Cl N O )].
20
16
2 4 6
2H O (%):C, 41.37; H, 3.47; N, 9.65; Zn, 11.26. Found
2
−1
(9.38 g, 55 mM), hydrazine hydrate (8.25 g, 165 mM) was
(%): C, 41.39; H, 3.43; N, 9.67; Zn, 11.28. IR (cm ): amide
added slowly with stirring and refluxed for 8 hours. The
hydrazide separated was filtered, washed with methanol and
air dried. (Yield = 90%).
carbonyl ν(C5 = O3) 1658, azomethine ν(C = N) 1597,
1
νasym(C3O2O1) 1597, νsym(C3O2O1) 1361. H NMR
13
(500 MHz, DMSO‐d , ppm): 11.20 (s, N2H, 1H).
C
6
NMR (500 MHz, DMSO‐d , ppm): 174.66 (C3), 166.81
(C5), 137.06 (C4).
6
Synthesis
propanoate (IV)
‐Chlorobenzohydrazide (8.36 g, 49 mM), ethyl pyruvate
5.68 g, 49 mM) in methanol (100 ml) was added and stirred
of
ethyl 2‐(2‐(2‐chlorobenzoyl)hydrazono)
2
(
2.3 | X‐ray crystallography
for 4 hours. The white colored product obtained was washed
thoroughly with cold methanol and air dried to get Ethyl
Single crystal X‐ray diffraction study of ethyl 2‐(2‐(2‐
chlorobenzoyl)hydrazono)propanoate was taken up at
296 K using BRUKER SMART APEX‐CCD diffractometer
with Mo Kα radiation (λ =0.71073). All the non‐hydrogen
atoms were refined with anisotropic temperature factors.
The structures were solved using SHELXL‐97 and refined
2‐(2‐(2‐nitrobenzoyl)hydrazono)propanoate. The purity of
the compound was checked by TLC on precoated silica gel
plates (Rf: 0.45, Hexane: Ethyl acetate 3:7).
Yield 94%; m.p. 144–146 °C; white solid. Anal. Calcd
for C H ClN O (%): C, 53.64; H, 4.88; N, 10.43. Found
2
using Full‐matrix least squares F . All the calculations were
1
2
13
2 3
−
1
(
%): C, 53.61; H, 4.85; N, 10.43. IR (cm ): amide carbonyl
performed using WinGX crystallographic software. Molecu-
lar geometry calculations were made using PARST and the
structure drawing was obtained with ORTEP‐3 for windows
ν(C5 = O3) 1692, pyruvate carbonyl ν(C3 = O2) 1717,
1
azomethine ν(C = N) 1614. H NMR (E and Z isomers)
[
18–20]
(
1
500 MHz, DMSO‐d , ppm): 11.21/11.47 (s, N2H, 1H),
.26/1.04 (t, C1, 3H), 4.22/4.01 (q, C2, 2H). C NMR (E
program.
6
1
3
and Z isomers) (500 MHz, DMSO‐d , ppm): 170.38/170.32
6
2.4 | Biological studies
(C3), 164.67/163.80 (C5), 134.94/135.48 (C4), 14.16/13.89
(C1), 61.30/60.82 (C2).
The following biological studies have been undertaken to
evaluate the activity of newly synthesized compounds.
2
.2.2 | General procedure for the synthesis of
transition metal complexes
2.4.1 | DNA binding studies
A suspension of ligand (0.50 g, 1.8 mM) in methanol (20 ml)
was added to a methanolic solution of appropriate transition
metal chlorides (CoCl .6H O, NiCl .6H O, CuCl .2H O
The interaction of the prepared compounds with DNA was
evaluated extensively through different techniques.
2
2
2
2
2
2
and ZnCl ) in 1:1 molar ratio and refluxed for 7 hours. 2–3
drops of ammonia was added to the reaction mixture. The
precipitate formed was filtered under reduced pressure,
UV absorption studies
UV Absorption experiments were performed by maintaining
a constant ligand and metal complex concentration (20 μM)
2

