New palladium(II) hydrazone complexes: Synthesis, structure and biological evaluation

Article abstract of DOI:10.1016/j.jphotobiol.2016.08.003

Two new palladium(II) complexes of 4-hydoxy-benzoic acid (5-bromo-2-hydroxy-benzylidene)-hydrazide (H₂L) (1) with triphenylphosphine and triphenylarsine as coligand have been synthesized and characterized by the aid of various spectral techniqu

Full text of DOI:10.1016/j.jphotobiol.2016.08.003

New palladium(II) hydrazone complexes: Synthesis, structure and biological
evaluation
Ganesan Ayyannan, Maruthachalam Mohanraj, Gunasekaran Raja, Nan-
jan Bhuvanesh, Raju Nandhakumar, Chinnasamy Jayabalakrishnan
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DOI:
Reference:
S1011-1344(16)30502-4
doi: 10.1016/j.jphotobiol.2016.08.003
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1 August 2016
1 August 2016
Please cite this article as: Ganesan Ayyannan, Maruthachalam Mohanraj, Gunasekaran
Raja, Nanjan Bhuvanesh, Raju Nandhakumar, Chinnasamy Jayabalakrishnan, New pal-
ladium(II) hydrazone complexes: Synthesis, structure and biological evaluation, (2016),
doi: 10.1016/j.jphotobiol.2016.08.003
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New palladium(II) hydrazone complexes: Synthesis, structure and
biological evaluation
Ganesan Ayyannana, Maruthachalam Mohanraja, Gunasekaran Rajaa, Nanjan Bhuvaneshb,
Raju Nandhakumarb, Chinnasamy Jayabalakrishnana*
aPost-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, Coimbatore - 641 020, Tamil Nadu, India.
bDepartment of Chemistry, Karunya University, Karunya nagar, Coimbatore - 641 114,
Tamil Nadu, India.
*Corresponding author contact details, e-mail address: drcjbstar@gmail.com
(C.Jayabalakrishnan), Tel.: +0422 2692461, Fax: 0422 2693812.
Abstract
Two new palladium(II) complexes of 4-hydoxy-benzoic acid (5-bromo-2-hydroxy-
benzylidene)-hydrazide (H2L) (1) with triphenylphosphine and triphenylarsine as coligand
have been synthesized and characterized by the aid of various spectral techniques. The
structure of the ligand and complexes was confirmed by single crystal X-ray diffraction
studies. The hydrazone ligand acts as a tridendate ligand with ONO as the donor sites and is
preferably found in the enol form in all the complexes. The structural analysis of 2 and 3
confirms the square planar geometry of the two complexes. The DNA binding of these
complexes and ligand calf thymus DNA (CT-DNA) was investigated by using various
methods, which revealed that the compounds interacted with CT-DNA through intercalation.
Binding properties of the free ligand and its complexes with bovine serum albumin (BSA)
protein have been investigated using UV-visible and fluorescence spectroscopic methods
which indicated the stronger binding nature of the palladium complexes to BSA than the free
hydrazone ligand. In addition, concentration dependent free radical scavenging potential of
all the synthesised compounds (1-3) was also carried out under in vitro conditions. Further,
the in vitro cytotoxicity of the compounds was examined on a HeLa and MCF-7 cell lines,
which revealed that complex 2 exhibited a superior cytotoxicity than complex 3 and ligand 1.
Keywords:
Palladium(II) complexes; Nucleotide and protein interaction; Antioxidant; Cytotoxicity.

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Introduction
In recent years, design and synthesis of transition metal complexes and to find the interaction
ability with DNA and protein is an interest field of medicinal chemistry. Due to metal
complexes binding to DNA or protein, are commonly considered as the main molecular
targets in the action of basis of designing and discovering new and more efficient anticancer
drugs [1-5]. Cisplatin is widely used and well-known metal based drug for cancer therapy,
represents a major landmark in the history of metal-based anticancer drugs. However, the
clinical success of cisplatin is limited by significant side effects that originate from its
binding mode with DNA and the formation of covalent cross-links [6-8]. Therefore, much
attention has been paid to the design and synthesis of new metal-based drugs bearing metal
ions other than platinum with more-efficacious, target-specific, less-toxic and non-covalent
DNA-binding [9, 10]. Small molecules of drug can interact with DNA through the following
three non-covalent modes: intercalation, groove binding and external static electronic effects.
Intercalation is one of the most important DNA binding modes because it invariably leads to
cellular degradation. The planarity, coordination geometry, and type of donor atom present in
ligand play key roles in determining the intercalating ability of metal complexes with DNA
[11]. The metal ion type and its valence, which are responsible for the geometry of the
complexes, also influence the intercalating ability of metal complexes. In this regard, the
square-planar geometry of the palladium complexes showed a remarkable intercalation
binding properties with DNA.
Based on the aspect of the metallic ion, because of the similar coordination modes and
chemical properties of palladium(II) and platinum(II), they both form square planer
complexes. On the basis of the structural and thermodynamic analogy between platinum(II)
and palladium(II) complexes, much attention has been paid to palladium(II) compounds as
potential anticancer agents [12]. Palladium(II) complexes undergo aquation and ligand
exchange reactions 105 times faster than the corresponding platinum(II) complexes [13].
Furthermore, number of mixed ligand palladium(II) complexes are tested and proved to be an
efficient compounds of antitumor drugs [14-19 ]. In addition, hydrazones are important class
of ligands with interesting ligation properties due to the presence of several coordination sites
and are widely applied in the field of insecticides, medicines and analytical reagents due to
their excellent bioactivity [20-24]. The formation of palladium complexes with hydrazone
ligand plays an important role to enhance their biological activity.

