Biological evaluation of organometallic palladium(II) complexes containing 4-hydroxybenzoic acid (3-ethoxy-2-hydroxybenzylidene)hydrazide: Synthesis, structure, DNA/protein binding, antioxidant activity and cytotoxicity

Article abstract of DOI:10.1002/aoc.3599

New palladium(II) complexes, [Pd(PPh₃)L] (2) and [Pd(AsPh₃)L] (3), were synthesized using 4-hydroxybenzoic acid (3-ethoxy-2-hydroxybenzylidene)hydrazide (1) ligand (H₂L), and characterized using various physicochemical techniques. The molecular structures of 2 and 3 were determined using single-crystal X-ray diffraction, which reveals a square planar geometry around the palladium(II) metal ion. In vitro DNA binding studies were conducted using UV–visible absorption spectroscopy, emission spectroscopy, cyclic voltammetry and viscosity measurements, which suggest that the metal complexes act as efficient DNA binders. The interaction of ligand H₂L and complexes 2 and 3 with bovine serum albumin (BSA) was investigated using UV–visible and fluorescence spectroscopies. Absorption and emission spectral studies indicate that complexes 2 and 3 interact with BSA protein more strongly than the parent ligand. The free radical scavenging potential of all the synthesised compounds (1–3) was also investigated under in vitro conditions. In addition, the in vitro cytotoxicity of the complexes to tumour cells lines (HeLa and MCF-7) was examined using the MTT assay method.

Full text of DOI:10.1002/aoc.3599

Received: 21 July 2016
DOI 10.1002/aoc.3599
Accepted: 6 August 2016
F U L L PA P E R
Biological evaluation of organometallic palladium(II) complexes
containing 4‐hydroxybenzoic acid (3‐ethoxy‐2‐
hydroxybenzylidene)hydrazide: Synthesis, structure, DNA/protein
binding, antioxidant activity and cytotoxicity
Ganesan Ayyannan1 | Perumal Veerasamy1 | Maruthachalam Mohanraj1 |
Gunasekaran Raja1 | Arumugam Manimaran2 | Marappan Velusamy3 | Nanjan Bhuvanesh4 |
4
1
*
Raju Nandhakumar | Chinnasamy Jayabalakrishnan
1 Post‐Graduate and Research Department of
Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, Coimbatore 641020,
Tamil Nadu, India
2 Department of Chemistry, Kongunadu Arts and
Science College, Coimbatore 641029, Tamil Nadu,
India
New palladium(II) complexes, [Pd(PPh3)L] (2) and [Pd(AsPh3)L] (3), were synthe-
sized using 4‐hydroxybenzoic acid (3‐ethoxy‐2‐hydroxybenzylidene)hydrazide (1)
ligand (H2L), and characterized using various physicochemical techniques. The
molecular structures of 2 and 3 were determined using single‐crystal X‐ray diffrac-
tion, which reveals a square planar geometry around the palladium(II) metal ion. In
vitro DNA binding studies were conducted using UV–visible absorption spectros-
copy, emission spectroscopy, cyclic voltammetry and viscosity measurements,
which suggest that the metal complexes act as efficient DNA binders. The interac-
tion of ligand H2L and complexes 2 and 3 with bovine serum albumin (BSA) was
investigated using UV–visible and fluorescence spectroscopies. Absorption and
emission spectral studies indicate that complexes 2 and 3 interact with BSA protein
more strongly than the parent ligand. The free radical scavenging potential of all the
synthesised compounds (1–3) was also investigated under in vitro conditions. In
addition, the in vitro cytotoxicity of the complexes to tumour cells lines (HeLa
and MCF‐7) was examined using the MTT assay method.
3 Department of Chemistry, North Eastern Hill
University, Shillong, Meghalaya 793022, India
4 Department of Chemistry, Karunya University,
Karunya Nagar, Coimbatore 641114, Tamil Nadu,
India
Correspondence
C. Jayabalakrishnan, Post‐Graduate and Research
Department of Chemistry, Sri Ramakrishna
Mission Vidyalaya College of Arts and Science,
Coimbatore – 641020, Tamil Nadu, India.
Email: drcjbstar@gmail.com
KEYWORDS
antioxidant, crystal structure, cytotoxic activity, DNA/BSA binding, palladium(II)
complexes
1
|
INTRODUCTION
non‐covalent DNA binding.[4,5] DNA is the primary intracel-
lular target for anticancer drug design and remains one of the
Platinum‐based metallodrugs, e.g. cisplatin, oxaliplatin,
carboplatin, have been successfully used as effective antican-
cer drugs in the clinical treatment of cancer. These deriva-
tives are being used for more than 50% of treatment
regimes worldwide for cancer patients.[1–3] However, their
remarkable success is marred by clinical limitations, includ-
ing acquired or intrinsic resistance problems, limited spec-
trum of activity, less target specificity and high toxicity
leading to side effects. These limitations have stimulated us
to design and synthesize new metal‐based drugs with more
effectiveness, less toxicity, target specificity and preferably
most promising biological receptors for the development of
chemotherapeutic agents.[6,7] The interaction between the
small molecules of metal‐based drugs and DNA can cause
DNA damage in cancer cells, consequently blocking the divi-
sion of cancer cells and resulting in cell death. It is commonly
known that metal complexes can interact with DNA non‐
covalently through electrostatic interactions, groove binding
and intercalation.[8] Among these non‐covalent binding
modes, the intercalation mode attracts considerable attention
due to its strong binding ability and various applications in
cancer therapy and molecular biology.[9] The intercalating
Appl. Organometal. Chem. 2016; 1–13
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
AYYANNAN ET AL.
ability of a molecule with DNA is determined by numerous
factors, for example coordination geometry, metal ion size
and nature of the ligand.
2
|
EXPERIMENTAL
2.1 | Materials and physical measurements
Furthermore, proteins are also considered to be one of the
main molecular targets in the action of anticancer agents.
Serum albumins are most extensively studied because of their
efficient role in drug delivery and remarkable binding proper-
ties with metal complexes.[10,11] Proteins play an important
role in the transport and deposition of a variety of endoge-
nous and exogenous substances in blood. The interactions
of drugs with them result in the formation of stable drug–pro-
tein complexes, which can exert an important effect on the
distribution, free concentration and metabolism of a drug in
the bloodstream, etc. The binding affinity of drug to serum
albumin is pivotal in the design of new drugs.[12,13] Studies
of the binding of drugs to albumins are of great importance
for obtaining information on structural features that deter-
mine the therapeutic effect of drugs. In this regard, bovine
serum albumin (BSA) has been extensively studied, due to
its structural homology with human serum albumin. Also,
some metal ions present in blood plasma affect the binding
between drugs and serum albumins and could participate in
many biochemical processes. Therefore, knowledge of the
reactivity of metal complexes towards DNA and protein
BSA is useful for the design and synthesis of metal‐based
anticancer therapeutics.
