New Palladium(II) complexes with ONO chelated hydrazone ligand: Synthesis, characterization, DNA/BSA interaction, antioxidant and cytotoxicity

Article abstract of DOI:10.1016/j.ica.2020.119868

There is a continuous finding of suitable metal complexes for biomedical and biological applications since the discovery of cisplatin. Two biologically active complexes (2 and 3) are synthesized by using 2-chloro-benzoic acid (3,5-dichloro-2-hydroxy-benzylidene)-hydrazide (1) ligand. The ligand (1) and complexes (2 & 3) are characterized by various spectroscopic tools. The molecular structures of (1) and (3) are confirmed via., XRD data. The spectroscopic and XRD data reveals that the ligand coordinates to palladium through ONO chelation and the geometry of the complexes is distorted square planar. The in vitro interaction of the synthesized compounds with calf-thymus DNA (CT-DNA) are analysed through different experiments which shows the intercalation mode of binding. Further, the interaction studies of the compounds with bovine serum albumin protein (BSA) show that, the compounds binds strongly with BSA. Also, antioxidant studies have been carried out with the compounds. Finally, MTT assay reveals that, the Pd(II) complexes displayed significant activity against HeLa and MCF-7 cancer cell lines.

Full text of DOI:10.1016/j.ica.2020.119868

Journal Pre-proofs
Review article
New Palladium(II) complexes with ONO chelated hydrazone ligand: Synthe‐
sis, characterization, DNA/BSA interaction, antioxidant and cytotoxicity
Ganesan Ayyannan, Maruthachalam Mohanraj, Mayakrishnan Gopiraman,
Ramasamy Uthayamalar, Gunasekaran Raja, Nanjan Bhuvanesh, Raju
Nandhakumar, Chinnasamy Jayabalakrishnan
PII:
DOI:
Reference:
S0020-1693(20)31067-7
https://doi.org/10.1016/j.ica.2020.119868
ICA 119868
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Inorganica Chimica Acta
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Accepted Date:
6 May 2020
5 July 2020
6 July 2020
Please cite this article as: G. Ayyannan, M. Mohanraj, M. Gopiraman, R. Uthayamalar, G. Raja, N. Bhuvanesh,
R. Nandhakumar, C. Jayabalakrishnan, New Palladium(II) complexes with ONO chelated hydrazone ligand:
Synthesis, characterization, DNA/BSA interaction, antioxidant and cytotoxicity, Inorganica Chimica Acta
(2020), doi: https://doi.org/10.1016/j.ica.2020.119868
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New Palladium(II) complexes with ONO chelated hydrazone ligand: Synthesis,
characterization, DNA/BSA interaction, antioxidant and cytotoxicity
Ganesan Ayyannana, Maruthachalam Mohanraja,b, Mayakrishnan Gopiramanc, Ramasamy
Uthayamalara,d, Gunasekaran Rajaa, Nanjan Bhuvaneshe, Raju Nandhakumare, Chinnasamy
Jayabalakrishnana,f*
aDepartment of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts and Science,
Coimbatore – 641 020, Tamil Nadu, India.
bDepartment of Chemistry, Kalaignarkarunanidhi Institute of Technology, Coimbatore-641402,
Tamil Nadu, India.
cDepartment of Applied Bioscience, College of Life & Environment Science, Konkuk University, 120
Neungdong-ro, Gwangjin-gu, Seoul 05029, South Korea
dDepartment of Science and Humanities, Sri Krishna College of Technology, Coimbatore-641042,
Tamil Nadu, India.
eDepartment of Chemistry, Karunya Institute of Technology and Sciences, Karunya Nagar,
Coimbatore 641 114, Tamil Nadu, India.
fPPG College of Arts and Science, Coimbatore – 641 035, Tamil Nadu, India.
*Corresponding author’s contact details, e-mail address: drcjbstar@gmail.com (C.
Jayabalakrishnan), Tel.: +91 9442001793.
Abstract
There is a continuous finding of suitable metal complexes for biomedical and biological
applications since the discovery of cisplatin. Two biologically active complexes (2 and 3) are
synthesized by using 2-chloro-benzoic acid (3,5-dichloro-2-hydroxy-benzylidene)-hydrazide (1)
ligand. The ligand (1) and complexes (2 & 3) are characterized by various spectroscopic tools. The
molecular structures of (1) and (3) are confirmed via., XRD data. The spectroscopic and XRD data
reveals that the ligand coordinates to palladium through ONO chelation and the geometry of the