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On the other hand, binding of drugs with proteins have also attracted enormous
research interest as a prime molecular target [25]. The magnitude of the albumin interactions
with the drug is essential since it plays a dominant role in drug disposition and efficacy. The
bound drug can act as a depot while the unbound drug produces the desired pharmacological
effect. Therefore, the interactions with proteins play an important role in the absorption,
transportation and deposition of a variety of endogenous and exogenous substances such as
fatty acids drugs and metal ions in blood stream [26]. Therefore interaction studies of the
metal complexes with biomolecules like DNA and BSA become very important to develop
new drugs with great potential. In this respect, we have synthesis and characterization of two
new palladium(II) hydrazone complexes [Pd(PPh3)L] (2) and [Pd(AsPh3)L] (3). The
molecular structure of palladium(II) complexes are determined by single crystal X-ray
diffraction method. Further, a series of experiments have been carried out to study the
interaction of palladium(II) complexes and free ligand with nucleic acid and BSA. Also, in
vitro free radical scavenging and cytotoxic activities has also been carried out.
Experimental section
Materials and methods
All the reagents used were of analytical or chemically pure grade. Solvents were purified and
dried according to standard procedures [27]. Doubly distilled water was used to prepare
buffers. Ethidium bromide (EB), bovine serum albumin (BSA), calf thymus DNA (CT-DNA)
were purchased from Sigma-Aldrich and used as received. The ligand and the starting
complexes [PdCl2(PPh3)2] [PdCl2(AsPh3)2] were prepared according to the previous report
[28, 29]. Microanalyses (C, H and N) were performed on a Vario EL III CHNS analyser. IR
spectra were recorded as KBr pellets in the 400-4000 cm-1 region using a Perkin Elmer FT-IR
8000 spectrophotometer. Electronic spectra were recorded in DMSO solution with a
Systronics double beam UV-vis spectrophotometer 2202 in the range 200-800 nm.
Fluorescence spectral data were performed on a JASCO FP-8200 fluorescence
1
13
31
spectrophotometer at room temperature. H, C and P NMR spectra were recorded on a
Bruker AV III 500 MHZ instrument using TMS and ortho phosphoric acid as an internal
standard. Melting points were recorded with Veego VMP-DS heating table.
Synthesis of hydrazone ligand (H2L) (1)
The ligand, 4-hydoxy-benzoic acid (5-bromo-2-hydroxy-benzylidene)-hydrazide (H2L) (1)
was prepared by refluxing an equimolar mixture of 4-hydroxy benzoic acid hydrazide (0.152

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g; 1 mmole) and 5-bromo-2-hydroxy benzaldehyde (0.201g; 1 mmole) in 50 ml methanol for
6 h (Scheme 1). The reaction mixture was cooled to room temperature and the solid obtained
was filtered, washed several times and recrystallized from methanol. White coloured crystals
suitable for X-ray diffraction analysis were obtained by slow evaporation of its methanolic
solution. Yield: 84%; Melting point: 259°C. Elemental Analysis: Found (calculated) (%)
C14H11BrN2O3: C, 51.93 (50.17); H, 3.11 (3.31); N, 8.18 (8.36). IR (KBr, cm-1): 3302 ν(NH);
1610 ν(C=O); 1599 ν(C=N); 1103 ν(N-N); UV-vis (DMSO), λmax (nm) (ε M-1 cm-1): 304
(6240), 358 (3925) (π → π*, n→ π*). 1H NMR (DMSO-d6): δ 12.01 (s, 1H, hydrazine NH), δ
8.58 (s, 1H, H-C=N), δ 11.42 (s, 1H, o-OH), δ 10.19 (s, 1H, p-OH), δ 7.84 (d, 1H, aromatic
CH), δ 7.77 (d, 1H, aromatic CH), δ 7.43 (d, 1H, aromatic CH), δ 7.41 (d, 2H, aromatic CH),
13
δ 6.9 (d, 2H, aromatic). C NMR: δ 163 (C=O), δ 161 (C=N), δ 110.44, 115.21, 118.68,
121.31, 123.16, 129.90, 130.80, 133.35, 134.40, 145.29 (aromatic).
Synthesis of [Pd(L2)(PPh3)] (2)
To a warm methanolic solution (25 ml) of H2L (0.0335g; 0.1 mmole), dichloromethane
solution of [PdCl2(PPh3)2] (0.0701 g, 0.1 mmole) was added and followed by two drops of
triethylamine. The reaction mixture was refluxed 1 h and kept at room temperature for
crystallization. Needle like orange red crystals suitable for X-ray studies were obtained on
slow evaporation. Yield: 79%, Melting point: 221°C. Elemental Analysis: Found (calculated)
(%) C32H24BrN2O3PPd: C, 52.56 (54.76); H, 3.76 (3.45); N, 3.90 (3.99). Selected IR bands
(cm-1): 1590 ν(C=N); 1353 ν(enolic C-O); 1423, 1095, 690 (for PPh3). UV-vis (DMSO), λmax
(nm) (ε M-1 cm-1): 302 (9170), 368 (8221) (intra-ligand transitions); 409 (7253), 424 (6560)
(LMCT). 1H NMR (DMSO-d6): δ 8.53 (s, 1H, H–C=N), δ 9.93 (s, 1H, p-OH), δ 7.70 (d, 1H,
aromatic CH), δ 7.68 (d, 1H, aromatic CH), δ 7.66 (d, 1H, aromatic CH), δ 7.65 (d, 2H,
aromatic CH), δ 7.63 (d, 2H, aromatic CH), δ 7.32 (d, 6H, aromatic CH), δ 6.75 (d, 6H,
aromatic CH), δ 6.64 (d, 3H, aromatic CH), 13C NMR: δ 158 (C-O), δ 171 (C=N), δ 144.61,
134.65, 134.55, 133.21, 132.31, 131.16, 130.78, 130.61, 128.78, 128.31, 127.31, 126.22,
118.89, 114.21 (aromatic). 31P NMR: δ 20.8.
Synthesis of [Pd(L1)(AsPh3)] (3)
It was prepared as described for 2 by the reaction of [PdCl2(AsPh3)2] (0.0789 g, 0.1 mmole)
with ligand (0.0290g; 0.1 mmole). Dark orange coloured crystals obtained were found to be
suitable for X-ray diffraction. C32H24BrN2O3AsPd (3): Yield: 81%, MP: 280 °C. Anal. calcd.
(%): C, 51.53; H, 3.45; N, 3.24. Found (%): C, 50.68; H, 3.37; N, 4.13. Selected IR bands
(cm-1): 1593 ν(C=N); 1356 ν(enolic C-O); 1423, 1084, 693 (for AsPh3). UV-vis (DMSO),