Reagent‐grade chemicals PdCl2⋅2H2O, triphenylphosphine,
triphenylarsine, 3‐ethoxysalicylaldehyde and 4‐
hydroxybenzoic acid hydrazide were purchased from
Sigma‐Aldrich and used as received. Calf‐thymus DNA
(CT‐DNA) was purchased from Bangalore Genei, Bangalore,
India. BSA was purchased from Himedia Company. All the
other chemicals and reagents used for DNA binding, protein
binding, antioxidant and cytotoxicity assays were of high
quality. Microanalyses (C, H and N) were performed with a
Vario EL III CHNS analyser. Infrared (IR) spectra were
recorded as KBr pellets in the range 400–4000 cm−1 using
a PerkinElmer FT‐IR 8000 spectrophotometer. Electronic
spectra were recorded in dimethylsulfoxide (DMSO) solution
with a Systronics 2202 double‐beam UV–visible spectropho-
tometer in the range 200–800 nm. Fluorescence spectral data
were acquired with a JASCO FP‐8200 fluorescence spectro-
1
photometer at room temperature. H NMR, 13C NMR and
31P NMR spectra were recorded with a Bruker AV III
500 MHz instrument using tetramethylsilane and
orthophosphoric acid as internal standards. Melting points
were recorded with a Veego VMP‐DS heating table.
Because of the similar coordination modes and chemical
properties of palladium(II) and platinum(II), they both form
square planar complexes. On the basis of the structural and
thermodynamic analogy between platinum(II) and palla-
dium(II) complexes, much attention has been paid to palla-
dium(II) compounds as potential anticancer agents.[14]
Palladium(II) complexes undergo aquation and ligand
exchange reactions 105 times faster than the corresponding
platinum(II) complexes.[15] Furthermore, a number of
mixed‐ligand palladium(II) complexes have been tested and
proved to be efficient compounds of antitumor drugs.[16–22]
In addition, hydrazones are an important class of ligands with
interesting ligation properties due to the presence of several
coordination sites and are widely applied in the field of insec-
ticides, medicines and analytical reagents due to their excel-
lent bioactivity.[23,24] The formation of palladium complexes
with hydrazone ligand plays an important role in enhancing
their biological activity. Herein, we present an investigation
of the synthesis, structure, DNA interaction, protein binding,
anti‐oxidative activity and cytotoxicity of mononuclear palla-
dium complexes of 4‐hydroxybenzoic acid (3‐ethoxy‐2‐
hydroxybenzylidene)hydrazide. The crystal structures of the
new palladium(II) complexes were determined using X‐ray
crystallography. Biological properties of the prepared com-
plexes (binding properties with calf thymus DNA and com-
petitive binding studies with ethidium bromide) were
investigated. The protein binding, cytotoxicity and radical
scavenging were also investigated to understand the biologi-
cal efficacy of the new palladium(II) complexes.
2.2 | Synthesis of hydrazone ligand (H2L) (1)
The hydrazone ligand 4‐hydroxybenzoic acid (3‐ethoxy‐2‐
hydroxybenzylidene)hydrazide (1) was prepared by refluxing
an equimolar mixture of 3‐ethoxysalicylaldehde (0.166 g;
1 mM) with 4‐hydroxybenzoic acid hydrazide (0.152 g;
1 mM) in 50 ml of methanol for 6 h, as shown in Scheme 1.
The reaction mixture was then cooled to room temperature
and the solid obtained was filtered, washed several times with
distilled water and recrystallized from methanol to afford the
ligand in pure form in good yield.
Yield 84%; m.p. 175°C. Anal. Calcd for C16H16N2O4
(%): C, 63.99; H, 5.37; N, 9.33. Found (%): C, 62.81; H,
5.48; N, 9.10. IR (KBr, cm−1): 3370 ν(NH); 1640 ν(C═O);
1612 ν(C═N). UV–visible (DMSO, λmax, nm): 304, 358
1
(π → π*, n → π*). H NMR (DMSO‐d6, δ, ppm): 11.91 (s,
1H, hydrazine NH), 8.61 (s, 1H, H─C═N), 11.17 (s, 1H,
o‐OH), 10.20 (s, 1H, p‐OH), 4.02 (q, OCH2), 1.35 (t, CH3),
6.81–7.84 (m, 7H, aromatic), 8.47 (s, H─C═N), 9.93 (s,
OH), 3.77 (q, O‐CH2), 1.04 (t, CH3), 6.61–7.95 (m, aro-
matic). 13C NMR (δ, ppm): 162 (C═O), 160 (C═N), 64
(O─CH2), 14 (CH3), 115–147 (aromatic).
2.3 | Synthesis of palladium precursor complexes
The starting complexes [PdCl2(PPh3)2] and [PdCl2(AsPh3)2]
were prepared according to previous reports.[25,26]

AYYANNAN ET AL.
3
2.5 | Crystal structure determination
Single‐crystal X‐ray diffraction data of 2 and 3 were col-
lected at room temperature with a Bruker AXS KAPPA
APEX2 CCD diffractometer equipped with a fine focused
sealed tube. The unit cell parameters were determined and
the data collections of 2 and 3 were performed using graph-
ite‐monochromated Mo Kα radiation (k = 0.71073 Å) by u
and x scans. The data collected were reduced using the
SAINT program[27] and the empirical absorption corrections
were carried out using the SADABS program.[28] The struc-
ture of the ligand and complexes was solved by direct
methods[29] using SHELXS‐97, which revealed the position
of all non‐hydrogen atoms, and was refined by full‐matrix
least squares on F2 (SHELXL‐97).[30] All non‐hydrogen
atoms were refined anisotropically, while the hydrogen atoms
were placed in calculated positions and refined as riding
atoms.
SCHEME 1 Synthesis of ligand and palladium(II) complexes.