complexes is distorted square planar. The in vitro interaction of the synthesized compounds with
calf-thymus DNA (CT-DNA) are analysed through different experiments which shows the
intercalation mode of binding. Further, the interaction studies of the compounds with bovine serum
albumin protein (BSA) show that, the compounds binds strongly with BSA. Also, antioxidant studies
have been carried out with the compounds. Finally, MTT assay reveals that, the Pd(II) complexes
displayed significant activity against HeLa and MCF-7 cancer cell lines
Keywords:
Hydrazone; Palladium(II) complexes; crystal structure; DNA/BSA interaction; antioxidant;
cytotoxicity.
1. Introduction
Metal complexes have gained considerable development over the past decades in medicinal
chemistry. Among the metal complexes, transition-metal complexes are the most widely used
chemotherapeutic agents and continue to play a major role in cancer-treatment since the discovery of
cisplatin [1-5]. The cisplatin-based compounds have a vast expansion of interest in the use of
inorganic compounds for the treatment of tumors [6-8]. Even though cisplatin is most active drug
used as a therapeutic agent for a wide range of cancers, it has a number of limitations such as side
effects and acquired cell resistance [9, 10]. Therefore, particular effort has been devoted to develop
the novel complexes (non-platinum antitumor compounds) that are alternative to cisplatin with
higher potency, higher cancer cell selectivity, lower resistance, particularly, that non-platinum
complexes bind to DNA under physiological conditions [11].
In recent years, many transition metal complexes were synthesized and evaluated for their
antitumor properties [12-16]. Among them, palladium-based complexes have occupied a definite role
in chemotherapy since they accumulates in tumors due to specific permeability through cancer cell
membranes [17-19]. Moreover, palladium(II) complexes are supposed to be potential anticancer

agents due to close resemblance of Pd complexes with Pt complexes [20]. In addition, the
coordination chemistry of the palladium(II) complexes have similar properties with platinum(II)
complexes but the higher liability to ligand exchange at the Pd centre (105 times greater than Pt)
causes rapid hydrolytic processes leading to the dissociation of the complex and the formation of
very reactive species, which leads to reduce the biological activity. The above mentioned drawback
could be overcome by using bulky hydrazone and chelating ligands. Hydrazone ligand has more
chelating capability and structural flexibility that can provide more attention to synthesize metal
complexes [21, 22]. Nitrogen and oxygen congaing hydrazone ligands are well biological active
compounds [23]. The hydrazone with metal complexes enhances the biological properties than the
free hydrazone ligands [24, 25]. In this connection, it is well known that hydrazone type ligand
represents a significant class of compounds for chemotherapeutic applications with great potential.
However, a very few reports on palladium(II) complexes containing 3,5-dichloro-2-hydroxy-
benzaldehyde hydrazone are available in the literature.
In order to explain the antitumor properties of the palladium complexes, better understanding
on the binding nature between complexes and DNA is necessary [26, 27], which can be used for the
design of new types of antitumor drugs [28-30]. For interaction of metal complexes with DNA, the
intercalation mode of binding [31-33] is important because, it influences the anticancer activity of
the complexes. In addition, there is interest in the study about interaction between drug-protein
interactions that can influence the absorption, distribution, metabolism of amino acids, and excretion
potential of metal drugs. The investigation on interaction of proteins with compounds is very useful
as it could provide information to determine the therapeutic efficiency and pharmacological response
of drugs [34]. Therefore, evaluation of the metal drug molecules towards DNA and BSA are very
helpful in order to design and discover the metal-based anticancer therapeutics. Herein, we report the
synthesis of hydrazone complexes of palladium by the reaction of [PdCl2(EPh3)2] with H2L (where E
= P or As) in 1:1 metal to ligand stoichiometry ratio. The synthesized ligands and complexes were

characterized by various spectroscopic techniques via., UV-Visible, FT-IR and NMR. The crystal
structure of ligand and complex 3 was confirmed by X-ray crystallography technique. Finally, the
binding properties of compounds with calf thymus (CT-DNA) and bovine serum albumin (BSA)
were investigated by different techniques. The radical scavenging and cytotoxicity activities of the
synthesized compounds were analysed under in vitro conditions.
2. Experimental
2.1. Materials and instrumentation
Palladium chloride, triphenyl phosphine, triphenyl arsine, 2-chlorobenzohydrazide and 3,5-
dichloro salicyladehyde were purchased from sigma Aldrich. Solvents were used after purification
[35]. Bovine serum albumin (BSA) and calf thymus DNA (CT-DNA) (stored at -20 ºC) (Genei) were
used as received. Elemental analysis (C, H and N) was carried on a Vario EL III CHNS analyser.
Fourier-transform infrared spectroscopy (FT-IR, a Perkin Elmer FT-IR 8000 spectrophotometer) was
used to record IR spectra of ligand and complexes. Absorption spectra of compounds were recorded
on a Systronics double beam UV-vis spectrophotometer 2202. Emission spectra were recorded on a
JASCO FP-8200 spectrophotometer. NMR (1H, 13C and 31P) spectra of the compounds were
recorded using Bruker AV III 500 MHZ instrument.
2.2. Synthesis
2.2.1. Synthesis of hydrazone ligand (1)
Ligand (H2L) was prepared by refluxing equimolar solutions of 3,5-dichloro-2-hydroxy
benzaldehyde (0.1910 g; 1mM) and 2-chlorobenzohydrazide (0.1706 g; 1 mM) in 50 mL of methanol
for 6 h. Then the reaction mixture was kept in room temperature until the formation of white colour
precipitate. The resultant solid was recrystallized by using methanol/chloroform mixture and the high
pure ligand crystal (1) was obtained. Yield: 86%; M.P: 178°C. Anal. calcd. for C14H9Cl3N2O2 (%):