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λmax (nm) (ε M-1 cm-1): 305 (7673), 368 (7111) (intra-ligand transitions); 406 (5995), 432
1
(5880) (LMCT). H NMR (DMSO-d6): δ 8.52 (s, 1H, H–C=N), δ 9.97 (s, 1H, p-OH), δ 7.71
(d, 1H, aromatic CH), δ 7.65 (d, 1H, aromatic CH), δ 7.59 (d, 1H, aromatic CH), δ 7.37 (d,
2H, aromatic CH), δ 7.36 (d, 2H, aromatic CH), δ 7.31 (d, 6H, aromatic CH), δ 6.76 (d, 6H,
aromatic CH), δ 6.70 (d, 3H, aromatic CH), 13C NMR: δ 159 (C-O), δ 173 (C=N), δ 144.84,
135.13, 134.95, 133.32, 132.72, 131.69, 130.93, 130.11, 129.42, 128.89, 121.19, 115.11,
109.12, 105.87 (aromatic).
Single crystal X-ray diffraction studies
Single crystal X-ray diffraction data of 1, 2 and 3 were collected at room temperature on 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 1, 2 and 3 were
performed using a graphite-mono chromate Mo Kα(k = 0.71073 Å) radiation by u and
x scans. The data collected were reduced SAINT program [30] and the empirical absorption
corrections were carried out using the SADABS program [31]. The structure of the ligand
and complexes was solved by direct methods [32] using SHELXS-97, which revealed the
position of all non-hydrogen atoms, and was refined by full-matrix least squares on F2
(SHELXL- 97) [33]. All non-hydrogen atoms were refined anisotropically, while the
hydrogen atoms were placed in calculated positions and refined as riding atoms.
DNA binding experiments
Absorption titration
In order to identify the binding mode of the synthesized compounds (1-3) with CT-DNA, the
UV-visible titration method is used. In absorption titration experiments, the test compounds
with CT-DNA were carried out in double distilled water with aminomethane (Tris, 5 mmole)
and sodium chloride (50 mmole) ( pH = 7.2). The UV absorbance at 260 nm and 280 nm of
the CT-DNA solution gave a ratio of 1.8-1.9, indicating that the DNA was sufficiently free of
protein [34, 35]. The molar absorption coefficient, ε260, was taken as 6600 M−1cm−1. A stock
solution of CT-DNA was stored at 277 K and used after no more than 4 days. Absorption
titration experiments were performed with a fixed concentration of the compounds ()
while gradually increasing the concentration of DNA (0-50 M). The blank Tris-HCl buffer
(2.5 ml) solution and the compounds (25 M) were placed in two cuvettes, respectively. To
eliminate the absorbance of DNA, an equal amount of DNA was added to both the test
solution and the reference solution. After each addition of DNA to the test compounds, the

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readings were noted. The magnitude of the binding strength of the test 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 absorption maxima (λmax
with increasing concentrations of CT-DNA and is given by the equation.
[DNA]/[εa- εf] = [DNA]/[εa- εf] + 1/Kb [εb- εf]
)
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 the
intercept.
Competitive binding experiments
The competitive DNA binding studies of the new synthesized compounds (1-3) with ethidium
bromide (EB) was investigated with fluorescence spectroscopy in order to inspect whether
the compound could displace bound EB from the DNA-EB complex by the addition of the
solution of the respective compounds (1-3) to the Tris-HCl buffer of the DNA-EB mixture.
Before measurements were taken, the mixture was shaken up and recorded. The fluorescence
spectra of DNA bound EB were obtained in the excitation (λex) and emission (λem)
wavelengths of 515 and 602 nm, respectively. Before measurements, the system was
shaken and incubated at room temperature for 5 min. The emission was recorded at
530-750 nm. The quenching constants of compounds (1-3) were evaluated qualitatively by
employing Stern-Volmer equation [36],
F0 / F = Kq / [Q] + 1
where F0 is the emission intensity in the absence of complex, F is the emission intensity in
the presence of complex, Kq is the quenching constant, and [Q] is the concentration of the
compound. The Kq value has been obtained as a slope from the plot of F0 / F versus
[Q]. Further, the binding constant (Kapp) value obtained for the compounds using the
following equation.
KEB[EB] = Kapp[compound]
where the compound concentration is the value at a 50% reduction in the fluorescence
intensity of EB, KEB (1.0 ×107M−1) is the DNA binding constant of EB, [EB] is the
concentration of EB = 10 μM.

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Cyclic voltammetry assay
Voltammetric techniques are complementary to other related biophysical techniques that are
applied to study the interaction between the redox active molecules and biomolecules. This
electrochemical study were performed on a CHI 604A electrochemical analyzer with three
electrode system of glassy carbon as the working electrode, a platinum wire as auxiliary
electrode and Ag/AgCl as the reference electrode. Solutions were deoxygenated by purging
with N2 prior to measurements. The supporting electrolyte was used 50 mmole NaCl and 5
mmole Tris-HCl buffer at pH 7.2. In order to determine diffusion coefficients before and after
the addition of DNA with palladium(II) complexes, voltammetric responses were recorded at
various scan rates.
Viscosity measurements
The viscosity of a DNA solution has been measured in the presence of increasing
amounts of synthesized compounds. Flow time was measured with a digital stop watch three
times for each sample and an average flow time was calculated. The relative viscosities for
DNA in the absence (ηo) and presence (η) of the compounds was calculated using the relation
η = (t-to)/to, where t and to are the observed flow time for each sample and buffer [37]. The
values of relative viscosity (η/ηo)1/3 were plotted against R, where R= [DNA]/[compound].
Protein binding studies
The binding of synthesized compounds with bovine serum albumin (BSA) were studied using
fluorescence spectra recorded at affixed excitation wavelength corresponding to bovine
serum albumin (BSA) as 280 nm and monitoring the emission at 345 nm. The excitation and
emission slit widths and scan rates were constantly maintained for all the experiments.
Samples were carefully degassed using pure nitrogen gas for 15 min by using quartz cells
(4 × 1 × 1 cm) with high vacuum Teflon stopcocks. Stock solution of BSA was prepared in
50 mM tris-HCl buffer (pH = 7.2) and stored in the dark at 4ºC for further use. Concentrated
stock solutions of ligand and complexes were prepared by dissolving them in DMSO: tris-
HCl buffer (1:100) and diluted suitably with buffer to required concentrations. 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.
Antioxidant assays
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the synthesized
compounds was measured according to the method of Blois [38]. The DPPH radical is a