2.4 | Synthesis of new palladium(II) hydrazone
complexes [Pd(PPh3)L] (2) and [Pd(AsPh3)L] (3)
Complex 2 was prepared by refluxing equimolar quantities of
2.6 | DNA binding studies
[PdCl2(PPh3)2] (0.7019 g;
1
mM) in 20 ml of
dichloromethane and the hydrazone ligand H2L (0.3000 g;
1 mM) in 20 ml of methanol (Scheme 1). After a few minutes
of mixing the above reactants, two drops of triethylamine
were added and continuously refluxed for 6 h. The reaction
mixture was then cooled to room temperature, and the
resulting product was filtered off, washed with methanol
and dried under vacuum. Slow evaporation of 2 from a
MeOH–CHCl3 mixture afforded single crystals suitable for
X‐ray diffraction studies.
Experiments involving the interaction of the free hydrazone
ligand and its palladium(II) complexes with DNA were
carried out in double‐distilled water containing tris
(hydroxymethyl)aminomethane buffer (Tris; 5 mM) and
NaCl (50 mM) and the pH was adjusted to 7.2 with
hydrochloric acid. Stock solution of CT‐DNA was stored at
277 K and used only for a period of four days. The concentra-
tion of CT‐DNA was determined by UV absorbance at
260 nm and its molar absorption coefficient was taken as
6600 M−1 cm−1 [31]
Solutions of CT‐DNA in Tris–HCl buffer
.
Complex 2 (C34H29N2O4PPd). Yield 81%; m.p. 182°C.
Anal. Calcd (%): C, 61.22; H, 4.38; N, 4.20; Found (%): C,
61.65; H, 4.23; N, 4.42. Selected IR bands (cm−1): 1596
ν(C═N); 1350 ν(enolic C─O); 1425, 1033, 691 (for PPh3).
UV–visible (DMSO, λmax, nm): 302, 368 (intraligand transi-
gave a ratio of UV absorbance at 260 and 280 nm of approx-
imately 1.8–1.9, indicating that the DNA was sufficiently free
of protein. Concentrated stock solutions of ligand 1 and pal-
ladium(II) complexes 2 and 3 were prepared in DMSO and
diluted with Tris–HCl buffer to required concentrations.
Absorption spectral titration was performed by keeping the
concentration of the test compounds constant while varying
the CT‐DNA concentration. Equal volume of CT‐DNA solu-
tion was added to the reference solution to eliminate the
absorbance of CT‐DNA itself.
1
tions); 409, 424 (ligand‐to‐metal charge transfer). H NMR
(DMSO‐d6, δ, ppm): 8.51 (s, 1H, H─C═N), 9.89 (s, 1H,
p‐OH), 3.71 (q, OCH2), 1.00 (t, CH3), 6.59–7.77 (m, 22H,
aromatic). 13C NMR (δ, ppm): 159 (C─O), 172 (C═N),
64 (O─CH2), 14 (CH3), 114–152 (aromatic). 31P NMR (δ,
ppm): 17.29.
Complex 3 was synthesised as described above for 2 by
utilizing [PdCl2(AsPh3)2] (0.7918 g; 1 mM) and the
hydrazone ligand H2L (0.3000 g; 1 mM) as in Scheme 1.
Slow evaporation of 3 from a MeOH–CHCl3 mixture
afforded single crystals suitable for X‐ray diffraction studies.
Complex 3 (C34H29N2O4AsPd). Yield 78%; m.p. 191°C.
Anal. Calcd (%): C, 57.44; H, 4.11; N, 3.94. Found (%): C,
56.73; H, 4.32; N, 4.06. Selected IR bands (cm−1): 1598
ν(C═N); 1357 ν(enolic C─O); 1435, 1080, 693 (for AsPh3).
UV–visible (DMSO, λmax, nm): 305, 368 (intraligand transi-
Competitive studies of each compound with ethidium
bromide (EB) were conducted with fluorescence spectros-
copy in order to examine whether the compound can displace
EB from the DNA–EB complex. The DNA–EB complex was
prepared by adding 10 μM EB and CT‐DNA in buffer. The
binding effect of ligand and complexes with the DNA–EB
complex was studied by adding a certain amount of a solution
of the test compound step by step into a solution of the DNA–
EB complex. The influence of the addition of each compound
to the DNA–EB complex solution was determined by record-
ing the variation of fluorescence emission spectra. Electro-
chemical titration experiments were performed by keeping
the concentration of the test compounds constant while
varying the CT‐DNA concentration using Tris–HCl buffer
as electrolyte. The relative viscosities for DNA in the absence
1
tions); 406, 432 (ligand‐to‐metal charge transfer). H NMR
(DMSO‐d6, δ, ppm): 8.57 (s, 1H, H─C═N), 9.93 (s, 1H,
p‐OH), 3.77 (q, OCH2), 1.04 (t, CH3), 6.61–7.95 (m, 22H,
aromatic). 13C NMR (δ, ppm): 159 (C─O), 172 (C═N),
63 (O─CH2), 14 (CH3), 115–152 (aromatic).

4
AYYANNAN ET AL.
(η0) and presence (η) of the compounds was calculated using
the relation η= (t − t0)/t0, where t and t0 are the observed flow
time for each sample and buffer. The values of relative vis-
cosity (η/η0)1/3 were plotted against R, where R = [DNA]/
[compound].
Assay of nitric oxide scavenging activity was based on
the Green method,[35] where sodium nitroprusside in aqueous
solution at physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite ions that
can be estimated using Greiss reagent. Scavengers of nitric
oxide compete with oxygen leading to reduced production
of nitrite ions. For the experiment, sodium nitroprusside
(10 mM) in phosphate‐buffered saline (PBS) was mixed with
a fixed concentration of the test compound and incubated at
room temperature for 150 min. After the incubation period,
0.5 ml of Griess reagent containing 1% sulfanilamide, 2%
2.7 | BSA binding studies
The protein binding study was performed by tryptophan fluo-
rescence quenching experiments using BSA. The excitation
wavelength of BSA at 280 nm and the quenching of the emis-
sion intensity of tryptophan residues of BSA at 345 nm were
monitored using the complexes as quenchers with increasing
concentrations.[32] The excitation and emission slit widths
and scan rates were kept constant for all experiments. A stock
solution of BSA was prepared using 50 mM NaCl/Tris–HCl
(pH = 7.2) and stored in the dark at 4°C for further use. Con-
centrated stock solutions of ligand and complexes were pre-
pared by dissolving them in DMSO/Tris–HCl buffer (1:100)
and diluted suitably with buffer to required concentrations.
An amount of 2.5 ml of BSA solution (1 μM) was titrated
by successive additions of a 25 ml stock solution of
compounds (0–25 μM) using a micropipette.