4
of 19
HEGDE ET AL.
and varying nucleotide concentration (0–60 μM) in buffer.
The concentration of CT‐DNA was calculated from its
known extinction coefficient at 260 nm (6600 M−1 cm ).
Solutions of calf thymus DNA in phosphate buffer gave a
ratio of UV absorbance at 260 and 280 nm (A260/A280)
ethidium bromide (EtBr) bound CT‐DNA solution in
phosphate buffer. The changes in fluorescence intensities
at 596 nm (546 nm excitation) of EtBr bound to DNA
were recorded with an increasing amount of ligand and
metal complex concentration. EtBr was non‐emissive in
phosphate buffer due to fluorescence quenching of the free
EtBr by the solvent molecules. In the presence of DNA,
EtBr showed enhanced emission intensity due to its
intercalative binding to DNA. A competitive binding of
copper complex to CT‐DNA resulted in the displacement
−1
1.8–1.9, indicating that the DNA was pure and sufficiently
free of contaminants like the proteins and RNA. The solution
of DNA and desired compounds were kept to equilibrate at
25 °C for 20 min, after which absorption readings were
noted. The data were then fit to following equation (1) to
[21–23]
[23–25]
obtain intrinsic binding constant Kb.
of the bound EtBr, decreasing its emission intensity.
Addition of a second DNA binding molecule would
quench the EtBr emission by either replacing the DNA‐
bound EtBr (if it binds to DNA more strongly than EtBr)
or accepting an excited state electron from EtBr. The
apparent binding constant (Kapp) has been calculated from
the equation (2).
½
DNAꢀ=½εa–εfꢀ ¼ ½DNAꢀ=½εb–εfꢀ þ 1=Kb½εb–εfꢀ
(1)
Where, [DNA] = concentration of DNA in base pairs,
εa is the extinction coefficient observed for the MLCT
absorption band at the given DNA concentration,
εf is the extinction coefficient of the complex without
DNA, and
εb is the extinction coefficient of the complex when fully
bound to DNA.
A plot of [DNA]/[ε ‐ε ] v/s [DNA] gave a slope 1/[ε ‐ ε ]
and intercept equal to (1/Kb)[ε ‐ε ]. The intrinsic binding
KEtBr½EtBrꢀ ¼ Kapp½Compoundꢀ
(2)
7
−1
where K[EtBr] is 1 x10 M and the concentration of
EtBr is 20 μM; [complex] is the concentration of the
complex causing 50% reduction in the emission intensity
of EtBr.
a
f
a
f
b
f
constant Kb is the ratio of slope to the intercept.
Viscosity measurements
DNA cleavage studies
Viscosity experiments were carried out using Ostwald
microviscometer maintained at 26 °C in a thermostatic water
bath. Flow time of solutions in phosphate buffer was
recorded in triplicate and an average flow time was
The DNA cleavage study of the synthesized compounds was
determined by agarose gel electrophoresis using the E.coli
pBR322 plasmid as a target. The synthesized compounds
(100 μM) were dissolved in 6% DMSO (dimethylsulfoxide)
and mixed with the target plasmid (1:1). The mixture was
then incubated at 37 °C for 2 h. After the incubation period,
the plasmid and the compound mixture was mixed with the
tracking dye, bromophenol blue (1:1). It was then loaded into
0
1/3
calculated. Data was presented as (η/η ) versus binding
ratio, where η is the viscosity of CT‐DNA in the presence
0
[21,22,24,25]
of complex and η is the viscosity of DNA alone.
Thermal denaturation study
1
% agarose gel (containing 0.5 μg/mL ethidium bromide)
DNA melting experiments were carried out in phosphate
wells along with two control wells, the first one with the
untreated plasmid and the second one with the plasmid
treated with DMSO solvent. Finally it was electrophoresed
at a constant voltage of 50 V constant voltage for about 30
buffer on a HITACHI U‐3310 spectrophotometer equipped
with
5 °C). Solutions of E. coli DNA (Escherichia coli DNA) in
phosphate buffer gave a ratio of UV absorbance at 260 and
80 nm (A260/A280) as 1.8–1.9, indicating that the DNA
a
temperature‐controlling programmer ETC‐717
(
[
22–25]
mins using Tris‐EDTA (TAE) buffer (pH = 8.0).
The
2
bands were visualized by UV light and photographed for
analysis. The extent of cleavage of the supercoiled DNA
was determined by measuring intensities of the bands using
Molecular Imager Geldoc gel‐XR imaging system
was pure and sufficiently free of contaminants like the
proteins and RNA. UV melting profiles were obtained by
scanning A260 absorbance monitored at a heating rate of
5
°C/min for solutions of E. coli DNA (100 μM) in the
absence and presence of ligand and metal complexes
20 μM) from 25 to 85 °C. The melting temperature Tm,
(BIORAD).
(
Experimental procedure for MTT assay
which is defined as the temperature where half of the total
The cytotoxicity is performed by 3‐(4,5‐dimethylthiazol‐2‐
yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. This is a
colorimetric assay that measures the reduction of yellow
3‐(4,5‐dimethythiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide
by mitochondrial succinate dehydrogenase. The MTT enters
the cells and passes into the mitochondria where it is reduced
base pairs are unbound, was determined from the midpoint
of the melting curves.
[21,23,25]
Competitive binding fluorescence measurements
The apparent binding constants (Kapp) of the complexes
were determined by fluorescence spectral technique using