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stable free radical having a λmax at 517 nm. A fixed concentration 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 ºC for 30 min in the dark. The decrease in absorbance of DPPH was measured at 517 nm.
The hydroxyl (OH) radical scavenging activities of the complexes have been
investigated using the Nash method [39]. In vitro hydroxyl radicals were generated by
Fe3+/ascorbic acid system. The detection of hydroxyl radicals was carried out by measuring
the amount of formaldehyde formed from the oxidation 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 sulphate 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 ºC for 15 min on a water bath. After
incubation, the reaction was terminated by the addition of 1.0 mL of ice-cold TCA (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
spectrophotometrically at 412 nm against a reagent blank.
Assay of nitric oxide scavenging activity is based on the Green method [40], 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 mmole) in phosphate buffered
saline was mixed with a fixed concentration of the compound and incubated at room
temperature for 150 min. After the incubation period, 0.5 mL of Griess reagent containing
1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride
were added. The absorbance of the chromophore formed was measured at 546 nm.
For each of the above assay, tests were done in triplicate by varying the
concentration of the complexes ranging from 10-50 µM. The percentage activity was
calculated by using the 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 percentage activity.

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Evaluation of cytotoxicity
Cytotoxicity of the ligand and palladium complexes was evaluated on human cervical (HeLa)
and human breast (MCF-7) cell lines. Cell viability was carried out using the 3-[4, 5-
dimethylthiazol-2-yl] 2, 5-diphenyltetrazolium bromide (MTT) assay method [41]. The
MCF-7 and HeLa cells were grown in Eagles minimum essential medium containing 10%
fetal bovine serum (FBS). 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
was 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 MTT
[5 mg mL-1 phosphate buffered saline (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 DMSO. The absorbance was then measured at 570 nm using micro plate reader. The
% cell inhibition was determined using the following formula, % cell inhibition=100-Abs
(sample)/Abs (control) × 100. The IC50 values were determined from the graph plotted
between % cell inhibition and concentration.
RESULTS & DISCUSSION
Synthesis
The stoichiometric reactions of [PdCl2(EPh3)2] (where E = P or As) with hydrazone ligand
(H2L) in 1:1 methanol/dichloromethane resulted in the formation of new complexes
[Pd(PPh3)L] (2) and [Pd(AsPh3)L] (3) (Scheme 2), where the hydrazone ligand acted as a
tridentate ONO ligand. The analytical data confirms the stoichiometry of the complexes. The
structures of the ligand (1) and complexes (2 and 3) were confirmed by the X-ray
crystallographic study. All of the three synthesized compounds are quite stable in air, light
and soluble in most of the organic solvents, such as methanol, ethanol, CH2Cl2, CHCl3, DMF
and DMSO.
OH
OH
H
Methanol
N
H
CHO
OH
N
N
H2N
After reflux 6 h
O
OH
O
Br
Br
Scheme 1. Synthesis of hydrazone ligand.

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OH
OH
N
H
[PdCl2(EPh3)2]
N
N
N
O
Pd
CH2Cl2/MeOH
After reflux 1 h
O
O
OH
Br
E
Br
Where E = P (or) As
Scheme 2. Synthesis of the palladium(II) complexes
Spectroscopic studies
The electronic spectra of ligand and Palladium(II) complexes have been recorded in DMSO
solvent and they displayed two to three bands in the region around 302-432 nm. Electronic
spectra of 1 showed two strong absorption bands at 304 and 358 nm, which are assigned to π-
π* and n-π* transitions respectively. In the electronic spectra of the complexes, the bands
appeared in the region 302-368 nm have been assigned to intra ligand transition [42] and the
bands around 406-432 nm have been assigned to metal to ligand charge transfer transition
[43].
The infrared spectra of the palladium(II) complexes (2 and 3) compared with that of
the free ligand (H2L) provide significant information regarding to the coordination mode of
ligand in new metal complexes. The IR spectra of the ligand showed a strong absorption at
1599 cm-1 due to the presence of azomethine group. In complexes this has been shifted to
lower frequency (1590 and 1593 cm-1) indicating the coordination of azomethine nitrogen to
palladium [44]. The (C=O) and (N-H) bands appeared at 1660 and 3302 cm-1 respectively in
the free ligand completely disappeared in the complexes and a new band (C-O) appeared at
1353 and 1356 cm-1 for the complexes (2) and (3) respectively, which indicating the
enolisation of NH-C=O group and subsequent coordination through the deprotonated oxygen
atom [45]. In addition, the bands confirming the presence of triphenyl phosphine and arsine
in were observed in the expected region [46]. All these facts suggested that the ligand H2L
were coordinated to palladium(II) ion via the phenolate oxygen, the azomethine nitrogen and
the imidolate oxygen in complexes.
1
The H NMR spectra of the ligand H2L and the corresponding palladium(II)
complexes were recorded in DMSO-d6 solvent. In the spectra of H2L, a sharp singlet
corresponding to the phenolic -OH group has appeared at 11.42 ppm. However, this singlet
completely disappeared in the two complexes confirmed the involvement of phenolic oxygen
in coordination with Pd(II) ion. Also, a weak singlet appeared at 12.01 ppm has been

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assigned to the NH proton. In the spectrum of the complexes (2 and 3) there was no such
resonance attributable to NH, which indicates the coordination of the enolate oxygen atom
after enolisation and subsequent deprotonation [47]. The spectrum of H2L showed a sharp
singlet at 8.58 ppm corresponding to azomethine proton. However, in case of the complexes
(2 and 3), the singlet at 8.58 ppm gets shifted (for 2 and 3 are 8.53 and 8.52 ppm,
respectively) due to the coordination of azomethine N with Pd(II) ion. The spectra of 1, 2 and
3 show multiplets at 6.58-7.89 ppm due to the presence of aromatic protons [48].
The 13C NMR spectra of the ligand, displayed well defined signal at δ 163
corresponding to the carbonyl carbon (C=O), which disappears in the spectra of the
complexes and appears as a new signal at 159 and 158 ppm (C-O) indicating the coordination
of oxygen via deprotonation. The spectra of the ligand show a single resonance at 161 ppm
due to the azomethine carbon atom, the downfield shift of the complexes signal at 171 and
173 ppm clearly indicates the coordination of C=N [49]. The aromatic carbons of the free
ligand and complexes show signal in the regions 105-147 ppm [50].
31
In addition, to confirm the presence of triphenylphosphine group, P NMR spectra
were recorded for the complex 2. A sharp singlet observed at 20.8 ppm for the complex 2 was
due to the presence of phosphorous atom and thus suggesting the presence of
triphenylphosphine group in complex 2 [51].
Based on IR, UV-vis, 1H, 13C and 31P NMR data, the hydrazone ligand is coordinated
to the palladium ion by square planar geometry in a tridentate fashion by replacing both
chloride ions and the triphenylphosphine/triphenylarsine from the starting precursors. Further
the geometry of the complexes confirmed by X-ray diffraction studies.
X-ray crystallography
Needle shaped crystals suitable for X-ray diffraction studies were grown from a solution of
H2L in methanol. The summary of the data collected and the refinement parameters are given
in Table 1. The selected bond lengths and bond angles are given in Table 2. An ORTEP
representation of 1 is shown in Figure 1. The crystals of hydrazone ligand 1 in the
orthorhombic space group Pna21 and unit cell comprises of four molecules. The azomethine
C(7)-N(1) bond length, 1.278(4) Å is in conformity with a formed C=N double bond length,
1.28 Å. The bond distances in the hydrazone side chain agree well with the values observed
for other hydrazones where the C(8)-O(2) group is present in the keto form 1.229(3) Å. And
the bond distances for N(1)-N(2) at 1.378(3), and for C(8)-N(2) at 1.354(4) Å are closer to N-