H3PO4
and
0.1%
N‐(1‐naphthyl)ethylenediamine
dihydrochloride was added. The absorbance of the chromo-
phore formed was measured at 546 nm.
For each of the above assays, tests were done in triplicate
by varying the concentration of the complexes ranging from
10 to 50 μM. The percentage activity was calculated by using
the following formula: activity (%) =[(A0 − Ac)/A0]× 100,
where A0 and Ac represent the absorbance in the absence
and presence of the test compounds, respectively. The 50%
activities (IC50) were calculated from the results of percent-
age activity.
2.9 | Cytotoxicity studies
Cytotoxicity of the ligand and palladium(II) complexes was
evaluated against human cervical (HeLa) and human breast
(MCF‐7) cell lines. Cell viability was determined using the
2.8 | Antioxidant assays
The 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scaveng-
ing activity of the synthesized compounds was measured
according to the method of Blois.[33] The DPPH radical is a
stable free radical having λmax at 517 nm. A fixed concentra-
tion of the experimental compounds was added to a solution
of DPPH in methanol (125 μM, 2 ml), and the final volume
was made up to 4 ml with double‐distilled water. The solution
was incubated at 37°C for 30 min in the dark. The decrease in
absorbance of DPPH was measured at 517 nm.
The hydroxyl radical scavenging activities of the com-
pounds were investigated using the Nash method.[34] In vitro
hydroxyl radicals were generated by Fe3+/ascorbic acid sys-
tem. The detection of hydroxyl radicals was carried out by
measuring the amount of formaldehyde formed from the oxi-
dation reaction with DMSO. The formaldehyde produced
was detected spectrophotometrically at 412 nm. A mixture
of 1.0 ml of iron–EDTA solution (0.13% ferrous ammonium
sulfate and 0.26% EDTA), 0.5 ml of EDTA solution (0.018%)
and 1.0 ml of DMSO (0.85% DMSO (v/v) in 0.1 M
phosphate buffer, pH = 7.4) were sequentially added in the
test tubes. The reaction was initiated by adding 0.5 ml of
ascorbic acid (0.22%) and incubated at 80–90°C for 15 min
in a water bath. After incubation, the reaction was terminated
by the addition of 1.0 ml of ice‐cold trichloroacetic acid
(17.5% w/v). Subsequently, 3.0 ml of Nash reagent was added
to each tube and left at room temperature for 15 min. The
intensity of the colour formed was measured spectrophoto-
metrically at 412 nm against a reagent blank.
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bro-
mide (MTT) assay method.[36] The MCF‐7 and HeLa cells
were grown in Eagle's minimum essential medium containing
10% foetal bovine serum. For screening experiment, the cells
were seeded into 96‐well plates in 100 ml of respective
medium. The test compounds (1–3) dissolved in DMSO were
seeded to the wells. Triplication was maintained, and the
medium without the compounds served as the control. After
24 h, the wells were treated with 20 μl of MTT (5 mg ml−1
PBS) and incubated at 37°C for 4 h. The medium with
MTT was then removed separately and the formed formazan
crystals were dissolved in 100 ml of DMSO. The absorbance
was then measured at 570 nm using a microplate reader. The
percentage cell inhibition was determined using the following
equation: cell inhibition (%) =100 − Abs(sample)/Abs(con-
trol) × 100. The IC50 values were determined from a graph
of percentage cell inhibition plotted against concentration.
3
| RESULTS AND DISCUSSION
3.1 | Synthesis of complexes
The reaction of H2L with [PdCl2(PPh3)2] and [PdCl2(AsPh3)
2] (1:1) affords new complexes [Pd(PPh3)L] (2) and [Pd
(AsPh3)L] (3) (Scheme 1). It is found that the ligand coordi-
nates in a dibasic tridentate nature. All the synthesized com-
pounds were characterized using elemental analysis and IR,

AYYANNAN ET AL.
5
1
UV–visible, H NMR, 13C NMR and 31P NMR spectros-
copies; data are given in Section 2. The structures of 2 and
3 were confirmed from the X‐ray crystallographic study.
coordination mode of benzoylhydrazone ligand to palla-
dium(II) ion via the phenolate oxygen, the azomethine nitro-
gen and imidolate oxygen and also the presence of PPh3/
AsPh3 groups.
The 13C NMR spectrum of the ligand demonstrates a sig-
nal at 162 ppm corresponding to the carbonyl carbon (C═O),
which disappears for the complexes and a new signal at
159 ppm (C─O) is observed, which indicates the coordina-
tion of oxygen via deprotonation. The ligand displays a single
resonance at 160 ppm due to the azomethine carbon atom.
The downfield shift of this signal for the complexes at
172 ppm clearly indicates the coordination of the
azomethaine group. The aromatic carbons of the free ligand
and complexes show signals in the region 114–152 ppm.
The signals due to CH2 and CH3 carbons of ligand and com-
plexes appear at 64 and 14 ppm, respectively.
In order to confirm the presence of triphenylphosphine
group, the 31P NMR spectrum was recorded for complex 2.
A sharp singlet is observed at 17.29 ppm which shows the
presence of phosphorous atom and thus suggesting the pres-
ence of triphenylphosphine group in complex 2.[44]
3.2 | Spectroscopic studies
The characteristic IR bands of the new palladium(II) com-
plexes were compared with those of the free ligand (H2L)
and provide significant information regarding the coordina-
tion mode of ligand in metal complexes. The spectrum of the
free ligand displays a peak at 1640 cm−1 for C═O and it is
absent in the spectra of the complexes. This observation may
be attributed to the enolization of ─NH─C═O and subse-
quent coordination through the deprotonated oxygen.[37] The
bands observed at 1350 and 1357 cm−1 (2 and 3) due to C─O
further confirm the coordination through the deprotonated
oxygen. The ligand shows a strong band at 1612 cm−1 which
is characteristic of the azomethine group (>C═N). Coordina-
tion of the ligand to the palladium ion through azomethine
nitrogen atom is expected to reduce the electron density in
the azomethine link and thus lowers the C═N band frequency
in the region 1596 and 1598 cm .