C, 48.94; H, 2.64; N, 8.15. Found (%): C, 46.77; H, 2.14; N, 9.6. IR (KBr, cm-1): 3380 ν(OH); 2946
ν(NH); 1656 ν(C=O); 1595 ν(C=N). UV–vis (DMSO), λmax (nm): 298, 362 (π → π*, n→ π*).
1H NMR (500 MHz, DMSO-d6) δH: 12.56 (s, 1H, -NH), 12.21 (s, 1H, -OH), 8.46 (s, 1H, CH=N),
7.69 (d, 1H, J = 2.5 Hz, -Ar-H), 7.60 (d, 1H, J = 1 Hz, -Ar-H), 7.57 (dd, 1H, J = 1.5 & 7.5 Hz, -Ar-
13
H), 7.54 (t, 1H, J = 5.0 Hz), 7.48-7.51 (m, 2H, -Ar-H). C NMR (125 MHz, DMSO-d6) δC: 168.18
(C=O), 162.46 (C-OH), 152.19 (C=N), 121.19-134.96 (m, -Ar-C).
H
N
Cl
CHO
OH
Cl
H
MeOH/DCM
Reflux 6h
N
N
H2N
O
Cl
OH
O
Cl
Cl
Cl
H2L
Scheme 1. Synthesis of ONO hydrazone ligand (1)
2.2.2. Synthesis of palladium(II) complexes [Pd(PPh3)L] (2) and [Pd(AsPh3)L] (3)
Initially, the starting compounds, [PdCl2(PPh3)2] and [PdCl2(AsPh3)2], were prepared
according to reported literature [36]. In a typical preparation of complex (2), two different solutions
were prepared; (a) [PdCl2(PPh3)2] (0.7019 g; 1 mM) in dichloromethane (20 mL), and hydrazone
ligand (H2L, 0.3000 g; 1 mM) in methanol (20 mL) (Scheme 2). The above two solutions were
stirred with the addition of 2 drops of triethylamine (TEA) and refluxed for 6 h. After reflection, the
reaction mixture was concentrated to 5ml and cooled to room temperature. The formed solid was
filtered, washed well with methanol and dried under vacuum. Similarly, complex 3 was synthesised
by using [PdCl2(AsPh3)2] (0.7918 g; 1 mM) and the ligand (1) (0.3000 g; 1 mM). Slow evaporation
of complexes 2 and 3 from the mixture of methanol/chloroform afforded single-crystals. However,
complex 3 is suitable for X-ray diffraction studies whereas complex 2 is not suitable.
C32H22Cl3N2O2PPd ([Pd(PPh3)L], 2): Yield: 84%, MP: 159 °C, Anal. calcd. (%): C, 54.11; H,
3.12; N, 3.94; Found (%): C, 55.86; H, 4.15; N, 4.72. Selected IR bands (cm-1): 1592 ν(C=N); 1320

ν(enolic C–O); 1426, 1031, 691 (for PPh3). UV-vis (DMSO), λmax(nm): 297, 361 (intra-ligand
1
transitions); 408, 419 (LMCT). H NMR (500 MHz, CDCl3) δH: 8.21 (s, 1H, CH=N), 7.73-7.77 (m,
6H, -Ar-H), 7.64 (dd, 1H, J = 1.5 & 7.5 Hz, -Ar-H), 7.49-7.53 (m, 3H, -Ar-H), 7.41-7.45 (m, 6H, -
Ar-H), 7.39 (dd, 1H, J = 1.0 & 8.0 Hz, -Ar-H), 7.37 (d, 1H, J = 2.5 Hz), 7.28-7.31 (m, 2H, -Ar-H),
13
7.23 (dd, 1H, J = 1.0 & 7.5 Hz, -Ar-H). C NMR (125 MHz, CDCl3) δC: 174.91 (C=O), 155.91
(C=N), 146.73 (C-OH), 120.71-134.65 (m, -Ar-C). 31P NMR: δ 20.16.
C32H22Cl3N2O2AsPd ([Pd(AsPh3)L], 3): Yield: 78%, MP: 192 °C. Anal. calcd. (%): C, 50.96;
H, 2.94; N, 3.71.Found (%): C, 51.42; H, 3.57; N, 3.13. Selected IR bands (cm-1): 1591 ν(C=N);
1323 ν(enolic C–O); 1420, 1095, 687 (for AsPh3). UV-vis (DMSO), λmax(nm): 273, 313, 332 (intra-
ligand transitions); 408 (LMCT). 1H NMR (500 MHz, DMSO-d6) δH: 8.21 (s, 1H, CH=N), 7.84 (s,
1H, -Ar-H), 7.69 (d, 8H, J = 7.0 Hz, -Ar-H), 7.51-7.64 (m, 7H, -Ar-H), 7.46 (t, 2H, J = 7.0 Hz), 7.37
(d, 2H, J = 15.5 Hz, -Ar-H), 7.30 (s, 1H, -Ar-H). 13C NMR (125 MHZ, DMSO-d6) δC: 176.42
(C=O), 154.49 (C=N), 147.20 (C=O), 118.13-133.39 (m, -Ar-C).
2.4. Crystal structure determination
A Bruker AXS KAPPA APEX2 CCD diffractometer was performed at room temperature for
data collection, crystal screening, and unit cell determination. A graphite-mono chromate Mo Kα
radiation (λ = 0.71073 Å) by u and x scans was used for the data collection. The crystallographic
data collected were reduced by SAINT program and indexing the reflections and determining the
lattice parameters. The SAINT [35] program was adopted for the integration of intensity of
reflections and scaling; and the SHELXTL-97 program was ran for space group and structure
determination, and full-matrix least‐squares refinements on F2. All non-hydrogen atoms were refined
anisotropically, while the hydrogen atoms were placed in calculated by using the HFIX instruction.