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N and N-C normal single bonds respectively. The oxygen O(2) atom and the hydrazine
nitrogen N(1) are in a trans position with respect to the C(8)-N(2) bond 1.354(4) Å.
The mononuclear structure of the newly synthesized palladium(II) complexes 2 and 3
are determined by single crystal X-ray diffraction in order to identify the exact coordination
mode of hydrazone ligand in the complexes (Fig. 2 and 3). The selected bond lengths and
bond angles are summarised in Table 3. The single crystal X-ray study reveals that the
complexes 2 and 3 crystallized in monoclinic space group P21/c with four molecules per unit
cell. In the complexes (2 and 3), the Pd(II) ion is coordinated to the dibasic tridentate ligand
through enolate oxygen (Pd-O bond distances of 1.9961(19) A˚ and 1.993(2) A˚
respectively), phenolic oxygen (Pd-O bond distances of 1.9625(19) A˚ and 1.958(2) A˚
respectively) and the nitrogen atom (Pd-N bond distances of 1.981(2) A˚ and 1.970(3) A˚
respectively). The remaining coordination site is occupied by triphenylphosphine and
triphenylarsine satisfying the fourth coordination site in 2 and 3 respectively. In complexes,
the Pd–P and Pd–As bond lengths of 2.2864(8) and 2.3920(5) Å for 2 and 3 respectively are
close to the reported value of palladium(II) complexes [53]. The triphenylphosphine and
triphenylarsine in the complexes N(1) nitrogen are mutually trans to each other. The trans
angles are N(1)-Pd(1)-P(1), 171.44(6)°, and O(3)-Pd(1)-O(1), 172.74(8)° (for 2), N(1)-Pd(1)-
As(1), 171.14(8)°, and O(3)-Pd(1)-O(1), 172.74(8)°(for 3) which deviate considerably from
the ideal angle of 180°, suggesting distortion in the square planar coordination geometry of
the complexes. The C=O bond distance in C(8)-O(2) of the free ligand is found to be
1.229(3) Å. Whereas, upon complexation, the value of C(1)-O(1); 1.308(3) Å for 2 and C(7)-
O(1); 1.303(4) Å for 3 due to the enolization of oxygen. There is an increase in the C=N bond
lengths C(8)-N(1); 1.278(4) Å for 2 and 1.285(4) Å for 3 compared to ligand 1 C(7)-N(1);
1.278(3) Å due to azomethine nitrogen involved in coordination. The selected bond lengths
and bond angles agree very well with those that are reported for other palladium(II)
complexes [54].
Nucleic acid binding studies
Electronic absorption titration
Electronic absorption spectroscopy is employed to determine the binding mode of test
compounds with DNA. The absorption spectra of compounds (1-3) were recorded with a
fixed concentration of the compounds in the absence and presence of different concentrations
of CT-DNA and the results are shown in Fig. 4. The absorption spectra of ligand (1) mainly
consist of two resolved bands [Intra ligand transitions (IL)] centered at 291 and 355 nm. As

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the DNA concentration is increased, the bands at 291 and 355 nm showed 18.32% and
12.91% hypochromism, respectively without any shift in the absorption maxima. For
complex 2, upon addition of DNA, the intra ligand band at 265 and 323 nm exhibited
hypochromism of 61.39% and 60.12% with a red shift of 1 and 4 nm respectively. The CT
band at 413 nm showed 63.53% hypochromism with a red shift of 3nm in the absorption
maxima. Similarly, complex 3 exhibited hypochromism (IL) at 281 (25.59%) and 328 nm
(66.89%) with a red shift of 3 and 4 nm, respectively. In addition, complex 3 showed
hypochromism (CT) at 409 nm (22.31%). Generally, a compound binding to DNA through
intercalation mode results in hypochromism with or without a small red or blue shift, due to a
strong stacking interaction between the planar aromatic chromophore of the compound and
the base pairs of DNA [55, 56]. These results revealed that the compounds 1, 2 and 3 with
bind to the DNA helix via intercalation. After the compounds intercalate to the base pairs of
DNA, the π* orbital of the intercalated compounds could couple with π orbital of the base
pairs, thus decreasing the π→π* transition energies, resulted hypochromism. The complexes,
2 and 3 showed more hypochromicity with red shift than the ligand (H2L), indicating that the
binding strength of the palladium(II) complexes is much stronger than that of the free ligand.
The magnitudes of the intrinsic binding constants (Kb) were calculated to be
9.96 × 103M−1for the ligand (H2L) and 6.78 × 104M−1 and 2.04 × 104M−1 for complexes 2 and
3, respectively (Fig. 5 and Table 4). The observed values of Kb revealed that the ligand and
the palladium(II) complexes bind to DNA via an intercalative mode. These results are similar
with the earlier reported for the intercalative mode of various metallointercalators [57]. From
the above results obtained, it has been found that both the palladium complexes (2 and 3)
exhibit a good binding affinity to DNA, greater than that of the corresponding free ligand.
This is mainly due to the chelation of the palladium metal with the ligand. It has been found
that complex 2 strongly bound with CT-DNA relative to that with 1 and 3, and the order of
binding affinity is 2>3>1. The different DNA-binding properties of the palladium complexes
2 and 3 are due to the two different co-ligands present.
Ethidium bromide displacement assay
The competitive DNA binding of compounds has been studied by monitoring changes in
emission intensity of ethidium bromide (EtBr) bound to CT-DNA by the addition of
compound concentration (1-3) as quenchers. The fluorescence emission intensity of EB can
be quenched by the addition second DNA binding molecule by either replacing the DNA
bound EB or accepting an excited-state electron from EB. Fig. 6 shows the fluorescence