−1 [38,39] The spectrum of the
ligand shows a medium to strong band at around 3370 cm−1
which is characteristic of N─H functional group. This band
disappeared in the spectra of the complexes due to deproton-
ation of phenolic oxygen prior to coordination to the palla-
dium(II) ion. In addition, the palladium(II) hydrazone
complexes show strong vibrations is the expected region
confirming the presence of triphenylphosphine and arsine.[40]
The electronic spectra of ligand and complexes show two to
four bands in the region 302–432 nm. The bands appearing
in the region 302–368 nm are assigned to intraligand transi-
tion[41] and the bands around 406–432 nm are assigned to
metal‐to‐ligand charge transfer transition.[42]
3.3 | X‐ray crystallography
The molecular structures of complexes 2 and 3 were deter-
mined using single‐crystal X‐ray diffraction to find out the
coordination mode of the hydrazone ligand in the complexes
and stereochemistry of the complexes. The ORTEP views of
complexes 2 and 3 are shown in Figures 1 and 2, respectively.
A summary of data collection and refinement parameters is
given in Table 1, whereas selected bond lengths and bond
angles are given in Table 2. Complex 2 crystallizes in triclinic
space group P1 whereas the complex 3 crystallizes in mono-
clinic space group P21/c. The hydrazone ligand coordinates
with the metal at O, N and O forming one five‐membered
and one six‐membered chelate ring with bite angles of
79.72(7)° N(28)–Pd(1)–O(31) and 93.97(7)° N(28)–Pd(1)–
O(35) and bond lengths of 1.9744(18) Å Pd(1)–N(28),
1
The H NMR spectra of all the synthesized compounds
were recorded in DMSO‐d6 to confirm the binding of the
benzoylhydrazone ligand to the palladium(II) ion. The free
ligand shows a broad signal at 11.17 ppm due to ortho‐OH
group. In the spectra of complexes, the signal of the phenolic
proton is absent due to deprotonation. However, ligand and
complexes demonstrate a singlet at 9.93–10.20 ppm which
indicates the non‐participation of para‐OH from coordina-
tion. The NMR spectrum of the ligand shows a singlet at
11.91 ppm corresponding to ─NH group. Interestingly, in
the spectra of the complexes, there is no resonance attribut-
able to ─NH, indicating the coordination of ligand in the
anionic form upon deprotonation.[43] A sharp singlet at
8.61 ppm is due to the azomethine proton. The positions of
the azomethine signal in the spectra of the complexes are
slightly upfield when compared with those of the free ligand,
suggesting that the azomethine proton is coordinated to palla-
dium ion. In the spectra of ligand and complexes, the multi-
plet observed at around 6.59–7.95 ppm is assigned to
aromatic
protons
of
the
phenyl
group
of
triphenylphosphine/triphenylarsine. The results confirm the
FIGURE 1 ORTEP diagram of 2 with thermal ellipsoids at 50% probability.

6
AYYANNAN ET AL.
remaining
binding
site
is
occupied
by
the
triphenylphosphine unit Pd(1)–P(8) bond distance of
2.2888(6) Å and triphenylarsine unit Pd(1)–As(8) bond dis-
tance 2.3920(5) Å for 2 and 3, respectively. The bond
angles O(31)–Pd(1)–O(35) 173.62(6)°, N(28)–Pd(1)–P(8)
176.67(5)° for complex 2, and O(31)–Pd(1)–O(35) 174.63
(13)°, N(28)–Pd(1)–As(8) 179.51(10)° for complex 3 devi-
ate considerably from the ideal angle of 180° causing sig-
nificant distortion in the square planar geometry of the
complexes. The selected bond lengths and bond angles
agree very well with those reported for other palladium(II)
complexes.[45]
3.4 | DNA binding studies
FIGURE 2 ORTEP diagram of 3 with thermal ellipsoids at 50% probability.
3.4.1
| Electronic absorption titration
Both covalent and non‐covalent interactions are involved
between transition metal complexes and DNA. In the case
of covalent binding, the labile ligands of complexes is
replaced by a nitrogen base of DNA such as guanine N7
while the non‐covalent DNA interactions include
intercalative, electrostatic and groove binding of metal
1.9867(16) Å Pd(1)–O(31) and 1.9684(16) Å Pd(1)–O(35)
for complex 2, and bite angles of 79.79(13)° N(28)–Pd
(1)–O(31) and 95.06(13)° N(28)–Pd(1)–O(35) and bond
lengths of 1.971(4) Å Pd(1)–N(28), 1.993(3) Å Pd(1)–O
(31) and 1.963(3) Å Pd(1)–O(35) for complex 3. The
TABLE 1 Crystal and structure refinement data
2
3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C
34H31N2O5PPd
C37H36AsN3O5Pd
784.01
685.02
295.3(3)
0.71073
Triclinic
P1
296
0.71073
Monoclinic
P21/c
9.6660(4)
12.3195(5)
13.3136(6)
103.326(4)
91.705(4)
90.266(3)
1541.91(12)
2
11.5575(11)
26.1997(17)
11.6320(7)
90
b (Å)
c (Å)
α (°)
β (°)
98.039(6)
90
γ (°)
Volume (Å3)
3487.6(5)
4
Z
Density (calculated) (Mg m−3
)
1.4754
1.4988
Absorption coefficient (mm−1
)
0.698
1.522
F(000)
698.5
1588.9
Crystal size (mm3)
0.23 × 0.23 × 0.21
6.56 to 57.48
0.69 × 0.59 × 0.56
6.22 to 57.5
θ range for data collection (°)
Index ranges
−13 ≤ h ≤ 12, −15 ≤ k ≤ 15, −17 ≤ l ≤ 16
12 172
−14 ≤ h ≤ 12, −31 ≤ k ≤ 32, −15 ≤ l ≤ 15
10 998
Reflections collected
Independent reflections
Refinement method
Data/restraints/parameters
Goodness‐of‐fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
6969 [R(int) = 0.0195]
Full‐matrix least‐squares on F2
6969/0/392
6670[R(int ) = 0.0336] = 0.0336]
Full‐matrix least‐squares on F2
6670/0/427
1.068
1.080
R1 = 0.0311, wR2 = 0.0851
R1 = 0.0394, wR2 = 0.0780
0.52 and −0.64
R1 = 0.0495, wR2 = 0.0456
R1 = 0.0796, wR2 = 0.1123
1.02 and −0.80
Largest diff. Peak and hole (e Å−3
)

AYYANNAN ET AL.