2.5. Stability test
Complexes 2 and 3 were first dissolved in DMSO (0.5% of the final volume) and then diluted
with tris-HCl buffer (99.5% of the final volume) to a required concentration. The hydrolysis profiles
of these complexes were recorded by monitoring the electronic spectra of the resulting mixture over
24 h.
2.6. Biological Evaluation
2.6.1. DNA binding studies
The interaction of synthesized compounds with DNA was studied by using absorption
spectroscopy. In fact, it is very useful technique to understand the mode of binding between the drug
molecule and DNA and to calculate the binding constant value (Kb). Initially stock solution of CT-
DNA was prepared in tris(hydroxymethyl)amino methane (Tris, 5mM) and NaCl (50 mM), and the
PH was adjusted to 7.2 with HCl. The stock solutions of compounds were prepared in 5% of dimethyl
sulfoxide (DMSO) and diluted with 95% of tris-HCL buffer solution to the required concentration.
The absorption experiment was carried out by keeping the concentration of the synthesized
compounds constant (25µM) and varying the concentration of DNA (0-50µM). The absorption was
recorded for each addition [36]. The binding constant (Kb) was calculated using Wolfe-Shimer
equation.
The competitive ethidium bromide studies were performed in the absence and presence of
synthesized compounds. Addition of test compound solutions to EtBr-DNA resulted in the decrease
of the emission intensity. From the emission intensity values, the quenching constant value was
calculated using the following equation,
F0/F = Kq [Q] + 1
Electrochemical technique was used to explore the interaction between DNA and synthesized
compounds and to verify the interaction mode. In this experiment the addition of DNA to complex

solution and the electrical potential of the complex were recorded. In addition, the viscosity of
synthesized compounds was carried out in absence (η0) and presence (η) of compounds. The binding
properties of the compounds were found out using η = (t-t0)/t0, where t and t0 are the obtained flow
time for the compounds.
2.6.2. BSA binding studies
The binding ability of synthesized ligand and complexes with BSA protein was determined
by using absorption and fluorescence spectroscopic techniques. The BSA stock solution (1µM) was
prepared with tris-HCl buffer (pH=7.2) and it was stored in a dark room for further analysis. The
fluorescence quenching spectra were recorded at room temperature with constant concentration of
protein while varying the concentration of compounds (1-3). Further, the BSA interaction with
compounds was studied using absorption spectra. The concentration of compounds and BSA protein
was from 0-25 µM and 1µM, respectively.
2.6.3. Antioxidant assays
The radical (DPPH, NO and OH) scavenging activity of the synthesized compounds was
studied by using previously reported methods [37-39]. The experiments were carried out in triplicate
by varying the concentration of compounds (10-50 µM). The activity percentage was calculated by
using the relation, % activity = [A0-Ac] × 100, where A0 and Ac represent the absorbance in the
absence and presence of compounds. In addition, ascorbic acid and methanol was used for positive
and negative controls, respectively. From the percentage of activity, the 50% of antioxidant potential
(IC50) value was calculated.
2.6.4. MTT assay
In vitro cytotoxicity of synthesized compounds was analyzed on human cervical (HeLa) and
human breast cancer cell (MCF-7) [40]. For this assay, the test compounds were dissolved in DMSO
and blank samples containing same volume of DMSO were taken as controls to identify the activity