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spectra of DNA-bound EB by compounds and as the concentration of the compounds
increases, the emission band at 613 nm exhibited hypochromism up to 14.88, 20.20 and
21.11% with blue shifts of 3, 2, and 3 nm of the initial fluorescence intensity for 1-3
respectively. This indicates that both the complexes and the free ligand could compete with
EB in binding to DNA and complex 2 binds to DNA stronger than other 1 and 3. From the
results observed, decrease in the fluorescence intensity clearly indicates that the EB
molecules are displaced from their DNA binding sites and are replaced by the complexes
under investigation [58].
Further, the analysis of the quenching data using Stern-Volmer equation gave the
corresponding Kq values and the apparent binding constant (Kapp) values (Fig.7 and Table 4).
The quenching constant (Kq) values for 1, 2 and 3 were found to be 2.54 × 104 M-1, 3.66 ×
104 M-1 and 3.01 × 104 M-1, respectively. Furthermore, the apparent binding constant (Kapp)
values obtained for the corresponding complexes 1 and 2 were found to be 1.8 × 105 M−1,
2.7 × 105 M−1 and 2.2 × 105 M−1, respectively. The results suggest that complex 2 intercalated
more strongly than complex 3. The binding activities of the palladium(II) complexes may be
better than that of the free hydrazone ligand.
Cyclic voltammetry assay
The electrochemical method to study about binding of transitional metal complexes to DNA
is a useful complement to other investigation methods, such as UV-visible and fluorescence
spectroscopies. In general, the electrochemical potential of a small molecule will shift
positively when it intercalates into DNA double helix, and it will shift to a negative direction
in the case of electrostatic interaction with DNA [58, 59]. Cyclic voltammograms (CV) of the
palladium(II) complexes in the absence and presence of CT-DNA in tris-HCl buffer solution
are shown in Fig. 8. Upon addition of CT-DNA to a complex solution, no new peaks
appeared and the current intensity decreased, these results suggesting the existence of an
interaction between each complex and CT-DNA. The decrease in current intensity may be
attributed to slow diffusion of the palladium(II) complex to the electrode surface. For
addition of CT-DNA complex solution, both the cathodic (Epc) and the anodic (Epa) potentials
of 2 and 3 exhibit a positive shift, suggesting an intercalative mode of binding between the
complexes and CT-DNA base pairs [60].

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Viscosity Measurements
Viscosity measurements are sensitive to changes in length of DNA therefore; its
measurement upon addition of a compound is often concerned to clarify the interaction mode
of a synthesized compound with DNA. Generally, titrations of DNA by classical intercalating
compounds would show increased DNA viscosity, as the DNA helix must lengthen when
base pairs are separated to host the binding compounds. And also, a partial or non-classical
ligand intercalation causes a compaction in the DNA helix and reducing its effective length of
DNA and thereby its viscosity decreases. The effects of the ligand (1) and complexes (2 and
3) on the viscosity of CT-DNA are shown in Fig. 9. On increasing the amounts of
compounds, the relative viscosities of CT-DNA solution increase steadily. The increased
degree of viscosity, which may depend on its affinity of compounds to DNA, followed the
order of 2>3>1. These results further proved that the two palladium(II) complexes and free
hydrazone can bind to DNA in the mode of intercalation [61].
On the basis of the above discussion of DNA binding studies, we concluded that the
free ligand and palladium(II) complexes can bind to CT-DNA in an intercalative mode and
Pd(II) complexes bind to CT-DNA more strongly than the free ligand. The positive results of
DNA binding studies for the synthesized compounds prompt us to explore their protein
BSA-binding activity and in vitro anticancer activity.
BSA-binding properties
UV-vis spectroscopy
The quenching process can occur by different mechanisms, which classified as dynamic
quenching and static quenching. In a dynamic quenching mechanism, with the addition of a
quencher, only the excited state fluorescence molecule is affected, which results in no change
in the absorption spectra of BSA. While, in a static quenching, a new compound is formed
between BSA and the quencher, therefore, the absorption spectra of BSA would be
considerably affected. It appears that the UV absorption peak goes up or down and with or
without any shift of wavelength. The absorption spectra of BSA in the absence and presence
of compounds (1-3) are shown in Fig. 10. The absorption band obtained for the BSA at
278 nm in the presence of compounds showed an increase in the intensity of absorption after
the addition of compounds without any shift, reveals quenching of BSA by the compounds
are static quenching processes [62].

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Fluorescence quenching studies
Fluorescence quenching techniques have been widely used to study the interaction of small
molecules with protein molecules. Fluorescence quenching measurement refers to any
process that decreases the fluorescence intensity of a fluorophore due to a variety of
molecular interactions including excited-state reactions, molecular rearrangements, energy
transfer, ground-state complex formation and collision quenching. Changes in molecular
environment in the vicinity of fluorophore can be accessed by the changes in fluorescence
spectra in the absence and presence of the test compound and hence provide information to
the nature of the binding. The interaction of BSA with the ligand and Pd(II) complexes were
studied by fluorescence measurement at room temperature. A solution of BSA () was
titrated with various concentrations of the compound (0-25µM). Fluorescence spectra were
recorded in the range of 290-450 nm upon excitation at 280 nm. The changes observed on the
fluorescence emission spectra of solution of BSA on the addition of increasing amounts of
the compounds are given in Fig. 11. Upon the addition of the new compounds to the BSA
solution, a significant decrease of the fluorescence intensity of BSA at 345 nm of up to 70, 82
and 76% from the initial fluorescence intensity of BSA accompanied by a bathochromic shift
of 4, 2 and 3 nm for the compounds 1-3 respectively have been observed. The observed red
shift is mainly due to the fact that the active site in protein is buried in a hydrophobic
environment. This result suggested a definite interaction of all of the compounds with the
BSA protein [63].
Further, the fluorescence quenching data were analyzed with the Stern-Volmer
equation and the Scatchard equation. From the plot of Fo/F versus [Q], the quenching
constant (Kq) can be calculated (Fig. 12). It is assumed that the binding of compounds with
BSA occurs at equilibrium and the equilibrium binding constant can be analyzed according to
the Scatchard equation.
log[ (Fo – F) / F ] = log Kbin + n log[Q]
where Kbin is the binding constant of the compound with BSA and n is the number of binding
sites. The binding constant (Kbin) and the number of binding sites (n) have been calculated
from the plot of log[(Fo−F)/F] versus log[Q] (Fig. 13). The calculated Kq, Kbin, and n values
are listed in Table 5. The calculated value of binding site 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. The values of Kq and Kbin for all of the compounds suggested that the complexes