7
TABLE 2 Selected bond lengths (Å) and angles (°) of complexes 2 and 3
2
3
Bond lengths
Pd(1)–N(28)
Pd(1)–O(31)
Pd(1)–O(35)
Pd(1)–P(8)
1.9744(18)
1.9867(16)
1.9684(16)
2.2888(6)
1.395(2)
Pd(1)–N(28)
Pd(1)–O(31)
Pd(1)–O(35)
Pd(1)–As(8)
N(28)–N(29)
N(28)–C(27)
O(35)–C(22)
O(31)–C(30)
N(29)–C(30)
1.971(4)
1.993(3)
1.963(3)
2.392(5)
1.379(5)
1.273(5)
1.307(5)
1.317(5)
1.300(6)
N(28)–N(29)
N(28)–C(27)
O(35)–C(26)
O(31)–C(30)
N(29)–C(30)
Bond angles
1.281(3)
1.319(3)
1.298(3)
1.301(3)
O(31)–Pd(1)–O(35)
N(28)–Pd(1)–P(8)
N(28)–Pd(1)–O(35)
N(28)–Pd(1–O(31)
173.62(6)
176.67(5)
93.97(7)
79.72(7)
O(31)–Pd(1)–O(35)
N(28)–Pd(1)–As(8)
N(28)–Pd(1)–O(31)
N(28)–Pd(1)–O(35)
174.63(13)
179.51(10)
79.79(13)
95.06(13)
complexes outside of the DNA helix. An absorption titration
experiment was carried out to study the DNA binding proper-
ties of the new synthesized compounds (1–3). The absorption
spectra of the compounds at a constant concentration
(25 μM) in the presence of various concentrations of CT‐
DNA (10–50 μM) are shown in Figure 3. Upon incremental
additions of DNA to the ligand solution, the absorption bands
of H2L observed at 228 and 309 nm exhibit a hypochromism
of about 31 and 5% without any shift in the band positions.
This reveals that there is an interaction between the ligand
and DNA through intercalation or some other mode of bind-
ing. However, complex 2 exhibits a hypochromism of about
30, 18 and 14% with a hypsochromic shift of 3, 2 and 2 nm
at 267, 330 and 404 nm. Similarly, absorption bands of com-
plex 3 found at 267, 329 and 403 nm exhibit the same phe-
nomenon of hypochromism of about 33, 32 and 23%,
respectively, with a hypsochromic shift of about 2 nm. These
results suggest an intimate association of complexes 2 and 3
with CT‐DNA, and it is also likely that they bind to the
DNA helix via intercalation.[46] After the compounds interca-
late to the base pairs of DNA, the π* orbital of the interca-
lated compounds could couple with π orbitals of the base
pairs, thus decreasing the π → π* transition energies,
resulting in hypochromism. Complexes 2 and 3 show more
hypochromicity with red shift than the ligand, indicating that
the binding strength of the palladium(II) complexes is much
stronger than that of the free ligand. The magnitude of the
binding strength of the compounds with CT‐DNA can be
estimated through the binding constant Kb, which can be
obtained by monitoring the changes in the absorbance of
the corresponding λmax with increasing concentrations of
CT‐DNA and is given by
FIGURE 3 Electronic spectra of compounds 1, 2 and 3 in Tris–HCl buffer
upon addition of CT‐DNA. [Compound] = 25 μM, [DNA] = 0–50 μM.
Arrows show that the absorption intensities decrease with increasing DNA
concentration.
Where [DNA] is the concentration of DNA in base
pairs, εa is the extinction coefficient observed at a given
DNA concentration, εf is the extinction coefficient of the
free complex in solution and εb is the extinction coefficient
of the complex when fully bound to DNA. In plots of
[DNA]/(εa − εf) versus [DNA], Kb is given by the ratio of
slope to intercept.
½DNAꢀ ½DNAꢀ
1
The magnitudes of the intrinsic binding constants (Kb)
¼
þ
were calculated to be 1.42 × 104 M−1 for the H2L),
εa−εf
εb−εf
Kbðεb−εfÞ

8
AYYANNAN ET AL.
7.09 × 104 M−1 for complex 2 and 3.71 × 104 M−1 for com-
plex 3 (Figure 4 and Table 3). The observed values of Kb
reveal that the ligand and palladium(II) complexes bind to
DNA via an intercalative mode. These results are similar to
previously reported values for the intercalative mode of
various metallointercalators.[47,48] The larger aromatic moiety
in complexes 2 and 3 facilitates its potential intercalative
DNA binding. Based on the Kb value, we can arrange the
compounds in the following order: 2 > 3 > 1.
TABLE 3 DNA binding constants (Kb), quenching constants (Kq) and
apparent binding constants (Kapp
)
Compound
Kb (M−1
)
Kq (M−1
)
Kapp (M−1
)
1
2
3
1.42 × 104
7.09 × 104
3.71 × 104
2.53 × 104
1.07 × 105
2.82 × 104
1.8 × 105
8.1 × 105
3.1 × 105
3.4.2
| EB displacement study
The binding of compounds 1–3 to CT‐DNA was studied by
evaluating the fluorescence emission intensity of EB bound
to DNA (EB–DNA) upon the addition of the compounds.
EB is one of the most sensitive fluorescence probes that
can bind with DNA. The fluorescence of EB increases after
intercalating into DNA. If a metal complex intercalates into
DNA, it leads to a decrease in the binding sites of DNA
available for EB, resulting in decrease in the fluorescence
intensity of the CT‐DNA–EB system. The extent of decrease
in the fluorescence intensity (quenching) of CT‐DNA–EB
reflects the extent of interaction of the complex with CT‐
DNA. Figure 5 shows the fluorescence spectra of EB–DNA
with increasing amounts of the test compounds. As the con-
centration of the compounds increases, the emission band at
613 nm exhibits hypochromism up to 60, 84 and 68% with
blue shifts of 2 nm of the initial fluorescence intensity for
1, 2 and 3, respectively. The EB–DNA quenching results also
indicate that many of the EB molecules have been displaced
from the EB–DNA complex by each quencher at the approx-
imate titration end point and that the intercalation takes place
between the test compounds and DNA. Comparatively, free
ligand can compete with EB in binding to DNA and complex
2 binds to DNA stronger than 1 and 3.
The quenching data were analysed according to the
Stern–Volmer equation, and the Kq value is obtained as the
FIGURE 5 Fluorescence quenching curves of EB bound to DNA for 1, 2
and 3. [DNA] = 10 μM, [EB] = 10 μM, [complex] = 0–50 μM. Arrows
show the emission intensity changes with increasing complex concentration.
FIGURE 4 Plots of [DNA]/(εa − εf) versus [DNA] for compounds 1–3 with
CT‐DNA.