HEGDE ET AL.
5 of 19
to an insoluble, colored (dark purple) formazan product.
The cells are then solubilized with an organic solvent
3 | RESULTS AND DISCUSSION
(
DMSO) and the released, solubilised formazan reagent is
The newly synthesized ligand was characterized by
1
13
measured spectrophotometrically using ELISA. Since
reduction of MTT can only occur in metabolically active
cells the level of activity is a measure of the viability of
the cells.
The cell lines were cultured in DMEM medium which
was supplemented with 10% FBS and 1% Antibiotic‐
Antimycotic 100X solution. The cells were seeded at a
density of approximately 5 × 10 cells/well in a 96‐well
flat‐bottom micro plate and maintained at 37 °C in 95%
humidity and 5% CO for overnight. Different concentrations
elemental analysis, IR, H NMR, C NMR and 2D
CH‐COSY NMR spectral studies. The ligand is soluble in
chloroform, hot methanol, DMF and DMSO, where as
complexes are soluble in DMF and DMSO. All the
complexes are non‐hygroscopic and stable in both solid
and solution phases at room temperature. Analytical data
of all synthesised compounds (Table S1) is within the
acceptable range and confirm their structure. The lower
molar conductance values of all the complexes suggest
their non‐electrolytic nature. White crystals suitable for
XRD studies were isolated by slow evaporation of
methanolic solution of ligand. Attempts to grow single
crystals of metal complexes were unsuccessful. However,
the complexes were comprehensively characterized by
various established physico‐chemical techniques. The
ligand has undergone hydrolysis and converted into its
carboxylic acid in presence of metal salts in‐situ and later
formed the corresponding metal complexes.
3
2
(200, 100, 50, 25, 12.5, 6.5, 3.125 μg/300 μl) of compounds
were treated. The cells were incubated for another 72 hours.
The cells in well were washed twice with phosphate buffer
solution, and 20 μl of the MTT staining solution (5 mg/ml
in phosphate buffer solution) was added to each well and
plate was incubated at 37 °C. After 4 h, 100 μl of
dimethylsulfoxide (DMSO) was added to each well to
dissolve the formazan crystals, and absorbance was recorded
[
26]
at 570 nm using micro plate reader (ELISA reader).
results are compared with that of Cisplatin used as reference
drug against KB(Oral), A431(Skin), MIA‐Pa‐Ca
Pancreases), K‐549 (Lung), K‐562 (Leukemia), MCF‐
The
TABLE 1 Crystal refinement data for ligand
CCDC no.
1479628
13ClN
268.70
296(2) K
0.71073 Å
(
7
Empirical formula
Formula weight
Temperature
C
12
H
2 3
O
(Breast) and VERO cell lines. The IC50 values were
calculated using graph Pad Prism Version 5.1. The
surviving cells and inhibiting cells are calculated by using
following formula.
Surviving cells (%) = Mean OD of test compound /Mean
OD of negative control ×100 Inhibiting cells (%) =100‐
surviving cells.
Wavelength
Crystal system
Space group
Orthorhombic
P n a 21
Unit cell dimensions
a = 8.3158(3) Å α = 90°
b = 8.5171(3) Å β = 90°
c = 18.5995(7) Å γ = 90°
Dual Acridine orange/ethidium bromide (AO/EB)
fluorescent staining
The cell line oral (KB) was further tested for its apoptotic
behavior after showing cytotoxicity activity at desired
concentration. The DMEM culture medium containing
3
Volume
1317.34(8) Å
Z
4
Reflections collected
Goodness‐of‐fit on F2
F(000)
6142
10% fetal calf serum was added to each well of a 96‐well
0.951
564
plate (100 μl/well). Cells were added to a final concentra-
4
tion of 2 × 10 /ml and the plates were incubated. The
samples, ligand and copper complex treated cells are
seeded in a 24‐well plate after being cultured for 4 days,
Final R indices [1776 data;
I > 2σ(I)]
R1 = 0.0370, wR2 = 0.1048
2
0 μl of trypsin was added into each well. When cells
Final R indices all data
Weighting scheme
R1 = 0.0447, wR2 = 0.1116
had sloughed off, suspensions (25 μl) were transferred to
glass slides. Dual fluorescent staining solution (1 μl)
containing 100 μg/ml AO and 100 μg/ml EB (AO/EB)
was added to each suspension and then covered with a
coverslip. The morphology of apoptotic cells was observed
and 260 cells were counted within 10 min using a
fluorescent microscope (Carl Zeiss AxioVert S100). Dual
acridine orange/ethidium bromide (AO/EB) staining
2
2
2
w = 1/[σ (F
o
) + (0.0788P)
+ 0.0494P],
2
2
Where P = (F
o
c
+ 2F )/3
Theta range for data collection
Index ranges
2.19 to 25.18°
‐9 < =h < =9, −8 < =k < =10,
19 < =l < =22
−
Data / restraints / parameters
Density (calculated)
2097 / 1 / 165
3
[27]
1.360 Mg/cm
method was done in triplicate.

6
of 19
HEGDE ET AL.
TABLE 2 Selective bond lengths (Å) for the ligand obtained by crystal structure
Atom label
Bond length
Atom label
Bond length
Atom label
Bond length
Cl1‐C11
O1‐C2
O3‐C3
N1‐N2
N2‐H2
1.715
1.452
1.194
1.365
0.860
O1‐C3
O2‐C5
N1‐C4
N2‐C5
C1‐C2
1.321
1.213
1.281
1.354
1.423
C4‐C12
C6‐C7
C3‐C4
C5‐C6
C6‐C11
1.486
1.366
1.495
1.489
1.387
o
TABLE 3 Selective bond angles ( ) for the ligand obtained by crystal structure
Atoms
Bond angle
Atoms
Bond angle
Atoms
Bond angle
C3‐O1‐C2
C5‐N2‐N1
N1‐N2‐H2
O3‐C3‐C4
C4‐N1‐N2
N1‐C4‐C12
117.4
118.6
120.7
122.4
117.5
127.1
C5‐N2‐H2
C1‐C2‐O1
O1‐C2‐H2A
O1‐C2‐H2B
O3‐C3‐O1
N1‐C4‐C3
120.7
110.1
109.6
109.6
124.4
115.8
O1‐C3‐C4
C9‐C10‐C11
C11‐C10‐H10
O2‐C5‐C6
O2‐C5‐N2
N2‐C5‐C6
113.2
119.4
120.3
122.9
123.4
113.6
FIGURE 1 ORTEP diagram of ligand
FIGURE 3 Possible planes in the ligand structure
FIGURE 4 Intramolecular and intermolecular interactions in ligand
FIGURE 2 Packing diagram of ligand
structure