of solvent in experiments. These solutions were incubated for 4 hours. Percentage of inhibition was
calculated by using following equation, % cell inhibition = 100-Abs (sample)/Abs(control) × 100.
The IC50 values were calculated from the cell viability curve of the graph plotted between % of
inhibition and compound concentration.
3. Results and discussion
3.1. Synthesis and spectroscopic investigation
Palladium(II) complexes were synthesized by refluxing the methanolic mixture of 2-chloro-
benzoic acid (3,5-dichloro-2-hydroxy-benzylidene)-hydrazide, dichloromethane solutions of
palladium precursors and triethylamine for 6 h. It was confirmed that the obtained compounds are air
stable and have good solubility in DMF, DMSO, methanol and 1% DMSO/50 mM Tris-HCl buffer
solution. The molecular structure of the ligand (1) and complexes (2 and 3) was determined by
1
elemental analysis and various techniques, viz. FT-IR, UV-Visible, H NMR, 13C NMR and 31P
NMR spectroscopy. The molecular geometry of 1 and 3 was confirmed by the XRD study.
Cl
N
[PdCl2(EPh3)2]
Cl
N
H
O
Cl
N
Pd
CH2Cl2/MeOH, TEA
Reflux 6h
N
O
O
Cl
Cl
E
OH
Cl
Where E = P (or) As
Scheme 2. Synthesis of complexes
1
13
The spectroscopic data (FT-IR, UV-visible, H and C NMR) were collected for ligand and
complexes (2 and 3) and it was used to determine the mode of coordination of the hydrazone ligand
to palladium ion in complexes. The ligand showed strong bands at 2946 (N-H) and 1656 cm-1 (C=O)
whereas these bands disappeared in FT-IR spectra of the complexes. Similarly, free hydrazone
displays band at 1595 cm-1 corresponding to the azomethaine (C=N), whereas, in the case of

complexes, the peak shifted towards lower values [1592 (2) and 1591 cm-1 (3)]. The NH peak
disappeared completely in complexes (former), indicating the involvement of enol form of oxygen in
coordination with the palladium(II) ion [41]. The later proves the coordination of C=N group to the
palladium centre [42]. Complexes have characteristic vibrations appeared for triphenyl phosphine
and triphenyl arsine in expected region. The electronic spectra of ligand and complexes showed two
to four bands in the region 293-419 nm. The bands of complexes (2 and 3) at 297-273 nm and 361-
332 nm are due to the π-π* and n-π* intra ligand transition, respectively. The enolization followed by
the deprotonation of the ligand during complexation was confirmed by observing a shift in the n-π*
transition which appears in the region of 298 nm. Absorption spectra of complexes showed broad
band with less intensity (408 and 419 nm), which can be assigned to the ligand to metal charge
transfer transition (LMCT). Broadness of the bands may be due to the overlapping of the LMCT
transitions of O → Pd and N → Pd [43, 44].
1
In the H NMR spectrum of ligand H2L, the chemical shift for the hydroxyl proton (-OH)
appears at 12.21 ppm, whereas, the complexes show no signal in the region. Alike, a weak singlet
appears at 12.56 ppm (due to NH proton), whereas, no such resonance was attributed to NH proton
signal for complexes 2 and 3. These observations reveal that phenolic oxygen is involved in the
complexation with Pd(II) ion and also the enolate oxygen atom coordinated to the metal centre after
enolisation followed by deprotonation [45]. Further, the singlet at 8.46 ppm confirmed the presence
of azomethine proton (-CH=N). In case of complexes 2 and 3 the singlet at 8.45 ppm was shifted to
8.21 ppm in both the complexes due to the coordination of azomethine N with Pd(II) ion [46]. The
region of 7.20-7.83 ppm is ascribed to the aromatic protons for both in ligand and complexes. The
13C NMR spectrum of ligand H2L showed the chemical shift of carbonyl carbon (C=O) at 168 ppm
which is completely disappeared for complexes. Whereas new signals were noticed for the Pd-
complexes at 155 and 154 ppm (C-O), indicates the coordination of oxygen via deprotonation [47].
The ligand displayed a single resonance at 162 ppm due to the azomethine carbon atom [48]. The

downfield shift of this signal for the complexes at 174 and 176 ppm clearly indicates the
coordination of the azomethaine group. Signals in the regions of 118-152 ppm confirmed the
aromatic carbons of the ligand and complexes. Further 31P NMR spectrum was recorded for the
complex 2. A sharp singlet was observed at 20.8 ppm, thus suggested the presence of
triphenylphosphine group in complex 2 [49].
Overall, the spectroscopic results confirmed the coordination mode of benzoylhydrazone
ligand to palladium (II) ion via the phenolate oxygen (O), the azomethine nitrogen (N) and imidolate
oxygen (O) and also the presence of PPh3/AsPh3 groups.
3.2. Crystal structure description of H2L (1) and complex (3)
The single crystal X-ray diffraction studies confirmed the structure of the ligand 1 and the
molecular structure of the ligand is shown in Fig. 1. The selected bond distances and angles are listed
in Table 2. The ligand crystallizes in monoclinic C2/c space group and eight molecules present in a
unit cell. The successful formation of hydrazone ligand is confirmed by using the hydrazone C(8)-
N(2) bond length, 1.278(2) Å which confirmed the formation of azomethine C=N bond. The
presence of hydrazinic (N-H) hydrogen and bond distances (C(7)-O(1), 1.229(2) Å) in the ligand
confirms that the hydrazone exists in keto form. The N(1)-N(2) (1.3713(19) Å) and N(1)-C(7)
(1.346(2) Å) bond distances in H2L are in between the ideal values corresponding single bonds [N-N,
1.45 A; C-N, 1.47 A] and double bonds [N=N, 1.25 A, C=N, 1.28 Å], as obvious for an extended π-
delocalization throughout the ligand molecule. The hydrazine nitrogen N(1) and the oxygen O(1)
atoms are presented in a tans manner with corresponding to C(7)-N(1); with the bond length of
1.346(2) Å.
ORTEP diagram (Fig. 2) shows ONO pincer type of ligand coordinated with central metal
palladium ion via the phenolate oxygen, azomethine nitrogen and deprotonated imidole oxygen, each
forming five membered and six membered chelate rings. Complex (3) was crystallized in C2/c space