AYYANNAN ET AL.
9
slope of a plot of I0/I versus [Q]. The quenching plots illus-
trate that the quenching of EB bound to CT‐DNA by the
complexes are in good agreement with the linear Stern–
Volmer equation. The Kq values for the compounds are given
in Table 3. Further, the binding constant (Kapp) value
obtained for the compounds using the following equation
are given in Table 3:
intercalates into a DNA double helix, and it will shift in a
negative direction in the case of electrostatic interaction with
DNA.[50] Upon incremental addition of CT‐DNA to a com-
plex solution, new redox peaks do not appear and the current
intensity decreases, suggesting the occurrence of interaction
between each metal complex and CT‐DNA. The decrease in
current intensity can be explained in terms of an equilibrium
mixture of free and DNA‐bound complex at the electrode sur-
face.[51] The cyclic voltammograms of Pd(II) complexes 2
and 3 in the absence and presence of CT‐DNA are shown in
Figure 7. In the presence of DNA, both the cathodic (Epc) and
the anodic (Epa) potentials of 2 and 3 exhibit a positive shift,
while the current peaks decrease significantly suggesting the
existence of an interaction between each complex and CT‐
DNA. The above results reveal an intercalative mode of
binding between the complexes and CT‐DNA base pairs.
KEB½EBꢀ ¼ Kapp½compoundꢀ
where the compound concentration is the value at a 50%
reduction in the fluorescence intensity of EB, KEB
(1.0 × 107 M−1) is the DNA binding constant of EB and
[EB] is 10 μM.
The apparent binding constants are found to be 1.8 × 105,
8.1 × 106 and 3.1 × 105 M−1 for 1, 2 and 3, respectively
(Figure 6). These results suggest that the interaction of the
palladium(II) complexes with CT‐DNA is stronger than that
of the free ligand, which is consistent with the absorption
spectral observations. The quenching constants and binding
constants of the ligand and Pd(II) complexes suggest that
the interaction of all the compounds with DNA should be
of an intercalative nature.[49] On the basis of all the spectro-
scopic studies, we conclude that the free ligand and Pd(II)
complexes can bind to CT‐DNA via an intercalative mode
and also that complexes 2 and 3 bind to CT‐DNA more
strongly than the free ligand. This result also indicates that
compounds 1–3 bind strongly to the DNA probably by
intercalative interaction which is supported by the absorption
titration experiment.
3.4.4
| Viscosity experiments
To further confirm the interaction mode of the complexes
with DNA, a viscosity study was carried out. The addition
of the compounds into DNA solution results in a significant
increase of the relative viscosity of DNA; this increase may
be explained by an increase of the overall DNA length caused
3.4.3
| Cyclic voltammetry
The application of electrochemical methods to the study of
interaction of metal complexes to DNA provides a useful
complement to other methods, such as UV–visible and fluo-
rescence spectroscopies. In general, the electrochemical
potential of a small molecule will shift positively when it
FIGURE 6 Stern–Volmer plots of EB–DNA fluorescence titrations for
compounds 1–3.
FIGURE 7 Cyclic voltammograms of complexes 2 and 3 in the absence and
presence (inner curve) of DNA (10 μM). Scan rate: 100 mV s−1
.

10
AYYANNAN ET AL.
by the insertion of the complexes in between the DNA base
pairs which increases the separation of base pairs at intercala-
tion sites.[52] The relative viscosity of DNA changes with the
amount of compounds (Figure 8). These results suggest that
the viscosity of DNA increases on addition of compounds
1–3. In general, the viscosity of DNA increases steadily when
compounds intercalate between adjacent DNA base pairs.
The increased degree of viscosity, which may depend on
affinity to DNA, follows the order 2 > 3 > 1.
On the basis of the above discussion of DNA binding for
the ligand and two complexes, it has been shown, as a result
of binding energy, that the complexes have a greater DNA‐
binding affinity than the free ligand. The positive results of
DNA binding studies for the ligand and complexes prompted
us to study their protein BSA‐binding activity.
at room temperature. A solution of BSA (1 μM) was titrated
with various concentrations of the complexes (0–25 μM).
Fluorescence spectra were recorded in the range
290–500 nm upon excitation at 280 nm. The changes
observed in the fluorescence emission spectra of a solution
of BSA on the addition of increasing amounts of test com-
pounds are shown in Figure 9. As can be seen, the fluores-
cence emission intensities of BSA at 348 nm show
significant decreasing trends with increasing concentration
of the test compounds, indicating that the interaction of the
3.5 | BSA binding properties
3.5.1
| Fluorescence quenching of BSA by compounds
Proteins are important chemical substances and major targets
for many types of medicines. Interaction of metal complexes
with proteins is becoming increasingly important for
interpreting metabolism and transport processes. In the pres-
ent work, BSA was chosen as the model protein due to its
structural homology with human serum albumin, its avail-
ability, low cost and unusual ligand binding properties.
BSA contains three fluorophores, i.e. tryptophan, tyrosine
and phenylalanine, and the intrinsic fluorescence of BSA is
mainly due to tryptophan alone and changes in the emission
spectra of tryptophan are common in response to protein con-
formational transitions, subunit associations, substrate bind-
ing or denaturation. Furthermore, the binding of metal
complexes to proteins may provide information on the rela-
tionship between structures and functions of proteins. There-
fore, the binding of the synthesized compounds to BSA can
be studied by examining fluorescence spectra.
The binding activities of the ligand and complexes with
BSA protein were studied using fluorescence measurements
FIGURE 8 Effect of compounds 1–3 on viscosity of CT‐DNA.
FIGURE 9 Fluorescence titrations of 1–3 (0–25 μM) with BSA (1 μM).

AYYANNAN ET AL.
11
compounds with BSA could cause conformational changes in
protein structure, leading to changes in the tryptophan micro-
environment of BSA. Upon addition of the compounds to the
BSA solution, a significant decrease of the fluorescence
intensity of BSA at 348 nm up to 66, 79 and 68% of the initial
fluorescence intensity of BSA accompanied by
a
bathochromic shift of 1, 2 and 3 nm for compounds 1–3,
respectively, are observed. The observed red shift is mainly
due to the binding of compounds with the active site in
BSA.[53] These results suggest a definite interaction of all
the complexes with the BSA protein.