HEGDE ET AL.
7 of 19
3
.1 | Crystallographic study of ethyl 2‐(2‐(2‐
The ORTEP diagram, molecular packing diagram and
molecular planes are presented in the Figures 1–3
respectively. The frames were integrated with the Bruker
SAINT Software package using a narrow‐frame algorithm.
The single crystal X‐ray diffraction of the new ligand was
chlorobenzoyl)hydrazono)propanoate
The crystal structure refinement details, selected bond length
and bond angles are presented in Tables 1–3 respectively.
TABLE 4 Diagnostic IR bands (cm− ) of ligand and its metal complexes
1
ν (C3O2O1)
Compound
name
ν (C5 = O3)
(amide)
ν (C3 = O2)
(pyruvate)
νasym(COO)
ν
sym(COO)
ν (C4 = N1)
L
1692
1649
1620
1647
1658
1717
‐‐‐‐
‐‐‐‐
1614
1570
1570
1583
1570
CoL
NiL
CuL
ZnL
Disappeared
Disappeared
Disappeared
Disappeared
1593
1592
1593
1597
1376
1384
1383
1361
1
13
TABLE 5 H and C NMR spectral data for ligand and its zinc complex (shifts in ppm)
Ligand
Zn‐complex
1
13
1
13
Position
H–NMR
C–NMR
H–NMR
C–NMR
N2H
N2H′
C1
11.21 (s,1H)
11.47 (s,1H)
1.26 (t,3H)
1.04 (t,3H)
4.22 (q,2H)
4.01 (q,2H)
‐
‐
11.20 (s,1H)
‐
‐
‐
‐
‐
‐
14.16
C1′
C2
13.89
61.30
‐
‐
‐
C2′
C3
60.82
170.38
170.32
134.94
135.48
164.67
163.80
130.70
130.47
128.95
128.95
129.61
129.61
131.61
131.61
127.23
126.72
145.38
145.38
12.89
174.66
C3′
C4
‐
‐
‐
137.06
C4′
C5
‐
‐
‐
‐
166.81
C5′
C6
‐
‐
‐
‐
‐
129.88
C6′
C7
‐
‐
‐
7.38 (d,1H)
7.38 (d,1H)
7.43 (t,1H)
7.43 (t,1H)
7.45 (t,1H)
7.45 (t,1H)
7.53 (d,1H)
7.53 (d,1H)
‐
7.35 (t,1H)
130.20
C7′
C8
‐
‐
7.42(t,1H)
130.46
C8′
C9
‐
‐
7.45 (t,1H)
131.27
C9′
C10
C10′
C11
C11′
C12
C12′
‐
‐
7.63 (d,1H)
126.64
‐
‐
‐
151.15
‐
‐
‐
2.48 (s,3H)
2.48 (s,3H)
7.33 (s,3H)
12.30
12.89
‐
‐

8
of 19
HEGDE ET AL.
found to be orthorhombic having the space group Pna 21
with a = 8.31 Å, b = 8.51 Å, c = 18.59 Å, α = 90°,
β = 90°, γ = 90°, V = 1317.34 Å and Z = 4 for the formula
hydrogens H2, H12A and carbon C7 as shown in
Figure 4. Because of these interactions the bond angle
of N2‐C5‐C6 deviated from 120° to 113.65° and bond
angle N1‐N2‐C5 reduced to 118.60°, it is further
supported by the intermolecular interaction of N1 with
H2 and involvement of hydrogen H2 bonded to N2 in
intermolecular interaction with N1 and O2. The bond
angle of O1‐C3‐C4 also deviates from 120° to 113.25°
and is because oxygen O3 is involved in interaction with
H8 of another molecule.
3
C H ClN O .
1
2
13
2 3
The molecule shows two molecular planes having C6‐C11
atoms in one plane and acetylhydrazonopropanoate part in
another plane forming an angle 69.46° between the two
planes. In this molecule amide carbonyl oxygen O2 is
involved in intramolecular interaction with phenyl chlorine
and intermolecular interactions with nitrogen N2,
3.2 | Infrared spectral studies
The insitu convertion of ligand into its carboxylic acid and its
further coordination to the metal ion can be supported by
their IR spectral data. The diagnostic IR bands of ligand
and its complexes are compiled in Table 4.
The strong band due to azomethine ν(C4=N1)
functional group in the uncoordinated ligand was observed
at 1614 cm− and has shifted to lower wave number ligand
on complex formation, indicating the coordination of through
1
[
28]
azomethine nitrogen to the metal ion.
1
The strong band at
717 cm− in ligand spectrum assigned to carbonyl
1
ν(C3 = O2) stretching frequency has disappeared on complex
formation. Simultaneously two new bands have appeared in
the range of 1592–1597 cm− and 1361–1384 cm , and
1
−1
FIGURE 5 1H NMR spectrum of ligand
were assigned to the respective asymmetric and symmetric
FIGURE 6 13C NMR spectrum for ligand

HEGDE ET AL.
9 of 19
FIGURE 7 2D CH COSY NMR spectrum for ligand
FIGURE 8 1H NMR spectrum for ZnL