group in the monoclinic crystal system. Selected refinement data are represented in table 1. ORTEP
representation of the complex (3), is shown in Fig. 2 and unit cell packing diagram of the complex 3
is provided in ESI. In complex 3 bond lengths are 1.971(3) Å Pd(1)-N(1), 1.971(2) Å Pd(1)-O(1) and
1.993(2) Å Pd(1)-O(2) and 2.4121(4) Å Pd(1)-As(1). The trans angles of the complexes O(1)-Pd(1)-
O(2) and N(1)-Pd(1)-As(2) are 174.60(10) and 177.68(8) respectively. The selected bond lengths and
bond angles confirmed the square planar geometry around the central metal palladium with
significant distortion. Bond length and bond angle of complex 3 are in well agreement with the
previous data of palladium(II) complexes [50]. The spectroscopic and single crystal XRD studies
strongly suggested that the hydrazone ligand is coordinated to the Pd ion through ONO in a tridentate
fashion.
3.3. Stability Assays
Stability of synthesized compounds was investigated in DMSO Tris-HCl buffer as a solvent.
In fact, the DMSO is the most common solvent used to prepare stock solutions for many biological
testing and it can act as a ligand in some transition metal complexes (can coordinate the metal centre
via either the S or O atom). The time-dependent UV-vis spectral scans found that the present ligand
is stable in aqueous DMSO even after extended time intervals, indicating its suitability for cellular
studies in an aqueous medium. The UV-visible absorption spectra of the synthesized complexes 2
and 3 were recorded in buffer-DMSO solution (Fig S6†). From absorption spectra no changes (either
in both intensity and position of bands) were observed after a 24 h of period, which strongly indicate
the stability of the complexes in buffer-DMSO solution.
3.4. Nucleic acid binding studies
UV-Vis spectrophotometry, fluorescence, electrochemical, and relative viscosity
measurement were used to confirm the interactions of the synthesized compounds with CT-DNA.

The binding ability of the compounds with CT-DNA was investigated by absorption titration
technique. Fig. 3 shows the absorption spectra of the ligand and complexes in absence and presence
of CT-DNA. Increasing the amount of CT-DNA to the ligand (1) the spectrum exhibited a
hypochromism of about 31% (at 292 nm) and 37% (357 nm) with 4 nm red shifts. Interestingly,
complex 2 exhibited a hypochromism of about 58%, 56% and 57% with a red shift of 7, 4 and 1 nm
at 264, 315 and 420 nm and absorption bands of 3 at 267, 339 and 319 nm exhibited the same
incident of hypochromism of about 31%, 30% and 29% respectively with a red shift of about 1, 5, 1
nm. From the absorption changes of parent ligand and complexes, the binding to CT-DNA was
confirmed [51, 52]. In comparison to ligand, the complexes showed better hypochromicity with red
shift. This phenomenon is obviously due to the better ability of Pd(II) complexes (2 and 3) than the
parent ligand. Binding properties were evaluated through the binding constant Kb which can be
obtained from the following equation,
[DNA]/[ɛa-ɛf] = [DNA]/[ɛb-ɛf] + 1/Kb[ɛb-ɛf]
where [DNA] is the concentration of DNA in base pairs, ɛa, ɛb and ɛf are the extinction
coefficient of apparent, fully bound and free compounds in solution respectively. From the
absorption values plot of [DNA]/[ɛa-ɛf] versus [DNA] assigned as a 1/[ɛb-ɛf] while y-intercept is
1/Kb[ɛb-ɛf]. The binding constant Kb is the ratio of slope/ y-intercept (Fig. S7†). The intrinsic binding
constant (Kb) values are 1.3×104 M-1 (ligand 1), 7.1×104 M-1 (2) and 6.3×104 M-1 (3). From the
binding constant values shows intercalation mode of binding between the CT-DNA and compounds.
The Kb values of the present Pd-complexes are confirm that bind to DNA helix via intercalative
mode [53].
Further ethidium bromide (EtBr) fluorescence quenching experiment was carried out for
compounds (1-3) and the data is shown in Fig.4. The addition of compounds to EtBr causes a
significant amount of decrease in emission intensities, indicating that the compounds competitively