In addition, the fluorescence quenching data were
analysed with the Stern–Volmer equation and the Scatchard
equation. From a plot of F0/F versus [Q], the quenching con-
stant (Kq) can be calculated (Figure 10). It is assumed that the
binding of the compounds with BSA occurs at equilibrium
and the equilibrium binding constant can be analysed
according to the Scatchard equation:
FIGURE 11 Plots of log[Q] versus log[(F0 − F)/F].
TABLE 4 Quenching constants (Kq), binding constants (Kbin) and number
of binding sites (n) for interactions of compounds 1–3 with BSA
ꢀ
ꢁ
F0−F
F
log
¼ logKbin þ n log½Qꢀ
Compound
Kq (M−1
)
Kbin (M−1
)
n
1
2
3
5.2 × 104
1.5 × 105
6.9 × 104
6.3 × 104
8.4 × 105
7.0 × 104
0.89
1.21
0.94
where Kbin is the binding constant of the compound with
BSA and n is the number of binding sites. Kbin and n have
been calculated from plots of log[(F0 − F)/F] versus log[Q]
(Figure 11). The calculated Kq, Kbin and n values are listed in
Table 4. The calculated value of n is around 1 for all of the
compounds, indicating the existence of just a single binding
site in BSA for all of the compounds. Also, the results show
that complex 2 interacts with BSA more strongly than
complex 3 and ligand 1.
intensity of BSA with the addition of the test compounds and
there is a small red shift of about 1 nm for compounds 1, 2
and 3. From the results obtained it is concluded that only a
static interaction takes place between the tested compounds
1–3 and BSA.[54]
3.5.2
compounds
| UV absorption spectra of BSA in the presence of
3.6 | Antioxidant activity
Results of UV absorption spectral titrations of BSA in the
presence and absence of ligand and complexes are shown in
Figure 12. It is seen that there is a decrease in the absorption
The antioxidative properties of hydrazone ligand H2L and its
metal complexes have attracted a lot of interest recently and
have been extensively investigated, mainly in in vitro sys-
tems. The antioxidant potential of the new palladium com-
plexes along with that of standard ascorbic acid in cell‐free
systems has been examined with reference to DPPH radicals,
nitric oxide radicals (NO) and hydroxyl radicals (OH) and
their corresponding IC50 (determination of 50% activity)
values are given in Table 5. The IC50 values of all test com-
pounds obtained from different types of assay experiments
strongly support that the complexes investigated in this work
possess excellent antioxidant activities, which are better than
that of the standard antioxidant ascorbic acid. The antioxidant
activity of the free ligand H2L is observed to be less effective
on the free radicals. The new complexes 2 and 3 having sim-
ilar structure show significant antioxidant activities. Among
the Pd(II) complexes, that containing triphenylphosphine as
a co‐ligand shows better activities than that containing
triphenylarsine. From these results, it is clear that the scav-
enging effects of the free ligand are significantly less when
compared to the corresponding Pd(II) complexes, which is
FIGURE 10 Stern–Volmer plots of fluorescence titrations of 1–3 with
BSA.

12
AYYANNAN ET AL.
The results indicate that both of the palladium complexes
exhibit anti‐tumour activities against the human cancer cell
lines. The enhanced cytotoxic properties of the complexes
over the ligand may be due to the extended planar structure
induced by the π → π* conjugation resulting from the che-
lation of the hydrazone ligand to the palladium ion in the
complexes.[56] The in vitro cytotoxic activity studies indicate
that the two complexes have better activities than the corre-
sponding ligand but show significantly less activity than
standard cisplatin. Finally, the observed ordering of the
cytotoxicity among the two complexes is very similar to that
of the DNA and BSA binding activities, showing the higher
potential for complex 2.
4
| CONCLUSIONS
FIGURE 12 Absorption titrations of 1–3 with BSA.
In summary, a hydrazone ligand and its palladium(II) com-
plexes were synthesized and characterized. The dibasic
tridentate coordination of the ligand with central metal
and the molecular structures of the ligand and both new
complexes 2 and 3 were authenticated by X‐ray diffraction
studies. DNA binding of the ligand and Pd(II) complexes
suggests that the two complexes bind to DNA more
strongly than the ligand. The strong binding affinity of the
complexes is attributed to the extension of the π system
of the intercalated ligand which leads to a greater planar
area of the complexes than the free ligand enabling the
complexes to penetrate more deeply into, and stack more
strongly with, the base pairs of DNA. In addition, the pro-
tein interaction properties of the new compounds were stud-
ied using UV–visible and fluorescence spectroscopies, and
the results indicated that complex 2 has stronger binding
compared with complex 3 and the ligand. The results
obtained from three different antioxidant assays showed
the significant efficacy in scavenging radicals and the
assumed patterns of activity decreased in the following
order: 2 > 3 > 1. The cytotoxicity of the compounds
against a panel of cancer cells demonstrated that the new
complexes 2 and 3 showed promising tumour cell growth‐
inhibiting activity compared to the free ligand. Among the
trivalent palladium hydrazone complexes, that containing
triphenylphosphine as a co‐ligand exhibited superior perfor-
mance than its arsine‐containing counterpart.
TABLE 5 Antioxidant activity of ligand, complexes and standard ascorbic
acid
IC50 (μM)
Compound
DPPH•
OH•
NO•
1
95.11
43.01
52.34
27.71
90.73
17.15
19.45
5.12
85.92
58.37
58.73
37.52
2
3
Ascorbic acid
mainly due to the chelation of the organic ligand with the Pd
(II) ion.[55]
3.7 | In vitro cytotoxicity assays
Cytotoxicity assays of ligand and complexes were
performed against two tumour cell lines, human cervical
cancer cell line (HeLa) and human breast cancer cell line
(MCF‐7), using MTT assay. The results were analysed by
means of cell inhibition expressed as (50% activity) IC50
values (concentration of compounds required to achieve
50% cell death) and are summarized in Table 6. From the
biological assays of the ligand and Pd(II) complexes, the
complexes exhibit more significant activities than the ligand
against cancer cells, which confirms that the chelation of
ligand with the Pd(II) ion is the only responsible factor for
the observed cytotoxic properties of the new complexes.
The outcome of this study would be helpful for under-
standing the mechanism of interactions of Pd(II) hydrazone
complexes with serum albumin and nucleic acid and also in
the development of potential probes for BSA and DNA struc-
ture and conformation, or new therapeutic agents for many
diseases.
TABLE 6 Cytotoxic activity of ligand and complexes against the HeLa and
MCF‐7 cancer cell lines
IC50 (μM)
Compound
HeLa
MCF‐7
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