1
0 of 19
HEGDE ET AL.
FIGURE 9 13C NMR spectrum for ZnL
stretching frequencies of coordinated carboxylate ion. This
observation indicates that, the ester group of ligand was
converted to carboxylic acid group before complex forma-
tion in‐situ and the later same carboxylic acid moiety was
involved in coordination to the metal ion. To confirm this
phenomenon, the ligand was converted into sodium salt of
its carboxylic acid by hydrolysis using NaOH and its IR
spectrum was compared with the spectra of metal com-
oxygen in coordination and the ligand behaves as monobasic
tridentate towards all the metal ions.
3.3 | NMR studies
1
13
The H and C NMR spectral assignments of ligand and its
Zn(II) complex are compiled in Table 5, and the numbering
scheme followed for the NMR assignment is presented in
1
13
plexes. This suggests that, carboxylic acid has coordinated
Scheme 1. The H, C and 2D CH COSY NMR spectra of
ligand are depicted in Figures 5–7, and those of Zn (II)
complex are presented in Figures 8–10.
−
to metal ion. The difference between ν
(COO ) and
asym
−
νsym(COO ) frequencies in all complexes were found to be
greater than the difference observed in the sodium salt of
the ligand. This implies that, carboxylate group of ligand
has coordinated to metal ion in unidentate fashion as sug-
1
13
The ligand has shown two set of signals in the H,
C
and 2D CH COSY NMR spectra which corresponds to its
cis and trans isomeric forms aroused due to restricted rotation
along N1 = C4 double bond. On complex formation, only
one set of signals were observed indicating the presence of
[
29,30]
gested by Deacon.
The amide carbonyl stretching frequency ν(C5 = O3)
−
1
[31,32]
observed at 1692 cm in ligand has shifted to 1649, 1620,
single form of ligand in the complex.
−
1
1
647 and 1658 cm in CoL, NiL, CuL and ZnL respectively
The singlet at 11.21 ppm assigned to N2H proton has
remained almost unaltered on complex formation,
suggesting its noninvolvement in enolisation. The protons
corresponding to C1 and C2 in ligand observed at 1.26/
suggesting the involvement of amide carbonyl oxygen in
coordination. A sharp band of medium intensity in the ligand
−1
spectrum at 3194 cm assigned ν(N2H) has remained unal-
tered/slightly changed in the spectra of complexes suggesting
its non‐involvement in the enolisation with adjacent carbonyl
group. This was further confirmed by presence of N2H peak
1
1.04 (t, 3H) and 4.22/4.01 (q, 2H) were absent in H NMR
spectrum of its zinc complex, suggesting its hydrolysis and
13
formation of carboxylate ion. In C NMR spectrum of the
ligand, the signals due to C1 and C2 appeared at 14.16/
1
in H NMR of zinc complex.
13
Thus, IR spectral analyses suggest the involvement of
amide carbonyl oxygen, azomethine nitrogen and carboxylate
13.89 and 61.30/60.82 ppm are absent in C NMR spectrum
of the zinc complex. This is further confirmed by 2D CH

HEGDE ET AL.
11 of 19
FIGURE 10 2D CH COSY NMR spectrum for ZnL
COSY NMR spectral study. The downfield shift of signals
due to C3, C4 and C5 suggests the involvement of
carboxylate oxygen, azomethine nitrogen and amide
carbonyl oxygen of the ligand in coordination respectively
3.4 | Electronic spectral studies of ligand and
its complexes
Electronic spectra of ligand and their complexes were
recorded in order to assign the plausible geometry around
the metal ions. Representative electronic spectra of ligand
and nickel complex are shown in Figures 11 and 12
[
33]
with zinc (II).
The remaining signals are observed in the
expected regions and confirm the structure of the ligand and
its zinc complex.
FIGURE 11 UV–visible spectrum of ligand
FIGURE 12 UV–visible spectrum of NiL

1
2 of 19
HEGDE ET AL.
respectively. The band at 274 nm in the ligand spectrum is
3.5 | Mass spectral studies of ligand and its
assigned to π ! π* transition. The cobalt complex shows a
complexes
4
4
peak at 514 nm and was attributed to T1g(F) ! T (P),
1g
suggesting an octahedral structure. The bands at 618, 759
and 1007 nm in the spectrum of nickel(II) complex are
The mass spectrum of ligand shows molecular ion peak at
+
269 (M ) which corresponds to its molecular weight. The
3
3
3
3
3
assigned to A (F)! T (P), A (F)! T (F) and A
ESI mass spectra of cobalt, nickel, copper and zinc
2g
1g
2g
1g
2g
3
+
(
F) ! T (F) suggesting octahedral geometry. The typical
complexes show molecular ion peaks at m/z 537 (M‐1) ,
2g
2
2
+
+
+
d‐d absorption band due to Eg ! T was observed at
537 (M ), 543 (M + 1) and 544 (M ) corresponding to their
respective molecular weights. Mass spectrum of the ligand
and ESI‐mass spectra of cobalt complex are shown in
2
g
6
01 nm and is suggestive of a distorted octahedral structure
[34]
for copper(II) complex.
FIGURE 13 GC‐mass spectrum for ligand
FIGURE 14 ESI mass spectrum of CoL