bound to CT-DNA with EtBr. Further, the emission intensity values were utilized according to the
Stern-Volmer equation.
F0/F= Kq [Q] + 1
where F0 and F is the emission intensity of absence and presence of compounds, Kq is the quenching
constant and [Q] is the concentration of the compound. The Kq Value is obtained as a slope from the
plot of F0/F versus [Q] (Fig. S8†). The Kq values for compounds were 1.58 x 104 M-1, 4.75 x 104 M-1
and 3.03 x 104 M-1 for the compounds (1-3) respectively. In addition apparent binding values (Kapp)
are calculated by following equation.
KEB[EB] = Kapp [compound]
where the DNA binding constant of EB, [EB] is the concentration of EB=10µM and they were (1.1 ×
105 M-1), (3.5 ×107 M-1) and (2.2 ×107M-1) for compound 1, 2 and 3 respectively. The above the
results show that complex 2 has more ability to bind with DNA. The binding and apparent constant
values express the interaction between DNA and complexes should be intercalation [54].
Electrochemical techniques have been used to study the binding properties of complexes with
DNA in order to verify the DNA modes suggested by the spectroscopic studies. DNA is added to
complexes, the new redox peak did not appear while intensity of current decreases, this changes
reveals the interaction between DNA and complexes are confirmed (Fig.5). The current intensity is
decreased in both the complexes due to the electrode surface bound with equilibrium mixture of free
DNA and palladium complex. Moreover addition of DNA showed a positive shift at the cathodic
(∆Epc) potential and anodic (∆Epa) potential of the complexes which evidences the presence of
intercalative binding properties [55]. The electrochemical studies are well acknowledged with
absorption and fluorescence studies.

Viscosity studies were useful to understand about binding properties of the ligand and
complex to DNA. Viscosity of DNA depends on both the size and shape of DNA. Viscosity of DNA
increases due to an increase in the DNA helix lengths. In fig. 6 shown that presence and absence of
compounds (1-3) in DNA solution. The relative viscosity of DNA bind to ligand and complexes
increases with increases in concentration. Increase in degree of viscosity indicates the intercalative
mode of binding of the compounds in the order of 2>3>1. Therefore, above DNA binding studies
confirmed that the metal complexes have more binding affinity than free ligand due to complexes
has more planar properties than ligand.
3.5. BSA binding studies
3.5.1. Fluorescence quenching mechanism
Protein binding properties of the ligand and complexes (1-3) with BSA protein studies were
carried out [56]. Change in fluorescence spectra in absence and presence of quencher provides the
information about nature of the binding properties. Fluorescence binding experiment was studied by
increasing the concentration of H2L and palladium(II) complexes (0-25µM) to BSA (1µM) at room
temperature. Experiments exhibits peak at 346 nm due to tryptophan residue it excited at 296 nm
were measured using the compounds as quenchers with increasing concentrations (Fig. 7). Upon
addition of test compound solution to BSA, the fluorescence intensity of tryptophan decreased
significantly which confirm the interaction between test compounds and BSA. Fluorescence intensity
of tryptophan moiety decrease in order to 65%, 72%, and 70% were obtained for compound 1, 2 and
3 respectively. Emission intensity changes would provide the interaction of compounds with BSA. It
could cause conformational changes in BSA fluorophore tryptophan environment [57]. Further
fluorescence quenching is expressed by the Stern-Volmer relation,
F0/F = 1 + Kq [Q]

where F0 and F are the absence and presence of quencher fluorescence intensities, Kq is quenching
constant and [Q] quencher concentration. Kq value of the test compounds calculated from the plot of
F0/F versus [Q] (Fig. S9†) in respect to ligand and complexes 1, 2 and 3 were found to be 5.4 × 104
M-1, 1.2 × 105 M-1 and 7.3 × 104 M-1, respectively. The quenching constant value strongly suggests
the interaction of the compounds with BSA biomolecule [58, 59].
3.5.2 Binding constant and binding sites
Further, the binding constant and number of binding sites can be calculated by using
following Scatchard equation,
log [(F0-F)/F] = log Kbin + n log[Q]
where Kbin is the binding constant of synthesized compounds with BSA and n is the number of
binding sites. The binding constant (Kbin) and the binding sites (n) have been obtained from the plot
of log[F0-F] versus log[Q] (Fig. S10†). From slope values, the binding constant Kbin are calculated,
5.9 × 104 M-1 (1), 7.7 ×105 M-1 (2), 7.1 × 104 M-1 (3) and binding sites are 0.92 (1), 1.45 (2) and 1.21
(3) respectively. The binding constant (Kbin) values suggested that the synthesized complexes have
more binding affinity than ligand. The obtained binding site value n (~1) strongly supported the
existence of single binding site in BSA protein. Calculated Kq and Kbin values are proved that
complex 2 has more interaction with BSA due to presence of triphenylphosphine as a co-ligand and
thus showed a higher binding activity.
3.5.3. UV-Vis absorption
Absorption spectroscopic technique is very simple and easy way for exploring the type of
quenching. Generally, BSA solution showed a strong band around 200 nm due to protein backbone
and another one band shown around 278 nm that is related to absorption of aromatic amino acids
(Trp, Tyr, and Phe). The absorption spectrum of the BSA solution and BSA-compounds were shown