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interact with BSA more strongly than the ligand. Among the two Pd(II) complexes, the
complex (2) has better interaction with BSA than the complex (3).
Antioxidant activity
Newly synthesized ligand and Pd(II) complexes exhibit good DNA and protein binding
ability, it was further considered to study their ability to antioxidant properties of the
compounds. Hence, we carried out experiments to investigate the free radical scavenging
ability of the ligand and Pd(II) hydrazone complexes against standard antioxidant ascorbic
acid (Aca) with DPPH, OH and NO radicals. The IC50 values indicated that no significant
radical scavenging activities were observed for free ligand under the same experimental
conditions. From the experimental results, the IC50 value of the complexes with DPPH, OH
and NO radical assays were found to be comparable radical scavenging activity with respect
to standard antioxidant (Aca) (Table 6). The IC50 values show that the ligand and Pd(II)
complexes exhibit antioxidant activity in the order of 2˃3˃1 in all of the experiments.
The palladium complexes having higher antioxidant activity when compared to free
ligand, which is due to chelation of the organic ligand with the Pd(II) ion and planarity of
the complexes plays an important role in determining the antioxidant properties of the
compounds [64]. Moreover, from the results obtained for the two Pd(II) complexes, it can be
inferred that the difference in the nature of the complex and the co-ligand present in the
complexes are likely to induce variations in antioxidant activities. The complex containing
triphenylphosphine as a co-ligand showed better activities than the complex containing
triphenylarsine.
In vitro cytotoxicity
From the previous biological studies namely, DNA binding, BSA binding and antioxidant
studies the positive results obtained of compounds 1, 2 and 3 encouraged us to test their
cytotoxicity against a panel of human cancer cell lines (human cervical cancer cell line
(HeLa) and human breast cancer cell line (MCF-7)) by using MTT assay. The IC50 values of
the free ligand and palladium hydrazone complexes are given in table 7 and fig. 14. These
results observed that the ligand did not show any inhibition of the cell growth even up to
100 µM and clearly indicates chelation of the organic ligand with palladium ion is
responsible for the observed cytotoxicity properties of the complexes [65]. The observed
result indicates that the palladium complexes 2 and 3 possess great selectivity towards HeLa
and MCF-7 cells and display potential application in cancer chemotherapy and less toxic
drug. The IC50 value showed that both the Pd(II) complexes 2 and 3 exhibited excellent

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activity against HeLa and MCF-7 cell lines which was lower than that of the well known
anticancer drug cisplatin. The observed higher efficiency of the complex 2 is correlated
to the nature of the substitution of the co-ligand, triphenylphosphine coordinated to the
central palladium ion. This is due to the liphophilic effect of triphenylphosphine, which
favours its permeation through the lipid layer of the cell membrane in the complex and
could enhance the activity [66, 67]. The enhanced cytotoxic properties of Pd(II) complexes
over the ligand, which may be due to an increase in chelation of the ligand with the Pd(II)
ion, cationic nature, and enhanced planarity of the complexes [68, 69]. Hence, these
complexes are active against the tumor cell lines under in vitro conditions, and the results are
comparable to those obtained for other palladium(II) complexes [70]. Though the synthesized
complexes were active against tumor cell lines under in vitro cytotoxicity experiments, the
IC50 values are comparable with standard drug cisplatin. The findings of the in vitro cytotoxic
activities confirm the binding of the complexes to DNA/BSA, which consequently leads to
cell death.
Conclusion
Reactions of palladium(II) precursors [PdCl2(EPh3)2] (where E = P or As) with
4-hydoxy-benzoic acid (5-bromo-2-hydroxy-benzylidene)-hydrazide (H2L) yielded a two
new [Pd(EPh3)L] complexes. The molecular structure of the ligand H2L and complexes were
confirmed by single crystal X-ray diffraction studies. The XRD results of complexes
confirmed the distorted square planar geometry around the Pd ion through ONO coordination
of 1. The DNA binding properties of free ligand and palladium(II) complexes were evaluated
by the UV-Vis spectra, fluorescence spectra, electrochemical and viscosity measurements
which suggest their involvement in intercalative DNA interaction with different binding
affinities. From the BSA binding studies, the mechanism of quenching of BSA was found to
be a static one indicating that the complexes did effectively bind to BSA interaction than the
ligand. All the synthesized compounds exhibit excellent antioxidant activities and the activity
of complex 2 showed greater IC50 values than the other. In addition, the in vitro cytotoxicity
assay have been conducted with a cancer cell line demonstrated that both the complexes and
free ligand are active against the HeLa and MCF-7 cancer cell line. In particular, complex 2
showed more activity than complex 3 and ligand 1. This significant activity of 2 might be due
to the presence of the phosphine ligand, which is supposed to provide a better cytotoxicity by
enhancing lipophilicity and consequently permeability through the lipid layer of the cell
membrane. The outcome of this study would be helpful to understand the mechanism of

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interactions of palladium(II) hydrazone complexes with nucleic acid and serum albumin and
also in the development of potential probes for DNA and BSA structure and conformation of
new chemotherapeutic agents.
Acknowledgements
One of the authors, G.Raja gratefully acknowledges University Grants Commission (UGC),
Hyderabad, India for financial assistance (NO.F MRP-5925/15 (SERO/UGC)).
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and K. Natarajan, RSC Adv.,2014,4, 2004
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Photobiol., 2016, 158,164-173.
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12.

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Compound
1
2
3
Table 1.