HEGDE ET AL.
13 of 19
Figures 13, 14 respectively. (ESI‐mass spectra of NiL and
ZnL are shown in Figures S1‐S2 respectively).
weight loss of 6.14 to 6.11%, observed in the range of
68–108 °C and 75–110 °C corresponds to the loss of two
[
35]
lattice held water molecules.
The loss of lattice held water molecules is followed by the
loss of ligands in all the complexes. The weight loss of
85.98%, 85.99% and 82.71% in the temperature range of
3
.6 | Thermal studies of ligand and its
complexes
1
05–631 °C, 120–628 °C and 108–568 °C assignable to the
In order to study the thermal stability and decomposition
pattern, we have undertaken the thermogravimetric studies
of all synthesized complexes in the temperature range
loss of two ligand in CoL, NiL and CuL complexes. But in
ZnL, second step of weight loss of 82.38% is observed in
the range 350–637 °C. The plateau obtained after the loss
of ligand corresponds to the formation of metal residue.
25–800 °C and decomposition details are presented in
Table 6 and representative thermogram in Figure 15.
All the complexes exhibited a weight loss around 110 °C
indicating the presence of lattice held water molecules. In the
thermograms of CoL and NiL complexes, a weight loss in the
range of 3.17–3.5% was observed between 70–105 °C and
3.7 | DNA binding studies
3.7.1 | Absorption spectral studies
75–120 °C respectively, suggesting presence of one lattice
Examining the changes in absorption spectrum of the metal
complexes upon addition of increasing amounts of DNA is
held water molecule. In case of CuL and ZnL complexes,
TABLE 6 Thermal decomposition details of the complexes
Weight loss in %
Comp.
Temp. Range (in °C)
Found
Calc.
Weight loss due to
CoL
70–105
3.17
85.98
10.85
3.50
3.24
86.35
10.41
3.23
One lattice held water molecule
Two ligands
1
05–631
31–800
75–120
20–628
28–800
68–108
08–568
68–800
75–110
50–637
37–800
6
Metal residue
NiL
CuL
ZnL
One lattice held water molecules
Two ligands
1
6
85.99
10.51
6.14
86.20
10.57
6.21
Metal residue
Two lattice held water molecules
Two ligands
1
5
82.71
11.15
6.11
82.80
10.99
6.19
Metal residue
Two lattice held water molecules
Two ligands
3
6
82.38
11.51
82.54
11.27
Metal residue
FIGURE 15 Thermogram for CuL

1
4 of 19
HEGDE ET AL.
one of the most widely used methods to know the interaction
of metal complexes with DNA and for determining overall
binding constants. The absorption spectra of ligand and com-
plexes in the presence and absence of CT‐DNA are shown in
the Figure 16. A complex binding to DNA through intercala-
tion usually results in hypochromism and bathochromism
involving strong stacking interaction between an aromatic
chromophore and the base pairs of DNA. Upon addition of
calf‐thymus DNA to ligand and its complexes, there is a
decrease in molar absorptivity (hypochromic shift) of the
intense intraligand absorption bands (240–400 nm) of com-
plexes. The hypochromic shift in case of ligand (264 nm),
cobalt complex (268 nm), nickel complex (266 nm), copper
complex (268 nm) and zinc complex (262 nm) is indicating
strong binding of ligand and complexes with DNA. In order
to compare the DNA‐binding affinities of these complexes
quantitatively, their intrinsic binding constants with CT‐DNA
were obtained by monitoring the changes in absorption at
intraligand band with increasing concentrations of DNA
4
using equation 1 and were found to be 0.97 x 10 , 0.95 x
4
4
4
4
−1
1
0 , 0.75 x 10 , 2.63 x 10 and 0.78 x 10 M for ligand,
cobalt, nickel, copper and zinc complexes respectively,
indicating their binding interaction to DNA. Among all
the compounds copper complex has shown a strong
binding to DNA.
3.7.2 | DNA thermal denaturation
Interacalation of small molecules into the double helix is
known to increase the helix melting temperature (Tm), the
temperature at which the double helix denatures into single‐
stranded DNA. The extinction coefficient of DNA bases at
2
60 nm in the double strand is much less than that in the
single‐stranded form. Hence, melting of the helix leads to
an increase in the absorption at this wavelength. E. coli
DNA was seen to melt at 55–60 °C (phosphate buffer) in
the absence of complex. The melting temperature of DNA
for ligand, and nickel complex were found to be in the
region 60–65 °C, for cobalt and zinc complexes it is found
in the range 65–70 °C and for copper complex it is found in
the region 70–75 °C (Figure S3). This indicates that copper
complex bind strongly compared too other compoubnds
tested. This behavior is comparable to classical
[
36]
intercalators.
3.7.3 | Ethidium bromide displacement assay
The competitive DNA binding of complexes has been studied
by examining changes in emission intensity of ethidium bro-
mide (EtBr) bound to CT‐DNA as a function of added com-
[
37]
plex concentration.
Though the emission intensity of EtBr
in buffer medium is quenched by the solvent molecules, it is
enhanced by its stacking interaction between adjacent DNA
base pairs. When ligand and complexes were added to
DNA pretreated with EtBr {[DNA]/[EtBr] = 1:1}, the
DNA‐induced emission intensity of EtBr has decreased
FIGURE 16 Changes in the electronic absorption spectra of L(a),
CoL(B), NiL(C) and ZnL(D)(20uM) with increasing concentrations
6
−1
(
6
Figure 17). The Kapp values 5.00x10 M for ligand,
6
−1
6
−1
.66x10 M for CoL, NiL, 10.00x10 M for CuL and
(
0‐50uM) of CT‐DNA(phosphate buffer); the inset shows a fitting of the
absorbance data at 264 nm for L, 268 nm for CoL, 266 nm for NiL and
62 nm for ZnL used to obtain binding constants
6
−1
4.00x10 M for ZnL. The higher values of Kapp indicate
that these complexes bind to DNA by intercalation.
2