in Fig. 8. From the spectra, the intensity range of BSA was enhanced and small blue shifts were
observed about 1, 3 and 2 nm for 1, 2 and 3 respectively. The results revealed the existence of a
static quenching mechanism in the complexes formation between BSA and palladium(II) complexes
[60].
3.6. Antioxidant activities
The antioxidant potentials (as IC50 values) of the present test compounds against reactive
radical species (DPPH, OH and NO) are given in table 3 (Fig. S11†). The IC50 value shows that the
synthesized ligand and palladium(II) complexes exhibit antioxidant properties in the order of 2>3>1
in all three radicals. In comparison to the free ligand, the Pd-complexes possess higher antioxidant
activity. This is due to chelation of the hydrazone ligand with the Pd(II) ion and more planarity of the
complexes [61-63]. Present significant aspect of the investigation is complex 2 produced excellent
antioxidant activities that are comparable with those of standard antioxidant ascorbic acid (Ac).
3.7. In vitro cytotoxicity MTT Assay
The in vitro cytotoxicity results of ligand and complexes are summarized in table 4. From the
results, ligand has poor cytotoxicity value in all the tested tumour cell lines. The palladium(II)
complexes (2 and 3) exhibited significant cytotoxic effect in lower concentration. In particular,
complex 2 have better activity than other. The good cytotoxicity activity of new complexes are
attributed to the extended planar structure induced by the π→π* conjugation resulting from the
chelation of the ligand to complexes [64, 65]. Furthermore, the high lipophilic character owing to the
presence of triphenyl phosphine group may also be the reason for the better cytotoxicity of complex
2. Moreover, comparison of the results inferred that the present palladium(II) complexes have more
activity than reported results [66].

4. CONCLUSION
Two square planar Pd(II) complexes were successfully synthesized from 2-chloro-benzoic
acid (3,5-dichloro-2-hydroxy-benzylidene)-hydrazide ligand and characterized. Their biological
properties were also investigated. From UV−vis spectroscopic titration, EtBr displacement assay,
electrochemical and viscosity studies, it is confirmed that the ligand and complexes bind with CT-
DNA through the intercalation mode due to higher planar aromatic rings and π→π* stacking
interactions, which initiate the intercalation in to the base pair of DNA double helices. Absorption
and fluorescence emission study indicates that the complexes have good binding properties with
BSA. The ligand and the complexes (2 and 3) have been screened for their in vitro free radical
scavenging activity. The complexes exhibit more ability to scavenge DPPH, OH and NO radicals and
significantly high scavenging activity was observed against superoxide and hydroxyl radicals.
Complex 2 is the most active radical scavenger than the other. The in vitro cytotoxicity of the ligand
and complexes on tumour cell lines (HeLa and MCF-7) have been assessed by MTT and the results
indicates that complex 2 shows better antitumor activity than ligand 1 and complex 3. Hence, the
complexes 2 and 3 display significant biological properties compared to the previous reports and
may be helpful to understand the mechanism of complexes with biomolecules, and also useful in the
development of new potential anticancer agents. In particular, the complex containing
triphenylphosphine coligand was found to be extremely effective.
Supporting information:
Electronic supplementary information (ESI) available: Crystallographic data for the
structures reported in this paper have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication numbers (CCDC 1504793 and CCDC 1504704, for 1
and 3 respectively). e-mail: deposit@ccdc.cam.ac.uk ; Web site http://www.ccdc.cam.ac.uk).

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Manuscript Title:
New Palladium(II) complexes with ONO chelated hydrazone ligand: Efficient candidates for
DNA/BSA interaction, antioxidant and cytotoxicity
All the authors declared no conflict of interest
Highlights
 Two palladium(II) hydrazone complexes were synthesized and characterized.

 Molecular structure of ligand (1) and complex (3) was confirmed by X-ray diffraction study.
 The complexes exhibited intercalation binding ability on DNA/BSA.
 The synthesized complexes showed good antioxidant activities.
 The cytotoxicity in vitro of synthesized compounds against two tumor cell lines was
investigated.
Fig. 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability
Fig. 2. ORTEP diagram of 3 with thermal ellipsoid at 50% probability

Fig. 3. 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

Fig. 4. Fluorescence quenching curves of ethidium bromide bound to DNA: 1, 2 and 3.
[DNA] = 10μM, [EB] = 10μM, and [complex] = 0-50 μM. Arrow shows the emission intensity
changes upon increasing complex concentration.

Fig. 5. Cyclic voltammogram of complex 2 and 3 in the absence and presence (inner line) of DNA
(10 μM). Scan rate: 100 mV s-1
Fig. 6. Effect of the compounds (1-3) on the viscosity of CT-DNA.