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Empirical formula
CCDC
C14H11BrN2O3
1482486
335.16
296(2) K
0.71073Å
Orthorhombic
Pna21
C32H24BrN2O3PPd C32H24BrN2O3AsPd
1482487
701.81
1482488
745.76
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α ( )
296(2) K
0.71073 Å
Monoclinic
P 21/c
296(2) K
0.71073Å
Monoclinic
P21/c
8.0392(7)
25.680(3)
6.2125(5)
90
15.3568(5)
9.0646(2)
21.4790(6)
90
15.7334(6)
9.1057(2)
21.2449(7)
90
β ( )
90
90
1282.5(2)
108.729(10)
90
2831.62(14)
109.0330(10)
90
2877.23(16)
γ ( )
Volume (Å3)
Z
4
4
4
Density (calculated) Mg/m3
1.736
1.646
1.722
Absorption coefficient mm-1
F(000)
3.214
672
2.160
1400
3.211
1472
Crystal size/mm3
Theta range for data
collection (°)
0.35 x 0.30 x 0.30 0.30 x 0.25 x 0.20 0.35 x 0.30 x 0.30
1.59 to 28.28
1.40 to 28.34
1.37 to 28.32
Index ranges
-10 <= h <= 10,
-21 <= k <= 34,
-8 <= l <= 7
7340
-18 <= h <= 20,
-11 <= k <= 12,
-28 <= l <= 28
21142
-20 <= h<= 19,
-12 <=k <= 12,
-26 <= l <= 28
21646
Reflections collected
Independent reflections
3099
6978
7151
[R(int) = 0.0265] [R(int) = 0.0266]
[R(int) = 0.0287]
Refinement method
Full-matrix least- Full-matrix least- Full-matrix least-
squares on F2
3099/1/181
1.018
squares on F2
6978/0/361
1.118
squares on F2
7151/0/361
1.114
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R1 = 0.0297,
wR2 = 0.0699
R1 = 0.0398,
wR2 = 0.0760
0.274and -0.329
R1 = 0.0302,
wR2 = 0.0759
R1 = 0.0513,
wR2 = 0.0996
0.690 and -0.864
R1 = 0.0353,
wR2 = 0.0846
R1 = 0.0623,
wR2 = 0.1098
0.740 and -0.851
R indices (all data)
Largest diff. peak and
-3
hole e.Å
Crystal and structure refinement data.
Table 2. Selected bond lengths [Å] and bond angles [°] for ligand 1.
Bond lengths
1

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N(2)-C(8)
C(7) N(1)
N(1) N(2)
C(8) O(2)
C(8)-N(2)
1.354(4)
1.278(4)
1.378(3)
1.229(3)
1.354(4)
1.356(3)
1.356(3)
C(1) O(1)
C(12)-O(3)
Bond angles
O(1)-C(1)-C(6)
C(7)-N(1)-N(2)
O(2)-C(8)-N(2)
N(1)-C(7)-C(6)
N(2)-C(8)-C(9)
122.7(3)
116.1(2)
122.4(3)
121.8(3)
115.5(2)
Table 3. Selected bond lengths [Å] and angles [°] of palladium(II) complexes 2 and 3.
Bond lengths
Pd(1)-N(1)
Pd(1)-O(1)
Pd(1)-O(3)
Pd(1)-P(1)
2
3
1.981(2)
1.9961(19)
1.9625(19)
2.2864(8)
1.308(3)
1.285(3)
Pd(1)-N(1)
Pd(1)-O(1)
Pd(1)-O(3)
Pd(1)-As(1)
O(1)-C(7)
C(8)-N(1)
1.970(3)
1.993(2)
1.958(2)
2.392(5)
1.303(4)
1.284(4)
O(1)-C(1)
C(8)-N(1)
Bond angles
N(2)-N(1)-Pd(1)
O(3)-Pd(1)-N(1)
N(1)-Pd(1)-P(1)
O(3)-Pd(1)-O(1)
O(3)-Pd(1)-P(1)
114.45(17)
94.35(9)
171.44(6)
172.74(8)
90.17(6)
N(2)-N(1)-Pd(1)
O(3)-Pd(1)-N(1)
N(1)-Pd(1)-As(1)
O(3)-Pd(1)-O(1)
O(3)-Pd(1)-As(1)
114.5(2)
95.02(11)
171.14(8)
172.74(8)
89.34(8)
Table 4. DNA binding constant (Kb), quenching constant (Kq) and apparent binding constant
(Kapp) values
Compounds
Kb (M−1)
Kq (M−1)
Kapp (M−1)
9.96 × 103 2.54 × 104 1.8 × 105
6.78 × 104 3.66 × 104 2.7 × 105
2.04 × 104 3.01 × 104 2.2 × 105
1
2
3
Table 5. Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n)
for the interactions of compounds (1-3) with BSA

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Compounds
Kq (M−1)
3.4 × 104
9.4 × 104
5.2 × 104
Kbin (M−1)
9.0 × 104
2.3 × 105
1.0 × 105
n
0.95
1.42
1.15
1
2
3
Table.6. Antioxidant activity of the ligand and new palladium(II) complexes with standard
ascorbic acid.
IC50 values (µM)
Compounds
DPPH∙
OH∙
NO∙
98.73 ± 1.2
42.84 ± 0.9
48.63 ± 1.4
27.71 ± 0.7
43.97 ± 1.4
14.06 ± 1.2
19.74 ± 1.8
5.12 ± 0.2
84.72 ± 0.7
53.64 ± 2.5
62.16 ± 2.8
37.52 ± 0.5
1
2
3
Ascorbic acid (Asc)
Table 7. Cytotoxic activity of the ligand and palladium(II) complexes against the HeLa and
MCF-7 cancer cell lines.
IC50 values (µM)
Compounds
HeLa
MCF-7
96.76 ± 2.3
31.64 ± 1.2
44.62 ± 1.1
16.21 ± 0.9
99.23 ± 2.8
30.32 ± 0.8
40.13 ± 0.6
15.35 ± 1
1
2
3
Cisplatin

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Figure 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability
Figure 2. ORTEP diagram of 2 with thermal ellipsoid at 50% probability

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Figure 3. ORTEP diagram of 3 with thermal ellipsoid at 50% probability

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Figure 4. Electronic spectra of the compounds (1), (2) and (3) in Tris-HCl buffer upon
addition of CT-DNA. [Compound] = 25μM, [DNA] = 0-50μM. Arrow shows that the
absorption intensities decrease upon increasing DNA concentration