HEGDE ET AL.
15 of 19
FIGURE 17 Effect of addition of L(a), CoL(B), NiL(C), CuL(D), ZnL(E) on the emission intensity of the CT‐DNA bound ethidium bromide
20uM) at different concentrations in phosphate buffer(pH = 7.2). (F) plots of relative integrated emission intensity versus [DNA]/[compound] for
LH, col, nil, CuL and ZnL
(
3
.7.4 | Viscosity measurements
Absorption and emission studies generally provide necessary,
but not sufficient clues to support interacalative binding
mode. To further clarify the nature of the interaction between
the complexes and DNA, viscosity measurements were
carried out. Hydrodynamic measurements are sensitive to
change in length of DNA and considered to be the most crit-
ical and least ambiguous tests in evaluating binding modes in
solution in the absence of crystallographic structural data.
Lengthening of DNA helix occurs on intercalation as base
pairs are separated to accommodate the binding ligand lead-
[38]
ing to increase in DNA viscosity.
Partial or on classical
intercalation of ligand may bend or kink the DNA helix,
thereby decreasing its effective length and subsequently vis-
cosity. The values of relative specific viscosities of DNA in
the absence and presence of complexes are plotted against
FIGURE 18 Effect of increasing amount of the EtBr, L, CoL, NiL,
CuL and ZnL on the relative viscosities of CT‐DNA at 28 °C,
[DNA] = 200uM. The results are the mean of three independent
experiments carried out under identical conditions
[complex]/[DNA]. The relative viscosities of CT‐DNA
bound to ligand and its complexes have increased with

1
6 of 19
HEGDE ET AL.
increasing concentration (Figure 18).similar to some known
intercalators, indicative of a classical intercalation.
the newly synthesized compounds. The results suggest that
the compounds exhibit good cytotoxic activities in a
concentration dependent manner. Cisplatin was used as
reference drug. The cytotoxicity activities of the most
potent derivatives were also tested in MCF‐7, KB, A431,
Mia‐Pa‐Ca‐2, K‐562 cell lines and were treated with
different concentrations of each compound for 72 h and
it was observed that all compounds prevent cell growth
in a dose dependent manner. The IC50 values (concentra-
tion that inhibits 50% of cell growth) for all compounds
were determined by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐
Diphenyltetrazolium Bromide (MTT).
[39]
3
.7.5 | DNA cleavage studies
Many studies have shown that the clinical efficacies of many
drugs correlate with their ability to induce DNA cleavage.
The extent to which the newly synthesized ligands and their
metal complexes could function as DNA cleavage agents is
examined using supercoiled (SC) plasmid pBR322 DNA as
a target. The agarose gel electrophoresis method is employed
to study the efficiency of cleavage by the synthesized
compounds. The photograph representing the cleavage of
DNA by various samples is presented in Figure 19.
As shown in Graphs 1 to 6, copper complex exhibited
enhanced cytotoxic activity with all the six cell lines
The characterization of DNA recognition by transition
metal complex has been aided by the DNA cleavage chemis-
try that is associated with redox‐active or photoactivated
metal complexes. The electrophoretic analysis clearly reveals
that the new molecule and their metal complexes have acted
on DNA as there has been a difference in molecular weight
between the control and the treated DNA samples. Usually
the pBR 322 plasmid shows nicked/open circular, linear and
[40,41]
supercoiled DNA bands.
It is observed that at a given
concentration, there is diminishing of bands with a prominent
streak indicating more cleavage activity of copper complex.
But the ligand, cobalt, nickel and zinc complexes are not
showing significant cleavage of bands.
GRAPH 1 Cytotoxicity against Oral cancer cell lines(KB)
GRAPH 2 Cytotoxicity against Skin cancer cell lines (A431)
GRAPH 3 Cytotoxicity against Pancreas cancer cell lines (MIAPACA)
3
3
.8 | Anticancer studies
.8.1 | MTT assay
The anticancer activities of ligand, and its copper complex
were screened for their in vitro cytotoxicity against KB
(
(
Oral), A431 (Skin), MIA‐Pa‐Ca (Pancreases), K‐549
Lung), K‐562 (Leukemia), MCF‐7 (Breast) and VERO cell
lines, to obtain information about the anticancer potential of
FIGURE 19 Lane 1, DNA control; Lane2, DNA+ solvent; Lane3,
DNA + L(100uM); Lane4, DNA + CoL (100uM); Lane5,
DNA + NiL(100uM); Lane6, DNA + CuL(100uM); Lane7,
DNA + ZnL(100uM). Graphic 1. Cytotoxicity against oral cancer cell
lines(KB). Graphic 2. Cytotoxicity against Skin cancer cell lines
(
(
(
A431). Graphic 3. Cytotoxicity against pancreas cancer cell lines
MIAPACA). Graphic 4. Cytotoxicity against Lung cancer cell lines
A549). Graphic 5. Cytotoxicity against Leukemia cell lines (K562).
Graphic 6 cytotoxicity against Breast cancer cell line (MCF‐7)

HEGDE ET AL.
17 of 19
attached ligand. The antiproliferative activity of both ligand
and copper complex on tested cell lines are in the order
KB > A‐431 > Mia‐Pa‐Ca > A‐549 > K‐562 > MCF‐
7
> VERO. The cytotoxicity of these compounds on VERO
cell line showed IC50 value 560 μg/ml, which suggest that
these compounds have a low toxicity to VERO cell lines.
The IC50 values are compared between L and CuL for their
action on KB (Oral), A431 (Skin), MIA‐Pa‐Ca (Pancreases),
K‐549 (Lung), K‐562 (Leukemia), MCF‐7 (Breast) and
VERO cell lines and are compiled in Table 7.
While tumor cells undergo apoptosis in the presence
of anticancer drugs, normal cells become necrotic if the
drug is toxic. MTT assays cannot differentiate between
these mechanisms of cell death. Therefore, the effects of
drug may primarily be toxic, poisoning normal cells.
These drugs have limited clinical application. Therefore,
detection of tumor cell apoptosis is more valuable than
generally assessing tumor cell viability. Cisplatin in the
treatment of human cancers as one of the most active
chemotherapeutic drugs, was designated as a positive
control.
GRAPH 4 Cytotoxicity against Lung cancer cell lines (A549)
3
.8.2 | Acridine orange ethidium bromide
(
AO/EB) staining
GRAPH 5 Cytotoxicity against Leukemia cell lines (K562)
KB, the oral cell line was labeled by AO/EB. Dual
staining was examined under a fluorescent microscope.
No significant apoptosis was detected in the negative
control group. Primary stage apoptotic cells, marked by
granular yellow green AO nuclear staining, were noticed
in the experimental group. In ligand treated group the
numbers of primary stage apoptotic cells were increased
with increasing concentrations and treatment period. In
the CuL treated cells, the apoptotic cells at final stage
were also detected with rising concentration and period
of treatment by EB stain which showed localized orange
nuclear cells. Thus, results depicted raise in necrotic cells
and exhibited uneven orange‐red fluorescence at their
periphery. Double AO/EB fluorescent staining can detect
basic morphological changes in apoptotic cells. In
addition, it permits to distinguish between normal,
primary, late apoptotic and necrotic cells. Morphological
evidence of apoptosis by AO/EB dual staining in L and
CuL treated cells are shown in Figure 20.
GRAPH 6 Cytotoxicity against Breast cancer cell line (MCF‐7)
compared to ligand alone. In this case, the enhancement of
antiproliferative activity, with respect to other compounds,
might be related to their different structural characteristics
and mainly attributed to redox active metal ion and the
TABLE 7 IC50 values of L, CuL and cisplatin (μg/ml)
Compound
KB
A431
MIAPACA
A549
K‐562
MCF‐7
Vero
L
5.42
2.14
4.2
4.55
1.49
5.2
15.19
4.98
4.2
50.65
16.60
6.4
168.84
55.35
5.2
4.31
2.24
4.8
562.82
184.50
6.8
CuL
Cisplatin

1
8 of 19
HEGDE ET AL.
2
FIGURE 21 Tentative structures for the complexes
We guess that AO entered normal and primary
apoptotic cells without damaging membranes, fluorescing
green when attached to DNA. EB only entered cells with
broken membranes, such as final stage apoptotic and
deceased cells, radiating orange‐red fluorescence when
bound to concentrated DNA fragment or apoptotic
[
42,43]
forms.
Likewise, double AO/EB staining is capable
to detect minor DNA damages. Hence, to distinguish
normal, primary apoptotic, late apoptotic cells and the
dead cells, nuclear morphology must be assessed.
Fluorescent staining using Acridine Orange alone has
been used earlier; though, revealing of cell apoptosis
using AO/EB doubled staining is
a
comparatively
different method, and very few articles have stated its
[
44]
usage.
In contrast to Acridine orange staining, the
combined AO/EB method increases the rate of detection
of apoptosis and can distinguish between different stages
of apoptosis.
4
| CONCLUSION
The transition metal complexes of novel ligand ethyl‐2‐(2‐(2‐
chlorobenzoyl)hydrazono)propanoate were synthesized and
characterized using various physico‐chemical techniques.
The ligand behaves as a monobasic tridentate ligand with
ONO donor sites. Elemental analyses, thermal and spectral
studies confirm the proposed composition of metal
complexes. The octahedral geometry was assigned for all
the complexes and tentative structures proposed for them
are presented in Figure 21. The strong ability of copper
complex to bind to DNA (CT‐DNA) has been suggested by
absorption, emission, viscosity and thermal denaturation
studies with E. coli DNA. The gel electrophoresis method
confirms the higher ability of copper complex to cleave
pBR322 DNA. The anticancer studies suggest that the
cytotoxic activity of ligand was enhanced on formation of
copper complex towards KB, MCF‐7, A‐431, Mia‐Pa‐Ca‐2,
A‐549 K‐562 and VERO cell lines. Copper complex has
shown better activity against KB, A431 and MCF‐7 cell lines
compared to the standard used.
FIGURE 20 Morphological evidence of apoptosis by AO/EB dual
staining L, CuL and. Cisplatin. (a) negative control group (untreated
cells)‐the circular nucleus homogeneously spread in the central region of
the cell. (b) ligand treated cells showing early stage of apoptosis i.E cell
shrinkage (c) positive control, cisplatin treated cells showing tigtly
packed cells undergoing apoptosis. (d) CuL treated cells showing
intense cell shrinkage showing strong apoptosis

HEGDE ET AL.
19 of 19
ACKNOWLEDGEMENT
[25] A. Barve, A. Kumbhar, M. Bhat, B. Joshi, R. Butcher, U.
Sonawane, R. Joshi, Inorg. Chem. 2009, 48, 9120.
The authors thank the USIC, Karnatak University, Dharwad for
providing the spectral facilities and thankful to UGC for provid-
ing UGC‐UPE fellowship and UPE‐FAR‐I program. Authors are
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